The mode of action of cytotoxic and antitumor 1-nitroacridines. I. The 1-nitroacridines do not exert their cytotoxic effects by physicochemical binding with DNA

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.     

Curing of the killer character of Saccharomyces cerevisiae with acridine orange

Acridine orange, an intercalating dye usually employed in the curing of bacterial plasmids, was tested for its ability to cure K1 and K2 killer strains (laboratory and wine strains). The results showed a high curing percentage of the killer character. This was demonstrated by the loss of M1 or M2 dsRNAs (responsible for toxin production and resistance to it) and because the meiotic products exhibited non-Mendelian segregation. The curing percentages varied, depending on the strain but not on the killer type, and showed similar efficiency as compared with other known curing agents.     

Intracellular distribution of acridine derivatives in platelets and their suitability for cytoplasmic pH measurements

The site and mechanism of accumulation of acridine derivatives into platelets and their isolated organelles were investigated. In addition, their suitability as indicators of cytoplasmic pH was analysed. Direct microscopic observation showed that quinacrine and 9-aminoacridine are concentrated inside organelles in platelets. Using fractionation studies, the acridine derivatives were found to accumulate particularly in dense and α-granules. Uptake into these organelles is driven by a pH differential across their membrane (acidic inside). Because of their cellular distribution, acridine derivatives were found to be poor indicators of cytoplasmic pH. In contrast, a poorly permeant dicarboxylated fluorescein derivative, generated in situ by cytosolic enzymes, is shown to be a more reliable probe of intracellular pH. The results are compared with previous reports of the use of 9-aminoacridine as a cytoplasmic pH probe in platelets and of quinacrine as a selective dense-granule marker.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Synthesis of novel acridine and bis acridine sulfonamides with effective inhibitory activity against the cytosolic carbonic anhydrase isoforms II and VII

4-Amino-N-(4-sulfamoylphenyl)benzamide was synthesized by reduction of 4-nitro-N-(4-sulfamoylphenyl)benzamide and used to synthesize novel acridine sulfonamide compounds, by a coupling reaction with cyclic-1,3-diketones and aromatic aldehydes. The new compounds were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely the cytosolic isoforms hCA I, II and VII. hCA I was inhibited in the micromolar range by the new compounds (K_Is of 0.16–9.64 μM) whereas hCA II and VII showed higher affinity for these compounds, with K_Is in the range of 15–96 nM for hCA II, and of 4–498 nM for hCA VII. The structure–activity relationships for the inhibition of these isoforms with the acridine–sulfonamides reported here were also elucidated.     

Subcellular localization of proflavine derivative and induction of oxidative stress—In vitro studies

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem. 2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells.     

Development of acridine derivatives as selective Mycobacterium tuberculosis DNA gyrase inhibitors

In this study we have designed p-phenylene diamine linked acridine derivative from our earlier reported quinoline–aminopiperidine hybrid MTB DNA gyrase inhibitors with aiming more potency and less cardiotoxicity. We synthesized thirty six compounds using four step synthesis from 2-chloro benzoic acid. Among them compound 4-chloro-N-(4-((2-methylacridin-9-yl)amino)phenyl)benzenesulphonamide (6) was found to be more potent with MTB DNA gyrase super coiling IC₅₀ of 5.21 ± 0.51 μM; MTB MIC of 6.59 μM and no zHERG cardiotoxicity at 30 μM and 11.78% inhibition at 50 μM against mouse macrophage cell line RAW 264.7.     

Structure–activity relationships in platinum–acridinylthiourea conjugates: effect of the thiourea nonleaving group on drug stability, nucleobase affinity, and in vitro cytotoxicity

The synthesis, cytotoxicity, and nucleoside binding of some platinum–acridinylthiourea conjugates derived from the prototypical compound [PtCl(en)(ACRAMTU)](NO₃)₂ {PT-ACRAMTU; en=ethane-1,2-diamine, ACRAMTU=1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, protonated form} are reported. To establish structure–activity relationships within this class of compounds, systematic changes were made to the thiourea nonleaving group, which links the intercalator to platinum. Three new derivatives of ACRAMTU, one di-, one tri-, and one tetraalkylated, were generated, where the degree of alkylation indicates the number of alkyl groups attached to the SCN₂ framework. Subsequent reaction of the tri- and tetraalkylated derivatives with activated [PtCl₂(en)] yielded the corresponding platinum conjugates. The dialkylated thiourea gave an unstable complex, which was not included in the studies. The crystal structure of PT-ACRAMTU·MeOH has been determined. In the solid state, one axial position of the square-planar platinum coordination sphere is partially shielded by the bulky thiourea group, providing a strong rationale for the kinetic inertness of the compound. The cytotoxicity of the prototype, the two new conjugates, and cisplatin was assessed in ovarian (A2780, A2780/CP), lung (NCI-H460), and colon (RKO) cancer cell lines using clonogenic survival assays. The derivatives containing trialkylated thiourea groups showed activity similar or superior to cisplatin, with IC₅₀ values in the low micromolar concentration range. The complex modified with the tetraalkylated (bulkiest) thiourea was significantly less active, possibly due to the greatly decreased rate of binding to nucleobase nitrogen (¹H NMR spectroscopy), but was most efficient at overcoming cross resistance to cisplatin in A2780/CP. Possible consequences of the reported structural modifications for the mechanism of action of these agents are discussed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations ACRAMTU 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea - Boc t-butyl carbamate - dGuo 2-deoxyguanosine - dien N¹-(2-aminoethyl)ethane-1,2-diamine - en ethane-1,2-diamine - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PT-ACRAMTU [PtCl(en)(ACRAMTU)](NO₃)₂ - TSP 3-(trimethylsilyl)propionate, sodium salt     

Pharmacophore-based discovery of triaryl-substituted imidazole as new telomeric G-quadruplex ligand

Discovery of potent and selective ligands for telomeric G-quadruplex DNA is a challenging work. Through a combination approach of pharmacophore model construction, model validation, database virtual screening, chemical synthesis and interaction evaluation, we discovered and confirmed triaryl-substituted imidazole TSIZ01 to be a new telomeric G-quadruplex ligand with potent binding and stabilizing activity to G-quadruplex DNA, as well as a 8.7-fold selectivity towards telomeric G-quadruplex DNA over duplex DNA.