Novel Fluorinated Prodrugs for Activation by Carboxypeptidase G2 Showing Good in Vivo Antitumor Activity in Gene-Directed Enzyme Prodrug Therapy Lawrence C. Davies, Frank Friedlos, Douglas Hedley, Jan Martin, Lesley M. Ogilvie, Ian J. Scanlon, and Caroline J. Springer* Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom Received March 9, 2005 Sixteen novel polyfluorinated benzoic acid mustards have been synthesized for use in gene- directed enzyme prodrug therapy (GDEPT). Eight of these were benzoic acid L-glutamate mustards for evaluation as prodrugs and the other eight were the active drugs formed by the action of the bacterial enzyme carboxypeptidase G2 (CPG2). All of the di- and trifluorinated prodrugs were efficiently cleaved by the enzyme. In contrast, the tetrafluorinated prodrugs were found to be competitive inhibitors of CPG2, the first such inhibitors to have been described. The di- and trifluorinated prodrugs were differentially cytotoxic to human breast carcinoma cells (MDA MB 361) expressing CPG2, compared to control cells that did not express the enzyme. The difluorinated prodrug {4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid and its iodoethylamino analogue were effective substrates for the enzyme and showed excellent therapeutic activity in CPG2-expressing MDA MB 361 xenografts, either curing or greatly inhibiting tumor growth and extending the life of the animals. Introduction Gene-directed enzyme prodrug therapy (GDEPT) is a two-step approach to the therapy of cancer. 1 The foreign gene for a prodrug-activating enzyme is selec- tively introduced into the tumor by a vector, such as a virus or liposome, by either local or systemic delivery. Time is allowed for the enzyme to be expressed in the tumor, and then a nontoxic prodrug is administered. This prodrug is converted by the enzyme within the tumor to a toxic drug. This localized activation reduces the normal tissue toxicity associated with conventional cytotoxic chemotherapy. A number of enzyme/prodrug systems have been evaluated for GDEPT. 2 In our laboratory, we have concentrated on the enzyme car- boxypeptidase G2 (CPG2, glutamate carboxypeptidase, EC 3.4.17.11) derived from Pseudomonas RS16. This enzyme catalyses the cleavage of amide bonds between a carboxyl-, phenol-, or aniline-substituted aromatic ring and L-glutamic acid, an activity unknown in any mam- malian enzyme. Our GDEPT system has significant benefits in that prodrugs can be designed that release several different types of effector molecule, including aromatic mustards and antibiotics. Moreover, the active cytotoxic compound is released from the prodrug in a single step without requiring subsequent processing by host enzymes that may be lacking in tumor cells. The benzoic acid mustard-derived prodrug (2-chloroethyl)- (2-(methylsulfonyloxyethyl)amino)aminobenzyl-L-glutam- ic acid (1) is a substrate for CPG2 that releases the cytotoxic alkylating agent 4-(2-chloroethyl)(2-(methyl- sulfonyloxyethyl)amino)benzoic acid (1a) (Scheme 1). Prodrug 1 has been shown to be effective both in vitro and in vivo against tumor cells expressing CPG2, especially when the enzyme had been engineered to be expressed externally, tethered to the outer membrane of the cell. 3 When CPG2 was expressed in the cytoplasm, the efficacy was much reduced (Table 1). This has been shown to be due to poor transport of the prodrug through the cell membrane. 3 We have synthesized novel benzoic acid prodrugs (21-28) for use in GDEPT with di-, tri-, and tetrafluorinated benzene rings. These prodrugs are designed to be more lipophilic than 1 with the aim of improving intracellular access and efficacy. The substitution of a single fluorine atom at the 3-posi- tion of the aromatic ring has been shown previously by our group to improve efficacy in ADEPT (antibody- directed enzyme prodrug therapy). 4 The prodrugs were tested for their chemical half-life, as substrates of CPG2, and for cytotoxicity against a human breast carcinoma cell line (MDA MB 361) expressing CPG2, either cyto- plasmically or tethered to the outer surface of the cell membrane. Prodrugs active in the latter assay were tested for therapeutic efficacy in xenografts of the same cell line, growing in nude mice. * To whom correspondence should be addressed. Phone: 44 20 73528133x4214. Fax: 44 20 87224046. E-mail: caroline.springer@ icr.ac.uk. Scheme 1. Hydrolysis of Prodrug 1 by Carboxypeptidase G2 5321 J. Med. Chem. 2005, 48, 5321-5328 10.1021/jm0502182 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/12/2005