FIELD OF THE INVENTION

The present invention relates to an improved process for the preparation of Bivalirudin (I).

BACKGROUND OF THE INVENTION

Bivalirudin, also known as hirulog-8, is a synthetic peptide and is a 20-amino acids polypeptide. It is chemically known as D-phenylalanyl-L-prolyl-L-arginyl-L-prolyl-glycyl-glycyl-glycyl-glycyl-L-asparaginyl-glycyl-L-alpha-aspartyl-L-phenylalanyl-L-alpha-glutamyl-L-alpha-glutamyl-L-isoleucyl-L-prolyl-L-alpha-glutamyl-L-alpha-glutamyl-L-tyrosyl-L-leucine trifluoroacetate hydrate.
Bivalirudin is a specific and reversible direct thrombin inhibitor. It directly inhibits thrombin by specifically binding both to the catalytic site and to the anion-binding exosite of circulating and clot-bound thrombin. Bivalirudin is approved as an anticoagulant under the brand name Angiomax®.

US patent No. 5,196,404 disclosed Bivalirudin and pharmaceutically acceptable salts thereof. US '404 discloses a process for the preparation of Bivalirudin, wherein sequential approach of adding Boc-protected amino acids on divinylbenzene resin. The peptide sequence obtained was fully de-protected and cleaved from the resin using anhydrous HF, /?-cresol, ethyl methyl sulfate, followed by Lyophilization. The crude peptide was purified by HPLC using a Vydac C-18 column to give pure Bivalirudin.

The major disadvantage with the above process is the cleavage of the peptide from the resin; it requires strong acidic conditions, to de-protect globally which results in undesired side reactions.

US 2007/0093423 disclose a process for the preparation of Bivalirudin by fragment coupling strategy.

US 2008/0051558 discloses a process for the preparation of Bivalirudin that uses a resin, which is selected from trityl chloride resin, 4-methyltrityl chloride resin, 4-methoxytrityl chloride resin, 2-C1 trityl chloride resin, or Wang resin. The stepwise coupling of protected amino acids according to their sequence, followed by cleavage and deprotection of side-chain protecting groups provides Bivalirudin.
US 2009/0062511 discloses a process for the preparation of Bivalirudin, wherein the "C" terminal Amino Acid is anchored to a resin, followed by capping was performed after the amino acid attachment.

US 2011/0160431 discloses a process for the preparation of Bivalirudin, wherein dipeptide (Fmoc-Gly-Gly-OH) was used in place of coupling of two single residues of Gly to produce Bivalirudin.

The synthesis of Bivalirudin by sequential (stepwise) method results in the formation of impurities , which is selected from endoGly Bivalirudin and desGly Bivalirudin, which will be difficult to separate from Bivalirudin. Formation of these impurities will be avoided by employing the dipeptide (Fmoc-Gly-Gly-OH) for the incorporation of four glycines in the Bivalirudin molecule.

The present invention involves coupling of fragments; dipeptide(Y-Gly-Gly-OH) and a protected tetrapeptide (Y-D-Phe-Pro-Arg(X)-Pro-OH), wherein X, Y represent same or different protecting groups. This process minimizes the formation of such impurities as desD-Phe, desPro, endoPro, endoGly, desGly and desArg, and requires less quantity of Y-Arg(X)-OH during the synthesis of Bivalirudin.

OBJECTIVE OF INVENTION

The main objective of the present invention is to develop an improved process for the preparation of Bivalirudin of high purity and yield for commercial scale of manufacturing.

SUMMARY OF THE INVENTION

In one of our embodiments, the present invention provides an improved process for the preparation of Bivalirudin which comprises:

a) providing a protected dipeptide of formula (II) Y-Gly-Gly-OH - (II)
wherein, Y represents protecting group;
b) coupling of the protected dipeptide in step (a) two times to a protected
dodecapeptide on a solid support having the following formula (III),
(X)Asn-Gly-Asp-Phe-Glu-GIu-Ileu-Pro-Glu-Glu-Tyr-Leu-resin-(III)
Wherein, X represents protecting group; to produce a protected hexadecapeptide fragment of the following formula (IV);
(Y)Gly-Gly-Gly-GIy-Asn-Gly-Asp-Phe-GIu-Glu-Ileu-Pro-Glu-Glu-Tyr-Leu-resin-(IV)
c) coupling of a protected tetrapeptide of following formula (V),
(Y)D-Phe-Pro-Arg(X)-Pro-OH - (V)
Wherein X,Y represent same or different protecting groups; to the hexadecapeptide of formula (IV) in step (b) to yield Bivalirudin on resin;
d) concurrent cleaving and de-blocking of the peptide from the resin yields Bivalirudin;
e) Optionally, purifying crude Bivalirudin to produce pure Bivalirudin.

In another embodiment, the present invention provides a process for the preparation of protected tetra peptide having the formula:
(Y)D-Phe-Pro-Arg(X)-Pro-OH- (V),

The process comprises:

a) loading (X)Pro-OH to the resin;
b) de-protection of (X)Pro-resin and coupling of second protected amino acid (Y)Arg(X)-OH), wherein X,Y are same or different protecting groups;
c) repeating step (b) with another amino acid according to the sequence;
d) Cleaving and isolating the protected tetra peptide from the resin.
In another embodiment, the present invention provides a process for the preparation of Bivalirudin, which comprises:

a) providing a protected tetra peptide having the formula (V);
(Y)D-Phe-Pro-Arg(X)-Pro-OH- (V) wherein, X,Y are same or different protecting groups;
b) Employing the tetra peptide fragment (V) in the process for the preparation of Bivalirudin (I).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an improved process for the preparation of pure Bivalirudin.

The process comprises of providing a protected dipeptide Y-Gly-Gly-OH (II), which is coupled two times to a protected dodecapeptide (X)-Asn-Gly-Asp-Phe-Glu-Glu-Ileu- Pro-Glu-Glu-Tyr-Leu-resin-(III) supported on a resin, wherein X, Y represent same or different protecting groups , which is selected from 9-flurenyl-methyloxycarbonyl (Fmoc), tert-butoxycarbonyl(Boc), benzyl(Bzl), benzyloxycarbonyl(Cbz), 2-(4- biphenylyl)-2-propyloxcarbonyl(Bpoc), and propargyloxcarbonyl (Poc), pentamethyldihydrobenzofuransulfonyl (Pbf), tert-buryl ester (tBu) and trityl (Trt) etc., to produce a protected hexadecapeptide (X)-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu- Glu-Ileu-Pro-Glu-Glu-Tyr-Leu-resin-(IV). The coupling is carried out in presence of a coupling agent selected form o-benzotriazole-l-yl-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIG), 2-( 1 H-benzotriazole-1 -yl)-1,1,3,3- tetramethyluronium (HBTU), benzotriazole-1 -yl-oxy-tris(dimethylamino)phosphonium (BOP), benzotriazole-1-yl-oxy-tris-(pyrrolidino)-phosphonum (PyBOP), bromo-tris-pyrrolidino-phosphoniumhexaflurophosphate (PyBrOP), tris(pyrrolidino)-phosphonium hexaflurophosphate (pyCOP), ethyl cyanoglyoxylate-2-oxime (Oxyma Pure), 0-(6-
chloro-1 -hydrocibenzotriazol-1 -yl)-1,1,3,3 –tetramethyluronium tetrafluoroborate (TCTU), 2-ethoxycarbonyl-l,2-dihydropoquinoline (EEDQ), 2-(lH-7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uronium hexafluorophosphate (HATU), l-cyano-2-ethoxy-2- oxoethydenminooxy)dimethylamino-morpholion-carbenium hexafluorophosphate (COMU) performed active esters either individually or as a combination thereof. The amount of individual coupling agent used varies from 1 molar equivalent to 5 molar equivalents per molar equivalent of amino acid used.

The coupling reaction may be carried out in a solvent, which is selected from dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrrolidone, N-methylmorpholine or a mixture of solvents. The temperature at which the coupling is carried out may range from 10°C to 50°C. The overall process preferably is carried out in an inert atmosphere, which is selected from nitrogen or argon.
The protected amino acid anchored to the resin is selectively deprotected by method known in the art. For example, using a secondary base, which is selected from 20% piperidine, pyrrolidine, piperazine, morpholine, and thiomorpholine etc., in a solvent, which is selected from dimethylformamide (DMF), dichloromethane (DCM), N-methylmorpholine (NMM), l-methyl-2-pyrrolidinone (NMP) or mixtures thereof. The process of selective deprotection is carried out at a temperature in the range of 5°C to 50°C. However, the temperature may vary from amino acid to amino acid. The process of selective de-protection further comprises of washing the deprotected peptide resin with a solvent, which is selected from dichloromethane, dimethylformamide, isopropyl alcohol (IPA) or mixture thereof, removed residual reagents and byproducts.

The protected dodecapeptide (III) used in the above coupling is obtained by sequential addition of a amino acids starting with the first amino acid, L-leucine, anchored to the acid-labile resin, which is selected from 2-Cl-Trt-Cl resin, 4-methyltrityl chloride resin, 4-methoxytrityl chloride resin, or TentaGel TGA, TentaGel S PHB, TentaGel S AC, ChemMatrix Wang, Wang, Wang resin or HMPB Chem Matrix etc., wherein the amino terminus of L-leucine is blocked by a protecting group. The resin is suspended in an organic solvent, which is selected from methylene chloride, tetrahydrofuran, N,N-dimethylformamide or N-methylpyrrolidone, N-methylmorpholine or mixture thereof. The resin is then treated with N-protected amino acid in presence of a coupling agent for a desired period of time to affect the coupling. The coupling is carried out in the presence of a base , which is selected from N-methylmorpholine (NMM), di-isopropylethyamine (DIPEA), triethylamine (TEA), diethyl azodicarboxylate (DEAD), dimethylaminopyridine (DMAP) or l-(2-mesitylenesulfonyl)-3-nitro-IH-l,2,4-triazole (MSNT) and N-methylimidazole. After completion of the reaction, the resin is optionally washed with a solvent, which is selected from dichloromethane, dimethylformamide, dimethyformamide or mixture thereof to remove residual reagents and byproducts. After the first amino acid is attached to the resin, and the resin was encamped by a suitable reagent.

Coupling of the next N-protected amino acid {(X)L-Tyrosine}, wherein X is a protecting group as defined above; with the free amino terminus of L-leucine-resin obtained from the selective deprotection of amino protecting group of the resin. The coupling is carried out in presence of a coupling agent selected from TBTU, DCC, DIC, HBTU, BOP, PyBOP, PyBrOP, PyCOP, Oxyma Pure, TCTU, EEDQ, HATU, COMU or performed active esters either individually or as a combination thereof. The amount of individual coupling agent used may range from 1 molar equivalent to 5 molar equivalents, per molar equivalent of amino acid used. The coupling efficiency is monitored at each coupling step during the synthesis by a Kaiser test or any other suitable test. The above process is repeated for the remaining amino acids i.e. Glu, Glu, Pro, lieu, Glu, Glu, Phe, Asp, Gly, Asn to produce the protected dodecapeptide fragment (III).

The coupling reaction may be carried out in a solvent selected from dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrrolidone, N-methylmorpholine or a mixture of solvents. The temperature at which the coupling is carried out may range from 10°C to 50°C. The overall process may be carried out in an inert atmosphere, which is selected from nitrogen or argon.

The protected amino acid anchored to the resin is selectively deprotected by method known in the art. For example, a base, which is selected from 20% piperidine, pyrrolidine, piperazine, morpholine, and thiomorpholine etc. or mixture thereof, is used in presence of a solvent, which is selected from DMF, DCM, NMM and NMP or mixture thereof. The process of selective deprotection is carried out at a temperature in the range of 5°C to 50°C. However, the temperature may vary from amino acid to amino acid. The process of selective de-protection further comprises of washing the deprotected peptide resin with a solvent, which is selected from dichloromethane, dimethylformamide, Isopropyl alcohol (IPA) or moisture thereof removed residual reagents and byproducts.

A protected tetra peptide (Y)-D-Phe-Pro-Arg(X)-OH (V) is coupled to the protected hexadecapeptide (IV) on resin, wherein X and Y represent same or different protecting groups as defined above to produce Bivalirudin on resin. The process is carried out in presence of a coupling agent selected form TBTU, DCC, DIC, HBTU, BOP, PyBOP, PyBrOP, PyCOP, Oxyma Pure, TCTU, EEDQ, HATU,COMU or performed active esters either individually or as a combination thereof. The amount of individual coupling agent used varies from 1 molar equivalent to 5 molar equivalents per molar equivalent of amino acid used.
The coupling reaction may be carried out in a solvent, which is selected from dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrrolidone, N-methylmorpholine or mixture thereof. The temperature for the coupling varies from 10°C to 50°C. The overall process may be carried out in an inert atmosphere, which is selected from nitrogen or argon. After completion of the reaction, the resin is optionally washed with a solvent, which is selected from dichloromethane, dimethylformamide or mixture thereof to remove residual reagents and byproducts. The process can be repeated, if desired.

Concurrent cleavage and deprotection of protected Bivalirudin on the resin is accomplished by any one of conventional methods. The overall process may be carried out in an inert atmosphere i.e. nitrogen or argon. The cleavage and de-blocking of the peptide from the peptide resin involves treating of the protected peptide resin with one of the following cocktail mixtures:

> Trifluoro acetic acid (TFA)/phenol/water/triisopropyplsilane (TIS),
> TFA/TIS/Water,
> TFA/phenol/thioanisole/water/TIS
> TFA/TIS/Water/DCM
> w-cresol/ethanedithiol(EDT)/thioanisole/trimethylsilylbromide(TMSBr)/
TFA/DCM
> TF A/water
> TFA/p-cresol/water
> TFA / EDT/p-cresol/ water
> TFA /EDT/phenol/thioanisole/water etc.,

The temperature range for the cleavage and global de-protection is from 15°C to 40°C. After completion of the reaction, the reaction mixture is filtered and washed with an acid , which is selected from TFA or an organic solvent, which is selected from DCM.

Bivalirudin obtained above is isolated by precipitating with a solvent, which is selected from diethyl ether, diisopropyl ether, tert-butyl methyl ether, tert-butyl ethyl ether, tert-amyl methyl ether, isopropyl ether or mixtures thereof, followed by filtration and drying the product under vacuum.

Bivalirudin thus obtained is optionally purified by any known methods. For example the peptide is purified by reverse phase HPLC (RP-HPLC) using a binary gradient. The binary gradient consist of buffer or dilute TFA in water and one organic solvent, which is selected from acetonitrile, methanol, ethanol, n-propanol, or isopropanol, TFA. The resulting purified product is isolated by freeze-drying or spray-drying technologies.

In another embodiment, the present invention provides a process for the preparation of tetraprptide(Y)D-Phe-Pro-Arg(X)-Pro-OH-(V).

The process comprises of a stepwise Solid Phase Peptide Synthesis (SPPS) procedure starting with loading of a protected (X)Pro-OH wherein, X is a protecting group as defined above, on to a resin , which is selected from tritylchloride resin, 4-methyltrityl chloride resin, 4-methoxytrityl chloride resin, 2-Cl-trityl chloride resin. The protected amino acid anchored to the resin is selectively deprotected by method known in the art. For example, a base, which is selected from Piperidine, pyrrolidine, piperazine, morpholine, and thiomorpholine, DBU (1, 8-Diazabicyclo [5.4.0] undec-7-ene) etc. or mixture thereof, is used in presence a solvent, which is selected from DMF, DCM, NMM and NMP or mixture thereof. The process of selective deprotection is carried out at a temperature in the range of 5°C to 50°C. However, the temperature may vary from amino acid to amino acid. The process of selective de-protection further comprises of washing the deprotected peptide resin with a solvent, which is selected from dichloromethane, dimethylformamide, Isopropyl alcohol (IPA) or mixture thereof. Residual reagents and byproducts are removed. The second protected amino acid (Y)-Arg(X)-OH is introduced wherein X, Y are the same or different protecting groups as defined above. The protected amino acid is activated in-situ using coupling agent, which is selected from DIC & HOBt, DCC & HOBt, DCC & DMAP, PPh3 & DEAD. The coupling efficiency after each coupling step may be monitored during synthesis by means of a Kaiser test or any other suitable test. This cycle is repeated for each amino acid according to the sequence of the peptide. At the end of synthesis, the peptide resin is washed with a solvent, which is selected from DMF, DCM or mixture thereof, and dried under vacuum. Peptide thus obtained is cleaved from the resin using an acid, which is selected from diluted TFA in a solvent selected from DCM, by three repeated washings. The protected peptide solution is neutralized with a base, which is selected from pyridine, diisopropylethylamine to produce required protected tetrapeptide fragment.

In another embodiment, the present invention provides a process for the preparation of Bivalirudin in which protected tetra peptide (Y)D-Phe-Pro-Arg(X)-Pro-OH (V) is employed to produce Bivalirudin (I) or its pharmaceutically acceptable salts or esters or hydrates thereof.

The following examples illustrate the nature of the invention and are provided for illustrative purposes only and should not be construed to limit the scope of the invention.

Example 1:

Preparation of high purity Bivalirudin by using fragment based Solid Phase
I. Synthetic Procedure for the Protected Tetra Peptide from N-Term:

Step-1: Synthesis of Fmoc-Pro- Chloro Trityl Chloride Resin:

2-ChloroTrityl Chloride Resin (CTC Resin) (lOOg) (1.6mmol/g, 100-20Qmesh, B.No:GLSl 10921-48101) was transferred to a glass reaction vessel containing a sintered disk. Anhydrous dichloromethane (500 ml) was added to the glass vessel and drained after two min. A clear solution of Fmoc-Pro-OH (68.24g, 1.5eqv) dissolved in dry DCM (400ml) and N,N di-isopropylethylamine (224ml) was added. The reaction mixture was stirred mechanically for 3hrs. After 3hrs the solution was drained out and the resin was washed with 1% DIPEA in DCM (400ml) for 5min and then drained. The peptide resin was washed twice with a mixture of 1:1 [10% DIPEA: Methanol] (500ml) for 5min and drained. The peptide resin was washed with 1%DIPEA in DCM (two times). Finally the peptide resin was washed with 0.5% DIPEA in MTBE and dried under vacuum for 12 hrs. After 12hrs vacuum drying the peptide resin weight was 132g. Resin Substation calculated by UV method was 0.9mmol/g.

Step-2: Synthesis of Fmoc-Arg (Pbf)-Pro- CTC Resin:

Fmoc-Pro-CTC Resin from step-1, was swelled in DCM (400 ml) for 20min and DMF (700 ml) for 20min. 20% Piperidine in DMF (500mL) (5+15min) was added to the Fmoc-Pro- CTC Resin. The resin was washed with DMF (700ml) twice, IPA (350ml) twice and DMF (700ml) twice. A mixture consisting of Fmoc-Arg(Pbf)-OH (128g; 1.5eqv), HOBT (18.4 g) (FW.135.1) and DIC (42ml) was dissolved in DMF(600 ml) was added to the peptide resin, and the reaction was allowed under 25 °C for 3hrs, followed by washed with DMF (600ml) twice to obtain Fmoc-Arg(Pbf)-Pro-CTC Resin. Coupling reaction was monitored by Kaiser Test and chloranil test.

Step-3 Synthesis of Fmoc-Pro-Arg (Pbf)-Pro- CTC Resin:

20% Piperidine in DMF (200 ml) (5+15min) was added to Fmoc-Arg (Pbf)-Pro-CTC Resin obtained from step-(2) and washed with DMF (600ml) twice, IPA(350ml) twice, and DMF(600ml) twice. A mixture consisting of Fmoc-Pro-OH (61g; 2 eqv); HOBT |(24.4 g) (FW: 135.1) and DIC (42ml), which was dissolved in DMF (600 ml). The reaction was allowed under 25 °C for 3hours and washed with DMF (400ml) twice to obtain Fmoc-Pro-Arg(Pbf)-Pro- CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step-4: Synthesis of Fmoc-D-phe-Pro-Arg (Pbf)-Pro-CTC Resin.

20% Piperidine in DMF (500 ml) (5+15min) was added to the Fmoc-Pro-Arg(Pbf)-Pro-CTC Resin obtained from step-3 and washed with DMF(700ml) twice, IPA (350ml) twice, and DMF(700ml) twice. A mixture consisting of Fmoc-D-Phe-OH (68g; 2 eqv), HOBT (24.4 g) (FW: 135.1) and DIC (42ml) was dissolved in DMF (600 ml), at 25°C for 3hours, followed by washed with DMF (400ml) twice to obtain Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-CTC Resin. Coupling reaction was monitored by Kaiser Test and chloranil test.

After completion of the synthesis the peptide-resin was washed with DMF(500ml,5min) twice, followed by DCM(400ml,5min), MeOH(350ml) twice and MTBE(300ml) twice, and dried under vacuum for 20hrs. After drying the peptide-resin weight was 162g.

Step-5: Cleavage:

The peptide was cleaved from the resin using a 5% TFA solution in DCM by three repeated washings (15 min each). The acidic peptide solution was neutralized by using 10% DIPEA in DCM solution. The DCM solution was washed with by water (300ml) two times; the organic layer was dried under sodium sulphate and evaporated under reduced pressure. The protected peptide was precipitated by the addition of 4 volumes of MTBE, filtered and dried in vacuum to obtain 69g powder. It was identified as Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-OH by LCMS and HPLC.

Color: White Powder
HPLC Purity: 95.0%
MS calculated: 990.01
MS observed: 990.44

II. Preparation of diPeptide (Fmoc-Gly-Gly-OH) on solution Phase:

Step-1: Synthesis of Fmoc-Gly-Gly-OtBu:

Fmoc-Gly-OH (30gm; l.Oeqv) was dissolved in DMF (400ml) solution at room temperature. TBTU (35.4g; 1.1 eqv) was added in cold condition (5°C-10°C), followed by DIPEA (39.36ml; 2.2eqv) was added at cooling condition (pH 8-8.5). A solution of H-Gly-OtBu (18.5gms;l.leqv) in DMF (200ml) was added the above clear solution at 8°C. After completion of addition, the reaction mixture was maintained at RT for 3 .5hrs and checked TLC.mobile phase

(CHCl3:MeOH:CH3COOH:9:0.8:0.2). After conformation of TLC that SM was consumed distilled the RM solution to 80% and pour the RM solution in to ice Pieces under stirring stirred at overnight. White color powder was observed and the RM was filtered, DM water was added two times to the white color solid. The slurry solution of the compound was washed with MTBE for 10 min, filtered and dried.
Result:
Color: White Powder

HPLC Purity: 99.65% Yield : 36.7gm(88.6%)

Step-(2): Synthesis of Fmoc-Gly-Gly-OH

Fmoc-Gly-Gly-OtBu (40.5g) obtained in step-(l) was dissolved in DM water (10ml) and cooled with ice and then added TFA(200 ml) slowly at cooling and stirred for 30 min after the complete the addition of TFA. The reaction mixture was stirred it at RT for 2hrs and the TLC.mobile phase (Ethyl acetate: n-hexane:6:3) was checked. After completion of the reaction, distilled TFA and MTBE (200ml) were added and stirring for 10-15 min, then filtered and washed with MTBE (100ml) and dried for 10 min under vacuum. The compound was dissolved in ethyl acetate (2.5L) and washed three times with water. The organic layer was washed with brine solution (100ml) twice. The organic layer was washed with sodium sulphate and distilled ethylacetate solvent up to 80%. The solution was cooled for 3-4 hrs at 4°C, followed by filtered and washed with ethyl acetate (50ml) and dried completely under vacuum. The product was confirmed by LCMSandHlNMR.
Result:

Colour: White amorphous powder
Yield: 30.2gm (86.3%)
HPLC Purity: 99.18%

III. Preparation of protected Bivalirudin on resin (Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu- CTC Resin)

Step (1) Preparation of Fmoc-Leu-CTC Resin:

2-C1 Trityl Chloride Resin (50 g) (100-400, 1.6mmol/g) was swelled with DCM (500 ml) for 5 minutes. A clear solution of Fmoc-Leu-OH (19.1g) dissolved in dry DCM (300ml) and N, N di-isopropylethylamine (50ml) was added to the resin. The reaction mixture was stirred mechanically for 3hr. The solution was drained and the resin was washed with 1% DIPEA in DCM twice and 10%DIPEA in 1:1 mixture of DCM: MeOH twice. Finally the resin was washed with 0.5% MTBE and dried under vacuum for 12hrs to produce the peptide resin (68gm). As per weight gain and UV estimation the resin substation was 0.83 mmol/g.

Step (2) Preparation of Fmoc-Tyr (tBu)-Leu-CTC Resin

Fmoc-Leu-Resin was swelled in DCM for 20min and DMF 20min. 20% Piperidine in DMF (500 ml) (5+15min) was added to the Fmoc-Leu- CTC Resin and washed with DMF (400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Tyr(tBu)-OH (42 g) (459.6), HOBT (12.3 g) (FW:135.1) and DIC (21ml) which is dissolved in DMF (400 ml) was added and reaction was allowed at 25°C for 2 hours and dried. The obtained compound was washed with DMF (2X400ml) to obtain Fmoc-Tyr (tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (3) Preparation of Fmoc-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin
20% Piperidine in DMF(5+15min) was added to Fmoc-Tyr(OtBu)-Leu-CTC Resin obtained in step(2) and washed with DMF(400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Glu(tBu)-OH (38.3 g) (425.5), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25°C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (4): Preparation of Fmoc-GIu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin
20% Piperidine in DMF(5+15min) was added to Fmoc-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(3) and washed with DMF(400ml) twice, IPA(250ml) twice, and DMF(400ml) twice. A mixture consisting of Fmoc-Glu(tBu)-OH (38.3 g) (425.5), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml) was added and the reaction was allowed under 25°C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (5): Preparation of Fmoc-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:

20% Piperidine in DMF(5+15min) was added to Fmoc-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(4) and washed with DMF(400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Pro-OH (30.3 g) (337.12), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25 °C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (6): Preparation of Fmoc-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin

20% Piperidine in DMF(5+15min) was added to Fmoc-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(5) and washed with DMF(400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Ile-OH (31.8) (337.12), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25 °C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (7): Preparation of Fmoc-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:

20% Piperidine in DMF(5+15min) was added to Fmoc-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(6) and washed with DMF(400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Glu(tBu)-OH (38.3 g) (425.5), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25°C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (8): Preparation of Fmoc-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin

20% Piperidine in DMF(5+15min) was added to Fmoc-Glu(OtBu)-Ile-Pro-Glu(OtBu)- Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(7) and washed with DMF(400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Glu(tBu)-OH (38.3 g) (425.5), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25°C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc- Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin.

Coupling reaction was monitored by Kaiser Test.

Step (9): Preparation of Fmoc-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin.

20% Piperidine in DMF(5+15min) was added to Fmoc-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(8) and washed with DMF(400ml) twice, IPA(250ml) twice, DMF(400ml) twice. A mixture consisting of Fmoc-Phe-OH (34.9 g) (387.4), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25°C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.
Step (10): Preparation of Fmoc-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:

20% Piperidine in DMF(5+15min) was added to Fmoc-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(9) and washed with DMF(400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Asp(OtBu)-OH (37 g) (411.5), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25°C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (11): Preparation of Fmoc-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-IIe-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:

20% Piperidine in DMF(5+15min) was added to Fmoc-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(10) and washed with DMF(400ml) twice, IPA(250ml) twice, DMF(400ml) twice.

A mixture consisting of Fmoc-Gly-OH (40 g) (297.3), HOBT (12.3 g) (FW:135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25° C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)- Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (12): Preparation of Fmoc-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:
20% Piperidine in DMF(5+15min) was added to Fmoc-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step(ll) and washed with DMF(400ml) twice, IPA(250ml) twice and DMF(400ml) twice. A mixture consisting of Fmoc-Asn(Trt)-OH (80 g) (596.7), HOBT (12.3 g) (FW: 135.1) and DIC (21ml) dissolved in DMF (400 ml), was added and the reaction was allowed under 25° C for 2 hours and dried, followed by washed with DMF (400ml) twice to obtain Fmoc-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (13): Preparation of Fmoc-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:
20% Piperidine in DMF (500 ml)(5+15min) was added to a Fmoc-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step (12), allowed the reaction under 25°C, and washed with DMF(450ml) twice, IPA(300ml) twice and DMF(450ml) twice. A mixture consisting of Fmoc-Gly-Gly-OH (48 g), of HOBT (12.3 g) (FW:135.1) and DIC (21ml), which was dissolved in DMF (400 ml) was added and allowed the reaction under 25° C for 3 hours, followed by washed the resin with DMF (400ml) twice to obtain a Fmoc-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.

Step (14): Preparation of Fmoc-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-GIu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:
20% Piperidine in DMF (500 ml)(5+15min) was added to a Fmoc-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step (13), allowed the reaction under 25°C, and washed with DMF(450ml) twice, IPA(300ml) twice and DMF(450ml) twice. A mixture consisting of Fmoc-Gly-Gly-OH (48 g), of HOBT (12.3 g) (FW:135.1) and DIC (21ml), which was dissolved in DMF (400 ml) was added and allowed the reaction under 25°C for 3 hours, followed by washed the resin with DMF (400ml) twice to obtain a Fmoc-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-He-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. Coupling reaction was monitored by Kaiser Test.
Step (15): Preparation of Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin:

20% Piperidine in DMF (500 ml) (5+15min) was added to Fmoc-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin obtained in step-(14) at 25°C and washed with DMF(450ml) twice, IPA(300ml) twice, DMF(450ml) twice. A mixture consisting of tetra peptide (Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-OH) (69 g), HOBT (9.2 g) (FW:135.1) and DIC (17.5 ml) which was dissolved in DMF (600 ml) was added at 25° C and maintained for 10 hours. The peptide resin was washed with DMF (400ml) twice to obtain Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-CTC Resin. The peptide-resin was deprotected by 20% Piperidine in DMF (5+15 min) for 20 min. At the end of the synthesis the peptide-resin was washed with DMF, followed by DCM, MTBE and dried under vacuum to obtain 160g of dry peptide-resin.

Step (16): Concurrent cleavage and deprotection of Peptide-resin:
The peptide- resin (160g) obtained in step-(15) was added to the reactor containing a cold solution of 90% TFA, 5% TIS, 5% H20 (1.2L). The mixture was mixed for 2 hours at room temperature. After two hours the solution was filtered and the resin was washed with TFA one time and then filtered. The filtrates were combined and concentrated on Rotavapour at 25°C. The concentrated TFA solution (500ml) was precipitated by the addition of 8 volumes of ether (MTBE; 4L), then filtered and dried in vacuum to obtain crude product (93g).
Step (17): Purification of Crude Peptide:

The crude peptide (65 g) obtained in step-2, was dissolved in aqueous solution
18 contained 5% acetonitrile. The resulting solution was loaded on a C RP-HPLC column and purified to obtain fractions containing Bivalirudin at a purity of >98.22%. The pure fractions were collected and lyophilized to obtain a final dry peptide (25 g), which is at least 98.8% pure (HPLC). It contained not more than 0.53% [Asp9-Bivalirudin] and not more than 0.5% of any impurity. The purity of the Bivalirudin was determined with HPLC on a Synergi CI2 Max-RP (250x4.6 mm, 5 um) column. The mobile phase A was 0.025% (v/v) OPA in water and the mobile phase B 1:1 ethanol: acetonitrile. The following gradient was applied to the column loaded with 20 \x\ of sample, at tO: A=80%, B=20%, at t30 A=60%, B=40%, at t30.1 A=80%, B=20%, and at t35 A=80%, B=20%. The flow rate was 1.0 ml/min at an oven temperature of 45°C. The UV-detector was set at 210 nm.

WE CLAIM:

1. A process for the preparation of Bivalirudin, which comprises:

a) providing a protected dipeptide of formula (II), Y-Gly-Gly-OH - (II) wherein, Y represents protecting group;
b) coupling of the protected dipeptide in step (a) two times to a protected
dodecapeptide on a solid support having the following formula (HI),
(X)Asn-Gly-Asp-Phe-Glu-Glu-Ileu-Pro-GIu-Glu-Tyr-Leu-resin-(III)
wherein, X represents protecting group; to produce a protected hexadecapeptide fragment of the following formula (IV),
(Y)Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ileu-Pro-Glu-Glu-Tyr-Leu-resin-(IV)
c) coupling of a protected tetrapeptide of following formula (V),
(Y)D-Phe-Pro-Arg(X)-Pro-OH - (V)
Wherein X and Y represent the same or different protecting groups; to the hexadecapeptide of formula (IV) in step (b) to yield Bivalirudin on resin;
d) concurrent cleaving and de-blocking of the peptide from the resin yields Bivalirudin;
e) Optionally, purifying Bivalirudin to produce pure Bivalirudin.

2. The process according to claim 1, wherein the protecting group comprises 9-flurenyl-methyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyl (Bzl), benzyloxycarbonyl (Cbz), 2-(4-biphenylyl)-2-propyloxcarbonyl (Bpoc), and propargyloxcarbonyl (Poc), pentamethyldihydrobenzofuransulfonyl (Pbf), tert-buryl ester (tBu) and trityl (Trt) etc.,
3. The process according to claim 1, wherein the coupling reaction is carried out using a coupling agent comprises o-benzotriazole-l-yl-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU), 1,3-dicyclohexylcarbodiimide
(DCC),diisopropylcarbodiimide (DIC), 2-( 1 H-benzotriazole-1 -yl)-1,1,3,3-tetramethyluronium (HBTU), benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium (BOP), benzotriazole-1 -yl-oxy-tris-(pyrrolidino)- phosphonum (PyBOP), bromo-tris-pyrrolidino-phosphoniumhexaflurophosphate (PyBrOP), tris(pyroolidino)-phosphonium hexaflurophosphate (pyCOP), ethyl cyanoglyoxylate-2-oxime (Oxyma Pure), 0-(6-chloro-l-hydrocibenzotriazol-l-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), 2-ethoxycarbonyl-l,2- dihydropoquinoline (EEDQ), 2-( 1 H-7-azabenzotriazol-1 -yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU), 1-cyano-2-ethoxy-2- oxoethydenminooxy)dimethylamino-morpholion-carbenium hexafluorophosphate (COMU) performed active esters either individually or as a combination thereof.

4. The process according to claim 1, wherein the coupling reaction is carried out using a solvent comprises dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrrolidone, N-methylmorpholine or mixtures thereof.

5. The process according to claim 1, wherein the deprotection of protecting groups is carried out by using a base comprises 20% piperidine, pyrrolidine, piperazine, morpholine, thiomorpholine or a mixture thereof.

6. The process according to claim 5, wherein the deprotection is carried in presence of a solvent comprises dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrrolidone, N-methylmorpholine or mixtures thereof.

7. The process according to claim 1, wherein the cleavage and de-blocking of the peptide from the peptide resin is carried out by treating of the protected peptide resin with a cocktail mixture comprises Trifluoro acetic acid (TFA)/phenol/water/triisopropyplsilane(TIS), TFA/TIS/Water, TFA/phenol/thioanisole/water/TIS, TFA/TIS/Water/DCM, w-cresol/ ethanedithiol(EDT)/thioanisole/trimethylsilylbromide(TMSBr)/ TFA/DCM TFAAvater, TFA/p-cresol/water, TFA/EDT/p-cresol/ water, TFA /EDT/phenol/thioanisole/water or mixtures thereof.

8. The process according to claim 1, wherein the purification of Bivalirudin is carried out by reverse phase HPLC (RP-HPLC) using a binary gradient, comprises a buffer or dilute TFA in water and one organic solvent comprises acetonitrile, methanol, ethanol, n-propanol, or isopropanol or mixtures thereof.

9. A process for the preparation of protected tetra peptide having the formula:
(Y)D-Phe-Pro-Arg(X)-Pro-OH- (V) which comprises:

a) loading (X)Pro-OH to the resin;
b) de-protection of Pro and coupling of second protected amino acid (Y)-Arg-(X)-OH), wherein X and Y are same or different protecting groups;
c) repeating step (b) with another amino acid according to the sequence;
d) Cleaving and isolating the protected tetra peptide from the resin.

10. A process for the preparation of Bivalirudin, which comprises:

a) providing a protected tetra peptide having the formula (V):

(Y)D-Phe-Pro-Arg(X)-Pro-OH- (V) wherein, X and Y are same or different protecting groups;

b) Employing the tetra peptide fragment (V) in the process for the preparation of Bivalirudin (I).