Biological activities of nitromethylene analogues of imidacloprid with a 4,5-dimethylated imidazolidine ring.

Four nitromethylene analogues of imidacloprid (CH-IMIs) having a 4,5-dimethylated (diMe) imidazolidine ring were stereospecifically synthesized to evaluate their affinity for the nicotinic acetylcholine receptors of the housefly Musca domestica. Among the analogues, the 4S,5R-diMe analogue showed the highest receptor affinity (Ki=0.39 nM). The insecticidal activity against M. domestica of the synthesized compounds was also measured under synergistic and nonsynergistic conditions. Under nonsynergistic conditions, the insecticidal activity of the 4S,5R-diMe analogue was the highest. The order of the insecticidal potency of the four diMe-CH-IMIs (4S,5R->4R,5S-=4R,5R->4S,5S-diMe analogues) was the same as that of the receptor affinity. Piperonyl butoxide (PBO) did not synergize with the test compounds, but both PBO and NIA16388 applications strengthened the activity of analogues other than the 4S,5S-diMe analogue. This suggests that the configuration of the substituents on the imidazolidine ring should influence the metabolism process of CH-IMI in houseflies.

Effect of further substitutions at 5-, 6-, 7-, or 8-position of 3-[3-(4-methoxyphenyl)-1-hydroxyprop-2-yl]coumarin on phytotoxicity.

Derivatives of the coumarin ring in (R)-3-[3-(4-methoxyphenyl)-1-hydroxyprop-2-yl]coumarin 2, which is a lignan structure, were synthesized to clarify their structure-phytotoxicity relationships. The growth-inhibitory activity of the 8-OCH3 derivative 8 (IC50=228 µM) was more potent against the roots of lettuce seedlings than the compound without substituents 2. As for the roots of Italian ryegrass seedlings, the presence of the methoxy group at the 7- or 8-position was extremely effective for inhibiting growth (7-OCH3 7: IC50=121 µM, 8-OCH3 8: 56.7 µM). Methyl derivatives at the 5- or 8-position showed activity levels similar to those of the compound without substituents 2 (5-CH3 13: IC50=214 µM, 8-CH3 16: IC50=225 µM). The activities of OH- and F-derivatives were not observed or were lower.

Syntheses of Tetrahydropyran Type 8,7'-Neolignans Using a Ring-Expansion Reaction and Tetrahydrofuran Type 8,7'-Neolignans to Discover a Novel Phytotoxic Neolignan.

One novel tri-substituted tetrahydropyran type 8,7'-neolignan and its enantiomer with higher enantiomeric excess were synthesized from all cis-tetra-substituted tetrahydrofuran with an iodomethyl group by a hydride or H2 ring-expansion reaction. The normal hydride reductions of C-I bonds of tetra-substituted tetrahydrofurans bearing iodomethyl groups were observed in other 2,3-cis-stereoisomers of tetra-substituted tetrahydrofurans to give tetra-substituted tetrahydrofurans bearing 7,8-cis and 8,7'-neolignan structures. The phytotoxicities of their synthesized compounds were compared with previously synthesized 7,8-trans-8,7'-neolignans bearing tetra-substituted tetrahydrofurans to find out the highest phytotoxic tri-substituted tetrahydropyran type 8,7'-neolignan.

Regiospecific and Enantiospecific Effects of the ß-Benzyl-α-benzylidene-γ-butyrolactone Structure on Phytotoxic Fungi.

Novel derivatives of E-ß-benzyl-α-benzylidene-γ-butyrolactone (3-benzyl-2-benzylidene-4-butanolide) with lignano-9,9'-lactone structures were developed as anti-phytopathogenic fungal compounds. Their regiospecific and enantiospecific characteristics were determined, with the E-form and 3R-configuration showing higher activities against the Alternaria alternata Japanese pear pathotype. By the syntheses of benzyl compounds instead of benzylidene and aromatic derivatives, followed by an bioassay experiment, the importance of the benzylidene structure and effects of the substituents of the aromatic ring were clarified. The (2-OCH3, 4'-CH3/4'-CF3)-derivatives, 19 and 25, and (2-OCH3, 6-CH3/6-F/6-Br, 4'-OCH3)-derivatives, 34, 38, and 42, were more effective with EC50 values of 0.1-0.3 µM. It was assumed that the 2-OCH3 group, a hydrophobic group at the 6-position, and some size of the hydrophobic group at the 4'-position were necessary for the increased activity.

Crystal Structure of l-2,4-Diketo-3-deoxyrhamnonate Hydrolase Involved in the Nonphosphorylated l-Rhamnose Pathway from Bacteria.

2,4-Diketo-3-deoxy-l-rhamnonate (L-DKDR) hydrolase (LRA6) catalyzes the hydrolysis reaction of L-DKDR to pyruvate and l-lactate in the nonphosphorylated l-rhamnose pathway from bacteria and belongs to the fumarylacetoacetate hydrolase (FAH) superfamily. Most of the members of the FAH superfamily are involved in the microbial degradation of aromatic substances and share low sequence similarities with LRA6, by which the underlying catalytic mechanism remains unknown at the atomic level. We herein elucidated for the first time the crystal structures of LRA6 from Sphingomonas sp. without a ligand and in complex with pyruvate, in which a magnesium ion was coordinated with three acidic residues in the catalytic center. Structural, biochemical, and phylogenetic analyses suggested that LRA6 is a close but distinct subfamily of the fumarylpyruvate hydrolase (FPH) subfamily, and amino acid residues at equivalent position to 84 in LRA6 are related to different substrate specificities between them (Leu84 and Arg86 in LRA6 and FPH, respectively). Structural transition induced upon the binding of pyruvate was observed within a lid-like region, by which a glutamate-histidine dyad that is critical for catalysis was arranged sufficiently close to the ligand. Among several hydroxylpyruvates (2,4-diketo-5-hydroxycarboxylates), L-DKDR with a C6 methyl group was the best substrate for LRA6, conforming to the physiological role. Significant activity was also detected in acylpyruvate including acetylpyruvate. The structural analysis presented herein provides a more detailed understanding of the molecular evolution and physiological role of the FAH superfamily enzymes (e.g., the FAH like-enzyme involved in the mammalian l-fucose pathway).

Plant Growth Suppressive Activity of (R)-3-(7'-Aryl-9'-hydroxyprop-8'-yl)coumarin, Structural Isomer of Z-2-Hydroxybenzylidene-γ-butyrolactone-type Lignan.

3-(7'-Aryl-9'-hydroxyprop-8'-yl)coumarin, which is a structural isomer of a Z-2-hydroxybenzylidene-γ-butyrolactone-type lignan, was stereoselectively synthesized and subjected to plant growth regulation examination. (R)-4'-Methoxyphenyl derivative 3 showed stereospecific plant growth suppressive activity. The significance of the presence of hydroxy group at the 9'-position for the activity was clarified. The effect of the substituent at the 7'-aryl group was also shown. The 3'-methoxy, 4'-methoxy, and 4'-trifluoromethyl derivatives 10, 3, and 22 led to the most significant growth suppression of Italian ryegrass roots. The 2'-methoxy derivative 9 and 4'-methoxy derivative 3 provided the most growth suppressive activity against lettuce shoots and roots, respectively.

Germination Stimulant Activity of Isothiocyanates on Phelipanche spp.

The root parasitic weed broomrapes, Phelipanche spp., cause severe damage to agriculture all over the world. They have a special host-dependent lifecycle and their seeds can germinate only when they receive chemical signals released from host roots. Our previous study demonstrated that 2-phenylethyl isothiocyanate is an active germination stimulant for P. ramosa in root exudates of oilseed rape. In the present study, 21 commercially available ITCs were examined for P. ramosa seed germination stimulation, and some important structural features of ITCs for exhibiting P. ramosa seed germination stimulation have been uncovered. Structural optimization of ITC for germination stimulation resulted in ITCs that are highly active to P. ramosa. Interestingly, these ITCs induced germination of P. aegyptiaca but not Orobanche minor or Striga hermonthica. P. aegyptiaca seeds collected from mature plants parasitizing different hosts responded to these ITCs with different levels of sensitivity. ITCs have the potential to be used as inducers of suicidal germination of Phelipanche seeds.

Structure-activity relationship of the aromatic moiety of 6-substituted 5,6-dihydro-2H-pyran-2-one to find the novel compound showing higher plant growth inhibitory activity.

In the course of our research on the structure-activity relationship of 5,6-dihydro-2H-pyran-2-one, (S)-6-[(R)-2-hydroxy-6-(4-fluorophenyl)hexyl]-5,6-dihydro-2H-pyran-2-one was found to show 2-3-fold more potent plant growth inhibitory activity against Italian ryegrass shoots (IC50 = 95 µm) and roots (IC50 = 17 µm) than compound bearing unsubstituted phenyl group. The small and electron withdrawing atom at 4-position of the benzene ring caused the higher activity.

Aqueous Extract from Leaves of Citrus unshiu Attenuates Lipopolysaccharide-Induced Inflammatory Responses in a Mouse Model of Systemic Inflammation.

Inflammation is related to various life-threatening diseases including cancer, neurodegenerative diseases, and metabolic syndrome. Because macrophages are prominent inflammatory cells, regulation of macrophage activation is a key issue to control the onset of inflammation-associated diseases. In this study, we aimed to evaluate the potential anti-inflammatory activity of Citrus unshiu leaf extract (CLE) and to elucidate the mechanism underlying its anti-inflammatory effect. We found the inhibitory activity of CLE on the secretion of proinflammatory cytokines and a chemokine from mouse macrophage-like RAW 264.7 cells and mouse peritoneal macrophages. The inhibitory activity of CLE was attributed to downregulated JNK, p38 MAPK, and NF-κB signaling pathways, leading to suppressed gene expression of inflammation-associated proteins. Oral administration of CLE significantly decreased the serum level of proinflammatory cytokines IL-6 and TNFα and increased that of anti-inflammatory cytokine IL-10 in lipopolysaccharide-induced systemic inflammation mice. In addition, oral administration of CLE decreased secretion and gene expression of several proinflammatory proteins in the liver and spleen of the model mice. Overall results revealed that C. unshiu leaf is effective to attenuate inflammatory responses in vitro and in vivo.

Stereocontrolled syntheses of (-)- and (+)-γ-diisoeugenol along with optically active eight stereoisomers of 7,8'-epoxy-8,7'-neolignan.

It was shown that reduction of the tertiary benzylic hydroxy group of (2R,3S,4R,5S)-3,5-bis(4-benzyloxy-3-methoxyphenyl)-2,4-dimethyltetrahydro-3-furanol 17 followed by the intramolecular Friedel-Crafts reaction gave exclusively indane with (7S,7'S,8R,8'R)-2,7'-cyclo-7,8'-neolignan structure 18 along with (7S,7'R,8S,8'R)-7,8'-epoxy-8,7'-neolignan structure 19. Indane 18 was converted to (-)-γ-diisoeugenol ((-)-4). On the other hand, (2S,3R,4R,5S)-3,5-bis(4-benzyloxy-3-methoxyphenyl)-2,4-dimethyltetrahydro-3-furanol 22 did not afford indane, but the tetrahydrofuran structure with (7S,7'S,8S,8'S)-7,8'-epoxy-8,7'-neolignan structure 23 and 7'-epi-23.

Syntheses of all eight stereoisomers of conidendrin.

All eight stereoisomers of conidendrin were synthesized from (1 R,2 S,3 S)-1-(4-benzyloxy-3-methoxyphenyl)-3-(4-benzyloxy-3-methoxybenzyl)-2- hydroxymethyl-1,4-butanediol ((+)-4) and its enantiomer with high optical purity. The configurations at 4-positions of the conidendrin stereoisomers were constructed by intramolecular Friedel-Crafts reaction of protected 4. After conversion to tetrahydronaphthalene intermediate 7a, the 2- and 3-position of tetrahydronaphthalene structure 7a were converted to 3a- and 9a-position of (+)-α-conidendrin (3a), respectively. By the epimerization process of 2- or 3-position of 7a, the other diastereomers were obtained. All enantiomers were also synthesized from (-)-4.

Discovery of stereospecific cytotoxicity of (8R,8'R)-trans-arctigenin against insect cells and structure-activity relationship on aromatic ring.

One of the arctigenin stereoisomers, (8R,8'R)-trans-form 1, showed stereospecific cytotoxicity against insect cells, Sf9 and NIAS-AeAl-2 cells. By the comparison with other stereoisomers, the most importance of the 8'R stereochemistry for the higher activities was clarified. On the other hand, the wider range of activity level among stereoisomers against cancer cells, HL-60, was not observed. The structure-activity relationship research using derivatives bearing (8R,8'R)-trans-form was performed to show the same level of activities of 3-iodo, 4-iodo, and 3,4-methylenedioxy derivatives 28, 29, and 36 as (8R,8'R)-trans-arctigenin 1. In the examination of thiono derivatives, 4-iodo thiono and 3,4-methylenedioxy thiono derivatives 66, 67 showed similar level of activities to that of (8R,8'R)-trans-arctigenin 1. The expression of ribosomal 28S rRNA gene of Sf9 cells was increased by (8R,8'R)-trans-arctigenin 1, whereas a degradation of DNA was not observed.

Hydroxyl carlactone derivatives are predominant strigolactones in Arabidopsis.

Strigolactones (SLs) regulate important aspects of plant growth and stress responses. Many diverse types of SL occur in plants, but a complete picture of biosynthesis remains unclear. In Arabidopsis thaliana, we have demonstrated that MAX1, a cytochrome P450 monooxygenase, converts carlactone (CL) into carlactonoic acid (CLA) and that LBO, a 2-oxoglutarate-dependent dioxygenase, can convert methyl carlactonoate (MeCLA) into a metabolite called [MeCLA + 16 Da]. In the present study, feeding experiments with deuterated MeCLAs revealed that [MeCLA + 16 Da] is hydroxymethyl carlactonoate (1'-HO-MeCLA). Importantly, this LBO metabolite was detected in plants. Interestingly, other related compounds, methyl 4-hydroxycarlactonoate (4-HO-MeCLA) and methyl 16-hydroxycarlactonoate (16-HO-MeCLA), were also found to accumulate in lbo mutants. 3-HO-, 4-HO-, and 16-HO-CL were detected in plants, but their expected corresponding metabolites, HO-CLAs, were absent in max1 mutants. These results suggest that HO-CL derivatives may be predominant SLs in Arabidopsis, produced through MAX1 and LBO.

Functional and structural characterization of a novel L-fucose mutarotase involved in non-phosphorylative pathway of L-fucose metabolism.

Mutarotases catalyze the α-ß anomeric conversion of monosaccharide, and play a key role in utilizing sugar as enzymes involved in sugar metabolism have specificity for the α- or ß-anomer. In spite of the sequential similarity to l-rhamnose mutarotase protein superfamily (COG3254: RhaM), the ACAV_RS08160 gene in Acidovorax avenae ATCC 19860 (AaFucM) is located in a gene cluster related to non-phosphorylative l-fucose and l-galactose metabolism, and transcriptionally induced by these carbon sources; therefore, the physiological role remains unclear. Here, we report that AaFucM possesses mutarotation activity only toward l-fucose by saturation difference (SD) NMR experiments. Moreover, we determined the crystal structures of AaFucM in the apo form and in the l-fucose-bound form at resolutions of 2.21 and 1.75 Å, respectively. The overall structural folding was clearly similar to the RhaM members, differed from the known l-fucose mutarotase (COG4154: FucU), strongly indicating their convergent evolution. The structure-based mutational analyses suggest that Tyr18 is important for catalytic action, and that Gln87 and Trp99 are involved in the l-fucose-specific recognition.

Docosahexaenoyl ethanolamide mitigates IgE-mediated allergic reactions by inhibiting mast cell degranulation and regulating allergy-related immune cells.

Docosahexaenoic acid (DHA) is a long-chain polyunsaturated fatty acid mainly found in fish oil. Although several studies have suggested that it can alleviate allergy symptoms, its mechanism of action remains to be elucidated. In the present study, we found that docosahexaenoyl ethanolamide (DHEA), a metabolite of DHA produced in the human body, exerts the anti-allergic activity in vitro and in vivo. DHEA suppressed degranulation of rat basophilic leukemia RBL-2H3 cells and bone marrow-derived mast cells in a dose-dependent manner without cytotoxicity. This occurred due to a decrease in Ca2+ influx, which is critical for mast cell degranulation. DHEA also suppressed IgE-mediated passive cutaneous anaphylaxis reaction in mice. In addition, DHEA was demonstrated to lessen an allergic symptom in a mouse model of pollinosis and to alter the production of IgE and cytokines secreted by splenocytes collected from the pollinosis mice. Taken together, this study indicates that DHEA is a promising anti-allergic agent as it inhibits mast cell degranulation and modulates other immune cells.

Cytotoxicity against HL60 Cells of Ficifolidione Derivatives with Methyl, n-Pentyl, and n-Heptyl Groups.

Ficifolidione, a natural insecticidal compound isolated from the essential oils of Myetaceae species, is a spiro phloroglucinol with an isobutyl group at the C-4 position. We found that ficifolidione showed cytotoxicity against cancer cells via apoptosis. Replacement of the isobutyl group by n-propyl group did not influence the potency, but the effect of the replacement of this group by a shorter or longer alkyl group on the biological activity remains unknown. In this study, ficifolidione derivatives with alkyl groups such as methyl, n-pentyl, and n-heptyl group-instead of the isobutyl group at the C-4 position-were synthesized to evaluate their cytotoxicity against the human promyelocytic leukaemia cell line HL60 and their insecticidal activity against mosquito larvae. The biological activities of their corresponding 4-epimers were also evaluated. As a result, the conversion of the isobutyl group to another alkyl group did not significantly influence the cytotoxicity or insecticidal activity. In HL60 cells treated with the n-heptyl-ficifolidione derivative, the activation of caspase 3/7 and the early stages of apoptosis were detected by using immunofluorescence and flow cytometric techniques, respectively, suggesting that the cytotoxicity should be induced by apoptosis even though the alkyl group was changed.

Syntheses and Phytotoxicity of All Stereoisomers of 6-(2-Hydroxy-6-phenylhex-1-yl)-5,6-dihydro-2H-pyran-2-one and Determination of the Effect of the α,ß-Unsaturated Carbonyl Structure and Hydroxy Group Bonding to Chiral Carbon.

All four stereoisomers of naturally occurring 6-(2-hydroxy-6-phenylhex-1-yl)-5,6-dihydro-2H-pyran-2-one (1) were synthesized by employing yeast-reduction products with high optical purity [from 95% enantiomeric excess (ee) to more than 99% ee], and then their phytotoxicities against lettuce and Italian ryegrass were evaluated. In the Italian ryegrass seedlings test, (6S,2'R)-1 showed the most potent and stereospecific activity against the shoots (IC50 = 260 µM) and roots (IC50 = 43.2 µM), with a significant difference from other stereoisomers. The highest seed germination inhibitory activity against Italian ryegrass seed was also observed in (6S,2'R)-1, showing a 53% germination ratio from the control at 1000 µM. This advantageous (6S,2'R) stereochemistry was employed in the syntheses of α,ß-dihydro, 2'-dehydroxy, and 2'-methoxy derivatives 13-15. By the test using these derivatives, the importance of the α,ß-unsaturated double bond and hydroxy group bonding to a chiral center on the 6-alkyl chain of 5,6-dihydro-α-pyrone for phytotoxicity was determined. In the test against lettuce, the 6S configuration and (6S,2'S) configuration were necessary for growth inhibition (IC50 = ca. 60 µM) and germination inhibition (63% germination ratio at 1000 µM), respectively.

Structure-Activity Relationships of Fish Oil Derivatives with Antiallergic Activity in Vitro and in Vivo.

A series of unsaturated fatty acids in fish oil and their corresponding ethanolamide metabolites were explored to find active fish oil components of antiallergic activity in vitro. Ethanolamides of omega-3 fatty acids (α-linolenic acid, EPA, and DHA) were found to possess promising antiallergic activity, whereas free fatty acids and ethanolamides of other fatty acids exhibited no or weak potency. Based on this finding, structure-activity relationships of DHA-ethanolamide (DHEA) derivatives were investigated to yield better fatty acid derivatives with enhanced antiallergic activity in vitro and in vivo. When the ethanolamide moiety of DHEA was replaced by the substituted sulfonamide functionality, highly promising potency was provided in vitro. Compound 59 showed improved antiallergic activity in vivo over DHEA. The results indicate that optimized DHEA derivatives have enhanced antiallergic activity in vitro and in vivo, and the resulting structures will be an important basis for further development of bioavailable derivatives with promising allergy suppressive activity.

Rosmarinic acid and its ester derivatives for enhancing antibacterial, α-glucosidase inhibitory, and lipid accumulation suppression activities.

Rosmarinic acid (RA), commonly found in Nepetoidae subfamily of Lamiaceae family, possesses various biological activities. To expand its application, RA was modified by esterification with methyl (me), propyl (pro), and hexyl (hex) alcohols and then tested antibacterial, α-glucosidase inhibitory, and lipid accumulation suppression activities. Consequently, RA derivatives enhanced antibacterial activity, especially the RA-pro and RA-hex, which effectively inhibited the growth of Bacillus cereus rather than tannic acid, a natural antibacterial agent. RA-hex also inhibited α-glucosidase inhibitory activity greater than luteolin. By computational molecular docking, dihydroxyphenyl group and hexyl group were selected as essential groups for interaction with the active site of α-glucosidase through hydrogen bonding and hydrophobic interaction, contributing to the great inhibitory activity. Furthermore, RA-pro and RA-hex effectively suppressed lipid accumulation of 3T3-L1 cells, superior to EGCG, a well-known anti-obesity phytochemical. These biological effects of RA derivatives commonly attributed to hydrophobicity, hydrogen bonding, and steric bulkiness of the side chain. PRACTICAL APPLICATIONS: Rosmarinic acid (RA), a fundamental compound in the family Lamiaceae, is one of powerful naturally occurring antioxidants as well as other biological activities. Furthermore, its abundance in nature was also high in amount in the plant kingdom. So, natural RA can be one of possible natural resources for creating potent natural drugs and biologically useful substances after chemical modification. Studies on various biological activities may intensively expand usage and application of RA. In this study, RA was derivatized to corresponding ester such as methyl, propyl, and hexyl alcohols with higher hydrophobicity, and found great antibacterial, α-glucosidase inhibitory, and lipid accumulation suppression activities. RA-pro and RA-hex significantly suppressed lipid accumulation and cell differentiation. Therefore, RA derivatives with multiple biological activities have the potential to be applied in the food and pharmaceutical industries as food ingredients and supplements.

Design of 92 New 9-Norlignan Derivatives and Their Effect on Cell Viabilities of Cancer and Insect Cells.

Ninety-two new 9-norlignan derivatives containing more effective compounds against both cancer and insect cells than lead compounds were synthesized. Against HeLa cells, 7-(3,4-dimethoxyphenyl)-7'-(3'-hydroxy-4'-methoxyphenyl) derivative 63 (IC50 = 0.9 ± 0.2 µM) was to be around 6-fold more potent than lead compound 5. Moreover, against HL-60 cells, 7-(4-trifluoromethylphenyl)-7'-(3'/4'-hydroxyphenyl) derivatives 78 and 79 (IC50 = 2.2 ± 0.4 µM and 2.4 ± 0.6 µM) were 3-fold more potent than lead compound 5. Furthermore, against Sf9 cells from the common cutworm, the 7-(4-trifluoromethylphenyl) derivatives bearing electron-withdrawing groups 76-96 showed a wider range of activity (around 20-fold difference), giving valuable information on the structure-activity relationship. The 7-(4-trifluoromethylphenyl)-7'-(2'/3'-hydroxyphenyl) derivatives 77 and 78 (IC50 = 4.7 ± 0.6 µM and 4.9 ± 0.9 µM) had around 2-fold higher activity against Sf9 cells than lead compound 5. The 7-(4-trifluoromethylphenyl)-7'-(3'-hydroxyphenyl) derivative 78 was also effective against mosquito NIAS-AcAl-2 cells with an IC50 value of 5.4 ± 0.3.