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 atosiban acetate.
2. APPLICANT(S)
(a) NAME: Emcure Pharmaceuticals Ltd
(b) NATIONALITY: an Indian Company (b) ADDRESS: P-l, LT-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 over the disclosed process of the of application no. 1265/MUM/2010, with ease of manufacturing due to use of novel process steps. Atosiban (l-(3-mercaptopropanoic acid)-2-(0-ethyl-D-tyrosine)-4-L-threonine-8-L-ornitliine-oxytocin) is a member of the class of synthetic peptide molecules that are 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 and improved 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 process 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 and steps disclosed are addition to the main invention of preparation of atosiban as disclosed in 1265/MUM/2010.
Atosiban is prepared by various strategies as known in the prior art. In this improved invention, novel steps for the preparation of atosiban acetate are disclosed having several advantages over the existing processes and the process disclosed in the main invention. 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 fragment 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. In the second step, peptide of formula (1) is condensed with the tripeptide of formula (2) leading to formation of the heptapetide of formula (2a). The peptide of formula (2) is separately prepared by liquid phase peptide synthesis and isolated to high purity. Then the peptide of formula (2a) is deprotected at c-termtims to obtain the peptide of formula (3). In the

fourth 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 fifth 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.
Table 1: List of protecting groups.

Position Moiety
Na and/or Nw Protection Boc, Bpoc, Fmoc, Alloc, Troc
Thiol Group Acm, PhAcm, Trt, Mtt, Mmt, Tmob, Xan
C-terminus -OH All, OMe

Scheme 1

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:
All = allyl
Alloc - allyloxycarbonyl
Boc = t-butyloxycarbonyl
Bpoc = 2-(4-biphenyl)isopropoxycarbonyl
DCC = Dicydohexylcarbodiimide
DIPEA = diisopropylethylamine
DMF = dimethylformamide
Fmoc = 9-fluorenylmethoxycarbonyl
HOBt = 1-hydroxybenzotriazole
IBCF = isobutylchloroformate
NMM = N-methylmorpholine
MDC = Methylene dichloride
TEA = triethylamine
TFA = trifluoroacetic acid
THF = Tetrahydrofuran
Trt - triphenyl methyl (trityl)
Troc = 2,2,2-trichloroethyloxycarbonyl
Example 1: Preparation of Boc-Pro-OH
To acetronitrile (100 mL) add proline (50 gm) and DM water (100 mL). To
this add 'methyl amine (52.62 gm) and stir the reaction mass at 0-5 °C.

Then drop-wise add Boc-anhydride (142.33 gm) and stir the reaction at to 25 °C till completion of the reaction. Then remove acetronitrile at 40 °C and cool the reaction at 10-12 °C. Then add 2N HC1 (400 mL) to the reaction mass and filter the solid. Wash the solid with water and cyclohexane to get the product of about 95% purity with yield of 85 gm.
Example 2: Preparation of Boc-Pro-OAll
To acetronitrile (450 mL) add Boc-Pro-OH (100 gm) and and cooled to 10-15 °C. Then add potassiumcarbonate (83.42 gm) followed by allyl bromide (67.52 gm) and stir the reaction at to 25 oC till completion of the reaction. Then remove acetronitrile and extract the compound in ethyl acetate. Remove ethyl acetate and collect pale yellow liquid of Boc-Pro-OAll of about 90% purity and yield of 100 mL.
Example 3: Preparation of L-threonine methyl ester HC1 To dry methanol (412 mL) add L-threonine (51.5 gm) at 25-30 °C and gradually cool the mixture to 0 °C. To this add thionyl chloride (103 gm) and stir the reaction mass at 0 °C for 10 min. Raise the temperature to 25 °C and keep for 18 h. Check reaction by TLC for the formation of product. Then concentrate the product under vacuum at 45 °C to get Triturated L-threonine methyl ester HCL Then use the oily product as such in the next reaction.
Example 4: Preparation of Boc-Ile-Thr-OMe
To THF (100 mL) solution add Boc-L-isoleucine (50 gm) at 25-30 °C and
stir tb dissolve under nitrogen, cool the mixture to 0 °C. To this add N-

methylmorpholine (21.89 gm) and stir the reaction mass at 2 °C for 10 min. Cool reaction to -10 °C and add drop-wise IBCF (29.5 gm) with stirring for 10 min. Separately, to dry DMF (70 mL) dissolve L-threonine methyl ester (from Example 3) and neutralise it with NMM (43 mL) at 10-15 °C Make slurry of obtained white syrup and add to Boc-L-isoleucine reaction mass drop-wise at -10 °C. The 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 ethyl acetate. Wash with HC1 (1 N) twice, followed by Na2CO3 solution (1 N) twice. Add Na2SO4 and distil out ethylacetate. To obtained clear syrup add cyclohexane at 20-25 °C and stir. Collect white powder, add cyclohexane (200 mL) and stir for 2-3 h. Filter the solid and dry under vacuum for 5-6 h. The product yield of 75% and purity of 95% by HPLC is obtained.
Example 5: Preparation of Boc-D-Tyr(Et)-Ile-Thr-OMe To MDC (100 mL) add Boc-Ile-Thr-OMe (50 gm) at 25-30 °C and stir. To this add TFA (100 mL) at 0 °C and stir at 25 °C for 2 h. Check removal of Boc by HPLC. Concentrate the reaction mass at 40-45 °C. To this add toluene (100 mL) and distil under vacuum at 45 °C to get oily mass. diossolve this mass in DMF (75 mL) and neutralise with NMM. Separately, take Boc-Tyr(Et)-OH (37.33 gm) in dry DMF (75 mL). Cool the solution at 0 °C, under nitrogen add HOBt and stir for 5 min. To this add above amine component 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 (1300 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 95 % by HPLC is obtained.
Example 6: Preparation of Mpa(Trt)-D-Tyr(Et)-Ile-Thr-OH To MDC (100 mL) solution take Boc-D-Tyr(Et)-Ile-The-OMe (50 gm) at 5 °C and stir. To this add TFA (100 mL) at 5 °C and stir at 25 °C for 2-3 h. Check removal of Boc by HPLC. Concentrate the reaction mass at 40-45 °C. To this add toluene (100 mL) and concentrate under vacuum at 45 °C. to this add DMF (54 mL) and neutralise with NMM. Separately, take 3Mpa(trt)-OH (27 gm) in dry DMF (54 mL). Cool the solution at 0 °Q under nitrogen add HOBt and stir for 5 min. To this add above free compound in DMF 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 5-6 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 75% and purity of 95 % by HPLC is obtained.
Example 7: Preparation of Boc-Asn(Trt)-Cys(Acm)-Pro-OAll To 10% HC1 in ethyl acetate (250 mL) solution add Boc-Pro-OAll (50 gm) at 0-5 °C and stir for 4 h at 25 °C. Remove ethyl acetate at 40 °C. Then dissolve the sticky oil in DMF and neutralize with N-methylmorpholine. In another RBF, take Boc-Cys(ACM)-OH (38.25 gm) and add ethyl acetate, HOBt and neutralise Pro-OAll. Then add EDAC-HC1 and stir the reaction

mass at 25 °C till reaction is complete. After workup collect sticky mass of Boc-Cys(ACM)-Pro-OAll. Disolve this compound in 10% HC1 in ethyl acetate (280 mL) and stir for 4 h. Remove ethyl acetate at 40 °C and dissolve the reaction mass in DMF and neutralize with N-methylmorpholine. In another RBF, take Boc-Asn(Trt)-OH (41.39 gm) and add DMF and HOBt, cool the mass to 0-5 °C and add neutralized Cys-(Acm)-Pro-OAll to it. Then add EDAC-HC1 and stir it at 25 °C till the reaction is complete. The product [Boc-Asn-(Trt)-Cys(Acm)-Pro-OAll] yield of 93% and purity of 93 % by HPLC is obtained.
Example 8: Preparation of Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-OH
The peptides obtained in Examples 6 and 7 are ligated to form the heptapeptide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-OAll. The procedure used for the preparation of said heptapeptide is briefly as: Tripeptide Boc-Asn(Trt)-Cys(Acm)-Pro-OAll is ligated to tetrapeptide Mpa(Trt)-D-Tyr(Et)-Ile-Thr-OH leading to formation of Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-OAll. This product is deproteced with tetrakistriphenyl phosphine palladium leading to the formation of helptapetide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-OH. The product yield of 85% and purity of 85% by HPLC is obtained.
Example 9: Preparation of Fmoc-Orn(Boc)-Gly-NH2
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 NMM and add this to the reaction mass. To this add EDAC (3.4 gm) dissolved in DMF (5 mL). Allow the reaction to proceed at 0 °C for 20 min under nitrogen. Then allow the reaction at 25 °C for 4 h. Check formation of the product with TLC Then extract the product in ethyl acetate and isolate as crude viscous compound after washing in cyclohexane. The product yield of 82% and purity of 93% by HPLC is obtained.
Example 10: Preparation of Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-Orn(Boc)-Gly-NH2
The peptides obtained in Examples 8 and 9 are ligated to form the nonapeptide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Acm)-Pro-Orn(Boc)-Gly- NH2. 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)-Ile-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-NH2. The product yield of 85% and purity of 80% by HPLC is obtained.
Example 11: Preparation of (N -1) atosiban TFA salt
To MDC (6 mL) solution add protected atosiban salt (1 gm) of Example 10 and cool to 0 °C To this add TFA (6 mL) and triethylsilane [or 1,2-ethandithiol or triisoporpylsilane] at 0-10 °C and stir at RT till the completion of reaction. 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)-Ile-Thr-Asn-Cys(Acm)-Pro-Orn-Gly-NH2 is obtained. The product yield of 85% and purity of >75% by HPLC is obtained.
Example 12: Preparation of crude cyclic atosiban TFA salt Dissolve the peptide of Example 11 (1 gm) in water (600 mL) and add I2 (0.21 gm) with acetic acid (1 mL) and stir for 3 h. then quench the reaction with ascorbic acid (100 mg), then filter through HYFLO bed and 0. 45 u filters. check completion of reaction by HPLC.
Example 13: Purification of crude atosiban TFA sait
The crude atosiban salt obtained in Example 12 is purified by preparative HPLC chromatography. On a preparative HPLC column (YMC, 50 x 300 mm; 10 µ; 100 A) atosiban salt is loaded for total running time of 260 min. Gradient mobile phase used contains Sol. A: 0.1% TFA in DM water and Sol. B: 0.1% TFA in ACN. Sol. B amount is raised to 100% during running from 0% at the beginning and coming back to 0% at me end. Fractions are analysed with HPLC and fractions with >98% hroduct purity are isolated and ACN evaporated.
Example 14: Conversion of atosiban TFA salt to atosiban acetate The crude atosiban salt obtained by preparative HPLC is changed to atosiban acetate. In the first step, pool with >98% purity is loaded onto a column (YMC, 50 x 300 mm; 10 µ; 100 A) and wash with ammonium acetate (3%), followed by acetic acid (0.1%). Then gradient elute using

mobile phases A: 0.1% acetic acid in water and B: 0.1% acetic acid in ACN over a programme of 130 min. Fractions with ^98% atosiban are collected and lyophilised.

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-Thr-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-Tyr(Et)-Ile-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)-Ile-Thr-OH;
e) reacting tetrapeptide of step d) with tripeptide methyl ester of sequence NH2-Asn-Cys(Acm)-Pro-OAll leading to the formation of heptapeptide allyl carboxyl of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-OAll;

i) hydrolysing heptapeptide allyl carboxyl of step e) with a acid leading to the formation of heptapeptide of sequence Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-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-NH2;
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)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2 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 tetrapepfade 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-OA11 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.