In vitro inhibition of human immunodeficiency virus (HIV) type 1 replication by C2 symmetry-based HIV protease inhibitors as single agents or in combinations.

C2 symmetry-based human immunodeficiency virus (HIV) protease inhibitors were examined in vitro as single agents or in combination with 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine for activity against HIV type 1 (HIV-1). Ten C2 symmetry-based or pseudo-C2 symmetry-based HIV protease inhibitors were active against a laboratory strain (HIV-1IIIB) in the HIV-1 cytopathic effect inhibition assay. Three inhibitors, A75925, A76928, and A77003, selected to represent a range of aqueous solubility and antiviral activity, were active against four different HIV-1 strains tested. These three inhibitors exhibited a significant inhibition of the cytopathic effect of HIV-1 against the CD4+ ATH8 cell line, with 90% inhibitory concentrations ranging from 0.1 to 4 microM. Cellular toxicity was negligible at up to 20 microM. Furthermore, they completely inhibited the replication of monocytotropic strain HIV-1Ba-L in purified monocytes and macrophages at 0.75 to 2 microM. Potent inhibitory activity against a primary HIV-1 isolate and an AZT-resistant HIV-1 variant was also observed for all three inhibitors in phytohemagglutinin-activated peripheral blood mononuclear cells. When these three HIV protease inhibitors and AZT or 2',3'-dideoxyinosine were used in combinations against a primary HIV isolate in phytohemagglutinin-activated peripheral blood mononuclear cells and the results were analyzed with the COMBO program package, their antiviral activities were identified to be synergistic in some cases and additive in others. The present data warrant further investigations of these compounds as potential antiviral agents for the therapy of HIV infections.     

Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology.

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.     

Oxidative Modifications of Kynostatin-272, a Potent Human Immunodeficiency Virus Type 1 Protease Inhibitor: Potential Mechanism for Altered Activity in Monocytes/Macrophages

Previous studies have indicated that human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) are less active at blocking viral replication in HIV-1 infected peripheral blood monocytes/macrophages (M/M) than in HIV-1-infected T cells. We explored the hypothesis that oxidative modification and/or metabolism of the PIs in M/M might account for this reduced potency. We first tested the susceptibility of several PIs (kynostatin-272 [KNI-272], saquinavir, indinavir, ritonavir, or JE-2147) to oxidation after exposure to hydrogen peroxide (H(2)O(2)): only KNI-272 was highly susceptible to oxidation. Treatment of KNI-272 with low millimolar concentrations of H(2)O(2) resulted in mono-oxidation of the sulfur in the S-methyl cysteine (methioalanine) moiety, as determined by reversed-phase high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS). Higher concentrations of H(2)O(2) led to an additional oxidation of the sulfur in the thioproline moiety of KNI-272. None of the PIs were metabolized or oxidized when added to T cells and cultured for up to 12 days. However, when KNI-272 was added to M/M, the concentration of the original KNI-272 steadily decreased with a corresponding increase in the production of three KNI-272 metabolites as identified by RP-HPLC/MS. The structures of these metabolites were different from those produced by H(2)O(2) treatment. The two major products of M/M metabolism of KNI-272 were identified as isomeric forms of KNI-272 oxidized solely on the thioproline ring. Both metabolites had reduced capacities to inhibit HIV-1 protease activity when tested in a standard HIV-1 protease assay. These studies demonstrate that antiviral compounds can be susceptible to oxidative modification in M/M and that this can affect their antiviral potency.     

Metabolic characterization of a tripeptide human immunodeficiency virus type 1 protease inhibitor, KNI-272, in rat liver microsomes

KNI-272 is a tripeptide protease inhibitor for treating human immunodeficiency virus type 1 (HIV-1). In in vitro stability studies using rat tissue homogenates, KNI-272 concentrations in the liver, kidney, and brain decreased significantly with time. Moreover, in tissue distribution studies, KNI-272 distributed highly to the liver, kidney, and small intestine in vivo. From these results and reported physiological parameters such as the tissue volume and tissue blood flow rate, we considered the liver to be the main organ which takes part in the metabolic elimination of KNI-272. Then the hepatic metabolism of KNI-272 was more thoroughly investigated by using rat liver microsomes. KNI-272 was metabolized in the rat liver microsomes, and five metabolites were found. The initial metabolic rate constant (kmetabolism) tended to decrease when the KNI-272 concentration in microsomal suspensions increased. The calculated Michaelis-Menten constant (K(m)) and the maximum velocity of KNI-272 metabolism (Vmax), after correction for the unbound drug concentration, were 1.12 +/- 0.09 micrograms/ml (1.68 +/- 0.13 microM) and 0.372 +/- 0.008 microgram/mg of protein/min (0.558 +/- 0.012 nmol/mg of protein per min), respectively. The metabolic clearance (CLint,metabo), calculated as Vmax/K(m), was 0.332 ml/mg of protein per min. Moreover, by using selective cytochrome P-450 inhibitors and recombinant human CYP3A4 fractions, KNI-272 was determined to be metabolized mainly by the CYP3A isoform. In addition, ketoconazole, a representative CYP3A inhibitor, inhibited KNI-272 metabolism competitively, and the inhibition constant (Ki) was 4.32 microM.     

Protein binding of human immunodeficiency virus protease inhibitor KNI-272 and alteration of its in vitro antiretroviral activity in the presence of high concentrations of proteins.

KNI-272 represents a peptide-based protease inhibitor having potent antiretroviral activity against human immunodeficiency virus (HIV) in vitro. The structure contains allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] with a hydroxymethylcarbonyl isostere. We asked whether this experimental anti-HIV agent could exert its activity in vitro in the presence of relatively high concentrations of fetal calf serum (FCS) and assessed its protein-binding properties by using fresh human plasma preparations. The 50 and 75% inhibitory concentrations of KNI-272 against HIV type 1 replication in vitro were 3- to 5-fold and 5-fold higher in the presence of 50% FCS and 15- to 25-fold and 25- to 100-fold higher in the presence of 80% ECS, respectively, than those with 15% FCS, whereas the antiviral activity of 2',3'-dideoxyinosine was not significantly affected by FCS concentrations in the culture. Detailed studies of the protein binding of KNI-272 suggest that in human plasma binding occurs predominantly to alpha 1-acid glycoprotein and that KNI-272 is probably extensively (approximately 98 to 99%) protein bound at concentrations likely to be achieved in the circulation. Thus, higher levels of KNI-272 in plasma may be required when this compound undergoes clinical trials relative to those inferred from in vitro data involving the use of 10 to 15% FCS-containing culture media. The current data may have a relevance to other antiretroviral drugs that are under development and that have a high protein-binding capacity.     

A novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI), GRL-98065, is potent against multiple-PI-resistant human immunodeficiency virus in vitro

We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC(50)], 0.0002 to 0.0005 microM) with minimal cytotoxicity (50% cytotoxicity, 35.7 microM in CD4(+) MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1(NL4-3) variants exposed to and selected by up to a 5 microM concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 microM concentration of lopinavir or atazanavir (EC(50), 0.0015 to 0.0075 microM), although it was less active against HIV-1(NL4-3) selected by amprenavir (EC(50), 0.032 microM). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active-site amino acids (Asp29 and Asp30) is important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.     

Antiviral properties of aminodiol inhibitors against human immunodeficiency virus and protease.

A series of aminodiol inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were identified by using an in vitro peptide cleavage assay. BMS 182,193, BMS 186,318, and BMS 187,071 protected cells against HIV-1, HIV-2, and simian immunodeficiency virus infections, with 50% effective doses ranging from 0.05 to 0.33 microM, while having no inhibitory effect on cells infected with unrelated viruses. These compounds were also effective in inhibiting p24 production in peripheral blood mononuclear cells infected with HIV-1 IIIB and against the zidovudine-resistant HIV-1 strain A018C. Time-of-addition studies indicated that BMS 182,193 could be added as late as 27 h after infection and still retain its antiviral activity. To directly show that the activity of these compounds in culture was due to inhibition of proteolytic cleavage, the levels of HIV-1 gag processing in chronically infected cells were monitored by Western blot (immunoblot) analysis. All compounds blocked the processing of p55 in a dose-dependent manner, with 50% effective doses of 0.4 to 2.4 microM. To examine the reversibility of BMS 186,318, chronically infected CEM-SS cells were treated with drug and virions purified from the culture medium. Incubation of HIV-1 particles in drug-free medium indicated that inhibition of p55 proteolysis was slowly reversible. The potent inhibition of HIV-1 during both acute and chronic infections indicates that these aminodiol compounds are effective anti-HIV-1 compounds.     

Cyclobut-A and cyclobut-G, carbocyclic oxetanocin analogs that inhibit the replication of human immunodeficiency virus in T cells and monocytes and macrophages in vitro.

Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (cyclobut-G) were tested for activity against the infectivity of human immunodeficiency virus (HIV) in vitro. A number of other carbocyclic oxetanocin analogs failed to exert good antiretroviral effects. Both cyclobut-A and cyclobut-G protected CD4+ ATH8 cells against the infectivity and cytopathic effect of HIV type 1 (HIV-1) and suppressed proviral DNA synthesis in ATH8 cells exposed to HIV-1 in vitro at concentrations of 50 to 100 microM. These compounds also inhibited the in vitro infectivity of another human pathogenic retrovirus, HIV-2. Furthermore, both compounds completely suppressed the replication of a monocytotropic strain of HIV-1 in monocytes and macrophages at concentrations as low as 0.5 microM, as assessed by inhibition of HIV-1 p24 gag protein production. We also found that 2'-deoxyguanosine readily reversed the antiretroviral activity of cyclobut-G in our system, whereas the activity of cyclobut-A was hardly reversed by 2'-deoxyadenosine or 2'-deoxycytidine. We noted, however, that these compounds inhibited the proliferation of peripheral blood mononuclear cells at concentrations of greater than or equal to 100 microM in vitro. Although both cyclobut-A and cyclobut-G appear to have a certain level of in vitro toxicity, our observations may have theoretical and clinical implications in understanding the structure-activity relationships of antiretroviral agents active against HIV.     

Pharmacokinetics of the Protease Inhibitor KNI-272 in Plasma and Cerebrospinal Fluid in Nonhuman Primates after Intravenous Dosing and in Human Immunodeficiency Virus-Infected Children after Intravenous and Oral Dosing

KNI-272 is a human immunodeficiency virus (HIV) protease inhibitor with potent activity in vitro. We studied the pharmacokinetics of KNI-272 in the plasma and cerebrospinal fluid (CSF) of a nonhuman primate model and after intravenous and oral administration to children with HIV infection. Plasma and CSF were sampled over 24 h after the administration of an intravenous dose of 50 mg of KNI-272 per kg of body weight (approximately 1,000 mg/m²) to three nonhuman primates. The pharmacokinetics of KNI-272 were also studied in 18 children (9 males and 9 females; median age, 9.4 years) enrolled in a phase I trial of four dose levels of KNI-272 (100, 200, 330, and 500 mg/m² per dose given four times daily). The plasma concentration-time profile of KNI-272 in the nonhuman primate model was characterized by considerable interanimal variability and rapid elimination (clearance, 2.5 liters/h/kg; terminal half-life, 0.54 h). The level of drug exposure achieved in CSF, as measured by the area under the KNI-272 concentration-time curve, was only 1% of that achieved in plasma. The pharmacokinetics of KNI-272 in children were characterized by rapid elimination (clearance, 276 ml/min/m²; terminal half-life, 0.44 h), limited (12%) and apparently saturable bioavailability, and limited distribution (volume of distribution at steady state, 0.11 liter/kg). The concentrations in plasma were maintained above a concentration that is active in vitro for less than half of the 6-h dosing interval. There was no significant increase in CD4 cell counts or decrease in p24 antigen or HIV RNA levels. The pharmacokinetic profile of KNI-272 may limit the drug’s efficacy in vivo. It appears that KNI-272 will play a limited role in the treatment of HIV-infected children.     

Antimalarial activity of human immunodeficiency virus type 1 protease inhibitors

Aspartic proteases play key roles in the biology of malaria parasites and human immunodeficiency virus type 1 (HIV-1). We tested the activity of seven HIV-1 protease inhibitors against cultured Plasmodium falciparum. All compounds inhibited the development of parasites at pharmacologically relevant concentrations. The most potent compound, lopinavir, was active against parasites (50% inhibitory concentration [IC50], 0.9 to 2.1 microM) at concentrations well below those achieved by ritonavir-boosted lopinavir therapy. Lopinavir also inhibited the P. falciparum aspartic protease plasmepsin II at a similar concentration (IC50, 2.7 microM). These findings suggest that use of HIV-1 protease inhibitors may offer clinically relevant antimalarial activity.     

NP-06: a novel anti-human immunodeficiency virus polypeptide produced by a Streptomyces species.

From an extract of a Streptomyces culture, we identified and purified a novel compound, NP-06, which is active against human immunodeficiency virus (HIV) in vitro. Analyses indicate that NP-06 is a hydrophobic 21-mer oligopeptide, N terminally cyclized through the side chain of Asp-9, containing two intramolecular cystine linkages with a molecular weight of 2,163.4. The 50% inhibitory concentrations were 2.8 and 1.3 microM when NP-06 was tested for in vitro anti-HIV-1 activity in ATH8 cells and phytohemagglutinin-activated peripheral blood mononuclear cells, respectively, NP-06 appears to block the early stage of HIV-1 infection, most likely at the stage of virus-cell fusion.     

Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture.

Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polyproteins in cultures of human immunodeficiency virus type 1 (HIV-1)-infected T lymphocytes and, as a result, inhibit the infectivity of HIV-1 for such cultures. The ability of HIV-1 protease inhibitors to suppress replication of the C-type retrovirus Rauscher murine leukemia virus (R-MuLV) and the HIV-related lentivirus simian immunodeficiency virus (SIV) was examined in plaque reduction assays and syncytium reduction assays, respectively. Three of seven compounds examined blocked production of infectious R-MuLV, with 50% inhibitory concentrations of < or = 1 microM. Little or no cellular cytotoxicity was detectable at concentrations up to 100 microM. The same compounds which inhibited the infectivity of HIV-1 also produced activity against SIV and R-MuLV. Electron microscopic examination revealed the presence of many virions with atypical morphologies in cultures treated with the active compounds. Morphometric analysis demonstrated that the active compounds reduced the number of membrane-associated virus particles. These results demonstrate that synthetic peptide analog inhibitors of retroviral proteases significantly inhibit proteolytic processing of the gag polyproteins of R-MuLV and SIV and inhibit the replication of these retroviruses. These results are similar to those for inhibition of HIV-1 infectivity by these compounds, and thus, R-MuLV and SIV might be suitable models for the in vivo evaluation of the antiretroviral activities of these protease inhibitors.     

CGP 53437, an orally bioavailable inhibitor of human immunodeficiency virus type 1 protease with potent antiviral activity.

CGP 53437 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease containing a hydroxyethylene isostere. The compound inhibited recombinant HIV-1 protease with a Ki of 0.2 nM. The inhibition constant versus human cathepsin D and human cathepsin E was 4 nM. Human pepsin and gastricsin were inhibited with Kis of 8 and 500 nM, respectively, and human renin was inhibited with a Ki of 190 microM. The replication of HIV-1/LAV, HIV-1/Z-84, and HIV-1/pLAI was inhibited with a 90% effective dose of 0.1 microM in acutely infected MT-2 cells. The 50% cytotoxic dose was 100 microM. Similar antiviral activity was observed when the compound was added up to 10 h after infection. At the effective concentration, processing of Gag precursor protein p55 was greatly reduced, confirming an action on the late stage of the virus life cycle, as expected. The efficacy of the inhibitor was also demonstrated by using primary human peripheral blood lymphocytes infected with the HIV-1/LAV strain, low-passage clinical isolates obtained from HIV-1-seropositive individuals (including a zidovudine-resistant strain), and HIV-2/ROD. In these cells, CGP 53437 delayed the onset of HIV replication in a dose-dependent fashion (substantial effects with concentrations of > or = 0.1 microM) as long as the inhibitor was maintained in the culture. CGP 53437 was orally bioavailable in mice. Concentrations in plasma 10-fold in excess of the in vitro antiviral 90% effective dose could be sustained for several hours after oral application of 120 mg/kg. Therefore, CGP 53437 has the potential to be a therapeutically useful anti-HIV agent for the treatment of AIDS.     

GRL-02031, a novel nonpeptidic protease inhibitor (PI) containing a stereochemically defined fused cyclopentanyltetrahydrofuran potent against multi-PI-resistant human immunodeficiency virus type 1 in vitro

We generated a novel nonpeptidic protease inhibitor (PI), GRL-02031, by incorporating a stereochemically defined fused cyclopentanyltetrahydrofuran (Cp-THF) which exerted potent activity against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) isolates, including multidrug-resistant HIV-1 variants. GRL-02031 was highly potent against laboratory HIV-1 strains and primary clinical isolates, including subtypes A, B, C, and E (50% effective concentration [EC(50)] range, 0.015 to 0.038 microM), with minimal cytotoxicity (50% cytotoxic concentration, >100 microM in CD4(+) MT-2 cells), although it was less active against two HIV-2 strains (HIV-2(EHO) and HIV-2(ROD)) (EC(50), approximately 0.60 microM) than against HIV-1 strains. GRL-02031 at relatively low concentrations blocked the infection and replication of each of the HIV-1(NL4-3) variants exposed to and selected by up to 5 microM of saquinavir, amprenavir, indinavir, nelfinavir, or ritonavir and 1 microM of lopinavir or atazanavir (EC(50) range, 0.036 to 0.14 microM). GRL-02031 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to the conventional antiretroviral regimens that then existed, with EC(50)s ranging from 0.014 to 0.042 microM (changes in the EC(50)s were less than twofold the EC(50) for wild-type HIV-1). Upon selection of HIV-1(NL4-3) in the presence of GRL-02031, mutants carrying L10F, L33F, M46I, I47V, Q58E, V82I, I84V, and I85V in the protease-encoding region and G62R (within p17), L363M (p24-p2 cleavage site), R409K (within p7), and I437T (p7-p1 cleavage site) in the gag-encoding region emerged. GRL-02031 was potent against a variety of HIV-1(NL4-3)-based molecular infectious clones containing a single primary mutation reported previously or a combination of such mutations, although it was slightly less active against HIV-1 variants containing consecutive amino acid substitutions: M46I and I47V or I84V and I85V. Structural modeling analysis demonstrated a distinct bimodal binding of GRL-02031 to protease, which may provide advantages to GRL-02031 in blocking the replication of a wide spectrum of HIV-1 variants resistant to PIs and in delaying the development of resistance of HIV-1 to GRL-02031. The present data warrant the further development of GRL-02031 as a potential therapeutic agent for the treatment of infections with primary and multidrug-resistant HIV-1 variants.     

Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing

More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.     

Isothiazole derivatives as novel HIV replication inhibitors

A series of 3,4,5-trisubstituted isothiazoles has been screened against HIV-1 (IIIB) and HIV-2 (ROD) at sub-toxic concentrations in acutely infected MT-4 cells. Among the tested compounds, only 3-mercapto-5-phenyl-4-isothiazolecarbonitrile was found to inhibit the replication of HIV-1 (IIIB) and HIV-2 (ROD) at 50% effective concentrations (EC50) of 7.8 and 9.7 microg/ml, respectively. The presence of a thioalkyl chain or dialkylamino function in the 3-position caused a loss of anti-HIV activity. New 4-cyano-5-phenylisothiazoles with other substituents in the 3-position have also been synthesized and studied as potential anti-HIV agents. Our results have demonstrated that 5-phenyl-3-(4-cyano-5-phenylisothiazol-3-yl) disulphanyl-4-isothiazolecarbonitrile and S-(4-cyano-5-phenylisothiazol-3-yl)-O-ethyl thiocarbonate are effective against both HIV-1 (IIIB) (EC50=13.6 and 15.2 microg/ml, respectively) and HIV-2 (ROD) (EC50=17.4 and 13.4 microg/ml, respectively).