Triazene Drug Metabolites. Part 17: Synthesis and Plasma Hydrolysis of Acyloxymethyl Carbamate Derivatives of Antitumour Triazenes Emõ Â lia Carvalho, a, * Ana Paula Francisco, a Jim Iley b and Eduarda Rosa a, * a Centro de Estudos de Cie Ãncias Farmace Ãuticas (CECF), Faculdade de Farma Âcia, Universidade de Lisboa, Avenida das Forc Ë as Armadas, 1600 Lisbon, Portugal b Chemistry Department, The Open University, Milton Keynes, MK7 6AA, UK Received 4 December 1999; accepted 15 March 2000 AbstractÐA series of 3-acyloxymethyloxycarbonyl-1-aryl-3-methyltriazenes 5 was synthesised by the sequential reaction of 1-aryl- 3-methyltriazenes with (i) chloromethyl chloroformate, (ii) NaI in dry acetone, and (iii) either the silver carboxylate or the car- boxylic acids in the presence of silver carbonate. The hydrolysis of these compounds was studied in pH 7.7 isotonic phosphate buer and in human plasma. Triazene acyloxycarbamates demonstrated their ability to act as substrates for plasma enzymes. For compound 5f, a pH±rate pro®le was obtained which showed the hydrolysis to involve acid±base catalysis. The reaction is also buer catalysed. Thus, at pH 7.7, pH-independent, base-catalysed and buer-catalysed processes all contribute to the hydrolysis reaction. The sensitivity of the hydrolysis reaction to various structural parameters in the substrates indicates that hydrolysis occurs at the ester rather than the carbamate functionality. In plasma, the rates of hydrolysis correlate with partition coecients, the most lipo- philic compounds being the most stable. An aspirin derivative suers two consecutive enzymatic reactions, the scission of the aspirin acetyl group being followed by the scission of the acyloxy ester group. These results indicate that triazene acyloxymethyl carbamates are prodrugs of the antitumour monomethyltriazenes. They combine chemical stability with a rapid enzymatic hydro- lysis, and are consequently good candidates for further prodrug development. Moreover, this type of derivative allowed the synthesis of mutual prodrugs, associating the antitumour monomethyltriazenes with anti-in¯ammatory NSAIDs as well as with the anticancer agent butyric acid. # 2000 Elsevier Science Ltd. All rights reserved. Introduction For many years dacarbazine 1 (Fig. 1) has been one of the most widely used drugs to treat malignant melanoma. The incidence of this disease has increased recently and is expected to continue in the near future. 1 The biological action of dacarbazine and, in general, the anticancer 1-aryl-3,3-dimethyltriazenes (2, in Fig. 2) is a consequence of their capacity to alkylate DNA. These compounds suer metabolic oxidation by cytochrome P450 enzymes to give hydroxymethyltriazenes 3, which, by loss of formaldehyde, generate the cytotoxic mono- methyltriazenes 4. These are known alkylating agents, capable of methylating DNA and RNA (Fig. 2). 2,3 With the objective of ®nding suitable prodrugs that by-pass the need for oxidative metabolism, 4 various derivatives of 3 and 4 have been synthesised, such as esters, 5 ethers, 6 thioethers, 7 and acyl derivatives. 8,9 None of these possess ideal prodrug characteristics. More recently, an imidazotetrazine Ð temozolomide Ð has been found to be active against several tumours in phase I clinical studies. Its activity is thought to be due to the genera- tion of an alkylating monomethyltriazene by reaction with water. 10 Anticancer triazenes containing amino acid carriers have also been studied. 11,12 One such com- pound, an N-acetylalanyl derivative, displayed chemical stability in isotonic phosphate buer yet rapid hydro- lysis to the monomethyltriazene in plasma, indicating it to be a promising candidate for prodrug development. As part of our ongoing work directed toward the development of triazene prodrugs, we have extended our investigations to encompass the acyloxymethyl car- bamates 5 (Fig. 3). The acyloxymethyl carbamate func- tionality has been proposed as a promoiety for primary and secondary amines. Such compounds suer esterase- catalysed hydrolysis of the ester function, producing an 0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(00)00100-0 Bioorganic & Medicinal Chemistry 8 (2000) 1719±1725 *Corresponding author. Tel.: +351-217946477; fax: +351-21794670; e-mail: merosa@.ul.pt