Synthesis and evaluation of nevirapine analogs to study the metabolic activation of nevirapine.

Nevirapine (NVP) is widely used as a non-nucleoside reverse transcriptase inhibitor of HIV-1, however, it is associated with severe skin and liver injury. The mechanisms of these adverse reactions are not yet clear, but the metabolic activation of NVP is thought to be related to the injury process. Until now, several metabolic activation pathways of NVP have been reported. In this study, in order to identify the reactive metabolite of NVP mainly responsible for CYP inhibition and liver injury, we synthesized five NVP analogs designed to avoid the proposed bioactivation pathway and evaluated their metabolic stabilities, CYP3A4 time-dependent inhibitory activities, and cytotoxicity. As a result, only a pyrimidine analog of NVP, which could avoid the formation of a reactive epoxide intermediate, did not inhibit CYP3A4. Outside of this compound, the other synthesized compounds, which could avoid the generation of a reactive quinone-methide intermediate, inhibited CYP3A4 equal to or stronger than NVP. The pyrimidine analog of NVP did not induce cytotoxicity in HepG2 and transchromosomic HepG2 cells, expressing major four CYP enzymes and CYP oxidoreductase. These results indicated that the epoxide intermediate of NVP might play an important role in NVP-induced liver injury.

2'-Deoxythymidine adducts from the anti-HIV drug nevirapine.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used against HIV-1. Currently, NVP is the most widely used anti-HIV drug in developing countries, both in combination therapy and to prevent mother-to-child transmission of HIV. Despite its efficacy against HIV, NVP produces a variety of toxic responses, including hepatotoxicity and skin rash. It is also associated with increased incidences of hepatoneoplasias in rodents. In addition, epidemiological data suggest that NNRTI use is a risk factor for non-AIDS-defining cancers in HIV-positive patients. Current evidence supports the involvement of metabolic activation to reactive electrophiles in NVP toxicity. NVP metabolism includes oxidation to 12-hydroxy-NVP; subsequent Phase II sulfonation produces an electrophilic metabolite, 12-sulfoxy-NVP, capable of reacting with DNA to yield covalent adducts. Since 2'-deoxythymidine (dT) adducts from several alkylating agents are regarded as having significant mutagenic/carcinogenic potential, we investigated the formation of NVP-dT adducts under biomimetic conditions. Toward this goal, we initially prepared and characterized synthetic NVP-dT adduct standards using a palladium-mediated Buchwald-Hartwig coupling strategy. The synthetic standards enabled the identification, by LC-ESI-MS, of 12-(2'-deoxythymidin-N3-yl)-nevirapine (N3-NVP-dT) in the enzymatic hydrolysate of salmon testis DNA reacted with 12-mesyloxy-NVP, a synthetic surrogate for 12-sulfoxy-NVP. N3-NVP-dT, a potentially cytotoxic and mutagenic DNA lesion, was also the only dT-specific adduct detected upon reaction of dT with 12-mesyloxy-NVP. Our data suggest that N3-NVP-dT may be formed in vivo and play a role in the hepatotoxicity and/or putative hepatocarcinogenicity of NVP.

Efficient synthesis of nevirapine analogs to study its metabolic profile by click fishing.

Knowledge of the biotransformation and pharmacokinetics of the antiretroviral agent nevirapine is still insufficient. In order to trace rash inducing metabolites of nevirapine, we devised a short and efficient multi-gram synthesis of a nevirapine analog that can be coupled to azide containing compounds by click chemistry.

Identification of a process impurity formed during synthesis of a nevirapine analogue HIV NNRT inhibitor using LC/MS and forced degradation studies.

Impurities in pharmaceutical products do not enhance the desired therapeutic effect and may, of course, have adverse effects. Impurities must therefore be limited or controlled for quality and safety considerations. Structural identification of an impurity is the first step in understanding the chemistry of its formation and subsequently controlling the impurity. In this article, the chemical structure of an unknown by-product formed during the synthesis of a nevirapine analogue HIV NNRT inhibitor was identified using a combination of low resolution, high resolution and H/D exchange LC/MS and LC/MS/MS. The origin of the impurity was investigated through a series of photo- and oxidative stress studies. It was concluded that this impurity is formed via a side-reaction of the last intermediate with the oxidant used in the synthesis.

Synthesis and anti-HIV activity of nevirapine prodrugs.

The synthesis, in vitro anti-HIV activity and stability studies of the N-Mannich bases of nevirapine are reported. Among the synthesized compounds, 5-{[4-(4-chlorophenyl)piperazin-1 -yl]methyl}-1-cyclopropyl-4-methyl-5,11-dihydro-6H-dipyrido[2,3-e:3',2'-b][1,4]diazepin-6-one (3) was found to be the most potent compound with EC50 of 0.0159 microM against HIV-1 replication and CC50 of >1000 microM against CEM cell lines with selectivity index of >62893. Compound 3 was five times more active than nevirapine (EC50 of 0.09 microM). In vitro hydrolysis of the Mannich bases in phosphate buffer (pH 7.4) indicated that these agents were relatively stable with t1/2 ranging from 15 to 240 min.

Nevirapine derivatives with broad-spectrum chemotherapeutic properties for the effective treatment of HIV/AIDS.

A series of nevirapine derivatives has been synthesized in an effort to enhance the spectrum of chemotherapeutic properties for the effective treatment of AIDS and AIDS-related opportunistic infections. The nevirapine derivative bearing isoniazid moiety (3a) was found to be the most potent compound with EC50 of<0.0636 microM, CC50 of>1000 microM and selectivity index of>15,723 which also exhibited 90% inhibition against Mycobacterium tuberculosis at 6.25 microg/ml. Compound 3c showed 100% inhibition against M. tuberculosis and also exhibited potent antibacterial activity against 24 pathogenic bacteria with MIC less than 1 microg/ml.

Novel nevirapine-like inhibitors with improved activity against NNRTI-resistant HIV: 8-heteroarylthiomethyldipyridodiazepinone derivatives.

A series of 8-heteroarylthiomethyldipyridodiazepinone derivatives were prepared and evaluated for their antiviral profile against wild type virus and the important K103N/Y181C mutant as an indicator for broad activity. 2,6-Dimethylpyridine derivative 16 was found to have a good pharmacokinetic profile in spite of poor metabolic stability in rat liver microsomes.

1,5-Benzodiazepines. Part XII. Synthesis and biological evaluation of tricyclic and tetracyclic 1,5-benzodiazepine derivatives as nevirapine analogues.

A number of properly substituted 5H-pyrimido[4,5-b][1,5]benzodiazepines (2) and pyrazolo[3,4-b][1,5]benzodiazepines (3 and 4), as well as compounds 5-7, which are derivatives of new tetracyclic systems, were prepared as nevirapine analogues through multistep synthetic routes. The cytotoxic and anti-HIV-1 properties of compounds 2-7 were evaluated in cell-based assays, together with their inhibitory activity against the HIV-1 recombinant reverse transcriptase (rRT) in enzyme assays. The modifications introduced into nevirapine heterocyclic skeleton proved to have a negative effect for the anti-HIV-1 activity. It is worth noting that some of the new derivatives proved to be cytotoxic in the low micromolar range.

Novel nonnucleoside inhibitors of HIV-1 reverse transcriptase. 8. 8-Aryloxymethyl- and 8-arylthiomethyldipyridodiazepinones.

Nevirapine (I) is the first human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor to reach regulatory approval. As a result of a second generation program around the tricyclic core system of nevirapine, 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-(2-(pyridin-4-yl)ethyl)-6H-dipyrido[3, 2-b:2',3'-e][1,4]diazepin-6-one (II)1a and 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-phenylethyl-6H-dipyrido[3,2-b:2', 3'-e][1,4]diazepin-6-one (III)1a were identified as broad spectrum HIV-1 RT inhibitors. A detailed examination of replacing either of the methylenes of the 8-ethyl linker of II or III is presented. It was found that 8-aryloxymethyl and 8-arylthiomethyl are the preferred pattern of substitution for potency against RT. The most potent compounds were further evaluated against a panel of clinically significant mutant RT enzymes (K103N, V106A, G190A, P236L) and in cytotoxicity and in vitro metabolism assays. The most potent compound was 2-chloro-8-phenylthiomethyl analogue 37 which displayed sub-100 nM activity against all HIV-1 RT enzymes tested.

A dipyrido [2,3-b:3',2'-f]azepine analog of the HIV-1 reverse transcriptase inhibitor nevirapine.

The syntheses of 11-ethyl and 11-cyclopropyldipyrido[2,3-b:3',2'-f]azepines, analogs of the HIV-1 reverse transcriptase inhibitor nevirapine 1, are described. These compounds exhibit potency equivalent to nevirapine in the inhibition of wild-type and some mutant RT enzymes.