Title of Invention

A PROCESS FOR PRODUCTION OF THE SOMATOSTATIN ANALOG, OCTREOTIDE

Abstract

The present invention relates to a process for commercial production of octreotide using solution peptide chemistry and inexpensive amino acid derivatives. Thus the hexapeptide (Boc) D-Phe-Cys(Acm)-Phe-D-Trp-Lys(Boc)-Thr-OMe is synthesised by condensation of two tripeptide fragments, saponified and condensed with Cys(Acm)-Thr-OL to give the linear octapeptide alcohol. The linear peptide alcohol is trated with iodine, after removal of Boc groups, to give the cyclic peptide octreotide. The linear octapeptide alcohol can alternately be made by condensation of the protected hexapeptide acid with the dipeptide Cys(Acm)-Thr-OMe, followed by reduction with borohydride.

Full Text

FORM 2 THE PATENT ACT 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See Section 10 and rule13) 1. TITLE OF THE INVENTION: A PROCESS FOR PRODUCTION OF THE SOMATOSTATIN ANALOG, OCTREOTIDE 2. APPLICANT (S) (a) NAME: Wockhardt Limited (b) NATIONALITY: Indian (c) ADDRESS: Wockhardt Towers, Bandra-Kurla Complex, Bandra (East), Mumbai-400051, India 3. PREAMBLE TO THE DESCRIPTION This invention discloses process for the preparation of Somatostatin Analog Octreotide by solution peptide chemistry and use of inexpensive amino acid derivative. The following specification particularly describes the invention and the manner in which it is to be performed. 31 MAY 2005 A PROCESS FOR PRODUCTION OF THE SOMATOSTATIN ANALOG, OCTREOTIDE Field Of The Invention The present invention relates to a process for the commercial production of the somatostatin analog, octreotide (I) and its pharmaceutically acceptable salts, using solution peptide chemistry, in high yield and purity. I The present invention also relates to the intermediate compounds useful in the synthesis of octreotide. Background Of The Invention Octreotide is a highly potent and pharmacologically selective analog of somatostatin. It inhibits growth hormone for long duration and is therefore indicated for acromegaly to control and reduce the plasma level of growth hormone. The presence of D-Phe at the N-terminal and an amino alcohol at the C -terminal, along with D-tryptophan and a cyclic structure makes it very resistant to metabolic degradation. The only solution synthesis reported in literature is by Bauer,W. and Pless, J. in Pat. No. U.S. 4,395,403 and EP029579. Several solid phase syntheses have been subsequently described viz. Patent Nos. EP0953577A1 and U.S. 5,889,146 and in various research publications. Mergler et al (Proceedings of the 12th American Peptide Symposium) have used 2 aminomethyl resin and Fmoc-butyl protection scheme for synthesis of octreotide. Alsina et al. Tetrahedron Letters, 38, 883, 1997) have used an active carbonate resin and Boc-Bzl protection scheme, necessitating the use of hydrogen fluoride/anisole for final deprotection. Edwards et al (J. Med. Chem., 37, 3749, 1994)) have described another synthesis using Fmoc-butyl protection and HMP resin, and Berta et al (EP 0 953 577A1) a synthesis using 2-chlorotrityl- type resin and Fmoc-butyl protection scheme. All the solid phase syntheses described are useful only for the manufacture of small quantities of octreotide (100-300 mg). These procedures are not suitable for commercial manufacture of octreotide because they use costly resins and costly Fmoc-butyl protected amino acids in 2 to 4 times excess at every step. In one synthesis the final deprotection is carried out with hydrogen fluoride, a destructive and hazardous reagent. The solution synthesis described by Bauer and Pless in Patent No. U.S. 4,395,403 and EP029579 uses BTFA/TFA to remove the methoxybenzyl group protecting the thiol group of cysteine, followed by cyclization. Decomposition of tryptophan is frequently known to occur during such harsh acid treatment for removal of protecting groups. Summary Of The Invention This invention describes a process for obtaining octreotide scalable upwards to kilogram quantities by solution chemistry methods using mild reagents and giving good yields. The process includes the following: 1) Cysteine thiol groups are protected by acetamidomethyl (Acm) groups. Treatment of the Cys(Acm)- containing linear novel octapeptide (XVI) of the 3 invention with iodine, in one step removes the Acm groups and simultaneously effects cyclization to give octreotide (I) in 80-90 per cent yield. H-(D)-Phe1-Cys(Acm)2-Phe3-(D)-Trp4-Lys5-Thr6-Cys(Acm)7-Thr8-OL.2TFA XVI H-(D)-Phe1-Cys2-Phe3-(D)-Trp4-Lys-5-Thr6-Crs7-Thr8-OL I I I 2) The suitably protected octapeptide alcohol (XVI) of the invention is prepared by either of the following processes. a) Sodium borohydride reduction of the novel C-terminal dipeptide methyl ester (X) to obtain the dipeptide alcohol (XI) (step 10). The Boc group is removed and the dipeptide (XII) on condensation with the hexapeptide acid (XIV), followed by deprotection, gives the novel intermediate (XVI) (Method 1-Steps 14 and 15) b) Sodium borohydride reduction of the octapeptide XIX with methyl ester at C-terminal to give the novel intermediate XVI (Method 2-Step 19) The novel hexapeptide fragment XIV is prepared by condensation of two appropriately protected novel tripeptide fragments V and IX followed by saponification (Steps 12 and 13). 4 Detailed Description Of The Invention The process for the synthesis of H-(D)-Phe-Cys-Phe-(D)-Trp-Lys-Thr-Cys-Thr-OL. of formula 1 comprises the synthesis of appropriate peptide fragments using the standard processes of peptide chemistry, known to the practitioners in the art. Thus amino functions of amino acids are protected with one of the commonly employed protecting groups like t-butyloxycarbonyl or benzyloxycarbonyl, and the carboxyl functions of amino acids are protected with alkyl groups like methyl, ethyl, or aralkyl groups like benzyl. The condensation of the carboxyl group of the amino protected amino acid with the amino group of carboxyl protected amino acid is typically carried out by dissolving the respective appropriately protected amino acids in approximately equimolar quantities in a nonpolar solvent preferably like tetrahydrofuran, dichloromethane, chloroform, and adding a condensing agent such as N,N-dicyclocarbodiimide (DCCI), 1-hydroxy-benzotriazole (HOBt) or Benzotriazole-1-yl-oxy-tris-(dimethylamino) -phosphonium hexafluorophosphate ( BOP ) in approximately equimolar quantity at a temperature of -10°C to + 10°C and stirring at 20°C -30°C for 4 to 24 hrs. Alternately the carboxyl function of the amino protected aminoacid is activated as mixed anhydride by addition of an alkylchloro-formates wherein alkyl means methyl, ethyl, propyl, etc, preferably isobutylchloroformate, a tertiary amine such as TEA, DIEA, NMM, preferably NMM, in approximately equimolar quantities in a nonpolar solvent like dichloromethane, THF, chloroform, preferably THF at a temperature of-10°C to + 10°C following by addition of carboxyl protected amino 5 acid carrying a free amino group and stirring at 20°C - 30°C for 4 to 24 hrs. The amino or the carboxyl protecting group is then selectively removed by the use of appropriate deprotecting agents known to those skilled in the art and condensed as desired with another amino acid derivative in an iterative procedure until the desired sequence is obtained. The basic difference from other procedures already described is that a) the cysteine thiol groups are protected by acetamidomethyl (Acm) groups, b) the N-terminal hexapeptide has been synthesised by condensation of two tripeptide fragments, c) the C-terminal dipeptide alcohol is generated by sodium borohydride reduction of dipeptide methyl ester X instead of using threoninol as the starting material, d) treatment of the linear octapeptide alcohol XVI of the invention with iodine, in one step, removes the Acm groups and simultaneously effects cyclization to give octreotide in good yield, and e) the key intermediate octapeptide alcohol XVI could also be prepared by sodium borohydride reduction of octapeptide methyl ester XIX. The Process for Producing OCTREOTIDE H-(D)-Phe1-Cys2-Phe3-(D)-Trp4-Lys-5-Thr6-Crs7-Thr8-OL I Step-1 Boc-Cys(Acm)-OH+H-Phe-OMe.HCl ►Boc-Cys(Acm)-Phe-OMe II Step-2 Boc-Cys(Acm)-Phe-OMe ► H-Cys(Acm)-Phe-OMe . TFA III 6 Step-3 Boc-D-Phe-OH + H-Cys(Acm)-Phe-OMe .TFA ► Boc-D-Phe-Cys(Acm)-Phe-OMe IV Step-4 Boc-D-Phe-Cys(Acm)-Phe-OMe ► Boc-D-Phe-Cys(Acm)-Phe-OH V Step-5 Z-Lys(Boc)-OH + H-Thr-OMe.HCl ► Z-Lys(Boc)-Thr-OMe VI Step-6 Z-Lys(Boc)-Thr-OMe ►H-Lys(Boc)-Thr-OMe. AcOH VII Step-7 H-Lys(Boc)-Thr-OMe.AcOH Z-D-Trp-OH + ► Z-D-Trp-Lys(Boc)-Thr-OMe VIII Step-8 Z-D-Trp-Lys(Boc)-Thr-OMe ► H-D-Trp-Lys(Boc)-Thr-OMe IX Step-9 Boc-Cys(Acm) + H-Thr-OMe.HCl ► Boc-Cys(Acm)-Thr-OMe X Step-10 Boc-Cys(Acm)-Thr-OMe ► Boc-Cys(Acm)-Thr-OL XI Step-11 Boc-Cys(Acm)-Thr-OL ► H-Cys(Acm)-Thr-OL. TFA XII 7 8 Method 2 Step-16 Step-17 .TFA Step-18 Step-19 Preparation Of Octreotide From Novel Linear Octapeptide Alcohol Di Trifluoroacetate XVI Step-20 9 Abbreviations: Acm = Acetamidomethyl Boc = tert. -Butyloxycarbonyl Bzl = Benzyl BTFA= Boron-tris-trifluoroacetate tBu = tert-Butyl DCCI = Dicyclohexylcarbodiimide DCM = Dichloromethane DIEA = Diisopropylethylamine DMAc = Dimethylacetamide DMSO = Dimethylsulfoxide ESMS = Electrospray Mass Spectrometry EtOH = Ethanol Fmoc = Flourenylmethoxycarbonyl HF = Hydrogen fluoride HOBt = 1-Hydroxybenzotriazole IBCF = Isobutylchloroformate NMM = N-methylmorpholine TEA = Triethylamine TFA = Trifluoroacetic acid THF = Tetrahydrofuran Trt = Triphenyl methyl (Trityl) Z = Benzyloxycarbonyl The following preferred embodiment is described. As shown in step 9, cysteine carrying Boc group for Na and Acm group for side chain SH protection may be treated with IBCF, NMM, H-Thr-OMe.HCl and TEA, all in approximately equimolar amounts in THF at - 10°C to give dipeptide methyl ester X. The C-terminal methyl ester is converted into alcohol function by reduction with sodium 10 borohydride (2 equivalents) in 90% EtOH at 0°C to give dipeptide alcohol XI (step 10). The Boc group is removed by treatment with TFA at 0°C and the resultant compound XII is condensed with appropriately protected hexapeptide XIV, using DCC/HOBt as the condensing agents, in approximately equimolar amounts in THF/DMAc at 0°C, to give the protected octapeptide alcohol XV (step 14). Boc groups are removed by treatment with TFA at 0°C to give the novel octapeptide alcohol XVI (step 15) which is cyclised with iodine (5-10 equivalents) in 90% MeOH to give octreotide I (Step 20). Alternately as shown in step 17 (method 2) Boc protection is removed from dipeptide methyl ester X by treatment with TFA at 0°C and the resulting t dipeptide ester XVII is condensed with the hexapeptide XIV, using DCC/HOBt as the condensation agents in approximately equimolar amounts in THF at 0°C ,to give the protected octapeptide methyl ester XVIII. Boc groups are removed by treatment with TFA at 0°C and the octapeptide methyl ester XIX on reduction with sodium borohydride (5 to 6 equivalents), in 90% EtOH gives the novel octapeptide alcohol XVI (see steps 18 and 19) which is cyclized with iodine (5 to 10 equivalents) to octreotide I (Step 20). The hexapeptide acid XIV of the invention is synthesised by condensation of two appropriately protected tripeptide fragments V and IX followed by saponification as shown in steps 12 and 13. For synthesis of tripeptide fragment V, cysteine carrying Boc group for Nα and Acm group for side chain SH protection is treated with IBCF, NMM, H-Phe-OMe.HCl and TEA, all in approximately equimolar amounts in THF/DMSO at -10°C to give dipeptide II (step 1). The Boc group is removed from the protected dipeptide methyl ester II by treatment with TFA at 0°C to give III (step 2) which on condensation with (D)-phenyl-alanine carrying Boc group as Nα protection and 11 using IBCF and NMM to make the mixed anhydride, in approximately equimolar amounts in THF at -10° gives protected tripeptide methyl ester IV (step 3). The saponification of IV gives the protected tripeptide acid V (step 4). Similarly for synthesis of tripeptide fragment IX, lysine carrying Z and Boc group at Na and Ns respectively as protecting groups is treated with IBCF, NMM, H-Thr-OMe.HCl and TEA, all in approximately equimolar amounts in THF/DMSO at -10° to give dipeptide VI (step 5). The Z group is removed from dipeptide VI by hydrogenation over Pd/C to give VII (step 6) which on condensation with (D)-tryptophan carrying Z group as Nα protection and using IBCF and NMM in approximately equimolar amounts in THF/DMSO to make the mixed anhydride, at -10°C gives protected tripeptide methyl ester VIII (step 7). Removal of Z group from VIII by hydrogenation over Pd/C provides the fragment IX (step 8). The hexapeptide methyl ester XIII is then made by condensing the tripeptide V carrying the free carboxyl function and the tripeptide IX, carrying the free amino function and using DCCI and HOBt as the condensation reagents in approximately equimolar amounts in THF/DMA at 0° (step 12). The methyl ester group of XIII is removed by saponification to give the hexapeptide acid XIV (step 13). Examples H-(D)-Phe1-Cys2-Phe3-(D)-Trp4-Lys5-Thr6-Cys7-Thr8-OL. I I I A solution of linear octapeptide XVI H-(D)-Phe1-Cys(Acm)2-Phe3-(D)-Trp4--Lys5-Thr6-Cys(Acm)7-Thr8-OL.2TFA(5.6g, 4mmol) in 90% methanol (200ml ) is added dropwise to a solution of 90% methanol (1 lit) containing iodine (5.0g, 20mmol) over a period of an hour at 30°. The stirring is continued at 30° till the completion of reaction. The reaction mixture is cooled to 5°C, 20ml 1M sodium bisulphite is 12 added followed by the addition of 40ml 1M sodium hydroxide and 2ml acetic acid. The solvent is evaporated under vacuum and the crude material is purified by column chromatography to give compound I. Yield 3.7g (84 %), [a]D20 = -41.8° (c=l in 95% acetic acid, Merck Index -42°), ESMS= 1019 (M+H). The novel starting materials which also form an embodiment of this invention may be obtained as follows: Boc-Cys(Acm)-Phe-OMe (II) IBCF (13.0ml, lOOmmol) is added to a solution of Boc-Cys(Acm)-OH (29.2, lOOmmol) and NMM (11.0ml, lOOmmol) in THF (300ml) at -10°C. To the reaction mixture is added slowly, after 5 mins, a cold solution of of H-Phe-OMe.HCl(21.5g, lOOmmol) and TEA (14.0ml, lOOmmol) in DMSO (10 ml) and THF (50ml). The stirring is continued at same temperature for one hour and then overnight at 30°C. The solvent is evaporated under vacuum at 40°C and diluted with ethyl acetate (300ml). The organic layer is washed with saturated sodium bicarbonate solution (3 x 100ml), 0.5M ice cold hydrochloric acid (3 x 100ml), brine (3x100ml), dried on sodium sulphate and evaporated to dryness to give compound II. Yield 43.0g (94%), m. p. = 99°C, [a]D20 = -21.7° (c=l in methanol), ESMS= 454.1 (M+H), 476.13 (M+Na). H-Cys(Acm)-Phe-OMe. TFA (III) The dipeptide II (40.8g, 90mmol) is suspended in DCM (40ml) and stirred at 0°C. TFA (160ml) is added and stirring continued for one hour. The TFA and DCM are evaporated under vacuum, ether (200ml) is added to the residue under stirring, the precipitate filtered, washed with diethyl ether and dried in vacuum to give compound III in 98% yield. ESMS= 354.1 (M+H). Boc-(D)-Phe-Cys(Acm)-Phe-OMe(IV) IBCF (11.8ml, 90mmol) is added to a solution of Boc-D-Phe-OH (24.0g, 90mmol) and NMM (9.9ml, 90mmol) in THF (100ml) at -10°C. To the reaction mixture is added ,after 5 mins, a cold solution of compound III ( 80mm, 38g ) and TEA (12.7ml, 80mmol) in DMSO (25ml) and THF (50ml). The stirring is continued for 13 one hour at same temperature and then overnight at 30°C. The reaction mixture is concentrated in vacuum, and the residue is dissolved in ethyl acetate (300ml). The organic layer is washed with saturated sodium bicarbonate solution ( 3 x 100ml), 0.5M ice cold hydrochloric acid (3 x 100ml), brine ( 3 x 100ml), dried on sodium sulphate and evaporated to dryness to give compound IV. Yield 48.8g (90%), m. p. = 134-135°C, [α]D20=-31.6° (c =0.5 in methanol), ESMS= 601 (M+H). Boc-(D)-Phe-Cys(Acm)-Phe-OH (V) 1M sodium hydroxide solution (80.0ml) is added in 15 minutes to the cold solution of tripeptide methyl ester IV (48.0g, 80mmol) dissolved in methanol (300ml). The solution is stirred at 30° till the completion of reaction. pH is brought to 7 by addition of IN hydrochloric acid, the solution is concentrated in vacuum, and acidified further with 1M hydrochloric acid (80ml) under cooling to pH 3, and ethyl acetate (300ml) is added. The organic layer is separated and water layer is re-extracted with ethyl acetate (200ml). The organic layers are combined, washed with brine (2 x 100ml), dried on sodium sulphate and evaporated to give compound V. Yield 43.5g (93%), m.p. = 141°- 145°C, [α]D20 = -20.8° (c =1 in methanol), ESMS= 587 (M+H). Z-Lys(Boc)-Thr-OMe (VI) IBCF (26.0ml, 200mmol) is added to a solution of Z-Lys(Boc)-OH (76g, 200mmol) and NMM (22.0ml, 200mmol) in THF (400ml) at -10°C. To the reaction mixture is added, after 5 minutes, a cold solution of H-Thr-OMe.HCl (40.6g, 240mmol) and TEA (34.0ml, 240mmol) in DMSO (50 ml) and THF(100ml). The stirring is continued at same temperature for an hour and then overnight at 30°C. The solvent is evaporated under vacuum at 40°C and diluted with ethyl acetate(700ml). The organic layer is washed with saturated sodium bicarbonate solution( 3 x 100ml), 0.5M ice cold hydrochloric acid (3 x 100ml, brine (3 x 100ml), dried on sodium sulphate and evaporated to dryness to give compound VI. Yield 83.Og (84%), m. p. = 77-78°C [α]D20= -10.2° (c=l in DMF), ESMS= 496 (M+H). 14 H-Lys(Boc)-Thr-OMe. AcOH (VII) The dipeptide VI (80.0g, 160mmol) is dissolved in methanol ( acid (12.0ml) and hydrogen gas bubbled in the presence of Pal (8.0g, 10%). When the hydrogenation is complete, the solution celite bed, evaporated to dryness to yield compound VII in 96" 362.1 (M+H). Z-(D)-Trp-Lys(Boc)-Thr-OMe (VIII) IBCF (20.8ml, 160mmol) is added to a solution of Z-(D 160mmol) and NMM (17.6ml, 160mmol) in THF (400ml) a reaction mixture is added, after 5 minutes, a cold solution of cc 160 mm) and TEA (22.4ml, 160mmol) in DMSO (50ml) and " stirring is continued for an hour at same temperature and then The reaction mixture is concentrated in vacuum and the resid ethyl acetate (700ml). The organic layer is washed with bicarbonate solution (3 x 100ml), 0.5M ice cold hydrochloric brine (3 x 100ml), dried on sodium sulphate and evaporated compound VIII. Yield 78g (70%), m. p. = 112-114°C, [a]D20 = ; ESMS= 682 (M+H). H-(D)-Trp-Lys(Boc)-Thr-OMe (IX) The tripeptide VIII (50.0g, 73mmol) is dissolved in meth hydrogen gas bubbled through in the presence of Palladium . 10%). After the hydrogenation is complete, the solution is filte bed, evaporated to dryness to yield compound IX in 98% y (M+H), 570 (M+Na). Boc-Cys(Acm)-Thr-OMe (X) IBCF (39.0ml, 300mmol) is added to a solution of Boc-Cy; 300mmol) and NMM (33.0ml, lOOmmol) in THF(400ml) reaction mixture is added, after 5 minutes, a cold solution of (59.3g, 350mmol) and (TEA 49.4ml, 350mmol) in DMSO (3 15 (150ml). The stirring is continued at same temperature for an hour and then overnight at 30°C. The solvent is evaporated under vacuum at 40°C and diluted with ethyl acetate (700ml). The organic layer is washed with saturated sodium bicarbonate solution (3 x 100ml), 0.5M ice cold hydrochloric acid (3 x 100ml), brine (3 x 100ml), dried on sodium sulphate and evaporated to dryness to give compound X . Yield 110.Og (90%), ESMS= 408 (M+H). Boc-Cys(Acm)-Thr-OL (XI) NaBH4 (7.4g, 200mmol) dissolved in 90% ethanol (50ml) is added dropwise to a solution of compound X (40.7g, lOOmmol) in 90% ethanol (150ml) at 0°C. The stirring is continued for 3-4 hours till the reaction is complete as monitored by tic. The solution is concentrated and desalted by HPLC to give compound XI in 95% yield . ESMS= 380 (M+H), 402(M+Na). H-Cys(Acm)-Thr-OL. TFA (XII) The dipeptide XI (7g, 18mmol)is dissolved in DCM (10ml) at 0°C under stirring. (TFA 30ml) is added and continue the stirring for an hour. The TFA and DCM are evaporated under vacuum, diethyl ether (100ml) is added to the residue under stirring, ether is decanted, repeated twice and dried in vacuum to give compound XII in 96% yield . ESMS= 280 (M+H), 302 (M+Na). Boc-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys(Boc)-Thr-OMe(XIII) The tripeptide acid V (38.0g, 65mmol) and HOBt (9.9g, 65mmol) is added to a solution of tripeptide IX (35.6g, 65mmol) in THF (200ml) and DMA (30ml). The solution is cooled to 0°C and DCCI (13.4g, 65mmol) is added. The reaction mixture is stirred at same temperature for 1-2 hours followed by overnight stirring at 30°C. Dicyclohexylurea is filtered, the filtrate is concentrated in vacuum and diluted with (500ml) ethyl acetate. The organic layer is washed with saturated sodium bicarbonate solution (3 x 100ml), 0.5M ice cold hydrochloric acid (3 x 100ml), brine (3 x 100ml), dried on sodium sulphate and evaporated to dryness to give compound XIII: Yield 55.2g (76.3%), m. p. = 141-143°C , [α]D20 = -29.8° (c=l in in methanol), ESMS= 1116 (M+H), 1138(M+Na). 16 Boc-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys(Boc)-Thr-OH (XIV) 1M sodium hydroxide (45.0ml) solution is added in 15 minutes to the cold solution of hexapeptide methyl ester XIII (50.2g, 45mmol) in methanol (500ml). The solution is stirred at 30°C till the reaction is complete. pH is brought to 7 by addition of IN hydrochloric acid, the solution is concentrated in vacuum, and acidified further with 1M hydrochloric acid (total 45ml) under cooling to pH 3, and ethyl acetate (300ml) is added. The organic layer is separated and water layer is re-extracted ethyl acetate (200ml). The organic layers are combined, washed with brine (2 x 100ml), dried on sodium sulphate and evaporated to give title compound XIV. Yield 46.1g (97%) m. p. = 134-135°C, [α]D20 = -24.7° (c=lin methanol), ESMS= 1102 (M+l), 1124 (M+Na). Preparation Of Novel Linear Octapeptide Alcohol XVI Method 1 Boc-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys(Boc)-Thr-Cys(Acm)-Thr-OL(XV) A solution of protected hexapeptide acid XIV (ll.Og, lOmmol) and HOBt (1.5g, lOmmol) in DMAc (30 ml) is added to a solution of dipeptide alcohol TFA salt XII (7.0g, 18mmol) and TEA (2.5ml, 18mmol) in THF (50ml) and stirred at 0°C. DCCI (2.2g, 11mmol) is added at 0°C and the reaction mixture is stirred at 0°C for an hour followed by overnight stirring at 30°. Dicyclohexylurea is filtered and the filtrate concentrated in vacuum followed by addition of diethyl ether (50ml). The precipitate is filtered, washed with ethyl acetate, chloroform and dried in vacuum to give the title compound XV. Yield 12g ( 88%), ESMS= 1363.95 (M+H). [0001]H-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys-Thr-Cys(Acm)-Thr-OL.2TFA (XVI) The protected octapeptide alcohol XV (16g) and anisole (2.3ml) and mercaptoethanol (2.0ml) are suspended in DCM (20ml) under N2. The suspension 17 is cooled to 0°C and TFA (80ml) is added. Stirring is continued for one and half hour at same temperature. The TFA and DCM are evaporated under vacuum at 30°C, ether (200ml) is added to the residue under stirring. The precipitate is filtered, washed with diethyl ether (300ml), dried in vacuum and purified by HPLC to give compound XVI; Yield 9.7g (80%), m. p. - 161-163°C, [α]D20 = -61.2° (c= 0.25 in methanol), ESMS= 1163 (M+H). Method 2 H-Cys(Acm)-Thr-OMe. TFA (XVII) The dipeptide methyl ester X (8.1g, 20mmol) is dissolved in DCM (10ml) at 0°C under stirring. TFA (30ml) is added and continue the stirring for an hour. The TFA and DCM are evaporated under vacuum, diethyl ether (50ml) is added to the residue under stirring, ether is decanted, repeated twice and dried in vacuum to give compound XVII in 97% yield . ESMS= 308 (M+H). Boc-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys(Boc)-Thr-Cys(Acm)-Thr-OMe(XVIII) To a solution of dipeptide XVII (8.1g, 20mmol) and TEA (2.8ml, 20mmol) in THF(50ml) is added, hexapeptide acid XIV (ll.Og, lOmmol) and HOBt (1.5g, lOmmol). DCCI (2.2g, 1 lmmol) is added at 0°C . The reaction mixture is stirred at 0°C for an hour followed by overnight stirring at 30°C. Dicyclohexylurea is filtered and the filtrates are concentrated in vacuum followed by addition of ether (100ml). The precipitate is filtered, washed with ethyl acetate, water, diethyl ether and dried in vacuum to give the title compound. Yield 11 g (79%) . ESMS= 1391 (M+H). H-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys-Thr-Cys(Acm)-Thr-OMe. 2TFA (XIX) The octapeptide ester XVIII (llg, 7.9mm anisole, 2 ml) and mercaptoethanol (2 ml, ) are suspended in DCM (20ml) under N2. The suspension is cooled to 0°C and TFA (80ml) is added. Stirring is continued for one and half hour at same temperature. The TFA and DCM are evaporated under vacuum at 30°C, ether 18 (200ml) is added to the residue under stirring. The precipitates are filtered, washed with diethyl ether (300ml) and dried in vacuum to give compound XIX: Yield lO.Og (91%). ESMS= 1191 (M+H). H-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys-Thr-Cys(Acm)-Thr-OL. 2TFA (XVI) Sodiumborohydride (1.5g, 40mmol) dissolved in 90% ethanol (20ml) is added dropwise to crude compound XIX (lOg, 7mm) dissolved in 90% ethanol (50ml at 0°C. The stirring is continued at same temperature till the reaction is complete. Acetic acid is added to the reaction mixture which is concentrated and purified on column chromatography to yield compound XVI. Yield 6.8g (69% ), m. p. = 161-163°C, [α]D20= -60.6° (c= 0.25 in methanol), ESMS= 1163 (M+H). 19 5. Claims: We Claim: 1. A solution phase process for preparing H - (D) - Phe1 - Cys2 - Phe3 - (D) - Trp4 - Lys5 - Thr6 - Cys7 - Thr8 -OL comprising the steps of a) Condensing Boc - Cys(Acm) - OH and H - Phe - OMe. HCl to obtain Boc -Cys (Acm) -Phe - OMe (II); b) Condensing Boc - Cys(-Acm) - Phe - OMe and TFA to obtain H-Cys (Acm) -Phe - Ome. TFA; c) Condensing Boc - D - Phe - OH and H-Cys(Acm) - Phe - OMe TFA to obtain Boc - D - Phe - Cys (Acm) - Phe - OMe; d) saponifying Boc - D - Phe - Cys(Acm) - Phe - OMe to obtain Boc - D - Phe -Cys (Acm) - Phe - OH; e) Condensing Z-Lys (Boc) - OH and H -Thr - OMe. HCl to obtain Z- Lys (Boc) - Thr - Ome (VI) where Z is benzyloxy carbonyl; f) hydrogenating Z-Lys (Boc) - Thr - OMe (VI) to obtain H - Lys (Boc) - Thr -OMe, AcOH; g) condensing Z - (D) - Trp - OH and H - Lys (Bpc) - Thr - OMeAcOH (VII) to obtain Z - D - Trp - Lys (Boc) - Thr - OMe (VIII); h) hydrogenating Z - D - Trp- Lys (Boc) - Thr - OMe to obtain H - Trp - Lys(Boc) - Thr - OMe (IX); i) Condensing Boc - Cys (Acm) and H - Thr - OMe. HCl to obtain Boc - Cys (Acrn) - Thr - OMe (X); j) reducing Boc - Cys(Acm) - Thr- OMe (X) with sodium borphydride to obtain Boc - Cys (Acrn) - Thr - OL (XI); k) treating Boc - Cys(Acm) - Thr- OL (XI) with TFA to obtain H - Cys(Acm) - Thr-OL (XII); 1) condensing Boc - D - Phe Cys(Acm) - Phe - OH (V) and D - Trp - Lys (Boc) - Thr - OMe (IX) to obtain Boc - D - Phe Cys(Acm) - Phe -(D )- Trp - Lys(Boc) - Thr - OMe (XIII); 20 m) saponifying Bpc - D - Phe - Cys(Acm) - Phe - D - Trp - Lys (BPC) - Thr -OMe (XIII) with a base to obtain Bpc - (D) - Phe - Cys(Acm) - Phe - (D) - Trp - Lys(Bpc) - Thr - OH (XIV); n) treating Boc - Cys(Acm) - Thr - OMe (X) with TFA to obtain H-Cys (Acm) -Thr-OMe. TFA (XVII); o) condensing Boc - D - Phe - Cys(Acm) - Phe -DTrp - Lys (Boc) - Thr - OH (XIV) and H - Cys(Acm) - Thr - OMe. TFA (XVII) to obtain Boc -D-Phe-Cys(Acm) - f he - D - Trp - Lys (Boc) - Thr - Cys(Acm) - Thr - OMe (XVIII); ,p) treating treating Boc - D - Phe - Cys(Acm) - Phe - D - Trp - Lys (Boc) - Thr -Cys(Acm) - Thr - OMe (XVIII) with TFA to obtain H - D - Phe - Cys(Acm) -Phe -D- Trp - Lys - Thr - Cys(Acm) - Thr - OMe. 2 TFA (XIX); q) reducing H - D - Phe - Cys(Acm) - Phe - D - Trp - Lys - Thr - Cys (Acm) -Thr - OMe. 2 TFA (XIX) with sodium borohydride to obtain H - D - Phe -Cys (Acm) - Phe -D - Trp - Lys - Thr - Cys (Acm) - Thr - OL. 2 TFA (XVI); r) treating H - (D) - Phe1 - Cys(Acrn)2 - Phe3 - (D) - Trp4 - Lys5 - Thr6 - Cys(Acm)7 - Thr8 - OL. 2 TFA (XVI) with iodine to obtain H - (D) - Phe1 - Cys2 - Phe3 - (D) - Trp4 - Lys5 - Thr6 - Cys7 -Thr8-OL I I A process as claimed in claim 1 step (a) wherein Boc-Cys(Acm)-Phe-OMe was prepared by treating Bpc-Cys(Acm)-OH with H-Phe-OMe.HCl in presence of alkylchloroforrhates such as methylchloroformate, ethylchlprpformate, isobutylchlerpfprmate and N-methylmorpholine and organic base such as triethylamine, pyridine, imidazole, diispprppylethylainine in ether solvent such as diethyl ether, diisopropyl ether, tetrahydrofuran, dipxane by stirring at temperature between -30°C to 50°C for 1 to 24 hours. A propess as claimed in claim 1 step (b) wherein H-Cys(Acm)-Phe-OMe.TFA was prepared by treating Boc-Cys(Acm)-Phe-OMe with organic acids such as trifluoroacetic acid in chlprinated solvent such as methyl chloride, chloroform, carbon tetrachloride by stirring at a temperature between -30°C to 30°C for 1 to 3 hours. 21 4. A process as claimed in claim 1 step (c) wherein Boc-D-Phe-Cys(Acm)-Phe-OMe was prepared by treating Bpc-D-Phe-OH with H-Cys(Acm)-Phe-QMe.TFA in presence of alkylchlorpformates such as methylchloropformate, ethylchloroformate, isobutylchloroformate and N-methylmorpholine and organic base such as triethylamine, pyridine, imidazole, diisopropylethylamine in ether solvent such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane by stirring at temperature between -30° to 50°C for 1 to 24 hours. 5. A process as chimed in claim 1 step (d) wherein Bpc-D-Cys(Acm)-Phe-QH was prepared by treating Boc-D-Phe-Cys(Acm)-Phe-OMe with alkali metal hydroxide such as sodium hydroxide, potassium hydrpxide, lithium hydroxide in alcohol solvent such as methanol, ethanpl, isopropanol. 6. A process as claimed in claim 1 step (e) wherein Z-Lys(Boc)-Thr-OMe was prepared by condensation of Z-Lys(Bpc)-OH with Thr.OMe.HCl in presence of alkylchloroformates such as methylchlprpformate, ethylchloroformate, isobutylchloroformate and N-methylmorpholine and organic base such as triethy lamine, pyridine, imidazole, diisopropylethylamine in ether solvent such as diethyl ether, diisppropyl ether, tetrahydrofuran, dioxane by stirring at temperature between -30° to 50°C for 1 to 24 hours. 7. A prpcess as claimed in claim 1 step (f), wherein H-Lys(Boc)-Thr-OMe.AcOH was prepared by hydrogenation of Z-Lys(Boc)-Thr-OMe using hydrogen gas in presence of catalyst such as 5% or 10% palladium on charcoal or platinum on charcoal in alcohol solvent such as. methanol, ethanol, isopropanol and acetic acid- 8. A process as claimed in claim 1 step (g) wherein Z-D-Trp-Lys(Boc)-Thr-OMe was prepared by condensation of Z-(D)-Trp-OH with Lys-(Boc)-Thr-OMe.AcOH in presence of alkylchlorpforrnates such as methylchloroformate, ethylchloroformate, isobutylchlproformate and N-methylmorphpline and organic base such as triethylarnine, pyridine, imidazole, diisopropylethylamine in ether splvent such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane by stirring at temperature between -30°C to 50°C for 1 to 24 hours. 9. A process as claimed in claim 1 step (h) wherein H-D-Trp-Lys(Boc)-Thr-OMe was prepared by hydrogination of Z-D-Trp-Lys(Boc)-Thr-OMe using hydrogen gas in presence of catalyst such as 5% or 10 % palladium on charcoal or platinum on charcoal in alcohol solvent such.as methanol, ethanol, isopropanol and acetic acid. 10. A process as claimed in claim 1 step (i) wherein Boc-Cys(Acm)-Thr-OMe was prepared by condensation of Boc-Cys(Acm)-QH with H-Thr-OMe.HCl in presence of alkylchloroformates such as memylchlproformate, ethylchlorpformate, isobutylchloroformate and N-methylmorpholine and organic base such as triethylamine, pyridine, imidazole, diisopropylethylamine in ether solvent such as diethyl ether, diisppropyl ether, tetrahydrofuran, dioxane by stirring at temperature between -30°C to 50°C for 1 to 24 hpurs, 11. A process as claimed in claim 1 step (j) wherein Bpc-D-Phe-Cys(Acm)-Phe-(D)-Trp-Lys(Bpc)-Thr-OMe was prepared by treating Boc-D-Phe-Cys(Acm)-Phe-OH with D-Trp-Lys(Boc)-Thr-OMe in presence of coupling reagent such as dicyclphexylcarbpdiimide in ether splvent such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane by stirring at temperature between -30oC to 50°C for 1 to 24 hours. 12. A process as claimed in claiin 1 step (k) wherein Boc-(D)-Phe-Cys(Acm)-Phe-(D)-Trp-Lys(Boc)-Thr-OH was prepared by reacting Boc-D-Phe-Cys(Acm)-Phe-D-Trp-Lys-Thr-OMe with alkali metal hydroxide such as sodium hydroxide, potassium hydroxide, lithium hydroxide in alcohol solvent such as methanol, ethanol, isopropanpl. 13. A process as claimed in claim.1 step (1) wherein H-Cys(Acm)-Thr-OMe.TFA (XVII) was prepared by treating Boc-Cys(Acm)-Thr-OMe with organic acid such as trifluoroacetic acid in the chlorinated solvent such as methylene chloride, chloroform, carbon tetrachloride by stirring at temperature between -30°C to 30°C for 1 to 3 hours. 14. A process as claimed in claim 1 step(m) wherein Boc-D-Phe-Cys(Acm)-Phe-D-Trp-Lys(Boc)-Thr-Cys(Acm)-Thr-OMe was prepared by reacting Boc-D-Phe-Cys(Acm)-Phe-D-Tip-LystBoc-Thr-OH, with H-Cys(Acm)-Thr-OMe.TFA in presence of organic base amines such as imidazole, pyridine, triethylarnine, diethylamine, diisppropyl amine, 1-hydroxybenzotriazole and coupling reagents such as dicyclphexylcarbpdiimide in ether solvent such as diethylether, diisopropyl ether, tetrahydrofuran, dioxane by stirring at temperature between -30°C to 50°C for 1 to 3 hours. 15. A process as claimed in claim 1 step (n) wherein Boc-Cys(Acm)-Thr-OMe is treated with triflurpacetic acid to obtain H-Cys(Acm)-thr-OMe.TFA in chlorinated solvent such as methylene chloride, chloroform, carbon tetrachloride by stirring at temperature between -30°C to 30°C for 1 to 3 hours. 16. A process as claimed in claim 1 step (p) wherein Bpc-D-Phe-Cys(Acm)-Phe-D-Lys(Boc)-Thr-OH is condensed with H-Cys(Acm)-Thr-OMe.TFA in presence of organic base amines such as imidazole, pyridine, triethylamine, diefhylamine, diisopropylamine, 1-hydroxybenzotriazole and coupling reagents such as dicyclphexylcarbpdiirnide in ether splvent such as diethylether, diisppropyl ether, tetrahydrofuran, dioxane by stirring at temperature between -30°C to 50°C for 1 to 3 hours. 17. A process as claimed in claim 1 step (p) wherein Boc-D-Phe-Cys(Acm)-Phe-D-Trp-Lys(Boc)-Thr-Cys(Acm)-Thr-OMe was treated with TFA to obtain H-D-Phe-Cys(Acm)-Phe-D-Trp-Lys(Boc)-Thr-Cys(Acm)-Thr-OMe.2TFA in chlorinated solvent such as methylene chloride, chloroform, carbon tetrachloride by stirring at temperature between -30°C to. 30°C for 1 to 3 hours. 18. A process as claimed in claim 1 step (q) wherein H-D-Phe-Cys(Acfn)-Phe-D-Trp-Lys-Thr-Cys(Acm)-"Thr-OMe.2TFA was reduced with sodium borohydride to pbtain H-D-Phe-Cye(Acm)-Phe-D-Trp-Lys-Cys(Acm)Thr-OL.2TFA in alcoholic solvent such as methanol, ethanol, isPprppanpl at temperature between -3O°C to 30°C for 1 tp 3 hpurs. 19. A process as claimed in claim 1 step (r) wherein H-D-Phe-Cys(Acm)-Phe-D-Trp-Lys-Tbx^Cys(Acm)-Thr-OMe.2TFA was oxidized with iodine to obtain claim 1 product in alcoholic splvent such as methanol, ethanol, isppropanpl at. temperature between -30°C tp 30°C for 1 tp 3 hpurs. Dated this 26th day of May 2005 Dr. Krishna K Dubey Research Scientist-Intellectual Property

Documents:

457-mum-2000-abstract(31-05-2005).doc

457-mum-2000-claims(granted)-(31-05-2005).doc

457-mum-2000-form 2(granted)-(31-05-2005).doc

457-mum-20004cancelled pages(31-05-2005).pdf

457-mum-2004-abstract(31-05-2005).pdf

457-mum-2004-claims(granted)-(31-05-2005).pdf

457-mum-2004-correspondence(31-05-2005).pdf

457-mum-2004-correspondence-ipo(24-02-2005).pdf

457-mum-2004-form 1(15-04-2004).pdf

457-mum-2004-form 13(31-05-2005).pdf

457-mum-2004-form 19(15-04-2004).pdf

457-mum-2004-form 2(granted)-(31-05-2005).pdf

457-mum-2004-form 3(15-04-2004).pdf

457-mum-2004-form 3(15-04-2005).pdf

457-mum-2004-form 3(31-05-2005).pdf

457-mum-2004-petition under rule 137(31-05-2005).pdf

457-mum-2004-petition under rule 81(1)(31-05-2005).pdf