Synthesis and antiviral activities of the major metabolites of the HIV protease inhibitor ABT-378 (Lopinavir).

The HIV protease inhibitor ABT-378 (Lopinavir) is metabolized rapidly and extensively by CYP-3A4 catalyzed oxidation. Three of the major metabolites identified were synthesized and their antiviral (HIV) activities determined.

ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease.

The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 microM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, 50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.

Human serum attenuates the activity of protease inhibitors toward wild-type and mutant human immunodeficiency virus.

The potency of therapeutic regimens containing human immunodeficiency virus (HIV) protease inhibitors is related to the ability to maintain concentrations of drug in the plasma of patients that are sufficient for blocking viral replication. The estimation of concentrations required for in vivo activity using in vitro assays is complicated by the fact that extensive binding of many protease inhibitors to serum proteins attenuates their antiviral potency. To provide insight into the relative in vivo potency of current protease inhibitors, we assayed their in vitro activity against wild-type and mutant HIV in the presence of human serum (HS). Using this assay, ABT-378, a new protease inhibitor with trough levels in humans far in excess of the EC50 in the presence of 50% HS, was identified. The antiviral activity of ABT-378 was only modestly attenuated by HS, in contrast to ritonavir, saquinavir, and nelfinavir. Examination of the effect of individual serum components suggested that the activity of ABT-378 is affected predominantly by binding to alpha1-acid glycoprotein (AGP) while the activity of ritonavir is modulated by both AGP and albumin. The method described here may provide insight into the in vivo potency of protease inhibitors and be useful for the preclinical evaluation and selection of new protease inhibitors for clinical studies.

Discovery of ritonavir, a potent inhibitor of HIV protease with high oral bioavailability and clinical efficacy.

The structure-activity studies leading to the potent and clinically efficacious HIV protease inhibitor ritonavir are described. Beginning with the moderately potent and orally bioavailable inhibitor A-80987, systematic investigation of peripheral (P3 and P2') heterocyclic groups designed to decrease the rate of hepatic metabolism provided analogues with improved pharmacokinetic properties after oral dosing in rats. Replacement of pyridyl groups with thiazoles provided increased chemical stability toward oxidation while maintaining sufficient aqueous solubility for oral absorption. Optimization of hydrophobic interactions with the HIV protease active site produced ritonavir, with excellent in vitro potency (EC50 = 0.02 microM) and high and sustained plasma concentrations after oral administration in four species. Details of the discovery and preclinical development of ritonavir are described.

Potent piperazine hydroxyethylamine HIV protease inhibitors containing novel P3 ligands.

The 2-isopropyl thiazolyl group is a highly optimized P3 ligand for C2 symmetry-based HIV protease inhibitors, as exemplified in the drug ritonavir. Here we report that incorporation of this P3 ligand into a piperazine hydroxyethylamine series also yielded novel, highly potent inhibitors. In tissue culture assays, the presence of human serum was less deleterious to the activity of these inhibitors than to that of ritonavir. Furthermore, potent activity against ritonavir resistant HIV was observed.

Novel azacyclic ureas that are potent inhibitors of HIV-1 protease.

A series of novel, azacyclic ureas which are highly potent inhibitors of the HIV-1 protease (IC50 = 4.1 to < 0.5 nM) were synthesized. Aqueous solubilities of this series of compounds were improved by incorporating polar functional groups at the P1' P2 and P2' positions. These compounds also possess good anti-viral activity by inhibition of the cytopathic effect of HIV-13B in MT-4 cells in vitro.

Ordered accumulation of mutations in HIV protease confers resistance to ritonavir.

Analysis of the HIV protease gene from the plasma of HIV-infected patients revealed substitutions at nine different codons selected in response to monotherapy with the protease inhibitor ritonavir. Mutants at valine-82, although insufficient to confer resistance, appeared first in most patients. Significant phenotypic resistance required multiple mutations in HIV protease, which emerged subsequently in an ordered, stepwise fashion. The appearance of resistance mutations was delayed in patients with higher plasma levels of ritonavir. Early mutants retained susceptibility to structurally diverse protease inhibitors, suggesting that dual protease inhibitor therapy might increase the duration of viral suppression.

A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease.

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.

Potent inhibitors of the HIV-1 protease with good oral bioavailabilities.

A series of novel pseudo-symmetrical and unsymmetrical inhibitors based on the backbone modification of a peptidomimetic were synthesized and found to be highly potent inhibitors of the HIV-1 protease (IC50 = 2.9 to < 0.5 nM). These compounds also possess good antiviral activity in vitro as measured by inhibition of the cytopathic effect of HIV-1(3B) in MT-4 lymphocytes. Importantly, some of these compounds also have good oral bioavailabilities in rats (F = 30.6% to 100%). One of these compounds 4C, also has good oral bioavailability in beagle dogs and cynomolgus monkeys.

ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans.

Examination of the structural basis for antiviral activity, oral pharmacokinetics, and hepatic metabolism among a series of symmetry-based inhibitors of the human immunodeficiency virus (HIV) protease led to the discovery of ABT-538, a promising experimental drug for the therapeutic intervention in acquired immunodeficiency syndrome (AIDS). ABT-538 exhibited potent in vitro activity against laboratory and clinical strains of HIV-1 [50% effective concentration (EC50) = 0.022-0.13 microM] and HIV-2 (EC50 = 0.16 microM). Following a single 10-mg/kg oral dose, plasma concentrations in rat, dog, and monkey exceeded the in vitro antiviral EC50 for > 12 h. In human trials, a single 400-mg dose of ABT-538 displayed a prolonged absorption profile and achieved a peak plasma concentration in excess of 5 micrograms/ml. These findings demonstrate that high oral bioavailability can be achieved in humans with peptidomimetic inhibitors of HIV protease.

Design of orally bioavailable, symmetry-based inhibitors of HIV protease.

A series of novel inhibitors of HIV-1 protease with excellent oral bioavailability is described. Differential acylation of the two amino groups of symmetry-based diamine core groups 2-5 led to unsymmetrically substituted inhibitors 17-43, many of which inhibited HIV protease at subnanomolar concentrations. Anti-HIV activity in vitro was observed at 0.1-1 microM. A systematic evaluation of the pharmacokinetic behavior of these inhibitors in rats identified the influence of aqueous solubility, molecular size and hydrogen-bonding functionality. Compound 30 (A-80987) was selected for further evaluation based on a favorable Cmax/ ED50 ratio (> 20) and half-life (> 2 h).

Pseudo-symmetrical difluoroketones. Highly potent and specific inhibitors of HIV-1 protease.

A series of novel, pseudo-symmetrical difluoroketones which are highly potent inhibitors of the HIV-1 protease (IC50 = 1.55-0.02 nM) were synthesized. These compounds also possess good antiviral activity by inhibition of the cytopathic effect of HIV-13B in MT-4 cells in vitro.

Symmetry-based inhibitors of HIV protease. Structure-activity studies of acylated 2,4-diamino-1,5-diphenyl-3-hydroxypentane and 2,5-diamino-1,6-diphenylhexane-3,4-diol.

The structure-activity relationships in two series of novel, symmetry-based inhibitors of HIV protease, the enzyme responsible for maturation of the human immunodeficiency virus, are described. Beginning with lead compounds 3-6, the effect of adding polar, heterocyclic end groups to one or both ends of the symmetric or pseudosymmetric inhibitors was probed. Aqueous solubility was enhanced > 1000-fold while maintaining potent inhibition of purified HIV-1 protease and anti-HIV activity in vitro. Pharmacokinetic studies in rats indicated a substantial difference in the absorption properties of mono-ol-based and diol-based inhibitors. The oral bioavailability of inhibitor 19 in rats was 19%; however, the Cmax obtained failed to exceed the anti-HIV EC50 in vitro. Substantial plasma levels of potent inhibitors of the diol class were not obtained after oral administration in rats; however, the optimal combination of aqueous solubility and in vitro antiviral activity of several inhibitors support their potential use in intravenous therapy.

Antiviral and pharmacokinetic properties of C2 symmetric inhibitors of the human immunodeficiency virus type 1 protease.

Specific processing of the human immunodeficiency virus (HIV) gag and gag-pol polyprotein gene products by the HIV protease is essential for the production of mature, infections progeny virions. Inhibitors of HIV protease block this maturation and thus prohibit the spread of HIV in vitro. Previously, we reported a series of novel, symmetric inhibitors of HIV protease designed to match the C2 symmetric structure of the active site of the enzyme. In response to the poor aqueous solubility of those lead compounds, we designed a series of analogs with substantially improved (greater than 10(4) fold) solubility. These inhibitors showed anti-HIV activity in H9 and MT4 cells at 0.05 to 10 microM, and in most cases, they were noncytotoxic at concentrations in excess of 100 microM. Further examination of one inhibitor (A-77003) revealed broad-spectrum activity against both HIV types 1 and 2, including azidothymidine-resistant HIV, in a variety of transformed and primary human cell lines. After administration of the inhibitors to rats, short half-lives and, with two notable exceptions, moderate oral bioavailability were observed. Additional pharmacokinetic studies in dogs and monkeys revealed the potential utility of A-77003 as an intravenous anti-HIV agent.

Potent HIV-1 protease inhibitors with antiviral activities in vitro.

A series of novel difluoroketones with low molecular weight (less than 600 m.u.) and which are potent inhibitors of the HIV-1 protease (IC50 = 1.0 to 21 nM) were synthesized. These compounds also exhibited antiviral activity by inhibition of the cytopathic effect of HIV-1(3)B in MT-4 cells in vitro.