SYNTHESIS OF A NOVEL ESTERASE-SENSITIVE CYCLIC PRODRUG OF A HEXAPEPTIDE USING AN (ACYLOXY)ALKOXY PROMOIETY

Synthetic methodology for preparing novel esterase-sensitive cyclic pr odrugs of peptides with increased protease stability and cell membrane permeability compared to linear peptides is described. Cyclic prodrug 1 of the hexapeptide H-Trp-Ala-Gly-Gly-Asp-Ala-OH linked by the N-ter minal amino group to the C-terminal carboxyl group via an (acyloxy)alk oxy promoiety was synthesized. A convergent synthetic approach involvi ng Boc[[(alaninyloxy)methyl]carbonyl]-N-tryptophan (2) and H-Ala-Gly-G ly-Asp(OBzl)-OTce (3) was used. The key fragment 2 has the promoiety i nserted between the Ala and the Trp residues. Fragment 3 was synthesiz ed by a solution-phase approach using standard Boc-amino acid chemistr y. These fragments were coupled to produce the protected linear hexape ptide, which after deprotection was cyclized using standard high-dilut ion techniques to yield cyclic prodrug 1. In pH 7.4 buffer (HBSS) at 3 7 degrees C, cyclic prodrug 1 was shown to degrade quantitatively to t he hexapeptide (t(1/2) = 206 +/- 11 min). The rate of hydrolysis of cy clic prodrug 1 was significantly faster in human blood (t(1/2) = 132 /- 4 min) than in HBSS. Paraoxon, a known inhibitor of esterases, slow ed this hydrolysis of cyclic prodrug 1 to a value (t(1/2) = 198 +/- 9 min) comparable to the chemical stability. In human blood, cyclic prod rug 1 was shown to be 25-fold more stable than the linear hexapeptide.