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Three series of indeno[1,2-c]isoquinolines bearing a ferrocenyl entity were synthesized and evaluated for DNA interaction, topoisomerase I and II inhibition, and cytotoxicity against breast human cancer cell lines. In the first and second series, the ferrocenyl scaffold was inserted as a linker between the two nitrogen atoms. In the last series, it was introduced at the end of the carbon chain. The present study showed that the ferrocenyl entity enhanced the topoisomerase II inhibition. Most compounds showed a potent growth inhibitory effect on MDA-MB-231 cell line with the IC50 in μM range., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Three series of indeno[1,2-c]isoquinolin-5,11-dione-amino acid conjugates were designed and synthesized. Amino acids were connected to the tetracycle through linkers with lengths of n=2 and 3 atoms using ester (series I), amide (series II), and secondary amine (series III) functions. DNA binding was evaluated by thermal denaturation and fluorescence measurements. Lysine and arginine substituted derivatives with n=3 provided the highest DNA binding. Arginine derivative 32 (n=2, series II) and glycine derivative 34 (n=2, series III) displayed high topoisomerase II inhibition. Incrementing the length of the N-6 side chain from two to three methylene units provided a significant increase in DNA affinity but a substantial loss in topoisomerase II inhibition. The most cytotoxic compounds toward HL60 leukemia cells were 19, 33, and 34 displaying micromolar IC(50) values. When tested with the topoisomerase II-mutated HL60/MX2 cell line, little variation of IC(50) values was found, suggesting that topoisomerase II might not be the main target of these compounds and that additional targets could be involved., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A series of amino- and glycoconjugates of pyrido[4,3,2-kl]acridine and pyrido[4,3,2-kl]acridin-4-one have been prepared. The most active molecules, the amino conjugates 7 and 11, display a cytostatic activity against HT-29 cancer cells at micromolar concentration. This activity correlates well with a strong DNA binding. The molecules, amino or glycoconjugates, bind DNA by intercalation, the amino or glyco substituent being located in one groove. None of the molecules inhibits topoisomerase activity.

We have identified a novel series of indenoindole derivatives endowed with potent cytotoxic activities toward cancer cells. Five compounds containing a 8-[2-(dialkylamino)ethoxy]-2,3-dimethoxy-5H-10H-indeno[1,2-b]indol-10-one-O-propynyl-oxime core substituted with a phenyl, furanyl, or a methyl substituent on the propynyl side chain have been synthesized and their mechanism of action was investigated using a panel of complementary biophysical and biochemical techniques. The compounds were shown to intercalate into DNA with a preference for AT-rich sequences. They have no effect on topoisomerase I but they strongly stimulate DNA cleavage by topoisomerase II. Their capacity to stabilize topoisomerase II-DNA covalent complexes is comparable to that of the reference drug etoposide. The nature and orientation of the substituent on the propynyl chain modulate the DNA binding and topoisomerase II inhibitory properties of the compounds and, apparently, there is a correlation between the cytotoxic potential and the molecular action at the DNA-topoisomerase II level. The growth of human K562 leukemia cells is strongly reduced in the presence of the indenoindoles (IC(50) in the 50nM range) which maintain a high cytotoxic activity toward the adriamycin-resistant K562adr cells line in vitro. The low resistance indexes measured with the indenoindoles (RRI = 10-30) compared to adriamycin (RRI = 1000) suggest that our new compounds are weakly or not sensitive to drug efflux mediated by glycoprotein-P and/or multidrug resistance (MDR) protein pumps. Finally, we also show that these indenoindoles arrest K562 cells in the G2/M phase of the cell cycle and promote apoptosis, as indicated by the appearance of internucleosomal DNA cleavage. One compound in the series was tested for in vivo antitumor activity against the colon 38 model and at 25mg/kg it showed 100% complete tumor regression in the treated mice, without significant body weight loss. Altogether, the results reported here establish that our indenoindole derivatives represent a novel interesting series of DNA-targeted cytotoxic agents.

Fagaronine and nitidine are natural benzo[c] phenanthridinium alkaloids, which display antileukemic activity. Both act as topoisomerase I and topoisomerase II inhibitors. The objective of the present study was to prepare noncharged isosters of these compounds, with replacement of the aromatic A ring by a pyridine ring, present in other topoisomerase I inhibitors. Various 7,8- and 8,9-dimethoxy and metylenedioxy benzo[c][1,7] and [1,8]phenanthrolines were readily synthesized by benzyne-mediated cyclization of the corresponding substituted N-(2-halobenzyl)-5-quinolinamines or 5-isoquinolinamines. In both series, compounds bearing oxygenated substituents at positions 8 and 9 exhibited cytotoxic properties towards L1210 murine leukemia cells, which may result from their capacities to intercalate into DNA. Topoisomerase I inhibition was observed for all active compounds.

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.

In the course of structure-activity relationship studies, new rebeccamycin derivatives substituted in 3,9-positions on the indolocarbazole framework, and a 2',3'-anhydro derivative were prepared by semi-synthesis from rebeccamycin. The antiproliferative activities against nine tumor cell lines were determined and the effect on the cell cycle of murine leukemia L1210 cells was examined. Their DNA binding properties and inhibitory properties toward topoisomerase I and three kinases PKCzeta, CDK1/cyclin B, CDK5/p25 and a phosphatase cdc25A were evaluated. The 3,9-dihydroxy derivative is the most efficient compound of this series toward CDK1/cyclin B and CDK5/p25. It is also characterized as a DNA binding topoisomerase I poison. Its broad spectrum of molecular activities likely accounts for its cytotoxic potential. This compound which displays a tumor cell line-selectivity may represent a new lead for subsequent drug design in this series of glycosylated indolocarbazoles.

In recent years, several glycosyl indolocarbazole derivatives have been developed as antitumor agents targeting the topoisomerase I-DNA complex and a few of them were evaluated in clinical trials. The lead drug in the series is compound A which bears a formylamino substituent on the N-imide F-ring. This compound has shown promising antitumor activities in vivo and was tested clinically but it has been recently replaced with a more active analogue, J-107088, bearing a (hydroxymethyl-2-hydroxy) ethylamino substituent on the N-imide F-ring. We have compared the plasma stability of two molecules in this series, compounds A and D, which only differ by the nature of the group on the imide ring. The conversion of the compounds into the anhydride species B was studied by HPLC and the resulting metabolite, formed both in human plasma ultrafiltrate and in water, was characterized by NMR and mass spectrometry. Absorption measurements provided a facile method to follow the conversion of compounds A and D into their metabolite product B. Altogether, the experimental data demonstrate that the replacement of the NHCHO substituent of compound A with a hydrophilic NHCH(CH(2)OH)(2) chain preserves the intact imide function that is known to be essential for topoisomerase I inhibition and cytotoxicity. The transformation of compound A into the anhydride metabolite B (or its diacid open form) occurs much more slowly compared to compound D. Half-life parameter t(1/2) of 67 and 245 min(-1) were calculated for compounds A and D, respectively. A molecular modeling analysis, performed to compare the conformation and electronic properties of compounds A and D, offers a rational explanation for the gain of chemical stability of the indolocarbazole derivative D. The data provide important information for the rational design of antitumor indolocarbazole derivatives.

Ascididemin (ASC) is a pentacyclic DNA-intercalating agent isolated from the Mediterranean ascidian Cystodytes dellechiajei. This marine alkaloid exhibits marked cytotoxic activities against a range of tumor cells, but its mechanism of action remains poorly understood. We investigated the effects of ASC on DNA cleavage by human topoisomerases I and II. Relaxation assays using supercoiled DNA showed that ASC stimulated double-stranded cleavage of DNA by topoisomerase II, but exerted only a very weak effect on topoisomerase I. ASC is a conventional topoisomerase II poison that significantly promoted DNA cleavage, essentially at sites having a C on the 3' side of the cleaved bond (-1 position), as observed with etoposide. The stimulation of DNA cleavage by topoisomerase I in the presence of ASC was considerably weaker than that observed with camptothecin. Cytotoxicity measurements showed that ASC was even less toxic to P388 leukemia cells than to P388CPT5 cells resistant to camptothecin. In addition, the marine alkaloid was found to be equally toxic to HL-60 leukemia cells sensitive or resistant to mitoxantrone. It is therefore unlikely that topoisomerases are the main cellular targets for ASC. This alkaloid was found to strongly induce apoptosis in HL-60 and P388 leukemia cells. Cell cycle analysis showed that ASC treatment was associated with a loss of cells in the G1 phase accompanied with a large increase in the sub-G1 region. Cleavage experiments with poly(ADP-ribose) polymerase (PARP) revealed that caspase-3 was a mediator of the apoptotic pathway induced by ASC. The DNA of ASC-treated cells was severely fragmented. Collectively, these findings indicate that ASC is a potent inducer of apoptosis in leukemia cells.