Sequence:

1ADKLPNIVIL ATGGTIAGSA ATGTQTTGYK AGALGVDTLI NAVPEVKKLA51NVKGEQFSNM ASENMTGDVV LKLSQRVNEL LARDDVDGVV ITHGTDTVEE101SAYFLHLTVK SDKPVVFVAA MRPATAISAD GPMNLLEAVR VAGDKQSRGR151GVMVVLNDRI GSARYITKTN ASTLDTFKAN EEGYLGVIIG NRIYYQNRID201KLHTTRSVFD VRGLTSLPKV DILYGYQDDP EYLYDAAIQH GVKGIVYAGM251GAGSVSVRGI AGMRKAMEKG VVVIRSTRTG NGIVPPDEEL PGLVSDSLNP301AHARILLMLA LTRTSDPKVI QEYFHTY

>Protein sequence for asparaginase (Erwinia chrysanthemi) monomer
ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNM
ASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAA
MRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKAN
EEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQH
GVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNP
AHARILLMLALTRTSDPKVIQEYFHTY

References:

1. Therapeutic Targets Database: TTD Biologic drug sequences in fasta format [Link]

Asparaginase erwinia chrysanthemi (recombinant)-rywn

JZP458-201

JZP458

CAS Registry Number 1349719-22-7

Protein Chemical FormulaC₁₅₄₆H₂₅₁₀N₄₃₂O₄₇₆S₉

Protein Average Weight 140000.0 Da

Rylaze, FDA APPROVED 6/30/2021, BLA 761179

L-Asparaginase (ec 3.5.1.1, L-asparagine amidohydrolase) erwinia chrysanthemi tetramer alpha4Asparaginase (Dickeya chrysanthemi subunit) 

Other Names

• Asparaginase Erwinia chrysanthemi
• Crisantaspase
• Cristantaspase
• Erwinase
• Erwinaze
• L-Asparagine amidohydrolase (Erwinia chrysanthemi subunit)

D733ET3F9O

1349719-22-7

Asparaginase erwinia chrysanthemi [USAN]

UNII-D733ET3F9O

L-Asparaginase (erwinia)

Erwinia asparaginase

L-Asparaginase, erwinia chrysanthemi

Asparaginase (erwinia chrysanthemi)

Erwinase

Asparaginase erwinia chrysanthemi

Erwinaze

Crisantaspase

Crisantaspase [INN]

L-Asparaginase (ec 3.5.1.1, L-asparagine amidohydrolase) erwinia chrysanthemi tetramer alpha4

Asparaginase erwinia sp. [MI]

Asparaginase erwinia chrysanthemi (recombinant) [USAN]

Asparaginase erwinia chrysanthemi (recombinant)

JZP-458

A hydrolase enzyme that converts L-asparagine and water to L-aspartate and NH3.

NCI: Asparaginase Erwinia chrysanthemi. An enzyme isolated from the bacterium Erwinia chrysanthemi (E. carotovora). Asparagine is critical to protein synthesis in leukemic cells, which cannot synthesize this amino acid due to the absence of the enzyme asparagine synthase. Asparaginase hydrolyzes L-asparagine to L-aspartic acid and ammonia, thereby depleting leukemic cells of asparagine and blocking protein synthesis and tumor cell proliferation, especially in the G1 phase of the cell cycle. This agent also induces apoptosis in tumor cells. The Erwinia-derived product is often used for those patients who have experienced a hypersensitivity reaction to the E. Coli formulation. (NCI Thesaurus)

• Treatment of Acute Lymphoblastic Leukemia (ALL)
• Antineoplastic Agents

Label (PDF)
Letter (PDF)

Label (PDF)

PATENT

WO 2011003633

https://patents.google.com/patent/WO2011003633A1/en

The present invention concerns a conjugate of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol, particularly wherein the polyethylene glycol has a molecular weight less than or equal to about 5000 Da, particularly a conjugate wherein the protein is a L-asparaginase from Erwinia, and its use in therapy.Proteins with L-asparagine aminohydrolase activity, commonly known as L- asparaginases, have successfully been used for the treatment of Acute Lymphoblastic Leukemia(ALL) in children for many years. ALL is the most common childhood malignancy (Avramis and Panosyan, Clin. Pharmacokinet. (2005) 44:367-393).[0003] L-asparaginase has also been used to treat Hodgkin’s disease, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).The anti-tumor activity of L-asparaginase is believed to be due to the inability or reduced ability of certain malignant cells to synthesize L-asparagine (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669). These malignant cells rely on an extracellular supply of L-asparagine. However, the L-asparaginase enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia, thereby depleting circulating pools of L-asparagine and killing tumor cells which cannot perform protein synthesis without L-asparagine (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).[0004] L-asparaginase from E. coli was the first enzyme drug used in ALL therapy and has been marketed as Elspar® in the USA or as Kidrolase® and L-asparaginase Medac® in Europe. L- asparaginases have also been isolated from other microorganisms, e.g., an L-asparaginase protein from Erwinia chrysanthemi, named crisantaspase, that has been marketed as Erwinase® (Wriston Jr., J.C. (1985) “L-asparaginase” Meth. Enzymol. 113, 608-618; Goward, CR. et al. (1992) “Rapid large scale preparation of recombinant Erwinia chrysanthemi L-asparaginase”, Bioseparation 2, 335-341). L-asparaginases from other species of Erwinia have also been identified, including, for example, Erwinia chrysanthemi 3937 (Genbank Accession#AAS67028), Erwinia chrysanthemi NCPPB 1125 (Genbank Accession #CAA31239), Erwinia carotovora (Genbank Accession #AAP92666), and Erwinia carotovora subsp. Astroseptica (Genbank Accession #AAS67027). These Erwinia chrysanthemi L-asparaginases have about 91-98% amino acid sequence identity with each other, while the Erwinia carotovora L- asparaginases have approximately 75-77% amino acid sequence identity with the Erwinia chrysanthemi L-asparaginases (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).[0005] L-asparaginases of bacterial origin have a high immunogenic and antigenic potential and frequently provoke adverse reactions ranging from mild allergic reaction to anaphylactic shock in sensitized patients (Wang, B. et al. (2003) “Evaluation of immunologic cross reaction of anti- asparaginase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients),Leukemia 17, 1583-1588). E. coli L-asparaginase is particularly immunogenic, with reports of the presence of anti-asparaginase antibodies to E. coli L-asparaginase following i.v. or i.m. administration reaching as high as 78% in adults and 70% in children (Wang, B. et al. (2003) Leukemia 17, 1583-1588).[0006] L-asparaginases from Escherichia coli and Erwinia chrysanthemi differ in their pharmacokinetic properties and have distinct immunogenic profiles, respectively (Klug Albertsen, B. et al. (2001) “Comparison of intramuscular therapy with Erwinia asparaginase and asparaginase Medac: pharmacokinetics, pharmacodynamics, formation of antibodies and influence on the coagulation system” Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown that antibodies that developed after a treatment with L-asparaginase from E. coli do not cross react with L-Asparaginase from Erwinia (Wang, B. et al., Leukemia 17 (2003) 1583-1588). Thus, L-asparaginase from Erwinia (crisantaspase) has been used as a second line treatment of ALL in patients that react to E. coli L-asparaginase (Duval, M. et al. (2002) “Comparison of Escherichia co/z-asparaginase with £Vwzm^‘α-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment ofCancer, Children’s Leukemia Group phase 3 trial” Blood 15, 2734-2739; Avramis and Panosyan,Clin. Pharmacokinet. (2005) 44:367-393).[0007] In another attempt to reduce immunogenicity associated with administration of microbial L-asparaginases, an E. coli L-asparaginase has been developed that is modified with methoxy- polyethyleneglycol (mPEG). This method is commonly known as “PEGylation” and has been shown to alter the immunological properties of proteins (Abuchowski, A. et al. (1977) “Alteration of Immunological Properties of Bovine Serum Albumin by Covalent Attachment of Polyethylene Glycol,” J.Biol.Chem. 252 (11), 3578-3581). This so-called mPEG-L- asparaginase, or pegaspargase, marketed as Oncaspar® (Enzon Inc., USA), was first approved in the U.S. for second line treatment of ALL in 1994, and has been approved for first- line therapy of ALL in children and adults since 2006. Oncaspar® has a prolonged in vivo half-life and a reduced immunogenicity/antigenicity.[0008] Oncaspar® is E. coli L-asparaginase that has been modified at multiple lysine residues using 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S. Patent No. 4,179,337). SS-PEG is aPEG reagent of the first generation that contains an instable ester linkage that is sensitive to hydro lysis by enzymes or at slightly alkaline pH values (U.S. Patent No. 4,670,417; Makromol. Chem. 1986, 187, 1131-1144). These properties decrease both in vitro and in vivo stability and can impair drug safety.[0009] Furthermore, it has been demonstrated that antibodies developed against L-asparaginase from E. coli will cross react with Oncaspar® (Wang, B. et al. (2003) “Evaluation of immunologic cross-reaction of anti-asparaginase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients),” Leukemia 17, 1583-1588). Even though these antibodies were not neutralizing, this finding clearly demonstrated the high potential for cross-hypersensitivity or cross-inactivation in vivo. Indeed, in one report 30-41% of children who received pegaspargase had an allergic reaction (Wang, B. et al. (2003) Leukemia 17, 1583-1588).[0010] In addition to outward allergic reactions, the problem of “silent hypersensitivity” was recently reported, whereby patients develop anti-asparaginase antibodies without showing any clinical evidence of a hypersensitivity reaction (Wang, B. et al. (2003) Leukemia 17, 1583-1588). This reaction can result in the formation of neutralizing antibodies to E. coli L-asparaginase and pegaspargase; however, these patients are not switched to Erwinia L-asparaginase because there are not outward signs of hypersensitivity, and therefore they receive a shorter duration of effective treatment (Holcenberg, J., J. Pediatr. Hematol. Oncol. 26 (2004) 273-274).[0011] Erwinia chrysanthemi L-asparaginase treatment is often used in the event of hypersensitivity to E. co/z-derived L-asparaginases. However, it has been observed that as many as 30-50% of patients receiving Erwinia L-asparaginase are antibody-positive (Avramis andPanosyan, Clin. Pharmacokinet. (2005) 44:367-393). Moreover, because Erwinia chrysanthemi L-asparaginase has a significantly shorter elimination half-life than the E. coli L-asparaginases, it must be administered more frequently (Avramis and Panosyan, Clin. Pharmacokinet. (2005) 44:367-393). In a study by Avramis et al., Erwinia asparaginase was associated with inferior pharmacokinetic profiles (Avramis et al., J. Pediatr. Hematol. Oncol. 29 (2007) 239-247). E. coli L-asparaginase and pegaspargase therefore have been the preferred first-line therapies for ALL over Erwinia L-asparaginase.[0012] Numerous biopharmaceuticals have successfully been PEGylated and marketed for many years. In order to couple PEG to a protein, the PEG has to be activated at its OH terminus. The activation group is chosen based on the available reactive group on the protein that will bePEGylated. In the case of proteins, the most important amino acids are lysine, cysteine, glutamic acid, aspartic acid, C-terminal carboxylic acid and the N-terminal amino group. In view of the wide range of reactive groups in a protein nearly the entire peptide chemistry has been applied to activate the PEG moiety. Examples for this activated PEG-reagents are activated carbonates, e.g., p-nitrophenyl carbonate, succinimidyl carbonate; active esters, e.g., succinimidyl ester; and for site specific coupling aldehydes and maleimides have been developed (Harris, M., Adv. Drug – A -DeI. Rev. 54 (2002), 459-476). The availability of various chemical methods for PEG modification shows that each new development of a PEGylated protein will be a case by case study. In addition to the chemistry the molecular weight of the PEG that is attached to the protein has a strong impact on the pharmaceutical properties of the PEGylated protein. In most cases it is expected that, the higher the molecular weight of the PEG, the better the improvement of the pharmaceutical properties (Sherman, M. R., Adv. Drug Del. Rev. 60 (2008), 59-68; Holtsberg, F. W., Journal of Controlled Release 80 (2002), 259-271). For example, Holtsberg et al. found that, when PEG was conjugated to arginine deaminase, another amino acid degrading enzyme isolated from a microbial source, pharmacokinetic and pharmacodynamic function of the enzyme increased as the size of the PEG attachment increased from a molecular weight of 5000Da to 20,000 Da (Holtsberg, F.W., Journal of Controlled Release 80 (2002), 259-271).[0013] However, in many cases, PEGylated biopharmaceuticals show significantly reduced activity compared to the unmodified biopharmaceutical (Fishburn, CS. (2008) Review “The Pharmacology of PEGylation: Balancing PD with PK to Generate Novel Therapeutics” J. Pharm. Sd., 1-17). In the case of L-asparaginase from Erwinia carotovora, it has been observed that PEGylation reduced its in vitro activity to approximately 57% (Kuchumova, A.V. et al. (2007) “Modification of Recombinant asparaginase from Erwinia carotovora with Polyethylene Glycol 5000” Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 1, 230-232). The L-asparaginase from Erwinia carotovora has only about 75% homology to the Erwinia chrysanthemi L-asparaginase (crisantaspase). For Oncaspar® it is also known that its in vitro activity is approximately 50% compared to the unmodified E. coli L-asparaginase.[0014] The currently available L-asparaginase preparations do not provide alternative or complementary therapies— particularly therapies to treat ALL— that are characterized by high catalytic activity and significantly improved pharmacological and pharmacokinetic properties, as well as reduced immunogenicity. L-asparaginase protein has at least about 80% homology or identity with the protein comprising the sequence of SEQ ID NO:1, more specifically at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the protein comprising the sequence of SEQ ID NO:1. SEQ ID NO:1 is as follows:ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGE QFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTV KSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSA RYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKV DILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY (SEQ ID NO:1) [0048] The term “comprising the sequence of SEQ ID NO:1” means that the amino-acid sequence of the protein may not be strictly limited to SEQ ID NO:1 but may contain additional amino-acids.ExamplesExample 1 : Preparation of Recombinant Crisantaspase [0100] The recombinant bacterial strain used to manufacture the naked recombinant Erwinia chrysanthemi L-asparaginase protein (also referred to herein as “r-crisantaspase”) was an E. coli BL21 strain with a deleted ansB gene (the gene encoding the endogenous E. coli type II L- asparaginase) to avoid potential contamination of the recombinant Erwinia chrysanthemi L- asparaginase with this enzyme. The deletion of the ansB gene relies on homologous recombination methods and phage transduction performed according to the three following steps:1) a bacterial strain (NMI lOO) expressing a defective lambda phage which supplies functions that protect and recombine electroporated linear DNA substrate in the bacterial cell was transformed with a linear plasmid (kanamycin cassette) containing the kanamycin gene flanked by an FLP recognition target sequence (FRT). Recombination occurs to replace the ansB gene by the kanamycin cassette in the bacterial genome, resulting in a ΛansB strain; 2) phage transduction was used to integrate the integrated kanamycin cassette region from the ΛansB NMI lOO strain to the ansB locus in BL21 strain. This results in an E. coli BL21 strain with a deleted ansB gene and resistant to kanamycin; 3) this strain was transformed with a FLP -helper plasmid to remove the kanamycin gene by homologous recombination at the FRT sequence. The genome of the final strain (BL21 ΛansB strain) was sequenced, confirming full deletion of the endogenous ansB gene.[0101] The E. co/z^‘-optimized DNA sequence encoding for the mature Erwinia chrysanthemi L- asparaginase fused with the ENX signal peptide from Bacillus subtilis was inserted into an expression vector. This vector allows expression of recombinant Erwinia chrysanthemi L- asparaginase under the control of hybrid T5/lac promoter induced by the addition of Isopropyl β- D-1-thiogalactopyranoside (IPTG) and confers resistance to kanamycin.[0102] BL21 ΛansB strain was transformed with this expression vector. The transformed cells were used for production of the r-crisantaspase by feed batch glucose fermentation in Reisenberg medium. The induction of the cell was done 16h at 23°C with IPTG as inducer. After cell harvest and lysis by homogenization in 1OmM sodium phosphate buffer pH6 5mM EDTA (Buffer A), the protein solution was clarified by centrifugation twice at 1500Og, followed by 0.45μm and 0.22μm filtration steps. The recombinant Erwinia chrysanthemi L-asparaginase was next purified using a sequence of chromatography and concentration steps. Briefly, the theoretical isoelectric point of the Erwinia chrysanthemi L-asparaginase (7.23) permits the recombinant enzyme to adsorb to cation exchange resins at pH6. Thus, the recombinant enzyme was captured on a Capto S column (cation exchange chromatography) and eluted with salt gradient in Buffer A. Fractions containing the recombinant enzyme were pooled. The pooled solution was next purified on Capto MMC column (cation exchange chromatography) in Buffer A with salt gradient. . The eluted fractions containing Erwinia chrysanthemi L-asparaginase were pooled and concentrated before protein separation on Superdex 200pg size exclusion chromatography as polishing step. Fractions containing recombinant enzymes were pooled, concentrated, and diafiltered against 10OmM sodium phosphate buffer pH8. The purity of the final Erwinia chrysanthemi L-asparaginase preparation was evaluated by SDS-PAGE (Figure 1) and RP-HPLC and was at least 90%. The integrity of the recombinant enzyme was verified byN-terminal sequencing and LC-MS. Enzyme activity was measured at 37°C using Nessler’s reagent. The specific activity of the purified recombinant Erwinia chrysanthemi L-asparaginase was around 600 U/mg. One unit of enzyme activity is defined as the amount of enzyme that liberates lμmol of ammonia from L-asparagine per minute at 37°C. Example 2: Preparation of 10 kDa mPEG-L- Asparaginase Conjugates[0103] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer at pH 8.0, at a protein concentration between 2.5 and 4 mg/mL, in the presence of 150 mg/mL or 36 mg/mL 10 kDa mPEG-NHS, for 2 hours at 22°C. The resulting crude 10 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein-containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 10 kDa mPEG-L-asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as a reference, one corresponding to full PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N-terminus) residues being conjugated corresponding to PEGylation of 78% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (39% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 50% of accessible amino groups (e.g., lysine residues and/or the N-terminus)) . SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.Example 3: Preparation of 5 kDa mPEG-L-Asparaginase Conjugates[0104] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer at pH 8.0, at a protein concentration of 4 mg/mL, in the presence of 150 mg/mL or 22.5 mg/mL 5 kDa mPEG-NHS, for 2 hours at 22°C. The resulting crude 5 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein-containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 5 kDa mPEG-L- asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as a reference, one corresponding to full PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N-terminus) being conjugated corresponding to PEGylation of 84% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (36% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 43% of accessible amino groups (e.g., lysine residues and/or the N-terminus)). SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.Example 4: Preparation of 2 kDa mPEG-L-Asparaginase Conjugates[0105] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer pH 8.0 at a protein concentration of 4 mg/mL in the presence of150 mg/mL or 22.5 mg/mL 2 kDa mPEG-NHS for 2 hours at 22°C. The resulting crude 2 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 2 kDa mPEG-L- asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as reference, one corresponding to maximum PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N- terminus) being conjugated corresponding to PEGylation of 86% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (47% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 55% of accessible amino groups {e.g., lysine residues and/or the N-terminus)). SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.Example 5: Activity of mPEG-r-Crisantaspase Conjugates[0106] L-asparaginase aminohydrolase activity of each conjugate described in the proceeding examples was determined by Nesslerization of ammonia that is liberated from L-asparagine by enzymatic activity. Briefly, 50μL of enzyme solution were mixed with 2OmM of L-asparagine in a 50 mM Sodium borate buffer pH 8.6 and incubated for 10 min at 37°C. The reaction was stopped by addition of 200μL of Nessler reagent. Absorbance of this solution was measured at 450 nm. The activity was calculated from a calibration curve that was obtained from Ammonia sulfate as reference. The results are summarized in Table 2, below:Table 2: Activity of mPEG-r-crisantaspase conjugates

* the numbers “40%” and “100%” indicate an approximate degree of PEGylation of respectively 40-55% and 100% of accessible amino groups (see Examples 2-4, supra).** the ratio mol PEG / mol monomer was extrapolated from data using TNBS assay, that makes the assumption that all amino groups from the protein (e.g., lysine residues and the N-terminus) are accessible.[0107] Residual activity of mPEG-r-crisantaspase conjugates ranged between 483 and 543 Units/mg. This corresponds to 78-87% of L-asparagine aminohydrolase activity of the unmodified enzyme. Example 6: L-Asparagine-Depleting Effect of Unmodified Crisantaspase

PAPER

Biotechnology and Applied Biochemistry (2019), 66(3), 281-289.  |

https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bab.1723

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L⁻¹ H⁻¹, respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg⁻¹, 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5–9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a K_M value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL.

ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSN

MASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVV

FVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNAST

LDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEY

LYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEE

LPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

Figure S1 – Amino acid sequence of the enzyme crisantaspase without the signal peptide and with the lysines highlighted in red (Swiss-Prot/TrEMBL accession number: P06608|22-348 AA).

……………………………………………………………………………………………………………………………..

As a component of a chemotherapy regimen to treat acute lymphoblastic leukemia and lymphoblastic lymphoma in patients who are allergic to E. coli-derived asparaginase products
Press ReleaseFor Immediate Release:June 30, 2021

FDA Approves Component of Treatment Regimen for Most Common Childhood Cancer

Alternative Has Been in Global Shortage Since 2016

Today, the U.S. Food and Drug Administration approved Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) as a component of a chemotherapy regimen to treat acute lymphoblastic leukemia and lymphoblastic lymphoma in adult and pediatric patients who are allergic to the E. coli-derived asparaginase products used most commonly for treatment. The only other FDA-approved drug for such patients with allergic reactions has been in global shortage for years.

“It is extremely disconcerting to patients, families and providers when there is a lack of access to critical drugs for treatment of a life-threatening, but often curable cancer, due to supply issues,” said Gregory Reaman, M.D., associate director for pediatric oncology in the FDA’s Oncology Center of Excellence. “Today’s approval may provide a consistently sourced alternative to a pivotal component of potentially curative therapy for children and adults with this type of leukemia.”

Acute lymphoblastic leukemia occurs in approximately 5,700 patients annually, about half of whom are children. It is the most common type of childhood cancer. One component of the chemotherapy regimen is an enzyme called asparaginase that kills cancer cells by depriving them of substances needed to survive. An estimated 20% of patients are allergic to the standard E. coli-derived asparaginase and need an alternative their bodies can tolerate.

Rylaze’s efficacy was evaluated in a study of 102 patients who either had a hypersensitivity to E. coli-derived asparaginases or experienced silent inactivation. The main measurement was whether patients achieved and maintained a certain level of asparaginase activity. The study found that the recommended dosage would provide the target level of asparaginase activity in 94% of patients.

The most common side effects of Rylaze include hypersensitivity reactions, pancreatic toxicity, blood clots, hemorrhage and liver toxicity.

This review was conducted under Project Orbis, an initiative of the FDA Oncology Center of Excellence. Project Orbis provides a framework for concurrent submission and review of oncology drugs among international partners. For this review, FDA collaborated with Health Canada, where the application review is pending.

Rylaze received Fast Track and Orphan Drug designations for this indication. Fast Track is a process designed to facilitate the development and expedite the review of drugs to treat serious conditions and fulfill an unmet medical need. Orphan Drug designation provides incentives to assist and encourage drug development for rare diseases.

The FDA granted approval of Rylaze to Jazz Pharmaceuticals.

REF

https://www.prnewswire.com/news-releases/jazz-pharmaceuticals-announces-us-fda-approval-of-rylaze-asparaginase-erwinia-chrysanthemi-recombinant-rywn-for-the-treatment-of-acute-lymphoblastic-leukemia-or-lymphoblastic-lymphoma-301323782.html#:~:text=Jazz%20Pharmaceuticals%20Announces,details%20to%20follow

DUBLIN, June 30, 2021 /PRNewswire/ — Jazz Pharmaceuticals plc (Nasdaq: JAZZ) today announced the U.S. Food and Drug Administration (FDA) approval of Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) for use as a component of a multi-agent chemotherapeutic regimen for the treatment of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in pediatric and adult patients one month and older who have developed hypersensitivity to E. coli-derived asparaginase.¹ Rylaze is the only recombinant erwinia asparaginase manufactured product that maintains a clinically meaningful level of asparaginase activity throughout the entire duration of treatment, and it was developed by Jazz to address the needs of patients and healthcare providers with an innovative, high-quality erwinia-derived asparaginase with reliable supply.

“We are excited to bring this important new treatment to patients who are in critical need, and we are grateful to FDA for the approval of Rylaze based on its established safety and efficacy profile. We are pleased Rylaze was approved before the trial is complete and are diligently working to advance additional clinical trial data. We are committed to quickly engaging with FDA to evolve the Rylaze product profile with additional dosing options and an IV route of administration,” said Bruce Cozadd, chairman and CEO of Jazz Pharmaceuticals. “Thank you to our collaborators within the Children’s Oncology Group, the clinical trial investigators, patients and their families, and all of the other stakeholders who helped us achieve this significant milestone.”

Rylaze was granted orphan drug designation for the treatment of ALL/LBL by FDA in June 2021. The Biologics Licensing Application (BLA) approval followed review under the Real-Time Oncology Review (RTOR) program, an initiative of FDA’s Oncology Center of Excellence designed for efficient delivery of safe and effective cancer treatments to patients.

The company expects Rylaze will be commercially available in mid-July.

“The accelerated development and approval of Rylaze marks an important step in bringing a meaningful new treatment option for many ALL patients – most of whom are children – who cannot tolerate E. coli-derived asparaginase medicine,” said Dr. Luke Maese, assistant professor at the University of Utah, Primary Children’s Hospital and Huntsman Cancer Institute. “Before the approval of Rylaze, there was a significant need for an effective asparaginase medicine that would allow patients to start and complete their prescribed treatment program with confidence in supply.”

Recent data from a Children’s Oncology Group retrospective analysis of over 8,000 patients found that patients who did not receive a full course of asparaginase treatment due to associated toxicity had significantly lower survival outcomes – regardless of whether those patients were high risk or standard risk, slow early responders.²

About Study JZP458-201
The FDA approval of Rylaze, also known as JZP458, is based on clinical data from an ongoing pivotal Phase 2/3 single-arm, open-label, multicenter, dose confirmation study evaluating pediatric and adult patients with ALL or LBL who have had an allergic reaction to E. coli-derived asparaginases and have not previously received asparaginase erwinia chrysanthemi. The study was designed to assess the safety, tolerability and efficacy of JZP458. The determination of efficacy was measured by serum asparaginase activity (SAA) levels. The Phase 2/3 study is being conducted in two parts. The first part is investigating the intramuscular (IM) route of administration, including a Monday-Wednesday-Friday dosing schedule. The second part remains active to further confirm the dose and schedule for the intravenous (IV) route of administration.

The FDA approval of Rylaze was based on data from the first of three IM cohorts, which demonstrated the achievement and maintenance of nadir serum asparaginase activity (NSAA) greater than or equal to the level of 0.1 U/mL at 48 hours using IM doses of Rylaze 25 mg/m². The results of modeling and simulations showed that for a dosage of 25 mg/m² administered intramuscularly every 48 hours, the proportion of patients maintaining NSAA ≥ 0.1 U/mL at 48 hours after a dose of Rylaze was 93.6% (95% CI: 92.6%, 94.6%).¹

The most common adverse reactions (incidence >15%) were abnormal liver test, nausea, musculoskeletal pain, fatigue, infection, headache, pyrexia, drug hypersensitivity, febrile neutropenia, decreased appetite, stomatitis, bleeding and hyperglycemia. In patients treated with the Rylaze, a fatal adverse reaction (infection) occurred in one patient and serious adverse reactions occurred in 55% of patients. The most frequent serious adverse reactions (in ≥5% of patients) were febrile neutropenia, dehydration, pyrexia, stomatitis, diarrhea, drug hypersensitivity, infection, nausea and viral infection. Permanent discontinuation due to an adverse reaction occurred in 9% of patients who received Rylaze. Adverse reactions resulting in permanent discontinuation included hypersensitivity (6%) and infection (3%).¹

The company will continue to work with FDA and plans to submit additional data from a completed cohort of patients evaluating 25mg/m² IM given on Monday and Wednesday, and 50 mg/m² given on Friday in support of a M/W/F dosing schedule. Part 2 of the study is evaluating IV administration and is ongoing. The company also plans to submit these data for presentation at a future medical meeting.

Investor Webcast
The company will host an investor webcast on the Rylaze approval in July. Details will be announced separately.

About Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn)
Rylaze, also known as JZP458, is approved in the U.S. for use as a component of a multi-agent chemotherapeutic regimen for the treatment of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in pediatric and adult patients one month and older who have developed hypersensitivity to E. coli-derived asparaginase. Rylaze has orphan drug designation for the treatment of ALL/LBL in the United States. Rylaze is a recombinant erwinia asparaginase that uses a novel Pseudomonas fluorescens expression platform. JZP458 was granted Fast Track designation by the U.S. Food and Drug Administration (FDA) in October 2019 for the treatment of this patient population. Rylaze was approved as part of the Real-Time Oncology Review program, an initiative of the FDA’s Oncology Center of Excellence designed for efficient delivery of safe and effective cancer treatments to patients.

The full U.S. Prescribing Information for Rylaze is available at: <http://pp.jazzpharma.com/pi/rylaze.en.USPI.pdf>

Important Safety Information

RYLAZE should not be given to people who have had:

• Serious allergic reactions to RYLAZE
• Serious swelling of the pancreas (stomach pain), serious blood clots, or serious bleeding during previous asparaginase treatment

RYLAZE may cause serious side effects, including:

• Allergic reactions (a feeling of tightness in your throat, unusual swelling/redness in your throat and/or tongue, or trouble breathing), some of which may be life-threatening
• Swelling of the pancreas (stomach pain)
• Blood clots (may have a headache or pain in leg, arm, or chest)
• Bleeding
• Liver problems

Contact your doctor immediately if any of these side effects occur.

Some of the most common side effects with RYLAZE include: liver problems, nausea, bone and muscle pain, tiredness, infection, headache, fever, allergic reactions, fever with low white blood cell count, decreased appetite, mouth swelling (sometimes with sores), bleeding, and too much sugar in the blood.

RYLAZE can harm your unborn baby. Inform your doctor if you are pregnant, planning to become pregnant, or nursing. Females of reproductive potential should use effective contraception (other than oral contraceptives) during treatment and for 3 months following the final dose. Do not breastfeed while receiving RYLAZE and for 1 week after the final dose.

Tell your healthcare provider if there are any side effects that are bothersome or that do not go away.

These are not all the possible side effects of RYLAZE. For more information, ask your healthcare provider.

You are encouraged to report negative side effects of prescription drugs to the FDA. Visit www.fda.gov/medwatch, or call 1-800-FDA-1088 (1-800-332-1088).

About ALL
ALL is a cancer of the blood and bone marrow that can progress quickly if not treated.³ Leukemia is the most common cancer in children, and about three out of four of these cases are ALL.⁴  Although it is one of the most common cancers in children, ALL is among the most curable of the pediatric malignancies due to recent advancements in treatment.^5,6 Adults can also develop ALL, and about four of every 10 cases of ALL diagnosed are in adults.⁷  The American Cancer Society estimates that almost 6,000 new cases of ALL will be diagnosed in the United States in 2021.⁷ Asparaginase is a core component of multi-agent chemotherapeutic regimens in ALL.⁸  However, asparaginase treatments derived from E. coli are associated with the potential for development of hypersensitivity reactions.⁹

About Lymphoblastic Lymphoma
LBL is a rare, fast-growing, aggressive subtype of Non-Hodgkin’s lymphoma, most often seen in teenagers and young adults.⁸ LBL is a very aggressive lymphoma – also called high-grade lymphoma – which means the lymphoma grows quickly with early spread to different parts of the body.^10,11

About Jazz Pharmaceuticals plc
Jazz Pharmaceuticals plc (NASDAQ: JAZZ) is a global biopharmaceutical company whose purpose is to innovate to transform the lives of patients and their families. We are dedicated to developing life-changing medicines for people with serious diseases – often with limited or no therapeutic options. We have a diverse portfolio of marketed medicines and novel product candidates, from early- to late-stage development, in neuroscience and oncology. We actively explore new options for patients including novel compounds, small molecules and biologics, and through cannabinoid science and innovative delivery technologies. Jazz is headquartered in Dublin, Ireland and has employees around the globe, serving patients in nearly 75 countries. For more information, please visit www.jazzpharmaceuticals.com and follow @JazzPharma on Twitter.

About The Children’s Oncology Group (COG)
COG (childrensoncologygroup.org), a member of the NCI National Clinical Trials Network (NCTN), is the world’s largest organization devoted exclusively to childhood and adolescent cancer research. COG unites over 10,000 experts in childhood cancer at more than 200 leading children’s hospitals, universities, and cancer centers across North America, Australia, and New Zealand in the fight against childhood cancer. Today, more than 90% of the 14,000 children and adolescents diagnosed with cancer each year in the United States are cared for at COG member institutions. Research performed by COG institutions over the past 50 years has transformed childhood cancer from a virtually incurable disease to one with a combined 5-year survival rate of 80%. COG’s mission is to improve the cure rate and outcomes for all children with cancer.

Caution Concerning Forward-Looking Statements 
This press release contains forward-looking statements, including, but not limited to, statements related to Jazz Pharmaceuticals’ belief in the potential of Rylaze to provide a reliable therapeutic option for adult and pediatric patients to maximize their chance for a cure, plans for a mid-July 2021 launch of Rylaze, the availability of a reliable supply of Rylaze and other statements that are not historical facts. These forward-looking statements are based on Jazz Pharmaceuticals’ current plans, objectives, estimates, expectations and intentions and inherently involve significant risks and uncertainties. Actual results and the timing of events could differ materially from those anticipated in such forward-looking statements as a result of these risks and uncertainties, which include, without limitation, effectively launching and commercializing new products; obtaining and maintaining adequate coverage and reimbursement for the company’s products; delays or problems in the supply or manufacture of the company’s products and other risks and uncertainties affecting the company, including those described from time to time under the caption “Risk Factors” and elsewhere in Jazz Pharmaceuticals’ Securities and Exchange Commission filings and reports (Commission File No. 001-33500), including Jazz Pharmaceuticals’ Annual Report on Form 10-K for the year ended December 31, 2020 and future filings and reports by Jazz Pharmaceuticals. Other risks and uncertainties of which Jazz Pharmaceuticals is not currently aware may also affect Jazz Pharmaceuticals’ forward-looking statements and may cause actual results and the timing of events to differ materially from those anticipated. The forward-looking statements herein are made only as of the date hereof or as of the dates indicated in the forward-looking statements, even if they are subsequently made available by Jazz Pharmaceuticals on its website or otherwise. Jazz Pharmaceuticals undertakes no obligation to update or supplement any forward-looking statements to reflect actual results, new information, future events, changes in its expectations or other circumstances that exist after the date as of which the forward-looking statements were made.

Jazz Media Contact:
Jacqueline Kirby
Vice President, Corporate Affairs
Jazz Pharmaceuticals plc
CorporateAffairsMediaInfo@jazzpharma.com
Ireland, +353 1 697 2141
U.S. +1 215 867 4910

Jazz Investor Contact:
Andrea N. Flynn, Ph.D.
Vice President, Head, Investor Relations
Jazz Pharmaceuticals plc
investorinfo@jazzpharma.com  
Ireland, +353 1 634 3211

References

1. Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) injection, for intramuscular use Prescribing Information. Palo Alto, CA: Jazz Pharmaceuticals, Inc.
2. Gupta S, Wang C, Raetz EA et al. Impact of Asparaginase Discontinuation on Outcome in Childhood Acute Lymphoblastic Leukemia: A Report From the Children’s Oncology Group. J Clin Oncol. 2020 Jun 10;38(17):1897-1905. doi: 10.1200/JCO.19.03024
3. National Cancer Institute. Adult Acute Lymphoblastic Leukemia Treatment (PDQ^®)–Patient Version. Available at www.cancer.gov/types/leukemia/patient/adult-all-treatment-pdq. Accessed June 29, 2021
4. American Cancer Society. Key Statistics for Childhood Leukemia. Available at https://www.cancer.org/cancer/leukemia-in-children/about/key-statistics.html. Accessed June 29, 2021.
5. American Cancer Society. Cancer Facts & Figures 2019. www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2019.html. Accessed June 29, 2021.
6. Pui C, Evans W. A 50-Year Journey to Cure Childhood Acute Lymphoblastic Leukemia. Seminars in Hematology. 2013;50(3), 185-196.
7. American Cancer Society. Key Statistics for Acute Lymphocytic Leukemia (ALL). Available at https://cancerstatisticscenter.cancer.org/?_ga=2.8163506.1018157754.1621008457-1989786785.1621008457#!/data-analysis/NewCaseEstimates. Accessed June 29, 2021.
8. Salzer W, Bostrom B, Messinger Y et al. 2018. Asparaginase activity levels and monitoring in patients with acute lymphoblastic leukemia. Leukemia & Lymphoma. 59:8, 1797-1806, DOI: 10.1080/10428194.2017.1386305.
9. Hijiya N, van der Sluis IM. Asparaginase-associated toxicity in children with acute lymphoblastic leukemia. Leuk Lymphoma. 2016;57(4):748–757. DOI: 10.3109/10428194.2015.1101098.
10. Leukemia Foundation. Lymphoblastic Lymphoma. Available at https://www.leukaemia.org.au/disease-information/lymphomas/non-hodgkin-lymphoma/other-non-hodgkin-lymphomas/lymphoblastic-lymphoma/. Accessed June 29, 2021.
11. Mayo Clinic. Acute Lymphocytic Leukemia Diagnosis. Available at https://www.mayoclinic.org/diseases-conditions/acute-lymphocytic-leukemia/diagnosis-treatment/drc-20369083. Accessed June 29, 2021.

SOURCE Jazz Pharmaceuticals plc

Related Links

CLIP

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776285/

/////////////asparaginase erwinia chrysanthemi (recombinant)-rywn, Rylaze, Jazz Pharmaceuticals, JZP458-201, JZP458, FDA 2021, APPROVALS 2021, ORPHAN, Fast Track, Acute Lymphoblastic Leukemia, ALL, Antineoplastic Agents

https://chem.nlm.nih.gov/chemidplus/id/1349719227

https://go.drugbank.com/drugs/DB08886

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BELUMOSUDIL

MW 452.5

911417-87-3, SLx-2119, KD-025, KD 025, WHO 11343

2-[3-[4-(1H-indazol-5-ylamino)quinazolin-2-yl]phenoxy]-N-propan-2-ylacetamide

2-(3-(4-(lH-indazol-5-ylamino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide

Belumosudil mesylate

KD025 mesylate

2109704-99-4

Belumosudil mesylate is an orally available rho kinase 2 (ROCK 2) inhibitor being developed at Kadmon. In 2020, the drug candidate was submitted for a new drug application (NDA) in the U.S., under a real-time oncology review pilot program, for the treatment of chronic graft-versus-host disease (cGVHD). The compound is also in phase II clinical development for the treatment of idiopathic pulmonary fibrosis and diffuse cutaneous systemic sclerosis. Formerly, the company had also been conducting clinical research for the treatment of psoriasis and non-alcoholic steatohepatitis (NASH); however, no further development has been reported for these indications. Originally developed by Nano Terra, the product was licensed to Kadmon on an exclusive global basis in 2011. In 2019, Kadmon entered into a strategic partnership with BioNova Pharmaceuticals and established a joint venture, BK Pharmaceuticals, to exclusively develop and commercialize KD-025 for the treatment of graft-versus-host disease in China. The compound has been granted breakthrough therapy designation in the U.S. for the treatment of cGVHD and orphan drug designations for cGVHD and systemic sclerosis. In the E.U. belumosudil was also granted orphan drug status in the E.U. for the treatment of cGVHD.

Kadmon , under license from NT Life Sciences , is developing belumosudil as mesylate salt, a ROCK-2 inhibitor, for treating IPF, chronic graft-versus-host disease, hepatic impairment and scleroderma. In July 2021, belumosudil was reported to be in pre-registration phase.

Belumosudil (formerly KD025 and SLx-2119) is an experimental drug being explored for the treatment of chronic graft versus host disease (cGvHD), idiopathic pulmonary fibrosis (IPF), and moderate to severe psoriasis. It is an inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2; ROCK-II).^[1] Belumosudil binds to and inhibits the serine/threonine kinase activity of ROCK2. This inhibits ROCK2-mediated signaling pathways which play major roles in pro- and anti-inflammatory immune cell responses. A genomic study in human primary cells demonstrated that the drug also has effects on oxidative phosphorylation, WNT signaling, angiogenesis, and KRAS signaling.^[2] Originally developed by Surface Logix, Inc,^[1] Belumosudil was later acquired by Kadmon Corporation. As of July 2020 the drug was in completed or ongoing Phase II clinical studies for cGvHD, IPF and psoriasis.^[3]

cGvHD is a complication that can follow stem cell or hematopoietic stem cell transplantation where the transplanted cells (graft) attack healthy cells (host). This causes inflammation and fibrosis in multiple tissues. Two cytokines controlled by the ROCK2 signaling pathway, IL-17 and IL-21, have a major role in the cGvHD response. In a 2016 report using both mouse models and a limited human clinical trial ROCK2 inhibition with belumosudil targeted both the immunologic and fibrotic components of cGvHD and reversed the symptoms of the disease.^[4] In October 2017 KD025 was granted orphan drug status in the United States for treatment of patients with cGvHD.^[5]

IPF is a progressive fibrotic disease where the lining of the lungs become thickened and scarred.^[6] Increased ROCK activity has been found in the lungs of humans and animals with IPF. Treatment with belumosudil reduced lung fibrosis in a bleomycin mouse model study.^[7] Belumosudil may have a therapeutic benefit in IPF by targeting the fibrotic processes mediated by the ROCK signaling pathway.

Psoriasis is an inflammatory skin condition where patients experiences eruptions and remissions of thickened, erythematous, and scaly patches of skin. Down-regulation of pro-inflammatory responses was observed with KD025 treatment in Phase 2 clinical studies in patients with moderate to severe psoriasis.^[8]
“Substance Name:Substance Name: Belumosudil [USAN]”.

PATENT

https://patents.google.com/patent/WO2012040499A2/en

PATENT

https://patents.google.com/patent/CN106916145A/en

WO 2014055996, WO 2015157556

Patent

WO-2021129589

Novel crystalline polymorphic forms (N1, N2 and N15) of KD-025 (also known as belumosudil ), useful as a Rho A kinase 2 (ROCK-2) inhibitor for treating multiple sclerosis, psoriasis, rheumatoid arthritis, idiopathic pulmonary fibrosis (IPF), atherosclerosis, non-alcoholic fatty liver and systemic sclerosis. Represents the first filing from Sunshine Lake Pharma or its parent HEC Pharm that focuses on belumosudil.KD-025 is a selective ROCK2 (Rho-associated protein kinase 2, Rho-related protein kinase 2) inhibitor. It has multiple clinical indications such as the treatment of multiple sclerosis, psoriasis, rheumatoid arthritis, and Primary pulmonary fibrosis, atherosclerosis, non-alcoholic fatty liver, etc., among which many indications are in clinical phase I, and psoriasis and systemic sclerosis are in clinical phase II.
The structure of KD-025 is shown in the following formula (1).

Example 1 Preparation method of crystal form N1 of KD-025[0222]300mg of KD-025 solid was suspended and stirred in 10mL methanol at room temperature. After 22h, it was filtered, suction filtered and placed in a drying oven at 50°C under vacuum overnight to obtain 262mg of powder. The obtained crystal was detected by XPRD and confirmed to be KD-025 crystal form N1; its X-ray powder diffraction pattern was basically the same as that of Fig. 1, its DSC pattern was basically the same as that of Fig. 2, and the TGA pattern was basically the same as that of Fig. 3.

PATENT

WO2006105081 ,

Belumosudil product pat, 

protection in the EU states until March 2026, expires in the US in May 2029 with US154 extension.

Example 82
2-(3-(4-(lH-indazol-5-ylamino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide

[0257] A suspension of 2-(3-(4-(lH-indazol-5-ylamino)qumazolin-2-yl)ρhenoxy)acetic acid (70 mg, 0.14 mmol), PyBOP^® (40 mg, 0.077 mmol), DlEA (24 μL, 0.14 mmol) in dry CH₂Cl₂ : DMF (2 : 0.1 mL) was stirred at RT for 15 minutes. To this solution of activated acid was added propan-2-amine (5.4 mg, 0.091 mmol). After 30 minutes, 1.0 equivalent of DIEA and 0.55 equivalents of PyBOP^® were added. After stirring the solution for 15 minutes, 0.65 equivalents of propan-2-aminewere added and the mixture was stirred for an additional 30 minutes. The solvent was removed in vacuo and the crude product was purified using prep HPLC (25-50 90 rnins) to afford 2-(3-(4-(lH-indazol-5-ylamino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide. (40 mg, 0.086 mmol, 61 %).

References

1. ^ Jump up to:^a b Boerma M, Fu Q, Wang J, Loose DS, Bartolozzi A, Ellis JL, et al. (October 2008). “Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin”. Blood Coagulation & Fibrinolysis. 19 (7): 709–18. doi:10.1097/MBC.0b013e32830b2891. PMC 2713681. PMID 18832915.
2. ^ Park J, Chun KH (5 May 2020). “Identification of novel functions of the ROCK2-specific inhibitor KD025 by bioinformatics analysis”. Gene. 737: 144474. doi:10.1016/j.gene.2020.144474. PMID 32057928.
3. ^ “KD025 – Clinical Trials”. ClinicalTrials.gov. Retrieved 25 July 2020.
4. ^ Flynn R, Paz K, Du J, Reichenbach DK, Taylor PA, Panoskaltsis-Mortari A, et al. (April 2016). “Targeted Rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through a Stat3-dependent mechanism”. Blood. 127 (17): 2144–54. doi:10.1182/blood-2015-10-678706. PMC 4850869. PMID 26983850.
5. ^ Shanley M (October 6, 2017). “Therapy to Treat Transplant Complications Gets Orphan Drug Designation”. RareDiseaseReport. Retrieved 25 July 2018.
6. ^ “Pulmonary Fibrosis”. The Mayo Clinic. Retrieved July 25, 2018.
7. ^ Semedo D (June 5, 2016). “Phase 2 Study of Molecule Inhibitor for Idiopathic Pulmonary Fibrosis Begins”. Lung Disease News. BioNews Services, LLC. Retrieved 25 July 2018.
8. ^ Zanin-Zhorov A, Weiss JM, Trzeciak A, Chen W, Zhang J, Nyuydzefe MS, et al. (May 2017). “Cutting Edge: Selective Oral ROCK2 Inhibitor Reduces Clinical Scores in Patients with Psoriasis Vulgaris and Normalizes Skin Pathology via Concurrent Regulation of IL-17 and IL-10”. Journal of Immunology. 198 (10): 3809–3814. doi:10.4049/jimmunol.1602142. PMC 5421306. PMID 28389592.

////////////BELUMOSUDIL, SLx-2119, KD-025, KD 025, WHO 11343, PHASE 2, cGvHD, IPF,  psoriasis, Breakthrough Therapy, Orphan Drug Designation

CC(C)NC(=O)COC1=CC=CC(=C1)C2=NC3=CC=CC=C3C(=N2)NC4=CC5=C(C=C4)NN=C5

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DEUCRAVACITINIB

BMS-986165

CAS 1609392-27-9, C20H22N8O3, 425.46

6-(cyclopropanecarbonylamino)-4-[2-methoxy-3-(1-methyl-1,2,4-triazol-3-yl)anilino]-N-(trideuteriomethyl)pyridazine-3-carboxamide

6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

Tyk2-IN-4

UNII-N0A21N6RAU

N0A21N6RAU

GTPL10432

EX-A3154

BDBM50507816

NSC825520

s8879

• OriginatorBristol-Myers Squibb
• ClassAmides; Aniline compounds; Anti-inflammatories; Antipsoriatics; Antirheumatics; Cyclopropanes; Ethers; Hepatoprotectants; Organic deuterium compounds; Pyridazines; Skin disorder therapies; Small molecules; Triazoles
• Mechanism of ActionTYK2 kinase inhibitors

• Phase IIIPlaque psoriasis
• Phase IICrohn’s disease; Lupus nephritis; Psoriatic arthritis; Systemic lupus erythematosus; Ulcerative colitis
• Phase IAutoimmune disorders
• No development reportedInflammatory bowel diseases; Psoriasis

• 02 Jul 2021Bristol-Myers Squibb plans a phase I pharmacokinetics trial (In volunteers) in USA (PO, Tablet) in July 2021 (NCT04949269)
• 14 Jun 2021Bristol-Myers Squibb plans a phase III trial for Psoriatic arthritis (Treatment-naïve) in USA, Brazil, Colombia, Czech republic, Hungary, Italy, Mexico, Romania, Spain and Taiwan in July 2021 (NCT04908202) (EudraCT2020-005097-10)
• 02 Jun 2021Interim efficacy and adverse events data from the phase III POETYK-PSO-1 trial in Psoriatic psoriasis presented at the 22nd Annual Congress of the European League Against Rheumatism (EULAR-2021)

BMS , presumed to be in collaboration with Jinan University and Chinese Academy of Sciences , is developing deucravacitinib, a TYK2 inhibitor, for treating autoimmune diseases, primarily psoriasis. In July 2021, deucravacitinib was reported to be in phase 3 clinical development.

Deucravacitinib (BMS-986165) is a highly selective, orally bioavailable allosteric TYK2 inhibitor for the treatment of autoimmune diseases, which selectively binds to TYK2 pseudokinase (JH2) domain (IC₅₀=1.0 nM) and blocks receptor-mediated Tyk2 activation by stabilizing the regulatory JH2 domain. Deucravacitinib inhibits IL-12/23 and type I IFN pathways.

PAPER

https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.9b00444

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Bristol Myers Squibb Presents Positive Data from Two Pivotal Phase 3 Psoriasis Studies Demonstrating Superiority of Deucravacitinib Compared to Placebo and Otezla® (apremilast)

04/23/2021.. https://news.bms.com/news/details/2021/Bristol-Myers-Squibb-Presents-Positive-Data-from-Two-Pivotal-Phase-3-Psoriasis-Studies-Demonstrating-Superiority-of-Deucravacitinib-Compared-to-Placebo-and-Otezla-apremilast/default.aspx

Significantly more patients treated with deucravacitinib achieved PASI 75 and sPGA 0/1 compared to patients treated with placebo and Otezla at Week 16, with an increased benefit versus Otezla at Week 24 and maintained through Week 52

Deucravacitinib was well tolerated with a low rate of discontinuation due to adverse events

Deucravacitinib is a first-in-class, oral, selective tyrosine kinase 2 (TYK2) inhibitor with a unique mechanism of action

Results presented as late-breaking research at the 2021 American Academy of Dermatology Virtual Meeting Experience

PRINCETON, N.J.–(BUSINESS WIRE)– Bristol Myers Squibb (NYSE:BMY) today announced positive results from two pivotal Phase 3 trials evaluating deucravacitinib, an oral, selective tyrosine kinase 2 (TYK2) inhibitor, for the treatment of patients with moderate to severe plaque psoriasis. The POETYK PSO-1 and POETYK PSO-2 trials, which evaluated deucravacitinib 6 mg once daily, met both co-primary endpoints versus placebo, with significantly more patients achieving Psoriasis Area and Severity Index (PASI) 75 response and a static Physician’s Global Assessment score of clear or almost clear (sPGA 0/1) after 16 weeks of treatment with deucravacitinib. Deucravacitinib was well tolerated with a low rate of discontinuation due to adverse events (AEs).

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Deucravacitinib demonstrated superior skin clearance compared with Otezla^® (apremilast) for key secondary endpoints in both studies, as measured by PASI 75 and sPGA 0/1 responses at Week 16 and Week 24. Findings include:

PASI 75 Response in POETYK PSO-1 and POETYK PSO-2:

• At Week 16, 58.7% and 53.6% of patients receiving deucravacitinib achieved PASI 75 response, respectively, versus 12.7% and 9.4% receiving placebo and 35.1% and 40.2% receiving Otezla.
• At Week 24, 69.0% and 59.3% of patients receiving deucravacitinib achieved PASI 75 response, respectively, versus 38.1% and 37.8% receiving Otezla.
• Among patients who achieved PASI 75 response at Week 24 with deucravacitinib and continued treatment with deucravacitinib, 82.5% and 81.4%, respectively, maintained PASI 75 response at Week 52.

sPGA 0/1 Response in POETYK PSO-1 and POETYK PSO-2:

• At Week 16, 53.6% and 50.3% of patients receiving deucravacitinib achieved sPGA 0/1 response, respectively, versus 7.2% and 8.6% receiving placebo and 32.1% and 34.3% receiving Otezla.
• At Week 24, 58.4% and 50.4% of patients receiving deucravacitinib achieved sPGA 0/1 response, respectively, versus 31.0% and 29.5% receiving Otezla.

“In both pivotal studies, deucravacitinib was superior to Otezla across multiple endpoints, including measures of durability and maintenance of response, suggesting that deucravacitinib has the potential to become a new oral standard of care for patients who require systemic therapy and need a better oral option for their moderate to severe plaque psoriasis,” said April Armstrong, M.D., M.P.H., Associate Dean and Professor of Dermatology at the University of Southern California. “As many patients with moderate to severe plaque psoriasis remain undertreated or even untreated, it is also highly encouraging to see that deucravacitinib improved patient symptoms and outcomes to a greater extent than Otezla.”

Superiority of Deucravacitinib Versus Placebo and Otezla

Deucravacitinib demonstrated a robust efficacy profile, including superiority to placebo for the co-primary endpoints and to Otezla for key secondary endpoints. In addition to PASI 75 and sPGA 0/1 measures, deucravacitinib was superior to Otezla across both studies in multiple other secondary endpoints, demonstrating significant and clinically meaningful efficacy improvements in symptom burden and quality of life measures.

Safety and Tolerability

Deucravacitinib was well-tolerated and had a similar safety profile in both trials. At Week 16, 2.9% of 419 patients on placebo, 1.8% of 842 patients on deucravacitinib and 1.2% of 422 patients on Otezla experienced serious adverse events (SAEs) across both studies. The most common AEs (≥5%) with deucravacitinib treatment at Week 16 were nasopharyngitis and upper respiratory tract infection with low rates of headache, diarrhea and nausea. At Week 16, 3.8% of patients on placebo, 2.4% of patients on deucravacitinib and 5.2% of patients on Otezla experienced AEs leading to discontinuation. Across POETYK PSO-1 and POETYK PSO-2 over 52 weeks, SAEs when adjusted for exposure (exposure adjusted incidence per 100 patient-years [EAIR]) were 5.7 with placebo, 5.7 with deucravacitinib and 4.0 with Otezla. In the same timeframe across both studies, EAIRs for AEs leading to discontinuation were 9.4 with placebo, 4.4 with deucravacitinib and 11.6 with Otezla. No new safety signals were observed during Weeks 16‒52.

Across both Phase 3 trials, rates of malignancy, major adverse cardiovascular events (MACE), venous thromboembolism (VTE) and serious infections were low and generally consistent across active treatment groups. No clinically meaningful changes were observed in multiple laboratory parameters (including anemia, blood cells, lipids and liver enzymes) over 52 weeks.

“The findings from both studies affirm that deucravacitinib – a first-in-class, oral, selective TYK2 inhibitor with a unique mechanism of action that inhibits the IL-12, IL-23 and Type 1 IFN pathways –may become an oral treatment of choice for people living with psoriasis. We believe deucravacitinib has significant potential across a broad range of immune-mediated diseases, and we are committed to further advancing our expansive clinical program with this agent,” said Mary Beth Harler, M.D., head of Immunology and Fibrosis Development, Bristol Myers Squibb. “We are in discussions with health authorities with the goal of bringing this new therapy to appropriate patients as soon as possible. At Bristol Myers Squibb, we are committed to building an immunology portfolio that addresses pressing unmet needs that exist for those impacted by serious dermatologic conditions and other immune-mediated diseases, to ultimately deliver the promise of living a better life.”

These results are available as a late-breaking research presentation (Session S033 – Late-Breaking Research Abstracts) as part of the 2021 American Academy of Dermatology (AAD) Virtual Meeting Experience (VMX). Full results of both studies will be submitted to a medical journal for peer review. In November 2020 and February 2021, respectively, Bristol Myers Squibb announced positive topline results from POETYK PSO-1 and POETYK PSO-2.

Visit www.bms.com/media/medical-meetings/bms-at-aad-vmx.html for more information on Bristol Myers Squibb’s scientific approach and resources on psoriasis and immune-mediated diseases.

About Deucravacitinib

Deucravacitinib (pronounced doo-krav-a-sih-ti-nib) is a first-in-class, oral, selective tyrosine kinase 2 (TYK2) inhibitor with a unique mechanism of action. Deucravacitinib is the first and only TYK2 inhibitor in clinical studies across multiple immune-mediated diseases. Bristol Myers Squibb scientists designed deucravacitinib to selectively target TYK2, thereby inhibiting signaling of interleukin (IL)-12, IL-23 and Type 1 interferon (IFN), key cytokines involved in psoriasis pathogenesis. Deucravacitinib achieves a high degree of selectivity by uniquely binding to the regulatory, rather than the active, domain of TYK2, which is structurally distinct from the regulatory domains of Janus kinase (JAK) 1, 2 and 3. At therapeutic doses, deucravacitinib does not inhibit JAK1, JAK2 or JAK3. Due to the innovative design of deucravacitinib, Bristol Myers Squibb earned recognition with the 2019 Thomas Alva Edison Patent Award for the science underpinning the clinical development of deucravacitinib.

Deucravacitinib is being studied in multiple immune-mediated diseases, including psoriasis, psoriatic arthritis, lupus and inflammatory bowel disease. In addition to POETYK PSO-1 and POETYK PSO-2, Bristol Myers Squibb is evaluating deucravacitinib in three other Phase 3 studies in psoriasis: POETYK PSO-3 (NCT04167462); POETYK PSO-4 (NCT03924427); POETYK PSO-LTE (NCT04036435). Deucravacitinib is not approved for any use in any country.

About the Phase 3 POETYK PSO-1 and POETYK PSO-2 Studies

PrOgram to Evaluate the efficacy and safety of deucravacitinib, a selective TYK2 inhibitor (POETYK) PSO-1 (NCT03624127) and POETYK PSO-2 (NCT03611751) are global Phase 3 studies designed to evaluate the safety and efficacy of deucravacitinib compared to placebo and Otezla^® (apremilast) in patients with moderate to severe plaque psoriasis. Both POETYK PSO-1, which enrolled 666 patients, and POETYK PSO-2, which enrolled 1,020 patients, were multi-center, randomized, double-blind trials that evaluated deucravacitinib (6 mg once daily) compared with placebo and Otezla (30 mg twice daily). POETYK PSO-2 included a randomized withdrawal and retreatment period after Week 24.

The co-primary endpoints of both POETYK PSO-1 and POETYK PSO-2 were the percentage of patients who achieved Psoriasis Area and Severity Index (PASI) 75 response and those who achieved static Physician’s Global Assessment (sPGA) score of 0 or 1 at Week 16 versus placebo. Key secondary endpoints of the trials included the percentage of patients who achieved PASI 75 and sPGA 0/1 compared to Otezla at Week 16 and other measures.

About Psoriasis

Psoriasis is a widely prevalent, chronic, systemic immune-mediated disease that substantially impairs patients’ physical health, quality of life and work productivity. Psoriasis is a serious global problem, with at least 100 million people worldwide impacted by some form of the disease, including around 14 million people in Europe and approximately 7.5 million people in the United States. Up to 90 percent of patients with psoriasis have psoriasis vulgaris, or plaque psoriasis, which is characterized by distinct round or oval plaques typically covered by silvery-white scales. Despite the availability of effective systemic therapy, many patients with moderate to severe psoriasis remain undertreated or even untreated and are dissatisfied with current treatments. People with psoriasis report an impact on their emotional well-being, straining both personal and professional relationships and causing a reduced quality of life. Psoriasis is associated with multiple comorbidities that may impact patients’ well-being, including psoriatic arthritis, cardiovascular disease, metabolic syndrome, obesity, diabetes, inflammatory bowel disease and depression.

About Bristol Myers Squibb

Bristol Myers Squibb is a global biopharmaceutical company whose mission is to discover, develop and deliver innovative medicines that help patients prevail over serious diseases. For more information about Bristol Myers Squibb, visit us at BMS.com or follow us on LinkedIn, Twitter, YouTube, Facebook and Instagram.

Celgene and Juno Therapeutics are wholly owned subsidiaries of Bristol-Myers Squibb Company. In certain countries outside the U.S., due to local laws, Celgene and Juno Therapeutics are referred to as, Celgene, a Bristol Myers Squibb company and Juno Therapeutics, a Bristol Myers Squibb company.

Otezla^® (apremilast) is a registered trademark of Amgen Inc.

PATENT

WO-2021129467

Novel crystalline polymorphic forms (CSI and CSII) of deucravacitinib (also known as BMS-986165), useful a tyrosine kinase 2 pseudokinase domain (TYK2) inhibitor for treating psoriasis, systemic lupus erythematosus, and Crohn’s disease.Tyrosine kinase 2 (TYK2) is an intracellular signal transduction kinase that can mediate interleukin-23 (IL-23), interleukin-12 (IL-12) and type I interferon (IFN) These cytokines are involved in inflammation and immune response. 
BMS-986165 is the first and only new oral selective TYK2 inhibitor, clinically used to treat autoimmune and autoinflammatory diseases (such as psoriasis, psoriatic arthritis, lupus and inflammatory bowel disease, Crowe Graciousness, etc.). The results of a phase III clinical study of the drug announced in November 2020 showed that BMS-986165 has shown positive clinical effects in the treatment of moderate to severe plaque psoriasis. In addition, BMS-986165 also shows good therapeutic effects in the treatment of systemic lupus erythematosus and Crohn’s disease. 
The chemical name of BMS-986165 is 6-(cyclopropaneamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)benzene (Yl)amino)-N-(methyl-D3)pyridazine-3-carboxamide, the structural formula is shown below, and is hereinafter referred to as “compound I”: 

The crystal form is a solid in which the compound molecules are arranged in a three-dimensional order in the microstructure to form a crystal lattice. The phenomenon of drug polymorphism refers to the existence of two or more different crystal forms of the drug. Because of different physical and chemical properties, different crystal forms of the drug may have different dissolution and absorption in the body, which in turn affects the clinical efficacy and safety of the drug to a certain extent. Especially for poorly soluble solid drugs, the crystal form will have a greater impact. Therefore, drug crystal form must be an important content of drug research and also an important content of drug quality control. 
WO2018183656A1 discloses compound I crystal form A (hereinafter referred to as “crystal form A”) and a preparation method thereof. The crystalline form A disclosed in WO2018183656A1 is the only known free crystalline form of Compound I. The inventor of the present application repeated the preparation method disclosed in WO2018183656A1 to obtain and characterize the crystal form A. The results show that the crystal form A has poor compressibility and high adhesion. Therefore, there is still a need in the art to develop a compound I crystalline form with good stability, good compressibility, and low adhesion for the development of drugs containing compound I. 
The inventor of the present application has paid a lot of creative work and unexpectedly discovered the crystalline form CSI of compound I and the crystalline form CSII of compound I provided by the present invention, which have advantages in physical and chemical properties, preparation processing performance and bioavailability, for example, There are advantages in at least one aspect of melting point, solubility, hygroscopicity, purification, stability, adhesion, compressibility, fluidity, dissolution in vivo and in vitro, and bioavailability, especially good physical and chemical stability and mechanical stability It has good performance, good compressibility, and low adhesion, which solves the problems existing in the prior art, and is of great significance to the development of drugs containing compound I.

PATENT

US9505748 , a family member of WO2014074661 .

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014074661

Preparation 1

Step l Int1

Step 2 Int2 Step 3 Int3 Step 4 Int4

Example 52

Step 1

[00219] To a solution of 2-methoxy-3-(l-methyl-lH-l ,2,4-triazol-3-yl)aniline (10.26 g, 50.2 mmol) and Int8 (10.5 g, 50.2 mmol) in THF (120 mL) was added lithium bis(trimethylsilyl)amide (LiHMDS, 1M in THF, 151 mL, 151 mmol) in a dropwise manner using a pressure equalized addition funnel. The reaction was run for 10 minutes after the completion of the addition and then quenched with HCl (1M aq., 126 mL, 126 mmol). The reaction was concentrated on a rotary evaporator until the majority of the THF was removed and a precipitate prevailed throughout the vessel. Water (-500 mL) was then added and the slurry sonicated for 5 minutes and stirred for 15 min. The solid was filtered off, rinsing with water and then air dried for 30 minutes. The powder was collected and dissolved in dichloromethane. The organic layer was washed with water and brine and then dried over sodium sulfate, filtered and concentrated to provide the product (12.5 g, 66% yield) (carried on as is). 1H NMR (400MHz, DMSO-d₆) δ 11.11 (s, 1H), 9.36 (s, 1H), 8.56 (s, 1H), 7.72 (dd, J=7.8, 1.6 Hz, 1H), 7.60 (dd, J=7.9, 1.5 Hz, 1H), 7.29 (t, J=7.9 Hz, 1H), 7.19 (s, 1H), 3.95 (s, 3H), 3.72 (s, 3H). LC retention time 1.18 [E]. MS(E⁺) m/z: 377 (MH⁺).

Step 2

[00220] Intl3 (2.32 g, 6.16 mmol) and cyclopropanecarboxamide (1.048 g, 12.31 mmol) were dissolved in dioxane (62 mL) and Pd₂(dba)₃ (564 mg, 0.616 mmol), Xantphos (534 mg, 0.924 mmol) and cesium carbonate (4.01 g, 12.3 mmol) were added. The vessel was evacuated three times (backfilling with nitrogen) and then sealed and heated to 130 °C for 140 minutes. The reaction was filtered through CELITE® (eluting with ethyl acetate) and concentrated (on smaller scale this material could then be purified using preparative HPLC). The crude product was adsorbed onto CELITE® using dichloromethane, dried and purified using automated chromatography (100% EtOAc) to provide example 52 (1.22 g, 46% yield). 1H NMR (500MHz, chloroform-d) δ 10.99 (s, 1H), 8.63 (s, 1H), 8.18 (s, 1H), 8.10 (d, J=0.5 Hz, 2H), 7.81 (dd, J=7.9, 1.7 Hz, 1H), 7.51 (dd, J=7.9, 1.4 Hz, 1H), 7.33 – 7.20 (m, 7H), 4.01 (d, J=0.3 Hz, 3H), 3.82 (s, 3H), 1.73 -1.60 (m, 1H), 1.16 – 1.06 (m, 2H), 0.97 – 0.84 (m, 2H). LC retention time 6.84 [N]. MS(E⁺) m/z: 426 (MH⁺).

Example 53

[00221] To a homogeneous solution of Example 52 (50 mg, 0.12 mmol) in dichloromethane (3 mL) was added HCI (1M aq., 0.13 mL, 0.13 mmol) resulting in the solution turning yellow. The homogenous solution was concentrated down and then re-concentrated from dichloromethane twice to remove residual water, resulting in a white powder. The powder was suspended in dichloromethane and sonicated for 15 minutes, the powder was then collected via filtration, rinsing with dichloromethane to provide the corresponding HCI salt (38 mg, 70% yield). 1H NMR (500MHz, chloroform-d) δ 12.02 (s, 1H), 8.35 (s, 1H), 8.16 (s, 1H), 8.01 (dd, J=7.9, 1.5 Hz, 1H), 7.57 (br. s., 1H), 7.52 -7.46 (m, 1H), 7.36 (t, J=7.9 Hz, 1H), 4.03 (s, 3H), 3.83 (s, 3H), 2.05 – 1.95 (m, 1H), 1.16 – 1.09 (m, 2H), 1.03 (dd, J=7.4, 3.6 Hz, 2H). LC retention time 0.62 [j]. MS(E⁺) m/z: 426 (MH⁺).

[00222] Compare to NMR of parent free base: 1H NMR (500MHz, chloroform-d) δ 10.99 (s, 1H), 8.63 (s, 1H), 8.18 (s, 1H), 8.10 (d, J=0.5 Hz, 2H), 7.81 (dd, J=7.9, 1.7 Hz, 1H), 7.51 (dd, J=7.9, 1.4 Hz, 1H), 7.33 – 7.20 (m, 7H), 4.01 (d, J=0.3 Hz, 3H), 3.82 (s, 3H), 1.73 – 1.60 (m, 1H), 1.16 – 1.06 (m, 2H), 0.97 – 0.84 (m, 2H).

////////////DEUCRAVACITINIB, phase 3, BMS-986165, BMS 986165, psoriasis, systemic lupus erythematosus, Crohn’s disease,

CNC(=O)C1=NN=C(C=C1NC2=CC=CC(=C2OC)C3=NN(C=N3)C)NC(=O)C4CC4

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SAR-444727

1414354-91-8C₃₀ H₃₀ F N₇ O_{2 Molecular Weight539.60}1-Piperidinepropanenitrile, 3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-α-(2,2-dimethylpropylidene)-β-oxo-, (3R)-

(3R)-3-[4-Amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-α-(2,2-dimethylpropylidene)-β-oxo-1-piperidinepropanenitrile

2-(3-(4-amino~3-(2-fiuoro~4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile

• OriginatorPrincipia Biopharma
• ClassSmall molecules
• Mechanism of ActionAgammaglobulinaemia tyrosine kinase inhibitors

• Phase IAutoimmune disorders
• DiscontinuedArthritis

• 28 Sep 2020Principia Biopharma has been acquired by Sanofi
• 22 Jun 2020Principia Biopharma plans a pharmacokinetic phase I trial (In volunteers) for Hypersensitivity (for Immunoglobulin E-mediated allergies) in Australia (Topical) (ACTRN12620000693921)
• 10 Mar 2020Phase-I clinical trials in Autoimmune disorders (In volunteers) in Australia (Topical)
• US 8957080
• US 8673925
• WO 2014022569
• WO 2013191965
• WO 2012158764

Useful for treating pemphigus vulgaris, immune thrombocytopenia, inflammatory bowel disease, Sjogren’s syndrome, multiple sclerosis, chronic lymphocytic leukemia and ankylosing spondylitis. Principia Biopharma is developing a topical formulation PRN-473 (presumed to be SAR-444727), a reversible covalent bruton’s (BTK) tyrosine kinase inhibitor, developed based on Principia’s reversible, tailored covalency platform, for treating immune-mediated diseases [phase I, July 2021]. Principia Biopharma was also investigating BTK inhibitors , developed based on Principia’s reversible, tailored covalency platform, for treating hematologic malignancies [no development reported since July 2019]. At the time of publication, Zhu was also affiliated with Nurix Therapeutics , while By and Phiasivongsa were based at Rain Therapeutics and Kronos Bio , respectively.

PATENT

WO-2021142131

Novel crystalline polymorphic forms (I to V) of PRN-473 and their preparation method.

CRYSTALLINE FORMS OF 2- [3- [4- AMINO-3-(2- FLUORO-4-PHENOXY- PHENYL)-1H-PYRAZOLO[3,4-D]PYRIMIDIN-1-YL]PIPERIDINE-1-CARBONYL]- 4,4-DIMETHYLPENT-2-ENENITRILE

This application claims the benefit of priority to U.S. Provisional Application No. 62/958,389, filed January 8, 2020, the contents of which are incorporated by reference herein in their entirety.

Disclosed herein are crystalline forms of 2-(3-(4-amino~3-(2-fiuoro~4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile (Compound (I)), methods of using the same, and processes for making Compound (I), including its various crystalline forms. The crystalline forms of Compound (I) are inhibitors of Bruton’s tyrosine kinase (BTK). The enzyme BTK is a member of the Tec family non-receptor tyrosine kinases.

BTK is expressed in most hematopoietic cells, including B cells, mast cells, and macrophages. BTK plays a role in the development and activation of B cells and has been implicated in multiple signaling pathways across a wide range of immune-mediated diseases. BTK activity has been implicated in the pathogenesis of several disorders and conditions, such as B cel1-related hematological cancers (e.g,, non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia) and autoimmune diseases (e.g, rheumatoid arthritis,

Sjogren’s syndrome, pemphigus, IBD, lupus, and asthma).

Compound (I) and various solid forms thereof may inhibit BTK and be useful in the treatment of disorders and conditions mediated by BTK activity. Compound (I) is disclosed as, e.g., Compound 125A in Table 1 of WO 2012/158764 and has the following structure:

Example 1: Preparation of Crystalline Form (I) of Compound (I)

Methyl isobutyl ketone (MIBK; 6 mL) was added to amorphous (R)-2-(3-(4-amino-3- (2-fluoro-4-phenoxyphenyJ)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4- dimethylpent-2-enenitrile (1,0 g) and stirred to fonn a solution. After approximately five minutes of agitation, a precipitate began to form. Additional MIBK (10 mL) was charged, and the slurry was stirred. After approximately ten days, the solid was filtered and rinsed with MIBK (10 mL). The solid was dried under vacuum with heating to afford approximately 0.5 g of crystalline Form (I) of Compound (I) as a white solid.

PATENT

WO2012158764 , claiming BTK tyrosine kinase inhibitors, useful for treating cancer.

https://patents.google.com/patent/WO2012158764A1/en

WO 2012/158764 125A

PATENT

US20210205313

PATENT

US20210205312 ,

for concurrently published filings, claiming a gel composition comprising PRN-473 and use of another BTK tyrosine kinase inhibitor ie PRN1008 , respectively.

PATENT

WO2016100914 , claiming use of a BTK inhibitor ie PRN-473, alone or in combination with corticosteroid therapy, for treating pemphigus vulgaris.

PATENT

WO 2014022569

https://patents.google.com/patent/WO2014022569A1/en

//////// PRN-473,  PRN 473, SAR 444727, PHASE 1

CC(C)(C)C=C(C#N)C(=O)N1CCC[C@H](C1)n1nc(c2c(N)ncnc21)c1ccc(Oc2ccccc2)cc1F

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PRN 1008, Rilzabrutinib

CAS 1575591-66-0

C36H40FN9O3,

2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile

Anti-inflammatory disease, Autoimmune disease treatment

• OriginatorPrincipia Biopharma
• Class2 ring heterocyclic compounds; Amines; Anti-inflammatories; Fluorobenzenes; Nitriles; Phenyl ethers; Piperazines; Piperidines; Pyrazoles; Pyrimidines; Skin disorder therapies; Small molecules
• Mechanism of ActionAgammaglobulinaemia tyrosine kinase inhibitors
• Orphan Drug StatusYes – Idiopathic thrombocytopenic purpura; Pemphigus vulgaris

• Phase IIIIdiopathic thrombocytopenic purpura; Pemphigus vulgaris
• Phase IIAutoimmune disorders

• 02 Jun 2021Efficacy data from a phase IIa trial in Ankylosing spondylitis presented at the 22^nd Annual Congress of the European League Against Rheumatism (EULAR-2021)
• 07 Apr 2021Sanofi initiates enrollment in a phase I pharmacokinetics trial in healthy volunteers in Australia (PO, Tablet, Capsule) (NCT04748926)
• 31 Mar 2021Sanofi announces intention to seek regulatory approval for Idiopathic thrombocytopenic purpura in 2023 (Sanofi pipeline, May 2021)

CLIP

https://cen.acs.org/pharmaceuticals/drug-development/Sanofi-acquire-BTK-inhibitor-firm/98/web/2020/08

Sanofi to acquire BTK inhibitor firm Principia for $3.7 billion

Principia is testing its small-molecule compounds in multiple sclerosis and immune system diseases

Sanofi will pay $3.7 billion to acquire Principia Biopharma, a San Francisco-based biotech firm developing small molecules that inhibit Bruton tyrosine kinase (BTK). The price represents about a 75% premium over Principia’s stock market value in early July, before reports surfaced that Sanofi was interested in buying the firm.

BTK is a protein important for both normal B cell development and the proliferation of lymphomas, which are B cell cancers. AbbVie, AstraZeneca, and BeiGene all market BTK inhibitors for treating specific kinds of lymphomas. Sales of AbbVie’s inhibitor, Imbruvica, approached $4.7 billion in 2019.

Other drug firms have been eager to get in on the action as well. In January, Merck & Co. spent $2.7 billion to acquire ArQule, whose experimental noncovalent BTK inhibitor is designed to overcome resistance that some cancers develop after treatment with current covalent BTK inhibitors. Eli Lilly and Company’s $8 billion acquisition of Loxo Oncology in 2019 also included a noncovalent BTK inhibitor.

BTK is also linked to inflammation, and Principia focuses on developing BTK inhibitors for immune system diseases and multiple sclerosis. Its compound rilzabrutinib is currently in clinical trials for pemphigus and immune thrombocytopenia. In 2017, Sanofi struck a deal to develop Principia’s brain-penetrant BTK inhibitor, SAR442168, for multiple sclerosis.

Sanofi announced in April of this year that the inhibitor reduced formation of new lesions—the scarred nervous tissue that gives multiple sclerosis its name—by 85% in a Phase II clinical trial. A Phase III trial of the compound began in June.

Upon announcing its deal to acquire Principia, Sanofi said that both rilzabrutinib and SAR442168 have the potential to become a “pipeline in a product,” indicating they can be used for many immune-related and neurological diseases, respectively.

The anti-inflammatory effects of BTK inhibitors have raised interest in the drugs as treatments for people hospitalized with COVID-19. Notably, the US National Cancer Institute conducted a small study suggesting acalabrutinib may help reduce the respiratory distress and inflammation in people with COVID-19. Based on that preliminary study, AstraZeneca—which markets acalabrutinib as Calquence—is conducting a 60-person randomized trial of the drug for COVID-19.

Sanofi has not indicated interest in investigating Principia’s BTK inhibitors as COVID-19 treatments.Chemical & Engineering NewsISSN 0009-2347 
PATENTWO 2021127231https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021127231&tab=PCTDESCRIPTION&_cid=P20-KRA0I9-18818-1

SOLID FORMS OF 2-[3-[4-AMTNO-3-(2-FT,TTORO-4-PHENOXY- PHEN¥L)PYRAZOLO[3,4 D]PYRIMIDIN l~YL]PIPERIDINE~l~CARBON¥L] 4~

METHYL-4-[4-(OXETAN-3-YL)PIPERAZIN-l-YLjPENT-2-ENENITRILE

[11 This application claims the benefit of priority to U.S. Provisional Application

No 62/951,958, filed December 20, 2019, and U.S Provisional Application No. 63/122,309, filed December 7, 2020, the contents of each of which are incorporated by reference herein in their entirety.

[2] Disclosed herein are solid forms of 2-[3-[4~amino-3~(2~fluoro-4-phenoxy-plienyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l Carbonyl]~4-nietliyl-4~[4-(oxetaii~3-yl)piperazin-!~yi]pent-2~enenitriie (Compound (I)), methods of using the same, and processes for making Compound (I), including its solid forms. The solid forms of Compound (I) may be inhibitors of Bruton’s tyrosine kinase (BTK) comprising low residual solvent content.

[3| The enzyme BTK is a member of the Tec family non-receptor tyrosine kinases.

BTK is expressed in most hematopoietic cells, including B cells, mast cells, and macrophages BTK plays a role in the development and activation of B cells. BTK activity has been implicated in the pathogenesis of several disorders and conditions, such as B cell-related hematological cancers (e.g., non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia) and autoimmune diseases (e.g., rheumatoid arthritis, Sjogren’s syndrome, pemphigus, IBD, lupus, and asthma).

[4] Compound (I), pharmaceutically acceptable salts thereof, and solid forms of any of the foregoing may inhibit BTK and be useful in the treatment of disorders and conditions mediated by BTK activity. Compound (I) is disclosed in Example 31 of WO 2014/039899 and has the following structure:

where *C is a stereochemical center. An alternative procedure for producing Compound (!) is described in Example 1 of WO 2015/127310.

[5] Compound (I) obtained by the procedures described in WO 2014/039899 and WO 2015/127310 comprises residual solvent levels well above the limits described in the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (“ICH”) guidelines. In general, manufacturing processes producing residual solvent levels near or above the ICH limits are not desirable for preparing active pharmaceutical ingredients (APIs).

Example 1: Spray Drying Process A

[311] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was washed with pH 3 phosphate buffer to remove basic impurities that are more soluble than Compound (I) in the aqueous layer. The dichloromethane solution was then washed with pH 7 buffer and solvent exchanged into isopropyl acetate. The isopropyl acetate solution was then washed with pH 3 phosphate buffer, bringing Compound (I) into the aqueous layer and removing non-basic impurities. The pH of the aqueous layer was adjusted to pH 9 with 10% sodium hydroxide, and the aqueous layer was extracted with isopropyl acetate. Upon concentration under vacuum, Compound (I) was precipitated from heptane at 0 °C, filtered and dried to give a white amorphous solid as a mixture of the (E) and (Z) isomers, as wet Compound (I). Wet Compound (I) was dissolved in methanol and spray dried at dryer inlet temperature of 125 °C to 155 °C and dryer outlet temperature of 48 to 58 °C to obtain the stable amorphous Compound (I) free base with levels of isopropyl acetate and heptane below 0.5% and 0.05%, respectively.

Example 2: Spray Drying Process B

intermediate A

Compound (!)

[241] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe, and recirculating fluid chiller/heater was charged with Intermediate A (20.2 kg) and Intermediate B (13.6 kg, 1.5 equiv). DCM (361.3 kg, 14.5 vol) was charged to the reactor. The mixture was agitated, and the batch cooled to 0 °C to 5 °C. The reactor was charged with pyrrolidine (18.3 kg, 6 equiv) and then charged with TMSC1 (18.6 kg, 4 eq). Stirring was continued at 0 °C to 5 °C for 0.5 to 1 hour

[242] At 0 °C to 5 °C, acetic acid (2.0 equiv) was charged to the reactor followed by water (5 equiv). Stirring was continued at 0 °C to 5 °C for 1 to 1.5 hours. Water (10 equiv) was charged to the reactor, and the solution was adjusted to 20 °C to 25 °C. The internal temperature was adjusted to 20 °C to 25 °C and the biphasic mixture was stirred for 15 to 20 mins. Stirring was stopped and phases allowed to separate for at least 0.5 h. The lower aqueous layer was removed.

[243] Water (7 vol) was charged to the reactor. The pH was adjusted to 2.8-3.3 with a 10 wt. % solution of citric acid. Stirring was continued at 0 to 5 °C for 1 to 1.5 hours. Stirring was stopped and phases allowed to separate for at least 0.5 h. The lower aqueous layer was removed.

[244] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe, and recirculating fluid chiller/heater was charged with an approximately 9% solution of NaHCCri (1 vol) and the organic layer. The internal temperature was adjusted to 20 °C to 25 °C, and the biphasic mixture was stirred for 15 to 20 mins. Stirring was stopped and phases allowed to separate for at least 0.5 h. The lower aqueous layer was removed. The aqueous layer was measured to have a pH greater than 7.

[245] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe and recirculating fluid chiller/heater was charged with the organic layer. The organic phase ¾s distilled under vacuum at less than 25 °C to 4 total volumes. IP AC (15 vol) was charged to the reactor. The organic phase was distilled under vacuum at less than 25 °C to 10 total volumes. Water (15 vol) followed by pH 2.3 phosphate buffer were charged to the reactor at an internal temperature of 20 °C to 25 °C. The pH adjusted to 3 Stirring was stopped and phases allowed to separate for at least 0.5 h. The organic phase was removed.

[246] The following steps were repeated twice: IP AC (5 vol) was charged to the reactor containing the aqueous layer. Stirring was continued for 0.25 to 0.5 hours. Stirring was stopped and phases allowed to separate for at least 0.5 h. The organic phase was removed. [247] IP AC (15 vol) was charged to the reactor containing the aqueous layer. A pH 10 phosphate buffer was charged to the reactor and the pH adjusted to 10 with 14% NaOH solution. Stirring was continued for 1.5 to 2 hours. Stirring was stopped and phases allowed to separate for at. least 0.5 h. The aqueous layer was discarded. The organic layer was dried over brine.

[248] The organic solution was distilled under vacuum at less than 25 °C to 5 total volumes.

[249] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe and recirculating fluid chiller/heater was charged with n-heptane (20 vol). The internal temperature was adjusted to 0 to 5 °C, and the IP AC solution was added.

[250] The suspension was filtered. The filter cake was washed with n-heptane and the tray was dried at 35 °C. Compound (I) (24.6 kg) was isolated in 86% yield.

[251] Compound (1) was dissolved in methanol (6 kg) and spray dried to remove residual IP AC and n-heptane.

Example 3: Precipitation Process A

[252] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was quenched with acetic acid and water, followed by washing with pH 3 aqueous solution to remove basic impurities that are more soluble than Compound (1) in the aqueous layer. Washing was repeated as needed to reduce impurities. Methanesulfonic acid was added to the dichloromethane solution, and the dichloromethane solution was concentrated by distillation under reduced pressure, followed by addition of 1% NaCi aqueous solution and isopropyl acetate before adjustment of pH to approximately 3 with potassium hydroxide. The isopropyl acetate layer was removed and discarded. The aqueous layer containing Compound (I) was washed with isopropyl acetate to remove hydrophobic impurities. Washing was repeated as needed to reduce related substance impurities. Residual isopropyl acetate was removed by distillation under reduced pressure. The aqueous solution containing Compound (I) was cooled to 0 to 5°C before adjusting the pH to approximately 9 with potassium hydroxide. The free base of Compound (I) was allowed to precipitate and maturate at 20 °C for 20 hours. The mixture temperature was then adjusted to 20 °C to 25 °C, and the hydrate impurity was verified to be less than 0.3% (< 0.3%). The cake of the free base of Compound (I) was filtered and washed as needed to reduce conductivity. The cake was then allowed to dry on the filter under vacuum and nitrogen swept to reduce water content by Karl-Fischer (KF < 50%) before transferring to the oven for drying. The wet cake of the free base of Compound (1) was dried under vacuum at 25 °C until water content by Karl -Fischer was less than 1.5% (KF < 1.5%), and then dehmiped by milling to yield a uniform white amorphous solid as a mixture of the (E) and (Z) isomers, with no detectible levels of isopropyl acetate or heptane.

Example 4: Precipitation Process 3B

[253] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was quenched with acetic acid and water, followed by washing with pH 3 aqueous solution to remove basic impurities that are more soluble than Compound (I) in the aqueous layer. The washing was repeated as needed to reduce residual solvents and impurities. The dichloromethane solution was then washed with saturated sodium bicarbonate (pH > 7). Dichloromethane was removed by distillation under reduced pressure, followed by addition of water and isopropyl acetate. The pH of the aqueous layer was adjusted to pH to 2.8 – 3.3 with 2 M aqueous sulfuric acid (H2SQ4) at 0 – 5 °C, and the mixture rvas stirred and settled. After phase separation removal of the organic layer, the aqueous layer was washed with isopropyl acetate three times and the residual isopropyl acetate in aqueous layer was distilled out under vacuum at a temperature below 25 °C and the solution was basitied with 5% aqueous KOFI to pH 9 – 10 to a slurry . The resulting suspension was stirred and warmed up to 20 °C to 25 °C and aged for 20 h. The product was filtered and washed with water and dried to give white solid in 86% yield.

Example 5: Precipitation Process C

[254] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was quenched with acetic acid and water, followed by washing to remove basic impurities that are more soluble than Compound (I) in the aqueous layer. Washing was repeated as needed to reduce impurities. Methanesulfonic acid was added to the d chloromethane solution, and the dichloromethane solution was concentrated under reduced pressure to obtain a thin oil. The concentrated oil was cooled to approximately 5°C before washing with an aqueous solution of sodium chloride. The organic phase was discarded. Washing of the aqueous layer was repeated as needed with dichloromethane to remove low level impurities. The pH of the aqueous solution was adjusted to approximately 3 with an aqueous solution of potassium hydroxide. Residual dichloromethane was removed

under reduced pressure. The level of residual acetic acid was determined by, for example, titration. The aqueous solution containing Compound (I) was cooled to a temperature between 0°C and 5°C. Acetic acid was present at 0 wt % to 8 wt. %. Acetic acid level was 0 wt % if the aqueous acid solution was washed with aqueous sodium bicarbonate or another aqueous inorganic base. Optionally, additional acetic acid was added to achieve a 0 wt.% to 8 wt. % acetic acid level. An aqueous solution of potassium hydroxide was constantly charged to the aqueous solution to obtain a pH to approximately 9.5. The free base of Compound (I) was allowed to precipitate and maturate at approximately 20 °C for least 3 hours. The cake (wet solid) of the free base of Compound (I) was filtered and washed with water. The wet cake was then dried under reduced vacuum with slight heat. Alternatively, instead of washing the wet cake with water, the wet cake was reslurried with water at approximately 15 °C for at least 1 hour before filtering. The free base of Compound (I) in the fomi of a wet cake was dried under vacuum with slight heat at 25°C.

[255] FIGs. 12-15 are example SEM images showing the variable morphologies of particles of Compound (I) during the filtration step to isolate Compound (I) based on the amount acetic acid added during the initial step in the precipitation of Compound (Ϊ) (FIG. 12: at 0 wt. % acetic acid; FIG 13: at 3 wt. % acetic acid; FIG. 14: at 5 wt. % acetic acid; FIG 15: at 8 wt. % acetic acid). Filtration speed depended on the morphology and was the fastest for 0 wt. % acetic acid. At 1 wt. % acetic acid, the filtration speed diminished considerably, improving at 2 wt. % to 3 wt. % acetic acid. Morphologies with more open holes (such as, e.g., more porous particles) resulted in improved filtration speeds, whereas more compact particles resulted in decreased filtration speed.

Example 6: Conversion of a Crystalline Form of Compound (Ϊ) to an Amorphous Form

[256] 9.8 grams of a crystalline form of Compound (I) were dissolved in approximately 20 mL of dichloromethane and approximately 120 ml. of brine solution. Then, approximately 1 equivalent of methanesulfonic acid was added. The pH w¾s approximately 2. The layers were separated. The aqueous layer was concentrated at a temperature between 0°C and 5°C to remove residual dichloromethane before slowly adding aqueous KOI I solution (approximately 5%) to adjust the pH to a value between 9 and 10. During aqueous KOH addition, an amorphous form of Compound (I) precipitated out. The slurry was slowly warmed to room temperature and then was stirred for approximately 24 hours before filtering and rinsing the wet cake with water. The wet cake was dried under vacuum with slight heat at approximately 30°C to provide 7 grams of a white to an off-white solid (87% yield and 98 4% purity). XRPD showed that the product was an amorphous solid form of Compound (I).

Example 7: Micronization of Compound (I) Particles Obtained by Precipitation Processes

[257] A fluid jet mill equipment was used during lab scale jet milling trials. The fluid jet mill equipment includes a flat cylindrical chamber with 1.5” diameter, fitted with four symmetric jet nozzles winch are tangentially positioned in the inner wall. Prior to feeding material to the fluid jet mill in each trial, the material was sieved in a 355 iim screen to remove any agglomerates and avoid blocking of the nozzles during the feed of material to the micronization chamber. The material to be processed was drawn into the grinding chamber through a vacuum created by the venturi (P vent ~ 0 5 – 1 0 bar above P grind). The feed flow rate of solids (F_feed) was controlled by a manual valve and an infinite screw volumetric feeder. Compressed nitrogen was used to inject the feed material; compressed nitrogen was also used for the jet nozzles in the walls of the milling chamber. Compressed fluid issuing from the nozzles expands from P grind and imparts very’ high rotational speeds in the chamber. Accordingly, material is accelerated by rotating and expanding gases and subjected to centrifugal forces. Particles move outward and are impacted by high velocity jets, directing the particles radially inward at very high speeds. Rapidly moving particles impact the slower moving path of particles circulating near the periphery of the chamber. Attrition takes place due to the violent impacts of particles against each other. Particles with reduced size resulting from this sequence of impacts are entrained in the circulating stream of gas and swept against the action of centrifugal force toward the outlet at the center. Larger particles in the gas stream are subjected to a centrifugal force and returned to the grinding zone. Fine particles are carried by the exhaust gas to the outlet and pass from the grinding chamber into a collector.

[258] The feeder has continuous feed rate control; however, to more precisely control the feed rate, the full scale of feed rates was arbitrary divided in 10 positions. To calibrate F feed, the feeder was disconnected from milling chamber and 10 g of Compound (I) powder was fed through the feeder operating at various feed rate positions. The mass of powder flowing through the feeder over 6 minutes was marked. The resulting feed rate was directly proportional to feeder position. After processing each of the four trials, the jet mill was stopped, micronized product removed from the container, and the milling chamber checked for any powder accumulation.

Variables/Parameters

F_feed Feed flow rate of solids [kg/h]

P grind Grinding pressure inside the

drying chamber [bar]

P vent Feed pressure in the venturi [bar]

Example 8: Residual Solvent Levels

[251] Retention of process solvents (/.<?., res dual solvents) depends on van der Waal s’ forces that are unique to and an inherent property of each molecule. Additionally, solvent retention depends how the API solid is formed, isolated, washed, and dried (i.e., during the manufacturing process). Because residual solvents may pose safety risks, pharmaceutical processes should be designed to minimize residual solvent levels (e.g , to result in residual solvent levels below the limits established in the ICH guidelines).

[252] Residual solvent analysis was performed using gas chromatography-mass spectrometry. The residual solvent levels in solid forms of Compound (I) prepared by spray drying processes described herein and precipitation processes described herein are provided in Table 2. The residual solvent levels in crude Compound (I) listed in Table 2 are comparable to the residual solvent levels in crude Compound (I) prepared according to the procedures detailed in Example 31 of WO 2014/039899 and Example 1 of WO 2015/127310.

Table 2: Residual solvent levels in solid forms of Compound (I)

PATENTWO 2015127310https://patents.google.com/patent/WO2015127310A1/enExample 1Synthesis of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l- yl]-piperidine-l-carbonyl]-4-m iperazin-l-yl]pent-2-enenitrile

Step 1To a solution of 3-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin-l -yl]-l-piperidyl]-3-oxo-propanenitrile (15 g, 3.12mmol), 2-methyl-2-[4- (oxetan-3-yl)piperazin-l-yl]propanal (794.25mg, 3.74mmol) in DCM (40mL), pyrrolidine (1.54mL,18.71mmol) at 0-5 °C was added, which is followed by TMS-Cl (1.58mL,12.47mmol). The reaction mixture was stirred at 0-5 °C for 3 h and was quenched with 1 M potassium phosphate buffer (pH 3). Layers were separated and the organic layer was washed once more with 1 M potassium phosphate buffer (pH 3). The organic layer was extracted withl M potassium Phosphate buffer at pH 1.5. Layers were separated. The aqueous phase contained the desired product while the impurities stayed in the organic phase. The aqueous phase was neutralized with 1 M potassium phosphate (pH 7) and was extracted with isopropylacetate (10 volumes). Upon concentration 2-[(3R)-3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile was obtained as a foam having >99% HPLC purity. MS (pos. ion) m/z: 666 (M+l ).The foam containing high levels of residual solvent was dissolved in 2 M HC1 and the resulting solution was placed under vacuum to remove residual organic solvents. pH of the solution was then adjusted to ~ 7 and the resulting paste was filtered and dried in vacuum without heat. This resulted in isolation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin- l-yl]pent-2-enenitrile containing residual water up to 10%. Drying under vacuum without heat reduces the water level but lead to generation of impurities.Step 1AAlternatively, the isopropylacetate solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-l -yl]pent-2-enenitrile can be concentrated to 4 vol and added to heptane (20 volume) at 0 °C. The resulting suspension was stirred at 0 °C overnight and the product was filtered, washed twice with heptane and dried at 45 °C for 2 days under vacuum to give 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l – yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile in 85 – 90 % yield as a free flowing solid. However, the solids obtained by this method contained high residual solvents (3.9 wt% isopropylacetate and 1.7 wt% heptane). In addition, the free base form was not very stable as degradation products were observed during the drying process at less than 45 °C.Salt formationExample 2Preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4-d]pyrimidin- l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)-piperazin-l-yl]pent-2-enenitrile hemisulfate and sulfate saltHemisulfate: To the solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4- d]pyrimidin-l-yl]-piperidine -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)-piperazin-l-yl]pent-2- enenitrile (4.2 g) in EtOAc (60 mL, 15 vol) was added sulfuric acid (0.31 g, 0.17 mL, 0.5 eq) in EtOAc (20 mL, 5 vol) at ambient temperature. The suspension was stirred at ambient temperature for ~ 2 hr and then 40 °C for 4 hr and then at ambient temperature for at least 1 hr. After filtration and drying at ambient temperature under vacuum, 1.5 g of white powder was obtained. Solubility of the hemi-sulfate at ambient temperature was > 100 mg/mL in water.Sulfate saltTo the solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4- d]pyrimidin-l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)-piperazin-l-yl]pent-2- enenitrile (810 mg) in EtOAc (8 mL, 10 vol) was added sulfuric acid (0.06 mL, 1.0 equiv.) in EtOAc (2.5 mL, 5 vol) at ambient temperature. The resulting suspension was stirred at 40 °C for 2 hr and then cooled to ambient temperature for at least 1 hr. After filtration, solids were dried by suction under Argon for 1 h to give a white powder (0.68 g) in 69% yield.

Example 3Preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4- d]pyrimidin- 1 -yl]-piperidine- 1 -carbonyl] -4-methyl-4-[4-(oxetan-3-yl)-piperazin- 1 -yl]pent-2- enenitrile hydrochlorideTo a solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin- 1 -yl]pent-2- enenitrile (100 mg, 0.15 mmol) in CH2CI2 (1ml) at ambient temperature was added 2 equivalent of HC1 (0.3 mmol, 0.15 ml of 2M HC1 in 1 : 1 dioaxane:CH₂Cl₂). The resulting homogeneous solution was stirred at ambient temperature for 1 h and was added dropwise to 15 volumes of ethylacetate (as compared to CH₂C1₂) resulting in formation of a white solid. The mixtures was aged at ambient temperature for lh and placed at 2-8 C for 19 h. Upon filtration and washing of the filter cake with ethylacetate and drying a white solid was obtained. Analysis by XRPD indicated formation of an amorphous solid. Both Ή-NMR and IC analysis indicated formation of the salt. IC indicated formation mono-HCl salt.

Example 4General procedure for preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)- piperazin-l-yl]pent-2-enenitrile mono- and di-mesylate saltsTo a solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2- enenitrile (100 mg, 0.15 mmol) in CH₂C1₂ (1 ml) at ambient temperature was added either 1 equivalent of methanesulfonic acid (0.15 mmol, 0.2 ml of 74 mg/ml solution in CH₂C1₂) or 2 equivalent of methanesulfonic acid (0.3 mmol, 0.4 ml of 74 mg/ml solution in CH₂C1₂). The resulting homogeneous solution was stirred at ambient temperature for 1 h and was added dropwise to 10 volumes of antisolvents (ethylacetate, methyl tert-butylether (MTBE), or cyclohexane) (10 ml as compared to CH₂C1₂) resulting in formation of a white solid. The mixture was aged at ambient temperature for lh and placed at 2-8 °C for 19 h. Upon filtration and washing of the filter cake with the antisolvent and drying, a white solid was obtained. Analysis by XRPD indicated formation of an amorphous solid. Both Ή-NMR and IC analysis indicated formation of the salt as well as counterion ratio.Alternatively 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]- pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin- 1 -yl]pent-2- enenitrile can be dissolved in 4 volumes of isopropylacetate and added to 2 equivalent of methanesulfonic acid in 6 volumes of isopropylacetate at 0 °C to generate the dimesylate salt.

1. Theoretical mesylate content, monomesylate=12.6% and dimesylate=22.4%, NO- not determinedExample 5 General procedure for the preparation of carboxylate salt Approximately 20 mg of the compound (I) was dissolved in minimum amount of the allocated solvent system. These were then mixed with the appropriate number of equivalents of counterion dissolved or slurried in the allocated solvent.If compound (I) was insoluble in the selected solvent, slurry of the sample was used after adding 300 μί.If the acid was insoluble in the selected solvent, slurry of the acid was used after adding 300 xL.If the acid was a liquid, the acid was added to the dissolved/slurried compound (I) from a stock solution in the allocated solvent.The suspensions/ precipitates resulting from the mixtures of compound (I) were temperature cycled between ambient (ca. 22°C) and 40°C in 4 hour cycles for ca. 48 hrs (the cooling/heating rate after each 4 hour period was ca. 1 °C/min). The mixtures were visually checked and any solids present were isolated and allowed to dry at ambient conditions prior to analysis. Where no solid was present, samples were allowed to evaporate at ambient. Samples which produced amorphous material, after the treatment outlined above, were re- dissolved and precipitated using anti-solvent (ter/-butylmethylether) addition methods at ambient conditions (ca. 22°C). i.e. the selected anti-solvent was added to each solution, until no further precipitation could be observed visually or until no more anti-solvent could be added. The solvents used in this preparation were acetonitrile, acetone, isopropyl acetate, THF and MTBE. The acid used were oxalic acid, L-aspartic acid, maleic acid, malonic acid, L-tartaric acid, and fumaric acid.Example 6General procedure for preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)- piperazin-l-yl]pent-2-enenitrile hemicitrate saltTo a solution 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]- pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin- 1 -yl]pent-2- enenitrile (5 g, 7.5 mmol) in ethanol (50 ml) was added citric acid (720.5 mg, 3.76 mmol) dissolved in 2 ml of water. Mixture was stirred at ambient temperature for 15 min, additional 0.5 ml of water was added and the mixture was stirred for 1 h, concentrated in vacuo to a gum. Ethanol was added and the mixture was concentrated. This process was repeated twice more and then CH2CI2 was added to the mixture. Upon concentration a white solid was obtained which was tumble dried under reduced pressure at 40 C for 4 h, then in a vacuum oven for 19h to give 5.4 g of a solid. Analysis by XRD indicated formation of an amorphous solid 

PATENT

WO2014039899, Example 31

Rilzabrutinib (PRN1008) is an oral, reversible covalent inhibitor of Bruton’s tyrosine kinase (BTK) [1].

https://patents.google.com/patent/WO2014039899A1/enExample 31Synthesis of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)- 1 H-pyrazolo[3,4-d]pyrimidin- 1 -yl)piperidine- 1 -carbonyl)-4-methyl-4-(4-(oxetan-3-yl)piperazin- 1 -yl)pent-2-enenitrile

Step 1A solution of 2-bromo-2-methyl-propanal (696.6 mg, 4.61 mmol) in DCM (10 mL) was cooled with an ice bath and l -(oxetan-3-yl)piperazine (328 mg, 2.31 mmol), diluted with 5-10 mL of DCM, was slowly added via addition funnel over a 15 min period. Next, Hunig’s base (0.4 mL, 2.31 mmol) was added and then the cooling bath was removed. The reaction mixture was stirred at room temperature overnight and the DCM layer was washed three times with 0.5N HC1. The combined aqueous layer was neutralized with NaOH to pH 10-11 and extracted with DCM. The combined organic layer was washed with brine and dried over Na?S0₄. Filtration and removal of solvent afforded 2-methyl-2-[4-(oxetan-3-yl)piperazin-l- yl]propanal as a light yellow liquid, which was used directly in the next step without further purification.Step 2To a cooled (0 °C) solution of 3-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)- pyrazolo[3,4-d]pyrimidin-l-yl]-l-piperidyl]-3-oxo-propanenitrile (80 mg, 0.17 mmol), was added 2-methyl-2-[4-(oxetan-3-yl)piperazin-l-yl]propanal (-108 mg, 0.51 mmol) in DCM (10 mL) followed by pyrrolidine (0.08 mL, 1.02 mmol) and TMS-C1 (0.09 raL, 0.68 mmol.) The ice bath was removed, and the reaction stirred 1 hour. Most of the solvent was removed and the residues were purified by chromatography, using 95:5 CH₂Cl₂:MeOH to obtain 79 mg of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]-pyrimidin-l- yl)piperidine- 1 -carbonyl)-4-methyl-4-(4-(oxetan-3-yl)piperazin- 1 -yl)pent-2-enenitrile as a white solid. MS (pos. ion) m/z: 666 (M+l).

PAPER

https://www.sciencedirect.com/science/article/abs/pii/S0223523421001781?dgcid=rss_sd_all

Therapy based on Bruton’s tyrosine kinase (BTK) inhibitors one of the major treatment options currently recommended for lymphoma patients. The first generation of BTK inhibitor, Ibrutinib, achieved remarkable progress in the treatment of B-cell malignancies, but still has problems with drug-resistance or off-target induced serious side effects. Therefore, numerous new BTK inhibitors were developed to address this unmet medical need. In parallel, the effect of BTK inhibitors against immune-related diseases has been evaluated in clinical trials. This review summarizes recent progress in the research and development of BTK inhibitors, with a focus on structural characteristics and structure-activity relationships. The structure-refinement process of representative pharmacophores as well as their effects on binding affinity, biological activity and pharmacokinetics profiles were analyzed. The advantages and disadvantages of reversible/irreversible BTK inhibitors and their potential implications were discussed to provide a reference for the rational design and development of novel potent BTK inhibitors.

///////////////PRN-1008,  PRN 1008, Rilzabrutinib, リルザブルチニブ,
N#CC(=CC(N(C1COC1)C)(C)C)C(=O)N1CCCC1Cn1nc(c2c1ncnc2N)c1ccc(cc1F)Oc1ccccc1

Melarsoprol

MelarsoprolCAS Registry Number: 494-79-1 
CAS Name: 2-[4-[(4,6-Diamino-1,3,5-triazin-2-yl)amino]phenyl]-1,3,2-dithiarsolane-4-methanolAdditional Names:p-[(4,6-diamino-s-triazin-2-yl)amino]dithiobenzenearsonous acid 3-hydroxypropylene ester; 2-p-(4,6-diamino-s-triazin-2-ylamino)phenyl-4-hydroxymethyl-1,3,2-dithiarsoline; 2-(4-melamin-2-ylphenyl)-4-hydroxymethyl-1,3-dithia-2-arsolaneTrademarks: Mel B; Arsobal (Specia) 
Molecular Formula: C12H15AsN6OS2Molecular Weight: 398.34 
Percent Composition: C 36.18%, H 3.80%, As 18.81%, N 21.10%, O 4.02%, S 16.10%Literature References: Prepn: Friedheim, US2659723 (1953); US2772303 (1956).Properties: Practically insol in water, cold ethanol, methanol. Sol in propylene glycol. 
Therap-Cat: Antiprotozoal (Trypanosoma).Keywords: Antiprotozoal (Trypanosoma).

Melarsoprol is a medication used for the treatment of sleeping sickness (African trypanosomiasis).^[1] It is specifically used for second-stage disease caused by Trypanosoma brucei rhodesiense when the central nervous system is involved.^[1] For Trypanosoma brucei gambiense, eflornithine or fexinidazole is usually preferred.^[1] It is effective in about 95% of people.^[3] It is given by injection into a vein.^[2]

Melarsoprol has a high number of side effects.^[4] Common side effects include brain dysfunction, numbness, rashes, and kidney and liver problems.^[2] About 1-5% of people die during treatment.^[3] In those with glucose-6-phosphate dehydrogenase (G6PD) deficiency, red blood cell breakdown may occur.^[2] It has not been studied in pregnancy.^[2] It works by blocking pyruvate kinase, an enzyme required for aerobic metabolism by the parasite.^[2]

Melarsoprol has been used medically since 1949.^[1] It is on the World Health Organization’s List of Essential Medicines, the safest and most effective medicines needed in a health system.^[5] In regions of the world where the disease is common, melarsoprol is provided for free by the World Health Organization.^[4] It is not commercially available in Canada or the United States.^[2] In the United States, it may be obtained from the Centers for Disease Control and Prevention, while in Canada it is available from Health Canada.^[1][2]

Medical uses

People diagnosed with trypanosome-caused disease should be treated with an anti-trypanosomal. Treatment is based on stage, 1 or 2, and parasite,T. b. rhodesiense or T. b. gambiense. In stage 1 disease, trypanosomes are present only in the peripheral circulation. In stage 2 disease, trypanosomes have crossed the blood-brain barrier and are present in the central nervous system.^[6]

The following are considerable treatment options:^[6]

Melarsoprol is a treatment used during the second stage of the disease. So far, it is the only treatment available for late-stage T. b. rhodesiense.^[7]

Due to high toxicity, melarsoprol is reserved only for the most dangerous cases. Other agents associated with lower toxicity levels are used during stage 1 of the disease.^[8] The approval of the nifurtimox-eflornithine combination therapy (NECT) in 2009 for the treatment of T. b. gambiense limited the use of melarsoprol to the treatment of second-stage T. b. rhodesiense.^[9]

Failure rates of 27% in certain African countries have been reported.^[10] This was caused by both drug resistance and additional mechanisms that have not yet been elucidated. Resistance is likely due to transport problems associated with the P2 transporter, an adenine-adenosine transporter. Resistance can occur with point mutations within this transporter.^[11] Resistance has been present since the 1970s.^[12]

Mechanism of action

Melarsoprol is a prodrug, which is metabolized to melarsen oxide (Mel Ox) as its active form. Mel Ox is an phenylarsonous acid derivative that irreversibly binds to sulfhydryl groups on pyruvate kinase, which disrupts energy production in the parasite. The inability to distinguish between host and parasite PK renders this drug highly toxic with many side effects.

Mel Ox also reacts with trypanothione (a spermidine-glutathione adduct that replaces glutathione in trypanosomes). It forms a melarsen oxide-trypanothione adduct (Mel T) that competitively inhibits trypanothione reductase, effectively killing the protist.^[11]

Pharmacokinetics

The half-life of melarsoprol is less than one hour, but bioassays indicate a 35-hour half-life. This is commonly associated with pharmacologic agents that have active metabolites. One such metabolite, Mel Ox, reaches maximum plasma levels about 15 minutes after melarsoprol injection. Melarsoprol clearance is 21.5 ml/min/kg and the Mel Ox half-life is approximately 3.9 hours.^[13]

Dosage

Two arsenic-containing stereoisomers exist in a 3:1 molar ratio. Since melarsoprol is insoluble in water, dosage occurs via a 3.6% propylene glycol intravenous injection.^[11] To avoid the risk of injection site reactions, melarsoprol must be given slowly.^{[citation needed]}

Melarsoprol used for the treatment of African trypanosomiasis with CNS involvement is given under a complicated dosing schedule. The dosing schedule for children and adults is 2–3.6 mg/kg/day intravenously for three days, then repeated every seven days for a total of three series.^[6] To monitor for relapse, follow-up is recommended every six months for at least two years.^[3]

Side effects

Although melarsoprol cures about 96% of people with late stage disease, its toxicity limits its use.^[7] About 1-5% of people die during treatment.^[3] As a toxic organic compound of arsenic, melarsoprol is a dangerous treatment that is typically only administered by injection under the supervision of a licensed physician. Notable side effects are similar to arsenic poisoning. Among clinicians, it is colloquially referred to as “arsenic in antifreeze”.^[14] Severe and life-threatening adverse reactions are associated with melarsoprol. It is known to cause a range of side effects including convulsions, fever, loss of consciousness, rashes, bloody stools, nausea and vomiting. In approximately 5-10% of cases, it causes encephalopathy. Of those, about 50% die due to encephalopathy-related adverse reactions.^[6] Additional potentially serious side effects of melarsoprol include damage to the heart, presence of albumin in the urine that could be associated with kidney damage, and an increase in blood pressure.^[3]

Cautions

Numerous warnings must be examined before melarsoprol treatment can be initiated. Prior to initiation, the following must be noted: glucose-6-phosphate dehydrogenase deficiency, kidney or liver disease, cardiac problems (high blood pressure, irregular beating of the heart or arrhythmias, any damage to the heart muscles and potential signs of heart failure), preexisting nervous system disorders, and any signs of leprosy.

Routine laboratory testing is needed before and after melarsoprol initiation. Laboratory parameters for both therapeutic effects and toxic effects need to be evaluated.

Blood analysis is used to detect the presence of trypanosomes. An evaluation of the cerebrospinal fluid via a lumbar puncture is also used to determine an individual’s white blood count and level of protein. These are diagnostic criteria such that the presence of trypanosomes, an elevated white blood count greater than five per microliter, or a protein content greater than 40 mg are considered abnormal and initiation should be considered. Continuous cerebrospinal fluid evaluation should be repeated every six months for at least three years in individuals that have undergone melarsoprol treatment.

To assess potential concerns related to toxicity, the following should be completed: a complete blood count, an assessment of electrolyte levels, liver and kidney function tests, and a urinalysis to detect the appearance, concentration and content of the urine.

Melarsoprol should be given using glass syringes (if they can be reliably sterilised). The propylene glycol it contains is capable of dissolving plastic.^[15]

Pregnancy and breastfeeding

Currently, melarsoprol is not recommended for use in pregnant women. The World Health Organization suggests that treatment be deferred until immediately after delivery since the effects of the medication on the developing fetus have not yet been established.^[3]

Lactation guidelines associated with melarsoprol have not yet been established.

Society and culture

Melarsoprol is produced by Sanofi-Aventis and under an agreement with the WHO, they donate melarsoprol to countries where the disease is common.^{[medical citation needed]}

Melarsoprol was used to treat a patient with African trypanosomiasis on season 1 episode 7 “Fidelity” of the medical drama House MD.^[16]

PAPER

Journal of Organometallic Chemistry (2006), 691(5), 1081-1084.

https://www.sciencedirect.com/science/article/abs/pii/S0022328X05009344

Graphical abstract

(2-Phenyl-[1,3,2]dithiarsolan-4-yl)-methanol derivatives were tested on K562 and U937 human leukemia cell lines. Their systemic toxicity was estimated by the corresponding LD₅₀ on mice. The cytotoxic activity of each derivative was significantly better than that of arsenic trioxide and the therapeutic index (T.I. = LD₅₀/IC₅₀) was improved.

References

1. ^ Jump up to:^{a b c d e f} “Our Formulary Infectious Diseases Laboratories CDC”. http://www.cdc.gov. 22 September 2016. Archived from the original on 16 December 2016. Retrieved 7 December 2016.
2. ^ Jump up to:^{a b c d e f g h} “Melarsoprol Drug Information, Professional”. http://www.drugs.com. 20 December 1994. Archived from the original on 30 December 2016. Retrieved 7 December 2016.
3. ^ Jump up to:^{a b c d e f} “WHO Model Prescribing Information: Drugs Used in Parasitic Diseases – Second Edition: Protozoa: African trypanosomiasis: Melarsoprol”. WHO. 1995. Archived from the original on 2016-11-10. Retrieved 2016-11-09.
4. ^ Jump up to:^a b “Trypanosomiasis, human African (sleeping sickness)”. World Health Organization. February 2016. Archived from the original on 4 December 2016. Retrieved 7 December 2016.
5. ^ World Health Organization (2019). World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
6. ^ Jump up to:^a b c d CDC (2013). “Disease Control and Prevention: Parasites – African Trypanosomiasis”. Archived from the original on 2017-06-19.
7. ^ Jump up to:^a b “Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense (African Trypanosomiasis) – Infectious Disease and Antimicrobial Agents”. http://www.antimicrobe.org. Archived from the original on 2016-11-28. Retrieved 2016-11-17.
8. ^ Bisser S, N’Siesi FX, Lejon V, et al. (2007). “Equivalence trial of melarsoprol and nifurtimox monotherapy and combination therapy for the treatment of second-stage Trypanosoma brucei rhodesiense sleeping sickness”. J. Infect. Dis. 195 (3): 322–9. doi:10.1086/510534. PMID 17205469.
9. ^ Farrar J (2014). “Manson’s Tropical Diseases: Expert Consult-Online”. 23: 616.
10. ^ Kioy, D.; Jannin, J.; Mattock, N. (March 2004). “Human African trypanosomiasis”. Nature Reviews Microbiology. 2 (3): 186–187. doi:10.1038/nrmicro848. PMID 15751187. S2CID 36525641.
11. ^ Jump up to:^a b c Brunton L (2011). “Goodman & Gillman’s The Pharmacological Basis of Therapeutics”. McGraw Hill Medical: 1427–28.
12. ^ Brun, Reto; Schumacher, Reto; Schmid, Cecile; Kunz, Christina; Burri, Christian (November 2001). “The phenomenon of treatment failures in Human African Trypanosomiasis”. Tropical Medicine and International Health. 6 (11): 906–914. doi:10.1046/j.1365-3156.2001.00775.x. PMID 11703845. S2CID 21542129.
13. ^ Keiser J.; Ericsson O; Burri C (2000). “Investigations of the metabolites of the trypanocidal drug melarsoprol”. Clinical Pharmacology. 67 (5): 478–88. doi:10.1067/mcp.2000.105990. PMID 10824626. S2CID 24326873.
14. ^ Hollingham R (2005). “Curing diseases modern medicine has left behind”. New Scientist. 2005 (2482): 40–41. Archived from the original on 2015-05-11.
15. ^ “MELARSOPROL injectable – Essential drugs”. medicalguidelines.msf.org. Retrieved 6 December 2019.
16. ^ Holtz, Andrew (2006). The Medical Science of House, M.D.Penguin. p. 272. ISBN 1440628734. Retrieved 25 March 2020.

External links

• “Melarsoprol”. Drug Information Portal. U.S. National Library of Medicine.

/////////Melarsoprol

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Nangibotide

LQEEDAGEYGCM-amide

CAS 2014384-91-7

• Molecular FormulaC₅₄H₈₂N₁₄O₂₂S₂
• Average mass1343.439 Da
• 2014384‐91‐7

• L-Leucyl-L-glutaminyl-L-α-glutamyl-L-α-glutamyl-L-α-aspartyl-L-alanylglycyl-L-α-glutamyl-L-tyrosylglycyl-L-cysteinyl-L-methioninamide
• LR 12 peptide
• LQEEDAGEYG CM

L-Leucyl-L-glutaminyl-L-glutaminyl-L-α-glutamyl-L-α-aspartyl-L-alanylglycyl-L-α-glutamyl-L-tyrosylglycyl-L-cysteinyl-L-methionine
L-Methionine, L-leucyl-L-glutaminyl-L-glutaminyl-L-α-glutamyl-L-α-aspartyl-L-alanylglycyl-L-α-glutamyl-L-tyrosylglycyl-L-cysteinyl-нангиботидمانغيبوتيد南吉博肽

• OriginatorInotrem
• ClassAnti-infectives; Anti-inflammatories; Anti-ischaemics; Antivirals; Peptides
• Mechanism of ActionTREML1 protein inhibitors

• Phase II/IIICOVID 2019 infections
• Phase IISeptic shock
• Phase IMyocardial infarction

• 12 Jul 2021Inotrem has patents pending for nangibotide use in severe forms of COVID-19
• 12 Jul 2021Inotrem receives funding from French government by Bpifrance for nangibotide development in COVID-2019 infections
• 12 Jul 2021Inotrem receives authorization from both the French and Belgian authorities to proceed with clinical development of nangibotide up to registration in COVID-2019 infections

Nangibotide, also referred as LR12, is an antagonist of triggering receptor expressed on myeloid cells (TREM)-1, and was derived from residues 94 to 105 of TREM-like transcript-1 (TLT-1).

TREM-1 plays a crucial role in the onset of sepsis by amplifying the host immune response. TLT-1– and TLT-1–derived peptides therefore exhibit anti-inflammatory properties by dampening TREM-1 signalling.  LR12 blocks TREM-1 by binding to the TREM-1 ligand and provides protective effects during sepsis such as inhibiting hyper-responsiveness, organ damage, and death, without causing deleterious effects. The protective effects of modulating TREM-1 signalling are also evident in other models of inflammation such as: pancreatitis; haemorrhagic shock; inflammatory bowel diseases and inflammatory arthritis

Inotrem is developing the peptide nangibotide, a triggering receptor expressed on myeloid cells 1 inhibitor, for treating sepsis and septic shock. In July 2021, this drug was reported to be in phase 3 clinical development.

Nangibotide is an inhibitor of TREM-1, a receptor found on certain white blood cells. Activation of TREM-1 stimulates inflammation. Nangibotide is therefore being investigated as a treatment for the overwhelming inflammation typically seen in severe sepsis.

Mode of action

TREM-1 is a receptor found on neutrophils, macrophages and monocytes, key elements of the immune system. Activation of TREM-1 results in expression of NF-κB, which promotes systemic inflammation. Nangibotide inhibits TREM-1, thereby preventing the inflammatory activation. Absence of TREM-1 results in vastly reduced inflammation without impairing the ability to fight infection.^[2]

Animal models

LR17, a mouse equivalent of nangibotide, improves survival in mouse models of severe sepsis.^[3] In a pig model of sepsis, LR12 – another animal equivalent of nangibotide – resulted in significantly improved haemodynamics and less organ failure.^[4] In monkeys, LR12 also reduced the inflammatory and hypotensive effects of sepsis.^[5]

Human studies

Nangibotide has demonstrated safety in Phase 1 (healthy volunteers)^[6] and Phase 2 (sick patients with septic shock)^[7] studies. The ASTONISH trial will examine clinical efficacy in 450 patients with septic shock.^[8]

Inotrem Receives Approval to Expand Nangibotide Clinical Trial in Critically Ill COVID-19 Patients and Receives Additional Public Funding of €45 Million

• Inotrem’s phase 2/3 clinical trial “ESSENTIAL” will enroll up to 730 patients in Europe to demonstrate the safety and efficacy of nangibotide to treat critically ill COVID-19 patients with respiratory failure.
• Recent preclinical studies have strengthened the body of evidence for targeting the TREM-1 pathway which is activated in a subset of patients suffering from severe COVID-19.

July 12, 2021 03:00 AM Eastern Daylight Time

PARIS–(BUSINESS WIRE)–Inotrem S.A., a biotechnology company specializing in the development of immunotherapies targeting the TREM-1 pathway, announces that it has obtained authorization to pursue the clinical development of nangibotide up to registration in COVID-19 patients from both the French and Belgian competent authorities.

As part of this program, Inotrem receives additional 45 million euros in public funding under the “Capacity Building” Call for Expression of Interest, operated on behalf of the French government by Bpifrance, the French national investment bank, as part of the Programme d’investissements d’avenir (PIA) and the France Recovery Plan, bringing French state support for the project to a total of 52,5 million euros. This public funding will support Inotrem’s clinical program including the phase 2/3 study “ESSENTIAL” which aims to demonstrate the efficacy and safety of nangibotide in treating patients in respiratory distress with severe forms of COVID-19.

The primary endpoint is evaluation of the impact of nangibotide on the progression of disease in patients receiving ventilatory support due to COVID-19 as well as on the severity of the respiratory failure, duration of mechanical ventilation, length of stay in intensive care and mortality. In “ESSENTIAL”, a Phase 2/3 clinical program, up to 730 patients will be enrolled initially in France and Belgium and, possibly in other European countries. Pre-defined interim analyses will be conducted by an independent Data Monitoring Board to test futility and to allow for the study design to be adapted as necessary. “ESSNTIAL” is the continuation of a 60 patients phase 2a evaluating the safety and efficacy of nangibotide in patients suffering from severe COVID-19. In July 2020, the CoviTREM-1 consortium, which includes the Nancy and Limoges university hospitals and Inotrem, obtained public funding of 7,5 million euros under the “PSPC-COVID” call for projects, operated on behalf of the French government by Bpifrance

New pre-clinical studies with nangibotide have demonstrated that the administration of nangibotide in murine models infected with SARS-CoV-2 was associated with a decrease in inflammatory mediators and an improvement of clinical signs, in particular respiratory function, and survival. Inotrem also confirmed in 3 different and independent cohorts that sTREM-1, a marker of the activation of the TREM-1 biological pathway, is associated with both severity and mortality in critically ill COVID-19 patients.

Leveraging the results of these preclinical studies and the implications for the role of the TREM-1 pathway in COVID-19, Inotrem has filed additional patents to cover nangibotide use in severe forms of COVID-19 as well as the use of sTREM-1 as a biomarker and companion diagnostic. This significantly strengthens Inotrem’s already broad patent estate.

Jean-Jacques Garaud, Executive Vice-President, Head of Scientific and Medical Affairs and Inotrem’s co-founder said :“We are eager to pursue the development of nangibotide in these severe forms of COVID-19. Nangibotide is a TREM-1 inhibitor which has already demonstrated a trend towards efficacy in septic shock patients and has the potential to modulate the dysregulated immune response in critically ill COVID-19 patients. With this large clinical study, we can demonstrate efficacy for nangibotide in a further indication with the goals of reducing the duration of hospitalization and mortality.”

Sven Zimmerman, CEO of Inotrem, also declared: “The size of the financial support awarded to us as part of the French government’s initiative against COVID-19 is a testimony to the relevance of targeting the TREM-1 pathway with nangibotide in these severely ill patients. We are delighted by the confidence placed in our technology and our team. Everyone at Inotrem is fully committed to deliver on this ambitious program alongside nangibotide’s ongoing Phase 2b trial in septic shock patients.”

About Inotrem
Inotrem S.A. is a biotechnology company specialized in immunotherapy for acute and chronic inflammatory syndromes. The company has developed a new concept of immunomodulation that targets the TREM-1 pathway to control unbalanced inflammatory responses. Through its proprietary technology platform, Inotrem has developed the first-in-class TREM-1 inhibitor, LR12 (nangibotide), with potential applications in a number of therapeutic indications such as septic shock and myocardial infarction. In parallel, Inotrem has also launched another program to develop a new therapeutic modality targeting chronic inflammatory diseases. The company was founded in 2013 by Dr. Jean-Jacques Garaud, a former head of research and early development at the Roche Group, Prof. Sébastien Gibot and Dr. Marc Derive. Inotrem is supported by leading European and North American investors.

www.inotrem.com

About TREM-1 pathway
TREM-1 pathway is an amplification loop of the immune response that triggers an exuberant and hyperactivated immune state which is known to play a crucial role in the pathophysiology of septic shock and acute myocardial infarction.

About Nangibotide
Nangibotide is the formulation of the active ingredient LR12, which is a 12 amino-acid peptide prepared by chemical synthesis. LR12 is a specific TREM-1 inhibitor, acting as a decoy receptor and interfering in the binding of TREM-1 and its ligand. In preclinical septic shock models, nangibotide was able to restore appropriate inflammatory response, vascular function, and improved animals’ survival post septic shock.

About ESSENTIAL study:
The Efficacy and Safety Study Exploring Nangibotide Treatment in COVID-19 pAtients with ventiLatory support, is a randomized, double-blind, placebo-controlled confirmatory study with adaptive features that will be performed in Europe. This is a pivotal study and it is expected that based on its results, nangibotide could be registered in this indication. The first part of the study (i.e.: 60 patients) has been already finalized and assessed by an independent data monitoring committee with excellent safety results. The study will recruit up to 730 patients in up to 40 sites. Several interim and futility analyses are foreseen as part of the adaptive design of the study.

About Bpifrance
Bpifrance is the French national investment bank: it finances businesses – at every stage of their development – through loans, guarantees, equity investments and export insurances. Bpifrance also provides extra-financial services (training, consultancy.). to help entrepreneurs meet their challenges (innovation, export…).

PATENT

WO-2021144388

Process for preparing nangibotide by solid phase synthesis, useful for treating acute inflammatory disorders such as septic shock. Also claims novel peptide fragments, useful in the synthesis of nangibotide.

Example 1

Preparation of nangibotide by full SPPS (Reference)

Step 1 : Loading of the first amino acid onto the Rink Amide Resin

2 g of MBHA resin (1.0-1.3 mmol/g) was swelled using 16 mL of DMF for 30 min. 2 eq Fmoc-Met-OH (2.4 mmol, 2.67 g), 2 eq DIC (2.4 mmol, 1.136 mL) and 2 eq OxymaPure (2.4 mmol, 1.023 g) were dissolved in 8 mL of DMF at 0.3 M cone, and added to the resin after 5 min. All the coupling steps were conducted in this way unless described differently. The loading step was carried out for 1.5 hour. After the loading, the resin was filtered and washed 3 times with 12 mL of DMF. The Fmoc deprotection step was carried out by addition of 12 mL of 20% piperidine solution in DMF for two 10 min cycles. This step was performed analogously for all the amino acid residues. The loading, calculated by UV absorption for the peptidyl resin, was 0.8 mmol/g.

Step 2: peptide elongation

For the coupling of all the amino acids involved in the synthesis of nangibotide, 3 eq of each amino acid were activated by 3 eq of DIC and OxymaPure dissolved in DMF at 0.3 M cone. At the end of the peptide elongation, a final Fmoc deprotection, as already described, was performed before moving to the cleavage step.

Step 3: Cleavage and precipitation of crude nangibotide

The cleavage of nangibotide off the resin was carried out using a solution of 16 mL of TFA/DODT/TIPS/water in 90/4/3/3 ratio cooled at 0°C. The peptidyl resin was added portionwise in 30 min keeping the internal temperature under 25°C. The cleavage was run for 3.5 hours, then the resin was filtered and washed by 10 mL of TFA for 10 min.

DIPE was used for the precipitation of the peptide, adding 12 volumes (300 mL) dropwise to the peptide TFA solution, keeping the temperature under 20°C. The suspension with nangibotide was filtered on a gooch funnel, the peptide washed again with 100 mL of DIPE and then dried under vacuum overnight. Molar yield 40%. Purity 61%.

Example 2

Preparation of nangibotide by three-fragment condensation

In the approach using three fragments, only the cysteine residue was coupled to the methionine on rink amide resin to prepare fragment 11-12, whereas protected peptide fragments 1-7 and 8-10 were synthesized using 2-CTC resin.

Step 1: Synthesis of fragment 11-12

2 g of MBHA resin (1.0-1.3 mmol/g) was swelled using 16 mL of DMF for 30 min 2 eq of Fmoc-Met-OH (2.4 mmol, 2.67 g), 2 eq DIC (2.4 mmol, 1.136 mL) and 2 eq OxymaPure (2.4 mmol, 1.023 g) were dissolved in 8 mL of DMF at 0.3 M cone, and added to the resin. The loading step was carried out for 1 and half hour. After the loading, the resin was filtered and washed 3 times with 12 mL of DMF. The Fmoc deprotection step was carried out by

addition of 12 mL of a 20% piperidine solution in DMF for two 10 min cycles. Same procedure was repeated for the coupling of Fmoc-Cys(Trt)-OH to obtain resin-attached Fmoc-deprotected fragment 11-12. The loading, calculated by UV absorption for the peptidyl resin relative to the first amino acid inserted, was 0.8 mmol/g.

Step 2: Synthesis of fragments 1-7 and 8-10

For the synthesis of both fragments the loading of 2-chloro trityl chloride resin was performed on 5 g (1.6 mmol/g) using 0.8 eq Fmoc-Gly-OH (6.40 mmol, 1.90 g) dissolved in 30 mL of DCM and addition of 3 eq DIPEA (24 mmol, 4.19 mL). The loading step was carried out for 1 hour, then the resin was washed by 30 mL DCM for three times and eventual Cl-groups were capped by two different capping solutions: first by 30 mL of methanol/DIPEA/DCM (1:2:7) and then by 30 mL AC2O/DIPEA/DCM in the same ratio. After the treatment with these solutions for 15 min and subsequent washing with DCM, the resin was washed three times with DMF, before deprotection of Fmoc and evaluation of the resin loading. Generally, this protocol gave a resin loaded with 1.1 mmol/g Fmoc-Gly-OH. The Fmoc deprotection and coupling step protocols were equally performed with all the amino acids in the respective sequences: Fmoc-Tyr(tBu)-OH and Fmoc-Glu(tBu)-OH for fragment 8-10, and Fmoc-Ala-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH twice, Fmoc-Gln(Trt)-OH and Fmoc-Leu-OH for fragment 1-7.

For each coupling, 3 eq amino acid were activated by 3 eq DIC and 3 eq OxymaPure dissolved in DMF at 0.3 M cone.

Fragment Fmoc-Glu(tBu)-Tyr(tBu)-Gly-OH (8-10) was obtained by cleavage off the resin using 6 volumes (30 mL) of a TFA 1.5 % solution in DCM, 5 times for 2 min. The final TFA solution was neutralized by 1.2 eq pyridine (15.89 mmol, 1.3 mL) diluted in 30 mL methanol. The final solution was concentrated to 50 mL under vacuum then washed by water and brine. The organic layer was dried by anhydrous sodium sulphate, filtered and further concentrated before crystallization of the tripeptide with 5 volumes of petroleum ether at 0°C. The peptide was filtered, washed by petroleum ether and dried overnight in a vacuum oven at 37°C. Molar yield 65%. Purity 90%.

Fragment Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-OH (1-7) was obtained by cleavage off the resin using 6 volumes (30 mL) of a TFA 1.5 % solution in DCM, 5 times for 2 min. The final TFA solution was neutralized by 1.2 eq pyridine (15.89 mmol, 1.3 mL) diluted in 30 mL methanol. The DCM was evaporated and replaced by methanol, adding and evaporating 30 mL methanol a couple of times till one third of the volume. The peptide fragment was precipitated by adding 5 volumes (150 mL) water to the methanol solution at 0°C and filtered after stirring for 30 min. The full protected heptapeptide was washed by water and dried overnight in a vacuum oven at 37°C. Molar yield 85%. Purity 89%.

Step 3: Synthesis of fragment 8-12 (Fragment condensation 1)

The fragment condensation between Fmoc-Glu(tBu)-Tyr(tBu)-Gly-OH (8-10) and H-Cys(Trt)-Met-MBHA resin (11-12) was carried out activating 2 eq (1.6 mmol, 1.12 g) of fragment 8-10 dissolved in 6 mL of DMF at 40°C by using 2 eq OxymaPure (1.6 mmol, 0.22 g) and 2 eq DIC (1.6 mmol, 0.25 mL) for 10 min. The activated ester of tripeptide 8-10 was added to the resin-attached fragment 11-12 and stirred for 3 hours at 40°C. After filtration, the resin was washed three times by 15 mL DMF and then capped by 12 mL of AC2O 10% in DMF for 15 min. The resin was washed three timed by 12 mL DMF before deprotection of Fmoc to finally obtain resin-attached Fmoc-protected fragment 8-12. Molar yield 91%. Purity 89%.

Step 4: Synthesis of nanaibotide (Fragment condensation 2)

The fragment condensation between fragment 1-7 and H-Glu(OtBu)-Tyr(tBu)-Gly-Cys(Trt)-Met-MBHA resin (8-12) was carried out activating 1.5 eq (2.25 mmol, 2.64 g) of fragment 1-7 dissolved in 25 mL DMF at 40°C by using 2 eq OxymaPure (2.25 mmol, 0.32 g) and 2 eq DIC (2.25 mmol, 0.35 mL) for 15 min. The activated ester of fragment 1-7 was added to the resin-attached fragment 8-12 and stirred for 3.5 hours at 40°C. After filtration, the resin was washed three times by 12 mL DMF before deprotection of Fmoc with the standard procedure described above. After Fmoc deprotection, the resin was washed again by DMF and DCM and then dried at vacuum pump.

Step 5: Cleavage and precipitation of crude nanaibotide

The cleavage of nangibotide off the resin was carried out using a solution of 16 mL of TFA/DODT/TIPS/water in 90/4/3/3 ratio cooled at 0°C. The peptidyl resin was added portionwise in 30 min keeping the internal temperature under 25°C. The cleavage was run for 3.5 hours, then the resin filtered and washed by 10 mL of TFA for 10 min.

DIPE was used to precipitate the peptide, adding 12 volumes (300 mL) dropwise to the peptide TFA solution, keeping the temperature under 20°C. The suspension with nangibotide was filtered on a gooch funnel, the peptide washed again with 100 mL of DIPE and then dried at vacuum pump overnight. Molar yield 61%. Purity 73%.

Example 3

Preparation of nangibotide by two-fragment condensation

In the approach using two fragments, the SPPS elongation onto MBHA resin, as described in Example 2, step 1, was continued until Glu⁸ was attached to provide fragment 8-12, then fragment 1-7, synthesized on 2-CTC resin as described in example 2, step 2, was coupled to the resin-attached fragment 8-12 as described in example 2, step 4.

Step 1: Synthesis of fragment 8-12

2 g of MBHA resin (1.0-1.3 mmol/g) was swelled using 16 mL of DMF for 30 min 2 eq of Fmoc-Met-OH (2.4 mmol, 2.67 g), 2 eq DIC (2.4 mmol, 1.136 mL) and 2 eq OxymaPure (2.4 mmol, 1.023 g) were dissolved in 8 mL of DMF at 0.3 M cone, and added to the resin. The loading step was carried out for 1 and half hour. After the loading, the resin was filtered and washed 3 times with 12 mL of DMF. The Fmoc deprotection step was carried out by addition of 12 mL of a 20% piperidine solution in DMF for two 10 min cycles. Same procedure was repeated for the coupling of Fmoc-Cys(Trt)-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Gly-OH to obtain fragment 8-12. The loading, calculated by UV absorption for the peptidyl resin relative to the first amino acid inserted, was 0.8 mmol/g. Molar yield 88%. Purity 83%.

Step 2: Synthesis of nanaibotide (Fragment condensation 2)

The final fragment condensation was performed as described in example 2, step 4.

Step 3: Cleavage and precipitation of crude nanaibotide

The cleavage of nangibotide off the resin was carried out as described in example 2, step 5. Molar yield 60%. Purity 70%.

PAPER

Methods in enzymology (2000), 312, 293-304

 Journal of the American College of Cardiology (2016), 68(25), 2776-2793

PATENT

https://patents.google.com/patent/WO2011124685A1/en

Product pat, WO2011124685 ,protection in the EU states and the US  April 2031

References

1. ^ Cuvier V, Lorch U, Witte S, Olivier A, Gibot S, Delor I, Garaud JJ, Derive M, Salcedo-Magguilli M (2018). “A first-in-man safety and pharmacokinetics study of nangibotide, a new modulator of innate immune response through TREM-1 receptor inhibition”. Br J Clin Pharmacol. 84 (10): 2270–2279. doi:10.1111/bcp.13668. PMC 6138490. PMID 29885068.
2. ^ Weber B, Schuster S, Zysset D, Rihs S, Dickgreber N, Schürch C, Riether C, Siegrist M, Schneider C, Pawelski H, Gurzeler U, Ziltener P, Genitsch V, Tacchini-Cottier F, Ochsenbein A, Hofstetter W, Kopf M, Kaufmann T, Oxenius A, Reith W, Saurer L, Mueller C (2014). “TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance”. PLOS Pathog. 10 (1): e1003900. doi:10.1371/journal.ppat.1003900. PMC 3894224. PMID 24453980.
3. ^ Derive M, Bouazza Y, Sennoun N, Marchionni S, Quigley L, Washington V, Massin F, Max JP, Ford J, Alauzet C, Levy B, McVicar DW, Gibot S (1 June 2012). “Soluble TREM-like transcript-1 regulates leukocyte activation and controls microbial sepsis”. Journal of Immunology. 188 (11): 5585–5592. doi:10.4049/jimmunol.1102674. PMC 6382278. PMID 22551551.
4. ^ Derive M, Boufenzer A, Bouazza Y, Groubatch F, Alauzet C, Barraud D, Lozniewski A, Leroy P, Tran N, Gibot S (Feb 2013). “Effects of a TREM-like transcript 1-derived peptide during hypodynamic septic shock in pigs”. Shock. 39 (2): 176–182. doi:10.1097/SHK.0b013e31827bcdfb. PMID 23324887. S2CID 23583753.
5. ^ Derive M, Boufenzer A, Gibot S (April 2014). “Attenuation of responses to endotoxin by the triggering receptor expressed on myeloid cells-1 inhibitor LR12 in nonhuman primate”. Anaesthesiology. 120 (4): 935–942. doi:10.1097/ALN.0000000000000078. PMID 24270127. S2CID 10347527.
6. ^ Cuvier V, Lorch U, Witte S, Olivier A, Gibot S, Delor I, Garaud JJ, Derive M, Salcedo-Magguilli M (2018). “A first-in-man safety and pharmacokinetics study of nangibotide, a new modulator of innate immune response through TREM-1 receptor inhibition”. Br J Clin Pharmacol. 84 (10): 2270–2279. doi:10.1111/bcp.13668. PMC 6138490. PMID 29885068.
7. ^ François B, Wittebole X, Ferrer R, Mira JP, Dugernier T, Gibot S, Derive M, Olivier A, Cuvier V, Witte S, Pickkers P, Vandenhende F, Garaud JJ, Sánchez M, Salcedo-Magguilli M, Laterre PF (July 2020). “Nangibotide in patients with septic shock: a Phase 2a randomized controlled clinical trial”. Intensive Care Medicine. 46 (7): 1425–1437. doi:10.1007/s00134-020-06109-z. PMID 32468087. S2CID 218912723.
8. ^ “Efficacy, Safety and Tolerability of Nangibotide in Patients With Septic Shock (ASTONISH)”. ClinicalTrials.gov. US National Library of Medicine. Retrieved 13 July 2020.

Derive et al (2013) Effects of a TREM-Like Transcript 1–Derived Peptide During Hypodynamic Septic Shock in Pigs. Shock39(2) 176 PMID: 23324887

Derive et al (2014) Attenuation of Responses to Endotoxin by the Triggering Receptor Expressed on Myeloid Cells-1 Inhibitor LR12 in Nonhuman Primate. Anesthesiology120(4) 935 PMID: 24270127

Derive et al (2012) Soluble Trem-like Transcript-1 Regulates Leukocyte Activation and Controls Microbial Sepsis. J. Immunol.188(11) 5585 PMID: 22551551

//////////////Nangibotide, phase 3, нангиботид , مانغيبوتيد , 南吉博肽 , INOTREM, SEPTIC SHOCK, PEPTIDE

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OTESECONAZOLE

VT 1161

(2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol

Other Names

• (αR)-α-(2,4-Difluorophenyl)-β,β-difluoro-α-(1H-tetrazol-1-ylmethyl)-5-[4-(2,2,2-trifluoroethoxy)phenyl]-2-pyridineethanol
• (2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,3,4-tetrazol-1-yl)- 1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}propan-2-ol

 Oteseconazole, also known as VT-1161, is a tetrazole antifungal agent potentially for the treatment of candidal vaginal infection. VT-1161 Protects Immunosuppressed Mice from Rhizopus arrhizus var. arrhizus Infection. VT-1161 dosed once daily or once weekly exhibits potent efficacy in treatment of dermatophytosis in a guinea pig model.

Oteseconazole has been used in trials studying the treatment of Tinea Pedis, Onychomycosis, Candidiasis, Vulvovaginal, and Recurrent Vulvovaginal Candidiasis.

Mycovia Pharmaceuticals is developing oteseconazole, the lead from a program of metalloenzyme Cyp51 (lanosterol demethylase) inhibitors, developed using the company’s Metallophile technology, for treating fungal infections including onychomycosis and recurrent vulvovaginal candidiasis (RVVC). In July 2021, oteseconazole was reported to be in phase 3 clinical development. Licensee Jiangsu Hengrui Medicine is developing otesaconazole, as an oral capsule formulation, for treating fungal conditions, including RVVC, onychomycosis and invasive fungal infections, in Greater China and planned for a phase 3 trial in April 2021 for treating VVC.

• OriginatorViamet Pharmaceuticals
• DeveloperMycovia Pharmaceuticals; Viamet Pharmaceuticals
• ClassAntifungals; Foot disorder therapies; Pyridines; Small molecules; Tetrazoles
• Mechanism of Action14-alpha demethylase inhibitors

• PreregistrationVulvovaginal candidiasis
• Phase IIOnychomycosis
• No development reportedTinea pedis

• 01 Jun 2021Preregistration for Vulvovaginal candidiasis (In adolescents, In adults, In children, Recurrent) in USA (PO)
• 01 Jun 2021Mycovia intends to launch otesaconazole (Recurrent) for Vulvovaginal candidiasis in the US in early 2022
• 06 Jan 2021Interim efficacy and adverse events data from a phase III ultraVIOLET trial in Vulvovaginal candidiasis released by Mycovia Pharmaceuticals

PATENT

WO 2017049080

WO 2016149486

US 20150024938

WO 2015143172

WO 2015143184 

WO 2015143180

 WO 2015143142

 WO 2013110002

WO 2013109998

WO 2011133875 

PATENT

WO 2017049080,

PATENT

WO-2021143811

Novel crystalline polymorphic form of VT-1161 (also known as oteseconazole) phosphate disodium salt, useful as a prodrug of oteseconazole, for treating systemic fungal infection (eg Candida albicans infection) or onychomycosis.The function of metalloenzymes is highly dependent on the presence of metal ions in the active site of the enzyme. It is recognized that reagents that bind to and inactivate metal ions at the active site greatly reduce the activity of the enzyme. Nature uses this same strategy to reduce the activity of certain metalloenzymes during periods when enzyme activity is not needed. For example, the protein TIMP (tissue inhibitor of metalloproteinases) binds to zinc ions in the active sites of various matrix metalloproteinases, thereby inhibiting enzyme activity. The pharmaceutical industry has used the same strategy in the design of therapeutic agents. For example, the azole antifungal agents fluconazole and voriconazole contain 1-(1,2,4-triazole) group, which exists in the active site of the target enzyme lanosterol demethylase The heme iron binds, thereby inactivating the enzyme. Another example includes zinc-bound hydroxamic acid groups, which have been introduced into most of the published inhibitors of matrix metalloproteinases and histone deacetylases. Another example is the zinc-binding carboxylic acid group, which has been introduced into most of the published angiotensin converting enzyme inhibitors. 
VT-1161, the compound 2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2, 2,2-Trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol, is an antifungal drug developed by VIAMET, currently in the clinical research stage, its structure is as follows Shown:

This compound mainly acts on the CYP51 target of fungal cells. Compared with the previous triazole antifungal drugs, it has the advantages of wider antibacterial spectrum, low toxicity, high safety and good selectivity. However, this compound is not suitable for Liquid preparations (including or excluding the parenteral delivery carrier) are used to treat patients in need thereof. 
2-(2,4-Difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoro Ethoxy)phenyl)pyridin-2-yl)propan-2-yl dihydrogen phosphate is a prodrug of VT-1161. 
On the other hand, nearly half of the drug molecules are in the form of salts, and salt formation can improve certain undesirable physicochemical or biological properties of the drug. Relative to 2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2,2- Trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-yl dihydrogen phosphate, it is of great significance to develop salts with more excellent properties in terms of physical and chemical properties or pharmaceutical properties.To this end, the present disclosure provides a new pharmaceutically acceptable salt form of a metalloenzyme inhibitor.Example 1:[0161](R)-2-(2,4-Difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2, 2-Trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-yl phosphate disodium salt (Compound 1)[0162]

[0163](R)-2-(2,4-Difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2 ,2-Trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-yl phosphate (compound 1a, prepared according to the method of patent WO2013110002, 0.28g, 0.46mmol, 1.0eq) and ethanol (5mL ) Add to the reaction flask and stir evenly. A solution of NaOH (36.90 mg, 2.0 eq) dissolved in water (1 mL) was added dropwise into the above reaction flask, stirring was continued for 2 h, and concentrated to obtain compound 1, 300 mg of white solid.[0164]After X-ray powder diffraction detection, the XRPD spectrum has no sharp diffraction peaks, as shown in FIG. 10.[0165]Ms:608.10[M-2Na+3H] ⁺ .[0166]Ion chromatography detected that the sodium ion content was 6.23%.[0167]Example 2: (R)-((2-(2,4-Difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4 -(2,2,2-Trifluoroethoxy)phenyl)pyridin-2-yl)prop-2-yl)oxy)methyl phosphate disodium salt (compound 2)

[0169]Under ice-cooling, NaH (58mg, 0.87mmol) was added to the reaction flask, 1.5mL of N,N-dimethylformamide and 0.6mL of tetrahydrofuran were added, followed by iodine (38mg, 0.15mmol), and then Compound 2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2,2-tri Fluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol (2b, prepared according to the method of patent WO2013110002, 158mg, 0.3mmol) tetrahydrofuran (1ml) solution was added to the reaction solution, stirred and reacted for 1-4h , And then add compound 2a (519mg, 2.01mmol) in tetrahydrofuran (1ml) solvent to the reaction, stir until the reaction is complete, 10% aqueous ammonium chloride solution to quench the reaction, extract, concentrate and drain, the crude product 2c is directly used for the next One-step reaction, Ms: 750.0[M+H] ⁺ .[0170]

[0171]Under ice-bath cooling, add trifluoroacetic acid (0.5mL) to the crude product 2c (300mg) in dichloromethane (2mL) solution, stir until the reaction is complete, and after concentration, the target compound 2d, 82mg, Ms was separated by high performance liquid phase separation. :638.0[M+H] ⁺ .[0172]

Add compound 2d (0.29g, 0.46mmol, 1.0eq) and ethanol (5mL) obtained in the previous step into the reaction flask, stir, and add NaOH (36.90mg, 2.0eq) water (1ml) solution dropwise to the aforementioned reaction solution , Stirred for 2-5 h, and concentrated to obtain 2,313 mg of the target compound. 
Ms:638.10[M-2Na+3H] ⁺ .

PATENT

WO2011133875

https://patents.google.com/patent/WO2011133875A2/en

Product pat, WO2011133875 , protection in the EU states and the US April 2031.

PATENT

WO2015143184 ,

https://patents.google.com/patent/WO2015143184A1/en

Mycovia, claiming a process for preparing antifungal compounds, particularly oteseconazole.EXAMPLE 11

2-(2,4-Difluorophenyl)-l,l-difluoro-3-(lH-tetrazol-l-yl)-l-(5-(4-(2,2,2- trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol (11)Compound 11 was prepared using the conditions employed for 1: 0.33 g as a solid. The precursor l-bromo-4-(2,2,2-trifluoroethoxy)benzene was prepared as described below in one step.1H NMR (500 MHz, CDC1₃): δ 8.76 (s, 1 H), 8.70 (s, 1 H), 7.95 (d, / = 8.0 Hz, 1 H), 7.70 (s, 1 H), 7.64 (d, / = 8.5 Hz, 1 H), 7.54 (d, / = 8.5 Hz, 2 H), 7.42- 7.37 (m, 1 H), 7.08 (d, / = 8.5 Hz, 2 H), 6.79- 6.75 (m, 1 H), 6.69- 6.66 (m, 1 H), 5.58 (d, / = 14.0 Hz, 1 H), 5.14 (d, / = 14.0 Hz, 1 H), 4.44 – 4.39 (m, 2 H). HPLC: 99.1%. MS (ESI): m/z 528 [M⁺+l].Chiral preparative HPLC Specifications for (+)-ll:Column: Chiralpak IA, 250 x 4.6mm, 5uMobile Phase: A) w-Hexane, B) IPAIsocratic: A: B (65:35)Flow Rte: l.OO mL/minOptical rotation [a]_D: + 24° (C = 0.1 % in MeOH). 1 -Bromo-4-( 2,2,2-trifluoroethoxy )benzeneTo a stirred solution of trifluoroethyl tosylate (1.5 g, 5.8 mmol) in DMF (20 mL) was added K₂CO₃ (4 g, 29.4 mmol) followed by addition of p-bromo phenol (1.1 g, 6.46 mmol) at RT under inert atmosphere. The reaction mixture was stirred at 120 °C for 6 h. The volatiles were evaporated under reduced pressure; the residue was diluted with water (5 mL) and extracted with ethyl acetate (3 x 30 mL). The organic layer was washed with water, brine and dried over anhydrous Na₂S0₄, filtered and concentrated in vacuo. The crude compound was purified by silica gel column chromatography eluting with 5% EtOAc/hexane to afford the desired product (0.8 g, 3.13 mmol, 53.3%) as semi solid. 1H NMR (200 MHz, CDC1₃): δ 7.44 – 7.38 (m, 2 H), 6.86-6.80 (m, 2 H), 4.38- 4.25 (m, 2 H).ExamplesThe present invention will now be demonstrated using specific examples that are not to be construed as limiting.General Experimental ProceduresDefinitions of variables in the structures in schemes herein are commensurate with those of corresponding positions in the formulae delineated herein.Synthesis of 1 or la

A process to prepare enantiopure compound 1 or la is disclosed. Syntheses of lor la may be accomplished using the example syntheses that are shown below (Schemes 1-4). The preparation of precursor ketone 3-Br is performed starting with reaction of 2,5-dibromo- pyridine with ethyl 2-bromo-difluoroacetate to produce ester 2-Br. This ester can be reacted with morpholine to furnish morpholine amide 2b-Br, followed by arylation to provide ketone 3-Br. Alternatively, ketone 3-Br can be afforded directly from ester 2-Br as shown in Scheme 1. Scheme 1. Synthesis of ketone 3-Br r

Ketone 3 may be prepared in an analogous fashion as described in Scheme 1 starting from corresponding substituted 2-bromo-pyridines, which can be prepared according to synthetic transformations known in the art and contained in the references cited herein (Scheme 2).Scheme 2. Synthesis of ketone 3

R-i = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, – 0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S0₂)-alkyl, -0(S0₂)-substituted alkyl, – 0(S0₂)-aryl, or -0(S0₂)-substituted aryl.Alternatively, compound 1 can be prepared according to Scheme 3 utilizing diols 2-6b (or 2- 6d, the enantiomer of 2-6b, or mixtures thereof) or 2-6a (or 2-6c, the enantiomer of 2-6a, or mixtures thereof). Olefins 2-5a and 2-5 can be prepared by reacting ketones 3 and 1-4 under Wittig olefination conditions (e.g., Ph₃PCH₃Br and BuLi). Also, as indicated in Scheme 5, any of pyridine compounds, 3, 2-5a, 2-6b, 2-7b, 4*, 4b, or 6 can be converted to the corresponding 4-CF₃CH₂O-PI1 analogs (e.g., 1-4, 2-5, 2-6a, 2-7a, 5*, 1-6*, or 1 or the corresponding enantiomers, or mixtures thereof) by cross-coupling with 4,4,5, 5-tetramethyl-2- (4-(2,2,2-trifluoroethoxy)phenyl)-l,3,2-dioxaborolane (or the corresponding alkyl boronates or boronic acid or the like), in a suitable solvent system (e.g., an organic-aqueous solvent mixture), in the presence of a transition metal catalyst (e.g., (dppf)PdCl₂), and in the presence of a base (e.g., KHCO₃, K₂C0₃, Cs₂C0₃, or Na₂C0₃, or the like). Olefins 2-5a and 2-5 can be transformed to the corresponding chiral diols, 2-6b (or 2-6d, the enantiomer of 2-6b, or mixtures thereof) or 2-6a (or 2-6c, the enantiomer of 2-6a, or mixtures thereof), through exposure to Sharpless asymmetric dihydroxylation conditions: 1) commercially available AD- mix alpha or AD-mix beta with or without additional osmium oxidant and methanesulfonamide, 2) combination of a catalytic osmium oxidant (e.g., Os0₄ or K₂0sC>₂(OH)₄), a stoichiometric iron oxidant (e.g., K₃Fe(CN)₆), a base (e.g., KHCO₃, K2CO₃, Cs₂C0₃, or Na₂C0₃, or the like), and a chiral ligand (e.g., (DHQ)₂PHAL, (DHQD)₂PHAL, (DHQD)₂AQN, (DHQ)₂AQN, (DHQD)₂PYR, or (DHQ)₂PYR; preferably (DHQ)₂PHAL, (DHQD)₂PHAL, (DHQD)₂AQN, and (DHQD)₂PYR), or 3) option 2) with methanesulfonamide. The primary alcohol of the resultant chiral diols, 2-6b (or 2-6d, the enantiomer of 2-6b, or mixtures thereof) or 2-6a (or 2-6c, the enantiomer of 2-6a, or mixtures thereof), can then be activated to afford compounds 2-7b (or 2-7d, the enantiomer of 2-7b, or mixtures thereof) or 2-7a (or 2-7c, the enantiomer of 2-7a, or mixtures thereof). For example, the mesylates can be prepared by exposing chiral diols, 2-6b (or 2-6d, the enantiomer of 2-6b, or mixtures thereof) or 2-6a (or 2-6c, the enantiomer of 2-6a, or mixtures thereof), to methanesulfonyl chloride and a base. Epoxide formation can be affected by the base-mediated (e.g., KHCO₃, K2CO₃, CS2CO₃, or Na₂CC>₃, or the like) ring closure of compounds 2-7b (or 2- 7d, the enantiomer of 2-7b, or mixtures thereof) or 2-7a (or 2-7c, the enantiomer of 2-7a, or mixtures thereof) to provide epoxides 4* (or 4c*, the enantiomer of 4*, or mixtures thereof) and 5* (or 5-b*, the enantiomer of 5*, or mixtures thereof). The epoxides can then be converted into amino-alcohols 4b (or 4c, the enantiomer of 4b, or mixtures thereof) and 1-6* (or 1-7*, the enantiomer of 1-6*, or mixtures thereof) through ammonia-mediated epoxide opening using ammonia in a suitable solvent (e.g., MeOH, EtOH, or water). Subsequent treatment with TMS-azide in the presence of trimethylorthoformate and sodium acetate in acetic acid would yield compounds 6 (or 6a, the enantiomer of 6, or mixtures thereof) or 1 (or la, the enantiomer of 1, or mixtures thereof) (US 4,426,531).Scheme 3. Synthesis of 1 via Asymmetric Dihydroxylation Method

Y is -OS02-alkyl, -OS02-substituted alkyl, -OS02-aryl, -OS02- substituted aryl, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, – 0(C=0)-aryl, -0(C=0)-substituted aryl, or halogen

R-i = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, -0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S0₂)-alkyl, -0(S0₂)-substituted alkyl, -0(S0₂)-aryl, or -0(S0₂)-substituted aryl.Compound 1 (or la, the enantiomer of 1, or mixtures thereof) prepared by any of the methods presented herein can be converted to a sulfonic salt of formula IX (or IXa, the enantiomer of IX, or mixtures thereof), as shown in Scheme 4. This can be accomplished by a) combining compound 1 (or la, the enantiomer of 1, or mixtures thereof), a crystallization solvent or crystallization solvent mixture (e.g., EtOAc, i^‘PrOAc, EtOH, MeOH, or acetonitrile, or oZ-S-OHcombinations thereof), and a sulfonic acid o (e.g., Z = Ph, p-tolyl, Me, or Et), b) diluting the mixture with an appropriate crystallization co-solvent or crystallization co-solvent mixture (e.g., pentane, methyl i-butylether, hexane, heptane, or toluene, or combinations thereof), and c) filtering the mixture to obtain a sulfonic acid salt of formula IX (or IXa, the enantiomer of IX, or mixtures thereof). cheme 4. Synthesis of a Sulfonic Acid Salt of Compound 1 or la

The following describes the HPLC method used in assessing HPLC purity of the examples and intermediates presented below:Column: Waters XBridge Shield RP18, 4.6 x 150 mm, 3.5 μιηMobile Phase: A = 0.05% TFA/H₂0, B = 0.05% TFA/ACNAutosampler flush: 1 : 1 ACN/H₂0Diluent: 1:1 ACN/H₂0Flow Rate: 1.0 ml/minTemperature: 45 °CDetector: UV 275 nmPump Parameters:

EXAMPLE 1Preparation of ethyl 2-(5-bromopyridin-2-yl)-2,2-difluoroacetate (2-Br)

2-Br Dialkylated impurity In a clean multi-neck round bottom flask, copper powder (274.7 g, 2.05 eq) was suspended in dimethyl sulfoxide (3.5 L, 7 vol) at 20 – 35 °C. Ethyl bromodifluoroacetate (449 g, 1.05 eq) was slowly added to the reaction mixture at 20 – 25 °C and stirred for 1 – 2 h. 2, 5- dibromopyridine (500 g, 1 eq) was added to the reaction mixture and the temperature was increased to 35 – 40 °C. The reaction mixture was maintained at this temperature for 18 – 24 h and the reaction progress was monitored by GC.After the completion of the reaction, ethyl acetate (7 L, 14 vol) was added to the reaction mixture and stirring was continued for 60 – 90 min at 20 – 35 °C. The reaction mixture was filtered through a Celite bed (100 g; 0.2 times w/w Celite and 1L; 2 vol ethyl acetate). The reactor was washed with ethyl acetate (6 L, 12 vol) and the washings were filtered through a Celite bed. The Celite bed was finally washed with ethyl acetate (1 L, 2 vol) and all the filtered mother liquors were combined. The pooled ethyl acetate solution was cooled to 8 – 10 °C, washed with the buffer solution (5 L, 10 vol) below 15 °C (Note: The addition of buffer solution was exothermic in nature. Controlled addition of buffer was required to maintain the reaction mixture temperature below 15 °C). The ethyl acetate layer was washed again with the buffer solution until (7.5 L; 3 x 5 vol) the aqueous layer remained colorless. The organic layer was washed with a 1: 1 solution of 10 % w/w aqueous sodium chloride and the buffer solution (2.5 L; 5 vol). The organic layer was then transferred into a dry reactor and the ethyl acetate was distilled under reduced pressure to get crude 2-Br.The crude 2-Br was purified by high vacuum fractional distillation and the distilled fractions having 2-Br purity greater than 93 % (with the dialkylated not more than 2 % and starting material less than 0.5 %) were pooled together to afford 2-Br.Yield after distillation: 47.7 % with > 93 % purity by GC (pale yellow liquid). Another 10 % yield was obtained by re-distillation of impure fractions resulting in overall yield of ~ 55 – 60 %.*H NMR: δ values with respect to TMS (DMSO-d₆; 400 MHz): 8.85 (1H, d, 1.6 Hz), 8.34 (1H, dd, J = 2.0 Hz, 6.8 Hz), 7.83 (1H, d, J = 6.8 Hz), 4.33 (2H, q, J = 6.0 Hz), 1.22 (3H, t, J = 6.0 Hz). ¹³C NMR: 162.22 (i, -C=0), 150.40 (Ar-C-), 149.35 (t, Ar-C), 140.52 (Ar-C), 123.01 (Ar-C), 122.07 (Ar-C), 111.80 (t, -CF₂), 63.23 (-OCH₂-), 13.45 (-CH₂CH₃).EXAMPLE 2

Preparation of2-( 5-bromopyridin-2-yl )-l -(2,4-difluorophenyl )-2, 2-difluoroethanone ( 3-Br ) A. One-step Method

l-Bromo-2,4-difluorobenzene (268.7 g; 1.3 eq) was dissolved in methyl tert butyl ether (MTBE, 3.78 L, 12.6 vol) at 20 – 35 °C and the reaction mixture was cooled to -70 to -65 °C using acetone/dry ice bath. n-Butyl lithium (689 rriL, 1.3 eq; 2.5 M) was then added to the reaction mixture maintaining the reaction temperature below -65 °C (Note: Controlled addition of the n-Butyl Lithium to the reaction mixture was needed to maintain the reaction mixture temperature below – 65 °C). After maintaining the reaction mixture at this temperature for 30 – 45 min, 2-Br (300 g, 1 eq) dissolved in MTBE (900 rriL, 3 vol) was added to the reaction mixture below – 65 °C. The reaction mixture was continued to stir at this temperature for 60 – 90 min and the reaction progress was monitored by GC.The reaction was quenched by slow addition of 20 % w/w ammonium chloride solution (750 mL, 2.5 vol) below -65 °C. The reaction mixture was gradually warmed to 20 – 35 °C and an additional amount of 20 % w/w ammonium chloride solution (750 mL, 2.5 vol) was added. The aqueous layer was separated, the organic layer was washed with a 10 % w/w sodium bicarbonate solution (600 mL, 2 vol) followed by a 5 % sodium chloride wash (600 mL, 2 vol). The organic layer was dried over sodium sulfate (60 g; 0.2 times w/w), filtered and the sodium sulfate was washed with MTBE (300 mL, 1 vol). The organic layer along with washings was distilled below 45 °C under reduced pressure until no more solvent was collected in the receiver. The distillation temperature was increased to 55 – 60 °C, maintained under vacuum for 3 – 4 h and cooled to 20 – 35 °C to afford 275 g (73.6 % yield, 72.71 % purity by HPLC) of 3-Br as a pale yellow liquid.*H NMR: δ values with respect to TMS (DMSO-d₆; 400 MHz):8.63 (1H, d, 1.6 Hz, Ar-H), 8.07 – 8.01 (2H, m, 2 x Ar-H), 7.72 (1H, d, J = 6.8 Hz, Ar-H), 7.07 – 6.82 (1H, m, Ar-H), 6.81 – 6.80 (1H, m, Ar-H). ¹³C NMR: 185.60 (t, -C=0), 166.42 (dd, Ar-C-), 162.24 (dd, Ar-C),150.80 (Ar-C), 150.35 (Ar-C), 140.02 (Ar-C), 133.82 (Ar-C), 123.06 (Ar-C), 1122.33 (Ar-C), 118.44 (Ar-C), 114.07 (-CF₂-), 122.07 (Ar-C), 105.09 (Ar-C).

B. Two-step Method via 2b-Br

2-Br (147.0 g) was dissolved in n-heptane (1.21 L) and transferred to a 5-L reactor equipped with overhead stirrer, thermocouple, condenser and addition funnel. Morpholine (202 ml) was added. The solution was heated to 60 °C and stirred overnight. The reaction was complete by HPLC analysis (0.2% 2-Br; 94.7% 2b-Br). The reaction was cooled to room temperature and 1.21 L of MTBE was added. The solution was cooled to ~4 °C and quenched by slow addition of 30% citric acid (563 ml) to maintain the internal temperature <15 °C. After stirring for one hour the layers were allowed to settle and were separated (Aq. pH=5). The organic layer was washed with 30% citric acid (322 ml) and 9% NaHC0₃ (322 ml, aq. pH 7+ after separation). The organic layer was concentrated on the rotary evaporator (Note 1) to 454 g (some precipitation started immediately and increased during concentration). After stirring at room temperature the suspension was filtered and the product cake was washed with n-heptane (200 ml). The solid was dried in a vacuum oven at room temperature to provide 129.2 g (77%) dense powder. The purity was 96.5% by HPLC analysis.To a 1-L flask equipped with overhead stirring, thermocouple, condenser and addition funnel was added magnesium turnings (14.65 g), THF (580 ml) and l-bromo-2,4-difluorobenzene (30.2 g, 0.39 equiv). The mixture was stirred until the reaction initiated and self-heating brought the reaction temperature to 44 °C. The temperature was controlled with a cooling bath as the remaining l-bromo-2,4-difluorobenzene (86.1 g, 1.11 equiv) was added over about 30 min. at an internal temperature of 35-40 °C. The reaction was stirred for 2 hours while gradually cooling to room temperature. The dark yellow solution was further cooled to 12 °C.During the Grignard formation, a jacketed 2-L flask equipped with overhead stirring, thermocouple, and addition funnel was charged with morpholine amide 2b-Br (129.0 g) and THF (645 ml). The mixture was stirred at room temperature until the solid dissolved, and then the solution was cooled to -8.7 °C. The Grignard solution was added via addition funnel over about 30 min. at a temperature of -5 to 0 °C. The reaction was stirred at 0 °C for 1 hour and endpointed by HPLC analysis. The reaction mixture was cooled to -5 °C and quenched by slow addition of 2N HC1 over 1 hour at <10 °C. The mixture was stirred for 0.5 h then the layers were allowed to settle and were separated. The aqueous layer was extracted with MTBE (280 ml). The combined organic layers were washed with 9% NaHCC>3 (263 g) and 20% NaCl (258 ml). The organic layer was concentrated on the rotary evaporator with THF rinses to transfer all the solution to the distillation flask. Additional THF (100 ml) and toluene (3 x 100 ml) were added and distilled to remove residual water from the product. After drying under vacuum, the residue was 159.8 g of a dark brown waxy solid (>theory). The purity was approximately 93% by HPLC analysis.EXAMPLE 3Preparation of 3-amino-l-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-l,l-difluoropropan- -ol (±ib-Br)

4-Br (200g, 1 eq) was added into methanolic ammonia (8.0 L; 40 vol; ammonia content: 15 – 20 % w/v) in an autoclave at 10 – 20 °C. The reaction mixture was gradually heated to 60 – 65 °C and at 3 – 4 kg/cm² under sealed conditions for 10 – 12 h. The reaction progress was monitored by GC. After completion of the reaction, the reaction mixture was cooled to 20 – 30 °C and released the pressure gradually. The solvent was distilled under reduced pressure below 50 °C and the crude obtained was azeotroped with methanol (2 x 600 mL, 6 vol) followed by with isopropanol (600 mL, 2 vol) to afford 203 g (96.98 % yield, purity by HPLC: 94.04 %) of +4b-Br. EXAMPLE 4Preparation of3-amino-l-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-l,l-difluoropropan- -ol (4b-Br or 2c-Br)

Amino alcohol ±4b-Br (150 g, 1 eq) was dissolved in an isopropanol /acetonitrile mixture (1.5L, 8:2 ratio, 10 vol) and Di-p-toluoyl-L-tartaric acid (L-DPTTA) (84.05 g, 0.55 eq) was added into the reactor at 20 – 30 °C. The reaction mixture was heated to 45 – 50 °C for 1 – 1.5 h (Note: The reaction mixture becomes clear and then became heterogeneous). The reaction mixture was gradually cooled to 20 – 30 °C and stirred for 16 – 18 h. The progress of the resolution was monitored by chiral HPLC analysis.After the completion of the resolution, the reaction mixture was gradually cooled to 20 – 35 °C. The reaction mixture was filtered and the filtered solid was washed with a mixture of acetonitrile and isopropanol (8:2 mixture, 300 mL, 2 vol) and dried to afford 75 g of the L- DPTTA salt (95.37 % ee). The L-DPTTA salt obtained was chirally enriched by suspending the salt in isopropanol /acetonitrile (8:2 mixture; 750 mL, 5 vol) at 45 – 50 °C for 24 – 48 h. The chiral enhancement was monitored by chiral HPLC; the solution was gradually cooled to 20 – 25 °C, filtered and washed with an isoporpanol /acetonitrile mixture (8:2 mixture; 1 vol). The purification process was repeated and after filtration, the salt resulted in chiral purity greater than 96 % ee. The filtered compound was dried under reduced pressure at 35 – 40 °C to afford 62 g of the enantio-enriched L-DPPTA salt with 97.12% ee as an off-white solid. The enantio-enriched L-DPTTA salt (50 g, 1 eq) was dissolved in methanol (150 mL, 3 vol) at 20 – 30 °C and a potassium carbonate solution (18.05 g K2CO₃ in 150 mL water) was slowly added at 20 – 30 °C under stirring. The reaction mixture was maintained at this temperature for 2 – 3 h (pH of the solution at was maintained at 9). Water (600 mL, 12 vol) was added into the reaction mixture through an additional funnel and the reaction mixture was stirred for 2 – 3 h at 20 – 30 °C. The solids were filtered; washed with water (150 mL, 3 vol) and dried under vacuum at 40 – 45 °C to afford 26.5 g of amino alcohol 4b-Br or 4c-Br with 99.54 % chemical purity, 99.28 % ee as an off-white solid. (Water content of the chiral amino alcohol is below 0.10 % w/w).1H NMR: δ values with respect to TMS (DMSO-d₆; 400 MHz):8.68 (1H, d, J = 2.0 Hz, Ar- H), 8.16 (1H, dd, J = 8.0 Hz, 2.0 Hz, Ar-H), 7.49 – 7.43 (1H, m, Ar-H), 7.40 (1H, d, J = 8 Hz, Ar-H), 7.16 – 7.11 (1H, m, Ar-H), 7.11 – 6.99 (1H, m, Ar-H), 3.39 – 3.36 (1H, m, -OCH_AH_B– ), 3.25 – 3.22 (1H, m, -OCH_AH_B-).¹³C NMR: 163.87 -158.52 (dd, 2 x Ar-C-), 150.88 (Ar-C), 149.16 (Ar-C), 139.21 (Ar-C), 132.39 (Ar-C), 124.49 (Ar-C), 122.17 (Ar-C), 121.87 (d, Ar- C), 119.91 (t, -CF₂-), 110.68 (Ar-C), 103.97 (i, Ar-C), 77.41 (i,-C-OH), 44.17 (-CH₂-NH₂).EXAMPLE 5

Preparation of l-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-l,l-difluoro-3-(lH-tetrazol-l- yl)propan-2-ol (l-6*-Br or l-7*-Br)

4b-Br or 4c-Br (20.0 g, 1 eq.) was added to acetic acid (50 mL, 2.5 vol) at 25 – 35 °C followed by the addition of anhydrous sodium acetate (4.32 g, 1 eq), trimethyl orthoformate (15.08 g, 2.7 eq). The reaction mixture was stirred for 15 – 20 min at this temperature and trimethylsilyl azide (12.74 g, 2.1 eq) was added to the reaction mixture (Chilled water was circulated through the condenser to minimize the loss of trimethylsilyl azide from the reaction mixture by evaporation). The reaction mixture was then heated to 70 – 75 °C and maintained at this temperature for 2 -3 h. The reaction progress was monitored by HPLC. Once the reaction was complete, the reaction mixture was cooled to 25 – 35 °C and water (200 mL, 10 vol) was added. The reaction mixture was extracted with ethyl acetate (400 mL, 20 vol) and the aqueous layer was back extracted with ethyl acetate (100 mL, 5 vol). The combined organic layers were washed with 10 % potassium carbonate solution (3 x 200 mL; 3 x 10 vol) followed by a 10 % NaCl wash (1 x 200 mL, 10 vol). The organic layer was distilled under reduced pressure below 45 °C. The crude obtained was azeotroped with heptanes (3 x 200 mL) to get 21.5g (94 % yield, 99.26 5 purity) of tetrazole 1-6* or 1-7* compound as pale brown solid (low melting solid).1H NMR: δ values with respect to TMS (DMSO-d₆; 400 MHz NMR instrument): 9.13 (1H, Ar-H), 8.74 (1H, Ar-H), 8.22 – 8.20 (1H, m, Ar-H), 7.44 (1H, d, J = 7.2 Hz, Ar-H), 7.29 (1H„Ar-H), 7.23 – 7.17 (1H, m, Ar-H), 6.92 – 6.88 (1H, Ar-H), 5.61 (1H, d, J = 1 1.2 Hz, – OCHAHB-), 5.08 (1H, d, J = 5.6 Hz, -OCH_AH_B-).¹³C NMR: 163.67 -161.59 (dd, Ar-C-), 160.60 – 158.50 (dd, Ar-C-), 149.65 (Ar-C), 144.99 (Ar-C), 139.75 (Ar-C), 131.65 (Ar-C), 124.26 (Ar-C), 122.32 (d, Ar-C), 119.16 (t, -CF₂-), 118.70 (d, Ar-C), 1 11.05 (d, Ar-C) 104.29 (t, Ar-C), 76.79 (i,-C-OH), 59.72 (Ar-C), 50.23 (-OCH₂N-). EXAMPLE 6Preparation of 2-(2,4-difluorophenyl)-l , 1 -difluoro-3-( 1 H-tetrazol-1 -yl)-l -(5-(4-(2,2,2- trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol (1 or la)A. Preparation of 1 or la via l-6*-Br or l-7*-Br

Synthesis of 4,4,5, 5-tetramethyl-2-(4-(2,2,2-trifluoroethoxy)phenyl)-l,3,2-dioxaborolane Potassium carbonate (59.7 g, 2.2 eq.) was added to a slurry of DMF (190 mL, 3.8 Vol.), 4- Bromo phenol (37.4g, 1.1 eq.) and 2,2,2-trifluroethyl tosylate (50.0 g, 1.0 eq.) at 20 – 35 °C under an inert atmosphere. The reaction mixture was heated to 115 – 120 °C and maintained at this temperature for 15 – 18 h. The reaction progress was monitored by GC. The reaction mixture was then cooled to 20 – 35 °C, toluene (200 mL, 4.0 vol.) and water (365 mL, 7. 3 vol.) were added at the same temperature, stirred for 10 – 15 minutes and separated the layers. The aqueous layer was extracted with toluene (200 mL, 4.0 vol.). The organic layers were combined and washed with a 2M sodium hydroxide solution (175 mL, 3.5 vol.) followed by a 20 % sodium chloride solution (175 mL, 3.5 vol.). The organic layer was then dried over anhydrous sodium sulfate and filtered. The toluene layer was transferred into clean reactor, spurged with argon gas for not less than 1 h. Bis(Pinacolato) diborane (47 g, 1.1 eq.), potassium acetate (49.6 g, 3.0 eq.) and 1,4-dioxane (430 mL, 10 vol.) were added at 20 -35 °C, and spurged the reaction mixture with argon gas for at least 1 h. Pd(dppf)Cl₂ (6.88 g, 0.05eq) was added to the reaction mixture and continued the argon spurging for 10 – 15 minutes. The reaction mixture temperature was increased to 70 – 75 °C, maintained the temperature under argon atmosphere for 15 – 35 h and monitored the reaction progress by GC. The reaction mixture was cooled to 20 – 35 °C, filtered the reaction mixture through a Celite pad, and washed with ethyl acetate (86 mL, 2 vol.). The filtrate was washed with water (430 mL, 10 vol.). The aqueous layer was extracted with ethyl acetate (258 mL, 6 vol.) and washed the combined organic layers with a 10 % sodium chloride solution (215 mL, 5 vol.). The organic layer was dried over anhydrous sodium sulfate (43g, 1 time w/w), filtered and concentrated under reduced pressure below 45 °C to afford crude 4,4,5, 5-tetramethyl-2-(4-(2,2,2- trifluoroethoxy)phenyl)-l,3,2-dioxaborolane (65 g; 71 % yield with the purity of 85.18 % by GC). The crude 4,4,5,5-tetramethyl-2-(4-(2,2,2-trifluoroethoxy)phenyl)-l,3,2-dioxaborolane (65 g, 1 eq.) was dissolved in 10 % ethyl acetate – n-Heptane (455 mL, 7 vol.) and stirred for 30 – 50 minutes at 20 – 35 °C. The solution was filtered through a Celite bed and washed with 10 % ethyl acetate in n-Heptane (195 mL, 3 vol.). The filtrate and washings were pooled together, concentrated under vacuum below 45 °C to afford 4,4,5, 5-tetramethyl-2-(4-(2,2,2- trifluoroethoxy)phenyl)-l,3,2-dioxaborolane as a thick syrup (45.5 g; 70 % recovery). This was then dissolved in 3 % ethyl acetate-n-heptane (4 vol.) and adsorbed on 100 – 200 M silica gel (2 times), eluted through silica (4 times) using 3 % ethyl acetate – n- heptane. The product rich fractions were pooled together and concentrated under vacuum. The column purified fractions (> 85 % pure) were transferred into a round bottom flask equipped with a distillation set-up. The compound was distilled under high vacuum below 180 °C and collected into multiple fractions. The purity of fractions was analyzed by GC (should be > 98 % with single max impurity < 1.0 %). The less pure fractions (> 85 % and < 98 % pure fraction) were pooled together and the distillation was repeated to get 19g (32% yield) of 4,4,5, 5-tetramethyl-2-(4- (2,2,2-trifluoroethoxy)phenyl)-l,3,2-dioxaborolane as a pale yellow liquid.*H NMR: δ values with respect to TMS (DMSO-d⁶; 400 MHz):7.64 (2H, d, 6.8 Hz), 7.06 (2H, d, J = 6.4 Hz), 4.79 (2H, q, J = 6.8 Hz), 1.28 (12H, s).1³C NMR: 159.46 (Ar-C-O-), 136.24 (2 x Ar-C-), 127.77 – 120.9 (q, -CF₃), 122.0 (Ar-C-B), 114.22 (2 x Ar-C-), 64.75 (q, J = 27.5 Hz).Synthesis of 2-(2.4-difluorophenyl)-l.l-difluoro-3-(lH-tetrazol-l-yl)-l-(5-(4-(2.2.2- trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol (1 or la)l-6*-Br or l-7*-Br (14 g, 0.03 mol, 1 eq) was added to tetrahydrofuran (168 mL, 12 vol) at 25 – 35 °C and the resulting solution was heated to 40 – 45 °C. The reaction mixture was maintained at this temperature for 20 – 30 min under argon bubbling. Sodium carbonate (8.59 g, 0.08 mol, 2.5 eq) and water (21 mL, 1.5 vol) were added into the reaction mixture and the bubbling of argon was continued for another 20 – 30 min. 4,4,5, 5-tetramethyl-2-(4-(2,2,2- trifluoroethoxy)phenyl)-l,3,2-dioxaborolane (10.76 g, 1.1 eq) dissolved in tetrahydrofuran (42 mL, 3 vol) was added into the reaction mixture and argon bubbling was continued for 20 – 30 min. Pd(dppf)Cl₂ (2.65 g, 0.1 eq) was added to the reaction mixture under argon bubbling and stirred for 20 – 30 min (Reaction mixture turned into dark red color). The reaction mixture was heated to 65 – 70 °C and maintained at this temperature for 3 – 4 h. The reaction progress was monitored by HPLC. The reaction mixture was cooled to 40 – 45 °C and the solvent was distilled under reduced pressure. Toluene (350 mL, 25 vol.) was added to the reaction mixture and stirred for 10 – 15 min followed by the addition of water (140 mL, 10 vol). The reaction mixture was filtered through Hyflo (42 g, 3 times), the layers were separated and the organic layer was washed with water (70 mL, 5 vol) and a 20 % w/w sodium chloride solution (140 mL, 10 vol). The organic layer was treated with charcoal (5.6 g, 0.4 times, neutral chalrcoal), filtered through Hyflo. (lS)-lO-Camphor sulfonic acid (7.2 g, 1 eq.) was added to the toluene layer and the resulting mixture was heated to 70 – 75 °C for 2 – 3 h. The reaction mixture was gradually cooled to 25 – 35 °C and stirred for 1 – 2 h. The solids were filtered, washed with toluene (2 x 5 vol.) and then dried under vacuum below 45 °C to afford 18.0 g of an off white solid. The solids (13.5 g, 1 eq.) were suspended in toluene (135 mL, 10 vol) and neutralized by adding 1M NaOH solution (1.48 vol, 1.1 eq) at 25 – 35 °C and stirred for 20 – 30 min. Water (67.5 mL, 5 vol) was added to the reaction mixture and stirred for 10 – 15 min, and then the layers were separated. The organic layer was washed with water (67.5 mL, 5 vol) to remove the traces of CSA. The toluene was removed under reduced pressure below 45 °C to afford crude 1 or la. Traces of toluene were removed by azeotroping with ethanol (3 x 10 vol), after which light brown solid of crude 1 or la (7.5 g, 80% yield) was obtained.The crude 1 or la (5 g) was dissolved in ethanol (90 mL, 18 vol.) at 20 – 35 °C, and heated to 40 – 45 °C. Water (14 vol) was added to the solution at 40 – 45 °C, the solution was maintained at this temperature for 30 – 45 min and then gradually cooled to 20 – 35 °C. The resulting suspension was continued to stir for 16 – 18 h at 20 – 35 °C, an additional amount of water (4 vol.) was added and the stirring continued for 3 – 4 h. The solids were filtered to afford 4.0 g (80% recovery) of 1 or la (HPLC purity >98%) as an off-white solid.1H NMR: δ values with respect to TMS (DMSO-d₆; 400 MHz):9.15 (1H, s, Ar-H), 8.93 (1H, d, J = 0.8 Hz, Ar-H), .8.22 – 8.20 (1H, m, Ar-H), 7.80 (2H, d, J = 6.8 Hz, Ar-H), 7.52 (1H, d, J = 6.8 Hz, Ar-H), 7.29 (1H, d,J = 3.2Hz, Ar-H), 7.27 – 7.21 (1H, m, Ar-H), 7.23 – 7.21 (2H, d, J = 6.8 Hz, Ar-H), 7.19 (1H, d, J = 6.8 Hz, Ar-H), 6.93 – 6.89 (1H, m, Ar-H), 5.68 (1H, / = 12 Hz, -CH_AH_B), 5.12 (2H, d, J = 11.6 Hz, -CH_AH_B), 4.85 (2H, q, J = 1.6 Hz).1³C NMR: 163.93 – 158.33 (m, 2 x Ar-C), 157.56 (Ar-C), 149.32 (i, Ar-C), 146.40 (Ar-C), 145.02 (Ar-C), 136.20 (Ar-C), 134.26 (2 x Ar-C), 131.88 – 131.74 (m, AR-C), 129.72 (Ar-C), 128.47 (2 x Ar-C), 123.97 (q, -CF₂-), 122.41 (Ar-C), 119.30 (-CF₃), 118.99 (Ar-C), 115.65 (2 x Ar-C), 110.99 (d, Ar-C), 104.22 (i, Ar-C), 77.41 – 76.80 (m, Ar-C), 64.72 (q, -OCH₂-CF₃), 50.54 (-CH₂-N-).B. Preparation of 1 or la via 4b-Br or 4c-Br

Synthesis of 3-amino-2-(2.4-difluorophenyl)-l.l-difluoro-l-(5-(4-(2.2.2- trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol (8a or 8b)Potassium carbonate (30.4 g) and water (53.3 g) were charged to a 1-L flask equipped with overhead stirring, thermocouple, and nitrogen/vacuum inlet valve, and stirred until dissolved. The boronic acid (19.37 g), a solution of 4b-Br or 4c-Br in 2-butanol (103.5 g, 27.8 g theoretical 4b-Br or 4c-Br)) and 2-BuOH (147.1 g) were added and stirred to form a clear mixture. The flask was evacuated and refilled with nitrogen 3 times. Pd(d f)2Cl2 (0.30 g) was added and stirred to form a light orange solution. The flask was evacuated and refilled with nitrogen 4 times. The mixture was heated to 85 °C and stirred overnight and endpointed by HPLC analysis. The reaction mixture was cooled to 60 °C and the layers were allowed to settle. The aqueous layer was separated. The organic layer was washed with 5% NaCl solution (5 x 100 ml) at 30-40 °C. The organic layer was filtered and transferred to a clean flask with rinses of 2-BuOH. The combined solution was 309.7 g, water content 13.6 wt% by KF analysis. The solution was diluted with 2-BuOH (189 g) and water (10 g). Theoretically the solution contained 34.8 g product, 522 ml (15 volumes) of 2-BuOH, and 52.2 ml (1.5 volumes) of water. L-Tartaric acid (13.25 g) was added and the mixture was heated to a target temperature of 70-75 °C. During the heat-up, a thick suspension formed. After about 15 minutes at 70-72 °C the suspension became fluid and easily stirred. The suspension was cooled at a rate of 10 °C/hour to 25 °C then stirred at 25 °C for about 10 hours. The product was collected on a vacuum filter and washed with 10:1 (v/v) 2-BuOH/water (50 ml) and 2- butanol (40 ml). The salt was dried in a vacuum oven at 60 °C with a nitrogen purge for 2 days. The yield was 40.08 g of 8a or 8b as a fluffy, grayish-white solid. The water content was 0.13 wt% by KF analysis. The yield was 87.3% with an HPLC purity of 99.48%. Synthesis of 2-(2,4-difluorophenyl)-l,l-difluoro-3-(lH-tetrazol-l-yl)-l-(5-(4-(2,2,2- trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol (1 or la)To a 350 ml pressure bottle were charged acetic acid (73 ml), 8a or 8b (34.8 g), sodium acetate (4.58 g) and trimethylorthoformate (16.0 g). The mixture was stirred for 18 min. at room temperature until a uniform suspension was obtained. Azidotrimethylsilane (8.88 g) was added and the bottle was sealed. The bottle was immersed in an oil bath and magnetically stirred. The oil bath was at 52 °C initially, and was warmed to 62-64 °C over about ½ hour. The suspension was stirred at 62-64 °C overnight. After 20.5 hours the suspension was cooled to room temperature and sampled. The reaction was complete by HPLC analysis. The reaction was combined with three other reactions that used the same raw material lots and general procedure (total of 3.0 g additional starting material). The combined reactions were diluted with ethyl acetate (370 ml) and water (368 ml) and stirred for about ½ hour at room temperature. The layers were settled and separated. The organic layer was washed with 10% K₂C0₃ solution (370 ml/ 397 g) and 20% NaCl solution (370 ml/ 424 g). The organic layer (319 g) was concentrated, diluted with ethanol (202 g) and filtered, rinsed with ethanol (83 g). The combined filtrate was concentrated to 74 g of amber solution.The crude 1 or la solution in ethanol (74 g solution, containing theoretically 31.9 g 1 or la) was transferred to a 2-L flask equipped with overhead stirring, thermocouple, and addition funnel. Ethanol (335 g) was added including that used to complete the transfer of the 1 or la solution. The solution was heated to nominally 50 °C and water (392 g) was added over 12 minutes. The resulting hazy solution was seeded with 1 or la crystals and stirred at 50 °C. After about ½ hour the mixture was allowed to cool to 40 °C over about ½ hour during which time crystallization started. Some darker colored chunky solid separated out from the main suspension. The pH of the crystallizing mixture was adjusted from 4.5 to 6 using 41% KOH (1.7 g). After about 1 hour a good suspension had formed. Additional water (191 g) was added slowly over ½ hour. The suspension was heated to 50 °C and cooled at 5 °C/min to room temperature. After stirring overnight the suspension was cooled in a water bath to 16 °C and filtered after 1 hour. The wet cake was washed with 55:45 (v/v) water/ethanol (2 x 50 ml) and air-dried on the vacuum filter funnel overnight. Further drying at 40 °C in a vacuum oven with a nitrogen bleed resulted in no additional weight loss. The yield was 30.2 g of off-white fine powder plus some darker granular material. By in-process HPLC analysis there was no difference in the chemical purity of the darker and lighter materials. The purity was 99.4%. The water content was 2.16 wt% by KF analysis. The residual ethanol was 1.7 wt% estimated by ‘Ft NMR analysis. The corrected yield was 29.0 g, 91.0% overall yield for tetrazole formation and crystallization. The melting point was 65 °C by DSC analysis.

/////////OTESECONAZOLE, vt 1161, fungal infection,  Candida albicans infection, onychomycosis, PHASE 3,

C1=CC(=CC=C1C2=CN=C(C=C2)C(C(CN3C=NN=N3)(C4=C(C=C(C=C4)F)F)O)(F)F)OCC(F)(F)F

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MIRDAMETINIB

391210-10-9
Chemical Formula: C16H14F3IN2O4
Molecular Weight: 482.19

PD0325901; PD 0325901; PD-325901; mirdametinib

IUPAC/Chemical Name: (R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide

SpringWorks Therapeutics (a spin out of Pfizer ) is developing mirdametinib, a second-generation, non-ATP competitive, allosteric MEK1 and MEK2 inhibitor derived from CI-1040, for treating type 1 neurofibromatosis (NF1) and advanced solid tumors. In June 2021, a phase I/II trial was initiated in patients with low grade glioma.

• OriginatorPfizer
• DeveloperAstraZeneca; BeiGene; BIOENSIS; Pfizer; SpringWorks Therapeutics; St. Jude Childrens Research Hospital; University of Oxford
• ClassAniline compounds; Anti-inflammatories; Antineoplastics; Benzamides; Immunotherapies; Small molecules
• Mechanism of ActionMAP kinase kinase 1 inhibitors; MAP kinase kinase 2 inhibitors
• Orphan Drug StatusYes – Neurofibromatosis 1

• Phase IINeurofibromatosis 1
• Phase I/IIGlioma
• Phase ISolid tumours
• PreclinicalChronic obstructive pulmonary disease
• No development reportedCervical cancer
• DiscontinuedBreast cancer; Cancer; Colorectal cancer; Malignant melanoma; Non-small cell lung cancer

• 22 Jul 2021SpringWorks Therapeutics receives patent allowance for mirdametinib from the US Patent and Trademark Office for the treatment of Neurofibromatosis type 1-associated plexiform neurofibromas
• 16 Jun 2021SpringWorks Therapeutics and St. Jude Children’s Research Hospital agree to develop mirdametinib in USA for glioma
• 15 Jun 2021Efficacy and safety data from the phase IIb RENEU trial for Neurofibromatosis type 1-associated plexiform neurofibromas released by SpringWorks Therapeutics

PATENT

US-11066358

On July 20, 2021, SpringWorks Therapeutics announced that the United States Patent and Trademark Office (USPTO) has issued US11066358 , directed to mirdametinib , the Company’s product candidate in development for several oncology indications, including as a monotherapy for patients with neurofibromatosis type 1-associated plexiform neurofibromas (NF1-PN) and was assigned to Warner-Lambert Company (a subsidiary of Pfizer ).This patent was granted on July 20, 2021, and expires on Feb 17, 2041. Novel crystalline forms of mirdametinib and compositions comprising them are claimed.

Example 1: Production of Essentially Pure Form IV

Lab Scale Production of Essentially Pure Form IV

(MOL) (CDX)  (MOL) (CDX) Step 1: Preparation of “Side Chain”, PD-0337792

Step 2: Fluoride Displacement

(MOL) (CDX)

(MOL) (CDX)  (MOL) (CDX)

Pilot Plant Preparation of Essentially Pure Form IV

Step 1: Preparation of “Side Chain”, PD-0337792

Step 2: Preparation of PD-0315209

Step 3: Preparation of PD-0325901

(MOL) (CDX)

Polymorph Transformation

PATENT

WO2006134469 , claiming methods of preparing MEK inhibitor, assigned to Warner-Lambert Co .

https://patents.google.com/patent/WO2006134469A1/enThe compound Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide represented by formula 1

i is a highly specific non-ATP-competitive inhibitor of MEK1 and MEK2. The compound of formula ± (Compound I) is also known as the compound PD 0325901. Compound I is disclosed in WO 02/06213; WO 04/045617; WO 2005/040098; EP 1262176; U.S. Patent Application Pub. No. 2003/0055095 A1 ; U.S. Patent Application Pub. No. 2004/0054172 A1; U.S. Patent Application Pub. No. 2004/0147478 A1 ; and U.S. Patent Application No. 10/969,681, the disclosures of which are incorporated herein by reference in their entireties.Numerous mitogen-activated protein kinase (MAPK) signaling cascades are involved in controlling cellular processes including proliferation, differentiation, apoptosis, and stress responses. Each MAPK module consists of 3 cytoplasmic kinases: a mitogen-activated protein kinase (MAPK), a mitogen-activated protein kinase kinase (MAPKK), and a mitogen-activated protein kinase kinase kinase (MAPKKK). MEK occupies a strategic downstream position in this intracellular signaling cascade catalyzing the phosphorylation of its MAP kinase substrates, ERK1 and ERK2. Anderson et al. “Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase.” Nature 1990, v.343, pp. 651-653. In the ERK pathway, MAPKK corresponds with MEK (MAP kinase ERK Kinase) and the MAPK corresponds with ERK (Extracellular Regulated Kinase). No substrates for MEK have been identified other than ERK1 and ERK2. Seger et al. “Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells.” J. Biol. Chem., 1992, v. 267, pp. 14373-14381. This tight selectivity in addition to the unique ability to act as a dual-specificity kinase is consistent with MEK’s central role in integration of signals into the MAPK pathway. The RAF-MEK-ERK pathway mediates proliferative and anti-apoptotic signaling from growth factors and oncogenic factors such as Ras and Raf mutant phenotypes that promote tumor growth, progression, and metastasis. By virtue of its central role in mediating the transmission of growth- promoting signals from multiple growth factor receptors, the Ras-MAP kinase cascade provides molecular targets with potentially broad therapeutic applications.One method of synthesizing Compound I is disclosed in the above-referenced WO 02/06213 andU.S. Patent Application Pub. No. 2004/0054172 A1. This method begins with the reaction of 2-fluoro-4- iodo-phenylamine and 2,3,4-trifluoro-benzoic acid in the presence of an organic base, such as lithium diisopropylamide, to form 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid, which is then reacted with (R)-0-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of a peptide coupling agent (e.g., diphenylphosphinic chloride) and a tertiary amine base (e.g., diisopropylethylamine). The resulting product is hydrolyzed under standard acidic hydrolysis conditions (e.g., p-TsOH in MeOH) to provide Compound 1. (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine is prepared by reaction of [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol with N-hydroxyphthalimide in the presence of Ph₃P and diethyl azodicarboxylate.Another method of synthesizing Compound I, which is disclosed in the above-referenced U.S.Patent Application No. 10/969,681, comprises reaction of 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzoic acid with (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of N₁N¹– carbonyldiimidazole. The resulting product is hydrolyzed with aqueous acid and crystallized to provide polymorphic form IV of Compound I.Although the described methods are effective synthetic routes for small-scale synthesis of Compound I, there remains a need in the art for new synthetic routes that are safe, efficient and cost effective when carried out on a commercial scale.The present invention provides a new synthetic route including Steps I through Step III to the MEK inhibitor Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound I).Step I: Preparation of 0-{r(4RV2.2-dimethyl-1.3-dioxolan-4-ynmethyl}hydroxylanπine (6) The method of the present invention comprises a novel Step I of preparing of 0-{[(4R)-2,2- dimethyl-1 ,3-dioxolan-4-yl]methyl}hydroxylamine (6) from [(4S)-2,2-dimethyl-1 ,3-dioxoIan-4-yl]methanol (1) through the formation of [(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl trifluoromethanesulfonate (3) and its coupling with N-hydroxyphthalimide (4) to afford 2-{[(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methoxy}-1 H- isoindole-1 ,3(2H)-dione (5), which is subsequently de-protected to give 6 as shown in Scheme 1.Scheme 1

The reaction of compound (1) with trifluoromethanesulfonic anhydride (2) is carried out in the presence of a non-nucleophilic base, such as, for example, a tertiary organic amine, in an aprotic solvent at a temperature of from -5O⁰C to 5⁰C, preferably, at a temperature less than -15⁰C, to form triflate (3). A preferred tertiary organic amine is triethylamine, and a preferred solvent is toluene. Treatment of triflate (3) with N-hydroxyphthalimide (4) furnishes phthalimide (5), which can be isolated if desired. However, in order to minimize processing time and increase overall yield, 0-{[(4R)- 2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) can be prepared in a one-pot process with no phthalimide (S) isolation. Cleavage of the phthalimide function could be achieved by methods known in the art, for example, by hydrazinolysis. However, the use of less hazardous aqueous or anhydrous ammonia instead of methyl hydrazine (CH₃NHNH₂) is preferred.Step II: Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) As shown in Scheme 2, Step Il of the method of the present invention provides 3,4-difluoro-2-(2- fluoro-4-iodophenylamino)-benzoic acid (9).Scheme 2

Preparation of compound (9) can be carried out by reacting compound (7), wherein X is halogen, or O-SC^R^ or 0-P(³O)(OR^, wherein R^ is alkyl or aryl, with compound (8) optionally in a solvent, and in the presence of from about 1 mol equivalent to about 10 mol equivalents of at least one base, wherein the base is selected from: a Group I metal cation hydride or a Group 2 metal cation hydride, including lithium hydride, sodium hydride, potassium hydride, and calcium hydride, a Group I metal cation dialkylamide or a Group 2 metal cation dialkylamide, including lithium diisopropylamide, a Group I metal cation amide or a Group 2 metal cation amide, including lithium amide, sodium amide, potassium amide, a Group I metal cation alkoxide or a Group 2 metal cation alkoxide, including sodium ethoxide, potassium terf-butoxide, and magnesium ethoxide, and a Group I metal cation hexamethyldisilazide, including lithium hexamethyldisilazide; for a time, and at a temperature, sufficient to yield compound (9).Preferably, preparation of compound (9) is carried out by reacting compound (7), wherein X is halogen, more preferably, X is fluorine, in an aprotic solvent with compound (8) in the presence of from about 3 mol equivalents to about 5 mol equivalents of a Group I metal cation amide at a temperature of from 2O C to 55^°C, more preferably, at a temperature from 45^°C to 55^°C. A catalytic amount of Group I metal cation dialkylamide can be added if necessary. A preferred Group I metal cation amide is lithium amide, a preferred Group I metal cation dialkylamide is lithium diisopropylamide, and a preferred solvent is tetrahydrofuran. Preferably, the reaction is performed by adding a small amount of compound (7) and compound (8) to lithium amide in tetrahydrofuran followed by slow continuous addition of the remaining portion. This procedure minimizes the risk of reactor over-pressurization due to gas side product (ammonia) generation.Step III: Preparation of N-((RV2.3-dihydroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound I)Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using a carboxylic acid activating reagent such as, for example, COCI2, S(O)C^, S(O)2Cl2, P(O)Cl3, triphenylphosphine/diethylazodicarboxylate, diphenylphosphinic chloride, N, N’-dicyclohexylcarbodiimide, (benzotriazol-1 -yloxy)tripyrolidinophosphonium hexafluorophosphate, (benzotriazol-1 – yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, N-ethyl-N’-(3- dimethylaminopropyl)carbodiimide hydrochloride, or 1,1′-carbonyldiimidazole (CDI).A preferred carboxylic acid activating reagent is 1,1′-carbonyldimidazole (CDI) shown in Scheme 3. Preparation of the desirable polymorphic Form IV of Compound I using CDI is described in the above- referenced U.S. Patent Application No. 10/969,681.Scheme 3

10

10 11 Compound IIn according to the present invention, the method was modified to include the advantageous procedure for product purification and isolation, which procedure is performed in single-phase systems such as, for example, toluene/acetonitrile for the first isolation/crystallization and ethanol/toluene for the second recrystallization. Water addition, implemented in the previous procedure, was omitted to avoid the two-phase crystallization from the immiscible water-toluene system that caused inconsistent product purity. The one-phase procedure of the present invention provides consistent control and removal of un- reacted starting material and side products. Alternatively, Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using thionyl chloride (SOCI₂) as shown in Scheme 4.Scheme 4

Compound IExamplesThe reagents and conditions of the reactions described herein are merely illustrative of the wide variety of starting materials, their amounts and conditions which may be suitably employed in the present invention as would be appreciated by those skilled in the art, and are not intended to be limiting in any way.HPLC (Conditions A): 10 μL injection volume onto Agilent Zorbax RX-C18 150 mm x 4.6 mm x 3.5 μm column at 30^°C column temperature, 1.0 mL/min flow rate and detection at 246 nm. Mobile phase A (v/v): 25 mM Acetate Buffer, pH 6.0; Mobile phase B (v/v): Acetonitrile, and Linear Gradient Table:

Sample Preparation: Dilute 100 μL reaction mixture to 10 mL with acetonitrile. Mix in a vial 200 μL of this sample solution with 300 μL carbonate buffer pH 10.0 and 300 μL solution of 2-mercaptopyridine in acetonitrile (18 mM), heat the vial for 10 minutes at 50⁰C and dilute to 1:1 ratio in mobile phase A.GC (Conditions B): 1 μL injection onto an RTX-5 column (30 m x 0.25 mm x 0.25 μm) with initial oven temperature of 120^°C for 2 min. to final temperature of 250^°C in 15^°C/minute ramping and a final time of 2.33 min; Flow rate: 1 mL/min.HPLC (Conditions C): 5 μL injection onto Phenomenex Luna C18(2) 150 mm x 4.6 mm x 3μm column ; flow rate : 1.0 mL/min; detection at 225 nm; mobile phase A: 95/5 v/v Water/Acetonitrile with 0.1% Trifluoroacetic acid (TFA), mobile phase B: 5/95 v/v Water/Acetonitriie with 0.1% TFA; Linear Gradient Table:

Sample preparation: Dilute 1 ml_ reaction mixture to 100 mL with acetonitrile and dilute 1 mL of this solution to 10 mL with 50:50 Water/Acetonitrile.HPLC (Conditions D): 5 μL injection onto Waters SymmetryShield RP 18, 150 mm x 4.6 mm x 3.5 μm column; flow rate: 1.0 mL/min; detection at 235 nm; mobile phase A: 25 mM Acetate Buffer adjusted to pH 5.5, mobile phase B: Acetonitrile; Linear Gradient Table:

Sample preparation: Dilute 40 μL of reaction mixture in 20 mL acetonitrile.HPLC (Conditions E): 10 μL sample injection onto YMC ODS-AQ 5 μm, 250 mm x 4.6 mm column; flow rate: 1.0 ml_/min; detection at 280 nm; temperature 30^°C; mobile phase : 75/25 v/v Acetonitrile/Water with 0.1% Formic acid.Sample preparation: Quench reaction mixture sample with dipropylamine and stir for about 5 minutes before further dilution with mobile phase.DSC measurement was performed using a Mettler-Toledo DSC 822, temperature range 25^° to 150^°C with 5^°C/min heating rate in a 40 μL aluminum pan. Experimental Conditions for Powder X-Rav Diffraction (XRD):A Rigaku Miniflex+ X-ray diffractometer was used for the acquisition of the powder XRD patterns. The instrument operates using the Cu Ka₁ emission with a nickel filter at 1.50451 units. The major instrumental parameters are set or fixed at:X-ray: Cu / 30 kV (fixed) / 15 mA (fixed)Divergence Slit: Variable Scattering Slit: 4.2° (fixed) Receiving Slit: 0.3 mm (fixed) Scan Mode: FT Preset Time: 2.0 s Scan Width: 0.050° Scan Axis: 2Theta/Theta Scan Range: 3.000° to 40.000°Jade Software Version: 5.0.36(SP1) 01/05/01 (Materials Data, Inc.) Rigaku Software: Rigaku Standard Measurement for Windows 3.1 Version 3.6(1994-1995) Example 1. Preparation of 0-ffl4R)-2.2-dimethyl-1.3-dioxolan-4-vπmethyl}hvdroxylamine (6)A solution containing [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) (13.54 ml_, 0.109 mol) (DAISO Co., Ltd., CAS# 22323-82-6) and triethylamine (18.2 ml_, 0.131 mol) in 115 mL toluene was cooled to -15 C, then trifluoromethanesulfonic anhydride (2) (18.34 mL, 30.75 g, 0.109 mol) (Aldrich, Catalog # 17,617-6 ) was added drop wise while maintaining the temperature at less than -15^°C. The mixture was then stirred for 2 hours, and transferred to a separate flask containing a mixture (slurry) of N- hydroxyphthalimide (4) (18.99 g, 0.116 mol) (Aldrich, Catalog # H5.370-4) and 18.2 mL (0.13 mol) triethylamine in 95 mL toluene. The resulting mixture was warmed to 20-25^°C and stirred for at least 5 hours or until reaction completion (determined by HPLC (Conditions A)). Water (93 mL) was then added to quench the reaction mixture, the phases were separated, and the bottom aqueous layer was discarded. The water quench was repeated two more times resulting in a pale yellow organic layer. The organic layer was heated to 35 C and treated with 36.7 mL ammonium hydroxide solution (contains about 28-29% wt/wt ammonia). The mixture was stirred for at least 12 hours or until the reaction was deemed complete as determined by GC (Conditions B). The water was then removed under reduced pressure by co- distilling it with toluene to about half of the original volume at temperatures around 35-45 C. Toluene (170 mL) was added to the concentrated solution and the distillation was repeated. A sample was drawn for water content determination by Karl Fisher method (using EM Science Aquastar AQV-2000 Titrator with a sample injected to a pot containing methanol and salicylic acid). The distillation was repeated ifl water content was more than 0.1%. The concentrated solution was filtered to remove the white solid side product, and the filtrate was stored as 112mL (98 g) product solution containing 9.7% w/w compound 6 in toluene. This solution was ready for use in the final coupling step (Example 3). Overall chemical yield was 59%. A small sample was evaporated to yield a sample for NMR identification.¹H NMR (400 MHz, CDCI₃): δ 5.5 (bs, 2H), 4.35 (m, 1H), 4.07 (dd, 1H), 3.77 (m, 2H), 3.69 (dd, 1H), 1.44 (s, 3H), 1.37 (s, 3H).Example 2. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9)A solution of 2-fluoro-4-iodoaniline (8) (16.4 g, 0.069 mol) (Aldrich, Catalog # 30,660-6) and 2,3,4- trifluorobenzoic acid (7) (11.98 g, 0.068 mol) (Aldrich, Cat # 33,382-4) in 38 mL tetrahydrofuran (THF) was prepared and a portion (about 5%) of this solution was added to a stirring slurry of lithium amide (5 g, 0.22 mol) in 40 mL THF at 50-55 C. After about 15-30 min. an exotherm followed by gas release and color change are observed. The remaining portion of the (8) and (7) solution was added slowly over 1-2 hr while maintaining temperatures within 45-55^°C. The mixture was stirred until the reaction was deemed complete (by HPLC (Conditions C). The final mixture was then cooled to 20-25^°C and transferred to another reactor containing 6 N hydrochloric acid (47 mL) followed by 25 mL acetonitrile, stirred, and the bottom aqueous phase was discarded after treatment with 40 mL 50% sodium hydroxide solution. The organic phase was concentrated under reduced pressure and 57 mL acetone was added. The mixture was heated to 50^°C, stirred, and added with 25 mL warm (40-50^°C) water and cooled to 25-30^°C to allow crystallization to occur (within 1-4 hours). Once the crystallization occurred, the mixture was further cooled to 0 to -5^°C and stirred for about 2 hours. The solid product was filtered and the wet cake was dried in vacuum oven at about 55^°C. Overall chemical yield was 21.4 g, 80%. ¹H NMR (400 MHz, (CD₃)₂SO): δ 13.74 (bs, 1H), 9.15 (m, 1 H), 7.80 (dd, 1H), 7.62 (d, 1H), 7.41 (d, 1H), 7.10 (q, 1H), 6.81 (m, 1H).Example 2B. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) by the solid addition of lithium amide methodTo a stirring solution of 2,3,4-trifluorobenzoic acid (13) (5.0 g, 28.4 mmol) and 2-fluoro-4- iodoaniline (14) (6.73 g, 28.4 mmol) in MeCN (100 mL), under N₂ atmosphere was added lithium amide (2.61 g, 113.6 mmol) in small portions. The reaction mixture was heated to reflux for 45 minutes, cooled to ambient temperature and quenched with 1 N HCI and then water. The yellowish white precipitate was filtered, washed with water. The solid was triturated in CH₂CI₂ (30 mL) for 1h, filtered and dried in a vacuum oven at 45^°C for 14 hours to give 8.Og (72%) of compound (9) as an off-white solid, mp 201.5-203 ^°C.Example 3. Preparation of N-((R)-2.3-dihvdroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound \)3,4-Difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (20 g, 0.051 mol) in 100 mL acetonitrile was treated with 1,1′-carbonyldiimidazole (CDI) (8.66 g, 0.053 mol) (Aldrich, Cat # 11,553-3) and stirred for about 2 hours at 20-25^°C until the reaction was deemed complete by HPLC (Conditions D). 94 mL (84.9 g) of 9.7% w/w solution of O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) in toluene was then added and stirred for about 4 hours or until the reaction was deemed complete by HPLC (Conditions D). To this mixture was added 66 mL of 5.6 % hydrochloric acid solution, and after stirring, the bottom aqueous phase was discarded. Again 66 mL of 5.6 % hydrochloric acid solution was added to the organic phase and stirred at 20-25^°C for 12-18 hours or until the reaction was deemed complete by HPLC (Conditions D). The bottom layer was then discarded and the remaining organic layer was concentrated under reduced pressure to remove about 10-20% solvent, and the volume was adjusted to about 9-11 mL/g with toluene (80 mL). Crude product was then crystallized at 10-15^°C. The slurry was allowed to stir for about 2 hours and the crude solid product was filtered, and dried. The dried crude product was recharged to the reactor and dissolved into 150 mL of 5% v/v ethanol/toluene mixture at 55- 67^°C. The solution was then clarified at this temperature through filter (line filter) to remove any remaining particulate matter. The solution was then cooled slowly to 5^°C to crystallize and stirred for at least 2 h, filtered and dried. The dried solid product was redissolved in EtOH (60 mL) at 35^°C, and product was precipitated out by adding water (300 mL) at 35^°C followed by cooling to 20⁰C. The slurry was stirred for at least 2 hours to transform the crystals to the desired polymorphic Form IV as determined by DSC and Powder X-ray Diffraction pattern (PXRD). The slurry was filtered and dried under vacuum oven at 70- 90^°C to yield the final N-((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound I) product. Overall chemical yield was 13 g, 53%. Melting point (DSC): 112+1^° C. Appearance: White to off-white crystals.Shown in Figure 1, PXRD conforms to polymorphic crystal Form IV disclosed in the above mentioned U.S. Patent Application No. 10/969,681 ¹H NMR (400 MHz, (CD₃)₂SO): δ 11.89 (bs, 1H), 8.71 (bs, 1H), 7.57 (d, 1H), 7.37 (m, 2H), 7.20 (q, 1H), 6.67 (m, 1H), 4.84 (bs, 1H), 4.60 (m, 1H), 3.87 (m, 1 H), 3.7 (m, 2H), 3.34 (m, 2H).Example 4. Preparation of N-((R)-2.3-dihydroxypropoxyV3.4-difluoro-2-(2-fluoro-4-iodo-phenylanrιinoV benzamide (Compound \)To a stirring solution of 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (120 g, 0.30 mol) in a mixture of 1 mL N,N-dimethylformamide and 1000 mL toluene was added thionyl chloride (55 g, 0.462 mol). The mixture was heated to 50-65 C and stirred for 2 hours or until reaction completion as determined by HPLC (Conditions E). The final reaction mixture was then cooled and concentrated under reduced pressure to a slurry keeping the temperature below 35^°C. Toluene (600 mL) was added to dissolve the slurry and vacuum distillation was repeated. Additional toluene (600 mL) was added to the slurry dissolving all solids and the solution was then cooled to 5^° -10^°C. The solution was then treated with O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) (63 g, 0.43 mol) solution in 207 mL toluene followed by potassium carbonate (65 g) and water (200 mL), stirred for at least 2 hours at 20- 25^°C. The stirring was stopped to allow phase separation and the bottom phase was discarded. The remaining organic layer was treated with hydrochloric acid solution (7.4%, 240 mL) until pH was less than 1 and stirred for 2 hours. The final reaction mixture was slightly concentrated under vacuum collecting about 100 mL distillate and the resulting organic solution was cooled to 5^°C to crystallize the product and filtered. The filter cake was washed with toluene (1000 mL) followed by water (100 mL) and the wet cake (crude product Compound I) was charged back to the flask. Toluene (100 mL), ethanol (100 mL) and water (100 mL) are then added, stirred at 30-35^°C for about 15 min, and the bottom aqueous phase was discarded. Water (200 mL) was then added to the organic solution and the mixture was stirred at about 3O C to allow for crystallization. The stirring was continued for 2 hours after product crystallized, then it was further cooled to about 0^°C and stirred for at least 2 hours. The slurry was filtered and wet cake was dried under reduced pressure at 55-85^°C to yield the final product N-((R)-2,3-dihydroxypropoxy)-3,4- difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound I) product. Overall chemical yield was 86 g, 58%.

PATENT

WO2002/006213 describes crystalline Forms I and II. U.S. Pat. No. 7,060,856 (“the ‘856 patent”)

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2002006213

////////MIRDAMETINIB, Orphan Drug Status, Neurofibromatosis 1, PHASE 2, PD0325901, PD 0325901, PD-325901, 

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Ezutromid

945531-77-1
Chemical Formula: C19H15NO3S
Molecular Weight: 337.39

945531-77-1, SMT c1100, BMN-195, BMN 195,  C 1100

 5-(ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole

BMN-195; BMN 195; BMN195; SMTC-1100; SMTC1100; SMTC 1100; VOX-C1100; Ezutromid

Ezutromid, also known as BMN-195 and SMTC-1100, is a first orally bioavailable utrophin’s translation modulator. Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fibers leading to the gradual depletion of skeletal muscle.

Ezutromid is an orally administered small molecule utrophin modulator currently involved in a Phase 2 clinical trial produced by Summit Therapeutics for the treatment of Duchenne muscular dystrophy (DMD).^[1][2] DMD is a fatal x-linked recessive disease affecting approximately 1 in 5000 males and is a designated orphan disease by the FDA and European Medicines Agency.^[3] Approximately 1/3 of the children obtain DMD as a result of spontaneous mutation in the dystrophin gene and have no family history of the disease.^[3] Dystrophin is a vital component of mature muscle function, and therefore DMD patients have multifarious forms of defunct or deficient dystrophin proteins that all manifest symptomatically as muscle necrosis and eventually organ failure.^[3][4] Ezutromid is theorized to maintain utrophin, a protein functionally and structurally similar to dystrophin that precedes and is replaced by dystrophin during development.^[3][5] Utrophin and dystrophin are reciprocally expressed, and are found in different locations in a mature muscle cell.^[4][6] However, in dystrophin-deficient patients, utrophin was found to be upregulated and is theorized to replace dystrophin in order to maintain muscle fibers.^[7] Ezutromid is projected to have the potential to treat all patients suffering with DMD as it maintains the production of utrophin to counteract the lack of dystrophin to retard muscle degeneration.^[7][8] Both the FDA and European Medicines Agency has given ezutromid an orphan drug designation.^[5][9] The FDA Office of Orphan Products and Development offers an Orphan Drug Designation program (ODD) that allows drugs aimed to treat diseases that affect less than 200,000 people in the U.S. monetary incentives such as a period of market exclusivity, tax incentives, and expedited approval processes.^[5][10]

The Phase 2 clinical trial was ended in 2018 and the medication discontinued after it failed to show any benefit in slowing the disease.^[11]

Clinical trials

The first Phase 1b trial (NCT02056808) began on November 2013 and involved 12 patients aged 5–11 years old.^[12] The patients were divided into three groups given escalating oral doses testing the safety and tolerability after each increase over the course of 10 days.^[12]

Another completed Phase 1b trial (NCT02383511) began February 2015 and involved 12 patients aged 5–13 years old.^[13] The goal was to determine the safety, tolerability, and pharmacokinetic parameters by measuring plasma concentration and major metabolite levels over 28 days for three sequence groups.^[13] Each sequence involved placebo, 1250 mg, and 2500 mg BID (twice a day) doses given for one week each.^[4][13]

A PhaseOut DMD, Phase 2, Proof of Concept (NCT02858362) clinical trial is underway that tests the clinical safety and efficacy of an oral suspension of ezutromid.^[2] The 48-week open-label trial is enrolling 40 boys, ages 5–10, living in the U.K. or U.S.^[2] MRI leg muscle change will be measured as well as ezutromid plasma concentration levels, with a secondary goal of obtaining quantifiable images of utrophin membrane stained biopsies at baseline and either 24 or 48 weeks.^[2]

Commercial aspects

As of 2016, ataluren was the only approved drug in the EU to treat a specific subpopulation of patients with nmDMD, or DMD caused by a nonsense mutation.^[14] However, nonsense mutations only account for approximately 15% of all patients with DMD.^[15] Therefore, Summit Therapeutics projects to file for regulatory approval in the US and EU by 2019 and to reach market in 2020.^[8] They expect to profit just over £24,046 in 2020 and £942,656 in 2025, which amounts to ~10% CGR for the first 7 years on the basis of treating all DMD patients in the US, EU, Iceland, Norway, Switzerland and Russia.^[8]

Furthermore, Summit Therapeutics has entered an agreement with Sarepta Theraputics as of October 2016 regarding the commercialization of ezutromid.^[16] The agreement consists of a collaboration between Sarepta and Summit to share the research and developing costs for the development of novel therapies to treat DMD patients.^[16]

PAPER

https://onlinelibrary.wiley.com/doi/10.1002/anie.201906080

4-(ethylthio)Phenol S2: To a 250 mL round bottle, 4-mercaptophenol S1 (12.6 g, 100 mmol), K2CO3 (15.3 g, 110 mmol), acetone (100 mL) were added, then, iodoethane (15.6 g, 8.0 mL, 130 mmol) was added slowly at 0 oC. The system was stirred at room temperature overnight. After filtration, distillation of solvent, and flash chromatography, S2 (10.780 g) was obtained with 70% yield.

4-(ethylthio)-2-Nitrophenol S3: To a 250 mL round bottle, 4-(ethylthio)Phenol S2 (3.084 g, 20 mmol), 300-400 mesh silica gel (2 g), distilled water (2 g), and CH3CN (60 mL) was added. The system was then cooled by an ice water bath. Subsequently, citric acid (3.842 g, 20 mmol), NaNO2 (2.760 g, 40 mmol) were separately added slowly in portionwise. The system was reacted at room temperature overnight. After filtration and distillation of solvent, EA (50 mL) and water (50 mL) was added, after separation, the aqueous phase was extraction with EA (30 mL) twice. The combined organic phase was dried with MgSO4. Following by filtration and chromatography, S3 (3.590 g) was obtained with 90% yield.

4-(ethylthio)-2-Nitrophenol S4: To a 100 mL round bottle, S3 (2.46 g, 12.3 mmol), reductive iron powder (2.07 g, 36.9 mmol), and EtOH (50 mL) was added. Then, HCl (aq.) (0.15 M) (12 mL, 1.85 mmol) was added slowly. The system was refluxed overnight. After filtration, distillation of solvent, and flash chromatography, S4 (1.040 g) was obtained with 50% yield.

5-(ethylthio)-2-(naphthalen-2-yl)Benzo[d]oxazole S6 (Ezutromid-S): S4 (324 mg1.91 mmol), 2-naphthoyl chloride S5 (545.7 mg, 2.87 mmol), dry 1,4-dioxane (5 mL) was added into a sealing tube. Then, the system was vacuumed and filled with nitrogen for three times. Subsequently, the reaction was run at 160 oC for 10 hours. After distillation of solvent and flash chromatography, S6 (361.7 mg) was obtained with 62% yield. 1H NMR (500 MHz, Chloroform-d) δ 8.74 (s, 1H), 8.28 (dd, J = 8.5, 1.7 Hz, 1H), 7.96 (t, J = 7.5 Hz, 2H), 7.92 – 7.84 (m, 1H), 7.81 (d, J = 1.8 Hz, 1H), 7.57 (pd, J = 6.8, 3.4 Hz, 2H), 7.51 (d, J = 8.3 Hz, 1H), 7.39 (dd, J = 8.4, 1.8 Hz, 1H), 2.99 (q, J = 7.3 Hz, 2H), 1.33 (t, J = 7.3 Hz, 3H).13C NMR (126 MHz, Chloroform-d) δ 163.79, 149.70, 142.92, 134.76, 132.89, 132.38, 128.92, 128.78, 128.20, 127.87, 127.85, 126.91, 124.11, 123.84, 121.38, 110.72, 29.17, 14.40

Dibenzoate5-(ethylsulfone)-2-(naphthalen-2- yl)benzo[d]oxazole (Ezotrumid) 5a:

5- (ethylthio)-2-(naphthalen-2-yl)Benzo[d]oxazole (30.5 mg, 0.1 mmol), UO2(OAc)2 . 2H2O (0.8 mg, 0.002 mol), H2O (10 equiv., 36 μL), o-xylene (8.3 equiv., 0.2 mL), CH3CN (1 mL) were stirred under oxygen atmosphere (1 atm, balloon) at room temperature until the total consumption of sulfide and sulfoxide under the irradiation of three 2 w blue LEDs in a paralleled reactor. 5a (27.3 mg, 81%) was obtained through column chromatography (PE/EA = 20/1-5/1) as a white solid, Rf = 0.6 (PE/EA = 2/1);

1H NMR (500 MHz, Chloroform-d) δ 8.82 (s, 1H), 8.37 (s, 1H), 8.32 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.99 – 7.89 (m, 2H), 7.84 – 7.76 (m, 1H), 7.61 (t, J = 7.3 Hz, 2H), 3.28 – 3.08 (m, 2H), 1.32 (dt, J = 7.3, 3.6 Hz, 3H)..

13C NMR (126 MHz, Chloroform-d) δ 165.57, 153.87, 142.86, 135.26, 135.14, 132.86, 129.09, 128.97, 128.37, 127.99, 127.19, 125.35, 123.87, 123.34, 121.00, 111.36, 51.04, 7.62.

IR (KBr) 2933, 1507, 1498, 1258, 1064, 1046, 756, 474 cm-1 .

HRMS (ESI) Calcd for C19H16NO3S 338.0851 (M+H), Found 338.0865.

PATENT

WO 2007091106

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2007091107

PATENT

WO 2009021749

WO 2009019504

WO 2013167737 A

CN 110437170

CN 110483345

CN 110563619

PATENT

WO 2009021748

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2009021748

It has been discovered that the compound of formula I (5-(ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole) has excellent properties for the treatment of Duchenne muscular dystrophy (see, e.g., international patent application publication no. WO 2007/091106).

The compound of formula I (R = 5-ethylsulfonyl; R₉ = 2-naphthalen-2-yl) may be synthesised according to the following procedure, as disclosed in WO 2007/091106 (page 51):

Experimental

S nthesis of 5- eth lsulfon -2- na hthalen-2- l^‘benzo d oxazole

Procedure:
A vessel was equipped with a retreat blade stirrer and downward pumping turbine, a five necked flange lid, seal and clamp, stirrer gland and overhead stirrer, thermometer pocket, Dean- Stark trap, dropping funnel and condenser. The water to the condenser was then switched on.
The sodium hydroxide and 0.80 L of water were then mixed (whilst cooling in an ice bath until all the sodium hydroxide has dissolved – caution exothermic). The resulting solution was then transferred to a scrubber appropriately attached to the vessel.

The 2-amino-4-(ethylsulfonyl)phenol and 2.00 L of xylenes (mixed) were then transferred to the vessel, and the reagents and solvent were stirred at 100 rpm.
Then, the 2-naphtholyl chloride was dissolved in 2.00 L of xylenes (mixed) and transferred into the vessel. The stirring rate was increased to 150 rpm.

The temperature of the solution was gradually increased to 100°C over a period of not less than 30 mins, and then maintained at that level for 10 mins. (Caution: HCl gas is evolved during this process through the gas scrubber). The stirrer speed was then increased to 315 rpm and the temperature gradually increased over a period of 30 minutes until reflux (155°C) at which level it was maintained for 90 mins. (Caution: HCl gas is evolved during this process through the gas scrubber).
The methanesulfonic acid was then added drop-wise over a period of 30 mins and relux was maintained until no further water was being collected in the Dean-Stark apparatus (approx 15 mins).
The heat was then removed and the pipe adapter from the Dean- Stark apparatus disconnected. The resulting solution was allowed to cool to 90⁰C, and then filtered using Whatman 1 filter paper.
The resulting solution was then left at ambient temperature for 18h, after which time the product crystallised, and the product was separated by filtration using Whatman 1 filter paper. The product was then washed with Ix 1.0 L of tert-butyl methyl ether (TBME)

The product was then dried in a vacuum oven at 65°C at a pressure of 1 Ombar until constant weight was achieved (less than 0.5 g difference between consecutive measurements of mass which must be at least 1 h apart).
The product was obtained as a sandy-beige powder in a yield of 80%.

Characterisation:
5-(EthylsuIf onyl)-2-(naphthalen-2-yl)benzo [d] oxazole
LCMS RT= 6.94min, MH⁺ 338.1;
¹H NMR (DMSO): 8.90 (IH, br), 8.34 (IH, d, J 1.4 Hz), 8.30 (IH, dd, J 8.6 1.7 Hz), 8.24-8.05 (4H, m), 7.99 (IH, dd, J 8.5 1.8 Hz), 7.73-7.64 (2H, m), 3.41 (2H, q, J 7.3 Hz), 1.15 (3H, t, J7.3 Hz);

MP = 160-161⁰C.

Synthesis of polymorphic forms

1. Procedure
100 mg of the compound of formula I was dissolved in the minimum amount of good solvent and then the anti-solvent was added to induce crystallisation. The supernatant liquor was then removed, and the resulting solid was dried under vacuum for 12 his.

PAPER

Journal of medicinal chemistry (2011), 54(9), 3241-50

https://pubs.acs.org/doi/10.1021/jm200135z

Abstract

A series of novel 2-arylbenzoxazoles that upregulate the production of utrophin in murine H2K cells, as assessed using a luciferase reporter linked assay, have been identified. This compound class appears to hold considerable promise as a potential treatment for Duchenne muscular dystrophy. Following the delineation of structure–activity relationships in the series, a number of potent upregulators were identified, and preliminary ADME evaluation is described. These studies have resulted in the identification of 1, a compound that has been progressed to clinical trials.

PAPER

Angewandte Chemie, International Edition (2019), 58(38), 13499-13506

Angewandte Chemie, International Edition (2020), 59(3), 1346-1353.

PAPER

https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.9b01547

Journal of medicinal chemistry (2020), 63(5), 2547-2556.

Abstract

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

PAPER

https://www.sciencedirect.com/science/article/abs/pii/S004040201931227X

Abstract

Following on from ezutromid, the first-in-class benzoxazole utrophin modulator that progressed to Phase 2 clinical trials for the treatment of Duchenne muscular dystrophy, a new chemotype was designed to optimise its physicochemical and ADME profile. Herein we report the synthesis of SMT022357, a second generation utrophin modulator preclinical candidate, and an asymmetric synthesis of its constituent enantiomers. The pharmacological properties of both enantiomers were evaluated in vitro and in vivo. No significant difference in the activity or efficacy was observed between the two enantiomers; activity was found to be comparable to the racemic mixture.

Graphical abstract

References

1. ^ “About Summit Therapeutics – Summit”. Summit. Retrieved 2016-11-14.
2. ^ Jump up to:^a b c d Clinical trial number NCT02858362 for “PoC Study to Assess Activity and Safety of SMT C1100 (Ezutromid) in Boys With DMD” at ClinicalTrials.gov
3. ^ Jump up to:^a b c d “Duchenne Muscular Dystrophy – Summit”. Summit. Archived from the original on 2016-11-15. Retrieved 2016-11-14.
4. ^ Jump up to:^a b c Ricotti V, Spinty S, Roper H, Hughes I, Tejura B, Robinson N, et al. (2016-01-01). “Safety, Tolerability, and Pharmacokinetics of SMT C1100, a 2-Arylbenzoxazole Utrophin Modulator, following Single- and Multiple-Dose Administration to Pediatric Patients with Duchenne Muscular Dystrophy”. PLOS ONE. 11 (4): e0152840. Bibcode:2016PLoSO..1152840R. doi:10.1371/journal.pone.0152840. PMC 4824384. PMID 27055247.
5. ^ Jump up to:^a b c “Potential DMD Therapy, Ezutromid, Shows Promise in Upgraded Form”. Retrieved 2016-11-14.
6. ^ Janghra N, Morgan JE, Sewry CA, Wilson FX, Davies KE, Muntoni F, Tinsley J (2016-03-14). “Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies”. PLOS ONE. 11 (3): e0150818. Bibcode:2016PLoSO..1150818J. doi:10.1371/journal.pone.0150818. PMC 4790853. PMID 26974331.
7. ^ Jump up to:^a b “Home – Summit”. Summit. Retrieved 2016-11-14.
8. ^ Jump up to:^a b c Werther CA (2016). Ezutromid Has the Potential to Treat All Duchenne Patients; Initiating Coverage With a Buy. H.C. Wainwright & Co. pp. 1–29.
9. ^ “Search Orphan Drug Designations and Approvals”. http://www.accessdata.fda.gov. Retrieved 2016-11-14.
10. ^ Office of the Commissioner. “Developing Products for Rare Diseases & Conditions”. http://www.fda.gov. Retrieved 2016-11-14.
11. ^ Inacio P (2018-06-29). “Summit Therapeutics Ends Development of Ezutromid Therapy for DMD After Trial Failure”. Muscular Dystrophy News. Retrieved 2019-11-17.
12. ^ Jump up to:^a b Clinical trial number NCT02056808 for “A Phase 1b Study of SMT C1100 in Subjects With Duchenne Muscular Dystrophy (DMD)” at ClinicalTrials.gov
13. ^ Jump up to:^a b c Clinical trial number NCT02383511 for “Modified Diet Trial: A Study of SMT C1100 in Paediatric Patients With DMD Who Follow a Balanced Diet ” at ClinicalTrials.gov
14. ^ “PTC Therapeutics | ataluren”. PTC Therapeutics. Retrieved 2016-11-15.
15. ^ Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Howard MT, Sampson JB, et al. (March 2011). “Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene”. Human Mutation. 32 (3): 299–308. doi:10.1002/humu.21426. PMC 3724403. PMID 21972111.
16. ^ Jump up to:^a b Summit Therapeutics PLC. “Sarepta Therapeutics and Summit Enter Into Exclusive License and Collaboration Agreement for European Rights to Summit’s Utrophin Modulator Pipeline for the Treatment of Duchenne Muscular Dystrophy”. GlobeNewswire News Room. Retrieved 2016-11-15.

/////////Ezutromid, BMN-195, BMN 195, BMN195, SMTC-1100, SMTC1100, SMTC 1100, VOX-C1100, Ezutromid

O=S(C1=CC=C(OC(C2=CC=C3C=CC=CC3=C2)=N4)C4=C1)(CC)=O

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(Heavy chain)
EVQLVQSGAE VKKPGESLKI SCKGSGYIFT NYWIAWVRQM PGKGLESMGI IYPGDSDIRY
SPSFQGQVTI SADKSITTAY LQWSSLKASD TAMYYCARHD IEGFDYWGRG TLVTVSSAST
KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY
SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APEFEGGPSV
FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA SIEKTISKAK GQPREPQVYT LPPSREEMTK
NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE ALHNHYTQKS LSLSPGK
(Lihgt chain)
EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFFAWYQQK PGQAPRLLIY GASSRATGIP
DRLSGSGSGT DFTLTITRLE PEDFAVYYCQ QYDSSAITFG QGTRLEIKRT VAAPSVFIFP
PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL
TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC
(Disulfide bridge: H22-96, H144-H200, H220-L215, H226-H’226, H229-H’229, H261-H321, H367-H425, H’22-H’96, H’144-H’200, H’220-L’215, H’261-H’321, H’367-H’425, L23-L89, L135-L195, L’23-L’89, L’135-L’195)

Anifrolumab

FDA APPROVED 2021/7/30, Saphnelo

• MEDI 546

Monoclonal antibody

Treatment of systemic lupus erythematosus (SLE)

• OriginatorMedarex
• DeveloperAstraZeneca; Medarex; MedImmune
• ClassAntirheumatics; Monoclonal antibodies; Skin disorder therapies
• Mechanism of ActionInterferon alpha beta receptor antagonists

• RegisteredSystemic lupus erythematosus
• Phase IILupus nephritis
• DiscontinuedRheumatoid arthritis; Scleroderma

• 02 Jul 2021Phase-III clinical trials in Systemic lupus erythematosus in USA (SC) (NCT04877691)
• 25 Jun 2021AstraZeneca plans a phase III trial in Systemic lupus erythematosus (Adjunctive treatment) in the China, Hong Kong, South Korea, Philipines, Taiwan and Thailand (IV, Infusion), in July 2021 (NCT04931563)
• 02 Jun 2021Pharmacokinetic, efficacy and adverse events data from a phase II TULIP-LN1 trial in Lupus nephritis presented at the 22nd Annual Congress of the European League Against Rheumatism (EULAR-2021)

Anifrolumab, sold under the brand name Saphnelo, is a monoclonal antibody used for the treatment of systemic lupus erythematosus (SLE).^[1][2] It binds to the type I interferon receptor, blocking the activity of type I interferons such as interferon-α and interferon-β.^{[medical citation needed]}

Anifrolumab was approved for medical use in the United States in August 2021.^[1][3][4][5]

Anifrolumab is a monoclonal antibody that inhibits type 1 interferon receptors, indicated in the treatment of moderate to severe systemic lupus erythematosus.

Anifrolumab, or MEDI-546, is a type 1 interferon receptor (IFNAR) inhibiting IgG1κ monoclonal antibody indicated in the treatment of adults with moderate to severe systemic lupus erythematosus.^7,11 The standard therapy for systemic lupus erythematosus consists of antimalarials like hydroxychloroquine, glucocorticoids like dexamethasone, and disease modifying antirheumatic drugs like methotrexate.^8,11

Three monoclonal antibodies (anifrolumab, rontalizumab, and sifalimumab) that target the type 1 interferon pathway entered clinical trials as potential treatments for systemic lupus erythematosus, but so far only anifrolumab has been approved.³

The design of early clinical trials of anti-interferon treatments such as anifrolumab, rontalizumab, and sifalimumab have come under criticism.³ The design of the clinical trials use different definitions of autoantibody positivity, making comparison between trials difficult; all trials involve large portions of patients also using corticosteroids, which may alter patient responses in the experimental and placebo groups; and patient populations were largely homogenous, which may have increased the odds of success of the trial.³

Anifrolumab has also been investigated for the treatment of Scleroderma.¹

Anifrolumab was granted FDA approval on 30 July 2021.¹¹

Adverse effects

The most common adverse effect was shingles, which occurred in 5% of patients in the low-dose group, to 10% in the high-dose group, and to 2% in the placebo group. Overall adverse effect rates were comparable in all groups.^[6]

History

The drug was developed by MedImmune, a unit of AstraZeneca, which chose to move anifrolumab instead of sifalimumab into phase III trials for lupus in 2015.^[7][8][9]

Clinical trial results

Anifrolumab failed to meet its endpoint of significant reduction in disease as assessed by the SLE Responder Index 4 instrument in the TULIP 1 phase III trial.^[10] This multi-center, double-blind, placebo-controlled study followed adults with moderate to severe SLE over the course of one year. Preliminary results were announced on 31 August 2018.

Names

Anifrolumab is the international nonproprietary name (INN).^[11]

References

1. ^ Jump up to:^a b chttps://www.accessdata.fda.gov/drugsatfda_docs/label/2021/761123s000lbl.pdf
2. ^ Statement On A Nonproprietary Name Adopted By The USAN Council – Anifrolumab, American Medical Association.
3. ^https://www.accessdata.fda.gov/drugsatfda_docs/appletter/2021/761123Orig1s000ltr.pdf
4. ^ https://www.astrazeneca.com/media-centre/press-releases/2021/saphnelo-approved-in-the-us-for-sle.html
5. ^ “Saphnelo (anifrolumab) Approved in the US for Moderate to Severe Systemic Lupus Erythematosus” (Press release). AstraZeneca. 2 August 2021. Retrieved 2 August 2021 – via Business Wire.
6. ^ Spreitzer H (29 August 2016). “Neue Wirkstoffe – Anifrolumab”. Österreichische Apothekerzeitung (in German) (18/2016).
7. ^ “Press release: New Hope for Lupus Patients”. MedImmune. 11 August 2015. Archived from the original on 31 July 2017.
8. ^ “Anifrolumab”. NHS Specialist Pharmacy Service. Retrieved 31 July 2017.
9. ^ “Anifrolumab”. AdisInsight. Retrieved 31 July 2017.
10. ^ “Update on TULIP 1 Phase III trial for anifrolumab in systemic lupus erythematosus”. http://www.astrazeneca.com. Retrieved 2019-02-05.
11. ^ World Health Organization (2014). “International nonproprietary names for pharmaceutical substances (INN): recommended INN: list 71”. WHO Drug Information. 28 (1). hdl:10665/331151.

Further reading

• Anderson E, Furie R (April 2020). “Anifrolumab in systemic lupus erythematosus: current knowledge and future considerations”. Immunotherapy. 12 (5): 275–86. doi:10.2217/imt-2020-0017. PMID 32237942.

External links

• “Anifrolumab”. Drug Information Portal. U.S. National Library of Medicine.
• Clinical trial number NCT01438489 for “A Study of the Efficacy and Safety of MEDI-546 in Systemic Lupus Erythematosus” at ClinicalTrials.gov
• Clinical trial number NCT02446912 for “Efficacy and Safety of Two Doses of Anifrolumab Compared to Placebo in Adult Subjects With Active Systemic Lupus Erythematosus” at ClinicalTrials.gov
• Clinical trial number NCT02446899 for “Efficacy and Safety of Anifrolumab Compared to Placebo in Adult Subjects With Active Systemic Lupus Erythematosus” at ClinicalTrials.gov

1. Goldberg A, Geppert T, Schiopu E, Frech T, Hsu V, Simms RW, Peng SL, Yao Y, Elgeioushi N, Chang L, Wang B, Yoo S: Dose-escalation of human anti-interferon-alpha receptor monoclonal antibody MEDI-546 in subjects with systemic sclerosis: a phase 1, multicenter, open label study. Arthritis Res Ther. 2014 Feb 24;16(1):R57. doi: 10.1186/ar4492. [Article]
2. Peng L, Oganesyan V, Wu H, Dall’Acqua WF, Damschroder MM: Molecular basis for antagonistic activity of anifrolumab, an anti-interferon-alpha receptor 1 antibody. MAbs. 2015;7(2):428-39. doi: 10.1080/19420862.2015.1007810. [Article]
3. Massarotti EM, Allore HG, Costenbader K: Editorial: Interferon-Targeted Therapy for Systemic Lupus Erythematosus: Are the Trials on Target? Arthritis Rheumatol. 2017 Feb;69(2):245-248. doi: 10.1002/art.39985. [Article]
4. Furie R, Khamashta M, Merrill JT, Werth VP, Kalunian K, Brohawn P, Illei GG, Drappa J, Wang L, Yoo S: Anifrolumab, an Anti-Interferon-alpha Receptor Monoclonal Antibody, in Moderate-to-Severe Systemic Lupus Erythematosus. Arthritis Rheumatol. 2017 Feb;69(2):376-386. doi: 10.1002/art.39962. [Article]
5. Tummala R, Rouse T, Berglind A, Santiago L: Safety, tolerability and pharmacokinetics of subcutaneous and intravenous anifrolumab in healthy volunteers. Lupus Sci Med. 2018 Mar 23;5(1):e000252. doi: 10.1136/lupus-2017-000252. eCollection 2018. [Article]
6. Riggs JM, Hanna RN, Rajan B, Zerrouki K, Karnell JL, Sagar D, Vainshtein I, Farmer E, Rosenthal K, Morehouse C, de Los Reyes M, Schifferli K, Liang M, Sanjuan MA, Sims GP, Kolbeck R: Characterisation of anifrolumab, a fully human anti-interferon receptor antagonist antibody for the treatment of systemic lupus erythematosus. Lupus Sci Med. 2018 Apr 5;5(1):e000261. doi: 10.1136/lupus-2018-000261. eCollection 2018. [Article]
7. Bui A, Sanghavi D: Anifrolumab . [Article]
8. Trindade VC, Carneiro-Sampaio M, Bonfa E, Silva CA: An Update on the Management of Childhood-Onset Systemic Lupus Erythematosus. Paediatr Drugs. 2021 Jul;23(4):331-347. doi: 10.1007/s40272-021-00457-z. Epub 2021 Jul 10. [Article]
9. Ryman JT, Meibohm B: Pharmacokinetics of Monoclonal Antibodies. CPT Pharmacometrics Syst Pharmacol. 2017 Sep;6(9):576-588. doi: 10.1002/psp4.12224. Epub 2017 Jul 29. [Article]
10. Koh JWH, Ng CH, Tay SH: Biologics targeting type I interferons in SLE: A meta-analysis and systematic review of randomised controlled trials. Lupus. 2020 Dec;29(14):1845-1853. doi: 10.1177/0961203320959702. Epub 2020 Sep 22. [Article]
11. FDA Approved Drug Products: Saphnelo (Anifrolumab-fnia) Intravenous Injection [Link]

//////////Anifrolumab, Saphnelo, FDA 2021, APPROVALS 2021, peptide, Monoclonal antibody, アニフロルマブ (遺伝子組換え) , MEDI 546, AstraZeneca, Medarex, MedImmune

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Rutoside

RUTIN

• Molecular FormulaC₂₇H₃₀O₁₆
• Average mass610.518
• рутозид [Russian] [INN]ルチン [Japanese]روتوسيد [Arabic] [INN]芦丁 [Chinese] [INN]

CAS 153-18-4

• C.I. 75730
• NSC-9220

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one

Rutin trihydrate | CAS 250249-75-3 RutinCAS Registry Number: 153-18-4 
CAS Name: 3-[[6-O-(6-Deoxy-a-L-mannopyranosyl)-b-D-glucopyranosyl]oxy]-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one 
Additional Names: rutoside; quercetin-3-rutinoside; 3,3¢,4¢,5,7-pentahydroxyflavone-3-rutinoside; melin; phytomelin; eldrin; ilixathin; sophorin; globularicitrin; paliuroside; osyritrin; osyritin; myrticolorin; violaquercitrin 
Trademarks: Birutan (Merck KGaA) 
Molecular Formula: C27H30O16Molecular Weight: 610.52Percent Composition: C 53.12%, H 4.95%, O 41.93% 
Literature References: Identity with ilixanthin: Schindler, Herb, Arch. Pharm.288, 372 (1955). Found in many plants, especially the buckwheat plant (Fagopyrum esculentum Moench., Polygonaceae) which contains about 3% (dry basis): Couch et al.,Science103, 197 (1946). From tobacco (Nicotiana tabacum L., Solanaceae) Couch, Krewson, U.S. Dept. Agr., Eastern Regional Res. Lab.AIC-52 (1944). 
In forsythia [Forsythia suspensa (Thunb.) Vahl. var. fortunei (Lindl.) Rehd., Oleaceae], in hydrangea (Hydrangea paniculata Sieb., Saxifragaceae), in pansies (Viola sp., Violaceae).
General extraction procedure: BeilsteinXXXI, 376. From leaves of Eucalyptus macroryncha F. v. Muell., Myrtaceae: Attree, Perkin, J. Chem. Soc.1927, 234. 
Industrial production from Eucalyptus spp.: Humphreys, Econ. Bot.18, 195 (1964). 
Structure: Zemplén, Gerecs, Ber.68B, 1318 (1935). 
Synthesis: Shakhova et al.,Zh. Obshch. Khim.32, 390 (1962), C.A.58, 1426e (1963). Rutin is hydrolyzed by rhamnodiastase from the seed of Rhamnus utilis Decne, Rhamnaceae (Chinese buckthorn); emulsin is not effective: Bridel, Charaux, Compt. Rend.181, 925 (1925). Toxicity data: Harrison et al.,J. Am. Pharm. Assoc.39, 557 (1950). Book: J. Q. Griffith, Jr., Rutin and Related Flavonoids (Mack, Easton, Pa., 1955). 
Comprehensive description: T. I. Khalifa et al.,Anal. Profiles Drug Subs.12, 623-681 (1983).UV

Properties: Pale yellow needles from water, gradual darkening on exposure to light. The crystals contain 3 H2O and become anhydr after 12 hrs at 110° and 10 mm Hg. Anhydr rutin browns at 125°, becomes plastic at 195-197°, and dec 214-215° (with effervescence). [a]D23 +13.82° (ethanol); [a]D23 -39.43° (pyridine). Anhydr rutin is hygroscopic. One gram dissolves in about 8 liters water, about 200 ml boiling water, 7 ml boiling methanol. Sol in pyridine, formamide and alkaline solns; slightly sol in alcohol, acetone, ethyl acetate. Practically insol in chloroform, carbon bisulfide, ether, benzene, petr solvents. Dil solns give green color with ferric chloride. Rutin is colored brown by tobacco enzyme under experimental conditions: Neuberg, Kobel, Naturwissenschaften23, 800 (1935). LD50 i.v. in mice: 950 mg/kg (propylene glycol soln) (Harrison). 
Optical Rotation: [a]D23 +13.82° (ethanol); [a]D23 -39.43° (pyridine) 
Toxicity data: LD50 i.v. in mice: 950 mg/kg (propylene glycol soln) (Harrison)Therap-Cat: Capillary protectant.Keywords: Vasoprotectant. 
C13

MASS 

Rutin, also called rutoside, quercetin-3-O-rutinoside and sophorin, is the glycoside combining the flavonol quercetin and the disaccharide rutinose (α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranose). It is a citrus flavonoid found in a wide variety of plants including citrus.

Rutin, also called rutoside, is the glycoside flavonoid found in a certain fruits and vegetables. Most rutine-rich foods are capers, olives, buckwheat (whole grain flour), asparagus, raspberry.In a clinical trial, rutin was found to aid control of intraocular pressure in patients with primary open angle glaucoma. As a component of dietary supplement Phlogenzym, rutin is used for treatment of osteoarthritis. Rutin is also used for treatment of post-surgical swelling of the arm after breast cancer surgery. Traditionally, rutin is used to prevent mucositis due to cancer treatment, to treat blood vessel disease such as varicose veins, bleeding, hemorrhoids.

Occurrences

Rutin is one of the phenolic compounds found in the invasive plant species Carpobrotus edulis and contributes to the antibacterial^[3] properties of the plant.

Its name comes from the name of Ruta graveolens, a plant that also contains rutin.

Various citrus fruit peels contain 32 to 49 mg/g of flavonoids expressed as rutin equivalents.^[4]

Citrus leaves contain rutin at concentrations of 11 and 7 g/kg in orange and lime trees respectively.^[5]

Metabolism

The enzyme quercitrinase can be found in Aspergillus flavus.^[6] It is an enzyme in the rutin catabolic pathway.^[7]

In food

Rutin is a citrus flavonoid glycoside found in many plants including buckwheat,^[8] the leaves and petioles of Rheum species, and asparagus. Tartary buckwheat seeds have been found to contain more rutin (about 0.8–1.7% dry weight) than common buckwheat seeds (0.01% dry weight).^[8] Rutin is one of the primary flavonols found in ‘clingstone’ peaches.^[9] It is also found in green tea infusions.^[10]

Approximate rutin content per 100g of selected foods, in milligrams per 100 milliliters:^[11]

Research

Rutin (rutoside or rutinoside)^[12] and other dietary flavonols are under preliminary clinical research for their potential biological effects, such as in reducing post-thrombotic syndrome, venous insufficiency, or endothelial dysfunction, but there was no high-quality evidence for their safe and effective uses as of 2018.^{[12][13][14][needs update]} As a flavonol among similar flavonoids, rutin has low bioavailability due to poor absorption, high metabolism, and rapid excretion that collectively make its potential for use as a therapeutic agent limited.^[12]

Biosynthesis

The biosynthesis pathway of rutin in mulberry (Morus alba L.) leaves begins with phenylalanine, which produces cinnamic acid under the action of phenylalanine ammonia lyase (PAL). Cinnamic acid is catalyzed by cinnamic acid-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL) to form p–coumaroyl-CoA. Subsequently, chalcone synthase (CHS) catalyzes the condensation of p-coumaroyl-CoA and three molecules of malonyl-CoA to produce naringenin chalcone, which is eventually converted into naringenin flavanone with the participation of chalcone isomerase (CHI). With the action of flavanone 3-hydroxylas (F3H), dihydrokaempferol (DHK) is generated. DHK can be further hydroxylated by flavonoid 3´-hydroxylase (F3’H) to produce dihydroquercetin (DHQ), which is then catalyzed by flavonol synthase (FLS) to form quercetin. After quercetin is catalyzed by UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) to form isoquercitrin, finally, the formation of rutin from isoquercitrin is catalyzed by flavonoid 3-O-glucoside L-rhamnosyltransferase.^[15]

SYN

https://www.sciencedirect.com/science/article/abs/pii/S100184171300017X

The compound 2 was synthesized for the first time by highly selective esterification reaction and fully characterized. The by-products of the reaction were complex, which brought out many considerable difficulties in separation and purification of the target product. Our work was the first in using the improved pyrogallol autoxidation method to test the antioxidant activities of these two flavonoids compounds in vitro and discovered that the compound 2 was much more effective as a free radical scavenger than the compound 1.

SYN

Synthesis of Rutin

The synthesis of rutin can be achieved according to the following three schemes. These schemes differ in the synthesis of ouercetin (the aglycone moiety of rutin).

Scheme 1: Kostanecki — et al. 1904 (33 ). Based upon the Claisen reaction between 2-hydroxy4, 6-dimethoxyacetophenone [l] and 3, 4-dimethoxybenzaldehyde [2] to give the intermediate [3] which upon treatment with HC1, cyclization occurs to give 5, 7, 3 , which upon treatment with F2SO4 enolisation occurs to give 5, 7, 1’3 ,’4 -tetramethoxyflavonol [6]. tion with HI affords quercetin [7].

Scheme 2: Robinson et al. 1926 (34 ). , ‘4 -tetramethoxyflavonone [4]. Oximination affords [5] Demethyla- — Condensation ofw-methoxypholoroacetophenone [I] with veratric acid anhydride [2] in the presence of the potassium salt of veratric acid to give the diarylester [3]. On hydrolysis with alcoholic KOH affords 5, 7-dihydroxy-3, /3 , ‘4 -trimethoxyf lavone [ 41 , which on demethylation with HI gives quercetin [5].

Scheme 3: Shakhova et al. 1962 (35), complete synthesis of rutin. W-methoxyphloroacetophenone [2] was condensed with 0-benzylvanillinic acid, anhydride [ 13 in triethylamine to give 5 , 7-dihydroxy-4 -benzyloxy-3, /3 -dimethoxyf lavone [3]. On treatment with AcOH-HC1 mixture gave 5, 7, ‘4 -trihydroxy-3,’3 -dimethoxyflavone [4]. Demethylation of the latter with HI yielded (about 802) quercetin [5]. Ouercetin potassium salt [6] was produced upon treating [5] with AcOK in ethanol. Levoglucosan [7] was acetylated with Ac20 in the presence of AcONa to give 2, 3, 4-triacetyllevoglucosan [8] which with TIC14 gave 1-chloro-2, 3, 4-triacetyl Dglucose [9]. L-rhamnose tetraacetate [lo] treated with TiBr4 in CHC13 gave 1-bromo-2, 3, I-triacetyl-L-rhamnose [ll]. [lo] + [11] heated with Hg (OAC)~ in C6H6 gave (53x) CC – acetochloro-f3-l-L-rhamnosido-6-D-glucose [12]. [12] was treated with AgOAc and acetylated with Ac20 to prodilce (68.703 B-heptaacet yl-f3-1-L-rhamnos ido-6-D-glucose [13]. This with 33% HBr in AcOH gave (61%) d – acetobromo-~-l-L-rhamnosido-6-D-glucose [14]. [14] and quercetin potassium salt [6] were dissolved in NH40H which was evaporated and treated with methanol andpurified over a chromatographic column packed with polycaprolactum resin to give rutin [151. 

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References

1. ^ Merck Index, 12th Edition, 8456
2. ^ Krewson CF, Naghski J (Nov 1952). “Some physical properties of rutin”. Journal of the American Pharmaceutical Association. 41 (11): 582–7. doi:10.1002/jps.3030411106. PMID 12999623.
3. ^ van der Watt E, Pretorius JC (2001). “Purification and identification of active antibacterial components in Carpobrotusedulis L.”. Journal of Ethnopharmacology. 76 (1): 87–91. doi:10.1016/S0378-8741(01)00197-0. PMID 11378287.
4. ^ [1] p. 280 Table 1
5. ^ [2] p.8 fig. 7
6. ^ quercitrinase on www.brenda-enzymes.org
7. ^ Tranchimand S, Brouant P, Iacazio G (Nov 2010). “The rutin catabolic pathway with special emphasis on quercetinase”. Biodegradation. 21 (6): 833–59. doi:10.1007/s10532-010-9359-7. PMID 20419500. S2CID 30101803.
8. ^ Jump up to:^a b Kreft S, Knapp M, Kreft I (Nov 1999). “Extraction of rutin from buckwheat (Fagopyrum esculentumMoench) seeds and determination by capillary electrophoresis”. Journal of Agricultural and Food Chemistry. 47 (11): 4649–52. doi:10.1021/jf990186p. PMID 10552865.
9. ^ Chang S, Tan C, Frankel EN, Barrett DM (Feb 2000). “Low-density lipoprotein antioxidant activity of phenolic compounds and polyphenol oxidase activity in selected clingstone peach cultivars”. Journal of Agricultural and Food Chemistry. 48 (2): 147–51. doi:10.1021/jf9904564. PMID 10691607.
10. ^ Malagutti AR, Zuin V, Cavalheiro ÉT, Henrique Mazo L (2006). “Determination of Rutin in Green Tea Infusions Using Square-Wave Voltammetry with a Rigid Carbon-Polyurethane Composite Electrode”. Electroanalysis. 18 (10): 1028–1034. doi:10.1002/elan.200603496.
11. ^ “foods in which the polyphenol Quercetin 3-O-rutinoside is found”. Phenol-Explorer v 3.6. June 2015.
12. ^ Jump up to:^a b c “Flavonoids”. Micronutrient Information Center, Linus Pauling Institute, Oregon State University, Corvallis, Oregon. November 2015. Retrieved 25 February 2018.
13. ^ Morling, J. R; Yeoh, S. E; Kolbach, D. N (November 2018). “Rutosides for treatment of post-thrombotic syndrome”. Cochrane Database of Systematic Reviews. 11 (11): CD005625. doi:10.1002/14651858.CD005625.pub4. PMC 6517027. PMID 30406640.
14. ^ Martinez-Zapata, M. J; Vernooij, R. W; Uriona Tuma, S. M; Stein, A. T; Moreno, R. M; Vargas, E; Capellà, D; Bonfill Cosp, X (2016). “Phlebotonics for venous insufficiency”. Cochrane Database of Systematic Reviews. 4: CD003229. doi:10.1002/14651858.CD003229.pub3. PMC 7173720. PMID 27048768.
15. ^ Yu X, Liu J, Wan J, Zhao L, Liu Y, Wei Y, Ouyang Z. Cloning, prokaryotic expression, and enzyme activity of a UDP-glucose flavonoid 3-o-glycosyltransferase from mulberry (Morus alba L.) leaves. Phcog Mag 2020;16:441-7

Names
IUPAC name3′,4′,5,7-Tetrahydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyloxy]flavone
Preferred IUPAC name(42S,43R,44S,45S,46R,72R,73R,74R,75R,76S)-13,14,25,27,43,44,45,73,74,75-Decahydroxy-76-methyl-24H-3,6-dioxa-2(2,3)-[1]benzopyrana-4(2,6),7(2)-bis(oxana)-1(1)-benzenaheptaphane-24-one
Other namesRutoside (INN) Phytomelin Sophorin Birutan Eldrin Birutan Forte Rutin trihydrate Globularicitrin Violaquercitrin Quercetin rutinoside
Identifiers
CAS Number	153-18-4
3D model (JSmol)	Interactive image
ChemSpider	4444362
DrugBank	DB01698
ECHA InfoCard	100.005.287
KEGG	C05625
PubChem CID	5280805
RTECS number	VM2975000
UNII	5G06TVY3R7
CompTox Dashboard (EPA)	DTXSID3022326
showInChI
showSMILES
Properties
Chemical formula	C27H30O16
Molar mass	610.521 g·mol−1
Appearance	Solid
Melting point	242 °C (468 °F; 515 K)
Solubility in water	12.5 mg/100 mL[1] 13 mg/100mL[2]
Pharmacology
ATC code	C05CA01 (WHO)
Hazards
NFPA 704 (fire diamond)	200
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
verify (what is  ?)
Infobox references

/////////Rutoside, RUTIN, рутозид , ルチン , روتوسيد , 芦丁 , C.I. 75730, NSC 9220,

CC1C(C(C(C(O1)OCC2C(C(C(C(O2)OC3=C(OC4=CC(=CC(=C4C3=O)O)O)C5=CC(=C(C=C5)O)O)O)O)O)O)O)O

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TRK-700

CAS 1463432-16-7C₁₄ H₂₄ N₄ O264.371-Propanone, 1-[4-(dimethylamino)-1-piperidinyl]-3-(1-methyl-1H-imidazol-2-yl)-

1-[4-(dimethylamino)piperidin-1-yl]-3-(1-methylimidazol-2-yl)propan-1-one

• 1-[4-(Dimethylamino)-1-piperidinyl]-3-(1-methyl-1H-imidazol-2-yl)-1-propanone

• OriginatorToray Industries
• ClassAnalgesics
• Mechanism of ActionUndefined mechanism

• Phase IIPostherpetic neuralgia
• PreclinicalPeripheral nervous system diseases

• 12 Sep 2018Pharmacodynamics data from a preclinical trial in Peripheral neuropathy presented at the 17^th World Congress on Pain (WCP-2018)
• 01 Jul 2017Toray Industries completes a phase II trial for Postherpetic neuralgia (In adults, In the elderly) in Japan (PO) (NCT02701374)
• 21 May 2017Toray Industries completes a phase I drug-drug interaction trial in Healthy volunteers in Japan (PO) (NCT03043248)

developed by Toray for treating neuropathic pain and investigating for fibromyalgia. In August 2021, this drug was reported to be in phase 1 clinical development.

PATENT

WO 2016136944

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016136944

(Reference Example 22) Synthesis of (E) -methyl 3- (1-methyl-1H-imidazol-2-yl) acrylate:
[Chemical 56]


　1-methyl-1H-imidazol-2-carbaldehyde (10.0 g, Methyl (triphenylphosphoranylidene) acetate (33.4 g, 99.9 mmol) was added to a solution of 90.8 mmol) in dichloromethane (240 mL) at room temperature, and the mixture was stirred for 16 hours and then concentrated under reduced pressure. The residue was washed with a mixed solvent of hexane / dichloromethane = 19/1, and the washing liquid was concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give (E) -methyl 3- (1-methyl-1H-imidazol-2-yl) acrylate as a white solid (11.9 g, 71. 6 mmol, 79%).
¹ H-NMR (400 MHz, CDCl ₃ ) δ: 3.76 (3H, s), 3.81 (3H, s), 6.82 (1H, d, J = 15.6 Hz), 6.98 (1H, brs), 7.16 (1H, brs), 7.53 (1H, d, J = 15.6Hz).
ESI-MS: m / z = 167 (M + H) ⁺ .

(Reference Example 27) Synthesis of 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propan-1-one:
[Chemical 61]


　(E) )-Methyl 3- (1-methyl-1H-imidazol-2-yl) acrylate (0.180 g, 1.08 mmol) in ethanol (4.0 mL) solution of palladium-carbon (10% wet, 15 mg) at room temperature In a hydrogen atmosphere, the mixture was stirred for 4 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. Methanol (1.0 mL) was added to the obtained residue at room temperature to dissolve it, and the mixture was cooled to 0 ° C. An aqueous sodium hydroxide solution (1.0 N, 1.19 mL, 1.19 mmol) was added to the reaction solution at 0 ° C., the mixture was stirred at room temperature for 2 hours, and then concentrated under reduced pressure. Chloroform (10.0 mL) was added to the obtained residue at room temperature to dissolve it. Add diisopropylethylamine (0.568 mL, 3.25 mmol), HBTU (0.616 g, 1.63 mmol) and 4- (dimethylamino) piperidine (0.125 g, 0.975 mmol) to the reaction solution at room temperature, and add the reaction solution. The mixture was stirred at the same temperature for 16 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with a 10% aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (NH silica gel, chloroform / methanol) and 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propane. -1-one (0.179 g, 0.68 mmol, 63%) was obtained as a colorless oil.
^{1 1} H-NMR (400 MHz, CDCl ₃) δ: 1.29-1.43 (2H, m), 1.80-1.88 (2H, m), 2.27 (6H, s), 2.29-2.38 (1H, m), 2.54-2.63 (1H, m), 2.88-3.04 ( 5H, m), 3.62 (3H, s), 3.98-4.05 (1H, m), 4.57-4.65 (1H, m), 6.79 (1H, d, J = 1.2 Hz), 6.91 (1H, d, J = 1.2 Hz).
ESI-MS: m / z = 265 (M + H) ⁺ .

(Comparative Example 1) Synthesis of 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propan-1-one hydrochloride:
[Chemical 66]


　1- (4- (Dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propan-1-one (1.50 g, 5.67 mmol) diethyl ether (60) A dioxane solution of hydrogen chloride (4.0 M, 3.69 mL, 14.8 mmol) was added to the (0.0 mL) solution at 0 ° C. The reaction mixture was stirred at the same temperature for 1 hour and then at room temperature for 30 minutes. The precipitated white solid was collected by filtration, washed with diethyl ether (100 mL), dried at room temperature for 36 hours, and then 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-). Imidazole-2-yl) propan-1-one hydrochloride (1.41 g, 4.18 mmol, 74%) (hereinafter, the compound of Comparative Example 1) was obtained as a white solid.
^{1 1} H-NMR (400 MHz, D ₂ O) δ: 1.53-1.80 (2H, m), 2.12-2.23 (2H, m), 2.68-2.80 (1H, m), 2.88 (6H, s), 3.01- 3.08 (2H, m), 3.15-3.26 (3H, m), 3.47-3.58 (1H, m), 3.84 (3H, s), 4.08-4.16 (1H, m), 4.50-4.59 (1H, m), 7.29-7.33 (2H, m).
ESI-MS; 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) as propan-1-one : m / z = 265 (M + H) ⁺ .

(Comparative Example 2) Synthesis of 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propan-1-one sulfate monohydrate:
[Chemical 67]


　1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propan-1-one (6.72 g, 25.4 mmol) Concentrated sulfuric acid (2.49 g, 25.4 mmol), water (1.83 g, 102 mmol) and 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl) in a DMSO (100 mL) solution. Seed crystals (50 mg, 0.13 mmol) of -1H-imidazol-2-yl) propan-1-one sulfate monohydrate were added at 80 ° C. The reaction was stirred at the same temperature for 2.5 hours, at 50 ° C. for 2.5 hours and at room temperature for 15 hours. The precipitated white solid was collected by filtration, washed successively with DMSO (20 mL) and methyl ethyl ketone (40 mL), dried at room temperature, and then 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl). -1H-imidazol-2-yl) propan-1-one sulfate monohydrate (8.42 g, 22.1 mmol, 87%) (hereinafter, the compound of Comparative Example 2) was obtained as white crystals.
^{1 1} H-NMR (400 MHz, DMSO-d ₆₎) δ: 1.36 (1H, m), 1.58 (1H, m), 1.95 (2H, br), 2.44-2.57 (1H, m), 2.65 (6H, s), 2.74-2.88 (4H, m), 3.00 (1H, t, J = 12.0 Hz), 3.22 (1H, m), 3.61 (3H, s), 4.02 (1H, d, J = 14.0 Hz), 4.47 (1H, d, J = 12.8 Hz), 6.87 (1H, d, J = 1.2 Hz), 7.11 (1H, d, J = 1.2 Hz).
ESI-MS; 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-) As 1H-imidazol-2-yl) propan-1-one: m / z = 265 (M + H) ⁺ .

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PATENT

WO-2021153744

PATENT

WO-2021153743

Novel crystalline polymorphic form of 1-(4-(dimethylamino) piperidin-1-yl)-3-(1-methyl-1H-imidazol-2-yl)propan-1-one, useful as an analgesic in treating neuropathic pain and/or fibromyalgia.Pain is an experience with unpleasant sensations and emotions that occurs when or may cause tissue damage. Pain is mainly classified into nociceptive pain, neuropathic pain or psychogenic pain according to its cause. In addition, fibromyalgia is known as pain of unknown cause. 
　Neuropathic pain is pathological pain caused by dysfunction of the peripheral or central nervous system itself, and is caused by direct damage or compression of nervous tissue even though nociceptors are not stimulated. It refers to the pain that occurs. As a therapeutic agent for neuropathic pain, an anticonvulsant, an antidepressant, anxiolytic, or an antiepileptic drug such as gabapentin or pregabalin is used. 
　Fibromyalgia is a disease in which systemic pain is the main symptom and neuropsychiatric symptoms and autonomic nervous system symptoms are secondary symptoms. Pregabalin approved in the United States and Japan, duloxetine and milnacipran approved in the United States are mainly used as therapeutic agents for fibromyalgia, and non-approved agents for fibromyalgia are not approved. It has also been used for steroidal anti-inflammatory agents, opioid compounds, antidepressants, anticonvulsants and antiepileptic drugs. However, the therapeutic effects of non-steroidal anti-inflammatory drugs and opioid compounds are generally considered to be low (Non-Patent Document 1). 
　On the other hand, Patent Document 1 discloses that certain substituted piperidins have cardiotonic activity, and Patent Document 2 discloses that an imidazole derivative exhibits an FXa inhibitory effect. Patent Document 3 suggests that the substituted piperidins may have a medicinal effect on overweight or obesity, and Patent Documents 4 to 6 and Non-Patent Document 2 indicate that the imidazole derivative has an analgesic effect. It is disclosed. 
　In addition, the quality of pharmaceutical products needs to be maintained over a long period of time such as distribution and storage, and the compound as an active ingredient is required to have high chemical and physical stability. Therefore, as the active ingredient of a pharmaceutical product, a crystal that can be expected to have higher stability than an amorphous substance is generally adopted. Further, if crystals are obtained, a purification effect due to recrystallization during production can be expected. Further, it is preferable to have low hygroscopicity from the viewpoint of maintaining stability and handling during manufacturing, storage, formulation and analysis of the drug substance. In addition, since a drug needs to be dissolved in the digestive tract in order to exhibit its medicinal effect, it is preferable that the drug has excellent solubility, which is a physical property contrary to stability. 
　In order to obtain crystals of a compound that is an active ingredient of a pharmaceutical product, it is necessary to study various conditions for precipitating crystals from the solution. It is common to carry out crystallization under the condition of being dissolved in.

Patent documents

Patent Document 1: French Patent Invention No. 2567885
Patent Document 2: Japanese Patent Application Laid-Open No. 2006-0083664
Patent Document 3: International Publication No. 2003/031432
Patent Document 4: International Publication No. 2013/147160
Patent Document 5: International Publication No. 2015/046403
Patent Document 6: International Publication No. 2016/136944

Non-patent literature

Non-Patent Document 1: Okifuji et al., Pain and Therapy, 2013, Volume 2, p. 87-104
Non-Patent Document 2: Takahashi et al., Toxicological Pathology, 2019, Vol. 47. p. 494-503

Compound (I) was synthesized by the method described in the following reference example. For the compounds used in the synthesis of the reference example compounds for which the synthesis method is not described, commercially available compounds were used. 
(Reference Example 4) Synthesis of amorphous compound (I):
[Chemical formula 2] 2 of

crude ethyl 3- (1-methyl-1H-imidazol-2-yl) propanol (5.00 g, 27.4 mmol) Aqueous sodium hydroxide solution (1.0N, 30.2 mL, 30.2 mmol) was added to a solution of -propanol (55 mL) at 0 ° C., and the mixture was stirred at room temperature for 12 hours. 2-Propanol (220 mL) was added to the reaction solution at room temperature, and crude 4- (dimethylamino) piperidine (3.17 g, 24.7 mmol) and DMT-MM (8.35 g, 30.2 mmol) were added at room temperature to react. The liquid was stirred at the same temperature for 3 hours. A 10% aqueous sodium chloride solution and a 1.0N aqueous sodium hydroxide solution were added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound (I) (6.98 g) as an amorphous substance.
^{1 1} H-NMR (400 MHz, CDCl ₃ ) δ: 1.29-1.43 (2H, m), 1.80-1.88 (2H, m), 2.27 (6H, s), 2.29-2.38 (1H, m), 2.54-2.63 (1H, m), 2.88-3.04 (5H, m), 3.62 (3H, s), 3.98-4.05 (1H, m), 4.57-4.65 (1H, m), 6.79 (1H, d, J = 1.2 Hz) ), 6.91 (1H, d, J = 1.2 Hz).
ESI-MS: m / z = 265 (M + H) ⁺ .
(Reference Example 5) Synthesis of crude 4- (dimethylamino) piperidine:
[Chemical

formula 3] 1-benzyloxycarbonyl-4- (dimethylamino) piperidine (20.1 g, 77.0 mmol) in methanol (154.0 mL) Palladium-carbon (10% wet, 2.01 g) was added thereto, and the mixture was stirred at room temperature for 19 hours under a hydrogen atmosphere. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to give a crude product of 4- (dimethylamino) piperidine (9.86 g).
(Reference Example 6) Synthesis of crude ethyl 3- (1-methyl-1H-imidazol-2-yl) propanoate:
[Chemical

formula 4] Sodium hydride (55%, 4.36 g, 100 mmol) aqueous solution and tetrahydrofuran (150 mL) To the mixture was added triethylphosphonoacetate (19.1 mL, 95.0 mmol) at 0 ° C. After stirring the reaction solution for 20 minutes, a solution of 1-methyl-1H-imidazol-2-carbaldehyde (10.0 g, 91.0 mmol) in tetrahydrofuran (150 mL) was added at 0 ° C., and then ethanol (30 mL) was added in the same manner. The mixture was added at temperature and stirred at room temperature for 2 hours. A 10% aqueous sodium chloride solution was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, chloroform / methanol). After adding methanol (310 mL) to the residue, palladium-carbon (10% wet, 1.40 g) was added, and the mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain a crude product (14.2 g) of ethyl 3- (1-methyl-1H-imidazol-2-yl) propanoate.
(Reference Example 7) Synthesis of 1-benzyloxycarbonyl-4- (dimethylamino) piperidine:
[Chemical

formula 5] dichloromethane (55.7 mL) of 1-benzyloxycarbonyl-4-oxopiperidine (13.0 g, 55.7 mmol) ) Solution of dimethylamine in tetrahydrofuran (2.0 M, 34.8 mL, 69.7 mmol), acetic acid (0.32 mL, 5.6 mmol) and sodium triacetoxyborohydride (4.8 g, 22.6 mmol). Added at ° C. After stirring the reaction solution at the same temperature for 30 minutes, sodium triacetoxyborohydride (4.8 g, 22.6 mmol) was added at 0 ° C. The reaction mixture was stirred at the same temperature for 30 minutes, sodium triacetoxyborohydride (8.1 g, 38.2 mmol) was added at 0 ° C., and the mixture was stirred at room temperature for 12 hours. The reaction solution was cooled to 0 ° C. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, n-hexane / ethyl acetate) and then again by flash chromatography (silica gel, chloroform / methanol) to obtain 1-benzyloxycarbonyl-4- (dimethylamino) piperidine (dimethylamino) piperidine. 13.6 g, 51.8 mmol, 93%) was obtained as a colorless oil.
^{1 1} H-NMR (400 MHz, CDCl ₃) δ: 1.34-1.46 (2H, m), 1.78-1.86 (2H, m), 2.28 (6H, s), 2.29-2.34 (1H, m), 2.75-2.85 (2H, m), 4.14-4.28 ( 2H, m), 5.12 (2H, s), 7.29-7.36 (5H, m).
ESI-MS: m / z = 263 (M + H) ⁺ .
(Reference Example 8) Synthesis of 1-benzyloxycarbonyl-4-oxopiperidine:
[Chemical

formula 6] Hydrochloride (130 mL) and water (130 mL) of 4-piperidinone hydrochloride monohydrate (10.0 g, 65.1 mmol) Sodium carbonate (13.8 g, 130.2 mmol) and benzyl chloroformate (8.79 mL, 61.8 mmol) were added to the mixed solution with and at 0 ° C., and the mixture was stirred at room temperature for 3 hours. The reaction mixture was extracted with ethyl acetate. The organic layer was washed with 10% aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, n-hexane / ethyl acetate) to give 1-benzyloxycarbonyl-4-oxopiperidine (13.1 g, 56.2 mmol, 86%) as a colorless oil.
^{1 1} H-NMR (400 MHz, CDCl ₃ ) δ: 2.42-2.50 (4H, m), 3.78-3.82 (4H, m), 5.18 (2H, s), 7.32-7.38 (5H, m).
(Example 1) Production of A-type crystal of
compound (I): Amorphous compound (6.98 g) of compound (I) prepared in Reference Example 4 is purified and concentrated with chloroform / methanol by silica gel column chromatography. After that, the wall surface of the flask was rubbed with a spartel and mechanical stimulation was applied to obtain A-type crystals of compound (I) as a powder. For the obtained crystals, measurement of powder X-ray diffraction using a powder X-ray diffractometer (Rigaku Co., Ltd .; 2200 / RINT ultima + PC) and TG-DTA using a TG-DTA device (Rigaku Co., Ltd .; TG8120) Was done. The results of these measurements are shown in FIGS. 1 and 2.
Diffraction angle 2θ: 5.9, 16.5, 17.7, 20.8, 26.7 °
Endothermic peak: 55 ° C

PATENT

WO2013147160

Example 1 Synthesis of 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propan-1-one:
[Chemical 27]

(E) )-Methyl 3- (1-methyl-1H-imidazol-2-yl) acrylate (0.180 g, 1.08 mmol) in ethanol (4.0 mL) solution of palladium-carbon (10% wet, 15 mg) at room temperature In a hydrogen atmosphere, the mixture was stirred for 4 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. Methanol (1.0 mL) was added to the obtained residue at room temperature to dissolve it, and the mixture was cooled to 0 ° C. An aqueous sodium hydroxide solution (1.0 N, 1.19 mL, 1.19 mmol) was added to the reaction solution at 0 ° C., the mixture was stirred at room temperature for 2 hours, and then concentrated under reduced pressure. Chloroform (10.0 mL) was added to the obtained residue at room temperature to dissolve it. Add diisopropylethylamine (0.568 mL, 3.25 mmol), HBTU (0.616 g, 1.63 mmol) and 4- (dimethylamino) piperidine (0.125 g, 0.975 mmol) to the reaction solution at room temperature, and add the reaction solution. The mixture was stirred at the same temperature for 16 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with a 10% aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (NH silica gel, chloroform / methanol) and 1- (4- (dimethylamino) piperidine-1-yl) -3- (1-methyl-1H-imidazol-2-yl) propan- 1-one (0.179 g, 0.68 mmol, 63%) (hereinafter, the compound of Example 1) was obtained as a colorless oil.
^{1 1} H-NMR (400 MHz, CDCl ₃) δ: 1.29-1.43 (2H, m), 1.80-1.88 (2H, m), 2.27 (6H, s), 2.29-2.38 (1H, m), 2.54-2.63 (1H, m), 2.88-3.04 ( 5H, m), 3.62 (3H, s), 3.98-4.05 (1H, m), 4.57-4.65 (1H, m), 6.79 (1H, d, J = 1.2 Hz), 6.91 (1H, d, J = 1.2 Hz).
ESI-MS: m / z = 265 (M + H) ⁺ .

//////////TRK-700, phase 1, neuropathic pain, fibromyalgia, toray

O=C(CCc1nccn1C)N1CCC(CC1)N(C)C

Bemiparin

• AVE 5026
• Adomiparin
• Ardeparin
• Arteven
• Bemiparin

• CY 216
• CY 222
• Centaxarin
• Certoparin
• Clevarin
• Clivarin
• Clivarine
• Dalteparin
• Deligoparin
• F 202
• FR 860
• Fluxum
• Fragmin A
• Fragmin B
• Fraxiparin
• Gammaparin
• H 5284
• H 9399
• Hapacarin
• Heparin subcutan
• Heparin sulfate
• Heparinic acid
• Heparins
• KB 101
• Leparan
• LipoHep Forte
• Livaracine
• M 118
• M 118REH
• M 402
• M 402 (heparin)
• Mono-embolex
• Multiparin
• Nadroparin
• Nadroparine
• Necuparanib
• Novoheparin
• OP 386
• OP 622
• Octaparin
• Pabyrn
• Parnaparin
• Parvoparin
• Reviparin
• Sandoparin
• Semuloparin
• Subeparin
• Sublingula
• Tafoxiparin
• Tinzaparin
• Triofiban
• Vetren
• Vitrum AB
• α-Heparin

cas 91449-79-5

Bemiparin (trade names Ivor and Zibor, among others) is an antithrombotic and belongs to the group of low molecular weight heparins (LMWH).^[1]

Bemiparin is an ultra-low molecular weight heparin (ultra-LMWH) used to prevent thromboembolism following surgery and extracorporeal clotting during dialysis.

Rovi and Archimedes (a wholly owned subsidiary of ProStrakan), have developed and launched bemiparin, a Factor Xa inhibitor for the injectable treatment and prevention of thrombosis.

low or very low molecular weight heparins (eg bemiparin sodium) with a high anti-factor Xa activity for the treatment of deep vein thrombosis.

Bemiparin is an antithrombotic and belongs to the group of drugs known as the low molecular weight heparins (LMWH). Like semuloparin, bemiparin is classified as an ultra-LMH because of its low mean molecular mass of 3600 daltons, which is a unique property of this class ¹. These heparins have lower anti-thrombin activity than the traditional low molecular weight heparins and act mainly on factor-Xa, reducing the risk of bleeding due to selectivity for this specific clotting factor. Interestingly, current research is underway for the potential benefit of bemiparin in the treatment of tumors and diabetic foot ulcers ^12,1.

Laboratorios Farmaceuticos Rovi has developed and launched Enoxaparina Rovi, a biosimilar version of enoxaparin sodium, an injectable low-molecular-weight fraction of heparin, for the prophylaxis of venous thromboembolism.

PATENT

WO2018015463

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2018015463

claiming a method for analyzing glycosaminoglycans, heparins and their derivatives in a compound comprising a monosaccharide residues present in heparin (eg bemiparin sodium) chains by identification and relative quantification of its characteristic signals by1H NMR one-dimensional nuclear magnetic resonance and/or 1H-13C HSQC two-dimensional nuclear magnetic resonance, using dimethylmalonic acid as internal reference

PATENT

CN-110092848

https://patents.google.com/patent/CN110092848A/enEmbodiment 1Experimental raw used and instrument are as follows in embodiment 1:Refined heparin sodium (ZH160712 quality of lot meets CP2015), benzethonium chloride, purified water, 40% (W/V) trimethoxy Base methanolic ammonium hydroxide, methylene chloride, methanol, 10% (W/V) sodium acetate methanol solution, 30% hydrogen peroxide, medicinal second Alcohol, sodium chloride, glass reaction pot (5000ml) three-necked flask 500ml, digital display heat-collecting magnetic stirring device, beaker, freeze dryer (on Hai Dongfulong) etc..A kind of preparation method of Bemiparin sodium of the present invention, the following steps are included:1. at salt1.1 weigh, dissolution, react1.1.1 the refined heparin sodium for weighing 10g is poured into tank, and the purified water of 100ml is added into reactor tank, is stirred to molten Solution is complete.1.1.2 25g benzethonium chloride is added in beaker, 125ml purified water stirring and dissolving is added.1.1.3 benzethonium chloride solution is added slowly with stirring in the heparin sodium aqua in reactor tank, time for adding 4.5h controls 35 DEG C of feed liquid temperature, continues stirring 2 hours, stops stirring and stands 2 hours, then as far as possible by supernatant liquid Removing.1.2 washings, centrifugation, drying:1.2.1 300ml purified water is added into residue precipitating suspended matter to wash in three times, then starts to wash for the first time, 20 DEG C of feed liquid temperature of control is stirred 1 hour, is stopped stirring and is stood 2 hours, repeats the above operation twice.1.2.2 supernatant liquid is removed, filters and be washed with water under stirring, record slurry amount, collect sediment.1.2.3 final gained sediment is uniformly divided in stainless steel disc, is transferred in heated-air circulation oven, adjust temperature 40 DEG C of degree, dry 6h crushes solid with Universalpulverizer after then 60 DEG C of dry range estimations are not glued to solid, smashed solid Body continues to be transferred in heated-air circulation oven, until loss on drying≤2.0%.Rewinding obtains heparin-benzyl rope ammonium salt about 32g, does Dry weightless 1.5%.2. degradation2.1 weighingBy above-mentioned 30g heparin-benzyl rope ammonium salt in 500ml three-necked flask, the methylene chloride of 150ml is added into reactor tank It is added in three-necked flask.2.2 dissolutions: three-necked flask is put into digital display heat-collecting magnetic stirring device, is heated to 33 DEG C and is stirred to having dissolved Entirely.2.3 degradations: being added 40% (W/V) trimethoxy methanolic ammonium hydroxide of 20.4ml in Xiang Shangshu solution, puts down Respectively 4 additions, it is for 24 hours that interval time is added every time.It after the 4th is added, then reacts for 24 hours, amounts to reaction 96h, during reaction Maintain 34 DEG C of temperature.2.4 terminate reaction: above-mentioned reaction solution being cooled to 20 DEG C, 180ml10% (W/V) sodium acetate methanol is added thereto Solution stirs 30min, filters to obtain its precipitating.2.5 washings: washing above-mentioned sediment with 300ml methanol solution, dry bemiparin crude product about 9g.3. purification3.1 will be above-mentioned dry that 9g bemiparin crude product pours into tank, and the purified water of 90ml, stirring are added into reactor tank It is complete to dissolution.3.2 adjust material liquid pH 9.5 with 20% sodium hydroxide solution.0.54ml hydrogen peroxide is added to be stirred to react at 20 DEG C 7.5 hours, through 0.22 μm of micro porous filtration.3.3 1.8g sodium chloride is added into feed liquid, then uses 4mol/L hydrochloric acid flavouring liquid pH to 6.5, is added into feed liquid 450ml medicinal alcohol stops stirring after stirring 30 minutes, places 4 hours.3.4 take supernatant away, and 90ml purified water is added, and stirring adjusts PH6.5 to dissolving completely, through 0.22 μm of micro porous filtration, Sabot freeze-drying.After 3.5 freeze-drying 36h, collection material weighing 7g.Three, the primary quality measure statistics of gained bemiparin

Embodiment 2Experimental raw used and instrument are as follows in embodiment 1:Refined heparin sodium (ZH180912 quality of lot meets CP2015), benzethonium chloride, purified water, 40% (W/V) trimethoxy Base methanolic ammonium hydroxide, methylene chloride, methanol, 10% (W/V) sodium acetate methanol solution, 30% hydrogen peroxide, medicinal second Alcohol, sodium chloride, glass reaction pot (10000ml, 30000L), three-necked flask 500ml, digital display heat-collecting magnetic stirring device, beaker, Freeze dryer (Shanghai Dong Fulong) etc..A kind of preparation method of Bemiparin sodium of the present invention, the following steps are included: 1. one-tenth salt1.1 weigh, dissolution, react1.1.1 the refined heparin sodium for weighing 500g is poured into tank, the purified water of 5000ml is added into reactor tank, stirring is extremely Dissolution is complete.1.1.2 1250g benzethonium chloride is added in beaker, 6300ml purified water stirring and dissolving is added.1.1.3 benzethonium chloride solution is added slowly with stirring in the heparin sodium aqua in reactor tank, time for adding 5h controls 35 DEG C of feed liquid temperature, continues stirring 2 hours, stops stirring and stands 2 hours, then as far as possible by supernatant liquid It removes.1.2 washings, centrifugation, drying:1.2.1 5000ml purified water is added into residue precipitating suspended matter to wash in three times, then starts to wash for the first time, 30 DEG C of feed liquid temperature of control is stirred 1 hour, is stopped stirring and is stood 2 hours, repeats the above operation twice.1.2.2 supernatant liquid is removed, filters and be washed with water under stirring, record slurry amount, collect sediment.1.2.3 final gained sediment is uniformly divided in stainless steel disc, is transferred in heated-air circulation oven, adjust temperature 45 DEG C of degree, dry 6h crushes solid with Universalpulverizer after then 70 DEG C of dry range estimations are not glued to solid, smashed solid Body continues to be transferred in heated-air circulation oven, until loss on drying≤2.0%.Rewinding obtains heparin-benzyl rope ammonium salt about 1505g, Loss on drying 1.0%.2. degradation2.1 weighingBy above-mentioned 1500g heparin-benzyl rope ammonium salt in 30L glass reaction kettle, the methylene chloride of 7500ml is added thereto.2.2 dissolutions: leading to hot water for its interlayer, is heated to 33~36 DEG C and stirs complete to dissolving.2.3 degradations: being added 40% (W/V) trimethoxy methanolic ammonium hydroxide of 1020ml in Xiang Shangshu solution, puts down Respectively 4 additions, it is for 24 hours that interval time is added every time.It after the 4th is added, then reacts for 24 hours, amounts to reaction 96h, during reaction Maintain 35 DEG C of temperature.2.4 terminate reaction: above-mentioned reaction solution being cooled to 20 DEG C, 9000ml10% (W/V) sodium acetate first is added thereto Alcoholic solution stirs 30min, filters to obtain its precipitating.2.5 washings: washing above-mentioned sediment with 15000ml methanol solution, dry bemiparin crude product about 400g.3. purification3.1 will be above-mentioned dry that 400g bemiparin crude product pours into tank, and the purified water of 4000ml is added into reactor tank, Stirring is complete to dissolving.3.2 adjust material liquid pH 9.5 with 20% sodium hydroxide solution.24ml hydrogen peroxide is added, and at 30 DEG C to be stirred to react 7 small When, through 0.22 μm of micro porous filtration.3.3 8g sodium chloride is added into feed liquid, then uses 4mol/L hydrochloric acid flavouring liquid pH to 6.5, is added into feed liquid 20000ml medicinal alcohol stops stirring after stirring 30 minutes, places 4 hours.3.4 take supernatant away, and 4000ml purified water is added, and stirring adjusts PH6.5, through 0.22 μm of micropore mistake to dissolving completely Filter, sabot freeze-drying.After 3.5 freeze-drying 36h, collection material weighing 350g.Three, the primary quality measure statistics of gained bemiparin

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PATENT

WO-2021152192

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=9D96E01E1CE8B8107A83A95B4B344DD3.wapp2nC?docId=WO2021152192&tab=PCTDESCRIPTION
Use of a composition comprising low or very low molecular weight heparins (eg bemiparin sodium) with a high anti-factor Xa activity for the treatment of deep vein thrombosis. 

Heparin belongs to the glycosaminoglycan family and is a polysaccharide of animal origin, which is extracted from the intestine or lungs of mammals (cow, lamb, pig) and is used in human therapies for the prevention and treatment of thromboembolic diseases . It is well known that the use of heparin is accompanied by very annoying bleeding effects and its daily administration, three subcutaneous or intravenous injections, constitutes a very considerable inconvenience.

During the course of the last few years, different chemical methods have been used to depolymerize heparin, such as:

– treatment with sodium nitrite in an acid medium,

– alkaline treatment of asters,

– use of free radicals generated in the presence of hydrogen peroxide,

– treatment of a quaternary ammonium salt of heparin in a non-aqueous medium with a strong base according to a beta elimination mechanism.

These methods make it possible to obtain, with variable yields, mixtures of heparin fragments in which the average molecular weight and anticoagulant activity vary according to the procedure and operating conditions. Low molecular weight heparins (LMWH) described in the state of the art or commercialized are obtained according to different depolymerization procedures. Their average molecular weights (Mw) are in the range of 3,600 and 7,500 Daltons.

It is now recognized that the antithrombotic activity of LMWH is mainly due to its ability to activate antithrombin III, a plasma protein and potent inhibitor of activated factor X and thrombin. In this way, it is possible to measure the antithrombotic activity of heparin by means of specific tests to determine the inhibition of these factors.

Research carried out by different authors shows that heparin fragments or oligosaccharides, with short chains of average molecular weight <4,800 Daltons, have a selective action on activated factor X and not on thrombin, in determinations using methods of the Pharmacopoeia. .

It has been found that if very low molecular weight fragments are required that have strong anti-factor Xa activity, it is preferable to use a selective depolymerization technique in non-aqueous medium, as described in US patent 9,981,955, which respects the antithrombin III binding site.

The document EP 1070503 A1 describes the controlled depolymerization of heparin using a process in a non-aqueous medium that makes it possible to obtain a family of LMWH that are obtained enriched in low molecular weight oligosaccharides that have a high anti-factor Xa activity and a low anti-factor lia activity, and which can be represented by the general formula:

in which:

n can vary between 1 and 12,

Ri = H or S0 ₃ Na,

R ₂ = SOsNao COCH ₃ ,

Said very low molecular weight heparin is obtained by selective depolymerization of heparin in a non-aqueous medium according to a beta elimination procedure.

Medical uses

Bemiparin is used for the prevention of thromboembolism after surgery, and to prevent blood clotting in the extracorporeal circuit in haemodialysis.^[2]

Contraindications

The medication is contraindicated in patients with a history of heparin-induced thrombocytopenia with or without disseminated intravascular coagulation; acute bleeding or risk of bleeding; injury or surgery of the central nervous system, eyes or ears; severe liver or pancreas impairment; and acute or subacute bacterial endocarditis.^[2]

Interactions

No interaction studies have been conducted. Drugs that are expected to increase the risk of bleeding in combination with bemiparin include other anticoagulants, aspirin and other NSAIDs, antiplatelet drugs, and corticosteroids.^[2]

Chemistry

Like semuloparin, bemiparin is classified as an ultra-LMWH because of its low molecular mass of 3600 g/mol on average.^[3] (Enoxaparin has 4500 g/mol.) These heparins have lower anti-thrombin activity than classical LMWHs and act mainly on factor Xa, reducing the risk of bleeding.^[4]

References

1. ^ Chapman TM, Goa KL (2003). “Bemiparin: a review of its use in the prevention of venous thromboembolism and treatment of deep vein thrombosis”. Drugs. 63 (21): 2357–77. doi:10.2165/00003495-200363210-00009. PMID 14524738.
2. ^ Jump up to:^a b c Austria-Codex (in German). Vienna: Österreichischer Apothekerverlag. 2018. Ivor 2500 IE Anti-Xa/0,2 ml Injektionslösung in Fertigspritzen.
3. ^ Planès A (September 2003). “Review of bemiparin sodium–a new second-generation low molecular weight heparin and its applications in venous thromboembolism”. Expert Opinion on Pharmacotherapy. 4 (9): 1551–61. doi:10.1517/14656566.4.9.1551. PMID 12943485. S2CID 13566575.
4. ^ Jeske WP, Hoppensteadt D, Gray A, Walenga JM, Cunanan J, Myers L, Fareed J, Bayol A, Rigal H, Viskov C (October 2011). “A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin”. Thrombosis Research. 128 (4): 361–7. doi:10.1016/j.thromres.2011.03.001. PMID 21458847.

External links

• bemiparin at the US National Library of Medicine Medical Subject Headings (MeSH)

1. Chapman TM, Goa KL: Bemiparin: a review of its use in the prevention of venous thromboembolism and treatment of deep vein thrombosis. Drugs. 2003;63(21):2357-77. [Article]
2. Planes A: Review of bemiparin sodium–a new second-generation low molecular weight heparin and its applications in venous thromboembolism. Expert Opin Pharmacother. 2003 Sep;4(9):1551-61. [Article]
3. Jeske WP, Hoppensteadt D, Gray A, Walenga JM, Cunanan J, Myers L, Fareed J, Bayol A, Rigal H, Viskov C: A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin. Thromb Res. 2011 Oct;128(4):361-7. doi: 10.1016/j.thromres.2011.03.001. Epub 2011 Apr 2. [Article]
4. Sanchez-Ferrer CF: Bemiparin: pharmacological profile. Drugs. 2010 Dec 14;70 Suppl 2:19-23. doi: 10.2165/1158581-S0-000000000-00000. [Article]
5. Hoffman M, Monroe DM: Coagulation 2006: a modern view of hemostasis. Hematol Oncol Clin North Am. 2007 Feb;21(1):1-11. doi: 10.1016/j.hoc.2006.11.004. [Article]
6. Antonijoan RM, Rico S, Martinez-Gonzalez J, Borrell M, Valcarcel D, Fontcuberta J, Barbanoj MJ: Comparative pharmacodynamic time-course of bemiparin and enoxaparin in healthy volunteers. Int J Clin Pharmacol Ther. 2009 Dec;47(12):726-32. [Article]
7. Irish Medicines Board: Bemiparin [Link]
8. Hibor-Bemiparin Sodium [Link]
9. Zibor 2,500 IU Solution for Injection [Link]
10. Injectable drugs guide [Link]
11. Thrombosis Advisors- Factor Xa inhibitor [Link]
12. Anti-tumor effects of bemiparin in HepG2 and MIA PaCa-2 cells [Link]
13. Bemiparin, an effective and safe low molecular weight heparin: a review [Link]
14. Bemiparin sodium [Link]

Patent 

Publication numberPriority datePublication dateAssigneeTitleUS4981955A *1988-06-281991-01-01Lopez Lorenzo LDepolymerization method of heparinEP0293539B1 *1987-01-051994-06-08Laboratorios Farmaceuticos Rovi, S.A.Process for the depolymerization of heparin for obtaining heparin with a low molecular weight and having an antithrombotic activityCN1379781A *1999-10-222002-11-13阿文蒂斯药物股份有限公司Novel oligosaccharides, preparation method and pharmaceutical composition containing sameCN102399306A *2010-09-092012-04-04上海喜恩医药科技发展有限公司Preparation method of heparin-derived polysaccharide mixtureCN105693886A *2016-04-192016-06-22常州市蓝勖化工有限公司Preparation method of heparin sodiumCN106467577A *2015-08-212017-03-01苏州融析生物科技有限公司A kind of pulmonis Bovis seu Bubali Enoxaparin Sodium and preparation method and applicationCN106977627A *2017-05-162017-07-25苏州二叶制药有限公司A kind of Enoxaparin production method of sodiumCN109575156A *2018-11-052019-04-05上海宝维医药技术有限公司A kind of purification process of low molecular weight heparinFamily To Family Citations

////////////Bemiparin sodium, Bemiparin 

ONO-2910

CAS 2410177-35-2

3- [2-[(E) -5- [3- (benzenesulfonamide) phenyl] penta-4-enoxy] phenyl] propanoic acid

3- [2-[(E) -5- [3- (benzenesulfonamido) phenyl] penta-4-enoxy] phenyl] propanoic acidC₂₆ H₂₇ N O₅ S465.56Benzenepropanoic acid, 2-[[(4E)-5-[3-[(phenylsulfonyl)amino]phenyl]-4-penten-1-yl]oxy]-

ONO Pharmaceuticals is developing ONO-2910 , the lead from a program of novel transient receptor potential cation channel 4/5 inhibitors, for treating peripheral neuropathy. In April 2021, a phase II trial in patients with diabetic polyneuropathy was initiated.

PATENT

CN112513011-BENZENE DERIVATIVE

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PATENT

WO-2021153690

Novel crystalline forms of 3-[2-[(E)-5-[3-(benzenesulfonamide) phenyl] penta-4-enoxy] phenyl] propanoic acid act as neuroprotective, useful for treating neurological disorders eg chronic inflammatory demyelinating polyneuritis, Guillain-Barre syndrome and allergic angiitis.Example 1:
Sulfuric acid (0.26 mL) is added to a solution of isopropyl 3- (2-hydroxyphenyl) propanoate 3,4-dihydrocoumarin (50.0 g) in isopropyl alcohol (500 mL), and the reaction mixture is mixed at room temperature for 2 hours. Stirred. The reaction mixture was concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate. The mixture was washed with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure to give the title compound (73.2 g) having the following physical properties.
^{1 1} H-NMR (CDCl ₃ ): δ 1.20, 2.66-2.70, 2.87-2.91, 4.95-5.08, 6.86-6.91, 7.06-7.15, 7.35.

Example 2: Isopropyl 3- (2- (pent-4-in-1-yloxy) phenyl) propanoate In a　solution of the compound (3.00 g) prepared in Example 1 in N, N-dimethylacetamide (25 mL) at room temperature. Cesium carbonate (9.39 g) was added at the same temperature, and the mixture was stirred at the same temperature for 15 minutes. 5-Chloro-1-pentyne (CAS Registry Number: 14267-92-6) (1.63 g) was added to the reaction solution at room temperature, and the mixture was stirred at 60 ° C. for 3 hours. Water was added to the reaction solution, and the mixture was extracted with diethyl ether. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 0 → 5: 1) to give the title compound (2.40 g) having the following physical property values.
HPLC retention time (minutes): 1.13.Example 3: Isopropyl (E) -3- (2-((5- (4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) penta-4-en-1-yl) Il) Oxy) Phenyl) Propanoate In
a heptane (2 mL) solution of the compound (1.00 g) prepared in Example 2, 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1. 17 g) and 4-dimethylaminobenzoic acid (60.2 mg) were added, and the mixture was stirred at 100 ° C. for 4 hours. The reaction solution was cooled to room temperature and then concentrated. The obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 20: 1 → 4: 1) to give the title compound (503 mg) having the following physical characteristics.
HPLC retention time (minutes): 1.38.Example 3 (1):
Pyridine (0.95 mL), N, N-dimethyl in a solution of N- (3-bromophenyl) benzenesulfonamide 3-bromoaniline (1.02 g) in dichloromethane (20 mL) at 0 ° C. Aminopyridine (hereinafter abbreviated as DMAP) (72.4 mg) and benzenesulfonyl chloride (1.10 g) were added, and the mixture was stirred at room temperature for 2 hours. After concentrating the reaction solution, the obtained residue is purified by silica gel column chromatography (hexane: ethyl acetate = 9: 1 → 2: 1) to give the title compound (1.96 g) having the following physical properties. rice field.
HPLC retention time (minutes): 0.98.
Example 4: Isopropyl (E) -3-(2-((5- (3- (phenylsulfonamide) phenyl) penta-4-en-1-yl) oxy) phenyl) propanoate The
compound prepared in Example 3. In a solution of (180 mg) in THF (3 mL), the compound (168 mg) prepared in Example 3 (1), chloro (2-dicyclohexylphosphino-2′, 4′, 6′-triisopropyl-1,1′- Biphenyl) [2- (2′-amino-1,1′-biphenyl)] palladium (II) (0.035 g) and a 2M tripotassium phosphate aqueous solution (0.67 mL) were added, and the mixture was stirred at 60 ° C. for 1 hour. .. The reaction solution was cooled to room temperature, water was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 7: 1 → 2: 1) to give the title compound (113 mg) having the following physical characteristics.
HPLC retention time (minutes): 1.24 
Example 5: 3- [2-[(E) -5- [3- (benzenesulfonamide) phenyl] penta-4-enoxy] phenyl] propanoic acid 
[Chemical 2]

　A 1 M aqueous lithium hydroxide solution (0.5 mL) was added to a solution of the compound (146 mg) prepared in Example 4 in THF (0.5 mL) and methanol (0.1 mL), and the mixture was stirred at 50 ° C. for 8 hours. It was acidified by adding 1M hydrochloric acid and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give the title compound (105 mg) having the following physical characteristics.
Form: Amorphous
HPLC retention time (minutes): 1.101
¹ H-NMR (CD ₃ OD): δ 1.95-2.03, 2.41-2.46, 2.57-2.61, 2.92-2.95, 4.03-4.06, 6.24, 6.36, 6.86, 6.90-6.95, 7.06-7.08, 7.11-7.19, 7.45-7.49, 7.55, 7.75-7.78.

PATENT

WO2020027150

https://patents.google.com/patent/WO2020027150A1/en

Example 83: Isopropyl (E) -3- (2-((5- (3- (phenylsulfonamido) phenyl) penta-4-en-1-yl) oxy) phenyl) propanoate The compound prepared in Example 82 Compound (168 mg) prepared in Example 9 and chloro (2-dicyclohexylphosphino-2 ′, 4 ′, 6′-triisopropyl-1,1′-biphenyl) [180 mg) in THF (3 mL) solution were added. 2- (2′-Amino-1,1′-biphenyl)] palladium (II) (0.035 g) and a 2M aqueous solution of tripotassium phosphate (0.67 mL) were added, and the mixture was stirred at 60 ° C. for 1 hour. After cooling the reaction solution to room temperature, water was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 7: 1 → 2: 1) to give the title compound (113 mg) having the following physical data.
HPLC retention time (min): 1.24.Example 84: 3- [2-[(E) -5- [3- (benzenesulfonamido) phenyl] penta-4-enoxy] phenyl] propanoic acid

To a solution of the compound prepared in Example 83 (146 mg) in THF (0.5 mL) and methanol (0.1 mL) was added a 1 M aqueous lithium hydroxide solution (0.5 mL), and the mixture was stirred at 50 ° C. for 8 hours. The mixture was acidified with 1M hydrochloric acid and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give the title compound (105 mg) having the following physical data.
HPLC retention time (min): 1.10
¹ H-NMR (CD ₃ OD): δ 1.95-2.03, 2.41-2.46, 2.57-2.61, 2.92-2.95, 4.03-4.06, 6.24, 6.36, 6.86, 6.90-6.95, 7.06-7.08, 7.11-7.19, 7.45 -7.49, 7.55, 7.75-7.78.

///////////ONO-2910, ONO 2910, PHASE 2,

O=S(=O)(Nc1cc(\C=C\CCCOc2ccccc2CCC(=O)O)ccc1)c1ccccc1

QQGASRPGPR DAQAHPGRPR AVPTQCDVPP NSRFDCAPDK AITQEQCEAR GCCYIPAKQG
LQGAQMGQPW CFFPPSYPSY KLENLSSSEM GYTATLTRTT PTFFPKDILT LRLDVMMETE
NRLHFTIKDP ANRRYEVPLE TPRVHSRAPS PLYSVEFSEE PFGVIVHRQL DGRVLLNTTV
APLFFADQFL QLSTSLPSQY ITGLAEHLSP LMLSTSWTRI TLWNRDLAPT PGANLYGSHP
FYLALEDGGS AHGVFLLNSN AMDVVLQPSP ALSWRSTGGI LDVYIFLGPE PKSVVQQYLD
VVGYPFMPPY WGLGFHLCRW GYSSTAITRQ VVENMTRAHF PLDVQWNDLD YMDSRRDFTF
NKDGFRDFPA MVQELHQGGR RYMMIVDPAI SSSGPAGSYR PYDEGLRRGV FITNETGQPL
IGKVWPGSTA FPDFTNPTAL AWWEDMVAEF HDQVPFDGMW IDMNEPSNFI RGSEDGCPNN
ELENPPYVPG VVGGTLQAAT ICASSHQFLS THYNLHNLYG LTEAIASHRA LVKARGTRPF
VISRSTFAGH GRYAGHWTGD VWSSWEQLAS SVPEILQFNL LGVPLVGADV CGFLGNTSEE
LCVRWTQLGA FYPFMRNHNS LLSLPQEPYS FSEPAQQAMR KALTLRYALL PHLYTLFHQA
HVAGETVARP LFLEFPKDSS TWTVDHQLLW GEALLITPVL QAGKAEVTGY FPLGTWYDLQ
TVPIEALGSL PPPPAAPREP AIHSEGQWVT LPAPLDTINV HLRAGYIIPL QGPGLTTTES
RQQPMALAVA LTKGGEARGE LFWDDGESLE VLERGAYTQV IFLARNNTIV NELVRVTSEG
AGLQLQKVTV LGVATAPQQV LSNGVPVSNF TYSPDTKVLD ICVSLLMGEQ FLVSWC
(Disulfide bridge:26-53, 36-52, 47-71, 477-502, 591-602, 882-896)

Avalglucosidase alfa

アバルグルコシダーゼアルファ (遺伝子組換え)

Avalglucosidase alfa (USAN/INN);
Avalglucosidase alfa (genetical recombination) (JAN);
Avalglucosidase alfa-ngpt

To treat late-onset Pompe disease

FDA APPROVED Nexviazyme, 2021/8/6, Enzyme replacement therapy product
Treatment of Pompe disease

Biologic License Application (BLA): 761194
Company: GENZYME CORP

https://www.fda.gov/news-events/press-announcements/fda-approves-new-treatment-pompe-diseaseFor Immediate Release:August 06, 2021

Today, the U.S. Food and Drug Administration approved Nexviazyme (avalglucosidase alfa-ngpt) for intravenous infusion to treat patients 1 year of age and older with late-onset Pompe disease.

Patients with Pompe disease have an enzyme deficiency that leads to the accumulation of a complex sugar, called glycogen, in skeletal and heart muscles, which cause muscle weakness and premature death from respiratory or heart failure. Normally, glycogen—the stored form of glucose—breaks down to release glucose into the bloodstream to be used as fuel for the cells.

“Pompe disease is a rare genetic disease that causes premature death and has a debilitating effect on people’s lives,” said Janet Maynard, M.D., deputy director of the Office of Rare Diseases, Pediatrics, Urologic and Reproductive Medicine in the FDA’s Center for Drug Evaluation and Research. “Today’s approval brings patients with Pompe disease another enzyme replacement therapy option for this rare disease. The FDA will continue to work with stakeholders to advance the development of additional new, effective and safe therapies for rare diseases, including Pompe disease.”

Nexviazyme, an enzyme replacement therapy, is an intravenous medication that helps reduce glycogen accumulation. The effectiveness of Nexviazyme for the treatment of Pompe disease was demonstrated in a study of 100 patients who were randomized to take Nexviazyme or another FDA-approved enzyme replacement therapy for Pompe disease. Treatment with Nexviazyme improved lung function similar to the improvement seen with the other therapy.

The most common side effects included headache, fatigue, diarrhea, nausea, joint pain (arthralgia), dizziness, muscle pain (myalgia), itching (pruritus), vomiting, difficulty breathing (dyspnea), skin redness (erythema), feeling of “pins and needles” (paresthesia) and skin welts (urticaria). Serious reactions included hypersensitivity reactions like anaphylaxis and infusion-associated reactions, including respiratory distress, chills and raised body temperature (pyrexia). Patients susceptible to fluid volume overload or with compromised cardiac or respiratory function may be at risk for serious acute cardiorespiratory failure.

The FDA granted this application Fast Track, Priority Review and Breakthrough Therapy designations. Nexviazyme also received an orphan drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases. The FDA granted the approval of Nexviazyme to Genzyme Corporation.

###

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FDA grants priority review for avalglucosidase alfa, a potential new therapy for Pompe disease

• The FDA decision date for avalglucosidase alfa, an investigational enzyme replacement therapy, is set for May 18, 2021
• Regulatory submission based on positive data from two trials in patients with late-onset and infantile-onset Pompe disease, respectively
• Avalglucosidase alfa received FDA Breakthrough Therapy and Fast Track designations for the treatment of people with Pompe Disease
• Pompe disease, a rare degenerative muscle disorder, affects approximately 3,500 people in the U.S.
• Milestone reinforces 20+year commitment to Pompe disease community

PARIS – November 18, 2020 – The U.S. Food and Drug Administration (FDA) has accepted for priority review the Biologics License Application (BLA) for avalglucosidase alfa for long-term enzyme replacement therapy for the treatment of patients with Pompe disease (acid α-glucosidase deficiency). The target action date for the FDA decision is May 18, 2021.

Avalglucosidase alfa is an investigational enzyme replacement therapy designed to improve the delivery of acid alpha-glucosidase (GAA) enzyme to muscle cells, and if approved, would offer a potential new standard of care for patients with Pompe disease.

In October, the European Medicines Agency accepted for review the Marketing Authorization Application for avalglucosidase alfa for long-term enzyme replacement therapy for the treatment of patients with Pompe disease. The Medicines and Healthcare Products Regulatory Agency in the UK has granted Promising Innovative Medicine designation for avalglucosidase alfa.

“The hallmarks of Pompe disease are the relentless and debilitating deterioration of the muscles, which causes decreased respiratory function and mobility,” said Karin Knobe, Head of Development for Rare Diseases and Rare Blood Disorders at Sanofi. “Avalglucosidase alfa is specifically designed to deliver more GAA enzyme into the lysosomes of the muscle cells.  We have been greatly encouraged by positive clinical trial results in patients with late-onset and infantile-onset Pompe disease.”

Pompe disease is a rare, degenerative muscle disorder that can impact an individual’s ability to move and breathe. It affects an estimated 3,500 people in the U.S. and can manifest at any age from infancy to late adulthood.ⁱ

The BLA is based on positive data from two trials:

• Pivotal Phase 3, double-blind, global comparator-controlled trial (COMET), which evaluated the safety and efficacy of avalglucosidase alfa compared to alglucosidase alfa (standard of care) in patients with late-onset Pompe disease. Results from this trial were presented during a Sanofi-hosted virtual scientific session in June 2020 and in October 2020 at World Muscle Society and the American Association of Neuromuscular and Electrodiagnostic Medicine.
• The Phase 2 (mini-COMET) trial evaluated the safety and exploratory efficacy of avalglucosidase alfa in patients with infantile-onset Pompe disease previously treated with alglucosidase alfa. Results from this trial were presented at the WORLDSymposium, in February 2020.

Delivery of GAA to Clear Glycogen

Pompe disease is caused by a genetic deficiency or dysfunction of the lysosomal enzyme GAA, which results in build-up of complex sugars (glycogen) in muscle cells throughout the body. The accumulation of glycogen leads to irreversible damage to the muscles, including respiratory muscles and the diaphragm muscle supporting lung function, and other skeletal muscles that affect mobility.

To reduce the glycogen accumulation caused by Pompe disease, the GAA enzyme must be delivered into the lysosomes within muscle cells. Research led by Sanofi has focused on ways to enhance the delivery of GAA into the lysosomes of muscle cells by targeting the mannose-6-phosphate (M6P) receptor that plays a key role in the transport of GAA.

Avalglucosidase alfa is designed with approximately 15-fold increase in M6P content, compared to standard of care alglucosidase alfa, and aims to help improve cellular enzyme uptake and enhance glycogen clearance in target tissues.ⁱⁱ The clinical relevance of this difference has not been confirmed.

Avalglucosidase alfa is currently under clinical investigation and its safety and efficacy have not been evaluated by any regulatory authority worldwide.

/////////Avalglucosidase alfa, FDA 2021,  Nexviazyme, APPROVALS 2021, PEPTIDE, Enzyme replacement therapy ,  Pompe disease, アバルグルコシダーゼアルファ (遺伝子組換え), Fast Track, Priority Review,  Breakthrough Therapy,  orphan drug designation, genzyme, sanofi

IOHEXOLCAS Registry Number: 66108-95-0N1,N3-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetamido]-2,4,6-triiodobenzene-1,3-dicarboxamide 
CAS Name: 5-[Acetyl(2,3-dihydroxypropyl)amino]-N,N¢-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamideAdditional Names:N,N¢-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetamido]-2,4,6-triiodoisophthalamide 
Manufacturers’ Codes: Win-39424; Compd 545Trademarks: Omnipaque (GE Healthcare) 
Molecular Formula: C19H26I3N3O9Molecular Weight: 821.14Percent Composition: C 27.79%, H 3.19%, I 46.36%, N 5.12%, O 17.54% 
Literature References: Nonionic radio-contrast medium. Prepn: V. Nordal, H. Holtermann, DE2726196; eidem,US4250113 (1977, 1981 both to Nyegaard). HPLC-UV determn in plasma: R. S. Soman et al., J. Chromatogr. B816, 339 (2005). 
Pharmacology and toxicology: Acta Radiol.Suppl. 362, 1-134 (1980). Acute toxicity: S. Salvesen, ibid. 73. Fibrillatory potential in dogs: G. L. Wolf et al.,Invest. Radiol.16, 320 (1981).Comparative clinical studies in coronary angiography: G. B. J. Mancini et al.,Am. J. Cardiol.51, 1218 (1983); I. D. Sullivan et al.,Br. Heart J.51, 643 (1984); M. A. Bettmann et al.,Radiology153, 583 (1984). Review: T. Almén, Acta Radiol.Suppl. 366, 9-19 (1983). 
Properties: Crystals from butanol, mp 174-180°. Sol in water. Stable in aqueous solutions. Viscosity (cP): 6.2 at 37°; 12.6 at 20° (c = 200 mg Iodine/ml). LD50 in male, female rats, mice (g Iodine/kg): 15.0, 12.3, 24.3, 25.1 i.v. (Salvesen). 
Melting point: mp 174-180° 
Toxicity data: LD50 in male, female rats, mice (g Iodine/kg): 15.0, 12.3, 24.3, 25.1 i.v. (Salvesen)Therap-Cat: Diagnostic aid (radiopaque medium).Keywords: Diagnostic Aid (Radiopaque Medium). 

Synthesis ReferenceXiu C. Wang, Steve A. Chamberlin, Ashok V. Bhatia, Gregg E. Robinson, John Hufnagel, “Process for the preparation of iohexol.” U.S. Patent US5705692, issued December, 1985.

US5705692

Iohexol, sold under the trade name Omnipaque among others, is a contrast agent used for X-ray imaging.^[1] This includes when visualizing arteries, veins, ventricles of the brain, the urinary system, and joints, as well as during computed tomography (CT scan).^[1] It is given by mouth, injection into a vein, or into a body cavity.^[2]

Iohexol is a contrast agent for intrathecal administration used in myelography and contrast enhancement for computerized tomography.

Side effects include vomiting, skin flushing, headache, itchiness, kidney problems, and low blood pressure.^[1] Less commonly allergic reactions or seizures may occur.^[1] Allergies to povidone-iodine or shellfish do not affect the risk of side effects more than other allergies.^[3] Use in the later part of pregnancy may cause hypothyroidism in the baby.^[4] Iohexol is an iodinated non-ionic radiocontrast agent.^[1] It is in the low osmolar family.^[5]

Iohexol was approved for medical use in 1985.^[6] It is on the World Health Organization’s List of Essential Medicines.^[7][2]

Chemistry

The osmolality of iohexol ranges from 322 mOsm/kg—approximately 1.1 times that of blood plasma—to 844 mOsm/kg, almost three times that of blood.^[8] Despite this difference, iohexol is still considered a low-osmolality contrast agent; the osmolality of older agents, such as diatrizoate, may be more than twice as high.^[9]

Society and culture

Names

It is sold under the brand names Omnipaque^[10] and Hexopaque. It is also sold as a density gradient medium under the names Accudenz, Histodenz and Nycodenz.^[11][12]

Formulations

It is available in various concentrations, from 140^{[citation needed]} to 350^[13] milligrams of iodine per milliliter.

PATENT

https://patents.google.com/patent/WO2005003080A1/en#:~:text=Primary%20production%20of%20iohexol%20involves,and%20a%20thorough%20purification%20stage.&text=The%20solvent%20is%20then%20evaporated,and%20recrystallised%20twice%20from%20butanol.The present invention relates to a process for the manufacture of iohexol, 5-[N- (2,3- dihydroxypropyl) -acetamido]-N,N’-bis(2,3 -dihydroxypropyl)-2,4,6- triiodoisophtalamide.Iohexol is the non-proprietory name of the chemical drug substance of a non-ionic iodinated X-ray contrast agent marketed under the trade name OMNIPAQUE^®. OMNIPAQUE^® is one of the most used agents in diagnostic X-ray procedures.The manufacture of such non-ionic contrast agents involves the production of the chemical drug substance (referred to as primary production) followed by formulation into the drug product (referred to as secondary production). Primary production of iohexol involves a multistep chemical synthesis and a thorough purification stage. For a commercial drug product it is important for the primary production to be efficient and economical and to provide a drug substance fulfilling the specifications.The final step in the synthesis of iohexol is a N-alkylation step in which 5-(acetamido)-N,N’-bis(2,3-dihydroxypropyl)-2,4,6 triiodoisophtalamide (hereinafter 5- Acetamide) is reacted in the liquid phase with an alkylating agent to introduce the 2,3-dihydroxypropyl group at the nitrogen of the 5-acetamido group. Following this reaction, iohexol is isolated from the reaction mixture and purified by crystallisation and treatment with ion exchange resins.The manufacture of iohexol is disclosed for example in US-4,250,113 which is hereby incorporated by reference. In the last step of the multistep chemical synthesis crude iohexol is obtained from the reaction between 5-Acetamide and 1-chloro-2,3- propandiol at ambient temperature in propylene glycoi and in the presence of sodium methoxide. The solvent is then evaporated and crude iohexol is obtained. The crude product is evaporated to dryness and recrystallised twice from butanol.Several suggestions to improve the N-alkylation and the purification steps have been published. WO-A-98/08804 discloses the use of 2-methoxy-ethanol and optionally isopropanol both in the alkylation step of 5-Acetamide and in the purification of crude iohexol. WO-A-02/083623 discloses the purification of crude iohexol using 1- methoxy-2-propanol as the solvent optionally in a mixture with other solvents.The N-alkylation step where 5-Acetamide in solution is reacted with an alkylation agent such as e.g. 1-chloro-2,3-propandiol to introduce the 2,3-dihydroxypropyl group at the nitrogen of the 5-acetamido group is illustrated in Scheme 1 :

5-Acetamide Iohexol5-acatamido-N,N’-bis(2,3-dihydroxypropyl)- 5-[N-(2,3-dihydroxypropyl)acetamido]- 2,4,6-triiodoisophtalamide N,N’-bis(2,3-dihydroxypropyl)- 2,4,6-triiodoisophtalamideScheme 1.The N-alkylation step is challenging because O-alkylated by-products can also be formed when the alkylation occurs at the oxygen atoms of the hydroxy groups. It is therefore a desire to limit the formation of these O-alkylated by-products and thereby to limit their presence in the final purified iohexol. The upper limit for values for O- alkylated by-products in the end product is fixed by the European Pharmacopea to 0.6% (HPLC by area).The O-alkylated by-products are removed to the degree desired or necessary by recrystallisation steps. Further unidentified by-products also referred to as impurities are also formed during the alkylation reaction and must be reduced to a tolerable level. In addition the solvents used should be easily available, be environmentally friendly and be of low toxicity.There is therefore a need to identify a solvent that can be used in the N-alkylation reaction and that fulfil the desiderata mentioned above. It is further desired to improve the overall process including the N-alkylation step and the purification step in the manufacture of iohexol. If the crude product obtained by the N-alkylation step is to be re-crystallised from a solvent that is different from the solvent used in the N- alkylation step, then the reaction solvent must first be removed e.g. by evaporation to dryness. It is known from crystallisation theory and experience that even small quantities of residual solvents from previous steps may cause a crystallisation process to get out of control due to changes in its supersaturation conditions, and thorough removal of the reaction solvent is an important step. Solvent removal is an energy consuming operation which also risks degradation of the product due to exposure to elevated temperature.Example 1 : Synthesis of iohexol in 1-methoxy-2-propanol/methanol1-methoxy-2-propanol (44 ml), methanol (19 ml) and sodium hydroxide (4.87 g) was added to a jacketed glass reactor and stirred for about 15 minutes at 25°C. 5-Acetamide (70 g) was added to the reactor, and the mixture stirred overnight at 45°C, before it was allowed to cool to 25°C. 1-chloro-2,3-propanediol (12.43 g) was added to the solution. After 1.5 hours, more 1-chloro-2,3-propanediol (0.83 g) was added, and the reaction was allowed to proceed for 24 hours. HPLC analysis (water/acetonitrile) of the reaction mixture gave the following results:Iohexol 98.1 %5-Acetamide 1.17 % O-alkylated substances 0.58 %Other impurities 0.1 %Example 2: Synthesis of iohexol in 1 -methoxy-2-propanol/water1-methoxy-2-propanol (63 ml), water (7 ml) and sodium hydroxide (4.50 g) was added to a jacketed glass reactor and stirred for about 15 minutes at 25°C. 5-Acetamide (70 g) was added to the reactor, and the mixture stirred overnight at 45°C, before it was allowed to cool to 35°C. 1-chloro-2,3-propanediol (11.39 g) was added to the solution. After 3 hours, more 1-chloro-2,3-propanediol (0.83 g) was added, and the reaction was allowed to proceed for 24 hours. HPLC analysis (water/acetonitrile) of the reaction mixture gave the following results:Iohexol 98.3 % 5-Acetamide 0.68 %O-alkylated substances 0.81 %Other impurities 0.3 % Example 3: Alkylation and crystallisation in solutions containing 1-methoxy-2- propanol1-methoxy-2-propanol (63 L), methanol (27 L) and sodium hydroxide (6.96 kg) was added to a 500 L reactor and stirred until all solids were dissolved and the temperature was below 30°C. 5-Acetamide (100 kg) was added to the reactor, and the mixture stirred overnight at 45°C before it was allowed to cool to 25°C. 1-chloro- 2,3-propanediol (16.76 kg) was added to the clear solution. After 1.5 hours, more 1- chloro-2,3-propanediol (1.18 kg) was added, and the reaction was allowed to proceed for 30 hours. HPLC analysis (water/acetonitrile) of the reaction mixture gave the following results:Iohexol 97.9 % 5-Acetamide 0.9 %O-alkylated substances 0.83 %Other impurities 0.4 %The reaction was stopped by addition of hydrochloric acid (650 ml), and the reaction mixture diluted with a mixture of 1-methoxy-2-propanol (53 L) and methanol (13 L). The mixture was filtered, and the salts on the filter washed with methanol (3×10 L). The combined filtrate and wash was diluted with water (22 L) and treated with cationic ion exchange resin (AMB 200C, 80 L) and anionic ion exchange resin (IRA 67, 80 L) to a salt content of 0.006 w/w %. The solution was filtered, and the ion exchange resins washed in several stages with a mixture of water (160 L) and methanol (85 L). The combined filtrate and wash was concentrated under reduced pressure to a volume of 155 L. One half of this was taken further to crystallisation as described below.Water was removed from the solution by azeotropic distillation. The volume was held at a constant level by replacing the distillate by 1-methoxy-2-propanol (80 L). At water content of 0.16 Ukg iohexol, further 1-methoxy-2-propanol (159 L) was added, and the solution seeded with iohexol crystals (0.26 kg). After stirring at reflux overnight, the volume of the solution was reduced by 42 L by distillation under reduced pressure (300-600 mbar). The temperature was set to 90°C, which was held for 3 hours before cooling to 60°C over 3 hours. The crystallisation mixture was stirred overnight at 60°C, filtered and washed with isopropanol (90 L, 6 portions). The yield was 48.4 kg (as dry powder), corresponding to 88-weight % corrected for seeding material and samples. HPLC analysis (water/acetonitrile) of the crystals gave the following results:Iohexol 99.3 %5-Acetamide 0.15 %O-alkylated substances 0.45 %Other impurities 0.11 % 
PAPERhttps://www.quickcompany.in/patents/a-new-process-for-the-synthesis-of-high-pure-iohexol-and-its-intermediatesPATENThttps://patents.google.com/patent/WO2005003080A1/enThe present invention relates to a process for the manufacture of iohexol, 5-[N- (2,3- dihydroxypropyl) -acetamido]-N,N’-bis(2,3 -dihydroxypropyl)-2,4,6- triiodoisophtalamide.Iohexol is the non-proprietory name of the chemical drug substance of a non-ionic iodinated X-ray contrast agent marketed under the trade name OMNIPAQUE^®. OMNIPAQUE^® is one of the most used agents in diagnostic X-ray procedures.The manufacture of such non-ionic contrast agents involves the production of the chemical drug substance (referred to as primary production) followed by formulation into the drug product (referred to as secondary production). Primary production of iohexol involves a multistep chemical synthesis and a thorough purification stage. For a commercial drug product it is important for the primary production to be efficient and economical and to provide a drug substance fulfilling the specifications.The final step in the synthesis of iohexol is a N-alkylation step in which 5-(acetamido)-N,N’-bis(2,3-dihydroxypropyl)-2,4,6 triiodoisophtalamide (hereinafter 5- Acetamide) is reacted in the liquid phase with an alkylating agent to introduce the 2,3-dihydroxypropyl group at the nitrogen of the 5-acetamido group. Following this reaction, iohexol is isolated from the reaction mixture and purified by crystallisation and treatment with ion exchange resins.The manufacture of iohexol is disclosed for example in US-4,250,113 which is hereby incorporated by reference. In the last step of the multistep chemical synthesis crude iohexol is obtained from the reaction between 5-Acetamide and 1-chloro-2,3- propandiol at ambient temperature in propylene glycoi and in the presence of sodium methoxide. The solvent is then evaporated and crude iohexol is obtained. The crude product is evaporated to dryness and recrystallised twice from butanol.Several suggestions to improve the N-alkylation and the purification steps have been published. WO-A-98/08804 discloses the use of 2-methoxy-ethanol and optionally isopropanol both in the alkylation step of 5-Acetamide and in the purification of crude iohexol. WO-A-02/083623 discloses the purification of crude iohexol using 1- methoxy-2-propanol as the solvent optionally in a mixture with other solvents.The N-alkylation step where 5-Acetamide in solution is reacted with an alkylation agent such as e.g. 1-chloro-2,3-propandiol to introduce the 2,3-dihydroxypropyl group at the nitrogen of the 5-acetamido group is illustrated in Scheme 1 :

5-Acetamide Iohexol5-acatamido-N,N’-bis(2,3-dihydroxypropyl)- 5-[N-(2,3-dihydroxypropyl)acetamido]- 2,4,6-triiodoisophtalamide N,N’-bis(2,3-dihydroxypropyl)- 2,4,6-triiodoisophtalamideScheme 1.The N-alkylation step is challenging because O-alkylated by-products can also be formed when the alkylation occurs at the oxygen atoms of the hydroxy groups. It is therefore a desire to limit the formation of these O-alkylated by-products and thereby to limit their presence in the final purified iohexol. The upper limit for values for O- alkylated by-products in the end product is fixed by the European Pharmacopea to 0.6% (HPLC by area).The O-alkylated by-products are removed to the degree desired or necessary by recrystallisation steps. Further unidentified by-products also referred to as impurities are also formed during the alkylation reaction and must be reduced to a tolerable level. In addition the solvents used should be easily available, be environmentally friendly and be of low toxicity.There is therefore a need to identify a solvent that can be used in the N-alkylation reaction and that fulfil the desiderata mentioned above. It is further desired to improve the overall process including the N-alkylation step and the purification step in the manufacture of iohexol. If the crude product obtained by the N-alkylation step is to be re-crystallised from a solvent that is different from the solvent used in the N- alkylation step, then the reaction solvent must first be removed e.g. by evaporation to dryness. It is known from crystallisation theory and experience that even small quantities of residual solvents from previous steps may cause a crystallisation process to get out of control due to changes in its supersaturation conditions, and thorough removal of the reaction solvent is an important step. Solvent removal is an energy consuming operation which also risks degradation of the product due to exposure to elevated temperature.Example 1 : Synthesis of iohexol in 1-methoxy-2-propanol/methanol1-methoxy-2-propanol (44 ml), methanol (19 ml) and sodium hydroxide (4.87 g) was added to a jacketed glass reactor and stirred for about 15 minutes at 25°C. 5-Acetamide (70 g) was added to the reactor, and the mixture stirred overnight at 45°C, before it was allowed to cool to 25°C. 1-chloro-2,3-propanediol (12.43 g) was added to the solution. After 1.5 hours, more 1-chloro-2,3-propanediol (0.83 g) was added, and the reaction was allowed to proceed for 24 hours. HPLC analysis (water/acetonitrile) of the reaction mixture gave the following results:Iohexol 98.1 %5-Acetamide 1.17 % O-alkylated substances 0.58 %Other impurities 0.1 %Example 2: Synthesis of iohexol in 1 -methoxy-2-propanol/water1-methoxy-2-propanol (63 ml), water (7 ml) and sodium hydroxide (4.50 g) was added to a jacketed glass reactor and stirred for about 15 minutes at 25°C. 5-Acetamide (70 g) was added to the reactor, and the mixture stirred overnight at 45°C, before it was allowed to cool to 35°C. 1-chloro-2,3-propanediol (11.39 g) was added to the solution. After 3 hours, more 1-chloro-2,3-propanediol (0.83 g) was added, and the reaction was allowed to proceed for 24 hours. HPLC analysis (water/acetonitrile) of the reaction mixture gave the following results:Iohexol 98.3 % 5-Acetamide 0.68 %O-alkylated substances 0.81 %Other impurities 0.3 % Example 3: Alkylation and crystallisation in solutions containing 1-methoxy-2- propanol1-methoxy-2-propanol (63 L), methanol (27 L) and sodium hydroxide (6.96 kg) was added to a 500 L reactor and stirred until all solids were dissolved and the temperature was below 30°C. 5-Acetamide (100 kg) was added to the reactor, and the mixture stirred overnight at 45°C before it was allowed to cool to 25°C. 1-chloro- 2,3-propanediol (16.76 kg) was added to the clear solution. After 1.5 hours, more 1- chloro-2,3-propanediol (1.18 kg) was added, and the reaction was allowed to proceed for 30 hours. HPLC analysis (water/acetonitrile) of the reaction mixture gave the following results:Iohexol 97.9 % 5-Acetamide 0.9 %O-alkylated substances 0.83 %Other impurities 0.4 %The reaction was stopped by addition of hydrochloric acid (650 ml), and the reaction mixture diluted with a mixture of 1-methoxy-2-propanol (53 L) and methanol (13 L). The mixture was filtered, and the salts on the filter washed with methanol (3×10 L). The combined filtrate and wash was diluted with water (22 L) and treated with cationic ion exchange resin (AMB 200C, 80 L) and anionic ion exchange resin (IRA 67, 80 L) to a salt content of 0.006 w/w %. The solution was filtered, and the ion exchange resins washed in several stages with a mixture of water (160 L) and methanol (85 L). The combined filtrate and wash was concentrated under reduced pressure to a volume of 155 L. One half of this was taken further to crystallisation as described below.Water was removed from the solution by azeotropic distillation. The volume was held at a constant level by replacing the distillate by 1-methoxy-2-propanol (80 L). At water content of 0.16 Ukg iohexol, further 1-methoxy-2-propanol (159 L) was added, and the solution seeded with iohexol crystals (0.26 kg). After stirring at reflux overnight, the volume of the solution was reduced by 42 L by distillation under reduced pressure (300-600 mbar). The temperature was set to 90°C, which was held for 3 hours before cooling to 60°C over 3 hours. The crystallisation mixture was stirred overnight at 60°C, filtered and washed with isopropanol (90 L, 6 portions). The yield was 48.4 kg (as dry powder), corresponding to 88-weight % corrected for seeding material and samples. HPLC analysis (water/acetonitrile) of the crystals gave the following results:Iohexol 99.3 %5-Acetamide 0.15 %O-alkylated substances 0.45 %Other impurities 0.11 % 

PatentCN109134289https://patents.google.com/patent/CN109134289A/en

N-Acylation of 5-amino-N,N’-bis(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide (1) with acetic anhydride (2) in the presence of p-TsOH gives 5-(acetylamino)-N,N’-bis(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide (3) , which upon condensation with glycidol  using NaOMe in 2-methoxyethanol at 90 °C  or epichlorohydrin  by means of NaHCO3 in propylene glycol at 85 °C  or 3-chloropropane-1,2-diol (5) using aqueous NaOH furnishes the  iohexol .(7) synthesis of IodixanolModus ponens (I) compound (200g, 0.28mol) be added 1L there-necked flask in, thereto be added acetic anhydride (207g, 2.03mol), acetic acid (103.3mL), p-methyl benzenesulfonic acid monohydrate (1g, 5.42mmol), finishes reaction solution being heated to 60 DEG C Start to react, keep the temperature 30 minutes after reacting liquid temperature reaches 120-125 DEG C, cooling is concentrated into after can just stirring thereto It is added 50%v/v (600mL), is slowly added dropwise thereto into 50%w/v sodium hydrate aqueous solution, by adding in reaction process The mode of 50%w/v sodium hydrate aqueous solution keeps the pH of reaction solution between 11~12, and reaction temperature is maintained at 40-45 DEG C, Reaction is finished, and concentrated hydrochloric acid is added into reaction solution and adjusts pH3-4, and stirring filters after 3.0 hours, and filter cake is washed with water to neutrality, dries It is dry, obtain white solid 187g, yield 88.2%, HPLC98.14%.Go step obtained solid (150g, 0.2mol) be added there-necked flask in, thereto be added sodium hydroxide (14.4g, 0.36mol), purified water (300mL), epoxychloropropane (27.9g, 0.30mol) finish 30-35 DEG C of reaction 72.0 hours, instead It should finish, adjust pH3-4, Iodixanol HPLC purity 72.5%, Iohexol HPLC11.3% with concentrated hydrochloric acid.(4) synthesis of IohexolModus ponens (I) compound (200g, 0.28mol) be added 1L there-necked flask in, thereto be added acetic anhydride (432g, 4.23mol) flows back 3.0 hours, be then concentrated under reduced pressure into p-methyl benzenesulfonic acid monohydrate (1g, 5.42mmol), agitating and heating It can just stir, be added portionwise into reaction solution methanol (25g), methanol is added after 1.0 hours in stirring thereto again (140g) is finished and is stirred to react 1.0 hours, and being concentrated under reduced pressure into can just stir, and purified water (20g) then is added thereto, 60 DEG C are finished to be stirred overnight.Reaction solution is cooled to 30 DEG C hereinafter, extracting reaction solution 200mL, stirring is lower will with 50%w/v sodium hydrate aqueous solution Reaction solution pH is adjusted to 12, the addition 1- chloro- 2 into reaction solution, 3-propanediol (20g, 0.18mol), passes through benefit in reaction process The mode of 50%w/v sodium hydrate aqueous solution is added to keep the pH of reaction solution between 11~12, after reaction 12.0 hours thereto Add 1- chloro- 2,3-propanediol (3g, 29.29mmol) finishes that the reaction was continued 48.0 hours, and reaction solution samples HPLC detection, iodine Mykol purity is 89.9%.(5) synthesis of IoversolModus ponens (I) compound (200g, 0.28mol) is added in 1L there-necked flask, and N-Methyl pyrrolidone is added thereto Chloracetyl chloride (200mL) is added in (200mL) thereto under stirring, finish 50-53 DEG C and react 3.0 hours, and reaction is finished, and is cooled to 20 DEG C, reaction solution is slowly added in methanol (2000mL).It finishing, flows back 9.0 hours, reaction is finished, and is cooled to 25 DEG C, it filters, Filter cake is washed with methanol, and drying obtains white solid 177g, yield 79.8%, HPLC purity 98.3%.It takes previous step obtained solid (150g, 0.19mol) to be added in 1L there-necked flask, purified water 300mL is added thereto, Acetic acid sodium trihydrate (183g, 1.34mol) finishes back flow reaction, by adding 50%w/v sodium hydroxide water in reaction process The mode of solution keeps the pH of reaction solution between 5-6, and reaction is finished, and concentrated hydrochloric acid is added into reaction solution, adjusts pH3-4, stirring It being filtered after 3.0 hours, filter cake is with purifying water washing to neutrality, and drying obtains white solid 127g, yield 86.7%, HPLC98.4%.It takes step obtained solid (100g, 0.13mol), is added in 1L there-necked flask, purified water 300mL, chlorine are added thereto Change sodium (46.5g, 0.796mol), finish, be warming up to 50 DEG C, 10N sodium hydrate aqueous solution (39.3mL) and 2- are added thereto Chlorethanol (63.5g, 0.79mol) finishes 48-52 DEG C of heat preservation and reacts 5.0 hours, and reaction is finished, and concentrated hydrochloric acid is added thereto and adjusts PH6.5, reaction solution HPLC detection, Iohexol purity 89.7%.(6) synthesis of IopentolModus ponens (I) compound (200g, 0.28mol) be added 1L there-necked flask in, thereto be added acetic anhydride (432g, 4.23mol) flows back 3.0 hours, be then concentrated under reduced pressure into p-methyl benzenesulfonic acid monohydrate (1g, 5.42mmol), agitating and heating It can just stir, be added portionwise into reaction solution methanol (25g), methanol (140g) is added thereto again after stirring 1.0 hours, It finishes and is stirred to react 1.0 hours, being concentrated under reduced pressure into can just stir, and purified water (20g) then is added thereto, finishes 60 DEG C It is stirred overnight.Reaction solution is cooled to 30 DEG C hereinafter, extracting reaction solution 200mL, stirring is lower will with 50%w/v sodium hydrate aqueous solution Reaction solution pH is adjusted to 12, and the chloro- 3- methoxy-2-propanol (22.5g, 0.18mol) of 1-, reaction process are added into reaction solution In keep the pH of reaction solution between 11~12 by way of adding 50%w/v sodium hydrate aqueous solution, react 12.0 hours Add 1- chloro- 2 thereto afterwards, 3-propanediol (3.4g, 29.29mmol) finishes that the reaction was continued 48.0 hours, reaction solution sampling HPLC detection, Iopentol purity are 91.3%.(7) synthesis of IodixanolModus ponens (I) compound (200g, 0.28mol) be added 1L there-necked flask in, thereto be added acetic anhydride (207g, 2.03mol), acetic acid (103.3mL), p-methyl benzenesulfonic acid monohydrate (1g, 5.42mmol), finishes reaction solution being heated to 60 DEG C Start to react, keep the temperature 30 minutes after reacting liquid temperature reaches 120-125 DEG C, cooling is concentrated into after can just stirring thereto It is added 50%v/v (600mL), is slowly added dropwise thereto into 50%w/v sodium hydrate aqueous solution, by adding in reaction process The mode of 50%w/v sodium hydrate aqueous solution keeps the pH of reaction solution between 11~12, and reaction temperature is maintained at 40-45 DEG C, Reaction is finished, and concentrated hydrochloric acid is added into reaction solution and adjusts pH3-4, and stirring filters after 3.0 hours, and filter cake is washed with water to neutrality, dries It is dry, obtain white solid 187g, yield 88.2%, HPLC98.14%.Go step obtained solid (150g, 0.2mol) be added there-necked flask in, thereto be added sodium hydroxide (14.4g, 0.36mol), purified water (300mL), epoxychloropropane (27.9g, 0.30mol) finish 30-35 DEG C of reaction 72.0 hours, instead It should finish, adjust pH3-4, Iodixanol HPLC purity 72.5%, Iohexol HPLC11.3% with concentrated hydrochloric acid.To sum up, method of the invention is easy to operate, and (III) three obtained formula (I), formula (II) or formula intermediate can be made For the raw material for synthesizing diodone, not by-product truly；Importantly, general sieve of synthesis iodine that can be convenient Amine does not have the generation of two acylated by-products, and compared with original grinds the production technology of medicine, process route is entirely different, high income, cost It is low, a kind of very effective, completely new approach is provided for industrialized production Iopromide, is had a extensive future.

Patent

Publication numberPriority datePublication dateAssigneeTitleWO1998008804A1 *1996-08-291998-03-05Nycomed Imaging AsProcess for iohexol manufactureUS5847212A *1997-04-211998-12-08Abbott LaboratoriesProcess for the preparation of iohexolWO1999026916A1 *1997-11-261999-06-03Nycomed Imaging AsN-alkylation of 5-amino-2,4,6-triiodo-isophthalamidesFamily To Family CitationsITMI20010773A1 *2001-04-112002-10-11Chemi SpaProcess for the production of high purity iohexole

Non-Patent

TitleHAAVALDSEN J ET AL: “X-RAY CONTRAST AGENTS. I. SYNTHESIS OF SOME DERIVATIVES OF 5-AMINO-2, 4, 6-TRIIODOISOPHTHLAMIDE”, ACTA PHARMACEUTICA SUECICA, XX, XX, vol. 20, no. 3, 1983, pages 219 – 232, XP002052827, ISSN: 0001-6675 * 

Publication numberPriority datePublication dateAssigneeTitleWO2007013816A1 *2005-07-292007-02-01Ge Healthcare AsContinuous crystallisation process of iodinated phenyl derivativesWO2007060380A1 *2005-11-242007-05-31Hovione Inter LtdProcess for the manufacture of iohexolJP2009502910A *2005-07-292009-01-29ジーイー・ヘルスケア・アクスイェ・セルスカプMethod for continuous crystallization of iodinated phenyl derivativesCN101195587B *2006-12-192010-07-21浙江尖峰海洲制药有限公司Production method for lodixanol hydrolysateUS8766002B22009-11-262014-07-01Imax Diagnostic Imaging Holding LimitedPreparation and purification of iodixanolNO342021B1 *2005-07-292018-03-12Ge Healthcare AsContinuous crystallization processFamily To Family CitationsWO2011041275A1 *2009-09-302011-04-07Mallinckrodt Inc.Alkylation of triiodo-substituted arylamides in an aqueous mixed solvent systemES2680019T3 *2010-12-212018-09-03Ge Healthcare AsDesalination of a composition comprising a contrast agentUS20140065076A1 *2012-08-302014-03-06Otsuka Pharmaceutical Co. Ltd.Container with concentrated substance and method of using the same* Cited by examiner, † Cited by third party, ‡ Family to family citation

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References

1. ^ Jump up to:^{a b c d e} World Health Organization (2009). Stuart MC, Kouimtzi M, Hill SR (eds.). WHO Model Formulary 2008. World Health Organization. pp. 317–8. hdl:10665/44053. ISBN 9789241547659.
2. ^ Jump up to:^a b Hamilton, Richart (2015). Tarascon Pocket Pharmacopoeia 2015 Deluxe Lab-Coat Edition. Jones & Bartlett Learning. p. 171. ISBN 9781284057560.
3. ^ ACR Manual on Contrast Media v10.3. 2017 (PDF). American College of Radiology. 2017. p. 6. ISBN 9781559030120. Archived (PDF) from the original on 1 January 2018. Retrieved 1 January 2018.
4. ^ Briggs, Gerald G.; Freeman, Roger K.; Yaffe, Sumner J. (2011). Drugs in Pregnancy and Lactation: A Reference Guide to Fetal and Neonatal Risk. Lippincott Williams & Wilkins. p. 761. ISBN 9781608317080. Archived from the original on 1 January 2017.
5. ^ Sutton, David; Young, Jeremy W. R. (2012). A Short Textbook of Clinical Imaging. Springer Science & Business Media. p. 235. ISBN 9781447117551. Archived from the original on 1 January 2017.
6. ^ Broe, Marc E. de; Porter, George A.; Bennett, William M.; Verpooten, G. A. (2013). Clinical Nephrotoxins: Renal Injury from Drugs and Chemicals. Springer Science & Business Media. p. 325. ISBN 9789401590884. Archived from the original on 1 January 2017.
7. ^ World Health Organization (2019). World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
8. ^ GE Healthcare (May 2006). “Omnipaque (Iohexol) injection. Product label”. DailyMed. U.S. National Library of Medicine. Retrieved 28 March 2007.
9. ^ Amersham Health (April 2006). “Hypaque (Diatrizoate Meglumine and Diatrizoate Sodium) injection, solution. Product label”. DailyMed. U.S. National Library of Medicine. Archived from the original on 23 May 2011. Retrieved 29 March 2007.
10. ^ “Omnipaque” (PDF). Ireland: Health Products Regulatory Authority. January 2018. Retrieved 31 July 2020.
11. ^ “HistoDenz (D2158)” Archived 2015-11-20 at the Wayback Machine, product information sheet, Sigma-Aldrich. Accessed on line 19 November 2015.
12. ^ “Nycodenz®: A universal density gradient medium” Archived 2015-02-26 at the Wayback Machine, Axis-Shield Density Gradient Media. Accessed 19 November 2015.
13. ^ Haberfeld H, ed. (2020). Austria-Codex (in German). Vienna: Österreichischer Apothekerverlag. Omnipaque 350 mg J/ml Infusionsflasche.

External links

• “Iohexol”. Drug Information Portal. U.S. National Library of Medicine.
• “Iohexol Injection, Oral, Rectal Advanced Patient Information”. Drugs.com. 13 January 2019. Retrieved 3 February 2020.

////////////IOHEXOL, Win-39424, Compd 545, Omnipaque, Oraltag, GE Healthcare, X RAY CONTRAST AGENTS, WIN 39424

CC(=O)N(CC(O)CO)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I

PropofolCAS Registry Number: 2078-54-8 
CAS Name: 2,6-Bis(1-methylethyl)phenolAdditional Names: 2,6-diisopropylphenol; disoprofol 
Manufacturers’ Codes: ICI-35868 
Trademarks: Ansiven (Abbott); Diprivan (AstraZeneca); Disoprivan (AstraZeneca); Rapinovet (Schering-Plough Vet.)Molecular Formula: C12H18OMolecular Weight: 178.27Percent Composition: C 80.85%, H 10.18%, O 8.97% 
Literature References: Prepn: A. J. Kolka et al.,J. Org. Chem.21, 712 (1956); 22, 642 (1957); G. G. Ecke, A. J. Kolka, US2831898 (1958 to Ethyl Corp.); T. J. Kealy, D. D. Coffman, J. Org. Chem.26, 987 (1961); B. E. Firth, T. J. Rosen, US4447657 (1984 to Universal Oil Products). Chromatographic study: J. K. Carlton, W. C. Bradbury, J. Am. Chem. Soc.78, 1069 (1956). Animal studies: J. B. Glen, Br. J. Anaesth.52, 731 (1980).Pharmacokinetics: H. K. Adam et al.,ibid. 743; idem,ibid.55, 97 (1983). Determn in blood: eidem,J. Chromatogr.223, 232 (1981). Comparative studies vs other injectable anesthetics: B. Kay, D. K. Stephenson, Anaesthesia35, 1182 (1980); D. V. Rutter et al.,ibid. 1188. Use in i.v. anesthesia: E. Major et al.,ibid.37, 541 (1982). Cardiovascular effects: D. Al-Khudhairi et al.,ibid. 1007. Pharmacology of emulsion formulation: J. B. Glen, S. C. Hunter, Br. J. Anaesth.56, 617 (1984). Series of articles on pharmacology and clinical experience: Postgrad. Med. J.61, Suppl. 3, 1-169 (1985). 
Properties: bp30 136°. bp17 126°. mp 19°. nD20 1.5134. nD25 1.5111. d20 0.955.Melting point: mp 19°Boiling point: bp30 136°; bp17 126°Index of refraction:nD20 1.5134; nD25 1.5111Density: d20 0.955Therap-Cat: Anesthetic (intravenous).Therap-Cat-Vet: Intravenous anesthetic (dogs and cats).Keywords: Anesthetic (Intravenous).SYN

Prepn: A. J. Kolka et al., J. Org. Chem. 21, 712 (1956); 22, 642 (1957); G. G. Ecke, A. J. Kolka, US 2831898 (1958 to Ethyl Corp.); T. J. Kealy, D. D. Coffman, J. Org. Chem. 26, 987 (1961); B. E. Firth, T. J. Rosen, US 4447657 (1984 to Universal Oil Products).SYN

SYNhttps://pubs.acs.org/doi/pdf/10.1021/op400300t

A commercially viable manufacturing process for propofol (1) is described. The process avoids acid–base neutralization events during isolation of intermediate, 2,6-di-isopropylbenzoic acid (3) and crude propofol, and thus simplifies the synthesis on industrial scale to a considerable extent. Syntheses of five impurities/related substances (USP and EP) are also described. 

SYN

SYN

PATENThttps://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021156776&tab=PCTDESCRIPTION&_cid=P20-KSCZNY-60539-1

Propofol is used during surgeries for sedation and an injectable grade with purity > 99.90% is desired by the medical community. An embodiment of the present invention provides an economically feasible, industrial process for the manufacture of high purity injectable grade Propofol. An embodiment of the present invention relates to a process and novel strategy for purification of 2,6-diisopropylphenol (Propofol) and similar products.

[0003] Propofol is a sterile injectable drug that appears in the USP, EP and IP Monographs. Drug product is manufactured by using high purity drug substance 2,6-di-isopropylphenol commonly known as Propofol.

[0004] Propofol is used to put patients to sleep and keep them asleep during general anesthesia for surgery or other medical procedures. It is used in adults as well as children 2 months and older. Propofol is frequently used as a sedative, and has a rapid onset of action and a short recovery period. Propofol slows the activity of brain and nervous system. Propofol is also used to sedate a patient who is under critical care and needs a mechanical ventilator (breathing machine). Propofol is a hypnotic alkylphenol derivative. When formulated for intravenous induction of sedation and hypnosis during anaesthesia, Propofol facilitates inhibitory neurotransmission mediated by gamma- Aminobutyric acid (GABA). Propofol is associated with minimal respiratory depression and has a short half-life with a duration of action of 2 to 10 minutes.

[0005] Propofol is commonly used parenteral anesthetic agent in the United States, extensively used for minor and outpatient surgical procedures because of its rapid onset and reversal of action, and in intensive care units (ICUs) for maintaining coma. Propofol has been associated with rare instances of idiosyncratic acute liver injury; in addition, prolonged high dose Propofol therapy can cause the “Propofol infusion syndrome” which is marked by brady arrhythmias, metabolic acidosis, rhabdomyolysis, hyperlipidemia and an enlarged or fatty liver.

[0006] Friedel-Craft’s alkylation of phenol using propylene gas in the presence of Lewis acid (LA) catalysts is a commonly used method for the synthesis of Propofol and is well documented in a number of publications and patents [Ecke, G. G., Kolka, A. J. US 2,831,898 A, 1958. Firth, B. E., Rosen, T. J. US 4,447,657, 1984. Akio, T., Yoshiaki, I., Hidekichi, H., Kiyoji, K., Takashi, K., Masanobu, M. EP 0169359A1, 1986. Ecke, G. G., Kolka, A. J. US 3,271,314, 1966. Napolitano, J. P. US 3,367,981 A, 1968. Goddard L. E. US 3,766,276, 1973. Firth, B. E. US 4,275,248, 1981, etc.]

[0007] A number of patents and published literature describe the manufacture of Propofol. US. Pat. No. 4,275,248; W0200034218; EP169359; US. Pat. No. 3,367,981; US. Pat. No.

3,271,314; US. Pat. No. 3,766,276; US. Pat. No. 2,831,898; US.Pat.No.2,207,753; GB1318100; U.S. Pat. No. 4,391,998; US. Pat. No. 4,774, 368; US. Pat. No. 5,589,598; US. Pat. No. 6,362,234; etc. EP 0511947, discloses purification of Propofol that is obtained by alkylation of phenol and purified by crystallization at -10 to -20°C (melting point of Propofol is 18°C). This patent also describes purification using non-polar solvents such as Petroleum ether or Hexane, where solvent residue is removed by distillation or evaporation and finally Propofol is obtained using fractional distillation under high vacuum.

[0008] Continuous separation of a mixture of Propofol with phenolic impurities and methanol is described in an U.S. Pat. No. 5,264,085. U.S. Pat. No. 5,705,039 describes the purification of impure 2,6-diisopropylphenol first using continuous distillation and then distilling pure Propofol under high vacuum.

[0009] Patent CN103360219A describes purification wherein 2,6-diisopropyl phenol is reacted with benzoyl chloride to generate ‘benzoic acid-2, 6-diisopropyl benzene ester’, which is then purified to yield Propofol. The patent discloses that an adsorbent is added at the rectifying stage, so that impurities with similar chemical structures and boiling points are effectively removed; the content of a single impurity in the product is not higher than 0.01%; the total impurity is not higher than 0.05%.

[0010] CN105601477A describes purification of Propofol wherein crude Propofol is purified with three-stage distillation method; the crude Propofol enters feeding tank protected by nitrogen and is charged into first-stage film distillation system through pump; then the product is fed to second-stage molecular distillation system and low boiling point impurities are removed; finally, the processed product is charged into third-stage molecular distiller through a pump, high-boiling-point impurities are separated, and the colourless or yellowish high-purity Propofol is obtained.

[0011] In another prior art disclosure, after completion of the reaction, the final product is isolated and purified by high-vacuum distillation. Alkylation of phenol using propylene gas at high pressure and high temperature is reported. Several impurities like 2,4-diisopropyl and 2,4,6-triisopropyl phenol are the major side products along with the corresponding Isopropyl ether. All these impurities need to be controlled at a limit of NMT 0.05% or less in the final API for it to be pharmaceutically acceptable. In another prior art disclosure, isopropanol was used as the propylating agent instead of direct propylene gas. In this method propylene is generated in situ using IPA and strong acid like sulfuric acid and catalysts like Aluminoslicate [See Baltalksne, A. E.; Zitsmanis, A. H. SU 443019, 1974. Jain, K. P., Edaki, D. U., Minhas H. S., Minhas G. S. WO/2011/ 161687 Al, 2011. Wu, M. US 4,391,998, 1983]

[0012] Another method is to use of protected phenol, where 4-chloro or 4-carboxylic acid substituted phenol is used as starting material along with Isopropanol in sulfuric acid, followed by removal of the 4-substituent to give Propofol [Baltalksne, A. E.; Zitsmanis, A. H. SU 443019, 1974. Jain, K. P., Edaki, D. U., Minhas H. S., Minhas G. S. WO/2011/ 161687 Al, 2011. Wu, M. US 4,391,998, 1983. Tsutsumi, S.; Yoshizawa, T.; Koyama, K. Nippon Kagaku Zasshi 1956, 77, 737-738. Paiocchi, M. US 5,589,598, 1996. Nieminen, K., Essen, P. US 5,175,376, 1992. Keller, S., Schlegel, J. WO/2012/152665 Al, 2012.] The final purification is carried out by high- vacuum distillation to get highly pure Propofol. Since the para position is blocked, related impurities such as 2,4-isopropyl and 2,4,6-triisopropyl derivatives are avoided. In this approach, intermediate is purified before converting to crude Propofol using either de-chlorination by hydrogenation or de-carboxylation before vacuum distillation for final purification.

[0013] It is reported in the literature that 4-hydroxybenzoic acid is used as starting material for alkylation with isopropyl alcohol in sulfuric acid. In that method 2,6-diisopropyl-4-hydroxy benzoic acid gets formed, which is extracted in toluene either in presence of an acid or the impurities are extracted in toluene under alkaline condition. The decarboxylation is carried out using solvents like monoethylene glycol or ethoxyethanol at high temperature. At the end of decarboxylation, crude Propofol is isolated by extracting into toluene. The advantage is Propofol does not form sodium salt under the conditions, but all other acidic impurities form sodium salt and thus do not get extracted in toluene. The toluene containing Crude Propofol is distilled to recover toluene and then vacuum distilled to obtain pure Propofol. [Chen, T; Chen, X.; Bois-Choussy, M.; Zhu, J. J. Am. Chem. Soc. 2006, 128, 87-89. Lau, S.; Keay, B. Can. J. Chem. 2001, 79, 1541-1545]

[0014] In summary, strategies disclosed in prior art for the production of 2,6-diisopropylphenol (Propofol) predominantly involve synthesis starting from phenol or by using protected 4-position of phenol like, 4-hydroxybenzoic acid, 4-chlorophenol (references: Baltalksne, A. E.; Zitsmanis, A. H. SU 443019, 1974. Jain, K. P., Edaki, D. U., Minhas H. S., Minhas G. S. WO/2011/ 161687 Al, 2011. Wu, M. US 4,391,998, 1983. Tsutsumi, S.; Yoshizawa, T.; Koyama, K. Nippon Kagaku Zasshi 1956, 77, 737-738. Paiocchi, M. US 5,589,598, 1996. Nieminen, K., Essen, P. US 5,175,376, 1992. Keller, S., Schlegel, J. WO/2012/152665 Al, 2012). Processes described in the literature generally propose purification of crude 2,6-diisopropylphenol by ‘high vacuum distillation or molecular distillation’.

[0015] The phenols are susceptible to oxidation, formation of polymeric and color impurities. There are processes where repeated vacuum distillation has been carried out to obtain desired purity of product. Sometimes, to reduce the oxidized and colored impurities, reduction of impurities by catalytic hydrogenation is also used.

[0016] Propofol that does not meet Pharmaceutical grade may be manufactured by several processes generally known to persons of skill in the art and described in prior art, but purification of Propofol to consistently achieve high purity required for the injectable drug substance using an economical and industrial process remains a challenge.

Example 1:

[0033] Commercially available concentrated sulfuric acid (30 Kg) was diluted with water (2.26 Kg) at low temperature (0-15°C). Methyl 4-hydroxybenzoate (5 Kg 32.79 mol.) was added to this diluted sulfuric acid at 5 to 10 °C with stirring. After complete addition, isopropyl alcohol (5.9 Kg 98.16 mol.) was gradually added to the reaction content, controlling the temperature at 0-15 °C. The reaction mixture was then heated at 60-70°C and continued to complete di-isopropylation and ester hydrolysis to yield methyl-4-hydroxybenzoate. The conversion was checked on TLC or by HPLC for the complete conversion of methyl-4 hydroxybenzoate to 3, 5 -Diisopropyl 4-hydroxybenzoic acid.

[0034] The reaction contents were cooled at room temperature and carefully charged into a stirred, precooled mixture of water (50 L) and Toluene (40 L) at (0 to 5°C). The mixture was stirred and maintained below 15°C for about 30 to 60 minutes.

[0035] The content was then heated at 25 to 30°C, stirred for 30 min., allowed to settle into two layers. The water layer was extracted again with toluene and discarded. The toluene layers, containing the product 3, 5-Diisopropyl 4-hydroxybenzoic acid, were combined and extracted with about 25 L of 10 % NaOH. The aqueous layer containing the sodium salt of 3, 5 -Di-isopropyl 4-hydroxybenzoic acid was acidified with concentrated HC1 (about 9 Kg) to precipitate 3, 5-Diisopropyl 4-hydroxybenzoic acid, filtered, and washed with water (about 50 L) to yield 3, 5 -diisopropyl 4-hydroxybenzoic acid (about 45-60 %)

[0036] To the mixture of 3, 5-diisopropyl 4-hydroxybenzoic acid (3 Kg, 13.5 mol.) in ethylene glycol (5.0 Kg, 80.55 mol.) was added sodium hydroxide (1.25 Kg, 31.25 mol.) for decarboxylation. The reaction mixture was heated at 145 ± 5°C till completion of

decarboxylation by monitoring using TLC or HPLC (or solubility in bicarbonate of precipitated product). After complete decarboxylation, the reaction mixture was cooled at 40 to 45 °C, under nitrogen environment and diluted with water (about 15 L) and allowed to settle. The oily product layer was separated and washed with water (6L) to isolate crude Propofol (i.e., 2,6-diisopropyl phenol) and stored under nitrogen. The isolated volatile Crude Propofol (along with carry over ppm ethylene glycol and NaOH) was then subjected to steam distillation purification process as described below.

[0037] The Crude Propofol is purified by using one of the steam distillation processes as described below.

[0038] The Crude Propofol layer is added to purified water in a reactor (preferably glass lined reactor), and slowly heated to boiling to co-distil Pure Propofol along with water under normal atmospheric pressure and the high volatile initial fraction is isolated first. The biphasic layers of main distillate, are separated and the liquid layer of Propofol is treated with dehydrating agent to absorb dissolved moisture in Pure Propofol under nitrogen or argon. The transparent Pure Propofol liquid layer is then filtered through ultrafme Micron filter (0.45 and 0.2 micron) under nitrogen (or argon) pressure and isolated in controlled environment to obtain pharmaceutical injectable grade Pure Propofol of more than 99.90% purity.

[0039] The Crude Propofol liquid layer is charged into a reactor with steam distillation arrangement, like steam purging dip tube, column, heat exchanger and receivers. Pure steam is purged in the reactor at controlled pressure to co-distil Pure Propofol with water. The layers are allowed settle and water layer is kept aside for recirculation. The transparent Pure Propofol transparent liquid layer is then filtered through ultrafme Micron filter (0.45 and 0.2 micron) under nitrogen (or argon) pressure and isolated in controlled environment to obtain pharmaceutical injectable grade Pure Propofol of more than 99.90% purity.

[0040] The Crude Propofol layer is added to purified water in a reactor (preferably glass lined or Hastelloy reactor) and slowly heated at boiling to co-distil Pure Propofol along with water under mild vacuum. The biphasic layers are separated and the liquid layer of Propofol is treated with dehydrating agent to absorb dissolved moisture in Pure Propofol under nitrogen (or argon). The transparent Pure Propofol liquid layer is then filtered through ultrafme Micron filter (0.45 and 0.2 micron) under nitrogen (or argon) pressure and isolated in controlled environment to obtain pharmaceutical injectable grade Pure Propofol of more than 99.90% purity.

[0041] The Crude Propofol layer is added to reactor containing purified water and 0.1 to 1% antioxidant and 0.1 to 0.5% sodium hydroxide and slowly heated to boiling to co-distil Pure Propofol along with water. The biphasic layers are separated and the liquid layer of Propofol is treated or passed through column packed with dehydrating agent to absorb dissolved moisture in Pure Propofol. The transparent Pure Propofol liquid layer is then filtered through ultrafme Micron filter (0.45 and 0.2 micron) under nitrogen (or argon) pressure and isolated in controlled environment to obtain pharmaceutical injectable grade Pure Propofol of more than 99.90% purity.

[0042] The crude Propofol liquid layer is treated with preferably neutral or basic activated carbon (about 2-5%) and filtered under nitrogen. The filtered liquid is collected, under nitrogen, in distillation reactor containing purified water is slowly heated to boiling to co-distil Pure Propofol along with water under normal pressure or mild vacuum. The co-distilled biphasic layers are separated and the liquid layer of Propofol, is treated under nitrogen, with or passed through column packed with dehydrating agent to absorb dissolved moisture trapped in Pure Propofol. The transparent Pure Propofol liquid layer is then filtered through ultrafme Micron filter (0.45 and 0.2 micron) under nitrogen (or argon) pressure and isolated in controlled environment to obtain pharmaceutical injectable grade Pure Propofol of more than 99.90% purity.

Example No. 2:

[0043] Friedel-Crafts reaction was performed as described in Example 1. Decarboxylation was performed by using KOH instead of NaOH by following the same procedure as described in Example 1.

Example No. 3:

[0044] Decarboxylation was performed as per operations described in Example 1. After complete decarboxylation, the reaction mixture was cooled at 40 to 45°C, under nitrogen environment and diluted with water (about 15 L) The biphasic mixture subjected to steam distillation by any of the purification methods described in Example 1.

Example No. 4:

[0045] Friedel-Crafts reaction was performed as described in Example 1. The reaction contents were cooled at room temperature and carefully charged at 0 to 5°C into a sodium hydroxide solution to basic pH at stirred. The aqueous alkaline solution was extracted twice with toluene or hexane. The aqueous layer was acidified with HC1 to precipitate 3, 5-diisopropyl-4-hydroxybenzoic acid. The wet product was washed with water, dried and decarboxylated using sodium hydroxide in ethylene glycol as solvent at 145±5°C. The reaction contents were cooled to room temperature, diluted with water and acidified and then Crude Propofol was extracted twice in toluene. The toluene layer was washed with water, bicarbonate and with water then distilled to obtain crude oily layer of Propofol (>99% pure). This Crude Propofol was then purified by using purification steam distillation by any of the purification methods described in Example 1.

Example 5:

[0046] Continuous steam distillation of crude Propofol by purging pure steam. Continuous steam distillation of Crude Propofol was carried out using a feed pump for feeding liquid Crude Propofol (prepared by one of the processes described in this application or other literature) to the steam distillation system connected to a pure steam generator. Steam at 1-10 kg pressure was purged in the steam distillation system at controlled rate and the co-distilled Pure Propofol with water was cooled using heat exchanger and continuous separator. The residue was discharged from bottom valve at defined time intervals. The water layer was recycled to steam generator and Pure Propofol was dehydrated, filtered and collected in controlled environment as described in Example 1.

Propofol, marketed as Diprivan, among other names, is a short-acting medication that results in a decreased level of consciousness and a lack of memory for events.^[4] Its uses include the starting and maintenance of general anesthesia, sedation for mechanically ventilated adults, and procedural sedation.^[4] It is also used for status epilepticus if other medications have not worked.^[4] It is given by injection into a vein, and the maximum effect takes about two minutes to occur and typically lasts five to ten minutes.^[4] Propofol is also used for medical assistance in dying in Canada.^[5]

Common side effects of propofol include an irregular heart rate, low blood pressure, a burning sensation at the site of injection and the cessation of breathing.^[4] Other serious side effects may include seizures, infections due to improper use, addiction, and propofol infusion syndrome with long-term use.^[4] The medication appears to be safe for use during pregnancy but has not been well studied for use in this case.^[4] It is not recommended for use during a cesarean section.^[4] It is not a pain medication, so opioids such as morphine may also be used,^[6] however whether or not they are always needed is not clear.^[7] Propofol is believed to work at least partly via a receptor for GABA.^[4]

Propofol was discovered in 1977 and approved for use in the United States in 1989.^[4][8] It is on the World Health Organization’s List of Essential Medicines^[9] and is available as a generic medication.^[4] It has been referred to as milk of amnesia (a play on “milk of magnesia“), because of the milk-like appearance of the intravenous preparation, and because of its tendency to suppress memory recall.^[10][11] Propofol is also used in veterinary medicine for anesthesia.^[12][13]

Medical uses

Anesthesia

To induce general anesthesia, propofol is the drug used almost always, having largely replaced sodium thiopental.^[14][6] It can also be administered as part of an anesthesia maintenance technique called total intravenous anesthesia, using either manually programmed infusion pumps or computer-controlled infusion pumps in a process called target controlled infusion (TCI). Propofol is also used to sedate individuals who are receiving mechanical ventilation but not undergoing surgery, such as patients in the intensive care unit.^[15][16] In critically ill patients, propofol is superior to lorazepam both in effectiveness and overall cost.^[17] Propofol is relatively inexpensive compared to medications of similar use due to shorter ICU stay length.^[17] One of the reasons propofol is thought to be more effective (although it has a longer half-life than lorazepam) is because studies have found that benzodiazepines like midazolam and lorazepam tend to accumulate in critically ill patients, prolonging sedation.^[17] Propofol has also been suggested as a sleep aid in critically ill adults in the ICU, however, the effectiveness of this medicine at replicating the mental and physical aspects of sleep for people in the ICU are not clear.^[16]

Propofol is often used instead of sodium thiopental for starting anesthesia because recovery from propofol is more rapid and “clear”.^{[citation needed]}

Propofol can be run through a peripheral IV or central line. Propofol is frequently paired with fentanyl (for pain relief) in intubated and sedated people.^[18] Both are compatible in IV form.^[18]

Procedural sedation

Propofol is also used for procedural sedation. Its use in these settings results in a faster recovery compared to midazolam.^[19] It can also be combined with opioids or benzodiazepines.^[20][21][22] Because of its rapid induction and recovery time, propofol is also widely used for sedation of infants and children undergoing MRI.^[23] It is also often used in combination with ketamine with minimal side effects.^[24]

COVID-19

In March 2021, the U.S. Food and Drug Administration (FDA) issued an emergency use authorization (EUA) for Propofol‐Lipuro 1% to maintain sedation via continuous infusion in people greater than age sixteen with suspected or confirmed COVID‑19 who require mechanical ventilation in an intensive care unit ICU setting.^{[25][26][27][28]} In the circumstances of this public health emergency, it would not be feasible to require healthcare providers to seek to limit Fresenius Propoven 2% Emulsion or Propofol-Lipuro 1% only to be used for patients with suspected or confirmed COVID‑19; therefore, this authorization does not limit use to such patients.^[28]

Other uses

Executions

The US state of Missouri added propofol to its execution protocol in April 2012. However, Governor Jay Nixon halted the first execution by the administration of a lethal dose of propofol in October 2013 following threats from the European Union to limit the drug’s export if it were used for that purpose.^[29][30] The United Kingdom had already banned the export of medicines or veterinary medicines containing propofol to the United States.^[31]

Recreational use

Recreational use of the drug via self-administration has been reported^[32][33] but is relatively rare due to its potency and the level of monitoring required for safe use.^{[citation needed]} Critically, a steep dose-response curve makes recreational use of propofol very dangerous, and deaths from self-administration continue to be reported.^[34][35] The short-term effects sought via recreational use include mild euphoria, hallucinations, and disinhibition.^[36][37]

Recreational use of the drug has been described among medical staff, such as anesthetists who have access to the drug.^[38][39] It is reportedly more common among anesthetists on rotations with short rest periods, as usage generally produces a well-rested feeling.^[40] Long-term use has been reported to result in addiction.^[38][41]

Attention to the risks of off-label use of propofol increased in August 2009 due to the Los Angeles County coroner’s conclusion that music icon Michael Jackson died from a mixture of propofol and the benzodiazepine drugs lorazepam, midazolam, and diazepam on June 25, 2009.^{[42][43][44][45]} According to a July 22, 2009 search warrant affidavit unsealed by the district court of Harris County, Texas, Jackson’s physician, Conrad Murray, administered 25 milligrams of propofol diluted with lidocaine shortly before Jackson’s death.^[43][44][46] Even so, as of 2016, propofol was not on a US Drug Enforcement Administration schedule.^[40][47]

Side effects

One of propofol’s most common side effects is pain on injection, especially in smaller veins. This pain arises from activation of the pain receptor, TRPA1,^[48] found on sensory nerves and can be mitigated by pretreatment with lidocaine.^[49] Less pain is experienced when infused at a slower rate in a large vein (antecubital fossa). Patients show considerable variability in their response to propofol, at times showing profound sedation with small doses.

Additional side effects include low blood pressure related to vasodilation, transient apnea following induction doses, and cerebrovascular effects. Propofol has more pronounced hemodynamic effects relative to many intravenous anesthetic agents.^[50] Reports of blood pressure drops of 30% or more are thought to be at least partially due to inhibition of sympathetic nerve activity.^[51] This effect is related to the dose and rate of propofol administration. It may also be potentiated by opioid analgesics.^[52] Propofol can also cause decreased systemic vascular resistance, myocardial blood flow, and oxygen consumption, possibly through direct vasodilation.^[53] There are also reports that it may cause green discolouration of the urine.^[54]

Although propofol is heavily used in the adult ICU setting, the side effects associated with propofol seem to be of greater concern in children. In the 1990s, multiple reported deaths of children in ICUs associated with propofol sedation prompted the FDA to issue a warning.^[55]

As a respiratory depressant, propofol frequently produces apnea. The persistence of apnea can depend on factors such as premedication, dose administered, and rate of administration, and may sometimes persist for longer than 60 seconds.^[56] Possibly as the result of depression of the central inspiratory drive, propofol may produce significant decreases in respiratory rate, minute volume, tidal volume, mean inspiratory flow rate, and functional residual capacity.^[50]

Diminishing cerebral blood flow, cerebral metabolic oxygen consumption, and intracranial pressure are also characteristics of propofol administration.^[57] In addition, propofol may decrease intraocular pressure by as much as 50% in patients with normal intraocular pressure.^[58]

A more serious but rare side effect is dystonia.^[59] Mild myoclonic movements are common, as with other intravenous hypnotic agents. Propofol appears to be safe for use in porphyria, and has not been known to trigger malignant hyperpyrexia.^{[citation needed]}

Propofol is also reported to induce priapism in some individuals,^[60][61] and has been observed to suppress REM sleep stage and to worsen the poor sleep quality in some patients.^[62]

As with any other general anesthetic agent, propofol should be administered only where appropriately trained staff and facilities for monitoring are available, as well as proper airway management, a supply of supplemental oxygen, artificial ventilation, and cardiovascular resuscitation.^[63]

Because of its lipid base, some hospital facilities require the IV tubing (of continuous propofol infusions) to be changed after 12 hours. This is a preventive measure against microbial growth and infection.^[64]

Propofol infusion syndrome

Main article: Propofol infusion syndrome

A rare, but serious, side effect is propofol infusion syndrome. This potentially lethal metabolic derangement has been reported in critically ill patients after a prolonged infusion of high-dose propofol, sometimes in combination with catecholamines and/or corticosteroids.^[65]

Interactions

The respiratory effects of propofol are increased if given with other respiratory depressants, including benzodiazepines.^[66]

Pharmacology

Pharmacodynamics

Propofol has been proposed to have several mechanisms of action,^[67][68][69] both through potentiation of GABA_A receptor activity and therefore acting as a GABA_A receptor positive allosteric modulator, thereby slowing the channel-closing time. At high doses, propofol may be able to activate GABA_A receptors in the absence of GABA, behaving as a GABA_A receptor agonist as well.^[70][71][72] Propofol analogs have been shown to also act as sodium channel blockers.^[73][74] Some research has also suggested that the endocannabinoid system may contribute significantly to propofol’s anesthetic action and to its unique properties.^[75] EEG research upon those undergoing general anesthesia with propofol finds that it causes a prominent reduction in the brain’s information integration capacity.^[76]

Pharmacokinetics

A 20 ml ampoule of 1% propofol emulsion, as sold in Australia by Sandoz

Propofol is highly protein-bound in vivo and is metabolised by conjugation in the liver.^[77] The half-life of elimination of propofol has been estimated to be between 2 and 24 hours. However, its duration of clinical effect is much shorter, because propofol is rapidly distributed into peripheral tissues. When used for IV sedation, a single dose of propofol typically wears off within minutes. Propofol is versatile; the drug can be given for short or prolonged sedation, as well as for general anesthesia. Its use is not associated with nausea as is often seen with opioid medications. These characteristics of rapid onset and recovery along with its amnestic effects^[78] have led to its widespread use for sedation and anesthesia.

History

John B. Glen, a veterinarian and researcher at Imperial Chemical Industries (ICI) spent 13 years developing propofol, an effort which led to the awarding to him of the prestigious 2018 Lasker Award for clinical research. Propofol was originally developed as ICI 35868. It was chosen for development after extensive evaluation and structure–activity relationship studies of the anesthetic potencies and pharmacokinetic profiles of a series of ortho-alkylated phenols.^[79]

First identified as a drug candidate in 1973, clinical trials followed in 1977, using a form solubilised in cremophor EL.^[80] However, due to anaphylactic reactions to cremophor, this formulation was withdrawn from the market and subsequently reformulated as an emulsion of a soya oil/propofol mixture in water. The emulsified formulation was relaunched in 1986 by ICI (now AstraZeneca) under the brand name Diprivan. The currently available preparation is 1% propofol, 10% soybean oil, and 1.2% purified egg phospholipid as an emulsifier, with 2.25% glycerol as a tonicity-adjusting agent, and sodium hydroxide to adjust the pH. Diprivan contains EDTA, a common chelation agent, that also acts alone (bacteriostatically against some bacteria) and synergistically with some other antimicrobial agents. Newer generic formulations contain sodium metabisulfite or benzyl alcohol as antimicrobial agents. Propofol emulsion is a highly opaque white fluid due to the scattering of light from the tiny (about 150-nm) oil droplets it contains: Tyndall Effect.

Developments

A water-soluble prodrug form, fospropofol, has been developed and tested with positive results. Fospropofol is rapidly broken down by the enzyme alkaline phosphatase to form propofol. Marketed as Lusedra, this formulation may not produce the pain at injection site that often occurs with the conventional form of the drug. The U.S. Food and Drug Administration (FDA) approved the product in 2008.^[81] However fospropofol is a Schedule IV controlled substance with the DEA ACSCN of 2138 in the United States unlike propofol.^[82]

By incorporation of an azobenzene unit, a photoswitchable version of propofol (AP2) was developed in 2012, that allows for optical control of GABA_A receptors with light.^[83] In 2013, a propofol binding site on mammalian GABA_A receptors has been identified by photolabeling using a diazirine derivative.^[84] Additionally, it was shown that the hyaluronan polymer present in the synovia can be protected from free-radical depolymerization by propofol.^[85]

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