Phosphodiesterase inhibitors. Part 5: Hybrid PDE3/4 inhibitors as dual bronchorelaxant/anti-inflammatory agents for inhaled administration

(?)-6-(7-Methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (KCA-1490) exhibits moderate dual PDE3/4-inhibitory activity and promises as a combined bronchodilatory/anti-inflammatory agent. N-alkylation of the pyridazinone ring markedly enhances potency against PDE4 but suppresses PDE3 inhibition. Addition of a 6-aryl-4,5-dihydropyridazin-3(2H)-one extension to the N-alkyl group facilitates both enhancement of PDE4-inhibitory activity and restoration of potent PDE3 inhibition. Both dihydropyridazinone rings, in the core and extension, can be replaced by achiral 4,4-dimethylpyrazolone subunits and the core pyrazolopyridine by isosteric bicyclic heteroaromatics. In combination, these modifications afford potent dual PDE3/4 inhibitors that suppress histamine-induced bronchoconstriction in vivo and exhibit promising anti-inflammatory activity via intratracheal administration.     

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC₅₀ = 8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED₅₀ = 4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment     

Phosphodiesterase inhibitors. Part 6: Design,synthesis, and structure–activity relationships of PDE4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory activity

We previously identified KCA-1490 [(?)-6-(7-methoxy-2-trifluoromethyl-pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone], a dual PDE3/4 inhibitor. In the present study, we found highly potent selective PDE4 inhibitors derived from the structure of KCA-1490. Among them, N-(3,5-dichloropyridin-4-yl)-7-methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridine-4-carboxamide (2a) had good anti-inflammatory effects in an animal model.     

Brassinolide-2,3-acetonide: A brassinolide-induced rice lamina joint inclination antagonist

A novel chemical tool compound that is an antagonist of brassinolide (BL, 1)-induced rice lamina joint inclination was developed. Although 2-O-, 3-O-, 22-O-, or 23-O-methylation of BL causes a critical decrease in biological activity,⁵ a crystal structure of the extracellular leucine-rich repeat (LRR) domain of BRASSINOSTEROID-INSENSITIVE I (BRI1) bound to BL3, 4 indicates that the loss of activity of the O-methylated BL may result from not only the low affinity to BRI1, but also from blocking the interaction with another BR signaling factor, a partner protein of BRI1 (e.g., BRI1-ASSOCIATED KINASE 1, BAK1). On the basis of this hypothesis we synthesized the BL 2,3-acetonide 2, the 22,23-acetonide 3, and the 2,3:22,23-diacetonide 4 to assess the possibility of 2-O- and 3-O- or/and 22-O- and 23-O-alkylated BL as an antagonist in BR signaling evoked by exogenously applied BL. The 2,3-acetonide 2 more strongly inhibited the lamina inclination caused by BL relative to the 22,23-acetonide 3, whereas the diacetonide 4 had no effect most likely due to its increased hydrophobicity. This suggested that the 2,3-hydroxyl groups of BL play a more significant role in the interaction with a BRI1 partner protein rather than BRI1 itself in rice lamina joint inclination. Taken together it was demonstrated that BL, the most potent agonist of BRI1, is transformed into an antagonist by functionalization of the 2,3-dihydroxyl groups as the acetonide. This finding opens the door to the potential development of a chemical tool that modulates protein–protein interactions in the BR signaling pathway to dissect the BR-dependent processes.     

l-Serine Production Improved by Analogue-resistant Mutants of a Methanol-utilizing Bacterium

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

Streptococcus cremoris was cultivated for 7 days at 30°C in sterilized skim milk or in the sterilized 10% solution of dry skim milk. This skim milk culture was divided into precipitate and supernatant by centrifugation. The absorbancy at 280 mμ of the supernatant prepared from the skim milk culture of S. cremoris was higher than that of the control supernatant.

Casein prepared from the skim milk culture of S. cremoris was less hydrolyzed by rennet than control casein at pH 7.0.

According to the free boundary electrophoretic analysis of the treated casein in m/10 veronal buffer of pH 8.5 containing urea, α-casein seemed to be hydrolyzed by S. cremoris but β-casein did with more difficulty.     

Isolation and Identification of a Novel β-Carboline Derivative in Salted Radish Roots,Raphanus sativus L.

The methanol extract of salted radish roots contains several precursors of yellow pigment. The main compound was isolated by the use of Toyopearl HW-40S column chromatography, and its structure was determined to be 1-(2′-pyrrolidinethion-3′-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid on the basis of an elemental analysis, and IR, UV, FAB-MS and NMR spectroscopy. This compound is presumed to have been the condensation product from the degradation of 4-methylthio-3-butenyl isothiocyanate and l-tryptophan. This carboline compound is considered to play an important role in the formation of the yellow pigment in salted radish roots.     

Studies on the Proteolytic Action of Dairy Lactic Acicl Bacteria

Aseptic rennet curd prepared under the aseptic conditions and Str. cremoris- and L. helveticus-cheese prepared by sandwiching the cell pellets of Str. cremoris and L. helveticus between aseptic rennet curd, respectively, were ripened at 10°C for desired period.

Water soluble nitrogen (WSN) contents of both aseptic rennet curd and two kinds of cheese were determined. Gradual increase of WSN content of aseptic rennet curd was recognized all through the ripening preiod. WSN contents of both Str. cremoris- and L. helveticus-cheese were remarkably higher than those of aseptic rennet curd after 12 days ripening. This tendency was more remarkably recognized after 60 or 70 days ripening. α^s-Casein was mainly hydrolyzed by these lactic acid bacteria during ripening. α^s-Casein in two kinds of the cheese was more easily degradated by these lactic acid bacteria than that in aseptic rennet curd by rennet.

Judging from the results in previous and present reports, it was estimated that lactic acid bacteria used as a starter began to autolyze after 12 days ripening and that intracellular proteases released from their cells mainly hydrolyzed α^s-casein contained in Ca-paracaseinate of aseptic rennet curd to water soluble substances. This hydrolysis was also estimated from the viscous texture observed by scanning electron micrography.     

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

Studies on the Changes of Meat Fats by Various Processings

Data are presented to show the gas chromatographic identification of a total of 18 saturated aliphatic γ- and δ-lactones obtained from melted beef depot fat, namely, δ-C₆, γ-C₇, γ-C₈, γ-C₉, and a homologous series of γ- and δ-lactones of the even-carbon numbers C₁₀ to C₁₆ and of smaller amount of the odd-carbon numbers C₁₁ to C₁₅. These lactones were isolated by steam distillation and silicic acid adsorption chromatography, and identified through gas chromatography and infrared spectroscopy.

Lactones obtained had a peach-like flavor, and it was suggested that lactones were important in heated beef fat as the flavor compounds.