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A series of HIV protease inhibitors with modifications on the P3 position have been designed and synthesized. These compounds exhibit excellent antiviral activity against both the wild type enzyme and PI-resistant clinical viral isolates. The synthesis and biological activity of the compounds are described.

Efforts directed to identifying potent HIV protease inhibitors (PI) have yielded a class of compounds that are not only very active against wild-type (NL4-3) HIV virus but also very potent against a panel of PI-resistant viral isolates. Chemistry and biology are described.

HIV-1 protease inhibitors (PI's) bearing 1,3,4-oxadiazoles at the P1' position were prepared by a novel method involving the diastereoselective installation of a carboxylic acid and conversion to the P1' heterocycle. The compounds are picomolar inhibitors of native HIV-1 protease, with most of the compounds maintaining excellent antiviral activity against a panel of PI-resistant strains.

HIV-1 protease inhibitors (PI) with an N-arylpyrrole moiety in the P(3) position afforded excellent antiviral potency and substantially improved aqueous solubility over previously reported variants. The rapid in vitro clearance of these compounds in human liver microsomes prompted oral coadministration with indinavir to hinder their metabolism by the cyctochrome P450 3A4 isozyme and allow for in vivo PK assessment.

Transposition of the pyridyl nitrogen from the P(3) substituent to the P(1)' substituent in HIV-1 protease inhibitors (PI) affords compounds such as 3 with an improved inhibitory profile against multiple P450 isoforms. These compounds also displayed increased potency, with 3 inhibiting viral spread (CIC(95)) at <8 nM for every strain of PI-resistant HIV-1 tested. The poor to modest bioavailability of these compounds may correlate in part to their aqueous solubility.

A biaryl pyridylfuran P(3) substituent on the hydroxyethylene isostere scaffold affords HIV protease inhibitors (PI's) with picomolar (IC(50)) potency against the protease enzymes from PI-resistant HIV-1 strains. Inclusion of a gem-dimethyl substituent afforded compound 3 with 100% oral bioavailability (dogs) and more than double the t(1/2) of indinavir. Inhibition of multiple P450 isoforms is dependent on the regiochemistry of the pyridyl nitrogen in these compounds.

Replacement of the pyridylmethyl moiety in indinavir with a pyridyl oxazole yielded HIV-1 protease inhibitors (PI) with greatly improved potency against PI-resistant HIV-1 strains. A meta-methoxy group on the pyridyl ring and a gem-dimethyl methyl linkage afforded compound 10 with notable in vitro antiviral activity against HIV-1 viral strains with reduced susceptibility to the clinically available PIs. Compound 10 also demonstrated favorable in vivo pharmacokinetics in animal models.

A series of highly potent HIV protease inhibitors have been designed and synthesized. These compounds are active against various clinical viral isolates as well as wild-type virus. The synthesis and biological activity of these HIV protease inhibitors are discussed.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

Indinavir analogues with blocked metabolism sites show highly improved pharmacokinetic profiles in animals. The cis-aminochromanol substituted analogues exhibited excellent potency against both the wild-type (NL4-3) virus and protease inhibitor-resistant HIV strains.

Substitution of the t-butylcarboxamide substituent in analogues of the HIV protease inhibitor (PI) Indinavir with a trifluoroethylamide moiety confers greater potency against both the wild-type (NL4-3) virus and PI-resistant HIV. The trifluoroethyl substituent also affords a slower clearance rate in vivo (dogs); however, this may be due to more potent inhibition of at least two P450 isoforms.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.

Osmotic-swelling experiments were conducted on a variety of preparations of "uniform" unilamellar vesicle systems. The synthetic lipid preparations included both vesicles produced by extrusion through polycarbonate ultrafiltration membranes and vesicles produced by the pH-adjustment method. The vesicles were monitored by photon correlation spectroscopy during swelling as the osmolarity of the external solution was decreased. Contrary to our previously reported results [Aurora, T. S., Li, W., Cummins, H. Z., & Haines, T. H. (1985) Biochim. Biophys. Acta 820, 250-258; Li, W., & Haines, T. H. (1986) Biochemistry 25, 7477-7483; Li, W., Aurora, T. S., Haines, T. H., & Cummins, H. Z. (1986) Biochemistry 25, 8220-8229; Haines, T. H., Li, W., Green, M., & Cummins, H. Z. (1987) Biochemistry 26, 5439-5447] large unilamellar vesicles produced from acidic lipids by the pH-adjustment technique were highly polydisperse and did not swell in a manner that permitted the computation of a Young's modulus, presumably due to the polydispersity. Also contrary to our previous reports, membranes derived from bovine submitochondrial particles did not produce evidence of swelling when subjected to similar protocols. Analysis of osmotic swelling of extruded unilamellar vesicles has allowed us to assign Young's moduli for bilayers of dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol, in the range (5-8) x 10(8) and (3-6) x 10(8) dyn/cm2, respectively. The diameters and polydispersites obtained with electron microscopy and photon correlation spectroscopy were compared directly and with computer-modeling techniques. While excellent agreement was obtained for distributions with low polydispersity (approximately greater than 0.1), serious disagreement was found when the polydispersity exceeded approximately 0.2.