Background: From a collection of 250 benzophenones that were synthesized based on structure-activity relationship and modeling, analogs have been identified which are potent inhibitors of wild-type and NNRTI-resistant strains of HIV-1. Studies were undertaken in enzyme, cell-based anti-viral, and cytotoxicity assays to determine the suitability of 3 analogs, GW4751, GW4511 and GW3011, as leads for a new generation NNRTI capable of treating current NNRTI-resistant strains.

Methods: Wild-type HIV, 20 NNRTI-resistant and 7 NRTI-resistant strains were assessed in one or more in vitro assays including HeLa-MAGI, MT4, HOS and PBL. Activity against wild-type and NNRTI-resistant mutant reverse transcriptase (RT) enzyme was assessed in a continuous time-resolved fluorescence assay. Effect on IC₅₀ due to serum protein binding was determined in HeLa MAGI assays. Cell cytotoxicity was assessed in human leukemic cell lines (IM9, MT4, U-973, Molt-4) by MTT assay and in primary human bone marrow progenitor cells by colony formation. Serial passage was carried out in MT4 cells.

Results: The IC₅₀ values for GW4751, GW4511 and GW3011 were ≤ 2nM versus wild-type HIV-1 in the HeLa MAGI assay. The highest IC₅₀ in a panel of 20 NNRTI-resistant mutants was 16nM for GW4511, 21nM for GW3011, and 34nM for GW4751. These compounds inhibited wild-type and mutant RT enzyme in biochemical assays with IC₅₀s that ranged from < 3–17nM. Forty-five percent (45%) human serum and/or 0.8mg/ml a1-acid glycoprotein resulted in a modest increase in IC₅₀ (4-8X). Cytotoxicity (CCIC₅₀) of all 3 compounds was similar and ranged from 12uM to > 28uM (limited by solubility) among 4 human leukemic cell lines. Human bone marrow progenitor assays (CFU-GM and BFU-E) demonstrated a CCIC₅₀ > 10 uM (highest concentration tested) for all 3 compounds. Eight (8) passages in escalating concentrations of GW4511 selected for mutational changes at RT loci V106A, E138K, and F227L.

Conclusions: Benzophenone analogues GW4751, GW4511, and GW3011 are potent new NNRTIs active against both wild-type virus and a broad-spectrum of NNRTI-associated mutant viruses including those known to be highly resistant to currently available NNRTIs. The mutations selected during passage with GW4511 are known to be associated with NNRTI resistance. [j1] The effects on IC₅₀ of human serum protein binding were modest and cytotoxicity was low. These benzophenone compounds represent important lead molecules for the discovery of a potent new generation NNRTI.