Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

• the present invention relates to the efficient commercial synthesis for the making of pramilintide, a synthetic analog of human amylin which is a peptide hormone.
• Pramilintide is indicated to treat type 1 and type 2 diabetics who use insulin.
• the process for making pramilintide substantially comprises the syntheses of various fragments of the polypeptide and the coupling of the fragments to produce pramilintide.
• pramlintide is disclosed in U.S. Pat. No. 5,686,411, which is herein incorporated in its entirety by reference.
• Pramlintide is known to be prepared by solid phase synthesis that successively adds the desired amino acid to a growing peptide chain.
• an ⁇ -N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin support are reacted at room temperature in an inert solvent in the presence of coupling agents such as dicyclohexylcarbodiimide 1-hydroxybenzotriazole in the presence of a base.
• the ⁇ -N-carbamoyl protecting group is removed from the resultant peptide with a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid.
• a reagent such as trifluoroacetic acid or piperidine
• Suitable N-protecting groups are known in the art, with t-butyloxycarbonyl herein preferred.
• U.S. Pat. No. 5,424,394 provides a classical stepwise approach for the synthesis of amylin and amylin analogues. Single amino acid residues are covalently coupled to a growing peptide chain which is covalently linked to a solid resin support. The synthetic route is very lengthy and inefficiently since several coupling and deprotected steps have to be repeated.
• the present invention provides a more efficient synthesis of pramlintide and the yield and purity of final product will be improved in view of the prior art.
• the present invention provides for an efficient process for making pramlinitide that is high in yield and scalable for commercial production.
• the process comprises the stepwise synthesis of amino acid segments, and the coupling together of these segments to produce pramlinitide.
• the present invention provides for four novel intermediate amino acid segments for the preparation of pramlintide.
• the four segments are synthesized in solid phase synthesis and the coupling reaction is performed in solution phase.
• the segments are produced by coupling a protected designated amino acid to a growing peptide chain that is covalently linked to an insoluble solid resin support.
• the “protected designated amino acid” refers to single amino acid (having protected sidechains and amino termini) which generally proceed from the carboxy-terminal end to the amino-terminal end to give a peptide of specified sequence.
• a “growing peptide chain” refers to a general cycle of synthesis comprising deprotection of the ⁇ -amino group of the resin-bound amino acid or peptide, followed by reaction (coupling) of the free ⁇ -amino group with some carboxyl-activated form of the next ⁇ -amino protected amino acid to form a peptide linkage and to give a support-bound peptide.
• the Fmoc protecting group was removed by treatment with 20% piperidine in DMF twice for 10 min and 30 min, respectively.
• the second amino acid (Fmoc-Cys (Acm)-OH) was introduced to start the first coupling step.
• the Fmoc protected amino acid was activated in situ using 1:1:2 molar ratio of HBTU (O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate)/HOBt (N-Hydroxybenzotriazole)/DIEA in DMF and subsequently coupled to the growing peptide on resin for 3 h.
• the peptide was cleaved from the peptide on resin (8 g) prepared as described above, using 20% TFE solution in DCM for 2 h.
• the peptide solution was solvent replaced by MeOH and concentrated (30 mL).
• the concentrated residue was cooled and the product was precipitated by adding water (30 mL).
• the precipitated product was separated by filtration and washed with mixed solvent of MeOH/water (10 mL/10 mL) twice to give Boc-Lys(Boc)-Cys(Acm)-Asn(Trt)-Thr(tBu)-Ala-Thr(tBu)-Cys(Acm)-Ala-OH(S1a).
• the second amino acid (Fmoc-His(Trt)-OH) was introduced to start the first coupling step.
• the Fmoc protected amino acid was activated in situ using 1:1:2 molar ratio of HBTU/HOBt/DIEA in DMF and subsequently coupled to the growing peptide on resin for 3 h. Completion of the coupling was indicated by a Kaiser test.
• the Fmoc protecting group on the ⁇ -amine was removed with 20% piperidine in DMF twice for 10 min and 30 min, respectively. These steps were repeated each time with another amino acid according to peptide sequence. All amino acids used were Fmoc-N ⁇ protected.
• Trifunctional amino acids were side chain protected as follows: Asn(Trt)-OH, Arg(Pbf)-OH, Gln(Trt)-OH and Thr(tBu)-OH. Three equivalents of the activated amino acids were employed in the coupling reactions. At the end of the synthesis the growing peptide on resin was washed with DMF, MeOH followed by MTBE, and dried under vacuum to give dry peptide on resin.
• the peptide was cleaved from the peptide on resin (24 g) prepared as described above, using 20% TFE solution in DCM for 2 h.
• the peptide solution was solvent replaced by MeOH and concentrated (50 mL).
• the product was absolutely precipitated by adding cool MeOH (50 mL) to the concentrated residue.
• the product was separated by filtration and washed with cool MeOH (20 mL) twice to give S2 (Fmoc-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Trt)-Ser(tBu)-O H, 12 g).
• the Fmoc protected amino acid was activated in situ using 1:1:2 molar ratio of HBTU/HOBt/DIEA in DMF and subsequently coupled to the growing peptide on resin for 3 h. Completion of the coupling was indicated by a Kaiser test. After washing of the resin, the Fmoc protecting group on the ⁇ -amine was removed with 20% piperidine in DMF twice for 10 min and 30 min, respectively. These steps were repeated each time with another amino acid according to peptide sequence. All amino acids used were Fmoc-N ⁇ protected. Trifunctional amino acids were side chain protected as follows: Ser(tBu)-OH, and Asn(Trt)-OH. Three equivalents of the activated amino acids were employed in the coupling reactions. At the end of the synthesis the growing peptide on resin was washed with DMF, MeOH followed by MTBE, and dried under vacuum to give dry peptide on resin.
• the peptide was cleaved from the growing peptide on resin (10 g) prepared as described above, using 1% TFA solution in DCM for 1.5 h. After neutralizing with Pyridine the peptide solution was concentrated (15 mL). The product was precipitated by adding the concentrated residue into Heptanes (50 mL). The product was separated by filtration and washed with mixed solvent of DCM/Heptanes (1 mL/3 mL) three times to give S3 (Fmoc-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-OH, 5.2 g).
• the second amino acid (Fmoc-Asn(Trt)-OH) was introduced to start the first coupling step.
• the Fmoc protected amino acid was activated in situ using 1:1:2 molar ratio of HBTU/HOBt/DIEA in DMF and subsequently coupled to the growing peptide on resin for 3 h. Completion of the coupling was indicated by a Kaiser test.
• the Fmoc protecting group on the ⁇ -amine was removed with 20% piperidine in DMF twice for 10 min and 30 min, respectively. These steps were repeated each time with another amino acid according to peptide sequence. All amino acids used were Fmoc-N ⁇ protected.
• Trifunctional amino acids were side chain protected as follows: Ser(tBu)-OH, Asn(Trt)-OH and Thr(tBu)-OH. Three equivalents of the activated amino acids were employed in the coupling reactions. At the end of the synthesis the growing peptide on resin was washed with DMF, MeOH followed by MTBE, and dried under vacuum to give dry peptide on resin.
• the peptide was cleaved from the peptide on resin (23 g) prepared as described above, using 20% TFE solution in DCM for 2 h.
• the peptide solution was solvent replaced by MeOH and concentrated (60 mL).
• the product was precipitated by adding MeOH (50 mL) to the concentrated residue.
• the product was separated by filtration and washed with MeOH/water (20 mL) twice to give S4 (Fmoc-Thr(tBu)-Asn(Trt)-Val-Gly-Ser(tBu)-Asn(Trt)-Thr(tBu)-OH, 10.6 g)
• the reaction mixture is warmed to 20 to 30° C. and kept for 15 hr. Diethylamine (DEA) (0.42 Kg; 10.0 eq.) is charged while maintaining the temperature at 25° C.
• DEA Diethylamine
• the reaction mixture is stirred at 20 to 30° C. for 2 hr.
• Ethyl acetate (EA) (7.38 Kg) and softened potable water (SPW) (50.0 Kg) are slowly added to the reaction mixture while maintaining the temperature at 35° C. until the cloud point is observed and held at cloud point for 1 hr. The remained SPW is added while maintaining the temperature at 35° C.
• the wet cake is purged by nitrogen for 1 hr and dried at 50° C. for 5 hr to get M2 (Thr(tBu)Asn(Trt)ValGlySer(tBu)Asn(Trt)Thr(tBu) Tyr(tBu)-NH 2 ) (about 0.81 Kg).
• Ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride (0.30 Kg; 3.0 eq) is charged into the resulting mixture while maintaining the temperature at 20 to 30° C. and stirred for 3 hr.
• Ethyl acetate (EA) (5.1 Kg) and SPW (43.6 Kg) are slowly added into the reaction mixture while maintaining the temperature at 35° C. until the cloud point is observed and held at cloud point for 1 hr. The remained SPW is added while maintaining the temperature at 35° C.
• the solid is filtered and washed by MeOH twice. The wet cake is purged with nitrogen for 1 hr and dried at 50° C.
• the wet cake is purged with nitrogen for 1 hr and dried at 50° C. for 5 hr to provide M4 (Ser(tBu)Asn(Trt)Asn(Trt)PheGlyProlleLeuProProThr(tBu)Asn(Trt)ValGlySer(tBu) Asn(Trt)Thr(tBu)Tyr(tBu)-NH 2 ) (about 1.32 Kg).
• M5 (2.16 Kg; 1.0 eq) and dichloromethane (DCM) (28.7 Kg) are charged into a suitable reactor under nitrogen. Then piperidine (0.32 Kg; 10.0 eq) is charged while maintaining the temperature at 20 to 30° C. and stirred for 2 hr. Methyl-t-butyl ether (MTBE) (47.9 Kg) is slowly added while maintaining the temperature at 0 to 10° C. until the cloud point is observed and held at cloud point for 1 hr. The remained MTBE is added while maintaining the temperature at 0 to 10° C.
• MTBE Methyl-t-butyl ether
• the wet cake is purged with nitrogen for 1 hr and dried at 50° C. for 5 hr to provide M6 (Thr(tBu)Gln(Trt)Arg(Pbf)LeuAlaAsn(Trt)Phe LeuValHis(Trt)Ser(tBu)Ser(tBu)Asn(Trt)Asn(Trt)PheGlyProlleLeuProProThr(tBu) Asn(Trt)ValGlySer(tBu)Asn(Trt)Thr(tBu)Tyr(tBu)-NH 2 ) (about 1.87 Kg).
• 1-hydroxy-7-azabenzotriazole (0.14 Kg; 3.0 eq.) are charged into a suitable reactor under nitrogen.
• M7 (2.09 Kg) is charged into reactor I under nitrogen.
• the solid is kept at temperature 0 to 10° C.
• SPW 0.52 Kg
• TEA trifluoroacetic acid
• TIS triisopropylsilane
• the mixed solution in reactor II is cooled to 0 to 10° C. and charged into reactor I at 25° C.
• the reaction mixture is stirred at 20-30° C. for 3 hr.
• the reaction mixture is cooled to 0 to 10° C. and the pre-cooled (0 to 10° C.) methyl-t-butyl ether (MTBE) (61.85 Kg) is slowly charged at 15° C. and stirred for 1 hr.
• the solid product is filtered and washed with methyl-t-butylether (MTBE) twice and tetrahydrofuran (THF) twice.
• the wet cake is purged with nitrogen for 1 hr and dried at 50° C. for 6 hr to provide pramlintide acetate (about 1.21 Kg).

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