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To date, there are no specific treatment regimens for HIV-1-related central nervous system (CNS) complications, such as HIV-1-associated neurocognitive disorders (HAND). Here, we report that two newly generated CNS-targeting HIV-1 protease (PR) inhibitors (PIs), GRL-08513 and GRL-08613, which have a P1-3,5-bis-fluorophenyl or P1-para-monofluorophenyl ring and P2-tetrahydropyrano-tetrahydrofuran (Tp-THF) with a sulfonamide isostere, are potent against wild-type HIV-1 strains and multiple clinically isolated HIV-1 strains (50% effective concentration [EC(50)]: 0.0001 to ∼0.0032 μM). As assessed with HIV-1 variants that had been selected in vitro to propagate at a 5 μM concentration of each HIV-1 PI (atazanavir, lopinavir, or amprenavir), GRL-08513 and GRL-08613 efficiently inhibited the replication of these highly PI-resistant variants (EC(50): 0.003 to ∼0.006 μM). GRL-08513 and GRL-08613 also maintained their antiviral activities against HIV-2(ROD) as well as severely multidrug-resistant clinical HIV-1 variants. Additionally, when we assessed with the in vitro blood-brain barrier (BBB) reconstruction system, GRL-08513 and GRL-08613 showed the most promising properties of CNS penetration among the evaluated compounds, including the majority of FDA-approved combination antiretroviral therapy (cART) drugs. In the crystallographic analysis of compound-PR complexes, it was demonstrated that the Tp-THF rings at the P2 moiety of GRL-08513 and GRL-08613 form robust hydrogen bond interactions with the active site of HIV-1 PR. Furthermore, both the P1-3,5-bis-fluorophenyl- and P1-para-monofluorophenyl rings sustain greater contact surfaces and form stronger van der Waals interactions with PR than is the case with darunavir-PR complex. Taken together, these results strongly suggest that GRL-08513 and GRL-08613 have favorable features for patients infected with wild-type/multidrug-resistant HIV-1 strains and might serve as candidates for a preventive and/or therapeutic agent for HAND and other CNS complications.

We designed, synthesized, and identified two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL- 037 and GRL-044, containing P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), P1-benzene and P1-methoxybenzene, respectively, and P2'-isopropyl-aminobenzothiazole (Ip-Abt), based on the structure of the prototypic PI, darunavir (DRV). The 50% inhibitory concentrations (IC(50)s) of GRL-037 and GRL-044 against wild-type HIV-1(NL4-3) were 0.042 and 0.0028-0.0033 nM with minimal cytotoxicity profiles compared to the IC(50) values of four most potent FDA-approved PIs, ranging from 2.6 to 70 nM. GRL-044 was also potent against HIV-2EHO (IC(50)=0.0004 nM) and various PI-resistant HIV-1 variants (IC(50) ranging from 0.065 to 19 nM). In the selection assays we conducted, the emergence of HIV-1 variants resistant to GRL-044 was significantly delayed compared to that against DRV. Thermal stability test using differential scanning fluorimetry employing purified HIV-1 protease (PR) and SYPRO(®) Orange showed that both GRL-037 and GRL-044 tightly bound to PR. A28S substitution emerged in the homologous recombination-based selection assays with GRL-044. Structural analyses showed that the larger size of GRL-044 over DRV, enabling GRL-044 to fit better to the hydrophobic cavity of protease, contributed to the greater potency of GRL- 044 against HIV-1. Structural analyses also suggested that the van der Waals surface contact of GRL-044 with A28' appears to be better compared to that of DRV because of the larger surface of Ip-Abt of GRL-044, which may be partially responsible for the emergence of A28S. The present antiviral data and structural features of GRL-044 should provide molecular insights for further design and development of potent and "resistance-repellant" novel PIs.

There is currently no specific therapeutics for the HIV-1-related central nervous system (CNS) complications. Here we report that three newly designed CNS-targeting HIV-1 protease inhibitors (PIs), GRL-083-13, GRL-084-13, and GRL-087-13, which contain a P1-3,5-bis-fluorophenyl or P1-para-monofluorophenyl ring, and P2-bis-tetrahydrofuran (bis-THF) or P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), with a sulfonamide isostere, are highly active against wild-type HIV-1 strains and primary clinical isolates (50% effective concentration [EC(50)], 0.0002 to ∼0.003 μM), with minimal cytotoxicity. These CNS-targeting PIs efficiently suppressed the replication of HIV-1 variants (EC(50), 0.002 to ∼0.047 μM) that had been selected to propagate at high concentrations of conventional HIV-1 PIs. Such CNS-targeting PIs maintained their antiviral activity against HIV-2(ROD) as well as multidrug-resistant clinical HIV-1 variants isolated from AIDS patients who no longer responded to existing antiviral regimens after long-term therapy. Long-term drug selection experiments revealed that the emergence of resistant-HIV-1 against these CNS-targeting PIs was substantially delayed. In addition, the CNS-targeting PIs showed the most favorable CNS penetration properties among the tested compounds, including various FDA-approved anti-HIV-1 drugs, as assessed with the in vitro blood-brain barrier reconstruction system. Crystallographic analysis demonstrated that the bicyclic rings at the P2 moiety of the CNS-targeting PIs form strong hydrogen-bond interactions with HIV-1 protease (PR) active site. Moreover, both the P1-3,5-bis-fluorophenyl and P1-para-monofluorophenyl rings sustain greater van der Waals contacts with PR than in the case of darunavir (DRV). The data suggest that the present CNS-targeting PIs have desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 strains and might serve as promising preventive and/or therapeutic candidates for HIV-1-associated neurocognitive disorders (HAND) and other CNS complications.

We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1 WT ), with 50% effective concentrations (EC 50 s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC 50 ) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIV MDR ). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1 DRV R P51 ); the three compounds remained active against HIV-1 DRV R P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIV MDR . IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIV DRV R ) has recently been observed in vivo and in vitro . Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIV DRV R . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIV DRV R . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIV DRV R .

We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2′ cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC 50 s) were 2.5 to 30 nM against wild-type HIV-1 NL4-3 , 0.3 to 6.7 nM against HIV-2 EHO , and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIV MDR ). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIV DRV r p51 ), with EC 50 s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC 50 s of >1,000 nM) against HIV DRV r p51 . Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2′ Cp-Abt group forms strong hydrogen bonds with Asp30′. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIV MDR strains (HIV 11MIX ), HIV 11MIX became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV 11MIX failed to replicate at >0.08 μM and >0.2 μM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2′ Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1 NL4-3 and a wide spectrum of HIV MDR strains.

Design, synthesis, biological and X-ray crystallographic studies of a series of potent HIV-1 protease inhibitors are described. Various polar functionalities have been incorporated on the tetrahydropyranyl-tetrahydrofuran-derived P2 ligand to interact with the backbone atoms in the S2-subsite. The majority of the inhibitors showed very potent enzyme inhibitory and antiviral activity. Two high-resolution X-ray structures of 30b- and 30j-bound HIV-1 protease provide insight into ligand-binding site interactions. In particular, the polar functionalities on the P2-ligand appear to form unique hydrogen bonds with Gly48 amide NH and amide carbonyl groups in the flap region.

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors incorporating stereochemically defined fused tricyclic P2-ligands are described. Various substituent effects were investigated in order to maximize the ligand-binding site interactions in the protease active site. Inhibitors 16a and 16f showed excellent enzyme inhibitory and antiviral activity while incorporation of sulfone functionality resulted in a decrease in potency. Both inhibitors 16a and 16f have maintained activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 16a-bound HIV-1 protease revealed important molecular insights into the ligand-binding site interactions which may account for the inhibitor’s potent antiviral activity and excellent resistance profiles.

We investigated substituted bis-THF-derived HIV-1 protease inhibitors in order to enhance ligand-binding site interactions in the HIV-1 protease active site. In this context, we have carried out convenient syntheses of optically active bis-THF and C4-substituted bis-THF ligands using a [2,3]-sigmatropic rearrangement as the key step. The synthesis provided convenient access to a number of substituted bis-THF derivatives. Incorporation of these ligands led to a series of potent HIV-1 protease inhibitors. Inhibitor 23c turned out to be the most potent (Ki = 2.9 pM; IC50 = 2.4 nM) among the inhibitors. An X-ray structure of 23c-bound HIV-1 protease showed extensive interactions of the inhibitor with the protease active site, including a unique water-mediated hydrogen bond to the Gly-48 amide NH in the S2 site.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand-binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolution X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand-binding site interactions are possibly responsible for their potent activity.

Substituted benzo-fused 8-oxabicyclo[3.2.1]octanes and 9-oxabicyclo[4.2.1]nonanes are prepared from substituted electron-rich benzyl dihydrofurans and phenethyl dihydrofurans.

We investigated substituted bis-THF-derived HIV-1 protease inhibitors in order to enhance ligand-binding site interactions in the HIV-1 protease active site. In this context, we have carried out convenient syntheses of optically active bis-THF and C4-substituted bis-THF ligands using a [2,3]-sigmatropic rearrangement as the key step. The synthesis provided convenient access to a number of substituted bis-THF derivatives. Incorporation of these ligands led to a series of potent HIV-1 protease inhibitors. Inhibitor 23c turned out to be the most potent (K(i) = 2.9 pM; IC(50) = 2.4 nM) among the inhibitors. An X-ray structure of 23c-bound HIV-1 protease showed extensive interactions of the inhibitor with the protease active site, including a unique water-mediated hydrogen bond to the Gly-48 amide NH in the S2 site.

A new and convenient synthesis of benzo-fused 8-oxabicyclo[3.2.1]octane and 9-oxabicyclo[4.2.1]nonane derivatives are described. The reaction involved a TiCl(4)-mediated tandem carbonyl or imine addition followed by a Friedel-Crafts cyclization to provide these functionalized derivatives in good to excellent yields and high diastereoselectivity.