Synthesis and antiviral property of allophenylnorstatine-based HIV protease inhibitors incorporating D-cysteine derivatives as P2/P3 moieties

We designed several HIV protease inhibitors with various d-cysteine derivatives as P(2)/P(3) moieties based on the structure of clinical drug candidate, KNI-764. Herein, we report their synthesis, HIV protease inhibitory activity, HIV IIIB cell inhibitory activity, cellular toxicity, and inhibitory activity against drug-resistant HIV strains. KNI-1931 showed distinct selectivity against HIV proteases and high potency against drug-resistant strains, surpassing those of Ritonavir and Nelfinavir.     

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.     

Ultra-potent P1 modified arylsulfonamide HIV protease inhibitors: the discovery of GW0385

A novel series of P1 modified HIV protease inhibitors was synthesized and evaluated for in vitro antiviral activity against wild-type virus and protease inhibitor-resistant viruses. Optimization of the P1 moiety resulted in compounds with femtomolar enzyme activities and cellular antiviral activities in the low nanomolar range culminating in the identification of clinical candidate GW0385.     

Increased antiviral activity of cyclic urea HIV protease inhibitors by modifying the P1/P1' substituents.

A series of alkyl substituted P1/P1' analogs was prepared in an attempt to increase translation of the 3-aminoindazole class of HIV protease inhibitors. Increasing the lipophilicity of the P1/P1' residues dramatically improved translation of enzyme activity to antiviral activity in the whole cell assay.     

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.     

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.     

Synthesis and antiviral activity of P1' arylsulfonamide azacyclic urea HIV protease inhibitors

A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P1' were synthesized utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more potent inhibitors. The effects of substituting the P1' benzenesulfonyl group on antiviral activity and protein binding are described.     

Design, synthesis, and biological evaluation of HIV/FIV protease inhibitors incorporating a conformationally constrained macrocycle with a small P3' residue

A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tripeptide (Ala-Val-Phe), a motif with a small P3' residue elective against the FIV protease and the drug-resistant HIV proteases, has been synthesized. It was found that the macrocycle is important to the overall activity of the inhibitors. Certain inhibitors were developed expressing low nanomolar inhibitory activity against the HIV/FIV proteases and they are also effective against some drug-resistant as well as TL3-resistant HIV proteases.     

Hydrophobicity in the design of P2/P2' tetrahydropyrimidinone HIV protease inhibitors

As part of an ongoing effort in understanding the role of hydrophobicity in the design of nonpeptidic HIV protease inhibitors, the QSAR study on P2/P2' tetrahydropyrimidinone is presented in this report. Our results suggest that the balance of hydrophobicity and a volume- dependent polarizability term plays a key role in the inhibition of the viral protease by these inhibitors. The size of the substituent of ligands at particular positions that induce steric fit is crucial. The role of hydrophobicity in the design of tetrahydropyrimidinone is discussed. It has been found that a sufficient spread in the data is required to observe the optimum value of ClogP for these inhibitors.     

Unsymmetric nonpeptidic HIV protease inhibitors containing anthranilamide as a P2′ ligand

A series of novel unsymmetrical anthranilamide-containing HIV protease inhibitors was designed. The structure-activity studies revealed a series of potent P2–P3′ inhibitors that incorporate an anthranilamide group at the P2′ position. A reduction in molecular weight and lipophilicity is achieved by a judicious choice of P2 ligands (i.e., aromatic, heteroaromatic, carbamate, and peptidic). A systematic investigation led to the 5-thiazolyl carbamate analog 8m, which exhibited a favorable C_max/EC₅₀ ratio (>30), plasma half-life (>8 h), and potent in vitro antiviral activity (EC₅₀ = 0.2 uM).     

A rational approach in the search for potent inhibitors against HIV proteinase

Synthetic peptides described as dog renin inhibitors were found to effectively inhibit the aspartyl protease of human immunodeficiency virus (HIV). The selection of oligopeptides for the HIV protease inhibition study was based on 1) the current strategy of inhibiting aspartyl proteases with transition state analogs, and 2) our previous observations regarding optimal structural differentiation at the P2 position among human, dog, and rat renin inhibitors. In an in vitro assay system consisting of recombinant HIV protease and a synthetic decapeptide substrate (at pH 5.5), results show that HIV protease was unaffected by statine-containing analogs carrying histidine at the P2 position whereas analogs containing valine at the same position yielded anti-protease IC50 values ranging from 50 to 500 nM. As anticipated, some analogs were also shown to inhibit processing of recombinant polyprotein substrate by HIV protease in vitro. The anti-viral activity of three inhibitors was studied in HIV-infected CEM and MT-2 cells. Results showed that one compound, Ac-Naphthylalanyl-Pro-Phe-Val-Statine-Leu-Phe-NH2 (antiprotease IC50 value = 0.4 microM), protected the infected cells effectively with IC50 values (0.73 microM for CEM cells and 0.88 microM for MT-2 cells). This antiviral effect is comparable to those obtained with AZT and ddC in parallel studies of MT-2 cells.     

Optimization of pyrrolidinone based HIV protease inhibitors

Optimization of P1-substituted pyrrolidinone based HIV protease inhibitors has yielded analogs with very potent antiviral activity.     

HIV protease inhibitors with picomolar potency against PI-Resistant HIV-1 by extension of the P3 substituent

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.     

Novel P1 chain-extended HIV protease inhibitors possessing potent anti-HIV activity and remarkable inverse antiviral resistance profiles

A novel series of tyrosine-derived HIV protease inhibitors was synthesized and evaluated for in vitro antiviral activity against wild-type virus and two protease inhibitor-resistant viruses. All of the compounds had wild-type antiviral activities that were similar to or greater than several currently marketed HIV protease inhibitors. In addition, a number of compounds in this series were more potent against the drug-resistant mutant viruses than they were against wild-type virus.     

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.     

Structure-activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.     

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.     

Design and synthesis of broad-based mono- and bi- cyclic inhibitors of FIV and HIV proteases

Based on the substrate transition state and our strategy to tackle the problem of drug resistance, a series of HIV/FIV protease (HIV /FIV PR) monocyclic inhibitors incorporating a 15- or 17-membered macrocycle with an equivalent P3 or P3' group and a unique unnatural amino acid, (2R, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, have been designed and synthesized. In addition, based on the structure of TL3 with small P3/P3' group, we have synthesized two conformationally restricted bicyclic inhibitors containing the macrocycle, which mimic the P1/P1'-P3/P3' tripeptide [Phe-Val-Ala] of TL3. We have found that the contribution of the macrocycle in our monocyclic inhibitors is important to the overall activity, but the ring size does not affect the activity to a significant extent. Several inhibitors that were developed in this work, exhibit low nanomolar inhibitory activity against the wild-type HIV/FIV PR and found to be highly effective against some drug-resistant as well as TL3-resistant mutants of HIV PRs. Compound 15, in particular, is the most effective cyclic inhibitor in hand to inhibit FIV replication in tissue culture at a concentration of 1.0 micro g/mL (1.2 microM).     

Potency and selectivity of P2/P3-modified inhibitors of cysteine proteases from trypanosomes

A systematic study of P2 and P3 substitution in a series of vinyl sulfone cysteine protease inhibitors is described. The introduction of a methyl substituent in the P2 phenylalanine aryl ring had a favorable effect on protease inhibition and conferred modest selectivity for rhodesain over cruzain. Rhodesain selectivity could be enhanced further by combining these P2 modifications with certain P3 amide substituents.     

Design and synthesis of novel HIV-1 protease inhibitors incorporating oxyindoles as the P2'-ligands

A series of novel oxyindole-derived HIV-1 protease inhibitors were designed and synthesized based upon our X-ray crystal structure of inhibitor 2 (TMC-114) bound to HIV-1 protease. The effects of substituents, spirocyclic rings, and ring sizes have been investigated. A number of inhibitors exhibited low nanomolar inhibitory potencies against HIV protease.