In earlier work, we have described a novel chemotype, the pyridinioalkanoyl thioesters (PATEs) as anti-HIV-1 agents that selectively target the viral nucleocapsid protein zinc fingers without affecting other viral or host zinc fingers. Our efforts during the last several years have been devoted to finding compounds with substantially improved in vitro antiviral potencies. An entirely different class of ligand structures, based on simple aliphatic amino acid amides, was uncovered (Song et al. Bioorg Med Chem.10:1263-73, 2002; Goel et al. Bioorg Med Chem Lett.12:767-70, 2002). This past year, we have synthesized suitable S-acyl 2-mercaptobenzamide derivatives that are poor substrates for non-specific carboxyesterases (thioesterases) yet retain good antiviral activity. We have explored this possibility by introducing a variety of substituents on the acyl group and the benzamide head structure that modify the reactivity of the thioester bond through electronic influences and provide steric hindrance to the approach of enzymes. So far, we have prepared about 50 representatives of this chemotype and evaluated them for anti-HIV activity via the dose-response inhibition of p24 secreted in vitro by activated spleen cells taken from HIV-1 transgenic mice and via the in vitro XTT cytoprotection assay. All the compounds showed EC50 values between 1.9 and 31.8 mM, and exhibited a wide range of cellular toxicity (IC50) values between 22 and 790 mM in the XTT assay. EC50s between 2.2 and 15.2 mM and IC50s of 59 and >250 mM were obtained in the Tg assay. Introduction of substituents in the phenyl ring of the acyl group modified the reactivity of the thioester bond through an electronic effect and also provided steric hindrance that reduced hydrolysis of the thioester bond. The presence of an electron donating substituent such as OCH3 showed enhanced antiviral activity but the half-life was only 80 mins. On the other hand, the presence of the same substituent at a different position or at more than one position in the phenyl ring, allowed greater stability but decreased antiviral activity. Compounds where the substituents are bulky in nature, for example Cl or CH3 at position 2 and 3 of the phenyl ring, showed low EC50s and high toxicity. Moreover, a compound having a tertiary butyl moiety at the acyl group was highly stable with a half-life of 335 minutes, low EC50 value and moderate cytotoxicity. Thus we have accomplished one of our goals for this year through the identification of a new chemotype with promising potential for development as an HIV drug. Recently, some of the lead compounds were further evaluated for antiviral efficacy in combination with nucleoside reverse transcriptase inhibitors (NRTI; AZT, PMPA), nonnucleoside reverse transcriptase inhibitors (NNRTI; Efavirenz [EFV], Nevaripine [NVP]), Protease inhibitors (PI; Rotinavir [RTV], Indinavir [IDV]), Zn finger inhibitors (ZFI; azodicarbonamide [ADA]) and virus entry inhibitors (EI; Chicago Sky Blue [CSB] and T-20) using the Prichard and Shipman MacSynergy 2 proportional statistics model (Prichard and Shipman (1990) Antiviral Res. 14:181-206). Our results show that the PATE chemotype via targeting retroviral Zn fingers can interact synergistically with all classes of clinically relevant antivirals and that the Zn fingers inhibitors could potentially play an important role in antiviral therapy by providing a fourth class of antivirals for treatment. The discovery of this family of highly potent NCp7 inhibitors holds promise for inactivating all strains of HIV-1 without generating resistant strains.
