Pentamidine Analogues Research Articles

Efficient and specific cardiac IK1 inhibition by a new pentamidine analogue

In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK₁) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK₁ by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK₁ inhibitors based on the P structure. We analysed seven pentamidine analogues (PA-1 to PA-7) for IK₁ blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC₅₀ values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK₁ in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.

New Pentamidine Analogues in Medicinal Chemistry

Sixty years after its introduction, 1,5-bis(4-amidinophenoxy)pentane (Pentamidine) is still one of the most used drugs for the treatment of the first stage of Human African trypanosomiasis and other neglected diseases such as malaria and leishmaniasis. These protozoan infections are prevalent in the poorest world areas such as sub-saharian and developing countries, however the increasing immigration from these countries to the richest part of the world and the overlap of HIV with parasitic infections result in a growing number of cases in developed nations. A great effort has been made to develop new generations of diamidines for the treatment of these infections transmitted by insects. This review summarises the synthesis and evaluation of pentamidine analogues reported in the last years in the effort to find new drugs with better pharmaceutical activity, higher lipophilicity and lower citotoxycicty.

Analogs of pentamidine as potential anti-Pneumocystis chemotherapeutics

A series of 20 pentamidine analogs were prepared using 2 general Schemes that evaluated heteroatoms, sulfobenzene and alkanediamide groups in the aliphatic linker and methoxy substituents attached to the benzene rings for efficacy against the fungal pathogen, Pneumocystis carinii in an ATP bioassay. All but one of the 20 bisamidines reduced the ATP content of the P. carinii over the 72 h of the assay period. The highest activities were associated with the lack of methoxy groups and the presence of the O, N and S heteroatoms. Activity (IC 50) for compounds 1, 5, 6, 10 ranged from 1.1 to 2.13 μM. The compound 11 with similar activity (1.33 μM), bears a sulfobenzene group at a nitrogen in the aliphatic linker. The alkanediamide-linked bisbenzamidines showed a moderate inhibition of ATP. Generally, the inclusion of a heteroatom in the aliphatic linker and absence of methoxy groups at the benzene rings were associated with higher activities in this assay. Of note, most of the compounds had little to no cytotoxicity in mammalian cell cultures. Although not quite as potent as other pentamidine derivatives, these compounds hold promise for decreased side effects within the mammalian host.

Novel Diamidines with Activity against Babesia divergens In Vitro and Babesia microti In Vivo

Dicationic diamidines, such as diminazene and pentamidine, are well-studied chemotherapeutic agents with significant activity against parasitic diseases. The in vitro activities of novel diamidine compounds against the Babesia divergens strains 1903B and 4201 were investigated. The most potent compound, a diphenyl furan, had a 50% inhibitory concentration (IC(50)) of 1.5 ng/ml. In a murine model, several test compounds were effective enough to cure mice infected with Babesia microti at a dose of 12.5 and/or 25 mg/kg of body weight given by the subcutaneous route for 4 days. The best antibabesial properties were exhibited by terphenyls, benzimidazoles, diphenyl furans, pentamidine, and pentamidine analogues.

State of the art in African trypanosome drug discovery.

African sleeping sickness is endemic in sub-Saharan Africa where the WHO estimates that 60 million people are at risk for the disease. Human African trypanosomiasis (HAT) is 100% fatal if untreated and the current drug therapies have significant limitations due to toxicity and difficult treatment regimes. No new chemical agents have been approved since eflornithine in 1990. The pentamidine analog DB289, which was in late stage clinical trials for the treatment of early stage HAT recently failed due to toxicity issues. A new protocol for the treatment of late-stage T. brucei gambiense that uses combination nifurtomox/eflornithine (NECT) was recently shown to have better safety and efficacy than eflornithine alone, while being easier to administer. This breakthrough represents the only new therapy for HAT since the approval of eflornithine. A number of research programs are on going to exploit the unusual biochemical pathways in the parasite to identify new targets for target based drug discovery programs. HTS efforts are also underway to discover new chemical entities through whole organism screening approaches. A number of inhibitors with anti-trypanosomal activity have been identified by both approaches, but none of the programs are yet at the stage of identifying a preclinical candidate. This dire situation underscores the need for continued effort to identify new chemical agents for the treatment of HAT.

Three optimized and validated (using accuracy profiles) LC methods for the determination of pentamidine and new analogs in rat plasma

Three novel LC-UV methods for the determination of pentamidine (PTMD) and two of its new analogs in rat plasma are described. The chromatographic conditions (wavelength, acetonitrile percentage in the mobile phase, internal standard) were optimized to have an efficient selectivity. A pre-step of extraction was simultaneously developed for each compound. For PTMD, a solid phase extraction (SPE) with Oasis ® HLB cartridges was selected, while for the analogs we used protein precipitation with acetonitrile. SPE for PTMD gave excellent results in terms of extraction yield (99.7 ± 2.8) whereas the recoveries for the analogs were not so high but were reproducible as well (64.6 ± 2.6 and 36.8 ± 1.6 for analog 1 and 2, respectively). By means of a recent strategy based on accuracy profiles (β-expectation tolerance interval), the methods were successfully validated. β was fixed at 95% and the acceptability limits at ±15% as recommended by the FDA. The method was successfully validated for PTMD (29.6–586.54 ng/mL), analog 1 (74.23–742.3 ng/mL) and analog 2 (178.12–890.6 ng/mL). The first concentration level tested was considered as the LLOQ (lower limit of quantification) for PTMD and analog 1 whereas for analog 2, the LLOQ was not the first level tested and was raised to 178.12 ng/mL.

Solid state structural analysis of new pentamidine analogs designed as chemotherapeutics that target DNA by X-ray diffraction and 13C, 15N CP/MAS NMR methods

The paper presents the solid-state analysis of the crystalline form of 1,5- bis[(4-cyanophenyl)- N-methylamino]pentane ( 1) and polycrystalline powder sample of 1,5- bis[(4-amidinophenyl)- N-methylamino]pentane dihydrochloride ( 2). The methods used are X-ray diffraction technique and 13C, 15N CP/MAS NMR spectroscopy in an attempt to detect the effects of possible polymorphism. Both methods indicate that only single conformers exist in the solid-state for 1 and 2. 1,5- Bis[(4-cyanophenyl)- N-methylamino]pentane 1, crystallizes in the orthorhombic space group P2 12 12. The asymmetric unit contains one half of the ordered molecule. Only weak intermolecular interactions were found in solid-state, in which methyl groups are engaged.

Pharmacophore model for pentamidine analogs active against Plasmodium falciparum

Pentamidine and its analogs constitute a class of compounds that are known to be active against Plasmodium falciparum, which causes the most dangerous malarial infection. Malaria is a widespread disease known to affect hundreds of millions of people and presents a perceivable threat of spreading. Hence, there is a need for well-defined scaffolds that lead to new, effective treatment. Here we present a pentamidine-based pharmacophore constructed using GALAHAD that would aid targeted synthesis of leads with enhanced properties, as well as the development of lead scaffolds. The study was supported by high-quality biological in vitro data of 22 compounds against the P. falciparum strains NF54 and K1. The model established reveals the importance of hydrophobic phenyl rings with polar oxygen and amidine substituents and the hydrophobic linking chain for the activity against malaria.

Theoretical models of pentamidine analogs activity based on their DNA minor groove complexes

The B-DNA binding property of pentamidine analogs was analyzed employing molecular dynamic simulations and UV–vis absorption spectroscopy. Their AATT and ATAT sequence preferences were also examined. In theoretical energy computations, we considered two solvation models: the implicit model I, where the solvent is characterized by a dielectric constant and the explicit model E, where water molecules are treated as discrete entities. The binding energy correlates well with the increase of melting temperature of the 5′-d(CGCGAATTCGCG) 2-3′ and 5′-d(CGCGATATCGCG) 2-3′ dodecamers after complexation with ligands (Δ T m ), and deduced models predicted adequately the DNA affinities of the studied diamidines.

Synthesis and antiprotozoal activities of dicationic bis(phenoxymethyl)benzenes, bis(phenoxymethyl)naphthalenes, and bis(benzyloxy)naphthalenes

A series of 37 dicationically substituted bis(phenoxymethyl)benzene bis(phenoxymethyl)naphthalene, and bis(benzyloxy)naphthalene analogues of pentamidine was prepared and evaluated for antiprotozoal activities and cytotoxicity in in vitro. 1,3-Bis(4-amidinophenoxymethyl)benzene ( 1) was the most active against Trypanosoma brucei rhodesiense (IC 50 = 2.1 nM). 1,3-Bis[4-( N-isopropylamidino)phenoxymethyl]benzene ( 2) was most active against Plasmodium falciparum (IC 50 = 3.6 nM) and displayed a selectivity index more than 50 times greater than that of pentamidine. Several other compounds displayed lower antiplasmodial IC 50 values and higher selectivity indices relative to pentamidine. 1,4-Bis(4-amidinophenoxymethyl)benzene ( 14) was the most active against Leishmania donovani (IC 50 = 1.3 μM). Compound 2 displayed the greatest activity against T. b. rhodesiense in vivo, curing three of four infected mice dosed intraperitoneally at 5 mg/kg × 4 days.

Pentamidine Movement across the Murine Blood-Brain and Blood-Cerebrospinal Fluid Barriers: Effect of Trypanosome Infection, Combination Therapy, P-Glycoprotein, and Multidrug Resistance-Associated Protein

During the first stage of human African trypanosomiasis (HAT), Trypanosoma brucei gambiense is found mainly in the blood, and pentamidine treatment is used. Pentamidine is predominantly ineffective once the parasites have invaded the central nervous system (CNS). This lack of efficacy is thought to be due to the inability of pentamidine to cross the blood-brain barrier, although this has never been explored directly. This study addresses this using brain perfusion in healthy mice, P-glycoprotein-deficient mice, and in a murine model of HAT (T. brucei brucei). The influence of additional antitrypanosomal drugs on pentamidine delivery to the CNS also was investigated. Results revealed that [(3)H]pentamidine can cross the blood-brain barrier, although a proportion was retained by the capillary endothelium and failed to reach the healthy or trypanosome-infected brain (up to day 21 p.i.). The CNS distribution of pentamidine was increased in the final (possibly terminal) stage of trypanosome infection, partly because of loss of barrier integrity (days 28-35 p.i.) as measured by [(14)C]sucrose and [(3)H]suramin. Furthermore, pentamidine distribution to the CNS involved influx and efflux [via P-glycoprotein and multidrug resistance-associated protein (MRP)] transporters and was affected by the other antitrypanosomal agents, suramin, melarsoprol, and nifurtimox, but not eflornithine. These interactions could contribute to side effects or lead to the development of parasite resistance to the drugs. Thus, great care must be taken when designing drug combinations containing pentamidine or other diamidine analogs. However, coadministration of P-glycoprotein and/or MRP inhibitors with pentamidine or other diamidines might provide a means of improving efficacy against CNS stage HAT.

Synthesis and In Vitro Leishmanicidal Effects of Conformationally Restricted Analogues of Pentamidine

Four conformationally restricted analogues of pentamidine were prepared. Then, different concentrations (0.039, 0.078, 0.156, 0.312 and 0.625 mg/mL) of each compound and two positive controls (amphotericin B and pentamidine, 0.625 mg/mL), one negative control (culture medium) and one solvent control (DMSO) were prepared and placed in 24-well plates containing 50000 parasite per well. Promastigotes of Leishmania major were incubated over a period of 2 days at 25°C; subsequently, percent of viable parasite in each well determined spectrophotometrically using MTT assay. The average EC 50 for compounds 4a,b and 8a,b in DMSO was 0.098, 0.410, 0.150, 0.720 mg/mL, respectively. The average EC 50 for positive controls pentamidine and amphotericin B was found to be 0.062 and 0.026 mg/mL. The control

Inhibition by Dications of In Vitro Growth of Leishmania major and Leishmania tropica: Causative Agents of Old World Cutaneous Leishmaniasis

Old World cutaneous leishmaniasis is caused by infection with Leishmania major and Leishmania tropica. Pentamidine and related dications exhibit broad spectrum antiprotozoal activity. Based on the previously reported efficacy of these compounds against related organisms, 18 structural analogs of pentamidine were evaluated for in vitro antileishmanial activity, using pentamidine as the standard reference drug for comparison. Furan analogs and reversed amidine compounds were examined for activity against L. major and L. tropica promastigotes. The most active compounds against both Leishmania species were in the reversed amidine series. DB745 and DB746 exhibited the highest activity against L. major and DB745 was the most active compound against L. tropica. Both of these compounds exhibited 50% inhibitory concentrations (IC50) below 1 nM for L. major. Ten reversed amidines were also tested for their ability to inhibit growth in an axenic amastigote model. Nine of 10 reversed amidine analogs were active at concentrations below 1 nM. These results justify further study of dicationic compounds as potential new agents for treating cutaneous leishmaniasis.

Pentamidine Analogs: Syntheses, Structures in Solid State by 13C CP/MAS NMR Spectroscopy, and X-Ray Crystallography and their Preliminary Biological Screening Against Human Cancer

The synthesis of four new 1,5-bis(4-amidinophenoxy)-3-oxapentane analogs is described. The structures of the obtained bis-amidines and bis-nitriles in the solid state are evaluated on the basis of 13C CP/MAS NMR spectra and theoretical calculations at DFT level. A single crystal X-ray diffraction structure is presented for 1,5-bis(4-amidinophenoxy)-3-oxapentane. A preliminary anticancer assay against three cell lines is also given.

Electrochemical DNA Biosensor for Testing Pentamidine and Its Analogues as Potential Chemotherapeutics

AbstractElectrochemical DNA‐based biosensors are used as screening devices for a rapid evaluation of chemical compounds interacting with double helix of the nucleic acid. Pentamidine is one of minor groove binding chemotherapeutics clinically used as anti‐PCP agent. Therefore, we designed, synthesized and tested some pentamidine analogues with diethyl ether linker and 1,2‐diazine (pyridazine) or 1,3,5‐triazine derivatives with cyanophenoxy substituent, precursors of amidine groups to establish if they can be more efficacious DNA interacting compounds than the original pentamidine drug. The tested compounds were: 1,5‐bis(4‐amidinophenoxy)pentane isethionate‐pentamidine (1) used as template, 6‐chloro‐3‐(4‐cyanophenoxy)pyridazine (2), 4,6‐dichloro‐2‐(4‐cyanophenoxy)‐1,3,5‐triazine (3), 4‐amidinophenol dihydrate (4), 1,5‐bis(4‐amidinophenoxy)‐3‐oxapentane dihydrochloride‐γ‐oxapentamidine (5), 1,5‐bis(N‐cyclohexyl‐4‐amidinophenoxy)‐3‐oxapentane dihydrochloride (6). The results show that none of the tested analogues binds dsDNA more strongly than the original drug pentamidine (1). Compound 2 with pyridazine group interacts with the nucleic acid in a different way than the one with triazine (3) – the signals obtained using DNA‐based biosensor had opposite features. Close derivatives 5 and 6 of the main drug pentamidine (1) showed a weak influence on the double‐stranded nucleic acid which proves that even small changes in the structure of a molecule can strongly affect the character and properties of the above mentioned compounds in the presence of DNA. In this context atomic charge distribution in the studied molecules is also discussed.

Accumulation and Intracellular Distribution of Antitrypanosomal Diamidine Compounds DB75 and DB820 in African Trypanosomes

The aromatic diamidine pentamidine has long been used to treat early-stage human African trypanosomiasis (HAT). Two analogs of pentamidine, DB75 and DB820, have been shown to be more potent and less toxic than pentamidine in murine models of trypanosomiasis. The diphenyl furan diamidine, DB75, is the active metabolite of the prodrug DB289, which is currently in phase III clinical trials as a new orally active candidate drug to treat first-stage HAT. The new aza analog, DB820, is the active diamidine of the prodrug DB844, currently undergoing preclinical evaluation as a new candidate to treat HAT of the central nervous system. The exact mechanisms of antitrypanosomal activity of aromatic dications remain poorly understood, with multiple mechanisms hypothesized. Pentamidine is known to be actively transported into trypanosomes and binds to DNA within the nucleus and kinetoplast. A long-hypothesized mechanism of action has been that DNA binding ultimately leads to interference with DNA-associated enzymes. Both DB75 and DB820 are intensely fluorescent, which provides an important tool for determining the kinetics of accumulation and intracellular distribution in trypanosomes. We show in the current study that DB75 and DB820 rapidly accumulate and strongly concentrate within trypanosomes, with intracellular concentrations over 15,000-fold higher than mouse plasma concentrations. Both compounds initially accumulate in the DNA-containing nucleus and kinetoplast, but at later time points, they concentrate in non-DNA-containing cytoplasmic organelles. Analyses of the kinetics of uptake and intracellular distribution are necessary to begin to define antitrypanosomal mechanisms of action of DB75, DB820, and other aromatic diamidines.

Roles for mitochondria in pentamidine susceptibility and resistance in Leishmania donovani

Pentamidine resistant Leishmania donovani was raised in the laboratory by stepwise exposure to increasing drug pressure until a line capable of growth in 8 μM pentamidine (R8) had been selected. An IC 50 value of 40 μM was determined for this line, some 50-fold higher than that recorded for the parental wild-type line. The pentamidine resistant promastigotes were cross-resistant to other toxic diamidine derivatives but not to antimonials or substrates of multidrug resistance pumps. Decreased mitochondrial transmembrane potential was observed in pentamidine resistant promastigotes. A substantial net decrease in accumulation of [ 3H]-pentamidine accompanied the resistance phenotype. Inhibitors of P-glycoprotein pumps, including prochlorperazine and trifluoperazine, did not reverse this decreased drug uptake, which distinguishes the L. donovani resistant line studied here from L. mexicana promastigotes previously studied for pentamidine resistance. Kinetic analysis identified a carrier with an apparent K m value of 6 μM for pentamidine. No significant difference between wild-type and resistant parasites could be detected with respect to this transporter in rapid uptake experiments. However, in longer-term uptake experiments and also using concentrations of pentamidine up to 1 mM, it was demonstrated that wild-type cells, but not resistant cells, could continue to accumulate pentamidine after apparent saturation via the measured transporter had been reached. Agents that diminish the mitochondrial membrane potential inhibited this secondary route. A fluorescent analogue of pentamidine, 2,5-bis-(4-amidophenyl)-3,4-dimethylfuran (DB99), accumulated in the kinetoplast of wild-type but not resistant parasites indicating that uptake of this cationic compound into mitochondria of wild-type cells was more pronounced than in the resistant line. These data together indicate that resistance to pentamidine in L. donovani is associated with alterations to the mitochondria of the parasites, which lead to reduced accumulation of drug.

DB75, a novel trypanocidal agent, disrupts mitochondrial function in Saccharomyces cerevisiae.

The aromatic diamidines represent a class of compounds with broad-spectrum antimicrobial activity; however, their development is hindered by a lack of understanding of their mechanism of antimicrobial action. DB75 [2,5-bis(4-amidinophenyl)furan] is a trypanocidal aromatic diamidine that was originally developed as a structural analogue of the antitrypanosomal agent pentamidine. DB289, a novel orally active prodrug of DB75, is undergoing phase IIb clinical trials for early-stage human African trypanosomiasis, Pneumocystis jiroveci carinii pneumonia, and malaria. The purpose of this study was to investigate mechanisms of action of DB75 using Saccharomyces cerevisiae as a model organism. The results of this investigation suggest that DB75 inhibits mitochondrial function. Yeast cells relying upon mitochondrial metabolism for energy production are especially sensitive to DB75. DB75 localizes (by fluorescence) within the mitochondria of living yeast cells and collapses the mitochondrial membrane potential in isolated yeast mitochondria. Furthermore, addition of DB75 to yeast cells or isolated rat liver mitochondria results in immediate uncoupling of oxidative phosphorylation and subsequent inhibition of respiration. We conclude that the mitochondrion is a cellular target of DB75 in yeast cells and anticipate that the results of this study will aid in the target-based design of new antimicrobial aromatic diamidines.

Distribution and quantitation of the anti-trypanosomal diamidine 2,5-bis(4-amidinophenyl)furan (DB75) and its N-methoxy prodrug DB289 in murine brain tissue

The current epidemic of sleeping sickness, also known as human African trypanosomiasis in sub-Saharan Africa places nearly 60 million people at risk for developing this life threatening infection. Although effective treatments for early-stage sleeping sickness exist, these drugs usually require extended dosing schedules and intravenous administration. New treatments are also needed for cerebral (late) stage trypanosomiasis. 2,5-Bis(4-amidinophenyl)furan (DB75), a pentamidine analog, has potent in vitro and in vivo anti-trypanosomal activity. However, DB75 does not exhibit significant oral bioavailability and has proved to be ineffective against mouse models of late-stage sleeping sickness regardless of administration route. To circumvent the limited oral bioavailability of DB75, an N-methoxy prodrug 2,5-bis(4-amidinophenyl)furan-bis- O-methylamidoxime (DB289) was designed and developed initially as a compound to treat AIDS-related Pneumocystis carinii pneumonia (PCP). Despite excellent oral activity against early-stage sleeping sickness, oral administration of DB289 exhibited limited efficacy in mouse models of late-stage disease. DB289 has recently entered Phase II(b) clinical trials to treat primary-stage sleeping sickness in Central Africa. The current study takes advantage of the innate fluorescence of DB75 and DB289 along with specific and sensitive quantitative analyses to examine plasma and brain distribution of these compounds. Animals were dosed with intravenous DB75, oral DB289, and intravenous DB289. Following intravenous administration, DB75 was readily detectable in whole brain extracts and persisted for long periods. Fluorescence microscopy revealed that DB75 did not penetrate into brain parenchyma, however, but was sequestered within cells lining the blood–brain and blood–cerebrospinal fluid barriers. In contrast, brain tissue of mice treated with oral DB289 exhibited diffuse fluorescence within the brain parenchyma, suggesting that the prodrug was not trapped within blood–brain barrier cells (BBB). However, maximal brain concentrations of the active compound DB75 were very low (13 nmol/mg of tissue at 24 h). Intravenous administration of DB289 resulted in a qualitatively similar fluorescence pattern to oral DB289, indicating again that DB289 and DB75 were present within brain parenchyma, not only in barrier regions. Furthermore, peak DB75 tissue levels were higher (61 nmol/mg of tissue at 24 h) than with oral prodrug. The near five-fold increase in brain levels of DB289 combined with parenchymal localization of compound fluorescence after intravenous administration suggest that the unaltered prodrug penetrates the blood–brain barrier, and may be subject to in situ biotransformation. Intravenous administration of DB289 should be evaluated in mouse models of late-stage sleeping sickness.

Aromatic analogues of DNA minor groove binders—synthesis and biological evaluation

Nine carbocyclic analogues of mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted N-terminal amine group were synthesized. We have investigated the cytotoxic activity of new aromatic analogues of DNA binding ligands in MCF-7 breast cancer cells and assessed their ability to act as inhibitors of topoisomerase I and II. These studies indicate that aromatic analogues of bis-netropsin contain two identical units tethered by alkyloxyl chains are a potent catalytic inhibitor of both topoisomerases and exhibit moderate cytotoxicity in MCF-7 breast cancer cells.