Membrane phospholipids are susceptible to oxidation, which is involved in various pathological processes such as inflammation, atherogenesis, neurodegeneration, and aging. One enzyme that may help to remove oxidized phospholipids from cells is intracellular type II platelet-activating factor acetylhydrolase (PAF-AH (II)), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids. Overexpression of PAF-AH (II) in cells or tissues was previously shown to suppress oxidative stress-induced cell death. In this study we investigated the functions of PAF-AH (II) by generating PAF-AH (II)-deficient (Pafah2

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor γ-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific autoAbs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.

The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDC activity is tightly regulated by four members of a family of pyruvate dehydrogenase kinase isoforms (PDK1-4), which phosphorylate and inactivate PDC. Recently, the development of specific inhibitors of PDK4 has become an especially important focus for the pharmaceutical management of diabetes and obesity. In this study, crystal structures of human PDK4 complexed with either AMPPNP, ADP or the inhibitor M77976 were determined. ADP-bound PDK4 has a slightly wider active-site cleft and a more disordered ATP lid compared with AMPPNP-bound PDK4, although both forms of PDK4 assume open conformations with a wider active-site cleft than that in the closed conformation of the previously reported ADP-bound PDK2 structure. M77976 binds to the ATP-binding pocket of PDK4 and causes local conformational changes with complete disordering of the ATP lid. M77976 binding also leads to a large domain rearrangement that further expands the active-site cleft of PDK4 compared with the ADP- and AMPPNP-bound forms. Biochemical analyses revealed that M77976 inhibits PDK4 with increased potency compared with the previously characterized PDK inhibitor radicicol. Thus, the present structures demonstrate for the first time the flexible and dynamic aspects of PDK4 in the open conformation and provide a basis for the development of novel inhibitors targeting the nucleotide-binding pocket of PDK4.

Factor Xa (FXa) is a serine protease which plays a critical role in the coagulation cascade, serving as the point of convergence of intrinsic and extrinsic pathways.3-7) It represents an attractive target for anticoagulant drug development. [8][9][10][11][12][13][14] Most nonpeptide FXa inhibitors reported in the literature are dibasic compounds. Nagahara et al. 15,16) have reported the synthesis and evaluation of a series of bis(amidino)-derivatives, and through investigation, DX-9065a (1) [17][18][19][20][21] was found to be a selective FXa inhibitor. Further, YM-60828 (2), [22][23][24][25][26][27] which is closely related to 1 in terms of structure, was found to have a more potent inhibitory effect on FXa.On the other hand, as described in the preceding paper, 1) we have synthesized M55113 (3) as a potent inhibitor of FXa (IC 50 ϭ0.06 mM) with high selectivity for FXa over trypsin and thrombin. Compared with the structure of compound 3, the features of compound 1 and compound 2 include a functional acid group in the linker part of the molecule. According to the Xray structure of the complex of compound 1 in des- GlaFXa,28) it is clear that the nitrogen atom of Gln-192 forms a weak hydrogen bond with the acid function of compound 1.In order to obtain a compound which has powerful inhibitory activity on FXa, it is necessary to introduce a functional group into the position at which compound 3 and Gln-192 of FXa protein are expected to form a hydrogen bond.In this paper, we wish to report the continuing search for potent compounds that have a substituent at the piperazine and piperidine rings of compound 3. Thus, the structure-activity relationships of compound 3 derivatives were examined. ChemistryA key intermediate 7 was prepared from glycine ethyl ester hydrochloride, as illustrated in Chart 1. The compound 5, obtained by the reaction of glycine ethyl ester hydrochloride (4) with bromoacetaldehyde diethyl acetal in the presence of cesium carbonate (Cs 2 CO 3 ) 29,30) and sodium iodide, was treated with 6-chloro-2-naphthalenesulfonyl chloride under basic conditions, and the resulting sulfonamide 6 was hydrolyzed with aqueous trifluoroacetic acid (TFA) to give the formyl compound 7.Synthetic routes of compounds 12 and 17 are shown in Chart 2. Compound 9 was prepared by protection of the primary amino group with di-tert-butyl dicarbonate (Boc 2 O) and subsequent debenzylation of a secondary amino group with H 2 over Pd/C. Condensation of 9 with 4-chloropyridine 1-oxide under basic conditions gave the 4-piperidinopyridine 1-oxide derivative 10. Hydrogenolysis of 10 over Raney Ni 31) in methanol (MeOH), followed by treatment with HClMeOH, yielded compound 11. The final compound 12 was obtained smoothly by reductive condensation of 11 with the intermediate 7. 32)Reaction conditions in the preparation of compound 17 from compound 13 33) were not much different from the conversion of 8 to 12.Synthesis of 4-substituted piperidine derivatives containing another functional group (compound 18-20) in place of the hydroxyl...

Factor Xa (FXa), a trypsin-like serine protease, holds the central position that links the intrinsic and extrinsic mechanisms in the blood coagulation cascade. FXa is known to activate prothrombin to thrombin. Thrombin has several procoagulant functions that include the activation of platelets, feedback activation of other coagulation factors, and conversion of fibrinogen to insoluble fibrin clots. [4][5][6][7][8] Comparison of hirudin 9-13) (a thrombin inhibitor) and tick anticoagulant peptide [14][15][16][17][18][19][20] (a FXa inhibitor) suggests that inhibition of FXa may result in less bleeding risk, leading to a more favorable safety/efficacy ratio. [21][22][23][24] Direct inhibition to FXa has therefore emerged as an attractive strategy for the discovery of novel antithrombotic agents. [25][26][27][28][29][30][31] In preceding papers, 1,2) we reported the synthesis and evaluation of compounds in a series of 1-arylsulfonyl-3-piperazinone derivatives, of which M55113 (1) 4--2-piperazinecarboxylic acid were found to be potent inhibitors of FXa (IC 50 ϭ60 nM, 31 nM, 6 nM, respectively) with high selectivity for FXa over trypsin and thrombin.In more recent investigations, fixation of the conformation of testing compounds is believed to affect the strength of interaction between such compounds and the target enzyme. Accordingly, in the next stage of investigation our interest was focused on the synthesis of compounds containing a rigid structure in the central part of the compound (2, 3), and on comparison of the inhibitory activities of the compounds thus synthesized for FXa with those of previously reported compounds. A molecule with a spiro structure in between the piperidine moiety and piperazine moiety was therefore designed as the next candidate for further development of FXa inhibitor. The present paper concerns the synthesis of a series of compounds 4 with a spiro[5H-oxazolo[3,2-a]pyrazine-2(3H),4Ј-piperidin]-5-one skeleton, together with the FXa inhibitory activities of these new compounds. ChemistryFirst, acyclic precursor 9 was prepared as shown in Chart 1. Sulfonylamidation of glycine ethyl ester hydrochloride (5) with 6-chloro-2-naphthalenesulfonyl chloride (6) under traditional conditions yielded the corresponding naphthalenesulfonylamide 7. When 7 was treated with 1-acetoxy-3-chloroacetone (8) in DMF in the presence of potassium carbonate, 9 was obtained in good yield as expected.When 4-(aminomethyl)-1-benzyl-4-piperidinol (10) was allowed to react with acyclic precursor 9 under acidic conditions, a product 11 containing a spiro N,O-acetal structure on the piperazinone ring was obtained, as expected.The reaction pathway of the formation of the spiro skeleton from 9 and 10 is illustrated in Chart 2. In the first step, a Schiff base was formed by dehydration between a carbonyl group in 9 and a primary amino group in 10. Subsequent nucleophilic addition of a hydroxyl group to an azomethine Discovery Research Center, Mochida Pharmaceutical Co., Ltd.; 722 Jimba-aza-uenohara, Gotemba, Shizuoka 412-852...