Title of Invention

A PROCESS FOR THE PREPARATION OF ATOSIBAN ACETATE

Abstract

The present invention related to a process for the preparation of atosiban acetate using a novel non-linear liquid phase peptide synthesis scheme. This invention makes use of combing precursor peptides synthesised separately in high purity and yield over presently known processes. The disclosed process has several advances over known process like ease of scale-up and work-up as the process utilises liquid phase peptide preparation chemistry.

Full Text

FORM 2 THE PATENT ACT 1970 & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) 1. TITLE OF THE INVENTION A process for the preparation of atosibart acetate. 2. APPLICANT(S) (a) NAME: Emcure Pharmaceuticals Ltd (b) NATIONALITY: an Indian Company (b) ADDRESS: P-l, IT-BT Park MIDC Phase-2, Hinjwadi, Pune-411057, INDIA 3. PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be performed. 4. DESCRIPTION This invention relates to a process for the preparation of atosiban and its salts of higher yield and purity with ease of manufacturing due to use of a novel process route. Atosiban (l-(3-mercaptopropanoic acid)-2-(0-thyl-D-tyrosine)-4-L-threoriine-8-L-ornithrne-oxytocin) is a member of the class of synthetic peptide molecules that are the oxytocin mimetics. These molecules are indicated for the tocolytic therapy of preterm labour in pregnant women. The mechanism of action is through the inhibition of oxytocin function. Atosiban has been synthesized in the known art by solid state peptide synthesis methods. Several of the currently used synthesis schemes have limitations in that solvents and catalysts used are not eco-friendly and industrially economical. They also have problems with respect to scale-up and work-up of the current processes. These factors lead to formation of impurities that can limit the use of the final products in pharmaceutical compositions. Besides, solid phase synthesis methods are not routinely applied for atosiban synthesis as they have some limitation. Here an economical process for the preparation of atosiban is disclosed that uses non-linear convergent solution phase peptide synthesis approach. It is known to the skilled person in organic chemical synthesis that in order to obtain highly pure final products, it is a good strategy to avoid formation of by-products than develop purification methods to remove impurities from the final products. The principal object of this invention relates to an improved method of preparation of atosiban for use in pharmaceutical compositions with improved impurity profiles for peptide impurities formed during the preparation. Another object of this invention relates to the use of a novel method of non-linear / convergent synthesis of peptides required in preparation of atosiban in high purity separately of each other and then combining the precursor peptides into final cyclic peptide of atosiban. The disclosed scheme is previously not used in this type of synthesis with new chemistry that significantly increases the utility of the invention in industrial applications. Atosiban is prepared by various strategies as known in the prior art. In this invention, a novel process for the preparation of atosiban acetate is disclosed having several advantages over the existing processes. The method disclosed uses a convergent liquid phase synthesis of precursor peptide fragments, followed by condensation of the fragments leading to formation of cyclic atosiban in the final step. The first fragment of four amino acids in attached to the second fraginent of three amino acids leading to the formation of a seven amino acid fragment. This heptapeptide in then condensed with a third fragment of two amino acids leading to formation of nonapeptide followed by cyclisation leading to the formation of cyclic atosiban peptide. This strategy entirely employs liquid phase peptide synthesis having several advantages like easy scale-up of the preparation to kilogram scale and easy of handling the process steps. It is disclosed in the prior patent applications several methods of preparation of atosiban and similar peptides. All the known methods use solid phase peptide synthesis to produce atosiban and related peptides. Solid phase methods have limitations in that scale-up is not practical in the industrial settings where economy of the process is of importance. On the other hand liquid phase synthesis of peptides has several advantages in industrial set-ups. However; there are no examples of use of LPS of atosiban in the prior art except the patent application 284/MUM/2004; which discloses methods for preparation of atosiban using liquid phase synthesis schemes. Though, disclosed methods have different schemes for ligation of fragments of precursor peptides than the invention of this disclosure. This invention disclosed more advantageous and economical methods over the prior art with novel scheme of preparation and ligation of precursor peptides, creation of salts of the peptide and purification of * said peptide. In Scheme 1 in the first step, the protected tetrapeptide of formula (1) is prepared by liquid phase peptide synthesis and isolated to high purity by HPLC. In the second step, peptide of formula (1) is condensed with the tripeptide of formula (2) leading to formation of the heptapetide of formula (3). The peptide of formula (2) is separately prepared by liquid phase peptide synthesis and isolated to high purity by HPLC. In the third step, peptide of formula (3) is condensed with the dipeptide of formula (4), which is prepared separately, leading to formation of the nonapeptide of the formula (5). In the fourth step, peptide of formula (5) is deprotected in TFA leading to formation of peptide of formula (6). This linear nonapetide is further cyclised to form atosiban (7) having a 1-6 disulphide bond between amino acid 1 and 6 of the molecule, which is purified to high purity by chromatography. The atosiban so obtained in further converted to the acetate salt by chromatographic ion-exchange leading to formation of atosiban acetate of high purity. Many of the protecting groups/moieties used in Scheme 1 can be replaced with other groups as shown in Table 1. Scheme 1 is depiction of an example of the protecting strategy used for the preparation of atosiban and it is not limited by the use of protecting groups shown in said scheme, and other protecting groups may be used. As depicted in Table 1, different protecting moieties that may be used for protecting different functional groups during the preparation of atosiban and similar compounds of higher yield and purity. The invention detailed above is illustrated with the following examples for the purpose showing the utility of the invention. Embodiments below do not restrict the invention in any way from broader application of the reaction chemistry for preparation of peptides similar to atosiban. The teaching of this invention can also be used in the preparation of the peptides that are oxytocin analogues. List of abbreviations: Boc = t-butyloxycarbonyl Bzl = benzyl DCC = Dicyclohexylcarbodiimide DIPEA = diisopropylethylamine DMF = dimethylformamide Fmoc = flourenylmethoxycarbonyl HOBt = 1-hydroxybenzotriazole IBCF= isobutyfchforoformate NMM = N-methylmorpholine MDC = Methylene dichloride TEA = triethylamine TFA = trifluoroacetic acid THF = Tetrahydrofuran Trt = triphenyl methyl (trityl) Example 1: Preparation of L-threonine methyl ester HC1 To dry methanol (100 mL) add L-threonine (15.47gm) at 25-30 °C and gradually cool the mixture to 0 °C. To this add thionyl chloride (20.11 gm) and stix the reaction mass at 0 °C for 10 min. Raise the temperature to 25 °C and keep for 30 min. Reflux the reaction mass at 60 °C for 4-5 h. Check reaction by TLC for the formation of product. Triturated L-threonine methyl ester HC1 in diisopropylether and recover it. The product yield of 91% and purity of 91 % by HPLC is obtained. Example 2: Preparation of Boc-Ile-The-OMe To THF (40 mL) solution add Boc-L-isoleucine (20 gm) at 25-30 °C and stir to dissolve under nitrogen, cool the mixture to 0°C. To this add N-methylmorpholine (8.7 gm) and stir the reaction mass at 2 °C for 10 min. Cool reaction to -10 °C and add drop-wise IBCF with string for 10 min. Separately, to dry DMF (100 mL) dissolve L-threonine methyl ester (26.41 gm) and neutralise it with TEA (21.6 mL) at 10-15 °C. Make slurry of obtained white syrup in THF (140 mL) and add to Boc-L-isoleucine reaction mass drop-wise at -10 °C. pH should remain between 7 and 7.5. Stir reaction mass at 0 °C for 1 h under nitrogen. Then stir reaction at 25 °C for another 14-16 h. Check product development by TLC. Distil out THF and dissolve the compound in chloroform. Wash with HC1 (1 N) twice, followed by Na2CCfe (1 N) twice. Then wash chloroform with Brine's solution. Add NaiSCU and distil out chloroform. To obtained clear syrup add n-hexane at 10-15 °C and stir. Collect white powder, add n-hexane (80 mL) and stir for 2-3 h. Filter the solid and dry under vacuum for 5-6 h. The product yield of 80% and purity of 97 % by HPLC is obtained. Example 3: Preparation of Boc-D-Tyr(Et)-Ile-The-OMe To MDC (25 mL) solution add Boc-Ile-The-OMe (25 gm) at 25-30 °C and stir. To this add TFA (50 mL) at 0 °C and stir at 25 °C for 2 h. Check removal of Boc by TLC. Concentrate the reaction mass at 40-45 °C. To this add diisopropyl ether (100 mL) at 0 °C. Isolate the sticky compound and diossolve in DMF (25 mL) and neutralise with NMM. Separately, take Boc-Tyr(Et)-OH (19 gm) in dry DMF (50 mL). Cool the solution at 0 °C, under nitrogen add HOBt and stir for 5 min. To this add above neutral salt and stir the reaction mass at 0 °C for 5 min. Then add NMM to followed by EDAC-HC1 and proceed the reaction at RT for 30 min. Keep pH between 7 to 7.5. Check product formation by TLC Take DM water (60 mL) at 10 °C. Slowly add reaction mass to it in 10 min and stir vigorously for 20 min. Filter out the while precipitated product. Purify the product with ethyl acetate, then hexane, etc. The product yield of 90% and purity of 91 % by HPLC is obtained. Example 4: Preparation of Mpa(Trt)-D-Tyr(Et)-Ile-Thr-OH To MDC (20 mL) solution take Boc-D-Tyr(Et)-lle-The-OMe (10 gm) at 5 °C and stir. To this add TFA (20 mL) at 5 JC and stir at 25 °C for 90 min. Check removal of Boc by TLC. Concentrate the reaction mass at 40-45 °C. To this add diisopropylether (50 mL) at 5 °C. Isolate the sticky compound and dissolve in DMF (20 mL) and neutralise with NMM. Separately, take 3Mpa(trt)OH (5.18 gm) in dry DMF (30 mL). Cool the solution at 0 °C, under nitrogen add HOBt and stir for 5 min. To this add above neutral TFA salt and stir the reaction mass at 0 °C for 5 min. Then add NMM followed by EDAC-HC1 and proceed the reaction at RT for 30 min under nitrogen. Further allow the reaction at 25 °C for 14-16 h. Check with TLC for the completion of reaction. Then in the next step, obtained peptide methyl ester is hydrolysed leading to formation of the product. The product yield of 90% and purity of 95 % by HPLC is obtained. Example 5: Preparation of Boc-Asn(Trt)-Cys(Acm)-Pro-OMe To MDC (10 mL) solution add Boc-Cys(Acm)-Pro-OMe (12.75 gm) at 25-30 °C and stir. To this add TFA (26 mL) at 5 °C and stir at 25 °C for 2 h. Check removal of Boc by TLC. Concentrate the reaction mass at 40-42 °C. To this add diisopropylether (150 mL) at 0 °C. Isolate the compound and dissolve in DMF (25 mL) and neutralise with NMM. Separately, take Boc-Asn(Trt)-OH (10 gm) in dry DMF (50 mL). Cool the solution at 0 °C, under nitrogen add HOBt and stir for 10 min. To this add above neutral TFA salt and stir the reaction mass at 0 °C for 5 min. Then add NMM followed by EDAC-HC1 and proceed the reaction at RT for 30 min. Keep pH between 7 and 7.5. Further allow the reaction to complete at 25 °C for 18-22 h. Check product formation by TLC. Take DM water (420 mL) at 10 °C. Slowly add reaction mass to it in 10 min and stir vigorously for 20 min. Filter out the white precipitated product. Purify the product with ethyl acetate, then hexane, etc. The product yield of 93% and purity of 93 % by HPLC is obtained. Example 6: Preparation of Mpa(Trt)-D-Tyr(Et)-Ee-Thr-Asn(Trt)-Cys(Acm)-Pro-OH The peptides obtained in Examples 4 and 5 are ligated to form the heptapeptide of sequence Mpa(Trt)-D-Tyr(Et)-ne-Trir-Asn(Trt)-Cys(Acin)-Pro-OMe. The procedure used for the preparation of said heptapeptide is briefly as: Tripeptide Boc-Asn(Trt)-Cys(Acm)-Pro-OMe is deprotected by TFA and isolated using ether as salt. To this tetrapeptide Mpa(Trt)-D-Tyr(Et)-Ile-Thr-OH is ligated leading to formation of methyl ester of sequence Mpa(Trt)-D-Tyr(Et)-ne-Thr-Asn(Trt)-Cys(Aon)-Pro-OMe. This ester is hydrolysed with NaOH leading to the formation of helptapetide of sequence Mpa(Trt)-D-Tyr(Et)-He-Thr-Asn(Trt)-Cys(Acm)-Pro-OH. The product yield of 85% and purity of 75 % by HPLC is obtained. Example 7: Preparation of Fmoc-Orn(Boc)-Gly-NFb To DMF (30 mL) solution add Fmoc-Orn(Boc)-OH (5 gm) at 25-30 °C and stir under nitrogen. Cool the reaction mass to 0 °C. To this add HOBt (1.5 gm) at 0 °C and stir for 5 min. Separately, take glycinamide HC1 (1.6 gm) in dry DMF (10 mL) and neutralise with TEA and add this to the reaction mass. To this add DCC (3.4 gm) dissolved in DMF (5 mL). Allow the reaction to proceed at 0 for 20 min under nitrogen. Then allow the reaction at 25 for 12-15 h. Check formation of the product with TLC. Then extract the product in ethyl acetate and isolate as crude viscous compound. The product yield of 82% and purity of 93% by HPLC is obtained. Example 8: Preparation of Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-Orn(Boc)-Gly-NH2 The peptides obtained in Examples 6 and 7 are ligated to form the nonapepride of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-Orn(Boc)-Gly- NFL. The procedure used for the preparation of said nonapeptide is briefly as: Dipeptide Fmoc-Orn(Boc)-Gly-NH2 is deprotected by diethyl amine and isolated as free base. To this heptapeptide Mpa(Trt)-D-Tyr(Et)-ne-Thr-Asn(Trt)-Cys(Acm)-Pro-OH is ligated leading to formation of nonapetide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-Orn(Boc)-Gly-NFL. The product yield of 60% and purity of 73 % by HPLC is obtained. Example 9: Preparation of (N -1) atosiban TFA salt To MDC (2 mL) solution add protected atosiban salt (1 gm) of Example 8 and cool to Ofc. To this add TFA (4 mL) and tfiethylsilane at 0 °C and stir at RT for 4-5 h. Check product formation by TLC. After completion of reaction concentrate the reaction mass in a vacuum concentrator. Linear atosiban TFA salt of sequence Mpa-D-Tyr(Et)Jle-Thr-Asn-Cys Example 10: Preparation of crude cyclic atosiban TFA salt Take a solution of iodine-methanol (0.95 gm iodine in 25 mL 90% methanol). Separately, take the deprotected atosiban salt (0.8 gm) of Example 9 in acetic acid-mefhanol (2% acetic acid in 55 ml 90% methanol). Add second solution to the first solution drop-wise under stirring. Check completion of reaction by HPLC after about 6 h. Example 11: Purification of crude atosiban TFA salt The crude atosiban salt obtained in Example 10 is purified by preparative HPLC chromatography. On a preparative HPLC column (Kromasil, 21.2 x 250 mm; 10 \x; 100 A) atosiban salt is loaded for total running time of 160 min. Gradient mobile phase used contains Sol- A: 0.1% TFA in WFI and Sol. B: 0.1% TFA in ACN. Sol. B amount is raised to 90% during running from 4% at the beginning and coming back to 4% at the end. Fractions are analysed with HPLC and fractions with >98% product purity are isolated. Example 12: Conversion of atosiban TFA salt to atosiban acetate The crude atosiban salt obtained purified by preparative HPLC is changed to atosiban acetate by ion-exchange chromatography. In the first step, fractions with >98% product purity are loaded onto a column having buffer 1 made up of: Sol. A, 96%; Sol. B 4% (Sol A: 3% ammonium acetate in water; Sol B: 100% ACN) at flow rate of 10 mL/min for 1 h. In the second step, 1% acetic acid is passed to remove ammonium acetate through the column. In the third step, the compound was eluted with buffer 2 made up of: gradient of Sol. B = 4% to 90% in Sol. A (Sol A: 0.1% acetic acid in water; Sol B: 0.1% acetic acid in ACN) in 80 min. The purified atosiban acetate is lyophilised with purity of >98.5% by HPLC. 5. CLAIMS We claim: 1. A process of the preparation of atosiban acetate by non-linear liquid phase peptide synthesis, comprising the steps of: a) coupling of N-terminus protected Boc-L-isoleucine with L-threonine methyl ester, in the presence of a catalyst, leading to the formation of protected dipeptide methyl ester of sequence Boc-Ile-Thr-OMe; b) deprotecting the dipeptide methyl ester of step a) and coupling of said deprotected dipeptide methyl ester with Boc-O-ethyl-D-tyrosine leading to the formation of protected tripeptide methyl ester of sequence Boc-D-Tyr(Et)-Ile-Trir-OMe; c) deprotecting the tripeptide methyl ester of step b) and coupling of said deprotected tripeptide methyl ester with S-trityl-3-mercaptopropionic acid leading to the formation of another protected tetrapeptide methyl ester of sequence Mpa(Trt)-D-TyT(Et)-He-Thr-OMe; d) hydrolysing tetrapeptide methyl ester of step c) with a base leading to the formation of tetrapeptide of sequence Mpa(Trt)-D-Tyr(Et)-ne-Thr-OH; e) reacting tetrapeptide of step d) with tripeptide methyl ester of sequence NH2-Asn-Cys(Acm)-Pro-OMe leading to the formation of heptapeptide methyl ester of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-OMe; f) hydrolysing heptapeptide methyl ester of step e) with a base leading to the formation of heptapeptide of sequence Mpa(rrtVD-Tyr(EtVIle-Thr-Asi\-Cys(Acm)- Pro-OH; g) reacting heptapeptide of step f) with isolated NH2- Orn(Boc)-Gly-NH2 leading to the formation of nonapeptide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr~ Asn-Cys(Acm)-Pro-Orn(Boc)-Gly-NHz; h) deprotecting nonapeptide of step g) in the presence of an acid leading to the formation of deprotected nonapeptide of sequence Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-Orn-Gly-NH2; and i) oxidizing nonapeptide of step h) with iodine in a protic solvent leading to the formation of cyclic atosiban acetate of sequence Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2 having a disulphide bond (1-6) between Mpa and Cys residues. 2. A process of Claim 1, wherein the tetrapeptide obtained in step d) is isolated prior to step e); the heptapeptide obtained in step f) is isolated prior to step g); and nonapeptide obtained in step i) is isolated in high purity. 3. A process of Claim 2, wherein the isolation is by precipitation, crystallization, extraction, or chromatography. 4. A process of Claim 1, further comprising the steps of: a) reacting the protected nonapeptide obtained in step g) with trifluoroacetic acid solution containing methylene dichloride and triethylsilane; b) adding ether to obtain a precipitate of non-cyclic atosiban consisting of amino acids having the sequence of: Mpa-D-Tyr(Et)-ne-Thr-Asn(Acm)-Cys-Pro-Orn-Gly-NH2; c) cyclizing the non-cyclic atosiban; and d) isolating cyclic atosiban having the sequence of: Mpa-D-Tyr(Et)-ne-Thr-Asn-Cys-Pro-Om-Gly-NHz cyclic (1-6) disulfide. 5. A process according to Claim 1 or 4, further comprising the steps: a) purifying the atosiban by HPLC chromatography, and simultaneously replacing the counter-ion of the peptide with acetate ion to obtain atosiban acetate; and b) drying the solution of the peptide acetate so obtained. 6. A process of according to Claim 5, wherein the drying is by lyophilizing or spray drying. 7. Atosiban acetate obtained according to Claim 1 having a purity of at least about 98.0% as determined by HPLC method. 8. Atosiban acetate obtained according to Claim 1 contains no more than about 1% impurity of other peptide and no more than 2% total impurities. 9. A novel tetrapeptide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-OH according to Claim 1. 10. A novel tripeptide of sequence NH2-Asn-Cys(Acm)-Pro-OMe according to Claim 1. 11. A novel heptapeptide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-OH according to Claim 1. 12. A novel dipeptide of sequence NH2-Orn(Boc)-Gly-NH2 according to Claim 1. 13. A process for preparing a pharmaceutical formulation comprising combining atosiban acetate made by the process of Claim 1, with at least one pharmaceutically acceptable excipient. 14. A process for the preparation of atosiban acetate substantially as described with reference to the examples.

Documents:

1265-mum-2010-abstract.doc

1265-mum-2010-abstract.pdf

1265-MUM-2010-CLAIMS(AMENDED)-(5-9-2013).pdf

1265-MUM-2010-CLAIMS(MARKED COPY)-(5-9-2013).pdf

1265-mum-2010-claims.doc

1265-mum-2010-claims.pdf

1265-MUM-2010-CORRESPONDENCE(6-7-2011).pdf

1265-mum-2010-correspondence.pdf

1265-mum-2010-description(complete).pdf

1265-mum-2010-form 1.pdf

1265-MUM-2010-FORM 18(14-10-2010).pdf

1265-mum-2010-form 2(title page).pdf

1265-mum-2010-form 2.doc

1265-mum-2010-form 2.pdf

1265-mum-2010-form 3.pdf

1265-mum-2010-form 5.pdf

1265-MUM-2010-FORM 9(28-7-2010).pdf

1265-MUM-2010-REPLY TO EXAMINATION REPORT(5-9-2013).pdf