Sequence-selectivity of 5,11-dimethyl-5H-indolo[2,3-b]quinoline binding to DNA. Footprinting and molecular modeling studies.

Indolo[2,3-b]quinolines are a new family of the DNA intercalators showing significant cytotoxic activity. The mechanism of their action is based on the inhibition of DNA topoisomerase II activity. It depends on their ability to induce and stabilize drug-topII-DNA cleavable complexes. Site-specific intercalation of 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ) was analyzed in vitro by DNaseI footprinting and by molecular modeling. To model the DNA-intercalator complex, use was made of the CVFF and ESFF force fields implemented in Insight 97.0 software. Experimental results were verified using a simple statistical model. The DiMIQ molecule was found to bind preferentially to the pBR322 DNA plasmid in the 5'-TGCTAACGC-3' region between adjacent adenine bases.

Microbial conversion of methyl- and methoxy- substituted derivatives of 5H-indolo[2,3-b]quinoline as a method of developing novel cytotoxic agents.

In furtherance of our structure-activity relationship studies on the antitumor activity of indolo[2,3-b]quinolines, novel cytotoxic derivatives bearing methyl groups at N-5, C-11, C-2 and/or C-9, as well as methoxy-groups at C-2 and/or C-9, were synthesized by the modified Graebe-Ullmann reaction. To elucidate the metabolic pathways of these compounds, zygomycete fungus Cunninghamella elegans ATCC 9245 (which is known to produce drug metabolites that are also formed in mammals) was used as a mimetic organism. Simultaneously, biotransformation of the same substrates was carried out with a microsomal fraction of rat liver. Three forms of microbial conversion were observed: hydroxylation of the aromatic ring or hydroxylation of the methyl group, and O-demethylation. The reaction proceeded regioselectively, and only positions C-2 and C-9 were affected in the indolo[2,3-b]quinoline system. The products formed were found to be identical with the metabolites generated by rat liver microsomes. The metabolites obtained displayed a cytotoxic activity in vitro against colon adenocarcinoma SW-707 and lung carcinoma A-549 (ID50 in the range 0.27-3.04 microM), which was as strong as that of the substrates. In the course of the further metabolic pathway study of indolo[2,3-b]quinolines we found that metabolites with a hydroxyl group in the aromatic system were transformed to non-cytotoxic polymeric products by multicopper oxidases: human ceruloplasmin or fungal laccase (used as mimetic enzyme), whereas metabolites with a hydroxymethyl group did not undergo such bioconversion. The last mentioned compounds can be regarded as a novel type of cytotoxic indolo[2,3-b]quinoline derivatives formed in metabolic processes.

Synthesis, and cytotoxic activity of some novel indolo[2,3-b]quinoline derivatives: DNA topoisomerase II inhibitors.

A series of new 5H-indolo[2,3-b]quinoline derivatives bearing methoxy and methyl groups at C-2 and C-9 was synthesized (according to the modified Graebe-Ullmann reaction). These compounds were evaluated for their antimicrobial and cytotoxic activity and tested as inhibitors of DNA topoisomerase II. Lipophilic and calf thymus DNA binding properties of these compounds were also established. In the SAR studies we used quantum-mechanical methodology to analyze the molecular properties of the drugs. All of the 5H-indolo[2,3-b]quinolines tested were found to inhibit the growth of gram-positive bacteria and pathogenic fungi at MIC ranging between 2.0 and 6.0 microM. They showed also cytotoxic activity in vitro against several human cancer cell lines of different origin (ID50 varied from 0.6 to 1.4 microM), and stimulated the formation of topoisomerase-II-mediated pSP65 DNA cleavage at concentration between 0.2 and 0.5 microM. The most active indolo[2,3-b]quinolines which had the greatest contribution to the increase in the Tm of DNA displayed also the highest DNA binding constants and the highest cytotoxic activity. The differences in DNA binding properties and cytotoxic activity seem to be more related to steric than electrostatic effects.

Methoxy- and methyl-, methoxy-5,6,11-trimethyl-6H-indolo [2,3-b]quinolinium derivatives as novel cytotoxic agents and DNA topoisomerase II inhibitors.

New members of the cytotoxic indolo[2,3-b]quinoline family, with a methyl groups at N-5, N-6 (their presence stabilizes the positive charge of the molecule), were prepared using a modified Graebe-Ullmann reaction. The derivatives obtained were well soluble in water in a non-pH-dependent manner. They displayed strong antimicrobial activity against Gram-positive bacteria and pathogenic fungi (the MIC values fall between 0.0025 and 0.12 mM) and highly selective cytotoxicity in vitro against different human cancer cell lines: colon adenocarcinoma SW 707, lung carcinoma A 549, transitional cell carcinoma Hu 1703, and oral epidermoid carcinoma KB, in the range of 0.01 to 3.0 microM. They also stimulated the formation of topoisomerase-II-mediated DNA cleavage at concentration from 0.04 to 0.5 microM. These observations correspond well with the ability of the tested compounds to increase the melting temperature of calf thymus DNA (delta Tm being between 13 degrees C and 22 degrees C).

Synthesis and structure-activity relationship of methyl-substituted indolo[2,3-b]quinolines: novel cytotoxic, DNA topoisomerase II inhibitors.

In furtherance of our SAR study on the chemistry and antitumor activity of fused nitrogen heteroaromatic compounds, a series of linear, methyl-substituted derivatives of 5H- and 6H-indolo[2,3-b]quinolines were synthesized according to the modified Graebe-Ullmann reaction. To establish the relationship between the physicochemical and biological activities of indolo[2,3-b]quinolines, their lipophilic properties, cytotoxic and antimicrobial activity, and ability to induce topoisomerase II dependent pSP65 DNA cleavage in vitro were investigated. We found that the antimicrobial and cytotoxic activity of indolo[2,3-b]quinolines was strongly influenced by the position, and the number of methyl substituents and the presence of methyl group at pyridine nitrogen was essential for the cytotoxicity of these compounds. All indolo[2,3-b]quinolines belonging to the 5H series, i.e., bearing a methyl group on the pyridine nitrogen, showed significant activity against procaryotic and eucaryotic organisms. They inhibited the growth of Gram-positive bacteria and pathogenic fungi at MIC range 3 x 10(-2) to 2.5 x 10(-1) mumol/mL, displayed cytotoxicity against KB cells ID50 in the range 2 x 10(-3) to 9 x 10(-3) mumol/mL, and stimulated the formation of calf thymus topoisomerase II mediated DNA cleavage at concentration between 0.4 and 10 microM. None of the indolo[2,3-b]quinolines belonging to the 6H series, i.e., lacking a methyl group on the pyridine nitrogen, was active in analogous tests. Of the investigated compounds, the most active was 2,5,9,11-tetramethyl-5H-indolo[2,3-b]quinoline, a compound bearing the highest number of symmetrically distributed methyl groups. The interaction of indolo[2,3-b]quinolines with DNA was studied by measuring the increase of calf thymus DNA denaturating temperature (Tm). The delta Tm values for the 5H series were found to be about 10 times as high as those for the 6H compounds. Indolo[2,3-b]quinolines with the highest number of methyl groups had the greatest contribution to the increase in the Tm of calf thymus DNA. The values of delta Tm reached 19 degrees C and 1.6 degrees C for the most substituted compounds of both series.

Mutagenicity of carboline methyl derivatives in Salmonella.

A series of linear, methyl-substituted derivatives of 5H-indolo[2,3-b] quinolines was tested for mutagenic activity with the battery of Ames tester strains. Mutagenic activity of indoloquinolones was strongly influenced by the position and a number of methyl groups. All compounds tested, with one exception, act like frame-shift mutagens. Only two compounds among them were mutagenic in the strain detecting oxidative and cross-linking mutagens.

A carboline derivative as a novel mammalian DNA topoisomerase II targeting agent.

The DNA intercalating, ellipticine analog drug, 5,11-dimethyl-5H-indol[2,3-b]quinoline, is able to stabilize in vitro the topoisomerase II-DNA cleavable complex and to induce DNA breaks in BPV I episome in rat fibroblasts. Cytotoxicity studies with DC3F cells resistant to ellipticine strongly suggest that topoisomerase II is a cellular target involved in the mechanism of cytotoxic action of this carboline derivative.

Hemagglutinins of Beauveria bassiana strains: the effect of growth conditions on their production.

Forty strains of Beauveria bassiana were screened for their ability to produce hemagglutinins. It has been found that majority of mycelial extracts but not cultural broth display non specific hemagglutinating activity toward animal and human type AB and A, B, O erythrocytes when microorganisms are cultivated on rich in amino acids media supplemented with saccharose. The formation of hemagglutinins in mycelia is strongly dependent on composition of medium and associated with a production of extracellular lipases, chitinases and amylases. No monosaccharide binding specificity of obtained hemagglutinins toward glucose/mannose, N-acetylglucosamine, N-acetylgalactosamine/galactose, L-fucose and sialic acid was observed.

Purification and some properties of hemagglutinin from Beauveria bassiana.

A novel hemagglutinin produced by insect pathogen Beauveria bassiana was isolated from mycelium of the stationary growing microorganism and purified by adsorption on carboxymethyl cellulose followed by separation on Spherogel TSK--phenyl 5 PW column using high performance liquid chromatography. The purified hemagglutinin was homogeneous in polyacrylamide gel electrophoresis and isoelectric focusing. Its molecular weight was estimated to be around 25,000, isoelectric point was found at pH 8.6 +/- 0.2. Amino acid composition of purified B. bassiana hemagglutinin was determined by HPLC fluorometric analysis of o-phthaldialdehyde derivatives of protein hydrolysate. Purified hemagglutinin agglutinated some animal and all human erythrocytes independently of blood group ABO phenotype. The observed hemagglutination is not inhibited by glucose/mannose, N-acetylglucosamine, N-acetyl galactosamine, galactose, L-fucose and sialic acid.

Formation of beauvericin by selected strains of Beauveria bassiana.

Various strains of Beauveria bassiana were cultivated in submerged cultures and examined for their abilities to produce beauvericin, cyclodepsipeptide antibiotic displaying antibacterial and insecticidal properties. It has been found that among twenty four strains of Beauveria bassiana only three produced beauvericin. Apparently, the striking differences among tested strains concerning several secondary product formations have been observed.

Production of beauvericin by Beauveria bassiana on L-methionine enriched medium.

Beauveria bassiana strain isolated from curculionid beetle (Coleoptera) was cultivated in fermentation tank on the medium composed of protein hydrolysate and glucose. It has been found that L-methionine addition to the medium increases significantly the yield of beauvericin biosynthesis. Apparently, for the optimal antibiotic production, low aeration conditions are preferable.

New inhibitors for aminoglycoside-adenylyltransferase.

Two hydroxymethyltropolones and two tropolone acetate derivatives were found to inhibit an aminoglycoside-adenylyltranferase in a gentamicin-resistant Escherichia coli strain. The inhibitory effect of tropolones depends on the nature of the aminoglycoside antibiotic subject to adenylation. Combinations of hydroxymethyltropolones with tobramycin were more active compared with tropolone acetates against gentamicin-resistant strains displaying adenylyltransferase activity. On the contrary a combination of the investigated acetate with gentamicin was of lower activity. It could be shown that these inhibitors inhibit, to a varying degree, the transfer of radioactive ATP to different aminoglycoside molecules.

Microbial transformation of azacarbazoles. III. Conversion of methoxy- and phenyl- substituted alpha-carbolines to corresponding alpha-iso-carbolines by Kitasatosporia setae strain.

Microbial N-1 methylation of alpha-carboline derivatives substituted at position C-2, C-6 and C-8 with methoxy- and at position C-2 and C-6 with phenyl groups conducted with Kitasatosporia setae resulted in corresponding alpha-iso-carboline formation. The yield of obtained products is significantly dependent on the position of substituent in alpha-carboline molecule. Compounds, as 2- and 6-methoxy-alpha-carboline undergo N-1 microbial methylation with strikingly low yields of 5%, whereas products of biotransformation of 8-methoxy-alpha-carboline was formed in markedly higher amount, about 50%. Similar correlations were observed for subjected to bioconversion C-2 and C-6 phenyl-alpha-carbolines. The yields of formed products were estimated as 3% and 5%, respectively. All obtained alpha-iso-carbolines were found to be toxic to the transforming strain of Kitasatosporia setae at the range of 1.2-2.5 microM/ml as well as against KB tumor culture cells (ID50 0.04-0.6 microM/ml).

Microbial transformation of azacarbazoles. IV. Conversion of chloro-substituted alpha-carbolines by Kitasatosporia setae strain.

Biotransformation of alpha-carboline derivatives substituted at positions C-5, C-6, C-7 and C-8 with chlorine, carried out with Kitasatosporia setae strain yielded corresponding 1-methyl-alpha-iso-carbolines. The formation of products is dependent on the position of chlorine in substrate molecule. When chlorine is introduced at C-6, the yield of N-1 methylation is low, about 5%. Derivatives of alpha-carboline substituted with chlorine at C-7 and C-8 form corresponding alpha-iso-carbolines with yield up to 20% and 30%, respectively, whereas 5-chloro-alpha-carboline is converted into 5-chloro-1-methyl-alpha-iso-carboline with 60% yield. Apparently, additional pathway of microbial transformation of 2-chloro-alpha-carboline has been found. Primarily formed 2-chloro-1-methyl-alpha-iso-carboline subjected to complex enzymic conversion yields quantitatively 2-methoxy-1-methyl-alpha-iso-carboline-9-N-oxide. It has been found that 2-chloro-1-methyl-alpha-iso-carboline exhibit strong cytotoxic activity, against KB cells tissue culture, ID50 = 0.01 microM/ml and inhibits growth of Kitasatosporia setae strain, MIC = 0.5 microM/ml. Toxicity of formed 2-methoxy-1-methyl-alpha-iso-carboline-N-9-oxide is markedly lower, ID50 = 0.3 microM/ml and MIC = 3.5 microM/ml. The remaining C-5, C-6, C-7 and C-8 chloroderivatives of alpha-iso-carboline occur to be less active than 2-chloro-1-methyl-alpha-iso-carboline.

Microbial transformation of azacarbazoles. V. Studies on N-1 methylation of 4-methyl-alpha-carboline by Kitasatosporia setae strain.

N-1 methylation of an azacarbazole, 4-methyl-alpha-carboline by Kitasatosporia setae was associated with antifungal metabolite production. Incubation of 4-methyl-alpha-carboline with intact cells or homogenates of Kitasatosporia setae and L-/14CH3/-methionine or adenosyl-S-/14CH3/-L-methionine yielded radioactive products, 1,4-dimethyl-alpha-iso-carboline. The presence of 14C-labelled methyl groups in antifungal metabolite has been also observed. Mutants of Kitasatosporia setae with lost abilities to produce antifungal metabolite did not convert 4-methyl-alpha-carboline into its methyl derivative.

Microbial transformation of azacarbazoles. VII. Antitumor properties of benzo-alpha-iso-carbolines formed by Kitasatosporia setae strain from corresponding benzo-alpha-carbolines.

2,3-Benzo-alpha-carboline, 7,8-benzo-alpha-carboline and their 4-methyl derivatives were subjected to microbial conversion yielding corresponding benzo-alpha-iso-carbolines. All obtained products showed significant antimicrobial and cytotoxic properties. ID50 values were found to be at range 0.01-0.001 microM/ml, regarding in vitro KB tumor cells system data. It has been found that introduction of methyl group at para-position to the nitrogen of pyridine nucleus strongly increases cytotoxic and microbial activity of benzo-alpha-iso-carbolines. Apparently it has been indicated that antitumor activity of benzo-alpha-iso-carbolines is strongly dependent on shape and size of the molecule. Of all the compounds tested only 2,3-benzo-1,4-dimethyl-alpha-iso-carboline increases life span of leukemia P388 bearing mice up to 160%.

Microbial transformation of azacarbazoles. VI. Conversion of 6-hydroxy- and 6-amino-alpha-carbolines with copper oxidases.

alpha-Carboline substituted at C-6 with hydroxy-group is oxidized by human ceruloplasmin and fungal laccase into reactive intermediate which dimerizes, while 6-amino-alpha-carboline subjected to action of both enzymes yields the polymeric products. To prepare sufficient quantities of compounds needed for structure elucidation studies, laccase, immobilised in polyacrylamide gel was employed as a convenient reagent. The resulting products are suggested to be metabolities of 6-hydroxy- and 6-amino-alpha-carbolines formed in vivo.

Microbial transformation of azacarbazoles. VIII. Production of 14C-labelled benzo-alpha-iso-carbolines by permeabilized cells of Kitasatosporia setae strain.

N-1 methylation of benzo-alpha-carbolines performed with Kitasatosporia setae strain yields corresponding benzo-alpha-iso-carbolines, compounds demonstrating relatively strong cytostatic and antitumor properties. To obtain radioactive benzo-alpha-iso-carbolines required for the further studies of their mode of antitumor action, a simple and useful method of the preparation of 14C-labelled compounds by microbial means has been elaborated.

Microbial transformation of azacarbazoles. IX. Preliminary studies on hydroxylation of 2,3-benzo-1,4-dimethyl-alpha-iso-carboline by Paecilomyces flavinosus.

Microbial transformation of 2,3-benzo-1,4-dimethyl-alpha-iso-carboline performed with several strains of fungi Beauveria bassiana, Verticillum lecani and Paecilomyces flavinosus yielded common products which were expected to be hydroxylated derivatives of starting compound. Among the microorganisms tested, strain Paecilomyces flavinosus P-5 was selected to perform quantitative bioconversion of 2,3-benzo-1,4-dimethyl-alpha-iso-carboline for preparative scale.