RESUMEN

Human immunodeficiency virus (HIV) type I integrase (IN) active site, and viral DNA-binding residues K156 and K159 are predicted to interact both with strand transfer-selective IN inhibitors (STI), e.g. L-731,988, Elvitegravir (EVG), and the FDA-approved IN inhibitor, Raltegravir (RGV), and strand transfer non-selective inhibitors, e.g. dicaffeoyltartaric acids (DCTAs), e.g. L-chicoric acid (L-CA). To test posited roles for these two lysine residues in inhibitor action we assayed the potency of L-CA and several STI against a panel of K156 and K159 mutants. Mutagenesis of K156 conferred resistance to L-CA and mutagenesis of either K156 or K159 conferred resistance to STI indicating that the cationic charge at these two viral DNA-binding residues is important for inhibitor potency. IN K156N, a reported polymorphism associated with resistance to RGV, conferred resistance to L-CA and STI as well. To investigate the apparent preference L-CA exhibits for interactions with K156, we assayed the potency of several hybrid inhibitors containing combinations of DCTA and STI pharmacophores against recombinant IN K156A or K159A. Although K156A conferred resistance to diketo acid-branched bis-catechol hybrid inhibitors, neither K156A nor K159A conferred resistance to their monocatechol counterparts, suggesting that bis-catechol moieties direct DCTAs toward K156. In contrast, STI were more promiscuous in their interaction with K156 and K159. Taken together, the results of this study indicate that DCTAs interact with IN in a manner different than that of STI and suggest that DCTAs are an attractive candidate chemotype for development into drugs potent against STI-resistant IN.

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RESUMEN

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.

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Bioassay-directed fractionation of the organic extract of Mallotus philippinensis gave five compounds (1-5), the most active of which against Mycobacterium tuberculosis was a new compound, 8-cinnamoyl-5,7-dihydroxy-2,2-dimethyl-6-geranylchromene (1) for which the name mallotophilippen F is suggested. Compound (2), 8-cinnamoyl-2,2-dimethyl-7-hydroxy-5-methoxychromene, was isolated from a natural source for the first time, while the remaining three compounds, rottlerin (3), isoallorottlerin=isorottlerin (4) and the so-called "red compound," 8-cinnamoyl-5,7-dihydroxy-2,2,6-trimethylchromene (5), had been isolated previously from this plant. All compounds were identified by analysis of their spectra including 2D-NMR, which was used to correct the literature NMR spectral assignments of compounds 2-4. The C-13 NMR of 5 is reported for the first time.

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RESUMEN

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.

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Two compounds (3 and 10) were isolated from the aerial parts of Sida spinosa L. Their structures have been established as glyceryl-1-eicosanoate and 20-hydroxy, 24-hydroxymethylecdysone by 1D and 2D-NMR techniques. In addition 12 known compounds (1, 2, 4-9 and 11-14) have been isolated and identified.

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The reactions of seven thiophenes with benzyne generated from diphenyliodonium-2-carboxylate (DPIC) under a standard set of conditions led among other products to the formation of alpha- and beta-naphthyl phenyl sulfides 2a and 2b from thiophene (1a) and of 2c and 2d from 2-methylthiophene (1b). Dithienyl sulfides 4a-f were produced from the halothiophenes 1c-g. The structures of the naphthyl sulfides were proven by comparison with authentic samples of 2a-f, thus eliminating one of two possible mechanisms of formation. The remaining mechanism involves [4+2]-cycloaddition of benzyne to thiophene or to an S-phenylthiophenium ylide 10 to give the dipolar 2:1 benzyne/thiophene adduct 8 followed by ring-opening. Stevens-like rearrangements of 11, formed from 10 by proton transfer, may also explain the origin of arylated thiophenes such as 12 and 3 found in some reactions of benzynes with thiophene.

RESUMEN

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.

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