OXIDATION OF THE NEVIRAPINE METABOLITE, 2-HYDROXY-NEVIRAPINE, WITH FRÉMY’S SALT: UNUSUAL PYRIDINE RING CONTRACTION

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV transmission in developing countries. One of the limitations of NVP use is severe hepatotoxicity, which raises concerns about its chronic administration, particularly in the perinatal and pediatric settings. The reasons for the adverse effects of NVP administration are currently not clear, although there is increasing evidence that metabolic activation to reactive electrophiles capable of reacting with bionucleophiles is likely to be involved in the initiation of toxic responses. Phase I NVP metabolism involves oxidation of the 4-methyl substituent to 12-hydroxy-NVP, and the formation of phenolic derivatives that are conceivably capable of undergoing further metabolic oxidation to electrophilic quinoid species prone to react with bionucleophiles. The covalent adducts thus formed might be at the genesis of toxic responses. As part of a program aimed at evaluating the possible contribution of quinoid derivatives of Phase I phenolic NVP metabolites to the toxic responses elicited by the parent drug, we have investigated the oxidation of 2-hydroxy-NVP with dipotassium nitroso-disulfonate (Frémy’s salt), mimicking the one-electron oxidation involved in enzyme-mediated metabolic oxidations. We report herein the isolation and full structural characterization (by NMR, MS and X-ray diffraction) of a 1H-pyrrole-2,5-dione derivative as a major product, stemming from an unusual pyridine ring contraction.

Despite the high efficacy of the drug, favorable lipid profile [7] and suitability for use during pregnancy and breastfeeding [8,9], NVP therapy is associated with toxic events. Among these, skin rash is the most frequent and hepatotoxicity is the most severe [10]. These adverse side effects raise concerns about the chronic use of the drug, particularly in the perinatal and pediatric settings.
While the reasons for the adverse effects of NVP are still unclear, increasing evidence suggests that metabolic activation to highly reactive electrophiles, prone to react with bionucleophiles, has a role in the initiation of the toxic responses. In all species investigated, cytochrome P450 (CYP)-mediated Phase I metabolism of NVP yields 2-, 3-, 8-, and 12-hydroxy-NVP, and 4carboxy-NVP (2-6, Scheme 1) [11][12][13]. Glucuronidation, and subsequent renal excretion of the conjugates, is the major detoxification pathway for these metabolites.
Scheme 1 Structures of Nevirapine (1) and its Phase I metabolites.
It has been suggested that NVP oxidation to 12-hydroxy-NVP (5), probably involving subsequent Phase II activation, is the pathway responsible for a skin rash in rats that resembles the rash in humans [14][15][16]. However, other metabolic pathways may play a role in the generation of NVP-derived reactive electrophiles, as demonstrated by Srivastava et al. [17], who identified the mercapturate through the C3 position of NVP (7) in the urine of NVP-treated patients (cf. Scheme 2). This adduct was suggested to be formed by initial gluthatione (GSH) attack to an oxirane intermediate (8), yielding the GSH adduct 9 which underwent anabolism to 7. Alternatively phenolic NVP metabolites may undergo metabolic activation to quinone/semiquinone electrophiles (eg., 10) capable of reacting with bionucleophiles through Michael-type addition and/or Schiff-base formation, leading to covalent adduct formation catalysis, together with the presence of NVP in breast milk [19] and the frequent administration of the drug concurrently with breastfeeding, suggest that a quinone-imine-mediated pathway could be at the onset of adverse drug reactions in the perinatal setting. Despite our efforts toward a thorough characterization of all reaction products, at least one product from the oxidation of 2 with dipotassium nitrosodisulfonate (Frémy's salt), remained unidentified [18]. As a further contribution to understand the reactivity of quinoid derivatives from phenolic NVP metabolites, we report herein the isolation and full structural characterization (by NMR, MS and X-ray diffraction) of a 1H-pyrrole-2,5-dione derivative, stemming from an unusual pyridine ring contraction, as the major product of 2-hydroxy-NVP oxidation with Frémy's salt.

Experimental section
Chemicals. NVP was purchased from Cipla (Mumbai, India). All other commercially available reagents and enzymes were acquired from Sigma-Aldrich Química, S.A. (Madrid, Spain), unless specified otherwise, and were used as received. 2-hydroxy-NVP was prepared by reaction with silver acetate/iodine, followed by basic hydrolysis, as described in Antunes et al. [18]. Whenever necessary solvents were purified by standard methods [20].
Instruments Infrared (IR) spectra were recorded on a Perkin-Elmer 683 FTIR spectrometer; group frequencies are reported in cm All non-hydrogen atoms were refined anisotropically, and the hydrogen atoms were inserted in idealized positions, riding on the parent C atom, except for the methyl hydrogens, whose orientation was refined from electron density, allowing the refinement of both C-C torsion angles and C-H distances, and the hydrogen atoms bonded to nitrogens, which were found directly in the density map. Drawings were made with ORTEP3 for Windows [26].

Results and Discussion
As Similarly to what was observed with all other products from 2-hydroxy-NVP oxidation [18], an initial inspection of the 1 H and 13 C NMR spectra of product 14 (cf. Experimental section) promptly allowed the conclusion that ring C (Scheme 1) remained unchanged in this derivative, whereas a substantial degradation of rings A and B had occurred during the oxidation process.
The lack of aromatic protons on ring A and the 3-bond 1 H- 13 C correlations observed on the HMBC spectrum (Figure 1) between the methyl protons H6' (2.19 ppm) and carbon C5' (171.8 ppm), with a resonance compatible with a carbonyl group, initially suggested the formation of a quinone structure, which was in agreement with the presence of three carbonyl groups in the molecule, inferred from the IR and 13 C NMR data (cf. Experimental section). However, the presence of three labile protons in the 1 H NMR spectrum, together with the presence of only 13 distinct carbons in the 13 C NMR spectrum and the indication from the mass spectral data, obtained by electrospray ionization (ESI), that the protonated molecule had m/z 287, were not consistent with a quinone structure. Conclusive evidence for the structural assignment was only obtained by X-ray diffraction, which showed that 14 is composed of a nicotinamide framework with a cyclopropylamino substituent at position C2 and a methyl-2,5-dioxo-2,5-dihydro-1Hpyrrole substituent at the amide nitrogen (Figure 2), stemming from ring B opening and an unusual pyridine ring A contraction of the parent compound, 2-hydroxy-NVP.  The structure of 14 obtained by X-ray diffraction analysis was found to be in agreement with the remaining spectral data. The X-ray crystallographic data indicated that 14 crystallized in the triclinic P-1 space group, with two crystallographically independent molecules in the asymmetric unit. Both types of molecules of compound 14 include a large planar system, with bonds displaying angles close to 120º between them (see Table 1 (7) 61.55 (7) N8-C9-C10 120.07(11) 119.08(11) C9-C11-C10 59.97 (8) 60.00(9) C9-C10-C11 59.70 (8) 59.95(9) C10-C9-C11 60.33 (8) 60.05(9)

Conclusion
The oxidation of the phenolic NVP metabolite 2-hydroxy-NVP with Frémy's salt, both at pH 7.4 and pH10, yielded the 1H-pyrrole-2,5-dione derivative 14 as a major product, stemming from an unusual pyridine ring contraction. Although the significance of this NVP derivative in vivo remains to be established, the considerable structural degradation of the parent drug, leading to a mass increment inconsistent with that expected from direct oxidation alone, may explain why 14 has eluded detection in previous NVP metabolism studies, both in vitro and in vivo, which have been conducted with LC-MS detection. The availability of this fully characterized oxidation product is a valuable tool to assess its formation in vivo, as a further effort to establish the metabolic pathways that convert NVP into reactive electrophiles. Based upon structural considerations, reaction of 14 with bionucleophiles is conceivable, and a potential role for this compound in the onset of toxic responses elicited by NVP cannot be excluded. This new NVP derivative is now accessible for further molecular toxicology studies that are expected to clarify the relevance of phenolic NVP metabolites and their oxidation products to the toxic events associated with the parent drug.