Hepatitis C NS5B polymerase inhibitors: functional equivalents for the benzothiadiazine moiety.

A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothiadiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited potent activity in enzymatic and replicon assays.

Des-A-ring benzothiadiazines: inhibitors of HCV genotype 1 NS5B RNA-dependent RNA polymerase.

In our program to discover non-nucleoside, small molecule inhibitors of genotype 1 HCV polymerase, we investigated a series of promising analogs based on a benzothiadiazine screening hit that contains an ABCD ring system. After demonstrating that a methylsulfonylamino D-ring substituent increased the enzyme potency into the low nanomolar range, we explored a minimum core required for activity by truncating to a three-ring system. Described herein are the syntheses and structure-activity relationship of a set of inhibitors lacking the A-ring of an ABCD ring system. We observed that small aromatic rings and alkenyl groups appended to the 5-position of the B-ring were optimal, resulting in inhibitors with low nanomolar potencies.

Synthesis and SAR of novel 1,1-dialkyl-2(1H)-naphthalenones as potent HCV polymerase inhibitors.

A series of gem-dialkyl naphthalenone derivatives with varied alkyl substitutions were synthesized and evaluated according to their structure-activity relationship. This investigation led to the discovery of potent inhibitors of the hepatitis C virus at low nanomolar concentrations in both enzymatic and cell-based HCV genotype 1a assays.

Hepatitis C NS5B polymerase inhibitors: 4,4-Dialkyl-1-hydroxy-3-oxo-3,4-dihydronaphthalene-3-yl benzothiadiazine derivatives.

4,4-Dialkyl-1-hydroxy-3-oxo-3.4-dihydronaphthalene-3-yl benzothiadiazine derivatives were synthesized and evaluated as inhibitors of genotypes 1a and 1b HCV NS5B polymerase. A number of these compounds exhibited potent activity against genotypes 1a and 1b HCV polymerase in both enzymatic and cell culture activities. A representative compound also showed favorable pharmacokinetics in the rat.

Identification and characterization of mutations conferring resistance to an HCV RNA-dependent RNA polymerase inhibitor in vitro.

Compound A-837093, a non-nucleoside HCV RNA-dependent RNA polymerase inhibitor, displayed nanomolar potencies against HCV genotypes 1a and 1b replicons. It also exhibited an excellent metabolic profile and achieved high plasma and liver concentrations in animals. In order to characterize the development of resistance to this anti-HCV agent, HCV subgenomic 1b strain N replicon cells were cultured in the presence of A-837093 with G418. Mutations S368A, Y448H, G554D, Y555C, and D559G in the NS5B polymerase gene were identified that led to substantial decreases in the susceptibilities of 1b genotype replicons to the inhibitor A-837093. However, the resistant mutants remained susceptible to HCV protease inhibitor BILN-2061 and alpha interferon as well as to a different class of non-nucleoside HCV polymerase inhibitor. In addition, each single resistant mutation identified significantly reduced the replication capacity of mutant compared to wild-type replicon. These findings provide a strategic guide for the future development of non-nucleoside inhibitors of HCV NS5B polymerase.

Synthesis and biological characterization of B-ring amino analogues of potent benzothiadiazine hepatitis C virus polymerase inhibitors.

Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F = 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30 is a potent inhibitor in replicon assays, with EC(50) values of 10 and 6 nM against genotypes 1a and 1b, respectively.

Inhibitors of hepatitis C virus polymerase: synthesis and biological characterization of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines.

The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2 and 3. The most active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral bioavailability of 77%, and a peak liver concentration of 21.8 microg/mL.

Characterization of a novel human immunodeficiency virus type 1 protease inhibitor, A-790742.

A-790742 is a potent human immunodeficiency virus type 1 (HIV-1) protease inhibitor, with 50% effective concentrations ranging from 2 to 7 nM against wild-type HIV-1. The activity of this compound is lowered by approximately sevenfold in the presence of 50% human serum. A-790742 maintained potent antiviral activity against lopinavir-resistant variants generated in vitro as well as against a panel of molecular clones containing proteases derived from HIV-1 patient isolates with multiple protease mutations. During in vitro selection, A-790742 selected two primary mutations (V82L and I84V) along with L23I, L33F, K45I, A71V/A, and V77I in the pNL4-3 background and two other mutations (A71V and V82G) accompanied by M46I and L63P in the HIV-1 RF background. HIV-1 pNL4-3 clones with a single V82L or I84V mutation were phenotypically resistant to A-790742 and ritonavir. Taking these results together, A-790742 displays a favorable anti-HIV-1 profile against both the wild type and a large number of mutants resistant to other protease inhibitors. The selection of the uncommon V82L and V82G mutations in protease by A-790742 suggests the potential for an advantageous resistance profile with this protease inhibitor.

In vitro selection and characterization of human immunodeficiency virus type 2 with decreased susceptibility to lopinavir.

Lopinavir (LPV)-ritonavir has demonstrated durable antiviral activity in human immunodeficiency virus type 1 (HIV-1)-infected antiretroviral-naïve and protease inhibitor (PI)-experienced patients. However, information on LPV activity against HIV-2 and the patterns of mutations in HIV-2 in response to selection by LPV is limited. The activity of LPV against three strains of HIV-2 was assessed and compared to activity against a reference HIV-1 strain. LPV demonstrated activity similar to that observed against HIV-1 in two HIV-2 strains (HIV-2(MS) and HIV-2(CBL-23)) tested. On the other hand, approximately 10-fold-reduced susceptibility was observed with the third HIV-2 strain, HIV-2(CDC310319). Passage of HIV-2(MS) with increasing concentrations of LPV selected mutations V47A and D17N in the HIV-2 protease gene. The introduction of both 17N and 47A either individually or together into HIV-2(ROD) molecular infectious clones showed that the single V47A substitution in HIV-2 resulted in a substantial reduction in susceptibility to LPV. In contrast, this mutant retained wild-type susceptibility to other PIs and appeared to be hypersusceptible to atazanavir and saquinavir.

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of unsymmetrical 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives.

Substituted 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed. It was found that the saturated alkane dialkyl units provided the most active analogs.

Discovery of imidazolidine-2,4-dione-linked HIV protease inhibitors with activity against lopinavir-resistant mutant HIV.

A new series of HIV protease inhibitors has been designed and synthesized based on the combination of the (R)-(hydroxyethylamino)sulfonamide isostere and the cyclic urea component of lopinavir. The series was optimized by replacing the 6-membered cyclic urea linker with an imidazolidine-2,4-dione which readily underwent N-alkylation to incorporate various methylene-linked heterocycle groups that bind favorably in site 3 of HIV protease. Significant improvements compared to lopinavir were seen in cell culture activity versus wild-type virus (pNL4-3) and the lopinavir-resistant mutant virus A17 (generated by in vitro serial passage of HIV-1 (pNL4-3) in MT-4 cells). Select imidazolidine-2,4-dione containing PIs were also more effective at inhibiting highly resistant patient isolates Pt1 and Pt2 than lopinavir. Pharmacokinetic data collected for compounds in this series varied considerably when coadministered orally in the rat with an equal amount of ritonavir (5 mg/kg each). The AUC values ranged from 0.144 to 12.33 microg h/mL.

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-1-benzyl and N-1-[3-methylbutyl]-4-hydroxy-1,8-naphthyridon-3-yl benzothiadiazine analogs containing substituents on the aromatic ring.

A series of non-nucleoside HCV NS5B polymerase inhibitors based on the N-1-benzyl or N-1-[3-methylbutyl]-4-hydroxy-1,8-naphthyridon-3-yl benzothiadiazine core substituted in the D-ring aromatic moiety have been prepared and evaluated. Aromatic substituents extending from position 7 of the D-ring exhibited excellent potency against both genotypes 1a and 1b.

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides.

Substituted N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed.

Mutations conferring resistance to a hepatitis C virus (HCV) RNA-dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro.

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.