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The virus-encoded RNA-dependent RNA polymerase (RdRp) has emerged as a primary target in the search for selective inhibitors of Flaviviridae. Recently, we reported on the selective inhibition, in cell-based assays, of both BVDV (EC50 = 0.80 ± 0.06 μM) and HCV (EC50 = 1.11 ± 0.15 μM) by 2-{1-[2-(2,4-dimethoxyphenyl)-1H-benzimidazol-5-yl]ethylidene}hydrazinecarbothioamide (227G). Here we show that, in enzyme assays with recombinant enzymes, 227G inhibits, in a dose-dependent manner, the RdRp of both BVDV (IC50 = 0.0020 ± 0.0004 μM) and HCV (IC50 = 0.40 ± 0.04 μM). Furthermore, we report on the selection and molecular analysis of a BVDV-resistant mutant, characterized by the presence of the I261M mutation. By applying a multilevel computational approach, we identified different 227G binding sites on the two RdRps. They were further validated by the good agreement between the calculated affinities and those extrapolated from IC50 values. Our findings suggest different molecular mechanisms of inhibition of the HCV and BVDV RdRps by 227G and indicate the importance of understanding ligand-enzyme interactions at the molecular level for the rational design of new and more potent leads.

Within a project aimed at discovering new Flaviviridae inhibitors, new variously substituted 2-phenylbenzimidazoles were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against viruses representatives of the three genera of the Flaviviridae family, i.e.: Pestivirus (BVDV), Flavivirus (YFV) and Hepacivirus (HCV). Title compounds were also tested against RNA viruses representative of other single-stranded, positive-sense (ssRNA(+)) negative-sense (RNA(-)), or double-stranded (dsRNA) genomes, as well as against representatives of two DNA virus families. Nine compounds showed activity against BVDV (EC(50) = 0.8-8.0 μM), compound 31 being the most potent (EC(50) = 0.80 μM) and selective (SI = CC(50)/EC(50) = >100). When tested in an HCV replicon assay, compound 31 resulted again the most potent, displaying an EC(50) value of 1.11 μM and an SI of 100. Besides inhibiting BVDV, two compounds (35 and 38) showed a moderate activity also against YFV (EC(50) = 13 μM). Interestingly, 35 was moderately active also against RSV (EC(50) = 25 μM)., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

In this study three new classes of linear N-tricyclic compounds, derived by condensation of the quinoline nucleus with 1,2,3-triazole, imidazole or pyrazine, were synthesized, obtaining triazolo[4,5-g]quinolines, imidazo[4,5-g]quinolines and pyrido[2,3-g]quinoxalines, respectively. Title compounds were tested in cell-based assays for cytotoxicity and antiviral activity against RNA viruses representative of the three genera of the Flaviviridae family, that is BVDV (Pestivirus), YFV (Flavivirus) and HCV (Hepacivirus). Quinoline derivatives were also tested against representatives of other RNA virus families containing single-stranded, either positive-sense (ssRNA(+)) or negative-sense (RNA(-)), and double-stranded genomes (dsRNA), as well as against representatives of two DNA virus families. Some quinolines showed moderate, although selective activity against CVB-5, Reo-1 and RSV. However, derivatives belonging to all classes showed activity against BVDV. Among the most potent were the bis-triazoloquinoline 1m, the imidazoquinolines 2e and 2h, and the pyridoquinoxalines 4h, 4j and 5n (EC(50) range 1-5 μM). When tested in a replicon assay, compound 2h was the sole derivative to also display anti-HCV activity (EC(50)=3.1 μM). In enzyme assays, 1m, 2h, 5m and 5n proved to be potent inhibitors of the BVDV RNA-dependent RNA polymerase (RdRp), while only 2h also inhibited the recombinant HCV enzyme., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 microM). The most potent congener 50 (EC50 = 0.01 microM) 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 microM. 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.

A series of 11 new 9H-bis-[1,2,4]triazolo[4,3-a:3',4'-d] [1,5]benzodiazepine derivatives 8e-o was synthesized. Ten of these compounds (8e-m,o), along with four analogues (8a-d) (previously synthesized by us) were tested in vitro in order to evaluate their cytotoxic and anti-HIV-1 properties. In this connection other six original compounds, i.e., five 9-substituted compounds prepared starting from the 6,12-diphenylderivative 8c (compounds 10, 11, 12, 13a,b) and the bis-triazolone derivative 14, were synthesized and tested for the same purpose. While none of the 20 compounds tested exhibited any appreciable anti-HIV-1 activity, some of them exhibited interesting cytotoxic properties, the best results being shown by compounds 8c,d,k and 11 (CC(50) range=3-12 microM). Therefore, these four compounds were further evaluated for their antiproliferative activity against a panel of human tumor cell lines; actually, compounds 8d, 8k and 11 showed antiproliferative properties against either or both leukemia- and lymphoma-derived cell lines in the low micromolar range.

A series of 6-aryl-2,4-dioxo-5-hexenoic acids, were synthesized and tested against HIV-1 in cell-based assays and against recombinant HIV-1 integrase (rIN) in enzyme assays. Compound 8a showed potent antiretroviral activity (EC(50)=1.5 microM) and significant inhibition against rIN (strand transfer: IC(50)=7.9 microM; 3'-processing: IC(50)=7.0 microM). A preliminary molecular modeling study was carried out to compare the spatial conformation of 8a with those of L-731988 (4) and 5CITEP (7) in the IN core.

Three series of 5-substituted 1,3-diphenyl-6-(omega-dialkyl- and omega-cyclo-aminoalkyl)thio-2-thiobarbiturates (11-13) were synthesized as polysubstituted thioanalogues of merbarone, a topoisomerase II inhibitor acting on the catalytic site. To better understand pharmacophore requirements, a forth series of conformationally constrained analogues 14 was also prepared. Derivatives 11b,e, 14b,e,h,i,j were active in the low micromolar concentration range (IC(50): 3.3-4.3 microM), whereas compounds 11a,c,d,f,h,j and 13a,b,d,g,j and 14a,d,f showed IC(50) values between 10 and 15.5 microM. In contrast, compounds 12a-c,g-j, 13e,f,h and 14k were inactive. Cytotoxicity data provided from N.C.I. on selected compounds provided evidence that 11b,d, 13d,g and 14b,d,f,h,i,j were endowed with potent antiproliferative activity against leukemia and prostate cell lines (GI(50) up to 0.01 microM). In general, bicyclic derivatives 14 were up to 10-fold more potent than monocyclic counterparts against solid tumor-derived cell lines. SAR studies indicated that, in general, a certain tolerability in length of the alkyl side chains and in shape of distal amines is allowed in the four series, but in the monocyclic derivatives (11-13) antiproliferative activity was strongly affected by the nature of the 5-substituents (COOC(2)H(5)>COCH(3)>>C(6)H(5)). Compounds 11b and 14b were also evaluated against KB cell subclones expressing altered levels of topoisomerases or the multidrug resistance phenotype (MDR). In both cases the above compounds showed a decrease in potency. In enzyme assays, 11b and 14b turned out to be inhibitors of topoisonerase II as merbaron.

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 addressing 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 target. 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.

A number of properly substituted 5H-pyrimido[4,5-b][1,5]benzodiazepines (2) and pyrazolo[3,4-b][1,5]benzodiazepines (3 and 4), as well as compounds 5-7, which are derivatives of new tetracyclic systems, were prepared as nevirapine analogues through multistep synthetic routes. The cytotoxic and anti-HIV-1 properties of compounds 2-7 were evaluated in cell-based assays, together with their inhibitory activity against the HIV-1 recombinant reverse transcriptase (rRT) in enzyme assays. The modifications introduced into nevirapine heterocyclic skeleton proved to have a negative effect for the anti-HIV-1 activity. It is worth noting that some of the new derivatives proved to be cytotoxic in the low micromolar range.

Worldwide, the heterosexual route is the prevalent mode of transmission of AIDS; therefore, demands have been raised for measures that block sexual spreading of the HIV infection. Development of microbicides for topical use may represent an efficacious alternative to condoms. Several approaches are being investigated. Besides surfactants, which directly act on the virus particle, and measures that enhance natural defence mechanisms, promising new candidates appear to be drugs that block the early steps of HIV multiplication. We describe herein a long-term assay which enables the establishment of whether the above drugs reversibly (virustatic action) or irreversibly (virucidal action) inhibit HIV-1 multiplication, thus allowing screening for effective and potent microbicides. We validated our assay with nucleoside (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Following a chronic treatment, the NRTIs tested (didanosine, zalcitabine, stavudine and lamivudine) simply delayed the viral breakthrough with respect to infected, untreated controls. Under the same experimental conditions, non-nucleoside reveres transcriptase inhibitors (NNRTIs), such as MKC-442, alphaAPA, nevirapine, efavirenz and 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs) MC 1047 and MC 1220 suppressed HIV-1 replication for the entire experimental period (40 days). When cell culture samples were evaluated for the presence of infectious virus, p24 antigen and viral DNA sequences, none of them was detected up to day 40 post-infection (p.i.). Identical results were obtained after a treatment with the above NNRTIs limited to the first 4 days p.i. Under more selective experimental conditions, that is drug treatments limited to the first 4 h p.i., nevirapine and efavirenz proved to be virustatic; in fact, viral breakthrough ensued shortly after their removal from the culture medium. Conversely, DABO MC 1220 was endowed with potent virucidal activity; in fact, at 3.5 microM it was able to suppress HIV-1 multiplication in cultures acutely infected with a very high multiplicity of infection (5 CCID50/cell), thus allowing exponential cell multiplication as in uninfected cultures for the next 40 days.

Several 5-alkyl, 5-alkenyl, 5-iso-alkyl, 5-halo, 5-aminomethyl and 5-carboxy derivatives of S-DABOs (dihydro-alkyl (or cyclo-alkyl)thio-benzyloxopyrimidines), DATNOs (dihydro-alkylthionaphthylmethyl-oxopyrimidines) and F2-S-DABOs (dihydro-alkyl (or cyclo-alkyl)thio-2,6-difluorobenzyl-oxopyrimidines) have been prepared and tested as anti-HIV-1 agents. S-DABO derivatives bearing at C-6 position monosubstituted phenylmethyl or heteroarylmethyl units have also been synthesized. 2-Alkylthio-3,4-dihydropyrimidin-4(3H)-one derivatives of F2-S-DABO series bearing small alkyl groups at C-5 proved to be potent inhibitors of HIV-1 replication in vitro with selectivity indexes ranging from 250 to >2,500.

A number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a-g (7b-f) were synthesized and tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic concentrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c was also 8- and 125-fold more potent than amphotericin B and fluconazole, respectively. None of the compounds was active against bacteria. Preliminary structure-activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear to play a decisive role in modulating cytotoxicity and antifungal activity.

Using 2,6-dichloro-4-aminopyrimidine, a number of uracil and cytosine derivatives with both arylthio and alkoxy moieties were prepared. These novel pyrimidines share chemical similarities with DABOs and HEPTs, two classes of non-nucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitors (NNRTIs), which have been widely studied of late. All new derivatives were tested in MT-4 cells to explore their potential in vivo anti-HIV activity. Like other NNRTIs, they selectively inhibit HIV-1 but not HIV-2. The majority of test derivatives were found to have low potency and were sometimes more cytotoxic than zidovudine and emivirine (formerly MKC-442), used here as reference drugs. Uracil and cytosine derivatives bearing a sec-butoxy chain and a methyl-substituted benzenesulphonyl moiety were the most potent. Enzyme assays proved that these derivatives target RT. Structure-activity relationship studies established a correlation between the anti-HIV-1 activity and the meta substitution on the phenyl ring; furthermore, oxidation of sulphide to sulphone significantly increased the potency of certain derivatives.

Due to the capability of peptidyl derivatives of araC to behave as prodrugs of this antimetabolite, and because of the well known biological properties of peptide T and its analogues (in particular that of targeting CD4+ cells), new peptide T-araC conjugates were prepared and tested in vitro for antiproliferative activity. The aim was that of specifically delivering the antitumor drug to CD4+ cells. N4-(Acylpeptidyl)-derivatives of araC were synthesized by a new general approach involving solid-phase synthesis, which allows mild conditions, avoids the usually required protection of the glycoside portion of nucleosides and affords high yields of the final products. After the demonstration that peptide T-araC conjugates were able to activate chemotaxis by binding CD4 receptor on monocyte membranes, the antiproliferative activity was evaluated against a panel of leukemia lymphoma and carcinoma cell lines derived from human tumors, three CD4+ cell lines included. Title compounds resulted effective as antiproliferative agents at concentrations 4- to 10-fold higher than those of araC alone, did not preferentially inhibit CD4+ cells and proved stable not only in cell culture medium containing 20% FCS, but also in human plasma. All this suggests their potential utility in vivo.

Chemotherapy of HIV-1 infection/AIDS currently employs inhibitors of two products of the viral pol gene, the reverse transcriptase and protease enzymes. However, a third product of the pol gene is essential for retroviral multiplication, the integrase. As no cellular homologue of HIV integrase has been described, potential inhibitors could be relatively nontoxic. Development of HIV-1 integrase inhibitors could have favorable implication for combination therapy, including potential synergy with currently available inhibitors, as well as prevention of the chronic carrier state and the emergence of resistant mutants. Although several classes of putative integrase inhibitors that been described, still no clinically useful anti-integration drugs are available. It is the structural and functional complexity of the integration process together with the limitations of the available in vitro assays that has made it problematic to develop inhibitors of the HIV integrase. In this review we summarize current knowledge concerning the biology of this enzyme and of the integration process, and discuss major classes representatives of integrase inhibitors considering the obstacles to the development of true anti-integrase drugs.

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.

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=C-C=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.

In view of the biological relevance of triazole-based heterocyclic structures as antifungal, antiviral, and antitumor agents, we have synthesized a series of substituted pyrazolo[3,4-d]-1,2,3-triazoles (2e-h, 2j, 4b) which we evaluated for their cytostatic and antiviral (HIV-1 included) activity and for their capability to inhibit the multiplication of various human pathogenic fungi and bacteria. Moreover, the biological activities of a few compounds, namely pyrazolo[3,4-d]oxazoles (3a-e) and pyrazolo[3,4-d]-1,2,3-triazoles (2a-d, 4a, 5), previously obtained by us but not investigated for their biological activity, were also studied. Only compounds 3a-e were endowed with a significative antiproliferative activity on the human lymphoblastoid cell line MT-4 cells. All pyrazole derivatives proved ineffective in protecting cell cultures against the HIV-1-induced cytopathogenicity, and none of the compounds was active against the bacteria and fungi tested.

IPCAR is a pyrazole nucleoside analog which belongs to a class of compounds structurally related to the inosine monophosphate (IMP) dehydrogenase (IMPDH) inhibitors ribavirin, selenazofurin and tiazofurin. Unlike other anticancer drugs, IPCAR showed a potent and broad-spectrum antiproliferative activity in vitro coupled with low cytotoxicity for resting PBL and CFU-GM. IPCAR proved fully inhibitory against human nasopharyngeal carcinoma KB cells expressing the MDR phenotype, whereas IPCAR-resistant renal adenocarcinoma ACHN/R1 cells were fully susceptible to inhibition by a number of anticancer drugs, with the exception of 6TG, 6MP and 5FU towards which they showed a partial cross-resistance. In combinations studies, IPCAR proved synergistic with 6MP, 6TG, 5FU and ribavirin, and additive with ara-A, MTX, doxorubicin, taxol and tiazofurin. Antagonistic effects were never observed. Although the precise molecular target of IPCAR remains to be identified, the data presented herein suggest that, unlike ribavirin and tiazofurin, this drug inhibits a step of the de novo purine biosynthesis different from the conversion of IMP into GMP. In vivo, IPCAR showed low acute toxicity (DL10 > 1000 mg/kg) and was active against the L1210 murine lymphocytic leukemia model. Drug doses of 125 and 250 mg/kg on a day-1, -3 and -5 dosing schedule increased the life span (ILS) relative to untreated control mice of 36.4 and 68.2%, respectively, whereas administration of 500 mg/kg on days 1 and 3 resulted in a ILS of 86.4% and also increased the 30-day survival rate (25% of the mice).

The new of 1-(2-hydroxyethoxy)methylindole derivatives 3a-i were prepared in good yields. None of them showed any significant anti-HIV activity and therefore the benzocondensation between the 2 and 3 positions of the pyrrole ring definitely reduced the weak activity found in the analogues 1a-c.

The series of 1-(1,3-dihydroxy-2-propoxy)methylpyrroles 2a-o were prepared in good overall yields according to Scheme I. When evaluated for antiviral activity against HIV-1, only compounds of the triphenyl series (R3 = NH2, N3, Br) were found to inhibit the HIV-1 replication at concentrations that were very not cytotoxic for MT-4 cells, with selectivity index 1.5-9.3. None of these compounds showed antibacterial or antifungal activity.

Among a series of unmodified phosphodiester (PO)-oligodeoxynucleotides (PO-ODNs) complementary to some of the human immunodeficiency virus type 1 (HIV-1) regulatory genes, several PO-ODN sequences complementary to the vpr gene (PO-ODNs-a-vpr, where a-vpr is the antisense vpr sequence) emerged as potent inhibitors (at concentrations of 0.8 to 3.3 microM) of HIV-1 multiplication in de novo infected MT-4 cells, while they showed no cytotoxicity for uninfected cells at concentrations up to 100 microM. Unlike phosphorothioate counterparts, PO-ODN-a-vpr sequences were not inhibitory to HIV-2 multiplication in de novo infected C8166 cells and neither prevented the fusion between chronically infected and bystander CD4+ cells nor inhibited the activity of the HIV-1 reverse transcriptase in enzyme assays. Moreover, they were not inhibitory to HIV-1 multiplication in chronically infected cells. Delayed addition experiments showed that PO-ODNs-a-vpr inhibit an event in the HIV-1 replication cycle following adsorption to the host cell, but preceding reverse transcription. Structure-activity relationship studies indicated that the antiviral activity of the test PO-ODN-a-vpr sequences is not related to an antisense mechanism but to the presence, within the active sequences, of contiguous guanine residues. Physical characterization of the test PO-ODNs suggested that the active structure is a tetramer stabilized by G quartets (i.e., four G residues connected by eight hydrogen bonds).

2',3'-dideoxyadenosine (ddAdo) and 2',3'-dideoxyinosine (ddIno) are potent and selective inhibitors of the replication of the human immunodeficiency virus type 1 (HIV1) in several cell culture systems. Equipotent in terms of antiviral activity, both compounds selectively inhibit the reverse transcription of HIV-1 by virtue of their conversion into ddATP. In human lymphoid cells ddAdo is converted to the active metabolite, ddATP, but it also undergoes rapid deamination, via adenosine deaminase, to form ddIno. ddIno, like ddAdo, gives rise to dideoxynucleotides of the dideoxy-adenylate series (ddAMP, ddADP and ddATP), as well as to IMP and to adenylate ribonucleotides. With the main object of blocking the deamination of ddAdo, we studied its anti-HIV-1 activity in the presence of different adenosine deaminase inhibitors, namely Coformycin (CF), 9-(erythro-2-hydroxy-3-nonyl) adenine (EHNA) and some deaza-EHNA derivatives. In contrast with reports on 2'-deoxycoformycin (Cooney et al., 1987), the adenosine deaminase inhibitors tested by us showed a significant increase in the antiviral activity of ddAdo, but not of ddIno. Enhancement was obtained with EHNA and CF concentrations up to 250 and > 12,500 times lower than their respective maximum non toxic doses. In combination with EHNA or CF, ddAdo could be used at concentrations up to ten times lower than those required to obtain the same degree of inhibition when ddAdo (or ddIno) was used alone. The use of EHNA or CF in combination with ddAdo at concentrations that inhibit the multiplication of HIV-1, allowed uninfected cells to maintain their normal multiplication rates. In fact, in combination experiments, cytotoxic effects were evident only with doses of EHNA, or CF and ddAdo 10 to 100 or more times higher than those required to inhibit HIV-1 significantly. The in vivo implications of these results for anti-HIV chemotherapy are discussed.

As an approach to more extensive structural modifications of [(oxazolylphenoxy)alkyl]isoxazoles, we synthesized new compounds characterized by the replacement of the isoxazole nucleus with furan, pyrrole, and thiophene rings and by the presence of a ketocarbonyl group in the aliphatic chain connecting these pentatomic heterocycles to the 4-(4,5-dihydro-2-oxazolyl)phenoxy, 4-(ethoxycarbonyl)phenoxy, and 4-carboxyphenoxy moieties. Some pentamethylene derivatives were also prepared, and their antirhinovirus activity was compared to that of the corresponding ketomethylene derivatives. Syntheses were carried out by Friedel-Crafts acylation of the above pentatomic heterocycles and subsequent reaction of chloroalkyl ketones with the proper 4-substituted phenol. Reduction of the ketone function afforded the related polymethylene derivatives. The new compounds were tested for antirhinovirus activity and cytotoxicity in comparison with WIN 51711, used as reference drug. Inspection of the structure-activity relationships revealed that the thiophene ring and the carbonyl group are the structural components which to a large extent contribute to the positive biological profile in terms of both wideness of spectrum and low cytotoxicity. Among the various derivatives, compounds 8e,d showed in vitro the same potency of WIN 51711 but a cytotoxicity at least 10 times lower.

Novel 3,4-dihydro-6-benzyl-4-oxopyrimidines (DABOs), variously substituted at both the C-2 and C-5 positions of the pyrimidine ring, proved to be specific inhibitors of the human immunodeficiency virus type 1 (HIV-1) in vitro. Some compounds showed potency at micromolar doses, no cytotoxicity at the maximum testable doses and selectivity indexes comparable to that of 2'-3'-dideoxyinosine (ddI). Mode of action studies suggested that DABOs interfered with a step of the virus multiplication cycle following adsorption and preceding integration. Enzyme assays indicated that DABOs targeted HIV-1 reverse transcriptase: they inhibited the RNA-dependent DNA polymerase activity in a template-dependent manner and, to a lesser extent, the DNA-dependent DNA polymerase activity. No inhibition of the RNase-H associated activity was observed. When DABOs were assayed in combination with 3'-azido-3'-dideoxythymidine (AZT) or ddI against HIV-1 in cell cultures, a slightly synergistic inhibitory effect was observed. The combination of DABO 546 and AZTTP in enzyme assays showed that the two compounds were kinetically mutually exclusive.

N-acetyl-L-cysteine (NAC) is known to antagonize the PMA- or cytokine-stimulated HIV-1 replication in latently and acutely infected monocytic and lymphocytic cell lines, and to reduce the virus multiplication in acutely infected, PHA-stimulated PBMC. We here report on the modulatory effects of NAC on the HIV-1 multiplication in both chronically and acutely infected lymphocytes that produce high virus levels independently from cytokine activation. In both cases, NAC doses of 0.12 and 0.25 mM decreased, whereas doses of 0.5-2 mM increased the infectious HIV-1 yield. At these concentrations, the modulatory effect of NAC on the HIV-1 multiplication paralleled that on cell proliferation, suggesting a close correlation between the two phenomena; in fact, under conditions where NAC could not modulate the cell growth, the drug also failed to modulate the HIV-1 multiplication. High NAC concentrations (4-16 mM), which were able to increase the proliferative rate of both chronically infected H9/IIIB and normal T lymphocytes, increased up to 6-fold the virus multiplication in H9/IIIB cells but were inhibitory to HIV-1 in acutely infected cells. This inhibition was due to the fact that, like dextran sulfate, NAC interfered with an early event in the virus growth cycle. The finding that high NAC doses were also capable of preventing syncytium formation in H9/IIIB and C8166 (or MT-4) cocultures further indicated an interference of the drug with receptor-binding-related events.

Several pyrazole and pyrazolo[4,3-d]-1,2,3-triazin-4-one ribonucleosides were prepared and tested for antiviral/antitumor activities. Appropriate heterocyclic bases were prepared by standard methodologies. Glycosylation of pyrazoles 6a-e,g,i and of pyrazolo[4,3-d]-1,2,3-triazin-4-ones 12f-1 mediated by silylation with hexamethyldisilazane, with 1-beta-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose, gave in good yields the corresponding glycosides 7a-e,g, 8g,i, 13f,h,k, and 14f, but could not be applied to compounds 12g,i,j,l. To overcome this occurrence, a different strategy involving the preparation, diazotization, and in situ cyclization of opportune pyrazole glycosides 9 and 10 was required. Moreover derivatives having the general formula 5 were considered not only as synthetic intermediates in the synthesis of 3 but also as carbon bioisosteres of ribavirin 4. All compounds were evaluated in vitro for cytostatic and antiviral activity. The pyrazolo[4,3-d]-1,2,3-triazin-4-one nucleosides that resulted were substantially devoid of any activity; only 15h,k showed a moderate cytostatic activity against T-cells. However, pyrazole nucleosides 9b,c,e were potent and selective cytotoxic agents against T-lymphocytes, whereas 9e showed a selective, although not very potent, activity against coxsackie B1.

Xylosyl-methylthio-adenosine, a naturally occurring analogue of 5'-deoxy-5'-methylthio-adenosine, has been postulated to play a protective role during egg development in the mollusc Doris verrucosa. However, in vitro tests showed that this analogue is devoid of activity against fungi, bacteria and viruses.

Condensation between furyllithium reagents and pyrimidinones 1, 2, 3, and 4 has been studied. The product composition is strongly dependent upon the reaction conditions and purification methodologies. Cytostatic and antiviral activities of some substrates and reaction products is reported.

Herpes simplex virus type-1 (HSV-1) was grown in the presence of 5-iodo-2'-deoxyuridine (IdUrd), and the virion-DNA was isolated by isopycnic centrifugation in CsCl. Irradiation of IdUrd-containing HSV DNA with either 302 nm or 254 nm ultraviolet (UV) light introduced strand breakage into the DNA in a dose-dependent manner when analyzed by alkaline sucrose density gradient sedimentation. Irradiation of unsubstituted HSV DNA under similar conditions produced little strand breakage. These observations are in agreement with the proposed mechanism for photochemical generation of strand breakage in 5-halo-2'-deoxyuridine-containing DNA. Irradiation of IdUrd-substituted virions followed by analysis of the isolated DNA indicated less strand breakage than irradiation of isolated IdUrd-substituted DNA under equivalent conditions. The dosage of irradiation required to introduce DNA strand breakage in IdUrd-substituted virions was equivalent to that employed to affect greater than 99% loss of infectious virus activity in both control and IdUrd-containing virions. It is suggested that the relative UV insensitivity of IdUrd-substituted HSV may be due to the microstructure environment of the substituted HSV DNA which may favor recombination of the photochemically formed halogen-uracil radical pairs.

A number of aroylpyrroleacrylic acid derivatives were synthesized by standard procedures and evaluated for cytotoxicity in Vero cells and for capacity to inhibit the multiplication of viruses, bacteria, and fungi. While none of the test compounds showed any activity against bacteria and fungi, most of them inhibited the replication of some DNA viruses at concentrations allowing the exponential growth of uninfected cells. In particular three compounds (8, 9c, and 10h) showed an antiviral activity at doses that were from 4- to greater than 8-fold lower than the maximum nontoxic doses.

The effect of 3'-deoxythymidin-2'-ene (d4T) on the metabolism of exogenously supplied radiolabeled nucleosides was investigated. Following a 24-hr exposure to 250 microM d4T, we observed no significant effect on the incorporation of [3H]thymidine or [3H]deoxycytidine into DNA. In contrast, the amounts of [3H]uridine, [3H]deoxyuridine, and [3H]cytidine were significantly lower than those incorporated by control cultures. d4T had no significant effect on the incorporation of [3H]uridine or [3H]cytidine into RNA, or the incorporation of 3H-labeled amino acids into protein. In d4T-treated cells the relative proportions of [3H]dTMP, [3H]dTDP, and [3H]dTTP formed did not change but their absolute concentrations were increased. d4T significantly reduced the level of [3H]dUMP, and a parallel decrease in [3H]dTMP derived from [3H]dUMP was also evident. d4T increased the amounts of labeled deoxycytidine metabolites formed, with increased dCMP levels the most prominent. In a cell-free extract, [3H]d4T was phosphorylated at a rate of 1.6 pmol/30 min. Increasing concentrations of both thymidine and deoxyuridine inhibited the phosphorylation of [3H]d4T with IC50 values of 5.7 and 35 microM respectively. d4T was found to be a weak substrate for purified H9 cytosolic thymidine kinase (Km = 138 microM) and a weak competitive inhibitor of thymidine and deoxyuridine phosphorylation by this enzyme (Ki = 1.37 and 0.33 mM respectively).

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.

At present, only a few drugs have been approved by the FDA for therapy of viral infections in humans. There is a great need for antiviral drugs with increased potency and decreased toxicity, as well as drugs to treat viral diseases for which no drug or vaccine is currently available. Two approaches for development of antiviral drugs are described--an empirical strategy and a rational strategy--with several examples of each. Although many compounds have potent antiviral activity in cell culture, only a small fraction of these will go on to become antiviral drugs for use in humans. At this time, only seven synthetic compounds and alpha interferon have been approved by the FDA for therapy of viral infections in humans. None of these approved drugs are without toxicities, however, and hence there is a great need for antiviral drugs with increased potency and decreased toxicity, as well as for drugs to treat viral diseases for which no drug or vaccine is currently available. Two approaches for the development of antiviral drugs--the empirical and the rational strategies--and their applications and future directions are discussed.

A new pyrimidine analog, 5-(2-bromoethyl)-2'-deoxyuridine (BEUdR), was tested in vitro for antiviral activity on Herpes simplex virus types 1 and 2. As reference compounds, ACG, BVUdR and PAA were used. Compared to ACG and BVUdR, BEUdR resulted less potent on both HSV-1 and HSV-2. However, a 50% inhibition of the multiplication of uninfected cells could be obtained only at very high BEUdR concentration (ID50 = 8500 microM). This makes BEUdR the least toxic analog known and gives it a selective index comparable to, if not better, than of ACG and BVUdR.