ABT 494

cas 1310726-60-3

Tartrate form (C₁₇H₁₉F₃N₆( C4H₆06)).

(35^,,4R)-3-ethyl-4-(3H-imidazo[l,2-fl]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine- l-carboxamide.

(35^,,4R)-3-ethyl-4-(3H- imidazo[l,2-fl]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine- l-carboxamide,

(ci5^,)-3-ethyl-4-(3H-imidazo[l,2-fl]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-l-carboxamide

pharmaceutically acceptable salts thereof, stereoisomers thereof, and isomers thereof, is provided in U.S. Patent No. 8,426,411,

ABBOTT ……INNOVATOR

PATENT

WO2011068881

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011068881&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Novel Tricyclic Compounds [US2011311474]

2011-12-22

/////c21cnc4c(n1c(cn2)[C@@H]3[C@@H](CN(C3)C(=O)NCC(F)(F)F)CC)ccn4

IC50:Aldose reductase: IC₅₀ = 7 nM (bovine); Aldose reductase: IC₅₀ = 16 nM (rat); Aldose reductase: IC₅₀ = 21 nM (pig); Aldose Reductase: IC₅₀ = 21 nM (human); Rattus norvegicus:

Medicinal Chemistry, 2009, Vol. 5, No. 5,

Synthesis of ethyl 2-(3-oxo-1,3-dihydro-1-isobenzofurany liden)acetate (2) A solution of phthalic anhydride (1.0 equiv.) and ethyl 2- (1,1,1-triphenyl-5 -phosphanylidene)acetate (1.1 equiv.) in 300 ml of dichloromethane (DCM) was refluxed for 3 hr. DCM was removed by vacuum at 40-50 o C. 2×150 ml of hexane was added to the resulting sticky solid, stirred for 10 min and the un-reacted 2-(1,1,1-triphenyl-5 -phosphanylidene)acetate was removed by filtration. The organic solvent was removed under vacuum and the resulting crude semisolid was taken to next step without further purification. Yield: 84%. 1 H-NMR CDCl3; (ppm): 1.1 (t, 3H), 4.2 (q, 2H), 6.0 (s, 1H), 7.6 (t, 1H), 7.7 (t, 1H), 7.8 (d, 1H), 8.9 (d, 1H). S

Synthesis of ethyl 2-(4-oxo-3,4-dihydro-1-phthalazinyl) acetate (3) A mixture of 2 (1.0 equiv.), hydrazine hydrate (0.8 equiv) and PTSA (1.0 equiv.) was ground by pestle and mortar at room temperature for 8 min. On completion, as indicated by TLC, the reaction mixture was treated with water. The resultant product was filtered, washed with water and recrystallized from DMF to give 3 in high yields (86%).1 H-NMR CDCl3; (ppm): 1.1 (t, 3H), 3.9 ( s, 2H), 4.1 (q, 2H), 7.6

Synthesis of 2-[3-(4-bromo-2-fluorobenzyl)-4-oxo-3,4- dihydro-1-phthalazinyl]acetic acid (4)

A mixture of 3 (1.0 equiv.), NaOH (5.0 equiv.), and THF was stirred for 30 min at 40-50 o C. 4-bromo-1-bromomethyl-2-fluoro benzene (1.1 equiv.) was added to the reaction mixture and stirred for 2 hr at 50-60 o C. Water was added to the reaction mixture and stirred at room temperature for 1 hr. pH was adjusted to 2-3 using cold acetic acid. THF was removed and the aqueous phase was extracted with ethyl acetate (2×50 ml), washed with brine, dried over sodium sulphate and evaporated. The solid was crystallized with methanol to give 4 with 54 % yield.

1H-NMR (DMSOd6); (ppm): 3.98 (s, 2H), 5.3 (s, 2H), 7.17 (t, 1H), 7.35 ( dd, 1H, J1= 8.0, J2= 1.6), 7.55 (dd, 1H, J1= 8.0, J2= 1.6), 7.87 (t, 1H), 7.9 (t, 1H), 7.95 (t, 1H0, 8.29 (d, 1H).

///////////Ponalrestat, ICI-128436, MK-538, ICI-plc,

C1=CC=C2C(=C1)C(=NN(C2=O)CC3=C(C=C(C=C3)Br)F)CC(=O)O

VAL-083

(1R,2S)-1-((R)-oxiran-2-yl)-2-((S)-oxiran-2-yl)ethane-1,2-diol

Galactitol, 1,​2:5,​6-​dianhydro-

• 1,2:5,6-Dianhydrodulcitol
• 1,2:5,6-Dianhydrogalactitol
• 1,2:5,6-Diepoxydulcitol

Dianhydrodulcitol; Dianhydrogalactitol; VAL083; VAL 083, Dulcitol diepoxide, NSC 132313

CAS 23261-20-3

MF C6H10O4, MW 146.14

VAL-083 is a bi-functional alkylating agent; inhibit U251 and SF188 cell growth in monolayer better than TMZ and caused apoptosis

VAL-083 is a bi-functional alkylating agent, with potential antineoplastic activity. Upon administration, VAL-083 crosses the blood brain barrier (BBB) and appears to be selective for tumor cells. This agent alkylates and crosslinks DNA which ultimately leads to a reduction in cancer cell proliferation. In addition, VAL-083 does not show cross-resistance to other conventional chemotherapeutic agents and has a long half-life in the brain. Check for active clinical trials or closed clinical trials using this agent

Currently, VAL-083 is approved in China to treat chronic myelogenous leukemia and lung cancer, while the drug has also secured orphan drug designation in Europe and the US to treat malignant gliomas.

LAUNCHED CHINA FOR Cancer, lung

Del Mar Pharmaceuticals Inc……..Glioblastoma…………..PHASE2

DelMar and MD Anderson to accelerate development of anti-cancer drug VAL-083
DelMar Pharmaceuticals has collaborated with the University of Texas MD Anderson Cancer Center (MD Anderson) to speed up the clinical development of its VAL-083 anti-cancer drug.

VAL-083 is a BI-Functional alkylating agent; INHIBIT U251 and SF188 Cell Growth in monolayer Better than TMZ and Caused apoptosis. IC50 Value : 5 uM (INHIBIT U251, SF188, T98G Cell Growth in monolayer after 72h) [1]. in vitro :.. VAL-083 INHIBITED U251 and SF188 Cell Growth in monolayer and as neurospheres Better than TMZ and Caused apoptosis after 72 hr Formation Assay In the colony, VAL-083 (5 uM) SF188 Growth suppressed by about 95% are T98G cells classically TMZ-resistant and express MGMT, but VAL-083 inhibited their growth in monolayer after 72 hr in a dose-dependent manner (IC50, 5 uM). VAL-083 also inhibited the growth of CSCs (BT74, GBM4, and GBM8) . by 80-100% in neurosphere self-Renewal assays Conversely, there was minimal normal Effect on Human Neural stem cells [1]. in Vivo : Clinical Trial : Safety Study of VAL-083 in Patients With Recurrent Malignant glioma or Secondary Progressive Brain Tumor. Phase 1 / Phase 2

VAL-083 has demonstrated activity in cyclophosphamide, BCNU and phenylanine mustard resistant cell lines and no evidence of cross-resistance has been encountered in published clinical studies. Based on the presumed alkylating functionality of VAL-083, published literature suggests that DNA repair mechanisms associated with Temodar and nitrosourea resistance, such as 06-methylguanine methyltransferace (MGMT), may not confer resistance to VAL-083.  VAL-083 readily crosses the blood brain barrier where it maintains a long half-life in comparison to the plasma. Published preclinical and clinical research demonstrates that VAL-083 is selective for brain tumor tissue.  VAL-083 has been assessed in multiple studies as chemotherapy in the treatment of newly diagnosed and recurrent brain tumors. In published clinical studies, VAL-083 has previously been shown to have a statistically significant impact on median survival in high grade gliomas when combined with radiation vs. radiation alone. The main dose-limiting toxicity related to the administration of VAL-083 in previous clinical studies was myelosuppression

Glioblastoma is the most common form of primary brain cancer

DelMar Pharmaceuticals has collaborated with the University of Texas MD Anderson Cancer Center (MD Anderson) to speed up the clinical development of its VAL-083 anti-cancer drug.

VAL-083 is a small-molecule chemotherapeutic designed to treat glioblastoma multiforme (GBM), the most common and deadly cancer that starts within the brain.

Under the deal, MD Anderson will begin a new Phase II clinical trial with VAL-083 in patients with GBM at first recurrence / progression, prior to Avastin (bevacizumab) exposure.

During the trial, eligible patients will have recurrent GBM characterised by a high expression of MGMT, the DNA repair enzyme implicated in drug-resistance, and poor patient outcomes following current front-line chemotherapy.

The company noted that MGMT promoter methylation status will be used as a validated biomarker for enrollment and tumours must exhibit an unmethylated MGMT promoter for patients to be eligible for the trial.

DelMar chairman and CEO Jeffrey Bacha said: “The progress we continue to make with our research shows that VAL-083 may offer advantages over currently available chemotherapies in a number of tumour types.

“This collaboration will allow us to leverage world-class clinical and research expertise and a large patient population from MD Anderson as we extend and accelerate our clinical focus to include GBM patients, following first recurrence of their disease.

“We believe that VAL-083’s unique cytotoxic mechanism offers promise for GBM patients across the continuum of care as a potential superior alternative to currently available cytotoxic chemotherapies, especially for patients whose tumours exhibit a high-expression of MGMT.”

The deal will see DelMar work with the scientists and clinicians at MD Anderson to accelerate its research in order to transform the treatment of patients whose cancers fail or are unlikely to respond to existing treatments.

In more than 40 clinical trials, VAL-083 showed clinical activity against several cancers including lung, brain, cervical, ovarian tumours and leukemia both as a single-agent and in combination with other treatments.

PATENT

WO 2012024368

https://www.google.com/patents/WO2012024368A3?cl=en

Dianhydrogalactitol (DAG or dianhydrodulcitol) can be synthesized from dulcitol which can be produced from natural sources (such as Maytenus confertiflora) or commercial sources.The structure of DAG is given below as Formula (I).

One method for the preparation of dulcitol from Maytenus confertiflora is as follows: (1) The Maytenus confertiflora plant is soaked in diluted ethanol (50-80%) for about 24 hours, and the soaking solution is collected. (2) The soaking step is repeated, and all soaking solutions are combined. (3) The solvent is removed by heating under reduced pressure. (4) The concentrated solution is allowed to settle overnight and the clear supernatant is collected. (5) Chloroform is used to extract the supernatant. The chloroform is then removed under heat and reduced pressure. (6) The residue is then dissolved in hot methanol and cooled to allow crystallization. (7) The collected crystals of dulcitol are filtered and dried under reduced pressure. The purified material is dulcitol, contained in the original Maytenus confertiflora plant at a concentration of about 0.1% (1/1000).

DAG can be prepared by two general synthetic routes as described below:

Route 1 :

Dulcitol DAG

Route 2. Dulcitol

In Route 1 , “Ts” represents the tosyl group, or p-toluenesulfonyl group. PATENT

However, the intermediate of Route 1, 1,6-ditosy)dulcitol, was prepared with low yield (~36%), and the synthesis of 1,6-ditosyldulcitol was poorly reproducible. Therefore, the second route process was developed, involving two major steps: (1) preparation of dibromodulcitol from dulcitol; and (2) preparation of dianhydrodulcitol from dibromodulcitol.

Dibromodulcitol is prepared from dulcitol as follows: (1) With an aqueous HBr solution of approximately 45% HBr concentration, increase the HBr concentration to about 70% by reacting phosphorus with bromine in concentrated HBr in an autoclave. Cool the solution to 0° C. The reaction is:

2P+3Br₂→2PBr₃+H₂0→HBr†+H₃P0₄. (2) Add the dulcitol to the concentrated HBr solution and reflux at 80° C to complete the reaction. (3) Cool the solution and pour the mixture onto ice water. Dibromodulcitol is purified through recrystallization.

The results for the preparation of dibromodulcitol (DBD) are shown in Table 1, below.

TABLE 1

For the preparation of DAG from DBD, DBD was poorly dissolved in methanol and ethanol at 40° C (different from what was described in United States PATENT

Patent No. 3,993,781 to Horvath nee Lengyel et al., incorporated herein by this reference). At refluxing, DBD was dissolved but TLC showed that new impurities formed that were difficult to remove from DBD.

The DBD was reacted with potassium carbonate to convert the DBD to dianhydrogalactitol.

The results are shown in Table 2, below.

TABLE 2

In the scale-up development, it was found the crude yield dropped significantly. It is unclear if DAG could be azeotropic with BuOH. It was confirmed that t-BuOH is essential to the reaction. Using MeOH as solvent would result in many impurities as shown spots on TLC. However, an improved purification method was developed by using a slurry with ethyl ether, which could provide DAG with good purity. This was developed after a number of failed attempts at recrystallization of DAG.

Bromination of dulcitol with HBr at 80°C gives dibromodulcitol , which upon epoxidation in the presence of K2CO3 in t-BuOH or NaOH in H2O  or in the presence of ion exchange resin Varion AD (OH) (4) affords the target dianhydrogalactitol .

PATENT

US 20140155638

SCHEME 5

PATENT

CN 103923039

http://www.google.com/patents/CN103923039A?cl=en

The resulting Dulcitol 9g and 18ml mass percent concentration of 65% hydrobromic acid at 78 ° C under reflux for 8 hours to give 1,6-dibromo dulcitol, and the product is poured into ice crystals washed anhydrous tert-butyl alcohol, and dried to give 1,6-dibromo dulcitol crystal, then 10.0gl, 6- dibromo dulcitol sample is dissolved in t-butanol, adding solid to liquid 2 % obtained through refining process 1,6_ dibromo dulcitol seed stirred and cooled to 0 ° C, allowed to stand for seven days to give 1,6_ dibromo dulcitol crystal, anhydrous t-butanol, dried to give 1,6-dibromo dulcitol. 5g of the resulting 1,6_ dibromo Euonymus dissolved in 50ml tert-butanol containing 5g of potassium carbonate, the elimination reaction, at 80 ° C under reflux time was 2 hours, the resulting product was dissolved in t-butanol, Join I% stock solution to the water quality of 1,2,4,5_ two Dulcitol including through a purification step to get less than 1% of 1,2,5,6_ two to water Dulcitol seeded stirring, cooling to 0 ° C, allowed to stand for I-day, two to go get 1,2,5,6_ water Dulcitol crystals washed anhydrous tert-butyl alcohol, and dried to give 1,2,5,6 two to crystalline water Dulcitol and lyophilized to give two to water Dulcitol lyophilized powder, containing I, 2,4,5- two to water Dulcitol less than 0.3%.

PATENT

WO 2005030121

PATENT

US 20140066642

• DAG can be prepared by two general synthetic routes as described below:
• In Route 1, “Ts” represents the tosyl group, or p-toluenesulfonyl group.
• However, the intermediate of Route 1, 1,6-ditosyldulcitol, was prepared with low yield (˜36%), and the synthesis of 1,6-ditosyldulcitol was poorly reproducible. Therefore, the second route process was developed, involving two major steps: (1) preparation of dibromodulcitol from dulcitol; and (2) preparation of dianhydrodulcitol from dibromodulcitol.
• Dibromodulcitol is prepared from dulcitol as follows: (1) With an aqueous HBr solution of approximately 45% HBr concentration, increase the HBr concentration to about 70% by reacting phosphorus with bromine in concentrated HBr in an autoclave. Cool the solution to 0° C. The reaction is: 2P+3Br₂→2PBr₃+H₂O→HBr↑+H₃PO₄. (2) Add the dulcitol to the concentrated HBr solution and reflux at 80° C. to complete the reaction. (3) Cool the solution and pour the mixture onto ice water. Dibromodulcitol is purified through recrystallization.

PATENT

US 20150329511

 PAPER

Antibacterial and Anti-Quorum Sensing Molecular Composition Derived from Quercus cortex (Oak bark) Extract

REFERENCES

Currently, VAL-083 is approved in China to treat chronic myelogenous leukemia and lung cancer, while the drug has also secured orphan drug designation in Europe and the US to treat malignant gliomas.

[1]. Fotovati A, Hu KJ, Wakimoto H, VAL-083, A NOVEL N7 ALKYLATING AGENT, SURPASSES TEMOZOLOMIDE ACTIVITY AND INHIBITS CANCER STEM CELLS, PROVIDING A NEW POTENTIAL TREATMENT OPTION FOR GLIOBLASTOMA MULTIFORME. Neuro-oncology, 2012, 14, AbsET-37, Suppl. 6

[2]. Fotovati A, Hu KJ, Wakimoto H, VAL-083, A NOVEL AGENT N7 alkylating, SURPASSES temozolomide Inhibits TREATMENT ACTIVITY AND STEM CELLS, PROVIDING A NEW TREATMENT OPTION FOR POTENTIAL glioblastoma multiforme. Neuro-oncology, 2012, 14, AbsET-37, Suppl. 6

1: Szende B, Jeney A, Institoris L. The diverse modification of N-butyl-N-(4-hydroxybutyl) nitrosamine induced carcinogenesis in urinary bladder by dibromodulcitol and dianhydrodulcitol. Acta Morphol Hung. 1992;40(1-4):187-93. PubMed PMID: 1365762.

2: Anderlik P, Szeri I, Bános Z. Bacterial translocation in dianhydrodulcitol-treated mice. Acta Microbiol Hung. 1988;35(1):49-54. PubMed PMID: 3293340.

3: Huang ZG. [Clinical observation of 15 cases of chronic myelogenous leukemia treated with 1,2,5,6-dianhydrodulcitol]. Zhonghua Nei Ke Za Zhi. 1982 Jun;21(6):356-8. Chinese. PubMed PMID: 6957285.

4: Anderlik P, Szeri I, Bános Z, Wessely M, Radnai B. Higher resistance of germfree mice to dianhydrodulcitol, a lymphotropic cytostatic agent. Acta Microbiol Acad Sci Hung. 1982;29(1):33-40. PubMed PMID: 6211912.

5: Bános Z, Szeri I, Anderlik P. Effect of Bordetella pertussis vaccine on the course of lymphocytic choriomeningitis (LCM) virus infection in suckling mice pretreated with dianhydrodulcitol (DAD). Acta Microbiol Acad Sci Hung. 1979;26(2):121-5. PubMed PMID: 539467.

6: Bános Z, Szeri I, Anderlik P. Dianhydrodulcitol treatment of lymphocytic choriomeningitis virus infection in suckling mice. Acta Microbiol Acad Sci Hung. 1979;26(1):29-34. PubMed PMID: 484266.

7: Gerö-Ferencz E, Tóth K, Somfai-Relle S, Gál F. Effect of dianhydrodulcitol (DAD) on the primary immune response of normal and tumor bearing rats. Oncology. 1977;34(4):150-2. PubMed PMID: 335301.

8: Kopper L, Lapis K, Institóris L. Incorporation of 3H-dibromodulcitol and 3H-dianhydrodulcitol into ascites tumor cells. Autoradiographic study. Neoplasma. 1976;23(1):47-52. PubMed PMID: 1272473.

9: Bános S, Szeri I, Anderlik P. Combined phytohaemagglutinin and dianhydrodulcitol treatment of lymphocytic choriomeningitis virus infection in mice. Acta Microbiol Acad Sci Hung. 1975;22(3):237-40. PubMed PMID: 1155228.

Carbohydrate Research, 1982 ,  vol. 108, p. 173 – 180

Deryabin, Dmitry G.; Tolmacheva, Anna A.
Molecules, 2015 ,  vol. 20,  9  pg. 17093 – 17108

Gati; Somfai-Relle
Arzneimittel-Forschung/Drug Research, 1982 ,  vol. 32,   2  pg. 149 – 151

/////////////////

C1C(O1)C(C(C2CO2)O)O

O[C@H]([C@H]1OC1)[C@@H](O)[C@H]2CO2

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO …..FOR BLOG HOME CLICK HERE

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AM 7209

Amgen Inc. INNOVATOR

MF 747.700043 g/mol, C₃₇H₄₁Cl₂FN₂O₇S

US8952036

4-({[(3r,5r,6s)-1-[(1s)-2-(Tert-Butylsulfonyl)-1-Cyclopropylethyl]-6-(4-Chloro-3-Fluorophenyl)-5-(3-Chlorophenyl)-3-Methyl-2-Oxopiperidin-3-Yl]acetyl}amino)-2-Methoxybenzoic Acid;

4-[[2-[(3R,5R,6S)-1-[(1S)-2-tert-butylsulfonyl-1-cyclopropylethyl]-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl]acetyl]amino]-2-methoxybenzoic acid

MDM2 inhibitor that is useful as therapeutic agent, particularly for the treatment of cancers

DETAILS COMING…………

p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of numerous genes involved in cell cycle arrest, apoptosis, senescence, and DNA repair. Unlike normal cells, which have infrequent cause for p53 activation, tumor cells are under constant cellular stress from various insults including hypoxia and pro-apoptotic oncogene activation. Thus, there is a strong selective advantage for inactivation of the p53 pathway in tumors, and it has been proposed that eliminating p53 function may be a prerequisite for tumor survival. In support of this notion, three groups of investigators have used mouse models to demonstrate that absence of p53 function is a continuous requirement for the maintenance of established tumors. When the investigators restored p53 function to tumors with inactivated p53, the tumors regressed.

p53 is inactivated by mutation and/or loss in 50% of solid tumors and 10% of liquid tumors. Other key members of the p53 pathway are also genetically or epigenetically altered in cancer. MDM2, an oncoprotein, inhibits p53 function, and it is activated by gene amplification at incidence rates that are reported to be as high as 10%. MDM2, in turn, is inhibited by another tumor suppressor, p14ARF. It has been suggested that alterations downstream of p53 may be responsible for at least partially inactivating the p53 pathway in p53^WT tumors (p53 wildtype). In support of this concept, some p53^WT tumors appear to exhibit reduced apoptotic capacity, although their capacity to undergo cell cycle arrest remains intact. One cancer treatment strategy involves the use of small molecules that bind MDM2 and neutralize its interaction with p53. MDM2 inhibits p53 activity by three mechanisms: 1) acting as an E3 ubiquitin ligase to promote p53 degradation; 2) binding to and blocking the p53 transcriptional activation domain; and 3) exporting p53 from the nucleus to the cytoplasm. All three of these mechanisms would be blocked by neutralizing the MDM2-p53 interaction. In particular, this therapeutic strategy could be applied to tumors that are p53^WT, and studies with small molecule MDM2 inhibitors have yielded promising reductions in tumor growth both in vitro and in vivo. Further, in patients with p53-inactivated tumors, stabilization of wildtype p53 in normal tissues by MDM2 inhibition might allow selective protection of normal tissues from mitotic poisons.

The present invention relates to a compound capable of inhibiting the interaction between p53 and MDM2 and activating p53 downstream effector genes. As such, the compound of the present invention would be useful in the treatment of cancers, bacterial infections, viral infections, ulcers and inflammation. In particular, the compound of the present invention is useful to treat solid tumors such as: breast, colon, lung and prostate tumors; and liquid tumors such as lymphomas and leukemias. As used herein, MDM2 means a human MDM2 protein and p53 means a human p53 protein. It is noted that human MDM2 can also be referred to as HDM2 or hMDM2.

 PATENT

US8952036

http://www.google.com/patents/US20140243372

Example 4 2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetic acid

Step A. Methyl-4-chloro-3-fluorobenzoate

• A solution of 4-chloro-3-fluoro benzoic acid (450.0 g, 2.586 mol, Fluororochem, Derbyshire, UK) in methanol (4.5 L) was cooled to 0° C. and thionyl chloride (450.0 mL) was added over 30 minutes. The reaction mixture was stirred for 12 hours at ambient temperature. The reaction was monitored by TLC. Upon completion, the solvent was removed under reduced pressure and the residue was quenched with 1.0 M sodium bicarbonate solution (500 mL). The aqueous layer was extracted with dichloromethane (2×5.0 L). The combined organic layer was washed with brine (2.5 L), dried over anhydrous sodium sulfate and concentrated under reduced pressure afforded the title compound as light brown solid. The crude compound was used in the next step without further purification.
• ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.82-7.74 (m, 2H), 7.46 (dd, J=8.2, 7.5 Hz, 1H), 3.92 (s, 3H).

Step B. 1-(4-chloro-3-fluorophenyl)-2-(3-chlorophenyl)ethanone

• Sodium bis(trimethylsilyl)amide (1 M in tetrahydrofuran, 4 L, 4000 mmol) was added over 1 hour to a solution of 3-chlorophenyl acetic acid (250.0 g, 1465 mmol) in anhydrous tetrahydrofuran (1.75 L) at −78° C. under nitrogen. The resulting reaction mixture was stirred for an additional hour at −78° C. Then, a solution of methyl-4-chloro-3-fluorobenzoate (221.0 g, 1175 mmol, Example 4, Step A) in tetrahydrofuran (500 mL) was added over 1 hour at −78° C., and the resulting reaction mixture was stirred at the same temperature for 2 hours. The reaction was monitored by TLC. On completion, reaction mixture was quenched with 2 N hydrochloric acid (2.5 L) and aqueous phase was extracted with ethyl acetate (2×2.5 L). The combined organic layer was washed with brine (2.5 L), dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide the crude material which was purified by flash column chromatography (silica gel: 100 to 200 mesh, product eluted in 2% ethyl acetate in hexane) to afford the title compound as a white solid.
• ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.74 (ddd, J=10.1, 8.9, 1.8 Hz, 2H), 7.56-7.48 (m, 1H), 7.26 (t, J=6.4 Hz, 3H), 7.12 (d, J=5.7 Hz, 1H), 4.22 (s, 2H). MS (ESI) 282.9 [M+H]⁺.

Step C. Methyl 5-(4-chloro-3-fluorophenyl)-4-(3-chlorophenyl)-2-methyl-5-oxopentanoate

• Methyl methacrylate (125.0 g, 1097 mmol) and potassium tert-butoxide (1 M in tetrahydrofuran, 115 mL, 115 mmol) were sequentially added to a solution of 1-(4-chloro-3-fluorophenyl)-2-(3-chlorophenyl)ethanone (327.0 g, 1160 mmol, Example 4, Step B) in anhydrous tetrahydrofuran (2.61 L), at 0° C. The reaction mixture was stirred for 1 hour at 0° C. and then warmed to ambient temperature and stirred for 12 hours. On completion, the reaction was quenched with water (1.0 L) and extracted with ethyl acetate (2×2.5 L). The combined organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude material which was purified by flash column chromatography (silica gel: 60 to 120 mesh, product eluted in 4% ethyl acetate in hexane) affording the title compound (mixture of diastereomers) as light yellow liquid.
  ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.74-7.61 (m, 4H), 7.47-7.40 (m, 2H), 7.28-7.18 (m, 6H), 7.16-7.10 (m, 2H), 4.56 (m, 2H), 3.68 (s, 3H), 3.60 (s, 3H), 2.50-2.39 (m, 2H), 2.37-2.25 (m, 2H), 2.10-2.02 (m, 1H), 1.94 (ddd, J=13.6, 9.1, 4.2 Hz, 1H), 1.21 (d, J=7.0 Hz, 3H), 1.15 (d, J=7.0 Hz, 3H). MS (ESI) 383.0 [M+H]⁺.

Step D. (3S,5R,6R)-6-(4-Chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one and (3R,5R,6R)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

• In a 2000 mL reaction vessel charged with methyl 5-(4-chloro-3-fluorophenyl)-4-(3-chlorophenyl)-2-methyl-5-oxopentanoate (138.0 g, 360 mmol, Example 4, Step C) (which was cooled on ice for 10 minutes before transferring to a glove bag) anhydrous 2-propanol (500 mL), and potassium tert-butoxide (16.16 g, 144 mmol) were sequentially added while in a sealed glove bag under argon. This mixture was allowed to stir for 30 minutes. RuCl₂(S-xylbinap)(S-DAIPEN) (1.759 g, 1.440 mmol, Strem Chemicals, Inc., Newburyport, Mass., weighed in the glove bag) in 30.0 mL toluene was added. The reaction was vigorously stirred at room temperature for 2 hours. The vessel was set on a hydrogenation apparatus, purged with hydrogen 3 times and pressurized to 50 psi (344.7 kPa). The reaction was allowed to stir overnight at room temperature. On completion, the reaction was quenched with water (1.5 L) and extracted with ethyl acetate (2×2.5 L). The organic layer was washed with brine (1.5 L), dried over anhydrous sodium sulfate and concentrated under reduced pressure to get crude material which was purified by flash column chromatography (silica gel; 60-120 mesh; product eluted in 12% ethyl acetate in hexane) to provide a dark colored liquid as a mixture of diastereomers.
• The product was dissolved in (240.0 g, 581 mmol) in tetrahydrofuran (1.9 L) and methanol (480 mL), and lithium hydroxide monohydrate (2.5 M aqueous solution, 480.0 mL) was added. The reaction mixture was stirred at ambient temperature for 12 hours. On completion, the solvent was removed under reduced pressure and the residue was acidified with 2 N hydrochloric acid to a pH between 5 and 6. The aqueous phase was extracted with ethyl acetate (2×1.0 L). The combined organic layer was washed with brine (750 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide a dark colored liquid, which was used without further purification.
• A portion of the crude intermediate (25.4 g, predominantly seco acid) was added to a 500 mL round bottom flask, equipped with a Dean-Stark apparatus. Pyridinium p-toluenesulfonate (0.516 g, 2.053 mmol) and toluene (274 mL) were added, and the mixture was refluxed for 1 hour (oil bath temperature about 150° C.). The reaction was cooled to room temperature and concentrated under reduced pressure. The reaction was diluted with saturated aqueous sodium bicarbonate (150 mL), extracted with diethyl ether (2×150 mL), and washed with brine (150 mL). The combined organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification by flash column chromatography (divided into 3 portions, 330 g SiO₂/each, gradient elution of 0% to 30% acetone in hexanes, 35 minutes) provided the title compounds as a pale yellow solid and a 1:1.6 mixture of diastereomers at C2. MS (ESI) 353.05 [M+H]⁺.
• Step E. (3S,5R,6R)-3-Allyl-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one
• (3S,5R,6R)-6-(4-Chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one and (3R,5R,6R)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one (18 g, 51.0 mmol, Example 4, Step D) was added to an oven dried 500 mL round-bottom flask. The solid was dissolved in anhydrous toluene and concentrated to remove adventitious water. 3-Bromoprop-1-ene (11.02 mL, 127 mmol, passed neat through basic alumina prior to addition) in tetrahydrofuran (200 mL) was added and the reaction vessel was evacuated and refilled with argon three times. Lithium bis(trimethylsilyl)amide (1.0 M, 56.1 mL, 56.1 mmol) was added dropwise at −40° C. (dry ice/acetonitrile bath) and stirred under argon. The reaction was allowed to gradually warm to −10° C. and stirred at −10° C. for 3 hours. The reaction was quenched with saturated ammonium chloride (10 mL), concentrated, and the crude product was diluted in water (150 mL) and diethyl ether (200 mL). The layers were separated and the aqueous layer was washed twice more with diethyl ether (200 mL/each). The combined organic layer was washed with brine (100 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to a residue. The residue was purified by flash chromatography (2×330 g silica gel columns, gradient elution of 0% to 30% acetone in hexanes) to provide the title compound as a white solid. The product can alternatively be crystallized from a minimum of hexanes in dichloromethane. Enantiomeric excess was determined to be 87% by chiral SFC (90% CO₂, 10% methanol (20 mM ammonia), 5.0 mL/min, 100 bar (10,000 kPa), 40° C., 5 minute method, Phenomenex Lux-2 (Phenomenex, Torrance, Calif.) (100 mm×4.6 mm, 5 μm column), retention times: 1.62 min. (minor) and 2.17 min. (major)). The purity could be upgraded to >98% through recrystallization in hexanes and dichloromethane.
• ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.24-7.17 (m, 3H), 6.94 (s, 1H), 6.80 (d, J=7.5 Hz, 1H), 6.48 (dd, J=10.0, 1.9 Hz, 1H), 6.40 (d, J=8.3 Hz, 1H), 5.90-5.76 (m, 1H), 5.69 (d, J=5.2 Hz, 1H), 5.20-5.13 (m, 2H), 3.81 (dd, J=13.9, 6.9 Hz, 1H), 2.62 (dd, J=13.8, 7.6 Hz, 1H), 2.50 (dd, J=13.8, 7.3 Hz, 1H), 1.96 (d, J=8.4 Hz, 2H), 1.40 (s, 3H). MS (ESI) 393.1 [M+H]⁺.

Step F. (2S)-2-((2R)-3-(4-Chloro-3-fluorophenyl)-2-(3-chlorophenyl)-3-hydroxypropyl)-N—((S)-1-cyclopropyl-2-hydroxyethyl)-2-methylpent-4-enamide

• Sodium methoxide (25% in methanol, 60.7 ml, 265 mmol) was added to a solution of (S)-2-amino-2-cyclopropylethanol hydrochloride (36.5 g, 265 mmol, NetChem Inc., Ontario, Canada) in methanol (177 mL) at 0° C. A precipitate formed during the addition. After the addition was complete, the reaction mixture was removed from the ice bath and warmed to room temperature. The reaction mixture was filtered under a vacuum and the solid was washed with dichloromethane. The filtrate was concentrated under a vacuum to provide a cloudy brown oil. The oil was taken up in dichloromethane (150 mL), filtered under a vacuum and the solid phase washed with dichloromethane to provide the filtrate as a clear orange solution. The solution was concentrated under a vacuum to provide (S)-2-amino-2-cyclopropylethanol as a light brown liquid.
• (3S,5R,6R)-3-Allyl-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one (32 g, 81 mmol, Example 4, Step E) was combined with (S)-2-amino-2-cyclopropylethanol (26.7 g, 265 mmol) and the suspension was heated at 100° C. overnight. The reaction mixture was cooled to room temperature, diluted with ethyl acetate and washed with 1 N hydrochloric acid (2×), water, and brine. The organic layer was dried over magnesium sulfate and concentrated under vacuum to provide the title compound as a white solid.
• ¹H NMR (500 MHz, CDCl₃, δ ppm): 0.23-0.30 (m, 2H), 0.45-0.56 (m, 2H), 0.81 (m, 1H), 1.12 (s, 3H), 1.92-2.09 (m, 3H), 2.39 (dd, J=13.6, 7.2 Hz, 1H), 2.86 (br s, 1H), 2.95 (dtd, J=9.5, 6.3, 6.3, 2.9 Hz, 1H), 3.44 (dd, J=11.0, 5.6 Hz, 1H), 3.49 (m, 1H), 3.61 (dd, J=11.0, 2.9 Hz, 1H), 4.78 (d, J=5.6 Hz, 1H), 4.95-5.13 (m, 2H), 5.63 (m, 1H), 5.99 (d, J=6.4 Hz, 1H), 6.94-7.16 (m, 3H), 7.16-7.32 (m, 4H). MS (ESI) 494 [M+H]⁺.

Step G. (3S,5R,6S)-3-Allyl-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-1-((S)-1-cyclopropyl-2-hydroxyethyl)-3-methylpiperidin-2-one

• A solution of (2S)-2-((2R)-3-(4-chloro-3-fluorophenyl)-2-(3-chlorophenyl)-3-hydroxypropyl)-N—((S)-1-cyclopropyl-2-hydroxyethyl)-2-methylpent-4-enamide (40.2 g, 81 mmol, Example 4, Step F) in dichloromethane (80 mL) was added p-toluenesulfonic anhydride (66.3 g, 203 mmol) in dichloromethane (220 mL) at 0° C., and the reaction mixture was stirred for 10 minutes at same the temperature. 2,6-Lutidine (43.6 mL, 374 mmol, Aldrich, St. Louis, Mo.) was added dropwise via addition funnel at 0° C. The reaction mixture was slowly warmed to room temperature, and then it was stirred at reflux. After 24 hours, sodium bicarbonate (68.3 g, 814 mmol) in water (600 mL) and 1,2-dichloroethane (300 mL) were added in succession. The reaction mixture was heated at reflux for an hour and then cooled to room temperature. The layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with 1 N hydrochloric acid, water, and brine, then concentrated under reduced pressure. The residue was purified by flash chromatography (1.5 kg SiO₂ column, gradient elution of 10% to 50% ethyl acetate in hexanes) to provide the title compound as a white solid.
• ¹H NMR (500 MHz, CDCl₃, δ ppm): 0.06 (m, 1H), 0.26 (m, 1H), 0.57-0.67 (m, 2H), 0.85 (m, 1H), 1.25 (s, 3H), 1.85-2.20 (m, 2H), 2.57-2.65 (m, 2H), 3.09 (ddd, J=11.8, 9.8, 4.8 Hz, 1H), 3.19 (t, J=10.0 Hz, 1H), 3.36 (td, J=10.3, 4.6 Hz, 1H), 3.63 (dd, J=11.0, 4.6 Hz, 1H), 4.86 (d, J=10.0 Hz, 1H), 5.16-5.19 (m, 2H), 5.87 (m, 1H), 6.77 (dd, J=7.7, 1.6 Hz, 1H), 6.80-6.90 (m, 2H), 7.02 (t, J=2.0 Hz, 1H), 7.16 (dd, J=10.0, 7.7 Hz, 1H), 7.21 (dd, J=10.0, 1.6 Hz, 1H), 7.29 (t, J=10.0 Hz, 1H). MS (ESI) 476 [M+H]⁺.

Step H. (3S,5S,6R,8S)-8-Allyl-5-(4-chloro-3-fluorophenyl)-6-(3-chlorophenyl)-3-cyclopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium 4-methylbenzenesulfonate

• p-Toluenesulfonic acid monohydrate (30.3 g, 159 mmol, Aldrich, St. Louis, Mo.) was added to a solution of (3S,5R,6S)-3-allyl-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-1-((S)-1-cyclopropyl-2-hydroxyethyl)-3-methylpiperidin-2-one (73.6 g, 154 mmol) in toluene (386 mL). The reaction mixture was heated at reflux using a Dean-Stark apparatus. After 4 hours, the reaction was cooled and concentrated under reduced pressure to provide the title compound as a pale yellow syrup. The crude product was used in next step without further purification.
• ¹H NMR (500 MHz, CDCl₃, δ ppm): −0.25 to −0.10 (m, 2H), 0.08-0.18 (m, 1H), 0.33-0.50 (m, 2H), 1.57 (s, 3H), 1.92 (dd, J=3.7 and 13.9 Hz, 1H), 2.37 (s, 3H), 2.63 (dd, J=7.3 and 13.7 Hz, 1H), 2.72 (dd, J=7.6 and 13.7 Hz, 1H), 2.93 (t, J=13.7 Hz, 1H), 3.29 (m, 1H), 4.51 (t, J=8.6 Hz, 1H), 4.57-4.63 (m, 1H), 5.33 (d, J=17.1 Hz, 1H), 5.37 (d, J=10.5 Hz, 1H), 5.47 (dd, J=9.1 and 10.0 Hz, 1H), 5.75-5.93 (m, 2H), 6.80 (br s, 1H), 7.08 (s, 1H), 7.16-7.20 (m, 5H), 7.25-7.32 (m, 2H), 7.87 (d, J=8.3 Hz, 2H). MS (ESI) 458 [M+H]⁺.
• Step I. (3S,5R,6S)-3-Allyl-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methylpiperidin-2-one
• 2-Methyl-2-propanethiol (15.25 mL, 135 mmol, dried over activated 4 Å molecular sieves) was added to a solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (1.0 M, 135 mL, 135 mmol) at room temperature under argon in a 500 mL round-bottomed flask. The reaction mixture was heated to 60° C. After 30 minutes, a solution of (3S,5S,6R,8S)-8-allyl-5-(4-chloro-3-fluorophenyl)-6-(3-chlorophenyl)-3-cyclopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium 4-methylbenzenesulfonate (78 g, 123 mmol, Example 4, Step H) in anhydrous tetrahydrofuran (100 mL) was added via cannula. The reaction mixture was heated at 60° C. for 3 hours and then cooled to room temperature. The reaction mixture was quenched with water and extracted thrice with ethyl acetate. The organics were pooled, washed with brine, dried over magnesium sulfate, filtered and concentrated under a vacuum to provide a yellow foam. Purification by flash column chromatography (1.5 kg SiO₂ column, gradient elution with 5% to 30% ethyl acetate in hexanes provided the title compound as an off-white foam.
• ¹H NMR (400 MHz, CDCl₃, δ ppm): −0.89 to −0.80 (m, 1H), −0.15 to −0.09 (m, 1H), 0.27-0.34 (m, 1H), 0.41-0.48 (m, 1H), 1.28 (s, 3H), 1.35 (s, 9H), 1.70-1.77 (m, 1H), 1.86 (dd, J=3.1 and 13.5 Hz, 1H), 2.16 (t, J=13.7, 1H), 2.17-2.23 (m, 1H), 2.60-2.63 (m, 3H), 3.09 (dt, J=3.1 and 10.4 Hz, 1H), 3.62 (t, J=11.1 Hz, 1H), 4.70 (d, J=10.1 Hz, 1H), 5.16 (s, 1H), 5.19-5.21 (m, 1H), 5.82-5.93 (m, 1H), 6.65-6.80 (m, 1H), 6.80-6.83 (m, 1H), 6.84-6.98 (m, 1H), 7.05-7.07 (m, 1H), 7.12-7.18 (m, 2H), 7.19-7.26 (m, 1H). MS (ESI) 548.2 [M+H]⁺.

Step J. 2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetic acid

• Ruthenium(III) chloride hydrate (0.562 mg, 2.493 mmol) was added to a mixture of (3S,5R,6S)-3-allyl-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methylpiperidin-2-one (62.17 g, 113 mmol, Example 4, Step I) and sodium periodate (24.67 g) in ethyl acetate (216 mL), acetonitrile (216 mL) and water (324 mL) at 20° C. The temperature quickly rose to 29° C. The reaction mixture was cooled to 20° C. and the remaining equivalents of sodium periodate were added in five 24.67 g portions over 2 hours, being careful to maintain an internal reaction temperature below 25° C. The reaction was incomplete, so additional sodium periodate (13 g) was added. The temperature increased from 22° C. to 25° C. After stirring for an additional 1.5 hours, the reaction mixture was filtered under a vacuum and washed with ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The organics were pooled, washed with brine, dried over magnesium sulfate, filtered and concentrated under a vacuum to provide a dark green foam. Purification by flash column chromatography (1.5 kg SiO₂ column, gradient elution of 0% to 20% isopropanol in hexanes) provided an off-white foam. 15% Ethyl acetate in heptanes (970 mL) was added to the foam, and the mixture was heated at 80° C. until the foam dissolved. The solution was then cooled slowly, and at 60° C. the solution was seeded with previously obtained crystalline material. The mixture was cooled to room temperature and then allowed to stand at room temperature for 2 hours before collecting the solid by vacuum filtration to provide a white solid with a very pale pink hue (57.1 g). The mother liquor was concentrated under a vacuum to provide a pink foam (8.7 g). 15% ethyl acetate in heptanes (130 mL) was added to the foam, and it was heated at 80° C. to completely dissolve the material. The solution was cooled, and at 50° C., it was seeded with crystalline material. After cooling to room temperature the solid was collected by vacuum filtration to provide a white crystalline solid with a very pale pink hue.
• ¹H NMR (500 MHz, CDCl₃, δ ppm): −1.10 to −1.00 (m, 1H), −0.30 to −0.22 (m, 1H), 0.27-0.37 (m, 1H), 0.38-0.43 (m, 1H), 1.45 (s, 9H), 1.50 (s, 3H), 1.87 (dd, J=2.7 and 13.7 Hz, 1H), 1.89-1.95 (m, 1H), 2.46 (t, J=13.7, 1H), 2.69-2.73 (m, 1H), 2.78 (d, J=14.9 Hz, 1H), 2.93 (dd, J=2.0 and 13.7 Hz, 1H), 3.07 (d, J=14.9 Hz, 1H), 3.11 (dt, J=2.7 and 11.0 Hz, 1H), 4.30 (t, J=13.5 Hz, 1H), 4.98 (d, J=10.8 Hz, 1H), 6.75-6.87 (m, 1H), 6.88-6.90 (m, 1H), 6.98 (br s, 1H), 7.02-7.09 (m, 1H), 7.11-7.16 (m, 2H), 7.16-7.25 (m, 1H). MS (ESI) 598.1 [M+H]⁺.

Example 5 4-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoic acid

Step A. Methyl 4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoate

• N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 76 g, 398 mmol) was added to a mixture of 2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetic acid (79.4 g, 133 mmol, Example 4, Step J) and methyl 4-amino-2-methoxybenzoate (26.4 g, 146 mmol) in pyridine (332 mL) at 3° C. The mixture was allowed to warm to room temperature and was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0° C. and added to an ice-cold solution of 1 M hydrochloric acid (1 L). Ether (1 L) was added and the layers were agitated and then separated. The organic layer was washed with 1 M hydrochloric acid (6×500 mL), saturated aqueous sodium bicarbonate (500 mL), brine (500 mL), dried over magnesium sulfate, filtered and concentrated under a vacuum to provide an off-white foam.
• ¹H NMR (400 MHz, CDCl₃, δ ppm): −1.20 to −1.12 (m, 1H), −0.35 to −0.20 (m, 1H), 0.05-0.20 (m, 1H), 0.32-0.45 (m, 1H), 1.45 (s, 9H), 1.48 (s, 3H), 1.86-1.98 (m, 1H), 2.03 (dd, J=2.7 and 13.7 Hz, 1H), 2.43 (t, J=13.7, 1H), 2.64-2.75 (m, 1H), 2.80 (d, J=14.3 Hz, 1H), 2.89-2.96 (m, 2H), 3.24 (dt, J=2.5 and 10.8 Hz, 1H), 3.89 (s, 3H), 3.96 (s, 3H), 4.28-4.36 (m, 1H), 4.98 (d, J=10.8 Hz, 1H), 6.85-6.93 (m, 3H), 6.99 (br s, 1H), 7.06-7.18 (m, 4H), 7.82 (br s, 1H), 7.85 (d, J=8.4 Hz, 1H), 8.81 (br s, 1H). MS (ESI) 761.2 [M+H]⁺.

Step B. 4-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoic acid

• A solution of lithium hydroxide monohydrate (18.2 g, 433 mmol) in water (295 mL) was added to a solution of methyl 4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoate (164.9 g, 217 mmol, Example 5, Step A) in tetrahydrofuran (591 mL) and methanol (197 mL) at room temperature. After stirring for 15 hours at room temperature, a trace amount of the ester remained, so the reaction mixture was heated at 50° C. for 1 hour. When the reaction was complete, the mixture was concentrated under a vacuum to remove the tetrahydrofuran and methanol. The thick mixture was diluted with water (1 L) and 1 M hydrochloric acid (1 L) was added. The resulting white solid was collected by vacuum filtration in a Büchner funnel. The vacuum was removed, and water (1 L) was added to the filter cake. The material was stirred with a spatula to suspend it evenly in the water. The liquid was then removed by vacuum filtration. This washing cycle was repeated three more times to provide a white solid. The solid was dried under vacuum at 45° C. for 3 days to provide the title compound as a white solid.
• ¹H NMR (500 MHz, DMSO-d₆) δ ppm −1.30 to −1.12 (m, 1H), −0.30 to −0.13 (m, 1H), 0.14-0.25 (m, 1H), 0.25-0.38 (m, 1H), 1.30 (s, 3H), 1.34 (s, 9H), 1.75-1.86 (m, 1H), 2.08-2.18 (m, 2H), 2.50-2.60 (m, 1H), 2.66 (d, J=13.7, 1H), 3.02-3.16 (m, 2H), 3.40-3.50 (m, 1H), 3.77 (s, 3H), 4.05-4.20 (m, 1H), 4.89 (d, J=10.5 Hz, 1H), 6.90-6.93 (m, 3H), 7.19 (d, J=8.8 Hz, 1H), 7.22-7.26 (m, 3H), 7.40-7.50 (m, 1H), 7.54 (br s, 1H), 7.68 (d, J=8.6 Hz, 1H) 10.44 (s, 1H), 12.29 (br s, 1H). MS (ESI) 747.2 [M+H]⁺.

PAPER

Discovery of AM-7209, a Potent and Selective 4-Amidobenzoic Acid Inhibitor of the MDM2–p53 Interaction

Abstract

Structure-based rational design and extensive structure–activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2–p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (K_D from ITC competition was 38 pM, SJSA-1 EdU IC₅₀ = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED₅₀ = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED₅₀ = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1

AUTHORS

Experience

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c1(c(ccc(c1)NC(C[C@]2(C[C@@H]([C@H](N(C2=O)[C@H](CS(=O)(=O)C(C)(C)C)C3CC3)c4cc(c(cc4)Cl)F)c5cccc(c5)Cl)C)=O)C(=O)O)OC

CC1(CC(C(N(C1=O)C(CS(=O)(=O)C(C)(C)C)C2CC2)C3=CC(=C(C=C3)Cl)F)C4=CC(=CC=C4)Cl)CC(=O)NC5=CC(=C(C=C5)C(=O)O)OC

Pacritinib

A Jak2 inhibitor potentially for the treatment of acute myeloid Leukemia and myelofibrosis.

ONX-0803; SB-1518
CAS No. 937272-79-2

472.57868 g/mol, C₂₈H₃₂N₄O₃

S*Bio Pte Ltd. and concert innovator

11-(2-pyrrolidin-1-ylethoxy)-14,19-dioxa-5,7,26-triazatetracyclo(19.3.1.1(2,6).1(8,12))heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene

• (16E)-11-[2-(1-Pyrrolidinyl)ethoxy]-14,19-Dioxa-5,7,27-triazatetracyclo[19.3.1.12,6.18,12]heptacosa-1(25),2,4,6(27),8,10,12(26),16,21,23-decaene
• 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene
• SB-1518|||(16E)-11-[2-(1-Pyrrolidinyl)ethoxy]-14,19-dioxa-5,7,27-triazatetracyclo[19.3.1.12,6.18,12]heptacosa-1(25),2,4,6(27),8,10,12(26),16,21,23-decaene

Pacritinib (INN^[1]) is a macrocyclic Janus kinase inhibitor that is being developed for the treatment of myelofibrosis. It mainly inhibits Janus kinase 2 (JAK2). The drug is in Phase III clinical trials as of 2013.^[2] The drug was discovered in Singapore at the labs of S*BIO Pte Ltd. It is a potent JAK2 inhibitor with activity of IC₅₀ = 23 nM for the JAK2WT variant and 19 nM for JAK2V617F with very good selectivity against JAK1 and JAK3 (IC₅₀ = 1280 and 520 nM, respectively).^[3][4] The drug is acquired by Cell Therapeutics, Inc. (CTI) and Baxter international and could effectively address an unmet medical need for patients living with myelofibrosis who face treatment-emergent thrombocytopenia on marketed JAK inhibitors.^[5]

Pacritinib is an orally bioavailable inhibitor of Janus kinase 2 (JAK2) and the JAK2 mutant JAK2V617F with potential antineoplastic activity. Oral JAK2 inhibitor SB1518 competes with JAK2 for ATP binding, which may result in inhibition of JAK2 activation, inhibition of the JAK-STAT signaling pathway, and so caspase-dependent apoptosis. JAK2 is the most common mutated gene in bcr-abl-negative myeloproliferative disorders; the JAK2V617F gain-of-function mutation involves a valine-to-phenylalanine modification at position 617. The JAK-STAT signaling pathway is a major mediator of cytokine activity.

Pacritinib is an orally bioavailable inhibitor of Janus kinase 2 (JAK2) and the JAK2 mutant JAK2V617F with potential antineoplastic activity. Oral JAK2 inhibitor SB1518 competes with JAK2 for ATP binding, which may result in inhibition of JAK2 activation, inhibition of the JAK-STAT signaling pathway, and so caspase-dependent apoptosis. JAK2 is the most common mutated gene in bcr-abl-negative myeloproliferative disorders; the JAK2V617F gain-of-function mutation involves a valine-to-phenylalanine modification at position 617. The JAK-STAT signaling pathway is a major mediator of cytokine activity.

The compound 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene (Compound I) was first described in PCT/SG2006/000352 and shows significant promise as a pharmaceutically active agent for the treatment of a number of medical conditions and clinical development of this compound is underway based on the activity profiles demonstrated by the compound.

• In the development of a drug suitable for mass production and ultimately commercial use acceptable levels of drug activity against the target of interest is only one of the important variables that must be considered. For example, in the formulation of pharmaceutical compositions it is imperative that the pharmaceutically active substance be in a form that can be reliably reproduced in a commercial manufacturing process and which is robust enough to withstand the conditions to which the pharmaceutically active substance is exposed.
• In a manufacturing sense it is important that during commercial manufacture the manufacturing process of the pharmaceutically active substance be such that the same material is reproduced when the same manufacturing conditions are used. In addition it is desirable that the pharmaceutically active substance exists in a solid form where minor changes to the manufacturing conditions do not lead to major changes in the solid form of the pharmaceutically active substance produced. For example it is important that the manufacturing process produce material having the same crystalline properties on a reliable basis and also produce material having the same level of hydration.
• In addition it is important that the pharmaceutically active substance be stable both to degradation, hygroscopicity and subsequent changes to its solid form. This is important to facilitate the incorporation of the pharmaceutically active substance into pharmaceutical formulations. If the pharmaceutically active substance is hygroscopic (“sticky”) in the sense that it absorbs water (either slowly or over time) it is almost impossible to reliably formulate the pharmaceutically active substance into a drug as the amount of substance to be added to provide the same dosage will vary greatly depending upon the degree of hydration. Furthermore variations in hydration or solid form (“polymorphism”) can lead to changes in physico-chemical properties, such as solubility or dissolution rate, which can in turn lead to inconsistent oral absorption in a patient.
• Accordingly, chemical stability, solid state stability, and “shelf life” of the pharmaceutically active substance are very important factors. In an ideal situation the pharmaceutically active substance and any compositions containing it, should be capable of being effectively stored over appreciable periods of time, without exhibiting a significant change in the physico-chemical characteristics of the active substance such as its activity, moisture content, solubility characteristics, solid form and the like.
• In relation to 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene initial studies were carried out on the hydrochloride salt and indicated that polymorphism was prevalent with the compound being found to adopt more than one crystalline form depending upon the manufacturing conditions. In addition it was observed that the moisture content and ratio of the polymorphs varied from batch to batch even when the manufacturing conditions remained constant. These batch-to-batch inconsistencies and the exhibited hygroscopicity made the hydrochloride salt less desirable from a commercial viewpoint.
• Accordingly it would be desirable to develop one or more salts of 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene which overcome or ameliorate one or more of the above identified problems.

PATENT

US 2011263616

http://www.google.com/patents/US20110263616

PATENT

http://www.google.com/patents/US8415338

Representative Procedure for the Synthesis of Compounds Type (XVIIId) [3-(2-Chloro-pyrimidin-4-yl)-phenyl]-methanol (XIIIa2)

Compound (XIIIa2) was obtained using the same procedure described for compound (XIIIa1); LC-MS (ESI positive mode) m/z 221 ([M+H]⁺).

4-(3-Allyloxymethyl-phenyl)-2-chloro-pyrimidine (XVa2)

Compound (XVa2) was obtained using the same procedure described for compound (XVa1); LC-MS (ESI positive mode) m/z 271 ([M+H]⁺).

[4-(3-Allyloxymethyl-phenyl)-pyrimidin-2-yl]-[3-allyloxymethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (XVIId1)

Compound (XVIId1) was obtained using the same procedure described for compound (XVIIb1); LC-MS (ESI positive mode) m/z 501.

Macrocycle Example 3 Compound 13

Compound (13) was obtained using the same procedure described for compound (1) HPLC purity at 254 nm: 99%; LC-MS (ESI positive mode) m/z 473 ([M+H]⁺); ¹H NMR (MeOD-d₄) δ 8.79 (d, 1H), 8.46 (d, 1H), 8.34-8.31 (m, 1H), 7.98-7.96 (m, 1H), 7.62-7.49 (m, 2H), 7.35 (d, 1H), 7.15-7.10 (m, 1H), 7.07-7.02 (m, 1H), 5.98-5.75 (m, 2H, 2×=CH), 4.67 (s, 2H), 4.67 (s, 2H), 4.39-4.36 (m, 2H), 4.17 (d, 2H), 4.08 (d, 2H), 3.88-3.82 (m, 2H), 3.70 (t, 2H), 2.23-2.21 (m, 2H), 2.10-2.07 (m, 2H).

References

• “International Nonproprietary Names for Pharmaceutical Substances (INN) List 104” (PDF). WHO Drug Information 24 (4): 386. 2010.
• 2
• “JAK-Inhibitoren: Neue Wirkstoffe für viele Indikationen”. Pharmazeutische Zeitung (in German) (21). 2013.
• 3
• William, A. D.; Lee, A. C. -H.; Blanchard, S. P.; Poulsen, A.; Teo, E. L.; Nagaraj, H.; Tan, E.; Chen, D.; Williams, M.; Sun, E. T.; Goh, K. C.; Ong, W. C.; Goh, S. K.; Hart, S.; Jayaraman, R.; Pasha, M. K.; Ethirajulu, K.; Wood, J. M.; Dymock, B. W. (2011). “Discovery of the Macrocycle 11-(2-Pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene (SB1518), a Potent Janus Kinase 2/Fms-Like Tyrosine Kinase-3 (JAK2/FLT3) Inhibitor for the Treatment of Myelofibrosis and Lymphoma”. Journal of Medicinal Chemistry 54 (13): 4638–58. doi:10.1021/jm200326p. PMID 21604762.
• 4
• Poulsen, A.; William, A.; Blanchard, S. P.; Lee, A.; Nagaraj, H.; Wang, H.; Teo, E.; Tan, E.; Goh, K. C.; Dymock, B. (2012). “Structure-based design of oxygen-linked macrocyclic kinase inhibitors: Discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)”. Journal of Computer-Aided Molecular Design 26 (4): 437–50. doi:10.1007/s10822-012-9572-z. PMID 22527961.
• 5

http://www.pmlive.com/pharma_news/baxter_licenses_cancer_drug_from_cti_in_$172m_deal_519143

Pacritinib

Systematic (IUPAC) name
(16E)-11-[2-(1-Pyrrolidinyl)ethoxy]-14,19-dioxa-5,7,26-triazatetracyclo[19.3.1.12,6.18,12]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene
Clinical data
Legal status	Investigational
Routes of administration	Oral
Identifiers
ATC code	None
PubChem	CID: 46216796
ChemSpider	28518965
ChEMBL	CHEMBL2035187
Synonyms	SB1518
Chemical data
Formula	C28H32N4O3
Molecular mass	472.58 g/mol

///////

c1cc2cc(c1)-c3ccnc(n3)Nc4ccc(c(c4)COC/C=C/COC2)OCCN5CCCC5

C1CCN(C1)CCOC2=C3COCC=CCOCC4=CC=CC(=C4)C5=NC(=NC=C5)NC(=C3)C=C2

SB1578

ONX 0805

(9E)-15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12,25-trioxa-19,21,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14(26), 15,17,20,22-nonaene

7,​12,​26-​Trioxa-​19,​21,​24-​triazatetracyclo[18.​3.1.1^2,​5.1^14,​18]​hexacosa-​1(24)​,​2,​4,​9,​14,​16,​18(25)​,​20,​22-​nonaene, 15-​[2-​(1-​pyrrolidinyl)​ethoxy]​-​, (9E)​-

Phase 1 clinical trials

C₂₆ H₃₀ N₄ O₄

CAS 937273-04-6

CITRATE 1262279-15-1

HCL 1262279-16-2

S*Bio Pte Ltd INNOVATOR

US8153632

SB1578 (disclosed in WO2007058627 and in WO2011008172 as the citrate salt) is in ongoing phase I studies for the treatment of rheumatoid arthritis. SB 1578 is shown below.

SB1578, also known as ONX-0805, is a novel, orally bioavailable JAK2 inhibitor with specificity for JAK2 within the JAK family and also potent activity against FLT3 and c-Fms. SB1578 blocks the activation of these kinases and their downstream signaling in pertinent cells, leading to inhibition of pathological cellular responses. The biochemical and cellular activities of SB1578 translate into its high efficacy in two rodent models of arthritis. SB1578 not only prevents the onset of arthritis but is also potent in treating established disease in collagen-induced arthritis mice with beneficial effects on histopathological parameters of bone resorption and cartilage damage. SB1578 abrogates the inflammatory response and prevents the infiltration of macrophages and neutrophils into affected joints. It also leads to inhibition of Ag-presenting dendritic cells and inhibits the autoimmune component of the disease. In summary, SB1578 has a unique kinase spectrum, and its pharmacological profile provides a strong rationale for the ongoing clinical development in autoimmune diseases. ( J Immunol. 2012 Oct 15;189(8):4123-34)

Synonym: ONX 0805; ONX0805; ONX0805; SB1578; SB1578; SB 1578.

PATENT

WO 2011008172

http://www.google.im/patents/WO2011008172A1?cl=en

The compound 9E-15-(2-pyrrolidin-1-yl-ethoxy)-7,12,25-trioxa-19,21 ,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1 (24),2,4,9,14,16_l18(26)_l20,22-nonaene (Compound I) was first described in PCT/SG2006/000352 and shows significant promise as a pharmaceutically active agent for the treatment of a number of medical conditions. Pharmaceutical development of this compound is underway based on the activity profiles demonstrated by the compound.

Compound I

In the development of a drug suitable for mass production and ultimately commercial use acceptable levels of drug activity against the target of interest is only one of the important variables that must be considered. For example, in the formulation of pharmaceutical compositions it is imperative that the pharmaceutically active substance be in a form that can be reliably reproduced in a commercial

manufacturing process and which is robust enough to withstand the conditions to which the pharmaceutically active substance is exposed.

From a manufacturing perspective, it is important that the commercial manufacturing process of a pharmaceutically active substance is such that the same material is produced when the same manufacturing conditions are used. In addition, it is desirable that the pharmaceutically active substance exists in a solid form where minor changes to the manufacturing conditions do not lead to major changes in the solid form of the pharmaceutically active substance produced. For example, it is important that the manufacturing process produces material having the same crystalline properties on a reliable basis, and also that the process produces material having the same level of hydration.

In addition, it is important that the pharmaceutically active substance be stable to degradation, hygroscopicity and subsequent changes to its solid form. This is important to facilitate the incorporation of the pharmaceutically active ingredient into pharmaceutical formulations. If the pharmaceutically active substance is hygroscopic (“sticky”) in the sense that it absorbs water over time it is almost impossible to reliably formulate the pharmaceutically active substance into a drug as the amount of substance to be added to provide the same dosage will vary greatly depending upon the degree of hydration. Furthermore, variations in hydration or solid form (“polymorphism”) can lead to changes in physico-chemical properties, such as solubility or dissolution rate, which can in turn lead to inconsistent oral absorption in a patient.

Accordingly, chemical stability, solid state stability, and “shelf life” of the pharmaceutically active agent are very important factors. In an ideal situation the pharmaceutically active agent and any compositions containing it, should be capable of being effectively stored over appreciable periods of time without exhibiting a significant change in the physico-chemical characteristics of the active component such as its activity, moisture content, solubility characteristics, solid form and the like.

In relation to 9E-15-(2-pyrrolidin-1-yl-ethoxy)-7,12,25-trioxa-19,21 ,24-triaza-tetracyclo[18.3.1.1 (2,5).1(14,18)]hexacosa-1(24),2,4,9,14,16,18(26),20,22-nonaene

initial studies were carried out on the hydrochloride salt and indicated that polymorphism was prevalent, with the compound being found to adopt more than one crystalline form depending upon the manufacturing conditions. In addition it was observed that the ratio of the polymorphs varied from batch to batch even when the manufacturing conditions remained constant. These batch-to-batch inconsistencies made the hydrochloride salt less desirable from a commercial viewpoint.

Accordingly it would be desirable to develop salts of 9E-15-(2-pyrrolidin-1-yl-ethoxy)-7, 12,25-trioxa-i 9,21 ,24-triaza-tetracyclo[18.3.1.1 (2,5).1 (14,18)]hexacosa-1(24)_l2,4,9,14,16,18(26),20,22-nonaene which overcome or ameliorate one or more of the above identified problems.

Figure 22 shows a ¹H NMR spectrum for Batch 4 in d6-DMSO.

Figure 23 shows a ¹H NMR spectrum for Batch 4 in D₂O.

List of hydrochloride and citrate salt batches used for comparative studies

Example 4 – Formation of the Citrate salt (Batch 4) in THF as solvent:

The free base of compound 1 (0.30Og, 0.648mmoles, 1.eq) was added to 12mL of THF. The solution was heated to reflux until complete dissolution was observed and maintained for 1h. A solution of citric acid (0.149g, 0.778mmoles, 1.2eq) dissolved in 12mL THF was then added slowly at reflux conditions. The mixture was refluxed for a further 15min then cooled. Crystallization was observed on gradual cooling. The crystals were stirred at room temperature for 12h and filtered under vacuum. The product was dried under vacuum to afford 250mg.

PATENT

http://www.google.im/patents/WO2007058627A1?cl=en

Representative procedure for the synthesis of compounds type (XVIIIf)

5-(2-Chloro-pyrimidin-4-yl)-furan-2-carbaldehyde (XIIIfI)

(XIIfI) (XIIIH) .

Compound (XIIIfI) was obtained using the same procedure described for compound (XIIIeI); LC-MS (ESI positive mode) /τVz 209 ([M+H]⁺)

[5-(2-Chloro-pyrimidin-4-yl)-furan-2-yl]-methanol (Xlllf2)

Compound (Xlllf2) was obtained using the same procedure described for compound (XXIb); LC-MS (ESI positive mode) m/z 211 ([M+H]⁺).

4-(5-Allyloxymethyl-furan-2-yl)-2-chloro-pyrimidine (XVfI)

Compound (XVfI) was obtained using the same procedure described for compound (XXIIb); LC-MS (ESI positive mode) m/z 251 ([M+H]⁺).

^-(S-Allyloxymethyl-furan-Σ-yO-pyrimidin^-yll-IS-allyloxymethyl^^-pyrrolidin-i-yl- ethoxy)-phenyl]-amine (XVIIfI)

(XVIb2) (XVIIfI)

Compound (XVIIfI) was obtained using the same procedure described for compound (XVIIbI); LC-MS (ESI positive mode) m/z 491.

Macrocycle Example 6 (Compound 38)

(XVIIfI)

Compound (38) was obtained using the same procedure described for compound (1) HPLC purity at 254nm: 99%; LC-MS (ESI positive mode) m/z 463 ([M+H]⁺); ¹H NMR (MeOD-d₄) δ 8.90 (d, 1 H), 8.33 (d, 1 H), 7.37 (d, 1 H), 7.17 (d, 1 H), 7.14-7.11 (m, 1 H)₁ 7.04 (d, 1 H), 6.67 (d, 1 H), 6.04 (dt, 1 H, CH, J = 5.2Hz, J_trans = 15.8Hz), 5.96 (dt, 1 H, CH, J = 5.0Hz, J_tran_s = 15.8Hz), 4.65 (s, 2H), 4.62 (s, 2H), 4.37 (t, 2H), 4.14 (d, 2H), 4.09 (d, 2H), 3.81 (br s, 2H), 3.66 (t, 2H), 3.33 (s, 2H), 2.21-1.98 (m, 4H).

PAPER

Discovery of the Macrocycle (9E)-15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12,25-trioxa-19,21,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14(26),15,17,20,22-nonaene (SB1578), a Potent Inhibitor of Janus Kinase 2/Fms-LikeTyrosine Kinase-3 (JAK2/FLT3) for the Treatment of Rheumatoid Arthritis

http://pubs.acs.org/doi/abs/10.1021/jm201454n

Herein, we describe the synthesis and SAR of a series of small molecule macrocycles that selectively inhibit JAK2 kinase within the JAK family and FLT3 kinase. Following a multiparameter optimization of a key aryl ring of the previously described SB1518 (pacritinib), the highly soluble 14l was selected as the optimal compound. Oral efficacy in the murine collagen-induced arthritis (CIA) model for rheumatoid arthritis (RA) supported 14l as a potential treatment for autoimmune diseases and inflammatory disorders such as psoriasis and RA. Compound 14l (SB1578) was progressed into development and is currently undergoing phase 1 clinical trials in healthy volunteers.

(9E)-15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12,25-trioxa-19,21,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14(26), 15,17,20,22-nonaene (14l)

S*Bio Pte Ltd  

Phone:+65 6827 5000

S*BIO Pte Ltd. provides research and clinical development services for small molecule drugs for the treatment of cancer in Singapore. The company’s products include JAK2 inhibitors, such as SB1518 for leukemia/myelofibrosis, lymphoma, and polycythemia; and SB1578 for RA/psoriasis. The company also offers SB939, a histone deacetylases for MDS/AML+combo, prostate cancer, sarcoma, pediatric tumor, and myelofibrosis; SB2602, a mTOR inhibitor; SB2343, a mTOR/PI3K inhibitor; and SB1317, a CDK/Flt3 inhibitor. The company was founded in 2000 and is based in Singapore. S*BIO Pte Ltd. operates as a subsidiary of Chiron Corporation Limited.

AUTHOR’S

Anthony william

Doctor of Philosophy

Team Leader

Agency for Science, Technology…, Singapore ·

Institute of Chemical and Engineering Sciences (ICES)

Agency for Science, Technology and Research (A*STAR)

Agency for Science, Technology…, Singapore · Institute of Chemical and Engineering Sciences (ICES)

NEXT………..

Dr. Babita Madan,
Scientist,
S*BIO Pte Ltd,
Singapore.

SEE……..http://apisynthesisint.blogspot.in/2016/01/sb1578-onx-0805.html

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N3=C1NC(=CC=N1)c2oc(cc2)COCC=CCOCc5cc3ccc5OCCN4CCCC4

OR

C1(C2=CC=C(O2)COC/C=C/COCC3=CC(N4)=CC=C3OCCN5CCCC5)=NC4=NC=C1

(2S,4S,5S,7S)-5-amino-N-(2-carbamoyl-2,2-dimethylethyl)-4-hydroxy-7-{[4-methoxy-3-(3-methoxypropoxy)phenyl]methyl}-8-methyl-2-(propan-2-yl)nonanamide,  CAS 173334-57-1, base

CAS 173334-58-2,aliskiren hemifumarate

Aliskiren is a renin inhibitor. It was approved by the U.S. Food and Drug Administration in 2007 for the treatment of hypertension.

Tekturna contains aliskiren hemifumarate, a renin inhibitor, that is provided as tablets for oral administration. Aliskiren hemifumarate is chemically described as (2S,4S,5S,7S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-octanamide hemifumarate and its structural formula is

Molecular formula: C₃₀H₅₃N₃O₆ • 0.5 C₄H₄O₄

Aliskiren hemifumarate is a white to slightly yellowish crystalline powder with a molecular weight of 609.8 (free base- 551.8). It is soluble in phosphate buffer, n-octanol, and highly soluble in water.

Aliskiren (INN) (trade names Tekturna, US; Rasilez, UK and elsewhere) is the first in a class of drugs called direct renin inhibitors. Its current licensed indication is essential (primary) hypertension.

Aliskiren was co-developed by the Swiss pharmaceutical companies Novartis andSpeedel.^[1][2] It was approved by the US Food and Drug Administration in 2007 for the treatment of primary hypertension.^[3]

In December 2011, Novartis had to halt a clinical trial of the drug after discovering increased incidence of nonfatal stroke, renal complications, hyperkalemia, and hypotension in patients with diabetes and renal impairment (ALTITUDE Trial ).^[4] [5]

As a result, in April 20, 2012:

• A new contraindication was added to the product label concerning the use of aliskiren with angiotensin receptor blockers (ARBs) or angiotensin-converting enzyme inhibitors (ACEIs) in patients with diabetes because of the risk of renal impairment, hypotension, and hyperkalemia.
• A warning to avoid use of aliskiren with ARBs or ACEIs was also added for patients with moderate to severe renal impairment (i.e., where glomerular filtration rate is less than 60 ml/min).

Renin, the first enzyme in the renin-angiotensin-aldosterone system, plays a role in blood pressure control. It cleaves angiotensinogen to angiotensin I, which is in turn converted byangiotensin-converting enzyme (ACE) to angiotensin II. Angiotensin II has both direct and indirect effects on blood pressure. It directly causes arterial smooth muscle to contract, leading to vasoconstriction and increased blood pressure. Angiotensin II also stimulates the production of aldosterone from the adrenal cortex, which causes the tubules of the kidneys to increase reabsorption of sodium, with water following, thereby increasing plasma volume, and thus blood pressure. Aliskiren binds to the S3^bp binding site of renin, essential for its activity.^[6] Binding to this pocket prevents the conversion of angiotensinogen to angiotensin I. Aliskiren is also available as combination therapy withhydrochlorothiazide.^[7]

Many drugs control blood pressure by interfering with angiotensin or aldosterone. However, when these drugs are used chronically, the body increases renin production, which drives blood pressure up again. Therefore, doctors have been looking for a drug to inhibit renin directly. Aliskiren is the first drug to do so.^[8][9]

Aliskiren may have renoprotective effects independent of its blood pressure−lowering effect in patients with hypertension, type 2 diabetes, and nephropathy, who are receiving the recommended renoprotective treatment. According to the AVOID study, researchers found that treatment with 300 mg of aliskiren daily, as compared with placebo, reduced the mean urinary albumin-to-creatinine ratio by 20%, with a reduction of 50% or more in 24.7% of the patients who received aliskiren as compared with 12.5% of those who received placebo. Furthermore, the AVOID trial showed treatment with 300 mg of aliskiren daily reduces albuminuria in patients with hypertension, type 2 diabetes, and proteinuria, who are receiving the recommended maximal renoprotective treatment with losartan and optimal antihypertensive therapy. Therefore, direct renin inhibition will have a critical role in strategic renoprotective pharmacotherapy, in conjunction with dual blockade of the renin−angiotensin−aldosterone system with the use of ACE inhibitors and angiotensin II–receptor blockers, very high doses of angiotensin II−receptor blockers, and aldosterone blockade.^[10]

Aliskiren is a minor substrate of CYP3A4 and, more important, P-glycoprotein:

• It reduces furosemide blood concentration.
• Atorvastatin may increase blood concentration, but no dose adjustment is needed.
• Due to possible interaction with ciclosporin, the concomitant use of ciclosporin and aliskiren is contraindicated.
• Caution should be exercised when aliskiren is administered with ketoconazole or other moderate P-gp inhibitors (itraconazole, clarithromycin, telithromycin, erythromycin, or amiodarone).
• Doctors should stop prescribing aliskiren-containing medicines to patients with diabetes (type 1 or type 2) or with moderate to severe kidney impairment who are also taking an ACE inhibitor or ARB, and should consider alternative antihypertensive treatment as necessary.^[13]

• Aliskiren (I) is a second generation renin inhibitor with renin-angiotensin system (RAS) as its target. It’s used clinically in the form of Aliskiren hemifumarate (Rasilez®) and was approved by FDA in May, 2007.
• Aliskiren has the chemical name: (2S, 4S, 5S, 7S)-5-amino-N-(2-carbamoyl-2-methylpropyl)-4-hydroxy-2-isopropyl-7-[4-methoxy-3-(3-methoxypropoxy)benzyl]-8-methyloctanamide (CAS No.: 173334-57-1). Its chemical structure is illustrated with Formula I given below:
• The method of preparation for Aliskiren and its intermediates has been reported in US7132569 , WO0208172 , US5559111 (equivalent patent toCN1266118 ), US5606078 , CN101016253 , WO2007/045421 ,EP2062874 , Helvetica ChimicaActa (2005, 3263-3273).

• In US7132569 , WO0208172 et al., the preparation of Aliskiren (I) comprises the following steps as described in reaction scheme 1: coupling 2-(3-methoxypropoxy)-4-((R)-2-(bromomethyl)-3-methylbutyl)-1-methoxybenzene (II) with (2S, 4E)-5-chloro-2-isopropyl-4-pentenoic acid derivative (III) to obtain the compound of formula IV; halolactonization of the compound of formula IV to obtain the compound of formula V; then substituting the compound of formula V with azide to obtain the compound of formula VI; ring-opening the compound of formula VI with 3-amino-2,2-dimethylpropionamide (VII) in the presence of 2-hydroxypyridine and triethylamine to obtain the compound of formula VIII and a final catalytic hydrogenation of the compound of formula VIII to obtain Aliskiren (I). This preparation process is illustrated in Reaction Scheme 1.
• In the patented preparation described above, chiral starting materials with the compounds of formula II and III are utilized to obtain the compound of formula IV. However, the reactions followed after the preparation of the compound of formula IV, such as the halolactonization and especially the substitutive reaction between the compound of formula V and azide, have problems of low yields and numerous by-products, which is not conducive to industrial scale production.
• US5559111 (equivalent patent CN1266118 ) and US5606078 et al. report the preparation of the compound of formula XI via Grignard reaction with 4-bromo-1-methoxy-2-(3-methoxypropoxy)benzene (IX) and the compound of formula X as starting materials as illustated in Reaction Scheme 2:

• In the patented preparation described above, there are multiple reaction steps in the preparation of the compound of formula X from the compound of formula XII. The key steps, as described in Reaction Scheme 3, involve selective reduction agents such as sodium tri-tert-butoxyaluminum hydride and diisobutylaluminium hydride to prepare aldehyde and the reaction conditions need to be very well-controlled.
• [0009]
  The compound of formula XI prepared by reaction scheme 2 could then be converted into Aliskiren (I) after multiple catalytic hydrogenation, protection and de-protection. In this method of preparation, a stepwise catalytic hydrogenation, azido reduction and dehydroxylation were implemented to reduce by-products during the catalytic hydrogenation. In addition, it is necessary to protect and de-protect the free hydroxyl group during the preparation. This synthetic scheme has disadvantage of multiple synthetic steps, tedious operation, lengthy overall reaction duration, low yield and particularly high production cost for the starting compound of formula X.

• WO2007/045421 has reported an improved preparation method in which the starting material 4-bromo-1-methoxy-2-(3-methoxypropoxy)benzene (IX) firstly reacts with the compound of formula XIII via Grignard reaction to obtain the compound of formula XIV, and then followed by catalytic hydrogenation and ketone reduction to yield the compound of formula XV-A, as illustrated in Reaction Scheme 4:
• In the above preparation, expensive reagents, such as sodium tri-tert-butoxyaluminum hydride and diisobutylaluminium hydride were eliminated, but additional synthetic steps were introduced. In addition, the preparation of the compound of formula XV-A prepared from the compound of formula XIV via ketone reduction required extended reaction time, great amount of catalyst with multiple small addition and good operation skills.
• EP2062874A1 provides a method in preparing the compound of formula XVI. In this method, the compound of formula XVII is obtained from the compound of formula XVI via halogenation. A corresponding Grignard reagent is firstly prepared from the compound of formula IX or XVII reacting with magnesium, which is then couples with another chemical in the presence of the metal catalyst iron(III) acetylacetonate (Fe(acac)3) to obtain the compound of formula XVIII as described in Reaction Scheme 5:
• In EP2062874A1 , the compound of formula XVIII reacts with 3-amino-2,2-dimethylpropionamide (VII). The resulted product is then through reduction of the azio group to obtain Aliskiren (I). In this patent, detailed experimental protocol was not provided although N-methylpyrrolidone was mentioned as solvent. We found: 1) it is difficult to prepare the Grgnard reagent from the compound of formula IX; 2) the compounds of formula XVII and XVIII are not quite stable in the presence of iron(III) acetylacetonate. In addition, the yield in preparing the compound of formula XVIII was extremely low.

the spiro aldehyde (XLVII) is treated with N-benzylhydroxylamine in dichloromethane to give nitrone (LII), which is submitted to a Grignard reaction with the magnesium derivative of intermediate (XXX) in THF to afford the adduct (LIII) as a mixture of epimers at the amino group. Simultaneous N-dehydroxylation and cleavage of the spiro function of (LIII) by means of Zn, Cu (OAc) 2 in AcOH / water gives lactone (LIV), which is condensed with 3-amino- 2,2-dimethylpropionamide (XIX) by means of TEA and 2-hydroxypyridine giving the adduct (LV). Finally, the benzylamino group of (LV) is removed with H2 over Pd / C in methanol to yield a mixture of two epimers at the amino group, from which aliskiren is separated.

NMR

ALISKIREN BASE

EP2546243A1

MS m/z: 552.6 (M+H)⁺; 1H-NMR (400 MHz, CDCl₃) δ 6.88-6.75 (m, 3H), 4.08-4.04 (t, J = 6.3Hz, 2H), 3.79 (s, 3H), 3.60-3.55 (t, J = 6.3Hz, 2H), 3.30 (s, 3H), 3.30-3.25 (m, 3H), 2.69 (m, 2H), 2.49 (m, 1H), 2.27 (m, 1H), 2.04 (m, 2H), 1.78-1.35 (m, 7H), 1.10 (m, 6H), 0.90 (m, 12H) ppm.

Paper

A novel synthesis of the renin inhibitor aliskiren based on an unprecedented disconnection between C5 and C6 was developed, in which the C5 carbon acts as a nucleophile and the amino group is introduced by a Curtius rearrangement, which follows a simultaneous stereocontrolled generation of the C4 and C5 stereogenic centers by an asymmetric hydrogenation. Operational simplicity, step economy, and a good overall yield makes this synthesis amenable to manufacture on scale.

Convergent Synthesis of the Renin Inhibitor Aliskiren Based on C5–C6 Disconnection and CO₂H–NH₂ Equivalence

 

• Prescribing Information for Tekturna
• aliskiren at the US National Library of Medicine Medical Subject Headings (MeSH)
• Chemical synthesis

Drugs Fut2001, 26, (12): 1139

Tetrahedron Lett 2001, 42: 4819-23.

Tetrahedron Lett2000, 41, (51): 10085

EP 0678500; EP 0678503; JP 1996053434; JP 1996081430; US 5559111; US ​​5627182; US 5646143, WO 0109079; WO 0109083

Aliskiren

Systematic (IUPAC) name
(2S,4S,5S,7S)-5-amino-N-(2-carbamoyl-2,2-dimethylethyl)-4-hydroxy-7-{[4-methoxy-3-(3-methoxypropoxy)phenyl]methyl}-8-methyl-2-(propan-2-yl)nonanamide
Clinical data
AHFS/Drugs.com	monograph
MedlinePlus	a607039
Licence data	EMA:Link, US FDA:link
Pregnancy category	C in first trimester D in second and third trimesters
Legal status	UK: POM (Prescription only) US: ℞-only
Routes of administration	PO (oral)
Pharmacokinetic data
Bioavailability	Low (approximately 2.5%)
Metabolism	Hepatic, CYP3A4-mediated
Biological half-life	24 hours
Excretion	Renal
Identifiers
CAS Number	173334-57-1
ATC code	C09XA02 C09XA52 (with HCT)
PubChem	CID: 5493444
IUPHAR/BPS	4812
DrugBank	DB01258
ChemSpider	4591452
UNII	502FWN4Q32
KEGG	D03208
ChEBI	CHEBI:601027
ChEMBL	CHEMBL1639
Chemical data
Formula	C30H53N3O6
Molecular mass	551.758 g/mol

////

O=C(N)C(C)(C)CNC(=O)[C@H](C(C)C)C[C@H](O)[C@@H](N)C[C@@H](C(C)C)Cc1cc(OCCCOC)c(OC)cc1

AT9283, AT 9283

N-cyclopropyl-N’-[3-[6-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]urea

1-cyclopropyl-3-[(3Z)-3-[5-(morpholin-4-ylmethyl)benzimidazol-2-ylidene]-1,2-dihydropyrazol-4-yl]urea

CAS

896466-04-9, 896466-57-2 ((±)-Lactic acid), 896466-61-8 (HCl), 896466-55-0 (methanesulfonate)

MolFormulaC22H29N7O5

MolWeight471.5096

CAS 896466-76-5  L LACTATE

(2S)-2-Hydroxypropanoic acid compd. with N-cyclopropyl-N’-[3-[6-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]urea

Astex Therapeutics Ltd, INNOVATOR

AT-9283 is a potent AuroraA/AuroraB and multi-kinase inhibitor. AT-9283 has shown to inhibit growth and survival of multiple solid tumor cell lines and is efficacious in mouse xenograft models.

AT 9283 is a substance being studied in the treatment of some types of cancer. It is small molecule a multi-targeted c-ABL, JAK2, Aurora A and B inhibition with 4, 1.2, 1.1 ad approximate 3 nM for Bcr-Abl (T3151), Jak2 and Jak3 aurora A and B, respectively. It blocks enzymes (Aurora kinases) involved in cell division and may kill cancer cells

WO2006070195 to Astex Therapeuitcs discloses pyrazole compounds of the general structure shown below as kinase inhibitors.

The compound AT9283 is in phase II clinical trials for treating advanced or metastatic solid tumors or Non-Hodgkin’s Lymphoma. AT9283 is shown below.

a Reagents and conditions:

(a) SOCl2, THF, DMF; (b) morpholine, THF, Et3N;  ………FORMATION OOF ACID CHLORIDE AND COUPLING WITH MORPHOLINE

(c) NaBH4, BF3.OEt2, THF; …………..KETO TO CH2

(d) 10% Pd-C, H2, EtOH; TWO NITRO GPS TO TWO AMINO , REDN

(e) EDC, HOBt, DMF; (f) AcOH, reflux;COUPLING WITH 4-Nitro-lH-pyrazole-3-carboxylic acid

(g) 10%Pd-C, H2, DMF; NITRO GP TO  AMINO

(h) standard amide and urea coupling methods

WO2006070195

https://www.google.co.in/patents/WO2006070195A1?cl=en

Stage 10: Synthesis of l-cvclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- beiizoimidazol-2-ylV 1 H-pyrazol-4-yli -urea.

To a mixture of 7-morpholin-4-ylmethyl-2,4-dihydro- 1 ,2,4,5a, 10- pentaaza- cyclopenta[a]fluoren-5-one (10.7 g, 32.9 mmol) in NMP (65 mL) was added cyclopropylamine (6.9 mL, 99 mmol). The mixture was heated at 100 ⁰C for 5 h. LC/MS analysis indicated -75% conversion to product, therefore a further portion of cyclopropylamine (2.3 mL, 33 mmol) was added, the mixture heated at 100 ⁰C for 4 h and then cooled to ambient. The mixture was diluted with water (100 mL) and extracted with EtOAc (100 niL). The organic portion was washed with sat. aq. NH₄Cl (2 x 50 mL) and brine (50 rnL) and then the aqueous portions re-extracted with EtOAc (3 x 100 mL). The combined organic portions were dried over MgSO₄ and reduced in vacuo to give l-cycloρropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yl]-urea as an orange glassy solid (9.10 g).

Stage 11: Synthesis of l-cvclopropyl-S-P-fS-morpholin^-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yll-urea, L-lactate salt

To a solution of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea (9.10 g, 24 mmol) in EtOAc-iPrOH (1 :1, 90 mL) was added L-lactic acid (2.25 g, 25 mmol). The mixture was stirred at ambient temperature for 24 h then reduced in vacuo. The residue was given consecutive slurries using toluene (100 mL) and Et₂O (100 mL) and the resultant solid collected and dried (8.04 g).

This solid was purified by recrystallisation from boiling iPrOH (200 mL) to give after drying l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2-yl)- lH-pyrazol-4-yl]-urea, L-lactate salt (5.7 g) as a beige solid.

EXAMPLE 66

Stage 1: Preparation of (3,4-dinitrophenyl)-morpholin-4-yl-methanone

3,4-Dinitrobenzoic acid (1.000Kg, 4.71mol, l.Owt), tetiuhydrofuran (10.00L₅ lO.Ovol), and dimethylformamide (0.010L, O.Olvol) were charged to a flask under nitrogen. Thionyl chloride (0.450L, 6.16mol, 0.45vol) was added at 20 to 3O⁰C and the reaction mixture was heated to 65 to 7O⁰C. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO), typically in 3 hours. The reaction mixture was cooled to 0 to 5⁰C and triethylamine (1.25L, 8.97mol, 1.25vol) was added at 0 to 10⁰C. Morpholine (0.62L, 7.07mol, 0.62vol) was charged to the reaction mixture at 0 to 1O⁰C and the slurry was stirred for 30 minutes at 0 to 1O⁰C. Reaction completion was determined by H NMR analysis (d₆-DMSO). The reaction mixture was warmed to 15 to 2O⁰C and water (4.00L, 4.0vol) was added. This mixture was then charged to a 4OL flange flask containing water (21.0OL, 21.0vol) at 15 to 25⁰C to precipitate the product. The flask contents were cooled to and aged at 0 to 5⁰C for 1 hour and the solids were collected by filtration. The filter-cake was washed with water (4x 5.00L, 4x 5.0vol) and the pH of the final wash was found to be pH 7. The wet filter-cake was analysed by H NMR for the presence of triethylamine hydrochloride. The filter-cake was dried at 40 to 45⁰C under vacuum until the water content by KF <0.2%w/w, to yield (3,4-dinitrophenyl)-morpholin-4-yl-methanone (1.286Kg, 97.0%, KF 0.069%w/w) as a yellow solid.

Stage 2: Preparation of 4-(3,4-dinitro-benzyl)-morpholine

C₁₁H₁₁N₃O₆ C₁₁H₁₃N₃O₅

FW:281.22 FW:267.24

(3,4-DinitiOphenyl)-morpholin-4-yl-methanone (0.750Kg, 2.67mol, l.Owt) and tetrahydrofuran (7.50L, lO.Ovol) were charged to a flask under nitrogen and cooled to 0 to 5⁰C. Borontrifluoride etherate (0.713L, 5.63mol, 0.95vol) was added at 0 to 5⁰C and the suspension was stirred at this temperature for 15 to 30 minutes. Sodium borohydride (0.212Kg, 5.60mol, 0.282wt) was added in 6 equal portions over 90 to 120 minutes. (A delayed exotherm was noted 10 to 15 minutes after addition of the first portion. Once this had started and the reaction mixture had been re-cooled, further portions were added at 10 to 15 minute intervals, allowing the reaction to cool between additions). The reaction mixture was stirred at 0 to 5⁰C for 30 minutes. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO). Methanol (6.30L, 8.4vol) was added drop wise at 0 to 1O⁰C to quench the reaction mixture (rapid gas evolution, some foaming). The quenched reaction mixture was stirred at 0 to 1O⁰C for 25 to 35 minutes then warmed to and stirred at 20 to 3O⁰C (exotherm, gas/ether evolution on dissolution of solid) until gas evolution had slowed. The mixture was heated to and stirred at 65 to 7O⁰C for 1 hour. The mixture was cooled to 30 to 4O⁰C and concentrated under vacuum at 40 to 45⁰C to give crude 4-(3,4-dinitro-benzyl)-morpholine (0.702Kg, 98.4%) as a yellow/orange solid.

4-(3,4-Dinitro-benzyl)-niorpholme (2.815kg, 10.53mol, l.Owt) and methanol (12.00L, 4.3vol) were charged to a flask under nitrogen and heated to 65 to 7O⁰C. The temperature was maintained until complete dissolution. The mixture was then cooled to and aged at 0 to 5⁰C for 1 hour. The solids were isolated by filtration. The filter-cake was washed with methanol (2x 1.50L, 2x 0.5vol) and dried under vacuum at 35 to 45°C to give 4-(3,4-dinitro-benzyl)-morpholine (2.353Kg, 83.5% based on input Stage 2, 82.5% overall yield based on total input Stage 1 material,) as a yellow solid.

Stage 3: Preparation of 4-morpholin-4-yl-methyl-benzene-L2-diamine

C₁₁H₁₃N₃O₅ C₁₁H₁₇N₃O

FW:267.24 FW:207.27

4-(3,4-Dinitro-benzyl)-morρholine (0.800Kg, 2.99mol, l.Owt), and ethanol (11.20L, 14.0vol) were charged to a suitable flask and stirred at 15 to 25⁰C and a vacuum / nitrogen purge cycle was performed three times. 10% Palladium on carbon (10%Pd/C, 50%wet paste, 0.040Kg, 0.05wt wet weight) was slurried in ethanol (0.80L, l.Ovol) and added to the reaction. The mixture was cooled to 10 to 2O⁰C and a vacuum / nitrogen purge cycle was performed three times. A vacuum / hydrogen purge cycle was performed three times and the reaction was stirred under a hydrogen atmosphere at 10 to 2O⁰C. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO), typically 14 to 20 hours. A vacuum / nitrogen purge cycle was performed three times and the reaction mixture was filtered through glass microfibre paper under nitrogen. The filter-cake was washed with ethanol (3x 0.80L, 3x l.Ovol) and the combined filtrate and washes were concentrated to dryness under vacuum at 35 to 45⁰C to give 4-morpholin-4-yl-methyl-benzene-l,2- diamine (0.61 IKg 98.6%) as a brown solid.

Stage 4: Preparation of 4-nitiO-lH-pyrazole-3-carboxγlic acid methyl ester

C₄H₃N₃O₄ C₅H₅N₃O₄

FW: 157.09 FW: 171.11

4-Nitro-lH-pyrazole-3-carboxylic acid (1.00kg, 6.37mol, l.Owt) and methanol (8.00L, 8.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. The suspension was cooled to 0 to 5°C under nitrogen and thionyl chloride (0.52L, 7.12mol, 0.52vol) was added at this temperature. The mixture was warmed to 15 to 25°C over 16 to 24 hours. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO). The mixture was concentrated under vacuum at 35 to 45°C. Toluene (2.00L, 2.0vol) was charged to the residue and removed under vacuum at 35 to 45⁰C. The azeotrope was repeated twice using toluene (2.00L, 2.0vol) to give 4-nitro-lH-pyrazole-3-carboxylic acid methyl ester (1.071Kg, 98.3%) as an off white solid.

Stage 5: Preparation of 4-amino-lH-pyrazole-3-carboxylic acid methyl ester. O₂Me

C₅H ₅N₃O₄ C₅H₇N₃O₂ FW: 171.11 FW: 141.13

A suspension of 4-nitro-lH-pyrazole-3-carboxylic acid methyl ester (1.084Kg, 6.33mol, l.Owt) and ethanol (10.84L, lO.Ovol) was heated to and maintained at 30 to 35°C until complete dissolution occurred. 10% Palladium on carbon (10% Pd/C wet paste, 0.152Kg, 0.14wt) was charged to a separate flask under nitrogen and a vacuum / nitrogen purge cycle was performed three times. The solution of 4-nitro- lH-pyrazole-3-carboxylic acid methyl ester in ethanol was charged to the catalyst and a vacuum / nitrogen purge cycle was performed three times. A vacuum / hydrogen purge cycle was performed three times and the reaction was placed under an atmosphere of hydrogen. The reaction mixture was stirred at 28 to 30°C until deemed complete by ¹H NMR analysis (d₆-DMSO). The mixture was filtered under nitrogen and concentrated under vacuum at 35 to 45⁰C to give 4-amino-lH- pyrazole-3-carboxylic acid methyl ester (0.883Kg, 98.9%) as a purple solid.

Stage 6: Preparation of 4-fert-butoxycarbonylamino-lH-pyrazole-3-carboxylic acid

C₅H₇N₃O₂ C₉H₁₃N₃O₄

FW: 141.13 FW:227.22

4-Amino-lH-pyrazole-3-carboxylic acid methyl ester (1.024Kg, 7.16mol, l.Owt) and dioxane (10.24L, lO.Ovol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. 2M aq. Sodium hydroxide solution (4.36L, 8.72mol, 4.26vol) was charged at 15 to 25⁰C and the mixture was heated to 45 to 55⁰C. The temperature was maintained at 45 to 55⁰C until reaction completion, as determined by ¹H NMR analysis (d₆-DMSO). Di-te/Y-butyl dicarbonate (Boc anhydride, 1.667Kg, 7.64mol, 1.628wt) was added at 45 to 55°C and the mixture was stirred for 55 to 65 minutes. ¹H NMR IPC analysis (d₆-DMSO) indicated the presence of 9% unreacted intermediate. Additional di-fert-butyl dicarbonate (Boc anhydride, 0.141Kg, 0.64mol, 0.14wt) was added at 55°C and the mixture was stirred for 55 to 65 minutes. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO). The dioxane was removed under vacuum at 35 to 45⁰C and water (17.60L, 20.0vol) was added to the residue. The pH was adjusted to pH 2 with 2M aq. hydrochloric acid (4.30L, 4.20vol) and the mixture was filtered. The filter-cake was slurried with water (10.00L₃ 9.7vol) for 20 to 30 minutes and the mixture was filtered. The filter-cake was washed with heptanes (4.10L, 4.0vol) and pulled dry on the pad for 16 to 20 hours. The solid was azeodried with toluene (5x 4.00L, 5x 4.6vol) then dried under vacuum at 35 to 45°C to give 4-tert- butoxycarbonylamino-lH-pyrazole-3-carboxylic acid (1.389Kg, 85.4%) as a purple solid.

Stage 7: Preparation of [3-(2-amino-4-moipholin-4-ylmetliyl-phenylcarbamoviy lH-pyrazol-4-yl]-carbamic acid tert-butyl ester

C₉H₁₃N₃O₄ C₁₁H₁₇N₃O C₂₀H₂₈N₆O₄

FW: 227.22 FW: 207.27 FW: 416.48

+ regioisomer

4-førf-Butoxycarbonylamino-lH-pyrazole-3-carboxylic acid (0.750Kg, 3.30 mol, l.Owt), 4-morpholin-4yl-methyl-benzene-l,2-diamine (0.752Kg, 3.63mol, l.Owt) and N,N’-dimethylformamide (11.25L, 15.0vol) were charged under nitrogen to a flange flask equipped with a mechanical stirrer and thermometer. 1- Hydroxybenzotriazole (HOBT, 0.540Kg, 3.96mol, 0.72wt) was added at 15 to 25⁰C. N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC, 0.759Kg, 3.96mol, 1.01 wt) was added at 15 to 25⁰C and the mixture was stirred at this temperature for 16 to 24 hours. Reaction completion was determined by ¹H NMR analysis. The reaction mixture was concentrated under vacuum at 35 to 45°C. The residue was partitioned between ethyl acetate (7.50L, lO.Ovol) and sat. aq. sodium hydrogen carbonate solution (8.03L, 10.7vol) and the layers were separated. The organic phase was washed with brine (3.75L, 5.0vol), dried over magnesium sulfate (1.00Kg, 1.33wt) and filtered. The filter-cake was washed with ethyl acetate (1.50L, 2.0vol). The combined filtrate and wash were concentrated under vacuum at 35 to 45⁰C to give [3-(2-amino-4-morpholin-4-ylmethyl-phenylcarbamoyl)-lH-pyrazol- 4-yl]-carbamic acid tert-butyl ester (1.217Kg, 88.6%) as a dark brown solid.

Stage 8 : Preparation of 3 -f 5-morpholin-4-ylmethyl- 1 H-benzoimidazol-2-ylV 1 H- pyrazol-4-ylamme

C₁₅H₁₉N₆O

FW: 298.35

As a mixture of two regioisomers

[3-(2-Amino-4-morpholin-4-ylmethyl-phenylcarbamoyl)-lH-pyrazol-4-yl]- carbamic acid tert-butyl ester (1.350Kg, 3.24 mol, l.Owt) and ethanol (6.75L, 5.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. Cone. aq. hydrochloric acid (1.10L, 13.2 mol, 0.80vol) was added at 15 to 3O⁰C under nitrogen and the contents were then heated to 70 to 8O⁰C and maintained at this temperature for 16 to 24 hours. A second portion of hydrochloric acid (0.1 IL, 1.32 mol, O.OSOvol) was added at 70 to 8O⁰C and the reaction was heated for a further 4 hours. Reaction completion was determined by HPLC analysis. The reaction mixture was cooled to 10 to 20⁰C and potassium carbonate (1.355Kg, 9.08mol, l.Owt) was charged portionwise at this temperature. The suspension was stirred until gas evolution ceased and was then filtered. The filter-cake was washed with ethanol (1.35L, l.Ovol) and the filtrates retained. The filter-cake was slurried with ethanol (4.00L, 3.0vol) at 15 to 25⁰C for 20 to 40 minutes and the mixture was filtered. The filter-cake was washed with ethanol (1.35L₃ 1.Ovol) and the total combined filtrates were concentrated under vacuum at 35 to 45⁰C. Ethanol (4.00L, 3. Ovol) was charged to the residue and removed under vacuum at 35 to 45⁰C. Tetrahydrofuran (5.90L, 4.4vol) was added to the residue and stirred for 10 to 20 minutes at 15 to 25°C. The resulting solution was filtered, the filter-cake was washed with tetrahydrofuran (1.35L, l.Ovol) and the combined filtrates were concentrated under vacuum at 35 to 45⁰C. Tetrahydrofuran (5.40L, 4. Ovol) was charged to the concentrate and removed under vacuum at 35 to 45⁰C. Tetrahydrofuran (5.40L, 4. Ovol) was charged to the concentrate and removed under vacuum at 35 to 45°C to give the desired product, 3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-ylamine (0.924Kg, 95.5%, 82.84% by HPLC area) as a purple foam.

Stage 9: Preparation of 7-morpholin-4-ylmethyl-2,4-dihydro- 1,2,4,5a ,10-pentaaza- cyclopentaFal fluoren-5 -one

C₁₅H₁₈N₆O C₁₆H₁₆N₆O₂ FW: 298.35 FW: 324.34

As a mixture of two regioisomers

3-(5-Morpholin-4-ylmethyl-lH-benzoimidazol-2-yl)-lH-pyrazol-4-ylamine (0.993Kg, 3.33 mol, l.Owt) and tetrahydrofuran (14.0L, 15.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. The contents were stirred under nitrogen at 15 to 25°C and l,l ‘-carbonyldiimidazole (0.596Kg, 3.67 mol, O.όOwt) was added. The contents were then heated to 60 to 70⁰C and stirred at this temperature for 16 to 24 hours. Reaction completion was determined by TLC analysis. The mixture was cooled to 15 to 20⁰C and filtered. The filter-cake was washed with tetrahydrofuran (4.00L, 4. Ovol) and pulled dry for 15 to 30 minutes. The solid was dried under vacuum at 35 to 45⁰C to yield 7- morpholin-4-ylmethyl-2,4-dihydro- 1 ,2,4,5a, 10-pentaaza-cyclopenta[a]fluoren-5- one (0.810Kg, 75.0%th, 92.19% by HPLC area) as a purple solid. Stage 10: Preparation of l-cvclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-vD- 1 H-pyrazol-4-yll -urea

C₁₆H₁₆N₆O₂ C₁₉H₂₃N₇O₂

FW: 324.34 FW: 381.44

As a mixture of two regioisomers

7-Morpholin-4-ylmethyl-2₅4-dihydro-l,2,4,5a,10-pentaaza-cyclopenta[a]fluoren-5- one (0.797Kg, 2.46mol, l.Owt) and l-methyl-2-pyrrolidinone (2.40L, 3.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. Cyclopropylamine (0.279Kg, 4.88mol, 0.35 lwt) was added at 15 to 30°C under nitrogen. The contents were heated to 95 to 105°C and stirred at this temperature for 16 to 24 hours. Reaction completion was determined by ¹H NMR analysis. The reaction mixture was cooled to 10 to 20⁰C and ethyl acetate (8.00L, lO.Ovol) and sat. aq. sodium chloride (2.50L, 3.0vol) were charged, the mixture was stirred for 2 to 5 minutes and the layers separated. The organic phase was stirred with sat. aq. sodium chloride (5.00L, ό.Ovol) for 25 to 35 minutes, the mixture filtered and the filter-cake washed with ethyl acetate (0.40L, 0.5vol). The filter-cake was retained and the filtrates were transferred to a separating funnel and the layers separated. The procedure was repeated a further 3 times and the retained solids were combined with the organic phase and the mixture concentrated to dryness under vacuum at 35 to 45⁰C. The concentrate was dissolved in propan-2-ol (8.00L, lO.Ovol) at 45 to 55°C and activated carbon (0.080Kg₅ O.lwt) was charged. The mixture was stirred at 45 to 55⁰C for 30 to 40 minutes and then hot filtered at 45 to 55°C. The filter-cake was washed with propan-2-ol (0.40L, 0.5vol). Activated carbon (0.080L, O.lwt) was charged to the combined filtrates and wash and the mixture stirred at 45 to 55⁰C for 30 to 40 minutes. The mixture was hot filtered at 45 to 55⁰C and the filter-cake washed with propan-2-ol (0.40L, 0.5vol). The filtrates and wash were concentrated under vacuum at 35 to 45⁰C. Ethyl acetate (8.00, lO.Ovol) and water (2.20L, 3.0vol) were charged to the concentrate at 25 to 35⁰C and the mixture stirred for 1 to 2 minutes. The layers were separated and the organic phase was concentrated under vacuum at 35 to 45°C. Ethyl acetate (4.00L, 5.0vol) was charged to the residue and concentrated under vacuum at 35 to 45⁰C. Ethyl acetate (4.00L, 5.0vol) was charged to the residue and the mixture was stirred for 2 to 20 hours at 15 to 25⁰C. The mixture was cooled to and aged at 0 to 5°C for 90 to 120 minutes and then filtered. The filter-cake was washed with ethyl acetate (0.80L, l.Ovol) and pulled dry for 15 to 30 minutes. The solid was dried under vacuum at 35 to 45⁰C to yield l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yl]-urea (0.533Kg, 56.8%, 93.20% by HPLC area) as a brown solid.

Several batches of Stage 9 product were processed in this way and the details of the quantities of starting material and product for each batch are set out in Table IA.

Table IA – Yields from urea formation step – Stage 10

Stage 11 : Preparation of l-cyclopiOpyl-3-r3-(5-moipholin-4-ylmethyl-lH- benzoimidazol-2-yl^s)-lH-pyrazol-4-yll-urea £-lactic acid salt L-Lactic acid

acid

C₁₉H₂₃N₇O₂ C₂₂H₂₉N₇O₅

FW: 381.44 FW: 471.52 l-Cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2-yl)-lH-ρyrazol- 4-yl]-urea (1.859Kg, 4.872mol, l.Owt), propan-2-ol (9.00L₅ 5.0vol) and ethyl acetate (8.0OL, 4.5vol) were charged to a flange flask equipped with a mechanical stirrer and thermometer. The contents were stirred under nitrogen and L-lactic acid (0.504Kg, 5.59mol, 0.269wt) was added at 15 to 25°C followed by a line rinse of ethyl acetate (0.90L, 0.5vol). The mixture was stirred at 15 to 25°C for 120 to 140 minutes. The solid was isolated by filtration, the filter-cake washed with ethyl acetate (2x 2.00L, 2x l.Ovol) and pulled dry for 20 to 40 minutes. The filter-cake was dissolved in ethanol (33.00L, 17.7vol) at 75 to 85⁰C, cooled to 65 to 70⁰C and the solution clarified through glass microfibre paper. The filtrates were cooled to and aged at 15 to 25⁰C for 2 to 3 hours. The crystallised solid was isolated by filtration, the filter-cake washed with ethanol (2x 1.00L, 2x 0.5vol) and pulled dry for at least 30 minutes. The solid was dried under vacuum at 35 to 45°C to yield 1- cyclopropyl-3 – [3-(5 -morpholin-4-ylmethyl- 1 H-benzoimidazol-2-yl)- 1 H-pyrazol-4- yl]-urea l-lactic acid salt (1.386Kg, 58.7%th, 99.47% by HPLC area,) as a dark pink uniform solid.

The infra-red spectrum of the lactate salt (KBr disc method) included characteristic peaks at 3229, 2972 and 1660 cm^“1.

Without wishing to be bound by any theory, it is believed that the infra red peaks can be assigned to structural components of the salt as follow:

Peak: Due to:

3229 cm^“1 N-H

2972 cm^“1 aliphatic C-H

1660 cm^“1 urea C=O EXAMPLE 67

Synthesis of Crystalline Free Base And Crystalline Salt Forms Of l-Cyclopropyl-3-

[3-(5-Morpholin-4-ylmethyl-lH-Benzoimidazol-2-vπ-lH-Pyrazol-4-yll-Urea

A. Preparation of l-Cvclopropyl-3-[3-f5-Moφholm-4-ylmethyl-lH- Benzoimidazol-2-yl)-lH-Pyrazol-4-yll-Urea free base

A sample of crude l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol- 2-yl)-lH-pyrazol-4-yl]-urea free base was prepared as outlined in Example 60 and initially purified by column chromatography on silica gel, eluting with EtOAc- MeOH (98:2 – 80:20). A sample of the free base obtained was then recrystallised from hot methanol to give crystalline material of l-cyclopropyl-3-[3-(5-morpholin- 4-ylmethyl- 1 H-benzoimidazol-2-yl)- 1 H-pyrazol-4-yl] -urea free base.

B. Preparation of l-Cyclopropyl-S-rS-fS-Morpholin^-ylmethyl-lH-Benzoimidazol- 2-yl)-lH-Pyrazol-4-yl]-Urea free base dihydrate

A sample of crude l-cyclopropyl-3-[3-(5-moφholm-4-ylmethyl-lH-benzoimidazol- 2-yl)-l H-pyrazol-4-yl] -urea free base was dissolved in THF and then concentrated in vacuo to a minimum volume (~4 volumes). To the solution was added water dropwise (2 – 4 volumes) until the solution became turbid. A small amount of THF was added to re-establish solution clarity and the mixture left to stand overnight to give a crystalline material which was air-dried to give l-cyclopropyl-3-[3-(5- morpholin-4-ylmethyl- 1 H-benzoimidazol-2-yl)- 1 H-pyrazol-4-yl] -urea free base dihydrate.

C. Preparation of l-Cyclopl^pyl-3-[3-(5-Morpholm-4-ylmethyl-lH-Benzoimidazol- 2-ylVlH-Pyrazol-4-yl]-Urea hydrochloride salt

A sample of crude l-cyclopropyl-3-[3-(5-moφholin-4-ylmethyl-lH-benzoimidazol- 2-yl)-l H-pyrazol-4-yl] -urea free base was dissolved in the minimum amount of MeOH and then diluted with EtOAc. To the solution at 0 °C was slowly added 1.1 equivalents of HCl (4M solution in dioxane). Following addition, solid precipitated from solution which was collected by filtration. To the solid was added MeOH and the mixture reduced in vacuo. To remove traces of residual MeOH the residue was evaporated from water and then dried at 60 ⁰C/ 0.1 mbar to give the hydrochloride salt.

D. Preparation of l-Cyclopropyl-3-[3-(^‘5-Morpholm-4-ylmethyl-lH- Benzoimidazol-2-yiyiH-Pyrazol-4-yl1-Urea ethanesulfonate salt

To a solution of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea free base in MeOH-EtOAc was added 1 equivalent of ethanesulfonic acid. The mixture was stirred at ambient temperature and then reduced in vacuo. The residue was taken up in MeOH and to the solution was added Et₂O. Mixture left to stand for 72 h and the solid formed collected by filtration and dried to give l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yl]-urea ethanesulfonate salt.

E. Preparation of l-Cvclopropyl-3-[3-(^‘5-Morpholm-4-ylmethyl-lH-Benzoimidazol- 2-yl)-lH-Pyrazol-4-yl]-Urea methanesulfonate salt

To a solution of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea free base (394 mg) in MeOH-EtOAc was added 1 equivalent of methanesulfonic acid (67 μl). A solid was formed which was collected by filtration, washing with EtOAc. The solid was dissolved in the minimum amount of hot MeOH, allowed to cool and then triturated with Et₂O. The solid was left to stand for 72 h and then collected by filtration, washing with MeOH, to give l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea methanesulfonate salt.

EXAMPLE 68

Characterisation of l-Cvclopropyl-3-[3-(5-Morpholin-4-ylmethyl-lH-

Benzoimidazol-2-yl)-lH-Pyrazol-4-yll-Urea Free Base and Salts

Various forms of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol- 2-yl)-lH-pyrazol-4-yl]-urea were characterised. The forms selected for characterisation were identified from studies which primarily investigated extent of polymorphism and salt stability. The salts selected for further characterisation were the L-lactate salt, Free base dihydrate, Esylate salt, Free base and Hydrochloride salt.

Paper

Fragment-Based Discovery of the Pyrazol-4-yl Urea (AT9283), a Multitargeted Kinase Inhibitor with Potent Aurora Kinase Activity^†

Abstract

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC₅₀ ≈ 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

1-Cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-1H-benzoimidazol-2-yl)-1H-pyrazol-4-yl]urea (16)

 16 as a pale-yellow solid (8.19 g, 87%). ¹H NMR (400 MHz, Me-d₃-OD): 8.07 (s, 1H), 7.58 (s, 2H), 7.26 (d, J = 8 Hz, 1H), 3.74−3.69 (m, 4H), 3.67 (s, 2H), 2.74−2.69 (m, 1H), 2.55−2.50 (m, 4H), 1.02−0.93 (m, 2H), 0.72−0.65 (m, 2H). LC/MS: t_R = 1.08 min, m/z = 382 [M + H]⁺.

1-Cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-1H-benzoimidazol-2-yl)-1H-pyrazol-4-yl]urea (16), Hydrochloride Salt

 ¹H NMR (400 MHz, DMSO-d₆): 13.26−13.07 (m, 2H), 11.05−10.80 (m, 1H), 9.64 (s, 1H), 8.08 (s, 1H), 7.98−7.19 (4H, m), 4.44 (s, 2H), 3.94 (d, J = 12.4 Hz, 2H), 3.77 (t, J = 12.3 Hz, 2H), 3.28−3.20 (m, 2H), 3.17−3.05 (m, 2H), 2.65−2.57 (m, 1H), 0.96−0.79 (m, 2H), 0.63−0.51 (m, 2H).

///////////

C1CC1NC(=O)NC2=CNNC2=C3N=C4C=CC(=CC4=N3)CN5CCOCC5

A simple and practical one-pot, two-directional approach to access olefinic esters through simultaneous breaking and making of olefins using ozonolysis of alkenyl aryl selenides is disclosed. The scope of the method with a variety of examples is demonstrated, and the end products obtained here are useful building blocks. As a direct application of the present method, the macrocyclic core of potent anti-inflammatory natural cyclic peptides, solomonamides, is synthesized.

Breaking and Making of Olefins Simultaneously Using Ozonolysis: Application to the Synthesis of Useful Building Blocks and Macrocyclic Core of Solomonamides

 

!e™A!a™Trp™leu™Va!~-Arg~~GIy-~Arg~~Gly~~OH

Formula (I)

LIRAGLUTIDE

Dr Reddy’s Laboratories Ltd, New patent, WO 2016005960,  Liraglutide

Process for preparation of liraglutide

Kola, Lavanya; Ramasamy, Karthik; Thakur, Rajiv Vishnukant; Katkam, Srinivas; Komaravolu, Yagna Kiran Kumar; Nandivada, Giri Babu; Gandavadi, Sunil Kumar; Nariyam Munaswamy, Sekhar; Movva, Kishore Kumar

Improved process for preparing liraglutide, by solid phase synthesis, useful for treating type 2 diabetes.

It having been developed and launched by Novo Nordisk, under license from Scios and Massachusetts General Hospital.

Liraglutide, marketed under the brand name Victoza, is a long-acting glucagon like peptide agonist developed by Novo Nordisk for the treatment of type 2 diabetes.

Liraglutide is an injectable drug that reduces the level of sugar (glucose) in the blood. It is used for treating type 2 diabetes and is similar to exenatide (Byetta). Liraglutide belongs to a class of drugs called incretin mimetics because these drugs mimic the effects of incretins. Incretins, such as human-glucagon-like peptide-1 (GLP-1 ), are hormones that are produced and released into the blood by the intestine in response to food. GLP-1 increases the secretion of insulin from the pancreas, slows absorption of glucose from the gut, and reduces the action of glucagon. (Glucagon is a hormone that increases glucose production by the liver.)

All three of these actions reduce levels of glucose in the blood. In addition, GLP-1 reduces appetite. Liraglutide is a synthetic (man-made) hormone that resembles and acts like GLP-1 . In studies, Liraglutide treated patients achieved lower blood glucose levels and experienced weight loss.

Liraglutide, an analog of human GLP-1 acts as a GLP-1 receptor agonist. The peptide precursor of Liraglutide, produced by a process that includes expression of recombinant DNA in Saccharomyces cerevisiae, has been engineered to be 97% homologous to native human GLP-1 by substituting arginine for lysine at position 34. Liraglutide is made by attaching a C-16 fatty acid (palmitic acid) with a glutamic acid spacer on the remaining lysine residue at position 26 of the peptide precursor.

The molecular formula of Liraglutide is Ci₇₂H₂65N₄30₅i and the molecular weight is 3751 .2 Daltons. It is represented by the structure of formula (I)

!e™A!a™Trp™leu™Va!~-Arg~~GIy-~Arg~~Gly~~OH

Formula (I)

U.S. Patent No. 7572884 discloses a process for preparing Liraglutide by recombinant technology followed by acylation and removal of N-terminal extension.

U.S. Patent No. 7273921 and 6451974 discloses a process for acylation of Arg-³⁴GLP-1 (7-37) to obtain Liraglutide.

U.S. Patent No. 8445433 discloses a solid phase synthesis of Liraglutide using a fragment approach.

International Application publication No. WO2013037266A1 discloses solid phase synthesis of Liraglutide, characterized in that comprises A) the presence of the activator system, solid phase carrier and by resin Fmoc protection N end obtained by coupling of glycine (Fmoc-Gly-OH) Fmoc-Gly-resin; B) by solid phase synthesis, prepared in accordance with the sequentially advantage Liraglutide principal chain N end of the coupling with Fmoc protected amino acid side chain protection and, wherein the lysine using Fmoc-Lys (Alloc)-OH; C) Alloc getting rid of the lysine side chain protecting group; D) by solid phase synthesis, the lysine side chain coupling Palmitoyl-Glu-OtBu; E) cracking, get rid of protecting group and resin to obtain crude Liraglutide ; F) purification, freeze-dried, to obtain Liraglutide.

Even though, the above mentioned prior art discloses diverse processes for the preparation of Liraglutide, they are often not amenable on commercial scale because of expensive amino acid derivatives such as pseudo prolines used in those processes.

Hence, there remains a need to provide simple, cost effective, scalable and robust processes for the preparation of Liraglutide involving commercially viable amino acid derivatives and reagents.

EXAMPLE 1 :

Stage I Preparation of Wang resin-Gly-Arg(pbf)-Gly-Arg(pbf)-Val-Leu-Trp(Boc)-Ala-lleu-Phe-Glu(Otbu)-Lys-{Glu(OH)-NH(palmitoyl)}-Ala-Ala-Gln(trt)-Gly-OH-Glu(Otbu)-Leu-Tyr(Otbu)-Ser(Otbu)-Ser(Otbu)-Val-Asp(Otbu)-Ser(Otbu)-Thr(Otbu)-Phe-Thr(Otbu)-Gly-Glu(Otbu)-Ala-Boc-His(trt)-OH.

Wang resin (50gm) is swelled in DCM (500ml) for 1 hr in a sintered flask. DCM was filtered using Vacuum. Fmoc-Glycine (44.6 gm, 150 mmol) was dissolved in dichloromethane (250 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-1 H-1 ,2,4 triazole (44.4 gm, 150 mmol) and 1 -methyl imidazole (9 ml, 1 12 mmol) was then added. The reaction mixture was added to wang resin and stirred for 3hrs at about 25° C. The resin was washed with DCM and a second lot of Fmoc-Glycine (27 gm, 90 mmol) was dissolved in dichloromethane (250 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-1 H-1 ,2,4 triazole (26.6 gm, 90 mmol) and 1 -methyl imidazole (5.3 ml, 90 mmol) was then added and stirred for 3hrs. The resin was washed with DCM and a sample of resin beads were checked for UV analysis. The capping was carried out using acetic anhydride (15 ml) DCM (120 ml) and pyridine (120 ml). The resin was washed with dichloromethane and DMF. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF. The

resin was washed repeatedly with DMF. The next amino acid Fmoc-Arg(pbf)-OH (52 gm, 80 mmol) dissolved in 250 ml DMF was then added. The coupling was carried out by addition of HOBt (10.8gm, 80 mmol) and DIC (6.2ml, 80 mmol) in DMF. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group was removed with 20% piperidine in DMF. These steps were repeated each time with the respective amino acid according to the peptide sequence. After coupling 12^th amino acid Fmoc-Lys (Alloc)-OH, deprotection of alloc group is carried out with palladium tetrakis and phenyl silane in DCM. The resin was washed repeatedly with DMF. The next amino acid H-Glu(OH)-NH(palmitoyl)-Otbu (9.9 gm, 0.023 moles) dissolved in 250 ml DMF was then added. The coupling was carried out by addition of HOBt (10.8gm, 80 mmol) and DIC (6.2ml, 80 mmol) in DMF. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group of Lys was removed with 20% piperidine in DMF. The next amino acid Fmoc-Ala-OH (52 gm, 80 mmol) dissolved in 250 ml DMF was then added. The coupling was carried out by addition of HOBt (10.8gm, 80 mmol) and DIC (6.2ml, 80 mmol) in DMF. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group was removed with 20% piperidine in DMF. These steps were repeated each time with the respective amino acid according to the peptide sequence. The resin was washed repeatedly with DMF, Methanol and MTBE and dried under vacuum.

Stage II: Cleavage of Liraglutide from resin along with global deprotection

45gms of resin obtained in stage I was treated with cleavage cocktail mixture of TFA (462.5ml), TIPS (12.5ml), Water (12.5ml), and Phenol (12.5 ml), stirred at 0°C for 30 min. and at 25°C for 3hrs at 200RPM. Then the reaction mixture was filtered, repeatedly wash the resin with TFA and the filtrate was concentrated on Rotary evaporator at 30°C. Pour the concentrated solution to MTBE (2L) at 4°C slowly and stir for 1 hr. The precipitate obtained is filtered and dried in a vacuum tray drier to afford 18 gm of Liraglutide crude with a purity of 27.5%.

Stage III: Purification of crude Liraglutide using RP HPLC.

The crude Liraglutide (4 gm) of purity around 27.5% is dissolved in 10 mM Tris buffer (120ml) of pH: 8.00 and 0.5 N NaOH is further added drop wise to the solution for making the crude solid completely dissolved. The solution is further passed through 0.2 micron filter. The Reverse phase C 18 – 150 Angstrom media (C18 silica media – 10 micron particle size) is equilibrated with 10mM Tris buffer of pH: 8.0 The crude solution is loaded onto the column and the gradient elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 1 : Gradient program for pre purification

The desired fractions are collected in the gradient range of and the fractions (F1 , F2, F3, F4 and F5) whose purity > 80% are pooled. The pooled fractions are then subjected to further purification.

The Pooled fractions having purity >80% are then subjected to C18 RPHPLC silica media (5 micron particle size) for further purification. The pooled fractions – Feed is diluted with purified water in the ratio of 1 :2 (one part of pooled fraction to two parts of purified water) as a part of sample preparation before loading into the column. The media C18 is first equilibrated with 0.1 % TFA for 3 column volumes (1 CV = bed volume of media). After equilibration, the sample is loaded onto the column and the gradient

elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 2: Gradient program for second purification

The desired fractions are collected in the gradient range of and the fraction whose purity > 96% are pooled together and lyophilized to afford 220mg of Liraglutide trifluoro acetate salt. The pooled fractions and their purity by HPLC are listed in the below table.

The pooled fractions with the purity of average 97% are subjected further to de solvation to remove the Acetonitrile content by Rota vapor. The final solution was filtered through 0.2 micron filter and lyophilized to get Liraglutide API.

EXAMPLE 2:

Stage I Preparation of Tentagel SPHB resin-Gly-Arg(pbf)-Gly-Arg(pbf)-Val-Leu-Trp(Boc)-Ala-lleu-Phe-Glu(Otbu)-Lys-{Glu(OH)-NH(palmitoyl)}-Ala-Ala-Gln(trt)-Gly-OH-Glu(Otbu)-Leu-Tyr(Otbu)-Ser(Otbu)-Ser(Otbu)-Val-Asp(Otbu)-Ser(Otbu)-Thr(Otbu)-Phe-Thr(Otbu)-Gly-Glu(Otbu)-Ala-Boc-His(trt)-OH using Fragment approach.

Fragments used are as follows

1 . Fmoc-Arg(pbf)-Gly-OH.

2. Fmoc-Leu-Ala-Arg(pbf)-OH.

3. Fmoc-lle-Ala-Trp(boc)-OH.

4. Fmoc-Glu(Otbu)-Phe-OH.

5. Fmoc-Glu(Otbu)-Phe-OH.

6. Fmoc-Lys-Glu-Palmitic acid.

7. Fmoc-Gly-Gln(trt)-Ala-Ala-OH.

8. Fmoc-Tyr(Otbu)-Leu-Glu(Otbu)-OH.

9. Fmoc-Val-Ser(Otbu)-Ser(Otbu)-OH.

10. Fmoc-Phe-Thr(Otbu)-Ser(Otbu)-Asp(Otbu)-OH

1 1 . Fmoc-Gly-Thr(Otbu)-OH.

12. Boc-His(Trt)-Ala-Glu(Otbu)-OH.

Tentagel SPHB resin (30gm) is swelled in DCM (300ml) for 1 hr in a sintered flask. DCM was filtered using Vacuum. Fmoc-Glycine (13.8 gm, 46.8 moles) was dissolved in dichloromethane (150 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-1 H-1 ,2,4 triazole (13.8 gm, 46.8 moles) and 1 -methyl imidazole (2.4 ml, 29.25 moles) was then added. The resulting solution was added to tentagel resin and stirred for 2hrs at about 25° C. The resin was washed with DCM and a second lot of Fmoc-Glycine (13.8 gm, 46.8 moles) was dissolved in dichloromethane (150 ml). 1 -(2-mesitylene sulfonyl)-3-nitro-I H-1 ,2,4 triazole (13.8 gm, 46.8 moles) and 1 -methyl imidazole (2.4 ml, 29.25 moles) was then added and stirred for 2hrs. The resin was washed with DCM and a sample of resin beads were checked for UV analysis. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF. The resin was washed repeatedly

with DMF. The next amino acid fragment 1 Fmoc-Gly-Arg(pbf)-OH (8.25 gm, 1 1 .7 moles) dissolved in 150 ml DMF was then added. The coupling was carried out by addition of HOBt (2.1 gm, 1 1 .7 moles) and DIC (2.5ml, 1 1 .7 moles) in DMF for 2hrs. The completion of the coupling was confirmed by a ninhydrin test. After washing the resin, the Fmoc protecting group was removed with 20% piperidine in DMF. These steps were repeated each time with the respective amino acid fragments according to the peptide sequence. The resin was washed repeatedly with DMF, Methanol and MTBE and dried under vacuum.

Stage II: Cleavage of Liraglutide from resin along with global deprotection

58gms of resin obtained from stage I was treated with cleavage cocktail mixture of TFA (555ml), TIPS (15ml), Water (15ml), and Phenol (15 ml) and stirred at 0°C for 30 min. at 25°C for 3hrs at 200RPM. Then filter the reaction mixture, repeatedly wash the resin with TFA and concentrate on Rotary evaporator at 30°C. Pour the concentrated solution to MTBE at 4°C slowly and stirred for 1 hr. The precipitate obtained was filtered and dried in a vacuum tray drier to afford 23.12 gm of crude Liraglutide with a purity of 36.89%.

Stage III: Purification of crude Liraglutide using RP HPLC.

The crude Liraglutide (4 gm) of purity around 27.5% is dissolved in 10 mM Tris buffer (120ml) of pH: 8.00 and 0.5 N NaOH is further added drop wise to the solution for making the crude solid completely dissolved. The solution is further passed through 0.2 micron filter. The Reverse phase C 18 – 150 Angstrom media (Irregular C18 silica media – 10 micron particle size) is equilibrated with 10mM Tris buffer of pH: 8.0 The crude solution is loaded onto the column and the gradient elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 1 : Gradient program for pre purification

60 40 30

55 45 30

52 48 30

51 49 60

The desired fractions are collected in the gradient range of and the fractions (F1 , F2, F3, F4 and F5) whose purity > 80% are pooled. The pooled fractions then subjected to further purification.

The Pooled fractions having purity >80% are then subjected to C18 RPHPLC silica media (5 micron particle size) for further purification. The pooled fractions – Feed is diluted with purified water in the ratio of 1 :2 (one part of pooled fraction to two parts of purified water) as a part of sample preparation before loading into the column. The media C18 is first equilibrated with 0.1 % TFA for 3 column volumes (1 CV = bed volume of media). After equilibration, the sample is loaded onto the column and the gradient elution is performed as per the below tabular column against the mobile phase B (Acetonitrile).

Table 2: Gradient program for second purification

The desired fractions are collected in the gradient range and the fraction whose purity > 96% are pooled together and Lyophilized to afford 865 mg of Liraglutide trifluoro acetate salt. The pooled fractions and their purity by HPLC are listed in the below table.

The pooled fractions with the purity of average 97% are subjected further to de solvation to remove the Acetonitrile content by Rota vapor. The final solution was filtered through 0.2 micron filter and lyophilized to get Liraglutide API.

G.V. Prasad, chairman, Dr Reddy’s Laboratories.

REFERENCE

IN2014CH3453 INDIAN PATENT

WO 2016005960, CLICK FOR PATENT

//////

Formula (1)

Lupin Ltd, New patent, Pitavastatin, WO 2016005919

MANE, Narendra, Dattatray; (IN).
NEHATE, Sagar, Purushottam; (IN).
GODBOLE, Himanshu, Madhav; (IN).
SINGH, Girij, Pal; (IN)

The present invention is directed to polymorphic forms of Pitavastatin sodium and processes for preparation of the same

Novel crystalline polymorphic forms (I and II) and an amorphous form of pitavastatin, useful for treating hyperlipidemia and mixed dyslipidemia.

Also claims a method for preparing the crystalline and amorphous forms of pitavastatin. In January 2016, Newport Premium™ reported that Lupin holds an active US DMF for pitavastatin calcium since July 2013.

Nissan Chemical Industries and licensee Kowa, with sub-licensees Sankyo, Eli Lilly, Esteve, JW Pharmaceutical, Recordati, Laboratorios Delta and Zydus-Cadila, have developed and launched pitavastatin.

WO2014203045, claiming a process for preparing an intermediate useful in the synthesis of statins (eg pitavastatin).

Pitavastatin is a cholesterol lowering agent of the class of HMG-CoA reductase inhibitor. The HMG-CoA reductase enzyme catalyzes the conversions of HMG- CoA to mevalonate. Inhibitors of HMG-CoA reductase are commonly referred to as “statins.” Statins are therapeutically effective drugs used for reducing low density lipoprotein (LDL) particle concentration in the blood stream of patients at risk for cardiovascular disease.

Pitavastatin is one of the synthetic statins which is chemically known as (3R, 5S, 6E)-7-[2-cyclopropyl-4-(4-fluorophenyl) quinoline-3-yl]-3, 5-dihydroxy-6- heptenoic acid represented by structural formula (1):

Formula (1)

Pitavastatin and its pharmaceutically acceptable salts are described in US 5,753,675 patent and US 5,856,336 patent, respectively.

Processes for the preparation of Pitavastatin are well documented in the literature. European patents, EP 0304063 and EP 1099694 and reports by Miyachi et al (Tetrahedron Letters

(1993) vol. 34, pages 8267-8270) and Takahashi et al (Bull. Chem. Soc. Japan (1995) Vol. 68, 2649-2656) describe processes for preparation of Pitavastatin.

US 5,872,130 patent discloses sodium salt of Pitavastatin. This patent, however, is silent about the solid state form of Pitavastatin Sodium.

It is generally known in the art that active pharmaceutical ingredients frequently do not exhibit the range of physical properties that makes them directly suitable for development. One of the approaches that is used to modify the characteristics of drug substances is to employ a salt form of the substance, since salts enable one to modify aqueous solubility, dissolution rate, solution pH, crystal form, hygroscopicity, chemical stability, melting point and even mechanical properties. The beneficial aspects of using salt forms of active pharmaceutical ingredients are well known and represent one of the means to increase the degree of solubility of otherwise intractable substances and to increase bioavailability.

Although the known salts of Pitavastatin like sodium, potassium, magnesium, calcium etc. and their polymorphic forms may address some of the deficiencies in terms of formulated product and its manufacturability. There remains a need for yet further improvement in these properties as well as improvements in other properties such as flowability, and solubility.

Polymorphism is a known phenomenon among pharmaceutical substances. It is commonly defined as the ability of any substance to exist in two or more crystalline phases that have a different arrangement and/or conformation of the molecules in the crystal lattice. Different polymorphic forms of the same pharmaceutically active moiety also differ in their physical properties such as melting point, solubility, chemical reactivity, etc. These properties may also appreciably influence pharmaceutical properties such as dissolution rate and bioavailability.

Further, the discovery of new polymorphic forms and solvates of an active pharmaceutical ingredient provides broader scope to a formulation scientist for formulation optimization, for example by providing a product with different properties, e.g., better processing or handling characteristics, improved dissolution profile, or improved shelf-life. For at least these reasons, there is a need for polymorphs of Pitavastatin salts such as Pitavastatin sodium.

New polymorphic forms and hydrates and/or solvates of a pharmaceutically acceptable salt of Pitavastatin can also provide an opportunity to improve the performance characteristics of a pharmaceutical product.

Therefore, there is a scope to prepare novel polymorphic forms of Pitavastatin sodium and hydrates and/or solvates.

Example-1: Preparation of Pitavastatin Sodium (Form-I)

A mixture of 40.0 gm Pitavastatin acid and 120 ml water was cooled to 15-20 °C temperature. Thereafter aqueous solution of sodium hydroxide (4.0 gm) in water (20 ml) was added to the reaction mixture. The reaction mixture was stirred for 30-45 min at 15-20 °C temperature. Ethyl acetate (80ml) was added into the reaction mixture at 15-20 °C temperature, stirred for 15-20 min and the layers were separated. The aqueous layer was filtered and acetonitrile (1200 ml) was gradually added to the aqueous layer under stirring till the precipitation was completed. The reaction mixture was cooled to 5-8 °C temperature and stirred for 2-3 hours at 5-8 °C temperature. The precipitated solid was filtered, washed with acetonitrile (40ml) and dried at 45-50 °C temperature under vacuum for 10-12 hours to afford the title compound (28.0 gm).

Yield (w/w): 0.70 (66.0%)

HPLC purity: 99.70 %

Example-2: Preparation of Pitavastatin Sodium (Form-II)

A mixture of 40.0 gm of Pitavastatin acid and 120 ml of water was cooled to 15-20°C temperature under stirring. Thereafter aqueous solution of sodium hydroxide (4.0 gm) in water (20 ml) was added to the reaction mixture. The reaction mixture was stirred for 30-45 min at 15-20 °C temperature. Ethyl acetate (80ml) was added to the reaction mixture at 15-20 °C temperature, stirred for 15-20 min and the layers were separated. The aqueous layer was filtered and acetonitrile (1200 ml) was gradually added to the aqueous layer under stirring till the precipitation was completed. The reaction mixture was cooled to 5-8 °C temperature and stirred for 2-3 hours at 5-8 °C temperature. The precipitated solid was filtered, washed with acetonitrile (40ml) and dried at 45-50 °C temperature under vacuum for 10-12 hours and kept in a petri dish at 25-30 °C and 60 ± 5 RH (relative humidity) for 18-24 hours to afford the title compound (31.6 gm).

Yield (w/w): 0.79 (65.8%)

HPLC purity: 99.70 %

Example-3: Preparation of Pitavastatin Sodium Amorphous

Pitavastatin sodium (3.0 gm) and ethanol (60 ml) were taken in a round bottomed flask at 25-30 °C temperature. The reaction mixture was filtered and the solvent was distilled off on rotatory evaporator under vacuum maintaining bath temperature at 45-50 °C temperature. Thereafter the reaction mixture was degassed under vacuum for 2-3 hours to afford the title compound (2.8gm).

HPLC purity: 99.70 %.

SEE……..https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016005919&redirectedID=true

/////////Lupin Ltd, New patent, Pitavastatin, WO 2016005919, statins, POLYMORPH

Lupin Ltd, Patent, Pitavastatin, WO2014203045

A NOVEL, GREEN AND COST EFFECTIVE PROCESS FOR SYNTHESIS OF TERT-BUTYL (3R,5S)-6-OXO-3,5-DIHYDROXY-3,5-O-ISOPROPYLIDENE-HEXANOATE

ROY, Bhairabnath; (IN).
SINGH, Girij, Pal; (IN).
LATHI, Piyush, Suresh; (IN).
AGRAWAL, Manoj, Kunjabihari; (IN).
MITRA, Rangan; (IN).
TRIVEDI, Anurag; (IN).
PISE, Vijay, Sadashiv; (IN).
RUPANWAR, Manoj; (IN)

The present invention describes an eco-friendly and cost effective process for the synthesis of teri-butyl (3R,5S)-6-oxo-3,5-dihydroxy-3,5-0-isopropylidene-hexanoate [I]

PITAVASTATIN

TEXT

tert-b tyl (3R,5S)-6-oxo-3,5-dihydroxy-3,5-0-isopropylidene-hexanoate [I] [CAS No. 124752-23-4] is key intermediate for the preparation of statins such as Atorvastatin (Tetrahedron 63, 2007, 8124 -8134), Cerivastatin (Journal of Labeled Compounds and Radiopharmaceuticals, 49, 2006 311-319), Fluvastatin [WO2007125547; US 4739073], Pitavastatin [WO2007/132482; US2012/22102 Al, WO2010/77062 A2; WO2012/63254 Al ; EP 304063; Tetrahedron Letters, 1993, 34, 513 – 516; Bulletin of the Chemical Society of Japan, 1995, 68, 364 – 372] and Rosuvastatin [WO2007/125547 A2; WO2011/132172 Al ; EP 521471]. Statins are used for treatment of hypercholesterolemia, which reduces the LDL cholesterol levels by inhibiting activity of HMG-CoA reductase enzyme, which is involved in the synthesis of cholesterol in liver.

[I]

Compound [I] is generally obtained by various methods of oxidation of teri-butyl 2- ((4R,65)-6-(hydroxymethyl)-2,2-dimethyl-l,3-dioxan-4-yl)acetate [compound II] and are discussed in details hereinafter. In addition, various methods for synthesis of compound [II] are also elaborated below.

[II]

[II]

A) tert-butyl2-((4«,6.S)-6-(hydroxymethyl)-2,2-dimethyl-l,3-dioxan-4-yl)acetate

[compound II]

US patent Number 5278313 describes a process for synthesis of compound [II]

(Schemel). In the said process, (5)-methyl 4-chloro-3-hydroxybutanoate has been obtained in only 70% yield through whole cell enzymatic reduction of methyl 4-chloro-3- oxobutanoate, which has a necessity of special equipment such as fermenters as well as other microbial facilities such as sterile area, autoclaves, incubator for growing seed culture, etc.

(S)-mefhyl 4-chloro-3-hydroxybutanoate upon reaction with teri-butyl acetate in presence of LiHMDS or LDA at -78°C, yielded (S)-ieri-butyl 6-chloro-5-hydroxy-3- oxohexanoate, which was further transformed to corresponding diol through syn selective reduction in presence of methoxydiethyl borane/sodium borohydride at -78°C. The diol thus obtained was converted to compound [II] .

The overall yield for this process is low and required special equipment such as fermenters, etc and in addition to that, this process is not cost effective due to use of costly reagent such as methoxydiethyl borane.

Moreover, methoxydiethylborane is highly pyrophoric (Encyclopedia for organic synthesis, editor in chief L. Paquette; 2, 5304; Published by John and Wiley Sons;

Organic Process Research & Development 2006, 10, 1292-1295) and hence safety is a major concern.

Scheme 1

EP 1282719 B l (PCT application WO 01/85975 Al ) discloses a process for synthesis of compound ( R, 5S)-tert-bv y\ 3,5,6-trihydroxyhexanoate from (S)-tert-b tyl-5,6-dihydroxy-3-oxohexanoate through a) asymmetric hydrogenation in presence of a chiral catalyst e.g. di-mu-chlorobis-[(p-cymene)chlororuthenium(II)] along with an auxiliary such as (IS, 2S)-(+)-N- (4-toluenesulfonyl)-l ,2-diphenylethylenediamine as ligand, which gave desired product only in 70% diastereomeric excess (de); b) Whole cell enzymatic reduction of (S)-tert- butyl 5,6-dihydroxy-3-oxohexanoate to obtain compound (3R, 5S)-tert-bv y\ 3,5,6-trihydroxyhexanoate in 99% de (80% yield).

It is needless to mention that it has necessity of fermenter and other microbiological equipment (Scheme 2).

Moreover, conversion of (2>R,5S)-tert-bv y\ 6-acetoxy-3,5-dihydroxyhexanoate to tert-bv yl 2-((4R,65)-6-(acetoxymethyl)-2,2-dimethyl-l ,3-dioxan-4-yl)acetate was accomplished in only 25% yield and also required the flash chromatography for isolation of desired product.

Thus, overall yield for this process is poor and process is not operation friendly especially at large scale hence cannot be considered feasible for commercial manufacturing.

Scheme 2

EP1317440 Bl (PCT Application WO 02/06266 Al) has disclosed the process for synthesis of compound [II] from 6-chloro-2,4,6-trideoxy-D-erythro-hexose (Scheme 3) .

In the said patent application 6-chloro-2,4,6-trideoxy-D-erythro-hexose was converted to (4R, 65)-4-hydroxy-6-chloromethyl-tetrahydropyran-2one with excess of bromine in presence of potassium bicarbonate, which liberates environmentally undesired gas i.e. carbon dioxide.

Moreover, starting material i.e. 6-chloro-2,4,6-trideoxy-D-erythro-hexose is not commercially available and conversion efficiency of starting material at large scale towards (4R, 65)-4-hydroxy-6-chloromethyl-tetrahydropyran-2-one is only 67%.

Scheme 3

US Patent No. 6689591 B2 has demonstrated the whole cell enzymatic reduction of teri-butyl 6-chloro-3,5-dioxohexanoate to compound [II] (Scheme 4).

In the said process, whole cell enzymatic reduction is not specific; yield for desired product is only 34% and other partially reduced products are also obtained.

Hence, further purification is required for obtaining the desired compound. Thus, this process is not suitable for commercial scale.

Scheme 4

Tatsuya et al (Tetrahedron Letters; 34, 1993,513 – 516) has reported synthesis of compound [I] from derivative of L-tartatric acid (Scheme 5).

In the said process, tartaric acid di-i^‘sopropyl ester is doubly protected by tert-butyldimethylsilyl group, which was reacted with dianion of teri-butyl acetoacetate to give β, δ-diketo ester compound.

β,δ-diketo ester was reacted with 2 equivalent of diisobutylaluminium hydride (which is a pyrophoric reagent) to afford -hydroxy,8-keto ester in only 60% yield.

This process is not industrially viable as overall yield is very low and also because of use of costly and pyrophoric reagents/chemicals.

Scheme 5

US7205418 (PCT application WO03/053950A1) has described the process for synthesis of compound [II] from (S)-ieri-butyl-3,4-epoxybutanoate (Scheme 6).

The overall yield for this process is very low and moreover, it required the diastereomeric separation of teri-butyl 2-(6-(iodomethyl)-2-oxo-l,3-dioxan-4-yl)acetate by flash chromatography.

Since overall requirement of title compound is very high, any operation involving flash chromatography will tend to render the process commercially unviable.

Scheme 6

Fengali et al (Tetrahedron: Asymmetry 17; 2006; 2907-2913) has reported the process for synthesis of compound [II] from racemic epichlorohydrin (Scheme 7).

In this process, racemic epichlorohydrin was converted to corresponding nitrile intermediate through reaction with sodium cyanide; nitrile intermediate thus obtained was further resolved through lipase catalyzed stereo-selective esterification to obtain (5)-4-(benzyloxy)-3-hydroxybutanenitrile and (R)-l-(benzyloxy)-3-cyanopropan-2-yl acetate;

separation of desired product i.e. (S)-4-(benzyloxy)-3-hydroxybutanenitrile having 98% de (40% yield) was done by column chromatography.

Needless to mention a commodity chemical like compound [I] cannot be manufactured by such a laboratory method, which involved number of steps.

Scheme 7

Bode et al (Organic letters, 2002, 4, 619-621) has reported diastereomer- specific hydrolysis of 1,3-diol-acetonides (Scheme 8).

In this publication, duration of the reaction for diastereomer- specific hydrolysis of 1,3, diol-acetonides is reported to be 4 h, however, in our hand it was observed that hardly any reaction took place in 4 h, which made it non-reproducible.

In addition to that, separation of desired product is achieved by flash chromatography and it is needless to mention that any process which involved flash chromatography would render the process to be commercially unviable.

Hence, additional innovation needs to be put in for making the process industrially viable.

Scheme 8

CN 101613341A has reported the process for synthesis of compound [II] (Scheme

9).

In the same patent application tert-b tyl (S)-6-chloro-5-hydroxy-3-oxohexanoate was synthesized through Blaise condensation of (5)-4-chloro-3-hydorxy-butanenitrile with zinc enolate of tert butyl bromo acetate.

In the literature, synthesis of tert-bv yl (S)-6-chloro-5-hydroxy-3-oxohexanoate was reported through Blaise condensation of silyl protected (5)-4-chloro-3-(trimethylsilyl)oxy-butanenitrile with zinc enolate of tert butyl bromo acetate, in good yield (Synthesis 2004, 16, 2629-2632). Thus, protection of hydroxy group in (5)-4-chloro-3-hydorxy-butanenitrile is imperative.

In the said Chinese patent application, in claim 7, it was mentioned that solvent used for conversion of tert-bv yl (5)-6-chloro-5-hydroxy-3-oxohexanoate to ( R,5S)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate is anyone or mixture of more than one from tetrahydrofuran, ether, methanol, ethanol, n-propanol, /so-propanol and ethylene glycol.

However, in enablement the only example using mixture of solvent was that of THF-methanol (Experimental section, Example 4: The preparation of (R,5)-6-chloro-3,5- dihydroxyhexanoate) and same outcome was expected in other individual or mixture of solvents.

Claim 8 of CN 101613341A mentioned that reduction was carried out by any one or mixture of more than one reducing agents such as sodium borohydride, potassium borohydride, lithium aluminum hydride, diethylmethoxy borane, triethyl borane and tributyl borane.

It implies that either any one of the reducing agents or a mixture of the same can be employed. From reaction mechanism it is very much clear that diethylmethoxy borane, triethyl borane and tributyl borane form the six membered complex between optically active hydroxyl and carbonyl group, which gets reduced by sodium borohydride, signifying that individually diethylmethoxy borane, triethyl borane and tributyl borane are not reducing agents

Moreover, in claims 12 and 13 (Experimental section, Example 4: The preparation of (R,S)-6-chloro-3,5-dihydroxyhexanoate), it is mentioned that reduction should be carried out in temperature range -80 °C to -60 °C, implying that reaction would not work beyond this temperature range i.e. it would work in the temperature window of -80 °C to -60 °C only.

Summarizing, the teachings of the application are not workable.

Scheme 9

Wolberg et al (Angewandte Chemie International Edition, 2000, 4306) has reported that diastereomeric excess for syn selective reduction using mixture of diethyl methoxy borane/sodium borohydride of compound [VI] gave 93% de for compound [VIII], which required further re-crystallization to obtain compound [VIII] in 99% de and 70% yield.

Thus, all the reported methods for stereo-selective hydride reduction of compound [VI] were achieved through mixture of trialkyl borane or diethyl methoxy borane & sodium borohydride in THF, at -78°C. As mentioned earlier, trialkyl borane or diethyl methoxy borane are pyrophoric in nature; in addition to that anhydrous THF is costly and moreover, reaction required large dilution.

Hence, there is need for developing efficient, environment friendly, cost effective and green process for stereo-selective reduction compound [VI].

B) The process of Oxidation of compound [II] to compound [I] has been discussed in following literature processes.

1) Swern oxidation (US4970313; Tetrahedron Letters, 1990, 2545

Synthetic Communications, 2003, 2275 – 2284).

2) Parrkh-Doering oxidation (J. Am. Chem. Soc, 1967, 89, 5505-5507)

3) TEMPO/NaOCl oxidization (EP2351762)

4) Trichloroisocyanuric acid/ TEMPO (CN 101747313A)

5) Oxidation of compound [II] to compound [I] through IBX [CN101475558A].

It would be evident that most of the reported methods are not “green” and

environmentally benign; none of the reported methods use molecular oxygen as oxidizing agent in presence of metal catalyst/co-catalyst.

Example 18: Process for synthesis of tert-butyl 2-((4R,6S)-6-formyl-2,2-dimethyl-l,3-dioxan-4-yl)acetate [I]

A reactor was charged with 1.1 g of copper (I) chloride and 10 mL of acetonitrile. 2-2′ Bipyridyl (156 mg) and TEMPO (156 mg) were added to the reactor under oxygen environment at 25°C. A solution of (6-Hydroxymethyl-2,2-dimethyl-[l,3]dioxan-4-yl)-acetic acid tert-butyl ester 2.6 g in 26 mL DCM was added dropwise over a period of 10 min into it. The reaction mass was stirred at 40°C and progress of reaction was monitored on GLC, which shows that 90% conversion for desired product.

Example 19: Process for synthesis of tert-butyl 2-((4R,6S)-6-formyl-2,2-dimethyl-l,3-dioxan-4-yl)acetate [I]

A reactor was charged with 1.1 g of copper (I) chloride and 10 mL of dichlorome thane. 2-2′ Bipyridyl (156 mg) and TEMPO (156 mg) were added to the reactor under oxygen environment at 25°C. A solution of (6-Hydroxymethyl-2,2-dimethyl-[l,3]dioxan-4-yl)-acetic acid tert-butyl ester 2.6 g in 26 mL DCM was added dropwise over a period of 10 min into it. The reaction mass was stirred at 40°C and progress of reaction was monitored on GLC, which shows that 90% conversion for desired product.

AUTHORS

SEE………https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014203045&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCT+Biblio

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FDA´s Emerging Technology Applications Program – Draft Guidance

The FDA recently published a draft guidance for industry on the “Advancement of Emerging Technology Applications”. The draft guidance provides recommendations to pharmaceutical companies interested in participating in a program involving the submission of CMC information containing emerging manufacturing (including testing, packaging and labeling, and quality control) technology to FDA. Find out more about the draft guidance for industry “Advancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Base“..

http://www.gmp-compliance.org/enews_05164_FDA%B4s-Emerging-Technology-Applications-Program—Draft-Guidance_15455,15149,15153,Z-PDM_n.html

On December 23, 2015, the FDA published a draft guidance for industry “Advancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Base“. Comments and suggestions regarding this draft document should be submitted within 60 days of publication.

The draft guidance provides recommendations to pharmaceutical companies interested in participating in a program involving the submission of CMC (chemistry, manufacturing, and controls) information containing emerging manufacturing (including testing, packaging and labeling operations, and quality control) technology to FDA.

The program is open for new drug applications (INDs), original or supplemental new drug application (NDA), abbreviated new drug application (ANDA), or biologic license application (BLA). It only affects the quality section of a submission (CMC and facility-related information).

The development of emerging manufacturing technology, like, for example, aseptic manufacturing facilities with highly automated systems and isolators, may lead to improved manufacturing, and therefore improved product quality and availability throughout a product´s lifecycle.

Pharmaceutical companies can submit questions and proposals about the use of these technologies to a group within CDER (Emerging Technology Team – ETT).

The draft guidance is a follow-on to the FDA guidance for industry “PAT – A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance” which describes the concept that quality cannot be tested into products. It should be built-in or should be present by design. Through the ETT, FDA intends to encourage the adoption of innovative approaches by leveraging existing resources of FDA to facilitate regulatory reviews of submissions.

Examples of emerging technology elements include an innovative or novel:

• Product manufacturing technology, such as the dosage form;
• Manufacturing process (e.g., design, scale-up, and/or commercial scale);
• Testing technology.

Interested parties should submit a written meeting request to participate in the ETT program at least three months prior to the planned application (IND, ANDA, BLA, NDA) submission date. In addition to the items outlined in the FDA guidance “Formal Meetings Between the FDA and Sponsors or Applicants” the request should also include the following items:

• A brief description of the proposed testing, process, and/or proposed technology;
• A brief explanation why the proposed testing, process, and/or technology are substantially novel and unique;
• A description of how the proposed testing and/or technology could modernize pharmaceutical manufacturing and thus improve product safety, identity, strength, quality, or purity;
• A summary of the development plan and any perceived roadblocks to technical or regulatory implementation;
• A timeline for submission.

The request should generally not exceed five pages and FDA expects to notify companies of its decision regarding acceptance into the program within 60 days of receipt of the request. Once accepted into the program, the participant can engage with ETT and CMC in accordance with existing meeting procedures and guidances (e.g. above mentioned FDA guidance on Formal Meetings).

For further information, please find all the details in the draft guidance “Advancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Base“.

WO2016005874, PROCESS FOR THE PREPARATION OF REGORAFENIB AND ITS CRYSTALLINE FORMS

SHILPA MEDICARE LIMITED [IN/IN]; 10/80,Second Floor,Rajendra Gunj, Raichur, ರಾಯಚೂರು , karnataka 584102 (IN)

RAMPALLI, Sriram; (IN).
UPALLA, Lav Kumar; (IN).
RAMACHANDRULA, Krishna Kumar; (IN).
PUROHIT, Prashant; (IN).
AKSHAY KANT, Chaturvedi; (IN)

The present invention relates to a process for the preparation of 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2- carboxamide or Regorafenib (I): Formula (I). The present invention further relates to a process for the purification of 4-[4-({[4-chloro-3-(trifluoromethyl) phenyl] carbamoyl} amino)-3-fluorophenoxy]-N-methylpyridine-2- carboxamide or Regorafenib (I) to provide highly pure material. The present invention further relates to a process for the preparation stable crystalline material of 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]- N-methyl pyridine-2-carboxamide or Regorafenib (I) useful in the preparation of pharmaceutical compositions for the treatment of cancer.

4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide or Regorafenib is low molecular weight, orally available, inhibitor of multiple protein kinases, including kinases involved in tumour angiogenesis (VEGFR1, -2, -3, TIE2), oncogenesis (KIT, RET, RAF-1, BRAF, BRAFV600E), and the tumour microenvironment (PDGFR, FGFR). In preclinical studies regorafenib has demonstrated antitumour activity in a broad spectrum of tumour models including colorectal tumour models which is mediated both by its antiangiogenic and antiproliferative effects. Major human metabolites (M-2 and M-5) exhibited similar efficacies compared to Regorafenib both in vitro and in vivo models.

Regorafenib was approved by USFDA in 2012 and is marketed under the brand name Stivarga^®, is an important chemotherapeutic agent useful for the treatment of adult patients with metastatic colorectal cancer (CRC) who have been previously treated with, or are not considered candidates for, available therapies. These include fluoropyrimidine-based chemotherapy, an anti-VEGF therapy and an anti-EGFR therapy.

Regorafenib is chemically known as 4-[4-({[4-chloro-3-(trifluoromethyl) phenyl] carbamoyl} amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide (I). Regorafenib is a white to slightly pink or slightly brownish solid substance with the empirical formula C2iHi₅ClF₄N₄03 and a molecular weight of 482.82. Regorafenib is practically insoluble in water, dilute alkaline solution, dilute acid solution, n-heptane, glycerine and toluene. It is slightly soluble in acetonitrile, dichloromethane, propylene glycol, methanol, 2-propanol, ethanol and ethyl acetate. It is sparingly soluble in acetone and soluble in PEG 400 (macrogol). Regorafenib is not hygroscopic.

Regorafenib is generically disclosed in US 7351834, and specifically disclosed in US 8637553. US ‘553 disclose a process for the preparation of Regorafenib starting from 3-fluoro-4-nitrophenol. The process is as demonstrated below:

The present inventors has repeated the above process and found the following disadvantages:

Unwanted reactions are observed during the formation of Regorafenib, due to the involvement of prolonged time in process.

> Incomplete reactions were observed with excessive impurity formations due to incomplete conversion.

Removal of impurities from final product

US 2010173953 disclose Regorafenib monohydrate and crystalline Form I of Regorafenib. This patent application further discloses that crystalline Form I of Regorafenib stated in this application is obtained as per the process disclosed in WO 2005009961 A2 (Equivalent to US ‘553). The compound obtained was having a melting point of 186-206° C.

This patent publication discloses a process for the preparation of Regorafenib monohydrate comprises dissolving Regorafenib Form I obtained as per WO ‘961 in acetone

and the solution is filtered, followed by addition of water until precipitation, which was filtered and dried at room temperature

US 2010/0113533 discloses crystalline Form II of Regorafenib, comprises dissolving Regorafenib Form I obtained as per WO ‘961 in ethyl acetate, the suspension was heated to 40-45°C, addition of isocyanate solution (isocyanate in ethyl acetate) and is cooled to room temperature to yield the crystals, which was filtered, washed with ethyl acetate and dried at room temperature.

US 2010/0063112 discloses Form III of Regorafenib, process comprises of heating

Regorafenib monohydrate at 100°C or 60 min, and further 15 min at 110°C, followed by cooling to room temperature.

As polymorphism has been given importance in the recent literatures owing to its relevance to the drugs having oral dosage forms due to its apparent relation to dose preparation/suitability in composition steps/ bioavailability and other pharmaceutical profiles, stable polymorphic form of a drug has often remained the clear choice in compositions due to various reasons of handling, mixing and further processing including bioavailability and stability.

Exploring new process for these stable polymorphic forms which are amenable to scale up for pharmaceutically active / useful compounds such as 4-[4-({[4-chloro-3-(trifluoro methyl)phenyl]carbamoyl } amino)-3 -fluorophenoxy] -N-methylpyridine-2 -carboxamide or Regorafenib may thus provide an opportunity to improve the drug performance characteristics of such products.

Hence, inventors of the present application report a process for the preparation of a stable and usable form of 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluoi phenoxy]-N-methylpyridine-2-carboxamide or Regorafenib, which may be industrially amenable and usable for preparing the corresponding pharmaceutical compositions. The present invention provides an improved process for the preparation of 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fiuorophenoxy]-N-methylpyridine-2-carboxamide or Regorafenib crystalline forms specifically for crystalline polymorphic forms Form I and Form III. Crystalline polymorphic forms of 4-[4-({[4-chloro-3-(trifluoromethyl) phenyl] carbamoyl } amino)-3 -fluorophenoxy] -N-methylpyridine-2 -carboxamide or Regorafenib obtained by the process of the present invention is non-hygroscopic and chemically stable and has good dissolution properties.

The process related impurities that appear in the impurity profile of the Regorafenib may be substantially removed by the process of the present invention resulting in the formation of highly pure material. The process of the present invention is as summarized below:

Example 1

Preparation of 4-(4-amino-3-fluorophenoxy) pyridine-2-carboxylic acid methyl amide

4-Amino-3-fiuorophenol (l lg, 0.08 moles) and of 4-Chloro-N-methyl-2-pyridinecarboxamide (8.85 g, 0.05 moles) was added to a reaction flask containing N, N-dimethylacetamide (55 ml) at 25-30°C and stirred for 15 minutes. The reaction mixture was heated to 110-115°C and then potassium tert-butoxide in tetrahydrofuran (60 ml, 0.06 moles) was added slowly over a period of 3 to 4hours. Distill off solvent at same temperature, cooled the reaction mass to 25-30°Cand water(110 ml) was added slowly over a period of 15min. and cooled the reaction mass to 0-5°C . Adjust the pH of the reaction mass in between 7 and 7.5 by using 10% aqueous hydrochloric acid (~7 ml). Stir the reaction mass for 30min at the same temperature. Filter the product, washed with water (22 mL) and Dried at 50-55 °C for 12hrs. The obtained crude material was added to the flask containing Ethyl acetate (55 mL).The reaction mass was heated to reflux to get a clear solution and stirred for 15min at reflux. Cooled to 0-5°C, stir for 2hrs at the same temperature. Filter the product, washed with Toluene (9 mL) and dried at 50-55°C for 3-5hrs.

Above recrystallized material was added to the reaction flask containing methylene dichloride (270 mL) at 25-30°C and stirred for 10-15 min. Activated carbon (1 g) and silica gel (4.4 g) was added to the reaction mass and stir for lh at the same temperature. Filter the reaction mass through hyflow bed and wash with methylene dichloride (18 mL).Distill off solvent still~l-2 volumes of methylene dichloride remains in the flask and then cooled to 25-30°C. Toluene (20 mL) was added and stirred for 30min at the same temperature. Filtered the product, washed with Toluene (9 mL) and dried at 50-55°C for 12h.

Yield: 9 gm

Chromatographic Purity (By HPLC): 98%

Example 2

Preparation of Regorafenib

4-(4-amino-3-fluorophenoxy) pyridine-2-carboxylic acid methyl amide (4g, 0.01 moles) was added in to a reaction flask containing acetone (20 ml) at 25-30°C and stirred for 15 minutes. 4-chloro-3-trifluoromethylisocyanate (6.1g, 0.02 moles) was added slowly over a period of 5 to 10 minutes and stirred the reaction mixture 3 to 4 hours. Toluene (20 n L) was added to the reaction mass and stirred for 30 min at 25-30°C.The obtained reaction mass was filtered and washed with toluene (8 mL). Dried the material still constant weight appears to yield title product a crystalline material.

Yield: 5.5 gm

Chromatographic Purity (By HPLC): 97%

Example 3

Purification of Regorafenib using acetone and toluene mixture

4- [4-( { [4-chloro-3 -(trifluoromethyl)phenyl] carbamoyl } amino)-3 -fluorophenoxy] -N-methylpyridine-2-carboxamide (I) or Regorafenib (1 g) was added slowly in to the reaction flask containing acetone (2 mL) and toluene (3 mL) at 25-30°C and stirred for 15 minutes.

The reaction mixture was heated to 50-55°C and stirred the reaction mixture for 30 minutes.

Cooled the reaction mass to 25-30°C and stirred for 1 hour. Filter the material, washed with toluene (2 mL) and suck dried for 15 min, followed by drying at 50-55°C for 10-12h to yield

Pure 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methyl pyridine-2-carboxamide (I) or Regorafenib.

Yield: 0.88gm

Chromatographic Purity (By HPLC): 99.3 %

Example 4

Purification of Regorafenib using acetone

4-[4-({[4-chloro-3-(trifluoromethyl) phenyl] carbamoyl} amino)-3 -fluorophenoxy] -N-methylpyridine-2-carboxamide (I) or Regorafenib (1 g) was added slowly in to the reaction flask containing acetone (5 mL) at 25-30°C and stirred for 15 minutes. The reaction mixture was heated to 50-55°C and stirred the reaction mixture for 30 minutes. Cooled the reaction mass to 0-5°C and stirred for 1 hour. Filter the material, washed with acetone (1 mL) and suck dried for 15 min. The obtained wet cake was added in to the reaction flask containing acetone (5 mL) at 25-30°C and stirred for 15 minutes. The reaction mixture was heated to 50- 55°C and stirred the reaction mixture for 30 minutes. Cooled the reaction mass to 0-5°C and stirred for 1 hour. Filter the material, washed with acetone (1 mL) and dried at 60-65°C for 12 h to yield Pure 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methyl pyridine -2-carboxamide (I) or Regorafenib.

Yield: 0.7 gm

Chromatographic Purity (By HPLC): 99.77%

Example 5

Double – Purification of Regorafenib using acetone and toluene mixture

4-[4-({[4-chloro-3-(trifluoromethyl) phenyl] Carbamoyl} amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide (I) or Regorafenib (1 g) was added slowly in to the reaction flask containing acetone (2 mL) and toluene (3 mL) at 25-30°C and stirred for 15 minutes. The reaction mixture was heated to 50-55°C and stirred the reaction mixture for 30 minutes. Cooled the reaction mass to 25-30°C and stirred for 1 hour. Filter the material, washed with toluene (2 mL) and suck dried for 15 min. The obtained wet cake was added in to the reaction flask containing acetone (2 mL) and toluene (3 mL) mixture at 25-30°C and stirred for 15 minutes. The reaction mixture was heated to 50-55°C and stirred the reaction mixture for 30 minutes. Cooled the reaction mass to 25-30°C and stirred for 1 hour. Filter the material, washed with toluene (2 mL) and dry at 60-65°C for 12h.

Yield: 0.80gm

Chromatographic Purity (By HPLC): 99.79 %

Moisture content: 0.09%

Impurity-A: 0.03%

Impurity-B: Not detected

Impurity-C: 0.02%

Example 6

Preparation of Regorafenib Form I

4-(4-amino-3-fluorophenoxy) pyridine-2-carboxylic acid methyl amide (1.3 g, 0.004 moles) was added in to a reaction flask containing acetone (13 mL) at 25-30°C and stirred for 15 minutes.4-chloro-3-trifluoromethylisocyanate (6.6 g, 0.006 moles) wasadded slowly over a period of 15 to 20 minutes and stirred the reaction mixture 3 to 4 hours. The obtained reaction mass was filtered and washed with acetone. Dried the material still constant weight appears to yield title product a crystalline material.

Yield: 1.9 g

Chromatographic Purity (By HPLC): 98.4 %

XRPD was found to resemble similar to Fig-1.

Omprakash Inani – Chairman, Vishnukant C Bhutada – Managing Director, Namrata Bhutada – Non Executive Director, Ajeet Singh Karan – Independent Director, Carlton Felix Pereira – Independent Director, Pramod Kasat – Independent Director, Rajender Sunki Reddy – Independent Director, N P S Shinh – Independent Director,

Mr. Omprakash Inani		Mr. Omprakash Inani – CHAIRMAN Mr. Omprakash Inani has more than 30 years of Business experience. He monitors business and functional aspects of the Company along with the operations of all the plants. Additionally, he is member of Audit and Remuneration committee of Shilpa Medicare Group of Companies. Currently he is also a council Member in “Academy of Medical Education, Dental College & V.L. College of Pharmacy”, “Taranath Shikshana Samsthe, Raichur” and a trustee in “Akhil Bhartiya Maheshwari Education Trust, Pune”. Mr. Omprakash Inani is also Managing Committee Member of “Karnataka State Cotton Assn., Hubli”.
Mr. Vishnukant C. Bhutada		Mr. Vishnukant C. Bhutada – MANAGING DIRECTOR Mr. Vishnukant has vast and diverse Business experience of API and Intermediates and presently leads the core Business and functional teams which accelerate growth and performance by Innovating for Affordable solutions at Shilpa Medicare Group of Companies. He is the key decision maker with the teams for Shilpa Group for successful API and Generics formulation strategies. His untiring efforts have led the company to a leadership position in the Indian pharmaceutical domain and helped create a prominent presence for Oncology APIs globally. For his efforts on APIs Business, Mr. Vishnukant was awarded “Best Entrepreneur Award” by Late Dr Shankar Dayal Sharma – President of India in 1995. Subsequently, various state honours were conferred upon him -like -“Best Entrepreneur” from Karnataka State Govt. in 1996; “Excellence in Exports” from Vishweshwarayya Industrial Trade Centre, Bangalore 1996; and Export Excellence Award-2006” by FKCCI, Bangalore. Success has never stopped coming his way- as he was awarded “First runner up” at the Emerging India Awards London 2008 by CNBC TV18. Recently, his efforts in the Shilpa Group for environment sustainability, has led to “Best National Energy Conservation Award in Drugs & Pharmaceutical Sector for the year 2012” by Hon’ble President of India, Dr. Pranab Mukherjee.
Dr. Vimal Kumar Shrawat		Dr. Vimal Kumar Shrawat – CHIEF OPERATING OFFICER Dr. Shrawat by qualification holds degrees of M.Sc (Organic Chemistry), Ph.D. (from Delhi University) and joined Shilpa Medicare in 2009. He has vast experience of more than 25 years of working in large pharma industries like Ranbaxy/ Dabur Pharma- presently known as Fresenius Kabi Oncology Ltd., spanning across activities of R&D, Pilot and Plant Productions, QA/QC, Administration, CRAMS, Project management etc. Presently, Dr. Shrawat is spearheading the entire Operations/ Control of Shilpa Medicare. His vision of team work and time bound approach always guides and motivates teams at all operational sites. His keen interest and consistent efforts for R&D has led him to become one of key contributor in large number of Patent/applications of Shilpa Medicare.
Dr. Pramod Kumar		Dr. Pramod Kumar – MANAGING DIRECTOR(LOBA FEINCHEMIE GMBH AUSTRIA), SENIOR VICE-PRESIDENT (SHILPA MEDICARE LTD) Dr. Pramod Kumar, who by qualification holds degrees of M.Pharm, Ph.D (Pharmaceutical chemistry) and a PGDBA, joined Shilpa Medicare in 1989. Since 2009 he is Managing Director of Loba FeinchemieGmBH, Austria and driving all R&D driven commercial processes. Dr. Pramod Kumar has more than 25 years of experience in Pharmaceutical industry, spanning across activities of production, QA/QC, administration, import/export, CRAMS etc. His efforts in CRAMS have led to the formation of Joint venture company RAICHEM MEDICARE Pvt LTD with Italian companies ICE SPA / P.C.A SPA.
Mr. Prashant Purohit		Mr. Prashant Purohit – VICE-PRESIDENT-CRD Mr. Prashant Purohit by qualification holds degrees of, M.Sc.(Organic Chemistry) and Diploma in Business Management and joined Shilpa Medicare in 1996. He is presently heading Chemical R&D wings of Shilpa Medicare Group. He has vast experience of handling CRAMS and Generics APIs R&D. His vast experience of nearly 35 years in R & D and production in Pharmaceutical Industry has consistently enriched the portfolio of Shilpa Medicare Group of Companies. He is one of key contributor in large number of Patent/applications of Shilpa Medicare.
Dr. Akshay Kant Chaturvedi		Dr. Akshay Kant Chaturvedi – HEAD- CORPORATE IPM & LEGAL AFFAIRS Dr. Akshay Kant by qualification holds degrees of M.Sc, Organic Chemistry (Univ. Gold Medalist), Ph.D. (Medicinal Chem), LL.B., M.B.A. and joined Shilpa Medicare in Jun 2012. Besides above qualifications, he is a Registered Patent Agent (IN-PA-1641) at Indian patent Office. He has various certificates of Advanced Courses of IP from WIPO-Geneva, which include Patent Searching/ Drafting of Patents/ Arbitration and Mediation through WIPO/ Copyrights in Publishing Industries/ Patent Management/ Biotech IP etc. He has vast experience of about 21 years of working in large pharma industries like Jubilant Organosys Ltd./Dabur Pharma Ltd.- presently known as Fresenius Kabi Oncology Ltd./ DrReddys Labs, spanning across activities of R&D and IP-Patenting etc. Presently, Dr. Akshay is spearheading the entire IP portfolio management/ Legal Affairs of Contractual Business of Shilpa Medicare Group. His vision of innovative and creative thinking, team work and time bound approach always guide and motivate teams at all locations.His keen interest and consistent efforts for R&D has led him to become one of key contributor in large number of Patent/applications of Shilpa Medicare.
Dr. Seshachalam U.		Dr. Seshachalam U. -ASSOCIATE VICEPRESIDENT- QUALITY AND RA Dr. Seshachalam by qualification holds M.Sc (Chemistry) and Ph.D. (Chemistry) and joined Shilpa Medicare in 2008. He is presently heading Regulatory Affairs wings of Shilpa Medicare Group of Companies. He has vast experience of handling regulatory affairs related to Generics APIs. Being instrumental in Shilpa Medicare’s efforts to achieve recognition of different authorities, his key contribution in successful inspection and audit by various regulatory authorities is one of the core strength to the organization’s aims and objectives.
Mr. Sharath Reddy		Mr. Sharath Reddy – VICE-PRESIDENT PROJECTS & OPERATIONS Mr. Sharath Reddy by qualification holds M.Pharm from BITS Pilani and has overall experience of about 22 years predominately in the field of pharmaceuticals new projects and operations. His expertise of Oncology specialized equipment and Utilities designing has boosted organizations confidence to takeover new endeavors of upcoming projects with faster pace of time. His efforts have led to successfully executing Energy Saving projects of Shilpa Medicare Group of Companies and registration of the project under Clean Development Mechanism with UNFCC (Under Kyoto Protocol).
Mr. R K Somani		Mr. R K Somani – VICE-PRESIDENT FORMULATION -BUSINESS DEVELOPMENT Mr. R. K. Somani is a professional Chartered Accountant and holds a Diploma in Central Excise.He has overall business experience of more than 21 years predominately in the field of pharmaceuticals. Mr. Somani is one of the key drivers of Formulation business besides handling various key Contract Businesses of advanced oncology/ Non-Oncology APIs. He is known for successfully building formulations portfolio and spearheading the Generic sales operation.

Shilpa Medicare Limited
1st Floor, 10/80,
Rajendra Gunj,
RAICHUR ರಾಯಚೂರು – 584 102.
Karnataka, India.
Telephone: +91-8532-236494
Fax: +91-8532-235876
Email: info@vbshilpa.com

RAICHUR, ರಾಯಚೂರು Karnataka, India

Historical Stone Elephants in Malayabad, Raichur Taluk …

///Regorafenib, SHILPA MEDICARE LIMITED, new patent, WO 2016005874, raichur, ರಾಯಚೂರು , karnataka, india

BMS 911543

N,N-dicyclopropyl-4-((1,5-dimethyl-1H-pyrazol-3-yl)amino)-6-ethyl-1-methyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-carboxamide

cas 1271022-90-2
Chemical Formula: C23H28N8O
Exact Mass: 432.23861

UNII-7N03P021J8;

N,N-dicyclopropyl-4-((1,5-dimethyl-1H-pyrazol-3-yl)amino)-6-ethyl-1-methyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-carboxamide

Bristol-Myers Squibb Company  innovator

BMS-911543 is an orally available small molecule targeting a subset of Janus-associated kinase (JAK) with potential antineoplastic activity. JAK2 inhibitor BMS-911543 selectively inhibits JAK2, thereby preventing the JAK/STAT (signal transducer and activator of transcription) signaling cascade, including activation of STAT3. This may lead to an induction of tumor cell apoptosis and a decrease in cellular proliferation. JAK2, often upregulated or mutated in a variety of cancer cells, mediates STAT3 activation and plays a key role in tumor cell proliferation and survival.

The JAK2 selective compound BMS911543 (WO2011028864) is in phase II clinical trials for the treatment of m elofibrosis. BMS91 1543 is shown below.

PAPER

ACS Medicinal Chemistry Letters (2015), 6(8), 850-855

Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile

MS (ESI) m/z 434.3 (M+H). 1H NMR (CDCl3) δ: 7.96 (s, 1H), 7.65 (s, 1H), 6.83 (s, 1H), 4.67 (q, J = 7.1 Hz, 2H), 4.01 (s, 3H), 3.82 (s, 3H), 2.77 – 2.84 (m, 2H), 2.43 (s, 3H), 1.48 (t, J = 7.2 Hz, 3H), 0.79 – 0.86 (m, 4H), 0.71 – 0.77 (m, 4H).

PAPER

Journal of Organic Chemistry (2015), 80(12), 6001-601

http://pubs.acs.org/doi/suppl/10.1021/acs.joc.5b00572/suppl_file/jo5b00572_si_001.pdf

Ni-Catalyzed C–H Functionalization in the Formation of a Complex Heterocycle: Synthesis of the Potent JAK2 Inhibitor BMS-911543

BMS-911543 is a complex pyrrolopyridine investigated as a potential treatment for myeloproliferative disorders. The development of a short and efficient synthesis of this molecule is described. During the course of our studies, a Ni-mediated C–N bond formation was invented, which enabled the rapid construction of the highly substituted 2-aminopyridine core. The synthesis of this complex, nitrogen-rich heterocycle was accomplished in only eight steps starting from readily available materials.

N,N-Dicyclopropyl-4-((1,5-dimethyl-1H-pyrazol-3-yl)amino)-6-ethyl-1-methyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-carboxamide, 1

PATENT

WO 2015031562

These Schemes are illustrative and are not meant to limit the possible techniques one skilled in the art may use to manufacture compounds disclosed herein.

As shown below in Scheme 1, the general preparation of compound 7 is described. Trichloroacetyl pyrrole (Compound 1) is reacted with a halogenating agent to give the C4-bromo pyrrole (Compound 2). Alcoho lysis occurs in the presence of an alcohol and base to generate ester (Compound 3), which can be selectively nitrated through contact with an appropriate nitrating agent (defined as a species that generates N0₂ ), yielding C5-nitro pyrrole (Compound 4). Compound 4 can be isolated as its free form, or optionally as a salt with an appropriate base. Ethylation with an appropriate alkylating agent generates the N-ethyl pyrrole (Compound 5), which in the presence of an imidazole, base, palladium and an appropriate phosphine ligand, will undergo a coupling process to form Compound 6. Reduction of the nitro-group of Compound 6 in the presence of hydrogen, a metal catalyst and optionally a base will produce Compound 7.

Scheme 1

As shown below in Scheme 2, the preparation of Compound 13 is described. Trichloroacetyl pyrrole is treated with NBS in acetonitrile to produce Compound 8. Treatment with sodium ethoxide in EtOH yields the ethyl ester Compound 9. This may be treated with a range of nitrating systems, in this example, NaNC /SCVPy, to generate nitro-pyrrole Compound 10, which can be isolated directly or as a salt form with an appropriate base, preferably dibenzylamine. Ethylation with ethyl iodide generates Compound 11 which may be isolated, or optionally telescoped directly into the arylation with Compound 32. Arylation proceeds in the presence of palladium, Xantphos, potassium pivylate and Hunig’s base to generate Compound 12. Hydrogenation presence of Pt/C followed by cyclization with NaOEt yields Compound 13.

Scheme 2

Another process of the invention is disclosed in Scheme 3 shown below. Compound 14 is prepared from Compound 3 in the presence of an alkylating agent. Treatment with a suitable diboron reagent produces Compound 15, which can then be coupled with a suitably functionalized imidazole derivative to yield Compound 16. Amino lysis with a suitable nitrogen donor produces Compound 17, which can cyclize under appropriate conditions to produce Compound 7.

Scheme 3

Step 3 Step 4 Step 5

As shown below in Scheme 4, ethylation of Compound 9 with ethyl iodide produces Compound 18. This may be directly reacted with dipinacol-diboron in the presence of Pd(OAc)₂ and tricyclohexylphosphin hexafluorophosphate and

tetramethylammonium acetate to yield Compound 19. Subsequent coupling with 5-Br-imidazole derivative yields Compound 20. Treatment with hydroxylamine hydrochloride in the presence of triethylamine yields the Compound 21. Subsequent cyclization with Piv₂0 in the presence of PRICAT™ and hydrogen yields Compound 13.

Scheme 4

77% isolated over 2-steps%

18

Step 5 Pd(OAc)₂

PPh₃

78%

As shown below in Scheme 5, Compound 23 may be converted to Compound 26 by two pathways. In one option, Compound 23 can be treated with palladium, ligand and a mild base to prepare Compound 25. Reaction of Compound 25 with a metal hydroxide produces Compound 26.

Alternately, Compound 23 can be treated with palladium and ligand in the presence of a soluble hydroxide base, followed by treatment with the metal counter-ion to prepare Compound 26 directly. Once Compound 26 is formed, it can be coupled to Compound 27 to form compound I.

A solution of Compound 1 in acetonitrile (1238.0 kg, 264.9 kg after correction) was charged into a 5000 L glass-lined reactor at a temperature of 20-30 °C. The mixture was added with stirring over about 2 h and then cooled to 0 °C. NBS (221.8 kg) was charged into the mixture at intervals of 20-30 min at 0-20 °C. The mixture was cooled to 0-5 °C and reacted until the content of Compound 8was < 1.0%. Additional NBS (4.0 kg) was charged into the mixture at 0-20 °C. The mixture was reacted over 3 h until the content of Compound 8 was < 1.0%. Purified water (2650.0 kg) was added over about 1.5 – 2.5 h at 0-20 °C. The mixture was cooled to 0-5 °C and then stirred for about 1 h for crystallization. The mixture was filtered and the filter cake was rinsed with water.

Example 2

While maintaining the temperature at 20-30 °C, anhydrous ethanol (950.0 kg) was charged into a 3000 L glass-lined reactor followed by Compound 8 (342.7 kg). The mixture was cooled to 0-5 °C over about 2 h. Sodium alcoholate solution in ethanol (21%, 36.4 kg) was added dropwise over about 1-1.5 h at 0-5 °C. The reaction mixture was then heated to about 25-30 °C and tested until the content of Compounds 8/9 was < 1.0%. The reaction mixture was concentrated at a temperature < 50 °C until about 1.3-1.4 volume of Compound 8 was left. The concentrated mixture was cooled at 25-30 °C. The mixture was quenched into cooled water (3427.0 kg) over about 2 h. After addition, the mixture was stirred at 0-5 °C over about 2 h for crystallization. The mixture was filtered and the filter cake was rinsed. The solid was dried at 30-40 °C over 40-45 h to afford 234.3 kg of Compound 9 , 99.9% purity and 91.3% yield.

Example 3

9 10

A mixture of NaN0₃, NaHS0₄, and Na₂S0₄ in CH₃CN is wet-milled to constant particle size of -50 micron. To the slurry of inorganic salts is added S0₃ -pyridine and Compound 9. The reaction mixture is agitated at 25 °C until 90-95% conversion is achieved. The reaction is quenched with aqueous sodium hydroxide and the spent inorganic salts are removed by filtration. The filtrate is passed through a carbon pad and distilled under constant volume distillation and diluted with water to a target 15

volumes/kg of Compound 9 and a target ratio 1.0:2.0 vol/vol MeCN to water. The resulting solids are deliquored, washed, and dried to afford Compound 10.

Example 4

Toluene (10 L/Kg)

65 °C

Compound 10 (1.0 eq) and TBABr (1.0 eq) were added to a biphasic mixture of toluene (8 L/kg 10) and potassium carbonate (1.5 eq) in water (5 L/kg 10). The batch temperature was held at 25 °C. The resulting triphasic slurry was heated to 60-65 °C and diethylsulfate (1.5 eq, in a solution of toluene 2 L/kg 10) was slowly added over ~ 1 h. The reaction was aged until less than 1 RAP of Compound 10 (10:11) remained. The resulting homogeneous biphasic mixture was cooled to 20 °C and the lean aq. phase was removed. The rich organic phase was washed with water (2×7 L/kg 10) and concentrated to 6 mL/g 10. The concentrated stream was dried via azeotropic, constant volume distillation with toluene until the water content of the stream was <0.1 wt %. The resulting stream was telescoped into the subsequent direct arylation reaction.

Example 5

11 28 12

To the toluene stream of Compound 11, with potassium pivalate (1.5 equiv.) was charged, followed by DIPEA (3 eq.), Compound 28 (3 eq.) and Pd(Xantphos)Cl₂ (0.04 eq.). The vessel was evacuated to < 200 torr and backfilled with nitrogen (3 X) followed by heating to 95 °C until residual Compound 11 was less than 1 RAP (11: 12). The reaction mixture was cooled to 25 °C and diluted with ethyl acetate (15 mL/g vs input pyrrole) and aq. N-acetylcysteine (0.2 eq., 5 wt % solution, 1.8 mL/g vs. input pyrrole) and heated to 50 °C for 1 h. The biphasic mixture was cooled to 25 °C. The lower aqueous layer was removed. The ethyl acetate stream was washed with water (2×7 mL/g vs. input pyrrole). The rich organic phase was polish filtered followed by a vessel/polish filter rinse with ethyl acetate (2 mL/g vs. input pyrrole). The rich organic stream was concentrated to 4 mL/g vs. input pyrrole via vacuum distillation, while maintaining the batch temperature above 50 °C. If spontaneous nucleation did not occur, Compound 12 seeds (1 wt %) were charged, followed by aging for 30 min at temperature. MTBE (5 mL/g vs. 11) was charged to the slurry over 1 hour while maintaining the batch temperature above 40 °C, followed by aging at 40 °C for 1 h. The slurry was cooled to 0 °C over 6 h and aged at 0°C for 6 h. The slurry was filtered and washed with

EtO Ac : Toluene : MTBE (1.5: 1.0: 1.5, 2 mL/g vs. input 11 ). The wet cake was dried (50 °C, 100 torr) until LOD was < 1 wt %.

Example 6

Compound 12 (1 eq., limiting reagent (LR)) is dissolved in THF/NMP (20 Vol wrt LR, 9/1 ratio) and submitted to hydrogenation using 10 wt% (wrt LR) Pt/C (5 wt%) at 25 to 40° C for 5-10 h. The reaction containing the corresponding amine is filtered. The rich organic stream is concentrated to Compound 12 Vol (wrt LR) and subjected to 0.1 eq of 21 wt% NaOEt/EtOH for 5 h at 20-25 °C, upon which Compound 13 forms. The stream is cooled to 0-10 °C, and water (5L/Kg, wrt to LR) is added and then filtered to isolate Compound 13. The product is dried at 50 °C under vacuum.

Example 7

in toluene solution

9

18

Compound 18 was prepared by treating the pyrrole with ethyl iodide and pulverized potassium carbonate in DMF at 25-30°C under inert atmosphere. After the reaction was completed, the batch mass was cooled to 15°C to 20°C and quenched by slow addition of water then MTBE. The MTBE layer was separated and washed with water. The MTBE layer was distilled to 4 Vol and solvent swapped with toluene. The toluene stream was then taken into the next step.

Example 8

18 19

Tetra-methyl ammonium acetate in toluene slurry was heated to 75-80°C to get a clear solution. The mass was cooled to below 30°C and pyrrole in toluene and bis (pinacolato) diborane were added. The reactor was inerted by nitrogen purging then the reaction was heated to 75-80°C. A freshly prepared catalyst/ligand complex (0.0 leq of palladium acetate, 0.025eq of tricyclohexyl phosphino hexafluoroborate and 0.2eq of tetra methyl ammonium acetate in toluene) was charged under nitrogen atmosphere at RT and stirred for 2h. The mass was then stirred at 75-80°C under nitrogen atmosphere. After the reaction was completed, the mixture was cooled below 30°C and quenched with aq. sodium bisulphate solution. The organic layer was polish filtered through a Celite bed and the filtrate was washed with water. The solvent swapped to ethanol until the toluene content became less than 0.5 %. The solution was cooled to 0-5°C and water was added for crystallization. The product was then isolated by filtration.

Example 9

Compound 20 was prepared by treating Compound 19 with Compound 34 in the presence of palladium acetate, triphenyl phosphine and potassium carbonate in dimethyl acetamide with the water mixture as the solvent. Dimethyl acetamide, water, potassium carbonate and the two starting materials were charged into the reactor. The mixture was made inert with nitrogen for 30 min and then charged with freshly prepared catalyst mixture (palladium acetate, triphenyl phosphine and potassium carbonate in dimethyl acetamide). The temperature was raised to 78-83 °C then the mass was stirred at this temperature. After the reaction was completed, the reaction mass was cooled to ambient temperature and purified water was added slowly into the mass for product

crystallization. The mass was stirred for a period of 3 h and filtered. The wet cake was washed with purified water and dried in VTD at 50-55 °C under vacuum.

Example 10

Compound 21 was prepared by treating Compound 20 with hydroxylamine hydrochloride and triethyl amine using ethanol as the solvent. Compound 20 was added into ethanol (15 Vol) and the reaction mass was heated to 38-40 °C. Hydroxylamine hydrochloride was charged and stirred for 10 min, then triethyl amine was added slowly at 38-40 °C over a period of lh. The above mass was stirred at 38-40 °C until Compound 20 becomes less than 5.0%, typically in about 15 h. After the reaction was completed, the above reaction mass was cooled to ambient temperature (below 30 °C) and filtered. The wet cake was washed with purified water (4 Vol) and dried under vacuum in VTD at 55-60 °C.

Example 11

Initially Compound 21 was treated with pivalic anhydride using toluene and acetic acid mixture as solvent under inert atmosphere until Compound 21 becomes less than 3.0% with respect to Compound 21, typically in about 30 min. PRICAT Nickel was then added under nitrogen atmosphere. The reaction mass was inerted with nitrogen for three cycle times and then degassed with hydrogen gas for three cycle times. Following this, 3.0 kg/cm² hydrogen pressure was applied to the reaction mass which was stirred for about 12h. After the reaction was completed, the reaction mixture was filtered through a sparkler filter. The filtrate was distilled and the solvent exchanged with toluene until the ratio of acetic acid & toluene reaches 1 :20. At this time, n-Heptane was charged and cooled to 15°C. Then the product was filtered and the wet cake was dried in VTD at 50-55°C under vacuum.

Compound 30 was prepared by the coupling of Compound 22 with Compound 29, 3 -bromo- 1,5 -dimethyl- lH-pyrazole in the presence of

Tris(dibenzylideneacetone)dipalladium chloroform adduct, t-Brettphos and potassium phosphate in tert-amyl alcohol at 98-103 °C under inert atmosphere. After completion of the reaction (typical level of Int.9 -5% & typical reaction hrs 20 h), the mass was cooled to ambient temperature and t-amyl alcohol (4 Vol) and 20 Vol of water were charged into the reaction mass. The reaction mass was stirred for 15 min. and then phase split. The organic layer was diluted with 10 Vol of MTBE and product was extracted with 20 Vol of 1M methane sulphonic acid. The MSA stream was treated with 15 wt % charcoal to reduce the residual palladium numbers. The filtrate was cooled to below 20 °C and the pH was adjusted to 1.7-1.9 using IN NaOH for product crystallization and then iltered. The wet cake was washed with purified water (3 x 5 Vol), followed by methanol (5 Vol). The cake was vacuum dried for 3 h. then the wet cake and dimethyl sulfoxide (20 Vol) were charged into a reactor. The mass was heated to 120-125 °C to get clear solution then the mass was cooled to ambient temperature and stirred for 2 h, then filtered. The wet cake was washed with methanol (3x 4.0 Vol) and vacuum dried for 2 h. The wet cake was dried in VTD at below 55°C under vacuum.

Example 13

Compound 30 , ethanol (16.5 Vol), water and aq sodium hydroxide solution were charged into a reactor then the mass was heated to 70-75 °C and stirred until Compound 30 becomes less than 1.0%. After the reaction was completed, the mass was diluted with ethanol for complete product precipitation at 65-75 °C. Then the mass was cooled to 50 °C for a period of lh and stirred for lh at 50 °C. The mass was further cooled to 20 °C and stirred for lh at 20 °C and then filtered. The wet cake was washed with 5 Vol of 15% aqueous ethanolic solution followed by THF. The wet cake was dried under vacuum at 70-75 °C till LOD comes to less than 5.0 %, typically in about 40 h.

Example 14

In a vessel 36.5 mmol (-42.6 mL) of Compound 29 solution in 2-methyl-2-butanol was combined with 30.7g (65.1 mmol) tetrabutylammonium hydroxide (55 wt% in water), 8.01g (27.0 mmol) Compound 13 , and 10 mL 2-methyl-2-butanol. The mixture was heated at 70 °C until hydrolysis of Compound 13 was complete (full dissolution, <15 min). The solution was cooled to 60 °C and 1.12g (2.22 mmol) of tBuBippyPhos followed by 384 mg (1.028 mmol) allylpalladium chloride dimer (L:Pd = 1 :1) was added. The mixture was heated to 80 °C and was aged at this temperature for 20h before cooling to 22 °C.

Water was added and the mixture concentrated, a constant volume distillation was then performed to swap to ethanol (40-55 °C, 150 mbar). The resulting solution was passed through a 5 micron filter to remove any particulates. The solution was heated to 55 °C and 8.10 mL (40.52 mmol, 1.5 equiv) 5N NaOH (aq) was added dropwise over a 3 h period. Crystals of Compound 31 began to form, and after aging for an additional lh, the mixture was cooled to 20 °C over 3 h. After an additional 6h of aging, crystals were collected on a frit and the cake was washed with 40 mL of 90: 10 ethanol: water, followed by 48 mL acetone. After drying at 80 °C in a vacu-oven for 16 h, Compound 31 was collected as an off-white solid (8.89g, 85%).

Example 15

Compound 31 was added into dichloromethane (20 Vol) and cooled to 15-20 °C. The reaction mass was charged with DMC in DCM solution (1.4 eq of DMC in 5.0 Vol of DCM). The mixture was stirred until Compound 31 becomes less than 2.0% with respect to the corresponding acid chloride, typically in about lh. After completion of the reaction, Compound 27 (1.4 eq) and N,N-diisopropylethyleneamine (3.0 eq) were charged and the mixture was stirred. After completion of the reaction, the mass was quenched with 12 Vol of water then the layers were separated. The organic layer was washed with water and filtered through a celite bed. The filtrate was concentrated to ~6.0 vol and then the mass was cooled to 35 °C. To the resulting solution was added THF, followed by seeds of product, then stirred for 3 h. The solvent was swapped with THF until

dichloromethane becomes less than 2 wt% (wrt THF). The mass was cooled to -5 to 0 °C over a period of 2 h and stirred for 2 h. The reaction mass was then filtered under a nitrogen atmosphere. The material was slurried with pre-cooled THF (2*2 Vol) and filtered. The wet cake was dried in VTD at 60 °C under vacuum till LOD becomes < 1%, typically in about 20 h.

Example 16

DC , RT

I

To a slurry of Compound 31 (15.00 g, 40.0 mmol) in dichloromethane (300 ml) was added diphenylphosphinic chloride (12.29 g, 51.9 mmol). The mixture was stirred at room temperature for 2 h and Ν,Ν-diisopropylethylamine ( 16.53 g, 127.9 mmol) was then added and stirred for another 30 min. Compound 27 (6.94 g, 51.9 mmol) and 4-dimethylaminopyridine (0.49 g, 4.0 mmol) were subsequently added and stirred for 16 h until the reaction was completed. The reaction mixture was treated with N-acetyl-L-cysteine (3.26 g, 20.0 mmol) and citric acid (10.10 g, 48.0 mmol) in deionized water (180 ml) for 2 h. After phase split, the dichloromethane phase was washed once with 0.42 N NaOH solution (180 ml) and washed twice with deionized water (180 ml each). The final dichloromethane phase was concentrated (to 90 ml) and acetone (30 ml) was added. The solution was cooled to 35 °C and N-2 form seed of Compound 1 ( 150 mg ) was added and aged for 1 h. The resulting slurry was solvent-swapped to acetone (DCM < 10% v/v), and cooled to 0 °C. The solid was filtered and washed with cold acetone and dried to afford 14.69 g (85%) of Compound I (HPLC AP 99.8) as off-white crystals.

Patent

WO 2011028864

http://www.google.com/patents/WO2011028864A1?cl=en

Compounds of general formula I in which the R group is thiazole (as in Ial) and R¹ and R² groups are CF₃ or alkyl or cycloalkyl or combine to form a saturated carbocyclic or heterocyclic ring or where R2 group is COOR^b could be prepared using the general method depicted in Scheme 1. Dichloro intermediate II (prepared using procedure reported in WO200612237) could be combined with a 2,4-dimethoxybenzyl and the resulting secondary amine is capped with suitable protective group (Boc) (III). The second chlorine atom could be converted into the

corresponding amine (IV) through the benzophenone imine intermediate. The amino compound could be halogenated to intermediate V. V could be subjected to transition metal mediated indole ring formation and the resulting indole nitrogen is capped with ethyl iodide to afford VI. Ester hydrolysis followed by amide bond formation and cleavage of protective groups with acid treatment would yield amine VII. Amine VII could be converted into thiourea VIII by first coupling with benzoyl isothiocyanate followed treatment with aqueous base. Formation of thiazole could be achieved by condensation with an a-bromoketone derivative (R^HBrCOR²).

a) 2,4-dimethoxybenzylamine, heat; b) NaHMDS, Boc₂0; c) (Ph)₂=NH; d) HCl; e) NIS; f) Pd₂(dba)₃, ethyl pyruvate; g) Etl, Cs₂C0₃; h) NaOH (aq); i) dicyclopropylamine HCl, HATU, DIPEA; j) TFA; k) Benzoyl isothiocyanate;

1) NaOH (aq); m) I^CHBrCOR¹

Scheme 1

Compounds of general formula Ia2 in which the R¹ group is CONR^aR^a could be made using Scheme 2. Thiourea intermediate (VIII) could be combined with Et0₂CCHBrCOR¹ to afford the thiazole ester (IX). The ester could be hydrolyzed and the acid could be coupled with amine to afford thiazole amide derivative (la)

a) Et0₂CCHBrCOR¹; b) NaOH (aq); c) HNR^aR^a, HATU, DIPEA

Scheme 2

Similarly, compounds of general formula Ia3 in which the R¹ group is CONR^aR^a could be prepared using the general protocol depicted in Scheme 3.

a) R²CHBrCOC0₂Me; b) NaOH (aq); c) HNR^aR^a, HATU, DIPEA

Scheme 3

Compounds of general formula la in which R¹ is halogen (CI, Br or I) could be prepared by condensing an a,a’-dihaloketone as depicted in Scheme 4.

a) R²COCH(Hal)₂

Scheme 4

Alternatively, thiourea derivative VIII could be converted to room temperature into C-5 un-substituted thiazole XI and then directly halogenated using electrophilic halogen source or through metallation followed by quenching with an electrophilic halogenating agent (Scheme 5).

a) BrCH₂COR²; b) Selectfluor or NCS or NBS or NIS or tBuLi followed Selectfluor or NBS or NCS

Scheme 5

Compounds of general formula Ia5 in which R¹ is S0₂R^b could be synthesized using the general synthetic approach shown in Scheme 6

a) Br2-acetic acid; b) EtOH, heat

Scheme 6

Compounds with general formula la in which R¹ and R² combine to form an aromatic or heteroaromatic ring could be prepared using Scheme 7.

X = hal, -S0₂Me

a) Pd(0) catalyst, NaOtBu, phosphine ligand, heat

Scheme 7

Alternatively, these compounds could be made by first coupling aniline or heteroaniline (XVI) with the isothiocyanate (XV) followed by oxidative cyclization (Scheme 8).

a) 1, 1 ‘-Thiocarbonyldi-2( 1 H)-pyridone; b) NaH; c) NIS

Scheme 8

Compounds of general formula Ibl could be prepared using the general synthetic approach depicted in Scheme 9. Aniline VII could be combined with γ-dithiomethylketone compound XVII, (prepared using the procedure reported at room temperature in Synlett, p 2331 (2008)) under basic condition to afford XVIII.

Stepwise condensation of the Boc-protected hydrazine derivative would give the required pyrazole Ibl.

a) NaH, THF; b) R¹N(Boc)NH₂, AcOH, 35-40°C; c) HCO₂H or TFA, 60°C

Scheme 9

Compounds of general formula Ibl or Ifl and If could also be prepared by coupling C-4 halo derivative (XIX) with an appropriately substituted 2-aminopyrazole derivative (XX) using a transition metal catalyzed reaction (Scheme 10).

a) isoamyl nitrite, CH2I2 or isoamyl nitrite, CH₂Br₂; b) Pd2(dba)₃, Xanphos, Cs₂C0₃

Scheme 10

Compounds of general formula Ib2 in which R² group is CONR^aR^a could be synthesized using Scheme 11. Aniline VII could be combined with γ-dithiomethylketone derivative XXII, (prepared using the procedure from

Tetrahedron, p 2631 (2003)) to afford intermediate XXIII. Stepwise condensation of Boc-protected hydrazine derivative would give the required pyrazole aldehyde XXIV. Aldehyde could be oxidized using oxone or sodium hypochlorite to furnish carboxylic acid XXV. Coupling of acid XXV with amine would give pyrazole amide Ib2.

a) NaH, THF, heat; b) R¹N(Boc)NH2, AcOH; c) TFA; d) oxone or sodium hypochlorite; e) HNR^aR^a, HATU, DIPEA

Scheme 11

Compounds of general formula Icl could be prepared using the general protocol as shown in Scheme 12. Aniline VII could be coupled with chloroacetyl chloride and the resulting amide could be treated with thioamide (R²CS H₂) to furnish thiazole Icl .

a) chloroacetyl chloride, base; b) R²CSNH₂

Scheme 12

00120] Compounds of general formula ldl could be made as per Scheme 13. Previously described isothiocyanate derivative XV could be combined with amidine XXV under dehydrating reaction conditions to give 1,2,4-thiadiazole (ldl).

Scheme 13

Compounds of general formula lei could be prepared using a synthetic approach as shown in Scheme 14. Isothiocyanate XV could be combined with azide XXVI in the presence of phosphine to yield 1,3-oxazole Iel .

Scheme 14

Compounds of general formula lgl could be prepared using a synthetic approach as shown in Scheme 15. Amine VII could be combined with acyl isothiocyanate XXVII. The acylthioureaido could be condensed with hydrazine derivative to yield the 1,2,4-triazol derivative lgl.

igi

Scheme 15

without a methyl

Preparation of 7V,7V-dicyclopropyl-6-ethyl-l-methyl-4-(5-m ethyl- lH-pyrazol-3- ylamino)-l,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-carboxamide

[00437] Prepared using similar protocol as for example 72 from hydrazine.

[00438] MS (ESI) m/z 419.3 (M+H)

[00439] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.70 (br s, 1 H), 7.91 (br s, 1 H), 6.87 (s, 1 H), 6.09 (br s, 1 H), 4.64 (q, 2 H, J= 7.03 Hz), 4.08 (s, 3 H), 2.74 -2.95 (m, 2 H), 2.41 (s, 3 H), 1.51 (t, 3 H, J= 7.15 Hz), 0.81 – 0.95 (m, 4 H), 0.70 -0.81 (m, 4 H)

with an ethyl

7V,iV-dicyclopropyl-6-ethyl-4-(l-ethyl-5-methyl-lH-pyrazol-3-ylamino)-l-methyl- 1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridine-7-carboxamide

74A Preparation of fe/t-butyl l,3-dioxoisoindolin-2-yl(ethyl)carbamate

Diisopropyl azodicarboxylate (2.92 mL, 15.00 mmol) was added in one portion to a solution of tert-butyl l,3-dioxoisoindolin-2-ylcarbamate (2.62 g, 10 mmol, prepared following the procedure described by Nicolas Brosse et al. in Eur. J. Org. Chem. 4757-4764, 2003), triphenylphosphine (3.93 g, 15.00 mmol) and ethanol (0.691 g, 15.00 mmol) in THF (20 mL) at 0 °C and the reaction solution was stirred at room temperature for lh (monitored by TLC until completion). Solvent was evaporated and the residue was purified by flash chromatography on silica gel using an automated ISCO system (80 g column, eluting with 5-35% ethyl acetate / hexanes) to provide tert-butyl l,3-dioxoisoindolin-2-yl(ethyl)carbamate (2.6 g, 90 % yield) as a white solid which was used as it in the next step

74B Preparation of fe/t-butyl l-ethylhydrazinecarboxylate

Boc

H₂N-N

\

Methylhydrazine (1.415 niL, 26.9 mmol) was added to a solution oi tert-butyl l,3-dioxoisoindolin-2-yl(ethyl)carbamate (example 74A, 5.2 g, 17.91 mmol) in THF (40 mL) at 0 °C and the reaction mixture was stirred at room temperature overnight. A white precipitate formed and was filtered off through a pad of Celite, The filtrate was concentrated in vacuo. The residue was dissolved in ethyl acetate (50 ml) and extracted with IN HC1 (3×30 ml), the acid layer was washed with ethyl acetate (50 ml) and basified to pH 10 by addition of 20% NaOH. The basic solution was then extracted with ethyl acetate (3×50 ml) and the combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo to give tert-butyl 1 -ethylhydrazinecarboxylate (2.5 g, 87 % yield) as colorless oil.

^XH NMR (400 MHz, CDC1₃) δ: 3.90 (br. s., 2H), 3.35 (q, J = 7.0 Hz, 2H), 1.42 (s, 9H), 1.07 (t, J = 7.0 Hz, 3H)

74 Preparation of N.N-dicyclopropyl-6-ethyl-4-(l-ethyl-5-methyl-lH-pyrazol-3-ylamino)-l-methyl-l ,6-dihydroimidazor4,5-d1pyrrolor2,3-b1pyridine-7-carboxamide

A mixture of (Z)-N,N-dicyclopropyl-6-ethyl- 1 -methyl-4-( 1 -(methylthio)-3-oxobut-l-enylamino)-l,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-carboxamide (example 74B, 70 mg, 0.155 mmol) and tert-butyl 1-ethylhydrazinecarboxylate (49.6 mg, 0.309 mmol) in acetic acid (1 mL) wan stirred at 35 °C for 4 h (monitored by LC/MS until no starting material left). Formic acid (1 mL) was added and the reaction mixture stirred at 60 °C for 6 h. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel using an automated ISCO system (12 g column, eluting with 2-10% methanol / dichloromethane). The material was further purified by preparative HPLC to afford N,N-dicyclopropyl-6-ethyl-4-( 1 -ethyl-5-methyl- lH-pyrazol-3-ylamino)- 1 -methyl- 1 ,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-carboxamide (38 mg, 53.4 % yield) as an off-white solid.

MS (ESI) m/z 447.3 (Μ+Η).

^XH NMR (500 MHz, CDC1₃) δ: 8.08 (s, 1H), 7.61 (s, 1H), 6.93 (s, 1H),

6.84 (s, 1H), 4.66 (q, J = 7.1 Hz, 2H), 4.02 (q, J = 7.2 Hz, 2H), 3.98 (s, 3H), 2.79 – 2.85 (m, 2H), 2.34 (s, 3H), 1.49 (t, J = 7.1 Hz, 3H), 1.41 (t, J = 7.2 Hz, 3H), 0.82 -0.87 (m, 4H), 0.72 – 0.78 (m, 4H).

Patent

JAK2 INHIBITORS AND THEIR USE FOR THE TREATMENT OF MYELOPROLIFERATIVE DISEASES AND CANCER [US8202881]2011-03-102012-06-19

JAK2 inhibitors and their use for the treatment of myeloproliferative diseases and cancer [US8673933]2012-04-302014-03-18

: Purandare AV, McDevitt TM, Wan H, You D, Penhallow B, Han X, Vuppugalla R, Zhang Y, Ruepp SU, Trainor GL, Lombardo L, Pedicord D, Gottardis MM, Ross-Macdonald P, de Silva H, Hosbach J, Emanuel SL, Blat Y, Fitzpatrick E, Taylor TL, McIntyre KW, Michaud E, Mulligan C, Lee FY, Woolfson A, Lasho TL, Pardanani A, Tefferi A, Lorenzi MV. Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2. Leukemia. 2012 Feb;26(2):280-8. doi: 10.1038/leu.2011.292. Epub 2011 Oct 21. PubMed PMID: 22015772.

Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2http://www.nature.com/leu/journal/vaop/ncurrent/full/leu2011292a.html

GRAPHS

http://pubs.acs.org/doi/suppl/10.1021/acs.joc.5b00572/suppl_file/jo5b00572_si_001.pdf

//////BMS 911543, phase 2, bms,

Elotuzumab

Approved nov 30 2012

A SLAMF7-directed immunostimulatory antibody used to treat multiple myeloma.

(Empliciti®)

HuLuc-63;BMS-901608

915296-00-3

Elotuzumab (brand name Empliciti, previously known as HuLuc63) is a humanized monoclonal antibody used in relapsed multiple myeloma.^[1] The package insert denotes its mechanism as a SLAMF7-directed (also known as CD 319) immunostimulatory antibody.^[2]

Approvals and indications

In May 2014, it was granted “Breakthrough Therapy” designation by the FDA. ^[3] On November 30, 2015, FDA approved elotuzumab as a treatment for patients with multiple myeloma who have received one to three prior medications.^[1] Elotuzumab was labeled for use with lenalidomide and dexamethasone. Each intravenous injection of elotuzumab should be premedicated with dexamethasone, diphenhydramine, ranitidine and acetaminophen.^[2]

Elotuzumab is APPROVED for safety and efficacy in combination with lenalidomide and dexamethasone.

Monoclonal antibody therapy for multiple myeloma, a malignancy of plasma cells, was not very clinically efficacious until the development of cell surface glycoprotein CS1 targeting humanized immunoglobulin G1 monoclonal antibody – Elotuzumab. Elotuzumab is currently APPROVED in relapsed multiple myeloma.

Elotuzumab (HuLuc63) binds to CS1 antigens, highly expressed by multiple myeloma cells but minimally present on normal cells. The binding of elotuzumab to CS1 triggers antibody dependent cellular cytotoxicity in tumor cells expressing CS1. CS1 is a cell surface glycoprotein that belongs to the CD2 subset of immunoglobulin superfamily (IgSF). Preclinical studies showed that elotuzumab initiates cell lysis at high rates. The action of elotuzumab was found to be enhanced when multiple myeloma cells were pretreated with sub-therapeutic doses of lenalidomide and bortezomib. The impressive preclinical findings prompted investigation and analysis of elotuzumab in phase I and phase II studies in combination with lenalidomide and bortezomib.

Elotuzumab As Part of Combination Therapy: Clinical Trial Results

Elotuzumab showed manageable side effect profile and was well tolerated in a population of relapsed/refractory multiple myeloma patients, when treated with intravenous elotuzumab as single agent therapy. Lets’ take a look at how elotuzumab fared in combination therapy trials,

In phase I trial of elotuzumab in combination with Velcade/bortezomib in patients with relapsed/refractory myeloma, the overall response rate was 48% and activity was observed in patients whose disease had stopped responding to Velcade previously. The trial results found that elotuzumab enhanced Velcade activity.
A phase I/II trial in combination with lenalidomide and dexamethasone in refractory/relapsed multiple myeloma patients showed that 82% of patients responded to treatment with a partial response or better and 12% of patients showed complete response. Patients who had received only one prior therapy showed 91% response rate with elotuzumab in combination with lenalidomide and dexamethasone.

Phase I/II trials of the antibody drug has been very impressive and the drug is currently into Phase III trials. Two phase III trials are investigating whether addition of elotuzumab with Revlimid and low dose dexamethasone would increase the time to disease progression. Another phase III trial (ELOQUENT 2) is investigating and comparing safety and efficacy of lenalidomide plus low dose dexamethasone with or without 10mg/kg of elotuzumab in patients with relapsed/refractory multiple myeloma.

Elotuzumab is being investigated in many other trials too. It is being evaluated in combination with Revlimid and low-dose dexamethasone in multiple myeloma patients with various levels of kidney functions, while another phase II study is investigating elotuzumab’s efficacy in patients with high-risk smoldering myeloma.

The main target of multiple myeloma drug development is to satisfy the unmet need for drugs that would improve survival rates. Elotuzumab is an example that mandates much interest in this area and should be followed with diligence.

Elotuzumab

Monoclonal antibody
Type	Whole antibody
Source	Humanized
Target	SLAMF7 (CD319)
Clinical data
Trade names	Empliciti
Pregnancy category	US: X (Contraindicated) X (used with lenalidomide)
Legal status	US: ℞-only
Routes of administration	IV
Pharmacokinetic data
Bioavailability	100% (IV)
Identifiers
CAS Number	915296-00-3
ATC code	None
IUPHAR/BPS	8361
UNII	1351PE5UGS
Chemical data
Formula	C6476H9982N1714O2016S42
Molecular mass	145.5 kDa

References

1 “Press Announcement—FDA approves Empliciti, a new immune-stimulating therapy to treat multiple myeloma”. U.S. Food and Drug Administration. Retrieved 3 December 2015.

2“Empliciti (elotuzumab) for Injection, for Intravenous Use. Full Prescribing Information” (PDF). Empliciti (elotuzumab) for US Healthcare Professionals. Bristol-Myers Squibb Company, Princeton, NJ 08543 USA.

3 “Bristol-Myers Squibb and AbbVie Receive U.S. FDA Breakthrough Therapy Designation for Elotuzumab, an Investigational Humanized Monoclonal Antibody for Multiple Myeloma” (Press release). Princeton, NJ & North Chicago, IL: Bristol-Myers Squibb. 2014-05-19. Retrieved 2015-02-05.

///////

BIA 10-2474

3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide

BIA 10-2474 is an experimental fatty acid amide hydrolase inhibitor^[1] developed by the Portuguese pharmaceutical company Bial-Portela & Ca. SA. The drug was developed to relieve pain,^[2][3] to ease mood and anxiety problems, and to improve movement coordination linked to neurodegenerative illnesses.^[4] It interacts with the human endocannabinoid system.^[5][6] It has been linked to severe adverse events affecting 5 patients in a drug trial in Rennes, France, and at least one death, in January 2016.^[7]

 Synthesis coming…….

Structure and action

French newspaper Le Figaro has obtained Bial study protocol documents listing the the chemical name of BIA-10-2474 as 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide.^[8] A Bial news release described BIA-10-2474 as “a long-acting inhibitor of FAAH”.^[9]

Fatty acid amide hydrolase (FAAH) is an enzyme which degrades endocannabinoid neurotransmitters like anandamide,^[10] which relieves pain and can affect eating and sleep patterns.^[11][12] FAAH inhibitors have been proposed for a range of nervous-system disorders including anxiety, alcoholism, pain and nausea.

The Portuguese pharmaceutical company Bial holds several patents on FAAH enzyme inhibitors.^{[12][13][14][15]}

No details of the preclinical testing of this molecule have been made public by the manufacturer Bial. However, the French newspaper Le Figaro has obtained and published an apparently legitimate copy of the full clinical trial protocol (BIA-102474-101).^[8] The protocol presents a summary of what appears to be a full package of pharmacodynamic, pharmacokinetic and toxicological studies that might be expected to support a first-in-man study, including safety pharmacology studies in two species (rat, dog) and repeated dose toxicity studies in four species (13 week sub-chronic studies in mouse, rat, dog and monkey). The summary presented however includes no assessment of the relevance of the animal species selected for study (that is, in terms of physiological and genetic similarities with humans and the mechanism of action of the study drug).

Of note, few adverse events were observed in any of the studies, with the 13-week oral No Observed Adverse Effect Level (NOAEL) varying between 10 mg/kg/day in mice to 75 mg/kg/day in monkeys. The authors suggest that these were the maximum doses tested in these studies, though it is not clear. The authors also report no effects of significance in the animal models used for the CNS safety pharmacology studies, which studied a dose of up to 300 mg/kg/day.^[8]

Notably absent from the protocol are calculations of receptor occupancy; predictions of in vivo ligand binding saturation levels; measures of target affinity; or assessment of the molecule’s activity in non-target tissues or non-target binding interactions as suggested by the European guidance for Phase I studies,^[16] assuming BIA 10-2474 could be considered ‘high risk’).^[8]

The trial protocol makes no reference to chimpanzee studies (only monkeys) which contradicts a previous statement to the media in which the French Health Minister stated that the drug had been tested on animals including chimpanzees.^{[4][17] [18]} Some experts had remarked that drug testing in chimpanzees was unlikely.^[19]

These findings provide no explanation for the type and severity of events observed in Rennes. In describing the rationale for the starting dose, the authors conclude that:

No target organ was identified during toxicology studies and few adverse clinical findings were observed at the highest dose tested. For the single ascending dose part [of the clinical trial], a starting dose of 0.25 mg was judged to be safe for a first-in-human administration. [8]

The protocol defines no starting dose for the multi-dose treatment groups, noting that this will be based on the outcome of the single dose portion of the trial (an approach known as adaptive trial design). The authors note that nonetheless, the starting dose will not exceed 33% of the maximum tolerated dose (MTD) identified in the single dose groups (or 33% of the maximum administered dose if the MTD is not reached).^[8]

Death and serious adverse events during phase I clinical trial

In July 2015 Biotrial, a contract research organization, began testing the drug in a human phase one clinical trial for the manufacturer. The study was approved by French regulatory authority, the Agence Nationale de Sécurité du Médicament (ANSM), on June 26, 2015, and by the Brest regional ethics committee on July 3, 2015.^[20] The trial commenced on July 9, 2015,^[21] in the city of Rennes, and recruited 128 healthy volunteers, both men and women aged 18 to 55. According to French authorities, the study employed a three-stage design with 90 of the volunteers having received the drug during the first two stages of the trial, with no serious adverse events being reported .^[17][20] Participants of the study were to receive €1,900 and, in turn, asked to stay at Biotrial’s facility for two weeks during which time they would take the drug for ten days and undergo tests.^[22]

In the third stage of the trial evaluating multiple doses, six male volunteers received doses by mouth, starting on 7 January 2016. The first volunteer was hospitalized at the Rennes University Hospital on January 10, became brain dead,^{[17][23][24][25]} and died on January 17.^[26] The other five men in the same dosage group were also hospitalized, in the period of January 10 through January 13^[27] four of them suffering injuries including deep hemorrhagic and necrotic lesions seen on brain MRI.^[7] The six men who were hospitalised were the group which received the highest dose.^[26] A neurologist at the University of Rennes Hospital Center, Professor Pierre-Gilles Edan, stated in a press conference with the French Minister for Health, that 3 of the 4 men who were displaying neurological symptoms “already have a severe enough clinical picture to fear that even in the best situation there will be an irreversible handicap” and were being given corticosteroids to control the inflammation.^[27] The sixth man from the group was not showing adverse effects but had been hospitalized for observation.^[25][28][29] Biotrial stopped the experiment on January 11, 2016.^[4]

No details of the trial have been made public by the manufacturer Bial. The study does not appear in searches of any of the key clinical trial registries, including EudraCT and ClinicalTrials.gov which would normally contain details of approved clinical studies.^{[30][31][32][33]} The trial protocol published by Le Figaro provides extensive detail on what was planned for the study, but many details of the key multi-dose part are not included and were to have been finalised at the conclusion of the single-dose part of the trial.^[8]

The French health minister Marisol Touraine called the event “an accident of exceptional gravity” and promised to investigate the matter.^[4] On January 18 it was reported authorities were investigating if a manufacturing or transport error might be involved.^[34]

Other pharmaceutical companies, including Merck, Pfizer, Johnson & Johnson, Sanofi and Vernalis, have previously taken other FAAH inhibitors into clinical trials without experiencing such adverse events (e.g. respectively, MK-4409,^[35][36] PF-04457845, JNJ-42165279,^[37] SSR411298 and V158866.^[38][39] Related enzyme inhibitor compounds such as URB-597 and LY-2183240 have been sold illicitly as designer drugs,^[40][41] all without reports of this type of toxicity emerging, so the mechanism of the toxicity observed with BIA 10-2474 remains poorly understood.

Following the events in Rennes, Janssen announced that it was temporarily suspending dosing in two Phase II clinical trials with its own FAAH inhibitor JNJ-42165279, headlining the decision as “precautionary measure follows safety issue with different drug in class”. Janssen was emphatic that no serious adverse events had been reported in any of the clinical trials with JNJ-42165279 to date. The suspension is to remain in effect until more information is available about the BIA 10-2474 study.^[42]

References

• 1 “BRIEF-Bial says firmly committed to ensure wellbeing of test participants”. Reuters. 15 January 2016.
• 2 “BIA 10-2474”. Biocentury.com. Retrieved 2016-01-17.
• 3 “BIA 102474”. Adisinsight/springer.com. Retrieved 2016-01-17.
• 4 Adamson B (January 15, 2016). “Botched Drug Trial Leaves 1 Brain Dead, 5 in Hospital”. ABC, AP. Retrieved January 16, 2016.
• 5 Angeline Benoit,Makiko Kitamura (15 January 2016). “France Ties Brain-Dead Person to Tests of Bial-Portela Drug”. Bloomberg.com.
• 6 “France/Monde – Essai thérapeutique : 90 personnes ont pris la molécule”. Ledauphine.com. Retrieved 2016-01-17.
• 7 Enserink, Martin (2016). “More Details Emerge on Fateful French Drug Trial” (online). Science (January 16). Retrieved 16 January 2016.
• 8 “Drame de Rennes : le protocole de l’essai clinique en accusation”. sante.lefigaro.fr. Retrieved 2016-01-21.
• 9 “News Release – Phase I Clinical Trial Rennes”. http://www.bial.com. Retrieved 2016-01-21.
• 10 Debora Mackenzie. “Six in hospital after French pain relief drug trial goes wrong”. New Scientist.
• 11 “The Discovery and Development of Inhibitors of Fatty Acid Amide Hydrolase (FAAH)”. PubMed Central.
• 12 “Patent WO2015012708A1 – Imidazolecarboxamides and their use as faah inhibitors – Google Patents”. Google.com. Retrieved 2016-01-17.
• 13 “Patent WO2015016729A1 – Urea compounds and their use as faah enzyme inhibitors”. Google.com. Retrieved 2016-01-17.
• 14 “PT2014000052 UREA COMPOUNDS AND THEIR USE AS FAAH ENZYME INHIBITORS”. Patentscope.wipo.int. Retrieved 2016-01-17.
• 15 WO application 2015016729, Laszlo Erno KISS, Rita GUSMÃO DE NORONHA, Carla Patrícia ROSA DA COSTA PEREIRA, Rui PINTO, “Urea compounds and their use as faah enzyme inhibitors”, published Feb 5, 2015, assigned to BIAL
• 16″Strategies to identify and mitigate risks for first-in-human clinical trials with investigational medicinal products (CHMP/SWP/28367/07)” (PDF). European Medicines Agency. 1 September 2007. Retrieved 22 January 2016.
• 17 “Accident grave dans le cadre d’un essai clinique – Intervention de Marisol Touraine à Rennes”. Ministère des Affaires Sociales, de la Santé et des Droits des Femmes, France. 15 January 2016.
• 18Barbara Casassus (23 January 2016). “France investigates drug trial disaster” (PDF). The Lancet 387 (10016): 326. doi:10.1016/S0140-6736(16)00154-9.
• 19
• “Expert reaction to French drug trial – reports of one patient dying and five others in hospital and of the Paris prosecutor’s office having opened an investigation into what happened”. Science Media Centre, London. 16 January 2016. Retrieved 21 January 2016.
• 20
• “La survenue d’effets graves ayant entraîné l’hospitalisation de 6 patients, dont un en état de mort cérébrale, a conduit à l’arrêt prématuré d’un essai clinique du laboratoire BIAL – Point d’information”. Agence Nationale de Sécurité du Médicament, France (ANSM). 15 January 2016.
• 21
• “Six hospitalized in Bial clinical trial in France”. BioWorld.com. Retrieved 2016-01-17.
• 22
• Enserink M (January 16, 2016). “More details emerge on fateful French drug trial”. Science Magazine. Retrieved January 18, 2016.
• 23
• “France clinical trial: 90 given drug, one man brain-dead”. BBC. January 15, 2016. Retrieved January 16, 2016.
• 24
• “Ce que l’on sait de l’accident survenu lors d’un essai clinique à Rennes”, Le Monde, 15 January 2016
• 25
• Blamont M (January 15, 2016). “French drug trial disaster leaves one brain dead, five injured”. Reuters. Retrieved January 16, 2016.
• 26
• Clarisse Lucas, AFP (January 17, 2016). “Man dies after being left brain-dead in French drug trial”. Yahoo. Retrieved January 17, 2016.
• 27
• “Accident “inédit” lors d’un essai clinique: un homme en état de mort cérébrale, cinq hospitalisés”. La Depeche. January 15, 2016. Retrieved January 18, 2016.
• 28
• “France clinical trial: ‘No known antidote’ to drug”. BBC News. 15 January 2016. Retrieved 18 January 2016.
• 29
• Enserink, Martin (2016). “What We Know So Far About the Clinical Trial Disaster in France” (online). Science (January 15). Retrieved 16 January 2016.
• 30
• “EU Clinical Trials Register”. European Medicines Agency. Retrieved 20 January 2016.
• 31
• “WHO International Clinical Trials Registry Platform”. World Health Organization. Retrieved 20 January 2016.
• 32
• “ANSM – Répertoire public des essais cliniques de médicaments”. Agence Nationale de Sécurité du Médicament et des Produits de Santé, France. Retrieved 20 January 2016.
• 33
• “ClinicalTrials.gov Registry”. U.S. National Institutes of Health. Retrieved 20 January 2016.
• 34
• “Drug trial volunteer dies as ‘manufacturing error’ theory investigated”. The Independent. January 18, 2016. Retrieved January 18, 2016.
• 35
• Chobanian; et al. (10 April 2014). “Discovery of MK-4409, a Novel Oxazole FAAH Inhibitor for the Treatment of Inflammatory and Neuropathic Pain”. ACS Med. Chem. Lett.
• 36
• Merck (15 October 2009). “Merck Pipeline, Oct 2009” (PDF). Merck.
• 37
• “Seven studies found for: jnj-42165279”. Clinicaltrials.gov. Retrieved 2016-01-19.
• 38
• “2 studies found for: V158866”. Clinicaltrials.gov. Retrieved 2016-01-19.
• 39
• Bisogno T, Maccarrone M. Latest advances in the discovery of fatty acid amide hydrolase inhibitors. Expert Opin Drug Discov. 2013 May;8(5):509-22. PMID 23488865 doi: 10.1517/17460441.2013.780021.
• 40
• Shanks KG, Behonick GS, Dahn T, Terrell A. Identification of novel third-generation synthetic cannabinoids in products by ultra-performance liquid chromatography and time-of-flight mass spectrometry. J Anal Toxicol. 2013 Oct;37(8):517-25. PMID 23946450 doi: 10.1093/jat/bkt062.
• 41
• Uchiyama N, Matsuda S, Kawamura M, Shimokawa Y, Kikura-Hanajiri R, Aritake K, Urade Y, Goda Y. Characterization of four new designer drugs, 5-chloro-NNEI, NNEI indazole analog, α-PHPP and α-POP, with 11 newly distributed designer drugs in illegal products. Forensic Sci Int. 2014 Oct;243:1-13. PMID 24769262 doi: 10.1016/j.forsciint.2014.03.013.

External links

• Bial pipeline
• Urea compounds and their use as faah enzyme inhibitors – Patent
• Selection patent

BIA 10-2474

Systematic (IUPAC) name
3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide
Clinical data
Legal status	Investigational New Medicine
Routes of administration	Oral
Identifiers
PubChem	CID: 46831476
Chemical data
Formula	C16H20N4O2   Molecular mass 300.36 g·mol−1 SMILES O=C(N1C=C(C2=C[N+]([O-])=CC=C2)N=C1)N(C3CCCCC3)C

/////////

C1C(CCCC1)N(C)C(=O)n2cc(nc2)c3ccc[n+](c3)O

GCC-4401C ( GC-2107), Nokxaban

In phase 1 for treating thrombosis

5-chloro-N-({(5S)-2-oxo-3-[4-(5,6-dihydro-4H-[1,2,4]triazin-1-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide methanesulfonate

5-chloro-N-[[3-[4-(5,6-dihydro-2H-1,2,4-triazin-1-yl)phenyl]-2-oxo-1,3-oxazolidin-5-yl]methyl]thiophene-2-carboxamide

CB02-0133; GC-2107; GC4401; GCC-2107; GCC-4401; GCC-4401C; I Fxa – LegoChem Biosciences; LCB02-0133; Nokxaban

WO2010002115; LegoChem Bioscience INNOVATOR

DEVELOPER

CAS NO FREE FORM

CAS 1159610-29-3, 159610-29-3, C₁₈ H₁₈ Cl N₅ O₃ S

2-​Thiophenecarboxamide​, 5-​chloro-​N-​[[(5S)​-​3-​[4-​(5,​6-​dihydro-​1,​2,​4-​triazin-​1(2H)​-​yl)​phenyl]​-​2-​oxo-​5-​oxazolidinyl]​methyl]​-

Molecular Formula: C₁₈H₁₈ClN₅O₃S Molecular Weight: 419.88522 g/mol

METHANE SULFONATE

CAS 1261138-12-8, C₁₈ H₁₈ Cl N₅ O₃ S . C H₄ O₃ S,

2-​Thiophenecarboxamide​, 5-​chloro-​N-​[[(5S)​-​3-​[4-​(5,​6-​dihydro-​1,​2,​4-​triazin-​1(2H)​-​yl)​phenyl]​-​2-​oxo-​5-​oxazolidinyl]​methyl]​-​, methanesulfonate (1:1)

HYDROCHLORIDE

CAS 1261138-08-2., C₁₈ H₁₈ Cl N₅ O₃ S . Cl H, 2-​Thiophenecarboxamide​, 5-​chloro-​N-​[[(5S)​-​3-​[4-​(5,​6-​dihydro-​1,​2,​4-​triazin-​1(2H)​-​yl)​phenyl]​-​2-​oxo-​5-​oxazolidinyl]​methyl]​-​, hydrochloride (1:1)

SUMMARY

• 09 Jan 2015GC 2107 is available for licensing as of 09 Jan 2015. http://www.greencross.com
• 01 May 2014Green Cross Corporation completes a phase I trial in Healthy volunteers in USA (NCT01954238)
• 26 Sep 2013Green Cross initiates enrolment in a phase I trial in Healthy volunteers in USA (NCT01954238)

Used as factor Xa antagonist for treating coronary artery disease, inflammatory disease, myocardial infarction and thrombosis.

Green Cross Corp in collaboration with LegoChem Bioscience, is developing GCC-4401C ( phase I), for treating thrombosis including venous thromboembolism

KDDF-201210-04

PATENT

WO 2016010178

GREEN CROSS CORPORATION [KR/KR]; 107, Ihyeon-ro 30beon-gil, Giheung-gu, Yongin-si, Gyeonggi-do 446-770 (KR).
LEGOCHEM BIOSCIENCES, INC. [KR/KR]; 8-26, Munpyeongseo-ro, Daedeok-gu, Daejeon 306-220 (KR)

The present invention relates to a novel crystalline form of 5-chloro-N-({(5S)-2-oxo-3-[4-(5,6-dihydro-4H-[1,2,4]triazin-1-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide methanesulfonate and a pharmaceutical composition containing the same. The novel crystalline form of a compound according to the present invention exhibits excellent stability even in high-temperature and humidity environments, and thus can be favorably used to prevent or treat diseases, such as thrombosis, myocardial infarction, atherosclerosis, inflammation, stroke, angina pectoris, restenosis after angioplasty, and thromboembolism.

According to the present invention 5-chloro -N – ({(5 S) -2- oxo-3- [4- (5,6-dihydro the -4H- [1, 2, 4] triazine-1-yl) phenyl] -1, 3-oxazolidin-5-yl} methyl) thiophene-2-mid copy methane sulfonic acid salt (hereinafter referred to as a new crystal form has excellent solubility referred to) in “GCO4401C”, Ko Un and wet environments It is excellent in stability.

Novel crystalline forms of GCC-4401C of the present invention, the organic solvent under reduced pressure crystallization method, a cooling crystallization method or solvent-can be easily obtained by the anti-solvent crystallization process.

Ateumyeo GCC-4401C is used as a reaction raw material can be prepared according to the procedure described in PCT Publication No. W02011 / 005029 No., dissolving the starting compound in an organic solvent the semi-adding a solvent after filtration to determine the resulting mixture was cooled and then dried to give the novel crystalline form can be a compound according to the invention.

PATENT

http://www.google.com/patents/WO2011005029A2?cl=en

5-Chloro-N-( {(5S)-2-oxo-3-[4-(5,6-dihydro-4H-[ 1 ,2,4]triazin- 1-yl)phenyl]-l,3-oxazolidin-5-yl}-methyl)thiophene-2-carboxamide of formula (A) has been known as an inhibitor of blood coagulation factor Xa and used for treating and preventing thrombosis, myocardial infarction, arteriosclerosis, inflammation, stroke, angina pectoris, recurrent stricture after angioplasty, and thromboembolism such as intermittent claudication.

Korea Patent No. 2008-64178, whose application has been filed by the present invetors, discloses a use of the compound as an inhibitor of blood coagulation factor Xa and a preparation method thereof. The preparation method comprises the step of preparing a cyclic amidrazone starting from 4-nitroaniline, as shown in reaction scheme 1 :

Reaction Scheme 1

Specifically, the cyclic amidrazone (A) is prepared by the steps of: preparing the compound (B) using 4-nitroaniline; treating the compound (B) with a t-butoxycarbonyl amine protecting group to prepare the compound (C); introducing a nitroso group into the compound (C) using NaNO₂, followed by reduction using zinc to prepare the compound (D); and treating the compound (D) successively with hydrochloric acid and an ortho-formate.

However, the above preparation method is complicated and gives a low yield of the compound (A) (e.g., a total yield of 9 %), and it also requires the use of a column chromatography purification step, which limits mass production of the cyclic amidrazone. In particular, the step for preparing the compound (D) from the compound (C) is required to use a harmful heavy metal-containg materal such as zinc amalgam which gives an unsatisfactorily low yield, and the isolation step of the compound (D) does not proceed easily.

Reaction Scheme 2

Reaction Scheme 3

Example 1: Preparation of Ethyl formimidate hydrochloride

To a solution of benzoyl chloride (1212 g, 8.62 mol, 1 eq) in anhydrous ether (5.8 L) was added dropwise a solution of formamide (388 g, 8.62 mol, 1 eq) in EtOH (396 g, 8.60 mol, 0.998 eq) at 0 ^°C for lhr. The mixture thus obtained was stirred at 0 ^°C for 30min. The solid was filtered off, washed with ether (3 L) and EA (3 L). The solid was dried under high vacuum.

Yield : 625 g (66%)

Example 1: 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-lH-[l,2,4]triazin-4-yl)phenyl]-l,3-oxazolidin-5-yl}-methyl)-2-thiophene carboxamide hydrochloride

Step 1: Preparation of 2- [N-(4-nitro-phenyl)-hydrazino]-ethanol

l-Fluoro-4-nitrobenzene (7.1 g, 50 mmol) was dissolved in CH₃CN (70 ml), 2-hydroxyethylhyrazine (purity: 90 %, Aldrich, 5.0 g, 66 mmol) and K₂CO₃ (7.6 g, 55 mmol) were added thereto. The suspension thus obtained was stirred for 4 hrs with reflux. The resulting orange-colored suspension was concentrated under reduced pressure (reflux condenser, 10 torr, 40 ^°C) and ethylacetate (EA, 90 ml) and water (18 ml) were added thereto. The resulting mixture was stirred strongly at r.t. for 10 min. The organic layer was extracted and washed with the saturated brine (10 ml). The resulting solution was cooled to 10 °C and 48 % HBr solution (3.7 ml) was added thereto dropwise with stirring. The pale yellow colored solid thus obtained was filtered off and dried under high vacuum (1 torr, 40 “C) to obtain the title compound as an intermediate.

Yield: 7.1 g (51 %).

TLC : Rf= 0.62 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (600 MHz, DMSO-J₆) δ 8.17 (d, J = 9.0 Hz, 2H), 7.12 (d, J = 9.0 Hz, 2H), 3.82 (t, J= 5.4 Hz, 2H), 3.69 (t, J= 5.4 Hz, 2H)

LCMS: 198 (M+H⁺) (C₈H₁₁N₃O₃)

Step 2: Preparation of l-bromo-2-[N-(4-nitro-phenyl)-hydrazino] -ethane

The compound obtained in Step 1 (38.9 g, 0.140 mol) was suspended in anhydrous 1 ,2-dimethoxyethane (585 ml). The resultant suspension was cooled to 0 ^°C and PBr₃ (15.9 ml, 0.168 mol) was added thereto dropwise for 30 min. The mixture thus obtained was stirred at 60 ^°C for 4 hrs. The pale yellow colored solution thus obtained was concentrated under reduced pressure (reflux condenser, 10 torr, 45 ^°C). The resultant residue (oil) was suspended with water (150 ml) and stirred. Aq. sat’d NaHCO₃ solution (150 m) was added to the resultant suspension to be pH 4. The resulting mixture was stirred for 30 min to precipitate the pale yellow colored precipitates. The precipitates were filtered off and washed with water (100 ml). The resulting solid was mixed with water (100 ml), aq. sat’d NaHCO₃ solution (70 ml) and CH₂Cl₂ (500 ml). The resulting mixture was stirred for 10 min and stood to separate organic and aqueous layers. The organic layer was dried over 20 g of MgSO₄ and filtered off. The resulting filterate was concentrated under reduced pressure (reflux condenser, 10 torr, 40 ^°C) to obtain the title compound as a pale yellow solid.

Yield : 31.3 g (86 %)

TLC : Rf= 0.91 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (600 MHz, CDCl₃) δ 8.14 (d, J = 10.2 Hz, 2H), 6.92 (d, J= 10.2 Hz, 2H), 4.00 (t, J= 7.2 Hz, 2H), 3.65 (t, J= 7.2 Hz, 2H)

LCMS: 261 (M+H⁺) (C₈H₁₀BrN₃O₂)

Step 3: Preparation of 4-(5,6-dihydro-4H-[l,2,4]triazin-l-yl)-l-nitrobenzene

The compound obtained in Step 2 (13.0 g, 50.0 mmol) was completely dissolved in anhydrous 1,2-dimethoxyethane (200 ml) which is prepared by mixing 1,2-dimethoxyethane (purity: 99 %, Junsei Co. Ltd) with an desired amount of molecular sieve 4A and standing for 5 hrs or more with stirring at times. Ethyl formimidate HCl salt (5.8 g, 52.5 mmol) was added thereto. The suspension thus obtained was stirred at 25 ^°C for 10 min. Anhydrous sodium acetate (NaOAc, 8.6 g, 105 mmol) was added thereto and stirred for 15 hrs with reflux. The orange colored suspension thus obtained was concentrated under reduced pressure (10 torr, 50 “C). The orange colored residue thus obtained was mixed with IN HCl (140 ml), EA (50 ml) and hexane (100 ml), and stirred at r.t for 10 min. A small amount of insoluble suspended solids was remained in aqueous layer and filtered off. The resulting aqueous layer was washed with a mixture of EA (30 ml) and hexane (60 ml). 12 g of sodium carbonate was added to the resulting solution to be pH 8.5. The orange colored solid thus obtained was filtered off under reduced pressure, washed with water (15 ml) and dried under vacuum to obtain the title compound .

Yield : 7.7 g (75 %).

TLC : R/= 0.45 (EA/MeOH/AcOH = 20/1/0.5)

HPLC : R, = 8.65 (Gradient A), purity 91.1%

¹H NMR (400 MHz, DMSO-^₆) δ 8.03 (d, J= 9.6 Hz, 2H), 7.16 (d, J = 9.6 Hz, 2H), 7.12 (br s, IH), 7.01 (d, J= 4.0 Hz, 2H), 3.77 (t, J= 5.2 Hz, 2H), 3.43-3.40 (m, 2H)

LCMS: 207 (M+H⁺) (C₉H₁₀N₄O₂)

Step 4: Preparation of 4-(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yl)-1-nitrobenzene

To the orange colored suspension prepared by suspending the compound obtained in Step 3 (12.4 g, 60 mmol) in tetrahydrofurane (THF, 200 ml), 4-dimethylaminopyridine (DMAP, 0.367 g, 3 mmol) and di-tert-butyl dicarbonate

(BoC₂O, 19.6 g, 90 mmol) were added and stirred with reflux for 1.5 hrs. The yellow colored suspension thus obtained was concentrated under reduced pressure

(reflux condenser, 10 torr, 40 ^°C) to remove the solvent. The resulting yellow colored residue was completely dissolved in CH₂Cl₂ (700 ml) and washed with IN HCl (700 ml). The organic layer was extracted, dried over 25 g of MgSO₄, and concentrated under reduced pressure (condenser, 10 torr, 40 ^°C). The resultant yellow colored residue was dissolved in cyclohexane (250 ml) and stirred strongly at r.t. for 30 min. The resulting mixture was concentrated under reduced pressure to obtain yellow colored solids. The solids were dried (1 torr, 50 ^°C ) to obtain a disried compound.

Yield: 15.6 g (85 %)

TLC : R/= 0.93 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (600 MHz, DMSO-J₆) δ 8.14 (d, J= 9.6 Hz, 2H), 7.62 (br s, IH), 7.30 (d, J = 9.6 Hz, 2H), 3.89 (br s, 2H), 3.79 (br s, 2H), 1.50 (s, 9H)

LCMS: 307 (M+H⁺) (C₁₄H₁₈N₄O₄)

Step 5: Preparation of 4-(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yl)aniline

To the yellow colored suspension prepared by suspending the compound obtained in Step 4 (19.9 g; 65 mmol) in methanol (200 ml), 10 % palladium on carbon (4.0 g) was added. The resulting mixture was subjected to vacuum outgassing and stirred at r.t., for 2 hrs in the flask connected with hydrogen bollum. The resulting mixture was filtered through celite 545 under redued pressure to remove the palladium on carbon. The fϊlterate was concentrated under reduced pressure (reflux condenser, 10 torr, 40 °C). The resulting pale brown colored residue was dissolved in isopropylalcohol (140 ml) and refluxed to dissolve completely. The resulting solution was stood at 0 ^°C for 2 hrs to cool, stirred for 30 min and filtered off under redued pressure. The resulting ivory crystalline solid was dried in vacuo to obtain the title compound (15.8 g, 88 %).

TLC : R_f= 0.38 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, DMSO-(I₆) δ 7.34 (br s, IH), 6.91 (d, J = 12.0 Hz, 2H), 6.51 (d, J = 12.0 Hz, 2H), 6.64 (br s, 2H), 3.74 (br s, 2H), 3.41 (br s, 2H), 1.48 (s, 9H)

LCMS: 277 (M+H⁺) (C₁₄H₂₀N₄O₂)

Step 6: Preparation of N-(3-(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yI)anilino-(2R)-2-hydroxypropyI)-5-chloro-2-thiophene carboxamide

The compound obtained in Step 5 (19.3 g, 70 mmol) and 5-chloro-N-(((S)-oxiran-2-yl)methyl)thiophene-2-carboxamide (19.1 g, 88 mmol) were suspended in isobutyl alcohol (350 ml) and stirred for 18 hrs with reflux. The dark blue colored solution thus obtained was concentrated under reduced pressure (reflux condenser, 10 torr, 50 °C). To the yellow solid residue thus obrained, ethylacetate (200 ml) was added and the resulting mixture was stirred at r.t. for 30 min and further stirred strongly at 0 ^°C for 30 min. The suspended solid thus obtained was filtered off under reduced pressure and dried in vaccum (1 torr, 50 °C ) to obtain the title compound as ivory crude.

Yield : 25.9 g (75 %)

TLC : R_/= 0.34 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR of a crude sample (600 MHz, DMSO-</₆) δ 8.62 (t, J = 5.4 Hz, IH), 7.69 (d, J = 3.6 Hz, IH), 7.36 (br s, IH), 7.18 (d, J = 4.2 Hz, IH), 6.95 (d, J = 9.0 Hz, 2H), 6.54 (d, J = 9.0 Hz, 2H), 5.10 (t, J = 6.6 Hz, IH), 5.05 (d, J = 5.4 Hz, IH), 3.81-3.75 (m, 3H), 3.44 (br s, 2H), 3.37-3.34 (m, IH), 3.25-3.21 (m, IH), 3.08-3.04 (m, IH), 2.94-2.89 (m, IH), 1.48 (s, 9H)

LCMS: 494 (M+H⁺) (C₂₂H₂₈ClN₅O₄S)

Step 7: Preparation of 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yl)phenyl]-l,3-oxazolidin-5-yI}-methyl)-2-thiophene carboxamide

The compound obtained in Step 6 (25.2 g, 51 mmol) was completely dissolved in THF (325 ml), and Ll’-carbonyldiimidazole (10.8 g, 66 mmol) and DMAP (0.31 mg, 2.6 mmol) were added thereto. The resulting mixture was stirred with reflux for 18 hrs. The resulting pale yellow colored suspension was cooled to r.t, concentrated under reduced pressure and dried in vacuo (1 torr, 50 ^°C) to obtain the title compound as an ivory solid.

Yield : 23.3 g (88 %)

TLC : R/= 0.75 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, DMSO-J₆) δ 8.97 (t, J = 5.4 Hz, IH), 7.69 (d, J= 4.2 Hz, IH), 7.43 (br s, IH), 7.41 (d, J = 9.0 Hz, 2H), 7.20 (d, J = 4.2 Hz, IH), 7.19 (d, J= 9.0 Hz, 2H), 4.82-4.77 (m, IH), 4.12 (t, J= 9.0 Hz, IH), 3.80-3.78 (m, 3H), 3.62 (br s, 2H), 3.59 (t, J= 6.0 Hz, 2H), 1.49 (s, 9H)

LCMS: 520 (M+H⁺) (C₂₃H₂₆ClN₅O₅S)

Step 8: Preparation of 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4H-[l,2,4]triazin-l-yl)phenyl]-l,3-oxazolidin-5-yl}-methyl)-2-thiophene

carboxamide hydrochloride

The compound obtained in Step 7 (16.1 g, 31 mmol) was completely

dissolved in THF (193 ml), 3N HCl (193 ml) was added thereto. The resulting solution was stirred with reflux for 1 hr. The white suspension thus obtained was cooled tq r.t, concentrated under reduced pressure and dried in vacuo (1 torr, 40 ^°C ) to obtain the title compound as a white solid.

Yield : 13.4 g (95 %)

TLC : R/= 0.82 (MC/MeOH/AcOH = 10/1/0.5)

HPLC : R, = 12.39 (Gradient A), purity 99.5%

¹H NMR (600 MHz, OMSO-d₆) δ 12.12 (br s, IH), 10.20 (br s, IH), 9.08

(t, J = 6.0 Hz, IH), 8.60 (d, J = 5.2 Hz, IH), 7.74 (d, J= 4.2 Hz, IH), 7.53 (d, J = 9.0 Hz, 2H), 7.20 (d, J= 4.2 Hz, IH), 7.13 (d, J= 9.0 Hz, 2H), 4.85-4.81 (m, IH),

4.15 (t, J = 8.8 Hz, IH), 3.85 (dd, J = 6.0, 9.2 Hz, IH), 3.66 (t, J = 4.8 Hz, 2H),

3.63-3.56 (m, 2H), 3.19 (br s, 2H)

LCMS: 420 (M+H⁺) (C₁₈H₁₈ClN₅O₃S)

Example 2: Preparation of 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4H-[l,2,4]triazin-l-yI)phenyl]-l,3-oxazolidin-5-yl}-methyI)-2-thiophene

carboxamide

The HCl salt obtained in Example 1 (6.9 g, 15 mmol) was completely dissolved in 33 % methanol aqueous solution (1.1 L) and heated to 50 ^°C while stirring. To the resulting colorlessness solution, 0.6M aq. Na₂CO₃ solution (25 ml) was added and the white suspension thus obtained was stood at 0 ^°C for 0.5 hr to cool. The white solid thus obtained was concentrated under reduced pressure, wished with H₂O (150 ml) and dried in vacuo (1 torr, 40 “C) to obtain the title compound (yield: 5.5 g, 87 %). The title compound was dissolved in methanol (330 ml) and stirred with reflux. The pale yellow colored solution thus obtained was stood at 0 ^°C for 2 hrs to cool. The resulting white solid was concentrated under reduced pressure, washed with methanol (10 ml), and dried in vacuo (1 torr, 40 ^“C) to obtain a crystal of the title compound (yield: 5.0 g, 80 %).

HPLC : R, = 12.37 (Gradient A), purity 99.7 %

¹H NMR (400 MHz, DMSO-^₆) δ 8.97 (t, J = 6.0 Hz, IH), 7.69 (d, J = 4.0 Hz, IH), 7.32 (d, J = 9.2 Hz, 2H), 7.20 (d, J = 4.0 Hz, IH), 7.12 (d, J = 9.2 Hz, 2H), 6.79 (d, J = 4.0 Hz, IH), 6.52 (br s, IH), 4.80-4.75 (m, IH), 4.10 (t, J = 8.8 Hz, IH), 3.77 (dd, J= 6.0, 9.2 Hz, IH), 3.58 (t, J= 5.6 Hz, 2H), 3.33 (s, 4H)

LCMS: 420 (M+H⁺) (C₁₈H₁₈ClN₅O₃S)

Example 3: Preparation of 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4H-[l,2,4]triazin-l-yl)phenyl]-l,3-oxazolidin-5-yI}-methyI)-2-thiophene carboxamide methane sulfonate

To the compound obtained in Example 2 (3.3 g, 7.9 mmol), a mixture solution of MeOH/CH₂Cl₂ (1/4 v/v, 70 ml) was added and stirred with reflux. The pale yellow colored solution thus obtained was cooled to 0 ^°C and methylsulfonic acid (0.56 ml, 8.6 mmol) was added thereto. The resulting mixture was concentrated under reduced pressure (reflux condenser, 10 torr, 40 ^°C) to obtain pale yellow foamy solid. To the resultant solid, absolute ethanol (20 ml) was added and the resulting mixture was stirred with reflux to dissolve solid clearly. The resulting solution was cooled to 0 ^°C to 2 hrs. The resulting white solid was concentrated under reduced pressure, washed with absolute EtOH (5 ml), and dried in vacuo (1 torr, 40 “C) to obtain a crystalline methane sulfonate.

Yield : 3.8 g (93 %)

HPLC : R, – 12.35 (Gradient A), purity 99.8%

¹H NMR (400 MHz, DMSO-CZ₆) δ 11.97 (br s, IH), 10.07 (br s, IH), 8.99

(t, J= 6.0 Hz, IH), 8.59 (U₁ J= 6.0 Hz, IH), 7.70 (d, J= 4.0 Hz, IH), 7.53 (d, J =

9.2 Hz, 2H), 7.20 (d, J= 4.0 Hz, IH), 7.13 (d, J= 9.2 Hz, 2H), 4.86-4.80 (m, IH),

4.16 (t, J = 9.2 Hz, IH), 3.82 (dd, J = 6.0, 9.2 Hz, IH), 3.67 (m, 2H), 3.60 (t, J = 5.6 Hz, 2H), 3.20 (br s, 2H), 2.31 (s, 3H)

LCMS: 420 (M⁺H⁺)(C₁₈H₁₈ClN₅O₃S)

Example 4: (S)-5-chloro-N-((3-(4-(5,6-dihydro-l,2,4-triazin-l(4H)-yl)phenyI)-2-oxooxazolidin-5-yl)methyl)thiophene-2-carboxamide methane sulfonate

Step 1: Preparation of (2-[N-(4-nitro-phenyl)-hydrazinyl]-ethanol) hydrobromide

l-Flouro-4-nitrobenzene (428 g, 3.03 mol, Aldrich Fl 1204) was dissolved in CH₃CN (4.3 L), and 2 -hydroxy ethylhyrazine (300 g, 3.94 mol, 1.3 eq, imported from China, >98 %) and K₂CO₃ (461 g, 3.34 mol, 1.1 eq, Aldrich

347825) were added thereto. The mixture thus obtained was stirred at 80 ^°C for

19 hrs. The mixture was cooled to r.t. and evaporated to remove solvent. The residue was dissolved with EA (1.5 L) and H₂O (1 L). The organic layer was extracted and washed with H₂O (500 mL) and brine (200 mL). The extracted

EA layer was cooled to 0 °C and 48 % HBr solution (360 mL, Aldrich 244260) was added thereto dropwise at 0 °C with stirring. The resultant mixture was stirred at 0 ^°C for 1 hr. The solid thus obtained was filtered off and washed with

EA (5 L). The obtained solid was dried under high vacuum to obtain the title compound.

Yield : 531 g (63 %)

TLC : Rf= 0.62 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, OMSO-d₆) δ 7.94 (d, J = 9.6 Hz, 2H), 7.12 (br s, 2H), 6.63
5.8 Hz, 2H) LCMS: 198 (M+H⁺) (C₈H₁₁N₃O₃)

Step 2: Preparation of l-bromo-2-[N-(4-nitro-phenyl)-hydrazino]-ethane

The compound obtained in Step 1 (531 g, 1.90 mol) was suspended in

anhydrous 1,2-dimethoxyethane (4.5 L). The resultant suspension was cooled to 0 ^°C and PBr₃ (220 niL, 2.29 mol, 1.2 eq, Aldrich 256536) was added thereto dropwise at 0 ^°C . The mixture thus obtained was warmed up to r.t. and stirred at 6O ⁰C for l5 hrs.

The mixture was cooled to r.t., and filtered off to remove remained insoluble solid. The filter cake thus obtained was washed with 1,2- dimethoxyethane (700 mL) and the filtrate was concentrated in vacuo. The resultant residue was suspended with H₂O (2.5 L), stirred and cooled to 0 ^°C . Aq. 2N NaOH solution (1.7 L) was added thereto at 0^°C to neutralize the suspension mixture (pH 6-7). The solid was filtered off and washed with H₂O (5 L). The filtered solid was air-dried for 5 hrs.

The air-dried solid was dissolved with CH₂Cl₂ (3 L), and aq. sat’d

NaHCO₃ solution (1.5 L) and H₂O (700 mL) were added thereto. The resultant

– mixture was stirred for 15 min and stood to separate organic and aqueous layers. Insoluble solid which was not dissolved in organic layer and H₂O was remained in the mixture. The mixture was filtered off to remove insoluble solid and the filter cake was washed with CH₂Cl₂ (700 mL). The organic layer was extracted, dried over MgSO₄, filtered off, and concentrated in vacuo. The resultant solid was dried under high vacuum to obtain the title compound.

Yield : 383 g (77% : When product was dissolved in CDCl₃ to check the

¹H NMR spectroscopy, insoluble solid was stilled remained in CDCl₃)

TLC : Rf= 0.91 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, CDCl₃) δ 8.12 (d, J = 9.6 Hz, 2H), 6.92 (d, J = 9.2 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H)

LCMS: 261 (M+H⁺) (C₈H₁₀BrN₃O₂)

Step 3: Preparation of 4-(5,6-dihydro-4H-[l,2,4]triazin-l-yl)-l-nitrobenzene

Ethyl formimidate HCI, NaOAc

1 ,2-dimethoxyethane

The compound obtained in Step 2 (384 g, 1.48 mol) was dissolved in anhydrous 1,2-dimethoxyethane (4 L) and ethyl formimidate HCl salt (322 g, 2.94 mol, 2 eq) was added thereto at r.t. The resultant mixture was stirred at r.t. for 30 min. NaOAc (364 g, 4.44 mol, 3.0 eq, Aldrich 110191) was added to the mixture and the mixture was stirred at 75 ^°C for 15 hrs.

The mixture was cooled to r.t. and evaporated to remove solvent. The resultant residue was suspended in EA (2 L) and 1,2-dimethoxyethane (I L). Aq.

3N HCl solution (2.5 L) was added to the suspension. Insoluble solid was remained in resultant mixture. The solid was filtered off two times to remove insoluble solid. Ether (3 L) was added to the filtrate to separate organic and aqueous layers effectively. Aqueous layer was separated and washed with mixed organic solution (EA (1 L) + Hexane (500 mL)). The combined organic layer should be kept to recover the product.

(The treatment of aqueous layer)

The aqueous layer was cooled to 0 ^°C and aq. 6N NaOH solution (2.2 L) was added thereto slowly to basify the H₂O layer (pH ~ 9). The resultant suspension was stirred at r.t. for 12 hrs. The solid was filtered off and washed with H₂O (3 L) and dried under high vacuum.

(The treatment of combined organic layer)

The combined organic layer was concentrated in vacuo. The resultant residue was acidified with aq. 3N HCl solution (500 mL). Filtration was carried out to remove insoluble solid. The filtrate (H₂O layer) thus obtained was washed with ether (700 mL X 2). The aqueous layer was stirred and cooled to 0 ^°C . Aq. 5N NaOH solution (1 L) was added to the cooled aqueous layer to basify (pH ~9). The mixture thus obtained was stirred at r.t. for 12 hrs. The solid thus obtained was filtered off and washed with H₂O (1.5 L). The solid was dried under high vacuum to obtain the title compound.

Yield : 187 g (62 %)

TLC : Rf= 0.45 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, DMSO-</₆) δ 7.99 (d, J = 9.6 Hz, 2H), 7.16 (d, J =

9.6 Hz, 2H), 7.09 (br s, IH), 6.97 (d, J = 3.6 Hz, 2H), 3.73 (t, J = 5.0 Hz, 2H), 3.45-3.46 (m, 2H)

LCMS: 207 (M+H⁺) (C₉H₁₀N₄O₂)

Step 4: Preparation of 4-(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yl)- 1-nitrobenzene

The compound obtained in Step 3 (187g, 0.907 mol) was suspended in anhydrous THF (2.2 L), and BoC₂O (30Og, 1.36 mol, 1.5 eq, Aldrich 205249) and DMAP (6g, 0.045 mol, 0.05 eq, Aldrich 107700) were added thereto. The mixture thus obtained was stirred at 65 ^°C for 5 hrs.

The mixture was cooled to 0 ^°C . MeOH (1.5 L) was added to the mixture at 0 ^°C and stirred at 0 °C for 1 hr. The solid thus obtained was filtered off, washed with MeOH (750 niL) and dried under high vacuum.

Filtrate thus obtained was concentrated in vacuo. MeOH (1 L) was added to the resultant residue with stirring. The mixture thus obtained was stirred at r.t for 12 hrs. Solid thus obtained was filtered off, washed with MeOH (500 mL), and dried under high vacuum to obtain the title compound.

Yield : 182 g (65 %)

TLC : R_f= 0.93 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, DMSO-J₆) δ 8.17 (d, J= 9.6 Hz, 2H), 7.57 (br s, IH), 7.19 (d, J= 9.6 Hz, 2H), 3.93-3.86 (m, 2H), 3.83-3.745 (m, 2H), 1.56 (s, 9H)

LCMS: 307 (M+H⁺) (C₁₄H₁₈N₄O₄)

Step 5: Preparation of 4-(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yl)aniline

The compound obtained in Step 4 (134 g, 438 mmol) was suspended in

MeOH (1.3 L) at r.t., and NH₄Cl (12 g, 0.5 eq, Aldrich A4514) and Zn (15 g, 0.5 eq, Aldrich 209988) were added 6 times at intervals of 15 min at r.t. (total amounts Of NH₄Cl = 73 g (1356 mmol, 3.1 eq) and total amounts of Zn = 88 g

(1356 mmol, 3.1 eq))

Temperature of the resultant mixture was risen gradually to 65 ^°C and the mixture was stirred at 65 ^°C for 12 hrs. The mixture was cooled to 40 ^°C and NH₄Cl (12 g, 0.5 eq, Aldrich A4514) and Zn (15 g, 0.5 eq, Aldrich 209988) were added thereto. Temperature of the resultant mixture was risen gradually to 65 ^°C and the mixture was stirred at 65 “C for 1 hr.

The mixture was cooled to r.t. and filtered off through celite pad. The filter cake was washed with MeOH (700 mL) and THF (700 mL) and the filtrate was concentrated. The crude product thus obtained was dried under high vacuum and used without further purification.

Yield : 124 g (quantitative)

TLC : Rf= 0.38 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, OMSO-d₆) δ 7.31 (br s, IH), 6.86 (d, J = 12.0 Hz, 2H), 6.48 (d, J = 12.0 Hz, 2H), 4.60 (s, 2H), 3.71 (br s, 2H), 3.38 (br s, 2H), 1.44 (s, 9H)

LCMS: 277 (M+H⁺) (C₁₄H₂₀N₄O₂)

Step 6: Preparation of N-(3-(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yl)anilino-(2R)-2-hydroxypropyl)-5-chloro-2-thiophene carboxamide

The compound obtained in Step 5 (120 g, 435 mmol) and 5-chloro-N-(((S)-oxiran-2-yl)methyl)thiophene-2-carboxamide (123 g, 566 mmol, 1.3 eq, purchased from RStech (Daejeon, Korea) was suspended in absolute EtOH (1450 mL). The mixture thus obtained was stirred at 85 ^°C for 16 hrs. The mixture was cooled to r.t. and evaporated in vacuo to remove solvent. The resultant residue was dried under high vacuum for 18 hrs. The dried solid was suspended in EA (2 L). The suspension thus obtained was stirred at r.t. for 1 hr. The solid thus obtained was filtered off and washed with EA (500 mL) and ether (500 mL). The filtered solid was dried under high vacuum to obtain the title compound.

Aniline (starting material), epoxide, over-reacted by product were contained in crude product.

Yield : 158 g (74 %)

TLC : Rf= 0.34 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR of a crude sample (400 MHz, DMSO-^₆) δ 8.57 (t, J = 5.4 Hz,

IH), 7.65 (d, J = 3.6 Hz, IH), 7.32 (br s, IH), 7.14 (d, J = 4.2 Hz, IH), 6.90 (d, J

= 9.0 Hz, 2H), 6.51 (d, J = 9.0 Hz, 2H), 5.04 (t, J = 6.6 Hz, IH), 5.00 (d, J = 5.4 Hz, IH), 3.87-3.65 (m, 3H), 3.40 (br s, 2H), 3.37-3.34 (m, IH), 3.25-3.21 (m, IH),

3.17-2.96 (m, IH), 2.94-2.84 (m, IH), 1.44 (s, 9H)

LCMS: 494 (M+H⁺) (C₂₂H₂₈ClN₅O₄S)

Step 7: Preparation of 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4-t-butoxycarbonyl-[l,2,4]triazin-l-yl)phenyl]-l,3-oxazolidin-5-yl}-methyl)-2-thiophene carboxamide

The compound obtained in Step 6 (158 g, 320 mmol) was suspended in

THF (1000 niL), and 1,1-carbonyldiimidazole (68 g, 416 mmol, 1.3 eq, Aldrich 115533) and DMAP (2 g, 16 mmol, 0.05 eq, Aldrich 107700) were added thereto. The mixture thus obtained was stirred at 75 ^°C for 3 hrs, cooled to r.t, and evaporated in vacuo to remove solvent. The resultant residue was suspended in EtOH (1300 mL). The suspension thus obtained was stirred at 0 ^°C for 1 hr. The solid thus produced was filtered off and washed with cold EtOH (800 mL) and cold MeOH (300 mL). The filtered solid was dried under high vacuum to obtain the title compound.

Yield : 101 g (61 %)

TLC : R/= 0.75 (EA/MeOH/AcOH = 20/1/0.5)

¹H NMR (400 MHz, DMSO-^₆) δ 8.93 (t, J= 5.4 Hz, IH), 7.66 (d, J= 4.2 Hz, IH), 7.43-7.33 (m, 3H),7.29-7.12 (m, 3H), 4.82-4.73 (m, IH), 4.09 (t, J = 9.0 Hz, IH), 3.82-3.70 (m, 3H), 3.65-3.52 (m, 4H), 1.45 (s, 9H)

LCMS: 520 (M+H⁺) (C₂₃H₂₆ClN₅O₅S)

Step 8: Preparation of 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4H-[l,2,4]triazin-l-yl)phenyl]-l,3-oxazolidin-5-yl}-methyl)-2-thiophene

carboxamide hydrochloride

The compound obtained in Step 7 (101 g, 194 mmol) was suspended in aq.

3N HCl solution (1.1 L) and THF (1.1 L), and stirred at 80 “C for 3 hrs. The mixture thus obtained was cooled to r.t. The solid thus produced was filtered off, washed with THF (700 mL) and dried under high vacuum to obtain the title compound.

Yield : 75 g (85 %)

TLC : Rf= 0.82 (MC/MeOH/AcOH = 10/1/0.5)

¹H NMR (400 MHz, DMSO-J₆) δ 12.12 (br s, IH), 10.32 (br s, IH), 9.13

(t, J = 6.0 Hz, IH), 8.57 (d, J= 5.2 Hz, IH), 7.75 (d, J = 4.2 Hz, IH), 7.49 (d, J =

9.0 Hz, 2H), 7.15 (d, J= 4.2 Hz, IH), 7.09 (d, J= 9.0 Hz, 2H), 4.85-4.74 (m, IH), 4.11 (t, J = 8.8 Hz, IH), 3.85 (dd, J = 6.0, 9.2 Hz, IH), 3.62 (t, J = 4.8 Hz, 2H),

3.59-3.49 (m, 2H), 3.15 (br s,2H)

LCMS: 420 (M+H⁺) (C₁₈H₁₈ClN₅O₃)

Example 5: Preparation of 5-chloro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4H-[l,2,4]triazin-l_^yl)phenyl]-l,3-oxazolidin-5-yl}-methyl)-2-thiophene

carboxamide

The compound obtained in Example 4 (20 g, 43.8 mmol) was suspended in MeOH/H₂O (1/2 wt/wt, 3.2 L) and stirred at 100 ^°C until the compound obtained in Example 4 was dissolved clearly. 0.6M aq. Na₂CO₃ solution (75 mL) was added thereto. The mixture thus obtained was stood at 0 ^°C for 2 hrs. The solid thus produced was filtered off, washed with H₂O (400 mL) and dried

under high vacuum to obtain the title compound.

Yield : 17 g (93 %)

¹H NMR (400 MHz, DMSO-J₆) δ 8.93 (t, J = 6.0 Hz, IH), 7.66 (d, J = 4.0 Hz, IH), 7.29 (d, J = 9.2 Hz, 2H), 7.16 (d, J = 4.0 Hz, IH), 7.08 (d, J = 9.2 Hz, 2H), 6.76 (d, J = 4.0 Hz, IH), 6.48 (br s, IH), 4.78-4.69 (m, IH), 4.07 (t, J = 8.8 Hz, IH), 3.74 (dd, J = 6.0, 9.2 Hz, IH), 3.54 (t, J = 5.6 Hz, 2H), 3.38 (s, 4H)

LCMS: 420 (M+H⁺) (C₁₈H₁₈ClN₅O₃)

Example 6: Preparation of 5-chIoro-N-({(5S)-2-oxo-3-[(5,6-dihydro-4H-[l,2,4]triazin-l-yl)phenyI]-l,3-oxazolidin-5-yl}-methyl)-2-thiophene

carboxamide methane sulfonate

The compound obtained in Example 5 (16.7 g, 39.8 mmol) was suspended in MeOH/CH₂Cl₂ (1/4 v/v, 350 mL) and stirred at 50 ^°C until the compound obtained in Example 5 was dissolved clearly. The mixture thus obtained was cooled to 0 ^°C and methylsulfonic acid (2.9 mL, 43.8 mmol, 1.3 eq, Aldrich 471356) was added thereto at 0 ^°C . The resulting mixture was evaporated in vacuo to remove solvent. The resultant solid was suspended in absolute EtOH (100 mL) and the suspension was stirred at 90 ^°C to dissolve solid clearly. The resulting mixture was cooled to 0 ^°C and stirred at 0 ^°C for 2 hrs. The solid thus produced was filtered off, washed with absolute EtOH (100 mL), and dried under high vacuum to obtain the title compound.

Yield : 18.4 g (89.7 %)

¹H NMR (400 MHz, DMSO-J₆) δ 11.93 (br s, IH), 10.03 (br s, IH), 8.94 (t, J = 6.0 Hz, IH), 8.55 (d, J = 6.0 Hz, IH), 7.66 (d, J = 4.0 Hz, IH), 7.49 (d, J = 9.2 Hz, 2H), 7.16 (d, J = 4.0 Hz, IH), 7.08 (d, J = 9.2 Hz, 2H), 4.93-4.87 (m, IH), 4.10 (t, J = 9.2 Hz, IH), 3.77 (dd, J = 6.0, 9.2 Hz, IH), 3.63 (m, 2H), 3.57 (t, J = 5.6 Hz, 2H), 3.16 (br s, 2H), 2.28 (s, 3H)

LCMS: 420 (M+H⁺) (C₁₈H₁₈ClN₅O₃)

PATENT

WO2010002115

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2010002115

[Reaction Scheme 1] [96] A., O

^NCO^NH2 + &J\ – NC NC- boc IPA, reflux O*B£.H. .κ> boc DMAP boc 2

Example 10: Preparation of compound 109

Compound 15a (450 mg, 0.88 mmol) obtained in Manufacturing Example 3 was dissolved in dichloromethane (10 mL), to which HCl (4 M 1,4-dioxane solution) (10 mL) was added, followed by stirring at room temperature for 1 hour. The reactant was concentrated under reduced pressure and dried to give light yellow solid compound (425 mg, 0.88 mmol, 100%). This compound (392 mg, 0.81 mmol) was dissolved in acetic acid (4 mL), to which trimethylorthoformate (2 mL) was added, followed by reflux with stirring. 10 hours later, after solvent was evaporated all, column chromatography (dichlorome thane/me thanol(v/v) 20/1 → 12/1) was performed to give the title compound 109 as a light yellow solid (215 mg, 5.12 mmol, 63 %).

¹H NMR (400 MHz, CDCl₃) δ 7.35 (d, J = 9.2 Hz, 2H), 7.33 (d, J = 4.4 Hz, IH), 7.14 (d, J = 9.2 Hz, 2H), 7.01 (t, J = 6.4 Hz, IH), 6.88 (s, IH), 6.85 (d, J = 4.4 Hz, IH), 4.87-4.79 (m, IH), 4.06 (t, J = 9 Hz, IH), 3.86 (ddd, J = 14.4 ,6, 3 Hz, IH), 3.81 (dd, J = 9, 6.4 Hz, IH), 3.69 (dt, J = 14.4, 6 Hz, IH), 3.62-3.58 (m, 2H), 3.55-3.51 (m, 2H); LCMS: 420 (M+H⁺) to Ci₈H₁₈ClN₅O₃S

REFERENCES

https://clinicaltrials.gov/ct2/show/NCT01954238

SEE EARLIER MOLECULE   LCB01-0371…..http://newdrugapprovals.org/2014/03/31/lcb01-0371-new-oxazolidinone-has-improved-activity-against-gram-positive-pathogens/

////////////////phase 1, Green Cross Corp,  LegoChem Bioscience, GCC 4401C, thrombosis, venous thromboembolism, GC 2107, CB02-0133, GC-2107, GC4401, GCC-2107, GCC-4401, GCC-4401C, I Fxa – LegoChem Biosciences, LCB02-0133, Nokxaban

O=C(NC[C@H]3CN(c1ccc(cc1)N2CCNC=N2)C(=O)O3)c4ccc(Cl)s4.CS(=O)(=O)O   METHANE SULFONATE

O=C(NC[C@H]3CN(c1ccc(cc1)N2CCNC=N2)C(=O)O3)c4ccc(Cl)s4      FREE FORM

C1CN(NC=N1)C2=CC=C(C=C2)N3CC(OC3=O)CNC(=O)C4=CC=C(S4)Cl

Fresolimumab
GC 1008, GC1008
UNII-375142VBIA

cas 948564-73-6

Structure

• immunoglobulin G4, anti-(human transforming growth factors beta-1, beta-2 (G-TSF or cetermin) and beta-3), human monoclonal GC-1008 γ4 heavy chain (134-215′)-disulfide with human monoclonal GC-1008 κ light chain, dimer (226-226”:229-229”)-bisdisulfide
• immunoglobulin G4, anti-(transforming growth factor β) (human monoclonal GC-1008 heavy chain), disulfide with human monoclonal GC-1008 light chain, dimer

For Idiopathic Pulmonary Fibrosis, Focal Segmental Glomerulosclerosis,and Cancer

An anti-TGF-beta antibody in phase I clinical trials (2011) for treatment-resistant primary focal segmental glomerulosclerosis.

A pan-specific, recombinant, fully human monoclonal antibody directed against human transforming growth factor (TGF) -beta 1, 2 and 3 with potential antineoplastic activity. Fresolimumab binds to and inhibits the activity of all isoforms of TGF-beta, which may result in the inhibition of tumor cell growth, angiogenesis, and migration. TGF-beta, a cytokine often over-expressed in various malignancies, may play an important role in promoting the growth, progression, and migration of tumor cells.

Fresolimumab (GC1008) is a human monoclonal antibody^[1] and an immunomodulator. It is intended for the treatment of idiopathic pulmonary fibrosis (IPF), focal segmental glomerulosclerosis, and cancer^[2][3] (kidney cancer and melanoma).

It binds to and inhibits all isoforms of the protein transforming growth factor beta (TGF-β).^[2]

History

Fresolimumab was discovered by Cambridge Antibody Technology (CAT) scientists^[4] and was one of a pair of candidate drugs that were identified for the treatment of the fatal condition scleroderma. CAT chose to co-develop the two drugs metelimumab (CAT-192) and fresolimumab with Genzyme. During early development, around 2004, CAT decided to drop development of metelimumab in favour of fresolimumab.^[5]

In February 2011 Sanofi-Aventis agreed to buy Genzyme for US$ 20.1 billion.^[6]

As of June 2011 the drug was being tested in humans (clinical trials) against IPF, renal disease, and cancer.^[7][8] On 13 August 2012, Genzyme applied to begin a Phase 2 clinical trial in primary focal segmental glomerulosclerosis^[9] comparing fresolimumab versus placebo.

As of July 2014, Sanofi-Aventis continue to list fresolimumab in their research and development portfolio under Phase II development.^[10]

References

1 WHO Drug Information

2 National Cancer Institute: Fresolimumab

• 3 Statement On A Nonproprietary Name Adopted By The USAN Council – Fresolimumab
• 4 Grütter, Christian; Wilkinson, Trevor; Turner, Richard; Podichetty, Sadhana; Finch, Donna; McCourt, Matthew; Loning, Scott; Jermutus, Lutz; Grütter, Markus G. (2008-12-23). “A cytokine-neutralizing antibody as a structural mimetic of 2 receptor interactions”. Proceedings of the National Academy of Sciences 105 (51): 20251–20256. doi:10.1073/pnas.0807200106. ISSN 0027-8424. PMC 2600578. PMID 19073914.
• 5 http://www.independent.co.uk/news/business/news/cat-may-abandon-skin-drug-after-trial-results-disappoint-569445.html
• 6 http://www.bbc.co.uk/news/business-12477750
• 7 http://www.genengnews.com/gen-news-highlights/scientists-trigger-white-fat-to-become-brown-fat-like-to-treat-obesty-and-type-2-diabetes/81245389/
• 8 Clinicaltrials.gov for Fresolimumab
• 9 http://clinicaltrials.gov/show/NCT01665391
• 10 http://en.sanofi.com/rd/rd_portfolio/rd_portfolio.aspx

Fresolimumab

Monoclonal antibody
Type	Whole antibody
Source	Human
Target	TGF beta 1, 2 and 3
Clinical data
Legal status	Investigational
Identifiers
CAS Number	948564-73-6
ATC code	None
ChemSpider	none
KEGG	D09620
Chemical data
Formula	C6392H9926N1698O2026S44
Molar mass	144.4 kDa

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