FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 & Rule 13)
1. Title of the Invention
"An Improved Process for Fmoc Synthesis of Eicosapeptide"
2. Applicant (s)
Name: USV Limited
Nationality: Indian company incorporated under the Companies Act, 1956.
Address: B.S.D. Marg, Station Road, Govandi, Mumbai 400 088, Maharashtra, India.
3. Preamble to the Description
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of Invention:
The present invention relates to an improved, simple, environmentally benign and cost effective process for the synthesis of linear eicosapeptide having an amino acid sequence of D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH as set forth in Formula I.
Background of the invention
Bivalirudin (CAS# 128270-60-0, Mol.FnC98H138N24O33) is a reversible, direct thrombin inhibiting eicosapeptide that was developed via rational drug design as a hirudin analogue (hirulog-8, Hirulog). Similar to hirudin, it is a bivalent thrombin inhibitor, as its 20 amino acid structure combines a carboxy-terminal region that recognises thrombin's fibrin(ogen) - binding site, and an amino terminal tetrapeptide that inhibits the active site of thrombin, connected by a tetraglycine spacer. The D-FPRP moiety binds to the active site cleft and the NGDFEEIPEEYL moiety has affinity for the positively charged domain of thrombin. It is a thrombin substrate, degraded by thrombin at a rate of Kcat=0.015' and has an inhibition constant of 1.9 nM. It has better pharmacological advantages over hirudin, specially in enzymic metabolism (less dependance on renal clearance) and low immunogenicity (reduced potential for anaphylaxis) as compared to recombinant hirudin. Bivalirudin is approved for use as an alternative anticoagulant in percutaneous transluminal coronary angioplasty (PTCA), also shows beneficial effect as well as cost-effectiveness in heparin induced thrombocytopenia (HIT), elective percutaneous coronary intervention (PCI), in patients with non-ST-elevation active coronary syndrome (NSTEACS) as well as myocardial infarction (MI) patients and is undergoing active investigation for anticoagulation during cardiac surgery, both 'off pump' and with cardiopulmonary bypass ('on pump') with high rate success in control bleeding episodes. The advantage
2

of bivalirudin is that, it is a direct thrombin inhibitor for both free and clot bound thrombin, with very little immunogenicity and given as an IV injection and infusion (Bolus 1 mg/Kg followed by a 4 hour IV infusion at a rate of 2.5 mg/Kg/h) for upto 20 hours. Discontinuation of the drug leads to gradual reduction in anticoagulation effect primarily due to drug metabolism. The molecule is not known to bind any other serum protein. As compared to heparin, it has better safety end point (% hemorrhage) and better efficacy end points (% procedural failures, % revascularization failures) in PTCA. The safety and efficacy in other indications are being aggressively followed by clinicians.Anecdotal 'off-label' experience for the treatment of heparin-induced thrombocytopenia shows promise. In all these cases bivalirudin is not inferior to unfractionated heparin and GpIIb/IIIa inhibitors and has shown consistent stability and efficacy in improving antithrombic activity. Other antithrombic peptidomimetic direct thrombin inhibitors in this domain include argatroban, ximelagatran and dabigatran.To meet the unmet clinical need, it is a continuous endeavour to produce high quantities of bivalirudin in a cost effective way. The production of a high purity peptide product is a highly desired but difficult to achieve goal. Development of specially designed and highly optimized processes to produce high purity bivalirudin is the only solution to achieve the target. The present invention provides an improved, simple and cost-effective way to synthesize bivalirudin with commercially feasible yield and purity of > 99% with good stability.
US 5196404 (herein after referred as '404 patent) discloses the novel biologically active molecules which bind to and inhibit thrombin, compositions, combinations and method for the synthesis of the same. '404 patent explicitly discloses the synthesis of Hirulog-8 peptide by solid phase synthesis using BOC-L-Leucine-O-divinyl-benzene resin.'404 patent discloses the use of t-BOC amino acid in the synthesis which include
3

BOC-0-2,6-dichlorobenzyl tyrosine, BOC-L-glutamic acid(7-benzyl ester), BOC-L-proline, BOC-L-isoleucine, BOC-L-phenylalanine, BOC-L-aspartic acid (fi-benzyl ester), BOC-glycine, BOC-L-asparagine, BOC-L-phenylalanine (from Beckman Biosciences Inc..Philadelphia), and BOC-L-arginine. '404 patent teaches the manual addition of additional BOC-glycylglycine to achieve higher yields in synthesis cycle by attaching the (Gly)4 linker segment in two cycles thereby increasing the cost of production. Further, '404 patent discloses the use of HF:p-cresol:ethylmethyl sulfate (10:1:1 ,v/v/v) for deprotection and uncoupling of the fully protected peptide from the divinylbenzene resin after completion of the synthesis. '404 patent advocates the purification of the said peptide, after being lyophilized to dryness, by RP-HPLC employing an Applied Biosystems 151A liquid chromatographic system and a Vydac CI8 column (2.2x25 cm).The used column is equilibrated in 0.1 %TFA/water and developed with linear gradient of increasing acetonitrile concentration from 0 to 80% over 45 minutes at a flow rate of 4.0 ml/min and monitored for absorbance at 229 nm. '404 patent reports a recovery yield of 15-20 mg of pure peptide from 25-30mg (about 60% to 66% recovery)of crude Hirulog-8 by HPLC. A common problem associated with HF cleavage of resin-peptide following solid phase peptide synthesis is side reactions caused by prolonged contact of the peptide with HF. In order to avoid "bumping", or a sudden surge of HF/resin slurry, the process is carried out very slowly, thereby prolonging the exposure of peptide to HF and causing the above noted side reactions. The problem is even more pronounced in a large scale cleavage, e.g., greater than one litre, when a large quantity of HF cannot rapidly be removed after a proper reaction time has elapsed. Furthermore, constant monitoring and adjustment of vacuum level are required to control the process. Also HF is a toxic,corrosive gas (boiling point 19°C), and it must always be used in an adequate fume hood. Because it attacks glass very rapidly, with an exothermic reaction, all equipment for handling HF
4

must be made exclusively of plastic or noncorrosive metal. It requires an infrastructure development of fluorocarbon vacuumlines and extremely good hoods to avoid inhalation of the vapors which severely restricts the usage on commercial scale. Moreover, the use of strongly acidic conditions can produce deleterious changes in the structural integrity of peptides containing fragile sequences. The present invention avoids the use of HF by following Fmoc/tBu strategy which is operationally simple and chemically less complex not only because of the Fmoc base lability and orthogonal nature relative to the acid-labile protecting groups, but also allowing an element of chemical versatility in solid phase strategies. Example 1 in '404 elicits the use of harsh chemicals like GuCl at 6M concentration in RP-HPLC environment which can be deleterious to a preparative CI8 column.
WO98/50563 discloses a recombinant method for production of various peptides, including Hirulog. The method comprises expressing the peptide as part of a fusion protein, followed by the release of the peptide from the fusion protein by an acyl acceptor, such as a sulphur containing reductant by hydrolysis. The hydrolysis step as well as addition of the recombinant peptide by ammonia would generate a lot of related peptides and further conjugation of this hydrolysed peptide to a synthetic small peptide by thio ester bond is cumbersome and complicated. Regulatory hurdles impose several restrictions on the recombinant synthesis whereas the solid phase peptide synthesis (SPSS) has several advantages well documented in the literature. The primary advantage of SPPS is high yield by employing modern SPPS instrumentation. SPPS is also much quicker than conventional step-by-step solution synthesis. The attractiveness of solid phase chemistry can be attributed to the elimination of time-consuming work-up steps and the fact that the reactions can be driven to completion by using large excess of reagents. These features make solid phase chemistry
5

potentially attractive also for chemical development purposes. Also solution phase route is more cumbersome as compared to the solid phase route as after each coupling the peptide formed has to be isolated, whereas in the solid phase synthesis the excess reagents and by-products are washed off by simple filtration. But in both, the desired peptide compound is prepared by step-wise addition of amino acid moieties to a building peptide chain.
WO2006/045503 discloses an improved method of solid phase peptide synthesis of the anticoagulant peptide Bivalirudin. WO2006/045503 teaches the solid phase synthesis of bivalirudin on 2-Chloro trityl resin preloaded with Fmoc-Leu-OH, with a loading density of about 0.60 mmol/g. The further assembly of the peptide on the resin is carried out by coupling of Fmoc protected L-amino acids with the exception of D-Phe as readily Boc-protected Boc-D-Phe. However, the trityl chloride resin is extremely moisture sensitive, and presence of even traces of moisture will result in hydrolysis into the alcohol form. With 2-Chloro trityl resin, as the chloride form of the resin is exceedingly moisture sensitive and requires handling and storage under inert conditions, it poses a challenge for handling at the commercial levels.
WO2006/045503 exemplifies the use of TCTU (N-[(lH-6 Chlorobenzotriazoloa-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide) in dichloromethane/N-methylpyrrolidone (NMP) as coupling agent in the presence of Hunig base (diisopropyl-ethyl-amine, DIEA). Also a molar excess ratio of 1.5 equivalent of Fmoc-amino acids is used with an exception of Arg(Pbf) in molar excess of 2.5 equivalent. Fmoc deprotection is carried out with 20% piperidine in NMP at 30°C. The cleavage of the assembled linear peptide is accomplished by 2% (w/w) TFA, 1% w/w triethylsilane (TES) in dichloromethane. The global deprotection is
6

targeted by employing a cleavage cocktail of TFA/thioanisole/phenol/water/TES in the mixing ratio (% w/w):89:2.5:2.5:5.0:1.0. The deprotected peptide is recovered by addition of methyl-tertbutyl-ether, by cooling the reaction down to 0°C in a water bath for 30 minutes under stirring and filtrating off the salt precipitate formed. The crude peptide is rinsed several times with methyl tertiary butyl ether (MTBE) and dried at room temperature with 55% cleavage yield (jointly from cleavage and global deprotection steps) and purity of 55% by HPLC.
It is well known in the art that in Fmoc SPPS, the cleavage is carried out by treating the peptidyl resin with TFA which generates highly reactive cationic species from the protecting groups and the handles on the resin which unless trapped, reacts and modifies the amino acid residues inclusive of Tip, Tyr,Cys and Met which contain nucleophilic functional groups. To quench these generated cations, nucleophilic reagents also known as scavengers are added to the TFA used in the cleavage cocktail. Reagent K (TFA/water/phenol/thioanisole/EDT (82.5:5:5:5:2.5)) is an universal cleavage mixture well known in the art. Alternatively, use of trialkylsilanes such as triiisopropyl silane (TIS) and triethyl silane (TES) are equally effective as scavengers and substitute the odorous and toxic thiols e.g. thioanisole, ethane dithiols selectively. The present invention employs the use of simple, non-toxic and non-odorous cleavage cocktail comprising TFA/TIS/water without compromising on the purity and yield of the target peptide. An optimised strategy for the solid phase synthesis of the target peptide by use of Arg(Pmc) or Arg(Pbf) as the side chain protecting group eliminates the use of complex mixtures as reagent K used as cleavage cocktails which contain toxic and malodorous reagents.WO2006/045503 acknowledges that upon global deprotection of side chains under strongly acidic conditions, in usually aqueous medium, bystander alkylation of deprotected Tyr is not observed with Pmc especially
7

Pbf and also that Pbf s cleavage rate is highest ever.
There are numerous cleavage cocktails cited in the literature for cleavage of peptides from Wang's resin. Reagent K(TFA/phenol/water/thooanisole/EDT; 82.5:5:5:5:2.5) and Reagent R (TFA/thioanisole/EDT/anisole; 90:5:3:2) are reported to give higher cleavage yields but the incorporation of strongly stinking thiols as scavengers is highly noxious and may not be preferable to handle on commercial scale. The present invention relates to the synthesis of linear eicosapeptide devoid of intermediate polarity amino acids Tip, His with the exception of Tyr and also devoid of hydrophobic amino acids inclusive of Cys, Met, Ala, Val, Met but with the exception of Ile,Leu and Phe.
WO2006/045503 invention scores over the erratic alkylation of Tyr residue upon cleavage from Wang resin by employing an acid labile 2-chloro trityl resin as the solid support. Further the invention stresses the negative effect of Tyr alkylation on product purity. However it also exemplifies the use of Wang resin for highlighting the modification at Tyr residue but has no supportive data for the same. WO2006/045503 discusses the proximity effect for juxtaposition Tyr at the C terminal end in bivalirudin which is prone to alkylation during global deprotection under strongly acidic conditions and discloses a two step cleavage to eliminate the Tyr alkylation impurities wherein the peptide is cleaved from the solid support under weakly acidic conditions (0.01% v/v to 30% v/v TFA in DCM) preventing alkylation by segregation of the different deprotection events in time and further globally deprotecting the peptide under strongly acidic conditions (50% v/v TFA in DCM) . WO2006/045503 advocates the solid phase synthesis of bivalirudin to be completely devoid of diketopiperazine side reaction, a side reaction known in the art wherein the Leu and Tyr amino acids at the C terminal end being most sensitive to the same.
8

Prior art teaches inevitably the use of thioanisole, EDT as essential scavenging reagents against Tyr alkylation, a major side reaction affecting the product purity. The present invention surprisingly found a solution to the above addressed problem by employing an environmentally benign cleavage cocktail devoid of thioanisole, EDT with concomitant cleavage of the peptide from the resin with comparable cleavage yield and higher purity. Also the present invention employs tBu as side chain protecting group for Tyr meaning that the tyrosyl side chain is converted to a tertiary butyl ether which requires strongly acidic conditions for efficient removal achieved by a single step cleavage and deprotection used in the present invention. The amino acids most vulnerable to the consequences of TFA deprotection are Met, Tyr, and Trp. Met is susceptible to oxidation or tertiary butylation but is not part of the eicosapeptide. Tyr may also be tertiary butylated during TFA cleavage or sulfonated by a Pmc group. However, Lebl and co workers, in Int. J.Pept.Protein Res., 43, 31(1994) disclose study results for correlation between the proximity of Trp to Arg in a sequence and extent of modification of Trp by Pmc, wherein the side reaction is the greatest when Trp and Arg were separated by one residue or when the vulnerable sequence Trp-Xxx-Arg was located at the C terminus rather than at the N terminus, the extent of modification increased. The proximity effect is not pronounced during synthesis of the eicosapeptide of the present invention as Tyr is placed at the C terminal spaced from Arg at the N terminal by 16 amino acid residues. The present invention also encompasses the Tyr alkylation during the cleavage by incorporation of triisopropylsilane and water as effective scavengers yielding a purity of atleast 65% which is commercially feasible to operate. Literature reports TIS to be used as a carbocation scavenger by acting as a hydride donor and quenching highly stable Trt cation which are not irreversibly scavenged by thiols.
9

Further, WO2006/045503 discusses the modification of the thrombin cleavage site -Arg(pbf)-Pro- by formation of pseudoscissile bond by replacement of an amide bond with -CH2NH and designated as -Arg(psiCH2NH)Pro- rendering the bivalirudin moiety resistant to thrombin proteolysis. WO2006/045503 teaches away from the synthesis of the peptide of present invention wherein the same discloses a modified peptide. WO2006/045503 also discloses the advantage of the handle or linker 2-chloro trityl, 4-methoxy trityl, 4 4'-dimethoxy trityl, 4-methyltrityl, preferably 2-chloro trityl grafted to the polystyrene base polymer via a PEG spacer moiety which provides more amphilic resin and hence leads to better handling e.g. in DCM/TFA mixtures for one step detachment and deprotection. The process of the present invention is an improved and efficient process over the one described in WO2006/045503 PCT Patent application as herein mentioned below:
- Coupling of first amino acid on Wang's resin or HMPA resin or Knorr resin efficiently by esterification with high substitution minimising epimerisation.
- Precise selection of cost-effective and readily available coupling agents and use of suitable base and solvents with improved solvation properties preventing racemization of amino acids.
- Single step cleavage and deprotection achieved with low content of byproducts, saving solvents and time
- Absence of stinking and noxious thiols as scavengers or HF with improved cleavage yield of atleast 90% as against 55% and crude peptide purity of atleast 65% as against 55%.
- Use of robust solid support against moisture-sensitive solid support aiding in ease of handling and storage.
Okayama et al. (1996, Chem Pharm. Bull. 44:1344-1350) is directed towards
10

designing an anticoagulant decapeptide (Suc-Phe-Glu-Pro-Ile-Glu-Tyr-Tyr-X-OH) (X= bond, Leu or Leu-Gln) possessing two O-sulfated tyrosine residues at position 62 and 63 based on C-terminal functional domain of hirudin, a 65 mer peptide with 20 fold activity as against hirudin. It is well known in the art that deletion of amino acid residues at position 64 and/or 65, as well as removal of the acidic functionality at position 62 in hirudin causes a decrease in the anticoagulant activity. Okayama et al. report positive contribution of N-succinylation and substitution of Glu with Pro at position 58 to overcome the disadvantages of the structural modification made by removal of acidic functionality of Glu and replacing it with Tyr. The disclosed decapeptide NF22 by Okayama et al. is a potent and stable peptide anticoagulant among the hirudin analogs with a IC50 value of 0.3mM. Okayama et al. also disclose the solid phase synthesis of Hirudin (54-65),hirugen and MDL-28050 using Fmoc chemistry on Wang's resin as the solid support, performed by using DCC (dicyclohexylcarbodiimide)/ WSCI (water soluble carbodiimide - l-ethyl-3-(3-dimethylaminopropyl) carbodiimide)) in presence of HOBt as coupling agent. It discusses about hirulog-1 but does not disclose hirulog-8 which is bivalirudin. Further, it discloses the acylation of the peptide synthesized on the resin by using a mixture of the corresponding acid anhydride and DIEA and subsequent cleavage of the peptide from the resin with the concomitant removal of all protecting groups by treatment with TFA at room temperature for 1.5 hours in the presence of 5% water and 5% anisole. Still further it discloses the purification of the isolated crude peptide by RP-HPLC. Use of stinking thiols poses an environmental threat in handling and disposal of the same at the commercial scale. DCC is incompatible with automated solid phase synthesis because the dicyclohexylurea is not soluble in common solvents. Use of dicyclohexylcarbodiimide (DCC) as coupling reagent is plagued because of its high reactivity by formation of O-acylisourea which may undergo rearrangement to N-acylurea which is not reactive or undergo an intramolecular cyclization to give
11

oxazolone which is less reactive than O-acylisourea. Use of HOBt as an additive to DCCAVSCI has an advantage over the carbodiimide which encounter low yields and
undesired side reactions. Moreover the active OBt esters formed are though less reactive, more stable and less prone to racemize. However, use of HOBt is restricted due to storage, transportation, and user safety problem due to imidazole ring which makes the structure unstable with relatively high sensitivity to friction, spark and electrostatic discharge resulting in burning or explosion.
Steinmetzer et al.(1999, Eur.J.Biochem.265,598-605) disclose the synthesis of two bivalent thrombin inhibitors consisting of a benzamidine-based active site blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments by solid phase synthesis on Wang's resin using Fmoc chemistry and using benzotriazol-l-yloxy-tris(pyrrolidino)phosphoniumhexafluorophosphate (PyBop) as coupling agent in the presence of 6 equivalents of N-diisopropylethylamine(DIEA) in dimethylfonnamide, for 2 hours at room temperature. Further it discloses the use of reagent K ie; 82.5% TFA, 5% water, 5% phenol, 5% thioanisole and 2.5% ethanedithiol as cleavage cocktail to cleave the resin from the support. Literature reports the use of benzotriazole derivative PyBOP, which is specially useful for cyclization steps or for the activation of hindered amino acids, where the use of aminium salts can lead to the formation of guanidine derivatives but prohibitive for industrial purposes due to its extremely high price.
WO2007/033383 relates to method for the preparation of high purity bivalirudin by solid phase and/or solution phase using hyper acid labile 2-Chloro trityl resin using Fmoc chemistry. Further, the same exemplifies the activation of Fmoc amino acids using TBTU/HOBt with a coupling time of 50 minutes, DIPEA as an organic base,
12

20% piperidine in DMF for deprotection, side chain protecting groups as Arg(Pbf), Tyr(tBu), Asp(OtBu), Glu (OtBu). Still further, the assembled linear peptide is cleaved from the resin employing 95%TFA, 2.5%TIS, 2.5%EDT as cleavage cocktail, the peptide is isolated by precipitation with methyl tertiary butyl ether (MTBE), and the crude peptide purified by RP-HPLC using gradient mode to a purity of 99% containing not more than 0.5% (Asp9-Bivalirudin) and 0.5% of any other impurity. The aminium salt TBTU has tetrafluoroborate as the counterion and HBTU has hexafuorophosphate as the counterion, but prior art reports of a comparison study between HBTU and TBTU which showed that the counterion had no significant influence on the coupling rate or racemisation. The present invention discloses use of HBTU, the onium salt component as an efficient coupling agent having stabilised structure thus safe to handle at commercial scale. HBTU have been shown to give superior results in terms of both coupling efficiency and suppression of enantiomerization (Fmoc Solid Phase Peptide Synthesis by Chan W.C. And White P.D., Oxford University Press, 2000, p.41-74). Use of HBTU provides high yield and high purity. It saves time in the activation step with no cooling required. WO2007/033383 discloses that the crude peptide is dissolved in aqueous acetonitrile. The present invention involves the use of 30% acetic acid prior to RP-HPLC purification and a reverse osmosis step for concentration.
US20060241282 discloses the extreme acid lability of 2-chlorotrityl resin leading to a premature cleavage of the peptide from the resin during chain assembly. Swelling characteristics of the 2-chlorotrityl resin,, renders it unsuitable and inefficient in the assembly of long peptides. 2-chlorotrityl resin is generally recommended for the synthesis of relatively short ( e.g. <20 residues) peptides.
Use of aqueous TFA with silanes as scavengers for efficient cleavage of bivalirudin
13

from the solid support with atleast 90% yield and with atleast 65 % purity of the crude
peptide has not been reported so far in literature. Moreover, the purity of the product depends on the chemistry and various process related parameters which employs extensive variety of starting materials which might be potentially contaminated due to many possible side reactions. The improved process of the present invention, also shows commercially feasible overall yield of 20% to 30% and is easily scalable for industrial production of therapeutic grade bivalirudin. The bivalirudin synthesized by the present invention lacks the disadvantages of the prior art and yields the peptide of high purity. One of the advantage of the process of the present invention is that all synthetic steps are performed under mild conditions providing a low content of byproducts and thereby a high yield and high purity of the final bivalirudin peptide. Another advantage is that it uses regular commercially available and relatively cheap protected amino acids.
SUMMARY OF INVENTION:
One aspect of the present invention discloses an improved process for synthesis of linear eicosapeptide as set forth in Formula I by an orthogonal Fmoc/tBu strategy comprising of:
D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH
Formula I
i. covalently linking a Fmoc-Leu-OH to a p-alkoxybenzyl alcohol
(Wang's resin)solid support through benzyl ester linkage,
ii. removing the N-a -NH2 protecting group from Fmoc-Leu-wang resin to
obtain an N-a -NH2 group,
iii. coupling the second Fmoc-Tyr(tBu) to the Leu-Wang resin by
14

activating the amino acid by HBTU/NMM in the presence of polar
aprotic solvent,
iv. deprotecting the Fmoc group by deprotectant,
v. repeating steps ii),iii),iv) for assembling the eicosapeptide (Boc)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-
Wang resin,
vi. cleaving and deprotecting said eicosapeptide of step v) using cleavage
cocktail characterized wherein said cleavage cocktail consists of
aqueous TFA and a non thiol scavenger, vii. obtaining cleavage yield of at least 90% and crude peptide purity of at
least 65%,
viii. isolating the crude eicosapeptide as set forth in Formula I by
precipitation, filtration and drying under vacuum,
ix. purifying the eicosapeptide of step viii) by chromatography wherein
said eicosapeptide has a purity of 99%,
x. concentrating the purified eicosapeptide of step ix) by reverse osmosis, xi. lyophilizing the concentrated purified eicosapeptide of step x.
Another aspect of the present invention discloses the use of HBTU/NMM as coupling agent in the presence of DMF as solvent.
Still another aspect of the present invention relates to the use of 20% piperidine in DMF for the deprotection of Fmoc group.
Yet another aspect of the invention focusses on the cleavage cocktail consisting of TFA in the range of 80% to 98%, water in the range of 0% to 5% and TIS in the range
15

of 0 % to 5%.
Further aspect of the invention is purification of the crude eicosapeptide to a purity of > 99% by chromatography, preferably RP-HPLC by isocratic and/or gradient mode.
Additionally,other aspect of the invention relates to an eluant for RP-HPLC purification of the crude eicosapeptide by gradient mode, wherein the eluant comprises of aqueous TFA and acetonitrile as an organic phase with isolated yield of 40% and purity of atleast 90%.
Additionally, another aspect of the invention relates to an eluant for RP-HPLC purification of the crude eicosapeptide by isocratic mode, wherein the eluant comprises of phosphoric acid buffer as the aqueous phase and acetonitrile as an organic phase with isolated yield of 60% to 70% and purity of atleast 99%.
Still further aspect of the present invention relates to the concentration of the pure peptide by reverse osmosis wherein the recovery is atleast 90%, preferably 98%.
Still further aspect of the present invention relates to eicosapeptide of formula I obtained by an improved process wherein the total impurity is not more 1% and the single largest impurity is not more than 0.5% .
One aspect of the invention also relates to a pharmaceutical composition comprising eicosapeptide of formula I obtained by an improved process and atleast one pharmaceutically acceptable excipient.
The present invention encompasses improved methods of synthesizing the Bivalirudin peptide on Wang's resin using Fmoc/tBu strategy.
16

BRIEF DISCUSSION OF THE ACCOMPANYING DRAWINGS:
Fig.l shows analytical RP-HPLC profile of chemically synthesized crude bivalirudin after cleavage by the process of the present invention. Column used:250 X 4.6mm, 5m, CI 8, wave length:210 nm; Mobile phase: A-0.1% H3PO4, B-0.1% H3PO4 and Acetonitrile; Flow rate: 1 ml/minute; Injection volume: 20ml.
Fig. 2 shows mass spectra of crude bivalirudin after cleavage from Wang's resin with bivalirudin peak at 2180 Da.
Fig.3 shows analytical RP-HPLC profile of pure bivalirudin purified by the process of the present invention. Column used:250 X 4.6mm, 5u, C18, wave length:210 nm; Mobile phase: A-0.1% H3PO4, B-0.1% H3PO4 and Acetonitrile; Flow rate:l ml/minute; Injection volume: 20ml.
Fig.4 shows mass spectra of pure bivalirudin at 2180 Da.
Fig.5 shows analytical RP-HPLC profile of chemically synthesized deamidated impurity. Column used:250 X 4.6mm, 5m, CI8, wave length:210 nm; Mobile phase: A-0.1% H3PO4, B-0.1% H3PO4 and Acetonitrile; Flow rate:l ml/minute; Injection volume: 20ml.
Fig.6 shows analytical RP-HPLC profile of chemically synthesized deamidated impurity with bivalirudin. Column used:250 X 4.6mm, 5m, CI8, wave length:210 nm; Mobile phase: A-0.1% H3PO4, B-0.1% H3PO4 and Acetonitrile; Flow rate:l ml/minute; Injection volume: 20ml.
17

Fig. 7 shows analytical RP-HPLC profile of chemically synthesized deamidated impurity spiked with bivalirudin.Column used:250 X 4.6mm, 5|x, CI8, wave length:210 nm; Mobile phase: A-0.1% H3PO4, B-0.1% H3PO4 and Acetonitrile; Flow rate:l ml/minute; Injection volume: 20ml.
Fig. 8 shows the bioactivity of the eicosapeptide of the present invention as compared with the innovator for inhibition of thrombin activity.
Fig.9 shows stability studies (at -20°C after 12 months) analytical RP-HPLC profile of eicosapeptide of the present invention.
DETAILED DESCRIPTION OF THE INVENTION:
One embodiment of the present invention is directed to an improved process for synthesis of linear eicosapeptide as set forth in Formula I by an orthogonal Fmoc/tBu strategy comprising of:
D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH
Formula I
i. covalently linking a Fmoc-Leu-OH to a p-alkoxybenzyl alcohol (Wang's
resin)solid support through benzyl ester linkage,
ii. removing the N-a -NH2 protecting group from Fmoc-Leu-wang resin to obtain
an N-a -NH2 group,
iii. coupling the second Fmoc-Tyr(tBu) to the Leu-Wang resin by activating the
amino acid by HBTU/NMM in the presence of polar aprotic solvent,
iv. deprotecting the Fmoc group by deprotectant,
v. repeating steps ii),iii),iv) for assembling the eicosapeptide (Boc)D-Phe-Pro-
18

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-Wang resin,
vi. cleaving and deprotecting said eicosapeptide of step v) using cleavage cocktail
characterized wherein said cleavage cocktail consists of aqueous TFA and a
non thiol scavenger,
vii. obtaining cleavage yield of at least 90% and crude peptide purity of at least
65%,
viii. isolating the crude eicosapeptide as set forth in Formula I by precipitation,
filtration and drying under vacuum,
ix. purifying the eicosapeptide of step viii) by chromatography wherein said
eicosapeptide has a purity of ³ 99%,
x. concentrating the purified eicosapeptide of step ix) by reverse osmosis,
xi. lyophilizing the concentrated purified eicosapeptide of step x.
Second embodiment of the present invention relates to the use of HBTU/NMM as coupling agent in the presence of DMF as solvent.
Third embodiment of the present invention relates to the use of 20% piperidine in DMF for the deprotection of Fmoc group.
Fourth embodiment of the invention is directed to cleavage cocktail consisting of TFA in the range of 80% to 98%, water in the range of 0% to 5% and TIS in the range of 0% to 5%.
Fifth embodiment of the present invention relates to purification of the crude eicosapeptide to a purity of ³ 99% by chromatography, preferably RP-HPLC by
19

isocratic and/or gradient mode.
Sixth embodiment of the invention is directed to RP-HPLC purification of the crude eicosapeptide by gradient mode, wherein the eluant comprises of aqueous TFA and acetonitrile as an organic phase with isolated yield of 40% and purity of atleast 90%.
Seventh embodiment of the invention is directed to RP-HPLC purification of the crude eicosapeptide by isocratic mode, wherein the eluant comprises of phosphoric acid buffer as the aqueous phase and acetonitrile as an organic phase with isolated yield of 60% to 70% and purity of atleast 99%.
Eighth embodiment of the present invention relates to the concentration of the pure peptide by reverse osmosis wherein the recovery is atleast 90%, preferably 98%.
Ninth embodiment of the present invention relates to bivalirudin obtained by an improved process wherein the total impurity is not more 1% and the single largest impurity is not more than 0.5% .
Tenth embodiment of the invention also relates to a pharmaceutical composition comprising eicosapeptide of formula I obtained by an improved process and atleast one pharmaceutically acceptable excipient.
Glossary of terms used in the Specification:
HBTU: N-[(lH-benzotriazol-l-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide
TBTU: 2-(l -H-benzotriazol-1 -yl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate TCTU:N-((IH-6 Chlorobenzotriazolo-1 -yl) (dimethylamino)methylene)-N-
20

methylmethanaminium tetrafluoroborate N-oxide)
PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino phosphonium hexaflurophosphate DCC:1,3- dicyclohexylcarbodiimde
HOBt: 1-hydroxybenzotriazole
DIPEA: N,N-diisopropyl ethyl amine
DIC: 1,3-diisopropyl carbodiimide
Boc: tert-butyloxycarbonyl Z benzyloxycarbonyl
Fmoc: 9-fluorenylmethyloxycarbonyl
TFA: Trifluoroacetic acid
HF: Hydrofluoric acid
DCM: Dichioromethane
EDT: Ethanedithiol
DMF: Dimethylformamide
DIPE:Diisopropyl ether
MeOH: Methanol
THE: Tetrahydrofuran
DMAP: 4-(N,N-dimethylamino)pyridine
TIS: Triisopropylsilane
TES:Triethylsilane
TPP: Triphenylphosphine
TFE: Trifluoroethanol
MTBE: Methyl tertiary butyl ether
NMM: N-methymorpholine
NMP: N-methylpyrrolidone
Pbf:2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulphonyl Pmc:2,2,5,7,8-pentamethylchroman-6-sulphonyl
OtBu:O-tert.butyl
21

Trt:trityl
tBu:tert-butyl
D-Phe: D-Phenyl alanine
Pro: Proline
Arg:Arginine
Gly:Glycine
Asp: Aspartic acid
Glu: Glutamic acid
He: Isoleucine
Tyr: Tyrosine
Leu: Leucine
Tip: Tryptophan
SOLV: Solvent
DEP: Deprotection
ACT: Activation
RV: Reaction vessel
AA: Amino acid
RP-HPLC: Reverse Phase High Performance Liquid Chromatography As used herein the term "eicosapeptide" refers to an oligopeptide with 20 amino acid residues.
As used herein the term "deprotectant" refers to any reagent used for removing the N-a-amino protecting group, in the present invention reference is hereby made to Fmoc. As used herein the term "orthogonal Fmoc/tBu strategy" refers to an approach which uses the base-labile N-Fmoc group for protection of the a-amino function, and acid-labile side chain protecting groups.
22

General Processes for Peptide Synthesis
In general protected amino acids are added to a growing peptide chain for the synthesis
of peptides. Either the amino group or the carboxyl group as well as any
reactive group in the side chain of the first amino acid are protected. This protected
amino acid is either coupled to an inert support or it can also be used in solution. The
next amino acid in the peptide sequence is appropriately protected under conditions
which favour the formation of an amide bond and is added to the first. After all desired
amino acids have been coupled in the correct sequence, the protective groups and
optionally the support phase are cleaved. The crude polypeptide that is obtained is
reprecipitated and preferably purified chromatographically to form the final product.
A preferred method for synthesizing physiologically active polypeptides with fewer than forty amino acids comprises a solid phase peptide synthesis. In this method the N-alpha-amino functions and any reactive side chains are protected with acid-labile or base-labile groups. The protective groups that are used should be stable under the conditions for linking amide bonds but it should be possible to readily cleave them without impairing the polypeptide chain that has formed. Suitable protective groups for the N-alpha-amino function include the following groups but are not limited to these: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), o-chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl, tert.-amyloxycarbonyl (Amoc), .alpha.,.alpha.-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl, 2-cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), l-(4,4-dimethyl-2,6-dioxocylohex-l-ylidene)ethyl (Dde) and the like. 9-Fluorenylmethoxycarbonyl (Fmoc) is preferably used as the N-alpha amino protective group.
Suitable side chain protective groups include the following but are not limited to these:
23

acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl, 2-chlorobenzyl, 2-chlorobenzyloxycarbonyl (2-CIZ), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)ethyl (Dde), isopropyl, 4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr), 2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulphonyl (Pbf), pivalyl, tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl, trimethylsilyl and trityl (Trt) and the like.
In the solid phase synthesis the C-terminal amino acid is coupled as the first to a suitable support material. Suitable support materials are those which are inert towards the reagents and reaction conditions for the stepwise condensation and cleavage reactions and which do not dissolve in the reaction media that are used. Examples of commercially available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol and also chloromethylated styrene/divinylbenzene copolymers, hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers and the like. Polystyrene (1%)-divinylbenzene or TentaGel.RTM. (Rapp Polymere, Tubingen) derivatized with 4-benzyloxybenzyl-alcohol (Wang-anchor (Wang, S. S. 1973)) or 2-chlorotrityl chloride (Barlos, K. et al. 1989) is preferably used if it is intended to prepare the peptidic acid. In the case of the peptide amide, polystyrene (1%) divinylbenzene or TentaGel.RTM. derivatized with 5-(4'-aminomethyl)-3',5'-dimethoxyphenoxy)valeric acid (PAL-anchor) (Albericio, F. et al. 1987) or p-(2,4-dimethoxyphenyl-amino methyl)-phenoxy group (Rink-Amid anchor (Rink, H. 1987)) is preferred.
The linkage to the polymeric support can be achieved by reacting the C-terminal
24

Fmoc-protected amino acid with the support material with the addition of an activation reagent in ethanol, acetonitrile, N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone or similar solvents preferably in DMF at room temperature or elevated temperatures e.g. between 40°C and 60°C, preferably at room temperature and with reaction times of 2 to 72 hours.The coupling of the N alpha protected amino acid preferably the Fmoc amino acid to the PAL, Wang or Rink anchor can for example be carried out with the aid of coupling reagents such as N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or other carbodiimides, 2-( 1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or N-(l H-benzotriazol-l-yl)(dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate N-oxide or other uronium salts, o-acyl-ureas, benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) or other phosphonium salts, N-hydroxysuccinimides, other N-hydroxyimides or oximes in the presence or also in the absence of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole preferably with the aid of TBTU with addition of HOBt, with or without the addition of a base such as for example diisopropylethylamine (DIEA), triethylamine or N-methylmorpholine, preferably diisopropylethylamine with reaction times of 2 to 72 hours, preferably 3 hours in a 1.5 to 3-fold excess of the amino acid and the coupling reagents, preferably in a 2-fold excess and at temperatures between about 10°C and 50°C , preferably 25°C in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, preferably dimethylformamide. Instead of the coupling reagents it is also possible to use the active esters (e.g. pentafluorophenyl, p-nitrophenyl or the like), the symmetric anhydride of the N alpha -Fmoc-amino acid, its acid chloride or acid fluoride under the conditions described above. The successive coupling of the protected amino acids can be carried out according to conventional methods in peptide synthesis typically in an automated peptide
25

synthesizer. After cleavage of the N alpha. -Fmoc protective group of the coupled amino acid on the solid phase by treatment with piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes, the next protected amino acid in a 1.5 to 5-fold excess, is coupled to the previous amino acid in an inert, non-aqueous, polar solvent such as dichloromethane, DMF or mixtures of the two, preferably DMF and at temperatures between about 10°C and 50°C, preferably at 25°C. The reagents that have already been mentioned for coupling the first N alpha-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable as coupling reagents. Active esters of the protected amino acid, or chlorides or fluorides or symmetric anhydrides thereof can also be used as an alternative.
At the end of the solid phase synthesis the peptide is cleaved from the support material while simultaneously cleaving the side chain protecting groups. Cleavage can be carried out with trifluoroacetic acid or other strongly acidic media with addition of 0%-5% v/v scavengers such as dimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol, anisole ethanedithiol, phenol or water, preferably 2.5% v/v . Peptides with fully protected side chains are obtained by cleaving the wang resin anchor with TFA/TIS/water. The protected peptide can be purified by chromatography on silica gel.
The acidic solution that is obtained is admixed with a 3 to 20-fold amount of cold ether or n-hexane, preferably a 10-fold excess of diethyl ether, in order to precipitate the peptide and hence to separate the scavengers and cleaved protective groups that remain in the ether. A further purification can be carried out by re-precipitating the peptide several times from glacial acetic acid. The precipitate that is obtained is taken up in water or tertiary butanol or mixtures of the two solvents, preferably a 1:1 mixture of tert-butanol/water and freeze-dried. The peptide obtained can be purified by some or all of the following chromatographic
26

methods: ion exchange over a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on non-derivatized polystyrene/divinylbenzene copolymers (e.g. Amberlite.RTM. XAD); adsorption chromatography on silica gel; ion exchange chromatography e.g. on carboxymethyl cellulose; distribution chromatography e.g. on Sephadex.RTM. G-25; countercurrent distribution chromatography; or high pressure liquid chromatography (HPLC) in particular reversed-phase HPLC on octyl or octadecylsilylsilica (ODS) phases.
In summary part of the present invention encompasses Fmoc processes using Wang's resin for the preparation of eicosapeptide of the present invention thereof. These processes which lead to physiologically active eicosapeptide of formula 1 comprise processes for the sequential condensation of protected amino acids on Wang's support, methods for cleaving the support and protective groups and for purifying the crude peptides that are obtained.
Wang resin is the most widely used solid phase support for acid substrates. The linker attached to the polystyrene core is a 4-hydroxybenzyl alcohol (p-benzyloxybenzyl alcohol) moiety. The linker is bound to the resin through a phenyl ether bond and the \ substrate is generally attached to the linker by a benzylic ester or ether bond. This linkage has good stability to a variety of reaction conditions, but can be readily cleaved by moderate treatment with an acid, generally trifluoroacetic acid. Impurities can form if a portion of the linker is attached to the resin through the benzylic position leaving a reactive phenolic site. This can occur during attachment of the linker if exact reaction condition are not maintained. Commercial Wang's resin of high purity are readily available.
Addition of the substrate is generally accomplished by coupling the nucleophilic resin
27

with a desired electrophile or by a Mitsunobu reaction. Care should be taken when loading optically active substrates, such as a-amino acids derivatives, because the activation step can lead to racemization. Many techniques have been developed to minimise this problem. Solvents such as DMF, DCM are commonly used because of the large swelling factors associated with these solvents. In situations where the substrate has base labile protecting groups such as Fmoc or Fm, it is important to either use amine free DMF or avoid the use of this solvent together. Partial deprotection during the coupling process leads to oligomerization and subsequent impurities in the final product. Commercially available finest quality Wang resin overcomes all the above problems.
The single largest advantage of using Wang's resin is complete and one step cleavage and global deprotection of the side chain protecting groups using 95% aqueous TFA. It is well documented that greater resistance to acid cleavage is due to the presence of hydrophobic residues at the C terminal extremities of the peptide which affects the cleavage yield also adversely. The greatest threat during cleavage of the peptide from the solid support is its incomplete removal which amounts to considerable loss but can be overcome by establishing the appropriate combination of resin type, linker group and cleavage protocol used. Wang's resin supports complete removal of peptide from the solid support within a much smaller time period as compared to the other solid supports including Rink acid, HMPA, and Knorr resins and minimises peptidyl side chain modifications.
The anchoring of the first amino acid to the solid support by esterification is often more difficult, and even hazardous, for some residues and can lead to epimerization, dipeptide formation and low substitution. For hydroxymethyl-based resins, formation of the ester linkage is easier with unhindered resins such as Wang resin compared with
28

Sasrin and HMPB resins which withdraw methoxy groups. The most commonly used esterification process is the symmetrical anhydride method. In case of difficult anchoring, the esterification step can be repeated with fresh reactants. After anchoring, unreacted resin bound hydroxyl groups should be capped by acetic anhydride.
It is obvious for a person skilled in the art, to adopt highly effective coupling reagents resulting in avoiding double coupling, deletions and modified peptides which will cut the solvent and protected amino acid usage favouring the purification step and decreasing the production cost. Selection of coupling agent mainly depends on industrial applicability, cost effectiveness, safety and whether it is user friendly. The use of aminium salts are usually the most preferred ones in view of its greater efficiency, faster process and low degree of racemization and comparatively lower costs as compared to the phosphonium salts. It is well known in the art that HBTU reacts with the amino component of the Arg residue leading to the guanidino derivative causing termination of the peptide. Most importantly, this side reaction is not a problem during the coupling of single protected amino acid, because activation is fast and HBTU is rapidly consumed and the present invention claims the advantage of the same by employing a sequential solid phase approach for eicosapeptide assembly on the solid support. The problem is commonly encountered during much slower activation of hindered amino acids or protected peptide segments where HBTU reacts with the amino group. HBTU also has a high convertion rate at room temperature and is soluble in all currently used solvents like NMM and DIPEA and also can be used at high concentrations. HBTU is a conventional aminium salt which is readily available and hence can be easily used for commercial upscaling of the process.HBTU converts the N-protected amino acids into their corresponding OBt esters. A tertiary amine is
required to produce the carboxylate of N-protected amino acids which reacts with
29

HBTU. As the tertiary amine, there can be used triethylamine, triethanolamine, trimethylamine, N-methyl morpholine, diisopropyl ethyl amine. The most preferred are DIEA and N-methyl morpholine.
As 99% of coupling sites are not at the surface but inside the resin beads, swelling of beads carrying the growing peptide chain is essential for the optimal permeation of activated N-protected amino acids within the polymer matrix, thus improving coupling yields. Before starting the solid phase synthesis, the resin has to be swollen in an adequate solvent such as DMF or DCM for 20 to 30 minutes. For coupling steps, polar aprotic solvents such as DMF or NMP are preferred to improve solubility of reactants. DMF inspite of being hygroscopic offers a cheaper alternative and is readily available.
Certain amino acids can cause problems during TFA cleavage and deprotection. Amino acids like Tyr whose protecting groups are easily removed but the deprotected side-chains are especially labile in acid conditions. Amino acids like Arg (Pmc) or Arg(Pbf) need more than two hours for complete removal of the side chain protecting groups, and also side chains once removed are extremely reactive and must be scavenged to prevent reattachment or modification of the deprotected side chains. Although unlikely, the side chain of tyrosine can be alkylated by unscavenged protecting groups released by other side chains, e.g. Pmc group protecting the side chain of Arg. Peptides containing C-terminal tyrosine may undergo reattachment to the resin.
Dehydration of unprotected asparagine can occur during coupling leading to deamidation. Therefore, a protecting group is recommended. Trt group is preferred as
it is readily removed and easily scavenged but N-terminal Asn(Trt) may need extended
30

cleavage times. Arg(Pmc)or Arg(Pbf) protection greatly reduces deprotection times, but it can still take more than 4 hours when multiple Arg(Pmc) are present. Pmc is also difficult to scavenge and has a tendency to reattach or alkylate sensitive residues. The use of Pbf-protection for arginine is therefore strongly recommended, since deprotection times, even with multiple Arg(Pbf) present are 2 hours usual and the Pbf group is easily scavenged.
Peptides synthesized on supports with a Wang linker are cleaved in high percentage of TFA to yield fully deprotected peptides in a single step. Literature cites numerous cleavage cocktails for cleavage of peptide from the solid support.
The present invention finds a solution to all the cited prior art problems by preparing bivalirudin, inexpensively and in commercially feasible amount with an overall yield of 20% to 30% by an improved solid phase process using Fmoc/tBu strategy. The present invention also provides a simple,easy, environment friendly and cost effective process of preparing the Bivalirudin peptide with purity of> 99%.
The preferred embodiments and examples are given to illustrate the scope and spirit of the present invention. These embodiments and examples will make apparent to those skilled in the art other embodiments and examples. These other embodiments are also examples within the contemplation of the present invention. Therefore, the present invention should be limited only by the appended claims. The following examples only represent an illustrative selection of the inventive thought and not a limitation of the subject matter of the invention.
31

EXAMPLES
Example (1): Chemical synthesis of bivalirudin using HBTU/NMM
(Boc)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-Wang resin
Formula (II)
General Procedure:The assembly of the peptide chain is carried out in the following manner.The resin is transferred to the RV of the peptide synthesizer (CS936, CS BIO, Calf., Peptide synthesizer), and the protected linear peptide is assembled on it using 1.5-4.0 times molar excess amino acid derivatives, on the peptide synthesizer. The first starting amino acid, Fmoc-Leu-OH, was loaded on the Wang resin with 0.5-0.8 mmol/gm of loading. This, Fmoc-Leu-Wang resin was then used for all the further couplings. The second amino acid Fmoc-Tyr(tBu)-OH, was coupled to the resin by deprotecting the Fmoc-group on the resin, followed by the activation of the amino acid, Fmoc-Tyr(tBu)-OH with HBTU, NMM. The Fmoc group of the growing peptidyl resin was deprotected using 20% piperidine solution, followed by the activation of the next amino acid by HBTU in the presence of NMM. This cycle was repeated by incorporating the appropriate amino acids at their respective positions, till the entire linear peptide chain was assembled. Each coupling was carried out for 45-90 minutes. After all the couplings were completed, the peptidyl resin was washed with organic solvent/s, which were selected from the range of DMF, TV-Methyl pyrrolidone, DCM, MeOH, preferably DMF, followed by DCM, and then drying under vacuum. The linear peptide of the formula (II) was thus obtained.
The peptide was synthesized as the peptide acid by the solid phase peptide synthesis technology on the Wang resin using Fmoc/t-Bu chemistry.
32

Instrument Resin CS936, CS BIO, Calf. Peptide synthesizer Wang resin
Side chain protecting Groups Arg(Pbf/Pmc), Asn(Trt), Asp(OtBu), Glu(OtBu),Tyr(tBu)
Activator HBTU/NMM
Solvent DMF
Deprotection of Fmoc group 20% Piperidine in DMF
(i) Coupling of the first amino acid, Fmoc-Leu-OH to Wang resin (1 mm/g)
The pre-swollen resin (l0mmol, 1.0 mm/g) was washed twice with DMF. The Fmoc-Leu-OH (20 mmol, 2 equivalents) and DMAP, DIC, were dissolved in NMM and added to the resin. The coupling was carried out at 4°C for 60 minutes. The resin was washed with DMF. The loading obtained by the above procedure was 0.71 mmol/g.
(ii) Coupling of the Fmoc-Tyr(tBu)-OH to Fmoc-Leu-Wang resin ( 0.71 mm/g)
Coupling of Fmoc-Tyr(tBu)-OH, to Fmoc-Leu-Wang-Resin was performed by deprotecting the Fmoc group on the resin with 20% piperidine followed by coupling with Fmoc-Tyr(tBu)-OH in the presence of HBTU, NMM. The synthesis cycle was programmed as follows.The synthesis cycle was programmed as follows:

The couplings of the remaining amino acids were carried out in a similar way, by repeating the above cycle, till the desired sequence length was attained. The assembly of the peptide chain is carried out in the following manner.
The resin (14.0 g of Fmoc Leu-Wang resin, l0mmole) was transferred to the RV of the CS936. The resin was allowed to swell in DMF for about 45 minutes. The Fmoc-Tyr(tBu)-OH, was then coupled to the resin by deprotecting the Fmoc-group on the resin, followed by the activation of the amino acid, Fmoc-Tyr(tBu)-OH by HBTU/NMM. The linear peptide was assembled on it using 1.5 - 4.0 times molar excess of the amino acid derivatives, on the peptide synthesizer. The Fmoc group of the growing peptidyl resin was deprotected using 20% piperidine solution, followed by the activation of the next amino acid by HBTU in the presence of NMM. This cycle was repeated by incorporating the appropriate amino acids at their respective positions, till the entire linear peptide chain was assembled. Each coupling is carried out for 45-90 minutes. After all the couplings were completed, the peptidyl resin was washed with organic solvent/s which were selected from the range of DMF, N-Methyl pyrrolidone, DCM, MeOH, preferably DMF followed by DCM, and then drying under vacuum. The linear peptide of the formula (II) was thus obtained. The peptide was synthesized as the peptide acid by the solid phase peptide synthesis technology on the Wang resin using Fmoc/t-Bu chemistry.

34

EXAMPLE (2): Chemical synthesis of bivalirudin using HBTU/DIPEA
The synthesis of Bivaluridine has also been carried out using HBTU, DIPEA as a combination for the coupling reactions. The details are described below, (i) Coupling of the Fmoc-Tyr(tBu)-OH to the Fmoc-Leu-Wang resin (0.71 mm/g).Coupling of Fmoc-Tyr(tBu)-OH, to Fmoc-Leu-Wang-Resin was performed by deprotecting the Fmoc group on the resin with 20% piperidine followed by coupling with Fmoc-Tyr(tBu)-OH/HBTU/DIPEA. The synthesis cycle was programmed as follows.

Steps Reagent SOLVDEP SOLVACTAA SOLV Time Repeat Activity
1 10 min 5min X3 WASHES RESIN
2 X2 DEP-N-TERMINUS
3 30 sec X6 WASHES RESIN
4 30sec XI DISSOLVES Fmoc-Tyr(tBu)-OH/ HBTU/ DIPEA
5 45min XI | Fmoc-Tyr(tBu)-OH COUPLING
6 30 sec X3 WASHES RESIN
The couplings of the remaining amino acids were carried out in a similar way, by repeating the above cycle, till the desired sequence length was attained. The assembly of the peptide chain is carried out in the following manner.
The resin (14.0 g of Fmoc Leu Wang resin, l0mmole) was transferred to the RV of the CS936. The resin was allowed to swell in DMF for about 45 minutes. The Fmoc-Tyr(tBu)-OH, was then coupled to the resin by deprotecting the Fmoc-group on the resin, followed by the activation of the amino acid, Fmoc-Tyr(tBu)-OH by HBTU/DIPEA. The linear peptide was assembled on it using 1.5 - 4.0 times molar excess of the amino acid derivatives, on the peptide synthesizer. The Fmoc group of the growing peptidyl resin was deprotected using 20% piperidine solution, followed by the activation of the next amino acid by HBTU in the presence of DIPEA. This cycle
35

was repeated by incorporating the appropriate amino acids at their respective positions, till the entire linear peptide chain was assembled. Each coupling is carried out for 45-90 minutes. After all the couplings were completed, the peptidyl resin was washed with organic solvent/s which were selected from the range of DMF, N-Methyl pyrrolidone, DCM, MeOH, preferably DMF followed by DCM, and then drying under vacuum. The linear peptide of the formula (II) was thus obtained. The peptide was synthesized as the peptide acid by the solid phase peptide synthesis technology on the Wang resin using Fmoc/t-Bu chemistry.

Instrument
Resin
Side chain protecting
Groups
Activator
Solvent
Deprotection of
Fmoc group

CS936, CS BIO, Calf. Peptide synthesizer
Fmoc-Leu-Wang resin
Arg(Pmc), Asn(Trt), Asp(OtBu), Glu(OtBu),Tyr(tBu)
HBTU/DIPEA
DMF
20% Piperidine in DMF

Example (3): Chemical synthesis of bivalirudin using DIC/HOBt:
The synthesis of Bivaluridine has also been carried out using DIC, HOBt as a combination for the coupling reactions. The details are described below, (i) Coupling of the Fmoc-Tyr(tBu)-OH to the Fmoc-Leu-Wang resin (0.71 mm/g): Coupling of Fmoc-Tyr(tBu)-OH, to Fmoc-Leu-Wang-Resin was performed by deprotecting the Fmoc group on the resin with 20% piperidine followed by coupling with DIC/HOBt.
36

The synthesis cycle was programmed as follows:

Steps Reagent Time Repeat Activity
1 SOLV 10min X3 WASHES RESIN
2 DEP 5min X2 DEP-N-TERMINUS
3 SOLV 30 sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Fmoc-Tyr(tBu)-OH/ DIC/ HOBt
5 AA 45min XI Fmoc-Tyr(tBu)-OH COUPLING
6 SOLV 30 sec X3 WASHES RESIN
The couplings of the remaining amino acids were carried out in a similar way, by repeating the above cycle, till the desired sequence length was attained.
The assembly of the peptide chain is carried out in the following manner. The resin (14.0 g of Fmoc Leu Wang resin, l0mmole) was transferred to the RV of the CS936. The resin was allowed to swell in DMF for about 45 minutes. The Fmoc-Tyr(tBu)-OH, was then coupled to the resin by deprotecting the Fmoc-group on the resin, followed by the activation of the amino acid, Fmoc-Try(tBu)-OH by DIC/HOBt. The linear peptide was assembled on it using 1.5 - 4.0 times molar excess of the amino acid derivatives, on the peptide synthesizer. The Fmoc group of the growing peptidyl resin was deprotected using 20% piperidine solution, followed by the activation of the next amino acid by 0.5M DIC in the presence of HOBt. This cycle was repeated by incorporating the appropriate amino acids at their respective positions, till the entire linear peptide chain was assembled. Each coupling is carried out for 45-90 minutes. After all the couplings were completed, the peptidyl resin was washed with organic solvent/s which were selected from the range of DMF, N-Methyl pyrrolidone, DCM, MeOH, preferably DMF followed by DCM, and then drying under vacuum. The linear peptide of the formula (II) was thus obtained.
The peptide was synthesized as the peptide acid by the solid phase peptide synthesis technology on the Wang resin using Fmoc/t-Bu chemistry.
37

Instrument CS936, CS BIO, Calf. Peptide synthesizer
ResinSide chain protecting Groups Fmoc-Leu-Wang resinArg(Pmc/Pdf), Asn(Trt), Asp(OtBu), Glu(OtBu), Tyr(tBu)
Activator DIC/HOBt
Solvent DMF
Deprotection of Fmoc group 20% Piperidine in DMF
Example (4) : Cleavage of the peptide from the resin to give the peptide, D-Phe-Pro-
Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-
COOH
To the dried peptidyl-resin was added 660ml of cleavage mixture (TFA : 95%; H2O: 2.5%;TIS: 2.5%) which was cooled to 4°C, prior to addition. The cleavage mixture was stirred for 2.5 hours at ambient temperature with the resin, to cleave the side chain protecting groups from the peptide and the peptide from the resin. The reaction mixture was filtered under vacuum and washed with TFA (3 X 90ml). The filtrate was pooled, and the TFA was evaporated under vacuum to ~80ml. The peptide was then precipitated with cold dry DIPE (800ml) and was let to stand at -20°C for 4 hrs. The peptide was isolated by filtering through sintered funnel and washing the precipitate with cold, dry DIPE (100ml x3). The crude peptide purity was analysed by HPLC (Fig.l) and the mass spectra was analysed by ESI-MS (Fig.2).The crude peptide was dried under vacuum. The weight and the purity by RP-HPLC of the crude peptide:
Example 1 ( HBTU/NMM) Yield = 95% purity = 67%
Example 2 (HBTU/DIPEA) Yield = 92.6% purity = 50%
Example 3 (DIC/HOBt) Yield = 93% purity = 30%
38

Example (5): Purification of the crude peptide:
The crude peptide was dissolved with 30% acetic acid while maintaining a concentration of the peptide to about 125mg/ml. The sample was then loaded on to a CI8 (250 x 50 mm, l0m) column. The peptide was eluted using 0.1% TFA as the aqueous medium and acetonitrile as the organic solvent with the formation of a gradient. The fractions were then analyzed on an analytical RP-HPLC, which displayed purity of the purified peptide as about 90%, with the crude isolated yield of about 40%. The amount of crude peptide in solution: 3.3 g.
Example (6): Purification of the peptide
The peptide was loaded on to the CI8 (250 x 50 mm, 10m) column and purified using an isocratic run. Phosphoric acid buffer was used as the aqueous phase and acetonitrile as the organic solvent. The fractions were analyzed by analytical RP-HPLC, which displayed purity greater than 99%, with no single impurity >0.5% and with crude yield of about 60- 70%.(amount of peptide 5.6 g) (Fig.3).Fig.4 shows the MALDI-TOF analysis of the pure peptide.
Example(7): Buffer exchange of the peptide
The peptide to be converted to the trifluroacetate salt was loaded onto a RP-HPLC column, which was pre equilibrated with aqueous TFA. The peptide was eluted using a acetonitrile, isocratic run. The fractions collected were pooled and were analysed by RP-HPLC, which displayed the purity >99%, with the isolated yield of 80%. These fractions were pooled and were concentrated. The concentrated fractions were then dried on a lyophilizer.
Example 8 Concentration and desalting using Reverse osmosis:
39

The peptide was concentrated and desalted using Reverse Osmosis system with cartridges from millipore( Nanomax 50) or spiral wound membrane from Novasep/Nishotech. The peptide solution was circulated for -10 min to remove any air bubble from the RO system. Slowly the pressure was increased to 6.8 to 7 bar by using pressure regulator valve and the mixture was concentrated to half volume. Given diafiltration with water 1 x 103 ml each time to concentrate, the pressure was slowly released by using pressure regulator valve (i.e. By opening the retentate valve). The circulation pump was then stopped. The retentate was removed in a cleaned retentate reservoir through drain valve of feed tank. After complete removal of retentate the drain valve of feed tank was closed. The feed tank was charged with 1000 ml water and the circulation pump was started, by circulating the water for 5 minutes. The circulation pump was then stopped. The retentate was drained through drain valve of feed tank and pooled with the main sample. After complete removal of retentate the drain valve of feed tank was closed. The peptide recovery was > 98%.
Example 9:Impurity Identification and synthesis :
Bivalimdin API (active pharmaceutical ingredient) of the present invention was
subjected to controlled forced degradation with 0.01N NaOH. Deamidated impurity at
relative retention time (RRT) 1.03 was observed (Fig.6).
Impurity (Bivalimdin Asp9) was synthesized using the same process as that in example
1, except the Fmoc- asparagine (Trt)-OH was replaced by Fmoc-Aspartic acid (OtBu)- •
OH at the 9th position from N-terminal of formula II. The peptide was cleaved and
analyzed separately by HPLC (Fig.5).
Retention time for this Bivalimdin Asp9 and Deamidated impurity was found to be
exactly same. Synthesized Impurity(Bivalirudin Asp-9) was spiked with forced
degradation sample and area of peak at RRT 1.03 was found to be increased (Fig.7).
The stability study of the pure, lyophilised eicosapeptide of formula I was carried out
40

and the said eicosapeptide was stable. Fig.9 shows the stability of the eicosapeptide of formula I carried at -20°C at 12 months period.
Example 10: Thrombin Inhibition Bioassay Using Bivalirudin
The Bivalirudin sample was diluted in the range of 0.9 ng to 1000 ng in Tris buffer (50 mM Tris,pH 8.3, 227 mM NaCl) and added to the 96-well plate. Thrombin substrate was used as positive control, and Tris buffer (50 mM Tris,pH 8.3, 227 mM NaCl) as blank . 25ml substrate Chromozyme TH solution (5 mg/4ml) was added in all wells including blank and positive control wells. 25 ul of Thrombin solution (0.66 NIH Units) was then added in all wells except blank wells.The plate was incubated for 10-15 minutes for colour to develop. The absorbance was read using a Spectrophotometer at 405 nm. The bioassay is based on the following principle

Bivalirudin inhibits thrombin mediated release of 4-nitrilaline from Chromozyme TH peptide. The rate of increase of 4-nitraline gives thrombin activity in U/ml at 405 nm.Percent inhibition of Thrombin activity at different concentration of Bivalirudin was calculated using positive control as 100 %. A graph of linear log concentration of Bivalirudin vs. % inhibition of Thrombin activity was plotted (Figure 8). ED50 of Bivaluridin ranged from 0.012 to 0.031 ug for all samples.
Example 11 inhibition of Thrombin Induced Platelet Aggregation by Bivalirudin using Laser Aggregometer (Chronolog )
20-25 ml peripheral blood was collected from five consented volunteers in 50 ml sterile centrifuge tubes containing 3.8 % sodium citrate. 2/3 volume of blood was transferred to fresh sterile 15 ml centrifuge tubes and spun at 600 rpm for 10 minutes at 10°C to obtain Platelet Rich Plasma (PRP). The remaining blood was centrifuged at
41

4000 rpm for 30 minutes at 10°C to obtain Platelet Poor Plasma (PPP). The Platelet Count was adjusted to 140 x 106/ 450ul using PPP. 140 x 106 PRP was taken in cuvette and measured baseline value Aaggregating concentration of Thrombin was added to the cuvette and the aggregation checked. This aggregation was used as 100 % for calculation of percent inhibition. For checking the inhibition, 140 x 106PRP was taken in cuvette and inhibitory concentration of Bivalirudin, was added and incubated at 37°C for 15 minutes at room temparature.. After the incubation with Bivalirudin, aggregating concentration of Thrombin was added to the same cuvette and the aggregation measured.
The percent inhibition by Bivalirudin,was calculated and mean percent inhibition was calculated.
Table 1: Bivalirudin Platelet aggregation / inhibition Bioassay

ND- Not done
Inhibition of thrombin induced aggregation by Innovator's and USV's sample was
comparable.
* High SD is a feature of biological variation whch is generally seen in platelet
aggregation assay.
42

We Claim,
1) An improved process for synthesis of linear eicosapeptide as set forth in Formula l by an orthogonal Fmoc/tBu strategy comprising of:
D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH
Formula I
i. covalently linking a Fmoc-Leu-OH to a p-alkoxybenzyl alcohol
(Wang's resin) solid support through benzyl ester linkage,
ii. removing the N-a -NH2 protecting group from Fmoc-Leu-Wang resin to
obtain an N-a -NH2 group,
iii. coupling the second Fmoc-Tyr(tBu) to the Leu-Wang resin by
activating the amino acid by HBTU/NMM in the presence of polar
aprotic solvent,
iv. deprotecting the Fmoc group by deprotectant,
v. repeating steps ii),iii),iv) for assembling the eicosapeptide (Boc)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-
Wang resin,
vi. cleaving and deprotecting said eicosapeptide of step v) using cleavage
cocktail characterized wherein said cleavage cocktail consists of
aqueous TFA and a non thiol scavenger,
vii. obtaining cleavage yield of at least 90% and crude peptide purity of at
least 65%,
viii. isolating the crude eicosapeptide as set forth in Formula I by
precipitation, filtration and drying under vacuum,
43

ix. purifying the eicosapeptide of step viii) by chromatography wherein
said eicosapeptide has a purity of ³ 99%,
x. concentrating the purified eicosapeptide of step ix) by reverse osmosis, xi. lyophilizing the concentrated purified eicosapeptide of step x.
2) The process as claimed in claim 1, wherein the polar aprotic solvent is selected from a group consisting of DMF, Methanol, THF, DCM, preferably DMF.
3) The process as claimed in claim 1, wherein the N-a NH2 protected amino acids are added in 1.5 to 5 molar excess for synthesis of said eicosapeptide.
4) The process as claimed in claim 1, wherein the deprotectant is piperidine, preferably 20% piperidine in DMF.
5) The process as claimed in claim 1, wherein said cleavage cocktail consists of TFA in the range of 80% to 98%, water in the range of 0% to 5% and TIS in the range of 0% to 5%.
6) The process as claimed in claim 1, wherein the purification of the crude eicosapeptide to a purity of ³ 99% carried by chromatography is by RP-HPLC by isocratic and/or gradient mode.
7) The process as claimed in claim 6, wherein the eluant for RP-HPLC purification by gradient mode comprises of TFA as an aqueous phase and acetonitrile as an organic phase with isolated yield of 40% and purity of atleast 90%.
8) The process as claimed in claim 6, wherein the eluant for RP-HPLC purification by isocratic mode comprises of phosphoric acid buffer as an
44

aqueous phase and acetonitrile as an organic phase with isolated yield of 60% to 70% and purity of atleast 99%.
9) The process as claimed in claim 1, wherein the recovery by reverse osmosis is
atleast 90%, preferably 98%.
10) The process as claimed in claim 1, wherein the total impurity of the pure
eicosapeptide of formula 1 is not more than 1% and the single largest impurity
is not more than 0.5% .
11 )The process as claimed in claim 12, wherein the single largest impurity is Asp9 not more than 0.3%.
12) An eicosapeptide of formula I obtained by the process as claimed in claim 1.
13) An eicosapeptide of formula I as claimed in claim 12, which shows a mass of 2180 Da.
14) An eicosapeptide of formula I as claimed in claim 12, with an ED50 in the range of 0.012 to 0.031 meg.
15) An eicosapeptide of formula I as claimed in claim 12, wherein said eicosapeptide elicits anti-platelet aggregation effect.
16) A pharmaceutical composition comprising eicosapeptide of formula I as claimed in claim 12 and atleast one pharmaceutically acceptable excipient.
45

ABSTRACT:
The present invention relates to an improved, simple, environmentally benign and cost effective process for the synthesis of linear eicosapeptide having an amino acid sequence of D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH as set forth in Formula I. The present invention specifically relates to an improved process for synthesis of linear eicosapeptide as set forth in Formula I by an orthogonal Fmoc/tBu strategy, said process comprising the steps of:
D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-
Pro-Glu-Glu-Tyr-Leu-COOH
Formula I
i. covalently linking a Fmoc-Leu-OH to a p-alkoxybenzyl alcohol (Wang's
resin)solid support through benzyl ester linkage,
ii. removing the N-a -NH2 protecting group from Fmoc-Leu-wang resin to obtain
an N-a -NH2 group,
iii. coupling the second Fmoc-Tyr(tBu) to the Leu-Wang resin by activating the
amino acid by HBTU/NMM in the presence of polar aprotic solvent, iv. deprotecting the Fmoc group by deprotectant,
v. repeating steps ii),iii),iv) for assembling the eicosapeptide (Boc)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-Wang resin,
vi. cleaving and deprotecting said eicosapeptide of step v) using cleavage cocktail characterized wherein said cleavage cocktail consists of aqueous TFA and a non thiol scavenger, vii. obtaining cleavage yield of at least 90% and crude peptide purity of at least
65%,
viii. isolating the crude eicosapeptide as set forth in Formula I by precipitation,
filtration and drying under vacuum,
ix. purifying the eicosapeptide of step viii) by chromatography wherein said

eicosapeptide has a purity of > 99%,
x. concentrating the purified eicosapeptide of step ix) by reverse osmosis, xi. lyophilizing the concentrated purified eicosapeptide of step x.