METHOD OF RETARDING AN ETHYLENE RESPONSE

Abstract

A compound of Formula I, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently hydrogen, halogen, an unsubstituted alkyl, alkenyl, alkynyl, cydoalkyi, cydoalkylalkyi, aryl, phenyl, or naphthyl group, and a substituted alkyl, alkenyl, alkynyl, cydoalkyi, cydoalkylalkyi, aryl, phenyl, or naphthyl group having as a substituent a halogen, alkoxy, substituted phenoxy, unsubstituted phenoxy group or a heteroatom such as oxygen, sulfur, nitrogen, phosphorus and boron.

##STR00001##

Claims

1. A compound of Formula I: ##STR00027## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently hydrogen, halogen, an unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, phenyl, or naphthyl group, and a substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, phenyl, or naphthyl group having as a substituent a halogen, alkoxy, substituted phenoxy, unsubstituted phenoxy group or a heteroatom such as oxygen, sulfur, nitrogen, phosphorus and boron, or a salt thereof.

2. A composition comprising a compound of Formula I: ##STR00028## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently hydrogen, halogen, an unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, phenyl, or naphthyl group, and a substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, phenyl, or naphthyl group having as a substituent a halogen, alkoxy, substituted phenoxy, unsubstituted phenoxy group or a heteroatom such as oxygen, sulfur, nitrogen, phosphorus and boron, or a salt thereof.

3. The compound or salt in accordance with claim 1, wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently alcohol.

4. The compound or salt in accordance with claim 1, wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently polyols.

5. The compound or salt in accordance with claim 4, wherein each polyol is independently selected from the group consisting of a sugar alcohol and a glycol such as ethylene glycol.

6. (canceled)

7. The compound or salt in accordance with claim 3, wherein the alcohol is a polymerised alcohol such as polyethylene glycol.

8. The compound or salt in accordance with claim 1, wherein R.sub.1 and R.sub.2 are independently selected from an alkene, a ketone, a halogen and hydrogen.

9. (canceled)

10. (canceled)

11. The compound or salt in accordance with claim 1, wherein R.sub.3 and R.sub.6 are hydrogen.

12. The compound or salt in accordance with claim 11, wherein R.sub.4 and R.sub.5 are substituted.

13. The compound or salt in accordance with claim 1, wherein the compound comprises at least one substituted or unsubstituted aromatic and/or nonaromatic ring formed between positions R.sub.1 and R.sub.2.

14. The compound or salt in accordance with claim 13, wherein the ring is a carbocyclic or heterocyclic ring.

15. The compound or salt in accordance with claim 1, wherein the compound comprises at least one substituted or unsubstituted aromatic and/or nonaromatic ring formed between positions R.sub.3 and R.sub.4, R.sub.4 and R.sub.5 and/or R.sub.5 and R.sub.6.

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. The compound or salt in accordance with claim 1, wherein there is provided a compound of Formula II: ##STR00029## wherein X is hydrogen, fluorine and/or chlorine.

21. The compound or salt in accordance with claim 1, wherein there is provided a compound of Formula III: ##STR00030## wherein X is hydrogen, fluorine and/or chlorine.

22. The compound or salt in accordance with claim 1, wherein there is provided a compound of Formula IV: ##STR00031## wherein X is hydrogen, fluorine and/or chlorine.

23. The compound or salt in accordance with claim 1, wherein there is provided a compound of Formula V: ##STR00032## wherein X is hydrogen, fluorine and/or chlorine.

24. The compound or salt in accordance with claim 1, wherein there is provided a compound of Formula VI: ##STR00033## wherein X is hydrogen, fluorine and/or chlorine.

25. The compound or salt in accordance with claim 1, wherein the compound of the invention is water soluble.

26. (canceled)

27. The compound or salt in accordance with claim 261, which is provided in the form of a salt selected from the group comprising phosphate, acetate, formate, carbonate, hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, trifluoroacetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate and laurylsulfonate salts.

28. A method for one or more of: retarding an ethylene response in a plant; retarding ripening of fruit; retarding ripening of vegetables; retarding senescence of a plant or plant part; retarding abscission of a plant or plant part; extending the life of a cut plant; and extending the storage life of fresh horticultural produce; the method comprising the step of contacting the plant with an effective ethylene response retarding amount of a compound of Formula I: ##STR00034## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently hydrogen, halogen, an unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, phenyl, or naphthyl group, and a substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, phenyl, or naphthyl group having as a substituent a halogen, alkoxy, substituted phenoxy, unsubstituted phenoxy group or a heteroatom such as oxygen, sulfur, nitrogen, phosphorus and boron, or a salt thereof.

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:

[0072] FIG. 1 is the chemical structures of benzocyclopropene and naphtho[b]cyclopropane;

[0073] FIG. 2 depicts the concentration of ethylene and CO.sub.2 on the day of climacteric peak;

[0074] FIG. 3 depicts the concentration of ethylene (A) and CO.sub.2 (B) on the day of climacteric peak in Fortune plum fruit treated with different concentration of naphtho[b]cyclopropane (NC); and

[0075] FIG. 4 depicts the percent of flower/bud abscission after two days of treatment with antagonist.

DESCRIPTION OF EMBODIMENTS

[0076] Throughout this specification, unless the context requires otherwise, the word comprise or variations such as comprises or comprising, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0077] Benzocyclopropene was prepared as shown in Scheme 1.

##STR00021##

[0078] A solution of 1,3-cyclohexadiene (80.5 g, 0.5 mol), cetyltrimethylammonium bromide (2.00 g) in 50% aqueous sodium hydroxide (200 g) was cooled (0-25 C.) and stirred under nitrogen. Ethanol (5 mL) and chloroform (80 mL) were added successively in one portion. The solution was stirred for 1 hour at 0 C. and allowed to warm to room temperature for a further 1 hour. Water was added to the reaction mixture and extracted. The organic phase was washed with water (2100 mL), dried (CaCl.sub.2) and concentrated under reduced pressure to provide an oil. The oil was purified by flash chromatography to afford the 7,7-dichlorobicyclo[4.1.0]-hept-2-ene as a colourless oil (42 g, 26%).

[0079] Potassium t-butoxide was added in portions to a solution of 7,7-dichlorobicyclo[4.1.0]-hept-2-ene (1.00 g, 6.1 mmol) in anhydrous DMSO (30 mL) under nitrogen. The dark brown mixture was stirred for 30 minutes. A vacuum was applied to the reaction mixture and the volatiles collected in an 86 C. trap. The distillate was diluted in petroleum spirits, and washed with brine (460 mL) and water (230 mL), dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure at 0 C. to afford benzocyclopropene as an oil (80 mg, 14%). 1H NMR (CDCl.sub.3) 3.17 (2H, s), 7.21 (s, 4H).

[0080] Naphtho[b]cyclopropene was prepared as shown in Scheme 2..sup.1 .sup.1W. E. Billups and C. Y. Chow J. Am. Chem. Soc. 1973, 95, 4099.

##STR00022##

[0081] Small pieces of sodium metal (15 g) were added to solution of naphthalene (30 g) in anhydrous THF (100 mL). The solution turned to a deep green colour during this time. A solution of tert-butanol (24 mL) and THF (24 mL) in water was added dropwise over 20 minutes. The resulting solution was stirred for a further 3 hours. The excess sodium metal was removed by filtration and the filtrate washed with water (250 mL), dried and concentrated under reduced pressure to give pure 1,4-dihydronaphthalene as a colourless solid (17.5 g, 57%).

[0082] A solution of 1,4-dihydronaphthalene (17.0 g, 0.131 mol), cetyltrimethylammonium bromide (0.567 g, 1.6 mmol) in 50% aqueous sodium hydroxide (50 g) was cooled (0-25 C.) and stirred under nitrogen. Ethanol (1.6 mL) followed by chloroform (23 mL) was added. The solution was stirred for 1 hour at 0 C. and allowed to warm to room temperature for a further 1 hour. Water (100 mL) was added to the reaction mixture and extracted. The organic phase was washed with water (250 mL), dried (CaCl.sub.2) and concentrated under reduced pressure to provide an oil. The oil was purified by flash chromatography to afford the adduct (7.56 g, 27%).

[0083] Potassium tert-butoxide (11.0 g, 98.2 mmol) was added in portions to a solution of 1,1-dichloro-1a,2,7,7a-tetrahydro1H-cyclopropa[b]naphthalene (4.78 g, 29.3 mmol) in anhydrous THF (60 mL) under nitrogen at room temperature and stirred for a further 18 hour. The resulting mixture was diluted in petroleum spirits (20 mL), washed with brine (420 mL) and water (210 mL), dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The residue was purified by flash chromatography to afford naphtho[b]cyclopropene as a colourless solid (1.11 g, 42%).

##STR00023##

[0084] A solution of dimethyl acetylenedicarboxylate (1.00 mL, 0.008 mol), butadiene sulfone (5.40 g, 0.045 mol) and xylene (8 mL) was heated under reflux under nitrogen atmosphere for 2.5 hr. After 2.5 hr, the yellow reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (10% ethyl acetate/petroleum spirits) to give dimethyl-1,4-cyclohexadiene-1,2-dicarboxylate as a yellow liquid (0.75 g, 47%). .sup.1H NMR: 5.93 (m, 1H), 3.78 (s, 3H), 3.00 (d, J=1.2 Hz, 4H).

##STR00024##

[0085] 1,4-Cyclohexadiene-1,2-dicarboxylate (0.20 g, 0.1 mmol) was added to a solution of sodium trichloroacetate (3 g, 16 mmol) and tetrabutylammonium iodide (0.005 g, 0.014 mmol) in chloroform (20 mL) and the reaction mixture was heated under reflux under nitrogen overnight. The reaction was allowed to cool to room temperature and diluted in chloroform (20 mL), washed with water (525 mL), dried under anhydrous calcium chloride and the solvent was removed under reduced pressure. The residue was purified by column chromatography (10% ethyl acetate/petroleum spirits) to afford dichlorocarbene adduct as a colourless solid (0.172 g, 87%). .sup.1H NMR: 3.76 (s, 6H); 2.87-2.75 (m, 2H); 2.55 (d, J=7.6 Hz, 2H); 1.98-1.94 (m, 2H). .sup.13C NMR : 168.11 (C); 131.68 (C); 64.06 (C); 52.37 (CH); 23.84 (CH.sub.2); 21.56 (CH.sub.3). IR: 3058, 2873, 1736, 1628, 1600, 1469, 1390, 1352, 1258, 1216, 1104, 970, 838, 747. Microanalysis: Calculated: C=47.33, H=4.33%, Found: C=47.32, H=4.11%.

##STR00025##

[0086] Diisobutylaluminium hydride (1.364 g, 0.01 mol) and toluene solution was added dropwise to a solution of the diester (0.10 g, 0.0004 mol) in dry THF (10 mL) at 78 C. The reaction was stirred for 30 minutes at 78 C., and then it was allowed to warm to room temperature overnight. 1 M HCl solution (10 mL) was added to the reaction mixture and extracted with ethyl acetate (320 mL). The combined organic extracts were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (40% ethyl acetate in petroleum spirit) to give 7,7-Dichloro-bicyclo[4,1,0]hept-3-ene-1,2-dimethanol as colourless solid (0.038 g, 46%). .sup.1H NMR: 4.23 (m, 4H); 2.74-2.58 (m, 2H); 2.34 (d, J=10.9 Hz, 2H); 1.89 (d, J=10.9 Hz, 2H). .sup.13C NMR : 13104 (C), 65.31 (C), 62.55 (CH), 24.83 (CH), 23.53 (CH.sub.2). IR: 3291, 2878, 2830, 1679, 1422, 1261, 1086, 995, 784. Microanalysis: Calculated: C=48.45, H=5.42%; Found: C=48.32, H=5.33%.

##STR00026##

[0087] 7,7-Dichloro-bicyclo[4,1,0]hept-3-ene-1,2-dimethanol (0.0485 g, 0.2 mmol) was added to a solution of potassium tert-butoxide (0.154 g, 1.4 mmol) in dry THF (8 mL) the reaction mixture was stirred for 36 hrs. Petroleum spirits (10 mL) was added to the brown mixture and washed with brine water (310 mL) and water (110 mL) and then the organic phase was dried under anhydrous magnesium sulfate and the solvent was removed under reduced pressure to give 1H-cyclopropabenzene-3,4-dimethanol. .sup.1H NMR: 7.27 (s, 2H); 4.79 (s, 4H); 3.28 (s, 4H).

[0088] The chemical structures of benzocyclopropene (BC) and naphtho[b]cyclopropene (NC) are provided at FIG. 1. Advantageously, benzocyclopropene is a liquid at room temperature, making it easier to handle than 1-methylcyclopropene. Advantageously, naphthocyclopropene is a solid at room temperature, making it easier to handle than 1-methylcyclopropene.

[0089] Advantageously, benzocyclopropene and naphtho[b]cyclopropane are stable at room temperature for several months.

[0090] Benzocyclopropene and naphtho[b]cyclopropene are only partially soluble in water. To prepare substantially aqueous solutions of these compounds, the lead compounds where dissolved in ethanol and then diluted with water.

[0091] Various experiments were conducted using Tegan Blue and Fortune Japanese plums, Arctic Pride nectarine, Fuji and Pink Lady apples, WX7, WX17, WX39, WX 56, WX 58, WX73 and WX107, WXFU, hybrid, Revelation, Purple Pride and Jenny wax flowers (Chamelaucium Desf.).

[0092] Mature Tegan Blue and Fortune Japanese plum and Arctic Pride nectarine fruits of uniform size and maturity, free from visual blemishes and diseases were harvested in early morning from a commercial orchard in Western Australia. Following the harvest, the fruit were brought to the Horticulture Research Laboratory, Curtin University, using a temperature controlled vehicle at 20-25 C. The fruit were treated by fumigation with 0 to 100 nLL.sup.1 of BC and 1000 nLL.sup.1 of 1-MCP (Tegan Blue plum) for 18 hr at ambient conditions (201 C. and 655% RH) by using hermetically sealed plastic containers of 60 L volume. Whatman filter paper (number 2) soaked with specific concentrations of BC and 1-MCP were kept along with the fruit and 30 g of soda lime inside each container. A small battery operated fan was used to ensure equal distribution of the vapours from the chemicals. Half of the treated fruit was exposed to ethylene (10 L L.sup.1) for 24 hr following BC and 1-MCP treatment.

[0093] In a second set of experiments, Fortune plum fruit were treated with 0 to 1000 nIL.sup.1 of NC as described above. The treated fruit were kept in the ambient conditions for ripening and endogenous level of ethylene and CO.sub.2 was determined. The experiments were laid out by following completely randomized design (CRD) with four replications for each treatment and 10 fruit in each replication.

[0094] To evaluate the effects of BC and NC on flower abscission, flowering stems of Wax flower (Chamelaucium Desf.) (WX17, WX73 and WX107) were collected from mature bushes grown at Department of Agriculture and Food Western Australia (DAFWA), Perth (31 58 55 S/115 51 47 E). Collected stems were immediately placed upright in buckets with water and recut at 20-25 cm in length (from the cut end to the most extreme opened-flowers). The flower stalks were treated similarly as the fruits with BC (0-100 nLL.sup.1) or NC (0-100 nLL.sup.1) and ethylene. The experiments were laid out by following CRD design, having five replications for each treatment and five stalks in each replication. During the treatment period, the flower stalks were placed in small plastic bottles with distilled water. A cone made of nylon mesh was placed at the base of the stalks to check the number of abscised flowers.

[0095] The endogenous level of ethylene was determined by using the Sensor Sense (Sensor sense B.V, Nijmegen, The Netherlands). The Sensor Sense includes an ETD 300 ethylene detector, a set of valve controllers with an option of six valves connected to six separate cuvettes [1.0 L air-tight jar, fitted with a rubber septum (SubaSeal, Sigma-Aldrich Co., St. Louis, USA)]. The continuous flow method was used with coarse mode (conversion factor 99818, capacity to measure ethylene concentration at 0-500 ppm, sensitivity at <1%) of analysis. Each sample was run for 20 minutes with a flow rate of 4.0 L hour.sup.1 and the average reading of last 15 minutes was considered to calculate the concentration of ethylene and expressed as mol kg.sup.1 h.sup.1.

[0096] Respiration rate was determined as carbon dioxide (CO.sub.2) production from the fruit during ripening period a using CO.sub.2 analyser. The headspace gas sample (2.0 mL) was taken through rubber septum (SubaSeal, Sigma-Aldrich Co., St. Louis, USA) using a syringe from the air tight jar with sample fruit and injected into an infrared gas analyser [Servomex Gas Analyzer, Analyzer series 1450 Food Package Analyzer, Servomex (UK) Ltd., East Sussex, UK]. The respiration rate was calculated on the basis of the peak areas of 2.0 mL gas sample and CO.sub.2 standard (8.520.17%) and expressed as mmoL CO.sub.2 kg.sup.1 h.sup.1.

[0097] Assessment of floral organs abscission (%): Following ethylene treatment (2-4 days), the flower stalks were taken out from the treatment container and gently beaten against a collection tray to calculate the percentage of abscised flowers and buds.

[0098] The experimental data were analysed following one-way analysis of variance (ANOVA) by using Genstat 13 (release 13.1; Lawes Agricultural Trust, Rothamsted Experimental Station, Harpenden, UK). The effects of various treatments and their interactions were assessed and least significant differences (Fisher's LSD) were calculated by F test at 5% level of significance.

[0099] The level of climacteric ethylene in Tegan Blue plum fruit was significantly (P 0.05) suppressed by 100 nLL.sup.1 BC+ethylene and 1000 nLL.sup.1 1-MCP+ethylene (0.80- and 0.70-fold respectively) in comparison to the solely ethylene treated fruit where the ethylene concentration was 4.73 mol kg.sup.1 h.sup.1 (FIG. 2A).

[0100] Similarly, BC (50 nLL.sup.1)+ethylene treated Arctic Pride nectarine fruit exhibited significantly suppressed (0.63-fold) levels of ethylene than the solely ethylene treated fruit (0.414 mol kg.sup.1 h.sup.1) (FIG. 2 B).

[0101] The NC (100-1000 nLL-1) also showed antagonistic effect by significantly suppressing the level of climacteric ethylene (0.81-fold) than the solely ethylene treated fruit in Fortune plum fruit (FIG. 3).

[0102] The climacteric respiration was also suppressed in BC (100 nLL.sup.1)+ethylene and 1-MCP (1000 LL.sup.1)+ethylene treated Tegan Blue plum fruit (0.83- and 0.77-fold respectively) than the solely ethylene treated fruit (0.72 mmol CO.sub.2 kg.sup.1 h.sup.1) (FIG. 2C). On the other hand, significant suppression of respiration climacteric was observed in both BC (50 nLL.sup.1) and BC (50 nLL.sup.1)+ethylene treated Arctic Pride nectarine fruit (0.71- and 0.77-fold respectively) in comparison to the solely ethylene treated fruit (0.31 mmol CO.sub.2 kg.sup.1 h.sup.1) (FIG. 2D).

[0103] The fumigation of BC (100 nLL.sup.1) followed by ethylene exposure (10 LL.sup.1) significantly reduced the rate of flower/bud abscission in WX17 (6.05%). Whilst 50 and 100 nL L.sup.1 BC followed by ethylene treatment significantly lowered the rate of abscission at 22.43% and 28.40% respectively in WX73 wax flower as compared to ethylene treatment alone (FIGS. 4 A and B).

[0104] The treatment of NC (100 nL L.sup.1) also significantly (P0.05) suppressed the rate of flower/bud abscission in WX73 (0%) and WX107 (22.82%) wax flowers in comparison to the ethylene treated flowers. Suppressed flower/bud abscission was also observed in WX73 and WX107 wax flowers (38.11% and 25.51% respectively), even when the NC treatment was followed by ethylene treatment (10 L L.sup.1) (FIG. 5). The highest level of flower/bud abscission in all genotypes was noted from the ethylene treated flowers.

[0105] Fruit and flower stalks treated with BC or NC (50-100 nL L.sup.1) followed by ethylene treatment (10 L L.sup.1) significantly (P0.05) reduced the rate of flower abscission and concentration of climacteric ethylene and CO.sub.2 production than the solely ethylene exposed flowers and fruits which suggests that the inhibition of ethylene action by the BC and NC was not only exogenous but also at endogenous. This is the first disclosure on the effects of BC and NC on antagonising ethylene action during fruit ripening and floral organs abscission processes.

[0106] Similar effects to 1-MCP have been observed for BC and NC which has been reflected through the non-significant differences among the effects of BC (100 nL L.sup.1) and 1-MCP (1000 nL L.sup.1) on climacteric ethylene in Tegan Blue and Fortune Japanese plum fruits (FIGS. 2 A and 3 A) Arctic Pride nectarine (FIG. 2 B) and respiration in Tegan Blue plum (FIG. 2C) and Arctic Pride nectarine (FIG. 2 D).

[0107] BC and NC fumigation exhibited ethylene antagonistic effects on ripening of climacteric fruits such as plums, nectarines and abscission of floral organs in wax flowers.

[0108] In the tested chemicals (BC and NC), the cyclopropene portion of the molecule is thought to make a potential bond at or near the ethylene binding site of the receptor. As the flowers/fruit were exposed to the BC and NC treatment shortly after collection and completely blocked the ethylene receptor to prohibit ethylene activity for a period of time, so it worked at a non-competitive basis.