TOXIN-TARGETED DESIGN FOR ANTICANCER THERAPY .1. SYNTHESIS AND BIOLOGICAL EVALUATION OF NEW THIOIMIDATE HETEROBIFUNCTIONAL REAGENTS

In an effort to obtain a more potent and specific immunotoxin for canc er therapy, we designed a series of heterobifunctional linkers charact erized by a thioimidate group linked to a S-acetyl thiol (4, 5) or sub stituted aryldithio group (6-10). These ligands were synthesized by a Pinner-type process from the corresponding nitrile derivatives obtaine d by thiol-disulphide exchange reaction, reaction with substituted ben zene-sulphenyl chloride, or other known procedures. To check the reage nt of choice for immunoconjugate preparation, we studied thioldisulphi de exchange kinetics between the intermediate nitrile derivatives and cysteine. Among the tested aryldithio derivatives (6-1 0), we selected ethyl 3-(4-carboxamido-phenyldithio)propionthioimidate (CDPT, 9) for further studies. By analyzing the rate of incorporation of the linkers 4, 5, and 9 in a model immunoglobulin G protein, we found similar res ults with CDPT 9 and ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT, 5) because both reagents showed a linear correla tion between the number of introduced thiol groups and factors such as time and protein and reagent concentrations. Comparison of the two ac etylthio-derivative ligands 4 and 5 showed that AMPT 5 was more stable toward deacetylation than ethyl S-acetyl 2-mercaptopropionthioimidate ester hydrochloride (AMAT, 4). By comparing the kinetic and biologica l parameters of seven new thioimidate linkers, we found that two of th ese (CDPT and AMPT) could be superior ligands for protein-protein conj ugation. They offer advantages over the commercially available compoun ds, such as minimal perturbation of the protein structure, controlled reactivity, and good stability.