Regorafenib, SHILPA MEDICARE LIMITED, New patent, WO 2016005874

WO2016005874, 

PROCESS FOR THE PREPARATION OF REGORAFENIB AND ITS CRYSTALLINE FORMS

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

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

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

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

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

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

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

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

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

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

Removal of impurities from final product

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

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

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

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

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

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

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

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

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

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

Example 1

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

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

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

Yield: 9 gm

Chromatographic Purity (By HPLC): 98%

Example 2

Preparation of Regorafenib

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

Yield: 5.5 gm

Chromatographic Purity (By HPLC): 97%

Example 3

Purification of Regorafenib using acetone and toluene mixture

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

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

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

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

Yield: 0.88gm

Chromatographic Purity (By HPLC): 99.3 %

Example 4

Purification of Regorafenib using acetone

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

Yield: 0.7 gm

Chromatographic Purity (By HPLC): 99.77%

Example 5

Double – Purification of Regorafenib using acetone and toluene mixture

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

Yield: 0.80gm

Chromatographic Purity (By HPLC): 99.79 %

Moisture content: 0.09%

Impurity-A: 0.03%

Impurity-B: Not detected

Impurity-C: 0.02%

Example 6

Preparation of Regorafenib Form I

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

Yield: 1.9 g

Chromatographic Purity (By HPLC): 98.4 %

XRPD was found to resemble similar to Fig-1.

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

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

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

RAICHUR ರಾಯಚೂರು, Karnataka, India

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

Lupin Ltd, Patent, Pitavastatin, WO2014203045

Lupin Ltd, Patent, Pitavastatin, WO2014203045

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

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

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

PITAVASTATIN

TEXT

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

[I]

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

[II]

[II]

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

[compound II]

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

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

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

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

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

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

Scheme 1

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

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

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

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

Scheme 2

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

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

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

Scheme 3

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

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

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

Scheme 4

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

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

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

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

Scheme 5

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

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

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

Scheme 6

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

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

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

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

Scheme 7

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

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

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

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

Scheme 8

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

9).

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

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

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

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

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

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

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

Summarizing, the teachings of the application are not workable.

Scheme 9

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

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

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

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

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

Synthetic Communications, 2003, 2275 – 2284).

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

3) TEMPO/NaOCl oxidization (EP2351762)

4) Trichloroisocyanuric acid/ TEMPO (CN 101747313A)

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

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

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

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

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

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

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

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AUTHORS

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Lupin Ltd, New patent, Pitavastatin, WO 2016005919

Formula (1)

Lupin Ltd, New patent, Pitavastatin, WO 2016005919

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

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

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

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

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

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

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

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

Formula (1)

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

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

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

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

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

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

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

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

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

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

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

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

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

HPLC purity: 99.70 %

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

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

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

HPLC purity: 99.70 %

Example-3: Preparation of Pitavastatin Sodium Amorphous

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

HPLC purity: 99.70 %.

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

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

 

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

Formula (I)

LIRAGLUTIDE

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

Process for preparation of liraglutide

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

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

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

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

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

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

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

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

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

Formula (I)

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

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

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

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

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

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

EXAMPLE 1 :

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

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

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

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

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

Stage III: Purification of crude Liraglutide using RP HPLC.

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

Table 1 : Gradient program for pre purification

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

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

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

Table 2: Gradient program for second purification

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

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

EXAMPLE 2:

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

Fragments used are as follows

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

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

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

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

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

6. Fmoc-Lys-Glu-Palmitic acid.

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

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

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

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

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

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

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

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

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

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

Stage III: Purification of crude Liraglutide using RP HPLC.

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

Table 1 : Gradient program for pre purification

60 40 30

55 45 30

52 48 30

51 49 60

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

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

Table 2: Gradient program for second purification

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

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

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

REFERENCE

IN2014CH3453 INDIAN PATENT

WO 2016005960, CLICK FOR PATENT

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