CPG a metalloenzyme derived from sp was the
CPG2, a metalloenzyme derived from sp., was the elected enzyme for the first pilot-scale clinical trial of ADEPT. This enzyme has no mammalian homologue and activates glutamic SB 747651A dihydrochloride prodrug derivatives of several nitrogen mustards alkylating agents., , , , , A bond cleavable by CPG2 is essential and was constructed between the active drug and the glutamic acid moiety in the prodrug. Prodrugs with linkages –OCO– (oxycarbonyl) for the phenol nitrogen mustards and –NHCO– (carbamoyl) for the aniline nitrogen mustards have been shown to be substrates for CPG2. Triazene compounds of clinical interest, dacarbazine and temozolomide , are a group of alkylating agents with similar properties. Their mechanism of action is mainly related to methylation of DNA, mediated by methyldiazonium ion, a highly reactive metabolite of the two compounds.Of all the chemotherapeutic agents tested against this malignancy, dacarbazine (DTIC), , remains the reference drug in the treatment of metastatic melanoma., More recently a new compound has been approved by FDA, the vemurafenib which is an orally inhibitor of mutated BRAF, for the treatment of latest stages of melanoma. Related 1-aryl-3,3-dimethyltriazenes, (), are also documented as anticancer drugs, but with reduced selectivity for tumour cells. The biological action of DTIC and the dimethyltriazenes , is a consequence of their capacity to alkylate DNA and RNA via the corresponding monomethyltriazene following metabolic oxidation by cytochrome P450 enzymes ()., , , Thus, the treatment of melanoma can be improved by developing of a prodrug strategy that can specifically deliver the ultimate methylating agent to malignant cells. Since monomethyltriazenes are good leaving groups as alcohols, we considered that coupling them to carboxypeptidase substrates would identify suitable prodrugs capable of regenerating the alkylating metabolite via the pathway depicted in ., Additionally, triazenes as small molecules with adequate lipophilicity will be free to diffuse throughout the tumor. We report here the synthesis, the chemical stability studies and the evaluation of triazene prodrugs, derived from glutamic acid , as potential molecules to be converted into the cytotoxic drug MMT by CPG2 activation. Attempts to synthesize the desired compounds – proceeds via the carbamate derivatives –, these having been previously efficiently synthesized by reaction of the corresponding monomethyltriazenes () with 4-nitrophenyl chloroformate, according to the published procedure (). The reaction of pure carbamates with -glutamic acid di--butyl ester in the presence of triethylamine produced very good yields of – compounds (77–100%). Finally, the deprotection of to the potential prodrugs was achieved with trifluoracetic acid (TFA) and anisole at −10°C. The presence of anisole appears to be important for the latter reaction, since it acts like a scavenger for cations. Moreover, the presence of anisole prevents side reactions. Derivatives – were prepared in good yields and with correct spectral data described in . The chemical hydrolysis of triazene derivatives –, to the corresponding 1-aryl-3-methyltriazenes, was performed in isotonic phosphate buffer at 37°C. The reactions of the and compounds were followed by the monitoring of both the loss of the starting materials and the formation of products (MMT and amine) by HPLC () as detailed in . Prodrugs and proved to be too unstable to be studied by HPLC so their hydrolysis reactions were followed by UV. The kinetics of the hydrolysis of each prodrug followed pseudo-first-order kinetics. Half-lives of the hydrolysis in PBS are shown in . The analysis of half-lives for prodrugs reveals that their reactivity is nearly not influenced by the nature of the substituent −X (=0.65, =0.99) being the faster hydrolyzed compounds those containing electron-withdrawing substituents ().