In cancer treatment, targeting the cellular DNA or its nucleotide precursors has been a viable method for inhibiting the synthesis of new genetic material or causing irreparable damage to the DNA itself. For example, agents such as bleomycins oxidatively degrade DNA, whereas cisplatin forms interstrand cross-links within DNA. Issues of selectivity and delivery remain a major problem in the development of new reactive agents. Typically, covalent reactions are irreversible, suppressing the potency of an agent. Reversible covalent reactions, in contrast, provide a strategy for recovering the reactive potential despite initial interaction with non-targeted constituents. Adding bioactivation to a reversible agent should ensure better selectivity and control for chemotherapy. Quinone methide precursors (QMP) are appropriate for this approach. Reactive and transient QMs and related electrophiles are formed during the metabolic activation of cytotoxic and genotoxic agents and may lead to DNA alkylation. Adducts formed between QMs and deoxyadenosine, deoxyguanosine, deoxycytosine, thymidine have been identified; reversible reaction dominates the initial product profile. We have elected to use dC which forms one reversible adduct to measure the relative reversibility of QM reaction vs bioactivity. We synthesized a series of EHBPs and determined the time-dependent evolution of their dC adduct.


QMP: para-series, (agent 1) 4-acetyloxybenzyl diethylphosphate; (agent 2) 3,5-dimethyl-4-acetyloxybenzyl diethylphosphate; meta-series, (agent 3) 3-acetyloxybenzyl diethylphosphate; (agent 4) 2-chloro-3-acetyloxybenzyl diethylphosphate were synthesized, purified, and NMR verified. Adduct analysis: reverse phase-HPLC, unsubstituted and substituted QMPs were incubated ± esterase at 37 °C over 0.5 - 120 h.


Retention times for dC, internal standard, and all putative QMPs were first determined. Agent 1: Time course hydrolysis by the esterase was essentially complete in 4 hr, resulting in formation of dC and water adducts. The dC adduct formation was maximum at 6 h, thereafter slowly decomposing, with a half-life of approx. 21 h. Time course for formation of the water adduct showed that the product yield remained constant for 6-8 hrs indicating a stable product. These results are consistent with a rate determining hydrolysis of carboxylic ester group by the esterase giving a phenol that in turn rapidly undergoes a 1,6-elimination reaction to a QM. This then reacts with water and dC competitively to produce the identified adducts. Agent 2: Esterase reaction gave similar products to agent 1. However, the half-life for the decomposition of the dC adduct was significantly less (about 10 h) underscoring the effect of the electron releasing groups. Agent 3: Esterase hydrolysis yielded the phenol derivative which underwent so benzylic substitution with water yielding the corresponding benzyl alcohol, dC-N3 adduct did not form. Agent 4: Similar results to Agent 3, but the hydrolysis appeared to be much faster and incomplete.


dC adduct formation was reversible, electron donating/withdrawing groups significantly affect the rate of adduct formation/decomposition. The meta analog did not form a QM. These results underscore the potential use of EHBPs as chemotherapeutic agents.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):B109.

Seventh AACR International Conference on Frontiers in Cancer Prevention Research-- Nov 16-19, 2008; Washington, DC