Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and Topoisomerase IV

We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 μM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.     

Synthesis and potent anticonvulsant activities of 4-oxo-imidazo[1,2-a]indeno[1,2-e]pyrazin-8- and -9-carboxylic (acetic) acid AMPA antagonists

The over-stimulation of excitatory amino acid receptors such as the glutamate AMPA receptor has been suggested to be associated with neurodegenerative disorders. Here we describe an original series of readily water soluble 4-oxo-imidazo[1,2-a]indeno[1,2-e]pyrazin-8- and -9-carboxylic (acetic) acid derivatives. One of these compounds, 4f, exhibited nanomolar binding affinity, potent competitive antagonism at the ionotropic AMPA receptor and a long duration of anticonvulsant activity after administration by parenteral route in vivo.     

Rational Design of Pyrrolo[1,2-a]benzimidazole-Based Antitumor Agents Targeting the DNA Major Groove

Models have been developed for the interaction of the pyrrolo[1,2-a]benzimidazole (PBI) antitumor agents with the two-electron activating enzyme DT-diaphorase and the DNA major groove. The DT-diaphorase model and experimental results indicate that the S-enantiomer of 3-carbamido PBI can enantioselect ovarian cancers. The reduced PBI interacts with the DNA major groove at AT base pairs by forming Hoogsteen-like hydrogen bonds. The reduced 3-amino PBI forms three hydrogen bonds in the major groove with the amino group acting as an H-bond donor to the thymine carbonyl. The DNA-binding model will permit the design of major groove recognition agents.     

Synthesis and antibacterial activity of a new series of 3-[3-(substituted phenyl)-1-phenyl-1H-pyrazol-5-yl]-2H-chromen-2-one derivatives

A series of 3-[3-(substituted phenyl)-1-phenyl-1H-pyrazol-5-yl]-2H-chromen-2-one (4a–k) were synthesized by reaction of 3-[2,3-dibromo-3-(substituted phenyl)propanoyl]-2H-chromen-2-one (3 a-k) with phenyl hydrazine in presence of triethylamine in absolute ethanol, characterized by spectral data and screened for their in vitro antibacterial activity against gram-positive and gram-negative bacteria. Among the series, compounds 4d, 4h and 4i displayed an encouraging antibacterial activity profile as compared to reference standard drug ciprofloxacin against tested bacterial strains.     

Determination of 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo[1,5-a]quinoxalin-4(5H)-one in serum by high-performance liquid chromatography

A high-performance liquid chromatographic assay with solid-phase extraction (SPE) for the rapid and sensitive quantitation of 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)imidazo[1,5-a]quinoxalin-4(5H)-one (I, U-78875) in serum is described. The validation results indicated that the present method had excellent intra- and inter-assay precision (≤ 9.5%, mean ± S.D. = 3.9±3.0%, n = 25) and accuracy (≤ 10.0%, mean ± S.D. = 3.0 ± 2.9%, n = 25), as well as improved sensitivity (2 ng/ml, using a 100-μl injection). Each chromatographic run is only 10 min and the organic solvent for the extraction of I and internal standard (U-82217) from serum was only 300 μl. The application results obtained from the SPE method were in good agreement with the advanced automated sample preparation method.     

3-[Benzimidazo- and 3-[benzothiadiazoleimidazo-(1,2-c)quinazolin-5-yl]-2H-chromene-2-ones as potent antimicrobial agents

A series of 3-[benzimidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one (6a-6f) and 3-[benzothiadiazole- imidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one derivatives (7a-7f) that incorporate a variety of substituents at the 6- and/or 8-positions of the coumarin moieties have been synthesized utilizing cellulose sulfuric acid as an efficient catalyst under both conventional heating and microwave irradiation procedures. These analogs were evaluated for their antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes (Gram-positive bacteria), Escherichia Coli, Klebsiella pneumonia, Salmonella typhimurium (Gram-negative bacteria), and Aspergillus niger, Candida albicans, and Aspergillus flavus (Fungi). Two analogs, 6c (a 6,8-dichloro analog, MIC([SA]) = 2.5 μg/mL; MIC([ST]) = 2.5 μg/mL) and 7d (a 6,8-dibromo analog, MIC([ST]) = 2.5 μg/mL) were identified as potent antibacterial agents, and two analogs, 6b (a 6-bromo analog, MIC([AF]) = 10 μg/mL) and 6d (a 6,8-dibromo analog, MIC([AF]) = 15 μg/mL; MIC([CA]) = 15μg/mL), were identified as potent antifungal agents. Based on the MIC data, analogs 6b, 6c, 6d, and 7d were identified as the most potent antimicrobial agents in the series.     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Synthesis of N-substituted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]naphthyridines as novel topoisomerase I-targeting antitumor agents

Several N-alkyl and N,N-dialkyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones have been identified as topoisomerase I-targeting agents with potent antitumor activity. In the present study, the impact on biological activity of substitution of a trifluoromethyl, cyano, aminocarbonyl, or ethynyl group on a N-methyl substituent of N,N-dimethyl-, N-methyl-N-ethyl-, and N-methyl-N-isopropyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones was assessed.     

Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy-5(R)-[2(R,S)-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2(R,S)-yl-methyl)-[1,3]dioxolan-4(S)-yl]-5H-furan-2-one and 3-O-octadecyl derivatives

Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of α-tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a–d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic α-tocopherol analogue (5) and natural α-tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a–d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 μM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.     

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.     

S-[2-(N7-guanyl)ethyl]glutathione, the major DNA adduct formed from 1,2-dibromoethane

The reaction of 1,2-dibromoethane and glutathione with DNA in the presence of glutathione S-transferase results in the formation of a single major DNA adduct, which can be released by thermal hydrolysis at neutral pH and separated by octadecylsilyl and propylamino high-performance liquid chromatography. The same DNA adduct is the only major one formed in livers of rats treated with 1,2-dibromo[1,2-14C]ethane. The DNA adduct was identified as S-[2-(N7-guanyl)ethyl]glutathione: (1) The chromatographic behavior was altered by treatment with gamma-glutamyl transpeptidase or Streptomyces griseus protease. (2) The molecular ions observed in positive and negative mode fast atom bombardment mass spectrometry were those expected for the structure when either glycerol or a mixture of dithiothreitol and dithioerythritol was used as the bombardment matrix. (3) The two-dimensional 1H NMR correlated spectroscopy spectrum of the DNA adduct was compared to the spectra of glutathione, oxidized glutathione, and N7-methylguanine and found to be consistent with the assigned structure. No evidence for in vitro or in vivo opening of the guanyl imidazole ring was observed under these conditions. The structure of the adduct supports a pathway involving enzyme-catalyzed conjugation of 1,2-dibromoethane with glutathione, non-enzymatic dehydrohalogenation of the resulting half-mustard to form a cyclic episulfonium ion, and attack of the N7 nitrogen of DNA guanine on the episulfonium ion to generate this major DNA adduct, which may be related to the carcinogenicity of this chemical.     

Solution structure of 2-(pyrido[1,2-e]purin-4-yl)amino-ethanol intercalated in the DNA duplex d(CGATCG)2

The solution structure of the complex formed between d(CGATCG)(2) and 2-(pyrido[1,2-e]purin-4-yl)amino-ethanol, a new antitumor drug under design, has been resolved using NMR spectroscopy and restrained molecular dynamic simulations. The drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the minor groove. Analysis of NMR data establishes a weak stacking interaction between the intercalated ligand and the DNA bases; however, the drug/DNA affinity is enhanced by a hydrogen bond between the hydroxyl group of the end of the intercalant side chain and the amide group of guanine G6. Unrestrained molecular dynamic simulations performed in a water box confirm the stability of the intercalation model. The structure of the intercalated complex enables insight into the structure-activity relationship, allowing rationalization of the design of new antineoplasic agents.     

Novel tetrahydropyran analogs as dipeptidyl peptidase IV inhibitors: Profile of clinical candidate (2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine (23)

A series of novel tri-2,3,5-substituted tetrahydropyran analogs were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the series provided inhibitors with good DPP-4 potency and selectivity over other peptidases (QPP, DPP8, and FAP). Compound 23, which is very potent, selective, efficacious in the diabetes PD model, and has an excellent pharmacokinetic profile, is selected as a clinical candidate.     

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid in seeds of Lonchocarpus sericeus

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid have been isolated from seeds of Lonchocarpus sericeus. The concentration of each compound was ca 0.5 % of the fresh seed weight.     

Chair-form [Ag2(1,2-bimb)2] in silver(I) complexes containing the ditopic ligand 1,2-bis(1-imidazolylmethyl)benzene (1,2-bimb)

The ditopic ligand 1,2-bis(1-imidazolylmethyl)benzene (1,2-bimb) and its silver(I) complexes [Ag₂(1,2-bimb)₂](PF₆)₂ (1) and {[Ag₂(1,2-bimb)₂]₂(SbF₆)₄}_n (2) were prepared and their structures characterized by X-ray crystallography. Both complexes contain the chair-form unit [Ag₂(1,2-bimb)₂]²⁺ with Ag(I) linearly coordinated by N_Im (Im=1-imidazolyl) from the Im groups of two 1,2-bimb. However, the [Ag₂(1,2-bimb)₂]²⁺ units are positioned differently in forming 1D infinite chains through weak argentophilic interactions: linear chains in 1 with the units oriented in same direction are formed via Ag?π interactions, while polymeric chains constructed via Ag?Ag interactions in 2 are observed with the units arranged in alternate directions. Differences in supramolecular structures may be a result of different size of the anions.     

Design and synthesis of 2-[4-[4-(m-(ethylsulfonamido)-phenyl) piperazin-1-yl]butyl]-1,3-dioxoperhydropyrrolo[1,2-c]imidazole (EF-7412) using neural networks. A selective derivative with mixed 5-HT1A/D2 antagonist properties.

A test series of 32 phenylpiperazines III with affinity for 5-HT1A and alpha1 receptors was subjected to QSAR analysis using artificial neural networks (ANNs), in order to get insight into the structural requirements that are responsible for 5-HT1A/alpha1 selectivity. Good models and predictive power were obtained for 5-HT1A and alpha1 receptors. A comparison of these models gives information for the design of the new ligand EF-7412 (5-HT1A:Ki(nM)= 27; alpha1: Ki(nM) > 1000). This derivative displayed affinity for dopamine D2 receptor (Ki = 22 nM) and is selective for all other receptor examined (5-HT2A, 5-HT3, 5-HT4 and Bz). EF-7412 acts an antagonist in vivo in pre- and postsynaptic 5-HT1A receptor sites and as an antagonist in dopamine D2 receptor.     

Gene within gene configuration and expression of the Drosophila melanogaster genes lethal(2)neighbour of tid [l(2)not] and lethal(2)relative of tid [l(2)rot]

In this paper, we describe the structure and temporal expression pattern of the Drosophila melanogaster genes l(2)not and l(2)rot located at locus 59F5 vis à vis the tumor suppressor gene l(2)tid described previously and exhibiting a gene within gene configuration. The l(2)not protein coding region, 1530 nt, is divided into two exons by an intron, 2645 nt, harboring the genes l(2)rot, co-transcribed from the same DNA strand, and l(2)tid, co-transcribed from the opposite DNA strand, located vis à vis. To determine proteins encoded by the genes described in this study polyclonal rabbit antibodies (Ab), anti-Not and anti-Rot, were generated. Immunostaining of developmental Western blots with the anti-Not Ab resulted in the identification of a 45-kDa protein, Not45, which is smaller than the Not56 protein predicted from the sequence. Its localization in endoplasmic reticulum (ER) was established by immunoelectron microscopy of Drosophila melanogaster Schneider 2 cells. Not45 shows significant homology to yeast ALG3 protein acting as a dolichol mannosyltransferase in the asparagine-linked glycosylation. It is synthesized ubiquitously throughout embryonic life. The protein predicted from the l(2)rot sequence, Rot57, shows a homology to the NS2B protein of the yellow fever virus1 (yefv1). The results of l(2)rot RNA analysis by developmental Northern blot and by in situ RNA localization, as well as the results of the protein analysis via Western blot and immunohistochemistry suggest that l(2)rot is transcribed but not translated. Since RNAs encoded by the genes l(2)tid and l(2)rot are complementary and l(2)rot is presumably not translated we performed preliminary experiments on the function of the l(2)rot RNA as a natural antisense RNA (asRNA) regulator of l(2)tid expression, expressed in the same temporal and spatial manner as the l(2)tid− and l(2)not RNA. l(2)tid knock-out by antisense RNA yielded late embryonic lethality resulting from multiple morphogenetic defects.