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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 29 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 29 year tenure till date Aug 2016, Around 30 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 25 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 13 lakh plus views on New Drug Approvals Blog in 212 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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FDA approves Xermelo (telotristat ethyl) for carcinoid syndrome diarrhea


ChemSpider 2D Image | Telotristat ethyl | C27H26ClF3N6O3Image result for telotristat ethyl

 

Telotristat ethyl

Molecular Formula, C27-H26-Cl-F3-N6-O3,

Molecular Weight, 574.9884,

RN: 1033805-22-9
UNII: 8G388563M

LX 1032

(2S)-2-Amino-3-[4-[2-amino-6-[[(1R)-1-[4-chloro-2-(3-methylpyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl]oxy]pyrimidin-4-yl]phenyl]propionic acid ethyl ester

Ethyl-4-(2-amino-6-{(1R)-1-[4-chlor-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluorethoxy}-4-pyrimidinyl)-L-phenylalaninat

L-Phenylalanine, 4-[2-amino-6-[(1R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethoxy]-4-pyrimidinyl]-, ethyl ester
SEE……………
Image result for Telotristat etiprate,LX1606 Hippurate.png
Telotristat etiprate,
(S)-ethyl 2-amino-3-(4-(2-amino-6-((R)-1-(4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoate 2-benzamidoacetate .
CAS: 1137608-69-5 (etiprate), LX 1606
Chemical Formula: C36H35ClF3N7O6
Molecular Weight: 754.16
L-Phenylalanine, 4-[2-amino-6-[(1R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethoxy]-4-pyrimidinyl]-, ethyl ester, compd. with N-benzoylglycine (1:1)
  • LX 1032 hippurate
  • LX 1606
SEE ALSO………….
Telotristat, also known as LX1033, 1033805-28-5 CAS OF ACID FORM
 Arokiasamy Devasagayaraj
02/28/2017
The U.S. Food and Drug Administration today approved Xermelo (telotristat ethyl) tablets in combination with somatostatin analog (SSA) therapy for the treatment of adults with carcinoid syndrome diarrhea that SSA therapy alone has inadequately controlled.
February 28, 2017
The U.S. Food and Drug Administration today approved Xermelo (telotristat ethyl) tablets in combination with somatostatin analog (SSA) therapy for the treatment of adults with carcinoid syndrome diarrhea that SSA therapy alone has inadequately controlled.

Carcinoid syndrome is a cluster of symptoms sometimes seen in people with carcinoid tumors. These tumors are rare, and often slow-growing. Most carcinoid tumors are found in the gastrointestinal tract. Carcinoid syndrome occurs in less than 10 percent of patients with carcinoid tumors, usually after the tumor has spread to the liver. The tumors in these patients release excess amounts of the hormone serotonin, resulting in diarrhea. Complications of uncontrolled diarrhea include weight loss, malnutrition, dehydration, and electrolyte imbalance.

“Today’s approval will provide patients whose carcinoid syndrome diarrhea is not adequately controlled with another treatment option,” said Julie Beitz, M.D., director of the Office of Drug Evaluation III in the FDA’s Center for Drug Evaluation and Research.

Xermelo, in a regimen with SSA therapy, is approved in tablet form to be taken orally three times daily with food. Xermelo inhibits the production of serotonin by carcinoid tumors and reduces the frequency of carcinoid syndrome diarrhea.

The safety and efficacy of Xermelo were established in a 12-week, double-blind, placebo-controlled trial in 90 adult participants with well-differentiated metastatic neuroendocrine tumors and carcinoid syndrome diarrhea. These patients were having between four to 12 daily bowel movements despite the use of SSA at a stable dose for at least three months. Participants remained on their SSA treatment, and were randomized to add placebo or treatment with Xermelo three times daily. Those receiving Xermelo added on to their SSA treatment experienced a greater reduction in average bowel movement frequency than those on SSA and placebo. Specifically, 33 percent of participants randomized to add Xermelo on to SSA experienced an average reduction of two bowel movements per day compared to 4 percent of patients randomized to add placebo on to SSA.

The most common side effects of Xermelo include nausea, headache, increased levels of the liver enzyme gamma-glutamyl transferase, depression, accumulation of fluid causing swelling (peripheral edema), flatulence, decreased appetite and fever. Xermelo may cause constipation, and the risk of developing constipation may be increased in patients whose bowel movement frequency is less than four bowel movements per day. Patients treated with a higher than recommended dosage of Xermelo developed severe constipation in clinical trials. One patient required hospitalization and two other patients developed complications of either intestinal perforation or intestinal obstruction. Patients should be monitored for severe constipation. If a patient experiences severe constipation or severe, persistent or worsening abdominal pain, they should discontinue Xermelo and contact their healthcare provider.

The FDA granted this application fast track designation and priority review. The drug also received orphan drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.

Xermelo is manufactured by Woodlands, Texas-based Lexicon Pharmaceuticals, Inc.

SYNTHESIS…….WO 2011100285

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011100285&recNum=142&docAn=US2011024141&queryString=((serotonin)%2520OR%2520(HT2C)%2520OR%2520(&

5.67. Synthesis of (S)-2-Amino-3-[4-(2-amino-6-{R-l-[4-chloro-2-(3-methyl-pyrazol-l-yll- phenyll-2,2,2-trifluoro-ethoxy)-pyrimidin-4-yl)-phenyll-propionic acid ethyl ester

The title compound was prepared stepwise, as described below:

Step 1: Synthesis of l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone. To a 500 ml 2 necked RB flask containing anhydrous methanol (300 ml) was added thionyl chloride (29.2 ml, 400 mmol) dropwise at 0-5°C (ice water bath) over 10 minutes. The ice water bath was removed, and 2-bromo-4-chloro-benzoic acid (25 g, 106 mmol) was added. The mixture was heated to mild reflux for 12h. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was concentrated. Crude product was dissolved in dichloromethane (DCM, 250 ml), washed with water (50 ml), sat. aq. NaHC03 (50 ml), brine (50 ml), dried over sodium sulfate, and concentrated to give the 2- bromo-4-chloro-benzoic acid methyl ester (26 g, 99 %), which was directly used in the following step.

2-Bromo-4-chloro-benzoic acid methyl ester (12.4 g, 50 mmol) in toluene (200 ml) was cooled to -70°C, and trifluoromethyl trimethyl silane (13 ml, 70 mmol) was added.

Tetrabutylamonium fluoride (1M, 2.5 ml) was added dropwise, and the mixture was allowed to warm to room temperature over 4h, after which it was stirred for 10 hours at room temperature. The reaction mixture was concentrated to give the crude [l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-l-methoxy-ethoxy]-trimethyl-silane. The crude intermediate was dissolved in methanol (100 ml) and 6N HCI (100 ml) was added. The mixture was kept at 45-50°C for 12h. Methanol was removed, and the crude was extracted with dichloromethane (200 ml). The combined DCM layer was washed with water (50 ml), NaHC03 (50 ml), brine (50 ml), and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography, using 1-2% ethyl acetate in hexane as solvent, to afford l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone (10 g, 70%). !H-NMR (300 MHz, CDC ): δ (ppm) 7.50 (d,lH), 7.65(d,lH), 7.80(s,lH).

Step 2: Synthesis of R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol. To catechol borane (1M in THF 280 ml, 280 mmol) in a 2L 3-necked RB flask was added S-2-methyl-CBS oxazaborolidine (7.76 g, 28 mmol) under nitrogen, and the resulting mixture was stirred at room temperature for 20 min. The reaction mixture was cooled to -78°C (dry ice/acetone bath), and 1-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone (40 g, 139 mmol) in THF (400 ml) was added dropwise over 2 hours. The reaction mixture was allowed to warm to -36°C, and was stirred at that temperature for 24 hours, and further stirred at -32 °C for another 24h. 3N NaOH (250 ml) was added, and the cooling bath was replaced by ice-water bath. Then 30 % hydrogen peroxide in water (250 ml) was added dropwise over 30 minutes. The ice water bath was removed, and the mixture was stirred at room temperature for 4 hours. The organic layer was separated, concentrated and re-dissolved in ether (200 ml). The aqueous layer was extracted with ether (2 x 200 ml). The combined organic layers were washed with IN aq. NaOH (4 x 100 ml), brine, and dried over sodium sulfate. Removal of solvent gave crude product which was purified by column chromatography using 2 to 5% ethyl acetate in hexane as solvent to give desired alcohol 36.2 g (90 %, e.e. >95%). The alcohol (36.2 g) was crystallized from hexane (80 ml) to obtain R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol 28.2 g (70 %; 99-100 % e.e.). !H-NMR (400 MHz, CDCIs) δ (ppm) 5.48 (m, 1H), 7.40 (d, 1H), 7.61 (d, 2H).

Step 3: Synthesis of R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyll-2.2.2-trifluoro-ethanol. R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol (15.65 g, 54.06 mmol), 3-methylpyrazole (5.33 g, 65 mmol), Cul (2.06 g, 10.8 mmol), 2CO3 (15.7 g, 113.5 mmol), (lR,2R)-N,N’-dimethyl-cyclohexane-l,2-diamine (1.54 g, 10.8 mmol) and toluene (80 ml) were combined in a 250 ml pressure tube and heated to 130°C (oil bath temperature) for 12 hours. The reaction mixture was diluted with ethyl acetate and washed with H2O (4 x 100 ml), brine, and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography using 5-10 % ethyl acetate in hexane as solvent to get R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (13.5 g; 86 %). i-H-NMR (400 MHz, CDC ): δ (ppm) 2.30(s, 3H), 4.90(m, 1H), 6.20(s, 1H), 6.84(d, 1H), 7.20(s, 1H), 7.30(d, 1H), 7.50(d, 1H).

Step 4: Synthesis of (S)-2-Amino-3- 4-(2-amino-6-fR-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyll^^^-trifluoro-ethoxyl-pyrimidin^-yll-phenvD-propionic acid ethyl ester. R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (17.78 g, 61.17 mmol), (S)-3-[4-(2-amino-6-chloro-pyrimidine-4-yl)-phenyl]-2-tert-butoxycarbonylamino-propionic acid (20.03 g, 51 mmol), 1,4-dioxane (250 ml), and CS2CO3 (79.5 g, 244 mmol) were combined in a 3-necked 500 ml RB flask and heated to 100°C (oil bath temperature) for 12-24 hours. The progress of reaction was monitored by LCMS. After the completion of the reaction, the mixture was cooled to 60°C, and water (250 ml) and THF (400 ml) were added. The organic layer was separated and washed with brine (150 ml). The solvent was removed to give crude BOC protected product, which was taken in THF (400 ml), 3N HCI (200 ml). The mixture was heated at 35-40 °C for 12 hours. THF was removed in vacuo. The remaining aqueous layer was extracted with isopropyl acetate (2x 100 ml) and concentrated separately to recover the unreacted alcohol (3.5 g). Traces of remaining organic solvent were removed from the aqueous fraction under vacuum.

To a 1L beaker equipped with a temperature controller and pH meter, was added H3PO4 (40 ml, 85 % in water) and water (300 ml) then 50 % NaOH in water to adjust pH to 6.15. The temperature was raised to 58 °C and the above acidic aqueous solution was added dropwise into the buffer with simultaneous addition of 50 % NaOH solution in water so that the pH was maintained between 6.1 to 6.3. Upon completion of addition, precipitated solid was filtered and washed with hot water (50-60°C) (2 x 200 ml) and dried to give crude (S)-2-amino-3-[4-(2-amino-6-[R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethoxy}-pyrimidin-4-yl)-phenyl}^ propionic acid (26.8 g; 95 %). LCMS and HPLC analysis indicated the compound purity was about 96-97 %.

To anhydrous ethanol (400 ml) was added SOC (22 ml, 306 mmol) dropwise at 0-5°C.

Crude acid (26.8 ) from the above reaction was added. The ice water bath was removed, and the reaction mixture was heated at 40-45°C for 6-12 hours. After the reaction was completed, ethanol was removed in vacuo. To the residue was added ice water (300 ml), and extracted with isopropyl acetate (2 x 100 ml). The aqueous solution was neutralized with saturated Na2C03 to adjust the pH to 6.5. The solution was extracted with ethyl acetate (2 x 300 ml). The combined ethyl acetate layer was washed with brine and concentrated to give 24 g of crude ester (HPLC purity of 96-97 %). The crude ester was then purified by ISCO column chromatography using 5 % ethanol in DCM as solvent to give (S)-2-amino-3-[4-(2-amino-6-{R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethoxy}-pyrimidin-4-yl)-phenyl}-propionic acid ethyl ester (20.5g; 70 %; HPLC purity of 98 %). LCMS M+l = 575. !H-NMR (400 MHz, CDsOD): δ (ppm) 1.10 (t, 3H), 2.25 (s, 3H), 2.85 (m, 2H), 3.65 (m, IH), 4.00 (q, 2H), 6.35 (s, IH), 6.60 (s, IH), 6.90 (m, IH), 7.18 (d, 2H), 7.45 (m, 2H), 7.70 (d, IH), 7.85 (m, 3H).

SYNTHESIS OF INTERMEDIATE

WO 2009048864

https://google.com/patents/WO2009048864A1?cl=en

6.15. Preparation of 6SV3-(4-(2-Amino-6-chloropyrimidin-4-yl)phenyl)-2- (fert-butoxycarbonylamino)propanoic Acid Using the Lithium Salt of (S)-2-(te^-butoxycarbonylamino)-3-(4-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)phenyl)propanoic Acid

Figure imgf000021_0001

During preparation of compound 7, the isolation of the free acid can be optionally omitted. Thus, an aqueous solution of the lithium salt of compound 7 in 100 ml water, prepared from 5.0 g of Boc-Tyr-OMe (4, 17 mmol), was mixed 2-amino-4,6- dichloropyrimidine (3.3 g, 1.2 eq), potassium bicarbonate (5.0 g, 3 eq), bis(triphenylphosphine)palladium(II) dichloride (60 mg, 0.5 mol%), and 100 ml ethanol. The resulting mixture was heated at 700C for 5 hours. Additional 2-amino-4,6- dichloropyrimidine (1.1 g, 0.4 eq) was added and heating was continued at 7O0C for an additional 2 hours. HPLC analysis showed about 94% conversion. Upon cooling and filtration, the filtrate was analyzed by HPLC against a standard solution of compound 8. The assay indicated 3.9 g compound 8 was contained in the solution (59% yield from compound 4).

6.16. Alternative Procedure for Preparation of (S)-3-(4-f2-Amino-6- chloropyrimidin-4-yl)phenyl)-2-(fe^-butoxycarbonylamino)propanoic Acid Using Potassium Carbonate as Base

Figure imgf000021_0002

The boronic acid compound 11 (Ryscor Science, Inc., North Carolina, 1.0 g, 4.8 mmol) and potassium carbonate (1.32 g, 2 eq) were mixed in aqueous ethanol (15 ml ethanol and 8 ml water). Di-ter£-butyldicarbonate (1.25 g, 1.2 eq) was added in one portion. After 30 minutes agitation at room temperature, HPLC analysis showed complete consumption of the starting compound 11. The 2-amino-4,6- dichloropyrimidine (1.18 g, 1.5 eq) and the catalyst bis(triphenylphosphine)palladium(II) dichloride (34 mg, 1 mol%) were added and the resulting mixture was heated at 65-700C for 3 hours. HPLC analysis showed complete consumption of compound 12. After concentration and filtration, HPLC analysis of the resulting aqueous solution against a standard solution of compound 8 showed 1.26 g compound 8 (67% yield).

6.17. Alternative procedure for preparation of (5)-3-(4-(2-Amino-6-

Figure imgf000022_0001

The boronic acid compound 11 (10 g, 48 mmol) and potassium bicarbonate (14.4 g, 3 eq) were mixed in aqueous ethanol (250 ml ethanol and 50 ml water). Oi-tert- butyldicarbonate (12.5 g, 1.2 eq) was added in one portion. HPLC analysis indicated that the reaction was not complete after overnight stirring at room temperature. Potassium carbonate (6.6 g, 1.0 eq) and additional di-te/t-butyldicarbonate (3.1 g, 0.3 eq) were added. After 2.5 hours agitation at room temperature, HPLC analysis showed complete consumption of the starting compound 11. The 2-amino-4,6-dichloropyrimidine (11.8 g, 1.5 eq) and the catalyst bis(triphenylphosphine)-palladium(II) dichloride (0.34 g, 1 mol%” were added and the resulting mixture was heated at 75-8O0C for 2 hours. HPLC analysis showed complete consumption of compound 12. The mixture was concentrated under reduced pressure and filtered. The filtrate was washed with ethyl acetate (200 ml) and diluted with 3 : 1 THF/MTBE (120 ml). This mixture was acidified to pH about 2.4 by 6 N hydrochloric acid. The organic layer was washed with brine and concentrated under reduced pressure. The residue was precipitated in isopropanol, filtered, and dried at 500C under vacuum to give compound 8 as an off-white solid (9.0 g, 48% yield). Purity: 92.9% by HPLC analysis. Concentration of the mother liquor yielded and additional 2.2 g off-white powder (12% yield). Purity: 93.6% by HPLC analysis

PATENT

https://www.google.com/patents/WO2013059146A1?cl=en

This invention is directed to solid pharmaceutical dosage forms in which an active pharmaceutical ingredient (API) is (S)-ethyl 2-amino-3-(4-(2-amino-6-((R)-l-(4-chloro-2-(3- methyl-lH-pyrazol-l-yl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoate

(telotristat):

Figure imgf000004_0001

or a pharmaceutically acceptable salt thereof. The compound, its salts and crystalline forms can be obtained by methods known in the art. See, e.g., U.S. patent no. 7,709,493.

PATENT

http://www.google.co.in/patents/WO2008073933A2?cl=en

6.19. Synthesis of (S)-2-Amino-3-r4-q-amino-6-{R-l-r4-chloro-2-(3-methyl- Pyrazol-l-yl)-phenyll-2,2,2-trifluoro-ethoxy}-pyrimidin-4-yl)-phenyll- propionic acid ethyl ester

Figure imgf000042_0001

The title compound was prepared stepwise, as described below: Step 1 : Synthesis of l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone. To a 500 ml 2 necked RB flask containing anhydrous methanol (300 ml) was added thionyl chloride (29.2 ml, 400 mmol) dropwise at 0-50C (ice water bath) over 10 min. The ice water bath was removed, and 2-bromo-4-chloro-benzoic acid (25 g, 106 mmol) was added. The mixture was heated to mild reflux for 12h. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was concentrated. Crude product was dissolved in dichloromethane (DCM, 250 ml), washed with water (50 ml), sat. aq. NaHCO3 (50 ml), brine (50 ml), dried over sodium sulfate, and concentrated to give the 2- bromo-4-chloro-benzoic acid methyl ester (26 g, 99 %), which was directly used in the following step.

2-Bromo-4-chloro-benzoic acid methyl ester (12.4 g, 50 mmol) in toluene (200 ml) was cooled to -700C, and trifluoromethyl trimethyl silane (13 ml, 70 mmol) was added. Tetrabutylamonium fluoride (IM, 2.5 ml) was added dropwise, and the mixture was allowed to warm to room temperature over 4h, after which it was stirred for 1Oh at room temperature. The reaction mixture was concentrated to give the crude [l-(2-bromo-4-chloro-phenyl)-2,2,2- trifluoro-l-methoxy-ethoxy]-trimethyl-silane. The crude intermediate was dissolved in methanol (100 ml) and 6N HCl (100 ml) was added. The mixture was kept at 45-500C for 12h. Methanol was removed, and the crude was extracted with dichloromethane (200 ml). The combined DCM layer was washed with water (50 ml), NaHCO3 (50 ml), brine (50 ml), and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography, using 1-2% ethyl acetate in hexane as solvent, to afford 1- (2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone (10 g, 70%). 1H-NMR (300 MHz, CDCl3): δ (ppm) 7.50 (d,lH), 7.65(d,lH), 7.80(s,lH).

Step 2: Synthesis of R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol. To catechol borane (IM in THF 280 ml, 280 mmol) in a 2L 3-necked RB flask was added S-2- methyl-CBS oxazaborolidine (7.76 g, 28 mmol) under nitrogen, and the resulting mixture was stirred at room temperature for 20 min. The reaction mixture was cooled to -78°C (dry ice/acetone bath), and l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone (40 g, 139 mmol) in THF (400 ml) was added dropwise over 2h. The reaction mixture was allowed to warm to -36°C, and was stirred at that temperature for 24 h, and further stirred at -32°C for another 24h. 3N NaOH (250 ml) was added, and the cooling bath was replaced by ice-water bath. Then 30 % hydrogen peroxide in water (250 ml) was added dropwise over 30 minutes. The ice water bath was removed, and the mixture was stirred at room temperature for 4h. The organic layer was separated, concentrated and re-dissolved in ether (200 ml). The aqueous layer was extracted with ether (2 x 200 ml). The combined organic layers were washed with IN aq. NaOH (4 x 100 ml), brine, and dried over sodium sulfate. Removal of solvent gave crude product which was purified by column chromatography using 2 to 5% ethyl acetate in hexane as solvent to give desired alcohol 36.2 g (90 %, e.e. >95%). The alcohol (36.2 g) was crystallized from hexane (80 ml) to obtain R-l-(2-bromo-4-chloro- phenyl)-2,2,2-trifiuoro-ethanol 28.2 g (70 %; 99-100 % e.e.). 1H-NMR (400 MHz, CDCl3) δ (ppm) 5.48 (m, IH), 7.40 (d, IH), 7.61 (d, 2H). Step 3: Synthesis of R-l-r4-chloro-2-(3-methyl-pyrazol-l-vπ-phenyl1-2.2.2-trifluoro- ethanol. R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol (15.65g, 54.06 mmol), 3- methylpyrazole (5.33 g, 65 mmol), CuI (2.06 g, 10.8 mmol), K2CO3 (15.7 g, 113.5 mmol), (lR,2R)-N,N’-dimethyl-cyclohexane-l,2-diamine (1.54 g, 10.8 mmol) and toluene (80 ml) were combined in a 250 ml pressure tube and heated to 1300C (oil bath temperature) for 12 h. The reaction mixture was diluted with ethyl acetate and washed with H2O (4 x 100 ml), brine, and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography using 5-10 % ethyl acetate in hexane as solvent to get R-I- [4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (13.5 g; 86 %). 1H-NMR (400 MHz, CDCl3): δ (ppm) 2.30(s, 3H), 4.90(m, IH), 6.20(s, IH), 6.84(d, IH), 7.20(s, IH), 7.30(d, IH), 7.50(d, IH).

Step 4: Synthesis of (S)-2-Amino-3- r4-(2-amino-6- (R-I- r4-chloro-2-(3-methyl- pyrazol- 1 -ylVphenyl~|-2,2.,2-trifluoro-ethoxy| -pyrimidin-4-yl)-phenyU -propionic acid ethyl ester. R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (17.78 g, 61.17 mmol), (S)-3-[4-(2-amino-6-chloro-pyrimidine-4-yl)-phenyl]-2-tert- butoxycarbonylamino-propionic acid (20.03 g, 51 mmol), 1,4-dioxane (250 ml), and Cs2CO3 (79.5 g, 244 mmol) were combined in a 3-necked 500 ml RB flask and heated to 1000C (oil bath temperature) for 12-24 h. The progress of reaction was monitored by LCMS. After the completion of the reaction, the mixture was cooled to 600C, and water (250 ml) and THF (400 ml) were added. The organic layer was separated and washed with brine (150 ml). The solvent was removed to give crude BOC protected product, which was taken in THF (400 ml), 3N HCl (200 ml). The mixture was heated at 35-400C for 12h. THF was removed in vacuo. The remaining aqueous layer was extracted with isopropyl acetate (2x 100 ml) and concentrated separately to recover the unreacted alcohol (3.5 g). Traces of remaining organic solvent were removed from the aqueous fraction under vacuum.

To a IL beaker equipped with a temperature controller and pH meter, was added H3PO4 (40 ml, 85 % in water) and water (300 ml) then 50 % NaOH in water to adjust pH to 6.15. The temperature was raised to 58°C and the above acidic aqueous solution was added dropwise into the buffer with simultaneous addition of 50 % NaOH solution in water so that the pH was maintained between 6.1 to 6.3. Upon completion of addition, precipitated solid was filtered and washed with hot water (50-600C) (2 x 200 ml) and dried to give crude (S)-2- amino-3-[4-(2-amino-6-{R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro- ethoxy}-pyrimidin-4-yl)-phenyl} -propionic acid (26.8 g; 95 %). LCMS and HPLC analysis indicated the compound purity was about 96-97 %. To anhydrous ethanol (400 ml) was added SOCl2 (22 ml, 306 mmol) dropwise at 0-

5°C. Crude acid (26.8 g ) from the above reaction was added. The ice water bath was removed, and the reaction mixture was heated at 40-450C for 6-12h. After the reaction was completed, ethanol was removed in vacuo. To the residue was added ice water (300 ml), and extracted with isopropyl acetate (2 x 100 ml). The aqueous solution was neutralized with saturated Na2CO3 to adjust the pH to 6.5. The solution was extracted with ethyl acetate (2 x 300 ml). The combined ethyl acetate layer was washed with brine and concentrated to give 24 g of crude ester (HPLC purity of 96-97 %). The crude ester was then purified by ISCO column chromatography using 5 % ethanol in DCM as solvent to give (S)-2-amino-3-[4-(2- amino-6- (R- 1 -[4-chloro-2-(3-methyl-pyrazol- 1 -yl)-phenyl]-2,2,2-trifluoro-ethoxy} – pyrimidin-4-yl)-phenyl} -propionic acid ethyl ester (20.5g; 70 %; HPLC purity of 98 %). LCMS M+l = 575. 1H-NMR (400 MHz, CD3OD): δ (ppm) 1.10 (t, 3H), 2.25 (s, 3H), 2.85 (m, 2H), 3.65 (m, IH), 4.00 (q, 2H), 6.35 (s, IH), 6.60 (s, IH), 6.90 (m, IH), 7.18 (d, 2H), 7.45 (m, 2H), 7.70 (d, IH), 7.85 (m, 3H).

PATENT

WO 2011056916

https://www.google.com/patents/WO2011056916A1?cl=en

PATENT

WO 2010065333

https://www.google.com/patents/WO2010065333A1?cl=en

CLIP,……..PL CHECK ERROR

CONFUSION ON CODES, CLEAR PIC BELOW……LINK
Description of Telotristat Etiprate
Telotristat etiprate is the hippurate salt of telotristat ethyl.
Telotristat ethyl, also known as LX1032, has the chemical name, CAS identifier, and chemical structure shown below:
Chemical name: (S)-ethyl 2-amino-3-(4-(2-amino-6-((R)-1-(4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoate
CAS Registry number: 1033805-22-9
Chemical structure:
Telotristat etiprate, also known as LX1606, is the hippurate salt of telotristat ethyl, and has the chemical name, CAS identifier, and chemical structure shown below:
Chemical Name: (S)-ethyl 2-amino-3-(4-(2-amino-6-((R)-1-(4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoate 2-benzamidoacetate
CAS Registry number: 1137608-69-5
Chemical Structure:
Description of LX1033
Telotristat, also known as LX1033, has the chemical name, CAS identifier and chemical structure shown below:
Chemical Name: (S)-2-amino-3-(4-(2-amino-6-((R)-1-(4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
CAS Registry number: 1033805-28-5
Chemical Structure:

REFERENCES

Kulke, M.H.; Hoersch, D.; Caplin, M.E.; et al.
Telotristat ethyl, a tryptophan hydroxylase inhibitor for the treatment of carcinoid syndrome
J Clin Oncol 2017, 35(1): 14

WO2010056992A1 * Nov 13, 2009 May 20, 2010 The Trustees Of Columbia University In The City Of New York Methods of preventing and treating low bone mass diseases
US7709493 May 20, 2009 May 4, 2010 Lexicon Pharmaceuticals, Inc. 4-phenyl-6-(2,2,2-trifluoro-1-phenylethoxy)pyrimidine-based compounds and methods of their use
US20090088447 * Sep 25, 2008 Apr 2, 2009 Bednarz Mark S Solid forms of (s)-ethyl 2-amino-3-(4-(2-amino-6-((r)-1-(4-chloro-2-(3-methyl-1h-pyrazol-1-yl)phenyl)-2,2,2-trifluoroethoxy)-pyrimidin-4-yl)phenyl)propanoate and methods of their use
Citing Patent Filing date Publication date Applicant Title
US9199994 Sep 5, 2014 Dec 1, 2015 Karos Pharmaceuticals, Inc. Spirocyclic compounds as tryptophan hydroxylase inhibitors
US9512122 Sep 1, 2015 Dec 6, 2016 Karos Pharmaceuticals, Inc. Spirocyclic compounds as tryptophan hydroxylase inhibitors

///////////telotristat ethyl, fast track designation,priority review,orphan drug designation, Xermelo ,  Woodlands, Texas-based,  Lexicon Pharmaceuticals, Inc, fda 2017, LX 1606, LX 1032

O=C(OCC)[C@@H](N)Cc1ccc(cc1)c2cc(nc(N)n2)O[C@H](c3ccc(Cl)cc3n4ccc(C)n4)C(F)(F)F

O=C(OCC)[C@@H](N)CC1=CC=C(C2=NC(N)=NC(O[C@H](C3=CC=C(Cl)C=C3N4N=C(C)C=C4)C(F)(F)F)=C2)C=C1.O=C(O)CNC(C5=CC=CC=C5)=O

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Deflazacort


Deflazacort structure.svgChemSpider 2D Image | Deflazacort | C25H31NO6

Deflazacort

  • CAS 14484-47-0
  • Molecular Formula C25H31NO6
  • Average mass 441.517 Da
(11b,16b)-21-(Acetyloxy)-11-hydroxy-2′-methyl-5’H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione
11b,21-Dihydroxy-2′-methyl-5’bH-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione 21-acetate
2-[(4aR,4bS,5S,6aS,6bS,9aR,10aS,10bS)-5-Hydroxy-4a,6a,8-trimethyl-2-oxo-2,4a,4b,5,6,6a,9a,10,10a,10b,11,12-dodecahydro-6bH-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]oxazol-6b-yl]-2-oxoethyl acetate
  • 5’βH-Pregna-1,4-dieno[17,16-d]oxazole-3,20-dione, 11β,21-dihydroxy-2′-methyl-, 21-acetate (8CI)
  • (11β,16β)-21-(Acetyloxy)-11-hydroxy-2′-methyl-5’H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione
  • 2H-Naphth[2′,1′:4,5]indeno[1,2-d]oxazole, 5’H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione deriv.
  • Azacort
  • Azacortinol
  • Calcort
  • DL 458IT
  • Deflan
Optical Rotatory Power +62.3 ° Conc: 0.5 g/100mL; Solv: chloroform (67-66-3); Wavlength: 589.3 nm

…………..REF, “Drugs – Synonyms and Properties” data were obtained from Ashgate Publishing Co. (US)Hoechst Marion Roussel (now Aventis Pharma) has developed and launched Deflazacort (Dezacor; Flantadin; Lantadin; Calcort) a systemic corticosteroid developed for the treatment of a variety of inflammatory conditions .

In March 1990, the drug was approved in Spain, and by January 2013, the drug had been launched by FAES Farma . By the end of 1999, the product had been launched in Germany, Italy, Belgium, Switzerland and South Korea

Deflazacort is a corticosteroid first launched in 1985 by Guidotti in Europe for the oral treatment of allergic asthma, rheumatoid arthritis, arthritis, and skin allergy.

In 2017, an oral formulation developed at Marathon Pharmaceuticals was approved by the FDA for the treatment of Duchenne’s muscular dystrophy in patients 5 years of age and older.

Deflazacort (trade name Emflaza or Calcort among others) is a glucocorticoid used as an anti-inflammatory and immunosuppressant.

In 2013, orphan drug designation in the U.S. was assigned to the compound for the treatment of Duchenne’s muscular dystrophy. In 2015, additional orphan drug designation in the U.S. was assigned for the treatment of pediatric juvenile idiopathic arthritis (JIA) excluding systemic JIA.

Also in 2015, deflazacort was granted fast track and rare pediatric disease designations in the U.S. for the treatment of Duchenne’s muscular dystrophy.

Deflazacort is a glucocorticoid used as an anti-inflammatory and immunosuppressant. It was approved in February, 2017 by the FDA for use in treatment of Duchenne muscular dystrophy (trade name Emflaza).
  • Aventis Pharma (Originator), Lepetit (Originator), Guidotti (Licensee), Shire Laboratories (Licensee)

Image result for deflazacort

February 9, 2017 FDA approved

The U.S. Food and Drug Administration today approved Emflaza (deflazacort) tablets and oral suspension to treat patients age 5 years and older with Duchenne muscular dystrophy (DMD), a rare genetic disorder that causes progressive muscle deterioration and weakness. Emflaza is a corticosteroid that works by decreasing inflammation and reducing the activity of the immune system.

Corticosteroids are commonly used to treat DMD across the world. This is the first FDA approval of any corticosteroid to treat DMD and the first approval of deflazacort for any use in the United States.

Image result for Deflazacort

“This is the first treatment approved for a wide range of patients with Duchenne muscular dystrophy,” said Billy Dunn, M.D., director of the Division of Neurology Products in the FDA’s Center for Drug Evaluation and Research. “We hope that this treatment option will benefit many patients with DMD.”

DMD is the most common type of muscular dystrophy. DMD is caused by an absence of dystrophin, a protein that helps keep muscle cells intact. The first symptoms are usually seen between 3 and 5 years of age and worsen over time. The disease often occurs in people without a known family history of the condition and primarily affects boys, but in rare cases it can affect girls. DMD occurs in about one of every 3,600 male infants worldwide.

People with DMD progressively lose the ability to perform activities independently and often require use of a wheelchair by their early teens. As the disease progresses, life-threatening heart and respiratory conditions can occur. Patients typically succumb to the disease in their 20s or 30s; however, disease severity and life expectancy vary.

The effectiveness of deflazacort was shown in a clinical study of 196 male patients who were 5 to 15 years old at the beginning of the trial with documented mutation of the dystrophin gene and onset of weakness before age 5. At week 12, patients taking deflazacort had improvements in a clinical assessment of muscle strength across a number of muscles compared to those taking a placebo. An overall stability in average muscle strength was maintained through the end of study at week 52 in the deflazacort-treated patients. In another trial with 29 male patients that lasted 104 weeks, deflazacort demonstrated a numerical advantage over placebo on an assessment of average muscle strength. In addition, although not statistically controlled for multiple comparisons, patients on deflazacort appeared to lose the ability to walk later than those treated with placebo.

The side effects caused by Emflaza are similar to those experienced with other corticosteroids. The most common side effects include facial puffiness (Cushingoid appearance), weight gain, increased appetite, upper respiratory tract infection, cough, extraordinary daytime urinary frequency (pollakiuria), unwanted hair growth (hirsutism) and excessive fat around the stomach (central obesity).

Other side effects that are less common include problems with endocrine function, increased susceptibility to infection, elevation in blood pressure, risk of gastrointestinal perforation, serious skin rashes, behavioral and mood changes, decrease in the density of the bones and vision problems such as cataracts. Patients receiving immunosuppressive doses of corticosteroids should not be given live or live attenuated vaccines.

The FDA granted this application fast track designation and priority review. The drug also received orphan drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.

The sponsor is receiving a rare pediatric disease priority review voucher under a program intended to encourage development of new drugs and biologics for the prevention and treatment of rare pediatric diseases. A voucher can be redeemed by a sponsor at a later date to receive priority review of a subsequent marketing application for a different product. This is the ninth rare pediatric disease priority review voucher issued by the FDA since the program began.

Emflaza is marketed by Marathon Pharmaceuticals of Northbrook, Illinois.

Medical uses

The manufacturer lists the following uses for deflazacort:[1]

In the United States, deflazacort is only FDA-approved for the treatment of Duchenne muscular dystrophy in people over the age of 5.

Image result for DeflazacortImage result for Deflazacort

Image result for DeflazacortImage result for Deflazacort

Adverse effects

Deflazacort carries the risks common to all corticosteroids, including immune suppression, decreased bone density, and endocrine insufficiency. In clinical trials, the most common side effects (>10% above placebo) were Cushing’s-like appearance, weight gain, and increased appetite.[2]

Pharmacology

Mechanism of action

Deflazacort is an inactive prodrug which is metabolized rapidly to the active drug 21-desacetyldeflazacort.[3]

Relative potency

Deflazacort’s potency is around 70–90% that of prednisone.[4] A 2017 review found its activity of 7.5 mg of deflazacort is approximately equivalent to 25 mg cortisone, 20 mg hydrocortisone, 5 mg of prednisolone or prednisone, 4 mg of methylprednisolone or triamcinolone, or 0.75 mg of betamethasone or dexamethasone. The review noted that the drug has a high therapeutic index, being used at initial oral doses ranging from 6 to 90 mg, and probably requires a 50% higher dose to induce the same demineralizing effect as prednisolone. Thus it has “a smaller impact on calcium metabolism than any other synthetic corticosteroid, and therefore shows a lower risk of growth rate retardation in children and of osteoporosis” in the elderly, and comparatively small effects on carbohydrate metabolism, sodium retention, and hypokalemia.[5]

History

In January 2015, the FDA granted fast track status to Marathon Pharmaceuticals to pursue approval of deflazacort as a potential treatment for Duchenne muscular dystrophy, a rare, “progressive and fatal disease” that affects boys.[6] Although deflazacort was approved by the FDA for use in treatment of Duchenne muscular dystrophy on February 9, 2017,[7][8] Marathon CEO announced on February 13, 2017 that the launch of deflazacort (Emflaza) would be delayed amidst controversy over the steep price Marathon was asking for the drug – $89,000-a-year. In Canada the same drug can be purchased for around $1 per tablet.[9] Marathon has said that Emflaza is estimated to cost $89,000/year which is “roughly 70 times” more than it would cost overseas.[10] Deflazacort is sold in the United Kingdom under the trade name Calcort;[4] in Brazil as Cortax, Decortil, and Deflanil; in India as Moaid, Zenflav, Defolet, DFZ, Decotaz, and DefZot; in Bangladesh as Xalcort; in Panama as Zamen; Spain as Zamene; and in Honduras as Flezacor.[11]

SYNTHESIS

Worlddrugtracker drew this

1 Protection of the keto groups in pregna-1,4-diene derivative  with NH2NHCOOMe using HCOOH, yields the corresponding methyl ester.

2 Cleavage of epoxide  with NH3 in DMAc/DMF gives amino-alcohol,

3 which on esterification with acetic anhydride in the presence of AcOH furnishes acetate.

4 Cyclization of amine using NaOH, Na2CO3 or K2CO3 produces oxazoline derivative ,

5 which is finally deprotected with HCl to afford Deflazacort 

SYNTHESIS FROM CHEMDRUG

The cyclization of 17alpha-azido-3beta,16alpha-acetoxy-5alpha-pregnane-11,20-dione (I) by hydrogenation with H2 over Pt in methanol, followed by a treatment with 10% HCl gives 3beta-hydroxy-5alpha-pregnane-11,20-dione-[17alpha,16alpha-d]-2′-methyloxazoline (II), which is converted into the semicarbazone (III) by treatment with semicarbazide hydrochloride (A) and pyridine in refluxing methanol. The reduction of one ketonic group of (III) with NaBH4 in refluxing ethanol yields the dihydroxy-semicarbazone (IV), which is hydrolyzed with 10% HCl in refluxing methanol to afford the ketodiol (V). The oxidation of (V) with cyclohexanone and aluminum isopropoxide in refluxing toluene gives 11beta-hydroxy-5alpha-pregnane-3,20-dione-[17alpha,16alpha-d]-2′-methyloxazoline (VI). The dehydrogenation of (VI) by treatment with Br2 in dioxane-acetic acid, followed by treatment with Li2CO3 in DMF at 140 C yields the corresponding 1,4-diene derivative (VII). Finally, the reaction of (VII) with I2 by means of azobisisobutyronitrile in CH2Cl2 affords the corresponding 21-iodo compound, which is then acetylated with triethylammonium acetate in refluxing acetone.

The monoacetylation of (V) with acetic anhydride and pyridine at 100 C gives the 3-acetoxy-11-hydroxy compound (IX), which is dehydrated by treatment with methanesulfonyl chloride and then with sodium acetate yielding 3beta-acetoxy-5alpha-pregn-9(11)-ene-20-one-[17alpha,16alpha-d]-2′-methyloxazoline (X). The hydrolysis of (X) with KOH in refluxing methanol affords the corresponding hydroxy compound (XI), which is acetoxylated by treatment with I2 and AZBN as before giving the iodo derivative (XII), and then with triethylammonium acetate also as before, yielding 3beta-hydroxy-21-acetoxy-5alpha-pregn-9(11)-ene-20-one-[17alpha,16alpha-d]-2′-methyloxazoline (XIII). The oxidation of (XIII) with CrO3 in acetone yields the 3,20-diketone (XIV), which by treatment with Br2 and Li2CO3 as before is dehydrogenated affording the 1,4,9(11)-pregnatriene (XV). Finally, the reaction of (XV) with N-bromoacetamide in THF yields 9alpha-bromo-11beta-hydroxy-21-acetoxy-5alpha-pregna-1,4-dieno-3,20-dione-[17alpha,16alpha-d]-2′-methyloxazoline (XVI), which is then debrominated by reaction with chromous acetate and butanethiol in DMSO.

PAPER

Journal of Medicinal Chemistry (1967), 10(5), 799-802

Steroids Possessing Nitrogen Atoms. III. Synthesis of New Highly Active Corticoids. [17α,16α,-d]Oxazolino Steroids

J. Med. Chem., 1967, 10 (5), pp 799–802
DOI: 10.1021/jm00317a009

PATENT

CN 105622713

PATENT CN 106008660

MACHINE TRANSLATED FROM CHINESE may seem funny

Description of the drawings

[0007] Figure 1 is a map of the traditional method of the combination process;

Figure 2 is a two-step method of the present invention.

detailed description

[0008] In order to more easily illustrate the gist and spirit of the present invention, the following examples illustrate:

Example 1

A: Preparation of hydroxylamine

In a 100 ml three-necked flask, 20 g of 16 (17) a-epoxy prednisolone, 30 ml of DMF, 300 ml of chloroform was added and incubated at 30-35 ° C with 8 g of ammonia gas at 1-2 atmospheres Reaction 16 ~ 20 hours, TLC detection reaction end point, after the reaction, the vacuum exhaust ammonia gas, add 3x100ml saturated brine washing 3 times, plus 10ml pure water washing times, then, under reduced pressure to chloroform to dry, add 200ml Ethyl acetate, Ig activated carbon, stirring reflux 60-90 minutes, cooling to 50-55 degrees, hot filter, l-2ml ethyl acetate washing carbon, combined filtrate and lotion, and then below 500C concentrated under pressure 95 % Of ethyl acetate, the system cooled to -5-0 ° C, stirring crystallization 2 ~ 3 hours, filter, 0.5-lml ethyl acetate washing, lotion and filtrate combined sets of approved; filter cake below 70 ° C Drying, get hydroxylamine 18.2g, HPLC content of 99.2%, weight loss of 91%.

[0009] B: Preparation of terracavir

Add 10 g of hydroxylamine, 150 ml of glacial acetic acid and 150 ml of acetic anhydride in a 100 ml three-necked flask. Add 5 g of concentrated sulfuric acid under stirring at room temperature. The reaction was carried out at 30-35 ° C for 12-16 hours. TLC confirmed the end of the reaction. Add 500ml of pure water, and adjust the pH of 7.5.5 with liquid alkali, cool to 10 ~ 15 ° C, stirring crystallization 2-3 hours, filtration, washing to neutral, combined filtrate and lotion, pretreated into Waste water treatment tank, filter cake below 70 V drying, Texaco can be special crude 112.5g, HPLC content of 98.2%, the yield of 112.5% ο the above terracotta crude dissolved in 800ml of alcohol, add 5g activated carbon, Decolorization 1-1.5 hours, hot filter, 10ml alcohol detergent cake, lotion and filtrate combined, atmospheric pressure recovery of about 90% of the alcohol, and then cooled to -5-0 ° C, frozen crystal 2-3 hours, Filtration, filter cake with 4-5ml alcohol washing, 70 ° C below drying, digoxin special product 89.2g, melting point 255.5-256.0 degrees, HPLC content of 99.7%, yield 89.2%. The mother liquor is recycled with solvent and crude.

[0010] Example II

A: Preparation of hydroxylamine

In a 100 ml three-necked flask, 20 g of 16 (17) a-epoxy prednisolone, 120 ml of toluene was added and incubated at 30-35 ° C with 8 g of ammonia and 16 to 20 at atmospheric pressure The reaction was carried out in the presence of 3 x 50 ml of saturated brine and 50 ml of pure water was added. Then, the toluene was dried under reduced pressure to dryness, and 200 ml of ethyl acetate, Ig activated carbon was added, and the mixture was stirred. Reflux 60-90 minutes, cool to 50-55 ° C, hot filter, l2ml ethyl acetate wash carbon, combined filtrate and lotion, and then below 500C under reduced pressure 95% ethyl acetate, the system cooling To 5-0C, stirring crystallization 2 ~ 3 hours, filter, 0.5-lml ethyl acetate washing, lotion and filtrate combined sets of the next batch; filter cake 70 ° C below drying, hydroxylamine 18.0g, HPLC content 99.1%, 90% by weight.

[0011] B: Preparation of terracavir

Add 10 g of hydroxylamine, 500 ml of chloroform and 150 ml of acetic anhydride in a 100 ml three-necked flask, add 5 g of p-toluenesulfonic acid under stirring at room temperature, and incubate at 30-35 ° C for 12-16 hours. TLC confirms the reaction end, After the addition of 500ml of pure water, and with the liquid alkali pH 7.55, down to 10 ~ 15 ° C, stirring 0.5_1 hours, separate the water layer, washed to neutral, combined with water and lotion, pretreated into Waste water treatment tank, organic layer under reduced pressure concentrated chloroform to near dry, adding 200ml hexane, reflux 0.5-1 hours, slowly cooling to -5 ~ O0C, stirring crystallization 2-3 hours, filter, filter cake with 4-5ml Alcohol washing, the filtrate and lotion combined apply to the next batch, the filter cake below 70 ° C drying, Texaco can crude 110.5g, HPLC content of 98.4%, the yield of 110.5%. The above-mentioned diltiazem crude product dissolved in 800ml alcohol, add 5g activated carbon, temperature reflux bleaching 1-1.5 hours, hot filter, 10ml alcohol washing cake, lotion and filtrate combined, atmospheric pressure recovery of about 90% of the alcohol And then cooled to -500C, frozen crystallization for 2-3 hours, filtration, filter cake with 4-5ml alcohol washing, 70 ° C the following drying, digester can special products 88.6g, melting point 255.0-256.0 degrees, HPLC content of 99.5%, the yield of 88.6%. The mother liquor is recycled with solvent and crude.

[0012] Example 3

A: Preparation of hydroxylamine

Add 20 g of 16 (17) a-epoxy prednisolone to 120 ml of ethanol in a 100 ml three-necked flask and incubate at 30-35 ° C with stirring to give Sg ammonia at 16 to 20 hours , TLC test reaction end point, after the reaction, vacuum exhaust ammonia gas, concentrated ethanol to the near dry, cooling, adding 300ml chloroform, stirring dissolved residue, and then add 3x100ml saturated brine washing, plus 10ml pure water washing, washing And then concentrated to reduce the chloroform to dry, add 200ml of ethyl acetate, Ig activated carbon, stirring reflux 60-90 minutes, cooling to 50-55 ° C, hot filter, l2ml ethyl acetate washing carbon, combined filtrate and lotion And then concentrated below 50 ° C to 95% ethyl acetate under reduced pressure. The system was cooled to -5-0 0C, stirred for 2 to 3 hours, filtered, 0.5-l of ethyl acetate, washed and filtrate The filter cake was dried at 70 ° C, 18.6 g of hydroxylamine, 99.5% of HPLC, and 93% by weight.

[0013] B: Preparation of terracavir

In a 100ml three-necked flask, add 10g of hydroxylamine, 500ml toluene, 150ml acetic anhydride, stirring at room temperature by adding 5g concentrated sulfuric acid, insulation at 30-35 degrees stirring reaction 12-16 hours, TLC confirmed the end of the reaction, after the reaction, Add 500ml of pure water, and liquid pH adjustment pH 7.5, cooling to 1 ~ 15 ° C, stirring 0.5-1 hours, the water layer, washed to neutral, combined with water and lotion, pretreated into the wastewater The cells were dried and the organic layer was concentrated to dryness under reduced pressure. 200 ml of hexane was added and refluxed

0.5-1 hours, slowly cool to -5 ~ O0C, stirring crystallization 2-3 hours, filtration, filter cake with 4-5ml hexane, the filtrate and lotion combined apply to the next batch, filter cake below 70 ° C Drying, digoxin crude 112.5g, HPLC content of 97.4%, the yield of 112,5% ο will be the above terracotta crude dissolved in 800ml of alcohol, add 5g activated carbon, heating reflux bleaching 1-1.5 hours, while Hot filter, 10ml alcohol detergent cake, lotion and filtrate combined, atmospheric pressure recovery of about 90% of the alcohol, and then cooled to -500C, frozen crystallization for 2-3 hours, filter, filter cake with 4-5ml alcohol Washing, 70 ° C below the dry, Diges can special products 86.2g, melting point 255.5-256.0 degrees, HPLC content of 99.8%, the yield of 86.2%. The mother liquor is recycled with solvent and crude.

PATENT

https://www.google.com/patents/CN101418032A?cl=en

Example 1

21- bromo -ll (3- hydroxy – pregna–l, 4- diene -3, 20-dione [170, 16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask was added 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), N- bromosuccinimide (9.79 g; Fw: 178.00; 55 mmol), 150 ml of ether; then ammonium acetate (0.39 g; Fw: 77.08; 0.005 mmol) added to the system. System continues to stir at 20 ° C 0.5 h, the reaction is complete. After completion of the reaction was filtered to remove the white precipitate cake was washed with 50 mL of dichloromethane, and the combined organic Xiangde pale yellow clear liquid, the solvent was evaporated under reduced pressure to give a pale yellow solid 21.27 g, yield: 92%, HPLC content of greater than 95%.

Example 2

21- bromo -lip- hydroxy – pregna–l, 4- diene -3, 20-dione [17 “16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask were added sequentially 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), N- bromosuccinimide (9.79 g; Fw : 178.00; 55 mmol), 150 ml of toluene; then ammonium acetate (0.39 g; Fw: 77.08; 0.005 mmol) added to the system. System continues to stir at 110 ° C 5 h, the reaction is complete. After completion of the reaction was cooled to room temperature, the white precipitate was removed by filtration cake was washed with 50 mL of dichloromethane, and the combined organic Xiangde pale yellow clear liquid, concentrated under reduced pressure to remove the solvent to give a pale yellow solid 19.65 g, yield: 85%, HPLC content greater than 95%.

Example 3

21 Jie bromo -11 – hydroxy – pregna-1,4-diene -3, 20-dione [17a, 16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask were added sequentially 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), 1,3- dibromo-5,5-dimethyl- Hein (35.74 g; Fw: 285.94; 125 mmol), 150 ml of ether; then ammonium acetate (0.39 g; Fw: 77.08; 0.005 mmol) added to the system. System Stirring was continued at reflux for 3 h, the reaction was completed. After completion of the reaction a white precipitate was removed by filtration and the cake was washed with 50 mL of diethyl ether, and the combined organic Xiangde pale yellow clear liquid, concentrated under reduced pressure to remove the solvent to give a pale yellow solid 16.18 g, yield: 70%, HPLC content greater than 92%.

Example 4

21- bromo -11 Jie – hydroxy – pregna-1,4-diene -3, 20- dione [17c, 16o-d] -2′- methyl-oxazoline (4) Preparation:

A dry fitted with a thermometer, a reflux condenser, magnetically stirred flask were added sequentially 250mL three compound (2) (19.17 g; Fw: 383.48; 50 mmol), 1,3- dibromo-5,5-dimethyl- Hein (35.74 g; Fw: 285.94; 125 mmol), 150 ml dichloromethane; followed by ammonium acetate (0.039 g; Fw: 77.08; 0.0005 mmol) added to the system. System Stirring was continued at reflux for 24 h, the reaction was completed. After completion of the reaction a white precipitate was removed by filtration and the cake was washed with 50 mL of diethyl ether, and the combined organic Xiangde pale yellow clear liquid, concentrated under reduced pressure to remove the solvent to give a pale yellow solid 16.41 g, yield: 71%, HPLC content of greater than 92. / 0.

Example 5

Deflazacort Preparation:

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of sodium acetate (8.20g; Fw: 82.03; lOOmmol), 50 mL methanol was added to the system.

Then tetrabutylammonium bromide (O. 81g; Fw: 322.38; 2.5 mmol). Warmed to 50 ° C with stirring

48 h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase washed with 10% aqueous sodium carbonate paint 3 times, saturated sodium chloride once. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, purified ethyl acetate to give the product 9.93g, yield 90%, HPLC content> 990/0.

Example 6

Deflazacort Preparation –

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of anhydrous potassium acetate (3.68g; Fw: 98.14; 37.5 mmol), 50 mL acetone was added to the system. Followed by tetrabutylammonium iodide (0.10g; Fw: 369.37; 0.25 mmol). Heated to reflux with stirring 2h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase was washed 3 times with 10% aqueous sodium carbonate , washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, ethyl acetate was purified to give the product 10.93 g, yield 99%, HPLC content> 99%.

Example 7

Deflazacort Preparation:

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of anhydrous potassium acetate (3.68g; Fw: 98.14; 37.5 mmol), 50 mL acetonitrile was added to the system. Followed by tetrabutylammonium iodide (0.10g; Fw: 369.37; 0.25 mmol). Heated to reflux with stirring 2h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase was washed 3 times with 10% aqueous sodium carbonate , washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, ethyl acetate was purified to give the product 10.93 g, yield 99%, HPLC content> 99%.

Example 8

Deflazacort Preparation:

In a nitrogen-filled dry fitted with a thermometer, magnetic stirring and a reflux condenser 100 mL three-necked flask was charged with Compound (4) (11.56 g; Fw: 462.38; 25 mmol), followed by addition of anhydrous potassium acetate (2.45g; Fw: 98.14; 25 mmol), the N, N- dimethylformamide, 50 mL added to the system. Followed by tetrabutylammonium iodide (O.IO g; Fw: 369.37; 0.25 mmol). Warmed to 120. C stirring 2h. Until after the completion of the reaction was cooled to room temperature. After completion of the reaction, temperature of the system was cooled to room temperature, the system was supplemented with chloroform 50mL, filtered, and the filter cake was washed with small amount of chloroform and then to confirm that no product was dissolved, and the combined organic phases, the organic phase was washed 3 times with 10% aqueous sodium carbonate , washed once with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate, the inorganic salt was removed to give a pale yellow liquid, was concentrated to dryness, ethyl acetate was purified to give the product 10.93 g, yield 99%, HPLC content> 99o / q.

PATENT

https://www.google.com/patents/WO1997021722A1?cl=zh

compound (llβ,16β)-21-(acetyloxy)-11- hydroxy-2 ‘ -methyl-5 ‘H-pregna-1, -dieno[17 , 16-d Joxazole- 3,20-dione, also known, and hereinafter referred to, with the INN (International Nonproprietary Name) deflazacort. Deflazacort is represented by the following formula I

Figure imgf000003_0001

Deflazacort is employed in therapy aince some years as a calcium-sparing corticoid agent. This compound belongs to the more general class of pregneno-oxazolines, for which anti-inflammatory, glucocorticoid and hormone-like pharmacological activities are reported. Examples of compounds of the above class, comprising deflazacort, are disclosed in US 3413286, where deflazacort is referred to as llβ-21-dihydroxy-2 ‘ -methyl-5 ‘ βH-pregna-1,4-dieno.17 , 16- d]oxazole-3,20-dione 21-acetate.

According to the process disclosed by US 3413286, deflazacort is obtained from 5-pregnane-3β-ol-ll , 20- dione-2 ‘-methyloxazoline by 2 , -dibromination with Br2– dioxane, heating the product in the presence of LiBr- iC03 for obtaining the 1,4-diene, and converting this latter into the 21-iodo and then into the desired 21- acetyloxy compound. By hydrolysis of deflazacort, the llβ-21-dihydroxy-2 ‘ -methyl-5 ‘βH-pregna-1, -dieno[ 17 , 16- d-]oxazoline-3, 20-dione of formula II is obtained:

Figure imgf000004_0001

The compound of formula II is preferably obtained according to a fermentation process disclosed in

EP-B-322630; in said patent, the compound of formula II is referred to as llβ-21-dihydroxy-2 ‘-methyl-5 ‘ βH- pregna-1,4-dieno[17,16-d-]oxazoline-3,20-dione.

The present invention provides a new advantageous single-step process for obtaining deflazacort, by acetylation of the compound of formula II.

CLIP

Image result for Deflazacort NMR

tructure of deflazacort and its forced degradation product (A), chromatogram plot of standard deflazacort (B), contour plot of deflazacort (C). Deflazacort was found to be a stable drug under stress condition such as thermal, neutral and oxidative condition. However, the forceddegradation study on deflazacort showed that the drug degraded under alkaline, acid and photolytic conditions.

Mass fragmentation pathway for degradant product of deflazacort.

PATENT

CN 103059096

Figure CN103059096AD00051

Example 1: Protective reaction To the reaction flask was added 20 g of 1,4-diene-11? -hydroxy-16,17-epoxy_3,20-dione pregnone (Formula I) 20% of the aqueous solution of glacial acetic acid 300g, stirring 5 minutes, temperature 10 ° C ~ 15 ° C, adding ethyl carbazate 14g, temperature control 30 ° C reaction 6 hours; TLC detection reaction is complete, cooling to 0 ° C ~ 5 ° C for 2 hours, until dry, washed to neutral; 60 ° C vacuum dry to dry creatures 20. 5g; on P, oxazoline ring reaction The above protective products into the reaction bottle, add 41ml Of the DMAC dissolved, temperature 25 ~ 30 ° C, access to ammonia, to keep the reaction bottle micro-positive pressure, the reaction of 32 hours, atmospheric pressure exhaust ammonia and then decompression pumping ammonia for 30 minutes; 5 ° C, temperature 5 ~ 0 ° C by adding 5ml glacial acetic acid, then add 21ml acetic anhydride, heated to 35 ° C reaction 4 hours, the sample to confirm the reaction completely; slowly add 5% sodium hydroxide solution 610ml and heated to 60 ~ 70 ° C reaction 2 hours; point plate to confirm the end of the reaction, cooling to 50 ° C, half an hour by adding refined concentrated hydrochloric acid 40ml, insulation 50 ~ 55 ° C reaction 10 hours; to the end of the reaction temperature to room temperature, chloroform Extraction, drying and filtration, concentration of at least a small amount of solvent, ethyl acetate entrained twice, leaving a small amount of solvent, frozen crystallization filter high purity [17a, 16a-d] terfu Kete intermediate. Example 2: Protective reaction 20 g of 1,4-diene-l1-la-hydroxy-16,17-epoxy_3,20_dione progestin (Formula I) was added to the reaction flask and 15% Formic acid solution 300g, stirring for 5 minutes, temperature 10 ~ 15 ° C, adding methyl carbazate 12g, temperature control 30 ° C reaction 5 hours to test the end of the reaction, cooling to O ~ 5 ° C stirring 2 hours crystallization, Suction to dry, washed to neutral; 60 ° C vacuum drying to dry protection of 20g; on P, oxazoline ring reaction The protection of the reaction into the reaction flask, add 30ml of DMF dissolved, temperature control 25 ~ 30 ° C, access to ammonia, keep the reaction bottle in the micro-positive pressure, reaction 30 hours, atmospheric pressure exhaust ammonia and then decompression pumping ammonia for 30 minutes, ice water cooled to 5 ° C, temperature 5 ~ 10 ° C add 5ml of glacial acetic acid, then add 20ml acetic anhydride, heated to 30 ° C reaction for 5 hours to confirm the reaction is complete; slowly add 20% sodium carbonate aqueous solution 500ml and heated to 60 ~ 70 ° C reaction 4 hours, the point plate to confirm the reaction The temperature of 55 ~ 60 ° C for 10 hours; to be the end of the reaction temperature to room temperature, chloroform extraction, drying and filtration, concentration of a small amount of solvent, acetic acid isopropyl The ester was entrained twice, leaving a small amount of solvent, frozen and crystallized to obtain high purity [17a, 16a-d] oxazoline residues. [0024] Example 3: Protective reaction 20 g of I, 4-diene-16,17-epoxy-3,11,20-triketone pregnone (Formula I) was added to the reaction flask and 20% Formic acid solution 300g, stirring for 5 minutes, temperature 10 ~ 15 ° C, adding hydrazine carbamate 15g, temperature control 30 ° C reaction 5 hours to test the end of the reaction, cooling to O ~ 5 ° C stirring 2 hours crystallization, To the dry, washed to neutral; 60 ° C vacuum drying to dry protection of 22g; on P, oxazoline ring reaction of the protection of the reaction into the bottle, add 30ml of DMAC dissolved temperature control 35 ~ 40 ° C, access to ammonia, keep the reaction bottle in the micro-positive pressure, reaction 40 hours, atmospheric pressure exhaust ammonia and then decompression pumping ammonia for 30 minutes, ice water cooling to 5 ° C, temperature 5 ~ 10 ° C add 5ml of glacial acetic acid, then add 20ml acetic anhydride, heated to 40 ° C reaction 5 hours to confirm the reaction is complete; slowly add 20% potassium carbonate aqueous solution 500ml and heated to 60 ~ 70 ° C reaction 7 hours, the point plate to confirm the reaction The temperature of the reaction to the end of the temperature to room temperature, chloroform extraction, drying filter, concentrated to a small amount of solvent, acetic acid isopropyl The ester was entrained twice, leaving a small amount of solvent, frozen and crystallized to obtain high purity [17a, 16a-d] oxazoline residues.

PATENT

CN 102936274

Figure CN102936274BD00041

xample 1

[0028] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 15 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10-15 ° C), 30 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give product 30.6 g, 102% mass yield, product by HPLC , a purity of 95.2%.

[0029] Example 2

[0030] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mL of pyridine were mixed, added pressure reactor, stirring ammonia gas to the reactor pressure to 0. 15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 15 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give product 28.6 g, yield 95% by mass, product by HPLC , a purity of 94.8%.

[0031] Example 3

[0032] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction.Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction for 6 hours. The reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 31.2 g, yield 104% quality products by HPLC , a purity of 95.4%.

[0033] Example 4

[0034] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.5 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 31. I g, 102% mass yield, product by by HPLC, the purity was 95.2%.

[0035] Example 5

[0036] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 60 mL of acetic acid, 15 g of acetic anhydride, The reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 29. 5 g, yield 98% by mass, the product of by HPLC, purity of 95%.

[0037] Example 6

[0038] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. The reaction was complete, the material was transferred to a glass reaction flask until the material temperature drops below 10 ° C, plus acetic acid to adjust the pH to 5 to 6, the solvent was removed under reduced pressure; the reaction flask was added 30 mL of acetic acid, 30 g of maleic dianhydride, the reaction temperature was controlled at 30 ° C, the reaction 6 hours, the reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 30 g, 100% mass yield, product by HPLC purity of 95.2%.

[0039] Example 7

[0040] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of propionic anhydride, The reaction temperature was controlled at 30 ° C, the reaction for 6 hours. The reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 27.6 g, 92% yield of quality products by HPLC , a purity of 93.5%.

[0041] Example 8

[0042] A 30 g 16, 17 α- epoxy – pregn -20- substituting methyl hydrazine -3-acetyl-1,4-diene, 11- dione (a) and 150 mL of chloroform and 30 mLDMF mixed, pressure reactor, stirring ammonia gas to the reactor pressure to 0.15 MPa (during ventilation control the reaction temperature at 10~15 ° C), 40 ° C heat reaction, TLC track the progress of the reaction. Completion of the reaction, the material was transferred to a glass reaction flask, the temperature of the material to be reduced to below 10 ° C, add acetic acid adjusted to pH 5 to 6, the solvent was removed under reduced pressure; reaction flask was added 30 mL of acetic acid, 30 g of acetic anhydride, The reaction temperature is controlled at 50 ° C, the reaction for 6 hours. The reaction mixture was poured into cold 500 mL10% sodium hydroxide solution, stirred for 1 hour, filtration to give the product 29.8 g, 99% yield of quality products by HPLC , a purity of 94.8%.

References

  1. Jump up^ “Refla: deflazacort” (PDF).
  2. Jump up^http://www.accessdata.fda.gov/drugsatfda_docs/label/2017/208684s000,208685s000lbl.pdf
  3. Jump up^ Möllmann, H; Hochhaus, G; Rohatagi, S; Barth, J; Derendorf, H (1995). “Pharmacokinetic/pharmacodynamic evaluation of deflazacort in comparison to methylprednisolone and prednisolone”. Pharmaceutical Research. 12 (7): 1096–100. PMID 7494809.
  4. ^ Jump up to:a b “Calcort”. electronic Medicines Compendium. June 11, 2008. Retrieved on October 28, 2008.
  5. Jump up^ Luca Parente (2017). “Deflazacort: therapeutic index, relative potency and equivalent doses versus other corticosteroids”. BMC Pharmacol Toxicol. doi:10.1186/s40360-016-0111-8.
  6. Jump up^ Ellen Jean Hirst (January 19, 2015), Duchenne muscular dystrophy drug could get OK for U.S. sales in 2016, The Chicago Tribune, retrieved February 13, 2017,has been shown to prolong lives … a progressive and fatal disease that has no drug treatment available in the US
  7. Jump up^ “FDA approves drug to treat Duchenne muscular dystrophy”. http://www.fda.gov. 2017-02-09. Retrieved 2017-02-10.
  8. Jump up^ “Marathon Pharmaceuticals to Charge $89,000 for Muscular Dystrophy Drug”. http://www.wsj.com. 2017-02-10. Retrieved 2017-02-10.
  9. Jump up^ Clifton Sy Mukherjee (February 10, 2017). “Brainstorm Health Daily”. Retrieved February 13, 2017.
  10. Jump up^ Joseph Walker and Susan Pulliam (February 13, 2017), Marathon Pharmaceuticals to Charge $89,000 for Muscular Dystrophy Drug After 70-Fold Increase, The Wall Street Journal, retrieved February 13, 2017,FDA-approved deflazacort treats rare type of disease affecting boys
  11. Jump up^ “Substâncias: DEFLAZACORT” (in Portuguese). Centralx. 2008. Retrieved on October 28, 2008.
Deflazacort
Deflazacort structure.svg
Clinical data
Trade names Emflaza, Calcort, others
AHFS/Drugs.com International Drug Names
Routes of
administration
By mouth
ATC code
Legal status
Legal status
Pharmacokinetic data
Protein binding 40%
Metabolism By plasma esterases, to active metabolite
Biological half-life 1.1–1.9 hours (metabolite)
Excretion Renal (70%) and fecal (30%)
Identifiers
CAS Number
PubChem CID
DrugBank
ChemSpider
UNII
KEGG
ChEMBL
ECHA InfoCard 100.034.969
Chemical and physical data
Formula C25H31NO6
Molar mass 441.517 g/mol
3D model (Jmol)
CN102746358A * Apr 22, 2011 Oct 24, 2012 天津金耀集团有限公司 Novel technology for synthesis of pregnane 21-bit bromide
CN102746358B * Apr 22, 2011 Feb 10, 2016 天津金耀集团有限公司 一种合成孕甾21位溴化物的工艺
CN102936274A * Nov 12, 2012 Feb 20, 2013 浙江仙居君业药业有限公司 Preparation method for [17alpha, 16alpha-d] methyl oxazoline
CN102936274B * Nov 12, 2012 Apr 1, 2015 江西君业生物制药有限公司 Preparation method for [17alpha, 16alpha-d] methyl oxazoline

///////FDA 2017, Emflaza, Calcort, Deflazacort, orphan drug designation, FAST TRACK

[H][C@@]12C[C@@]3([H])[C@]4([H])CCC5=CC(=O)C=C[C@]5(C)[C@@]4([H])[C@@]([H])(O)C[C@]3(C)[C@@]1(N=C(C)O2)C(=O)COC(C)=O

VT 1129


str1

Image result for VT1129

str1

VT 1129

1340593-70-5 CAS
MF C22 H14 F7 N5 O2, MW 513.37
2-Pyridineethanol, α-(2,4-difluorophenyl)-β,β-difluoro-α-(1H-tetrazol-1-ylmethyl)-5-[4-(trifluoromethoxy)phenyl]-, (αR)-
R ISOMER
ROTATION +
  • Originator Viamet Pharmaceuticals
  • Class Antifungals; Small molecules
  • Mechanism of Action 14-alpha demethylase inhibitors
  • Orphan Drug Status Yes – Cryptococcosis
  • On Fast track Cryptococcosis
  • Phase I Cryptococcosis
  • Most Recent Events

    • 01 Jun 2016 VT 1129 receives Fast Track designation for Cryptococcosis [PO] (In volunteers) in USA
    • 30 May 2016 Viamet Pharmaceuticals plans a phase II trial for Cryptococcal meningitis in USA (Viamet Pharmaceuticals pipeline; May 2016)
    • 27 May 2016 Phase-I clinical trials in Cryptococcosis (In volunteers) in USA (PO) before May 2016 (Viamet Pharmaceuticals pipeline; May 2016)

Image result for Viamet Pharmaceuticals Holdings LLC

William J. Hoekstra, Stephen William Rafferty,Robert J. Schotzinger
Applicant Viamet Pharmaceuticals, Inc.

Image result for VT1129

Viamet, in collaboration with Therapeutics for Rare and Neglected diseases, is investigating VT-1129, a small-molecule lanosterol demethylase inhibitor, developed using the company’s Metallophile technology, for treating fungal infections, including Cryptococcus neoformans meningitis.

VT-1129 is a novel oral agent that we are developing for the treatment of cryptococcal meningitis, a life-threatening fungal infection of the brain and the spinal cord that occurs most frequently in patients with HIV infection, transplant recipients and oncology patients. Without treatment, the disease is almost always fatal.

VT-1129VT-1129 has shown high potency and selectivity in in vitro studies and is an orally administered inhibitor of fungal CYP51, ametalloenzyme important in fungal cell wall synthesis. In preclinical studies, VT-1129 has demonstrated substantial potency against Cryptococcus species, the fungal pathogens that cause cryptoccocal meningitis, and has also been shown to accumulate to high concentrations within the central nervous system, the primary site of infection.

In in vitro studies, VT-1129 was significantly more potent against Cryptococcus isolates than fluconazole, which is commonly used for maintenance therapy of cryptococcal meningitis in the United States and as a primary therapy in the developing world. Oral VT-1129 has also been studied in a preclinical model of cryptococcal meningitis, where it was compared to fluconazole.  At the conclusion of the study, there was no detectable evidence of Cryptococcus in the brain tissue of the high dose VT-1129 treated groups, in contrast to those groups treated with fluconazole. To our knowledge, this ability to reduce the Cryptococcus pathogen in the central nervous system to undetectable levels in this preclinical model is unique to VT-1129.

Opportunity

An estimated 3,400 hospitalizations related to cryptococcal meningitis occur annually in the United States and the FDA has granted orphan drug designation to VT-1129 for the treatment of this life-threatening disease. In addition, the FDA has granted Qualified Infectious Disease Product designation to VT-1129 for the treatment of Cryptococcus infections, which further underscores the unmet medical need. In developing regions such as Africa, cryptococcal meningitis is a major public health problem, with approximately one million cases and mortality rates estimated to be as high as 55-70%.

Current Status

VT-1129 has received orphan drug and Fast Track designations for the treatment of cryptococcal meningitis and has been designated a Qualified Infectious Disease Product (QIDP) by the U.S. Fod and Drug Administration.  We are currently conducting a Phase 1 single-ascending dose study of VT-1129 in healthy volunteers.

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Conclusions

• VT-1129 has robust activity against Cryptococcus isolates with elevated fluconazole MICs and may be a viable option in persons infected with such strains.

• A Phase 1 study of VT-1129 in healthy volunteers is scheduled to begin by the end of 2015. Phase 2 trials in persons with cryptococcal meningitis are targeted to begin by the end of 2016.

Image result for VT 1129

Living organisms have developed tightly regulated processes that specifically import metals, transport them to intracellular storage sites and ultimately transport them to sites of use. One of the most important functions of metals such as zinc and iron in biological systems is to enable the activity of metalloenzymes. Metalloenzymes are enzymes that incorporate metal ions into the enzyme active site and utilize the metal as a part of the catalytic process. More than one-third of all characterized enzymes are metalloenzymes.

The function of metalloenzymes is highly dependent on the presence of the metal ion in the active site of the enzyme. It is well recognized that agents which bind to and inactivate the active site metal ion dramatically decrease the activity of the enzyme. Nature employs this same strategy to decrease the activity of certain metalloenzymes during periods in which the enzymatic activity is undesirable. For example, the protein TIMP (tissue inhibitor of metalloproteases) binds to the zinc ion in the active site of various matrix metalloprotease enzymes and thereby arrests the enzymatic activity. The pharmaceutical industry has used the same strategy in the design of therapeutic agents. For example, the azole antifungal agents fluconazole and voriconazole contain a l-(l,2,4-triazole) group that binds to the heme iron present in the active site of the target enzyme lanosterol demethylase and thereby inactivates the enzyme.

In the design of clinically safe and effective metalloenzyme inhibitors, use of the most appropriate metal-binding group for the particular target and clinical indication is critical. If a weakly binding metal-binding group is utilized, potency may be suboptimal. On the other

hand, if a very tightly binding metal-binding group is utilized, selectivity for the target enzyme versus related metalloenzymes may be suboptimal. The lack of optimal selectivity can be a cause for clinical toxicity due to unintended inhibition of these off-target metalloenzymes. One example of such clinical toxicity is the unintended inhibition of human drug metabolizing enzymes such as CYP2C9, CYP2C19 and CYP3A4 by the currently- available azole antifungal agents such as fluconazole and voriconazole. It is believed that this off-target inhibition is caused primarily by the indiscriminate binding of the currently utilized l-(l,2,4-triazole) to iron in the active site of CYP2C9, CYP2C19 and CYP3A4. Another example of this is the joint pain that has been observed in many clinical trials of matrix metalloproteinase inhibitors. This toxicity is considered to be related to inhibition of off-target metalloenzymes due to indiscriminate binding of the hydroxamic acid group to zinc in the off-target active sites.

Therefore, the search for metal-binding groups that can achieve a better balance of potency and selectivity remains an important goal and would be significant in the realization of therapeutic agents and methods to address currently unmet needs in treating and preventing diseases, disorders and symptoms thereof. Similarly, methods of synthesizing such therapeutic agents on the laboratory and, ultimately, commercial scale is needed. Addition of metal-based nucleophiles (Zn, Zr, Ce, Ti, Mg, Mn, Li) to azole-methyl substituted ketones have been effected in the synthesis of voriconazole (M. Butters, Org. Process Res. Dev.2001, 5, 28-36). The nucleophile in these examples was an ethyl-pyrimidine substrate. Similarly, optically active azole-methyl epoxide has been prepared as precursor electrophile toward the synthesis of ravuconazole (A. Tsuruoka, Chem. Pharm. Bull.1998, 46, 623-630). Despite this, the development of methodology with improved efficiency and selectivity is desirable.

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011133875

Scheme 1

EXAMPLE 7

2-(2, 4-Difluorophenyl)-l, l-difluoro-3-(lH-tetrazol-l-yl)-l-(5-(4- (trifluoromethoxy) phenyl) pyridin-2-yl) propan-2-ol (7)

To a stirred solution of bromo epoxide C (0.5 g, 1.38 mmol) in THF (30 mL) and water (14 mL) were added 4-(trifluoromethoxy) phenylboronic acid (0.22 g, 1.1 mmol), Na2C03 (0.32 g, 3.1 mmol) and Pd(dppf)2Cl2 (0.28 g, 0.34 mmol) at RT under inert atmosphere. After purged with argon for a period of 30 min, the reaction mixture was heated to 75°C and stirring was continued for 4 h. Progress of the reaction was monitored by TLC. The reaction mixture was cooled to RT and filtered through a pad of celite. The filtrate was concentrated under reduced pressure; obtained residue was dissolved in ethyl acetate (30 mL). The organic layer was washed with water, brine and dried over anhydrous Na2S04 and concentrated under reduced pressure. The crude compound was purified by column chromatography to afford the coupled product (0.45 g, 1.0 mmol, 73%) as solid. 1H NMR (200 MHz, CDC13): δ 8.87 (s, 1 H), 7.90 (dd, / = 8.2, 2.2 Hz, 1 H), 7.66-7.54 (m, 3 H), 7.49-7.34 (m, 3 H), 6.90-6.70 (m, 2 H), 3.49 (d, / = 5.0 Hz, 1 H), 3.02-2.95 (m, 1 H). Mass: m/z 444 [M++l].

To a stirred solution of the coupled product (0.45 g, 1.0 mmol) in DMF (10 mL) was added K2C03 (70 mg, 0.5 mmol) followed by IH-tetrazole (70 mg, 1.0 mmol) at RT under inert atmosphere. The reaction mixture was stirred for 4 h at 80 °C. The volatiles were removed under reduced pressure and obtained residue was dissolved in water (15 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with water, brine and dried over anhydrous Na2S04 and concentrated under reduced pressure. The crude compound was purified by column chromatography to afford 7 (0.19 g, 0.37 mmol, 36 %) as white solid. 1H NMR (500 MHz, CDC13): δ 8.76 (s, 1 H), 8.70 (s, 1 H), 7.97 (dd, / = 8.0, 2.0 Hz, 1 H), 7.68 (d, / = 8.5 Hz, 1 H), 7.60-7.56 (m, 3 H), 7.43-7.36 (m, 3 H), 6.80-6.76 (m, 1 H), 6.70-6.67 (m, 1 H), 5.57 (d, / = 14.5 Hz, 1 H), 5.17 (d, / = 14.5 Hz, 1 H). HPLC: 98.3%. Mass: m/z 513.9 [M++l].

Chiral preparative HPLC of enantiomers:

The enantiomers of 7 (17.8 g, 34.6 mmol) were separated by normal-phase preparative high performance liquid chromatography (Chiralpak AD-H, 250 x 21.2 mm, 5μ; using (A) n-hexane – (B) IPA (A:B : 70:30) as a mobile phase; Flow rate: 15 mL/min) to obtain 7(+) (6.0 g) and 7(-) (5.8 g).

Analytical data for 7 (+):

HPLC: 99.8%.

Chiral HPLC: Rt = 9.88 min (Chiralpak AD-H, 250 x 4.6mm, 5μ; mobile phase (A) n-Hexane (B) IPA (7/3): A: B (70:30); flow Rate: 1.00 mL/min)

Optical rotation [a]D25: + 19° (C = 0.1 % in MeOH).

Patent

WO2015143137,

https://patentscope.wipo.int/search/ko/detail.jsf;jsessionid=61AAA66F887FDBB9CFC3F752AFF04016.wapp2nC?docId=WO2015143137&recNum=303&office=&queryString=&prevFilter=%26fq%3DICF_M%3A%22C07D%22&sortOption=%EA%B3%B5%EA%B0%9C%EC%9D%BC(%EB%82%B4%EB%A6%BC%EC%B0%A8%EC%88%9C)&maxRec=58609

Examples

The present invention will now be demonstrated using specific examples that are not to be construed as limiting.

General Experimental Procedures

Definitions of variables in the structures in schemes herein are commensurate with those of corresponding positions in the formulae delineated herein.

Synthesis of 1 or la

A process to prepare enantiopure compound 1 or la is disclosed. Syntheses of 1 or la may be accomplished using the example syntheses that are shown below (Schemes 1-9). The preparation of precursor ketone 8 is performed starting with reaction of dibromo-pyridine 2-Br with ethyl 2-bromo-difluoroacetate to produce ester 3-Br. This ester is reacted with tetrazole reagent 4 via Claisen reaction to furnish 5-Br. Decarboxylation of 5-Br via a two-step process produces compound 6-Br. Suzukin coupling of 6-Br with boronate 7 furnishes 8.

Scheme 1. Synthesis of ketone 8

Ketone 8 may be prepared in an analogous fashion as described in Scheme 1 starting from corresponding substituted 2-bromo-pyridines, which can be prepared using according to synthetic transformations known in the art and contained in the references cited herein (Scheme 2).

Scheme 2. Synthesis of ketone 8

= halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, – 0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S02)-alkyl, -0(S02)-substituted alkyl, – 0(S02)-aryl, or -0(S02)-substituted aryl.

Compounds 6 or 8 may be reacted with a series of metallated derivatives of 2,4-difluoro-bromobenzene and chiral catalysts/reagents (e.g. BINOL) to effect enantiofacial-selective addition to the carbonyl group of 6 or 8 (Scheme 3). These additions can be performed on 6 or 8 to furnish 9 (or 9a, the enantiomer of 9, or mixtures thereof) or 1 (or la, the enantiomer of 1, or mixtures thereof), respectively.

Scheme 3. Synthesis of 1 or la

R-i = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, -0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S02)-alkyl, -0(S02)-substituted alkyl, -0(S02)-aryl, or -0(S02)-substituted aryl.

Alternatively, ketone 8 can be synthesized from aldehyde 10 (Scheme 4). Aldehyde 10 is coupled with 7 to produce 11. Compound 11 is then converted to 12 via treatment with diethylaminosulfurtrifluoride (DAST).

Scheme 4. Alternate synthesis of ketone 8

Scheme 5 outlines the synthesis of precursor ketone 15-Br. The ketone is prepared by conversion of 2-Br to 3-Br as described above. Next, ester 3-Br is converted to 15-Br by treatment via lithiation of 2,4-difluoro-bromobenzene.

Scheme 5. Synthesis of ketone 15-Br

Ketone 15 may be prepared in an analogous fashion as described for 15-Br in Scheme 5 starting from corresponding substituted 2-bromo-pyridines, which can be prepared using according to synthetic transformations known in the art and contained in the references cited herein (Scheme 6).

Scheme 6. Synthesis of ketone 15

F = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, – 0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S02)-alkyl, -0(S02)-substituted alkyl, – 0(S02)-aryl, or -0(S02)-substituted aryl.

Ketone 15 may be used to prepare 9 (or 9a, the enantiomer of 9, or mixtures thereof) or 1 (or la, the enantiomer of 1, or mixtures thereof) by the following three-step process (Scheme 7). In the presence of a chiral catalyst/reagent (e.g. proline derivatives), base-treated nitromethane is added to 15 or 16 to furnish 17 (or 17a, the enantiomer of 17, or mixtures thereof) or 18 (or 18a, the enantiomer of 18, or mixtures thereof), respectively. Reduction of 17 (or 17a, the enantiomer of 17, or mixtures thereof) or 18 (or 18a, the enantiomer of 18, or mixtures thereof) (e.g. lithium aluminum hydride) produces 19 (or 19a, the enantiomer of 19, or mixtures thereof) or 20 (or 20a, the enantiomer of 20, or mixtures thereof). Annulation of 19 (or 19a, the enantiomer of 19, or mixtures thereof) or 20 (or 20a, the enantiomer of 20, or mixtures thereof) by treatment with sodium azide/triethylorthoformate furnishes tetrazoles 9 (or 9a, the enantiomer of 9, or mixtures thereof) or 1 (or la, the enantiomer of 1, or mixtures thereof). Suzuki coupling of 9 (or 9a, the enantiomer of 9, or mixtures thereof) with 4-trifluoromethoxyphenyl-boronic acid produces 1 (or la, the enantiomer of 1, or mixtures thereof).

Scheme 7. Asymmetric Henry reaction

R-ι = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, 0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S02)-alkyl, -0(S02)-substituted a 0(S02)-aryl, or -0(S02)-substituted aryl.

Ketone 21 may be employed to prepare optically-active epoxides via Horner-Emmons reaction of a difluoromethyl substrate to produce 22 or 22a. Ketones related to 21 have been prepared (M. Butters, Org. Process Res. Dev. 2001, 5, 28-36). Nucleophilic addition of metalated 5-(4-trifluoromethoxy)phenyl-2-pyridine (M = metal) to epoxide 22 or 22a may furnish compound

1 or la.

Scheme 8. Enantioselective epoxidation strategy

Ketone 15 or 16 may be used to prepare 9 (or 9a, the enantiomer of 9, or mixtures thereof) or 1 (or la, the enantiomer of 1, or mixtures thereof) by an alternative three-step process to Scheme 7 (Scheme 9). In the presence of a chiral catalyst/reagent, trimethylsilyl-cyanide is added to 15 or 16 to furnish 23 (or 23a, the enantiomer of 23, or mixtures thereof) or 24 (or 24a, the enantiomer of 24, or mixtures thereof), respectively (S.M. Dankwardt, Tetrahedron Lett. 1998, 39, 4971-4974). Reduction of 23 (or 23a, the enantiomer of 23, or mixtures thereof) or 24 (or 24a, the enantiomer of 24, or mixtures thereof) (e.g. lithium aluminum hydride) produces 19 (or 19a, the enantiomer of 19, or mixtures thereof) or 20 (or 20a, the enantiomer of 20, or mixtures thereof). Annulation of 19 (or 19a, the enantiomer of 19, or mixtures thereof) or 20 (or 20a, the enantiomer of 20, or mixtures thereof) by treatment with sodium azide/triethylorthoformate furnishes tetrazoles 9 (or 9a, the enantiomer of 9, or mixtures thereof) or 1 (or la, the enantiomer of 1, or mixtures thereof). Suzuki coupling of 9 (or 9a, the enantiomer of 9, or mixtures thereof) with 4-trifluoromethoxyphenyl-boronic acid produces 1 (or la, the enantiomer of 1, or mixtures thereof).

Scheme 9. Asymmetric cyanohydrin strategy

R’ = H or trimethylsilyl

Suzuki

R-i = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, -0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S02)-alkyl, -0(S02)-substituted alkyl, -0(S02)-aryl, or -0(S02)-substituted aryl.

1

2-(2, 4-Difluorophenyl)-l, l-difluoro-3-(lH-tetrazol-l-yl)-l-(5-(4-(trifluoromethoxy) phenyl) pyridin-2-yl) propan-2-ol (1 or la)

White powder: *H NMR (500 MHz, CDC13): δ 8.76 (s, 1 H), 8.70 (s, 1 H), 7.97 (dd, J = 8.0, 2.0 Hz, 1 H), 7.68 (d, / = 8.5 Hz, 1 H), 7.60-7.56 (m, 3 H), 7.43-7.36 (m, 3 H), 6.80-6.76 (m, 1 H), 6.70-6.67 (m, 1 H), 5.57 (d, J = 14.5 Hz, 1 H), 5.17 (d, J = 14.5 Hz, 1 H). HPLC: 98.3%. Mass: m/z 513.9 [M++l]. HPLC: 99.8%. Optical rotation [a]D25: + 19° (C = 0.1 % in MeOH).

INTERMEDIATE 3-Br Ri = Br)

To a clean and dry 100 L jacketed reactor was added copper powder (1375 g, 2.05 equiv, 10 micron, sphereoidal, SAFC Cat # 326453) and DMSO (17.5 L, 7 vol). Next, ethyl bromodifluoroacetate (2.25 kg, 1.05 equiv, Apollo lot # 102956) was added and the resulting slurry stirred at 20-25 °C for 1-2 hours. Then 2,5-dibromopyridine (2-Br, 2.5 kg, 1.0 equiv, Alfa Aesar lot # F14P38) was added to the batch and the mixture was immediately heated (using the glycol jacket) to 35 °C. After 70 hours at 35 °C, the mixture was sampled for CG/MS analysis. A sample of the reaction slurry was diluted with 1/1 CH3CN/water, filtered (0.45 micron), and the filtrate analyzed directly. Ideally, the reaction is deemed complete if <5% (AUC) of 2,5-dibromopyridine remains. In this particular batch, 10% (AUC) of 2,5-dibromopyridine remained. However due to the already lengthy reaction time, we felt that prolonging the batch would not help the conversion any further. The reaction was then deemed complete and diluted with EtOAc (35 L). The reaction mixture was stirred at 20-35 °C for 1 hour and then the solids (copper salts) were removed by filtration through a pad of Celite. The residual solids inside the reactor were rinsed forward using EtOAc (2 x 10 L) and then this was filtered through the Celite. The filter cake was washed with additional EtOAc (3 x 10 L) and the EtOAc filtrates were combined. A buffer solution was prepared by dissolving NH4CI (10 kg) in DI water (100 L), followed by the addition of aqueous 28% NH4OH (2.0 L) to reach pH = 9. Then the combined EtOAc filtrates were added slowly to a pre-cooled (0 to 15 °C) solution of NH4C1 and NH4OH (35 L, pH = 9) buffer while maintaining T<30 °C. The mixture was then stirred for 15-30 minutes and the phases were allowed to separate. The aqueous layer (blue in color) was removed and the organic layer was washed with the buffer solution until no blue color was discernable in the aqueous layer. This experiment required 3 x 17.5 L washes. The organic layer was then washed with a 1/1 mixture of Brine (12.5 L) and the pH = 9 NH4C1 buffer solution (12.5 L), dried over MgS04, filtered, and concentrated to dryness. This provided crude compound 3-Br [2.29 kg, 77% yield, 88% (AUC) by GC/MS] as a yellow oil. The major impurity present in crude 3-Br was unreacted 2,5-dibromopyridine [10% (AUC) by GC/MS]. ‘ll NMR (CDC13) was consistent with previous lots of crude compound 3-Br. Crude compound 3-Br was then combined with similar purity lots and purified by column chromatography (5/95 EtO Ac/heptane on S1O2 gel).

INTERMEDIATE 15-Br (R, = Br)

To a clean and dry 72 L round bottom flask was added l-bromo-2,4-difluorobenzene (1586 g,

1.15 equiv, Oakwood lot # H4460) and MTBE (20 L, 12.6 vol). This solution was cooled to -70 to -75 °C and treated with n-BuLi (3286 mL, 1.15 equiv, 2.5 M in hexanes, SAFC lot # 32799MJ), added as rapidly as possible while maintaining -75 to -55 °C. This addition typically required 35-45 minutes to complete. (NOTE: If the n-BuLi is added slowly, an white slurry will form and this typically gives poor results). After stirring at -70 to -65 °C for 45 minutes, a solution of compound 3-Br (2000 g, 1.0 equiv, AMRI lot # 15CL049A) in MTBE (3 vol) was added rapidly (20-30 min) by addition funnel to the aryl lithium solution while maintaining -75 to -55 °C. After stirring for 30-60 minutes at -75 to -55 °C, the reaction was analyzed by GC/MS and showed only trace (0.5% AUC) l-bromo-2,4-difluorobenzene present. The reaction was slowly quenched with aqueous 2 M HC1 (3.6 L) and allowed to warm to room temperature. The mixture was adjusted to pH = 6.5 to 8.5 using NaHCC>3 (4 L), and the organic layer was separated. The MTBE layer was washed with brine (5% NaCl in water, 4 L), dried over MgS04, filtered, and concentrated. In order to convert the intermediate hemi-acetal to 4-Br, the crude mixture was heated inside the 20 L rotovap flask at 60-65 °C for 3 hours (under vacuum), at this point all the hemi-acetal was converted to the desired ketone 4 by !Η NMR (CDC13). This provided crude compound 4-Br [2.36 kg, 75% (AUC) by HPLC] as a brown oil that solidified upon standing. This material can then be used “as-is” in the next step without further purification.

Image result for VT1129

PATENT FOR VT1161    SIMILAR TO VT 1129

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016149486&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Synthesis of 1 or la

EXAMPLE 1

Preparation of Compound 1 X-Hydrate

Compound 1 and its preparation are described in the art, including in US Patent 8,236,962 (incorporated by reference herein). Compound 1 can then be partitioned between ethanol and water to afford Compound 1 X-hydrate.

EXAMPLE 2

Compound 1 Anhydrous Form Recrystallization

Compound 1 X-hydrate (29.1 g, 28.0 g contained 1) was suspended in 2-propanol (150 ml) and heated to 56 °C. The solution was filtered through a 0.45 μιη Nylon membrane with 2-propanol rinses. The combined filtrate was concentrated to 96.5 g of a light amber solution. The solution was transferred to a 1-L flask equipped with overhead stirring, thermocouple and addition funnel, using 2-propanol (30 ml total) to complete the transfer. The combined solution contained about 116 ml 2-propanol.

The solution was heated to 50 °C and n-heptane (234 ml) was added over 22 minutes. The resulting hazy mixture was seeded with 1 anhydrous form. After about 1 hour a good

suspension had formed. Additional n-heptane (230 ml) was added over 48 minutes. Some granular material separated but most of the suspension was a finely divided pale beige solid. After about ½ hour at 50 °C the suspension was cooled at 10 °C/h to room temperature and stirred overnight. The product was collected at 22 °C on a vacuum filter and washed with 1:4 (v/v) 2-PrOH/ n-heptane (2 x 50 ml). After drying on the filter for 1-2 hours the weight of product was 25.5 g. The material was homogenized in a stainless steel blender to pulverize and blend the more granular solid component. The resulting pale beige powder (25.37 g) was dried in a vacuum oven at 50 °C. The dry weight was 25.34 g. The residual 2-propanol and n- heptane were estimated at <0.05 wt% each by 1H NMR analysis. The yield was 90.5% after correcting the X-hydrate for solvent and water content. Residual Pd was 21 ppm. The water content was 209 ppm by KF titration. The melting point was 100.7 °C by DSC analysis.

Table 1: Data for the isolated and dried Compound 1 – X-hydrate and anhydrous forms

M.P. by DSC; Pd by ICP; Purity by the API HPLC method; Chiral purity by HPLC; water content by KF titration; residual solvent estimated from :H NMR.

Table 2: Characterisation Data for Compounds 1 (X-hydrate) and 1 (anhydrous)

Needle like crystals Needle like crystals and agglomerates

PLM

particle size >100μιη particle size range from 5μπι-100μιη

0.59%w/w water uptake at 90%RH. 0.14%w/w water uptake at 90%RH.

GVS

No sample hysteresis No sample hysteresis

XRPD

No form change after GVS experiment No form change after GVS experiment post GVS

KF 2.4%w/w H20 Not obtained

<0.001mg/ml <0.001mg/ml

Solubility

pH of saturated solution = 8.6 pH of saturated solution = 8.7

Spectral Pattern 1 Spectral Pattern 2

Charcteristic bands/ cm“1: Charcteristic bands/ cm 1:

FT-IR 3499, 3378, 3213, 3172 3162

1612, 1598, 1588, 1522, 1502 1610, 1518, 1501 931, 903, 875, 855, 828, 816 927, 858, 841, 829, 812

The structure solution of Compound 1 anhydrous form was obtained by direct methods, full-matrix least-squares refinement on F 2 with weighting w‘1 = <52{F02) + (0.0474P)2 + (0.3258P), where P = (F02+2F 2)/3, anisotropic displacement parameters, empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. Final wR2

= {∑[w(F02-Fc2)2]/∑[w(F02)2]m} = 0.0877 for all data, conventional Ri = 0.0343 on F values of 8390 reflections with F0 > 4a( F0), S = 1.051 for all data and 675 parameters. Final Δ/a (max) 0.001, A/a(mean), 0.000. Final difference map between +0.311 and -0.344 e A“3.

Below shows a view of two molecules of Compound 1 in the asymmetric unit of the anhydrous form showing the numbering scheme employed. Anisotropic atomic displacement ellipsoids for the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. The absolute configuration of the molecules has been determined to be R.

EXAMPLE 3

Compound 1 Ethanol Solvate Recrystallization

Compound 1 X-hydrate (50 mg) was suspended in -40 volumes of 15% H20/EtOH. The suspension was then placed in an incubation chamber for maturation. The maturation protocol involved treating the suspension to a two-temperature cycle of 50 °C/ ambient temperature at 8 hours per cycle for 3 days with constant agitation. After maturation, the suspension was cooled in a fridge at 4°C for up to 2 days to encourage the formation of crystals. Then, the solvent was removed at RT and the sample was vacuum dried at 30°C -35°C for up to 1 day. Suitable crystals formed on cooling were harvested and characterized.

Table 4: Single Crystal Structure of 1 Ethanol solvate

Molecular formula C25H22F7N5O3

The structure solution of Compound 1 ethanol solvate was obtained by direct methods, full-matrix least-squares refinement on F 2 with weighting w‘1 = σ2^2) + (0.0450P)2 + (0.5000P), where P = (F02+2F 2)/3, anisotropic displacement parameters, empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. Final wR2 = {∑[w(F02-F 2)2]/∑[w(F02)2]m} = 0.0777 for all data, conventional Ri = 0.0272 on F values of 4591 reflections with F0 > 4σ( F0), S = 1.006 for all data and 370 parameters. Final Δ/σ (max) 0.000, A/a(mean), 0.000. Final difference map between +0.217 and -0.199 e A“3.

Below shows a view of the asymmetric unit of the ethanol solvate from the crystal structure showing the numbering scheme employed. Anisotropic atomic displacement ellipsoids for the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. The asymmetric unit shows stoichiometry of 1 : 1 for solvent of crystallisation to Compound 1.

EXAMPLE 4

Compound 1 1.5 Hydrate Recrystallization

Compound 1 X-hydrate (50 mg) was suspended in -40 volumes of 15% Η20/ΙΡΑ. The suspension was then placed in an incubation chamber for maturation. The maturation protocol involved treating the suspension to a two-temperature cycle of 50 °C/ ambient temperature at 8 hours per cycle for 3 days with constant agitation. After maturation, the suspension was cooled in a fridge at 4°C for up to 2 days to encourage the formation of crystals. Then, the solvent was removed at RT and the sample was vacuum dried at 30°C -35°C for up to 1 day. Suitable crystals formed on cooling were harvested and characterized.

Table 5: Single Crystal Structure of 1 1.5 Hydrate

The structure solution of Compound 1 1.5 hydrate was obtained by direct methods, full-matrix least-squares refinement on F 2 with weighting w‘1 = ^(F 2) + (0.1269P)2 + (0.0000P), where P = (F02+2F 2)/3, anisotropic displacement parameters, empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. Final wR2 = {∑[w(F 2-F 2)2]/∑[w(F 2)2] m} = 0.1574 for all data, conventional Ri = 0.0668 on F values of 2106 reflections with F0 > 4σ( F0), S = 1.106 for all data and 361 parameters. Final Δ/σ (max) 0.000, A/a(mean), 0.000. Final difference map between +0.439 and -0.598 e A“3.

Below shows a view of the asymmetric unit of the 1.5 hydrate from the crystal structure showing the numbering scheme employed. Anisotropic atomic displacement ellipsoids for the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. The asymmetric unit shows stoichiometry of 1.5: 1 for water to Compound 1.

EXAMPLE 5

Human Pharmacokinetic Comparison of Compound 1 X-Hydrate and Compound 1 Anhydrous Form

Table 6 compares human multiple-dose pharmacokinetic (PK) parameters between dosing with Compound 1 X-hydrate and Compound 1 Anhydrous form. Compound 1 X-hydrate was dosed at 600 mg twice daily (bid) for three days followed by dosing at 300 mg once daily (qd) for 10 days. Compound 1 Anhydrous form was dosed at 300 mg qd for 14 days. Despite the higher initial dosing amount and frequency (i.e., 600 mg bid) of Compound 1 X-hydrate, Compound 1 Anhydrous form surprisingly displayed higher maximal concentration (Cmax) and higher area-under-the-curve (AUC) than Compound 1 X-hydrate.

Table 6. Comparison of Multiple Dose PK between Compound 1 X-Hydrate and Compound 1

Anhydrous Polymorph

Further characterization of the various polymorph forms of compound 1 are detailed in the accompanying figures.

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015143154

Examples

General Experimental Procedures

Definitions of variables in the structures in schemes herein are commensurate with those of corresponding positions in the formulae delineated herein.

Synthesis of 1 or la

la

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

Scheme 1. Synthesis of ketone 3-Br

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

Scheme 2. Synthesis of ketone 3

R1 = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)-substituted aryl, -0(C=0)-0-alkyl, – 0(C=0)-0-substituted alkyl, -0(C=0)-0-aryl, -0(C=0)-0-substituted aryl, -0(S02)-alkyl, -0(S02)-substituted alkyl, 0(S02)-aryl, or -0(S02)-substituted aryl.

Alternatively, compound 1 (or la, the enantiomer of 1, or mixtures thereof) can be prepared according to Scheme 3 utilizing amino-alcohols ±4b or ±1-6. Epoxides 4 and 5 can be prepared by reacting ketones 3 and 1-4 with trimethylsulfoxonium iodide (TMSI) in the presence of a base (e.g., potassium i-butoxide) in a suitable solvent or a mixture of solvents (e.g., DMSO or THF). Also, as indicated in Scheme 3, any of pyridine compounds, 3, 4, ±4b, 4b, or 6, can be converted to the corresponding 4-CF3O-PI1 analogs (e.g., 1-4, 5, ±1-6, 1-6*, or 1 or the corresponding enantiomers, or mixtures thereof) by cross-coupling with (4-trifluoromethoxyphenyl)boronic acid (or the corresponding alkyl boronates or pinnacol boronates or the like), in a suitable solvent system (e.g., an organic-aqueous solvent mixture), in the presence of a transition metal catalyst (e.g., (dppf)PdCl2; dppf = 1,1′-(diphenylphosphino)ferrocene), and in the presence of a base (e.g., KHCO3, K2CO3, CS2CO3, or Na2CC>3, or the like). Epoxides 4 and 5 can then be converted into amino-alcohols ±4b and ±1-6 through ammonia-mediated epoxide opening using ammonia in a suitable solvent (e.g., MeOH, EtOH, or water). Racemic amino-alcohols ±4b and ±1-6 can then be enantio-enriched by exposure to a chiral acid (e.g., tartaric acid, di-benzoyltartaric acid, or di-p-toluoyltartaric acid or the like) in a suitable solvent (e.g., acetonitrile, isopropanol, EtOH, or mixtures thereof, or a mixture of any of these with water or MeOH; preferably acetonitrile or a mixture of acetonitrile and MeOH, such as 90:10, 85: 15, or 80:20 mixture) to afford compounds 4b (or 4c, the enantiomer of 4b, or mixtures thereof) or 1-6* (or 1-7*, the enantiomer of 1-6*, or mixtures thereof). Subsequent treatment with TMS-azide in the presence of trimethylorthoformate and sodium acetate in acetic acid would yield compounds 20 (or 20a, the enantiomer of 20, or mixtures thereof) or 1 (or la, the enantiomer of 1, or mixtures thereof) (US 4,426,531).

Scheme 3. Synthesis of 1 or la via TMSI Epoxidation Method

R-ι = halo, -0(C=0)-alkyl, -0(C=0)-substituted alkyl, -0(C=0)-aryl, -0(C=0)- substituted aryl, -0(C=0)-0-alkyl, -0(C=0)-0-substituted alkyl, -0(C=0)-0- aryl, -0(C=0)-0-substituted aryl, -0(S02)-alkyl, -0(S02)-substituted alkyl, – 0(S02)-aryl, or -0(S02)-substituted aryl.

Compound 1 (or la, the enantiomer of 1, or mixtures thereof) prepared by any of the methods presented herein can be converted to a sulfonic salt of formula IX (or IXa, the enantiomer of

IX, or mixtures thereof), as shown in Scheme 4. This can be accomplished by a) combining compound 1 (or la, the enantiomer of 1, or mixtures thereof), a crystallization solvent or crystallization solvent mixture (e.g., EtOAc, iPrOAc, EtOH, MeOH, or acetonitrile, or o

Z-S-OH

combinations thereof), and a sulfonic acid o (e.g., Z = Ph, p-tolyl, Me, or Et), b) diluting the mixture with an appropriate crystallization co-solvent or crystallization co-solvent mixture (e.g., pentane, methyl i-butylether, hexane, heptane, or toluene, or combinations thereof), and c) filtering the mixture to obtain a sulfonic acid salt of formula IX (or IXa, the enantiomer of IX, or mixtures thereof).

Scheme 4. Synthesis of a Sulfonic Acid Salt of Compound 1 or la

EXAMPLE 1: Preparation of l-(2,4-difluorophenyl)-2,2-difluoro-2-(5-(4- (trifluoromethoxy)phenyl)pyridin-2-yl)ethanone (1-4).

la. ethyl 2-(5-bromopyridin-2-yl)-2,2-difluoroacetate (2)

2-Br
Typical Procedure for Preparing 2-Br

Copper ( 45μιη, 149g, 0.198moles, 2.5 equiv) was placed into a 3L, 3-neck round bottom flask equipped with a condenser, thermocouple, and an overhead stirrer. DMSO (890 mL, 4.7 vol. based on ethyl 2-bromo-2,2-difluoroacetate) and 14mL of concentrated sulfuric acid was added and the mixture stirred for 30 minutes. The mixture self-heated to about 31°C during the stir time. After cooling the contents to 23°C, 2,5-dibromopyridine 1 (277g, 1.17 moles, 1.5 eq) was added to the reaction mixture. The temperature of the contents decreased to 16°C during a 10 minute stir time. 2-bromo-2,2-difluoroacetate (190 g, 0.936 moles, 1.0 eq) was added in one portion and the mixture stirred for 10 min. The flask contents were warmed to 35°C and the internal temperature was maintained between 35-38° for 18 h. In-process HPLC showed 72% desired 2-Br. The warm reaction mixture was filtered through filter paper and the collected solids washed with 300mL of 35°C DMSO. The solids were then washed with 450mL of n-heptane and 450mL of MTBE. The collected filtrate was cooled to about 10°C and was slowly added 900mL of a cold 20% aqueous NH4C1 solution, maintaining an internal temperature of <16°C during the addition. After stirring for 15 minutes, the layers were settled and separated. The aqueous layer was extracted 2 X 450mL of a 1: 1 MTBE: n-heptane mixture. The combined organic layers were washed 2 X 450mL of aqueous 20% NH4CI and with 200mL of aqueous 20% NaCl. The organic layer was dried with 50g MgS04 and the solvent removed to yield 2-Br as a dark oil. Weight of oil = 183g ( 70% yield by weight) HPLC purity ( by area %) = 85%. *H NMR (400 MHz, d6-DMSO) : 58.86 (m, 1H), 8.35 ( dd, J= 8.4, 2.3Hz, 1H), 7.84 (dd, J= 8.3, 0.6Hz, 1H), 4.34 ( q, J= 7.1Hz, 2H), 1.23 ( t, J= 7.1Hz, 3H). MS m/z 280 ( M+H+), 282 (M+2+H+).

lb. 2-(5-bromopyridin-2-yl)-2,2-difluoro-l-morpholinoethanone (2b-Br)

Table 2 illustrates the effects of the relative proportions of each of the reagents and reactants, and the effect of varying the solvent had on the overall performance of the transformation as measured by the overall yield and purity of the reaction.

Table 2. Process Development for the Preparation of compound 2b-Br

Note: All reactions were conducted at 22- 25°C

Typical Procedure for Converting 2-Br to 2b-Br

Crude ester 2-Br (183g, 0.65moles) was dissolved in 1.5L of n-heptane and transferred to a 5L 3-neck round bottom flask equipped with a condenser, an overhead stirrer and a thermocouple. Morpholine ( 248g, 2.85 moles, 4.4 equiv.) was charged to the flask and the mixture warmed to 60°C and stirred for 16 hours. In-process HPLC showed <1 % of ester 2-Br. The reaction mixture was cooled to 22-25 °C and 1.5L of MTBE was added with continued cooling of the mixture to 4°C and slowly added 700mL of a 30%, by weight, aqueous citric acid solution. The temperature of the reaction mixture was kept < 15°C during the addition. The reaction was stirred at about 14°C for one hour and then the layers were separated. The organic layer was washed with 400mL of 30%, by weight, aqueous citric acid solution and then with 400mL of aqueous 9% NaHC03. The solvent was slowly removed until 565g of the reaction mixture

remained. This mixture was stirred with overhead stirring for about 16 hours. The slurry was filtered and the solids washed with 250mL of n-heptane. Weight of 2b-Br = 133g. HPLC purity (by area %) 98%.

This is a 44% overall yield from 2,5-dibromopyridine.

*H NMR (400 MHz, d6-DMSO): 58.86 (d, J= 2.3Hz, 1H), 8.34 (dd, J= 8.5, 2.3Hz, 1H), 7.81 (dd, J = 8.5, 0.5Hz, 1H), 3.63-3.54 ( m, 4H), 3.44-3.39 (m, 2H), 3.34-3.30 ( m, 2H). MS m/z 321 (M+H+), 323 (M+2+H+).

lc. 2-(5-bromopyridin-2-yl)-l-(2,4-difluorophenyl)-2,2-difluoroethanone (3-Br)

Process Development

Table 3 illustrates the effects of the relative proportions of each of the reagents and reactants, and the effect of varying the temperature had on the overall performance of the transformation as measured by the overall yield and purity of the reaction.

Table 3. Process Development for the Preparation of bromo-pyridine 3-Br

Typical Procedure for Converting 2b-Br to 3-Br

Grignard formation:

Magnesium turnings (13.63 g, 0.56 moles) were charged to a 3-neck round bottom flask equipped with a condenser, thermocouple, addition funnel, and a stir bar. 540 mL of anhydrous tetrahydrofuran was added followed by l-Bromo-2,4-difluorobenzene (16.3 mL, 0.144 moles). The contents were stirred at 22-25°C and allowed to self -heat to 44°C. 1- Bromo-2,4-difluorobenzene ( 47mL, 0.416 moles) was added to the reaction mixture at a rate that maintained the internal temperature between 40-44°C during the addition. Once the addition was complete, the mixture was stirred for 2 hours and allowed to cool to about 25° during the stir time.

This mixture was held at 22-25°C and used within 3-4 hours after the addition of l-bromo-2,4-difluorobenzene was completed.

Coupling Reaction

Compound 2b-Br (120 g, 0.0374 moles) was charged to a 3-neck round bottom flask equipped with a condenser, thermocouple, and an overhead stirrer. 600 mL of anhydrous

tetrahydrofuran was added. The flask contents were stirred at 22°C until a clear solution was obtained. The solution was cooled to 0-5°C. The previously prepared solution of the Grignard reagent was then added slowly while maintaining the reaction temperature at 0-2°C. Reaction progress was monitored by HPLC. In-process check after 45 minutes showed <1% amide 2b-Br remaining. 2 N aqueous HC1 (600 mL, 3 vol) was added slowly maintaining the temperature below 18°C during the addition. The reaction was stirred for 30 minutes and the layers were separated. The aqueous layer was extracted with 240mL MTBE. The combined organic layers were washed with 240mL of aqueous 9% NaHCC>3 and 240mL of aqueous 20% NaCl. The organic layer was dried over 28g of MgS04 and removed the solvent to yield 3-Br (137g) as an amber oil.

HPLC purity ( by area %) = -90%; *H NMR (400 MHz, d6-DMSO) : 58.80 (d, J= 2.2Hz, 1H), 8.41 ( dd, J= 8.3, 2.3Hz, 1H), 8.00 (m, 2H), 7.45 ( m, 1H), 7.30 ( m, 1H). MS m/z 348 (M+H+), 350 (M+2+H+).

Id. l-(2,4-difluorophenyl)-2,2-difluoro-2-(5-(4-(trifluoromethoxy)phenyl)pyridin-2-yl)ethanone (1-4)

Typical Procedure for Converting 3-Br to 1-4

Into a 250 mL reactor were charged THF (45 mL), water (9.8 mL), bromo-pyridine 3-Br (6.0 g, 17.2 mmoles), 4-(trifluoromethoxy)phenylboronic acid (3.57 g, 17.3 mmoles), and Na2CC>3 (4.55 g, 42.9 mmoles). The stirred mixture was purged with nitrogen for 15 min. The catalyst (Pd(dppf)Cl2 as a CH2C12 adduct, 0.72 g, 0.88 mmoles) was added, and the reaction mixture was heated to 65 °C and held for 2.5 h. The heat was shut off and the reaction mixture was allowed to cool to 20-25 °C and stir overnight. HPLC analysis showed -90% ketone 1-4/hydrate and no unreacted bromo-pyridine 3-Br. MTBE (45 mL) and DI H20 (20 mL) were added, and the quenched reaction was stirred for 45 min. The mixture was passed through a plug of Celite (3 g) to remove solids and was rinsed with MTBE (25 mL). The filtrate was transferred to a separatory funnel, and the aqueous layer drained. The organic layer was washed with 20% brine (25 mL). and split into two portions. Both were concentrated by rotovap to give oils (7.05 g and 1.84 g, 8.89 g total, >100% yield, HPLC purity -90%). The larger aliquot was used to generate hetone 1-4 as is. The smaller aliquot was dissolved in DCM (3.7 g, 2 parts) and placed on a pad of Si02 (5.5 g, 3 parts). The flask was rinsed with DCM (1.8 g), and the rinse added to the pad. The pad was eluted with DCM (90 mL), and the collected filtrate concentrated to give an oil (1.52 g). To this was added heptanes (6 g, 4 parts) and the mixture stirred. The oil crystallized, resulting in a slurry. The slurry was stirred at 20-25 °C overnight. The solid was isolated by vacuum filtration, and the cake washed with heptanes (-1.5 mL). The cake was dried in the vacuum oven (40-45 °C) with a N2 sweep. 0.92 g of ketone 1-4 was obtained, 60.1% yield (corrected for aliquot size), HPLC purity = 99.9%.

TMSI Epoxidation Method

3d. 2-((2-(2,4-difluorophenyl)oxiran-2-yl)difluoromethyl)-5-(4-(trifluoromethoxy)phenyl)pyridine (5)

Typical Procedure for Converting 1-4 to 5

i-BuOK (2.22 g, 19.9 mmoles), TMSI (4.41 g, 20.0 mmoles), and THF (58.5 mL) were charged to a reaction flask, and the cloudy mixture was stirred. DMSO (35.2 mL) was added, and the clearing mixture was stirred at 20-25°C for 30 min before being cooled to 1-2°C.

Ketone 1-4 (crude, 5.85 g, 13.6 mmoles) was dissolved in THF (7.8 mL), and the 1-4 solution was added to the TMSI mixture over 12.75 min, maintaining the temperature between 1.5 and 2.0°C. The reaction was held at 0-2°C. After 1 h a sample was taken for HPLC analysis, which showed 77.6% epoxide 5, and no unreacted ketone 1-4. The reaction was quenched by the slow addition of 1 N HC1 (17.6 mL), keeping the temperature below 5°C. After 5 min 8% NaHCC>3 (11.8 mL) was added slowly below 5°C to afford a pH of 8. The reaction mixture was transferred to a separatory funnel, and the layers were separated. The aqueous layer was extracted with MTBE (78 mL), and the combined organic layers were washed with 20% NaCl (2 x 20 mL). After concentration, 7.36 g of a dark oil was obtained. HPLC of the crude oil shows it contained 75% epoxide 5. The oil was dissolved in DCM (14.7 g, 2 parts) and the solution placed on a pad of Si02 (22 g, 3 parts). The flask was rinsed with DCM (7.4 g, 1 part) and the rinse placed on the pad. The pad was eluted with DCM (350 mL) to give an amber filtrate. The filtrate was concentrated by rotovap, and when space in the flask allowed, heptane (100 mL) was added. The mixture was concentrated until 39.4 g remained in the flask, causing solid to form. The suspension was stirred for 70 min at 20-25°C. Solid was isolated by vacuum filtration, and the cake washed with heptane (10 mL) and pulled dry on the funnel. After drying in a vacuum oven (40-45 °C) with a N2 sweep, 3.33 g solid was obtained, 55.1% yield from bromo-pyridine 3, HPLC purity = 99.8%.

3e. 3-amino-2-(2,4-difluorophenyl)-l,l-difluoro-l-(5-(4-(trifluoromethoxy)phenyl)pyridin-2-yl)propan-2-ol (±1-6)

Process Development

Table 8 illustrates the effects of the relative proportions of each of the reagents and reactants, the effect of varying the solvent, and the effect of varying the temperature had on the overall performance of the transformation as measured by the overall yield and purity of the reaction. Table 8. Process Development for the Preparation of ±1-6

Typical Procedure for Converting 5 to +1-6

Epoxide 5 (2.17 g, 4.89 mmoles) was combined in a glass pressure tube with methanol (48 mL) and aqueous ammonia (19.5 mL). The tube was sealed and placed in an oil bath held at 54°C, with stirring. After 15 h the tube was removed from the bath, cooled, and the reaction sampled for HPLC, which showed 93.6% amino-alcohol ±1-6 and 6.0% di-adducts. To the reaction were added MTBE (48 mL) and 20% NaCl (20 mL). The layers were separated and the aqueous layer extracted with MTBE (20 mL). The combined organic layers were washed with H20 (20 mL) and transferred to a rotovap flask. Heptane (20 mL) was added, and the solution was concentrated until 16.9 g remained in the flask. An H20 layer appeared in the flask, and was pipetted out, leaving 12.8 g. Compound 1-6 seed was added, and the crystallizing mixture was stirred at 20-25 °C overnight. The flask was cooled in an ice bath for 2 h prior to filtration, and the isolated solid was washed with cold heptane (5 mL), and pulled dry on the funnel. After drying in a vacuum oven (40-45°C) for several hours 1.37 g of amino-alcohol ±1-6 was obtained, 60.8% yield, HPLC purity = 98.0%.

3f . 3-amino-2-(2,4-difluorophenyl)- 1 , 1-difluoro- 1 -(5-(4-(trifluoromethoxy)phenyl)pyridin-2- yl)propan-2-ol (1-6* or 1-7*)

Process Development

Table 9 illustrates the initial screen performed surveying various chiral acid/solvent combinations. All entries in Table 9 were generated using 0.1 mmoles of amino-alcohol ±1-6, 1 equivalent of the chiral acid, and 1ml of solvent.

Table 9. Resolution of ±1-6 (Initial Screen)

Since the best results from Table 9 were generated using tartaric acid and di-p-toluoyltartaric acid, Table 10 captures the results from a focused screen using these two chiral acids and various solvent combinations. All entries in Table 10 were performed with 0.2 mmoles of amino-alcohol ±1-6, 87 volumes of solvent, and each entry was exposed to heating at 51 °C for lh, cooled to RT, and stirred at RT for 24h.

Table 10. Resolution of ±1-6 (Focused Screen)

Each of the three entries using di-p-toluoyltartaric acid in Table 10 resulted in higher levels of enantio-enrichment when compared to tartaric acid. As such, efforts to further optimize the enantio-enrichment were focusing on conditions using di-p-toluoyltartaric acid (Table 11).

Ό.6 equivalents used

ee sense was opposite from the other entries in the table (i.e., enantiomer of 1-6*)

Typical Procedure for Converting +1-6 to 1-6* or 1-7*

(This experimental procedure describes resolution of ±1-6, but conditions used for DPPTA resolution of 1-6 or 1-7 are essentially the same.)

Amino-alcohol ±1-6 (7.0 g, 15 mmoles) was dissolved in a mixture of acetonitrile (84 mL) and methanol (21 mL). (D)-DPTTA (5.89 g, 15 mmoles) was added, and the reaction was warmed to 50°C and held for 2.5 h. The heat was then removed and the suspension was allowed to cool and stir at 20-25 °C for 65 h. The suspension was cooled in an ice bath and stirred for an additional 2 h. Solid was isolated by vacuum filtration, and the cake was washed with cold 8:2 ACN/MeOH (35 mL). After drying at 50°C, 5.18 g of 1-6* or l-7*/DPPTA salt was isolated, HPLC purity = 99.0, ee = 74.

The 1-6* or l-7*/DPPTA salt (5.18 g) was combined with 8:2 ACN/MeOH (68 mL) and the suspension was heated to 50°C and held for 20 min. After cooling to 20-25 °C the mixture was stirred for 16 h. Solids were isolated by vacuum filtration, and the cake washed with cold 8:2 ACN/MeOH (30 mL), and pulled dry on the funnel. 2.82 g of 1-6* or l-7*/DPPTA salt was obtained, 44.4% yield (from crude ±1-6), ee = 97.5. The resulting solids were freebased to provide 1-6* or 1-7* with the same achiral and chiral purity as the DPPTA salt.

EXAMPLE 4: Preparation of 2-(2.4-difluorophenyl -l.l-difluoro-3-(lH-tetrazol-l-yl -l-(5-(4-(trifluoromethoxy)phenyl)pyridin-2-yl)propan-2-ol (1 or la).

The procedure used to generate compound 1 or la is as described in US 4,426,531. Table 13 illustrates the efficient and quantitative nature of this procedure as performed on amino- alcohol 1-6* or 1-7* produced from both the TMS-cyanohydrin method and the TMSI- epoxidation method.

Table 13. Formation of Compound 1 or la

EXAMPLE 5: 2-(2.4-difluorophenyl -l.l-difluoro-3-(lH-tetrazol-l-yl -l-(5-(4- (trifluoromethoxy)phenyl)pyridin-2-yl)propan-2-ol benzenesulfonate (1 or la-BSA).

Typical Procedure for Converting 1 or la to 1 or la-BSA

46.6 g of compound 1 or la was dissolved in ethylacetate (360ml). The solution was filtered through a glass microfiber filter and placed in a 2 L reaction flask equipped with an overhead stirrer, condenser, and a J-Kem thermocouple. Pharma-grade benzenesulfonic acid (BSA, 14.39g, leq) was dissolved in ethyl acetate (100ml). The BSA solution was filtered through a glass microfiber filter and added to the stirred 1 or la solution in one portion. The mixture was warmed to 60-65 °C; precipitation of the 1 or la/BSA salt occurred during the warm up period. The slurry was held for 60 minutes at 60-65 °C. The suspension was allowed to slowly cool to 22 °C and was stirred at 20-25 °C for 16 hours. n-Heptane (920ml) was charged in one portion and the suspension was stirred at 22 °C for an additional 90 minutes. The slurry was filtered and the collected solids washed with n-heptane (250ml). The isolated solids were placed in a vacuum oven at 50 °C for 16 hours. 52.26g (86% yield) of 1 or la

benzenesulfonate was obtained.

*H NMR (400 MHz, DMSO-d6 + D20): 89.16 (s, 1H), 8.95 (d, J = 2.1 Hz, 1H), 8.26 (dd, J = 8.2, 2.3 Hz, 1H), 7.96-7.89 (m, 2H), 7.66-7.61 (m, 2H), 7.59 (dd, J = 8.3, 0.4 Hz, 1H), 7.53 (br d, J = 8.0 Hz, 2H), 7.38-7.15 (m, 5H), 6.90 (dt, J = 8.3, 2.5 Hz, 1H), 5.69 (d, J = 14.8 Hz, 1H), 5.15 (d, J = 15.2 Hz, 1H).

Further results are in Table 14.

Table 14. Formation of 1 or la-BSA

( ) (%ee) Yield Purity (%) ee

97.9 95.9 84% 98.2 97.1

Figures 1-2 contain the analytical data for 1 or la-BSA prepared by the TMSI-epoxidation process.

EXAMPLE 6: 5-bromo-2-((2-(2,4-difluorophenyl)oxiran-2-yl)difluoromethyl)pyridine -Br).

Typical Procedure for Converting 3-Br to 4-Br

KOtBu ( 41.7g, 0.372moles, 1.05 equiv) and trimethylsulfoxonium iodide ( 85.7g,

0.389moles, 1.1 equiv) were charged to a 3L 3-neck round bottom flask equipped with an overhead stirrer, a thermocouple and an addition funnel. 1.2L of anhydrous THF and 740mL of DMSO were added to the flask and stirred at 22-25 °C for 70 minutes. The contents were cooled to 0°C. Crude ketone 3 was dissolved in 250mL of anhydrous THF and slowly added the ketone 3-Br solution to the reaction mixture over 20 minutes while maintaining a reaction temperature at < 3°C during the addition and stirred at 0°C for one hour. In-process HPLC showed <1% ketone 3-Br remaining. 200mL of IN HC1 was slowly added maintaining a reaction temperature of < 6°C during the addition. After stirring for 30 minutes the layers were separated and the aqueous layer was extracted with 375mL of MTBE. The combined organic layers were washed with 375mL of aqueous 9% NaHCC>3 and with 375mL of aqueous 20% NaCl. The solvent was removed to yield 4-Br as a brown waxy solid.

Weight of crude epoxide 4-Br = 124.6g; *H NMR (400 MHz, d6-DMSO) : 58.82 (d, J= 2.3Hz, 1H), 8.21 ( dd, J= 8.3, 2.3Hz, 1H), 7.50 (dd, J= 8.3, 0.5Hz, 1H), 7.41 ( m, 1H), 7.25 ( m, 1H), 7.10 (m,lH), 3.40 ( d, J= 4.5Hz, 1H), 3.14 ( m, 1H). MS m/z 362 (M+H+), 364 (M+2+H+).

EXAMPLE 7: 3-amino-l-(5-bromopyridin-2-yl)-2-(2,4-difluorophenyl)-l,l-difluoropropan-2-ol (4b-Br).

Typical Procedure for Converting 4-Br to 4b-Br

Crude epoxide 4-Br ( 54.4g, 0.15moles) was placed into a Schott autoclave bottle equipped with a stir bar. 550mL of MeOH was added to the bottle and stirred for 90 minutes at 22-25 °C. Concentrated NH4OH ( 550mL, 7.98 moles, 53 equiv) was added to the epoxide 4-Br

solution. The bottle was sealed and placed in an oil bath at 55 °C. The mixture was stirred at 55°C for 17 hours. The bottle was removed from the oil bath and cooled to 22-25°C. In-process HPLC showed <1% epoxide 4-Br remaining. The solvent was removed via rotary evaporation until 362g ( 37%) of the reaction mass remained. 500mL of MTBE was added and cooled the mixture to 8°C. 500mL of 6N HCl was slowly added maintaining the reaction temperature between 8 – 12°C during the addition. After stirring for 10 minutes, the layers were separated. The MTBE layer was extracted with 350mL of 6N HCl. The combined aqueous layers were washed with 250mL MTBE and 2 X 250mL heptane. MTBE, 250mL, was added to the aqueous layer and the mixture was cooled to 2°C. 344g of KOH was dissolved in 500mL of water. The KOH solution was slowly added to the reaction mixture over one hour while maintaining the temperature at <19°C. After stirring for 15 minutes, the layers were separated. The aqueous layer was extracted with 250mL MTBE. The combined organic layers were washed with 250mL of aqueous 20% NaCl and the solvent was removed to yield ±4b-Br as a dark oil. Weight of crude amino alcohol ±4b-Br = 46.0g. HPLC purity ( by area %) = 92%; *H NMR (400 MHz, d6-DMSO) : 58.67 (d, J= 2.2Hz, 1H), 8.15 ( dd, J= 8.6, 2.4Hz, 1H), 7.46 (m, 1H), 7.40 ( dd, J= 8.5, 0.7Hz, 1H), 7.10 ( m, 1H), 7.00 (m,lH), 3.37 (dd, J= 13.7, 2.1Hz, 1H), 3.23 ( dd, J= 13.7, 2.7, 1H). MS m/z 379 (M+H+), 381 (M+2+H+).

EXAMPLE 8: 3-amino-l-(5-bromopyridin-2-yl -2-(2.4-difluorophenyl -l.l-difluoropropan-2-ol (4b-Br or 4c-Br).

Typical Procedure for Converting 4-Br to 4b-Br or 4c-Br

Crude amino alcohol ±4b-Br ( 42.4, O. llmoles) was dissolved in 425mL of 8:2 IPA: CH3CN. The solution was charged to a 1L 3-neck round bottom flask equipped with a condenser, overhead stirrer and a thermocouple. Charged di-p-toluoyl-L-tartaric acid ( 21.6g, 0.056moles, 0.5 equiv) to the flask and warmed the contents to 52°C. The reaction mixture was stirred at 52°C for 5 hours, cooled to 22-25°C and stirred for 12 hours. The slurry was cooled to 5-10°C and stirred for 90 minutes. The mixture was filtered and collected solids washed with 80mL of cold CH3CN. The solids were dried in a vacuum oven 45-50°C. Weight of amino alcohol/ DPTTA salt = 17.4g

Chemical purity by HPLC ( area %) = 98.5%; Chiral HPLC= 98.0% ee.

13.60g of the amino alcohol/ DPTTA salt was placed into a 250mL flask with a stir bar and to this was added lOOmL of MTBE and lOOmL of 10% aqueous K2CO3solution. The reaction was stirred until complete dissolution was observed. The layers were separated and the aqueous layer was extracted with 50mL of MTBE. The combined MTBE layers were washed with 50mL of 20% aqueous NaCl and the solvent removed to yield 8.84 (98%) of 4b-Br or 4c-Br as a light yellow oil.

EXAMPLE 9: 3-amino-2-(2,4-difluorophenyl)-l J-difluoro-l-(5-(4-(trifluoromethoxy)phenyl)pyridin-2-yl)propan-2-ol (1-6* or 1-7*).

Typical Procedure for Converting 4b-Br or 4c-Br to 1-6* or 1-7*

Amino alcohol 4b-Br or 4c-Br (8.84g, 0.023moles, 1 equiv) was dissolved in 73mL of n-propanol. The solution was transferred to a 250mL 3-neck round bottom flask equipped with a condenser, thermocouple, stir bar and septum. 17mL of water was added and stirred at 22-25°C for 5 minutes. To the reaction was added K2CO3 ( 9.67g, 0.07moles, 3 equiv), 4-(trifluoromethoxy)phenylboronic acid ( 5.76g, 0.028moles, 1.2 equiv.) and Pd(dppf)Cl2 as a CH2Cl2 adduct ( 0.38g, 0.47mmoles, 0.02 equiv) to the flask. After the mixture was purged with nitrogen for 10 minutes, the reaction was then warmed to 85-87°C and stirred at 85-87°C for 16 hours. HPLC analysis showed < 1% of the amino alcohol 4b-Br or 4c-Br remaining. The mixture was cooled to 22-25 °C, then 115mL of MTBE and 115mL of water were added and stirred for 30 minutes. The layers were separated and the organic layer was washed with 2 X 60mL of 20% aqueous NaCl. The solvent was removed to yield 12.96g ( 121% yield) of 1-6* or 1-7* as a crude dark oil. It should be noted that the oil contains residual solvent, Pd and boronic acid impurity.

‘ll NMR (400 MHz, d6-DMSO) : 58.90 (d, J= 2.2Hz, 1H), 8.22 ( dd, J= 8.3, 2.3Hz, 1H), 7.91 (m, 2H), 7.54 ( m, 4H), 7.14 ( m, 1H), 7.02 (m,lH), 3.41 (m, 1H), 3.27 ( dd, J= 14.0, 2.7, 1H). MS m/z 461 (M+H+)

CLIP

Med. Chem. Commun., 2016,7, 1285-1306

DOI: 10.1039/C6MD00222F

Fungal infections directly affect millions of people each year. In addition to the invasive fungal infections of humans, the plants and animals that comprise our primary food source are also susceptible to diseases caused by these eukaryotic microbes. The need for antifungals, not only for our medical needs, but also for use in agriculture and livestock causes a high demand for novel antimycotics. Herein, we provide an overview of the most commonly used antifungals in medicine and agriculture. We also present a summary of the recent progress (from 2010–2016) in the discovery/development of new agents against fungal strains of medical/agricultural relevance, as well as information related to their biological activity, their mode(s) of action, and their mechanism(s) of resistance.

 

Graphical abstract: A complex game of hide and seek: the search for new antifungals
CLIP
Design and optimization of highly-selective fungal CYP51 inhibitors
  • Viamet Pharmaceuticals Inc., Durham, NC 27703, USA

Image for figure Scheme 1

able 3.Antifungal activity of difluoromethyl-pyridyl-benzenes

Antifungal activity of difluoromethyl-pyridyl-benzenes
Compound R C. albicans MICa T. rubrum MICa CYP3A4 IC50b Selectivity indexc
7a Cl ⩽0.001 0.004 36 9000
7b CF3 ⩽0.001 0.002 54 27,000
7c

VT 1129

OCF3 ⩽0.001 ⩽0.001 79 >79,000
7d

VT 1161

OCH2CF3 ⩽0.001 ⩽0.001 65 >65,000
Itraconazole 0.016 0.062 0.07 1.1
aMinimum concentration that achieved 50% inhibition of fungal growth; MIC units in μg/mL.5
bInhibition of CYP3A4 measured in microsomes obtained from pooled human hepatocytes, IC50 units in μM.8
cIn vitro selectivity calculated as CYP3A4 IC50/T. rubrum MIC.
(R)-(+)-Enantiomers (7a7d) were isolated from racemates using chiral chromatography.
16 Hoekstra, W.J.; Schotzinger, R.J.; Rafferty, S.W. U.S. Patent 8,236,962 issued Aug. 7, 2012.

 

////////VT 1129,  VIAMET, WO 2016149486,  Viamet Pharmaceuticals,  Antifungals,  Small molecules,  14-alpha demethylase inhibitors, Orphan Drug Status, Cryptococcosis, On Fast track, PHASE 1, VT-1129

O[C@@](Cn1cnnn1)(c2ccc(F)cc2F)C(F)(F)c3ccc(cn3)c4ccc(OC(F)(F)F)cc4

FDA grants accelerated approval to first drug for Duchenne muscular dystrophy


Image result for Exondys 51

Image result for eteplirsen

CAS 1173755-55-9
eteplirsen, eteplirsén [Spanish], étéplirsen [French] , eteplirsenum [Latin], этеплирсен [Russian], إيتيبليرسان [Arabic]

Structure credit http://lgmpharma.com/eteplirsen-still-proves-efficacious-duchenne-drug/

FDA grants accelerated approval to first drug for Duchenne muscular dystrophy
New therapy addresses unmet medical need

The U.S. Food and Drug Administration today approved Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD.

Read more

Image result for Duchenne muscular dystrophy

FDA grants accelerated approval to first drug for Duchenne muscular dystrophy

September 19, 2016

Release

The U.S. Food and Drug Administration today approved Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD.

“Patients with a particular type of Duchenne muscular dystrophy will now have access to an approved treatment for this rare and devastating disease,” said Janet Woodcock, M.D., director of the FDA’s Center for Drug Evaluation and Research. “In rare diseases, new drug development is especially challenging due to the small numbers of people affected by each disease and the lack of medical understanding of many disorders. Accelerated approval makes this drug available to patients based on initial data, but we eagerly await learning more about the efficacy of this drug through a confirmatory clinical trial that the company must conduct after approval.”

DMD is a rare genetic disorder characterized by progressive muscle deterioration and weakness. It is the most common type of muscular dystrophy. DMD is caused by an absence of dystrophin, a protein that helps keep muscle cells intact. The first symptoms are usually seen between three and five years of age, and worsen over time. The disease often occurs in people without a known family history of the condition and primarily affects boys, but in rare cases it can affect girls. DMD occurs in about one out of every 3,600 male infants worldwide.

People with DMD progressively lose the ability to perform activities independently and often require use of a wheelchair by their early teens. As the disease progresses, life-threatening heart and respiratory conditions can occur. Patients typically succumb to the disease in their 20s or 30s; however, disease severity and life expectancy vary.

Exondys 51 was approved under the accelerated approval pathway, which provides for the approval of drugs that treat serious or life-threatening diseases and generally provide a meaningful advantage over existing treatments. Approval under this pathway can be based on adequate and well-controlled studies showing the drug has an effect on a surrogate endpoint that is reasonably likely to predict clinical benefit to patients (how a patient feels or functions or whether they survive). This pathway provides earlier patient access to promising new drugs while the company conducts clinical trials to verify the predicted clinical benefit.

The accelerated approval of Exondys 51 is based on the surrogate endpoint of dystrophin increase in skeletal muscle observed in some Exondys 51-treated patients. The FDA has concluded that the data submitted by the applicant demonstrated an increase in dystrophin production that is reasonably likely to predict clinical benefit in some patients with DMD who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping. A clinical benefit of Exondys 51, including improved motor function, has not been established. In making this decision, the FDA considered the potential risks associated with the drug, the life-threatening and debilitating nature of the disease for these children and the lack of available therapy.

Under the accelerated approval provisions, the FDA is requiring Sarepta Therapeutics to conduct a clinical trial to confirm the drug’s clinical benefit. The required study is designed to assess whether Exondys 51 improves motor function of DMD patients with a confirmed mutation of the dystrophin gene amenable to exon 51 skipping. If the trial fails to verify clinical benefit, the FDA may initiate proceedings to withdraw approval of the drug.

The most common side effects reported by participants taking Exondys 51 in the clinical trials were balance disorder and vomiting.

The FDA granted Exondys 51 fast track designation, which is a designation to facilitate the development and expedite the review of drugs that are intended to treat serious conditions and that demonstrate the potential to address an unmet medical need. It was also granted priority review and orphan drug designation.Priority review status is granted to applications for drugs that, if approved, would be a significant improvement in safety or effectiveness in the treatment of a serious condition. Orphan drug designation provides incentives such as clinical trial tax credits, user fee waiver and eligibility for orphan drug exclusivity to assist and encourage the development of drugs for rare diseases.

The manufacturer received a rare pediatric disease priority review voucher, which comes from a program intended to encourage development of new drugs and biologics for the prevention and treatment of rare pediatric diseases. This is the seventh rare pediatric disease priority review voucher issued by the FDA since the program began.

Exondys 51 is made by Sarepta Therapeutics of Cambridge, Massachusetts.

Image result for Exondys 51 (eteplirsen) injection

ChemSpider 2D Image | eteplirsen | C364H569N177O122P30

CAS 1173755-55-9 [RN]
eteplirsén [Spanish] [INN]
étéplirsen [French] [INN]
eteplirsenum [Latin] [INN]
этеплирсен [Russian] [INN]
إيتيبليرسان [Arabic] [INN]
Eteplirsen
Systematic (IUPAC) name
(P-deoxy-P-(dimethylamino)](2′,3′-dideoxy-2′,3′-imino-2′,3′-seco)(2’a→5′)(C-m5U-C-C-A-A-C-A-m5U-C-A-A-G-G-A-A-G-A-m5U-G-G-C-A-m5U-m5U-m5U-C-m5U-A-G),5′-(P-(4-((2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)carbonyl)-1-piperazinyl)-N,N-dimethylphosphonamidate) RNA
Clinical data
Routes of
administration
Intravenous infusion
Legal status
Legal status
  • Investigational
Identifiers
CAS Number 1173755-55-9
ATC code None
ChemSpider 34983391
UNII AIW6036FAS Yes
Chemical data
Formula C364H569N177O122P30
Molar mass 10305.738

///////////Exondys 51, Sarepta Therapeutics, Cambridge, Massachusetts, eteplirsen,  Orphan drug designationPriority reviewfast track designation, Duchenne muscular dystrophy, этеплирсен ,  إيتيبليرسان ,

Evofosfamide, эвофосфамид , إيفوفوسفاميد , 艾伏磷酰胺 ,


str1

TH-302.svg

Evofosfamide, HAP-302 , TH-302, TH 302

эвофосфамид ,  إيفوفوسفاميد ,  艾伏磷酰胺 ,

  • Molecular Formula C9H16Br2N5O4P
  • Average mass 449.036 Da

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl-N,N’-bis(2-bromethyl)phosphorodiamidat
918633-87-1

TH-302 is a nitroimidazole-linked prodrug of a brominated derivative of an isophosphoramide mustard previously used in cancer drugs

  • Originator Threshold Pharmaceuticals
  • Developer Merck KGaA; Threshold Pharmaceuticals
  • Class Antineoplastics; Nitroimidazoles; Phosphoramide mustards; Small molecules
  • Mechanism of Action Alkylating agents
  • Orphan Drug Status Yes – Soft tissue sarcoma; Pancreatic cancer
  • On Fast track Pancreatic cancer; Soft tissue sarcoma
  • Suspended Glioblastoma; Leukaemia; Malignant melanoma; Multiple myeloma; Non-small cell lung cancer; Solid tumours
  • Discontinued Pancreatic cancer; Soft tissue sarcoma

Most Recent Events

  • 01 Aug 2016 Threshold plans a clinical trial for Solid tumours
  • 01 Aug 2016 Threshold announces intention to submit NDA to the Pharmaceuticals and Medical Device Agency in Japan
  • 16 Jun 2016 Merck KGaA terminates a phase II trial in Soft tissue sarcoma (Combination therapy, Inoperable/Unresectable, Metastatic disease, Late-stage disease) in Japan (IV) due to negative results from the phase III SARC021 trial (NCT02255110)

Evofosfamide (first disclosed in WO2007002931), useful for treating cancer.

Image result for Evofosfamide

Threshold Pharmaceuticals and licensee Merck Serono are codeveloping evofosfamide, the lead in a series of topoisomerase II-inhibiting hypoxia-activated prodrugs and a 2-nitroimidazole-triggered bromo analog of ifosfamide, for treating cancer, primarily soft tissue sarcoma and pancreatic cancer (phase 3 clinical, as of April 2015).

In November 2014, the FDA granted Fast Track designation to the drug for the treatment of previously untreated patients with metastatic or locally advanced unresectable soft tissue sarcoma.

Evofosfamide (INN,[1] USAN;[2] formerly known as TH-302) is an investigational hypoxia-activated prodrug that is in clinical development for cancer treatment. The prodrug is activated only at very low levels of oxygen (hypoxia). Such levels are common in human solid tumors, a phenomenon known as tumor hypoxia.[3]

Evofosfamide is being evaluated in clinical trials for the treatment of multiple tumor types as a monotherapy and in combination with chemotherapeutic agents and other targeted cancer drugs.

Dec 2015 : two Phase 3 trials fail, Merck will not apply for a license

Collaboration

Evofosfamide was developed by Threshold Pharmaceuticals Inc. In 2012, Threshold signed a global license and co-development agreement for evofosfamide with Merck KGaA, Darmstadt, Germany (EMD Serono Inc. in the US and Canada), which includes an option for Threshold to co-commercialize evofosfamide in the United States. Threshold is responsible for the development of evofosfamide in the soft tissue sarcoma indication in the United States. In all other cancer indications, Threshold and Merck KGaA are developing evofosfamide together.[4] From 2012 to 2013, Merck KGaA paid 110 million US$ for upfront payment and milestone payments to Threshold. Additionally, Merck KGaA covers 70% of all evofosfamide development expenses.[5]

Mechanism of prodrug activation and Mechanism of action (MOA) of the released drug[edit]

Evofosfamide is a 2-nitroimidazole prodrug of the cytotoxin bromo-isophosphoramide mustard (Br-IPM). Evofosfamide is activated by a process that involves a 1-electron (1 e) reduction mediated by ubiquitous cellular reductases, such as the NADPH cytochrome P450, to generate a radical anion prodrug:

  • A) In the presence of oxygen (normoxia) the radical anion prodrug reacts rapidly with oxygen to generate the original prodrug and superoxide. Therefore, evofosfamide is relatively inert under normal oxygen conditions, remaining intact as a prodrug.
  • B) When exposed to severe hypoxic conditions (< 0.5% O2; hypoxic zones in many tumors), however, the radical anion undergoes irreversible fragmentation, releasing the active drug Br-IPM and an azole derivative. The released cytotoxin Br-IPM alkylates DNA, inducing intrastrand and interstrand crosslinks.[6]

Evofosfamide is essentially inactive under normal oxygen levels. In areas of hypoxia, evofosfamide becomes activated and converts to an alkylating cytotoxic agent resulting in DNA cross-linking. This renders cells unable to replicable their DNA and divide, leading to apoptosis. This investigational therapeutic approach of targeting the cytotoxin to hypoxic zones in tumors may cause less broad systemic toxicity that is seen with untargeted cytotoxic chemotherapies.[7]

The activation of evofosfamide to the active drug Br-IPM and the mechanism of action (MOA) via cross-linking of DNA is shown schematically below:

Activation of eofosfamide to the active drug Br-IPM, and mechanism of action via cross-linking of DNA

Drug development history

Phosphorodiamidate-based, DNA-crosslinking, bis-alkylator mustards have long been used successfully in cancer chemotherapy and include e.g. the prodrugs ifosfamide andcyclophosphamide. To demonstrate that known drugs of proven efficacy could serve as the basis of efficacious hypoxia-activated prodrugs, the 2-nitroimidizole HAP of the active phosphoramidate bis-alkylator derived from ifosfamide was synthesized. The resulting compound, TH-281, had a high HCR (hypoxia cytotoxicity ratio), a quantitative assessment of its hypoxia selectivity. Subsequent structure-activity relationship (SAR) studies showed that replacement of the chlorines in the alkylator portion of the prodrug with bromines improved potency about 10-fold. The resulting, final compound is evofosfamide (TH-302).[8]

Synthesis

Evofosfamide can be synthesized in 7 steps.[9][10]

  1. CPhI.cn: Synthetic routes to explore anti-pancreatic cancer drug Evofosfamide, 22 Jan 2015
  2.  Synthetic route Reference: International patent application WO2007002931A2

Formulation

The evofosfamide drug product formulation used until 2011 was a lyophilized powder. The current drug product formulation is a sterile liquid containing ethanol,dimethylacetamide and polysorbate 80. For intravenous infusion, the evofosfamide drug product is diluted in 5% dextrose in WFI.[11]

Diluted evofosfamide formulation (100 mg/ml evofosfamide, 70% ethanol, 25% dimethylacetamide and 5% polysorbate 80; diluted to 4% v/v in 5% dextrose or 0.9% NaCl) can cause leaching of DEHP from infusion bags containing PVC plastic.[12]

Clinical trials

Overview and results

Evofosfamide (TH-302) is currently being evaluated in clinical studies as a monotherapy and in combination with chemotherapy agents and other targeted cancer drugs. The indications are a broad spectrum of solid tumor types and blood cancers.

Evofosfamide clinical trials (as of 21 November 2014)[13] sorted by (Estimated) Primary Completion Date:[14]


Both, evofosfamide and ifosfamide have been investigated in combination with doxorubicin in patients with advanced soft tissue sarcoma. The study TH-CR-403 is a single arm trial investigating evofosfamide in combination with doxorubicin.[35] The study EORTC 62012 compares doxorubicin with doxorubicin plus ifosfamide.[36] Doxorubicin and ifosfamide are generic products sold by many manufacturers.Soft tissue sarcoma

The indirect comparison of both studies shows comparable hematologic toxicity and efficacy profiles of evofosfamide and ifosfamide in combination with doxorubicin. However, a longer overall survival of patients treated with evofosfamide/doxorubicin (TH-CR-403) trial was observed. The reason for this increase is probably the increased number of patients with certain sarcoma subtypes in the evofosfamide/doxorubicin TH-CR-403 trial, see table below.

However, in the Phase 3 TH-CR-406/SARC021 study (conducted in collaboration with the Sarcoma Alliance for Research through Collaboration (SARC)), patients with locally advanced unresectable or metastatic soft tissue sarcoma treated with evofosfamide in combination with doxorubicin did not demonstrate a statistically significant improvement in OS compared with doxorubicin alone (HR: 1.06; 95% CI: 0.88 – 1.29).

Metastatic pancreatic cancer

Both, evofosfamide and protein-bound paclitaxel (nab-paclitaxel) have been investigated in combination with gemcitabine in patients with metastatic pancreatic cancer. The study TH-CR-404 compares gemcitabine with gemcitabine plus evofosfamide.[39] The study CA046 compares gemcitabine with gemcitabine plus nab-paclitaxel.[40] Gemcitabine is a generic product sold by many manufacturers.

The indirect comparison of both studies shows comparable efficacy profiles of evofosfamide and nab-paclitaxel in combination with gemcitabine. However, the hematologic toxicity is increased in patients treated with evofosfamide/gemcitabine (TH-CR-404 trial), see table below.

In the Phase 3 MAESTRO study, patients with previously untreated, locally advanced unresectable or metastatic pancreatic adenocarcinoma treated with evofosfamide in combination with gemcitabine did not demonstrate a statistically significant improvement in overall survival (OS) compared with gemcitabine plus placebo (hazard ratio [HR]: 0.84; 95% confidence interval [CI]: 0.71 – 1.01; p=0.0589).

Drug development risks

Risks published in the quarterly/annual reports of Threshold and Merck KGaA that could affect the further development of evofosfamide (TH-302):

Risks related to the formulation

The evofosfamide formulation that Threshold and Merck KGaA are using in the clinical trials was changed in 2011[43] to address issues with storage and handling requirements that were not suitable for a commercial product. Additional testing is ongoing to verify if the new formulation is suitable for a commercial product. If this new formulation is also not suitable for a commercial product another formulation has to be developed and some or all respective clinical phase 3 trials may be required to be repeated which could delay the regulatory approvals.[44]

Risks related to reimbursement

Even if Threshold/Merck KGaA succeed in obtaining regulatory approvals and bringing evofosfamide to the market, the amount reimbursed for evofosfamide may be insufficient and could adversely affect the profitability of both companies. Obtaining reimbursement for evofosfamide from third-party and governmental payors depend upon a number of factors, e.g. effectiveness of the drug, suitable storage and handling requirements of the drug and advantages over alternative treatments.

There could be the case that the data generated in the clinical trials are sufficient to obtain regulatory approvals for evofosfamide but the use of evofosfamide has a limited benefit for the third-party and governmental payors. In this case Threshold/Merck KGaA could be forced to provide supporting scientific, clinical and cost effectiveness data for the use of evofosfamide to each payor. Threshold/Merck KGaA may not be able to provide data sufficient to obtain reimbursement.[45]

Risks related to competition

Each cancer indication has a number of established medical therapies with which evofosfamide will compete, for example:

  • If approved for commercial sale for pancreatic cancer, evofosfamide would compete with gemcitabine (Gemzar), marketed by Eli Lilly and Company; erlotinib (Tarceva), marketed by Genentech and Astellas Oncology; protein-bound paclitaxel (Abraxane), marketed by Celgene; and FOLFIRINOX, which is a combination of generic products that are sold individually by many manufacturers.
  • If approved for commercial sale for soft tissue sarcoma, evofosfamide could potentially compete with doxorubicin or the combination of doxorubicin and ifosfamide, generic products sold by many manufacturers.[46]

Risks related to manufacture and supply

Threshold relies on third-party contract manufacturers for the manufacture of evofosfamide to meet its and Merck KGaA’s clinical supply needs. Any inability of the third-party contract manufacturers to produce adequate quantities could adversely affect the clinical development and commercialization of evofosfamide. Furthermore, Threshold has no long-term supply agreements with any of these contract manufacturers and additional agreements for more supplies of evofosfamide will be needed to complete the clinical development and/or commercialize it. In this regard, Merck KGaA has to enter into agreements for additional supplies or develop such capability itself. The clinical programs and the potential commercialization of evofosfamide could be delayed if Merck KGaA is unable to secure the supply.[47]

History

Date Event
Jun 2005 Threshold files evofosfamide (TH-302) patent applications in the U.S.[48]
Jun 2006 Threshold files an evofosfamide (TH-302) patent application in the EU and in Japan[49]
Sep 2011 Threshold starts a Phase 3 trial (TH-CR-406) of evofosfamide in combination with doxorubicin in patients with soft tissue sarcoma
Feb 2012 Threshold signs an agreement with Merck KGaA to co-develop evofosfamide
Apr 2012 A Phase 2b trial (TH-CR-404) of evofosfamide in combination with gemcitabine in patients with pancreatic cancer meets primary endpoint
Jan 2013 Merck KGaA starts a global Phase 3 trial (MAESTRO) of evofosfamide in combination with gemcitabine in patients with pancreatic cancer
Dec 2015 two Phase 3 trials fail, Merck will not apply for a license

CLIP

CLIP

Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate Evofosfamide (TH-302)

*Corresponding authors
aDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, UK
E-mail: stuart.conway@chem.ox.ac.uk
bCancer Research UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, UK
Org. Chem. Front., 2015,2, 1026-1029

DOI: 10.1039/C5QO00211G

http://pubs.rsc.org/en/content/articlelanding/2015/qo/c5qo00211g/unauth#!divAbstract

http://www.rsc.org/suppdata/c5/qo/c5qo00211g/c5qo00211g1.pdf

Hypoxia, regions of low oxygen, occurs in a range of biological environments, and is involved in human diseases, most notably solid tumours. Exploiting the physiological differences arising from low oxygen conditions provides an opportunity for development of targeted therapies, through the use of bioreductive prodrugs, which are selectively activated in hypoxia. Herein, we describe an improved method for synthesising the most widely used bioreductive group, 2-nitroimidazole. The improved method is applied to an efficient synthesis of the anti-cancer drug Evofosfamide (TH-302), which is currently in Phase III clinical trials for treatment of a range of cancers.

Graphical abstract: Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate Evofosfamide (TH-302)

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(1-Methyl-2-nitro-1H-imidazol-5-yl)-N,N–bis(2-bromoethyl) phosphordiamidate (TH- 302)

The residue was then purified by semi-preparative HPLC on a Phenomenex Luna (C18(2), 10 µm, 250 × 10 mm) column, eluting with H2O and methanol (50 – 70% methanol over 10 min, then 1 min wash with methanol, 5 mL/min flow rate) to afford TH-302 as a yellow gum: vmax (solid) cm-1 : 3212 (br), 1489 (m), 1350 (m), 1105 (m), 1004 (s); δH (DMSO-D6, 400 MHz) 7.25 (1H, s, CH), 5.10–4.90 (2H, m, NHCH2CH2Br), 4.98 (2H, d, J 7.8, CH2O), 3.94 (3H, s, CH3), 3.42 (4H, t, J 7.0, NHCH2CH2Br), 3.11 (4H, dt, J 9.8, 7.2, NHCH2CH2Br); δC (DMSO-D6, 126 MHz) 146.1, 134.2 (d, J 7.5, OCH2CN), 128.2, 55.6 (d, J 4.6, CH2O), 42.7, 34.2 (d, J 26.4, CH2Br), 34.1; δP (DMSO-D6, 202 MHz) 15.4; HRMS m/z (ESI− ) [found; (M-H)− 447.9216, C9H16 79Br81BrN5O4P requires (M-H)− 447.9213]; m/z (ESI+ ) 448.0 ([M-H]− , 60%, [C9H15 79Br81BrN5O4P] − ), 493.9 ([M+formate] − , 100%, [C10H17 79Br81BrN5O6P] − ). These data are in good agreement with the literature values.4

4 J.-X. Duan, H. Jiao, J. Kaizerman, T. Stanton, J. W. Evans, L. Lan, G. Lorente, M. Banica, D. Jung, J. Wang, H. Ma, X. Li, Z. Yang, R. M. Hoffman, W. S. Ammons, C. P. Hart and M. Matteucci, J. Med. Chem., 2008, 51, 2412–2420.

J. Med. Chem., 2008, 51, 2412–2420/……………….1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N-bis(2-bromoethyl)
phosphordiami-date (3b). Compound 3b was synthesized by a procedure similar to that described for 3a and obtained as an off-white solid in 47.6% yield.

1H NMR (DMSO-d6) δ: 7.22 (s, 1H), 5.10–5.00 (m, 2H), 4.97 (d, J ) 7.6 Hz, 2H), 3.94 (s, 3H), 3.42 (t, J ) 7.2 Hz, 4H), and 3.00–3.20 (m, 4H).

13C NMR (DMSOd6)δ: 146.04, 134.16 (d, J ) 32 Hz), 128.17, 55.64, 42.70, 34.33,and 34.11 (d, J ) 17.2 Hz).

31P NMR (DMSO-d6) δ: -11.25.
HRMS: Calcd for C9H16N5O4PBr2, 446.9307; found, 446.9294.

CLIP

Synthesis Route reference WO2007002931A2

Med J.. Chem. 2008, 51, 2412-2420

From compound S-1 starting aminoacyl protection is S-2 , a suspension of NaH grab α -proton, offensive, ethyl, acidification, introduction of an aldehyde group, S-3followed by condensation with the amino nitrile, off N- acyl ring closure, migration rearrangement amino imidazole compound S-. 8 , the amino and sodium nitrite into a diazonium salt, raising the temperature, nitrite anion nucleophilic attack diazonium salt obtained nitro compound S-9, under alkaline conditions ester hydrolysis gives acid S-10 , followed by NEt3 under the action of isobutyl chloroformate and the reaction mixed anhydride formed by of NaBH 4 reduction to give the alcohol S-. 11 , [use of NaBH 4 reduction of the carboxyl group is another way and the I 2 / of NaBH 4 ] , to give S-11 later, the DIAD / PPh3 3 under the action via Mitsunobu linking two fragments obtained reaction Evofosfamide

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PATENT

http://www.google.co.in/patents/WO2015051921A1?cl=en

EXAMPLE 1

1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (Rt = 7,7 min): 97,9% (a/a).

HPLC data was obtained using Agilent 1100 series HPLC from agilent technologies using an Column: YMC-Triart CI 8 3μ, 100 x 4,6 mm Solvent A: 950 ml of ammonium acetate/acetic acid buffer at pH = 6 + 50 ml acetonitril; Solvent B: 200 ml of ammonium acetate/acetic acid buffer at pH = 6 + 800 ml acetonitril; Flow: 1,5 ml/min; Gradient: 0 min: 5 % B, 2 min: 5 % B, 7 min: 20 % B, 17 min: 85% B, 17, 1 min: 5% B, 22 min: 5% B.

PATENT

WO2007002931

http://www.google.com/patents/WO2007002931A2?cl=en

Example 8

Synthesis of Compounds 25, 26 [0380] To a solution of 2-bromoethylammmonium bromide (19.4 g) in DCM (90 mL) at – 1O0C was added a solution OfPOCl3 (2.3 mL) in DCM (4 mL) followed by addition of a solution of TEA (14.1 mL) in DCM (25 mL). The reaction mixture was filtered, the filtrate concentrated to ca. 30% of the original volume and filtered. The residue was washed with DCM (3×25 mL) and the combined DCM portions concentrated to yield a solid to which a mixture of THF (6 mL) and water (8 mL) was added. THF was removed in a rotary evaporator, the resulting solution chilled overnight in a fridge. The precipitate obtained was filtered, washed with water (10 mL) and ether (30 mL), and dryed in vacuo to yield 2.1 g of:

Figure imgf000127_0001

Isophosphoramide mustard

Figure imgf000127_0002

can be synthesized employing the method provided in Example 8, substituting 2- bromoethylammmonium bromide with 2-chloroethylammmonium chloride. Synthesis of Isophosphoramide mustard has been described (see for example Wiessler et al., supra).

The phosphoramidate alkylator toxin:

Figure imgf000127_0003

was transformed into compounds 24 and 25, employing the method provided in Example 6 and the appropriate Trigger-OH.

Example 25

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

Figure imgf000143_0002

A suspension of the nitro ester (39.2 g, 196.9 rnmol) in IN NaOH (600 mL) and water (200 mL) was stirred at rt for about 20 h to give a clear light brown solution. The pH of the reaction mixture was adjusted to about 1 by addition of cone. HCl and the reaction mixture extracted with EA (5 x 150 mL). The combined ethyl acetate layers were dried over MgS O4 and concentrated to yield l-N-methyl-2-nitroimidazole-5-carboxylis acid (“nitro acid”) as a light brown solid (32.2 g, 95%). Example 26

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

Figure imgf000144_0001

A mixture of the nitro acid (30.82 g, 180.23 mmol) and triethylamine (140 niL, 285 mmol) in anhydrous THF (360 mL) was stirred while the reaction mixture was cooled in a dry ice-acetonitrile bath (temperature < -20 0C). Isobutyl chloroformate (37.8 mL, 288 mmol) was added drop wise to this cooled reaction mixture during a period of 10 min and stirred for 1 h followed by the addition of sodium borohydride (36 g, 947 mmol) and dropwise addition of water during a period of 1 h while maintaining a temperature around or less than O0C. The reaction mixture was warmed up to O0C. The solid was filtered off and washed with THF. The combined THF portions were evaporated to yield l-N-methyl-2- nitroimidazole-5-methanol as an orange solid (25 g) which was recrystallized from ethyl acetate.

PATENT

WO-2015051921

EXAMPLE 1

1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (Rt = 7,7 min): 97,9% (a/a).

PATENT

WO 2016011195

http://google.com/patents/WO2016011195A1?cl=en

Figure 1 provides the differential scanning calorimetry (DSC) data of crystalline solid form A of TH-302.

Figure 2 shows the 1H-NMR of crystalline solid form A of TH-302.

Figure 5 shows the Raman Spectra of TH-302 (Form A)

Scheme 1 illustrates a method of preparing TH-302.

Scheme 1: Process for the Preparation of TH-302

NaOH (RGT)

Step 1. Imidazole Purified water (SLV)

Carboxylic Acid IPC: NMT 1.0% SM by HPLC

HCI (RGT)

IPC: pH 1.0 ± 0.5

IPC: NMT 1.0% water by KF

TH-302

MW = 449.0

SM = Starting Material INT = Intermediate IPC = In-process Control RGT = Reagent SLV = Solvent MW = Molecular Weight LOD = Loss on drying NMT = Not more than NLT = Not less than

TH-302 can be prepared by hydro lyzing (l-methyl-2-nitro-lH-imidazol-5-yl) ethyl ester above for example under aqueous conditions with a suitable base catalyst (e.g. NaOH in water at room temperature). The imidazole carboxylic acid prepared by this method can be used without further purification. However, it has been found that treating the dried crude intermediate product with a solvent such as acetonitrile, ethyl acetate, n-heptane, acetone, dimethylacetamide, dimethylformamide, 1, 4-dioxane, ethylene glycol, 2-propanol, 1-propanol, tetrahydrofuran (1 : 10 w/v) or combinations thereof in a vessel with heating, followed by cooling and filtration through a filtration aid with acetone decreased the number and levels of impurities in the product. The number and levels of impurities could be further reduced by treating the dried crude product with water (1 :5.0 w/v) in a vessel with heating followed by cooling and filtration through a filtration aid with water.

The carboxylic acid of the imidazole can then be reduced using an excess of a suitable reducing agent (e.g. sodium borohydride in an appropriate solvent, typically aqueous. The reaction is exothermic (i.e. potentially explosive) releasing borane and hydrogen gases over several hours. It was determined that the oxygen balance of the product imidazole alcohol is about 106.9, which suggests a high propensity for rapid decomposition. It has been found that using NaOH, for example 0.01M NaOH followed by quenching the reaction with an acid. Non-limiting examples of acids include, but are not limited to water, acetic acid, hydrobromic acid, hydrochloric acid, sodium hydrogen phosphate, sulfuric acid, citric acid, carbonic acid, phosphoric acid, oxalic acid, boric acid and combinations thereof. In some embodiments, the acid may diluted with a solvent, such as water and/or tetrahydrofuran. In some embodiments, acetic acid or hydrochloric acid provide a better safety profile, presumably because it is easier to control the temperature during the addition of the reducing agent and the excess reducing agent is destroyed after the reaction is complete. This also results in improved yields and fewer impurities, presumably due to reduced impurities from the reducing agent and decomposition of the product. Using this process, greater than 98.5% purity could be achieved for this intermediate. The formation of ether linkage can be accomplished by treating the product imidazole alcohol with solution of N,N’-Bis(2-bromoethyl)phosphorodiamidic acid (Bromo IPM), a trisubstituted phosphine and diisopropyl azodicarboxylate in tetrahydrofuran at room temperature to afford TH-302. It has been found that by recrystallizing the product from a solvents listed in the examples, one could avoid further purfication by column chromatography, which allowed for both reduced solvent use especially on larger scales.

Scheme 2 illustrates an alternative method of preparing TH-302.

Scheme 2: Process for the Preparation of TH-302

(SM)

ethylamine mide (SM) 04.9 ) SLV) , RGT) ter by KF

NT)

MW = 449.0

Example 1: Synthesis of TH-302

Step 1 – Preparation intermediate imidazole carboxylic

I T)

Crude imidazole carboxylic acid ethyl ester (1 : 1.0 w/w) was taken in water (1 : 10.0 w/v) at 25± 5°C and cooled to 17± 3°C. A 2.5 N sodium hydroxide solution (10 V) was added slowly at 17±3°C. The reaction mass was warmed to 25±5°C and monitored by HPLC. After the completion of reaction, the reaction mass was cooled to 3±2°C and pH of the reaction mass adjusted to 1=1=0.5 using 6 M HC1 at 3±2°C. The reaction mass was then warmed to 25±5°C and extracted with ethyl acetate (3 x 10 V). The combined organic layers

were washed with water (1 x 10 V) followed by brine (1 x 10 V). The organic layer was dried over sodium sulfate (3 w/w), filtered over Celite and concentrated. n-Heptane (1.0 w/v) was added and the the reaction mixture was concentrated below 45°C to 2.0 w/v. The reaction mass was cooled to 0±5°C. The solid was filtered, and the bed was washed with n-heptane (1 x 0.5 w/v) and dried at 35±5°C. In a vessel, acetone (1 : 10 w/v) was added. Dry crude imidazole carboxylic acid (ICA) from 1.12 was added to the acetone. The mixture was warmed to 45±5°C and was stirred for 30 minutes. The mass was cooled to 28±3°C and filtered through a Celite bed. The filter bed was washed with 1 : 1.0 w/v of acetone. Water (1 :5.0 w/v) was added to the filtrate and the mixture was concentrated. The concentrated mass was cooled to 5±5°C and stirred for 30 minutes. The material was filtered and the solid was washed 2 x 1 : 1.0 w/v of water at 3±2°C. The product was dried for 2 hours at 25±5°C and then at 45±5°C. As can be seen below, the number and levels of impurities are decreased.

Table I: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Imidazole alcohol:

CI^Oi-Bu

T

o

Imidazole carboxylic acid (1.0 w/w) was taken in tetrahydrofuran (10 w/v) under nitrogen atmosphere at 25±5°C. The reaction mass was cooled to -15±5°C. Triethylamine (1 : 1.23 w/v) was added slowly over a period of 1 hour maintaining the temperature at – 15±5°C. The reaction mass was stirred at -15±5°C for 15-20 min. Isobutylchloroformate (1 : 1.14 w/v) was added slowly over a period of 1 hour maintaining the temperature at – 15±5°C. The reaction mass was stirred at -15±5°C for 30-40 min. A solution of sodium borohydride (1 : 1.15 w/w) in 0.01 M aqueous sodium hydroxide (2.2 w/v) was divided into 6 lots and added to the above reaction mass while maintaining the temperature of the reaction mass between 0±10°C for 40-60 min for each lot. The reaction mass was warmed to 25±5°C and stirred until imidazole carboxylic acid content < 5.0 % w/w. The reaction mass was filtered and the bed was washed with tetrahydrofuran (1 :2.5 w/v). The filtrate was quenched with 10 % acetic acid in water at 25±5°C. Reaction mass stirred for 50-60 minutes at 25±5°C. The filtrate was concentrated below 45°C until no distillate was observed. The mass was cooled to 5±5°C and stirred for 50-60 minutes. The reaction mass was filtered and the solid was taken in ethanol (1 :0.53 w/v). The reaction mass was cooled 0±5°C and stirred for 30-40 min. The solid was filtered and the bed was washed ethanol (1 :0.13 w/v). The solid was dried at 40±5 °C.

Step 3 – Synthesis of intermediate Br-IPM:

P

o

M
W = 286.7 MW = 204.9 Purified water (SLV, RGT)

Acetone (SLV)

IPC: NMT 1.0% water by KF

2-Bromoethylamine hydrobromide (1 : 1.0 w/w) and POBr^ (1 :0.7 w/w) were taken in DCM (1 :2 w/v) under nitrogen atmosphere. The reaction mixture was cooled to -70±5°C. Triethylamine (1 : 1.36 w/v) in DCM (1 :5 w/v) was added to the reaction mass at -70±5°C. The reaction mass was stirred for additional 30 min at -70±5°C. Reaction mass was warmed to 0±3°C and water (1 :1.72 w/v) was added. The reaction mixture was stirred at 0±3°C for 4 hrs. The solid obtained was filtered and filter cake was washed with ice cold water (2 x 1 :0.86 w/v) and then with chilled acetone (2 x 1 :0.86 w/v). The solid was dried in at 20±5°C.

Step 4 Synthesis ofTH-302

TH-302

MW = 449.0

Imidazole alcohol (IA) (1 : 1.0 w/w), Bromo-IPM (1 :2.26 w/w) and

triphenylphosphine (1 :2.0 w/w) were added to THF (1 : 13.5 w/v) at 25±5°C. The reaction

mass was cooled to 0±5°C and DIAD (1.5 w/v) was added. The reaction mixture warmed to 25±5°C and stirred for 2 hours. Progress of the reaction was monitored by HPLC. Solvent was removed below 50°C under vacuum. Solvent exchange with acetonitrile (1 :10.0 w/v) below 50°C was performed. The syrupy liquid was re-dissolved in acetonitrile (1 : 10.0 w/v) and the mixture was stirred at -20±5°C for 1 hour. The resulting solid was filtered and the filtrate bed was washed with chilled acetonitrile (1 : 1.0 w/v). The acetonitrile filtrate was concentrated below 50°C under vacuum. The concentrated mass was re-dissolved in ethyl acetate (1 : 10.0 w/v) and concentrated below 50°C under vacuum. The ethyl acetate strip off was repeated two more times. Ethyl acetate (1 : 10.0 w/v) and silica gel (230-400 mesh, 1 :5.3 w/w) were added to the concentrated reaction mass. The mixture was concentrated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was charged to the above mass and the mixture was evaporated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was again added to the above mass and the solid was filtered and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in a mixture oftoluene (1 :7.1 w/v) and n-heptane (1 :21.3 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane

(1 : 1.0 w/v). The solid was re-suspended in a mixture of toluene (1 : 10.6 w/v) and n-heptane (1 : 10.6 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in acetone (1 : 19.0 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with acetone (1 : 1.0 w/v). The acetone washes were repeated 3 more times. Filtrates from the above acetone washings were combined and concentrated below 40°C under vacuum. The residue dissolved in ethyl acetate (1 : 10.0 w/v) and concentrated below 40°C under vacuum. The ethyl acetate strip off was repeated one more time. The residue was re-dissolved in ethyl acetate (1 :5.5 w/v), cooled to 0±3°C and stirred at 0±3°C for 2 h and then at -20±5°C for 2 h. The solid was filtered and the solid was washed with ethyl acetate (1 :0.10 w/v). The solid was dissolved in ethyl acetate (1 : 10.0 w/v) at 50±5°C and the resulting solution was filtered through a cartridge filter. The filtrate was concentrated to ~4.0 w/w and stirred at 0±3°C for 4 hours. The solid was filtered and washed with ethyl acetate (1 :0.10 w/v). The crystallization from ethyl acetate was repeated and TH-302 was dried at 25±5°C. Table 2 shows how the process reduces solvent use.

Table 2: Solvent and Silica Gel Usage for 10 kg Column and 10 kg Column-free Purification

“Amounts are estimated from a 5 kg batch

b Amounts are estimated

Example 2: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel, water (1 :7.0 w/v) was added. Dry crude ICA was added to the water. The reaction mixture was heated to 85±5°C until a clear solution was obtained. The reaction mass was cooled to 20±5°C and filtered through a Celite bed. The filter bed was washed with 2 x 5.0 of n-heptane. The material was dried for 2 hours at 25±5°C and then 45±5°C. As can be seen below, the number and levels of impurities decreased.

Table 3: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 3: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel

ethanol (1 :30.0 w/v) and ICA (1 : 1.0 w/w) were mixed. The reaction mixture was stirred at

25±5°C for 30 minutes and filtered. Water (1 :50.0 w/v) was added and the mixture was

stirred at 50±5°C for 30 minutes. The reaction mass was cooled to 20±5°C and filtered. The isolated solid was dried at 25±5°C for 24 hours. As can be seen below, the number and levels

of impurities generally decreased.

Table 4: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 4: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel

acetonitrile (1 :20.0 w/v) and ICA (1 : 1.0 w/w) were mixed at 25±5°C for one hour. The

reaction mixture was filtered and the solution was concentrated to ~ 6 volumes. The mixture

was then cooled to 0±5°C, stirred at this temperature for one hour and filtered. The isolated

solid was dried at 25±5°C for 24 hours. As can be seen below the number of impurities

decreased and except for TH-2717, the amounts also decreased.

Table 5: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 5: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with dimethylacetamide and water.

Example 6: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with dimethylforamide and water.

Example 7: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0109] Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a 1,4-dioxane and water mixture.

Example 8: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a mixture of ethylene glycol and water.

Example 9: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with 2-propanol and water.

Example 10: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0112] Crude ICA is prepared according to the method described in Example 1 and purified by treatment with 1-propanol and water.

Example 11: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0113] Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a mixture of tetrahydrofuran and water.

Example 12: Synthesis ofTH-302 using alternative procedure to quench IA:

[0114] The reduction of ICA to IA was carried out according to Example 1 except that after reaction completion and filtration of the inorganics, the filtrate was quenched with 1.5 M hydrochloric acid.

Example 13: Synthesis ofTH-302 using alternative procedure to quench IA:

[0115] The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with 1.5 M

hydrobromic acid.

Example 14: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with

hydrobromic acid in acetic acid.

Example 15: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was treated with sodium

hydrogen phosphate.

Example 16: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with 10% acetic

acid in tetrahydrofuran.

Example 17: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with water.

Example 18: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with sulfuric acid.

Example 19: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with citric acid.

Example 20: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is treated with carbonic acid.

Example 21: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is treated with phosphoric

acid.

Example 22: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with oxalic acid.

Example 23: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after reaction completion and filtration of the inorganics, the filtrate is quenched with boric acid.

Example 24: Synthesis ofTH-302 using alternative procedure to purify TH-302:

[0126] Coupling of bromo-IPM and IA was performed according to Example 1 except that after concentration of the reaction mixture, ethyl acetate (1 : 10 w/v) was added to the concentrated mass. The mixture was stirred at -55±5°C for 2 hours. The resulting solid was filtered and washed with chilled EtOAc (1 :2.0 w/v). The solid was reslurried in ethyl acetate (1 : 10 w/v) at -55±5°C for 2 hours, filtered and the solid was washed with chilled ethyl acetate (1 : 1.0 w/v). The filtrates from both filtrations were combined and treated with silica gel (1 :5.3 w/w) of silica gel (230-400 mesh). The mixture was concentrated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was again added to the above mass and the solid was filtered and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in a mixture of toluene (1 :7.1 w/v) and n-heptane (1 :21.3 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was re-suspended in a mixture of toluene (1 : 10.6 w/v) and n-heptane (1 :10.6 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in acetone (1 : 19.0 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with acetone (1 : 1.0 w/v). The acetone washes were repeated 3 more times. Filtrates from the above acetone washings were combined and concentrated below 40°C under vacuum. The residue dissolved in ethyl acetate (1 :5.5 w/v), cooled to 0±3°C and stirred at 0±3°C for 2 h and then at -20±5°C for 2 h. The solid was filtered and the solid was washed with ethyl acetate (1 :0.10 w/v). The solid was dissolved in ethyl acetate (1 :27 w/v), stirred at 50±5°C and filtered through Celite. The filtrate was concentrated to ~4.0 w/w and stirred at 0±5°C for 4 hours. The recrystallization from ethyl acetate was repeated and TH- 302 was dried at 25±5°C. Table 4 shows how the process reduced solvent use.

Table 4: Estimated Solvent and Silica Gel Usage for Column and 10 kg Column-free

(EtOAc) Purification

References

  1.  WHO Drug Information; Recommended INN: List 73
  2.  Adopted Names of the United States Adopted Names Council
  3.  Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M; et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q.PMID 18257544.
  4. Jump up^ Threshold Pharmaceuticals and Merck KGaA Announce Global Agreement to Co-Develop and Commercialize Phase 3 Hypoxia-Targeted Drug TH-302 – Press release from 3 February 2012
  5. Jump up^ Threshold Pharmaceuticals Form 8-K from 3 Nov 2014
  6. Jump up^ Weiss, G.J., Infante, J.R., Chiorean, E.G., Borad, M.J., Bendell, J.C., Molina, J.R., Tibes, R., Ramanathan, R.K., Lewandowski, K., Jones, S.F., Lacouture, M.E., Langmuir, V.K., Lee, H., Kroll, S., Burris, H.A. (2011) Phase 1 Study of the Safety, Tolerability, and Pharmacokinetics of TH-302, a Hypoxia-Activated Prodrug, in Patients with Advanced Solid Malignancies. Clinical Cancer Research 17, 2997–3004.doi:10.1158/1078-0432.CCR-10-3425
  7.  J. Thomas Pento (2011). “TH-302”. Drugs of the Future. 36 (9): 663–667.doi:10.1358/dof.2011.036.09.1678337.
  8. Jump up^ Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M; et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q.PMID 18257544.
  9. Jump up^ CPhI.cn: Synthetic routes to explore anti-pancreatic cancer drug Evofosfamide, 22 Jan 2015
  10.  Synthetic route Reference: International patent application WO2007002931A2
  11. Jump up^ FDA Advisory Committee Briefing Materials Available for Public Release, TH-302: Pediatric oncology subcommittee of the oncologic drugs advisory committee (ODAC) meeting, December 4, 2012
  12. Jump up^ AAPS 2014 – Measurement of Diethylhexyl Phthalate (DEHP) Leached from Polyvinyl Chloride (PVC) Containing Plastics by Infusion Solutions Containing an Organic Parenteral Formulation – Poster W4210, Nov 5, 2014
  13. Jump up^ ClinicalTrials.gov
  14.  The Primary Completion Date is defined as the date when the final subject was examined or received an intervention for the purposes of final collection of data for the primary outcome.
  15. Jump up^ Detailed Results From Positive Phase 2b Trial of TH-302 in Pancreatic Cancer at AACR Annual Meeting – Press release from 30 March 2012
  16. Jump up^ TH-302 Plus Gemcitabine vs. Gemcitabine in Patients with Untreated Advanced Pancreatic Adenocarcinoma. Borad et al. Presentation at the European Society for Medical Oncology (ESMO) 2012 Congress, September 2012. (Abstract 6660)
  17. Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  18.  Updated Phase 2 Results Including Analyses of Maintenance Therapy With TH-302 Following Induction Therapy With TH-302 Plus Doxorubicin in Soft Tissue Sarcoma – Press release from 15 November 2012
  19.  TH-302 Maintenance Following TH-302 Plus Doxorubicin Induction: The Results pf a Phase 2 Study of TH-302 in Combination with Doxorubicin in Soft Tissue Sarcoma. Ganjoo et al. Connective Tissue Oncology Society (CTOS) 2012 Meeting, November 2012
  20. Jump up^ Chawla, S.P., Cranmer, L.D., Van Tine, B.A., Reed, D.R., Okuno, S.H., Butrynski, J.E., Adkins, D.R., Hendifar, A.E., Kroll, S., Ganjoo, K.N., 2014. Phase II Study of the Safety and Antitumor Activity of the Hypoxia-Activated Prodrug TH-302 in Combination With Doxorubicin in Patients With Advanced Soft Tissue Sarcoma. Journal of Clinical Oncology 32, 3299–3306.doi:10.1200/JCO.2013.54.3660
  21. Jump up^ Follow-Up Data From a Phase 1/2 Clinical Trial of TH-302 in Solid Tumors – Press release from 12 October 2010
  22.  TH-302 Continues to Demonstrate Promising Activity in Pancreatic Cancer Phase 1/2 Clinical Trial – Press release from 24 January 2011
  23. Jump up^ TH-302, a tumor selective hypoxia-activated prodrug, complements the clinical benefits of gemcitabine in first line pancreatic cancer. Borad et al. ASCO Gastrointestinal Cancers Symposium, January 2011
  24. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  25. Jump up^ Borad et al., ESMO Annual Meeting, October 2010
  26. Jump up^ Video interview of Stefan Oschmann, CEO Pharma at Merck – Merck Serono Investor & Analyst Day 2014 – 18 Sept 2014 – 2:46 min – Youtube
  27. Jump up^ The Phase 3 Trial of TH-302 in Patients With Advanced Soft Tissue Sarcoma Will Continue as Planned Following Protocol-Specified Interim Analysis – Press release from 22 September 2014
  28. Jump up^ Threshold Pharmaceuticals’ Partner Merck KGaA, Darmstadt, Germany, Completes Target Enrollment in the TH-302 Phase 3 MAESTRO Study in Patients With Locally Advanced or Metastatic Pancreatic Adenocarcinoma – Press release from 3 November 2014
  29.  Data From Ongoing Phase 1/2 Trial of TH-302 Plus Bevacizumab (Avastin(R)) in Patients With Recurrent Glioblastoma – Press release from 30 May 2014
  30. Jump up^ Phase 1/2 Study of Investigational Hypoxia-Targeted Drug, TH-302, and Bevacizumab in Recurrent Glioblastoma Following Bevacizumab Failure. Brenner, et al. 2014 ASCO, 7 – 30 May 2014
  31. Jump up^ Phase 1/2 Interim Data Signaling Activity of TH-302 Plus Bevacizumab (Avastin(R)) in Patients With Glioblastoma – Press release from 17 November 2014
  32. Jump up^ Threshold Pharmaceuticals’ Partner Merck KGaA, Darmstadt, Germany, Completes Target Enrollment in the TH-302 Phase 3 MAESTRO Study in Patients With Locally Advanced or Metastatic Pancreatic Adenocarcinoma – Press release from 3 November 2014
  33. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  34. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  35. Jump up^ Chawala SP, et al. J Clin Oncol. 2014 (54) 3660 doi:10.1200/JCO.2013.54.3660
  36. Jump up^ Judson I, et al. Lancet Oncol. 2014 Apr;15(4):415-23doi: 10.1016/S1470-2045(14)70063-4
  37. Jump up^ Judson I, et al. Lancet Oncol. 2014 Apr;15(4):415-23doi: 10.1016/S1470-2045(14)70063-4
  38. Jump up^ Chawala SP, et al. J Clin Oncol. 2014 (54) 3660 doi:10.1200/JCO.2013.54.3660
  39. Jump up^ Borad, M. J. et al. Randomized Phase II Trial of Gemcitabine Plus TH-302 Versus Gemcitabine in Patients With Advanced Pancreatic Cancer. Journal of Clinical Oncology (2014). doi: 10.1200/JCO.2014.55.7504
  40. Jump up^ Von Hoff, D. D. et al. Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine. New England Journal of Medicine 369, 1691–1703 (2013). doi:10.1056/NEJMoa1304369
  41. Jump up^ Von Hoff, D. D. et al. Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine. New England Journal of Medicine 369, 1691–1703 (2013). doi:10.1056/NEJMoa1304369
  42. Jump up^ Borad, M. J. et al. Randomized Phase II Trial of Gemcitabine Plus TH-302 Versus Gemcitabine in Patients With Advanced Pancreatic Cancer. Journal of Clinical Oncology (2014). doi: 10.1200/JCO.2014.55.7504
  43. Jump up^ Threshold Pharmaceuticals 10-K Annual report 2011 from 15 Mar 2012
  44. Jump up^ Threshold Pharmaceuticals 10-Q Quarterly report Q3/2014 from 3 Nov 14
  45. Jump up^ Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  46. Jump up^ Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  47.  Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  48.  Phosphoramidate alkylator prodrugs US8003625B2,US8507464B2, US8664204B2
  49.  Phosphoramidate alkylator prodrugs EP1896040B1and JP5180824B2
WO2007002931A2 * Jun 29, 2006 Jan 4, 2007 Threshold Pharmaceuticals, Inc. Phosphoramidate alkylator prodrugs
WO2008083101A1 * Dec 21, 2007 Jul 10, 2008 Threshold Pharmaceuticals, Inc. Phosphoramidate alkylator prodrugs for the treatment of cancer
WO2010048330A1 * Oct 21, 2009 Apr 29, 2010 Threshold Pharmaceuticals, Inc. Treatment of cancer using hypoxia activated prodrugs
WO2015051921A1 * Oct 10, 2014 Apr 16, 2015 Merck Patent Gmbh Synthesis of 1-alkyl-2-amino-imidazol-5-carboxylic acid ester via calpha-substituted n-alkyl-glycine ester derivatives
Reference
1 * DUAN, J.-X. ET AL.: “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs“, JOURNAL OF MEDICINAL CHEMISTRY, vol. 51, 2008, pages 2412 – 2420, XP008139620, DOI: doi:10.1021/jm701028q
Evofosfamide
TH-302.svg
Names
IUPAC name

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate
Other names

TH-302; HAP-302
Identifiers
918633-87-1 Yes
ChemSpider 10157061 Yes
Jmol-3D images Image
PubChem 11984561
Properties
C9H16Br2N5O4P
Molar mass 449.04 g·mol−1
6 to 7 g/l

///////////Orphan Drug Status, soft tissue sarcoma,  Pancreatic cancer, Fast track,  TH-302, TH 302, эвофосфамид ,  إيفوفوسفاميد ,  艾伏磷酰胺 , Evofosfamide, 918633-87-1, PHASE 3

O=[N+]([O-])c1ncc(COP(=O)(NCCBr)NCCBr)n1C

Biafungin, CD 101, a Novel Echinocandin for Vulvovaginal candidiasis


STR1

str1

str1as  CH3COOH salt

UNII-W1U1TMN677.png

CD 101

Several structural representations above

Biafungin™; CD 101 IV; CD 101 Topical; CD101; SP 3025, Biafungin acetate, Echinocandin B

UNII-G013B5478J FRE FORM,

CAS 1396640-59-7 FREE FORM

MF, C63-H85-N8-O17, MW, 1226.4035

Echinocandin B,

1-((4R,5R)-4-hydroxy-N2-((4”-(pentyloxy)(1,1′:4′,1”-terphenyl)-4-yl)carbonyl)-5-(2-(trimethylammonio)ethoxy)-L-ornithine)-4-((4S)-4-hydroxy-4-(4-hydroxyphenyl)-L-allothreonine)-

Treat and prevent invasive fungal infections; Treat and prevent systemic Candida infections; Treat candidemia

2D chemical structure of 1631754-41-0

Biafungin acetate

CAS 1631754-41-0 ACETATE, Molecular Formula, C63-H85-N8-O17.C2-H3-O2, Molecular Weight, 1285.4472,

C63 H85 N8 O17 . C2 H3 O2
1-[(4R,5R)-4-hydroxy-N2-[[4”-(pentyloxy)[1,1′:4′,1”-terphenyl]-4-yl]carbonyl]-5-[2-(trimethylammonio)ethoxy]-L-ornithine]-4-[(4S)-4-hydroxy-4-(4-hydroxyphenyl)-L-allothreonine]-, acetate (1:1)

UNII: W1U1TMN677

CD101 – A novel echinocandin antifungal C. albicans (n=351) MIC90 = 0.06 µg/mL C. glabrata (n=200) MIC90 = 0.06 µg/mL  Echinocandins have potent fungicidal activity against Candida species

  • Originator Seachaid Pharmaceuticals
  • Developer Cidara Therapeutics
  • Class Antifungals; Echinocandins; Small molecules
  • Mechanism of Action Glucan synthase inhibitors

 

BIAFUNGIN, CD 101

Watch this space as I add more info…………….

U.S. – Fast Track (Treat candidemia);
U.S. – Fast Track (Treat and prevent invasive fungal infections);
U.S. – Orphan Drug (Treat and prevent invasive fungal infections);
U.S. – Orphan Drug (Treat candidemia);
U.S. – Qualified Infectious Disease Program (Treat candidemia);
U.S. – Qualified Infectious Disease Program (Treat and prevent invasive fungal infections)

Fungal infections have emerged as major causes of human disease, especially among the immunocompromised patients and those hospitalized with serious underlying disease. As a consequence, the frequency of use of systemic antifungal agents has increased significantly and there is a growing concern about a shortage of effective antifungal agents. Although resistance rates to the clinically available antifungal agents remains low, reports of breakthrough infections and the increasing prevalence of uncommon fungal species that display elevated MIC values for existing agents is worrisome. Biafungin (CD101, previously SP 3025) is a novel echinocandin that displays chemical stability and long-acting pharmacokinetics that is being developed for once-weekly or other intermittent administration (see posters #A-693 and A- 694 for further information). In this study, we test biafungin and comparator agents against a collection of common Candida and Aspergillus species, including isolates resistant to azoles and echinocandins.

The echinocandins are an important class of antifungal agents, but are administered once daily by intravenous (IV) infusion. An echinocandin that could be administered once weekly could facilitate earlier hospital discharges and could expand usage to indications where daily infusions are impractical. Biafungin is a highly stable echinocandin for once-weekly IV administration. The compound was found to have a spectrum of activity and potency comparable to other echinocandins. In chimpanzees single dose pharmacokinetics of IV and orally administered biafungin were compared to IV anidulafungin, which has the longest half-life (T1/2 ) of the approved echinocandins.

Background  Vulvovaginal candidiasis (VVC) is a highly prevalent mucosal infection  VVC is caused by Candida albicans (~85%) and non-albicans (~15%)  5-8% of women have recurrent VVC (RVVC) which is associated with a negative impact on work/social life  Oral fluconazole prescribed despite relapse, potential DDIs and increased risk to pregnant women  No FDA-approved therapy for RVVC and no novel agent in >20 years

str1

Cidara Therapeutics 6310 Nancy Ridge Drive, Suite 101 San Diego, CA 92121

The incidence of invasive fungal infections, especially those due to Aspergillus spp. and Candida spp., continues to increase. Despite advances in medical practice, the associated mortality from these infections continues to be substantial. The echinocandin antifungals provide clinicians with another treatment option for serious fungal infections. These agents possess a completely novel mechanism of action, are relatively well-tolerated, and have a low potential for serious drug–drug interactions. At the present time, the echinocandins are an option for the treatment of infections due Candida spp (such as esophageal candidiasis, invasive candidiasis, and candidemia). In addition, caspofungin is a viable option for the treatment of refractory aspergillosis. Although micafungin is not Food and Drug Administration-approved for this indication, recent data suggests that it may also be effective. Finally, caspofungin- or micafungin-containing combination therapy should be a consideration for the treatment of severe infections due to Aspergillus spp. Although the echinocandins share many common properties, data regarding their differences are emerging at a rapid pace. Anidulafungin exhibits a unique pharmacokinetic profile, and limited cases have shown a potential far activity in isolates with increased minimum inhibitory concentrations to caspofungin and micafungin. Caspofungin appears to have a slightly higher incidence of side effects and potential for drug–drug interactions. This, combined with some evidence of decreasing susceptibility among some strains ofCandida, may lessen its future utility. However, one must take these findings in the context of substantially more data and use with caspofungin compared with the other agents. Micafungin appears to be very similar to caspofungin, with very few obvious differences between the two agents.

Echinocandins are a new class of antifungal drugs[1] that inhibit the synthesis of glucan in the cell wall, via noncompetitive inhibition of the enzyme 1,3-β glucan synthase[2][3] and are thus called “penicillin of antifungals”[4] (a property shared with papulacandins) as penicillin has a similar mechanism against bacteria but not fungi. Beta glucans are carbohydrate polymers that are cross-linked with other fungal cell wall components (The bacterial equivalent is peptidoglycan). Caspofungin, micafungin, and anidulafungin are semisynthetic echinocandin derivatives with clinical use due to their solubility, antifungal spectrum, and pharmacokinetic properties.[5]

List of echinocandins:[17]

  • Pneumocandins (cyclic hexapeptides linked to a long-chain fatty acid)
  • Echinocandin B not clinically used, risk of hemolysis
  • Cilofungin withdrawn from trials due to solvent toxicity
  • Caspofungin (trade name Cancidas, by Merck)
  • Micafungin (FK463) (trade name Mycamine, by Astellas Pharma.)
  • Anidulafungin (VER-002, V-echinocandin, LY303366) (trade name Eraxis, by Pfizer)

History

Discovery of echinocandins stemmed from studies on papulacandins isolated from a strain of Papularia sphaerosperma (Pers.), which were liposaccharide – i.e., fatty acid derivatives of a disaccharide that also blocked the same target, 1,3-β glucan synthase – and had action only on Candida spp. (narrow spectrum). Screening of natural products of fungal fermentation in the 1970s led to the discovery of echinocandins, a new group of antifungals with broad-range activity against Candida spp. One of the first echinocandins of the pneumocandin type, discovered in 1974, echinocandin B, could not be used clinically due to risk of high degree of hemolysis. Screening semisynthetic analogs of the echinocandins gave rise to cilofungin, the first echinofungin analog to enter clinical trials, in 1980, which, it is presumed, was later withdrawn for a toxicity due to the solvent system needed for systemic administration. The semisynthetic pneumocandin analogs of echinocandins were later found to have the same kind of antifungal activity, but low toxicity. The first approved of these newer echinocandins was caspofungin, and later micafungin and anidulafungin were also approved. All these preparations so far have low oral bioavailability, so must be given intravenously only. Echinocandins have now become one of the first-line treatments for Candida before the species are identified, and even as antifungal prophylaxis in hematopoietic stem cell transplant patients.

CIDARA THERAPEUTICS DOSES FIRST PATIENT IN PHASE 2 TRIAL OF CD101 TOPICAL TO TREAT VULVOVAGINAL CANDIDIASIS

SAN DIEGO–(BUSINESS WIRE)–Jun. 9, 2016– Cidara Therapeutics, Inc. (Nasdaq:CDTX), a biotechnology company developing novel anti-infectives and immunotherapies to treat fungal and other infections, today announced that the first patient has been dosed in RADIANT, a Phase 2 clinical trial comparing the safety and tolerability of the novel echinocandin, CD101, to standard-of-care fluconazole for the treatment of acute vulvovaginal candidiasis (VVC). RADIANT will evaluate two topical formulations of CD101, which is Cidara’s lead antifungal drug candidate.

“There have been no novel VVC therapies introduced for more than two decades, so advancing CD101 topical into Phase 2 is a critical step for women with VVC and for Cidara,” said Jeffrey Stein, Ph.D., president and chief executive officer of Cidara. “Because of their excellent safety record and potency against Candida, echinocandin antifungals are recommended as first line therapy to fight systemic Candida infections. CD101 topical will be the first echinocandin tested clinically in VVC and we expect to demonstrate safe and improved eradication of Candida with rapid symptom relief for women seeking a better option over the existing azole class of antifungals.”

RADIANT is a Phase 2, multicenter, randomized, open-label, active-controlled, dose-ranging trial designed to evaluate the safety and tolerability of CD101 in women with moderate to severe episodes of VVC. The study will enroll up to 125 patients who will be randomized into three treatment cohorts. The first cohort will involve the treatment of 50 patients with CD101 Ointment while a second cohort of 50 patients will receive CD101 Gel. The third cohort will include 25 patients who will be treated with oral fluconazole.

The primary endpoints of RADIANT will be the safety and tolerability of a single dose of CD101 Ointment and multiple doses of CD101 Gel in patients with acute VVC. Secondary endpoints include therapeutic efficacy in acute VVC patients treated with CD101. Treatment evaluations and assessments will occur on trial days 7, 14 and 28.

The RADIANT trial will be conducted at clinical trial centers across the United States. More information about the trial is available at www.clinicaltrials.gov, identifier NCT02733432.

About VVC and RVVC

Seventy-five percent of women worldwide suffer from VVC in their lifetime, and four to five million women in the United Statesalone have the recurrent form of the infection, which is caused by Candida. Many women will experience recurrence after the completion of treatment with existing therapies. Most VVC occurs in women of childbearing potential (the infection is common in pregnant women), but it affects women of all ages. In a recent safety communication, the U.S. Food and Drug Administration(FDA) advised caution in the prescribing of oral fluconazole for yeast infections during pregnancy based on a published study concluding there is an increased risk of miscarriage. The Centers for Disease Control and Prevention (CDC) guidelines recommend using only topical antifungal products to treat pregnant women with vulvovaginal yeast infections. Vaginal infections are associated with a substantial negative impact on day-to-day functioning and adverse pregnancy outcomes including preterm delivery, low birth weight, and increased infant mortality in addition to predisposition to HIV/AIDS. According to the CDC, certain species of Candida are becoming increasingly resistant to existing antifungal medications. This emerging resistance intensifies the need for new antifungal agents.

About CD101 Topical

CD101 topical is the first topical agent in the echinocandin class of antifungals and exhibits a broad spectrum of fungicidal activity against Candida species. In May 2016, the FDA granted Qualified Infectious Disease Product (QIDP) and Fast Track Designation to CD101 topical for the treatment of VVC and the prevention of RVVC.

About Cidara Therapeutics

Cidara is a clinical-stage biotechnology company focused on the discovery, development and commercialization of novel anti-infectives for the treatment of diseases that are inadequately addressed by current standard-of-care therapies. Cidara’s initial product portfolio comprises two formulations of the company’s novel echinocandin, CD101. CD101 IV is being developed as a once-weekly, high-exposure therapy for the treatment and prevention of serious, invasive fungal infections. CD101 topical is being developed for the treatment of vulvovaginal candidiasis (VVC) and the prevention of recurrent VVC (RVVC), a prevalent mucosal infection. In addition, Cidara has developed a proprietary immunotherapy platform, Cloudbreak™, designed to create compounds that direct a patient’s immune cells to attack and eliminate pathogens that cause infectious disease. Cidara is headquartered inSan Diego, California. For more information, please visit www.cidara.com.

REF http://ir.cidara.com/phoenix.zhtml?c=253962&p=irol-newsArticle&ID=2176474

CLIP

Cidara Therapeutics raises $42 million to develop once-weekly anti-fungal therapy

Cidara Therapeutics (formerly K2 Therapeutics) grabbed $42 million in a private Series B funding round Wednesday to continue developing its once-weekly anti-fungal therapy. Just in June 2014, the company completed a $32 million Series A financing led by 5AM Ventures, Aisling Capital, Frazier Healthcare and InterWest Partners, which was the fourth largest A round in 2014 for innovative startups[1]. FierceBiotech named the company as one of 2014 Fierce 15 biotech startups.

Cidara has an impressive executive team. The company was co-founded by Kevin Forrest, former CEO of Achaogen (NASDAQ: AKAO), and Shaw Warren. Jeffrey Stein, former CEO of Trius Therapeutics (NASDAQ: TSRX) and Dirk Thye, former president of Cerexa, have joined Cidara as CEO and CMO, respectively. Trius successfully developed antibiotic tedizolid and was acquired in 2013 by Cubist Pharmaceuticals (NASDAQ: CBST) for $818 million.

Cidara’s lead candidate, biafungin (SP3025), was acquired from Seachaid Pharmaceuticals for $6 million. Biafungin’s half-life is much longer than that of similar drugs known as echinocandins (e.g., caspofungin, micafungin, anidulafungin), which may allow it to be developed as a once-weekly therapy, instead of once daily. The company is also developing a topical formulation of biafungin, namely topifungin. Cidara intends to file an IND and initiate a Phase I clinical trial in the second half of 2015.

Merck’s Cancidas (caspofungin), launched in 2001, was the first of approved enchinocandins. The drug generated annual sales of $596 million in 2008. The approved echinocandins must be administered daily by intravenous infusion. Biafungin with improved pharmacokinetic characteristics has the potential to bring in hundreds of millions of dollars per year.

[1] Nat Biotechnol. 2015, 33(1), 18.

CLIP

Biafungin is a potent and broad-spectrum antifungal agent with excellent activity against wild-type and troublesome azole- and echinocandin-resistant strains of Candida spp. The activity of biafungin is comparable to anidulafungin. • Biafungin was active against both wild-type and itraconazole-resistant strains of Aspergillus spp. from four different species. • In vitro susceptibility testing of biafungin against isolates of Candida and Aspergillus may be accomplished by either CLSI or EUCAST broth microdilution methods each providing comparable results. • The use of long-acting intravenous antifungal agents that could safely be given once a week to select patients is desirable and might decrease costs with long-term hospitalizations. Background: A novel echinocandin, biafungin, displaying long-acting pharmacokinetics and chemical stability is being developed for once-weekly administration. The activities of biafungin and comparator agents were tested against 173 fungal isolates of the most clinically common species. Methods: 106 CAN and 67 ASP were tested using CLSI and EUCAST reference broth microdilution methods against biafungin (50% inhibition) and comparators. Isolates included 27 echinocandin-resistant CAN (4 species) with identified fks hotspot (HS) mutations and 20 azole nonsusceptible ASP (4 species). Results: Against C. albicans, C. glabrata and C. tropicalis, the activity of biafungin (MIC50, 0.06, 0.12 and 0.03 μg/ml, respectively by CLSI method) was comparable to anidulafungin (AND; MIC50, 0.03, 0.12 and 0.03 μg/ml, respectively) and caspofungin (CSP; MIC50, 0.12, 0.25 and 0.12 μg/ml, respectively; Table). C. krusei strains were very susceptible to biafungin, showing MIC90 values of 0.06 μg/ml by both methods. Biafungin (MIC50/90, 1/2 μg/ml) was comparable to AND and less potent than CSP against C. parapsilosis using CLSI methodology. CLSI and EUCAST methods displayed similar results for most species, but biafungin (MIC50, 0.06 μg/ml) was eight-fold more active than CSP (MIC50, 0.5 μg/ml) against C. glabrata using the EUCAST method. Overall, biafungin was two- to four-fold more active against fks HS mutants than CSP and results were comparable to AND. Biafungin was active against A. fumigatus (MEC50/90, ≤0.008/0.015 μg/ml), A. terreus (MEC50/90, 0.015/0.015 μg/ml), A. niger (MEC50/90, ≤0.008/0.03 μg/ml) and A. flavus (MEC50/90, ≤0.008/≤0.008 μg/ml) using CLSI method. EUCAST results for ASP were also low for all echinocandins and comparable to CLSI results. Conclusions: Biafungin displayed comparable in vitro activity with other echinocandins against common wild-type CAN and ASP and resistant subsets that in combination with the long-acting profile warrants further development of this compound. 1. Arendrup MC, Cuenca-Estrella M, Lass-Florl C, Hope WW (2013). Breakpoints for antifungal agents: An update from EUCAST focussing on echinocandins against Candida spp. and triazoles against Aspergillus spp. Drug Resist Updat 16: 81-95. 2. Castanheira M, Woosley LN, Messer SA, Diekema DJ, Jones RN, Pfaller MA (2014). Frequency of fks mutations among Candida glabrata isolates from a 10-year global collection of bloodstream infection isolates. Antimicrob Agents Chemother 58: 577-580. 3. Clinical and Laboratory Standards Institute (2008). M27-A3. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: third edition. Wayne, PA: CLSI. 4. Clinical and Laboratory Standards Institute (2008). M38-A2. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi: Second Edition. Wayne, PA: CLSI. 5. Clinical and Laboratory Standards Institute (2012). M27-S4. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: 4th Informational Supplement. Wayne, PA: CLSI. 6. European Committee on Antimicrobial Susceptibility Testing (2014). Breakpoint tables for interpretation of MICs and zone diameters. Version 4.0, January 2014. Available at: http://www.eucast.org/clinical_breakpoints/. Accessed January 1, 2014. 7. Pfaller MA, Diekema DJ (2010). Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36: 1-53. 8. Pfaller MA, Diekema DJ, Andes D, Arendrup MC, Brown SD, Lockhart SR, Motyl M, Perlin DS (2011). Clinical breakpoints for the echinocandins and Candida revisited: Integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria. Drug Resist Updat 14: 164-176. ABSTRACT Activity of a Novel Echinocandin Biafungin (CD101) Tested against Most Common Candida and Aspergillus Species, Including Echinocandin- and Azole-resistant Strains M CASTANHEIRA, SA MESSER, PR RHOMBERG, RN JONES, MA PFALLER JMI Laboratories, North Liberty, Iowa, USA C

PATENT

https://www.google.com/patents/WO2015035102A2?cl=en

BIAFUNGIN ACETATE IS USED AS STARTING MATERIAL

Example 30b: Synthesis of Compound 31

Step a. Nitration of Biafungin Acetate

To a stirring solution of biafungin (1 00 mg, 0.078 mmol) in glacial acetic acid(1 .5 ml_) was added sodium nitrite (1 1 mg, 0.159 mmol) and the reaction was stirred at ambient temperature for 20 hours. The mixture was applied directly to reversed phase H PLC (Isco CombiFlash Rf; 50g RediSep C1 8 column, 5 to 95% acetonitrile in Dl water containing 0.1 % formic acid: 15 minute gradient). The pure fractions were pooled and lyophilized to yield 85 mg of the desired product as a light yellow solid, formate salt. 1 H-NMR (300 M Hz, Methanol-d4) δ 8.58 (d, 1 H, J = 1 1 .7 Hz), 8.47 (t, 2H, J = 8.7Hz), 8.05 (d, 1 H, J = 2.1 Hz), 7.99 (d, 2H, J = 9.3 Hz), 7.82 (d, 2H, J = 8.7 Hz), 7.79-7.60 (m, 12H), 7.1 7 (d, 1 H, J = 8.7 Hz), 7.03 (d, 2H, J = 9 Hz), 5.48 (d, 1 H, J = 6 Hz), 5.08 (dd, 1 H, J = 1 .2, 5.7 Hz), 4.95-4.73 (m, 5H), 4.68-4.56 (m, 2H), 4.53 (d, 1 H, J = 5.7 Hz), 4.48-4.39 (m, 2H), 4.31 -3.79 (m, 6H), 4.04 (t, 2H, J = 5.7 Hz), 3.72-3.44 (m,3H), 3.1 8 (s, 9H), 2.60-1 .99 (m, 5H), 1 .83 (m, 2H, J = 8.7 Hz), 1 .56-1 .35 (m, 5H), 1 .28 (d, 6H, J = 4.2 Hz), 1 .09 (d, 3H, J = 1 0.2 Hz), 0.99 (t, 3H, J = 8.7 Hz) ; LC/MS, [M/2+H]+: 635.79, 635.80 calculated.

Step b. Reduction of Nitro-Biafungin To Amino-Biafungin

To a stirring solution of Nitro-Biafungin (1 00 mg, 0.075 mmol) in glacial acetic acid(1 .5 ml_) was added zinc powder (50 mg, 0.77 mmol) and the reaction was stirred at ambient temperature for 1 hour. The mixture was filtered and applied directly to reversed phase HPLC (Isco CombiFlash Rf, 50g Redisep C18 column; 5 to 95% acetonitrile in Dl water containing 0.1 % formic acid: 15 minute gradient). The pure fractions were pooled and lyophilized to yield 55 mg of the desired product as a white solid, formate salt. 1 H-NMR (300 MHz, Methanol-d4) 5 8.47 (bs, 1 H), 7.99 (d, 2H, J = 1 0.8Hz), 7.82 (d, 2H, J = 7.5 Hz), 7.80-7.67 (m, 6H), 7.62 (d, 2H, J = 8.7 Hz), 7.03 (d, 2H, J = 7.5 Hz), 6.77 (d, 1 H, J = 1 .9 Hz), 6.68 (d, 1 H, J = 8.2 Hz), 6.55 (dd, 2H, J = 8.2, 1 .9 Hz), 5.43 (d, 1 H, J = 2.5 Hz), 5.05 (d, 1 H, J = 3 Hz), 4.83-4.73 (m, 2H), 4.64- 4.56 (m, 2H), 4.43-4.34 (m, 2H), 4.31 -4.15 (m, 4H), 4.03-4.08 (m, 1 H), 4.1 1 -3.89 (m, 8H), 3.83 (d, 1 H, J = 1 0.8 Hz), 3.68-3.47 (m, 3H), 3.1 7 (s, 9H), 2.57-2.42 (m, 2H), 2.35-2.27 (m, 1 H), 2.14-1 .98 (m, 2H), 1 .83 (m, 2H, J = 6 Hz), 1 .56-1 .38 (m, 4H), 1 .28 (dd, 6H, J = 6.5, 2 Hz), 1 .09 (d, 3H, J = 7 Hz), 0.986 (t, 3H, J = 7 Hz); High Res LC/MS: [M+H]+ 1241 .61 63; 1241 .6136 calculated.

Step c. Reaction of Amino-Biafungin with lnt-2 to Produce Compound 31

To a stirring solution of Amino-Biafungin (50 mg, 0.04 mmol) in DM F (1 ml_) was added formyl-Met-Leu-Phe- -Ala-OSu (lnt-2) (36 mg, 0.06 mmol) and DI PEA (7 uL, 0.04 mmol). The reaction was stirred at ambient temperature for 1 8 hours. The mixture was applied directly to reversed phase HPLC (Isco CombiFlash Rf; 50g Redisep C1 8 column; 5 to 95% acetonitrile in Dl water containing 0.1 % formic acid: 15 minute gradient). The pure fractions were pooled and lyophilized to yield 26 mg of a white solid as a formate salt. 1 H-NMR (300 M Hz, Methanol-d4) 5 8.55 (bs, 1 H), 8.44 (t, 1 H, J = 10 Hz), 8.1 8 (d, 1 H, J = 6 Hz), 8.1 1 (s, 1 H), 7.99 (d, 2H, J = 1 0 Hz), 7.84-7.70 (m, 6H), 7.63 (d, 2H, J = 7.8 Hz), 7.32-7.1 9 (m, 6H), 7.03 (d, 4H, J = 9 Hz), 6.87 (d, 1 H, J = 8.1 Hz), 5.44 (d, 1 H, J = 1 0.5 Hz), 5.05 (d, 1 H, J = 4.5 Hz), 4.83-4.74 (m, 2H), 4.66-4.50 (m, 6H), 4.45-4.29 (m, 10H), 4.1 9-3.82 (m, 1 0H), 3.67-3.57 (m, 6H), 3.1 7 (s, 9H), 2.64-2.46 (m, 6 H), 2.14-1 .92 (m, 6H), 1 .84 (m, 4H, J = 6 Hz), 1 .62-1 .40 (m, 8H), 1 .32-1 .22 (m, 6H), 1 .09 (d, 3H, J = 9 Hz), 0.99 (t, 3H, J = 7.5 Hz), 0.88 (m, 6H, J = 6.8 Hz) ; High Res LC/MS, [M/2+H]+ 865.4143, 865.4147 calculated.

REFERENCES

  1. Denning, DW (June 2002). “Echinocandins: a new class of antifungal.”. The Journal of antimicrobial chemotherapy 49 (6): 889–91. doi:10.1093/jac/dkf045. PMID 12039879.
  2.  Morris MI, Villmann M (September 2006). “Echinocandins in the management of invasive fungal infections, part 1”. Am J Health Syst Pharm 63 (18): 1693–703.doi:10.2146/ajhp050464.p1. PMID 16960253.
  3. Morris MI, Villmann M (October 2006). “Echinocandins in the management of invasive fungal infections, Part 2”. Am J Health Syst Pharm 63 (19): 1813–20.doi:10.2146/ajhp050464.p2. PMID 16990627.
  4. ^ Jump up to:a b “Pharmacotherapy Update – New Antifungal Agents: Additions to the Existing Armamentarium (Part 1)”.
  5.  Debono, M; Gordee, RS (1994). “Antibiotics that inhibit fungal cell wall development”.Annu Rev Microbiol 48: 471–497. doi:10.1146/annurev.mi.48.100194.002351.

17 Eschenauer, G; Depestel, DD; Carver, PL (March 2007). “Comparison of echinocandin antifungals.”. Therapeutics and clinical risk management 3 (1): 71–97. PMC 1936290.PMID 18360617.

///////////Biafungin™,  CD 101 IV,  CD 101 Topical,  CD101,  SP 3025, PHASE 2, CIDARA, Orphan Drug, Fast Track Designation, Seachaid Pharmaceuticals,  Qualified Infectious Disease Product, QIDP, UNII-G013B5478J, 1396640-59-7, 1631754-41-0, Vulvovaginal candidiasis, Echinocandin B, FUNGIN

FREE FORM

CCCCCOc1ccc(cc1)c2ccc(cc2)c3ccc(cc3)C(=O)N[C@H]4C[C@@H](O)[C@H](NC(=O)[C@@H]5[C@@H](O)[C@@H](C)CN5C(=O)[C@@H](NC(=O)C(NC(=O)[C@@H]6C[C@@H](O)CN6C(=O)C(NC4=O)[C@@H](C)O)[C@H](O)[C@@H](O)c7ccc(O)cc7)[C@@H](C)O)OCC[N+](C)(C)C

AND OF ACETATE

CCCCCOc1ccc(cc1)c2ccc(cc2)c3ccc(cc3)C(=O)N[C@H]4C[C@@H](O)[C@H](NC(=O)[C@@H]5[C@@H](O)[C@@H](C)CN5C(=O)[C@@H](NC(=O)C(NC(=O)[C@@H]6C[C@@H](O)CN6C(=O)[C@@H](NC4=O)[C@@H](C)O)[C@H](O)[C@@H](O)c7ccc(O)cc7)[C@@H](C)O)OCC[N+](C)(C)C.CC(=O)[O-]

Three antifungal drugs approved by the United States Food and Drug Administration, caspofungin, anidulafungin, and micafungin, are known to inhibit β-1 ,3-glucan synthase which have the structures shown below.

caspofungin

Anidulafungin

Other exemplary p-1 ,3-glucan synthase inhibitors include,

echinocandin B

cilofungin

pneumocandin A0

pneumocandin B0

L-705589

L-733560

A-174591

or a salt thereof,

Biafungin


or a salt thereof,

Amino-biafungin


or a salt thereof,

Amino-AF-053

ASP9726

Yet other exemplary p-1 ,3-glucan synthase inhibitors include, without limitation:

Papulacandin B

Ergokonin

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

Oliceridine


TRV130.svg

Oliceridine.png

Oliceridine

N-[(3-methoxythiophen-2-yl)methyl]-2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine

[(3-Methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9- yl]ethyl})amine

Phase III

A mu-opioid receptor ligand potentially for treatment of acute postoperative pain.

TRV-130; TRV-130A

CAS No.1401028-24-7

Molecular Formula: C22H30N2O2S
Molecular Weight: 386.5508 g/mol
  • Originator Trevena

Trevena, Inc.

  • Class Analgesics; Small molecules
  • Mechanism of Action Beta arrestin inhibitors; Opioid mu receptor agonists
  • Orphan Drug Status No
  • On Fast track Postoperative pain
    • Phase III Postoperative pain
    • Phase II Pain

    Most Recent Events

    • 09 Mar 2016Trevena intends to submit NDA to US FDA in 2017
    • 22 Feb 2016Oliceridine receives Breakthrough Therapy status for Pain in USA
    • 19 Jan 2016Phase-III clinical trials in Postoperative pain in USA (IV) (NCT02656875)

Oliceridine (TRV130) is an opioid drug that is under evaluation in human clinical trials for the treatment of acute severe pain. It is afunctionally selective μ-opioid receptor agonist developed by Trevena Inc. Oliceridine elicits robust G protein signaling, with potencyand efficacy similar to morphine, but with far less β-arrestin 2 recruitment and receptor internalization, it displays less adverse effectsthan morphine.[1][2][3]

In 2015, the product was granted fast track designation in the U.S. for the treatment of moderate to severe acute pain. In 2016, the compound was granted FDA breakthrough therapy designation for the management of moderate to severe acute pain.

Oliceridine (TRV130) is an intravenous G protein biased ligand that targets the mu opioid receptor. Trevena is developing TRV130 for the treatment of moderate to severe acute pain where intravenous therapy is preferred, with a clinical development focus in acute postoperative pain

TRV 130 HCl is a novel μ-opioid receptor (MOR) G protein-biased ligand; elicits robust G protein signaling(pEC50=8.1), with potency and efficacy similar to morphine, but with far less beta-arrestin recruitment and receptor internalization.

NMR

STR1

Oliceridine (TRV130) – Mu Opioid Biased Ligand for Acute Pain

Target Indication Lead
Optimization
Preclinical
Development
Phase
1
Phase
2
Phase
3
Ownership
Oliceridine (TRV130) Mu-receptor Moderate to
Severe Pain
intravenous Trevena Logo

Oliceridine (TRV130) is an intravenous G protein biased ligand that targets the mu opioid receptor. Trevena is developing TRV130 for the treatment of moderate to severe acute pain where intravenous therapy is preferred, with a clinical development focus in acute postoperative pain.

Recent TRV130 News

Opioid receptors (ORs) mediate the actions of morphine and morphine-like opioids, including most clinical analgesics. Three molecularly and pharmacologically distinct opioid receptor types have been described: δ, κ and μ. Furthermore, each type is believed to have sub-types. All three of these opioid receptor types appear to share the same functional mechanisms at a cellular level. For example, activation of the opioid receptors causes inhibition of adenylate cyclase, and recruits β-arrestin.

When therapeutic doses of morphine are given to patients with pain, the patients report that the pain is less intense, less discomforting, or entirely gone. In addition to experiencing relief of distress, some patients experience euphoria. However, when morphine in a selected pain-relieving dose is given to a pain-free individual, the experience is not always pleasant; nausea is common, and vomiting may also occur. Drowsiness, inability to concentrate, difficulty in mentation, apathy, lessened physical activity, reduced visual acuity, and lethargy may ensue.

There is a continuing need for new OR modulators to be used as analgesics. There is a further need for OR agonists as analgesics having reduced side effects. There is a further need for OR agonists as analgesics having reduced side effects for the treatment of pain, immune dysfunction, inflammation, esophageal reflux, neurological and psychiatric conditions, urological and reproductive conditions, medicaments for drug and alcohol abuse, agents for treating gastritis and diarrhea, cardiovascular agents and/or agents for the treatment of respiratory diseases and cough.

 PAPER

Structure activity relationships and discovery of a g protein biased mu opioid receptor ligand, ((3-Methoxythiophen-2-yl)methyl)a2((9R)-9-(pyridin-2-y1)-6-oxaspiro-(4.5)clecan-9-yl)ethylpamine (TRV130), for the treatment of acute severe pain
J Med Chem 2013, 56(20): 8019

Structure–Activity Relationships and Discovery of a G Protein Biased μ Opioid Receptor Ligand, [(3-Methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-[4.5]decan-9-yl]ethyl})amine (TRV130), for the Treatment of Acute Severe Pain

Trevena, Inc., 1018 West 8th Avenue, Suite A, King of Prussia, Pennsylvania 19406, United States
J. Med. Chem., 2013, 56 (20), pp 8019–8031
DOI: 10.1021/jm4010829
Publication Date (Web): September 24, 2013
Copyright © 2013 American Chemical Society
*Phone: 610-354-8840. Fax: 610-354-8850. E-mail: dchen@trevenainc.com.

Abstract

Abstract Image

The concept of “ligand bias” at G protein coupled receptors has been introduced to describe ligands which preferentially stimulate one intracellular signaling pathway over another. There is growing interest in developing biased G protein coupled receptor ligands to yield safer, better tolerated, and more efficacious drugs. The classical μ opioid morphine elicited increased efficacy and duration of analgesic response with reduced side effects in β-arrestin-2 knockout mice compared to wild-type mice, suggesting that G protein biased μ opioid receptor agonists would be more efficacious with reduced adverse events. Here we describe our efforts to identify a potent, selective, and G protein biased μ opioid receptor agonist, TRV130 ((R)-30). This novel molecule demonstrated an improved therapeutic index (analgesia vs adverse effects) in rodent models and characteristics appropriate for clinical development. It is currently being evaluated in human clinical trials for the treatment of acute severe pain.

http://pubs.acs.org/doi/abs/10.1021/jm4010829

[(3-Methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl] ethyl})amine ((R)-30)

Using a procedure described in method A, (R)-39e was converted to (R)-30 as a TFA salt. 1H NMR (400 MHz, CDCl3) δ 11.70 (brs, 1H), 9.14 (d, J = 66.6, 2H), 8.72 (d, J = 4.3, 1H), 8.19 (td,J = 8.0, 1.4, 1H), 7.70 (d, J = 8.1, 1H), 7.63 (dd, J = 7.0, 5.8, 1H), 7.22 (d, J = 5.5, 1H), 6.78 (d,J = 5.6, 1H), 4.08 (m, 2H), 3.80 (m, 4H), 3.69 (dd, J = 11.2, 8.7, 1H), 2.99 (d, J = 4.8, 1H), 2.51 (t, J = 9.9, 1H), 2.35 (m, 3H), 2.18 (td, J = 13.5, 5.4, 1H), 1.99 (d, J = 14.2, 1H), 1.82 (m, 2H), 1.65 (m, 1H), 1.47 (m, 4H), 1.14 (m, 1H), 0.73 (dt, J = 13.2, 8.9, 1H). LC-MS (API-ES) m/z = 387.0 (M + H).

Patent

WO 2012129495

http://www.google.com/patents/WO2012129495A1?cl=en

Scheme 1: Synthesis of Spirocyclic Nitrile

NCCH2C02CH3 AcOH, NH4OAc

Figure imgf000050_0001
Figure imgf000050_0002

1-5 1-6 1-7

Chiral HPLC separation n=1-2

R= phenyl, substituted phenyl, aryl,

Figure imgf000050_0003

s

Scheme 2: Converting the nitrile to the opioid receptor ligand (Approach 1)

Figure imgf000051_0001

2-4

Scheme 3: Converting the nitrile to the opioid receptor ligand (Approach 2)

Figure imgf000051_0002

1-8B 3-1 3-2 n=1-2

In some embodiments, the same scheme is applied to 1 -7 and 1 -8A. Scheme 4: Synthesis of Non-Spirocyclic Nitrile

Figure imgf000052_0001

4-1 4-2 4-3

KOH, ethylene glycol R= phenyl, substituted phenyl, aryl,

substituted aryl, pyridyl, substituted pyridyl, heat heteroaryl, substituted heteroaryl,

Figure imgf000052_0002

carbocycle, heterocycle and etc.

In some embodiments, 4-1 is selected from the group consisting of

Figure imgf000052_0003

4-1 A 4-1 B 4-1 C 4-1 D 4-1 E

Scheme 5: Synthesis of Other Spirocyclic Derived Opioid Ligands

Figure imgf000053_0001

5-1 5-2 5-3

Scheme 6: Allyltrimethylsilane Approach to Access the Quaternary Carbon Center

RMgX, or RLi

Figure imgf000053_0002

Scheme 7: N-linked Pyrrazole Opioid Receptor Ligand

Figure imgf000054_0001
Figure imgf000055_0001

[(3-Methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9- yl]ethyl})amine

Figure imgf000144_0001

Into a vial were added 2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-l -amine (500 mg, 1.92 mmole), 18 mL CH2C12 and sodium sulfate (1.3 g, 9.6 mmole). The 3- methoxythiophene-2-carboxaldehyde (354 mg, 2.4 mmole) was then added, and the misture was stirred overnight. NaBH4 (94 mg, 2.4 mmole) was added to the reaction mixture, stirred for 10 minutes, and then MeOH (6.0 mL) was added, stirred l h, and finally quenched with water. The organics were separated off and evaporated. The crude residue was purified by a Gilson prep HPLC. The desired fractions collected and concentrated and lyophilized. After lyophilization, residue was partitioned between CH2C12 and 2N NaOH, and the organic layers were collected. After solvent was concentrated to half of the volume, 1.0 eq of IN HC1 in Et20 was added,and majority of solvent evaporated under reduced pressure. The solid obtained was washed several times with Et20 and dried to provide [(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2- yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine monohydrochloride (336 mg, 41% yield, m/z 387.0 [M + H]+ observed) as a white solid. The NMR for Compound 140 is described herein.

Example 15: Synthesis of [(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9- (pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine (Compound 140).

Methyl 2-cyano-2-[6-oxaspiro[4.5]decan-9-ylidene]acetate (mixture of E and Z isomers)

Figure imgf000141_0001

A mixture of 6-oxaspiro[4.5]decan-9-one (13.74 g, 89.1 mmol), methylcyanoacetate (9.4 ml, 106.9 mmol), ammonium acetate (1.79 g, 26.17.mmol) and acetic acid (1.02 ml, 17.8 mmol) in benzene (75 ml) was heated at reflux in a 250 ml round bottom flask equipped with a Dean-Stark and a reflux condenser. After 3h, TLC (25%EtOAc in hexane, PMA stain) showed the reaction was completed. After cooling, benzene (50 ml) was added and the layer was separated, the organic was washed by water (120 ml) and the aqueous layer was extracted by CH2CI2 (3 x 120 ml). The combined organic was washed with sat’d NaHCCb, brine, dried and concentrated and the residual was purified by flash chromatography (340 g silica gel column, eluted by EtOAc in hexane: 5% EtOAc, 2CV; 5-25%, 14CV; 25-40%,8 CV) gave a mixture of E and Z isomers: methyl 2-cyano-2-[6- oxaspiro[4.5]decan-9-ylidene]acetate ( 18.37 g, 87.8 % yield, m/z 236.0 [M + H]+ observed) as a clear oil. -cyano-2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetate

Figure imgf000141_0002

A solution of 2-bromopyridine (14.4 ml, 150 mmo) in THF (75 ml) was added dropwise to a solution of isopropylmagnesium chloride (75 ml, 2M in THF) at 0°C under N2, the mixture was then stirred at rt for 3h, copper Iodide(2.59 g, 13.6 mmol) was added and allowed to stir at rt for another 30 min before a solution of a mixture of E and Z isomers of methyl 2-cyano-2-[6-oxaspiro[4.5]decan-9-ylidene]acetate (16 g, 150 mmol) in THF (60 ml) was added in 30 min. The mixture was then stirred at rt for 18h. The reaction mixture was poured into a 200 g ice/2 N HC1 (100 ml) mixture. The product was extracted with Et20 (3×300 ml), washed with brine (200 ml), dried (Na2S04) and concentrated. The residual was purified by flash chromatography (100 g silica gel column, eluted by EtOAc in hexane: 3% 2CV; 3-25%, 12 CV; 25-40% 6CV gave methyl 2-cyano-2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetate (15.44 g, 72% yield, m/z 315.0 [M + H]+ observed) as an amber oil .

-[9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile

Figure imgf000142_0001

Ethylene glycol (300 ml) was added to methyl 2-cyano-2-[9-(pyridin-2-yl)-6- oxaspiro[4.5]decan-9-yl]acetate( 15.43 g, 49 mmol) followed by potassium hydroxide (5.5 g , 98 mmol), the resulting mix was heated to 120oC, after 3 h, the reaction mix was cooled and water (300 ml) was added, the product was extracted by Et20(3 x 400 ml), washed with water(200 ml), dried (Na2S04) and concentrated, the residual was purified by flash chromatography (340 g silica gel column, eluted by EtOAc in hexane: 3% 2CV; 3-25%, 12 CV; 25-40% 6CV to give 2-[9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9- yl]acetonitrile (10.37 g, 82% yield, m/z 257.0 [M + H]+ observed).

-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile

Figure imgf000142_0002

racemic 2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile was separated by chiral HPLC column under the following preparative-SFC conditions: Instrument: SFC-80 (Thar, Waters); Column: Chiralpak AD-H (Daicel); column temperature: 40 °C; Mobile phase: Methanol /CO2=40/60; Flow: 70 g/min; Back pressure: 120 Bar; Cycle time of stack injection: 6.0min; Load per injection: 225 mg; Under these conditions, 2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile (4.0 g) was separated to provide the desired isomer, 2-[(9R)-9-(Pyridin-2-yI)-6- oxaspiro[4.5]decan-9-yl]acetonitrile (2.0 g, >99.5% enantiomeric excess) as a slow- moving fraction. The absolute (R) configuration of the desired isomer was later determined by an X-ray crystal structure analysis of Compound 140. [0240] -[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-l-amine

Figure imgf000143_0001

LAH (1M in Et20, 20ml, 20 mmol) was added to a solution of 2-[(9R)-9-(pyridin-2-yl)- 6-oxaspiro[4.5]decan-9-yl]acetonitrile (2.56 g, 10 mmol) in Et20 (100 ml, 0.1M ) at OoC under N2. The resulting mix was stirred and allowed to warm to room temperature. After 2 h, LCMS showed the reaction had completed. The reaction was cooled at OoC and quenched with water ( 1.12 ml), NaOH (10%, 2.24 ml) and another 3.36 ml of water. Solid was filtered and filter pad was washed with ether (3 x 20 ml). The combined organic was dried and concentrated to give 2-[(9R)-9-(Pyridin-2-yl)-6- oxaspiro[4.5]decan-9-yl]ethan-l -amine (2.44 g, 94% yield, m/z 260.6 [M + H]+ observed) as a light amber oil.

Alternatively, 2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-l -amine was prepared by Raney-Nickel catalyzed hydrogenation.

An autoclave vessel was charged with 2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4,5]decan-9- yl] acetonitrile and ammonia (7N solution in methanol). The resulting solution was stirred at ambient conditions for 15 minutes and treated with Raney 2800 Nickel, slurried in water. The vessel was pressurized to 30 psi with nitrogen and agitated briefly. The autoclave was vented and the nitrogen purge repeated additional two times. The vessel was pressurized to 30 psi with hydrogen and agitated briefly. The vessel was vented and purged with hydrogen two additional times. The vessel was pressurized to 85-90 psi with hydrogen and the mixture was warmed to 25-35 °C. The internal temperature was increased to 45-50 °C over 30-60 minutes. The reaction mixture was stirred at 45-50 °C for 3 days. The reaction was monitored by HPLC. Once reaction was deemed complete, it was cooled to ambient temperature and filtered through celite. The filter cake was washed with methanol (2 x). The combined filtrates were concentrated under reduced pressure at 40-45 °C. The resulting residue was co-evaporated with EtOH (3 x) and dried to a thick syrupy of 2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-l -amine.

References

  1.  Chen XT, Pitis P, Liu G, Yuan C, Gotchev D, Cowan CL, Rominger DH, Koblish M, Dewire SM, Crombie AL, Violin JD, Yamashita DS (October 2013). “Structure-Activity Relationships and Discovery of a G Protein Biased μ Opioid Receptor Ligand, [(3-Methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-[4.5]decan-9-yl]ethyl})amine (TRV130), for the Treatment of Acute Severe Pain”. J. Med. Chem. 56 (20): 8019–31.doi:10.1021/jm4010829. PMID 24063433.
  2.  DeWire SM, Yamashita DS, Rominger DH, Liu G, Cowan CL, Graczyk TM, Chen XT, Pitis PM, Gotchev D, Yuan C, Koblish M, Lark MW, Violin JD (March 2013). “A G protein-biased ligand at the μ-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine”. J. Pharmacol. Exp. Ther. 344 (3): 708–17.doi:10.1124/jpet.112.201616. PMID 23300227.
  3.  Soergel DG, Subach RA, Sadler B, Connell J, Marion AS, Cowan C, Violin JD, Lark MW (October 2013). “First clinical experience with TRV130: Pharmacokinetics and pharmacodynamics in healthy volunteers”. J Clin Pharmacol 54(3): 351–7. doi:10.1002/jcph.207. PMID 24122908.

External links

Patent ID Date Patent Title
US2015246904 2015-09-03 Opioid Receptor Ligands And Methods Of Using And Making Same
US8835488 2014-09-16 Opioid receptor ligands and methods of using and making same
US2013331408 2013-12-12 Opioid Receptor Ligands and Methods of Using and Making Same
Oliceridine
TRV130.svg
Systematic (IUPAC) name
N-[(3-methoxythiophen-2-yl)methyl]-2-[(9R)-9-pyridin-2-yl-6-oxaspiro[4.5]decan-9-yl]ethanamine
Clinical data
Routes of
administration
IV
Legal status
Legal status
Identifiers
CAS Number 1401028-24-7
ATC code none
PubChem CID 66553195
ChemSpider 30841043
UNII MCN858TCP0
ChEMBL CHEMBL2443262
Synonyms TRV130
Chemical data
Formula C22H30N2O2S
Molar mass 386.55 g·mol−1

////////TRV-130; TRV-130A, Oliceridine, Phase III, Postoperative pain, trevena, mu-opioid receptor ligand, fast track designation, breakthrough therapy designation

COc1ccsc1CNCC[C@]2(CCOC3(CCCC3)C2)c4ccccn4

AVORALSTAT


2D chemical structure of 918407-35-9

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 Logo

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).

2D chemical structure of 918407-35-9

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)

Conditions Interventions Phases Recruitment Sponsor/Collaborators
Hereditary Angioedema|HAE Drug: BCX4161|Drug: Placebo Phase 2|Phase 3 Recruiting BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161|Drug: Placebo Phase 2 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals

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.

Avoralstat & next generation kallikrein inhibitors for HAE

Avoralstat

Avoralstat is being developed as an oral prophylactic treatment for patients suffering from Hereditary Angioedema (HAE). Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

In May 2014 BioCryst, announced that the OPuS-1 (OralProphylaxiS-1) Phase 2a proof of concept clinical trial met its primary efficacy endpoint, several secondary endpoints and all other objectives established for the trial. OpuS-1 enrolled 24 HAE patients with a history of HAE attack frequency of at least 1 per week. Treatment with avoralstat demonstrated a statistically significant mean attack rate reduction of 0.45 attacks per week versus placebo, p<0.001. The mean attack rate per week was 0.82 on BCX4161 treatment, compared to 1.27 on placebo.

In December 2014, BioCryst initiated enrollment in OPuS-2 (Oral ProphylaxiS-2). OPuS-2 is a blinded, randomized, 12-week, three-arm, parallel cohort design trial evaluating the efficacy and safety of two different dose regimens of avoralstat administered three-times daily, 300 mg and 500 mg, compared with placebo. The primary efficacy endpoint for the trial will be the mean angioedema attack rate, which will be reported for each avoralstat dose group compared to placebo. The trial is being conducted in the U.S., Canada and Europe. On October 8, 2015, announced that it has completed enrollment of approximately 100 HAE patients with a history of moderately frequent to very frequent attacks in OPuS-2. BioCryst expects to report the OPuS-2 trial results in early 2016.

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 C13H16 204: 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

C27H2eN206: 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 Rf 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-de) 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 ❤ °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 (Tjackel= -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.

6582

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.

46582

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 ❤ °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 (Tjacke,= -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).

/////avoralstat, BCX4161, Fast Track, Treat hereditary angioedema (HAE), Orphan Drug, PRECLINICAL

COc1cc(c(cc1C=C)C(=O)Nc2ccc(cc2)C(=N)N)c3cc(ncc3C(=O)O)C(=O)NCC4CC4

GALETERONE


 

 

File:Galeterone.svg

Galeterone

SYNTHESIS SEE BELOW

A SARM potentially for the treatment of prostate cancer.

Research Code, TOK-001; VN; 124; 124-1; 1241

TOK-001; Galeterone; 851983-85-2; VN/124; UNII-WA33E149SW; VN/124-1;

CAS No. 851983-85-2(Galeterone)

(3S,8R,9S,10R,13S,14S)-17-(benzimidazol-1-yl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15-decahydro-1H-cyclopenta[a]phenanthren-3-ol

Fast track 2012 f

Molecular Formula: C26H32N2O
Molecular Weight: 388.54508 g/mol

 

Galeterone (TOK-001 or VN/124-1) is a novel steroidal antiandrogen under development by Tokai Pharmaceuticals for the treatment of prostate cancer. It possesses a unique dual mechanism of action, acting as both an androgen receptor antagonist and an inhibitor of CYP17A1, an enzyme required for the biosynthesis of the androgens.[1] It shows selectivity for 17,20-lyase over 17-hydroxylase.[2]

As of 2016, galeterone is being compared to enzalutamide in a phase III clinical trial (ARMOR3-SV) for AR-V7-expressing metastatic castration-resistant prostate cancer.[3][4]

Specific Androgen Receptor Modulator CYP17 Inhibitor TOK-001 is an orally bioavailable small-molecule androgen receptor modulator and CYP17 lyase inhibitor with potential antiandrogen activity. Galeterone exhibits three distinct mechanisms of action: 1) as an androgen receptor antagonist, 2) as a CYP17 lyase inhibitor and 3) by decreasing overall androgen receptor levels in prostate cancer tumors, all of which may result in a decrease in androgen-dependent growth signaling. Localized to the endoplasmic reticulum (ER), the cytochrome P450 enzyme CYP17 (P450C17 or CYP17A1) exhibits both 17alpha-hydroxylase and 17,20-lyase activities, and plays a key role in the steroidogenic pathway that produces progestins, mineralocorticoids, glucocorticoids, androgens, and estrogens.

About Galeterone

Tokai’s lead product candidate is galeterone, a highly-selective, oral small molecule with the potential to transform the treatment of prostate cancer. We are focusing our late-stage development of galeterone on the treatment of men with metastatic, castration-resistant prostate cancer, or CRPC, whose prostate tumor cells express the AR-V7 splice variant.


We are conducting ARMOR3-SV, a Phase 3 clinical trial of galeterone evaluating whether administration of galeterone results in a statistically significant increase in radiographic progression-free survival as compared to Xtandi® (enzalutamide), an oral therapy currently approved for the treatment of CRPC, in AR-V7 positive metastatic CRPC patients. ARMOR3-SV is the first pivotal trial in prostate cancer to employ a precision medicine approach for patient selection. For more information regarding ARMOR3-SV, click here.

Galeterone has been studied in over 250 subjects in Phase 1 and Phase 2 clinical trials, including in CRPC patients with and without the AR-V7 splice variant. In these trials, galeterone demonstrated good tolerability and showed clinically meaningful reductions in levels of prostate specific antigen, or PSA, a biochemincal marker used to evaluate prostate cancer patients for signs of response to therapy.

We are currently focusing our late-stage development of galeterone on AR-V7 positive metastatic CRPC patients because it represents an unmet need in prostate cancer and our precision medicine approach provides an efficient development path. Based on the data we and our collaborators have produced to date, we also believe there is rationale for the broader clinical exploration of galeterone in the future.


Galeterone acts by disrupting the androgen receptor signaling pathway. This pathway is activated by the binding of male hormones (also known as androgens), such as testosterone and dihydrotestosterone (DHT) to androgen receptors in prostate cancer cells.

Galeterone disrupts the activation of the androgen receptor pathway in three ways:

  • Androgen receptor degradation, which reduces the amount of androgen receptor protein in tumor cells. There are no currently marketed drugs whose mechanism of action entails degradation of the androgen receptor. Therefore, galeterone represents a potential first-in-class therapeutic opportunity.
  • CYP17 enzyme inhibition, which blocks the synthesis of testosterone. This mechanism has been validated clinically by Zytiga (abiraterone). Zytiga must be co-administered with the steroid prednisone in order to minimize the risk of a potentially fatal side effect called mineralocorticoid excess. Unlike Zytiga, galeterone has not been shown in clinical trials to cause mineralocorticoid excess and, as a result, does not require co-administration of steroids. As a result, we believe that galeterone may be easier to administer, provide convenience for patients and enhance patient compliance.
  • Androgen receptor inhibition, which blocks the binding of testosterone or DHT with the androgen receptor. This mechanism has been validated clinically by Xtandi® (enzalutamide), which is also currently approved for the treatment of CRPC. Xtandi™ has shown a risk of grand mal seizures in clinical trials. We have not had any reports of seizures in clinical trials of galeterone and, therefore, galeterone may have certain safety advantages over Xtandi.

 


Tokai retains global rights to galeterone. We intend to commercialize galeterone in the United States on our own, and to seek a partner to further develop and commercialize galeterone outside of the United States.

Galeterone has been granted Fast Track designation by U.S. Food and Drug Administration for the treatment of CRPC. Fast Track designation is designed to facilitate the development and expedite review of drugs intended to treat serious or life-threatening conditions and that demonstrate the potential to address unmet medical needs.

Androgen receptor degradation, which reduces the amount of androgen receptor protein in the tumor cells.

Androgen receptor antagonism, which blocks the binding of testosterone or DHT with the androgen receptor.

Inhibition of the enzyme CYP17, which blocks the synthesis of testosterone.

Figure 3: The structures of abiraterone, orteronel and galeterone.

From CYP17 inhibitors—abiraterone, C17,20-lyase inhibitors and multi-targeting agents

Nature Reviews Urology 11,32–42 (2014)
doi:10.1038/nrurol.2013.274

 SYNTHESIS

str1
CN 104098638

DETAILED DESCRIPTION

1J loss reaction.

  (1) raw material specifications to match.

acetate pregnancy dehydropregnenolone: ​​toluene + ethanol: Batch steep: hydrochloric acid amine light = 1: 3: 0 4: 0.213, which pregnenolone acetate pregnancy 160kg, toluene + ethanol 320kg + 160kg, approved Steep 64kg, hydrochloric acid amine light 34kg.

  (2) process operation.

  In the first input 1000L tank oximation with hydroxylamine hydrochloride in pyridine, and then pumped into a mixed solvent of toluene and ethanol, the reaction solution was stirred and heated to complete dissolution, pregnancy-dehydropregnenolone acetate was added and heated under reflux for 3 hours, cooling and crystallization, The Department conducted into the centrifuge centrifugal drying, apply a recovery from the mother liquor, rinse with warm water mixture to no foam, centrifugal drying, drying to a moisture at 0.2% or less, that acetic acid in pregnancy dehydropregnenolone oxime (oxime compounds) 163kg, content of 99%, a melting point of 202-204 ° C, a yield of about 102% (for pregnenolone acetate pregnancy weight ratio).

2, heavy drain hydrolysis reaction.

  (1) raw material specifications to match.

  acetate pregnancy dehydropregnenolone waning: Benzene: Batch steep: phosphorus oxychloride and toluene: HCl + water = 1: 6 5: 0 4: 1: 3.5, which acetate pregnancy alcohol one hand 163kg, benzene 1060kg, batch steep 64kg, phosphorus oxychloride and toluene 80kg + 80kg, hydrochloric acid + water 245kg + 325kg.

(2) process operation.

The first drying 2000L rearrangement reaction tank, then pumped to the reaction tank benzene, alcohol into acetate pregnancy oxime, pulls out into benzene, stirring heated to reflux until the reaction mixture is completely dissolved, cooling to 1 (TC When, pyridine, of the reaction liquid at temperatures down to 6 ° C, start dropping a mixed solution of previously prepared phosphorus oxychloride and toluene (1: 1 mass ratio), slowly dropping, dropping control, first After slow fast reaction when dropping liquid temperature control in 4-8 ° C, the addition was complete, the reaction solution at 9-12 ° C for 3 hours the first time under.

After incubation, a solution has been a mixed solution of hydrochloric acid and water, good preparation, while dropping the reaction liquid temperature is controlled at 15-25 ° C, the addition was complete, the reaction solution at 15-25 ° C under a second Insulation 1. 5-2 hours. After incubation, stand 40 minutes, then points to lower acidic water layer, the remaining upper layer was added 0.3 times the amount of 30-35 ° C in the brine and let stand 20 minutes, a second watershed, sub lower aqueous layer was then allowed to stand for 30 minutes, a third water diversion, to give the final weight of the upper layer reaction solution was drained.

  3, the red Dingding steam distillate process.

The rearrangement reaction liquid was pumped to punch distillate tank, conduct atmospheric distillate punch, has been rushed to the reaction mixture was distilled benzene mixed solvent only, at the start of the steam valve not to open too much, so as not to rush material, distillation after cooling discharge, centrifugal drying, washing with tap water to neutral, and then into the oven dried to a moisture in the square. 5% acetic acid in dehydroepiandrosterone (rearrangement thereof) The crude product is about 142kg, content of about 97.5%, a melting point of 160 ° C _165 ° C or so, yield about 88% (for acetate pregnancy dehydropregnenolone weight ratio).

  4, refining processes.

  The drying in acetic acid Dehydroepiandrosterone crude into refined tin, adding 8 times the weight of the crude methanol and 0.10 times the weight of activated carbon, heat, stirring to dissolve, reflux billion. 5 hours, filtered , concentrated, cooled to about 5 ° C, the discharge

str1
JMC 2005, 48, 2972-84
The most potent CYP17 inhibitors were 3β-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (5, code named VN/124-1), 3β-hydroxy-17-(51-pyrimidyl)androsta-5,16-diene (15)
PAPER
JMC 1998 41, 902-12
The most potent compounds are 3β-hydroxy-17-(1H-imidazol-1-yl)androsta-5,16-diene (17), 3β-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (19), and 17-(1H-imidazol-1-yl)androsta-4,16-dien-3-one (28), with Ki values of 1.2, 1.4, and 1.9 nM, respectively,
Discovery and Development of Galeterone (TOK-001 or VN/124-1)
for the Treatment of All Stages of Prostate Cancer…….http://pubs.acs.org/doi/pdf/10.1021/jm501239f
str1
str1
 CLICK ON PIC FOR CLEAR VIEW
Patent ID Date Patent Title
US2011034428 2011-02-10 Treatment of Prostate Cancer
US7875599 2011-01-25 C-17-heteroaryl steroidal CYP17 inhibitors/antiandrogens, in vitro biological activities, pharmacokinetics and antitumor activity
US2010137269 2010-06-03 Novel C-17-Heteroaryl Steroidal Cyp17 Inhibitors/Antiandrogens: Synehesis, In Vitro Biological Activities, Pharmacokinetics and Antitumor Activity
US2010048914 2010-02-25 Novel C-17-Heteroaryl Steroidal Cyp17 Inhibitors/Antiandrogens, In Vitro Biological Activities, Pharmacokinetics and Antitumor Activity
US2010048913 2010-02-25 Novel C-17-Heteroaryl Steroidal CYP17 Inhibitors/Antiandrogens Synthesis In Vitro Biological Activities, Pharmacokinetics and Antitumor Activity
US2010048912 2010-02-25 Novel C-17-Heteroaryl Steroidal CYP17 Inhibitors/Antiandrogens, In Vitro Biological Activities, Pharmacokinetics and Antitumor Activity
US2010048524 2010-02-25 Novel C-17-Heteroaryl Steroidal CYP17 Inhibitors/Antiandrogens Synthesis In Vitro Biological Activities, Pharmacokinetics and Antitumor Activity
US2010047338 2010-02-25 Novel C-17-Heteroaryl Steroidal CYP17 Inhibitors/Antiandrogens, In Vitro Biological Activities, Pharmacokinetics and Antitumor Activity
Patent ID Date Patent Title
US2013336962 2013-12-19 AZIRIDINE BISPHENOL ETHERS AND RELATED COMPOUNDS AND METHODS FOR THEIR USE
US8569393 2013-10-29 UV-LED curable compositions and inks
US2013203615 2013-08-08 ANTIANDROGEN THERAPY MONITORING METHODS AND COMPOSITIONS
US2012309861 2012-12-06 PHOTOINITIATORS FOR UV-LED CURABLE COMPOSITIONS AND INKS
US2012237502 2012-09-20 METHOD FOR TREATING BREAST CANCER AND OVARIAN CANCER
US2011319369 2011-12-29 COMBINATION OF A 17 ALPHA-HYDROXYLASE/C17, 20-LYASE INHIBITOR WITH AN ADDITIONAL THERAPEUTIC AGENT
US2011312924 2011-12-22 NOVEL STEROIDAL CYP17 INHIBITORS/ANTIANDROGENS
US2011312916 2011-12-22 NOVEL PRODRUGS OF STEROIDAL CYP17 INHIBITORS/ANTIANDROGENS
US2011118219 2011-05-19 NOVEL PRODRUGS OF C-17-HETEROARYL STEROIDAL CYP17 INHIBITORS/ANTIANDROGENS: SYNTHESIS, IN VITRO BIOLOGICAL ACTIVITIES, PHARMACOKINETICS AND ANTITUMOR ACTIVITY
US2011105445 2011-05-05 ANDROGEN RECEPTOR INACTIVATION CONTRIBUTES TO ANTITUMOR EFFICACY OF CYP17 INHIBITORS IN PROSTATE CANCER
Patent ID Date Patent Title
US2015051179 2015-02-19 NOVEL STEROIDAL CYP17 INHIBITORS/ANTIANDROGENS
US2015005265 2015-01-01 METHODS AND COMPOSITIONS FOR COMBINATION THERAPY USING P13K/MTOR INHIBITORES
US2014371261 2014-12-18 INDOMETHACIN ANALOGS FOR THE TREATMENT OF CASTRATE-RESISTANT PROSTATE CANCER
US2014371181 2014-12-18 NOVEL PRODRUGS OF STEROIDAL CYP17 INHIBITORS/ANTIANDROGENS
US2014343024 2014-11-20 TREATMENT OF PROSTATE CANCER
US2014288037 2014-09-25 NOVEL COMPOSITIONS AND METHODS FOR TREATING PROSTATE CANCER
US2014288036 2014-09-25 NOVEL C-17-HETEROARYL STEROIDAL CYP17 INHIBITORS/ANTIANDROGENS, IN VITRO BIOLOGICAL ACTIVITIES, PHARMACOKINETICS AND ANTITUMOR ACTIVITY
US2014274983 2014-09-18 NOVEL PRODRUGS OF C-17-HETEROARYL STEROIDAL CYP17 INHIBITORS/ANTIANDROGENS: SYNTHESIS, IN VITRO BIOLOGICAL ACTIVITIES, PHARMACOKINETICS AND ANTITUMOR ACTIVITY
US2014107085 2014-04-17 Bifunctional AKR1C3 Inhibitors/Androgen Receptor Modulators and Methods of Use Thereof
US2013336962 2013-12-19 AZIRIDINE BISPHENOL ETHERS AND RELATED COMPOUNDS AND METHODS FOR THEIR USE

str1

CN101691392A * Sep 17, 2009 Apr 7, 2010 扬州市天平化工厂有限公司 Method for preparing 3beta-acetoxyl group-5androstene-17ketone
CN102212099A * Apr 2, 2011 Oct 12, 2011 邵阳市科瑞化学品有限公司 Synthesis method for dehydroepiandrosterone
CN102603839A * Jan 13, 2012 Jul 25, 2012 宜城市共同药业有限公司 Preparation method of dehydroepiandrosterone
CN102746356A * Jul 17, 2012 Oct 24, 2012 湖北芳通药业股份有限公司 Process for producing dehydroepiandrosterone acetate through homogeneous phase method
1 * 石诚等: “5-雄甾烯-3β-醇-17-酮-3-醋酸酯的工艺研究“, 《山东化工》, vol. 41, no. 1, 31 December 2012 (2012-12-31)
2 * 石诚等: “醋酸妊娠双烯醇酮肟的工艺研究“, 《广州化工》, vol. 39, no. 23, 31 December 2011 (2011-12-31), pages 78 – 79

References

 

Silberstein, John L.; Taylor, Maritza N.; Antonarakis, Emmanuel S. (2016-04-01). “Novel Insights into Molecular Indicators of Response and Resistance to Modern Androgen-Axis Therapies in Prostate Cancer”. Current Urology Reports 17 (4): 29. doi:10.1007/s11934-016-0584-4. ISSN 1534-6285. PMID 26902623.

 

Galeterone
Galeterone.svg
Systematic (IUPAC) name
17-(1H-benzimidazol-1-yl)androsta-5,16-dien-3β-ol
Clinical data
Routes of
administration
Oral
Identifiers
CAS Number 851983-85-2
PubChem CID 11188409
ChemSpider 9363493
KEGG D10125 Yes
Chemical data
Formula C26H32N2O
Molar mass 388.25

///////

C[C@]12CC[C@@H](CC1=CC[C@@H]3[C@@H]2CC[C@]4([C@H]3CC=C4N5C=NC6=CC=CC=C65)C)O

CC12CCC(CC1=CCC3C2CCC4(C3CC=C4N5C=NC6=CC=CC=C65)C)O

Avoralstat


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 Logo

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).

2D chemical structure of 918407-35-9

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)

Conditions Interventions Phases Recruitment Sponsor/Collaborators
Hereditary Angioedema|HAE Drug: BCX4161|Drug: Placebo Phase 2|Phase 3 Recruiting BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161|Drug: Placebo Phase 2 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals
Hereditary Angioedema Drug: BCX4161 Phase 1 Completed BioCryst Pharmaceuticals

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.

Avoralstat & next generation kallikrein inhibitors for HAE

Avoralstat

Avoralstat is being developed as an oral prophylactic treatment for patients suffering from Hereditary Angioedema (HAE). Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

In May 2014 BioCryst, announced that the OPuS-1 (OralProphylaxiS-1) Phase 2a proof of concept clinical trial met its primary efficacy endpoint, several secondary endpoints and all other objectives established for the trial. OpuS-1 enrolled 24 HAE patients with a history of HAE attack frequency of at least 1 per week. Treatment with avoralstat demonstrated a statistically significant mean attack rate reduction of 0.45 attacks per week versus placebo, p<0.001. The mean attack rate per week was 0.82 on BCX4161 treatment, compared to 1.27 on placebo.

In December 2014, BioCryst initiated enrollment in OPuS-2 (Oral ProphylaxiS-2). OPuS-2 is a blinded, randomized, 12-week, three-arm, parallel cohort design trial evaluating the efficacy and safety of two different dose regimens of avoralstat administered three-times daily, 300 mg and 500 mg, compared with placebo. The primary efficacy endpoint for the trial will be the mean angioedema attack rate, which will be reported for each avoralstat dose group compared to placebo. The trial is being conducted in the U.S., Canada and Europe. On October 8, 2015, announced that it has completed enrollment of approximately 100 HAE patients with a history of moderately frequent to very frequent attacks in OPuS-2. BioCryst expects to report the OPuS-2 trial results in early 2016.

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 C13H16 204: 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

C27H2eN206: 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 Rf 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-de) 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 ❤ °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 (Tjackel= -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.

6582

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.

46582

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

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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 ❤ °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 (Tjacke,= -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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COc1cc(c(cc1C=C)C(=O)Nc2ccc(cc2)C(=N)N)c3cc(ncc3C(=O)O)C(=O)NCC4CC4

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