Hayato Fukuda

Determination of ω-6 and ω-3 PUFA metabolites in human urine samples using UPLC/MS/MS.

The ω-6 and ω-3 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are the precursors of various bioactive lipid mediators including prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acid, isoprostanes, lipoxins, and resolvins (Rvs). These lipid mediators play important roles in various physiological and pathological processes. The quantitative determination of PUFA metabolites seems necessary for disease research and for developing biomarkers. However, there is a paucity of analytical methods for the quantification of ω-6 and ω-3 PUFA metabolites—the specialized pro-resolving mediators (SPMs) present in the human urine. We developed a method for the quantification of ω-6 and ω-3 PUFA metabolites present in human urine using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). The developed method shows good linearity, with a correlation coefficient >0.99 for all of the analytes. The validation results indicate that our method is adequately reliable, accurate, and precise. The method was successfully used to examine urine samples obtained from 43 healthy volunteers. We could identify 20 PUFA metabolites, and this is the first report of the quantitative determination of RvD1, 17(R)-RvD1, 11-dehydro thromboxane B3, RvE2, and 5(S)-HETE in human urine. The urinary 8-iso PGF2α and PGE2 levels were significantly higher in the men smokers than in the men nonsmokers (p < 0.05). In this study, we developed an accurate, precise, and novel analytical method for estimating the ω-6 and ω-3 PUFA metabolites, and this is the first report that the SPMs derived from EPA and DHA are present in human urine.

Palladium-Nanoparticle-Catalyzed 1,7-Palladium Migration Involving C–H Activation, Followed by Intramolecular Amination: Regioselective Synthesis of N1-Arylbenzotriazoles and an Evaluation of Their Inhibitory Activity toward Indoleamine 2,3-Dioxygenase

A sulfur-modified gold-supported palladium material (SAPd) has been developed bearing palladium nanoparticles on its surface. Herein, we report for the first time the use of SAPd to affect a Pd-nanoparticle-catalyzed 1,7-Pd migration reaction for the synthesis of benzotriazoles via C-H bond activation. The resulting benzotriazoles were evaluated in terms of their inhibitory activity toward indoleamine 2,3-dioxygenase.

Conformationally Restricted GABA with Bicyclo[3.1.0]hexane Backbone as the First Highly Selective BGT-1 Inhibitor.

On the basis of the three-dimensional diversity-oriented conformational restriction strategy using key chiral cyclopropane units, we previously identified 3 ((2S,3R)-4-amino-3,4-methanobutyric acid) with a chiral trans-cyclopropane structure as a γ-aminobutyric acid (GABA) transporter inhibitor selective for GABA transporter (GAT) subtypes GAT-3 and BGT-1 (betaine/GABA transporter-1). Further conformational restriction of 3 with the rigid bicyclo[3.1.0]hexane backbone led to the successful development of the first highly potent and selective BGT-1 inhibitor 4 (IC50 = 0.59 μM). The bioactive conformation of 3 for BGT-1 was also identified.

One‐Pot Ring‐Closing Metathesis/1,3‐Dipolar Cycloaddition through Assisted Tandem Ruthenium Catalysis: Synthesis of a Dye with Isoindolo[2,1‐a]quinoline Structure

Assisted tandem catalytic reactions are defined as catalyzed reaction sequences that proceed through more than one mechanism, but with just one precatalyst. In these reactions, the catalyst of the first cycle is transformed into the catalyst of the second cycle by a chemical initiator, for example, an additive that induces an organometallic transformation in situ. Over the past decade, several reaction sequences comprising an olefin-metathesis step and a subsequent nonmetathesis transformation of the newly generated carbon= carbon bond were developed. For example, olefin metathesis can be combined with hydrogenation or isomerization by in situ conversion of a Ru–carbene into a Ru–hydride. Ruthenium–alkylidene-catalyzed tandem transformations that were developed to date include olefin metathesis, followed by cyclopropanation, hydrovinylation, hydroarylation, the aza-Michael reaction, the hetero-Pauson– Khand reaction, or oxidation. On the other hand, [RuClCp*] and the “first generation” Grubbs metathesis complex A (Figure 1) catalyze an azide– alkyne cycloaddition reaction to give 1,5-substituted triazoles, and an intramolecular [3+2] cycloaddition of alk-5ynylidenecyclopropanes to give bicyclo[3.3.0]octane, respectively. In our search for novel and efficient Ru-catalyzed reactions, 11d, 14] we developed a one-pot ring-closing metathesis/oxidation methodology to produce various 2-quinolones from N-allyl-2-vinylaniline derivatives (Scheme 1). Oxidation of the a-methylene group of amines to the corresponding amides is very difficult. The key intermediate in this reaction might be the azomethine ylide I, equivalent to 1,2-dihydroquinoline. If the azomethine ylide is generated as the intermediate, we envisaged 1,3-dipolar cycloaddition of azomethine ylide from the 1,2-dihydroquinoline, generated by a ruthenium–alkylidene-catalyzed ringclosing metathesis (RCM) of an N-alkyl-N-allyl-2-vinylaniline derivative as the first step in the tandem reaction, with 1,3-dipolarophile would be proceeded by the active ruthenium species derived from the catalyst precursor, the ruthenium–alkylidene catalyst. Considering the importance of streamlining syntheses toward complex molecular targets, we report herein a new tandem process that combines a ruthenium-catalyzed RCM with a ruthenium-catalyzed intermolecular 1,3-dipolar cycloaddition to afford an isoindolo[2,1-a]quinoline core. These heterocycles are novel solution-processable p-conjugated small molecules whose color can be altered dramatically by exchanging a substituent on the core. Our tandem-catalysis strategy was first examined using Nallyl-N-benzyl-2-vinylaniline derivative 1a, dipolarophile 3a, and second-generation Grubbs catalyst B under various reaction conditions (Table 1). Compound 1a was first treated with B (10 mol %) in benzene (reflux) for 30 min to form the corresponding 1,2-dihydroquinoline derivative 2a. When Figure 1. Ruthenium alkylidenes. Cy = cyclohexyl, Mes = 2,4,6-trimethylphenyl.

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

Design and Synthesis of Cyclic ADP-4-Thioribose as a Stable Equivalent of Cyclic ADP-Ribose, a Calcium Ion-Mobilizing Second Messenger†

Cyclic ADP-ribose (cADPR, 1, Scheme 1), originally isolated from sea urchins by Lee and co-workers,1 is a general mediator of intracellular Ca2+ ion signaling.2 Analogues of cADPR have been extensively designed and synthesized3, 4 because of their potential usefulness for investigating the mechanisms of cADPR-mediated Ca2+ release and application as lead structures for the development of drug candidates.2

Identification of a Potent and Selective GPR4 Antagonist as a Drug Lead for the Treatment of Myocardial Infarction.

GPR4, a pH-sensing G protein-coupled receptor, is highly expressed in endothelial cells and may be activated in myocardial infarction due the decreased tissue pH. We are interested in GPR4 antagonists as potential effective pharmacologic tools and/or drug leads for the treatment of myocardial infarction. We investigated the structure-activity relationship of a known GPR4 antagonist 1 as a lead compound to identify 3b as the first potent and selective GPR4 antagonist, whose effectiveness was demonstrated in a mouse myocardial infarction model.

Synthesis of 7-Deaza-cyclic Adenosine-5'-diphosphate-carbocyclic-ribose and Its 7-Bromo Derivative as Intracellular Ca(2+)-Mobilizing Agents.

Cyclic ADP-carbocyclic-ribose (cADPcR, 3) is a biologically and chemically stable equivalent of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. We became interested in the biological activity of the 7-deaza analogues of cADPcR, i.e., 7-deaza-cADPcR (7) and its 7-bromo derivative, i.e., 7-deaza-7-Br-cADPcR (8), because 7-deazaadenosine is an efficient bioisostere of adenosine. The synthesis of 7 and 8 required us to construct the key N1-carbocyclic-ribosyl-7-deazaadenosine structure. Therefore, we developed a general method for preparing N1-substituted 7-deazaadenosines by condensing a 2,3-disubstituted pyrrole nucleoside with amines. Using this method, we prepared the N1-carbocyclic ribosyl 7-deazaadenosine derivative 10a, from which we then synthesized the target 7-deaza-cADPcR (7) via an Ag(+)-promoted intramolecular condensation to construct the 18-membered pyrophosphate ring structure. The corresponding 7-bromo derivative 8, which was the first analogue of cADPR with a substitution at the 7-position, was similarly synthesized. Biological evaluation for Ca(2+)-mobilizing activity in the sea urchin egg homogenate system indicated that 7-deaza-cADPcR (7) and 7-deaza-7-Br-cADPcR (8) acted as a full agonist and a partial agonist, respectively.

Entry to Chiral 1,1,2,3-Tetrasubstituted Arylcyclopropanes by Pd(II)-Catalyzed Arylation via Directing Group-Mediated C(sp3)-H Activation

Here we report the construction of highly functionalized chiral 1,1,2,3-tetrasubstituted arylcyclopropanes of medicinal chemical importance using Pd(II)-catalyzed arylation via directing group-mediated C(sp3)-H activation. The key aspect for the effective arylation was control of the substrate conformation based on the characteristic steric and stereoelectronic features of cyclopropane by manipulating the protecting group at the hydroxyl. The arylation with good functional group tolerance is pivotal as the first entry to chiral 1,1,2,3-tetrasubstituted arylcyclopropanes with wide variety of aryl groups, including heteroaryl groups.

Preparation of Chiral Bromomethylenecyclopropane and Its Use in Suzuki–Miyaura Coupling: Synthesis of the Arylmethyl-(Z)-cyclopropane Structure Core

A preparative method for an optically active bromomethylenecyclopropane unit and its practical conversion to (Z)-cyclopropane-containing chiral compounds via Suzuki-Miyaura coupling were established.