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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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FOTAGLIPTIN


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FOTAGLIPTIN

CAS 1312954-58-7

342.37, C17 H19 F N6 O

Benzonitrile, 2-[[3-[(3R)-3-amino-1-piperidinyl]-6-methyl-5-oxo-1,2,4-triazin-4(5H)-yl]methyl]-4-fluoro-

(R)-2-((3-(3-amino-piperidin-1-yl)-6-methyl-5-oxo-1,2,4-piperazine-4(5H)-yl)methyl)-4-fluorobenzonitrile,

BENZOATE 1403496-40-1

(R) 2- Methyl-5-oxo-1,2,4-triazin-4 (5H) -yl) methyl) -4-fluorobenzonitrile (3- benzoate (compound benzoate A), of the formula: the C . 17 the H 19 the FN . 6 O · the C . 7 the H . 6 O 2 , molecular weight: 464.49.

useful as a dipeptidyl peptidase IV (DPPIV) inhibitor for treating diabetes, particularly type 2 diabetes

Dipeptidyl peptidase IV inhibitor,

a DPPIV inhibitor, being developed by Chongqing Fochon, with licensee Shenzhen Salubris Pharmaceuticals, for treating type 2 diabetes mellitus. In January 2017, fotagliptin benzoate was reported to be in phase 1 clinical development. The compound of the present invention was first disclosed in WO2011079778. See WO2015110078 and WO2015110077, claiming crystalline polymorphic form of the DPPIV inhibitor.

  • Originator Chongqing Fochon Pharmaceutical
  • Class Antihyperglycaemics
  • Mechanism of Action CD26 antigen inhibitors
  • Shanghai Fosun Pharma Transfers Development Rights in New Diabetes & Cancer Therapies to Swiss-Greek Firm
Shanghai Fosun Pharma Transfers Development Rights in New Diabetes & Cancer Therapies to Swiss-Greek Firm
On 23 October 2013, leading Chinese healthcare company Shanghai Fosun Pharmaceutical Group Co., Ltd. signed an agreement with Sellas Life Science Group, a Switzerland based Greek pharmaceutical R&D company. According to the agreement, Fosun Pharma transfers to Sellas the global rights (excluding China) in development, commercialisation, marketing and distribution of Fotagliptin Benzoate and Pan-HER Inhibitors, two novel compounds owned by Fosun Pharma’s subsidiary Chongqing Fochon Pharmaceutical Co. Ltd.
Fotagliptin Benzoate is developed by Chongqing Fochon independently and has a prospect of developing into type 2 diabetes medicines, whereas Pan-HER Inhibitors, a receptor inhibitor of which Chongqing Fochon owns the proprietary IP rights, is a potential therapy for curing lung, breast and other cancers. Chongqing Fochon has filed application for international patent under the Patent Cooperation Treaty in respect of the two compounds.
The estimated total consideration for the transaction of approximately RMB3.248 billion will be paid by installment. In addition, upon the compounds obtaining relevant approvals in the US and/or Europe, Chongqing Fochon will be entitled to a 10% royalty in these regions on net revenue sales for eight years.
SYNTHESIS
PAPER
Research Article

Development and validation of a UPLC–MS/MS method for simultaneous determination of fotagliptin and its two major metabolites in human plasma and urine

Zhenlei Wang1, Ji Jiang1, Pei Hu1 & Qian Zhao*,1

*Author for correspondence:

Aim: Fotagliptin is a novel dipeptidyl peptidase IV inhibitor under clinical development for the treatment of Type II diabetes mellitus. The objective of this study was to develop and validate a specific and sensitive ultra-performance liquid chromatography (UPLC)–MS/MS method for simultaneous determination of fotagliptin and its two major metabolites in human plasma and urine. Methodology & results: After being pretreated using an automatized procedure, the plasma and urine samples were separated and detected using a UPLC-ESI–MS/MS method, which was validated following the international guidelines. Conclusion: A selective and sensitive UPLC–MS/MS method was first developed and validated for quantifying fotagliptin and its metabolite in human plasma and urine. The method was successfully applied to support the clinical study of fotagliptin in Chinese healthy subjects.

PATENT

WO2011079778

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011079778&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

PATENT

WO2015110078

compound A can be prepared according to the method disclosed in PCT / CN2010 / 080370, the specific synthesis route and the main reaction conditions are as follows:
Example 1 Preparation of 1-bromo-4-fluoro-2- (isothiocyanatomethyl) benzene (2)
To a DMF solution (20 ml) of 1-bromo-2- (bromomethyl) -4-fluorobenzene (1,5.36 g, 20.0 mmol) was added sodium iodide (1.20 g, 8.00 mmol) and potassium thiocyanate (3.88 g, 40.0 mmol). After the mixture was heated to 80C under nitrogen atmosphere for 12 hours, it was cooled to room temperature, 100 ml of water was added thereto, and extracted with ethyl acetate (50 mL x 2). The combined organic layers were washed with saturated brine, dried over anhydrous magnesium sulfate, The concentrate was concentrated by suction to give a crude product, and the residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 1-bromo-4-fluoro-2- (isothiocyanatomethyl) benzene (2).
Example 2 Preparation of N- (2-bromo-5-fluorobenzyl) hydrazinocarbothioamide (3)
A solution of hydrazine hydrate (80%, 2.22 g, 35.5 mmol) in 1,4-dioxane (20 mL) was cooled to 0 ° C and 1-bromo-4-fluoro-2- (isothiocyanate Yl) benzene (2,3.16 g, 12.8 mmol) in 1,4-dioxane (5 ml). The mixture was stirred at room temperature for 2 h, to which was added 100 ml of ice water, solid precipitated, filtered, washed with water and dried over phosphorus pentoxide overnight to give N- (2-bromo-5-fluorobenzyl) hydrazinothiocarb Amide (3).
MS: m / z, 278 (100%, M + 1), 280 (100%), 300 (10%, M + 23), 302 (10%).
Example 3 Preparation of methyl 2- (2- (2-bromo-5-fluorobenzylaminothioformamide) hydrazino) propionate (4)
N- (2-bromo-5-fluorobenzyl) hydrazinocarbothioamide (3, 1.12 g, 4.00 mmol) was added successively to a solution of pyruvic acid (352 mg, 4.00 mmol) in methanol And the residue was extracted with ethyl acetate (150 ml). The organic layer was washed successively with water, saturated sodium bicarbonate solution and saturated brine, and dried over anhydrous magnesium sulphate (MgSO4). The organic layer was washed with water, Dried, and concentrated by suction filtration to give methyl 2- (2- (2-bromo-5-fluorobenzylaminothioformamide) hydrazino) propionate (4).
MS: m / z, 362 (100%, M + 1), 364 (100%), 384 (60%, M + 23), 386 (60%).
Example 4 4- (2-Bromo-5-fluorobenzyl) -6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin- (5)
Sodium methoxide (0.4 M), freshly prepared from sodium (273 mg, 11.88 mmol) and dry methanol (30 ml), was dissolved in 30 ml of methanol, and methyl 2- (2- (2-bromo-5-fluorobenzylamino sulfide The mixture was heated to reflux for 22 h. Most of the solvent was distilled off. The residue was diluted with 100 ml of water, adjusted to pH = 1-2 with 2N concentrated hydrochloric acid, and the residue was extracted with ethyl acetate. The extract was washed with brine, dried over anhydrous sodium sulfate and concentrated by suction to give a crude product which was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 20% -30%) to give 4- (2-bromo-5-fluorobenzyl) -6-methyl-3-thioxo-3,4-dihydro- ) -one (5).
MS: m / z, 330 (65%, M + 1), 332 (60%, M + 23).
Example 5 Preparation of 4- (2-bromo-5-fluorobenzyl) -6-methyl-3- (methylthio) -1,2,4-triazin-5 (4H) preparation
A mixture of 4- (2-bromo-5-fluorobenzyl) -6-methyl-3-thioxo-3,4-dihydro- , 914 mg, 2.77 mmol) was suspended in ethanol (15 ml), followed by addition of sodium hydroxide (111 mg, 2.77 mmol) and methyl iodide (787 mg, 5.54 mmol). The reaction mixture was diluted with 100 ml of water and extracted with ethyl acetate (30 ml x 2). The combined layers were washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated by suction, and the residue was recrystallized from the residue. Silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 20-25%) afforded 4- (2-bromo-5-fluorobenzyl) -6-methyl-3- (methylthio) -l, 2,4-triazin-5 (4H) -one (6).
1 the H NMR (400MHz, of DMSO, ppm by): [delta] 7.73 (m, IH), 7.16 (br, IH), 7.05 (D, IH), 5.09 (S, 2H), 2.56 (S, 3H), 2.32 ( S, 3H).
MS: m / z, 344 (100%, M + l), 346 (100%).
Example 6 (R) -tert-Butyl 1- (4- (2-bromo-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- -triazin-3-yl) piperidine-3-carbamate (8)
A solution of 4- (2-bromo-5-fluorobenzyl) -6-methyl-3- (methylthio) -1,2,4-triazin-5 (4H) Mmol) and (R) -tert-butylpiperidine-3carbamate (7,208 mg, 1.04 mmol) for 5 min and heated to 135 ° C for 13 h under nitrogen. The reaction mixture was purified by column chromatography on silica gel (R) -tert-Butyl 1- (4- (2-bromo-5-fluorobenzyl) -6-methyl-5- Oxo-4,5-dihydro-1,2,4-triazin-3-yl) piperidine-3-carbamate (8).
MS: m / z, 496 (100%, M + l), 498 (100%).
Example 7 (R) -tert-Butyl 1- (4- (2-cyano-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- Triazin-3-yl) piperidine-3-carbamate (9)
To a mixture of sodium carbonate (53 mg, 0.50 mmol), palladium acetate (3 mg, 0.013 mmol) and N-methylpyrrolidone 0.5 ml was added 3 drops of isopropanol and 2 drops of water, and the mixture was stirred at room temperature for 5 minutes, (R) -tert-Butyl 1- (4- (2-bromo-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- – triazin-3-yl) piperidine-3-carbamate (8,246mg, 0.496mmol) in NMP (1.0mL), and heated to 140 ℃, then add the K 4 [of Fe (the CN) . 6 ] 3H · 2 O (209mg, 0.496 mmol), was heated at 140 ℃ 12h, cooled to room temperature, water was added 10ml, extracted with ethyl acetate (20mL × 2), the combined organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, (R) -tert-Butyl l- (4- (2-cyano-5- (2-fluoro-4-methoxyphenyl) Fluoro-benzyl) -6-methyl-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl) piperidine-3-carbamate (9).
MS: m / z, 418 (20%), 443 (100%, M + 1), 465 (95%, M + 23).
Example 5 Preparation of compound A (R) -2 – ((3- (3-aminopiperidin- 1 -yl) -6-methyl- -yl) methyl) -4-fluorobenzonitrile (10)
To a solution of (R) -tert-Butyl 1- (4- (2-cyano-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- Yl) piperidine-3-carbamate (9,37 mg) in 1 ml of methylene chloride was added 0.5 ml of trifluoroacetic acid and the mixture was stirred at room temperature for 1 hour, neutralized with a saturated sodium hydrogencarbonate solution, (Eluent: dichloromethane / methanol / aqueous ammonia = 92: 6: 2), in order to obtain (10ml × 3), the organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to give the crude product, which was purified by silica gel column chromatography Methyl) -5-oxo-1,2,4-triazin-4 (5H) -yl) methyl) -4-fluorobenzonitrile (10), i.e. Compound A.
1 the H NMR (400MHz, of DMSO, ppm by): [delta] 7.96 (m, IH), 7.36 (br, IH), 7.29 (D, IH), 5.23 (S, 2H), 3.15 (m, 3H), 2.72 ( 2H), 2.23 (s, 3H), 1.78 (d, 1H), 1.64 (d, 1H), 1.47 (m, 1H), 1.12 (m, 1H).
MS: m / z, 343 (100%, M + l).
Methyl-5-oxo-1,2,4-triazin-4 (5H) -yl) -2-oxoquinoline-3- Methyl) -4-fluorobenzonitrile benzoate (Compound A benzoate)
Configuration 95% ethanol solution: 500mL beaker by adding 228mL ethanol, add 12mL of water, stir well, spare.
60g of 95% ethanol, 120mL of 95% ethanol, stirring, dissolving, filtering, washing with 95% ethanol 18ml; to make the 500mL reaction flask, The ethanolic solution of benzoic acid was added dropwise at an internal temperature of 15 ° C. After completion of the dropwise addition, 95% ethanol was washed and dried under reduced pressure to constant weight to give 42.4 g of (R) -2- (3- (3-aminopiperidin-1-yl) -6-methyl- 1,2,4-triazin-4 (5H) -yl) methyl) -4-fluorobenzonitrile benzoate (the product).
Melting point determination: Instrument: Tianjin University Precision Instrument Factory YRT-3 melting point instrument.
Detection method: Take appropriate amount of this product, small study, 60 ° C, 2 hours of vacuum drying, according to the Chinese Pharmacopoeia 2010 edition two appendix Ⅵ C determination of the product melting point of 95 ℃ -115 ℃.
(5H) -benzoic acid was isolated from (R) -2- (3- (3-aminopiperidin-l- yl) -6-methyl- Methyl) -4-fluorobenzonitrile benzoate 0.1g, according to the Chinese Pharmacopoeia 2010 edition of two Appendix Ⅲ “General Identification Test” under the “benzoate” test method for testing, set 10ml volumetric flask, Add water and dilute the solvent to the mark, shake, the precise amount of 5ml to 10ml beaker, adjust the solution of phenolphthalein was neutral, drop of ferric chloride solution, were observed ocher precipitation. At the same time do blank control test, the results: multiple batches of samples of benzoic acid identification test results were positive, reagent blank does not interfere with the determination of specificity.
Identification HPLC: chromatographic conditions for the introduction of the Eclipse Plus C the Agilent 18 column (5μm, 4.6х250mm), detection wavelength of 229nm, mobile phase of acetonitrile: 0.1% phosphoric acid = 7: 3, a flow rate of 1.0ml / min, The injection volume was 20μl.
The compound A (7.5 mg) of Example 8 was dissolved in a 50 mL volumetric flask, diluted with 70% aqueous acetonitrile and diluted to the mark, shaken as a solution of the compound A reference substance; and 12.5 mg of benzoic acid in a 25 mL volumetric flask, With a volume ratio of 70% acetonitrile aqueous solution and diluted to the mark, take 1mL in 25mL volumetric flask, with volume ratio of 70% acetonitrile aqueous solution and diluted to the mark, shake, as benzoic acid reference substance solution; take this product 10mg In a 50mL volumetric flask, with a volume ratio of 70% acetonitrile aqueous solution dissolved and diluted to the mark, shake, as the product A benzoic acid salt of the test solution. Respectively, the precise amount of the reference solution and the test solution 20μl, according to high performance liquid chromatography (Chinese Pharmacopoeia 2010 edition two Appendix VD), according to the chromatographic conditions of injection, chromatogram shown in Figure 1, Method.
The results showed that the retention time of the main peak was the same as the retention time of the reference substance, and the content of compound A and benzoic acid was calculated by the peak area. The molar ratio of compound A and benzoic acid was 1: 1.
Infrared absorption spectrum identification: the United States NICOLET AVATAR 330FT-IR infrared spectrometer, in accordance with the Chinese Pharmacopoeia 2010 edition two Appendix IVC correction, take the amount of goods, using KBr tablet method for determination of the product of the infrared diffraction pattern (Figure 2 shown) to wave number cm & lt -1 , he said in 3419.75cm -1 , 2936.46cm -1 , 2230.38cm -1 , 1683.28cm -1 , 1609.47cm -1 , 1511.65cm -1 , 1419.44cm -1 , 829.18cm -1 , 722.67cm -1 characteristic absorption peak, 0.2cm error is ± -1 .

NEW PATENT

WO-2017008684

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017008684&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=FullText

Shenzhen Salubris Pharmaceuticals Co Ltd, α-Crystal form of compound A, preparation method thereof, and pharmaceutical composition comprising same

Dipeptidyl peptidase IV (DPP-IV) is a serine protease that specifically hydrolyzes the N-terminal Xaa-Pro or Xaa-Ala dipeptide of a polypeptide or protein. DPP-IV is an atypical serine protease whose Ser-Asp-His catalytic triad at the C-terminal region is different from a typical serine protease in reverse order.
DPP-IV has a variety of physiologically relevant substrates, such as inflammatory chemokines, normal T-cell expressed and secreted (RANTES), eotaxin and macrophage Cell-derived chemokines, neuropeptides such as neuropeptide Y (NPY) and P5 substances, vasoactive peptides, incretin such as glucagon-like peptide-1 (GLP-1) And glucose-dependent insulinotropic polypeptide (GIP).
Inhibition of DPP-IV in vivo resulted in increased levels of endogenous GLP-1 (7-36) and decreased production of its antagonist GLP-1 (9-36). Thus, DPP-IV inhibitors may be effective in diseases associated with DPP-IV activity such as type 2 diabetes, diabetic dyslipidemia, impaired Glucose Tolerance (IGT), impaired Fasting Plasma Glucose (IFG ), Metabolic acidosis, ketosis, appetite regulation and obesity.
DPP-IV inhibitor Alogliptin (Alogliptin) clinically for type 2 diabetes showed good therapeutic effect, approved in the United States market. Therefore, DPP-IV inhibitors are currently considered to be novel therapeutic approaches for the treatment of type 2 diabetes
PCT / CN2010 / 080370 describes a series of DPP-IV inhibitors with neo-nuclear structure. (R) -2 – ((3- (3-aminopiperidin- 1 -yl) -6-methyl-5-oxo-l, 2,4- tris piperazine -4 (5H) – yl) methyl) -4-fluorobenzonitrile (using the prior art process to obtain the product as a yellow oil), molecular formula: the C . 17 the H 19 the FN . 6 O, molecular weight: 342 chemical formula The following formula (I)
In order to improve the medicinal properties of the compound, studies with favorable stability properties can be effectively used in the treatment of patients with pathological conditions by inhibiting DPP-IV in pharmaceutical compositions.
Summary of the Invention
It is an object of the present invention to provide a stable crystalline form of a stable competitive inhibitor compound D of a reversible dipeptidyl peptidase-IV (DPP-IV).
The chemical name of compound A is: (R) -2 – ((3- (3-aminopiperidin- 1 -yl) -6-methyl-5-oxo-1,2,4-triazin- 5H) – yl) methyl) -4-fluorobenzonitrile, molecular formula: the C . 17 the H 19 the FN . 6 O, molecular weight: 342, the chemical structure of formula a compound of the following formula (the I),
compound A can be prepared according to the method disclosed in PCT / CN2010 / 080370, the specific synthesis route and the main reaction conditions are as follows:
EXAMPLE 1 Preparation of Compound A.
Compounds A were prepared according to the procedures of PCT / CN2010 / 080370 Examples 2 and 3 using the following synthetic route:
The resulting compound of the A, 1 the H-NMR (400MHz, of DMSO, ppm by): [delta] 7.96 (m, IH), 7.36 (br, IH), 7.29 (D, IH), 5.23 (S, 2H), 3.15 (m, 3H), 2.72 (m, 2H), 2.23 (s, 3H), 1.78 (d, 1H), 1.64 (d, , 343 (100%, M + l).
Specific preparation steps are as follows:
Step A. 1-bromo-4-fluoro-2- (isothiocyanatomethyl) benzene (2)
To a DMF solution (20 mL) of 1-bromo-2- (bromomethyl) -4-fluorobenzene (1,5.36 g, 20.0 mmol) was added sodium iodide (1.20 g, 8.00 mmol) and potassium thiocyanate (3.88 g, 40.0 mmol). The mixture was heated to 80 ° C under nitrogen atmosphere for 12 hours, cooled to room temperature, and 100 mL of water was added thereto. The mixture was extracted with ethyl acetate (50 mL × 2). The combined organic layers were washed with saturated brine, dried over anhydrous magnesium sulfate, The concentrate was concentrated by suction to give a crude product, and the residue was purified by silica gel column chromatography (eluent: petroleum ether) to give 1-bromo-4-fluoro-2- (isothiocyanatomethyl) benzene (2).

Step BN- (2-Bromo-5-fluorobenzyl) hydrazinocarbothioamide (3)

Dioxane solution (20 mL) of hydrazine hydrate (80%, 2.22 g, 35.5 mmol) was cooled to 0 ° C, and thereto was added 1-bromo-4-fluoro-2- (isothiocyanate Yl) benzene (2,3.16 g, 12.8 mmol) in 1,4-dioxane (5 mL). The mixture was stirred at room temperature for 2 h, and 100 mL of ice water was added thereto. The solid was precipitated, filtered, washed with water and dried over phosphorus pentoxide overnight to give N- (2-bromo-5-fluorobenzyl) hydrazinothiazepine Amide (3). MS: m / z, 278 (100%, M + 1), 280 (100%), 300 (10%, M + 23), 302 (10%).
Step C. Methyl 2- (2- (2-bromo-5-fluorobenzylaminothiocarboxamide) hydrazino) propanoate (4)
N- (2-bromo-5-fluorobenzyl) hydrazinocarbothioamide (3, 1.12 g, 4.00 mmol) was added successively to a solution of pyruvic acid (352 mg, 4.00 mmol) in methanol And the residue was extracted with ethyl acetate (150 mL). The organic layer was washed successively with water, saturated sodium hydrogencarbonate solution and saturated brine, and dried over anhydrous magnesium sulphate (MgSO4). The organic layer was washed with water, Dried and concentrated by suction filtration to give methyl 2- (2- (2-bromo-5-fluorobenzylaminothioformamide) hydrazino) propionate (4). MS: m / z, 362 (100%, M + 1), 364 (100%), 384 (60%, M + 23), 386 (60%).
Step D. 4- (2-Bromo-5-fluorobenzyl) -6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin- (4)
Sodium methoxide (0.4 M), freshly prepared from sodium (273 mg, 11.88 mmol) and dry methanol (30 mL), was dissolved in 30 mL of methanol and methyl 2- (2- (2-bromo-5-fluorobenzylamino sulfide The mixture was heated to reflux for 22 h. Most of the solvent was distilled off. The residue was diluted with 100 mL of water and the pH was adjusted to 1 to 2 with concentrated hydrochloric acid (2N). The solvent was evaporated under reduced pressure. The extract was washed with brine, dried over anhydrous sodium sulfate and concentrated by suction to give a crude product which was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 20% 4- (2-bromo-5-fluorobenzyl) -6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5 (2H ) -one (5), MS: m / z, 330 (65%, M + 1), 332 (60%, M + 23).
(4H) -one (6) & lt; EMI ID = 36.1 & gt; [0161] Step 4. 4- (2-Bromo-5-fluorobenzyl) -6 -methyl-
Methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5 (2H) -one (5,914 (111 mg, 2.77 mmol) and methyl iodide (787 mg, 5.54 mmol) were added successively to 15 mL of ethanol. The reaction mixture was diluted with 100 mL of water and extracted with ethyl acetate (30 mL × 2). The combined layers were washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated by suction filtration, and the residue was recrystallized from the residue. (2-bromo-5-fluorobenzyl) -6-methyl-3- (methylthio) – (2-bromo-5-fluorobenzyl) -2-methylbenzene was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 20-25% 1,2,4-triazine -5 (4H) – one (. 6). 1 the H NMR (400MHz, of DMSO, ppm by): [delta] 7.73 (m, IH), 7.16 (br, IH), 7.05 (D, 1H), 5.09 (s, 2H), 2.56 (s, 3H), 2.32 (s, 3H). MS: m / z, 344 (100%, M + 1), 346 (100%).
Step F. Preparation of (R) -tert-Butyl 1- (4- (2-bromo-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- – three -3-yl) piperidin-3-ylcarbamate (8)
A solution of 4- (2-bromo-5-fluorobenzyl) -6-methyl-3- (methylthio) -1,2,4-triazin-5 (4H) -one (6,180 mg, 0.523 mmol ) And (R) -tert-butylpiperidine-3-carbamate (7, 208 mg, 1.04 mmol) for 5 min and heated to 135 ° C under nitrogen for 13 h. The reaction mixture was purified by silica gel column chromatography (R) -tert-Butyl 1- (4- (2-bromo-5-fluorobenzyl) -6-methyl-5-oxo-propan-1- (8). MS: m / z, 496 (100%, M + l), 498 (M + l) (100%).
Step G. Preparation of (R) -tert-Butyl 1- (4- (2-cyano-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- – three -3-yl) piperidine-3-carbamate (9)
To a mixture of sodium carbonate (53 mg, 0.50 mmol), palladium acetate (3 mg, 0.013 mmol) and 0.5 mL of N-methylpyrrolidone was added 3 drops of isopropanol and 2 drops of water, and the mixture was stirred at room temperature for 5 minutes, (R) -tert-Butyl 1- (4- (2-bromo-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- 3-yl) piperidine-3-carbamate (8,246mg, 0.496mmol) in NMP (1.0mL), and heated to 140 ℃, then add the K 4 [of Fe (the CN) . 6 ] .3H 2 O (209 mg, 0.496 mmol), heated at 140 ° C for 12 h, cooled to room temperature, and 10 mL of water was added thereto. The mixture was extracted with ethyl acetate (20 mL × 2). The combined organic layers were washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated by suction filtration to give (R) -tert-Butyl 1- (4- (2-cyano-5-fluorobenzyl) – (2-cyano-5-fluorophenyl) -carbamic acid ethyl ester 6-methyl-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl) piperidine-3- carbamate (9). MS: m / z, 418 (20%), 443 (100%, M + 1), 465 (95%, M + 23).
Methyl-5-oxo-1,2,4-triazin-4 (5H) -ylidene-2-methyl- ) methyl ) -4-fluorobenzonitrile (10, compound A)
To a solution of (R) -tert-Butyl 1- (4- (2-cyano-5-fluorobenzyl) -6-methyl-5-oxo-4,5-dihydro- Yl) piperidine-3-carbamate (9,37 mg) in dichloromethane was added 0.5 mL of trifluoroacetic acid and the mixture was stirred at room temperature for 1 hour, neutralized with saturated sodium hydrogencarbonate solution, (Eluent: dichloromethane / methanol / aqueous ammonia = 92: 6: 2) to obtain (R (10mL × 3), the combined organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to give a crude product, which was purified by silica gel column chromatography Methyl-5-oxo-1,2,4-triazin-4 (5H) -yl) methyl) – 2- Fluorobenzonitrile (10 as a yellow oil).
1 the H NMR (400MHz, of DMSO, ppm by): [delta] 7.96 (m, IH), 7.36 (br, IH), 7.29 (D, IH), 5.23 (S, 2H), 3.15 (m, 3H), 2.72 ( (M, 2H), 2.23 (s, 3H), 1.78 (d, 1H), 1.64 (d, 1H), 1.47 , M + 1).
Patent
CN 104803972
REFERENCES
CN 104803972
CN 104803971
US 20110160212

//////////FOTAGLIPTIN BENZOATE, FOTAGLIPTIN , PHASE 1, 1403496-40-1, 1312954-58-7

N[C@@H]1CCCN(C1)C3=NN=C(C)C(=O)N3Cc2cc(F)ccc2C#N

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NEW PATENT, SUGAMMADEX, WO 2016194001


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NEW PATENT, SUGAMMADEX, WO 2016194001
WO2016194001,  PROCESSES FOR PREPARATION OF SUGAMMADEX AND INTERMEDIATES THEREOF
ALAPARTHI, Lakshmi Prasad; (IN).
PAL, Palash; (IN).
GINJUPALLI, Sadasiva Rao; (IN).
SHARMA, Uday; (IN).
CHOWDARY, Talluri Bhushaiah; (IN).
MANTRI, Anand Vijaykumar; (IN).
GADE, Bharath Reddy; (IN).
KULKARNI, Gaurav; (IN)
LINK

Sugammadex (Org 25969, Bridion) is chemically known as Cyclooctakis-(l-→4)-[6-S-(2-carboxyethyl)-6-thio-a-D-glucopyranosyl]. Sugammadex is an agent for reversal of neuromuscular blockade by the neuromuscular blocking agents (NMBAs) rocuronium, vecuronium, pancuronium in general anesthesia. It is the first selective relaxant binding agent (SRBA). SRBAs are a new class of drugs that selectively encapsulates and binds NMBAs.

The word Sugammadex is derived from Su= Sugar and Gamma cyclodex = Cyclodextrin. Sugammadex is inert chemically and does not bind to any receptor. It acts by rapidly encapsulating steroidal NMBDs to form a stable complex at a 1 : 1 ratio and thus decreasing the free concentration of the drug from the plasma. This creates a concentration gradient favoring the movement of the remaining rocuronium molecules from the neuromuscular junction back into the plasma, where they are encapsulated by free Sugammadex molecules. The latter molecules also enter the tissues and form a complex with rocuronium. Therefore, the neuromuscular blockade of rocuronium is terminated rapidly by the diffusion of rocuronium away from the neuromuscular junction back into the plasma.

NMBDs are quaternary ammonium compounds with at least one charged nitrogen atom. Cyclodextrins have a lipophilic center but a hydrophilic outer core, attributable to negatively charged ions on their surface. These negatively charged ions on the surface of Sugammadex attract the positive charges of the quaternary ammonium relaxant, drawing the drug in to the central core of the cyclodextrin. The binding of the guest molecule into the host cyclodextrin occurs because of vander waal’s forces, hydrophobic and electrostatic interactions. The structure of the cyclodextrin is such that all four hydrophobic rings of the steroidal relaxant fit tightly within the concentric doughnut forming an inclusion complex. This has been confirmed by calorimetry and X-ray crystallography. Such a reaction occurs in the plasma not at the neuromuscular junction and the concentration of free rocuronium in the plasma decrease rapidly after Sugammadex administration.

[0004] US 6670340 disclose process for preparation of Sugammadex sodium. The process as disclosed in example 4 of this patent involves reaction of iodo γ-cyclodextrin intermediate with 3-mercapto propionic acid in presence of sodium hydride and DMF to give 6-per-deoxy-6-per-(3-carboxyethyl)thio-Y-cyclodextrin, sodium salt (Sugammadex sodium). The preparation of iodo intermediate, 6-per-deoxy-6-per-iodo-y-cyclodextrin is as given in example 3 which involves reaction of γ-cyclodextrin with iodine in presence of triphenylphosphine (PPh3) and DMF. In practice, and to develop a process that has to be taken from lab scale to manufacturing scale, purity is one of the most important criteria. Since this process involves use of triphenylphosphine reagent there is formation of triphenylphosphine oxide as a by-product. Removal of triphenylphosphine oxide from the reaction mass is very difficult as it requires repeated washing with the solvent, which leads to inconsistency in yield of final product Sugammadex sodium. Furthermore, the product was dialysed for 36 hours to get pure compound. The dialysis purification is expensive and provides product in lower yield and hence such processes are not feasible and economical at industrial scale.

[0005] Another process for preparing the intermediate compound, 6-perdeoxy-6-per-chloro gamma cyclodextrin as disclosed in WO2012025937 involves use of phosphorous halide in particular, phosphorous pentachloride. WO2012025937 also disclose process for preparation of Sugammadex sodium using this intermediate which involves a) reaction of gamma-cyclodextrin with phosphorous pentachloride and dimethylformamide to obtain 6-perdeoxy-6-per-chloro gamma cyclodextrin and b) reaction of 6-perdeoxy-6-per-chloro gamma cyclodextrin with 3-mercapto propionic acid in presence of alkali metal hydrides and an organic solvent to give Sugammadex sodium. Preparation of chloro gamma cyclodextrine intermediate using phosphorous pentachloride is associated with formation of phosphorous impurities during the reaction, which are difficult to remove and also it involves tedious workup procedure.

[0006] WO2014125501 discloses preparation of 6-perdeoxy-6-per-chloro gamma cyclodextrin using phosphorous pentachloride (see example 1). The process as given in example 1 of this patent application was repeated by the present inventors. The first step provided yellow to brown mass which lacked the powder form and the flow properties. The mass was pasty at times and difficult to filter. Thus the process was unclean and tedious. Overall, no consistent product was obtained. WO2014125501 also disclose preparation of Sugammadex sodium using this intermediate which involves reaction of 6-perdeoxy-6-per-halo-gamma-cyclodextrin with 3-mercapto propionic acid in presence of alkali metal alkoxide such as sodium methoxide and organic solvent, the drawback of this this reaction is that it needs anhydrous conditions for completion of the reaction.

[0007] It has been reported that the generation of impurities and obtaining less pure compounds are major concerns with Sugammadex. Applicant Nippon Organon K.K.in their “Report on the Deliberation Results” submitted to Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, mentions as follows:

For related substances, specifications for 14 different related substances (Related Substance A, Org 48301, Related Substance B, Related Substance D, Related Substance E, Related Substance F, Related Substance G, Related Substance H, Related Substance I, Related Substance J, Related Substance K, Related Substance L, Related Substance M, Related Substance N), other individual related substances, and total related substances have been set. In the course of regulatory review, the specifications limit for 4 different related substances (Related Substance A, Related Substance D, Related Substance F, Related Substance G) have been changed based on the results of batch analyses. For related substances (degradation products), specifications for Related Substance E, Related Substance I, Related Substance C, Related Substance G, Related Substance D, Related Substance K, other individual degradation products, and total degradation products have been established. In the course of regulatory review, a specification for Impurity A which arises in *** (hidden part) step has been newly set and the specification limits for individual degradation products have been changed based on the results of batch analyses and stability studies.

The cause for change of the colour of the drug product (the light yellow-brown colour darkened) was investigated using liquid chromatography -ultraviolet-visible spectrophotometry (LC-UV/VIS) and liquid chromatography-mass spectrometry (LC-MS), which suggested that trace amounts of varieties of unspecified degradation products (unidentified), instead of a single degradation product, were involved and in addition to *** investigated in formulation development, *** and *** content of the drug substance, *** and *** during the manufacture of the drug product, and *** were considered to affect the color of the drug product. Therefore, *** and *** have been included in the drug substance specification and the relevant manufacturing process steps have been improved.

[0008] In view of the above it is clear that Sugammadex is not only prone to degradation but traces of degradation impurities affect and change the colour to yellowish brown and makes it unacceptable in quality. Therefore, it is crucial to carefully select the process to prepare pure Sugammadex sodium.

[0009] The reported purification techniques for Sugammadex sodium employ column chromatographic and membrane dialysis which are costly and not convenient in large scale operations. Therefore, the reported processes for preparation of Sugammadex sodium as discussed herein are time consuming and not economically and industrially viable.

Thus, there exist a need to provide a process of preparation of Sugammadex sodium which is simple, convenient, with easy work up procedure, economically efficient and the one which provides Sugammadex sodium in good yield and high purity.

str0

Figure 2 is 1HNMR of 6-perdeoxy-6-per-chloro gamma cyclodextrin

str0

Figure 6 is 1HNMR of Sugammadex prepared according to example 6

str0

Figure 7 is 13CNMR of Sugammadex prepared according to example 6

str0

Figure 12 is 1HNMR of Sugammadex prepared according to example 8

SEE PATENT PLEASE

Figure 13 is HPLC profile of Sugammadex prepared according to process of example 1 of WO2014125501.

scheme 1.

scheme 2.

the process for preparation of Sugammadex sodium comprising reaction of 6-perdeoxy-6-per-chloro gamma cyclodextrin (Formula II) with 3-mercaptopropionic acid in presence of alkali metal amide selected from lithium amide, sodium amide (sodamide) or potassium amide to get Sugammadex sodium.

Sugammadex Sodium

scheme 4.

the present invention provides process for preparation of Sugammadex comprising reacting the acid of Sugammadex of formula (IV) with sodium hydroxide to form Sugammadex sodium of formula (I).

Formula IV Formula I

Scheme 6

scheme 7.

scheme 8.

scheme 9.

Examples

Example 1

[0079] Preparation of 6-perdeoxy-6-per-chloro gammacyclodextrin

In a four-neck round bottomed flask (2L) equipped with mechanical stirrer, thermometer pocket in a tub charged anhydrous DMF (250ml) under nitrogen atmosphere. Triphosgene (36.5g, 0.123mol) was added to the flask at 0-15°C and the mixture was stirred for lh. Dry gamma cyclodextrin (20g, 0.015mol) was added to the obtained slurry with stirring for 30 min followed by addition of DMF (50ml). The reaction mixture was heated at 65-70°C 16 h. After the completion of reaction, the reaction mixture was cooled and diisopropyl ether (800ml) was charged to the mixture to precipitate out the material. The solvent mixture of DMF and diisopropyl ether was decanted off from the reaction mixture to obtain gummy brown mass. The reaction mass was treated with saturated sodium bicarbonate solution (800ml) which leads to precipitation of the solid. The precipitated solid was filtered, washed with the water (250x3ml) and dried. This compound was used for the next step without any purification.

Yield: 95%, HPLC Purity: 99%

Example 2

[0080] Preparation of 6-perdeoxy-6-per-chloro gamma-cyclodextrin

In a 5L four-necked flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket, oxalyl chloride (293.8g, 198.5ml, 2315mmol) was added to DMF (1200 ml) and maintained the mixture at 0-5°C under nitrogen followed by stirring at 20-25°C for lhr. A solution of gamma-cyclodextrin (lOOg, 77.16mmol) in DMF (500ml) was added to above mixture at 5-10°C under nitrogen. The mixture was stirred at 65-70°C for 14- 16 hr. After the completion of reaction, the reaction mixture was cooled to 20-25°C and diluted with diisopropyl ether (1.2L). The organic layer was decanted and the viscous residue was treated with 10% NaOH solution at 5- 10°C until PH = 8. The resulting slurry was stirred for one hour at 20-25°C. The slurry was filtered under vacuum and the solid was washed with water (3 x 500ml) and dried under vacuum. The crude material was suspended in methanol (750ml), stirred for 30min, filtered under vacuum and washed with diisopropyl ether (500ml). The solid obtained was dried at 55- 60°C in an oven for 12-16hr to afford the titled compound (95g).

Yield: 85%, Purity: 98%, melting point: 226-228°C

lH NMR (400 MHz, DMSO-d6): δ 6.0 (br s., 16 H), 4.99 (m, 8 H), 4.04 (d, J = 10 Hz, 8 H), 3.87

– 3.78 (m, 16H), 3.64 – 3.56 (m, 8 H), 3.46 – 3.34 (m, 16 H) ppm.

13C NMR (100 MHz, DMSO-d6): δ 101.98, 82.93, 72.30, 72.16, 71.11, 44.92 ppm.

Mass: m/z (M+Na)+ calcd for
1463.14; found: 1463.06.

Example 3

[0081] Preparation of 6-perdeoxy-6-per-chloro gamma-cyclodextrin

In a clean, dried 50L glass reactor equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket was charged anhydrous dimethylformamide (15L, moisture content NMT 0.4%) while maintaining the temperature at 0-5°C (using dry ice acetone bath). Oxalyl chloride (2L, 23635mmol, 30eq) was added slowly over a period 4-5hr (while maintaining the temperature below 5°C) and stirring was continued for lhr at the same temperature. A solution of dry gamma-cyclodextrin (1.0kg, 770.94mmol) dissolved in dimethylformamide (5L) was added slowly into the above reaction mixture. The solution was heated at 65-70°C for 16hr. The reaction was monitored by TLC at regular intervals. After the completion of reaction, the reaction mixture was cooled to room temperature and diisopropyl ether (10L) was added to the reaction mixture with stirring. The gummy solid precipitate out. The upper layer solvent was decanted, the gummy brown material was cooled to 0 to 5°C and was neutralized (pH 8.0) with slow addition of aqueous sodium hydroxide solution (20%, 5L) with stirring. The slurry obtained was stirred for lhr at temperature 0 to 5°C. The precipitate was filtered, washed with the water (3 x 2L) and dried under vacuum. The wet cake was suspended into methanol (10L), stirred, filtered, washed with diisopropyl ether (2L) and dried in oven at 60°C for 14-16hr to give the titled compound (980g). Yield: 87.9%, Purity: 98.1% as measured by HPLC.

Example 4

[0082] Preparation of Sugammadex sodium

In a four-neck round bottomed flask (3L) equipped with mechanical stirrer, thermometer pocket in a tub under the nitrogen atmosphere, anhydrous DMF (300ml) and 3-Mercaptopropionic acid (18.3g, 0.172mol) were charged at 0-5°C followed by addition of sodamide (20g, O.38mol). The reaction mixture was stirred at the same temperature for lh. 6-perdeoxy-6-per-chloro gamma cyclodextrin (25g, 0.017mol, as obtained in example 1) was charged slowly. The reaction mixture was heated at 90-95°C for 16h. After completion of reaction, the reaction mixture was cooled to room temperature and methanol (300ml) was added to it. The mixture was stirred and the precipitated material was filtered off. The precipitated material was dissolved in a mixture of methanol (50ml) and water (50ml) and re-precipitated with the excess addition of methanol (450ml). The solid was filtered and dried. Yield: 76%

The dried solid was purified by the preparative HPLC method using formic acid buffer in mixture of acetonitrile and water (80:20%) followed by lyophilization to get acid of Sugammadex which is further converted to Sugammadex sodium using sodium hydroxide.

Example 5

[0083] Preparation of Sugammadex sodium

In a four-neck round bottomed flask (5L) equipped with mechanical stirrer, thermometer pocket in a tub under the nitrogen atmosphere, anhydrous DMF (1500ml) and 3-mercaptopropionic acid (HOg, 1038mmol) were charged at 0-5°C followed by addition of sodamide (81g, 2077mmol). The mixture was stirred at the same temperature for lh. 6-perdeoxy-6-per-chloro gamma cyclodextrin (lOOg, 69.25mmol, as obtained in example 1) was charged slowly. Extra DMF (500ml) was added to the mixture. The temperature of the mixture was raised to 80-85°C and maintained for 16h. After completion of reaction, the reaction mixture was cooled to room temperature and methanol (1500 ml) was added to it. The mixture was stirred and the precipitated material was filtered off. The precipitated material (wet cake) was dissolved in a mixture of methanol (800ml) and water (800ml). Charcoal (50g) was added and the mixture was stirred for 30mins at 50-55°C. The solution was filtered off through a pad of celite. Methanol (2500ml) was added the solution and precipitated solid was filtered and dried furnishing the titled compound (105g). Yield: 69.6%, Purity: 85.3%.

Example 6

[0084] Preparation of Sugammadex sodium

A clean, dried 10L four neck flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket, was charged with a solution of sodium hydroxide (83g, 2077mmol) dissolved in water (100ml) followed by addition of anhydrous DMF (2L) maintained under inert atmosphere using nitrogen. A solution of 3-mercapto propionic acid (HOg, 1037mmol) in DMF (1L) was added slowly under nitrogen maintaining the temperature between 0-5°C. The mixture was stirred for another lhr at this temperature. A mixture of 6-deoxy-6-chloro gamma cyclodextrin (lOOg, 69mmol) in DMF (1L) was added slowly at 5-10°C. The resulting mixture was heated to 75-80°C for 16-20hr. After the completion of reaction, the reaction mixture was cooled to 25-30°C and methanol (1.5L) was added into the reaction mixture, the resulting precipitate was stirred at 20-25°C, filtered, and dried under vacuum. The dried solid was dissolved in water (1L), treated with activated carbon (50 g, 5%) at 50°C, stirred and filtered through celite. The filtrate was stirred at 60°C and excess methanol (2.5L) was added slowly to the filtrate to get the precipitate. The precipitated material was filtered under vacuum as white solid, washed with methanol (500ml) and dried in oven to give pure Sugammadex sodium (90 g).

Yield: 90 g, Purity: 91.2%.

lU NMR (400 MHz, D20): δ 5.09 (m, 8H); 3.98-3.94 (m, 8H); 3.88-3.83 (m, 8H); 3.58-3.52 (m, 16H); 3.07-3.01 (m, 8H); 2.92-2.87 (m, 8H); 2.78-2.74 (m, 16H); 2.34-2.47 (m, 16H) ppm.

13C NMR (100 MHz, D20): δ 180.18, 100.60, 81.96, 72.14, 71.84, 70.72, 37.24, 32.83, 29.06 ppm. Mass: m/z (M-Na7+H6)+ calcd for C72HnoNa048S8: 2023.12; found: 2023.39.

Example 7

Preparation of Sugammadex acid (Compound of formula IV)

In a clean, dried 5L four neck flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket was charged dimethylformamide (1500ml) followed by addition of potassium hydroxide (194.0 g, 3464mmol) and the mixture maintained at 0-5°C. A solution of 3-mercapto propionic acid (186.35g, 153.0ml, 1756mmol) in DMF (500ml) was added to the reactor over a period of 30 minutes under nitrogen while maintaining the temperature between 0-5°C. The

resulting mixture was stirred at this temperature for 60 minutes. A solution of 6-deoxy-6-chloro gamma cyclodextrin (lOOg, 69.22mmol) in DMF (500ml) was added to the flask. The resulting mixture was heated at 110-120°C for 1.5-2hr while monitoring the progress of the reaction through HPLC. After completion of the reaction, the temperature of the reaction mixture was brought to 40-50°C and methanol (1000ml) was added to the mixture. The resulted precipitate was stirred at 20-25°C for lhr, filtered under vacuum and washed with methanol (500ml). The wet solid was dissolved in water (2000ml) with vigorous stirring and the solution was acidified with concentrated hydrochloric acid to give the white solid precipitate. The precipitated solid was filtered and suspended in ethyl acetate (500 ml), stirred for 30 minutes and filtered. The solid was dried to afford the titled compound (75g).

Yield: 55%, Purity: 95.8% as measured by HPLC.

lH NMR (400 MHz, DMSO-d6): δ 5.94 (br. s, 16H), 3.82-3.73 (m, 8H), 3.63-3.54 (m, 8H), 3.43-3.32 (m, 16H), 3.08-3.02 (m, 8H), 2.89-2.81 (m, 8H), 2.78-2.72 (m, 16H), 2.55-2.43 (m, 16H) ppm.

13C NMR (100 MHz, DMSO-d6): δ 173.00, 102.01, 83.94, 72.45, 72.33, 71.36, 34.53, 33.08, 27.87 ppm.

Mass: m/z (M-H2+K) + calcd for C72Hno048S8K: 2039.24; found: 2039.26.

Example 8

Preparation of Sugammadex Sodium

In a clean, dried 3L four neck flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket, the compound (75g) as obtained in example 4 was dissolved in solution of sodium hydroxide (37.5g, 0.937mol) in water (100ml) and methanol (100ml). The pH of resultant mixture was maintained between 8-10. To this mixture methanol (1.5L) was slowly added at room temperature and the mixture was stirred for additional 30 minutes. The precipitated white solid was filtered off under vacuum and thoroughly washed with methanol (500ml). The solid was dried at 50°C under vacuum oven for 24hr to afford Sugammadex sodium (79g).

Yield: 96.9%, Purity: 95.5% measured by HPLC.

Citarinostat


2D chemical structure of 1316215-12-9

str0

Citarinostat

Treatment of Hematological Malignancies, 

Molecular Formula, C24-H26-Cl-N5-O3, Molecular Weight, 467.9544,
RN: 1316215-12-9
UNII: 441P620G3P

  • 2-[(2-Chlorophenyl)phenylamino]-N-[7-(hydroxyamino)-7-oxoheptyl]-5-pyrimidinecarboxamide

2-((2-Chlorophenyl)phenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)-5-pyrimidinecarboxamide

5-Pyrimidinecarboxamide, 2-((2-chlorophenyl)phenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)-

ACY-241; HDAC-IN-2

Histone deacetylase-6 inhibitor

Acute myelogenous leukemia; Cancer; Mantle cell lymphoma; Multiple myeloma

Image result for ACY 241

  • Mechanism of ActionHDAC6 protein inhibitors

Highest Development Phases

  • Phase IIMultiple myeloma
  • Phase IMalignant melanoma; Non-small cell lung cancer; Solid tumours

Most Recent Events

  • 12 Dec 2016Chemical structure information added
  • 04 Dec 2016Efficacy and safety data from a phase Ia/Ib clinical trial in Multiple myeloma released by Acetylon
  • 03 Jun 2016Phase-II clinical trials in Multiple myeloma in USA (PO)

In December 2016, citarinostat was reported to be in phase 1 clinical development. The drug appears to be first disclosed in WO2011091213, claiming reverse amide derivatives as HDAC-6 inhibitors useful for treating multiple myeloma, Alzheimers disease and psoriasis.

HDAC-IN-2.png

Duzer John H. Van, Ralph Mazitschek, Walter Ogier, James Elliott Bradner, Guoxiang Huang, Dejian Xie, Nan Yu, Less «
Applicant Acetylon Pharmaceuticals

The identification of small organic molecules that affect specific biological functions is an endeavor that impacts both biology and medicine. Such molecules are useful as therapeutic agents and as probes of biological function. Such small molecules have been useful at elucidating signal transduction pathways by acting as chemical protein knockouts, thereby causing a loss of protein function. (Schreiber et al, J. Am. Chem. Soc, 1990, 112, 5583; Mitchison, Chem. and Biol., 1994, 15 3) Additionally, due to the interaction of these small molecules with particular biological targets and their ability to affect specific biological function (e.g. gene transcription), they may also serve as candidates for the development of new therapeutics.

One biological target of recent interest is histone deacetylase (HDAC) (see, for example, a discussion of the use of inhibitors of histone deacetylases for the treatment of cancer: Marks et al. Nature Reviews Cancer 2001, 7,194; Johnstone et al. Nature Reviews Drug Discovery 2002, 287). Post-translational modification of proteins through acetylation and deacetylation of lysine residues plays a critical role in regulating their cellular functions. HDACs are zinc hydrolases that modulate gene expression through deacetylation of the N-acetyl-lysine residues of histone proteins and other transcriptional regulators (Hassig et al Curr. Opin. Chem. Biol. 1997, 1, 300-308). HDACs participate in cellular pathways that control cell shape and differentiation, and an HDAC inhibitor has been shown effective in treating an otherwise recalcitrant cancer (Warrell et al J. Natl. Cancer Inst. 1998, 90, 1621-1625). At this time, eleven human HDACs, which use Zn as a cofactor, have been identified (Taunton et al. Science 1996, 272, 408-411 ; Yang et al. J. Biol. Chem. 1997, 272, 28001-28007. Grozinger et al. Proc. Natl. Acad. Sd. U.S.A. 1999, 96, 4868-4873; Kao et al. Genes Dev. 2000, 14, 55-66. Hu et al J. Biol. Chem. 2000, 275, 15254-15264; Zhou et al. Proc. Natl. Acad. Scl U.S.A. 2001, 98, 10572-10577; Venter et al. Science 2001, 291, 1304-1351) these members fall into three classes (class I, II, and IV). An additional seven HDACs h ave been identified which use NAD as a cofactor. To date, no small molecules are known that selectively target any particular class or individual members of this family ((for example ortholog- selective HDAC inhibitors have been reported: (a) Meinke et al. J. Med. Chem. 2000, 14, 4919-4922; (b) Meinke, et al Curr. Med. Chem. 2001, 8, 211-235). There remains a need for preparing structurally diverse HDAC and tubulin deacetylase (TDAC) inhibitors particularly ones that are potent and/or selective inhibitors of particular classes of HDACs or TDACs and individual HDACs and TDACs.

Recently, a cytoplasmic histone deacetylase protein, HDAC6, was identified as necessary for aggresome formation and for survival of cells following ubiquitinated misfolded protein stress. The aggresome is an integral component of survival in cancer cells. The mechanism of HDAC6-mediated aggresome formation is a consequence of the catalytic activity of the carboxy-terminal deacetylase domain, targeting an uncharacterized non-histone target. The present invention also provides small molecule inhibitors of HDAC6. In certain embodiments, these new compounds are potent and selective inhibitors of HDAC6.

The aggresome was first described in 1998, when it was reported that there was an appearance of microtubule-associated perinuclear inclusion bodies in cells over- expressing the pathologic AF508 allele of the cystic fibrosis transmembrane conductance receptor (CFTR). Subsequent reports identified a pathologic appearance of the aggresome with over-expressed presenilin-1 (Johnston JA, et al. J Cell Biol. 1998;143:1883-1898), parkin (Junn E, et al. J Biol Chem. 2002; 277: 47870-47877), peripheral myelin protein PMP22 (Notterpek L, et al. Neurobiol Dis. 1999; 6: 450-460), influenza virus nucleoprotein (Anton LC, et al. J Cell Biol. 1999;146:113-124), a chimera of GFP and the membrane transport protein pi 15 (Garcia- Mata R, et al. J Cell Biol. 1999; 146: 1239-1254) and notably amyloidogenic light chains (Dul JL, et al. J Cell Biol. 2001;152:705-716). Model systems have been established to study ubiquitinated (AF508 CFTR) (Johnston JA, et al. J Cell Biol. 1998;143:1883-1898) and non-ubiquitinated (GFP -250) (Garcia-Mata R, et al. J Cell Biol. 1999;146:1239-1254) protein aggregate transport to the aggresome. Secretory, mutated, and wild-type proteins may assume unstable kinetic intermediates resulting in stable aggregates incapable of degradation through the narrow channel of the 26S proteasome. These complexes undergo active, retrograde transport by dynein to the pericentriolar aggresome, mediated in part by a cytoplasmic histone deacetylase, HDAC6 (Kawaguchi Y, et al. Cell. 2003;1 15:727-738).

Histone deacetylases are a family of at least 11 zinc -binding hydrolases, which

catalyze the deacetylation of lysine residues on histone proteins. HDAC inhibition results in hyperacetylation of chromatin, alterations in transcription, growth arrest, and apoptosis in cancer cell lines. Early phase clinical trials with available nonselective HDAC inhibitors demonstrate responses in hematologic malignancies including multiple myeloma, although with significant toxicity. Of note, in vitro synergy of conventional chemotherapy agents (such as melphalan) with bortezomib has been reported in myeloma cell lines, though dual proteasome-aggresome inhibition was not proposed. Until recently selective HDAC inhibitors have not been realized.

HDAC6 is required for aggresome formation with ubiquitinated protein stress and is essential for cellular viability in this context. HDAC6 is believed to bind ubiquitinated proteins through a zinc finger domain and interacts with the dynein motor complex through another discrete binding motif. HDAC6 possesses two catalytic deacetylase domains. It is not presently known whether the amino-terminal histone deacetylase or the carboxy-terminal tubulin deacetylase (TDAC) domain mediates aggresome formation.

Aberrant protein catabolism is a hallmark of cancer, and is implicated in the stabilization of oncogenic proteins and the degradation of tumor suppressors (Adams J. Nat Rev Cancer. 2004;4:349-360). Tumor necrosis factor alpha induced activation of nuclear factor kappa B (NFKB) is a relevant example, mediated by NFKB inhibitor beta (1KB) proteolytic degradation in malignant plasma cells. The inhibition of 1KB catabolism by proteasome inhibitors explains, in part, the apoptotic growth arrest of treated myeloma cells (Hideshima T, et al. Cancer Res. 2001;61:3071-3076). Multiple myeloma is an ideal system for studying the mechanisms of protein degradation in cancer. Since William Russell in 1890, cytoplasmic inclusions have been regarded as a defining histological feature of malignant plasma cells. Though the precise composition of Russell bodies is not known, they are regarded as ER-derived vesicles containing aggregates of monotypic immunoglobulins

(Kopito RR, Sitia R. EMBO Rep. 2000; 1 :225-231) and stain positive for ubiquitin (Manetto V, et al. Am J Pathol. 1989;134:505-513). Russell bodies have been described with CFTR over-expression in yeast (Sullivan ML, et al. J. Histochem. Cytochem. 2003;51 :545-548), thus raising the suspicion that these structures may be linked to overwhelmed protein catabolism, and potentially the aggresome. The role of the aggresome in cancer remains undefined.

Aberrant histone deacetylase activity has also been linked to various neurological and neurodegenerative disorders, including stroke, Huntington’s disease, Amyotrophic Lateral Sclerosis and Alzheimer’s disease. HDAC inhibition may induce the expression of antimitotic and anti-apoptotic genes, such as p21 and HSP-70, which facilitate survival. HDAC inhibitors can act on other neural cell types in the central nervous system, such as reactive astrocytes and microglia, to reduce inflammation and secondary damage during neuronal injury or disease. HDAC inhibition is a promising therapeutic approach for the treatment of a range of central nervous system disorders (Langley B et al., 2005, Current Drug Targets— CNS & Neurological Disorders, 4: 41-50).

Histone deacetylase is known to play an essential role in the transcriptional machinery for regulating gene expression, induce histone hyperacetylation and to affect the gene expression. Therefore, it is useful as a therapeutic or prophylactic agent for diseases caused by abnormal gene expression such as inflammatory disorders, diabetes, diabetic

complications, homozygous thalassemia, fibrosis, cirrhosis, acute promyelocytic leukaemia (APL), organ transplant rejections, autoimmune diseases, protozoal infections, tumors, etc.

Thus, there remains a need for the development of novel inhibitors of histone deacetylases and tubulin histone deacetylases. In particular, inhibitors that are more potent and/or more specific for their particular target than known HDAC and TDAC inhibitors. HDAC inhibitors specific for a certain class or member of the HDAC family would be particularly useful both in the treatment of proliferative diseases and protein deposition disorders and in the study of HDACs, particularly HDAC6. Inhibitors that are specific for HDAC versus TDAC and vice versa are also useful in treating disease and probing biological pathways. The present invention provides novel compounds, pharmaceutical compositions thereof, and methods of using these compounds to treat disorders related to HDAC6 including cancers, inflammatory, autoimmune, neurological and neurodegenerative disorders

Image result for ACY 241

Rocilinostat (ACY-1215)

Image result for ACY 241

PATENT

WO2011091213

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011091213

Patent

US20160355486

WO 2013013113

WO 2015061684

WO 2015054474

US 20150099744

PATENT

CITARINOSTAT BY ACTYLON

WO-2016200919

Crystalline forms of a histone deacetylase inhibitor

Novel crystalline polymorphic forms of citarinostat, useful for treating cancer, eg multiple myeloma, mantle cell lymphoma or acute myelogenous leukemia. Also claims a method for preparing the crystalline form of citarinostat. Acetylon is developing citarinostat, a next generation selective inhibitor of HDAC6, for treating multiple myeloma and solid tumors, including melanoma.

Provided herein are crystalline forms of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide (CAS No. 1316215-12-9), shown as Compound (I) (and referred to herein as “Compound (I)”):

Compound (I) is disclosed in International Patent Application No.

PCT/US2011/021982 and U.S. Patent No. 8,609,678, the entire contents of which are incorporated herein by reference.

Accordingly, provided herein are crystalline forms of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide. In particular, provided herein are the following crystalline forms of Compound (I): Form I, Form II, Form III, Form IV, Form V, Form VI, Form VII, Form VIII, and Form IX. Each of these forms have been characterized by XRPD analysis. In an embodiment, the crystalline form of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide can be a hydrate or solvate (e.g., dichloromethane or methanol).

EXAMPLES

Example 1: Synthesis of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7- oxoheptyl)pyrimidine-5-carboxamide (Compound (I))

I. Synthesis of 2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide:

Synthesis of Intermediate 2: A mixture of aniline (3.7 g, 40 mmol), compound 1 (7.5 g, 40 mmol), and K2C03 (11 g, 80 mmol) in DMF (100 ml) was degassed and stirred at 120 °C under N2 overnight. The reaction mixture was cooled to r.t. and diluted with EtOAc (200 ml), then washed with saturated brine (200 ml χ 3). The organic layers were separated and dried over Na2S04, evaporated to dryness and purified by silica gel chromatography (petroleum ethers/EtOAc = 10/1) to give the desired product as a white solid (6.2 g, 64 %).

Synthesis of Intermediate 3: A mixture of compound 2 (6.2 g, 25 mmol), iodobenzene (6.12 g, 30 mmol), Cul (955 mg, 5.0 mmol), Cs2C03 (16.3 g, 50 mmol) in TEOS (200 ml) was degassed and purged with nitrogen. The resulting mixture was stirred at 140 °C for 14 hrs. After cooling to r.t., the residue was diluted with EtOAc (200 ml). 95% EtOH (200 ml) and H4F-H20 on silica gel [50g, pre-prepared by the addition of H4F (lOOg) in water (1500 ml) to silica gel (500g, 100-200 mesh)] was added, and the resulting mixture was kept at r.t. for 2 hrs. The solidified materials were filtered and washed with EtOAc. The filtrate was evaporated to dryness and the residue was purified by silica gel chromatography (petroleum ethers/EtOAc = 10/1) to give a yellow solid (3 g, 38%).

Synthesis of Intermediate 4: 2N NaOH (200 ml) was added to a solution of compound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture was stirred at 60 °C for 30min. After evaporation of the solvent, the solution was neutralized with 2N HCl to give a white precipitate. The suspension was extracted with EtOAc (2 χ 200 ml), and the organic layers were separated, washed with water (2 χ 100 ml), brine (2 χ 100 ml), and dried over Na2S04. Removal of the solvent gave a brown solid (2.5 g, 92 %).

Synthesis of Intermediate 6: A mixture of compound 4 (2.5 g, 8.58 mmol), compound 5 (2.52 g, 12.87 mmol), HATU (3.91 g, 10.30 mmol), and DIPEA (4.43 g, 34.32 mmol) was stirred at r.t. overnight. After the reaction mixture was filtered, the filtrate was evaporated to dryness and the residue was purified by silica gel chromatography (petroleum ethers/EtOAc = 2/1) to give a brown solid (2 g, 54 %).

Synthesis of 2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide: A mixture of the compound 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20 mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0 °C for 10 min. Hydroxylamine (50%) (10 ml) was cooled to 0 °C and added to the mixture. The resulting mixture was stirred at r.t. for 20 min. After removal of the solvent, the mixture was neutralized with 1M HCl to give a white precipitate. The crude product was filtered and purified by pre-HPLC to give a white solid (950 mg, 48%).

II. Synthetic Route 1 : 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptvDpyrimidine-5-carboxamide

Synthesis of Intermediate 2: A mixture of aniline (3.7 g, 40 mmol), ethyl 2-chloropyrimidine-5-carboxylate 1 (7.5 g, 40 mmol), K2C03 (11 g, 80 mmol) in DMF (100 ml) was degassed and stirred at 120 °C under N2 overnight. The reaction mixture was cooled to rt and diluted with EtOAc (200 ml), then washed with saturated brine (200 ml x 3). The organic layer was separated and dried over Na2S04, evaporated to dryness and purified by silica gel

chromatography (petroleum ethers/EtOAc = 10/1) to give the desired product as a white solid (6.2 g, 64 %).

Synthesis of Intermediate 3: A mixture of compound 2 (69.2 g, 1 equiv.), l-chloro-2-iodobenzene (135.7 g, 2 equiv.), Li2C03 (42.04 g, 2 equiv.), K2C03 (39.32 g, 1 equiv.), Cu (1 equiv. 45 μπι) in DMSO (690 ml) was degassed and purged with nitrogen. The resulting mixture was stirred at 140 °C for 36 hours. Work-up of the reaction gave compound 3 at 93 % yield.

Synthesis of Intermediate 4: 2N NaOH (200 ml) was added to a solution of the compound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture was stirred at 60 °C for 30min. After evaporation of the solvent, the solution was neutralized with 2N HC1 to give a white precipitate. The suspension was extracted with EtOAc (2 x 200 ml), and the organic layer was separated, washed with water (2 x 100 ml), brine (2 x 100 ml), and dried over Na2S04. Removal of solvent gave a brown solid (2.5 g, 92 %).

Synthesis of Intermediate 5: A procedure analogous to the Synthesis of Intermediate 6 in Part I of this Example was used.

Synthesis of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide: A procedure analogous to the Synthesis of 2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide in Part I of this Example was used.

III. Synthetic Route 2: 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide

(I)

Step (1): Synthesis of Compound 11: Ethyl 2-chloropyrimidine-5-carboxylate (7.0 Kgs), ethanol (60 Kgs), 2-Chloroaniline (9.5 Kgs, 2 eq) and acetic acid (3.7 Kgs, 1.6 eq) were charged to a reactor under inert atmosphere. The mixture was heated to reflux. After at least 5 hours the reaction was sampled for HPLC analysis (method TM-113.1016). When analysis indicated reaction completion, the mixture was cooled to 70 ± 5 °C and N,N-Diisopropylethylamine (DIPEA) was added. The reaction was then cooled to 20 ± 5°C and the mixture was stirred for an additional 2-6 hours. The resulting precipitate is filtered and washed with ethanol (2 x 6 Kgs) and heptane (24 Kgs). The cake is dried under reduced pressure at 50 ± 5 °C to a constant weight to produce 8.4 Kgs compound 11 (81% yield and 99.9% purity.

Step (2): Synthesis of Compound 3: Copper powder (0.68 Kgs, 1 eq, <75 micron), potassium carbonate (4.3 Kgs, 1.7 eq), and dimethyl sulfoxide (DMSO, 12.3 Kgs) were added to a reactor (vessel A). The resulting solution was heated to 120 ± 5°C. In a separate reactor (vessel B), a solution of compound 11 (2.9 Kgs) and iodobenzene (4.3 Kgs, 2 eq) in DMSO (5.6 Kgs) was heated at 40 ± 5°C. The mixture was then transferred to vessel A over 2-3 hours. The reaction mixture was heated at 120 ± 5°C for 8-24 hours, until HPLC analysis (method TM-113.942) determined that < 1% compound 11 was remaining.

Step (3): Synthesis of Compound 4: The mixture of Step (2) was cooled to 90-100 °C and purified water (59 Kgs) was added. The reaction mixture was stirred at 90-100 °C for 2-8 hours until HPLC showed that <1% compound 3 was remaining. The reactor was cooled to 25 °C. The reaction mixture was filtered through Celite, then a 0.2 micron filter, and the filtrate was collected. The filtrate was extracted with methyl t-butyl ether twice (2 x 12.8 Kgs). The aqueous layer was cooled to 0-5 °C, then acidified with 6N hydrochloric acid (HC1) to pH 2-3 while keeping the temperature < 25°C. The reaction was then cooled to 5-15 °C. The precipitate was filtered and washed with cold water. The cake was dried at 45-55 °C under reduced pressure to constant weight to obtain 2.2 kg (65% yield) compound 4 in 90.3% AUC purity.

Step (4): Synthesis of Compound 5: Dichloromethane (40.3 Kgs), DMF (33g, 0.04 eq) and compound 4 (2.3 Kg) were charged to a reaction flask. The solution was filtered through a 0.2 μπι filter and was returned to the flask. Oxalyl chloride (0.9 Kgs, 1 eq) was added via addition funnel over 30-120 minutes at < 30 °C. The batch was then stirred at < 30°C until reaction completion (compound 4 ❤ %) was confirmed by HPLC (method TM-113.946. Next, the dichloromethane solution was concentrated and residual oxalyl chloride was removed under reduced pressure at < 40 °C. When HPLC analysis indicated that < 0.10% oxalyl chloride was remaining, the concentrate was dissolved in fresh dichloromethane (24 Kgs) and transferred back to the reaction vessel (Vessel A).

A second vessel (Vessel B) was charged with Methyl 7-aminoheptanoate

hydrochloride (Compound Al, 1.5 Kgs, 1.09 eq), DIPEA (2.5 Kgs, 2.7 eq), 4

(Dimethylamino)pyridine (DMAP, 42g, 0.05 eq), and DCM (47.6 Kgs). The mixture was cooled to 0-10 °C and the acid chloride solution in Vessel A was transferred to Vessel B while maintaining the temperature at 5 °C to 10 °C. The reaction is stirred at 5-10 °C for 3 to 24 hours at which point HPLC analysis indicated reaction completion (method TM-113.946, compound 4 <5%). The mixture was then extracted with a 1M HC1 solution (20 Kgs), purified water (20 Kgs), 7% sodium bicarbonate (20 Kgs), purified water (20 Kgs), and 25% sodium chloride solution (20 Kgs). The dichloromethane was then vacuumdistilled at < 40 °C and chased repeatedly with isopropyl alcohol. When analysis indicated that <1 mol% DCM was remaining, the mixture was gradually cooled to 0-5 °C and was stirred at 0-5 °C for an at least 2 hours. The resulting precipitate was collected by filtration and washed with cold isopropyl alcohol (6.4 Kgs). The cake was sucked dry on the filter for 4-24 hours, then was further dried at 45-55 °C under reduced pressure to constant weight. 2.2 Kgs (77% yield) was isolated in 95.9% AUC purity method and 99.9 wt %.

Step (5): Synthesis of Compound (I): Hydroxylamine hydrochloride (3.3 Kgs, 10 eq) and methanol (9.6 Kgs) were charged to a reactor. The resulting solution was cooled to 0-5 °C and 25% sodium methoxide (11.2 Kgs, 11 eq) was charged slowly, maintaining the temperature at 0-10 °C. Once the addition was complete, the reaction was mixed at 20 °C for 1-3 hours and filtered, and the filter cake was washed with methanol (2 x 2.1 Kgs). The filtrate (hydroxylamine free base) was returned to the reactor and cooled to 0±5°C.

Compound 5 (2.2 Kgs) was added. The reaction was stirred until the reaction was complete (method TM-113.964, compound 5 < 2%). The mixture was filtered and water (28 Kgs) and ethyl acetate (8.9 Kgs) were added to the filtrate. The pH was adjusted to 8 – 9 using 6N HC1 then stirred for up to 3 hours before filtering. The filter cake was washed with cold water (25.7 Kgs), then dried under reduced pressure to constant weight. The crude solid compound (I) was determined to be Form IV/ Pattern D.

The crude solid (1.87 Kgs) was suspended in isopropyl alcohol (IP A, 27.1 Kg). The slurry was heated to 75±5 °C to dissolve the solids. The solution was seeded with crystals of Compound (I) (Form I/Pattern A), and was allowed to cool to ambient temperature. The resulting precipitate was stirred for 1-2 hours before filtering. The filter cake was rinsed with IPA (2 x 9.5 Kgs), then dried at 45-55°C to constant weight under reduced pressure to result in 1.86 kg crystalline white solid Compound (I) (Form I/Pattern A) in 85% yield and 99.5% purity (AUC%, HPLC method TM-113.941).

HPLC Method 113.941

Column Zorbax Eclipse XDB-C18, 4.6 mm x 150 mm, 3.5 μπι

Column Temperature 40°C

UV Detection Wavelength Bandwidth 4 nm, Reference off, 272 nm

Flow rate 1.0 mL/min

Injection Volume 10 μΐ. with needle wash

Mobile Phase A 0.05% trifluoroacetic acid (TFA) in purified water

Mobile Phase B 0.04% TFA in acetonitrile

Data Collection 40.0 min

Run Time 46.0 min

Gradient Time (min) Mobile Phase A Mobile Phase B

0.0 98% 2%

36.0 0% 100%

40.0 0% 100%

40.1 98% 2%

46.0 98% 2%

Example 2: Summary of Results and Analytical Techniques

Table 1. Summary of the Isolated Crystalline Forms of Compound (I)

Patent ID Patent Title Submitted Date Granted Date
US2016030458 TREATMENT OF LEUKEMIA WITH HISTONE DEACETYLASE INHIBITORS 2015-07-06 2016-02-04
US2015176076 HISTONE DEACETYLASE 6 (HDAC6) BIOMARKERS IN MULTIPLE MYELOMA 2014-12-19 2015-06-25
US2015150871 COMBINATIONS OF HISTONE DEACETYLASE INHIBITORS AND IMMUNOMODULATORY DRUGS 2014-12-03 2015-06-04
US2015119413 TREATMENT OF POLYCYSTIC DISEASES WITH AN HDAC6 INHIBITOR 2014-10-24 2015-04-30
US2015105358 COMBINATIONS OF HISTONE DEACETYLASE INHIBITORS AND IMMUNOMODULATORY DRUGS 2014-10-07 2015-04-16
US2015105383 HDAC Inhibitors, Alone Or In Combination With PI3K Inhibitors, For Treating Non-Hodgkin’s Lymphoma 2014-10-08 2015-04-16
US2015105384 PYRIMIDINE HYDROXY AMIDE COMPOUNDS AS HISTONE DEACETYLASE INHIBITORS 2014-10-09 2015-04-16
US2015105409 HDAC INHIBITORS, ALONE OR IN COMBINATION WITH BTK INHIBITORS, FOR TREATING NONHODGKIN’S LYMPHOMA 2014-10-07 2015-04-16
US2015099744 COMBINATIONS OF HISTONE DEACETYLASE INHIBITORS AND EITHER HER2 INHIBITORS OR PI3K INHIBITORS 2014-10-06 2015-04-09
US2015045380 REVERSE AMIDE COMPOUNDS AS PROTEIN DEACETYLASE INHIBITORS AND METHODS OF USE THEREOF 2014-10-22 2015-02-12
Patent ID Patent Title Submitted Date Granted Date
US2014378385 Histone Deacetylase 6 Selective Inhibitors for the Treatment of Bone Disease 2012-07-20 2014-12-25
US2014142117 REVERSE AMIDE COMPOUNDS AS PROTEIN DEACETYLASE INHIBITORS AND METHODS OF USE THEREOF 2013-11-11 2014-05-22
US8609678 Reverse amide compounds as protein deacetylase inhibitors and methods of use thereof 2012-04-02 2013-12-17
US8148526 Reverse amide compounds as protein deacetylase inhibitors and methods of use thereof 2011-12-02 2012-04-03
US2011300134 REVERSE AMIDE COMPOUNDS AS PROTEIN DEACETYLASE INHIBITORS AND METHODS OF USE THEREOF 2011-12-08

Acetylon Crafts New Buyout Deal With Celgene, Spins Out Startup Regenacy

Acetylon Crafts New Buyout Deal With Celgene, Spins Out Startup Regenacy

In the deal, Summit, NJ-based Celgene (NASDAQ: CELG) will get partial rights to two drug candidates developed by Acetylon: citarinostat (also known as ACY-241), and ricolinostat (ACY-1215). Specifically, Celgene will get worldwide rights to develop both drugs for cancer, neurodegenerative diseases, and autoimmune diseases, but nothing else.

Regenacy meanwhile, will also have partial rights to these two drugs, but only for other disease types, such as nerve pain. It also gets access to other preclinical drugs Acetylon has been developing for blood diseases like sickle cell disease and beta-thalassemia.

[Updated w/comments from CEO] Acetylon CEO Walter Ogier—who will be the president and CEO of Regenacy—said via e-mail that Celgene was only interested in the parts of Acetylon that fit with its current portfolio. Acetylon’s shareholders and executives, meanwhile, wanted to push the rest of the company’s experimental products forward. So the two companies let the original deal expire and came up with the new transaction.

“The remaining assets are exciting enough to create a new company to advance,” Ogier said.

Other “key members” of Acetylon’s executive team will switch over to the new company as well, according to the announcement. Ogier said Regenacy has acquired Acetylon’s remaining cash in the deal—he didn’t say how much—to get itself started.

Both citarinostat and ricolinostat interfere with what are known as histone deacetylases (HDACs), enzymes that help regulate gene expression and are implicated in a number of cancers. HDACs are a well-known molecular target, but Acetylon’s drugs are part of a newer breed of HDAC-blocking agents meant to be more precise, and thus less toxic, than their predecessors. Acetylon’s lead drug ricolinostat, for instance, is meant to block only the specific enzyme HDAC6. Citarinostat is a pill version of ricolinostat,

With Celgene’s help, Acetylon has been developing these drugs as potential treatments for breast cancer and the blood cancer multiple myeloma. It has been testing the drug in combination with Celgene’s own experimental drugs, like the myeloma drug pomalidomide (Pomalyst) and the breast cancer drug nab-paclitaxel (Abraxane).

[Updated w/CEO comments] Citarinostat, for instance, is being tested as a multiple myeloma treatment in a Phase 1b trial in combination with pomalidamide and dexamethasome in multiple myeloma. Acetylon and Celgene just reported early data at the American Society of Hematology’s annual meeting. Ricolinostat is in a mid-stage study in multiple myeloma as well as several investigator-sponsored studies in lymphoma, chronic lymphocytic leukemia, and ovarian and breast cancer, according to Ogier.

Regenacy will take ricolinostat into a Phase 2 trial in peripheral neuropathy next year, he says.

The two companies aren’t disclosing the terms of the deal. Co-founder and chairman Marc Cohen said in a statement that the deal is a “favorable outcome” for Acetylon’s shareholders—an unusual mix of private financiers, non-profits, public companies, and federal grant sources including Celgene itself, Kraft Group (the holding company founded by New England Patriots owner Robert Kraft), Cohen, and the Leukemia & Lymphoma Society. (All of those shareholders aside from Celgene will be the owners of Regenacy.)

But it’s a different outcome than Acetylon and Celgene anticipated when they signed a broad deal in 2013. At that time, Celgene paid Acetylon $100 million for the option to buy it outright for at least an additional $500 million (the actual price was to be tied to an independent valuation). The deal included another $1.1 billion in “bio-bucks,” future payments tied to clinical progress that may or may not materialize. All told, that meant the Celgene deal could have been worth $1.7 billion to Acetylon and its shareholders. Acetylon raised $55 million from shareholders before it struck that deal with Celgene.

Celgene extended its partnership with Acetylon in the summer of 2015, but that included a contingency that the relationship would end in May 2016 if it didn’t buy Acetylon. A regulatory filing in July showed that’s exactly what happened: the collaboration between the two companies ended this year, and that Celgene was no longer on the hook for any future payments related to 2013 deal.

Though that deal is now history, Acetylon shareholders were at least able to generate some type of return—and take another shot on some of the same assets. Ogier said these shareholders have “ample capacity” to make further investments in Regenacy, though the company will try to find new partners to help move its programs forward as well.

“We are excited to continue Acetylon’s legacy through the receipt of rights to many of Acetylon’s most promising compounds and the continued advancement of these clinical and preclinical programs in disease indications outside of Celgene’s areas of strategic focus, where we believe patients may especially benefit from selective HDAC inhibition,” he said in a statement.

REFERENCES

http://www.acetylon.com/docs/ACE-MM-200_Poster_Final%20Draft.pdf

References:
[1].  Quayle SN, Almeciga-Pinto I, Tamang D, et al. Selective HDAC inhibition by ricolinostat (ACY-1215) or ACY-241 synergizes with IMiD® immunomodulatory drugs in Multiple Myeloma (MM) and Mantle Cell Lymphoma (MCL) cells. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research, 2015, Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5380.
[2].  Huang P, Almeciga-Pinto I, Jordan M, et al. Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor models. In: Proceedings of the 2015 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, Massachusetts. Philadelphia (PA): AACR

NMR

str0

HPLC

str0

////////////ACY-241,  HDAC-IN-2, PHASE 1, CITARINOSTAT, 1316215-12-9

ONC(=O)CCCCCCNC(=O)c1cnc(nc1)N(c2ccccc2)c3ccccc3Cl

 

update……….

Image result for Acetylon Pharmaceuticals Inc

WO 2016200930, New patent, Citarinostat, Acetylon Pharmaceuticals Inc

citarinostat

Acetylon Pharmaceuticals Inc

(WO2016200930) METHODS OF MAKING PROTEIN DEACETYLASE INHIBITORS

(I)

Compound (I) is disclosed in U.S. Patent No. 8,148,526 as an HDAC inhibitor.

Example 2 of U.S. Patent Application Publication No. 2015/0099744 discloses a synthesis of compound (I). As detailed herein in Example 3, this synthesis procedure resulted in the formation of significant amounts of de-chlorination and chlorine-migration side products. These impurities have solubilities that are similar to the solubilities of the desired

intermediates. Removal of the impurities is very challenging, requiring lengthy work-ups, involving numerous washes, triturations and crystallizations. Triturations, in particular, are known to be inefficient and unscalable processes. When compound (I) was prepared according to Example 2, the necessary purification steps resulted in a significant loss of desired intermdiates, led to a modest overall yield, and rendered further industrial scale up of the synthesis route unpractical. There remains a need for new methods for the synthesis of compound (I), and related compounds, that minimize the formation of impurities, and that are amenable to industrial scale-up.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a generic synthesis of compound (I) according to the improved method described herein.

Figure 2 depicts a specific synthesis of compound (I) according to the improved method described herein.

Figure 6 depicts 1HNMR data for compound (I).

str1 str2 str3

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Acetylon president and CEO Walter Ogier

Example 1: Comparative Synthesis of 2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl) pyrimidine-5-carboxamide

Reaction Scheme

Synthesis of Intermediate 2: A mixture of aniline (3.7 g, 40 mmol), compound 1 (7.5 g, 40 mmol), and K2C03 (11 g, 80 mmol) in DMF (100 ml) was degassed and stirred at 120 °C under N2 overnight. The reaction mixture was cooled to r.t. and diluted with EtOAc (200 ml), then washed with saturated brine (200 ml χ 3). The organic layers were separated and dried over Na2S04, evaporated to dryness and purified by silica gel chromatography (petroleum ethers/EtOAc = 10/1) to give the desired product as a white solid (6.2 g, 64 %).

Synthesis of Intermediate 3: A mixture of compound 2 (6.2 g, 25 mmol), iodobenzene (6.12 g, 30 mmol), Cul (955 mg, 5.0 mmol), Cs2C03 (16.3 g, 50 mmol) in TEOS (200 ml) was degassed and purged with nitrogen. The resulting mixture was stirred at 140 °C for 14 hrs.

After cooling to r.t., the residue was diluted with EtOAc (200 ml). 95% EtOH (200 ml) and H4F-H20 on silica gel [50g, pre-prepared by the addition of H4F (lOOg) in water (1500 ml) to silica gel (500g, 100-200 mesh)] was added, and the resulting mixture was kept at r.t. for 2 hrs. The solidified materials were filtered and washed with EtOAc. The filtrate was evaporated to dryness and the residue was purified by silica gel chromatography (petroleum ethers/EtOAc = 10/1) to give a yellow solid (3 g, 38%).

Synthesis of Intermediate 4: 2N NaOH (200 ml) was added to a solution of compound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture was stirred at 60 °C for 30min. After evaporation of the solvent, the solution was neutralized with 2N HCl to give a white precipitate. The suspension was extracted with EtOAc (2 χ 200 ml), and the organic layers were separated, washed with water (2 χ 100 ml), brine (2 χ 100 ml), and dried over Na2S04. Removal of the solvent gave a brown solid (2.5 g, 92 %).

Synthesis of Intermediate 6: A mixture of compound 4 (2.5 g, 8.58 mmol), compound 5 (2.52 g, 12.87 mmol), HATU (3.91 g, 10.30 mmol), and DIPEA (4.43 g, 34.32 mmol) was stirred at r.t. overnight. After the reaction mixture was filtered, the filtrate was evaporated to dryness and the residue was purified by silica gel chromatography (petroleum ethers/EtOAc = 2/1) to give a brown solid (2 g, 54 %).

Synthesis of 2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide: A mixture of the compound 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20 mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0 °C for 10 min. Hydroxylamine (50%) (10 ml) was cooled to 0 °C and added to the mixture. The resulting mixture was stirred at r.t. for 20 min. After removal of the solvent, the mixture was neutralized with 1M HCl to give a white precipitate. The crude product was filtered and purified by pre-HPLC to give a white solid (950 mg, 48%).

Example 2: Comparative Synthesis of 2-((2-chlorophenyl)(phenyl)amino)-N-(7- (hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide – Compound (I)

Reaction Scheme

Step (1)

Synthesis of Intermediate 2: A mixture of aniline (3.7 g, 40 mmol), ethyl 2-chloropyrimidine-5-carboxylate 1 (7.5 g, 40 mmol), K2C03 (11 g, 80 mmol) in DMF (100 ml) was degassed and stirred at 120 °C under N2 overnight. The reaction mixture was cooled to rt and diluted with EtOAc (200 ml), then washed with saturated brine (200 ml x 3). The organic layer was separated and dried over Na2S04, evaporated to dryness and purified by silica gel

chromatography (petroleum ethers/EtOAc = 10/1) to give the desired product as a white solid (6.2 g, 64 %).

Step (2)

Synthesis of Intermediate 3: A mixture of compound 2 (69.2 g, 1 equiv.), l-chloro-2-iodobenzene (135.7 g, 2 equiv.), Li2C03 (42.04 g, 2 equiv.), K2C03 (39.32 g, 1 equiv.), Cu (1 equiv. 45 μπι) in DMSO (690 ml) was degassed and purged with nitrogen. The resulting mixture was stirred at 140 °C for 36 hours. Work-up of the reaction gave compound 3 at 93 % yield.

Step (3)

Synthesis of Intermediate 4: 2N NaOH (200 ml) was added to a solution of the compound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture was stirred at 60 °C for 30min. After evaporation of the solvent, the solution was neutralized with 2N HCl to give a white precipitate. The suspension was extracted with EtOAc (2 x 200 ml), and the organic layer was separated, washed with water (2 x 100 ml), brine (2 x 100 ml), and dried over Na2S04. Removal of solvent gave a brown solid (2.5 g, 92 %).

Step (4)

Synthesis of Intermediate 5: A procedure analogous to the Synthesis of Intermediate 6 in Example 1 was used.

Step (5)

Synthesis of 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide: A procedure analogous to the Synthesis of 2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide in Example 1 was used.

Exam le 3: Process development for Steps 2-3 of Example 2

Table 2. Reactants and reagents

(13.8, leq)

(22.2g, 2eq) Cu

5 24.3g (l.Oeq) 47.7g (2.0eq) 240mL 140 °C

K2C03 (1.0 ε¾45μπι)

(19.65, leq)

(42.04g, 2eq) Cu

6 69.2g (l.Oeq) 135.7g (2.0eq) 690mL 140 °C

K2C03 (1.0 ε¾45μπι)

(39.32g, leq)

Table 3. Results

Table 4. Purification of Compound 4 by extraction and slurry

MTBE/Heptane (lOvol/lOvol) 2.83% 2.67% 92.57%

MEK/Heptane (3vol/6vol) 4.42% 3.16% 90.00%

93.48%

EtoAc 3.87% 1.43%

iProAc 3.91% 2.81% 90.91%

Example 4: Improved synthesis of Compound (I)

Reaction Scheme

4 5 (I)

Step (1)

Synthesis of Compound 11: Ethyl 2-chloropyrimidine-5-carboxylate (ACY-5, 7.0 Kgs), ethanol (60 Kgs), 2-Chloroaniline (9.5 Kgs, 2 eq) and acetic acid (3.7 Kgs, 1.6 eq) were charged to a reactor under inert atmosphere. The mixture was heated to reflux. After at least 5 hours the reaction was sampled for HPLC analysis (method TM-113.1016). When analysis indicated reaction completion (< 1% ACY-5), the mixture was cooled to 70 ± 5 °C and N,N-Diisopropylethylamine (DIPEA) was added. The reaction was then cooled to 20 ± 5°C and the mixture was stirred for an additional 2-6 hours. The resulting precipitate is filtered and washed with ethanol (2 x 6 Kgs) and heptane (24 Kgs). The cake is dried under reduced pressure at 50 ± 5 °C to a constant weight to produce 8.4 Kgs compound 11 (81% yield and 99.9% purity (method TM-113.1016)). See 1HNMR data in Figure 3.

Step (2)

Synthesis of Compound 3: Copper powder (0.68 Kgs, 1 eq, <75 micron), potassium carbonate (4.3 Kgs, 3.0 eq), and dimethyl sulfoxide (DMSO, 12.3 Kgs) were added to a reactor (vessel A). The resulting solution was heated to 120 ± 5°C. In a separate reactor (vessel B), a solution of compound 11 (2.9 Kgs) and iodobenzene (4.3 Kgs, 2 eq) in DMSO (5.6 Kgs) was

heated at 40 ± 5°C. The mixture was then transferred to vessel A over 2-3 hours. The reaction mixture was heated at 120 ± 5°C for 8-24 hours, until HPLC analysis (method TM-113.942) determined that < 1% compound 11 was remaining.

Step (3)

Synthesis of Compound 4: The mixture of Step (2) was cooled to 90-100 °C and purified water (59 Kgs) was added. The reaction mixture was stirred at 90-100 °C for 2-8 hours until HPLC (method TM-113.942-see step 2) showed that <1% compound 3 was remaining. The reactor was cooled to 25 °C. The reaction mixture was filtered through Celite, then a 0.2 micron filter, and the filtrate was collected. The filtrate was extracted with methyl t-butyl ether twice (2 x 12.8 Kgs). The aqueous layer was cooled to 0-5 °C, then acidified with 6N hydrochloric acid (HC1) to pH 2-3 while keeping the temperature < 25°C. The reaction was then cooled to 5-15 °C. The precipitate was filtered and washed with cold water. The cake was dried at 45-55 °C under reduced pressure to constant weight to obtain 2.2 kg (65% yield) compound 4 in 90.3% AUC purity (method TM-113.942-see step 2). No dechlorinated product or Cl-migration product (i.e., de-Cl-4 or m-Cl-4) was observed. See 1HNMR data in Figure 4.

Step (4)

Synthesis of Compound 5: Dichloromethane (40.3 Kgs), DMF (33g, 0.04 eq) and compound 4 (2.3 Kg) were charged to a reaction flask. The solution was filtered through a 0.2 μπι filter and was returned to the flask. Oxalyl chloride (0.9 Kgs, 1 eq) was added via addition funnel over 30-120 minutes at < 30 °C. The batch was then stirred at < 30°C until reaction completion (compound 4 ❤ %) was confirmed by HPLC (method TM-113.946). Next, the dichloromethane solution was concentrated and residual oxalyl chloride was removed under reduced pressure at < 40 °C. When HPLC analysis (method TM-113.946) indicated that < 0.10%) oxalyl chloride was remaining, the concentrate was dissolved in fresh

dichloromethane (24 Kgs) and transferred back to the reaction vessel (Vessel A).

A second vessel (Vessel B) was charged with Methyl 7-aminoheptanoate

hydrochloride (Compound Al, 1.5 Kgs, 1.09 eq), DIPEA (2.5 Kgs, 2.7 eq), 4

(Dimethylamino)pyridine (DMAP, 42g, 0.05 eq), and DCM (47.6 Kgs). The mixture was cooled to 0-10 °C and the acid chloride solution in Vessel A was transferred to Vessel B while maintaining the temperature at 5 °C to 10 °C. The reaction is stirred at 5-10 °C for 3 to 24 hours at which point HPLC analysis indicated reaction completion (method TM-113.946, compound 4 <5%). The mixture was then extracted with a 1M HC1 solution (20 Kgs), purified water (20 Kgs), 7% sodium bicarbonate (20 Kgs), purified water (20 Kgs), and 25% sodium chloride solution (20 Kgs). The dichloromethane was then vacuumdistilled at < 40 °C and chased repeatedly with isopropyl alcohol. When analysis indicated that <1 mol% DCM was remaining, the mixture was gradually cooled to 0-5 °C and was stirred at 0-5 °C for an at least 2 hours. The resulting precipitate was collected by filtration and washed with cold isopropyl alcohol (6.4 Kgs). The cake was sucked dry on the filter for 4-24 hours, then was further dried at 45-55 °C under reduced pressure to constant weight. 2.2 Kgs (77% yield) was isolated in 95.9% AUC purity (method TM-113.953) and 99.9 wt %. See 1HNMR data in Figure 5.

Step (5)

Synthesis of Compound (I): Hydroxylamine hydrochloride (3.3 Kgs, 10 eq) and methanol (9.6 Kgs) were charged to a reactor. The resulting solution was cooled to 0-5 °C and 25% sodium methoxide (11.2 Kgs, 11 eq) was charged slowly, maintaining the temperature at 0-10 °C. Once the addition was complete, the reaction was mixed at 20 °C for 1-3 hours and filtered, and the filter cake was washed with methanol (2 x 2.1 Kgs). The filtrate (hydroxylamine free base) was returned to the reactor and cooled to 0±5°C. Compound 5 (2.2 Kgs) was added. The reaction was stirred until the reaction was complete (method TM-113.964, compound 5 < 2%). The mixture was filtered and water (28 Kgs) and ethyl acetate (8.9 Kgs) were added to the filtrate. The pH was adjusted to 8 – 9 using 6N HC1 then stirred for up to 3 hours before filtering. The filter cake was washed with cold water (25.7 Kgs), then dried under reduced pressure to constant weight. The crude solid compound (I) was determined to be Form IV/ Pattern D.

The crude solid (1.87 Kgs) was suspended in isopropyl alcohol (IP A, 27.1 Kg). The slurry was heated to 75±5 °C to dissolve the solids. The solution was seeded with crystals of Compund (I) (Form I/Pattern A), and was allowed to cool to ambient temperature. The resulting precipitate was stirred for 1-2 hours before filtering. The filter cake was rinsed with IPA (2 x 9.5 Kgs), then dried at 45-55°C to constant weight under reduced pressure to result in 1.86 kg crystalline white solid Compound (I) (Form I/Pattern A) in 85% yield and 99.5% purity. See 1HNMR data in Figure 6.

Example 5: Alternative synthesis of Compound (I)

Reaction Scheme

(I)

Step (1)

Synthesis of Compound 11: Ethyl 2-chloropyrimidine-5-carboxylate (ACY-5, 250g), ethanol (2179 g), 2-Chloroaniline (339.3 g, 2 eq) and acetic acid (132.1 g, 1.6 eq) were charged to a reactor under inert atmosphere. The mixture was heated to reflux. After at least 5 hours the reaction was sampled for HPLC analysis. When analysis indicated reaction completion (< 1% ACY-5), the mixture was cooled to 70 ± 5 °C and Ν,Ν-Diisopropylethylamine (DIPEA, 553.6 g, 3.2 eq) was added. The reaction was then cooled to 20 ± 5°C and the mixture was stirred for an additional 2-6 hours. The resulting precipitate is filtered and washed with ethanol (2 x 401 g) and heptane (2 x 428 g). The cake is dried under reduced pressure at 50 ± 5 °C to a constant weight to produce 307. lg compound 11 (82.5% yield and 99.7% purity.

Step (2)

Synthesis of Compound 3: Cuprous iodide (17.5g, 8 eq), potassium carbonate (373.8 g, 3 eq), L-Prolin (11.4 g, 0.11 eq.) and dimethyl sulfoxide (DMSO, and 1180 g ) were added to a reactor (vessel A). The resulting solution was heated to 90 ± 5°C. In a separate reactor (vessel B), a solution of compound 11 (250g) and iodobenzene (1469.5 g, 8 eq) in DMSO (402.5 g) was heated at 40 ± 5°C. The mixture was then transferred to vessel A over 2-3 hours. The reaction mixture was heated at 90 ± 5°C for 8-24 hours, until HPLC analysis determined that < 1%) compound 11 was remaining.

Step (3)

Synthesis of Compound 4: The mixture of Step (2) was cooled to 40-50 °C and water (500g) and potassium hydroxide solution 10% (700.0 g, 2.8 eq) were added. The reaction mixture was stirred at 40-50 °C for 2-8 hours until HPLC showed that <1% compound 3 was remaining. The reactor was cooled to 25 °C. The reaction mixture was filtered through Celite, then a 0.2 micron filter, and the filtrate was collected. The filtrate was extracted with toluene (3 x 150g). The aqueous layer was cooled to 0-5 °C, then acidified with hydrochloric acid (HC1) to pH 2-3 while keeping the temperature < 25°C. The reaction was then cooled to 5-15 °C. The precipitate was filtered and washed with cold water. The cake was dried at 45-55 °C under reduced pressure to constant weight to obtain 291 g (81% yield) compound 4 in 98% AUC purity. No dechlorinated product or Cl-migration product (i.e., de-Cl-4 or m-Cl-4) was observed.

Step (4)

Synthesis of Compound 5 :

Compound 4 (250.0 g), A-l (159.2 g, 1.06 eq) and Methy-THF (5113 g) were charged to the reactor. DIPEA (283.7 g, 2.85 eq), hydroxybenzotriazole (HOBt, 12.5 g, 0.11 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC.HC1, 216.3 g, 1.47 eq) were added. The reaction solution was stirred at ambient temperature for 6-24 hours, at which point HPLC analysis indicated reaction completion (compound 4 <3%). The mixture was then extracted with a 1M HC1 solution (2270 g), purified water (2270 g), 7% sodium bicarbonate (2270 g), purified water (2270 g), and 25% sodium chloride solution (2270 g). The Methyl-THF was then vacuumdi stilled at < 40 °C and chased repeatedly with isopropyl alcohol. When analysis indicated that <1 mol% methyl-THF was remaining, the mixture was gradually cooled to 0-5 °C and was stirred at 0-5 °C for an at least 2 hours. The resulting precipitate was collected by filtration and washed with cold isopropyl alcohol (700g). The cake was sucked dry on the filter for 4-24 hours, then was further dried at 45-55 °C under reduced pressure to constant weight. 294g (82% yield) was isolated in 99.6% AUC purity and 99.4 wt %.

Step (5)

Synthesis of Compound (I): Hydroxylamine hydrochloride (330g, 10 eq) and methanol (960g) were charged to a reactor. The resulting solution was cooled to 0-5 °C and 25% sodium methoxide (1120 g, 11 eq) was charged slowly, maintaining the temperature at 0-10 °C. Once

the addition was complete, the reaction was mixed at 20 °C for 1-3 hours and filtered, and the filter cake was washed with methanol (2 x 210 g). The filtrate (hydroxylamine free base) was returned to the reactor and cooled to 0±5°C. Compound 5 (220 g) was added. The reaction was stirred until the reaction was complete (compound 5 < 2%). The mixture was filtered and water (280 g) and ethyl acetate (890 g) were added to the filtrate. The pH was adjusted to 8 -9 using HC1 then stirred for up to 3 hours before filtering. The filter cake was washed with cold water (2570 g), then dried under reduced pressure to constant weight to yield 980 g crude solid in 83% yield. The crude solid compound (I) was determined to be Form IV/ Pattern D.

The crude solid (980 g) was suspended in 1-propanol (400 g) and purified water (220 g). The suspension was heated to 40°C. The batch was then cooled to 38°C over 30 minutes. The solution was seeded with crystals of Compund (I) (Form I/Pattern A, 2-5 wt %). The batch was kept at 37-38°C for 2-4 hours, then was gradually cooled to 20±2°C. Water (950 g) was charged over 3 -5 hours. The batch was cooled to 12°C and was stirred for 2 hrs at this temperature. The batch was filtered and washed with cold 1-propanol/water, then dried at 50±5°C to constant weight to yield 910 g purified compound (I) in 93% yield and 99.8% AUC purity.

“NEW DRUG APPROVALS” CATERS TO EDUCATION GLOBALLY, No commercial exploits are done or advertisements added by me. This article is a compilation for educational purposes only.

P.S. : The views expressed are my personal and in no-way suggest the views of the professional body or the company that I represent

/////////WO-2016200930, WO 2016200930, New patent, Citarinostat, Acetylon Pharmaceuticals Inc

CPP 115


str0

(+)-(1S,4S)-4-Amino-3-(difluoromethylene)-1-cyclopentanecarboxylic acid

640897-20-7 CAS

PHASE 1

NORTHWESTERN UNIVERSITY .INNOVATORS

Sponsor:
CPP-115 free base; UNII-5TD9324Z2U; CHEMBL146927; 640897-20-7; (1S,3S)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic acid; (+)-(1S,4S)-4-Amino-3-(difluoromethylene)-1-cyclopentanecarboxylic acid
Molecular Formula: C7H9F2NO2
Molecular Weight: 177.151 g/mol

Catalyst Pharmaceutical Partners

  • Originator Northwestern University
  • Developer Catalyst Pharmaceutical Partners
  • Class Aminobutyric acids; Antiepileptic drugs; Small molecules
  • Mechanism of Action 4-aminobutyrate transaminase inhibitors
  • Orphan Drug Status Yes – Infantile spasms
  • On Fast track Drug abuse
  • Cocaine Dependency

Highest Development Phases

  • Phase I Gilles de la Tourette’s syndrome; Infantile spasms; Partial epilepsies
  • Preclinical Drug abuse

Most Recent Events

  • 19 Sep 2016 Efficacy data from a phase I trial in Infantile spasms released by Catalyst Pharmaceuticals
  • 16 Dec 2015 Top-line adverse events and pharmacodynamics data from a phase Ib trial in Healthy volunteers released by Catalyst Pharmaceuticals
  • 13 Oct 2015Catalyst Pharmaceuticals receives patent allowance for CPP 115 in USA

Image result for SILVERMAN, Richard, BRichard B. Silverman, Ph.D.,
John Evans Professor of Chemistry, Northwestern University, Evanston, Illinois, USA.

Click here for structure editor

UNII-0285I2MVUA.png

CPP 115 HCl salt, cas 760947-97-5

UNII-0285I2MVUA; CPP-115; 760947-97-5; (1S,3S)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic acid hydrochloride; Cyclopentanecarboxylic acid, 3-amino-4-(difluoromethylene)-, hydrochloride, (1S,3S)-; 0285I2MVUA
Molecular Formula: C7H10ClF2NO2
Molecular Weight: 213.609 g/mol

Responsible Party:Catalyst Pharmaceuticals, Inc.ClinicalTrials.gov Identifier:NCT01493596     History of ChangesOther Study ID Numbers:CPP-115-0001 Study First Received:November 28, 2011Last Updated:May 10, 2012Health Authority:United States: Food and Drug Administration

Cpp-115: An Investigational Drug For Epilepsy

The fact that 1 in 12 people will have a seizure in their lifetime raises alarming signals to mitigate, prevent and cure epilepsy. The etiology is still unclear, but one of the pharmaceutical strategies to treat seizures is to replenish the local concentrations of GABA (gamma-aminobutyric acid, an inhibitory neurotransmitter in the human brain) that is degraded by an enzyme called GABA aminotransferase (GABA-AT). Mere consumption of GABA capsules is not effective, due to its inability to cross the blood-brain barrier (BBB). Therefore, an alternative strategy that involved stopping the function of GABA-AT was envisioned. Sabril is a first-in-class, FDA-approved antiepileptic drug; however, its daily dosage limit (1g – 3g) and adverse side effects, which include vision defects, call for further innovation.

Prof. Richard Silverman and his lab members at Northwestern University embarked on a scientific journey to identify BBB-penetrating antiepileptic compounds that would not cause visual defects. Through computational modeling and several cycles of optimization they discovered CPP-115 (chemical name: (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid; kinact/KI = 52 mM.min-1.)1 Mechanistically, CPP-115 binds to GABA-AT, undergoing product transformation that kills GABA-AT’s function. In rat studies, CPP-115 suppressed spasms at a much lower dose (0.1 mg/kg) than Sabril (>200 mg/kg) and exhibited better tolerance without visual defects.

CPP-115 (licensed to Catalyst Pharmaceuticals) elicited no cross-inhibition. It is metabolically more stable, with favorable PK characteristics (including rapid absorption and clearance). In a randomized, double-blind, single ascending dose phase I(a) study, CPP-115 was very well tolerated in all six doses (n=55 patients; maximum dose 500 mg, therapeutic dose 80 mg/day).2 Phase I(b) studies conducted in double-blind, placebo-controlled conditions demonstrated the safety and tolerability of CPP-115 in healthy volunteers. Intriguingly, an increase in brain GABA levels (150% to over 200%) was detected, accentuating CPP-115’s antiepileptic potential.2 Further clinical trials are currently in progress. CPP-115, with 12 years of unexpired patent life, has been granted orphan-drug designation in both the U.S. and EU for treating infantile spasms.

CPP-115 is one of a group of novel GABA-aminotransferase inhibitors discovered by scientists at Northwestern University. In 2009 Catalyst entered into a strategic collaboration with Northwestern University and in-licensed the worldwide rights to these inhibitors.

CPP-115 binds to GABA-AT (GABA-aminotransferase, also known as GABA transaminase or GABA-T), causing increased levels of GABA, gamma-aminobutyric acid, the chief inhibitory neurotransmitter in humans. It plays a role in regulating neuronal excitability throughout the nervous system. In humans, GABA is also directly responsible for the regulation of muscle tone.

In preclinical studies CPP-115 has been shown to have potentially significant advantages compared to the only approved and marketed current GABA-AT inhibitor (vigabatrin). CPP-115 may not cause the visual field defects associated with chronic administration of vigabatrin and it has been shown to be at least 200 times more potent in both in-vitro and animal model studies. The increased potency could enable the development of superior or alternative dosage forms and routes of administration. Catalyst hopes these important benefits will allow it to develop CPP-115 for a broad range of other central nervous system indications, such as infantile spasms, epilepsy, Tourette Syndrome and Post Traumatic Stress Disorder (PTSD). Additionally, Catalyst is exploring other selected diseases in which modulation of GABA levels might be beneficial. Catalyst believes that it controls all current intellectual property for GABA-aminotransferase inhibitors.

CPP-115 has received orphan drug designation in both the US and the EU for infantile spasms. Catalyst has begun the clinical development of CPP-115 by completing a randomized, double-blind, single ascending dose Phase I(a) study in normal healthy volunteers to evaluate the human safety characteristics of CPP-115, including CNS side effects and respiratory and cardiovascular safety. The Company reported results which indicated that CPP-115 was well tolerated at all six doses administered up to 500 mg, well above the anticipated therapeutic dose of up to 80 mg/day.

The hydrochloride salt of CPP-115 (PubChem CID 71252718) has been granted orphan drug designation by the EMA for the treatment of West syndrome, an epileptic disorder of young children which causes developmental problems. West syndrome is a long-term debilitating disease which may be life threatening as it can lead to severe damage to motor and cognitive functions. CPP-115 may have additional therapeutic applications for treating other neurological disorders, including drug addiction [4]. A single Phase I clinical trial has assessed CPP-115 as a treatment for cocaine addiction [3], but development has not progressed further.

Image result for CPP 115

Patent

WO 2016073983

NORTHWESTERN UNIVERSITY [–/US]; 633 Clark Street Evanston, IL 60208 (US)
Inventors: SILVERMAN, Richard, B.; (US).
ILAN, Yaron; (IL)

Example 8

[0067] (IS, 4S)-6-Difluoromethylenyl-2-(4′-methoxybenzyl)-2- azabicyclo[2.2.1]heptan-3-one (13). At -78 °C, T uLi (1.7 M in pentane, 1.73 mL, 2.94 mmol) was slowly added to a stirred solution of diethyl (difluoromethyl)phosphonate (0.48 mL, 2.94 mmol) in anhydrous THF (15 mL). After being stirred for 0.5 h at -78 °C, 12 (0.60g, 2.45 mmol) in anhydrous THF (20 mL) was slowly added via syringe. Stirring continued for 1 h at – 78 °C , then the solution was allowed to warm to room temperature and heated to reflux for 24 h. Compound 12 is known and available in the art, and can be prepared as described in Qiu, J.; Silverman, R.B. A New Class of Conformationally Rigid Analogues of 4-Amino-5- halopentanoic Acids, Potent Inactivators of γ-Aminobutyric Acid Aminotransferase. J. Med. Chem. 2000, 43, 706-720. After the reaction had cooled down, THF was evaporated, and saturated NH4C1 solution (20 mL) was added to the residue, which was extracted with EtOAc (3 x 20 mL). The organic layer was washed with brine (2 x 20 mL), dried over anhydrous Na2S04, and concentrated under reduced pressure. The residue was purified by flash column

chromatography, eluting with hexanes/ethyl acetate (2: 1) to give 13 (0.47 g, 68%) as a colorless oil: 1H NMR (400 MHz, CDC13) δ 7.18 (d, J 8.4 Hz, 2H), 6.07 (d, J 8.4 Hz, 2H), 4.63 (d, J 14.8 Hz, 1H), 4.14 (s, 1H), 3.80 (s, 3H), 3.78 (d, J 14.8 Hz, 1H), 3.00 (s, 1H), 2.50 (dt, J 15.2, 3.6 Hz, 1H), 2.27 (dd, J 15.2, 2.4 Hz, 1H), 2.00 (d, J 9.2 Hz, 1H), 1.53 (d, 9.6 Hz, 1H); 13C NMR (100 MHz, CDC13) δ 177.37, 159.13, 152.19 (dd, J 285.7, 281.2 Hz), 129.59, 128.47, 1 14.13, 88.95 (dd, J 25.6, 22.2 Hz), 58.38 (d, J 5.3 Hz), 55.50, 45.60, 44.59, 40.96, 27.43; 19F NMR (376 MHz, CDC13) δ 42.64 and 41.01 (2 dd, J 60.2, 2.3 Hz, 2F). HRMS (EI) Ci5Hi5N02F2 calcd M

279.1071 , found M 279.10701.

Example 10

 (IS, 3S)-3-Amino-4-difluoromethylenyl-l-cyclopentanoic acid (15) (i.e., compound 10, CPP-115, Figure 2). To lactam 14 (20.0 mg, 0.13 mmol) was added 4 mL of 4 N HCl. The solution was stirred at 70 °C for 10 h. After being washed with ethyl acetate (3 x 4 mL), the water layer was evaporated under reduced pressure to give a yellow solid. Recrystallization with ethanol/ether gave a white solid, which was then loaded on a cation- exchange column (AG50W-X8) and eluted with 0.2 N ammonium hydroxide to give the free amino acid 15 as a white solid (16 mg, 72%). 1H NMR (400 MHz, D20) δ 4.44 (s, 1H), 2.92 (m, 1H), 2.74 (m, 1H), 2.57 (dd, J 16.4, 3.6 Hz, 1H), 2.34 (m, 1H), 2.02 (d, J 14.8 Hz, 1H); 13C NMR (126 MHz, D20) δ 186.08, 155.30 (t, J 288.7 Hz), 92.19 (m), 53.16 (d, J 3.8 Hz), 48.01, 37.89, 32.45; 19F NMR (376 MHz, D20) δ -8.43 and -9.02 (2d, J 46.3 Hz, 2F); MS (ESI) C7H9N02F2 calcd M+H 178, found M+H 178.

PATENT

US 6794413

https://www.google.com/patents/US6794413

C7H11O2N, H% 7.85 C% 59.56 N% 9.92, found H% 7.88 C% 59.23 N% 9.62.

Example 5

(1S, 4S)-6-Difluoromethylenyl-2-(4′-methoxybenzyl)-2-azabicyclo [2.2.1]heptan-3-one (13). At −78° C., tBuLi (1.7 M in pentane, 1.73 mL, 2.94 mmol) was slowly added to a stirred solution of diethyl (difluoromethyl)phosphonate (0.48 mL, 2.94 mmol) in anhydrous THF (15 mL). After being stirred for 0.5 h at −78° C., 12 (0.60 g, 2.45 mmol) in anhydrous THF (20 mL) was slowly added via syringe. Stirring continued for 1 h at −78° C., then the solution was allowed to warm to room temperature and heated to reflux for 24 h. Compound 12 is known and available in the, art, and can be prepared as described in Qiu, J.; Silverman, R. B. A New Class of. Conformationally Rigid Analogues of 4-Amino-5-halopentanoic Acids, Potent Inactivators of γ-Aminobutyric Acid Aminotransferase. J. Med. Chem. 2000, 43, 706-720. After the reaction had cooled down, THF was evaporated, and saturated NH4Cl solution (20 mL) was added to the residue, which was extracted with EtOAc (3×20 mL). The organic layer was washed with brine (2×20 mL), dried4over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography, eluting with hexanes/ethyl acetate (2:1) to give 13 (0.47 g, 68%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 7.18 (d, J 8.4 Hz, 2H), 6.07 (d, J 8.4 Hz, 2H), 4.63 (d, J 14.8 Hz, 1H), 4.14 (s. 1H), 3.80 (s, 3H), 3.78 (d, J 14.8 Hz, 1H), 3.00 (s, 1H), 2.50 (dt, J 15.2, 3.6 Hz, 1H), 2.27 (dd, J 15.2, 2.4 Hz, 1H), 2.00 (d, J 9.2 Hz, 1H) 1.53 (d, 9.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 177.37, 159.13, 152.19 (dd, J 285.7, 281.2 Hz), 129.59, 128.47, 114.13, 88.95 (dd, J 25.6, 22.2 Hz), 58.38 (d, J 5.3 Hz), 55.50, 45.60, 44.59, 40.96, 27.43; 19F NMR (376 MHz, CDCl3) δ 42.64 and 41.01 (2 dd, J 60.2, 2.3 Hz, 2F). HRMS (EI) C15H15NO2F2 calcd M 279.1071, found M 279.10701.

Example 6

(1S, 4S)-6-Difluoromethylenyl-2-azabicyclo[2.2.1]heptan-3-one (14). Compound 13 (86.9 mg, 0.31 mmol) was dissolved in CH3CN (1.75 mL). A solution of ceric ammonium nitrate (512 mg, 0.93 mmol) in water (0.87 mL) was slowly added. The resulting solution was stirred at room temperature for 4 h. The reaction mixture was then diluted with ethyl acetate (20 mL), washed with brine (2×10 mL), and dried over anhydrous Na2SO4. After being concentrated under reduced pressure, the residue was purified by flash column chromatography, eluting with hexanes/ethyl acetate (1:1) to give the desired product as a colorless oil (33.6 mg, 68%). 1H NMR (400 MHz, CDCl3) δ 5.48 (br s, 1H), 4.40 (s, 1H), 2.93 (s, 1H), 2.54 (dd, J 15.2, 2.8 Hz, 1H), 2.32 (d, J 15.2 Hz, 1H), 2.15 (d, J 9.6 Hz, 1H), 1.64 (d, J 10.0 Hz, 1H); 19F NMR (376 MHz, CDCl3) δ 42.85 and 40.00 (2d, J 60.2 Hz, 2F); HRMS (EI) C7H7NOF2 calcd M 159.0496, found M 159.04673.

Example 7

(1S, 3S)3-Amino-4-difluoromethylenyl-1-cyclopentanoic acid (15). To lactam 14 (20.0 mg, 0.13 mmol) was added 4 mL of 4 N HCl. The solution was stirred at 70° C. for 10 h. After being washed with ethyl acetate (3×4 mL), the water layer was evaporated under reduced pressure to give a yellow solid. Recrystallization with ethanol/ether gave a white solid, which was then loaded on a cation-exchange column (AG50W-X8) and eluted with 0.2 N ammonium hydroxide to give the free amino acid 15 as a white solid (16 mg, 72%). 1H NMR (400 MHz, D2O) δ 4.44 (s, 1H), 2.92 (m, 1H), 2.74 (m, 1H), 2.57 (dd, J 16.4, 3.6 Hz, 1H), 2.34 (m 1H), 2.02 (d, J 14.8 Hz, 1H); 13C NMR (126 MHz, D2O) δ 186.08, 155.30 (t, J 288.7 Hz), 92.19 (m), 53.16 (d, J 3.8 Hz), 48.01, 37.89, 32.45; 19F NMR (376 MHz, D2O) δ −8.43 and −9.02 (2d, J 46.3 Hz, 2F); MS (ESI) C7H9NO2F2 calcd M+H 178, found M+H 178.

paper

Journal of Medicinal Chemistry (2003), 46(25), 5292-5293

Design, Synthesis, and Biological Activity of a Difluoro-Substituted, Conformationally Rigid Vigabatrin Analogue as a Potent γ-Aminobutyric Acid Aminotransferase Inhibitor

Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and Drug Discovery Program, Northwestern University, Evanston, Illinois 60208-3113
J. Med. Chem., 2003, 46 (25), pp 5292–5293
DOI: 10.1021/jm034162s
Publication Date (Web): November 11, 2003
Copyright © 2003 American Chemical Society

Abstract

Abstract Image

Previously it was found that a conformationally rigid analogue (2) of the epilepsy drug vigabatrin (1) did not inactivate γ-aminobutyric acid aminotransferase (GABA-AT). A cyclic compound with an exocyclic double bond (6) was synthesized and was found to inactivate GABA-AT, but only in the absence of 2-mercaptoethanol. The corresponding difluoro-substituted analogue (14) was synthesized and was shown to be a very potent time-dependent inhibitor, even in the presence of 2-mercaptoethanol.

1 to 6 of 6
Patent ID Patent Title Submitted Date Granted Date
US2015196522 METHODS OF USING (1S, 3S)-3-AMINO-4-DIFLUOROMETHYLENYL-1-CYCLOPENTANOIC ACID 2015-03-02 2015-07-16
US8969413 Methods of using (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid 2011-02-25 2015-03-03
US2014336256 METHOD OF TREATING TOURETTE’S DISORDER WITH GABA-AMINOTRANSFERASE INACTIVATORS 2014-07-25 2014-11-13
US2011237554 Combination therapies: inhibitors of GABA transaminase and NKCC1 2011-09-29
US7381748 Compounds and related methods for inhibition of gamma-aminobutyric acid aminotransferase 2008-06-03
US6794413 Compounds and related methods for inhibition of gamma-aminobutyric acid aminotransferase 2004-09-21

RICHARD B. SILVERMAN

PROFESSOR

Research Statement

The research in my group can be summarized as investigations of the molecular mechanisms of action, rational design, and syntheses of potential medicinal agents, particularly for neurodegenerative diseases. Numerous drugs are known to function as specific inhibitors of particular enzymes. When little is known about the enzyme’s molecular mechanism of action, chemical model studies are designed to determine reasonable nonenzymatic pathways applicable to the enzyme. Based on the proposed mechanism of enzyme action, inhibitors are designed and synthesized. Organic synthesis is a primary tool for this work. The enzymes are isolated from either mammalian tissue or from overexpressed cells containing recombinant enzymes. Active site labeling studies utilize MALDI TOF and electrospray ionization mass spectrometry as well as radiolabeled inactivators and peptide mapping. We also are synthesizing compounds to act as receptor antagonists for important receptors related to neurodegenerative diseases.

Recent Publications

Lee, H.; Doud, E. H.; Wu, R.; Sanishvili, R.; Juncosa, J. I.; Liu, D.; Kelleher, N. L.; Silverman, R. B. Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115). J. Am. Chem. Soc. 2015, 137, 2628-2640.

Zigmond, E.; Ya’acov, A. B.; Lee, H.; Lichtenstein, Y.; Shalev, Z.; Smith, Y.; Zolotarov, L.; Ziv, E.; Kalman, R.; Le, H. V.; Lu, H.; Silverman, R. B.; Ilan, Y. Suppression of hepatocellular carcinoma by inhibition of overexpressed ornithine aminotransferase. ACS Med. Chem. Lett. 2015, 6, 840-844.

Tang, W.; Li, H.; Doud, E. H.; Chen, Y.; Choing, S.; Plaza, C.; Kelleher, N. L.; Poulos, T. L.; Silverman, R. B. Mechanism of inactivation of neuronal nitric oxide synthase by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid. J. Am. Chem. Soc. 2015, 137, 5980-5989.

Le, H. V.; Hawker, D. D.; Wu, R.; Doud, E.; Widom, J.; Sanishvili, R.; Liu, D.; Kelleher, N. L.; Silverman, R. B. Design and mechanism of tetrahydrothiophene-based GABA aminotransferase inactivators. J. Am. Chem. Soc. 2015, 137, 4525-4533.

Huang, H.; Li, H.; Yang, S.; Chreifi, G.; Martásek, P.; Roman, L. J.; Meyskens, F. L.; Poulos, T. L.; Silverman, R. B. Potent and Selective Double-headed Thiophene-2-carboximidamide Inhibitors of Neuronal Nitric Oxide Synthase for the Treatment of Melanoma. J. Med. Chem. 2014, 57, 686-700.

Trippier, P. C.; Zhao, K. T.; Fox, S. G.; Schiefer, I. T.; Benmohamed, R.; Moran, J.; Kirsch, D. R.; Morimoto, R. I.; Silverman, R. B. Proteasome Activation is a Mechanism for Pyrazolone Small Molecules Displaying Therapeutic Potential in Amyotrophic Lateral Sclerosis. ACS Chem. Neurosci. 2014, 5, 823-829.

Holden, J. K.; Li, H.; Jing, Q.; Kang, S.; Richo, J.; Silverman, R. B.; Poulos, T. L. Structural and biological studies on bacterial nitric oxide synthase inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 18127-18131.

Kang, S.; Cooper, G.; Dunne, S. F.; Dusel, B.; Luan, C.-H.; Surmeier, D. J.; Silverman, R. B. CaV1.3-selective L-type calcium channel antagonists as potential new therapeutics for Parkinson’s disease. Nature Commun 2012, 3, 1146.

Silverman, R. B. The 2011 E. B. Hershberg Award for Important Discoveries in Medicinally Active Substances: (1S,3S)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115), a GABA Aminotransferase Inactivator and New Treatment for Drug Addiction and Infantile Spasms. J. Med. Chem. 2012, 55, 567-575.

Chen, T.; Benmohamed, R.; Kim, J.; Smith, K.; Amante, D.; Morimoto, R. I.; Kirsch, D. R.; Ferrante, R. J.; Silverman, R. B. ADME-Guided Design and Synthesis of Aryloxanyl Pyrazolone Derivatives to Block Mutant SOD1 Cytotoxicity and Protein Aggregation: Potential Application for the Treatment of Amyotrophic Lateral Sclerosis. J. Med. Chem. 2012, 55, 515-527.

Selected Honors/Awards

  • 2014 Fellow of the National Academy of Inventors
  • 2014 Northwestern University Trustee Medal for Faculty Innovation and Entrepreneurship
  • 2014 iCON Innovator Award (iBIO Institute)
  • 2014 Elected to American Academy of Arts & Sciences
  • 2014 Excellence in Medicinal Chemistry Prize of the Israel Chemical Society
  • 2013 Fellow of the Royal Society of Chemistry (UK)
  • 2013 Centenary Prize of the Royal Society of Chemistry
  • 2013 Bristol-Myers Squibb-Edward E. Smissman Award of the American Chemical Society (ACS)
  • 2013 Roland T. Lakey Award from Wayne State University
  • 2012 Sato Memorial International Award of the Pharmaceutical Society of Japan
  • 2011 Fellow of the ACS
  • 2011 E. B. Hershberg Award for Important Discoveries in Medicinally Active Substances of the ACS
  • 2011 Alumni Hall of Fame, Central High School of Central High School of Philadelphia
  • 2009 Medicinal Chemistry Hall of Fame of the American Chemical Society
  • 2009 Perkin Medal, Society of Chemical Industry
  • 2008 Alumni Fellow Award, Pennsylvania State University
  • 2003 Arthur C. Cope Senior Scholar Award of the American Chemical Society
  • 2000 Northwestern University Alumni Association Excellence in Teaching Award
  • 1999 E. LeRoy Hall Award for Teaching Excellence
  • 1999 Excellence in Chemistry Education Award from the Northwestern University Chapter of Alpha Chi Sigma Chemistry Fraternity
  • 1990 Fellow of the American Association for the Advancement of Science
  • 1985 Fellow of the American Institute of Chemists
  • 1982 NIH Research Career Development Awardee
  • 1981 Alfred P. Sloan Research Fellow
  • 1976 Du Pont Young Faculty Fellow
  • Silverman describes the structure of pregabalin.
    Silverman describes the structure of pregabalin.

In recognition of his outstanding work in applied chemistry, the Society of Chemical Industry 2009 Perkin Medal has been awarded to Richard B. (Rick) Silverman, the John Evans Professor of Chemistry at Northwestern University. The Perkin Medal, which was first awarded just over one century ago, is recognized as one of the chemical industry’s most prestigious awards.

Silverman’s research primarily focuses on medicinal chemistry: studying the molecular basis of drug action, reaction mechanisms of enzymes, and design and synthesis of pharmaceutical agents. He has worked to deepen understanding of several diseases, including epilepsy, cancer, Parkinson’s, and cerebral palsy.

Among Silverman’s many scientific accomplishments, designing pregabalin and discovering the medicinal properties of that compound stand out for catapulting him and Northwestern to pharmaceutical fame and fortune. Pregabalin, a γ-aminobutyric acid analog, is the active substance in Lyrica, a pain and epilepsy medication commercialized by drug giant Pfizer.

In 2007, after Northwestern collected more than $70 million in royalties for the drug, the university sold a portion of its royalty rights for an additional $700 million (C&EN, March 10, 2008, page 56). Around the same time, Silverman and his family donated a portion of their earnings from the drug to fund construction of a new Northwestern science building. The facility, which is scheduled to open this fall, will house chemistry, biology, and engineering research groups devoted to biomedical science.

Silverman has published more than 250 papers in organic chemistry, medicinal chemistry, and enzymology. He is also the author of three books, including “The Organic Chemistry of Drug Design and Drug Action,” and holds 40 patents.

The Perkin Medal is named for Sir William Henry Perkin (1838–1907), who was honored by SCI in 1906 for developing the first synthetic dye, Perkin mauve. This year’s medal will be presented at SCI’s Perkin Medal banquet in Philadelphia in September.

The Legacy Of Lyrica

November 18, 2013

Northwestern’s Richard Silverman, professor of chemistry, developed pregabalin, the chemical that Pfizer now markets as Lyrica.  The drug is one of the two approved treatments for fibromyalgia, epilepsy, and the most effective treatment for seizures as well.

In his laboratory, Silverman’s research team studied chemicals made in the brain. Of particular interest was GABA, a neurotransmitter that inhibits certain brain functions. When GABA levels fall too low in some people, it can trigger epileptic seizures. His group studied enzymes that affect GABA levels, looking for ways to keep GABA elevated.  In 1989, the Parke-Davis unit of Warner-Lambert was interested in the research findings. Among the 17 chemical analogs that Silverman sent to Parke-Davis, only pregabalin showed effects in mice.

Serendipity played a huge part in shaping this success story, as most chemicals that affect cells in lab experiments do not survive inside an animal. Another outcome of the research was that the compound was effective for a reason entirely different from Silverman’s initial goal of producing more GABA. In another stroke of luck, the molecule happened to be of the right shape to be transported directly into the brain with nearly 90 percent efficacy.

Lyrica has been a tremendous medical and commercial success that has validated the nearly 15 year process from invention to market launch in 2005. In 2004 Lyrica was approved for use in adults for the treatment of various peripheral neuropathic pain indications as well as therapy for partial epilepsy in more than 60 countries outside of the United States. In 2006 Lyrica was also approved for the treatment of generalized anxiety disorder in Europe. The drug brought in $1.2 billion in sales in 2006 and in 2010 was approved in Europe to treat central neuropathic (nerve) pain. This is expected to push profits from the blockbuster drug to climb even higher.

Northwestern sold a sizeable amount of royalty interest in 2007 to Royalty Pharma, a company that specializes in acquiring cash-generating intellectual property, for $700 million to help the university’s endowment. This deal has been termed the largest sale ever of a royalty stream for a pharmaceutical product.

To learn more about Lyrica visit the product website at www.lyrica.com.

Originally Appeared:

////////Cocaine Dependency, CPP 115, PHASE 1, CATALYST, NORTHWESTERN UNIVERSITY, ORPHAN DRUG, 640897-20-7, 760947-97-5

C1C(CC(=C(F)F)C1N)C(=O)O

CB-618


str1

CB-618, CB-238618

CAS 1463520-70-8
C8 H10 N4 O6 S, 290.25
Sulfuric acid, mono[(1R,2S,5R)-2-(1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl] ester
(25, 5R)-sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester

SODIUM SALT

sodium salt of (2S,5R)-sulfuric acid mono-(2-[1,3,4]oxadiazol-2-yl-7-oxo-1,6-diaza-bicyclo[3.2.1]oct-6-yl)ester

CAS 1628207-16-8
C8 H10 N4 O6 S . Na
Sulfuric acid, mono[(1R,2S,5R)-2-(1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl] ester, sodium salt (1:1)
PATENTS

WO2013149121

US 20140275001

US 20150094472

WO 2016081452

Infection, multidrug resistant bacteria (MDR) in  phase 1 at  Merck
CB-618 is in phase I clinical trails by Cubist for the treatment of resistant bacterial infections, including carbapenem-resistant Enterobacteriaceae and Klebsiella pneumonia carbapenemases infection.

CB-618 is a beta-Lactamase inhibitor in phase I clinical trials at Merck & Co. for the treatment of multidrug resistant bacterial infections, including those caused by carbapenem-resistant Enterobacteriaceae and Klebsiella pneumoniae carbapenemases.

The product was originally developed at Cubist. In 2015, Merck & Co. acquired the company

  • Originator Cubist Pharmaceuticals
  • Class Antibacterials
  • Mechanism of Action Beta lactamase inhibitors

Highest Development Phases

  • Phase I Gram-negative infections

Most Recent Events

  • 01 Apr 2015 Cubist Pharmaceuticals completes a phase-I clinical trial in Gram-negative infections in USA (IV) (NCT02341599)
  • 21 Jan 2015 Cubist Pharmaceuticals has been acquired by Merck & Co
  • 14 Jan 2015 Phase-I clinical trials in Gram-negative infections in USA (IV)

Bacterial resistance to β-lactam antibiotics, especially in Gram-negative bacteria, is most commonly mediated by β-lactamases. β-lactamases are enzymes that catalyze the hydrolysis of the β-lactam ring, which inactivates the antibacterial activity of the β-lactam antibiotic and allows the bacteria to become resistant. Inhibition of the β-lactamase with a BLI slows or prevents degradation of the β-lactam antibiotic and restores β-lactam antibiotic susceptibility to β-lactamase producing bacteria. Many of these β-lactamases are not effectively inhibited by BLIs currently on the market rendering the β-lactam antibiotics ineffective in treating bacteria that produce these β-lactamases. There is an urgent need for novel BLIs that inhibit β-lactamases that are not effectively inhibited by the current clinical BLIs (e.g. KPC, class C and class D β-lactamases) and that could be used in combination with β-lactam antibiotics to treat infections caused by β-lactam resistant bacteria.

PATENT

WO2013149121

Yu Gui Gu, Yong He, Ning Yin, Dylan C. ALEXANDER, Jason B. CROSS, Chester A. Metcalf, Robert Busch
Applicant Cubist Pharmaceuticals, Inc.

Example 3: Synthesis of (2S,5R)-2-(l ,3,4-oxadiazol-2-yl)-7-oxo-l ,6- diazabicyclo[3.2.1 loctan-6-yl hydrogen sulfate (Compound 701 )

Figure imgf000068_0001

Step 1: Ι,Γ-Carbonyldiimidazole (5.8 g, 36.2 mmol) was added to a 0 °C solution of (2S,5R)- 6-(benzyloxy)-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carboxylic acid (5.0 g, 18.1 mmol) in dry THF (200 mL). The reaction mixture was allowed to warm to rt then was stirred at rt for 3 hrs. Formohydrazide (5.4 g, 90.5 mmol) was added in one portion, and the reaction mixture was stirred for additional 3 hrs. The mixture was then diluted with saturated sodium chloride and exatracted with EtOAc (3x). The combined organic layer was washed with saturated sodium chloride (2x), dried over Na2S04, and concentrated to afford crude (25,5 ?)- 6-(benzyloxy)-N-formyl-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carbohydrazide (-11 g), which was directly used in the next step. ESI-MS (Ef , m/z): 319.1 [M+H]+.

Step 2: To a -10 °C solution of (25′,5«)-6-(benzyloxy)-N-formyl-7-oxo-l,6- diazabicyclo[3.2.1]octane-2-carbohydrazide (11 g) in dry DCM (200 mL) was added pyridine (28 mL), followed by dropwise addition of (CF3S02)20 (28 mL). The reaction mixture was allowed to warm to rt and was stirred for 3 hrs. The reaction mixture was then cooled to -10 °C and quenched with sat. NaHCC>3. The organic layer was separated and the aqueous layer was extracted with EtOAc (3x). The combined organic layer was dried over Na2S04, concentrated and purified by silica gel column chromatography (gradient elution 1 :3 to 2: 1 EtOAc/hexanes) to give (25,5/?)-6-(benzyloxy)-2-(l,3,4-oxadiazol-2-yl)-l ,6- diazabicyclo[3.2.1]octan-7 -one (4.6 g, 86% for two steps) as a slightly yellow solid. ESI-MS (EI+, m/z): 301.0 [M+H]+.

Step 3: To a solution of (25,5/?)-6-(benzyloxy)-2-(l,3,4-oxadiazol-2-yl)-l ,6- diazabicyclo[3.2.1]octan-7-one (4.6 g, 15.3 mmol) in THF (150 mL) was added 10% Pd/C (1 g). The mixture was stirred under H2 atmosphere at rt for 3 hrs. The reaction mixture was then filtered and concentrated to afford (25,5/?)-6-hydroxy-2-(l,3,4-oxadiazol-2-yl)-l,6- diazabicyclo[3.2.1]octan-7-one (2.9 g, 91 %), which was used directly in the next step. ESI- MS (EI+, m/z): 211.1 [M+H]+. Step 4: To a solution of (25,5fl)-6-hydroxy-2-(l,3,4-oxadiazol-2-yl)-l,6- diazabicyclo[3.2.1]octan-7-one (2.9 g, 13.8 mmol) in dry pyridine (60 mL) was added SC>3- Py (11.0 g, 69.0 mmol). The reaction mixture was stirred at rt for 8 hrs and then concentrated under vacuum. The residue was re-dissolved in aqueous NaH2PC>4 (1.5 M, 100 mL) then tetrabutylammonium hydrogensulphate (5.88 g, 17.3 mmol) was added. The mixture was stirred at rt for 20 minutes, then was extracted with EtOAc (4x). The combined organic layer was dried and concentrated and the residue was purified by silica gel column chromatography (gradient elution 10:1 to 2:1 DCM/acetone) to afford tetrabutylammonium (25,5/?)-2-(l ,3,4-oxadiazol-2-yl)-7-oxo-l,6-diazabicyclo[3.2.1]octan-6-yl sulfate (4.1 g, 97%) as a white solid. ESI-MS (EL, m/z): 289.0 [M-H]\ lH NMR (400 MHz, CDC13): δ 8.48 (s, 1H), 4.75 (d, / = 6.5 Hz, 1H), 4.40 (br s, 1H), 3.34-3.26 (m, 9H), 2.82 (d, / = 12.0 Hz, 1H), 2.37-2.25 (m, 3H), 2.06-1.98 (m, 1H), 1.71-1.65 (m, 8H), 1.49-1.42 (m, 8H), 1.01 (t, / = 7.5 Hz, 12H).

Step 5: Resin Exchange: Tetrabutylammonium (25, 5R)-2-(l, 3, 4-oxadiazol-2-yl)-7-oxo-l, 6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (4.1 g, 7.72 mmol) was dissolved in a minimum amount of HPLC grade water (~ 40 mL) and passed through a column of 80 g of DOWEX 50WX 8 Na+ resin (the resin was prewased with >4 L of HPLC grade water) and eluted with HPLC grade water to afford sodium (25,5fl)-2-(l,3,4-oxadiazol-2-yl)-7-oxo-l,6- diazabicyclo[3.2.1]octan-6-yl sulfate (2.2 g, 91 %) as a white solid after lyophilization. ESI- MS (EI+, m/z): 291.2 [M+H]+. lH NMR (300 MHz, D20) δ 8.92 (s, 1H), 4.84 (d, J = 6.1 Hz, 1H), 4.20 (br s, 1H), 3.25-3.16 (m, 1H), 2.92 (d, / = 12.3 Hz, 1H), 2.41-2.26 (m, 1H), 2.26- 2.11 (m, 2H), 2.04-1.89 (m, 1H).

PATENT

WO-2016157057

Wockhardt Ltd

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016157057&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

A compound of Formula (I), chemically known as sodium salt of 2S, 5R) mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diazabicyclo[3.2.1 ]oct-6-yl)ester has antibacterial properties and is disclosed in PCT International Patent Application No. PCT/US2013/034562. The compound of Formula (I) is also generically disclosed in PCT International Patent Application No. PCT/IB2012/054296. The present invention discloses a process for preparation of a compound of Formula (I).

Formula (I)

Scheme 1.

(VI) Compound of Formula (I)

Example 1

Sodium salt of (25, 5R) sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester

Step I: Synthesis of (25,5R)-2-(iV’-formyl-hydrazinocarbonyl)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1] octane (III):

To a turbid solution of sodium salt of (2<S’,5i?)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1 ] octane-2-carboxylic acid (II, 20 g, 0.067 mol) (prepared according to process disclosed in PCT/IB2013/059264) in dimethylformamide (200 ml) was added EDC hydrochloride (19.44 g, 0.10 mol) followed by formyl hydrazide (4.02 g, 0.067 mol) and N-hydroxybenzotriazole (9 g, 0.67 mol) at about 25°C under stirring. Diisopropylethylamine (35.62 ml, 0.20 mol) was added to the reaction mixture and stirred at 25°C temperature for 18 hours. The reaction mixture was evaporated under vacuum to provide a residue. The residue was dissolved in ethyl acetate (500 ml) and washed with water (500 ml χ 2), followed by saturated aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulphate and evaporated under vacuum to provide a crude intermediate, which was purified by silica gel column chromatography to provide 11 g of the titled compound as solid in 52% yield.

Analysis:

Mass: 319.1 (M+l); for Molecular Formula of C15H18N4O4 and Molecular Weight of 318.34;

H1 NMR (DMSO-d6): δ 9.93 (s, 1H), 9.87 (s, 1H), 8.01 (s, 1H), 7.36-7.46 (m, 5H), 4.91-4.97 (dd, 2H), 3.83-3.84 (br s, 1H), 3.70 (s, 1H), 3.15-3.18 (br s, 1H), 2.90-2.95 (m, 1H), 1.99-2.03(m, 1H), 1.86(br s, 1H), 1.73-1.75 (m, 1H), 1.66 (m, 1H).

Step II: Synthesis of (25,5R)-2-([l,3,4]-oxadiazol-2-yl)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1] octane (IV):

To a clear solution of (2<S’,5i?)-2-(N’-formyl-hydrazinocarbonyl)-6-benzyloxy-7-oxo-l ,6-diaza-bicyclo[3.2.1 ] octane (III, 11 g, 0.0345 mol) in chloroform (120 ml) was added diisopropylethylamine (18.31 ml, 0.1035 mol) and p-tolylsulfonylchloride (9.83 g, 0.0517 mol). The solution was stirred at 60°C for 15 hours. Reaction mixture was cooled to room temperature and water (100 ml) was added. Organic layer was dried over anhydrous sodium sulphate and evaporated under vacuum to provide a crude residue, which was purified by silica gel column chromatography to provide 7 g of the titled compound as a solid in 68% yield.

Analysis:

Mass: 301.3 (M+l); for Molecular Formula of Ci5Hi6N403 and Molecular Weight of 300.32;

H1 NMR (CDC13): δ 8.45 (s, 1H), 7.25-7.44 (m, 5H), 5.07-5.10 (dd, 1H), 4.92-4.95 (dd, 1H), 4.76-4.78 (br s, 1H), 3.37 (br s, 1H), 2.93-.95 (br s, 1H), 2.75-2.77 (m, 1H), 2.32-2.33 (m, 2H), 2.13-2.16 (m, 1H), 1.93-2.01 (m, 1H).

Step III: Synthesis of (25,5R)-2-([l,3,4]-oxadiazol-2-yl)-6-hydroxy-7-oxo-l,6-diaza-bicyclo[3.2.1] octane (V):

To a clear solution of (2<S’,5i?)-2-([l,3,4]-oxadiazol-2-yl)-6-benzyloxy-7-oxo-l,6-diaza-bicyclo[3.2.1 ] octane (IV, 7.0 g, 0.0233 mmol) in methanol (70 ml) was added 10% palladium on carbon (2.5 g). The suspension was stirred under atmospheric hydrogen pressure at a temperature 25° C for 2 hrs. The catalyst was filtered over a celite bed and the bed was washed with methanol (30 ml). The filtrate was concentrated under vacuum to provide an oily residue. The residue was triturated with cyclohexane (100 ml) to effect solid formation. The suspension was filtered under suction and the wet cake was washed with additional cyclohexane (50 ml). The soild was dried under vacuum to provide 4.5 g of the titled compound as a whitish solid in 92% yield, which was used for the next reaction immediately.

Analysis:

Mass: 211.2 (M+l); for Molecular Formula of C8Hi0N4O3 and Molecular Weight of 210.19; 1H NMR (DMSO-d6): δ 9.88 (br s, 1H), 9.29 (s, 1H), 4.65 (d, 1H ), 4.64 (br s, 1H), 2.94-2.97 (br d, 1H), 2.63-2.66 (d, 1H), 1.89-2.09 (m,3H), 1.82-1.86 (m, 1H).

Step IV: Synthesis of tetrabutylammonium salt of (25, 5R)-sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester (VI):

To a clear solution of (2<S’,5i?)-2-([l,3,4]-oxadiazol-2-yl)-6-hydroxy-7-oxo-l ,6-diaza-bicyclo[3.2.1 ] octane (V, 4.5 g, 0.0214 mol) in dichloromethane (50 ml) was added triethylamine (9 ml, 0.642 mol), followed by the addition of sulfur trioxide pyridine complex (6.83 g, 0.428 mol). The resulting reaction mixture was stirred for 2 hours. Tetrabutylammonium hydrogen sulfate (7.26 g,

0.0214 mol) was added to the reaction mixture and it was stirred for 1.5 hours. A solution of aqueous 0.5 N KH2PO4 (100 ml) was added to the reaction mixture. Layers were separated and the aqueous layer was washed with dichloromethane (125 ml). Combined organic layer was dried over Na2S04, and was evaporated under vacuum to yield crude foam, which was purified on silica gel column chromatography to give 7 g of the titled compound as white foam in 98% yield.

Analysis:

Mass: 289.1 (M-l); for Molecular Formula
and Molecular Weight of 517.26;

1H NMR (DMSO-d6): δ 9.30 (s, 1H), 4.69 (d, 1H), 4.06 (br s, 1H ), 3.14-3.18 (m, 8H), 2.94-2.97 (br d, 1H), 2.67-2.70 (d, 1H), 1.98-2.05 (m,lH), 2.85-2.92 (m, 1H), 1.53-1.60 (m, 8H), 1.27-1.36 (m, 8H), 0.91-0.95 (m, 12H).

Step V: Sodium salt of (25, 5R)-sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester (I):

The compound sodium salt of (2S, 5i?)-sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester of Formula (I) was prepared by loading tetrabutylammonium salt of sulfuric acid mono-(2-[l,3,4]oxadiazol-2-yl-7-oxo-l,6-diaza-bicyclo[3.2.1]oct-6-yl) ester (VI, 7 g) on a column packed with Amberlite IR 120 Na form of resin, and by eluting the column with methanol water mixture (9: 1). Fractions containing compound were collected and solvent was evaporated under vacuum below 40°C, to provide formula- 1 compound in 4 gm (62%) quantity as a white solid.

Analysis:

Mass: 289.3 (M-l) as free acid; for Molecular Formula
and Molecular Weight 290.26;

H1 NMR (DMSO-d6): δ 9.29 (s, 1H), 4.70(d, 1H), 4.061 (d, 1H), 2.95 (d, 1H), 2.69 (d, 1H), 2.19 (m, 1H), 2.07 (m, 2H), 1.90 (m, 1H);

Purity as determined by HPLC: 89 86%;

WO2010056827A1 * Nov 12, 2009 May 20, 2010 Protez Pharmaceuticals, Inc. Beta-lactamase inhibitors
WO2010118361A1 * Apr 9, 2010 Oct 14, 2010 Sopharmia, Inc. Beta lactamase inhibitors
US20110294777 * Jan 15, 2009 Dec 1, 2011 Merck Sharp & Dohme Corp. Beta-lactamase inhibitors
Reference
1 * CROMPTON, I. E. ET AL.: “Beta-Lactamase inhibitors: The inhibition of serine beta-lactamases by specific boronic acids“, BIOCHEM. J., vol. 251, 1988, pages 453 – 459, XP055170895
2 * See also references of EP2831075A4

//////////CB-618, phase 1

O=S(=O)(O)ON3[C@H]1C[N@]([C@@H](CC1)c2nnco2)C3=O

BMS-986115


Figure imgf000170_0002

BMS-986115
CAS 1584647-27-7

(2R,3S)-N-((3S)-5-(3-Fluorophenyl)-9-methyl-2-oxo-2,3-dihydro-lH-l,4-benzodiazepin- 3-yl)-2, -bis(3,3,3-trifluoropropyl)succinamide

MW: 574.4945,  C26-H25-F7-N4-O3, UNII: LSK1L593UU

10-Nitrooleate, CTK3B7458, CTK3C3167, 9-Octadecenoic acid, 10-nitro-, 875685-46-4, AG-L-63109, 9-Octadecenoic acid, 10-nitro-, (9E)-, 88127-53-1

FOR advanced solid tumors

  • Originator Bristol-Myers Squibb
  • Class Antineoplastics
  • Mechanism of Action Amyloid precursor protein secretase inhibitors; Notch signalling pathway inhibitors
  • Phase I Solid tumours

Most Recent Events

  • 30 Aug 2016Bristol-Myers Squibb terminates a phase I trial for Solid tumours (late-stage disease, second-line therapy or greater) in USA, Australia and Canada (NCT01986218)
  • 25 Jan 2016Bristol-Myers Squibb completes enrolment in its phase I trial for Solid tumours in USA, Australia and Canada (NCT01986218)
  • 31 Dec 2013Phase-I clinical trials in Solid tumours (late-stage disease) in Canada & Australia (Oral)

DETAILS WILL BE UPDATED SOON………….

BMS-986115 is an orally bioavailable, gamma secretase (GS) and pan-Notch inhibitor, with potential antineoplastic activity. Upon administration, GS/pan-Notch inhibitor BMS 986115 binds to GS and blocks the proteolytic cleavage and release of the Notch intracellular domain (NICD), which would normally follow ligand binding to the extracellular domain of the Notch receptor. This prevents both the subsequent translocation of NICD to the nucleus to form a transcription factor complex and the expression of Notch-regulated genes. This results in the induction of apoptosis and the inhibition of growth of tumor cells that overexpress Notch. Overexpression of the Notch signaling pathway plays an important role in tumor cell proliferation and survival

 

Bristol-Myers Squibb
Ashvinikumar V. Gavai, George V. Delucca,Daniel O’MALLEY, Patrice Gill, Claude A. Quesnelle, Brian E. Fink, Yufen Zhao,Francis Y. Lee,
Applicant Bristol-Myers Squibb Company

str2

Ashvinikumar Gavai

Claude Quesnelle

Claude Quesnelle
Senior Research Investigator/Chemist at Bristol-Myers Squibb

str2

RICHARD LEE

 

 

 

Patrice Gill

Patrice Gill

Research scientist at BMS

Dan O’Malley (Rice University)
Currently: Bristol-Myers Squibb

PICTURES WILL BE UPDATED………….

Useful for the treatment of conditions related to the Notch pathway, such as cancer and other proliferative diseases.

Notch signaling has been implicated in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, apoptosis, and angiogenesis. (Bray, Nature Reviews Molecular Cell Biology, 7:678-689 (2006); Fortini, Developmental Cell 16:633-647 (2009)). The Notch proteins are single-pass heterodimeric transmembrane molecules. The Notch family includes 4 receptors, NOTCH 1-4, which become activated upon binding to ligands from the DSL family (Delta-like 1, 3, 4 and Jagged 1 and 2).

The activation and maturation of NOTCH requires a series of processing steps, including a proteolytic cleavage step mediated by gamma secretase, a multiprotein complex containing Presenilin 1 or Presenilin 2, nicastrin, APH1, and PEN2. Once NOTCH is cleaved, NOTCH intracellular domain (NICD) is released from the membrane. The released NICD translocates to the nucleus, where it functions as a transcriptional activator in concert with CSL family members (RBPSUH, “suppressor of hairless”, and LAG1). NOTCH target genes include HES family members, such as HES- 1. HES- 1 functions as transcriptional repressors of genes such as HERP 1 (also known as HEY2), HERP2 (also known as HEY1), and HATH1 (also known as ATOH1).

The aberrant activation of the Notch pathway contributes to tumorigenesis. Activation of Notch signaling has been implicated in the pathogenesis of various solid tumors including ovarian, pancreatic, as well as breast cancer and hematologic tumors such as leukemias, lymphomas, and multiple myeloma. The role of Notch inhibition and its utility in the treatment of various solid and hematological tumors are described in Miele, L. et al, Current Cancer Drug Targets, 6:313-323 (2006); Bolos, V. et al, Endocrine Reviews, 28:339-363 (2007); Shih, I.-M. et al, Cancer Research, 67: 1879- 1882 (2007); Yamaguchi, N. et al., Cancer Research, 68: 1881-1888 (2008); Miele, L., Expert Review Anti-cancer Therapy, 8: 1 197-1201 (2008); Purow, B., Current Pharmaceutical Biotechnology, 10: 154-160 (2009); Nefedova, Y. et al, Drug Resistance Updates, 1 1 :210-218 (2008); Dufraine, J. et al, Oncogene, 27:5132-5137 (2008); and Jun, H.T. et al, Drug Development Research, 69:319-328 (2008).

There remains a need for compounds that are useful as Notch inhibitors and that have sufficient metabolic stability to provide efficacious levels of drug exposure. Further, there remains a need for compounds useful as Notch inhibitors that can be orally or intravenously administered to a patient.

U.S. Patent No. 7,053,084 Bl discloses succinoylamino benzodiazepine compounds useful for treating neurological disorders such as Alzheimer’s Disease. The reference discloses that these succinoylamino benzodiazepine compounds inhibit gamma secretase activity and the processing of amyloid precursor protein linked to the formation of neurological deposits of amyloid protein. The reference does not disclose the use of these compounds in the treatment of proliferative diseases such as cancer.

Applicants have found potent compounds that have activity as Notch inhibitors and have sufficient metabolic stability to provide efficacious levels of drug exposure upon intravenous or oral administration. These compounds are provided to be useful as pharmaceuticals with desirable stability, bioavailability, therapeutic index, and toxicity values that are important to their drugability.

Image result for BMS 906024

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PATENTS

US-20150166489-A1

https://patentscope.wipo.int/search/en/detail.jsf?docId=US137591635&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

PATENT

US-20140087992-A1

https://www.google.com/patents/US20140087992

Example 1(2R,3S)—N-((3S)-5-(3-Fluorophenyl)-9-methyl-2-oxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamideFigure US20140087992A1-20140327-C00138

Intermediate 1A: (2S,3R)-tert-Butyl 6,6,6-trifluoro-3-(((S)-5-(3-fluorophenyl)-9-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate

Figure US20140087992A1-20140327-C00139

In a 100 mL round-bottomed flask, a solution of Intermediate B-1 (1683 mg, 5.94 mmol), Et3N (1.656 mL, 11.88 mmol), and Intermediate S-1 in DMF (20 mL) was treated with o-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (3815 mg, 11.88 mmol) and stirred at room temperature for 1 hour. The reaction mixture was diluted with water and saturated aqueous NaHCO3. An off white precipitate formed and was filtered and washed with water. The resulting solid was dried on the filter under a stream of nitrogen to give Intermediate 1A (3.7 g, 99% yield). MS (ES): m/z=632.4[M+H+]; HPLC: RT=3.635 min Purity=98%. (H2O/MeOH with TFA, CHROMOLITH® ODS S5 4.6×50 mm, gradient=4 min, wavelength=220 nm). 1H NMR (400 MHz, methanol-d4) δ 7.53 (t, J=4.5 Hz, 1H), 7.46-7.30 (m, 3H), 7.28-7.23 (m, 1H), 7.23-7.18 (m, 2H), 5.37 (s, 1H), 2.88 (td, J=10.4, 3.4Hz, 1H), 2.60 (td, J=10.2, 4.1 Hz, 1H), 2.54-2.40 (m, 1H), 2.47 (s, 3H), 2.33-2.12 (m, 3H), 1.98-1.69 (m, 4H), 1.51 (s, 9H).

Intermediate 1B: (2S,3R)-6,6,6-Trifluoro-3-(((S)-5-(3-fluorophenyl)-9-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid

Figure US20140087992A1-20140327-C00140

In a 250 mL round-bottomed flask, a solution of Intermediate 1A (3.7 g, 5.86 mmol) in DCM (25 mL) was treated with TFA (25 mL) and the resulting pale orange solution was stirred at room temperature for 1.5 hours. The reaction mixture was then concentrated to give Intermediate 1B. HPLC: RT=3.12 min (H2O/MeOH with TFA, CHROMOLITH® ODS S5 4.6×50 mm, gradient=4 min, wavelength=220 nm). MS (ES): m/z=576.3 (M+H)+. 1H NMR (400 MHz, methanol-d4) δ 7.54 (t, J=4.5 Hz, 1H), 7.49-7.29 (m, 3H), 7.28-7.15 (m, 3H), 5.38 (br. s., 1H), 2.89 (td, J=10.3, 3.7 Hz, 1H), 2.67 (td, J=9.9, 4.2Hz, 1H), 2.56-2.38 (m, 1H), 2.48 (s, 3H), 2.34-2.13 (m, 3H), 2.00-1.71 (m, 4H).

Example 1

In a 250 mL round-bottomed flask, a solution of Intermediate 1B (4.04 g, 5.86 mmol) in THF (50 mL) was treated with ammonia (2M in iPrOH) (26.4 mL, 52.7 mmol), followed by HOBT (1.795 g, 11.72 mmol) and EDC (2.246 g, 11.72 mmol). The resulting white suspension was stirred at room temperature overnight. The reaction mixture was diluted with water and saturated aqueous NaHCO3. The resulting solid was filtered, rinsed with water and then dried on the filter under a stream of nitrogen. The crude product was suspended in 20 mL of iPrOH and stirred at room temperature for 20 min and then filtered and washed with iPrOH and dried under vacuum to give 2.83 g of solid. The solid was dissolved in refluxing EtOH (100 mL) and slowly treated with 200 mg activated charcoal added in small portions. The hot mixture was filtered through CELITE® and rinsed with hot EtOH. The filtrate was reduced to half volume, allowed to cool and the white precipitate formed was filtered and rinsed with EtOH to give 2.57 g of white solid. A second recrystallization from EtOH (70 mL) afforded Example 1 (2.39 g, 70% yield) as a white solid. HPLC: RT=10.859 min (H2O/CH3CN with TFA, Sunfire C18 3.5 μm, 3.0×150 mm, gradient=15 min, wavelength=220 and 254 nm); MS (ES): m/z=575.3 [M+H+]; 1H NMR (400 MHz, methanol-d4) δ 7.57-7.50 (m, 1H), 7.47-7.30 (m, 3H), 7.29-7.15 (m, 3H), 5.38 (s, 1H), 2.85-2.75 (m, 1H), 2.59 (td, J=10.5, 4.0 Hz, 1H), 2.53-2.41 (m, 4H), 2.31-2.10 (m, 3H), 1.96-1.70 (m, 4H).

 

PATENT

WO-2014047372-A1

https://www.google.com/patents/WO2014047372A1?cl=en

Figure imgf000041_0001

Figure imgf000042_0001

Scheme 3

Figure imgf000044_0001
Figure imgf000045_0001

XII XI

Scheme 4

Figure imgf000047_0001

Intermediate S-l : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000053_0001

Intermediate S-IA: 3,3,3-Trifluoro ropyl trifluoromethanesulfonate

Figure imgf000053_0002

[00180] To a cold (-25 °C) stirred solution of 2,6-lutidine (18.38 mL, 158 mmol) in DCM (120 mL) was added Tf20 (24.88 mL, 147 mmol) over 3 min, and the mixture was stirred for 5 min. To the reaction mixture was added 3,3,3-trifluoropropan-l-ol (12 g, 105 mmol) over an interval of 3 min. After 2 hr, the reaction mixture was warmed to room temperature and stirred for 1 hr. The reaction mixture was concentrated to half its volume, then purified by loading directly on a silica gel column (330g ISCO) and the product was eluted with DCM to afford Intermediate S-IA (13.74 g, 53%) as a colorless oil. 1H NMR (400 MHz, CDC13) δ ppm 4.71 (2 H, t, J= 6.15 Hz), 2.49-2.86 (2 H, m).

Intermediate S-1B: (4S)-4-Benzyl-3-(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2-one

Figure imgf000054_0001

[00181] To a stirring solution of 5,5,5-trifluoropentanoic acid (14.76 g, 95 mmol) and DMF (0.146 rriL) in DCM (50 mL) was slowly added oxalyl chloride (8.27 mL, 95 mmol). After 2h, the mixture was concentrated to dryness. A separate flask was changed with (S)-4-benzyloxazolidin-2-one (16.75 g, 95 mmol) in THF (100 mL) and then cooled to -78 °C. To the solution was slowly added n-BuLi (2.5M, 37.8 mL, 95 mmol) over 10 min, stirred for 10 min, and then a solution of the above acid chloride in THF (50 mL) was slowly added over 5 min. The mixture was stirred for 30 min, and then warmed to room temperature. The reaction was quenched with sat aq NH4C1. Next, 10% aq LiCl was then added to the mixture, and the mixture was extracted with Et20. The organic layer was washed with sat aq NaHC03 then with brine, dried (MgSC^), filtered and concentrated to dryness. The residue was purified by Si02 chromatography (ISCO, 330 g column, eluting with a gradient from 100% hexane to 100% EtOAc) to afford the product Intermediate S-IB; (25.25 g, 85%): 1H NMR (400 MHz, CDC13) δ ppm 7.32-7.39 (2 H, m), 7.30 (1 H, d, J= 7.05 Hz), 7.18-7.25 (2 H, m), 4.64-4.74 (1 H, m), 4.17-4.27 (2 H, m), 3.31 (1 H, dd, J= 13.35, 3.27 Hz), 3.00-3.11 (2 H, m), 2.79 (1 H, dd, J= 13.35, 9.57 Hz), 2.16-2.28 (2 H, m), 1.93-2.04 (2 H, m).

Intermediate S-IC: tert- utyl (3R)-3-(((4S)-4-benzyl-2-oxo-l,3-oxazolidin-3- yl)carbonyl)-6,6,6-trifluoroh xanoate

Figure imgf000054_0002

[00182] To a cold (-78 °C), stirred solution of Intermediate S-IB (3.03 g, 9.61 mmol) in THF (20 mL) was added NaHMDS (1.0M in THF) (10.6 mL, 10.60 mmol) under a nitrogen atmosphere. After 2 hours, tert-butyl 2-bromoacetate (5.62 g, 28.8 mmol) was added neat via syringe at -78 °C and stirring was maintained at the same temperature. After 6 hours, the reaction mixture was warmed to room temperature. The reaction mixture was partitioned between saturated NH4C1 and EtOAc. The organic phase was separated, and the aqueous phase was extracted with EtOAc (3x). The combined organics were washed with brine, dried (Na2s04), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO

CombiFlash Rf, 5% to 100% solvent A/B = hexanes/EtOAc, REDISEP® Si02 120g). Concentration of the appropriate fractions provided Intermediate S-1C (2.79 g, 67.6%) as a colorless viscous oil: 1H NMR (400 MHz, CDC13) δ ppm 7.34 (2 H, d, J= 7.30 Hz), 7.24-7.32 (3 H, m), 4.62-4.75 (1 H, m, J= 10.17, 6.89, 3.43, 3.43 Hz), 4.15-4.25 (3 H, m), 3.35 (1 H, dd, J= 13.60, 3.27 Hz), 2.84 (1 H, dd, J= 16.62, 9.57 Hz), 2.75 (1 H, dd, J = 13.35, 10.07 Hz), 2.47 (1 H, dd, J= 16.62, 4.78 Hz), 2.11-2.23 (2 H, m), 1.90-2.02 (1 H, m), 1.72-1.84 (1 H, m), 1.44 (9 H, s).

Intermediate S-ID: (2R)-2-( -tert-Butoxy-2-oxoethyl)-5,5,5-trifluoropentanoic acid

Figure imgf000055_0001

[00183] To a cool (0 °C), stirred solution of Intermediate S-1C (2.17 g, 5.05 mmol) in THF (50 mL) and water (15 mL) was added a solution of LiOH (0.242 g, 10.11 mmol) and H202 (2.065 mL, 20.21 mmol) in H20 (2 mL). After 10 min, the reaction mixture was removed from the ice bath, stirred for lh, and then cooled to 0 °C. Saturated aqueous NaHCC”3 (25 mL) and saturated aqueous Na2s03 (25 mL) were added to the reaction mixture, and the mixture was stirred for 10 min, and then partially concentrated. The resulting mixture was extracted with DCM (2x), cooled with ice and made acidic with cone. HC1 to pH 3. The mixture was saturated with solid NaCl, extracted with EtOAc (3x), and then dried over MgS04, filtered and concentrated to a colorless oil to afford Intermediate S-ID, 1.2514g, 92%): 1H NMR (400 MHz, CDCI3) δ ppm 2.83-2.95 (1 H, m), 2.62-2.74 (1 H, m), 2.45 (1 H, dd, J= 16.62, 5.79 Hz), 2.15-2.27 (2 H, m), 1.88-2.00 (1 H, m), 1.75-1.88 (1 H, m), 1.45 (9 H, s). Intermediate S-l : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and Intermediate S-1E: (2R,3R)-3-(tert-butoxycarbonyl)- 6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

Figure imgf000056_0001

(S-1E)

[00184] To a cold (-78 °C) stirred solution of Intermediate S-1D (5 g, 18.50 mmol) in THF (60 mL) was slowly added LDA (22.2 mL, 44.4 mmol, 2.0M) over 7 min. After stirring for 2 hr, Intermediate S- 1 A (6.38 g, 25.9 mmol) was added to the reaction mixture over 3 min. After 60 min, the reaction mixture was warmed to -25 °C

(ice/MeOH/dry ice) and stirred for an additional 60 min at which time sat aq NH4C1 was added. The separated aqueous phase was acidified with IN HC1 to pH 3, and then extracted with Et20. The combined organic layers were washed with brine (2x), dried over MgS04, filtered and concentrated to provide a 1 :4 (II :I1E) mixture (as determined by 1H NMR) of Intermediate S-l and Intermediate S-1E (6.00 g, 89%) as a pale yellow solid. 1H NMR (500 MHz, CDC13) δ ppm 2.81 (1 H, ddd, J = 10.17, 6.32, 3.85 Hz), 2.63- 2.76 (1 H, m), 2.02-2.33 (4 H, m), 1.86-1.99 (2 H, m), 1.68-1.85 (2 H, m), 1.47 (9 H, s).

[00185] To a cold (-78 °C), stirred solution of a mixture of Intermediate S-l and Intermediate S-1E (5.97 g, 16.30 mmol) in THF (91 mL) was added LDA (19 mL, 38.0 mmol, 2.0M in THF/hexane/ethyl benzene) dropwise via syringe over 10 min (internal temperature never exceeded -65 °C, J-KEM® probe in reaction solution). The mixture was stirred for 15 min, and then warmed to room temperature (24 °C water bath), stirred for 15 min, and then cooled to -78 °C for 15 min. To the reaction mixture was added Et2AlCl (41 mL, 41.0 mmol, 1M in hexane) via syringe (internal temperature never exceeded -55 °C), and the mixture was stirred for 10 min, and then warmed to room temperature (24 °C bath) for 15 min and then back to -78 °C for 15 min. Meanwhile, a 1000 mL round bottom flask was charged with MeOH (145 mL) and precooled to -78 °C. With vigorous stirring the reaction mixture was transferred via cannula over 5 min to the MeOH. The flask was removed from the bath, ice was added followed by the slow addition of IN HC1 (147 mL, 147 mmol). Gas evolution was observed as the HC1 was added. The reaction mixture was allowed to warm to room temperature during which the gas evolution subsided. The reaction mixture was diluted with EtOAc (750 mL), saturated with NaCl, and the organic phase was separated, washed with a solution of potassium fluoride (8.52 g, 147 mmol) and IN HC1 (41 mL, 41.0 mmol) in water (291 mL), brine (100 mL), and then dried (Na2s04), filtered and concentrated under vacuum. 1H NMR showed the product was a 9: 1 mixture of Intermediate S-l and Intermediate S- 1E. The enriched mixture of Intermediate S-l and Intermediate S-1E (6.12 g, >99% yield) was obtained as a dark amber solid: 1H NMR (400 MHz, CDC13) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s).

Alternate procedure to make Intermediate S-l :

Intermediate S-IF: (2R,3 -1 -Benzyl 4-tert-butyl 2,3-bis(3,3,3-trifluoropropyl)succinate

Figure imgf000057_0001

[00186] To a stirred solution of a 9: 1 enriched mixture of Intermediate S-l and Intermediate S-1E (5.98 g, 16.33 mmol) in DMF (63 mL) were added potassium carbonate (4.06 g, 29.4 mmol) and benzyl bromide (2.9 mL, 24.38 mmol), the mixture was then stirred overnight at room temperature. The reaction mixture was diluted with EtOAc (1000 mL), washed with 10% LiCl (3×200 mL), brine (200 mL), dried (Na2S04), filtered, concentrated, and then dried under vacuum. The residue was purified by Si02 chromatography using a toluene:hexane gradient. Diastereomerically purified

Intermediate S-IF (4.81g, 65%) was obtained as a colorless solid: 1H NMR (400 MHz, chloroform-d) δ 7.32-7.43 (m, 5H), 5.19 (d, J= 12.10 Hz, 1H), 5.15 (d, J= 12.10 Hz, 1H), 2.71 (dt, J= 3.52, 9.20 Hz, 1H), 2.61 (dt, J= 3.63, 9.63 Hz, 1H), 1.96-2.21 (m, 4H), 1.69-1.96 (m, 3H), 1.56-1.67 (m, 1H), 1.45 (s, 9H).

Intermediate S-l : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000058_0001

[00187] To a solution of Intermediate S-1F (4.81 g, 10.54 mmol) in MeOH (100 mL) was added 10% palladium on carbon (wet, Degussa type, 568.0 mg, 0.534 mmol) in a H2– pressure flask. The vessel was purged with N2 (4x), then purged with H2 (2x), and finally, pressurized to 50 psi and shaken overnight. The reaction vessel was

depressurized and purged with nitrogen. The mixture was filtered through CELITE®, washed with MeOH and then concentrated and dried under vacuum. Intermediate S-1 (3.81 g, 99% yield)) was obtained as a colorless solid: 1H NMR (400 MHz, chloroform-d) δ 2.62-2.79 (m, 2H), 2.02-2.40 (m, 4H), 1.87-2.00 (m, 2H), 1.67-1.84 (m, 2H), 1.48 (s, 9H).

Alternate procedure to make Intermediate S-1 :

Intermediate S-1 : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000058_0002

[00188] Intermediate S-1 as a mixture with Intermediate S-IE was prepared in a similar procedure as above from Intermediate S-1D to afford a 1 :2.2 mixture of

Intermediate S-1 and Intermediate S-IE (8.60 g, 23.48 mmol), which was enriched using LDA (2.0 M solution in THF, ethyl benzene and heptane, 28.2 mL, 56.4 mmol) and diethyl aluminum chloride (1.0 M solution in hexane, 59 mL, 59.0 mmol) in THF (91 mL). After workup as described above, the resulting residue was found to be a 13.2: 1 (by 1H NMR) mixture of Intermediate S-1 and Intermediate S-IE, which was treated as follows: The crude material was dissolved in MTBE (43 mL). Hexanes (26 mL) were slowly charged to the reaction mixture while maintaining a temperature below 30 °C. The reaction mixture was stirred for 10 min. Next, tert-butylamine (2.7 mL, 1.1 eq) was charged slowly over a period of 20 minutes while maintaining a temperature below 30 °C. This addition was observed to be exothermic. The reaction mixture was stirred for 2 hrs below 30 °C and then filtered. The solid material was washed with 5:3 MTBE: hexane (80 mL), and the filtrate was concentrated and set aside. The filtered solid was dissolved in dichloromethane (300 mL), washed with IN HC1 (lOOmL), and the organic layer was washed with brine (100 mL x 2), and then concentrated under reduced pressure below 45 °C to afford Intermediate S-l (5.46 g, 64%).

A second alternate procedure for preparing Intermediate S-l :

Intermediate S-1G: tert- utyl 5,5,5-trifluoropentanoate

Figure imgf000059_0001

[00189] To a stirred solution of 5,5,5-trifluoropentanoic acid (5 g, 32.0 mmol) in THF (30 mL) and hexane (30 mL) at 0 °C, was added tert-butyl 2,2,2-trichloroacetimidate (11.46 mL, 64.1 mmol). The mixture was stirred for 15 min at 0 °C. Boron trifluoride etherate (0.406 mL, 3.20 mmol) was added and the reaction mixture was allowed to warm to room temperature overnight. To the clear reaction mixture was added solid NaHC03 (5 g) and stirred for 30 min. The mixture was filtered through MgSC^ and washed with hexanes (200 mL). The solution was allowed to rest for 45 min, and the resulting solid material was removed by filtering on the same MgSC^ filter again, washed with hexanes (100 mL) and concentrated under reduced pressure without heat. The volume was reduced to about 30 mL, filtered through a clean fritted funnel, washed with hexane (5 mL), and then concentrated under reduced pressure without heat. The resulting neat oil was filtered through a 0.45μιη nylon membrane filter disk to provide Intermediate S-1G (6.6 g, 31.4 mmol 98% yield) as a colorless oil: 1H NMR (400 MHz, CDC13) δ ppm 1.38 (s, 9 H) 1.74-1.83 (m, 2 H) 2.00-2.13 (m, 2 H) 2.24 (t, J= 7.28 Hz, 2 H). Intermediate S-1H: (4S)-4-(Propan-2-yl)-3-(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2- one

Figure imgf000060_0001

[00190] To a stirred solution of 5,5,5-trifluoropentanoic acid (5.04 g, 32.3 mmol) in DCM (50 mL) and DMF (3 drops) was added oxalyl chloride (3.4 mL, 38.8 mmol) dropwise over 5 min. The solution was stirred until all bubbling subsided. The reaction mixture was concentrated under reduced pressure to give pale yellow oil. To a separate flask charged with a solution of (4S)-4-(propan-2-yl)-l,3-oxazolidin-2-one (4.18 g, 32.4 mmol) in THF (100 mL) at -78 °C was added n-BuLi (2.5M in hexane) (13.0 mL, 32.5 mmol) dropwise via syringe over 5 min. After stirring for 10 min, the above acid chloride, dissolved in THF (20 mL), was added via cannula over 15 min. The reaction mixture was warmed to 0 °C, and was allowed to warm to room temperature as the bath warmed and stirred overnight. To the reaction mixture was added saturated NH4C1, and the mixture was extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na2s04), filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 60% solvent A/B = hexanes/EtOAc, REDISEP® Si02 120g). Concentration of the appropriate fractions provided Intermediate S-1H (7.39 g, 86%) as a colorless oil: 1H NMR (400 MHz, CDC13) δ ppm 4.44 (1 H, dt, J= 8.31, 3.53 Hz), 4.30 (1 H, t, J= 8.69 Hz), 4.23 (1 H, dd, J= 9.06, 3.02 Hz), 2.98-3.08 (2 H, m), 2.32-2.44 (1 H, m, J= 13.91, 7.02, 7.02, 4.03 Hz), 2.13-2.25 (2 H, m), 1.88-2.00 (2 H, m), 0.93 (3 H, d, J= 7.05 Hz), 0.88 (3 H, d, J= 6.80 Hz).

Intermediate S-1I: (2S,3R)-tert-Butyl 6,6,6-trifluoro-3-((S)-4-isopropyl-2- oxooxazolidine-3-carbonyl)-2-(3,3,3-trifluoropropyl)hexanoate, and Intermediate S-U: (2R,3R)-tert-Butyl 6,6,6-trifluoro-3-((S)-4-isopropyl-2-oxooxazolidine-3-carbonyl)-2- (3 ,3 ,3 -trifluoropropyl)hexanoate

Figure imgf000061_0001

[00191] To a cold (-78 °C), stirred solution of diisopropylamine (5.3 mL, 37.2 mmol) in THF (59 mL) under a nitrogen atmosphere was added n-BuLi (2.5M in hexane) (14.7 mL, 36.8 mmol). The mixture was then warmed to 0 °C to give a 0.5M solution of LDA. A separate vessel was charged with Intermediate S-1H (2.45 g, 9.17 mmol). The material was azeotroped twice with benzene (the RotoVap air inlet was fitted with a nitrogen inlet to completely exclude humidity), and then toluene (15.3 mL) was added. This solution was added to a flask containing dry lithium chloride (1.96 g, 46.2 mmol). To the resultant mixture, cooled to -78 °C, was added the LDA solution (21.0 mL, 10.5 mmol) and the mixture was stirred at -78 °C for 10 min, then warmed to 0 °C for 10 min., and then cooled to -78 °C. To a separate reaction vessel containing Intermediate S-1G (3.41 g, 16.07 mmol), also azeotroped twice with benzene, was added toluene (15.3 mL), cooled to -78 °C and LDA (37.0 mL, 18.5 mmol) was added. The resulting solution was stirred at -78 °C for 25 min. At this time the enolate derived from the ester was transferred via cannula into the solution of the oxazolidinone enolate and stirred at -78 °C for an additional 5 min, at which time the septum was removed and solid powdered bis(2- ethylhexanoyloxy)copper (9.02 g, 25.8 mmol) was rapidly added to the reaction vessel and the septum was replaced. The vessel was immediately removed from the cold bath and immersed into a warm water bath (40 °C) with rapid swirling and with a concomitant color change from the initial turquoise to brown. The reaction mixture was stirred for 20 min, was then poured into 5% aqueous NH4OH (360 mL) and extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na2s04), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 60% solvent A/B = hexanes/EtOAc, REDISEP® Si02 120g). Concentration of the appropriate fractions provided a mixture of Intermediate S- II and Intermediate S-1J (2.87 g, 66%) as a pale yellow viscous oil. 1H NMR showed the product was a 1.6: 1 mixture of diastereomers S-1LS-1J as determined by the integration of the multiplets at 2.74 and 2.84 ppm: 1H NMR (400 MHz, CDC13) δ ppm 4.43-4.54 (2 H, m), 4.23-4.35 (5 H, m), 4.01 (1 H, ddd, J= 9.54, 6.27, 3.51 Hz), 2.84 (1 H, ddd, J = 9.41, 7.28, 3.64 Hz), 2.74 (1 H, ddd, J= 10.29, 6.27, 4.02 Hz), 2.37-2.48 (2 H, m, J = 10.38, 6.98, 6.98, 3.51, 3.51 Hz), 2.20-2.37 (3 H, m), 1.92-2.20 (8 H, m), 1.64-1.91 (5 H, m), 1.47 (18 H, s), 0.88-0.98 (12 H, m). Intermediate S-1 : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and Intermediate S-IE: (2R,3R)-3-(tert-Butoxycarbonyl)- 6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

Figure imgf000062_0001

(S-IE)

[00192] To a cool (0 °C), stirred solution of Intermediate S-1I and Intermediate S-1 J (4.54 g, 9.51 mmol) in THF (140 mL) and water (42 mL) were sequentially added hydrogen peroxide (30% in water) (10.3 g, 91 mmol) and LiOH (685.3 mg, 28.6 mmol). The mixture was stirred for 1 hr. At this time the reaction vessel was removed from the cold bath and then stirred for 1.5 hr. To the reaction mixture were added saturated NaHC03 (45 mL) and saturated Na2s03 (15 mL), and then the mixture was partially concentrated under reduced pressure. The resulting crude solution was extracted with DCM (3x). The aqueous phase was acidified to pH~l-2 with IN HC1, extracted with DCM (3x) and then EtOAc (lx). The combined organics were washed with brine, dried (Na2s04), filtered and concentrated under reduced pressure to provide a mixture of Intermediates S-1 and S-IE (3.00 g, 86%) as a colorless oil: 1H NMR (400 MHz, CDC13) δ ppm 2.76-2.84 (1 H, m, diastereomer 2), 2.64-2.76 (3 H, m), 2.04-2.35 (8 H, m), 1.88- 2.00 (4 H, m), 1.71-1.83 (4 H, m), 1.48 (9 H, s, diastereomer 1), 1.46 (9 H, s,

diastereomer 2); 1H NMR showed a 1.7: 1 mixture of S-1E:S-1F by integration of the peaks for the t-butyl groups. Intermediate S-1 : (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and Intermediate S-IF: (2R,3R)-3-(fert-Butoxycarbonyl)- 6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

Figure imgf000063_0001

[00193] To a cold (-78 °C) stirred solution of diisopropylamine (1.7 mL, 11.93 mmol) in THF (19 mL) under a nitrogen atmosphere was added n-BuLi (2.5M in hexanes) (4.8 mL, 12.00 mmol). The mixture was stirred for 5 min and then warmed to 0 °C. In a separate vessel, to a cold (-78 °C) stirred solution of the mixture of Intermediates S-1 and S-1E (1.99 g, 5.43 mmol) in THF (18 mL) was added the LDA solution prepared above via cannula slowly over 25 min. The mixture was stirred for 15 min, then warmed to room temperature (placed in a 24 °C water bath) for 15 min, and then again cooled to -78 °C for 15 min. To the reaction mixture was added Et2AlCl (1M in hexane) (11.4 mL, 11.40 mmol) via syringe. The mixture was stirred for 10 min, warmed to room

temperature for 15 min and then cooled back to -78 °C for 15 min. Methanol (25 mL) was rapidly added, swirled vigorously while warming to room temperature, and then concentrated to ~l/4 the original volume. The mixture was dissolved in EtOAc and washed with IN HC1 (50 mL) and ice (75 g). The aqueous phase was separated and extracted with EtOAc (2x). The combined organics were washed with a mixture of KF (2.85g in 75 mL water) and IN HC1 (13 mL) [resulting solution pH 3-4], then with brine, dried (Na2s04), filtered and concentrated under reduced pressure to give a 9: 1 (S-LS-1E) enriched diastereomeric mixture (as determined by 1H NMR) of Intermediate S-1 and Intermediate S-1E (2.13 g, >99%) as a pale yellow viscous oil: 1H NMR (400 MHz, CDC13) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s).

Intermediate S-2: (2R,3S)-3-(fert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3- fluoropropyl)hexanoic acid

Figure imgf000064_0001

Intermediate S-2: (2R,3S)-3-(tert-Butoxycarbonyl)-7,7,7-trifluoro-2-(3,3,3- trifluoropropyl)heptanoic acid, and Intermediate S-2A: (2R,3R)-3-(tert-Butoxycarbonyl)- 7,7,7-trifluoro-2-(3,3,3-trifluoropropyl)heptanoic acid

Figure imgf000064_0002

(S-2A)

[00194] To a cold (-78 °C), stirred solution of Intermediate S-1D (1.72 g, 6.36 mmol) in THF (30 mL) was slowly added LDA (7.32 mL, 14.6 mmol) over 7 min. After stirring for 1 h, 4,4,4-trifluorobutyltrifluoromethanesulfonate (2.11 g, 8.11 mmol) was added to the reaction mixture over 2 min. After 15 min, the reaction mixture was warmed to -25 °C (ice/MeOH/dry ice) for lh, and then cooled to -78 °C. After 80 min, the reaction was quenched with a saturated aqueous NH4C1 solution (10 mL). The reaction mixture was further diluted with brine and the solution was adjusted to pH 3 with IN HC1. The aqueous layer was extracted with ether. The combined organics were washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to provide a mixture of Intermediates S-2 and S-2A (2.29 g, 95%) as a colorless oil. 1H NMR (400MHz, chloroform-d) δ 2.83-2.75 (m, 1H), 2.64 (ddd, J = 9.9, 6.7, 3.6 Hz, 1H), 2.32-2.03 (m, 5H), 1.98-1.70 (m, 3H), 1.69-1.52 (m, 3H), 1.50-1.42 (m, 9H). 1H NMR showed a 1 :4.5 mixture (S-2:S-2A) of diastereomers by integration of the peaks for the t- Bu groups.

Intermediate S-2: (2R,3S)-3-(fert-Butoxycarbonyl)-7,7,7-trifluoro-2-(3,3,3- trifluoropropyl)heptanoic acid, and Intermediate S-2A: (2R,3R)-3-(tert-Butoxycarbonyl)- 7,7,7-trifluoro-2-(3,3,3-trifluoropropyl)heptanoic acid

Figure imgf000065_0001

[00195] A mixture of Intermediate S-2 and Intermediate S-2A (2.29 g, 6.02 mmol) was dissolved in THF (38 mL) to give a colorless solution which was cooled to -78 °C. Then, LDA (7.23 mL, 14.5 mmol) (2.0M in heptane/THF/ethylbenzene) was slowly added to the reaction mixture over 3 min. After stirring for 15 min, the reaction mixture was placed in a room temperature water bath. After 15 min the reaction mixture was placed back in a -78 °C bath and then diethylaluminum chloride (14.5 mL, 14.5 mmol) (1M in hexane) was added slowly over 5 min. The reaction mixture was stirred at -78 °C. After 15 min, the reaction mixture was placed in a room temperature water bath for 10 min, and then cooled back to -78 °C. After 15 min, the reaction was quenched with MeOH (30.0 mL, 741 mmol), removed from the -78 °C bath and concentrated. To the reaction mixture was added ice and HC1 (60.8 mL, 60.8 mmol) and the resulting mixture was extracted with EtOAc (2x 200 mL). The organic layer was washed with potassium fluoride (3.50g, 60.3 mmol) in 55 mL H20 and 17.0 mL of IN HC1. The organics were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to provide an enriched mixture of Intermediate S-2 and Intermediate S-2A (2.25g, 98% yield) as a light yellow oil. 1H NMR (400MHz, chloroform-d) δ 2.83-2.75 (m, 1H), 2.64 (ddd, J= 9.9, 6.7, 3.6 Hz, 1H), 2.32-2.03 (m, 5H), 1.98-1.70 (m, 3H), 1.69-1.52 (m, 3H), 1.50-1.42 (m, 9H). 1H NMR showed a 9: 1 ratio in favor of the desired diastereomer Intermediate S-2.

Intermediate S-2B: (2R,3S)-1 -Benzyl 4-tert-butyl 2,3-bis(4,4,4-trifluorobutyl)succinate

Figure imgf000065_0002

[00196] To a stirred 9: 1 mixture of Intermediate S-2 and Intermediate S-2A (2.24 g, 5.89 mmoL) and potassium carbonate (1.60 g, 11.58 mmoL) in DMF (30 mL) was added benzyl bromide (1.20 mL, 10.1 mmoL)). The reaction mixture was stirred at room temperature for 19 h. The reaction mixture was diluted with ethyl acetate (400 mL) and washed with 10% LiCl solution (3 x 100 mL), brine (50 mL), and then dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under vacuum. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash 0%> to 100% solvent A/B = hexane/EtOAc, REDISEP® Si02 220 g, detecting at 254 nm, and monitoring at 220 nm). Concentration of the appropriate fractions provided Intermediate S-2B (1.59 g, 57.5%). HPLC: RT = 3.863 min (CHROMOLITH® SpeedROD column 4.6 x 50 mm, 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm), 1H NMR (400MHz, chloroform-d) δ 7.40-7.34 (m, 5H), 5.17 (d, J= 1.8 Hz, 2H), 2.73-2.64 (m, 1H), 2.55 (td, J= 10.0, 3.9 Hz, 1H), 2.16-1.82 (m, 5H), 1.79-1.57 (m, 3H), 1.53-1.49 (m, 1H), 1.45 (s, 9H), 1.37-1.24 (m, 1H).

Intermediate S-2: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(4,4,4- trifluorobutyl)hexanoic acid

Figure imgf000066_0001

[00197] To a stirred solution of Intermediate S-2B (1.59 g, 3.37 mmoL) in MeOH (10 mL) and EtOAc (10 mL) under nitrogen was added 10%> Pd/C (510 mg). The atmosphere was replaced with hydrogen and the reaction mixture was stirred at room temperature for 2.5 h. The palladium catalyst was filtered off through a 4 μΜ polycarbonate film and rinsed with MeOH. The filtrate was concentrated under reduced pressure to give intermediate S-2 (1.28 g, 99%). 1H NMR (400MHz, chloroform-d) δ 2.76-2.67 (m, 1H), 2.65-2.56 (m, 1H), 2.33-2.21 (m, 1H), 2.17-2.08 (m, 3H), 1.93 (dtd, J= 14.5, 9.9, 5.2 Hz, 1H), 1.84-1.74 (m, 2H), 1.70-1.52 (m, 3H), 1.48 (s, 9H).

Intermediate A- 1 : (2-Amino-3 -methylphenyl)(3 -fluorophenyl)methanone

Figure imgf000067_0001

Intermediate A-1 A: 2-Amino- -methoxy-N,3-dimethylbenzamide

Figure imgf000067_0002

[00198] In a 1 L round-bottomed flask was added 2-amino-3-methylbenzoic acid (11.2 g, 74.1 mmol) and Ν,Ο-dimethylhydroxylamine hydrochloride (14.45 g, 148 mmol) in DCM (500 mL) to give a pale brown suspension. The reaction mixture was treated with Et3N (35 mL), HOBT (11.35 g, 74.1 mmol) and EDC (14.20 g, 74.1 mmol) and then stirred at room temperature for 24 hours. The mixture was then washed with 10% LiCl, and then acidified with IN HCl. The organic layer was washed successively with 10%> LiCl and aq NaHC03. The organic layer was decolorized with charcoal, filtered, and the filtrate was dried over MgSC^. The mixture was filtered and concentrated to give 13.22 g (92% yield) of Intermediate A-1A. MS(ES): m/z = 195.1 [M+H+]; HPLC: RT = 1.118 min. (H20/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm); 1H NMR (500MHz, chloroform-d) δ 7.22 (dd, J= 7.8, 0.8 Hz, 1H), 7.12-7.06 (m, 1H), 6.63 (t, J= 7.5 Hz, 1H), 4.63 (br. s., 2H), 3.61 (s, 3H), 3.34 (s, 3H), 2.17 (s, 3H).

Intermediate A- 1 : (2-Amino-3 -methylphenyl)(3 -fluorophenyl)methanone

Figure imgf000067_0003

[00199] In a 500 mL round-bottomed flask, a solution of l-fluoro-3-iodobenzene (13.61 mL, 116 mmol) in THF (120 mL) was cooled in a -78 °C bath. A solution of n- BuLi, (2.5M in hexane, 46.3 mL, 116 mmol) was added dropwise over 10 minutes. The solution was stirred at -78 °C for 30 minutes and then treated with a solution of

Intermediate A-1 A (6.43 g, 33.1 mmol) in THF (30 mL). After 1.5 hours, the reaction mixture was added to a mixture of ice and IN HCl (149 mL, 149 mmol) and the reaction flask was rinsed with THF (5 ml) and combined with the aqueous mixture. The resulting mixture was diluted with 10% aq LiCl and the pH was adjusted to 4 with IN NaOH. The mixture was then extracted with Et20, washed with brine, dried over MgS04, filtered and concentrated. The resulting residue was purified by silica gel chromatography (220g ISCO) eluting with a gradient from 10% EtOAc/hexane to 30% EtOAc/hexane to afford Intermediate A-l (7.11 g, 94% yield) as an oil. MS(ES): m/z = 230.1 [M+H+]; HPLC: RT = 2.820 min Purity = 99%. (H20/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm).

Intermediate B-1 : (S)-3-Amino-5-(3-fluorophenyl)-9-methyl-lH-benzo[e][l,4]diazepin- 2(3H)-one

Figure imgf000085_0001

Intermediate B-1 A: (S)-Benzyl (5-(3-fluorophenyl)-9-methyl-2-oxo-2,3-dihydro benzo[e] [ 1 ,4]diazepin-3-yl)carbamate

Figure imgf000085_0002

(B-1A)

[00225] In a 1 L round-bottomed flask, a solution of 2-(lH-benzo[d][l,2,3]triazol-l- yl)-2-((phenoxycarbonyl)amino)acetic acid (J. Org. Chem., 55:2206-2214 (1990)) (19.37 g, 62.0 mmol) in THF (135 mL) was cooled in an ice/water bath and treated with oxalyl chloride (5.43 mL, 62.0 mmol) and 4 drops of DMF. The reaction mixture was stirred for 4 hours. Next, a solution of Intermediate A- 1 (7.11 g, 31.0 mmol) in THF (35 mL) was added and the resulting solution was removed from the ice/water bath and stirred at room temperature for 1.5 hours. The mixture was then treated with a solution of ammonia, (7M in MeOH) (19.94 mL, 140 mmol). After 15 mins, another portion of ammonia, (7M in MeOH) (19.94 mL, 140 mmol) was added and the resulting mixture was sealed under N2 and stirred overnight at room temperature. The reaction mixture was then concentrated to ~l/2 volume and then diluted with AcOH (63 mL) and stir at room temperature for 4 hours. The reaction mixture was then concentrated, and the residue was diluted with 500 mL water to give a precipitate. Hexane and Et20 were added and the mixture was stirred at room temperature for 1 hour to form an orange solid. Et20 was removed under a stream of nitrogen and the aqueous layer was decanted. The residue was triturated with 40 mL of iPrOH and stirred at room temperature to give a white precipitate. The solid was filtered and washed with iPrOH, then dried on a filter under a stream of nitrogen to give racemic Intermediate B-1A (5.4 g, 41.7%yield).

[00226] Racemic Intermediate B-1A (5.9 g, 14.3 mmol) was resolved using the Chiral SFC conditions described below. The desired stereoisomer was collected as the second peak in the elution order: Instrument: Berger SFC MGIII, Column: CHIRALPAK® IC 25 x 3 cm, 5 cm; column temp: 45 °C; Mobile Phase: C02/MeOH (45/55); Flow rate: 160 mL/min; Detection at 220 nm.

[00227] After evaporation of the solvent, Intermediate B-1A (2.73 g, 46% yield) was obtained as a white solid. HPLC: RT = 3.075 min. (H20/MeOH with TFA,

CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm).

Chiral HPLC RT: 8.661 min (AD, 60% (EtOH/MeOH)/heptane) > 99%ee. MS(ES): m/z = 418.3 [M+H+];1H NMR (500MHz, DMSO-d6) δ 10.21 (s, 1H), 8.38 (d, J= 8.3 Hz, 1H), 7.57-7.47 (m, 2H), 7.41-7.29 (m, 8H), 7.25-7.17 (m, 2H), 5.10-5.04 (m, 3H), 2.42 (s, 3H).

Intermediate B-l : (S)-3-Amino-5-(3-fluorophenyl)-9-methyl-lH-benzo[e][l,4]diazepin- 2(3H)-one.

[00228] In a 100 mL round-bottomed flask, a solution of Intermediate B-1A (2.73 g, 6.54 mmol) in acetic acid (12 mL) was treated with HBr, 33% in HOAc (10.76 mL, 65.4 mmol) and the mixture was stirred at room temperature for 1 hour. The solution was diluted with Et20 to give a yellow precipitate. The yellow solid was filtered and rinsed with Et20 under nitrogen. The solid was transferred to 100 mL round bottom flask and water was added (white precipitate formed). The slurry was slowly made basic with saturated NaHC03. The resulting tacky precipitate was extracted with EtOAc. The organic layer was washed with water, dried over MgS04, and then filtered and

concentrated to dryness to give Intermediate B-l (1.68 g, 91% yield) as a white foam solid. MS(ES): m/z = 284.2 [M+H+]; HPLC: RT = 1.72 min (H20/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm). 1H NMR (400MHz, DMSO-d6) δ 10.01 (br. s., 1H), 7.56-7.44 (m, 2H), 7.41-7.26 (m, 3H), 7.22-7.11 (m, 2H), 4.24 (s, 1H), 2.55 (br. s., 2H), 2.41 (s, 3H). [00229] The compounds listed below in Table 6 (Intermediates B-2 to B-3) were prepared according to the general synthetic procedure described for Intermediate B-l , using the starting materials Intermediate A- 10 and Intermediate A-4, respectively.

 

Example 1

(2R,3S)-N-((3S)-5-(3-Fluorophenyl)-9-methyl-2-oxo-2,3-dihydro-lH-l,4-benzodiazepin- 3-yl)-2, -bis(3,3,3-trifluoropropyl)succinamide

Figure imgf000098_0001

Intermediate 1A: (2S,3R)-tert-Butyl 6,6,6-trifluoro-3-(((S)-5-(3-fluorophenyl)-9-methyl- 2-0X0-2, 3-dihydro-lH-benzo[e][l,4]diazepin-3-yl)carbamoyl)-2-(3,3 ,3- trifluoropropyl)hexanoat

Figure imgf000098_0002

[00240] In a 100 mL round-bottomed flask, a solution of Intermediate B-l (1683 mg, 5.94 mmol), Et3N (1.656 mL, 11.88 mmol), and Intermediate S-l in DMF (20 mL) was treated with o-benzotriazol-l-yl-A .A .N’.N’-tetramethyluronium tetrafluoroborate (3815 mg, 11.88 mmol) and stirred at room temperature for 1 hour. The reaction mixture was diluted with water and saturated aqueous NaHC03. An off white precipitate formed and was filtered and washed with water. The resulting solid was dried on the filter under a stream of nitrogen to give Intermediate 1A (3.7 g, 99% yield). MS(ES): m/z =

632.4[M+H+]; HPLC: RT = 3.635 min Purity = 98%. (H20/MeOH with TFA,

CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm). 1H NMR (400MHz, methanol-d4) δ 7.53 (t, J = 4.5 Hz, 1H), 7.46-7.30 (m, 3H), 7.28-7.23 (m, 1H), 7.23-7.18 (m, 2H), 5.37 (s, 1H), 2.88 (td, J = 10.4, 3.4 Hz, 1H), 2.60 (td, J =

10.2, 4.1 Hz, 1H), 2.54-2.40 (m, 1H), 2.47 (s, 3 H), 2.33-2.12 (m, 3H), 1.98-1.69 (m, 4H), 1.51 (s, 9H). Intermediate IB: (2S,3R)-6,6,6-Trifluoro-3-(((S)-5-(3-fluorophenyl)-9-methyl-2-oxo-

2,3-dihydro-lH-benzo[e][l,4]diazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid

Figure imgf000099_0001

[00241] In a 250 mL round-bottomed flask, a solution of Intermediate 1A (3.7 g, 5.86 mmol) in DCM (25 mL) was treated with TFA (25 mL) and the resulting pale orange solution was stirred at room temperature for 1.5 hours. The reaction mixture was then concentrated to give Intermediate IB. HPLC: RT = 3.12 min (H20/MeOH with TFA, CHROMOLITH® ODS S5 4.6 x 50 mm, gradient = 4 min, wavelength = 220 nm).

MS(ES): m/z = 576.3 (M+H)+. 1H NMR (400MHz, methanol-d4) δ 7.54 (t, J= 4.5 Hz, 1H), 7.49-7.29 (m, 3H), 7.28-7.15 (m, 3H), 5.38 (br. s., 1H), 2.89 (td, J= 10.3, 3.7 Hz, 1H), 2.67 (td, J= 9.9, 4.2 Hz, 1H), 2.56-2.38 (m, 1H), 2.48 (s, 3 H), 2.34-2.13 (m, 3H), 2.00-1.71 (m, 4H).

Example 1 :

[00242] In a 250 mL round-bottomed flask, a solution of Intermediate IB (4.04 g, 5.86 mmol) in THF (50 mL) was treated with ammonia (2M in iPrOH) (26.4 mL, 52.7 mmol), followed by HOBT (1.795 g, 11.72 mmol) and EDC (2.246 g, 11.72 mmol). The resulting white suspension was stirred at room temperature overnight. The reaction mixture was diluted with water and saturated aqueous NaHC03. The resulting solid was filtered, rinsed with water and then dried on the filter under a stream of nitrogen. The crude product was suspended in 20 mL of iPrOH and stirred at room temperature for 20 min and then filtered and washed with iPrOH and dried under vacuum to give 2.83 g of solid. The solid was dissolved in re fluxing EtOH(100 mL) and slowly treated with 200 mg activated charcoal added in small portions. The hot mixture was filtered through CELITE® and rinsed with hot EtOH. The filtrate was reduced to half volume, allowed to cool and the white precipitate formed was filtered and rinsed with EtOH to give 2.57 g of white solid. A second recrystallization from EtOH (70 mL) afforded Example 1 (2.39 g, 70% yield) as a white solid. HPLC: RT = 10.859 min (H20/CH3CN with TFA, Sunfire C18 3.5μπι, 3.0x150mm, gradient = 15 min, wavelength = 220 and 254 nm); MS(ES): m/z = 575.3 [M+H+]; 1H NMR (400MHz, methanol-d4) δ 7.57-7.50 (m, 1H), 7.47-7.30 (m, 3H), 7.29-7.15 (m, 3H), 5.38 (s, 1H), 2.85-2.75 (m, 1H), 2.59 (td, J= 10.5, 4.0 Hz, 1H), 2.53-2.41 (m, 4H), 2.31-2.10 (m, 3H), 1.96-1.70 (m, 4H).

 

SEE

WO2012129353A1 *Mar 22, 2012Sep 27, 2012Bristol-Myers Squibb CompanyBis(fluoroalkyl)-1,4-benzodiazepinone compounds

 

PAPER RELATED

Structure–activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors

Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, United States
Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States
§ Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,United States
ACS Med. Chem. Lett., 2015, 6 (5), pp 523–527
*Phone: 609-252-5091. E-mail: ashvinikumar.gavai@bms.com.
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Patent

http://www.google.co.in/patents/WO2012129353A1?cl=en

 

PATENT RELATED

US-20160060232-A1

https://patentscope.wipo.int/search/en/detail.jsf?docId=US159930181&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

 

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Clip RELATED

For some disease targets, an indirect approach may be best. Or so Ashvinikumar V. Gavai and his colleagues atBristol-Myers Squibbfound in their quest toward a potential cancer drug. Gavai unveiled BMS-906024, which is an experimental—and slightly roundabout—treatment for a number of cancers, including breast, lung, and colon cancers, and leukemia.

Cancers have a tendency to relapse or to become resistant to treatments that once worked. Research at BMS and elsewhere had suggested that a family of proteins called Notch is implicated in that resistance and in cancer progression more generally. Gavai, director of oncology chemistry at BMS in Princeton, N.J., and his team set out to block Notch family signaling.

Notch family members lack enzymatic activity, so blocking them directly is difficult. Instead, BMS developed inhibitors of an enzyme that is essential for activating Notch signaling—γ-secretase.

09116-cover-bms906024

Company: Bristol-Myers Squibb

Target: pan-Notch

Disease: breast, lung, colon cancer; leukemia

Interfering with Notch, even in this indirect way, can have detrimental effects on the gastrointestinal tract. Only two of the four Notch family members are linked to that side effect, Gavai says. But he and his team think their drug will be most effective if it acts on all four family members roughly equally—a so-called pan-Notch inhibitor. By selecting a molecule that’s well tolerated in animals and carefully scheduling doses of the drug in humans, it could be possible to minimize side effects, he says.

The BMS team relied on Notch signaling assays in leukemia and breast cancer cell lines to find leads. They soon learned that for their molecules to work, three chiral centers had to be in the S,R,Sconfiguration. After that, they strove to make the molecules last in the bloodstream. They removed an isobutyl group and tweaked some other parts of their candidate’s succinamide side chain. It was tough to retain both a long half-life and activity against Notch, Gavai told C&EN. “You’d optimize one and lose the other.”

His team threaded the needle with BMS-906024. Their studies with mice suggest that a dose of 4–6 mg once a week could be effective in people. That’s lower than doses being tested for other Notch-targeted agents, according to the website clinicaltrials.gov. The mouse studies also back the idea that Notch is involved in cancer drug resistance and suggest that Notch could be a target for taking on cancer stem cells, which are notoriously resistant to chemotherapy.

BMS-906024 is in Phase I clinical trials, both alone and in combination with other agents. Patients with colon, lung, breast, and other cancers are receiving intravenous doses of the compound to determine its safety and optimum dose ranges.

09116-cover-BMScxd

(From left, front row) Gavai, Weifeng Shan, (second row) Aaron Balog, Patrice Gill, Gregory Vite, (third row) Francis Lee, Claude Quesnelle, (rear row) Wen-Ching Han, Richard Westhouse.

Credit: Catherine Stroud Photography

http://cen.acs.org/articles/91/i16/BMS-906024-Notch-Signaling-Inhibitor.html

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PAPER RELATED

Abstract Image

An enantioselective synthesis of (S)-7-amino-5H,7H-dibenzo[b,d]azepin-6-one (S1) is described. The key step in the sequence involved crystallization-induced dynamic resolution (CIDR) of compound 7 using Boc-d-phenylalanine as a chiral resolving agent and 3,5-dichlorosalicylaldehyde as a racemization catalyst to afford S1 in 81% overall yield with 98.5% enantiomeric excess.

Crystallization-Induced Dynamic Resolution toward the Synthesis of (S)-7-Amino-5H,7H-dibenzo[b,d]-azepin-6-one: An Important Scaffold for γ-Secretase Inhibitors

Department of Discovery Synthesis, Biocon Bristol-Myers Squibb Research Centre, Biocon Park, Bommasandra IV Phase, Jigani Link Road, Bengaluru 560099, India
Bristol-Myers Squibb Company, P.O Box 4000, Princeton, New Jersey 08543-4000, United States
Org. Process Res. Dev., Article ASAP
Cited Patent Filing date Publication date Applicant Title
WO2000007995A1 * Aug 7, 1999 Feb 17, 2000 Du Pont Pharmaceuticals Company SUCCINOYLAMINO LACTAMS AS INHIBITORS OF Aβ PROTEIN PRODUCTION
WO2000038618A2 * Dec 23, 1999 Jul 6, 2000 Du Pont Pharmaceuticals Company SUCCINOYLAMINO BENZODIAZEPINES AS INHIBITORS OF Aβ PROTEIN PRODUCTION
WO2001060826A2 * Feb 16, 2001 Aug 23, 2001 Bristol-Myers Squibb Pharma Company SUCCINOYLAMINO CARBOCYCLES AND HETEROCYCLES AS INHIBITORS OF Aβ PROTEIN PRODUCTION
US6737038 * May 17, 2000 May 18, 2004 Bristol-Myers Squibb Company Use of small molecule radioligands to discover inhibitors of amyloid-beta peptide production and for diagnostic imaging
US7053084 Feb 17, 2000 May 30, 2006 Bristol-Myers Squibb Company Succinoylamino benzodiazepines as inhibitors of Aβ protein production
US7456172 Jan 13, 2006 Nov 25, 2008 Bristol-Myers Squibb Pharma Company Succinoylamino benzodiazepines as inhibitors of Aβ protein production
US20030134841 * Nov 1, 2002 Jul 17, 2003 Olson Richard E. Succinoylamino lactams as inhibitors of A-beta protein production
US20120245151 * Mar 22, 2012 Sep 27, 2012 Bristol-Myers Squibb Company Bisfluoroalkyl-1,4-benzodiazepinone compounds

 

//////////BMS-986115, BMS 986115, 3,5-dichlorosalicylaldehyde, Alzheimer’s disease, Boc-D-phenylalanine, CIDR;dibenzoazepenone DKR; Notch inhibitorsNotch inhibitor, SAR T-acute lymphoblastic leukemia, triple-negative breast cancer, γ-secretase inhibitor, PHASE 1, BMS, Bristol-Myers Squibb,  Ashvinikumar Gavai1584647-27-7, UNII: LSK1L593UU

Cc1cccc2c1NC(=O)[C@H](N=C2c3cccc(c3)F)NC(=O)[C@H](CCC(F)(F)F)[C@H](CCC(F)(F)F)C(=O)N

BMS 906024


BMS-906024.pngBMS-906024.svg

 

Figure imgf000065_0001

BMS 906024

cas 1401066-79-2

  • MF C26H26F6N4O3
  • MW 556.500

(2R,3S)-N-[(3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]-2,3-bis(3,3,3-trifluoropropyl)succinamide

Butanediamide, N1-((3S)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluorophenyl)-, (2R,3S)-

(2R,35)-N-((35)-l-Methyl-2-oxo-5-phenyl-2,3-dihydro-lH-l,4-benzodiazepin-3-yl)-3- (2,2,2-trifluoroethyl)-2-(3,3,3-trifluoropropyl)succinamide

Claude Quesnelle, Soong-Hoon Kim, Francis Lee, Ashvinikumar Gavai
Applicant Bristol-Myers Squibb Company

 

str2

Ashvinikumar Gavai

 

 

Claude Quesnelle

Claude Quesnelle
Senior Research Investigator/Chemist at Bristol-Myers Squibb

str2

RICHARD LEE

BMS-906024 is a novel, potent Notch receptor inhibitor . Cancers have a tendency to relapse or to become resistant to treatments that once worked. A family of proteins called Notch is implicated in that resistance and in cancer progression more generally. BMS-906024 is in Phase I clinical trials, both alone and in combination with other agents. Patients with colon, lung, breast, and other cancers are receiving intravenous doses of the compound to determine its safety and optimum dose ranges.

New Phase I drug structure by Bristol-Myers Squibb disclosed at the spring 2013 American Chemical Society meeting in New Orleans to treat breast, lung, and colon cancers and leukemia.[1] The drug works as an pan-Notch inhibitor. The structure is one of a set patented in 2012,[2] and it currently being studied in clinical trials.[3][4]

useful for the treatment of conditions related to the Notch pathway, such as cancer and other proliferative diseases.

Notch signaling has been implicated in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, apoptosis, and angiogenesis. (Bray, Nature Reviews Molecular Cell Biology, 7:678-689 (2006); Fortini, Developmental Cell 16:633-647 (2009)). The Notch proteins are single-pass heterodimeric transmembrane molecules. The Notch family includes 4 receptors, NOTCH 1-4, which become activated upon binding to ligands from the DSL family (Delta-like 1, 3, 4 and Jagged 1 and 2).

The activation and maturation of NOTCH requires a series of processing steps, including a proteolytic cleavage step mediated by gamma secretase, a multiprotein complex containing Presenilin 1 or Presenilin 2, nicastrin, APH1, and PEN2. Once NOTCH is cleaved, NOTCH intracellular domain (NICD) is released from the membrane. The released NICD translocates to the nucleus, where it functions as a transcriptional activator in concert with CSL family members (RBPSUH, “suppressor of hairless”, and LAG1). NOTCH target genes include HES family members, such as HES- 1. HES- 1 functions as transcriptional repressors of genes such as HERP 1 (also known as HEY2), HERP2 (also known as HEY1), and HATH1 (also known as ATOH1).

The aberrant activation of the Notch pathway contributes to tumorigenesis. Activation of Notch signaling has been implicated in the pathogenesis of various solid tumors including ovarian, pancreatic, as well as breast cancer and hematologic tumors such as leukemias, lymphomas, and multiple myeloma. The role of Notch inhibition and its utility in the treatment of various solid and hematological tumors are described in Miele, L. et al, Current Cancer Drug Targets, 6:313-323 (2006); Bolos, V. et al, Endocrine Reviews, 28:339-363 (2007); Shih, I.-M. et al, Cancer Research, 67: 1879- 1882 (2007); Yamaguchi, N. et al., Cancer Research, 68: 1881-1888 (2008); Miele, L., Expert Review Anti-cancer Therapy, 8: 1 197-1201 (2008); Purow, B., Current Pharmaceutical Biotechnology, 10: 154-160 (2009); Nefedova, Y. et al, Drug Resistance Updates, 1 1 :210-218 (2008); Dufraine, J. et al, Oncogene, 27:5132-5137 (2008); and Jun, H.T. et al, Drug Development Research, 69:319-328 (2008).

There remains a need for compounds that are useful as Notch inhibitors and that have sufficient metabolic stability to provide efficacious levels of drug exposure. Further, there remains a need for compounds useful as Notch inhibitors that can be orally or intravenously administered to a patient.

U.S. Patent No. 7,053,084 Bl discloses succinoylamino benzodiazepine compounds useful for treating neurological disorders such as Alzheimer’s Disease. The reference discloses that these succinoylamino benzodiazepine compounds inhibit gamma secretase activity and the processing of amyloid precursor protein linked to the formation of neurological deposits of amyloid protein. The reference does not disclose the use of these compounds in the treatment of proliferative diseases such as cancer.

Applicants have found potent compounds that have activity as Notch inhibitors and have sufficient metabolic stability to provide efficacious levels of drug exposure upon intravenous or oral administration. These compounds are provided to be useful as pharmaceuticals with desirable stability, bioavailability, therapeutic index, and toxicity values that are important to their drugability.

Image result for BMS 906024Image result for BMS 906024

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PAPER

Abstract Image

Structure–activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors

Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, United States
Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States
§ Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,United States
ACS Med. Chem. Lett., 2015, 6 (5), pp 523–527
*Phone: 609-252-5091. E-mail: ashvinikumar.gavai@bms.com.
(2R,3S)-N-((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4- benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide
colorless solid: HPLC: RT = 9.60 min (HPLC Method D). Chiral LC/Analytical SFC conditions: Column: LuxCellulose-2 (0.46 x 25cm), Mobile phase: 10% methanol in CO2, Flow rate: 3 mL/min, wavelength: 220 nm; Temp.: 35C. RT = 9.21 min, Purity = 99.95%.
MS (ES): m/z = 557 [M+H]+ ;
1H NMR (400 MHz, DMSO-d6)  9.54 (1H, d, J = 7.28 Hz), 7.71 – 7.80 (1H, m), 7.68 (2H, d, J = 8.78 Hz), 7.50 – 7.62 (3H, m), 7.45 (2H, t, J = 7.28 Hz), 7.29 – 7.40 (2H, m), 7.15 (1H, s), 5.30 (1H, d, J = 7.28 Hz), 3.39 (3H, s), 2.74 – 2.86 (1H, m), 2.02 -2.32 (3H, m), 1.45 – 1.79 (4H, m);
[]D = -107.0° (5.73 mg/mL, DMSO).
Elemental analysis: Theoretical: C: 54.11%; H: 4.70%; N: 10.06%; Actual: C: 54.06%; H: 4.90%; N: 10.08%.
Karl Fisher Moisture: 0.48.
HPLC Method D: Sunfire C18 3.5um, 3.0x150mm column, solvent A: 5% acetonitrile – 95% water – 0.05% TFA, solvent B: 95% acetonitrile – 5% water – 0.05% TFA, flow=0.5 mL/min, gradient from 10%B to 100%B over 15min, 254 nm detector.
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Patent

http://www.google.co.in/patents/WO2012129353A1?cl=en

Example 1

(2R,35)-N-((35′)-l-Methyl-2-oxo-5-phenyl-2,3-dihydro-lH-l,4-benzodiazepin-3-yl)-2,3- b -trifluoropropy l)succinamide

Figure imgf000065_0001

Preparation 1A: tert-Butyl 5, -trifluoropentanoate

Figure imgf000065_0002

[00219] To a stirred solution of 5,5,5-trifluoropentanoic acid (5 g, 32.0 mmol) in THF (30 mL) and hexane (30 mL) at 0 °C, was added tert-butyl 2,2,2-trichloroacetimidate (11.46 mL, 64.1 mmol). The mixture was stirred for 15 min at 0 °C. Boron trifluoride etherate (0.406 mL, 3.20 mmol) was added and the reaction mixture was allowed to warm to room temperature overnight. To the clear reaction mixture was added solid aHC03 (5 g) and stirred for 30 min. The mixture was filtered through MgS04 and washed with hexanes (200 mL). The solution was allowed to rest for 45 min, and the resulting solid material was removed by filtering on the same MgS04 filter again, washed with hexanes (100 mL) and concentrated under reduced pressure without heat. The volume was reduced to about 30 mL, filtered through a clean fritted funnel, washed with hexane (5 mL), and then concentrated under reduced pressure without heat. The resulting neat oil was filtered through a 0.45μηι nylon membrane filter disk to provide tert-butyl 5,5,5- trifluoropentanoate (6.6 g, 31.4 mmol 98% yield) as a colorless oil: XH NMR (400 MHz, CDC13) δ ppm 1.38 (s, 9 H) 1.74-1.83 (m, 2 H) 2.00-2.13 (m, 2 H) 2.24 (t, J=7.28 Hz, 2 H).

Preparation IB: (45)-4-(Propan-2- l)-3-(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2-one

Figure imgf000066_0001

[00220] To a stirred solution of 5,5,5-trifluoropentanoic acid (5.04 g, 32.3 mmol) in DCM (50 mL) and DMF (3 drops) was added oxalyl chloride (3.4 mL, 38.8 mmol) dropwise over 5 min and the solution was stirred until all bubbling subsided. The reaction mixture was concentrated under reduced pressure to give pale yellow oil. To a separate flask charged with a solution of (45)-4-(propan-2-yl)-l,3-oxazolidin-2-one (4.18 g, 32.4 mmol) in THF (100 mL) at -78 °C was added n-BuLi (2.5M in hexane) (13.0 mL, 32.5 mmol) dropwise via syringe over 5 min. After stirring for 10 min, the above acid chloride dissolved in THF (20 mL) was added via cannula over 15 min. The reaction mixture was warmed to 0 °C, and was allowed to warm to room temperature as the bath warmed and stirred overnight. To the reaction mixture was added saturated NH4CI, and then extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na2S04), filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 60% solvent A/B=hexanes/EtOAc, REDISEP® S1O2 120g). Concentration of appropriate fractions provided Preparation IB (7.39 g, 86%) as a colorless oil: XH NMR (400 MHz, CDC13) δ ppm 4.44 (1 H, dt, J=8.31, 3.53 Hz), 4.30 (1 H, t, J=8.69 Hz), 4.23 (1 H, dd, J=9.06, 3.02 Hz), 2.98-3.08 (2 H, m), 2.32-2.44 (1 H, m, J=13.91, 7.02, 7.02, 4.03 Hz), 2.13-2.25 (2 H, m), 1.88-2.00 (2 H, m), 0.93 (3 H, d, J=7.05 Hz), 0.88 (3 H, d, J=6.80 Hz). Preparation 1C: (25′,3R)-tert-Butyl 6,6,6-trifluoro-3-((5)-4-isopropyl-2-oxooxazolidine- 3 -carbonyl)-2-(3 ,3,3 -trifluoropropyl)hexanoate, and

Preparation ID: (2R,3R)-tert-Butyl 6,6,6-trifluoro-3-((5)-4-isopropyl-2-oxooxazolidine- 3 -carbonyl)- -(3 ,3 ,3 -trifluoropropyl)hexanoate

Figure imgf000067_0001

(1 C) (1 D)

[00221] To a cold (-78 °C), stirred solution of diisopropylamine (5.3 mL, 37.2 mmol) in THF (59 mL) under nitrogen atmosphere was added n-BuLi (2.5M in hexane) (14.7 mL, 36.8 mmol), then warmed to 0 °C to give a 0.5M solution of LDA. A separate vessel was charged with Preparation IB (2.45 g, 9.17 mmol), the material was azeotroped twice with benzene (the RotoVap air inlet was fitted with nitrogen inlet to completely exclude humidity) then toluene (15.3 mL) was added. This solution was added to a flask containing dry lithium chloride (1.96 g, 46.2 mmol). To the resultant mixture, cooled to -78 °C, was added LDA solution (21.0 mL, 10.5 mmol) and stirred at -78 °C for 10 min, warmed to 0 °C for 10 min then recooled to -78 °C. To a separate reaction vessel containing Preparation 1A (3.41 g, 16.07 mmol), also azeotroped twice with benzene, was added toluene (15.3 mL), cooled to -78 °C and LDA (37.0 mL, 18.5 mmol) was added, the resulting solution was stirred at -78° for 25 min. At this time the enolate derived from the ester was transferred via cannula into the solution of the oxazolidinone enolate, stirred at -78 °C for an additional 5 min at which time the septum was removed and solid powdered bis(2-ethylhexanoyloxy)copper (9.02 g, 25.8 mmol) was rapidly added to the reaction vessel and the septum replaced. The vessel was immediately removed from the cold bath and immersed into a warm water bath (40 °C) with rapid swirling with a concomitant color change from the initial turquoise to brown. The reaction mixture was stirred for 20 min, was poured into 5% aqueous NH4OH (360 mL) and extracted with EtOAc (2x). The combined organics were washed with brine, dried (Na2S04), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 60% solvent A/B=hexanes/EtOAc, REDISEP® S1O2 120g). Concentration of appropriate fractions provided Preparation 1C (2.87 g, 66%) as pale yellow viscous oil. XH NMR showed the product was a 1.6: 1 mixture of diastereoisomers 1C: 1D as determined by the integration of the multiplets at 2.74 & 2.84 ppm: XH NMR (400 MHz, CDC13) δ ppm 4.43-4.54 (2 H, m), 4.23-4.35 (5 H, m), 4.01 (1 H, ddd, J=9.54, 6.27, 3.51 Hz), 2.84 (1 H, ddd, J=9.41, 7.28, 3.64 Hz), 2.74 (1 H, ddd, J=10.29, 6.27, 4.02 Hz), 2.37-2.48 (2 H, m, J=10.38, 6.98, 6.98, 3.51, 3.51 Hz), 2.20-2.37 (3 H, m), 1.92-2.20 (8 H, m), 1.64-1.91 (5 H, m), 1.47 (18 H, s), 0.88-0.98 (12 H, m). Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000068_0001

(1 E) (1 F)

[00222] To a cool (0 °C), stirred solution of Preparation 1C and ID (4.54 g, 9.51 mmol) in THF (140 mL) and water (42 mL) was sequentially added hydrogen peroxide (30% in water) (10.3 g, 91 mmol) and LiOH (685.3 mg, 28.6 mmol) and the mixture was stirred for 1 hr. At this time the reaction vessel was removed from the cold bath and then stirred for 1.5 hr. The reaction was judged complete by HPLC. To the reaction mixture was added saturated NaHC03 (45 mL) and saturated a2S03(15 mL), and then partially concentrated under reduced pressure. The resulting crude solution was extracted with DCM (3x). The aqueous phase was acidified to pH~l-2 with IN HC1, extracted with DCM (3x) and EtOAc (lx). The combined organics were washed with brine, dried (Na2S04), filtered and concentrated under reduced pressure to provide a mixture of Preparation IE and IF (3.00 g, 86%) as colorless oil: XH NMR (400 MHz, CDC13) δ ppm 2.76-2.84 (1 H, m, diastereoisomer 2), 2.64-2.76 (3 H, m), 2.04-2.35 (8 H, m), 1.88-2.00 (4 H, m), 1.71-1.83 (4 H, m), 1.48 (9 H, s, diastereoisomer 1), 1.46 (9 H, s, diastereoisomer 2); XH NMR showed a 1.7: 1 mixture of 1E: 1F by integration of the peaks for the ?-butyl groups.

Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000069_0001

(1 E) (1 F)

[00223] To a cold (-78 °C), stirred solution of diisopropylamine (1.7 mL, 11.93 mmol) in THF (19 mL) under nitrogen atmosphere was added n-BuLi (2.5M in hexanes) (4.8 mL, 12.00 mmol). The mixture was stirred for 5 min and then warmed to 0 °C. In a separate vessel, to a cold (-78 °C) stirred solution of the mixture of Preparation IE and IF (1.99 g, 5.43 mmol) in THF (18 mL) was added the LDA solution prepared above via cannula slowly over 25 min. The mixture was stirred for 15 min, then warmed to room temperature (placed in a 24 °C water bath) for 15 min, and then again cooled to -78 °C for 15 min. To the reaction mixture was added Et2AlCl (1M in hexane) (11.4 mL, 1 1.40 mmol) via syringe, stirred for 10 min, warmed to room temperature for 15 min and then cooled back to -78 °C for 15 min. Methanol (25 mL) was rapidly added, swirled vigorously while warming to room temperature, then concentrated to ~l/4 original volume. The mixture was dissolved in EtOAc and washed with IN HCl (50 mL) and ice (75 g). The aqueous phase was separated, extracted with EtOAc (2x). The combined organics were washed with a mixture of KF (2.85g in 75 mL water) and IN HCl (13 mL) [resulting solution pH 3-4], then with brine, dried (Na2S04), filtered and concentrated under reduced pressure to give a 9: 1 (IE: IF) enriched diastereoisomeric mixture (as determined by XH NMR) of Preparation IE and Preparation IF (2.13 g, >99%) as a pale yellow viscous oil: XH NMR (400 MHz, CDC13) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s). Preparation 1 G: (35)-3 -Amino- 1 -methyl-5-phenyl- 1 ,3 -dihydro-2H- 1 ,4-benzodiazepin-2- one, and

Preparation 1H: (3R)-3 -Amino- 1 -methyl-5-phenyl- 1 ,3-dihydro-2H- 1 ,4-benzodiazepin-2- one

Figure imgf000070_0001

(1G) (1 H)

[00224] Racemic 3-amino-l-methyl-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2- one (Rittle, K.E. et al, Tetrahedron Letters, 28(5):521-522 (1987)) was prepared according to the literature procedure. The enantiomers were separated under chiral-SFC conditions using the following method: CHIRALPAK® AS-H 5×25; Mobile phase: 30% MeOH+ 0.1% DEA in C02; Flow rate: 280 mL/min; Pressure: 100 bar; Temperature: 35 °C.

[00225] Obtained the S-enantiomer (Preparation 1G): HPLC: RT=1.75 min (30% MeOH + 0.1% DEA in C02 on CHIRALPAK® AS-H 4.6×250 mm, 3 mL/min, 35 °C, 100 bar, 230 nm, ΙΟμΙ injection); ¾ NMR (400 MHz, CDC13) δ ppm 7.58-7.63 (2 H, m), 7.55 (1 H, ddd, J=8.50, 7.1 1, 1.76 Hz), 7.40-7.47 (1 H, m), 7.34-7.40 (3 H, m), 7.31 (1 H, dd, J=7.81, 1.51 Hz), 7.14-7.22 (1 H, m), 4.46 (1 H, s), 3.44 (3 H, s), 3.42 (2 H, s); [a]D= -155° (c=1.9, MeOH) (Lit. Rittle, K.E. et al, Tetrahedron Letters, 28(5):521-522 (1987): [a]D=-236°).

[00226] Also obtained the R-enantiomer (Preparation 1H): HPLC: RT=1.71 min; [a]D=+165° (c=2.1, MeOH) (Lit [a]D= +227°).

Alternate procedure to make Preparation 1 G:

Preparation 1G»CSA salt: (35)-3-Amino-l-methyl-5-phenyl-l,3-dihydro-2H-l,4- benzodiazepin-2-one, (15)-(+)-10-camphorsulfonic acid salt

Figure imgf000071_0001

[00227] Preparation lG’CSA was prepared from racemic 3-amino-l-methyl-5-phenyl- l,3-dihydro-2H-l,4-benzodiazepin-2-one (9.98g, 37.6 mmol) (prepared according to the literature as shown above) according to the literature procedure (Reider, P.J. et al, J. Org. Chem., 52:955-957 (1987)). Preparation lG’CSA (16.91g, 99%) was obtained as a colorless solid: Optical Rotation: [a]D = -26.99° (c=l, H20) (Lit. [a]D = -27.8° (c=l,

H20))

Preparation II: tert-Butyl (25,,3R)-6,6,6-trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3- dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate, and Preparation 1J: tert-Butyl (2R,3R)-6,6,6-trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3- dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3-trifluoropropyl)hexanoate

Figure imgf000071_0002

(11) (U)

[00228] To a stirred solution of Preparation 1G (1.45 g, 5.47 mmol) and a 9: 1 mixture of Preparation IE and IF (1.989 g, 5.43 mmol) in DMF (19 mL) was added O- benzotriazol-l-yl-N,N,N’,N’-tetra-methyluronium tetrafluoroborate (1.79 g, 5.57 mmol) and triethylamine (3.0 mL, 21.52 mmol) and stirred overnight. The reaction was judged complete by LCMS. The reaction mixture was poured into water (125 mL) and the precipitated solid was collected by filtration, washed with water and air dried to provide an 8: 1 mixture of Preparation II and Preparation 1J (2.95 g, 89%) as a cream solid: MS (ES): m/z= 614 [M+H]+;XH NMR (400 MHz, CDC13) δ ppm 7.55-7.65 (3 H, m), 7.44- 7.52 (2 H, m), 7.35-7.45 (4 H, m), 5.52 (1 H, d, J=8.03 Hz), 3.48 (3 H, s), 2.63 (2 H, ddd, J=9.35, 3.95, 3.76 Hz), 2.14-2.25 (4 H, m), 1.90-2.03 (3 H, m), 1.69-1.82 (1 H, m), 1.51 (9 H, s).

Preparation IK: (25,,3R)-6,6,6-Trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3-dihydro- lH-l,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 1L: (2R,3R)-6,6,6-Trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3-dihydro- 1 H- 1 ,4-

Figure imgf000072_0001

(1 K) (1 L)

[00229] To a cool (0 °C), stirred solution of the above mixture of Preparation II and Preparation 1 J (2.95 g, 4.81 mmol) in DCM (20 mL) was added TFA (20 mL, 260 mmol). The reaction mixture was stirred for lhr, then allowed to warm to room temperature and stirred for 2.5 hr. The reaction was judged complete by LCMS. The reaction mixture was diluted with toluene (50 mL) and concentrated under reduced pressure. The residue mixture was redissolved in toluene (50 mL) and concentrated under reduced pressure then dried under high vacuum. The crude product was dissolved in DCM, S1O2 (15g) was added, concentrated, then was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 45% solvent A/B=DCM/EtOAc, REDISEP® S1O2 80g). Concentration of appropriate fractions provided a mixture of Preparation IK and Preparation 1L (2.00 g, 75%) as a cream solid: HPLC: RT=2.770 min

(CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 254 nm); MS (ES): m/z= 558 [M+H]+; XH NMR (400 MHz, CDC13) δ ppm 8.32 (1 H, d, J=8.03 Hz), 7.65-7.71 (1 H, m), 7.50-7.60 (3 H, m), 7.41-7.49 (2 H, m), 7.39 (1 H, dd, J=7.91, 1.63 Hz), 7.23-7.35 (2 H, m), 5.59 (1 H, d, J=8.03 Hz), 3.51 (3 H, s), 2.81 (1 H, ddd, J=10.54, 6.90, 3.64 Hz), 2.67-2.76 (1 H, m), 2.22-2.33 (3 H, m), 1.99-2.12 (3 H, m), 1.85-1.94 (1 H, m), 1.79 (1 H, ddd, J=13.87, 7.84, 3.64 Hz). Example 1 :

[00230] To a stirred solution of an 8: 1 mixture of Preparation IK and Preparation 1L (3.46 g, 6.21 mmol) in DMF (25 mL) under nitrogen atmosphere was added ammonium chloride (3.32 g, 62.1 mmol), EDC (3.55 g, 18.52 mmol), HOBT (2.85 g, 18.61 mmol), and triethyl amine (16 mL, 1 15 mmol) and stirred overnight. The reaction was judged complete by LCMS. The reaction mixture was poured into water (200 mL) with vigorous swirling and then allowed to sit. The solid was collected by filtration, washed with water, allowed to dry to afford 3.6 g colorless solid. The solid was purified by preparative SFC chromatography (Lux-Cellulose-2 (3x25cm), 8% methanol in CO2, 140ml/min @220nm and 35 °C; Sample: 3.6g in 50cc methanol, conc.=70mg/ml, Stack injection:

0.5cc/9.2min). Fractions containing product were concentrated, dried overnight under vacuum. Obtained Example 1 (2.74 g, 79%) as a colorless solid (Crystal Form -1): HPLC: RT=9.601 min (H20/CH3CN with TFA, Sunfire CI 8 3.5um, 4.6x150mm, 4.6x150mm, gradient = 15 min, wavelength = 220 and 254 nm). MS (ES): m/z= 557 [M+H]+; XH NMR (400 MHz, DMSO-d6) δ ppm 9.54 (1 H, d, J=7.28 Hz), 7.71-7.80 (1 H, m), 7.68 (2 H, d, J=8.78 Hz), 7.50-7.62 (3 H, m), 7.45 (2 H, t, J=7.28 Hz), 7.29-7.40 (2 H, m), 7.15 (1 H, br. s.), 5.30 (1 H, d, J=7.28 Hz), 3.39 (3 H, s), 2.74-2.86 (1 H, m), 2.02-2.32 (3 H, m), 1.45-1.79 (4 H, m); [a]D = -107.0° (5.73 mg/mL, DMSO).

[00231] Crystal Form A-2 was prepared by adding approximately 1 mg of Example 1 to approximately 0.7 mL of acetone/acetonitrile/water solution (2:2: 1). A mixture of colorless needles and thin blades crystals were obtained after one day of slow evaporation of the solution at room temperature. The thin blade crystals were separated to provide crystal Form A-2.

[00232] Crystal Form EA-3 was prepared by adding approximately 1 mg of Example 1 to approximately 0.7 mL of ethyl acetate/heptane solution (1 : 1). Colorless blade crystals were obtained after three days of slow evaporation of the solution at room temperature.

[00233] Crystal Form THF-2 was obtained by adding approximately 5 mg of Example 1 to approximately 0.7 mL of THF/water solution (4: 1). Colorless blade-like crystals were obtained after one day of solvent evaporation at room temperature.

Alternate Procedure to Make Example 1 : Preparation 1M: 3,3,3-Trifluoropropyl trifluoromethanesulfonate

Figure imgf000074_0001

[00234] To a cold (-25 °C), stirred solution of 2,6-lutidine (18.38 mL, 158 mmol) in CH2CI2 (120 mL) was added Tf20 (24.88 mL, 147 mmol) over 3 min, and stirred for 5 min. To the reaction mixture was added 3,3,3-trifluoropropan-l-ol (12 g, 105 mmol) over an interval of 3 min. After 2 hr, the reaction mixture was warmed to room temperature and stirred for 1 hr. The reaction mixture was concentrated to half volume, then purified by loading directly on silica gel column (330g ISCO) and eluted with CH2C12. Obtained Preparation 1M (13.74 g, 53%) as a colorless oil. XH NMR (400 MHz, CDCI3) δ ppm 4.71 (2 H, t, J=6.15 Hz), 2.49-2.86 (2 H, m).

Preparation IN: (45)-4-Benzyl- -(5,5,5-trifluoropentanoyl)-l,3-oxazolidin-2-one

Figure imgf000074_0002

[00235] Preparation IN was prepared from 5,5,5-trifluoropentanoic acid (3.35 g, 21.46 mmol) and (45)-4-benzyl-l,3-oxazolidin-2-one (3.80 g, 21.46 mmol) by the general methods shown for Preparation IB. Preparation IN (5.67 g, 84%) was obtained as a colorless viscous oil: XH NMR (400 MHz, CDC13) δ ppm 7.32-7.39 (2 H, m), 7.30 (1 H, d, J=7.05 Hz), 7.18-7.25 (2 H, m), 4.64-4.74 (1 H, m), 4.17-4.27 (2 H, m), 3.31 (1 H, dd, J=13.35, 3.27 Hz), 3.00-3.1 1 (2 H, m), 2.79 (1 H, dd, J=13.35, 9.57 Hz), 2.16-2.28 (2 H, m), 1.93-2.04 (2 H, m).

Preparation 10: tert-Butyl (3R)-3-(((45)-4-benzyl-2-oxo-l,3-oxazolidin-3-yl)carbonyl)- 6,6,6-trifluorohexanoate

Figure imgf000075_0001

[00236] To a cold (-78 °C), stirred solution of Preparation IN (3.03 g, 9.61 mmol) in THF (20 mL) was added NaHMDS (1.0M in THF) (10.6 mL, 10.60 mmol) under nitrogen atmosphere. After 2 hours, tert-butyl 2-bromoacetate (5.62 g, 28.8 mmol) was added neat via syringe at -78 °C and stirring was maintained at the same temperature. After 6 hours, the reaction mixture was warmed to room temperature. The reaction mixture was partitioned between saturated NH4C1 and EtOAc. The organic phase was separated, and the aqueous was extracted with EtOAc (3x). The combined organics were washed with brine, dried (Na2S04), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 100% solvent A/B=hexanes/EtO Ac, REDISEP® Si02 120g). Concentration of appropriate fractions provided Preparation 10 (2.79 g, 67.6%) as a colorless viscous oil: XH NMR (400 MHz, CDC13) δ ppm 7.34 (2 H, d, J=7.30 Hz), 7.24-7.32 (3 H, m), 4.62- 4.75 (1 H, m, J=10.17, 6.89, 3.43, 3.43 Hz), 4.15-4.25 (3 H, m), 3.35 (1 H, dd, J=13.60, 3.27 Hz), 2.84 (1 H, dd, J=16.62, 9.57 Hz), 2.75 (1 H, dd, J=13.35, 10.07 Hz), 2.47 (1 H, dd, J=16.62, 4.78 Hz), 2.1 1-2.23 (2 H, m), 1.90-2.02 (1 H, m), 1.72-1.84 (1 H, m), 1.44 (9 H, s). -2-(2-tert-Butoxy-2-oxoethyl)-5,5,5-trifluoropentanoic acid

Figure imgf000075_0002

[00237] Preparation IP was prepared from Preparation 10 (2.79 g, 6.50 mmol) by the general methods shown for Preparation IE. Preparation IP (1.45 g, 83%) was obtained as a colorless oil: XH NMR (400 MHz, CDC13) δ ppm 2.83-2.95 (1 H, m), 2.62-2.74 (1 H, m), 2.45 (1 H, dd, J=16.62, 5.79 Hz), 2.15-2.27 (2 H, m), 1.88-2.00 (1 H, m), 1.75-1.88 (1 H, m), 1.45 (9 H, s). Preparation IE: (2R,35′)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000076_0001

(1 E) (1 F)

[00238] To a cold (-78 °C), stirred solution of Preparation IP (5.44 g, 20.13 mmol) in THF (60 mL) was slowly added LDA (24.60 mL, 44.3 mmol) over 7 min. After stirring for 2 hr, Preparation 1M (6.44 g, 26.2 mmol) was added to the reaction mixture over 3 min. After 45 min, the reaction mixture was warmed to -25 °C bath (ice/MeOH/dry ice) for 1 hr, and then warmed to 0 °C. After 45 min, Preparation 1M (lg) was added and the reaction mixture was stirred for 20 min. The reaction was quenched with water and IN NaOH and was extracted with (¾(¾. The organic layer was again extracted with IN NaOH (2x) and the aqueous layers were combined. The aqueous layer was cooled in ice/water bath and then acidified with concentrated HCl to pH 2. Next, the aqueous layer was extracted with EtOAc. The combined organics were washed with brine, dried over anhydrous sodium sulphate, and concentrated under reduced pressure. The residue was dried under high vacuum to provide a 1 :5 (IE: IF) mixture (as determined by XH NMR) of Preparation IE and Preparation IF (5.925 g, 80%) as a pale yellow solid. XH NMR (500 MHz, CDC13) 8 ppm 2.81 (1 H, ddd, J=10.17, 6.32, 3.85 Hz), 2.63-2.76 (1 H, m), 2.02- 2.33 (4 H, m), 1.86-1.99 (2 H, m), 1.68-1.85 (2 H, m), 1.47 (9 H, s).

Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid, and

Preparation IF: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000077_0001

(1 E) (1 F)

[00239] A mixture of Preparation IE and Preparation IF (64 mg, 1.758 mmol) was taken in THF (6 mL) to give a colorless solution which was cooled to -78 °C. Then, LDA (2.149 mL, 3.87 mmol) (1.8M in heptane/THF/ethylbenzene) was slowly added to the reaction mixture over 10 min. After stirring for 15 min the reaction mixture was placed in a room temperature water bath. After 15 min the reaction mixture was placed back in -78 °C bath and then diethylaluminum chloride (3.87 mL, 3.87 mmol) (1M in hexane) was added slowly over 5 min. The reaction mixture was stirred at -78 °C. After 15 min the reaction mixture was placed in a room temperature water bath for 10 min and then cooled back to -78 °C bath. After 15 min the reaction was quenched with MeOH (8 mL, 198 mmol), removed from the -78 °C bath and concentrated. To the reaction mixture was added ice and HC1 (16 mL, 16.00 mmol), followed by extraction with EtOAc (2x). The organic layer was washed with potassium fluoride (920 mg, 15.84 mmol) (in 25 mL FLO) and HC1 (4.5 mL, 4.50 mmol). The organics were dried over anhydrous magnesium sulphate and concentrated under reduced pressure to provide a 9: 1 (IE: IF) enriched mixture of Preparation IE and Preparation IF (540 mg, 1.583 mmol, 90% yield) as light yellow/orange solid. ¾ NMR (400 MHz, CDC13) δ ppm 2.64-2.76 (2 H, m), 2.04-2.35 (4 H, m), 1.88-2.00 (2 H, m), 1.71-1.83 (2 H, m), 1.48 (9 H, s). It was converted to Example 1 by the sequence of reactions as outlined above.

Alternate procedure to make Preparation IE:

Preparation 1Q: (2R,35)- -Benzyl 4-tert-butyl 2,3-bis(3,3,3-trifluoropropyl)succinate

Figure imgf000077_0002

(1Q) [00240] A clean and dry 5 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler at room temperature was charged with Ν,Ν-dimethyl formamide (2.07 L), a 1.2: 1 mixture of Preparation IE and Preparation IF (207 g, 0.5651 moles), potassium carbonate (1 17.1 g, 0.8476 moles) followed by benzyl bromide (116 g, 0.6781 moles) over 15-20 min. The reaction mixture was stirred for 2-3 hr. After completion of the reaction, the reaction mixture was concentrated to dryness at 50-55 °C under vacuum. Ethyl acetate (3.1 L, 30 Vol.) was charged into the concentrated reaction mass and then washed with water (2.07 L), brine (0.6 L) then dried over anhydrous sodium sulfate (207 g), filtered and concentrated to dryness at 40-45 °C under vacuum. The residue was dissolved in dichloromethane (1.035 L, 5 vol.) and then absorbed onto silica gel (60-120) (607 g, 3.0 w/w), then was purified with column chromatography using petroleum ether and ethyl acetate as solvents. After pooling several batches, Preparation 1Q (235 g) was obtained. HPLC purity: 99.77%, Preparation IE: (2R,35)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000078_0001

[00241] A clean and dry 2 L autoclave was charged with methanol (540 mL) and was purged with nitrogen for 5-10 minutes. To the autoclave was added 10% palladium on carbon (12 g, 20%), purged with nitrogen once again for 5-10 min then was charged with Preparation 1Q (60g, 0.1315 moles), the autoclave was flushed with methanol (60mL) and stirred for 4-6 hr at 20-25 °C under 5Kg hydrogen pressure. After completion of the reaction, the reaction mass was filtered through CELITE®, washed with methanol (180 mL), dried with anhydrous sodium sulfate (60 g), filtered and concentrated to dryness at 45-50 °C under vacuum. Obtained Preparation IE (45.8 g, 95%) as a colorless solid: HPLC purity: 98.9%.

Alternate procedure to make Preparation IE: Preparation IE: (2R,35)-3-(te^Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3- trifluoropropyl)hexanoic acid

Figure imgf000079_0001

[00242] Preparation IE was prepared in a procedure identical as above from a mixture of Preparations IE and IF (200g, 0.5460 moles) using LDA (1.8 M solution in THF, ethyl benzene and heptane) (698mL, 2.3equiv.) and diethyl aluminum chloride (1.0 M solution in hexane) (1256mL, 2.3equiv) in THF (2.0L). After workup as explained above, the resulting residue was treated as follows: The crude material was added to a 2L four neck round bottom flask, followed by the addition of MTBE (1.0L) charged below 30 °C. The resulting mixture was stirred for 5-10 minutes to obtain a clear solution.

Hexanes (600mL) was charged to the reaction mixture at a temperature below 30 °C. The reaction mixture was stirred for 10 min. Next, tert-butylamine (43.8g, l. leq) was charged slowly over a period of 15 minutes below 30 °C. This addition was observed to be exothermic. The reaction mixture was stirred for 2 hrs below 30 °C and filtered. The solid material was washed with 5:3 MTBE: hexane (200mL), the filtrate was

concentrated and transferred to an amber color bottle. The filtered solid was dissolved in dichloromethane (2.0L), washed with IN HC1 (2.0), the organic layer was washed with brine (1.0L x 2), then was concentrated under reduced pressure below 45 °C. This material was found to be 91.12% pure. The material was repurified by the above t- butylamine crystallization purification procedure. Obtained Preparation IE (78 g, 39%): HPLC purity: 99.54%.

Alternate procedure to make Example 1 :

Preparation II: tert-Butyl (25,,3R)-6,6,6-trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3- dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate

Figure imgf000080_0001

[00243] A clean and dry 2 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler was charged with N,N- dimethylformamide (457 mL), Preparation IE (45.7g, 0.1248moles) and Preparation lG’CSA (62.08g, 0.1248moles) under nitrogen atmosphere at 20-25 °C. The reaction mixture was stirred for 15-20 minutes to make clear solution at 20-25 °C. To the reaction mixture was added TBTU (48.16g, 0.1498 moles) at 20-25 °C followed by triethylamine (50.51g, 0.4992 moles) over 15-20 minutes at 20-25 °C. The reaction mixture was stirred for 60-120 minutes at 20-25 °C under nitrogen atmosphere. After completion of the reaction, the reaction was quenched into water (1.37L, 30 Vol.) at 20-25 °C under stirring. The reaction mixture was stirred for 30 minutes at 20-25 °C. The reaction mixture was filtered and washed with water (228 mL). The resulting solid material was dissolved in ethyl acetate (457 mL), washed with water (2×137 mL), brine (137 mL), and then dried with anhydrous sodium sulfate (45.7g). Activated charcoal (9.14 g, 20%) was charged into the reaction mixture and stirred for 30 minutes. The mixture was filtered through CELITE® bed and 1 micron filter cloth, washed charcoal bed with ethyl acetate (137 mL), concentrated to 1.0 Vol. stage and then petroleum ether (457 mL, 10 Vol.) was charged and stirred for 30 minutes at 20-25 °C. The solid was collected by filtration, washed with petroleum ether (137 mL) and then dried under vacuum at 40-45 °C for 8 hr until loss on drying was less than 3.0%. Obtained Preparation II (65.2 g, 85%): HPLC purity: 98.26%.

Preparation IK: (25,,3R)-6,6,6-Trifluoro-3-(((35)-l-methyl-2-oxo-5-phenyl-2,3-dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoic acid

Figure imgf000081_0001

[00244] A clean and dry 3 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler was charged with dichloromethane (980 mL) under nitrogen atmosphere followed by Preparation II (140 g, 0.2282 moles) at 20-25 °C. The reaction mixture was cooled to 0-5 °C and trifluoroacetic acid (980 mL) was charged slowly for 30-40 minutes. The resulting mixture was stirred for 2 hr at 0-5 °C under nitrogen atmosphere. The reaction temperature was raised to 20 to 25 °C, and the reaction mixture was stirred for 1-2 hr at 20 to 25 °C. After completion of the reaction, the reaction mixture was concentrated to dryness at 50 to 55 °C under vacuum. Toluene (3×700 mL,) was charged into the concentrated reaction mass, and then distilled off at 50 to 55 °C under vacuum. After complete concentration from toluene, ethyl acetate (280 mL) was charged into the reaction mass at 20 to 25 °C, stirred for 60 minutes, then the solid was collected by filtration, washed with ethyl acetate (140 mL), dried under vacuum at 50 to 55 °C for 12 hr until loss on drying was less than 2.0%. Obtained Preparation IK (106 g, 84%): HPLC purity: 98.43%.

Example 1 :

[00245] A reaction vessel was charged with Preparation IK (30 g, 53.81 mmol), HOBt (8.7g, 64.38 mmol), and THF (150 mL) at room temperature. To the homogeneous solution was added EDCI (12.4g, 64.68 mmol), stirred for 15 min, then cooled to 8 °C. To the reaction mixture was added ammonia (2M in IP A) (81 mL, 162 mmol) over 5 min so as to maintain a temperature below 10 °C. The resulting heavy slurry was stirred for 10 min, warmed to room temperature over 30 min, then stirred for 4 hr. At the completion of the reaction, water (230 mL) was slowly added over 15 min to maintain a temperature below 20 °C, and then stirred for 2 hr. The solid was collected by filtration, washed with water (3X60 mL), then dried under vacuum 48 hr at 55 °C. The above crude product was charged into a 1 L 3 -necked round flask. IP A (200 mL) was added, then heated to 80 °C resulting in a homogeneous solution. Water (170 mL) was slowly added (15 min) to maintain an internal temperature >75 °C. The resulting slurry was stirred and cooled to room temperature for 2 hr. The solid was collected by filtration, washed with water (2 X 50 mL), then dried under vacuum (55 °C for 24 h, and 30 °C for 48 h).

Obtained Example 1 (23.4 g, 78% yield): HPLC purity: 99.43%.

Example 2 NOT SAME

WITHOUT METHYL GROUP

(2R,35)-N-((35)-2-Oxo-5-phenyl-2,3-dihydro-lH-l,4-benzodiazepin-3-yl)-2,3-bis(3,3,3- trifluoropropyl)succinamide

Figure imgf000082_0001

Preparation 2A: (35)-3-Amino-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2-one, and Preparation 2B: -3-Amino-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2-one

Figure imgf000082_0002

(2A) (2B)

[00246] Racemic 3-amino-5-phenyl-l,3-dihydro-2H-l,4-benzodiazepin-2-one (J. Med. Chem., 49:231 1-2319 (2006), compound# 5) was prepared according to the literature procedure. The enantiomers were separated on Berger SFC MGIII Column: Lux 25X3 cm, 5cm; Mobile phase: 30% MeOH+ 0.1% DEA in C02; Flow rate: 150 mL/min;

Temperature: 40 °C; Detector wavelength: 250 nM. Obtained the S-enantiomer

Preparation 2A as a white solid: XH NMR (400 MHz, DMSO-d6) δ ppm 10.67 (1 H, br. s.), 7.58 (1 H, td, J=7.65, 1.76 Hz), 7.37-7.53 (5 H, m), 7.23-7.30 (2 H, m), 7.14-7.22 (1 H, m), 4.23 (1 H, s), 2.60 (2 H, br. s.); HPLC: RT=3.0625 min (30% MeOH + 0.1% DEA in C02 on OD-H Column, 3 mL/min, 35 °C, 96 bar, 230 nm, ΙΟμΙ inj); [a]D = -208.3° (5.05 mg/niL, MeOH). Also obtained the R-enantiomer Preparation 2B as an off white solid: HPLC: RT=3.970 min; [a]D = 182.1° (2.01 mg/mL, MeOH).

Preparation 2C: tert-Butyl (25,,3R)-6,6,6-trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro- 1 H- 1 ,4-benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate, and

Preparation 2D: tert-Butyl (2R,3R)-6,6,6-trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro- 1 H- -benzodiazepin-3 -yl)carbamoyl)-2-(3 ,3 ,3 -trifluoropropyl)hexanoate

Figure imgf000083_0001

(2C) (2D)

[00247] Preparation 2C was prepared from Preparation 2A (564 mg, 2.244 mmol) and a mixture of Preparation IE and Preparation IF (822 mg, 2.244 mmol) according to the general procedure shown for Preparation II. Obtained Preparation 2C and Preparation 2D (1.31 g, 97%): HPLC: RT=3.443 min (CHROMOLITH® ODS 4.6 x 50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutes containing 0.% TFA, 4 mL/min, monitoring at 220 nm); MS (ES): m/z= 600.3 [M+H]+.

Preparation 2E: (25′,3R)-6,6,6-Trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro-lH-l,4- benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 2F: (2R,3R)-6,6,6-Trifluoro-3-(((35)-2-oxo-5-phenyl-2,3-dihydro-lH-l,4- benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid

Figure imgf000083_0002

(2E) (2F) [00248] A mixture of Preparation 2E and Preparation 2F was prepared from a mixture of Preparation 2C and Preparation 2D (1.3 lg, 2.185 mmol) by the general methods shown for Preparation IK. Obtained a mixture of Preparation 2E and Preparation 2F (1.18 g, 99%): HPLC: RT=2.885 min (CHROMOLITH® ODS 4.6 x 50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutes containing 0.% TFA, 4 mL/min, monitoring at 220 nm). MS (ES): m/z= 544.2 [M+H]+.

Example 2:

[00249] Example 2 was prepared from a mixture of Preparation 2E and Preparation 2F (354 mg, 0.651 mmol) by the general methods shown for Example 1. After separation of the diastereoisomers, Example 2 was obtained (188 mg, 52%) as a white solid: HPLC: RT=9.063 min (H20/CH3CN with TFA, Sunfire C18 3.5um, 4.6x150mm, 4.6x150mm, gradient = 15 min, wavelength = 220 and 254 nm); MS (ES): m/z= 543 [M+H]+; XH NMR (400 MHz, DMSO-d6) δ ppm 10.87 (1 H, br. s.), 9.50-9.55 (1 H, m), 7.62-7.69 (2 H, m), 7.40-7.57 (5 H, m), 7.29-7.36 (2 H, m), 7.22-7.28 (1 H, m), 7.16 (1 H, br. s.), 5.25 (1 H, d), 3.30-3.32 (1 H, m), 2.75-2.86 (1 H, m), 2.44-2.48 (1 H, m), 2.06-2.34 (3 H, m), 1.51- 1.77 (4 H, m); [a]D = -114.4° (8.04 mg/mL, DMSO).

[00250] Crystal Form M2- 1 was prepared by adding approximately 1 mg of Example 2 to approximately 0.7 mL of MeOH/fluorobenzene solution (3 : 1). Colorless plate-like crystals were obtained after 2 days of solvent evaporation at room temperature.

PATENT

US-20160060232-A1

https://patentscope.wipo.int/search/en/detail.jsf?docId=US159930181&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Example 1

(2R,3S)—N-((3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)-2,3-bis(3,3,3-trifluoropropyl)succinamide


Preparation 1A: tert-Butyl 5,5,5-trifluoropentanoate


      To a stirred solution of 5,5,5-trifluoropentanoic acid (5 g, 32.0 mmol) in THF (30 mL) and hexane (30 mL) at 0° C., was added tert-butyl 2,2,2-trichloroacetimidate (11.46 mL, 64.1 mmol). The mixture was stirred for 15 min at 0° C. Boron trifluoride etherate (0.406 mL, 3.20 mmol) was added and the reaction mixture was allowed to warm to room temperature overnight. To the clear reaction mixture was added solid NaHCO3 (5 g) and stirred for 30 min. The mixture was filtered through MgSO4 and washed with hexanes (200 mL). The solution was allowed to rest for 45 min, and the resulting solid material was removed by filtering on the same MgSO4 filter again, washed with hexanes (100 mL) and concentrated under reduced pressure without heat. The volume was reduced to about 30 mL, filtered through a clean fitted funnel, washed with hexane (5 mL), and then concentrated under reduced pressure without heat. The resulting neat oil was filtered through a 0.45 μm nylon membrane filter disk to provide tert-butyl 5,5,5-trifluoropentanoate (6.6 g, 31.4 mmol 98% yield) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ ppm 1.38 (s, 9H) 1.74-1.83 (m, 2H) 2.00-2.13 (m, 2H) 2.24 (t, J=7.28 Hz, 2H).

Preparation 1B: (4S)-4-(Propan-2-yl)-3-(5,5,5-trifluoropentanoyl)-1,3-oxazolidin-2-one


      To a stirred solution of 5,5,5-trifluoropentanoic acid (5.04 g, 32.3 mmol) in DCM (50 mL) and DMF (3 drops) was added oxalyl chloride (3.4 mL, 38.8 mmol) dropwise over 5 min and the solution was stirred until all bubbling subsided. The reaction mixture was concentrated under reduced pressure to give pale yellow oil. To a separate flask charged with a solution of (4S)-4-(propan-2-yl)-1,3-oxazolidin-2-one (4.18 g, 32.4 mmol) in THF (100 mL) at −78° C. was added n-BuLi (2.5M in hexane) (13.0 mL, 32.5 mmol) dropwise via syringe over 5 min. After stirring for 10 min, the above acid chloride dissolved in THF (20 mL) was added via cannula over 15 min. The reaction mixture was warmed to 0° C., and was allowed to warm to room temperature as the bath warmed and stirred overnight. To the reaction mixture was added saturated NH4Cl, and then extracted with EtOAc (2×). The combined organics were washed with brine, dried (Na2SO4), filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 60% solvent A/B=hexanes/EtOAc, REDISEP® SiO2 120 g). Concentration of appropriate fractions provided Preparation 1B (7.39 g, 86%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ ppm 4.44 (1H, dt, J=8.31, 3.53 Hz), 4.30 (1H, t, J=8.69 Hz), 4.23 (1H, dd, J=9.06, 3.02 Hz), 2.98-3.08 (2H, m), 2.32-2.44 (1H, m, J=13.91, 7.02, 7.02, 4.03 Hz), 2.13-2.25 (2H, m), 1.88-2.00 (2H, m), 0.93 (3H, d, J=7.05 Hz), 0.88 (3H, d, J=6.80 Hz).

Preparation 1C: (2S,3R)-tert-Butyl 6,6,6-trifluoro-3-((S)-4-isopropyl-2-oxooxazolidine-3-carbonyl)-2-(3,3,3-trifluoropropyl)hexanoate, and

Preparation 1D: (2R,3R)-tert-Butyl 6,6,6-trifluoro-3-((S)-4-isopropyl-2-oxooxazolidine-3-carbonyl)-2-(3,3,3-trifluoropropyl)hexanoate


      To a cold (−78° C.), stirred solution of diisopropylamine (5.3 mL, 37.2 mmol) in THF (59 mL) under nitrogen atmosphere was added n-BuLi (2.5M in hexane) (14.7 mL, 36.8 mmol), then warmed to 0° C. to give a 0.5M solution of LDA. A separate vessel was charged with Preparation 1B (2.45 g, 9.17 mmol), the material was azeotroped twice with benzene (the RotoVap air inlet was fitted with nitrogen inlet to completely exclude humidity) then toluene (15.3 mL) was added. This solution was added to a flask containing dry lithium chloride (1.96 g, 46.2 mmol). To the resultant mixture, cooled to −78° C., was added LDA solution (21.0 mL, 10.5 mmol) and stirred at −78° C. for 10 min, warmed to 0° C. for 10 min then recooled to −78° C. To a separate reaction vessel containing Preparation 1A (3.41 g, 16.07 mmol), also azeotroped twice with benzene, was added toluene (15.3 mL), cooled to −78° C. and LDA (37.0 mL, 18.5 mmol) was added, the resulting solution was stirred at −78° for 25 min. At this time the enolate derived from the ester was transferred via cannula into the solution of the oxazolidinone enolate, stirred at −78° C. for an additional 5 min at which time the septum was removed and solid powdered bis(2-ethylhexanoyloxy)copper (9.02 g, 25.8 mmol) was rapidly added to the reaction vessel and the septum replaced. The vessel was immediately removed from the cold bath and immersed into a warm water bath (40° C.) with rapid swirling with a concomitant color change from the initial turquoise to brown. The reaction mixture was stirred for 20 min, was poured into 5% aqueous NH4OH (360 mL) and extracted with EtOAc (2×). The combined organics were washed with brine, dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 60% solvent A/B=hexanes/EtOAc, REDISEP® SiO2 120 g). Concentration of appropriate fractions provided Preparation 1C (2.87 g, 66%) as pale yellow viscous oil. 1H NMR showed the product was a 1.6:1 mixture of diastereoisomers 1C:1D as determined by the integration of the multiplets at 2.74 & 2.84 ppm: 1H NMR (400 MHz, CDCl3) δ ppm 4.43-4.54 (2H, m), 4.23-4.35 (5H, m), 4.01 (1H, ddd, J=9.54, 6.27, 3.51 Hz), 2.84 (1H, ddd, J=9.41, 7.28, 3.64 Hz), 2.74 (1H, ddd, J=10.29, 6.27, 4.02 Hz), 2.37-2.48 (2H, m, J=10.38, 6.98, 6.98, 3.51, 3.51 Hz), 2.20-2.37 (3H, m), 1.92-2.20 (8H, m), 1.64-1.91 (5H, m), 1.47 (18H, s), 0.88-0.98 (12H, m).

Preparation 1E: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 1F: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid


      To a cool (0° C.), stirred solution of Preparation 1C and 1D (4.54 g, 9.51 mmol) in THF (140 mL) and water (42 mL) was sequentially added hydrogen peroxide (30% in water) (10.3 g, 91 mmol) and LiOH (685.3 mg, 28.6 mmol) and the mixture was stirred for 1 hr. At this time the reaction vessel was removed from the cold bath and then stirred for 1.5 hr. The reaction was judged complete by HPLC. To the reaction mixture was added saturated NaHCO3(45 mL) and saturated Na2SO3 (15 mL), and then partially concentrated under reduced pressure. The resulting crude solution was extracted with DCM (3×). The aqueous phase was acidified to pH-1-2 with 1N HCl, extracted with DCM (3×) and EtOAc (1×). The combined organics were washed with brine, dried (Na2SO4), filtered and concentrated under reduced pressure to provide a mixture of Preparation 1E and 1F (3.00 g, 86%) as colorless oil: 1H NMR (400 MHz, CDCl3) δ ppm 2.76-2.84 (1H, m, diastereoisomer 2), 2.64-2.76 (3H, m), 2.04-2.35 (8H, m), 1.88-2.00 (4H, m), 1.71-1.83 (4H, m), 1.48 (9H, s, diastereoisomer 1), 1.46 (9H, s, diastereoisomer 2); 1H NMR showed a 1.7:1 mixture of 1E:1F by integration of the peaks for the t-butyl groups.

Preparation 1E: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 1F: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid


      To a cold (−78° C.), stirred solution of diisopropylamine (1.7 mL, 11.93 mmol) in THF (19 mL) under nitrogen atmosphere was added n-BuLi (2.5M in hexanes) (4.8 mL, 12.00 mmol). The mixture was stirred for 5 min and then warmed to 0° C. In a separate vessel, to a cold (−78° C.) stirred solution of the mixture of Preparation 1E and 1F (1.99 g, 5.43 mmol) in THF (18 mL) was added the LDA solution prepared above via cannula slowly over 25 min. The mixture was stirred for 15 min, then warmed to room temperature (placed in a 24° C. water bath) for 15 min, and then again cooled to −78° C. for 15 min. To the reaction mixture was added Et2AlCl (1M in hexane) (11.4 mL, 11.40 mmol) via syringe, stirred for 10 min, warmed to room temperature for 15 min and then cooled back to −78° C. for 15 min. Methanol (25 mL) was rapidly added, swirled vigorously while warming to room temperature, then concentrated to ˜¼ original volume. The mixture was dissolved in EtOAc and washed with 1N HCl (50 mL) and ice (75 g). The aqueous phase was separated, extracted with EtOAc (2×). The combined organics were washed with a mixture of KF (2.85 g in 75 mL water) and 1N HCl (13 mL) [resulting solution pH 3-4], then with brine, dried (Na2SO4), filtered and concentrated under reduced pressure to give a 9:1 (1E:1F) enriched diastereoisomeric mixture (as determined by 1H NMR) of Preparation 1E and Preparation 1F (2.13 g, >99%) as a pale yellow viscous oil: 1H NMR (400 MHz, CDCl3) δ ppm 2.64-2.76 (2H, m), 2.04-2.35 (4H, m), 1.88-2.00 (2H, m), 1.71-1.83 (2H, m), 1.48 (9H, s).

Preparation 1G: (3S)-3-Amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, and

Preparation 1H: (3R)-3-Amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one


      Racemic 3-amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (Rittle, K. E. et al., Tetrahedron Letters, 28(5):521-522 (1987)) was prepared according to the literature procedure. The enantiomers were separated under chiral-SFC conditions using the following method: CHIRALPAK® AS-H 5×25; Mobile phase: 30% MeOH+0.1% DEA in CO2; Flow rate: 280 mL/min; Pressure: 100 bar; Temperature: 35° C.
      Obtained the S-enantiomer (Preparation 1G): HPLC: RT=1.75 min (30% MeOH+0.1% DEA in CO2 on CHIRALPAK® AS-H 4.6×250 mm, 3 mL/min, 35° C., 100 bar, 230 nm, 10 μl injection); 1H NMR (400 MHz, CDCl3) δ ppm 7.58-7.63 (2H, m), 7.55 (1H, ddd, J=8.50, 7.11, 1.76 Hz), 7.40-7.47 (1H, m), 7.34-7.40 (3H, m), 7.31 (1H, dd, J=7.81, 1.51 Hz), 7.14-7.22 (1H, m), 4.46 (1H, s), 3.44 (3H, s), 3.42 (2H, s); [α]D=−155° (c=1.9, MeOH) (Lit. Rittle, K. E. et al.,Tetrahedron Letters, 28(5):521-522 (1987): [α]D=−236°).
      Also obtained the R-enantiomer (Preparation 1H): HPLC: RT=1.71 min; [α]D=+165° (c=2.1, MeOH) (Lit [α]D=+227°).

Alternate Procedure to Make Preparation 1G

Preparation 1G•CSA salt: (3S)-3-Amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, (1 S)-(+)-10-camphorsulfonic acid salt


      Preparation 1G•CSA was prepared from racemic 3-amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (9.98 g, 37.6 mmol) (prepared according to the literature as shown above) according to the literature procedure (Reider, P. J. et al., J. Org. Chem., 52:955-957 (1987)). Preparation 1G•CSA (16.91 g, 99%) was obtained as a colorless solid: Optical Rotation: [α]D=−26.99° (c=1, H2O) (Lit. [α]D=−27.8° (c=1, H2O))

Preparation 1I: tert-Butyl (2S,3R)-6,6,6-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate, and

Preparation 1J: tert-Butyl (2R,3R)-6,6,6-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate

      To a stirred solution of Preparation 1G (1.45 g, 5.47 mmol) and a 9:1 mixture of Preparation 1E and 1F (1.989 g, 5.43 mmol) in DMF (19 mL) was added 0-benzotriazol-1-yl-N,N,N′,N′-tetra-methyluronium tetrafluoroborate (1.79 g, 5.57 mmol) and triethylamine (3.0 mL, 21.52 mmol) and stirred overnight. The reaction was judged complete by LCMS. The reaction mixture was poured into water (125 mL) and the precipitated solid was collected by filtration, washed with water and air dried to provide an 8:1 mixture of Preparation 1I and Preparation 1J (2.95 g, 89%) as a cream solid: MS (ES): m/z=614 [M+H]+; 1H NMR (400 MHz, CDCl3) δ ppm 7.55-7.65 (3H, m), 7.44-7.52 (2H, m), 7.35-7.45 (4H, m), 5.52 (1H, d, J=8.03 Hz), 3.48 (3H, s), 2.63 (2H, ddd, J=9.35, 3.95, 3.76 Hz), 2.14-2.25 (4H, m), 1.90-2.03 (3H, m), 1.69-1.82 (1H, m), 1.51 (9H, s).

Preparation 1K: (2S,3R)-6,6,6-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 1L: (2R,3R)-6,6,6-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid

      To a cool (0° C.), stirred solution of the above mixture of Preparation 1I and Preparation 1J (2.95 g, 4.81 mmol) in DCM (20 mL) was added TFA (20 mL, 260 mmol). The reaction mixture was stirred for 1 hr, then allowed to warm to room temperature and stirred for 2.5 hr. The reaction was judged complete by LCMS. The reaction mixture was diluted with toluene (50 mL) and concentrated under reduced pressure. The residue mixture was redissolved in toluene (50 mL) and concentrated under reduced pressure then dried under high vacuum. The crude product was dissolved in DCM, SiO2(15 g) was added, concentrated, then was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 0% to 45% solvent A/B=DCM/EtOAc, REDISEP® SiO2 80 g). Concentration of appropriate fractions provided a mixture of Preparation 1K and Preparation 1L (2.00 g, 75%) as a cream solid: HPLC: RT=2.770 min (CHROMOLITH® SpeedROD 4.6×50 mm (4 min grad) eluting with 10-90% aqueous MeOH over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 254 nm); MS (ES): m/z=558 [M+H]+; 1H NMR (400 MHz, CDCl3) δ ppm 8.32 (1H, d, J=8.03 Hz), 7.65-7.71 (1H, m), 7.50-7.60 (3H, m), 7.41-7.49 (2H, m), 7.39 (1H, dd, J=7.91, 1.63 Hz), 7.23-7.35 (2H, m), 5.59 (1H, d, J=8.03 Hz), 3.51 (3H, s), 2.81 (1H, ddd, J=10.54, 6.90, 3.64 Hz), 2.67-2.76 (1H, m), 2.22-2.33 (3H, m), 1.99-2.12 (3H, m), 1.85-1.94 (1H, m), 1.79 (1H, ddd, J=13.87, 7.84, 3.64 Hz).

Example 1

      To a stirred solution of an 8:1 mixture of Preparation 1K and Preparation 1L (3.46 g, 6.21 mmol) in DMF (25 mL) under nitrogen atmosphere was added ammonium chloride (3.32 g, 62.1 mmol), EDC (3.55 g, 18.52 mmol), HOBT (2.85 g, 18.61 mmol), and triethyl amine (16 mL, 115 mmol) and stirred overnight. The reaction was judged complete by LCMS. The reaction mixture was poured into water (200 mL) with vigorous swirling and then allowed to sit. The solid was collected by filtration, washed with water, allowed to dry to afford 3.6 g colorless solid. The solid was purified by preparative SFC chromatography (Lux-Cellulose-2 (3×25 cm), 8% methanol in CO2, 140 ml/min @220 nm and 35° C.; Sample: 3.6 g in 50 cc methanol, conc.=70 mg/ml, Stack injection: 0.5 cc/9.2 min). Fractions containing product were concentrated, dried overnight under vacuum. Obtained Example 1 (2.74 g, 79%) as a colorless solid (Crystal Form N-1): HPLC: RT=9.601 min (H2O/CH3CN with TFA, Sunfire C18 3.5 um, 4.6×150 mm, 4.6×150 mm, gradient=15 min, wavelength=220 and 254 nm). MS (ES): m/z=557 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ ppm 9.54 (1H, d, J=7.28 Hz), 7.71-7.80 (1H, m), 7.68 (2H, d, J=8.78 Hz), 7.50-7.62 (3H, m), 7.45 (2H, t, J=7.28 Hz), 7.29-7.40 (2H, m), 7.15 (1H, br. s.), 5.30 (1H, d, J=7.28 Hz), 3.39 (3H, s), 2.74-2.86 (1H, m), 2.02-2.32 (3H, m), 1.45-1.79 (4H, m); [α]D=−107.0° (5.73 mg/mL, DMSO).
      Crystal Form A-2 was prepared by adding approximately 1 mg of Example 1 to approximately 0.7 mL of acetone/acetonitrile/water solution (2:2:1). A mixture of colorless needles and thin blades crystals were obtained after one day of slow evaporation of the solution at room temperature. The thin blade crystals were separated to provide crystal Form A-2.
      Crystal Form EA-3 was prepared by adding approximately 1 mg of Example 1 to approximately 0.7 mL of ethyl acetate/heptane solution (1:1). Colorless blade crystals were obtained after three days of slow evaporation of the solution at room temperature.
      Crystal Form THF-2 was obtained by adding approximately 5 mg of Example 1 to approximately 0.7 mL of THF/water solution (4:1). Colorless blade-like crystals were obtained after one day of solvent evaporation at room temperature.

Alternate Procedure to Make Example 1

Preparation 1M: 3,3,3-Trifluoropropyl trifluoromethanesulfonate

      To a cold (−25° C.), stirred solution of 2,6-lutidine (18.38 mL, 158 mmol) in CH2Cl2 (120 mL) was added Tf2O (24.88 mL, 147 mmol) over 3 min, and stirred for 5 min. To the reaction mixture was added 3,3,3-trifluoropropan-1-ol (12 g, 105 mmol) over an interval of 3 min. After 2 hr, the reaction mixture was warmed to room temperature and stirred for 1 hr. The reaction mixture was concentrated to half volume, then purified by loading directly on silica gel column (330 g ISCO) and eluted with CH2Cl2. Obtained Preparation 1M (13.74 g, 53%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ ppm 4.71 (2H, t, J=6.15 Hz), 2.49-2.86 (2H, m).

Preparation 1N: (4S)-4-Benzyl-3-(5,5,5-trifluoropentanoyl)-1,3-oxazolidin-2-one

      Preparation 1N was prepared from 5,5,5-trifluoropentanoic acid (3.35 g, 21.46 mmol) and (4S)-4-benzyl-1,3-oxazolidin-2-one (3.80 g, 21.46 mmol) by the general methods shown for Preparation 1B. Preparation 1N (5.67 g, 84%) was obtained as a colorless viscous oil: 1H NMR (400 MHz, CDCl3) δ ppm 7.32-7.39 (2H, m), 7.30 (1H, d, J=7.05 Hz), 7.18-7.25 (2H, m), 4.64-4.74 (1H, m), 4.17-4.27 (2H, m), 3.31 (1H, dd, J=13.35, 3.27 Hz), 3.00-3.11 (2H, m), 2.79 (1H, dd, J=13.35, 9.57 Hz), 2.16-2.28 (2H, m), 1.93-2.04 (2H, m).

Preparation 1O: tert-Butyl (3R)-3-(((4S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl)carbonyl)-6,6,6-trifluorohexanoate

      To a cold (−78° C.), stirred solution of Preparation 1N (3.03 g, 9.61 mmol) in THF (20 mL) was added NaHMDS (1.0M in THF) (10.6 mL, 10.60 mmol) under nitrogen atmosphere. After 2 hours, tert-butyl 2-bromoacetate (5.62 g, 28.8 mmol) was added neat via syringe at −78° C. and stirring was maintained at the same temperature. After 6 hours, the reaction mixture was warmed to room temperature. The reaction mixture was partitioned between saturated NH4Cl and EtOAc. The organic phase was separated, and the aqueous was extracted with EtOAc (3×). The combined organics were washed with brine, dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (Teledyne ISCO CombiFlash Rf, 5% to 100% solvent A/B=hexanes/EtOAc, REDISEP® SiO2 120 g). Concentration of appropriate fractions provided Preparation 10 (2.79 g, 67.6%) as a colorless viscous oil: 1H NMR (400 MHz, CDCl3) δ ppm 7.34 (2H, d, J=7.30 Hz), 7.24-7.32 (3H, m), 4.62-4.75 (1H, m, J=10.17, 6.89, 3.43, 3.43 Hz), 4.15-4.25 (3H, m), 3.35 (1H, dd, J=13.60, 3.27 Hz), 2.84 (1H, dd, J=16.62, 9.57 Hz), 2.75 (1H, dd, J=13.35, 10.07 Hz), 2.47 (1H, dd, J=16.62, 4.78 Hz), 2.11-2.23 (2H, m), 1.90-2.02 (1H, m), 1.72-1.84 (1H, m), 1.44 (9H, s).

Preparation 1P: (2R)-2-(2-tert-Butoxy-2-oxoethyl)-5,5,5-trifluoropentanoic acid

      Preparation 1P was prepared from Preparation 1O (2.79 g, 6.50 mmol) by the general methods shown for Preparation 1E. Preparation 1P (1.45 g, 83%) was obtained as a colorless oil: 1H NMR (400 MHz, CDCl3) δ ppm 2.83-2.95 (1H, m), 2.62-2.74 (1H, m), 2.45 (1H, dd, J=16.62, 5.79 Hz), 2.15-2.27 (2H, m), 1.88-2.00 (1H, m), 1.75-1.88 (1H, m), 1.45 (9H, s).

Preparation 1E: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 1F: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

      To a cold (−78° C.), stirred solution of Preparation 1P (5.44 g, 20.13 mmol) in THF (60 mL) was slowly added LDA (24.60 mL, 44.3 mmol) over 7 min. After stirring for 2 hr, Preparation 1M (6.44 g, 26.2 mmol) was added to the reaction mixture over 3 min. After 45 min, the reaction mixture was warmed to −25° C. bath (ice/MeOH/dry ice) for 1 hr, and then warmed to 0° C. After 45 min, Preparation 1M (1 g) was added and the reaction mixture was stirred for 20 min. The reaction was quenched with water and 1N NaOH and was extracted with CH2Cl2. The organic layer was again extracted with 1N NaOH (2×) and the aqueous layers were combined. The aqueous layer was cooled in ice/water bath and then acidified with concentrated HCl to pH 2. Next, the aqueous layer was extracted with EtOAc. The combined organics were washed with brine, dried over anhydrous sodium sulphate, and concentrated under reduced pressure. The residue was dried under high vacuum to provide a 1:5 (1E:1F) mixture (as determined by 1H NMR) of Preparation 1E and Preparation 1F (5.925 g, 80%) as a pale yellow solid. 1H NMR (500 MHz, CDCl3) δ ppm 2.81 (1H, ddd, J=10.17, 6.32, 3.85 Hz), 2.63-2.76 (1H, m), 2.02-2.33 (4H, m), 1.86-1.99 (2H, m), 1.68-1.85 (2H, m), 1.47 (9H, s).

Preparation 1E: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid, and

Preparation 1F: (2R,3R)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

      A mixture of Preparation 1E and Preparation 1F (64 mg, 1.758 mmol) was taken in THF (6 mL) to give a colorless solution which was cooled to −78° C. Then, LDA (2.149 mL, 3.87 mmol) (1.8M in heptane/THF/ethylbenzene) was slowly added to the reaction mixture over 10 min. After stirring for 15 min the reaction mixture was placed in a room temperature water bath. After 15 min the reaction mixture was placed back in −78° C. bath and then diethylaluminum chloride (3.87 mL, 3.87 mmol) (1M in hexane) was added slowly over 5 min. The reaction mixture was stirred at −78° C. After 15 min the reaction mixture was placed in a room temperature water bath for 10 min and then cooled back to −78° C. bath. After 15 min the reaction was quenched with MeOH (8 mL, 198 mmol), removed from the −78° C. bath and concentrated. To the reaction mixture was added ice and HCl (16 mL, 16.00 mmol), followed by extraction with EtOAc (2×). The organic layer was washed with potassium fluoride (920 mg, 15.84 mmol) (in 25 mL H2O) and HCl (4.5 mL, 4.50 mmol). The organics were dried over anhydrous magnesium sulphate and concentrated under reduced pressure to provide a 9:1 (1E:1F) enriched mixture of Preparation 1E and Preparation 1F (540 mg, 1.583 mmol, 90% yield) as light yellow/orange solid. 1H NMR (400 MHz, CDCl3) δ ppm 2.64-2.76 (2H, m), 2.04-2.35 (4H, m), 1.88-2.00 (2H, m), 1.71-1.83 (2H, m), 1.48 (9H, s). It was converted to Example 1 by the sequence of reactions as outlined above.

Alternate Procedure to Make Preparation 1E

Preparation 1Q: (2R,3S)-1-Benzyl 4-tert-butyl 2,3-bis(3,3,3-trifluoropropyl)succinate

      A clean and dry 5 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler at room temperature was charged with N,N-dimethyl formamide (2.07 L), a 1.2:1 mixture of Preparation 1E and Preparation 1F (207 g, 0.5651 moles), potassium carbonate (117.1 g, 0.8476 moles) followed by benzyl bromide (116 g, 0.6781 moles) over 15-20 min. The reaction mixture was stirred for 2-3 hr. After completion of the reaction, the reaction mixture was concentrated to dryness at 50-55° C. under vacuum. Ethyl acetate (3.1 L, 30 Vol.) was charged into the concentrated reaction mass and then washed with water (2.07 L), brine (0.6 L) then dried over anhydrous sodium sulfate (207 g), filtered and concentrated to dryness at 40-45° C. under vacuum. The residue was dissolved in dichloromethane (1.035 L, 5 vol.) and then absorbed onto silica gel (60-120) (607 g, 3.0 w/w), then was purified with column chromatography using petroleum ether and ethyl acetate as solvents. After pooling several batches, Preparation 1Q (235 g) was obtained. HPLC purity: 99.77%,

Preparation 1E: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

      A clean and dry 2 L autoclave was charged with methanol (540 mL) and was purged with nitrogen for 5-10 minutes. To the autoclave was added 10% palladium on carbon (12 g, 20%), purged with nitrogen once again for 5-10 min then was charged with Preparation 1Q (60 g, 0.1315 moles), the autoclave was flushed with methanol (60 mL) and stirred for 4-6 hr at 20-25° C. under 5 Kg hydrogen pressure. After completion of the reaction, the reaction mass was filtered through CELITE®, washed with methanol (180 mL), dried with anhydrous sodium sulfate (60 g), filtered and concentrated to dryness at 45-50° C. under vacuum. Obtained Preparation 1E (45.8 g, 95%) as a colorless solid: HPLC purity: 98.9%.

Alternate Procedure to Make Preparation 1E

Preparation 1E: (2R,3S)-3-(tert-Butoxycarbonyl)-6,6,6-trifluoro-2-(3,3,3-trifluoropropyl)hexanoic acid

      Preparation 1E was prepared in a procedure identical as above from a mixture of Preparations 1E and 1F (200 g, 0.5460 moles) using LDA (1.8 M solution in THF, ethyl benzene and heptane) (698 mL, 2.3 equiv.) and diethyl aluminum chloride (1.0 M solution in hexane) (1256 mL, 2.3 equiv) in THF (2.0 L). After workup as explained above, the resulting residue was treated as follows: The crude material was added to a 2 L four neck round bottom flask, followed by the addition of MTBE (1.0 L) charged below 30° C. The resulting mixture was stirred for 5-10 minutes to obtain a clear solution. Hexanes (600 mL) was charged to the reaction mixture at a temperature below 30° C. The reaction mixture was stirred for 10 min. Next, tert-butylamine (43.8 g, 1.1 eq) was charged slowly over a period of 15 minutes below 30° C. This addition was observed to be exothermic. The reaction mixture was stirred for 2 hrs below 30° C. and filtered. The solid material was washed with 5:3 MTBE: hexane (200 mL), the filtrate was concentrated and transferred to an amber color bottle. The filtered solid was dissolved in dichloromethane (2.0 L), washed with 1N HCl (2.0), the organic layer was washed with brine (1.0 L×2), then was concentrated under reduced pressure below 45° C. This material was found to be 91.12% pure. The material was repurified by the above t-butylamine crystallization purification procedure. Obtained Preparation 1E (78 g, 39%): HPLC purity: 99.54%.

Alternate Procedure to Make Example 1

Preparation 1I: tert-Butyl (2S,3R)-6,6,6-trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoate

      A clean and dry 2 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler was charged with N,N-dimethylformamide (457 mL), Preparation 1E (45.7 g, 0.1248 moles) and Preparation 1G•CSA (62.08 g, 0.1248 moles) under nitrogen atmosphere at 20-25° C. The reaction mixture was stirred for 15-20 minutes to make clear solution at 20-25° C. To the reaction mixture was added TBTU (48.16 g, 0.1498 moles) at 20-25° C. followed by triethylamine (50.51 g, 0.4992 moles) over 15-20 minutes at 20-25° C. The reaction mixture was stirred for 60-120 minutes at 20-25° C. under nitrogen atmosphere. After completion of the reaction, the reaction was quenched into water (1.37L, 30 Vol.) at 20-25° C. under stirring. The reaction mixture was stirred for 30 minutes at 20-25° C. The reaction mixture was filtered and washed with water (228 mL). The resulting solid material was dissolved in ethyl acetate (457 mL), washed with water (2×137 mL), brine (137 mL), and then dried with anhydrous sodium sulfate (45.7 g). Activated charcoal (9.14 g, 20%) was charged into the reaction mixture and stirred for 30 minutes. The mixture was filtered through CELITE® bed and 1 micron filter cloth, washed charcoal bed with ethyl acetate (137 mL), concentrated to 1.0 Vol. stage and then petroleum ether (457 mL, 10 Vol.) was charged and stirred for 30 minutes at 20-25° C. The solid was collected by filtration, washed with petroleum ether (137 mL) and then dried under vacuum at 40-45° C. for 8 hr until loss on drying was less than 3.0%. Obtained Preparation 11 (65.2 g, 85%): HPLC purity: 98.26%.

Preparation 1K: (2S,3R)-6,6,6-Trifluoro-3-(((3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)carbamoyl)-2-(3,3,3-trifluoropropyl)hexanoic acid

      A clean and dry 3 L four neck round bottom flask equipped with mechanical stirring, thermometer socket and nitrogen bubbler was charged with dichloromethane (980 mL) under nitrogen atmosphere followed by Preparation 1I (140 g, 0.2282 moles) at 20-25° C. The reaction mixture was cooled to 0-5° C. and trifluoroacetic acid (980 mL) was charged slowly for 30-40 minutes. The resulting mixture was stirred for 2 hr at 0-5° C. under nitrogen atmosphere. The reaction temperature was raised to 20 to 25° C., and the reaction mixture was stirred for 1-2 hr at 20 to 25° C. After completion of the reaction, the reaction mixture was concentrated to dryness at 50 to 55° C. under vacuum. Toluene (3×700 mL,) was charged into the concentrated reaction mass, and then distilled off at 50 to 55° C. under vacuum. After complete concentration from toluene, ethyl acetate (280 mL) was charged into the reaction mass at 20 to 25° C., stirred for 60 minutes, then the solid was collected by filtration, washed with ethyl acetate (140 mL), dried under vacuum at 50 to 55° C. for 12 hr until loss on drying was less than 2.0%. Obtained Preparation 1K (106 g, 84%): HPLC purity: 98.43%.

Example 1

      A reaction vessel was charged with Preparation 1K (30 g, 53.81 mmol), HOBt (8.7 g, 64.38 mmol), and THF (150 mL) at room temperature. To the homogeneous solution was added EDCI (12.4 g, 64.68 mmol), stirred for 15 min, then cooled to 8° C. To the reaction mixture was added ammonia (2M in IPA) (81 mL, 162 mmol) over 5 min so as to maintain a temperature below 10° C. The resulting heavy slurry was stirred for 10 min, warmed to room temperature over 30 min, then stirred for 4 hr. At the completion of the reaction, water (230 mL) was slowly added over 15 min to maintain a temperature below 20° C., and then stirred for 2 hr. The solid was collected by filtration, washed with water (3×60 mL), then dried under vacuum 48 hr at 55° C. The above crude product was charged into a 1 L 3-necked round flask. IPA (200 mL) was added, then heated to 80° C. resulting in a homogeneous solution. Water (170 mL) was slowly added (15 min) to maintain an internal temperature>75° C. The resulting slurry was stirred and cooled to room temperature for 2 hr. The solid was collected by filtration, washed with water (2×50 mL), then dried under vacuum (55° C. for 24 h, and 30° C. for 48 h). Obtained Example 1 (23.4 g, 78% yield): HPLC purity: 99.43%.

PATENTS

US-20150284342-A1

US-20140357605-A1

US-20140100365-A1

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For some disease targets, an indirect approach may be best. Or so Ashvinikumar V. Gavai and his colleagues atBristol-Myers Squibbfound in their quest toward a potential cancer drug. Gavai unveiled BMS-906024, which is an experimental—and slightly roundabout—treatment for a number of cancers, including breast, lung, and colon cancers, and leukemia.

Cancers have a tendency to relapse or to become resistant to treatments that once worked. Research at BMS and elsewhere had suggested that a family of proteins called Notch is implicated in that resistance and in cancer progression more generally. Gavai, director of oncology chemistry at BMS in Princeton, N.J., and his team set out to block Notch family signaling.

Notch family members lack enzymatic activity, so blocking them directly is difficult. Instead, BMS developed inhibitors of an enzyme that is essential for activating Notch signaling—γ-secretase.

09116-cover-bms906024

Company: Bristol-Myers Squibb

Target: pan-Notch

Disease: breast, lung, colon cancer; leukemia

Interfering with Notch, even in this indirect way, can have detrimental effects on the gastrointestinal tract. Only two of the four Notch family members are linked to that side effect, Gavai says. But he and his team think their drug will be most effective if it acts on all four family members roughly equally—a so-called pan-Notch inhibitor. By selecting a molecule that’s well tolerated in animals and carefully scheduling doses of the drug in humans, it could be possible to minimize side effects, he says.

The BMS team relied on Notch signaling assays in leukemia and breast cancer cell lines to find leads. They soon learned that for their molecules to work, three chiral centers had to be in the S,R,Sconfiguration. After that, they strove to make the molecules last in the bloodstream. They removed an isobutyl group and tweaked some other parts of their candidate’s succinamide side chain. It was tough to retain both a long half-life and activity against Notch, Gavai told C&EN. “You’d optimize one and lose the other.”

His team threaded the needle with BMS-906024. Their studies with mice suggest that a dose of 4–6 mg once a week could be effective in people. That’s lower than doses being tested for other Notch-targeted agents, according to the website clinicaltrials.gov. The mouse studies also back the idea that Notch is involved in cancer drug resistance and suggest that Notch could be a target for taking on cancer stem cells, which are notoriously resistant to chemotherapy.

BMS-906024 is in Phase I clinical trials, both alone and in combination with other agents. Patients with colon, lung, breast, and other cancers are receiving intravenous doses of the compound to determine its safety and optimum dose ranges.

09116-cover-BMScxd

(From left, front row) Gavai, Weifeng Shan, (second row) Aaron Balog, Patrice Gill, Gregory Vite, (third row) Francis Lee, Claude Quesnelle, (rear row) Wen-Ching Han, Richard Westhouse.
Credit: Catherine Stroud Photography

http://cen.acs.org/articles/91/i16/BMS-906024-Notch-Signaling-Inhibitor.html

Image result for BMS 906024 synthesis

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Image result for BMS 906024

BMS-906024
Company: Bristol-Myers Squibb
Meant to treat: cancers including breast, lung, colon, and leukemia
Mode of action: pan-Notch inhibitor
Medicinal chemistry tidbit: The BMS team used an oxidative enolate heterocoupling en route to the candidate– a procedure from Phil Baran’s lab at Scripps Research Institute. JACS 130, 11546
Status in the pipeline: Phase I
Relevant documents: WO 2012/129353

PAPER

Abstract Image

An enantioselective synthesis of (S)-7-amino-5H,7H-dibenzo[b,d]azepin-6-one (S1) is described. The key step in the sequence involved crystallization-induced dynamic resolution (CIDR) of compound 7 using Boc-d-phenylalanine as a chiral resolving agent and 3,5-dichlorosalicylaldehyde as a racemization catalyst to afford S1 in 81% overall yield with 98.5% enantiomeric excess.

Crystallization-Induced Dynamic Resolution toward the Synthesis of (S)-7-Amino-5H,7H-dibenzo[b,d]-azepin-6-one: An Important Scaffold for γ-Secretase Inhibitors

Department of Discovery Synthesis, Biocon Bristol-Myers Squibb Research Centre, Biocon Park, Bommasandra IV Phase, Jigani Link Road, Bengaluru 560099, India
Bristol-Myers Squibb Company, P.O Box 4000, Princeton, New Jersey 08543-4000, United States
Org. Process Res. Dev., Article ASAP
 1. Quesnelle, Claude; Kim, Soong-Hoon; Lee, Francis; Gavai, Ashvinikumar. Bis(fluoroalkyl)-1,4-benzodiazepinone compounds as Notch receptor inhibitors and their preparation and use in the treatment of cancer. PCT Int. Appl. (2012), WO 2012129353 A1 20120927.
Patent ID Date Patent Title
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US2014357605 2014-12-04 BIS(FLUOROALKYL)-1, 4-BENZODIAZEPINONE COMPOUNDS
US8822454 2014-09-02 Bisfluoroalkyl-1, 4-benzodiazepinone compounds
US8629136 2014-01-14 Bisfluoroalkyl-1, 4-benzodiazepinone compounds
BMS-906024
BMS-906024.svg
Systematic (IUPAC) name
(2R,3S)-N-[(3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]-2,3-bis(3,3,3-trifluoropropyl)succinamide
Identifiers
PubChem CID 66550890
ChemSpider 28536138
Chemical data
Formula C26H26F6N4O3
Molar mass 556.500 g/mol

///////////////3,5-dichlorosalicylaldehyde, Alzheimer’s disease, Boc-D-phenylalanine, CIDR;dibenzoazepenone DKR; Notch inhibitorsNotch inhibitor, SAR T-acute lymphoblastic leukemia, triple-negative breast cancer, γ-secretase inhibitor, PHASE 1, BMS, Bristol-Myers Squibb, 1401066-79-2, Ashvinikumar Gavai

CN1c2ccccc2C(=N[C@@H](C1=O)NC(=O)[C@H](CCC(F)(F)F)[C@H](CCC(F)(F)F)C(=O)N)c3ccccc3

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Patent US8377886 – Use of gamma secretase inhibitors and notch …

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Figure US08377886-20130219-C00003. gamma secretase inhibitor

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RO4929097 | γ-secretase inhibitor – Cellagen Technology

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RO4929097 | γ-secretase inhibitor
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VT 1129


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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)

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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.

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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.

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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

Nacubactam, A diazabicyclooctane beta-lactamase inhibitor, for treating bacterial infection


 

Nacubactam

RG-6080,  FPI-1459,  OP-0595, WK ?, WK-?, WK?

 CAS 1452458-86-4,  MF C9 H16 N4 O7 S, MW 324.31
Sulfuric acid, mono[(1R,2S,5R)-2-[[(2-aminoethoxy)amino]carbonyl]-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl] ester,

(2S,5R)-N-(2-amino ethoxy)-6-(sulfooxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide

Beta lactamase inhibitor

Roche, under license from Meiji Seika Pharma and Fedora Pharmaceuticals is developing nacubactam hydrate

Meiji Seika Pharma Co., Ltd., Meiji Seikaファルマ株式会社

A diazabicyclooctane beta-lactamase inhibitor, for treating bacterial infection. In July 2016, nacubactam was reported to be in phase 1 clinical development

PATENTS , IN2015MU287, WO2016116878WO 2016120752, INDICATE INTEREST FROM WOCKHARDT

 

Sulfuric acid, mono[(1R,2S,5R)-2-[[(2-aminoethoxy)amino]carbonyl]-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl] ester

A β-lactamase inhibitor potentially for the treatment of bacterial infections.

RG-6080; FPI-1459; OP-0595

CAS No. 1452458-86-4

Molecular Formula C9 H16 N4 O7 S
Formula Weight 324.31
  • Originator Fedora Pharmaceuticals
  • Developer Meiji Seika Pharma
  • Class Antibacterials; Azabicyclo compounds
  • Mechanism of Action Beta lactamase inhibitors
  • Phase I Bacterial infections

Most Recent Events

  • 13 Jan 2015  OP 0595 licensed to Roche worldwide, except Japan ,
  • 30 Nov 2014 Meiji Seika Pharma completes a phase I trial in Healthy volunteers in Australia (NCT02134834)
  • 01 May 2014 Phase-I clinical trials in Bacterial infections (in volunteers) in Australia (IV)

In September 2014, preclinical data were presented at the 54th ICAAC Meeting in Washington, DC. Nacubactam hydratedemonstrated Ki values of 0.24, 3 and 0.79 microM against AmpC P99 derived from Enterobacter cloacae, KPC-3, and CTX-M-15 enzymes, respectively; the Ki values were lower than that of cefepime

Bacterial infections continue to remain one of the major causes contributing towards human diseases. One of the key challenges in treatment of bacterial infections is the ability of bacteria to develop resistance to one or more antibacterial agents over time. Examples of such bacteria that have developed resistance to typical antibacterial agents include: Penicillin-resistant Streptococcus pneumoniae, Vancomycin-resistant Enterococci, and Methicillin-resistant Staphylococcus aureus. The problem of emerging drug-resistance in bacteria is often tackled by switching to newer antibacterial agents, which can be more expensive and sometimes more toxic. Additionally, this may not be a permanent solution as the bacteria often develop resistance to the newer antibacterial agents as well in due course. In general, bacteria are particularly efficient in developing resistance, because of their ability to multiply very rapidly and pass on the resistance genes as they replicate.

The persistent exposure of bacterial strains to a multitude of beta- lactam antibacterial agents has led to overproduction and mutation of beta-lactamases. These new extended spectrum beta-lactamases (ESBL) are capable of hydrolyzing penicillins, cephalosporins, monobactams and even carbapenems. Such a wide spread resistance to many of the existing beta-lactam antibacterial agents, either used alone or in combination with other agents, is posing challenges in treating serious bacterial infections.

Due to various reasons, the oral therapeutic options for treating bacterial infections (including those caused by ESBL strains) are limited. For example, a combination of amoxicillin and clavulanic acid is effective against Class A ESBLs producing bacteria. However, the usefulness of this combination is compromised against bacteria producing multiple or mixed beta-lactamase enzymes (such as, for example, bacteria producing Class A and Class C ESBLs concurrently), and Klebsiella pneumoniae carbapenemases (KPCs). Therefore, oral antibacterial agents or combinations with activity against a range of bacterial strains (including those producing multiple ESBLs and KPCs) are urgently desired.

Cephalosporin antibacterial agents are known for treatment for various bacterial infections. Surprisingly, it has been found that pharmaceutical compositions comprising a cephalosporin antibacterial agent and certain nitrogen containing bicyclic compound (disclosed in PCT/IB2013/053092, PCT/JP2013/064971 and PCT/IB 2012/002675) exhibit unexpectedly synergistic antibacterial activity, even against highly resistant bacterial strains.

SYNTHESIS

WO 2015046207,

STR1

CONTD…………………..

STR1

CONTD………………………………..

STR1

Patent

The novel heterocyclic compound in Japanese Patent 4515704 (Patent Document 1), preparation and shown for their pharmaceutical use, sodium trans-7-oxo-6- (sulfooxy) as a representative compound 1,6-diazabicyclo [3 .2.1] discloses an octane-2-carboxamide (NXL104). Preparation in regard to certain piperidine derivatives which are intermediates Patent 2010-138206 (Patent Document 2) and JP-T 2010-539147 (Patent Document 3) are shown at further WO2011 / 042560 (Patent Document 4) NXL104 to disclose a method for producing the crystals.

 In Patent 5038509 (Patent Document 5) (2S, 5R) -7- oxo -N- (piperidin-4-yl) -6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane – 2- carboxamide (MK7655) is shown, discloses the preparation of certain piperidine derivatives with MK7655 at Patent 2011-207900 (Patent Document 6) and WO2010 / 126820 (Patent Document 7).

 The present inventors also disclose the novel diazabicyclooctane derivative represented by the following formula (VII) in Japanese Patent Application 2012-122603 (Patent Document 8).

Patent Document 1: Japanese Patent No. 4515704 Pat

Patent Document 2: Japanese Patent Publication 2010-138206 Pat

Patent Document 3: Japanese patent publication 2010-539147 Pat

Patent Document 4: International Publication No. WO2011 / 042560 Patent

Patent Document 5: Japanese Patent No. 5038509 Pat

Patent Document 6: Japanese Patent Publication 2011-207900 Pat

Patent Document 7: International Publication No. WO2010 / 126820 Patent

Patent Document 8: Japanese Patent application 2012-122603 Pat.

[Chemical formula 1] (In the formula, R 3 are the same as those described below)

Reference Example

5 of 5 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VII-1)

Formula 43]

step 1 tert-butyl {2 – [({[( 2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl } amino) oxy] ethyl} carbamate  (IV-1)(2S, 5R)-6-(benzyloxy) -7-oxo-1,6-diazabicyclo [3.2.1] octane-2-carboxylic acid (4 .30g, dehydrated ethyl acetate (47mL) solution of 15.56mmol) was cooled to -30 ℃, isobutyl chloroformate (2.17g, washing included dehydration ethyl acetate 1mL), triethylamine (1.61g, washing included dehydration ethyl acetate 1 mL), successively added dropwise, and the mixture was stirred 1 hour at -30 ° C.. To the reaction solution tert- butyl 2-dehydration of ethyl acetate (amino-oxy) ethyl carbamate (3.21g) (4mL) was added (washing included dehydration ethyl acetate 1mL), raising the temperature over a period of 1.5 hours to 0 ℃, It was further stirred overnight. The mixture of 8% aqueous citric acid (56 mL), saturated aqueous sodium bicarbonate solution (40 mL), sequentially washed with saturated brine (40 mL), dried over anhydrous magnesium sulfate, filtered, concentrated to 5 mL, up to 6mL further with ethanol (10 mL) It was replaced concentrated. Ethanol to the resulting solution (3mL), hexane the (8mL) in addition to ice-cooling, and the mixture was stirred inoculated for 15 minutes. The mixture was stirred overnight dropwise over 2 hours hexane (75 mL) to. Collected by filtration the precipitated crystals, washing with hexane to give the title compound 5.49g and dried in vacuo (net 4.98 g, 74% yield). HPLC: COSMOSIL 5C18 MS-II 4.6 × 150 mm, 33.3 mM phosphate buffer / MeCN = 50/50, 1.0 mL / min, UV 210 nm, Retweeted 4.4 min; 1 H NMR (400 MHz, CDCl 3 ) [delta] 1.44 (s, 9H), 1.56-1.70 (m, 1H), 1.90-2.09 (m, 2H), 2.25-2.38 (m, 1H), 2.76 (d, J = 11.6 Hz, 1H), 3.03 (br.d., J = 11.6 Hz , 1H), 3.24-3.47 (m, 3H), 3.84-4.01 (m, 3H), 4.90 (d, J = 11.6 Hz, 1H), 5.05 (d, J = 11.6 Hz, 1H), 5.44 (br. . s, 1H), 7.34-7.48 (yd, 5H), 9.37 (Br.S., 1H); MS yd / z 435 [M + H] + .

Step 2

tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate

(V-1) tert-butyl {2 – [({[( 2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl ] carbonyl} amino) oxy] ethyl} carbamate (3.91 g, to a methanol solution (80 mL) of 9.01mmol), 10% palladium on carbon catalyst (50% water, 803 mg) was added, under hydrogen atmosphere and stirred for 45 minutes . The reaction mixture was filtered through Celite, after concentrated under reduced pressure to give 3.11g of the title compound (quantitative).

HPLC: COSMOSIL 5C18 MS-II 4.6 × 150 mm, 33.3 mM phosphate buffer / MeCN = 75/25, 1.0 mL / min, UV 210 nm, Retweeted 3.9 from min; 1 H NMR (400 MHz, CD 3 OD) [delta] 1.44 (s, 9H) , 1.73-1.83 (m, 1H), 1.86-1.99 (m, 1H), 2.01-2.12 (m, 1H), 2.22 (br.dd., J = 15.0, 7.0 Hz, 1H), 3.03 (d, J= 12.0 Hz, 1H), 3.12 (br.d., J = 12.0 Hz, 1H), 3.25-3.35 (m, 2H), 3.68-3.71 (m, 1H), 3.82-3.91 (m, 3H); MS M / Z 345 [M Tasu H] Tasu .

Step 3

Tetrabutylammonium tert- butyl {2 – [({[( 2S, 5R) -7- oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl } amino) oxy] ethyl} carbamate

(VI-1) tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct 2-yl] carbonyl} amino) oxy] ethyl} carbamate (3.09g, in dichloromethane (80mL) solution of 8.97mmol), 2,6- lutidine (3.20mL), sulfur trioxide – pyridine complex (3 .58g) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was poured into half-saturated aqueous sodium bicarbonate solution, washed the aqueous layer with chloroform, tetrabutylammonium hydrogen sulfate to the aqueous layer and (3.47 g) chloroform (30 mL) was added and stirred for 10 minutes. The aqueous layer was extracted with chloroform, drying the obtained organic layer with anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the title compound 5.46g (91% yield).

HPLC: COSMOSIL 5C18 MS-II 4.6X150mm, 33.3MM Phosphate Buffer / MeCN = 80/20, 1.0ML / Min, UV210nm, RT 2.0 Min; 1 H NMR (400 MHz, CDCl 3 ) Deruta 1.01 (T, J = 7.4 Hz, 12H), 1.37-1.54 (m , 8H), 1.45 (s, 9H), 1.57-1.80 (m, 9H), 1.85-1.98 (m, 1H), 2.14-2.24 (m, 1H), 2.30- 2.39 (m, 1H), 2.83 (d, J = 11.6 Hz, 1H), 3.20-3.50 (m, 11H), 3.85-3.99 (m, 3H), 4.33-4.38 (m, 1H), 5.51 (br s , 1H), 9.44 (Br.S., 1H); MS yd / z 425 [M-Bu 4 N + 2H] + .

Step 4 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VII-1)

tetra butylammonium tert- butyl {2 – [({[( 2S, 5R) -7- oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate (5.20g, 7.82mmol) in dichloromethane (25mL) solution of ice-cold under trifluoroacetic acid (25mL), and the mixture was stirred for 1 hour at 0 ℃. The reaction mixture was concentrated under reduced pressure, washed the resulting residue with diethyl ether, adjusted to pH7 with aqueous sodium bicarbonate, subjected to an octadecyl silica gel column chromatography (water), after freeze drying, 1.44 g of the title compound obtained (57% yield).

HPLC: COSMOSIL 5C18 MS-II 4.6X150mm, 33.3MM Phosphate Buffer / MeCN = 99/1, 1.0ML / Min, UV210nm, RT 3.1 Min; 1 H NMR (400 MHz, D 2O) Deruta 1.66-1.76 (M, 1H), 1.76-1.88 (m, 1H ), 1.91-2.00 (m, 1H), 2.00-2.08 (m, 1H), 3.02 (d, J = 12.0 Hz, 1H), 3.15 (t, J = 5.0 Hz , 2H), 3.18 (br d , J = 12.0 Hz, 1H), 3.95 (dd, J = 7.8, 2.2 Hz, 1H), 4.04 (t, J = 5.0 Hz, 2H), 4.07 (dd, J = 6.4 3.2 Hz &, 1H); MS yd / z 325 [M + H] + .

PATENT

Example 

64 tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy ] ethyl} carbamate (V-1) 

[of 124] 

tert- butyl {2 – [({[(2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl } carbamate (example 63q, net 156.42g, 360mmol) in methanol solution (2.4L) of 10% palladium carbon catalyst (50% water, 15.64g) was added, under an atmosphere of hydrogen, stirred for 1.5 hours did. The catalyst was filtered through celite, filtrate was concentrated under reduced pressure until 450mL, concentrated to 450mL by adding acetonitrile (1.5 L), the mixture was stirred ice-cooled for 30 minutes, collected by filtration the precipitated crystals, washing with acetonitrile, and vacuum dried to obtain 118.26g of the title compound (net 117.90g, 95% yield). Equipment data of the crystals were the same as those of the step 2 of Reference Example 3.

Example

65 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VI-1)

 

 tert- butyl {2 – [({[(2S, 5R) -1,6- -6- hydroxy-7-oxo-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate (example 64,537.61g, 1.561mol) in acetonitrile (7.8L) solution of 2,6-lutidine (512.08g), sulfur trioxide – pyridine complex (810.3g) was added, at room temperature in the mixture was stirred overnight. Remove insolubles and the mixture was filtered, the filtrate concentrated to 2.5 L, diluted with ethyl acetate (15.1L). The mixture was extracted with 20% phosphoric acid 2 hydrogencarbonate aqueous solution (7.8L), the resulting aqueous layer into ethyl acetate (15.1L), added tetrabutylammonium hydrogen sulfate (567.87g), was stirred for 20 min. The organic layer was separated layers, dried over anhydrous magnesium sulfate (425 g), after filtration, concentration under reduced pressure, substituted concentrated tetrabutylammonium tert- butyl with dichloromethane (3.1L) {2 – [({[(2S, 5R ) -7-oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate was obtained 758g (net 586.27g, Osamu rate 84%).

 The tetra-butyl ammonium salt 719g (net 437.1g, 0.656mol) in dichloromethane (874mL) solution was cooled to -20 ℃, dropping trifluoroacetic acid (874mL) at 15 minutes, 1 the temperature was raised to 0 ℃ It was stirred time. The reaction was cooled to -20 ° C. was added dropwise diisopropyl ether (3.25L), and the mixture was stirred for 1 hour the temperature was raised to 0 ° C.. The precipitate is filtered, washed with diisopropyl ether to give the title compound 335.36g of crude and vacuum dried (net 222.35g, 99% yield).

 The title compound of crude were obtained (212.99g, net 133.33g) and ice-cold 0.2M phosphate buffer solution of pH5.3 mix a little at a time, alternating between the (pH6.5,4.8L). The solution was concentrated under reduced pressure to 3.6L, it was adjusted to pH5.5 at again 0.2M phosphate buffer (pH6.5,910mL). The solution resin purification (Mitsubishi Kasei, SP207, water ~ 10% IPA solution) is subjected to, and concentrated to collect active fractions, after lyophilization, to give the title compound 128.3 g (96% yield). Equipment data of the crystals were the same as those of step 3 of Reference Example 3.

PATENT

US 20140288051

WO 2014091268

WO 2013180197

US 20130225554

PATENT

IN2015MU287

PATENT

WO2013180197

Example 59
(2S, 5R) -N- (2- aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (II-059)

Figure JPOXMLDOC01-appb-C000130

Step 1
tert- butyl {2 – [({[(2S, 5R) -6- Benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl } carbamate

Figure JPOXMLDOC01-appb-C000131

Acid of Example 9 or 16 (6b, 1.34g, 4.87mmol) in methylene chloride (35mL) solution of triethylamine (2.71mL), N- ethyl -N ‘- (3- dimethylaminopropyl) carbodiimide hydrochloride (1.41g), 1- hydroxybenzotriazole monohydrate (1.15g), were added tert- butyl of Reference Example 9, wherein 2- (amino-oxy) ethyl carbamate (1.12g), room temperature It was stirred overnight Te.Water was added to the reaction solution to a residue obtained by concentration under reduced pressure, and extracted with ethyl acetate. The resulting organic layer with 0.1M hydrochloric acid, saturated aqueous sodium bicarbonate solution, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated.The resulting residue was purified by silica gel column and purified by chromatography (hexane / ethyl acetate = 8 / 2-0 / 10) to give the title compound 1.77g (84% yield).
[Α] D 20 -0.08 ° (c 0.29, CHCl 3); 1 H NMR (400 MHz, CDCl 3), δ: 1.44 (s, 9H), 1.56-1.70 (m, 1H), 1.90-2.09 (m , 2H), 2.25-2.38 (m, 1H), 2.76 (d, J = 11.6 Hz, 1H), 3.03 (br d, J = 11.6 Hz, 1H), 3.24-3.47 (m, 3H), 3.84-4.01 (m, 3H), 4.90 (d, J = 11.6 Hz, 1H), 5.05 (d, J = 11.6 Hz, 1H), 5.44 (br s, 1H), 7.34-7.48 (m, 5H), 9.37 (br s, 1H); MS m / z 435 [M + H] +; enantiomeric excess of 99.9% or higher ee (CHIRALPAK AD-H, 4.6x150mm, hexane / ethanol = 2/1, UV210nm, flow rate 1mL / min, retention time 4.95min (2R, 5S), 6.70min (2S, 5R).

Step 2
tert- butyl {2 – [({[(2S, 5R) -1,6- -6- hydroxy-7-oxo-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate

Figure JPOXMLDOC01-appb-C000132

Compound of the above Step 1 (3.91g, 9.01mmol) in methanol (80mL), 10% palladium on carbon catalyst (50% water, 803mg) was added, under hydrogen atmosphere and stirred for 45 minutes. The reaction mixture was filtered through Celite, then concentrated under reduced pressure, to give 3.11g of the title compound (quantitative).
1 H NMR (400 MHz, CD 3 OD), δ: 1.44 (s, 9H), 1.73-1.83 (m, 1H), 1.86-1.99 (m, 1H), 2.01-2.12 (m, 1H), 2.22 ( br dd, J = 15.0, 7.0 Hz, 1H), 3.03 (d, J = 12.0 Hz, 1H), 3.12 (br d, J = 12.0 Hz, 1H), 3.25-3.35 (m, 2H), 3.68-3.71 (m, 1H), 3.82-3.91 (m, 3H); MS m / z 345 [M + H] +.

Step 3
(2S, 5R) -N- (2- aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide The above step 2 compound (3. 09g, in methylene chloride (80mL) solution of 8.97mmol), 2,6- lutidine (3.20mL), sulfur trioxide – was added pyridine complex (3.58g), and stirred at room temperature overnight. The reaction mixture was poured into half-saturated aqueous sodium bicarbonate solution, and washed the aqueous layer with chloroform, and tetrabutylammonium hydrogen sulfate (3.47g) and chloroform (30mL) was added to the aqueous layer and stirred for 10 minutes. After extracting the aqueous layer with chloroform, drying the resulting organic layer over anhydrous sodium sulfate, filtered, concentrated under reduced pressure tetrabutylammonium tert- butyl {2 – [({[(2S, 5R) -7- oxo – 6- (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate was obtained 5.46g (91% yield).
1 H NMR (400 MHz, CDCl 3), δ: 1.01 (t, J = 7.4 Hz, 12H), 1.37-1.54 (m, 8H), 1.45 (s, 9H), 1.57-1.80 (m, 9H), 1.85-1.98 (m, 1H), 2.14-2.24 (m, 1H), 2.30-2.39 (m, 1H), 2.83 (d, J = 11.6 Hz, 1H), 3.20-3.50 (m, 11H), 3.85- 3.99 (m, 3H), 4.33-4.38 (m, 1H), 5.51 (br s, 1H), 9.44 (br s, 1H); MS m / z 425 [M-Bu 4 N + 2H] +.

The tetrabutyl ammonium salt (5.20g, 7.82mmol) in methylene chloride (25mL) solution of under ice-cooling trifluoroacetic acid (25mL), and the mixture was stirred for 1 hour at 0 ℃. The reaction mixture was concentrated under reduced pressure, washed resulting residue with diethyl ether, at aqueous sodium bicarbonate was adjusted to pH7, it performs an octadecyl silica gel column chromatography (water), after freeze-drying, 1.44g of the title compound The obtained (57% yield).
[Α] D 24 -63.5 ° (c 0.83, H 2 O); 1 H NMR (400 MHz, D 2 O), δ: 1.66-1.76 (m, 1H), 1.76-1.88 (m, 1H), 1.91 -2.00 (m, 1H), 2.00-2.08 (m, 1H), 3.02 (d, J = 12.0 Hz, 1H), 3.15 (t, J = 5.0 Hz, 2H), 3.18 (br d, J = 12.0 Hz , 1H), 3.95 (dd, J = 7.8, 2.2 Hz, 1H), 4.04 (t, J = 5.0 Hz, 2H), 4.07 (dd, J = 6.4, 3.2 Hz, 1H); MS m / z 325 [ M + H] +.

PATENT

WO2016116878

ANTIBACTERIAL COMPOSITIONS OF A BETA-LACTAMASE INHIBITOR WITH A CEPHALOSPORINAbstract:

Pharmaceutical compositions comprising: (a) at least one cephalosporin antibacterial agent and (b) a compound of Formula (I) or a stereoisomer or a pharmaceutically acceptable derivative thereof are disclosed. Formula (I)

PATENT

WO 2016120752, WOCKHARDT, NEW PATENT, Nacubactam

Formula (I), chemically known as (25, 5i?)-N-(2-aminoethoxy)-6-(sulfooxy)-7-oxo-l ,6-diazabicyclo[3.2.1 ]octane-2-carboxamide has antibacterial properties and is disclosed in PCT International Patent Application No. PCT/IB2013/053092, PCT/JP2013/064971 and PCT/IB2012/002675. The present invention discloses a process for preparation of a compound of Formula (I).

Formula (I)

 

(VII) (VIII) (IX)

Scheme 2

Example 1

Synthesis of fert-butyl-r2-(aminooxy) ethyllcarbamate (III)

Preparation of fert-butyl-2-hydroxy ethylcarbamate (VIII):

Formula (VIII)

To a stirred solution of ethanolamine (50.0 g, 0.8186 mol) in dichloromethane (1000 ml), was added triethylamine (124 g, 1.228 mol) at 0°C. After 10 minutes, di-teri-butyl dicarbonate (VII, 214.15 g, 0.9823 mol) was added drop wise at 0°C under continuous stirring. Then reaction mass was allowed to warm to 25°C and stirred further for 3 hours. After completion of reaction, the resulting reaction mixture was poured into water (250 ml) and the organic layer was separated and dried over anhydrous sodium sulfate. The dried organic layer was concentrated under reduced pressure to obtain 130 g of the titled product as colorless oil in 98% yield.

Analysis:

Mass: 162 (M+l); for Molecular Weight of 161.2 and Molecular Formula of C7H15NO3.

1H NMR (400MHz, CDC13): δ 4.92(br s,lH), 3.72-3.68(q,2H), 3.30-3.26(q,2H), 2.33(br s,lH), 1.44(s,9H).

Preparation of A7-Boc-2-(2-aminoethoxy)isoindoline-l,3-dione (IX):

To a stirred solution of teri;butyl-2-hydroxy-ethylcarbamate (VIII, 50 g, 0.3106 mol) in tetrahydrofuran (500 ml), was added triphenylphosphine (89.5 g, 0.3416 mol) at 25°C. After stirring for 10 minutes, a solution of N-hydroxyphthalimide (50.66 g, 0.3106 mol) in dichloromethane (250 ml) was added to the reaction mass at 25 °C over a period of 10 minutes. After stirring for further 10 minutes, diisopropyl azodicarboxylate (69.1 g, 0.3416 mol) was added to the reaction mass in small portions (exothermic reaction was observed up to 34°C). The resulting reaction mass was stirred further at 25°C. After 16 hours, the reaction mass was concentrated under reduced pressure to obtain colorless oily material. The oily residue was diluted with diisopropyl ether (200 ml) and stirred for 30 minutes. The separated solid was filtered under suction. The filtrate was evaporated under reduced pressure and the residue subjected to di-isopropyl ether treatment (200 ml). This procedure was repeated once again. The filtrate was concentrated to obtain a solid product. The obtained solid was washed with diisopropyl ether (50 ml) and dried under reduced pressure. This solid contains small amount of triphenylphosphine oxide, along with the product. This was used as such for the next reaction without further purification.

Analysis:

Mass: 307.2 (M+l); for Molecular Weight of 306.3 and Molecular Formula of Ci5Hi8N205; 1H NMR of purified material (400MHz, CDC13): 7.85-7.25 (m,4H), 5.62(br s,lH), 4.26-4.23(t,2H), 3.46-3.42(q,2H), 1.46(s,9H).

Step 3: Preparation of fert-butyl-[ -(aminooxy) ethyl]carbamate (III):

Formula (III)

To a stirred solution of N-Boc-2-(2-aminoethoxy)isoindoline-l ,3-dione (IX, 97 g, 0.3167 mol) in dichloromethane (970 ml) was added hydrazine hydrate (31.7 g, 0.6334 mol) , at 0°C, drop wise, over a period of 45 minutes and the stirring continued further. After 2 hours, the reaction mass was filtered under suction. Filtrate was washed with water (485 ml), and the organic layer was diluted with an aq. solution of 10% potassium hydrogen sulfate (485 ml) and stirred for 15 minutes. The aqueous layer was separated, neutralized with solid sodium hydrogen carbonate and extracted with dichloromethane (2 x 485 ml). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain colorless oil, this was used as such for further reaction immediately (28g, overall yield of step II and step III was 60%)

Analysis:

Mass: 177.2 (M+l) for Molecular Weight of 176.2 and Molecular Formula of C7H16N2O3.

Example 2

Synthesis of (25,5R)-jV-(2-aminoethoxy)-6-(sulfooxy)-7-oxo-l,6-diaza-bicvclor3.2.11octane-2- carboxamide (I)

Step 1: Preparation of (25,5R)-iV-(2-Boc-aminoethoxy)-6-(benzyloxy)-7-oxo-l,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (IV):

To a clear solution of sodium (25,5i?)-6-(benzyloxy)-7-oxo-l,6-diazabicyclo[3.2.1]octane-2-carboxylate (II, 42.67 g, 0.143 mol; prepared according to the procedure disclosed in Indian Patent Application No. 699/MUM/2013) in water (426 ml) was added EDC.HC1 (67.1 g, 0.349 mol) at 15°C

under stirring. After 10 minutes, a solution of teri-butyl-[2-(aminooxy) ethyl]carbamate (III, 28.0g, 0.159 mol; prepared as per the literature procedure depicted in Scheme 2) in dimethylformamide (56 ml) was added drop wise at 10°C under continuous stirring. The temperature of the reaction mass was allowed to warm to 25°C and then HOBt (21.5g, 0.159 mol) was added in small portions over a period of 15 minutes and the resulting mixture was further stirred at room temperature for 16 hours. The reaction was continuously monitored using thin layer chromatography using mixture of acetone and hexane (35 :65) as solvent system. After completion of reaction, the resulting mixture was filtered and the residue was washed with water (130 ml). The obtained white residue was suspended in water (130 ml) and the mixture stirred at 50°C for 3 hours. The resulting suspension was filtered, the residue dried under reduced pressure to obtain 51 g of (2S,5R)-N-(2-Boc-aminoethoxy)-6-(benzyloxy)-7-oxo-l ,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (IV) as off white solid in 73% yield.

Analysis:

Mass: 433.4 (M-l ); for Molecular Weight of 434.5 and Molecular Formula of C21H30N4O6;

1H-NMR (400MHz, CDC13): δ 9.32 (br s, 1H), 7.41 -7.26(m,5H), 5.41(br s, 1H), 5.06-4.88(dd, 2H), 3.98-3.96(d,lH), 3.91-3.90(m,2H), 3.39(m, 1H), 3.31-3.26(m, 2H), 3.04-3.01(d,lH), 2.77-2.74(d, 1H), 2.33-2.28(m, 1H), 2.03-1.93(m, 2H), 1.67-1.64(m, 1H), 1.44(s, 9H);

Purity as determined by HPLC: 99.4%.

Step 2: Preparation of (2S,5R)-iV-(2-Boc-aminoethoxy)-6-(hydroxy)-7-oxo-l,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (V):

A solution of (25,5i?)-N-(2-Boc-aminoethoxy)-6-(benzyloxy)-7-oxo-l ,6-diaza-bicyclo[3.2.1] octane-2-carboxamide (IV, 38 g, 0.0875 mol) in a mixture of dimethylformamide and dichloromethane (2: 8, 76 ml: 304 ml), containing 10% Pd/C (7.6 g, 50% wet) was hydrogenated at 50 psi hydrogen atmosphere at 25°C for 3 hours. The resulting mixture was filtered through a celite pad. The residue was washed with dichloromethane (75 ml). The solvent from the combined filtrate was evaporated

under reduced pressure to obtain 30 g (25,5i?)-N-(2-Boc-aminoethoxy)-6-(hydroxy)-7-oxo-l ,6-diaza-bicyclo[3.2.1 ]octane-2-carboxamide (V) as an oil, which was used as such for the next reaction without further purification.

Analysis:

Mass: 343.3 (M-l ) for Molecular Weight of 344.3 and Molecular Formula of C14H24N4O6.

Step 3: Preparation of (25,5R)-iV-(2-Boc-aminoethoxy)-6-(sulfooxy)-7-oxo-l,6-diaza-bicyclo[3.2.1]octane-2-carboxamide,tetrabutyl ammonium salt (VI):

To a stirred solution of (25,5i?)-N-(2-Boc-aminoethoxy)-6-(hydroxy)-7-oxo-l ,6-diaza-bicyclo[3.2.1 ]octane-2-carboxamide (V, 30.0 g, 0.0875 mol) in dimethylformamide (150 ml) was added sulphur trioxide dimethylformamide complex (16.06 g, 0.105 mol) in one portion, at 10°C. The reaction mass was stirred at the same temperature for 30 minutes and then allowed to warm to room temperature. After 2 hours, a solution of tetrabutylammonium acetate (31.6 g, 0.105 mol) in water (95 ml) was slowly added to the reaction mixture and stirred for another 2 hours. The solvent from the reaction mixture was evaporated under reduced pressure to obtain an oily residue. The oily mass was co-evaporated with xylene (2 x 60 ml) to obtain thick mass. This mass was partitioned between 1 : 1 mixture of dichloromethane (300 ml) and water (300 ml). The organic layer was separated and the aqueous layer re-extracted with dichloromethane (150 ml). The combined organic extracts were washed with water (3 x 150 ml) and dried over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure and the resulting oily mass was triturated with ether (3 x 60 ml). Each time the ether layer was decanted and the residue was finally concentrated under reduced pressure to obtain the sticky mass. The so obtained material was purified by column chromatography over silica gel using mixture of methanol and dichloromethane as elution solvent. The solvent from the combined fractions was evaporated to obtain 47.5 g of (25,5i?)-N-(2-Boc-aminoethoxy)-6-(sulfooxy)-7-oxo-l ,6-diaza-bicyclo[3.2.1 ]octane-2-carboxamide,tetrabutyl ammonium salt as white foam in 70% yield.

Analysis:

Mass: 423.4 (M-l) as free sulphonic acid; for Molecular Weight of 665.9 and Molecular Formula of C30H59N5O9 S;

1H- NMR (400MHz, CDC13): δ 9.52(br s, 1H), 5.53(br s, 1H), 4.33(s, 1H), 3.95-3.92(m,3H), 3.37-3.27(m, 1 1H), 2.87-2.84(d, 1H), 2.35-2.30(m, 1H), 2.17(m, 1H), 1.96-1.88(m, 2H), 1.74-1.60(m,8 H), 1.47-1.40(m, 17H), 1.02-0.98(m, 12H).

Step 4: Preparation of (2S R)-iV-(2-aminoethoxy)-6-(sulfooxy)-7-oxo-l,6-diaza-bicyclo[3.2.1]octane-2-carboxamide (I):

Formula (I)

To a stirred solution of (2S,5i?)-N-(2-Boc-aminoethoxy)-6-(sulfooxy)-7-oxo-l ,6-diaza-bicyclo[3.2.1 ]octane-2-carboxamide, tetrabutyl ammonium salt (VI, 17 g, 0.0225 mol) in dichloromethane (85 ml) was added trifluoroacetic acid (85 ml) drop wise at -10°C over a period of 45 minutes. The resulting mass was further stirred at same temperature for 1 hour. The resulting reaction mixture was poured into cyclohexane (850 ml), stirred well for 30 minutes and the separated oily layer was collected. This procedure was repeated one more time and finally the separated oily layer was added to tert-butyl methyl ether (170 ml) under vigorous stirring at 25°C. The ether layer was removed by decantation from the precipitated solid. This procedure was repeated twice again with tert-butyl methyl ether (2 x 170 ml). The solid thus obtained was stirred with fresh dichloromethane (170 ml) for 30 minutes and filtered. The residual solid was dried at 45°C under reduced pressure to yield 7.3g of the titled compound in crude form. The obtained solid was further dissolved in water, (7.3 ml) and to this solution was added basic resin (Amberlyst A-26 -OH ion exchange resin, 4.4 g) under stirring. After 0.5 hour, the resin was filtered and to the filtrate isopropanol (51 ml) was added slowly at 25°C. The solution was further stirred for 12 hours. The separated solid was filtered and washed with additional isopropanol (7.5 ml) and dried under reduced pressure to obtain 4.3 g of (2S ,5R)-N-(2-aminoethoxy)-6-(sulfooxy)-7-oxo-l ,6-diaza-bicyclo[3.2.1 ]octane-2-carboxamide as off-white solid in 52 % yield.

Analysis:

Mass: 323.1 (M-l); for Molecular Weight of 324.31 and Molecular Formula of C9H16N4O7S; 1H-NMR (400MHz, D20): δ 4.07-4.06(d, 1H), 4.05-4.03(t, 2H), 3.96-3.94(d, 1H), 3.20(br s, 1H), 3.16-3.13(t, 2H), 3.02-2.99(d, 1H), 2.04-1.68(m, 4H);

Purity as determined by HPLC: 94.88%.

REF

http://www.pewtrusts.org/~/media/assets/2015/02/antibioticsinnovationproject_datatable_201502_v3.pdf?la=en

WO2015110969A3 * Jan 21, 2015 Nov 26, 2015 Wockhardt Limited Nitrogen containing compounds and their use as antibacterial agents
WO2015150941A1 * Mar 12, 2015 Oct 8, 2015 Wockhardt Limited A process for preparation of sodium (2s, 5r)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate
WO2016088863A1 * Dec 4, 2015 Jun 9, 2016 Meiji Seikaファルマ株式会社 Method for producing crystals of diazabicyclooctane derivative and stable lyophilized preparation
EP2931723A4 * Dec 11, 2012 Jun 1, 2016 Fedora Pharmaceuticals Inc New bicyclic compounds and their use as antibacterial agents and -lactamase inhibitors
US8933232 Mar 29, 2013 Jan 13, 2015 Cubist Pharmaceuticals, Inc. 1,3,4-oxadiazole and 1,3,4-thiadiazole beta-lactamase inhibitors
US8933233 Mar 29, 2013 Jan 13, 2015 Cubist Pharmaceuticals, Inc. 1,3,4-oxadiazole and 1,3,4-thiadiazole β-lactamase inhibitors
US8940897 Mar 29, 2013 Jan 27, 2015 Cubist Pharmaceuticals, Inc. 1,3,4-oxadiazole and 1,3,4-thiadiazole β-lactamase inhibitors
US8962843 Mar 29, 2013 Feb 24, 2015 Cubist Pharmaceuticals, Inc. 1,3,4-oxadiazole and 1,3,4-thiadiazole beta-lactamase inhibitors
US8962844 Mar 29, 2013 Feb 24, 2015 Cubist Pharmaceuticals, Inc. 1,3,4-oxadiazole and 1,3,4-thiadiazole β-lactamase inhibitors
US9120795 Mar 14, 2014 Sep 1, 2015 Cubist Pharmaceuticals, Inc. Crystalline form of a β-lactamase inhibitor
US9120796 Oct 2, 2014 Sep 1, 2015 Cubist Pharmaceuticals, Inc. B-lactamase inhibitor picoline salt
US9309245 Apr 2, 2013 Apr 12, 2016 Entasis Therapeutics Limited Beta-lactamase inhibitor compounds
US9393239 Apr 15, 2014 Jul 19, 2016 Fedora Pharmaceuticals Inc. Bicyclic compounds and their use as antibacterial agents and betalactamase inhibitors

/////////////IN2015MU287, WO-2016120752, nacubactam, WOCKHARDT, NEW PATENT, WK ?, WK-?, WK?,  CAS 1452458-86-4C9 H16 N4 O7 S, 324.31, Beta lactamase inhibitor, Roche, Meiji Seika Pharma,  Fedora Pharmaceuticals, nacubactam hydrate , PHASE 1, A diazabicyclooctane beta-lactamase inhibitor, bacterial infection, July 2016,  phase 1 clinical development, RG-6080, 1452458-86-4, FPI-1459,  OP-0595, Phase I ,  β-lactamase inhibitor, bacterial infections, Fedora parmaceuticals, Meiji Seika Pharma

NCCONC(=O)[C@@H]2CC[C@@H]1C[N@]2C(=O)N1OS(=O)(=O)O

RG 6080, Nacubactam


STR1

RG-6080

Sulfuric acid, mono[(1R,2S,5R)-2-[[(2-aminoethoxy)amino]carbonyl]-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl] ester

Phase I

A β-lactamase inhibitor potentially for the treatment of bacterial infections.

RG-6080; FPI-1459; OP-0595

CAS No. 1452458-86-4

Molecular Formula C9 H16 N4 O7 S
Formula Weight 324.31
  • Originator Fedora Pharmaceuticals
  • Developer Meiji Seika Pharma
  • Class Antibacterials; Azabicyclo compounds
  • Mechanism of Action Beta lactamase inhibitors
  • Phase IBacterial infections

Most Recent Events

  • 13 Jan 2015 OP 0595 licensed to Roche worldwide, except Japan ,
  • 30 Nov 2014 Meiji Seika Pharma completes a phase I trial in Healthy volunteers in Australia (NCT02134834)
  • 01 May 2014 Phase-I clinical trials in Bacterial infections (in volunteers) in Australia (IV)

SYNTHESIS

WO 2015046207,

STR1

CONTD…………………..

STR1

CONTD………………………………..

STR1

Patent

WO 2015053297

The novel heterocyclic compound in Japanese Patent 4515704 (Patent Document 1), preparation and shown for their pharmaceutical use, sodium trans-7-oxo-6- (sulfooxy) as a representative compound 1,6-diazabicyclo [3 .2.1] discloses an octane-2-carboxamide (NXL104). Preparation in regard to certain piperidine derivatives which are intermediates Patent 2010-138206 (Patent Document 2) and JP-T 2010-539147 (Patent Document 3) are shown at further WO2011 / 042560 (Patent Document 4) NXL104 to disclose a method for producing the crystals.
 In Patent 5038509 (Patent Document 5) (2S, 5R) -7- oxo -N- (piperidin-4-yl) -6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane – 2- carboxamide (MK7655) is shown, discloses the preparation of certain piperidine derivatives with MK7655 at Patent 2011-207900 (Patent Document 6) and WO2010 / 126820 (Patent Document 7).
 The present inventors also disclose the novel diazabicyclooctane derivative represented by the following formula (VII) in Japanese Patent Application 2012-122603 (Patent Document 8).
Patent Document 1: Japanese Patent No. 4515704 Pat
Patent Document 2: Japanese Patent Publication 2010-138206 Pat
Patent Document 3: Japanese patent publication 2010-539147 Pat
Patent Document 4: International Publication No. WO2011 / 042560 Patent
Patent Document 5: Japanese Patent No. 5038509 Pat
Patent Document 6: Japanese Patent Publication 2011-207900 Pat
Patent Document 7: International Publication No. WO2010 / 126820 Patent
Patent Document 8: Japanese Patent application 2012-122603 Pat.
[Chemical formula 1] (In the formula, R 3 are the same as those described below)

Reference Example
5 of 5 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VII-1)
Formula 43]
step 1 tert-butyl {2 – [({[( 2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl } amino) oxy] ethyl} carbamate  (IV-1)(2S, 5R)-6-(benzyloxy) -7-oxo-1,6-diazabicyclo [3.2.1] octane-2-carboxylic acid (4 .30g, dehydrated ethyl acetate (47mL) solution of 15.56mmol) was cooled to -30 ℃, isobutyl chloroformate (2.17g, washing included dehydration ethyl acetate 1mL), triethylamine (1.61g, washing included dehydration ethyl acetate 1 mL), successively added dropwise, and the mixture was stirred 1 hour at -30 ° C.. To the reaction solution tert- butyl 2-dehydration of ethyl acetate (amino-oxy) ethyl carbamate (3.21g) (4mL) was added (washing included dehydration ethyl acetate 1mL), raising the temperature over a period of 1.5 hours to 0 ℃, It was further stirred overnight. The mixture of 8% aqueous citric acid (56 mL), saturated aqueous sodium bicarbonate solution (40 mL), sequentially washed with saturated brine (40 mL), dried over anhydrous magnesium sulfate, filtered, concentrated to 5 mL, up to 6mL further with ethanol (10 mL) It was replaced concentrated. Ethanol to the resulting solution (3mL), hexane the (8mL) in addition to ice-cooling, and the mixture was stirred inoculated for 15 minutes. The mixture was stirred overnight dropwise over 2 hours hexane (75 mL) to. Collected by filtration the precipitated crystals, washing with hexane to give the title compound 5.49g and dried in vacuo (net 4.98 g, 74% yield). HPLC: COSMOSIL 5C18 MS-II 4.6 × 150 mm, 33.3 mM phosphate buffer / MeCN = 50/50, 1.0 mL / min, UV 210 nm, Retweeted 4.4 min; 1 H NMR (400 MHz, CDCl 3 ) [delta] 1.44 (s, 9H), 1.56-1.70 (m, 1H), 1.90-2.09 (m, 2H), 2.25-2.38 (m, 1H), 2.76 (d, J = 11.6 Hz, 1H), 3.03 (br.d., J = 11.6 Hz , 1H), 3.24-3.47 (m, 3H), 3.84-4.01 (m, 3H), 4.90 (d, J = 11.6 Hz, 1H), 5.05 (d, J = 11.6 Hz, 1H), 5.44 (br. . s, 1H), 7.34-7.48 (yd, 5H), 9.37 (Br.S., 1H); MS yd / z 435 [M + H] + .
Step 2
tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate
(V-1) tert-butyl {2 – [({[( 2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl ] carbonyl} amino) oxy] ethyl} carbamate (3.91 g, to a methanol solution (80 mL) of 9.01mmol), 10% palladium on carbon catalyst (50% water, 803 mg) was added, under hydrogen atmosphere and stirred for 45 minutes . The reaction mixture was filtered through Celite, after concentrated under reduced pressure to give 3.11g of the title compound (quantitative).
HPLC: COSMOSIL 5C18 MS-II 4.6 × 150 mm, 33.3 mM phosphate buffer / MeCN = 75/25, 1.0 mL / min, UV 210 nm, Retweeted 3.9 from min; 1 H NMR (400 MHz, CD 3 OD) [delta] 1.44 (s, 9H) , 1.73-1.83 (m, 1H), 1.86-1.99 (m, 1H), 2.01-2.12 (m, 1H), 2.22 (br.dd., J = 15.0, 7.0 Hz, 1H), 3.03 (d, J= 12.0 Hz, 1H), 3.12 (br.d., J = 12.0 Hz, 1H), 3.25-3.35 (m, 2H), 3.68-3.71 (m, 1H), 3.82-3.91 (m, 3H); MS M / Z 345 [M Tasu H] Tasu .
Step 3
Tetrabutylammonium tert- butyl {2 – [({[( 2S, 5R) -7- oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl } amino) oxy] ethyl} carbamate
(VI-1) tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct 2-yl] carbonyl} amino) oxy] ethyl} carbamate (3.09g, in dichloromethane (80mL) solution of 8.97mmol), 2,6- lutidine (3.20mL), sulfur trioxide – pyridine complex (3 .58g) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was poured into half-saturated aqueous sodium bicarbonate solution, washed the aqueous layer with chloroform, tetrabutylammonium hydrogen sulfate to the aqueous layer and (3.47 g) chloroform (30 mL) was added and stirred for 10 minutes. The aqueous layer was extracted with chloroform, drying the obtained organic layer with anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the title compound 5.46g (91% yield).
HPLC: COSMOSIL 5C18 MS-II 4.6X150mm, 33.3MM Phosphate Buffer / MeCN = 80/20, 1.0ML / Min, UV210nm, RT 2.0 Min; 1 H NMR (400 MHz, CDCl 3 ) Deruta 1.01 (T, J = 7.4 Hz, 12H), 1.37-1.54 (m , 8H), 1.45 (s, 9H), 1.57-1.80 (m, 9H), 1.85-1.98 (m, 1H), 2.14-2.24 (m, 1H), 2.30- 2.39 (m, 1H), 2.83 (d, J = 11.6 Hz, 1H), 3.20-3.50 (m, 11H), 3.85-3.99 (m, 3H), 4.33-4.38 (m, 1H), 5.51 (br s , 1H), 9.44 (Br.S., 1H); MS yd / z 425 [M-Bu 4 N + 2H] + .
Step 4 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VII-1)
tetra butylammonium tert- butyl {2 – [({[( 2S, 5R) -7- oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate (5.20g, 7.82mmol) in dichloromethane (25mL) solution of ice-cold under trifluoroacetic acid (25mL), and the mixture was stirred for 1 hour at 0 ℃. The reaction mixture was concentrated under reduced pressure, washed the resulting residue with diethyl ether, adjusted to pH7 with aqueous sodium bicarbonate, subjected to an octadecyl silica gel column chromatography (water), after freeze drying, 1.44 g of the title compound obtained (57% yield).
HPLC: COSMOSIL 5C18 MS-II 4.6X150mm, 33.3MM Phosphate Buffer / MeCN = 99/1, 1.0ML / Min, UV210nm, RT 3.1 Min; 1 H NMR (400 MHz, D 2O) Deruta 1.66-1.76 (M, 1H), 1.76-1.88 (m, 1H ), 1.91-2.00 (m, 1H), 2.00-2.08 (m, 1H), 3.02 (d, J = 12.0 Hz, 1H), 3.15 (t, J = 5.0 Hz , 2H), 3.18 (br d , J = 12.0 Hz, 1H), 3.95 (dd, J = 7.8, 2.2 Hz, 1H), 4.04 (t, J = 5.0 Hz, 2H), 4.07 (dd, J = 6.4 3.2 Hz &, 1H); MS yd / z 325 [M + H] + .

PATENT

WO 2015046207

Example
64 tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy ] ethyl} carbamate (V-1)
[of 124]

tert- butyl {2 – [({[(2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl } carbamate (example 63q, net 156.42g, 360mmol) in methanol solution (2.4L) of 10% palladium carbon catalyst (50% water, 15.64g) was added, under an atmosphere of hydrogen, stirred for 1.5 hours did. The catalyst was filtered through celite, filtrate was concentrated under reduced pressure until 450mL, concentrated to 450mL by adding acetonitrile (1.5 L), the mixture was stirred ice-cooled for 30 minutes, collected by filtration the precipitated crystals, washing with acetonitrile, and vacuum dried to obtain 118.26g of the title compound (net 117.90g, 95% yield). Equipment data of the crystals were the same as those of the step 2 of Reference Example 3.

Example
65 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VI-1)
[of 125]
 tert- butyl {2 – [({[(2S, 5R) -1,6- -6- hydroxy-7-oxo-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate (example 64,537.61g, 1.561mol) in acetonitrile (7.8L) solution of 2,6-lutidine (512.08g), sulfur trioxide – pyridine complex (810.3g) was added, at room temperature in the mixture was stirred overnight. Remove insolubles and the mixture was filtered, the filtrate concentrated to 2.5 L, diluted with ethyl acetate (15.1L). The mixture was extracted with 20% phosphoric acid 2 hydrogencarbonate aqueous solution (7.8L), the resulting aqueous layer into ethyl acetate (15.1L), added tetrabutylammonium hydrogen sulfate (567.87g), was stirred for 20 min. The organic layer was separated layers, dried over anhydrous magnesium sulfate (425 g), after filtration, concentration under reduced pressure, substituted concentrated tetrabutylammonium tert- butyl with dichloromethane (3.1L) {2 – [({[(2S, 5R ) -7-oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate was obtained 758g (net 586.27g, Osamu rate 84%).
 The tetra-butyl ammonium salt 719g (net 437.1g, 0.656mol) in dichloromethane (874mL) solution was cooled to -20 ℃, dropping trifluoroacetic acid (874mL) at 15 minutes, 1 the temperature was raised to 0 ℃ It was stirred time. The reaction was cooled to -20 ° C. was added dropwise diisopropyl ether (3.25L), and the mixture was stirred for 1 hour the temperature was raised to 0 ° C.. The precipitate is filtered, washed with diisopropyl ether to give the title compound 335.36g of crude and vacuum dried (net 222.35g, 99% yield).
 The title compound of crude were obtained (212.99g, net 133.33g) and ice-cold 0.2M phosphate buffer solution of pH5.3 mix a little at a time, alternating between the (pH6.5,4.8L). The solution was concentrated under reduced pressure to 3.6L, it was adjusted to pH5.5 at again 0.2M phosphate buffer (pH6.5,910mL). The solution resin purification (Mitsubishi Kasei, SP207, water ~ 10% IPA solution) is subjected to, and concentrated to collect active fractions, after lyophilization, to give the title compound 128.3 g (96% yield). Equipment data of the crystals were the same as those of step 3 of Reference Example 3.

PATENT

US 20140288051

WO 2014091268

WO 2013180197

US 20130225554

///////////RG-6080, 1452458-86-4, FPI-1459,  OP-0595, Phase I ,  β-lactamase inhibitor, bacterial infections, Fedora parmaceuticals, Meiji Seika Pharma

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