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.     

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.     

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.     

The mechanism of inhibition of bacteriophage T7 RNA synthesis by acridines, diacridines and actinomycin D.

A homologous series of diacridines, as well as 9-amino acridine, were assayed for their ability to interfere with the synthesis of RNA (bands U-VI) by bacteriophage T7 DNA-dependent RNA polymerase transcribing T7 DNA in vitro; their action was compared to that of actinomycin D. It was found that, in contrast to actinomycin D which inhibits chain elongation, the acridines tested inhibited chain initiation only; no evidence for inhibition of chain elongation was noted. No clear-cut differentiation between single and double intercalators on the mechanism of inhibition of RNA synthesis could be determined, except that the latter are more potent inhibitors. However, it appears that diacridines connected with a diethyldiamine and a butyldiamine chain are less inhibitory to the synthesis of the RNA of Bands III and IV. The results furthermore indicate that the estimation of the number average molecular weight alone, without identification of the product RNA, is a potentially misleading method of determining the mode of action of these drugs.     

Effects of acridines on DNA synthesis in vitro

Summary Acridines are known to induce with a very high efficiency the cytoplasmic petite mutation in yeast. Since in petite mutants the base composition of mitochondrial DNA is altered, these dyes should interfere in the replication of mitochondrial DNA. To approach this problem a detailed knowledge of the effect of acridines on DNA synthesis is necessary.The inhibitory effect of two acridines and of the related dye methylene blue on Escherichia coli DNA polymerase has been studied under different assay conditions. Several aspects of this inhibitory effect have been investigated including the extent of inhibition in the presence of several primers, the effect of acridines on the unprimed synthesis and on the addition of single nucleoside triphosphates to DNA 3-OH terminals, the effect of preincubation of DNA and of DNA polymerase with acridines and the base composition of DNA synthesized in the presence of the dyes.The extent of inhibition has not been found to be dependent on the base composition of the primer except in the case of primers very rich in A+T, while a strong effect on the unprimed synthesis is due to a substantial increase of the lag period. The A/C ratio in the product synthesized under strongly inhibitory conditions is shifted with respect to that of the primer.The results seem to indicate the preferential replication, in the presence of acridines, of A+T rich regions of DNA.     

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.     

Interaction of antitumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study.

The cytotoxic and mutagenic properties of antitumor drugs such as adriamycin, acridines, diacridine, actinomycin D and Pt compounds are related to their interaction with nucleic acids and inhibition of protein synthesis. We have examined their interaction with human erythrocyte ghost membranes and murine mastocytoma cells using spin labeling techniques. These drugs induce changes in electron spin resonance of the spin labeled ghost membranes and in the mastocytoma cells. These alterations suggest that these drugs induce changes in protein conformation of the membranes. The membrane binding properties of these drugs may be important in their mechanism of action.     

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.     

Conversion of a powerful frameshifter acridine to a base-pair substitution analog.

The frameshift mutagenic mechanism for acridines has been attributed to the intercalative type of association between acridines and nucleic acids. However, it appears that these molecular details are insufficient to explain the frameshifting process. In order to design an effective drug probe to analyze the $\text{in vivo}$ interactions of acridines leading to frameshifting, an azide analog of 9-aminoacridine was studied in Ames' $\text{Salmonella}$ strains. The surprising findings were that by substituting an amino group at the 9 ring position with an azido group, the mutagenicity was converted from frameshifter to base-pair substitution.     

The induction of chromosomal aberrations and SCE by visible light in combination with dyes. I. The effect of hypoxia during light exposure in unsynchronized Chinese hamster ovary cells, sensitized with acridines

A comparison has been made between the ability of different acridine compounds to act as sensitizers for visible light (400-700 nm) induced chromosomal aberrations and sister-chromatid exchanges (SCE) in unsynchronized Chinese hamster ovary (CHO) cells. Cells were treated for 20 min with acridines (0.1-5.0 microgram/ml), washed free of excess dye and subsequently exposed to visible light (2 x 40 W/8 W m-2) either in air or in nitrogen for 5-15 min. The 4 acridines tested, proved to be effective sensitizers for the induction of both chromosomal aberrations and SCE by visible light. The most pronounced effect was observed when the light exposure of the fluorochrome-pretreated cells was performed in air. Hypoxic conditions during light exposure reduced the effect dramatically, especially in the case of induced chromosomal aberrations. The order of efficiency for the induction of both chromosomal aberrations and SCE was acridine orange greater than acridine yellow greater than proflavine greater than 3,6-diamino-10-methylacridine. The results are discussed in terms of S-independent versus S-dependent mechanisms for inducing chromosomal alterations and the potential involvement of oxygen-derived free radicals in this process.     

DNA-binding compounds--synthesis and intercalating properties of a peptide-diamino diacridine

A novel diacridine has been prepared in which two acridines are linked by a flexible peptide chain composed of gamma-aminobutyric acid, tyrosine, lysine and glycine. Synthesis of N-[9-acridinyl)-4-aminobutanoyl-tyrosyl-lysyl-lysyl-glycyl)-N'-(9- acridinyl)-1, 3-diaminopropane (VII) was achieved in 8% overall yield by a solution phase stepwise procedure. This compound binds to DNA by intercalation of both chromophores with at least a 140-fold enhancement of affinity compared to 9-aminoacridine.     

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.     

A comparison of the bactericidal and photobactericidal activities of aminoacridines and bis(aminoacridines)

An attempt was made to increase the activity of some antibacterial acridines in two ways: (a) via the use of bis-aminacrines (analogues of 1,6-bis-[9-acridinamino]hexane), in order to increase the potential for DNA intercalation and (b) via photoactivation, as several acridines are known to produce the cytotoxin singlet oxygen on irradiation. The bactericidal activity of the simple aminoacridines was much higher than that of the corresponding bis-acridine compounds. In addition, on low-power illumination of the compounds in liquid culture, many of the monomeric compounds exhibited marked increases in activity against both Gram-negative and Gram-positive bacteria, whereas the bis-acridines showed little or no increase.     

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.     

Mechanism of acridine uptake by submitochondrial particles.

Acridines were compared regarding their ability to be taken up by submitochondrial particles under energized conditions. pH dependence of uptake was explored, and it was found that acridines fell into three classes independently of their pK_a value: acridines which are not taken up, acridines taken up at all pH values, and acridines taken up only at alkaline pH. Partition measurements between H₂O and chloroform phase showed a similar pattern, and affinity for the organic phase seemed to parallel uptake. Acridines which are taken up by submitochondrial particles at acidic pH under energized conditions despite a high pK_a value could also be extracted into chloroform at acidic pH, thus implying that the dye's uncharged form has a high affinity for the organic phase. By supplementing the aqueous medium with lipophilic anions, the dye may also be extracted in its charged form. The data support a mechanism for acridine uptake in which diffusion of the uncharged form across the membranes is an obligatory step. Some previously reported inhibitory anion effects on uptake may be explained by ion pair formation, which allows release of the accumulated charged form.     

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.     

The effect of proflavine on pyruvate kinase I of Escherichia coli B.

Proflavine (PF) inhibited glucose use in sensitive but not resistant Escherichia coli B. Glucose transport (as measured by alpha-methylglucoside accumulation) was only partly inhibited by PF concentration that completely blocked glucose use. Fructose 1,6-diphosphate-(FDP)-regulated pyruvate kinase (PK1) (EC 2.7.1.40), the only glycolytic enzyme affected by PF, was completely inhibited by a dye concentration of 0.8 mM. The inhibition curve for PF was sigmoidal, suggesting that PF was acting as an allosteric inhibitor. PF increased the K 1/2 for phosphoenolpyruvate (PEP) and lowered the V; however, it had no effect on the Hill number for PEP. PF inhibition was partially reversed by FDP but not by cyclic AMP, AMP, ATP, fuctose 6-phosphate, or dithiothreitol. Studies with a variety of acridines indicated that those substituted at the 3-position are the most effective inhibitors and also that hydrophobic interactions may be involved in PF inhibition of PK I. PK I for E. coli B/Pr was also strongly inhibited by PF, indicating that PF resistance does not lie at the level of this enzyme. Ribose-5-phosphate-regulated pyruvate kinase (EC 2.7.1.40) was much less sensitive that PK I to the inhibitory effects of PF. A role for PF as a molecular probe for PK I has been proposed.     

3,4-Dihydro-1H-[1,3]oxazino[4,5-c]acridines as a new family of cytotoxic drugs

A series of [1,3]oxazino fused acridines has been prepared as precursors of cytotoxic 3-amino-4-hydroxymethylacridine 2. Their cytotoxic activity has been evaluated against HT29 colon carcinoma cell line and was shown to be dependent on the nature of the substituent located on position 2 of the oxazine ring. Additionally, the nitrophenyl derivative 3f is activated by nitroreductase, indicating its potency as prodrug for either gene-directed or antibody-directed enzyme prodrug therapies.     

Stimulation of incorporation of nucleic acid precursors into HeLa cells caused by provaline

1. The effect of proflavine and other acridines on the incorporation of precursors into the nucleic acids of HeLa cells was examined. 2. Relatively low concentrations (50mum) of proflavine completely inhibited incorporation of precursors into DNA, but allowed a small extent of incorporation into RNA. 3. Acridine-resistant incorporation into RNA was unaffected by actinomycin D at 2mug./ml. and persisted even at high concentrations (500mum) of many acridines. 4. A few combinations of acridine and precursor, notably 250mum-proflavine and [(14)C]adenine, caused a stimulation of incorporation. 5. The proflavine-stimulated incorporation was into alkali-stable di- and tri-nucleotides. 6. It was concluded that the effect was due to the preferential inhibition of degradation of a fraction of RNA that normally turned over, thus allowing small radioactive oligonucleotides to accumulate in the cells.     

Rational design,synthesis and biological evaluation of thiadiazinoacridines: a new class of antitumor agents

A series of potential DNA-binding antitumor agents, 3-[omega-(alkylamino)alkyl]-6-nitro-thiadiazino[3,4,5-kl]acridines 12 and 1,3-di[omega-(alkylamino)alkyl]-6-nitro-thiadiazino[3,4,5-kl]acridines 13, has been prepared by cyclization with SOCl(2) of 1-[[omega-(alkylamino)alkyl]amino]-9-imino-4-nitro-9,10-dihydroacridines 16 or 1-[[omega-(alkylamino)alkyl]amino]-9-[omega-(alkylamino)alkyl]imino-4-nitro-9,10-dihydroacridines 17, respectively. The non-covalent DNA-binding properties of 12, 13 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives toward six tumor cell lines, including human colon adenocarcinoma (HT29) and human ovarian carcinoma (A2780 sensitive, A2780cisR cisplatin-resistant, CH1, CH1cisR cisplatin-resistant, and SKOV-3) cells, are described and compared to that of reference drugs. In vivo antitumor activity of some selected derivatives, endowed with relevant cytotoxic activity against murine leukemia P388 are reported. The 3-[2-(dimethylamino)ethyl]-6-nitro-2,7-dihydro-3H-2 lambda(4)-thiadiazino[3,4,5-kl]acridin-2-one (12d) has been identified as a new lead in the development of anticancer tetracyclic acridine derivatives.