Synthetic lanostane-type triterpenoids as inhibitors of DNA topoisomerase II

DNA topoisomerase (Topo) II is one of the target enzymes for chemotherapeutic drug development. Lanostane-type triterpenoids with various functional groups (-Cl, -Br, -OMe, -CHO, -CN, -COOH, and -COOMe) at C-2 were synthesized from 3-oxolanost-9(11)-en-24S,25-diol (9) isolated from Pinus luchuensis and their inhibitory effects on Topo II activity and cytotoxic activities against A549 cells were examined. All the derivatives showed Topo II inhibitory effects with IC50 values ranging from 1.86 to 149.97 microM and cytotoxic activities with ED50 values ranging from 3.96 to 38.15 microM.     

Facile one-pot synthesis of thio and selenourea derivatives: a new class of potent urease inhibitors

A facile, one-pot synthesis of thio and selenourea derivatives from amines using tetrathiomolybdate 1 and tetraselenotungstate 2 as sulfur and selenium transfer reagents, respectively, is reported. The compounds were tested for their activity as urease inhibitors and some of the compounds showed potent activity in the nanomolar range towards jack bean urease.     

The synthesis of amino-acid functionalized beta-carbolines as topoisomerase II inhibitors

The synthesis and biological activity of amino acid functionalized beta-carboline derivatives, which are structurally related to azatoxin and the tryprostatins, are reported. These compounds were assayed for their growth inhibition properties in H520 and PC3 cell lines and were examined for their abilities to inhibit topoisomerase II-mediated DNA relaxation.     

Some benzoxazoles and benzimidazoles as DNA topoisomerase I and II inhibitors

Some novel fused heterocyclic compounds of 2, 5-disubstituted-benzoxazole and benzimidazole derivatives, which were previously synthesized by our group, were investigated for their inhibitory activity on both eukaryotic DNA topoisomerase I and II in a cell free system. 2-Phenoxymethylbenzimidazole (17), 5-amino-2-(p-fluorophenyl)benzoxazole (3), 5-amino-2-(p-bromophenyl)benzoxazole (5), 5-nitro-2-phenoxymethyl-benzimidazole (18), 2-(p-chlorobenzyl)benzoxazole (10) and 5-amino-2-phenylbenzoxazole (2) were found to be more potent as eukaryotic DNA topoisomerase I poisons than the reference drug camptothecin having IC(50) values of 14.1, 132.3, 134.1, 248, 443.5, and 495 microM, respectively. 5-Chloro-2-(p-methylphenyl)benzoxazole (4), 2-(p-nitrobenzyl)benzoxazole (6) and 5-nitro-2-(p-nitrobenzyl)benzoxazole (8) exhibited significant activity as eukaryotic DNA topoisomerase II inhibitors, having IC(50) values of 22.3, 17.4, 91.41 microM, respectively, showing higher potency than the reference drug etoposide.     

7-(4-Alkylidenylpiperidinyl)-quinolone bacterial topoisomerase inhibitors

Novel antibacterial fluoroquinolone agents bearing a 4-alkylidenylpiperidine 7-position substituent are active against quinolone-susceptible and quinolone-resistant gram-positive bacteria, including Streptococcus pneumoniae and MRSA. Analogs 22b, 23c, and 24 demonstrated superior in vitro and in vivo efficacy to ciprofloxacin against these cocci.     

New potential inhibitors of DNA topoisomerase. Part II: Design and synthesis of α-lapachone derivatives under microwave irradiation

A new series of potential inhibitors of DNA topoisomerase II were synthesized from facile materials (aromatic aldehydes, Meldrum’s acid and 2-hydroxynaphthalene-1,4-dione) under microwave irradiation. The method provides a valuable tool in designing new and more potent cytotoxic analogues. This procedure is advantageous both economically and environmentally.     

Inhibition of DNA synthesis and cytotoxic effects of some DNA topoisomerase II and gyrase inhibitors in Chinese hamster V79 cells

In this study, some DNA topoisomerase II and gyrase inhibitors have been identified as inhibitors of polymerization of deoxyribonucleotides [novobiocin (NVB), nalidixic acid (NDA), oxolinic acid (OXA)], or inhibitors of replicon initiation and DNA-chain elongation [etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)methansulfon-m-anisidine (m-AMSA), ellipticine (ELT)]. The inhibitors of deoxyribonucleotide polymerization produced a significant (greater than 85%) suppression of [3H]thymidine incorporation into V79 cells within 20 min of treatment, followed by a rapid recovery of DNA synthesis, and reduced cell killing. In contrast, the inhibitors of replicon initiation and DNA-chain elongation needed about 60 min to induce a partial, but irreversible inhibition of DNA replication, associated with extensive cell killing.     

Conformational properties of topoisomerase II inhibitors and sequence specificity of DNA cleavage

The sequence specificity of topoisomerase-II-mediated DNA cleavage, stimulated by 2-methyl-9-hydroxy ellipticinium and 4′, 5′,7-trihydroyflavone (genistein) was investigated by sequencing analysis of DNA cleavage sites and molecular modeling techniques. The former drug exhibits a marked preference for a T base at the position immediately preceding the cleavage site (?1). The latter shares the preference for the same base, with an additional preference for a thymine at position +1. The cleavage intensity patterns for the two drugs differ considerably. From a conformational point of view, ellipticinium and genistein exhibit similar overall shape and dimensions. However, the fused ring system in the former generates a planar structure whereas the single bond, connecting the two aromatic portions in the latter, allows internal rotation. The most stable conformation of genistein corresponds to a deviation of about 40° from planarity. A computer-assisted analysis was carried out to compare the steric and electrostatic properties of the two compounds. Two types of preferred (energetically almost degenerate) alignment for the two molecules were found. One corresponds to overlapping of the 9-hydroxyl containing ring of ellipticinium with the 4′-hydroxyphenyl moiety of genistein, the other envisages the same moiety of ellipticine superimposed to the hydroxyl-benzopyrone portion of genistein. The structural similarities of the test drugs might account for the common preference for stimulation of DNA cleavage at position +1, whereas the different possible arrangements of genistein in the cleavable complex could explain both the additional +1 specificity exhibited by this compound and the differences in cleavage intensity patterns observed in comparison to ellipticinium.     

DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: Synthesis and antibacterial activity

The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.     

Triple helix-forming oligonucleotides conjugated to new inhibitors of topoisomerase II: synthesis and binding properties

Triplex-forming oligonucleotides (TFOs) are among the most specific DNA ligands and represent an important tool for specific regulation of gene expression. TFOs have also been used to target DNA-modifying molecules to obtain irreversible modifications on a specific site of the genome. A number of molecules have been recognized to target topoisomerase II and stabilize double-stranded cleavage mediated by this enzyme thus determining permanent DNA damage. Among these poisons, etoposide (VP16), a 4'-demethylepipodophyllotoxin derivative, is widely used in cancer chemotherapy. In the aim to design DNA site-specific molecules, three analogues of VP16 (1, 2, and 3), recently described (Duca et al. J. Med. Chem. 2005, 48, 596-603), were attached to TFOs, together with a fourth one, of which the synthesis is reported here. Two different oligonucleotides, differing by the length (a 16-mer and a 20-mer), and two different linker arms between the oligonucleotide and the drug were used. The coupling reaction between the drug and the TFO was further improved. For the first time, we also report the synthesis of TFO conjugates bearing two molecules of inhibitor linked to the same oligonucleotide end. In total, 16 new conjugates were synthesized and evaluated for their ability to form triple helices. The loss in triplex stability due to the conjugation of the TFO to compounds that do not interact with DNA is compensated by the presence of the ethylene glycol linker arm. This stabilization effect is more pronounced at the 3' end than at the 5' end. All conjugates form a stable triplex selectively on the DNA target at 37 degrees C and pH 7.2.     

Antitumor agents. Part 230: C4'-esters of GL-331 as cytotoxic agents and DNA topoisomerase II inhibitors

Three C4'-acyl derivatives (5-7) of GL-331 (4) were synthesized and evaluated for cytotoxic and DNA topoisomerase II (topo II) inhibitory activity. All three compounds were cytotoxic against KB and KB-7d cells. Compounds 5 and 7, but not 6, were potent inhibitors of the DNA topoisomerase II in vitro and this relative activity ranking was maintained for cytotoxicity, in vitro cell growth inhibition, and ability to induce cellular double-strand DNA breaks. These results provided new information on the structure-activity relationships of the C4' molecular area of 4-analogues.     

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE), Part II: Development of inhibitors with broad spectrum,Gram-negative antibacterial activity

The structurally related bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as prime candidates for the development of broad spectrum antibacterial agents. However, GyrB/ParE targeting antibacterials with spectrum that encompasses robust Gram-negative pathogens have not yet been reported. Using structure-based inhibitor design, we optimized a novel pyrrolopyrimidine inhibitor series with potent, dual targeting activity against GyrB and ParE. Compounds were discovered with broad antibacterial spectrum, including activity against Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli. Herein we describe the SAR of the pyrrolopyrimidine series as it relates to key structural and electronic features necessary for Gram-negative antibacterial activity.     

Synergistic interactions between tumor necrosis factor and inhibitors of DNA topoisomerase I and II

TNF is a pleiotropic cytokine that mediates diverse cellular responses, including cytotoxicity, cytostasis, proliferation, differentiation, and the expression of specific genes. Many of these processes require the activity of DNA topoisomerases I and II. We have investigated the interactions of TNF with inhibitors of both topoisomerases in 16-h assays using the murine L929 and human ME-180 cell lines, which undergo a cytotoxic TNF response. Camptothecin, a specific inhibitor of topoisomerase I, enhanced TNF cytotoxicity 150-fold against both cell lines. The topoisomerase II inhibitors VM-26 and VP-16, which stabilize covalent DNA-topoisomerase intermediates, greatly enhance TNF cytotoxicity against both cell lines. The most effective, VM-26, can lower the TNF LD50 to femtomolar levels. In contrast, the topoisomerase II inhibitors novobiocin and coumermycin, which bind to the enzyme ATPase site, protect L929 cells from TNF cytotoxicity but enhance TNF cytotoxicity in ME-180 cells. The large changes in TNF sensitivity induced by drug concentrations that by themselves show no effect, and the opposing synergistic effects of inhibitors with different inhibitory mechanisms (in L929 cells), suggest the active involvement of topoisomerases in TNF-mediated cytotoxicity. The correlation of cytotoxic synergy with the stabilization of DNA strand breaks indicates that DNA damage may play a significant role in TNF-mediated cytotoxicity.     

Design and synthesis of Rho kinase inhibitors (II)

In a previous study, we identified several structurally unrelated scaffolds of the Rho kinase inhibitor using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. 1H-Indazole is one of the candidate scaffolds on which a new series of potent Rho kinase inhibitors could be developed. In this study, the detailed structure-activity relationship of 1H-indazole analogues was studied. During this study, we found that the cell-free enzyme inhibitory potential of Rho kinase inhibitors having the 1H-indazole scaffold did not necessarily correlate with their inhibitory potential toward the chemotaxis of cultured cells. The choice of the linker substructure was shown to be an important factor for the 1H-indazole analogues to inhibit the chemotaxis of cells. Optimization of the 1H-indazole inhibitors with respect to the in vitro inhibition of monocyte chemotaxis induced by MCP-1 was carried out. The inhibitory potential was improved both in the cell-free enzyme assay and in the chemotaxis assay.     

Stilbenoids of Kobresia nepalensis (Cyperaceae) exhibiting DNA topoisomerase II inhibition

Resveratrol oligomers, nepalensinol A, B and C, were isolated from the stem of Kobresia nepalensis (Cyperaceae). The structures were established on the basis of chemical properties and spectroscopic evidence including 2D NMR spectroscopic analysis. Nepalensinol A, B and C showed a potent inhibitory effect on topoisomerase II - stronger than etoposide (VP-16), a topoisomerase II inhibitor used as an anti-cancer drug. Nepalensinol B, in particular, exhibited the most potent activity with an IC₅₀ of 0.02 μg/ml.     

Different effects of eubacterial and eukaryotic DNA topoisomerase II inhibitors on chloroplasts ofEuglena gracilis

Inhibitors of eubacterial and eukaryotic DNA topoisomerases type II exhibited different effects on chloroplasts of the flagellateEuglena gracilis. Antibacterial agents (cinoxacin, nalidixic and oxolinic acids, ciprofloxacin, enoxacin, norfloxacin and ofloxacin) from the group of quinolones and coumarins (coumermycin A₁, clorobiocin and novobiocin) — all inhibitors of prokaryotic DNA topoisomerase II — were very potent eliminators of chloroplasts fromE. gracilis. In contrast, antitumor drugs (adriamycin, etoposide, teniposide and mitoxantrone) — antagonists of the eukaryotic counterpart — did not affect these semiautonomous photosynthetic organelles. These findings point out again the close evolutionary relationships between eubacteria and chloroplasts and are in agreement with the hypothesis of an endosymbiotic origin of chloroplasts.     

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.     

Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase

The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Antitumor agents. Part 235: Novel 4'-ester etoposide analogues as potent DNA topoisomerase II inhibitors with improved therapeutic potential

Eight 4'-ester epipodophyllotoxin derivatives (9-16) were designed and synthesized with the aim to overcome drug-resistance and improve water-solubility simultaneously. These compounds were superior to etoposide (1) in causing cellular protein-linked DNA breaks and inhibiting KB and 1-resistant KB-7d cell replication. Compounds 9 and 10 showed significant inhibitory activity against DNA topoisomerase II in vitro. Compound 10 also exhibited an in vitro DNA cleavage pattern similar to that of GL-331 (5). A hypothetical model on the action mode of 1-analogues is proposed based on the results.