WO2016047533A1 - アブシナゾール - Google Patents
アブシナゾール Download PDFInfo
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- WO2016047533A1 WO2016047533A1 PCT/JP2015/076336 JP2015076336W WO2016047533A1 WO 2016047533 A1 WO2016047533 A1 WO 2016047533A1 JP 2015076336 W JP2015076336 W JP 2015076336W WO 2016047533 A1 WO2016047533 A1 WO 2016047533A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
- A01N43/647—Triazoles; Hydrogenated triazoles
- A01N43/653—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
Definitions
- the present invention relates to absinazole.
- Abscisic acid is a plant hormone that plays an important role in seed dormancy and environmental stress response.
- the inactivation of abscisic acid by catabolism is mainly controlled by abscisic acid 8 'hydroxylase CYP707A.
- the plant dwarfing agent S-uniconazole exerts its function by inhibiting ent-kauren oxidase CYP701A, an enzyme involved in gibberellin biosynthesis, while S-uniconazole also inhibits CYP707A. Accordingly, a group of compounds was developed in which CYP701A inhibitory activity was removed from the function of S-uniconazole and CYP707A inhibitory activity remained, and this group of compounds was named absinazole.
- An object of the present invention is to provide a new absinazole which is an inhibitor of CYP707A.
- the present invention provides a compound represented by the formula (I) or a salt thereof.
- R is a C1-6 alkyl group, and the C1-6 alkyl group may have a substituent selected from the group consisting of a halogen atom and a C1-6 alkoxy group.
- R may be an n-butyl group or a 2-methoxyethyl group.
- the above compound or a salt thereof can be used as an inhibitor of CYP707A, and can also be used as a plant growth regulator.
- the compound represented by the formula (I) or a salt thereof has a CYP707A inhibitory action and can be a plant growth regulator.
- FIG. 6 is a graph showing the results of examining the effects of the compounds of Examples 1 to 4 on seed germination of Arabidopsis thaliana.
- 2 is a photograph showing the results of examining the effects of the compounds of Examples 1 to 4 on seed germination of Arabidopsis thaliana.
- 2 is a photograph showing the results of examining the effects of the compounds of Examples 1 and 2 and the compound of Comparative Example 1 on seed germination of Arabidopsis.
- 2 is a graph showing the results of examining the effects of the compounds of Examples 1 to 4 on the elongation of the second leaf sheath of rice.
- halogen atom means a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc., preferably a fluorine atom or a chlorine atom.
- C 1-6 alkyl group means a linear or branched alkyl group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group.
- the C1-6 alkyl group is more preferably a C1-4 alkyl group which is an alkyl group having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, or an n-butyl group.
- C1-6 alkoxy group means an oxy group to which a C1-6 alkyl group is bonded, for example, a methoxy group, an ethoxy group, a 1-propyloxy group, a 2-propyloxy group. Group, 1-butyloxy group, 2-methylpropyloxy group, 1-methylpropyloxy group, 1,1-dimethylethoxy group and the like.
- the compound according to this embodiment is represented by the formula (I). Hereinafter, it may be expressed as compound (I).
- R is a C1-6 alkyl group, and the C1-6 alkyl group may have a substituent selected from the group consisting of a halogen atom and a C1-6 alkoxy group.
- R is a C1-6 alkyl group having a substituent
- the position of the substituent may be any, and the number of substituents may be one or more.
- the substituent is a C 1-6 alkoxy group, the number of substituents is preferably 1.
- R is preferably a C1-6 alkyl group substituted with a C1-6 alkyl group or a C1-6 alkoxy group.
- R is more preferably a methyl group, ethyl group, n-propyl group, n-butyl group, methoxymethyl group, 2-methoxyethyl group, 3-methoxypropyl group, 4-methoxybutyl group, ethoxymethyl group, 2 -Ethoxyethyl group, 3-ethoxypropyl group or 4-ethoxybutyl group.
- R is most preferably an n-butyl group or a 2-methoxyethyl group.
- the bonding position of the substituent on the benzene ring of compound (I) may be any of the ortho position, meta position and para position, preferably the meta position or para position, and most preferably the meta position. That is, compound (I) is preferably compound (Ia) or compound (Ib) shown below, and most preferably compound (Ia).
- the preferred embodiment of R in compound (Ia) and compound (Ib) is the same as the preferred embodiment of R in compound (I) described above.
- the 3-position carbon atom of the pent-1-ene chain of compound (I) is an asymmetric carbon atom, and compound (I) has an optical isomer.
- Compound (I) may be R-form, S-form, racemate, or any mixture of R-form and S-form.
- Compound (I) may be in the form of a salt.
- the salt include inorganic acid salts and organic acid salts.
- inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like.
- organic acid salts include acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, ethanesulfonate , P-toluenesulfonate, benzenesulfonate and the like.
- Compound (I) can be produced by the following reaction scheme. [Wherein Ts represents a tosyl group. ]
- step 1 1,2,4-triazole and 1-bromo-3,3-dimethyl-2-butanone are reacted in the presence of a base such as potassium carbonate to produce 3,3-dimethyl-1- (1H -1,2,4-triazol-1-yl) butan-2-one.
- a base such as potassium carbonate
- Specific reaction conditions include, for example, the reaction conditions shown in Production Example 1.
- Step 2 is a step of reacting 3,3-dimethyl-1- (1H-1,2,4-triazol-1-yl) butan-2-one and iodobenzaldehyde to obtain compound (VI).
- Specific reaction conditions include, for example, the reaction conditions shown in Production Examples 2 and 7.
- Step 3 is a step of obtaining the compound (VI-E) from the compound (VI).
- the compound (VI-E) which is the E form may be separated from the compound (VI) which is the mixture of the EZ form, and the compound (VI) which is the mixture of the EZ form is irradiated with UV light to give the compound (E) VI-E) may be made excessive and then separated.
- usual separation means such as column chromatography can be used.
- Specific conditions for UV irradiation and separation include, for example, the conditions shown in Production Examples 3 and 8.
- Step 4 is a step of reducing compound (VI-E) to obtain compound (V).
- a reducing agent such as sodium borohydride or lithium aluminum hydride
- Specific reaction conditions include, for example, the reaction conditions shown in Production Examples 4 and 9.
- Step 5 is a step of obtaining compound (II) by reacting compound (V) with compound (IV).
- Specific reaction conditions include, for example, the reaction conditions shown in Production Examples 6 and 10.
- Compound (IV) can be easily produced by those skilled in the art using commercially available compounds as raw materials.
- the compound (IV) can be produced according to the reaction conditions shown in Production Example 5.
- Step 6 is a step in which compound (II) and compound (III) are reacted to obtain compound (I).
- Specific reaction conditions include, for example, the reaction conditions shown in Examples 1 to 3.
- the obtained compound (I) may be subjected to optical resolution as necessary.
- the optical resolution can be performed by a method well known to those skilled in the art. For example, when optical resolution is performed by the chiral HPLC method, it can be performed with reference to the conditions shown in Examples 1 to 3.
- the ( ⁇ ) and (+) isomers of compound (I) are optically resolved by the method shown in the following reaction scheme, and the ( ⁇ ) isomer having higher activity than the (+) isomer is obtained. Can be acquired. According to the following reaction scheme, fractionation with an expensive chiral column is unnecessary, and the ( ⁇ ) isomer of compound (I) can be obtained simply and at low cost.
- Step 7 is a step of reacting compound (I) with N- (p-toluenesulfonyl) -L-phenylalanyl chloride to obtain a ( ⁇ ) isomer of compound (I) and compound (VII).
- the equivalent ratio of compound (I) and N- (p-toluenesulfonyl) -L-phenylalanyl chloride is reacted at 1:10, almost all of the (+) form of compound (I) reacts. On the other hand, about half of the ( ⁇ ) form remains unreacted. Unreacted ( ⁇ )-(I) and compound (VII) can be easily separated by open column chromatography. For this reason, the ( ⁇ ) isomer of compound (I) having higher activity can be obtained more easily than the optical resolution using chiral HPLC by the above method.
- Specific reaction conditions include, for example, the reaction conditions shown in Example 5.
- step 8 compound (VII) is separated into diastereomers by HPLC using an ODS column, and then hydrolyzed with an alkaline aqueous solution such as an aqueous sodium hydroxide solution to give ( ⁇ ) isomer of compound (I) or (+ ) It is a process of obtaining a body.
- Specific reaction conditions include, for example, the reaction conditions shown in Example 6.
- Compound (I) has a CYP707A inhibitory action, and therefore can suppress the degradation of abscisic acid by CYP707A. Therefore, when abscisic acid is synthesized by stress, the plant to which compound (I) has been administered can maintain the effect of abscisic acid and enhance the effect of abscisic acid. Thereby, Compound (I) can regulate the growth of plants. In particular, considering the action mechanism, stress tolerance can be imparted to the plant. For example, even if a plant is exposed to stress such as dryness, high temperature, and low temperature, it is possible to prevent the plant from withering by administering the compound (I) to the plant.
- the target plant is not particularly limited, and may be a seed plant, a fern plant, or a moss plant.
- the seed plant may be a gymnosperm or an angiosperm.
- the angiosperm may be a monocotyledonous plant or a dicotyledonous plant.
- the target plant organ is not particularly limited, and may be any of roots, stems, leaves, flowers, reproductive organs, and seeds, and may be cultured cells.
- the concentration of the compound (I) applied to the plant and the contact method can be appropriately adjusted according to the type of the target plant, its organ and purpose.
- the plant growth regulator containing compound (I) contains, in addition to compound (I), a fungicide, a fungicide, an insecticide, or a compound having a plant growth regulating action other than compound (I). Also good. Furthermore, you may contain the well-known formulation additive. As such a formulation additive, for example, an excipient, an emulsifier, and a wetting agent can be used.
- the dosage form of the plant growth regulator of the present invention is not particularly limited.
- emulsion emulsion, wettable powder, aqueous solvent, liquid, granule, powder, microcapsule, fumigant, fumigant, aerosol, flowable agent , Pastes, tablets, coating agents, microdispersing agents, oils, and compound fertilizers, which can be appropriately selected by the user according to the target plant, its organ, purpose, and the like.
- a plant growth regulator of dosage form can be produced by a known method.
- the extract was washed with 2 mL of a saturated aqueous sodium chloride solution, 2 mL of water twice, 2 mL of a saturated aqueous sodium chloride solution, dehydrated with sodium sulfate, and concentrated under reduced pressure to obtain 756.9 mg of a brown oily substance.
- This was subjected to silica gel column chromatography (65 g, 2.4 cm inner diameter ⁇ 27 cm length) using hexane-ethyl acetate (4: 6) as an eluent, and 426.9 mg (0.7710 mmol) of the title compound as a pale yellow oily substance. Yield 71%).
- Example 1 (E) -1- (3- (2,5,8,11-tetraoxatetrades-13-in-14-yl) phenyl) -4,4-dimethyl-2- (1H-1 , 2,4-Triazol-1-yl) pent-1-en-3-ol Under an argon stream, 323.5 mg (8.088 mmol) of 60% sodium hydride was added to 3 mL of 2-methoxyethanol, and the mixture was stirred at room temperature for 15 minutes.
- Example 2 (E) -1- (3- (3- (2- (2-butoxyethoxy) ethoxy) prop-1-yn-1-yl) phenyl) -4,4-dimethyl-2- (1H Synthesis of -1,2,4-triazol-1-yl) pent-1-en-3-ol The reaction was conducted in the same manner as described in Example 1 using 1-butanol in place of 2-methoxyethanol to give the title compound (total yield 26%).
- the title compound was optically resolved by chiral HPLC (column, CHIRAL CEL OD-H (10 mm inner diameter ⁇ 250 mm length); 10% 2-propanol / hexane; flow rate 4.5 mL / min; detection, UV254 nm).
- the compound eluted earlier was the ( ⁇ ) isomer, and the compound eluted later was the (+) isomer.
- (-) Body ee> 99.96 (%) [ ⁇ ] 32 D -4.4 (MeOH; c0.5) (+) Body ee 99.96 (%) [ ⁇ ] 32 D +3.8 (MeOH; c0.5)
- Example 3 (E) -1- (4- (2,5,8,11-tetraoxatetrades-13-in-14-yl) phenyl) -4,4-dimethyl-2- (1H-1 , 2,4-Triazol-1-yl) pent-1-en-3-ol Under an argon stream, 75.6 mg (1.89 mmol) of 60% sodium hydride was added to 2 mL of 2-methoxyethanol, and the mixture was stirred at room temperature for 20 minutes.
- Total yield 16%) This was purified by HPLC (column, YMC-Pack Hydrosphere C18 (20 mm inner diameter ⁇ 150 mm length); flow rate 8 mL / min; detection UV 254 nm) using 65% methanol / water as an eluent. An oily material was obtained.
- Example 4 (E) -1- (3- (3- (2- (2-butoxyethoxy) ethoxy) prop-1-yn-1-yl) phenyl) -4,4-dimethyl-2- (1H Synthesis of -1,2,4-triazol-1-yl) pent-1-en-3-ol The reaction was carried out in the same manner as described in Example 3 using 1-butanol instead of 2-methoxyethanol to obtain the title compound (total yield: 15%).
- Example 5 ( ⁇ )-(S)-(E) -1- (3- (2,5,8,11-tetraoxatetrades-13-in-14-yl) phenyl) -4,4- Synthesis of dimethyl-2- (1H-1,2,4-triazol-1-yl) pent-1-en-3-ol Under an argon stream, (E) -1- (3- (2,5,8,11-tetraoxatetrades-13-in-14-yl) phenyl) -4,4-dimethyl-2- (1H-1 , 2,4-Triazol-1-yl) pent-1-en-3-ol 57.0 mg (0.12 mmol) was dissolved in 5 mL of toluene, and N- (para-toluenesulfonyl) -L-phenylalanyl chloride was dissolved.
- Example 6 ( ⁇ )-(S)-(E) -1- (3- (2,5,8,11-tetraoxatetrades-13-in-14-yl) phenyl) -4,4- Synthesis of dimethyl-2- (1H-1,2,4-triazol-1-yl) pent-1-en-3-ol Under an argon stream, (E) -1- (3- (2,5,8,11-tetraoxatetrades-13-in-14-yl) phenyl) -4,4-dimethyl-2- (1H-1 , 2,4-Triazol-1-yl) pent-1-en-3-ol was dissolved in 5 mL of toluene, and N- (para-toluenesulfonyl) -L-phenylalanyl chloride was dissolved.
- Test Example 1 Arabidopsis seed germination inhibition test (1) Arabidopsis thaliana (Col-0) seeds were immersed in 500 ⁇ L of 70% ethanol aqueous solution for 30 minutes, then in 100 ⁇ l ethanol 500 ⁇ L for 1 minute, washed three times with 1 mL of distilled water, and then the seeds were immersed in distilled water. Spring treatment was performed at 4 ° C. for 3 days in the dark. Put the methanol solution of the sample and the methanol solution of ABA into a 1.5 mL microtube, remove the methanol under reduced pressure, add 0.5% agar aqueous solution (containing 1/2 MS medium inorganic salts) at 70 ° C, and stir. did.
- Test Example 2 Arabidopsis seed germination inhibition test (2) Using the ( ⁇ ) body of Example 1, the ( ⁇ ) body of Example 2, and the ( ⁇ ) body of Comparative Example 1 as samples, the same test as in Test Example 1 was performed. The state of germination 120 hours after sowing is shown in FIG. Compared with the ( ⁇ ) body of Comparative Example 1, it was found that the ( ⁇ ) body of Example 1 and the ( ⁇ ) body of Example 2 can inhibit seed germination even at lower concentrations.
- Test Example 3 Rice Second Leaf Sheath Elongation Inhibition Test Rice (Oryza sativa L. cv. Nihonbare) seeds were immersed in ethanol for 5 minutes, then washed 10 times with water, immersed in water at 30 ° C. under continuous light. Cultured for 3 days to germinate. A methanol solution of the sample was placed in a culture tube, and after removing methanol under reduced pressure, 2 mL of deionized water was added. Seven seeds of germinating seeds were put in this, the lid was covered, and after culturing at 30 ° C. for 7 days under continuous light, the second leaf sheath length was measured, and the inhibition rate was calculated according to the following formula (2).
- Elongation inhibition rate (%) ⁇ 1 ⁇ (second leaf sheath length of each specimen / second leaf sheath length of control) ⁇ ⁇ 100 (2)
- Test Example 4 Abscisic acid 8′-hydroxylase CYP707A3 inhibition test In a 1.5 mL microtube, 10 ⁇ L of a 250 ⁇ M ABA potassium phosphate buffer (KPB) solution, 5 ⁇ L of a sample dimethylformamide solution, 435 ⁇ L of KPB Then, 40 ⁇ L of abscisic acid 8′-hydroxylase CYP707A3 + NADPH-P450 reductase ATR1 was added and heated at 30 ° C. for 10 minutes. Enzymatic reaction was started by adding 10 ⁇ L of 5 mg / mL NADPH KPB solution. The mixture was reacted at 30 ° C.
- KPB ABA potassium phosphate buffer
- phaseinic acid PA
- the enzyme reaction solution was applied to an OASIS HLB cartridge (1 cc, 30 mg; washed with 1 mL of methanol (1% acetic acid) and equilibrated with 1 mL of water (1% acetic acid)). 1 mL of 10% methanol / water (1% acetic acid) After washing, phaseinate was eluted with 1 mL of methanol (1% acetic acid).
- Test Example 5 ent-kaurene oxidase CYP701A6 inhibition test 8 ⁇ L of a 45% cyclodextrin aqueous solution of 500 ⁇ M ent-kaurene as a substrate and 8 ⁇ L of a sample dimethylformamide solution were placed in a 1.5 mL microtube. Thereto were added 10 ⁇ L of 2.32 ⁇ M ent-kaurene oxidase CYP701A6 and 10 ⁇ L of 4.6 unit / mL NADPH-P450 reductase ATR2, which had been heated at 30 ° C. for 5 minutes in advance. The enzyme reaction was started by adding 304 ⁇ L of ultrapure water that had been heated at 30 ° C.
- ent-kaurenoic acid which is an enzyme metabolite.
- the CYP701A6 inhibition rate by the sample was calculated according to the following formula (4).
- the peak area of ent-kaurenoic acid was calculated from the peak area of m / z 273, and it was confirmed that it was ent-kaurenoic acid from the area ratio of m / z 257,213.
- the peak area of abietic acid was calculated from the area of m / z 256, and it was confirmed that it was abietic acid from the area ratio of m / z 316.
- Enzyme inhibition rate (%) ⁇ 1- (ent-kaurenoic acid peak area of each sample / abietic acid peak area of each sample) / (control ent-kaurenoic acid peak area / control abietic acid peak area) ⁇ ⁇ 100 ... (4)
- Test Example 6 Arabidopsis Growth Test Five 2-week-old Arabidopsis thaliana (Col-0) grown in Gelzan medium (containing MS medium mixed salts and 1% sucrose), vermiculite and promix 1: The mixture was transplanted into a 200 mL plastic pot containing the culture soil mixed in 1 and grown for 2 weeks under conditions of 22 ° C., 16 hours light period to 8 hours dark period. At this stage, the water content was adjusted so that the mass of the pot containing the plant body and the culture soil was about 150 g.
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Abstract
Description
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)1.28(9H,s,t-Bu),5.20(2H,s,H-1),7.96(1H,s,H-3’),8.15(1H,s,H-5’)
製造例2で得られたEZ体の混合物100.1mg(0.263mmol)をメタノールに溶解し、氷冷しながらUVランプ(UVP BLACK-RAY LONGWAVE ULTRAVIOLET LAMP,MODEL B-100A)を照射した。2時間後、UV照射を止めて減圧濃縮し、淡黄色油状物質100.2mgを得た。これをヘキサン-酢酸エチル(75:25)を溶離液としたシリカゲルカラムクロマトグラフィー(12g,1.2cm内径×20cm長さ)に供し、EZ混合物10.3mg及びE体97.2mg(0.255mmol)を淡黄色油状物質として得た(収率97%)。
Z体
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)1.22(9H,s,t-Bu),6.68-6.71(1H,m,H-6’’),6.98(1H,t,J=7.9Hz,H-5’’),7.27(1H,m,H-2’’),7.46(1H,s,H-1),7.64-7.68(1H,m,H-4’’),7.98(1H,s,H-3’),8.18(1H,s,H-5’)
E体
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)1.03(9H,s,t-Bu),7.08(1H,s,H-1)7.12(1H,t,J=7.9Hz,H-5’’),7.30-7.33(1H,m,H-6’’),7.67-7.70(1H,m,H-2’’and4’’),8.07(1H,brs,H-3’),8.26(1H,brs,H-5’)
高分解能MS:C15H16I1N3Na1O1に対する計算値404.02357;実測値404.02399
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)0.68(9H,s,t-Bu),4.26(1H,d,J=8.6Hz,OH),4.53(1H,d,J=8.6Hz,H-3),6.88(1H,s,H-1),7.16(1H,t,J=7.9Hz,H-5’’),7.34-7.37(1H,m,H-6’’),7.68-7.76(2H,m,H-4’’and2’’),8.05(1H,s,H-3’),8.48(1H,s,H-5’)
高分解能MS:C15H18I1N3Na1O1に対する計算値406.03922;実測値406.03914
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)0.58(9H,s,t-Bu),2.37(3H,s,H-14’’’),3.57-3.67(6H,m,H-4’’’,5’’’and6’’’),4.10-4.13(2H,m,H-7’’’),4.34(2H,s,H-3’’’),4.47(1H,brs,H-3),6.85(1H,s,H-1),7.19-7.38(6H,m,H-5’’,6’’,9’’’,10’’’,12’’’,13’’’),7.72-7.75(2H,m,H-2’’and4’’),7.98(1H,s,H-3’),8.44(1H,brs,H-5’)
高分解能MS:C29H35N3Na1O6S1に対する計算値576.21443;実測値576.21478
Z体
1H-NMR(270MHz,CD3OD):δ(ppm)0.99(9H,s,t-Bu),7.13(1H,m,H-2’’or6’’),7.16(1H,m,H-2’’or6’’),7.24(1H,brs,H-1),7.73(1H,m,H-3’’or5’’),7.77(1H,m,H-3’’or5’’),8.12(1H,s,H-3’),8.92(1H,s,H-5’)
E体
1H-NMR(270MHz,CD3OD):δ(ppm)1.26(9H,s,t-Bu),6.62(1H,m,H-2’’or6’’),6.66(1H,m,H-2’’or6’’),7.64(1H,m,H-3’’or5’’),7.68(1H,m,H-3’’or5’’),7.73(1H,brs,H-1),8.23(1H,s,H-3’),8.45(1H,s,H-5’)
1H-NMR(270MHz,CD3OD):δ(ppm)0.56(9H,s,t-Bu),4.54(1H,s,H-3)6.95(1H,s,H-1),7.09(1H,m,H-2’’or6’’),7.12(1H,m,H-2’’or6’’),7.68(1H,m,H-3’’or5’’),7.70(1H,m,H-3’’or5’’),7.99(1H,s,H-3’),8.82(1H,s,H-5’)
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)0.66(9H,s,t-Bu),2.44(3H,s,H-14’’’),3.64-3.74(6H,m,H-4’’’,5’’’and6’’’),4.19(2H,t,J=4.6Hz,H-7’’’),4.41(2H,s,H-3’’’),4.58(1H,brs,H-3),6.93(1H,s,H-1),7.34(4H,m,H-2’’,6’’,10’’’and12’’’),7.49(2H,m,H-3’’and5’’),7.81(2H,m,H-9’’’and13’’’),8.06(1H,brs,H-3’),8.49(1H,brs,H-5’)
高分解能MS:C29H35N3Na1O6S1に対する計算値576.21443;実測値576.21416
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)0.66(9H,s,t-Bu),3.38(3H,s,H-10’’’),3.49-3.80(12H,m,H-4’’’,5’’’,6’’’,7’’’,8’’’and9’’’),4.26(1H,d,J=8.9Hz,OH),4.44(2H,s,H-3’’’),4.54(1H,d,J=8.9Hz,H-3),6.92(1H,s,H-1),7.31-7.45(4H,m,H-2’’,3’’,4’’and6’’),8.05(1H,s,H-3’),8.50(1H,s,H-5’)
UV λmax(MeOH) nm(ε):241(33000)
高分解能MS:C25H35N3Na1O5に対する計算値480.24744;実測値480.24698
(-)体
ee>99.96(%)
[α]29 D -2.8(MeOH;c0.52)
(+)体
ee=99.96(%)
[α]30 D +3.6(MeOH;c0.54)
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)0.66(9H,s,t-Bu),0.91(3H,t,J=7.3Hz,H-11’’’),1.22-1.43(2H,m,H-10’’’),1.52-1.62(2H,m,H-9’’’),3.47(2H,t,J=6.6Hz,H-8’’’),3.59-3.81(8H,m,H-4’’’,5’’’,6’’’and7’’’),4.27(1H,d,J=8.6Hz,OH),4.45(2H,s,H-3’’’),4.55(1H,d,J=8.6Hz,H-3),6.92(1H,s,H-1),7.31-7.45(4H,m,H-2’’,3’’,4’’and6’’),8.05(1H,s,H-3’),8.50(1H,s,H-5’)
UV λmax(MeOH) nm(ε):241(35000)
高分解能MS:C26H37N3Na1O4に対する計算値478.26818;実測値478.26751
(-)体
ee>99.96(%)
[α]32 D -4.4(MeOH;c0.5)
(+)体
ee=99.96(%)
[α]32 D +3.8(MeOH;c0.5)
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)0.66(9H,s,t-Bu),3.38(3H,s,H-10’’’),3.54-3.80(12H,m,H-4’’’,5’’’,6’’’,7’’’,8’’’and9’’’),4.27(1H,d,J=8.9Hz,OH),4.45(2H,s,H-3’’’),4.57(1H,d,J=8.9Hz,H-3),6.93(1H,s,H-1),7.32-7.35(2H,m,H-2’’and6’’),7.47-7.50(2H,m,H-3’’and5’’),8.05(1H,s,H-3’),8.48(1H,s,H-5’)
UV λmax(MeOH)nm(ε):270(24000)
高分解能MS:C25H35N3Na1O5に対する計算値480.24744;実測値480.24734
(-)体
ee>99.84(%)
[α]32 D -35(MeOH;c0.81)
(+)体
ee=99.84(%)
[α]32 D +36(MeOH;c0.82)
1H-NMR(500MHz,CD3OD):δ(ppm)0.22(9H,s,t-Bu),0.48(3H,t,J=7.6Hz,H-11’’’),0.90-0.98(2H,m,H-10’’’),1.08-1.14(2H,m,H-9’’’),3.04(2H,t,J=6.7Hz,H-8’’’),3.14-3.16(2H,m,H-7’’’),3.20-3.22(2H,m,H-6’’’),3.25-3.26(2H,m,H-5’’’),3.31-3.33(2H,m,H-4’’’),4.00(2H,s,H-3’’’),4.24(1H,s,H-3),6.66(1H,s,H-1),6.97-6.98(2H,m,H-2’’and6’’),7.05-7.06(2H,m,H-3’’and5’’),7.65(1H,s,H-3’),8.48(1H,s,H-5’)
UV λmax(MeOH) nm(ε):271(24000)
高分解能MS:C26H37N3Na1O4に対する計算値478.26818;実測値478.26763
(-)体
ee>99.9(%)
[α]27 D -36(MeOH;c0.78)
(+)体
ee>99.9(%)
[α]28 D +32(MeOH;c0.75)
1H-NMR(270MHz,CDCl3,TMS):δ(ppm)0.52(9H,s),2.23(3H,s),2.89(1H,dd),3.10(1H,dd,J=13.8and6.9Hz),3.34(3H,s),3.49-3.84(12H,m),4.36(1H,dd,J=7.9and7.3Hz),4.46(2H,s),5.82(1H,s),7.08-7.63(14H,m),8.14(1H,s),8.80(1H,s)
1H NMR(270MHz,CDCl3,TMS):δ(ppm)0.52(9H,s),2.23(3H,s),2.89(1H,dd),3.10(1H,dd,J=13.8and6.9Hz),3.34(3H,s),3.49-3.84(12H,m),4.36(1H,dd,J=7.9and7.3Hz),4.46(2H,s),5.82(1H,s),7.08-7.63(14H,m),8.14(1H,s),8.80(1H,s)
シロイヌナズナ(Arabidopsis thaliana,Col-0)種子を70%エタノール水溶液500μLに30分、続いて100%エタノール500μLに1分浸漬し、蒸留水1mLで3回洗浄した後に、種子を蒸留水に浸漬させ、暗所下、4℃で3日間春化処理した。試料のメタノール溶液及びABAのメタノール溶液を1.5mL容マイクロチューブに入れ、減圧下にてメタノールを除去し、70℃の0.5%寒天水溶液(含1/2MS培地無機塩類)を加えて撹拌した。それを96穴プレートに入れ、サンプルを含む培地とした。ここに春化処理したシロイヌナズナ種子を20-30粒ずつ播種し、連続光下、22℃で培養してその発芽数を経時的に観察した。下記式(1)にしたがって発芽率を計算した。なお,メタノールを用いて同様に培地を調製したものを無処理区とし、播種72時間後に発芽率100%を示した。播種72時間後の発芽率を図1に示し、120時間後のシロイヌナズナの初期生育の様子を図2に示す。実施例1~4の化合物の投与により種子の発芽が阻害されることが明らかとなった。
発芽率(%)=発芽数(個)/播種数(個)×100…(1)
試料に、実施例1の(-)体、実施例2の(-)体及び比較例1の(-)体を用いて、試験例1と同様の試験を行った。播種120時間後の発芽の様子を図3に示す。比較例1の(-)体と比較して、実施例1の(-)体及び実施例2の(-)はより低濃度でも種子の発芽を阻害できることが明らかとなった。
イネ(Oryza sativa L. cv. Nihonbare)種子をエタノールに5分間浸漬した後、水で10回洗浄し、水に浸漬して連続光下、30℃で3日間培養し発芽させた。試料のメタノール溶液を培養管に入れ、メタノールを減圧下で除去した後、脱イオン水2mLを加えた。これに発芽種子を7粒ずつ入れ、蓋をして連続光下、30℃で7日間培養後に第二葉鞘長を測定し、下記式(2)にしたがって阻害率を算出した。なお、脱イオン水のみで培養したものをコントロールとした。結果を図4に示す。実施例1~4の化合物の高濃度投与によってのみ葉鞘の伸長が阻害されることが明らかとなった。
伸長阻害率(%)={1-(各検体の第二葉鞘長/コントロールの第二葉鞘長)}×100…(2)
1.5mLマイクロチューブに、基質である250μM ABAのカリウム・リン酸緩衝液(KPB)溶液を10μL、試料のジメチルホルムアミド溶液を5μL、KPB435μL、アブシジン酸8’位水酸化酵素CYP707A3+NADPH-P450還元酵素ATR1 40μLを加え、30℃で10分間加温した。5mg/mLのNADPHのKPB溶液を10μL添加して酵素反応を開始した。30℃、1000rpmで10分間反応し、1M水酸化ナトリウム水溶液を25μL加えて反応を停止した。4℃で30分静置した後、1M塩酸50μLを加えて酵素代謝物をファゼイン酸(PA)に変換した。OASIS HLBカートリッジ(1cc,30mg;メタノール(1%酢酸)1mLで洗浄し、水(1%酢酸)1mLで平衡化したもの)に酵素反応溶液を供し、10%メタノール/水(1%酢酸)1mLで洗浄した後、メタノール(1%酢酸)1mLでファゼイン酸を溶出した。減圧濃縮し(トルエンを加えて酢酸を共沸)、メタノール50μLに溶解して20μLをHPLC(カラム、YMC-Pack HydrosphereC18(6mm内径×150mm長さ);流速1mL/min;検出、UV254nm)分析に供した。下記式(3)にしたがって試料によるCYP707A3阻害率を算出した。なお、コントロールには試料溶液の代わりにジメチルホルムアミドを用いた。各試料について2種類の濃度について試験を行い、その酵素阻害率から50%阻害濃度(IC50)を算出した。その結果を表1に示す。
酵素阻害率(%)={1-(各試料のPAピーク面積/コントロールのPAピーク面積)}×100…(3)
1.5mLマイクロチューブに、基質である500μM ent-カウレンの45%シクロデキストリン水溶液を8μL、試料のジメチルホルムアミド溶液を8μL入れた。そこへ、予め30℃で5分間加温しておいた2.32μMのent-カウレンオキシダーゼCYP701A6を10μLと4.6unit/mLのNADPH-P450還元酵素ATR2を10μL添加した。予め30℃で10分間加温しておいた超純水304μL、500mM KPB40μL、20mM NADPH水溶液20μLを添加して酵素反応を開始した。30℃、1000rpmで30分間反応し、1M塩酸を100μL加えて反応を停止した。内部標準として10μMのアビエチン酸5μLを添加して酢酸エチル400μLで抽出し、硫酸ナトリウムで脱水した後、減圧濃縮した。メタノール50μLと2Mトリメチルシリルジアゾメタン5μLを添加してカルボン酸をメチル化し、窒素乾固した。ヘキサン150μLに溶解して1μLをGC-MS分析に供し、酵素代謝物であるent-カウレン酸を検出した。下記式(4)にしたがって試料によるCYP701A6阻害率を算出した。なお、ent-カウレン酸のピーク面積はm/z273のピーク面積から算出し、m/z257,213の面積比からent-カウレン酸であることを確認した。アビエチン酸のピーク面積はm/z256の面積から算出し、m/z316の面積比からアビエチン酸であることを確認した。コントロールには試料溶液の代わりにジメチルホルムアミドを用いた。各試料について2種類の濃度について試験を行い、その酵素阻害率から50%阻害濃度(IC50)を算出した。その結果を表2に示す。
酵素阻害率(%)={1-(各試料のent-カウレン酸ピーク面積/各試料のアビエチン酸ピーク面積)/(コントロールのent-カウレン酸ピーク面積/コントロールのアビエチン酸ピーク面積)}×100…(4)
Gelzan培地(MS培地混合塩類と1%ショ糖を含む)で生育させた2週齢のシロイヌナズナ(Arabidopsis thaliana,Col-0)5個体を、バーミキュライトとプロミックスを1:1で混合させた培養土を入れた200mL容のプラスチックポットに移植し、22℃、16時間明期-8時間暗期の条件で2週間生育させた。この段階で、植物体と培養土を含むポットの質量が約150gになるように水分量を調整した。27ポット(合計135個体のシロイヌナズナ実生)に、実施例1の(-)体の50μM水溶液(0.05%Tween-20と0.2%のMeOHを含む)50mLを、1日1回ずつ3日間、地上部に散布した。上記化合物を含まない水溶液(0.05%Tween-20と0.2%のMeOHを含む)50mLを、1日1回ずつ3日間、地上部に散布したポットをコントロールとして用いた。12日間の乾燥処理後に灌水し、3日後の生存個体数を測定し、下記式(5)にしたがって、生存率を算出した。コントロールのポットでは、乾燥処理後のシロイヌナズナの生存率が15%であったのに対し、実施例1の(-)体50μM水溶液を散布されたポットでは、乾燥処理後のシロイヌナズナの生存率は75%であった。シロイヌナズナに実施例1の(-)体の化合物を散布することにより、乾燥による枯死が緩和されることが明らかとなった。
生存率(%)=生存個体数(個)/全個体数(個)×100…(5)
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JP2013231014A (ja) * | 2012-05-01 | 2013-11-14 | Chiba Univ | 植物の乾燥耐性付与方法及びそれに用いられる植物乾燥耐性付与剤 |
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Title |
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OKAZAKI,M. ET AL.: "Abscinasole-E2B, a practical and selective inhibitor ob ABA 8'-hydroxylase", CYP707A, BIOORGANIC & MEDICINAL CHEMISTRY, vol. 20, no. Iss.10, 2012, pages 3162 - 3172, XP028422750, DOI: doi:10.1016/j.bmc.2012.03.068 * |
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