Coumarin 7-hydroxylation in long-term adherents of a strict uncooked vegan diet

Objective: Coumarin 7-hydroxylation was investigated in 21 Finnish vegans (20 females, one male) consuming a strict, uncooked vegan diet (“living food diet”) and in their matched omnivorous controls, by means of an in vivo coumarin test. Method: A capsule containing 5?mg of coumarin (Venalot^®) was taken after an overnight fast, and urine samples were collected before and 2, 4 and 6?h after the drug administration. The extent and rate of urinary excretion of 7-hydroxycoumarin was determined using HPLC. Results: The total urinary excretion of 7-hydroxycoumarin during 6?h was 58 (range 23–85) and 64 (range 39–92)% of the administred dose in the vegan and control groups. The coumarin index (excretion of 7-hydroxycoumarin during the first 2?h as percentage of total excretion) was 72% in the vegan and 78% in the control groups. A negative correlation was observed between the coumarin index and the consumption of wheatgrass juice by the vegans (r?=??0.60, P?n?=?21). Proportion of slow hydroxylators (excreting 7-hydroxycoumarin after 4?h) was not statistically different between the groups (5/21 in the vegans vs 8/20 in the controls). Conclusion: According to the present study, the clearly different dietary patterns and nutrient intakes between the vegans and the omnivores resulted in similar extent and rate of 7-hydroxycoumarin formation, indicating only a minor effect on coumarin hydroxylase (CYP2A6) activity by the plant substances in the uncooked vegan diet.     

Metabolism of coumarin by rat,gerbil and human liver microsomes

1. o-Hydroxyphenylacetaldehyde was the major metabolite of coumarin (1 mM) in rat, gerbil and human liver microsomes.

2. Treatment of rats with phenobarbitone (PB) or β-naphthoflavone increased the o-hydroxyphenylacetaldehyde formed. 3-Hydroxycoumarin was the other main metabolite produced by rat liver microsomes.

3. Liver microsomal metabolism of coumarin in gerbil was extensive with 3-, 5-, 6-, 7-and 8-hydroxycoumarins, and 3,7- and 6,7-dihydroxycoumarins produced, in addition to o-hydroxyphenylacetaldehyde. The profile of the hydroxy metabolites was altered by in vivo treatment of gerbils with cytochrome P-450 inducers, but there was no increase of coumarin metabolism.

4. Coumarin was metabolized by human liver microsomes to o-hydroxyphenylacetaldehyde, 7-hydroxycoumarin, 3-hydroxycoumarin, and trace amounts of 5-, 6-and 8-hydroxycoumarins.

5. At low substrate concentrations (0-10 μM) hepatic microsomal metabolism of coumarin in gerbil resembled that in man, with 7-hydroxycoumarin being a major metabolite. However, the production of o-hydroxyphenylacetaldehyde was greater in gerbil than human liver microsomes.

6. At higher substrate concentrations (1 mM) metabolism of coumarin by liver microsomes from PB-treated gerbils most closely resembled that by human liver microsomes.

7. The gerbil would appear to be a more appropriate animal model than rat for studies to assess the toxicological hazard of coumarin for man.     

Hologram Quantitative Structure Activity Relationship,Docking, and Molecular Dynamics Studies of Inhibitors for CXCR4

CXCR4 plays a crucial role as a co-receptor with CCR5 for HIV-1 anchoring to mammalian cell membrane and is implicated in cancer metastasis and inflammation. In the current work, we study the relationship of structure and activity of AMD11070 derivatives and other inhibitors of CXCR4 using HQSAR, docking and molecular dynamics (MD) simulations. We obtain an HQSAR model (q² = 0.779), and the HQSAR result illustrates that AMD11070 shows a high antiretroviral activity. As HQSAR only provides 2D information, we perform docking and MD to study the interaction of It1t, AMD3100, and AMD3465 with CXCR4. Our results illustrate that the binding are affected by two crucial residues Asp97 and Glu288. The butyl amine moiety of AMD11070 contributes to its high antiretroviral activity. Without a butyl amine moiety, (2,7a-Dihydro-1H-benzoimidazol-2-ylmethyl)-methyl-(5,6,7,8-tetrahydro-quinolin-8-yl)-amine (compound 5a ) shows low antiretroviral activity. Our results provide structural details about the interactions between the inhibitors and CXCR4, which are useful for rational drug design of CXCR4.     

Structural characterization of human CRTh2: a combined homology modeling,molecular docking and 3D-QSAR-based in silico approach

Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) is an important therapeutic target for the treatment of asthma, chronic obstructive pulmonary disease, allergic rhinitis and atopic dermatitis. In this study, we report the binding site analysis of CRTh2 through molecular docking and quantitative structure–activity relationship (QSAR) studies to explore the interaction of 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl) acetic acid derivatives. Various comparative models were generated using the available crystal structure of human delta opioid receptor (4N6H) as the template, and benzylthio acetic acid derivatives were docked into the predicted binding site region of human CRTh2 receptor. Surflex docking studies enabled us to identify that K5.43, Y4.60, N185, Y6.51, Q5.36, E6.58, T7.38 and H6.52 residues were the most crucial amino acids interacting with the ligand. In addition to docking, atom-by-atom matching and structure-based 3D-QSAR method CoMFA was performed. Based on better q ² and r ² _pred values, the best predictions were obtained for the ligand-based (q ² = 0.552, r ² _pred = 0.636) and for receptor-based (q ² = 0.507, r ² _pred = 0.541) QSAR model, whose robustness and predictability were verified by external test set validation. The results correlate well with the previously reported mouse model, and our study serves as a guide for mutational studies of human CRTh2 and further experimental investigations on the synthesis of new CRTh2 antagonist.     

Inhibitory effects of 6-alkoxycoumarin and 7-alkoxycoumarin derivatives on lipopolysaccharide/interferon γ-stimulated nitric oxide production in RAW264 cells

Coumarin and its derivatives are well known for their anti-inflammatory and anti-oxidative effects. In this study, we synthesized 32 coumarin derivatives from commercially available 6-hydroxycoumarin (6HC) and 7-hydroxycoumarin (7HC) and examined their effects on lipopolysaccharide/interferon γ (LPS/IFNγ)-stimulated nitric oxide (NO) production in murine macrophage RAW264 cells. Among these derivatives, 6HC-8 (6-(3-phenylpropoxy)coumarin), 6HC-14 (6-(2-octynyloxy)coumarin), 7HC-14 (7-(2-octynyloxy)coumarin), and 7HC-16 (7-(3,5-dimethoxybenzyloxy)coumarin) markedly suppressed NO production at low concentration (25 μM). These synthesized coumarin derivatives also markedly inhibited inducible NO synthase (iNOS) protein and mRNA expression, as assessed by western blotting and quantitative real time-polymerase chain reaction (RT-PCR).     

Substitutionsreaktionen an 6-Amino-4-methyl-salicylsäureestern, 1. Mitt.: Nitrosierungen und Nitrierungen

Substitution Reactions of 6-Amino-4-methylsalicylates, I: Nitrosation and Nitration The nitrosation of methyl 4-methyl-6-morpholinosalicylate ( 2a ) yields the 3-nitroso derivative 1a , whereas the 5-nitroso derivatives 9d and 9c are obtained from the esters of 6-anilino- or 6-(1-propylamino)-4-methylsalicyclic acid ( 7d, 7c ) via the nitrosamines 8 by Fischer-Hepp-rearrangement. Nitration of methyl 4-methyl-6-morpholinosalicylate ( 2a ) affords the 3- and 5-mononitro derivatives 5a and 6a as well as the 3,5-dinitro-derivative 3a . Methyl 4-methyl-6-(1-propylamino)-salicylate 7c yields the dinitronitramine 4c . With ammonium cerium(IV)nitrate, methyl 6-dimethylamino-4-methylsalicylate 2b is converted with demethylation into the dinitronitramine 4b . The structures of the monosubstitution products were confirmed by ¹³C-nmr spectroscopy and the analysis of all coupling constants.     

Structure–activity relationship studies of 4-methylcoumarin derivatives as anticancer agents

Context: Cancer is a leading cause of death worldwide and novel chemotherapeutic agents with better efficacy and safety profiles are much needed. Coumarins are natural polyphenolic compounds with important pharmacological activities, which are present in many dietary plants and herbal remedies.

Objectives: The objective of this study is to investigate natural and synthetic coumarin derivatives with considerable anticancer capacity against three human cancer cell lines.

Materials and methods: We synthesized 27 coumarin derivatives (mostly having 4-methyl moiety) and examined their cytotoxic effect on three human cancer cell lines, K562 (chronic myelogenous leukemia), LS180 (colon adenocarcinoma), and MCF-7 (breast adenocarcinoma) by MTT reduction assay. Screened compounds included 7-hydroxy-4-methylcoumarins (7-HMCs), 7-acetoxy-4-methylcoumarins (7-AMCs), and different dihydroxy-4-methylcoumarin (DHMC) and diacetoxy-4-methylcoumarin (DAMC) derivatives. Some compounds with methoxy, amine, and bromine substitutions were also examined.

Results: 7,8-DHMCs bearing alkyl groups at C3 position were the most effective subgroup, and of which, the most potent is compound 11, with an n-decyl chain at C3, which had IC₅₀ values of 42.4, 25.2, and 25.1?µM against K562, LS180, and MCF-7 cells, respectively. The second most active subgroup was 7,8-DAMCs containing ethoxycarbonylmethyl and ethoxycarbonylethyl moieties at C3 position. Compound 27 (6-bromo-4-bromomethyl-7-hydroxycoumarin), the only derivative containing bromine also showed reasonable cytotoxic activities (IC₅₀ range: 32.7–45.8?µM).

Discussion and conclusion: This structure–activity relationship (SAR) study of 4-methylcoumarins shows that further investigation of these derivatives may lead to the discovery of novel anticancer agents.     

Synthesis,spectroscopic characterization,X-ray structure,and in vivo neurotropic activity of new 1,5-benzodiazepin-2-ones

The paper reports the synthesis and in vivo pharmacological studies of a series of N-alkyl-1,5-benzodiazepine-2-ones. In this work, 19 novel benzodiazepine derivatives have been prepared and characterized by spectroscopic methods including 2D nuclear magnetic resonance techniques. Crystal structure of 1-benzyl-8-methyl-4-phenyl-1H-benzo[b][1,4]diazepin-2(3H)-one has also been determined by X-ray diffraction. Prediction of activity spectra for substances prediction and docking studies onto human serum albumin were conducted. Two compounds under these investigation showed high antihypoxic, tranquilizing, and anticonvulsant activity in vivo.     

Coumarin derivatives,but not coumarin itself,cause skin irritation via topical delivery

Coumarin and its derivatives are widely employed as a fragrance in cosmetics and skin care products. The skin absorption level and possible disruption to the skin by topical application of coumarins were evaluated in this study. Percutaneous absorption of osthole, daphnoretin, coumarin, byakangelicin, and 7-hydroxycoumarin was assessed in vitro and in vivo. Skin physiology measurements and immunoblotting were utilized as methodologies for validating toxicity. The relationship between structures and permeation/toxicity of coumarins was elucidated. Both equimolar concentration and saturated solubility in 30% ethanol were used as the applied dose. Osthole with the most lipophilic characteristic demonstrated the greatest skin accumulation, followed by coumarin and 7-hydroxycoumarin. Coumarin was the permeant with the highest flux across the skin. The trend of in vivo deposition was consistent with that of the in vitro profiles. Skin uptake of osthole was 8-fold higher than that of coumarin. Hair follicles played a significant role as a pathway for transport of coumarin according to the examination of follicular accumulation. Osthole and 7-hydroxycoumarin slightly, but significantly, enhanced transepidermal water loss after a consecutive 5-day administration. The immunoblotting profiling verified the role of proliferation in skin damage induced by osthole, byakangelicin, and 7-hydroxycoumarin. The proliferation-related proteins examined in this work included glucose-regulated proteins, cytokeratin, and C-myc. Daphnoretin and coumarin showed a negligible alteration on protein biomarkers. The experimental results suggested that skin irritation caused by coumarins was mainly derived from the analogs but not from coumarin itself.     

Hetero-annulated coumarins as new AChE/BuChE inhibitors: synthesis and biological evaluation

A series of chromene-fused coumarins known as 10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-diones 4a–o were synthesized through one-pot reaction of appropriate benzaldehydes, dimedone, and 4-hydroxycoumarin in the presence of nano-silica sulfuric acid under solvent-free condition in good yields. The in vitro anticholinesterase assay revealed that the 3-hydroxyphenyl analog 4e showed the highest inhibitory activity against both acetylcholinesterase and butyrylcholinesterase, possessing IC₅₀ values of 3.28 and 2.19?µM, respectively. The structure-activity relationships study demonstrated that the selectivity for acetylcholinesterase over butyrylcholinesterase could be modulated by introducing second hydroxyl or methoxy substituent on the para-position of the 3-hydroxyphenyl pendent group. The docking study of compound 4e with acetylcholinesterase confirmed π–π stacking interaction between the coumarin moiety and Trp279 as well as the formation of hydrogen bonding between hydroxyl group and Asn85.     

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

1. Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways.

2. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays.

3. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion.

4. The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

     

Determination of 7-hydroxycoumarin and its glucuronide and sulphate conjugates in liver slice incubates by capillary zone electrophoresis

The simultaneous detection of the phase I metabolite of coumarin, 7-hydroxycoumarin, and the two phase II metabolites, 7-hydroxycoumarin-glucuronide and 7-hydroxycoumarin-sulphate by capillary electrophoresis with UV detection using liver slice incubations was investigated. Separation was carried out on an untreated fused silica capillary with detection at 320 nm. Separation was achieved in under 6 min with a total run time of 8 min. Both phase two metabolites were produced following an in vitro incubation of liver slices in Krebs-Hanseleit buffer with 100 μM 7-hydroxycoumarin. Limits of detection were 5.52 μM (2 μg ml⁻¹) for the glucuronide, 2.21 μM (0.5 μg ml⁻¹) for the sulphate and 6.17 μM (1 μg ml)⁻¹ for 7-hydroxycoumarin. Mean inter- and intra-assay results are presented for all three analytes, respectively.     

A combined pharmacophore modeling, 3D QSAR,virtual screening,molecular docking,and ADME studies to identify potential HDAC8 inhibitors

The World Cancer Report 2014 shows that cancer is a leading cause of death globally, and cancer related death is likely to go up by about 70?% in the next two decades. Among several target receptors of cancer, histone deacetylases are the promising therapeutic target for many cancers. The current study deals with a primary goal of identification of novel non-hydroxamic acid histone deacetylase 8 inhibitors. In this context, six featured pharmacophoric hypotheses with hydrogen bond acceptors (A), hydrogen bond donor (D), and aromatic ring (R) were generated using 36 reported histone deacetylase 8 inhibitors. Virtual screening of database using two pharmacophore hypothesis AAAADR.161 and AAADDR.189 resulted 2000 hits each having fitness score >1.503. The pharmacophore AAADDR.189 yielded statistically significant three-dimensional quantitative structure-activity relationship model with training set (R ²: 0.9756, SD: 0.0680, F: 151.6, N: 26) and test set (Q ²: 0.6922, Pearson R: 0.8705, N: 10) molecules. The R ² _pred value of the model was 0.6951, which confirmed the good predictive ability of the model for external data set. Hits resulted from virtual screening and known inhibitors were subjected to molecular docking study to identify the binding affinity of inhibitors with active site amino acid residues. Finally, absorption, distribution, metabolism, and excretion study were undertaken to determine the drug likeness properties of identified hits. On the basis of fitness score, predicted activities, XP Glide score, interacting amino acid residues of known inhibitors and absorption, distribution, metabolism, and excretion properties, ten structurally diverse hits are reported in this paper as potential histone deacetylase 8 inhibitors which reduce the cost of histone deacetylase 8 inhibitor discovery and enhance the process prior synthesis.     

Synthesis,molecular docking,antiproliferative, and antimicrobial activity of novel pyrano[3,2-<Emphasis Type="Italic">c</Emphasis>]carbazole derivatives

A new series of pyrano[3,2-c]carbazole derivatives which are biologically valuable and synthetically challenging frameworks have been synthesized by domino Knoevenagel-hetero-Diels-Alder reaction. The key strategy involve copper iodide catalyzed cyclization of O-propargyl derivative of N-Boc-carbazole-3-carboxaldehyde with cyclic 1,3-diketones or pyrazol-5-ones to afford N-Boc-pyrano[3,2-c]carbazole derivatives. The antiproliferative activity of the all synthesized compounds on three cancer cell lines such as PANC 1 (pancreatic), HeLa (cervical), and MDA-MB-231 (breast cancer) was investigated. The results clearly demonstrated that compounds 11b, 11d, and 13d were displayed pronounced antriproliferative activity. In addition, antimicrobial activity of these compounds also assayed against three representative gram-positive organisms and gram-negative organisms. Specifically, compound 11c exhibited significant antimicrobial activity. Molecular docking studies revealed that compound 13d selectively occupy the colchicine binding site of the tubulin polymer.     

Ultrasound-promoted synthesis, biological evaluation and molecular docking of novel 7-(2-chloroquinolin-4-yloxy)-4-methyl-2H-chromen-2-one derivatives

A series of quinoline-based coumarin derivatives have been synthesized by one pot dehydrochlorination of 2,4-dichloroquinolines (1a–g); 7-hydroxy-4-methyl-2H-chromen-2-one (2) under ultrasonic irradiation method with high regio selectivity. All the synthesized compounds were characterized through spectral data and screened against representative antibacterial and antioxidant activities. Some of the compounds are found to be equipotent or more potent than that of standard drugs. Molecular docking studies show that the binding energy value of the compounds is very less than that of standard chloroquine and amodiaquine drugs.     

Characteristics of coumarin metabolism by liver microsomes from cobalt-pretreated mice

1. Cobalt pretreatment of male mice leads to increased liver microsomal mixed-function oxidation of various coumarin derivatives; 7-ethoxycoumarin, 7-hydroxycoumarin and coumarin are metabolized three to four times more rapidly than in control microsomes.

2. In Arrhenius plots of 7-ethoxycoumarin de-ethylation, the temperature at which phase transition occurs is not changed by cobalt pretreatment. However, at high temperature, above the transition point, the slope indicates that cobalt lowers the apparent activation energy of the reaction to 65% of control value.

3. Metabolism of 7-hydroxycoumarin by hepatic microsomes from cobalt-treated mice gave 3,7-, 6,7- and 7,8-dihydroxycoumarin, identified by g.l.c. and mass spectrometry.     

Design,synthesis, and molecular docking studies of novel quinoxaline derivatives as anticancer agents

As lung cancer was placed foremost part among other types of cancer in terms of mortality. Recent researches are widely focused on developing multi-targeted and site-specific targeted drug designs. In the present study, we designed and developed a series of quinoxaline pharmacophore derivatives as active EGFR inhibitors for the treatment of non-small cell lung cancer. The compounds were synthesized through a condensation reaction between hexane-3,4-dione and methyl 3,4-diaminobenzoate as a first step. Their structures were confirmed by ¹H-NMR, ¹³C-NMR, and HRMS spectroscopic methods. Cytotoxicity (MTT) were applied to determine anticancer activity of the compounds against breast (MCF7), fibroblast (NIH3 T3), and lung (A549) cell lines as EGFR inhibitors. Doxorubicin was used as a reference agent, compound 4i exhibited a significant effect among other derivatives with IC₅₀ = 3.902 ± 0.098 μM value against A549 cell line. The docking study showed that the best position on EGFR receptor could be observed with 4i . From the obtained evaluations of the designed series, compound 4i was a promising agent as EGFR inhibitor for further investigation and evaluation studies in the future.     

Grapefruit juice inhibits 7-hydroxylation of coumarin in healthy volunteers

The effect of grapefruit juice on the urinary excretion of 7-hydroxycoumarin after oral administration of 10 mg coumarin, as an index of cytochrome P450 dependent coumarin metabolism, has been investigated in an open, randomised cross over study in 13 healthy volunteers (7 female, 6 male).The percentage of 7-hydroxycoumarin found in urine was significantly decreased up to 8 h after simultaneous intake of 300 ml grapefruit juice. If the same volume of juice was swallowed 30 min prior to the administration of coumarin, 7-hydroxycoumarin excretion was delayed by up to 6 h. MRT_excr. of coumarin was 70 % extended by coadministration of grapefruit juice.It appears that grapefruit flavonoids inhibit cytochrome P450 2A dependent metabolic pathways. The mechanism of cytochrome P450 inhibition by these flavonoids is still poorly understood.     

Synthesis,biological screening and molecular docking studies of novel 4,6-pyrimidine derivatives as EGFR-TK inhibitors

Novel 4, 6-disubstituted pyrimidine derivatives (5–16) were synthesized in four steps starting from 2,4-dichloropyrimidine and screened for their cytotoxicity using brine shrimp (Artemia Salina) lethality bioassay. The compounds such as 6, 11, 14 and 15 were found to be more toxic. The compounds were also studied for in vitro anticancer properties using six different cancer cell lines viz SIHA, PANC-1, MDA-MB-231, IMR-32, DU145 and A549. The compound 14 was effective inhibitor of SIHA and DU145, whereas compound 16 in Panc 1 and A549, compound 7 in MDA-MB-231 and compound 6 in IMR 32 respectively. Molecular docking studies were carried out using an X-ray crystallographic structure of epidermal growth factor receptor tyrosine kinase to explore the possible mode of action of compounds as epidermal growth factor receptor tyrosine kinase inhibitors.     

Synthesis,biological evaluation,and molecular modeling of (<Emphasis Type="Italic">E</Emphasis>)-2-aryl-5-styryl-1,3,4-oxadiazole derivatives as acetylcholine esterase inhibitors

A library of 2,5-disubstituted 1,3,4-oxadiazole derivatives of (E)-2-aryl-5-(3,4,5-trimethoxystyryl)-1,3,4-oxadiazoles 4(a–o) and (E)-2-aryl-5-(2-benzo[d][1,3]dioxol-5-yl)vinyl)-1,3,4-oxadiazoles 5(a–q) were synthesized and evaluated for their in vitro acetylcholinesterase (AChE) inhibitory activity. All the synthesized compounds exhibited moderate to good inhibitory activity toward the AChE enzyme. Among the oxadiazole derivatives examined, compounds 4a, 4g, 5c, and 5m (IC₅₀ values of 24.89, 13.72, 37.65, and 19.63 μM, respectively) were found to be promising inhibitors of AChE. Molecular protein–ligand docking studies were examined for these compounds using GOLD docking software and their binding conformations were determined and the simultaneous interactions mode was also established for the potent derivatives.