Photodynamic therapy of multidrug resistant leukemic murine cells by 3,6-bis(alkylthiourea)acridine hydrochlorides.

Efforts to overcome multidrug resistance in cancer have led to the development of several novel strategies including photodynamic therapy (PDT). PDT is based on the use of photosensitizers (PSs) photoactivation, which causes the formation of reactive oxygen species that can induce cell death. In the last decade, the development of new PSs has been significantly accelerated. Recently, acridine-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been investigated as a new group of PSs and we have shown that PDT/AcrDTUs caused cell death of mouse leukemic cells L1210. In this study, we investigated the efficacy of PDT/AcrDTUs for the treatment of L1210/VCR cells as a model of chemoresistant cells (overexpressing P-glycoprotein, P-gp). The photoactivation (365 nm, 1.05 J/cm2) increased the cytotoxicity of AcrDTUs 10-15 times. Inhibition of P-gp (verapamil) has been shown to have no significant effect on the accumulation of propyl-AcrDTU (the most potent derivative) in L1210/VCR cells. The intracellular distribution of this acridine derivative has been studied. Prior to irradiation of the resistant cells, propyl-AcrDTU was sequestered mainly in the cytosol, partly in the mitochondria, and, unlike in the sensitive cells, the AcrDTU was not found in the lysosomes. PDT with 1 µM propyl-AcrDTU induced cell shrinkage and "ladder DNA" formation, and although a drastic decrease of the intracellular ATP level was observed at the same time, there was no increase in extracellular LDH activity. AIF in the nucleus can induce DNA fragmentation and we have actually observed a mitochondrio-nuclear translocation of AIF. We concluded that AcrDTUs are photocytotoxic against L1210/VCR cells and that mitochondria play an important role in cell death induced by PDT.

Mechanochemical Synthesis and Isomerization of N-Substituted Indole-3-carboxaldehyde Oximes †.

Performing solution-phase oximation reactions with hydroxylamine hydrochloride (NH2OH·HCl) carries significant risk, especially in aqueous solutions. In the present study, four N-substituted indole-3-carboxaldehyde oximes were prepared from the corresponding aldehydes by solvent-free reaction with NH2OH·HCl and a base (NaOH or Na2CO3) using a mechanochemical approach, thus minimizing the possible risk. In all cases, the conversion to oximes was almost complete. The focus of this work is on 1-methoxyindole-3-carboxaldehyde oxime, a key intermediate in the production of indole phytoalexins with useful antimicrobial properties. Under optimized conditions, it was possible to reach almost 95% yield after 20 min of milling. Moreover, for the products containing electron-donating substituents (-CH3, -OCH3), the isomerization from the oxime anti to syn isomer under acidic conditions was discovered. For the 1-methoxy analog, the acidic isomerization of pure isomers in solution resulted in the formation of anti isomer, whereas the prevalence of syn isomer was observed in solid state. From NMR data the syn and anti structures of produced oximes were elucidated. This work shows an interesting and possibly scalable alternative to classical synthesis and underlines environmentally friendly and sustainable character of mechanochemistry.

Proliferation inhibition of novel diphenylamine derivatives.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs in the world but some NSAIDs such as diclofenac and tolfenamic acid display levels of cytotoxicity, an effect which has been attributed to the presence of diphenylamine contained in their structures. A novel series of diphenylamine derivatives were synthetised and evaluated for their cytotoxic activities and proliferation inhibition. The most active compounds in the cytotoxicity tests were derivative 6g with an IC50 value of 2.5 ±â€¯1.1 × 10-6 M and derivative 6f with an IC50 value of 6.0 ±â€¯3.0 × 10-6 M (L1210 cell line) after 48 h incubation. The results demonstrate that leukemic L1210 cells were much more sensitive to compounds 6f and 6g than the HEK293T cells (IC50 = 35 × 10-6 M for 6f and IC50 > 50 × 10-6 M for 6g) and NIH-3T3 (IC50 > 50 × 10-6 M for both derivatives). The IC50 values show that these substances may selectively kill leukemic cells over non-cancer cells. Cell cycle analysis revealed that a primary trend of the diphenylamine derivatives was to arrest the cells in the G1-phase of the cell cycle within the first 24 h. UV-visible, fluorescence spectroscopy and circular dichroism were used in order to study the binding mode of the novel compounds with DNA. The binding constants determined by UV-visible spectroscopy were found to be in the range of 2.1-8.7 × 104 M-1. We suggest that the observed trend for binding constant K is likely to be a result of different binding thermodynamics accompanying the formation of the complexes.

Low-dimensional compounds containing bioactive ligands. Part VIII: DNA interaction, antimicrobial and antitumor activities of ionic 5,7-dihalo-8-quinolinolato palladium(II) complexes with K+ and Cs+ cations.

Starting from well-defined NH2(CH3)2[PdCl2(XQ)] complexes, coordination compounds of general formula Cat[PdCl2(XQ)] have been prepared by cationic exchange of NH2(CH3)2+ and Cat cations, where XQ are biologically active halogen derivatives of quinolin-8-ol (5-chloro-7-iodo-quinolin-8-ol (CQ), 5,7-dibromo-quinolin-8-ol (dBrQ) and 5,7-dichloro-quinolin-8-ol (dClQ)) and Cat is K+ or Cs+. The cation exchange of all prepared complexes, K[PdCl2(CQ)] (1), K[PdCl2(dClQ)] (2), K[PdCl2(dBrQ)] (3), Cs[PdCl2(CQ)] (4), Cs[PdCl2(dClQ)] (5) and Cs[PdCl2(dBrQ)] (6) was approved using IR spectroscopy, their structures in DMSO solution were elucidated by one- and two-dimensional NMR experiments, whereas their stability in solution was verified by UV-VIS spectroscopy. Interaction of complexes to ctDNA was investigated using UV-VIS and fluorescence emission spectroscopy. The minimum inhibitory concentration and the minimum microbicidal concentration values were detected against 15 bacterial strains and 4 yeast strains to examine the antimicrobial activity for the complexes. The in vitro antitumor properties of the complexes were studied by testing the complexes on leukemic cell line L1210, ovarian cancer cell line A2780 and non-cancerous cell line HEK293. The majority of the prepared compounds exhibited moderate antimicrobial and very high cytotoxic activity.

Mechanochemical approach for the capping of mixed core CdS/ZnS nanocrystals: Elimination of cadmium toxicity.

The wet mechanochemical procedure for the capping of the CdS and CdS/ZnS quantum dot nanocrystals is reported. l-cysteine and polyvinylpyrrolidone (PVP) were used as capping agents. When using l-cysteine, the dissolution of cadmium(II) was almost none for CdS/ZnS nanocrystals. Moreover, prepared CdS- and CdS/ZnS-cysteine nanosuspensions exhibited unimodal particle size distributions with very good stability, which was further supported by the zeta potential measurements. The Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy showed the successful embedment of cysteine into the structure of the nanocrystals. Additionally, the optical properties were examined, and the results showed that the cysteine nanosuspension has promising fluorescence properties. On the other hand, PVP was not determined to be a very suitable capping agent for the present system. In this case, the release of cadmium(II) was higher in comparison to the l-cysteine capped samples. The nanosuspensions were successfully used for in vitro studies on selected cancer cell lines. Using fluorescence microscopy, it was evidenced that the nanocrystals enter the cell and that they can serve as imaging agents in biomedical applications.

New spiro tria(thia)zolidine-acridines as topoisomerase inhibitors, DNA binders and cytostatic compounds.

Three new diphenylsubstituted spirotriazolidine- and thiazolidinone-acridines were prepared and their interaction with calf thymus DNA investigated with UV-vis, fluorescence, circular dichroism spectroscopy and viscometry. The binding constants K were estimated to range from 0.34 to 0.93 × 10(4) M(-1). UV-vis, fluorescence and circular dichroism measurements indicated that the compounds act as effective DNA-interacting agents. Electrophoretic separation proved that ligands inhibited topoisomerase I and II. The biological activity of compounds 3, 5 &6 at several different concentrations (10, 20 and 50 µM) was evaluated both 48 h and 72 h following their addition to HL-60 cancer cells. The results were analysed using various different techniques (MMP detection, changes in metabolic activity/viability and analysis of cell cycle distribution). Acridine was also used as the positive control in these assays. The results from MMP analysis demonstrate the strong effect of 3-diphenylamino-2-(acridin-9-yl)imino-1,3-thiazolidin-4-one (5) on mitochondrial physiology. Cell viability analysis showed that acridine derivatives 3 and 6 were less effective than derivative 5 and the acridine control.

Prediction by (13)C NMR of regioselectivity in 1,3-dipolar cycloadditions of acridin-9-yl dipolarophiles.

Strong correlation was found between (13)C NMR chemical shifts of dipolarophilic CH=CH carbons and regioselectivity in 1,3-dipolar cycloadditions of new acridin-9-yl dipolarophiles with stable benzonitrile oxides (BNO). Accordingly, two starting dipolarophiles, (acridin-9-yl)-CH=CH-R (R = COOCH3 or Ph), reacted with three BNOs (2,4,6-trimethoxy, 2,4,6-trimethyl, and 2,6-dichloro) to give a mixture of two target isoxazoline regioisomers in which the acridine was bound either to isoxazoline C-4 carbon (4-Acr) or C-5 one (5-Acr). Methyl 3-(acridin-9-yl)propenoate afforded major 4-(acridin-9-yl)-isoxazoline-5-carboxylates (4-Acr) and minor 5-(acridin-9-yl)-4-carboxylates (5-Acr). 9-(2-Styryl)acridine regiospecifically afforded only 4-Acr cycloadducts. The ratios of regioisomers were compared with analogous reactions of acridin-4-yl dipolarophiles. Regioselectivity was dependent on a polarity of the CH=CH bond, donor effects in BNO, and stabilization by stacking of aromatic substituents in the products.

Strong deshielding in aromatic isoxazolines.

Very strong proton deshielding was found in di/tri-aromatic isoxazoline regioisomers prepared from acridin-4-yl dipolarophiles and stable benzonitrile oxides (BNO). Three alkenes, (acridin-4-yl)-CH=CH-R (R = COOCH3, Ph, and CONH2), reacted with three BNO dipoles (2,4,6-trimethoxy, 2,4,6-trimethyl, 2,6-dichloro) to give pairs of target isoxazolines with acridine bound to C-4 or C-5 carbon of the isoxazoline (denoted as 4-Acr or 5-Acr). Regioselectivity was dependent on both the dipolarophile and dipole character. The ester and amide dipolarophile displayed variable regioselectivity in cycloadditions whereas the styrene one afforded prevailing 4-Acr regioisomers. 2,4,6-Trimethoxy-BNO was most prone to form 5-Acr isoxazolines while mesitonitrile oxide gave major 4-Acr isoxazolines. Basic hydrolysis of the amide cycloadduct led to an unexpected isoxazolone product. The structure of the target compounds was studied by NMR, MS, and X-ray crystallography.

Synthesis and biological evaluation of novel tacrine derivatives and tacrine-coumarin hybrids as cholinesterase inhibitors.

A series of novel tacrine derivatives and tacrine-coumarin heterodimers were designed, synthesized, and biologically evaluated for their potential inhibitory effect on both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Of these compounds, tacrine-coumarin heterodimer 7c and tacrine derivative 6b were found to be the most potent inhibitors of human AChE (hAChE), demonstrating IC50 values of 0.0154 and 0.0263 µM. Ligands 6b, 6c, and 7c exhibited the highest levels of inhibitory activity against human BuChE (hBuChE), demonstrating IC50 values that range from 0.228 to 0.328 µM. Docking studies were performed in order to predict the binding modes of compounds 6b and 7c with hAChE/hBuChE.

Novel trisubstituted acridines as human telomeric quadruplex binding ligands.

A novel series of trisubstituted acridines were synthesized with the aim of mimicking the effects of BRACO19. These compounds were synthesized by modifying the molecular structure of BRACO19 at positions 3 and 6 with heteroacyclic moieties. All of the derivatives presented in the study exhibited stabilizing effects on the human telomeric DNA quadruplex. UV-vis spectroscopy, circular dichroism, linear dichroism and viscosimetry were used in order to study the nature of the DNA binding in more detail. The results show that all of the novel derivatives were able to fold the single-stranded DNA sequences into antiparallel G-quadruplex structures, with derivative 15 exhibiting the highest stabilizing capability. Cell cycle analysis revealed that a primary trend of the "braco"-like derivatives was to arrest the cells in the S- and G2M-phases of the cell cycle within the first 72h, with derivative 13 and BRACO19 proving particularly effective in suppressing cell proliferation. All studies derivatives were less toxic to human fibroblast cell line in comparison with HT 29 cancer cell line.

Novel tacrine/acridine anticholinesterase inhibitors with piperazine and thiourea linkers.

A new series of substituted tacrine/acridine and tacrine/tacrine dimers with aliphatic or alkylene-thiourea linkers was synthesized and the potential of these compounds as novel human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE) inhibitors with nanomolar inhibition activity was evaluated. The most potent AChE inhibitor was found to be homodimeric tacrine derivative 14a, which demonstrated an IC50 value of 2 nM; this value indicates an activity rate which is 250-times higher than that of tacrine 1 and 7500-times higher than 7-MEOTA 15, the compounds which were used as standards in the study. IC50 values of derivatives 1, 9, 10, 14b and 15 were compared with the dissociation constants of the enzyme-inhibitor complex, Ki1, and the enzyme-substrate-inhibitor complex, Ki2, for. A dual binding site is presumed for the synthesized compounds which possess two tacrines or tacrine and acridine as terminal moieties show evidence of dual site binding. DFT calculations of theoretical desolvation free energies, ΔΔGtheor, and docking studies elucidate these suggestions in more detail.

Coumarins of Matricaria chamomilla L.: aglycones and glycosides.

The identity and quantity of coumarin-like compounds in leaves and anthodia of Matricaria chamomilla L. were studied by LC-DAD and NMR. So far, two monosubstituted coumarins, herniarin and umbelliferone, and two herniarin precursors were identified therein. In this paper, two other coumarin glycosides and one aglycone were confirmed. Skimmin (umbelliferone-7-O-ß-d-glucoside), daphnin (daphnetin-7-O-ß-d-glucoside) and daphnetin (7,8-dihydroxycoumarin) were found for the first time in diploid and tetraploid leaves and anthodia of M. chamomilla L. Daphnetin is known as a strong sensitizer, so this compound and its glycosidic derivative can contribute to the allergic potential of chamomile. Commercial chamomile preparations were tested for their presence.

Binding of glyco-acridine derivatives to lysozyme leads to inhibition of amyloid fibrillization.

While amyloid-related diseases are at the center of intense research efforts, no feasible cure is currently available for these diseases. The experimental and computational techniques were used to study the ability of glyco-acridines to prevent lysozyme amyloid fibrillization in vitro. Fluorescence spectroscopy and atomic force microscopy have shown that glyco-acridines inhibit amyloid aggregation of lysozyme; the inhibition efficiency characterized by the half-maximal inhibition concentration IC50 was affected by the structure and concentration of the derivative. We next investigated relationship between the binding affinity and the inhibitory activity of the compounds. The semiempirical quantum PM6-DH+ method provided a good correlation pointing to the importance of quantum effects on the binding of glyco-acridine derivatives to lysozyme. The contribution of linkers may be explained by the valence bond theory. Our data provide a basis for the development of new small molecule inhibitors effective in therapy of amyloid-related diseases.

DNA binding acridine-thiazolidinone agents affecting intracellular glutathione.

Three new acridine-thiazolidinone derivatives (2a-2c) have been synthesized and their interactions with calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovarian cancer cell lines A2780) were studied. The compounds 2a-2c possessed high affinity to calf thymus DNA and their binding constants determined by spectrofluorimetry were in the range of 1.37 × 10(6)-5.89 × 10(6) M(-1). All of the tested derivatives displayed strong cytotoxic activity in vitro, the highest activity in cytotoxic tests was found for 2c with IC(50) = 1.3 ± 0.2 µM (HL-60), 3.1 ± 0.4 µM (L1210), and 7.7 ± 0.5 µM (A2780) after 72 h incubation. The cancer cells accumulated acridine derivatives very fast and the changes of the glutathione level were confirmed. The compounds inhibited proliferation of the cells and induced an arrest of the cell cycle and cell death. Their influence upon cells was associated with their reactivity towards thiols and DNA binding activity.

3,6-bis(3-alkylguanidino)acridines as DNA-intercalating antitumor agents.

A series of 3,6-bis(3-alkylguanidino) acridines was prepared and the interaction of these novel compounds with calf thymus DNA was investigated with UV-vis, fluorescence and circular dichroism spectroscopy, in addition to DNA melting techniques. The binding constants K were estimated to range from 1.25 to 5.26 × 10(5) M(-1), and the percentage of hypochromism was found to be 17-42% (from spectral titration). UV-vis, fluorescence and circular dichroism measurements indicated that the compounds act as effective DNA-intercalating agents. Electrophoretic separation proved that ligands 6a-e relaxed topoisomerase I at a concentration of 60 µM, although only those with longer alkyl chains were able to penetrate cell membranes and suppress cell proliferation effectively. The biological activity of novel compounds was assessed using different techniques (cell cycle distribution, phosphatidylserine externalization, caspase-3 activation, changes in mitochondrial membrane potential) and demonstrated mostly transient cytostatic action of the ethyl 6c and pentyl 6d derivatives. The hexyl derivative 6e proved to be the most cytotoxic. Different patterns of cell penetration were also observed for individual derivatives. Principles of molecular dynamics were applied to explore DNA-ligand interactions at the molecular level.

Synthesis, design and biological evaluation of novel highly potent tacrine congeners for the treatment of Alzheimer's disease.

New tacrine derivatives 5a-d, 6a-d with piperazino-ethyl spacer linked with corresponding secondary amines and tacrine homodimer 8 were synthesized and tested as cholinesterase inhibitors on human acetylcholinesterase (hAChE) and human plasmatic butyrylcholinesterase (hBChE). In most cases the majority of synthesized derivatives exhibit a high AChE and BChE inhibitory activity with IC(50) values in the low-nanomolar range, being clearly more potent than the reference standard tacrine (9-amino-1,2,3,4-tetrahydroacridine, 1) and 7-MEOTA (7-methoxy-9-amino-1,2,3,4-tetrahydroacridine). Among them, inhibitors 8 and 5c, showed a strong inhibitory activity against hAChE, with an IC(50) value of 4.49 nM and 4.97, nM resp., and a high selectivity to hAChE. The compound 5d acted as the most potent inhibitor against hBChE with an IC(50) value of 33.7 nM and exhibited also a good selectivity towards hBChE. The dissociation constants K(i) of the selected inhibitors were compared with their IC(50) values. Molecular modeling studies were performed to predict the binding modes between individual derivatives and hAChE/hBChE.

Accumulation of tetracoumaroyl spermine in Matricaria chamomilla during floral development and nitrogen deficiency.

The new natural polyamine conjugate 1N,5N,10N,14N-tetracoumaroyl spermine (tetracoumaroyl spermine) recently isolated from chamomile (Matricaria chamomilla L.) flower heads is applicable for the treatment of several human disorders such as depression and anxiety. High variability in the level of tetracoumaroyl spermine is found in commercial tisanes. Accumulation of tetracoumaroyl spermine was tested during floral development, and nitrogen deficiency was chosen as its putative limiting environmental factor. It was observed that tetracoumaroyl spermine is present mainly in tubular flowers, reaching its maximal content during the 3rd phase of flowering when the corollae of tubular flowers start to open. The later observed decrease could result from a release of pollen that also contains a considerable amount of tetracoumaroyl spermine. It is likely that tetracoumaroyl spermine plays an important role in pollen development, and so, despite overall N-deficiency in the plants, tetracoumaroyl spermine is accumulated at the same or even higher rate than in the flowers of the N-sufficient control.

Structure-activity relationship of acridine derivatives to amyloid aggregation of lysozyme.

BACKGROUND: Amyloid-related diseases (such as Alzheimer's disease or diabetes type II) are associated with self-assembly of protein into amyloid aggregates. METHODS: Spectroscopic and atomic force microscopy were used to determine the ability of acridines to affect amyloid aggregation of lysozyme. RESULTS: We have studied the effect of acridine derivatives on the amyloid aggregation of lysozyme to investigate the acridine structure-activity relationship. The activity of the effective planar acridines was characterized by the half-maximum depolymerization concentration DC(50) and half-maximal inhibition concentration IC(50). For the most effective acridine derivatives we examined their interaction with DNA and their effect on cell viability in order to investigate their eventual influence on cells. We thus identified planar acridine derivatives with intensive anti-amyloid activity (IC(50) and DC(50) values in micromolar range), low cytotoxicity and weak ability to interfere with the processes in the cell. CONCLUSIONS: Our findings indicate that both the planarity and the tautomerism of the 9-aminoacridine core together with the reactive nucleophilic thiosemicarbazide substitution play an important role in the anti-amyloid activities of studied derivatives. GENERAL SIGNIFICANCE: The present findings favor the application of the selected active planar acridines in the treatment of amyloid-related diseases.