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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.)

We have carried out a stratified phase II study of sorafenib (So) in patients with advanced angiosarcoma (n = 32) and epithelioid hemangioendothelioma (n = 13). This report concerns the correlative analysis of the predictive values of circulating pro/anti-angiogenetic biomarkers. Using the ELISA method (R&D Systems), circulating biomarkers (VEGF-A, in picograms per milliliter), thrombospondin-1 (TSP1, in micrograms per milliliter), stem cell factor (SCF, in picograms per milliliter), placental growth factor (PlGF, in picograms per milliliter), VEGF-C (in picograms per milliliter), and E-selectin (in nanograms per milliliter) were measured before So treatment and after 7 days. VEGF-A (mean value 475 vs. 541, p = 0.002), TSP1 (16 vs. 24, p = 0.0002), and PlGF (20.9 vs. 40.7, p = 0.0001) significantly increased during the treatment. Treatment did not affect the levels of SCF, VEGF-C, and E-selectin. Only two biomarkers were associated with better outcome as follows: VEGF-A and PlGF. Best objective response and non-progression at 180 days were associated with low level of VEGF-A at baseline (p = 0.04 and 0.03, respectively). There was a correlation between the circulating level of VEGF-A and time to progression (TTP) (r = -0.47, p = 0.001). Best objective response and non-progression at 180 days were not associated with baseline level of PIGF, but there was a correlation between the circulating level of PIGF at baseline and TTP. Low level of VEGF-A at baseline (<500) was significantly associated with better outcome.

Background: At this center, therapeutic drug monitoring of methotrexate (MTX) used to be performed by fluorescence polarization immunoassay (FPIA). We observed an increasing number of unusual high MTX concentrations at 48 and 72 hours during a couple of years. This study aimed to identify the causes of this variation., Methods: A retrospective analysis was conducted on 272 patients hospitalized between January 2008 and October 2012. The whole MTX use system was analyzed using Ishikawa's method. The proportion of MTX concentrations ≤0.2 μmole/L at 48 (P48h) and 72 hours (P72h) was recorded and compared between both FPIA and EMITSiemens assays. A χ or a Fisher exact test was used (α = 0.05)., Results: Because of an announced withdrawal of the FPIA reagent, the method was switched in 2009 to an immunoenzymatic technique (EMITSiemens). Both P48h and P72h dropped significantly after 2009 (P48h: 45% versus 5% and P72h: 91% versus 47%; P < 0.0001). The replacement of the EMITSiemens reagent by the EMITARK Diagnostics reagent in 2012 led to an increase in both P48h and P72h. No significant difference was found in the proportions of MTX ≤0.2 μmole/L concentrations between FPIA and EMITARK Diagnostics at 48 (45% and 40%; P = 0.556) and 72 hours (91% and 100%; P = 0.231). Both internal and external quality control assessments gave regular satisfactory results during the study period. Furthermore, the interassay comparisons that were performed with internal quality controls and spiked serum samples showed similar results at the time of both shifts. The other changes observed in the MTX circuit were not associated with MTX concentration variations., Conclusions: The overestimation of the plasma concentration of MTX was concluded to be because of the assay reagent. A further study is consequently necessary to assess the impact of this analytical pitfall on the patients' survival.

The synthesis of new acridinone and dioxophenothiazine derivatives along with their tubulin polymerization inhibitory and antiproliferative activities is reported. The analysis of correlation for cytotoxic and antitubulin potential of tested compounds showed that 4-methoxyphenylethyl derivatives 18a and 19a were highly cytotoxic but were regarded to have no significant antitubulin activity. However, the introduction of a 3-hydroxy substituent leading to compounds 18e and 19e, strongly increased the antitubulin potential but was associated with a loss of the antiproliferative activity. Modeling studies, topoisomerase inhibition assays and cell cycle analysis have been performed to better investigate the mechanism of action of such compounds., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A library of substituted chromeno[3,4-b]indoles was developed as Lamellarin isosters. Synthesis was achieved from indoles after a four-step pathway sequence involving C-3 iodination, a Suzuki cross-coupling reaction, and a one pot deprotection/lactonisation step. Twenty final compounds were tested in order to determine their activity against topoisomerase I and kinases, the two major biological activities of Lamellarins. One newly synthesized derivative exhibited a strong topoisomerase activity comparable to reference compounds such as campthotecin and Lamellarin with only a weak kinase inhibition. Two other lead compounds were identified as new nanomolar DYRK1A inhibitors and several other drugs affected the kinases in the sub-micromolar range. These results will enable us to use the chromeno[3,4-b]indole as a pharmacophore to develop potent treatments for neurological or oncological disorders in which DYRK1A is fully involved., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A number of mono- or diaminoalkylated indeno[1,2-c]isoquinolin-5,11-diones analogs of 1 were synthesized and evaluated for their DNA binding affinities, topoisomerase inhibition properties and antiproliferative activities against human cancer cell lines (HL60). Impact of the side chain connected to the aromatic D ring and to the N6 lactam position on the biological profile will be discussed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

New N-alkylanilinoquinazoline derivatives 5, 12, 20, and 22 have been prepared from 4-chloro-6,7-dimethoxyquinazoline 3, 4-chloro-6,7-methylenedioxyquinazoline 19, and commercially available anilines. Differents classes of compounds substituted by an aryloxygroup (6a-c, 16a,b, and 17a,b), (aminophenyl)ureas (12a,b and 13a-f), anilines (4a-m, 20a,b), N-alkyl(aniline) (5a-m, 21a,b, 22a,d), and N-aminoalkyl(aniline) (22e-g) have been synthesized. These molecules were evaluated for their cytotoxic activities and as potential DNA intercalating agents. We studied the strength and mode of binding to DNA of these molecules by DNA melting temperature measurements, fluorescence emission, and circular dichroism. The results of various spectral and gel electrophoresis techniques obtained with the different compounds, in particular compounds 5g and 22f, revealed significant DNA interaction. These experiments confirm that the N-aminoalkyl(anilino)-6,7-dimethoxyquinazoline nucleus is an efficient pharmacophore to trigger binding to DNA, via an intercalative binding process.

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.)

New 1,5-dihydro-4-(substituted phenyl)-3H-furo[3,4-b]carbazol-3-ones were synthesised via a key step Diels-Alder reaction under microwave irradiation. 3-Formylindole was successfully used in a 6-step synthesis to obtain those complex heterocycles. The Diels-Alder reaction generating the carbazole ring was optimised under thermal conditions or microwave irradiation. After cleavage of functional groups, DNA binding, topoisomerase inhibition and cytotoxic properties of the new-formed furocarbazoles were investigated. These carbazoles do not present a strong interaction with the DNA, and do not modify the relaxation of the DNA in the presence of topoisomerase I or II except for one promising compound. This compound is a potent topoisomerase II inhibitor, and its cellular activity is not moderated compared to etoposide. The synthesis of these molecules allowed the generalisation of the method using indole and 5-OBn indole and several benzaldehydes. The synthesis of these molecules produced chemical structures endowed with promising cytotoxic and topoisomerase II inhibition activities., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

A short route, involving a tetramolecular condensation reaction and a Pd/C catalyst-H(2)-mediated reductive N-heteroannulation as the key-steps, has been found for the synthesis of some new penta- and heptacyclic indolo- (12), quinolino- (13) and indoloquinolinocarbazole (11) derivatives. HF-DFT (B3LYP) energy profiles and NMR calculations were carried out to help in the understanding of the experimental results. N-Alkylated indoloquinolinocarbazoles (16b, 17a, 17b and 18) were prepared and screened essentially toward some cancer-(G-quadruplex, DNA, topoisomerase I) and CNS-related (kinases) targets. Biological results evidenced 13 as a potent CDK-5 and GSK-3β kinases inhibitor, while di- or triaminopropyl-substituted indoloquinolinocarbazoles 17b or 18 targeted rather DNA-duplex or telomeric G-quadruplex structures, respectively.

Two new heterocycles, pyrimido[4,5-c]carbazole and pyrimido[5,4-b]indole, were prepared in three steps from 3-aminocarbazole and 3-aminoindole, respectively. The key Friedel-Crafts intramolecular cyclization was realized under microwave irradiation using montmorillonite K-10 clay as a catalyst. The pyrimido[4,5-c]carbazole derivative shows significant micromolar IC(50) against cancer cell lines. Unlike similar carbazole and indolocarbazole compounds, the molecule does not interfere with topoisomerase activity., (2010 Elsevier Ltd. All rights reserved.)

The biological evaluation of some novel thiazoloindolo[3,2-c]quinoline, 8-substituted-11H-indolo[3,2-c]quinolines is described. These compounds were obtained via Graebe-Ullmann thermal cyclization from appropriated N-arylated benzotriazoles. 7H-4,7-Diaza-benzo[de]anthracene, a reaction by-product structurally closed to the pyridoacridine skeleton was also identified. All thiazolobenzotriazole intermediates were tested in vitro for their capacity to inhibit the growth of two breast cancer cell lines, MCF-7 and MDA-MB-231. In parallel, the newly synthesized skeletons were evaluated for DNA interaction, topoisomerases' inhibition, and cytotoxicity against HL60 and HL60/MX2 human leukemia cells. Most compounds showed a potent growth inhibitory effect on all the tested cell lines, with IC(50) in the muM range.

Indeno[2,1- c]quinolin-7-ones and 6 H-indeno[1,2- c]isoquinolin-5,11-diones, bearing two cationic aminoalkyl side chains, were synthesized and evaluated for DNA interaction, topoisomerases inhibition, and cytotoxicity against human cancer cell lines. They displayed strong interaction with DNA and one indeno[1,2- c]isoquinolin-5,11-dione bearing side chains at N-6 and C-8 positions ( 6a) was a potent human topoisomerase II inhibitor with high cytotoxicity toward HL60 cells. An increased topoisomerase II inhibition is found with (a) a cationic aminoalkyl side chain at the C-8 rather than at the C-9 position, (b) a dimethylaminoethoxy side chain at the C-8 position introduced on the N-6 monosubstituted derivative, going with suppression of topoisomerase I poisoning, and (c) a dimethylaminoethyl rather than a dimethylaminopropyl side chain at the N-6 position. The cytotoxicity was only partially reduced when using the topoisomerase II-mutated mitoxantrone-resistant HL60/MX2 cell line, suggesting that additional targets are involved in their mechanism of action. These indeno[1,2- c]isoquinolin-5,11-dione derivatives represent new DNA-topoisomerase II interfering anticancer molecules.

We report the synthesis and biological evaluation of new oxophenylarcyriaflavins designed as potential anticancer agents. An efficient synthesis involving palladium-catalyzed Suzuki and Stille reactions is presented, without any indolic protective group. The central ring closure of the scaffold was performed through an electrophilic reaction on the position C-2 of the indole ring. The use of indole and 5-benzyloxyindole, along with substituted phenyl rings, generated three different scaffolds, which were successively exploited to modulate the structure. The cytotoxicity of the newly designed compounds on four cancer cell lines and activities against three kinases (CDK1, CDK5 and GSK3) were evaluated. Several compounds showed a marked cytotoxicity with IC(50) values in the sub-micromolar range, and induced important cell cycle perturbations, with a G2/M arrest. Some compounds revealed DNA binding properties and were found to inhibit topoisomerase-mediated DNA relaxation of supercoiled DNA, but these properties are not mandatory for a cytotoxic action. A novel lead compound () has been identified and warrants further investigations.

In the context of the design and synthesis of minor groove binding and intercalating DNA ligands some new oligopyrrole carboxamides were synthesized. These hybrid molecules (combilexins) possess a variable and conformatively flexible spacer at the N-terminal end. As intercalating tricyclic systems acridone, acridine, anthraquinones and in a special case iminostilbene terminate the N-terminal end of the pyrrole chain. The cytotoxicity was examined by the NCI antitumor screening, furthermore, biophysical as well as biochemical studies were performed in order to get some information about the DNA binding properties and topoisomerase inhibition effect of this new series of molecules.

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.

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

A series of 2,6-diphenylpyrazine derivatives was synthesized from 2,6-dichloropyrazine and 4-methoxyphenylboronic acid using palladium(0) as catalyst in a Suzuki methodology. After deprotection of the hydroxyl, alkylation reactions with different halides afforded compounds 5-8 bearing hydrophilic chains. DNA binding and cytotoxic properties were investigated. Compound 11 bearing imidazoline terminal groups was found to be a potent AT-specific DNA minor groove binder but there was no relationship between DNA interaction and cytotoxicity. However, in all cases the incorporation of the pyrazine ring was found to promote the cytotoxicity of the molecules compared to the corresponding pyridine analogues, previously synthesized.

Pre-mRNA splicing is an essential step of the expression of most metazoan protein-coding genes, which is often regulated in a cell type-specific or developmental manner. We have demonstrated previously that human DNA topoisomerase I, an extensively studied target for anticancer drugs, also has an intrinsic protein kinase activity that specifically phosphorylates proteins involved in splice site selection. Therefore, DNA topoisomerase I was recently shown to play a critical role in alternative splicing. Here, we have exploited these novel properties of DNA topoisomerase I to develop entirely novel diospyrin derivatives targeting its protein kinase activity and thereby modulating pre-mRNA splicing. Although some derivatives indeed inhibit kinase activity of topoisomerase I, they did not block reactions of topoisomerase I on DNA. However, these drugs interfere with camptothecin-dependent topoisomerase I-mediated DNA cleavage, implying that diospyrin derivatives mediate a conformational change of topoisomerase I. It is note-worthy that in vitro splicing reactions revealed that diospyrin derivatives alter various steps of splicing. Some diospyrin derivatives inhibit either the first or the second catalytic step of splicing but not spliceosome assembly, whereas diospyrin itself prevents the formation of full spliceosome. Our data revealed for the first time that diospyrin derivatives are able to stall the dynamic assembly of the spliceosome and open the exciting possibility of using these derivatives to correct aberrant splicing in human genetic diseases.

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.

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.

Cassytha filiformis (Lauraceae), a widely distributed parasitic plant, contains several aporphine alkaloids and is often used in African folk medicine to treat cancer, African trypanosomiasis and other diseases. In a previous investigation, we showed that the alkaloid plant extract and the isolated aporphines possessed in vitro cytotoxic properties. In this paper, we evaluated the in vitro activity of the alkaloid extract (IC50 = 2.2 microg/mL) and its three major aporphine alkaloids (actinodaphnine, cassythine, and dicentrine) on Trypanosoma brucei brucei as well as four related commercially available aporphines (bulbocapnine, glaucine, isocorydine, boldine). Only the three alkaloids from Cassytha filiformis were active on the trypanosomes in vitro (IC50 = 3-15 microM). Additionally, we compared the cytotoxicity of these seven compounds on HeLa cells. Glaucine was the most cytotoxic compound on HeLa cells (IC50 = 8.2 microM) in the series. In order to elucidate their mechanism of action, the binding mode of these molecules to DNA was studied by UV absorption, circular and linear dichroism spectroscopy. The results of the optical measurements indicated that all seven aporphines effectively bind to DNA and behave as typical intercalating agents. Biochemical experiments showed that actinodaphnine, cassythine and dicentrine also interfere with the catalytic activity of topoisomerases in contrast to the four other aporphines. These interactions with DNA may explain, at least in part, the effects observed on cancer cells and on trypanosomes.

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.

Oxovanadium(IV) complexes of hydroxysalen derivatives have been prepared and tested as DNA reactive agents. The nuclease activity has been investigated under oxidative or reducing conditions, on the basis of the various oxidation states of vanadium: V(III), V(IV) and V(V). In the absence of an activating agent, none of the compounds tested was able to induce cleavage of DNA, whereas in the presence of mercaptopropionic acid (MPA) or Oxone the four complexes induced DNA modifications. Under both conditions, the para-hydroxy complex was found to be the most active compound. Reaction of these salen complexes with DNA occurs essentially at guanine residues and is more efficient in the presence of Oxone than under reducing conditions. The extent of Oxone-mediated DNA oxidation by the four vanadyl complexes was clearly superior to VOSO(4) and was observed without piperidine treatment. EPR studies provided information on the reactive metal-oxo species involved under each conditions and a mechanism of reaction with DNA is discussed.

Burasaine is a plant alkaloid isolated from the roots of several species of the Burasaia genus endemic to Madagascar. It exhibits in vitro antiplasmodial activities but the molecular basis of this biological activity is not known. The strong structural similarity with the alkaloid berberine prompted us to postulate that burasaine could interact with DNA. To test this hypothesis, we investigated the mode of binding of burasaine to DNA and tested its cytotoxic potential toward human HL-60 leukemia cells. Its inhibitory activity toward topoisomerases I and II was also studied. Absorption and melting temperature measurements attested that burasaine forms stable complexes with DNA. The results of electric linear dichroism (ELD) spectroscopy may be interpreted either by an intercalation or by an external stacking parallel to the base pairs. The affinity of burasaine for DNA is slightly lower than that of berberine and this translates at the cellular level by a reduced cytotoxicity. Burasaine does not promote DNA cleavage by human topoisomerases I or II and this likely accounts for its very weak cytotoxic potential and its very modest effects on the cell cycle progression observed at high concentrations. The study identifies DNA as a potential bioreceptor for burasaine and contributes to a better understanding of the mechanism of action of benzoquinolizine alkaloids.

In the course of a program aimed at discovering novel DNA-targeted antiparasitic drugs, the phenylfuran-benzimidazole unfused aromatic dication DB293 was identified as the first diamidine capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Its preferred binding sequence encompasses the tetranucleotide 5'-ATGA.5'-TCAT to which DB293 binds tightly with a strong positive cooperativity. Here we have investigated the influence of the DNA sequence on drug binding using two complementary technical approaches: surface plasmon resonance and DNase I footprinting. The central dinucleotide of the primary ATGA motif was systematically varied to represent all of the eight possible combinations (AXGA and ATYA, where X or Y = A, T, G, or C). Binding affinities for each site were precisely measured by SPR, and the extent of cooperative drug binding was also determined. The sequence recognition process was found to be extremely dependent on the nature of the central dinucleotide pair. Modification of the central TG step decreases binding affinity by a factor varying from 2 to over 500 depending on the base substitution. However, the diminished binding affinity does not affect the unique binding mode. In nearly all cases, the SPR titrations revealed a positive cooperativity in complex formation which reflects the ease of the dication to form stacked dimeric motifs in the DNA minor groove. DNase I footprinting served to identify additional binding sites for DB293 in the context of long DNA sequences offering a large variety of randomly distributed or specifically designed sites. The ATGA motif provided the best receptor for the drug, but lower affinity sequences were also identified. The design of two DNA fragments composed of various targeted tetranucleotide binding sites separated by an "insulator" (nonbinding) sequence allowed us to delineate further the influence of DNA sequence on drug binding and to identify a novel high-affinity site: 5'-ACAA.5'-TTGT. Collectively, the SPR and footprinting results show that the consensus sequence 5'-(A/T)-TG-(A/T) represents the optimal site for cooperative dimerization of the heterocyclic diamidine DB293.

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.

Control of gene expression is a cherished goal of cancer chemotherapy. Small ligand molecules able to bind tightly to DNA in a well-defined configuration are being actively searched for. With this goal in mind, we have designed and synthesized the trifunctional molecule R-132, which combines a bispyrrole skeleton for minor groove DNA recognition and two different chromophores, anilinoacridine and ethidium. The affinity and mode of binding of R-132 to DNA were studied by a combination of complementary biochemical and biophysical techniques, which included absorption and fluorescence spectroscopy and circular and linear dichroism. A surface plasmon resonance biosensor analysis was also performed to quantify the kinetic parameters of the drug-DNA interaction process. Altogether, the results demonstrate that the three moieties of the hybrid molecule are engaged in the interaction process, thus validating the rational design strategy. At the biological level, R-132 stabilizes topoisomerase-II-DNA covalent complexes and displays potent cytotoxic activities, which are attributable to its DNA-binding properties. R-132 easily enters and accumulates in cell nuclei, as evidenced by confocal microscopy. R-132 therefore provides a novel lead compound for the design of gene-targeted anticancer agents.

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.

In the course of a medicinal chemistry program aimed at discovering novel tumour-active rebeccamycin derivatives targeting DNA and/or topoisomerase I, a series of analogues with the sugar residue linked to the two indole nitrogens was recently developed. Two promising drug candidates in this staurosporine-rebeccamycin hybrid series were selected for a DNA-binding study reported here. The DNA interaction of the cationic indolocarbazole glycosides MP059 bearing a N,N-diethylaminoethyl side chain and MP072 containing a sugar bearing an amino group was compared with that of the uncharged analogue MP024. The results show that the addition of a cationic substituent, either directly on the indolocarbazole chromophore or on the carbohydrate residue, significantly reinforces the interaction of the drugs with nucleic acids. The two cationic molecules MP059 and MP072 recognise preferentially sequences containing GpT.ApC and TpG.CpA steps but they do not inhibit topoisomerase I, in contrast to the parent uncharged derivative MP024 which stimulates DNA single strand breaks by topoisomerase I. The cytotoxic activity of the indolocarbazole derivatives bearing positively charged groups is one order of magnitude higher than that of the neutral compound MP024. The high cytotoxic potential can be attributed to the enhanced DNA binding and sequence recognition capacity of the cationic compounds. The study provides useful information for further structure-activity relationship studies in the indolocarbazole series.

We report the synthesis of an asymmetric Tröger base containing the two well characterised DNA binding chromophores, proflavine and phenanthroline. The mode of interaction of the hybrid molecule was investigated by circular and linear dichroism experiments and a biochemical assay using DNA topoisomerase I. The data are compatible with a model in which the proflavine moiety intercalates between DNA base pairs and the phenanthroline ring occupies the DNA groove. DNase I cleavage experiments were carried out to investigate the sequence preference of the hybrid ligand and a well resolved footprint was detected at a site encompassing two adjacent 5'-GTC.5-GAC triplets. The sequence preference of the asymmetric molecule is compared to that of the symmetric analogues.

To achieve a sequence-specific DNA cleavage by topoisomerase I, derivatives of the antitumor drug camptothecin have been covalently linked to triple helix-forming oligonucleotides that bind in a sequence-specific manner to the major groove of double-helical DNA. Triplex formation at the target sequence positions the drug selectively at the triplex site, thereby stimulating topoisomerase I-mediated DNA cleavage at this site. In a continuous effort to optimize this strategy, a broad set of conjugates consisting of (i) 16-20-base-long oligonucleotides, (ii) alkyl linkers of variable length, and (iii) camptothecin derivatives substituted on the A or B quinoline ring were designed and synthesized. Analysis of the cleavage sites at nucleotide resolution reveals that the specificity and efficacy of cleavage depends markedly on the length of both the triple-helical structure and the linker between the oligonucleotide and the poison. The optimized hybrid molecules induced strong and highly specific cleavage at a site adjacent to the triplex. Furthermore, the drug-stabilized DNA-topoisomerase I cleavage complexes were shown to be more resistant to salt-induced reversal than the complexes induced by camptothecin alone. Such rationally designed camptothecin conjugates could provide useful antitumor drugs directed selectively against genes bearing the targeted triplex binding site. In addition, they represent a powerful tool to probe the molecular interactions in the DNA-topoisomerase I complex.

The brominated anilinoquinazoline derivative PD153035 exhibits a very high affinity and selectivity for the epidermal growth factor receptor tyrosine kinase (EGF-R TK) and shows a remarkable cytotoxicity against several types of tumor cell lines. In contrast, its N-methyl derivative, designated EBE-A22, has no effect on EGF-R TK but maintains a high cytotoxic profile. The present study was performed to explore the possibility that PD153035 and its N-methyl analogue might interact with double-stranded DNA, which is a primary target for many conventional antitumor agents. We studied the strength and mode of binding to DNA of PD153035 and EBE-A22 by means of absorption, fluorescence, and circular and linear dichroism as well as by a relaxation assay using human DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to show that both drugs bind to DNA and behave as typical intercalating agents. In particular, EBE-A22 unwinds supercoiled plasmid, stabilizes duplex DNA against heat denaturation, and produces negative CD and ELD signals, as expected for an intercalating agent. Extensive DNase I footprinting experiments performed with a large range of DNA substrates show that EBE-A22, but not PD153035, interacts preferentially with GC-rich sequences and discriminates against homooligomeric runs of A and T which are often cut more readily by the enzyme in the presence of the drug compared to the control. Altogether, the results provide the first experimental evidence that DNA is a target of anilinoquinazoline derivatives and suggest that this N-methylated ring system is a valid candidate for the development of DNA-targeted cytotoxic compounds. The possible relevance of selective DNA binding to activity is considered. The unexpected GC-selective binding properties of EBE-A22 entreat further exploration into the use of N-methylanilinoquinazoline derivatives as tools for designing sequence-specific DNA binding ligands.

We report the synthesis and pharmacological evaluation of a novel homocamptothecin (hCPT) derivative, 12-Cl-hCPT, which contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone found in camptothecin (CPT) and bears a chloro substituent at position 12. The capacity of 12-Cl-hCPT to inhibit DNA topoisomerase I was compared with that of SN-38, the active metabolite of the clinically used antitumour prodrug CPT-11. In the DNA relaxation assay, 12-Cl-hCPT proved to be slightly more potent than SN-38 at stimulating the formation of nicked plasmid DNA molecules. A series of radiolabelled DNA restriction fragments were employed to identify and compare the position of the DNA cleavage sites induced by topoisomerase I in the presence of 12-Cl-hCPT and SN-38. These sequencing studies confirm that both 12-Cl-hCPT and SN-38 strongly promote DNA cleavage by topoisomerase I and reveal that the majority of the cleavage sites are located at the same nucleotide positions for the two drugs. However, a certain number of DNA cleavage sites were found to be specific to 12-Cl-hCPT. These sites, previously characterized with unsubstituted hCPT, generally correspond to 5'-CG sites whereas the sites common to the 12-Cl-hCPT and SN-38 essentially correspond to 5'-TG sites. We also quantified the formation of drug-induced protein-DNA complexes formed in HT29 human colon carcinoma cells. Trapping of endogenous proteins onto DNA was found to be much more efficient with 12-Cl-hCPT than with SN-38. These data provide a molecular basis to account for the enhanced antiproliferative activity of 12-Cl-hCPT compared with that of SN-38. Biological evaluation on a panel of sensitive and drug-resistant cell lines revealed 12-Cl-hCPT to be more cytotoxic to tumour cells than SN-38. 12-Cl-hCPT proved 14- and 23-fold more active than SN-38 toward the K562adr and T24anp multidrug-resistant cell lines, respectively. The marked topoisomerase I inhibitory properties of 12-Cl-hCPT coupled with its interesting antiproliferative activity, in particular against cancer cells presenting multidrug resistance phenotype with overexpression of P-glycoprotein, makes 12-Cl-hCPT a valid candidate for subsequent preclinical evaluation. Collectively, the data strengthen homocamptothecin as an extremely promising template to generate novel and potent antitumour agents.

Cryptolepine and neocryptolepine are two indoloquinoline alkaloids isolated from the roots of the African plant Cryptolepis sanguinolenta. Both drugs have revealed antibacterial and antiparasitic activities and are strongly cytotoxic to tumour cells. We have recently shown that cryptolepine can intercalate into DNA and stimulates DNA cleavage by human topoisomerase II. In this study, we have investigated the mechanism of action and cytotoxicity of neocryptolepine, which differs from the parent isomer only by the orientation of the indole unit with respect to the quinoline moiety. The biochemical and physicochemical results presented here indicate that neocryptolepine also intercalates into DNA, preferentially at GC-rich sequences, but exhibits a reduced affinity for DNA compared with cryptolepine. The two alkaloids interfere with the catalytic activity of human topoisomerase II but the poisoning activity is slightly more pronounced with cryptolepine than with its isomer. The data provide a molecular basis to account for the reduced cytotoxicity of neocryptolepine compared with the parent drug.