Synthesis of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives and evaluation of their topoisomerase I and II inhibitory activity,cytotoxicity, and structure–activity relationship

A series of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives were designed, synthesized, and evaluated for their topoisomerase I and II inhibition and cytotoxic activity against several human cancer cell lines. Compounds 10–19 showed moderate topoisomerase I and II inhibitory activity and 20–29 showed significant topoisomerase II inhibitory activity. Structure–activity relationship study revealed that 4-(5-chlorofuran-2-yl)-2-(thiophen-3-yl) moiety has an important role in displaying topoisomerase II inhibition.     

Cytotoxicity and DNA topoisomerase inhibitory activity of benz[f]indole-4,9-dione analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50<20.0 microg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50=0.4 microg/ml)) compared to colon (IC50>20.0 microg/ml) and stomach (IC50>20.0 microg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.     

Design, synthesis, and biological evaluation of cytotoxic 11-aminoalkenylindenoisoquinoline and 11-diaminoalkenylindenoisoquinoline topoisomerase I inhibitors

The cytotoxic indenoisoquinolines are a novel class of noncamptothecin topoisomerase I inhibitors having certain features that compare favorably with the camptothecins. A new strategy was adopted to attach aminoalkenyl substituents at C-11 of the indenoisoquinoline ring system, which, according to molecular modeling, would orient the side chains toward the DNA minor groove. All of the newly synthesized compounds were more cytotoxic than the parent indenoisoquinoline NSC 314622. Despite an imperfect correlation between cytotoxicities and topoisomerase I inhibition results, the hypothetical structural model of the cleavage complex presented here provides a conceptual framework to explain the structure-activity relationships.     

Cytotoxicity and DNA Topoisomerase Inhibitory Activity of Benz[f]indole-4,9-dione Analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC₅₀<20.0 μg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC₅₀=0.4 μg/ml)) compared to colon (IC₅₀>20.0 μg/ml) and stomach (IC₅₀>20.0 μg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.     

Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays

We have characterized the topoisomerase I and II activities in nuclear extracts from immature embryos of Zea mays and the effect of the treatment with 2,4-dichlorophenoxyacetic acid (2,4-D) and abscisic acid (ABA). These extracts were shown to be essentially devoid of protease and nuclease activities and they were tested for their ability to relax supercoiled DNA, unknotting P4 DNA and catenate circular duplex DNA under catalytic conditions. Unknotting and catenation reactions are strictly magnesium- and ATP-dependent, but not the relaxation of circular supercoiled DNA allowing the detection of both topoisomerase I and II activities. Two cytotoxic drugs, camptothecin, a plant alkaloid that inhibits cukaryotic topoisomerase I, and epipodophyllotoxin VM-26 (teniposide) that inhibits topoisomerase II, have been assayed in our extracts showing similar inhibitory effects on topoisomerase enzymes. Alkaline phosphatase treatment of nuclear extracts abolishes both topoisomerase activities. Nuclear extracts from embryos treated with 2,4-D showed 200% increase on topoisomerase II activity as compared with untreated ones, but only residual activity was detected in ABA-treated embryos. Nuclear extracts from hormone-treated and untreated embryos showed similar topoisomerase I activity with deviations of less than 25%. These differences are discussed in terms of possible post-translational modifications of the enzymes associated with the increase in proliferation activity of calli.     

Dihydroindenoisoquinolines function as prodrugs of indenoisoquinolines

Dihydroindenoisoquinolines are analogs of cytotoxic indenoisoquinoline topoisomerase I (Top1) inhibitors, exhibiting potent cytotoxicity but weak inhibitory activity toward Top1. Through COMPARE analysis, cytotoxicity studies in Top1-deficient cells, chemical synthesis and biological evaluation of methylated dihydroindenoisoquinoline 5, we demonstrated that dihydroindenoisoquinolines function as prodrugs of indenoisoquinolines in cancer cells.     

Synthesis of 6-chloroisoquinoline-5,8-diones and pyrido[3,4-b]phenazine-5,12-diones and evaluation of their cytotoxicity and DNA topoisomerase II inhibitory activity

The substituted chloroisoquinolinediones and pyrido[3,4-b]phenazinediones were synthesized, and the cytotoxic activity and topoisomerase II inhibitory activity of the prepared compounds were evaluated. Chloroisoquinolinediones have been prepared by the reported method employing 6,7-dichloroisoquinoline-5,8-dione. The cyclization to pyrido[3,4-b]phenazinediones was achieved by adding the aqueous sodium azide solution to the dimethylformamide solution of corresponding chloroisoquinoline-5,8-dione. The cytotoxicity of the synthesized compounds was evaluated by a SRB (Sulforhodamine B) assay against various cancer cell lines such as A549 (human lung cancer cell line), SNU-638 (human stomach cancer cell), Col2 (human colon cancer cell line), HT1080 (human fibrosarcoma cell line), and HL-60 (human leukemia cell line). Almost all the synthesized pyrido[3,4-b]phenazinediones showed greater cytotoxic potential than ellipticine (IC(50)=1.82-5.97 microM). In general, the cytotoxicity of the pyrido[3,4-b]phenazinediones was higher than that of the corresponding chloroisoquinolinediones. The caco-2 cell permeability of selected compounds was 0.62 x 10(-6)-35.3 x 10(-6)cm/s. The difference in cytotoxic activity among tested compounds was correlated with the difference in permeability to some degree. To further investigate the cytotoxic mechanism, the topoisomerase II inhibitory activity of the synthesized compounds was estimated by a plasmid cleavage assay. Most of compounds showed the topoisomerase II inhibitory activity (28-100%) at 200 microM. IC(50) values for the most active compound 6a were 0.082 microM. However, the compounds were inactive for DNA relaxation by topoisomerase I at 200 microM.     

Topoisomerases of kinetoplastid parasites: why so fascinating?

DNA topoisomerases are the key enzymes involved in carrying out high precision DNA transactions inside the cells. However, they are detrimental to the cell when a wide variety of topoisomerase-targeted drugs generate cytotoxic lesions by trapping the enzymes in covalent complexes on the DNA. The discovery of unusual heterodimeric topoisomerase I in kinetoplastid family added a new twist in topoisomerase research related to evolution, functional conservation and their preferential sensitivity to Camptothecin. On the other hand, structural and mechanistic studies on kinetoplastid topoisomerase II delineate some distinguishing features that differentiate the parasitic enzyme from its prokaryotic and eukaryotic counterparts. This review summarizes the recent advances in research in kinetoplastid topoisomerases, their evolutionary significance and the death of the unicellular parasite Leishmania donovani induced by topoisomerase I inhibitor camptothecin.     

Analogues of antifungal tjipanazoles from rebeccamycin

Analogues of antifungal tjipanazoles were obtained by semi-synthesis from rebeccamycin, an antitumor antibiotic isolated from cultures of Saccharothrix aerocolonigenes. The antiproliferative activities of the new compounds were evaluated in vitro against nine tumor cell lines. The effect on the cell cycle of murine leukemia L1210 cells was examined and the antimicrobial activities against two Gram positive bacteria, a Gram negative bacterium and a yeast were determined. The inhibitory properties toward four kinases and toward topoisomerase I were evaluated. The most cytotoxic compound in the series was a dinitro derivative characterized as a potent topoisomerase I inhibitor.     

Synthesis, topoisomerase I inhibition and antitumor cytotoxicity of 2,2':6',2"-, 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives

For the development of new anticancer agents, 2,2':6',2"-, 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives were designed and evaluated for their topoisomerase I inhibitory activity and antitumor cytotoxicity. Structure-activity relationship studies indicated that 2,2':6',2"-terpyridine derivatives were highly cytotoxic toward several human tumor cell lines, whereas 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives were potent topoisomerase I inhibitors.     

Synthesis, cytotoxic activities and structure-activity relationships of topoisomerase I inhibitors: indolizinoquinoline-5,12-dione derivatives

A series of indolizinoquinoline-5,12-dione derivatives (IQDs) are synthesized and evaluated for their cytotoxic activities toward human lung adenocarcinoma (GLC-82), large-cell lung carcinoma (NCI-H460), promyelocytic leukemia (HL-60) and breast carcinoma (MCF-7) cells by MTT method. Most of the IQDs show significant cytotoxic potency. In addition, the evaluation of structure-activity relationships indicated that the incorporation of electron-withdrawing substituents at the C or D ring will enhance the activities of the target compounds distinctly. The topoisomerase I inhibitory activity is also measured.     

Phosphate ester derivatives of homocamptothecin: Synthesis,solution stabilities and antitumor activities

Homocamptothecins (hCPTs) represents a new promising class of topoisomerase I inhibitors with enhanced stability and superior antitumor activity. Some phosphodiesters and phosphotriesters homocamptothecin derivatives were designed and synthesized based on our previous synthetic route. The cytotoxicity in vitro on three cancer cell lines and antitumor activity in vivo, and inhibitory properties of topoisomerase I of these derivatives were evaluated. Among them compounds 24e and 24f exhibited higher cytotoxic activity than IRT and the former exhibited the best antitumor activity in vivo and solution stability both at pH 7.4 and pH 3.0.     

New insight for fluoroquinophenoxazine derivatives as possibly new potent topoisomerase I inhibitor

Fluoroquinolones, represented by ciproxacin and norfloxacin, are well-known clinical antimicrobial agents, and their phenyl ring expanded quinophenoxazines are reported as possible antitumor active compounds. These quinophenoxazines are known to inhibit DNA topoisomerase II essential for cell replication cycle. But there were no reports for topoisomerase I inhibition study for these compounds. In this report, we have prepared a few quinophenoxazine analogues and tested their topoisomerases I and II inhibitory activities and cytotoxicity. From the result, we found that quinophenoxazine analogues possessed strong topoisomerase I inhibitory capacity as well as topoisomerase II inhibition. Among the compounds prepared, A-62176 analogues showed strong topoisomerases I and II inhibitory activities. Interestingly, compound 8 missing the 3-aminopyrrolidine moiety at C2 position has similar potent inhibitory capacity against topoisomerases I and II at higher concentrations (20 and 10 microM, respectively). But compound 8 inhibited topoisomerase I function more selectively at lower concentration, 2 microM. Our observation might strongly implicate that fluoroquinophenoxazines can be developed as efficient topoisomerase I inhibitor with the elaborate modification.     

Synthesis and topoisomerase II inhibitory and cytotoxic activity of oxiranylmethoxy- and thiiranylmethoxy-chalcone derivatives

In order to find potential anticancer drug candidate targeting topoisomerases enzyme, we have designed and synthesized oxiranylmethoxy- and thiiranylmethoxy-retrochalcone derivatives and evaluated their pharmacological activity including topoisomerases inhibitory and cytotoxic activity. Of the compounds prepared compound 25 showed comparable or better cytotoxic activity against cancer cell lines tested. Compound 25 inhibited MCF7 (IC₅₀: 0.49 ± 0.21 μM) and HCT15 (IC₅₀: 0.23 ± 0.02 μM) carcinoma cell growth more efficiently than references. In the topoisomerases inhibition test, all the compounds were inactive to topoisomerase I but moderate inhibitors to topoisomerase II enzyme. Especially, compound 25 inhibited topoisomerase II activity with comparable extent to etoposide at 100 μM concentrations. Correlation between cytotoxicity and topoisomerase II inhibitory activity implies that compound 25 can be a possible lead compound for anticancer drug impeding the topoisomerase II function.     

Topoisomerase I inactivation by a novel thiol reactive naphthoquinone

The naphthoquinone adduct 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) contains structural features of both the anthracycline and isoquinone chemotherapeutics. An initial characterization showed TU100 is cytotoxic to mammalian cells and can inhibit topoisomerase I and II. Analysis using topoisomerase I now reveals TU100 is a slow acting inhibitor targeting the enzyme in the absence of DNA. Diluting pre-incubated TU100 and topoisomerase I failed to alleviate inhibition, suggesting the enzyme is being covalently modified. Critical cysteine thiols were identified as the possible target based on the ability of reducing agents to reverse TU100 inhibition. Consistent with this idea, TU100 protected topoisomerase I from inactivation by the sulfhydryl modifying agent N-ethylmaleimide (NEM). Unlike agents nonspecifically reacting with thiols, however, TU100 is specific for topoisomerase because it failed to inhibit a cysteine dependent protease. These results indicate TU100 is a novel naphthoquinone that inactivates free topoisomerase I via alkylation of cysteine residues.     

Synthesis and cytotoxic activity of substituted luotonin A derivatives

Luotonin A is a cytotoxic alkaloid that has been shown to inhibit topoisomerase I via stabilization of the binary complex topoisomerase-DNA in the same fashion as camptothecin. The synthesis and the cytotoxic activity on the lung carcinoma cell line H460 of a series of derivatives of Luotonin A is reported. The compounds inhibit topoisomerase I but show weak cytotoxic activity, thus confirming the peculiarity of ring E of camptothecin for antitumor activity.     

Synthesis, topoisomerase I inhibition and structure-activity relationship study of 2,4,6-trisubstituted pyridine derivatives

For the development of new anticancer agents, phenyl, 2-pyridyl, 2-furyl, 2-thienyl, 2-furylvinyl and 2-thienylvinyl substituted derivatives on 2,4,6-position in pyridine moiety were prepared and evaluated for their topoisomerase I inhibitory activity. Among the thirteen prepared compounds, four compounds exhibited strong topoisomerase I inhibitory activity. A structure-activity relationship study indicated that the 2-thienyl-4-furylpyridine skeleton was important for topoisomerase I inhibitory activity.     

Topoisomerase I‐Mediated Antiproliferative Activity of 10‐Substituted and 12‐Substituted Homocamptothecins

Homocamptothecin (hCPT) is an E‐ring modified camptothecin (CPT) analogue, which showed pronounced inhibitory activity of topoisomerase I. In search of novel hCPT‐type anticancer agents, two series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines. The results indicated that the 10‐substituted hCPT derivatives had a considerably higher cytotoxic activity than the 12‐substituted ones. Among the 10‐substituted compounds, 8a, 8b, 9b , and 9i showed an equivalent or even more potent activity than the positive control drug topotecan against the lung cancer cell line A‐549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory activity than CPT at a concentration of 100 μM .     

Topoisomerase I-mediated DNA cleavage as a guide to the development of antitumor agents derived from the marine alkaloid lamellarin D: triester derivatives incorporating amino acid residues

The marine alkaloid lamellarin D (LAM-D) has been recently characterized as a potent poison of human topoisomerase I endowed with remarkable cytotoxic activities against tumor cells. We report here the first structure-activity relationship study in the LAM-D series. Two groups of triester compounds incorporating various substituents on the three phenolic OH at positions 8, 14 and 20 of 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-6-one pentacyclic planar chromophore typical of the parent alkaloid were tested as topoisomerase I inhibitors. The non-amino compounds in group A showed no activity against topoisomerase I and were essentially non cytotoxic. In sharp contrast, compounds in group B incorporating amino acid residues strongly promoted DNA cleavage by human topoisomerase I. LAM-D derivatives tri-substituted with leucine, valine, proline, phenylalanine or alanine residues, or a related amino side chain, stabilize topoisomerase I-DNA complexes. The DNA cleavage sites detected at T downward arrow G or C downward arrow G dinucleotides with these molecules were identical to that of LAM-D but slightly different from those seen with camptothecin which stimulates topoisomerase I-mediated cleavage at T downward arrow G only. In the DNA relaxation and cleavage assays, the corresponding Boc-protected compounds and the analogues of the non-planar LAM-501 derivative lacking the 5-6 double bond in the quinoline B-ring showed no effect on topoisomerase I and were considerably less cytotoxic than the corresponding cationic compounds in the LAM-D series. The presence of positive charges on the molecules enhances DNA interaction but melting temperature studies indicate that DNA binding is not correlated with topoisomerase I inhibition or cytotoxicity. Cell growth inhibition by the 41 lamellarin derivatives was evaluated with a panel of tumor cells lines. With prostate (DU-145 and LN-CaP), ovarian (IGROV and IGROV-ET resistant to ecteinascidin-743) and colon (LoVo and LoVo-Dox cells resistant to doxorubicin) cancer cells (but not with HT29 colon carcinoma cells), the most cytotoxic compounds correspond to the most potent topoisomerase I poisons. The observed correlation between cytotoxicity and topoisomerase I inhibition strongly suggests that topoisomerase I-mediated DNA cleavage assays can be used as a guide to the development of superior analogues in this series. LAM-D is the lead compound of a new promising family of antitumor agents targeting topoisomerase I and the amino acid derivatives appear to be excellent candidates for a preclinical development.     

Furanocoumarins: Novel topoisomerase I inhibitors from Ruta graveolens L.

Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors.