Jan Heeres

TMC125, a Novel Next-Generation Nonnucleoside Reverse Transcriptase Inhibitor Active against Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Human Immunodeficiency Virus Type 1

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1); however, currently marketed NNRTIs rapidly select resistant virus, and cross-resistance within the class is extensive. A parallel screening strategy was applied to test candidates from a series of diarylpyrimidines against wild-type and resistant HIV strains carrying clinically relevant mutations. Serum protein binding and metabolic stability were addressed early in the selection process. The emerging clinical candidate, TMC125, was highly active against wild-type HIV-1 (50% effective concentration [EC50] = 1.4 to 4.8 nM) and showed some activity against HIV-2 (EC50 = 3.5 μM). TMC125 also inhibited a series of HIV-1 group M subtypes and circulating recombinant forms and a group O virus. Incubation of TMC125 with human liver microsomal fractions suggested good metabolic stability (15% decrease in drug concentration and 7% decrease in antiviral activity after 120 min). Although TMC125 is highly protein bound, its antiviral effect was not reduced by the presence of 45 mg of human serum albumin/ml, 1 mg of α1-acid glycoprotein/ml, or 50% human serum. In an initial screen for activity against a panel of 25 viruses carrying single and double reverse transcriptase amino acid substitutions associated with NNRTI resistance, the EC50 of TMC125 was <5 nM for 19 viruses, including the double mutants K101E+K103N and K103N+Y181C. TMC125 also retained activity (EC50 < 100 nM) against 97% of 1,081 recent clinically derived recombinant viruses resistant to at least one of the currently marketed NNRTIs. TMC125 is a potent next generation NNRTI, with the potential for use in individuals infected with NNRTI-resistant virus.

The medicinal chemistry of the DATA and DAPY series of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs)

This contribution presents an historical account of the medicinal chemistry of the DATA (diaryltriazine) and DAPY (diarylpyrimidine) series of inhibitors of HIV-1 reverse transcriptase (NNRTIs). The history extends over a period of more than 18 years, starting from 1987, and is still ongoing. The authors attempt to describe the often tortuous and unpredictable route that led from the virological screening of compounds, over serendipitous chemistry, X-ray crystallography, molecular modelling, animal pharmacokinetics, biochemistry and physical chemistry to the design and further development of highly potent anti-HIV compounds, three of which are now in advanced stages of clinical studies. The emphasis in this historical account is on the medicinal chemistry that led to the DATA and DAPY series of NNRTIs.

Diaryltriazine non-nucleoside reverse transcriptase inhibitors are potent candidates for pre-exposure prophylaxis in the prevention of sexual HIV transmission

OBJECTIVES Pre-exposure prophylaxis and topical microbicides are important strategies in the prevention of sexual HIV transmission, especially since partial protection has been shown in proof-of-concept studies. In search of new candidate drugs with an improved toxicity profile and with activity against common non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV, we have synthesized and investigated a library of 60 new diaryltriazine analogues. METHODS From this library, 15 compounds were evaluated in depth using a broad armamentarium of in vitro assays that are part of a preclinical testing algorithm for microbicide development. Antiviral activity was assessed in a cell line, and in primary human cells, against both subtype B and subtype C HIV-1 and against viruses resistant to therapeutic NNRTIs and the candidate NNRTI microbicide dapivirine. Toxicity towards primary blood-derived cells, cell lines originating from the female reproductive tract and female genital microflora was also studied. RESULTS AND CONCLUSIONS We identified several compounds with highly potent antiviral activity and toxicity profiles that are superior to that of dapivirine. In particular, compound UAMC01398 is an interesting new candidate that warrants further investigation because of its superior toxicity profile and potent activity against dapivirine-resistant viruses.

Development and in vitro evaluation of a vaginal microbicide gel formulation for UAMC01398, a novel diaryltriazine NNRTI against HIV-1

Diaryltriazines (DATAs) constitute a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are being investigated for use as anti-HIV microbicides. The aim of the present study was (1) to assess the biopharmaceutical properties of the DATA series, (2) to select the lead candidate as vaginal microbicide and (3) to develop and evaluate gel formulations of the lead candidate. First, the vaginal tissue permeation potential of the different DATAs was screened by performing permeability and solubility measurements. To obtain a suitable formulation of the lead microbicide candidate, several hydroxyethylcellulose-based gels were assessed for their cellular toxicity, stability and ability to enable UAMC01398 epithelial permeation. Also, attention was given to appropriate preservative selection. Because of its favourable in vitro activity, safety and biopharmaceutical profile, UAMC01398 was chosen as the lead microbicide candidate among the DATA series. Formulating UAMC01398 as a vaginal gel did not affect its anti-HIV activity. Safe and chemically stable gel formulations of UAMC01398 (0.02%) included a non-solubilizing gel and a gel containing sulfobutyl ether-β-cyclodextrin (SBE-βCD, 5%) as solubilizing excipient. Inclusion of SBE-βCD in the gel formulation resulted in enhanced microbicide flux across HEC-1A epithelial cell layers, to an extent that could not be achieved by simply increasing the dose of UAMC01398. The applied rational (pre)formulation approach resulted in the development of aqueous-based gel formulations that are appropriate for further in vivo investigation of the anti-HIV microbicide potential of the novel NNRTI UAMC01398.

Novel diarylpyridinones, diarylpyridazinones and diarylphthalazinones as potential HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs)

In this Letter, we report on diarylpyridinone, diarylpyridazinone and diarylphthalazinone analogs as potential inhibitors of HIV-1 nonnucleoside reverse transcriptase (NNRTIs). The most promising compounds in these series are three diarylpyridazinones 25a, 25l and 25n which demonstrated submicromolar activity against wild-type HIV-1 and moderate activity against the single mutant strain Ba-L V106A.

Synthesis, evaluation and structure-activity relationships of triazine dimers as novel antiviral agents.

This letter reports the synthesis and structure-activity relationship (SAR) study of a series of triazine dimers as novel antiviral agents. These compounds were obtained through a bivalent ligand approach in which two triazine moieties are covalently connected by suitable linkers. Several compounds showed submicromolar activity against wild-type HIV-1 and moderate activity against single mutant strains.

2,6-Di(arylamino)-3-fluoropyridine Derivatives as HIV Non-Nucleoside Reverse Transcriptase Inhibitors

New non-nucleoside reverse transcriptase inhibitors (NNRTI), which are similar in structure to earlier described di(arylamino)pyrimidines but featuring a 2,6-di(arylamino)-3-fluoropyridine, 2,4-di(arylamino)-5-fluoropyrimidine, or 1,3-di(arylamino)-4-fluorobenzene moiety instead of a 2,4-disubstituted pyrimidine moiety, are reported. The short and practical synthesis of novel NNRTI relies on two sequential Pd-catalyzed aminations as the key steps. It is demonstrated through direct comparison with reference compounds that the presence of a fluorine atom increases the in vitro anti-HIV activity, both against the wild type virus and drug-resistant mutant strains.

Resistance and cross-resistance profile of the diaryltriazine NNRTI and candidate microbicide UAMC01398

OBJECTIVES The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398. METHODS Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied. RESULTS We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost. CONCLUSIONS These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention.

From human immunodeficiency virus non-nucleoside reverse transcriptase inhibitors to potent and selective antitrypanosomal compounds

The presence of a structural recognition motif for the nucleoside P2 transporter in a library of pyrimidine and triazine non-nucleoside HIV-1 reverse transcriptase inhibitors, prompted for the evaluation of antitrypanosomal activity. It was demonstrated that the structure-activity relationship for anti-HIV and antitrypanosomal activity was different. Optimization in the diaryl triazine series led to 6-(mesityloxy)-N2-phenyl-1,3,5-triazine-2,4-diamine (69), a compound with potent in vitro and moderate in vivo antitrypanosomal activity.

Development of triazine non-nucleoside reverse transcriptase inhibitors for microbicidal applications

Background In search of antiretrovirals with microbicide potential, we have synthesized a library of non-nucleoside reverse transcriptase inhibitors (NNRTIs), encompassing 71 triazine analogues. We present data on the anti-HIV activity and toxicity using a broad armamentarium of in vitro assays and models. Materials and methods In a primary screen, the anti-HIV activity against the laboratory strain Ba-L and against a primary subtype C isolate was determined in the TZM-bl cell line. Cellular toxicity on TZM-bl cells was evaluated using WST-1. Subsequently, a selection o f1 7 compounds was further evaluated for anti-HIV activity in different primary cells, including peripheral blood mononuclear cells, dendritic cells and CD4+ T lymphocytes. In addition, the activity against NNRTI-resistantviruses (V106A, Y181C, L100I/ K103N) was tested. The toxicity profile was further investigated using blood cells and epithelial cells originating from the female genital tract (FGT) and in a dual chamber assay modeling the FGT and underlying mucosae. Finally, toxicity towards vaginal flora (reference strains of L.vaginalis, L. iners, L. jensenii, L. gasseri, L. crispatus ,A vaginae, G. vaginalis) was measured for the lead molecules UAMC00838 and UAMC01009. Dapivirine (TMC120) was used as a bench mark throughout the study. Results