IMPROVED PROCESS FOR PREPARATION OF BIVALIRUDIN FIELD OF INVENTION

The invention relates to a method of preparing bivalirudin by solid phase peptide synthesis employing Fmoc methodology.

BACK GROUND OF INVENTION

Bivalirudin (I) is a polypeptide consisting of 20 amino acid residues. It is used as an anticoagulant in the treatment of unstable angina for patients undergoing percutaneous coronary intervention / percutaneous transluminal coronary angioplasty (PCI/PTCA). Its mechanism action is by direct thrombin inhibition. The amino acid sequence of the peptide is D-Phenyalalanine-L-prolyl-L-argininyl-L-prolyl-glycyl-glycyl-glycyl-glycyl- L-asparginyl-glycyl-L-alpha-aspartylL-phenylsalanyl-L-alpha-glutamyl-L- alphaglutamyl-L-isoleucyl-
L-prolyl-L-tyrosyl-L-leucine and drug is used as trifluoroacetate hydrate.

D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr- Leu (I)

Bivalirudin is prepared by three different peptide synthetic methodologies. These are solution phase peptide synthesis, solid phase peptide synthesis and hybrid synthesis involving both the solution and solid phase synthesis.

Solution phase synthesis of bivalirudin is described in the recent patents, US2010/0056755(2010), CN101475631 (2009). The solution synthesis for long peptide of 20 amino acids is laborious, time consuming.

Hybrid synthesis is combination of solid and solution peptide synthesis. In these methodologies a part of synthesis is done in solid phase and other part is done solution phase. Recent patents WO 2007/033383, US20070093423A describe synthesis of bivalirudin by hybrid methodology. The methodology involves synthesis of fragments by solid phase technique, then purifying by HPLC and finally coupling the purified fragments by solution phase peptide synthesis. Overall, the process is laborious and difficult to implement on commercial scale as one needs to facilities for both solution and solid phase syntheses. Creating cGMP production facilities for both solution and solid phase synthesis is highly expensive and not feasible.

The most popular, convenient and faster method to synthesize long peptides such bivalirudin is by solid phase peptide synthesis. US patent US 5196404 describes a series of hirulog analogs as thrombin inhibitors. One of them is hirulog-8, which is later known as bivalirudin is synthesized in this patent by Boc chemistry. As compared to Boc chemistry, Fomc based solid phase peptide synthesis chemistry uses milder reaction conditions and is very convenient

Fmoc based solid phase peptide synthesis of bivalirudin are described in CN 101555274, US2010/0292436 (2010), CN101538317 (2009), US2009/0062511 (2009), WO 2008/109079 (2008), WO 2007/033383 (2007), WO 2006/045503. However, still there is a need for improved, cost effective, convenient process for large scale commercial operations. The present application describes one such process.

SUMMARY OF INVENTION

The main objective of the present invention is to prepare highly pure bivalirudin by fmoc solid phase peptide synthesis. The invention consists of following steps:

i. Attaching Fmoc -Leu-OH to the solid support followed by coupling other amino acids as in the sequence to yield resin bound peptide. The resin bound peptide is cleaved from the resin to afford novel nitro protected bivalirudin in which guanidine group of arginine residue is protected by nitro group. The sequence of which is;

D-Phe-Pro-Arg(N02)-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu (II)

ii. The nitro protecting group is then cleaved by formic acid involving reduction reaction in presence of Pd/C to yield crude bivalirudin. The crude bivalirudin is purified by preparative HPLC and lyophilized to get bivalirudin with a purity of more than 99%.

DETAILED DESCRIPTION OF INVENTION

The present application relates to an improved, commercially viable process for preparation of bivalirudin. The process basically consists of following step:

1. Preparation of resin bound protected peptide

2. Cleavage of resin bound peptide to yield protected bivalirudin in which guanidine group of arginine is protected by nitro group

3. Deblocking of nitro group protected bivalirudin by reduction to yield crude bivalirudin

4. HPLC purification of crude peptide

The first step in solid phase synthesis involves anchoring the carboxyl group of last amino acid in the chain to solid support. A wide variety of solid supports such as Wang resin, chlorotrityl chloride resin, TentaGel TGA, TentGel S AC, TentaGel S PHB, ChemMatrix Wang or HMPB Chem Matrix. However chlorotrityl chloride resin is preferred due to better and easy anchoring procedures and commercial availability. The first amino acid to anchor is Fmoc-Leu-OH using DIEA as catalyst in DMF solvent. After anchoring of the first amino acid, fmoc group is cleaved using 1-50% piperidine in DMF, DMSO, diethyl acetamide or any other suitable aprotic solvent. The fmoc cleaving agent may be selected from other secondary bases such DBU, diethyl amine etc. either as reagent single or in combination form 1-50%, combination of other secondary bases such as DBU, diethyl amine, etc. After cleaving fmoc protecting group, the free amine group is coupled to next amino acid in the sequence, which is Fmoc-Tyr(tBu)-OH
in a solvent. The peptide coupling reagent could be DIC, HBTU, TBTU, PyBoP, HATU, HCTU, with tertiary bases such as DIEA, N-methylmorphiline. The fmoc-amino acids and coupling agent are always taken in excess. It could be 1-8 equivalents to resin substitution. After the coupling of second amino acid, the attached finoc group is cleaved as above, then next amino acid, Glu(OtBu)-OH in the sequence is coupled and cleaved. Similarly, other amino acids in the sequence are coupled and cleaved in the following sequence. Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc- Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc- Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(N02)-OH, Fmoc-Pro-OH, Boc-D-Phe-OH. The amino acids are used in 1-8 equivalents to substitution.

Coupling and decoupling are monitored by Kaiser test and chloranil test. It has been observed that after attaching 10 amino acids, Kaiser and chloranil tests are found to be unsatisfactory. Kaiser and chloranil tests provide only a qualitative idea of coupling or decoupling efficiencies and never lead to any quantitative idea of coupling or decoupling. Hence to test completion of coupling/decoupling, a small sample of peptide attached to resin is cleaved using cocktail reagent and the sample is examined by HPLC analysis to check quantitatively coupling and decoupling efficiencies. As a part of this protocol, initially a small amount of resin bound peptide is taken, washed with solvent ether to remove adhering DMF and left overnight for drying. Then 10 mg of resin is treated with 0.5 ml cocktail mixture for 3h and sample is evaporated with nitrogen gas and treated with solvent ether. The precipitated product is washed 3 times with ether and residue is dissolved in mobile phase buffer and injected to HPLC. Initially, at pilot level samples are taken from amino acids 2-20, cleaved with reagent and after work up are run on HPLC. This study showed that couplings are not complete at amino acids 6, 7, 8, 9, 10, 11, 16, 17, 18, 19, 20. In order to increase coupling efficiency the coupling step is repeated 1-5 times.

After the completion of attachment of all the amino acids, the peptide chain is cleaved from the resin by treating with cocktail reagent consisting TFA (trifluoroacetic acid), ethanethiol, TIS (triisopropyl silane), p-cresol, water in various ratios. Preferred rations are TFA, TIS, cresol, water (92.5, 2.5, 2.5, and 2.5) for l-6h at 0-40°C. The cleaved peptide in TFA is concentrated to about 1/10 th volume, treated with suitable solvent such as diethyl ether, diisopropyl ether, methylisobutyl tertiary ether and left for l-24h at -10°C to precipitate the product. The precipitated product is filtered and washed with ether 3-5 times and dried. This product is nitro protected bivalirudin; here guanidine of arginine is protected by nitro group. Cleavage of nitro group of D-Phe-Pro-Arg(N02> Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu (II)

In order to cleave nitro group, we conducted series of pilot reactions such as hydrogenation in Pd/C in various solvents, like trifluoromethane sulfonic acid, HBr- acetic acid, HCl-acetic acid, formic acid, formic acid- Pd/C, ammonium formate -Pd/C . Hydrogenation in Pd/C is carried out in various solvents, trifluoromethane sulfonic acid, HBr-acetic acid, HCl-acetic acid etc. Many of these reagents failed cleave nitro group to yield bivalirudin. However, the reaction is observed to proceed efficiently with formic acid- Pd/C, and ammonium formate -Pd/C. Conversion of nitro protected bivalirudin to bivalirudin progressed better in formic acid-Pd/C when compared to ammonium formate- Pd/C. To further optimize the reaction conditions, various concentrations formic acid in water and solvents such as methanol, ethanol, isopropyl alcohol, ethyl acetate are tried. The best conversion has been achieved in water- formic acid mixtures. To see the effect of temperature, the reduction is carried out at various temperature settings in the range of 0 -55°C, for various time intervals. The best reaction conditions are 45°C for l-6h.

ADVANTAGES OF THE PRESENT INNOVATION

The hitherto known processes uses very expensive and commercially not easily available building block Fmoc-Arg(Pmc/Pbf)-OH for synthesis, whereas in the present invention the building block is substituted by cheap and easily available Fmoc-Arg(N02)-OH. This replacement has reduced the overall cost of production of bivalirudin appreciably. The present process also uses environmentally friendly, non-hazardous, industrially convenient nitro cleavage reagent formic acid-Pd/C in place of the normally used hydrogenation at high pressure.

EXAMPLE 1
Preparation of resin bound peptide:

The synthesis of peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure staring from chlorotrity chloride resin (lOg, substitution 1.1 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer (Sonata, Protein technologies, USA) and the synthesis was carried out using 2 molar excess protected amino acids with TBTU/DIEA activator. The first amino acid leucine was attached to resin in presence of 2 molar excess DIEA. After attaching the first amino acid excess chloride groups on chloritryl resin were neutralized by treating with methanol. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(N02)-OH, Fmoc-Pro-OH, Boc-D-Phe- OH. The coupling and decoupling of amino acids was monitored by Kaiser test. The coupling process was repeated till the Kaiser test is negative as in the case of arginine. Yield of resin bound peptide is 37.0g.

D-Phe-Pro-Arg(N02)-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu- Glu-Tyr-Leu-Resin (III)

EXAMPLE 2 #
Cleavage of resin bound peptide to yield nitro protected bivalirudin (II)
The assembled peptide on the resin was treated with 300 ml cleaver of cocktail mixture consisting of TFA(95%), TES (2.5), water (2.5%) for 3 h at 25-30°C and filtered to remove resin. The cocktail mixture was subjected to distillation, treated with ether and precipitated nitro protected peptide is filtered and dried (yield 19.0g).

EXAMPLE 3:

Deprotection of nitro group of nitro protected bivalirudin
Nitroprotected bivalirudin (16g) was treated with Pd/C (8g), formic acid (300ml) and stirred at 45°C for 4-6h. This reaction was monitored by HPLC for completion of reaction. After completion of reaction, the reaction mass is filtered to separate Pd/C and the product, crude bivalirudin in formic acid was subjected to reversed phase chromatography.

EXAMPLE 4
Purification of crude bivalirudin

i. Primary Purification The above crude bivalirudin solution was loaded on prep C-18 column (50x 250mm, 100A). Fractions with >90%purity were pooled and taken up for secondary purification using ammonium acetate buffer (Buffer A: 0.1M ammonium acetate, pH 4.5; Buffer B: acetonitrile).

ii. Secondary purification

Fractions with purity greater than 90% were pooled and loaded again onto prep C-18 column (5Ox 250mm, 100A°) using TEAP buffer (Buffer; .0.01M potassium dihydrogen phosphate with 2.0 ml triethyl amine, pH 3.0: Buffer B; acetonitrile). The fractions with more than 99% purity were pooled and taken up for salt exchange.

iii. Salt exchange
Fractions with purity greater than 99% were pooled and loaded again onto prep C-18 column (50x 250mm, 100A0 ) using trifluoroacetic acid buffer (BufferA; 0.1% trifluoroacetic acid in water, Buffer B; 0.1% trifluroacetic acid in acetonitrile) The fractions with more than 99% purity were pooled and lyophilized to get purified final product (2.4g).

CLAIMS We Claim:

1. A process for preparation of bivalirudin (I) using a novel nitro protected peptide(II) by fmoc solid phase synthesis technique D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu- Glu-Tyr-Leu (I)

D-Phe-Pro-Arg(NO2)-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro- Glu-Glu-Tyr-Leu (II) The said process comprising,

a) Assembling of peptide chain consisting of 20 protected amino acids on a solid support by coupling to one another to obtain a peptide bound resin (III) Boc-D-Phe-Pro-Arg(NO2)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe- Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(OtBu)-Leu-Resin (III)

b) Cleaving and deprotecting of all of protecting groups to obtain novel nitro protected bivalirudin (II)

c) Reducing the nitro group on arginine residue of novel protected bivalirudin to yield crude bivalirudin (I)

d) Purifying the crude bivalirudin by chromatography

2. The process of claim 1, wherein the solid support is chloritrityl chloride resin or Wang resin.

3. The process of claim 1, wherein the cleavage of the resin with the linker leads to the release of assembled novel nitro protected bivalirudin (II)

4. The process of claim 1, wherein the nitro group in nitro protected bivalirudin (II) is cleaved using hydrogen gas to yield crude bivalirudin.

5. The process of claim 4 wherein the hydrogenation is done using reagents H2- Pd/C, hydrogen donor formic acid-Pd/C , hydrogen donor sodium formate-Pd/C , hydrogen donor ammonium formate-Pd/C at a temperature in the range of 0-55°C.

6. The process of claim 4, wherein crude bivalirudin is purified by reverse phased chromatography to yield pure bivalirudin.

7. The process of claim 6, wherein the purity of bivalirudin is greater than 99%.

8. The process for preparation of bivalirudin essentially as described in the examples.