Microwave-assisted synthesis of medicinally relevant indoles.

Indoles represent an important structural class in medicinal chemistry with broad spectrum of biological activities. The synthesis of indoles, therefore, has attracted enormous attention from synthetic chemists. Microwave methods for the preparation of indole analogs have been developed to speed up the synthesis, therefore, microwave assisted organic synthesis (MAOS) in controlled conditions is an invaluable technique for medicinal chemistry. In this review, indole forming classical reactions such as Fischer, Madelung, Bischler-Mohlau, Batcho-Leimgruber, Hemetsberger-Knittel, Graebe-Ullmann, Diels-Alder and Wittig type reactions using microwave radiation has been summarized. In addition, metal mediated cyclizations along with solid phase synthesis of indoles have been discussed.

Assessing potential effects of inulin and probiotic bacteria on Fe availability from common beans (Phaseolus vulgaris L.) to Caco-2 cells.

Inulin, a prebiotic, may enhance intestinal Fe absorption. Our objective was to assess the effects of supplemental inulin and 2 probiotic bacteria (B. infantis and L. acidophillus) on Fe availability to Caco-2 cells from common white and red beans (Phaseolus vulgaris L.). Cooked beans were mixed or not with supplemental inulin (4%, w/w), and then subjected to simulated gastrointestinal digestion (pepsin, pH 2; pancreatin, pH 7.2). Subsequently, the digests were incubated overnight with and without B. infantis or L. acidophilus. Ferritin formation in Caco-2 cells was used to evaluate Fe uptake. Total soluble phenols (Folin-Ciocalteau) and phytate (HPLC-electrochemical detection) were quantified, and the flavonoids profile (HPLC-PDA/UV detection) was monitored in the digests. Supplemental inulin did not affect Fe uptake from white nor red beans. Incubation with B. infantis increased total soluble phenols (TSP) in the digests and decreased Fe uptake. Incubation with L. acidophilus decreased TSP in the digest and increased Fe uptake. Variations in Fe uptake were not associated with soluble phytate concentrations in the digests. The largest change in flavonoids profile were found in the digests incubated with L. acidophilus, which decreased the soluble concentration of astragalin (kaempferol-3-O-glucoside). These results suggest that certain probiotics could increase Fe uptake from common beans.

Glutamine nitrogen and ammonium nitrogen supplied as a nitrogen source is not converted into nitrate nitrogen of plant tissues of hydroponically grown pak-choi (Brassica chinensis L.).

Many vegetables, especially leafy vegetables, accumulate NO(-) (3)-N in their edible portions. High nitrate levels in vegetables constitute a health hazard, such as cancers and blue baby syndrome. The aim of this study was to determine if (1) ammonium nitrogen (NH(+) (4)-N) and glutamine-nitrogen (Gln-N) absorbed by plant roots is converted into nitrate-nitrogen of pak-choi (Brassica chinensis L.) tissues, and (2) if nitrate-nitrogen (NO(-) (3)-N) accumulation and concentration of pak-choi tissues linearly increase with increasing NO(-) (3)-N supply when grown in nutrient solution. In experiment 1, 4 different nitrogen treatments (no nitrogen, NH(+) (4)-N, Gln-N, and NO(-) (3)-N) with equal total N concentrations in treatments with added N were applied under sterile nutrient medium culture conditions. In experiment 2, 5 concentrations of N (from 0 to 48 mM), supplied as NO(-) (3)-N in the nutrient solution, were tested. The results showed that Gln-N and NH(+) (4)-N added to the nutrient media were not converted into nitrate-nitrogen of plant tissues. Also, NO(-) (3)-N accumulation in the pak-choi tissues was the highest when plants were supplied 24 mM NO(-) (3)-N in the media. The NO(-) (3)-N concentration in plant tissues was quadratically correlated to the NO(-) (3)-N concentration supplied in the nutrient solution.

Lysophosphatidic acid-induced platelet shape change revealed through LPA(1-5) receptor-selective probes and albumin.

Lysophosphatidic acid (LPA), a component of mildly-oxidized LDL and the lipid rich core of atherosclerotic plaques, elicits platelet activation. LPA is the ligand of G protein-coupled receptors (GPCR) of the EDG family (LPA(1-3)) and the newly identified LPA(4-7) subcluster. LPA(4), LPA(5) and LPA(7) increase cellular cAMP levels that would induce platelet inhibition rather than activation. In the present study we quantified the mRNA levels of the LPA(1-7) GPCR in human platelets and found a rank order LPA(4) = LPA(5) > LPA(7) > LPA(6) = LPA(2) >> LPA(1) > LPA(3). We examined platelet shape change using a panel of LPA receptor subtype-selective agonists and antagonists and compared them with their pharmacological profiles obtained in heterologous LPA(1-5) receptor expression systems. Responses to different natural acyl and alkyl species of LPA, and octyl phosphatidic acid analogs, alpha-substituted phosphonate analogs, N-palmitoyl-tyrosine phosphoric acid, N-palmitoyl-serine phosphoric acid were tested. All of these compounds elicited platelet activation and also inhibited LPA-induced platelet shape change after pre-incubation, suggesting that receptor desensitization is likely responsible for the inhibition of this response. Fatty acid free albumin (10 microM) lacking platelet activity completely inhibited platelet shape change induced by LPA with an IC(50) of 1.1 microM but had no effect on the activation of LPA(1,2,3,&5) expressed in endogenously non-LPA-responsive RH7777 cells. However, albumin reduced LPA(4) activation and shifted the dose-response curve to the right. LPA(5) transiently expressed in RH7777 cells showed preference to alkyl-LPA over acyl-LPA that is similar to that in platelets. LPA did not increase cAMP levels in platelets. In conclusion, our results with the pharmacological compounds and albumin demonstrate that LPA does not induce platelet shape change simply through activation of LPA(1-5), and the receptor(s) mediating LPA-induced platelet activation remains elusive.

Dietary inulin affects the expression of intestinal enterocyte iron transporters, receptors and storage protein and alters the microbiota in the pig intestine.

Inulin, a linear beta fructan, is present in a variety of plants including chicory root and wheat. It exhibits prebiotic properties and has been shown to enhance mineral absorption and increase beneficial bacteria in the colon. The aim of the present study was to assess the effect of dietary inulin on the gene expression of selected intestinal Fe transporters and binding proteins. Anaemic piglets at age 5 weeks were allocated to a standard maize-soya diet (control) or the same diet supplemented with inulin at a level of 4 %. After 6 weeks, the animals were killed and caecum contents and sections of the duodenum and colon were removed. Segments of the genes encoding for the pig divalent metal transporter 1 (DMT1) and duodenal cytochrome-b reductase (Dcytb) were isolated and sequenced. Semi-quantitative RT-PCR analyses were performed to evaluate the expression of DMT1, Dcytb, ferroportin, ferritin, transferrin receptor (TfR) and mucin genes. DMT1, Dcytb, ferroportin, ferritin and TfR mRNA levels in duodenal samples were significantly higher in the inulin group (P < or = 0.05) compared with the control. In colon, DMT1, TfR and ferritin mRNA levels significantly increased in the inulin group. Additionally, the caecal content microflora was examined using 16S rDNA targeted probes from bacterial DNA. The Lactobacillus and Bifidobacterium populations were significantly increased in the inulin group (P < or = 0.05) compared with the control group. These results indicate that dietary inulin might trigger an up regulation of genes encoding for Fe transporters in the enterocyte. The specific mechanism for this effect remains to be elucidated.

Rapid screening of doping agents in human urine by vacuum MALDI-linear ion trap mass spectrometry.

Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.

Antagonism by imidazoline-type drugs of muscarinic and other receptors in the guinea-pig ileum.

1 Several imidazolines were examined for the antagonism of muscarinic (M3) and other receptors on the isolated ileum of guinea-pig. The effect of the muscarinic agonist, carbachol was competitively antagonized by oxymetazoline at 10(-5) m. A dissociation constant (KB) of 3.6 microm for the antagonist was calculated. At higher concentrations, 3 x 10(-5) and 10(-4) m, of the antagonist, the agonist dose-response curve was shifted to the right with a decrease in the maximum effect. Thus, a non-competitive block occurred at higher concentrations of oxymetazoline. Blockade of histamine H, and serotonin receptor-mediated responses by oxymetazoline were also of a non-competitive type. 2 Naphazoline at 10(-4) m shifted the dose-response curves of carbachol and serotonin to the right by two- and 15-fold, respectively. The maximum contraction of the agonist was not affected. Tolazoline also had a weak antihistaminic activity. At similar concentration; tetrahydrozoline clonidine and phentolamine at 10(-5) m produced two-, three- and four-fold shift of the carbachol dose-response curve without significant changes in the maxima. Neither methoxamine, p-amino-clonidine nor cimetidine blocked the responses of carbachol. 3 The isosteric nature of the alpha-adrenoceptor agonist, oxymetazoline and some imidazolines with carbachol, in part, explains its molecular competition at the muscarinic M3 receptor of the guinea-pig ileum. Surprisingly, contractile effects of carbachol (M3), histamine (H1) or serotonin (5HT3/5HT4) were not influenced by methoxamine, tetrahydrozoline, p-amino clonidine and cimetidine.

Calcium, zinc, and iron bioavailabilities from a commercial human milk fortifier: a comparison study.

Adding human milk fortifiers (HMF) to human milk (HM) is one way of overcoming the nutrient deficits found in the latter. In this study, the bioavailabilities of calcium, zinc, and iron in S-26/SMA HMF added to HM were compared with those in HM fortified with various bovine milk proteins: alpha-lactalbumin, colostrum, caseinate, casein phosphopeptides, and whey protein concentrate. The bioavailability of each mineral was assessed using an in vitro digestion/Caco-2 cell culture model. Calcium and zinc uptake by the cells was traced with radioisotopes; iron uptake was assessed via cell ferritin levels. Samples were prepared on an equal protein content basis and with added calcium, but no zinc or iron was added. Results revealed that calcium uptake from HM + S-26/SMA was not different from any of the HM fortified with the bovine milk proteins, except for unfortified HM and HM + colostrum in which calcium uptake was significantly lower (-89 and -38%, respectively). Uptake of zinc and iron were significantly higher for HM + S-26/SMA than for the other HM + fortifiers.

Pharmacokinetics of S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro- 3-trifluoromethyl-phenyl)-propionamide in rats, a non-steroidal selective androgen receptor modulator.

1: S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide (also known as S-4) is a non-steroidal selective androgen receptor modulator demonstrating tissue-selective androgenic and anabolic effects. The purpose of the present study was to examine the systemic pharmacokinetics, elimination and oral bioavailability of S-4 in rats. 2: Thirty-five male Sprague-Dawley rats weighing approximately 250 g were randomly assigned to one of seven treatment groups. Intravenous doses of 0.5, 1, 10, and 30 mg kg(-1) were given via a jugular catheter. Oral doses of 1, 10 and 30 mg kg(-1) were administered via gavage. Plasma concentrations were determined using a validated high-performance liquid chromatography or by a high-performance liquid chromatography/mass spectrometry method. 3: Clearances ranged between 1.0 and 2.1 ml min(-1) kg(-1) and varied with dose. The volume of distribution was approximately 0.448 l kg(-1) in all treatment groups. Oral bioavailability was also dose dependent, with the lower doses showing complete oral bioavailability. The half-life of S-4 over the dose range tested was between 2.6 and 5.3 h. 4: It was demonstrated that S-4 is rapidly absorbed, slowly cleared, and has a moderate volume of distribution in rats. The pharmacokinetics and oral bioavailability of S-4 indicate that it is an excellent candidate for clinical development.

Structural elements of the osteopontin SVVYGLR motif important for the interaction with alpha(4) integrins.

The osteopontin SVVYGLR motif binds the integrins alpha(4)beta(1) and alpha(9)beta(1). We show that alpha(4)beta(7) also interacts with this motif and that an SVVYGLR-OH peptide antagonises the alpha(4)beta(7) MAdCAM interaction. The important elements of this motif required to bind alpha(4)beta(1) and alpha(4)beta(7) were probed using a series of mutated peptides based around SVVYGLR. Leu167 is important for the interaction with alpha(4) integrins, as is the C-terminal carboxylic acid of Arg168 exposed by thrombin cleavage. The importance of the acidic group means that SVVYGLR has structural elements in common with other alpha(4) integrin-binding motifs and suggests why thrombin cleavage activates this motif.

Comparison of relative coronary Doppler flow velocity reserve to stress myocardial perfusion imaging in patients with coronary artery disease.

To compare relative coronary artery vasodilator reserve (rCVR = CVRtarget/CVRreference) to myocardial perfusion stress imaging, 48 patients with coronary artery stenoses (61% +/- 16%; mean, +/- SD; range, 30%-91%) had measurements of target and reference vessel CVR (Doppler-tipped guidewire). rCVR was computed and compared to stress 201thallium or (99m)technetium-sestamibi myocardial tomography. Compared to 24 patients with negative stress imaging studies, 24 patients with positive stress studies had angiographically more severe stenoses (74% +/- 13% vs. 44% +/- 24%; P = 0.0005) with lower CVR(target) (1.68 +/- 0.55 vs. 2.46 +/- 0.74; P = 0.002) and lower rCVR (0.72 +/- 0.22 vs. 1.0 +/- 0.26; P < 0.003). Based on receiver-operator characteristic (ROC) cut points (CVR > 1.9; rCVR > 0.75), compared to CVR, rCVR had similar agreement (Kappa 0.54 vs. 0.50), sensitivity (63% vs. 71%), specificity (88% vs. 83%), and positive predictive value (83% vs. 81%) with myocardial perfusion tomography. A concordant CVRtarget/rCVR only slightly increased sensitivity, specificity, and positive predictive values (77%, 90%, and 87%, respectively). Although rCVR, like CVR, correlates with stress myocardial perfusion imaging results, rCVR did not have significant incremental prognostic value over CVR alone for myocardial perfusion imaging. However, rCVR does provide additional information regarding the status of the microcirculation in patients with coronary artery disease and complements the CVR for lesion assessment.

Significance of hydrogen bonding at the S(1)' subsite of calpain I.

alpha-Ketohydroxamates were synthesized as bioisosteres of alpha-ketoamides. The alpha-ketohydroxamates were generally more potent than the corresponding alpha-ketoamides. The potency of the compounds suggests that hydrogen bonding and steric bulk of substituents on the nitrogen atom of the ketoamide moiety influence calpain inhibition.

Iron loading and large doses of intravenous ascorbic acid promote lipid peroxidation in whole serum in guinea pigs.

Large doses of ascorbic acid may mobilise Fe from Fe-binding proteins in vivo which in turn could catalyse lipid peroxidation, a process associated with degenerative diseases. This hypothesis was tested in vitro in the serum of Fe-loaded animals. Eighteen male guinea pigs weighing about 500 g on arrival were allocated to two groups of nine. Fe loading was induced in one group by two intraperitoneal injections of 200 mg iron dextran given on days 1 and 5. Blood (6 ml) was drawn from all animals on day 12 by cardiac puncture. Serum and LDL were separated. Serum was tested for loosely-bound Fe (bleomycin assay) and lipid peroxidation (thiobarbituric acid reactive substances (TBARS) assay) and LDL for susceptibility to in vitro oxidation (TBARS and conjugated diene assays). On day 12, another intraperitoneal injection of 200 mg iron dextran was given to the animals in the Fe-loaded group. On day 19, all animals were given 75 mg ascorbic acid by intraperitoneal injection. Blood (6 ml) was drawn 4 h later by cardiac puncture. Serum and LDL assays were repeated. Ascorbic acid increased loosely-bound Fe and in vitro oxidation in the serum from animals of the Fe-loaded group but not in the serum from animals of the control group. Susceptibility of LDL to in vitro oxidation increased after the ascorbic acid injection in the control group but there was no further increase in the Fe-loaded group. These data suggest that large doses of ascorbic acid promote Fe mobilisation and in vitro oxidation in the serum of Fe-loaded animals.

Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry.

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.

Homology modeling using multiple molecular dynamics simulations and docking studies of the human androgen receptor ligand binding domain bound to testosterone and nonsteroidal ligands.

To facilitate the rational design of novel and more potent androgen receptor ligands, three-dimensional models for the human androgen receptor ligand binding domain bound to testosterone have been developed. These models of the androgen receptor were based on the crystal structure of the highly homologous human progesterone receptor ligand binding domain. The homology modeled androgen receptor was refined using unrestrained multiple molecular dynamics simulations in explicit solvent. Key H-bonding partners with the 17-hydroxy group and 3-keto group of testosterone are Asn705 and Thr877, and Gln711 and Arg752, respectively. These models show the presence of a unique unoccupied cavity within the androgen receptor binding pocket which may be valuable in the development of novel selective androgen receptor ligands. A qualitative analysis of amino acid mutations within the hAR binding pocket that affect ligand binding are consistent with these androgen receptor models. In addition to testosterone, the binding modes of several hydroxyflutamide-like nonsteroidal ligands for the androgen receptor are investigated using flexible docking with FlexX followed by refinement of the initial complexes with molecular dynamics simulations. These docking studies indicate that Asn705 is an important determinant in binding hydroxyflutamide and its derivatives by participating in H-bond interactions with the alpha-hydroxy moiety of these ligands. In addition, the nitro functionality mimics the 3-keto group of the natural ligand testosterone and is involved in H-bonding interactions with Gln711 and Arg752. From these docking studies, we suggest a mechanism for the enantioselective binding of chiral hydroxyflutamide derivatives and expand upon the previously reported structure-activity relationship for hydroxyflutamide and its derivatives.