Design of low molecular weight hematoregulatory agents from the structure-activity relationship of a dimeric pentapeptide.

We report herein, a new class of simple hematoregulatory semipeptides, formally derived from the cystine-dimerized peptide pGlu-Glu-Asp-Cys-Lys-OH, where the disulfide bond has been replaced by an isosteric dicarba bridge. The structure-activity relationship (SAR) of a series of analogues incorporating replacements at positions 1 and 2 of peptide 1 led to the design of active conformationally constrained cyclic peptides (12, 13). Ring closure was achieved by cyclization of the N-terminal amino groups at position 2 of peptide 2 using pyrazine-2,3-dicarboxylic acid. Subsequent excision of the putative C-terminal scaffold domain from the active cyclic peptides resulted in the discovery of a new class of low molecular weight hematoregulatory agents exemplified by compound 16. This semipeptide analogue, comprising two D-Ser residues connected via amide bonds to the acid groups of pyrazine-2,3-dicarboxylic acid, had comparable biological activity to the lead peptide 1. The stereochemical requirements for the observed biological activity of these novel compounds were examined. Furthermore, the hematopoietic synergistic activity induced by compound 16 in stromal cell cultures was blocked by an antibody known to neutralize the hematoregulatory effect of 1, indicating a common mechanistic end point. Compounds of the class typified by 16 may form the basis for the development of novel therapeutic agents within the area of immunoregulation.

Unexpected lability of cysteine acetamidomethyl thiol protecting group. Tyrosine ring alkylation and disulfide bond formation upon acidolysis.

Cleavage and deprotection of the peptidyl resin H-Asn-Gly-Gly-Cys (Acm)-Glu(OBu(t))-Gln-Tyr(Bu(t))-Cys(Acm)-Ser(Bu(t))-Asp( OBU(t))-[(p-alkoxy)benzyloxy polystyrene resin] using standard conditions with various trifluoroacetic acid-containing mixtures were found to result in partial removal of ordinarily acid-stable S-Acm groups. Thus, apart from the desired peptide H-Asn-Gly-Gly-Cys (Acm)-Glu-Gln-Tyr-Cys(Acm)-Ser-Asp-OH, a disulfide-cyclic peptide derivative was also isolated. Furthermore, it was found that in another major by-product of the peptide resin cleavage the tyrosine side chain had been alkylated with an Acm group in a position ortho to the phenolic function. The formation of both by-products could be suppressed by carrying out the cleavage/deprotection reaction at higher dilution and by inclusion of scavengers such as phenol. An authentic sample of the disulfide-cyclic peptide was obtained by oxidation of H-Asn-Gly-Gly-Cys-Glu-Gln-Tyr-Cys-Ser-Asp-OH using Ellman's reagent.