Self-immolative nitrogen mustard prodrugs for suicide gene therapy

Four new potential self-immolative prodrugs derived from phenol and aniline nitrogen mustards, four model compounds derived from their corresponding f luoroethyl analogues and two new self-immolative linkers were designed and synthesized for use in the suicide gene therapy termed GDEPT (gene-directed enzyme prodrug therapy). The self-immolative prodrugs were designed to be activated by the enzyme carboxypeptidase G2 (CPG2) releasing an active drug by a 1,6-elimination mechanism via an unstable intermediate. Thus, N-[(4-{ [4-(bis{2-chloroethyl}amino)phenoxycarbonyloxy]methyl}phenyl)carbamoyl]-L-g lutamic acid (23), N-[(4-{[4-(bis{2-chloroethyl}amino)phenoxycarbonyloxy]me thyl}phenoxy)carbonyl]-L-glutamic acid (30), N-[(4-{[N-(4-{bis[2-chloroethy l]amino}phenyl)carbamoyloxy]methyl}phenoxy)carbonyl]-L-glutamic acid (37), and N-[(4-{[N-(4-{bis[2-chloroethyl]amino}phenyl)carbamoyloxy]methyl}phenyl )carbamoyl]-L-glutamic acid (40) were synthesized. They are bifunctional al kylating agents in which the activating effects of the phenolic hydroxyl or amino functions are masked through an oxycarbonyl or a carbamoyl bond to a benzylic spacer which is itself linked to a glutamic acid by an oxycarbony l or a carbamoyl! bond. The corresponding fluoroethyl compounds 25, 32, 42, and 44 were also synthesized. The rationale was to obtain model compounds with greatly reduced alkylating abilities that would be much less reactive with nucleophiles compared to the corresponding chloroethyl derivatives. Th is enabled studies of these model compounds as substrates for CPG2, without incurring the rapid and complicated decomposition pathways of the chloroet hyl derivatives. The prodrugs were designed to be activated to their corres ponding phenol and aniline nitrogen mustard drugs by CPG2 for use in GDEPT. The synthesis of the analogous novel parent drugs (21b, 51) is also descri bed. A colorectal cell line was engineered to express CPG2 tethered to the outer cell surface. The phenylenediamine compounds were found to behave as prodrugs, yielding IC50 prodrug/IC50 drug ratios between 20- and 33-fold (f or 37 and 40) and differentials of 12-14-fold between CPG2-expressing and c ontrol LacZ-expressing clones. The drugs released are up to 70-fold more po tent than 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoic acid that result s from the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-gluta mic acid (CMDA) which has been used previously for GDEPT. These data demons trate the viability of this strategy and indicate that self-immolative prod rugs can be synthesized to release potent mustard drugs selectively by cell s expressing CPG2 tethered to the cell surface in GDEPT.