Fluorine Modifications Contribute to Potent Antiviral Activity against Highly Drug-Resistant HIV-1 and Favorable Blood-Brain Barrier Penetration Property of Novel Central Nervous System-Targeting HIV-1 Protease Inhibitors In Vitro.

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 [EC50]: 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 (EC50: 0.003 to ∼0.006 µM). GRL-08513 and GRL-08613 also maintained their antiviral activities against HIV-2ROD 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.

Novel Central Nervous System (CNS)-Targeting Protease Inhibitors for Drug-Resistant HIV Infection and HIV-Associated CNS Complications.

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 [EC50], 0.0002 to ∼0.003 µM), with minimal cytotoxicity. These CNS-targeting PIs efficiently suppressed the replication of HIV-1 variants (EC50, 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-2ROD 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.

A Modified P1 Moiety Enhances In Vitro Antiviral Activity against Various Multidrug-Resistant HIV-1 Variants and In Vitro Central Nervous System Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413.

We report here that GRL-10413, a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a hydroxyethylamine sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50] of 0.00035 to 0.0018 µM), with minimal cytotoxicity (50% cytotoxic concentration [CC50] = 35.7 µM). GRL-10413 blocked the infectivity and replication of HIV-1NL4-3 variants selected by use of atazanavir, lopinavir, or amprenavir (APV) at concentrations of up to 5 µM (EC50 = 0.0021 to 0.0023 µM). GRL-10413 also maintained its strong antiviral activity against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that against APV. In addition, GRL-10413 showed favorable central nervous system (CNS) penetration properties as assessed with an in vitro blood-brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants, with favorable CNS penetration capability, and that the newly modified P1 moiety may confer desirable features in designing novel anti-HIV-1 PIs.

A novel tricyclic ligand-containing nonpeptidic HIV-1 protease inhibitor, GRL-0739, effectively inhibits the replication of multidrug-resistant HIV-1 variants and has a desirable central nervous system penetration property in vitro.

We report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0019 to 0.0036 µM), with minimal cytotoxicity (50% cytotoxic concentration [CC50], 21.0 µM). GRL-0739 blocked the infectivity and replication of HIV-1NL4-3 variants selected by concentrations of up to 5 µM ritonavir or atazanavir (EC50, 0.035 to 0.058 µM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2ROD variant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novel in vitro blood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles.

GRL-04810 and GRL-05010, difluoride-containing nonpeptidic HIV-1 protease inhibitors (PIs) that inhibit the replication of multi-PI-resistant HIV-1 in vitro and possess favorable lipophilicity that may allow blood-brain barrier penetration.

We designed, synthesized, and identified two novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-04810 and GRL-05010, containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, bis-tetrahydrofuranylurethane (bis-THF), and a difluoride moiety, both of which are active against the laboratory strain HIV-1LAI (50% effective concentrations [EC50s], 0.0008 and 0.003 µM, respectively) with minimal cytotoxicity (50% cytotoxic concentrations [CC50s], 17.5 and 37.0 µM, respectively, in CD4(+) MT-2 cells). The two compounds were active against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to various antiviral regimens. GRL-04810 and GRL-05010 also blocked the infectivity and replication of each of the HIV-1NL4-3 variants selected by up to 5 µM lopinavir (EC50s, 0.03 and 0.03 µM, respectively) and atazanavir (EC50s, 0.02 and 0.04 µM, respectively). Moreover, they were active against darunavir (DRV)-resistant variants (EC50 in 0.03 to 0.034 µM range for GRL-04810 and 0.026 to 0.043 µM for GRL-05010), while DRV had EC50s between 0.02 and 0.174 µM. GRL-04810 had a favorable lipophilicity profile as determined with the partition (log P) and distribution (log D) coefficients of -0.14 and -0.29, respectively. The in vitro blood-brain barrier (BBB) permeability assay revealed that GRL-04810 and GRL-05010 may have a greater advantage in terms of crossing the BBB than the currently available PIs, with apparent penetration indexes of 47.8 × 10(-6) and 61.8 × 10(-6) cm/s, respectively. The present data demonstrate that GRL-04810 and GRL-05010 exert efficient activity against a wide spectrum of HIV-1 variants in vitro and suggest that two fluorine atoms added to their bis-THF moieties may well enhance their penetration across the BBB.

GRL-0519, a novel oxatricyclic ligand-containing nonpeptidic HIV-1 protease inhibitor (PI), potently suppresses replication of a wide spectrum of multi-PI-resistant HIV-1 variants in vitro.

We report that GRL-0519, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing tris-tetrahydrofuranylurethane (tris-THF) and a sulfonamide isostere, is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0005 to 0.0007 µM) with minimal cytotoxicity (50% cytotoxic concentration [CC50], 44.6 µM). GRL-0519 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to a 5 µM concentration of ritonavir, lopinavir, or atazanavir (EC50, 0.0028 to 0.0033 µM). GRL-0519 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, highly darunavir (DRV)-resistant variants, and HIV-2ROD. The development of resistance against GRL-0519 was substantially delayed compared to other PIs, including amprenavir (APV) and DRV. The effects of nonspecific binding of human serum proteins on GRL-0519's antiviral activity were insignificant. Our analysis of the crystal structures of GRL-0519 (3OK9) and DRV (2IEN) with protease suggested that the tris-THF moiety, compared to the bis-THF moiety present in DRV, has greater water-mediated polar interactions with key active-site residues of protease and that the tris-THF moiety and paramethoxy group effectively fill the S2 and S2' binding pockets, respectively, of the protease. The present data demonstrate that GRL-0519 has highly favorable features as a potential therapeutic agent for treating patients infected with wild-type and/or multi-PI-resistant variants and that the tris-THF moiety is critical for strong binding of GRL-0519 to the HIV protease substrate binding site and appears to be responsible for its favorable antiretroviral characteristics.