Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC₅₀ of 200 nM with a therapeutic index of >1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. C_max and absolute bioavailability of 34S were higher and half-life and time above EC₉₅ were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.     

Cyclic sulfamide HIV-1 protease inhibitors, with sidechains spanning from P2/P2' to P1/P1'

Previous studies of HIV protease inhibitors have shown that it is possible to elongate the P1/P1' sidechains to reach the S3/S3' binding sites. By analogy, we expected that it would be possible to design inhibitors reaching between the S1/S1' and S2/S2' binding sites. Molecular modeling suggested that this could be achieved with appropriate ortho-substitution of the P2/P2' benzyl groups in our cyclic sulfamide inhibitors. Four different spacer groups were investigated. The compounds were smoothly prepared from tartaric acid in five steps and exhibit low to moderate activity, the most potent inhibitor possessing a Ki value of 0.53 microM.     

Isomeric acetoxy analogues of rofecoxib: a novel class of highly potent and selective cyclooxygenase-2 inhibitors

A group of isomers possessing a 2-, 3-, or 4-acetoxy moiety on the 3-phenyl substituent of rofecoxib were synthesized that exhibit highly potent, and selective, COX-2 inhibitory activity that have the potential to acetylate the COX-2 isozyme.     

Synthesis and activity of novel HIV protease inhibitors with improved potency against multiple PI-resistant viral 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.     

4-[6-(2-Aminoethyl)naphthalen-2-yl]benzonitriles are potent histamine H3 receptor antagonists with high CNS penetration

4-[6-(2-Tertiaryaminoethyl)naphthalen-2-yl]benzonitriles are conformationally constrained histamine H3 receptor antagonists with high potency and selectivity. The analogs were designed around a naphthalene core, with the goal of enhancing lipophilicity and CNS penetration, as compared to a previously reported benzofuran series. The SAR of the tertiary amine moiety is similar to that reported for the benzofuran series, with analogs bearing a 2-methylpyrrolidine substituent possessing the greatest rat and human H3 receptor binding affinities.     

Potent inhibitors of the hepatitis C virus NS3 protease: use of a novel P2 cyclopentane-derived template

The HCV NS3 protease is essential for replication of the hepatitis C virus (HCV) and therefore constitutes a promising new drug target for anti-HCV therapy. Several potent and promising HCV NS3 protease inhibitors, some of which display low nanomolar activities, were identified from a series of novel inhibitors incorporating a trisubstituted cyclopentane dicarboxylic acid moiety as a surrogate for the widely used N-acyl-(4R)-hydroxyproline in the P2 position.     

Synthesis of novel 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-ones from thiosemicarbazide precursors

To develop novel biologically active organic compounds possessing a sugar moiety, a series of 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-one were synthesized via reaction of the thiosemicarbazide with ethyl bromoacetate. Their chemical structures were characterized by (1)H and (13)C NMR spectroscopy, elemental analysis and MS. The bioassay results indicated that some of these compound exhibit moderate fungicidal and herbicidal activities. Furthermore, the effect of various solvents at reflux temperature on the reactions of ethyl bromoacetate with the related thiosemicarbazides was investigated.     

Combinatorial diversification of indinavir: in vivo mixture dosing of an HIV protease inhibitor library

An efficient combination solution-phase/solid-phase route enabling the diversification of the P1', P2', and P3 subsites of indinavir has been established. The synthetic sequence can facilitate the rapid generation of HIV protease inhibitors possessing more favorable pharmacokinetic properties as well as enhanced potencies. Multiple compound dosing in vivo may also accelerate the identification of potential drug candidates.     

Design, synthesis, and biological evaluation of (E)-3-(4-methanesulfonylphenyl)-2-(aryl)acrylic acids as dual inhibitors of cyclooxygenases and lipoxygenases

A group of (E)-3-(4-methanesulfonylphenyl)acrylic acids possessing a substituted-phenyl ring (4-H, 4-Br, 3-Br, 4-F, 4-OH, 4-OMe, 4-OAc, and 4-NHAc) attached to the acrylic acid C-2 position were prepared using a stereospecific Perkin condensation reaction. A related group of compounds having 4- and 3-(4-isopropyloxyphenyl)phenyl, 4- and 3-(2,4-difluorophenyl)phenyl and 4- and 3-(4-methanesulfonylphenyl)phenyl substituents attached to the acrylic acid C-2 position were also synthesized, using a palladium-catalyzed Suzuki cross-coupling reaction, for evaluation as dual cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors. (E)-2-(3-Bromophenyl)-3-(4-methanesulfonylphenyl)acrylic acid (9h), and compounds having 4-(4-isopropyloxyphenyl-, 2,4-difluorophenyl-, or 4-methylsulfonylphenyl)phenyl moieties at the acrylic acid C-2 position (11a,b,d), were particularly potent COX-2 inhibitors with a high COX-2 selectivity index (COX-2 IC50 approximately 0.32 microM, SI > 316) similar to the reference drug rofecoxib (COX-2 IC50 = 0.5 microM, SI > 200). Acrylic acid analogs with a C-2 4-hydoxyphenyl (9d, IC50 = 0.56 microM), or 4-acetamidophenyl (9g, IC50 = 0.11 microM), substituent were particularly potent 5-LOX inhibitors that may participate in an additional specific hydrogen-bonding interaction. A number of compounds possessing a C-2 substituted-phenyl moiety (4-Br, 4-F, and 4-OH), or a 4- or 3-(2,4-difluorophenyl)phenyl moiety, showed potent 15-LOX inhibitory activity (IC50 values in the 0.31-0.49 microM range) relative to the reference drug luteolin (IC50 = 3.2 microM). Compounds having a C-2 4-acetylaminophenyl, or 4-(2,4-difluorophenyl)phenyl, moiety exhibited anti-inflammatory activities that were equipotent to aspirin, but less than that of celecoxib. The structure-activity data acquired indicate the acrylic acid moiety constitutes a suitable scaffold (template) to design novel acyclic dual inhibitors of the COX and LOX isozymes.     

The GB virus C (GBV-C) NS3 serine protease inhibits HIV-1 replication in a CD4+ T lymphocyte cell line without decreasing HIV receptor expression

Introduction

Persistent infection with GBV-C (GB Virus C), a non-pathogenic virus related to hepatitis C virus (HCV), prolongs survival in HIV infection. Two GBV-C proteins, NS5A and E2, have been shown previously to inhibit HIV replication in vitro. We investigated whether the GBV-C NS3 serine protease affects HIV replication.

Results

GBV-C NS3 protease expressed in a human CD4+ T lymphocyte cell line significantly inhibited HIV replication. Addition of NS4A or NS4A/4B coding sequence to GBV-C NS3 increased the effect on HIV replication. Inhibition of HIV replication was dose-dependent and was not mediated by increased cell toxicity. Mutation of the NS3 catalytic serine to alanine resulted in loss of both HIV inhibition and protease activity. GBV-C NS3 expression did not measurably decrease CD4 or CXCR4 expression.

Conclusion

GBV-C NS3 serine protease significantly inhibited HIV replication without decreasing HIV receptor expression. The requirement for an intact catalytic serine at the active site indicates that inhibition was mediated by proteolytic cleavage of an unidentified target(s).     

Structure-activity and structure-metabolism relationships of HIV protease inhibitors containing the 3-hydroxy-2-methylbenzoyl-allophenylnorstatine structure

A series of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing substituted all-phenylnorstatine [APNS: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were designed and synthesized. From the structure-metabolism relationship of this type of HIV protease inhibitors, the compounds having para substitution of the phenyl ring of Apns and/or 2,6-disubstitution of the P2' benzylamine were found to be able to avoid the P2 phenol glucuronidation that occurs with SM-319777 (formerly named JE-2147, KNI-764); one of the main metabolic pathways of SM-319777. These new analogues, such as SM-322377, had more desirable pharmacokinetic profiles and more potent antiviral activity against not only wild type HIV-1 but also the multi-drug-resistant HIV-1 than SM-319777.     

Potent cyclic urea HIV protease inhibitors with benzofused heterocycles as P2/P2′ groups

A series of benzofused heterocycles was examined to replace the metabolically unstable benzyl alcohol P2/P2′ groups of DMP 323 (1). Extremely potent inhibitors of HIV protease (Ki < 0.01 nM) and excellent antiviral activity (IC₉₀ = 8 nM) were found. An X-ray crystal structure of benzimidazolone 5a bound to HIV protease showed H-bonds to Asp30 and a bridging water molecule to Gly48.     

Synthesis and biological evaluation of 1,3-diphenylprop-2-en-1-ones possessing a methanesulfonamido or an azido pharmacophore as cyclooxygenase-1/-2 inhibitors

A group of (E)-1,3-diphenylprop-2-en-1-one derivatives (chalcones) possessing a MeSO(2)NH, or N(3), COX-2 pharmacophore at the para-position of the C-1 phenyl ring were synthesized using a facile stereoselective Claisen-Schmidt condensation reaction. In vitro COX-1/COX-2 structure-activity relationships were determined by varying the substituents on the C-3 phenyl ring (4-H, 4-Me, 4-F, and 4-OMe). Among the 1,3-diphenylprop-2-en-1-ones possessing a C-1 para-MeSO(2)NH COX-2 pharmacophore, (E)-1-(4-methanesulfonamidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7b) was identified as a selective COX-2 inhibitor (COX-2 IC(50)=1.0 microM; selectivity index >100) that was less potent than the reference drug rofecoxib (COX-2 IC(50)=0.50 microM; SI>200). The corresponding 1,3-diphenylprop-2-en-1-one analogue possessing a C-1 para-N(3) COX-2 pharmacophore, (E)-1-(4-azidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7f), exhibited potent and selective COX-2 inhibition (COX-1 IC(50)=22.2 microM; COX-2 IC(50)=0.3 microM; SI=60). A molecular modeling study where 7b and 7f were docked in the binding site of COX-2 showed that the p-MeSO(2)NH and N(3) substituents on the C-1 phenyl ring are oriented in the vicinity of the COX-2 secondary pocket (His90, Arg513, Phe518, and Val523). The structure-activity data acquired indicate that the propenone moiety constitutes a suitable scaffold to design new acyclic 1,3-diphenylprop-2-en-1-ones with selective COX-1 or COX-2 inhibitory activity.     

Advances toward new antidepressants beyond SSRIs: 1-aryloxy-3-piperidinylpropan-2-ols with dual 5-HT1A receptor antagonism/SSRI activities. Part 4

A series of 1-(1H-indol-4-yloxy)-3-(4-arylpiperidinyl)propan-2-ols possessing potent dual 5-HT(1A) receptor antagonism and serotonin reuptake inhibition was discovered. The fused aryl ring moiety contributed to the robust dual activities irrespective of the regiochemistry associated with its connectivity to the piperidine central ring.     

Structure-activity relationships of novel HIV-1 protease inhibitors containing the 3-amino-2-chlorobenzoyl-allophenylnorstatine structure

A series of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing substituted allophenylnorstatine (Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) were designed and synthesized. From the structure-activity relationship of this series of compounds, SM-309515 was found to have potent antiviral activity against wild-type and resistant HIV-1s and to possess a desirable pharmacokinetic profile in dogs.     

Synthesis and SAR studies of potent HIV protease inhibitors containing novel dimethylphenoxyl acetates as P2 ligands

Isopropyl substituted 4-thioazolyl valine side chains are highly optimized P(2)-P(3) ligands for C2 symmetry-based HIV protease inhibitors, as exemplified by the drug ritonavir. Replacement of the side chain with the conformationally constrained hexahydrofurofuranyloxy P(2) ligand in combination with a dimethylphenoxyacetate on the other end of the ritonavir core diamine yielded highly potent HIV protease inhibitors. The in vitro antiviral activity in MT4 cells increased by 10- and 20-fold, respectively, in the absence and presence of 50% human serum compared to ritonavir. The structure-activity relationships of inhibitor series with this combination of ligands were investigated. Preliminary pharmacokinetic studies in rats indicated rapid elimination of the inhibitors from the blood, and the plasma levels were not significantly enhanced by coadministration with ritonavir. However, the novel structural features and the high intrinsic antiviral potency of this series provides potential for the future exploration of prodrug strategies.     

Loss of protease dimerization inhibition activity of darunavir is associated with the acquisition of resistance to darunavir by HIV-1

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.     

Development of functional imidazole derivatives: the influence of alkaline metal cations on the rates of CL reactions of 2-(phenyl and 4-dimethylaminophenyl)-4-hydroperoxy-4-3',4'-(15-crown-5)phenyl-5-3'-4'-(15-crown-5)phenyl-4H-isoimidazoles.

Although several investigations have focused on luminescence modulation by chelation with metal cations using bidentate ligands or crown ether systems, a bis(crown ether) system has not yet been used for modulation of chemiluminescence (CL) reactions. In the CL reaction of 2-(phenyl and 4-dimethylaminophenyl)-4-hydroperoxy-4-3',4'-(15-crown-5)phenyl-5-3',4'(15-crown-5)phenyl-4H-isoimidazoles 2a and b possessing a bis(15-crown-5 ether) moiety, the rate acceleration was observed in the presence of K(+), Rb(+) and Cs(+) due to the holding effect of the bis-crown moiety, but no rate acceleration was observed by Li(+) and Na(+) due to the template effect of the crown moiety. The acceleration of the CL reaction rates is ascribable to the conformational change induced by the scissor-like motion of the bis-crown moiety assisted by the holding effect.     

A novel 3-0 sulfatase from human urine acting on methyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3-sulfate

Human urine contains a novel sulfatase which is specific for the 3-0 sulfate ester of sulfaminoglucopyranoside 3-sulfate. Of the three isomeric sulfamate derivatives, 3,4 and 6-0 sulfate esters, only the 3-0 ester is hydrolyzed. Enzymatic activity requires that the amino group be sulfated; sulfate is not released if the amino group is free or acetylated. The enzyme has been purified 70-fold. It has a pH optimum of 6.3 and is inhibited by inorganic sulfate and phosphate. The specificity of this enzyme suggests that a 3-0 sulfated glucosamine moiety may have a role in the physiological activity of heparin or heparan sulfate.