Toxin-targeted design for anticancer therapy. I: Synthesis and biological evaluation of new thioimidate heterobifunctional reagents.
In an effort to obtain a more potent and specific immunotoxin for cancer therapy, we designed a series of heterobifunctional linkers characterized by a thioimidate group linked to a S-acetyl thiol (4, 5) or substituted aryldithio group (6-10). These ligands were synthesized by a Pinner-type process from the corresponding nitrile derivatives obtained by thiol-disulphide exchange reaction, reaction with substituted benzene-sulphenyl chloride, or other known procedures. To check the reagent of choice for immunoconjugate preparation, we studied thioldisulphide exchange kinetics between the intermediate nitrile derivatives and cysteine. Among the tested aryldithio derivatives (6-10), 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 results with CDPT 9 and ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT, 5) because both reagents showed a linear correlation between the number of introduced thiol groups and factors such as time and protein and reagent concentrations. Comparison of the two acetylthio-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 biological parameters of seven new thioimidate linkers, we found that two of these (CDPT and AMPT) could be superior ligands for protein-protein conjugation. They offer advantages over the commercially available compounds, such as minimal perturbation of the protein structure, controlled reactivity, and good stability.
Delprino
L
L
Istituto di Chimica Farmaceutica Applicata, University of Turin, Italy.
Giacomotti
M
M
Dosio
F
F
Brusa
P
P
Ceruti
M
M
Grosa
G
G
Cattel
L
L
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D003640:Dealkylation; D004220:Disulfides / Q000138:chemical synthesis / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007096:Imidoesters / Q000138:chemical synthesis* / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D000922:Immunotoxins / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction
D003432:Cross-Linking Reagents; D004220:Disulfides; D007096:Imidoesters; D007074:Immunoglobulin G; D000922:Immunotoxins; C082811:3-(4-carboxamidophenyldithio)propionthioimidate; C082812:ethyl S-acetylpropionthioimidate