Synthesis, growth inhibitory activity against tumor cells and structure-activity relationship of CGK733 and its analogs.

CGK733 was reported as a compound that inhibited ATM/ATR kinase activities and blocked their checkpoint signaling pathways with great selectivity. However, this paper was subsequently retracted, and the truth about the activity of CGK733 remains unclear. We synthesized various analogs of CGK733 with a modification of the carboxylic acid moiety and/or the aniline derivative moiety to accumulate knowledge of the structure-activity relationship of this compound. Growth inhibitory activity of CGK733 and novel 35 analogs against HeLa S3 cells was evaluated, and the structure-activity relationship revealed that analogs with 2-naphthyl or 4-fluorophenyl group instead of the benzhydryl group have activity comparable to CGK733 and that the 3-nitro group on the aniline moiety significantly affects the activity.

Synthesis and structure-activity relationship of kujigamberol B, a dinorlabdane diterpenoid isolated from an ancient Kuji amber.

The versatile methodology was developed for synthesizing kujigamberol B, a dinorlabdane diterpenoid isolated from the methanol extract of Kuji amber. A highly efficient intramolecular cyclization is followed by a Sonogashira-coupling reaction during the total synthesis. The synthesized compounds were evaluated for the growth-restoring activity against the mutant yeast (zds1Δ erg3Δ pdr1Δ pdr3Δ) and for the degranulation of RBL-2H3 cells. We found that in both activities, primary alcohol and secondary alcohol analogs are as active as kujigamberol B.

Synthesis and structure-activity relationship of violaceoid D, a cytotoxic alkylated phenol isolated from Aspergillus violaceofuscus Gasperini.

The enantioselective synthesis of violaceoid D, a cytotoxic phenolic compound isolated from the culture broth of Aspergillus violaceofuscus Gasperini, was achieved. The total synthesis involves stereoselective construction of the stereogenic center of violaceoid D via Sharpless asymmetric dihydroxylation, followed by Smiles rearrangement. The absolute configuration of natural violaceoid D was determined to be R from the specific rotation value. Synthesized violaceoid D and its analogs were evaluated for cytotoxicity against two human cancer cell lines, Jurkat and HCT116. Because the enantiomer of violaceoid D showed no cytotoxicity, it is plausible that violaceoid D binds selectively to specific target molecules, such as proteins in the cancer cells.

Synthesis and DFT-NMR-guided structure revision of cremenolide.

The structure of an anti-plant pathogenic and plant growth-promoting nonenolide, namely cremenolide, was revised by an efficient combination of DFT-based theoretical NMR calculations and synthesis of a target diastereomer. Initially, the planar structure of cremenolide was reconsidered by an individual analysis of the reported NMR spectra. Subsequently, the relative configuration was predicted using NMR calculations of all possible diastereomers based on the ωB97X-D functional. Finally, the relative configuration of cremenolide was unambiguously confirmed by preparation of the proposed structure.

Synthesis and antifungal activity of the proposed structure of a volatile compound isolated from the edible mushroom Hypsizygus marmoreus.

We synthesized the proposed structure of an antifungal compound detected in the culture broth of the edible mushroom Hypsizygus marmoreus. Using the Evans aldol and Abiko-Masamune aldol reactions as the key steps, we synthesized all of the stereoisomers of the compound with high stereoselectivity. The GC retention times and the fragmentation patterns in the mass spectra of the synthesized isomers did not match those of the natural product. Therefore, this result may imply that it is necessary to reisolate the natural product and reconsider its structure. All of the synthesized isomers were found to exhibit antifungal activity against the phytopathogenic fungus Alternaria brassicicola. Due to their simple structures, the obtained isomers could be lead compounds for new pesticides.

Identification of biosynthetic intermediates for the mating hormone α2 of the plant pathogen Phytophthora.

The heterothallic group of the plant pathogen Phytophthora can sexually reproduce between the cross-compatible mating types A1 and A2. The mating hormone α2, produced by A2 mating type and utilized to promote the sexual reproduction of the partner A1 type, is known to be biosynthesized from phytol. In this study, we identified 2 biosynthetic intermediates, 11- and 16-hydroxyphytols (1 and 2), for α2 by administering the synthetic intermediates to an A2-type strain to produce α2 and by administering phytol to A2 strains to detect the intermediates in the mycelia. The results suggest that α2 is biosynthesized by possibly 2 cytochrome P450 oxygenases via 2 hydroxyphytol intermediates (1 and 2) in A2 hyphae and secreted outside.

Sulfoquinovosylglyceryl ether, a new group of ether lipids from lake ball-forming green alga Aegagropilopsis moravica (family Pithophoraceae).

Ether lipids are a minor group of glycerolipids but widespread in nature, playing a vital function as membrane lipids, signalling molecules, or buoyant material. We have discovered sulfoquinovosylchimyl alcohol (1), a sulfonate-substituted glyceroglycolipid, from a lake ball-forming green alga Aegagropilopsis moravica (family Pithophoraceae), with the guidance of antimicrobial activity. The structure of 1, including absolute configurations of all sterogenic centers, was established by extensive NMR analysis, chemical degradation studies, and finally by total synthesis. Lipid 1 is an ether variant of a lyso-form of sulfoquinovosyldiacylglycerol, a chloroplast-specific membrane lipid, and thus represents a new lipid class, sulfoquinovosylglyceryl ether. A high occurrence of mobile life form in the family Pithophoraceae and a unique behaviour of chloroplasts reported in closely related Aegagropila linnaei, the famous lake-ball alga, implies a possible role of lipid 1 or its acyl derivatives in ecological adaptation to dysphotic niches.

Synthesis of both enantiomers of lycoperdic acid, an unusual mushroom-derived amino acid.

An efficient synthesis of both enantiomers of lycoperdic acid, a 4-hydroxyglutamic acid derivative from edible mushroom Lycoperdon perlatum, was achieved from a chiral aminoalcohol. The key steps were a stereoselective introduction of a C3 unit into a bicyclic ketone and oxidative cleavage of a cyclic vicinal diol into a dicarboxylic acid. This report provides the first synthesis of (-)-lycoperdic acid.

Enantioselective synthesis and stereochemical determination of the highly reduced polyketide ishigamide.

Ishigamide was isolated as a metabolite of a recombinant strain of Streptomyces sp. MSC090213JE08 and its unsaturated fatty acid moiety has been confirmed in vitro to be synthesized by a type II PKS. Biosynthesis of such a highly reduced polyketide by a type II PKS is worthy of note. However, absolute configuration of ishigamide remained unknown. (R)-Ishigamide was synthesized enantioselectively employing Stille coupling and Wittig reaction between three units, vinyl iodide, stannyldienal, and Wittig salt. Stereochemistry of natural ishigamide was determined to be R by chiral HPLC analysis comparing with the synthesized standard.

Disproof of the Proposed Structures of Bradyoxetin, a Putative Bradyrhizobium japonicum Signaling Molecule, and HMCP, a Putative Ralstonia solanacearum Quorum-Sensing Molecule.

First, we revisited the reported NMR data of bradyoxetin, a putative cell density factor of Bradyrhizobium japonicum, and found some inconsistencies in the proposed structure. To elucidate the correct structure, we synthesized model oxetane compounds and confirmed that the NMR data of the synthetic compounds did not match those of the reported bradyoxetin. After reinterpreting the reported NMR data, we concluded that bradyoxetin must be chloramphenicol. Next, some derivatives of 2-hydroxy-4-((methylamino)(phenyl)methyl)cyclopentanone (HMCP), which is a putative quorum-sensing molecule of Ralstonia solanacearum, were synthesized. The NMR spectra of the synthesized compounds were completely different from those of the reported natural products. Based on theoretical studies, including the estimation of 1H and 13C NMR chemical shifts using density functional theory calculations, we confirmed the correctness of the structure of the synthesized compound. These results strongly suggest that the proposed structure of HMCP could be incorrect.

In planta functions of cytochrome P450 monooxygenase genes in the phytocassane biosynthetic gene cluster on rice chromosome 2.

In response to environmental stressors such as blast fungal infections, rice produces phytoalexins, an antimicrobial diterpenoid compound. Together with momilactones, phytocassanes are among the major diterpenoid phytoalexins. The biosynthetic genes of diterpenoid phytoalexin are organized on the chromosome in functional gene clusters, comprising diterpene cyclase, dehydrogenase, and cytochrome P450 monooxygenase genes. Their functions have been studied extensively using in vitro enzyme assay systems. Specifically, P450 genes (CYP71Z6, Z7; CYP76M5, M6, M7, M8) on rice chromosome 2 have multifunctional activities associated with ent-copalyl diphosphate-related diterpene hydrocarbons, but the in planta contribution of these genes to diterpenoid phytoalexin production remains unknown. Here, we characterized cyp71z7 T-DNA mutant and CYP76M7/M8 RNAi lines to find that potential phytoalexin intermediates accumulated in these P450-suppressed rice plants. The results suggested that in planta, CYP71Z7 is responsible for C2-hydroxylation of phytocassanes and that CYP76M7/M8 is involved in C11α-hydroxylation of 3-hydroxy-cassadiene. Based on these results, we proposed potential routes of phytocassane biosynthesis in planta.

The Phytophthora mating hormone α2 is an antagonist of the counterhormone α1.

The crop destroyer Phytophthora uses mating hormones α1 and α2 to commence its sexual reproduction. The α1-induced sexual reproduction of the A2 mating type was unexpectedly found to be interfered with by the counterhormone α2 that the A2 type itself produces to induce the sexual reproduction of the A1 type. A plausible mechanism is proposed based on structure-activity relationships.

Ubiquitin-specific peptidase 5, a target molecule of vialinin A, is a key molecule of TNF-α production in RBL-2H3 cells.

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.

Vialinin A is a ubiquitin-specific peptidase inhibitor.

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin.

Inhibitory effects of vialinin A and its analog on tumor necrosis factor-α release and production from RBL-2H3 cells.

Vialinin A is an extremely potent inhibitor of tumor necrosis factor (TNF)-α release from RBL-2H3 cells. The present study investigated in detail the inhibitory effects of vialinin A and its analog, 5',6'-dimethyl-1,1':4',1″-terphenyl-2',3',4,4″-tetraol (DMT), on TNF-α. Vialinin A and DMT inhibited the release of TNF-α from RBL-2H3 cells in a dose-dependent manner, but had no effect on ß-hexosaminidase activity. Also, vialinins had little effect on TNF-α mRNA levels. Intriguingly, vialinins inhibited TNF-α production at low concentrations, but not shown a dose-dependency. The potent inhibitory activities of vialinins against TNF-α production and release suggest promising new candidate pathways for anti-inflammatory agents.

Structure-activity relationship of α hormones, the mating factors of phytopathogen Phytophthora.

The mating hormones α1 and α2 induce sexual reproduction of the phytopathogenic genus Phytophthora. To demonstrate the structural elements responsible to hormonal activity, 17 derivatives of α1 and α2 were synthesized and their hormonal activity (oospore-inducing activity) was evaluated. The terminal ester derivatives of α1 (diacetate and dibenzoate) retained the hormonal activity, whereas a dicarbamate derivative completely suppressed the activity. Even monocarbamates showed weak activities; among them the 1-O-carbamate was less active than 16-O-carbamate, suggesting that the 1-OH group is a little more important than 16-OH. Dihydro, dehydro, and demethyl derivatives exhibited the minimum level of activity. Surviving activity of 15-epi-α1 suggested a less importance of this stereochemistry. Contrary to α1, not only the terminal diacetate derivative but also monoacetates of α2 exhibited no or little activity. Among the monoacetates, 1-O-acetyl-α2 exhibited little yet relatively better activity than the others. No activity was observed for mono- and dicarbamoyl derivatives of α2. Dihydro α2 with the saturated double bond lost most of the activity. These findings suggest that both the mating hormones α1 and α2 require most of the functional (hydroxyl, keto, and olefinic) groups they possess in their natural form for inducing the sexual reproduction of Phytophthora.

Synthesis of microbial signaling molecules and their stereochemistry-activity relationships.

Microbial signaling molecules such as autoinducers and microbial hormones play important roles in intercellular communication in microorganisms. Information transfer between the individual cells of a microorganism is one of the most important biological events among them. Researchers often suffer from extremely low levels of microbial signaling molecule contents, which prevent them from understanding chemistry and biology of intercellular communication in microorganisms. Chemical synthesis is a powerful tool to obtain sufficient amounts of sample and to clarify the structure of a molecule. This review focuses on the synthesis and stereochemistry-bioactivity relationships of five microbial signaling molecules, Vibrio cholerae autoinducer-1 (CAI-1), AI-2 precursor (DPD), an acylhomoserine lactone from Rhizobium leguminosarum (small bacteriocin), a diffusible extracellular factor of Xanthomondas campestris pv. campestris, and Phytophthora mating hormone α1.

The second Phytophthora mating hormone defines interspecies biosynthetic crosstalk.

The heterothallic species of the agricultural pest Phytophthora use mating hormones α1 and α2 to regulate their sexual reproduction. Here we describe the absolute stereostructure of the second mating hormone α2 as defined by spectroscopic analysis and total synthesis. We have uncovered not only the interspecies universality of α hormones but also the pathway by which α2 is biosynthesized from phytol by A2-mating type strains and metabolized to α1 by A1 strains.

Synthesis of (2RS,8R,10R)-YM-193221 and an improved approach to tyroscherin, bioactive natural compounds from Pseudallescheria sp.

Short-step syntheses of (2RS,8R,10R)-YM-193221 (1) and tyroscherin (2), which are biologically active compounds isolated from Pseudallescheria sp., were accomplished in six and eight steps from L-tyrosine. The relative stereochemistry of natural YM-193221 was determined to be 8R*,10R*.

Contact sex pheromone components of the seed beetle, Callosobruchus analis (F.).

Callosobruchus analis (Coleoptera: Chrysomelidae: Bruchinae), found throughout tropical Asia and Africa, is a pest of stored legumes. Previous work has shown that females of this species produce a contact sex pheromone that elicits copulatory behavior in males. Comparisons of copulatory activity between any two of four congeneric species suggest that the contact sex pheromones are species specific. In laboratory bioassays, male C. analis exhibited copulatory behavior to a female dummy to which a crude extract of virgin females was applied. The extract had been collected by a filter paper method and was purified by acid-base partition and chromatographic techniques. Gas chromatography-mass spectrometry (GC-MS) analyses of active fractions revealed that the active compounds were 2,6-dimethyloctane-1,8-dioic acid (1) and callosobruchusic acid, (E)-3,7-dimethyl-2-octene-1,8-dioic acid (2), previously identified as contact sex pheromones of Callosobruchus maculatus (F.) and C. chinensis (L.), respectively. The stereoisomeric and chemical compositions were determined by the 2D-HPLC-Ohrui-Akasaka method as (2S,6R)-1:(S)-2=1.8:1, which meant that both compounds in C. analis were stereochemically pure, unlike the case of C. maculatus and C. chinensis. An examination of the influence of synthetic pheromone compounds on male copulatory activity revealed that (2S,6R)-1 is the main component, and that (S)-2 has an additive effect. In the examination of the stereochemistry-activity relationship, no copulatory behavior was elicited by (2R,6S)-1, and, furthermore, the enantiomer significantly masked the pheromonal activity of (2S,6R)-1. Glass rod dummy assays also suggested the presence of synergists. These results could elucidate the specificity of mate recognition in C. analis.