Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part II. Effect of chromophore precursors

Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.     

Directed biosynthesis of novel derivatives of echinomycin by Streptomyces echinatus. I. Effect of exogenous analogues of quinoxaline-2-carboxylic acid on the fermentation

Streptomyces echinatus A8331 cultured on a maltose minimal salts medium normally produces a single antibiotic, echinomycin (quinomycin A), containing two quinoxaline-2-carbonyl chromophores. Echinomycin is powerfully active against experimental tumours and can be assayed by its activity against Gram-positive bacteria. Grown in the presence of aromatic carboxylic acids related to quinoxaline, S. echinatus responds in favourable circumstances by incorporating the added material into analogues of the natural antibiotic having replacement chromophores. Both mono- and bis-substituted derivatives are formed. With quinoline-2-carboxylic acid as precursor, large quantities of analogues are produced, and the time course of synthesis, extraction, purification, assay, and characterization of the derivatives are described. Twenty-two other aromatic acids have been tested as potential substrates for antibiotic analogue biosynthesis. Half of them did not significantly affect growth and echinomycin production. Five appeared to stimulate antibiotic synthesis, while the remainder proved inhibitory. New biologically active antibiotics were detected in cultures supplemented with 7-chloroquinoxaline-2-carboxylic acid; 1,2,4-benzo-as-triazine-3-carboxylic acid; thieno[3,2-b]pyridine-5-carboxylic acid; and 6-methylquinoline-2-carboxylic acid.     

Inhibition of Src kinase activity by 7-[(2,4-dichloro-5-methoxyphenyl)amino]-2-heteroaryl-thieno[3,2-b]pyridine-6-carbonitriles

7-[(2,4-Dichloro-5-methoxyphenyl)amino]thieno[3,2-b]pyridine-6-carbonitriles with various heteroaryl groups at C-2 are inhibitors of Src kinase activity. Of these new analogs, compounds substituted at C-2 by a 3,5-furan or a 2,5-pyridine had the best activity in the Src enzyme and cell assays.     

4-Methyl-3-hydroxyanthranilic acid activating enzyme from actinomycin-producing Streptomyces chrysomallus

A 4-methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme was purified 24-fold from a crude protein extract of Streptomyces chrysomallus . The enzyme catalyzes both 4-MHA-dependent ATP/PPi exchange and the formation of the corresponding adenylate. No AMP was formed during the reaction, indicating that no covalent binding of 4-MHA takes place. Besides 4-MHA, the enzyme also catalyzes the formation of adenylates from 3-hydroxyanthranilic acid (3-HA), anthranilic acid (AA), benzoic acid (BA), 3-hydroxybenzoic acid (3-HB), 4-methyl-3-hydroxybenzoic acid (4-MHB), 4-methyl-3-methoxybenzoic acid (4- MMB ), and 4-aminobenzoic acid (4-AB). No such adenylates were formed from 2-aminophenol (2-AP), 2-hydroxybenzoic acid (2-HB), 3-hydroxykynurenine (3-HK), and tryptophan (Trp). 3-HA, 4-MHB, and 4-AB were among the structural analogues of 4-MHA that were the most effective for adenylate synthesis. In the case of 3-HA, considerable AMP release was observed, most probably due to nonenzymatic hydrolysis of the corresponding adenylate. A molecular weight between 53 000 and 57 000 was estimated. The specific activity of the enzyme was correlated with the titer of antibiotic in the cultures, and feeding experiments with whole mycelium of S. chrysomallus showed that 4-MHB was a strong inhibitor of actinomycin synthesis in vivo. The data strongly suggest that the enzyme is involved in the biosynthesis of actinomycin.     

Thieno[3,2-b]thiophene-2-carboxylic acid derivatives as GPR35 agonists

The optimization of a series of thieno[3,2-b]thiophene-2-carboxylic acid derivatives for agonist activity against the GPR35 is reported. Compounds were optimized to achieve β-arrestin-biased agonism for developing probe molecules that may be useful for elucidating the biology and physiology of GPR35. Compound 13 was identified to the most potent GPR35 agonist, and compounds 30 and 36 exhibited the highest efficacy to cause β-arrestin translocation.     

Formation of thieno[3,2-g]pterines from the molybdenum cofactor

A fluorescent oxidation product of the molybdenum cofactor was isolated from Escherichia coli nitrate reductase (EC 1.9.6.1) and bovine milk xanthine oxidase (EC 1.2.3.2), which showed a visible absorption band at 395 nm and was dephosphorylated by alkaline phosphatase but not by phosphodiesterase I. The dephosphorylated species was oxidized by periodate to thieno[3,2-g]pterin-2-carbaldehyde which was quantitatively converted to thieno[3,2-g]pterin-2-carboxylic acid by subsequent treatment with Ag2O in 2 N NaOH. These results indicate that the oxidation product of the molybdenum cofactor is a thieno[3,2-g]pterin derivative with an unidentified side chain in the 2 position.     

Synthesis and biological evaluation of tetracyclic thienopyridones as antibacterial and antitumor agents

A facile synthesis of model 4-oxopyrido[3',2':4,5]thieno[3,2-b]indole-3-carboxylic acids 9a-e was achieved via Stille arylation of 2-chloro-3-nitro-4-oxothieno[2,3-b]pyridine-5-carboxylate and a subsequent microwave-assisted phosphite-mediated Cadogan reaction. The new compounds were tested for their in vitro antimicrobial and antiproliferative activity. Compounds 9a-c and 9e exhibited very high potency against Gram positive Bacillus subtilis and Bacillus megaterium at concentrations 0.000015-0.007 μg/mL. They also displayed excellent activity towards other Gram positive bacilli and staphylococci and Gram negative Haemophilus influenzae, being in most cases superior or equal to commercial fluoroquinolones. Both 9a and 9c were inhibitors of the DNA gyrase activity. As concerns antitumor properties, compounds 9b-e showed growth inhibition of MCF-7 breast tumor and A549 non-small cell lung cancer cells with IC(50) 1.6-2.8 μM and 2.6-6.9 μM, respectively, coupled with absence of cytotoxicity towards normal cells. These compounds are promising as dual acting chemotherapeutics.     

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.     

Chromophore activating enzyme involved in the biosynthesis of the mikamycin B antibiotic etamycin from Streptomyces griseoviridus

A 3-hydroxypicolinic acid activating enzyme from etamycin producing Streptomyces griseoviridus has been purified to apparent homogeneity. Etamycin is a member of mikamycin B antibiotics, chromopeptide lactones, which contain 3-hydroxypicolinic acid (3-HPA) as the chromophoric group. The enzyme catalyzes both the 3-HPA-dependent ATP-pyrophosphate exchange and the formation of 3-HPA adenylate from 3-HPA and ATP. SDS-polyacrylamide gel electrophoresis indicates that the enzyme is a single polypeptide chain with a Mr between 56,000 and 58,000. The molecular mass of the native enzyme was in the same range. In addition to 3-HPA, the enzyme catalyzes the formation of adenylates from picolinic acid, nicotinic acid, and 2-pyrazinecarboxylic acid. Nicotinic acid and picolinic acid when added externally to etamycin producing S. griseoviridus cultures gave rise to the formation of etamycin analogues each containing nicotinic acid or picolinic acid instead of the genuine 3-HPA. The data strongly suggest that the enzyme is involved in the biosynthesis of the chromopeptide lactone etamycin and possibly in that of other mikamycin B antibiotics.     

Purification and characterization of actinomycin synthetase I, a 4-methyl-3-hydroxyanthranilic acid-AMP ligase from Streptomyces chrysomallus.

Actinomycin synthetase I was purified to homogeneiety from actinomycin-producing Streptomyces chrysomallus. The purified enzyme is a single polypeptide chain of M(r) 45,000. It catalyzes the formation of the adenylate of 4-methyl-3-hydroxyanthranilic acid (4-MHA) from the free acid and ATP in an equilibrium reaction. 4-MHA is the precursor of the chromophoric part of actinomycin. By using the 4-MHA analogue, 4-methyl-3-hydroxybenzoic acid, as a model substrate it could be established that the equilibrium constant Keq is independent on enzyme concentration, which suggests that no stoichiometric acyladenylate-enzyme complex is formed in contrast to observations made with aminoacyl adenylates formed by aminoacyl-tRNA synthetases or multifunctional peptide synthetases. Actinomycin synthetase I does not charge itself with substrate carboxylic acid via a covalent thioester bond as is usual for amino acid activation in non-ribosomal peptide synthesis. In addition, the enzyme does not act as an acyl-coenzyme A ligase as revealed by its inability to release AMP in the presence of 4-MHA or other structurally related aromatic carboxylic acids, coenzyme A and ATP. Additional analysis of the activation reaction showed that it is exothermic, whereas the free enthalpy change delta G0 is positive due to a negative entropy change indicating a strong influence of restriction of random motion on the course of the reaction. Determinations of Km and kcat of various substrate carboxylic acids revealed the highest kcat/Km ratio for the natural substrate 4-MHA. From these properties, actinomycin synthetase I represents the prototype of novel chromophore activating enzymes involved in non-ribosomal synthesis of chromopeptide lactones in streptomycetes.     

The synthesis and vasopressin (AVP) antagonist activity of a novel series of N-aroyl-2,4,5,6-tetrahydropyrazolo[3,4-d]thieno[3,2-b]azepines

Synthesis and SAR of N-[4-[(4,5-dihydropyrazolo[3,4-d]thieno[3,2-b]azepin-6(2H)-y l)carbonyl]phenyl]benzamides as arginine vasopressin (AVP) receptor antagonists are discussed. Potent orally active AVP receptor antagonists are produced when the benzamide moiety contains a phenyl group at the 2-position. Similar analogues of 4,6,7,8-tetrahydro-5H-thieno[3,2-b]azepine and VPA-985 are reported.     

Synthesis of substituted indeno[1,2-b]quinoline-6-carboxamides, [1]benzothieno[3,2-b]quinoline-4-carboxamides and 10H-quindoline-4-carboxamides: evaluation of structure-activity relationships for cytotoxicity

New substituted indeno[1,2-b]quinoline-6-carboxamides, [1]benzothieno[3,2-b]quinoline-4-carboxamides and 10H-quindoline-4-carboxamides were prepared from methyl 2-amino-3-formylbenzoate by a new Friedlander synthesis. Evaluation of these carboxamides for cytotoxicity in a panel of cell lines showed that small lipophilic substituents in the non-carboxamide ring, in a pseudo-peri position to the side chain, significantly increased cytotoxic potency while retaining a pattern of cytotoxicity consistent with a non-topo II mode of action. The methyl-substituted indeno[1,2-b]quinoline-6-carboxamide demonstrated substantial effectiveness (20-day growth delays) in a sub-cutaneous colon 38 in vivo tumor model. This is comparable to that reported for the dual topo I/II inhibitor DACA that is in clinical trial.     

Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid

Various 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid derivatives were synthesized from 2-aminothiophenol by a five-step reaction, evaluated for in-vitro and in-vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC2), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the thirty-four synthesized compounds, 2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid (7l) was found to be the most active compound in vitro with MIC of 0.18 and 0.08 microM against MTB and MTR-TB, respectively. Compound 7l was found to be 2 and 570 times more potent than isoniazid against MTB and MDR-TB, respectively. In the in-vivo animal model 7l decreased the bacterial load in lung and spleen tissues with 2.78 and 3.12-log10 protections, respectively, at the dose of 50 mg/kg body weight.     

Synthesis and anthelmintic activity of 7-substituted 3,4a-dimethyl-4a,5a,8a,8b-tetrahydro-6H-pyrrolo[3',4':4,5]furo[3,2-b]pyridine-6,8(7H)-diones

The racemic 7-substituted 3,4a-dimethyl-4a,5a,8a,8b-tetrahydro-6H-pyrrolo[3',4':4,5]furo[3,2-b]pyridine-6,8(7H)-diones represent novel tricyclic compounds with strong in vivo efficacy against the parasitic nematode Haemonchus contortus Rudolphi in sheep. Here we report on the synthesis of tricyclic endo-2,3-dihydro[3,2-b]pyridine-type cycloadducts and describe the separation of the racemic 3,4a-dimethyl-7-ethyl-4a,5a,8a,8b-tetrahydro-6H-pyrrolo[3',4':4,5]furo[3,2]pyridine-6,8(7H)-dione into the enantiomers by HPLC. The absolute configuration of the most anthelmintically active (4aS,5aS,8aS,8bR)-enantiomer was determined by single crystal X-ray analysis using its stable (4aS,5aS,8aS,8bR)-enantiomer-CuCl2 (2:1)-complex.     

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.     

Synthesis and antiproliferative evaluation of certain pyrido[3,2-g]quinoline derivatives

The present report describes the synthesis and evaluation of tricyclic pyrido[3,2-g]quinoline derivatives in which an additional pyridine ring is linearly fused on the antibacterial quinoline-3-carboxylic acid. Among them, only diethyl 4,6-diamino-10-methylpyrido[3,2-g]quinoline-3,7-dicarboxylate (9a) and diethyl 4,6-bis-(3-dimethylaminopropylamino)-10-methylpyrido[3,2-g]quinoline-3,7-dicarboxylate (9d) were able to completely inhibit cell proliferation of MCF7 (Breast), NCI-H460 (Lung), and SF-268 (CNS) implying either amino or dimethylaminopropyl moiety at C-4 and C-6 positions is crucial for the antiproliferative activity of pyrido[3,2-g]quinoline derivatives. Compounds 9a-9d were further evaluated for their activity against the growth of MCF-7 and two prostate cancer cell lines, LNCaP and PC-3. Results indicated the antiproliferative activity decreased in an order 9d>9a>9b and 9c. Compound 9d was the most cytotoxic with an IC50 value of 5.63 and 3.96 microM, respectively, against LNCaP and MCF-7. Flow cytometric analyses revealed that growth inhibition of LNCaP by 9d was due to cell cycle arrest in G1 phase, and followed by apoptosis.     

Actinomycin synthetases. Multifunctional enzymes responsible for the synthesis of the peptide chains of actinomycin

Two enzymes were purified from actinomycin-synthesizing Streptomyces chrysomallus which could be identified as peptide synthetases involved in the biosynthesis of actinomycin. Actinomycin synthetase II activates the first two amino acids of the peptide chains of the peptide lactone antibiotic, threonine and valine (or isoleucine), as thioesters via their corresponding adenylates. It is a single polypeptide chain of Mr 225,000. Similarly, actinomycin synthetase III activates proline, glycine, and valine (the remaining three amino acids in the antibiotic) as thioesters and is a single polypeptide chain of about Mr 280,000. It also carries the methyltransferase function(s) for N-methylation of thioesterified glycine and valine. In addition, it catalyzes the formation of cyclo(sarcosyl-N-methyl-L-valine) from glycine, L-valine, and S-adenosyl-L-methionine at the expense of ATP. Although the cell-free synthesis of the peptide lactone was not as yet accomplished, the data provide evidence that together with the 4-methyl-3-hydroxyanthranilic acid-activating enzyme (now designated as actinomycin synthetase I) all amino acid-activating protein components of the actinomycin-synthesizing enzyme complex are identified.     

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.     

Synthesis of new aromatic pyrrolo[2,1-c] [1,4]benzodiazepines and pyrrolo[1,2-a]thieno[3,2-e] [1,4]diazepines as anti-tumoral agents

Diazepine analogs of thieno[2,3-b]pyrrolizin-8-ones were synthesized by aromatization of 2-hydroxypyrrolo[1,2-a]thieno[3,2-e][1,4]diazepines. These compounds were evaluated in vitro for their antiproliferative activity against the L1210 leukemia cell line. The activity of these compounds was in the micromolar range, the best result being for the mixture of the isomers 5 and 6 which showed a 0.35 microM IC50 against cell growth.     

[(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives; HIF prolyl 4-hydroxylase inhibitors as oral erythropoietin secretagogues

A new series of PHD (HIF prolyl 4-hydroxylase) inhibitors was designed based on the X-ray co-crystal structure of FG-2216. Using a lead generation process, a series of [(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives was developed as potent PHD2 inhibitors. This class of compounds also showed the ability to stabilize HIF-α, to stimulate EPO secretion in in vitro studies, and to increase hematocrit, red blood cell count, and hemoglobin levels in an animal efficacy study.