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Several analogues of the general formulae 2-methoxy-9-substituted acridine and 6-chloro-2-methoxy-9-substituted acridine were synthesized and evaluated in vitro at 6.25 microg/mL against M. tuberculosis H37Rv. Compounds 15 and 17 showed potential antitubercular activity with 100% inhibition to the virulent mycobacterium.
DACA [N-[2-(dimethylamino)ethyl]acridine-4-carboxamide], an acridine derivative that is highly active against solid tumours in mice, is currently in clinical trial. The ability of DACA to overcome "atypical" (topoisomerase II-mediated) multidrug resistance has been hypothesised to stem from its dual topoisomerase I/II specificity. We investigated the topoisomerase specificity of DACA and its 7-chloro derivative (C1-DACA) using camptothecin and amsacrine as control compounds. In cell-free assays employing supercoiled plasmid DNA, C1-DACA at 5 microM induced topoisomerase I-mediated DNA breakage, indicating cleavable complex formation (poisoning), and at 10 microM it inhibited relaxation of DNA, consistent with suppression (self-inhibition) of poisoning. In this assay, DACA provided no evidence of poisoning of this enzyme but inhibited its function at concentrations above 10 microM. In DNA cleavage assays utilising purified topoisomerase II, DACA induced breakage of supercoiled plasmid DNA at 5 microM whereas C1-DACA showed very weak poisoning at 1 microM and inhibition at 5 microM. Under conditions required for the assay of DNA relaxation, C1-DACA, but not DACA, inhibited topoisomerase II action at 5 microM. The actions of DACA and C1-DACA could also be distinguished by their ability to form DNA-protein cross-links in H460 human lung carcinoma cells as measured by precipitation of DNA-protein complexes with sodium dodecyl sulfate and potassium chloride. Both drugs stimulated the formation of complexes at low concentrations but inhibited formation at high concentrations. In survival assays with H460 cells, both drugs demonstrated biphasic responses with self-inhibition of cytotoxicity at intermediate drug concentrations. It was concluded that although both drugs have dual topoisomerase I/II specificity, DACA preferentially poisons topoisomerase II and C1-DACA preferentially poisons topoisomerase I. In addition, drug-induced inhibition of topoisomerase action at higher drug concentrations may mask poisoning in the cell-free assays as well as masking cytotoxicity in cultured cells. A model in which drug binding occludes topoisomerase-binding sites on the DNA can explain this self-inhibition of cytotoxic action.
DACA [N-[2-(dimethylamino)ethyl]acridine-4-carboxamide] is an acridine derivative with high activity against solid tumours in mice and a dual mode of cytotoxic action involving topoisomerases I and II. The plasma pharmacokinetics of DACA were studied in 28 patients with solid tumours in a phase I trial. A single dose was given every 3 weeks, being escalated from a starting dose of 18 mg/m2 (as the dihydrochloride trihydrate salt) to a maximal dose, limited by severe pain in the infusion arm, of 1000 mg/m2. Drug was given by constant intravenous infusion with a target delivery period of 3 h. Blood samples were taken from the contralateral arm before, during and for up to 72 h after the infusion. DACA was separated from plasma by solid-phase extraction and was analysed by reversed-phase high-performance liquid chromatography (C18 column) using fluorescence detection. A two-compartment pharmacokinetic model provided the best fit for the concentration-time profiles obtained for most patients showing clearance of 1.00+/-0.36 l h(-1) kg(-1), a volume of distribution of the central compartment of 0.72+/-0.55 l/kg, an initial half-life of 0.28+/-0.19 h and a terminal half-life of 2.04+/-0.94 h. All pharmacokinetic parameters were independent of dose, indicating first-order kinetics. As DACA binds strongly to alpha1-acid glycoprotein, plasma concentrations of this protein were determined and used to estimate free-drug fractions in plasma. Estimated values for the free fraction varied from 0.9% to 3.3% and were lower than those determined by equilibrium dialysis for mice and rats (15% and 16%, respectively). At the maximum tolerated dose (MTD) of 750 mg/m2, the area under the drug concentration-time curve (AUC) was 46.2+/-4.4 microM h, exceeding that obtained in mice treated at the MTD (23.4 microM h). On the other hand, the corresponding free-drug AUC was 0.92+/-0.03 microM h, much lower than the corresponding value (3.5 microM h) determined for mice. These results suggest that free-drug rather than total drug concentrations are more appropriate for interspecies dose comparisons when significant differences exist in the free plasma fraction.
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.
The strong binding of various acridine dyes to DNA has been studied by the measurements of flow dichroism, flow polarized fluorescence and viscosity. Negative flow dichroism and percentage change in polarized fluorescence intensity show that intercalated dye molecules are oriented rather perpendicularly to the main axis of the DNA helix, like base pairs. On the other hand, viscosity measurements show that the increase of the contour length of DNA depends on the dye structure, being much smaller in the case of dyes with bulky substituents compared to that of the other dyes. This may be attributed to the formation of the outside bound complex. Further, the introduction of bulk substituents to the acridine ring leads to a little smaller values of the reduced dichroism and intensity change of polarized fluorescence. The results may be qualitatively understood if we assume that the outside bound dye lies in the groove of the DNA helix and the plane of the dye tilts from the perpendicular direction relative to the main axis of the helix.
Oxidation of the experimental anti-tumour agent N-[(2'-dimethylamino)ethyl]acridine-4-carboxamide (AC; NSC 601316; acridine carboxamide) to the 9(10H)acridone, followed by ring hydroxylation and glucuronidation, appears to be the main pathway of detoxication of AC in the rat and mouse. The acridone formation has been further characterized in vitro using an enzyme-enriched fraction where activity per milligram protein is increased approximately 10-fold compared with the cytosolic fraction. Inhibition by amsacrine [4'-(9-acridinylamino)methanesulphon-m-anisidide; NSC 249992] and menadione (50% inhibition at 6.4 and 1.8 microM, respectively) but not allopurinol (to 30 microM) indicates that the activity is due to aldehyde oxidase, without the involvement of xanthine oxidase. Interestingly, acridone formation in both the cytosolic and enzyme-enriched fractions is highly sensitive to the classical cytochrome P450 inhibitor SKF-525A [proadifen hydrochloride; 2'-(diethylamino)ethyl 2,2-diphenylpentenoate] (50% inhibition at 9.2 and 1.9 microM, respectively). Further analysis indicates mixed non-competitive type inhibition by SKF-525A (K(is), 0.3 microM; K(ii), 4.9 microM). Little or no inhibition was seen with cimetidine, metyrapone or methimazole. No NADPH-dependent acridone formation was observed with the microsomal fraction. These data indicate that acridone formation previously observed in isolated rat hepatocytes and in vivo is most likely due to aldehyde oxidase rather than cytochrome P450.
The effect of dimeric DNA intercalating compounds was assayed on a purified AP endonuclease from Microccoccus luteus using apurinic supercoiled PM2 DNA as a substrate. Binding on apurinic sites was estimated through the competition with the intercalating compound, 9-NH2-ellipticine, which displays great specificity for apurinic sites. An acridine dimer with a spermine linker is at 0.1 microM the best inhibitor of cleavage at the apurinic site induced either by the AP endonuclease or by 9-NH2-ellipticine. Bisintercalating agents are more effective inhibitors of AP endonuclease than monointercalating ones. Most effective inhibitors among dimers have acridine residues.
Specific mitochondrial incorporation of 10 N-nonyl acridine orange (NAO) is demonstrated by subcellular fractionation of rat hepatocytes. Moreover, comparative studies with NAO and rhodamine 123 (Rh 123) prove that acridine orange-derivative uptake is independent of transmembrane mitochondrial potential, a property allowing its utilization for the assessment of mitochondrial membrane mass modifications under various physiological states. Using NAO and Rh 123, we have respectively followed the biosynthesis of mitochondrial membrane and its assembly under a functional state during the L1210 cell cycle. Their evolution occurs in two stages according to a well-defined sequential order. Mitochondrial biogenesis, as revealed by NAO incorporation, occurs essentially in the G1 phase (probably mitochondrion enlargement) but also starts in late S phase (probably mitochondrion division). The increased amount of functional mitochondrial membrane, monitored by Rh 123 uptake, is emphasized in late G1 (prerequisite to DNA synthesis) and during G2M phases (prerequisite to mitosis). This alternative succession of phases displays the existence of a time-lag between the biosynthesis of mitochondrial membrane and its functional organization. Such an analysis confirms the potential of the NAO probe to evaluate mitochondrial membrane mass changes in various biological fields.
Acridine orange staining differentiates the cystic and the central body forms of Blastocystis hominis and offers a very convenient and easy method to observe the internal structure of the parasite. Acridine orange stains the nuclei and the central body of the rounded vacuolar forms of the parasite bright and dull green, respectively. The colour changes to yellow and then to flaming red-orange when the rounded central body forms of the parasite become cystic.
Benz[c]acridine and many of its related compounds have been shown to exhibit carcinogenic activity. Unfortunately, these compounds are continually being found in many natural and environmental samples in widely divergent geographical locations. A review of chromatographic methods for mainly benz[c]acridine and its analogues is presented.
The antimutagenic effects of selenium as sodium selenite were investigated using the Ames Salmonella/microsome mutagenicity test. The compounds examined were acridine orange and 7,12-dimethylbenz[alpha]anthracene. Selenium (22 ppm) reduced the number of histidine revertants caused by 20 microgram acridine orange and 20 microgram 7,12-dimethylbenz[alpha]anthracene by 52 and 74%, respectively. Increasing the quantity of selenium added to the plates further suppressed the mutagenicity of the test compounds. The antimutagenic effects of selenium cannot be explained by lethality of Salmonella typhimurium.
Using the ADAPT and CHEMLAB-II systems for structure-activity analysis, computer-calculated electronic properties of molecules were used to derive structure-activity relationships for predicting the mutagenicity of a set of substituted acridines in strain TA1537 of the Ames Salmonella assay. A collection of 40 acridines, with a variety of substituents, was examined. A set of 4 electronic descriptors was found which could be used to correctly classify all but two of the compounds as mutagenic or nonmutagenic. A negative correlation was found between the sum of the Hammett aromatic substituent parameters and the level of mutagenicity of the structures, expressed as log(number of revertants/plate + 1) at a 20-micrograms dose. This correlation, however, was not high enough to allow precise estimation of the mutagenicity values.
Acridine Orange is commonly used as a fluorescent counterstain in fluorescent tract tracing techniques. Here we describe a method in which the substitution of the standard washing solutions (i.e., 0.9% saline) for a diluted solution of Acridine Orange (0.001%) during the perfusion of the animal before fixation provides a fluorescent counterstaining compatible with Fast Blue fluorescent retrograde labeling. In contrast to other fluorescent counterstaining methods, this procedure minimizes the diminution in the fluorescence of the tracer during the handling of sections.
Acridine Orange fluorescent staining was applied to the micronucleus test in mice and rats. Micronuclei emitted bright green fluorescence and were easily distinguished from micronucleus-like inclusions or contaminants. In rat bone-marrow cells, micronuclei with green fluorescence could be easily distinguished from granules accidentally dispersed from broken mast cells, which showed bright red fluorescence. Therefore, it is recommended that the Acridine Orange staining method be used to provide more reliable data in the micronucleus test.
Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluorescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D equals 10...
The application of scaled particle theory to the gels formed by DNA in the ultracentrifuge has provided values for the effective length and the effective radius of the DNA particle. Ethidium bromide has been shown to cause extensive lengthening of the DNA in dilute salt. Acridine orange interaction with DNA resulted in modest changes in DNA dimensions. These results are explained in terms of binding for acridine orange and of denaturation of DNA by ethidium bromide.
The effects of haematoporphyrin (Hp), haematoporphyrin derivative (HpD), coumarin, 7-hydroxycoumarin, 4-hydroxycoumarin, amino-methyl-coumarin acetic acid and acridine orange, with and without photoradiation, on Landschutz ascites tumour (LAT) cells were examined. Hp, HpD and acridine orange showed a significant effect on LAT cell viability following photoradiation. However, coumarin or its derivatives had no significant effect on LAT cell viability, with or without photoradiation, over 18 h. Coumarin alone had a significant effect on viability of LAT cells, in vitro, over a period of 9 days. This effect was verified by transplantability studies. Coumarin and 7-hydroxycoumarin both exhibited cytostatic effects on LAT cell growth.
A comparison of the relative efficiency of acridine orange with some Romanowsky staining procedures indicated that acridine orange is superior in demonstrating the presence of Haemobartonella felis in feline blood. Of the Romanowsky staining procedures, that of May-Grunwald-Giemsa was at least one and a half and about twice as efficient as the Giemsa and Leishman methods, respectively.