Search

The transcription factor HIF-1α regulates the adaptive response of cells to hypoxia and oxidative stress. In addition, an important regulatory role for HIF-1α in immune reactions and inflammation is suggested. The present study attempts to investigate the effect of the gaseous signalling molecule hydrogen sulphide (H2S) on HIF-1α in THP-1 macrophages using the slow H2S releasing donor GYY4137. We found that H2S induced HIF-1α protein accumulation in THP-1 macrophages in a concentration-dependent manner. Western blot analysis of cell fractions showed that HIF-1α protein translocates into the nucleus and leads to an increase of its target protein glucose transporter-1 (GLUT-1). Activation of nuclear factor-κB (NF-κB), as well as secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced in the presence of H2S. These findings indicate that HIF-1α accumulation due to H2S was not triggered by the NF-κB pathway. The antioxidant pathway Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1) was activated by H2S. Inhibition of the p38 mitogen-activated protein kinase (MAPK) reversed H2S mediated effects, suggesting that the p38 MAPK pathway may be involved in H2S induced HIF-1α/Nrf2 signalling pathways.

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.

P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an important role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed the highest activity (IC50 = 42 nM), which is one order of magnitude higher than the activity for an equilipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors, methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric features influencing P-gp inhibitory activity.

Graphical abstract, The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABAA) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated. GABAA receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (IGABA) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-voltage-clamp technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABAA receptor subtypes (EC50 range: 42.8 ± 7.6 μM (α2β2)–59.6 ± 12.3 μM (α3β2)). IGABA modulation by piperine did not require the presence of a γ2S-subunit, suggesting a binding site involving only α and β subunits. IGABA activation was slightly more efficacious on receptors formed from β2/3 subunits (maximal IGABA stimulation through α1β3 receptors: 332 ± 64% and α1β2: 271 ± 36% vs. α1β1: 171 ± 22%, p

The role of butyrylcholinesterase (BChE) in the progression of Alzheimer’s disease (AD) has recently become more crucial. In the AD brain, selective BChE inhibitors have been demonstrated to have a beneficial effect in vivo , probably by recovering cholinergic activity and/or by restoring AChE:BChE activity ratios to the levels observed in the healthy brain. Thienothiazines are compounds sharing some structural features with phenothiazines, which are known to be potent BChE inhibitors. Thus, in this contribution 45 thienothiazines were investigated for their BChE inhibitory activity. Six of them were proven to be potent and selective inhibitors of equine BChE’s hydrolase activity. Structure–activity relationships were laid out, and a tentative pharmacophore model for BChE inhibitors of the thienothiazine type was proposed. The most active compound, 3f , displayed a mixed type of inhibition and was also active against the human BChE (huBChE) with an IC 50 huBChE of 0.51 ± 0.07 μM. Computational studies suggested that 3f likely binds to the catalytic site and nearby to the peripheral site of the huBChE in an extended form. In addition, the chemical space occupied by the active thienothiazines, as opposed to phenothiazines and other representative chemical classes of BChE inhibitors, was explored with the aid of ChemGPS-NP, and the relevant chemical space regions were identified. This study shows for the first time that thienothiazines represent a new group of BChE inhibitors that can be used as molecular probes for studying the role of BChE in the brain or for developing newer drug leads for AD therapy.

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with (18)F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 (4a-e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line overexpressing human Pgp (MDCKII-MDR1). 1-[(18)F]fluoroelacridar ([(18)F]4b) was synthesized in a decay-corrected radiochemical yield of 1.7±0.9% by a 1-step no-carrier added nucleophilic aromatic (18)F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [(18)F]4b was performed in naïve rats, before and after administration of unlabelled 1 (5 mg/kg, n=3), as well as in wild-type and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3). In PET experiments in rats, administration of unlabelled 1 increased brain activity uptake by a factor of 9.5 (p=0.0002, 2-tailed Student's t-test), whereas blood activity levels remained unchanged. In Mdr1a/b((-/-))Bcrp1((-/-)) mice, the mean brain-to-blood ratio of activity at 60 min after tracer injection was 7.6 times higher as compared to wild-type animals (p=0.0002). HPLC analysis of rat brain tissue extracts collected at 40 min after injection of [(18)F]4b revealed that 93±7% of total radioactivity in brain was in the form of unchanged [(18)F]4b. In conclusion, the in vivo behavior of [(18)F]4b was found to be similar to previously described [(11)C]1 suggesting transport of [(18)F]4b by Pgp and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo defluorination will limit the utility of [(18)F]4b as a PET tracer.

Recently we presented a series of benzanilide derivatives with a selective spasmolytic effect on terminal ileum preparations of the guinea pig. In this report we demonstrate a further development of these compounds. The exchange of the amide oxygen against a sulfur atom resulted in an up to 325 fold increase of the antispasmodic activity of the thiobenzanilide (IC 50 of 0.1 μM) compared to its benzanilide derivative. Considering their mode of action the compounds interacted with several molecular targets, suggesting that we identified a chemical identity able to modulate multiple targets simultaneously. Furthermore, based on this data set, we present a structure–activity relationship study supporting the important role of the sulfur atom.

A series of dithiobenzanilide derivatives was synthesized and each compound was evaluated for its ability to reduce KCl-induced contractions of smooth muscle preparations of the guinea pig. Starting from a recent publication describing benzanilide derivatives as antispasmodic agents, structure–activity guided synthesis was performed to obtain compounds with improved spasmolytic activity. First, compounds with two amide bonds were designed and second, both amide oxygens were replaced by two sp2 sulfur atoms resulting in dithiobenzanilide derivatives. The most potent antispasmodic dithiobenzanilide 19 showed improved activity with an IC50 value of 0.4 μM. Moreover, the study also demonstrated that these active compounds were able to antagonize the effect of spasmogens like acetylcholine and phenylephrine and that the activity is not mediated by activation of ATP-dependent potassium channels (KATP-channels) or inhibition of endothelial nitric oxide synthase (eNOS).

BACKGROUND AND PURPOSE Subunit-specific modulators of γ-aminobutyric acid (GABA) type A (GABAA) receptors can help to assess the physiological function of receptors with different subunit composition and also provide the basis for the development of new drugs. Valerenic acid (VA) was recently identified as a β2/3 subunit-specific modulator of GABAA receptors with anxiolytic potential. The aim of the present study was to generate VA derivatives as novel GABAA receptor modulators and to gain insight into the structure–activity relation of this molecule. EXPERIMENTAL APPROACH The carboxyl group of VA was substituted by an uncharged amide or amides with different chain length. Modulation of GABAA receptors composed of different subunit compositions by the VA derivatives was studied in Xenopus oocytes by means of the two-microelectrode voltage-clamp technique. Half-maximal stimulation of GABA-induced chloride currents (IGABA) through GABAA receptors (EC50) and efficacies (maximal stimulation of IGABA) were estimated. Anxiolytic activity of the VA derivatives was studied in mice, applying the elevated plus maze test. KEY RESULTS Valerenic acid amide (VA-A) displayed the highest efficacy (more than twofold greater IGABA enhancement than VA) and highest potency (EC50= 13.7 ± 2.3 µM) on α1β3 receptors. Higher efficacy and potency of VA-A were also observed on α1β2γ2s and α3β3γ2s receptors. Anxiolytic effects were most pronounced for VA-A. CONCLUSIONS AND IMPLICATIONS Valerenic acid derivatives with higher efficacy and affinity can be generated. Greater in vitro action of the amide derivative correlated with a more pronounced anxiolytic effect in vivo. The data give further confidence in targeting β3 subunit containing GABAA receptors for development of anxiolytics.

The multidrug efflux transporter breast cancer resistance protein (BCRP) is highly expressed in the blood-brain barrier (BBB), where it limits brain entry of a broad range of endogenous and exogenous substrates. Methyl is a recently discovered BCRP-selective inhibitor, which is structurally derived from the potent P-glycoprotein (P-gp) inhibitor tariquidar. The aim of this study was to develop a new PET tracer based on 1 to map BCRP expression levels in vivo.Compound 1 was labelled with (11)C in its methyl ester function by reaction of the corresponding carboxylic acid 2 with [(11)C]methyl triflate. Positron emission tomography (PET) imaging of [(11)C]-1 was performed in wild-type, Mdr1a/b((-/-)), Bcrp1((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3 per mouse type) and radiotracer metabolism was assessed in plasma and brain.Brain-to-plasma ratios of unchanged [(11)C]-1 were 4.8- and 10.3-fold higher in Mdr1a/b((-/-)) and in Mdr1a/b((-/-))Bcrp1((-/-)) mice, respectively, as compared to wild-type animals, but only modestly increased in Bcrp1((-/-)) mice. [(11)C]-1 was rapidly metabolized in vivo giving rise to a polar radiometabolite which was taken up into brain tissue.Our data suggest that [(11)C]-1 preferably interacts with P-gp rather than BCRP at the murine BBB which questions its reported in vitro BCRP selectivity. Consequently, [(11)C]-1 appears to be unsuitable as a PET tracer to map cerebral BCRP expression.

Pancreatic cancer is a very aggressive malignant disease due to lack of early diagnosis and chemotherapeutic resistance of the tumor cells. There is distinct evidence that food derived polyphenols possess chemopreventive effects in the development of several cancers including pancreatic carcinoma. Resveratrol is one of those phenolic compounds found in grape skins and other fruits with known anticancer activity. Various polymethoxylated resveratrol derivatives showed stronger antiproliferative effects than resveratrol in tumor cell lines. The aim of our study was to evaluate the cytotoxic and biochemical effects of a newly synthesized polymethoxylated resveratrol analogue, N-hydroxy-N'-(3,4,5-trimethoxphenyl)-3,4,5-trimethoxy-benzamidine (KITC) in two human pancreatic cancer cell lines. The human pancreatic cancer cell lines, AsPC-1 and BxPC-3 were used to test the potential inhibitory effect of the resveratrol derivative on cell proliferation and the underlying mechanisms of this effect. After 7 days of incubation, KITC inhibited the growth of AsPC-1 and BxPC-3 cells with IC(50) values of 9.6 and 8.7 microM, respectively. KITC (40 microM) arrested cells in the G0/G1 phase and depleted cells in the S phase of the cell cycle (-105% and -35% of control, respectively). KITC induced dose-dependent apoptosis in both pancreatic cancer cell lines and was found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of DNA synthesis. Employing growth inhibition assays, KITC acted synergistically with gemcitabine in both cell lines. In summary, we found that KITC exerted considerable antitumor activity against human pancreatic cancer cells and could be a promising candidate for further investigations to establish a new chemotherapeutic regimen.

The following study describes the synthesis of new benzanilide derivatives and their pharmacological investigation on smooth muscle preparations of guinea pigs. All compounds were synthesized in good yields and showed a spasmolytic activity without significant effect on vascular smooth muscles and heart muscle preparations. Moreover, further pharmacological investigations as well as in silico studies were performed to elucidate the mechanism of action. Compound 3 showed the most potent spasmolytic activity with an IC(50) of 3.25microM.

In continuation of our studies on compounds with a resveratrol-based scaffold two compounds with N-containing functional groups have been synthesized and screened for their inhibitory effect on the growth of the human cancer cell lines HT29, 518A2, AsPC-1, BxPC-3, and PC-3. Compound 4, but not 1, demonstrated pronounced in vitro cytotoxicity against all these cancer cell lines, thus making this compound a promising candidate for further preclinical in vivo studies.

Resveratrol ((E)-3,4',5-trihydroxy-stilbene), a phytoalexin found in various plants, shows non-selective cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibition. In order to find more selective COX inhibitors a series of bridged stilbene derivatives was synthesized and evaluated for their ability to inhibit both COX-1 and COX-2 in vitro. The compounds showed a high rate of COX-1 inhibition with the most potent compounds exhibiting submicromolar IC(50) values and high selectivity indices. A prediction model for COX-inhibiting activity was also developed using the classical LIE approach resulting in consistent docking data for our molecule sample. Phenyl substituted 1,2-dihydronaphthalene derivatives and 1H-indene derivatives therefore represent a novel class of highly selective COX-1 inhibitors and land promising candidates for in vivo studies.

In this contribution the development of a new class of vasodilating compounds obtained by lead structure optimization is described. Three groups of compounds were synthesized and tested for their activity on various smooth muscle preparations of the guinea pig. Beside the lead compound 3a, the most interesting derivative was 1H-imidazole-1-carbothioic acid O-cyclohexyl ester hydrochloride (5b) with a good selective vasodilating potential on aorta and pulmonary artery rings (EC50 14 μM and 24 μM, respectively). Due to the properties of small molecules the hydrolysis behavior of the compounds can be easily adapted hence opening a new route in terms of duration of the agent’s effect. With the aid of structure–activity relationship studies, structural motifs influencing the biological activity on isolated smooth muscle cell preparations of the synthesized compounds were proposed. The presented compounds offer good tools in identifying promising molecules as emergency therapy in myocardial infarction.

Objective Resveratrol (3,4′,5,-trihydroxystilbene, RV), an ingredient of wine, is an inhibitor of the proliferation-linked enzyme ribonucleotide reductase (RR) and shows a broad spectrum of cytotoxic effects against human cancer cells. In order to enhance these effects, we introduced additional hydroxyl moieties into the molecule. In the present study, the activity of a novel RV analog, 3,3′,4,4′,5,5′-hexahydroxystilbene (M8), was investigated in HL-60 human promyelocytic leukemia cells. Methods Cytotoxicity of M8 alone or in combination with Ara-C was assessed employing growth inhibition assays. Effects of M8 on nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) were examined by HPLC. The apoptotic potential of M8 and RV was compared using a specific double-staining method and inhibition of TNF-α-induced activation of NF-κB was studied. Cell-cycle distribution was analyzed by FACS. Results Addition of ascorbic acid decreased the IC 50 value of M8 from 6.25 μM to 2 μM. M8 depleted dATP and dTTP pools to 41% and 21% of control values, whereas dCTP pools increased to 199% of untreated controls. In addition, TTP, ATP, CTP, and GTP concentrations were decreased while UTP concentrations increased. M8 induced apoptosis at concentrations significantly lower than RV and could remarkably inhibit the activation of NF-κB. M8 arrested cells in the S phase of the cell cycle while depleting cells in the G2-M phase and exhibited synergistic combination effects when applied simultaneously with Ara-C. Conclusion Due to these promising results, this novel polyhydroxylated stilbene derivative might become an additional option for the treatment of leukemia and therefore deserves further preclinical and in vivo testing.

A one-pot radiosynthesis method to prepare the new fluorine-18-labelled fluoropyridine derivatives 5-[18F]fluoro-2-pyridinamine and 6-[18F]fluoro-2-pyridinamine in two to three reaction steps was developed. The first step consisted of no-carrier-added nucleophilic aromatic substitution of commercially available halogen-substituted 2-pyridinecarboxamide or 2-pyridinecarbonitrile derivatives with K[18F]F-K222 in DMSO at 150–180°C. The [18F]fluoride incorporation yields ranged from 67 to 98% for all studied precursor molecules. It is remarkable that 5-bromo-2-pyridinecarbonitrile gave almost quantitative [18F]fluoride incorporation at the meta-position (5-position) of the pyridine ring after only 5 min of heating at 150°C. After base-catalysed hydrolysis of the [18F]fluorinated pyridinecarbonitriles into their corresponding carboxamides, the latter were transformed in a Hofmann-type rearrangement reaction into the respective amines by treatment of crude reaction mixtures with bromine and aqueous base (20–30% conversion yield). Reaction mixtures were purified by reversed-phase semipreparative HPLC followed by strong cation exchange solid-phase extraction to afford 5-[18F]fluoro-2-pyridinamine and 6-[18F]fluoro-2-pyridinamine in non-decay-corrected radiochemical yields of 6–10% in a total synthesis time of 83–112 min. The preparation of 5-[18F]fluoro-2-pyridinamine is one of very few examples demonstrating the feasibility of nucleophilic meta-[18F]fluorination of a pyridine derivative. Both 5-[18F]fluoro-2-pyridinamine and 6-[18F]fluoro-2-pyridinamine are new potentially useful radiolabelled synthons for radiopharmaceutical chemistry. Copyright © 2006 John Wiley & Sons, Ltd.

Cyclic hydroxamic acids like 2,4-dihydroxy-7-methoxy-2H-1 ,4-benzoxazin-3(4H)-one (DIM BOA) and 2,4-dihydroxy-2H-1 ,4-benzoxazin-3(4H)-one (DIBOA) are found in several plants playing an important role in the defense-system of plants against a variety of enemies. To investigate new mechanism and effects we synthesized the molecules using known synthetic pathways. Since the chemical data of DIMBOA are not complete or even false, we decided to publish the missing ones in this journal.

Resveratrol (trans-3,4',5-trihydroxystilbene), a naturally occurring hydroxystilbene, is considered an essential antioxidative constituent of red wine possessing chemopreventive properties. However, resveratrol and even more its metabolite piceatannol were reported to have also cytostatic activities. In order to find out whether this is related to antioxidative properties of those compounds, we synthesized five other polyhydroxylated resveratrol analogues and studied structure-activity relationships between pro-/antioxidant properties and cytotoxicity. Radical scavenging experiments with O(2)(*-) (5,5-dimethyl-1-pyrroline-N-oxide/electron spin resonance (DMPO/ESR)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (photometry) revealed that 3,3',4',5-tetrahydroxystilbene (IC(50): 2.69microM; k(9): 443000M(-1)s(-1)), 3,4,4',5-tetrahydroxystilbene (IC(50): 41.5microM; k(9): 882000M(-1)s(-1)) and 3,3',4,4',5,5'-hexahydroxystilbene (IC(50): 5.02microM), exerted a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues. Furthermore, in HL-60 leukemic cells hydroxystilbenes with ortho-hydroxyl groups exhibited a more than three-fold higher cytostatic activity compared to hydroxystilbenes with other substitution patterns. Oxidation of ortho-hydroxystilbenes in a microsomal model system resulted in the existence of ortho-semiquinones, which were observed by ESR spectroscopy. Further experiments revealed that these intermediates undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxygen radicals. In contrast to compounds with other substitution patterns hydroxystilbenes with one or two resorcinol groups (compounds 1 and 3) did not show an additional oxygen consumption or semiquinone formation. These findings suggest that the increased cytotoxicity of ortho-hydroxystilbenes is related to the presence of ortho-semiquinones formed during metabolism or autoxidation.

Objective Resveratrol, a naturally occurring stilbene derivative, is a potent free-radical scavenger causing a number of biochemical and antineoplastic effects. It was shown to induce differentiation and apoptosis in leukemia cells. Resveratrol was also identified as an inhibitor of ribonucleotide reductase (RR), a key enzyme of DNA synthesis. We report about the biochemical effects of resveratrol on the concentration of deoxyribonucleoside triphosphates (dNTPs), the products of RR, and on the incorporation of 14C-labeled cytidine into the DNA of HL-60 human promyelocytic leukemia cells. Materials and methods Incorporation of 14C-labeled cytidine into the DNA of resveratrol-treated HL-60 cells was measured. Concentration of dNTPs was determined by a HPLC method. Cytotoxic effects of resveratrol, Ara-C, and tiazofurin were analyzed using growth inhibition and clonogenic assays. Induction of apoptosis was studied using a Hoechst/propidium iodide staining method. Results We found that resveratrol effectively inhibited incorporation of 14C-labeled cytidine into DNA. Furthermore, incubation of HL-60 cells with resveratrol significantly decreased intracellular dCTP, dTTP, dATP, and dGTP concentrations. Based on these results, we investigated the combination effects of resveratrol with Ara-C or tiazofurin, both antimetabolites, which are known to exhibit synergistic effects in combination with other inhibitors of RR. In growth inhibition, apoptosis, and clonogenic assays, resveratrol acted synergistically with both Ara-C and tiazofurin in HL-60 cells. Conclusions We conclude that resveratrol could become a viable candidate as one compound in the combination chemotherapy of leukemia and therefore deserves further testing.

Under strong acidic conditions, 2,5-dimethylthiophene forms a tetracyclic trimer almost quantitatively whose peculiar structure could be elucidated by X-ray analysis. In addition, by means of quantum mechanical calculations, a sound mechanistic picture of the reaction pathway is proposed.

To prepare required thiolactarns. initial experiments were done with the lactarns 1 - 3 and Lawessol reagent. However in preliminary trials. treatment of the compounds failed to give the desired molecules. Modification of the reaction conditions. paticularly with regard to the solvent and the reaction temperature. did not permit the preparation of the corresponding thiolactams. The keto function in position 6 was thought to be responsible for this atypical behaviour. Therefore. as the following step. the acetyl group in compounds 1 - 3 should be reduced to the corresponding ethyl derivatives. This reaction did not give the needed bicycles. Reduction of compounds 4 - 6 with triethylsilanel trifluoroacetic acid brought up compounds 7 - 9.

GABA-receptor-ligands are still very interesting in drug-development. Usually benzodiazepines are used in the treatment but they have serious side-effects. Thus, a recently synthesized quioxaline derivative which showed reduced side-effects in an animal model was used as a model-substance. The cyclus was modified to optimize the pharmacological profile. Accordingly, a series of imidazo-thieno-thiazines was synthesized starting from 5-acetyl-2-chloro-3-nitrothiophene to yield 6-ethyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thiazine-2-one. Reaction with potassium tert-butoxide and diethylchlorophosphate gave an intermediate, which resulted in the desired ring system after adding the corresponding isocyanides and potassium tert-butoxide.

Some novel imidazolyl and pyrazolyl thiophene derivatives were synthesized. Different substituted N-containing heterocycles were chosen with the aim of enhancing the activity or selectivity of the products towards inhibition of one of the three isoforms of nitric oxide synthase. One series of substances was prepared by substitution reactions of 3-bromo-2-nitrothiophene with various heterocycles. In the other series introduction of a second heterocyclic substituent to pyrazolylthiophene compounds was attempted.

GABA-receptor-ligands are still very interesting in drug-development. Usually benzodiazepines are used in the treatment but they have serious side-effects. Thus, a recently synthesized quioxaline derivative which showed reduced side-effects in an animal model was used as a model-substance. The cyclus was modified to optimize the pharmacological profile. Accordingly, a series of imidazo-thieno-thiazines was synthesized starting from 5-acetyl-2-chloro-3-nitrothiophene to yield 6-ethyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thiazine-2-one. Reaction with potassium tert-butoxide and diethylchlorophosphate gave an intermediate, which resulted in the desired ring system after adding the corresponding isocyanides and potassium tert-butoxide.

A new series of substituted thieno[1,4]thiazines with an urea moiety has been designed. The synthesis and results of replacement of the quinoline and indoline moieties of lead structure 1 are described. The key step in preparation was the substitution with 4-nitrophenyl chloroformate to obtain the required reactivity for substitution with diamines. Structural modifications of the amino side chain with the aim of finding tissue specific compounds were carried out. Pharmacological testings explored the presumed calcium-channel-antagonistic and potassium-channel-opening activities.

In this study novel substituted 6-benzyl-thieno[2,3-b][1,4]thiazines with an urea moiety were synthesized. Structural modifications of the amino side chain were carried out with the aim of finding tissue specific compounds. The effects on papillary muscles, right atria, aortic strips, and terminal ilea were investigated. Compounds 10c and 10d showed the most potent negative inotropic effect. The calcium antagonism of all derivatives occurred in a non-competitive manner, which may indicate that they also have potassium channel opening activities.

Substituted thieno[1,4]thiazine derivatives have been synthesized. They are thienoanalogues of compound 3 [2], a potent cerebral protectant and calcium antagonist. Research activities concentrate on the preparation of structurally modified compounds of 3 to minimize its undesirable symptoms.

3-Bromo-2-nitrothiophene is reacted with bifunctional reagents like methyl 3-mercaptopropionate to yield the corresponding thioether derivatives. Reduction of the nitro function followed by ring closure provides a possibility to synthesize the hitherto unknown thieno [3,2-b][1,4]oxazines as well as thieno [3,2-b][1,4]thiazines. In a similar manner, the new thienodithiazepine ring system is obtained.

KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl) ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine (1). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC50 = 23 microM, n = 7), and a negative inotropic effect in papillary muscles (IC50 = 2.7 microM, n = 7) and left atria (IC50 = 4 microM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC50: 70 +/- 5 microM) and low (IC50: 129 +/- 34 microM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC50: 80 +/- 13 microM) in chick dorsal root ganglion neurons. In addition nicotine-induced currents were potently inhibited (IC50: 6 +/- 0.7 microM) in bovine chromaffin cells.

Genistein, a naturally occurring isoflavone, possesses many beneficial health effects. To improve the bioactivity of the natural compound, we designed and synthesized the genistein prodrug FEHH6-1. In the present study, we evaluated the biological effects of FEHH6-1 on mouse RAW264.7 macrophages and compared them with those obtained with the parent drug genistein. The characteristics of FEHH6-1 were determined by melting point, nuclear magnetic resonance spectroscopy (NMR), and mass spectrometric analysis. The effects of FEHH6-1 on cell proliferation, apoptosis, and pro-inflammatory cytokine expression were monitored by XTT-assay, Annexin-V/7-AAD staining, Western blotting, and ELISA. FEHH6-1 showed NMR spectra and relative molecular mass in agreement with the designed structure. In mouse RAW264.7 macrophages, FEHH6-1 inhibited proliferation, induced apoptotic cell death and blocked interleukin 6 and tumor necrosis factor alpha synthesis. At low concentrations, FEHH6-1 induced phosphorylation of AKT1, a kinase involved in cell proliferation and survival. Our data demonstrate that the genistein prodrug FEHH6-1 is a bioactive molecule but its solubility and therefore also its efficacy was significantly lower compared with genistein.

Studies on the Chemistry of Thieno-Annulated O,N- and S,N-Containing Heterocycles, 8[1].–Synthesis of a Racemic, Thieno Analogue Diltiazem with Potential Ca2+Antagonistic Activity The synthesis of the thieno analogue diltiazem cis-8-[2-(di-methylamino)ethyl]-5,6,7,8-tetrahydro-5-(4-methoxyphenyl)-7-oxothieno[3,2-b][1,4]thiazepin-6-yl acetate (1) is described. Reaction of 3-bromo-2-nitrothiophene with Na2S · 9H2O yields the intermediate 3-mercapto-2-nitrothiophene (6), which reacts with the racemic glycidate 7 to give the racemic threo compound 10. Reduction and cyclization of 10 furnish the thieno [3,2-b][1,4]thiazepine derivative 12.N-Alkylation and acetylation of the hydroxy group of 12 leads to 1.

HM30181, a potent and selective inhibitor of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp), was shown to enhance oral bioavailability and improve antitumour efficacy of paclitaxel in mouse tumour models. In search for a positron emission tomography (PET) radiotracer to visualise Pgp expression levels at the blood-brain barrier (BBB), we examined the ability of HM30181 to inhibit Pgp at the murine BBB. HM30181 was shown to be approximately equipotent with the reference Pgp inhibitor tariquidar in inhibiting rhodamine 123 efflux from CCRF-CEM T cells (IC(50), tariquidar: 8.2 ± 2.0 nM, HM30181: 13.1 ± 2.3 nM). PET scans with the Pgp substrate (R)-[(11)C]verapamil in FVB wild-type mice pretreated i.v. with HM30181 (10 or 21 mg/kg) failed to show significant increases in (R)-[(11)C]verapamil brain uptake compared with vehicle treated animals. PET scans with [(11)C]HM30181 showed low and not significantly different brain uptake of [(11)C]HM30181 in wild-type, Mdr1a/b((-/-)) and Bcrp1((-/-)) mice and significantly, i.e. 4.7-fold (P0.01), higher brain uptake, relative to wild-type animals, in Mdr1a/b((-/-))Bcrp1((-/-)) mice. This was consistent with HM30181 being at microdoses a dual substrate of Pgp and breast cancer resistance protein (Bcrp). In vitro autoradiography on low (EMT6) and high (EMT6Ar1.0) Pgp expressing murine breast tumour sections showed 1.9 times higher binding of [(11)C]HM30181 in EMT6Ar1.0 tumours (P0.001) which was displaceable with unlabelled tariquidar, elacridar or HM30181 (1 μM). Our data suggest that HM30181 is not able to inhibit Pgp at the murine BBB at clinically feasible doses and that [(11)C]HM30181 is not suitable as a PET tracer to visualise cerebral Pgp expression levels.

Substituted thieno[2,3-b][1,4]benzoxazepine derivatives as well as thieno[3,2-b][1,4]benzoxazepine deriva- tives have been synthesized. They are thienoanalogues of loxapine, a potent antipsychotic drug. Research activities concentrate on the preparation of structurally modified compounds of loxapine to minimize its undesirable extrapyramidal symptoms.

The reactivity of 5-acetyl-2-chloro-3-nitrothiophene with α-hydroxyesters is described. The influence of the solvent (DMF, tetrahydrofurane, toluene) and of the used base is discussed. These reactions provide an access to substituted thiophene derivatives, which were cyclized after reduction by iron powder/glacial acetic acid to give thieno [2,3-b][1,4]oxazine derivatives.

In this contribution, a chemical collection of aromatic compounds was screened for inhibition on butyrylcholinesterase (BChE)’s hydrolase activity using Ellman’s reaction. A set of diarylimidazoles was identified as highly selective inhibitors of BChE hydrolase activity and amyloid β (Aβ) fibril formation. New derivatives were synthesized resulting in several additional hits, from which the most active was 6c, 4-(3-ethylthiophenyl)-2-(3-thienyl)-1H-imidazole, an uncompetitive inhibitor of BChE hydrolase activity (IC50 BChE = 0.10 μM; Ki = 0.073 ± 0.011 μM) acting also on Aβ fibril formation (IC50 = 5.8 μM). With the aid of structure–activity relationship (SAR) studies, chemical motifs influencing the BChE inhibitory activity of these imidazoles were proposed. These bifunctional inhibitors represent good tools in basic studies of BChE and/or promising lead molecules for AD therapy.

A new goup of acridone derivatives, obtained by reaction of acridone-4-carboxylic acid derivatives with aromatic amines, was tested to determine the inhibitory properties toward the NS3 helicase of hepatitis C virus (HCV). Six compounds inhibited the NS3 helicase at low concentrations (IC(50) from 1.5 to 20 microM). The acridone derivatives probably act via intercalation into double-stranded nucleic acids with a strong specificity for double-stranded RNA, although an interaction with the enzyme cannot be excluded. Testing in the subgenomic HCV replicon system revealed that compounds 10 and 13 are efficient RNA replication inhibitors, with EC(50) of 3.5 and 1 microM and therapeutic indexes of28 and 20, respectively. Compound 16, with EC(50)1 microM and TI1000, is extremely specific and practically noncytotoxic at the concentrations tested, proving that the acridone derivatives may be regarded as potential antiviral agents. Although the mechanism of action of 16 in the replicon system remains unclear, it is the key lead compound for further development of anti-HCV drugs.

2-Vinylbenzoxazole (3) is obtained by condensation of acrylic acid with 2-aminophenol in a mixture of phosphorus pentoxide/methansulfonic acid. Interaction of 3 with N-, O-, and C-nucleophiles yields 2-substituted 2-ethyl-benzoxazoles. Compounds 8a, 8b and 8e show analgetic activity in mice. Moreover, fungicide, insecticide and nematocide activity of some compounds is found.

In continuation of our studies on compounds with a resveratrol-based scaffold two compounds with N-containing functional groups have been synthesized and screened for their inhibitory effect on the growth of the human cancer cell lines HT29, 518A2, AsPC-1, BxPC-3, and PC-3. Compound 4, but not 1, demonstrated pronounced in vitro cytotoxicity against all these cancer cell lines, thus making this compound a promising candidate for further preclinical in vivo studies.

The development of chemoresistant breast cancer is poorly understood and second treatment options are barely investigated. The term 'chemoresistance' is ill-defined and thus, our experimental analyses aimed to disentangle the resistance to cell cycle arrest from the resistance to trigger apoptosis, both of which are important mechanisms to be targeted by anticancer therapy. Therefore, an MCF-7 array, which encompassed clones harboring distinct genetically- and pharmacologically-induced stages of resistance, was established. For this, MCF-7 cells were stably transfected with erbB2 cDNA and a dominant negative p53 mutation and the two clones were subjected to long-term treatment with the clinical agents 2'-deoxy-5-fluorouridine (5-FdUrd) or arabinosylcytosine (AraC) to develop specific chemoresistance. This array was tested with 3,4',5-trihydroxy-trans-stilbene (resveratrol) and the methoxylated paired stilbene analogue 3,4',5-trimethoxy-trans-stilbene (M5) to investigate whether these agents can overcome genetically- and pharmacologically-induced chemoresistance and to correlate the structure-activity relationship of resveratrol and M5. In all conditions tested, M5 exhibited stronger anticancer activity than resveratrol, but the cell cycle inhibitory properties of the tested drugs were dependent on the genetic background and the chemoresistant phenotype. In contrast, the proapoptotic properties were rather similar in the distinct genetic backgrounds of the clone array and therefore, apoptotic triggers and cell cycle checkpoints were distinctly affected and are thus independent of each other. The study demonstrates the merits or virtues of the genotypically- and phenotypically-defined clones of the MCF-7 array as a testing tool for novel drugs, which discriminates the two types of chemoresistance mechanisms.

Resveratrol (3,4',5,-trihydroxystilbene, RV), an ingredient of wine, exhibits a broad spectrum of antiproliferative effects against human cancer cells. In order to develop a derivative with comparable effects, we modified the molecule by introducing additional methoxyl groups. The resulting novel RV analog, N-hydroxy-N'-(3,4,5-trimethoxyphenyl)-3,4,5-trimethoxybenzamidine (KITC), was investigated in HL-60 human promyelocytic leukemia cells. The induction of apoptosis was determined employing a specific Hoechst/propidium iodide double staining method and cell cycle distribution was evaluated by FACS. KITC's influence on the concentration of deoxyribonucleoside triphosphates, the products of ribonucleotide reductase (RR), was determined using the HPLC method. In addition, we analyzed the effects of KITC treatment on the incorporation of 14C-cytidine into the DNA of tumor cells in order to quantify the loss of RR in situ activity. To reveal a potential value of KITC for supporting conventional chemotherapy, we also examined whether a combination of KITC with arabinofuranosylcytosine (Ara-C) could yield synergistic growth inhibitory effects. KITC caused a dose-dependent induction of apoptosis, whereas no remarkable changes of the cell cycle distribution were observed. Incubation with KITC resulted in a significant depletion of intracellular dTTP and dATP pools and was also found to remarkably reduce the in situ activity of RR, the key enzyme of de novo DNA synthesis. In addition, KITC exhibited synergistic combination effects when applied sequentially with Ara-C. Due to these promising results, KITC deserves further preclinical and in vivo testing.

Reduced nitric oxide (NO) availability is associated with the development of atherosclerosis. Upregulation of endothelial nitric oxide synthase (eNOS) activity is pursued as a strategy for the prevention of cardiovascular diseases. The polyphenol hydroxytyrosol (HT) which is present in olive oil and red wine, is regarded to be partly responsible for the beneficial effects associated with olive oil consumption and has shown antiatherogenic activity in vitro and in vivo. To elucidate the underlying molecular mechanisms, we investigated possible effects of HT on the endothelial nitric oxide synthase (eNOS). We used human endothelial cells (EA.hy926) and examined eNOS on three different levels, addressing eNOS promoter transactivation, eNOS enzyme activity and nitric oxide availability. Cells were treated with a broad range of HT concentrations (from 10 nM to 100 microM) and for different incubation times (15 min to 24 h). HT did not exert significant positive effects on eNOS in any of our assay systems. Neither did we find evidence for a possible synergism between the red wine polyphenol resveratrol and HT. We conclude that a direct modulation of eNOS is unlikely to account for the antiatherogenic properties of HT under non-inflammatory conditions.

Background and objectives Resveratrol is a naturally occuring polyphenol produced in many plants (red grapes, raspberries, blueberries, etc.). The positive health effects of resveratrol are due its potent anti-inflammatory, anti-oxidant and anti-proliferative activities in many types of cancer cells. Due to the fact that resveratrol is sensitive to oxygen and is almost insoluble in water, its therapeutic application is very limited. Therefore, work is going on designing resveratrol analogues to improve the solubility and bioavailability of the natural resveratrol. Recently, we designed and synthesised a resveratrol prodrug (FEHH4–1) which is only active within the cells. Through esterification of all three phenolic groups, stability as well as lipophilicity of the natural resveratrol could be improved. In the present study, we compared the effects of resveratrol with FEHH4–1 on Jurkat T- cells. Material and methods Jurkat T- cells were stimulated with PMA/PHA in the absence or presence of different concentrations of resveratrol or FEHH4–1. Modulation in IL-2 promoter activity was monitored by a IL-2 luciferase reporter assay. IL-2 release was quantified by enzyme-linked immunosorbent assay (ELISA). The effects of resveratrol and FEHH4–1 on cell proliferation and apoptosis were evaluated by XTT assay, Annexin-V/7-AAD staining and Western blot. Results Both resveratrol and FEHH4–1 effectively attenuated IL-2 promoter activity and IL-2 expression in stimulated Jurkat T- cells. At a concentration of 25 µM, resveratrol blocked IL-2 production at about 50%, FEHH4–1, however, at about 80%. Additionally, we observed that FEHH4–1 reduced cell proliferation and induced apoptotic cell death at significantly lower concentrations than resveratrol. Conclusion The resveratrol prodrug FEHH4–1 significantly amplified the beneficial effects of resveratrol in Jurkat T- cells. In comparison to resveratrol, FEHH4–1 enhanced downregulation of IL-2 production, and decreased proliferation rate in Jurkat T- cells. These data indicate that FEHH4–1 could be a substitute for the natural resveratrol in the future.

Resveratrol (3,4',5-trihydroxystilbene, RV) exerts remarkable cytostatic and cytotoxic effects against a multitude of human cancer cell lines. Since the introduction of additional hydroxyl groups was supposed to increase the biological activity of RV, we have synthesized a number of polyhydroxylated stilbene analogues as potential antitumor agents. In this study, the activity of 3,3',4,4',5,5'-hexahydroxystilbene (M8) was investigated in HL-60 human promyelocytic leukemia cells. Employing a growth inhibition assay, incubation with M8 and RV resulted in IC50 values of 6.25 and 12 microM, respectively. Using a specific Hoechst/propidium iodide double staining method, we found that M8 was able to induce apoptosis in concentrations significantly lower than those of RV. In addition, M8 arrested cells in the S phase and totally depleted cells in the G2-M phase of the cell cycle (143% and 0% of control after treatment with 12.5 microM M8, respectively). We therefore believe that this promising agent deserves further preclinical and in vivo testing.

trans-Resveratrol (t-RES) is one of the most relevant and extensively investigated stilbenes with a broad spectrum of biological activities. In contrast to the detailed knowledge of t-RES activities in biological systems, much less is known about the effects of higher hydroxylated stilbenes. Therefore, the aim of this study was to evaluate the protective effects (antioxidant activities) of t-RES and two analogues: the natural metabolite piceatannol (PCA) and the synthesized 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (HHS) against H2O2-induced DNA damage in leukemic L1210, K562 and HL-60 cells using single-cell gel electrophoresis (SCGE). After 24 h pre-treatment of cells all compounds investigated significantly inhibited the incidence of DNA single strand breaks induced by H2O2. The protective effects of PCA and HHS in L1210 cells and of HHS in HL-60 cells were significantly higher compared to the activity of t-RES (+P < 0.05). In K562 cells the differences of the antioxidant activities of PCA and HHS, and of PCA in HL-60 cells were of much higher significance when compared to t-RES (++P < 0.01). In conclusion, we can prove that all stilbenes investigated, t-RES, PCA, and HHS, manifested potent antioxidant effects on three leukemic cell lines and the presence of ortho-dihydroxy structures enhanced the protective effect against DNA damage caused by .OH radicals.