Uptake of acridinecarboxamide derivatives by L1210 cells.

The uptake of six 9-aminoacridinecarboxamide derivatives by L1210 cells in relation to their lipophilicity and cytotoxic activity was studied. The amount of acridines taken up by cells was estimated by fluorimetric measurements. It was found that the uptake efficiency of this class of compounds by cells depends on the size of carboxamide residue as well as on position of the substituent. The increase of size of carboxamide chain resulted in the loss of capability of acridines to penetrate cell membrane. Cytotoxic effects of acridines were well correlated with the level of drugs accumulated by cells, whereas no clear correlation between uptake and lipophilicity was observed. It is concluded that uptake of 9-aminoacridinecarboxamides is the most important factor determining their antiproliferative activity.     

Synthesis and antileishmanial activities of 4,5-di-substituted acridines as compared to their 4-mono-substituted homologues

Newly synthesized 4,5-di-substituted acridines were assessed for in vitro antileishmanial activities as compared to those of their 4-mono-substituted homologues. Mono-substituted acridines exhibited a weak specificity for Leishmania parasites. Di-substituted acridines, on the contrary, displayed interesting amastigote-specific activities through a mechanism of action that might not involve intercalation to DNA. This antileishmanial property, associated with a low antiproliferative activity towards human cells, led to the identification of a new class of promising acridine derivatives such as 4,5-bis(hydroxymethyl)acridine with a nonclassical mechanism of action based on the inhibition of Leishmania internalization within macrophages. In the meantime, the effects of experimental lighting on the biological properties of acridines were assessed: experimental lighting did not significantly improve the antileishmanial activity of the compounds since it produced a greater toxicity against human cells.     

Acridine derivatives inhibit lysozyme aggregation

We have screened a library of structurally distinct acridine derivatives (19 compounds) for their ability to inhibit lysozyme amyloid aggregation in vitro. Studied acridines were divided into three structurally different groups depending on the molecule planarity and type of the side chain-planar acridines, spiroacridines and tetrahydroacridines. Thioflavine T fluorescence assay and transmission electron microscopy were used for monitoring the inhibiting activity of acridines. We have found that both the structure of the acridine side chains and molecule planarity influence their antiamyloidogenic activity. The planar acridines inhibited lysozyme aggregation effectively. Spiroacridines and tetrahydroacridines had no significant effect on the prevention of lysozyme fibrillization, probably resulting from the presence of the heterocyclic 5-membered ring and non-planarity of molecule. Moreover, in the presence of some tetrahydroacridines the enhanced extent of aggregation was detected. We identified the most active acridine derivates from studied compound library characterized by low micromolar IC(50) values, which indicate their possible application for therapeutic purpose.     

Interaction of aminoacridines with deoxyribonucleic acid: viscosity of the complexes

The intrinsic, viscosities at zero shear rate of defined complexes of proflavine, 9-aminoacridine, and 9-amino-l,2,3,4-tetrahydroaeridine with calf thymus DNA have been determined at, various ionic strengths by means of rotating cylinder viscometers. By controlled adjustment, of the composition of the mixtures, the amount of bound acridine (r moles/g.-atom DNA phosphorus) was maintained constant at different dilutions. The intrinsic viscosities of the complexes increased with r up to r values (ca. 0.16–0.20) corresponding to the end of the process of strong binding of the acridinium cations. However, complex formation between the acridines and thermally denatured DNA caused either a marked decrease in viscosity (at the low ionic strengths of 0.0015 and 0.005) or no change at all (ionic strength 0.1). These results are discussed in the light of presently available hydrodynamic theories relating the intrinsic, viscosity of DNA to its molecular extension.     

The genetic toxicology of acridines

Acridine and its derivatives are planar polycyclic aromatic molecules which bind tightly but reversibly to DNA by intercalation, but do not usually covalently interact with it. Acridines have a broad spectrum of biological activities, and a number of derivatives are widely used as antibacterial, antiprotozoal and anticancer drugs. Simple acridines show activity as frameshift mutagens, especially in bacteriophage and bacterial assays, by virtue of their intercalative DNA-binding ability. Acridines bearing additional fused aromatic rings (benzacridines) show little activity as frameshift mutagens, but interact covalently with DNA following metabolic activation (forming predominantly base-pair substitution mutations). Compounds where the acridine acts as a carrier to target alkylating agents to DNA (e.g. the ICR compounds) cause predominantly frameshift as well as base-pair substitution mutations in both bacterial and mammalian cells. Nitroacridines may act as simple acridines or (following nitro group reduction) as alkylating agents, depending upon the position of the nitro group. Acridine-based topoisomerase II inhibitors, although frameshift mutagens in bacteria and bacteriophage systems, are primarily chromosomal mutagens in mammalian cells. These mutagenic activities are important, since the compounds have considerable potential as clinical antitumour drugs. Although evidence suggests that simple acridines are not animal or human carcinogens, a number of the derived compounds are highly active in this capacity.     

Synthesis and evaluation of acridine- and acridone-based anti-herpes agents with topoisomerase activity

The discovery of new non-nucleoside antiviral compounds is of significant and growing interest for treating herpes virus infections due to the emergence of nucleoside-resistant strains. Using a whole cell virus-induced cytopathogenic assay, we tested a series of substituted triaryl heterocyclic compounds including acridones, xanthones, and acridines. The compounds which showed activity against Herpes Simplex-1 and/or Herpes Simplex-2 were further assayed for inhibition of topoisomerase activity to gain insight into the mechanism of action. The results indicate that the acridine analogs bearing substituted carboxamides and bulky 9-amino functionalities are able to inhibit herpes infections as well as inhibit topoisomerase II relaxation of supercoiled DNA. Given the mechanism of action of amsacrine (a closely related, well-studied 9-amino substituted acridine), the compounds were further tested in a DNA topoisomerase II cleavage assay to determine if the compounds function as poisons. The results show that the acridines synthesized in this study function through a different mechanism to that of amsacrine, most likely by blocking topoisomerase binding to DNA (akin to that of aclarubicin). This not only suggests a unique mechanism of action in treating herpes virus infections, but also may be of great interest in the development of anticancer agents that target topoisomerase II activity.     

Substituted benz[a]acridines and benz[c]acridines as mammalian topoisomerase poisons

Coralyne and several other synthetic benzo[a,g]quinolizium derivatives related to protoberberine alkaloids have exhibited activity as topoisomerase poisons. These compounds are characterized by the presence of a positively charged iminium group, which has been postulated to be associated with their pharmacological properties. The objective of the present study was to devise stable noncharged bioisosteres of these compounds. Several similarly substituted benz[a]acridine and benz[c]acridine derivatives were synthesized and their relative activity as topoisomerase poisons was determined. While the benz[c]acridine derivatives evaluated as part of this study were devoid of topoisomerase poisoning activity, several dihydrobenz[a]acridines were able to enhance DNA cleavage in the presence of topo I. In contrast to certain protoberberine derivatives that did exhibit activity as topo II poisons, none of the benz[a]acridines derivatives enhanced DNA cleavage in the presence of topo II. Among the benz[a]acridines studied, 5,6-dihydro-3,4-methylenedioxy-9,10-dimethoxybenz[a]acridine, 13e, was the most potent topo I poison, with comparable potency to coralyne. These data suggest that heterocyclic compounds structurally related to coralyne can exhibit potent topo I poisoning activity despite the absence of an iminium cation within their structure. In comparison to coralyne or other protoberberine derivatives, these benz[a]acridine derivatives possess distinctly different physicochemical properties and represent a novel series of topo I poisons.     

The mutagenic effects of diacridines and diquinolines in microbial systems

Two series of difunctional DNA-intercalating agents (diacridines and diquinolines) were tested for mutagenic properties in Salmonella typhimurium strain TA1537, and for 'petite' mutagenesis activity in Saccharomyces cerevisiae, and also compared in terms of their structural, lipophilic and DNA-binding properties. Diacridines with only a short chain length were monointercalators, while those with an alkyl linker chain longer than C6 were bisintercalators. Although the bisintercalators especially bound very tightly to DNA, none of these compounds was as effective a frameshift mutagen in TA1537 as the parent chromophore 9-aminoacridine. However, the two (monointercalating) diacridines of shortest chain length were still able to cause frameshifts, and this ability returned (albeit weakly) in the bisintercalators of longest chain length. Although 9-aminoacridine showed no ability for 'petite' mutagenesis, the diacridines of longer chain length were very effective in causing this mitochondrial event. In the quinoline series, both the parent chromophore (4-aminoquinoline) and all the diquinolines were weak monointercalators. None of these compounds showed any ability for frameshift mutagenesis, although some were very weak mitochondrial mutagens. It is concluded that linking two acridines produces compounds whose mutagenic properties might have been predicted from our current knowledge of the parent molecules. However, despite a similar ability to intercalate DNA, the diquinolines show no resemblance to acridines in their mutagenic properties.     

Effect of Deoxyribonucleic Acid Ligands on Deoxyribonucleases and Deoxyribonucleic Acid Polymerase I of Escherichia coli K-12

Endonuclease I, exonuclease I, and exonuclease II-deoxyribonucleic acid (DNA) polymerase I activities are not vital functions in Escherichia coli, although the latter two enzymes have been indirectly shown to be involved in DNA repair processes. Acridines such as acridine orange and proflavine interfere with repair in vivo, and we find that such compounds inhibit the in vitro activity of exonuclease I and DNA polymerase I but stimulate endonuclease I activity and hydrolysis of p-nitrophenyl thymidine-5′-phosphate by exonuclease II. Another acridine, 10-methylacridinium chloride, binds strongly to DNA but is relatively inert both in vivo and in vitro. These experiments suggest that acridines affect enzyme activity by interacting with the enzyme directly as well as with DNA. Resulting conformational changes in the DNA-dependent enzymes might explain why similar acridines which form similar DNA complexes have such a wide range of physiological effects. Differential sensitivity of exonuclease I and DNA polymerase I to acridine inhibition relative to other DNA-dependent enzymes may contribute to the acridine sensitivity of DNA repair.     

Inhibition and enhancement of phleomycin-induced DNA breakdown by aromatic tricyclic compounds.

Cationic aromatic tricyclic compounds including triphenylmethane dyes, phenazines, phenoxazines, acridines, phenothiazines, phenanthridinium compounds, anthracenes and xanthene dyes, which amplify cell killing in phleomycin-treated Escherichia coli B cells also modified phleomycin-induced breakdown of DNA to acid-soluble fragments. A plot of DNA breakdown as a function of concentration was bell-shaped for each of the active compounds, i.e. as the concentration increased, DNA breakdown was enhanced initially, but above a certain concentration, the proportion of DNA degraded declined, often to zero. One of the compounds, acriflavine, when tested also inhibited DNA breakdown following ultraviolet irradiation. A study, by sedimentation methods, of DNA single-strand breakage in phleomycin-treated E. coli cells, using 3 representative compounds, Crystal Violet, 3,6-diaminoacridine and Methylene Blue, revealed a consistent increase in DNA strand breaks as concentration of compound increased. In similar experiments with ethidium bromide the breakage yield/concentration curve exhibited a maximum. In general, however, it seems that the inhibition of DNA-breakdown observed at higher concentrations of these amplifying compounds is not explicable by an effect on the primary breakage event, but is due to suppression of exonucleolytic activity in the cells.     

Structure and Stability of Human Telomeric G-Quadruplex with Preclinical 9-Amino Acridines

G-quadruplexes are higher-order DNA structures formed from guanine-rich sequences, and have been identified as attractive anticancer drug targets. Elucidating the three-dimensional structure of G-quadruplex with 9-amino acridines and the specific interactions involved in binding selectivity are the key to understanding their mechanism of action. Fluorescence titration assays, competitive dialysis and NMR studies have been used to study the binding specificity of 9-amino acridines to DNA. Structural models of the complexes with the telomeric DNA G-quadruplex based on NMR measurements were developed and further examined by molecular dynamics simulations and free energy calculations. Selective binding of 9-amino acridines for G-quadruplex sequences were observed. These compounds bind between A and G-tetrads, involving significant π-π interactions and several strong hydrogen bonds. The specific interactions between different moieties of the 9-amino acridines to the DNA were examined and shown to play a significant role in governing the overall stabilities of DNA G-quadruplex complexes. Both 9-amino acridines, with similar binding affinities to the G-quadruplex, were shown to induce different level of structural stabilization through intercalation. This unique property of altering structural stability is likely a contributing factor for affecting telomerase function and, subsequently, the observed differences in the anticancer activities between the two 9-amino acridines.     

Differences in the interactions of liver alcohol dehydrogenases with probes binding into the substrate pocket

The interactions of three groups of probes (berberine alkaloids, tricyclic psychopharmaca and acridine derivatives) with isoenzymes of horse liver alcohol dehydrogenase and with rat liver alcohol dehydrogenase have been examined. These compounds inhibit the activity of the EE isoenzyme of horse liver alcohol dehydrogenase but differ in their behaviour towards the steroid-active enzymes (i.e. the ES isoenzyme of horse liver alcohol dehydrognase and alcohol dehydrogenase from rat liver): psychopharmaca inhibit, acridines activate and berberines do not bind. The ligands differ also in their influence on the modification of the EE isoenzyme by iodoacetate. Polarities (expressed as Kosower's Z values) of the respective binding sites on the EE isoenzyme were estimated from optical properties of bound probes. Berberines bind into a very hydrophobic area of the enzyme molecule, the binding site for psychopharmaca is moderately hydrophobic and that for acridines is rather polar. Steric arrangements of the binding sites are also discussed. The data presented confirm the existence of three distinct binding sites for these ligands in the substrate pocket of liver alcohol dehydrogenase.     

Synthesis and evaluation of unsymmetrical bis(arylcarboxamides) designed as topoisomerase-targeted anticancer drugs.

Symmetrical dimers of lipophilic intercalating chromophores linked by cation-containing chains have recently been shown to have broad-spectrum in vivo anticancer activity. We report the preparation and evaluation of a series of both symmetric and unsymmetric dimers of a variety of intercalating chromophores of varied DNA binding strength, including naphthalimides, acridines, phenazines, oxanthrenes and 2-phenylquinolines. The unsymmetrical dimers were prepared by sequential coupling of the chromophores to linkers with selectively protected primary terminal amines to ensure high yields and unequivocal product. Protection of the internal (secondary) amines as BOC derivatives was used to ensure complete structural specificity, and was also an aid to the purification of these very polar compounds. The growth inhibitory abilities (as IC(50) values) of the compounds in a range of cell lines showed that the nature of the linker chain was important, and independent of the nature of the chromophore, with compounds containing the dicationic linker [-(CH2)2NH(CH2)2NH(CH2)2-] being on average 30-fold more potent than the corresponding compounds containing the monocationic linker [-(CH2)3NMe(CH2)3-]. However, the chromophores also play a role in determining biological activity, with the cytotoxicities of symmetric and unsymmetric dicationic dimers correlating with the overall DNA binding abilities of the chromophores.     

In vitro efficiency of 9-(N-cinnamoylbutyl)aminoacridines against blood- and liver-stage malaria parasites

Novel 9-aminoacridine derivatives were synthesized by linking the heteroaromatic core to different cinnamic acids through an aminobutyl chain. The test compounds demonstrated mid-nanomolar in vitro activity against erythrocytic stages of the chloroquine-resistant W2 strain of the human malaria parasite Plasmodium falciparum. Two of the most active derivatives also showed in vitro activity against liver-stage Plasmodium berghei, with activity greater than that of the reference liver-stage antimalarial primaquine. The compounds were not toxic to human hepatoma cells at concentrations up to 5 μM. Hence, 9-(N-cinnamoylbutyl)aminoacridines are a new class of leads for prevention and treatment of malaria.     

Modulation of mutagenic properties in a series of DNA-directed alkylating agents by variation of chain length and alkylator reactivity.

Four series of aniline mustards linked to a DNA-affinic acridine chromophore by alkyl chains of varying length (2-5 carbon atoms) have been studied for their mutagenic properties, as estimated in four strains of Salmonella typhimurium and in Saccharomyces cerevisiae strain D5. The four series have very different mustard reactivities, as determined by the aniline link group (-O-, -CH2-, -S- or -SO2-). Some of the derived compounds cause frameshift mutagenesis which can be detected in TA98 and also "petite" mutagenesis activity, neither of which occur to significant extents with the parent mustards or with 9-aminoacridine. None of the derived compounds are as effective as the parent mustards in mitotic crossing-over, nor do they show ability for frameshift mutagenesis in S. typhimurium TA1977 which is typical of acridines. Some of the compounds have comparable frameshift activity to compounds such as ICR-191, but appear to have a different base-pair preference. The results indicate clear structure-activity relationships for the spectrum of mutagenic activity, which relate to both chain length and alkylator reactivity, for these compounds.     

Ce−ZSM-5: A Highly Capable Catalyst for the Construction of Acridines in Water and Their Computational Study against DNA Gyrase Protein

Cerium doped ZSM-5 (Ce−ZSM-5) as an environmentally benign and reusable catalyst for the construction of acridines in aqueous medium. This method produced corresponding acridines with good yields and shorter reaction times. Also avoids the use of hazardous solvents and involves a simple work-up process. The solid catalyst was prepared by doping Ce ions with ZSM-5 (Zeolite Socony Mobil-5) and confirmed by XRD, BET SA-PSD and SEM. The synthesised acridine derivatives were confirmed by ¹H-NMR, ¹³C-NMR and FT-IR spectroscopic data. The docking studies of the synthesised compounds are performed by the PyRx auto dock tool against DNA gyrase protein. The products 5a and 6d are found to be the best fit ligands against DNA gyrase protein.     

Frameshift mutagenesis by acridines in wild-type, uvrB and polA strains of Salmonella typhimurium with and without plasmid pKM101

A large range of acridines, including several anilinoacridines which are active as antitumour agents, have been studied for their ability to revert derivatives of Salmonella typhimurium strains carrying the frameshift marker hisC3076. The strains used all carried deep-rough (rfa) mutations, and were either wild-type with respect to DNA-repair capacity or carried uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without the mutation-enhancing N group plasmid pKM101 were also used. 9-Aminoacridine and other acridines appeared similar to the anilinoacridines for the most part, in that frameshift mutagenesis and toxicity appeared to be unaffected by the uvrB mutation or by the presence of plasmid pKM101. Exceptions were ICR191, 3-NO2-acridine and 1- or 3-NO2-anilinoacridine derivatives in which mutagenesis was increased in uvrB strains and also when pKM101 was present. These compounds were slightly more toxic in the uvrB background, but less toxic when pKM101 was present in either the uvrB or wild-type backgrounds. Mutagenesis by most compounds was reduced by the polA1 mutation and virtually eliminated (except in the case of ICR191) by the polA3 mutation. Plasmid pKM101 occasionally enhanced mutagenesis in the polA1 strain, whereas in the polA3 it appeared to have no effect whatsoever. Again, there were no obvious differences in toxicity between Pol+ and Pol- strains.