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Continuing our program of research concerning the antiviral activity of a wide series of new angular and linear azolo bicyclic and tricyclic derivatives, now we have simplified and modified the 4-chloro-2-(4-nitrophenyl)-3H-imidazo[4,5-g]quinoline 1, which previously resulted the most active derivative, through either the elimination of the central ring or the opening of the imidazole ring, obtaining various imidazopyridines and N-benzylidenequinolinamines respectively. Title compounds were tested in cell-based assays for cytotoxicity and antiviral activity against representatives of two DNA virus families as wells as against representatives of RNA virus families containing single-stranded, either positive-sense (ssRNA(+)) or negative-sense (ssRNA(-)), and double-stranded genomes (dsRNA). Some imidazo[4,5-b]pyridines emerged as new derivatives endowed with antiviral activity against Vaccinia Virus (VV) at concentrations ranging from 2 to 16 μM. In particular, compound 2b demonstrate to be about 10 times more potent than Cidofovir, used as reference drug. Similarly, the imidazo[4,5-c]pyridines and N-benzylidenequinolinamines derivatives resulted active against Bovine Viral Diarrhoea virus (BVDV), at concentrations ranging from 1.2 to 28 μM. Above all compounds 1, 3a and 3f showed an EC50 of the same order of magnitude of the reference drug, the 2'-C-methyl-guanosine. Moreover, several N-benzylidenequinolinamines showed an interesting activity against Respiratory Syncytial Virus (RSV) at concentrations between 12 and 26 μM.

Several diamino quinoxalines were designed, synthesized and evaluated as anti-tumor agents. Two compounds showed the most potent cytotoxic activities against the leukemia CCRF-CEM cell line (GI(50)0.01microM) and the ovarian cancer cell line OVCAR-4 (GI(50)=0.03microM), respectively, with comparable/better activities than Methotrexate (MTX). Docking calculations of the complexes of hDHFR with the most active compounds identified the binding mode of the described molecules with respect to MTX.

A series of new benzoylquinoxaline derivatives (7-26) was synthesized and evaluated for antitumor activity against a panel of 60 human cell lines at the NCI of Bethesda. Among the compounds which have passed the preliminary screening, compound 23 exhibited the best profile and growth inhibition activity at 100 - 10 μM. The compounds were then tested towards a folate-dependent enzymes bio-library including Thymidylate synthases enzymes and human Dihydrofolate reductase at 10 μM. The most of compounds exhibited a moderate inhibitory activity towards all or some of the enzymes tested with detectable inhibition constants (Ki) values in the range of 0.6-70 μM. Compounds 21, 23, 24 showed Ki in the range of 10-38 μM against both hDHFR and hTS.

A new series of 3-alkyl-, 3-trifluoromethyl-, 3-carboxyethyl- and 3-bromomethylquinoxaline-2-ones and 2,3-bis(bromomethyl)quinoxalines bearing Cl, CF3, morpholine on the benzo-moiety, were synthesised and submitted to a preliminary in vitro evaluation for antibacterial and anticandida activities. Results of the screening showed that compounds 9b, 14b and 19b (MIC=62.5 μg/ml) and 10b (MIC=15.6 μg/ml) were the most active against Vibrio alginolyticus.

Twenty eight pyrrolo[1,2-a]quinoxalines bearing at position 4 various substituents related to the moieties present in classical and non classical antifolic agents were prepared and evaluated in vitro for antiproliferative activity. In an in vitro screening performed at NCI, several compounds emerged as potent antiproliferative agents at concentrations ranging between 10 and 100 microM. Interestingly, some of these compounds proved active also against bovine and murine DHFR (Farmaco 53 (1998) 480). More recently, a compound of classical antifolate type has been reported to be a potent inhibitor of hDHFR in vitro (Farmaco 58 (2003) 51). We then synthesized new derivatives that, in our hands, were endowed with in vitro antiproliferative activities as low as 3.4 microM against a panel of cell lines derived from hematological and solid tumours. In addition, a complete screening of cytotoxicity, antiretroviral HIV-1 and antimicrobial activity has been carried out.

Among a new series of 28 3-carboxy or carbethoxy quinoxalines bearing a substituted benzylamino or N-[4-(aminomethyl)benzoyl]glutamate group on position 2 of the ring and various substituents at C-6, 7 positions, 21 were selected at the National Cancer Institute for evaluation of their in vitro anticancer activity. The results obtained seem to confirm that the carboxy or carbethoxy group on position 3 is not helpful, with a few exceptions, for the anticancer activity.

A new series of quinoxalinones 6/7-trifluoromethyl or nitro- and 6,7-difluoro substituted bearing various side-chains (alkyl, halogenoalkyl, benzyl and phenyl groups) at C-3 of the ring system was synthesized and submitted to preliminary in vitro evaluation for antibacterial, antifungal, antimycobacterial, anticancer and anti-HIV activities. Results of these screenings showed that compounds 23-28 exhibited a good inhibition activity against various strains of Candida. Compound 24 showed also an interesting in vitro anticancer activity.

A new set of 35 3-alkyl and 3-ethoxycarbonylalkyl 6- and/or 7-substituted-2-quinoxalinones was prepared and submitted to a preliminary in vitro investigation of their antimicrobial, anticancer and anti-HIV activities. Results are referred.

Thirty quinoxalines bearing a substituted phenoxy or hydroxybenzoylglutamate group on position 2, a carboethoxy or carboxy group on position 3 and a trifluoromethyl group on position 6 or 7 of the heterocycle were prepared in order to evaluate the in vitro anticancer activity. Screening over 21 compounds selected at the National Cancer Institute (Bethesda, MD) showed that only few derivatives exhibited a moderate growth inhibition activity on various tumor panel cell lines at 10 −4 molar concentration. The acid derivatives showed no growth inhibition activity. The results obtained in this series seem to indicate that in general carboxy or carboethoxy groups close to O-link with phenyl or benzoyl glutamates on position 2 are detrimental for anticancer activity.

Eighteen quinoxalines bearing a methyleneanilino or methyleneaminobenzoylglutamate group on position 6 of the ring and various lipophilic substituents on positions 2 and 3 were prepared in order to discover if their structural analogy with both trimetrexate (TMQ) and 10-propargyl-5,8-dideazafolic acid (CB 3717) might display in vitro anticancer activity. Among these, 12 compounds were selected at the National Cancer Institute, Bethesda, MD, USA; they exhibited moderate (4b,d,i,l,m and 8) to strong (4f,h and 5a,e) cell-growth inhibition at a concentration of 10−4 M. Interesting selectivities were also recorded between 10−8 and 10−6 M. These analogues proved to be less potent inihibitors of tumor cells than other classical and non-classical antifolate analogues previously described by us.

Among twenty-eight novel compounds (twenty-two 2,3-disubstituted-6-[(substituted-phenoxy)methyl-quinoxalines and six 4-[(2,3-disubstituted-quinoxalin-6-yl)methoxy]benzoylglutamates ) only thirteen were selected at NCI for evaluation of their in vitro anticancer activity. The results have shown that compounds 3l,c,b,e and 4b were endowed with significantly high values of percent tumor growth inhibition on several tumor cell lines at 10(-4) M, while compound 3t was characterized by a high selectivity, being still strongly inhibiting on three cell lines at 10(-5) M. Comparison of the presently observed activity with that of the previously described aza-analogues confirms that the effected isosteric substitution is highly valuable in some cases.

The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compound library which showed significantly improved selectivity for the parasite's folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was observed for compound 5b. This work represents a step forward in the identification of effective antileishmania agents.

Several compounds possessing imidazo[4,5-b]pyridine and benzimidazole structure bearing substituents in position 2 were prepared in order to evaluate an anti-ulcer and gastroprotective activity in rat pylorus ligature, in comparison with omeprazole. Among sixteen compounds taken as representatives of the synthetised series only one (3d) showed a good activity by i.m. administration at 50 mg/kg, while by oral administration of 100 mg/kg a certain number was active and in some cases this activity was quite superior to that of omeprazole.

Twenty compounds possessing benzimidazole, imidazo[4,5-b]pyridine and quinoxaline structure bearing either a substituted arylmethylmercapto- or an arylmethylsulfinyl group in position 2 were prepared in order to evaluate an antiulcer and gastroprotective activity in rat pylorus ligature, in comparison with omeprazole at the dose of 50 mg/kg after i.m. administration. One third of these compounds showed a moderate activity, being about half potent as omeprazole.

Thirty-five quinoxalines bearing a substituted aniline group on position 2 and various substituents on positions 3,6,7 and 8 were prepared in order to evaluate in vitro anticancer activity. Structural elucidation of some isomeric quinoxalinones formed by ring closure of 4-substituted-1,2-diaminobenzenes with dicarbonyl compounds was achieved by comparison with one isomer coming from an unambiguous independent route. Preliminary in vitro screening at NCI showed that many compounds exhibited a moderate to strong growth inhibition activity on various cell lines between 10(-5) and 10(-4) molar concentrations.

Thirty compounds possessing quinoxaline structure bearing either substituted arylmethylmercapto-, arylmethylsulfinyl group or a piperazinyl moiety in position 2 were prepared in order to evaluate an antiulcer and gastroprotective activity in rat pylorus ligature, in comparison with omeprazole and ranitidine at the dose of 100 mg/kg after oral administration. Among the compounds of the first group one third showed a moderate activity being about half potent as omeprazole whereas in the second group compound 5b exibited an activity superior to that of ranitidine accompanied with the lowest incidence of lesions and mortality and another compound (5i) was equiactive as ranitidine.

Among twenty-eight novel compounds (twenty-two 2,3-disubstituted-6-[(substituted-phenoxy)methyl-quinoxalines and six 4-[(2,3-disubstituted-quinoxalin-6-yl)methoxy]benzoylglutamates ) only thirteen were selected at NCI for evaluation of their in vitro anticancer activity. The results have shown that compounds 3l,c,b,e and 4b were endowed with significantly high values of percent tumor growth inhibition on several tumor cell lines at 10(-4) M, while compound 3t was characterized by a high selectivity, being still strongly inhibiting on three cell lines at 10(-5) M. Comparison of the presently observed activity with that of the previously described aza-analogues confirms that the effected isosteric substitution is highly valuable in some cases.

Thirty quinoxalines bearing a substituted phenoxy or hydroxybenzoylglutamate group on position 2, a carboethoxy or carboxy group on position 3 and a trifluoromethyl group on position 6 or 7 of the heterocycle were prepared in order to evaluate the in vitro anticancer activity. Screening over 21 compounds selected at the National Cancer Institute (Bethesda, MD) showed that only few derivatives exhibited a moderate growth inhibition activity on various tumor panel cell lines at 10 −4 molar concentration. The acid derivatives showed no growth inhibition activity. The results obtained in this series seem to indicate that in general carboxy or carboethoxy groups close to O-link with phenyl or benzoyl glutamates on position 2 are detrimental for anticancer activity.

A new series of quinoxalinones 6/7-trifluoromethyl or nitro- and 6,7-difluoro substituted bearing various side-chains (alkyl, halogenoalkyl, benzyl and phenyl groups) at C-3 of the ring system was synthesized and submitted to preliminary in vitro evaluation for antibacterial, antifungal, antimycobacterial, anticancer and anti-HIV activities. Results of these screenings showed that compounds 23-28 exhibited a good inhibition activity against various strains of Candida. Compound 24 showed also an interesting in vitro anticancer activity.

Among a new series of 28 3-carboxy or carbethoxy quinoxalines bearing a substituted benzylamino or N-[4-(aminomethyl)benzoyl]glutamate group on position 2 of the ring and various substituents at C-6, 7 positions, 21 were selected at the National Cancer Institute for evaluation of their in vitro anticancer activity. The results obtained seem to confirm that the carboxy or carbethoxy group on position 3 is not helpful, with a few exceptions, for the anticancer activity.

A series of novel 5,7-diamino-3-phenyl-2-benzylamino, 2-phenoxy, and 2-thiophenyl substituted quinoxalines has been designed, synthesized and evaluated for their in vitro antitumor activity towards cell lines of nine different types of human cancers. Some of these compounds exhibited inhibitory effects on the growth of a wide range of cancer cell lines generally at 10−6 M, in some cases at 10−7 M and 10−8 M concentrations. Within this series the benzylamino quinoxaline derivatives 1b–7b were the most active, whereas compound 2c showed the highest MG_MD value (−5.66).

A series of novel 5,7-diamino-3-phenyl-2-benzylamino, 2-phenoxy, and 2-thiophenyl substituted quinoxalines has been designed, synthesized and evaluated for their in vitro antitumor activity towards cell lines of nine different types of human cancers. Some of these compounds exhibited inhibitory effects on the growth of a wide range of cancer cell lines generally at 10−6 M, in some cases at 10−7 M and 10−8 M concentrations. Within this series the benzylamino quinoxaline derivatives 1b–7b were the most active, whereas compound 2c showed the highest MG_MD value (−5.66).

Following the antiviral screening of a wide series of new angular and linear N -tricyclic systems both in silico and in vitro, the [4,7]phenantroline nucleus emerged as a new ring system endowed with activity against viruses containing single-stranded, positive-sense RNA genomes (ssRNA + ). Here, we report our new pathway to the synthesis of this nucleus and of several related derivatives, as well as the results of both cell-based antiviral assays and molecular dynamics simulations. In the antiviral screening, several compounds ( 9 and 16 – 20 ) showed to be fairly active against BVDV, CVB-2, and Polio 1 (EC 50 , 6–25 μM). According to molecular dynamics simulations, compounds ( 15 ) and ( 17 ) emerged for its potency against the HCV NS5B, with a calculated IC 50 of 11–12 μM.

A new series of 3-alkyl-, 3-trifluoromethyl-, 3-carboxyethyl- and 3-bromomethylquinoxaline-2-ones and 2,3-bis(bromomethyl)quinoxalines bearing Cl, CF3, morpholine on the benzo-moiety, were synthesised and submitted to a preliminary in vitro evaluation for antibacterial and anticandida activities. Results of the screening showed that compounds 9b, 14b and 19b (MIC=62.5 μg/ml) and 10b (MIC=15.6 μg/ml) were the most active against Vibrio alginolyticus.

Fourteen out of 21 quinoxaline derivatives described in the present paper were selected at NCI for evaluation of their in vitro anticancer activity. Preliminary screening showed that some derivatives exhibited a moderate to strong growth inhibition activity on various tumor panel cell lines between 10 –5 and 10 –4 M concentrations. Interesting selectivities were also recorded between 10 –8 and 10 –6 M for the compounds 9 and 13 .

We report on an extension of our previous discovery of in vitro anticancer activity of trifluoromethylquinoxalines as analogues of classical and non-classical antifolic methotrexate and trimetrexate. In this case a small number of Schiff bases were obtained from the reaction of 2-bromethyl-3-R-6(7)trifluoromethylquinoxaline and ethyl p-aminobenzoylglutamate, ethyl p-aminobenzoate, p-toluidine instead of the expected 4-[2-quinoxalyl]methyl-N-methylanilino derivatives, which in turn formed with N-methylanilino derivatives. The reaction mechanism has been put forward. Structure elucidation of both Schiff bases and N-methylanilino analogues was achieved by a combination of 1H and 13C NMR spectra and hetcor experiments. Compounds 3a, 3b, 3c, 8, 11, 12, 13, Ie were tested in antifolic enzyme assay [Lactobacillus casei (LcTS), Leishmania major (LmTs), human Thymidylate synthase (hTs), human TS, human dihydrofolate reductase (hDHFR)] while compounds 3a, 3b, 3c were tested for anticancer activity. These results seem to indicate that the Schiff bases are somewhat active either as anticancer or as folate inhibitors, while compound Ie was selectively active against hDHFR with an inhibition constant (Ki) of 200 nM with a specificity of about 1000-folds with respect to hTS.

Among a new series of 26 4-(3-substituted-2-quinoxalylamino)phenylacetates and 4-(3-substituted-2-quinoxalylamino)phenylacetyl- l -glutamates, eight were selected at NCI for evaluation of their in vitro anticancer activity. The results obtained in comparison with the corresponding nor-compounds series seem to indicate that this type of homologation is not helpful.

A series of variously substituted quinoxalin-3-ones bearing an ethoxycarbonyl or carboxy group in the C-2 position has been prepared and their structures proved by 1H NMR spectroscopy. The obtained compounds were investigated in vitro for antimicrobial and anticancer activities. Preliminary results showed a moderate activity against a few strains of bacteria but no significant anticancer and anti-HIV activity.

Twenty-four out of twenty-nine quinoxalines were selected at the National Cancer Institute, Bethesda, Md, USA, for in vitro anticancer screening. Among these, 10 derivatives exhibited high values of percent tumor growth inhibition at a concentration of 10(-4) M in all cancer cell lines. Four of these compounds maintained these values at 10(-5) M, whereas a certain number exhibited significant values of percent inhibition at the most diluted concentrations (10(-8)-10(-6) M). Inhibitory activity against dihydrofolate reductase (DHFR) (bovine and rat liver) was determined for the most active compounds. This test showed that this type of quinoxaline exhibited an appreciable activity in comparison with the previously described aza analogues. In the other test (Lactobacillus casei, thymidylate synthase (TS), human HTS) no or poor activity was detected in both series of compounds.

A number of 7-amino and 7-acylamino substituted 4,4a-dihydro-5H-indeno[1,2-c]pyridazin-3-ones have been synthesized as rigid congeners of hypotensive 6-aryl-5-methyl-4,5-dihydro-3(2H)-pyridazinones and tested as antihypertensive, antithrombotic, antiulcer, and antiinflammatory agents. Unlike the previously described 7-cyano derivative, which displayed only antiinflammatory action, the new series exhibited significant antihypertensive and antithrombotic properties. In this respect, the 7-amino (2b) and the 7-acetylamino (2c) derivatives were found to be the most potent and long lasting in reducing the blood pressure in spontaneously hypertensive rats and in protecting mice from the induction of thrombosis. These compounds, as well as the 7-(2-chloropropionyl) derivative 2d, also exhibited antiinflammatory activity; in addition, 2c,d were highly effective in inhibiting indomethacin-induced ulcers in the rat.

A number of 7-amino and 7-acylamino substituted 4,4a-dihydro-5H-indeno[1,2-c]pyridazin-3-ones have been synthesized as rigid congeners of hypotensive 6-aryl-5-methyl-4,5-dihydro-3(2H)-pyridazinones and tested as antihypertensive, antithrombotic, antiulcer, and antiinflammatory agents. Unlike the previously described 7-cyano derivative, which displayed only antiinflammatory action, the new series exhibited significant antihypertensive and antithrombotic properties. In this respect, the 7-amino (2b) and the 7-acetylamino (2c) derivatives were found to be the most potent and long lasting in reducing the blood pressure in spontaneously hypertensive rats and in protecting mice from the induction of thrombosis. These compounds, as well as the 7-(2-chloropropionyl) derivative 2d, also exhibited antiinflammatory activity; in addition, 2c,d were highly effective in inhibiting indomethacin-induced ulcers in the rat.

Representative terms of the new heterocyclic system 9H-indeno[2,1-c]pyridazine have been synthesized. Their antiinflammatory, analgesic and antipyretic activities were evaluated, in order to compare the pharmacological profiles with those of the isomeric series of 5H-indeno[1,2-c]pyridazine previously investigated. The new compounds were, in general, equiactive as analgesic but less active as antiinflammatory and antipyretic agents with respect to their 5H-isomers.

The synthesis of the 6-fluoro (I a), 6-chloro (I b) and 6-bromo (I c) derivatives of the known antiinflammatory agent 2-(3-benzoylphenyl)propionic acid (ketoprofen) is reported. The procedure employed involves the direct phase-transfer methylation of the appropriate arylacetonitriles and the subsequent hydrolysis of the alpha-methyl arylacetonitriles (V) thus obtained. It is noteworthy that (V b) and (V c) were not accompanied by alpha, alpha-dimethylated contaminants, owing to the steric hindrance of the chloro and bromine substituent. The pharmacological evaluation of (I a-c) showed that the presence of the halogen unexpectedly abolished the antiinflammatory and antipyretic activities of the model drug. The 6-ethoxy derivative (I d), isolated as by-product of the synthesis of (I a) was also found inactive. A hypothesis has been formulated on the loss of activity of these compounds.