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Background: One major microbiological problem is the widespread antibiotic resistance. There is an urgent need for new antibiotics and ways to treat multi-drug-resistant infections. Inhibition of bacterial quorum sensing (QS) systems could be an effective alternative in a smuch as they regulate a broad spectrum of cell functions, including, virulence factor production, biofilm organisation and motility. Influx and efflux bacterial systems involved in quorum sensing (QS) are known to depend on the proton motive force (PMF). Thus, a new series of 12 trifluoromethyl ketones (TFs) known to inhibit the PMF, was investigated for effects on the efflux pump of a QS responding bacterium, for its subsequent effect on the response to a QS signal and its direct inhibition of the response to a QS signal., Materials and Methods: Chromobacterium violaceum 026 (CV026) was used as the indicator strain to evaluate the QS inhibitory effect of TFs. This strain responds to the presence of short carbon chain acyl-homoserine lactones (AHLs) by the development of a purple pigment. Effect on the QS response of CV026 to externally added AHLs was evaluated. In addition, the specific activity of the TFs on the efflux pump system of the CV026 strain and a wild-type Escherichia coli strain was assessed with the aid of the automated real-time ethidium bromide method., Results: From the 12 compounds, 6 proved to be effective inhibitors of the QS response by CV026, as well as inhibit the efflux pumps of CV026 and Escherichia coli., Conclusion: Our results show that TFs have QS inhibitory properties that are mediated through their inhibition of efflux pumps that extrude the noxious QS signal before it reaches its intended target. Because the TFs also inhibit the efflux pump of a pathogenic bacterium, the method used for the evaluation of the TFs in the current study has clinical relevance and may be exploited for the prevention of QS responses of infecting bacteria.

Background: In order to extend the search for tumour-targeting compounds, this study performed a quantitative structure-activity relationship (QSAR) analysis of 26 newly synthesised trihaloacetylazulenes., Materials and Methods: The value of 50% cytotoxic concentration (CC(50)) of trihaloacetylazulenes against human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human promyelocytic leukaemia (HL-60) cell lines was calculated from the dose-response curve by the MTT method. CONFLEX/CAChe PM5 was used for the calculation of 11 physico-chemical features for each compound., Results: When all 26 compounds were analysed together, the CC(50) values correlated well with the dipole moment, the lowest unoccupied molecular orbital energy and the reactivity index, and somewhat with the heat of formation, the stability of hydration and the absolute electron negativity, but not with other features. When these 26 compounds were separated into two groups with or without substituents of the 7-membered ring, the correlation coefficient was increased. When the 26 compounds were separated into a different set of two groups with different halogen atoms in the 5-membered ring, no improvement of correlation coefficient was observed., Conclusion: The present study suggests that appropriate grouping of test compounds may further increase the correlation coefficient. The QSAR approach was useful in the design of azulene compounds that are expected to show higher potency.

We have previously reported the cytotoxicity and type of cell death induced by twenty trihaloacetylazulenes in human tumor cell lines. We determined for the first time the most-stable chemical structures from their reported structures, using the semiempirical molecular-orbital method (CONFLEX/PM5), and then delineated the relationship between their cytotoxicity (evaluated by 50% cytotoxic concentration, CC(50)) and a total of twelve parameters. The parameters used are the molecular weight and eleven chemical descriptors: the heat of formation (COSMO, non-COSMO), stability of hydration (=COSMO - nonCOSMO (DeltaH)), dipole moment (D), hydrophobicity (log P), highest occupied molecular orbital energy (E(HOMO)), lowest unoccupied molecular orbital energy (E(LUMO)), absolute hardness [eta=(E(LUMO)-E(HOMO))/2], absolute electron negativity [chi=-(E(LUMO)+E(HOMO))/2], reactivity index (omega=chi(2)/2eta) and surface area (A(2)), and volume of the molecule (A(3)). There was good correlation between the CC(50) value and all descriptors except for absolute hardness in HL-60 cells. There was also a good correlation between the CC(50) value and EHOMO, chi, omega, surface area, volume and molecular weight in HSC-2, HSC-3 and HSC-4 cells. The descriptors determined by the present method are useful in evaluating the biological activity of trihaloacetylazulenes.

A semiempirical molecular-orbital method (CONFLEX/PM5) was applied to delineate the relationship between the cytotoxicity (evaluated by 50% cytotoxic concentration, CC(50)) of twenty-four tropolone-related compounds and their molecular weight or one of the following eleven chemical descriptors: the heat of formation (COSMO, non-COSMO; kcal/mole), stability of hydration (=COSMO-nonCOSMO (DeltaH); kcal/mole), dipole moment (D), hydrophobicity (log P), highest occupied molecular orbital energy (E(HOMO); eV), lowest unoccupied molecular orbital energy (E(LUMO); eV), absolute hardness [eta=(E(LUMO)-E(HOMO))/2; eV)], absolute electron negativity [chi=-(E(LUMO)+E(HOMO))/2; eV], reactivity index (omega=chi(2)/2eta; eV), surface area (A(2)) and volume (A(3)) of the molecule. No good correlation was found with the unseparated twenty-four compounds all together, but modest to high correlation was found after separation into three different groups of compounds, depending on the structural similarity. Particular descriptors could be used to evaluate the biological activity of newly synthesized tropolones.

Cdr1p is the major ATP-binding cassette multidrug transporter conferring resistance to azoles and other antifungals in Candida albicans. In this study, the identification of new Cdr1p inhibitors by use of a newly developed high-throughput fluorescence-based assay is reported. The assay also allowed monitoring of the activity and inhibition of the related transporters Pdr5p and Snq2p of Saccharomyces cerevisiae, which made it possible to compare its performance with those of previously established procedures. A high sensitivity, resulting from a wide dynamic range, was achieved upon high-level expression of the Cdr1p, Pdr5p, and Snq2p transporters in an S. cerevisiae strain in which the endogenous interfering activities were further reduced by genetic manipulation. An analysis of a set of therapeutically used and newly synthesized phenothiazine derivatives revealed different pharmacological profiles for Cdr1p, Pdr5p, and Snq2p. All transporters showed similar sensitivities to M961 inhibition. In contrast, Cdr1p was less sensitive to inhibition by fluphenazine, whereas phenothiazine selectively inhibited Snq2p. The inhibition potencies measured by the new assay reflected the ability of the compounds to potentiate the antifungal effect of ketoconazole (KTC), which was detoxified by the overproduced transporters. They also correlated with the 50% inhibitory concentration for inhibition of Pdr5p-mediated transport of rhodamine 6G in isolated plasma membranes. The most active derivative, M961, potentiated the activity of KTC against an azole-resistant CDR1-overexpressing C. albicans isolate.

A total of twenty benzo[b]cyclohept[e] [1,4]oxazines and their S-analogs, and 2-aminotropone derivatives were investigated for their cytotoxicity against three human normal cells and four tumor cell lines. These compounds showed moderate tumor-specific cytotoxicity. The cytotoxicity was enhanced by bromination at the tropone ring and replacement by formylbenzene. The cytotoxicity of 2-(2-hydroxyanilino) tropone was enhanced by introduction of bromine or isopropyl group to the tropone ring. The presence of a hydroxyl group at ortho or para-position should be necessary for the appearance of cytotoxicity and tumor-specificity. The highly active derivatives, 7-bromo-2-(4-hydroxyanilino)tropone [16] and 4-isopropyl-2-(2-hydroxyanilino)tropone [20], induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in human promyelocytic leukemia HL-60 cells, but only at concentrations twice or four times higher than CC(50) values. These compounds induced no discernible DNA fragmentation, and activated caspases much more weakly in human oral squamous cell carcinoma HSC-2 cells. Both [16] and [20] failed to induce the production of acidic organelles, a marker of autophagy, in contrast to the nutritional starvation. These data demonstrated that 2-aminotropones showed relatively higher tumor-specificity than benzo[b]cyclohept[e] [1,4]oxazine, and that 2-aminotropones induced little or no apoptotic cell death in oral squamous cell carcinoma, in contrast to HL-60 cells.

A total of thirty-nine naphtho[2,3-b]furan-4,9-diones and related compounds were tested for their cytotoxicity against three human normal oral cells (gingival fibroblast, HGF, pulp cell, HPC, periodontal ligament fibroblast, HPLF) and four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). 2-Acetylnaphtho[2,3-b]furan-4,9-dione [1] was highly cytotoxic to both normal and tumor cells, yielding low tumor-specificity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan [4], the 2-(3-furanoyl) benzoic acids [5, 6] and the 1,4-naphthoquinones [7, 8] showed much reduced cytototoxicity and low tumor-specificity. The introduction of phenoxy [18], isopropylamino [23] or 2-methylpiperidino [33] groups to the 2-position of naphtho[2,3-b]furan-4,9-dione yielded compounds that showed the greatest tumor-specificity. These compounds, at twice or four times higher concentrations than CC50, induced the activation of caspase-3, caspase-8 and caspase-9 in the HSC-2 and HL-60 cells, but not so apparently in the HSC-4 cells. However, they did not induce internucleosomal DNA fragmentation in the HSC-2 and HSC-4 cells even after 24 hours incubation and only slightly induced DNA fragmentation in the HL-60 cells. Compound [18] induced the production of annexin-positive cells, but did not induce microtubule-associated protein light chain 3 (LC3) accumulation in autophagosomes in LC3-green fluorescent protein (GFP)-transfected HSC-2 cells. These data suggested that naphtho[2,3-b]furan-4,9-diones may induce the early apoptotic marker, without induction of caspase activation and DNA fragmentation in oral squamous cell carcinoma cell lines. Quantitative structure-activity relationship (QSAR) analysis suggests the applicability of the theoretical calculations such as frontier molecular orbital, dipole moments and hydrophobicity in predicting their cytotoxic activity.

Twenty-six benzocycloheptoxazine derivatives were investigated for their effect on nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. Benzo[b]cyclohepta[e][1,4]thiazine most effectively inhibited the LPS-stimulated NO production at noncytotoxic concentrations. 6H-Benzo[b]cyclohepta[e][1,4]-diazine cation, and benzo[b]cyclohepta[e][1,4]oxazine and its 6-bromo derivative also efficiently inhibited the LPS-stimulated NO production. Another sixteen benzo[b]cyclohepta[e]-[1,4]oxazine derivatives, 14H-[1,4]benzoxazino[3',2' :3,4]-cyclohepta[1,2-b][1,4]benzoxazine and its 7-bromo- and 7-isopropyl derivatives were slightly less active (selectivity index (SI)=83-66). Bromination of benzo[b]cyclohepta[e][1,4]-thiazine, benzo[b]cyclohepta[e][1,4]oxazine and 2-methylbenzo[b]cyclohepta[e][1,4]oxazine at C-6, C-8 or C-10 positions resulted in the significant reduction of the inhibitory activity. The observed inhibitory activity of benzo[b]cyclohepta-[e][1,4]thiazine and its 6,8-dibromo derivatives were not due to the reduction of the intracellular level of inducible NO synthase protein (based on Western blot analysis), nor to NO scavenging activity (based on ESR spectroscopy). These results suggest the possible anti-inflammatory action of benzocyclo-heptoxazines via inhibition of LPS-activated macrophages.

Twenty-six benzocycloheptoxazine derivatives were investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) and four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, HSC-4, promyelocytic leukemia HL-60). Benzo[b]cyclohepta[e][1,4]thiazine [1] exhibited very weak cytotoxicity, whereas its 6,8,10-tribromo derivative [3] exhibited higher cytotoxicity and tumor specificity (TS = 5.6). 6H-Benzo[b]cyclohepta[e][1,4]diazine [4] and its cation [5] exhibited no tumor specificity. Among eighteen benzo[b]cyclohepta[e][1,4]oxazine derivatives [6-23], 6,8,10-triboromo- [9], 6-bromo-2-methyl- [20], and 6-bromo-2-chloro- [21] derivatives showed the highest tumor-specific cytotoxicity (TS = 12.5, 9.1 and 11.5, respectively). 14H-[1,4]Benzoxazino[3',2':3,4]cyclohepta[1, 2-b][1,4]benzoxazine [24] and its 7-bromo- [25] and 7-isopropyl- [26] derivatives had much lower cytotoxicity and tumor-specificity. Compounds [9, 20, 21] at 50% cytotoxic concentration (CC50) induced internucleosomal DNA fragmentation and caspase activation in HL-60 cells. On the other hand, these compounds induced apoptosis only at concentrations higher than CC50 in HSC-2 cells and failed to induce apoptosis in HSC-4 cells. Compounds [9, 20, 21] induced the formation of acidic organelles as measured by acridine orange staining. Transmission electron microscopy demonstrated the induction of moderate enlargement of mitochondria, the endoplasmic reticulum and nuclear membrane, and the vacuolation of the endoplasmic reticulum and the presence of a number of lamellar body-like organelles. These results indicate the diversity of the type of cell death induced by benzocycloheptoxazine derivatives in human tumor cell lines.

The effect of 20 trihaloacetylazulene derivatives with one halogen atom, on nitric oxide (NO) production by mouse macrophage-like cells Raw 264.7 was investigated. 2-Methoxyazulenes and 2-ethoxyazulenes exhibited comparable cytotoxicity. Trichloroacetylazulenes generally exhibited higher cytotoxicity, as compared with the corresponding trifluoroacetylazulenes. Substitution of chloride, bromide or iodine at the C-3 position further enhanced their cytotoxicity. All of these compounds failed to stimulate the Raw 264.7 cells to produce detectable amounts of NO, but did inhibit NO production by LPS-activated Raw 264.7 cells to different extents. 1-Trichloroacetyl-2-methoxyazulene and 1-trichloroacetyl-2-ethoxyazulene, with less cytotoxic activity, inhibited NO production to the greatest extent, producing the highest selectivity index (SI) of >24.7 and >28.7, respectively. This was accompanied by the efficient inhibition of inducible NO synthase (iNOS) mRNA expression, but not by iNOS protein abundance. Electron spin resonance (ESR) spectroscopy showed that neither of these compounds produced radicals, nor scavenged NO, superoxide anion or diphenyl-2-picrylhydrazyl radicals. The present study suggests that the inhibitory effects of trifluoroacetylazulenes and trichloroacetylazulenes on NO production by activated macrophages might be derived from the perturbation of NO anabolism (inhibition of iNOS mRNA expression and possibly the inactivation of iNOS protein) rather than NO catabolism (NO scavenging).

A semiempirical molecular-orbital method (CAChe) was applied to delineate the relationship between the cytotoxicity (evaluated by 50% cytotoxic concentration, CC50) of fifteen phenoxazine derivatives and eleven physical parameters (descriptors). Most of the phenoxazine derivatives had extended and planar structure. The cytotoxic activity of phenoxazines against the human oral squamous cell carcinoma HSC-2 and HSC-4 cells correlated to electron affinity, absolute hardness (eta), absolute electron negativity (chi) and octanol-water distribution coefficient (log-P). However, only log-P was correlated to CC50 in the HSC-3 cells, whereas only heat of formation and log-P were correlated to CC50 in the human promyelocytic leukemia HL-60 cells. The cytotoxic activity of the phenoxazine derivatives became maximum at the log-P = 5.9. Their cytotoxicity strongly depended on the chi value, but not on the eta value. Compounds with relatively higher cytotoxicity showed higher chi value (chi > 5.28), whereas compounds with relatively lower cytotoxicity showed lower chi value (chi < 4.27). These data suggest that appropriate chemical descriptors should be selected to estimate the cytotoxicity of phenoxazines, depending on the target cells.

Phenoxazines have shown diverse biological activities, but tumor-specific cytotoxic activity has not been investigated. A total of 24 phenoxazine derivatives (WM1-24) was investigated for their relative cytotoxicity against human tumor cell lines vs. normal cells. WM7 and WM8 showed the highest tumor-specificity index of 4.3 and 4.8, respectively. Considerable difference in drug-sensitivity was found among these tumor cell lines. Human promyelocytic leukemia HL-60 cells showed the highest sensitivity to both WM7 and WM8, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), and human gingival fibroblast (HGF), pulp cell (HPC) and periodontal ligament fibroblast (HPLF) were the most resistant. WM7 and WM8 induced little or no internucleosomal DNA fragmentation, and activated caspase-3 in HSC-2, HSC-4 and human glioblastoma T98G cells. These compounds failed to induce autophagic cell death, as judged by acridine orange and microtubule-associated protein 1 light chain 3 (LC3)-GFP assays. These results suggested that the higher cytotoxicity of WM7 and WM8 are derived from the positively-charged quaternary nitrogen substituents on the phenoxazine ring and the electron density of nitrogen at N12, and that inhibition of autophagy is not always coupled with apoptosis induction.

The relationship between the structure and cytotoxic activity of azulenequinones and trihaloacetylazulenes was investigated based on theoretical calculations. Four different dipole moments (mu(G), mu(ESP-G), mu(W) and mu(ESP-W)) and heats of formation (DeltaH(f)) of the azulenequinones [1-27] and trihaloacetylazulenes [28a,b-40a,b] were separately calculated in gas phase and aqueous solution using the conductor-like screening model/parametric method 3 (COSMO/PM3) method. The cytotoxic activity of azulenequinones was well correlated to DeltaDeltaH(f) HOMO energy and mu(ESP-w). The cytotoxic activity of trihaloacetylazulenes was correlated to DeltaDeltaH(f) LUMO energy and mu(ESP-W). QSAR may be applicable to predict the cytotoxicity of azulenequinones and trihaloacetylazulenes.

The cardiovascular drug lacidipine was screened in vitro for possible antibacterial activity with respect to 389 Gram-positive and Gram-negative bacterial strains. It was noticed that most bacteria (233) failed to grow at 50-200 microg/mL concentrations of the drug. Some strains were inhibited at even lower concentrations. The bacteria could be arranged according to their decreasing order of sensitivity as follows: Staphylococcus aureus, Vibrio cholerae, Salmonella spp., Shigellae, Escherichia coli, Bacillus spp., Klebsiellae and Pseudomonas spp. Lacidipine was found to be bacteriostatic in nature against S. aureus and V cholerae. When administered to Swiss strain of white mice at doses of 30 and 60 microg/mouse, lacidipine significantly protected the animals challenged with 50 MLD of S. typhimurium NCTC 74. According to the chi-square test, the in vivo data were highly significant (p<0.001).

Cardiac effects of 3,5-dibenzoyl-4-(3-phenoxyphenyl)-1,4-dihydro-2,6-dimethylpyridine (DP7), a novel multidrug resistance (MDR) inhibitor, in Langendorff-perfused rat heart have been investigated and compared to that of nifedipine. Nifedipine decreased concentration-dependently (IC50=8.89+/-1.09x10(-8) M) left ventricular pressure leaving unaltered coronary perfusion pressure, whereas DP7 did not affect both parameters. Nifedipine did not modify both QRS and QT intervals of electrocardiogram (ECG). Second-degree atrioventricular block or ventricular rhythm occurred in presence of nifedipine, however, in 4 out of 6 hearts. DP7, up to 30 microM, failed to alter ECG parameters. In conclusion, DP7, may represent a lead compound for the development of potent dihydropyridine MDR chemosensitizers devoid of cardiac effects.

Twenty trihaloacetylazulene derivatives with one atom of fluorine, chlorine, bromine or iodine was investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (gingival fibroblast, HGF; pulp cell, HPC; periodontal ligament fibroblast, HPLF) and four human tumor cell lines (squamous cell carcinoma, HSC-2, HSC-3, HSC-4; promyelocytic leukemia, HL-60). There was no apparent difference in the cytotoxic activity between 2-methoxyazulenes [1a-1e, 2a-2e] and 2-ethoxyazulenes [3a-3e, 4a-4e]. Trichloroacetylazulenes [2a-2e, 4a-4e] generally showed higher cytotoxicity and tumor-specificity (expressed as a TS value) as compared with the corresponding trifluoroacetylazulenes [1a-1e, 3a-3e]. Substitution of chloride [1c, 2c, 3c. 4c], bromide [1d, 2d, 3d, 4d] or iodine [1e, 2e, 3e, 4e] at the C-3 position further enhanced cytotoxic activity against four tumor cell lines, especially HL-60 cells. Among twenty trihaloacetylazulene derivatives, two compounds [2d] and [4c] showed the highest tumor specificity (TS = > 3.5 and > 2.5, respectively). Compounds [2d] and [4c] induced apoptotic cell death characterized by caspase-3, -8 and -9 activation and internucleosomal DNA fragmentation in HL-60 cells. On the other hand, compounds [2d] and [4c] induced autophagic cell death characterized by lower activation of caspases, lack of DNA fragmentation, vacuolization and autophagosome formation detected by acridine orange and LC3-GFP fluorescence, without the decline of the intracellular concentration of three major polyamines in HSC-4 cells. The cytotoxic activity of [4c], but not [2d], was slightly reduced by 3-methyladenine, an inhibitor of autophagy. These results suggest the diversity of cell death type induced in human tumor cell lines by trihaloacetylazulene derivatives.

Phenothiazines, a kind of sulfur-containing tricyclic compounds, have diverse biological activities including tranquilizer, antibacterial, antitumor and antihelmintic activities due to the relatively lower cytotoxicity. Phenothiazines have been used for clinical treatments as psychotropics. In contrast to the psychotropic preparations, their information of other biological activities of phenothiazines and their related compounds has been limited. This review article summarizes the interaction with DNA (using quantum calculation), antitumor activity, differentiation or apoptosis-inducing activity, tumor necrosis factor (TNF)-induction, antiproliferative activity, radical scavenging activity, antimutagenic activity, antiplasmid activity, antibacterial activity, reversal of multidrug resistance (MDR), blast transformation activity of phenothiazines, benz[c]acridines and benzo[a]phenothiazines.

The interactions of six newly synthesized phenoxazine derivatives with lipid bilayers were studied by means of calorimetry, fluorescence spectroscopic methods and electron spin resonance. Depending on their structure studied compounds decreased membrane fluidity and increased lipid order in liquid-crystalline bilayers to different degrees. These studies showed also that phenoxazine molecules are located close to the polar/apolar interface of bilayer. The results allow to conclude that phenoxazines rather weakly interact with lipid bilayers.

Several new 3-formylchromone derivatives proved to be modifiers of multidrug resistance in mouse lymphoma cells and in human Colo320 colon cancer cells. There is apparently a structure-activity relationship between the antiproliferative multidrug resistance-reversing effect and the chemical structure of the 3-formylchromones. The total polar surface areas and the ground state dipole moments of the molecules are presumed to play a key role in the multidrug resistance-reversing effect. The log P values can provide an adequate explanation for the selective cytotoxicity against cancer cells.

E. coli is the main agent of uncomplicated urinary tract infections (UTIs) and accounts for more than 85% of recurrent cystitis and at least 35% of recurrent pyelonephritis. Despite the widespread availability of antibiotics, UTIs remain the most common bacterial infection in the human population. It is currently advised that the clinical administration of antibiotics against the pathogenic bacteria should be prohibitted due to the emergence of multidrug resistant (MDR) bacterial strains. Therefore, newer and more effective antimicrobials are in demand to treat such cases. One hundred and thirty six urine samples were collected from UTI patients. E. coli was isolated from 85 samples, out of which 33% were resistant to common antibiotics. The isolates were decreasingly resistant to ampicillin, tobramycin, augmentin, nalidixic acid, cefuroxime, nitrofurantoin, kanamycin, pipemidic acid, chloramphenicol, cefotaxime, cefamendol, ofloxacin, ceftizoxime, norfloxacin and amikacin. The anti-inflammatory drug diclofenac exhibited significant antibacterial activity against common bacterial strains both in vitro and in vivo. The present work was conducted to evaluate the in vitro inhibitory effect of this drug on the clinically isolated strains of E. coli in hospitals. All the isolates were sensitive to diclofenac, with MIC values ranging from 5-50 microg/mL. The MIC90 value of the drug was 25 microg/mL. Therefore, it may be suggested that diclofenac has the capacity to treat UTI caused by E. coli.

The ability of 41 1,4-diphenyl-1,4-dihydropyridine derivatives to inhibit the transport activity of P-glycoprotein were studied by flow cytometry in a multidrug-resistant human colon cancer cell line (COLO320) and in human mdr1 gene-transfected mouse lymphoma cells (L 5178 Y). The cytotoxicities of these compounds were also examined against human normal and cancer cell lines. The majority of the tested compounds proved to be effective inhibitors of rhodamine 123 outward transport, but their cytotoxicities were not negligible. Some dihydropyridine derivatives displayed cytotoxic activity against four human oral tumour cell lines and against three normal human oral cell lines. There was no clear-cut relationship between the multidrug-resistance activity or cytotoxicity and the chemical structures of the compounds. New ring substituents could prevent the oxidation of the ring of the aromatic compound.

Luminescence studies on a series of new 12H-benzo[a]phenothiazines (BPHTs), possessing potentially useful antitumor therapeutic properties, are reviewed. The electronic absorption and fluorescence spectral properties of BPHTs, as well as their triplet- and singlet-excited states luminescence quenching are reviewed. Ground-state and singlet-excited state dipole moments and solvatochromic relationships are also described for these compounds. Studies on the formation of inclusion complexes between BPHTs and cyclodextrins (CDs), including CD-enhanced fluorescence, and thermodynamic constants and molecular geometry of these complexes, are discussed. The BPHTs antitumor properties in relation to their pi-electron density, and the physico-chemical and analytical applications based on their fluorescence and photophysical properties are also presented. This review article is based on selected literature data published in the last ten years (1993-2004).

In earlier experiments, the MDR (multidrug resistance)-reversal activities of Anastasia Black (Russian black sweet pepper) extracts had been analysed. Recently, the most effective MDR reversing extracts and fractions have been separated by HPLC (high-performance liquid chromatography, for carotenoids) and LC-MS-MS (HPLC combined with mass spectrometry, for phenolic compounds) methods. As a result of the analytical studies, the following flavonoids had been identified: feruloyl glucopyranoside, quercetin rhamnopyranoside glucopyranoside, luteolin glucopyranoside arabinopyranoside, apigenin glucopyranoside arabinopyranoside, quercetin rhamnopyranoside, luteolin arabinopyranoside diglucopy-ranoside, hesperidine and luteolin glucuronide. According to the literature, the aglycones of these phenolic compounds exhibit MDR-reversal activity in vitro, and the connection between the phenolic content of Anastasia Black and MDR-reversal action was therefore studied by different analytical methods. The results of this study revealed that the identified flavonoids of Anastasia Black may be only partially responsible for the modulation of the MDR of mouse lymphoma cells. Other lipophilic compounds, most probably carotenoids, present in Russian black sweet pepper may act as inhibitors of MDR reversal.

Phenothiazine derivatives (PDS) readily react with several metal ions in acid or buffer media to yield colored species which could be followed spectrophotometrically. Reaction conditions have been optimized to get colored species of maximum stability and intensity. The effects of foreign ions have been investigated. The results of all the methods were supported by statistical analysis. The proposed methods have been successfully applied for the analysis of various samples containing the interested metal ion(s). In addition, some extractive spectrophotometric methods based on the formation of ion-association complexes (extractable into organic solvents) and gravimetric methods have been discussed.

The effects of 26 trihaloacetylazulene derivatives on nitric oxide (NO) production by the mouse macrophage-like Raw 264.7 cells was investigated. The trichloroacetylazulenes [1b-13b] generally showed higher cytotoxicity as compared with the corresponding trifluoroacetylazulenes [1a-13a]. All the compounds inhibited NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells to various extents. 3-Trifluoroacetylguaiazulene [8a], 1-trifluoroacetyl-4,6,8-trimethylazulene [10a], 3-methyl-l-trichloroacetylazulene [2b] and 3-ethyl-1-trichloroacetylazulene [3b] showed lower cytotoxic activity and most effectively inhibited NO production. Western blot analysis revealed that compounds [8a, 1Oal dose-dependently reduced the intracellular concentration of inducible NO synthase (iNOS), whereas compounds [2b, 3b] only marginally affected the iNOS protein expression. RT-PCR analysis showed that compounds [8a, 2b] reduced the iNOS mRNA expression by approximately 50%. These compounds affected cyclooxygenase-2 protein and mRNA expression, depending on the concentrations. ESR spectroscopy revealed that compounds [8a, 10a, 2b, 3b] neither produced radical, nor scavenged NO, superoxide anion or diphenyl-2-picrylhydrazyl radicals. The present study showed the inhibitory effects of trifluoroacetylazulenes and trichloroacetylazulenes on NO production by activated macrophages.

Background: Knowledge of the structure-activity relationships of multidrug resistance protein 1 (MRP1, ABCC1) inhibitors may aid in developing potent inhibitors that can be used to circumvent MRP1-mediated multidrug resistance., Materials and Methods: Six stilbenes were examined for their ability to inhibit MRP1-mediated transport of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes and into inside-out erythrocyte membrane vesicles (IOVs). The concentrations of stilbenes decreasing BCPCF transport by 50% during 60 min of incubation at 37 degrees C (IC50) were determined from dose-response curves., Results: Stilbenes inhibited BCPCF transport in cells in the rank order (+)-alpha-viniferin (IC50 = 0.8 microM) > sophorastilbene A (IC50 = 3.1 microM) > (-)-epsilon-viniferin (IC50 = 8.9 microM) > piceatannol (IC50 = 57 microM). Resveratrol and rhaponticin were ineffective. (+)-alpha-Viniferin (IC50 = 0.8 microM), sophorastilbene A (IC50 = 3.7 microM) and (-)-epsilon-viniferin (IC50 = 3.5 microM) were also efficient BCPCF transport inhibitors in IOVs., Conclusion: Stilbenes may efficiently inhibit MRP1-mediated organic anion transport. This inhibitory potency of stilbenes increases with oligomerisation. The membrane is not a strong barrier for the inhibitory activity of the trimeric stilbenes.

Twenty-six trihaloacetylazulene derivatives were investigated for their tumor-specific cytotoxicity and apoptosis-inducing activity against three human normal cells (HGF, HPC, HPLF) and four human tumor cell lines (HSC-2, HSC-3, HSC-4, HL-60). The trichloroacetylazulenes [1b-13b] generally showed higher cytotoxicity as compared to the corresponding trifluoroacetylazulenes [1a-13a]. The trichloroacetylazulenes [1b-13b] also showed higher tumor-specific cytotoxicity (expressed as TS value) than the corresponding trifluoroacetylazulenes [1a-13a]. Especially, 2,3-dimethyl-1-trichloroacetylazulene [5b] and 1,3-ditrichloroacetyl-4,6,8-trimethylazulene [11b] showed the highest cytotoxicity and tumor specificity (TS > 35.6 and > 44.1, respectively). These compounds induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 and HSC-3 cells, but activated caspase-3, -8 and -9 in all of these cells, suggesting the activation of both mitochondria-independent (extrinsic) and dependent (intrinsic) pathways. Western blot analysis showed that two compounds [5b, 11b] slightly increased the intracellular concentration of pro-apoptotic proteins (Bad, Bax) in HSC-2 cells. None of the 26 compounds showed anti-HIV activity. These results suggest [5b] and [11b] as possible candidates for future cancer chemotherapy.

The structure-activity relationship of the cytotoxic activity of azulene and azulene derivatives was discussed, using theoretically calculated results. In order to clearly divide the azulenes into three groups according to their functional groups, the CC50, four different dipole moments (muG, muESP-G, muwand muESP-W) and heats of formation (deltaHf) of the azulenes [1-24] were separately calculated in two states, gas-phase and water, by the conductor-like screening model/parametric method 3 (COSMO/PM3). For the halogenated azulenes and isopropyl azulenes, the cytotoxic activity might follow the three quantitative structure-activity relationship (QSAR) parameters: deltadeltaHf, HOMO energy and muw Whereas, for the other ten compounds [3-5, 7-8, 10, 15-18], the cytotoxic activity might be related to the three QSAR parameters, deltadeltaHf, LUMO energy and muG

A structure-activity relationship of the cytotoxic activity of tropolone derivatives was discussed, using theoretical calculations. In order to clearly divide the tropolones into two structurally analogous groups, four different dipole moments (muG, muESP-G, muW and muESP-W) and heats of formation (deltaHf) of the tropolones [1-21] were calculated in the gas-phase and in water-solution by the conductor-like screening model/parametric method 3 (COSMO/PM3). The cytotoxic activities of the tropolones and 2-methoxytropones seem to be related to the three QSAR parameters deltadeltaHf, HOMO energy (EH) and muw. The cytotoxic activity of the five tropone derivatives [17-21] might depend on the QSAR parameters deltadeltaHf, LUMO energy (EL) and muESP-G. The results of the present study suggest the applicability of theoretical calculations such as frontier molecular orbital, dipole moments and deltadeltaHf in the prediction of the cytotoxic activity of tropolone derivatives.

A proton pump-deleted mutant E. coli, AG100 A, had greater sensitivity to ampicillin, tetracycline and erythromycin than the wild-type parent E. coli AG100 containing the proton pump. This antibiotic sensitivity was further increased by resistance modifiers such as the Ca2+ channel blocker (+/-) verapamil (VP) and the calmodulin antagonist promethazine (PMZ). Whereas the newly-synthesized trifluoromethyl-ketone (TF) enhanced the activity of these antibiotics against the wild-type strain, it did not enhance the activity of ampicillin against the proton pump-deleted mutant. These results suggested that TF14 had an inhibitory effect on the proton pump. Elimination of plasmids from another strain of E. coli, K12, was promoted by PMZ and 9-amino-acridine (9-AA), but not by TF14 alone. However, combinations of TF14 with either PMZ or 9-AA enhanced the plasmid elimination capacity of the latter compounds. The combination of TF14, PMZ and VP proved that the Ca2+ channel blocker was not effective by itself These results collectively suggest that TF14 inhibited the proton pump of E. coli and that it was this pump which, when inhibited by TF14, allowed more PMZ to reach its plasmid elimination target.

The ability of fifteen novel phenoxazine derivatives (four phenoxazines and eleven benzo[a]phenoxazines) to modulate multidrug resistance (MDR) in a P-gp-overexpressing mouse T lymphoma cell line (L5178 MDR) was studied. A flow cytometric functional test, based on the differential accumulation of rhodamine 123 by sensitive and multidrug-resistant cells, was employed. Seven benzo[a]phenoxazines were observed to increase the amount of rhodamine 123 accumulated by resistant cells, i.e. to be new effective MDR modulators. The results allowed us to draw preliminary conclusions about the structural features of benzo[a]phenoxazines which are important for MDR modulation.

Thepiperazine chlorcyclizine HCl (CCZ), possessing significant antimetabolic as well as virucidal and virustatic activities against the human immunodeficiency virus (HIV) and other retroviruses, was selected to determine its anticarcinogenic potential The anticancer activity of CCZ was evaluated against procarcinogen n-diethylnitrosamine (NDA)-initiated hepatocarcinogenesis, which was subsequently promoted by phenobarbital (PB) in male Sprague-Dawley rats. The anticancer efficacy of CCZ was monitored by estimating some potential markers of neoplastic and preneoplastic hepatic conditions, e.g., glutathione (GSH), glutathione-S-transferase (GST) and gamma-glutamyl transpeptidase (gammaGTP). CCZ exhibited antineoplastic activity on a long-term therapeutic basis. Furthermore, this drug restricted the exponential increase of the antioxidant markers in the hyperplastic nodule and the surrounding liver tissues in comparison with the carcinogen-controlled rats during the entire period of treatment. A decrease in the number of nodules was observed in the CCZ-treated group.

The expression of multidrug resistance-associated protein (MRP1) results in ATP-dependent reduction of drugs' concentration in cancer cells, i.e., multidrug resistance (MDR). Since the majority of projects are concentrated on the search of the new MDR modulators, there are very few reports on drug-induced stimulation of MDR transporters activity. In the present work, by means of functional fluorescence assay we have shown that MRP1-mediated efflux of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) out of human erythrocytes is stimulated by phenothiazine maleates that have been already identified as P-glycoprotein inhibitors. Phenothiazine maleates-induced stimulation of ATP-dependent uptake of 2',7'-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) into inside-out membrane vesicles prepared from erythrocyte membranes has been also demonstrated. Moreover, it was shown that phenothiazine maleates exerted stimulating effect on ATPase activity measured in erythrocyte membranes. To our best knowledge, this report is the first one demonstrating that compounds able to inhibit transport activity of P-glycoprotein can stimulate MRP1 transporter. We conclude that phenothiazine maleates probably exert their stimulatory effect on MRP1 by direct interaction with the protein at the site different from the substrate binding site.

Azulenequinone derivatives have been reported to display a broad spectrum of biological activities, but study at the cellular level has been limited. The effect of twenty-seven azulenequinone derivatives on nitric oxide (NO) production by mouse macrophage-like cells Raw 264.7 was investigated in this study. All of these compounds failed to stimulate the Raw 264.7 cells to produce detectable amounts of NO, but did inhibit NO production by lipopolysaccharide (LPS)-activated Raw 264. 7 cells to varying extents. Compounds [7, 8, 9, 13, 16, 25, 27], which showed lesser cytotoxic activity (CC50 = 425, 381, 482, 179, 119, 235, 225 microM, respectively), inhibited NO production to the greatest extent [selectivity index (SI) = 15.4, 26.2, 3.9, 21.6, 3.1, 6.0, 8.4, respectively]. Western blot and RT-PCR analyses demonstrated that the most active derivatives, 3-morpholino-1, 5-azulenequinone [8] and 3,7-dibromo-1, 5-azulenequinone [13], significantly reduced both the intracellular concentration of iNOS protein and the expression of iNOS mRNA. ESR spectroscopy showed that compounds [8, 13] weakly scavenged NO produced by NOC-7, possibly via their general reducing activity. These data suggest that the inhibitory effect of NO production by compounds [8, 13] might be generated mostly via the inhibition of iNOS expression, rather than the radical-mediated mechanism.

The influence of novel synthetic and plant origin flavonoids on activity of multidrug resistance-associated protein (MRP1) was investigated in human erythrocytes used as a cell model expressing MRP1 in plasma membrane. The fluorescent probe, BCPCF (2', 7'-bis-(3-carboxy-propyl)-5-(and-6)-carboxyfluorescein), was applied as a substrate for MRP1 multidrug resistance transporter. The effect of compounds belonging to different classes of natural flavonoids: flavone, flavonol, isoflavones and flavanolignan was compared with action of new synthetic derivatives of genistein. Most of the flavonoids showed strong or moderate ability to inhibit transport carried out by MRP1. Inhibitory properties of flavonoids were compared to the effects of indomethacin, probenecid and MK-571 known as MRP1 inhibitors. Studying the influence of new synthetic genistein derivatives on BCPCF transport we have found that the presence of hydrophobic groups substituting hydrogen of hydroxyl group at the position 4' in ring B of isoflavone is more important for inhibitory properties than hydrophobic substitution at the position 7 in ring A. In case of naturally occurring isoflavones the replacement of hydrogen at position 4' by hydrophobic ring structure seems also to be favourable for inhibition potency.

Carotenoid fractions were extracted from red paprika, Valencia orange peel and the peel of Golden delicious apple. Thus, hypophasic carotenoids of paprika (PM1), orange (PM3) and apple (PM4), and epiphasic extractions of paprika (PM2) and apple (PM5) were obtained by extraction, saponification and partition between MeOH-H(2)O (9:1) (hypophasic) and hexane (epiphasic). A high content of capsanthin was quantified in hypophasic carotenoids (PM1) from red spice paprika, whereas the hypophasic fractions from orange (PM3) and apple (PM4) were mainly composed of violaxanthin, zeaxanthin and lutein. On the other hand, a high content of beta,beta-carotene and beta-cryptoxanthin was found in epiphasic fractions (PM2 and PM5). The extracts were studied for their anti-Helicobacter pylori (H. pylori), anti-human immunodeficiency virus (HIV), cytotoxic, multidrug resistance (MDR) reversal and radical scavenging activity. Among five PM extracts and beta,betacarotene, PM4 showed potent anti-H. pylori activity (MIC(50) = 36 microg/mL), comparable to metronidazole (MIC(50) = 45 microg/mL). The extracts were inactive against HIV. PM3 and PM4 showed slightly higher cytotoxic activity against three human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG) and human promyelocytic leukemic HL-60 cells than against three normal human oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), suggesting a tumor-specific cytotoxic activity. PM1, PM3 and PM4 displayed much higher MDR-reversing activity than (+/-)-verapamil. ESR spectroscopy demonstrated that PM1-5 and beta,beta-carotene produced little or no detectable radical under alkaline conditions and did not scavenge the O(2) (-) produced by the hypoxanthine and xanthine oxidase reaction. On the other hand, PM1 and PM2 scavenged efficiently 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, whereas singlet oxygen was also quenched efficiently by PM5 and PM2. The data suggest the potential importance of carotenoids as possible anti-H. pylori and MDR reversal agents. The active principles in the carotenoid extract might differ, depending upon the types of fruits and vegetables., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

A preliminary exploration of coumarin derivatives as novel multidrug resistance (MDR) modulators was carried out to determine the basic features of the structure responsible for the MDR reversal activity. Among 44 coumarins, 14 compounds moderately induced the reversal of MDR (fluorescence activity ratio (FAR) values > 1). The most active compound, 6-hydroxy-3-(2-hydroxyethyl)-4-methyl- 7-methoxycoumarin [C34], was equally potent as a MDR modulator verapamil. These data show a relationship between the chemical structure and MDR-reversal effect on tumor cells. All coumarins tested were more cytotoxic against tumor cells than normal cells. Several compounds displayed potent cytotoxic activities (CC50 15 - 29 microg/mL in HSC cells), comparable with that of gallic acid (CC50 = 24 microg/mL). Both 6-hydroxy- 7-methoxy-4-methyl-3-isopropylcoumarin [C43] and 3-ethyl-6-hydroxy- 7-methoxy-4-methylcoumarin [C44] showed the highest tumor-specific cytotoxicity (SI value = 4.1 and 3.6, respectively). We conclude that coumarins are potentially potent new MDR modulators with low toxicity against normal cells. A deeper understanding of the relationship between their structures and their potency will contribute to the design of optimal agents.

Anastasia Black (Russian sweet pepper) of Capsicum annuum L. var. angulosum Mill. (Solanaceae) was successively extracted with hexane, acetone, methanol and 70% methanol, and the extracts were further separated into a total of twenty-three fractions by silica gel or octadecylsilane (ODS; C18) column chromatography. These extracts and fractions were investigated for their cytotoxicity, anti-human immunodeficiency virus (HIV), anti-Helicobacter pylori (H. pylori), urease inhibition and multidrug resistance (MDR) reversal activity. Some fractions of hexane and acetone extracts showed higher cytotoxic activity against three human oral tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG) than against three normal human oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), suggesting a tumor-specific cytotoxic activity. No fractions displayed anti-HIV activity, but some hydrophobic fractions showed higher anti-H. pylori activity, urease inhibition activity and MDR reversal activity. The higher MDR activity of these fractions against MDR gene-transfected L5178 mouse lymphoma T cells may possibly be due to their higher content of carotene or polyphenol. These data suggest that Anastasia Black should be further investigated as a potent supplement for cancer chemotherapy.

Methyl-L-DOPA, an antihypertensive agent, has significant in vitro activity against a variety of atypical mycobacteria such as the Mycobacterium avium complex, M. scrofulaceum, M. xenopi and M. marinum, and rare pathogens like M. fortuitum. In the present investigation, the screening of the in vitro activity was further extended by testing the in vitro activity against a total of 53 different strains of mycobacteria, including 34 clinical isolates of both drug-sensitive and drug-resistant Mycobacterium tuberculosis. Most of the strains were inhibited at 10-25 microg/mL concentrations of the drug. When methyl-L-DOPA was injected into male mice at a concentration of 10 microg/g body weight (20 g each), methyl-L-DOPA significantly protected them when challenged with a 50 median lethal dose of M. tuberculosis H37Rv102. According to the chi2 test, the in vivo data were highly significant (p<0.01).

The choice of cell lines for multidrug resistance (MDR) modulators screening may affect the results obtained. Screening is most often performed in model systems which employ cell lines derived from haematological malignancies. Cell lines originating from solid tumours are far less popular. In the present work, we aimed to test the usefulness of the drug-sensitive human sarcoma cell line MES-SA, and its multidrug-resistant counterpart MES-SA/Dx5, as a model system for modulators' anti-MDR potency evaluation. Overexpression of P-glycoprotein in the resistant but not in the sensitive cell line was confirmed by flow cytometry and confocal microscopy. Flow cytometry demonstrated that verapamil and trifluoperazine reduced MDR in MES-SA/Dx5 cells as assessed by the rhodamine 123 accumulation test. Both modulators also restored in MES-SA/Dx5 cells the drug accumulation pattern typical for sensitive cells, as judged by confocal microscopy. We conclude that the MES-SA and MES-SA/Dx5 cell line pair constitute a good model for MDR modulators study.

We investigated twenty-seven azulenequinone derivatives for their relative cytotoxicity against three human normal cell lines (HGF, HPC, HPLF) and four human tumor cell lines (HSG, HSC-2, HSC-3, HL-60). Parent 1,5-azulenequinone showed potent and some tumor-specific cytotoxicity. Halogenated derivatives of 1,5- and 1,7-azulenequinone showed potent cytotoxicity, but lower tumor-specific cytotoxicity. In contrast to other azulenequinones, amino derivatives such as 3-amino-1,5- and 1, 7-azulenequinones showed relatively lower cytotoxic activity. The 3-Phenoxy-1,5-azuleneqinone derivative showed higher cytotoxicity than the 3-phenoxy-1, 7-azulenequinone derivative. 1,5- and 1,7-Azulenequinones generally showed higher cytotoxicity, as compared with tropolones and azulene derivatives. 3- (3-Guaiazulenyl)-1, 5-azulenequinone [12] and 7-isopropyl-3- (4-methylanilino)-2-methyl- 1, 5-azulenequinone [24] showed a relatively higher TS value and induced apoptosis (internucleosomal DNA fragmentation, activation of caspases 3, 8 and 9) in HL-60 and HSC-2 cells, possibly via the activation of both mitochondria-independent (extrinsic) and -dependent (intrinsic) pathways. Western blot analysis showed that [24] slightly increased the intracellular concentration of pro-apoptotic proteins (Bad, Bax) in HSC-2 cells, whereas [12] was much less active. None of the twenty-seven azulenequinones showed anti-HIV activity. These results suggest [12] and [24] as possible candidates for future cancer chemotherapy.

We investigated the effect of twenty-seven azulenes on nitric oxide (NO) production by mouse macrophage-like Raw 264.7 cells. No azulene derivative alone induced NO production by the Raw 264.7 cells, but inhibited lipopolysaccharide (LPS)-stimulated NO production to various extents. The ability of azulenes to inhibit NO generation by activated macrophages was generally increased when their cytotoxic activity declined. Western blot and RT-PCR analyses demonstrated that the most potent compound, 1,3-difluoroazulene [11], slightly inhibited the expression of inducible NO synthase (iNOS), but only at extremely high concentrations. ESR spectroscopy showed that [11] did not produce radical under alkaline condition, nor scavenged O2- (generated by HX-XOD reaction) or NO (generated by NOC-7). These data suggest that the inhibitory effect of [11] may be produced via a mechanism other than iNOS induction and a radical-mediated mechanism.

We investigated the effect of 27 tropolones on nitric oxide (NO) production by mouse macrophage-like Raw 264.7 cells. All of these compounds failed to stimulate the Raw 264. 7 cells to produce detectable amounts of NO, but inhibited NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells to various extents. Generally, the ability of tropolones to inhibit LPS-stimulated NO production was inversely related to their cytotoxic activity. Western blot and RT-PCR analyses demonstrated that the most active compound, 2,4-dibromo-7-methoxytropone [21], significantly reduced both the intracellular concentration of iNOS protein and the expression of iNOS mRNA. ESR spectroscopy showed that [21] did not produce radicals under alkaline condition, nor scavenged NO, produced by NOC-7. These data suggested that the inhibitory effect of [21] on NO production might be generated via the inhibition of iNOS expression, rather than a radical-mediated mechanism.

In the present study, we showed the inhibition of motility by trifluoromethyl ketone (TF) derivatives (1-8) in Proteus vulgaris (P. vulgaris) cultures. Among them, 1-(2-benzoxazoyl)-3,3,3-trifluoro-2-propanone (1) showed a much stronger inhibitory effect on the motility of P. vulgaris than other TF compounds at 10% MIC. Our results suggest the possibility of an inhibitory action of TF compounds on the proton motive forces by affecting the action of biological motor and proton efflux in the membranes, resulting in a reduction of the ratio of running and the increased number of tumbling and non-motile cells.

A structure-activity relationship of dopamine and 3-benzazepine derivatives is discussed, using theoretically calculated results. In order to clearly divide dopamines and 3-benzazepines into a strongly active and a weakly active group, the CC50, two different dipole moments (microESP-G and microESP-W) and heat of formation (deltaHf) of dopamine [1-13] and 3-benzazepine derivatives [14-23] were separately calculated in two states of gas-phase and water-solution by the COSMO/PM3 method. It was found that ten derivatives [1-3, 9, 12-13 and 20-23] (CC50: 0.056 to 2.5 mM) showed the strongest cytotoxic activity with small delta(deltaHf) values, whereas thirteen derivatives [4-8, 10-11, 14-19] (CC50: > 3.6 mM) showed the weakest cytotoxic activity with large delta(deltaHf) values.

We have recently found that 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone [TF18] exhibited the most potent antibacterial activity among 30 trifluoromethyl ketones against various prokaryotes, such as Escherichia coli (E. coli). In the present study, the inhibition of E. coli motility by TF18 was investigated. TF18 showed the lowest minimum inhibitory concentration (MIC) and highest inhibitory effect on the motility of E. coli strains. The wild-type E. coli was more sensitive to inhibition of motility than its proton pump-deleted mutant strain at subinhibitory concentrations. These data suggest that one of the targets of the antibacterial effect of the trifluoromethyl ketone is the proton pump system.

Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-[methanesulfonylamid]buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures. Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones., (Copyright 2003 Elsevier B.V)

We previously reported that a trifluoromethyl ketone derivative, 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18), exhibited the potent antibacterial activity against Helicobacter pylori, but had no urease activity. In order to clarify the mechanism of anti-H. pylori action of TF18, we evaluated the growth and motility of TF18 on clarithromycin-susceptible H. pylori (CSHP) and -resistant H. pylori (CRHP). An effective proton pump inhibitor (TF18) had remarkable dose-dependent antibacterial activity and was able to inhibit the flagellar motor of both CSHP and CRHP isolates. The antimotility effect of TF18 was more pronounced at subinhibitory concentration in CRHP than in CSHP. The swimming (the forward motion) was more sensitive to the inhibition than the tumbling. Based on the results, it is supposed that TF18 works as an uncoupler similar to the 'clutch' in a biological motor, in which counterclockwise rotation is more sensitive to the effect of TF18 than the clockwise rotation., (Copyright 2004 Elsevier B.V.)

We investigated 2 isoflavones and 9 isoflavanones from Sophora species for their cytotoxic activity against 3 normal human cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) and 2 human tumor cell lines (squamous cell carcinoma HSC-2, submandibular gland carcinoma HSG). Compounds with 2 isoprenyl groups (one in A-ring and the other in B-ring) such as tetrapterol G [YS31] and isosophoranone [YS24], and those with alpha,alpha-dimethylallyl group at C-5' of B-ring [YS26 (secundifloran), YS27 (secundiflorol A), YS28 (secundiflorol D), YS29 (secundiflorol E)] showed relatively higher cytotoxic activity. When hydrophobicity was assessed by octanol-water partition coefficient (log P), the maximum cytotoxic activity was observed at a log P value around 4. Compounds with intermediate cytotoxic activity [YS27, genistein, YS28, YS29, YS30 (secundiflorol F)] showed relatively higher tumor specificity. All isoflavones and isoflavanones did not stimulate the nitric oxide (NO) production by mouse macrophage-like Raw 264.7 cells, but almost completely inhibited the NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells. ESR spectroscopy showed that YS26 and YS28, which are the most inhibitory for NO production, efficiently scavenged superoxide anion and NO radicals. These data suggest that the inhibition of macrophage NO production by these isoflavanones may, at least in part, be explained by their radical scavenging or reduction activity.

The partition coefficients (K(p)) between lipid bilayer of phosphatidylcholine (PC) vesicles and buffer for five new phenothiazines were determined using the second derivatives of ultraviolet absorption spectra. The lambda(max) of absorption band for each of the investigated phenothiazine derivatives (PDs) was shifted to the longer wavelengths in the presence of PC vesicles with increasing of lipid concentration. As a result of light scattering in liposome suspension no isosbestic point could be observed in absorption spectra. However, the background signal could be eliminated using the method of second derivative of absorption spectra. In the second derivative of absorption spectra two isosbestic points were observed. Changes of intensity (Delta D) of second derivative of absorption spectra at the lambda(max) (wavelength of absorption maximum for drug in buffer) caused by the increase in lipid concentration were measured for set of phenothiazine derivatives. K(p) for these drugs were calculated from the relationship between Delta D and lipid concentration. The K(p) values for all studied phenothiazine derivatives are in the order of magnitude of 10(5) and they increase about 1.7-fold when length of the alkyl phenothiazine chain was enhanced by addition of the each next one (-CH(2)) group. Substitution of -H atom by -CF(3) group at position 2 of phenothiazine ring results in 3.5-fold increase in K(p) values.