The VIZIER project: preparedness against pathogenic RNA viruses.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

2-Arylbenzimidazoles as antiviral and antiproliferative agents. Part 1.

Being involved in an anti-Flaviviridae Project, and because of the role played by benzimidazole derivatives as promising inhibitors of the HCV helicase and RNA polymerase, as well as of the Zn finger transcription factor, we synthesized a new series of 2-arylbenzimidazoles and evaluated them for antiviral activity, as well as for antiproliferative activity. Compounds were tested in cell-based assays against viruses representative of: i) two of the three genera of the Flaviviridae family, i.e. Flaviviruses and Pestiviruses; ii) other RNA virus families, such as Retroviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae and Reoviridae; iii) two DNA virus families (Herpesviridae and Poxviridae). Compounds 15, 28 and 29 resulted moderately active only against Yellow Fever Virus (a Flavivirus) (range 6-27 microM), whereas none of the title benzimidazoles showed any antiviral activity at concentrations not cytotoxic for the resting cell monolayers. Compounds were also tested for antiproliferative activity against a panel of exponentially growing cell lines derived from human haematological and solid tumors. Several new benzimidazoles turned out active. Among them, compound 27 was the most potent against human haematologic and solid tumor cells and turned out to be as potent as Etoposide and more potent than 6-mercaptopurine (6-MP), used as reference antitumor agents.

Synthesis and anti-picornaviridae in vitro activity of a new class of helicase inhibitors the N,N'-bis[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl] alkyldicarboxamides.

A series N,N'-bis[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides (3a-f and 5a-j) were prepared starting from their already known (1a-d) and (4a-c) or new (4d) amine parents. Because of the antiviral activity of several N-[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides previously reported, title compounds were evaluated in vitro for cytotoxicity and antiviral activity against viruses representative of Picornaviridae, [i.e. Enterovirus Coxsackie B2 (CVB-2) and Polio (Sb-1)] and of two of the three genera of the Flaviviridae [Bovine Viral Diarrhea Virus (BVDV) and Yellow Fever Virus (YFV)]. Furthermore, because of the in silico activity against the RNA-dependent RNA-helicase of Polio 1 previously reported, title compounds were evaluated against the 3D model of the Sb-1 helicase and against the 2D model of the CVB-2 helicase. As a reference we used the antiviral and in silico activities of an imidazo counterpart of the title compounds, N,N'-bis[4-(2-benzimidazolyl)phenyl]alkyldicarboxamides (III) that other authors reported to be able to inhibit the corresponding enzyme of Hepatitis C Virus (HCV). In cell-based antiviral assays, N,N'-bis[4-(1H-benzotriazol-1-yl)phenyl]alkyldicarboxamides (3a-f) resulted completely inactive whereas the bis-5,6-dimethyl-benzotriazol-2-yl derivatives (5d-f) exhibited good activity against the Enteroviruses, (EC(50)s ranged between 7 and 11 microM against CVB-2 and 19-52 against Sb-1). Interestingly, bis-5,6-dichloro-benzotriazol-2-yl derivatives (5h-j) showed very selective activity against CVB-2 (EC(50)s = 4-11 microM) whereas they resulted completely inactive against all the other viruses screened. In general, all title compounds showed a good cytotoxicity profile in MT-4 cells. Molecular modeling investigations showed that active compounds may interact with the binding site of the Sb-1 helicase and that their free binding energy values are in agreement with their EC(50)s values.

A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems.

In this work, we present and discuss a comprehensive set of both newly and previously synthesized compounds belonging to 5 distinct molecular classes of linear aromatic N-polycyclic systems that efficiently inhibits bovine viral diarrhea virus (BVDV) infection. A coupled in silico/in vitro investigation was employed to formulate a molecular rationale explaining the notable affinity of all molecules to BVDV RNA dependent RNA polymerase (RdRp) NS5B. We initially developed a three-dimensional common-feature pharmacophore model according to which two hydrogen bond acceptors and one hydrophobic aromatic feature are shared by all molecular series in binding the viral polymerase. The pharmacophoric information was used to retrieve a putative binding site on the surface of the BVDV RdRp and to guide compound docking within the protein binding site. The affinity of all compounds towards the enzyme was scored via molecular dynamics-based simulations, showing high correlation with in vitro EC50 data. The determination of the interaction spectra of the protein residues involved in inhibitor binding highlighted amino acids R295 and Y674 as the two fundamental H-bond donors, while two hydrophobic cavities HC1 (residues A221, I261, I287, and Y289) and HC2 (residues V216, Y303, V306, K307, P408, and A412) fulfill the third pharmacophoric requirement. Three RdRp (K263, R295 and Y674) residues critical for drug binding were selected and mutagenized, both in silico and in vitro, into alanine, and the affinity of a set of selected compounds towards the mutant RdRp isoforms was determined accordingly. The agreement between predicted and experimental data confirmed the proposed common molecular rationale shared by molecules characterized by different chemical scaffolds in binding to the BVDV RdRp, ultimately yielding compound 6b (EC50 = 0.3 µM; IC50 = 0.48 µM) as a new, potent inhibitor of this Pestivirus.

Synthesis of 5-aza-7-deazaguanine nucleoside derivatives as potential anti-flavivirus agents.

Coupling suitable sugars (D- or L-ribofuranose, 2' or 3-deoxysugar, branched sugars) with 2-aminoimidazo[1,2-a]-s-triazin-4-one was carried out using the different reaction conditions: 1) condensation in the presence of sodium hydride; or 2) condensation using Vorbrüggen's methods. The 5-aza- 7-deazaguanine nucleoside analogues obtained were evaluated in cell culture experiments for the inhibition of the replication of a number of RNA viruses, including BVDV, YFV, and WNV.

L-nucleosides containing modified nucleobases.

The synthesis of base modified L-nucleosides is described with pyrrolo[2,3-d]pyrimidines, pyrazolo[3,4-d]pyrimidines, benzimidazoles, and imidazo[1,2-a]-s-triazines as nucleobases. The conformation of the nucleosides is studied and the antiviral activity is evaluated.

Antiviral properties from plants of the Mediterranean flora.

Natural products are a successful source in drug discovery, playing a significant role in maintaining human health. We investigated the in vitro cytotoxicity and antiviral activity of extracts from 18 traditionally used Mediterranean plants. Noteworthy antiviral activity was found in the extract obtained from the branches of Daphne gnidium L. against human immunodeficiency virus type-1 (EC50 = 0.08 µg/mL) and coxsackievirus B5 (EC50 = 0.10 µg/mL). Other relevant activities were found against BVDV, YFV, Sb-1, RSV and HSV-1. Interestingly, extracts from Artemisia arborescens L. and Rubus ulmifolius Schott, as well as those from D. gnidium L., showed activities against two different viruses. This extensive antiviral screening allowed us to identify attractive activities, offering opportunities to develop lead compounds with a great pharmaceutical potential.

2-Sulfonyl-4-chloroanilino moiety: a potent pharmacophore for the anti-human immunodeficiency virus type 1 activity of pyrrolyl aryl sulfones.

The synthesis and the evaluation of cytotoxicity and anti-HIV-1 activity of new aryl pyrrolyl (8) and aryl indolyl (9) sulfones are reported. Preparation of above sulfones was achieved by reacting arylsulfonyl chlorides with substituted pyrroles and indoles or by condensing sulfonamides with 2,5-dimethoxytetrahydrofuran in glacial acetic acid according to the Clauson-Kaas method. Chemical requisites relevant to the anti-HIV-1 activity of these compounds are both a 2-sulfonyl-4-chloroanilino moiety and an alkoxycarbonyl group at position 2 of the pyrrole ring. The best activity and selectivity were obtained with ethoxycarbonyl and isopropoxycarbonyl substituents. Substitutions at the amino group of the pharmacophore moiety led to inactive products (alkylation) or weakened (acylation) anti-HIV-1 activity. Among test derivatives, 16 compounds showed EC50 values ranging between 10 and 1 microM, and five (8b',d',f',h'j') showed EC50S in the sub-micromolar range. The compounds were active against HIV-1, both wild type and AZT-resistant strains, but not against HIV-2. Moreover, in enzyme assays they potently inhibited the HIV-1 recombinant reverse transcriptase, were 10 times less active against enzymes from nevirapine- and TIBO-resistant strains, and were totally inactive against the HIV-2 recombinant enzyme. Interestingly, some compounds (8r'-y') were inactive against the recombinant reverse transcriptase while being active in tissue culture.

[[[(Thienylcarbonyl)alkyl]oxy]phenyl]- and [[[(pyrrylcarbonyl)alkyl]oxy]phenyl]oxazoline derivatives with potent and selective antihuman rhinovirus activity.

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.

Synthesis and antiviral activity of 8-aza analogs of chiral [2-(phosphonomethoxy) propyl]guanines.

(R)- And (S)-8-aza-9(-)[2-(phosphonomethoxy)propyl]guanine [(R)-and (S)-8-aza-PMPG] were synthesized and tested in vitro for anti-human immunodeficiency virus (HIV) activity. The synthesis of the above compounds and of (R)-9(-)[2-(phosphonomethoxy)propyl]guanine [(R)-PMPG] was carried out through the alkylation of 8-azaguanine or guanine with (R)- and (S)-2-O(-)[(diisopropylphosphono)methyl]-1-O-(tolylsulfonyl) -1,2-propanediol followed by deprotection of the phosphonic moiety. A different, even more convenient synthesis of (R)-8-aza-PMPG starting from 2-amino-6-chloro-5-nitro-4(3H)-pyrimidinone and (R)(-)[2(-)[(diisopropylphosphono)-methoxy]propyl]amine is also reported. Both (R)-8-aza-PMPG and (R)-PMPG demonstrated anti-HIV activity in the MTT assay with EC50 values of 12 and 4.5 microM, respectively. The corresponding S enantiomers were found to be less potent. When evaluated in combination with AZT, ddI, or DABO 603, (R)-8-aza-PMPG gave additive, additive, and synergistic anti-HIV-1 effects, respectively.

Synthesis and anti-HIV-1 activity of thio analogues of dihydroalkoxybenzyloxopyrimidines.

Various thio analogues of dihydroalkoxybenzyloxopyrimidines (DABOs), a new class of non-nucleoside reverse transcriptase inhibitors, were found to selectively inhibit the HIV-1 multiplication in vitro. Among the C-5 H-substituted 6-benzyl-3,4-dihydro-4-oxopyrimidines, the introduction of alkylthio or cycloalkylthio substituents at C-2 of the pyrimidine ring led to derivatives (S-DABOs) which were up to 10-fold more potent than the alkyloxy or cycloalkyloxy counterparts. The further introduction of a methyl group at the 3'-position of the benzyl portion of 2-(alkylthio)-6-benzyluracils reduced the cytotoxicity leading to more selective compounds. Among C-5 methyl-substituted S-DABOs, numerous derivatives showed EC50 values as low as 0.6 microM and lacked cytotoxicity at doses as high as 300 microM. In the C-5 double methyl-substituted series, a more pronounced cytotoxicity was observed and the further introduction of a methyl at the 3'-position in the benzylidene group resulted in total loss of antiviral activity. S-DABOs, namely 2-(alkylthio)-6-benzyl-3,4-dihydro-4-oxopyrimidines, were synthesized by reacting proper methyl (phenylacetyl)acetates or their 2-methyl compounds with thiourea to afford 6-benzyl-4-oxo-1,2,3,4-tetrahydro-2-thiaoxopyrimidines or the related 5-methyl derivatives. Treatment of the latter derivatives with alkyl or cycloalkyl halides in alkaline medium gave the required title compounds.

Pyrazole-related nucleosides. Synthesis and antiviral/antitumor activity of some substituted pyrazole and pyrazolo[4,3-d]-1,2,3-triazin-4-one nucleosides.

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.

Dihydro(alkylthio)(naphthylmethyl)oxopyrimidines: novel non-nucleoside reverse transcriptase inhibitors of the S-DABO series.

Novel compounds related to 2-(cyclohexylthio)-3,4-dihydro-5-methyl-6-(3-methylbenzyl)-4-ox opyrimidine (3c, MC 639) have been synthesized and tested as inhibitors of human immunodeficiency virus type-1 (HIV-1). Reaction of thiourea with ethyl arylmethylacetoacetates furnished 5-alkyl-6-(arylmethyl)-3,4-dihydro-2-mercapto-4-oxopyrimidines which were then alkylated at the sulfur atom to afford the required 2-alkylthio or 2-cycloalkylthio derivatives (S-DABOs). Chemical modifications at N-3, C-4, and C-6 of the pyrimidine ring were attempted with the aim of improving antiretroviral activity. In particular, replacement of the benzyl group with the 1-naphthylmethyl moiety enhanced the activity of S-DABOs, whereas N-3 alkylation and C=O transformation into C=S at position 4 of the pyrimidine ring led to compounds devoid of anti-HIV-1 activity. Lower activity was generally observed when 1-naphthylmethyl was replaced by the isomeric 2-naphthylmethyl moiety. The most active compounds showed activity in the low micromolar range with EC50 values comparable to that of nevirapine.

Structure-based design, synthesis, and biological evaluation of novel pyrrolyl aryl sulfones: HIV-1 non-nucleoside reverse transcriptase inhibitors active at nanomolar concentrations.

Pyrrolyl aryl sulfones (PASs) have been recently reported as a new class of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitors acting at the non-nucleoside binding site of this enzyme (Artico, M.; et al. J. Med. Chem. 1996, 39, 522-530). Compound 3, the most potent inhibitor within the series (EC(50) = 0.14 microM, IC(50) = 0.4 microM, and SI > 1429), was then selected as a lead compound for a synthetic project based on molecular modeling studies. Using the three-dimensional structure of RT cocrystallized with the alpha-APA derivative R95845, we derived a model of the RT/3 complex by taking into account previously developed structure-activity relationships. Inspection of this model and docking calculations on virtual compounds prompted the design of novel PAS derivatives and related analogues. Our computational approach proved to be effective in making qualitative predictions, that is in discriminating active versus inactive compounds. Among the compounds synthesized and tested, 20 was the most active one, with EC(50) = 0.045 microM, IC(50) = 0.05 microM, and SI = 5333. Compared with the lead 3, these values represent a 3- and 8-fold improvement in the cell-based and enzyme assays, respectively, together with the highest selectivity achieved so far in the PAS series.

Synthesis and evaluation of the anti-HIV activity of aza and deaza analogues of isoddA and their phosphates as prodrugs.

Some aza and deaza analogues of the anti-HIV agent 2',3'-dideoxy-3'-oxoadenosine (isoddA) (8-aza-, 8-aza-1-deaza, 8-aza-3-deaza-, 1-deaza-, and 3-deaza-isoddA) were synthesized and found inactive against HIV in vitro. The hypothesis that the inactivity of these isonucleosides might be due to their poor affinity for cellular nucleoside kinases was checked by the synthesis of a series of 5'-[bis(2,2,2-trichloroethyl) phosphate] triesters and 5'-phenyl phosphoramidate derivatives which, acting as membrane soluble prodrugs, could release the free phosphate form inside the cell. The 5'-(phenylmethoxy)alaninyl phosphate derived from 8-aza-isoddA was found active against HIV-1 and HIV-2 with a potency similar to that of isoddA, while the anti-HIV potency of 5'-(phenylmethoxy)alaninyl phosphate of isoddA proved remarkably higher than that of isoddA, in particular against HIV-2, being similar to that of AZT. Further evidence that 8-aza-isoddA could behave as anti-HIV agent, provided that it is activated as phosphate, was obtained by the synthesis of its 5'-triphosphate derivative, which proved to be an active inhibitor of HIV-1 recombinant reverse transcriptase.

5-Alkyl-2-(alkylthio)-6-(2,6-dihalophenylmethyl)-3, 4-dihydropyrimidin-4(3H)-ones: novel potent and selective dihydro-alkoxy-benzyl-oxopyrimidine derivatives.

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.

Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling.

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.

Synthesis and antimicrobial and cytotoxic activities of pyrrole-containing analogues of trichostatin A.

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.

Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine with potent antitumor and antimicrobial activity.

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.

Decomposition pathways and in vitro HIV inhibitory effects of isoddA pronucleotides: toward a rational approach for intracellular delivery of nucleoside 5'-monophosphates.

The decomposition pathways and kinetics in various biological media and the in vitro anti-HIV-1 and anti-HIV-2 activities of four derivatives of the 5'-mononucleotide of isoddA incorporating carboxylate esterase-labile transient phosphate protecting groups are reported and compared: namely, two mononucleoside aryl phosphoramidate derivatives 1a,b and two mononucleoside phosphotriester derivatives incorporating two S-acyl-2-thioethyl groups 2a,b. All four compounds show better antiviral activity, compared to the parent nucleoside analog isoddA. The results highlight that both types of compounds act as pronucleotides, i.e. they exert their antiviral effect via intracellular delivery of the 5'-mononucleotide of isoddA. The results may give insights for the design of new more efficient pronucleotides.