Fibroblast growth factor antagonists

Claim 1

A process for production of a peptide compound of either of the general forumlae (l) and (ll) below or a biologically active fragment thereof which functions as an FGF antagonist, binds with heparin or binds with the FGF receptor:- (l) Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-­Pro-Asp-Gly-Arg-Val-Asp-R₄₂-Val-Arg-Glu-Lys-R₄₇-Asp-Pro-­His-Ile-Lys-Leu-Gln-Leu-Gln-Leu-Ala-Glu-Glu-Arg-Gly-Val-Val-­Ser-Ile-Lys-Gly-Val-Y (ll) Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-­Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-­Lys-Arg-Y wherein Y is OH or NH₂, R₄₂ is Gly, Ala or Sar and R₄₇ is Ser, Ala or Thr which process comprises (i) forming a peptide intermediate having at least one protecting group and either of the general formulae (la) and (lla) below:- (la) X¹-Tyr(X²)-Cys(X⁴)-Lys(X⁷)-Asn(X⁸)-Gly-Gly-Phe-­Phe-Leu-Arg(X⁶)-Ile-His(X⁹)-Pro-Asp(X³)-Gly-Arg(X⁶)-Val-­Asp(X³)-R₄₂-Val-Arg(X⁶)-Glu(X³)-Lys(X⁷)-R₁47(X⁵)-Asp(X⁵)-Asp(X³)-Pro-­His(X⁹)-Ile-Lys(X⁷)-Leu-Gln(X⁸)-Leu-Gln(X⁸)-Ala-Glu(X³)-­Glu(X³)-Arg(X⁶)-Gly-Val-Val-Ser(X⁵)-Ile-Lys(X⁷)-Gly-Val-­X¹⁰ (lla) X¹-Phe-Phe-Phe-Glu(X³)-Arg(X⁶)-Leu-Glu-(X³)-­ Ser(X⁵)-Asn(X⁸)-Asn(X⁸)-Tyr(X²)-Asn(X⁸)-Thr(X⁵)-Tyr(X²)-­Arg(X⁶)-Ser(X⁵)-Arg(X⁶)-Lys(X⁷)-Tyr(X²)-Ser(X⁵)-Ser(X⁵)-­Trp-Tyr(X²)-Val-Ala-Leu-Lys(X⁷)-Arg(X⁶)-X¹⁰. wherein X¹ is hydrogen or an alpha-amino protecting group, X² is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, Bzl, CBZ, 4Br-CBZ and 2,6-dichlorobenzyl; X³ is hydrogen or an ester-forming protecting group for the carboxyl group of Asp or Glu and is selected from the group consisting of Bzl, cyclohexyl, cycloheptal, 2,6-­dichlorobenzyl, methyl and ethyl; X⁴ is hydrogen or a protecting group for Cys selected from the group consisting of p-methoxy-benzyl(MeOBzl), p-­methylbenzyl, acetamidomethyl, trityl and Bzl; X⁵ is hydrogen or a protecting group for the hydroxyl group of Thr and Ser and is selected from the group consisting of acetyl, benzoyl, tert-butyl, trityl, tetrahydropyranyl, Bzl, 2,6-dichlorobenzyl and CBZ; X⁶ is a protecting group for the guanido group of Arg selected from the group consisting of nitro, Tos, CBZ, adamantyloxycarbonyl, and BOC, or is hydrogen; X⁷ is hydrogen or a protecting group for the side chain amino substituent of Lys; X⁸ is a hydrogen or a protecting group for the side chain amido group of Gln and/or Asn; X⁹ is a hydrogen or a protecting group for the imidazole nitrogen of His; and X¹⁰ is selected from the class consisting of OH, OCH₃, esters, amides, hydrazides, -O-CH₂-resin support and -NH-­resin support; provided that at least one of X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, and X¹⁰ is a protecting group; and (ii) splitting off the protecting group(s) from said intermediate or cleaving a bond between the sequence and a resin support to produce the peptide, the process optionally including the step of converting said peptide to a non-toxic addition salt thereof.

Claim 3

A method of producing an FGF antagonist comprising: obtaining a DNA chain that encodes a polypeptide containing either of the following sequences: (l) Tyr-­Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-­Arg-Val-Asp-R₄₂-Val-Arg-Glu-Lys-R₄₇-Asp-Pro-His-Ile-Lys-­Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-­Gly-Val, wherein R₄₂ is Gly, Ala or Sar and R₄₇ is Ser, Ala or Thr; or (ll) Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-­Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-­Val-Ala-Leu-Lys-Arg or a biologically active fragment of either which functions as an FGF antagonist, binds with heparin or binds with the FGF receptor. inserting said DNA chain into a cloning vector in proper relationship to DNA sequences which promote expression of said encoded polypeptide, transforming an organism or cell line with said cloning vector having said inserted DNA chain, culturing said transformed organism or cell line, and obtaining said FGF polypeptide produced thereby.

Claim 4

A method of producing an FGF antagonist comprising: obtaining a DNA chain that encodes a polypeptide containing either of the following sequences: (l) Tyr-­Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-­Arg-Val-Asp-R₄₂-Val-Arg-Glu-Lys-R₄₇-Asp-Pro-His-Ile-Lys-­Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-­Gly-Val, wherein R₄₂ is Gly, Ala or Sar and R₄₇ is Ser, Ala or Thr; or (ll) Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-­Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-­Val-Ala-Leu-Lys-Arg or a biologically active fragment of either which functions as an FGF antagonist, binds with heparin or binds with the FGF receptor. inserting said DNA chain into a cloning vector in proper relationship to DNA sequences which promote expression of said encoded polypeptide, transforming an organism or cell line with said cloning vector having said inserted DNA chain, culturing said transformed organism or cell line, and obtaining said FGF polypeptide produced thereby.