Avoralstat, BCX4161,

CAS  918407-35-9
UNII: UX17773O15

513.5513, C28-H27-N5-O5

2-Pyridinecarboxylic acid, 3-(2-(((4-(aminoiminomethyl)phenyl)amino)carbonyl)-4-ethenyl-5-methoxyphenyl)-6-(((cyclopropylmethyl)amino)carbonyl)-

3-(2-((4-Carbamimidoylphenyl)carbamoyl)-4-ethenyl-5-methoxyphenyl)-6-((cyclopropylmethyl)carbamoyl)pyridine-2-carboxylic acid

Hereditary angioedema (HAE)

Kallikrein inhibitor

BioCryst Pharmaceuticals

BioCryst is also investigating second-generation plasma kallikrein inhibitors to avoralstat, for treating HAE (in February 2016, this program was listed as being in preclinical development).

Prevent acute attacks in patients with hereditary angioedema (HAE); Treat hereditary angioedema (HAE)

U.S. – Fast Track (Treat hereditary angioedema (HAE));
U.S. – Orphan Drug (Prevent acute attacks in patients with hereditary angioedema (HAE))

26 Feb 2016Clinical trials in Hereditary angioedema (Prevention) in USA (PO, Hard-gelatin capsule) before February 2016

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in France (PO, Soft-gelatin capsule)

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in Germany (PO, Soft-gelatin capsule)

Avoralstat, also known as BCX-4161, is a potent and orally active Kallikrein inhibitor and Bradykinin inhibitor. Avoralstat may be potentially useful for treatment for Hereditary angioedema. Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

Selective inhibitor of plasma kallikrein that subsequently suppresses bradykinin production

Hereditary angioedema (HAE) is a serious and potentially life-threatening rare genetic illness, caused by mutations in the C1-esterase inhibitor (C1 INH) gene, located on chromosome 11q. HAE is inherited as an autosomal dominant condition, although one quarter of diagnosed cases arise from a new mutation. HAE has been classed as an orphan disease in Europe, with an estimated prevalence of 1 in 50,000. Individuals with HAE experience recurrent acute attacks of painful subcutaneous or submucosal edema of the face, larynx, gastrointestinal tract, limbs or genitalia which, if untreated, may last up to 5 days. Attacks vary in frequency, severity and location and can be life-threatening. Laryngeal attacks, with the potential for asphyxiation, pose the greatest risk. Abdominal attacks are especially painful, and often result in exploratory procedures or unnecessary surgery. Facial and peripheral attacks are disfiguring and debilitating.

HAE has a number of subtypes. HAE type I is defined by C1 INH gene mutations which produce low levels of C1 -inhibitor, whereas HAE type II is defined by mutations which produce normal levels of ineffective C1 protein. HAE type III has separate pathogenesis, being caused by mutations in the F12 gene which codes for the serine protease known as Factor XII. Diagnostic criteria for distinguishing the subtypes of HAE, and distinguishing HAE from other angioedemas, can be found in Ann Allergy Asthma Immunol 2008; 100(Suppl 2): S30-S40 and J Allergy Clin Immunol 2004; 114: 629-37, incorporated herin by reference.

Current treatments for HAE fall into two main types. Older non-specific treatments including androgens and antifibrinolytics are associated with significant side effects, particularly in females. Newer treatments are based on an understanding of the molecular pathology of the disease, namely that C1 INH is the most important inhibitor of kallikrein in human plasma and that C1 INH deficiency leads to unopposed activation of the kallikrein-bradykinin cascade, with bradykinin the most important mediator of the locally increased vascular permeability that is the hallmark of an attack.

Approved therapies include purified plasma-derived C1 INH (Cinryze®, Berinert), the recombinant peptide kallikrein inhibitor ecallantide (Kalbitor®), and the bradykinin receptor B2 inhibitor iticabant (Firazyr®). All of the currently available targeted therapies are administered by intravenous or subcutaneous injection. There is currently no specific targeted oral chronic therapy for HAE.

There are many delivery routes for active pharmaceutical ingredients (APIs). Generally, the oral route of administration is favored. Oral administration provides a number of advantages, such as, but not limited to, patient convenience, flexibility of timing of administration, location of administration and non-invasiveness. Oral administration also provides more prolonged drug exposure compared with intermittent intravenous infusion, which may be important for drugs with schedule-dependent efficacy. For example, a drug with a short half-life can achieve a greater exposure time by either continuous infusion or by continuous oral dosing. The use of oral therapy further has the potential to reduce the cost of healthcare resources for inpatient and ambulatory patient care services.

In the pharmaceutical arts, it is known that a number of APIs cannot be administered effectively by the oral route. The main reasons why these compounds cannot be administered by the oral route are: a) rapid enzymatic and metabolic degradation; b) chemical and/or biological instability; c) low solubility in aqueous medium; and/or d) limited permeability in the gastrointestinal tract. For such compounds, non-oral routes of delivery, such as parenteral administration, mainly via intramuscular or subcutaneous injections, may be developed. However, non-oral administration poses a disadvantage for the patient as well as healthcare providers, and for this reason, it is important to develop alternative routes of administration for such compounds, such as oral routes of administration.

While the oral route of administration is the most convenient for the patient and the most economical, designing formulations for administration by the oral route involves many complications. Several methods are available to predict the ease by which an API may be formulated into a formulation suitable for administration by the oral route. Such methods include, but are not limited to, and Lipinski rule (also referred to as the Rule of Five) and the Biopharmaceutical Drug Disposition Classification System (BDDCS).

The BDDCS divides APIs into four classifications, depending on their solubility and permeability. Class I APIs have high solubility and high permeability; Class II APIs have low solubility and high permeability; Class III APIs have high solubility and low permeability; and Class IV APIs have low solubility and low permeability. APIs in higher classes in the BDDCS face greater challenges in formulating into an effective, pharmaceutically acceptable product than those in lower classes. Of the four classes, APIs falling into Class IV are the most difficult to formulate into a formulation for administration by the oral route that is capable of delivering an effective amount of the API as problems of both solubility and permeability must be addressed (note the BDDCS does not inherently address chemical stability). The role of BDDCS in drug development is described generally in L.Z. Benet J Pharm Sci. 2013, 102(1), 34-42.

Lipinski’s rule (described in Lipinski et al. Adv. Drug Deliv. Rev. 46 (1-3): 3-26) states, in general, that in order to develop a successful formulation for administration by the oral route, an API can have no more than one violation of the following criteria:

i) not more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms)

ii) not more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms) iii) a molecular mass less than 500 daltons

iv) an octanol-water partition coefficient log P not greater than 5.

J. Zhang et al. Medicinal Chemistry, 2006, 2, 545-553, describes a number of small molecule amidine compounds which have activity as inhibitors of kallikrein. The molecules described in this document fall into Class IV of the BDDCS as described above. The compounds are poorly soluble in aqueous and physiological fluids, and are poorly permeable as demonstrated by oral dosing in rats and in vitro experiments with Caco-2 cells.

Furthermore, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, one of the compounds described in Zhang et al., is a Class IV API and violates criteria iii) and iv) as set forth in the Lipinski Rule.

Furthermore, the compounds described in Zhang et al., including 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, exhibit poor stability with respect to oxidation in air, to light

(photodegradation) and in aqueous and physiological fluids, as well as to elevated temperatures.

Therefore, the compounds described by Zhang et al. including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, not only exhibit poor solubility and permeability characteristics, but also poor stability characteristics. As a result, such compounds are predicted to be especially difficult to formulate into an effective, orally deliverable

pharmaceutical composition that is capable of delivering an effective amount of the compound to a subject.

Polymorphism, the occurrence of different crystal forms, is a property of some molecules. A single molecule may give rise to a variety of polymorphs having distinct crystal structures and physical properties, such as, but not limited to, melting point, thermal behaviors (e.g. measured by thermogravimetric analysis (TGA), or differential scanning calorimetry (DSC), x-ray diffraction pattern, infrared absorption fingerprint, and solid state NMR spectrum. One or more of these techniques may be used to distinguish different polymorphic forms of a compound.

Discovering new polymorphic forms and solvates of a pharmaceutical product can provide alternate forms of the compound that display a number of desirable and advantageous properties, such as, but not limited to, ease of handling, ease of processing, ease of formulation, storage stability, and/or ease of purification. Further, new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof may further provide for improved pharmaceutical products, by providing compounds that are more soluble in a set of pharmaceutical excipients. Still further, the provision of new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof enlarges the repertoire of compounds that a formulation scientist has available for formulation optimization, for example by providing a pharmaceutical product with different properties, such as, but not limited to, improved processing characteristics, improved handling characteristics, improved solubility profiles, improved dissolution profile and/or improved shelf-life. Therefore, there is a need for additional polymorphs of pharmaceutically useful compounds, such as, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid and the compounds disclosed herein.

In one aspect, the present invention provides an oral formulation that is capable of delivering an effective amount of the amidine compounds described by Zhang et al. to a subject. In particular, the present invention provides an oral formulation that is capable of delivering an effective amount of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid to a subject. In one specific aspect, the 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid is present in a particular crystal form designated Form A. In light of the art suggesting the difficulties in formulating such an oral formulation, this result was unexpected.

As described herein, the amidine compounds described in Zhang et al., including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (specifically including particular crystal Form A), may now be conveniently used in oral administration and further used in oral administration for the treatment of a number of diseases and conditions in a subject, such as, but not limited to, HAE as described herein.

PATENT

WO200234711

http://www.google.com/patents/WO2002034711A1?cl=en

PATENT

WO2015134998

PATENT

WO2016029214

Examples

Example 1 – Synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl- phenyll-6-(cvclopropylmethyl-carbarnoyl)-pyridine-2-carboxylic acid

The synthesis of the above compound and intermediates is described below. In this section, the following abbreviations are used:

The synthesis of starting material, (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) is described in Scheme 1.

f ^0HCY ° ^ΒΓΥΥ°

Preparation of 6-bromobenzofdl[1,3ldioxole-5-carbaldehvde (1b)

1a 1b

To a mixture of piperonal (1a) (498 g, 3.32 mol) in glacial acetic acid (1000 mL) was added a solution of bromine (200 mL, 3.89 mol) in glacial acetic acid (500 mL) over a period of 30 min and stirred at room temperature for 24h. The reaction mixture was poured into water (2000 mL) and the solid that separated was collected by filtration. The solid was dissolved in boiling ethanol (4000 mL) and cooled to room temperature. The solid obtained on cooling was collected by filtration to furnish 6-bromobenzo[d][1 ,3]dioxole-5-carbaldehyde (lb) (365 g, 48 %) as a white solid, MP 126 °C; HNMR (300 MHz, DMSO-d₆): δ 10.06 (s, 1 H), 7.42 (s,1 H), 7.29 (s, 1 H), 6.20 (d, J=12.3, 2H); IR (KBr) 3434, 2866, 1673,1489, 1413, 259, 1112, 1031 , 925 cm^“1; Analysis calculated for CeH5BrO3.O 25H C, 41.15; H, 2.37; Found: C, 41.07; H, 2.11.

Preparation of 2-bromo-5-hvdroxy-4-methoxybenzaldehyde (1c)

1c

A solution of potassium tert-butoxide (397 g, 3.36 mol) in DMSO (1.5 L) was heated at 50 °C for 30 min. Methanol (1.5 L) was added to it and continued heating at 50 °C for additional 30 min. To the hot reaction mixture was added 6-bromo-benzo[d][1,3]dioxole-5-carbaldehyde (1 b) (350g, 1.53 mol) and continued heating at 50 °C for 30 min. The reaction mixture was cooled to room temperature and quenched with water (2.3 L) and sodium hydroxide (61.2 g, 1.53 mol). The reaction mixture was washed with ether (2 x 1.5 L), acidified to pH 2 using cone. HCI and extracted with ethyl acetate ( 1 L). The ethyl acetate layers were combined and concentrated under vacuum to dryness. The residue obtained was treated with water (1.5 L) and ethyl acetate (1 L). The solid obtained was collected by filtration to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (97 g, 27.5% as a first crop). The layers from the filtrate were separated and aqueous layer was extracted with ethyl acetate (200 ml_). The ethyl acetate layers were combined dried over MgS0₄ and concentrated under vacuum to dryness to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (192 g, 54.4%, second crop) as an orange solid, MP 108 °C; ‘HNMR (300MHz, DMSO-cfe): S 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H); IR (KBr) 3477, 2967, 2917,

2837, 2767, 2740, 1657, 1595, 1428, 1270, 1210, 1164, 1022 cm^“‘; Analysis calculated for C₈H₇Br03_.H₂0: C, 38.58; H, 3.64: Found: C, 38.60; H, 3.60.

Preparation of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehvde ( d)

To a solution 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (120 g, 520 mmol) in DMF (1000 mL) was added potassium carbonate (79 g, 572 mmol) and benzyl bromide (68 mL, 572 mmol). The reaction mixture was stirred at room temperature overnight and quenched with water (3000 mL). The solid obtained was collected by filtration, washed with ether and dried under vacuum to furnish 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (113.19 g, 67.9%) as a white solid, MP 144 °C;¹HNMR (300 MHz, DMSO-c/₆): δ 10.06 (s, 1H), 7.47-7.34 (m, 7H), 5.17 (s, 2H), 3.92 (s, 3H); IR (KBr) 2898, 2851 , 1673, 1592, 1502, 1437, 1402, 1264, 1210, 1158, 1017, 754 cm^“1; Analysis calculated for C ₅H₁₃Br0₃: C, 56.10; H, 4.08; Found: C, 55.44; H, 4.08.

Preparation of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e)

15 046578

146

1d 1e

To a solution of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (100 g, 311 mmol) in

ethanol (1500 mL) was added triethyl orthoformate (103 mL, 622 mmol), ammonium nitrate

(7.5 g, 93.3 mmol) and stirred at room temperature overnight. The reaction mixture was

treated with ether (1200 mL) and stirred for 15 min before filtration. The filtrate was

concentrated under vacuum to dryness to give 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (134 g) as a brown syrup; The product was used in the next step

without further purification; ¹H N R (300 MHz, DMSO-cf₆) δ 7.45 – 7.37 (m, 4H), 7.36 – 7.33

(m, 1 H), 7.17 – 7.14 (m, 1 H), 7.10 (s, 1 H), 5.10 (s, 2H), 3.80 (s, 3H), 3.58 – 3.33 (m, 5H),

1.13 – 1.07 (m, 6H); IR (KBr) 2974, 2879, 1601 , 1503, 1377, 1260, 1163, 1060 cm^“1;

Analysis calculated for C₁₉H₂₃Br0₄: C, 57.73; H, 5.86; Found: C, 57.21 ; H, 5.94.

acid (1fi

To a solution of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (120 g,

300 mmol) in dry ether (1000 mL) at -78 °C was added n-butyllithium (1.6 M solution in

hexanes, 244 mL, 390 mmol) over a period of 30 min and further stirred at -78 °C for 30 min.

A solution of tri-n-butylborate (110 mL, 405 mmol) in dry ether (300 mL) was added to this

solution at -78 °C over a period of 30 min. The reaction mixture was further stirred for 2 h at -78 °C and warmed to 0 °C. The reaction mixture was quenched with 3N HCI (300 mL) at 0

°C and heated at reflux for 1 h. After cooling to room temperature, the solid obtained was

collected by filtration washed with water (250 mL) dried in vaccum to afford (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (30.85 gm, 37.6% as a white solid. The organic

layer from above filtrate was extracted with 1.5 N NaOH (3 x 200 mL). The combined basic

extracts were acidified with cone. HCI (pH about 4). The solid obtained was collected by

filtration, washed with water and dried under vacuum to furnish a second crop of (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (22.3 g, 26%) as a light orange solid

MP 158 °C; ¹H NMR (300 MHz, DMSO-cfe) δ 10.08 (s, 1 H), 7.52 (s, 1 H), 7.48 – 7.33 (m, 5H),

7.24 (s, 1H), 5.18 (s, 2H), 3.89 (s, 3H); ¹H NMR (300 MHz, DMSO-d₆/D₂0) δ 10.06 (s, 1H),

7.52 (s, 1H), 7.49 – 7.32 (m, 5H), 7.23 (s, 1 H), 5.18 (s, 2H), 3.89 (s, 3H); MS (ES+) 309.1 (M+Na); IR (KBr) 3335, 2937, 1647, 1545, 1388, 1348, 1268, 1146, 1095 cm^-1; Analysis calculated for C₁₅H₁₅BO₅.0.25H₂O: C, 62.00; H, 5.38; Found: C, 61.77; H, 5.19.

Synthesis of methyl-6-(cvclopropylmethylcarbamoyl¾-3-ftrifluoromethylsulfonyloxyVpicolinate

The synthesis of the intermediate methyl 6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethyl sulfonyloxy)picolinate (2h) is described in Scheme 2.

Preparation of 2-bromo-3-hvdroxy-6-methylpyridine (2b)

H₃C N Br

2a 2b

To a solution of 3-hydroxy-6-methylpyridine (2a) (3000 g, 27.5 mol) in pyridine (24 L) cooled to 15 °C was added a solution of bromine (4.83 kg, 1.55 L, 30.2 mol) in pyridine (3 L) over a period of 50 min maintaining the internal temperature between 20 to 25 ^DC. After stirring for 19 h at room temperature the solvent was removed under vacuum and the residue was triturated with water. The solid separated was collected by filtration, washed with water and dried under vacuum to give 2-bromo-3-hydroxy-6-methylpyridine (2b) (3502 g, 67.7 %) as a light brown solid which was used as such without further purification; ¹H NMR (300 MHz, DMSO-d₆) δ 10.43 (s, 1H), 7.18 (d, J = 8.0 Hz, 1 H), 7.08 (d, J

MS (ES+) 188.35, 186.36 (M+1).

(2c)

2b ^2c

A mixture of 2-bromo-3-hydroxy-6-methylpyridine (2b) (3000 g, 15.96 mol), anhydrous potassium carbonate (3308 g, 23.94 mol), and iodomethane (2.491 kg, 1.09 L, 17.556 mol) in 30 L of acetone was heated at 40 °C overnight. The reaction mixture was cooled to room temperature and filtered through Celite. Evaporation of the solvent followed by silica gel chromatography (Hexane: ethyl acetate = 7:3) afforded the desired compound, 2-bromo-3-methoxy-6-methylpyridine (2c) which was used as such for the next step; ¹H NMR (300 MHz, DMSO-cfe) δ 7.42 (dd, J = 8.3, 1.5 Hz, 1H), 7.29 – 7.19 (m, 1H), 3.84 (d, J = 1.6 Hz, 3H), 2.37 (d, J = 1.7 Hz, 3H).

2c

2d

To a solution of 2-bromo-3-methoxy-6-methylpyridine (2c) (310 g, 1.53 mol) in 6000 mL of water at 60 °C was added KMnO, (725 g, 4.59 mol) in small portions over a 90 min period with vigorous mechanical stirring. A dark purple solution resulted. This solution was kept at 90 °C for a further 3 h and filtered through Celite while still hot to give a colourless filtrate.

After cooling, the aqueous solution was acidified to pH 1-2 by adding 6 N HCI. The white solid obtained was collected by filtration to give on drying 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (302g, 85%) of product, which was used as such in the next reaction without further purification. An analytical sample was obtained by recrystallization from methanol to give 6-bromo-5-methoxy-2-pyridinecarboxylic acid; ¹H NMR (300 MHz, DMSO-tfe) δ 7.40 – 7.28 (m, 1H), 7.17 (d, J = 8.3 Hz, 1 H), 3.83 (d, J = 1.7 Hz, 3H).

Preparation of 6-bromo-N-(cvclopropylmethyl)-5-methoxypicolinamide (2e)

To a solution of 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (12 g, 52 mol) in pyridine (70 mL) was added EDCI (11.5 g, 59 mmol) and cyclopropylmethylamine (3.6 g, 52 mmol). The reaction mixture was stirred at room temperature overnight and then concentrated under vacuum. The reaction mixture was diluted with water (100 mL) and ethyl acetate (100 mL). The organic layer was separated and the water layer was extracted with ethyl acetate (2 x 100 mL). The organic layers were combined and washed with water (2 x 50 mL), brine (500 mL), dried over magnesium sulphate, filtered and concentrated under vacuum to furnish 10.43g of crude product. The crude product was converted into a slurry (silica gel 20 g) and purified by flash column chromatography (silica gel 230 g, eluting with 0-100% ethyl acetate in hexane) to yield compound 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (8.02 g, 54%) as off white solid, mp 67-70 °C; ¹HNMR (300 MHz, DMSO-d₆) δ 8.51 (t, J = 5.8, 1 H), 8.02 (d, J = 8.4, 1 H), 7.65 (d, J = 8.5, 1 H), 3.96 (s, 3H), 3.14 (t, J = 6.5, 2H), 1.11 -0.99 (m, 1 H), 0.47 – 0.36 (m, 2H), 0.27 – 0.20 (m, 2H); MS (ES+) 307.0, 309.0 (100%

M+Na)

Preparation of methyl 6-(cvclopropylmethylcarbamoyl)-3-methoxypicolinate (2f)

To a solution of 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (7.5 g, 27.6 mol) in methanol (300 mL) in a 2-L stainless steel bomb was added Pd(OAc)₂(750 mg), 1 ,1-bis(diphenylphosphino)-ferrocene (750 mg), and triethylamine (3.9 mL, 27.6 mmol). The reaction mixture was vacuum flushed and charged with CO gas to 150 psi. The reaction mixture was and heated with stirring at 150°C overnight and cooled to room temperature. The catalyst was filtered through a pad of celite, and concentrated to dryness to furnish crude product. The crude was purified by flash column chromatography (silica gel 150 g,

eluting with, 0%, 5%, 10%, 20%, 30%, 50% ethyl acetate/hexanes (250 mL each) as eluents to give methyl 6-(cyclopropylmethyl-carbamoyl)-3-methoxypicolinate (2f) (6.29 g, 86.1 %) as a salmon coloured solid, MP 107 °C; ¹HNMR (300 MHz, DMSO-cfe) δ 8.28 (t, J = 6.0, 1H), 7.91 (d, J = 8.8, 1H), 7.55 (d, J = 8.8, 1 H), 3.68 (s, 3H), 3.64 (s, 3H), 2.90 (t, J = 6.5, 2H), 0.89 – 0.68 (m, 1 H), 0.26 – 0.09 (m, 2H), 0.08 – 0.00 (m, 2H); MS (ES+) 287.1 (M+Na); IR (KBr) 3316, 2921 , 1730, 1659, 1534, 1472, 1432, 1315, 1272, 1228, 1189, 1099, 1003, 929, 846, 680 cm^“1; Analysis calculated for C₁₃H_{16 2}0₄: C, 59.08; H, 6.10; N, 10.60; Found: C, 58.70; H, 5.97; N, 10.23.

Preparation of 6-(cvclopropylmethylcarbamoyl 3-hvdroxypicolinic acid (2q)

2f 2g

Aluminium chloride method:

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-methoxypicolinate (2f) (0.16 mmol) in dichloromethane (840 mL) was added AICI₃ (193 g, 1.5 mol). The reaction mixture was heated at reflux for 12 h under nitrogen. After slowly adding ~2L of 1 N HCI, the organic layer was separated. The aqueous layer was re-extracted several times with ethyl acetate/DME. The combined organic layer was washed with brine, dried (MgSO.4), and evaporated in vacuo to furnish crude 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid. To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid was added a solution of acetyl chloride (1 10 mL) in methanol (1.1 L). The reaction mixture was stirred for 12 h at room temperature and then concentrated to dryness in vacuo. After co-evaporating once with methanol, the compound was purified by flash-column chromatography (silica gel, 500 g, eluted with chloroform and 3% methanol in chloroform) to furnish 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g).

Boron tribromide method:

To a stirring solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-ethoxypicolinate (2f) (58.0 g, 208 mmol) was added BBr₃ (79 mL, 834 mmol) in CH₂CI₂ (1.3 L) at 0-5 °C. The reaction mixture was allowed to warm to room temperature and stirred for 18h. The reaction mixture was evaporated to dryness and anhydrous methanol (1 L) was added to the light yellowish solid residue. Insoluble solid was collected by filtration (36 g). Mother liquor was evaporated and co-evaporated with MeOH (2 x 200 mL). The insoluble solid (36 g) was treated with MeOH (500 mL) and acetyl chloride (50 mL) and stirred at room temperature for 18 h (at this point reaction mixture was clear). The mixture was evaporated to dryness and diluted with water and extracted with EtOAc. White solid that separated out from EtOAc layer was collected by filtration, washed with water (2 x 20 mL), dried in vacuo at 50 °C to afford 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (5.36 g, 10 %) as a white solid, MP 92-95 °C. ¹HNMR (DMSO-cfe) δ 11.04 (s, 1 H, exchangeable with D₂0), 8.37 (t, J = 6.0, 1 H, exchangeable with D₂0), 8.12 (d, J = 8.7 Hz, 1 H), 7.57 (d, J = 8.7 Hz, 1 H), 3.90 (m, 3 H), 3.15 (m, 2 H), 1.04 ( m, 1 H), 0.41 (m, 2 H), 0.24 (m, 2 H). IR (KBr): 3346, 3205, 1684 cm^“1; MS (ES+): 251.1 (M+1); Analysis calculated for C₁₂H₁₄N2O4.0.1 H₂O: C, 57.18; H, 5.67; N, 11.14; Found: C, 57.11 ; H, 5.61; N, 11.09.

Preparation of methyl-6-(cvclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy) picolinate (2h

To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (28 mmol) in DMF (200 mL) were added triethylamine (12 mL, 84 mmol) and N-phenyl-bis(trifluoromethanesulfonimide) (12 g, 34 mmol). The reaction mixture was stirred for 1.5 h at room temperature and then poured into ice. After diluting with water and extracting with ethyl acetate, the aqueous phase was re-extracted, and then the combined organic layer was washed with water and concentrated under vacuum to give methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)picolinate (2h), which was used in the next step without purification.

¹H NMR (300 MHz, CDCI₃) δ 8.50 (d, J = 8.6, 1 H), 8.07 (s, 1 H), 7.88 (d, J = 8.6, 1 H), 4.09 (d, J = 12.6, 3H), 3.48 – 3.24 (m, 2H), 1.18 – 1.01 (m, 1 H), 0.69 – 0.44 (m, 2H), 0.42 – 0.20 (m, 2H). MS (ES*): 405.17, 100%, M+Na.

Synthesis of 3-f2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid:

The synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i) is described as shown in Scheme 3.

3-f4-Benzyloxy-2-formyl-5-methoxy-phenylV6-(cvcloDroDvlmethvl-carbarnovn-pyridine-2-carboxylic acid methyl ester (3a)

5 046578

153

3a

To a solution of methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)

picolinate (2h) (24.3g, 63 mmol) in DME (225 mL) were added water (25 mL), (4- (benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (27.3 g, 95 mmol), NaHC0₃(15.9 g,

5 189 mmol), and bis(triphenylphosphine)palladium(ll) chloride (0.885 g). The reaction

mixture was stirred at 70°C overnight under nitrogen. After extracting with ethyl acetate, the organic layer was washed with water and brine and dried (MgSO^), and then concentrated

under vacuum. The compound was purified by flash-column chromatography (silica gel, 300 g, eluting with 10%, 20%, 30% and 40% ethyl acetate in hexane) to furnish 3-(4-benzyloxy- 10 2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

methyl ester (3a) (25 g, 83%) as off white solid, MP 48-50°C: ¹H NMR (300 MHz, DMSO-cfe) δ 9.61(s, 1 H), 8.40 (d, J= 7.9 Hz, 1H), 8.14 (t, J= 5.0 Hz, 1H), 7.87 (d, J= 8.1 Hz, 1 H), 7.58

(s, 1H), 7.54-7.30 (m, 5H), 6.71 (s, 1 H), 5.24 (s, 2H), 3.93 (s, 3H), 3.70 (s, 3H), 3.45-3.34 (m,

2H), 1.19-1.05 (m, 1 H), 0.64-0.54 (m, 2H), 0.37-0.30 (m, 2H); IR ( Br) 1735, 1678, 1594,

15 1513, 1437, 1283, 1217, 1141, 1092 cm^“1; MS (ES+) 497.29 (M+Na); Analysis calculated for

C₂₇H_2eN₂0₆: C, 68.34; H, 5.52; N, 5.90; Found; C, 68.16; H, 5.62; N, 5.80.

2-(6-(Cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-vn-4-methoxy-5- vinylbenzoic acid (3b)

To a solution of 3-(4-benzyloxy-2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl- carbamoyl)-pyridine-2-carboxylic acid methyl ester (3a) (24g, 50.6 mmol) in acetonitrile (50

mL), 2-methyl-2-propanol (350 mL), and water (125 mL) were added sodium dihydrogen

phosphate (12.5 g) and 2-methyl-2-butene (55 mL, 519 mmol). The reaction mixture was cooled in an ice bath and then sodium chlorite (28 g) was added. After stirring for 1 h, the reaction mixture was extracted with ethyl acetate and washed with water. The aqueous layer was re-extracted and then the combined organic layers were dried (MgS0₄). The solvent was evaporated in vacuo to furnish 5-(benzyloxy)-2-(6- ((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (29 g) which was used for the next step. MS (ES⁺): 513.24, (M+Na(; (ES ): 489.26, M-1.

Methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxytoarbonyltohenyl)-6-(cvclopropylmethylcarbamovnpicolinate (3c)

To a mixture of 5-(benzyloxy)-2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxy-carbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (31 g, 63.2 mmol), and triethylamine (17.7 mL, 126.4 mmol) in dichloromethane (300 mL), was added MEM-chloride (9.03 mL, 79 mmol), and stirred at room temperature overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with water and dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, 40 g) to furnish methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 89%) as a thick gum; H NMR (300 MHz, CDCI₃) δ 8.35 (d, J = 8.0 Hz, 1 H), 8.15 (t, J = 5.7 Hz, 1 H), 7.78 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.49 (d, J = 6.8 Hz, 2H), 7.36 (ddd, J = 7.5, 14.8, 22.4 Hz, 3H), 6.66 (s, 1 H), 5.37-5.13 (m, 4H), 3.90 (s, 3H), 3.69 (s, 3H), 3.60-3.49 (m, 2H), 3.49 (s, 2H), 3.39 (dd, J = 4.4, 8.4 Hz, 2H), 3.34 (s, 3H), 1.19-1.00 (m, 1H), 0.57 (q, J = 5.8 Hz, 2H), 0.38-0.25 (m, 2H). MS (ES⁺): 601.24 (M+Na); (ES^“): 577.27 (M-1);¹H NMR (300 MHz, DMSO-cfe) δ 8.69 (t, 7 = 6.1 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1 H), 7.63 (s, 1H), 7.41 (m, 5H), 6.92 (s, 1 H), 5.20 (m, 4H), 3.83 (s, 3H), 3.57 (s, 3H), 3.44 (m, 2H), 3_:33 (m, 2H), 3.21 (m, 5H), 1.14 (m, 1H), 0.44 (m, 2H), 0.27 (m, 2H). IR (KBr):

1732, 1671 cm^“1. MS (ES+): 601.1(M+Na); Analysis calculated for C₃₁H ₂Oe: C, 64.35; H, 5.92; N, 4.84; Found: C, 64.27; H, 6.04; N, 4.79.

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(4-hvdroxy-5-methoxy-2-(((2-methoxyethoxy¾methoxy)carbonyl)phenyl)picolinate (3d)

3c 3d

To a solution of methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)-carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 56.68 mmol) in ethanol (650 mL) was added 10% Pd/C (4 g) and hydrogenated at 45 psi for 5 h. The catalyst was removed by filtration through Celite and the filtrate was concentrated under vacuum to yield methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)picolinate (3d) (31.87 g, 86%), which was pure enough to be used as such for the next step. An analytical sample of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) was obtained by purification of 350 mg of above crude using flash column chromatography (silica gel, eluting with ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethyl-carbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-phenyl)picolinate (3d) as a clear gum; ¹HNMR (300 MHz, DMSO-d₆) δ 9.74 (s, 1 H), 8.68 (t, J = 6.1 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1 H), 7.95 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 6.83 (s, 1H), 5.19 (s, 2H), 3.77 (m, 3H), 3.58 (s, 3H), 3.44 (m, 2H), 3.34 (m, 2H), 3.21 (m, 5H), 1.04 (m, 1 H), 0.44 (m, 2H), 0.27 (m, 2H); IR (KBr): 1731 , 1664 cm^‘1. MS (ES^*): 489.0 (M+1); Analysis calculated for C^e^O,,: C, 59.01; H, 5.78; N, 5.73; Found: C, 58.92; H, 6.15; N, 5.29.

6-(Cvclopropylmethylcarbamovn-3-(5-methoxy-2-(((2-methoxyethoxy^methoxy)-carbonyl)-4- (trifluoromethylsulfonyloxy)phenyl)picolinate (3e)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2- methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) (14.3 g, 29.3 mmol) in dichloromethane (150 mL) were added pyridine (12 mL, 146 mmol) and triflic anhydride (7.5 mL g, 44 mmol). After stirring overnight at room temperature under N₂. the reaction mixture was poured into ice water and then extracted twice with dichloromethane. After washing the combined organic extracts with water and drying (MgS0 ), the solvent was evaporated in vacuo. The compound was purified by flash chromatography over silica gel column using ethyl acetate: hexane to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)-carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (1 g, 93%); H NMR (300 MHz, CDCy a 8.41 (d, J = 8.0, 1H), 8.17 (s, 1H), 8.03 (s, 1H), 7.79 (d, J = 8.0, 1 H), 6.82 (s, 1H), 5.32 (q, J = 6.1, 2H), 3.97 (s, 3H), 3.74 (s, 3H), 3.67 – 3.57 (m, 2H), 3.55 – 3.45 (m, 2H), 3.41 (dd, J = 8.2, 14.5, 2H), 3.34 (s, 3H), 1.36 – 1.17 (m, 1H), 0.58 (d, J = 7.1 , 2H), 0.33 (d, J = 5.1 , 2H).

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(5-methoxy-2-f((2-methoxyethoxy)- methoxy)carbonvn-4-vinylphenyl)picolinate (3f)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (37.4

g, 60.30 mmol) and potassium vinyltrifluoroborate (16.87 g, 120.6 mmol) in DMF (450 mL) and water (45 mL) was bubbled N₂ for 5 min. To this mixture was added NaHC0₃ (20.26 g, 241.2 mmol) and dichloro-bis(triphenylphosphine)palladium (II) (6.34 g, 9.0 mmol). The reaction mixture was stirred at 70 °C for 20 h under N₂(reaction progress was checked by ¹H N R because product and starting material had same R_f in TLC). The reaction mixture was cooled down to room temperature and diluted with ethyl acetate. The organic layer was separated, washed with water, brine, dried ( gS0₄) and filtered. The filtrate was concentrated under vacuum to yield crude methyl 6-(cyclopropylmethyl-carbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-4-vinylphenyl)-picolinate (3f). The crude product was purified by flash column chromatography (silica gel, 1 kg, eluting with 0-100% ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate [31) (26.54 g, 88%) as an amber gum; H NMR (300 MHz, DMSO-c¾ δ 8.70 (t, J = 6.1 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1 H), 8.12 (s, 1 H), 8.00 (d, J = 8.0 Hz, 1 H), 6.98 (m, 2H), 5.94 (dd, J = 1.2, 17.8 Hz, 1H), 5.43 (d, J = 12.5 Hz, 1 H), 5.21 (d, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.64 (s, 3H), 3.48 (d, J = 3.1 Hz, 2H), 3.35 (m, 5H), 3.22 (m, 2H), 1.11 (s, 1H), 0.44 (dt, J = 4.9, 5.5 Hz, 2H), 0.28 (q, J = 4.8 Hz, 2H). IR (KBr); 1732, 1670 cm^“1. MS (ES+) 499.1 (M+1).

2-(6-(cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzolc acid (3g)

A mixture of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate (3f) (27.4 mmol) in DME (160 mL) and 6N HCI (40 mL) was stirred at room temperature for 6 h or till TLC showed complete conversion. The solvent was removed under vacuum. The residue obtained was suspended in water, the solid separated out was collected by filtration, washed with water and dried under vacuum to give 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (7.0 g, 63%) as a white

solid MP 40 – 42 °C; H NMR (300 MHz, DMSO-d_e) δ 8.69 (t, J= 6.0 Hz, 1H, NH), 8.20 (d, J= 7.9 Hz, 1H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1H), 6.97 (dd, J= 18.0, 11.3 Hz, 1H), 6.88 (s, 1H), 5.92 (d, J= 7.9 Hz, 1H), 5.38 (d, J= 11.1 Hz, 1H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); IR (KBr): 3084, 1728, 1650, 1533, 1212, 1143 cm-1; MS (ES+) 433.26 (M+Na); (ES-): 409.28 (M-1); Analysis calculated for θ₂2Η22Ν₂Ο₆.0.25Η₂Ο; C, 63.68; H, 5.47; N, 6.75; Found C, 63.75; H, 5.56; N, 6.65

Methyl-3-(2-(4-carbamimidoylprienylcarbamoyl)-5-metrioxy-4-vinylphenyl)-6- (cvclopropylmethylcarbamoyl)picolinate (3h)

To a solution of 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (2.35 g, 5.7 mmol) and 4-aminobenzimidamide dihydrochloride (3j) (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 mL) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS04, filtered and purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 in CMA 50) yielding methyl-3-(2-(4-carbamimidoylphenyl-carbamoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (2.2 g, 65%) as a white solid MP 266 °C; ¹H NMR (300 MHz, DMSO-c/₆) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 – 7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for
C, 58.93; H, 5.63; N,11.85; Found: C, 58.75; H, 5.65; N, 11.92.

46578

159

3-r2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy -vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i)

3h 3i

To a solution of methyl-3-(2-(4-carbamirriidoylphenylcarbarnoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (1 g, 1.9 mmol) in methanol (10 mL) and THF

(10 mL) was added 2 N NaOH (10 mL). The reaction mixture was stirred at room

temperature for 3 h, and concentrated in vacuo to remove methanol and THF. The aqueous layer was acidified with 6N HCI to pH 6-7 and the solid obtained was collected by filtration

washed with water and ether to furnish on drying 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

(3i)(0.775 g, 80%) as the hydrochloride salt as an off white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 12.67 (s, 1 H), 9.11 (s, 2H), 8.97 (s, 2H), 8.74 (s, 1 H), 7.90

(d, J = 7.8, 1 H), 7.80 (s, 1 H), 7.72 – 7.58 (m, 4H), 6.99 (dd, J = 11.3, 17.7, 1 H), 6.78 (s, 1H),

5.95 (d, J = 17.2, 1H), 5.38 (d, J = 11.9, 1H), 3.82 (s, 3H), 3.18 (s, 2H), 1.06 (s, 1 H), 0.43 (d,

J = 7.9, 2H), 0.25 (d, J = 4.7, 2H); MS (ES+) 514.0 (M+1 ); Analysis calculated for

C2eH27N5O5.HCI.H₂O: C, 59.21; H, 5.32; N, 12.33; Found: C, 59.43; H, 5.21; N, 12.06.

Example 1A- Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride in Form

C

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 to -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1.6 kg) and water (0.57 kg). The mixture was heated to 46 °C. Smopex-234 (21 g) and Acticarbone 2SW (10 g) were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 “C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature <3 °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The

suspension was stirred at 0-3.0 “for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (T_jackel= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 11 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C.

Example-1 B: Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbartiovQpyridine-2-carboxylic acid hydrochloride in Form A

The procedure was carried out in an identical manner to Example 1 A, with the exception that after the final filtration the filter cake was rinsed with 2.87 kg methyl ierf-butyl ether instead of 2.87 kg water, and pulled dry. The product was dried at 40-43 °C and 50 mbar to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) as Form A.

PATENT

WO 2016029216

Methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (compound 6a) is (I) (pages 85 and 86). Avoralstat hydrochloride (compound of formula XVIII) is (II) (claim 40, page 109). A Markush structures is presented (claim 1, page 99).

The synthesis of (II) via intermediate (I) is described (example 1, pages 80-93).

A synthesis of the compound 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (Compound 3i) is described in Schemes A-C.

O y OHCk n ^Br^ ^OCH₃

B Brr₂₂,, AAccOOHH Y^ V”^“ \ \ tt–BBuuOOKK

OHC^^^{^}O ” _Br^\^0 MeOH ” OHC

1a 1b 66%

1d 95% ^{1 e}

1f

Scheme A



3h 31

Scheme C

Examples. In this section, the following abbreviations are used:

Example-1 : Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b)

7b

Step (1): Preparation of 6-Bromobenzo 1 ,3]dioxole-5-carbaldehyde (1 b):

1b

A solution of bromine (33.0 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to a solution of piperonal (1a) (29.9 kg, 199.16 mol) in acetic acid (105 L) at room

temperature over a period of 50 min and the reaction mixture was stirred at room temperature for 14.2 h. Additional solution of bromine (33 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to the reaction mixture over a period of 2 h and the reaction mixture was stirred for 22 h. The reaction mixture was quenched by addition of ice water (500 L) with stirring over a period of 6 h and continued stirring for additional 1.25 h. The mixture was allowed to settle and most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (600 L) was added to the solid, stirred, mixture was allowed to settle and then most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (100 L) was added to the decanted mixture, stirred for 15 min and the solid obtained was collected by filtration using a centrifuge. The solid was washed with water (2 x 100 L) and air-dried in a tray drier for 3.75 h to afford the crude product 1 b (52 kg). The crude product (51.2 kg) was stirred in n-hexane (178 L) for 3 h, collected by filtration, washed with n-hexane (25 L) and dried to afford 6-bromobenzo[1 ,3]dioxole-5-carbaldehyde (1b) (40.1 1 kg, 87.9%) as a light brown solid. MP: 109-112°C. ¹H NMR (300 MHz, CDCI₃) δ 10.21 (s, 1 H), 7.37 (s, 1 H), 7.07 (s, 1 H), 6.10 (s, 2H); HNMR (DMSO-cf₆): δ 10.06 (s, 1 H), 7.42 (s, 1 H), 7.29 (s, 1 H), 6.20 (d, J =12.3 Hz, 2H)

The process is also illustrated in Fig. 1.

Average yield of isolated 1 b from step-1 is 78 – 88%.

Step (2): Preparation of 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c)

A solution of potassium terf-butoxide (10.7 kg, 95.36 mol) in DMSO (49 L) was stirred at 50 °C for 30 min. Methanol (49 L) was added slowly over a period of 4.25 h and stirred at 50 °C for 30 min. 6-Bromobenzo[1 ,3]dioxole-5-carbaldehyde (1 b) (9.91 kg, 43.27 mol) was added to the reaction mixture in small portions over a period of 45 min and stirred at 50 °C for 1 h. The reaction mixture was cooled to room temperature and split into two equal portions. Each portion was quenched with water (50.9 L) and basified with 50% aqueous NaOH solution (2.4 L). Each portion was extracted with MTBE (4 x 36 L) to remove impurities. The aqueous layer was acidified with cone. HCI to pH ~ 3 to obtain

product as a yellow solid. The solid was collected by filtration using a centrifuge, washed with water (2 x 35 L) and air-dried to afford 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) (4.37 kg, 40.7%, contains 7 % water); Mp: 100-102°C; ¹HNMR (300MHz, DMSO-d₆): δ 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H).

The process is also illustrated in Fig. 2.

Average yield of isolated product 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) from step-2 is 40-50%.

Step (3): 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-y benzaldehyde (4a)

2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) [1.3 kg (93%, 7% water content), 5.25 mol] was dissolved in toluene (13 L) in a reaction flask equipped with a Dean Stark apparatus. The solution was heated at reflux with stirring to distil off about 25% of the toluene along with water (90 ml_). The solution was cooled to 90 °C then

bis(pinacolato)diboron (1.5 kg, 5.82 mol), KOAc (772.6 g, 7.87 mol) and Pd(PPh₃) (24.3 g, 0.02 mol) were added and the reaction mixture was heated at reflux for 10h. After confirming the completion of reaction by TLC (mobile phase: 100% DCM), the reaction mixture was cooled to room temperature and was kept standing overnight. The reaction mixture was filtered through celite and the celite cake was washed with toluene (4 L). The filtrate of this batch was mixed with the filtrate of another batch (batch size 1.3 kg obtained from an identical reaction). The mixed filtrate was washed with water (17.5 L), brine (17.5 L), dried over Na₂S0₄, filtered and the solution was passed through a pad of silica gel (2 kg, mesh size 230-400). The silica gel pad was washed with toluene. The combined filtrate and washing was concentrated under reduced pressure and the residual crude product was stirred with n-hexane (23 L) for 1 h to obtain a solid product. The solid was collected by filtration, washed with n-hexane (5 L) and dried to afford 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-yl)benzaldehyde (4a) (2.47 kg, 84.6%). H NMR (300 MHz, CDCI₃) δ 10.54 (s, 1 H), 7.57 (s, 1 H), 7.33 (s, 1 H), 5.89 (s, 1 H), 4.01 (s, 3H), 1.37 (s, 12H); ¹H NMR (300 MHz, DMSO-d₆) δ 10.35 (s, 1 H), 9.95 (s, 1 H), 7.33 (s, 1 H), 7.23 (s, 1 H), 3.87 (s, 3H), 1.33 (s, 12H); MS (ES+) 301.1 (M+Na); 579.1 (2M+Na); Analysis calculated for C₁₄H₁₉B0₅: C, 60.46; H, 6.89; Found: C, 60.60; H, 6.87

The average yield of 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) from step (3) is 78 – 90%.

The process is also illustrated in Fig. 3.

Step (4): Preparation of 3-Bromo-2,6-dimethylpyridine (5b)

2,6-lutidine (5a) (115 kg, 1073.3 mol) was added into pre-chilled oleum (20-23%, 1015 kg, 2276.7 mol) at 0 °C over a period of 4.5 h (temperature r6ached 14 °C during the addition). Bromine (88.18 kg, 1103.6 mol) was then added at 5-10 °C over a period of 1 h. The reaction mixture was slowly heated to 150 °C over a period of 12h. TLC analysis indicated about 40-50% conversion to product and the formation of a dimer by-product (5%). The reaction mixture was cooled to room temperature and then additional bromine (88.18 kg, 1103.6 mol) was added slowly. The reaction mixture was slowly heated to maintain a temperature of 65-75 °C over a period of 15h. TLC analysis indicated a 65-70 % conversion to product and the formation of 5% dimer by product. The reaction mixture was quenched by addition of water (500L) while maintaining the reaction temperature below 20 °C. The mixture was basified with 6.6 M NaOH (3800 L) while maintain the temperature at < 40 °C. EtOAc (220 L) was added and the mixture was stirred for 1 h then allowed to settle over a period of 2 h. The layers were separated and the aqueous layer was treated with NaOH (10 kg) in water (10 L) and extracted with EtOAc (160 L). The organic extracts were combined washed with brine (100 L), dried over Na₂S0₄ (50.0 kg), filtered and the solvent was evaporated under atmospheric pressure. The residue was vacuum distilled and the desired product 3-bromo-2,6-dimethylpyridine (5b) was collected at 58-60 °C, 2 mmHg (98.45 kg, 49.2 %) as a colorless liquid.

The process is also illustrated in Fig. 4.

Step (5): Preparation of 3-Bromopyridine-2,6-dicarboxylic acid (5c)

5b 5c

To a stirred solution of 3-bromo-2,6-dimethylpyridine (5b) (98 kg, 5326 mol) in water (1310 L) was added KMn0 (225 kg, 1423.6 mol) in 5 equal portions in 1 h intervals at 70 °C. After stirring for 1 h at 70 °C, additional KMn0₄ (225 Kg, 1423.6 mol) was added in 5 equal portion in 1 h intervals at 90 °C. The reaction mixture was stirred for 12 h at 90 °C. The suspension was filtered hot through celite to obtain a clear solution. The solvent was distilled off to remove about 30% of the total volume. The remaining concentrated solution was chilled to 0 °C and made acidic (to pH 3-4) by the addition of cone. HCI (120 L). The white precipitate obtained was collected by filtration and dried at 70 °C to afford 3-bromopyridine-2,6-dicarboxylic acid (5c) as a white solid (109 kg, 84%).

The process is also illustrated in Fig. 5.

Step (6): Preparation of Dimethyl 3-Bromopyridine-2,6-dicarboxylate (5d)

To a stirred solution of 3-bromopyridine-2,6-dicarboxylic acid (5c) (20.0 kg, 81.29 mol) in methanol (100 L) was added cone. H₂S0₄ (4.4 L) over a period of 30 min. The reaction mixture was heated to 65 °C and maintained at that temperature for 5 h (the reaction was monitored by TLC analysis to determine completion of reaction). The reaction mixture was cooled to room temperature basified by careful addition of aqueous NaHC0₃ solution (prepared from 10 kg NaHC0₃ in 120 L of water) and further diluted with water (120 L). The white solid obtained was collected by filtration, washed with plenty of water and then oven-dried at 40 °C to obtain dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (9.2 kg, 41.3%) as a white solid; ¹HNMR (300 MHz, DMSO-cf₆) δ 8.47 (d, J = 8.4, 1 H), 8.08 (dd, J = 4.5, 8.4, 1 H), 3.95 (s, 3H), 3.91 (s, 3H); MS (ES+) 570.6 (2M+Na); Analysis calculated for C₉H₈BrN0₄: C_, 39.44; H, 2.94; Br, 29.15 N, 5. 1 ;

Found: C, 39.52; H, 2.92; Br, 29.28; N, 5.03.

The process is also illustrated in Fig. 6.

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Step (7): Preparation of Methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (

To a stirred solution of dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (27 kg, 98.52 mol) in ierf-butanol (135 L) was added at room temperature cyclopropylmethanamine (7.83 kg, 110.1 mol). The reaction mixture was heated at 65 °C for 17 h. The progress of reaction was monitored by TLC and HPLC (HPLC analysis showed the formation of 74% of the product 5e after 17 h. The reaction mixture was cooled to room temperature and then cone. HCI (2.7 L) was added slowly and the mixture was stirred for 15 min. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in hot /-PrOH (54 L) filtered through a celite pad. The filtrate was cooled with stirring to 10 °C to obtain a white precipitate. The solid obtained was collected by filtration, washed with cold

i-PrOH (13 kg), n-hexane (15 L) and dried to provide pure methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e) (15.7 kg, 50.9%). The filtrate was concentrated under reduced pressure and the crude product can be purified by silica gel column chromatography eluting with tert-butanol in hexanes to furnish additional 10% methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e). HNMR (300 MHz, DMSO-cf₆) δ 8.83 (t, J = 5.9, 1 H), 8.47 – 8.41 (m, 1 H), 8.06 (d, J = 8.4, 1 H), 3.96 (s, 3H), 3.16 (t, J = 6.5, 2H), 1.14 – 0.99 (m, 1 H), 0.42 (m, 2H), 0.30 -0.19 (m, 2H); MS (ES+) 337.0 (M+23), 650.8 (2M+23); Analysis calculated for

C₁₂H₁₃BrN₂0₃: C, 46.03; H, 4.18; N, 8.95; Br, 25.52; Found: C, 46.15; H, 4.17; N, 8.72; Br, 25.26.

The average isolated yield for step (7) is 50% to 60%.

The process is also illustrated in Fig. 7.

Step (8): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a)

2

6a

THF (37.5 L) was charged to a 100 L reactor followed by ethyl 3-bromo-6- (cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate (5e) (2.5 kg, 7.98 mol) under a nitrogen atmosphere. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) (2.88 kg, 10.36 mol) was added, followed by the addition of PPh₃ (53.13 g, 0.20 mol), PdCI₂(PPh₃)₂ (120.4 g, 0.17 mol) and a solution of Na₂C0₃(2.12 kg, 20.00 mol) in demineralized water (10.0 L) under nitrogen atmosphere. The reaction mixture was degassed again two times by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 6.5 h, cooled to room temperature and filtered through a Celite bed. Water (75 L) was added to the filtrate and the product was extracted with ethyl acetate (75 L). The aqueous layer was back extracted with ethyl acetate (2 ^χ 60 L). The combined ethyl acetate extract was divided into two equal portions and each portion was washed with brine (37 L), dried over Na₂S0₄, filtered and concentrated under reduced pressure to give crude methyl 6- ((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) as a reddish viscous material (-4.5 Kg) which was used as such for the next step without further purification. An analytical sample was prepared by purification of a small sample by flash column chromatography (silica gel, eluting with 0-100% ethyl acetate in hexane) to furnish methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)-picolinate (6a) as an off-white solid; HNMR (300 MHz, DMSO-d₆) δ 9.89 (s, 1 H), 9.52 (s, 1 H), 8.79 (t, J = 6.1 Hz, 1 H), 8.23 (d, J = 8.0 Hz, 1 H), 8.09 (d, J = 8.0 Hz, 1 H), 7.34 (s, 1 H), 6.90 (s, 1 H), 3.85 (s, 3H), 3.62 (s, 3H), 3.22 (m, 2H), 1.16 -1.02 (m, 1 H), 0.49 – 0.38 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 791.0 (2M+Na), (ES-) 382.7 (M-1), 767.3 (2M-1); Analysis calculated for C₂₀H₂₀N₂O₆.0.25 H₂0: C, 61.77; H, 5.31 ; N, 7.20; Found: C, 61.54; H, 5.13; N, 7.05.

The process is also illustrated in Fig. 8.

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Step (9): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b)

6a 6b

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) (2.11 kg, estimated about 3.83 mol from step-8) in dichloromethane (16.0 L) and pyridine (1.4 L, 17.4 mol) cooled to -10°C and maintained at that temperature for 1 h was added a solution of triflic anhydride (980.0 ml_, 5.8 mol) in dichloromethane (6.0 L) drop wise over a period of 3 h at -10 °C. The reaction mixture was stirred at -5°C for 1.3 h, quenched with saturated aqueous NaHCO₃(10.4 L) and stirred for 30 mins. The organic layer was separated, washed successively with saturated aqueous NaHC0₃ (10.4 L), 1 HCI (2 x 16.6 L), water (13.2 L), brine (13.2 L), dried over MgS0₄, filtered and concentrated under reduced pressure to give the crude product. The crude product was stirred with 15% ethyl acetate in n-hexane (7.0 L) for 1 h. The solid obtained was collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (7.0 L) for 1 h, was collected by filtration and washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (8.0 L) for 1 h, collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was dried to afford methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)-oxy)phenyl)picolinate (6b) as a light brown solid (1.7 kg, 86% yield, for combined steps 8 & 9). Average isolated yield for combined steps 8 and 9 was 70% to 86%; Ή NMR (300 MHz, DMSO-cf₆): δ 9.64 (s, 1 H), 8.78 (t, J = 6.1 , 1 H), 8.29 (d, J = 8.0, 1 H), 8.16 (d, J = 8.0, 1 H), 8.03 (s, 1H), 7.39 (s, 1 H), 4.00 (s, 3H), 3.63 (s, 3H), 3.22 (m, 2H), 1.11 (m, 1 H), 0.52 – 0.39 (m, 2H), 0.28 (m, 2H); MS (ES+) 538.9 (M+Na). The process is also illustrated in Fig. 9.

Step (10): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c)

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4- (((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b) (12 kg, 23.24 mol) in DME (106 L) was charged into reactor under nitrogen. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. Potassium trifluoro(vinyl)borate (3.9 kg, 29.1 1 mol), PdCI₂(PPh₃)₂ (815 g, 1.13 mol), KHC0₃ (4.65 g, 46.44 mol) and demineralized water (12 L) was then added under a N₂ atmosphere. The reaction mixture was degassed by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 5 h. The reaction mixture was cooled to room temperature and then filtered through a Celite bed. Demineralized water (118 L) was added to the filtrate followed by ethyl acetate (124 L). The mixture was stirred for 20 min and then the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 95 L). The combined organic extract was washed with brine (95 L), dried over Na₂S0₄, and filtered. The solvent was evaporated under reduced pressure to give the crude product. The crude product was purified by column chromatography (silica gel, 120 kg, 230-400 mesh size, eluting with ethyl acetate in n-hexane) to obtain methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (6 kg, 72%). ¹H NMR (300 MHz, CDCI₃): δ (ppm) 9.64 (s, 1 H), 8.35 (d, J = 7.8 Hz, 1 H), 8.06-8.03 (m, 2H), 7.78(d, J = 7.8 Hz, 1 H), 7.02-6.92 (m, 1 H), 6.61 (s, 1 H), 5.86 (d, J = 17.7 Hz, 1 H), 5.38 (d, J = 1 1.4 Hz, 1 H), 3.84 (s, 3H), 3.67 (s, 3H), 3.35-3.29 (m, 2H),1.08-1.03 (m, 1H), 0.55-0.49 (m, 2H), 0.29-0.2 4(m, 2H). ¹HNMR (300 MHz, DMSO-d₆) 6 9.68 (s, 1 H), 8.77 (t, J = 6.1 , 1 H), 8.35 – 8.21 (m, 1 H), 8.16 – 8.01 (m, 2H), 7.14 -6.87 (m, 2H), 6.01 (dd, J = 1.2, 17.8, 1 H), 5.45 (dd, J = 1.1 , 1 1.3, 1 H), 3.91 (s, 3H), 3.64 (s, 3H), 3.23 (m, 2H), 1.21 – 1.01 (m, 1H), 0.51 – 0.40 (m, 2H), 0.34 – 0.20 (m, 2H). MS

(ES+) 417.0 (M+Na); Analysis calculated for C₂2H₂₂N₂0₅: C, 66.99; H, 5.62; N, 7.10;

Found: C, 66.75; H, 5.52; N, 7.06.

The process is also illustrated in Fig. 10.

Step (1 1): Preparation of 2-(6-((cyclopropylmethyl)carbamoyl)-2- (methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d)

To a stirred solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (1.57 kg, 3.80 mol) in acetonitrile (15.4 L) was added ferf-butyl alcohol (22.2 L), demineralized water (3.2 L) and sodium dihydrogen phosphate monohydrate (323.74 g, 2.346 mol). The reaction mixture was cooled to 0 °C and added 2-methyl-2-butene (5.3 L, 50.0 mol) and stirred at 0 °C for 30 min. A solution of 80% sodium chlorite (1.36 kg, 12.0 mol) in demineralized water (5.2 L) was added to the reaction mixture over a period of 2.5 h at 0 °C [temperature rises to 7 °C during the addition]. The reaction mixture was stirred at 0 °C for 2 h, diluted with water (40 L) and ethyl acetate (24 L). After stirring the mixture, it was allowed to settle and the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 20 L) then acidified with 5.9 % aqueous acetic acid (2 L) and extracted once with ethyl acetate (10 L). The organic extracts were combined washed with water (2 x 20 L), a solution of acetic acid (125 mL) in water (20.0 L), brine (2 ^χ 20 L), dried over Na₂S0₄, filtered and concentrated under reduced pressure (vapor temperature below 40 °C). The residue obtained was dissolved in acetone (7 L) (residue didn’t dissolve completely). The solution was poured slowly into a reactor containing stirred n-hexane (70.0 L) to precipitate the solid product and the mixture was stirred for 2 h. The solid obtained was collected by filtration, washed with 10% acetone in n-hexane (6.3 L), AJ-hexane (6.3 L), dried to afford 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4- methoxy-5-vinylbenzoic acid (6d) as an off-white solid (1.29 Kg, yield: 79.0%). Average isolated yield for step 1 1 is 74% to 84%. ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 12.50 (brs, 1 H), 8.69(t, J= 6.0 Hz, 1 H, NH), 8.20 (d, J= 7.9 Hz, 1 H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1 H), 6.97 (dd, J= 18.0, 1 1.3 Hz, 1 H), 6.88 (s, 1 H), 5.92 (d, J= 7.9 Hz, 1 H), 5.38 (d, J= 1 1.1 Hz, 1 H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); MS (ES+) 433.26, (M+Na); (ES-) 409.28 (M-1). The process is also illustrated in Fig. 1 1.

Step (12): Preparation of Methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate methanesulfonate (7a

Pyridine (3.8 L, 47.17 mol) and EDCI (5.31 kg, 27.66 mol) were sequentially added to a cooled solution (0 °C) of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (9 kg, 21.92 mol) and 4-aminobenzamidine dihydrochloride (5.13 kg, 24.65 mol) in /-PrOH (90 L). The reaction mixture was allowed to warm to room temperature and stirred for 2 h. TLC analysis indicated incomplete reaction. Additional EDCI (1.08 kg, 5.6 mol) was added and the reaction mixture was stirred for 8 h. The reaction was still incomplete as indicated by TLC analysis, additional EDCI (0.54 kg, 2.8 mol) was added and the reaction mixture was stirred for 5 h. TLC analysis indicated there was trace amount of unreacted starting material remaining. The reaction mixture was cooled to 0 °C and a solution of

methanesulfonic acid (MSA) (9.13 kg, 95 mol) in MeOH (38.7 L) was added to the cooled mixture over a period of 4 h. The reaction mixture was allowed to warm to room temperature and stirred for 15 h. The product was collected by filtration, washed with a mixture of /^‘-PrOH and MeOH (4:1 , 45 L). The wet cake was slurried in a mixture of /-PrOH and MeOH (2:1 , 135 L) stirred for 1 h and the product was collected by filtration and washed with a mixture of /^‘-PrOH and MeOH (4:1 , 46.8 L). The product was dried in

2015/046582

a vacuum oven at 45 °C to afford methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) as a pink-colored solid (12.71 kg, 93%). Average isolated yield for this step: >90%.

¹H NMR (300 MHz, DMSO-c/₆) δ 10.71 (s, 1 H), 9.16 (s, 2H), 8.80 (s, 2H), 8.68 (t, J = 6.1 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1 H), 7.93 (s, 1H), 7.84 – 7.72 (m, 4H), 7.12 – 6.97 (m, 2H), 6.04 (dd, J = 17.8, 1.3 Hz, 1 H), 5.45 (d, J = 12.6 Hz, 1H), 3.91 (s, 3H), 3.60 (s, 3H), 3.25 – 3.16 (m, 2H), 2.32 (s, 3H), 1.10 – 1.01 (m, 1 H), 0.48 – 0.37 (m, 2H), 0.30 – 0.22 (m, 2H); MS (ES+) 528.0 (M+1); Analysis calculated for

C₂₉H₂₉N₅O5.CH₃SO₃H.2H₂O. C, 54.62; H, 5.65; N, 10.62; S, 4.86; Found: C, 54.95; H, 5.55; N, 10.61 ; S, 4.87.

The process is also illustrated in Fig. 12.

Step (13): Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-rnethoxy-4- vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate

(3i) ^{,a 3i}

A pre-cooled (0-5 °C) aq. NaOH solution [prepared from solid NaOH (4 kg, 100 mol) in water (86 L)] was added to a suspension of methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) (28.7 kg, 46 mol) in acetonitrile (86 L) cooled to 0 to 5 °C over a period of 25 mins. The reaction mixture was stirred at 0 to 5 °C for 2.5 h (TLC analysis showed the reaction was complete). The reaction mixture was filtered through a sparkler filter, washed with a mixture of 1 :1 CH₃CN / H₂0 ( 57.4 L). Acetic acid (3.2 L, 55.9 mol) in water (56 L) was added to the filtrate at room temperature over a period of 25 mins and the resulting mixture was stirred at room temperature for 2.5 h. The solid product obtained was collected by filtration, washed with a 1 :4 mixture of CH₃CN / H₂0 (57.5 L). The solid was dried at 45°C in a vacuum oven to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate (3i) as an off-white solid (12,77 kg, 54.1%). Average yield for this step is 50% to 75%. Mp: >200°C; H NMR (300 MHz, DMSO-d₆): δ 13.49 (s, 1 H), 8.94 (bs, 4H), 8.56 (t, 1 H), 7.82 – 7.71 (m, 2H), 7.67 -7.56 (m, 4H), 7.51 (d, J = 7.8, 1 H), 6.98 (dd, J = 11.3, 17.8, 1 H), 6.68 (s, 1 H), 5.92 (d, J = 16.6, 1 H), 5.36 (d, J = 12.4, 1 H), 3.80 (s, 3H), 3.16 (m, 2H), 1.05 (m, 1 H), 0.43 (m, 2H), 0.24 (m, 2H); MS (ES+) 514.1 (M+1), 536.1 (M+Na), (ES-) 512.1 ; Analysis calculated for C28H27N5O5.3H2O: C, 59.25; H, 5.86; N, 12.34; Found C, 59.50; H,

5.75; N, 12.05. If needed this material can be crystallized from a mixture of acetone and water.

The process is also illustrated in Fig. 13.

Step 14: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b

A pre-cooled (5-8 °C) aqueous NaOH solution (prepared from solid NaOH (1.97 kg, 49.25 mol) in demineralized water (41 L) was added to a pre-cooled (0-5 °C) suspension of (3i) (13.8 kg, 26.9 mol) in acetonitrile (41 L). The reaction mixture was stirred at 0-5 °C for 30 min (until the reaction mixture becomes homogeneous). The reaction mixture was filtered through a sparkler filter washed with 50% acetonitrile in demineralized water (4.4 L). The filtrate was charged into a reactor and cooled to 0-5 °C. Aqueous HCI [prepared from cone. HCI (9.3 L) in demineralized water (36 L)] was added slowly with stirring to keep the reaction temperature at or below 15 °C, the resulting mixture was stirred at 10-15 °C for 13 h. The reaction mixture was cooled to 0-5 °C and stirred for 1 h. The solid obtained was collected by filtration and washed with demineralized water (36 L). The solid product was suspended in water (69 L) stirred for 30 mins and collected by filtration washed twice with water (20 L each). The solid product was dried in a vacuum oven at 45°C to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethyl carbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (1 1.21 Kg, 75.77%). Mp: >200°C; ¹H NMR (300 MHz, DMSO-ci₆): δ 12.98 (br s, 1 H), 10.86 (s, 1 H), 9.24 (s, 3H), 9.04 (s, 2H), 8.22 (d, J = 7.8 Hz, 1 H), 7.96 (d, J = 5.7 Hz, 2H), 7.78 (s, 4H), 7.09-6.99 (m, 2H), 6.07 (d, J = 17.7 Hz, 1 H), 5.45(d, J = 11.4 Hz, 1 H), 3.88 (s, 3H), 3.26-3.24 (m, 2H), 1.09 (m, 1 H), 0.47 (m, 2H), 0.28 (m, 2H).

Average isolated yield for this step varies from 63% to 80%.

The process is also illustrated in Fig. 14.

Example-2: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

6d 8a

To a solution of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (2.35 g, 5.7 mmol) and 4-aminobenzamidine dihydrochloride (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 ml_) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The

reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS0₄, filtered and concentrated in vacuum. The residue obtained was purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 and 0-100% chloroform in CMA 50) to furnish methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)-carbamoyl)picolinate hydrochloride (8a) (2.2 g, 65%) as a white solid; MP 266 °C; ¹HNMR (300 MHz, DMSO-d₆) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 -7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1 H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1 H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for C₂₉H₂₉N₅0₅ (H₂0)_{1 5} (HCI): C, 58.93; H, 5.63; N, 1 1.85; Found: C, 58.75; H, 5.65; N, 1 1.92.

Step-2: preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

8a 8b j₀ a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml), was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with a solution of sulfuric acid (0.483 ml, 9.00 mmol) in water (5 mL) and stirred for 10 min at room temperature. To this cold water (5 ml) was added and stirred at room temperature until product crystallized out. Cold water (5 mL) was added to the slurry and stir for additional 20 min, additional cold water (5 mL) was added prior to filtration of solid. The solid obtained was collected by filtration washed with water (5 mL and 2.5 mL), dried under vacuum overnight to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b) (1.103 g, 90 % yield) as a white solid; MP 221.7 °C; H NMR (300 MHz, DMSO-d₆) δ 12.30 – 10.91 (bs, 1 H, D₂0 exchangeable), 10.69 (bs, 1 H, D₂0 exchangeable), 9.24 (t, J = 6.0 Hz, 1 H), 9.16 (s, 2H, D2O exchangeable), 8.78 (s, 2H, D2O exchangeable), 8.24 (d, J = 8.0 Hz, 1 H), 8.04 – 7.91 (m, 2H), 7.84 – 7.67 (m, 4H), 7.13 – 6.94 (m, 2H), 6.03 (dd, J = 17.8, 1 .4 Hz, 1 H), 5.51 – 5.37 (m, 1 H), 3.88 (s, 3H), 3.24 (t, J = 6.4 Hz, 2H), 1.16 – 1.01 (m, 1 H), 0.52 – 0.41 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for: C₂₈H₂7N₆0₅ 1.0H₂SO₄ 1.5H₂0: C, 52.66; H, 5.05; N, 10.97; S, 5.02; Found: C, 52.81 ; H, 4.95; N, 10.94; S, 4.64.

Example-3: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane s

To a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml) was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with methanesulfonic acid (0.584 ml, 9.00 mmol) and stirred for 1 h at room temperature. Cold water (5.00 ml) was added to the reaction mixture and stirred at room temperature until product crystallized out. To the slurry was added water (5 ml) of water stirred for additional 20 min, followed by the addition of water (5 ml) prior to filtration. The solid obtained was collected by filtration washed with water (5 ml and 2.5 ml), dried under vacuum to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane sulfonate salt (8c)

(1 .138 g, 1.867 mmol, 93 % yield) as a white solid; MP 221.2 °C; ¹ H NMR (300 MHz,

DMSO-d₆) δ 12.89 (s, 1 H, D2O exchangeable), 10.69 (s, 1 H, D2O exchangeable), 9.24

(t, J = 6.0 Hz, 1 H), 9.16 (s, 2H,), 8.85 (s, 2H), 8.24 (d, J = 8.0 Hz, 1 H), 8.06 – 7.91 (m, 2H), 7.86 – 7.70 (m, 4H), 7.15 – 6.96 (m, 2H), 6.03 (dd, J = 17.8, 1.4 Hz, 1 H), 5.52 – 5.35 (m, 1 H), 3.88 (s, 3H), 3.25 (t, J = 6.3 Hz, 2H), 2.34 (s, 3H), 1.17 – 1.01 (m, 1 H), 0.53 -0.43 (m, 2H), 0.32 – 0.23 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for:

CzeH^NsOsCHsSOsH 1.5H₂0: C, 54.71 ; H, 5.38; N, 11.00; S, 5.04; Found: C, 54.80; H, 5.14; N, 10.94; S, 4.90.

Example-4: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) in Form C (Compound XX)

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled

46582

to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 – -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1 .6 kg) and 0.57 kg water. The mixture was heated to 46 °C. 21 g of Smopex-234 and 10 g Acticarbone 2SW were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 °C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature <3 °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The suspension was stirred at 0-3.0 °for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (T_jacke,= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 1 17 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C (Compound XX).

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AZD 7594

$AZN‘s asthma candidate

AZ13189620; AZD-7594

Bayer Pharma Aktiengesellschaft, Astrazeneca Ab

3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)propoxy]indazol-1-yl]-N-(oxolan-3-yl)benzamide

Cas 1196509-60-0

AZD-7594 is in phase II clinical trials by AstraZeneca for the treatment of mild to moderate asthma.

It is also in phase I clinical trials for the treatment of chronic obstructive pulmonary disorder (COPD).

https://clinicaltrials.gov/ct2/show/NCT02479412

• Phase II Asthma
• Phase I Chronic obstructive pulmonary disease

• 01 Feb 2016 AstraZeneca completes a phase II trial in Asthma in Bulgaria and Germany (Inhalation) (NCT02479412)
• 09 Jan 2016 AstraZeneca plans to initiate a phase I trial in Healthy volunteers in USA (IV and PO) (NCT02648438)
• 01 Jan 2016 Phase-I clinical trials in Chronic obstructive pulmonary disease (In volunteers) in USA (PO, IV, Inhalation) (NCT02648438)

PATENT

http://www.google.com/patents/WO2009142569A1

PATENT

US20100804345

UNWANTED ISOMER

WANTED COMPD

PATENT

WO 2009142571

Example 6

WANTED ISOMER

3-(5- { TC 1 R,2SV2-r(2,2-difluoropropanoyl)aminol- 1 -(2,3-dihydro-l ,4-benzodioxin-6-5 yDpropylioxy) – 1 H-indazol- 1 -ylVN-[(3R)-tetrahydrofuran-3-vnbenzamide. APCI-MS: m/z 607 [MH⁺] ¹H NMR ^OO MHz, DMSOd₆) δ 8.71 (IH, d), 8.65 (IH, d), 8.24 (IH, s), 8.18 (IH, s), 7.90 – 7.84 (2H, m), 7.77 (IH, d), 7.65 (IH, t), 7.21 (IH, dd), 7.13 (IH, d), 6.89 – 6.78 (3H, m), 5.17 (IH, d), 4.48 (IH, m), 4.23 – 4.10 (5H, m), 3.89 – 3.82 (2H, m), 3.72 (IH, td), 3.61 (IH, dd), 2.16 (IH, m), 1.94 (IH, m), 1.55 (3H, t), 1.29 (3H, d). LC (method A) rt = 12.03 min LC (method B) rt = 11.13 min Chiral SFC (method B) rt = 4.71 min M.p. = 177 °C

UNWANTED

o 3-(5- { IY 1 R,2S V2-r(2,2-difluoropropanoyl^‘)amino^‘|- 1 -(2,3-dihydro- 1 ,4-benzodioxin-6- yl)propyl]oxy } – 1 H-indazol- 1 -yP-N-IO S)-tetrahydrofuran-3 -yl^“|benzamide

APCI-MS: m/z 607 [MH⁺]

¹H NMR (400 MHz, DMSO-J₆) δ 8.71 (IH, d), 8.65 (IH, d), 8.24 (IH, s), 8.18 (IH, s),

7.90 – 7.84 (2H, m), 7.77 (IH, d), 7.65 (IH, t), 7.21 (IH, dd), 7.13 (IH, d), 6.89 – 6.78 (3H,s m), 5.17 (IH, d), 4.48 (IH, m), 4.24 – 4.11 (5H, m), 3.90 – 3.81 (2H, m), 3.72 (IH, td), 3.61

(IH, dd), 2.16 (IH, m), 1.94 (IH, m), 1.55 (3H, t), 1.29 (3H, d).

LC (Method A) rt = 12.02 min

LC (Method B) rt = 11.12 min

Chiral SFC (method B) rt = 5.10 min o M.p. = 175 ⁰C

PATENT

WO 2011061527

http://www.google.com/patents/WO2011061527A1?cl=en

Intermediate 12

( 1 R,2S)-2-amino- 1 -(2,3 -dihydrobenzo b [ 1 ,41dioxin-6-yl)propan- 1 -ol hydrochloride. (12)

5-6 N HC1 in 2-propanol (8 mL, 40-48 mmol) was added to tert-butyl (lR,2S)-l-(2,3- dihydrobenzo[b][l,4]dioxin-6-yl)-l-hydroxypropan-2-ylcarbamate (I2a) (3.1 g, 10.02 mmol) in ethyl acetate (40 mL) at 40°C and stirred for 3 hours. The reaction mixture was allowed to reach r.t. and was concentrated by evaporation. Ether was added and the salt was filtered off and washed with ether. The salt was found to be hygroscopic. Yield 2.10 g (85%)

APCI-MS: m/z 210 [MH⁺-HC1]

1H-NMR (300 MHz, DMSO-^): δ 8.01 (brs, 3H), 6.87-6.76 (m, 3H), 5.93 (brd, 1H), 4.79 (brt, 1H), 4.22 (s, 4H), 3.32 (brm, 1H), 0.94 (d, 3H).

tert-butyl (1R,2S)- 1 -(2,3-dihvdrobenzorbl Γ 1 ,41dioxin-6-yl)- 1 -hvdroxypropan-2-ylcarbamate.

The diastereoselective catalytic Meerwein-Ponndorf-Verley reduction was made by the method described by Jingjun Yin et. al. J. Org. Chem. 2006, 71, 840-843.

(S)-tert-butyl 1 -(2,3-dihydrobenzo[b] [ 1 ,4]dioxin-6-yl)- 1 -oxopropan-2-ylcarbamate (I2b) (3.76 g, 12.23 mmol), aluminium isopropoxide (0.5 g, 2.45 mmol) and 2-propanol (12 mL, 157.75 mmol) in toluene (22 mL) were stirred at 50°C under argon for 16 hours. The reaction mixture was poured into 1M HC1 (150 mL) and the mixture was extracted with ethyl acetate (250 mL). The organic phase was washed with water (2×50 mL) and brine (100 mL), dried over Na₂SC”4, filtered and concentrated. The crude product was purified by flash- chromatography on silica using ethyl acetate/hexane (1/2) as eluent. Fractions containing product were combined. Solvent was removed by evaporation to give the desired product as a colourless solid. Yield 3.19 g (84%) APCI-MS: m/z 236, 210, 192 [MH -tBu-18, MH -BOC, MH -BOC- 18]

1H NMR (300 MHz, DMSO-^): δ 6.80-6.70 (m, 3H), 6.51 (d, IH), 5.17 (d, IH), 4.36 (t, IH),

4.19 (s, 4H), 3.49 (m, IH), 1.31 (s, 9H), 0.93 (d, 3H).

(S)-tert-butyl 1 -(2,3-dihydrobenzo[bl [ 1 ,41dioxin-6-yD- 1 -oxopropan-2-ylcarbamate. (I2b)

A suspension of (S)-tert-butyl l-(methoxy(methyl)amino)-l-oxopropan-2-ylcarbamate (3 g, 12.92 mmol) in THF (30 mL) was placed under a protective atmosphere of argon and cooled down to -15 to -20°C. Isopropylmagnesium chloride, 2M in THF (6.5 mL, 13.00 mmol), was added keeping the temperature below -10°C. The temperature was allowed to reach 0°C. A freshly prepared solution of (2,3-dihydrobenzo[b][l,4]dioxin-6-yl)magnesium bromide, 0.7M in THF (20 mL, 14.00 mmol) was added. The temperature was allowed to reach r.t. overnight. The reaction mixture was poured into ice cooled IN HC1 (300 mL). TBME (300 mL) was added and the mixture was transferred to a separation funnel. The water phase was back extracted with TBME (200 mL). The ether phases were washed with water, brine and dried (Na₂S0₄). The crude product was purified by flash chromatography using TBME /Heptane 1/2 as eluent. Fractions containing the product were combined and solvents were removed by evaporation to give the subtitle compound as a slightly yellow sticky oil/gum. Yield 3.76g

(95%)

APCI-MS: m/z 208 [MH⁺ – BOC]

1H NMR (300 MHz, DMSO-^): δ 7.50 (dd, IH), 7.46 (d, IH), 7.24 (d, IH), 6.97 (d, IH), 4.97 (m, IH), 4.30 (m, 4H), 1.36 (s, 9H), 1.19 (d, 3H).

Intermediate 13

(lR,2S)-2-amino-l-(4H-benzo[dl[l,31dioxin-7- l)propan-l-ol hydrochloride (13)

Tert-butyl ( 1 R,2S)- 1 -(4H-benzo[d] [ 1 ,3]dioxin-7-yl)- 1 -hydroxypropan-2-ylcarbamate (I3b) (403 mg, 1.30 mmol) was dissolved in ethyl acetate (5 mL) and 5-6 N HC1 solution in 2- propanol (1.5 mL, 7.5-9 mmol) was added. The mixture was stirred at 50 °C for 1.5 hours. The solvents was removed by evaporation. The residual sticky gum was treated with ethyl acetate and evaporated again to give a solid material that was suspended in acetonitrile and stirred for a few minutes. The solid colourless salt was collected by filtration and was found to be somewhat hygroscopic. The salt was quickly transferred to a dessicator and dried under reduced pressure. Yield 293 mg (92%)

APCI-MS: m/z 210 [MH⁺ -HC1]

1H NMR (300 MHz, DMSO-^) δ 8.07 (3H, s), 7.05 (IH, d), 6.92 (IH, dd), 6.85 (IH, d), 6.03 (IH, d), 5.25 (2H, s), 4.87 (3H, m), 3.42 – 3.29 (IH, m), 0.94 (3H, d).

(4S.5R -5-(4H-benzordiri.31dioxin-7-vn- -methyloxazolidin-2-one (I3a

A mixture of (lR,2S)-2-amino-l-(4H-benzo[d][l,3]dioxin-7-yl)propan-l-ol hydrochloride (I3b) (120 mg, 0.49 mmol), DIEA (0.100 mL, 0.59 mmol) and CDI (90 mg, 0.56 mmol) in THF (2 mL) was stirred at r.t. for 2 hours. The reaction mixture was concentrated by evaporation and the residual material was partitioned between ethyl acetate and water. The organic phase was washed with 10% NaHS0₄, dried over MgS0₄, filtered and evaporated. The crude product was analysed by LC/MS and was considered pure enough for further analysis by NMR. Yield 66 mg (57%)

The relative cis conformation of the product was confirmed by comparing the observed 1H- NMR with the literature values reported for similar cyclised norephedrine (Org. Lett. 2005 (07), 13, 2755-2758 and Terahedron Assym. 1993, (4), 12, 2513-2516). In a 2D NOESY experiment a strong NOE cross-peak was observed for the doublet at 5.64 with the multiplet at 4.19 ppm. This also confirmed the relative czs-conformation.

APCI-MS: m/z 236 [MH⁺]

1H NMR (400 MHz, CDC1₃) δ 6.99 (d, J= 8.0 Hz, IH), 6.88 (dd, J= 8.0, 1.4 Hz, IH), 6.83 (s, IH), 5.81 (brs,lH), 5.64 (d, J= 8.0 Hz, IH), 5.26 (s, 2H), 4.91 (s, 2H), 4.19 (m, IH), 0.85 (d, J = 6.4 Hz, 3H). Tert-butyl ( 1 R,2S)- 1 -(4H-benzord1 Γ 1 ,31dioxin-7-yl)- 1 -hvdroxypropan-2-ylcarbamate (I3b)

A mixture (S)-tert-butyl l-(4H-benzo[d][l,3]dioxin-7-yl)-l-oxopropan-2-ylcarbamate (I3c) (680 mg, 2.21 mmol), triisopropoxyaluminum (140 mg, 0.69 mmol) and propan-2-ol (3 mL, 38.9 mmol) in toluene (3 mL) was stirred at 65 °C for 15 hours. The reaction mixture was allowed to cool down, poured into 1M HC1 (50 mL) and extracted with ethyl acetate (2×50 mL). The organic phase was washed with water, brine, dried over MgS0₄, filtered and solvents were removed by evaporation to afford a colourless solid. The crude product was purified by flash chromatography, (solvent A = Heptane, solvent B = EtOAc + 10% MeOH. A gradient of 10%B to 50%B in A was used). The obtained product was crystallised from DCM / heptane to afford the subtitle compound as colourless needles. Yield 414 mg (60%)

APCI-MS: m/z 210 [MH⁺ -BOC]

1H NMR (400 MHz, DMSO- ¾ δ 6.97 (1H, d), 6.88 (1H, d), 6.77 (1H, s), 6.56 (1H, d), 5.27 (1H, d), 5.22 (2H, s), 4.83 (2H, s), 4.44 (1H, t), 3.53 (1H, m), 1.32 (9H, s), 0.93 (3H, d). (S)-Tert-butyl 1 -(4H-benzord1 Γ 1 ,31dioxin-7-vD- 1 -oxopropan-2-ylcarbamate (I3c)

7-Bromo-4H-benzo[d][l,3]dioxine (1 g, 4.65 mmol) was dissolved in THF (5 mL) and added to magnesium (0.113 g, 4.65 mmol) under a protective atmosphere of argon. One small iodine crystal was added. The coloured solution was heated with an heat gun in short periods to initiate the Grignard formation. When the iodine colour vanished the reaction was allowed to proceed at r.t. for 1.5 hours.

In a separate reaction tube (S)-tert-butyl l-(methoxy(methyl)amino)-l-oxopropan-2- ylcarbamate (1 g, 4.31 mmol) was suspended in THF (5 mL) and cooled in an ice/acetone bath to below -5 °C. Isopropylmagnesium chloride, 2M solution in THF (2.5 mL, 5.00 mmol) was slowly added to form a solution. To this solution was added the above freshly prepared Grignard reagent. The mixture was allowed to reach r.t. and stirred for 4 hours. The reaction mixture was slowly poured into ice-cold 150 mL 1M HC1. Ethyl acetate (150 mL) was added and the mixture was stirred for a few minutes and transferred to a separation funnel. The organic phase was washed with water and brine, dried over MgS04, filtered and concentrated. The obtained crude product was further purified by flash chromatography using a prepacked 70g silica column with a gradient of 10% TBME to 40% TBME in heptane as eluent. The subtitle compound was obtained as a colourless solid. Yield 790 mg (59%>)

APCI-MS: m/z 208 [MH⁺ -BOC]

1H NMR (400 MHz, DMSO-^) δ 7.53 (IH, dd), 7.39 (IH, s), 7.30 (IH, d), 7.22 (IH, d), 5.30 (2H, s), 4.98 (IH, m), 4.95 (2H, s), 1.35 (9H, s), 1.20 (3H, d).

Preparation 4

3-(5-([(lR,2S)-2-[(2,2-difluoropropanoyl)aminol-l-(2,3-dihydro-l,4-benzodioxin-6- yl)propyl]oxy| – 1 H-indazol- 1 -yl)-N-[(3R)-tetrahydrofuran-3-yllbenzamide

TEA (2.0 g, 20.65 mmol) was added to a mixture of 3-(5-((lR,2S)-2-(2,2- difluoropropanamido)- 1 -(2,3-dihydrobenzo[b] [ 1 ,4]dioxin-6-yl)propoxy)-l H-indazol-1 – yl)benzoic acid (14) (3.6 g, 6.70 mmol), (R)-tetrahydrofuran-3 -amine hydrochloride (0.99 g, 8.0 mmol) and HBTU (2.65 g, 6.99 mmol) in DCM (15 mL). The reaction was stirred at r.t. for 3h, then quenched by addition of a mixture of water and ethyl acetate. The mixture was shaken and the organic layer was collected. The water phase was extracted twice with ethyl acetate. The combined organic layers were washed with a small portion of water and dried over magnesium sulphate. The product was purified by flash chromatography (silica, eluent: a gradient of ethyl acetate in heptane). The residue was crystallized by dissolving in refluxing acetonitrile (50 mL) and then allowing to cool to r.t. over night. The solid was collected by filtration, washed with a small volume of acetonitrile and dried at 40°C in vaccum to give the title compound (2.5 g, 61%).

APCI-MS: m/z 607 [MH⁺]

1H NMR (400 MHz, DMSO-d₆) δ 8.71 (IH, d), 8.65 (IH, d), 8.24 (IH, s), 8.18 (IH, s), 7.90 – 7.84 (2H, m), 7.77 (IH, d), 7.65 (IH, t), 7.21 (IH, dd), 7.13 (IH, d), 6.89 – 6.78 (3H, m), 5.17 (IH, d), 4.48 (IH, m), 4.23 – 4.10 (5H, m), 3.89 – 3.82 (2H, m), 3.72 (IH, td), 3.61 (IH, dd), 2.16 (IH, m), 1.94 (IH, m), 1.55 (3H, t), 1.29 (3H, d).

LC (method A) rt = 12.03 min

LC (method B) rt = 11.13 min

Chiral SFC (method B) rt = 4.71 min

M.p. = 177 °C

REFERENCES

https://www.astrazeneca.com/content/dam/az/press-releases/2014/Q2/Pipeline-table.pdf

////////AZD 7594, AZ13189620, AZD-7594 , phase 2, astrazeneca, 1196509-60-0

c21cc(ccc1n(nc2)c3cc(ccc3)C(=O)NC4COCC4)O[C@H](c5cc6c(cc5)OCCO6)[C@@H](NC(=O)C(F)(F)C)C

CC(C(C1=CC2=C(C=C1)OCCO2)OC3=CC4=C(C=C3)N(N=C4)C5=CC=CC(=C5)C(=O)NC6CCOC6)NC(=O)C(C)(F)F

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THE VIEWS EXPRESSED ARE MY PERSONAL AND IN NO-WAY SUGGEST THE VIEWS OF THE PROFESSIONAL BODY OR THE COMPANY THAT I REPRESENT, amcrasto@gmail.com, +91 9323115463 India.

I , Dr A.M.Crasto is writing this blog to share the knowledge/views, after reading Scientific Journals/Articles/News Articles/Wikipedia. My views/comments are based on the results /conclusions by the authors(researchers). I do mention either the link or reference of the article(s) in my blog and hope those interested can read for details. I am briefly summarising the remarks or conclusions of the authors (researchers). If one believe that their intellectual property right /copyright is infringed by any content on this blog, please contact or leave message at below email address amcrasto@gmail.com. It will be removed ASAP

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GLPG 1690

2-[[2-ethyl-6-[4-[2-(3-hydroxyazetidin-1-yl)-2-oxoethyl]piperazin-1-yl]-8-methylimidazo[1,2-a]pyridin-3-yl]-methylamino]-4-(4-fluorophenyl)-1,3-thiazole-5-carbonitrile

5- ​Thiazolecarbonitrile​, 2-​[[2-​ethyl-​6-​[4-​[2-​(3-​hydroxy-​1-​azetidinyl)​-​2-​oxoethyl]​-​ 1-​piperazinyl]​-​8-​methylimidazo[1,​2-​a]​pyridin-​3-​yl]​methylamino]​-​4-​(4-​fluorophenyl)​-

CAS 1628260-79-6

$GLPG compound for treating idiopathic pulmonary fibrosis

http://files.glpg.com/docs/website_1/Poster_ERS_2015_final.pdf

http://www.glpg.com/docs/view/56b360a81f6b2-en

Phase I Idiopathic pulmonary fibrosis

• Originator Galapagos NV
• Class Anti-inflammatories; Small molecules
• Mechanism of Action ENPP2 protein inhibitors

• 23 Sep 2015 Pharmacodynamics data from a preclinical trial in Indiopathic pulmonary fibrosis released by Galapagos
• 22 Sep 2015 Pharmacokinetics data from a phase I trial in healthy volunteers released by Galapagos
• 22 Sep 2015 Updated adverse events data from a phase I trial in healthy volunteers released by Galapagos

GLPG1690

GLPG1690 is a selective autotaxin inhibitor discovered by Galapagos, with potential application in idiopathic pulmonary disease (IPF). In a Phase 1 study in healthy human volunteers, GLPG1690 demonstrated favorable safety and tolerability, as well as a strong pharmacodynamic signal implying target engagement. Galapagos is currently preparing a Phase 2 study in IPF, to be filed for approval before the end of 2015. GLPG1690 is fully proprietary to Galapagos.

• Fully owned and proprietary clinical asset for pulmonary fibrosis
• GLPG1690 acts on autotaxin target
• Novel mode of action, originating from Galapagos target discovery engine
• Filing for Phase 2 clinical trial in 2015

MECHELEN, Belgium, March 16, 2015 (GLOBE NEWSWIRE) — Galapagos NV (Euronext: GLPG) announced that Janssen Pharmaceutica NV and Galapagos have mutually agreed to terminate the inflammation alliance and option agreements between the companies.  Galapagos views the molecules emerging from the alliance as strong additions to its growing proprietary pipeline.  Among others, all rights to candidate drug GLPG1690, a selective autotaxin inhibitor, return to Galapagos.  Galapagos has successfully completed a First-in-Human Phase 1 trial for GLPG1690 and is preparing a Phase 2 clinical trial in idiopathic pulmonary fibrosis (IPF).

“We are pleased to regain the rights to GLPG1690 to pursue the most suitable clinical application of autotaxin inhibition.  There is a large unmet medical need in IPF, and our pre-clinical data with GLPG1690 supports its potential as a competitive and novel approach in this disease area,” said Dr Piet Wigerinck, Chief Scientific Officer of Galapagos.  “The alliance with Janssen has been underway since October 2007 and has generated three clinical molecules, two of which are now proprietary Phase 2 assets of Galapagos: GLPG1205 and GLPG1690.  This program is a valuable component of our development portfolio, and regaining the rights is a next step in our transformation into a mature biotech company with a proprietary product pipeline.”

Galapagos identified autotaxin as playing a key role in inflammation, using an inflammation assay in its unique target discovery platform.  Pharmacology and translational studies published by other parties in the literature since then suggest autotaxin may play a key role in metabolic disease, arthritic pain, oncology, and lung disease.

GLPG1690 is a potent and selective inhibitor of autotaxin.  In a Phase 1 study in healthy human volunteers, GLPG1690 demonstrated favorable safety and tolerability, as well as a strong pharmacodynamic signal implying target engagement.  Galapagos is currently preparing a Phase 2 study in IPF, to be filed for approval before the end of 2015.

About IPF
Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease characterized by a progressive decline in lung function.  Pulmonary fibrosis involves scarring of lung tissue and is the cause of shortness of breath.  Fibrosis is usually associated with a poor prognosis.  The term “idiopathic” is used because the cause of pulmonary fibrosis is still unknown.  Estimated incidence of IPF is up to 16.3 per 100,000 persons in the US and 7.4 per 100,000 persons in Europe, with approximately 30,000-35,000 new patients diagnosed with IPF worldwide each year.  The goals of treatment in IPF are essentially to reduce the symptoms, slow down disease progression, reduce acute exacerbations, and prolong survival.  Approved treatments thus far have improved the overall survival of IPF patients, but unwanted side effects with these treatments are common, presenting an unmet need for effective treatments with safer side effect profiles.

MECHELEN, Belgium, Feb. 16, 2015 (GLOBE NEWSWIRE) — Galapagos NV (Euronext: GLPG) announced today that GLPG1690, a first-in-class molecule for pulmonary disease, has demonstrated target engagement, a good safety profile, and favorable drug properties in a Phase 1 study.  Galapagos is developing GLPG1690 within its alliance with Janssen Pharmaceutica NV.

The aim of the Phase 1 study was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of oral single and multiple ascending doses of GLPG1690.  The randomized, double-blind, placebo-controlled, single center study was conducted in 40 healthy volunteers in Belgium.  In the first part of the study, single ascending doses were evaluated.  In the second part, the new compound was administered daily for 14 days.

GLPG1690 proved to be safe and well-tolerated over a wide dose range in healthy volunteers.  Engagement of the thus far undisclosed novel target was confirmed using a relevant biomarker. GLPG1690 displayed a favorable pharmacokinetic and pharmacodynamic profile.  The data shown in Phase 1 encourage Galapagos to explore a Phase 2 study design in pulmonary disease.

“GLPG1690 is the first molecule against this target ever to be evaluated clinically, and we are pleased with the outcome of the Phase 1 study,” said Dr Piet Wigerinck, CSO of Galapagos.  “Galapagos continues to deliver novel therapeutics from its unique target and drug discovery engine.”

In 2007, Galapagos announced an alliance agreement with Janssen Pharmaceutica NV providing the option to worldwide, commercial licenses to certain Galapagos internal inflammatory disease programs.  These programs are based on novel targets for inflammatory disorders that were identified and validated by Galapagos using its proprietary target discovery engine.  Subsequent Galapagos research led to the discovery of GLPG1690, a first-in-class molecule that entered the clinic for inflammatory disorders.  Galapagos is responsible for execution of Phase 1 and Phase 2A studies with GLPG1690.

SYNTHESIS

INTRODUCTION

relates to compounds that are inhibitors of autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2), that is involved in fibrotic diseases, proliferative diseases, inflammatory diseases, autoimmune diseases, respiratory diseases, cardiovascular diseases, neurodegenerative diseases, dermatological disorders, and/or abnormal angiogenesis associated diseases. The present invention also provides methods for the production of a compound of the invention, pharmaceutical compositions comprising a compound of the invention, methods for the prophylaxis and/or treatment of diseases involving fibrotic diseases, proliferative diseases, inflammatory diseases, autoimmune diseases, respiratory diseases, cardiovascular diseases, neurodegenerative diseases, dermatological disorders, and/or abnormal angiogenesis associated diseases by administering a compound

STAGE 1

STAGE2

STAGE 3

STAGE4

STAGE 5

FINAL

PATENT

US2014303140

http://www.google.com/patents/US20140303140

1.2.4.4. Illustrative Synthesis of Intermediate Gen-3-e: N-(6-bromo-2-ethyl-8-methylimidazo[1,2-a]pyridin-3-yl)-N-methylformamide

• To a suspension of formamide Gen-2-d (720 g, 2.55 mol, 1 eq.) in 5 L of acetone were added potassium carbonate (1 kg, 7.66 mol, 3 eq.) and methyl iodide (700 g, 4.93 mol, 1.9 eq.). The reaction mixture was heated to 40° C. overnight. Additional methyl iodide (25 g, 0.18 mol, 0.07 eq.) was then introduced and stirring continued for 1 h at 40° C. The reaction mixture was filtered and washed with acetone (2×300 mL) and DCM (2×300 mL). The filtrate was concentrated in vacuo and the residue was partitioned between DCM (3 L) and water (1 L). The aqueous layer was further extracted with DCM. The combined organic layers were then washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The solid was triturated with Et₂O (1 L) at r.t. for 1 h, filtered off and dried to afford Intermediate Gen-3-e.
• Rotamer A (Major): ¹H NMR δ (ppm) (400 MHz, CDCl₃): 8.19 (1H, s), 7.78 (1H, s), 7.15 (1H, s), 3.24 (3H, s), 2.72 (2H, q), 2.59 (3H, s), 1.31 (3H, t)
• Rotamer B (Minor): ¹H NMR δ (ppm) (400 MHz, CDCl₃): 8.49 (1H, s), 7.65 (1H, s), 7.08 (1H, s), 3.36 (3H, s), 2.72 (2H, q), 2.59 (3H, s), 1.31 (3H, t)
• LC-MS: MW (calcd): 295 (⁷⁹Br), 297 (⁸¹Br); m/z MW (obsd): 296 (⁷⁹Br M+1), 298 (⁸¹Br M+1)

1.2.5.2. Illustrative Synthesis of Intermediate Gen-4-d: (6-Bromo-2-ethyl-8-methyl-imidazo[1,2-a]pyridin-3-yl)-methyl-amine

• Intermediate Gen-3-e (80 g, 270 mmol, 1 eq.) was dissolved in a 1.25 M HCl solution in MeOH (540 mL, 2.5 eq.) and the resulting mixture was refluxed overnight. 270 mL of 1.25 M HCl solution in MeOH were added and heating continued overnight. After 48 h, additional 70 mL of the 1.25 M HCl solution in MeOH were introduced in the reaction mixture. Heating was maintained overnight until conversion was complete. The crude mixture was then concentrated in vacuo and the residue was partitioned between EtOAc (300 mL) and water (700 mL). A saturated NaHCO₃ solution was added until pH reached 8-9. The aqueous layer was extracted twice with EtOAc (2×300 mL). The combined organic layers were then washed with brine (200 mL), dried over Na₂SO₄, filtered and concentrated in vacuo to give Intermediate Gen-4-d (6-bromo-2-ethyl-8-methyl-imidazo[1,2-a]pyridin-3-yl)-methyl-amine) as a free base.
• ¹H NMR δ (ppm) (400 MHz, CDCl₃): 8.05 (1H, s), 7.04 (1H, s), 2.84-2.78 (5H, m), 2.60 (3H, s), 1.35 (3H, t)
• LC-MS: MW (calcd): 267 (⁷⁹Br), 269 (⁸¹Br); m/z MW (obsd): 268 (⁷⁹Br M+1), 270 (⁸¹Br M+1)

1.2.6.4. Illustrative Synthesis of Intermediate Gen-5-t: 2-[(6-Bromo-2-ethyl-8-methyl-imidazo[1,2-a]pyridin-3-yl)-methyl-amino]-4-(4-fluoro-phenyl)-thiazole-5-carbonitrile

• To a solution of amine Gen-4-d (4.4 g, 16.6 mmol, 1 eq.) in THF (44 mL) under argon was slowly added NaH (60% in oil suspension, 2.0 g, 50.0 mmol, 3 eq.). The reaction mixture was heated at 90° C. for 30 min then cooled to 40° C. before adding the chlorothiazole Gen-12-a (4.74 g, 19.9 mmol, 1.2 eq.). The reaction mixture was stirred at 90° C. overnight. After cooling to r.t. the mixture was slowly quenched by addition of water and then diluted with EtOAc. The organic layer was separated and the aqueous layer extracted with EtOAc. The combined organic layers were then washed with water and brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was triturated in Et₂O, filtered and washed with Et₂O and MeCN. Recrystallization was performed in MeCN (180 mL) to afford Intermediate Gen-5-t (2-[(6-Bromo-2-ethyl-8-methyl-imidazo[1,2-a]pyridin-3-yl)-methyl-amino]-4-(4-fluoro-phenyl)-thiazole-5-carbonitrile).
• ¹H NMR δ (ppm) (400 MHz, CDCl₃): 8.15 (2H, dd), 7.80 (1H, s), 7.22-7.14 (3H, m), 3.62 (3H, s), 2.77 (2H, q), 2.64 (3H, s), 1.35 (3H, t)
• LC-MS: MW (calcd): 469 (⁷⁹Br), 471 (⁸¹Br); m/z MW (obsd): 470 (⁷⁹Br M+1), 472 (⁸¹Br M+1)

1.2.7.1.4. Illustrative Synthesis of 4-(3-{[5-Cyano-4-(4-fluoro-phenyl)-thiazol-2-yl]-methyl-amino}-2-ethyl-8-methyl-imidazo[1,2-a]pyridin-6-yl)-piperazine-1-carboxylic acid tert-butyl ester

• To a solution of Intermediate Gen-5-t (24.2 g, 51.5 mmol, 1 eq.) in toluene under argon were successively added N-Boc piperazine (14.4 g, 77.3 mmol, 1.5 eq.), sodium tert-butoxide (9.9 g, 103 mmol, 2 eq.), JohnPhos (1.54 g, 5.15 mmol, 0.1 eq.) and Pd₂(dba)₃ (2.36 g, 2.58 mmol, 0.05 eq.). The reaction mixture was heated at 115° C. for 1 h. After cooling to r.t., the crude product was filtered on Celpure® P65 and the residue dissolved in EtOAc and washed with water. The organic layer was further washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (elution with heptane/EtOAc:90/10 to 20/80) to afford the expected product.
• ¹H NMR δ (ppm) (400 MHz, CDCl₃): 8.16 (2H, dd), 7.17 (2H, app t), 6.99 (2H, bs), 3.62-3.53 (4H, m), 3.60 (3H, s), 3.04-2.93 (4H, m), 2.74 (2H, q), 2.62 (3H, s), 1.47 (9H, s), 1.33 (3H, t).
• LC-MS: MW (calcd): 575; m/z MW (obsd): 576 (M+1)

1.2.7.8.4. Illustrative Synthesis of Compound 1: 2-[(2-Ethyl-8-methyl-6-piperazin-1-yl-imidazo[1,2-a]pyridin-3-yl)-methyl-amino]-4-(4-fluoro-phenyl)-thiazole-5-carbonitrile

• 4-(3-{[5-Cyano-4-(4-fluoro-phenyl)-thiazol-2-yl]-methyl-amino}-2-ethyl-8-methyl-imidazo[1,2-a]pyridin-6-yl)-piperazine-1-carboxylic acid tert-butyl ester was prepared from intermediate Gen-5-t using Boc-piperazine and method Flb.
• To a solution of 4-(3-{[5-Cyano-4-(4-fluoro-phenyl)-thiazol-2-yl]-methyl-amino}-2-ethyl-8-methyl-imidazo[1,2-a]pyridin-6-yl)-piperazine-1-carboxylic acid tert-butyl ester (24.4 g, 42 mmol, 1 eq.) in MeOH (100 mL) was added a 2 M HCl solution in Et₂O (127 mL, 254 mmol, 6 eq.). The reaction mixture was stirred at r.t. for 3.5 h then concentrated in vacuo. The residue was partitioned between EtOAc and water. The aqueous layer was extracted twice with EtOAc. A 2 M NaOH solution was added to the aqueous layer until pH reached 8-9 and further extraction with EtOAc was performed. The combined organic layers were then washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The solid was triturated with heptane (100 mL) at r.t. overnight, filtered off, washed with heptane and Et₂O, and dried to afford the expected compound.
• ¹H NMR δ (ppm) (400 MHz, CDCl₃): 8.17 (2H, dd), 7.18 (2H, app t), 6.99 (2H, bs), 3.61 (3H, s), 3.09-2.98 (8H, m), 2.75 (2H, q), 2.61 (3H, s), 1.34 (3H, t).
• LC-MS: MW (calcd): 475; m/z MW (obsd): 476 (M+1)

////////////GLPG 1690, idiopathic pulmonary fibrosis, PHASE 1, GALAPAGOS, 1628260-79-6

n12c(c(nc1c(cc(c2)N3CCN(CC3)CC(=O)N4CC(C4)O)C)CC)N(C)c5nc(c(s5)C#N)c6ccc(cc6)F

CCC1=C(N2C=C(C=C(C2=N1)C)N3CCN(CC3)CC(=O)N4CC(C4)O)N(C)C5=NC(=C(S5)C#N)C6=CC=C(C=C6)F

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DISCLAIMER

I , Dr A.M.Crasto is writing this blog to share the knowledge/views, after reading Scientific Journals/Articles/News Articles/Wikipedia. My views/comments are based on the results /conclusions by the authors(researchers). I do mention either the link or reference of the article(s) in my blog and hope those interested can read for details. I am briefly summarising the remarks or conclusions of the authors (researchers). If one believe that their intellectual property right /copyright is infringed by any content on this blog, please contact or leave message at below email address amcrasto@gmail.com. It will be removed ASAP

.

Picture credit….Bethany Halford

GDC 0853

GDC-0853; RG 7845

2-[3-(hydroxymethyl)-4-[1-methyl-5-[(7-methyl-6,8-dihydro-5H-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one

3-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-[2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl]amino]-6-oxopyridin-3-yl]pyridin-2-yl]-7,7-dimethyl-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-4-one

3-[3-(hydroxymethyl)-4-[5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-1H-pyridin-3-yl]-2-pyridyl]-7,7-dimethyl-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-4-one

2H-​Cyclopenta[4,​5]​pyrrolo[1,​2-​a]​pyrazin-​1(6H)​-​one, 2-​[1,​6-​dihydro-​3′-​(hydroxymethyl)​-​1-​methyl-​5-​[[5-​[(2S) ​-​2-​methyl-​4-​(3-​oxetanyl)​-​1-​piperazinyl]​-​2-​pyridinyl]​amino]​ -​6-​oxo[3,​4′-​bipyridin]​-​2′-​yl]​-​3,​4,​7,​8-​tetrahydro-​7,​7-​ dimethyl-

s ISoMER 1434048-34-6 desired

r iSoMER 1434048-57-3 undesired

Phase 1

Patients with Patients with Resistant B-Cell Lymphoma or Chronic Lymphocytic Leukemia..

@genentech‘s Btk inhibitor

https://clinicaltrials.gov/ct2/show/NCT01991184

Bruton tyrosine kinase inhibitor

• 01 Sep 2015 Phase-I clinical trials in Autoimmune disorders (In volunteers) in USA (PO, Capsule and Tablet) (NCT02699710)
• 16 Oct 2014 Discontinued – Phase-I for Non-Hodgkin’s lymphoma (Second-line therapy or greater) in USA (unspecified route)
• 16 Oct 2014 Discontinued – Phase-I for Chronic lymphocytic leukaemia (Second-line therapy or greater) in USA (unspecified route)

BTK inhibitor GDC-0853 An orally available inhibitor of Bruton’s tyrosine kinase (BTK) with potential antineoplastic activity. Upon administration, GDC-0853 inhibits the activity of BTK and prevents the activation of the B-cell antigen receptor (BCR) signaling pathway. This prevents both B-cell activation and BTK-mediated activation of downstream survival pathways, which leads to the inhibition of the growth of malignant B-cells that overexpress BTK. BTK, a member of the Src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed in B-cell malignancies; it plays an important role in B-lymphocyte development, activation, signaling, proliferation and survival.

Patent

WO 2013067274

https://www.google.co.in/patents/WO2013067274A1?cl=en

part

Example 271a (S)-tert-Butyl 4-(6-(5-Chloro-2-methoxypyridin-3-ylamino)pyridin-3-yl)-3-methylpiperazine-1-carboxylate 271a

A 100-mL single-neck round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 1,4-dioxane (40 mL), (S)-tert-butyl 4-(6-amino pyridin-3-yl)-3-methylpiperazine-1-carboxylate 101h (2.04 g, 7.0 mmol), 3-bromo-5-chloro-2-methoxypyridine (2.8 g, 12.6 mmol), Pd₂(dba)₃ (640 mg, 0.70 mmol), XantPhos (404.6 mg, 0.70 mmol), and cesium carbonate (4.56 g, 14.0 mmol). After three cycles of vacuum/argon flush, the mixture was heated at 100 °C for 4 h. After this time the reaction was cooled to room temperature. It was then filtered and the filtrate was evaporated under reduced pressure. The residue was purified by silica-gel column chromatography eluting with 1:3 ethyl acetate/petroleum ether to afford 271a (1.7 g, 57%) as a yellow solid. MS-ESI: [M+H]⁺ 434.2

Example 271btert-Butyl (3S)-4-(6-{[5-(2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^2,6]dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4-yl)-2-methoxypyridin-3-yl] amino}pyridin-3-yl)-3-methylpiperazine-1-carboxylate 271b

A 100-mL single-neck round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 271a (650 mg, 1.50 mmol), {3-[(acetyloxy)methyl]-2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^2,6]dodeca-2(6),7-dien-10-yl}pyridin-4-yl}boronic acid 199e (1.79 g, 4.5 mmol), Pd₂(dba)₃ (137.2 mg, 0.15 mmol), P(cy)₃(167.4 mg, 0.60 mmol), Cs₂CO₃ (978 mg, 3.0 mmol), dioxane (20 mL), and water (0.5 mL). After three cycles of vacuum/argon flush, the mixture was heated at 110°C for 16 h. After this time the reaction was cooled to room temperature. Lithium hydroxide monohydrate (1.89 g, 45 mmol) and water (2.0 mL) were added. The resulting mixture was stirred at 45°C for 4 h. It was then filtered and the filtrate was evaporated under reduced pressure. The residue was purified by silica-gel column chromatography eluting with 3:1 ethyl acetate/petroleum ether to afford 271b (290 mg, 27%) as a yellow solid. MS-ESI: [M+H]⁺ 709.3

Example 271c 10-[3-(Hydroxymethyl)-4-[5-({5-[(2S)-2-methylpiperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]pyridin-2-yl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^2,6]dodeca-2(6),7-dien-9-one 271c

A solution of 271b (286.6 mg, 0.40 mmol) in dioxane/HCl (30 mL) was stirred at 50 °C for 2 h. It was evaporated under reduced pressure to afford 271c (450 mg, crude) as a black solid. MS-ESI: [M+H]⁺ 595.3

Example 271 3-[3-(hydroxymethyl)-4-[5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-1H-pyridin-3-yl]-2-pyridyl]-7,7-dimethyl-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-4-one 271

To a solution of 271c (450 mg, 0.75 mmol) in methanol (10 mL) was added oxetan-3-one (162 mg, 2.25 mmol), NaBH₃CN (141.8 mg, 2.25 mmol), and ZnCl₂ (306 mg, 2.25 mmol). The reaction was stirred at room temperature for 3 h. The mixture was evaporated under reduced pressure and the residue was diluted with water (5 mL). It was then extracted with dichloromethane (3 X 10 mL) and the combined dichloromethane extract was concentrated under reduced pressure. The residue was purified by reverse-phase prep-HPLC to afford 271 (23.0 mg, 8.8%, over two steps) as a yellow solid. MS-ESI: [M+H]⁺651.3. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 1H), 8.74 (d, J = 2.0 Hz, 1H), 8.53 (d, J = 5.0 Hz, 1H), 7.99 (d, J = 3.0 Hz, 1H), 7.84 (s, 1H), 7.73 (s, 1H), 7.41 (d, J = 4.5 Hz, 1H), 7.35 (dd, J = 2.5 Hz, 8.5 Hz, 1H), 6.87 (s, 1H), 6.85 (d, J = 9.0 Hz, 1H), 5.16-5.13 (m, 1H), 4.72-4.69 (m, 5H), 4.54-4.53 (m, 1H), 4.36-4.35 (m, 1H), 4.19-4.17 (m, 2H), 3.89-3.87 (m, 1H), 3.56-3.49 (m, 2H), 3.11-3.09 (m, 2H), 2.60-2.48 (m, overlap, 7H), 2.24-2.21 (m, 1H), 1.29 (s, 6H), 1.02 (d, J = 6.0 Hz, 3H)

271

………………………..

syn of 191 j

is intermediatenot product, is acid

To a mixture of 4-chloro-2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^2,6]dodeca-2(6),7-dien-10-yl}pyridine-3-carbaldehyde 108a (500 mg, 1.46 mmol), tert-butyl alcohol (20 mL), and dichloromethane (5 mL) was added 2-methyl-2-butene (3066 mg, 43.8 mmol). An aqueous solution (8 mL) of NaClO₂ (263 mg, 2.92 mmol) and NaH₂PO₄·2water (683 mg, 4.38 mmol) was added dropwise at -10°C and the reaction mixture was stirred at -10 °C for overnight. It was concentrated under reduced pressure and the residue was extracted with ethyl acetate (4 × 20 mL). The combined organic extract was dried over MgSO₄ and concentrated. The residue was purified with reverse-phase prep-HPLC to afford 210a (315 mg, 60%) as a pale yellow solid. MS-ESI: [M+H]⁺ 360.1

Example 210b 2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^2,6]dodeca-2(6),7-dien-10-yl} -4-[1-methyl-5-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]pyridine-3-carboxylic Acid 210b

A 25-mL round-bottomed flask equipped with a reflux condenser was charged with 210a (400 mg, 1.1 mmol), (S)-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one 191j (536 mg, 1.1 mmol), PdCl₂(dppf) (81 mg, 0.11 mmol), K₃PO₄ (466 mg, 2.2 mmol), sodium acetate (216 mg, 2.2 mmol), acetonitrile (10 mL), and water (0.2 mL). After three cycles of vacuum/argon flush, the mixture was heated at 100°C for 3 h. It was then filtered and the filtrate was evaporated in vacuo. The residue was purified by silica-gel column chromatography eluting with 1:3 petroleum/ethyl acetate to afford 210b as a yellow solid (306 mg, 41%). MS-ESI: [M+H]⁺ 679.3

construction, use your discretion

Example 130a (3S)-tert- utyl 3-methyl-4-(6-nitropyridin-3-yl)piperazine-l-carboxylate 130a

130a

Following the procedures as described for compound lOlg, reaction of 5-bromo-2-nitropyridine (10.5 g, 50 mmol), and (JS)-tert-butyl-3 -methylpiperazine- 1 -carboxylate (10.0 g, 50 mmol) afforded 130a as a yellow solid (8.05 g, 50%). LCMS: [M+H]⁺ 323

Example 130b (3 S)-tert-butyl-4-(6-aminopyridin-3 -yl)-3 -methylpiperazine- 1 -carboxylate 130b

130b

Following the procedures as described for compound lOlh, hydrogenation of 130a (5.8 g) afforded 130bas a brown solid (4.9 g, 96%). LCMS: [M+H]⁺ 293

Example 130c (3 S)-tert-Butyl-4-(6-(5 -bromo- 1 -methyl -2 -oxo- 1,2-dihydropyridin-3 -yl amino) pyridine-3 -yl)-3 -methylpiperazine- 1 -carboxylate 130c

N

Following the procedures as described for compound lOli, reaction of 130b (4.0 g) and 3,5-dibromo-l-methylpyridin-2(lH)-one (5.5 g) afforded 130c as a yellow solid (5.4 g, 83%). LCMS: [M+H]⁺ 478

Example 130d (3 S)-5 -Bromo- 1 -methyl-3 -(5 -(2-methylpiperazin- 1 -yl)pyridin- 2-ylamino)pyridine-2(lH)-one 130d

Following the procedures as described for compound lOlj, acidic hydrolysis of the Boc group of 130c (3.1 g) afforded 130d as a yellow solid (2.3 g, 95%). LCMS: [M+H]⁺ 380.

Example 130e (3 S)-5 -Bromo- 1 -methyl-3 -(5 -(2 -methyl-4-(ox etan-3-yl)piperazin-l-yl) pyridine -2-ylamino)pyridin-2(lH)-one 130e

Following the procedures as described for compound 101k, reductive amination of 130d (2.35 g) with oxetan-3-one (0.4 mL) afforded 130e as a yellow solid (2.6 g, 98%). LCMS: [M+H]⁺ 434.

Example 13 Of (3S)-l-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-l-yl)pyridin-2-ylamino) -5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridin-2(lH)-one 130f

check pyridine ring position

A 100 mL single-neck round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 130e (1.0 g, 1.0 eq., 2.3 mmol), Pin₂B₂ (1.46 g, 2.50 eq., 5.75 mmol), Pd₂(dba)₃ (105 mg, 0.05 eq., 0.125 mmol), X-Phos (93 mg, 0.1 eq., 0.23 mmol), AcOK (676 mg, 3.0 eq., 6.9 mmol), and dioxane (50 mL). After three cycles of vacuum/argon flush, the mixture was heated at 90 °C for 4 hrs, then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was washed with 3: 1 PE/EA (80 mL) to afford 130f as yellow solid (1.0 g, 90%). MS: [M+H]⁺ 482.

check pyridine ring position, use your discretion

Example 191h ( 3S)-5 -Bromo- 1 -methyl-3 -(5 -(2-methylpiperazin- 1 -yl)pyridin- -ylamino)pyridine-2(lH)-one 191h

Following the procedure described for compound lOlj and starting with (3S)-tert-butyl 4-(6-(5 -bromo- 1 -methyl-2-oxo- 1 ,2-dihydropyridin-3 -ylamino)pyridine-3 -yl)-3 -methyl-piperazine-l-carboxylate 191g (3.1 g, 6.5 mmol) afforded 191h as a yellow solid (2.3 g, 94%). MS-ESI: [M+H]⁺ 378.

Example 1 1 i (S)-5 -Bromo- 1 -methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin- 1 -yl)pyridin-2-ylamino)pyridin-2(lH)-one 191i

A mixture of (5)-5-bromo-l-methyl-3-(5-(2-methylpiperazin-l-yl)pyridin-2-ylamino)pyridin-2(lH)-one 191h (40.0 g, 106 mmol), oxetan-3-one (1 1.4 g, 159 mmol), NaBH₃CN (10.0 g, 159 mmol), and zinc chloride (21.3 g, 159 mmol) in methanol (700 mL) was stirred at 50°C for 5 hours. The mixture was added to water (100 mL) and concentrated under reduced pressure. The residue was extracted with dichloromethane (200 mL x 3). The combined organic layer was concentrated under reduced pressure and the residue was purified by silica-gel column chromatography eluting with 40: 1 dichloromethane /methanol to afford 191i (35 g, 73%). MS: [M+H]⁺ 434.

Example 191j (J5)-l-Methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-l-yl)-pyridin- -ylamino) -5-(4,4,5,5-tetramethyl-l ,3,2-dioxaborolan-2-yl)pyridin-2(lH)-one 191j

191 i 191j

A 100-mL single-neck round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with (5)-tert-butyl-4-(6-(5-bromo-l-methyl-2-oxo-l ,2-dihydropyridin-3-ylamino)pyridine-3-yl)-3-methylpiperazine-l-carboxylate 191i (1.0 g, 1.0 eq., 2.3 mmol), Pin₂B₂ (1.46 g, 2.50 eq., 5.75 mmol), Pd₂(dba)₃ (105 mg, 0.05 eq., 0.125 mmol), X-Phos (93 mg, 0.1 eq., 0.23 mmol), potassium acetate (676 mg, 3.0 eq., 6.9 mmol), and dioxane (50 mL). After three cycles of vacuum/argon flush, the mixture was heated at 90°C for 4 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was washed with 3 : 1 petroleum ether/ethyl acetate (80 mL) to afford 191j as yellow solid (1.0 g, 90%). MS: [M+H]⁺ 482.

pipeline

http://www.gene.com/medical-professionals/pipeline

Pictrelisib, GDC-0941, RG7321 and GNE0941

//////GDC 0853, genentech,  Btk inhibitor, phase 1, Patients with Resistant B-Cell Lymphoma,  Chronic Lymphocytic Leukemia, Bruton tyrosine kinase inhibitor,  GDC-0853,  RG 7845, 1434048-34-6

N1(CCN(CC1C)C2COC2)c3cnc(cc3)NC=4C(N(\C=C(/C=4)c5c(c(ncc5)N6CCn7c(C6=O)cc8CC(Cc78)(C)C)CO)C)=O

CC1CN(CCN1C2=CN=C(C=C2)NC3=CC(=CN(C3=O)C)C4=C(C(=NC=C4)N5CCN6C7=C(CC(C7)(C)C)C=C6C5=O)CO)C8COC8

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I , Dr A.M.Crasto is writing this blog to share the knowledge/views, after reading Scientific Journals/Articles/News Articles/Wikipedia. My views/comments are based on the results /conclusions by the authors(researchers). I do mention either the link or reference of the article(s) in my blog and hope those interested can read for details. I am briefly summarising the remarks or conclusions of the authors (researchers). If one believe that their intellectual property right /copyright is infringed by any content on this blog, please contact or leave message at below email address amcrasto@gmail.com. It will be removed ASAP

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PF 06650808

$PFE compound inspired by auristatins

https://clinicaltrials.gov/ct2/show/NCT02129205

http://www.pfizer.com/sites/default/files/product-pipeline/8_7_2014_Pipeline_Update.pdf

ALL DATA COMING………

Notch-3 receptor antagonists

Neoplasms
Breast

Cancer

PF-06650808, is currently being examined in a Ph1 clinical trial (Protocol B7501001).

Notch3
Researchers are also exploring the use of Notch3 targeting. “The Notch pathway plays an important role in the growth of several solid tumours, including breast and ovarian cancer and melanoma,” explained Joerger. “In particular, Notch3 alterations such as gene amplification and upregulation are associated with poor patient survival. Research using Notch3 targeting as an innovative approach to treat solid malignancies included 27 patients unselected for Notch3 who received increasing doses of the anti-Notch3 antibody-drug conjugate PF-06650808. Responses were seen in two breast cancer patients (LBA 30). While preliminary, targeting Notch3 may become a new treatment approach in patients with selected solid tumours.”

The anti-Notch3 antibody-drug conjugate PF-06650808 is being developed by Pfizer.

• 31 Jul 2014 Phase-I clinical trials in Solid tumours (Late-stage disease) in USA (Parenteral)
• 30 Apr 2014 Preclinical trials in Solid tumours in USA (Parenteral)
• 30 Apr 2014 Pfizer plans a phase I trial for Solid tumours (late-stage disease, second-line therapy or greater) in USA (NCT02129205)

251st Am Chem Soc (ACS) Natl Meet (March 13-17, San Diego) 2016, Abst MEDI 262

/////////PF 06650808, PF-06650808, PF-6650808, monoclonal antibody, pfizer, phase 1, Solid tumours , Notch-3 receptor antagonists

C1(C(N(C(C1)=O)CCCCCC(=O)NC([C@H](C)C)C(=O)NC(C(=O)Nc2ccc(cc2)COC(=O)NC(C)(C)C(=O)N[C@@H](C(C)C)C(=O)[N@](C)C(C(CC)C)[C@@H](OC)CC(=O)N3CCC[C@H]3C(OO)C(C)C(=O)N[C@H](c4nccs4)CC)CCCNC(=O)N)=O)SC

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I , Dr A.M.Crasto is writing this blog to share the knowledge/views, after reading Scientific Journals/Articles/News Articles/Wikipedia. My views/comments are based on the results /conclusions by the authors(researchers). I do mention either the link or reference of the article(s) in my blog and hope those interested can read for details. I am briefly summarising the remarks or conclusions of the authors (researchers). If one believe that their intellectual property right /copyright is infringed by any content on this blog, please contact or leave message at below email address amcrasto@gmail.com. It will be removed ASAP

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BMS 986120

Originator Bristol-Myers Squibb

Bristol-Myers Squibb Company, Université de Montréal

4-[4-[[6-methoxy-2-(2-methoxyimidazo[2,1-b][1,3,4]thiadiazol-6-yl)-1-benzofuran-4-yl]oxymethyl]-5-methyl-1,3-thiazol-2-yl]morpholine

4-(4-(((6-Methoxy-2-(2-methoxyimidazo[2,l-b][l,3,4]thiadiazol-6-yl)benzofuran-4-yl) oxy)methyl)-5-methylthiazol-2-yl)morpholine

Imidazo[2,​1-​b] ​-​1,​3,​4-​thiadiazole, 2-​methoxy-​6-​[6-​methoxy-​4-​[[5-​methyl-​2-​(4-​morpholinyl)​-​4-​ thiazolyl]​methoxy]​-​2-​benzofuranyl]​-

CAS 1478712-37-6

Phase I Thrombosis

• 02 Apr 2015 Bristol-Myers Squibb plans a phase I trial in Thrombosis (In volunteers) in United Kingdom (NCT02439190)
• 01 Aug 2014 Preclinical trials in Thrombosis in USA (PO)

https://clinicaltrials.gov/ct2/show/NCT02208882

https://clinicaltrials.gov/ct2/show/NCT02439190

Class Imidazoles; Small molecules; Thiadiazoles

$BMY antithrombic compound 

PATENT

http://www.google.com/patents/WO2013163279A1?cl=en

Thromboembolic diseases remain the leading cause of death in developed countries despite the availability of anticoagulants such as warfarin (COUMADIN®), heparin, low molecular weight heparins (LMWH), synthetic pentasaccharides, and antiplatelet agents such as aspirin and clopidogrel (PLAVIX®).

Current anti-platelet therapies have limitations including increased risk of bleeding as well as partial efficacy (relative cardiovascular risk reduction in the 20 to

30% range). Thus, discovering and developing safe and efficacious oral or parenteral antithrombotics for the prevention and treatment of a wide range of thromboembolic disorders remains an important goal.

Alpha-thrombin is the most potent known activator of platelet aggregation and degranulation. Activation of platelets is causally involved in atherothrombotic vascular occlusions. Thrombin activates platelets by cleaving G-protein coupled receptors termed protease activated receptors (PARs). PARs provide their own cryptic ligand present in the N-terminal extracellular domain that is unmasked by proteolytic cleavage, with subsequent intramolecular binding to the receptor to induce signaling (tethered ligand mechanism; Coughlin, S.R., Nature, 407:258-264 (2000)). Synthetic peptides that mimic the sequence of the newly formed N-terminus upon proteolytic activation can induce signaling independent of receptor cleavage. Platelets are a key player in atherothrombotic events. Human platelets express at least two thrombin receptors, commonly referred to as PARI and PAR4. Inhibitors of PARI have been investigated extensively, and several compounds, including vorapaxar and atopaxar have advanced into late stage clinical trials. Recently, in the TRACER phase III trial in ACS patients, vorapaxar did not significantly reduce cardiovascular events, but significantly increased the risk of major bleeding (Tricoci, P. et al, N. Eng. J. Med., 366(l):20-33 (2012). Thus, there remains a need to discover new antiplatelet agents with increased efficacy and reduced bleeding side effects.

There are several early reports of preclinical studies of PAR4 inhibitors. Lee, F-Y. et al., “Synthesis of l-Benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole Analogues as Novel Antiplatelet Agents”, J. Med. Chem., 44(22):3746-3749 (2001) discloses in the abstract that the compound

58

“was found to be a selective and potent inhibitor or protease-activated receptor type 4 (PAR4)-dependent platelet activation. ”

Compound 58 is also referred to as YD-3 in Wu, C-C. et al, “Selective Inhibition of Protease-activated Receptor 4-dependent Platelet Activation by YD-3”, Thromb. Haemost., 87: 1026-1033 (2002). Also, see Chen, H.S. et al, “Synthesis and platelet activity”, J. Bioorg. Med. Chem., 16: 1262-1278 (2008).

EP1166785 Al and EP0667345 disclose various pyrazole derivatives which are useful as inhibitors of platelet aggregation.\

IB. 5-(Benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][l,3]dioxin-4-one

A solution of 5-hydroxy-7-methoxy-2,2-dimethyl-4H-benzo[d][l,3]dioxin-4- one (30.00 g, 0.134 mol, see Kamisuki, S. et al, Tetrahedron, 60:5695-5700 (2004) for preparation) in N,N-dimethylformamide (400 mL) was treated with powdered anhydrous potassium carbonate (19.41 g, 0.14 mol) added all at once. The resulting mixture was stirred in vacuo for 10 min. and then flushed with nitrogen. The reaction flask was placed in a water bath (22 °C) and treated with benzyl bromide (24.03 g, 0.14 mol) added dropwise over 15 min. The resulting mixture was then stirred at 22 °C for 18 h (no starting material left by tic). The solid was filtered and washed with N,N- dimethylformamide. The filtrate was evaporated in vacuo and the residual oil was diluted with ethyl acetate (500 mL), washed with cold 0.1 N hydrochloric acid, saturated sodium bicarbonate and brine. After drying over anhydrous magnesium sulfate, evaporation of the solvent gave a thick syrup. Crystallization form ethyl acetate (50 mL) and hexane (150 mL) gave 35.17 g of 5-(benzyloxy)-7-methoxy-2,2-dimethyl-4H- benzo[d][l ,3]dioxin-4-one as large colorless prisms. Chromatography of the mother liquors on silica gel (4 x 13 cm, elution toluene – ethyl acetate 0-5%) gave 6.64 g of additional material to afford a total yield of 41.81 g (99%). HRMS(ESI) calcd for

Ci₈Hi₉0₅ [M+H]⁺ m/z 315.1227, found 315.1386. 1H NMR (CDC1₃, 600 MHz) δ 1.68 (s, 6H), 3.77 (s, 3H), 5.19 (s, 2H), 5.19 (s, 2H), 6.04 (d, J = 2.03 Hz, 1H), 6.15 (d, J = 2.03 Hz, 1H), 7.27 (broad t, 1H), 7.36 (broad t, 2H), 7.52 (broad d, 2H).

1 C. 2-(Benzyloxy)-6-hydroxy-4-methoxybenzaldehyde

A solution of 5-(benzyloxy)-7-methoxy-2,2-dimethyl-4H-benzo[d][l ,3]dioxin- 4-one (Example IB, 6.76 g, 21.5 mmol) in dichloromethane (120 mL) was cooled to -78 °C and treated with 43 mL (64.5 mmol) of a 1.5 M solution of diisobutylaluminum hydride in toluene added dropwise over 20 min. The resulting mixture was then stirred at -78 °C for 3 h. The reaction mixture was quenched by the careful addition of methanol (5 mL) added dropwise over 15 min, followed by IN hydrochloric acid (50 mL) added dropwise over 15 min. The cooling bath was then removed and an additional 150 mL of IN hydrochloric acid was added over 20 min. The mixture was then stirred at 22 °C for 2 h and diluted with dichloromethane (400 mL). The organic phase was collected and the aqueous phase (pH ~1) was extracted with dichloromethane (3 x 50 mL). The combined organic extracts were washed with brine, dried over anhydrous magnesium sulfate and concentrated in vacuo. The residual oil was diluted with tetrahydrofuran (70 mL), treated with 10 mL of 0.1N hydrochloric acid and stirred at 20 °C for 2 h. The reaction mixture was diluted with ethyl acetate (300 mL), washed with brine, dried over anhydrous magnesium sulfate, evaporated in vacuo to give a clear oil. Chromatography on silica gel (4 x 13 cm, elution toluene) gave 4.08 g (73% yield) of the title aldehyde as a clear oil which solidified on standing. LC (Method C): 2.237 min. HRMS(ESI) calcd for Ci₅Hi₅0₄ [M+H]⁺ m/z 259.0965, found 259.1153. 1H NMR (CDC1₃, 600 MHz) δ 3.80 (s, 3H), 5.07 (s, 2H), 5.97 (d, J= 2.1 Hz, 1H), 6.01 (d, J= 2.1 Hz, 1H), 7.3 – 7.4 (m, 5 H), 10.15 (s, 1H), 12.49 (s, 1H).

ID. 1 -(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)ethanone

A solution of 2-(benzyloxy)-6-hydroxy-4-methoxybenzaldehyde (Example 1C, 3.46 g, 13.4 mmol) in N,N-dimethylformamide (50 mL) was treated with powdered anhydrous cesium carbonate (4.58 g, 14.05 mmol) added all at once. The resulting mixture was stirred in vacuo for 10 min. and then flushed with nitrogen. The reaction flask was placed in a water bath (22 °C) and treated with chloroacetone (1.74 g, 18.7 mmol) added dropwise over 5 min. The resulting mixture was then stirred at 22 °C for 18 h (no starting aldehyde left by tic and formation of the intermediate alkylated aldehyde). The solid was filtered and washed with N,N-dimethylformamide. The filtrate was evaporated in vacuo and the residual oil was diluted with ethyl acetate (300 mL), washed with cold 0.1 N hydrochloric acid, saturated sodium bicarbonate and brine. After drying over anhydrous magnesium sulfate, evaporation of the solvent gave a thick syrup. This syrup was diluted with tetrahydrofuran (50 mL) and ethyl acetate (50 mL), treated p- toluenesulfonic acid monohydrate (0.2 g) and stirred at 20 °C for 1 h (tic indicated complete cyclization of the intermediate alkylated aldehyde to the benzofuran). The reaction mixture was diluted with ethyl acetate (300 mL), washed with saturated sodium bicarbonate and brine. After drying over anhydrous magnesium sulfate, evaporation of the solvent gave a thick syrup. Chromatography on silica gel (4 x 12 cm, elution toluene – ethyl acetate 2-4%) gave 3.51 g (88% yield) of the title benzofuran as a yellow solid. Recrystallization from ethyl acetate (10 mL) and hexane (20 mL) gave the title material as large yellow prisms (3.15 g). LC (Method D): 2.148 min. HRMS(ESI) calcd for Ci₈Hiv0₄ [M+H]⁺ m/z 297.1121, found 297.1092. 1H NMR (CDC1₃, 600 MHz) δ 2.51 (s, 3H), 3.82 (s, 3H), 5.13 (s, 2H), 6.37 (d, J= 1.77 Hz, 1H), 6.63 (broad s, 1H), 7.34 (broad t, 1H), 7.39 (broad t, 2H), 7.44 (broad d, 2H), 7.55 (d, J = 0.7 Ηζ,ΙΗ). IE. l-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-2-bromoethanone

A 250-mL, three-necked flask is equipped with a magnetic stirring bar and purged with a nitrogen atmosphere was charged with anhydrous tetrahydrofuran (25 mL) followed by 9.3 mL (9.3 mmol) of a 1M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran. The mixture was cooled to -78 °C and treated with a solution of l-(4- (benzyloxy)-6-methoxybenzofuran-2-yl)ethanone (Example ID, 2.40 g, 8.1 mmole) in tetrahydrofuran (20 mL) added dropwise over 10 min. The resulting mixture was then stirred at -78 °C for 45 min. Then chlorotrimethylsilane (1.18 mL, 9.31 mmol) was added dropwise over 5 min and the resulting solution was stirred at -78 °C for another 20 min. The cooling bath was then removed and the mixture is allowed to warm to room temperature over 30 min. The reaction mixture was then quenched by addition to a cold solution of ethyl acetate (200 mL), saturated sodium bicarbonate (30 mL) and ice. The organic phase was rapidly dried over anhydrous magnesium sulfate (magnetic stirring) and evaporated in vacuo to give the silyl enol ether as an oil which is co-evaporated with toluene (20 mL). The silyl enol ether was then dissolved in dry tetrahydrofuran (40 mL), cooled to -20 °C and treated with solid sodium bicarbonate (0.10 g) followed by N- bromosuccinimide (1.44 g, 8.1 mmol) added in small portions over 15 min. The reaction mixture was allowed to warm to 0 °C over 2h and then quenched by addition of ethyl acetate (300 mL) and saturated sodium bicarbonate. The organic phase was washed with brine, dried over anhydrous magnesium sulfate and evaporated to give an orange oil. Chromatography on silica gel (4 x 12 cm, elution toluene – ethyl acetate 0-5%) gave 2.62 g (86% yield) of the title bromomethylketone as a yellow solid. Recrystallization from ethyl acetate (10 mL) and hexane (20 mL) gave yellow prisms (2.30 g). LC (Method E): 1.977 min. HRMS(ESI) calcd for Ci₈Hi₆Br0₄ [M+H]⁺ m/z 375.0226, found 375.0277. 1H NMR (CDCls, 600 MHz) δ 3.84 (s, 3H), 4.33 (s, 2H), 5.14 (s, 2H), 6.38 (d, J = 1.76 Hz, 1H), 6.64 (broad s, 1H), 7.35 (broad t, 1H), 7.40 (broad t, 2H), 7.44 (broad d, 2H), 7.70 (s, 1H). 1 EE. 1 -(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-2-chloroethanone

Benzyltrimethylammonium dichloroiodate (117 g, 169 mmol) was added to a solution of l-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)ethanone (Example ID, 50 g, 170 mmol) in THF (500 mL) in a 1 L multineck round bottom flask under nitrogen atmosphere. The reaction mixture was stirred at RT for 6 h, cooled to 0 °C and quenched with 10% NaHCC”3 solution. The organic layer was washed with 1 M sodium thiosulphate solution, water, and brine, dried over Na₂S0₄, and concentrated in vacuo (bath temperature <45 °C). The residue was triturated with 5% EtOAc in pet. ether and dried to obtain the title chloromethylketone as a pale yellow solid (48 g, 130 mmol, 78%). 1H NMR (300 MHz, DMSO-d₆) δ 3.84-3.82 (d, J =4.5Hz, 3H) 4.98 (s, 2H), 5.27(s, 2H), 6.62 -6.61 (d, J = 1.8Hz, 1H), 6.92-6.93 (m, 1H), 7.54-7.36 (m, 5H), 8.10-8.09 (d, J = 3Hz, 1H); MS m/z: [M+H]⁺ 331.0. IF. 6-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-2-bromoimidazo[2, 1 – b] [ 1 ,3 ,4]thiadiazole

A mixture of l-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2-bromoethanone (Example IE, 3.00 g, 8.0 mmol) and 5-bromo-l,3,4-thiadiazol-2-amine (1.65 g, 9.16 mmol) in isopropanol (100 mL) was heated in a pressure flask equipped with a magnetic stirring bar at 78-80 °C for 18 h (homogeneous after 20 min and then formation of a precipitate after 2 h). The cooled mixture is then transferred into five 20 mL microwave vials and then heated in a microwave apparatus to 150 °C for 30 min. Each vial was then diluted with dichloromethane (250 mL) washed with saturated sodium bicarbonate (25 mL) and brine (25 mL), dried over anhydrous magnesium sulfate. The fractions were combined and concentrated in vacuo. Chromatography of the orange-brown residual solid on silica gel (4 x 10 cm, slow elution with dichloromethane due to poor solubility) gave 2.96 g of the title imidazothiadiazole contaminated with some l-(4-(benzyloxy)-6- methoxybenzofuran-2-yl)ethanone. The solid material was triturated with ethyl acetate (20 mL), filtered, washed with ethyl acetate (10 ml) and dried in vacuo to give 2.34 g (64% yield) of pure title imidazothiadiazole as an off white solid which is used as such for the next step. LC (Method E): 2.188 min. HRMS(ESI) calcd for C₂oHi₅BrN30₃S [M+H]⁺ m/z 456.00175, found 456.00397. 1H NMR (CDC1₃, 600 MHz) δ 3.82 (s, 3H), 5.16 (s, 2H), 6.38 (d, J= 1.67 Hz, 1H), 6.66 (broad s, 1H), 7.15 (s, 1H), 7.31 (broad t, 1H), 7.38 (broad t, 2H), 7.45 (broad d, 2H), 8.02 (s, 1H).

Alternatively, Example IF, 6-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2- bromoimidazo[2,l-b][l,3,4]thiadiazole, was prepared as follows:

A 1000-mL, three-necked flask equipped with a magnetic stirring bar and purged with a nitrogen atmosphere was charged with dry NMP (200 mL) followed by 1- (4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2-chloroethanone (Example 1EE, 50 g, 150 mmol) and 5-bromo-l,3,4-thiadiazol-2-amine (27.2 g, 151 mmol). The resulting mixture was stirred at 80 °C for 8h. TLC (8:2 dichloromethane/pet. ether) and LC/MS showed intermediate uncyclized material (m/z 476) and the reaction mixture was stirred at 120 °C for 3h. The reaction mixture was cooled to RT, quenched with water and extracted with EtOAc (3X). The combined organic layers were washed with brine, dried over Na₂S0₄, and concentrated in vacuo. The thick brown residue was purified by silica gel chromatography (0 to 100% dichloromethane in pet. ether) to give a brown solid. This material was triturated with EtOAc and dried to obtain the title imidazothiadiazole (24 g, 50 mmol, 33%>) as a light brown solid. (See the procedure set forth above for analytical data).

1 G. 6-(4-(Benzyloxy)-6-methoxybenzofuran-2-yl)-2-methoxyimidazo[2, 1 – b][l,3,4]thiadiazole

A solution of 6-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2- bromoimidazo[2,l-b][l,3,4]thiadiazole (Example IF, 2.30 g, 5.04 mmol) in a mixture of dichloromethane (180 mL) and methanol (45 mL) was treated at 22 °C with 4.2 mL of a 25 wt.% solution of sodium methoxide in methanol (0.2 mmol) added in one portion. More methanol (45 mL) was added and the mixture was stirred for 1 h. The reaction mixture was quenched by the addition of 25 mL of IN hydrochloric acid followed by 20 ml of saturated sodium bicarbonate. The solvent was evaporated under reduced pressure and the residue was diluted with dichloromethane (400 mL), washed with brine, dried over anhydrous magnesium sulfate and evaporated in vacuo. Chromatography of the residue on silica gel (3 x 10 cm, elution with dichloromethane – ethyl acetate 0-4%) gave 1.70 g (83% yield) of the title compound as a white solid. This material was recrystallized from ethyl acetate (30 mL per gram, 80% recovery) to give white needles. LC (Method

D): 2.293 min. HRMS(ESI) calcd for C₂₁H₁₈N₃O₄S [M+H]⁺ m/z 408.1013, found 408.1024. 1H NMR (CDC1₃, 600 MHz) δ 3.81 (s, 3H), 4.18 (s, 3H), 5.16 (s, 2H), 6.37 (d, J = 1.75 Hz, 1H), 6.67 (broad s, 1H), 7.07 (s, 1H), 7.31 (broad t, 1H), 7.37 (broad t, 2H), 7.45 (broad d, 2H), 7.81 (s, 1H).

1H. 6-Methoxy-2-(2-methoxyimidazo[2,l-b][l,3,4]thiadiazol-6-yl)benzofuran-4-ol

A mixture of 6-(4-(benzyloxy)-6-methoxybenzofuran-2-yl)-2- methoxyimidazo[2,l-b][l,3,4]thiadiazole (Example 1G, 1.250 g, 3.06 mmol) and pentamethylbenzene (3.17 g, 21.4 mmol) in dichloromethane (200 mL) was cooled to -78 °C under a nitrogen atmosphere and then treated immediately (to avoid crystallization) with 8 mL (8 mmol) of a 1 M solution of boron trichloride in dichloromethane added dropwise over 3 min. The resulting mixture was stirred at -78 °C for 1 h. The reaction mixture was then quenched by the addition of a solution of sodium bicarbonate (6 g) in water (100 mL) added in one portion. The cooling bath was removed and the resulting mixture was stirred at room temperature for 1 h. The solid formed was filtered, washed successively with water (50 m) and dichloromethane (50 mL). The filter cake was allowed to soak with anhydrous ethanol (15 ml) and then sucked dry. The white solid obtained was then dried under vacuum for 24 h to give 0.788 g (80%> yield) of pure title material (> 95% by hplc). The combined filtrate and washings were diluted with dichloromethane (600 mL) and stirred in a warm water bath till the organic phase was clear with no apparent solid in suspension. The organic phase was collected, dried over anhydrous magnesium sulfate and rapidly filtered while still warm. The filtrate was evaporated and the residue (product and pentamethylbenzene) was triturated with toluene (20 mL), the solid collected and washed with toluene (20 mL) to give 0.186 g (19% yield, 99% combined yield) of title material as a tan solid (> 95% by hplc). LC (Method E): 1.444 min. HRMS(ESI) calcd for C₁₄H₁₂N₃O₄S [M+H]⁺ m/z 318.0543, found 318.0578. 1H NMR (DMSO-de, 600 MHz) 5 3.71 (s, 3H), 4.16 (s, 3H), 6.21 (d, J = 1.87 Hz, 1H), 6.61 (broad s, 1H), 6.95 (s, 1H), 8.29 (s, 1H), 9.96 (s, 1H).

Example 94

4-(4-(((6-Methoxy-2-(2-methoxyimidazo[2,l-b][l,3,4]thiadiazol-6-yl)benzofuran-4-yl) oxy)methyl)-5-methylthiazol-2-yl)morpholine

94 A. Methyl 5-methyl-2-morpholinothiazole-4-carboxylate [00258] A solution of methyl 2-bromo-5-methylthiazole-4-carboxylate (2.80 g, 11.86 mmol) and morpholine (4.5 mL, 51.7 mmol) in THF (10 mL) was heated at reflux under nitrogen for 18 h. The volatiles were then removed under reduced pressure and the crude product was purified on the ISCO using a REDISEP® 40 g column (0 to 40% EtOAc- DCM), to give the title compound (2.20 g, 77%) as a yellow solid. LCMS (APCI): calcd for CioHisNzOsS [M+H]⁺ m/z 243.07, found 243.1. 1H NMR (CDC1₃, 400 MHz) δ ppm: 3.89 (s, 3H), 3.77-3.83 (m, 4H), 3.41-3.47 (m, 4H), 2.64 (s, 3H). [00259] Alternatively, Example 94A, methyl 5-methyl-2-morpholinothiazole-4- carboxylate, was prepared as follows:

94AA. Methyl 3-bromo-2-oxobutanoate

A 5L 4-neck round bottom flask equipped with a mechanical stirrer, temperature thermocouple, condenser and a 1L addition funnel, was charged copper(II) bromide (962 g, 4310 mmol) and ethyl acetate (2 L). A solution of methyl 2-ketobutyrate (250 g, 2150 mmol) in CHC1₃ (828 mL) was added dropwise. A scrubber (400 mL 1 N NaOH) was connected and the reaction mixture was heated to reflux (75 °C). The reaction started as a dark green color and as heating progressed, it became a light green with a white precipitate forming. NMR after one hour at reflux indicated that the reaction was complete. The reaction was cooled to RT and filtered through a pad of CELITE®. The filtrate was concentrated to an oil, dissolved in methylene chloride (500 mL) and filtered again through CELITE®. The filtrate was then passed through a pad of silica gel and eluted with ethyl acetate. Concentration of the filtrate provided the title bromoketoester (399 g, 2040 mmol, 95%) as a yellow oil. 1H NMR (400MHz, CDC1₃) δ 5.18 (q, J = 6.7 Hz, 1H), 3.94 (s, 3H), 1.83 (d, J = 6.8 Hz, 3H). 94AAA. Morpholine-4-carbothioamide

To a solution of morpholine (199 g, 2280 mmol) in CHC1₃ (1 L) was added isothiocyanatotrimethylsilane (150 g, 1140 mmol) dropwise. A white precipitate formed almost immediately, and the reaction was stirred for 1 h at RT. The reaction was then filtered and the resulting solid was washed with additional CHC1₃ and dried in vacuo to give the title thiourea as a white solid. (137 g, 937 mmol, 82%). 1H NMR (400MHz, DMSO-de) δ 3.81 – 3.71 (m, 2H), 3.17 – 3.08 (m, 2H).

94 A. Methyl 5-methyl-2-morpholinothiazole-4-carboxylate

To a solution of morpholine-4-carbothioamide (Example 94 AAA, 175 g, 1200 mmol) in methanol (500 mL) was charged methyl 3-bromo-2-oxobutanoate (Example 94AA, 233 g, 1200 mmol). The reaction was then heated to reflux for 1 hour, cooled to RT, and filtered. The filtrate was concentrated and the crude product was purified on by silica gel chromatography. The title thiazole (206g, 850 mmol, 71%) was isolated as a yellow oil. (See the procedure set forth above for analytical data).

(5-Methyl-2-morpholinothiaz l-4-yl)methanol

The compound was prepared according to the protocol described for Example 92B. The crude product was purified on the ISCO using a REDISEP® Gold 24 g column (0 to 50% EtOAc-DCM) to give the title compound as a white solid (0.086 g, 51%). LCMS (APCI): calcd for C₉Hi₅N₂0₂S [M+H]⁺ m/z 215.08, found 215.1. 1H NMR (CDCI3, 400 MHz) δ ppm: 4.48 (d, J= 4.7 Hz, 2H), 3.77-3.83 (m, 4H), 3.37-3.43 (m, 4H), 2.30 (t, J= 4.7 Hz, 1H), 2.28 (s, 3H).

Example 94. 4-(4-(((6-Methoxy-2-(2-methoxyimidazo[2, 1 -b] [ 1 ,3,4]thiadiazol-6-yl) benzofuran-4-yl)oxy)methyl)-5 -methylthiazol-2-yl)morpholine

The title compound was prepared according to the protocol described for Example 86. The crude product was purified on the ISCO using a REDISEP® 4 g column (0 to 40% EtOAc-DCM) and the obtained solid was suspended in MeOH, sonicated, filtered and dried to give the title compound as an off-white solid (0.094 g, 53%). LC (Method C): 2.314 min. HRMS(ESI): calcd for C₂₃H₂₄N₅0₅S₂ [M+H]⁺ m/z 514.122, found 514.126. 1H NMR (CDC1₃, 400 MHz) δ ppm: 7.83 (s, 1H), 7.06 (d, J = 0.8 Hz, 1H), 6.69 (d, J= 0.8 Hz, 1H), 6.50 (d, J= 2.0 Hz, 1H), 5.05 (s, 2H), 4.21 (s, 3H), 3.85 (s, 3H), 3.78- 3.84 (m, 4H), 3.39- 3.46 (m, 4H), 2.37 (s, 3H).

ABSTRACT

251st Am Chem Soc (ACS) Natl Meet (March 13-17, San Diego) 2016, Abst MEDI 263

////////BMS 986120, phase 1, Bristol-Myers Squibb ,  Imidazoles,  Small molecules,  Thiadiazoles, 1478712-37-6

c1(sc2nc(cn2n1)c3cc4c(cc(cc4o3)OC)OCc5nc(sc5C)N6CCOCC6)OC

CC1=C(N=C(S1)N2CCOCC2)COC3=C4C=C(OC4=CC(=C3)OC)C5=CN6C(=N5)SC(=N6)OC

,deletion of the 6-OH in the sugar moiety of dapagliflozin led to the discovery of a more potent SGLT2 inhibitor, 6-deoxydapagliflozin (1, ). In an in vitro assay, 1 was a more active SGLT2 inhibitor, with IC ₅₀ = 0.67 nM against human SGLT2 (hSGLT2), as compared with 1.1 nM for dapagliflozin, leading to the identification of 1 as the most active SGLT2 inhibitor discovered so far in this field. Also in an in vivo assay, 1 also introduced more urinary glucose in a rat urinary glucose excretion test (UGE) and exhibited more potent blood glucose inhibitory activity in a rat oral glucose tolerance test (OGTT) than dapagliflozin.

 Tianjin Institute Of Pharmaceutical Research,天津药物研究院

SPECTRAL DATA of Tianagliflozin

1 as a white solid (3.65 g, 93 %). R _f = 0.35 (EtOAc);

m.p.: 148–149 °C;

¹H NMR (400 MHz, DMSO-d ₆): δ = 7.35 (d, 1H, J = 8.4 Hz), 7.25 (s, 1H), 7.18 (d, 1H, J = 8.0 Hz), 7.08 (d, 2H, J = 8.4 Hz), 6.81 (d, 2H, J = 8.4 Hz), 4.95 (d, 1H, J = 5.2 Hz, OH), 4.90 (d, 1H, J = 4.4 Hz, OH), 4.79 (d, 1H, J = 5.6 Hz, OH), 3.92–4.01 (m, 5H), 3.24–3.29 (m, 1H), 3.18–3.22 (m, 1H), 3.09–3.15 (m, 1H), 2.89–2.95 (m, 1H), 1.29 (t, 3H, J = 7.0 Hz, CH₂ CH ₃ ), 1.15 (d, 3H, J = 6.0 Hz, CHCH ₃ ) ppm;

¹³C NMR (100 MHz, DMSO-d ₆): δ = 156.85, 139.65, 137.82, 131.83, 131.16, 130.58, 129.52, 128.65, 127.14, 114.26, 80.71, 77.98, 75.77, 75.51, 74.81, 62.84, 37.55, 18.19, 14.62 ppm;

IR (KBr): v¯¯¯ = 3,564, 3,385 (s), 2,981 (s), 2,899 (s), 2,861 (s), 1,613 (m), 1,512 (s), 1,477 (m), 1,247 (s), 1,102 (s), 1,045 (s), 1,012 (s) cm⁻¹;

HR–MS: calcd for C₂₁H₂₉ClNO₅ ([M + NH₄]⁺) 410.1729, found 410.1724.

PATENT

 CN 103864737

http://www.google.com/patents/CN103864737A?cl=en

PATENT

WO 2014094544

http://www.google.com/patents/WO2014094544A1?cl=en

-27-

1 D1 -6 Optionally, the step (7 ‘) is the step (7’) in place:

LS l- [4 – D (I- Dl- 6)

A.

(DMSO-d _6, 400 MHz), δ 7.35 (d, 1H, J = 8.0 Hz), 7.28 (d, 1H, J ‘. 2.0 Hz), 7.17 (dd, IH, / = 2.0 Hz and 8.4 Hz), 7.05 (d, 2H, _J: 8.8 Hz), 6.79 (d, 2H, 8.8 Hz): 4.924,95 (m, 2H), 4,81 (d, IH, 6,0 Hz), 3.93- 3.99 (m, 5H), 3,85 (d, 1H, J = 10,4 Hz), 3,66 (dd, IH, 5,2 Hz and 11,6 Hz), 3.17-3,28 (m, 3H), 3.02-3.08 _(m: IH), 1.28 (t, 3H, J = 7,0 Hz), 0,80 (s, 9H), -0.05 (s, 3H), -0.09 (s, 3H) .

PATENT

CN 104045614

[0066] The added 100mL dried over anhydrous methanol 0. 5g of sodium metal, nitrogen at room temperature with stirring, until the sodium metal disappeared. Followed by addition of 5. 2g (10mmol) of compound 6, stirring was continued at room temperature for 3 hours. To the reaction system was added 5g strong acid cation exchange resin, stirred at room temperature overnight, the reaction mixture until pH = 7. The resin was removed by suction, and the filtrate evaporated to dryness on a rotary evaporator, the residue was further dried on a vacuum pump to give the product I-D1-6, as a white foamy solid.

PATENT

 WO 2014139447

PATENT related

http://www.google.com/patents/WO2013044608A1?cl=en

http://link.springer.com/article/10.1007%2Fs40242-014-4043-9#/page-1

Design of SGLT2 Inhibitors for the Treatment of Type 2 Diabetes: A History Driven by Biology to Chemistry.

http://www.ncbi.nlm.nih.gov/pubmed/25557661

Paper

Discovery of 6-Deoxydapagliflozin as a Highly Potent Sodium-dependent Glucose Cotransporter 2 (SGLT2) Inhibitor for the Treatment of Type 2 Diabetes

Authors: Zhang, Lingyu; Wang, Yuli; Xu, Huaqiang; Shi, Yongheng; Liu, Bingni; Wei, Qunchao; Xu, Weiren; Tang, Lida; Wang, Jianwu; Zhao, Guilong

Source: Medicinal Chemistry, Volume 10, Number 3, May 2014, pp. 304-317(14)

http://www.ingentaconnect.com/content/ben/mc/2014/00000010/00000003/art00009?crawler=true

CLIP

A facile synthesis of 6-deoxydapagliflozin

The synthetic route to the target compound 1 is shown in Scheme 3. The starting material methyl 2,3,4-tri-O-benzyl-6-deoxy-6-iodo-α–d-glucopyranoside (3) was prepared from commercially available methyl α–d-glucopyranoside (2) according to a known method [5, 6].

Iodide 3 was reductively deiodinated to give 4 in 91 % yield under hydrogenolytic conditions using 10 % Pd/C as catalyst in the presence of Et₃N as base in THF/MeOH at room temperature.

when the iodide 3 was treated with Barton–McCombie reagent (n-Bu₃SnH/AIBN) [7] in toluene at room temperature no reaction occurred; however, when the reaction was carried out at elevated temperatures, such as reflux, a complex mixture formed with only a trace amount (3 %, entry 1) of the desired product 4.

When the iodide 3 was treated with LiAlH₄ in THF at 0 °C to room temperature, another complex mixture was produced with only a trace amount (2 %, entry 2) of 4.

When Pd(OH)₂ was used as the hydrogenolysis catalyst instead of 10 % Pd/C, the desired 4 was indeed formed (14 %, entry 4), but most of the starting material was converted to a few more polar byproducts, which were believed to result from the cleavage of at least one of the benzyl groups.

pdf available

Monatshefte für Chemie – Chemical Monthly

December 2013, Volume 144, Issue 12, pp 1903-1910

http://download.springer.com/static/pdf/721/art%253A10.1007%252Fs00706-013-1053-0.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs00706-013-1053-0&token2=exp=1458808857~acl=%2Fstatic%2Fpdf%2F721%2Fart%25253A10.1007%25252Fs00706-013-1053-0.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs00706-013-1053-0*~hmac=bd1c3c2bdc3712f5540267c99f732b2f7588020a868aa23021792a2a2a58d65e

////////IND Filing, SGLT-2 inhibitor, type 2 diabetes, Tianagliflozin, taigeliejing, 6-deoxydapagliflozin, 1461750-27-5

Clc1c(cc(cc1)C2[C@@H]([C@H]([C@@H]([C@H](O2)C)O)O)O)Cc3ccc(cc3)OCC

Avik Das | TNN | Mar 28, 2016, 02.52 PM IST

http://timesofindia.indiatimes.com/business/india-business/Biocons-Insulin-Glargine-gets-approval-in-Japan/articleshow/51583333.cms

BENGALURU: Biopharmaceutical company Biocon said it got approval from Japan’s health ministry to sell its biosimilar Insulin Glargine in the country.

The product, which is a ready-to-use, prefilled disposable pen with 3 ml of 100IU Insulin Glargine, is expected to be launched in Japan in the first quarter of 2017 with its commercial partner FUJIFILM Pharma Co. Ltd, Biocon said on Monday.

The move will help Biocon capture a significant share of the Japanese Glargine market, which is about $144 million and second largest market outside of North America & Europe.
“The Insulin Glargine approval in the highly regulated market like Japan, marks a huge credibility milestone for Biocon. We see this as a significant achievement in our journey of making global impact in diabetes management through our affordable biosimilar insulins,” chairperson and managing director Kiran Mazumdar-Shaw said.

Kiran Mazumdar–Shaw

Biosimilars are biologic products, made inside living cells and has no clinical differences in terms of safety and effectiveness from the main product. They are however not considered duplicates, like generics, by regulators as it is impossible to manufacture exact copies of biotech drugs.

Biocon Limited

Type	Public company
Traded as	BSE: 532523 NSE: BIOCON
Industry	Biotechnology
Founded	1978
Founder	Kiran Mazumdar-Shaw
Headquarters	Bangalore, Karnataka, India
Key people	Kiran Mazumdar-Shaw, (Chairman & MD)
Products	Pharmaceuticals Enzymes
Revenue	₹22.41 billion (US$330 million) (2014–15)[1]
Net income	₹3.61 billion (US$54 million) (2014–15)
Number of employees	5,585 (Mar 2011)[1]
Subsidiaries	Syngene Clinigene
Website	www.biocon.com

//////Biocon,  Insulin Glargine, approval,  Japan

TRIM24/BRPF1 bromodomain inhibitor

IACS-9571; IACS 9571; IACS9571.

N-[6-[3-[4-(dimethylamino)butoxy]-5-propoxyphenoxy]-1,3-dimethyl-2-oxobenzimidazol-5-yl]-3,4-dimethoxybenzenesulfonamide

BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM

IACS-9571 is a potent and selective inhibitor TRIM24 and BRPF1. The bromodomain containing proteins TRIM24 (Tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). IACS-9571 has low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), IACS-9571 is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo. (J Med Chem. 2015 Jun 10. [Epub ahead of print] )

PAPER

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b00405

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC K_d = 31 nM and ITC K_d = 14 nM, respectively). With its excellent cellular potency (EC₅₀ = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

TFA salt of 8i (106 mg, 57%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12–3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77–1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2, 126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9, 66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI) m/z 644 [M + H]+.

NMR

IACS -9571

CLICK ON IMAGE

ABSTRACT

251st ACS National Meeting & Exposition

MEDI 5

Discovery and development of a potent dual TRIM24/BRPF1 bromodomain inhibitor, IACS -9571, using structure- based drug design Wylie S. Palmer 1 , wpalmer@mdanderson.org, Guillaume Poncet -Montagne 1 , Gang Liu 1 , Alessia Petrocchi 1 , N aphtali Reyna 1 , Govindan Subramanian 1 , Jay Theroff 1 , Maria Kost -Alimova 1 , Jennifer Bardenhagen 1 , Elisabetta Leo 1 , Hannah Sheppard 1 , Trang Tieu 1 , Shi Xi 1 , Yanai Zhan 1 , Shuping Zhao 1 , Michelle Barton 2 , Giulio Draetta 1 , Carlo Toniatti 1 , Philip Jones 1 , Mary Ge ck Do 1 , Jannik Andersen 1 . (1) Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States (2) Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

Bromodomains are an important class of chromatin remodeling proteins that recognize acetylated lysine residues on histone tails. As epigenetic targets they regulate gene transcription and offer a new way to treat diseas es, particularly in inflammation and oncology. The bromodomain and extra- terminal (BET) family has emerged as an important and druggable example of this class of proteins with the successful entry of small- molecule inhibitors into the clinic. Other families of bromodomains are only starting to be explored, such as the Tripartite Motif -containing 24 protein (TRIM24) and bromodomain- PHD finger protein 1 (BRPF1). Both proteins contain a dual PHD -bromo motif which have a role in recognizing specific histone mar ks. TRIM24 recognizes the dual histone marks of unmodified H3K4 and acetylated- H3K23 within the same histone tail. TRIM24 is a potent co- activator of ER -alpha and overexpression of TRIM24 has been linked to poor survival rates in breast cancer patients.

This presentation will describe the structure guided development of a series of N,N- dimethyl -benzimidazolones through the iterative use of X -ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor (IACS -9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC 50 = 50 nM) and favorable pharmacokinetic properties, IACS -9571 is a high- quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo

PATENT

WO-2016033416-A1

Synthesis of Intermediates:

N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide (Intermediate 1):

Step 1 : 5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 4-nitrobenzene- 1 ,2-diamine (44 g, 285 mmol) in 80 mL of DMF was added l, l’-carbonyldiimidazole (70 g, 428 mmol). The reaction mixture was stirred at RT for 4 h, then water (250 mL) was added. The resulting suspension was filtered, and the collected solids were washed with water (200 mL) and dried to give 5-nitro-lH- benzo[d]imidazol-2(3H)-one as a yellow solid (45 g, 88%). MS (ES+) C7H5N3O3 requires: 179, found: 180 [M+H]⁺.

Step 2: l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a solution of 5-nitro-lH-benzo[d]imidazol-2(3H)-one (55 g, 309 mmol) in 150 mL of DMF was added K2CO3 (85 g, 618 mmol), the reaction mixture was cooled to 0 °C, then iodomethane (109 g, 772 mmol) was slowly added. The reaction mixture was stirred at RT overnight, then water was added to the reaction mixture. The resulting suspension was filtered and the collected solids were washed with water (200 mL) and dried to give 1,3- dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one as a yellow solid (55 g, 86%). MS (ES+) C9H9N3O3 requires: 207, found: 208 [M+H] ⁺.

Step 3: 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

 To a solution of l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one (50 g, 240 mmol) in 200 mL of EtOAc under an inert atmosphere was added 10% palladium on activated carbon (5 g, 24 mmol). The reaction mixture was then charged with hydrogen and stirred at RT under an ¾ atmosphere overnight. The reaction mixture was filtered through a pad of celite then concentrated to give 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)- one as a yellow solid (32 g, 68%). MS (ES+) C9H11N3O requires: 177, found: 178 [M+H]⁺.

Step 4: 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

 To a 0 °C solution of 5-amino-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (4 g, 22.6 mmol) in 25 mL of CHCI3 and 25 mL of AcOH was slowly added drop wise bromine (3.5 g, 22.6mmol). The mixture was stirred at RT for 30 min, then concentrated and purified by silica gel chromatography (1 : 1 EtOAc/ hexanes) to afford 5-amino-6-bromo-l ,3-dimethyl- lH-benzo[d]imidazol-2(3H)-one as a yellow solid (3.2 g, 69%). MS (ES+) C₉HioBrN₃0 requires: 256, found: 257 [M+H]⁺.

Step 5: N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide:

To a 0 °C solution of 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (1.50 g, 5.9 mmol) in DCM (45 ml) was added DMAP (72 mg, 0.59 mmol), triethylamine (1.63 ml, 11.7 mmol) and trifluoroacetic anhydride (0.91 ml, 6.4 mmol). The reaction mixture was stirred for 2 h and warmed to RT. The reaction mixture was then quenched with water and the organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated to give N-(6-bromo-l,3-dimethyl-2-oxo-2,3-dihydro-lH- benzo[d]imidazol-5-yl)-2,2,2-trifluoroacetamide (Intermediate 1) as a yellow solid (2.20 g, 100%). MS (ES+) CiiH₉BrF₃N302 requires: 352, found 353 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route A):

To a mixture of 5-amino-6-(3-(benzyloxy)phenoxy)-l,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one (400 mg, 1.07 mmol) in DCM (20 mL) at -78 °C was added tribromoborane (5.3 mL, 5.3 mmol). The mixture was warmed up to room temperature gradually, then quenched by methanol dropwise, concentrated, and purified by column chromatography (20-100% EtOAc/hexanes and then 0-40% methanol/EtOAc) to give 5- amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one as a solid (240 mg, 79%). MS (ES+) C15H15N3O3 requires: 285, found: 286 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route B):

Step 2

Step 1: 3-[(ieri-butyldimethylsilyl)oxy]phenol:

A mixture of lH-imidazole (2.25 g, 33.1 mmol), ieri-butylchlorodimethylsilane (3.83 g, 25.4 mmol) and resorcinol (5.6 g, 51 mmol) in THF (30 ml) was stirred at 80 °C for 5 h. The resulting suspension of the cooled reaction mixture was filtered and the collected filtrate was concentrated and purified by silica-gel chromatography (20:80 to 0:100, EtOAc/hexanes) to give 3-((ieri-butyldimethylsilyl)oxy)phenol (2.78 g, 49%). MS (ES+) C12H20O2S1 requires: 224, found 225 [M+H]⁺.

Step 2: 5-amino-6-(3-((ier^butyldimethylsilyl)oxy)phenoxy)-l ,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one:

 A mixture of 3-((ieri-butyldimethylsilyl)oxy)phenol (1.39 g, 6.20 mmol), quinolin-8-ol (79 mg, 0.55 mmol), copper(I) chloride (20 mg, 0.21 mmol), potassium phosphate (526 mg, 2.48 mmol) and 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (529 mg, 2.07 mmol) in diglyme (20 ml) in a 100 mL round-bottom flask was degassed under a nitrogen atmosphere and heated to 120 °C for 24 h. To the cooled reaction mixture was added silica gel, stirred for 2 min, then the mixture was filtered through a pad of silica gel. The collected filtrate was concentrated and purified by column chromatography (20:80 to 0: 100, EtOAc/hexanesthen 0: 100 to 40:60, MeOH/EtOAc) to give 5-amino-6-(3- ((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (521 mg, 63%). MS (ES+) C21H29N3O3S1 requires: 399, found 400 [M+H]⁺.

Step 3: 5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 5-amino-6-(3-((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3- dimethyl-lH-benzo[d]imidazol-2(3H)-one (623 mg, 1.56 mmol) in THF was added a solution of ieira-butylammonium fluoride (0.90 mL, 3.1 mmol) in THF, the reaction mixture was allowed to warm up to RT and then stirred for 1-2 h. The reaction mixture was quenched with 1 M hydrogen chloride (0.10 mL, 3.1 mmol) and then partitioned between EtOAc and water. The seperated organic layer was washed with water twice, then concentrated and purified by column chromatography (20-80% EtOAc/hexanes and 0-40% MeOH/DCM) to give 5-amino-6-(3-hydroxyphenoxy)-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (120 mg, 27%) as a solid. MS (ES+) C15H15N3O3 requires: 285, found 286 [M+H]⁺.

EXAMPLE 10: N-(6-(3-(4-(dimethylamino)butoxy)-5-propoxyphenoxy)-l,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2-

To a solution of N-(6-(3-(4-aminobutoxy)-5-propoxyphenoxy)-l ,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2- trifluoroacetate (180 mg, 0.247 mmol) in methanol (3.0 ml) was added triethylamine (0.034 ml, 0.25 mmol), acetic acid (0.028 ml, 0.49 mmol), formaldehyde (0.054 ml, 2.0 mmol), and sodium triacetoxyborohydride (131 mg, 0.618 mmol). The reaction mixture was stirred at room temperature and checked by LCMS every 30 minutes. After 3 h the reaction was complete by LCMS. The reaction was quenched with a few drops of TFA and concentrated under reduced pressure. The residue was purified by prep-HPLC using a gradient of 20-60% ACN/water containing 0.1% TFA to afford N-(6-(3-(4-(dimethylamino)butoxy)-5- propoxyphenoxy)-l,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4- dimethoxybenzenesulfonamide 2,2,2-trifluoroacetate (106 mg, 57%) as a white solid. MS (ES+) C32H42N4O8S requires: 642, found 643 [M+H]⁺. ¾ NMR (600 MHz, DMSO-ifc) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, 7 = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, 7 = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, 7 = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12-3.05 (m, 2H), 2.78 (d, 7 = 4.7 Hz, 6H), 1.77-1.63 (m, 6H), 0.95 (t, 7 = 7.3 Hz, 3H)

References

1: Palmer WS, Poncet-Montange G, Liu G, Petrocchi A, Reyna N, Subramanian G, Theroff J, Yau A, Kost-Alimova M, Bardenhagen JP, Leo E, Shepard HE, Tieu TN, Shi X, Zhan Y, Zhao S, Draetta G, Toniatti C, Jones P, Geck Do M, Andersen JN. Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor. J Med Chem. 2015 Jun 10. [Epub ahead of print] PubMed PMID: 26061247.

US-20160060260-A1

Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

The University of Texas MD Anderson Cancer Center | University of Texas System

The new Institute for Applied Cancer Science will be located at the south campus of M.D.

Draetta arrived at MD Anderson in 2011 to direct the Institute for Applied Cancer Science. He oversees the moon shots platforms

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

///////IACS-9571, TRIM24, BRPF1 bromodomain inhibitor, IACS-9571,  IACS 9571,  IACS9571, BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM
CAS BASE 1800477-30-8
CAS OF 1:1 TRIFLUOROACETATE 1883598-69-3

c1(cc(cc(c1)OCCC)Oc3cc2N(C(N(c2cc3NS(=O)(=O)c4cc(c(cc4)OC)OC)C)=O)C)OCCCCN(C)C

CCCOC1=CC(=CC(=C1)OC2=C(C=C3C(=C2)N(C(=O)N3C)C)NS(=O)(=O)C4=CC(=C(C=C4)OC)OC)OCCCCN(C)C

TFA salt of 8i (106 mg, 57%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12–3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77–1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2, 126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9, 66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI) m/z 644 [M + H]+.

Gly-Pro-Glu

Synonym: GPE, Glycyl-prolyl-glutamic acid, (1-3)IGF-1

Pfizer (Originator)
Neuren Pharmaceuticals (Originator)

Glypromate; glycine-proline-glutamate (neuroprotectant), Neuren

• CAS Number 32302-76-4
• Empirical Formula C₁₂H₁₉N₃O₆
• Molecular Weight 301.30
• Psychiatric Disorders (Not Specified)
  Neurologic Drugs (Miscellaneous)
  Cognition Disorders, Treatment of
  Antiepileptic Drugs
  Antidepressants Biochem/physiol Actions

Gly-Pro-Glu is a neuroprotective compound and the N-terminal tripeptide of IGF-1. Gly-Pro-Glu is neuroprotective after central administration in animal models of neurodegenerative processes, such as Huntington’s, Parkinson’s, Alzheimer’s diseases, and varies acute brain injury animal models. The neuroprotective activity is not related to its affinity to glutamate receptor. Findings indicate that GPE mimics insulin-like growth factor I effects on the somatostatin system through a mechanism independent of β-amyloid clearance that involves modulation of calcium and glycogen synthase kinase 3β signaling.

GPE is a naturally occurring peptide fragment which had been in phase III clinical trials at Neuren Pharmaceuticals for use as prophylactic neuroprotection for patients undergoing coronary artery bypass graft (CABG) and valvuloplasty surgery. Although clinical evaluation in Australia continues, phase III trials evaluating the compound in the U.S. were discontinued based on negative results. The compound is found in normal brain tissue and, when injected intravenously, has been shown to act by multiple pathways to protect brain tissue from injury. The drug was originally developed by Pfizer, but rights were transferred to Neuren pursuant to a proprietary agreement between the companies.

The tripeptide Glycyl-L-Prolyl-L-Glutamate (Gly-Pro-Glu or GPE) is a naturally occurring peptide, which is proteolytically cleaved from insulin-like growth factor-1 (IGF-1). IGF-1 is a potent neurotrophic factor produced endogenously in damaged regions of the brain. It has been postulated that some of the neuroprotective actions of IGF-1 are mediated by GPE although the precise mechanism of action remains unclear. GPE has a different mode of action to IGF-1 as GPE does not bind to the IGF-1 receptor. Rather, GPE has been shown to bind with low affinity to the N-methyl-D-aspartate (NMDA) receptor and also elicit a biological response via other mechanisms. GPE facilitates the release of dopamine through interaction with the NMDA receptor but GPE stimulated acetylcholine release is via an unknown, non-NMDA pathway.

It has been demonstrated that GPE can act as a neuronal rescue agent following brain injury or disease, including hypoxic-ischemic brain injury, NMDA challenge, chemical toxins and in animal models of Parkinson’s and Alzheimer’s disease. Analogs of GPE are thus of interest in the development of novel pharmaceutical agents for the treatment of central nervous system (CNS) injuries and neurodegenerative disorders among others.

CURRENT STATUS

SYDNEY, Australia, Sept. 4 /PRNewswire-FirstCall/ — Neuren Pharmaceuticals today announced that physicians from Madigan Army Medical Center (Madigan) in Tacoma, Washington, will conduct an investigator- initiated Phase 2 trial to determine the safety and efficacy of Glypromate(R) in reducing brain injury caused by out of hospital cardiac arrest. The trial will start in mid-2007 and will be managed by The Henry M. Jackson Foundation for the Advancement of Military Medicine (Jackson Foundation) in consultation with the clinical investigators at Madigan.

The proposed study will be an investigator-initiated study which means that the Investigational New Drug (IND) application will be submitted to the FDA by the Army investigators rather than by Neuren. Neuren will provide the drug product as well as access to preclinical, clinical and regulatory documents related to Glypromate(R). The Company’s only financial commitment will be compensation to the Jackson Foundation for administrative costs incurred in coordinating the study. Neuren will retain all commercial rights to Glypromate(R) in these indications.

Cardiac arrest involves the sudden, complete cessation of heart function and circulation leading rapidly to neurological and other organ system damage. Among patients who survive, the consequences of neurological damage resulting from lack of blood flow and oxygen to the brain represent the primary adverse outcomes. This occurs in up to 80% of survivors and causes cognitive impairment such as occurs in patients undergoing major cardiac surgery, the focus for Neuren’s upcoming Phase 3 study with Glypromate(R). However recovery without residual neurological damage after cardiac arrest is rare.

There are no drugs approved to reduce the neurological damage caused by cardiac arrest. Neuren believes that Glypromate(R) for this indication will be eligible for Orphan Drug designation. Orphan Drug designation provides for a period of market exclusivity following approval as well as possible access to US government grants. In addition, because of the serious nature of neurological impairment resulting from cardiac arrest and the lack of available drug therapy, Neuren intends to apply for Fast Track designation which provides for accelerated clinical development and review.

While the Army’s investigator-initiated trial will focus on out of hospital cardiac arrest, if this trial is successful, Neuren, the Jackson Foundation and the Army investigators are considering additional trials of Glypromate(R) to reduce brain damage resulting from related conditions including in-hospital cardiac arrest and treatment of patients with ventricular fibrillation, the heart rhythm disturbance associated with more than 75% of cardiac arrests.

Under the agreement, the Jackson Foundation will provide support to the Army investigators in clinical trial preparations, protocol development, obtaining human subjects clearance, coordination of patient enrolment, data management and analysis, and preparation of study reports.

Mr David Clarke, CEO of Neuren said: “This is a very important development for Neuren in that it reflects a growing appreciation of the potential for Glypromate(R) to reduce neurological damage. It also, of course, reinforces the value and strength of Neuren’s relationship with the US Army physicians and scientists. Cardiac arrest is a devastating clinical event and one for which a drug to reduce the neurological consequences is clearly needed. The addition of this trial will now give Neuren a very strong and cost effective portfolio of clinical trials in 2007 — a Phase 3 and a Phase 2 for Glypromate(R) and the two Phase 2 trials with NNZ-2566.”

Approximately 300,000 deaths result from cardiac arrest in the US each year, making cardiac arrest one of the leading causes of death. According to the American Heart Association, each year approximately 160,000 people in the US experience sudden cardiac arrest outside of a hospital or in a hospital emergency department.

Neuren estimates that the number of patients in the US that could be treated for out of hospital cardiac arrest and related indications is approximately 400,000 which could represent a potential market of US$800 million.

About Madigan Army Medical Center

Madigan Army Medical Center, located in Tacoma, Washington, is one of the major US Army medical centers, providing clinical care to over 120,000 active, reserve and retired military personnel and dependents. The hospital has a medical staff of more than 1,000 with 200 physicians and nurses in training. Madigan’s Department of Clinical Investigations, which is dedicated to writing, performing, and regulating clinical research, is conducting approximately 200 clinical trials across a wide spectrum of indications from Phase I to IV.

About the Jackson Foundation

The Jackson Foundation is a private, not-for-profit organisation that supports the US military in conducting medical research and clinical trials and has established relationships with more than 160 military medical organisations worldwide. It was founded in 1983, in part, to foster cooperative relationships between the military medical community and the private sector, including pharmaceutical sponsors. The Jackson Foundation manages Phase I – IV clinical trials utilizing an established network of military medical centers across the United States.

About Glypromate(R)

Glypromate(R) is a peptide fragment of IGF-1 and is being developed by Neuren as a potential therapeutic candidate for diseases caused by some forms of chronic or acute brain injury. Glypromate(R) has been shown to act by multiple pathways to protect brain tissue from injury. Neuren has successfully completed a Phase I safety study and a Phase IIa safety and pharmacokinetics study and plans to initiate a Phase III study in late 2006.

About Neuren Pharmaceuticals

Neuren Pharmaceuticals is a biotechnology company developing novel therapeutics in the fields of brain injury and diseases and metabolic disorders. The Neuren portfolio consists of six product families, targeting markets with large unmet needs and limited competition. Neuren has three lead candidates, Glypromate(R) andNNZ-2566, presently in the clinic in development to treat a range of acute neurological conditions, and NNZ-2591, in preclinical development for Parkinson’s and other chronic conditions. Neuren has commercial and development partnerships with the US ArmyWalter Reed Army Institute of Research, Metabolic Pharmaceuticals,UCLA Medical Center and the National Trauma Research Institute in Melbourne.

For more information, please visit Neuren’s website at http://www.neurenpharma.com

Company David Clarke CEO of Neuren T: 1800 259 181 (Australia) T: +64 9 3 367 7167 ext 82308 (New Zealand) M: +64 21 988 052 Media and investor relations Rebecca Piercy Buchan Consulting T: +61 9827 2800 M: +61 422 916 422

CONTACT: David Clarke, CEO of Neuren, 1-800-259-181(Australia), or
+64-9-3-367-7167 ext 82308 (New Zealand), or +64-21-988-052 (mobile); or
Media and investor relations – Rebecca Piercy of Buchan Consulting,
+61-9827-2800, +61-422-916-422 (mobile)

Web site: http://www.neurenpharma.com/

REFERENCES

1 EP 0366638

2 WO 2005042000

3 WO 2008153929

4 WO 2009033805

5 WO 2009033806

Synthesis off isotopically labelled glycyl-L-prolyl-L-glutamic acid (Glypromate(R)) and derivatives
J Label Compd Radiopharm 2006, 49(6): 571

An efficient fmoc solid-phase synthesis of an amphiphile of the neuroprotective agent glycyl-prolyl-glutamic acid
Synlett (Stuttgart) 2014, 25(15): 2221

Intracellular pathways activated by Insulin-like growth factor 1 and its derivates
40th Annu Meet Soc Neurosci (November 13-17, San Diego) 2010, Abst 167.13

//////Gly-Pro-Glu, GPE, Glycyl-prolyl-glutamic acid,  32302-76-4, Tripeptide,  Glycyl-L-Prolyl-L-Glutamate, Glypromate®, (1-3)IGF-1 , PHASE 3, Glypromate,  glycine-proline-glutamate, neuroprotectant, Neuren

Neuren’s NNZ-2566 shows clinical benefit in Rett syndrome trial

Promising results in Phase 2 clinical trial

This isn’t a Fragile X trial, but the Neuren compound, NNZ-2566, that is in trials now for Fragile X has shown significant positive effects in a Phase 2 trial for Rett syndrome.

The results of the trial are interesting, in that improvement was seen a Rett syndrome-specific rating scale compared to placebo, and there was also improvement noted on the CGI-I (Clinical Global Impression of Improvement) and Caregiver Top 3 Concerns. However, there was no effect seen on ABC scores (Aberrant Behavior Checklist) compared to placebo. Many in the Fragile X field have noted the inadequacies of the ABC; indeed, it was never designed or intended to be an outcome measure for clinical trials. In this case, a Rett-specific rating scale called the Motor-Behavior Assessment (MBA) showed a statistically significant and clinically meaningful treatment effect at the highest dose of the Neuren compound compared to placebo.

This is great news for those of us in the Fragile X community for several reasons:

• It shows that this compound really does something—it seems to have useful properties in actual patients, and that’s not trivial.
• It demonstrates that disease-specific symptoms can improve significantly on the drug, and that improvement can be measured in a relatively short clinical trial.
• It shows that a drug can have beneficial effects on core features of a genetically based developmental disorder, even if the more general rating scales (like the ABC) show no change.

–
This last point is strongly reminiscent of the experience of many families and clinicians in recent Fragile X clinical trials, where the drugs showed no advantage compared to placebo based on rating scales, but genuine improvement was noted in many subjects, with significant deterioration upon discontinuation of the drugs. Thus the calls for improved rating scales which can “capture” these core, disease-specific therapeutic effects. The NeurenFragile X trial is using some Fragile X-specific outcome measures which will hopefully lead to similar positive results.

The fact that this result is good news for Neuren also means that the company should remain financially viable for longer, so that they can continue the development of this compound for a number of indications—more “shots on goal”.

Of course, the usual caveats apply: this was a small study, and these results need to be replicated in a larger Phase 3 trial. Still, there’s a realistic possibility that we may see a similar result in Fragile X!

DS-1040

Daiichi Sankyo Co Ltd

Ischemic stroke

(2S)-5-amino-2-[[1-(4-methylcyclohexyl)imidazol-4-yl]methyl]pentanoic acid

1H-​Imidazole-​4-​propanoic acid, α-​(3-​aminopropyl)​-​1-​(trans-​4-​methylcyclohexyl)​-​, (αS)​-

(2S)-5-amino-2-{[1-(trans-4-methylcyclohexyl)-1H-imidazol-4-yl]methyl}pentanoic acid

free form cas 1335138-62-9

1:1 TOSYLATE 1335138-89-0

1335138-90-3  1:1:1 TOSYLATE HYDRATE

phase 2, Ischemic stroke

TAFIa inhibitors, useful for treating myocardial infarction, angina, pulmonary hypertension and deep vein thrombosis.

In March 2016, DS-1040 was reported to be in phase 2 C clinical development, and the study was expected to complete in June 2017.

https://clinicaltrials.gov/ct2/show/NCT02560688

• 01 Feb 2016Daiichi Sankyo initiates a phase I trial in Healthy volunteers in United Kingdom (NCT02647307)
• 09 Jan 2016Daiichi Sankyo plans a phase I trial in Healthy volunteers in United Kingdom (NCT02647307)
• 29 Sep 2015Daiichi Sankyo plans a drug-interaction phase I trial in Healthy volunteers in United Kingdom (IV) (NCT02560688)

SYNTHESIS

COMPLETE SYNTHESIS

Patent

WO201111506

PATENT

WO2013039202

PATENT

WO 2016043254

PATENT

WO2016043253

COMPLETE SYN……….

WO2016043253

(2S)-5-amino-2 – Optical purity measurement conditions {[1- (trans-4- methylcyclohexyl)-lH-imidazol-4-yl] methyl} valerate p- toluenesulfonate (A5 Step) :
column: CHIRLCEL OZ-H 4.6mmI. D. × 250mm (5μm),
mobile phase: hexane / ethanol / methanol / isopropanol / trifluoroacetic acid / triethylamine = 860/100/20/2/2
temperature: 30 ℃
flow rate: 1.0mL / min
detection method: UV at 220nm
retention time: R body: 16.1 minutes, S body: 13.0 minutes 　(example 　1) (1-1) 5 – [(Tert- butoxycarbonyl) amino] -2-methoxy-carbonyl) valeric acid morpholine salt

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¹ H-NMR _{(DMSO-d- . 6} ) [delta]: 6.79 (1H, t, J = 5.5 Hz), 3.61 (4H, t, J = 4.9 Hz), 3.58 The (3H, s) , 3.14 (1H, t, J = 7.8Hz), 2.90-2.80 (6H, m), 1.74-1.59 (2H, m), 1.37 (9H, s) , 1.34-1.25 (2H, m).

[Of 12]

(1-3) (2E) -5 – [(tert- butoxycarbonyl) amino] -2 – {[1-trans-4- methylcyclohexyl]-lH-imidazol-4-yl} methylidene} methyl valerate

[Of 13]

[Of 14]

[Of 15]

[Of 16]

[Of 17]

PATENT

Daiichi Sankyo Company,Limited, 第一三共株式会社

WO2011115064…..

http://www.google.co.in/patents/WO2011115064A1?cl=en

[Reference Example 1] 5 – [(tert- butoxycarbonyl) amino] -2- (diethoxyphosphoryl) valeric acid tert- butyl

Diethylphosphonoacetate tert- butyl (20.0g) was dissolved in tetrahydrofuran (500mL), sodium hydride (63%, 3.32g) was added at 0 ℃, 15 min at 0 ℃, and stirred for 1 hour at room temperature . (3-bromopropyl) tetrahydrofuran carbamic acid tert- butyl (20.0g) (20mL) was slowly at room temperature, and the mixture was stirred at room temperature for 18 hours. A saturated aqueous solution of ammonium chloride was added to the reaction solution, the organic matter was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to give the solvent was distilled off under reduced pressure the crude product. This silica gel column chromatography and purified (eluent hexane / ethyl acetate = 1/1-ethyl acetate) to give the title compound (26.6g).
¹ H-NMR (CDCl ₃₎ ^δ: 1.31-1.36 (6H, m), 1.44 (9H, m), 1.48 (9H, m), 1.51-1.59 (2H, m), 1.78-2.00 (2H, m) , 2.83 (1H, ddd, J = 22.9, 10.7, 4.4 Hz), 3.06-3.18 (2H, m), 4.10-4.18 (4H, m), 4.58 (1H, br).

[Reference Example 2] 5 – [(tert- butoxycarbonyl) amino] -2- (1H- imidazol-4-ylmethyl) valeric acid tert- butyl

In acetonitrile (100mL) solution of the compound obtained in Reference Example 1 (8.35g), at room temperature 1,8-diazabicyclo [5.4.0] undec-7-ene (4.58mL) and lithium chloride (1 .30g) and we were added. The suspension was added with 1-trityl–1H- imidazole-4-carbaldehyde (6.90g) was stirred at room temperature overnight, under vacuum, and the solvent was evaporated. After the solution separated by adding ethyl acetate and 10% citric acid aqueous solution, an organic layer, saturated brine, and then washed with a saturated aqueous sodium bicarbonate solution and brine. Dried over anhydrous sodium sulfate, (2E) -5 – [(tert- butoxycarbonyl) amino] -2 – [(1-trityl–1H- imidazol-4-yl) methylene] valeric acid tert- butyl and (2Z) -5 – obtain [(1-trityl–1H- imidazol-4-yl) methylene] valeric acid tert- butyl mixture (11.3g) – [(tert- butoxycarbonyl) amino] -2. The mixture was suspended in methanol (500mL), 10% palladium-carbon catalyst (water content, 4g) was added and stirred for 3 days at room temperature under hydrogen atmosphere. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. Silica gel chromatography gave (eluting solvent: methylene chloride / methanol = 9/1) the title compound (5.60g).
¹ H-NMR (CDCl ₃₎ ^δ: 1.41 (9H, s), 1.44 (9H, s), 1.48-1.57 (3H, m), 1.57-1.66 (1H, m), 2.58-2.68 (1H, m) , 2.73 (1H, dd, J = 14.7, 5.3 Hz), 2.89 (1H, dd, J = 14.7, 8.4 Hz), 3.02-3.19 (2H, m), 4.67 (1H, br s), 6.79 (1H, s), 7.54 (1H, s).

[Reference Example 3] 5 – [(tert- butoxycarbonyl) amino] -2- (methoxycarbonyl) valeric acid

Sodium methoxide in dimethyl malonate (102mL) – methanol (28%, 90.4mL) was added at room temperature and stirred at 60 ℃ 30 minutes. After cooling the white suspension solution to room temperature, (3-bromopropyl) was added carbamic acid tert- butyl (106g) in one portion and stirred at room temperature for 12 hours. Water was added to the reaction solution and the organics extracted with diethyl ether. The organic layer was successively washed with 1 N sodium hydroxide aqueous solution and saturated brine, dried over anhydrous sodium sulfate, filtered and the solvent was distilled off under reduced pressure {3 – [(tert- butoxycarbonyl) amino] propyl} malonic I got acid dimethyl of crude product. The resulting ester (94g) was dissolved in methanol (100mL), water lithium hydroxide monohydrate (13.6g) (300mL) – was added to methanol (300mL) solution at 0 ℃, 15 h stirring at room temperature It was. The methanol was distilled off under reduced pressure and the organics were extracted with ethyl acetate. 2N hydrochloric acid (160mL) was added to the aqueous layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to give the solvent was distilled off under reduced pressure the crude product. This silica gel column chromatography: – is purified (eluent methylene chloride methylene chloride / methanol = 10/1) to give the title compound (69.1g).
¹ H-NMR (CDCl ₃₎ ^δ: 1.44 (9H, m), 1.50-1.60 (2H, m), 1.86-2.01 (2H, m), 3.07-3.20 (2H, m), 3.43 (1H, m) , 3.77 (3H, s), 4.64 (1H, br).

[Reference Example 4] 1- (trans-4- methylcyclohexyl) -1H- imidazole-4-carbaldehyde [Step 1] 1- (trans-4- methylcyclohexyl) -1H- imidazole-4-carboxylic acid ethyl

Was dissolved in 3- (dimethylamino) -2-isocyanoethyl ethyl acrylic acid (Liebigs Annalen der Chemie, 1979 years 1444 pages) (1.52g) and the trans-4- methyl cyclohexylamine (3.07g), 70 ℃ in it was stirred for 4 hours. A saturated aqueous solution of ammonium chloride was added to the reaction solution, the organic matter was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and filtered to give the solvent was distilled off under reduced pressure the crude product. This silica gel column chromatography and purified (eluent hexane / ethyl acetate = 2 / 1-1 / 2) to give the title compound (1.90g).
¹ H-NMR (CDCl ₃₎ ^δ: 0.96 (3H, d, J = 6.6 Hz), 1.13 (2H, m), 1.39 (3H, d, J = 7.0 Hz), 1.47 (1H, m), 1.68 ( 2H, m), 1.88 (2H, m), 2.12 (2H, m), 3.91 (1H, tt, J = 12.1, 3.9 Hz), 4.36 (2H, q, J = 7.0 Hz), 7.54 (1H, s ), 7.66 (1H, s).

[Step 2] [1- (trans-4- methylcyclohexyl) -1H- imidazole-4-yl] methanol

Lithium aluminum hydride (92%, 0.31g) it was suspended in tetrahydrofuran (6mL). The compound obtained in Step 1 of this reference example (1.50g) was dissolved in tetrahydrofuran (6mL), it was slowly added dropwise to the suspension at 0 ℃.0 After stirring for 30 min at ℃, the reaction solution was diluted with diethyl ether, it was added a saturated aqueous solution of sodium sulfate. After stirring for 1 hour at room temperature, the resulting inorganic salt was removed by filtration through Celite. The filtrate to give the crude product was concentrated under reduced pressure. Mixed solvent of this from hexane and ethyl acetate: water (5 1), to give the title compound (1.09g).
¹ H-NMR (CDCl ₃₎ ^δ: 0.95 (3H, d, J = 6.6 Hz), 1.04-1.17 (2H, m), 1.44 (1H, m), 1.59-1.73 (2H, m), 1.81-1.89 (2H, m), 2.04-2.13 (2H, m), 2.78 (1H, br), 3.84 (1H, tt, J = 12.1, 3.9 Hz), 4.59 (2H, s), 6.91 (1H, s), 7.49 (1H, s).

[Step 3] 1- (trans-4- methylcyclohexyl) -1H- imidazole-4-carbaldehyde

The compound obtained in Step 2 of this reference example (1.04g) was dissolved in toluene (10mL). Aqueous solution of sodium hydrogen carbonate (1.35g) (5mL), iodine (2.72g) and 2,2,6,6-tetramethyl-1-sequential piperidinyloxy (84mg) was added and stirred for 2 hours at room temperature It was. The reaction solution was added saturated aqueous sodium thiosulfate solution and the organics were extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and filtered to give the solvent was distilled off under reduced pressure the crude product. This silica gel column chromatography and purified (eluent hexane / ethyl acetate = 1 / 1-1 / 2) to give the title compound (0.900g).
¹ H-NMR (CDCl ₃₎ ^δ: 0.97 (3H, d, J = 6.8 Hz), 1.09-1.19 (2H, m), 1.48 (1H, m), 1.65-1.75 (2H, m), 1.87-1.93 (2H, m), 2.11-2.18 (2H, m), 3.95 (1H, tt, J = 12.2, 3.9 Hz), 7.62 (1H, s), 7.68 (1H, s), 9.87 (1H, s).

[Example 15] (2R) -5- amino -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazole-4-yl] methyl} valeric acid and (2S) -5- amino-2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} valeric acid [Step 1] 5 – [(tert- butoxycarbonyl) amino] -2 – {[1- (trans 4-methylcyclohexyl) -1H- imidazole-4-yl] methyl} methyl valerate

The compound obtained in Reference Example 4 (300mg) and the compound obtained in Reference Example 3 (860mg) was suspended in cyclohexane (10mL). Piperidine (0.154mL) and cyclohexane propionic acid (0.116mL) and (10mL) solution was added, and the mixture was heated under reflux for 48 hours. After cooling, aqueous potassium carbonate solution was added to the reaction solution, and the organic matter was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The obtained crude product was dissolved in ethanol (12mL), 10% palladium-carbon catalyst (water, 250mg) was added and atmospheric pressure hydrogen atmosphere at room temperature for 4 hours and stirred at 60 ℃ 2.5 hours. After Celite filtration, to give the crude product and the filtrate was concentrated under reduced pressure. This silica gel column chromatography and purified (eluent hexane / ethyl acetate = 2 / 1-1 / 3) to give the title compound (562mg).
¹ H-NMR (CDCl ₃₎ ^δ: 0.94 (3H, d, J = 6.6 Hz), 1.02-1.15 (2H, m), 1.34-1.69 (7H, m), 1.43 (9H, s), 1.80-1.87 (2H, m), 1.99-2.09 (2H, m), 2.69 (1H, dd, J = 13.7, 6.3 Hz), 2.79 (1H, m), 2.88 (1H, dd, J = 13.7, 7.4 Hz), 3.03-3.13 (2H, m), 3.63 (3H, s), 3.79 (1H, tt, J = 12.1, 3.9 Hz), 4.76 (1H, br), 6.67 (1H, s), 7.47 (1H, s) .

[Step 2] (2R) -5 – [(tert- butoxycarbonyl) amino] -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} methyl valerate and ( 2S) -5 – [(tert- butoxycarbonyl) amino] -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} methyl valerate

The compound obtained in Step 1 of this Example (40mg) was dissolved in hexane (1.5mL) and ethanol (0.5mL), using CHIRALPAK IA semi-preparative column (2.0cm × 25.0cm) It was optically resolved by high performance liquid chromatography. Flow rate: 15mL / min, elution solvent: hexane / ethanol = 75/25, detection wavelength: 220nm.

The solvent of the divided solution was evaporated under reduced pressure to give both enantiomers each (15mg). Both enantiomers were confirmed to be optically pure by analytical HPLC. Column: CHIRALPAK IA (0.46cm × 25.0cm), flow rate: 1mL / min, elution solvent: hexane / ethanol = 80/20 <v / v>, detection wavelength: 220nm, retention time: (2R) -5- [(tert- butoxycarbonyl) amino] -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} methyl valerate (7.2 min), (2S) -5 – [(tert- butoxycarbonyl) amino] -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} methyl valerate (11.2 min).

[Step 3] (2R) -5- amino -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazole-4-yl] methyl} valerate

Obtained in Step 2 of this Example (2R) -5 – [(tert- butoxycarbonyl) amino] -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl } the methyl valerate (15.0mg) was added to 5 N hydrochloric acid (2mL), and the mixture was heated under reflux for 4 hours. After cooling, the solvent it was evaporated under reduced pressure. The resulting crude hydrochloride salt was dissolved in methanol, was added DOWEX50WX8-200. After the resin was washed with water and eluted with 4% aqueous ammonia. The eluate was concentrated, the crude product was washed with acetone to give the title compound (2.2mg).

[Step 4] (2S) -5- amino -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazole-4-yl] methyl} valerate

Obtained in Step 2 of this Example (2S) -5 – [(tert- butoxycarbonyl) amino] -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl } the methyl valerate (15.0mg) was added to 5 N hydrochloric acid (2mL), and the mixture was heated under reflux for 4 hours. After cooling, the solvent it was evaporated under reduced pressure. The resulting crude hydrochloride salt was dissolved in methanol, was added DOWEX50WX8-200 (200mg). After the resin was washed with water, ammonia water (4%, 80mL) and eluted with. The eluate was concentrated, the crude product was washed with acetone to give the title compound (1.8mg).

[Example 16] 5-amino -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazole-4-yl] methyl} valeric acid benzyl hydrochloride [Step 1] 5 – [(tert- butoxycarbonyl) amino] -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} valerate

The compound obtained in step 1 of Example 15 (7.00g) was dissolved in a mixed solvent consisting of tetrahydrofuran (70mL) and water (14mL), lithium hydroxide monohydrate and (1.26g) at room temperature The mixture was stirred overnight.The reaction solution 2 N hydrochloric acid (8.6mL) was added to neutralize, followed by distilling off the solvent under reduced pressure. The resulting residue was dried with anhydrous sodium sulfate added methylene chloride was to give the crude product was distilled off the solvent under reduced pressure the title compound. This it was used in the next reaction.
MS (ESI) m / z 394 [M ⁺ H] +.

[Example 40] (2S) -5- Amino -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} valerate · p- toluenesulfonate, anhydrous

The compound obtained in Step 4 of Example 15 (2.04g) was suspended stirring in tetrahydrofuran (15mL), p- toluenesulfonate monohydrate (1.32g) was added, at room temperature for 1 day the mixture was stirred. The precipitated crystals were collected by vacuum filtration to obtain dried in one day like the title compound (3.01g).
¹ H-NMR (CD ₃ OD) ^δ: 0.95 (3H, d, J = 6.5 Hz), 1.11-1.21 (2H, m), 1.43-1.79 (7H, m), 1.83-1.89 (2H, m), 2.05-2.10 (2H, m), 2.37 (3H, s), 2.57-2.64 (1H, m), 2.70 (1H, dd, J = 14.5, 5.5 Hz), 2.85-2.95 (3H, m), 4.07 ( 1H, tt, J = 11.7, 3.9 Hz), 7.18 (1H, s), 7.23 (2H, d, J = 7.8 Hz), 7.70 (2H, d, J = 8.2 Hz), 8.22 (1H, s).
Elemental _{analysis: C 16} H ₂₇ N _{3 O 2} · C 7 H ₈ O ₃ S,
Theoretical value: C; 59.33, H; 7.58, N; 9.02, O; 17.18, S; 6.89,
Measured value: C; 59.09, H; 7.53, N; 8.92, O; 17.22, S; 6.78.
———————————-.

[Example 41] (2S) -5- Amino -2 – {[1- (trans-4- methylcyclohexyl) -1H- imidazol-4-yl] methyl} valerate · p- toluenesulfonate & 1 Water hydrate

The obtained compound (101.6mg) in 6% water-containing tetrahydrofuran (600μL) was added in Example 40, was dissolved by heating at 60 ℃. Was allowed to stand at room temperature for 1 day, it was collected by filtration and the precipitated crystals were obtained by dried for one day wind the title compound (79.3mg).
Elemental _{analysis: C 16 H 27} N ₃ O ₂ · C 7 H 8 O ₃ S · _{1H 2} O,
Theoretical value: C; 57.12, H; 7.71, N; 8.69, O; 19.85, S; 6.63,
Measured value: C; 56.90, H; 7.69, N; 8.67, O; 19.81, S; 6.42.

References

Study to Assess the Safety, Pharmacokinetics, and Pharmacodynamics of DS-1040b in Subjects With Acute Ischemic Stroke (NCT02586233

Phase I Study to Evaluate the Safety and Tolerability of DS-1040b Intravenous Infusion With Clopidogrel in Healthy Subjects (NCT02560688)

Study of the Effects of Ethnicity on the Pharmacokinetics, Pharmacodynamics and Safety of DS-1040b (NCT02647307)

Edo, N.; Noguchi, K.; Ito, Y.; Morishima, Y.; Yamaguchi, K.
Hemorrhagic risk assessment of DS-1040 in a cerebral ischemia/reperfusion model of rats with hypertension and hyperglycemia
41st Int Stroke Conf (February 17-19, Los Angeles) 2016, Abst TP283

Noguchi, K.; Edo, N.; Ito, Y.; Morishima, Y.; Yamaguchi, K.
Improvement of cerebral blood flow with DS-1040 in a rat thromboembolic stroke model
41st Int Stroke Conf (February 17-19, Los Angeles) 2016, Abst TP271

Lapchak, P.A.; Boitano, P.D.; Noguchi, K.
DS-1040 an inhibitor of the activated thrombin activatable fibrinolysis inhibitor improves behavior in embolized rabbits
41st Int Stroke Conf (February 17-19, Los Angeles) 2016, Abst WP282 

A first-in-human, single ascending dose study of DS-1040, an inhibitor of the activated form of thrombinactivatable fibrinolysis inhibitor (TAFIa), in healthy subjects
25th Congr Int Soc Thromb Haemost (ISTH) (June 20-25, Toronto) 2015, Abst PO621-MON

Dow, J.; Puri, A.; McPhillips, P.; Orihashi, Y.; Dishy, V.; Zhou, J.
A drug-drug interaction study of DS-1040 and aspirin in healthy subjects
25th Congr Int Soc Thromb Haemost (ISTH) (June 20-25, Toronto) 2015, Abst PO603-TUE

Noguchi, K.; Edo, N.; Ito, Y.; Yamaguchi, K.
Effect of DS-1040 on endogenous fibrinolysis and impact on bleeding time in rats
25th Congr Int Soc Thromb Haemost (ISTH) (June 20-25, Toronto) 2015, Abst AS145

Noguchi, K.; Edo, N.; Ito, Y.; Maejima, T.; Yamaguchi, K.
DS-1040: A novel selective inhibitor of activated form of thrombin-activatable fibrinolysis inhibitor
25th Congr Int Soc Thromb Haemost (ISTH) (June 20-25, Toronto) 2015, Abst PO203-MON

DS1040b/Aspirin Drug/Drug Interaction Study (NCT02071004)
ClinicalTrials.gov Web Site 2014, February 26

//////DS-1040, DS 1040, phase 2, Daiichi Sankyo Co Ltd, Ischemic stroke

O=C(O)[C@@H](CCCN)Cc1cn(cn1)[C@@H]2CC[C@@H](C)CC2

O=S(=O)(O)c1ccc(C)cc1.O=C(O)[C@@H](CCCN)Cc1cn(cn1)[C@@H]2CC[C@@H](C)CC2

Glaxo to Stop Seeking Drug Patents in Low-Income Countries

Drugmaker says move could help poor nations access cheaper copycat versions of its medicines

GlaxoSmithKline’s CEO Andrew Witty below

By

Denise Roland

March 31, 2016 12:58 p.m. ET

http://www.wsj.com/articles/glaxo-to-stop-seeking-drug-patents-in-low-income-countries-1459443494?mod=pls_whats_news_us_business_f&utm_content=buffer1d705&utm_medium=social&utm_source=twitter.com&utm_campaign=buffer

LONDON— GlaxoSmithKline PLC said it would stop seeking patents for its drugs in low-income countries, a move the drugmaker said could help the world’s poorest people access copycat versions of its medicines at affordable prices.

The U.K.-based company said it would take this approach in low-income and least-developed countries, a group totaling around 85 nations. In so-called lower-middle-income countries, a group of 51 nations that includes Vietnam, Cameroon and Sri Lanka, it said it would file patents but aim to grant licenses to generic manufacturers to supply low-cost versions of its drugs in those markets in return for a small royalty.

Glaxo previously filed patents in most lower-middle-income countries, and in low-income nations where a patent office exists. But that “patchwork” approach meant that generic drugmakers held back from manufacturing copycat medicines for these markets owing to the risk of being sued by pharmaceutical companies, according to Glaxo Chief Executive Andrew Witty.,,,,,,,,,continue reading

http://www.wsj.com/articles/glaxo-to-stop-seeking-drug-patents-in-low-income-countries-1459443494?mod=pls_whats_news_us_business_f&utm_content=buffer1d705&utm_medium=social&utm_source=twitter.com&utm_campaign=buffer

/////////Glaxo Chief Executive,  Andrew Witty, filed patents, low income,poor nations, cheaper,  copycat versions, medicines, GlaxoSmithKline

Lomitapide mesylate

Was developed and launched by Aegerion, under license from the University of Pennsylvania (which acquired rights from BMS).

US-20160083345

Sanjay Jagdish DESAI
Brij KHERA
Jagdish Maganlal PATEL
Harshita Bharatkumar SHAH
Arunkumar Shyam Narayan UPADHYAY
Sureshkumar Narbheram AGRAVAT

Polymorphic forms of lomitapide and its salts and processes for their preparation

Zydus Cadila Healthcare Ltd

The present invention relates to various polymorphic forms of lomitapide or its salts and processes for preparation thereof. The present invention provides Lomitapide mesylate in solid amorphous form and process for preparation thereof. The invention also provides an amorphous solid dispersion of lomitapide mesylate. Further, various crystalline forms of lomitapide mesylate like A, B and C and process for preparation thereof are provided. The invention also provides crystalline forms of lomitapide free base, in particular Form I and Form-II and their preparation. The invention further provides compositions comprising various forms of lomitapide and its salts.

A novel amorphous form of lomitapide mesylate (having >98% of purity and 0.5% of residual solvent and particles size D90 of >250 µm, D50 of >100 µm and D10 of >50 µm), a process for it preparation and a composition comprising it is claimed. Also claimed is an amorphous solid dispersion of lomitapide mesylate and a carrier (eg hydroxypropylmethyl cellulose acetate succinate). Further claimed are crystalline forms of lomitapide mesylate (designated ad Forms A, B, C, I, II and free base of lomitapide in amorphous form), processes for their preparation and compositions comprising them. Lomitapide is known to act as a microsomal triglyceride transfer protein inhibitor, useful for treating familial hypercholesterolemia.

Lomitapide is a synthetic lipid-lowering agent for oral administration. It is a microsomal triglyceride transfer protein inhibitor approved as Juxtapid® in US and as Lojuxta® in Europe as an adjunct to a low-fat diet and other lipid-lowering treatments, including LDL apheresis where available, to reduce low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apo B), and non-highdensity lipoprotein cholesterol (non-HDL-C) in patients with homozygous familial hypercholesterolemia (HoFH). The approved drug product is a mesylate salt of lomitapide, chemically known as N-(2,2,2-trifluoroethyl)-9-[4-[4-[[[4′(trifluoromethyl)[1,1′-biphenyl]-2-yl]carbonyl]amino]-1-piperidinyl]butyl]-9H-fluorene-9carboxamide methanesulfonate [“lomitapide mesylate” herein after] and has the structural formula

(MOL) (CDX)

As per the approved label for Juxtapid® (US) “Lomitapide mesylate is a white to off-white powder that is slightly soluble in aqueous solutions of pH 2 to 5. Lomitapide mesylate is freely soluble in acetone, ethanol, and methanol; soluble in 2-butanol, methylene chloride, and acetonitrile; sparingly soluble in 1-octanol and 2-propanol; slightly soluble in ethyl acetate; and insoluble in heptane”.

As per Public Assessment Report for Lojuxta® (Europe) “Polymorphism has been observed for lomitapide mesylate. Of the different solid-state forms, hydrates, and solvates identified in the polymorph studies, only 2 desolvated solid-state forms, Form I and Form II, were identified in batches after drying to final drug substance.” The report further states, under the heading Manufacture, that “The final particle size distribution is controlled during the crystallisation step” (emphasis added) suggesting that the approved drug product lomitapide mesylate is a crystalline compound

U.S. Pat. No. 5,712,279 A discloses the lomitapide compound and a process for its preparation. It also discloses a process for preparation of lomitapide monohydrochloride.

U.S. Pat. No. 5,883,109 A discloses lomitapide mesylate specifically but no solid form was disclosed.

The reference article Synthesis and Applications of Isotopically Labelled Compounds, Vol. 8, Pg. 227-230 (2004) discloses the preparation of Deuterium labelled [d₄]BMS-201038, [³H]BMS-201038, [¹⁴C]BMS-201038 wherein BMS-201038 is lomitapide mesylate.

International (PCT) Publication No. WO 2015/121877 A2 discloses lomitapide crystalline Form I and Form II as well as amorphous form of Lomitapide mesylate and processes for their preparation.

There is still a need to provide a novel polymorph of lomitapide or its salts which is suitable for pharmaceutical preparations. Therefore, the present invention provides new crystalline forms of lomitapide free base and lomitapide mesylate. The present invention also provides amorphous form of lomitapide free base and lomitapide mesylate, which is stable and useful for pharmaceutical preparations.

EXAMPLES

Example-1

Preparation of Lomitapide Mesylate

In a 250 mL round bottom flask, equipped with a mechanical stirrer, thermometer and an addition funnel, 10 g lomitapide and 20 mL methanol were added and stirred to obtain a solution. 1.5 g methane sulfonic acid dissolved in 20 mL water was added slowly to the above solution under stirring. The reaction mixture was stirred till maximum salt formation was achieved. 50 mL water was added to the mixture, stirred for 15-20 min, filtered and washed with water. The product was dried further to obtain lomitapide mesylate.

EXAMPLE 2

Preparation of Amorphous Form of Lomitapide Mesylate

10 g lomitapide mesylate, 50 mL acetone and 150 mL ethyl acetate were heated in a 500 mL round bottom flask, equipped with a mechanical stirrer, thermometer and an addition funnel at 50-55° C. and stirred to obtain clear solution. The solution was subjected to spray drying in JISL Mini spray drier LSD-48 with feed pump running at 30-35 rpm, inlet temperature 50-55° C., out let temperature 45-50° C., aspiration rate 1200-1300 rpm, hot air supply 1.8-2.2 Kg/cm² and vacuum for conveying the dry product 80 mmHg. The product was collected from cyclone and characterized to an amorphous form by x-ray powder diffraction. The product was further dried to obtain the amorphous form of lomitapide mesylate

/////////////New patent, Lomitapide mesylate , Zydus Cadila Healthcare Ltd, US 20160083345, Amorphous

Acitretin

PDT PATENT US4105681

WO-2016042573

Process for preparation of acitretin

Emcure Pharmaceuticals Ltd

EMCURE PHARMACEUTICALS LIMITED [IN/IN]; an Indian company at EMCURE HOUSE, T-184, MIDC., Bhosari, Pune – 411 026 Maharashtra (IN)

GURJAR MUKUND KESHAV; (IN).
JOSHI SHASHIKANT GANGARAM; (IN).
BADHE SACHIN ARVIND; (IN).
KAMBLE MANGESH GORAKHANATH; (IN).
MEHTA SAMIT SATISH; (IN)

The present invention Provides a process for preparation of {(2E, 4E, 6E, 8E) -9- (4-methoxy-2,3,6-trimethyl) phenyl-3,7-dimethyl-nona-2,4,6 , 8} tetraenoate, acitretin year intermediate of formula (VI) with trans isomer ≥97%, comprenant of Reacting 3-formyl-Crotonic acid butyl ester of formula (V) Substantially free of impurities, with 5- (4-methoxy- 2,3,6-trimethylphenyl) -3-methyl-penta-2,4-diene-l-triphenyl phosphonium bromide of formula (IV) and isolating resulting compound of formula (VI) Treating the filtrate with iodine for isomerization of the Undesired cis intermediate and finally Obtaining acitretin (I), with trans isomer Desired ≥97%.

Samit Satish Mehta holds the position of the President – Research & Development

Acitretin of formula (I), chemically known as (2E,4E,6E,8E)-9-(4-methoxy-2,3,6- trimethyl)phenyl-3,7-dimethyl-nona-2,4,6,8-tetraenoic acid, is a second generation retinoid a roved by USFDA in 1996, for the treatment of psoriasis.

Acitretin (I)

The process for preparation of acitretin (I) was first disclosed in US 4,105,681 wherein the intermediate, 5-(4-methoxy-2,3,6-trimethylphenyl)-3-methyl-penta-2,4-diene-l-triphenyl phosphonium bromide was reacted with 3-formyl-crotonic acid butyl ester in presence of sodium hydride as base and dimethylformamide as solvent. The resultant ester derivative was obtained with a trans is (E/Z) ratio of around 55:45 which was subjected to hydrolysis in presence of potassium hydroxide and ethyl alcohol to obtain acitretin.

Use of hazardous, highly pyrophoric and moisture sensitive reagent like sodium hydride, along with cumbersome work-up and successive crystallizations to obtain the desired isomer rendered the process unviable for commercial scale.

Indian patent application 729/MUM/2012 discloses use of organic bases such as triethyl amine or pyridine for the reaction of 3-formyl-crotonic acid butyl ester and 5-(4-methoxy-2,3,6-trimethylphenyl)-3-methyl-penta-2,4-diene-l -triphenyl phosphonium bromide for the synthesis of acitretin. The process utilizes a large excess of the organic base (2.85:1.0) with respect to the reactant phosphonium bromide derivative. Further, there is no mention of the ratio of cis and trans geometric isomers of the product thus obtained either at the intermediate or final stage. The trans: cis (E/Z) ratio of the intermediate significantly impacts the final yield and purity of the product as several purifications and crystallizations are required to obtain the desired trans isomer.

The present inventors have experimentally observed that use of organic base in such large quantities severely hampers the removal of the undesired side product triphenyl phosphonium oxide formed in significant amounts. Also, the intermediate is obtained with a very modest trans: cis (E/Z) ratio.

WO2012/155796 discloses another method wherein alkali metal alkoxides are used as bases in the reaction of 5-(4-methoxy-2,3,6-trimethylphenyl)-3 -methyl -penta-2,4-diene-l -triphenyl phosphonium bromide with 3-formyl-crotonic acid. The obtained reaction mass, after adjusting pH to 7-8 with acid, is directly subjected to catalytic isomerization using catalysts such as Pd(OAc)₂ or Pd(NH₃)₂Cl_2. The reaction mixture so obtained is quenched with water, neutralized and filtered to get the desired product, which is further recrystallized from ethyl acetate. Although this procedure avoids the hydrolysis step and attempts in-situ isomerization, however the use of expensive, soluble palladium catalyst which cannot be recycled from the reaction mass coupled with lengthy reaction time of 25-30 hours and large solvent volumes make the process unviable.

It may be noted that in the synthesis of acitretin, the key reaction of 5-(4-methoxy-2,3,6-trimethylphenyl)-3 -methyl-penta-2 ,4-diene- 1 -triphenylphosphoniumbromide with 3 -formyl crotonic acid or its ester in presence of either strong inorganic bases such as sodium hydride, alkali metal alkoxides or organic bases like triethylamine is common to almost all synthetic routes disclosed in the prior art. Hence, all these routes suffer from the inherent problems of formation of undesired impurities including cis-isomeric compounds and their separation from the desired all-trans product which necessitates various purification methods ranging from column chromatography, multiple crystallizations etc.

Thus, there still exists a need for a convenient, easy-to-scale up process for synthesis of acitretin (I) which avoids use of pyrophoric strong bases and provides a robust method which affords acitretin having desired isomeric purity in high yield.

5-(4-methoxy,2,3,6 trimethylphenyl)- 3-formyl crotonic acid butyl glyoxalate L(+) tartaric acid

3-methyl-penta-2,4-dien-1-triphenyl butyl ester (V) dibutyl ester

phosphonium bromide (IV)

Acitretin (I)

Satish Mehta,CEO, Above and here Inspiring the participants

EXAMPLES

Example 1: Preparation of 4-(4-methoxy-2,3,6-trimethylphenyl)-but-3-en-2-one (II)

Acetone (6000 ml) was added to 4-methoxy-2,3,6 trimethyl benzaldehyde (500.3 g) and the mixture was stirred at 20-30°C. Aqueous solution of sodium hydroxide (134.8 g in 500 ml water) was gradually added to it and the resulting mixture was heated to 45-50°C with continued stirring. After completion of the reaction, as monitored by HPLC, the reaction mass was cooled and acetic acid was added till pH 4.5 to 5.5. Distillation of acetone, followed by addition of cyclohexane to the residue, followed by washing with water, separation and concentration of the organic layer gave 4-(4-methoxy-2,3,6 trimethylphenyl)-but-3-en-2-one of formula (II).

Yield: 80-84%

Example 2: Preparation of 5-(4-methoxy-2,3,6-trimethylphenyl)-3-methyl-penta-2,4-diene- 1-triphenyl phosphonium bromide (IV)

4-(4-Methoxy-2,3,6-trimethylphenyl)-but-3-en-2-one (II; 500 g) dissolved in toluene (2000 ml) was gradually added to a mixture of vinyl magnesium bromide (3500 ml; 1 molar solution in THF) and lithium chloride (4.8 g) and stirred at 20-30 C till completion of the reaction as monitored by HPLC. The reaction mixture was quenched with water and concentrated hydrochloric acid was added till the pH was between 3 and 4. The organic layer was separated and concentrated to give residue containing 5-(4-methoxy-2,3,6 trimethylphenyl)-3 -methyl -penta l,4-dien-3-ol (III). Methyl isobutyl ketone (3500 ml) was added to the residue, followed by gradual addition of triphenyl phosphine hydrobromide (745.3 g) at room temperature. The reaction mixture was heated to 50-60°C till completion of the reaction. The reaction mixture was cooled and filtered to give 5-(4-methoxy-2,3,6-trimethylphenyl)-3-methyl-penta-2,4-diene-l-triphenyl phosphonium bromide of formula (IV).

Yield: 1000 g (76%)

Example 3: Preparation of 3-formyl crotonic acid butyl ester (V)

Dibutyl-L- tartrate (500 g) was dissolved in isopropanol (3500 ml) at room temperature, and water (750 ml) was added to it. The reaction mixture was cooled to 15-25°C and sodium metaperiodate (448.5 g) was gradually added to it with stirring. The reaction was continued at 20-30°C till completion of the reaction based on GC analysis. The reaction mixture was filtered and the filtrate was concentrated. The resulting residue was dissolved in toluene (1000 ml), stirred and filtered to obtain the filtrate containing butyl glyoxylate. Propionaldehyde (221.0 g) was added to the filtrate and heated to around 60°C, followed by gradual addition of piperidine (26.4 g, dissolved in toluene). The reaction mixture was further heated and stirred at 110-120°C till completion of the reaction, as monitored by GC. After completion, the reaction mass was cooled, washed with aqueous sulfuric acid, water and finally with aqueous sodium bicarbonate solution. The organic layer was concentrated and the residue was distilled to give 3-formyl crotonic acid butyl ester (V)

Yield: 230-280 g (35-43%)

Example 4. Preparation of butyI{(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethyl) phenyl-3,7-dimethyl-nona-2,4,6,8}tetraenoate (VI)

Sodium carbonate (297. lg), was added to the mixture of 5-(4-Methoxy-2,3,6-trimethylphenyl)-3-methyl-penta-2,4-diene-l-triphenyl-phosphoniumbromide (IV; 1000 g) in toluene (5000 ml) followed by gradual addition of 3-formyl crotonic acid butyl ester (330 g) at room temperature. The stirred reaction mixture was heated to 60-70°C till completion of the reaction as monitored by HPLC. The reaction mass was cooled, quenched with water. The organic layer was separated, concentrated and n-heptane was added to the residue. The mass was stirred, filtered and 40% aqueous methanol (2000 ml) was added to it with stirring. Layer separation, concentration of the organic layer, and crystallization of the resulting residue from isopropyl alcohol, optionally with seeding followed by filtration gave crop I of butyl {{(2E,4E,6E,8E)— 9-(4-methoxy-2,3,6 trimethyl)phenyl-3,7 dimethyl -nona-2,4,6,8} tetraenoate (VI),.

Yield: 45-50%;

Cis: Trans isomer ratio (2.0:98.0)

The filtrate was concentrated, the residue was dissolved in toluene (2000 ml) and treated with iodine (4.5 g) at room temperature. After completion of the reaction, as monitored by HPLC, the reaction mixture was stirred with aqueous sodium thiosulfate solution. Separation and concentration of the organic layer and crystallization of the resulting residue from isopropyl alcohol, optionally with seeding, gave crop II of butyl {{(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethyl)phenyl-3,7-dimethyl-nona-2,4,6,8} tetraenoate (VI).

Yield (crop II): 15 to 20%.

Cis: Trans isomer ratio (2.0:98.0)

Total yield (crop I+II): 60-70%.

Example 5: Preparation of acitretin (I)

Aqueous solution of potassium hydroxide (155.2 g in 600 ml water) was added to a solution of butyl {(2E,4E,6E,8E)-9-(4-methoxy-2,3 ,6-trimethyl) phenyl-3 ,7-dimethyl-nona- 2,4,6,8}tetraenoate, VI (300.0 g) in ethanol (1800 ml) at 25-30°C and the reaction mixture was stirred at reflux temperature till completion of the reaction. After completion, as monitored by HPLC, the reaction mixture was quenched with water, and hydrochloric acid was added till pH was between 2.5 and 3.5. The mass was heated at 70°C, stirred, cooled to 40-50°C and filtered. Recrystallization of the resulting solid from tetrahydrofuran gave acitretin (I).

Yield: 154.0 g (60%)

Desired trans isomer: > 98%

India’s hockey stars Sardara Singh and Sandeep Singh with Emcure Pharmaceuticals COO, Arun Khanna

HE Dr. Kenneth Kaunda, First President of Zambia interacting with Mr. A. K. Khanna, COO & ED, Emcure at Emcure booth at AIDS 2012 conference, Washington

Mr. Sunil Mehta is an Executive Director and Senior Director (Projects)

Arun Khanna is the Chief Operating Officer and Executive Director on the Board of Emcure Pharmaceuticals Limited.

//////New patent, WO 2016042573,  Acitretin,   Emcure Pharmaceuticals Ltd

(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid

Acitretin is an oral retinoid effective in the treatment of psoriasis. It is the major metabolite of ETRETINATE with the advantage of a much shorter half-life when compared with etretinate.

• 1 “Important Safety Information for SORIATANE”. soriatane.com. Retrieved 31 October 2015.
• 2
• AABB Technical Manual, American Association of Blood Banks
• 3
• “Soriatane”. WebMD. Retrieved 15 August 2015.
• 4
• “Soriatane Side Effects”. Drugs.com. Retrieved 15 August 2015.
• 5

“Soriatane (Acitretin) Drug Information: Description, User Reviews, Drug Side Effects, Interactions – Prescribing Information at RxList”. RxList. Retrieved 15 August 2015.

Literature References:

Synthetic retinoid; free acid form and major metabolite of etretinate, q.v. Prepn: W. Bollag et al., DE 2414619; eidem, US 4105681 (1974, 1978 both to Hoffmann-La Roche).

Teratogenicity study: A. Kistler, H. Hummler, Arch. Toxicol. 58, 50 (1985).

HPLC determn in plasma: N. R. Al-Mallah et al., Anal. Lett. 21, 1603 (1988).

Pharmacokinetics in humans: F. G. Larsen et al., Pharmacol. Toxicol. 62, 159 (1988).

Clinical evaluation in cutaneous lupus erythematosus: T. Ruzicka et al., Arch. Dermatol. 124, 897 (1988).

Review of clinical pharmacology: A. Vahlquist, O. Rollman, Dermatologica 175, Suppl. 1, 20-27 (1987).

Review of clinical studies in psoriatic and nonpsoriatic dermatoses: J.-M. Geiger, B. M. Czarnetzki, ibid. 176, 182-190 (1988).

SORIATANE (acitretin), a retinoid, is available in 10-mg, 17.5-mg, and 25-mg gelatin capsules for oral administration. Chemically, acitretin is all-trans-9-(4-methoxy-2,3,6trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoic acid. It is a metabolite of etretinate and is related to both retinoic acid and retinol (vitamin A). It is a yellow to greenish-yellow powder with a molecular weight of 326.44. The structural formula is:

so

Each capsule contains acitretin, black monogramming ink, gelatin, maltodextrin (a mixture of polysaccharides), microcrystalline cellulose, and sodium ascorbate.

Gelatin capsule shells contain gelatin, iron oxide (yellow, black, and red), and titanium dioxide. They may also contain benzyl alcohol, carboxymethylcellulose sodium, edetate calcium disodium.

SYNTHESIS

Synthetic retinoid; free acid form and major metabolite of etretinate, q.v. Prepn: W. Bollag et al., DE 2414619; eidem, US 4105681 (1974, 1978 both to Hoffmann-La Roche).

NMR, IR SEE………….https://www.iarc.fr/en/publications/pdfs-online/prev/handbook4/Handbook4_Retinoids-6.pdf

https://www.iarc.fr/en/publications/pdfs-online/prev/handbook4/Handbook4_Retinoids-6.pdf

http://nopr.niscair.res.in/bitstream/123456789/19745/1/IJBB%2039%281%29%2022-27.pdf

Acitretin

Systematic (IUPAC) name
(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid
Clinical data
Trade names	Soriatane, Neotigason
AHFS/Drugs.com	monograph
MedlinePlus	a601010
Pregnancy category	AU: X (High risk) US: X (Contraindicated)
Routes of administration	Oral
Legal status
Legal status	AU: S4 (Prescription only) CA: ℞-only UK: POM (Prescription only) US: ℞-only
Pharmacokinetic data
Bioavailability	60%
Protein binding	>99.9%
Metabolism	Hepatic
Biological half-life	49 hours
Excretion	Faeces & urine
Identifiers
CAS Number	55079-83-9
ATC code	D05BB02 (WHO)
PubChem	CID 5284513
IUPHAR/BPS	7598
DrugBank	DB00459
ChemSpider	4447573
UNII	LCH760E9T7
KEGG	D02754
ChEBI	CHEBI:50173
ChEMBL	CHEMBL1131
Chemical data
Formula	C21H26O3
Molar mass	326.429 g/mol

////////////CC1=CC(=C(C(=C1C=CC(=CC=CC(=CC(=O)O)C)C)C)C)OC

WO 2016042441, Mankind Research Centre, Silodosin, New patent

WO-2016042441

Mankind Research Centre

MANKIND RESEARCH CENTRE [IN/IN]; 191-E, Sector 4-II, IMT-Manesar, Haryana 122050 (IN)

A novel process for the preparation of considerably pure silodosin

GANGWAR, Kuldeep Singh; (IN).
KUMAR, Anil; (IN).
BHASHKAR, Bhuwan; (IN)

The present invention relates to a novel, improved, commercially viable and industrially advantageous process for the preparation of Silodosin of Formula (I), its pharmaceutically acceptable salts or solvates thereof. The invention relates to the preparation of considerably pure Silodosin with high yield.

Silodosin, l-(3-hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2-trifluoroethoxy)phenoxy] ethyl} amino)propyl]-2,3-dihydro-lH-indole-7-carboxamide of Formula (I) is an indoline antidysuric which has a selectively inhibitory effect against urethra smooth muscle constriction, and decreases urethra internal pressure without great influence on blood pressure. Silodosin is available under trade names RAPAFLO^® or UROREC^®. Silodosin was first disclosed in EP 0600675 as a therapeutic agent for the treatment of dysuria associated with benign prostatic hyperplasia, where a process for producing the compound is also disclosed.

Formula (I)

Since, Silodosin is an optically active compound having a complex chemical structure; its synthesis is relatively complex and requires a sequence of multiple steps.

US patent no. 6,310,086, discloses a process for preparing Silodosin analogue compound from reaction of (R)-3-{5-(2-aminopropyl)-7-cyano-2,3-dihydro-lH-indol-l-yl} propylbenzoate with 2-(2-ethoxyphenoxy)ethyl methanesulfonate and finally isolated as a crude compound which is purified by column chromatography. The said process has a major disadvantage of using column chromatography which is not feasible at plant scale production.

PCT application no. WO 2012147019, discloses the preparation of Silodosin as shown in scheme- 1, wherein the Ν,Ν-dialkyl impurity of Formula (Ila) formed during condensation of 3-{7-cyano-5-[(2R)-2-aminopropyl]-2,3-dihydro-lH-indol-l-yl}propyl benzoate of Formula (III) with 2-(2-(2,2,2-trifluoroethoxy)phenoxy)ethyl methanesulfonate of Formula (IV); is removed through preparation of monotartarate salt to give compound of Formula (VI). The compound of Formula (VI) is base hydrolyzed followed by cyano hydrolysis to give crude Silodosin of Formula (VIII) which is then further purified by crystallization to get desired pure Silodosin.

Scheme- 1:

Major drawback of above said reaction process is that multiple isolations and crystallizations are required to get pure Silodosin.

Similarly, US 7,834,193 discloses monooxalate salt represented by Formula Via having 0.9% of dialkyl impurity represented by Formula Ila. The oxalate salt so obtained is subjected to alkaline hydrolysis followed by transformation of the nitrile to an amide.

Formula (Ila)

Similarly, PCT application no. WO 2012147107, discloses the method wherein Silodosin is prepared by condensation of 3-{7-cyano-5-[(2R)-2-aminopropyl]-2,3-dihydro-lH-indol-l-yl} propyl benzoate with 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methanesulfonate in solvent using base and phase transfer catalyst wherein, dialkyl impurity is formed up to 11%, followed by hydroxyl deprotection in protic solvent using base and phase transfer catalyst which is then subjected to purification to remove N,N-dialkyl impurity represented by Formula (lib) up to 0.6% through the preparation of acetate salt. This process suffers from a serious drawback i.e., accountable formation of dialkyl impurity and even after purification the impurity is reduced to only up to 0.6%. Secondly, the process requires multiple isolations and purifications ensuing into time engulfing workups and purifications and hence incurring solvent wastage. This makes process lengthy, uneconomical and tedious to be performed at plant scale.

Another PCT application no. WO 2012131710, discloses the preparation of Silodosin in which the chiral compound (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) is reacted with 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methane sulfonate in isopropyl alcohol using sodium carbonate as base. The reaction is completed in 40-50h and about 9-11% of dimer is formed during condensation. After completion of reaction, it is subjected to hydroxyl deprotection and the crude compound so obtained is purified to remove the Ν,Ν-dialkyl impurity of Formula (lib). The pure compound is then reacted with hydrogen peroxide in dimethyl sulfoxide to give Silodosin. The major drawback of this process is that the process is a multistep process wherein the condensation reaction is long-drawn-out resulting into countable amount of dimer formation during the process.

Thus, the prior art methods of preparing Silodosin require multiple and repeated purifications to synthesize DMF (Drug Master File) grade Silodosin. None of the prior art produces compound of Formula (VI) or (VII) with Ν,Ν-dialkyl impurity of Formula (Ila) or (lib) in an amount less than 0.6% to 0.5% even after purification. Therefore to prepare highly pure Silodosin, there is a need to explore new synthetic schemes that could be more economical and scalable. The present invention provides a novel, improved, commercially viable and industrially advantageous process for the synthesis of Silodosin and its pharmaceutically acceptable salts or solvates thereof. The present invention focus on preparation of highly pure Silodosin in appreciable yields with minimal use of solvents wherein the Silodosin is isolated with purity >99.5% having Ν,Ν-dialkyl impurity less than 0.03% and other individual impurities below 0.1%.

Ramesh Juneja (seated), founder of Mankind Pharma, with brother Rajeev, who is senior director (marketing & sales)

Mankind Pharma Chairman and Founder RC Juneja

In accordance to one embodiment of the present invention, the process of the preparation of Silodosin represented by Formula (I)

comprises the steps of:

a) condensing chiral compound represented by Formula (III)

Formula (III)

wherein, Bz represents to Benzoyl group with compound represented by Formula (IV)

Formula (IV)

wherein, Ms represents to Methanesulfonyl group in presence of base and phase transfer catalyst in an organic solvent to give intermediate represented by Formula (V)

Formula (V)

wherein, n is an integer of 1 and 2 and Bz is as defined above, wherein the compound having n=2 is formed in an amount of less than 5%;

b) optionally isolating compound of Formula (V),

c) without purification converting it to de-protected compound represented by Formula (IX) in an organic solvent;

Formula (IX)

wherein, n is as defined above;

d) optionally isolating compound of Formula (IX), and

e) without purification converting it to compound represented by Formula (X)

Formula (X)

wherein n is as defined above;

f) subjecting compound of Formula (X) to purification by converting to acid salt for removal of Ν,Ν-dialkyl impurity represented by Formula (lie);

Formula (He)

g) hydrolysis of the said acid salt to get Silodosin of Formula (I) with purity >99.5%.

Examples

The invention is explained in detail in the following examples which are given solely for the purpose of illustration only and therefore should not be construed to limit the scope of the invention.

Example 1

Preparation of Crude Silodosin:

Method A:

To the solution of lOg (0.0275 mol) of (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) in 100ml of toluene was added 14.3g (0.0826 mol) of dipotassium hydrogen phosphate and 8.20g (0.0261 mol) of 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methane sulfonate followed by addition of 2.0g (0.0055 mol) of tetrabutyl ammonium iodide and stirred the reaction mass at 85-90°C for 10-12h. Cooled the reaction mass, added de-mineralized water and separated the toluene layer followed by distillation to get crude viscous mass. Added 110ml of dimethyl sulfoxide and a solution of 1.51g (0.0415 mol) of sodium hydroxide dissolved in 8.52ml of water followed by addition of 6.42g (0.0567 mol) of 30% w/w of hydrogen peroxide. Stirred the reaction mass at 20-25°C till completion and added sodium sulfite solution. Extracted the compound in ethylacetate, washed the organic layer with brine solution and concentrated to get 10.2g of crude Silodosin.

Ν,Ν-dialkyl impurity is 3.2% as per HPLC.

Method B:

To the solution of lOg (0.0275 mol) of (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) in 100ml of toluene was added 14.3g (0.0826 mol) of dipotassium hydrogen phosphate and 8.20g (0.0261 mol) of 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methane sulfonate followed by addition of 2.0g (0.0055 mol) of tetrabutyl ammonium iodide and stirred the reaction mass at 85-90°C for 10-12h. Added solution of 4.4g of sodium hydroxide dissolved in 10ml of water and stirred the reaction at ambient temperature till completion. Quenched the reaction mass with water and separated the layers. Washed the toluene layer with brine and concentrated under reduced pressure to get crude mass. Dissolved the crude mass so obtained in 110ml of dimethyl sulfoxide and added a solution of 1.95g (0.0488 mol) of sodium hydroxide dissolved in 7.95ml of water followed by addition of 7.5g (0.066 mol) of 30% w/w of hydrogen peroxide. Stirred the reaction mass at room temperature followed by addition of 210ml of aqueous solution of sodium sulfite and extracted the compound in ethyl acetate. Washed the organic layer with brine and concentrated under reduced pressure to get 10. lg of crude Silodosin.

Ν,Ν-dialkyl impurity is 3.0% as per HPLC

Method C:

To the solution of lOg (0.0275 mol) of (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) in 100ml of dimethyl sulfoxide was added 14.3g (0.0826 mol) of dipotassium hydrogen phosphate and 8.20g (0.0261 mol) of 2-[2-(2,2,2-trifluoroethoxy)phenoxy] ethyl methane sulfonate followed by addition of 2.0g (0.0055 mol) of tetrabutyl ammonium iodide and stirred the reaction mass at 85-90°C for 2-3h. Added 100ml of water and 50ml of toluene and stirred the reaction mass at room temperature for half an hour. Separated the toluene layer and concentrated under reduced pressure. To the crude mass so obtained was added 110ml of dimethyl sulfoxide and a solution of 4.4g of sodium hydroxide dissolved in 10ml of water followed by addition of 7.5g (0.066 mol) of 30% w/w of hydrogen peroxide. Stirred the reaction mass at room temperature followed by addition of 210ml of aqueous solution of sodium sulfite and extracted the compound in ethyl acetate. Washed the organic layer with brine and concentrated under reduced pressure to get 9.8 g of crude Silodosin.

Ν,Ν-dialkyl impurity is 2.1% as per HPLC

Method D:

To the solution of 20g (0.055 mol) of (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) in 200ml of toluene was added 28.6g (0.165 mol) of dipotassium hydrogen phosphate and 16.4g (0.0522 mol) of 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methane sulfonate followed by addition of 4.0g (0.11 mol) of tetrabutyl ammonium iodide and stirred the reaction mass at 85-90°C for 10-12h. Added de-mineralized water and stirred at room temperature for half an hour. Separated the toluene layer to which was added a solution of 8.8g of sodium hydroxide dissolved in 20ml of water and stirred the reaction at ambient temperature till completion. Quenched the reaction mass with water and separated the layers. Washed the toluene layer with brine and concentrated under reduced pressure to get crude mass. Dissolved the crude mass so obtained in 200ml of dimethyl sulfoxide and added a solution of 3.9g (0.0976 mol) of sodium hydroxide dissolved in 16ml of water followed by addition of 15g (0.132 mol) of 30% w/w of hydrogen peroxide. Stirred the reaction mass at room temperature followed by addition of 400ml of aqueous solution of sodium sulfite and extracted the compound in ethyl acetate. Washed the organic layer with brine and concentrated under reduced pressure to get 21. Og of crude Silodosin.

Ν,Ν-dialkyl impurity is 2.8% as per HPLC

Method E:

To the solution of 2g (0.0055 mol) of (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) in 20ml of was dimethyl sulfoxide was added 2.87g (0.0165 mol) of dipotassium hydrogen phosphate and 1.64g (0.0052 mol) of 2-[2-(2,2,2-trifluoroethoxy)phenoxy] ethyl methane sulfonate followed by addition of 0.29g (0.0011 mol) of 16-crown ether and stirred the reaction mass at 85-90°C for 10-12h. Added a solution of 0.88g of sodium hydroxide dissolved in 2ml of water and stirred the reaction at ambient temperature till completion. Added de-mineralized water and toluene and stirred at room temperature for half an hour. Separated the toluene layer and concentrated under reduced pressure and to the solid mass so obtained were added 20ml of dimethyl sulfoxide and a solution of 0.38g (0.0231 mol) of sodium hydroxide dissolved in 1.6ml of water followed by addition of 1.5g (0.0132 mol) of 30% w/w of hydrogen peroxide. Stirred the reaction mass at room temperature followed by addition of aqueous solution of sodium sulfite and extracted the compound in ethyl acetate. Washed the organic layer with brine and concentrated under reduced pressure to get 2.1g of crude Silodosin.

Ν,Ν-dialkyl impurity is 2.2% as per HPLC

Method F:

To the solution of lOg (0.0275 mol) of (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) in 100ml of was acetonitrile was added 14.3g (0.0826 mol) of dipotassium hydrogen phosphate and 8.20g (0.0261 mol) of 2-[2-(2,2,2-trifluoroethoxy)phenoxy] ethyl methane sulfonate followed by addition of 2.0g (0.0055 mol) of tetra butyl ammonium iodide and stirred the reaction mass at 85-90°C for 10-12h. Added a solution of 4.4g of sodium hydroxide dissolved in 10ml of water and stirred the reaction at ambient temperature till completion. Added de-mineralized water and toluene and stirred at room temperature for half an hour. Separated the toluene layer and concentrated under reduced pressure and to the solid mass so obtained were added 110ml of dimethyl sulfoxide and a solution of 1.95g (0.0488 mol) of sodium hydroxide dissolved in 7.95ml of water followed by addition of 7.5g (0.066 mol) of 30% w/w of hydrogen peroxide. Stirred the reaction mass at room temperature followed by addition of 210ml of aqueous solution of sodium sulfite and extracted the compound in ethyl acetate. Washed the organic layer with brine and concentrated under reduced pressure to get 9.5g of crude Silodosin.

Ν,Ν-dialkyl impurity is 3.1% as per HPLC

Method G:

To the solution of lOg (0.0275 mol) of (3-(5-((R)-2-aminopropyl)-7-cyanoindolin-l-yl)propyl benzoate) in 100ml of was Dimethyl sulfoxide was added 14.3g (0.0826 mol) of dipotassium hydrogen phosphate and 8.20g (0.0261 mol) of 2-[2-(2,2,2-trifluoroethoxy)phenoxy] ethyl methane sulfonate followed by addition of 4.0g (0.0055 mol) of tetra butyl ammonium iodide and stirred the reaction mass at 85-90°C for 10-12h. Added a solution of 4.4g of sodium hydroxide dissolved in 10ml of water and stirred the reaction at ambient temperature till completion. Added de-mineralized water and toluene and stirred at room temperature for half an hour. Separated the toluene layer and concentrated under reduced pressure and to the solid mass so obtained were added 110ml of dimethyl sulfoxide and a solution of 1.95g (0.0488 mol) of sodium hydroxide dissolved in 7.95ml of water followed by addition of 7.5g (0.066 mol) of 30% w/w of hydrogen peroxide. Stirred the reaction mass at room temperature followed by addition of 210ml of aqueous solution of sodium sulfite and extracted the compound in ethyl acetate. Washed the organic layer with brine and concentrated under reduced pressure to get 10.4g of crude Silodosin.

Ν,Ν-dialkyl impurity is 1.83% as per HPLC

Example 2

Purification of Crude Silodosin:

To the lOg (0.0080 mol) of crude mass of Silodosin was added 110ml of isopropyl alcohol followed by addition of 1.75g of oxalic acid at ambient temperature. Stirred the solution 6-8h and filtered the precipitates. Added ethyl acetate and water in the ratio of 1: 1 to the above solid followed by addition of 5ml of liquor ammonia. Stirred the reaction mass at ambient temperature for 15 min and separated the layers. Concentrated the organic layer to ¼ of its volume and left undisturbed overnight. Filtered the precipitates and recrystallized with ethyl acetate followed by drying under reduced pressure to get 5.1g of pure Silodosin. The amount of impurities and the percent impurity of the Silodosin obtained was as follows:

Ν,Ν-dialkyl impurity: undetectable amount

Other impurities: 0.03 to 0.09%

Silodosin purity: 99.65% (HPLC)

////WO 2016042441, Mankind Research Centre, Silodosin, New patent

RP 6503

phase 1

RP 6503

Mass: 619.1 (M⁺+l). MP: 175-178° C Specific optical rotation (C=l in chloroform, at 25°C) : [a]_D = + 147.16.

A1

RP 6503

(S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl) ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide

(S)-N-[5-[4-amino-1-[1-[5-fluoro-3-(3-fluorophenyl)-4-oxochromen-2-yl]ethyl]pyrazolo[3,4-d]pyrimidin-3-yl]-2-methoxyphenyl]methanesulfonamide

Novartis to develop and commercialize Rhizen’s inhaled dual PI3K-delta gamma inhibitor and related compounds worldwide

The immune pipeline includes ‘dual PI3K inhibitors for various indications’ licensed to Novartis

‘inhaled dual inhibitor’,

Phosphoinositide-3 kinase delta inhibitor; Phosphoinositide-3 kinase gamma inhibitor

WO2011055215A2 and WO2012151525A1 and U.S. Publication Nos. US20110118257 and US20120289496

Rhizen Pharmaceuticals Sa   INNOVATOR

PATENT

http://www.google.com/patents/WO2011055215A2?cl=en

PATENT

http://www.google.com/patents/WO2012151525A1?cl=en

scheme 1A:

Ste -1

Step-2

Scheme 2

SCHEME 3

SCHEME4

List of Intermediates

Intermediate 27: 2-( l -(4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin- l – yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one: To a solution of 3-iodo- l H- pyrazolo[3,4-d]pyrimidin-4-amine (0.800 g, 2.88 mmol) in DMF (5 ml), potassium carbonate (0.398 g, 2.88 mmol) was added and stirred at RT for 30 min. To this mixture intermediate 22 (0.500 g, 1.44 mmol) was added and stirred for 12h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography with methanol: dichloromethane to afford the title compound as a off-white solid (0.300 g, 38%). Ή-NMR (5 ppm, DMSO-d₆₃, 400 MHz): 8.02 (s, 1 H), 7.94 (s, 1 H), 7.84 (dt, J = 8.4,5.7 Hz, 1H), 7.47 (d, 7 = 8.6 Hz, 1H), 7.29 (m, 3H), 7.09 (dt, 7 = 8.8,2.3 Hz, 1 H), 6.87 (s, 2H), 5.88 (q, 7 = 7.0 Hz, 1H), 1.82 (d, 7 = 7.0 Hz, 3H).

SYNTHESIS

MAIN PART

PATENT

http://www.google.com/patents/WO2015198289A1?cl=en

Prashant Kashinath Bhavar, Swaroop Kumar Venkata Satya VAKKALANKA

The present invention relates to a selective dual delta (δ) and gamma (γ) PI3K protein kinase modulator (S)-N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H- chromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl) methane sulfonamide, methods of preparing them, pharmaceutical compositions containing them and methods of treatment, prevention and/or amelioration of PI3K kinase mediated diseases or disorders with them.

compound of formula (Al):

(Al).

The process comprises the steps of:

(a) subjecting (R)-5-fluoro-3-(3-fluorophenyl)-2-(l-hydroxyethyl)-4H-chromen-4-one:

to a Mitsunobu reaction with 3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-4-amine:

(for example, in the presence of triphenylphosphine and diisopropylazodicarboxylate) to give (S)-2-(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one (Intermediate 3):

Intermediate 3;

(b) reducing Intermediate 3, for example with a reducing agent such as Raney Ni, to give (S)-2-(l-(4-amino-3-(3-amino-4-methoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin- l-yl)ethyl)-5-fluoro-3-( -fluorophenyl)-4H-chromen-4-one (Intermediate 4):

Intermediate 4;

The intermediates described herein may be prepared by the methods described in International Publication Nos. WO 11/055215 and WO 12/151525, both of which are hereby incorporated by reference.

Intermediate 1: N-(5-bromo-2-methoxyphenyl)methanesulfonamide:

To a solution of 5-bromo-2-methoxyaniline(1.00 g, 4.94 mmol) in dichloromethane (10 ml), pyridine (0.800 ml, 9.89 mmol) was added and cooled to 0°C. Methane sulphonyl chloride (0.40 ml, 5.19 mmol) was added and stirred for 30 min. The reaction mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was chromatographed with ethyl acetate : petroleum ether to afford the title compound as a reddish solid (1.20 g, 87%).

Intermediate 2: N-(2-methoxy-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)methanesulfonamide: Potassium acetate (0.841 g, 8.57 mmol) and bis(pinacolato)diboron (1.190 g, 4.71 mmol) were added to a solution of intermediate 1 (1.20 g, 4.28 mmol) in dioxane (17.5 ml) and the solution was degassed for 30 min.[l, -Bis(diphenylphosphino)ferrocene]dichloro palladium(II).CH2Ci2 (0.104 g, 0.128 mmol) was added under nitrogen atmosphere and heated to 80°C. After 2h the

reaction mixture was filtered through celite and concentrated. The crude product was purified by column chromatography with ethyl acetate : petroleum ether to afford the title compound as a yellow solid (1.00 g, 71%).^JH-NMR (δ ppm, CDCb, 400 MHz): 7. 91 (d, / = 1.2Hz, 1H), 7. 62 (dd, / = 8.1, 1.2Hz, 1H), 6. 92 (d, / = 8.1Hz, 1H), 6.73 (s, 1H), 3.91 (s, 3H), 2.98 (s, 3H), 1.32 (s, 12H).

Intermediate 3: (S)-2-(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one: (S)-2-(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one: To a solution of (R)-5-fluoro-3-(3-fluorophenyl)-2-(l-hydroxyethyl)-4H-chromen-4-one (0.500 g, 1.64 mmol) in THF (5 ml), 3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-4-amine (0.564 g, 1.97 mmol) and triphenylphosphine (0.649 g, 2.47 mmol) were added followed by the addition of diisopropylazodicarboxylate (0.50 ml, 2.47 mmol). ((R)-5-fluoro-3-(3-fluorophenyl)-2-(l-hydroxyethyl)-4H-chromen-4-one can be prepared as described for Intermediates 23, 25, and 26 in International Publication No. WO 2012/0151525.). After 4h at room temperature, the mixture was concentrated and the residue was purified by column chromatography with ethyl acetate : petroleum ether to afford the title compound as a brown solid (0.270 g, 29%). ^JH-NMR (δ ppm, DMSO-d6, 400 MHz): 8.04 (s, 1H), 7.83 (m, 1H), 7.63-7.50 (m, 3H), 7.29 (m, 2H), 7.06 (dt, J = 8.7,2.2Hz, 1H), 6.94 (m, 2H), 6.75 (dd, J = 8.1,2.1Hz, 1H), 5.95 (q, J = 7.0Hz, 1H), 4.98 (s, 2H), 3.81 (s, 3H), 1.86 (d, J = 7.0 Hz, 3H).

[109] Intermediate 4: (S)-2-(l-(4-amino-3-(3-amino-4-methoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one:

(S)-2-(l-(4-amino-3-(3-amino-4-methoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one : To a solution of Intermediate 3 (0.260 g, 0.455 mmol) in ethanol (5 ml), Raney Ni (0.130 g) was added and hydrogeneated at 20psi at 50°C for 24h. The reaction mixture was passed through celitepad and concentrated to afford the title compound as a brown solid (0.150 g, 60%). Mass : 540.8 (M⁺).

Example A

N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lH- pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide

To a solution of 2-(l-(4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one (0.200 g, 0.366 mmol) in DME (2.1 ml) and water (0.67 ml), intermediate 2 (0.179 g, 0.550 mmol) and sodium carbonate (0.116 g, 1.10 mmol) were added and the system was degassed for 30 min. (2-(l-(4-amino-3-iodo-lH^yrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one can be prepared as described for Intermediates 23, 25, and 26 in International Publication No. WO 2012/0151525). Bis(diphenylphosphino) ferrocene]dichloropalladium(II) (0.059 g, 0.075 mmol) was added and kept under microwave irradiation (microwave power = 100W, temperature = 100 °C) for 45 min. The reaction mixture was Celite filtered, concentrated and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography with methanol: dichloromethane to afford the title compound as a brown solid (0.080 g, 35%). MP: 216-218 °C. ¾-NMR (δ ppm, CDCb, 400 MHz): 8.20 (s, 1H), 7.73 (s, 1H), 7.53 (m, 2H), 7.31 (m, 2H), 7.07-6.73 (m, 6H), 6.07 (q, / = 6.2 Hz, 1H), 3.98 (s, 3H), 3.14 (s, 3H), 2.01 (d, / = 6.0Hz, 3H).

Example Al and A2

Method A

(S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)- lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide

and (R)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2- yl)ethyl)-lH-p anesulfonamide

The two enantiomerically pure isomers were separated by preparative SFC (supercritical fluid) conditions from N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide (0.500 g) on a CHIRALPAK AS-H column (250 x 30 mm; 5μπι) using methanol : CO2 (55:45) as the mobile phase at a flow rate of 80g / min.

Example Al (S-isomer): Brown solid (0.247 g). Enantiomeric excess: 97.4%. Retention time: 2.14 min. Mass: 619.1 (M⁺+l). MP: 156-158° C.

Example A2 (R-isomer): Brown solid (0.182 g). Enantiomeric excess: 99.3%. Retention t: 3.43 min. Mass: 619.1 (M⁺+l). MP: 168-171° C.

Method Al

(S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)- lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide

and (R)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2- yl)ethyl)-lH-p anesulfonamide

The two enantiomerically pure isomers were separated by preparative SFC (supercritical fluid) conditions from N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl) methanesulfonamide (15.0 g) on a CHIRALPAK AS-H column (250 x 20 mm; 5μπι) using methanol : CO2 (45:55) as the mobile phase at a flow rate of 120g / min.

Example Al (S-isomer): Enantiomeric excess: 100 %. Retention time: 2.21 min. Mass: 619.1 (M⁺+l). MP: 175-178° C Specific optical rotation (C=l in chloroform, at 25°C) : [a]_D = + 147.16.

Example A2 (R-isomer): Enantiomeric excess: 99.3%. Retention t: 3.72 min. Mass: 619.1 (M⁺+l). MP: 154-157° C. Specific optical rotation (C=l in chloroform, at 25°C) : [a]_D = – 159.54.

Method B

Example Al

(S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)- lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide

To a solution of Intermediate 4 (0.500 g, 0.923 mmol) in dichloromethane (5 ml) cooled to 0°C, pyridine (0.200 ml, 1.84 mmol) was added and stirred for 10 min. Methanesulphonyl chloride (0.100 ml, 0.923 mmol) was added stirred for 30 min. The reaction mixture was quenched with water, extracted with dichloromethane and dried over sodium sulphate. The crude product was column chromatographed with methanol : dichloromethane to afford the title compound as an off-white solid (0.240 g, 42%). MP: 211-213°C. ¾-NMR (δ ppm, DMSO-d6, 400 MHz): 9.15 (s, 1H), 8.06 (s, 1H), 7.83 (m, 1H), 7.49 (m, 4H), 7.28 (m, 4H), 7.08 (dt, / = 8.6, 1.7 Hz, 1H), 6.92 (s, 2H), 5.98 (q, / = 6.9 Hz, 1H), 3.88 (s, 3H), 2.99 (s, 3H), 1.88 (d, / = 7.0 Hz, 3H). Enantiomeric excess: 85.4% as determined by HPLC on a chiralpak AS-3R column, enriched in the fast eluting isomer (retention time = 7.46 min.).

CLIPS

La Chaux-de-Fonds, Switzerland, Sept. 6, 2013  — La Chaux-de-Fonds, Switzerland (6 September 2013): Rhizen Pharmaceuticals S.A. announces a scientific poster presentation on the pre-clinical characterization of its lead calcium release activated channel (CRAC) inhibitor, RP3128, for the treatment of respiratory disorders and an oral presentation on the pharmacological profile of its novel, dual Phosphoinositide-3 kinase (PI3K) delta/gamma inhibitor, RP6503, in the pulmonary disease systems, at the European Respiratory Society Annual Congress (ERS), to be held from 7-11 September 2013, at Barcelona, Spain.

RP6503 is a novel, potent and selective inhibitor of the delta and gamma isoforms of PI3K. It is to be delivered via the inhalation route and has a long duration of action along with excellent PI3K isoform selectivity, which is expected to result in better safety. RP3128 has been optimized with high potency for CRAC channel inhibition, selectivity over the other voltage gated channels and excellent oral bioavailability. Rhizen intends to move both these compounds to the clinic in 2014.

Details of the presentations:

1.      Abstract of the Poster Presentation: “Pre-clinical characterization of RP3128, a novel and potent CRAC channel inhibitor for the treatment of respiratory disorders”

Time and Location- 8 September 2013 between 14.45-16.45 in Room 3.6, at Poster Discussion: New drugs in respiratory medicine, at FIRA BARCELONA, Convention Centre de Gran Via, Barcelona, Spain

2.      Abstract of Oral Presentation: “In vitro and in vivo pharmacological profile of RP6503, a novel dual PI3K delta/gamma inhibitor, in pulmonary disease systems”

Time and Location- 11 September 2013 at 8.45 in Room 3.9; Session 8.30-10.30, at the Oral Presentation: Emerging new targets for the treatment of respiratory diseases, at FIRA BARCELONA, Convention Centre de Gran Via, Barcelona, Spain

CLIPS

La Chaux-de-Fonds, Switzerland , Dec. 09, 2015  — Rhizen Pharmaceuticals S.A. announced today that they have entered into an exclusive, worldwide license agreement with Novartis for the development and commercialization of Rhizen’s, inhaled dual PI3K-delta gamma inhibitor and its closely related compounds for various indications.

Under the terms of the agreement, Rhizen will receive an upfront payment and is eligible to receive development, regulatory and sales milestones payments. In addition Rhizen is also eligible to receive tiered royalties on annual nets sales.

The lead compound is a novel, potent, and selective dual PI3K-delta gamma inhibitor with demonstrated anti-inflammatory and immuno-modulatory activity in pre-clinical systems and models representative of respiratory diseases. With a favorable ADME and PK profile and high therapeutic index in animals, the inhaled dual PI3K-delta gamma inhibitor holds promise in the treatment of human airway disorders.

About Rhizen Pharmaceuticals S.A.:

Rhizen Pharmaceuticals is an innovative, clinical-stage biopharmaceutical company focused on the discovery and development of novel therapeutics for the treatment of cancer, immune and metabolic disorders. Since its establishment in 2008, Rhizen has created a diverse pipeline of proprietary drug candidates targeting several cancers and immune associated cellular pathways. Rhizen is headquartered in La-Chaux-de-Fonds, Switzerland. For additional information, please visit Rhizen’s website, http://www.rhizen.com.

SEE

https://newdrugapprovals.org/2015/12/10/alembic-pharma-advances-1-on-rhizen-novartis-license-agreement/

WO-2015181728 

WO-2015001491 

WO-2014072937 

WO-2014006572 

http://www.atsjournals.org/doi/abs/10.1164/ajrccm-conference.2013.187.1_MeetingAbstracts.A3880

///////RP 6503, Novartis, develop, commercialize,  Rhizen, inhaled dual PI3K-delta gamma inhibitor, PHASE 1, RP-6503

c21c(cccc1O/C(=C(\C2=O)c3cc(ccc3)F)C(C)n4c6ncnc(c6c(n4)c5cc(c(cc5)OC)NS(=O)(=O)C)N)F

CC(C1=C(C(=O)C2=C(O1)C=CC=C2F)C3=CC(=CC=C3)F)N4C5=C(C(=N4)C6=CC(=C(C=C6)OC)NS(=O)(=O)C)C(=NC=N5)N

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