PEPTIDES

Abstract: Octapeptide are described of the formula: wherein each A₁ and A₂, independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, R₁CO (where R₁ is C₁₋₂₀ alkyl, C₃₋₂₀ alke­ nyl, C₃₋₂₀ alkinyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl), or R₂OCO (where R₂ is C₁₋₁₀ alkyl or C₇₋₁₀ phenylalkyl), provided that when one of A₁ or A₂ is R₁CO or R₂OCO, the other must be H; A₃ is CH₂-A₆ (where A₆ is pentafluoro, naphthyl, pyridyl, or phenyl which is optionally ring-­ substituted with a halogen, C₁₋₃ alkyl, NO₂, C₁₋₃ alkoxy, NH₂, or OH); A₄ is o-m- or; more preferably, p substituted X-Phe (where X is a halogen, H, NO₂, OH, NH₂, or C₁₋₃ alkyl), pentafluoroalanine, or naphthylalanine; and A₅ is Thr, Ser, Phe, Val, or Ile; or a pharmaceutically acceptable salt thereof.

Description: PEPTrDES This invention relates to peptides. A number of somatostatin analogs exhibiting GH-releaseinhibiting activity have been described in the literature, including analogs containing fewer than the naturally occurring fourteen amino acids. For example, Coy et al U.S. Patent No. 4,485,101, the disclosure of which is hereby incorporated by reference, describes dodecapeptides having an N-terminal acetyl group, a C-terminal NH2, D-Trp at position 6, and p-Cl-Phe at position 4. (herein, when no designation of configuration is given, the L-isomer is intended). In general, the invention features an octapeptide of the formula: EMI1.1 wherein each A, and A2 , independently, is H, C1-12 alkyl, C7-10 phenylalkyl, R1CO (where R1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkinyl, phenyl, naphthyl, or C7-10 phenylalkyl), or R20CO (where R2 is C1-10 alkyl or C7-lo phenylalkyl) provided that when one of A1 or A2 is R1CO or R20CO, the other must be H; A3 is CH2-A6 (where A6 is pentafluoro, naphthyl, pyridyl, or phenyl which is optionally ringsubstituted with a halogen, C1-3 alkyl, NO2, C1-3 alkoxy, NH2, or OH); A4 is o-m-or, more preferably, p-substituted X-Phe (where X is a halogen, H, NH2, N02, OH, or C1-3 alkyl), pentafluoroalanine, or naphthylalanine; and A5 is Thr, Ser, Phe, Val, or Ile; or a pharmaceutically acceptable salt thereof. In the formula given above, the configuration of the molecule at the carbon atom to which A3 is bonded is not given, to indicate that the amino acid residue of which A3 is a substituent can have the D- or L- configuration. Preferred compounds of the invention include EMI2.1 In other preferred embodiments, a therapeutically effective amount of the therapeutic compound and a pharmaceutically acceptable carrier substance, e.g. magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle, together form a therapeutic composition, e.g. a pill, tablet, capsule, or liquid for oral administration to a human patient, a spreadable cream, gel, lotion, or ointment for application to the skin of a human patient in need of the compound, a liquid capable of being administered nasally as drops or spray, or a liquid capable of intravenous, parenteral, subcutaneous, or intraperitoneal administration. The pill, tablet or capsule can be coated with a substance capable of protecting the composition from the gastric acid in the patient's stomach for a period of time sufficient to allow the composition to pass undisintegrated into the patients small intestine. The therapeutic composition can also be in the form of a biodegradable sustained release formulation for intramuscular administration. The compounds of the invention are active in inhibiting the secretion of GH, insulin, and glucagon. Further, the aromatic lipophilic N-terminal end can provide long-lasting in vivo activity. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof. Structure The compounds of the invention have the general formula recited above. They are all octapeptide analogs of somatostatin which have D-Trp at position 4; and optional modifications at positions 3 (A4) and 6 (A5). It has been found that D-Phe, D-p-Cl-Phe , or most preferably, D-B-naphthylalanine, at position 1; Tyr at position 3; and Val at position 6 are modifications which particularly enhance activity. The compounds can be provided in the form of pharmaceutically acceptable salts. Examples of preferred salts are those with therapeutically acceptable organic acids, e.g., acetic, lactic, maleic, citric, malic, ascorbi-c,- succinic, benzoic, salicylic, methanesulfonic, toluenesulfonic, or pamoic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as the hydrohalic acids, e.g;, hydrochloric acid, sulfuric acid, or phosphoric acid. Synthesis The synthesis of two octapeptides follows. Other octapeptides of the invention can be prepared by making appropriate modifications, within the ability of someone of ordinary skill in this field, of the following synthetic method. The first step in the preparation of D-p-Cl EMI4.1 was the preparation of the intermediate tert-butylcarbonyl-D-p-Cl-Phe-S-methylbenzyl Cys-Tyr-D-Trp-NE-benzyloxycarbonyl-Lys-Val-S-methylbenzyl-Cys- O-benzyl-Thr-benzyhydrylamine resin, as follows. Benzhydrylamine-polystyrene resin (Vega Biochemicals, Inc.) (1.30 g, 0.5 mmole) in the chloride ion form was placed in the reaction vessel of a Beckman 990B peptide synthesizer progrÅammed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f) 10% triethylamine in chloroform. The neutralized resin was stirred with Boc-O-benzyl-threonine and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 h and the resulting amino acid resin was then cycled through steps (a) to (g) in the above wash program. The following amino acids (1.5-mmole) were then coupled successively by the same procedure: Boc-S-methylbenzyl-Cys, Boc-Val, Boc-Ne-benzyloxycarbonyllysine, Boc-D-Trp, Boc-Ty.r, .Boc-S-methylbenzyl-Cys , Boc-4-chloro-Phe. After washing and drying, the completed resin weighed 1.89 g. The resin (1.89 g, O.Smmole) was then mixed with anisole (4 ml) and anhydrous hydrogen fluoride (36 ml) at 0 C and stirred for 45 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide was then dissolved in 800 ml of 90% acetic acid to which was added Iz in methanol until a permanent brown color was present. The solution was then stirred for 1 h before removing the solvent in vacuo. The resulting oil was dissolved in a minimum volume of 50% acetic acid and eluted on a column (2.5 X 100 mm) of Sephadex G-25. Fractions containing a major component by uv absorption and thin layer chromatography were then pooled, evaporated to a small volume, and applied to a column (2.5 X 50 cm) of Whatman LRP-1 octadecylsilane (15-20 uM). The column was eluted with a linear gradient of 10-50 ó acetonitrile in 0.1% trifluoroacetic acid in water. Fractions were examined by thin layer chromatography and analytical high performance liquid chromatography and pooled to give maximum purity. Repeated lyophilization of the solution from water gave 138 mg of the product as a white, fluffy powder. The product was found to be homogeneous by Kple and Tlc. Amino acid analysis of-an acid hydrolysate confirmed the composition of the octapeptide. From the above-described resin (tert-butylcarbonyl-D-p -Cl-Phe-S-methylbenzyl-Cys-Tyr-D-Trp-N-benzyloxycarbonyl-Lys -Val-S-methylbenzyl-Cys-O-benzyl-Thr-benzyl-Thr-benzhydrylamine resin), there can be prepared, according to conventional techniques, an additional compound of the invention, of the formula tert-butylcarbonyl EMI6.1 Thr-NH2. An additional octapeptide of the invention, D ss-naphthylalanins EMI6.2 was synthesized as follows. The first step was the synthesis of the intermediate tert-butyloxycarbonyl-D- B -naphthylalanic-S-methylbenzyl-Cys- Tyr-D-Trp-NE-benzyloxycarbonyl-Lys-Val-S-methylbenzyl-Cys-Obenzyl-Thr-benzyhydrylaminic resin, according to the procedure previously described for the above intermediate, except that, in the final coupling step, Boc-D-I3-naphthylalanine was substituted for p-Cl-Phe. The resin, which weighed 1.78 g, was then subjected to hydrogen fluoride cleavage and Iz cyclization, as described above to produce 170 mg of lyophilized octapeptide, which was found to be homogeneous by Hplc and Tlc. Amino acid analysis of an acid hydrolysate confirmed the composition of the octapeptide. From the above-described resin (tert-butyloxycarbonyl-D- -nphthylalanic-S-methylbenzyl-Cys -Tyr-D-Trp-N E -benzyloxycarbonyl-Lys-Val-S-methylbenzyl-Cys-O Benzyl-Thr-benzhydrylaminic resin), there can be prepared, according to conventional techniques, an additional compound of the invention, of the formula tert-butyloxycarbonyl-D B-naphthylalanine EMI7.1 The octapeptide of the invention having the formula EMI7.2 was made according to a method analogous to those described above. Use When administered to mammals (e.g. orally, topically, intravenously, parenterally in a sustained release, biodegradable form, nasally, or by suppository), the compounds can be effective to inhibit GH release as well as to inhibit insulin, glucagon, and pancreatic exocrine secretion, and to therapeutically affect the central nervous system. The compounds can be administered to a mammal, e.g. a human, in the dosages used for somatostatin or, because of their greater potency, in smaller dosages. The compounds of the invention can be used for the treatment of cancer, particularly growth hormone-dependent cancer (e.g., bone, cartilage, pancreas (endocrine and exocrine), prostate, or breast), acromegaly and related hypersecretroy endocrine states, or of bleeding ulcers in emergency patients and in those suffering from pancreatitis or diarrhea. The compounds can also be used in the management of diabetes and to protect the liver of patients suffering from cirrhosis or hepatitis. The compounds can also be used to treat Alzheimer's disease, as analgesics to treat pain by acting specifically on certain opiate receptors, and as gastric cytoprotective compounds for ulcer therapy. The compounds can also be used to treat certain types of mushroom poisoning. The compounds can also be used to treat diabetes-related retinopathy. The anti-cancer activity of the compounds may be related to their ability to antagonize cancer-related growth factors such as epidermal growth factor. The compounds can be administered to a mammal, e.g., a human, in a dosage of 0.01 to 1000 mcg/kg/day, preferably 0.1 to 100 mcg/kg/day.

Claims: CLAIMS 1. An octapeptide of the formula: EMI9.1 wherein each A1 and A2, independently, is H, C1-12 alkyl, C7-10 pnehylalkyl, R1CO (where R1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkinyl, phenyl, naphthyl, or C7-10 phenylalkyl), or R20CO (where R2 is C1-10 alkyl or C7-10 phenylalkyl), provided that when one of A1 or A2 is R1CO or R20CO, the other must be H; A3 is CH2-A6 (where A6 is pentafluoro, naphthyl, pyridyl, or phenyl which is optionally ringsubstituted with a halogen, Cl-3 alkyl, NO2, C1-3 alkoxy, NH2, or OH); A4 is o-m-or, more preferably, p substituted X-Phe (where X is a halogen, H, NO2, OH, NH2, or C1-3 alkyl), pentafluoroalanine, or naphthylalanine; and As is Thr, Ser, Phe, Val, or Ile; or a pharmaceutically acceptable salt thereof. 2. An octapeptide according to Claim 1, wherein EMI9.2 is D-8-naphthylalanine. 3. An octapeptide according to Claim 1, wherein EMI9.3 is D-8-naphthylalanine, D-p-Cl-Phe, or D-Phe; A4 iS Tyr; and As is Val. 4. An octapepti de according to Claim 1, of the formula tert-butyl oxycarbonyl EMI9.4 Thr-NH2, or a pharmaceutically acceptable salt thereof. 5. An octapeptide according to Claim 1, of the formula tert-butyloxycarbonyl-D-ss-naphthylalanine-Cys-Tyr-D-Trp- Lys-Val-Cys-Thr-NH2, or a pharmaceutically acceptable salt thereof. 6. A therapeutic composition comprising a therapeutically effective amount of an octapeptide according to any preceding claim, together with a pharmaceutically acceptable carrier substance. 7. A therapeutic composition according to Claim 6, wherein said composition is in the form of a pill, tablet, or capsule for oral administration, optionally coated with a substance capable of protecting said composition from the gastric acid in the stomach of a patient for a period of time sufficient to allow said composition to pass undisintegrated into the small intestine of said patient; in the form of a liquid for oral administration; in the form of a cream, gel, spray, or ointment for application to the skin; in the form of a liquid capable of being administered nasally as drops or spray; in the form of a liquid for intravenous, subcutaneous, parenteral, or i ntrape ri ti oneal administration; or in the form of a biodegradable sustained release composition for intramuscular administration. 8. An octapeptide according to any of Claims 1 to 5 for use in therapy in inhibiting the release of or causing a reduction in growth hormone, insulin, glucagon, or pancreatic exocrine secretion, in the treatment of cancer, in the treatment of bleeding ulcers, in the management of diabetes, in the protection of the liver in patients suffering from cirrhosis or hepatitis, in Alzheimer's disease, as an analgesic, in ulcer therapy, in mushroom poisoning, or in diabetes-related retinopathy. 9. A method of synthesizing an octopeptide of the formula: EMI11.1 wherein each A1 and A2, independently, is H, C1-12 alkyl C7-10 pnehylalkyl, RlCO (where R1 is C1-20 alkyl, C3-20 alkenyl, C3-20 alkinyl , phenyl, naphthyl, or C7-10 phenylalkyl), orR20CO (where R2 is C1-10 alkyl or C7-10 phenylalkyl), provided that when one of A1 or A2 is R1CO or R20CO, the other must be H; A3 is CH2-A6 (where A6 is pentafluoro, naphthyl, pyridyl, or phenyl which is optionally ringsubstituted with a halogen, C1-3 alkyl, NO2, C1-3 alkoxy, NH2, or OH); A4 is o-m-or, more preferably, p substituted X-Phe (where X is a halogen, H, NO2, OH, NH2, or C1-3 alkyl), pentafluoroalanine, or naphthylalanine; and As is Thr, Ser, Phe, Val, or Ile; or a pharmaceutically acceptable salt thereof. comprising, forming a blocked amino acid resin of threonine, coupling successively to said threonine resin the blorkod amino acid resins cysteine, A5, lysine, D-tryptophan, A4, cysteine, and EMI11.2 washing and drying the product of said coupling, treating said washed and dried product to remove sald blocking group, and purifying said unblocked product.

Inventors: COY, DAVID H., HEIMAN, MARK L., MURPHY, WILLIAM A.
Applicants: ADMINISTRATORS OF THE  TULANE EDUCATIONAL FUND
Application year: 1985
Main classification: C07K7
IPC codes: C07K14, A61K38, C07K7, G07D9