LY207702 (1) is a difluorinated purine nucleoside that exhibits impressive antitumor activity in preclinical models. This agent, however, also possesses cardiotoxicity which limits the potential clinical utility of this novel drug candidate. We therefore developed linker chemistry whereby regioselective N6-tritylation of LY207702 (1) allowed this drug to be coupled to epsilon-lysine amino groups of mAb's reactive with human tumor-associated antigens. The resulting immunoconjugates 3 possessed conjugation ratios ranging from 5 to 7 mol of LY207702/mol of mAb, minimal aggregate content (5-10%), and good immunoreactivity. The electronic nature of substituents on the aromatic rings of the trityl group dictated the degree of acid lability of the trityl linker. Increased electronic stabilization of the transient trityl carbocation led to increase in the release rate of free drug, i.e., m-DMT 10a = p-DMT 10b > p-MMT 10d > p-T 10f. Consequently, the more acid labile DMT conjugates 3a and 3b proved to be the most potent cytotoxic agents, and the most stable p-T conjugate 3f exhibited the least antitumor activity when evaluated in vitro and in vivo. p-MeT-linked conjugate 3e, the most stable construct that retained excellent in vivo antitumor activity, was selected for more extensive evaluation. No detectable free drug or metabolite was observed in mouse plasma at a single intravenous dose of p-MeT conjugate 3e, which was consistent with its predicted stability under physiological conditions. This construct did, however, exhibit significant antigen-mediated antitumor activity in vivo. No cardiotoxicity was detected in mice dosed with conjugate 3e (6 mg/kg free drug content per day for 21 days) equivalent to approximately 8 times the total dose required for complete regression of well-established (approximately 1 g) HC1 human colon tumor xenografts in nude mice. Cardiotoxicity was induced in 20% of free drug 1 treated group at the equivalent dose. Cardiomyopathy was, however, observed when the dose of conjugate 3e was increased to 8 mg/kg per day for 21 days. These data suggest that antitumor activity of LY207702 (1) was maintained and its cardiotoxic potential reduced when this agent was administered to human tumor xenograft bearing nude mice as COL1-N6-p-MeT-207702 conjugate 3e.

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The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.

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