Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/655—Somatostatins
  • C07K14/6555—Somatostatins at least 1 amino acid in D-form

Definitions

• the present invention relates to a novel processes for the preparation of pharmaceutical grade octreotide (1) using classical solution phase peptide synthesis in high yield and purity.
• Octreotide is a cyclic octapeptide; it is highly potent and pharmacologically selective analog of somatostatin. It possesses excellent biological activity both in vitro and in vivo Pless J., Metabolisim, 41, 5-6, (1992).
• octreotide is D-phenylalanyl-L- Cysteinyl- L- phenylalanyl -D-tryptophyl - L-lysyl - threonyl - N- [2 -hydroxy - 1- (hydroxymethyl)propyl]- L-Cysteinamide cyclic (2-7)-disulfide (1).
• Octreotide inhibits growth hormone for a long period and is therefore indicated for acromegaly to control and reduce the plasma level of growth hormone. It is also indicated for the symptomatic treatment of patients with metastatic carcinoid tumors and vasoactive intestinal peptide tumors.
• octreotide may be divided two main types, direct solid phase peptide synthesis and solution-phase peptide synthesis.
• Direct Solid phase synthesis comprises attachment of a C-terminal amino acid to resin, and step by step elongation of the peptide chain, with pre activated amino acid.
• Several patents describe solid phase synthesis of octreotide, for example EP 953,577; US 5889146 disclose synthesis using 2-chlorotrityl resin and Fmoc-butyl protection.
• Solution phase synthesis comprises condensation of amino acids in solution. Several segments/blocks containing from 2 to 5 amino acids synthesized independently, followed by condensation of these segments/blocks to each other in the required sequence.
• the advantage of this type of process is that it is inexpensive than the solid phase synthesis and this method is also more suitable for large scale production.
• the strategy involves coupling two tripeptide segments Boc- D - Phe - Cys (Acm) - Phe - OMe and Z-D-Trp-Lys(Boc)-Thr- OMe to yield hexapeptide Boc- D - Phe - Cys (Acm) - Phe - D-Trp-Lys(Boc)-Thr-OMe, further hexapeptide segment is coupled with dipeptide H-Cys(Acm)-Tlu>OMe/OL to yield octapeptide Boc- D - Phe - Cys (Acm) - Phe - D-Trp-Lys(Boc)-Thr-Cys(Acm)-Thr- OMe/OL.
• Oxidative cyclization of protected or non-protected sulfhydryl groups with formation of disulfide structures is usually carried out as the final synthetic step, the reason being substantial thermal and chemical ability of the disulfide linkage.
• the oxidation of open- chain peptide containing free and / or certain types of protected sulfhydryl groups with iodine in methanol or acetic acid, acetic acid/water reported. Iodine, however, is not without drawback as cyclization agent. For instance, tryptophan moieties present in peptide substrates are at risk of being iodinated, making the balance between full conversion of starting materials and minimizing side reactions a delicate one, which, in turn impact product purity. In the present invention this aspect has been rightfully tackled by not opting for iodine route for oxidative cyclization.
• the present invention hydrogen peroxide used for oxidative cyclization.
• This invention describe a process for obtaining octreotide or a pharmaceutically acceptable salt in commercial scale quantities by solution phase chemistry methods using mild reagents to yield high purity octreotide.
• the process includes the following
• the novel protected octapeptide alcohol (Segment- ABC, 20) of present invention is prepared from Fmoc dipeptide alcohol (Segment C, 17) and hexapeptide acid (Segment AB2, 19).
• the novel hexapeptide (segment-ABl, 18) is prepared by condensation of two protected novel segments, dipeptide (4) and tetra peptide (13) which on hydrolysis with sodium hydroxide afforded novel hexapeptide acid (19).
• Fmoc Flourenylmethoxycarbonyl
• Trt Triphenyl methyl (Trityl)
• NMM N-methylmorpholine
• HBTU O-Benzotriazole-N, N, N', N'-tetramethyl-uronium-hexafluoro-phosphate
• DCI N, N- diisopropylcarbodiimide
• HOBt 1 -Hydroxybenzotriazole
• Formula (1) comprises preparation of appropriate peptide fragments using the standard process of peptide chemistry, known to the practitioners in the art.
• amino functions of amino acids are protected with protecting groups like Boc, Z, Fmoc and the carboxyl functions of amino acids are protected with alkyl like methyl N-osu or arylphenol like nitrophenol.
• the condensation of the carboxyl group of the amino protected amino acid is by using active ester method.
• the ester could be N-hydroxysuccinimide or aromatic phenol like nitro phenol.
• the reaction is carried out by dissolving the carboxyl protected amino acid in polar aprotic solvent like, dimethylformamide, tetrahydrofuran, acetonitrile, in presence of tertiary amines such triethylamine, diisopropylethylamine, N- methymorpholine, at temperature of 10 to 40°C for 2-24 hours.
• polar aprotic solvent like, dimethylformamide, tetrahydrofuran, acetonitrile
• condensation of the carboxyl group of the amino protected amino acid is typically carried out by dissolving the appropriately protected amino acids in equimolar quantities in a solvent like dimethylformamide, tetrahydrofuran, dichloromethane and adding a condensing agent such as HBTU, DCI, HOBt in equimolar quantity at temperature of 0-10°C and stirring at 5 to 40°C for 2 to 20 hours.
• a condensing agent such as HBTU, DCI, HOBt in equimolar quantity at temperature of 0-10°C and stirring at 5 to 40°C for 2 to 20 hours.
• the protected group of amino /carboxyl is removed by the use of appropriate reagent known in the art. The basic differences from other procedures already described are;
• the N-terminal hexapeptide (segment -AB2) (19) is synthesized by condensation of di, tetra peptide fragments (segment -A, 4, segment -B, 13) and followed by saponification.
• Boc and Trityl groups are removed by treatment with trifluoroacetic acid /water/Triisopropylsilane at 25°C to obtain linear octapeptide which is cyclized with hydrogen peroxide in 1% ammonium acetate solution at pH 7-8. to give octreotide (1)
• the hexapeptide acid segment AB2 (19) of the present invention is prepared by condensation of appropriately protected dipeptide and tetrapeptide segments A (4) and segment B (13) followed by saponification as shown in the scheme.
• the protected dipeptide Boc - D- Phe - Cys (Trt) -OH (segment A, 4) can be prepared by any of the processes known in the art from commercially available raw materials.
• D- Phenylalanine (2) carrying Boc group for N a" protection and nitrophenol group for carboxyl group as activation is treated with S-Trityl L- cysteine (3) employing TEA as base in equimolar amounts in dimethylformamide at 25°C to obtain the protected dipeptide(4).
• D- tryptophan (5) carrying Z group as ⁇ ⁇ ' protection and succinidimyl group for carboxyl group activation is treated with Boc- L- lysine (6) using sodium bicarbonate as base in tetrahydrofuran at 0-5°C to give protected dipeptide Z-D- Trp -Lys (Boc) - OH (7).
• the resulting peptide is condensed with L-Thr-OMe.HCl (8) in presence of TEA, and HBTU in dimethylformamide at 25 C to obtain the protected tripeptide methyl ester Z-D- Trp -Lys (Boc) - Thr-OMe (9).
• the Z-group is removed from tripeptide ester by hydrogenation over 10% Pd/C and resulting peptide H-D- Trp -Lys (Boc) - Thr-OMe (10) is condensed with L-Phe-OH (11) carrying Fmoc group, using HBTU as condensation agent in dimethylformamide at 25°C to obtain the protected tetrapeptide methyl ester Fmoc-Phe - D- Trp -Lys (Boc) - Thr-OMe (12). Treatment with diethylamine in dichloromethane affords H-Phe -D- Trp -Lys (Boc) - Thr-OMe (segment B), (13).
• the process further consists of coupling of two segments AB2 (19) and segment C (17), in the presence of HBTU in an aprotic solvent such as DMF, DMA, NMP, and THF.
• an aprotic solvent such as DMF, DMA, NMP, and THF.
• the mass is treated with 10% hydrochloric acid solution, the separated solid was filtered, resulting solid material dissolved in ethyl acetate and treated with n-hexane and filtered to get the desired product segment ABC (20).
• the product is treated with trifluoroacetic acid in the presence of 0.25% of water/Triisopropylsilane as scavengers.
• the mixture is concentrated and the residue is treated with ether.
• the separated product is filtered and suspended in water followed by adjustment to pH 7-8 with bases selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate and calcium carbonate.
• bases selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate and calcium carbonate.
• the resultant solution is oxidized with hydrogen peroxide to afford the desired octreotide and further treated with acetic acid to obtain crude octreotide acetate salt.
• This crude peptide solution is further purified by employing preparative HPLC process to get the product purity of more than 99.0%.
• Example-1 Preparation of (Boc-D - Phe - Cys (Tit) - OH) (segment- A)
• reaction mass was distilled to remove tetrahydrofuran, pH was adjusted to 2-3 and extracted with ethyl acetate (300 mL) and washed with 10% sodium bicarbonate solution (2x200mL), 10% hydrochloric acid solution (2x200mL), and brine (2x200mL), dried with sodium sulfate and evaporated to yield brown oily mass.
• the mass was treated with hexane and separated solid was filtered, and dried to get product (7) Yield 27.6g (85%).
• Example-6 Preparation of H -Phe - D - Trp - Lys (Boc) - Thr - OMe, (Segment- B5)
• reaction mass was treated with 1M hydrochloric acid (600 mL) at 0-5°C, separated pale yellow solid material was filtered and, washed with 10% hydrochloric acid solution(2x250mL), to get product 16. Yield 20.0g (89%).
• Example-8 Preparation of H -Cys (Trt) - Thr - OL, (Segment-C2)
• Dipeptide 17 (6.1 g, 13.6mmol) was dissolved in DMF (lOO.OmL) and hexapeptide acid 19 (14.0g, l lmmol), HBTU (4.6g, 12.1mmol) were added at 0-5°C, then N- methylmorpholine (3.0mL, 24.0mmol) was added and stirred for 3 hours.
• the reaction mass was treated with 1M hydrochloric acid (600mL) at 0-5°C. The separated pale yellow solid material was filtered.
• Fraction with purity greater than 95-98% are pooled and loaded again onto to prep C-18 column (50x 250mm, 100A 0 ). The fraction with more than 99% purity were pooled and lyophilization.
• Buffer A 0.5% AcOH in water.
• Buffer B 0.5% AcOH in water + acetonitrile (50+50) Gradient program

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• 2012
  • 2012-03-09 WO PCT/IN2012/000164 patent/WO2013132505A1/fr not_active Ceased

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