Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design,synthesis, and biological evaluation

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory K_i value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.     

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.     

Combinatorial design of nonsymmetrical cyclic urea inhibitors of aspartic protease of HIV-1

The aspartic protease (PR) of the human immunodeficiency virus type 1 (HIV-1) is an important target for the design of specific antiviral agents dedicated to treatment of HIV-1 infection. We have employed computer-assisted combinatorial chemistry methods to design a small focused virtual library of nonsymmetrically substituted cyclic urea inhibitors of the PR. Nonsymmetrical compounds with decreased peptidic character were namely found to inhibit the PR with comparable inhibition potencies as their C2-pseudosymmetric counterparts and to possess superior pharmacokinetic properties. To generate the virtual library of fully nonsymmetrical cyclic urea analogs, diverse reagents were selected from databases of available chemicals with characteristics similar to those of the building blocks of known potent PR inhibitors. The X-ray structure of the protease-inhibitor complex PR-XV-638 was used as the receptor model in the structure-based focusing and in silico screening of the virtual library. A target-specific LUDI-type scoring function, parameterized for a QSAR training set of known cyclic urea inhibitors and validated on a set of compounds not included into the training set, was used to predict the inhibition constants (Ki) of the generated analogs toward the HIV-1 PR. The fragments most frequently occurring in the analogs with the highest predicted inhibition potencies (Ki*<10 pM) were then selected to constitute a highly focused library subset containing novel nonsymmetrical cyclic ureas with predicted Ki*s 1 order of magnitude lower than the most potent known cyclic urea inhibitors. ADME properties calculated for the most promising analogs suggested that the cyclic ureas are endowed with a wide range of favorable pharmacokinetic properties, which may favor the discovery of a potent orally administrable antiviral drug.     

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.     

Design and synthesis of potent HIV-1 protease inhibitors incorporating hydroxyprolinamides as novel P2 ligands

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different hydroxyprolinamide P2 ligands were designed and synthesized. Variation of substitutions at the P2 significantly affected the enzyme inhibitory potency of the inhibitors. Compounds 2a and 2d showed excellent enzyme inhibitory activity with IC₅₀ values in the nanomolar range. An active site binding model for inhibitors 2a and 2d was suggested based upon the computational-docking results of the ligand with HIV-1 protease. This model offers molecular insights regarding ligand-binding site interactions of the hydroxyprolinamide-derived novel P2-ligand.     

Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors.

HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 A resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the delta-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.     

Computational design of new cyclic urea inhibitors for improved binding of HIV-1 aspartic protease

We report in this paper the design, by means of computational techniques, of new cyclic urea inhibitors of the HIV aspartic protease. The relationship between the complexation energies of the enzyme with known inhibitors and the experimentally determined log K(i) have been studied and used to predict inhibition constants for new inhibitors.     

Potent P2Y1 urea antagonists bearing various cyclic amine scaffolds

A number of new amine scaffolds with good inhibitory activity in the ADP-induced platelet aggregation assay have been found to be potent antagonists of the P2Y₁ receptor. SAR optimization led to the identification of isoindoline 3c and piperidine 4a which showed good in vitro binding and functional activities, as well as improved aqueous solubility. Among them, the piperidine 4a showed the best overall profile with favorable PK parameters.     

A mechanistic study of 3-aminoindazole cyclic urea HIV-1 protease inhibitors using comparative QSAR

Comparative QSAR studies on P2/P2' and P1/P1' substituted symmetrical and nonsymmetrical 3-aminoindazole cyclic urea HIV-1 protease inhibitors were performed. The protease inhibitory activity of these compounds was found to decrease with larger and more hydrophobic molecules, whereas the antiviral potency and translation across the cell membrane increases with increase in hydrophobicity and size. These results provide mechanistic insight about the mode of interaction of these compounds with HIV-1 protease receptor and would help in further improving the biological activity.     

Discovery of potent HIV-1 protease inhibitors incorporating sulfoximine functionality

Based on the unique property of sulfoximine and the homodimeric C(2) structural symmetry of HIV-1 protease, a novel class of sulfoximine-based pseudosymmetric HIV-1 protease inhibitors was designed and synthesized. The sulfoximine moiety was demonstrated to be important for HIV-1 protease inhibitor potency. The most active stereoisomer (2S,2'S) displays a potency of 2.5 nM (IC(50)) against HIV-1 protease and an anti-HIV-1 activity of 408 nM (IC(50)). A possible mode of action is proposed.     

Modified solvent accessibility free energy prediction analysis of cyclic urea inhibitors binding to the HIV-1 protease

One of the most successful drug targets against AIDS in the last decade has been the HIV-1 protease (HIV-1 PR), an enzyme that processes the polyprotein gene products into active replicative viral proteins. In our quest for a wide-ranging, binding free energy function we have extended the solvent accessibility free energy predictor (SAFE_p) method, recently developed for peptidic HIV-1 PR inhibitors, to the study of the binding of cyclic urea (CU) HIV-1 PR inhibitors. Our results show that there is a need for a specific term depicting polar contacts to be added to the original SAFE_p analytical expression, an outcome not seen in our studies of HIV-1 PR peptidic inhibitors. Nevertheless, despite the higher profile of the electrostatic interactions in the binding of the CU inhibitors, our analysis indicates that CU inhibitor binding is still driven by the hydrophobic entropic contribution, as much as for the peptidic inhibitors.     

Unsymmetrical cyclic ureas as HIV-1 protease inhibitors: novel biaryl indazoles as P2/P2' substituents

The preparation of unsymmetrical cyclic ureas bearing novel biaryl indazoles as P2/P2' substituents was undertaken, utilizing a Suzuki coupling reaction as the key step. Compound 6i was equipotent to the lead compound of the series SE063.     

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.     

Potent and selective aggrecanase inhibitors containing cyclic P1 substituents

Anti-succinate hydroxamates with cyclic P1 motifs were synthesized as aggrecanase inhibitors. The N-methanesulfonyl piperidine 23 and the N-trifluoroacetyl azetidine 26 were the most potent aggrecanase inhibitors both having an IC(50)=3nM while maintaining >100-fold selectivity over MMP-1, -2, and -9. The cyclic moieties were also capable of altering in vivo metabolism, hence delivering low clearance compounds in both rat and dog studies as shown for compound 14.     

Synthesis,antiviral activity and pharmacokinetics of P1/P1' substituted 3-aminoindazole cyclic urea HIV protease inhibitors

A series of P1/P1' substituted cyclic urea analogues were prepared in an attempt to increase the intra-cellular antiviral potency of the nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851. The effect of alkyl substitution of the P1/P1' residues on cellular antiviral potency, protein binding, resistance profile and pharmacokinetics are described.     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Novel spirocyclic pyrrolidones as P2/P1 mimetics in potent inhibitors of HIV-1 protease

We have developed concise and efficient syntheses of novel spirocyclic pyrrolidones 1-3, which involve the alkylation of pyrrolidone precursor 13 with 1,5-dibromopentane, 16 and 15, followed by an in situ lactamization. Conjugates of 1 and 2 with P1'/P2' hydroxy-indanolamine moiety resulted in novel and potent inhibitors of HIV-1 protease 25 and 26, suggesting that 1 and 2 are novel P2/P1 HIV-PI mimetics.     

Adaptive inhibitors of the HIV-1 protease

A significant obstacle to the efficacy of drugs directed against viral targets is the presence of amino acid polymorphisms in the targeted molecules. Amino acid polymorphisms may occur naturally due to the existence of variations within and between viral strains or as the result of mutations associated with drug resistance. An ideal drug will be one that is extremely effective against a primary target and maintains its effectiveness against the most important variations of the target molecule. A drug that simultaneously inhibits different variants of the target will lead to a faster suppression of the virus, retard the appearance of drug-resistant mutants and provide more efficacious and, in the long range, more affordable therapies. Drug molecules with the ability to inhibit several variants of a target with high affinity have been termed adaptive drugs (Nat. Biotechnol. 20 (2002) 15; Biochemistry 42 (2003) 8459; J. Cell. Biochem. S37 (2001) 82). Current drug design paradigms are predicated upon the lock-and-key hypothesis, which emphasizes shape complementarity as a way to attain specificity and improved binding affinity. Shape complementarity is accomplished by the introduction of conformational constraints in the drug molecule. While highly constrained molecules do well against a unique target, they lack the ability to adapt to target variations like those originating from naturally occurring polymorphisms or drug-resistant mutations. Targeting an array of closely related targets rather than a single one while still maintaining selectivity, requires a different approach. A plausible strategy for designing high affinity adaptive inhibitors is to engineer their most critical interactions (for affinity and specificity) with conserved regions of the target while allowing for adaptability through the introduction of flexible asymmetric functionalities in places facing variable regions of the target. The fundamental thermodynamics and structural principles associated with this approach are discussed in this chapter.     

Dimerization inhibitors of HIV-1 protease

By targeting the highly conserved antiparallel beta-sheet formed by the interdigitation of the N- and C-terminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current active-site directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N- and C-terminal mimetics, lipopeptides and cross-linked interface peptides.