Discovery of HIV-1 protease inhibitors with picomolar affinities incorporating N-aryl-oxazolidinone-5-carboxamides as novel P2 ligands

Here, we describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors incorporating N-phenyloxazolidinone-5-carboxamides into the (hydroxyethylamino)sulfonamide scaffold as P2 ligands. Series of inhibitors with variations at the P2 phenyloxazolidinone and the P2' phenylsulfonamide moieties were synthesized. Compounds with the (S)-enantiomer of substituted phenyloxazolidinones at P2 show highly potent inhibitory activities against HIV-1 protease. The inhibitors possessing 3-acetyl, 4-acetyl, and 3-trifluoromethyl groups at the phenyl ring of the oxazolidinone fragment are the most potent in each series, with K(i) values in the low picomolar (pM) range. The electron-donating groups 4-methoxy and 1,3-dioxolane are preferred at P2' phenyl ring, as compounds with other substitutions show lower binding affinities. Attempts to replace the isobutyl group at P1' with small cyclic moieties caused significant loss of affinities in the resulting compounds. Crystal structure analysis of the two most potent inhibitors in complex with the HIV-1 protease provided valuable information on the interactions between the inhibitor and the protease enzyme. In both inhibitor - enzyme complexes, the carbonyl group of the oxazolidinone ring makes hydrogenbond interactions with relatively conserved Asp29 residue of the protease. Potent inhibitors from each series incorporating various phenyloxazolidinone based P2 ligands were selected and their activities against a panel of multidrug-resistant (MDR) protease variants were determined. Interestingly, the most potent protease inhibitor starts out with extremely tight affinity for the wild-type enzyme (K(i) = 0.8 pM), and even against the MDR variants it retains picomolar to low nanomolar K(i), which is highly comparable with the best FDA-approved protease inhibitors.     

Design and synthesis of potent HIV-1 protease inhibitors incorporating hexahydrofuropyranol-derived high affinity P(2) ligands: structure-activity studies and biological evaluation

The design, synthesis, and evaluation of a new series of hexahydrofuropyranol-derived HIV-1 protease inhibitors are described. We have designed a stereochemically defined hexahydrofuropyranol-derived urethane as the P2-ligand. The current ligand is designed based upon the X-ray structure of 1a-bound HIV-1 protease. The synthesis of (3aS,4S,7aR)-hexahydro-2H-furo[2,3-b]pyran-4-ol, (-)-7, was carried out in optically active form. Incorporation of this ligand provided inhibitor 35a, which has shown excellent enzyme inhibitory activity and antiviral potency. Our structure-activity studies have indicated that the stereochemistry and the position of oxygens in the ligand are important to the observed potency of the inhibitor. Inhibitor 35a has maintained excellent potency against multidrug-resistant HIV-1 variants. An active site model of 35a was created based upon the X-ray structure of 1b-bound HIV-1 protease. The model offers molecular insights regarding ligand-binding site interactions of the hexahydrofuropyranol-derived novel P2-ligand.     

HIV-1 protease inhibitors based on hydroxyethylene dipeptide isosteres: an investigation into the role of the P1' side chain on structure-activity.

A systematic investigation was undertaken to determine the role of the P1' sidechain in a series of hydroxyethylene isostere based inhibitors of HIV-1 protease. Substitution and homologation of the benzyl P1' side chain of the Phe-Phe isostere based pseudo peptides 1 (L-682,679) and 2 (L-685,434) with various heteroalkyl groups leads to a series of extremely potent inhibitors of the enzyme. Several examples of the most potent inhibitors were very effective in an ex vivo cell based viral spread assay using human H9 T-lymphocytes and the IIIb isolate of HIV-1. Compound 19 is 120 times more potent than 1 and 16 times more potent than 2 in inhibiting the spread of infection in this assay.     

Design and fast synthesis of C-terminal duplicated potent C(2)-symmetric P1/P1'-modified HIV-1 protease inhibitors.

An analysis of the X-ray structure of a complex of HIV-1 protease with a linear C(2)-symmetric C-terminal duplicated inhibitor guided the selection of a series of diverse target compounds. These were synthesized with the objective to identify suitable P1/P1' substituents to provide inhibitors with improved antiviral activity. Groups with various physical properties were attached to the para-positions of the P1/P1' benzyloxy groups in the parent inhibitor. A p-bromobenzyloxy compound, prepared in only three steps from commercially available starting materials, was utilized as a common precursor in all reactions. The subsequent coupling reactions were completed within a few minutes and relied on palladium catalysis and flash heating with microwave irradiation. All of the compounds synthesized exhibited good inhibitory potency in the protease assay, with K(i) values ranging from 0.09 to 3.8 nM. A 30-fold improvement of the antiviral effect in cell culture, compared to the parent compound, was achieved with four of the inhibitors. The differences in K(i) values were not correlated to the differences in antiviral effect, efficiency against mutant virus, or reduced potency in the presence of human serum. The poorest enzyme inhibitors in fact belong to the group with the best antiviral effect. The binding features of two structurally related inhibitors, cocrystallized with HIV-1 protease, are discussed with special emphasis on the interaction at the enzyme/water phase.     

Structure-based design, synthesis, and structure-activity relationship studies of HIV-1 protease inhibitors incorporating phenyloxazolidinones

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different phenyloxazolidinone P2 ligands were designed and synthesized. Variation of phenyl substitutions at the P2 and P2' moieties significantly affected the binding affinity and antiviral potency of the inhibitors. In general, compounds with 2- and 4-substituted phenyloxazolidinones at P2 exhibited lower binding affinities than 3-substituted analogues. Crystal structure analyses of ligand-enzyme complexes revealed different binding modes for 2- and 3-substituted P2 moieties in the protease S2 binding pocket, which may explain their different binding affinities. Several compounds with 3-substituted P2 moieties demonstrated picomolar binding affinity and low nanomolar antiviral potency against patient-derived viruses from HIV-1 clades A, B, and C, and most retained potency against drug-resistant viruses. Further optimization of these compounds using structure-based design may lead to the development of novel protease inhibitors with improved activity against drug-resistant strains of HIV-1.     

Renin inhibitors containing new P1-P1' dipeptide mimetics with heterocycles in P1'.

A series of renin inhibitors containing new P1-P1' dipeptide mimetics are presented. The P1-P1' mimetics were obtained from (4S,5S)-3-(tert-butoxycarbonyl)-4-(cyclohexylmethyl)-5-[(omega- mesyloxy)alkyl]-2,2-dimethyloxazolidines 5b, 9, and 11b by nucleophilic substitution of the mesylate groups with the sodium salts of mercapto- and hydroxyheterocycles. Removal of the protecting groups and stepwise acylations with amino acid derivatives provided renin inhibitors with a length of a tripeptide. Replacement of P2 histidine by other amino acids maintained or enhanced renin inhibitory potency. By alteration of P3 phenylalanine, compounds with IC50 values in the nanomolar range and stability against chymotrypsin were obtained. Finally, the effect of the C-terminal heterocycle on the renin inhibition was studied. Compound XVII was examined in vivo for its hypotensive effects. In salt-depleted cynomolgus monkeys, XVII inhibited plasma renin activity and lowered blood pressure after oral administration of a dose of 10 mg/kg.     

Synthesis and biological activity of potent HIV-1 protease inhibitors based on Phe-Pro dihydroxyethylene isosteres

Peptidomimetic inhibitors of HIV-1 PR are still a key resource in the fight against AIDS. Here we describe the synthesis and biological activity of HIV-1 PR inhibitors based on four novel dihydroxyethylene isosteres of the Phe-Pro and Pro-Pro dipeptides. The isosteres, containing four stereogenic centers, were synthesized in high yield and excellent stereoselectivity via the cyclization of epoxy amines derived from α-amino acids. The inhibitors were assembled by coupling the isosteres with suitable flanking groups and were screened against recombinant HIV PR showing activities in the subnanomolar to micromolar range. Two Phe-Pro-based inhibitors active at the nanomolar level were further investigated: both inhibitors combine the ability to suppress HIV-1 replication in infected MT-2 cells with low cytotoxicity against the same cells, thereby displaying a high therapeutic index. These results demonstrate the potential of the new Phe-Pro dihydroxyethylene isostere as a core unit of powerful HIV-1 PR inhibitors.     

Design,synthesis, and biological evaluation of monopyrrolinone-based HIV-1 protease inhibitors

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors [(-)-6, (-)-7, (-)-23, (+)-24] based upon the 3,5,5-trisubstituted pyrrolin-4-one scaffold is described. Use of a monopyrrolinone scaffold leads to inhibitors with improved cellular transport properties relative to the earlier inhibitors based on bispyrrolinones and their peptide counterparts. The most potent inhibitor (-)-7 displayed 13% oral bioavailability in dogs. X-ray structure analysis of the monopyrrolinone compounds cocrystallized with the wild-type HIV-1 protease provided valuable information on the interactions between the inhibitors and the HIV-1 enzyme. In each case, the inhibitors assumed similar orientations for the P2'-P1 substituents, along with an unexpected hydrogen bond of the pyrrolinone NH with Asp225. Interactions with the S2 pocket, however, were not optimal, as illustrated by the inclusion of a water molecule in two of the three inhibitor-enzyme complexes. Efforts to increase affinity by displacing the water molecule with second and third generation inhibitors did not prove successful. Lack of success with this venture is a testament to the difficulty of accurately predicting the many variables that influence and build binding affinity. Comparison of the inhibitor positions in three complexes with that of Indinavir revealed displacements of the protease backbones in the enzyme flap region, accompanied by variations in hydrogen bonding to accommodate the monopyrrolinone ring. The binding orientation of the pyrrolinone-based inhibitors may explain their sustained efficacy against mutant strains of the HIV-1 protease enzyme as compared to Indinavir.     

Inhibition of HIV-2 protease by HIV-1 protease inhibitors in clinical use

Over the past 10 years, protease inhibitors have been a key component in antiretroviral therapies for HIV/AIDS. While the vast majority of HIV/AIDS cases in the world are due to HIV-1, HIV-2 infection must also be addressed. HIV-2 is endemic to Western Africa, and has also appeared in European countries such as Portugal, Spain, and Estonia. Current protease inhibitors have not been optimized for treatment of HIV-2 infection; therefore, it is important to assess the effectiveness of currently FDA-approved protease inhibitors against the HIV-2 protease, which shares only 50% sequence identity with the HIV-1 protease. Kinetic inhibition assays were performed to measure the inhibition constants (K(i)) of the HIV-1 protease inhibitors indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, lopinavir, atazanavir, tipranavir, and darunavir against the HIV-2 protease. Lopinavir, saquinavir, tipranavir, and darunavir exhibit the highest potency with K(i) values of 0.7, 0.6, 0.45, and 0.17 nm, respectively. These K(i) values are 84, 2, 24, and 17 times weaker than the corresponding values against the HIV-1 protease. In general, inhibitors show K(i) ratios ranging between 2 and 80 for the HIV-2 and HIV-1 proteases. The relative drop in potency is proportional to the affinity of the inhibitor against the HIV-1 protease and is related to specific structural characteristics of the inhibitors. In particular, the potency drop is high when the maximum cap size of the inhibitors consists of very few atoms. Caps are groups located at the periphery of the molecule that are added to core structures to increase the specificity of the inhibitor to its target. The caps positioned on the HIV-1 protease inhibitors affect selectivity through interactions with distinct regions of the binding pocket. The flexibility and adaptability imparted by the higher number of rotatable bonds in large caps enables an inhibitor to accommodate changes in binding pocket geometry between HIV-1 and HIV-2 protease.     

Design and structure of symmetry-based inhibitors of HIV-1 protease

Summary The concept of active-site symmetry along with structural and mechanistic considerations of aspartic proteases has been used to design novel,C ₂-symmetry-based inhibitors of HIV PR. A comparative molecular-modeling strategy was used in the design of a pseudo-C ₂-symmetric diaminoalcohol series and later a diastereomeric series ofC ₂-symmetric diaminodiols. The structure of the pseudosymmetric alcohol A-74704, complexed with HIV PR, confirmed the proposed symmetric mode of binding, and also proved useful in subsequent efforts to improve the solubility of the more potent diol series. The crystal-structure analysis of a pseudosymmetricR,S-diol provided insights into the relative contributions of the two hydroxyl groups to the stability of the complex. A comparison of the binding modes for symmetric and asymmetric, substrate-based inhibitors indicates that the influence of symmetry on binding is mainly expressed at the protein level, since the overall subsite symmetry is maintained even with highly asymmetric inhibitors. Thus, while the incorporation ofC ₂ symmetry does not necessarily lead to compounds with improved potency over asymmetric compounds, this strategy has led to the design of structurally novel and perhaps pharmacologically desirable inhibitors. The growing number of examples of symmetry-based inhibitors is evidence that symmetry has become a useful paradigm for HIV protease inhibitor design and that structure-based approaches to inhibitor design can contribute in unique and critical ways to the conceptualization of medicinal chemistry strategies that can lead to useful clinical candidates for AIDS, cancer and other diseases.     

Design of HIV-1 protease inhibitors with C3-substituted hexahydrocyclopentafuranyl urethanes as P2-ligands: synthesis, biological evaluation, and protein-ligand X-ray crystal structure

We report the design, synthesis, biological evaluation, and the X-ray crystal structure of a novel inhibitor bound to the HIV-1 protease. Various C3-functionalized cyclopentanyltetrahydrofurans (Cp-THF) were designed to interact with the flap Gly48 carbonyl or amide NH in the S2-subsite of the HIV-1 protease. We investigated the potential of those functionalized ligands in combination with hydroxyethylsulfonamide isosteres. Inhibitor 26 containing a 3-(R)-hydroxyl group on the Cp-THF core displayed the most potent enzyme inhibitory and antiviral activity. Our studies revealed a preference for the 3-(R)-configuration over the corresponding 3-(S)-derivative. Inhibitor 26 exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray structure of 26-bound HIV-1 protease revealed important molecular insight into the ligand-binding site interactions.     

P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays.

The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1' benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 microM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1-100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.     

HIV-1 protease inhibitors: effects on HIV-2 replication and resistance

Novel antiretroviral drugs include protease (PR) inhibitors (e.g. atazanavir, tipranavir and darunavir) that block HIV-1 maturation and show remarkable antiviral potency on drug-resistant isolates. However, the strains used as prototypes in the design of the novel drugs belong to a specific clade (i.e. HIV-1 group M subtype B), which is the most prevalent in developed countries. At the same time, there is an increasing concern about the expansion of other HIV-1 clades as well as other related retroviruses, such as HIV-2. The HIV-2 PR is weakly inhibited by some PR inhibitors (e.g. amprenavir), and little is known of the mutational pathways leading to drug resistance in this virus. The design of specific PR inhibitors targeting HIV-2, or potent drugs showing broad specificity on HIV-1 and HIV-2 clades, remains a major challenge for the future.     

Flexible cyclic ethers/polyethers as novel P2-ligands for HIV-1 protease inhibitors: design, synthesis, biological evaluation, and protein-ligand X-ray studies

We report the design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors. The inhibitors incorporate stereochemically defined flexible cyclic ethers/polyethers as high affinity P2-ligands. Inhibitors containing small ring 1,3-dioxacycloalkanes have shown potent enzyme inhibitory and antiviral activity. Inhibitors 3d and 3h are the most active inhibitors. Inhibitor 3d maintains excellent potency against a variety of multi-PI-resistant clinical strains. Our structure-activity studies indicate that the ring size, stereochemistry, and position of oxygens are important for the observed activity. Optically active synthesis of 1,3-dioxepan-5-ol along with the syntheses of various cyclic ether and polyether ligands have been described. A protein-ligand X-ray crystal structure of 3d-bound HIV-1 protease was determined. The structure revealed that the P2-ligand makes extensive interactions including hydrogen bonding with the protease backbone in the S2-site. In addition, the P2-ligand in 3d forms a unique water-mediated interaction with the NH of Gly-48.     

Microwave-accelerated synthesis of P1'-extended HIV-1 protease inhibitors encompassing a tertiary alcohol in the transition-state mimicking scaffold

Two series of P1'-extended HIV-1 protease inhibitors comprising a tertiary alcohol in the transition-state mimic exhibiting Ki values ranging from 2.1 to 93 nM have been synthesized. Microwave-accelerated palladium-catalyzed cross-couplings were utilized to rapidly optimize the P1' side chain. High cellular antiviral potencies were encountered when the P1' benzyl group was elongated with a 3- or 4-pyridyl substituent (EC50 = 0.18-0.22 microM). X-ray crystallographic data were obtained for three inhibitors cocrystallized with the enzyme.     

Design of HIV-1 protease inhibitors active on multidrug-resistant virus

On the basis of structural data gathered during our ongoing HIV-1 protease inhibitors program, from which our clinical candidate TMC114 9 was selected, we have discovered new series of fused heteroaromatic sulfonamides. The further extension into the P2' region was aimed at identifying new classes of compounds with an improved broad spectrum activity and acceptable pharmacokinetic properties. Several of these compounds display an exceptional broad spectrum activity against a panel of highly cross-resistant mutants. Certain members of these series exhibit favorable pharmacokinetic profiles in rat and dog. Crystal structures and molecular modeling were used to rationalize the broad spectrum profile resulting from the extension into the P2' pocket of the HIV-1 protease.     

Design, synthesis, and X-ray crystallographic analysis of a novel class of HIV-1 protease inhibitors

In the present paper, design, synthesis, X-ray crystallographic analysis, and HIV-1 protease inhibitory activities of a novel class of compounds are disclosed. Compounds 28-30, 32, 35, and 40 were synthesized and found to be inhibitors of the HIV-1 protease. The crucial step in their synthesis involved an unusual endo radical cyclization process. Absolute stereochemistry of the three asymmetric centers in the above compounds have been established to be (4S,2'R,3'S) for optimal potency. X-ray crystallographic analysis has been used to determine the binding mode of the inhibitors to the HIV-1 protease.     

Design and synthesis of dipeptide nitriles as reversible and potent Cathepsin S inhibitors

The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.