WO2017036153A1 - 增强植物抗逆性的小分子化合物 - Google Patents

增强植物抗逆性的小分子化合物 Download PDF

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WO2017036153A1
WO2017036153A1 PCT/CN2016/080790 CN2016080790W WO2017036153A1 WO 2017036153 A1 WO2017036153 A1 WO 2017036153A1 CN 2016080790 W CN2016080790 W CN 2016080790W WO 2017036153 A1 WO2017036153 A1 WO 2017036153A1
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compound
formula
aba
preparation
plant
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PCT/CN2016/080790
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English (en)
French (fr)
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朱健康
曹民杰
张玉路
刘雪
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中国科学院上海生命科学研究院
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Priority to AU2016314390A priority Critical patent/AU2016314390B2/en
Priority to US15/756,075 priority patent/US10842151B2/en
Priority to EP16840582.7A priority patent/EP3342766B1/en
Publication of WO2017036153A1 publication Critical patent/WO2017036153A1/zh

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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
    • A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
    • 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
    • A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
    • A01N3/02—Keeping cut flowers fresh chemically
    • 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
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
    • A01N43/42—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings condensed with carbocyclic rings
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/88—Vitaceae, e.g. Vitus [grape]

Definitions

  • Abscisic Acid is a key factor in balancing the metabolism of endogenous hormones and related active substances in plants. It has the ability to promote the balanced absorption of water, fertilizer and coordination of metabolism in plants, and can effectively regulate root/crown and vegetative growth of plants. And reproductive growth plays an important role in improving the quality and yield of crops.
  • ABA Abscisic Acid
  • exogenous ABA can cause rapid closure of leaf stomata, inhibit transpiration, and can be used for flower preservation, or to prevent wilting during transportation of crop seedlings.
  • ABA can also control flower bud differentiation, regulate flowering, and has great application value in flower gardening.
  • ABA can improve the growth of crops in low temperature, drought, spring cold, salt and other adverse growth environments. Therefore, ABA has a wide range of applications and can be used in lawns, farmland, gardens, and especially in water-deficient areas such as the western region, which is of great significance for the development of China's agricultural industry.
  • R 1 is H, halogen, C 1 -C 3 alkyl, or C 1 -C 3 haloalkyl
  • R 4 is H, halogen, C 1 -C 3 alkyl, or C 1 -C 3 haloalkyl
  • R 0 is H, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, or halogen;
  • the compound has the structure of formula Ia, Ib or Ic:
  • R 0 , R 1 - R 6 , m are as defined above.
  • R 1 , R 2 , R 3 and R 4 are F.
  • the plant comprises intact plants, organs (such as roots, stems, leaves, branches, flowers, fruits, seeds), tissues (such as callus), or cells.
  • organs such as roots, stems, leaves, branches, flowers, fruits, seeds
  • tissues such as callus
  • the agricultural preparation contains 0.0001 to 99% by weight, preferably 0.1 to 90% by weight, of the component (i), based on the total weight of the agricultural preparation.
  • the dosage form of the agricultural preparation is selected from the group consisting of a solution, an emulsion, a suspension, a powder, a foaming agent, a paste, a granule, an aerosol, or a combination thereof.
  • the dose is from 2 to 100 g/ha, preferably from 4 to 80 g/ha, more preferably from 6 to 60 g/ha.
  • a process for the preparation of a compound of the formula I or a salt thereof comprising the steps of:
  • R 0, R 1, R 2, R 3, R 4, R 5, R 6, R 7, m and As defined above.
  • the acid binding agent is selected from the group consisting of potassium carbonate, triethylamine, pyridine, or a combination thereof.
  • the inert solvent is selected from the group consisting of DMF, DCM, acetonitrile or a combination thereof.
  • reaction time is from 0.1 to 72 hours, more preferably from 1 to 24 hours, more preferably from 2 to 20 hours.
  • the plant to be preserved is contacted with a compound of the formula I according to the first aspect of the invention, or a salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a precursor thereof, thereby Plant preservation.
  • the plant comprises whole plants, or plant organs (such as roots, stems, leaves, branches, flowers, fruits).
  • the plant preservative treatment comprises fresh-keeping of flowers.
  • a seventh aspect of the present invention provides a fruit coloring processing method comprising the steps of:
  • the fruit to be colored is contacted with a compound of the formula I according to the first aspect of the invention, or a salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a precursor thereof, thereby Fruit coloring.
  • the fruit comprises grapes.
  • the eighth aspect of the present invention provides a compound of the formula I according to the first aspect of the invention, or a salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a precursor thereof, or a precursor thereof Characterized by, for the preparation of (i) an agonist of an ABA receptor; and/or (ii) a seed germination inhibitor.
  • the agonist promotes the interaction of the ABA receptor PYL protein with the PP2C protein phosphatase.
  • Figure 1 shows that the two AMX compounds 0604c and 0918 of the present invention can bind to the Arabidopsis PYR/PYL receptor (PYR1, PYL1, PYL2 and PYL7)-HAB1 complex, thereby inhibiting the activity of the protein phosphatase HAB1.
  • PYR1, PYL1, PYL2 and PYL7-HAB1 complex At 1 ⁇ M concentration, the inhibitory effect was significantly better than ABA.
  • FIG. 2 shows dose response curves for six AMX compounds (0604c, 0918, 1125A, 1125B, 1127 and 1103B) and Arabidopsis PYR/PYL receptor agonists of ABA.
  • AMX compounds can promote the interaction of protein phosphatase HAB1 with four Arabidopsis PYR/PYL receptors (PYR1, PYL1, PYL2 and PYL7), and this effect has a dose-dependent effect.
  • FIG. 3 shows dose response curves for three AMX compounds (0925, 1020A and 1020B) and PYR/PYL receptor agonists of ABA.
  • the AMX compound promotes the interaction of the protein phosphatase HAB1 with three PYR/PYL receptors (PYR1, PYL1 and PYL2), and this interaction has a dose-dependent effect.
  • Figures 4a to 4i respectively show two-dimensional interaction of ABA(a), existing ABA analogs (b) or multiple AMX compounds of the invention (c to i) with multiple amino acid residues in the PYL2-HAB1 complex pocket structure structure.
  • the figure shows the water molecule, the nitrogen atom, the oxygen atom and the halogen atom, the dashed line represents the hydrogen bond, and the number on it represents the distance between two atoms/molecule (in angstroms, ).
  • the results show that the AMX compound of the present invention forms more hydrogen bonds with the amino acid residues in the PYL2 pocket structure than the existing ABA or its analog, which makes the binding of AMX to the PYL2-HAB1 complex more compact.
  • Figure 5 shows changes in the transcriptional level of stress-related genes induced by ABA in wild-type Arabidopsis thaliana after treatment with different AMX compounds of the present invention for 6 hours.
  • DMSO and ABA treatment were negative and positive control groups, respectively.
  • Figure 6 shows the effect of different AMX compounds and ABA on seed germination of Col-0 and pyr1; pyl1; pyl4 triple mutants at a concentration of 1 ⁇ M.
  • Col-0 was sown in the left half of each dish, and pyr1; pyl1; pyl4 triple mutant was seeded in the right half.
  • the photographs were pyr1; pyl1; pyl4 triple mutant seeds were photographed 4 days after germination (after 6 days of sowing).
  • DMSO treatment was used as a control group.
  • FIGS. 7a and 7b show that treatment of the AMX compound of the present invention significantly reduces the transpiration rate of Arabidopsis leaf surface, resulting in elevated leaf surface temperature.
  • Figure 7a shows that after 5 ⁇ M AMX compound and ABA treatment, the leaf surface temperature was significantly increased compared to DMSO treatment. Among them, 0918 and 1127 could significantly inhibit foliar transpiration after four days of treatment.
  • Figure 7b shows a concentration-dependent effect of the inhibitory effect of AMX compounds on foliar transpiration.
  • the inhibitory effect at the same concentration is 1127>0918>1125A.
  • Figure 8 shows the results of the soil drought experiment. Wild-type Arabidopsis plants (Col-0) grown for 3 weeks in a short-day environment were stopped from watering and sprayed with the corresponding concentrations of compounds. DMSO was used as a negative control.
  • Figure 8a shows the growth of plants on the day of the first spraying of the compound and after 17 days. The concentration of the compound used in the experiment was 10 ⁇ M.
  • Figure 8b shows the growth of the plants on the day of the first spraying of the compound, 18 and 20 days later. The concentration of the compound is 5 ⁇ M;
  • Figure 8c The growth of the plants on the day of the first spraying of the compound and after 14 days was shown, and the concentration of the compound used in the experiment was 10 ⁇ M.
  • FIG. 9 shows dose response curves of three AMX compounds (0604c, 0918 and 1127) and ABA for soybean PYR/PYL receptor agonists.
  • the AMX compound promotes the interaction of Arabidopsis protein phosphatase HAB1 with two soybean PYR/PYL receptors (GmPYL3 and GmPYL6), and this interaction has a dose-dependent effect.
  • Figure 10 shows that 50 ⁇ M AMX compound 1127 can significantly reduce the transpiration rate of Arabidopsis leaf surface after 3 days of treatment, resulting in elevated leaf surface temperature.
  • FIG 11 shows the results of soil drought experiments for soybean and corn.
  • the soybean plants with the same growth were selected and rehydrated after one week of drought.
  • the photos showed the overall growth after one month of rehydration.
  • the concentration of each test compound in the experiment was 50 ⁇ M.
  • the corn experiment method is the same as the soybean experiment.
  • the concentration of compound 0918 was 50 ⁇ M in the experiment.
  • the survival rate of soybean and corn treated with AMX after rewatering was significantly higher than that of the control group.
  • Figure 12 shows that the anthocyanin content of grape skin after treatment with AMX compound 1127 was significantly higher than that of the clear water control group. Anthocyanin content is positively correlated with grape skin coloration.
  • Figure 13 shows that the compound 0720B of the present invention can bind to the Arabidopsis PYR/PYL receptor (PYR1, PYL1, PYL2 and PYL7)-HAB1 complex, thereby inhibiting the activity of the protein phosphatase HAB1.
  • the inhibitory effect was significantly better than ABA.
  • Figure 14 shows dose response curves of Arabidopsis PYR/PYL receptor agonists of Compound 0720B of the present invention and ABA.
  • the 0720B compound promotes the interaction of the protein phosphatase HAB1 with the two Arabidopsis PYR/PYL receptors (PYR1 and PYL2), and this effect has a dose-dependent effect.
  • FIG 15 shows that treatment of AMX compound (0720B) significantly reduced the transpiration rate of soybean leaf surface, resulting in elevated leaf surface temperature. After 16 days of sowing, the soybean plants were stopped from watering, and the corresponding compounds were sprayed. Compared with the control group (DMSO), spraying 10/20/50 ⁇ M of compound 0720B significantly reduced the transpiration rate of soybean leaves, and the inhibition effect was better. Spray 50 ⁇ M ABA.
  • Figure 16 shows that AMX compound (0720B) treatment significantly reduced the transpiration rate of cotton leaf surface, resulting in elevated leaf surface temperature. After 23 days of sowing, the cotton plants were stopped from watering, and 50 ⁇ M of compound 0720B was sprayed. Compared with the control group (DMSO), 50 ⁇ M of compound 0720B significantly reduced the transpiration rate of cotton leaf surface, and the inhibition effect was better than 50 ⁇ M ABA.
  • FIG 17 shows the results of soil drought experiments in soybeans.
  • the soybean in Fig. 15 was rehydrated after 11 days of drought, and the photograph showed the growth of soybeans after one day of rehydration.
  • the growth of soybeans treated with 10/20/50 ⁇ M of compound 0720B was significantly better than that of the control (DMSO) or 50 ⁇ M ABA treated soybeans.
  • Figure 18 shows the results of soil drought experiments in cotton.
  • the photograph shows the growth of the cotton in Figure 16 after 10 days of drought.
  • Cotton treated with 50 ⁇ M compound 0720B was significantly better than control (DMSO) or 50 ⁇ M ABA treated cotton.
  • the inventors have developed a class of ABAs with high abscisic acid (ABA) activity for the first time through extensive and in-depth research.
  • Alternative (compound of the invention) The compounds of the present invention have significantly higher activity than existing ABA analogs and can significantly enhance a variety of plant resistance (e.g., drought resistance, cold tolerance, etc.).
  • the compound of the invention has the advantages of simple preparation method, excellent environmental friendliness and rapid action, and therefore has wide application prospects.
  • the present invention has been completed on this basis.
  • AMX compounds not only have better activity than abscisic acid (ABA) but also existing ABA analogs (such as 4-methyl-nitrogen-(1,2,3,4-tetra Hydrogen-2-carbonyl-1-propyl-6-quinolinyl)-benzylsulfonamide (4-methyl-N-(1,2,3,4-tetrahydro-2-oxo-1-propyl-6) -quinolinyl)-benzene methanesulfonamide)), and can bind to a variety of different PYL receptors, can significantly enhance the resistance of various plants.
  • ABA abscisic acid
  • C 1 -C 7 alkyl refers to a straight or branched alkyl group having from 1 to 7 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, iso Butyl, sec-butyl, tert-butyl, or the like.
  • C 2 -C 7 alkenyl refers to a straight or branched alkenyl group having 2 to 7 carbon atoms, such as ethenyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.
  • C 2 -C 7 alkynyl group means a straight or branched alkynyl chain having 2-7 carbon atoms, e.g. ethynyl, propynyl, or the like.
  • C 1 -C 3 alkyl refers to a straight or branched alkyl group having from 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, or the like. group.
  • C 1 -C 3 haloalkyl refers to a straight or branched alkyl group having from 1 to 3 carbon atoms in which hydrogen is substituted by one or more halogens, for example, a halomethyl group. , haloethyl, halopropyl, haloisopropyl, or the like.
  • C 3 -C 7 cycloalkyl refers to a cyclic alkyl group having 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or Similar group.
  • halogen means fluoro, chloro, bromo, or iodo, preferably fluoro and chloro.
  • halo refers to a group substituted by the same or different one or more of the above halogen atoms, and may be partially or fully halogenated, such as trifluoromethyl, pentafluoroethyl, heptafluoro. Isopropyl, or a similar group.
  • the compounds of the invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers.
  • the asymmetric center that can exist depends on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention.
  • the invention includes all isomeric forms of the compounds.
  • the terms "compound of the invention”, “AMX compound”, “compound AMX”, “the ABA of the invention” “Substitute”, “compounds of formula I” are used interchangeably and refer to a compound having the structure of formula I.
  • the term also includes salts, optical isomers, racemates, solvates of the compounds of formula I. (such as hydrates), and/or precursors,
  • R 0 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , m The definition is as described above.
  • each reaction is mostly carried out in an inert solvent at 0 ° C to 150 ° C (or reflux temperature) (preferably, 20-60 ° C, or 25-40 ° C) for a period of time. (eg 0.1-72 hours, preferably 2-20 hours).
  • the compounds of formula I of the present invention can be prepared by the following schemes and exemplary methods described in the Examples and related publications used by those skilled in the art.
  • the methods of preparing the compounds of Formula Ia and/or Ib of the present invention may include, but are not limited to, the following schemes.
  • Step 1 First, the compound of the formula Ia-1 is reacted with an alkyl halide at a certain temperature (for example, 25-40 ° C) for a certain period of time in the presence of a base (such as potassium carbonate, sodium hydride) in an inert solvent (such as DMF). A compound of formula Ia-2 is formed.
  • a base such as potassium carbonate, sodium hydride
  • an inert solvent such as DMF
  • Step 2 The compound of formula Ia-2 is reacted with potassium nitrate at a temperature (e.g., 0-20 ° C) for a period of time in the presence of an acid such as sulfuric acid to form a compound of formula Ia-3.
  • a temperature e.g., 0-20 ° C
  • Step 3 The compound of the formula Ia-3 is subjected to a reduction reaction at a certain temperature (for example, 10 to 40 ° C) in an inert solvent (e.g., methanol) using palladium on carbon as a catalyst to form a compound of the formula Ia-4.
  • a certain temperature for example, 10 to 40 ° C
  • an inert solvent e.g., methanol
  • Step 4 A compound of formula Ia-5 (such as p-methylbenzyl bromide or p-methylbenzyl chloride) is reacted with thiourea in an inert solvent such as ethanol or acetonitrile to form the reaction product. Then, in the presence of an acid (such as concentrated hydrochloric acid), the reaction product and sodium chlorite are reacted at a certain temperature (such as 0-20 ° C) for a period of time in an inert solvent (such as acetonitrile) to form Formula Ia. -6 compound.
  • an acid such as concentrated hydrochloric acid
  • Step 5 The compound of the formula Ia-6 is reacted with the compound of the formula Ia-4 at a certain temperature (for example, 25-40 ° C) for a while in an inert solvent such as DMF in the presence of an acid binding agent such as potassium carbonate. Thereby obtaining a compound of the formula Ia/Ib.
  • X 2 is a leaving group and is chlorine, bromine or iodine.
  • Other various substituents and groups are as defined in the specification. Represents a single or double key.
  • the method of preparing the compound of formula Ic of the present invention may include, but is not limited to, the following scheme:
  • Step 1 Hydrogenation of Ia-7 at a certain temperature (such as 50-80 ° C) in an inert solvent (such as acetic acid, ethyl acetate) in the presence of a catalyst (such as palladium carbon, platinum dioxide) for a period of time Forming a compound of formula Ia-8.
  • an inert solvent such as acetic acid, ethyl acetate
  • a catalyst such as palladium carbon, platinum dioxide
  • Step 2 in the presence of a base (such as potassium carbonate, sodium hydride) in an inert solvent (such as DMF), first react the compound of formula Ia-8 with an alkyl halide at a certain temperature (such as 25-40 ° C) for a period of time, A compound of formula Ia-9 is formed.
  • a base such as potassium carbonate, sodium hydride
  • an inert solvent such as DMF
  • Step 3 The compound of formula Ia-9 is reacted with potassium nitrate at a temperature (e.g., 0-20 ° C) for a period of time in the presence of an acid such as sulfuric acid to form a compound of formula Ia-10.
  • a temperature e.g., 0-20 ° C
  • Step 4 In an inert solvent (such as methanol), using palladium on carbon as a catalyst, at a certain temperature (such as 10-40 ° C), The Ia-10 compound is subjected to a reduction reaction to form a compound of the formula Ia-11.
  • an inert solvent such as methanol
  • a certain temperature such as 10-40 ° C
  • Step 5 A compound of formula Ia-5 (such as p-methylbenzyl bromide or p-methylbenzyl chloride) is reacted with thiourea in an inert solvent such as ethanol or acetonitrile to form the reaction product. Then, in the presence of an acid (such as concentrated hydrochloric acid), the reaction product and sodium chlorite are reacted at a certain temperature (such as 0-20 ° C) for a period of time in an inert solvent (such as acetonitrile) to form Formula Ia. -6 compound.
  • an acid such as concentrated hydrochloric acid
  • Step 6 reacting a compound of formula Ia-6 with a compound of formula Ia-11 at a temperature (eg 25-40 ° C) for a period of time in an inert solvent such as DMF in the presence of an acid binding agent such as potassium carbonate. Thereby obtaining a compound of the formula Ic.
  • X 2 is a leaving group and is chlorine, bromine or iodine.
  • Other various substituents and groups are as defined in the specification.
  • the active substance of the present invention (the compound of the formula I, or a salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a precursor thereof) can be prepared into an agricultural preparation by a conventional method, for example, Solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with active substances, microcapsules in polymers, bags for seeds Coating agent.
  • preparations can be produced by known methods, for example, by mixing the active compound with an extender which is a liquid or liquefied gas or solid diluent or carrier, and optionally a surfactant, an emulsifier and/or A dispersant and/or a foam former.
  • an extender which is a liquid or liquefied gas or solid diluent or carrier
  • a surfactant for example, an organic solvent can also be used as an auxiliary.
  • an organic solvent can also be used as an auxiliary.
  • a liquid solvent When a liquid solvent is used as the diluent or carrier, it is basically suitable, such as: aromatic hydrocarbons such as xylene, toluene or alkylnaphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons such as chlorobenzene, vinyl chloride Or methylene chloride; aliphatic hydrocarbons such as cyclohexane or paraffin, such as mineral oil fractions; alcohols such as ethanol or ethylene glycol and their ethers and lipids; ketones such as acetone, methyl ethyl ketone, methyl isobutyl Ketone or cyclohexanone; or less common polar solvents such as dimethylformamide and dimethyl sulfoxide, and water.
  • aromatic hydrocarbons such as xylene, toluene or alkylnaphthalene
  • chlorinated aromatic or chlorinated aliphatic hydrocarbons such as chlorobenzene, vinyl chloride Or
  • the diluent or carrier of the liquefied gas means a liquid which will become a gas at normal temperature and pressure, such as an aerosol push.
  • Ingredients such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.
  • the solid support can be ground natural minerals such as kaolin, clay, talc, quartz, activated clay, montmorillonite, or diatomaceous earth, and ground synthetic minerals such as highly dispersed silicic acid, alumina and silicates.
  • Solid supports for granules are ground and graded natural zircons such as calcite, marble, pumice, sepiolite and dolomite, as well as inorganic and organic coarse powder synthetic particles, and organic materials such as sawdust, coconut shell, Corn cobs and granules of tobacco stems.
  • Emulsified columns of nonionic and anionic agents can be used as emulsifiers and/or foam formers.
  • polyoxyethylene-fatty acid esters polyoxyethylene-fatty alcohol ethers, such as alkaryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates, and white Protein hydrolysate.
  • Dispersing agents include, for example, lignin sulfite waste liquid and methyl cellulose.
  • Binders such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or emulsions such as acacia, polyvinyl alcohol and polyvinyl acetate may be employed in the formulation.
  • Coloring agents such as inorganic dyes such as iron oxide, oxidized diamonds and Prussian blue; organic dyes such as organic dyes such as azo dyes or metal phthalocyanine dyes; and trace nutrients such as iron, lanthanum, boron, copper may be used. , cobalt, aluminum and zinc salts, etc.
  • the "agronomic preparation” is generally an agricultural plant growth regulator comprising a compound of the formula I or a salt thereof, an optical isomer, a racemate, a solvate or a precursor thereof for enhancing plant stress resistance.
  • An active ingredient such as drought resistance
  • an agriculturally acceptable carrier for enhancing plant stress resistance.
  • the "agriculturally acceptable carrier” is an agrochemical for the delivery of a compound of the formula I according to the invention, or a salt, optical isomer, racemate, solvate or precursor thereof, to a plant.
  • the carrier can be a liquid or a solid.
  • the agriculturally acceptable carrier suitable for use in the present invention is selected from the group consisting of water, buffer, DMSO, a surfactant such as Tween-20, or a combination thereof. Any agriculturally acceptable carrier known to those skilled in the art can be used in the present invention.
  • the agricultural preparation of the present invention may be combined with other drought-tolerant agents in their commercial preparations or in the preparation forms prepared from these preparations.
  • These other drought-resistant agents include, but are not limited to, drought-resistant seed coating agents, drought-resistant agents.
  • the agricultural preparations of the invention may also be formulated as a mixture with the synergist in their commercial preparations or in the dosage forms prepared from these preparations, which are compounds which enhance the action of the active compound, due to the active compound It is active in itself, and it is not necessary to add a synergist.
  • the dosage form of the agricultural preparation of the present invention may be various, as long as the active ingredient can be effectively brought into the dosage form of the plant, and from the standpoint of ease of preparation and application, the preferred agricultural preparation is a spray. Agent or solution preparation.
  • the application may be carried out by various methods known, for example, by spraying, spraying, dusting or spreading the compound or an agricultural preparation containing the compound on the plant leaf, the propagation material, or otherwise contacting the plant with the compound or containing the compound.
  • An agricultural preparation of a compound, if seeded, is coated, wrapped or otherwise treated.
  • Another method of directly treating plants or seeds prior to planting can also introduce the agricultural preparation of the present invention into soil or other medium to be seeded.
  • a carrier can also be used, which can be a solid, liquid state as described above.
  • the compound or an agricultural formulation containing the compound can also be delivered to the plant by spraying (e.g., by aircraft spraying) or irrigation.
  • AMX compounds not only have better activity than abscisic acid (ABA) but also existing ABA analogs (such as 4-methyl-nitrogen-(1,2,3,4-tetra Hydrogen-2-carbonyl-1-propyl-6-quinolinyl)-benzylsulfonamide (4-methyl-N-(1,2,3,4-tetrahydro-2-oxo-1-propyl-6) -quinolinyl)-benzene methanesulfonamide)), and can bind to a variety of different PYL receptors, can significantly enhance the resistance of various plants.
  • ABA abscisic acid
  • Compound AM1 an existing ABA analog, chemical name 4-methyl-nitro-(1,2,3,4-tetrahydro-2-carbonyl-1-propyl-6-quinolinyl)-benzene 4-methyl-N-(1,2,3,4-tetrahydro-2-oxo-1-propyl-6-quinolinyl)-benzene methanesulfonamide).
  • AMX compounds Each of the compounds described in the present invention (e.g., 0604c, etc.) are shown in Examples 1-17.
  • the growth temperature of Arabidopsis thaliana is 22 ° C.
  • the photoperiod of plants grown on plant growth media (such as seed germination experiments and gene expression analysis) is long-day (24-hour light), and plants growing in soil (such as foliage)
  • the photoperiod of the transpiration experiment and the soil drought experiment was short daylight (8 hours light/16 hours dark) with a light intensity of 75 ⁇ mol ⁇ m –2 ⁇ s –1 .
  • the soybean growth temperature was 26 ° C and the photoperiod was 16 hours light / 8 hours dark.
  • the growth temperature of corn is 27 ° C, the photoperiod is 11 hours light / 13 hours dark; the cotton growth temperature is 26 ° C, the photoperiod is 14 hours light / 10 hours dark, and the light intensity is 400 ⁇ mol ⁇ m -2 ⁇ s -1 .
  • the plant growth medium used in the experiments was a 1/2 MS (Murashige and Skoog) solid medium containing 1% (w/v) sucrose and 0.6% (w/v) agar, unless otherwise specified.
  • Arabidopsis gene PYL1 (amino acid sequence 36-211), PYL2 (amino acid sequence 14-188) and Arabidopsis gene HAB1 (amino acid sequence 172-511) carrying the Biotin tag sequence with the 6 ⁇ His and SUMO double-tag sequences
  • PYR1 Arabidopsis gene
  • PYL3, PYL4, PYL5, PYL6, PYL7, PYL8, PYL9 and PYL10 (all of the above 9 genes are The recombinant plasmid of the whole gene coding sequence) and the soybean PYL gene (including GmPYL3 and GmPYL6) was constructed in the same manner as PYL1/PYL2.
  • the above recombinant plasmid was transformed into competent cell E. coli BL21 (DE3), inoculated into 200 ml LB liquid medium containing Amp resistance, and cultured overnight at 37 ° C at 200 rpm; inoculated to 1: 2 with Amp resistance at 1:50-1:100
  • the recombinant plasmid of PYR1/PYL1/PYL2/PYL7 carrying the 6 ⁇ His and SUMO double-tag sequences was induced overnight with 100 ⁇ M IPTG, while the HAB1 recombinant plasmid carrying the Biotin tag sequence was simultaneously induced with 100 ⁇ M IPTG and 40 ⁇ M biotin.
  • the bacterial cells after 16 hours of induction were centrifuged in a low-speed large-capacity centrifuge to collect the cells, and centrifuged at 4000 rpm for 20 min at 4 °C.
  • Each 2 L of the bacterial solution was resuspended in 50 ml of extraction buffer (containing 20 mM Tris, pH 8.0, 200 mM NaCl, and 10% (v/v) glycerin), and then crushed 3-5 times at 1000 ° C at 4 ° C.
  • the disrupted cells were subjected to ultracentrifugation, centrifuged at 16000 rpm for 30 min, and repeated twice, and the supernatant was collected through an affinity chromatography column.
  • HAB1 protein with Biotin tag sequence over 50 ml MBP affinity chromatography column (purchased from GE); first use 10% buffer C (containing 20 mM Tris, pH 8.0, 200 mM NaCl, 10 mM Maltose and 10% glycerol) Balance 600ml, elute with 200ml 50% buffer C, and finally elute with 100ml 100% buffer C; collect the solution with eluate solution (containing 20mM Tris, pH8.0, 200mM NaCl and 10% glycerol) over HiLoad26/60Superdex200 The gel filtration column further separates and purifies the protein.
  • 10% buffer C containing 20 mM Tris, pH 8.0, 200 mM NaCl, 10 mM Maltose and 10% glycerol
  • the AlphaScreen kit (purchased from Perkin Elmer) was used as follows: 150 ⁇ l of the system containing a 10:10 dilution of 10 ⁇ buffer (50 mM MOPS, pH 7.4, 50 mM NaF, 50 mM CHAPS, 0.1 mg/ml bovine serum) Albumin), each 100 nM HAB1 with Biotin tag sequence and PYR1/PYL1/PYL2/PYL7/GmPYL3/GmPYL6 protein with 6 ⁇ His and SUMO double tag sequences, corresponding concentrations of (+)-ABA/AM1/AMX, 5ug/ Ml donor beads and acceptor beads (purchased from Perkin Elmer), incubated for 1.5 hours at room temperature in the dark, placed in Envision Plate Reader (purchased from Perkin Elmer) according to the set AlphaScreen program Take readings.
  • 10 ⁇ buffer 50 mM MOPS, pH 7.4, 50 mM NaF, 50 mM CHAPS, 0.1 mg/ml bo
  • the reaction system contains 50 mM imidazole, pH 7.2, 5 mM MgCl 2 , 0.1% ⁇ -mercaptoethanol, 0.5 ⁇ g ⁇ ml -1 BSA, 100 nM HAB1 protein with Biotin tag sequence, 500 nM band 6 ⁇ His-SUMO double tag sequence
  • the PYL receptor protein and the corresponding concentration of (+)-ABA/AM1/AMX were incubated at room temperature for 30 minutes, and then the reaction was continued by adding a phosphorylated polypeptide containing 11 amino acids as a substrate for 30 minutes.
  • the phosphorylated polypeptide was SnRK2.6 protein.
  • amino acid at position 170-180 of the kinase (sequence HSQPKpSTVGTP), purchased from Kingsray, with phosphorylated serine at position 175, is a known target site for dephosphorylation of HAB1.
  • a chromogenic reagent purchased from BioVision
  • the absorbance at a wavelength of 650 nm was read with a microplate reader (purchased from Molecular Device).
  • Seeds of Arabidopsis Col-0 ecotype and PYL receptor triple deletion mutant (pyr1; pyl1; pyl4) were sterilized with NaClO and placed at 4 °C for 3 days, then seeded at 1 ⁇ M (+)-ABA/AM1. /0604c/0918/1127 and other AMX compounds or 0.05% DMSO (control) on 1/2 MS solid medium. Two lines were simultaneously sown on each 6 cm diameter medium, and 15 seeds were sown per line, with 4 replicates for each compound. The medium was placed in a long day sunshine culture at 22 ° C and photographed after 5 days.
  • Soybean leaf transpiration experiments for 0604c, 0918, and 1127 were performed using a Williams82 ecotype at 26 °C long-day culture, and 3 groups of 3 leaves were sprayed once, containing 0.05% tween-20 and 50 ⁇ M DMSO (control) / (+ )-ABA/0604c/0918/1127 compound, used in an amount of 4 ml/pot, was imaged using a FLIR A655sc thermal imaging camera at the same time period before and after spraying.
  • the Arabidopsis Col-0 ecotype seeds were sterilized with NaClO and placed at 4 °C for 3 days and then sown on 1/2MS solid medium. After 6 days of growth, seedlings with good growth and uniform size were selected and transplanted into 8 ⁇ 7 filled with soil. ⁇ 6cm 3 in a flower pot. Each pot was filled with the same weight of soil and transferred to the same number of plants (six strains) to reduce the experimental error. All the pots were placed in a short daylight culture at 22 ° C. After two weeks, the watering was stopped for drought treatment.
  • a solution containing 0.02% tween-20 and the corresponding concentration of (+)-ABA/AM1/0604c/0918/1127 or 0.02% tween-20 and 0.05% DMSO (control) was sprayed onto the circumferential foliage, and the spray amount was 2 ml solution/ In the basin, the position of the flowerpot is changed every day during the drought to reduce the error caused by environmental factors. During the whole drought period, the solution is sprayed twice and photographed regularly. Watering was resumed after about three weeks.
  • the soil drought test for soybeans and corn for 0918 and 1127 is similar to that of Arabidopsis thaliana. Each pot contains only one plant. All soybean plants are cultured at 26 °C for long days, and after 3 groups of 3 leaves, watering is stopped. The consistent plants were treated with drought, and the corn was stopped in the small bell mouth for water treatment. A solution containing 0.05% tween-20 and 50 ⁇ M (+)-ABA/0918/1127 or 0.05% tween-20 and 0.05% DMSO (control) was sprayed onto the foliage every three days during the drought treatment. The spray amount was 4 ml/ Pot, change the position of the pot at the same time. Return to watering after one week.
  • Soybean and cotton used for the 0720B-related foliar transpiration experiment were also used for soil drought experiments. Each pot contained only one plant and was filled with the same weight of soil to reduce experimental error. All soybean plants were cultured at 26 °C for a long period of time. After 16 days of sowing, the watering was stopped and the plants with the same growth were selected for drought treatment. At the beginning of the drought, spray a solution containing 0.1% Tween-20 and the corresponding concentration of (+)-ABA/0720B or 0.1% Tween-20 and 0.05% DMSO (control) to the foliage, and spray once every 3 days. The spraying amount was 4 ml/pot, and the position of the flower pot was changed at the same time.
  • the grapes used in the experiment were giant varieties of the commercially available variety. According to the weather conditions, during the color change period of the grapes, select the ears with uniform size and growth, and use 6% AMX compound 1127 water suspension agent 2000mg/L, 1000mg/L, 500mg/L, respectively. Soak the ears at 100 mg/L for 10 seconds, soak in water for control. Each treatment was repeated 10 times, and each ear was repeated once. Samples were taken after ripe harvesting, and samples were taken from the upper, middle and lower parts of each ear to the smooth side and the back side, and 6 seeds were collected per ear. Bring it back to the laboratory and store it at -20 °C.
  • the content of anthocyanin was determined by pH differential method (calculated as cyanidin-3-glucoside equivalent), and each treatment was repeated three times, anthocyanin content (mg/g)
  • X ⁇ A ⁇ F ⁇ M / ⁇ ⁇ m, Where ⁇ A is the absorbance value; F is the dilution factor; M is the relative molecular mass of cyanidin-3-glucoside 449.2, ⁇ is the molar extinction coefficient of cyanidin-3-glucoside 26900; m is the sample mass.
  • the bacterial back-mutation experiment used strains of TA98 and TA100 strains of Salmonella typhimurium.
  • the experiment was carried out under standard non-metabolic activation system conditions (-S9) using standard plate incorporation method.
  • a total of 5g/dish, 15g/dish, 50g were set.
  • the preparation method of 3,5-difluoro-4-methylbenzylsulfonyl chloride is the same as that of the step 1.4 of the first embodiment, except that 3,5-difluoro-4-methylbenzyl bromide is used instead of 3-fluoro-4-. Methylbenzyl bromide.
  • 2,3-difluoro-4-methylbenzylsulfonyl chloride is the same as that of the step 1.4 of Example 1, except that 2,3-difluoro-4-methylbenzyl bromide is used instead of 3-fluoro-4- Methylbenzyl bromide.
  • the preparation method of 2,3,5,6-tetrafluoro-4-methylbenzylsulfonyl chloride is the same as that of the step 1.4 of the first embodiment, except that 2,3,5,6-tetrafluoro-4-methylbenzyl is used. Bromine instead of 3-fluoro-4-methylbenzyl bromide.
  • 3-Fluoro-4-trifluoromethylbenzylsulfonyl chloride was prepared in the same manner as in Step 1.4 of Example 1, except that 3-fluoro-4-trifluoromethylbenzyl bromide was used instead of 3-fluoro-4-methyl Benzyl bromide.
  • the preparation method of 2,3,5,6-tetrafluoro-4-trifluoromethylbenzylsulfonyl chloride is the same as that of the step 1.4 of the first embodiment, except that 2,3,5,6-tetrafluoro-4-trid is used. Fluoromethylbenzyl bromide was substituted for 3-fluoro-4-methylbenzyl bromide.
  • 3-Chloro-4-methylbenzylsulfonyl chloride was prepared in the same manner as in Step 1.4 of Example 1, except that 3-chloro-4-methylbenzyl bromide was used instead of 3-fluoro-4-methylbenzyl bromide.
  • 6-Amino-1-propyl-2(1H)-quinolinone was prepared in the same manner as in Steps 1.1, 1.2, and 1.3 of Example 1, except that 2-hydroxyquinoline was used instead of 3,4-dihydro- 2(1H)-quinolinone.
  • 6-Amino-4-methyl-1-propyl-2(1H)-quinolinone was prepared in the same manner as in Steps 1.1, 1.2 and 1.3 of Example 1, except that 2-hydroxy-4-methyl was used. Quinoline replaces 3,4-dihydro-2(1H)-quinolinone.
  • the AMX compound of the present invention has a high affinity binding ability to a plurality of PYL receptors as a broad-spectrum and high-efficiency PYL receptor agonist, and promotes PYL receptor binding and inhibits PP2C protein phosphatase. active.
  • HAB1 protein phosphatase activity assay using SnRK2.6 phosphorylated polypeptide as a substrate showed that AMX can promote the binding of PYL2 receptor to PP2C protein phosphatase (HAB1), thereby inhibiting the dephosphorylation of SHB2.6 phosphorylated polypeptide by HAB1. Its effect, and its effect is significantly better than ABA at the same concentration.
  • AMX compounds e.g., compound 0604c, compound 0918, and compound 1127
  • PYL receptor agonists that are more efficient than existing compounds such as ABA.
  • the foliar transpiration inhibition experiment on soybean showed that the plant dosage of 50 ⁇ M of compound 1127 was 0.2 ⁇ mol per plant, and the leaf surface temperature was still significantly higher than that of the control group sprayed with DMSO after three days.
  • the results showed that the transpiration of soybean leaf surface was still inhibited, and the leaf surface temperature of the same concentration of ABA was the same as that of the control group (Fig. 10). This indicates that Compound 1127 also has the same effect of inhibiting foliar transpiration as that of Arabidopsis thaliana in soybean.
  • the leaf temperature of the plants was no different from that of the control group (Fig. 15).
  • the foliar transpiration inhibition experiment on cotton showed that the leaf temperature of the plants sprayed with 50 ⁇ M 0720B was still significantly higher than that of the control group sprayed with DMSO two days later, and the leaf surface temperature of the plants sprayed with 50 ⁇ M ABA was The control group was no different ( Figure 16).
  • Soybeans and 23 days of cotton were sown separately for 16 days, and plants of the same size were selected for soil drought experiments. Soybean sprayed 10/20/50 ⁇ M of 0720B or 50 ⁇ M ABA aqueous solution every 3 days after the start of drought, and cotton sprayed 50 ⁇ M aqueous solution of ABA/0720B compound every 4 days after the start of drought, soybean and cotton drought
  • an aqueous solution containing 0.05% DMSO was used as a control group, and 0.1% (v/v) of a surfactant Tween-20 was added to the above solution to enhance the penetration of the spray on the leaf epidermis.
  • the activity of the compound of the formula I of the present invention is significantly higher than that of the control compound (1, 2), and the amount of the halogen atom is proportional to the activity of the compound.
  • the crystal structure of the PYL2-AMX-HAB1 complex formed by a plurality of AMX compounds of the present invention was examined by the protein crystal analysis method described in the general method.
  • the control is ABA and the existing ABA analog, and the partial schematic diagram of the two-dimensional structure of each complex is shown in FIG. 4 .
  • AMX exists in the pocket structure of PYL2, and four oxygen atoms in the ABA structure can form hydrogen bonds with PYL2 pocket structure and multiple amino acid residues of HAB1 through multiple water molecules (Fig. 4a), AMX.
  • the oxygen atom and the nitrogen atom on the compound sulfonylamino group and the oxygen atom on the quinoline ring can also form a hydrogen bond.
  • the halogen substituent on p-xylene can help the oxygen and nitrogen atoms on the sulfonyl group to form hydrogen bonds with more adjacent amino acid residues by steric hindrance, or directly form hydrogen bonds with the PYL2 pocket structure, such as Fluorine atoms on compound 0918 (Fig. 4d), compound 1127 (Fig. 4e), compound 1020A (Fig. 4f), compound 1020B (Fig. 4g), and compound 1125A (Fig. 4h).
  • the ortho-position of the fluorine atom forms a hydrogen bond
  • the meta-halogen atom utilizes the steric hindrance effect to assist the sulfonyl group.
  • the oxygen and nitrogen atoms on the hydrogen bond form a hydrogen bond with the more adjacent amino acid residue; and in the case where a halomethyl group is present in the para position, the meta fluorine atom can also form a hydrogen bond, such as compound 1020A (Fig. 4f).
  • the inventors analyzed the effect of exogenously added AMX on plant gene expression.
  • the maximum tolerated dose of SD rats in a single oral gavage test showed that the male and female rats in the 5000 mg/kg group were observed in clinical observation, body weight and macroscopic observation of various tissues/organs. No drug-related changes were observed, and the maximum tolerated dose (MTD) of AMX compound 1127 was greater than 5000 mg/kg.

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Abstract

本发明公开了一种增强植物抗逆性的化合物及其制法和用途。具体地，本发明公开了一种式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体，并且本发明化合物为ABA的替代物，能够显著提高植物的抗逆性，因此具有极其广泛的应用前景。

Description

增强植物抗逆性的小分子化合物 技术领域
本发明涉及植物学领域，具体地，涉及增强植物抗逆性的化合物及其制法和用途。
背景技术
脱落酸(Abscisic Acid，ABA)是平衡植物内源激素和有关生长活性物质代谢的关键因子，具有促进植物平衡吸收水、肥和协调体内代谢的能力，可有效调控植物的根/冠和营养生长与生殖生长，对提高农作物的品质、产量具有重要作用。通过施用ABA，在提高农产品品质等许多方面有着重要的生理活性作用和应用价值。此外，外源ABA能引起叶片气孔的迅速关闭，抑制蒸腾作用，可用于花的保鲜，或在作物幼苗移植栽培的运输过程中防止萎蔫。ABA还能控制花芽分化，调节花期，在花卉园艺上有很大的应用价值。
ABA可以改善作物在低温、干旱、春寒、盐渍等不良生长环境中的生长。因此，ABA具有广泛应用，可用于草坪、农田、园林，尤其是可用于西部地区等缺水地区，对于发展中国农业产业具有重大意义。
然而，天然活性的(+)-ABA不稳定且人工合成难度较大，生产成本很高。因此，ABA一直未被广泛应用于农业生产，各国科学家都在开发天然ABA的替代物。
目前虽然已经开发了一些ABA的替代物，然而这些替代物的活性尚不能令人满意，农业生产上的应用价值低。此外，一些替代物的环境友好性较差。
因此，本领域迫切需要开发环境友好的且可有效提高植物抗逆性的化合物。
发明内容
本发明的目的是提供一种环境友好的且可有效提高植物抗逆性的化合物及其制法和用途。
本发明第一方面提供了式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体，
Figure PCTCN2016080790-appb-000001
式中，
R1为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
R2为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
R3为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
R4为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
R5为卤素、C1-C3烷基、C1-C3卤代烷基或SF5；
R6为H、C1-C3烷基、或C1-C3卤代烷基；
R7为C1-C7烷基、C2-C7链烯基、C2-C7链炔基、C3-C7环烷基、或-R8-O-R9,其中，R8为C1-C2亚烷基而R9为H、C1-C3烷基；
R0为H、C1-C4烷基、C1-C4卤代烷基、或卤素；
m为1或2；
Figure PCTCN2016080790-appb-000002
表示单键或双键；
附加条件是，R1、R2、R3、R4中有1-4个为卤素。
在另一优选例中，所述的化合物具有式Ia、Ib或Ic结构：
Figure PCTCN2016080790-appb-000003
式中，R0，R1-R6，m的定义如上所述。
在另一优选例中，R1、R2、R3、R4中有2-4个为卤素。
在另一优选例中，R1、R2、R3、R4中有3或4个为卤素。
在另一优选例中，R1、R2、R3、R4中有4个为卤素。
在另一优选例中，所述的卤素包括F、Cl、Br或I。
在另一优选例中，所述的卤素包括F或Cl。
在另一优选例中，所述的卤素为F。
在另一优选例中，R1、R2、R3、R4中有2-4个为F。
在另一优选例中，R1、R2、R3、R4中有3或4个为F。
在另一优选例中，R1、R2、R3、R4中有4个为F。
在另一优选例中，R1和/或R3为F。
在另一优选例中，R2和/或R4为F。
在另一优选例中，R5为甲基、Cl、三氟甲基、或SF5。
在另一优选例中，R6为H。
在另一优选例中，R7为C3烷基、或C3链烯基。
在另一优选例中，R7为正丙基、或-CH2-CH＝CH2。
在另一优选例中，当m＝2时，R0相同或不同。
在另一优选例中，R0为H、或甲基。
在另一优选例中，所述的化合物选自下组：
Figure PCTCN2016080790-appb-000004
Figure PCTCN2016080790-appb-000005
本发明第二方面提供了第一方面所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体的用途，用于制备农用制剂或组合物，所述农用制剂或组合物用于(i)增强植物抗逆性；(ii)植物保鲜处理(如鲜花保鲜)；和/或(iii)果实着色处理(如葡萄着色)。
在另一优选例中，所述农用制剂或组合物用于一种或多种以下用途：
(i)促进ABA受体PYL蛋白与PP2C蛋白磷酸酶的相互作用；
(ii)减弱叶片的蒸腾作用；
(iii)抑制种子萌发。
在另一优选例中，所述抗逆性为ABA相关的非生物胁迫抗逆性。
在另一优选例中，所述抗逆性选自下组：抗旱性、耐冷性、耐盐碱、耐渗透压、耐热性、或其组合。
在另一优选例中，所述植物为含有PYR/PYL家族ABA受体的植物。
在另一优选例中，所述植物包括苔藓、蕨类、裸子植物、单子叶植物和双子叶植物。
在另一优选例中，所述的植物包括农业植物、园艺植物、林业植物。
在另一优选例中，所述的植物包括木本植物、草本植物。
在另一优选例中，所述的植物包括完整的植株、器官(如根、茎、叶、枝、花、果实、种子)、组织(如愈伤组织)、或细胞。
在另一优选例中，所述的植物选自下组：禾本科、菊科、百合科、十字花科、蔷薇科、豆科、茶科、梧桐科、松科、胡桃科、胡椒科、木兰科、杜鹃花科、猕猴桃科、葡萄科、秋海棠科、凤梨科、银杏科、八角茴香科、姜科、石榴科、夹竹桃科、小檗科、芸香科、茄科、柏科、冬青科、棕榈科植物、或其组合。
在另一优选例中，所述植物选自下组：拟南芥、烟草、棉花、生菜、水稻、小麦、玉米、花生、高粱、燕麦、黑麦、甘蔗、大豆、马铃薯、荞麦、胡椒、葡萄、梨、苹果、香蕉、人参、番茄、辣椒、茄子、花椰菜、大白菜、油菜、黄瓜、西瓜、洋葱、向日葵、百合、玫瑰、菊花、牡丹、康乃馨、樟树、梧桐、松树、或其组合。
本发明第三方面提供了一种农用制剂，所述农用制剂包括：
(i)本发明第一方面所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体；和
(ii)农业上可接受的载体。
在另一优选例中，组分(i)在农用制剂中的含量为0.1-1000μM，较佳地，1-200μM，更佳地，5-100μM。
在另一优选例中，所述农用制剂中，含有0.0001-99wt％，较佳地0.1-90wt％的组分(i)，以农用制剂的总重量计。
在另一优选例中，所述农用制剂还包括额外的抗旱剂(如抗旱种衣剂、抗旱保水剂、或抗旱喷洒剂)或其他农用活性成分。
在另一优选例中，所述的农用活性成分选自下组：杀真菌剂、除草剂、杀虫剂、杀线虫剂、杀昆虫剂、植物激活剂、增效剂、植物生长调节剂、杀螨剂。
在另一优选例中，所述农用制剂还包括表面活性剂(如阳离子型、阴离子型、两性、或非离子型表面活性剂)。
在另一优选例中，所述农用制剂的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
本发明第四方面提供了一种增强植物抗逆性的方法，给所述植物施用本发明第一方面所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体或施用本发明第三方面所述的农用制剂。
在另一优选例中，所述施用选自下组：喷洒或灌溉。
在另一优选例中，所述施用的剂量为2-100g/公顷，较佳地，4-80g/公顷，更佳地，6-60g/公顷。
在另一优选例中，所述施用的剂量为1-5000微克/株，较佳地，10-2500微克/株，更佳地，20-1000微克/株。
本发明第五方面提供了一种式I化合物或其盐的制法，包括步骤：
(a)在惰性溶剂中，将式Ia-6化合物与式Ia-4化合物进行反应，从而形成式I化合物；
Figure PCTCN2016080790-appb-000006
上述各式中，R0、R1、R2、R3、R4、R5、R6、R7、m和
Figure PCTCN2016080790-appb-000007
如上定义。
在另一优选例中，所述反应在缚酸剂的存在下进行。
在另一优选例中，所述缚酸剂选自下组：碳酸钾、三乙胺、吡啶或其组合。
在另一优选例中，所述惰性溶剂选自下组：DMF、DCM、乙腈或其组合。
在另一优选例中，所述反应温度为0-150℃(或回流温度)，较佳地，20-60℃，更佳地，25-40℃。
在另一优选例中，所述反应时间为0.1-72小时，更佳地，1-24小时，更佳地，2-20小时。
本发明第六方面提供了一种植物保鲜处理方法，包括步骤：
将待保鲜的植物与本发明第一方面所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体进行接触，从而对植物保鲜。
在另一优选例中，所述的植物包括完整的植株、或植物器官(如根、茎、叶、枝、花、果实)。
在另一优选例中，所述的植物保鲜处理包括鲜花保鲜。
本发明第七方面提供了一种果实着色处理方法，包括步骤：
将待着色的果实与本发明第一方面所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体进行接触，从而对果实着色。
在另一优选例中，所述的果实包括葡萄。
本发明第八方面提供了一种本发明第一方面所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体的用途，其特征在于，用于制备(i)ABA受体的激动剂；和/或(ii)种子萌发抑制剂。
在另一优选例中，所述的激动剂促进ABA受体PYL蛋白与PP2C蛋白磷酸酶的相互作用。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此 不再一一累述。
附图说明
图1显示本发明的两种AMX化合物0604c和0918可以结合于拟南芥PYR/PYL受体(PYR1、PYL1、PYL2和PYL7)-HAB1复合物,进而抑制蛋白磷酸酶HAB1的活性。在1μM浓度下，抑制效果均显著优于ABA。
图2显示了六种AMX化合物(0604c、0918、1125A、1125B、1127和1103B)以及ABA的拟南芥PYR/PYL受体激动剂的剂量响应曲线。AMX化合物可以促进蛋白磷酸酶HAB1与四种拟南芥PYR/PYL受体(PYR1、PYL1、PYL2和PYL7)的相互作用，且该作用存在剂量依赖效应。
图3显示三种AMX化合物(0925、1020A和1020B)和ABA的PYR/PYL受体激动剂的剂量响应曲线。AMX化合物可促进蛋白磷酸酶HAB1与三种PYR/PYL受体(PYR1、PYL1和PYL2)的相互作用，且该相互作用存在剂量依赖效应。
图4a至4i分别显示ABA(a)、现有ABA类似物(b)或本发明多个AMX化合物(c至i)与PYL2-HAB1复合体口袋结构内多个氨基酸残基相互作用的二维结构。图中显示了水分子、氮原子、氧原子和卤素原子，虚线代表氢键，其上的数字则表示两个原子/分子之间的距离(单位是埃，
Figure PCTCN2016080790-appb-000008
)。结果显示，与现有的ABA或其类似物相比，本发明的AMX化合物与PYL2口袋结构内的氨基酸残基形成更多氢键，这使得AMX与PYL2-HAB1复合体的结合更加紧密。
图5显示用本发明不同AMX化合物处理6小时后，野生型拟南芥体内受ABA诱导的胁迫相关基因的转录水平变化。DMSO和ABA处理分别为阴性和阳性对照组。
图6显示1μM浓度下，不同AMX化合物和ABA对于Col-0和pyr1；pyl1；pyl4三突变体种子萌发的影响。每个培养皿的左半边播种Col-0，右半边播种pyr1；pyl1；pyl4三突变体。照片为pyr1；pyl1；pyl4三突变体种子萌发4天后(播种6天后)拍摄。DMSO处理为对照组。
图7a和7b显示本发明AMX化合物处理显著降低了拟南芥叶面的蒸腾速率，导致叶面温度升高。
图7a显示了5μM AMX化合物和ABA处理后，叶面温度较DMSO处理显著增加。其中0918和1127在处理四天后仍能显著抑制叶面蒸腾作用。
图7b显示了AMX化合物对于叶面蒸腾作用的抑制效应存在浓度依赖效应，相同浓度下抑制效果排序为1127>0918>1125A。
图8显示土壤干旱实验结果。短日照环境中生长3周的野生型拟南芥植物(Col-0)停止浇水并喷施相应浓度的化合物。DMSO作为负对照。其中，图8a显示了首次喷施化合物当天和17天后的植物生长状况，实验中使用的化合物浓度为10μM；图8b显示了首次喷施化合物当天，18和20天后的植物生长状况，实验中使用的化合物浓度为5μM；图8c 显示了首次喷施化合物当天和14天后的植物生长状况，实验中使用的化合物浓度为10μM。
图9显示三种AMX化合物(0604c,0918和1127)以及ABA的大豆PYR/PYL受体激动剂剂量响应曲线。AMX化合物可以促进拟南芥蛋白磷酸酶HAB1与二种大豆PYR/PYL受体(GmPYL3和GmPYL6)的相互作用，且该相互作用存在剂量依赖效应。
图10显示了50μM AMX化合物1127处理3天后仍能显著降低拟南芥叶面的蒸腾速率，导致叶面温度升高。
图11显示了大豆和玉米的土壤干旱实验结果。大豆实验中，选取生长一致的大豆植株，在干旱一周后复水，照片显示了复水一个月后的整体生长状况。实验中各测试化合物浓度为50μM。玉米实验方法同大豆实验。实验中化合物0918浓度为50μM。复水后经AMX处理的大豆和玉米的存活率均要显著高于对照组。
图12显示了AMX化合物1127的水悬浮剂处理后的葡萄果皮花色苷含量显著高于清水对照组。花色苷含量与葡萄果皮上色成正相关。
图13显示了本发明化合物0720B可以结合于拟南芥PYR/PYL受体(PYR1、PYL1、PYL2和PYL7)-HAB1复合物，进而抑制蛋白磷酸酶HAB1的活性。在1μM浓度下，抑制效果均显著优于ABA。
图14显示了本发明化合物0720B和ABA的拟南芥PYR/PYL受体激动剂的剂量响应曲线。0720B化合物可以促进蛋白磷酸酶HAB1与两种拟南芥PYR/PYL受体(PYR1和PYL2)的相互作用，且该作用存在剂量依赖效应。
图15显示了AMX化合物(0720B)处理显著降低了大豆叶面的蒸腾速率，导致叶面温度升高。播种16天后的大豆植株停止浇水，同时喷施相应化合物，与对照组(DMSO)相比，喷施10/20/50μM的化合物0720B可显著降低大豆叶面的蒸腾速率，且抑制效果优于喷施50μM ABA。
图16显示了AMX化合物(0720B)处理显著降低了棉花叶面的蒸腾速率，导致叶面温度升高。播种23天后的棉花植株停止浇水，同时喷施50μM的化合物0720B，与对照组(DMSO)相比，50μM的化合物0720B均可显著降低棉花叶面的蒸腾速率，且抑制效果优于50μM ABA。
图17显示了大豆的土壤干旱实验结果。图15中的大豆在干旱11天后复水，照片显示了复水一天后的大豆生长状况。经10/20/50μM的化合物0720B处理的大豆长势要显著优于对照组(DMSO)或50μM ABA处理的大豆。
图18显示了棉花的土壤干旱实验结果。照片显示了图16中的棉花在干旱10天后的生长状况。经50μM化合物0720B处理的棉花长势要显著优于对照组(DMSO)或50μM ABA处理的棉花。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一类具有高脱落酸(ABA)活性的ABA 替代物(本发明化合物)。本发明化合物具有明显高于现有ABA类似物的活性，能够显著增强植物的多种抗逆性(如抗旱、耐冷等)。此外，本发明化合物制法简便，且具有优异的环境友好性和作用迅速等优点，因此，具有广泛的应用前景。在此基础上完成了本发明。
实验表明，本发明化合物(简称为AMX化合物)不仅活性优于脱落酸(Abscisic Acid，ABA)以及现有的ABA类似物(如4-甲基-氮-(1,2,3,4-四氢-2-羰基-1-丙基-6-喹啉基)-苯甲基磺酰胺(4-methyl-N-(1,2,3,4-tetrahydro-2-oxo-1-propyl-6-quinolinyl)-benzene methanesulfonamide))，而且可结合于多个不同PYL受体，可显著增强各种不同植物的抗逆性。
基团定义
如本文所用，术语“C1-C7烷基”是指具有1-7个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
如本文所用，术语“C2-C7链烯基”指具有2-7个碳原子的直链或支链的烯基，例如乙烯基、烯丙基、1-丙烯基、异丙烯基、1-丁烯基、2-丁烯基、或类似基团。
如本文所用，术语“C2-C7链炔基”是指具有2-7个碳原子的直链或支链的炔基，例如乙炔基、丙炔基、或类似基团。
如本文所用，术语“C1-C3烷基”是指具有1-3个碳原子的直链或支链烷基，例如甲基、乙基、正丙基、异丙基、或类似基团。
如本文所用，术语“C1-C3卤代烷基”是指氢被1个或1个以上的卤素取代的具有1-3个碳原子的直链或支链烷基，例如，卤代甲基、卤代乙基、卤代丙基、卤代异丙基、或类似基团。
如本文所用，术语“C3-C7环烷基”指具有3-7个碳原子的环状烷基，例如环丙基、环丁基、环戊基、环己基、环庚基、或类似基团。
如本文所用，术语“卤素”是指氟、氯、溴、或碘，优选氟和氯。
如本文所用，术语“卤代的”指被相同或不同的一个或多个上述卤原子取代的基团，可以部分卤代或全部卤代，例如三氟甲基、五氟乙基、七氟异丙基、或类似基团。
本发明的化合物可以含有一个或多个不对称中心，并因此以消旋体、外消旋混合物、单一对映体、非对映异构体化合物和单一非对映体的形式出现。可以存在的不对称中心，取决于分子上各种取代基的性质。每个这种不对称中心将独立地产生两个旋光异构体，并且所有可能的旋光异构体和非对映体混合物和纯或部分纯的化合物包括在本发明的范围之内。本发明包括化合物的所有异构形式。
式I化合物及其制备方法
如本文所用，术语“本发明化合物”、“AMX化合物”、"化合物AMX"、“本发明ABA 替代物”、“式I化合物”可互换使用，均指具有式I所示结构的化合物。此外，所述术语还包括式I化合物的盐、光学异构体、外消旋体、溶剂化物(如水合物)、和/或前体，
Figure PCTCN2016080790-appb-000009
其中，R0、R1、R2、R3、R4、R5、R6、R7、m、
Figure PCTCN2016080790-appb-000010
的定义如前所述。
下面更具体地描述本发明式I化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便的制得，这样的组合可由本发明所属领域的技术人员容易的进行。通常，在本发明的制备方法中，各反应大多在惰性溶剂中，在0℃至150℃(或回流温度)(较佳地，20-60℃，或25－40℃)下，反应一段时间(如0.1-72小时，较佳地2－20小时)。
较佳地，本发明式I化合物可以通过以下方案及实施例中所述的示例性方法以及本领域技术人员所用的相关公开文献操作完成。
典型地，本发明的式Ia和/或Ib化合物的制备方法可包括(但不限于)如下流程。
方案I(以R6＝H且R7＝丙基为例)
(1)制备6-氨基-1-丙基-喹啉酮
Figure PCTCN2016080790-appb-000011
步骤1：在碱(如碳酸钾，氢化钠)存在下，在惰性溶剂(如DMF)中，首先将式Ia-1化合物与卤代烷在一定温度(如25-40℃)下进行反应一段时间，形成式Ia-2化合物。
步骤2：在酸(如硫酸)存在下，式Ia-2化合物与硝酸钾在一定温度(如0-20℃)下进行反应一段时间，形成式Ia-3化合物。
步骤3：在惰性溶剂(如甲醇)中，以钯碳作催化剂，在一定温度(如10-40℃)下，将式Ia-3化合物进行还原反应，形成式Ia-4化合物。
(2)制备对甲基卤代苄磺酰氯
Figure PCTCN2016080790-appb-000012
步骤4：在惰性溶剂(如乙醇、乙腈)中，式Ia-5化合物(如对甲基苄溴或对甲基苄氯)与硫脲反应，形成反应产物。然后，在酸(如浓盐酸)存在下，在惰性溶剂(如乙腈)中，将所述反应产物和亚氯酸钠在一定温度(如0-20℃)下进行反应一段时间，形成式Ia-6化合物。
(3)制备式Ia/Ib化合物
Figure PCTCN2016080790-appb-000013
步骤5：在惰性溶剂(如DMF)中，在缚酸剂(如碳酸钾)存在下，式Ia-6化合物与式Ia-4化合物在一定温度(如25-40℃)下进行反应一段时间，从而得到式Ia/Ib化合物。
方案I中，X2为离去基团，为氯、溴、碘。其它各取代基和基团如说明书中所定义。
Figure PCTCN2016080790-appb-000014
表示单键或双键。
本发明的式Ic化合物的制备方法可包括(但不限于)如下流程：
方案II(以m＝1，R6为H，R7＝丙基且R0为甲基为例)
(1)制备6-氨基-4-甲基-1-丙基-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000015
步骤1：在催化剂(如钯碳，二氧化铂)存在下，在惰性溶剂(如乙酸，乙酸乙酯)中，在一定温度下(如50-80℃)下Ia-7进行氢化反应一段时间，形成式Ia-8化合物。
步骤2：在碱(如碳酸钾，氢化钠)存在下，在惰性溶剂(如DMF)中，首先将式Ia-8化合物与卤代烷在一定温度(如25-40℃)下进行反应一段时间，形成式Ia-9化合物。
步骤3：在酸(如硫酸)存在下，将式Ia-9化合物与硝酸钾在一定温度(如0-20℃)下进行反应一段时间，形成式Ia-10化合物。
步骤4：在惰性溶剂(如甲醇)中，以钯碳作催化剂，在一定温度(如10-40℃)下，将式 Ia-10化合物进行还原反应，形成式Ia-11化合物。
(2)制备对甲基卤代苄磺酰氯
Figure PCTCN2016080790-appb-000016
步骤5：在惰性溶剂(如乙醇、乙腈)中，式Ia-5化合物(如对甲基苄溴或对甲基苄氯)与硫脲反应，形成反应产物。然后，在酸(如浓盐酸)存在下，在惰性溶剂(如乙腈)中，将所述反应产物和亚氯酸钠在一定温度(如0-20℃)下进行反应一段时间，形成式Ia-6化合物。
(3)制备式Ic化合物
Figure PCTCN2016080790-appb-000017
步骤6：在惰性溶剂(如DMF)中，在缚酸剂(如碳酸钾)存在下，式Ia-6化合物与式Ia-11化合物在一定温度(如25-40℃)下进行反应一段时间，从而得到式Ic化合物。
方案II中，X2为离去基团，为氯、溴、碘。其它各取代基和基团如说明书中所定义。
农用制剂
可将本发明的活性物质(式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体)以常规的方法制备成农用制剂，例如溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、用活性物质浸渍的天然的和合成的材料、在多聚物中的微胶囊、用于种子的包衣剂。
这些制剂可用已知的方法生产，例如，将活性化合物与扩充剂混合，这些扩充剂就是液体的或液化气的或固体的稀释剂或载体，并可任意选用表面活性剂即乳化剂和/或分散剂和/或泡沫形成剂。例如在用水作扩充剂时，有机溶剂也可用作助剂。
用液体溶剂作稀释剂或载体时，基本上是合适的，如：芳香烃类，例如二甲苯，甲苯或烷基萘；氯化的芳香或氯化的脂肪烃类，例如氯苯，氯乙烯或二氯甲烷；脂肪烃类，例如环己烷或石蜡，例如矿物油馏分；醇类，例如乙醇或乙二醇以及它们的醚和脂类；酮类，例如丙酮，甲乙酮，甲基异丁基酮或环已酮；或不常用的极性溶剂，例如二甲基甲酰胺和二甲基亚砜，以及水。
就液化气的稀释剂或载体说，指的是在常温常压下将成为气体的液体，例如气溶胶推 进剂，如卤化的烃类以及丁烷，丙烷，氮气和二氧化碳。
固体载体可用研磨的天然矿物质，例如高岭土，粘土，滑石，石英，活性白土，蒙脱土，或硅藻土，和研磨合成的矿物质，例如高度分散的硅酸，氧化铝和硅酸盐。供颗粒用的固体载体是碾碎的和分级的天然锆石，例如方解石，大理石，浮石，海泡石和白云石，以及无机和有机粗粉合成的颗粒，和有机材料例如锯木屑，椰子壳，玉米棒子和烟草梗的颗粒等。
非离子的和阴离子的乳化列可用作乳化剂和/或泡沫形成剂。例如聚氧乙烯-脂肪酸酯类，聚氧乙烯-脂肪醇醚类，例如烷芳基聚乙二醇醚类，烷基磺酸酯类，烷基硫酸酯类，芳基磺酸酯类以及白蛋白水解产物。分散剂包括，例如木质素亚硫酸盐废液和甲基纤维素。
在制剂中可以用粘合剂，例如羧甲基纤维素和以粉末，颗粒或乳液形式的天然和合成的多聚物，例如阿拉伯胶，聚乙烯基醇和聚乙烯醋酸酯。
可以用着色剂例如无机染料，如氧化铁，氧化钻和普鲁士蓝；有机染料，如有机染料，如偶氮染料或金属钛菁染料；和用痕量营养剂，如铁，猛，硼，铜，钴，铝和锌的盐等。
在本发明中，所述“农用制剂”通常是农用植物生长调节剂，其含有式I化合物或其盐、其光学异构体、外消旋体、溶剂化物或前体作为增强植物抗逆性(如抗旱性)的活性成分；以及农业上可接受的载体。
如本文所用，所述“农业上可接受的载体”是用于将本发明的式I化合物或其盐、光学异构体、外消旋体、溶剂化物或前体传送给植物的农药学上可接受的溶剂、悬浮剂或赋形剂。载体可以是液体或固体。适用于本发明的农业上可接受的载体选自下组：水、缓冲液、DMSO、表面活性剂如Tween-20、或其组合。任何本领域技术人员已知的农业上可接受的载体均可用于本发明中。
本发明的农用制剂可与其他抗旱剂制成一种混合物存在于它们的商品制剂中或从这些制剂制备的使用剂型中，这些其他的抗旱剂包括(并不限于)：抗旱种衣剂、抗旱保水剂、或抗旱喷洒剂等。
此外，本发明的农用制剂也可与增效剂制成一种混合物存在于它们的商品制剂中或从这些制剂制备的使用剂型中，这些增效剂是提高活性化合物作用的化合物，由于活性化合物本身有活性，也可不必加增效剂。
本发明所述的农用制剂的剂型可以是多种多样的，只要能够使活性成分有效地到达植物体内的剂型都是可以的，从易于制备和施用的立场看，优选的农用制剂是一种喷雾剂或溶液制剂。
本发明所述的农用制剂通常含有占所述农用制剂总重量的0.0001-99wt％，较佳地0.1-90wt％的本发明化合物。商品制剂或使用剂型中的本发明化合物的浓度可在广阔的范围内变动。商品制剂或使用剂型中的本发明化合物的浓度可从0.0000001-100％(g/v)，最好在0.0001与1％(g/v)之间。
增强植物抗逆性的方法
本发明提供了一种增强植物抗逆性(如抗旱性)的方法，包括步骤：给植物施用式I化合物或其盐、其光学异构体、外消旋体、溶剂化物或前体、或其相应的的农用制剂。
施用可采用已知的各种方法，例如，通过在植物叶片、繁殖材料上喷洒、喷雾、喷粉或播撒该化合物或含有该化合物的农用制剂，或以其他方式使植物接触该化合物或含有该化合物的农用制剂，如果是种子，则通过涂布、包裹或以其他方式处理种子。另一种在种植前直接处理植物或种子的方法，还可将本发明的农用制剂引入土壤或其他待播种种子的培养基。在一些实施方案中，还可以使用载体，所述载体可以是如上所述的固态、液态。
在一优选实施方式中，还可以通过喷洒(如飞机喷洒)或灌溉将该化合物或含有该化合物的农用制剂递送给所述植物。
其他应用
本发明的化合物可以用作植物保鲜剂，所述植物包括(但不限于)：鲜花、园艺植物等，尤其对鲜花(如非洲菊)有着非常显著的保鲜效果。
本发明的化合物还可以用作果实着色剂，其中，所述果实包括(但不限于)葡萄。
本发明的主要优点包括：
首次开发了一类具有高脱落酸(ABA)活性的ABA替代物(本发明化合物)。本发明化合物具有明显高于现有ABA类似物的活性，能够显著增强植物的多种抗逆性(如抗旱、耐冷等)。此外，本发明化合物制法简便，且具有优异的环境友好性，因此，具有广泛的应用前景。在此基础上完成了本发明。
实验表明，本发明化合物(简称为AMX化合物)不仅活性优于脱落酸(Abscisic Acid，ABA)以及现有的ABA类似物(如4-甲基-氮-(1,2,3,4-四氢-2-羰基-1-丙基-6-喹啉基)-苯甲基磺酰胺(4-methyl-N-(1,2,3,4-tetrahydro-2-oxo-1-propyl-6-quinolinyl)-benzene methanesulfonamide))，而且可结合于多个不同PYL受体，可显著增强各种不同植物的抗逆性。
(1)本发明首次合成了天然脱落酸(Abscisic Acid，ABA)的高活性的替代物AMX化合物。本发明的AMX化合物能够显著增强植物多种抗逆性(如抗旱、耐冷)。并且，光学异构体或外消旋体的本发明化合物均具有高活性。
(2)本发明的AMX化合物的活性显著优于脱落酸(Abscisic Acid，ABA)以及现有的ABA类似物。
(3)本发明化合物AMX对于多个PYR/PYL受体蛋白与PP2C蛋白磷酸酶HAB1的相互作用均有促进作用，且对PYR1、PYL1、PYL2、PYL7的促进作用尤为显著。
(4)本发明化合物更容易被植物吸收，且作用迅速。
(5)本发明化合物对环境友好，对哺乳动物无害。
(6)本发明化合物的合成方法简单、成本低廉。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
材料和通用方法
材料
实验中使用的模式植物均为常规的或市售的品种，其中拟南芥(Arabidopsis thaliana)包括：哥伦比亚(Col-0)生态型和基于Col-0生态型的ABA合成突变体aba2-1和ABA受体PYL三突变体pyr1；pyl1；pyl4；大豆品种为Williams82，棉花品种为陆地棉R15，玉米品种为丹玉402。
化合物AM1：现有的ABA类似物，化学名称为4-甲基-氮-(1,2,3,4-四氢-2-羰基-1-丙基-6-喹啉基)-苯甲基磺酰胺(4-methyl-N-(1,2,3,4-tetrahydro-2-oxo-1-propyl-6-quinolinyl)-benzene methanesulfonamide)。
AMX化合物：本发明所述的各化合物(如0604c等)见实施例1-17。
植物生长
拟南芥生长温度为22℃，生长在植物生长培养基上的植物(如种子萌发实验和基因表达分析)的光周期为长日照(24小时光照)，生长在土壤中的植物(如叶面蒸腾实验和土壤干旱实验)的光周期为短日照(8小时光照/16小时黑暗)，光强为75μmol·m–2·s–1。
大豆生长温度为26℃，光周期为16小时光照/8小时黑暗。玉米的生长温度为27℃，光周期为11小时光照/13小时黑暗；棉花生长温度为26℃，光周期为14小时光照/10小时黑暗，光强均为400μmol·m-2·s-1。
如无特殊指明，实验中使用的植物生长培养基均为含1％(w/v)蔗糖和0.6％(w/v)琼脂的1/2MS(Murashige and Skoog)固体培养基。
蛋白表达纯化
带6×His和SUMO双标签序列的拟南芥基因PYL1(氨基酸序列36-211)，PYL2(氨基酸序列14-188)和带Biotin标签序列的拟南芥基因HAB1(氨基酸序列172-511)的重组质粒的构建方法详见“A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors”(Nature,Vol462,2009)，带6×His和SUMO双标签序列的其他拟南芥PYL基因，包括PYR1，PYL3，PYL4，PYL5，PYL6，PYL7，PYL8，PYL9和PYL10(以上9个基因均为 全基因编码序列)，以及大豆PYL基因(包括GmPYL3和GmPYL6)的重组质粒的构建方法与PYL1/PYL2相同。
将上述重组质粒转入感受态细胞大肠杆菌BL21(DE3)，接种到含有Amp抗性的200ml LB液体培养基，37℃200rpm培养过夜；按1:50-1:100接种至2L含有Amp抗性的LB液体培养基中进行扩大培养，37℃200rpm培养3-4小时，16℃低温培养至OD600＝0.8-1.0左右。带6×His和SUMO双标签序列的PYR1/PYL1/PYL2/PYL7的重组质粒用100μM IPTG诱导过夜，而带Biotin标签序列的HAB1重组质粒要用100μM IPTG和40μM biotin同时进行诱导。
将诱导16小时后的菌液在低速大容量离心机中离心收集菌体，4℃以4000rpm转速离心20min。每2L菌液用50ml提取缓冲液(含20mM Tris，pH8.0，200mM NaCl和10％(v/v)甘油)重悬菌体，然后在4℃进行1000Pa压力破碎3-5次。破碎后的细胞进行超速离心，16000rpm离心30min，重复2次，收集上清液过亲和层析柱。
对于带6×His和SUMO双标签序列的PYR/PYL蛋白，选择50ml亲和层析Ni柱(50ml Ni-NTA column，购自GE公司)；先用10％缓冲液B(含20mM Tris，pH8.0，200mM NaCl，500mM imidazole和10％甘油)平衡600ml，再用200ml50％缓冲液B洗脱，最后用100ml100％缓冲液B洗脱；用于晶体解析的蛋白与ulp1酶以1000：1的摩尔比混合进行酶切透析过夜；酶切后的蛋白再过一次亲和层析Ni柱；收集液用洗脱溶液(含25mM Tris，pH8.0，200mM ammonium acetate，1mM dithiotreitol和1mM EDTA)过HiLoad26/60Superdex200凝胶过滤柱(购自GE公司)进一步分离纯化蛋白。
对于带Biotin标签序列的HAB1蛋白，过50ml MBP亲和层析柱(购自GE公司)；先用10％缓冲液C(含20mM Tris，pH8.0，200mM NaCl，10mM Maltose和10％甘油)平衡600ml，再用200ml50％缓冲液C洗脱，最后用100ml100％缓冲液C洗脱；收集液用洗脱溶液(含20mM Tris，pH8.0，200mM NaCl和10％甘油)过HiLoad26/60Superdex200凝胶过滤柱进一步分离纯化蛋白。
蛋白晶体解析
结晶前，将酶切后的PYL2和HAB1蛋白与化合物按1:1:5的摩尔比混合，浓缩到6mg/ml用于点晶体。采用悬滴法进行点晶体；用于结晶的孔缓冲液(well buffer)包含0.1M Succinic acid和15％PEG3350、或0.2M丙二酸二钠(Di-sodium malonate)和20％PEG3350、或0.2M柠檬酸三钠(Tri-sodium Citrate)和20％PEG3350、或0.2M甲酸镁(Magnesium formate)和20％PEG3350。一天后可看到晶体出现，约3-4天可长到100-120μm。晶体用X射线衍射并收集衍射数据，再根据相关PYR/PYL受体结构模型来解析复合物结构。
AlphaScreen实验
采用AlphaScreen试剂盒(购自Perkin Elmer)，方法如下：150μl实验体系中含1:10稀释的10×buffer(50mM MOPS，pH7.4，50mM NaF，50mM CHAPS，0.1mg/ml bovine serum  albumin)，各100nM带Biotin标签序列的HAB1和带6×His和SUMO双标签序列的PYR1/PYL1/PYL2/PYL7/GmPYL3/GmPYL6蛋白，相应浓度的(+)-ABA/AM1/AMX，5ug/ml供体珠(donor beads)和受体珠(acceptor beads)(购自Perkin Elmer)，室温下避光孵育1.5小时后，置于Envision Plate Reader(购自Perkin Elmer)中按设定的AlphaScreen程序进行读数。
HAB1磷酸酶活性检测
反应体系中含50mM咪唑，pH7.2，5mM MgCl2，0.1％β-巯基乙醇，0.5μg·ml-1BSA，100nM带Biotin标签序列的HAB1蛋白，500nM带6×His-SUMO双标签序列的PYL受体蛋白及相应浓度的(+)-ABA/AM1/AMX，室温孵育30分钟，随后加入含11个氨基酸的磷酸化多肽作为底物继续反应30分钟，该磷酸化多肽为SnRK2.6蛋白激酶的170-180位氨基酸(序列HSQPKpSTVGTP)，购自金斯瑞，其中175位的磷酸化丝氨酸，为已知的HAB1脱磷酸化靶位点。30分钟后加入显色试剂(购自BioVision)，用酶标仪(购自Molecular Device)读取650nm波长的吸收值。
基因表达分析
取全植株或叶子，用常规方法进行RNA抽提、经逆转录后进行荧光定量PCR。每种处理取3个生物学重复并进行两次实验重复，ACT7基因被用作内参。
种子萌发和土壤干旱实验
(1)种子萌发
拟南芥Col-0生态型和PYL受体三缺失突变体(pyr1；pyl1；pyl4)的种子用NaClO消毒后置于4℃春化3天，然后播种在含1μM(+)-ABA/AM1/0604c/0918/1127等多种AMX化合物或0.05％DMSO(对照)的1/2MS固体培养基上。每个6cm直径的培养基上同时播种两个株系，每个株系播种15粒种子，每种化合物设4个重复。培养基置于22℃长日照培养，5天后拍照。
(2)植物叶面蒸腾实验
拟南芥叶面蒸腾实验使用ABA合成突变体aba2-1。在环境胁迫条件下，该突变体中的内源ABA含量并不增加，仅为同样条件下野生型拟南芥Col-0中的1/40，因此使用该突变体可以排除内源ABA对于蒸腾实验的影响。持续浇水三周后的植物一次性喷施含0.05％tween-20和相应浓度的DMSO(对照)/(+)-ABA/0918/1127化合物，使用量为1.2ml/盆，喷施前和喷施后每天在相同时间段使用FLIR A655sc红外热像仪成像。用于0604c，0918和1127的大豆叶面蒸腾实验使用Williams82生态型在26℃长日照培养，3组3叶后一次性喷施，含0.05％tween-20和50μM的DMSO(对照)/(+)-ABA/0604c/0918/1127化合物，使用量为4ml/盆，喷施前和喷施后每天在相同时间段使用FLIR A655sc红外热像仪成像。
用于0720B实验的大豆和棉花叶面蒸腾实验在26℃长日照进行，分别在播种16天和 23天后喷施含0.1％tween-20和相应浓度的(+)-ABA/0720B化合物或0.1％Tween-20和0.05％DMSO(对照)的溶液，使用量为4ml/盆，喷施前和喷施后每天在相同时间段使用FLIR A655sc红外热像仪成像。
(3)土壤干旱实验
拟南芥Col-0生态型种子用NaClO消毒后置于4℃春化3天后播种在1/2MS固体培养基上，生长6天后选取长势良好且大小一致的幼苗移入装满土的8×7×6cm3的花盆中。每个花盆装有相同重量的土并移入相同数目的植物(六株)以减少实验误差，所有的花盆均置于22℃短日照培养，两周后停止浇水进行干旱处理，期间每周向叶面喷洒一次含0.02％tween-20和相应浓度的(+)-ABA/AM1/0604c/0918/1127或0.02％tween-20和0.05％DMSO(对照)的溶液，喷施量为2ml溶液/盆，干旱过程中每天变换花盆位置以减少环境因素引起的误差，整个干旱期间总计喷施两次溶液，定期拍照记录。约三周后恢复浇水。
用于0918和1127的大豆和玉米的土壤干旱实验与拟南芥类似，每盆只包含一株植物，所有的大豆植株均在26℃长日照培养，3组3叶后停止浇水，选择长势一致的植株进行干旱处理，玉米则在小喇叭口期停止浇水进行干旱处理。干旱处理期间每三天向叶面喷洒一次含0.05％tween-20和50μM(+)-ABA/0918/1127或0.05％tween-20和0.05％DMSO(对照)的溶液，喷施量为4ml/盆，同时变换花盆位置。一周后恢复浇水。
用于进行0720B相关叶面蒸腾实验的大豆和棉花同时用于土壤干旱实验，每个花盆只包含一株植物并装有相同重量的土以减少实验误差。所有的大豆植株均在26℃长日照培养，播种16天后停止浇水，选择长势一致的植株进行干旱处理。干旱开始时向叶面喷施一次含0.1％Tween-20和相应浓度的(+)-ABA/0720B或0.1％Tween-20和0.05％DMSO(对照)的溶液，此后每3天喷施一次，喷施量为4ml/盆，同时变换花盆位置，干旱11天后复水并在复水一天后拍照。棉花干旱实验与大豆类似，播种23天后停止浇水，选择长势一致的植株进行干旱处理。干旱开始时向叶面喷施一次含0.1％Tween-20和50μM(+)-ABA/0720B或0.1％Tween-20和0.05％DMSO(对照)的溶液，此后每4天喷施一次，喷施量为4ml/盆，同时变换花盆位置，干旱10天后拍照。
鲜花保鲜实验
市售云南百合品种“西伯利亚”二年生花苞，每支5个花苞，每支留10片叶，每瓶1支进行试验；分别将花枝插入10ppm(22.5μM)的AMX化合物1127水悬浮剂和自来水中，处理前和处理20天后记录花朵状况。
葡萄着色实验
实验使用的葡萄为市售品种巨峰。根据天气情况，在葡萄转色期，选取大小、长势均匀一致的果穗，分别用6％AMX化合物1127水悬浮剂2000mg/L、1000mg/L、500mg/L、 100mg/L浸泡果穗10秒钟，清水浸泡作为对照。实验各处理重复10次，每个果穗为一次重复。成熟采收后取样，样品采集时分别从每个果穗的上、中、下3个部位的向光面、背光面两个方向进行采样，每穗采果6粒。带回实验室-20℃下保存备用。
用直径10mm的打孔器于葡萄果粒的赤道处上下均匀取30片果皮，混匀称其重量，进行果皮花色苷的测定。将果皮加入5ml含1％甲酸的甲醇溶液中，25℃下避光摇床萃取30min，然后用8000×g低温离心10min，收集上清液，残渣重复提取4次，合并上清液，30℃旋转蒸发除去甲酸和甲醇，然后用甲酸乙醇的水溶液(甲酸：乙醇：水＝2:10:88)溶解复溶，定容至5ml。用pH示差法测定花色苷的含量(以矢车菊苏-3-葡萄糖苷当量计算)，每个处理重复三次，花色苷含量(mg/g)X＝ΔA×F×M/ε×m，其中ΔA为吸光值；F为稀释倍数；M为矢车菊素-3-葡萄糖苷的相对分子质量449.2，ε为矢车菊素-3-葡萄糖苷的摩尔消光系数26900；m为样品质量。
AMX毒理实验
AMX毒理实验包括：1)细菌回复突变实验来检测受试物是否具有诱发突变的作用，2)体外微核实验检测受试物是否在非代谢活化系统条件(-S9)具有诱导中国仓鼠肺细胞(CHL)微核率升高的作用和3)通过SD大鼠单次经口灌胃受试物最大耐受量实验来初步估测毒性作用的靶器官和可能的毒作用机理。实验中使用的受试物为AMX化合物1127。
细菌回复突变实验使用菌株为TA98和TA100两种鼠伤寒沙门氏菌株，实验在非代谢活化系统条件(-S9)下采用标准平板掺入法进行实验，共设定5g/皿、15g/皿、50g/皿、150g/皿、500g/皿、1500g/皿和5000g/皿7个剂量以及阴性对照和阳性对照。
体外微核实验中使用CHL细胞首先进行IC50测定实验，根据IC50测定结果设定体外微核实验给药剂量，共设定1μg/mL、20μg/mL、30μg/mL和40μg/mL4个给药剂量，同时设定阴性对照和阳性对照，在-S9条件下作用CHL细胞24h后进行制片，通过计数每个剂量500个细胞中的单核细胞、双核细胞和多核细胞数来计算该剂量的胞质分裂阻滞增殖指数(cytokinesis-block proliferation index，CBPI)和细胞毒性。
SD大鼠单次经口灌胃受试物最大耐受量实验选用8只SD大鼠，随机分为2组，每组4雌4雄。大鼠单次经口灌胃给药后，连续观察14天，于第15天解剖。对下列指标进行了评价：临床观察、体重和病理学(肉眼形态学)检查。
实施例1化合物0604c的制备
1.1制备1-丙基-3，4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000018
将1.47g(10mmol)3,4-二氢-2(1H)-喹啉酮加入到20ml无水DMF，分批加入0.48g(60％in oil，12mmol)氢化钠，加毕搅拌0.5小时；滴加2.04g(12mmol)碘丙烷，室温反应16小时；冰浴下加入饱合氯化铵溶液淬灭反应，加入乙酸乙酯萃取。用饱和氯化钠水溶液洗涤，在加入无水硫酸钠干燥有机相。减压蒸去溶剂和过量的碘丙烷，得到油状1-丙基-3,4-二氢-2(1H)-喹啉酮，未经纯化直接用于下一步。
1.2制备6-硝基-1-丙基-3，4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000019
在冰水浴下将10ml浓硫酸加入到1.89g(10mmol)1-丙基-3,4-二氢-2(1H)-喹啉酮中，并剧烈搅拌0.5小时；用滴液漏斗缓慢滴加硝酸钾/硫酸溶液(1.01g KNO3/10ml H2SO4)，维持反应温度低于5℃，并反应2小时；倾倒反应液入冰水中搅拌0.5h。过滤并用大量的水洗涤滤饼。用乙醇对粗品进行重结晶。得淡黄色固体1.65g，收率70％。1H NMR(400MHz,CDCl3):δ8.16-7.05(m,3H),3.95(t,2H),3.00(t,2H),2.71(t,2H),1.72-1.63(m,2H),0.98(t,3H)ppm。
1.3制备6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000020
将1.18g(5mmol)6-硝基-1-丙基-3,4-二氢-2(1H)-喹啉酮加入到50ml甲醇中，再加入催化剂量钯碳。反应体系用氢气真空置换三次。室温搅拌8小时。反应液通过加入硅藻土的玻璃砂漏斗，滤去催化剂。浓缩滤液得6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮，未经纯化直接用于下一步。
1.4制备3-氟-4-甲基苄磺酰氯
Figure PCTCN2016080790-appb-000021
取2.02g(10mmol)3-氟-4-甲基苄溴和0.76g(10mmol)硫脲溶于50ml无水乙醇中，然后慢慢加热至回流。反应4-6小时。减压蒸去溶剂。加入30ml乙腈和10ml浓盐酸。控制温度低于20℃，在剧烈搅拌下分批加入5.4g(60mmol)亚氯酸钠。在15-20℃下反应8-16小时。加入冰水终止反应。乙酸乙酯萃取。浓缩萃取液得2.03g白色固体。3-氟-4-甲基苄磺酰氯未经纯化直接用于下一步。
1.5制备0604c
Figure PCTCN2016080790-appb-000022
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.33g(6mmol)3-氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，为淡黄色固体1.38g，收率71％。1H NMR(400MHz,CDCl3):δ7.58-6.90(m,6H),4.25(s,2H),3.85(t),2.83(t,2H),2.60(t,2H),2.22(s,3H),1.69-1.60(m,2H),0.95(t,3H)ppm。
实施例2化合物1125A的制备
2-氟-4-甲基苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用2-氟-4-甲基苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000023
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.33g(6mmol)2-氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，为淡黄色固体1.15g，收率60％。1H NMR(400MHz,CDCl3):δ7.34-6.91(m,6H),4.36(s,2H),3.89(t,2H),2.87(t,2H),2.65(t,2H),2.34(s,3H),1.71-1.63(m,2H),0.98(t,3H)ppm。
实施例3化合物1125B的制备
3，5-二氟-4-甲基苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用3，5-二氟-4-甲基苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000024
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.44g(6mmol)3，5-二氟4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物0.98g，为淡黄色固体，收率48％。1H NMR(400MHz, DMSO-d6):δ9.75(s,1H),7.09-6.96(m,5H),4.49(s,2H),3.80(t,2H),2.80(t,2H),2.53(t,2H),2.13(s,3H),1.60-1.49(m,2H),0.88(t,3H)ppm。
实施例4化合物0918的制备
2，3-二氟-4-甲基苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用2，3-二氟-4-甲基苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000025
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.44g(6mmol)2，3-二氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到淡黄色固体1.12g，收率55％。1H NMR(400MHz,CDCl3):δ7.09-6.79(m,5H),4.42(s,2H),3.89(t,2H),2.87(t,2H),2.65(t,2H),2.29(s,3H),1.73-1.63(m,2H),0.98(t,3H)ppm。
实施例5化合物1127的制备
2，3，5，6-四氟-4-甲基苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用2，3，5，6-四氟-4-甲基苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000026
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.66g(6mmol)2，3，5，6-四氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，为淡黄色固体1.56g，收率70％。1H NMR(400MHz,DMSO-d6):δ10.07(s,1H),7.08-7.05(m,3H),4.58(s,2H),3.81(t,2H),2.80(t,2H),2.53(t,2H),2.25(s,3H),1.57-1.48(m,2H),0.88(t,3H)ppm。
实施例6化合物1020A的制备
3-氟-4-三氟甲基苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用3-氟-4-三氟甲基苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000027
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.65g(6mmol)3-氟-4-三氟甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，淡黄色固体0.85g，收率39％。1H NMR(400MHz,DMSO-d6):δ10.02(s,1H),7.79-6.99(m,6H),4.63(s,2H),3.80(t,2H),2.78(t,2H),2.48(t,2H),1.56-1.47(m,2H),0.87(t,3H)ppm。
实施例7化合物1020B的制备
2，3，5，6-四氟-4-三氟甲基苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用2，3，5，6-四氟-4-三氟甲基苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000028
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.98g(6mmol)2，3，5，6-四氟-4-三氟甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，为黄色固体0.85g，收率35％。1H NMR(400MHz,DMSO-d6):δ10.17(s,1H),7.07-7.05(m,3H),4.74(s,2H),3.78(t,2H),2.79(t,2H),2.48(t,2H),1.56-1.49(m,2H),0.86(t,3H)ppm。
实施例8化合物1103B的制备
3-氯-4-甲基苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用3-氯-4-甲基苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000029
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.43g(6mmol)3-氯-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱 层析得到标题化合物，为黄色固体0.96g，收率42％。1H NMR(400MHz,DMSO-d6):δ10.17(s,1H),7.41-6.96(m,6H),4.41(s,2H),3.81(t,2H),2.78(t,2H),2.23(t,2H),1.99(s,3H),1.56-1.50(m,2H),0.87(t,3H)ppm。
实施例9化合物0925的制备
2-氟-4-氯苄磺酰氯的制备方法同实施例1的步骤1.4，不同点在于，用2-氟-4-氯苄溴代替3-氟-4-甲基苄溴。
Figure PCTCN2016080790-appb-000030
将1.02g(5mmol)6-氨基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1.45g(6mmol)2-氟-4-氯苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，为黄色固体0.83g，收率40％。1H NMR(400MHz,CDCl3):δ7.63-6.90(m,6H),4.39(s,2H),3.87(t,2H),2.87(t,2H),2.61(t,2H),1.71-1.61(m,2H),0.96(t,3H)ppm。
实施例10化合物0703B的制备
6-氨基-1-丙基-2(1H)-喹啉酮的制备方法同实施例1的步骤1.1，1.2，1.3，不同点在于：用2-羟基喹啉代替3,4-二氢-2(1H)-喹啉酮。
Figure PCTCN2016080790-appb-000031
将1.02g(5mmol)6-氨基-1-丙基-2(1H)-喹啉酮和1.33g(6mmol)3-氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，为淡黄色固体1.44g，收率75％。1H NMR(400MHz,DMSO-d6):δ10.00(s,1H),7.81-6.59(m,8H),4.47(s,2H),4.17(t,2H),2.28(s,3H),1.66-1.57(m,2H),0.96(t,3H)ppm。
实施例11化合物0717的制备
Figure PCTCN2016080790-appb-000032
将1.01g(5mmol)6-氨基-1-丙基-2(1H)-喹啉酮和1.66g(6mmol)2，3，5，6-四氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物，为黄色固体1.35g，收率64％。1H NMR(400MHz,DMSO-d6):δ10.29(s,1H),7.86-6.60(m,5H),4.64(s,2H),4.15(t,2H),2.19(s,3H),1.69-1.57(m,2H),0.96(t,3H)ppm。
实施例12化合物0707的制备
Figure PCTCN2016080790-appb-000033
将1.01g(5mmol)6-氨基-1-丙基-2(1H)-喹啉酮和1.33g(6mmol)2-氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物(1.23g)，收率64％。1H NMR(400MHz,DMSO-d6):δ9.90(s,1H),7.91-6.59(m,8H),4.49(s,2H),4.16(t,2H),2.18(s,3H),1.67-1.55(m,2H),0.95(t,3H)ppm。
实施例13化合物0714的制备
Figure PCTCN2016080790-appb-000034
将1.02g(5mmol)6-氨基-1-丙基-2(1H)-喹啉酮和1.44g(6mmol)2，3-二氟-4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到标题化合物(1.45g)，为黄色固体，收率71％。1H NMR(400MHz,DMSO-d6):δ10.07(s,1H),7.87-6.61(m,8H),4.55(s,2H),4.15(t,2H),2.57(s,3H),1.67-1.58(m,2H),0.95(t,3H)ppm。
实施例14化合物130925AMX的制备
6-氨基-1-烯丙基-3,4-二氢-2(1H)-喹啉酮的制备方法同实施例1的步骤1.1，1.2和 1.3，不同点在于：用3-溴丙烯代替3-碘丙烷。
Figure PCTCN2016080790-appb-000035
将1.01g(5mmol)6-氨基-1-烯丙基-3,4-二氢-2(1H)-喹啉酮和1.23g(6mmol)4-甲基苄磺酰氯加入到30mL DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到化合物130925，为淡黄色固体1.36g，收率73％。1H NMR(400MHz,CDCl3):δ7.22-6.93(m,7H),5.91-5.83(m，1H),5.25-5.13(dd,2H),4.54(s,2H),4.27(d,2H),2.89(t,2H),2.68(t,2H),2.32(s,3H)ppm。
类似地，重复上述步骤，用3-氟-4-甲基苄磺酰氯替代4-甲基苄磺酰氯，制得AMX化合物，即为130925AMX。
Figure PCTCN2016080790-appb-000036
实施例15 化合物140228AMX的制备
6-氨基-4-甲基-1-丙基-2(1H)-喹啉酮的制备方法同实施例1的步骤1.1，1.2和1.3，不同点在于：用2-羟基-4-甲基喹啉代替3,4-二氢-2(1H)-喹啉酮。
Figure PCTCN2016080790-appb-000037
将1.08g(5mmol)6-氨基-4-甲基-1-丙基-2(1H)-喹啉酮和1.23g(6mmol)4-甲基苄磺酰氯加入到30ml DMF中，再加入2.01g(15mmol)碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得到化合物140228，为淡黄色固体1.15g，收率62％。
类似地，重复上述步骤，用3-氟-4-甲基苄磺酰氯替代4-甲基苄磺酰氯，制得AMX化合物，即为140228AMX。
Figure PCTCN2016080790-appb-000038
实施例16化合物0720B的制备
16.1制备4-甲基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000039
将3.0g4-甲基喹啉酮加入到200mL乙酸中，氮气氛围下加入300mg钯碳，用氢气置换三次。升温到70℃，反应12小时；玻璃砂漏斗加入硅藻土真空抽滤，滤液减压浓缩，得到2.7g淡黄色固体。收率为90％。1HNMR(400MHz，DMSO-d6)：δ10.11(s，1H),6.84-7.20(m，4H),3.04(m，1H),2.59(dd，1H)，2.22(dd，1H)，1.17(d，3H)ppm。
16.2制备4-甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000040
将2.0g4-甲基-3,4-二氢-2(1H)-喹啉酮加入到80mL N，N-二甲基甲酰胺中，在冰水浴下搅拌分批加入1.05当量氢化钠，加完搅拌0.5小时；滴加1.1当量的碘丙烷，撤去冰水浴，反应12小时；加入饱和氯化铵溶液淬灭反应，加入乙酸乙酯萃取反应，合并有机相，用饱和氯化钠水溶液洗涤，在加入无水硫酸钠干燥有机相。减压蒸去溶剂和过量的碘丙烷，得到淡黄色油状液体2.2g。粗品未经过进一步纯化，粗收率为88％。
16.3制备4-甲基-6-硝基-1-丙基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000041
在冰水浴下将20mL硫酸加入到盛有2.0g4-甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮烧瓶中，并剧烈搅拌0.5小时；用滴液漏斗缓慢滴加1.1当量硝酸钾的硫酸溶液，维持冰水浴温度并反应1-2小时；倾倒反应液倒冰水中搅拌0.5小时。过滤并用大量的水洗涤滤饼。用乙醇对粗品进行重结晶。得1.8g4-甲基-6-硝基-1-丙基-3,4-二氢-2(1H)-喹啉酮，收率为77％。1HNMR(400MHz，DMSO-d6)：δ8.22-7,32(m,3H)，3.93(m,2H)，3.26(m,1H)，2.76(dd,1H)，2.44(dd,1H)，1.64(m,2H)，1.22(d,3H),0.89(t，3H)ppm。
16.4制备16-氨基-4-甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000042
将1.8g4-甲基-6-硝基-1-丙基-3,4-二氢-2(1H)-喹啉酮加入到甲醇中，再加入钯碳作为催化剂。反应体系用氢气置换三次。室温搅拌8小时。反应液通过加入硅藻土的玻璃砂漏斗，滤去固体。浓缩滤液得1.4g6-氨基-4-甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮。未进一步纯化直接进行下一步，收率为90％。
16.5制备2，3，5，6-四氟-4-甲基苄磺酰氯
Figure PCTCN2016080790-appb-000043
取2，3，5，6-四氟-4-甲基苄氯和1当量的硫脲溶于乙醇中，然后慢慢加热至回流。反应4-6小时后浓缩反应液。加入乙腈和浓盐酸。控制温度5-10℃下，在剧烈搅拌下分批加入1.5当量的亚氯酸钠。在15-20℃下反应8-16小时。加入冰水终止反应。乙酸乙酯萃取三次。浓缩萃取液得2，3，5，6-四氟-4-甲基苄磺酰氯。未进一步纯化直接进行下一步。
16.6制备化合物0720B
Figure PCTCN2016080790-appb-000044
将1.0g6-氨基-4-甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1当量的2，3，5，6-四氟-4-甲基苄磺酰氯加入到DMF中，再加入3当量碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品用硅胶柱层析得1.5g化合物0720B，收率为70％。
1HNMR(400MHz，DMSO-d6)：δ10.10(s,1H)，7.06-7.12(m,3H),4.59(s,2H),3.80(m,2H)，2.93(m,1H),2.61(dd,1H),2.31(dd,1H)，2.23(s,3H),1.60(m,2H)，1.11(d,3H),0.98(t，3H)ppm。
实施例17化合物0825A的制备
17.1制备3-甲基-N-苯基-丁烯酰胺
Figure PCTCN2016080790-appb-000045
将4.9g苯胺加入到100mL氯仿中，加入3摩尔当量的三乙胺。搅拌状态下滴加1.1摩尔当量的3-甲基丁烯酰氯，冰浴控温小于5摄氏度。滴加完毕，移去冰浴，缓慢升温至回流，反应2小时；减压蒸干得到8.2g3-甲基-N-苯基-丁烯酰胺。收率为89％。
17.2制备4,4-二甲基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000046
将7.0g3-甲基-N-苯基-丁烯酰胺加入到50mL甲苯中，加入18g无水三氯化铝。缓慢升温至80℃。反应2.5小时；减压蒸干，硅胶柱层析纯化得到4.7g淡黄色固体。收率为67％1HNMR(400MHz，DMSO-d6)：δ10.12(s,1H),6.86-7.29(m,4H),2.34(s,2H)，1.82(s，6H)ppm。
17.3制备4,4-二甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000047
将4.0g4,4-二甲基-3,4-二氢-2(1H)-喹啉酮加入到80mL N，N-二甲基甲酰胺中，在冰水浴下搅拌分批加入1.05当量氢化钠，加完搅拌0.5小时；滴加1.1当量的碘丙烷，撤去冰水浴，反应12小时；加入饱和氯化铵溶液淬灭反应，加入乙酸乙酯萃取反应。合并有机相，用饱和氯化钠水溶液洗涤，在加入无水硫酸钠干燥有机相。减压蒸去溶剂和过量的碘丙烷，得到4.4g淡黄色油状液体。收率为89％。
17.4制备4,4-二甲基-6-硝基-1-丙基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000048
在冰浴下将20mL硫酸加入到4.0g4，4-二甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮烧瓶中，并剧烈搅拌0.5小时；用滴液漏斗缓慢滴加1.1当量的硝酸钾硫酸溶液，维持冰水浴温度并反应1-2小时；倾倒反应液倒冰水中搅拌0.5小时。过滤,并用大量的水洗涤滤饼。红外灯下干燥。用乙醇对粗品进行重结晶。得3.6g4,4-二甲基-6-硝基-1-丙基-3,4-二氢-2(1H)-喹啉酮，收率为77％
17.5制备6-氨基-4,4-二甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮
Figure PCTCN2016080790-appb-000049
将4.0g4,4-二甲基-6-硝基-1-丙基-3,4-二氢-2(1H)-喹啉酮加入到50mL甲醇中，再加入300mg钯碳作为催化剂。反应体系用氢气置换三次。室温搅拌8小时。反应液通过加入硅藻土的玻璃砂漏斗，滤去固体。浓缩滤液得3.1g6-氨基-4，4-二甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮。未进一步纯化直接进行下一步，收率为89％。
17.6制备0825A
Figure PCTCN2016080790-appb-000050
将1.0g6-氨基-4，4-二甲基-1-丙基-3,4-二氢-2(1H)-喹啉酮和1当量的2，3，5，6-四氟-4-甲基苄磺酰氯加入到DMF中，加入3当量碳酸钾为缚酸剂。使反应保持在室温搅拌16小时。反应结束加入冰水，乙酸乙酯萃取。无水硫酸钠干燥。浓缩有机相。粗品经硅胶柱层析纯化得1.9淡黄色化合物0825A，收率为78％。
1HNMR(400MHz，DMSO-d6)：δ10.07(s,1H),7.09-7.18(m,3H),4.58(s,2H),3.87(t,2H)，2.41(s,2H),2.23(s,3H),1.61(m,2H),1.20(s,6H),0.91(t，3H)ppm。
实施例18 AMX化合物(化合物0604c、化合物0918和化合物1127)体外实验的活性测试
18.1体外生化实验和PP2C蛋白磷酸酶活性测试
体外生化实验表明，本发明的AMX化合物作为一系列广谱高效的PYL受体激动剂，与多个PYL受体具有高亲和性的结合能力，同时促进PYL受体结合并抑制PP2C蛋白磷酸酶活性。
以SnRK2.6磷酸化多肽为底物的HAB1蛋白磷酸酶活性实验表明，AMX可促进PYL2受体可与PP2C蛋白磷酸酶(HAB1)结合，从而抑制HAB1对于SnRK2.6磷酸化多肽的去磷酸化作用，且在相同浓度下其效果显著优于ABA。
其中化合物0604c和化合物0918的PYR/PYL受体结合能力广谱性实验表明，在1μM浓度下，其可与PYR1/PYL1/PYL2/PYL3/PYL4/PYL5/PYL7/PYL10结合，其中与PYR1/PYL1/PYL2/PYL7的结合能力显著高于ABA(图1)，上述结果表明，AMX是一种广谱高效的PYL受体激动剂。
18.2 AlphaScreen实验
用AlphaScreen技术检测AMX对于PYL受体和PP2C蛋白磷酸酶(HAB1)结合的促进能力。
实验结果表明，对于PYR1/PYL1/PYL2/PYL7，AMX显著优于ABA的PYL受体亲和性和结合能力(图2和图3)。在多个AMX化合物存在的条件下，上述四种PYL受体与HAB1均具有显著优于ABA和现有ABA类似物的亲和性，并且AMX的EC50值较ABA要小1-2个数量级，且四种PYL受体与HAB1的结合能力存在AMX的剂量依赖效应(图2和图3)。
此外，使用大豆GmPYL3(拟南芥PYL1的同源基因)和GmPYL6(拟南芥PYL2的同源基因)与拟南芥AtHAB1的实验表明，化合物0604c、化合物0918和化合物1127与大豆GmPYL蛋白同样具有显著高于ABA的亲和性(图9)。
上述结果表明，AMX化合物(如化合物0604c、化合物0918和化合物1127)是一系列比ABA等现有化合物更为高效的PYL受体激动剂。
实施例19 AMX化合物(化合物0720B和0825A)体外实验的活性测试
19.1体外生化实验和PP2C蛋白磷酸酶活性测试
体外生化实验表明，本发明的化合物0720B和0825A作为高效的PYL受体激动剂，与多个PYL受体具有高亲和性的结合能力，同时促进PYL受体结合并抑制PP2C蛋白磷酸酶活性。
以SnRK2.6磷酸化多肽为底物的HAB1蛋白磷酸酶活性实验表明，化合物0720B和0825A均可促进PYL2受体可与PP2C蛋白磷酸酶(HAB1)结合，从而抑制HAB1对于SnRK2.6磷酸化多肽的去磷酸化作用，且在同浓度下的效果显著优于同浓度的ABA(表1)。
化合物0720B的PYR/PYL受体结合能力广谱性实验表明，在1μM浓度下，其与PYR1/PYL1/PYL2/PYL7的结合能力显著高于ABA(图13)，上述结果表明，化合物0720B是一种广谱高效的PYL受体激动剂。
19.2 AlphaScreen实验
用AlphaScreen技术检测化合物0720B对于PYL受体和PP2C蛋白磷酸酶(HAB1)结合的促进能力。
实验结果表明，对于拟南芥PYL2受体和PYR1受体，化合物0720B显著优于ABA的PYL受体亲和性和结合能力(图2)，且0720B的EC50值较ABA要小1-2个数量级(表1)，表明这两种PYL受体与HAB1的结合能力存在0720B的剂量依赖效应(图14)。
上述结果表明，AMX化合物(如化合物0720B和0825A)是比ABA等现有化合物更为高效的PYL受体激动剂。
实施例20 AMX化合物(0604c、1125A、1125B、0918、1127)的生理活性测试
20.1对拟南芥种子萌发的抑制效果
结果如图6所示。播种6天后，1μM对照化合物AM1、化合物0604c、化合物0918、化合物1127和ABA同样能够抑制Col-0生态型种子的萌发，而无法抑制PYR/PYL三突变体pyr1；pyl1；pyl4种子的萌发。
结果表明，AMX化合物的萌发抑制效应是因为其激活了植物内在的ABA信号通路，而非对植物种子产生了毒性。其中两种AMX化合物(化合物0918和化合物1127)对Col-0生态型种子萌发的抑制效果显著优于对照化合物。
20.2拟南芥叶面蒸腾作用
本实验中，利用红外摄像机观察记录叶面的温度变化，从而反映出植物的蒸腾作用强弱。
拟南芥叶面蒸腾实验结果如图7a所示。5μM对照化合物ABA、对照化合物AM1和多种AMX化合物喷施拟南芥两天后，叶面温度均高于DMSO对照组，意味着化合物处理的植物蒸腾作用减弱；喷施4天后，化合物0918和化合物1127处理过的植物叶面温度仍要高于对照组，其余化合物处理过的植物叶面温度则已降至DMSO对照组水平。浓度梯度实验则显示AMX化合物对于蒸腾作用的抑制存在剂量效应(图7b)，其效果与使用化合物的浓度以及化合物中卤素原子的数量均呈正相关。
对大豆进行的叶面蒸腾抑制实验表明，喷施50μM的化合物1127的植株，每株的用量是0.2μmol，三天后其叶面温度仍显著高于喷施DMSO的对照组。结果表明，此时大豆叶面的蒸腾效应仍受到抑制，而喷施相同浓度ABA的植株叶面温度已与对照组无异(图10)。这表明化合物1127在大豆中也存在与拟南芥同样的抑制叶面蒸腾作用的效果。
20.3拟南芥的抗旱性
为了进一步探索AMX对于植株抗旱性的影响，在土壤中生长两周的拟南芥Col-0生态型停止给水，干旱期间每周向叶面喷洒一次含相应浓度的(+)-ABA/AM1/AMX或0.05％DMSO(对照)的溶液，共喷施两次，溶液中同时添加了0.02％(v/v)的表面活性剂Tween-20以增强喷剂对于叶片表皮的穿透作用。经过17天的干旱处理后，喷施DMSO的对照组和喷施10μM ABA和AM1的植物均已旱死，而喷施10μM 0604c的植物仍能存活(图8a)；浓度降至5μM后，喷施化合物0918、化合物1125b和化合物1127的植株在干旱18天后仍然存活，而喷施DMSO的对照组ABA的植株均已旱死，其中喷施化合物1127的植株在干旱20天后仍然保持良好的长势(图8b)。
利用结构相似的化合物(如化合物0604c、化合物0918和化合物1127)进行的实验表明，植物的抗旱性强弱与喷施化合物的卤素原子个数呈正相关，干旱2周后，卤素原子多的化合物(如化合物0918和化合物1127)处理后的植物较相同浓度下卤素原子少的化合物(如化合物0604c)处理后的植物有更强的抗旱性(图8c)。
20.4大豆和玉米的抗旱性
大豆在3组3叶期，玉米在小喇叭口期开始干旱，选取相同大小的植株进行土壤干旱实验，期间每隔两天喷施一次含50μM的各测试化合物AMX或各对照化合物的水溶液，溶液中同样添加了0.05％(v/v)的表面活性剂Tween-20。经过一周的干旱处理后复水，喷施50μM的化合物0918和化合物1127的大豆在复水一月后长势明显优于喷施DMSO的对照组和喷施50μM ABA的株系，其中喷施化合物0918与喷施ABA的株系存活率相当，而喷施化合物1127的株系存活率要高于上述两者(图11)。喷施化合物0918的玉米在复水后大部分存活，而喷施DMSO的对照组玉米在复水后亦无法复原(图11)。
上述结果表明，本发明的化合物可以显著提高单子叶(玉米)和双子叶(大豆)农作物的抗旱性。
本发明所述化合物活性(通过促进PYR/PYL受体与HAB1蛋白磷酸酶的结合从而抑制后者磷酸酶活的能力)的具体数据见表1。
实施例21 AMX化合物(化合物0720B)的抗旱活性测试
21.1对大豆和棉花叶面蒸腾作用的抑制
本实验中，利用红外摄像机观察记录叶面的温度变化，从而反映出植物的蒸腾作用强弱。
大豆和棉花的叶面蒸腾实验结果如图15和图16所示，对大豆进行的叶面蒸腾抑制实验表明，分别喷施10/20/50μM 0720B化合物的植株，在喷施一天后其叶面温度均显著高于喷施DMSO的对照组和喷施50μM ABA的植株，这意味着化合物处理的植物的蒸腾作用要弱于后者。其中喷施20或50μM 0720B化合物的植株其叶面温度在喷施两天后仍显著高于喷施DMSO的对照组，表明此时大豆叶面的蒸腾效应仍受到抑制，而此时喷施50μM ABA的植株叶面温度已与对照组无异(图15)。对棉花进行的叶面蒸腾抑制实验则表明，喷施50μM0720B化合物的植株，两天后其叶面温度仍显著高于喷施DMSO的对照组，而喷施50μM ABA的植株叶面温度此时已与对照组无异(图16)。
上述结果表明，0720B化合物对于大豆和棉花叶面蒸腾作用的抑制效果均显著优于ABA。
21.2对大豆和棉花抗旱性的增强
分别播种16天的大豆和23天的棉花，选取相同大小的植株进行土壤干旱实验。大豆在开始干旱后每3天喷施含10/20/50μM的0720B或50μM的ABA水溶液，棉花则在开始干旱后每4天喷施一次含50μM的ABA/0720B化合物的水溶液，大豆和棉花干旱实验均使用含0.05％DMSO的水溶液作为对照组，上述溶液中均添加了0.1％(v/v)的表面活性剂Tween-20以增强喷剂对于叶片表皮的穿透作用。大豆在干旱11天后复水，喷施10/20/50μM 0720B化合物的大豆复水后长势均明显优于喷施DMSO的对照组以及喷施50μM ABA的植株(图 17)。喷施50μM 0720B化合物的棉花在干旱10天后长势同样明显优于喷施DMSO的对照组以及喷施50μM ABA的植株(图18)。
本发明所述化合物活性(通过促进PYR/PYL受体与HAB1蛋白磷酸酶的结合从而抑制后者磷酸酶活的能力)的具体数据见表1。
表1式I化合物的代表化合物的活性列表
Figure PCTCN2016080790-appb-000051
从表1可以看出，本发明的式I化合物的活性显著高于对照化合物(1、2)，并且，卤素原子的数量与化合物的活性成正比。
实施例22 PYL2-AMX-HAB1复合物的结构
采用通用方法中所述的蛋白晶体解析方法，检测了多个本发明AMX化合物所形成的PYL2-AMX-HAB1复合物晶体结构。对照为ABA和现有的ABA类似物，各复合物的二维结构局部示意图如图4所示。
从图4看出，AMX存在于PYL2的口袋结构中，ABA结构上的四个氧原子可通过多个水分子与PYL2口袋结构以及HAB1的多个氨基酸残基形成氢键(图4a)，AMX化合物磺酰氨基团上的氧原子和氮原子以及喹啉环上的氧原子同样可以形成氢键。此外，对二甲苯上的卤素取代基可通过空间位阻效应帮助磺酰氨基团上的氧原子和氮原子与更邻近的氨基酸残基形成氢键，或直接与PYL2口袋结构形成氢键，如化合物0918(图4d)、化合物1127(图4e)、化合物1020A(图4f)、化合物1020B(图4g)、和化合物1125A(图4h)上的氟原子。
值得注意的是，在对位存在甲基或卤素原子(如氯原子)的情况下，邻位的氟原子多形成氢键，间位的卤素原子则多利用空间位阻效应帮助磺酰氨基团上的氧原子和氮原子与更临近的氨基酸残基形成氢键；而当对位存在卤素代甲基的情况下，间位的氟原子也可形成氢键，如化合物1020A(图4f)。
此外，通过比较三组结构相似的化合物(化合物0604c(图4c)/化合物0918(图4d)/化合物1127(图4e)，化合物1020A(图4f)/化合物1020B(图4g)以及化合物1125A(图4h)/化合物1125B(图4i))，可以发现卤素原子的数量与氢键的数量也呈正相关性。与对照化合物相比，化合物AMX与PYL2口袋结构之间形成的氢键数量更多或结合更强，因此，AMX表现出更强的PYL受体亲和性。
实施例23 AMX可诱导ABA响应的胁迫相关基因的表达
本发明人分析了外源添加AMX对于植物基因表达的影响。
基因表达分析的结果表明，AMX可诱导ABA响应的胁迫相关基因的表达，且表达水平大多可以达到或高于相同浓度外源ABA所诱导的表达水平(图5)。10μM的化合物AMX处理后，10天大的野生型拟南芥(Col-0)苗期植株中，7个已知的受ABA诱导的与环境胁迫相关基因的表达量显著增加，其中用化合物0604c和化合物0717处理6小时后，多数基因的表达水平显著超过10μM ABA处理相同时间后的水平，而10μM AM1处理6小时后，上述基因的表达水平则低于10μM ABA处理相同时间后的水平。此外，化合物0604c和化合物0918处理一天后，相关基因的表达水平亦显著超过10μM ABA处理相同时间后的水平(图5)。
综上，结果表明，AMX对于多数环境胁迫相关基因的诱导效果要显著优于ABA和AM1。
实施例24植物保鲜(鲜花保鲜)
结果表明，AMX化合物1127的水悬浮剂可显著延缓花苞凋谢和叶片枯萎的情况。自 来水对照组放置20天后花朵全部凋谢，叶片全部枯萎，而AMX化合物1127的水悬浮剂处理组尚有60％花朵正常开放，没有叶片枯萎且有70％叶片保持绿色。
实施例25葡萄着色
实验结果如图12所示。
结果表明，AMX化合物1127的水悬浮剂处理后的葡萄果皮花色苷含量显著高于清水对照组，因此，花色苷的含量与葡萄果皮上色成正相关。
实施例26毒性实验
细菌回复突变实验的结果显示，AMX化合物1127在各剂量下对TA98和TA100菌株均未发现明显的细菌毒性。
体外微核实验的结果显示，AMX化合物1127在-S9条件下不具有诱导CHL细胞微核率升高的作用。
SD大鼠单次经口灌胃受试物最大耐受量实验结果显示，给药量为5000mg/kg组的雌雄大鼠在临床观察、体重和各组织/脏器的肉眼形态学观察方面均未见药物相关性改变，AMX化合物1127的最大耐受量(MTD)大于5000mg/kg。
这表明，本发明的AMX化合物具有很高的安全性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体，
    Figure PCTCN2016080790-appb-100001
    式中，
    R1为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
    R2为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
    R3为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
    R4为H、卤素、C1-C3烷基、或C1-C3卤代烷基；
    R5为卤素、C1-C3烷基、C1-C3卤代烷基或SF5；
    R6为H、C1-C3烷基、或C1-C3卤代烷基；
    R7为C1-C7烷基、C2-C7链烯基、C2-C7链炔基、C3-C7环烷基、或-R8-O-R9,其中，R8为C1-C2亚烷基而R9为H、C1-C3烷基；
    R0为H、C1-C4烷基、C1-C4卤代烷基、或卤素；
    m为1或2；
    Figure PCTCN2016080790-appb-100002
    表示单键或双键；
    附加条件是，R1、R2、R3、R4中有1-4个为卤素。
  2. 如权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体，其特征在于，所述的化合物具有式Ia、Ib或Ic结构：
    Figure PCTCN2016080790-appb-100003
    Figure PCTCN2016080790-appb-100004
    式中，R0，R1-R6，m的定义如上所述。
  3. 如权利要求1或2所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体，其特征在于，R1、R2、R3、R4中有2-4个为卤素。
  4. 如权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体，其特征在于，所述的化合物选自下组：
    Figure PCTCN2016080790-appb-100005
    Figure PCTCN2016080790-appb-100006
  5. 一种权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体的用途，其特征在于，用于制备农用制剂或组合物，所述农用制剂或组合物用于(i)增强植物抗逆性；(ii)植物保鲜处理(如鲜花保鲜)；和/或(iii)果实着色处理(如葡萄着色)。
  6. 一种农用制剂，其特征在于，所述农用制剂包括：
    (i)权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体；和
    (ii)农业上可接受的载体。
  7. 一种增强植物抗逆性的方法，其特征在于，给所述植物施用权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体或施用权利要求6所述的农用制剂。
  8. 一种式I化合物或其盐的制法，其特征在于，包括步骤：
    (a)在惰性溶剂中，将式Ia-6化合物与式Ia-4化合物进行反应，从而形成式I化合物；
    Figure PCTCN2016080790-appb-100007
    上述各式中，R0、R1、R2、R3、R4、R5、R6、R7、m和
    Figure PCTCN2016080790-appb-100008
    如权利要求1中所定义。
  9. 一种植物保鲜处理方法，其特征在于，包括步骤：
    将待保鲜的植物与权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体进行接触，从而对植物保鲜。
  10. 一种果实着色处理方法，其特征在于，包括步骤：
    将待着色的果实与权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体进行接触，从而对果实着色。
  11. 一种权利要求1所述的式I化合物、或其盐、或其光学异构体、或其外消旋体、或其溶剂化物、或其前体的用途，其特征在于，用于制备(i)ABA受体的激动剂；和/或(ii)种子萌发抑制剂。
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AU2016380736B2 (en) * 2015-12-28 2019-11-14 CAS Center of Excellence in Molecular Plant Sciences High stress resistant plant growth regulator and preparation and use thereof

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