Nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b: 2',3'-e][1,4]diazepin-6-one, NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus (HIV). NVP is carcinogenic in experimental animals; however, the mechanism for its tumorigenicity is not known. NVP metabolism involves oxidation of the benzylic 4-methyl substituent to 4-hydroxymethyl-NVP (12-hydroxy-NVP), and ring hydroxylation to phenolic derivatives. Further metabolism, either through oxidation of the phenols to quinoid derivatives or Phase II esterification of the hydroxymethyl substituent, may produce electrophilic species capable of reacting with DNA to yield covalent adducts. These adducts could potentially be involved in the initiation of mutagenic and carcinogenic events. We have synthesized 12-O-mesyl-NVP to serve as a model electrophile for reactive esters of 12-hydroxy-NVP and investigated its reactivity towards deoxynucleosides and DNA. Reactions with the deoxynucleosides (deoxyguanosine, deoxyadenosine, and deoxycytidine) were conducted at 37ºC in tetrahydrofuran/dimethylformamide/water. In each instance, DNA adducts were isolated by preparative reversed-phase HPLC and fully characterized by proton and carbon-13 NMR, and mass spectrometry. Salmon testis DNA in 5 mM Bis-Tris, 0.1 mM EDTA (pH 7.1) was similarly reacted with 12-O-mesyl-NVP, enzymatically hydrolyzed to nucleosides, and chromatographed. DNA adducts were isolated and compared to those obtained from the reactions with deoxynucleosides, on the basis of HPLC retention times, UV and mass spectra and, whenever possible, NMR spectra. In parallel experiments, aimed at producing significant amounts of NVP-DNA adduct standards, we tested an alternative synthetic strategy by reacting 12-bromo-NVP with 3’,5’-O-bis(tert-butyldimethylsilyl)-deoxynucleosides in toluene, under palladium(0) catalysis and using cesium carbonate for catalyst regeneration. Following desilylation of the deoxyribosyl moiety with tetrabutylammonium fluoride, DNA adducts were isolated and characterized as described above. Multiple adducts from deoxyguanosine, deoxyadenosine, and deoxycytidine were isolated in all reactions. Regardless of the synthetic approach, the adduct structures consistently involved binding through C12 of NVP and either the exocyclic heteroatoms of the DNA bases or the endocyclic nitrogens of the purines, with subsequent depurination. Our results suggest that metabolism of NVP to 12-hydroxy-NVP could potentially be a factor in NVP carcinogenicity.

Ackowledgement: Fundação para a Ciência e a Tecnologia and FEDER are gratefully acknowledged for a research grant (POCI/QUI/56582/2004) and postdoctoral fellowships to AMMA and MPD. Support by the NTP is also acknowledged. AMMA is currently affiliated with REQUIMTE/CQFB, FCT-UNL, Caparica, Portugal.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA