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WO1994015467A1 - Use of compounds to confer low temperature tolerance to plants - Google Patents

Use of compounds to confer low temperature tolerance to plants Download PDF

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Publication number
WO1994015467A1
WO1994015467A1 PCT/CA1994/000005 CA9400005W WO9415467A1 WO 1994015467 A1 WO1994015467 A1 WO 1994015467A1 CA 9400005 W CA9400005 W CA 9400005W WO 9415467 A1 WO9415467 A1 WO 9415467A1
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Prior art keywords
loweralkyl
oxo
thio
hydroxy
halogen
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French (fr)
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Suzanne R. Abrams
Lawrence V. Gusta
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National Research Council of Canada
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National Research Council of Canada
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/38—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
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    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
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    • A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
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Definitions

  • the invention generally relates to conferring low temperature tolerance to plants.
  • the invention includes the use of compounds in agricultural compositions to be applied to plants in the field to enhance their overall freezing resistance and/or chilling tolerance.
  • Brassica and native trees require a growth period at low temperatures (0 to 10°C) to trigger the appropriate genes involved in acclimation to freezing stresses. During this period of cold acclimation, numerous biochemical, physiological and metabolic functions are altered in plants.
  • the freezing tolerance of tissue cultures can be enhanced by treating cultures with abscisic acid.
  • abscisic acid for example, bromegrass cell suspension cultures treated with 75 ⁇ M ABA for 7 days can withstand freezing to -40°C (Reaney and Gusta 1987, Plant Physiol., 83:423).
  • results on whole plants are conflicting in that ABA can increase, decrease or have no effect on freezing tolerance.
  • chilling injury usually occurs between the temperature range of 17 to 0°C. It is distinguished from frost injury which occurs at subzero temperatures and involves the crystallization of water.
  • frost injury which occurs at subzero temperatures and involves the crystallization of water.
  • One of the first indications of chilling injury is wilting of the plant but the temperature at which chilling occurs at depends on both the species and cultivar.
  • cultivated crops such as tomato and beans are sensitive to temperatures below 17°C. As a result of exposure to these low temperatures, growth is inhibited which results in a delay in flowering, fruiting and maturity. Often flower seeds abort, seed set is reduced and the quality of the product is often unacceptable in the market place.
  • Daie et al. ((1981) J. Am. Hortic. 106:11-13) and Daie and Campbell ((1981) Plant Physiol. 67:26-28) examined the effect of low temperature (above 0°C) on ABA accumulation in chilling-sensitive plant species. They found that chilling sensitive species exhibited higher levels of ABA when exposed to 10°C. It has repeatedly been shown that exogenous ABA protects plants against a chilling stress (see for example Rikin et al. (1976), Bot. Gaz. 137:307-312; Bowman and Jansson (1980), Physiol. Plant 56:207-212; Eamus and Wilson (1983), J. Exp. Bot. 34:1000-1006 and Eamus (1986), J. Exp. Bot.
  • ABA at concentrations as low as 10 -8 M acts as an antitranspirant in partially closing stomata (N. Kondo, I. Maruta and K. Sugahara, 1980, Plant Cell. Physiol., 21:817).
  • Stomata may remain partially closed for as long as 4 days after treatment with 10 -4 M ABA and an acetylenic ABA aldehyde analog (H. Schaudolf, 1987, J. Plant Physiol., 131:433). This analog decreases water use in Helianthus annuus. Triticum aestivum and Lycopersicon esculentum while maintaining yield.
  • abscisic acid is expensive to produce commercially and secondly, the desired effect is only observed for short periods of time beause ABA, a naturally occurring hormone, is rapidly degraded by microorganisms found on the plants or by the plants themselves.
  • the present invention first relates to a composition for enhancing low temperature tolerance in plants which comprises an effective amount of at least one compound having the following formula (I) :
  • R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally sbustituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
  • R 1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium;
  • R 3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
  • R 2 when R 2 is oxo or thio, R 2 may be linked to both C 1 and C 2 carbon atoms to form an epoxy or a thioepoxy ring;
  • R 4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, lower- alkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen
  • R 4 when R 4 is oxo or thio, R 4 may be linked to the carbon atom adjacent to R 5 to form an epoxy or thioepoxy ring;
  • R 5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R 5 is oxo, it may be linked to the carbon atom bearing R 3 ;
  • R 6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R 7 is oxo, it may be linked to the carbon atom bearing R 3 ; and wherein the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
  • R 1 or R 6 is absent if the dotted line adjacent to R 1 and R 6 is a single bond
  • R 2 is absent if either of the dotted lines adjacent to R 2 is a single bond
  • the alkyl group bearing R 7 is absent if the dotted line adjacent to the alkyl group bearing R 7 is a single bond, and isomers and functional derivatives thereof,
  • R, R 1 , R 2 , R 4 , R 5 , R 6 or R 7 are phosphate, sulfoxide or sulfone.
  • compositions of the present invention can be applied in combination with other fungicides and/or other growth regulators such as auxins, ethylene, gibberellins, cytokinins and brassinolides to form agricultural solutions possessing freezing and/or chilling tolerance as well as germination enhancing properties.
  • other growth regulators such as auxins, ethylene, gibberellins, cytokinins and brassinolides
  • the agricultural compositions of the present invention are useful to increase plant resistance to water loss through stomata by stimulating the closure of plant stomata, to promote plant emergence, to act as hardeners or dehardeners and to promote freeze resistance in plants and to improve plant resistance to low temperature injury.
  • compositions of the present invention to stimulate the closure of stomata is especially beneficial when transplanting plants.
  • plants can experience tremendous shock, wilt and die. Closing the stomata of plants prior to transplanting has proved to be efficient in promoting quick recovery.
  • compositions of the present invention provide the possibility to either enhance or reduce temperature resistance of plants, to enhance plant resistance to herbicides, to overcome both low and high temperature dormancy and to improve drought and freeze resistance in plants.
  • compositions of the present invention can be synthesized in short efficient sequences from inexpensive starting materials.
  • the structures and stereochemistry of the synthesized compounds can then be easily established.
  • R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
  • R 1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium;
  • R 3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
  • R 2 when R 2 is oxo or thio, R 2 may be linked to both C 1 and C 2 carbon atoms to form an epoxy or a thioepoxy ring;
  • R 4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen,
  • R 4 when R 4 is oxo or thio, R 4 may be linked to the carbon atom adjacent to R 5 to form an epoxy or thioepoxy ring;
  • R 5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyl- oxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R 5 is oxo, it may be linked to the carbon atom bearing R 3 ;
  • R 6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy-
  • loweralkyl acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R 7 is oxo, it may be linked to the carbon atom bearing R 3 ; and wherein
  • the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
  • R 1 or R 6 is absent if the dotted line adjacent to R 1 and R 6 is a single bond
  • R 2 is absent if either of the dotted lines adjacent to R 2 is a single bond
  • the alkyl group bearing R 7 is absent if the dotted line adjacent to the alkyl group bearing R 7 is a single bond, and isomers and functional derivatives thereof,
  • R 1 is CH 3
  • R 2 is oxo or OH
  • R 3 is CH 3
  • R 4 is oxo or H
  • R 5 is H
  • the present invention relates to a method for enhancing low temperature tolerance in plants which comprises treating plants with an effective amount of a solution comprising at least one compound having the following formula (I) :
  • R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxylowera lkyl , acetyl loweralkyl , loweralkanoyl , loweralkylamino , diloweralkylamino , loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
  • R 1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium;
  • R 3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
  • R 2 when R 2 is oxo or thio, R 2 may be linked to both C 1 and C 2 carbon atoms to form an epoxy or a thioepoxy ring;
  • R 3 when R 3 is oxo or thio, R 3 may be linked to the carbon atom adjacent to R 5 to form an epoxy or thioepoxy ring;
  • R 4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, lower- alkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkyl amino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl,
  • R 4 when R 4 is oxo or thio, R 4 may be linked to the carbon atom adjacent to R 5 to form an epoxy or thioepoxy ring;
  • R 5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R 5 is oxo, it may be linked to the carbon atom bearing R 3 ;
  • R 6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R 7 is oxo, it may be linked to the carbon atom bearing R 3 ; and wherein
  • the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
  • R 1 or R 6 is absent if the dotted line adjacent to R 1 and R 6 is a single bond
  • R 2 is absent if either of the dotted lines adjacent to R 2 is a single bond
  • the alkyl group bearing R 7 is absent if the dotted line adjacent to the alkyl group bearing R 7 is a single bond, and isomers and functional derivatives thereof,
  • an acceptable agricultural carrier comprising an agriculturally acceptable carrier cation when R, R 1 , R 2 , R 4 , R 5 , R 6 , or R 7 are phosphate, sulfoxide or sulfone, for the purpose of enhancing low temperature tolerance in plants.
  • a plant seed treated with the agricultural composition referred to above is also within the scope of the present invention.
  • Figure 1 represents the influence of compound PBI-11 on the emergence of Katepwa wheat seedlings at low temperature.
  • Figure 2 represents the influence of compound PBI-10 on the emergence of Tobin Canola seedlings
  • Figure 3 represents the influence of compound PBI-10 on the emergence of Westar Canola at low temperature.
  • Figure 4 represents the frost tolerance of winter rye treated with various compounds of the invention.
  • Figure 5 shows the regrowth of rye seedlings submitted to freezing conditions following a root drench treatment with ABA.
  • Figures 6a and 6b show the regrowth of rye seedlings submitted to freezing conditions following a root drench treatment with PBI-54.
  • Figure 7 represents the effect of chain bond order at C-4, C-5 and C-2', C-3' on freezing resistance of seedlings.
  • Figure 8 represents the effect of chain bond order at C-2', C-3' and C-1 of compounds of the invention on freezing resistance of rye seedlings.
  • Figure 9 represents the effect of chain bond order at C-4, C-5 and C-1 of compounds of the invention on freezing resistance of rye seedlings.
  • Figure 10 represents the effect of ring bond level at C-2', C-3' of compounds of the invention on freezing resistance of rye seedlings.
  • Figure 11 represents the effect of chain bond level at C-4, C-5 of compounds of the invention on freezing resistance of rye seedlings.
  • Figure 12 represents the effect of C-1 functionality of compounds of the invention on freezing resistance of rye seedlings.
  • Figure 13 represents the effect of ring bond order at C-2', C-3' , chain bond order at C-4, C-5 and functionality at C-1 of compounds of the invention on freezing resistance of rye seedlings.
  • Figure 14 represents the effect of ring bond order at C-2', C-3' and chain bond order at C-4, C-5 of compounds of the invention on freezing resistance of rye seedlings over time.
  • Figure 15 represents the effect of ring bond order at C-2', C-3' and of functionality at C-1 of compounds of the invention on freezing resistance of rye seedlings over time.
  • Figure 16 represents the effect of compounds of the invention on the induction of freezing tolerance in rye seedlings over time when applied as a foliar spray.
  • Figure 17 represents the effect of a foliar spray of seedlings of canola c.v. Touchdown with compounds of the invention on freezing resistance and low temperature growth.
  • Figure 18 represents the effect of a foliar spray of seedlings of canola c.v. Touchdown with compounds of the invention on low temperature growth.
  • halogen includes chlorine, bromine, iodine and fluorine.
  • loweralkyl, loweracyloxyloweralkyl, loweralkanoyl, loweralkoxycarbonyl, loweralkoxy and loweracyloxy wherever employed, include straight and branched alkyl, acyloxyloweralkyl, alkanoyl, alkoxy and acyloxy groups having 1 to 10 carbon atoms in the alkyl, acyloxy- loweralkyl, alkanoyl, alkoxycarbonyl, alkoxy or acyloxy moiety.
  • the invention relates to compounds which, when applied as a root drench or a foliar spray, are useful to confer low temperature tolerance to plants.
  • low temperature tolerance is intended to include temperatures associated with chilling injury (usually between 17 and 0°C), frost injury (subzero temperatures) or both.
  • a first group is useful as chilling tolerance enhancers
  • a second group is useful to confer freezing resistance to plants at subzero temperatures
  • a third group can be used either in chilling or freezing resistance applications.
  • all groups have the ability to enhance plant tolerance to low temperature injury, either at temperatures above or below freezing.
  • composition useful to provide low temperature tolerance to plants, comprises a compound having the following formula (I) :
  • R, R 1 , R 2 , R 4 , R 5 , R 6 or R 7 are phosphate, sulfoxide or sulfone.
  • R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkyxo having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio;
  • R 1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 2 is hydrogen, hydroxy, halogen or thio
  • R 3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweralkylhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl; and when R 2 is thio, R 2 may be linked to both C 1 and C 2 carbon atoms to form a thioepoxy ring;
  • R 4 is hydrogen, oxo, halogen, thio or amino
  • R 5 is hydrogen, oxo or nitrogen
  • R 6 is hydrogen
  • R 7 is hydrogen, oxo or nitrogen.
  • a more preferred group of compounds to be used in the composition of the present invention include those having the following formula IA:
  • R is hydroxy, aldehyde, carboxyl or loweralkoxyl
  • R 1 is loweralkyl
  • R 2 is hydroxy
  • R 3 is loweralkyl or loweralkylhalide
  • R 4 is oxo
  • R 5 and R 7 are hydrogen
  • the dotted line is optionally a single bond and the double dotted line is a double bond or a triple bond; and R 7 is absent when the dotted line adjacent to R 5 is a single bond.
  • composition of the present invention are the following compounds:
  • Some of the compounds used in the context of the present invention have chemical structures containing asymetric carbon atoms, and therefore can be obtained as optical isomers.
  • the present invention therefore intends to cover racemic mixtures as well as isolated optical isomers of the compounds of formulae I and IA, obtained through resolution techniques well-known to those skilled in the art. These isomers may also be obtained through appropriate chemical synthesis, some examples of which are set forth in the present application. Generally speaking, the compounds of formulae I and IA have been used as racemic mixtures unless otherwise indicated.
  • the ability of the agricultural compositions of the present invention to enhance low temperature tolerance in plants appears to be related to specific regions of the active compound they contain.
  • nature of the functional group of substituent R, the double bond configuration at C-2, C-3, the bond order at C-4, C-5 (for example either a trans double bond or a triple bond) as well as the saturation of the ring, especially the presence or absence of a double bond at C-2', C-3' can influence the low temperature tolerance activity.
  • the carbon numbering order used in the context of the present invention was taken from the usual carbon numbering of abscisic acid.
  • the nature of the other substituents on the molecule does not appear to be as critical in affecting overall low temperature tolerance activity even though some preferred substituents have been suggested above.
  • the activity associated with any one substituent can also be influenced by the presence of other substituents.
  • cis and trans compounds appear to be more active than their corresponding acetylenic analogs unless R is an ester.
  • cyclohexenones tend to demonstrate more activity than cyclohexanones regardless of the oxidation level at R.
  • other examples showed that an acetylenic side chain decreased the activity of cyclohenenones but increased the activity of cyclohexanones relative to their cis, trans counterparts.
  • the present invention also relates to novel compounds useful to confer low temperature tolerance to plants. These compounds have the following formula (I):
  • R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkoxy having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio;
  • R 1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 2 is hydrogen, hydroxy, halogen or thio
  • R 3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweralkylhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl; a preferred group of the formula I compounds are those in which
  • R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkoxy having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio;
  • R 1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
  • R 2 is hydrogen, hydroxy, halogen or thio
  • R 3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweralkylhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl;
  • R 2 when R 2 is thio, R 2 may be linked to both C 1 and C 2 carbon atoms to form a thioepoxy ring;
  • R 4 is hydrogen, oxo, halogen, thio or amino
  • R 5 is hydrogen, oxo or nitrogen
  • R 6 is hydrogen; R 7 is hydrogen, oxo or nitrogen.
  • novel compounds of the present invention are prepared by alkylation of an appropriate cyclohexanone derivative with an appropriate acetylide derivative. This method is well-known to those skilled in the art.
  • the compounds of the present invention can be applied to plant parts using various vehicles to insure that the chemicals are active.
  • the rate of application should be such that a sufficient amount of the composition containing the active ingredient is applied to the targeted plant part to obtain the desired plant response to low temperature exposure.
  • the excipients used in the composition of the present invention can be selected from a wide variety of agriculturally acceptable carriers.
  • a preferred carrier is water.
  • Agents having the ability to enhance chemical uptake such as surfactants in very small percentages in the range of 0.01 to 1% are preferably used in the composition of the present invention.
  • the rate of application depends on a number of factors, such as environmental conditions, type of crop and the like. It has also been found that timing and rate of application bear a relationship to one another and to the crop to which they are applied, such that the rate of application and the timing thereof bear a relationship to the yield increase. Also, it has been discovered that the activity of some of these compounds on plants is concentration dependent since the compounds seem to be interfering with the action of some of the plant's normal hormones.
  • compositions of the present invention vary from one species to another depending on the nature and concentration of the compound used and on the method of application of the compounds.
  • a given compound may possess germination enhancement properties in Canola while another compound may be active in wheat but not in Canola.
  • some of the compositions of the present invention are highly specific to certain plant species while others are highly specific to different plant species.
  • the compounds of the present invention once dissolved in the desired carrier, are preferably applied either as a root drench or as a foliar spray. As mentioned previously, the method of application can sometimes affect efficacy of the treatment.
  • the compounds be applied as a root drench rather than a foliar spray for optimal efficacy, although both methods can be used efficiently.
  • the composition can usually be applied at a rate of from about 0.000005 g to 1.5 kg per acre, in a total applied volume of from about 5 1 to 100 1 per acre.
  • Preferred concentrations of the active compound in the foliar composition usually range between 10 -2 ⁇ M and 10 -5 ⁇ M.
  • compositions are applied by means of a root drench, the concentrations of the active compounds are the same as in the case of a foliar spray. The only difference is the fact that the compositions are applied to the roots rather than the leaves. Even though root drench application appears to be more effective at least in some plant species, foliar sprays are sufficiently effective to be used in widespread commercial applications.
  • An indication of enhanced freezing resistance in plants through application of the composition of the present invention is the fact that the dry matter content of plant cells is increased during incubation at low temperatures. Increases of the plant's dry matter content is usually accompanied by decreases in the plant's water content. Decreases in water content usually signifies an increase in freezing resistance.
  • compositions of the present invention also possess the ability to enhance germination and emergence at low temperatures. Cool soils in the spring delay germination increase the risk of fungal infection and produce uneven stands.
  • the composition described above allows better plants yields at low germination temperatures. Any seed or plant treatment which enhances germination is of considerable importance for the establishment of grasses which are slow to germinate and only a certain percentage of the seedlings survive to produce a proper coverage.
  • compositions of the present invention also promote plant growth and development at suboptimal temperatures. This is especially important in countries such as Canada where springs are cool for prolonged periods of time. Promotion of growth and development in the spring results in earlier maturity and avoids the risks of early fall frost. Since the plants can grow and develop early in the spring, they avoid midsummer periods of heat and drought. This leads to an increase in both yield and crop quality.
  • compositions of the present invention when applied to plant roots, lead to a reduction of the plant's transpiration rate.
  • Water is pulled into the plant via its roots, when a plant transpires water from its leaves. If a plant is transpiring more water than can be pulled into the plant, an automatic mechanism in the plant closes the stomata before irreversible damage occurs. When water becomes available, the stomata open.
  • the transpiration rate of a plant is proportional to the degree at which the plant's stomata are opened. A low transpiration rate therefore indicates that the plant's stomata are practically closed. This property is useful to avoid shock when transplanting from one container to another or from a container to the field.
  • the compounds of the present invention are useful as seed treatment for agronomic, forestry and horticultural crops. As well, these compounds are useful in the malting and distilling industry, where high alpha- amylase activity in germinating barley is required.
  • Ketal 1.31 g, 5.5 mmol was reacted with Z-3-methylpent-2-en-4-yn-1-ol (0.65 g, 6.7 mmol) and n-butyllithium (1.6 M in hexane, 8 mL, 12.8 mmol) in dry THF by the procedure described for the preparation of Z-5-(5-hydroxy-3-methyl-pent-3-en-1-ynyl)-3,5,5- trimethyl-2-cyclohexen-1-one.
  • molybdenum hexacarbonyl could be used in the above procedure instead of vanadyl acetylacetonate but longer reaction time was required.
  • the ketodiol 17 (30 mg, 0.11 mmol) was oxidized with manganese oxide (420 mg, 4.8 mmol) to give the corresponding aldehyde by the procedure described in Example 17.
  • the crude aldehyde was usually oxidized to ester as described in Example 23 without purification.
  • the ketosilyl ether from Example 26 (5.7 g, 21 mmol) was treated with Z-3-methylpent-2-en-4-yn-1-ol (3.0 g, 32 mmol) and n-butyllithium (1.6 M in hexane, 40 mL, 63 mmol).
  • the keto-acetate (0.94 g, 3.2 mmol) was hydrolysed by stirring with 5M NaOH (1 mL) and methanol (25 mL) at room temperature for 1 h. After working up and purification, the desired product (+)-4(Z)-(4R,5S)-4- hydroxy-4-(5-hydroxy-3-methylpent-3-en-1-ynyl)-3,3,5- trimethylcyclohexanone was obtained as colorless crystals (0.80 g, 100%), mp 96.5-98.0°C; [a]D +22.3°C (c 0.53, CH3OH); ir, 1H and 13C nmr identical with those of the (-)-(4S, 5R) enantiomer .
  • (+)-4(Z)-(4R,5S)-4-Hydroxy-4-(5-hydroxy-3- methylpent-3-en-1-ynyl)-3,3,5-trimethylcyclohexanone was treated with 2,2-dimethylpropane-1,3-diol in benzene with a catalytic amount of p-toluenesulfonic acid to afford the ketal, [a]D +27.1°C (c 0.90, CH3OH).
  • the aldehyde was prepared by the oxidation of
  • (+)-(4S, 5S)-methyl dihydroabscisate was hydrolyzed with 2M KOH and methanol to give (+)-(4S, 5S)-dihydroabscisic acid as colorless crystals, 173-180°C; [a]D +63.5°C (c 1.17, CH3OH).
  • the product 36 was obtained pure by chromatography over silica (Chromatotron, elution with 50% ether 50% hexane, as an oil that gave: ir (CHCl3) 3600 (weak), 1710 (strong) cm-1; 1H nmr -
  • Example 40 Example 40
  • the ester 36 was saponified as for compound 30 to afford the enynoic acid 37 in 83% yield.
  • (+)-(1'R, 2'R)-5',6'-Dihydroabscisic alcohol PBI-91) .
  • IR n max cm -1 (neat): 3400 (OH), 1700 (C O).
  • Compound PBI-171 was characterized as the methyl ester PB-170, m.p. 138-148°C, which had the following spectral properties: IR n max cm -1 : 3600, 3450, 1700; EIMS: m/z 278 [M-18] + (7), 248 (5), 219 (14), 191 (100); CIMS (ammonia): m/z 314 [M+18] + (100), 297 [M+1] + (4), 296 [M] + (8), 279 [M-18+1] + (30); CIMS (isobutane) : m/z 297 [M+1] + (6), 279 [M-18+1] + (62), 249 (100); trimethylsilyl ether derivative CIMS (ammonia) : m/z 386 [M+18] + (27), 369 [M+1] + (9), 368 [M] + (12), 351 [M-18+1] + (45). Anal, found:
  • (-)-Methyl 7',7'-difluoroabscisate showed the following properties:
  • Example 48
  • the seeds were dried at 35°C to a moisture content of approximately 12 percent.
  • the seeds were then planted in a soil mixture of 1 part soil, 1 part peat and 1 part vermiculite at a uniform depth of 3 cm.
  • the seeds in the soil were then transferred to a ConViron Model E-15 controlled environment chamber maintained at 10°C, in the dark. The number of seeds which emerged was determined twice a day.
  • compositions containing compound PBI-10 on the emergence of Canola at 10°C.
  • 'Tobin' 7.4 g of 'Westar' canola were soaked for 8 hours at 25°C, in each of the following solutions: water; and one of 10, 1, or 0.1 ⁇ M PBI-10 in glass beakers. Beakers were sealed with aluminum foil to prevent evaporation and to exclude light. After incubation, solutions were removed and seeds were blotted dry with paper towels. Seeds were sandwiched between 4 layers of paper towels, which were daily changed and seeds were separated, and dried at 25°C, until their dried weight was close to their pre-soaking weight.
  • Compounds in Group 1 were ABA, PBI-01, PBI-04, PBI-05, PBI-06, PBI-07, PBI-10, PBI-11, PBI-14 and PBI-15; compounds in Group 2 were ABA, PBI-16, PBI-17, PBI-18, PBI-19, PBI-34, PBI-43, PBI-37, and PBI-47.
  • Compounds at the highest concentration 1000 or 100 ⁇ M
  • a control of Ericksson's media (Group 1) or Ericksson's media with and without 1% DMSO (Group 2) was used.
  • Bromegrass cells (1 gram), aseptically added to each concentration and control (s), were incubated for 1 week at 25°C in darkness on a rotary shaker at 150 rpm. Each treatment was repeated twice for Group 1 and 3 times for Group 2. After incubation, cells were removed and weighed to determine the growth of cells in each concentration. Cells were sampled for gm water / gm dry weight and for a Freeze Test to determine the lethal temperature for 50% (LT 50 ) of the cells.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • the LT 50 is that part of the curve where the absorbance value of the frozen treatment in less than one half the value of the unfrozen control.
  • Group 2 the categories were as follows: 1) the hardeners were 0.01 to 10 ⁇ M PBI-34 and all of PBI-43; 2) PBI-16 gave no response; and 3) the dehardeners were PBI-17, PBI-18, PBI-19, PBI-37, and PBI-47 (Table 2).
  • the compounds induced categories of response. These categories were, as follows: 1) hardeners (lower LT 50 than the control); 2) same as the control; and 3) dehardeners (higher LT 50 than control). Tables 1 and 2 also show that each response was dependent on the concentration.
  • the plants were tested for frost tolerance using a controlled freeze test as follows. The plants were held at -2°C for two hours to equilibrate and then the leaves of the plants were nucleated with small ice crystals to initiate freezing of water in the plant.
  • the plants were cooled to -3°C and allowed to equilibrate at that temperature for 1 hour. Plants were then removed from this temperature and allowed to thaw slowly at 4°C. The remaining plants were then cooled to -4, -5, -6, -7 and -9°C using the same protocol as described above. The frosted plants were compared to an unfrozen control after two weeks of regrowth in a glass house maintained at 25°C. The results of the freeze test are summarized in Table 3.
  • Seedlings of Brassica napus c.v. Delta (a spring type brassica) were treated with compounds PBI-01, PBI-16, PBI-37, PBI-38, PBI-40, PBI-252, PBI-260 and ABA as described in Example 54.
  • the plants were grown similar to the plants grown in Example 54 and subjected to a frost test as described in Example 54.
  • the plants were subjected to frosts two, three and four days following treatment with ABA and the compounds referred to above. The results of this test are described in Table 4.
  • Results are based on the activity of 16 compounds which were applied to the roots of tender rye seedlings. Compounds were applied as a 10-4 M root drench on two consecutive days. LT 50 values were calculated from freeze tests performed 3, 4 and 6 days after the first application.
  • Figure 7 represents the interaction between chain bond order at C-4, C-5 and ring bond order at C-2', C-3' based on the induction of freezing tolerance in rye seedlings after compounds were applied as a root drench. Values are the average of 3 freeze tests performed 3, 4 and 6 days after treatment. It can be seen from this figure that altering the ring bond order did not increase nor decrease the effect of chain bond order on compound activity, or vice versa. Compounds with ring double bonds were more effective than ones with ring single bonds, and compounds with chain triple bonds were more effective than ones with a trans double bond.
  • Figure 8 represents the interaction between ring bond order at C-2', C-3' and C-1 functionality based on the induction of freezing tolerance in rye seedlings after compounds were applied as a root drench. Values are the average of 3 freeze tests performed 3, 4 and 6 days after treatment.
  • the highly significant ring bond order by functionally at C-1 interaction stemmed primarily from esters for which activity was the same regardless of ring bond order.
  • C-1 was an acid, aldehyde or alcohol
  • the effect of ring bond order was additive with double bonds imparting greater activity than single bonds.
  • ring bond order at C-2', C-3' altered the effectiveness of the compounds over time.
  • Compounds with a C-2', C-3' double bond were more active initially than ones with a C-2', C-3' single bond.
  • the activity of compounds with a C-2', C-3' single bond however, increased over time and was equal to compounds with a C-2', C-3' double bond by day 6. Determinations were carried out with preplanned contrasts between bond level for day 3 and day 6.
  • esterification decreased the activity of cyclohexenones. but did not affect the activity of cyclohexanones.
  • the reason for this interaction is not clear, but may be related to esterase activity and rates of hydrolysis if a free carboxyl group was required for activity.
  • the C-1 has to be at the acid oxidation level to induce freezing tolerance in a bromegrass cell suspension culture.
  • the greater activity of analogs with a triple bond at C-4, C-5 may also partially relate to the rate of hydrolysis as acetylenic esters were of equal activity to acetylenic acids, but dienoic esters were less active than dienoic acids.
  • Seedlings of a Graminae species winter rye (Secale cereale) cv Puma were grown at 25°C in a glass house. At the two to three leaf stage, seedlings grown in vermiculite received a foliar application of 100 ⁇ M ABA analogs. A mesting bottle was used for foliar applications and approximately 0.5 ml of solution per plant was applied. Sixteen compounds were tested for increasing the freezing tolerance of the rye. The plants were tested for freezing tolerance as described in Example 54. Following the controlled freeze test the plants were evaluated for frost tolerance after three weeks growth in a glasshouse at 25°C. Results are summarized in Figure 16. When compared to the results from a root application of the same analogs, lesser compounds showed a consistent response to time when applied as a foliar spray. However, compounds such as PBI-54 and PBI-11 maintained more consistent activities over time.
  • Foliar applied ABA may be rapidly ion trapped in the phloem which has a pH of 7.5.
  • Studies with radiolabelled ABA have shown that foliar applied ABA is translocated very slowly to strong sinks such as young leaves.
  • root applied ABA rapidly accumulates in the xylem stream.
  • the xylem sap is typically pH 5.5 to 6.5, therefore, ABA is not ion trapped and has access to all tissues that are continuous with the apoplast.
  • the crown is the critical region for survival in cereals, therefore, ABA must come into contact with this tissue if there is to be a response.
  • the crown contains the meristems in cereal seedlings, which are strong sinks for photosynthate. Xylem vessels also pass through the crown, therefore, both methods of application would expose the crown to ABA.
  • the marked differences in response to the method of application must be due to either lesser foliar uptake or the location of the ABA receptor (s) governing acclimation.
  • the location of the ABA receptor (s) for cold hardening is unknown, the situation may be analogous to that of stomata.
  • Stomatal receptors are on the plasma membrane and are sensitive to the apoplastic concentration of ABA.
  • Phaseolus vulgaris (beans) grown at 25°C in a peat mixture were soil drenched with approximately 50 mls of either water, ABA and the compounds PBI-03, PBI-19, PBI-16 and PBI-11 (all chemicals at 10 -6 M). The plants were held at 25°C with a 16 hour photoperiod. After seven days the plants were transferred to 25°C and allowed to grow for an additional 14 days. The plants were evaluated for injury (visually) based on a scale of 0 to 5 when 0 indicates no injury and 5 indicates complete necrosis.
  • Seeds of tomato (Lycopersicon esculentum cv. Swift) were sown to a 1:2 mix of peat moss and vermiculite in a glass house maintained at 25°C. At the two leaf stage the seedlings were transplanted a "Styrofoam tray" containing holes of 3 cm in diameter by 12 cm deep. The roots of the seedlings were covered with greenhouse grade vermiculite. Plants were grown in the glass house at a light intensity of 350 ⁇ mol/sec/m 2 and fertilized every third day with a 10% solution of a 20-20-20 (N-P-K) fertilizer. When the plants were 20 to 30 cm in height
  • ABA analogs PBI-01, PBI-37, PBI-40 and PBI-53 at either 1 or 10 ⁇ M. Racemic ABA at equivalent concentrations was also added to the tomato plants as a comparison treatment.
  • Plants treated with PBI-16 and PBI-37 had the lowest value transpiration rates. Plants treated with PBI-40, PBI-16 and PBI-37 flowered 4 days earlier than the control. According to statistical analysis these results are significant (Table 3). Plants treated with PBI-40 and racemic ABA suffered the lowest losses due to transplant shock in the field.
  • Tomato plants cv. Swift were grown as described previously in Example 59. The plants were treated with the following compounds: -ABA, PBI-37, PBI-40, PBI-11, PBI-63 and PBI-51.
  • the tomato plants were chilled for 0, 2, 4 and 6 days in one trial and for 0, 2, 4, 7 and 9 days in a second trial. For both cases, the chilling temperatures was 5°C.
  • the results of the treatments on chilling injury to tomatoes is shown in Tables 4 and 5.
  • the effect of the treatments on chilling injury to tomatoes is shown in Tables 4 and 5.
  • the effect of the treatments is shown in Table 8.
  • Tomato plants exposed to chilling temperatures were protected for 2 days. The trend was to afford protection for also 4 days of chilling temperatures. Plants treated with -ABA also flowered 6 to 13 days earlier than the controls. The compound PBI-37 flowered 3 to 10 days earlier than the controls.

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Abstract

The invention disclosed is a composition for conferring low temperature tolerance to agricultural plants which comprises an effective amount of a compound having formula (I). The composition includes compounds, some of which are novel abscisic acid derivatives. Methods of treating plants and plant tissues such as seeds, with compositions according to the invention are also disclosed.

Description

TITLE OF THE INVENTION
Use of compounds to confer low temperature tolerance to plants.
This patent application is a continuation-in- part of co-pending patent application Serial Number 444,704 filed December 1, 1989, which is a continuation- in-part of patent application Serial Number 280,102 filed December 1, 1988 now abandoned, both hereby incorporated by reference.
FIELD OF THE INVENTION
The invention generally relates to conferring low temperature tolerance to plants. Particularly, the invention includes the use of compounds in agricultural compositions to be applied to plants in the field to enhance their overall freezing resistance and/or chilling tolerance.
BACKGROUND OF THE INVENTION
Cold tolerant plants such as winter wheat,
Brassica and native trees require a growth period at low temperatures (0 to 10°C) to trigger the appropriate genes involved in acclimation to freezing stresses. During this period of cold acclimation, numerous biochemical, physiological and metabolic functions are altered in plants.
Originally, it was demonstrated by Irvin and Lanphea ((1967) Plant Physiol. 42:1191-1196) that ABA could induce 3°C of freezing tolerance in Acer necrundo. Attempts to harden plants with exogenous ABA have had only marginal success until Chen and Gusta ((1983) Plant Physiol. 73:71-75) demonstrated a 30°C increase in freezing tolerance in bromegrass cell cultures treated with 75 μM ABA at non hardening temperatures. Subsequently, ABA has been reported to induce freezing tolerance at non hardening temperatures in cultural cells of Brassica napus ((1986) J. Plant Physiol. 126:23-32, Johnson-Flanagan et al. (1991) Plant Physiol. 95:1044- 1048) and Lotus corniculatus (Keith and McKersie, (1986) Plant Physiol. 80:766-770). Also, moderate increases in hardiness were obtained when hardened winter wheat plants were sprayed with ABA (Lalk and Dorffling, (1985) Plant Physiol. 63:287-292) or closely related ABA analogs (Flores et al. (1988) J. Plant Physiol. 132:363-369).
The freezing tolerance of tissue cultures can be enhanced by treating cultures with abscisic acid. For example, bromegrass cell suspension cultures treated with 75 μM ABA for 7 days can withstand freezing to -40°C (Reaney and Gusta 1987, Plant Physiol., 83:423). However, results on whole plants are conflicting in that ABA can increase, decrease or have no effect on freezing tolerance. No practical application of ABA or ABA analogs
for enhancing freezing tolerance of plants has been reported.
Most cultivated crops are sensitive to low temperatures and can be damaged as a result of what is usually referred to as chilling injury. Chilling injury usually occurs between the temperature range of 17 to 0°C. It is distinguished from frost injury which occurs at subzero temperatures and involves the crystallization of water. One of the first indications of chilling injury is wilting of the plant but the temperature at which chilling occurs at depends on both the species and cultivar. For example, cultivated crops such as tomato and beans are sensitive to temperatures below 17°C. As a result of exposure to these low temperatures, growth is inhibited which results in a delay in flowering, fruiting and maturity. Often flower seeds abort, seed set is reduced and the quality of the product is often unacceptable in the market place.
Daie et al. ((1981) J. Am. Hortic. 106:11-13) and Daie and Campbell ((1981) Plant Physiol. 67:26-28) examined the effect of low temperature (above 0°C) on ABA accumulation in chilling-sensitive plant species. They found that chilling sensitive species exhibited higher levels of ABA when exposed to 10°C. It has repeatedly been shown that exogenous ABA protects plants against a chilling stress (see for example Rikin et al. (1976), Bot. Gaz. 137:307-312; Bowman and Jansson (1980), Physiol. Plant 56:207-212; Eamus and Wilson (1983), J. Exp. Bot. 34:1000-1006 and Eamus (1986), J. Exp. Bot. 37:1854-1862. Flores and Dorffling ((1989), IRRN 14:25) also reported that three membered-ring compounds derived from ABA by BASF and coded LAB 173711 and LAB 144143 increased chilling tolerance cucumber, tomato, wheat and rice. Rice tolerance to chilling at 5°C was increased by a foliar application at 10-3 and 10--4 mol/liter.
ABA at concentrations as low as 10-8M acts as an antitranspirant in partially closing stomata (N. Kondo, I. Maruta and K. Sugahara, 1980, Plant Cell. Physiol., 21:817). Stomata may remain partially closed for as long as 4 days after treatment with 10-4M ABA and an acetylenic ABA aldehyde analog (H. Schaudolf, 1987, J. Plant Physiol., 131:433). This analog decreases water use in Helianthus annuus. Triticum aestivum and Lycopersicon esculentum while maintaining yield.
Over 80 percent of transplanted Capsicum annuum plants dipped into a solution of ABA prior to planting in dry soil survived while less than 60 percent of control plants survived (G.A. Berkowitz and J. Rabin, 1988, Plant Physiol. 86:344). Furthermore, the treated plants had a 30 percent higher yield.
The successful use of ABA in freezing and chilling resistance as well as in antitranspiration experiments has been reported. However, the use of ABA in
plants presents drawbacks. Firstly, abscisic acid is expensive to produce commercially and secondly, the desired effect is only observed for short periods of time beause ABA, a naturally occurring hormone, is rapidly degraded by microorganisms found on the plants or by the plants themselves.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a class of compounds useful to low temperature tolerance to plants and plant tissues and to promote the closure of plant stomata.
In general terms, the present invention first relates to a composition for enhancing low temperature tolerance in plants which comprises an effective amount of at least one compound having the following formula (I) :
Figure imgf000007_0001
wherein
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally sbustituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium;
R3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
when R2 is oxo or thio, R2 may be linked to both C1 and C2 carbon atoms to form an epoxy or a thioepoxy ring;
and when R3 is oxo or thio, R 3 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, lower- alkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
and when R4 is oxo or thio, R4 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R5 is oxo, it may be linked to the carbon atom bearing R3;
R6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R 7 is oxo, it may be linked to the carbon atom bearing R3; and wherein the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
R1 or R6 is absent if the dotted line adjacent to R1 and R6 is a single bond,
R2 is absent if either of the dotted lines adjacent to R2 is a single bond,
the alkyl group bearing R7 is absent if the dotted line adjacent to the alkyl group bearing R 7 is a single bond, and isomers and functional derivatives thereof,
in admixture with an acceptable agricultural carrier comprising an agriculturally acceptable carrier cation when R, R1, R2, R4, R5, R6 or R7 are phosphate, sulfoxide or sulfone.
The compositions of the present invention can be applied in combination with other fungicides and/or other growth regulators such as auxins, ethylene, gibberellins, cytokinins and brassinolides to form agricultural solutions possessing freezing and/or chilling tolerance as well as germination enhancing properties.
The agricultural compositions of the present invention are useful to increase plant resistance to water loss through stomata by stimulating the closure of plant stomata, to promote plant emergence, to act as hardeners or dehardeners and to promote freeze resistance in plants and to improve plant resistance to low temperature injury.
The use of the compositions of the present invention to stimulate the closure of stomata is especially beneficial when transplanting plants. When such an operation is performed, plants can experience tremendous shock, wilt and die. Closing the stomata of plants prior to transplanting has proved to be efficient in promoting quick recovery.
The hardening and dehardening properties of the compositions of the present invention provide the possibility to either enhance or reduce temperature resistance of plants, to enhance plant resistance to herbicides, to overcome both low and high temperature dormancy and to improve drought and freeze resistance in plants.
Most of the compounds comprised in the compositions of the present invention can be synthesized in short efficient sequences from inexpensive starting materials. The structures and stereochemistry of the synthesized compounds can then be easily established.
Also within the scope of the present invention is a novel compound having the following formula (I):
Figure imgf000011_0001
wherein
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium;
R3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
when R2 is oxo or thio, R2 may be linked to both C1 and C2 carbon atoms to form an epoxy or a thioepoxy ring;
and when R3 is oxo or thio, R3 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
and when R4 is oxo or thio, R4 may be linked to the carbon atom adjacent to R 5 to form an epoxy or thioepoxy ring; R5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyl- oxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R5 is oxo, it may be linked to the carbon atom bearing R3;
R6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy-
loweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R7 is oxo, it may be linked to the carbon atom bearing R3; and wherein
the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
R1 or R6 is absent if the dotted line adjacent to R1 and R6 is a single bond,
R2 is absent if either of the dotted lines adjacent to R2 is a single bond,
the alkyl group bearing R7 is absent if the dotted line adjacent to the alkyl group bearing R7 is a single bond, and isomers and functional derivatives thereof,
with the proviso that when R is -CHO, -CH2OH or -COOCH 3' R1 is CH3, R2 is oxo or OH, R3 is CH3, R4 is oxo or H and R5 is H, the following compounds are excluded from formula (I):
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Furthermore, the present invention relates to a method for enhancing low temperature tolerance in plants which comprises treating plants with an effective amount of a solution comprising at least one compound having the following formula (I) :
Figure imgf000017_0002
wherein
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxylowera lkyl , acetyl loweralkyl , loweralkanoyl , loweralkylamino , diloweralkylamino , loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium;
R3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
when R2 is oxo or thio, R2 may be linked to both C1 and C2 carbon atoms to form an epoxy or a thioepoxy ring;
and when R3 is oxo or thio, R3 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, lower- alkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkyl amino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
and when R4 is oxo or thio, R4 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R5 is oxo, it may be linked to the carbon atom bearing R3;
R6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R7 is oxo, it may be linked to the carbon atom bearing R3; and wherein
the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
R1 or R6 is absent if the dotted line adjacent to R1 and R6 is a single bond,
R2 is absent if either of the dotted lines adjacent to R2 is a single bond,
the alkyl group bearing R7 is absent if the dotted line adjacent to the alkyl group bearing R7 is a single bond, and isomers and functional derivatives thereof,
in admixture with an acceptable agricultural carrier comprising an agriculturally acceptable carrier cation when R, R1, R2, R4, R5, R6, or R7are phosphate, sulfoxide or sulfone, for the purpose of enhancing low temperature tolerance in plants.
Methods for stimulating the closure of plant stomata through treatment of plant roots or leaves with an agricultural solution comprising at least one compound of formula I also fall within the scope of the present invention.
Also within the scope of the present invention is a plant seed treated with the agricultural composition referred to above. A plant treated with the agricultural composition referred to above also falls within the scope of the present invention.
IN THE DRAWINGS
Figure 1 represents the influence of compound PBI-11 on the emergence of Katepwa wheat seedlings at low temperature.
Figure 2 represents the influence of compound PBI-10 on the emergence of Tobin Canola seedlings
at low temperature.
Figure 3 represents the influence of compound PBI-10 on the emergence of Westar Canola at low temperature.
Figure 4 represents the frost tolerance of winter rye treated with various compounds of the invention.
Figure 5 shows the regrowth of rye seedlings submitted to freezing conditions following a root drench treatment with ABA.
Figures 6a and 6b show the regrowth of rye seedlings submitted to freezing conditions following a root drench treatment with PBI-54.
Figure 7 represents the effect of chain bond order at C-4, C-5 and C-2', C-3' on freezing resistance of seedlings.
Figure 8 represents the effect of chain bond order at C-2', C-3' and C-1 of compounds of the invention on freezing resistance of rye seedlings.
Figure 9 represents the effect of chain bond order at C-4, C-5 and C-1 of compounds of the invention on freezing resistance of rye seedlings.
Figure 10 represents the effect of ring bond level at C-2', C-3' of compounds of the invention on freezing resistance of rye seedlings.
Figure 11 represents the effect of chain bond level at C-4, C-5 of compounds of the invention on freezing resistance of rye seedlings.
Figure 12 represents the effect of C-1 functionality of compounds of the invention on freezing resistance of rye seedlings.
Figure 13 represents the effect of ring bond order at C-2', C-3' , chain bond order at C-4, C-5 and functionality at C-1 of compounds of the invention on freezing resistance of rye seedlings.
Figure 14 represents the effect of ring bond order at C-2', C-3' and chain bond order at C-4, C-5 of compounds of the invention on freezing resistance of rye seedlings over time.
Figure 15 represents the effect of ring bond order at C-2', C-3' and of functionality at C-1 of compounds of the invention on freezing resistance of rye seedlings over time.
Figure 16 represents the effect of compounds of the invention on the induction of freezing tolerance in rye seedlings over time when applied as a foliar spray.
Figure 17 represents the effect of a foliar spray of seedlings of canola c.v. Touchdown with compounds of the invention on freezing resistance and low temperature growth.
Figure 18 represents the effect of a foliar spray of seedlings of canola c.v. Touchdown with compounds of the invention on low temperature growth.
As used herein, the term halogen includes chlorine, bromine, iodine and fluorine. The terms loweralkyl, loweracyloxyloweralkyl, loweralkanoyl, loweralkoxycarbonyl, loweralkoxy and loweracyloxy, wherever employed, include straight and branched alkyl, acyloxyloweralkyl, alkanoyl, alkoxy and acyloxy groups having 1 to 10 carbon atoms in the alkyl, acyloxy- loweralkyl, alkanoyl, alkoxycarbonyl, alkoxy or acyloxy moiety.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to compounds which, when applied as a root drench or a foliar spray, are useful to confer low temperature tolerance to plants. When used in the context of the present invention, the term "low temperature tolerance" is intended to include temperatures associated with chilling injury (usually between 17 and 0°C), frost injury (subzero temperatures) or both. Among the preferred compounds of the present invention, a first group is useful as chilling tolerance enhancers, a second group is useful to confer freezing resistance to plants at subzero temperatures and a third group can be used either in chilling or freezing resistance applications. However, all groups have the ability to enhance plant tolerance to low temperature injury, either at temperatures above or below freezing.
Agricultural compositions comprising compounds conferring low temperature tolerance to plants
The composition, useful to provide low temperature tolerance to plants, comprises a compound having the following formula (I) :
Figure imgf000024_0001
as defined above and isomers and functional derivatives thereof, in admixture with an acceptable agricultural carrier comprising an agriculturally acceptable carrier cation when R, R1, R2, R4, R5, R6 or R7 are phosphate, sulfoxide or sulfone.
Preferred embodiments falling within this generic class include the following compounds:
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
A preferred group of the formula I compounds are those in which
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkyxo having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, hydroxy, halogen or thio;
R3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweralkylhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl; and when R2 is thio, R2 may be linked to both C1 and C2 carbon atoms to form a thioepoxy ring;
R4 is hydrogen, oxo, halogen, thio or amino;
R5 is hydrogen, oxo or nitrogen;
R6 is hydrogen;
R7 is hydrogen, oxo or nitrogen.
A more preferred group of compounds to be used in the composition of the present invention include those having the following formula IA:
Figure imgf000046_0001
wherein
R is hydroxy, aldehyde, carboxyl or loweralkoxyl;
R1 is loweralkyl;
R2 is hydroxy;
R3 is loweralkyl or loweralkylhalide;
R4 is oxo;
R5 and R7 are hydrogen;
the dotted line is optionally a single bond and the double dotted line is a double bond or a triple bond; and R7 is absent when the dotted line adjacent to R5 is a single bond.
Some of the compounds that are to be used in the context of the present invention are not novel and have been previously synthesized. Known compounds that have been described as growth inhibiting agents but that have been unexpectedly found to possess the properties referred to above fall within the scope of the following composition that also includes novel abscisic acid-related compounds of the present invention.
Of particular interest for use in the composition of the present invention are the following compounds:
Figure imgf000047_0001
Figure imgf000048_0001
One may refer to the following publications that describe the synthesis of these and other compounds for which either racemic mixtures or a given isomer may be used: Agr. Biol. Chem. 46(3), 817-818, 1982, Agr Biol. Chem. Vol. 33, No. 2, p. 296-298 (1969), J. Chem. Soc. Pekin Trans. 1, 1984, 2147-2157, Planta 121: 263-272 (1974), Helv. Chim. Acta 59, 1424, (1976) and U.S.P. 4,153,615. The compounds represented in formula I shown above may therefore be combined with suitable agricultural carriers to provide compositions to be used for the treatment of plant including plant parts used in propagation.
Some of the compounds used in the context of the present invention have chemical structures containing asymetric carbon atoms, and therefore can be obtained as optical isomers. The present invention therefore intends to cover racemic mixtures as well as isolated optical isomers of the compounds of formulae I and IA, obtained through resolution techniques well-known to those skilled in the art. These isomers may also be obtained through appropriate chemical synthesis, some examples of which are set forth in the present application. Generally speaking, the compounds of formulae I and IA have been used as racemic mixtures unless otherwise indicated.
It is to be noted that the ability of the agricultural compositions of the present invention to enhance low temperature tolerance in plants appears to be related to specific regions of the active compound they contain. In general, nature of the functional group of substituent R, the double bond configuration at C-2, C-3, the bond order at C-4, C-5 (for example either a trans double bond or a triple bond) as well as the saturation of the ring, especially the presence or absence of a double bond at C-2', C-3', can influence the low temperature tolerance activity. The carbon numbering order used in the context of the present invention was taken from the usual carbon numbering of abscisic acid. The nature of the other substituents on the molecule does not appear to be as critical in affecting overall low temperature tolerance activity even though some preferred substituents have been suggested above.
It seems to be mostly preferred that a cis C-2,
C-3 double bond be present on the molecule to obtain enhancement in low temperature tolerance. Overall, acids and esters appear to be more active than aldehydes and alcohols, cyclohexenones seem more active than
cyclohexanones and dienoic and acetylenic compounds appear to be equally active.
The activity associated with any one substituent can also be influenced by the presence of other substituents. For example, cis and trans compounds appear to be more active than their corresponding acetylenic analogs unless R is an ester. Also, cyclohexenones tend to demonstrate more activity than cyclohexanones regardless of the oxidation level at R. Furthermore, other examples showed that an acetylenic side chain decreased the activity of cyclohenenones but increased the activity of cyclohexanones relative to their cis, trans counterparts.
From experiments conducted so far, it appears possible to suggest preferred trends for activity. Hence, the configuration at C-1 should be the same as in (S)-ABA, in dihydro analogs the C-2'-methyl and the side chain should be cis, small positional changes of the 7 '-methyl are tolerable, and the C-1 substituent should be such that it has an oxidation level close to or above the oxidation level of an acidic substituent. It is to be appreciated that even though scientific evidence gathered recently has led to the above suggestions with regard to structure- related activity, it is likely that other compounds of similar structure can be tailored for use as enhancers of low temperature tolerance.
One of the important advantages in using the compounds described above in agricultural compositions for enhancing low temperature tolerance is the fact that most of the compounds tested so far appear to gain activity over time. This tendency is contrary to what is observed when using abscisic acid, which rapidly loses its activity only a few days after having been applied to plants. It seems that the fact that most plants have the ability to metabolize ABA more rapidly than compounds which are structurally related to ABA could account for prolonged low temperature tolerance activity. Other factors which may not necessarily be metabolically related could also contribute to this enhancement. In northern climates where sudden sub-zero temperatures can be observed for several days in the spring for example, prolonged low temperature tolerance can have a beneficial effect on early rising crops.
Novel compounds useful in conferring chilling or freezing tolerance to plants
The present invention also relates to novel compounds useful to confer low temperature tolerance to plants. These compounds have the following formula (I):
(I)
Figure imgf000051_0001
as defined above and isomers and functional derivatives thereof ; with the proviso that when R is -CHO, -CH2OH or -COOCH3, R1 is CH3, R2 is oxo or OH, R3 is CH3, R4 is oxo or H and R5 is H, the same compounds listed above are excluded from formula (I).
A preferred group of the formula I compounds are those in which
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkoxy having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, hydroxy, halogen or thio;
R3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweralkylhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl; a preferred group of the formula I compounds are those in which
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkoxy having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio; R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, hydroxy, halogen or thio;
R3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweralkylhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl;
and when R2 is thio, R2 may be linked to both C1 and C2 carbon atoms to form a thioepoxy ring;
R4 is hydrogen, oxo, halogen, thio or amino;
R5 is hydrogen, oxo or nitrogen;
R6 is hydrogen; R7 is hydrogen, oxo or nitrogen.
Particularly, some of the more preferred novel compounds to be used in the context of the present invention have the following formulae:
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Generally speaking, the novel compounds of the present invention are prepared by alkylation of an appropriate cyclohexanone derivative with an appropriate acetylide derivative. This method is well-known to those skilled in the art.
It will also be understood by those skilled in the art that the compounds having the general formula described above can be found as geometric isomers having cis or trans configuration with respect to the double bond in the carbon chain. Furthermore, although the stereoisomeric configurations have not been indicated in the formulae examplified above, it is to be understood that all geometric isomers and stereoisomers of the compounds falling within the scope of formula I do fall within the scope of the present invention.
Application of the composition of the present invention to plant parts
The compounds of the present invention can be applied to plant parts using various vehicles to insure that the chemicals are active. The rate of application should be such that a sufficient amount of the composition containing the active ingredient is applied to the targeted plant part to obtain the desired plant response to low temperature exposure. The excipients used in the composition of the present invention can be selected from a wide variety of agriculturally acceptable carriers. A preferred carrier is water. Agents having the ability to enhance chemical uptake such as surfactants in very small percentages in the range of 0.01 to 1% are preferably used in the composition of the present invention.
The rate of application depends on a number of factors, such as environmental conditions, type of crop and the like. It has also been found that timing and rate of application bear a relationship to one another and to the crop to which they are applied, such that the rate of application and the timing thereof bear a relationship to the yield increase. Also, it has been discovered that the activity of some of these compounds on plants is concentration dependent since the compounds seem to be interfering with the action of some of the plant's normal hormones.
Furthermore, the tests performed in the field tend to demonstrate that the effects of the compositions of the present invention vary from one species to another depending on the nature and concentration of the compound used and on the method of application of the compounds. In other words, a given compound may possess germination enhancement properties in Canola while another compound may be active in wheat but not in Canola. Hence, some of the compositions of the present invention are highly specific to certain plant species while others are highly specific to different plant species. The compounds of the present invention, once dissolved in the desired carrier, are preferably applied either as a root drench or as a foliar spray. As mentioned previously, the method of application can sometimes affect efficacy of the treatment. For example, it seems preferred that the compounds be applied as a root drench rather than a foliar spray for optimal efficacy, although both methods can be used efficiently. In the case of a foliar spray mixture, the composition can usually be applied at a rate of from about 0.000005 g to 1.5 kg per acre, in a total applied volume of from about 5 1 to 100 1 per acre. Preferred concentrations of the active compound in the foliar composition usually range between 10 -2 μM and 10-5 μM.
If the compositions are applied by means of a root drench, the concentrations of the active compounds are the same as in the case of a foliar spray. The only difference is the fact that the compositions are applied to the roots rather than the leaves. Even though root drench application appears to be more effective at least in some plant species, foliar sprays are sufficiently effective to be used in widespread commercial applications.
An indication of enhanced freezing resistance in plants through application of the composition of the present invention is the fact that the dry matter content of plant cells is increased during incubation at low temperatures. Increases of the plant's dry matter content is usually accompanied by decreases in the plant's water content. Decreases in water content usually signifies an increase in freezing resistance.
The compositions of the present invention also possess the ability to enhance germination and emergence at low temperatures. Cool soils in the spring delay germination increase the risk of fungal infection and produce uneven stands. The composition described above allows better plants yields at low germination temperatures. Any seed or plant treatment which enhances germination is of considerable importance for the establishment of grasses which are slow to germinate and only a certain percentage of the seedlings survive to produce a proper coverage.
The compositions of the present invention also promote plant growth and development at suboptimal temperatures. This is especially important in countries such as Canada where springs are cool for prolonged periods of time. Promotion of growth and development in the spring results in earlier maturity and avoids the risks of early fall frost. Since the plants can grow and develop early in the spring, they avoid midsummer periods of heat and drought. This leads to an increase in both yield and crop quality.
Furthermore, the compositions of the present invention, when applied to plant roots, lead to a reduction of the plant's transpiration rate. Water is pulled into the plant via its roots, when a plant transpires water from its leaves. If a plant is transpiring more water than can be pulled into the plant, an automatic mechanism in the plant closes the stomata before irreversible damage occurs. When water becomes available, the stomata open. The transpiration rate of a plant is proportional to the degree at which the plant's stomata are opened. A low transpiration rate therefore indicates that the plant's stomata are practically closed. This property is useful to avoid shock when transplanting from one container to another or from a container to the field. Thus, the compounds of the present invention are useful as seed treatment for agronomic, forestry and horticultural crops. As well, these compounds are useful in the malting and distilling industry, where high alpha- amylase activity in germinating barley is required.
The present invention will be more readily illustrated by referring to the following examples which are introduced only to illustrate rather than limit the scope of the present disclosure.
Example 1
Preparation of (3E)-4-(5-acetoxy-3-methyl-1,3-pentadien-1- ylidene)-3,5,5-trimethyl-2-cyclohexen-1-one (PBI-27).
A solution of (2E)-5-(4-oxo-1-hydroxy-2,2,6- trimethylcyclohexyl)-3-methylpent-2-en-4-yn-1-ol (570 mg, 2.3 mmol) and potassium hydrogen sulfate (approx. 20 mg) in acetic acid (3.0 ml) and acetic anhydride (2.0 ml) was heated under argon for 2.5 h at 100°C. The solution was cooled to room temperature, water was added, and the product was extracted three times with ether. The combined ethereal phases were washed first with saturated sodium bicarbonate solution, then with sodium chloride solution and dried over anhydrous sodium sulfate. Evaporation of the solvent afforded an oil (483 mg) which was chromatographed over silica gel eluting with 30% ether / 70% hexane, yielding (3E)-4-(5-acetoxy-3-methyl-1,3- pentadien-1-ylidene)-3,5,5-trimethyl-2-cyclohexen-1-one (148 mg, 23%). The product gave a single spot on tic
(silica gel, 50% ether / 50% hexane, Rf 0.3); 1H NMR (360
MHz, CDCl3) : 6.31 (br s, H-2, 1H) , 5.89 (t, J=1.2Hz, H-
2, 1H) 5.61 (br t,J=7.0 Hz, H-4', 1H). 4.68 (d, J=7.0 Hz,
H-5', 2H), 2.38 (br s,H-6,2H_, 1.94 (d,J=1.2 Hz, C-3 methyl, 3H), 1.74 (br s, C-3' methyl, 3H), 1.17 (s, C-5 methyl, 3H), and 1.15 (s, C-5 methyl, 3H); IR (film)m ax
1910,1730,1650 and 1590 cm-1;GC/MS m/z 274(5), 232(7), 214(100) and 199(49); UV (hexane) 267 nm (24,400). Example 2
Alternative preparation of (3E)-4-(5-acetoxy-3-methyl-1,3- pentadien-1-ylidene)3,5,5-trimethyl-2-cyclohexen-1-one (PBI-27).
To a solution of (3E)-4-(5-hydroxy-3-methyl-1,3- pentadien-1-ylidene)-3,5,5-trimethyl-2-cyclohexen-1-one (1.2 g, 5.2 mmol) in acetic anydride (5.0 mL) and triethylamine (5.0 mL) cooled to 0°C, was added 4,4- dimethylaminopyridine (25 ng). After 15 min. water was added to the reaction mixture, and the product was extracted three times with ether. The combined ethereal extracts were washed with sodium chloride solution, and dried over anhydrous sodium sulfate. Evaporation of the solvent and chromatography over silica gel in the manner described in Example 1 above gave the desired product (680 mg, 48%).
Example 3
Preparation of (4E)-5-(2,6,6-trimethylcyclohex-1-enyl)-3- hydroxy-3-methylpentenoic acid (PBI-03).
The corresponding methyl ester of (4E)-5-(2,6,6- trimethylcyclohex-1-enyl)-3-hydroxy-3-methyl- pentenoic acid (10 g, 36 mmol) in ethanol (30 mL) was treated with sodium hydroxice solution (3 N, 250 mL) and the solution refluxed for 0.5 h. After cooling, the ethanol was removed at reduced pressure. The basic aqueous phase was extracted three times with ether to remove neutral components. The aqueous phase was then made acidic with hydrochloric acid and the product extracted three times with dichloromethane. The pooled organic extracts were washed with sodium chloride solution and then dried over anhydrous sodium sulfate. Evaporation of the solvent afforded 8.5 g of an oil. Treatment of an analytical sample with diazomethane afforded the starting ester. The acid was employed without further purification. 1H NMR (360 MHz, CDCl3) : 6.08 (d, J=16.1 Hz, H-5, 1H), 5.45 (d, J=16.1 Hz, H-4, 1H), 2.65 (s, H-2, 2H), 1.93 (m, H-3', 2H), 1.35-1.55 (m, H-4', H-5', 4H), 0.93, 0.92 (s, H-8, H- 9, 6H).
Example 4
Preparation of PBI-26 (III).
A solution of (2E)-5-(4-oxo-1-hydroxy-2,2,6- trimethylcyclohexyl)-3-methylpent-2-en-4-yn-1-ol (570 mg,
2.3 mmol) and potassium hydrogen sulfate (approx. 20 mg) in acetic acid (3.0 ml) and acetic anhydride (2.0 ml) was heated under argon for 2.5 h at 100°C. The solution was cooled to room temperature, water was added, and the product was extracted three times with ether. The combined ethereal phases were washed first with saturated sodium bicarbonate solution, then with sodium chloride solution and dried over anhydrous sodium sulfate.
Evaporation of the solvent afforded and oil (483 mg) which was chromatographed over silica gel eluting with 50% ether/50% hexane yielding compound III (48 mg, 9%).
Example 5
Z-5-(5-hydroxy-3-methyl-pent.-3-en-1-ynyl)-3,5,5- trimethyl-2-cyclohexen-1-one (1) (PBI-05). A solution of Z-3-methylpent-2-en-4-yn-1-ol
(Fluka, 7.9 g, 80 mmol) in dry THF (200 mL) under an argon atmosphere was cooled to about -60°C in a dry ice-acetone bath. n-Butyllithium (Aldrich, 1.6 M in hexane, 103 mL, 164 mmol) was added dropwise with stirring, followed after 0.5 h by a solution of oxoisophorone (Fluka, 6.1 g, 40 mmol) in dry THF (80 mL). A heavy precipitate was obtained at the end of the addition. The reaction mixture was stirred for 45 min. before it was poured into water and extracted three times with ether. The combined organic extracts were washed twice with saturated NaCl and dried over anhydrous Na2SO4. Evaporation of solvent gave a yellow oil (18 g) as the crude product. Purification of the product by flash column chromatography, with 50% ether + 50 % hexane followed by 100% ether as eluents, gave the alkylation product (6.9 g, 70% yield) as a yellow oil, ir: 3620 (sharp, medium, OH), 3420 (broad, medium, OH) , 2220 (weak, acetylene), 1660 (strong, C=O) cm-1; 1H nmr d: 5.83 (tq, J = 6.6, 1.5 Hz, 1H, =CH), 5.76 (q, J = 1.3 Hz, 1H, =CH), 4.17 (d, J = 6.6 Hz, 2H, CH2OH), 3.3 (1H, OH), 2.37 (m, 2H, CH2), 2.05 (d, J = 1.3 Hz, 3H, vinyl CH3), 1.79 (m, 3H, vinyl CH3), 1.12 and 1.03 (2s, 6H, 2CH3); 13C nmr d: 198.76 (s, C=O), 160.89 (s, =C), 136.74 (d, =CH), 125.75 (d, =CH), 120.02 (=C), 92.80 and 85.19 (2s, 2 acetylenic C), 74.59 (s, C-OH), 65.74 and 60.93 (2t, 2CH2), 41.79 (s, C), 25.11, 22.81, 19.74 and 15.12 (4q, 4CH3); ms m/z: 248 (M+, approx. 0.05), 230 (7), 192 (18), and 174 (100).
Example 6
9-Z-9-(5-hydroxy-3-methylpent-3-en-1-ynyl)-3,3,8,8,10- pentamethyl-1,5-dioxaspiro[5,5]undecan-9-ol (2). A mixture of 2,2,6-trimethyl-1,4-cyclo- hexanedione (1.07 g, 6.9 mmol), 2,2-dimethyl-1,3- propanediol (0.95 g, 9.1 mmol), p-toluenesulfonic acid (59 mg), and benzene (15 mL) was heated to reflux under a Dean-Stark water separator for 2 h. The reaction mixture was allowed to cool to room temperature before it was neutralized with saturated NaHCO3, washed with H2O, and dried over anhydrous Na2SO4. Evaporation of solvent gave a colorless oil (1.76 g) as the crude product, which was distilled using the Kugel-rohr apparatus (about 150°C, 0.5 mm Hg) to give pure ketal as a colorless oil (1.60 g, 96%), ir: 1710 (Strong, C=O) cm-1; 1H nmr d: 3.61 and 3.53 (2d, J = 11.4 Hz, 2 axial H of CH2O), 3.48 and 3.41 (2dd, J = 11.4, 1.6 Hz, 2 equatorial H of CH2O), 2.85 (m, 1H, CH), 2.47 (dd, J = 14.2, 3.7 Hz, 1H, equatorial H of CH2), 2.38 (ddd, J = 13.5, 5.3, 3.8 Hz, 1H, equatorial H of CH2), 1.58 (d, J = 14.2 Hz, axial H of CH2), 1.56 (dd, J = 13.5, 13.5 Hz, axial H of CH2), 1.16 (s, 3H, CH3), 0.97 (S, 6H, 2CH3), 0.91 (d, J = 6.6 Hz, 3H, CHCH3), 0.85 (s, 3H, CH3); ms m/e: 240 (M+, 0.58), 141 (27), 155 (98), 83 (27), 69 (100). Ketal 1.31 g, 5.5 mmol) was reacted with Z-3-methylpent-2-en-4-yn-1-ol (0.65 g, 6.7 mmol) and n-butyllithium (1.6 M in hexane, 8 mL, 12.8 mmol) in dry THF by the procedure described for the preparation of Z-5-(5-hydroxy-3-methyl-pent-3-en-1-ynyl)-3,5,5- trimethyl-2-cyclohexen-1-one. The crude product (yellow oil, 2.6 g) obtained was purified by flash column chromatography using 75% ether + 25% hexane as eluent and subsequent distillation using the Kugel-rohr apparatus (about 250°C, 0.06 mm Hg) to give ketal (2) (1.40 g, 77%), ir: 3610 (strong, sharp, OH), 3440 (broad, medium, OH), 1110 and 1090 (strong, C-O) cm-1; 1H nmr d: 5.50 (ddq, J = 6.7, 6.7, 1.5 Hz, 1H, =CH), 4.29 (broad s, 2H, CH2OH), 3.55 (d, J = 11.3 Hz, 2H, 2 axial H of CH2O), 3.38 and 3.36 (2dd, J = 11.3, 1.9 Hz, 2H, 2 equatorial H of CH2O), 2.51 (dd. J = 14.3, 3.2 Hz, 1H, equatorial H of CH2), 2.18 (m, 1H, CH), 1.98 (ddd, J = 13.8, 3.4, 3.4 Hz , 1H, equatorial H of CH2), 1.84 (m, 3H, vinyl CH3), 1.53-1.61 (m, 2H, 2 axial H Of CH2), 1.11, 1.09, 1.06, 1.05, 1.04 and 0.83 (15H, 5CH3); ms (trimethylsilyl ether) m/z: 408 (M+ of trimethylsilyl ether), 155 (100); high resolution ms (trimethylsilyl ether): calc. for C23H4004Si 408.2696, found 408.2718. Example 7
Z-4- (5-acetoxy-3-methyl-pent-4-en-1-ynyl) -3 , 5, 5-trimethyl- cyclohex-3-en-1-one (3 ) .
A mixture of (Z)-4-hydroxy-4-(5-hydroxy-3- methylpent-3-en-1-ynyl)-3,5,5-trimethylcyclohexanone 795 mg, 3.1 mmol), glacial acetic acid (5 mL), acetic anhydride (5 mL), and KHSO4 (440 mg, 3.2 mmol) was heated to 70°C under argon for 5 h. Then the mixture was cooled to room temperature and slowly added to a chilled (ice bath) and stirred mixture of hexane and saturated NaHCO3. More saturated NaHCO3 was added until the pH of the aqueous phase was about 6-7. The organic and aqueous layers were then separated, and the aqueous layer was extracted with hexane. The combined hexane layers were washed with saturated NaHCO3, H2O, and dried over anhydrous Na2SO4. Removal of solvent gave a yellow oil (737 mg) which on purification by flash column chromatography using 75% ether + 25% hexane as solvent gave compound (3) (600 mg, 70%) as a yellow oil, ir: 1730 (strong, broad) cm-1; 1H nmr d: 5.77 (ddq, J = 7.0, 7.0, 1.5 Hz, 1H, =CH), 4.76 (dd, J = 7.0, 1.0 Hz, 2H CH2O), 2.90 (broad s, 2H, CH2), 2.39 (s, 2H, CH2), 2.03 (s, 3H, CH3COO), 1.94 and 1.93 (2m, 6H, 2 vinyl CH3), 1.16 (s, 6H, 2CH3); ms m/z: 274 (M+, 30), 214 (100).
Example 8
Z-4-(5-acetoxy-3-methyl-pent-3-en-1-ynyl)-3,4-epoxy- 3,5,5-trimethylcyclohexan-1-ol (4).
To a solution of Z-4-(5-acetoxy-3-methyl-pent-3- en-lynyl)-3,5,5-trimethylcyclohex-3-en-1-ol (42 mg, 0.15 mmol) in toluene (6 mL) was added t-butyl hydroperoxide (3M solution in 2,2,4-trimethylpentane, 0.07 mL, 0.21 mmol) and vanadyl acetylacetonate (3 mg, 0.01 mmol). The reaction mixture, which was reddish orange in color, was stirred under argon at room temperature for 20 min. and then was heated to 70°C for 30 min. The color of the mixture changed to yellow. After cooling to room temperature, saturated NaHSO3 was added with stirring until there was no peroxide as indicated by peroxide test tapes. The organic and aqueous layers were separated. The aqueous layer was extracted three times with ether. The combined organic layers were washed with H2O, saturated NaHCO3, H2O and dried over anhydrous Na2SO4. Evaporation of solvent gave yellow oil (46 mg) which was purified using the Chromatotrontm sold by Harrison Scientific and with 75% ether + 25% hexane as solvent to give compound (4) as a colorless oil (17 mg, 38%), ir: 3620 (weak, sharp, OH), 3500 (weak, broad, OH), 1740 (strong, sharp, C=0) cm-1; 1H nmr d: 5.80 (ddq, J = 6.9, 6.9, 1.6 Hz, 1H, =CH), 4.71 (dd, 6.9, 1.0 Hz, 2H, CH2O), 3.83 (m, 1H, CHOH), 2.19 (ddd, J = 14.7, 6.8, 1.5 Hz, 1H, CH2), 2.03 (s, 3H, CH3COO), 1.89 (m, 3H, vinyl CH3), 1.81 (dd, J = 14.7, 8.9 Hz, 1H, CH2), 1.47 (s, CH3), 1.37 (ddd, J = 12.7, 4.0, 1.5 Hz, 1H, CH2), 1.18 (s, 6H, 2CH3); ms (isobutane CI) m/z: 293 (M+1), 331 (M+39); (NH4Cl CI): 310 (M+18), 275 (M-17), 233 (M-59).
Alternatively, molybdenum hexacarbonyl could be used in the above procedure instead of vanadyl acetylacetonate but longer reaction time was required.
Example 9
Z-4-(5-hydroxy-3-methyl-pent-3-en-lynyl)-3,4-epoxy- 3,5,5-trimethylcyclohexan-1-ol (5).
A mixture of Z-4-(5-acetoxy-3-methyl-pent-3- en-lynyl)-3,4-epoxy-3,5,5-trimethylcyclohexan-1-ol (36 mg, 0.12 mmol), K2CO3 (26 mg, 0.18 mmol), methanol (1 mL) and H2O (1 mL) was stirred at room temperature for 1 h. It was then concentrated by evaporation and the residue was diluted with H2O and extracted with CHCl3. The organic extract was dried over anhydrous Na2SO4. Evaporation of solvent gave compound (5) as a colorless oil (21 mg, 69%), ir: 3620 (sharp, medium, OH) and 3450 (broad, weak, OH) cm-1; 1H nmr d: 5.87 (ddq, J = 6.7, 6.7, 1.4 Hz, 1H, =CH), 4.27 (dd, J = 6.7, 1.0 Hz, 2H, CH2O), 3.82 (m, 1H, CHOH), 2.18 (ddd, J = 14.8, 6.8, 1.4 Hz, 1H, CH2), 1.86 (m, 3H, vinyl CH3), 1.79 (dd, J = 14.8, 9.0 Hz, 1H, CH2), 1.46 (s, CH3), 1.36 (ddd, J = 12.7, 4.1, 1.5 Hz, 1H, CH2), 1.16 and 1.17 (2s, 6H, 2CH3); 13C nmr d: 136.63 (d, =CH), 119.95 (s, =C), 90.95 and 83.99 (2s, 2 acetylenic C), 65.08 and 64.52 (2s, C-O-C), 63.29 (d, CHOH), 61.15,
41.84 and 38.16 (3t, 3 CH2), 34.83 (s, C), 27.03, 25.60,
22.98 and 22.92 (4q, 4CH3); ms (NH4Cl CI) m/z: 268 (M+18), 251 (M+1), 233 (M-17).
Example 10
Trans (-)-(4R,6R)-4-t-Butyldimethylsilyloxy-2,2,6-trime- thylcyclohexanone.
A mixture of trans (4R,6R)-4-hydroxy-2,2,6- trimethylcyclohexanone (115 mg, 0.73 mmol), t-butyldimethylsilyl chloride (Aldrich, 202 mg, 1.27 mmol), imidazole (100 mg, 1.47 mmol) and dry DMF (3 mL) was stirred at room temperature under an argon atmosphere for 1.5 h. Then water was added and the mixture was extracted three times with ether. The ether extract was washed with saturated NaCl and dried over anhydrous Na2SO4. Evaporation of solvent gave a colorless oil (325 g) which was distilled using the Kugel-rohr apparatus. After some forerun which was discarded, the desired silyl ether was collected as a colorless oil at 150-180°C, 9-10 mm Hg (156 mg, 80%). The product solidified on storage at -10°C to form colorless crystals, mp 29.5-31.5°C; [a]D -65.1°C (c 1.06, CH3OH); ir: 1710 cm-1; 1H nmr d: 0.06 and 0.07 (2s, 6H, CH3SiCH33, 0.89 (s, 9H, 3CH3), 0.98 (s, 3H, CH3), 0.98 (d, J = 6.4 Hz, 3H, CHCH3), 1.32 (s, 3H, CH3), 1.57 (ddd, J = 13.4, 13.2, 2.7 Hz, H-5ax), 1.65 (dd, J = 14.2, 3.4 Hz, 1H, H-3ax), 1.88 (ddd, J = 14.2, 3.3, 3.2 Hz, 1H, H-3eq), 1.99 (dddd, J = 13.0, 5.3, 3.3, 3.2 Hz, 1H, H-5eq), 3.16 (ddq, J = 13.2, 5.3, 6.4 Hz, 1H, H-6ax), 4.08 (q, J = 3.2 Hz, 1H, H-4eq); eims m/z: 255 (M+-15, 1), 213 (60), 171 (78), 121 (43), 75 (100); hrms: calc. for C15H30O2Si 270.2015, found 270.2015.
Example 11 (-)-1(Z)-(1S, 4R, 6R)- and (-)-1(Z)-(1R, 4R, 6R)-1- (5-Acetoxy-3-methylpent-3-en-1-ynyl) -2, 2, 6-tri- methylcyclohexan-1,4-diol (6 and 7).
A solution of Z-3-methylpent-2-en-4-yn-1-ol (Fluka, 1.60 g, 16.7 mmol) in dry THF (20 mL) under an argon atmosphere was cooled to about -60°C in a dry ice-acetone bath. n-Butyllithium (Aldrich, 1.6 M in hexane, 19 mL, 30.4 mmol) was added dropwise with stirring. After all the n-butyllithium had been added the reaction mixture, which was orange in color, was allowed to warm up to -5°C over 30 min. Then it was again cooled to -60°C and a solution of trans (-)-(4R,6R)-4-t-Butyldi- methylsilyloxy-2,2,6-trimethylcyclohexanone (2.7 g, 10 mmol) in dry THF (20 mL) was added dropwise. After the addition had been completed the reaction mixture was allowed to warm up to 0°C over 90 min. before it was poured into water and extracted three times with ether. The combined organic extracts were washed twice with saturated NaCl and dried over anhydrous Na2SO4. Evaporation of solvent gave a yellow oil (4.5 g) as the crude product. Purification of the product by flash column chromatography (75% ether + 25 % hexane as eluent) followed by distillation (Kugel-rohr, 180-200°C, 0.03 mmHg) gave a mixture of compounds as a yellow oil (2.82 g, 76% yield), gc retention times (DB1701 column, 70-240°C at 10°C min-1) 20.05 min. and 19.77 min., ratio of peak areas about 8:1, respectively.
The mixture (2.82 g, 7.6 mmol) was dissolved in pyridine (15 mL). A mixture of acetic anhydride (2.35 g,
23.0 mmol) and pyridine (5 mL) was added, followed by
4-dimethylaminopyridine (Aldrich, 32 mg, 0.26 mmol). The reaction mixture was stirred at room temperature for 1 h before it was worked up by pouring into water and extracting three times with hexane. The combined organic extract was washed with saturated NaCl and dried over anhydrous Na2SO4. Removal of solvent gave a mixture of acetates as a pale yellow oil (3.72g) which was desilated to give two hydroxyacetates by the procedure below without purification. GC analysis of the crude acetate mixture
(DB1701tm column, 70-240°C at 10°C min-1) showed two components in the ratio of 8:1 (retention times 20.26 min and 20.12 min, respectively). The crude acetates obtained in the above procedure was stirred with glacial acetic acid (30 mL) and H2O (10 mL), and the mixture was heated to 70°C under argon for 20 h. After cooling to room temperature, the reaction mixture was diluted with water and extracted three times with CHCl3. The organic extract was washed with H2O, saturated NaHC03 and dried over anhydrous Na2SO4. Evaporation of solvent gave a yellow oil (2.98 g) as the crude product. Separation by flash column chromatography. (75% ether + 25% hexane) followed by preparative tic (same eluent) gave the cis diol (1.26 g, 56% overall yield) and trans diol (0.15 g, 7% overall yield). (-)-1(Z)-(IS, 4R,6R)-1-(5-acetoxy-3-methylpent- 3-en-1-ynyl)-2,2,6-trimethylcyclohexan-1,4-diol, colorless oil; gc (DB5 column, 70-240°C at 10°C min-1) retention time 18.37 min.; tic (90% ether + 10% hexane) Rf about 0.30; [a]D -18.4°C (C 1.02, CH3OH); ir: 3610, 3400, 1735 cm-1; 1Hnmr d: 1.05 (d, J = 6.6 Hz, CHCH3), 1.08 and 1.22 (2s, 6H, 2CH3), 1.55-1.75 (m, 4H, 2CH2), 1.89 (m, 3H, Vinyl CH3), 2.03 (s, 3H, CH3COO), 2.34 (m, 1H, CHCH3), 4.02 (m, 1H, CHOH), 4.71 (dd, J = 7.0, 1.0 Hz, 2H, CH2O), 5.77 (ddq, J = 7.0, 7.0, 1.5 Hz, 1H, =CH); 13C nmr d: 16.05, 20.88, 23.08, 23.28 and 27.39 (5 CH3), 38.77 (C2), 31.92, 40.12 and 44.47 (CH2 and CH), 62.80 and 67.55 (CH2O and CHOH), 79.01 (COH), 84.89 and 95.33 (2 acetylenic C), 123.52 (=C), 130.03 (=CH), 170.83 (C=0); eims m/z: 234 (M+-60, 6), 178 (28), 148 (100).
(-)-1(Z)-(1R, 4R,6R)-1-(5-acetoxy-3-methylpent- 3-en-1-ynyl)-2,2,6-trimethylcyclohexan-1,4-diol , colorless oil; gc (DB5 column, 70-240°C at 10°C min-1) retention time 18.50 min.; tic (90% ether + 10% hexane)
Rf about 0.35; [a]D -28.3°C (c 0.92, CH3OH); ir: 3630, 3500, 1735 cm-1; 1H nmr d: 1.05 (s, 3H, CH3), 1.06 (d, J = 5.2 Hz, 3H, CHCH3), 1.27, (s, 3H, CH3), 1.43 (ddd, J = 14.6, 2.5, 2.5 Hz, 1H, H-3eq), 1.52 (dddd, J = 14.3, 3.9, 2.5, 2.5 Hz, 1H, H-5eq), 1.68 (ddd, J = 14.3, 12.6, 3.3 Hz, 1H, H-5ax), 1.76 (dd, J = 14.6, 3.5 Hz, 1H, H-3ax), 1.89 (m, 3H, vinyl CH3), 2.03 (S, 3H, CH3COO), 2.33 (m, 1H, CHCH3), 4.07 (m, 1H, CHOH), 4.72 (dd, J = 7.0, 1.0 Hz, 2H, CH2O), 5.76 (ddq, J = 7.0, 7.0, 1.5 Hz, 1H, =CH); 13C nmr d: 16.81, 20.91, 23.21, 26.78 and 27.13 (5 CH3), 38.17 (C2), 30.77, 35.75 and 40.29 (2 CH2 and CH), 62.79 and 67.04 (CH2O and CHOH), 76.53 (COH), 83.52 and 97.37 (2 acetylenic C), 123.62 (=C), 129.99 (=CH), 170.86 (C=O); eims m/z: 234 (M+-60, 3), 178 (10), 148 (100).
Example 12
(-)-4(Z)-(4R,5R)-4-Hydroxy-4-(5-hydroxy-3-methylpent-3- en-1-ynyl)-3,3,5-trimethylcyclohexanone (8).
(Z)-(1R,4R,6R)-1-(5-Acetoxy-3-methylpent-3-en- 1-ynyl)-2,2,6-trimethylcyclohexan-1,4-diol (140 mg, 0.47 mmol) was oxidized with pyridinium dichromate (850 mg, 2.26 mmol) in CH2Cl2 (15 mL) to give a keto-acetate as a colorless oil (90 mg, 70%), [a]D -26.0°C (c 0.78, CH3OH); ir: 3630, 1730 cm-1; 1H nmr d: 1.03 (s, 3H, CH3), 1.17 (d, J = 6.3 Hz, 3H, CHCH3), 1.19 (s, 3H, CH3), 1.89 (m, 3H, vinyl CH3), 1.95 (dd, J = 13.8, 2.3 Hz, 1H, H-2eq), 2.04 (S, 3H, CH3COO), 2.16 (ddd, J = 13.2, 3.8, 2.3 Hz, 1H, H-6eq), 2.31 (ddq, J = 12.4, 6.3, 3.8 Hz, 1H, CHCH3), 2.39 (dd, J = 13.2, 12.4 Hz, 1H, H-6ax), 2.73 (broad d, J = 13.8 Hz, 1H, H-2ax), 4.73 (dd, J = 7.0, 0.9 Hz, 2H, CH2O), 5.81 (ddq, J = 7.0, 7.0, 1.5 Hz, 1H, =CH); ms m/e: 232 (M+-60, 24), 176 (47), 148 (41), 120 (23), 106 (100); hrms (M+-60 peak): calc. for C15H20O2 232.1463, found 232.1485.
The keto-acetate (90 mg, 0.3 mmol) was hydrolyzed by treating with 5M KOH (5 drops) and methanol (10 mL) to give -)-4(Z)-(4R,5R)-4-Hydroxy-4-(5-hydroxy- 3-methylpent-3-en-1-ynyl)-3,3,5-trimethylcyclo-hexanone as a colorless oil (69 mg, 92%) {(the racemate crystallized on storage at -10°C to give colorless crystals, mp72.5-79.5°C}, ir: 3630, 1710 cm-1; 1H nmr d: 1.03 (s, 3H, CH3), 1.17 (d, J = 6.4 Hz, 3H, CHCH3), 1.19 (s, 3H, CH3), 1.88 (m, 3H, vinyl CH3), 1.96 (dd, J = 13.8, 2.3 Hz, 1H, H-2eq), 2.16 (ddd, J = 13.3, 3.4, 2.3 Hz, 1H, H-6eq), 2.32 (m, 1H, CHCH3), 2.39 (ddd, J = 13.3, 12.8, 0.8 Hz, 1H, H-6ax), 2.72 (ddd, J = 13.8, 0.8, 0.8 Hz, 1H, H-2ax), 4.29 (dd, J = 6.8, 1.0 Hz, 2H, CH2OH), 5.88 (ddq, J = 6.8, 6.8, 1.5 Hz, 1H, =CH); 13C nmr d: 16.76, 23.13, 24.98 and 25.41 (4q, 4CH3), 37.64 (d, CH), 43.06 (s, C3), 44.25, 49.48 and 61.27 (3t, 3CH2), 75.25 (s, COH), 84.47 and 94.85 (2s, 2 acetylenic C), 120.26 (s, =C), 136.08 (d, =CH), 210.86 (s, C=0); ms m/e: 250 (M+, very weak), 232 (5), 179 (23), 165 (66), 106 (100); hrms: calc. for C15H2203 250.1569, found 250.1570. Example 13
(-)-(9Z)-(9S,10R)-9-(5-Hydroxy-3-methylpent-3-en-1-ynyl)- 3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]undecan-9-ol (9).
A mixture of (-)-4(Z)-(4S,5R)-4-hydroxy-4-(5- hydroxy-3-methylpent-3-en-1-ynyl)-3,3,5-trimethylcyclo- hexanone (620 mg, 2.49 mmol), 2,2-dimethyl-1,3-propanediol (Aldrich, 460 mg, 4.4 mmol), pyridinium p-tosylate (Aldrich, 19 mg, 0.07 mmol) and benzene (18 mL) was heated to reflux under a Dean-Stark separator for 4 hrs. The reaction mixture was then allowed to cool to room temperature, washed with saturated Na2CO3, saturated NaCl and water. After drying over anhydrous Na2SO4 and evaporation of solvent a yellow oil was obtained as the crude product (1 g). Purification by flash column chromatography (75% ether + 25% hexane) gave the desired ketal as a pale yellow oil (750 mg, 90%), [a]D -29.4°C (c 1.02, CH3OH); ir: 3610, 3440, 1110 and 1090 cm-1; 1H nmr d: 0.83, 1.04, 1.05, 1.06, 1.09, and 1.11 (15H, 5 CH3), 1.53-1.61 (m, 2H, 2 axial H at C7 and C11), 1.84 (m, 3H, vinyl CH3), 1.98 (ddd, J = 13.8, 3.4, 3.4 Hz, 1H, H-11eq), 2.18 (m, 1H, CH), 2.51 (dd. J = 14.3, 3.2 Hz, 1H, H-7eq), 3.36 and 3.38 (2dd, J = 11.3, 1.9 Hz, 2H, 2 equatorial H at C2 and C4), 3.55 (d, J = 11.3 Hz, 2H, 2 axial H at C2 and C4), 4.29 (broad s, 2H, CH2OH), 5.50 (ddq, J = 6.7, 6.7, 1.5 Hz, 1H, =CH); eims (trimethylsilyl ether) m/z: 408 (M+ of trimethylsilyl ether), 155 (100); hrms (trimethylsilyl ether): calc. for C23H4004Si 408.2696, found 408.2718.
Example 14
(-)-9(1E,3Z)-(9R,10R)-9-(5-Hydroxy-3-methy1-1,3- pentadienyl)-3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]- undecan-9-ol (10).
A solution of (-)-(9Z)-(9S,10R)-9-(5-hydroxy-3- methylpent-3-en-1-ynyl)-3,3,8,8,10-pentamethyl-1,5- dioxaspiro[5,5]undecan-9-ol (730 mg, 2.2 mmol) in dry THF (50 mL) was stirred under an argon atmosphere and cooled with an ice-water bath. Sodium is(2-methoxyethoxy)- aluminium hydride (RedalR, Aldrich, 3.4M in toluene, 1.3 mL, 4.4 mmol) was added dropwise. Some frothing occurred as the RedalR was added. The reaction mixture was stirred at 0°C until the frothing subsided. Then another portion of RedalR (0.7 mL, 2.2 mmol) was added. After 1.5 h of stirring at 0°C followed by 1 h at room temperature, the reaction was worked up by pouring into H2O and extracting three times with ether. The combined organic extracts were washed with saturated NaCl and dried over anhydrous Na2SO4. Evaporation of solvent gave a colorless oil (about 1 g) as the crude product which was usually hydrolyzed by the procedure described in Example 15. A small amount of the crude product was purified on the Chromatotron (75% ether + 25% hexane) to give the desired ketal as a colorless oil, [a]D -64.4°C (c 1.02, CH3OH); ir: 3600, 1600, 1100, 975, 910 cm-1; 1H nmr d: 0.76, 0.78, 0.85, 1.05 and 1.12 (15H, 5 CH3), 1.34-1.42 (m, 2H, 2 axial H at C7 and C11), 1.85 (d, J = 0.9 Hz, 3H, vinyl CH3), 1.97 (ddd, J = 14.0, 3.5, 3.5 Hz, 1H, H-11eq), 2.16 (m, 1H, CH), 2.29 (dd, J = 14.6, 3.2 Hz, 1H, H-7eq), 3.40 (m, 2H, 2 equatorial H at C2 and C4), 3.57 and 3.58 (2d, J = 11.2, 11.4 Hz, respectively, 2H, 2 axial H at C2 and C4), 4.31 (d, J = 6.9 Hz, 2H, CH2OH) , 5.54 (t, J = 7 Hz, 1H, =CH), 5.94 (d, J = 15.6 Hz, 1H, =CH) , 6.68 (d, J = 15.6 Hz, 1H, =CH).
Example 15 (-)-4(1E,3Z)-(4R,5R)-4-Hydroxy-4-(5-hydroxy-3-methyl- 1,3-pentadienyl)-3,3,5-trimethylcyclohexanone (11).
The crude ketal obtained in the procedure described in Example 14 was hydrolyzed by stirring with 1M HCl (5 drops) and acetone (50 mL) at room temperature for 2 h. After concentration of the acetone solution, saturated NaHCO3 was added and the mixture was extracted with ether. The organic layer was dried with anhydrous Na2SO4 and concentrated to give a yellow oil as the crude product. Flash column chromatography using 90% ether + 10% hexane as eluent gave the desired compound as a colorless oil (338 mg, 60%) {The racemate crystallized on standing at room temperature and could be recrystallized from ether-hexane to give colorless crystals, mp 128°C}, [a]D -41.6°C (c 0.98, CH3OH); ir: 3610 , 3450, 1710, 1610, 975 cm-1; 1H nmr d: 0.84 (d, J = 6.3 Hz, CHCH3), 0.89 and 1.01 (2s, 6H, CH3), 1.88 (d, J = 0.8 Hz, 3H, vinyl CH3), 2.1-2.3 (m, 4H, H-2eq, CH2 at C6 and CHCH3), 2.46 (d, J = 15.8 Hz, 1H, H-2ax), 4.31 (d, J = 7.0 Hz, 2H, CH2OH), 5.59 (t, J = 7.0 Hz, 1H, =CH), 6.07 (d, J = 15.6 Hz, 1H, =CH), 6.81 (dd, J = 15.6, 0.4 Hz, 1H, =CH); 13C nmr d: 15.91, 20.78, 22.78 and 25.18 (4q, 4 CH3), 37.39 (d, CH), 41.64 (s, C3), 47.08, 52.88 and 58.34 (3t, 3CH2), 78.12 (s, COH), 128.36, 128.64 and 128.96 (3d, 3 =CH), 134.40 (s, =C), 209.55 (s, C=O); eims (trimethylsilyl ether) m/e: 324 (M+ of trimethylsilyl ether, 3), 73 (100); hrms (trimethylsilyl ether) : calc. for C18H32O3Si 324.2121, found 324.2109.
Example 16
(-)-4(1E,3Z)-(4R,5R)-4-Hydroxy-4-(5-oxo-3-methyl-1,3- pentadienyl)-3,3,5-trimethylcyclohexanone (12).
To a solution of ketoalcohol as described in Example 15 (300 mg, 1.2 mmol) in acetone (30 mL) was added manganese oxide (2.08 g, 24 mmol). The mixture was stirred at room temperature under a drying tube for 1 h before the manganese oxide was removed by filtration. The solid residue was rinsed with acetone and the rinsing was combined with the filtrate. Evaporation of acetone gave the crude aldehyde which was usually oxidized to ester without purification as described in Example 17. A small amount of aldehyde was purified by preparative tic (90% ether + 10% hexane) followed by recrystallization from ether-hexane to give colorless crystals, mp 106.0-108.5°C; [a]D -64.5 (c 0.38, CH3OH); ir: 3550, 1715.and 1665 cm-1; 1H nmr d: 0.89 (d, J = 6.4 Hz, 3H, CHCH3), 0.93 and 1.04 (2s, 6H, 2CH3), 2.11 (d, J = 1.1 Hz, 3H, vinyl CH3) , 2.15-2.42 (m, CH2 at C6 and CHCH3), 2.17 (dd, J = 14.9, 2.5 Hz, H-2eq), 2.48 (d, J = 14.9 Hz, 1H, H-2ax), 5.90 (d, J = 7.7 Hz, 1H, =CH), 6.49 and 7.50 (2d, J = 15.4 Hz, 2H, 2 =CH), 10.21 (d, J = 8.0 Hz, 1H, HC=O); high resolution ms: calc. for C15H22O3 250.1569, found 250.1575.
Example 17
(-)-(4R, 5R)-Methyl 2',3'-dihydroabscisate (13).
Crude aldehyde as described in Example 16 (about 1.2 mmol) obtained from the procedure described in Example
16 was dissolved in methanol (30 mL). Manganese oxide
(1.79 g, 20 mmol), sodium cyanide (87 mg, 1.7 mmol) and glacial acetic acid (0.10 g, 1.6 mmol) were added. The mixture was stirred at room temperature for 5 h before it was filtered through CeliteR. The manganese oxide residue and Celite were rinsed with methanol and the rinsing was combined with the filtrate. The combined rinsing and filtrate was then concentrated, and the residue was partitioned between ether and H2O. The ether layer was separated, dried over anhydrous Na2SO4, and concentrated to give a yellow oil (283 mg) as crude product.
Purification on the Chromatotrontm (4 mm silica gel plate, 90% ether + 10% hexane) gave (-)-(4R, 5R)-methyl dihydroabscisate as colorless crystals (199 mg, 70% overall yield). Part of the product was recrystallized from ether-hexane to give colorless needles, mp 117.0-119.5°C; [a]D -62.7°C (c 0.90, CH3OH); hplc {Chiracel OD column (18, 19), 10% isopropanol + 90% hexane at 1.0 mL min-1} retention time 10.7 min.; ir: 3600, 1710 cm-1; 1H nmr d: 0.88 (d, J = 6.3 Hz, 3H, CHCH3), 0.92 and
1.04 (2S, 6H, 2 CH3), 2.03 (d, J = 1.2 Hz, 3H, vinyl CH3), 2.15 (dd, J = 16.0, 15.0 Hz, H-6ax), 2.15 (dd, J = 15.0,
2.5 Hz, H-2eq), 2.30-2.40 (m, 2H, CHCH3 and H-6eq), 2.46 (d, J = 14.9 Hz, 1H, H-2ax), 3.69 (s, 3H, OCH3), 5.74 (s, 1H, =CH), 6.46 (d, J = 16.0 Hz, 1H, =CH), 7.91 (dd, J = 16.0, 0.6 Hz, 1H,=CH); 13C nmr d: 16.00, 21.28, 22.87 and 25.25 (4q, 4 CH3), 37.40 (d, CH), 41.71 (s, C3), 47.10 (t, CH2), 51.10 (q, CH3O), 52.92 (t, CH2), 78.14 (s, COH), 117.62, 129.41 and 135.15 (3d, 3 =CH), 149.41 (s, =C), 166.51 (s, OC=0), 209.12 (s, C=0); eims m/e: 280 (M+, 2), 192 (35), 164 (23), 123 (100); hrms: calc. for C16H24O4 280.1675, found 280.1664. Anal. calc. for C16H24O4: C 68.53%, H 8.63%; found: C 68.67%, H 8.74%.
Example 18
(-)-(4R, 5R)-2',3'-dihydroabscisic acid (14). A mixture of (-)-(4R, 5R)-methyl 2',3'- dihydroabscisate (166 mg, 0.6 mmol), 2M KOH (6 mL) and methanol (3 mL) was stirred at room temperature for 4 h. Most of the methanol was then evaporated. The residue was diluted with H2O, extracted with ether, and the ether extract was discarded. The aqueous layer was acidified with IM HCl and then extracted with CHCl3. The CHCl3 layer gave, after drying over anhydrous Na2SO4 and evaporation of solvent, (-)-(4R, 5R)-dihydroabscisic acid as white crystals (118 mg, 78%). Part of the product was recrystallized from CHCl3-hexane to give white crystals, mp 177-184°C {lit. mp of racemate 193.5°C}; [a]D -65.2°C (c 0.66, CH3OH); ir: 2800-3200, 1715, 1690 cm-1; 1H nmr d: 0.89 (d, J = 6.4 Hz, CHCH3), 0.93 and 1.06 (2s, 6H, 2 CH3), 2.08 (s, 3H, vinyl CH3), 2.14-2.43 (m, 4H, CHCH3, CH2 at C6 and H-2eq), 2.47 (d, J = 14.9 Hz, 1H, H-2ax), 5.77 (broad s, 1H, =CH), 6.50 and 7.88 (2d, J = 16.0 Hz, 2H, 2 =CH); eims (trimethylsilyl ether) m/z: 338 (M+, 2), 192 (30), 73 (100); hrms (trimethylsilyl ether): calc. for C18H30O4Si 338.1913, found 338.1921. Anal. calc. for C15H22O4: C 67.63%, H 8.33 %; found C 67.21 %, H 8.30 %.
Example 19
(-)-(9Z)-(9R,10R)-9-(5-Hydroxy-3-methylpent-3-en-1-ynyl) -3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]undecan-9-ol (15).
(-)-4(Z)-(4R,5R)-4-Hydroxy-4-(5-hydroxy-3- methylpent-3-en-1-ynyl)-3,3,5-trimethylcyclohexanone (130 mg, 0.52 mmol) was treated with a mixture of 2,2-dimethyl -1,3-propanediol (160 mg, 1.53 mmol), pyridinium p-tosylate (9 mg, 0.04 mmol) and benzene (4 mL) according to the procedure described in Example 13. The desired ketal was obtained as a pale yellow oil (149 mg, 89%), [a]D -27.8°C (c 1.2, CH3OH); ir: 3610, 3440, 1110 and 1090 cm-1; 1H nmr d: 0.84 (s), 1.04 (s), 1.08 (s), 1.09 (d, J = 6.8 Hz), and 1.16 (s) (15H, 5 CH3), 1.52 (d, J = 14.1 Hz, 1H, H-7ax), 1.58 (dd, J = 13.4, 13.2 Hz, 1H, H-1lax), 1.83 (ddd, J = 13.4, 3.7, 2.9 Hz, 1H, H-11eq), 1.87 (m, J = 1Hz, 3H, vinyl CH3), 2.11 (dd. J = 14.1, 2.8 Hz, 1H, H-7eq), 2.13-2.22 (m, 1H, CHCH3), 3.35-3.41 (m, 2H, 2 equatorial H at C2 and C4), 3.58 and 3.54 (2d, J = 11.9 and 12.6 Hz, respectively, 2H, 2 axial H at C2 and C4), 4.29 (d, J = 6.2 Hz, 2H, CH2OH), 5.84 (ddq, J = 6.7, 6.7, 1.5 Hz, 1H, =CH); hrms (M+-18 peak): calc. for C20H3003 318.2195, found 318.2188.
Example 20
(-)-9(1E, 3Z)-(9S,10R)-9-(5-Hydroxy-3-methyl-1,3- pentadienyl)-3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]- undecan-9-ol (16).
(-)-(9Z)-(9R,10R)-9-(5-Hydroxy-3-methylpent- 3-en-1-ynyl)-3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]- undecan-9-ol (149 mg, 0.44 mmol) was reduced with sodium bis (2-methoxyethoxy) aluminium hydride (RedalR, Aldrich, 3.4M in toluene, 0.8 mL, 2.48 mmol) by the procedure described in Example 14. The crude product obtained was usually hydrolyzed to give the ketodiol without purification. A small amount of the crude product was purified on the Chromatotron (75% ether + 25% hexane) to give the desired ketal as a colorless oil, ir: 3600, 1600, 1100, 975, 910 cm-1; 1H nmr d: 0.76 (d, J = 6.9 Hz), 0.83(s), 0.84 (s), 1.02 (s) and 1.06 (s) (15H, 5 CH3), 1.57 (d, J = 14.1 Hz, 1H, H-7ax), 1.59 (dd, J = 13.0, 12.7 Hz, 1H, H-11ax), 1.84 (ddd, J = 12.7, 3.6, 2.7 Hz, 1H, H-11eq), 1.85 (d, J = 0.9 Hz, 3H, vinyl CH3), 2.08 (dd, J = 14.1, 2.7 Hz, 1H, H-7eq), 2.16 (ddq, J = 13.0, 3.6, 6.8 Hz, 1H, CHCH3), 3.30-3.50 (m, 2H, 2 equatorial H at C2 and C4), 3.56 and 3.60 (2d, J = 11.7, 11.5 Hz, respectively, 2H, 2 axial H at C2 and C4), 4.30 (d, J = 7.0 Hz, 2H, CH2OH), 5.54 (dd, J = 7.0, 6.8 Hz, 1H, =CH), 5.70 (d, J = 15.8 Hz, 1H, =CH), 6.60 (d, J = 15.8 Hz, 1H, =CH). Example 21
(-)-4(1E, 3Z)-(4S,5R)-4-Hydroxy-4-(5-hydroxy-3-methyl- 1,3-pentadienyl)-3,3,5-trimethylcyclohexanone (17).
The crude ketal obtained in the procedure described in Example 20 was hydrolyzed by stirring with 1M HCl (4 drops) and acetone (5 mL) at room temperature for 1 h. Working up in the usual manner followed by purification on the Chromatotrontm (4mm silica gel plate, 90% ether + 10% hexane) gave the desired compound as a colorless oil (33 mg, 30% overall), [a]D -18.3°C (c 1.1, CH3OH); ir: 3620, 1700 cm-1; 1H nmr d: 0.86 (d, J = 6.5 Hz, CHCH3), 0.88 and 0.93 (2s, 6H, CH3), 1.86 (d, J = 1.1 Hz, 3H, vinyl CH3), 1.90 (dd, J = 13.6, 2.2 Hz, 1H, H-2eq), 2.18 (ddd, J = 13.5, 4.3, 2.2 Hz, 1H, H-6eq), 2.23-2.33 (m, 1H, CHCH3), 2.41 (t, J = 13.5 Hz, 1H, H-6ax), 2.82 (d, J = 13.6 Hz, H-2ax), 4.31 (m, 2H, CH2OH), 5.59 (dd, J = 7.0, 6.3 Hz, 1H, =CH), 5.73 (d, J = 15.7 Hz, 1H, =CH), 6.69 (d, J = 15.7 Hz, 1H, =CH); 13C nmr d: 16.03, 20.72, 24.51 and 24.59 (4q, 4 CH3), 36.83 (d, CH), 42.85 (s, C3), 45.11, 51.46 and 58.34 (3t, 3 CH2), 77.78 (s, COH), 126.70, 128.28 and 134.02 (3d, 3 =CH), 134.51 (s, =C), 211.45 (s, C=O); hrms: calc. for C15H24O3 252.1725, found 252.1708.
Example 22
(-)-4(1E,3Z)-(4S,5R)-4-Hydroxy-4-(5-oxo-3-methyl-1,3- pentadienyl)-3,3,5-trimethylcyclohexanone (18).
The ketodiol 17 (30 mg, 0.11 mmol) was oxidized with manganese oxide (420 mg, 4.8 mmol) to give the corresponding aldehyde by the procedure described in Example 17. The crude aldehyde was usually oxidized to ester as described in Example 23 without purification.
A small amount of the aldehyde 18 was purified by preparative tic (90% ether + 10% hexane) to give a colorless oil, [a]D -39.5 (c 0.77, CH3OH); ir: 3610, 3450, 1700 and 1665 cm-1; 1H nmr d: 0.89 (d, J = 6.4 Hz, 3H, CHCH3), 0.96 (s, 6H, 2CH3), 1.93 (dd, J = 13.6, 2.2 Hz, H-2eq), 2.08 (d, J = 1.1 Hz, 3H, vinyl CH3), 2.20-2.46 (m, CH2 at C6 and CHCH3), 2.84 (d, J = 13.6 Hz, 1H, H-2ax), 5.90 (d, J = 7.8 Hz, 1H, =CH), 6.16 (dd, J = 15.47, 0.4 Hz, 1H, =CH), 7.40 (d, J = 15.7 Hz , 1H, =CH), 10.20 (d, J = 7.8 Hz, 1H, HC=O).
Example 23
(-)-(4S, 5R)-2',3'-Methyl dihydroabscisate (19).
Crude (-)-4(1E, 3ZZ-(4S,5R)-4-Hydroxy-4-(5-oxo-
3-methyl-1,3-pentadienyl)-3,3,5-trimethylcyclohexanone (about 0.11 mmol) obtained from the procedure described in Example 22 was reacted with a mixture of methanol (5 mL), manganese oxide (310 mg, 3.56 mmol), sodium cyanide (35 mg, 0.71 mmol) and glacial acetic acid (35 mg, 0.58 mmol). The desired product (-)-(4S, 5R)-methyl dihydroabscisate was obtained as colorless crystals (21 mg, 70% overall yield). Part of the product was recrystallized from ether-hexane at 0°C to give colorless plates, mp 105-108°C; [a]D -40.6°C (c 1.03, CH3OH); ir: 1700 cm-1; 1H nmr d: 0.87 (d, J = 6.5 Hz, 3H, CHCH3), 0.94 and 0.96 (2S, 6H, 2 CH3), 1.91 (dd, J = 13.6, 2.2 Hz, 1H, H-2eq), 2.01 (d, J = 1.2 Hz, 3H, vinyl CH3), 2.20 (ddd, J = 13.6, 4.2, 2.2 Hz, H-6eq), 2.33 (m, 1H, CHCH3), 2.44 (dd, J = 13.6, 13.0 Hz, 1H, H-6ax), 2.86 (d, J = 13.6 Hz, 1H, H-2ax), 3.70 (s, 3H, OCH3), 5.72 (s, 1H, =CH), 6.09 (d, J = 15.9 Hz, 1H, =CH), 7.81 (d, J = 15.9 Hz, 1H, =CH); eims m/z: 280 (M+, 4), 192 (43), 164 (24), 123 (100); hrms: calc. for C16H24O4 280.1675, found 280.1649.
Example 24
(-)-(4S, 5R)-2',3'-dihydroabscisic acid (20)
(-)-(4S, 5R)-Methyl dihydroabscisate (15 mg, 0.05 mmol) was hydrolysed with 2M KOH (3 mL) and methanol (3 mL) to give (-)-(4S,5R)-dihydro- abscisic acid as a colorless oil (13 mg, 90%), [a]D -34.5°C (c 0.89, CH3OH); ir: 2800-3500, 1680 cm-1; 1H nmr d: 0.88 (d, J = 6.4 Hz, CHCH3), 0.95 and 0.96 (2s, 6H, 2 CH3), 1.91 (dd, J = 13.6, 2.1 Hz, 1H, H-2eq), 2.04 (d, J = 1.2 Hz, 3H, vinyl CH3), 2.21 (ddd, J = 13.4, 4.1, 2.1 Hz, H-6eq), 2.35 (m, 1H, CHCH3), 2.44 (dd, J = 13.4, 12.7 Hz, 1H, H-6ax), 2.86 (d, J = 13.6 Hz, 1H, H-2ax), 5.75 (s, 1H, =CH), 6.14 and 7.79 (2d, J = 16.1 Hz, 2H, 2 =CH); 13C nmr: 16.05, 21.48, 24.58 and 24.63 (4 CH3), 43.00 (C3), 36.67, 45.03 and 51.42 (CH and 2 CH2), 78.00 (COH), 151.77, 140.60, 127.72 and 116.99 (4 =C), 170.63 (COOH), 211.26 (C=O); eimsm/z: 266 (M+, about 2), 248 (5), 192 (29), 164 (67), 123 (100); hrms: calc. for C15H22O4 266.1518, found 266.1519.
Example 25
(+)-(4R, 6S)-4-Hydroxy-2,2,6-trimethylcyclohexanone (21)
A mixture of (-)-(4R,6R)-4-hydroxy-2,2,6- trimethylcyclohexanone (300 mg, 0.19 mmol), 5M NaOH (1 mL) and ethanol (10 mL) was heated to 85°C for 24 hrs under an argon atmosphere. Then most of the ethanol was removed by evaporation. The residue was dissolved in ether, and the solution was washed with water. After drying over anhydrous Na2SO4 and evaporation of the solvent, a pale yellow oil (255 mg) was obtained. Purification by flash chromatography (75% ether + 25% hexane) gave unreacted trans ketol (46 mg), [a]D -102.3°C (c 0.92, CH3OH), followed by the desired cis ketol as a colorless oil (195 mg, 73% based on starting material consumed) . The product solidified on storage at -10°C and was recrystallized from ether-hexane to give colorless needles, mp 48.0-50.0°C {lit. (13) mp 52-53°C}; [a]D +95.0°C (c 0.88, CH30H) {lit. (13) [a]D +107.4°C (c 0.8, CH30H) } ; 1H nmr d: 0.96 (d, J = 6.5 Hz, 3H, CHCH3), 1.00 and 1.14 (2s, 3H each, 2 CH3), 1.35 (ddd, J= 12.4, 12.4, 11.3 Hz, 1H, H-5ax), 1.53 (dd, J = 12.2, 11.5 Hz, 1H, H-3ax), 2.00 (ddd, J = 12.2, 4.2, 3.5 Hz, H-3eq), 2.22 (m, 1H, H-5eq), 2.67 (m. 1H, H-6ax), 4.25 (tt, J = 11.3, 4.3 Hz, 1H, H-4ax); eims m/z: 156 (M+, 10), 138 (8), 83 (66), 74 (50), 69 (60), 57 (100).
Example 26 (+)-(4R,6S)-4-t-Butyldimethylsilyloxy-2,2,6-trimethyl-cy- clo hexanone (22).
The cis ketol obtained in Example 25 (66 mg, 0.42 mmol) was treated with a mixture of t-butyldimethylsilyl chloride (130 mg, 0.86 mmol), imidazole (73 mg, 1.07 mmol) and dry DMF (2 mL). After working up and purification by distillation (kugel-rohr, 150-180°C, 8-10 mm Hg), the desired silyl ether was obtained as a colorless oil (111 mg, 99%); [a]D +58.0°C (c 0.98, CH3OH); ir: 1710 cm-1; 1H nmr d: 0.07 (s, 6H, CH3SiCH3), 0.87 (s, 9H, 3 CH3), 0.98 (d, J = 6.5 Hz, 3H, CHCH3), 1.03 and 1.17 (2s, 3H each, 2 CH3), 1.43 (ddd, J = 13.6, 12.8, 11.0 Hz, 1H, H-5ax), 1.58 (dd, J = 13.1, 11.1 Hz, 1H, H-3ax), 1.89 (ddd, J = 13.1, 4.3, 3.5 Hz, 1H, H-3eq), 2.11 (m, 1H, H-5eq), 2.68 (ddq, J = 12.8, 6.5, 6.5 Hz, 1H, H-6ax), 4.24 (tt, J = 11.0, 4.4 Hz, 1H, H-4ax); cims (isobutane) m/e: 271 (M++1); hrms: calc. for C15H3002Si 270.2015, found 270.2004. Example 27
(+)-1(Z)-(1R, 4R,6S)-4-t-Butyldimethylsilyloxy-1-(5- hydroxy-3-methylpent-3-en-1-ynyl)-2,2,6-trimethyl- cyclohexanol (23).
The ketosilyl ether from Example 26 (5.7 g, 21 mmol) was treated with Z-3-methylpent-2-en-4-yn-1-ol (3.0 g, 32 mmol) and n-butyllithium (1.6 M in hexane, 40 mL, 63 mmol). The desired product was obtained as a colorless oil (4.9 g, 64%) which solidified on storage at 0°C to give colorless crystals, gc (DB1701 column, 70-240°C at 10°C min-1) retention time 19.11 min.; mp 89-94°C; [a]D +19.1°C (c 0.82, CH3OH); ir: 3620 cm-1; 1H nmr d: 0.03 (s, 6H, CH3SiCH3), 0.86 (s, 9H, 3 CH3), 1.01 (s, 3H, CH3), 1.05 (d, J = 6.5 Hz, 3H, CHCH3), 1.11 (s, 3H, CH3), 1.40, 1.55 and 1.73 (3m, 2 CH2), 1.89 (d, J = 1.0 Hz, 3H, vinyl CH3), 1.92-2.00 (m, 1H, CHCH3), 3.83 (tt, J = 10.8, 5.2 Hz, 1H, CHOSi), 4.32 (m, 2H, CH2OH), 5.86 (ddd, J = 6.7, 6.7, 1.5 Hz, 1H, =CH); cims (isobutane) m/e: 367 (M++1), 349.
Example 28
(+)-1(Z)-(1R,4R,6S)-1-(5-Acetoxy-3-methylpent-3-en-1- ynyl)- 2,2,6-trimethylcyclohexan-1,4-diol (24). The dihydroxysilyl ether described in Example 27
(2.5 g, 6.9 mmol) was reacted with acetic anhydride (2.5 g, 18.6 mmol), pyridine (25 mL) and 4-dimethylamino- pyridine (32 mg, 0.2 mmol) by the procedure previously described. A small amount of the crude acetate obtained was purified on the Chromatotrontm (1mm silica gel plate, 50% ether + 50% hexane) to give a colorless oil, gc (DB1701 column, 70-240°C at 10°C min-1) retention time 19.37 min.; [a]D +17.6°C (c 1.07, CH3OH); ir: 3610, 3500, 1735 cm-1; 1H nmr d: 0.03 (s, 6H, CH3SiCH3), 0.86 (s, 9H, 3 CH3), 1.01 (s, 3H, CH3), 1.05 (d, J = 6.5 Hz, 3H, CHCH3), 1.11 (s, 3H, CH3), 1.37, 1.56 and 1.73 (3m, 2 CH2), 1.90 (d, J = 1.3 Hz, 3H, vinyl CH3), 1.96 (m, 1H, CHCH3), 2.03 (s, 3H, CH3C=O), 3.84 (tt, J = 11.0, 5.0 Hz, 1H, CHOSi), 4.74 (dd, J = 7.1, 0.9 Hz , CH2O), 5.79 (dd, J = 7.1, 7.1, 1.5 Hz, 1H, =CH); eims m/e: 348 (M+-60); cims (isobutane) m/e: 409 (M++1), 349. The crude acetate was desilated by heating
(80°C) with glacial acetic acid (30 mL) and water (10 mL) to give, after work up and purification, the desired product as a colorless oil (1.46 g, 73% overall yield), [a]D +23.8°C (c 0.4, CH3OH); ir: 3610, 1735 cm-1; 1H nmr d: 1.03 (s, 3H, CH3), 1.06 (d, J = 6.5 Hz, 3H, CHCH3), 1.12 (s, 3H, CH3), 1.37 and 1.57 (2m, H-3ax and CH2 at C5), 1.70 (ddd, J = 12.7, 4.6, 2.3 Hz, 1H, H-3eq), 1.91 (d, J = 1.3 Hz, vinyl CH3), 2.00 (m, 1H, CHCH3), 2.04 (s, 3H, CH3C=O), 3.84 (m, 1H, CHOH), 4.77 (d, J = 7.3 Hz, CH2O), 5.70 (ddd, J = 7.1, 7.1, 1.5 Hz, 1H, =CH); 13C nmr: 16.43, 20.73, 20.95, 23.17 and 26.96 (5 CH3), 39.92 (C2), 35.86, 41.76 and 46.38 (CH and 2 CH2), 62.89 and 66.10 (CHOH and CH2O), 78.31 (COH), 85.43 and 95.17 (2 acetylenic C), 123.68 (=C), 129.96 (=CH), 171.04 (C=O); eims m/e: 294 (M+, very weak), 234 (10), 148 (100).
Example 29
(+)-4(Z)-(4R,5S)-4-Hydroxy-4-(5-hydroxy-3-methylpent-3- en-1-ynyl)-3,3,5-trimethylcyclohexanone (25). The dihydroxy acetate obtained in Example 28
(1.44 g, 4.9 mmol) was oxidized with pyridinium dichromate (6.07 g, 16.1 mmol) in CH2C12 (30 mL). A keto-acetate was obtained as colorless needles (0.94 g, 70%), mp 118.0-120.0°C; [a]D +20.5°C (c 0.58, CH3OH); ir, 1H and 13C nmr identical with those of the (-)-enantiomer.
The keto-acetate (0.94 g, 3.2 mmol) was hydrolysed by stirring with 5M NaOH (1 mL) and methanol (25 mL) at room temperature for 1 h. After working up and purification, the desired product (+)-4(Z)-(4R,5S)-4- hydroxy-4-(5-hydroxy-3-methylpent-3-en-1-ynyl)-3,3,5- trimethylcyclohexanone was obtained as colorless crystals (0.80 g, 100%), mp 96.5-98.0°C; [a]D +22.3°C (c 0.53, CH3OH); ir, 1H and 13C nmr identical with those of the (-)-(4S, 5R) enantiomer .
Example 30
(+)-(9Z)-(9R,10S)-9-(5-Hydroxy-3-methylpent-3-en-1-ynyl)- 3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]undecan-9-ol (26).
(+)-4(Z)-(4R,5S)-4-Hydroxy-4-(5-hydroxy-3- methylpent-3-en-1-ynyl)-3,3,5-trimethylcyclohexanone was treated with 2,2-dimethylpropane-1,3-diol in benzene with a catalytic amount of p-toluenesulfonic acid to afford the ketal, [a]D +27.1°C (c 0.90, CH3OH).
Example 31
(+)-9(1E, 3Z)-(9S,10S)-9-(5-Hydroxy-3-methyl-1,3- pentadienyl)-3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]- undecan-9-ol and (+)-4(1E,3Z)-(4S,5S)-4-Hydroxy-4-(5- hydroxy-3-methyl-1,3-pentadienyl)-3,3,5-trimethylcyclo- hexanone (27).
Reduction of (+)-(9Z)-(9R,10S)-9-(5-Hydroxy-3- methylpent-3-en-1-ynyl)-3,3,8,8,10-pentamethyl-1,5- dioxaspiro[5,5]undecan-9-ol with RedalR as described in Example 14 afforded the dienoic system and the product was hydrolyzed with 1M HCl and acetone as previously described to give the desired product ketone, [a]D +42.6°C (c 1.03, CH3OH). Example 32
(+)-4-(1E,3Z)-(4S,5S)-4-Hydroxy-4-(5-oxo-3-methyl-1,3- pentadienyl)-3,3,5-trimethylcyclohexanone (29).
The aldehyde was prepared by the oxidation of
(+)-4(1E, 3Z)-(4S,5S)-4-Hydroxy-4-(5-hydroxy-3-methyl-1,3- pentadienyl)-3,3,5-trimethylcyclohexanone with manganese oxide and was further oxidized to (+)-(4S, 5S)-methyl 2',3'-dihydroabscisate without purification.
Example 33
(+)-(4S, 5S)-2',3'-Methyl dihydroabscisate (30).
The aldehyde obtained in Example 32 was reacted with MnO2, NaCN, methanol and glacial acetic acid according to the procedure previously described to give (+)-(4S, 5S) -methyl dihydroabscisate as colorless needles, mp 117.5-118.5°C; [a]D +65.7°C (c 0.9, CH3OH); hplc (Chiracel OD column, 10% isopropanol + 90% hexane at 1.0 mL min-1) retention time 8.7 min.
Example 34
(+)-(4S, 5S)-2',3'-Dihydroabscisic acid (31).
(+)-(4S, 5S)-methyl dihydroabscisate was hydrolyzed with 2M KOH and methanol to give (+)-(4S, 5S)-dihydroabscisic acid as colorless crystals, 173-180°C; [a]D +63.5°C (c 1.17, CH3OH). Example 35
(-)-(10R)-3,3,8,8,10-Pentamethyl-1,5-dioxaspiro[5,5]- undecan-9-one (32).
A mixture of (-)-(6R)-2,2,6-trimethyl-1,4- cyclohexandione (924 mg, 6.0 mmol), 2,2-dimethyl-1,3- propandiol (791 mg, 7.6 mmol), pyridinium p-tosylate (34 mg, 0.13 mmol), and benzene (15 mL) was heated to reflux under a Dean-Stark water separator for 4 h. The reaction mixture was allowed to cool to room temperature before it was washed with H2O, and dried over anhydrous Na2SO4. Evaporation of solvent gave a pale yellow oil (1.43 g) as the crude product, which was distilled using the Kugel- rohr apparatus (80-100°C, 0.03 mm Hg) to give pure ketal as a colorless oil (1.31 g, 91%), [a]D -87.7°C (c 1.10, CH3OH); ir: 1710 cm-1; 1H nmr d: 0.85 (s, 3H, CH3), 0.91 (d, J = 6.6 Hz, 3H, CHCH3), 0.97 (s, 6H, 2CH3), 1.16 (s, 3H, CH3), 1.56 (dd, J = 13.5, 13.5 Hz, H-11ax), 1.58 (d, J = 14.2 Hz, H-7ax), 2.38 (ddd, J = 13.5, 5.3, 3.8 Hz, 1H, H-11eq), 2.47 (dd, J = 14.2, 3.8 Hz, 1H, H-7eq), 2.85 (m, 1H, CHCH3), 3.41 and 3.48 (2dd, J = 11.4, 1.6 Hz, 2H, 2 equatorial H at C2 and C4), 3.53 and 3.61 (2d, J = 11.4 Hz, 2H, 2 axial H at C2 and C4); ms m/e: 240 (M+, 0.58), 141 (27), 155 (98), 83 (27), 69 (100).
Example 36
(-)-(9Z)-(9S,10R)-9-(5-Hydroxy-3-methylpent-3-en-1-ynyl) -3,3,8,8,10-pentamethyl-1,5-dioxaspiro[5,5]undecan-9-ol (33).
(-)-(10R)-3,3,8,8,10-Pentamethyl-l,5- dioxaspiro[5,5]undecan-9-one (1.31 g, 5.5 mmol) was reacted with Z-3- methylpent-2-en-4-yn-1-ol (0.65 g, 6.7 mmol) and n-butyllithium (1.6 M in hexane, 8 mL, 12.8 mmol) in dry THF. The crude product obtained (yellow oil, 2.6 g) was purified by flash column chromatography (75% ether + 25% hexane) followed by distillation using the Kugel-rohr apparatus (about 250°C, 0.06 mm Hg) to give the product as a colorless oil (1.40 g, 77%), [a]D -30.0°C (c 1.05, CH3OH); ir and 1H nmr identical to those reported above for its antipode.
Example 37
(±)-Z-4-hydroxy-4-(5-oxo-3-methylpent-3-en-1-ynyl)- 3,3,5-trimethylcyclohexanone (34) (PBI-18).
To pyridinium dichromate (1.14 g, 3.75 mmol) in dry DMF (6 mL), at 5-10°C was slowly added (±)-Z-4- hydroxy-4-(5-hydroxy-3-methylpent-3-en-1-ynyl)-3,3,5- trimethylcyclohexanone (750 mg, 3.0 mol) in DMF (5 mL). The reaction was maintained at 5°C for 2.5 h, and then water was added and the product extracted three times with ether. The combined ethereal phases were washed with water, then with saturated NaCl solution, then dried over Na2SO4 and the solvent evaporated to afford 570 mg of crude product which was crystallized from ether to give 410 mg (54%) aldehyde that gave m.p. 126-127°C; ir (CHCl3) 3600 strong, 2200 weak, 1710, 1670, 1600, 1100, 1060, and 1020 cm-1; 1H nmr -|| : 1.01 and 1.22 (s, gem CH3, 6H), 1.16 (d, J = 5.8 Hz, CHCH3, 3H), 2.16 (d, J = 1.5 Hz, =CCH3, 3H), 2.1-2.4 (m, 4H), 2.61 (d, J = 14.4 Hz, H-2ax, 1H), 6.22 (dq, J = 8.1, 1.5 Hz, =CH, 1H), and 10.03 (d, J = 8.1 Hz, CHO, 1H).
Example 38
(±)-E-4-hydroxy-4-(5-oxo-3-methylpent-3-en-1-ynyl)- 3,3,5-trimethylcyclohexanone (35) (PBI-19).
A mixture of (±)-E-4-hydroxy-4-(5-hydroxy-3- methylpent-3-en-1-ynyl)-3,3,5-trimethylcyclohexanone (2.0 g, 8.0 mol), manganese dioxide (14 g, 160 mmol), and acetone (50 mL) were combined and strred for 1.5 h. The mixture was filtered, the solvent removed by evaporation, and the residue chromatographed over silica eluting with 75% ether and 25% hexane to afford 1.17 g (±)-E-4-hydroxy- 4-(5-oxo-3-methylpent-3-en-1-ynyl)-3,3,5-trimethyl- cyclohexanone (58%), as an oil, that gave ir: 3600 (strong), 220 (weak), 1710, 1660, 900 cm-1; gc/eims: 248 (M+, 15%) 233 (9), 219 (19), 205 (16), 192 (42), 163 (95) and 121 (100); 1H nmr -||: 0.99 and 1.20 (s, gem CH3, 6H), 1.14 (d, J = 5.9 Hz, HCCH3, 3H), 2.11 (dd, J = 14.3, 2.3 Hz, H-2 eq, 1H), 2.18 (m, H-6eq, 1H), 2.3 (m, 2H), 2.32 (d, J = 1.5 Hz, =CCH3, 3H), 2.60 (d, J = 14.3 Hz, H-2ax, 1H), 6.22 (dq, J = 7.7, 1.5 Hz, =CH, 1H), and 10.03 (d, J = 7.7 Hz, CHO, 1H).
Example 39
Methyl 2-Z 5-(4-oxo-2,2,6-trimethylcyclohexan-1-ol)-3- methylpenten-4-ynoate (36) (PBI-41).
Z-4-hydroxy-4-(5-oxo-3-methylpent-3-en-1- ynyl)-3,3,5-trimethylcyclohexanone (34) (350 mg, 1.4 mmol) was treated with manganese dioxide (1.9 g, 22 mmol), sodium cyanide (165 mg, 3.4 mmol), acetic acid (80 uL, 1.4 mL) in methanol (15 mL). After 2 h the mixture was filtered, the solid washed with ether. The combined organic phases were washed twice with water, then saturated NaCl solution, dried over anhydrous Na2S04, and the solvent removed at reduced pressure. The product 36 was obtained pure by chromatography over silica (Chromatotron, elution with 50% ether 50% hexane, as an oil that gave: ir (CHCl3) 3600 (weak), 1710 (strong) cm-1; 1H nmr -|| : 0.99 and 1.23 (s, gem CH3, 6H), 1.16 (d, J = 6.3 Hz, HCCH3, 3H), 2.06 (d, J = 1.5 Hz, =CCH3, 3H), 2.1-2.6 (m, 4H), 2.86 (d, J = 14.3 Hz, H-3ax, 1H), 3.67 (s, OCH3, 3H), and 6.02 (q, J = 1.5 Hz, =CH, 1H); gc/eims m/z 278 (m+, 4), 247 (6), 219 (46) and 137 (100). Example 40
2-Z 5-(4-oxo-2,2,6-trimethylcyclohexan-1-ol)-3-methyl-pen- ten-4-ynoic acid (37) (PBI-40).
The ester 36 was saponified as for compound 30 to afford the enynoic acid 37 in 83% yield. The product gave ir (CHCl3) 3600 (weak), 3300 (br,strong) and 1690 (strong) cm-1; 1H nmr d: 0.99 and 1.21 (s, gem CH3, 6H), 1.14 (d, J = 6.2 Hz, HCCH3, 3H), 2.25-2.35 (m, 4H), 2.47 (d, J = 14.1 Hz, H-3eq, 1H) , 2.82 (d, J = 14.1 Hz, H-3ax, 1H), and 6.03 (q, J = 1.4 Hz, =CH, 1H).
Example 41
Preparation of 4 (Z)-(4R)-4-Hydroxy-4-(5-hydroxy-3- methylpent-3-en-1-ynyl)-3, 5, 5-trimethylcyclohex-2-enone (PBI-53).
This compound was prepared using the procedure described by Lamb and Abrams in 1990 Can. J. Chem. 68:1151-1162, hereby incorporated by reference, with the exception that oxo-isophorone was used as a starting material. Spectroscopic data is as follows: 1H NMR: δ 6.01 (d, 1H-3', J = 1Hz), 5.83 (d, 1H-2, J = 0.5 Hz), 3.67 (s, 3H-1), 3.34 (br s, 1H, OH), 2.58 (d, 1H-5', J = 16 Hz), 2.38 (d, 1H-5', J = 16 Hz), 2.12 (d, 3H-7', J = 1 Hz), 2.00 (d, 3H-6, J = 1 Hz), 1.21 (s, 3H-8'/9'), 1.09 (s, 3H-8'/9'). Example 42
Preparation of 4(Z)-(4R)-4-Hydroxy-4-(5-carboxy-3- methylbut-3-en-1-ynyl )-3, 5, 5-trimethylcyclohex-2-enone (BPI-54).
This compound was prepared using the procedure described by Lamb and Abrams in 1990 Can. J. Chem. 68:1157-1162, hereby incorporated by reference, with the exception that oxo-isophorone was used as a starting material. Spectroscopic data is as follows: 1H NMR: δ 6.03 (d, 1H-3', J = 1Hz), 5.86 (s , 1H-2), 2.63 (d, 1H-5', J = 16 Hz), 2.38 (d, 1H-5', J = 16 Hz), 2.10 (d, 3H-7', J = 1 Hz), 2.04 (d, 3H-6, J = 1 Hz), 1.21 (s, 3H-8'/9'), 1.09 (s, 3H-8'/9').
Example 43 Preparation of compounds PBI-209-211.
These compounds were prepared using the methods described by Lamb and Abrams in 1990 Can. J. Chem. 68:1151-1162, hereby incorporated by reference. The starting materials used were chosen from (8S*, 10S*)-8- Cyano-3,3,8,10-tetramethyl-1,5-dioxaspiro-[5, 5]-undecan- 9-one or from (2S*, 6S*) -2-Cyano-2 , 6-Di methyl--4,4-(2',2'- dimethylpropanedioxy) -cyc lohexanone which has the following spectroscopic data:
1H NMR: δ3.738 (d, 1H, J = 11.7Hz, OCH2), 3.574 (dd, 1H, J = 11.7, 1.7 Hz, OCH2), 3.513 (d, 1H, J = 11.4 Hz, OCH2), 3.416 (dd, 1H, J = 11.4, 1.7 Hz, OCH2), 3.266 (m, 1H, H-2), 3.082 (dd, 1H, J = 14.3, 4.2Hz, H-5e), 2.436 (ddd, 1H, J = 13.4, 5.3, 4.4 Hz, H-3e), 1.552 (d, 1H, J = 13.4 Hz, H- 3a), 1.520 (d, 1H, J = 14.3 Hz, H-5a), 1.420 (s, 3H, C-9, Me), 1.068 (s, 3H, Me), 1.090 (s, 3H, J = 6.5 Hz, C-7, Me), 0.881 (s, 3H, Me). HRMS: calcd for C14H21O3N (M+)
251.1550, found 251.1521.
Spectroscopic data for the resulting compounds is as follows:
(1'S*, 2'S*)-(2E, 4Z)-WethyI-5-(6'-cyano-2',6'-dimethy-1'- hydroxy-cyclohexan -4'-onyl)-3-methylpent-2,4-dienate
(PBI-209).
1H NMR: δ 8.064 (s, 1H, J =15.8 Hz, H-4), 6.095 (d, 1H, J = 15.8 Hz, H-5), 5.802 (s, 1H, H-2), 3.708 (s, 3H, OMe), 2.711 (dd, 1H, J =15.9, 2.3 Hz, H-5'e), 2.675-2.510 (overlap 2H, H-2', H-3'e), 2.475 (d, 1H, J = 15.9 Hz, H- 5'a), 2.418 (br s, 1H, OH), 2.226 (dd, 1H, J = 16.0, 13.5 Hz, H-3'a), 2.019 (d, 3H, J = 1.0 Hz, C-6, Me), 1.382 (s, 3H, C-9', Me), 0.977 (d, 3H, J = 6.5 Hz, C-7', Me). HRMS: calcd for C16H21O4N (M+) 291.1498, found 291.1471. (1'S*, 2'S*)-(2E, 4Z)-5-(6'-cyano-2',6'-dimethy-1'- hydroxy-cyclohexan-4'-onyl)-3-methylpent-2,4-dienoic acid (PBI-210).
1H NMR: δ7.818 (d, 1H, J = 15.9 Hz, H-4), 6.288 (d, 1H, J = 15.9 Hz, H-5), 5.718 (s, 1H, H-2), 2.123 (d, 1H, J = 11.3 Hz, H-5'), 2.053 (d, 3H, J = 1.1 Hz, C-6, Me), 1.990- 1.910 (overlap 2H, H-2', H-3'), 1.813 (dd, 1H, J = 11.3, 3.0 Hz, H5'), 1.718 (t, 1H, J = 14.2 Hz, H-3'), 1.062 (br s , 1H, OH), 1.047 (s , 3H, C-9, Me), 0.903 (d, 3H, J = 6.4 Hz, C-7', Me).
(1'S*, 2'S*)-(2E, 4Z)-5-(6'-σyano-2',6'-dijnethy-1'- hydroxy-cyclohexan-4'-onyl ) -3-methylpent-2 , 4-dien-1-al (PBI-211).
1H NMR: δ10.050 (d, 1H, J = 7.7 Hz, CHO), 6.811 (d, 1H, J = 15.5 Hz, H-4), 6.221 (d, 1H, J = 15.5 Hz, H-5), 6.077 (d, 1H, J = 7.7 Hz, H-2), 2.748 (dd, 1H, J = 16.0, 2.3 Hz, H-5'), 2.650-2.100 (overlap 4H, H-2', H-3', H-5'), 2.286 (d, 3H, J = 1.0 Hz, C-6, Me), 1.568 (s, 1H, OH), 1.373 (s, 3H, C-9', Me), 0.978 (d, 3H, J = 6.5 Hz, C-7', Me).
Example 44
Preparation of compounds PBI-250-253, PBI-258-260.
These compounds were prepared using the method described by Lamb and Abrams in 1990 Can. J. Chem. 68:1151-1162, hereby incorporated by reference. The starting material used was the following: 2 , 6-Dimethy-- 4, 4-ethylenedioxy-cyclohexa-2 , 5-dienone IR nmax cm-1: 1715 (C=0), 1630 (C=C); 1HNMR: 56.39 (s, 2H, H-3, H-4), 4.18 (s, 4H, OCH2CH2O), 1.86 (s, 6H, Me). 13C NMR: 5 186.4, 138.3 (2C), 135.5 (2C), 98.8, 65.3 (2C), 15.4 (2C). HRMS: calcd for C10H12O3 (M+) 180.0786, found 180.0786. Spectroscopic data for the resulting compounds is as follows:
(2E, 4Z)-5-(2',6'-dimethy-1'-hydroxy-cyclohexa-2',5'-dien- 4 ' -onyl )-3-methyIpent-2,4-dien-1-oI (PBI-250).
1H NMR: δ6.882 (d, 1H, J = 15.6 Hz, H-4), 5.997 (s, 2H, H- 3', H-5'), 5.607 (t, 1H, J = 6.9 Hz, H-2), 5.309 (d, 1H, J = 15.6 Hz, H-5), 4.310 (d, 2H, J = 6.9 Hz, OCH2), 2.053 (s, 1H, OH), 2.018 (s, 1H, OH), 1.946 (s, 6H, C-7', C-8', Me), 1.798 (s, 3H, C-6, Me).
(2E, 4Z)-5-(2',6'-dimethy-1'-hydroxy-cyclohexa-2 ',5'-dien- 4'-ony 1)-3-methylpent-2,4-dien-1-aI (PBI-251).
1H NMR: δ9.973 (d, 1H, J = 8.3 Hz, CHO), 7.421 (d, 1H, J = 15.5 Hz, H-4), 6.016 (s, 2H, H-3', H-5'), 5.821 (d, 1H, J = 8.3 Hz, H-2), 5.723 (d, 1H, J = 15.5 Hz, H-5), 2.703 (s, 1H, OH), 2.172 (s, 6H, C-7', C-8', Me), 1.986 (d, 3H, J = 1.0 Hz, C-6, Me). (2E, 4Z)-Methyl-5-(2', 6 ' -dimethy-1 ' -hydroxy-cyclohexa- 2 ' , 5'-dien-4 ' -onyl) -3-methylpent-2 , 4-dienate (PBI-252). 1H NMR: δ7.984 (d, 1H, J = 15.9 Hz, H-4), 5.996 (s, 2H, H- 3', H-5'), 5.708 (s, 1H, H-2), 5.619 (d, 1H, J = 15.9 Hz, H-5), 3.664 (s , 3H, OCH3), 2.004 (br s , 1H, OH), 1.955 (s, 6H, C-7', C-8', Me), 1.929 (d, 3H, J = 0.7 Hz, C-6, Me). (2E, 4Z)-5-(2',6'-dimethy-1'-hydroxy-cyclohexa-2',5'-dien- 4'-ony 1)-3-methylpent-2,4-dienoic acid (PBI-253).
1H NMR: δ7.957 (d, 1H, J = 15.5 Hz, H-4), 6.031(s, 2H, H- 3', H-5'), 5.763 (d, 1H, J = 15.5 Hz, H-5), 5.741 (s , 1H, H-2), 1.991 (s, 6H, C-7', C-8', Me), 1.965 (d, 3H, J = 0.94 Hz, C-6, Me). HRMS: calcd for C14H14O3 (M+-H2O)
230.0925, found 230.0943. (2E)-5-(2',6'-dimethy-1'-hydroxy-cyclohexa-2',5'-dien-4'- onyl )-3-methylpent-2-en-4-yn-1-ol (PBI-258).
1H NMR: δ 6.016 (s, 2H, H-3', H-5'), 5.945 (tm, 1H, J = 6.6 Hz, H-2), 4.259 (d, 2H, J = 6.6 Hz, OCH2), 2.733 (s, 1H, OH), 2.204 (s, 6H, C-7', C-8', Me), 1.864 (d, 3H, J = 1.1Hz, C-6, Me) . HRMS: calcd for C14H16O3 (M+) 232.1099, found 232.1093.
(2E)-5-(2',6'-dimethy-1'-hydroxy-cyclohexa-2',5'-dien-4'- onyl )-3-methylpent-2-en-4-yn-1-al (PBI-259).
1H NMR: δ 9.861 (d, 1H, J = 8.1 Hz, CHO), 6.187 (dm, 1H, J =8.1 Hz, H-2), 6.012 (s, 2H, H-3', H-5'), 4.187 (br s, 1H, OH), 2.205 (s, 6H, C-7', C-8', Me), 2.093 (d, 3H, J =1.4 Hz, C-6, Me). (2E)-Methyl-5-(2',6'-dimethy-1'-hydroxy-cyclohexa-2',5'- dien-4'-onyl)-3-methylpent-2-en-4-ynate (PBI-260).
1H NMR: δ6.022 (m, 1H, H-2), 5.99 (s , 2H, H-3', H-5'), 4.38 (br s, 1H, OH), 3.660 (s, 3H, OMe), 2.217 (s, 6H, C-7', C-8', Me), 1.981 (d, 3H, J = 1.5 Hz , C-6, Me) . HRMS: calcd for C15H16O4 (M+) 260.1049, found 260.1075.
Example 45
Preparation of compounds PBI-91, PBI-150, PBI-264, PBI- 268, PBI-270, PBI-276, PBI-277.
The compounds were prepared using the method described by Lamb and Abrams in 1990 Can. J. Chem. 68:1151-1162, hereby incorporated by reference. Spectroscopic data for these compounds is as follows:
(-)-(1'R, 2'R)-5',6'-Dihydroabscisic alcohol (PBI-276). 1H NMR: δ6.815 (d, 1H, J = 15.5 Hz, H-4), 6.096 (d, 1H, J = 15.5 Hz, H-5), 5.618 (t, 1H, J = 6.9 Hz, H-2), 4.339 (dd, 2H, J = 6.9, 0.6 Hz, OCH2), 2.479 (d, 1H, J = 14.8 Hz, H-5'), 2.378-2.113 (m, 4H, H-2', H-3', H-5'), 1.901 (d, 3H, J = 0.76 Hz, C-6, Me), 1.594 (brs, 2H, OH), 1.033 (s, 3H, C-8', Me), 0.909 (s, 3H, C-9', Me), 0.867 (s, 3H, C- 7', Me). [α]D -38.6_ (MeOH).
(-)-(1'R, 2'R)-(2E, 4Z)-5-(1'-hydroxy-2',6',6'-trimethy- cyclohexa-4'-onyl)-3-methylpent-2,4-dien-1-ol (PBI-277). 1H NMR: δ 6.457 (d, 1H, J = 15.7 Hz, H-4), 6.013 (d, 1H, J = 15.7 Hz, H-5), 5.736 (t, 1H, J = 6.8 Hz, H-2), 4.301 (d, 2H, J = 6.8 Hz, OCH2), 2.477 (d, 1H, J = 14.9 Hz, H-5'), 2.368-2.263 (m, 4H, H-2', H-3', H-5'), 1.825 (s, 3H, C-6, Me), 1.561( br s, 2H, OH), 1.024 (s, 3H, C-9', Me), 0.899 (s, 3H, C-8', Me), 0.857 (d, 3H, J = 6.2 Hz, C-7', Me). [α]D -51.0_ (MeOH).
(+)-(1'R, 2'R)-5',6'-Dihydroabscisic alcohol (PBI-91) . 1H NMR: δ 6.811 (d, 1H, J = 15.5 Hz, H-4), 6.088(d, 1H, J = 15.5 Hz, H-5), 5.618 (t, 1H, J = 7.1 Hz, H-2), 4.329 (d, 2H, J = 7.1, OCH2), 2.479 (d, 1H, J = 14.8 Hz, H-5'), 2.378-2.113 (overlap 3H, H-2', H-3', H-5'), 2.1279 (dd, 1H, J = 14.8, 2.0 Hz, H-5'a), 1.892 (s , 3H, C-6, Me), 1.594 (br s, 1H, OH), 1.024 (s, 3H, C-8', Me), 0.901 (s, 3H, C-9', Me), 0.860 (s, 3H, C-7', Me). HRMS: calcd for C15H24O3 (M+) 252.1744, found 252.1725 [α]D +42.6_ (c 1.03, MeOH).
(-)-(1'R, 2'R)-(2E)-5-(1'-hydroxy-2',6',6'-trimethy- cyclohexa-4'-onyl)-3-methylpent-2-en-4-yn-1-ol (PBI-150). IR nmax cm-1 (CHCl3) :3600 (OH), 1710 (C=0). 1H NMR: δ6.01
(ddq, 1H, J = 6.7, 6.7, 1.5 Hz, H-2), 4.23(dd, 2H, J = 5.8, 5.8 Hz, OCH2), 2.65 (d, 1H, J = 14.3 Hz, H-5'e), 2.29 (m, 3H, H-2', H-3'), 2.07 (dd, 1H, J = 14.3, 2.0 Hz, H- 5'a), 1.84 (m, 3H, C-6, Me), 1.47 (dd, 1H, J = 5.8, 5.8 Hz, OH), 1.18 (s, 3H, C-9', Me), 1.12 (m, 3H, C-7', Me), 0.96 (s, 3H, C-8', Me). HRMS: calcd for C13H18O3 (M+-28) 222.1256, found 222.1227. [α]D -22.5 (c 0.98, MeOH).
(-)-(1'R, 2'R)-(2E)-5-(1'-hydroxy-2',6',6'-trimethy- cyclohexa-4'-onyl)-3 -methylpent-2-en-4-yn-1-al (PBI-
264).
IR nmax cm-1 (CHCl3): 3610 (OH), 1720 (C=0),1670 (CHO), 1605 (C=C). 1H NMR: 510.030 (d, 1H, J = 7.8 Hz, CHO), 6.229 (dq, 1H, J = 7.8, 1.4 Hz, H-2), 2.607 (d, 1H, J = 14.4 Hz, H-5'e), 2.38-2.15 (overlap 3H, H-2', H-3'), 2.325 (d, 3H, J = 1.5 Hz, C-6, Me), 2.125 (dd, 1H, J = 14.4, 2.3 Hz, H-5'a), 2.090 (Jbr s, 1H, OH), 1.204 (s, 3H, C-9', Me), 1.145 (d, 3H, J = 5.7 Hz, C-7', Me), 0.996 (s, 3H, C- 8', Me). HRMS: calcd for C15H20O3 (M+-28) 248.1403, found 248.1412. [α]D -27.9 (c 1.01, MeOH).
(+)-(1'S, 2'S)-(2E)-5-(1'-hydroxy-2',6',6'-trimethy- cyclohexa-4'-onyl)-3-methylpent-2-en-4-yn-1-ol (PBI-267). IR nmax cm-1 (neat): 3400 (OH), 1700 (C=O). 1H NMR: 56.025 (tq, 1H, J = 6.8, 1.5 Hz, H-2), 4.242 (d, 2H, J = 6.8 Hz, H-2), 2.658 (d, 1H, J = 14.3 Hz , H-5'e), 2.34-2.23 (overlap 3H, H-2', H-3'), 2.077 (dd, 1H, J = 14.3, 1.8 Hz, H-5'a), 1.999 (Jbr s, 1H, OH), 1.849 (d, 3H, J = 0.6 Hz, C- 6, Me), 1.194 (s, 3H, C-9', Me), 1.132 (d, 3H, J = 5.8 Hz, C-7', Me), 0.997 (s, 3H, C-8', Me). HRMS: calcd for
C15H22O3 (M+) 250.1575, found 250.1569. [α]D +22.7 (c 1.0, MeOH). (+)-(1'S, 2'S)-(2E)-5-(1'-hydroxy-2',6',6'-trimethy- cyclohexa-4'-onyl)-3-methylpent-2-en-4-yn-1-al (PBI-268). IR nmax cm-1 (CHCl3): 3610 (OH), 1720 (C=O),1670 (CHO), 1605 (C=C). 1H NMR: 510.030 (d, 1H, J = 7.6 Hz, CHO), 6.229
(dq, 1H, J = 7.6, 1.4 Hz, H-2), 2.608 (d, 1H, J = 14.4 Hz, H-5'e), 2.38-2.15 (overlap 3H, H-2', H-3'), 2.325 (d, 3H, J = 1.5 Hz, C-6, Me), 2.126 (dd, 1H, J = 14.4, 2.3
Hz, H-5'a), 2.079 (br s, 1H, OH), 1.205 (s, 3H, C-9', Me), 1.146 (d, 3H, J = 5.8 Hz, C-7', Me), 0.997 (s, 3H, C-8', Me). HRMS: calcd for C15H20O3 (M+) 248.1403, found
248.1412. [α]D +24.1 (c 1.48, MeOH) .
(-)-(1'S, 2'S)-(2E, 4Z)-5-(1'-hydroxy-2',6',6'-trimethy- cyclohexa-4'-onyl)-3-methylpent-2,4-dien-1-ol (PBI-270). IR nmax cm-1 (neat): 3400 (OH), 1700 (C=0), 1625 (C=C). 1H NMR: 56.463 (d, 1H, J = 15.7 Hz, H-4), 6.046 (d, 1H, J = 15.7 Hz, H-5), 5.765 (t, 1H, J = 6.8 Hz, H-2), 4.312 (d, 2H, J = 6.8 Hz, OCH2), 2.484 (d, 1H, J = 14.9 Hz, H-5'), 2.36-2.18 (overlap 3H, H-2', H-3'), 2.135 (dd, 1H, J = 14.9, 2.5 Hz, H-5'), 1.836 (s, 3H, C-6, Me), 1.483 ( br s, 1H, OH), 1.033 (s, 3H, C-9', Me), 0.908 (s, 3H, C-8', Me), 0.867 (d, 3H, J = 6.2 Hz, C-7', Me). HRMS: calcd for C15H24O3 (M+) 252.1728, found 252.1725. [α]D +49,9 (c 2.89, MeOH).
Example 46
Preparation of compounds PBI-135, PBI-168-171, PBI-173- 175, PBI-281. These compounds were prepared using the method described by Lamb and Abrams in 1990 Can. J. Chem. 68:1151-1162, hereby incorporated by reference, except that the starting ring compound was (±)-(8R*, 10S*)-8- acetoxymethyl-3,3,8,10-tetramethyl-1,5-dioxaspiro[5, 5]undecan-9-one. Spectroscopic data for the resulting compounds is as follows:
(±)-9-(1E, 3Z)-(8R*, 9S*, 10S*)-8-Acetoxymethyl-9-(5- hydroxy-3-methyl-1,3-pentadienyl)-3,3,8,10-tetramethyl- 1,5-dioxaspiro[5, 5]undecan-9-ol (PBI-135).
1H NMR δ (ABA numbering): 6.71 (d, J = 15.5 Hz, 1H, H-5), 5.90 (d, J = 15.5 Hz, 1H, H-4), 5.56 (t, J = 7.1 Hz, 1H, H-2), 4.40 and 4.33 (2d, J = 11.1 Hz, H2C-8') overlaps with 4.33 (d, J = 7.1 Hz, H2C-1) (4H), 3.45 (m, 4H, 2CH2O-), 2.43 (dd, J = 14.8, 3.0 Hz, 1H, H-5'eq), 2.18 (m, H-2'ax), 2.06 (s, CH3COO), 1.86 (s, 3H, H3C-6), 1.33 (d, J = 14.9 Hz, H-5'ax) overlaps with 1.29-1.41 (m, H-3'eq, ax) (3H), 0.97, 0.88, 0.84 (3s, 3CH3) and 0.78 (d, J = 6.6 Hz, H3C- 7') (12H); 13C NMR 5: 170.9 (COO), 134.5, 129.6, 128.2 and 127.5 (4 olefinic C), 96.8 (O-C-O), 79.4, 70.5, 70.0, 68.6, 58.4, 41.7, 40.0, 37.7, 33.9, 30.0, 22.5, 22.4, 21.3, 21.1, 20.9, 15.3; CIMS (ammonia): m/z 414 [M+18]+ (100), 397 [M+1]+ (22). Anal, found: C, 66.70; H, 9.26. C22H36O6 requires: C, 66.62; H, 9.16.
(±)-9-(1E, 3Z)-(8R*, 9S*, 10S*) -8-Acetoxymethyl-9-(4- carbomethoxy-3-methyl-1,3-butadienyl)-3,3,8,10- tetramethyl-1,5-dioxaspiro[5, 5]undecan-9-ol (PBI-281) 1H NMR δ (ABA numbering): 7.80 (d, J = 6.0 Hz, 1H, H-4), 6.28 (d, J = 6.0 Hz, 1H, H-5), 5.69 (s, 1H, H-2), 4.42 and 4.30 (2d, J = 11.2 Hz, 2H, H2C-8'), 3.68 (s, 3H, CH3O), 3.45 (m, 4H, 2CH2O), 2.46 (dd, J = 14.8, 3.0 Hz, 1H, H- 5eq), 2.06 (s, CH3COO) and 2.00 (s, H3C-6) (6H), 0.98,
0.88, 0.85 (3s, 3CH3) and 0.80 (d, J = 6.6 Hz, H3C-7')
(12H); 3C NMR δ: 170.8 and 166.6 (2C00), 149.9, 135.9,
128.7 and 117.1 (4 olefinic C), 96.8 (0-C-O), 79.4, 70.5, 70.0, 68.5, 51.0, 41.7, 40.0, 37.9, 34.0, 30.1, 22.5,
22.4, 21.3, 21.2, 15.3; EIMS: m/z 424 [M] + (10), 393 [M-
31]+ (2), 364 [M-60]+ (10), 309 (95), 229 (60), 155 (100);
CIMS (ammonia): m/z 442 [M+18]+ (100), 407 [M-17]+ (28);
HRMS: [M]+ at m/z 424.2524 (C23H36O7 requires 424.2587). Anal, found: C, 65.27; H, 8.48. C23H36O7 requires: C, 65.06;
H, 8.55.
(±)-4-(1E, 3Z)-(3R*, 4S*, 5S*)-4-(4-carbomethoxy-3-methyl- 1,3-butadienyl)-4-hydroxy-3- hydroxymethyl-3,5- dimethylcyclohexan-1-one [PBI-168, or 8'-acetoxy-2', 3'- dihydroabscisic acid, methyl ester]
PBI-168 was obtained by recrystallization from CHCl3- hexane, m.p. 184-186°C. Anal, found: C, 63.68; H, 7.87. C18H26O6 requires: C, 63.87; H, 7.75%.
PBI-171 was reacted with ethereal diazomethane to give methyl ester PBI-169, m.p. 138-148°C, which had the following spectral properties: IR nmax cm-1: 3600, 3450, 1700; EIMS: m/z 278 [M-18]+ (7), 248 (5), 219 (14), 191 (100); CIMS (ammonia): m/z 314 [M+18]+ (100), 297 [M+1]+ (4), 296 [M]+ (8), 279 [M-18+1]+ (30); CIMS (isobutane): m/z 297 [M+1]+ (6), 279 [M-18+1]+ (62), 249 (100)); trimethylsilyl ether derivative CIMS (ammonia) : m/z 386 [M+18]+ (27), 369 [M+1]+ (9), 368 [M]+ (12), 351 [M-18+1]+ (45). Anal. found: C, 64.57; H, 8.43. C16H24O5 requires: C, 64.83; H 8.17%.
(±)-4-(1E, 3Z)-(3R*, 4S*, 5S*)-4-(4-carboxy-3-methyl-1,3- butadienyl)-4-hydroxy-3-hydroxymethyl-3,5-dimethyl- cyclohexan-1-one [(PBI-171, or 8'-hydroxy-2', 3'- dihydroabscisic acid] and methyl ester PBI-170
Compound PBI-171 was characterized as the methyl ester PB-170, m.p. 138-148°C, which had the following spectral properties: IR nmax cm-1: 3600, 3450, 1700; EIMS: m/z 278 [M-18]+ (7), 248 (5), 219 (14), 191 (100); CIMS (ammonia): m/z 314 [M+18]+ (100), 297 [M+1]+ (4), 296 [M]+ (8), 279 [M-18+1]+ (30); CIMS (isobutane) : m/z 297 [M+1]+ (6), 279 [M-18+1]+ (62), 249 (100); trimethylsilyl ether derivative CIMS (ammonia) : m/z 386 [M+18]+ (27), 369 [M+1]+ (9), 368 [M]+ (12), 351 [M-18+1]+ (45). Anal, found: C, 64.57; H, 8.43. C16H24O5 requires: C, 64.83; H 8.17%.
(+)-1-(1E, 3Z)-(1S, 4R, 6S)-1-(4-carboxy-3-methyl-1,3- butadienyl)-2,2,6-trimethylcyclohexan-1,4-diol (PBI-175) 1H NMR δ : 7.71 (d, J = 16.0 Hz, 1H, H-4), 6.43 (d, J = 16.0 Hz, 1H, H-5), 5.70 (s, 1H, H-2), 4.02 (dddd, J = 11.4, 11.4, 4.9 4.9 Hz, 1H, H-4'ax), 2.03 (d, J = 0.9 Hz , H3C-6) and 2.04 (m, H-2'ax) (4H), 1.88 (dddd, J = 12.2, 4.9, 3.2, 3.2 Hz, 1H, H-3'eq), 1.71 (ddd, J = 12.2, 4.7, 2.3 Hz, 1H, H-5'eq), 1.48 (dd, J = 12.2, 11.5 Hz, 1H, H- 5'ax), 1.24 (m, 1H, H-3'ax), 1.08 and 0.81 (2s, 3H each, H3C-8', 9'), 0.79 (d, J = 6.9 Hz, 3H, H3C-7'); HRMS: [M+] at m/z 268.1687 (C15H24O4 requires 268.1675).
PBI-174 gave identical NMR and MS data and m.p. 98-103°C, [α]D = +58.1° (c=0.78).
PBI-173 gave identical NMR and MS data and m.p. 151-156°C; [α]D = -63.2° (c=0.62).
Example 47 Preparation of compounds PBI-197-205.
These compounds were prepared using the method described by Lam and Abrams in 1992 Phytochemistry 31:1105-1110, hereby incorporated by reference. Spectroscopic data for these compounds is as follows: (2E)-5-(2-Difluoromethyl-4,4-ethylenedioxy-6,6- dimethylcyclohex-2-enyl)-3-methylpent-2-en-4-yn-1-ol (PBI 197). 1H NMR: δ 6.40 (t, 1H, J = 55.4 Hz, H-7'), 5.96 (s, 1H, H- 3'), 5.88 (m, 1H, H-2), 4.24 (d, 2H, J = 7 Hz , H-1), 3.95 (s, 4H, OCH2), 2.68 (bs, 1H, OH), 2.00 (d, 1H, J = 14.3 Hz, H-5'), 1.92 (d, 1H, J = 14.3 Hz, H-5'), 1.85 (s, 3H, C-6, CH3), 1.13 (s, 3H, C-8', CH3), 1.10 (s, 3H, C-9', CH3). (2Z, 4E)-5-(2-Difluoromethyl-4,4-ethylenedioxy-6,6- dimethylcyclo-hex-2-enyl)-3-methylpenta-2,4-dien-1-ol (PBI
198). 1H NMR: δ 6.63 (d, 1H, J = 15.8 Hz, H-5), 6.06 (t, 1H, J = 55.1 Hz, H-7', overlapping s, 1H, H-3'), 5.70 (d, 1H, J = 15.8 Hz, H-4), 5.59 (t, 1H, J = 7 Hz, H-2), 4.25-4.32 (m, 2H, H-1), 3.92-4.03 (m, 4H, OCH2), 1.97 (d, 1H, J= 14.3 Hz, H-5'), 1.83 (s, 3H, C-6, CH3), 1.77 (d, 1H, J= 14.3 Hz, H-5'), 1.06 (s, 3H, C-8', CH3), 0.91 (s, 3H, C-9', CH3).
(2Z,4E)-5-(2-Difluoromethy1-4,4-ethylenedioxy-6,6- dimethylcyclo-hex-2-enyl)-3-methylpenta-2,4-dien-1-al (PBI
199).
1H NMR: δ 10.17 (d, 1H, J = 8 Hz, H-1), 7.32 (d, 1H, J =
15.6 Hz, H-5), 6.11 (d, 1H, J = 15.6 Hz, H-4), 6.10 (t, 1H, J = 55 Hz, H-7'), 6.08 (s, 1H, H-3'), 5.88 (d, 1H, J =
8 Hz, H-2), 3.94-4.04 (m, 4H, OCH2), 2.06 (s, 3H, C-6, CH3), 1.93 (d, 1H, J = 15 Hz, H-5'), 1.83 (d, 1H, J = 15
Hz, H-5'), 1.09 (s, 3H, C-8', CH3), 0.94 (s, 3H, C-9',
CH3).
Methyl (2Z, 4E)-5-(2-difluoromethy1-4,4-ethylenedioxy-6,6- dimethyl-cyclohex-2-enyl)-3-methylpenta-2,4-dienoate (PBI
200).
1H NMR: δ 7.77 (d, 1H, J = 16.1 Hz, H-5), 6.11 (t, 1H, J = 55 Hz, H-7'), 6.06 (d, 1H, J = 16.1 Hz, H-4, overlapping s, 1H, H-3'), 5.70 (s, 1H, H-2), 3.93-4.04 (m, 4H, OCH2), 3.69 (s, 3H, CO2CH3), 1.97 (s, 3H, C-6, CH3), 1.93 (d, 1H, J = 14.5 Hz, H-5'), 1.82 (dd, 1H, J = 16.1, J' = 1 Hz, H- 5'), 1.08 (s, 3H, C-8', CH3), 0.92 (s, 3H, C-9', CH3).
Methyl 7',7'-difluoroabscisate (PBI 201 (racemic), 202 (+) and 203 (-)).
1H NMR: δ 7.84 (d, 1H, J = 16.1 Hz, H-5), 6.31 (t, 1H, J = 54.3 Hz, H-7'), 6.34 (s , 1H, H-3'), 6.11 (d, 1H, J = 16.1 Hz, H-4), 5.75 (s, 1H, H-2), 3.66 (s, 3H, CO2CH3), 2.54 (s, 1H, OH), 2.47 (d, 1H, J = 7.1 Hz, H-5'), 2.38 (d, 1H, J = 7.1 Hz, H-5'), 1.98(d, 3H, J = 1 Hz, C-6, CH3), 1.10 (s, 3H, C-8', CH3), 1.02 (s, 3H, C-9', CH3). (+)- Methyl 7',7'-difluoroabscisate showed the following properties: [α] = (+) 286.3; mp = 91-93º. (-)-Methyl 7',7'-difluoroabscisate showed the following properties:
[α] = (-) 306.76; mp = 90-91º.
(+) and (-)-7',7'-Difluoroabscisate (PBI 204 (+) and 205 (-)). 1H NMR: δ 7.79 (d, 1H, J = 16.2 Hz, H-5), 6.32 (t, 1H, J = 55.6 Hz, H-7'), 6.37 (s, 1H, H-3'), 6.14 (d, 1H, J = 16.3 Hz, H-4), 5.79 (s, 1H, H-2), 2.53 (d, 1H, J = 17.2 Hz, H-5'), 2.41 (d, 1H, J = 17.2 Hz, H-5'), 2.06 (d, 1H, J = 1 Hz, C-6, CH3), 1.14 (s, 3H, C-8', CH3), 1.06 (s, 3H, C-9', CH3).
(+)-7',7'-Difluoroabscisic acid showed the following properties: mp 155-156º, (hexane/Et2O); [α] = (+)-283.25. (-) -1 ',7'-Difluoroabscisic acid showed the following properties: [α] = (-)-296.19; mp 156-158º. Example 48
Preparation of compounds PBI-193-196, PBI-207, PBI-208, PBI-216-221.
These compounds were prepared using the method described by Rose et al. in 1992 Tetrahedron: Asymmetry 3, pp. 443-450, hereby incorporated by reference. Spectroscopic data for these compounds is as follows:
(-)-4(Z)-(4R)-4-Hydroxy-4-(5-hydroxy-3-methylpent-3-en-1- yny 1)-3, 5, 5-trimethylcyclohex-2-enone, (PBI 207) and (+) form, (PBI 216). 1HNMR: δ 5.90 (m, 1H-3'), 5.82 (s , 1H-2), 4.24 (d, 2H-1, J = 7 Hz), 2.47 (d, 1H-5', J = 16 Hz), 2.38 (d, 1H-5', J = 16 Hz), 2.10 (s, 3H-7'), 1.85 (s , 3H-6), 1.18 (s, 3H- 8'/9'), 1.08 (s, 3H-8'/9'); 13C NMR: d 198.4 (C=0), 160.3 (C=), 136.8 (C=)m 126.0 (C=), 120.0 (C=), 92.8, 85.3, 74.7, 61.1, 49.2, 41.8, 25.2, 22.9, 21.9, 19.7. PBI 216 has the following rotation: [α] = (+)-255.2 [MeOH, c 1.25]. PBI 207 showed the following rotation: [α] = (- )-264.6 [MeOH, c 1.05]. (-)-4(Z)-(4R)-4-Hydroxy-4-(5-oxo-3-methylpent-3-en-1- ynyl)-3 , 5, 5-trimethylcyclohex-2-enone, (PBI 208) and (+) form, (PBI 217).
1H NMR: δ 9.93 (d, 1H-1, J = 1, 8 Hz), 6.21 (dd, 1H-2, J = 1.5, 8 Hz), 5.87 (brd, 1H-3', J =2Hz), 2.94 (s, 1H, OH), 2.45 (d, 1H-5', J = 2 Hz), 2.11 (s, 6H-6,7'), 1.21 (s , 3H-8'/9'), 1.11 (s, 3H-8'/9'); 13C NMR: d 197.5 (C=O), 191.7 (C=O), 154.6 (C=), 140.7 (C=), 136.1 (C=), 126.6 (C=), 98.9, 83.6, 75.0, 49.1, 42.0, 25.2, 24.7, 21.9, 19.7. PBI 208 showed the following rotation: [α] = (-)- 294.4 [MeOH, c 1.21]. PBI 217 showed the following rotation: [α] = (+) -308.2 [MeOH, c 1.03].
(+)-4(Z)-(4R)-4-Hydroxy-4-(5-carboxymethyl-3-methylbut-3- en- 1-ynyl)-3, 5, 5-trimethylcyclohex-2-enone (PBI 194) and (-) form, (PBI 193).
1H NMR: δ 6.01 (d, 1H-3', J = 1Hz), 5.83 (d, 1H-2, J = 0.5 Hz), 3.67 (s, 3H-1), 3.34 (br s, 1H, OH), 2.58 (d, 1H-5', J = 16 Hz), 2.38 (d, 1H-5', J = 16 Hz), 2.12 (d, 3H-7', J = 1 Hz), 2.00 (d, 3H-6, J = 1 Hz), 1.21 (s , 3H-8'/9'), 1.09 (s, 3H-8'/9'). PBI 194 showed the following rotation:
[α] = (+)-238.3 [MeOH, c 1.26]. PBI 193 showed the following rotation: [α] = (-) -225.0 [MeOH, c 1.33] (+)-4(Z)-(4R)-4-Hydroxy-4-(5-carboxy-3-methylbut-3-en-1- ynyl)-3, 5, 5-trimethylcyclohex-2-enone, (PBI 196) and (-) form, (PBI 195).
1H NMR: δ 6.03 (d, 1H-3', J = 1Hz), 5.86 (s , 1H-2), 2.63 (d, 1H-5', J = 16 Hz), 2.38 (d, 1H-5', J = 16 Hz), 2.10 (d, 3H-7', J = 1 Hz), 2.04 (d, 3H-6, J = 1 Hz), 1.21 (s, 3H-8'/9'), 1.09 (s, 3H-8'/9'). PBI 196 showed the following rotation: [α] = (+)-283.5 [MeOH, c 0.45]. PBI 195 showed the following rotation: [o] = (-)-278.8 [MeOH, c 1.67]. (+)-Abscisyl alcohol, (PBI 218), and (-) form (PBI 220).
[α] = (+) -372.5 [MeOH, c 1.25]; HREIMS: [M+] at m/z 250.1575 (C15H22O3 requires 250.1569); IR umax cm-1: 3600 (w, O-H), 1660 (C=0); 1H NMR: 5 6.72 (d, 1H-5, J = 16 Hz), 5.89 (s, 1H-3'), 5.78 (d, 1H-4, J = 16 Hz), 5.60 (m , 1H- 2), 4.27 (d, 2H-1, J = 5.5 Hz), 2.43 (d, 1H-5', J = 17 Hz), 2.24 (d, 1H-5', J = 17 Hz), 1.87 (s, 3H-7'), 1.84 (s, 3H-6), 1.67 (bs, 1H, OH), 1.07 (s, 3H-8'/9'), 0.98 (s, 3H- 8'/9'); 13C NMR: 5 198.0 (C=0), 162.9 (C=), 134.1 (C=), 130.7 (C=), 129.5 (C=), 127.0 (C=), 126.8 (C=), 79.6, 59.2, 58.3, 49.8, 41.4, 24.2, 23.0, 20.6, 18.9, 12.8. PBI 220 has the following rotation: [α] = (-) -362.0 [MeOH, c 1.04]
(+)-Abscisyl aldehyde, (PBI 219), and (-) form (PBI 221).
[α] = (+) -451.7 [MeOH, c 1.38]; HREIMS: [M+] at m/z 248.1399 (C15H20O3 requires 248.1412); IR umax cm-1: 3600 (w, O-H), 1665 (C=O); 1H NMR: 5 10.16 (d, 1H-1, J = 8 Hz), 7.46 (d, 1H-5, J = 15.5 Hz), 6.18 (d, 1H-4, J = 15.5 Hz), 5.90 (m, 2H-2',3), 2.45 (d, 1H-5', J = 17 Hz), 2.31 (d, 1H-5', J = 17 Hz), 2.06 (d, 3H-6, J = 1 Hz), 1.89 (d, 3H-7', J = 1 Hz), 1.09 (s, 3H-8'/9'), 1.01 (s, 3H- 8'/9'); 13C NMR: 5 197.3 (C=O), 190.1 (C=O), 161.7 (C=), 153.0 (C=), 137.6 (C=), 129.3 (C=), 127.2 (C=), 126.0 (C=), 79.6, 49.7, 41.5, 24.3, 23.0, 21.5, 18.8. PBI 221 has the following rotation: [α] = (-)-451.5 [MeOH, c 1.04]. Example 49
Preparation of compounds PBI-262, PBI-263, PBI-274 and PBI-275.
These compounds were prepared by stirring the appropriate starting material with trimethylsilyl chloride and 1,8-diazobicyclo[5.4.0]undecane. Spectroscopic data for the resulting compounds is as follows:
(+)-4Z-4(R)-4-Trimethylsiloxy-4-(5-carboxymethyl-3-methyl- 2, 4-butadienyl)-3,5,5-trimethylcyclohex-2-enone. (PBI 262) and the (-) form, (PBI 263).
1H NMR: δ 7.56 (d, 1H, J = 16 Hz, HC=CH), 6.17 (d, 1H, J = 16 Hz, CH=CH), 6.01 (s, 1H, =CH), 5.71 (s, 1H, =CH), 3.64 (s, 3H, COOCH3), 2.22 (bs, 2H, CH2), 2.00 (s, 3H, =CCH3), 1.91 (s, 3H, =CCH3), 1.01 (s, 3H, C(CH3)2), 0.99 (s, 3H, C(CH3)2), 0.18 (s, 9H, Si(CH3)3).
(+)-4Z-4(R)-4-Trimethylsiloxy-4-(5-carboxy-3-methyl-2,4- buta dienyl)-3,5,5-trimethylcyclohex-2-enone. (PBI 274) and the (-) form (PBI 275).
1H NMR: δ 7.62 (d, 1H, J = 16 Hz, CH=CH), 6.21 (d, 1H, J = 16 Hz, CH=CH), 6.01 (d, 1H, J = 1 Hz, =CH), 5.75 (s, 1H, =CH), 2.24 (bs, 2H, CH2), 2.04 (d, 3H, J = 1 Hz, =CCH3), 1.91 (d, 3H, J = 1Hz, =CCH3), 1.01 (s, 3H, C(CH3)2), 1.00 (s, 3H, C(CH3)2), 0.18 (s, 9H, Si(CH3)3). Example 50
Preparation of compounds PBI-222, PBI-224 and PBI-225. These compounds were prepared through the reaction of abscisic acid with the appropriate diol and dicyclohexylcarbodiimide.
Spectroscopic data is as follows:
6-Hydroxyhexyl abscisate, (PBI 222) (racemic), (PBI 224) (+), and (PBI 225) (-).
1H NMR : δ 7.80 (d, 1H, J = 16 Hz, CH=CH), 6.12 (d, 1H, J = 16 Hz, CH=CH), 5.90 (d, 1H, J = 1 Hz, =CH), 5.72 (s, 1H, =CH), 4.09 (t, 2H, J = 7 Hz, CH2OH), 3.62 (t, 2H, J = 6.5 Hz, CH2OH), 2.44 (d, 1H, J = 17 Hz, CHH), 2.26 (d, 1H, J = 17 Hz, CHH), 1.98 (d, 3H, J = 1 Hz, =CCH3), 1.89 (d, 3H, J = 1Hz, =CCH3), 1.34-1.65 (m, 8H, (CH2)4), 1.07 (s, 3H, C(CH3)2), 0.98 (s, 3H, C(CH3)2).
Example 51
Influence of a composition containing compound PBI-11 on the enhancement of germination of Katepwa wheat seeds at low temperatures.
The effect of abscisic acid related compound PBI-11 on the emergence of Katepwa seedlings grown at 10°C was evaluated. Seeds of Katepwa wheat were steeped in PBI-11 made to a concentration of 10 -5 to 10-7 M in distilled water. The seeds were steeped for 4 hours at
22°C. Then the seeds were dried at 35°C to a moisture content of approximately 12 percent. The seeds were then planted in a soil mixture of 1 part soil, 1 part peat and 1 part vermiculite at a uniform depth of 3 cm. The seeds in the soil were then transferred to a ConViron Model E-15 controlled environment chamber maintained at 10°C, in the dark. The number of seeds which emerged was determined twice a day.
The results of this experiment which are set forth in Figure 1 demonstrate that PBI-11 at concentrations of 10 -5 and 10-7 M promoted the emergence of Katepwa wheat seedlings at 10°C with respect to the water controls (H2O C).
Example 52
Effect of compositions containing compound PBI-10 on the emergence of Canola at 10°C.
Methods
'Tobin' (7.3 g) and 7.4 g of 'Westar' canola were soaked for 8 hours at 25°C, in each of the following solutions: water; and one of 10, 1, or 0.1 μM PBI-10 in glass beakers. Beakers were sealed with aluminum foil to prevent evaporation and to exclude light. After incubation, solutions were removed and seeds were blotted dry with paper towels. Seeds were sandwiched between 4 layers of paper towels, which were daily changed and seeds were separated, and dried at 25°C, until their dried weight was close to their pre-soaking weight. About 100 seeds/treatment were sown, 2.5 cm deep in flats of 1:1:1 soil mix of peat, soil, and 'Vermiculite' in 4 rows 2.5 cm apart, and incubated at 10°C in darkness. Flats were watered with cold tap water to saturation point, before incubation, and as needed. Flats were examined at daily intervals, until plants began emerging, and at 12 and 8 h. intervals as emergence progressed. The number of plants which had emerged in each interval were recorded. Results are shown in Figures 2 and 3 wherein 10-5, 10-6 and 10-7 respectively correspond to concentrations of 10 μM, 1 μM and 0.1 μM of PBI-10 in solution.
Results
As shown in Figure 2, 'Tobin' seedlings from 10 μM PBI-10 treated seed emerged at 10°C more than a day earlier than those of the control (H2O). 'Westar' seedlings from the 10 and 1 μM PBI-10 treatments also emerged earlier than those of Water as shown in Figure 3. 'Westar' seeds probably responded to the 1 μM solution and 'Tobin' did not, because 'Westar' seeds are larger and could imbibe more 1μM solution, and consequently reach a "treshhold level" to respond. Emergence of the seedlings was promoted by these seed treatments, even though the soil was wet and the temperature less than optimal.
Example 53
Effect of various compounds on cold hardiness of bromegrass cells.
Methods
Suspension cell cultures of bromegrass (Brgmus inermis Leyss.) grown at 25°C in darkness for 1 week in liquid Ericksson's medium containing 75 μM ABA were hardy to -40°C. This method was the Model, on which the cold hardiness tests of the compounds, were based. However, these compounds are organic and contain hydrophobic components, which limit their solubility at high concentrations in water. The Tests were divided into 2 groups, as follows: 1) compounds were dissolved in water, and 2) compounds were dissolved in 1% dimethylsulfoxide (DMSO). Compounds in Group 1 were ABA, PBI-01, PBI-04, PBI-05, PBI-06, PBI-07, PBI-10, PBI-11, PBI-14 and PBI-15; compounds in Group 2 were ABA, PBI-16, PBI-17, PBI-18, PBI-19, PBI-34, PBI-43, PBI-37, and PBI-47. Compounds at the highest concentration (1000 or 100 μM) were serially diluted with water until a 0.01 μM concentration was reached. Five ml of each concentration was aseptically added to 45 ml of sterile liquid Eriksson's media (5 ml of 1000 μM 'X' in 45 ml media = 100 μM of Compound 'X'). A control of Ericksson's media (Group 1) or Ericksson's media with and without 1% DMSO (Group 2) was used. Bromegrass cells (1 gram), aseptically added to each concentration and control (s), were incubated for 1 week at 25°C in darkness on a rotary shaker at 150 rpm. Each treatment was repeated twice for Group 1 and 3 times for Group 2. After incubation, cells were removed and weighed to determine the growth of cells in each concentration. Cells were sampled for gm water / gm dry weight and for a Freeze Test to determine the lethal temperature for 50% (LT50) of the cells. About 0.25 g of cells was used to determine the gm water/gm dry weight of cells; about 0.1 g of cells was put into each test tubes, noted as 3, -3, -5, -7, -9, -11, -14, -17, -20, -25, -30, -35, and -40°C for each concentration of each compound plus the control. All tubes were sealed to prevent desiccation and held at -3°C except the Controls which were held at 3°C. After 1 hour at -3°C, the cells were nucleated and held at -3°C overnight. The temperature of the water bath was lowered at -3°C h-1, until -40°C was reached. Tubes were removed at each of the temperatures and thawed at 3°C overnight. Three ml of filtered, 0.08% 2,3,5-triphenyltetrazolium chloride (TTC), buffered with sodium phosphate to a pH of 7.5, was added to each test tube. Tubes were incubated for 24 h. at 25°C in darkness. TTC was removed and 3 ml of 95% ethanol was added to extract the red pigment from the cells. Cells were incubated for 48 h. at 25°C. Approximately 2 ml of the liquid was removed from the cells and used in a 'Beclcmann' spectrophotometer set at 486 nm to determine the absorbance value for each sample. TTC forms the red
pigment only with living cells, so a gradient curve of high to low absorbance values is formed. The LT50 is that part of the curve where the absorbance value of the frozen treatment in less than one half the value of the unfrozen control.
Results None of the compounds induced cells to -30°C as did 10 μM ABA, but PBI-04, PBI-05, PBI-34, and PBI-43 gave cells a lower LT50 than ABA at concentrations as low as 0.01 μM as shown in Tables 1 and 2. In Group 1, the categories were as follows: 1) the hardeners were 10 μM PBI-01, and all of both PBI-04 and PBI-05; 2) PBI-06, PBI-07, PBI-11, PBI-14, and PBI-15 were the same as control; and 3) the dehardeners were 0.1, 1, 10 and 100 μM PBI-10.
In Group 2, the categories were as follows: 1) the hardeners were 0.01 to 10 μM PBI-34 and all of PBI-43; 2) PBI-16 gave no response; and 3) the dehardeners were PBI-17, PBI-18, PBI-19, PBI-37, and PBI-47 (Table 2).
These categories were derived by consideration of the moisture content and growth rates, as well as the LT50 of the cells. Compounds, which are hardeners tend to reduce the weight of cells during incubation and the gm water/gm dry weight of cells is lower than that of the control. Dehardeners often increase the weight of cells during incubation, but the moisture content of the cells is always higher than that of the control. TABLE 1
Effects of concentration of various compounds on the LT50 (°C) of bromegrass cells incubated in a solution ccomprising one compound dissolved in water (Group 1)
Figure imgf000128_0001
x Hardener
* Dehardener.
TABLE 2
Effect of concentration of various compounds on the LT50 (ºC) of bromegrass cells incubated in a solution comprising one compound dissolved in 1% DMSO.
Figure imgf000129_0001
x Same as control .
* Hardener
The compounds induced categories of response. These categories were, as follows: 1) hardeners (lower LT50 than the control); 2) same as the control; and 3) dehardeners (higher LT50 than control). Tables 1 and 2 also show that each response was dependent on the concentration.
Example 54
Effect of various compounds on plant survivals after freezing at -7°C in Brassica napus c.v. Touchdown.
Seedlings of Brassica napus c.v. Touchdown; a winter type brassica grown at 10°C, 14 hour photoperiod received a foliar application of compounds PBI-01, PBI-16, PBI-37, PBI-38, PBI-39, PBI-40, PBI-41, PBI-53, PBI-54, PBI-252, PBI-260 and ABA at a concentration of 100 μM. Following treatment the plants were tested for frost tolerance using a controlled freeze test as follows. The plants were held at -2°C for two hours to equilibrate and then the leaves of the plants were nucleated with small ice crystals to initiate freezing of water in the plant. The plants were cooled to -3°C and allowed to equilibrate at that temperature for 1 hour. Plants were then removed from this temperature and allowed to thaw slowly at 4°C. The remaining plants were then cooled to -4, -5, -6, -7 and -9°C using the same protocol as described above. The frosted plants were compared to an unfrozen control after two weeks of regrowth in a glass house maintained at 25°C. The results of the freeze test are summarized in Table 3.
TABLE 3
Effect of various compounds on plant survival after freezing at -7°C in
Brassica napus cv Touchdown.
Figure imgf000131_0001
During the above experiment, the following compounds appeared to increase plant growth at 10°C: PBI-16, PBI-37, PBI-38 and PBI-54. These results were based on the morphological appearance of the control plants (plants sprayed, but not frozen) after three weeks at 10°C.
All of the leaves of control plants frozen to -7°C were killed by this severe frost. Plants treated with PBI-37 and PBI-40 had 95 and 64% viable leaves, respectively, following the severe frost. It is essential for plants to maintain their leaves following a frost for growth, development and vigor. Although the apex of all the plants was not affected by the severe frost, those plants treated with PBI-37 and 40 developed faster because their leaves were not destroyed by the frost. Often following a lethal frost to leaves, the maturity of the plant is delayed and yield is reduced.
Example 55
Effect of various compounds on percent plant survival after freezing Brassica napus c.v. Delta.
Seedlings of Brassica napus c.v. Delta (a spring type brassica) were treated with compounds PBI-01, PBI-16, PBI-37, PBI-38, PBI-40, PBI-252, PBI-260 and ABA as described in Example 54. The plants were grown similar to the plants grown in Example 54 and subjected to a frost test as described in Example 54. The plants were subjected to frosts two, three and four days following treatment with ABA and the compounds referred to above. The results of this test are described in Table 4.
TABLE 4
Effect of selected compounds on percent plant
survival after freezing Brassica napus cv
Delta (leaf survival at -4°C or -5°C; apex
survival at -7°C)
Figure imgf000133_0001
In non treated plants, only 7% of the total leaves were alive in plants subjected to a frost of -4°C. Approximately 36% of the plants had a viable growth apex. In plants tested for frost tolerance to -4°C two days after teatment with ABA analogs, ABA analogs PBI-01, 16, 37 and 40 resulted in significantly more viable leaves than the untreated plants. Also plants treated with PBI- 01, 16, 37, 38, 40, -252 and 260 had significantly more viable growth apexes than the controls. The frost protection afforded by the compounds persisted for at least 4 days after treatment, particularly so for PBI-16 and PBI-40 as determined by apex regrowth. In all cases PBI-40 was superior to ABA.
Example 56
Effects of root drench application of various compounds on freezing tolerance of winter rye.
Seedlings of a Graminae species winter rye
(Secale cereale) cv Puma were grown at 25°C in a glass house. At the two to three leaf stage, seedlings grown in vermiculite received a root application of 100 μM ABA analogs. Sixteen compounds were tested for increasing the freezing tolerance of the rye. The plants were tested for freezing tolerance as described in Example 54. Following the controlled freeze test the plants were evaluated for frost tolerance after three weeks growth in a glasshouse at 25°C. The results of this experiment are summarized in Figure 4.
As it can be seen from Figure 4, three days after treatment, plants treated with 100 μM ABA (PBI-57) were the most freezing tolerant, whereas the untreated plants were the least. Thereafter, the ABA treated plants declined in frost tolerance, however, plants treated with PBI-31, 54, 53, 16, 5, 40 and 41 increased in freezing tolerance with PBI-40 and PBI-54 could tolerate a frost of -8.5°C or better versus -2.5°C for the untreated plants. The results are discussed in further detail below.
Although most compounds induced freezing tolerance, differences existed as to the magnitude and induction profile over time as shown in Figure 4. ABA (57) and compounds 54, 53, 40 and 41 all induced tolerances of about -9°C, however, the lag time between application and expression of maximum tolerance differed. ABA treated plants showed maximal tolerance 3 days after treatment, but then rapidly dehardened to control levels 6 days after treatment. These hardening-dehardening trends are also evident from visual inspection of both the shoots (Figs. 5 and 6) and roots (Figs. 6a and 6b) treated with ABA and 54 following regrowth.
Molecular structure comparisons The main effects of chemical structure on freezing resistance are shown in Table 5.
TABLE 5
Summary of the mean and standard error LT50 values for the main effects of chemical
structure and time
- -
-
-
-
Figure imgf000136_0001
a Means separated with contrasts (*, P = 0.05; NS = not significant.
b Means separated with Fisher's LSD (Means followed by different letters are different at P = 0.05).
Results are based on the activity of 16 compounds which were applied to the roots of tender rye seedlings. Compounds were applied as a 10-4 M root drench on two consecutive days. LT50 values were calculated from freeze tests performed 3, 4 and 6 days after the first application. Figure 7 represents the interaction between chain bond order at C-4, C-5 and ring bond order at C-2', C-3' based on the induction of freezing tolerance in rye seedlings after compounds were applied as a root drench. Values are the average of 3 freeze tests performed 3, 4 and 6 days after treatment. It can be seen from this figure that altering the ring bond order did not increase nor decrease the effect of chain bond order on compound activity, or vice versa. Compounds with ring double bonds were more effective than ones with ring single bonds, and compounds with chain triple bonds were more effective than ones with a trans double bond.
Figure 8 represents the interaction between ring bond order at C-2', C-3' and C-1 functionality based on the induction of freezing tolerance in rye seedlings after compounds were applied as a root drench. Values are the average of 3 freeze tests performed 3, 4 and 6 days after treatment. The highly significant ring bond order by functionally at C-1 interaction stemmed primarily from esters for which activity was the same regardless of ring bond order. When C-1 was an acid, aldehyde or alcohol, the effect of ring bond order was additive with double bonds imparting greater activity than single bonds.
The effect of chain bond order at C-4, C-5 on activity was, to a certain extent, dependent upon C-1 functionality. The presence of a triple bond at C-4, C-5 caused a multiplicative increase in activity of acids, aldehydes and esters but did not alter the activity of alcohols as shown in Figure 9. The difference in activity between compounds with dienoic versus acetylenic side chains was most pronounced for acids and esters.
As shown in Figure 10, ring bond order at C-2', C-3' altered the effectiveness of the compounds over time. Compounds with a C-2', C-3' double bond were more active initially than ones with a C-2', C-3' single bond. The activity of compounds with a C-2', C-3' single bond, however, increased over time and was equal to compounds with a C-2', C-3' double bond by day 6. Determinations were carried out with preplanned contrasts between bond level for day 3 and day 6.
Compounds with a double bond at C-4, C-5 induced maximum freezing tolerance early (day 3), but were decreased in activity by day 4 and 6 as shown in Figure 11. In contrast, compounds with a triple bond at C-4, C-5 were of equal quality to compounds with a double bond at C-4, C-5 on day 3, but then increased in activity over time attaining a maximum on day 6. Determinations were carried out with preplanned contrasts between bond level for day 3 and day 6.
The effect of functionality at C-1 also varied with time as shown in Figure 12. All functional groups except the acid showed different hardening profiles with time. Esters increased in activity during all 3 test days, whereas aldehydes increased inirially and then decreased. Alcohols maintained a constant low activity on day 3 and 4 but decreased in activity by day 6. Determinations were carried out with preplanned contrasts between bond level for day 3 and day 6.
All three way interactions between molecular substituents were significant. As seen in Figure 13, the ring bond order by chain bond order by C-1 functionality interaction revealed that ring bond order at C-2', C-3' had a similar additive effect on dienoic compounds when the functional group at C-1 is an acid, alcohol or aldehyde but not when it was an ester. The effect of any given substituent on activity was more variable for acetylenic than for dienoic compounds. For acetylenic compounds, the effect of ring bond order at C-2', C-3' on activity was not additive as it was for dienoic compounds. Acetylenic acids and esters were of equal activity regardless of ring bond order. Replacing the double bond in the chain at C-4, C-5 with a triple bond increased the activity of the cyclohexanone acid and both the cyclohexanone and cyclohexenone ester.
The ring bond order at C-2', C-3' by chain bond order at C-4, C-5 interaction also interacted with time, as did both when viewed independently (see Figs. 9 and 10). The activity of all acetylenic compounds, regardless of ring bond order, increased with time, as shown in Figure 14. Dienoic compounds with a cyclohexenone ring decreased in activity with time, whereas ones with a cyclohexanone ring were relatively inactive and did not show a consistent trend with time.
The ring bond order at C-2', C-3' by C-1 functionality interaction was also affected by time. Cyclohexenone acids decreased in activity over time, whereas cyclohexanone acids increased in activity with time (see Figure 15). This explains why acids taken collectively did not interact with time since the changes in activity due to ring bond order cancel one another when averaged over time. Cyclohexenone alcohols showed consistent activity with time, whereas cyclohexanone alcohols showed lower activity, and decreased with time. For aldehydes, cyclohexenones did not vary in activity over time while cyclohexanones showed a slight increase. Esters showed a small but consistent increase in activity over time for both ring bond orders.
The main effects can be summarized as follows for the compounds tested: compounds with a trans double bond at C-2', C-3' in the ring were more active than compounds with a single bond in the ring; compounds with a triple bond at C-4, C-5 in the chain were more active than compounds with a double bond in the chain; and at C- 1, acid compounds were more active than ester compounds, whereas compounds alcohol and aldehyde based compounds were of lower but equal activity.
Relative to their respective acids, esterification decreased the activity of cyclohexenones. but did not affect the activity of cyclohexanones. The reason for this interaction is not clear, but may be related to esterase activity and rates of hydrolysis if a free carboxyl group was required for activity. The C-1 has to be at the acid oxidation level to induce freezing tolerance in a bromegrass cell suspension culture. The greater activity of analogs with a triple bond at C-4, C-5 may also partially relate to the rate of hydrolysis as acetylenic esters were of equal activity to acetylenic acids, but dienoic esters were less active than dienoic acids.
The inactivity of aldehyde and alcohol based analogs, regardless of other molecular substitutions, is in contrast to their high activity in numerous other assays. ABA alcohol was more active than ABA in a germination inhibition assay using cress seeds (Gusta et al. 1992). The high activity of ABA aldehyde, several acetals and alcohol in a transpiration assay was attributed to conversion to ABA (Walton, 1983; Schubert et al. 1991). The aldehyde and alcohol would be expected to be taken up faster than the acid since they are less polar. Therefore, inactivity may stem from slow conversion to ABA, or more stringent requirements at the receptor compared to transpiration. For example, stomatal closure was as rapid with both ABA alcohol and aldehyde as ABA suggesting that a single oxygen at C-1 was sufficient for activity. Although the molecular substituents native to ABA led to the most active compounds initially, these compounds quickly lost activity. For example, compounds with a double bond at C-2', C-3' lost activity within 6 days following application as did analogs with a double bond at C-4, C-5. Although there are numerous possible explanations for this phenomenon, it may stem from different specificity between the physiological response and metabolism.
Example 55
Effects of various compounds applied as a foliar spray on freezing tolerance of winter rye.
Seedlings of a Graminae species winter rye (Secale cereale) cv Puma were grown at 25°C in a glass house. At the two to three leaf stage, seedlings grown in vermiculite received a foliar application of 100 μM ABA analogs. A mesting bottle was used for foliar applications and approximately 0.5 ml of solution per plant was applied. Sixteen compounds were tested for increasing the freezing tolerance of the rye. The plants were tested for freezing tolerance as described in Example 54. Following the controlled freeze test the plants were evaluated for frost tolerance after three weeks growth in a glasshouse at 25°C. Results are summarized in Figure 16. When compared to the results from a root application of the same analogs, lesser compounds showed a consistent response to time when applied as a foliar spray. However, compounds such as PBI-54 and PBI-11 maintained more consistent activities over time.
The origin of differences in method of application is likely to relate to uptake, partitioning, or vascular transport, or combinations thereof, as the active sites would be the same.
A contributing factor to the differences observed between methods of application is partitioning of absorbed ABA. Foliar applied ABA may be rapidly ion trapped in the phloem which has a pH of 7.5. Studies with radiolabelled ABA have shown that foliar applied ABA is translocated very slowly to strong sinks such as young leaves. In contrast, root applied ABA rapidly accumulates in the xylem stream. The xylem sap is typically pH 5.5 to 6.5, therefore, ABA is not ion trapped and has access to all tissues that are continuous with the apoplast. The crown is the critical region for survival in cereals, therefore, ABA must come into contact with this tissue if there is to be a response. The crown contains the meristems in cereal seedlings, which are strong sinks for photosynthate. Xylem vessels also pass through the crown, therefore, both methods of application would expose the crown to ABA. The marked differences in response to the method of application must be due to either lesser foliar uptake or the location of the ABA receptor (s) governing acclimation. Although the location of the ABA receptor (s) for cold hardening is unknown, the situation may be analogous to that of stomata. Stomatal receptors are on the plasma membrane and are sensitive to the apoplastic concentration of ABA.
Example 58
Effect of various compounds applied as a foliar spray on freezing tolerance and growth at low temperature of canola c.v. Touchdown. Seedlings of canola c.v. Touchdown growing at
10°C were sprayed with 100 μM ABA and compounds PBI-37 and PBI-16. The plants were subjected to a frost of -3°C and then held at 10°C for 14 days after the frost. The frost had no effect on the plants. Furthermore, as shown in Figure 17, advanced growth of the seedlings was noted through the period during which they were maintained at 10°C. Also note the yellow, small leaves in the control which are starting to senescence.
Example 59
Effect of various compounds applied as a foliar spray on growth at low temperature of canola c.v. Touchdown.
Seedlings of canola c.v. Touchdown growing at 10°C in a controlled environment chamber were sprayed at the first leave stage with 100 μM ABA and various compounds at 10°C. A picture was taken 14 days after spraying and is shown in Figure 18. The plants were transferred to a greenhouse maintained at 23°C. Figure 18 demonstrates senescence of leaves and small leaves in the control compared to the healthy and robust plants sprayed with compounds PBI-37 and PBI-16. Table 6 demonstrates the effect of the various compounds on the development of plants. It can be seen from this table that plants treated with compound PBI-260 flowered 3.1 days earlier than the non-treated control plants.
TABLE 6
Effect of selected ABA analogs on flowering in
Brassica napus cv Delta after extended growth at 10°C. All values are given in days relative
to untreated plants at each temperature
Figure imgf000146_0001
Note: Plants treated with analogs were held at 10°C for
14 days following treatment and then transferred to a greenhouse maintained at 23°C. Plants treated with PBI-260 flowered 3.1 days earlier than the non-treated control plants.
Example 60
Effect of various compounds on the chilling injury of Phaseolus vulgaris after seven days at 5°C.
Phaseolus vulgaris (beans) grown at 25°C in a peat mixture were soil drenched with approximately 50 mls of either water, ABA and the compounds PBI-03, PBI-19, PBI-16 and PBI-11 (all chemicals at 10-6 M). The plants were held at 25°C with a 16 hour photoperiod. After seven days the plants were transferred to 25°C and allowed to grow for an additional 14 days. The plants were evaluated for injury (visually) based on a scale of 0 to 5 when 0 indicates no injury and 5 indicates complete necrosis.
The effect of ABA and compounds PBI-03, 11, 16 and 19 on the chilling injury of Phaselous vulgaris after seven days at 5°C.
Figure imgf000147_0001
The results demonstrate the ABA analogs PBI-19 and PBI-03 were equal to or superior to ABA. The analogs PBI-16 and PBI-11 increased injury to the low temperature treatment.
Example 61
Effect of various compounds on chilling injury in tomato seedlings.
Seeds of tomato (Lycopersicon esculentum cv. Swift) were sown to a 1:2 mix of peat moss and vermiculite in a glass house maintained at 25°C. At the two leaf stage the seedlings were transplanted a "Styrofoam tray" containing holes of 3 cm in diameter by 12 cm deep. The roots of the seedlings were covered with greenhouse grade vermiculite. Plants were grown in the glass house at a light intensity of 350 μmol/sec/m 2 and fertilized every third day with a 10% solution of a 20-20-20 (N-P-K) fertilizer. When the plants were 20 to 30 cm in height
(growth stage suitable for transplanting to the field) the plants were treated with 10 mis of either water, and the
ABA analogs PBI-01, PBI-37, PBI-40 and PBI-53 at either 1 or 10μM. Racemic ABA at equivalent concentrations was also added to the tomato plants as a comparison treatment.
Following treatment the plants were held in the glass house for 24 prior to transfer to a controlled environment chamber maintained at 11°C with a light intensity of 290 μmol/sec/m 2. The plants were held at these temperatures for either 0, 2, 4 or 6 and then transferred back to a glass house at 25°C. The plants were analyzed for water loss as determined by transpiration or water loss. Then the plants were transplanted to the field and evaluated for days to flowering and yield. Results are shown in Table 7.
TABLE 7
Effects of various compounds on FFD, standard error (SE), and co-efficient of variability
(CV), of tomato plants, soild-drenched with analogs, and chilled for 0 to 6 days.
Figure imgf000149_0001
%
means followed by the same letter are not significantly different by Turkey's HSD test (Critical
Range = 3.07, Alpha = .05, N = 56).
FFD = First Flowering Day (after transplant)
Four days of chilling treatment at 11°C were more harmful than either 2 or 6 days. Plants treated with PBI-16 and PBI-37 had the lowest value transpiration rates. Plants treated with PBI-40, PBI-16 and PBI-37 flowered 4 days earlier than the control. According to statistical analysis these results are significant (Table 3). Plants treated with PBI-40 and racemic ABA suffered the lowest losses due to transplant shock in the field.
Example 62
Effects of compounds on chilling injury and flowering in tomato seedlings. Tomato plants cv. Swift were grown as described previously in Example 59. The plants were treated with the following compounds: -ABA, PBI-37, PBI-40, PBI-11, PBI-63 and PBI-51. The tomato plants were chilled for 0, 2, 4 and 6 days in one trial and for 0, 2, 4, 7 and 9 days in a second trial. For both cases, the chilling temperatures was 5°C. The results of the treatments on chilling injury to tomatoes is shown in Tables 4 and 5. The effect of the treatments on chilling injury to tomatoes is shown in Tables 4 and 5. The effect of the treatments is shown in Table 8.
Figure imgf000151_0001
z 0 = no injury; 1 = less than 25% leaves injured; 2 = 25=50% leaves injured; 3 = 50-75% leaves injured; 4 = more than 75% leaves injured; 5 = whole plant died.
y mean separation in column (lowercase letters) by LSD, p = 0.05, n = 5.
Tomato plants exposed to chilling temperatures were protected for 2 days. The trend was to afford protection for also 4 days of chilling temperatures. Plants treated with -ABA also flowered 6 to 13 days earlier than the controls. The compound PBI-37 flowered 3 to 10 days earlier than the controls.
Claims to the invention follow.

Claims

1. A composition for enhancing low temperature tolerance in plants which comprises an effective amount of a compound having the following formula (I) :
Figure imgf000152_0001
wherein
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium; R3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
when R2 is oxo or thio, R2 may be linked to both C1 and C2 carbon atoms to form an epoxy or a thioepoxy ring;
and when R3 is oxo or thio, R3 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, lower- alkoxy carbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
and when R4 is oxo or thio, R4 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R5 is oxo, it may be linked to the carbon atom bearing R3;
R6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R7 is oxo, it may be linked to the carbon atom bearing R3; and wherein
the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
R1 or R6 is absent if the dotted line adjacent to R1 and R6 is a single bond,
R2 is absent if either of the dotted lines adjacent to R2 is a single bond,
the alkyl group bearing R7 is absent if the dotted line adjacent to the alkyl group bearing R7 is a single bond, and isomers and functional derivatives thereof,
in admixture with an acceptable agricultural carrier comprising an agriculturally acceptable carrier cation when
R, R1, R2, R4, R5, R6 or R7 are phosphate, sulfoxide or sulfone.
2. A composition for enhancing low temperature tolerance in plants which comprises an effective amount of at least one compound having the following formula (IA) :
Figure imgf000155_0001
wherein
R is hydroxy, aldehyde, carboxyl or loweralkoxyl;
R1 is loweralkyl;
R2 is hydroxy;
R3 is loweralkyl or loweralkylhalide;
R4 is oxo;
R5 and R7 are hydrogen;
the dotted line is optionally a single bond and the double dotted line is a double bond or a triple bond; and R7 is absent when the dotted line adjacent to R5 is a single bond.
3. A composition according to claim 2 wherein said compound of formula IA is selected from the group consisting of
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
4. A composition according to claim 3 wherein the concentration of said compound of formula IA ranges between 10-2 and 10-5M.
5. A method for enhancing low temperature tolerance in a plant, said method comprises treating said plant with an effective amount of a compound having the following formula (l):
wherein
Figure imgf000161_0001
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinly, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium; R2 is hydrogen, oxo, hydroxy, halogen or thio, phosphate, sulfoxide, sulfone or deuterium;
R3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy-loweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkylhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
when R2 is oxo or thio, R2 may be linked to both C1 and C2 carbon atoms to form an epoxy or a thioepoxy ring;
and when R3 is oxo or thio, R3 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkyIsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkyl amino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
and when R4 is oxo or thio, R4 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R5 is oxo, it may be linked to the carbon atom bearing R3;
R6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyl- oxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R7 is oxo, it may be linked to the carbon atom bearing R3; and wherein
the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
R1 or R6 is absent if the dotted line adjacent to R1 and R6 is a single bond, R2 is absent if either of the dotted lines adjacent to R2 is a single bond,
the alkyl group bearing R7 is absent if the dotted line adjacent to the alkyl group bearing R 7 is a single bond, and isomers and functional derivatives thereof,
in admixture with an acceptable agricultural carrier comprising an agriculturally acceptable carrier cation when
R, R1, R2, R4, R5, R6 or R7 are phosphate, sulfoxide or sulfone, for the purpose of enhancing low temperature tolerance in plants.
6. A method according to claim 5 wherein in said compound of formala I:
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxy- loweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkoxy having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, hydroxy, halogen or thio;
R3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkyl- amino, kiloweralkylamino, loweralkoxy, loweralkyIhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl; and when R2 is thio, R2 may be linked to both C1 and C2 carbon atoms to form a thioepoxy ring; R4 is hydrogen, oxo, halogen, thio or ammo;
R5 is hydrogen, oxo or nitrogen;
R6 is hydrogen;
R7 is hydrogen, oxo or nitrogen.
7. A method for enhancing low temperature tolerance in a plant, which comprises treating said plant with an effective amount of a compound having the following formula (IA):
Figure imgf000165_0001
wherein
R is hydroxy, aldehyde, carboxyl or loweralkoxyl;
R1 is loweralkyl;
R2 is hydroxy;
R3 is loweralkyl or loweralkyIhalide;
R4 is oxo; R5 and R7 are hydrogen;
the dotted line is optionally a single bond and the double dotted line is a double bond or a triple bond, for the purpose of enhancing low temperature tolerance in plants; and R7 is absend when the dotted line adjacent to R5 is a single bond.
8. A method according to Claim 7 wherein said compound of formula IA is selcted from the group consisting of:
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
9. A method according to claim 8 wherein the concentration of said compound of formula IA ranges between 10-2 and 10-5.
10. A compound having the following formula (I) :
Figure imgf000171_0001
wherein
R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, thio, phosphate, sulfoxide, sulfone, deuterium or cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, oxo, hydroxy, halogen, thio, phosphate, sulfoxide, sulfone or deuterium;
R3 is oxo, thio, carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyIhalide, loweralkyldeuterium, loweralkyl sulphonyl, loweralkyl sulphinyl, or carbonyl;
when R2 is oxo or thio, R2 may be linked to both C1 and C2 carbon atoms to form an epoxy or a thioepoxy ring;
and when R3 is oxo or thio, R3 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R4 is hydrogen, oxo, halogen, thio, phosphate, sulfoxide, sulfone, deuterium, hydroxy, loweralkylsiloxane, carboxyl, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkyl- amino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, cycloalkyl or cycloalkoxy having from 4 to 6 carbon atoms which is optionally substituted by loweralkyl, halogen, oxygen, hydroxy or loweralkoxy;
and when R4 is oxo or thio, R4 may be linked to the carbon atom adjacent to R5 to form an epoxy or thioepoxy ring; R5 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyl- oxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R5 is oxo, it may be linked to the carbon atom bearing R3;
R6 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R7 is carboxyl, hydroxy, aldehyde, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, acetoxyloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl, amino, carbonyl, halogen, hydrogen, oxo, thio, phosphate, sulfoxide, sulfone or deuterium, and when R7 is oxo, it may be linked to the carbon atom bearing R3; and wherein the dotted lines may each represent a single bond and the double dotted line represents either a double bond or a triple bond,
R1 or R6 is absent if the dotted line adjacent to R1 and R6 is a single bond,
R2 is absent if either the dotted line adjacent to R2 is a single bond,
the alkyl group bearing R7 is absent if the dotted line adjacent to the alkyl group bearing R7 is a single bond, and isomers and functional derivatives thereof,
with the proviso that when R is -CHO, -CH2OH or -COOCH-,
R1 is CH3, R2 is oxo or OH, R3 is CH3, R4 is oxo or H and R5 is H, the following compounds are excluded from formula (I) :
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
11. A compound according to claim 10, wherein R is carboxyl, aldehyde, hydroxy, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, cycloalkoxy having from 4 to 6 carbon atoms, amino, carbonyl, halogen or thio;
R1 is loweralkyl, hydrogen, oxo, hydroxyloweralkyl, loweralkoxy, halogen, thio, sulfoxide, sulfone, phosphate or deuterium;
R2 is hydrogen, hydroxy, halogen or thio;
R3 is carboxyl, aldehyde, loweralkyl, hydroxyloweralkyl, alkoxyloweralkyl, loweralkoxycarbonyl, loweracyloxyloweralkyl, acetylloweralkyl, loweralkanoyl, loweralkylamino, diloweralkylamino, loweralkoxy, loweralkyIhalide, loweracyloxy, loweralkylthio, loweralkyl sulphonyl, loweralkyl sulphinyl or carbonyl;
and when R2 is thi.o, R2 may be linked to both C1 and C2 carbon atoms to form a thioepoxy ring;
R4 is hydrogen, oxo, halogen, thio or ammo;
R5 is hydrogen, oxo or nitrogen;
R6 is hydrogen;
R7 is hydrogen, oxo or nitrogen.
12. A compound according to claim 10, characterized by having the following formula (IA)
Figure imgf000178_0001
wherein
R is hydroxy, aldehyde, carboxyl or loweralkoxyl;
R1 is loweralkyl;
R2 is hydroxy;
R3 is loweralkyl or loweralkyIhalide;
R4 is oxo;
R5 and R7 are hydrogen;
the dotted line is optionally a single bond and the double dotted line is a double bond or a triple bond; and R7 is absent when the dotted line adjacent to R5 is a single bond.
13. A compound according to claim 10, characterized by having the following formula (IA)
Figure imgf000178_0002
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
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