Synthesis, Antitumor and Antiviral In Vitro Activities of New Benzotriazole-Dicarboxamide Derivatives.

Cancer and viral infections continue to threaten humankind causing death worldwide. Hence, the discovery of new anticancer and antiviral agents still represents a major scientific goal. Heterocycles designed to mimic the chemical structure of natural pyrimidines and purines have been designed over the years, exerting their activity acting as false substrates on several different targets. We reported a series of bis-benzotriazole-dicarboxamide derivatives which inhibit viral helicase of poliovirus, and hence we planned structure modifications to obtain different series of new dicarboxamides. Here, the synthesis and characterization of 56 new compounds: 31 bis-benzotriazole dicarboxamides and 25 mono-substituted acidic derivatives are reported. The synthesized compounds were tested for their antiviral and antitumor activity. Mostly, compounds 4a, 4c and 4d showed antiviral activity against tested Picornaviruses, Coxsackievirus B5 and Poliovirus-1. Likewise, four derivatives (3b, 3d, 4d, 9b) showed notable antiproliferative activity inhibiting cell growth in two distinct antitumor screenings. Compound 3b was selected as the antitumor lead compound for the wide range of activity and the potency proved. The lead compound was proved to induce apoptosis in SK-MES1 tumor cells, in a dose-dependent manner.

Dichloro-Phenyl-Benzotriazoles: A New Selective Class of Human Respiratory Syncytial Virus Entry Inhibitors.

Human Respiratory Syncytial Virus (RSV) is the primary cause of bronchopneumonia in infants and children worldwide. Clinical studies have shown that early treatments of RSV patients with ribavirin improve prognosis, even if the use of this drug is limited due to myelosuppression and toxicity effects. Furthermore, effective vaccines to prevent RSV infection are currently unavailable. Thus, the development of highly effective and specific antiviral drugs for pre-exposure prophylaxis and/or treatment of RSV infections is a compelling need. In the quest of new RSV inhibitors, in this work we evaluated the antiviral activity of a series of variously substituted 5,6-dichloro-1-phenyl-1(2)H-benzo[d][1,2,3]triazole derivatives in cell-based assays. Several 1- and 2-phenyl-benzotriazoles resulted fairly potent (µM concentrations) inhibitors of RSV infection in plaque reduction assays, accompanied by low cytotoxicity in human highly dividing T lymphoid-derived cells and primary cell lines. Contextually, no inhibitory effects were observed against other RNA or DNA viruses assayed, suggesting specific activity against RSV. Further results revealed that the lead compound 10d was active during the early phase of the RSV infection cycle. To understand whether 10d interfered with virus attachment to target cells or virus-cell fusion events, inhibitory activity tests against the RSV mutant strain B1 cp-52-expressing only the F envelope glycoprotein-and a plasmid-based reporter assay that quantifies the bioactivity of viral entry were also performed. The overall biological results, in conjunction with in silico modeling studies, supported the conclusion that the RSV fusion process could be the target of this new series of compounds.

Synthesis, structural characterization and biological evaluation of 4'-C-methyl- and phenyl-dioxolane pyrimidine and purine nucleosides.

Nucleoside analogues play an important role in antiviral, antibacterial and antineoplastic chemotherapy. Herein we report the synthesis, structural characterization and biological activity of some 4'-C -methyl- and -phenyl dioxolane-based nucleosides. In particular, α and ß anomers of all natural nucleosides were obtained and characterized by NMR, HR-MS and X-ray crystallography. The compounds were tested for antimicrobial activity against some representative human pathogenic fungi, bacteria and viruses. Antitumor activity was evaluated in a large variety of human cancer cell-lines. Although most of the compounds showed non-significant activity, 23α weakly inhibited HIV-1 multiplication. Moreover, 22α and 32α demonstrated a residual antineoplastic activity, interestingly linked to the unnatural α configuration. These results may provide structural insights for the design of active antiviral and antitumor agents.

Unconventional Knoevenagel-type indoles: Synthesis and cell-based studies for the identification of pro-apoptotic agents.

A new series of indole-based analogues were recently identified as potential anticancer agents. The Knoevenagel-type indoles herein presented were prepared via a one-pot condensation of iminium salts with active methylene reagents and were isolated as single geometric isomers. Biological evaluation in different cell-based assays revealed an antiproliferative activity for some analogues already in the nanomolar range against leukaemia, breast and renal cancer cell lines. To explain these effects, the most promising analogues of the series were engaged in further cell-based studies. Compounds 5e, l, p and 6a, b highlighted a pro-apoptotic potential being able to induce apoptosis in HL60, K562 and MCF-7 cell lines in a dose and time-dependent manner. The ability of these compounds to arrest cell cycle at the G2/M phase inspired the immunofluorescence studies which allowed us to identify tubulin as a potential target for compounds 5l and 6b.

Further SAR studies on bicyclic basic merbarone analogues as potent antiproliferative agents.

Pyrimidopyrimidine derivatives 1 were prepared as rigid thioanalogues of merbarone (a catalytic topoisomerase II inhibitor) and screened as antiproliferative agents against different tumor cell lines. A number of the synthesized compounds emerged as cytotoxic in cell-based assays (MT-4, HeLa and MCF-7 cells) at low micromolar concentrations. In a National Cancer Institute screening, selected member of the series showed a broad spectrum of antiproliferative activity against various tumours (melanoma, renal, CNS, colon and breast cancers). The acid-base and steric properties of the substituent at position 7 of the pyrimidopyrimidine scaffold deeply affected potency. Enzymatic assays evidenced that a subset of tested derivatives efficiently inhibit topoisomerase IIα accordingly to merbarone mechanism of action. However this property does not fully rationalize the cytotoxicity data of the full ligand panel, suggesting that different target(s) should be additionally involved.

3-Aryl-2-[1H-benzotriazol-1-yl]acrylonitriles: a novel class of potent tubulin inhibitors.

During a screening for compounds that could act against Mycobacterium tuberculosis, a series of new cellular antiproliferative agents was identified. The most cytotoxic molecules were evaluated against a panel of human cell lines derived from hematological and solid human tumors. In particular, (E)-2-(1H-benzo[d] [1,2,3]triazol-1-yl)-3-(4-methoxyphenyl)acrylonitrile (1) was found to be of a potency comparable to etoposide and greater than 6-mercaptopurine in all cell lines tested. Accordingly, a synthesis of a new series of (E)-2-(5,6-dichloro-1H-benzo[d] [1,2,3]triazol-1-yl)-3-(4-R-phenyl)acrylonitriles was conducted in order to extend the studies of structure-activity relationship (SAR) for this class of molecules. With the aim to evaluate if 3-aryl-2-[1H-benzotriazol-1-yl]acrylonitriles were able to act like tubulin binding agents, the effects on cell cycle distribution of the most active compounds (1, 2a, 3 and 4) were analyzed in K562 cells. A detailed molecular modeling study of the putative binding mode of this series of compounds on tubulin is also reported.

Protective properties of non-nucleoside reverse transcriptase inhibitor (MC1220) incorporated into liposome against intravaginal challenge of Rhesus macaques with RT-SHIV.

In the absence of an effective vaccine against HIV, it is urgent to develop an effective alternative such as a microbicide. Single and repeated applications of MC1220 microbicide were evaluated in macaques. First, animals were given a single application of 0.5% or 1.5% MC1220-containing liposomal gel. A second group were treated with 0.5% MC1220 once a day for 4 days. The control groups were treated by liposomal gel alone. Thirty minutes after the last application, animals were challenged with RT-SHIV. In the first protocol, 2 of 4 animals treated by 0.5% of the MC1220 and 2 of 5 treated by 1.5% were protected. In the second protocol, 3 of 5 treated animals were protected and 5 of 5 controls were infected. The RNA viral load at necropsy was significantly lower (p=0.05) in treated-infected animals than in controls. In both protocols, the number of CD4+ T cells was lower at viremia peak in infected than in protected animals.

Platinum and palladium-triazole complexes as highly potential antitumor agents.

The palladium complexes [(dppe)Pd(L)(2)PdCl(2)], [(dppe)Pd(L)(2)PtCl(2)], [(dppp)Pd(L)(2)PdCl(2)], [(dppm) Pd(L)(2)NiCl(2)], and [(dppm)Pd(L)(2)SnCl(4)] 15-19 were prepared. The antiproliferative activity of the newly synthesized complexes as well as their previously prepared analogues 3-14 and 20-26 were screened against a large panel of human cancer cell lines derived from haematological CD4(+) human T-cells containing an integrated HTLV-1 genome (MT-4). The complex 12a, b exhibited remarkable antiproliferative activity against MT-4, CD4(+) human acute T-lymphoblastic leukemia (CCRF-CEM), human splenic B-lymphoblastoid cells (WIL-2NS), human acute B-lymphoblastic leukemia (CCRF-SB), skin melanoma (SK-MEL-28), and prostate carcinoma (DU145) cell lines (CC(50 )= 0.5 microM, 0.4 +/- 0.05 microM, 0.6 +/- 0.05 microM, 0.4 +/- 0.1 microM, and 0.8 +/- 0.2 microM, respectively), meanwhile, 9a, b, 14a, b, and 23 showed significant activity against the CCRF-SB cell lines (CC(50) = 0.6 +/- 0.06 microM, 0.7 +/- 0.05 microM, 0.6 +/- 0.05 microM, and 0.8 +/- 0.15 microM, respectively). Further, 19 exhibited activity against the CCRF-CEM cell line (CC(50 )= 0.4 +/- 0.05 microM).

Antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives.

Twelve aminoarylazocompounds (A-C) and 46 aryltriazene 7 derivatives (D-G) have been synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. Eight aminoazocompounds and 27 aryltriazene derivatives exhibited antiviral activity, sometimes of high level, against one or more viruses. A marked activity against BVDV and YFV was prevailing among the former compounds, while the latter type of compounds affected mainly CVB-2 and RSV. None of the active compounds inhibited the multiplication of HIV-1, VSV and VV. Arranged in order of decreasing potency and selectivity versus the host cell lines, the best compounds are the following; BVDV: 1>7>8>4; YFV: 7>5; CVB-2: 25>56>18; RSV: 14>20>55>38>18>19; HSV-1: 2. For these compounds the EC(50) ranged from 1.6 microM (1) to 12 microM (18), and the S. I. from 19.4 (1) to 4.2 (2). Thus the aminoarylazo and aryltriazene substructures appear as interesting molecular component for developing antiviral agents against ss RNA viruses, particularly against RSV and BVDV, which are important human and veterinary pathogens. Finally, molecular modeling investigations indicated that compounds of structure A-C, active against BVDV, could work targeting the viral RNA-dependent RNA-polymerase (RdRp), having been observed a good agreement between the trends of the estimated IC(50) and the experimental EC(50) values.

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

In prosecution of an anti-Flaviviridae project a new series of variously substituted 2-diphenyl-benzimidazoles were synthesized and tested in vitro for antiviral and antiproliferative activities. 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). The 5-Acetyl-2-(4'-nitrobiphenyl-4-yl)-1H-benzimidazole (24) emerged as potent active lead compound against Yellow Fever Virus (a Flavivirus) (EC(50) = 0.5 microM) and CVB-2 at 1 microM and was not cytotoxic, whereas the other title benzimidazoles showed no antiviral activity at concentrations not cytotoxic for the resting cell monolayers. Among the examined series, the most cytotoxic derivatives (11,12,14,16,18,19,20,21,23,25-30) against mock-infected MT-4 cells (CC50 < 8.0 microM) were evaluated against a panel of human cell lines derived from haematological and solid tumours,using 6-mercaptopurine (6-MP) and etoposide as reference drugs. In particular, compounds 26 and 28 showed a similar potency of 6-MP and etoposide.

Aryl nucleoside H-phosphonates. Part 16: synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters.

Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been developed for the preparation of this class of compounds. It was found that anti-HIV-1 activity of the phosphotriesters bearing an antiviral nucleoside moiety (AZT, ddA) and also ddU was due, at least partially, to intracellular conversion into the corresponding nucleoside 5'-monophosphates, and their efficiency correlated well with the pK(a) values of the aryloxy groups present.

Anti-HIV evaluation of benzo[d]isothiazole hydrazones.

The synthesis and the anti-HIV-1 activity of novel benzo[d]isothiazole hydrazones are reported. Target compounds tested in MT-4 cells cultures for their anti-HIV properties against wild type HIV-1 and HIV strains carrying clinically relevant mutations (EFV(R), Y181C and K103/Y181C) showed good activity against wild type HIV-1 and against the EFV(R) mutant. In terms of SAR the relevant result was that, in the class of benzisothiazole hydrazones, the benzo[d]isothiazol-3(2H)-one moiety (compounds 1 and 4) is an essential structural requirement for the antiretroviral activity.

N-acylated and N,N'-diacylated imidazolidine-2-thione derivatives and N,N'-diacylated tetrahydropyrimidine-2(1H)-thione analogues: synthesis and antiproliferative activity.

Fifty-one acylthioureas (ATUs) incorporating imidazolidine-2-thione or its upper cyclohomologue were prepared by parallel synthesis and evaluated against a high number of human cancer cell lines for antiproliferative activity. ATUs 1o (3,5-dichlorobenzoyl), 1s (2-furoyl), 3s (2-furoyl) and 1t (2-thenoyl) displayed activity against leukemia, melanoma LOX IMVI, non-small cell lung NCI-H522, renal 786-0, CAKI-1, SN12C, UO-31 and breast MCF7, MDA-MB-435, T-47D cancer cell lines in the 0.3-9.7 microM concentration range. Compound 14s exhibited selectivity for melanoma SK-MEL-5 (GI(50)<5 nM); 1s for leukemia MOLT-4 (GI(50): 300 nM); 1q, 3b and 3q for renal cancer UO-31 (GI(50): 70-200 nM); 8s, 9s for non-small cell lung cancer EKVX (GI(50): 300, 10 nM) and 3j for HOP-92 (GI(50): 700 nM) cell line.

In vitro antitumor and antiviral activities of new benzothiazole and 1,3,4-oxadiazole-2-thione derivatives.

A series of new benzothiazole derivatives 6a-h have been synthesized, in five steps, from substituted phenols via the 1,3,4-oxadiazole-2-thiones 5a-h. The in vitro antitumor activity of the compounds obtained was investigated and the benzothiazol derivatives 6d and 6e showed strong effects on leukaemia cell lines CCRF-CEM (CC50=12+/-2 micromol L(-1), 8+/-1 micromol L(-1), respectively). These compounds are leading candidates for further development. The title compounds were tested against representatives of several virus families containing single stranded RNA genomes, either positive-sense (ssRNA+), or negativesense (RNA-), and against double-stranded RNA genomes (dsRNA), as well as some Flaviviridae viruses.

2(3)-Aryl-thio(oxy)-methylquinoxaline derivatives: a new class of P-glycoprotein-mediated drug efflux inhibitors.

A series of quinoxalines variously substituted, namely 3-arylthiomethyl-1,6-dimethylquinoxalin-2-ones (6a-f), 3-arylthiomethyl-1-benzyl-7-trifluoromethylquinoxalin-2-ones (8a-g) and 2-arylthiomethyl-3-benzyloxy-6-trifluoro-methylquinoxalines (10a,b,e-h), were synthesized and compared with previous arylphenoxymethylquinoxalines (1a-f, 2a-f and 3a-b). The purpose was to verify whether the replacement of oxygen with sulphur atom and the insertion of different substituents on the phenyl side chain were able to improve the capability to inhibit the Pgp pump and restore the antiproliferative activity of clinically useful drugs, such as doxorubicin (Doxo), vincristine (VCR) and etoposide (VP16), in drug-resistant human nasopharyngeal carcinoma KB cells (KB(wt), KB(MDR), KB(7D) and KB(V20C)). Furthermore, 2,3-bis(aryloxy-methyl)-6-trifluoromethylquinoxalines (13a-c) were designed with the objective to evaluate the capability of the double side chain to potentiate the antiproliferative activity of the drugs tested. Biological assays showed that title compounds were, in general, endowed with good activity as Pgp inhibitors. In particular compound 3a, bearing 2-CONHPh substituent on phenoxymethyl side chain, resulted the most effective, while the double side chain (compound 13c) gives the ability to inhibit a different MRP pump (a membrane glycoprotein named mrp). Furthermore, we can conclude that replacement of oxygen with sulphur atom did not improve the biological activity.

Design, synthesis and anti flaviviridae activity of N(6)-, 5',3'-O- and 5',2'-O-substituted adenine nucleoside analogs.

During a random screening of representative libraries of nucleoside analogues we discovered that the adenine derivatives FEVB28 and FEG118 were Flaviviridae inhibitors endowed with potency comparable, if not superior, to that of ribavirin. Those studies prompted us to design a new class of protected nucleoside analogs, reported herein, which displays interesting anti-bovine viral diarrhea virus (BVDV) activity and low cytotoxicity in cell-based assays (4, 23, 29 EC(50): 14, 11, 26 microM respectively, CC(50)>100 microM) and appreciable activity in enzyme assays against the RNA dependent RNA polymerase (RdRp) of BVDV (4, 23, 29, RdRp inhibition activity 27, 16, 15 microM respectively). A molecular modeling study was also carried out to highlight the possible interactions between this compounds class and the corresponding hepatitis C virus (HCV) enzyme.

Synthesis and antiproliferative properties of N3/8-disubstituted 3,8-diazabicyclo[3.2.1]octane analogues of 3,8-bis[2-(3,4,5-trimethoxyphenyl)pyridin-4-yl]methyl-piperazine.

A series of novel N(3/8)-disubstituted-3,8-diazabicyclo[3.2.1]octanes in order to improve the in vitro activity of the prototype 3,8-bis[2-(3,4,5-trimethoxyphenyl)pyridyl-4-yl)methylpiperazine (1) were synthesized and evaluated by assays of growth inhibition against several tumor cell lines. Compounds 2a,b,f and m demonstrated not only growth-inhibitory activities against leukemia cancer cells, but also fairly good activities against the growth of certain solid tumors. Among them, 2a is the most potent one with IC(50) values in the low micromolar range. Moreover, compound 2a has been selected for in vitro testing on MCF-7 cell to evaluate the mode of action of this lead compound.

N, N' (4,5-dihydro-1h-imidazol-2-yl)3-aza-1,10-decane-diamine and N, N'(4,5-dihydro-1H-imidazol-2-yl)3-aza-1, 10-dodecane-diamine antagonize cell proliferation as selective ligands towards topoisomerase II.

New alkyl imidazoline derivatives have been synthesized as potential anti-cancer agents. The anti-proliferative activity of these compounds, evaluated against representative human haematological and solid neoplastic cell lines, showed that N, N'-di (4,5-dihydro-1H-imidazol-2-yl)3-aza-1,10-decane-diamine (8) and N, N'-di (4,5-dihydro-1H-imidazol-2-yl)3-aza-1,10-dodecane-diamine (9) were the most active compounds; in fact, they inhibited the cell proliferation at submicromolar concentrations. In enzyme assays, compound 9 turned out to be an inhibitor of topoisomerase II at concentrations comparable with those of the reference topoisomerase II inhibitor, etoposide.

Synthesis of variously substituted 3-phenoxymethyl quinoxalin-2-ones and quinoxalines capable to potentiate in vitro the antiproliferative activity of anticancer drugs in multi-drug resistant cell lines.

Two series of 1,6-dimethyl-3-phenoxymethylquinoxalin-2-ones and 1-benzyl-3-phenoxymethyl-7-trifluoromethylquinoxalin-2-ones, and a series of 2-benzyloxy-3-phenoxymethyl-7-trifluoromethylquinoxaline were synthesized. Their capability to restore/potentiate the antiproliferative activity of clinically useful drugs, such as doxorubicin (Doxo), vincristine (VCR) and etoposide (VP16), in drug-resistant human nasopharyngeal carcinoma KB cells (KB(WT), KB(MDR), KB(7D)and KB(V20C)) was evaluated. In vitro data show that many quinoxalin-2-ones and quinoxalines potentiate the antiproliferative activity of Doxo and VCR in tumor-derived MDR cell lines. In this series, 17a turned out to be the most potent quinoxaline derivative in potentiating the antiproliferative activity of doxorubicin and vincristine against KB(MDR) and KB(V20C) resistant cell lines, respectively.

Synthesis and antiproliferative activity of benzo[d]isothiazole hydrazones.

Several benzo[d]isothiazole hydrazones have been evaluated for their potential antiretroviral activity. Since a number of these compounds were found to be inactive against viruses, but showed cytotoxicity at micromolar concentrations against the human CD4+ lymphocytes (MT-4) that were used to support HIV-1 growth, they were further tested for antiproliferative activity. The compounds resulted as being cytotoxic for MT-4 cells and new derivatives which were rationally designed and synthesized, were tested for antiproliferative activity against several leukaemia and solid tumour cell lines. In addition, these compounds were evaluated against "normal" cell lines. Compound 2h proved to be the most active compound and the fragment -CO-NH-N=CH-2-hydroxyphenyl was identified as being very important for biological activity, suggesting intramolecular hydrogen bond formation or favourable mutual disposition between two important centres in the pharmacophore. 1H-NMR spectra have been explained with the support of a conformational analysis.