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Polycyclic or O-glycoconiugate polycyclic compounds 1a-g were previously tested for their in vitro antiproliferative activity. In this series of compounds, activity increases as log P decreases. Specifically, compounds 1d and 1g showed lower log P values together with the best antiproliferative profiles. With the aim of extending our understanding of the structure–activity relationship (SAR) of this class of compounds, we prepared new polycyclic derivatives 2a-c, which bear on each of the two phenyl rings hydrophilic substituents (OH, SO2NH2 or NHCOCH3). These substituents are able to form hydrogen bonds and to decrease the partition coefficient value as compared with compound 1d. Compound 2a was slightly more active than 1d, while 2b and 2c had antiproliferative activity comparable to that of 1d. Finally, the role of the two phenyl groups of polycycle derivatives 1 was also investigated. The analog 3, which bears two methyls instead of the two phenyls had a lower log P value (2.94 ± 1.22) than all the other compounds, but it had negligible antiproliferative activity at 10 µM. The analysis of the most active derivative 2a revealed a significant antiproliferative activity against the triple-negative breast cancer cell line MDA-MB231. After a 24 h treatment, an autophagic process was activated, as demonstrated by an increase in monodansylcadaverine-positive cells as well as by the appearance of the autophagic markers Beclin and LC3II. Prolonging the treatment to 48 h, 2a caused cytotoxicity through the activation of caspase-dependent apoptosis.

The ability of several N-phenyl-1H-pyrazole-4-carboxamide derivatives and other pyrazoles opportunely modified at the positions 3, 4 and 5, to reduce the formation of the biofilm in some Staphylococcus aureus strains (ATCC 29213, ATCC 25923 and ATCC 6538) were investigated. All the tested compounds were able, although to a different extent, to reduce the biofilm formation of the three bacterial strains considered. Among these, the 1-(2,5-dichlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carboxamide 14 resulted as the best inhibitor of biofilm formation showing an IC50 ranging from 2.3 to 32 μM, against all the three strains of S. aureus. Compound 14 also shows a good protective effect in vivo by improving the survival of wax moth larva (Galleria mellonella) infected with S. aureus ATCC 29213. These findings indicate that 14d is a potential lead compound for the development of new anti-virulence agents against S. aureus infections.

A natural like O -glycoconjugate polycyclic compound 4 was obtained by a multistep procedure starting from N -(3-methyl-1-(4-nitrophenyl)-1 H -pyrazol-5-yl)acetamide. The glycosyl derivative 4 showed antiproliferative activity against all the tumoral cell lines of the NCI panel in the range 0.47–5.43 μ M. Cytofluorimetric analysis performed on MDA-MB231, a very aggressive breast cancer cell line, which does not express estrogen, progesterone and HER-2/neu receptors, showed that 4 is able to induce prolonged cell cycle arrest at G2/M phase and morphological signs of differentiation. These events are correlated with down-regulation of both cyclin B1 and cdc2, the cyclins involved in G2/M transition, as well as up-regulation of cyclin-dependent kinase (CDK) inhibitor p21 Cip1/Waf1.

In the struggle against the emergence of the antibiotic resistance, new molecules targeting biofilm formation could be useful as adjuvant of conventional antibiotics. This study focused on a new class of 2-phenylhydrazinylidene derivatives as antivirulence agents. The compound 12e showed interesting activities against biofilm formation of all tested Staphylococcus aureus strains with IC50 ranging from 1.7 to 43 µM; compounds 12f and 13a resulted strong inhibitors of S. aureus ATCC 6538 and ATCC 29213 biofilm formation with IC50 of 0.9 and 0.8 µM, respectively. A preliminary study on the mechanism of action was carried on evaluating the inhibition of sortase A transpeptidase. Compound 12e resulted not to be toxic at 1 mg/ml by using an in vivo model (the wax moth larva model, Galleria mellonella).

Based on the encouraging results found for 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 7 previously tested by us, as well as the consideration that heterocycle fused tetrazepinones bearing the 2-chloroethyl substituent show a better cytotoxic profile than temozolomide and mitozolomide against human cancer cell lines which express the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), in this paper we report the multistep synthesis and the biological study of 3-(2-cloroethyl)-5-methyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 10. Like compound 7, it was active on P-glycoprotein expressing cells (MDR) HL60 and on K562 cell line that are resistant to apoptosis induced by different stimuli, showing GI50 values of 14 and 18 µM respectively. As an antiproliferative agent against the above cells compound 10 was about 2.2 times more active than compound 7. Compound 10 was also tested against WiDR cells which are overexpressing the DNA repair protein MGMT, showing a GI50 value of 2.3 µM. Finally, concerning the effect on cell cycle we observed an evident difference between compounds 7 and 10. In fact, compound 7 induces a block of cell cycle in G0-G1, therefore acting as phase-specific drug, in contrast, compound 10 is a not phase-specific agent. Both the compounds are able to increase the apoptotic sub G0-G1 peak of cell cycle.

In this review we report the recent advances in anticancer activity of the family of natural occurring flavonoids, covering the time span of the last five years. The bibliographic data will be grouped, on the basis of biological information, in two great categories: reports in which the extract plants bioactivity is reported and the identification of each flavonoid is present or not, and reports in which the anticancer activity is attributable to purified and identified flavonoids from plants. Wherever possible, the targets and mechanisms of action as well as the structure-activity relationships of the molecules will be reported. Also, in the review it was thoroughly investigated the recent discovery on flavonoids containing the 2-phenyl-4H-chromen-4-one system even if some examples of unusual flavonoids, bearing a non-aromatic B-ring or other ring condensed to the base structure are reported.

Several anthranilamide-based 2-phenylcyclopropane-1-carboxamides 13a-f, 1,1’-biphenyl-4-carboxamides 14a-f and 1,1’-biphenyl-2-carboxamides 17a-f were obtained by a multistep procedure starting from the (1S,2S)-2-phenylcyclopropane-1-carbonyl chloride 11, the 1,1'-biphenyl-4-carbonyl chloride 12 or the 1,1'-biphenyl-2-carbonyl chloride 16 with the appropriate anthranilamide derivative 10a-f. Derivatives 13a-f, 14a-f and 17a-f showed antiproliferative activity against human leukemia K562 cells. Among these derivatives 13b, 14b and 17b exerted a particular cytotoxic effect on tumor cells. Derivative 17b showed a better antitumoral effect on K562 cells than 13b and 14b. Analyses performed to explore 17b mode of action revealed that it induced an arrest in G2/M phase of cell cycle which was consequent to DNA lesions as demonstrated by the increase in phospho-ATM and γH2AX, two known markers of DNA repair response system. The effect of 17b was also related to ROS generation, activation of JNK and induction of caspase-3 dependent apoptosis.

The compound 2-((1,3-dimethyl-1H-pyrazol-5-yl)(methyl)carbamoyl)benzene-diazonium hydrogen sulfate (10) was reacted with copper sulfate and sodium chloride, in the presence of ascorbic acid as reducing agent, to afford a mixture of the chlorinated epimers 4′-chloro-2,2′,5′-trimethyl-2′,4′-dihydrospiro[isoindoline-1,3′-pyrazol]-3-one (18) and (19), the epimers 4′-hydroxy-2,2′,5′-trimethyl-2′,4′-dihydrospiro[isoindoline-1,3′-pyrazol]-3-one (20) and (21), and N-(1,3-dimethyl-1H-pyrazol-5-yl)benzamide (22). Under the foregoing conditions, diazonium salt 10 affords neither the 2-chloro-N-(1,3-dimethyl-1H-pyrazol-5-yl)-N-methylbenzamide (23) nor the tricyclic derivative 24, the classical products of the Sandmeyer and Pschorr reactions, respectively. Finally, by heating 20 at 210 °C the compound 1,3-dimethylisochromeno[4,3-c]pyrazol-5(1H)-one (24) was obtained. The transformation under the above conditions of 2-((4-chloro-3-methyl-1-phenyl- 1H-pyrazol-5-yl)(methyl)carbamoyl)benzendiazonium hydrogen sulphate (11) afforded 4′,4′-dichloro-2,5′-dimethyl-2′-phenyl-2′,4′-dihydrospiro[isoindoline-1,3′-pyrazol]-3-one (29) as the sole reaction product.

A FRET-based random screening assay was used to generate hit compounds as sortase A inhibitors that allowed us to identify ethyl 3-oxo-2-(2-phenylhydrazinylidene)butanoate as an example of a new class of sortase A inhibitors. Other analogues were generated by changing the ethoxycarbonyl function for a carboxy, cyano or amide group, or introducing substituents in the phenyl ring of the ester and acid derivatives. The most active derivative found was 3-oxo-2-(2-(3,4dichlorophenyl)hydrazinylidene)butanoic acid (2b), showing an IC50 value of 50 µM. For a preliminary assessment of their antivirulence properties the new derivatives were tested for their antibiofilm activity. The most active compound resulted 2a, which showed inhibition of about 60% against S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538 and S. epidermidis RP62A at a screening concentration of 100 µM.

New N-methyl and N-ethyl substitutions in the indazole nucleus are reported by reacting 3-(2-aminobenzamido)indazole and the appropriate trimethyl/triethyl orthobenzoate. Single crystal X-ray analysis confirms the N-ethylation position for the 3-(1-ethyl-1H-indazol-3-yl)-2-phenylquinazolin-4(3H)-one derivative 3f. Compounds 11a-d and 3a-d were tested to evaluate their antimicrobial, their antiproliferative activity and their COX inhibitory activities showing scarce or moderately antiproliferative activity and some inhibitory activity against COX-1 and COX-2.

The multistep synthesis of 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H)-one 15 has been carried out. The compound showed antiproliferative and apoptotic effects against K562, K562-R (imatinib mesilate resistant), HL60 and multidrug resistant (MDR) HL60 cell lines. Compound 15 showed a pro-apoptotic activity against HL60 and K562 resistant cell lines markedly higher than etoposide and busulfan, respectively. Flow cytometry studies carried out on K562 cells allowed to establish that 15 induces G0-G1 phase arrest followed by apoptosis.

Several new 2-(2-phenoxyacetamido)benzamides 17a-v, 21 and 22 were synthesized by stirring in pyridine the acid chlorides 16a-e and the appropriate5-R-4-R1-2-aminobenzamide 15a-e and initially evaluated in vitro for antiproliferative activity against the K562 (human chronic myelogenous leukemia) cell line. Some of synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell line panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). The most active compounds caused an arrest of K562 cells in the G0-G1 phase of cell cycle and induction of apoptosis, which was mediated by caspase activation.

In this review we report the recent advances in bioactive system containing pyrazole fused with a five membered heterocycle, covering the time span of the last decade. All of them are represented around the common structure of the pyrazole ring fused with another five membered heterocycle containing the nitrogen, sulfur and oxygen atoms in all their possible combinations. The classification we have used is based in terms of the therapeutic area providing, when possible, some general conclusions on the targets and mechanisms of action as well as the structure-activity relationships of the molecules.

The emergence of antibiotic resistance is currently considered one of the most important global health problem. The continuous onset of multidrug-resistant Gram-positive and Gram-negative bacterial strains limits the clinical efficacy of most of the marketed antibiotics. Therefore, there is an urgent need for new antibiotics. Pyrrolomycins are a class of biologically active compounds that exhibit a broad spectrum of biological activities, including antibacterial, antifungal, anthelmintic, antiproliferative, insecticidal, and acaricidal activities. In this review we focus on the antibacterial activity and antibiofilm activity of pyrrolomycins against Gram-positive and Gram-negative pathogens. Their efficacy, combined in some cases with a low toxicity, confers to these molecules a great potential for the development of new antimicrobial agents to face the antibiotic crisis.

The diazonium salt derived from 4-amino-N,1,3-trimethyl-N-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-1H-pyrazole-5-carboxamide (14) was reacted with a mixture of CuSO4 and NaCl, with ascorbic acid as an initiator to afford the planar derivative 4,6-dihydro-1,4,6,8-tetramethyl-3-phenyldipyrazolo[3,4-b:4′,3′-d]pyridin-5(3H)-one (16) and its unexpected isomer 4,6-dihydro-3,4,6,8-tetramethyl-1-phenyldipyrazolo[4,3-b:4′,3′-d]pyridin-5(1H)-one (17), as well as the epimers (3S,4S)- (or (3S,4R)-) and (3S,4R)- (or (3S,4S)-) 4-chloro-2,4-dihydro-1′,3′,5,5′-tetramethyl-2-phenylspiro[pyrazole-3,4′(1′H)-pyrrolo[3,4-c]pyrazol]-6′(5′H)-one (18a and b, respectively). Epimers 18a and b were converted under basic conditions to 4′-chloro-N,1,3,3′-tetramethyl-1′-phenyl-[4,5′-bi-1H-pyrazole]-5-carboxamide (19). The structures of isomers 16 and 17 determined by single-crystal X-ray analysis are also reported. Linear dichroism (LD) measurements for the above isomers suggest that 17 intercalates into DNA, and 17 exhibited antiproliferation activity against human NCI-H460 pulmonary carcinoma cells.

In order to study the influence of 3-substitution on the cytotoxic activity of 2-styrylquinazolinones, new 6-chloro-2-styryl-3-(heteroaryl)-4(3H)-quinazolinones were synthesized by refluxing equimolar amounts of 6-chloro-2-methyl-3-(heteroaryl)-4(3H)-quinazolinones and benzaldehyde in glacial acetic acid. At 1 microg ml(-1) concentration, almost all 2-styrylquinazolinones showed some cytotoxic activity against the L1210 and K562 leukemia cell lines. However, only 6-chloro-2-styryl-3-(pyrimidin-2yl)-4(3H)-quinazolinone inhibited the growth of these cells by over 50%. This last compound was also the only member of the series that inhibited tubulin polymerization, with an IC(50) value of 5.8 versus 3.2 microM for colchicine. It was also examined for effects on the growth of human MCF7 breast carcinoma cells and Burkitt lymphoma CA46 cells, which had IC(50) values of 0.34 and 1.0 microM, respectively. At 10 microM 6-chloro-2-styryl-3-(pyrimidin-2yl)-4(3H)-quinazolinone induced G2/M arrest (66%) in Burkitt cells, with a mitotic index of 20%. At 3.4 microM, it caused disruption of the cellular microtubule system of the MCF7 cells. Both these cellular effects are consistent with its mechanism of action resulting from its inhibitory effect on tubulin assembly.

The reaction under reflux between 1-phenyl-3-R-5-methylaminopyrazoles and 2,5-hexanedione lead to 5,7:7,13-dimethanopyrazolo[3,4-b]pyrazolo[3′,4′:2,3]azepino[4,5-f]azocine derivatives 3b–g. These unusual molecules show the structural complexity of many biologically active natural products and are endowed with the chemical diversity that is required in drug discovery. The compounds 3b,e were reduced by hydrogen in the presence of Palladium on activated charcoal to give the dihydro derivatives 5b,e. Compounds 3b–f and 5b,e were selected by the NCI to evaluate their in vitro antiproliferative activity against 60 human cell lines derived from nine clinically isolated cancer types (leukaemia, lung, colon, melanoma, renal, ovarian, brain, breast, and prostate). The most active compound of this series, caused a block in G0–G1 phase of cell cycle. Analysis of pRb expression showed that this compound favours pRb dephosphorylation.

Several new benzamides 4a–q were synthesized by stirring in pyridine the acid chlorides 3a–q with the appropriate anthranilamide derivatives 2a–g. Some of the synthesized compounds were evaluated for their in vitro antiproliferative activity against a panel of 5 human cell lines (K562 human chronic myelogenous leukemia cells, MCF-7 breast cancer cells, HTC-116 and HT26 colon cancer cells and NCI H460 non-small cell lung cancer cells).

Several new 4-diazopyrazole derivatives 6a – g and 9a – c were obtained by the reaction of 1-(R-substituted-phenyl)-3-(1,3-dimethyl-1 H -pyrazol-5-yl)ureas 5a – g and N -(1,3-dimethyl-1 H -pyrazol-5-yl)-2-(R-substituted-phenyl)acetamides 8a – c respectively with a sevenfold excess of nitrous acid in acetic acid solution. The compounds were assayed for their activity against the Staphylococcus aureus reference strains ATCC 25923, ATCC 29213 and ATCC 6538, as well as six veterinary strains. The best anti-staphylococcal profile was showed by [(R-substituted-phenyl)acetyl](4-diazonio-1,3-dimethyl-1 H -pyrazol-5-yl)azanides 9a , c . Compound 9c was also able at 3.1 μg mL −1 to inhibit of 45.7% the biofilm formation of the strains S. aureus ATCC 29213.

A series of isochromeno[4,3-c]pyrazole-5(1H)-one derivatives 7b–h were prepared and tested at 10 μM for their ability to displace specific [3H]flunitrazepam from bovine brain membranes. The substitution pattern of the above derivatives was shown to influence the receptor affinity. The most active compound of the series was 7e, showing a 54% inhibition of [3H]flunitrazepam binding. Compounds 7a–d,i were compared with the known isomers chromeno[4,3-c]pyrazole-4(1H)-ones 14a–d,i, showing that the isochromene/chromene isomerism influences the activity.

Several new 2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides 12a–s and 17t–v were synthesized by stirring in pyridine the (E)-3-(2-R1-3-R2-4-R3-phenyl)acrylic acid chlorides 11c–k and 11t–v with the appropriate anthranilamide derivatives 10a–c or the 5-iodoanthranilic acid 13. Some of the synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell line panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). COMPARE analysis, effects on tubulin polymerization in cells and with purified tubulin, and effects on cell cycle distribution for 17t, the most active of the series, indicate that these new antiproliferative compounds act as antitubulin agents.

Pyrazolylbenzotriazinones are endowed with structural analogy with the COX-2 selective inhibitor celecoxib. Considering that our research group has long been interested in the 3-pyrazolyl-substituted benzotriazinones as anti-inflammatory agents, six new pyrazolylbenzotriazinone derivatives 16a-c and 18a-c have been prepared by reacting the opportune ethyl 5-(2-aminobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylate or 5-(2-aminobenzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxyic acid with sodium nitrite in glacial acetic acid. The biological studies revealed a good pharmacological profile for some pyrazolylbenzotriazinones and, in the case of the ethyl 5-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-2-yl-1H-pyrazole-4-carboxylate 16a, a good COX-1/COX-2 selectivity. Molecular modeling studies confirmed the obtained biological results.

A series of new 3-amino-N-phenyl-1H-indazole-1-carboxamides 10 have been prepared from commercially available phenyl isocyanate precursors 8 and 3-aminoindazole 9. Some of the synthesized compounds were evaluated for their in vitro antineoplastic activity against 60 human cell lines derived from seven clinically isolated cancer types (lung, colon, melanoma, renal, ovarian, brain, and leukemia) according to the NCI standard protocol. The test results indicated that 3-amino-1H-indazole-1-carboxamides 10 were endowed with an interesting antiproliferative activity. The most active compounds of this series, 10d,e, were able to inhibit cell growth of many neoplastic cell lines at concentrations lower than 1 μM (0.0153 μM in SR leukemia) causing a block in G0–G1 phase of cell cycle. Analysis of pRb expression showed that these two compounds increased the ratio between underphosphorylated pRb and total pRb. The X-ray structure of 10w, confirmed the 3-amino-N-phenyl-1H-indazole-1-carboxamide structure of compounds 10.

The X-Ray crystal and molecular structures of the 4-pyrazol derivatives 3-methyl-4-diazo-5-benzamido-1H-pyrazole (4) and 3-benzamido-5-methyl-1H-pyrazole (3) have been determined. A dimeric structure has been found for the first and polymeric for the second. A comparison of 4 with 1,3-dimethyl-4-diazo-5-benzamido-lH-pyrazole (2) shows differences in the geometrical parameters of the pyrazole ring due to electron delocalization in 2 consequent to the nitrogen negative charge in the latter. Theoretical investigation at the density functional theory (DFT) level shows difference in the molecular electronic distribution of 2 and 4, in agreement with the structural parameters and the IR stretching frequencies of the respective carbonyl moieties.

Several triazenoindazoles and triazenopyrazoles were prepared transforming the appropriate aminoindazoles and aminopyrazoles in the corresponding diazonium salts which were reacted with dimethylamine, diethylamine and pyrrolidine. All the triazenes were tested for their antiproliferative activity against K562, HL60, L1210 and MCF7 cell lines. The biological data showed that the benzocondensation plays a positive role on the antiproliferative activity. The (1)H-NMR spectra showed that the rotational barrier around the N(2)-N(3) bond in the triazene group can be influenced both by the position of this group in the indazole nucleus and by the substitution pattern in the benzene moiety.

2-(Methyl(3-methyl-1-phenyl-1H-pyrazol-5-yl)carbamoyl)thiophene-3-diazonium hydrogen sulfate 20, 2-(methyl(3-methyl-isoxazol-5yl)carbamoyl)-benzenediazonium hydrogen sulfate 21 and 2-(methyl(phenyl)carbamoyl)-benzenediazonium hydrogen sulphate 22 were synthesized and reacted with a CuSO4/NaCl/ascorbic acid combination to give complex mixtures. The structures of the reaction products were elucidated, depending upon the pathways followed. Compound 20 almost exclusively afforded an Ar-5 cyclization product and trace amounts of the product derived from a competing Ar-6 Pschorr closure. In the case of compound 21, the Ar-6 cyclization was not observed, while the Ar-5 cyclization and 1,5-hydrogen radical transfer processes equally occurred. Finally, the Ar-6 cyclization was observed as the main process for compound 22, accompanied by the Ar-5 cyclization and, in a much lesser extent, by an 1,5-hydrogen radical transfer process.

The diazonium tetrafluoroborate 11 obtained from 2-amino-N-methyl-N-(1-phenyl-3- methylpyrazol-5-yl)benzamide was transformed in dry acetonitrile via an ionic or radical pathway. Differences were observed with respect to ionic or radical transformations in aqueous media of the analogous diazonium hydrogen sulfate 1 derived from the same amine. In acetonitrile solution, the ionic pathway was characterized by an increased yield of 1,4-dimethyl- 3-phenyl-pyrazolo(3,4-c)isoquinolin-5-one 4 and by the formation of its isomer, the new derivative 7,9-dimethyldibenzo(e,g)pyrazolo(1,5-a)(1,3)diazocin-10(9H)-one 12. When the reaction followed a radical pathway, the pyrazolo(3,4-c)isoquinoline derivative 4 and N-methyl- 2-(1-phenyl-3-methylpyrazol-5-yl)benzamide 17, the latter due to a 1,4-pyrazolyl transfer process, were isolated in low yields. Decomposition of the solid diazonium tetrafluoroborate at its melting point gave compounds 4, 12 and the N-(1-phenyl-3-methylpyrazol-5-yl)-2- fluorobenzamide 17. The crystal structure of compound 12 was also determined.

The multistep synthesis of two pyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H)-one derivatives, a new class of fused 1,2,3,5-tetrazepinones with potential antiproliferative activity, has been carried out. Owing to the instability of the above compounds, the last step of the synthesis was performed at -5/0 degrees C. The obtained tetrazepinones, when allowed to stand at r.t. for 24 h, afforded quantitatively 1-phenyl-3,6-dimethylpyrazolo [3,4-d][1,2,3] triazole.