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In this paper we describe our structure-based ligand design, synthetic strategy, and structure−activity relationship (SAR) studies that led to the identification of thiocarbamates (TCs), a novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), isosteres of phenethylthiazolylthiourea (PETT) derivatives. Assuming as a lead compound O-[2-(phthalimido)ethyl]phenylthiocarbamate 12, one of the precursors of the previously described acylthiocarbamates (Ranise, A.; et al. J. Med. Chem. 2003, 46, 768−781), two targeted solution-phase TC libraries were prepared by parallel synthesis. The lead optimization strategy led to para-substituted TCs 31, 33, 34, 39, 40, 41, 44, 45, and 50, which were active against wild-type HIV-1 in MT-4-based assays at nanomolar concentrations (EC50 range:  0.04−0.01 μM). The most potent congener 50 (EC50 = 0.01 μM) bears a methyl group at position 4 of the phthalimide moiety and a nitro group at the para position of the N-phenyl ring. Most of the TCs showed good selectivity indices, since no cytotoxic effect was detected at concentrations as high as 100 μM. TCs 31, 37, 39, 40, and 44 significantly reduced the multiplication of the Y181C mutant, but they were inactive against K103R and K103N + Y181C mutants. Nevertheless, the fold increase in resistance of 41 was not greater than that of efavirenz against the K103R mutant in enzyme assays. The docking model predictions were consistent with in vitro biological assays of the anti-HIV-1 activity of the TCs and related compounds synthesized.

Despite the unprecedented successes in the therapy of HIV infection, AIDS remains a major world health problem being the first cause of death in Africa and the fourth leading cause of death worldwide. Rapid emergence of drug-resistant HIV variants and severe side effects limit the efficacy of existing therapies. The intrinsic high variability of HIV calls for combining different drugs with distinct mode of action to achieve synergistic antiviral activity. Efforts are being made to develop agents address- ing new steps in HIV replication and to optimize both antiviral activity and pharmacokinetic of the current drugs targeting reverse transcriptase and protease. The class of viral entry inhibitors is undergoing evaluation for both systemic and topical administration, and compounds targeting the fusion step may be the first to reach the market. Identification of compounds unambiguously affecting HIV replication by targeting integrase supports the potential of this crucial viral enzyme as a drug targ et. Targeting HIV gene regulation, which could also lead to cellular toxicity, may also become an important discovery strategy, provided that inhibitors with sufficient specificity are identified. In this review we will summarize the current understanding of the key steps in HIV life cycle in the context of representative inhibitors based on their modes of action. We then present a summary of compounds under clinical development, with the aim of providing a picture of the current potential for targeting HIV.

Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine 5 were synthesized by diazotization of substituted 2-(2-aminophenyl)indoles followed by an intramolecular coupling reaction of the diazonium group with the indole nitrogen. To obtain the indolobenzotriazine system it was necessary to protect the 3 position of the indole nucleus to avoid cyclization into the indolo[3,2-c]cinnoline system 4. Indolobenzotriazines 5a−g were evaluated in vitro for antitumor activity against a panel of leukemia-, lymphoma-, carcinoma-, and neuroblastoma-derived cell lines. Some compounds inhibited the proliferation of T and B cell lines at submicromolar concentrations, whereas their activity against solid tumor cell lines was in the micromolar range. When evaluated for their antifungal potential 5a,d inhibited some of the fungi tested, although at concentrations very close to those inhibiting the proliferation of human cells. On the contrary, all indolobenzotriazines proved fairly potent and selective inhibitors of Streptococcus and Staphylococcus. In particular 5b,c,g were up to 80 times more potent than the reference drug streptomycin and inhibited the growth of the above Gram-positive bacteria at concentrations far lower than those cytotoxic for animal cells.

Molecular modeling analysis of compounds belonging to the recently published series of dihydro-alkoxy-benzyl-oxopyrimidines (DABOs), such as S-DABOs and DATNOs, gave support to the design of new 2,6-disubstituted benzyl-DABO derivatives as highly potent and specific inhibitors of the HIV-1 reverse transcriptase (RT). To follow up on the novel DABO derivatives, we decided to investigate the effect of electron-withdrawing substituents in the benzyl unit of the S-DABO skeleton versus their anti-HIV-1 activity. Such chemical modifications impacted the inhibitory activity, especially when two halogen units were introduced at positions 2 and 6 in the phenyl portion of the benzyl group bound to C-6 of the pyrimidine ring. Various 5-alkyl-2-(alkyl(or cycloalkyl)thio)-6-(2,6-dichloro(or 2,6-difluoro)phenylmethyl)-3,4-dihydropyrimidin-4(3H)-ones were then synthesized and tested as anti-HIV-1 agents in both cell-based and enzyme (recombinant reverse transcriptase, rRT) assays. Among the various mono- and disubstituted phenyl derivatives, the most potent were those containing a 6-(2,6-difluorophenylmethyl) substituent (F-DABOs), which showed EC50's ranging between 40 and 90 nM and selectivity indexes up to ≥5000. An excellent correlation was found between EC50 and IC50 values which confirmed that these compounds act as inhibitors of the HIV-1 RT. The structure−activity relationships of the newly synthesized pyrimidinones are presented herein.

The syntheses of 4-amino-1-(β-D-ribofuranosyl)-1H-1,2,3-triazolo[4,5-c]pyridine (8-aza-3-deazaadenosine, 1), 4-amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1H-1,2,3-triazolo[4,5-c]pyridine (2′-deoxy-8-aza-3-deazaadenosine, 2), and their N8and N7glycosylated analogues (12,13, 21,22) and 4-amino-1-(2,3-dideoxy-β-D-erythro-pentof uranosyl)-1H-1,2,3-triazolo [4,5-c]pyridine (2′,3′-dideoxy-8-aza-3-deazaadenosine, 3) were carried out by glycosylation of the 4-chloro-3H-1,2,3-triazolo[4,5-c]pyridine anion. The anomeric configuration as well as the position of glycosylation were determined by 1H-, 13C-NMR, UV and N.O.E. difference spectroscopy. Nucleoside (2) and its parent compound 2′-deoxy-3-deazaadenosine were found active against ASFV and VSV. The 4-chloro-2-(β-D-ribofuranosyl)-2H-1,2,3-triazolo[4,5-c] pyridine (9) was active against Coxsackie B1, whereas none of the 8-aza-3-deaza purine nucleosides, compound (3) included, was active against HIV-1. The 6-chloro derivatives of 8-aza-3-deazapurine ribo- and 2′-deoxyribonucleosides (11) and (20) showed some activity against LoVo human colon adenocarcinoma.

Various cinnammoyl-based structures were synthesized and tested in enzyme assays as inhibitors of the HIV-1 integrase (IN). The majority of compounds were designed as geometrically or conformationally constrained analogues of caffeic acid phenethyl ester (CAPE) and were characterized by a syn disposition of the carbonyl group with respect to the vinylic double bond. Since the cinnamoyl moiety present in flavones such as quercetin (inactive on HIV-1-infected cells) is frozen in an anti arrangement, it was hoped that fixing our compounds in a syn disposition could favor anti-HIV-1 activity in cell-based assays. Geometrical and conformational properties of the designed compounds were taken into account through analysis of X-ray structures available from the Cambridge Structural Database. The polyhydroxylated analogues were prepared by reacting 3,4-bis(tetrahydropyran-2-yloxy)benzaldehyde with various compounds having active methylene groups such as 2-propanone, cyclopentanone, cyclohexanone, 1,3-diacetylbenzene, 2,4-dihydroxyacetophenone, 2,3-dihydro-1-indanone, 2,3-dihydro-1,3-indandione, and others. While active against both 3‘-processing and strand-transfer reactions, the new compounds, curcumin included, failed to inhibit the HIV-1 multiplication in acutely infected MT-4 cells. Nevertheless, they specifically inhibited the enzymatic reactions associated with IN, being totally inactive against other viral (HIV-1 reverse transcriptase) and cellular (RNA polymerase II) nucleic acid-processing enzymes. On the other hand, title compounds were endowed with remarkable antiproliferative activity, whose potency correlated neither with the presence of catechols (possible source of reactive quinones) nor with inhibition of topoisomerases. The SARs developed for our compounds led to novel findings concerning the molecular determinants of IN inhibitory activity within the class of cinnamoyl-based structures. We hypothesize that these compounds bind to IN featuring the cinnamoyl residue C&dbd;C−C&dbd;O in a syn disposition, differently from flavone derivatives characterized by an anti arrangement about the same fragment. Certain inhibitors, lacking one of the two pharmacophoric catechol hydroxyls, retain moderate potency thanks to nonpharmacophoric fragments (i.e., a m-methoxy group in curcumin) which favorably interact with an “accessory” region of IN. This region is supposed to be located adjacent to the binding site accommodating the pharmacophoric dihydroxycinnamoyl moiety. Disruption of coplanarity in the inhibitor structure abolishes activity owing to poor shape complementarity with the target or an exceedingly high strain energy of the coplanar conformation.

The current model of poliovirus morphogenesis postulates a fundamental role for procapsid, 80S shells that, upon interaction with viral RNA and subsequent proteolytic cleavage, give rise to complete virus particles. Although 80S sedimenting particles can, indeed, be isolated from cytoplasmic extracts of infected cells, their physical properties differ from those reported for procapsids. Far from being stable structures, they can be dissociated by pH 8.5 and 0.1% sodium dodecyl sulfate into slower-sedimenting subunits. The reasons for this discrepancy were investigated, and two main modalities leading to the appearance of procapsids in vitro were identified. The first involves a temperature-mediated conversion of dissociable 80S particles into stable 80S procapsids, and the second involves the self-assembly of endogenous 14S subunits, also primed by an increase in the temperature of cytoplasmic extracts.

A previously undetected subviral particle, designated the 55S particle because of its position in sucrose density gradients, has been found in cytoplasmic extracts of poliovirus-infected cells. It contains no RNA, is composed of equimolar amounts of the structural polypeptides P1AB, P1C, and P1D, and is stable in vitro under a variety of conditions: presence or absence of EDTA, dilution in low- or high-ionic-strength buffers, suspension in buffers up to pH 10, incubation at 37 degrees C, and centrifugation to equilibrium in CsCl gradients (where it bands at a density of 1.285 g/cm3). Conventional pulse-chase experiments show that 55S particles are the products of the assembly of 14S subunits and the precursors of virions. These data led to the formulation of a model of poliovirus morphogenesis in which the conversion of capsomers into 73S empty capsids does not occur directly, but through the formation of an intermediate structure, the 55S particle.