From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of nitroaromatics in Salmonella typhimurium. Part II. Substituents far away from the nitro group

Derivatives of 4-nitrobiphenyl, 4-nitrosobiphenyl, 2-phenyl-5-nitropyridine (2-aza-4-nitrobiphenyl) and 2-nitrofluorene, bearing various alkyl substituents far away from the nitro group (4'-position in nitrobiphenyls, 7-position in 2-nitrofluorenes) were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. In the absence of S9 in both strains, mutagenicity of all4'-Ad (Ad=adamantyl). Changes of the molecular shape from 'planar' to non-planar caused by the bulk of the introduced substituents (without influencing the twisting of the nitro substituent or the phenyl rings in the biphenyl compounds) may be responsible for this effect by interfering with an efficient intercalation into DNA.A comparison between experimental and theoretical values as calculated from recently developed equations (QSAR) confirmed our previous results (see the preceding paper) that mutagenicity of alkyl-substituted nitroaromatics cannot be predicted by hydrophobicity and LUMO-energies alone without including steric parameters.     

Synthesis and in vitro antitrypanosomal activity of novel Nifurtimox analogues

Eight novel analogues of Nifurtimox, 4-[(5-nitrofurfurylidene)amino]-3-methylthiomorpholine-1,1-dioxide, containing alpha-beta unsaturated amides, were synthesized and evaluated for their in vitro activity against Trypanosoma cruzi epimastigotes. Four derivatives bearing a nitro group at the 5-position of the furan ring were the most active in inhibiting culture growth and provoking cell death, showing trypanocidal activity more than threefold the potency of Nifurtimox, our positive control. Two derivatives lacking a nitro group were less potent than the positive control. Active nitro derivatives very efficiently caused epimastigote death, which suggests a nitro reduction mechanism of action.     

The effects of 4'-alkyl substituents on the mutagenic activity of 4-amino- and 4-nitrostilbenes in Salmonella typhimurium

Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4'-position and six of the corresponding nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and TA100. Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl. More bulky substituents reduced the mutagenic potential in the order iso-propylethyl>iso-propyl>sec-butyl>tert-butyl). These trends have been compared with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes. The unexpected result for the tert-butyl nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway.     

2-Nitro analogues of adenosine and 1-deazaadenosine: synthesis and binding studies at the adenosine A1, A2A and A3 receptor subtypes

The influence of nitro substituents on the properties of adenosine and 1-deazaadenosine was studied. Combination of a nitro group at the 2-position with several N6 substituents such as cyclopentyl and m-iodobenzyl gave a series of analogues with good adenosine receptor affinity, showing directable selectivity for the A1, A2A and A3 adenosine receptor subtypes.     

Synthesis, biological evaluation, and molecular modeling of 3,5-substituted-N1-phenyl-N4,N4-di-n-butylsulfanilamides as antikinetoplastid antimicrotubule agents

Dinitroanilines are of interest as antiprotozoal lead compounds because of their selective activity against the tubulin of these organisms, but concern has been raised due to the potentially mutagenic nitro groups. Analogues of N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (GB-II-150, compound 2b), a selective antimitotic agent against African trypanosomes and Leishmania, have been prepared where the nitro groups are replaced with amino, chloro, cyano, carboxylate, methyl ester, amide, and methyl ketone moieties. Dicyano compound 5 displays IC(50) values that are comparable to 2b against purified leishmanial tubulin assembly (6.6 vs 7.4 microM), Trypanosoma brucei brucei growth in vitro (0.26 vs 0.18 microM), Leishmania donovani axenic amastigote growth in vitro (4.4 vs 2.3 microM), and in vitro toxicity against Vero cells (16 vs 9.7 microM). Computational studies provide a rationale for the antiparasitic order of activity of these analogues and further insight into the role of the substituents at the 3 and 5 positions of the sulfanilamide ring.     

Design and synthesis of methyl 2-methyl-7,7-dihalo-5-phenyl-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates with calcium channel antagonist activity

A group of methyl 2-methyl-7,7-dihalo-5-(substituted-phenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates were prepared by reaction of dihalocarbenes (:CX2, X = Br, Cl) with methyl 1-methyl-4-(substituted-phenyl)-1,4-dihydropyridine-3-carboxylates. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. The title compounds exhibited weaker CC antagonist activity (10(-5)-10(-6)M range) than the reference drug nifedipine (1.4 x 10(-8)M). Structure-activity relationship studies showed that the position of a nitro substituent on the C-5 phenyl ring where the relative potency order was ortho > meta > para, and the size and/or electronegativity of the C-7 geminal-dihalo substituents (Br > Cl), were determinants of calcium channel antagonist activity. This class of compounds did not exhibit any inotropic effect on guinea pig left atria. A dihalocyclopropyl moiety is a potential bioisostere for the 2-methyl-3-methoxycarbonylvinyl moiety present in the calcium channel antagonist nifedipine.     

Novel and potent oxazolidinone antibacterials featuring 3-indolylglyoxamide substituents

Novel oxazolidinone antibacterials bearing a variety of 3-indolylglyoxamide substituents have been explored in an effort to improve the spectrum and potency of this class of agents. A subclass of this series was also made with the diversity at C-5 terminus. These derivatives have been screened against a panel of clinically relevant Gram-positive pathogens and fastidious Gram-negative organisms. Several analogs in this series were identified with in vitro activity superior to linezolid (MIC=0.25-2 microg/mL). Compounds 10a, 10c, 10e and 10f displayed activity against linezolid resistant Gram-positive organisms (MIC=2-4 microg/mL). Selected oxazolidinones were evaluated for in vivo efficacy against a mouse systemic infection model.     

New N-(phenoxydecyl)phthalimide derivatives displaying potent inhibition activity towards α-glucosidase

Several members of a new family of non-sugar-type α-glucosidase inhibitors, bearing a phthalimide moiety connected to a variously substituted phenoxy ring by an alkyl chain, were synthesized and their activities were investigated. The efficacy of the inhibition activity appeared to be governed by the chain length of the substrate. Substrates possessing 10 carbons afforded the highest levels of activity, which were one to two orders of magnitude more potent than the known inhibitor 1-deoxynojirimycin (dNM). Furthermore, structure–activity relationship studies indicated a critical role of electron-withdrawing substituents at the phenoxy group for the activity. Derivatives bearing a chlorine atom along with a strong electron-withdrawing group, such as a nitro group, were the most potent of the series.     

Investigating the promiscuity of the chloramphenicol nitroreductase from Haemophilus influenzae towards the reduction of 4-nitrobenzene derivatives

Chloramphenicol nitroreductase (CNR), a drug-modifying enzyme from Haemophilus influenzae, has been shown to be responsible for the conversion of the nitro group into an amine in the antibiotic chloramphenicol (CAM). Since CAM structurally bears a 4-nitrobenzene moiety, we explored the substrate promiscuity of CNR by investigating its nitroreduction of 4-nitrobenzyl derivatives. We tested twenty compounds containing a nitrobenzene core, two nitropyridines, one compound with a vinylogous nitro group, and two aliphatic nitro compounds. In addition, we also synthesized twenty-eight 4-nitrobenzyl derivatives with ether, ester, and thioether substituents and assessed the relative activity of CNR in their presence. We found several of these compounds to be modified by CNR, with the enzyme activity ranging from 1 to 150% when compared to CAM. This data provides insights into two areas: (i) chemoenzymatic reduction of select compounds to avoid harsh chemicals and heavy metals routinely used in reductions of nitro groups and (ii) functional groups that would aid CAM in overcoming the activity of this enzyme.     

Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds.

A variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT), were reduced by hydrogen in the presence of enzyme preparations from Veillonella alkalescens. Consistent with the proposed reduction pathway, R-NO2 H2 leads to R-NO H2 leads to R-NHOH H2 leads to R-NH2, 3 mol of H2 was utilized per mol of nitro group. The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V. alkalescens extract were determined. The reactivity of the nitro groups depended on other substituents and on the position of the nitro groups relative to these substituents. In the case of the nitrotoluenes, the para-nitro group was the most readily reduced, the 4-nitro position of 2,4-dinitrotulene being reduced first. The pattern of reduction of TNT (disappearance of TNT and reduction products formed) depended on the type of preparation (cell-free extract, resting cells, or growing culture), on the species, and on the atmosphere (air or H2). The "nitro-reductase" activity of V. alkalescens extracts was associated with protein fractions, one having some ferredoxin-like properties and the other possessing hydrogenase activity. Efforts to eliminate hydrogenase from the reaction have thus far been unsuccessful. The question of whether ferredoxin acts as a nonspecific reductase for nitroaromatic compounds remains unresolved.     

Nitroquinolones with broad-spectrum antimycobacterial activity in vitro.

During search on quinolonecarboxylic acids we used a facile, convenient two- or three-step procedure to synthesize new quinolone analogs, bearing at the C-7 position alkylamino substituents, and at the C-6 position a fluorine or alternatively a nitro group. The new derivatives were tested against both Gram-positive and Gram-negative bacteria and against a number of different mycobacteria. In vitro assays showed 1-tert-butyl-7-tert-butylamino-6-nitro-1,4-dihydro-4-quinolone-3-carboxy lic acid to be a potent inhibitor of Streptococcus and Staphylococcus with potencies superior to those of ofloxacin and ciprofloxacin, used as reference drugs. Some 6-nitroquinolones were found to exert good inhibiting activities against Mycobacterium tuberculosis and various atypical mycobacteria, whereas the 6-fluoro counterparts showed poor or no activity against this bacterium.     

A novel opioid structure which accepts protonated as well as non-protonated nitrogen: a family of pure, delta receptor selective antagonists.

Conventional opioids including opioid peptides require an "opioid" nitrogen which exists in protonated state while interacting with the receptor. In the present paper we demonstrate that the Tyr-Pro-Gly-Phe-Leu-Thr hexapeptide sequence accepts N-terminal substituents such as N-t-Boc, N-phenylacetyl and N-diphenylacetyl where the N cannot become protonated, as well as "traditional" substitutions such as N,N-diallyl, where protonation is likely under physiological conditions. The opioid peptides bearing these substituents are pure antagonists of medium affinity (Ke values in the mouse vas deferens bioassay against [Met5]-enkephalin are in the 3 x 10(-7)-4 x 10(-6) M range) with a high delta receptor preference (50-350-fold delta over mu selectivity ratios).     

QSAR of adenosine A3 receptor antagonist 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives using chemometric tools

Considering the potential of selective adenosine A3 receptor subtype ligands in the development of prospective therapeutic agents, an attempt has been made to explore physicochemical requirements of 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives for A3 receptor binding. In this study, lipophilicity (logP), physicochemical substituent constants (pi, MR, sigma p) of phenyl ring substituents, and Wang-Ford charges of common atoms of the quinoxaline nucleus (calculated from molecular electrostatic potential surface of energy-minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best multiple linear regression (MLR) equation obtained from factor analysis (FA-MLR) as the preprocessing step could explain and predict 72.6% and 65.3%, respectively, of the variance of the binding affinity. The same equation also emerged as the best equation in the population of 100 equations obtained from genetic function approximation (GFA-MLR). The results suggested that presence of an electron-withdrawing group at the para position of the phenyl ring would be favorable for the binding affinity. Again, the presence of a nitro group at position R1 increases the binding affinity. When factor scores were used as predictor variables in the principal component regression analysis, the resultant model showed 78.6% explained variance and 63.1% predicted variance. The best equation derived from G/PLS could explain and predict 74.4% and 64.8%, respectively. The results have suggested the importance of Wang-Ford charges of atoms C15 and C19, apart from positive contributions of electron-withdrawing para substituents of the variance of the phenyl ring and nitro group at the R1 position.     

Synthesis, structure-activity relationship and in vitro biological evaluation of N-arylethyl isoquinoline derivatives as Coxsackievirus B3 inhibitors

Currently, there is no approved antiviral drug for the infection caused by enteroviruses. A series of novel N-arylethyl isoquinoline derivatives defined with substituents on the ring A and C were designed, synthesized and evaluated in vitro for their activities against Coxsackievirus B3 (CVB3). The primary structure-activity relationship revealed that substituents on the ring A were not beneficial for the activity. Among these analogs synthesized, compound 7f bearing a methylenedioxy at the R(4) and R(5) positions afforded an anti-CVB3 activity and a reasonable selectivity index (SI=26.8); furthermore, 7f exhibited a moderate activity against enterovirus 71 (EV71) with SI value of 9.0. Thus it has been selected as an anti-enteroviral lead compound for further investigation.     

NAD(P)H:flavin mononucleotide oxidoreductase inactivation during 2,4,6-trinitrotoluene reduction

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, K(N), of 394 microM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.     

Structural design,synthesis and substituent effect of hydrazone-N-acylhydrazones reveal potent immunomodulatory agents

4-(Nitrophenyl)hydrazone derivatives of N-acylhydrazone were synthesized and screened for suppress lymphocyte proliferation and nitrite inhibition in macrophages. Compared to an unsubstituted N-acylhydrazone, active compounds were identified within initial series when hydroxyl, chloride and nitro substituents were employed. Structure-activity relationship was further developed by varying the position of these substituents as well as attaching structurally-related substituents. Changing substituent position revealed a more promising compound series of anti-inflammatory agents. In contrast, an N-methyl group appended to the 4-(nitrophenyl)hydrazone moiety reduced activity. Anti-inflammatory activity of compounds is achieved by modulating IL-1β secretion and prostaglandin E2 synthesis in macrophages and by inhibiting calcineurin phosphatase activity in lymphocytes. Compound SintMed65 was advanced into an acute model of peritonitis in mice, where it inhibited the neutrophil infiltration after being orally administered. In summary, we demonstrated in great details the structural requirements and the underlying mechanism for anti-inflammatory activity of a new family of hydrazone-N-acylhydrazone, which may represent a valuable medicinal chemistry direction for the anti-inflammatory drug development in general.     

Inhibition of nucleoside transport by new analogues of nitrobenzylthioinosine

Nitrobenzylthioinosine (NBTI, 1) was systematically modified by attachment of substituents at positions C6 and N9, and also by substitution of N1 with C. These modifications were chosen to reduce the polarity of the new compounds. Incorporation of the nitro functionality into a benzoxadiazole ring system was considered first. These new nucleosides showed high affinity (1.5-10nM) towards the nucleoside transport protein as present on human erythrocyte ghosts. Next, modification of this benzoxadiazole ring system with C, S and O in different positions produced a number of less polar nucleosides with affinity in the higher nanomolar range. Modification of N9 was achieved with different alkyl and alcohol substituents. An n-butyl substituent proved best, although all variations yielded substantial decreases in affinity. Replacement of N1 by a carbon atom in combination with a 2-Cl substituent also resulted in a relatively potent NBTI derivative (47 nM).     

Synthesis and biological evaluation of 1,3-diphenylprop-2-en-1-ones possessing a methanesulfonamido or an azido pharmacophore as cyclooxygenase-1/-2 inhibitors

A group of (E)-1,3-diphenylprop-2-en-1-one derivatives (chalcones) possessing a MeSO(2)NH, or N(3), COX-2 pharmacophore at the para-position of the C-1 phenyl ring were synthesized using a facile stereoselective Claisen-Schmidt condensation reaction. In vitro COX-1/COX-2 structure-activity relationships were determined by varying the substituents on the C-3 phenyl ring (4-H, 4-Me, 4-F, and 4-OMe). Among the 1,3-diphenylprop-2-en-1-ones possessing a C-1 para-MeSO(2)NH COX-2 pharmacophore, (E)-1-(4-methanesulfonamidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7b) was identified as a selective COX-2 inhibitor (COX-2 IC(50)=1.0 microM; selectivity index >100) that was less potent than the reference drug rofecoxib (COX-2 IC(50)=0.50 microM; SI>200). The corresponding 1,3-diphenylprop-2-en-1-one analogue possessing a C-1 para-N(3) COX-2 pharmacophore, (E)-1-(4-azidophenyl)-3-(4-methylphenyl)prop-2-en-1-one (7f), exhibited potent and selective COX-2 inhibition (COX-1 IC(50)=22.2 microM; COX-2 IC(50)=0.3 microM; SI=60). A molecular modeling study where 7b and 7f were docked in the binding site of COX-2 showed that the p-MeSO(2)NH and N(3) substituents on the C-1 phenyl ring are oriented in the vicinity of the COX-2 secondary pocket (His90, Arg513, Phe518, and Val523). The structure-activity data acquired indicate that the propenone moiety constitutes a suitable scaffold to design new acyclic 1,3-diphenylprop-2-en-1-ones with selective COX-1 or COX-2 inhibitory activity.     

Radioprotective effectiveness and toxicity of 4,5-disubstituted tryptamines

In experiments on mice a study was made of different substituents in the 4th position of the indole ring of 5-methoxytryptamines (5-MOT) on toxicity and radioprotective efficiency of the compounds of this class. It was shown that the administration of the amino-group to a mexamine molecule increased the preparation toxicity; the nitro-group somewhat diminished the toxic properties, and the acetylamino group did not change 5-MOT toxicity. A 5-MOT derivative with a nitro group possessed the strongest radioprotective action. The radioprotective efficiency of these compounds persisted for 1-2 h.