Mutagenicity of some derivatives of dipyrido[1,2-a:3',2'-d]imidazoles in Salmonella typhimurium with metabolic activation by rat liver and small intestine subcellular fractions

The mutagenic effect of 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) was compared with that of the 3-amino, 3-nitro, or 3-N-hydroxylated derivatives of the same base ring with methyl groups at positions 4 and 6 of the molecule. The compounds were tested in Salmonella typhimurium strain TA98 without metabolic activation and in the presence of different concentrations of subcellular fractions from livers or small intestines of rats pretreated with different P448/P450 inducers. The 4,6-dimethyl compounds are always more mutagenic than Glu-P-2. Pretreatment with Aroclor 1254 (ARO) is the most effective inducer in the activation of the 2- and 3-amino compounds by liver S9, whereas the same fraction decreases the mutagenicity of the 3-nitro derivative. S9 from small intestine increased the mutagenic effect of the 3-nitro and 3-N-hydroxylated compounds, but it was unable to activate the amino compounds.     

Synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline and 5-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine derivatives,as potential bacterial multidrug resistance pump inhibitors

The synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline derivatives 1a-l is described in five or six steps starting from various substituted nitroanilines 2a-e. The bioisostere 5-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine 1m was also prepared. The new derivatives were evaluated as efflux pump inhibitors (EPIs) in a model targeting the NorA system of Staphylococcus aureus. The antibiotic susceptibility of two strains overproducing NorA, SA-1199B and SA-1, was determined alone and in combination with the neo-synthesised compounds by the agar diffusion method and MIC determination, in comparison with reserpine and omeprazole taken as reference EPIs. A preliminary structure-activity relationship study firstly allowed to clarify the influence of the substituents at positions 7 and/or 8 of the pyrrolo[1,2-a]quinoxaline nucleus. Methoxy substituted compounds, 1b and 1g, were more potent EPIs than the unsubstituted compounds (1a and 1f), followed by chlorinated derivatives (1c-d and 1h). Moreover, the replacement of the N,N-diethylamino group (compounds 1a-e) by a bioisostere such as pyrrolidine (compounds 1f-h) enhanced the EPI activity, in contrast with the replacement by a piperidine moiety (compounds 1i-k). Finally, the pyrrolo[1,2-a]thieno[3,2-e]pyrazine compound 1m exhibited a higher EPI activity than its pyrrolo[1,2-a]quinoxaline analogue 1a, opening the way to further pharmacomodulation.     

Four metal-organic networks based on benzene-1,4-dioxyacetic acid and dipyrido[3,2-a:2′,3′-c]phenazine ligand

Four structurally diverse complexes, [Cd(dppz)(bdoa)]_n (1), [Zn(dppz)(bdoa)(H₂O)]_n (2), [Fe(dppz)₂(bdoa)]_n·2nH₂O (3), and [Co₂(dppz)₂(bdoa)₂(H₂O)]_n·3nH₂O (4), where H₂bdoa = benzene-1,4-dioxyacetic acid and dppz = dipyrido[3,2-a:2′,3′-c]phenazine, have been hydrothermally synthesized. Compounds 1-4 feature chain structures. There exist π-π interactions in the structures of 1, 2 and 4. Two neighboring chains of 1 are linked through the π-π interactions into a double chain supramolecular structure. The chains of 2 and 4 are further extended by the π-π interactions to form 3D and 2D supramolecular structures, respectively. The structural differences among such complexes show that the transition metals have important influences on their structures. The photoluminescent property of complex 2 and the magnetic property of complex 4 have also been investigated.     

Metabolic activation of glutamic acid pyrolysis products, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole, by purified cytochrome P-450

Metabolic activation by cytochrome P-450 of glutamic acid pyrolysis products, 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1) and 2-amino-dipyrido(1,2,-a:3',2'-d)imidazole (Glu-P-2), to mutagenic metabolites was studied using Salmonella typhimurium TA98 as a tester strain. Cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH were essential requirements for the activation of these compounds. Of the four forms of cytochrome P-450 examined, polychlorinated biphenyls (PCB) P-448 and 3-methylcholanthrene (MC) P-448 purified from liver microsomes of rats treated with a PCB mixture and MC, respectively, showed high activity in the activation of both Glu-P-1 and Glu-P-2. The presence of three metabolites from Glu-P-1 or Glu-P-2 was demonstrated by high performance liquid chromatographic (HPLC) analysis. Among the metabolites of Glu-P-1, two metabolites were mutagenic without any further enzymatic activation. In accordance with the results of a mutation assay, PCB P-448 also exhibited higher activity to form the major mutagenic metabolite of Glu-P-1. The major active metabolite of Glu-P-1 was characterized as N-hydroxy-Glu-P-1 by chemical analysis using oxidizing and reducing reagents and by mass spectrometry.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

Synthesis and biological activity of pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one derivatives: in silico approach

Xanthine oxidase (XO) is responsible for the pathological condition called gout. Inhibition of XO activity by various pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidine-4-one derivatives was assessed and compared with the standard inhibitor allopurinol. Out of 10 synthesized compounds, two compounds, viz. 3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one (3b) and 3-amino-6-(4-chloro-2-hydroxy-5-methylphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one (3g) were found to have promising XO inhibitory activity of the same order as allopurinol. Both compounds and allopurinol inhibited competitively with comparable Ki (3b: 3.56?µg, 3g: 2.337?µg, allopurinol: 1.816?µg) and IC₅₀ (3b: 4.228?µg, 3g: 3.1?µg, allopurinol: 2.9?µg) values. The enzyme–ligand interaction was studied by molecular docking using Autodock in BioMed Cache V. 6.1 software. The results revealed a significant dock score for 3b (?84.976?kcal/mol) and 3g (?90.921?kcal/mol) compared with allopurinol (?55.01?kcal/mol). The physiochemical properties and toxicity of the compounds were determined in silico using online computational tools. Overall, in vitro and in silico study revealed 3-amino-6-(4-chloro-2-hydroxy-5-methylphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2–a]pyrimidin-4-one (3g) as a potential lead compound for the design and development of XO inhibitors.     

In vitro and in vivo anti-tumoral activities of imidazo[1,2-a]quinoxaline, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline derivatives

Imidazoquinoxaline and pyrazoloquinoxaline derivatives, analogues of imiquimod, were synthesized, and their in vitro cytotoxic and pharmacodynamic activities were evaluated. In vitro cytotoxicity studies were assessed against melanoma (A375, M4Be, RPMI-7591), colon (LS174T), breast (MCF7), and lymphoma (Raji) human cancer cell lines. In vivo studies were carried out in M4Be xenografted athymic mice. EAPB0103, EAPB0201, EAPB0202, and EAPB0203 showed significant in vitro activities against A375 compared to fotemustine and imiquimod used as references. These compounds were 6-110 and 2-45 times more active than fotemustine and imiquimod, respectively. EAPB0203 bearing phenethyl as substituent at position 1 and methylamine at position 4 showed the highest activity. EAPB0203 has also a more potent cytotoxic activity than imiquimod and fotemustine in M4Be and RPMI-7591 and interesting cytotoxic activity in other tumor cell lines tested. In vivo, EAPB0203 treatment schedules caused a significant decrease in tumor size compared to vehicle control and fotemustine treatments.     

2-Aminodipyrido[1,2-a:3',2'-d]imidazole-porphyrin(Fe) derivatives as sequence specific DNA cleaving reagents

Some porphyrin (Fe)-intercalators were synthesized. We chose 2-aminodipyrido[1, 2-a:3',2'-d]imidazoles (Glu-P's), which were muta-carcinogens isolated from a pyrolysate of L-glutamic acid, as intercalator moieties. These synthetic porphyrin (Fe)-intercalators cleave DNA at G-C and G-T sequences. The specificity for cleavage of base sequences of DNA is quite similar to that of bleomycin.     

Comparison of the reactivity of B-DNA and Z-DNA with two isosteric chemical carcinogens: 2-N,N-acetoxyacetylaminofluorene and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido-[1,2-a:3'',2'' -d] imidazole.

The reactivity of nucleic acids in various conformations and two isosteric chemical carcinogens 2-N,N-acetoxyacetylaminofluorene (N-AcO-AAF) and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido [1,2-a:3',2'-d] imidazole (N-AcO-AGlu-P-3) have been studied. Both carcinogens bind covalently to poly(dG-dC).poly(dG-dC) (B form) and to poly(dG-br5C).poly(dG-br5dC) (Z form). They also bind covalently to (dC-dG)16 and to (dG-dT)15 sequences inserted in plasmids when the inserts are in the B form but they do not bind to the inserts in the Z form. The reactivity of guanine residues at the B-Z junctions depends upon the superhelical density of the plasmids and upon the base sequences at the junction. The distribution of AGlu-P-3 modified guanines in a restriction fragment of pBR322 is not uniform and is different from that of AAF-modified guanines. The conclusion is that N-AcO-Glu-P-3 as N-AcO-AAF can probe at the nucleotide level the polymorphism of DNA. On the other hand, the non-reactivity of both chemical carcinogens and Z-DNA and the hyperreactivity of some junctions might have some importance in the understanding of chemical carcinogenesis.     

Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems

Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure–activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.     

Potency switch between CHK1 and MK2: Discovery of imidazo[1,2-a]pyrazine- and imidazo[1,2-c]pyrimidine-based kinase inhibitors

Chemistry has been developed to access both imidazo[1,2-a]pyrazines and imidazo[1,2-c]pyrimidines. Small structural modifications in both series led to a switch of potency between two kinases involved in mediating cell cycle checkpoint control, CHK1 and MK2.     

Excited state vibrational spectroscopy of metal complexes of dipyrido[3,2-a:2′,3′-c]phenazine

The use of vibrational spectroscopic methods to elucidate the states present in metal complexes of dipyrido[3,2-a:2,3-c]phenazine (dppz) are reviewed. The presence of the close lying b₁(ψ) and b₁(phz) molecular orbitals leads to a number of close lying intraligand and MLCT excited states. Using resonance Raman spectroscopy the nature of initial photoexcitation may be established as M→b₁(ψ). For Ru(II) complexes the lowest excited state is ³MLCT(phz) in nature. However, for [Re(CO)₃Cl] complexes the relaxation from the initial excited state may lead to population of a ³MLCT(phen), ³MLCT(phz) state, an state, or an equilibrium between these states. Time-resolved resonance Raman spectroscopy may be used to identify the presence of dppz^·- or the state and has also been used to identify features associated with intercalation of dppz complexes with DNA. The Raman methods are less effective at detecting the short time dynamics between these states. However, this may be accomplished using time-resolved infrared spectroscopy in which all three states may be unambiguously determined. The clearest picture of the dynamics in dppz complexes has been achieved by using a combination of time-resolved resonance Raman, time-resolved infrared and DFT calculations for rhenium(I) complexes.     

Synthesis and evaluation of the antiproliferative activity of novel isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives

A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity against leukemia and breast cancer cell lines in the 3- to 18-µM concentration range.     

A rapid and sensitive method for determination of covalent binding of benzo[a]pyrene to proteins

A method is presented for the quantitative determination of covalent binding of metabolically activated benzo[a]pyrene to microsomal proteins. After incubation of radiolabelled benzo[a]pyrene with microsomes and NADPH, the mixture is applied to filter paper discs. These are immersed in ethanol to precipitate the proteins. Unbound radiolabel is removed by repeated washes of the filters in organic solvents before scintillation counting. The method is simple, rapid, sensitive and accurate, and works both with 14C- and 3H-labelled compounds. The method is suitable for measuring the incorporation of other radiolabelled xenobiotics to proteins of both microsomes and other subcellular fractions and for the analysis of binding to isolated proteins.     

3-[2-Amino-2-imidazolin-4-yl] alanine, 2-[2-amino-2-imidazolin-4-yl] acetic acid, 2-aminoimidazole and other guanidine derivatives in the tephrosieae

The distribution of enduracididine, 2-[2-amino-2-imidazolin-4-yl] acetic acid, 2-aminoimidazole, canavanine, homoarginine, γ-hydroxyhomoarginine and other unidentified guanidino compounds in seeds of spp. of the Tephrosieae is described. Within Lonchocarpus enduracididine and 2-[2-amino-2-imidazolin-4-yl] acetic acid were found only in American spp. and canavanine only in African spp.     

QSAR of peripheral benzodiazepine receptor ligand 2-phenylimidazo-[1,2-a]pyridine derivatives with physico-chemical parameters

QSAR of the binding affinities of [2-phenylimidazo[1,2-a]pyridin-3-yl]acetamide derivatives (Fig. 1) with central and peripheral (from cortex and ovary) benzodiazepine receptors has been explored using physico-chemical parameters. Attempt has been made to explore the structural and/or physico-chemical requirements of the compounds that are responsible for the selective action against peripheral benzodiazepine receptors over central ones. The results indicate that the presence of bi-substitution on the carboxamido nitrogen, presence of substitutions at X and Y positions, especially, chloro substitution at X position, and presence of chloro substitution at Z position in presence of lipophilic X and/or Y substitutions increase selectivity for binding affinity with peripheral benzodiazepine receptors over central ones.     

Synthesis of new 4-[2-(alkylamino) ethylthio]pyrrolo[1,2-a]quinoxaline and 5-[2-(alkylamino) ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine derivatives, as potential bacterial multidrug resistance pump inhibitors

The synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline derivatives la-1 is described in five or six steps starting from various substituted nitroanilines 2a-e. The bioisostere 5-[2-(alkylamino)ethylthio]pyrrolo[1,2- a]thieno[3,2-e]pyrazine 1m was also prepared. The new derivatives were evaluated as efflux pump inhibitors (EPIs) in a model targeting the NorA system of Staphylococcus aureus. The antibiotic susceptibility of two strains overproducing NorA, SA-1199B and SA-1, was determined alone and in combination with the neo-synthesised compounds by the agar diffusion method and MIC determination, in comparison with reserpine and omeprazole taken as reference EPIs. A preliminary structure-activity relationship study firstly allowed to clarify the influence of the substituents at positions 7 and/or 8 of the pyrrolo[1,2-a]quinoxaline nucleus. Methoxy substituted compounds, 1b and 1g, were more potent EPIs than the unsubstituted compounds (1a and 1f), followed by chlorinated derivatives (1c-d and 1h). Moreover, the replacement of the N,N-diethylamino group (compounds 1a-e) by a bioisostere such as pyrrolidine (compounds 1f-h) enhanced the EPI activity, in contrast with the replacement by a piperidine moiety (compounds 1i-k). Finally, the pyrrolo[1,2-a]thieno[3,2-e]pyrazine compound 1m exhibited a higher EPI activity than its pyrrolo[1,2-a]quinoxaline analogue la, opening the way to further pharmacomodulation.     

Influence of 2-substituent on the activity of imidazo[1,2-a] pyridine derivatives against human cytomegalovirus

The synthesis of various 2-substituted imidazo[1,2-a]pyridine bearing a thioether side chain in position 3 was reported. The new compounds were characterized by 1H and 13C NMR spectra. A conformational study was obtained by X-ray crystallographic analysis for 2-biphen-4-ylimidazopyridine 7. The antiviral activity against human cytomegalovirus (HCMV) was investigated. It was strongly influenced by the nature of C-2 substituent.     

Design,synthesis, and evaluation of novel VEGFR2 kinase inhibitors: Discovery of [1,2,4]triazolo[1,5-a]pyridine derivatives with slow dissociation kinetics

For the purpose of discovering novel type-II inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase, we designed and synthesized 5,6-fused heterocyclic compounds bearing a anilide group. A co-crystal structure analysis of imidazo[1,2-b]pyridazine derivative 2 with VEGFR2 revealed that the N1-nitrogen of imidazo[1,2-b]pyridazine core interacts with the backbone NH group of Cys919. To retain this essential interaction, we designed a series of imidazo[1,2-a]pyridine, [1,2,4]triazolo[1,5-a]pyridine, thiazolo[5,4-b]pyridine, and 1,3-benzothiazole derivatives maintaining a ring nitrogen as hydrogen bond acceptor (HBA) at the corresponding position. All compounds thus designed displayed strong inhibitory activity against VEGFR2 kinase, and the [1,2,4]triazolo[1,5-a]pyridine 13d displayed favorable physicochemical properties. Furthermore, 13d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhibited platelet-derived growth factor receptor (PDGFR) kinases. Oral administration of 13d showed potent anti-tumor efficacy in DU145 and A549 xenograft models in nude mice.     

Sequence selective modification of DNA with muta-carcinogenic 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

2-Acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole binds covalently to the 8 position of guanine residues in DNA. Treatment of the modified DNA with aqueous piperidine causes the liberation of the modified nucleic acid base, 2-(C8-guanyl)amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, and cleavage of DNA at the sites of the modified guanylic acid residues. By use of 5'-end 32P-labelled DNA and sequence analysing gel electrophoresis, we discovered the base sequence specificity of DNA modification with 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. The guanine residues in G-C cluster-like regions are modified more frequently.