Synthesis and pharmacology of novel analogues of oxytocin and deaminooxytocin: directed methods for the construction of disulfide and trisulfide bridges in peptides.

Abstract

Using as models the neurohypophyseal nonapeptide hormone oxytocin and its analogue deaminooxytocin, several directed routes to formation of sulfur-sulfur bridges have been developed and evaluated. The linear sequences (through common octapeptide-resin intermediates) were assembled smoothly on tris(alkoxy)benzylamide (PAL) poly(ethylene glycol)-polystyrene (PEG-PS) graft supports, using stepwise Fmoc solid-phase chemistry. Side-chain protection of beta-mercaptopropionic acid (Mpa) and/or cysteine (Cys) was provided by S-2,4,6-trimethoxybenzyl (Tmob), S-acetamidomethyl (Acm), and/or a series of sulfenyl thiocarbonate and carbamoylsulfenyl protecting/activating groups: S-(methoxycarbonyl)sulfenyl (Scm), S-(methoxycarbonyl)disulfanyl (Sscm), S-(N-methyl-N-phenylcarbamoyl)sulfenyl (Snm), and S-(N-methyl-N-phenylcarbamoyl)disulfanyl (Ssnm). Thiolytic displacement of S-Snm (preferred) or S-Scm provided intramolecular cyclized peptide disulfides, and homologation of the chemistry with S-Ssnm (again preferred) and S-Sscm provided the corresponding trisulfides along with smaller amounts of disulfides and tetrasulfides. These chemistries could be implemented both in solution and in solid-phase modes. Various parameters were studied systematically and optimized, and the novel trisulfides of oxytocin and deaminooxytocin were synthesized and purified to homogeneity. The trisulfide compounds were evaluated in three assays: uterotonic in vitro, uterotonic in vivo, and pressor tests, and they showed substantial potencies, ranging from 5% to 40% of the parent (disulfide) activities, as well as protracted actions. The affinities of the peptide trisulfides to uterine membrane receptors were only 3.3-3.6-fold lower than those of the parent disulfides. Possible explanations of the biological results are discussed.