JP7572057B2 - Flower aging inhibitor - Google Patents

Description

The present invention relates to an agent for inhibiting aging of flowers, which contains a compound containing a tetrafluorophthalimide skeleton or a salt thereof as an active ingredient.

Petals or similar organs in flowers (the inner and outer perianth) have a mechanism for actively aging on their own. This aging generally means the wilting or detachment of the petals or similar organs. In general, petals or similar organs are organs that have developed to attract pollinators such as insects, and become unnecessary once pollination is successful. Petals or similar organs of flowers that are not pollinated will also senesce eventually. The period until petals or similar organs senesce is roughly determined by each plant species, and the aging of petals or similar organs is thought to be a programmed death process.
On the other hand, the "flower life" of commercially traded flowers is one of the most important quality components, and the biggest factor in losing commercial value is senescence of the petals or similar organs.
It is known that the plant hormone ethylene is involved in the senescence of some flowers. To date, genes involved in the biosynthesis, reception, and signaling of ethylene have been identified in several species of flowers, and it is known that the lifespan of flowers can be extended by recombination of ethylene signaling genes.

Patent No. 6044952

Veen, H. and van de Geijn, S. C., 1978, Planta, Vol. 140, p.93-96

For flowers such as carnations, whose senescence is controlled by ethylene (ethylene-dependent flowers), many drugs to suppress senescence, such as ethylene inhibitors containing silver ions as the main component, have been developed and are widely used (for example, Non-Patent Document 1, etc.).
In contrast, the control mechanism of senescence in flowers that do not depend on ethylene (ethylene-independent flowers), such as lilies, tulips, and irises, remains largely unknown, and no drugs have yet been developed to suppress senescence.
In this situation, the applicant has discovered a key gene (EPH1) that controls ethylene-independent senescence (Patent Document 1).
Therefore, an object of the present invention is to provide an agent for inhibiting senescence in ethylene-independent flowers by utilizing the above-mentioned key gene (EPH1).

As a result of intensive research conducted by the inventors to solve the above problems, they constructed a system that can select substances that inhibit the transcription factor function of the above key gene (EPH1), and discovered a group of compounds that have the function of inhibiting aging in flowers, thereby solving the above problems.

Specifically, the present invention relates to the following items.
1. An agent for inhibiting aging of flowers, comprising as an active ingredient one or more compounds selected from compounds containing a tetrafluorophthalimide skeleton or salts thereof.
2. The flower aging inhibitor according to 1., wherein the compound containing a tetrafluorophthalimide skeleton is a compound represented by the following general formula (1):
(Wherein,
Y represents a single bond or any one of the following structures Y-1 to Y-3,
Q: any of the following structures Q-1 to Q-4.
(In the formula, _R1 is independently any one of a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, and a hydroxymethyl group, "*" indicates the bonding position to the nitrogen atom in general formula (1), and "**" indicates the bonding position to Q.)
(In the formula, _R2 each independently represents an alkyl group having 1 to 10 carbon atoms, an acylamino group having 2 to 10 carbon atoms, a hydroxyl group, a halogen atom, or a phenyl group, "**" represents the bonding position with Y in general formula (1), and n represents 0, 1, 2, or 3.)
3. A method for inhibiting aging of flowers, comprising applying the flower aging inhibitor according to 1. or 2. to flowers.

The flower aging inhibitor of the present invention contains a compound containing a tetrafluorophthalimide skeleton or a salt thereof as an active ingredient, and inhibits the progression of cell death in the perianth and delays aging, thereby improving the "flower life" and extending the viewing period in all flowers, including flowers that exhibit ethylene-independent aging (ethylene-independent flowers).

1 is a photograph showing the appearance of petal slices treated with a compound in Test 1 for confirming aging of petal slices treated with a compound in Example 3. 1 is a graph showing the change in protein content of petals treated with a compound in senescence confirmation test-1 of petal slices treated with a compound in Example 3. 1 is a photograph showing the state of a petal slice 24 hours after flowering, which was treated with a compound in Test 2 to confirm aging of the petal slice treated with a compound in Example 4. 1 is a graph showing the protein content of petals 24 hours after anthesis treated with a compound in senescence confirmation test 2 of petal slices treated with a compound in Example 4. 1 is a photograph showing the state of cut flowers treated with a compound 24 hours after flowering in a test to confirm senescence of cut flowers treated with the compound of Example 5.

The flower aging inhibitor of the present invention contains as an active ingredient one or more compounds selected from compounds containing a tetrafluorophthalimide skeleton or salts thereof. The compound containing a tetrafluorophthalimide skeleton refers to an N-substituted 3,4,5,6-tetrafluorophthalimide having the following chemical structure:
The salt in the present invention means a salt that is acceptable for application to ornamental plants, and examples thereof include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, hydrobromide, hydroiodide, etc.; organic acid salts such as acetate, fumarate, maleate, oxalate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc.; and salts with inorganic or organic bases such as sodium ion, potassium ion, calcium ion, trimethylammonium, etc.

Among the compounds containing a tetrafluorophthalimide skeleton in the present invention, a compound represented by the following general formula (1) is preferred.
(Wherein,
Y represents a single bond or any one of the following structures Y-1 to Y-3,
Q: any of the following structures Q-1 to Q-4.
(In the formula, _R1 is independently any one of a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, and a hydroxymethyl group, "*" indicates the bonding position to the nitrogen atom in general formula (1), and "**" indicates the bonding position to Q.)
(In the formula, _R2 each independently represents an alkyl group having 1 to 10 carbon atoms, an acylamino group having 2 to 10 carbon atoms, a hydroxyl group, a halogen atom, or a phenyl group, "**" represents the bonding position with Y in general formula (1), and n represents 0, 1, 2, or 3.)
In the compound represented by general formula (1) in the present invention, when "Y" is Y-1, Y-2, or Y-3 and R ₁ is either an alkyl group having 1 to 3 carbon atoms or a hydroxymethyl group, the carbon to which R ₁ is bonded becomes an asymmetric carbon, and optically active substances derived therefrom exist. The compound containing a tetrafluorophthalimide skeleton in the present invention may be any of these optically active substances, or may be a racemic mixture containing equal amounts of each of these optically active substances, or may be a compound containing any of the optically active substances in excess.

In the present invention, the alkyl group having 1 to 3 carbon atoms for R ₁ means a linear, branched or cyclic alkyl group having 1 to 3 carbon atoms, and examples of such an alkyl group include a methyl group, an ethyl group, a normal propyl group, an isopropyl group and a cyclopropyl group. Among these, a methyl group, an ethyl group, a normal propyl group and an isopropyl group are preferred, and a methyl group and an ethyl group are particularly preferred.
In the present invention, the alkyl group having 1 to 10 carbon atoms for _R2 means a linear, branched or cyclic alkyl group having 1 to 10 carbon atoms. Among them, a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms is preferred, and a linear, branched or cyclic alkyl group having 1 to 3 carbon atoms, that is, any one of a methyl group, an ethyl group, a normal propyl group, an isopropyl group and a cyclopropyl group, is more preferred.
In the present invention, the acylamino group having 2 to 10 carbon atoms for R ₂ means an amino group substituted with an acyl group having 2 to 10 carbon atoms. The acyl group having 2 to 10 carbon atoms refers to an (alkyl)-C(═O)- group having 1 to 9 carbon atoms. The alkyl group having 1 to 9 carbon atoms may be linear, branched, or cyclic. Among these, an acylamino group having 2 to 6 carbon atoms is preferred, an acylamino group having 2 to 5 carbon atoms is more preferred, and an acetylamino group, a propionylamino group, an isopropionylamino group, a butyrylamino group, an isobutyrylamino group, a valerylamino group, an isovalerylamino group, and a pivaloylamino group are particularly preferred.
In the present invention, the halogen atom of _R2 is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, of which a chlorine atom is preferred.
The compound represented by general formula (1) of the present invention is preferably a compound in which "Y" in general formula (1) is a single bond, Y-1, or Y-3. "R ₁ " in Y-1 is preferably a methyl group or an ethyl group, and "R ₁ " in Y-2 is preferably a methyl group.

The compounds suitable as the compound represented by general formula (1) of the present invention are the compounds represented by general formulas (1a) to (1g).
The compound represented by the following general formula (1a) is a compound in which "Y" in general formula (1) is a single bond and "Q" is Q-1.
(In the formula, _R2 and n are defined as in general formula (1).)
As the compound represented by general formula (1a), a compound in which R ₂ is independently any one of an alkyl group having 1 to 10 carbon atoms, a hydroxyl group, a halogen atom, and a phenyl group, and n is 0, 1, 2, or 3 is preferable, and a compound in which R ₂ is independently any one of an alkyl group having 1 to 5 carbon atoms, a hydroxyl group, and a phenyl group, and n is 0, 1, 2, or 3 is more preferable.
Among the compounds represented by general formula (1a), the most suitable compounds are compounds 1a-1 to 1a-6 shown in the following Table 1. In Table 1, "Me" indicates a methyl group, "OH" indicates a hydroxyl group, "Ph" indicates a phenyl group, "iPr" indicates an isopropyl group, and "-" indicates no substitution.

The compound represented by the following general formula (1b) is a compound in which "Y" in general formula (1) is Y-1 and "Q" is Q-1.
(In the formula, R ₁ , R ₂ and n are defined as in general formula (1).)
As the compound represented by general formula (1b), a compound in which R ₁ is any one of a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, and a hydroxymethyl group, R ₂ are each independently an alkyl group having 1 to 10 carbon atoms, an acylamino group having 2 to 10 carbon atoms, and a halogen atom, and n is 0, 1, 2, or 3 is preferred, and a compound in which R ₁ is any one of an alkyl group having 1 to 3 carbon atoms, and a hydroxymethyl group, R ₂ are each independently an acylamino group having 2 to 5 carbon atoms, and a halogen atom, and n is 0, 1, or 2 is more preferred.
Of the compounds represented by general formula (1b), the most suitable compounds are compounds 1b-1 to 1b-7 shown in the following Table 2. In Table 2, "Me" indicates a methyl group, "Et" indicates an ethyl group, "nPr" indicates a normal propyl group, "tBu" indicates a tertiary butyl group, "H" indicates a hydrogen atom, "OH" indicates a hydroxyl group, "NH" indicates an amino group, "C=O" indicates a carbonyl group, "Cl" indicates a chlorine atom, and "-" indicates no substitution.

The compound represented by the following general formula (1c) is a compound in which "Y" in general formula (1) is Y-2 and "Q" is Q-1.
(In the formula, R ₁ , R ₂ and n are defined as in general formula (1).)
As the compound represented by general formula (1c), a compound in which R ₁ is any one of a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, and a hydroxymethyl group, R ₂ is any one of an alkyl group having 1 to 3 carbon atoms, and a halogen atom, and n is 0 or 1 is preferred, and a compound in which R ₁ is any one of a hydrogen atom and a hydroxymethyl group, R ₂ is any one of an alkyl group having 1 to 3 carbon atoms, and n is 0 or 1 is more preferred.
Of the compounds represented by formula (1c), the most suitable compound is compound 1c-1, in which R ₁ is a hydroxymethyl group and n=0.

The compound represented by the following general formula (1d) is a compound in which "Y" in general formula (1) is Y-3 and "Q" is Q-1.
(In the formula, R ₁ , R ₂ and n are defined as in general formula (1).)
As the compound represented by general formula (1d), a compound in which R ₁ is any one of a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, and a hydroxymethyl group, R ₂ is any one of an alkyl group having 1 to 5 carbon atoms, and a halogen atom, and n is 0 or 1 is preferred, and a compound in which R ₁ is any one of a hydrogen atom and a hydroxymethyl group, R ₂ is any one of an alkyl group having 1 to 3 carbon atoms, and n is 0 or 1 is more preferred.
Of the compounds represented by formula (1d), the most suitable compound is compound 1d-1, in which R ₁ is a methyl group and n=0.

The compound represented by the following general formula (1e) is a compound in which "Y" in general formula (1) is a single bond and "Q" is Q-4.
Among the compounds represented by general formula (1e), the most suitable compounds are compound 1e-1 and compound 1e-2 having the following chemical structures.

The compound represented by the following general formula (1f) is a compound in which "Y" in general formula (1) is Y-1 and "Q" is Q-2.
(In the formula, _R1 is defined as in general formula (1).)
As the compound represented by general formula (1f), a compound in which the bonding position to the naphthalene ring is the 1-position, the same as in the following compound (1f-1), is preferable. Among them, a compound in which R ₁ represents any one of a hydrogen atom and an alkyl group having 1 to 3 carbon atoms is more preferable, and a compound in which R ₁ represents any one of an alkyl group having 1 to 3 carbon atoms is even more preferable.
A particularly preferred compound represented by general formula (1f) is compound 1f-1 having the following chemical structure:

The compound represented by the following general formula (1g) is a compound in which "Y" in general formula (1) is Y-1 and "Q" is Q-3.
(In the formula, _R1 is defined as in general formula (1).)
As the compound represented by general formula (1g), a compound in which R ₁ represents any one of a hydrogen atom and an alkyl group having 1 to 3 carbon atoms is preferable, and a compound in which R ₁ represents any one of an alkyl group having 1 to 3 carbon atoms is more preferable.
Of the compounds represented by formula (1g), the most suitable is compound 1g-1, in which R ₁ is a methyl group.

In the present invention, the term "flowers" refers to ornamental plants in general, and the flower aging inhibitor of the present invention is effective in inhibiting the aging of the entire plant by applying it to the flower, and is particularly effective in inhibiting the aging of the perianth.
As mentioned above, many drugs, such as ethylene inhibitors, have been developed to suppress senescence in flowers whose senescence is controlled by ethylene (ethylene-dependent flowers), such as carnations. However, no drugs have yet been developed to suppress senescence in flowers whose senescence is not dependent on ethylene, such as lilies (ethylene-independent flowers).
However, the flower senescence inhibitor of the present invention contains as its active ingredient a group of compounds discovered by a system for selecting substances that inhibit the transcription factor function of a key gene (EPH1) that controls ethylene-independent senescence, and therefore exhibits excellent senescence inhibitory effects not only on ethylene-independent flowers but also on ethylene-dependent flowers.
The protein encoded by the key gene (EPH1) is a transcription factor, and the binding of the key gene (EPH1) protein to a DNA sequence in the expression control region of a target gene induces the transcription of the target gene. The target genes of the key gene (EPH1) protein include cell death-related genes such as protease genes, and the key gene (EPH1) protein binds to a DNA sequence in the expression control region of the cell death-related genes and activates the transcription of these genes, thereby promoting cell death (aging).
It is presumed that the flower aging inhibitor of the present invention acts directly on the key gene (EPH1) protein and inhibits the binding of the key gene (EPH1) protein with the DNA sequence, and as a result, the progression of cell death in the perianth is inhibited, and the effect of inhibiting perianth aging is expressed. Since proteins homologous to the key gene (EPH1) protein are generally present in angiosperms, it is believed that, according to the mechanism of action, the effect of the present invention will be expressed not only in the morning glory specifically shown in the examples, but also in flowers in general.
As the ornamental plant of the present invention, an ethylene-independent ornamental plant is preferable, and examples thereof include plants of the Convolvulaceae, Rosaceae, Liliaceae, Asteraceae, Iridaceae, etc., and among these, plants such as morning glory, rose, lily, tulip, chrysanthemum, sunflower, gerbera, margaret, iris, dahlia, gladiolus, iris, iris, freesia, Sandersonia, etc. are more preferable.

The flower aging inhibitor of the present invention is prepared by dissolving an active ingredient selected from a compound containing a tetrafluorophthalimide skeleton or a salt thereof in water to obtain a water-soluble solution, which is then applied to the target flower or plant as is or after further dilution with water. Methods for application to flowers include, for example, a method of spraying the entire flower or a method of sealing the entire flower in a container and fumigating the flower, and among these, when the target is cut flowers, a method of adding the agent to the water for the cut flowers, a method of spraying the flower or the entire cut flowers, a method of immersing the entire cut flowers, a method of sealing the cut flowers in a container and fumigating the cut flowers, and the like can be mentioned.
When the flower aging inhibitor of the present invention is applied to flowers as a water-soluble solution, the active ingredient concentration is generally preferably in the range of 0.01 μM to 100 mM, more preferably in the range of 0.05 μM to 50 mM, even more preferably in the range of 0.1 μM to 10 mM, and particularly preferably in the range of 0.1 μM to 5 mM.
In addition, the flower aging inhibitor of the present invention is usually preferably mixed with an inert carrier such as a liquid carrier or a solid carrier and formulated for use, and can be formulated into a water-soluble agent, a hydrated agent, a flowable agent, a microcapsule agent, an emulsion, a powder, a granule agent, etc. for use.
When the flower aging inhibitor of the present invention is formulated and used, it is usually applied after diluting with water so that the concentration of the active ingredient selected from a compound containing a tetrafluorophthalimide skeleton or a salt thereof is 0.1 to 10,000 ppm, preferably 0.1 to 100 ppm, and more preferably 1 to 10 ppm. When it is formulated into a dust or granule, it is preferable to apply it as it is.

Examples of liquid carriers include water, alcohols (methanol, ethanol, isopropyl alcohol, butanol, hexanol, benzyl alcohol, ethylene glycol, propylene glycol, phenoxyethanol, etc.), ketones (acetone, methyl ethyl ketone, cyclohexanone, etc.), aromatic hydrocarbons (toluene, xylene, ethylbenzene, dodecylbenzene, phenylxylylethane, methylnaphthalene, etc.), aliphatic hydrocarbons (hexane, cyclohexane, kerosene, diesel, etc.), esters (ethyl acetate, butyl acetate, isopropyl myristate, ethyl oleate, diisopropyl adipate, diisobutyl adipate, propylene glycol monomethyl ester, etc.), and the like. ether acetate, etc.), nitriles (acetonitrile, isobutyronitrile, etc.), ethers (diisopropyl ether, 1,4-dioxane, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, diethylene glycol monomethyl ether, propylene glycol monomethyl ether, dipropylene glycol monomethyl ether, 3-methoxy-3-methyl-1-butanol, etc.), amides (N,N-dimethylformamide (hereinafter referred to as "DMF"), N,N-dimethylacetamide, etc.), sulfoxides (dimethyl sulfoxide (hereinafter referred to as "DMSO"), etc.), propylene carbonate, and vegetable oils (soybean oil, cottonseed oil, etc.).
Examples of solid carriers include fine powders and granules of clays (kaolin clay, diatomaceous earth, bentonite, fubasami clay, acid clay, etc.), synthetic hydrous silicon oxide, talc, ceramics, other inorganic minerals (sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, etc.), chemical fertilizers (ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride, etc.), and synthetic resins (polyester resins such as polypropylene, polyacrylonitrile, polymethyl methacrylate, polyethylene terephthalate, etc., nylon resins such as nylon-6, nylon-11, nylon-66, etc., polyamide resins, polyvinyl chloride, polyvinylidene chloride, vinyl chloride-propylene copolymers, etc.).

When the flower aging inhibitor of the present invention is formulated, a surfactant and other formulation auxiliaries may be added as necessary.
Examples of the surfactant include nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene alkylaryl ethers, and polyethylene glycol fatty acid esters, and anionic surfactants such as alkyl sulfonates, alkylbenzene sulfonates, and alkyl sulfates.
Other formulation adjuvants include fixing agents, dispersing agents, colorants, stabilizers, and the like, specific examples of which include casein, gelatin, sugars (starch, gum arabic, cellulose derivatives, alginic acid, and the like), lignin derivatives, bentonite, synthetic water-soluble polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acids, and the like), PAP (isopropyl acid phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), and BHA (a mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol).

The present invention will be explained below with reference to examples, but the technical scope of the present invention is not limited to these examples.

Example 1: Construction of a system for selecting compounds having an effect of inhibiting senescence in flowers (Summary) Selection of compounds having an effect of inhibiting senescence in flowers was carried out based on a system constructed according to the following procedure.
(1) A DNA sequence that binds to EPH1 protein (target DNA sequence of EPH1 protein) was identified.
(2) EPH1 protein was synthesized using a cell-free protein synthesis system.
(3) A system was constructed to rapidly and sensitively detect in a test tube the strength of interaction between EPH1 protein synthesized in a cell-free protein synthesis system and a specified target DNA sequence.
(4) Using this detection system, compounds that inhibit the binding of EPH1 protein to the target DNA sequence were screened.

(1) Identification of EPH1 Protein-Binding DNA Sequence The DNA sequence to which EPH1 protein binds had not previously been elucidated, but as a result of extensive research in the present invention, it has now been identified.
Specifically, a group of genes whose expression levels increase during senescence of morning glory petals, like the EPH1 gene, were selected from approximately 20,000 genes by microarray analysis. As a result of comprehensive analysis of the DNA sequences of the expression control regions of these selected genes, a DNA sequence (VPE1: 5'-ATTTGTCTTGTTGCGTATATGATTGAGGT-3') predicted to bind to the EPH1 protein was found in the expression control region of the vacuolar processing enzyme gene (VPE gene) involved in protein degradation.
Analysis of the interaction between the EPH1 protein and the VPE1 sequence confirmed that the EPH1 protein binds to the VPE1 sequence.
(2) Synthesis of EPH1 protein by cell-free protein synthesis system Since the DNA sequence (VPE1 sequence) to which EPH1 protein binds was newly identified in the present invention, we attempted to construct a system that can monitor the interaction between EPH1 protein and a DNA sequence using EPH1 protein and VPE1 sequence. In screening of compounds, it is necessary to analyze the inhibitory activity of many compounds, and in order to obtain highly reproducible results, it is essential to mass-synthesize high-quality proteins and to have a high-speed and highly sensitive detection system. Generally, living cells such as Escherichia coli are used to mass-synthesize proteins. However, although the cause is unknown, it has been difficult to mass-synthesize EPH1 protein using E. coli. Therefore, we developed a system to mass-synthesize EPH1 protein with an AGIA tag (EPH1-AGIA protein) using a wheat cell-free protein synthesis system. Specifically, a DNA sequence encoding an AGIA polypeptide was bound to the 3' end of a DNA sequence containing the full-length EPH1, and the DNA sequence was incorporated into a vector for protein synthesis. The EPH1-AGIA protein was synthesized using the obtained protein synthesis vector pEU-EPH1-AGIA and WEPRO1240 expression kit (Cell-Free Sciences).
(3) Construction of an inhibitor compound selection system A detection system using AlphaScreen was developed to detect the interaction between the EPH1 protein and the VPE1 sequence. Specifically, the EPH1-AGIA protein, the biotin-labeled VPE1 sequence (Bio-VPE1 sequence), and the AlphaScreen detection reagent are mixed, and the luminescence signal generated from the mixture is detected using an EnVision plate reader (PerkinElmer). This led to the construction of a system that can detect the strength of the interaction between the EPH1 protein and the target DNA sequence in a test tube at high speed and with high sensitivity.
(4) Screening for Inhibitory Compounds Using the above-mentioned monitoring system, compounds that inhibit the binding of EPH1 protein to a target DNA sequence were screened.

<Example 2-1> Primary Screening of Inhibitory Compounds Primary screening of inhibitory compounds was performed using the Core Library containing 9600 compounds provided by the University of Tokyo Drug Discovery Initiative, using the above inhibitory compound selection system. This Core Library is a compound library containing 9600 samples selected in consideration of the structural diversity of the compounds.
Specifically, AlphaScreen Buffer (PerkinElmer), EPH1-AGIA protein, and Bio-VPE1 sequence were added to a 384-well Alphaplate into which a compound dissolved in DMSO had been dispensed, and the plate was left to stand at 26°C for 1 hour. Then, a detection reagent containing AlphaScreen donor beads and AlphaScreen acceptor beads (PerkinElmer) was added, and the plate was left to stand at 26°C for another 1 hour. The final concentration of the compound was 10 μM. The luminescence signal of AlphaScreen was measured using an EnVision plate reader (PerkinElmer). The inhibition rate (%) of the interaction between the EPH1 protein and the VPE1 sequence for each compound was calculated from the strength of the AlphaScreen signal.
As a result, compounds 1e-2 and 1b-6, which showed an inhibition rate of 75% or more and commonly contain a tetrafluorophthalimide skeleton, were obtained.

Example 2-2 Screening of inhibitory compounds Since both of the compounds 1e-2 and 1b-6 selected in Example 2-1 had a tetrafluorophthalimide skeleton, 27 compounds were selected by computer search from the compound database of the University of Tokyo Drug Discovery Center. Furthermore, two compounds having a tetrafluorophthalimide skeleton were purchased from Enamin.
A total of 31 compounds including compounds 1e-2 and 1b-6 were analyzed for the inhibition rate for each compound by the same method as in the primary screening in Example 2. The final concentration of the compound was 5 μM.
The inhibition rates of the 31 compounds are summarized in Table 3. As the final concentration was 5 μM, the compounds selected by this screening are a group of compounds that have an extremely high effect of inhibiting senescence in flowers.

The compounds with inhibition rates of 90% or more and 100% or less were 12 compounds having the following chemical structures: 1e-1, 1f-1, 1a-1, 1d-1b, 1b-1, 1a-2, 1g-1a, 1b-2, 1d-1a, 1a-3, 1a-4, and 1b-3a.
The compounds with inhibition rates of 80% or more and less than 90% were eight compounds having the following chemical structures: 1b-4b, 1e-2, 1b-3b, 1c-1a, 1b-4a, 1c-1b, 1b-5, and 1g-1b.

The compounds with an inhibition rate of 70% or more and less than 80% were the two compounds 1b-6 and 1a-5, which have the following chemical structures.
The compounds with an inhibition rate of 60% or more and less than 70% were two compounds, 1b-7a and 1b-7b, which have the following chemical structures.
There was no compound with an inhibition rate of 50% or more but less than 60%.
The compound with an inhibition rate of 40% or more and less than 50% was compound 1a-6 having the following chemical structure.
The compounds with an inhibition rate of 30% or more and less than 40% were two compounds 1h-1a and 1h-1b, which have the following chemical structures.
The compounds with an inhibition rate of 20% or more and less than 30% were compounds 1h-2 and 1h-3, which have the following chemical structures.
The compounds with an inhibition rate of 10% or more and less than 20% were two compounds, 1h-4a and 1h-4b, which have the following chemical structures.
As described above, the screening shown in Example 2-2 is set to a final concentration of 5 μM, and is intended to find a group of compounds with an extremely high effect of inhibiting flower aging. Therefore, although the inhibition rates of the above compounds 1h-1 to 1h-4 in this screening are low, at 10% or more and less than 40%, in practical situations, they are a group of compounds that exhibit a good effect of inhibiting flower aging.

Example 3: Senescence confirmation test of petal slices treated with compounds-1
(1) Effect of Compound Treatment on Petal Senescence In the above Examples 2-1 and 2-2, Compound 1e-1 and Compound 1b-2, which showed high inhibitory activity, were treated with slices of morning glory petals, and their effects on the progression of senescence were analyzed.
Morning glory cultivar "Murasaki" was grown in an incubator (temperature 24°C, relative humidity 70%, PPFD 100 μmol m ^-2 s ^-1 , 12-h light/dark cycle). At the start of the light period, the flowers were almost in full bloom, and this time point was designated as 0 h after flowering.
Compound 1e-1 and compound 1b-2 (20 mM) dissolved in DMSO were diluted with distilled water to final concentrations of 10 μM, 5 μM, and 1 μM. 5 mL of the diluted solution was placed in a 9 cm diameter Petri dish, and petal slices prepared from buds of morning glory "purple" 4 hours before flowering were floated on the diluted solution with the adaxial side of the petal facing up. The Petri dish was placed at an air temperature of 25°C under continuous fluorescent lighting (PPFD 15 μmol m ^-2 s ^-1 ) to observe the aging of the petals. Photographs showing the aging of petal slices treated with compound 1e-1 and compound 1b-2 at a final concentration of 5 μM are shown in Figure 1.
As shown in Figure 1, in the control group, waterlogging (petals becoming transparent) due to the progression of cell death began to be observed 18 hours after anthesis, whereas in the petals treated with the compound, no obvious waterlogging was observed even 36 hours after anthesis.
Similar results were also obtained in petal sections treated with 10 μM and 1 μM of compound 1e-1 and compound 1b-2.
From these results, it was revealed that compound 1e-1 and compound 1b-2, which are specific examples of the flower aging inhibitor of the present invention, inhibit the progress of petal aging.

(2) Changes in protein content It is known that the protein content of petals decreases during senescence. Therefore, changes in protein content in petals treated with the compounds were analyzed.
Compound 1e-1 and compound 1b-2 (20 mM) dissolved in DMSO were diluted with distilled water to a final concentration of 5 μM. 5 mL of the diluted solution was placed in a 9 cm diameter Petri dish, and petal slices prepared from buds of morning glory "purple" 4 hours before flowering were floated on the diluted solution with the adaxial side of the petal facing up. The Petri dish was placed at 25°C under continuous fluorescent lighting (PPFD 15 μmol m ^-2 s ^-1 ), and the petal slices were sampled every 8 hours. Protein content was measured using a TaKaRa Bradford Protein Assay Kit (manufactured by Takara Bio Inc.). The measurement results are shown in Figure 2.
As shown in the graph in Figure 2, in the control group (●), a rapid decrease in protein content was observed 16 hours after anthesis (to 28% of the protein content at 0 hours after anthesis), whereas in the petals treated with compound 1e-1 (□) and compound 1b-2 (△), the protein content maintained 88% and 70%, respectively, of the protein content at 0 hours after anthesis even 24 hours after anthesis.
These results confirmed that treatment with compound 1e-1 and compound 1b-2 suppressed the decrease in the protein content in petals.
This result clearly indicates that treatment with compound 1e-1 and compound 1b-2, which are specific examples of the flower aging inhibitor of the present invention, inhibits aging of petals.

Example 4: Senescence confirmation test of petal slices treated with compounds-2
(1) Effect of Compound Treatment on Petal Senescence The effects of 12 compounds, namely compounds 1e-2, 1a-3, 1a-5, 1a-1, 1a-4, 1f-1, 1e-1, 1c-1a, 1b-1, 1b-2, 1a-6, and 1a-2, which showed high inhibitory activity in the above Examples 2-1 and 2-2, on the progression of senescence in morning glory petal slices were analyzed.
Morning glory cultivar "Murasaki" was grown in an incubator (temperature 24°C, relative humidity 70%, PPFD 100 μmol m ^-2 s ^-1 , 12-h light/dark cycle). At the start of the light period, the flowers were almost in full bloom, and this time point was designated as 0 h after flowering.
As in Example 3 above, each of the 12 compounds was applied to petal slices of morning glory, and the effect on the progression of senescence was analyzed.
Compounds (20 mM) dissolved in DMSO were diluted with distilled water to a final concentration of 5 μM. 5 mL of the diluted solution was placed in a 9 cm diameter Petri dish, and petal slices prepared from buds of morning glory "purple" 4 hours before flowering were floated on the diluted solution with the adaxial side of the petal facing up. The Petri dish was placed at an air temperature of 25°C under continuous fluorescent lighting (PPFD 15 μmol m ^-2 s ^-1 ) to observe the aging of the petals. A photograph showing the aging of the petal slices 24 hours after flowering is shown in Figure 3.
As shown in FIG. 3, in the control group, cell death in the petals progressed and waterlogging became noticeable 24 hours after flowering, whereas in the petals treated with the compound that is a specific example of the flower aging inhibitor of the present invention, waterlogging was not observed.
This result clearly indicates that treatment with Compound 12, which is a specific example of the flower aging inhibitor of the present invention, inhibits the progress of petal aging.

(2) Changes in protein content As in Example 3, changes in protein content in petals treated with 10 compounds, namely compounds 1e-2, 1a-3, 1a-5, 1a-1, 1a-4, 1f-1, 1e-1, 1c-1a, 1b-1, and 1b-2, which showed high inhibitory activity in Examples 2-1 and 2-2, were analyzed.
Compounds (20 mM) dissolved in DMSO were diluted with distilled water to a final concentration of 5 μM. 5 mL of the diluted solution was placed in a 9 cm diameter Petri dish, and petal slices prepared from buds of morning glory "purple" 4 hours before flowering were floated on the diluted solution with the adaxial side of the petal facing up. The Petri dish was placed under continuous fluorescent lighting (PPFD 15 μmol m ^-2 s ^-1 ) at an air temperature of 25°C, and the petal slices were sampled every 8 hours. Protein content was measured using a TaKaRa Bradford Protein Assay Kit (Takara Bio). The measurement results are shown in Figure 4.
As shown in Figure 4, in the control group, the protein content 24 hours after anthesis decreased to 36% compared to 0 hours after anthesis, whereas in the petals treated with the compound, the protein content remained at 65 to 100% of that at 0 hours after anthesis even 24 hours after anthesis.
These results indicate that treatment with a compound which is a specific example of the flower aging inhibitor of the present invention inhibits the decrease in protein content in petals.
This result clearly indicates that the treatment with a compound which is a specific example of the flower aging inhibitor of the present invention inhibits the aging of petals.

Example 5: Senescence confirmation test of cut flowers treated with compounds Compound 1e-1 and compound 1b-2 were treated with cut morning glory flowers, and the effects on the progression of senescence were analyzed.
Compound 1e-1 and compound 1b-2 (20 mM) dissolved in DMSO were diluted with distilled water to a final concentration of 5 μM. 400 mL of the diluted solution was placed in a 500 mL beaker, and the whole cut flower of morning glory cut from the plant body at 0 hours after flowering was immersed in the diluted solution. Only the abaxial side of the petal of the cut flower was in contact with the treatment solution and floating. The beaker containing the cut flower was placed at an air temperature of 25° C. under continuous fluorescent lighting (PPFD 15 μmol m ⁻² s ⁻¹ ) and the state of aging of the petal was observed. A photograph showing the state of aging of the petal slice 24 hours after flowering is shown in FIG. 5.
As shown in Figure 5, in the control group, water-soaked petals (transparent state) due to the progression of cell death in the petals became noticeable 24 hours after anthesis, and the petals of the cut flowers removed from the treatment solution were completely wilted. In contrast, in the cut flowers treated with Compound 1e-1 and Compound 1b-2, water-soaked petals were not observed, and the petals of the cut flowers removed from the treatment solution did not wilt either.
These results show that treatment with compound 1e-1 and compound 1b-2, which are specific examples of the flower aging inhibitor of the present invention, inhibited the progression of aging in cut morning glory flowers.

The above-mentioned Examples 1 to 5 are all test examples using morning glories, but it was separately confirmed that similar effects could be obtained with other flowers such as petunias.

The flower senescence inhibitor of the present invention contains as its active ingredient a group of compounds discovered using a system for selecting substances that inhibit the transcription factor function of a key gene (EPH1) that controls ethylene-independent senescence, and the active ingredient is a compound containing a tetrafluorophthalimide skeleton or a salt thereof.
The flower aging inhibitor of the present invention is thought to act directly on the protein encoded by the key gene (EPH1) and inhibit the binding between the key gene (EPH1) protein and the DNA sequence in the expression control region of a cell death-related gene, thereby suppressing the progression of cell death in the perianth and exerting an aging inhibitory effect in flowers.Therefore, it can be said that a similar aging inhibitory effect is exerted on all flowers that have proteins homologous to the key gene (EPH1) protein.
In other words, the flower aging inhibitor of the present invention exerts the remarkable effect of improving the "flower life" and extending the viewing period in all flowers, including flowers that exhibit ethylene-independent aging (ethylene-independent flowers).

Claims (2)

An agent for inhibiting aging of flowers, comprising as an active ingredient one or more compounds selected from compounds having a tetrafluorophthalimide skeleton represented by the following general formula (1) or salts thereof:

(Wherein,
Y represents a single bond or any one of the following structures Y-1 to Y-3,
Q: any of the following structures Q-1 to Q-4.

(In the formula, _R1 is independently any one of a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, and a hydroxymethyl group, "*" indicates the bonding position to the nitrogen atom in general formula (1), and "**" indicates the bonding position to Q.)

(In the formula, _R2 each independently represents an alkyl group having 1 to 10 carbon atoms, an acylamino group having 2 to 10 carbon atoms, a hydroxyl group, a halogen atom, or a phenyl group, "**" represents the bonding position with Y in general formula (1), and n represents 0, 1, 2, or 3.) A method for inhibiting aging of flowers, comprising applying the flower aging inhibitor according to claim 1 to flowers.