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     

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae)

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d₃₁-hexadecanoic acid (16:0), d₂₇-octadecadienoic acid (18:2), produced from d₃₁-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d₂₇-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.     

Structure–activity-relationship studies on dihydrofuran-fused perhydrophenanthrenes as an anti-Alzheimer’s disease agent

As an extended study on development of anti-Alzheimer’s disease agent, we newly synthesized various dihydrofuran-fused perhydrophenanthrenes via o-quinodimethane chemistry. This study revealed that the introduction of carbon side-chain on 8-position or removal of the acetal moiety on 3-position arose a cytotoxicity on rat cortical neurons. On the other hand, the ethereal or thio-ethereal substituent on 8-position enhanced the elongation effect on Aβ-damaged neurons. The necessity of the cyano group on 10b position was also proved in this structure–activity-relationship study.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

In vitro mycorrhization and acclimatization of Amanita caesareoides and its relatives on Pinus densiflora

Amanita caesareoides is a sister species of Amanita caesarea, also known as Caesar’s mushroom and one of the most desirable edible mycorrhizal mushrooms. However, cultivation of Caesar’s mushrooms has not yet been successful due to the difficulties involved in establishing pure cultures. In this study, we established pure cultures of four Asian Caesar’s mushroom species, i.e., A. caesareoides, Amanita javanica, Amanita esculenta, and Amanita similis, which were identified by sequence analysis of their rDNA internal transcribed spacer (ITS) region. Five selected isolates in A. caesareoides, A. javanica, and A. esculenta were tested for ectomycorrhizal syntheses with axenic Pinus densiflora seedlings in vitro. Ectomycorrhizal tips of each fungal isolate tested were observed on pine lateral roots within 5 months of inoculation. Seventeen pine seedlings that formed ectomycorrhizas in vitro with these three Amanita species were acclimatized under non-sterile conditions. Seven months following acclimatization, ectomycorrhizal colonization by A. caesareoides was observed on newly grown root tips, which was confirmed by polymerase chain reaction restriction fragment length polymorphism analysis of the fungal rDNA ITS region. Two other Amanita species also survived during ectomycorrhizal acclimatization. These results suggest that the cultivation of A. caesareoides and its relatives can be attempted through mycorrhizal synthesis using P. densiflora as a host. This is the first report of in vitro mycorrhization of Asian Caesar’s mushrooms and their acclimatization under non-sterile conditions.     

Inactivation of Bacteriophage ϕX174 by d-Fructose 6-Phosphate

The inactivation of bacteriophage ?X174 by d-fructose 6-phosphate was investigated. This inactivation was inhibited by EDTA or reducing agents, and stimulated by Cu²⁺ but other metal ions could not be substituted for Cu²⁺. The reaction was also inhibited by superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and various free radical scavengers.

No detectable changes were observed in adsorption capacity of phage and in the conformation of the virion. The viral DNA in the virion was, however, found to be cleaved. This strand scission was also enhanced by Cu²⁺ and protected by catalase. Similar results were obtained when ?X174 DNA was directly treated with d-fructose 6-phosphate.

It is concluded that the inactivation of ?X174 is due to DNA strand scission in the virion by the free radical of d-fructose 6-phosphate or oxygen radicals generated during autoxidation of d-fructose 6-phosphate.     

Quantification of Physical and Cyto-physiological Conditions for the Electrofusion of Saccharomyces cerevisiae

Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated. An AC field of 400 V_p/cm and a DC field of 2 square pulses (7 kV/cm; 60/βsec each) at an interval of 0.5 sec were effective. Treatment with 0.2 (SH1509) or l.0 mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential. As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 m. The presence of CaCl₂ (up to 0.4 mm) or 0.1 mm CaCl₂ with 0.1 mm MgCl₂ enhanced the yield. The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28°C.     

Isolation of Permethylated Dicarbonyl Sugar Derivatives from Polysaccharides

Oxidation of methyl trimethyl glucopyranosides which were obtained by methanolysis of permethylated cellulose, laminarin, and dextran, was performed with dimethyl sulfoxide (DMSO)-phosphorus pentoxide to afford the corresponding ulose derivatives, methyl 2,3,6-tri-O-methyl-d-xylo-hexopyranosid-4-ulose, methyl 2,4,6-tri-O-methyl-d-ribo-hexopyranosid-3-ulose, and methyl 2,3,4-tri-O-methyl-d-gluco-hexodialdo-l,5-pyranoside, respectively, in good or moderate yields. As a new type of derivatives for the linkage analysis of polysaccharides the chromatographic and spectrometric properties of 2,4-dinitrophenylhydrazone of the ulose derivatives were investigated.