Design, synthesis, and biological evaluation of novel hybrid dicaffeoyltartaric/diketo acid and tetrazole-substituted L-chicoric acid analogue inhibitors of human immunodeficiency virus type 1 integrase.

Fourteen analogues of the anti-HIV-1 integrase (IN) inhibitor L-chicoric acid (L-CA) were prepared. Their IC(50) values for 3'-end processing and strand transfer against recombinant HIV-1 IN were determined in vitro, and their cell toxicities and EC(50) against HIV-1 were measured in cells (ex vivo). Compounds 1-6 are catechol/ß-diketoacid hybrids, the majority of which exhibit submicromolar potency against 3'-end processing and strand transfer, though only with modest antiviral activities. Compounds 7-10 are L-CA/p-fluorobenzylpyrroloyl hybrids, several of which were more potent against strand transfer than 3'-end processing, a phenomenon previously attributed to the ß-diketo acid pharmacophore. Compounds 11-14 are tetrazole bioisosteres of L-CA and its analogues, whose in vitro potencies were comparable to L-CA but with enhanced antiviral potency. The trihydroxyphenyl analogue 14 was 30-fold more potent than L-CA at relatively nontoxic concentrations. These data indicate that L-CA analogues are attractive candidates for development into clinically relevant inhibitors of HIV-1 IN.

Design, synthesis, and antiviral evaluation of some 3'-carboxymethyl-3'-deoxyadenosine derivatives.

3'-Carboxymethyl-3'-deoxyadenosine derivatives were prepared from 2'-O-TBDMS-3'-[(ethoxycarbonyl)methyl]-3'-deoxyadenosine (1) via simple and efficient procedures. Conversion of 1 to its 5'-azido-5'-deoxy derivative 5 was accomplished via a novel one-pot method employing 5'-activation (TosCl) followed by efficient nucleophilic displacement with tetramethylguanidinium azide. Compound 5 was converted to 5'-[(N-methylcarbamoyl)amino] derivative 8 via one-pot reduction/acylation employing H(2)/Pd-C followed by treatment with p-nitrophenyl N-methylcarbamate. N(6)-phenylcarbamoyl groups were introduced by treatment with phenylisocyanate, and an efficient new method for lactonization of 2'-O-TBDMS-3'-[(ethoxycarbonyl)methyl]-3'-deoxyadenosines to give corresponding 2',3'-lactones was also developed. Target compounds were evaluated for anti-HIV and anti-HIV integrase activities, but were not active at the concentrations tested.

Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56lck.

With current anti-HIV treatments targeting only 4 of the 15 HIV proteins, many potential viral vulnerabilities remain unexploited. We report small-molecule inhibitors of the HIV-1 protein Nef. In addition to expanding the anti-HIV arsenal, small-molecule inhibitors against untargeted HIV proteins could be used to dissect key events in the HIV lifecycle. Numerous incompletely characterized interactions between Nef and cellular ligands, for example, present a challenge to understanding molecular events during HIV progression to AIDS. Assays with phage-displayed Nef from HIV(NL4-3) were used to identify a series of guanidine alkaloid-based inhibitors of Nef interactions with p53, actin, and p56(lck). The guanidines, synthetic analogs of batzellidine and crambescidin natural products, inhibit the Nef-ligand interactions with IC(50) values in the low micromolar range. In addition, sensitive in vivo assays for Nef inhibition are reported. Although compounds that are effective in vitro proved to be too cytotoxic for cellular assays, the reported Nef inhibitors provide proof-of-concept for disrupting a new HIV target and offer useful leads for drug development.

L-chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro.

The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.

Replication kinetics for divergent type 1 human immunodeficiency viruses using quantitative SYBR green I real-time polymerase chain reaction.

A quantitative and sensitive measure of human immunodeficiency virus type 1 (HIV-1) replication is quantitative real-time polymerase chain reaction (PCR). Real-time PCR using SYBR green I and oligonucleotide primers that amplify early, intermediate, and late products of reverse transcription were optimized to measure HIV-1 replication of clade A, B, C, and D HIV-1 isolates in peripheral blood lymphocytes and in both transformed and viral-transformed CD4+ lymphocyte cell lines. Real-time PCR can detect HIV-1 replication as early as 1 hr postinfection and demonstrates that in established cell lines cDNA can be detected as early as 4 hr postinfection. The first round of HIV-1 replication in established cell lines is complete between 12 and 24 hr postinfection. Furthermore, real-time PCR can detect HIV-1 replication in fewer than 0.1% of cells. Patient isolates replicated at different rates in peripheral blood lymphocytes, with viral cDNA peaking between 48 and 120 hr, depending on the virus being studied. Real-time PCR differentiated the mechanisms of action of drugs targeted at HIV-1 entry, reverse transcription, and proteolytic processing and identified differences in the kinetics of reverse transcription between zidovudine-sensitive and zidovudine-resistant HIV in the presence of zidovudine. In summary, real-time PCR using SYBR green I dye is a sensitive, quantitative, and reproducible measure of replication kinetics for a variety of group M HIV-1 isolates.

Dicaffeoyltartaric acid analogues inhibit human immunodeficiency virus type 1 (HIV-1) integrase and HIV-1 replication at nontoxic concentrations.

The human immunodeficiency virus type 1 (HIV-1) is a major health problem worldwide. In this study, 17 analogues of L-chicoric acid, a potent inhibitor of HIV integrase, were studied. Of these analogues, five submicromolar inhibitors of integrase were discovered and 13 compounds with activity against integrase at less than 10 microM were identified. Six demonstrated greater than 10-fold selectivity for HIV replication over cellular toxicity. Ten analogues inhibited HIV replication at nontoxic concentrations. Alteration of the linkages between the two bis-catechol rings, including the use of amides, mixed amide esters, cholate, and alkyl bridges, was explored. Amides were as active as esters but were more toxic in tissue culture. Alkyl and cholate bridges were significantly less potent against HIV-1 integrase in vitro and were inactive against HIV-1 replication. Two amino acid derivates and one digalloylderivative of L-chicoric acid (L-CA) showed improved selectivity over L-CA against integration in cell culture. These data suggest that in addition to the bis-catechols and free carboxylic acid groups reported previously, polar linkages are important constituents for optimal activity against HIV-1 integrase and that new derivatives can be developed with increased specificity for integration over HIV entry in vivo.