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Bromo analogues of the natural metabolite rebeccamycin with and without a methyl substituent on the imide nitrogen were synthesized. The effects of the drugs on protein kinase C, the binding to DNA, and the effect on topoisomerase I were determined. The drugs' uptake and their antiproliferative activities against P388 leukemia cells sensitive and resistant to camptothecin, their antimicrobial activity against a Gram-positive bacterium (B. cereus), and their anti-HIV-1 activity were measured and compared to those of the chlorinated and dechlorinated analogues. Dibrominated imide 5 shows a remarkable activity against topoisomerase I, affecting both the kinase and DNA cleavage activity of the enzyme. The marked cytotoxic potency of this compound depends essentially on its capacity to inhibit topoisomerase I.

In the course of structure-activity relationships on rebeccamycin analogues, a series of compounds bearing a halogenoacetyl substituent were synthesized with the expectation of increasing the interaction with DNA, possibly via covalent reaction with the double helix. Two rebeccamycin analogues bearing an acetyl instead of a bromoacetyl substituent were prepared to gain an insight into the role of the halogen atom. The new compounds show very little effect on protein kinase C and no covalent reaction with DNA was detected. However, the drugs behave as typical topoisomerase I poisons, and they are significantly more toxic toward P388 leukemia cells than to P388/CPT5 cells resistant to camptothecin. The introduction of a bromo- or chloro-acetyl substituent does not affect the capacity of the drug to interfere with topoisomerase I either in vitro or in cells. One of the bromoacetyl derivatives, compound 8, is the most cytotoxic rebeccamycin derivative among the hundred of derivatives we have synthesized to date. In addition, we determined the antimicrobial activities against two Gram-positive bacteria, Bacillus cereus and Streptomyces chartreusis, and against the Gram-negative bacterium Escherichia coli. The effect of the drugs on Candida albicans yeast growth and their anti-HIV-1 activities were also measured.

Many antitumor agents contain a carbohydrate side chain appended to a DNA-intercalating chromophore. This is the case with anthracyclines such as daunomycin and also with indolocarbazoles including the antibiotic rebeccamycin and its tumor active analog, NB506. In each case, the glycoside residue plays a significant role in the interaction of the drug with the DNA double helix. In this study we show that the DNA-binding affinity and sequence selectivity of a rebeccamycin derivative can be enhanced by replacing the glucose residue with a 2'-aminoglucose moiety. The drug-DNA interactions were studied by thermal denaturation, fluorescence, and footprinting experiments. The thermodynamic parameters indicate that the newly introduced amino group on the glycoside residue significantly enhanced binding to DNA by increasing the contribution of the polyelectrolyte effect to the binding free energy, but does not appear to participate in any specific molecular contacts. The energetic contribution of the amino group of the rebeccamycin analog was found to be weaker than that of the sugar amino group of daunomycin, possibly because the indolocarbazole derivative is only partially charged at neutral pH. Topoisomerase I-mediated DNA cleavage studies reveal that the OH-->NH2 substitution does not affect the capacity of the drug to stabilize enzyme-DNA covalent complexes. Cytotoxicity studies with P388 leukemia cells sensitive or resistant to camptothecin suggest that topoisomerase I represents a privileged intracellular target for the studied compounds. The role of the sugar amino group is discussed. The study provides useful guidelines for the development of a new generation of indolocarbazole-based antitumor agents.

As a part of studies on structure-activity relationships, several potential topoisomerase I inhibitors were prepared. Different analogues of the antitumor antibiotic rebeccamycin substituted on the imide nitrogen with a methyl group were synthesized. These compounds bore either the sugar residue of rebeccamycin, with or without the chlorine atoms on the indole moieties, or modified sugar residues (galactopyranosyl, glucopyranosyl, or fucopyranosyl) linked to the aglycone via a beta- or alpha-N-glycosidic bond. Their inhibitory properties toward protein kinase C, topoisomerase I, and topoisomerase II were examined, and their DNA-binding properties were investigated. Their in vitro antitumor activities against murine B16 melanoma and P388 leukemia cells were determined. Their antimicrobial activities were tested against Gram-positive bacteria Bacillus cereus and Streptomyces chartreusis, Gram-negative bacterium Escherichia coli, and yeast Candida albicans. These compounds are inactive toward topoisomerase II but inhibit topoisomerase I. A substitution with a methyl group on the imide nitrogen led to a loss of proteine kinase C inhibition in the maleimide indolocarbazole series but did not prevent topoisomerase I inhibition. Compounds possessing a beta-N-glycosidic bond, which fully intercalated into DNA, were more efficient at inhibiting topoisomerase I than their analogues with an alpha-N-glycosidic bond; however, both were equally toxic toward P388 leukemia cells. Dechlorinated rebeccamycin possessing a methyl group on the imide nitrogen was about 10 times more efficient in terms of cytotoxicity and inhibition of topoisomerase I than the natural metabolite.

DNA topoisomerase I has been shown to be an important therapeutic target in cancer chemotherapy for the camptothecins as well as for indolocarbazole antibiotics such as BE-13793C and its synthetic derivatives NB-506 and ED-110 [Yoshinari et al. (1993) Cancer Res. 53, 490-494]. To investigate the mechanism of topoisomerase I inhibition by indolocarbazoles, we have studied the induction of DNA cleavage by purified mammalian topoisomerase I mediated by the antitumor antibiotic rebeccamycin and a series of 20 indolocarbazole derivatives. The compounds tested bear (i) various functional groups on the non-indolic moiety (X = CO, CH2, CHOH), (ii) a hydrogen or a chlorine atom at positions 1 and 11 (R2), and (iii) different substituents on the maleimido function (R1 = H, OH, NH2, NHCHO). Half of the ligands have the same carbohydrate moiety as rebeccamycin whereas the other ligands have no sugar residue. The inhibitory potency of the test compounds was assessed in vitro by comparing the cleavage of [32P]-labeled restriction fragments by the enzyme in the absence and presence of the drug. In addition, the DNA-binding properties of these compounds were investigated by means of complementary spectroscopic techniques including electric linear dichroism, and the DNA sequence selectivity was probed by DNase I footprinting. The study shows that the sugar residue attached to the indolocarbazole chromophore is critical for the drug ability to interfere with topoisomerase I as well as for the formation of intercalation complexes. Structure-activity relationships indicate that the presence of chlorine atoms significantly reduces the effects on topoisomerase I whereas the substituents on the maleimido function and the functional group on the non-indolic moiety can be varied without reduction of activity. The results suggest that the inhibition of topoisomerase I by indolocarbazoles arises in part from their ability to interact with DNA. Analysis of the base preferences around topoisomerase I cleavage sites in various restriction fragments indicated that, in a manner similar to camptothecin, the rebeccamycin analogue R-3 stabilized topoisomerase I preferentially at sites having a T and a G on the 5' and 3' sides of the cleaved bond, respectively. By analogy with models previously proposed for camptothecin and numerous topoisomerase II inhibitors which intercalate into DNA, a stacking model for the interaction between DNA, topoisomerase I and indolocarbazoles is proposed. These findings provide guidance for the development of new topoisomerase I-targeted antitumor indolocarbazole derivatives.

A series of compounds structurally related to staurosporine, rebeccamycin, and corresponding aglycones was synthesized, and their activities toward protein kinase C and topoisomerases I and II were tested together with their in vitro antitumor efficiency against murine B16 melanoma and P388 leukemia cells. Their antimicrobial activities were also examined against a Gram-negative bacterium (Escherichia coli), a yeast (Candida albicans), and three Gram-positive bacteria (Bacillus cereus, Streptomyces chartreusis, and Streptomyces griseus). To avoid side effects expected with protein kinase C inhibitors, we introduced substitution on the maleimide nitrogen and/or a sugar moiety linked to one of the indole nitrogens to obtain specific inhibitors of topoisomerase I with minimal activities on protein kinase C. As expected, these structures were inefficient on topoisomerase II, and some of them exhibited a strong activity against topoisomerase I. Generally, dechlorinated compounds were found to be more active than chlorinated analogues against both purified topoisomerase I and protein kinase C. On the other hand, opposite results were obtained in the cell antiproliferative assays. These results suggest lack of cell membrane permeability in the absence of the chlorine residue or cleavage of carbon-chlorine bonds inside the cell.

CPT-11 (irinotecan) is a water-soluble analogue of camptothecin (CPT), an antitumor drug extracted from the Chinese tree Camptotheca acuminata. SN-38 is an active metabolite of CPT-11 that contributes significantly to its activity. The antitumor effects of CPT-11 and SN-38 are exerted through a novel mechanism of action; inhibition of DNA topoisomerase I. CPT-11 and its metabolite have demonstrated potent inhibitory activity against a variety of cancer cell lines in vitro and against several murine and human tumors grafted in mice in vivo, including those that express multidrug resistance. CPT-11 has also shown synergistic activity in combination with 5-fluorouracil and cisplatin in vitro. No irreversible or unusual toxicities were observed with CPT-11 in animal toxicity studies. In summary, the preclinical profile of CPT-11 confirmed this drug to be an attractive candidate for clinical development.

There are at least two well-characterized mechanism of resistance to Topo I and II inhibitors: modifications of intracellular accumulation and reduced formation of cleavable complexes. Limited drug accumulation is usually due to P-glycoproteinMDR or to Multidrug Resistance associated Protein (MRP). Reduction of Topo I (or II) cleavable complexes not related to drug transport can either be due to decreased enzyme levels or enzyme mutations. For Topo II inhibitors, differential expression of the Topo II isoforms alpha and beta and changes in Topo II phosphorylation may also contribute to resistance. For dual Topo I and II inhibitors, resistance mechanisms are more complex to analyze but may also involve dual Topo I and II alterations. Finally, in a given cell line, several mechanisms are commonly associated in pleiotropic resistance to Topo inhibitors.

Molecular interactions of intoplicine, dual DNA-topoisomerases (Topo) I and II inhibitor, with topoisomerases, plasmid DNA, in ternary cleavable complexes with enzymes and plasmid DNA, and in the reversed cleavable complexes were examined by means of surface-enhanced Raman scattering (SERS) and CD spectroscopy and by biochemical techniques. Detailed spectral analysis of intoplicine derivatives allowed us to assign SERS vibrational modes of chromophores and to propose the models for these complexes. Intoplicine was found to be able to interact specifically with the Topo II alone, but with Topo I only when in the presence of DNA. It shows at least two modes of binding to the DNA: the first was found to be dominant for its derivative 1c (most potent Topo I inhibitor), and the second was dominant for derivative 2a (most potent Topo II inhibitor). The possibility of forming these two types of complexes simultaneously is suggested to be one of the main factors enabling the drug to be a dual Topo I and Topo II inhibitor. The "deep intercalation mode" of the drug from the DNA minor groove with the long axis of the chromophore oriented roughly parallel to the dyad axis has been suggested to be responsible for induction of distortions of the DNA structure by the intercalating drug. Being involved in the formation of Topo I-mediated cleavable ternary complex, the molecules participating in the deep intercalation mode within the DNA do not change their molecular interactions as compared with their complex with the DNA alone. The stabilization of the Topo I-mediated cleavable complex was shown to be followed by the local denaturation of DNA in the AT-rich regions of the helix. When the ternary cleavable complex was reversed, the drug was shown to be in the complex with the plasmid. The "outside binding mode" from the DNA major groove via the hydroxyl group of the A-ring of the chromophore has been suggested to be responsible for Topo II inhibition. These molecules did not induce significant distortions of the DNA structure. Being involved in the formation of Topo II-mediated cleavable ternary complex, the drug changed its molecular interactions as compared with the complex with DNA alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.

The mechanisms of action of intoplicine (RP-60475), a 7H-benzo[e]pyrido[4,3-b]indole derivative that is presently in early clinical trials, have been investigated. Intoplicine induced both topoisomerase I- and II-mediated DNA strand breaks, using purified topoisomerases. The topoisomerase cleavage site patterns induced by intoplicine were unique, relative to those of camptothecin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and other known topoisomerase inhibitors. Both topoisomerase I- and II-induced DNA breaks decreased at drug concentrations higher than 1 microM, which is consistent with the DNA-intercalating activity of intoplicine. DNA damage was investigated in KB cells in culture by using alkaline elution. Intoplicine induced single-strand breaks (SSB) in a bell-shaped manner with respect to drug concentration (maximum frequency at 1 microM approximately 220 rad-equivalents). SSB formation was fast, whereas reversal after drug removal was slow. Similar bell-shaped curves were obtained for DNA double-strand breaks (DSB) and DNA-protein cross-links. SSB and DNA-protein cross-link frequencies were approximately equal, and no protein-free breaks were detectable, indicating the protein concealment of the breaks, as expected for topoisomerase inhibition. Comparison of SSB and DSB frequencies indicated that intoplicine produced a significant amount of SSB not related to DSB, which is consistent with concomitant inhibition of both DNA topoisomerases I and II in cells. Data derived from resistant cell lines indicated that multidrug-resistant cells were cross-resistant to intoplicine but that m-AMSA- and camptothecin-resistant cells were sensitive to intoplicine. Hence, intoplicine might circumvent topoisomerase I-mediated and topoisomerase II-mediated resistance by poisoning both enzymes simultaneously.

With the aim of obtaining new inhibitors of topoisomerases, we have evaluated various heterocyclic quinone derivatives for their ability to induce topoisomerase I (Topo I)- or Topo II-associated DNA breaks, using P388 cell nuclear extract. Several compounds belonging to the indolo[3,2-c]quinoline-1,4-dione series have been shown to possess DNA-cleavage activity. Further analysis using purified Topo I and II preparations has indicated that the members of the series stimulate cleavable complex formation of both Topo I and II. 3-Methoxy-11H-pyrido[3',4':4,5]pyrrolo[3,2-c] quinoline-1,4-dione (AzalQD), one of the most active members of the series, stimulates cleavable complex formation and inhibits the catalytic activities of both eukaryotic Topo I and II, with, however, less potency than camptothecin and etoposide. Topo I cleavage site patterns for AzalQD and camptothecin were found to be nearly identical, with, however, some differences in cleavage site intensities. Use of filter binding assays also indicates that AzalQD is at least 10 times more potent against Topo I than against Topo II. Structure-activity relationships of indoloquinolinedione derivatives have been established and have shown that Topo I and II inhibitions are strongly linked, with a dose-selective preference towards Topo I. AzalQD does not display detectable DNA-unwinding properties. AzalQD induces a preferential cytotoxicity for the yeast strain JN2-134 bearing the human top1 gene under the control fo the GAL1 promoter, indicating that Topo I inhibition is responsible for the yeast cytotoxicity. These data indicate that AzalQD and its structural analogs represent a new distinct class of eukaryotic Topo I and II inhibitors.