BIOCON PATENT..........US20140288269) PROCESS FOR THE PREPARATION OF RANDOM POLYPEPTIDES AND EMPLOYING CIRCULAR DICHROISM AS A GUIDANCE TOOL FOR THE MANUFACTURE OF GLATIRAMER ACETATE .

PROCESS FOR THE PREPARATION OF RANDOM POLYPEPTIDES AND EMPLOYING CIRCULAR DICHROISM AS A GUIDANCE TOOL FOR THE MANUFACTURE OF GLATIRAMER ACETATE (Fri, 26 Sep 2014) 
CLICK read this at wipo


OWNER

BANGALORE: Biotech major Biocon today said its Chairperson and Managing Director Kiran Mazumdar-Shaw

Application Number: 14355504 Application Date: 26.10.2012

Publication Number: 20140288269 Publication Date: 25.09.2014

Publication Kind : A1

PCT Reference: Application Number:PCT/IN2012/000708 ; Publication Number: Click to see the data

IPC: C07K 1/04 C07K 1/14 Applicants: Biocon Limited Inventors: Venkata Srinivas Pullela Khedkar Anand Patil Nitin Sopanrao Ujire Sandhya Chatterjee Amarnath Janakiraman Ashwini Priority Data: 3781/CHE/2011 03.11.2011 IN Title: (EN) PROCESS FOR THE PREPARATION OF RANDOM POLYPEPTIDES AND EMPLOYING CIRCULAR DICHROISM AS A GUIDANCE TOOL FOR THE MANUFACTURE OF GLATIRAMER ACETATE Abstract: (EN) The present invention discloses novel process for the preparation of mixture of polypeptides comprising L-Glutamaic acid, L-Alanine, L-Tyrosine, and L-Lysine. by employing circular dichroism as a guidance tool.

Glatiramer is a peptide based polymer composed of four amino acids: L-Glutamaic acid, L-Alanine, L-Tyrosine, and L-Lysine. It's pharmaceutically acceptable salt Glatiramer acetate is approved by FDA and marketed as Copaxone® for the treatment of multiple sclerosis. Copaxone is also known as copolymer-1 and cop-1. Multiple sclerosis is an autoimmune disease affects the brain and central nervous system due to the damage to the myelin sheath of the nerve cells, which results as demyelination of axons. Glatiramer acetate is a synthetic polypeptide analogue of myelin basic protein (MBP). Pharmacologically, Copaxone is a non-interferon and non-steroidal immunomodulator, which arrests the multiple sclerosis aggression. Glatiramer acetate is administrated by subcutaneous injections.

Chemically, glatiramer acetate is designated L-glutamic acid polymer with L-alanine, L-lysine and L-tyrosine, acetate salt. Its structural formula is:

          (Glu, Ala, Lys, Tyr)_x.xCH₃COOH 
          (C₅H₉NO₄.C₃H₇NO₂.C₆H₁₄N₂O₂.C₉H₁₁NO₃)_x.xC₂H₄O₂

Average molecular weight of glatiramer acetate is 5,000-11,000 daltons and the average molar fractions of the respective amino acids are: 0.141, 0.427, 0.095, and 0.338.

U.S. Pat. No. 3,849,550 describes the observation of MBP arrest in experimental allergic encephalomyelitis (a disease similar to multiple sclerosis) by immunotherapy agents. With continuous endeavours, glatiramer acetate is resulted as an advanced analogue for the treatment of multiple sclerosis with improved safety and efficacy.

U.S. Pat. Nos. 5,800,808; 5,981,589; 6,048,898 describes the process preparation of glatiramer acetate employing the N-carboxyanhydrides (NCAs) derived from alanine, γ-benzyl glutamate, N-trifluoroacetyl lysine, and tyrosine. Following the steps: polymerization, sequential cleavage of the γ-benzyl ester of glutamate and N∈-trifluoroacetyl derivative of lysine, acetate salt formation and final purification. U.S. Pat. No. 6,620,847 describes a process for the preparation of glatiramer acetate using the aqueous piperidine for trifluoroacetyl cleavage of lysine. U.S. Pat. No. 7,049,399 describes the process for preparation of polypeptide-1 using the catalytic transfer hydrogenation for the cleavage of γ-benzyl ester of glutamate. E.P. Pat. No. 1,807,467 describes the processes for preparation of glatiramer using NCAs of alanine, tyrosine, N-t-butoxycarbonyl L-Lysine, and protected glutamic acid, where in the protecting group is selected from γ-methoxybenzyl and γ-benzyl. U.S. Pat. No. 7,495,072 describes the process for the preparation of mixtures of polypeptides using purified hydrobromic acid. The major drawback of all these processes is the generation of impurities, multiple steps of purification and the variability in the secondary structures amongst different batches manufactured using the same process. HO Biocon Directions Pharmaceutical Company Address: Door No. A - 210, Gokul Arcade, Garevan Chowk, Sahar Road, Vile Parle East, Mumbai - 400057 Phone:022 6691 9761

EXAMPLES

Example 1

Preparation of Glatiramer Acetate Using Potassium Tert Butoxide

Preparation of Protected Polymer 2

Protected copolymer (1 gm) was taken in a mixture of THF and water, to that Lewatit K 2629 resin (1 gm) was added and stirred at 65° C. for 24 h. The resin was filtered through buckner funnel and washed with THF (5 ml).The reaction mass was distilled to 3-4 volume stage and water was added and the precipitated product was filtered and dried in VTD for 24 h at 40-45° C. Yield: 0.6 gm

Preparation of Glatiramer Acetate

To the stirred solution of protected polymer 2 (0.6 g) in anhydrous methanol (9 ml) was added potassium tertiary butoxide (0.6 g) and stirred for 1 hour. Reaction mass was concentrated under reduced pressure (below 35□ C.). To the reaction mass water (0.6 mL) was added and pH was adjusted with Glacial acetic acid to 5.5. Crude glatiramer acetate was isolated by crystallising with acetone. Crystallised solid was filtered and suck dried. Yield: 0.4 g

Crude glatiramer acetate obtained is subjected for gel permeation chromatography for purification.

Example 2

Preparation of Glatiramer Acetate Using Sodium Methoxide

Preparation of Protected Polymer 2

Protected copolymer (1 gm) was taken in a mixture of THF (8 ml) and water (ml), to that Lewatit K 2629 resin (1 gm) was added and stirred at 65° C. for 24 h. The resin was filtered through buckner funnel and washed with THF (5 ml).The reaction mass was distilled to 3-4 volume stage and water was added and the precipitated product was filtered and dried in VTD for 24 h at 40-45° C. Yield: 0.6 gm

Preparation of Glatiramer Acetate

To the stirred solution of protected polymer 2 (0.6 g) in anhydrous methanol (6 ml) was added a solution of sodium methoxide (0.9 g) in anhydrous methanol (4.5 mL)and stirred for 7 hours. Reaction mass was concentrated under reduced pressure (below 35° C.). To the reaction mass water (0.6 mL) was added and pH was adjusted with Glacial acetic acid to 6. Crude glatiramer acetate was isolated by crystallising with acetone. Crystallised solid was filtered and suck dried. Yield: 0.4 g

Crude glatiramer acetate obtained is subjected for gel permeation chromatography for purification.

Example 3

Preparation of Glatiramer Acetate Using TMSCl/NaI Followed by Sodium Methoxide

Protected polymer 1 (20 g) was charged in THF (200 ml) under nitrogen atmosphere, added sodium iodide (1 g) was added followed by trimethylsilyl chloride (20 ml) at room temperature and stirred for 3 h. The reaction mass was quenched after the completion of reaction with water (20 ml). The solids were filtered, washed with water (100 ml) and dried under high vacuum to obtain protected copolymer 2 (10 g).

The resulted protected polymer 2 was suspended in anhydrous methanol (100 ml), solution of sodium methoxide (15 g)in anhydrous methanol (75 ml) was added and stirred at room temperature for 7 h. pH was adjusted after the completion of the reaction to 6 with glacial acetic acid, and the mass was purified to obtain glatiramer acetate (6 g).

Example 4

Preparation of Glatiramer Acetate from Protected Polymer 3

Solution of sodium methoxide (1.5 g) in anhydrous methanol (7.5 ml) was added to protected copolymer 3 (1 g) in anhydrous methanol (10 ml) at room temperature and stirred for 7 h. pH was adjusted to 5 after completion of the reaction with glacial acetic acid. The resulted mass was purified to obtain glatiramer acetate (0.6 g).

The Far UV CD spectra, of Glatiramer acetate synthesised using the processes described in examples 1-4 exhibits the presence of random coils in the wavelength region 195-215 nm and alpha helices in the wavelength region of 222 nm. (FIG. 5) OWNER BANGALORE: Biotech major Biocon today said its Chairperson and Managing Director Kiran Mazumdar-Shaw

BANGALORE

BENGALURU, INDIA,




































BULL TEMPLE

WO 2015059679 New patent on Eliglustat by Dr Reddys

WO-2015059679

http://newdrugapprovals.org/2015/05/08/eliglustat/

 

Eliglustat tartrate (Genz-1 12638) is a glucocerebroside (glucosylceramide) synthase inhibitor for the treatment of gaucher disease and other lysosomal storage disorders, which is currently under development.

Eliglustat is chemically known as 1 R, 2R-Octanoic acid [2-(2', 3'-dihydro-benzo [1 , 4] dioxin-6'-yl)-2-hydroxy-1 -pyrrolidin-1 -ylmethyl]-ethyl]-amide, having a structural formula I depicted here under.

Formula I

Eliglustat hemitartrate (Genz-1 12638) development by Genzyme, is a glucocerebroside (glucosylceramide) synthase inhibitor for the treatment of Gaucher disease and other lysosomal storage disorders. Eliglustat hemitartrate is orally active with potent effects on the primary identified molecular target for type 1 Gaucher disease and other glycosphingolipidoses, appears likely to fulfill high expectations for clinical efficacy. 

Gaucher disease belongs to the class of lysosomal diseases known as glycosphingolipidoses, which result directly or indirectly from the accumulation of glycosphingolipids, many hundreds of which are derived from glucocerebroside. The first step in glycosphingolipid biosynthesis is the formation of glucocerebroside, the primary storage molecule in Gaucher disease, via glucocerebroside synthase (uridine diphosphate [UDP] - glucosylceramide glucosyl transferase). Eliglustat hemitartrate is based on improved inhibitors of glucocerebroside synthase.

U.S. patent No. 7,196,205 (herein described as US'205) discloses a process for the preparation of eliglustat or a pharmaceutically acceptable salt thereof. In this patent, eliglustat was synthesized via a seven-step process involving steps in that sequence: 

(i) coupling S-(+)-2-phenyl glycinol with phenyl bromoacetate followed by column chromatography for purification of the resulting intermediate, 

(ii) reacting the resulting (5S)-5-phenylmorpholin-2-one with 1 , 4-benzodioxan-6-carboxaldehyde to obtain a lactone, 

(iii) opening the lactone of the oxazolo-oxazinone cyclo adduct via reaction with pyrrolidine, 

(iv) hydrolyzing the oxazolidine ring, (v) reducing the amide to amine to obtain sphingosine like compound, (vi) reacting the resulting amine with octanoic acid and N-hydroxysuccinimide to obtain crude eliglustat, (vii) purifying the crude eliglustat by repeated isolation for four times from a mixture of ethyl acetate and n-heptane.

U.S. patent No. 6855830, 7265228, 7615573, 7763738, 8138353, U.S. patent application publication No. 2012/296088 disclose processes for preparation of eliglustat and intermediates thereof.

U.S. patent application publication No. 2013/137743 discloses (i) a hemitartrate salt of eliglustat, (ii) a hemitartrate salt of eliglustat, wherein at least 70% by weight of the salt is crystalline, (iii) a hemitartrate salt of Eliglustat, wherein at least 99% by weight of the salt is in a single crystalline form.

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=234E6BE008E68831F6875FB703760826.wapp2nA?docId=WO2015059679&recNum=1&office=&queryString=FP%3A%28dr.+reddy%27s%29&prevFilter=%26fq%3DCTR%3AWO&sortOption=Pub+Date+Desc&maxRec=364


http://newdrugapprovals.org/2015/05/08/eliglustat/

WO-2015059679

Enhanced	Process for the preparation of eliglustat free base - comprising the reaction of S-(+)-phenyl glycinol with phenyl-alpha-bromoacetate to obtain 5-phenylmorpholin-2-one, which is further converted to eliglustat.
	Dr Reddy's Laboratories Ltd http://newdrugapprovals.org/2014/08/20/fda-approves-new-drug-cerdelga-eliglustat-to-treat-a-form-of-gaucher-disease/
	New crystalline eliglustat free base Form R1 and a process for its preparation are claimed. Also claimed is a process for the preparation of eliglustat free base which comprises the reaction of S-(+)-phenyl glycinol with phenyl-alpha-bromoacetate to obtain 5-phenylmorpholin-2-one, which is further converted to eliglustat.  Further eliglustat oxalate, its crystalline form, and a process for the preparation of crystalline eliglustat oxalate, are claimed. Formula II
	http://newdrugapprovals.org/2015/05/08/eliglustat/
	Example 1 : Preparation of 5-phenyl morpholine-2-one hydrochloride To a (S) + phenyl glycinol (100g) add N, N-diisopropylethylamine (314ml) and acetonitrile (2000ml) under nitrogen atmosphere at room temperature. It was cooled to 10- 15° C. Phenyl bromoacetate (172.4g) dissolved in acetonitrile (500ml) was added to the above solution at 15° C over a period of 30 min. The reaction mixture is allowed to room temperature and stirred for 16-20h. Progress of the reaction was monitored by thin layer chromatography. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at a water bath temperature less than 25° C to get a residue. The residue was dissolved in ethyl acetate (1000ml) and stirred for 1 h at 15-20°C to obtain a white solid. The solid material obtained was filtered and washed with ethyl acetate (200ml). The filtrate was dried over anhydrous sodium sulphate (20g) and concentrated under reduced pressure at a water bath temperature less than 25° C to give crude compound (1000g) as brown syrup. The Crude brown syrup is converted to HCI salt by using HCI in ethyl acetate to afford 5-phenyl morpholine-2-one hydrochloride (44g) as a white solid. Yield: 50%, Mass: m/z = 177.6; HPLC (% Area Method): 90.5% Example 2: Preparation of (1 R,3S,5S,8aS)-1 ,3-Bis-(2',3'-dihydro-benzo[1 ,4] dioxin-6'-yl)-5-phenyl-tetrahydro-oxazolo[4,3-c][1 ,4]oxazin-8-one. 5-phenyl morpholine-2-one hydrochloride (100g) obtained from above stage 1 is dissolved in toluene (2500ml) under nitrogen atmosphere at 25-30°C. 1 ,4-benzodioxane-6-carboxaldehyde (185.3g) and sodium sulphate (400g) was added to the above solution and the reaction mixture was heated at 100-105°C for 72h. Progress of the reaction was monitored by thin layer chromatography. After completion of reaction, the reaction mixture was concentrated under reduced pressure at a water bath temperature less than 25° C to get a residue. The residue was cooled to 10°C, ethyl acetate (2700ml) and 50% sodium bisulphate solution (1351 ml) was added to the residue and stirred for 1 h at 10°C to obtain a white solid. The obtained white solid was filtered and washed with ethyl acetate. The separated ethyl acetate layer was washed with water (1000ml), brine (1000ml) and dried over anhydrous sodium sulphate. The organic layer was concentrated under reduced pressure at a water bath temperature of 45-50°C to get a crude material. The obtained crude material is triturated with diethyl ether (1500ml) to get a solid material which is filtered and dried under vacuum at room temperature for 2-3h to afford (1 R,3S,5S,8aS)-1 ,3-Bis-(2',3'-dihydro-benzo[1 ,4]dioxin-6'-yl)-5-phenyl-tetrahydro-oxazolo[4,3-c][1 ,4]oxazin-8-one (148g) as a yellow solid. Yield: 54%, Mass: m/z = 487.7; HPLC (% Area Method): 95.4 % Example 3: Preparation of (2S,3R,1 "S)-3-(2',3'-(Dihydro-benzo[1 ,4]dioxin-6'-yl)-3-hydroxy-2-(2"-hydroxy-1 ''^henyl-ethy^ (1 R,3S,5S,8aS)-1 !3-Bis-(2'!3'-dihydro-benzo[1 ,4]dioxin-6'-yl)-5-phenyl-tetrahydro-oxazolo[4,3-c][1 ,4]oxazin-8-one (70g) obtained from above stage 2 was dissolved in chloroform (1400ml) at room temperature. It was cooled to 0-5°C and pyrrolidone (59.5ml) was added at 0-5°C over a period of 30 minutes. The reaction mixture was allowed to room temperature and stirred for 16-18h. Progress of the reaction was monitored by thin layer chromatography. After completion of reaction, the reaction mixture was concentrated under reduced pressure at a water bath temperature of 40-45°C to obtain a crude. The obtained crude was dissolved in methanol (1190ml) and 1 N HCI (1 190ml) at 10-15° C, stirred for 10 minutes and heated at 80-85°C for 7h. Progress of the reaction was monitored by thin layer chromatography. After completion of reaction, methanol was concentrated under reduced pressure at a water bath temperature of 50-55°C.The aqueous layer was extracted with ethyl acetate and the organic layer was washed with 1 N HCI (50ml). The aqueous layer was basified with saturated sodium bicarbonate solution up to pH 8-9 and extracted with ethyl acetate (3x70ml). The combined organic layers was washed with brine (100ml), dried over anhydrous sodium sulphate and concentrated under reduced pressure at a water bath temperature of 50-55°C to afford (2S,3R,1"S)-3-(2',3'-(Dihydro-benzo[1 ,4]dioxin-6'-yl)-3-hydroxy-2-(2"-hydroxy-1 "-phenyl-ethylamino)-1 -pyrrolidin-1 -yl-propan-1 -one (53g) as a yellow foamy solid. Yield: 90%, Mass: m/z = 412.7, HPLC (% Area Method): 85.1 % Example 4: Preparation of (1 R,2R,1 "S)-1-(2',3'-(Dihydro-benzo[1 ,4]dioxin-6'-yl)2-hydroxy-2-(2"-hydroxy-1 '-phenyl-ethylamino)-3-pyrrolidin-1-yl-propan-1-ol. (2S,3R,1 "S)-3-(2',3'-(Dihydro-benzo[1 ,4]dioxin-6'-yl)-3-hydroxy-2-(2"-hydroxy-1 "-phenyl-ethylamino)-1 -pyrrolidin-1 -yl-propan-1 -one (2.5g) obtained from above stage 3 dissolved in Tetrahydrofuran (106ml) was added to a solution of Lithium aluminium hydride (12.2g) in tetrahydrofuran (795ml) at 0°C and the reaction mixture was heated at 60-65°C for 10h. Progress of the reaction was monitored by thin layer chromatography. After completion of reaction, the reaction mixture was cooled to 5- 10°C and quenched in saturated sodium sulphate solution (100ml) at 5-10°C. Ethyl acetate was added to the reaction mass and stirred for 30-45 min. The obtained solid is filtered through celite bed and washed with ethyl acetate. Filtrate was dried over anhydrous sodium sulphate and concentrated under reduced pressure at a water bath temperature of 50°C to afford (1 R,2R, 1"S)-1 -(2',3'-(Dihydro-benzo[1 ,4]dioxin-6'-yl)2-hydroxy-2-(2"-hydroxy-1 '-phenyl-ethylamino)-3-pyrrolidin-1 -yl-propan-1 -ol (43.51 g) as a yellow gummy liquid. The crude is used for the next step without further purification. Yield: 85%, Mass: m/z = 398.7, HPLC (% Area Method): 77 % Example 5: Preparation of (1 R, 2R)-2-Amino-1-(2', 3'-dihydro-benzo [1 , 4] dioxin-6'-yl)-3-pyrrolidin-1 -yl-propan-1 -ol. (1 R,2R,1 "S)-1 -(2',3'-(Dihydro-benzo[1 ,4]dioxin-6'-yl)2-hydroxy-2-(2"-hydroxy-1 '-phenyl-ethylamino)-3-pyrrolidin-1 -yl-propan-1 -ol (40g) obtained from above stage 4 was dissolved in methanol (400ml) at room temperature in a 2L hydrogenation flask. Trifluoroacetic acid (15.5ml) and 20% Pd (OH) 2 (40g) was added to the above solution under nitrogen atmosphere. The reaction mixture was hydrogenated under H2, 10Opsi for 16-18h at room temperature. Progress of the reaction was monitored by thin layer chromatography. After completion of reaction, the reaction mixture was filtered through celite bed and washed with methanol (44ml) and water (44ml). Methanol was concentrated under reduced pressure at a water bath temperature of 50-55°C and the aqueous layer was washed with ethyl acetate. The aqueous layer was basified with 10M NaOH till the PH reaches 12-14 and then extracted with dichloromethane (2x125ml). The organic layer was dried over anhydrous sodium sulphate (3gm) and concentrated under reduced pressure at a water bath temperature of 45°C to obtain a gummy liquid. The gummy liquid was triturated with methyl tertiary butyl ether for 1 h to get a white solid, which is filtered and dried under vacuum at room temperature to afford (1 R, 2R)-2-Amino-1 -(2', 3'-dihydro-benzo [1 , 4] dioxin-6'-yl)-3-pyrrolidin-1 -yl-propan-1 -ol (23g) as a white solid. Yield: 82.3%, Mass (m/zj: 278.8, HPLC (% Area Method): 99.5%, Chiral HPLC (% Area Method): 97.9% Example 6: Preparation of Eliglustat {(1 R, 2R)-Octanoic acid[2-(2',3'-dihydro-benzo [1 , 4] dioxin-6'-yl)-2-hydroxy-1 -pyrrolidin-1-ylmethyl-ethyl]-amide}. (1 R, 2R)-2-Amino-1 -(2', 3'-dihydro-benzo [1 , 4] dioxin-6'-yl)-3-pyrrolidin-1 -yl-propan-1 -ol (15g) obtained from above stage 5 was dissolved in dry dichloromethane (150ml) at room temperature under nitrogen atmosphere and cooled to 10-15° C. Octanoic acid N-hydroxy succinimide ester (13.0 g)was added to the above reaction mass at 10-15° C and stirred for 15 min. The reaction mixture was stirred at room temperature for 16h-18h. Progress of the reaction was monitored by thin layer chromatography. After completion of reaction, the reaction mixture was cooled to 15°C and diluted with 2M NaOH solution (100 ml_) and stirred for 20 min at 20 °C. The organic layer was separated and washed with 2M sodium hydroxide (3x90ml).The organic layer was dried over anhydrous sodium sulphate (30g) and concentrated under reduced pressure at a water bath temperature of 45°C to give the crude compound (20g).The crude is again dissolved in methyl tertiary butyl ether (25 ml_) and precipitated with Hexane (60ml). It is stirred for 10 min, filtered and dried under vacuum to afford Eliglustat as a white solid (16g). Yield: 74%, Mass (m/zj: 404.7 HPLC (% Area Method): 97.5 %, ELSD (% Area Method): 99.78%, Chiral HPLC (% Area Method): 99.78 %. Example 7: Preparation of Eliglustat oxalate. Eliglustat (5g) obtained from above stage 6 is dissolved in Ethyl acetate (5ml) at room temperature under nitrogen atmosphere. Oxalic acid (2.22g) dissolved in ethyl acetate (5ml) was added to the above solution at room temperature and stirred for 14h. White solid observed in the reaction mixture was filtered and dried under vacuum at room temperature for 1 h to afford Eliglustat oxalate as a white solid (4g). Yield: 65.46%, Mass (m/zj: 404.8 [M+H] +> HPLC (% Area Method): 95.52 %, Chiral HPLC (% Area Method): 99.86 %
	http://newdrugapprovals.org/2015/05/08/eliglustat/