Development of triazene prodrugs for ADEPT strategy: New insights into drug delivery system based on carboxypeptidase G2 activation Verónica Capucha, Eduarda Mendes ⇑ , Ana Paula Francisco, M. Jesus Perry Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Professor Gama Pinto, 1649-003 Lisbon, Portugal article info Article history: Received 27 July 2012 Revised 6 September 2012 Accepted 10 September 2012 Available online 18 September 2012 Keywords: Triazenes ADEPT Carboxypeptidase G2 Intramolecular catalysis abstract Six novel urea triazene prodrugs have been synthesized to apply in antibody-directed enzyme prodrug therapy (ADEPT). The chemical and plasmatic stability of L-glutamate triazene prodrugs were evaluated and the chemical reactivity was mainly attributed to an intramolecular catalysis promoted by the neigh- bouring carboxylate group of the glutamic moiety. These prodrugs showed an elevated binding to plasma proteins. The L-glutamate triazenes were evaluated as prodrugs of the alkylating agent’s monomethyl- triazenes, by activation of the bacterial enzyme carboxypeptidase G2 (CPG2). The synthesized prodrugs have been shown to be good substrates for CPG2, and therefore new candidates for ADEPT strategy. Ó 2012 Elsevier Ltd. All rights reserved. Systemic cancer therapy is limited by a lack of tumor selectivity, which leads to harmful side effects in normal tissues, and by drug resistance. Antibody-directed enzyme prodrug therapy (ADEPT) is designed to overcome both problems. 1–4 ADEPT is a two step ap- proach for the treatment of cancer which seeks to generate a po- tent cytotoxic agent selectively at the tumor site or its metastases. In the first step, a tumor selective antibody chemically linked to an enzyme (antibody–enzyme conjugate) is administered and allowed to fix itself on the tumor site. In the second step after a suitable time, to allow the clearance of the antibody–enzyme con- jugate from other tissues, the process is followed by the adminis- tration of a relatively nontoxic prodrug. This prodrug will be enzymatically converted into the active drug only at the tumor site and the usually nonspecific cytotoxicity will be minimized. The ac- tive drug is generated extracellularly with a higher concentration and diffuses itself throughout the tumor mass, killing not only cells expressing tumor antigen but also neighboring antigen-negative tumor cells. Thus selectivity is achieved by the tumor specificity of the antibody and by delaying prodrug administration until there is a large differential between tumor and normal tissue enzyme levels. Drug resistance can be overcome by generating high levels of an alkylating agent in the tumor, and this is achieved through the capacity of each enzyme to convert many molecules of prodrug into drug. Such a complex system has already been applied in clin- ical trials and in the last few years, a number of new monoclonal antibodies-based drugs have been clinically approved (Rituxan, Herceptin, Erbitux, Avastin and Mylotarg), and numerous other mAb-based agents have also progressed to phase II/phase III clini- cal trials in cancer. 5,6 CPG2, a metalloenzyme derived from Pseudomonas sp., was the elected enzyme for the first pilot-scale clinical trial of ADEPT. This enzyme has no mammalian homologue and activates glutamic acid prodrug derivatives of several nitrogen mustards alkylating agents. 7–12 A bond cleavable by CPG2 is essential and was con- structed 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. 7 Triazene compounds of clinical interest, dacarbazine 1 and tem- ozolomide 2, are a group of alkylating agents with similar proper- ties. Their mechanism of action is mainly related to methylation of DNA, mediated by methyldiazonium ion, a highly reactive metab- olite of the two compounds. 13 1 N H N N N Me Me O N H 2 N 2 N H 2 O N O N Me N N N Of all the chemotherapeutic agents tested against this malignancy, dacarbazine (DTIC), 1, remains the reference drug in the treatment 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.09.029 ⇑ Corresponding author. Tel.: +351 21 7946400; fax: +351 21 7946470. E-mail address: ermendes@ff.ul.pt (E. Mendes). Bioorganic & Medicinal Chemistry Letters 22 (2012) 6903–6908 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl