3D-QSAR analysis on benzazole derivatives as eukaryotic topoisomerase II inhibitors by using comparative molecular field analysis method

Selective topoisomerase II inhibitors have created a great deal of interest in recent years for the design of new antitumoral compounds. 3D-QSAR analysis has been performed on a series of previously synthesized benzoxazole, benzimidazole, and oxazolo(4,5-b)pyridine derivatives, which are screened as eukaryotic topoisomerase II inhibitors, using comparative molecular field analysis (CoMFA) with partial least squares fit to predict the steric and electrostatic molecular field interactions for the activity. The CoMFA study was carried out using a training set of 16 compounds. The predictive ability of the model was assessed using a test set of 7 compounds. The analyzed 3D-QSAR CoMFA model has demonstrated a good fit, having r(2) value of 0.997 and cross-validated coefficient q(2) value as 0.435 for the model. The obtained model reveals that the electronegatively charged substituents such as NO(2) or COOCH(3) group on position R and/or R(1) at the heterocyclic ring system and positively charged atom and/or atom groups located between the benzazole moiety and 2-substituted phenyl ring as a bridge element improve the activity. On the other hand, a bulky substituent, such as methoxy group, attached to the ortho position of 2-phenyl-5-nitro-benzoxazole (1) enhances the activity similar to compound 13, which is both a meta and para substituent of the phenyl group attached to the 2-position of benzimidazole ring system, fit into the favored steric region to improve the activity.     

Some fused heterocyclic compounds as eukaryotic topoisomerase II inhibitors

Our previously synthesized 37 compounds, which are 2,5,6-substituted benzoxazole, benzimidazole, benzothiazole, and oxazolo(4,5-b)pyridine derivatives, were tested for their eukaryotic DNA topoisomerase II inhibitory activity in cell free system and 28 were found to inhibit the topoisomerase II at an initial concentration of 100 microg/ml. After further testing at a lower range of concentrations, 12 derivatives, which were considered as positive topoisomerase inhibitors, exhibited IC50 values between 11.4 and 46.8 microM. Etoposide was used as the standard reference drug to compare the inhibitor activity. Among these compounds, 2-phenoxymethylbenzothiazole (3f), 6-nitro-2-(2-methoxyphenyl)benzoxazole (1a), 5-methylcarboxylate-2-phenylthiomethylbenzimidazole (3c), and 6-methyl-2-(2-nitrophenyl)benzoxazole (1c) were found to be more active than the reference drug etoposide. Present results point out that, besides the very well-known bi- and ter-benzimidazoles, compounds with single bicycle fused ring systems in their structure such as benzimidazole, benzoxazole, benzothiazole, and/or oxazolopyridine derivatives also exhibit significant topoisomerase II inhibitory activity.     

Mutagenic activity and heterocyclic amine carcinogens in commercial pet foods

Twenty-five commercial pet foods were analyzed for mutagenic activity using the Ames/Salmonella test with strain TA98 and added metabolic activation. All but one gave a positive mutagenic response. Fourteen of these samples were analyzed for heterocyclic amine mutagens/carcinogens and all but one contained 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 10 of 14 contained 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as analyzed by HPLC and confirmed by photodiode array peak matching. From these findings it is hypothesized that there is a connection between dietary heterocyclic amines and cancer in animals consuming these foods.     

2,4-Diamino-9H-pyrimido[4,5-b]indol-5-ols: synthesis, in vitro cytotoxic activity, and QSAR investigations

A series of novel 2,4-diaminopyrimido[4,5-b]indol-6-ols has been synthesized and the in vitro cytotoxic activities were evaluated against four human cancer cell lines originating from solid tumors. An increase in activity was observed when a heteroaromatic ring was annulated on side g of the pyrimido[4,5-b]indole system to give compounds with activities comparable to ellipticine and cisplatin. To understand the experimental cytotoxic activities, QSAR investigations were performed, which showed a very good linearity between the experimental and predicted IC(50).     

Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.     

Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Resveratrol is a phytoalexin, that belongs to a family of naturally occurring stilbenes. It has been reported that resveratrol can inhibit chemical carcinogenesis in experimental animals and although the mechanisms involved are unknown, an anti-mutagen mechanism has been proposed. We have explored this hypothesis using mutagenicity assays based on bacterial (Salmonella typhimurium) and eukaryotic cells (Chinese hamster V79 cells). We found resveratrol to be potent in both systems, blocking the mutagenicity of the food-derived heterocyclic amines (HA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at micromolar concentrations. Furthermore, in cells capable of activating 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine to cytotoxic derivatives, resveratrol was able to attenuate cytotoxicity. Paradoxically, in cells lacking the ability to activate PhIP, resveratrol itself was toxic and co-incubation with PhIP reduced this toxicity. Our data confirm the potent anti-mutagenic activity of resveratrol and support its potential as a chemopreventative.     

Antibacterial profile against drug-resistant Staphylococcus epidermidis clinical strain and structure-activity relationship studies of 1H-pyrazolo[3,4-b]pyridine and thieno[2,3-b]pyridine derivatives

Antibacterial resistance is a complex problem that contributes to health and economic losses worldwide. The Staphylococcus epidermidis is an important nosocomial pathogen that affects immunocompromised patients or those with indwelling devices. Currently, there are several resistant strains including S. epidermidis that became an important medical issue mainly in hospital environment. In this work, we report the biological and theoretical evaluations of a 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acids series (1, 1a-m) and the comparison with a new isosteric ring nucleus series, 4-(arylamino)thieno[2,3-b]pyridine-5-carboxylic acids derivatives (2, 2a-m). Our results revealed the 1H-pyrazolo[3,4-b]pyridine derivatives significant antibacterial activity against a drug-resistant S. epidermidis clinical strain in contrast to the thieno[2,3-b]pyridine series. The minimal inhibitory concentration (MIC) of the most active derivatives (1a, 1c, 1e, and 1f) against S. epidermidis was similar to that of oxacillin and twofold better than chloramphenicol. Interestingly, the position of the functional groups has a great impact on the activity as observed in our structure-activity relationship (SAR) study. The SAR of 1H-pyrazolo[3,4-b]pyridine derivatives shows that the highest inhibitory activity is observed when the meta position is occupied by electronegative substituents. The molecular modeling analysis of frontier molecular orbitals revealed that the LUMO density is less intense in meta than in ortho and para positions for both series (1 and 2), whereas HOMO density is overconcentrated in 1H-pyrazolo[3,4-b]pyridine ring nucleus compared to the thieno[2,3-b]pyridine system. The most active derivatives of series 1 were submitted to in silico ADMET screening, which confirmed these compounds as potential antibacterial candidates.     

Binding activity of natto (a fermented food) and Bacillus natto isolates to mutagenic-carcinogenic heterocyclic amines.

The fermented food, whole meal Natto, viscous polymeric material from Natto, Natto bean, cooked soya bean, and 28 bacterial isolates from Natto were studied for their binding capacity to foodborne mutagenic-carcinogenic heterocyclic amines. The mutagenic heterocyclic amines used were Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole); Trp-P-2 (3-amino-1-methyl-5H-pyrido(4,3-b)indole); Glu-P-1 (2-amino-6-methyldipyrido(1,2-a:3'2'-d)imidazole); PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine); IQ (2-amino-3-methylimidazo(4,5-f)quinoline); MeIQ (2-amino-3,4-dimethylimidazo(4,5-f)quinoxaline); MeIQx (2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline); and MeAalphaC (2-amino-3-methyl-9H-pyrido(2,3)indole). The lyophilized Natto and other fractions of Natto exhibited high binding activity towards Trp-P-1, Trp-P-2, PhIP, and MeAalphaC, while Glu-P-1, IQ, and MeIQ were not effectively bound. The binding capacity of bacterial isolates (Bacillus natto) were isolate-mutagen dependent. Heat treated lyophilized cells, cell wall, and cytoplasmic contents of the bacterial isolate with the highest binding capacity were analyzed for their ability to bind different heterocyclic amines. The results indicate the importance of the cell wall in binding to heterocyclic amines, whereas the cytoplasmic contents were less effective. Heat-treated cells were not much different from that of viable cells in their binding. The impact of different factors, such as pH, incubation time, metal ions, different concentrations of sodium chloride and alcohol, various enzymes, and acetylation of mutagens on binding of Trp-P-1 and IQ, were discussed. The significance of the present results is also discussed from the viewpoint that Natto, a fermented food, is able to scavenge dietary mutagenic heterocyclic amines through binding.     

Effects of seasoning and heating device on mutagenicity and heterocyclic amines in cooked beef.

Pan-roasted beef showed a lower mutagenicity after various degrees of cooking than charcoaled one. The high mutagenicity of charcoaled beef was due to the formation of more heterocyclic amines, especially AalphaC (2-amino-9H-pyrido- [2,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) because of rapid and direct heating on the surface of the meat at a high temperature. Seasoning decreased mutagenicity of pan-roasted beef except the very well done sample with unchanged heterocyclic amine contents, but increased mutagenicity of charcoaled beef with decreased levels of AalphaC and PhIP, probably due to the change of heterocyclic amine precursors or alternatively to the occurrence of other mutagens.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.     

Effects of Seasoning and Heating Device on Mutagenicity and Heterocyclic Amines in Cooked Beef

Pan-roasted beef showed a lower mutagenicity after various degrees of cooking than charcoaled one. The high mutagenicity of charcoaled beef was due to the formation of more heterocyclic amines, especially AαC (2-amino-9 H-pyrido- [2,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) because of rapid and direct heating on the surface of the meat at a high temperature. Seasoning decreased mutagenicity of pan-roasted beef except the very well done sample with unchanged heterocyclic amine contents, but increased mutagenicity of charcoaled beef with decreased levels of AαC and PhIP, probably due to the change of heterocyclic amine precursors or alternatively to the occurrence of other mutagens.     

Inhibition of the metabolism of mutagens occurring in food by arachidonic acid.

Hepatic microsomal fractions (microsomes) were prepared from male Sprague-Dawley rats. The effect of arachidonic acid on the conversion of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay (indicator: Salmonella typhimurium TA98). Arachidonic acid inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA-repair processes within the bacterial cell. The activation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and aflatoxin B1 (AFB1) was also inhibited by this polyunsaturated fatty acid.     

Mutagenic and carcinogenic heterocyclic amines as affected by muscle types/skin and cooking in pan-roasted mackerel

The dependence on muscle types/skin and on degrees of cooking in the formation of heterocyclic amines (HCAs) of pan-roasted mackerel was studied. High levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were found in very well done skin and ordinary muscle, being 4.2 and 5.3 ng/g, followed by 2-amino-3,4-dimethylimidazo[4,5-f] quinoxaline (MeIQx), being 1.8 and 2.1 ng/g and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), being 1.2 and 2.8 ng/g, respectively. In pan-roasted mackerel, ordinary muscles contributed much more greatly to the formation of HCAs than skins due to its higher HCA contents and composition (76.1%).     

Effects of industrially produced flavours with pro- and antioxidative properties on the formation of the heterocyclic amine PhIP in a model system

PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is a heterocyclic aromatic amine belonging to a class of mutagens found in food. This project studied the effects of commercially available flavours of spices on the formation of PhIP, one of the most common heterocyclic aromatic amines in heated meat and fish products. The model reactions were carried out in diethylene glycol. Highest amounts of PhIP were obtained at 200 degrees C, a heating time of 60 min and an equivalent molar ratio of phenylalanine and creatinine. With this model system, the influence of Monascus red and flavours extracted from thyme, marjoram and rosemary on the formation of PhIP was tested. The flavours were added to the model system in different amounts. The oxidative properties were determined with the rancimat method. It was shown that all tested products, independent of their pro- or antioxidative properties, increased PhIP in the model system.     

Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding its antimutagenic properties against cooked food mutagens. Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent.     

8-Aza Analogues of Deaza Purine Nucleosides. Synthesis and Biological Evaluation of 8-Aza-1-deazaadenosine and 2′-Deoxy-8-aza-1-deazaadenosine

Abstract

The syntheses of 7-amino-3-(β-D-ribofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (8-aza-1-deazaadenosine) (2) and 7-amino-3-(2-deoxy-β-D-erythro-pentofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (2′-deoxy-8-aza-1-deazaadenosine) (3) by glycosylation of the anion of 7-chloro-3H-1,2,3-triazolo[4,5-b]pyridine are described. The anomeric configuration as well as the position of glycosylation were determined by ¹H, ¹³ NMR, UV and N.O.E. difference spectroscopy. The cytotoxicity of these nucleosides against several murine and human tumor cell lines is discussed. Compounds 2 and 3 proved to be good inhibitors of adenosine deaminase.     

Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane--a constituent of broccoli

Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines). These include imidazoazaarenes such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9. Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.     

Anti-genotoxic activity of Vitis coignetiae Pulliat towards heterocyclic amines and isolation and identification of caftaric acid as an antimutagenic component from the juice

Our study demonstrated that the formation of DNA adducts in liver, lungs, colon and kidneys of mice given a carcinogenic heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in the diet significantly decreased following the administration of the juice of Vitis coignetiae, purple berries from a vine tree. The juice of V. coignetiae significantly inhibited the clastogenicity and mutagenicity of heterocyclic amines in the micronucleus assay and the Ames test, and was an effective inhibitor of the activities of phase I enzymes (cytochrome P450 1A1 and cytochrome P450 1A2) and enhancer of the activities of phase II enzymes (uridine 5'-diphospho-glucuronosyltransferase and glutathione S-transferase). We investigated the purification and isolation of an active compound in the juice of V. coignetiae using antimutagenicity as a separation marker. Caftaric acid, a polyphenolic compound, was identified as a component responsible for antimutagenicity in the juice of V. coignetiae towards the carcinogenic heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). This is the first report of antimutagenicity of caftaric acid. Caftaric acid was reported as an inhibitor of the protein-protein interactions mediated by the Src-family kinases. The impact of the juice of V. coignetiae and its constituents on tumor initiation and promotion thus warrants further study.     

The formation of heart DNA adducts in F344 rat following dietary administration of heterocyclic amines

Most heterocyclic amines formed during the cooking of meat and fish have been shown to form adducts in the livers of rats. Recently, however, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), administered in the diet to Fischer 344 (F344) rats for 4 weeks, was shown to produce the highest levels of adducts in the heart. In the present study 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a:1',2'-d]imidazole (Glu-P-1) were given to F344 rats at carcinogenic dose levels (IQ 0.03%, MeIQx 0.04%, Trp-P-1 0.015%, Glu-P-1 0.05%) in the diet for 4 weeks. DNA adducts in the liver and heart were analyzed by 32P-postlabeling. DNA adducts were demonstrated to appear in the hearts of all animals exposed to heterocyclic amines at the following levels: IQ, 1.8 adducts/10(7) nucleotides, MeIQx, 3.8/10(7) ntd, Trp-P-1, 20/10(7) ntd and Glu-P-1, 7.2/10(7) ntd. Values for the heart were 10-20% of the respective liver adduct levels. Heart adducts increased linearly throughout the observed period when MeIQx was administered for up to 40 weeks. When MeIQx feeding was discontinued after 20 weeks and the animals subsequently given the basal diet, the adduct level at 20 weeks did not change during the following 20 weeks. A possible role for heart DNA alterations caused by food-borne heterocyclic amines in the development of age-related myopathies and cardiovascular disease is not inconceivable.