1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE INHIBITORS (ACO-I)

Abstract

This invention provides for use of a compound of formula (1) for inhibiting a post-germination ethylene production response in a plant or plant part and a method of inhibiting a post-germination ethylene production response of a plant or plant part comprising delivering a compound of formula (1) to a plant or plant part.

##STR00001##

Claims

1. A compound of formula (1), wherein formula (1) is: ##STR00029## or a salt or tautomer thereof, wherein: ring A is a six-membered aromatic or non-aromatic ring in which X.sup.1 and X.sup.2 are independently selected from O, CH, CH.sub.2, CH(C.sub.1-4 alkyl), and C(C.sub.1-4 alkyl).sub.2; R.sup.1a and R.sup.1b are independently selected from hydrogen and C.sub.1-4 alkyl or R.sup.1a and R.sup.1b together form a carbonyl group with the carbon atom of ring A to which they are attached; R.sup.2 and R.sup.3 are independently selected from hydrogen and C.sub.1-4 alkyl or are absent when the oxygen atom to which they are attached forms a carbonyl group with the carbon ring member of ring A; L.sup.1 is selected from a bond, CH.sub.2, CHCH and CH.sub.2CH.sub.2Ar.sup.1 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group optionally substituted by one or more substituents R.sup.4; and R.sup.4 is selected from hydroxy, halogen, OAr.sup.2, Hyd.sup.1, OHyd.sup.1, NH(Hyd.sup.1) and N(Hyd.sup.1).sub.2, wherein Hyd.sup.1 is a C.sub.1-4 hydrocarbon group optionally substituted with one or more fluorine atoms; Ar.sup.2 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group; wherein the compound is operative to inhibit a post-germination ethylene production response in a plant or in a plant part.

2. The compound of claim 1, wherein the Use of a compound is capable of modifying at least one physiological process of a plant or plant part selected from: a) preventing or slowing food ripening or crop maturation; b) preventing or slowing plant or plant part senescence; c) preventing or slowing flower senescence; d) improving crop quality whilst on the plant or following harvest; e) reducing a biotic or an abiotic stress response in a plant, for example a response to heat and drought stress; f) maintaining the freshness of plants or plant parts; and wherein formula (1) is ##STR00030## or a salt or tautomer thereof, wherein: ring A is a six-membered aromatic or non-aromatic ring in which X.sup.1 and X.sup.2 are independently selected from O, CH, CH.sub.2, CH(C.sub.1-4 alkyl), and C(C.sub.1-4 alkyl).sub.2; R.sup.1a and R.sup.1b are independently selected from hydrogen and C.sub.1-4 alkyl or R.sup.1a and R.sup.1b together form a carbonyl group with the carbon atom of ring A to which they are attached; R.sup.2 and R.sup.3 are independently selected from hydrogen and C.sub.1-4 alkyl or are absent when the oxygen atom to which they are attached forms a carbonyl group with the carbon ring member of ring A; L.sup.1 is selected from a bond, CH.sub.2, CHCH and CH.sub.2CH.sub.2Ar.sup.1 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group optionally substituted by one or more substituents R.sup.4; R.sup.4 is selected from hydroxy, halogen, OAr.sup.2, Hyd.sup.1, OHyd.sup.1, NH(Hyd.sup.1) and N(Hyd.sup.1).sub.2, wherein Hyd.sup.1 is a C.sub.1-4 hydrocarbon group optionally substituted with one or more fluorine atoms; Ar.sup.2 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group.

3. The compound according to claim 1, wherein the compound is not 2,2,4-trimethyl-6-(3-phenylpropanoyl) cyclohexane-1,3,5-trione.

4. The compound according to claim 1, wherein the compound is 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione.

5. The compound according to claim 1, wherein the compound is 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one.

6. The compound according to claim 1, wherein the compound is 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione, and wherein the compound is operative for modifying at least one physiological process of a plant or plant part selected from: a) preventing or slowing food ripening or crop maturation; b) preventing or slowing plant or plant part senescence; c) preventing or slowing flower senescence; d) improving crop quality whilst on the plant or following harvest; e) reducing a biotic or an abiotic stress response in a plant, for example a response to heat and drought stress; f) maintaining the freshness of plants or plant parts; g) preventing seed germination; and h) weed control.

7. The compound according to claim 1, wherein the compound is 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one, and wherein the compound is operative for modifying at least one physiological process of a plant or plant part selected from: a) preventing or slowing food ripening or crop maturation; b) preventing or slowing plant or plant part senescence; c) preventing or slowing flower senescence; d) improving crop quality whilst on the plant or following harvest; e) reducing a biotic or an abiotic stress response in a plant, for example a response to heat and drought stress; f) maintaining the freshness of plants or plant parts; g) preventing seed germination; and h) weed control.

8. A method of inhibiting a post-germination ethylene production response of a plant or plant part comprising contacting the plant or the plant part with a compound of formula (1), wherein formula (1) comprises: ##STR00031## or a salt or tautomer thereof, wherein: ring A is a six-membered aromatic or non-aromatic ring in which X.sup.1 and X.sup.2 are independently selected from O, CH, CH.sub.2, CH(C.sub.1-4 alkyl), and C(C.sub.1-4 alkyl).sub.2; R.sup.1a and R.sup.1b are independently selected from hydrogen and C.sub.1-4 alkyl or R.sup.1a and R.sup.1b together form a carbonyl group with the carbon atom of ring A to which they are attached; R.sup.2 and R.sup.3 are independently selected from hydrogen and C.sub.1-4 alkyl or are absent when the oxygen atom to which they are attached forms a carbonyl group with the carbon ring member of ring A; L.sup.1 is selected from a bond, CH.sub.2, CHCH and CH.sub.2CH.sub.2Ar.sup.1 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group optionally substituted by one or more substituents R.sup.4; and R.sup.4 is selected from hydroxy, halogen, OAr.sup.2, Hyd.sup.1, OHyd.sup.1, NH(Hyd.sup.1) and N(Hyd.sup.1).sub.2, wherein Hyd.sup.1 is a C.sub.1-4 hydrocarbon group optionally substituted with one or more fluorine atoms; and Ar.sup.2 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group; and whereby the compound is operative to inhibit a post-germination ethylene production response in the plant or in the plant part.

9. The method of claim 8, further comprising wherein the method is operative for modifying at least one physiological process of a plant or plant part selected from: a) preventing or slowing the food ripening or crop maturation; b) preventing or slowing plant or plant part senescence; c) preventing or slowing flower senescence; d) improving crop quality whilst on the plant or following harvest; e) reducing a biotic or an abiotic stress response in a plant including a response to heat and drought stress; f) maintaining the freshness of a plant or plant part; and wherein the method comprises delivering a compound of formula (1) to the plant or plant part.

10. The method according to claim 8, wherein the compound of formula (1) is applied to a medium in which the plant or plant part is located, for subsequent uptake of the compound into the plant or plant part.

11. The method according to claim 8, wherein the compound of formula (1) is applied to a medium in which the plant or plant part is located, for subsequent uptake of the compound into the plant or plant part, and wherein the medium is a liquid.

12. The method according to claim 8, wherein the compound of formula (1) is applied to a medium in which the plant or plant part is located, for subsequent uptake of the compound into the plant or plant part, and wherein the medium is a solid.

13. The method according to claim 8, wherein the plant part is selected from the group consisting of: a leaf, stem, flower, seed, fruit or any combination thereof.

14. The method according to claim 8, wherein the compound is not 2,2,4-trimethyl-6-(3-phenylpropanoyl)cyclohexane-1,3,5-trione.

15. The method according to claim 8, wherein the compound is 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione.

16. The method according to claim 8, wherein the compound is 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one.

17. The method according to claim 8, wherein the compound of formula (1) is applied to a medium in which the plant or plant part is located, for subsequent uptake of the compound into the plant or plant part, and wherein the concentration of the compound applied to the medium in which the plant or plant part is located is between 0.005 M and 50 M.

18. The method according to claim 8, wherein the compound of formula (1) is applied to a medium in which the plant or plant part is located, for subsequent uptake of the compound into the plant or plant part, and wherein the concentration of the compound applied to the medium in which the plant or plant part is located is between 5 M and 15 M.

19. The method according to claim 8, further comprising whereby an execution of the method results in at least one of the following processes: a) preventing or slowing food ripening or crop maturation; b) preventing or slowing plant or plant part senescence; c) preventing or slowing flower senescence; d) improving crop quality whilst on the plant or following harvest; e) reducing a biotic or an abiotic stress response in a plant, for example a response to heat and drought stress; f) maintaining the freshness of plants or plant parts; and wherein formula (1) is ##STR00032## or a salt or tautomer thereof, wherein: ring A is a six-membered aromatic or non-aromatic ring in which X.sup.1 and X.sup.2 are independently selected from O, CH, CH.sub.2, CH(C.sub.1-4 alkyl), and C(C.sub.1-4 alkyl).sub.2; R.sup.1a and R.sup.1b are independently selected from hydrogen and C.sub.1-4 alkyl or R.sup.1a and R.sup.1b together form a carbonyl group with the carbon atom of ring A to which they are attached; R.sup.2 and R.sup.3 are independently selected from hydrogen and C.sub.1-4 alkyl or are absent when the oxygen atom to which they are attached forms a carbonyl group with the carbon ring member of ring A; L.sup.1 is selected from a bond, CH.sub.2, CHCH and CH.sub.2CH.sub.2Ar.sup.1 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group optionally substituted by one or more substituents R.sup.4; and R.sup.4 is selected from hydroxy, halogen, OAr.sup.2, Hyd.sup.1, OHyd.sup.1, NH(Hyd.sup.1) and N(Hyd.sup.1).sub.2, wherein Hyd.sup.1 is a C.sub.1-4 hydrocarbon group optionally substituted with one or more fluorine atoms; and Ar.sup.2 is a 5- or 6-membered carbocyclic or heterocyclic aromatic group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0113] In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

[0114] FIG. 1 (A) is a graph of the growth of D. discoideum on exposure to MyA at difference concentrations; (B) is a graph of the normalised growth rate of D. discoideum on exposure to MyA; (C) illustrates a schematic representation of wild type developmental phenotypes under control conditions, showing different stages of development; (D) illustrates developmental phenotypes, under control conditions, showing fruiting body morphology at 18 and 24 hours, from top down view and individual fruiting bodies in the absence of MyA (control) and in the presence of 100 M of MyA.

[0115] FIG. 2 illustrates the development of wild type D. discoideum after 36 hours in the absence of MyA (control) and in the presence of 100 M of MyA.

[0116] FIG. 3 (A) illustrates the common size and domain structure of the D. discoideum (ACO) and Petunia hybrida (ACO) proteins; (B) illustrates the conserved catalytic residues necessary for Fe (II) binding, consistent with orthologous function; (C) is a graph of the growth sensitivity of wild type and ACO-mutant cells in the presence of MyA; (D) illustrates wild type and ACO-mutant D. discoideum cell development at 20 h in the presence of MyA and/or CEPA; (E) illustrates wild type and ACO-mutant D. discoideum cell development at 20 h in the presence of AIB (10 M) or POA (50 M); (F) is a schematic of the developmental programme of D. discoideum on expression of specific developmental genes including csA (Contact site A), cAR1 (cAMP receptor 1), pspA (prespore-specific protein A), and ecmA (extracellular matrix protein A) and the graphs to show the absolute copy number of the genes in the absence of MyA, on exposure to MyA (100 M) and on exposure to MyA and CEPA.

[0117] FIG. 4 is a graph which illustrates the wild type and ACO-mutant D. discoideum cell ethylene production over time in the absence and the presence of MyA.

[0118] FIG. 5 illustrates modelled structures of D. discoideum ACO protein wherein A shows the MyA bonded to the ACO protein, B shows (commonality of D. discoideum and Petunia (plant ACO protein in purple)) structure, C shows binding of MyA adjacent to the catalytic site, and D close up of binding.

[0119] FIG. 6A illustrates A. thaliana crop maturation, root growth and hypocotyl growth after 6 days following exposure to MyA and also exposure to ethylene response inhibitors AIB and POA at higher concentrations.

[0120] FIG. 6B illustrates and also exposure to A. thaliana crop maturation, root growth and hypocotyl growth after 6 days following exposure to 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione (ACOi-84-16-4 or 4B) and 3-[(2E)-3-(4-fluorophenyl)rop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (ACOi-74-12-16 or 16B).

[0121] FIG. 7 a graph depicting the flower size of carnations after 9 days and treatment with no compound (control), POA, AIB, 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione (4B) or 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (16B).

[0122] FIG. 8 End of test photos of barley plants following watering (control watered) or drought conditions in the absence of a treatment compound (control drought) or in the presence of AIB, POA or 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (16B) or 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione (4B).

[0123] FIG. 9 End of test photos of wheat plants following watering (control watered) or drought conditions in the absence of a treatment compound (control drought) or in the presence of AIB or 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (16B).

[0124] FIG. 10 a graph displaying the plant height of the plants of FIG. 9 wherein ACOi is 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one.

[0125] FIG. 11 End of test photos of rye plants following watering (control watered) or drought conditions in the absence of a treatment compound (control drought) or in the presence of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (16B) and following 19 days recovery post drought.

[0126] FIG. 12 End of test photos of rye plants following watering (control watered) or drought conditions in the absence of a treatment compound (control drought) or in the presence of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (16B) and following 23 days recovery post drought.

[0127] A first embodiment of a use of a compound of formula (1) for inhibiting a post-germination ethylene production response in a plant or plant part is provided wherein the compound of formula (1) is 2,2,4-trimethyl-6-(3-phenylpropanoyl) cyclohexane-1,3,5-trione or Myrigalone A (MyA) which is of the formula:

##STR00026##

Inhibition of D. discoideum Growth

[0128] The unicellular growth rate and multicellular of D. discoideum cells on exposure to MyA was tested and the results are illustrated in FIG. 1 and FIG. 2.

[0129] D. discoideum cells were divided by binary fission in nutrient-rich media, initially with a lag phase (0-120 h), and then an exponential phase. The D. discoideum cells were then exposed to MyA at concentrations of 0 m, 1 m, 10 m, 15 m, 25 m, 50 m and 100 m. FIG. 1A illustrates that the MyA treatment caused a concentration-dependant inhibition of unicellular growth with a significant reduction at 10 M (P<0.05) and a block in growth at 100 M. FIG. 1B illustrates secondary plot analysis whilst provided an IC50 of 7.6 M which shows that MyA is highly potent. This data demonstrates that D. discoideum growth is sensitive to the presence of low concentrations of MyA. This is advantageous because it shows that the effect of MyA on the model is potent.

[0130] The effect of MyA on multicellular development of D. discoideum was measured. The MyA was tested at a concentration of 100 M. The control D. discoideum cells aggregated and differentiated over a 24-hour period to form a multicellular fruiting body consisting of a spore head, a stalk and a basal disk (FIG. 1C and 1D). 100 M of MyA resulted in blocked cell growth and later stages of development were delayed.

[0131] FIG. 2 shows that after extended incubation (36 hours), MyA-treated cells developed into fully mature fruiting bodies, showing morphology similar to untreated cells. These results therefore suggest that high dose MyA is not lethal, but instead triggers a specific delay in development, likely caused by inhibition of molecular targets required for development rather than a generalized toxic effect. The data therefore shows a likely biochemical mechanism in the model to slow development, rather than a toxic effect to kill cells.

Impact of MyA on Ethylene Production Response

[0132] The ACO enzyme functions as the rate limiting step in ethylene synthesis in plants, which is required for the release of seed dormancy and plant growth. The D. discoideum ACO protein was identified in a genetic resistance screen as a potential target for MyA, and bioinformatics analysis determined that the protein is a likely ortholog of the plant ACO protein. FIG. 3A and 3B illustrate that both proteins are of similar size (319 and 368 aa) and contain a common domain structure, with a conserved 2-oxoglutalate (2OG) and Fe (II) dependent oxygenase superfamily domains, necessary for the oxidation of organic substrates such as ACC, and conserved Fe (II) binding residues required for enzyme function. These characteristics provide evidence that the D. discoideum ACO protein is a homologue of the plant ACO protein, and thus functions in ethylene synthesis in D. discoideum.

[0133] The cell and developmental role of the D. discoideum ACO protein were investigated. FIG. 3C illustrates the resistance to the growth and developmental inhibitory effect of MyA on wild type D. discoideum cells (WT) and ACO ablated D. discoideum cells (ACO-). The results show around a four-fold reduction in potency in the ACO-mutant (IC50 of 29.9 M) on exposure to MyA. This result is consistent with a loss of ACO protein as a primary target for MyA, where loss of the enzyme reduces sensitivity to growth inhibition.

[0134] The effect of ACO loss on multicellular development was assessed. ACO-D. discoideum mutants were engineered and compared to standard wild type D. discoideum cells. FIG. 3D shows the multicellular development of the ACO-D. discoideum mutant (ACO-), the standard wild type D. discoideum cell (WT) and the impact of the exposure of 100 M of MyA. ACO-mutant showed a block in multicellular development at the mound stage, after around 12 hours of development, suggesting a delay of around 6 hours. Interestingly, this developmental defect was identical to that shown in the treatment of wild type cells with MyA (100 M). To investigate this further, the development assays were repeated in the presence of exogenous ethylene, provided through the breakdown of 2-chloroethylphosphonic acid (CEPA) the results (ACO-+CEPA and WT+MyA+CEPA) showed that the developmental delay caused by both MyA treatment and ACO loss were partially rescued by the addition of ethylene (FIG. 3D).

[0135] A similar development delay is also evident following treatment with two structurally distinct ACO inhibitors, AIB (2-amino oxyisobutyric acid) and POA (pyrazinecarboxylic acid) (FIG. 3E), and an inhibitor of the plant ethylene receptors 1-methylcyclopropene.

[0136] Without being bound by theory, it is understood that this data shows that the D. discoideum protein is a functional ACO enzyme, that ethylene production is necessary for timely late development, and that the bioactivity of MyA in D. discoideum development is through the ACO inhibition to block ethylene production.

[0137] To provide quantitative analysis of the developmental effects of MyA, wild type cells were induced to develop on nitrocellulose filters for time periods between 0 and 20 hours, using cells under solvent only conditions, in the presence of MyA (100 M), or in the presence of MyA and CEPA. The results are shown in FIG. 3F. Developmental gene expression associated with early aggregation (csA), cAR1 mid development (PspA), and in late development (ecmA) were assessed using qPCR. Analysis of csA expression showed peak expression levels at around 4 hours in untreated wild type cells that was delayed with MyA treatment rescued by exogenous ethylene. Similar delays in peak gene expression were also seen for carA (peak 4 h), and pspA (peak 12 h), and ecmA (peak 20 h), following MyA treatment, and these delays were rescued by addition of exogenous ethylene. These experiments confirm a MyA-dependent delay in D. discoideum development that is rescued by exogenous ethylene application, consistent with a role for MyA in inhibiting ACO activity and slowing crop maturation.

[0138] Wild type cells in the presence or absence of MyA (500 M), or ACO-cells were maintained in sealed small flasks with limited head space over 36 h, and headspace gas was taken at 6-hour intervals and analysed by GCMS. The results are shown in FIG. 4. FIG. 4 shows that wild type cells showed increasing ethylene production after 6 hours, and levels increased more slowly up to 36 hours. MyA treatment significantly reduced ethylene production, causing a 39.5% decrease in ethylene production at 12 hours (P=0.031) which was maintained up to 36 hours (37.9% decrease, P=0.0025). Similarly, ACO-cells also showed a significantly reduced level of ethylene after 12 hours of 62.4% (p=0.0045) that remained low throughout the analysis. These findings show that D. discoideum cells produce ethylene during starvation, and ethylene production is reduced in the presence of MyA, consistent with the inhibition of ACO in the ethylene synthesis pathway.

Direct Binding of MyA to ACO To identify a potential direct mechanism of MyA dependent ACO inhibition, a range of molecular modelling techniques were used. The tertiary structure of D. discoideum ACO protein was predicted using phyre2 based upon the closest available crystal structure (Petunia ACO: PDB:5LUN) as a template (FIG. 5A). The D. discoideum ACO protein and P. hybridia ACO protein are predicted to share a common structure, featuring a double-stranded-helix jellyroll fold surrounded by alpha-helices, with superimposed structures provide a root-mean-square deviation of 1.016 angstroms over 282 aligned CA atoms (FIG. 5B). This analysis identified high 3D structural conservation between the key Fe.sup.2+ dioxygenase domain required for catalytic activity between both proteins. It is understood that despite a lack of crystal structure binding analysis, the ACO substrate, 1-aminocyclopropane-1-carboxylic acid (ACC) is likely to bind directly to Fe.sup.2+ via its carboxylate and amino groups during catalysis, and thus substrate access to the facial triad is essential for ACO activity. It was shown, via predictive docking assays, that MyA binds within the binding pocket of the facial triad, and via a hydrogen bonding to Lys 100 (FIG. 5C and 5D).

Preventing or Slowing Crop Maturation

[0139] A first embodiment of the use of a compound of formula (1) for modifying a physiological process of a plant or plant part according to the second aspect of the invention was provided wherein the compound of formula (1) is 2,2,4-trimethyl-6-(3-phenylpropanoyl)cyclohexane-1,3,5-trione or Myrigalone A (MyA) and the physiological process was preventing or slowing crop maturation.

[0140] A second embodiment of the use of a compound of formula (1) for modifying a physiological process of a plant or plant part according to the second aspect of the invention was provided wherein the compound of formula (1) is 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione and the physiological process was preventing or slowing crop maturation.

[0141] The formula of 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione is:

##STR00027##

[0142] Within this document, 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione may also be referred to as ACOi-84-16-4 or 4B.

[0143] A third embodiment of the use of a compound of formula (1) for modifying a physiological process of a plant or plant part according to the second aspect of the invention was provided wherein the compound of formula (1) is 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one and the physiological process was preventing or slowing crop maturation.

[0144] The formula of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one is:

##STR00028##

[0145] Within this document, 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one may also be referred to as ACOi-74-12-16 or 16B.

[0146] A model illustrating the binding of MyA to the active site of plant ACO enzymes is illustrated in FIG. 5. 16 compounds were selected and tested for bioassay efficacy analysis in this model (100 M) with the data presented in table 2.

TABLE-US-00002 TABLE 2 Name No. Activity* IUPAC Name ACOi-57-1 1 1,3-dimethyl-5-[2-oxo-2-(piperidin-1-yl)ethyl]-1,3,5- triazinane-2,4,6-trione ACOi-28-2 2 N-[(4-chlorophenyl)methyl]-2,6-dimethoxybenzamide ACOi-46-3 3 (2E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-phenylprop- 2-en-1-one ACOi-21-4 4 1,3-dimethyl-5-(3-oxobutanoyl)-1,3-diazinane-2,4,6-trione ACOi-70-5 5 2-(5,5-dimethyl-2,4-dioxo-1,3-oxazolidin-3-yl)-N- [(2-methoxyphenyl)methyl]acetamide ACOi-68-6 6 2-[(2E)-3-(4-iodophenyl)prop-2-enoyl]-2,3-dihydro-1H- indene-1,3-dione ACOi-77-7 7 * 5-benzoyl-4,5,6,7-tetrahydro-1-benzofuran-4-one ACOi-67-8 8 * methyl 5-acetyl-2,2-dimethyl-4,6-dioxocyclohexane-1-carboxylate ACOi-62-9 9 6-amino-1,3-dimethyl-5-{2-[(1-methyl-1H-1,2,3,4-tetrazol- 5-yl)-sulfanyl]acetyl}-1,2,3,4-tetrahydropyrimidine-2,4-dione ACOi-69-10 10 * 6-amino-5-{2-[(3,4-dichlorophenyl)amino]acetyl}-1,3- dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione ACOi-54-11 11 ** 2,6-dimethoxyphenyl 3-(5-methylfuran-2-yl)prop-2-enoate ACOi-74-12 12 *** 3-[3-(3-bromo-4-fluorophenyl)prop-2-enoyl]-6- methyl-3,4-dihydro-2H-pyran-2,4-dione ACOi-72-13 13 ND 3-[3-(3-chlorophenyl)prop-2-enoyl]-6-methyl-3,4- dihydro-2H-pyran-2,4-dione ACOi-25-14 14 (2E)-3-(4-chlorophenyl)-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one ACOi-39-15 15 ND (2E)-3-(2-chlorophenyl)-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one ACOi-84-16 16 *** 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione

[0147] Table 2 shows the effect of novel compounds on D. discoideum ACO-inhibition dependent development block at mound formation. In this assay, D. discoideum WT cells were starved on nitrocellulose filters at 100 M of indicated compounds, incubated for 20 hours (22 C.), and developmental block at the mound stage was assessed, where-indicates no effect, * indicates some effect, ** indicates strong effect, and *** indicates potent effect similar to MyA, and ND not determined.

[0148] 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (ACOi-84-16-4) and 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (ACOi-74-12-16) demonstrated potent efficacy in this model.

[0149] Since seed germination and root/hypocotyl extension of A. thaliana and other plant species are promoted by ACC and ethylene, a role for MyA in these processes was also investigated.

[0150] In these experiments, A. thaliana seeds were germinated and grown in 24 h light conditions for 6 days and root and hypocotyl length were recorded (FIG. 6A and 6B). FIG. 6A shows that both AIB and POA treatment provided a dose-dependent reduction in root and hypocotyl growth. MyA treatment produced a comparative reduction in both root and hypocotyl growth, but with greater potency than the established ACO inhibitors. In particular the lowest concentration of MyA which was shown to have an effect on growth (0.025 mM), was 20 times lower than the concentration of AIB, and 4 times lower than the concentration of POA.

[0151] FIG. 6B shows that 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (ACOi-84-16-4) and 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (ACOi-74-12-16) result in a dose dependent effect on root/hypocotyl growth from as low as 5 m compared to the control which was only exposed to DMSO and compared to AIB and POA when used at higher concentrations.

[0152] The IC50 values for the root or hypocotyl extension after 6 days for 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (ACOi-84-16-4), 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (ACOi-74-12-16) and MyA, compared to known compounds AIB and POA are presented in table 3. Images of the plant growth and the root hairs are shown in FIG. 6B for 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (ACOi-84-16-4) and 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (ACOi-74-12-16) and FIG. 6A for MyA and comparably for AIB and POA.

TABLE-US-00003 TABLE 3 Root extension or Compound hypocotyl extension IC50/M Control - AIB Root 1617 Control - AIB Hypocotyl 4293 Control - POA Root 213 Control - POA Hypocotyl 184 MyA Root 53.3 MyA Hypocotyl 59.4 ACOi-84-16-4 Root 6.9 ACOi-84-16-4 Hypocotyl 8.2 ACOi-74-12-16 Root 1.02 ACOi-74-12-16 Hypocotyl 0.79

[0153] The data in table 3 shows that the novel compounds of formula (1) (MyA, ACOi-84-16-4 and ACOi-74-12-16) result in a potent inhibition of hypocotyl or root extension wherein the potency of 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (ACOi-84-16-4) and 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one is over 5400-fold or 1580-fold compared to AIB respectively. All three compounds of formula (1) result in a prevention or slowing of crop maturation. In particular 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (ACOi-84-16-4) and 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (ACOi-74-12-16) are highly potent and therefore are advantageous because they can be used at significantly lower concentrations.

Preventing or Slowing Flower Senescence

[0154] A fourth embodiment of the use of a compound of formula (1) for modifying at least one physiological process of a plant or plant part according to the second aspect of the invention was provided wherein the compound of formula (1) is 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione and the physiological process was preventing or slowing flower senescence.

[0155] A fifth embodiment of the use of a compound of formula (1) for modifying at least one physiological process of a plant or plant part according to the second aspect of the invention was provided wherein the compound of formula (1) is 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one and the physiological process was preventing or slowing flower senescence.

[0156] The slowing of flower senescence in the presence of 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione or 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one was tested and compared to a control untreated sample and known ethylene production inhibitors POA and AIB.

[0157] Carnations were cut to approximately 5 cm long stems and placed in a solution wherein the solution comprised 5 mL water and either no additional components (control), 0.01 mM or 0.02 mM of 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (labelled as 4B) or 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (labelled as 16B) or 0.5 mM or 1 mM of POA or 10 mM or 5mM of AIB were added. The flower size coverage was measured and the petal shrinkage, or reduction in flower size, is used as an indicator of flower senescence. The results are shown in FIG. 7.

[0158] 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione and 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one showed significant effect compared to the control sample showing that both compounds effectively slow flower senescence at low concentrations.

[0159] 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione and 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one demonstrated comparable performance to significantly higher concentrations of known ethylene inhibitors POA and AIB thereby showing that they are more potent. This is advantageous because it means that the compounds can be used at much lower concentrations which may be cheaper for the consumer whilst successfully extending the lifetime of the cut flower compared to no treatment.

Reducing a Drought Stress Response in a Plant

[0160] A sixth embodiment of the use of a compound of formula (1) for modifying at least one physiological process of a plant or plant part according to the second aspect of the invention was provided wherein the compound of formula (1) is 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione and the physiological process was reducing an abiotic stress response in a plant, to enhance stress recovery, wherein the abiotic stress response is a response to heat and drought stress.

[0161] A seventh embodiment of the use of a compound of formula (1) for modifying at least one physiological process of a plant or plant part according to the second aspect of the invention was provided wherein the compound of formula (1) is 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one and the physiological process was reducing an abiotic stress response in a plant, to enhance stress recovery, wherein the abiotic stress response is a response to heat and drought stress.

[0162] The reduction of drought stress in the presence of 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione or 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one was tested and compared to a control untreated sample.

[0163] The plants tested were barley, wheat and rye plants.

[0164] The seeds of each plant were washed with 5% sodium hypochlorite for 15 minutes (with shaking at 120 rpm) and then washed three times in sterile distilled water. The seeds were placed on two layers of waterlogged 3 MM for 72 hours under lights at RT until hypocotyl emerged. 7-10 germinated seeds were incubated in sterile Magenta boxes containing 100 mL perlite per box, moistened with 60 ml sterile distilled water and autoclaved. The seeds were incubated in a growth cabinet with a 12 hour light/dark cycle at 22 C. until the first seedling reached approximately 5 cm height. This took approximately 4-5 days. The water was then replaced with 0.5Hoagland solution with the test compounds (or DMSO for control samples) for 24 hour (Hoagland's No2 Basal salt mixture, Sigma, H2395, use 1.6 g/L in sterile water and autoclaved), and returned to growth cabinet. The media was then removed, the perlite was rinsed twice with sterile distilled water and the solution was replace with 35 ml 0.5 Hoaglands solution and returned to the growth cabinet for 24 hours.

[0165] The no drought sample was then grown in 0.5Hoaglands solution and the solution was topped up daily.

[0166] The drought samples were exposed to drought conditions for 9 days by removing the media and returned to the growth cabinet. At recovery, 0.5Hoaglands solution was added back to the drought sample Magenta boxes, up to the top of the perlite and this level was maintained during recovery. The plant recovery over time (up to 19 days post rescue) was recorded and the photographs from the end of the experiment are illustrated in FIG. 8, FIG. 9 and FIG. 11.

[0167] 10 M of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one (labelled as 16B), 10 M of 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione (labelled as 4B) and 10,000 M 2,000 M of AIB and 500 M of POA were tested on barley seedlings. The results after 5 days of growth, 9 days of drought and 12 days of normal watered conditions are shown in FIG. 8. The barley treated with 500 M of POA did not show positive result as most of the seedlings died during the drought period and did not recover. The barley treated with 2,000 M of AIB did slightly better and the barley did better still with 10,000 M AIB however all samples treated with POA or AIB did not recover to the same seedling height as the watered control sample. The barley exposed to 10 M of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one showed comparable seedling height to the control sample which had been watered throughout the duration and therefore shows that 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one provides a significant benefit over known AIB or no compound in high stress, drought conditions. 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one also shows a high potency as it is shown to be effected at 10 M. The barley exposed to 10 M of 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione showed comparable growth to the same treated with 10,000 M AIB showing that 5,5-dimethyl-2-(2-phenylacetyl)cyclohexane-1,3-dione is significantly more potent than known POA or known AIB. This data shows that barley seedlings treated with compounds of formula (1) recover better after periods of drought, or high stress, which is advantageous for growing crops in areas with variable weather conditions and improving the crop yield in those areas.

[0168] The drought testing was repeated with wheat seedlings and the results are shown in FIGS. 9 and 10 wherein the data was measured following 5 days of growth, 9 days of drought and 19 days of recovery. The ACOi treated sample was treated with 10 M of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one after the initial 5 days of growth. The wheat seedlings showed improved recovery upon treatment compared to the control drought sample which was not treated. This is shown by the increased height and mass of the wheat seedlings as shown in FIG. 10 wherein the control sample was not exposed to the drought, the drought sample showed the shortest seedling height and the lowest seedling mass, and the drought +ACOi samples showed improved seedling growth. This improved seedling growth upon treatment with 10 M of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one, which is a low concentration of a compound of formula (1), reflects better drought stress recovery, and is advantageous because it would lead to improved crop yield following treatment by reducing the stress response of the plant following periods of drought.

[0169] The drought testing was repeated with rye seedlings and the results are shown in FIG. 11 wherein the data was measured following 5 days of growth, 9 days of drought and 19 days of recovery. The ACOi treated sample was treated with 10 M of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one after the initial 5 days of growth. The rye seedlings showed improved recovery upon treatment compared to the control drought sample which was not treated. The recovery period was extended to 23 days. The photos of the control sample and the treated sample after 23 days recover is shown in FIG. 12. The ACOi treated sample continues to recover and results in a number of green leaves as the plant grows, and the drought sample showed the shortest seedling height. This improved seedling growth upon treatment with 10 M of 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one, which is a low concentration of a compound of formula (1), reflects better drought stress recovery, and is advantageous because it would lead to improved crop yield following treatment by reducing the stress response of the plant following periods of drought.

[0170] The above testing demonstrates that the compounds 3-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-4-hydroxy-6-methyl-2H-pyran-2-one and 5,5-dimethyl-2-(2-phenylacetyl) cyclohexane-1,3-dione are suitable for reducing a post-germination abiotic stress response recovery in a plant, for example a response to heat and drought stress after germination, and can be used with a variety of plants, in particular a variety of crops. This is advantageous as changing climates are resulting in more countries experiencing drought periods and therefore crop shortages and therefore the compounds of formula (1) are advantageous to address this and improve consistency of crop supply in periods of unpredictable weather which may otherwise cause stress to the plant.

[0171] The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention as defined in the appended claims.