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Background We reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis. Method We induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca2+]i), and cell viability in these cells, when incubated with cholecystokinin (CCK). Results The expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca2+]i increase in AR42J-S100g cells. These effects were weak in AR42J-Anxa10 cells. Cell viability was not changed by incubation with cerulein. Conclusion In cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca2+ buffer in acute pancreatitis.

In order to clarify the role of the 1-substituent of quinazoline derivatives in their inhibitory activity against poly(ADP-ribose) polymerase (PARP), two novel inhibitors, 1 [8-hydroxy-1-(3-morpholinopropyl)-quinazoline-2,4(1H,3H)-dione] and 2 [8-hydroxy-1-(3-phenoxypropyl)-quinazoline-2,4(1H,3H)-dione], were synthesized and subjected to X-ray crystal analysis in complex with the PARP C-terminal catalytic domain (PARP-CD), which requires NAD+ coenzyme for biological function. The nicotinamide-mimicking part of the quinazoline skeleton of 1 and 2 were both located at the nicotinamide subsite of the NAD+-binding pocket in the same manner as previously reported inhibitors: three hydrogen bonds [(Gly-863)NH-O12, (Gly-863)O-HN3 and (Ser-904)O(gamma)-O12] and stacking interaction between the Tyr-907 phenol and the quinazoline ring. On the other hand, the N-morpholinoprop-3-yl moiety introduced at the 1-position of the quinazoline ring in 1 bridged the large gap between the donor site and the acceptor site through a (Met-890)NH-O20(morpholine) hydrogen bond, where the donor and the acceptor sites are classified as the binding sites of NAD+ and the ADP moiety of the poly(ADP-ribose) chain, respectively. In contrast, the N-phenoxyprop-3-yl moiety in 2 formed hydrophobic interactions close to the adenosine-binding site of NAD+, unlike the hydrogen bond such as in 1. As the inhibitory activities of 1 and 2 for PARP were much more potent than those of the unsubstituted nicotinamide analogues, these results suggest that the occupation of the proximal region of the ADP phosphate-and adenosine-binding subsite of the donor site or that of the gap between the donor and the acceptor site by the 1-substituent of quinazoline may increase the inhibitory activity considerably. The nearly equal inhibitory activities of 1 and 2, despite of their different binding modes at the active site, indicate that this 1-substituent is promising in improving the bioavailability of the inhibitor without compromising its inhibitory activity.

A novel sulfenylation method induced by aromatization of quinone mono-O,S-acetals is described. These sulfenylation reagents readily react with silyl enolethers or electron rich aromatic compounds to give sulfenylation products under mild conditions. In particular, O,S-acetal 2j, which possesses a pentafluorophenylthio function, is the most effective reagent from the standpoint of the adaptability for various substrates.

The Ile-Pro sequence of CA074, potent covalent-type inhibitor, is necessary to exhibit the specificity for cathepsin B, but not for papain. In order to elucidate how its sequence binds to papain and why such binding does not exhibit the specificity for papain at the atomic level, two CA074-related compounds, 1 (N-(L-3-carboxyloxirane-2-carbonyl)-L-isoleucyl-L-proline) and 2 (N-(L-3-carboxyloxirane-2-carbonyl)-L-isoleucyl-diethylamide), were designed and their structure--inhibitory activity relationship was investigated by the X-ray crystal analyses of the complexes with papain. The Ile-Pro moiety of 1 was located at the S2 and S3 subsites consisting of Val-133, Val-157, and Asp-158 and of Tyr-61, Gly-66, and Tyr-67 residues of papain, respectively, which is in contrast with the binding of CA074 to S'n (n = 1 approximately 2) subsites in the complex with cathepsin B. Although 2 in the complex with papain showed the similar binding pattern to 1, its inhibitory activity was about two-fold higher than of 1, suggesting the importance of tight S3-P3 hydrophobic interaction for the activity. The difference of the substrate specificity between papain and cathepsin B has also been discussed based on the X-ray results of the present and cathepsin B-inhibitor complexes.

A new platelet aggregation inhibitor, bassiatin, was isolated from the cultured broth of Beauveria bassiana which had been isolated from a soil sample collected in Yunnan province, China. The structure of bassiatin was determined to be (3S, 6R)-4-methyl-6-(1-methylethyl)-3-phenylmethyl-1, 4-perhydrooxazine-2,5-dione by NMR analysis, X-ray crystallographic analysis and chemical synthesis. Bassiatin inhibited ADP-induced aggregation of rabbit platelets with the IC50 being 1.9 x 10(-4) M.

In order to investigate the stereo specificity of papain Sn subsites (n = 1-4) at the atomic level, two kinds of covalent-type inhibitors were designed based on the previous results on papain-E-64 and papain-E-64-c interactions, and their complex crystals with papain were analyzed by X-ray diffraction. The results show that the hydrophobic regions consisting of Val-133, Val-157 and Asp-158 and of Tyr-61, Gly-66 and Tyr-67 residues interact with the hydrophobic P2 and P3 side chains of inhibitors, thus indicating the function of the former and latter binding pockets as S2 and S3 subsites, respectively. Furthermore, the X-ray analysis suggests that the papain has no definite Sn subsite of nor = 4, and the S3-P3 hydrophobic interaction is significantly affected by the Pn side chain (nor = 4) of both the substrate and the inhibitor.

Keramaphidin B (1), a novel cytotoxic alkaloid possessing a 1,4-etheno-2,7-decahydronaphthylidine core with two macrocyclic rings has been isolated from an Okinawan marine sponge Amphimedon sp., and the structure was elucidated by extensive 2D NMR investigation as well as a single crystal X-ray analysis. Keramaphidin B (1) might be a plausible biogenetic precursor of manzamine alkaloids and a very important key compound to clarify the biogenetic path.

Hantzsch cyclization of cyanoethyl 3-aminocrotonate and (E,Z)-4-dialkoxymethyl-2-benzylidene-acetoacetates (5a,b) afforded 3,4-trans-2-hydr

Enantiomeric (+)- and (–)-methy3-nitrooxypropyl 1,4-dihydro-2,6-dimethyl-4-(3- nitrophenyl) pyridine-3,5-dicarboxylate 1 were synthesized by esterification of the optically active monocarboxylic acids (+)-6 and (–)-6, which are available from racemate (±)-6 by optical resolution using cinchonidine and cinchonine. The absolute configuration of the key intermediates (S)-(+)-6 and (R)-(–)-6, was also unambiguously determined by the comparison with optical active (+)- and (–)-1 derived from (R)-(–)- and (S)-(+)-7 and (+)- and (–)-6, and X-ray crystallographic analysis of bromoethyl ester (R)-(–)-8 prepared from the acid (S)-(+)-7.

Hantzsch cyclization of cyanoethyl 3-aminocrotonate and (E,Z)-4,4-dialkoxy-2-benzylideneacetoacetates 12a–h afforded the corresponding 3,4-trans-2-hydroxy-1,2,3,4-tetrahydropyridines 14a–h with high stereoselectivity. 1H NMR and X-ray analyses of compound 14a established the configuration of 3-H and 4-H as trans and that of 3-H and 2-OH as trans also.

A new platelet aggregation inhibitor, bassiatin, was isolated from the cultured broth of Beauveria bassiana which had been isolated from a soil sample collected in Yunnan province, China. The structure of bassiatin was determined to be (3S, 6R)-4-methyl-6-(1-methylethyl)-3-phenylmethyl-1, 4-perhydrooxazine-2,5-dione by NMR analysis, X-ray crystallographic analysis and chemical synthesis. Bassiatin inhibited ADP-induced aggregation of rabbit platelets with the IC50 being 1.9 x 10(-4) M.

A novel sulfenylation method induced by aromatization of quinone mono-O,S-acetals is described. These sulfenylation reagents readily react with silyl enolethers or electron rich aromatic compounds to give sulfenylation products under mild conditions. In particular, O,S-acetal 2j, which possesses a pentafluorophenylthio function, is the most effective reagent from the standpoint of the adaptability for various substrates.

A new neuritogenic compound NGA0187 was isolated from the fermentation broth of Acremonium sp. TF-0356. The structure of NGA0187 was determined by means of spectroscopic analysis and X-Ray diffraction. NGA0187 induced significant neurite outgrowth in PC12 cells. However, survival effect of NGA0187 on the primary culture of cerebral cortical neurons was not observed.

In order to investigate the binding mode of E64-c (a synthetic cysteine proteinase inhibitor) to papain at the atomic level, the crystal structure of the complex was analysed by X-ray diffraction at 1.9 A (1 A is expressed in SI units as 0.1 nm) resolution. The crystal has a space group P2(1)2(1)2(1) with a = 43.37, b = 102.34 and c = 49.95 A. A total of 21,135 observed reflections were collected from the same crystal, and 14811 unique reflections of up to 1.9 A resolution [Fo > 3 sigma(Fo)] were used for the structure solution and refinement. The papain structure was determined by means of the molecular replacement method, and then the inhibitor was observed on a (2 magnitude of Fo-magnitude of Fc) difference Fourier map. The complex structure was finally refined to R = 19.4% including 207 solvent molecules. Although this complex crystal (Form II) was polymorphous as compared with the previously analysed one (Form I), the binding modes of leucine and isoamylamide moieties of E64-c were significantly different from each other. By the calculation of accessible surface area for each complex atom, these two different binding modes were both shown to be tight enough to prevent the access of solvent molecules to the papain active site. With respect to the E64-c-papain binding mode, molecular-dynamics simulations proposed two kinds of stationary states which were derived from the crystal structures of Forms I and II. One of these, which corresponds to the binding mode simulated from Form I, was essentially the same as that observed in the crystal structure, and the other was somewhat different from the crystal structure of Form II, especially with respect to the binding of the isoamylamide moiety with the papain S subsites. The substrate specificity for the papain active site is discussed on the basis of the present results.

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.

The novel 6-O-methyl tricyclic ketolides TE-802 and its analogs were synthesized by two successive cyclization reactions, 11,12-cyclic carbamate formation by intramolecular Michael addition and 9,11-diazaheptene ring construction by intramolecular dehydration reaction. These new tricyclic ketolides exhibited good in vitro antibacterial activity against not only erythromycin-susceptible strains but also erythromycin-resistant Staphylococcus aureus and Streptococcus pneumoniae, which are problematic pathogens of nosocomial and community-acquired respiratory tract infections, respectively.

This paper focuses on the inhibitory mechanism of E-64 and its derivatives (epoxysuccinyl-based inhibitors) with some cysteine proteases, based on the binding modes observed in the x-ray crystal structures of their enzyme-inhibitor complexes. E-64 is a potent irreversible inhibitor against general cysteine proteases, and its binding modes with papain, actinidin, cathepsin L, and cathepsin K have been reviewed at the atomic level. E-64 interacts with the Sn subsites of cysteine proteases. Although the Sn-Pn (n = 1-3) interactions of the inhibitor with the main chains of the active site residues are similar in respective complexes, the significant difference is observed in the side-chain interactions of S2-P2 and S3-P3 pairs because of different residues constituting the respective subsites. E-64-c and CA074 are representative derivatives developed from E-64 as a clinical usable and a cathepsin B-specific inhibitors, respectively. In contrast with similar binding/inhibitory modes of E-64-c and E-64 for cysteine proteases, the inhibitory mechanism of cathepsin B-specific CA074 results from the binding to the Sn' subsite.

The effect of pulverization on the dehydration behavior of 2-[(2-methylimidazol-1-yl)methyl]benzo[f]thiochromen-1-one monohydro-chloride hemihydrate (GK-128), a newly developed serotonin3 receptor antagonist, was studied using powder X-ray diffraction analysis, thermo-gravimetry (TG), and differential scanning calorimetry (DSC). The crystalline forms of GK-128 obtained by pulverizing with a jet mill and an agate mortar were each confirmed to be the same as that of intact GK-128, since the powder X-ray diffraction patterns of the two prepared samples were identical with that of intact GK-128. However, after pulverization by jet mill, the dehydration temperature of GK-128 was markedly lowered and the DSC endothermic peak due to dehydration disappeared. The activation energy for dehydration, calculated by the Ozawa method using TG data, decreased with decreasing particle size and/or crystallinity of GK-128 crystals, e.g., the activation energies for dehydration of intact and jet-milled GK-128 were 128.1 and 75.9 kJ/mol, respectively. The results of comparison of powder X-ray diffraction patterns of pulverized GK-128 and intact GK-128 and of determination of the crystal structure of GK-128 suggested that water molecules could be removed easily along the c-axis of GK-128 crystals.

2-Acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanonic acid (KE-298) is an antirheumatic agent. To elucidate the effects of optically active KE-298, we resolved the racemic acid and obtained the two optical isomers. (+)-KE-298 was converted to the 4-bromobenzyl ester derivative and the absolute structure was confirmed as (S) by X-ray crystallographic analysis. The pharmacological activities of the optical isomers and racemic KE-298 were compared by using the characteristic tests for KE-298. Though (+)-KE-298 showed a stronger suppressive effect on rat adjuvant arthritis than (-)-KE-298, no difference between the two isomers was detected in in vitro tests (enhancing effect on lymphocyte transformation, IL-1 antagonistic effect).

A series of (nitrooxy)alkyl apovincaminates has been synthesized and evaluated for their effects on vertebral and femoral blood flow. These derivatives were prepared from apovincaminic acid (4). In cerebral circulation, compound 5 (0.03-1.0 mg/kg iv) caused a dose-dependent increase in cerebral blood flow (CerBF) without affecting the blood pressure. It was more potent than vinpocetine (2). The structures of 2 and 5, determined by X-ray crystallography, showed differences in the electrostatic potential image and in the conformation of the ethyl group at the 16-position.

Two new cembrane-type diterpenoids, sarcodiol (1) and 3,4-epoxysarcophytonin (4), were isolated from Okinawan soft coral of the genus, Sarcophyton. Their structures were determined from spectroscopic measurements, X-ray analysis and chemical reaction.

The conformations of tricyclic ketolide TE-802 (1) and its analogue were determined by crystallographic analysis and NMR spectroscopy. The 3-carbonyl function of 1 took an 'exo' orientation against the lactone ring, and the orientation of the amino sugar at the 5-position differed from that in clarithromycin (CAM:2). On the other hand, di- and tricyclic ketolides and a tricyclic macrolide were found to exist in 'semi-fold-out (4H-downward)' conformation in deuteriochloroform. The conformation of 1 in deuteriochloroform was closely similar to that in D,O. The newly formed tetrahydrodiazepine ring existed in chair form or boat form, governed by the presence of substituent and its position.

A new method has been developed for the synthesis of chiral β-amido cyanides by means of the regioselective cleavage of β-lactams in the presence of a TMSOTf catalyst and RCN

Hantzsch cyclization of cyanoethyl 3-aminocrotonate and (E,Z)-4,4-dialkoxy-2-benzylideneacetoacetates 12a–h afforded the corresponding 3,4-trans-2-hydroxy-1,2,3,4-tetrahydropyridines 14a–h with high stereoselectivity. 1H NMR and X-ray analyses of compound 14a established the configuration of 3-H and 4-H as trans and that of 3-H and 2-OH as trans also.

Enantiomeric (+)- and (–)-methy3-nitrooxypropyl 1,4-dihydro-2,6-dimethyl-4-(3- nitrophenyl) pyridine-3,5-dicarboxylate 1 were synthesized by esterification of the optically active monocarboxylic acids (+)-6 and (–)-6, which are available from racemate (±)-6 by optical resolution using cinchonidine and cinchonine. The absolute configuration of the key intermediates (S)-(+)-6 and (R)-(–)-6, was also unambiguously determined by the comparison with optical active (+)- and (–)-1 derived from (R)-(–)- and (S)-(+)-7 and (+)- and (–)-6, and X-ray crystallographic analysis of bromoethyl ester (R)-(–)-8 prepared from the acid (S)-(+)-7.

The three-dimensional structure of the E-64-c-papain complex has been determined by X-ray crystal analysis at 2.5 Å resolution (conventional R = 26.9%). The structure determined indicates that: (i) the C2 atom of the oxirane ring of E-64-c is covalently bound by the Sγ atom of Cys-25 of papain; (ii) this covalent bond formation results in a configurational conversion of the oxirane C2 atom from the S- to the R-form; and (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the specific interaction of the E-64-c molecule with papain.

In order to elucidate the conformational characteristics of cysteine protease inhibitors contributing to their inhibitory activities, the conformation of E-64 (N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine), a potent inhibitor of papain, was determined by X-ray crystal structure analysis. The molecules were packed in the crystal through electrostatic forces and hydrogen bonding between the oppositely charged terminal groups and between the amide groups. Two crystallographically independent E-64 molecules both took a flattened and slightly curved structure, which is similar to that of loxistatin, a related cysteine protease inhibitor. Based on the present results, a possible inhibitory mechanism of E-64 is proposed, with reference to the binding mode observed in the crystal structure of papain-substrate analogue complex.

The first highly asymmetric Pummerer-type cyclization of chiral, non-racemic β-amido sulfoxides to enantiomerically enriched β-lactams (80-85% ee) is described. S- and R-Sulfoxides (S-2a-d and R-2a-c) were treated with O-methyl-O-tert-butyldimethylsilyl ketene acetal 1 in the presence of a catalytic amount of zinc chloride in methylene dichloride to give predominantly the corresponding 4R- and 4S-β-lactams (R-3a-d and S-3a-c) in more than 80% ee. These results show that the stereoinduction is governed by the absolute configuration of the sulfoxides. Optically pure R- and S-3c were readily obtained by simple recrystallization in about 60% yield. The usefulness of the chiral, non-racemic 4-tolylsulfanyl-β-lactams 3a-d has been shown by their conversion into the key intermediate 11 for the optically pure carbapenem antibiotic, (+)-PS-5.