SULFONAMIDES THAT ACTIVATE ABA RECEPTORS
20170231224 · 2017-08-17
Inventors
Cpc classification
A01N47/24
HUMAN NECESSITIES
A01N53/00
HUMAN NECESSITIES
International classification
Abstract
The present invention provides methods and compositions comprising agonist compounds that activate ABA receptors. In one aspect, the invention provides an agricultural formulation useful for inducing ABA responses in plant vegetative tissues, reducing abiotic stress in plants, and inhibiting germination of plant seeds. The compounds are also useful for inducing expression of ABA-responsive genes in cells that express endogenous or heterologous ABA receptors.
Claims
1. A method of increasing abiotic stress tolerance in a plant, the method comprising contacting a plant with an effective amount of a sulfonamide agonist compound to increase abiotic stress tolerance in the plant, thereby increasing abiotic stress tolerance; wherein the sulfonamide agonist compound is of Formula I: ##STR00048## wherein: A is selected from the group consisting of alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and cycloalkyl; each R.sup.1 is a substituent independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, fluoroalkyl, hydroxyl, hydroxyalkyl, alkoxy, fluoroalkoxy, alkoxyalkyl, amino, aminoalkyl, alkylthio, alkylthioalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido, and amidoalkyl; wherein the R.sup.1 cycloalkyl, heterocyclyl, aryl, or heteroaryl is additionally substituted with from 0 to 3 R.sup.6; or alternatively, two R.sup.1 substituents join to form an additional R.sup.1 ring, wherein the additional R.sup.1 ring is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl; and wherein the additional R.sup.1 ring is additionally substituted with from 0 to 3 R.sup.6; or alternatively, an R.sup.1 and a R.sup.2 substituent join to form an R.sup.1,2 ring, wherein the R.sup.1,2 ring is selected from the group consisting of cycloalkyl and heterocyclyl; and wherein the R.sup.1,2 ring is additionally substituted with from 0 to 3 R.sup.6; m is an integer selected from 0 to 5; wherein if A is not aryl or if at least two R.sup.1 are not halo, m is an integer selected from 0 to 3; L is a bond, —C(R.sup.2)(R.sup.2′)—, —O—, or —NR.sup.6—; wherein if L is a bond, A is not alkyl; R.sup.2 and R.sup.2′ are each a substituent independently selected from the group consisting of hydrogen, alkyl, fluoroalkyl, cycloalkyl, cycloalkenyl, heterocycyl, heteroaryl, and aryl; wherein the cycloalkyl, heterocycyl, heteroaryl, and aryl is additionally substituted with from 0 to 3 R.sup.6; or alternatively, an R.sup.2 and an R.sup.2′ join to form a geminal R.sup.2 ring, wherein the geminal R.sup.2 ring is selected from the group consisting of cycloalkyl, cycloalkenyl, and heterocyclyl; and wherein the geminal R.sup.2 ring is additionally substituted with from 0 to 4 R.sup.6; or alternatively, the R.sup.2 is joined into the R.sup.1,2 ring; R.sup.3 is a substituent selected from the group consisting of hydrogen, alkyl, and fluoroalkyl; and R.sup.4 and R.sup.4′ are each a substituent independently selected from the group consisting of hydrogen, alkyl, chloro, fluoro, and fluoroalkyl; or alternatively, an R.sup.4 and an R.sup.4′ join to form a geminal R.sup.4 ring, wherein the geminal R.sup.4 ring is selected from the group consisting of cycloalkyl, cycloalkenyl, and heterocyclyl; and wherein the geminal R.sup.4 ring is additionally substituted with from 0 to 4 R.sup.6; B is selected from the group consisting of aryl, heteroaryl, heterocyclyl, and cycloalkyl; each R.sup.5 is a substituent independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, halo, fluoroalkyl, nitro, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, aminoalkyl, alkylthio, alkylthioalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido, and amidoalkyl; or alternatively, two R.sup.5 join to form an additional R.sup.5 ring, wherein the additional R.sup.5 ring is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocyclyl; and wherein the additional R.sup.5 ring is additionally substituted with from 0 to 5 R.sup.6; n is an integer selected from 0 to 5; wherein if Bis not aryl or if at least two R.sup.5 are not halo, m is an integer selected from 0 to 3; and each R.sup.6 is a substituent independently selected from the group consisting of alkyl, aryl, halo, fluoroalkyl, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl, and amido.
2. The method of claim 1, wherein L is —O— or —C(R.sup.2)(R.sup.2′)—.
3. The method of claim 1, wherein A is aryl.
4. The method of claim 3, wherein A is phenyl, 4-cyanophenyl, 4-nitrophenyl, or 4-fluorophenyl.
5. The method of claim 1, wherein the sulfonamide agonist compound is of Formula II: ##STR00049## wherein: A is heteroaryl; p is an integer selected from 0 to 4; R.sup.4 and R.sup.4′ are independently hydrogen or lower alkyl; and B is heteroaryl.
6. The method of claim 1, wherein the sulfonamide agonist compound is of Formula IIIB: ##STR00050## wherein: p is an integer selected from 0 to 4; R.sup.4 and R.sup.4′ are independently hydrogen or lower alkyl; and each M is independently CH or N.
7. The method of claim 1, wherein the sulfonamide agonist compound is of Formula IV: ##STR00051## wherein: A is selected from the group consisting of 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, 2-pyrrolyl, and 3-pyrrolyl; and R.sup.6a and R.sup.6b are each independently selected from the group consisting of hydrogen, hydroxyl, alkoxy, alkyl, fluoroalkyl, and halo.
8. The method of claim 1, wherein the sulfonamide agonist compound is selected from the group consisting of ##STR00052##
9. The method of claim 1, wherein R.sup.2 is alkyl.
10. The method of claim 1, wherein R.sup.2 and R.sup.2′ join to form a geminal cyclopropyl, cyclobutyl, cyclopenyl, cyclohexyl, 4-methylcyclohexyl, 4,4-dimethylcyclohexyl, piperidinyl, N-acylpiperidinyl, N-alkylpiperidinyl, tetrahydro-2H-pyranyl, or cycloheptyl ring.
11. The method of claim 1, wherein R.sup.4 is hydrogen or alkyl.
12. The method of claim 1, wherein B is aryl.
13. The method of claim 1, wherein R.sup.5 is a para-substituent.
14. The method of claim 1, wherein R.sup.5 is a hydrogen bond acceptor.
15. The method of claim 14, wherein R.sup.5 is independently selected from the group consisting of hydroxyl, lower alkoxy, cyclopropyloxy, amino, lower alkylamino, carboxy, lower alkoxycarbonyl, lower amido, thio, and lower alkylthio.
16. The method of claim 1, wherein R.sup.5 is independently selected from the group consisting of cyano, fluoro, halo, and nitro.
17. The method of claim 1, wherein the abiotic stress tolerance comprises drought tolerance.
18. The sulfonamide agonist compound of the method of claim 1, with the proviso that the compound is not selected from the group consisting of: ##STR00053##
19. An agricultural formulation comprising the sulfonamide agonist compound of claim 18 and an agriculturally acceptable adjuvant.
20. The agricultural formulation of claim 19, further comprising at least one of a fungicide, an herbicide, a pesticide, a nematicide, an insecticide, a plant activator, a synergist, an herbicide safener, a plant growth regulator, an insect repellant, an acaricide, a molluscicide, or a fertilizer.
Description
DETAILED DESCRIPTION OF THE INVENTION
[0135] The present application provides selective abscisic acid (ABA) agonists. The agonists described herein activate the ABA pathway in plants (e.g., plant vegetative tissues) and induce abiotic stress tolerance. The new agonists can be used to induce stress tolerance in crop species of monocot or dicot plants.
[0136] Abscisic acid is a multifunctional phytohormone involved in a variety of phyto-protective functions including bud dormancy, seed dormancy or maturation, abscission of leaves and fruits, and response to a wide variety of biological stresses (e.g., cold, heat, salinity, and drought). ABA is also responsible for regulating stomatal closure by a mechanism independent of CO.sub.2 concentration. The PYR/PYL family of ABA receptor proteins mediate ABA signaling. Plants examined to date express more than one PYR/PYL receptor protein family member, which have at least somewhat redundant activity. PYR/PYL receptor proteins mediate ABA signaling as a positive regulator in, for example, seed germination, post-germination growth, stomatal movement and plant tolerance to stress including, but not limited to, drought.
[0137] A wide variety of wild-type (naturally occurring) PYR/PYL polypeptide sequences are known in the art. Although PYR1 was originally identified as an abscisic acid (ABA) receptor in Arabidopsis, in fact, PYR1 is a member of a group of at least 14 proteins (PYR/PYL proteins) in the same protein family in Arabidopsis that also mediates ABA signaling. This protein family is also present in other plants (see, e.g., SEQUENCE LISTING) and is characterized in part by the presence of one or more or all of a polyketide cyclase domain 2 (PF10604), a polyketide cyclase domain 1 (PF03364), and a Bet V I domain (PF03364). START/Bet v 1 superfamily domain are described in, for example, Radauer, BMC Evol. Biol. 8:286 (2008). In some embodiments, a wild-type PYR/PYL receptor polypeptide comprises any of SEQ ID NOs:1-119. In some embodiments, a wild-type PYR/PYL receptor polypeptide is substantially identical to (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% identical to) any of SEQ ID NOs:1-119. In some embodiments, a PYR/PYL receptor polypeptide is substantially identical to (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% identical to) any of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.
I. ABA Agonists
[0138] The present invention provides for small-molecule ABA agonists, i.e., compounds that activate PYR/PYL proteins. In some aspects, the present invention provides for a compound or a composition as set forth herein (i.e., in one or more of the other aspects, such as formulations or methods, and embodiments herein). In some aspects, the present invention provides a formulation comprising, consisting essentially of, or consisting of a compound as set forth herein. In some aspects, the present invention provides a method of using or use of a compound or formulation as set forth herein.
[0139] In one aspect, the present invention provides a method of increasing abiotic stress tolerance in a plant, the method comprising contacting a plant with an effective amount of a sulfonamide agonist compound to increase abiotic stress tolerance in the plant, thereby increasing abiotic stress tolerance,
[0140] wherein the sulfonamide agonist compound is of Formula I:
##STR00003##
[0141] wherein:
[0142] A is selected from the group consisting of alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and cycloalkyl;
[0143] each R.sup.1 is a substituent independently selected from the group including alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, fluoroalkyl, hydroxyl, hydroxyalkyl, alkoxy, fluoroalkoxy, alkoxyalkyl, amino, aminoalkyl, alkylthio, alkylthioalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido, and amidoalkyl; wherein the R.sup.1 cycloalkyl, heterocyclyl, aryl, or heteroaryl is additionally substituted with from 0 to 3 R.sup.6; or
[0144] alternatively, two R.sup.1 substituents join to form an additional R.sup.1 ring, wherein the additional R.sup.1 ring is selected from the group including aryl, heteroaryl, cycloalkyl, and heterocyclyl; and wherein the additional R.sup.1 ring is additionally substituted with from 0 to 3 R.sup.6; or
[0145] alternatively, an R.sup.1 and a R.sup.2 substituent join to form an R.sup.1,2 ring, wherein the R.sup.1,2 ring is selected from the group including cycloalkyl and heterocyclyl; and wherein the R.sup.1,2 ring is additionally substituted with from 0 to 3 R.sup.6;
[0146] m is an integer selected from 0 to 5; wherein if A is not aryl or if at least two R.sup.1 are not halo, m is an integer selected from 0 to 3;
[0147] L is a bond, —C(R.sup.2)(R.sup.2′)—, —O—, or —NR.sup.6—; wherein if L is a bond, A is not alkyl; R.sup.2 and R.sup.2′ are each a substituent independently selected from the group including hydrogen, alkyl, fluoroalkyl, cycloalkyl, heterocycyl, heteroaryl, and aryl; wherein the cycloalkyl, heterocycyl, heteroaryl, and aryl is additionally substituted with from 0 to 3 R.sup.6; or
[0148] alternatively, an R.sup.2 and an R.sup.2′ join to form a geminal R.sup.2 ring, wherein the geminal R.sup.2 ring is selected from the group including cycloalkyl, cycloalkenyl, and heterocyclyl; and wherein the geminal R.sup.2 ring is additionally substituted with from 0 to 4 R.sup.6; or
[0149] alternatively, the R.sup.2 is joined into the R.sup.1,2 ring;
[0150] R.sup.3 is a substituent selected from the group including hydrogen, alkyl, and fluoroalkyl; and
[0151] R.sup.4 and R.sup.4′ are each a substituent independently selected from the group including hydrogen, alkyl, chloro, fluoro, and fluoroalkyl; or
[0152] alternatively, an R.sup.4 and an R.sup.4′ join to form a geminal R.sup.4 ring, wherein the geminal R.sup.4 ring is selected from the group including cycloalkyl, cycloalkenyl, and heterocyclyl; and wherein the geminal R.sup.4 ring is additionally substituted with from 0 to 4 R.sup.6;
[0153] B is selected from the group including aryl, heteroaryl, heterocyclyl, and cycloalkyl;
[0154] each R.sup.5 is a substituent independently selected from the group including alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, fluoroalkyl, nitro, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, aminoalkyl, alkylthio, alkylthioalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido, and amidoalkyl; or
[0155] alternatively, two R.sup.5 join to form an additional R.sup.5 ring, wherein the additional R.sup.5 ring is selected from the group including aryl, heteroaryl, cycloalkyl, and heterocyclyl; and wherein the additional R.sup.5 ring is additionally substituted with from 0 to 5 R.sup.6;
[0156] n is an integer selected from 0 to 5; wherein if Bis not aryl or if at least two R.sup.5 are not halo, m is an integer selected from 0 to 3; and
[0157] each R.sup.6 is a substituent independently selected from the group including alkyl, aryl, halo, fluoroalkyl, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl, and amido.
[0158] In one aspect, the present invention provides a method of increasing abiotic stress tolerance in a plant, the method comprising a step of contacting a plant with an effective amount of a compound to increase abiotic stress tolerance in the plant, where the compound is of Formula IB:
##STR00004##
[0159] where each R.sup.1 is a substituent independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, fluoroalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, aminoalkyl, alkylthio, alkylthioalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido, or amidoalkyl; or, alternatively, two R.sup.1 substituents join to form an additional R.sup.1 ring, where the additional R.sup.1 ring is selected from aryl, heteroaryl, cycloalkyl, or heterocyclyl; and where the additional R.sup.1 ring is additionally substituted with from 0 to 3 R.sup.6; or an R.sup.1 and a R.sup.2 substituent join to form an R.sup.1,2 ring, where the R.sup.1,2 ring is selected from cycloalkyl or heterocyclyl; and where the R.sup.1,2 ring is additionally substituted with from 0 to 3 R.sup.6;
[0160] m is an integer selected from 0 to 3;
[0161] R.sup.2 and R.sup.2′ are each a substituent independently selected from hydrogen, alkyl,or fluoroalkyl;
[0162] or, alternatively, an R.sup.2 and an R.sup.2′ join to form a geminal R.sup.2 ring, where the geminal R.sup.2 ring is selected from cycloalkyl, cycloalkenyl, or heterocyclyl; and where the geminal R.sup.2 ring is additionally substituted with from 0 to 3 R.sup.6; or the R.sup.2 is joined into the R.sup.1,2 ring;
[0163] R.sup.3 is a substituent selected from hydrogen, alkyl, or fluoroalkyl;
[0164] R.sup.4 and R.sup.4′ are each a substituent independently selected from hydrogen, alkyl, chloro, fluoro, or fluoroalkyl;
[0165] each R.sup.5 is a substituent independently selected alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, fluoroalkyl, nitro, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, aminoalkyl, alkylthio, alkylthioalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido,or amidoalkyl; or, alternatively, two R.sup.5 join to form an additional R.sup.5 ring, wherein the additional R.sup.5 ring is selected from aryl, heteroaryl, cycloalkyl, or heterocyclyl; and wherein the additional R.sup.5 ring is additionally substituted with from 0 to 3 R.sup.6;
[0166] n is an integer selected from 0 to 3; and
[0167] each R.sup.6 is a substituent independently selected from alkyl, aryl, halo, fluoroalkyl, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl, or amido.
[0168] As previously discussed, the embodiments (and further aspects) set forth herein may generally be applied to either Formula I, IB, II, III, IIIB, or IV, except when such application is impossible (e.g., Formula IB does not include A or B as Markush groups) and may be further combined to create subgenera of the embodiments as disclosed.
[0169] In some embodiments, L is —O—. In some embodiments, L is —C(R.sup.2)(R.sup.2′)—. In some embodiments, L is a bond; in some embodiments, A is not alkyl. In some embodiments, L is —NR.sup.6—.
[0170] In some embodiments, A is alkyl or arylalkyl. In some embodiments, A is isobutyl. In some embodiments, A is lower alkyl (e.g., methyl).
[0171] In some embodiments, A is aryl. In some embodiments, A is phenyl, 4-cyanophenyl, 4-nitrophenyl, or 4-fluorophenyl.
[0172] In some embodiments, A is heteroaryl. In some embodiments, A is 2-, 3-, or 4-pyridyl.
[0173] In some embodiments, the sulfonamide agonist compound is
##STR00005##
[0174] In some embodiments, the sulfonamide agonist compound is of Formula II:
##STR00006##
[0175] wherein:
[0176] p is an integer selected from 0 to 4;
[0177] R.sup.4 and R.sup.4: are independently hydrogen or lower alkyl; and
[0178] B is heteroaryl.
[0179] In some embodiments, the sulfonamide agonist compound is of Formula III:
##STR00007##
wherein:
[0180] p is an integer selected from 0 to 4;
[0181] R.sup.4 and R.sup.4′ are independently hydrogen or lower alkyl; and each M is independently C or N.
[0182] In some embodiments, A is selected from the group including 2-thiophenyl, 3-thiophenyl, 2-furanyl, and 3-furanyl. In some embodiments, A is selected from the group including 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, 2-pyrrolyl, and 3-pyrrolyl. In some embodiments, A is selected from the group including 2-thiophenyl and 3-thiophenyl.
[0183] In some embodiments, the sulfonamide agonist compound is of Formula IIIB:
##STR00008##
wherein:
[0184] p is an integer selected from 0 to 4;
[0185] R.sup.4 and R.sup.4′ are independently hydrogen or lower alkyl; and each M is independently CH, CR.sup.5, or N.
[0186] In some embodiments, each M is independently CH or N. In some embodiments, each M is independently CH or CR.sup.5. In some embodiments, each M is CH.
[0187] In some embodiments, A is selected from the group including 2-thiophenyl, 3-thiophenyl, 2-furanyl, and 3-furanyl. In some embodiments, A is selected from the group including 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, 2-pyrrolyl, and 3-pyrrolyl. In some embodiments, A is selected from the group including 2-thiophenyl and 3-thiophenyl.
[0188] In some embodiments, R.sup.5 is a meta-substituent (e.g., 3-halo, such as 3-chloro).
[0189] In some embodiments, the sulfonamide agonist compound is of Formula IV:
##STR00009##
wherein R.sup.6a and R.sup.6b are each independently selected from the group including hydrogen, hydroxyl, alkoxy, alkyl, fluoroalkyl, and halo.
[0190] In some embodiments, the sulfonamide agonist compound is selected from the group including
##STR00010##
[0191] In some embodiments, A is cycloalkyl. In some embodiments, A is cyclohexyl.
[0192] In some embodiments, R.sup.1 is selected from the group including alkyl, fluoroalkyl, alkoxy, and cyano.
[0193] In some embodiments, R.sup.1 is a substituent independently selected from alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, heteroaryl (e.g., pyridyl), halo (e.g., fluoro, chloro), fluoroalkyl, hydroxyl, hydroxyalkyl, alkoxy, fluoroalkoxy, alkoxyalkyl, amino, aminoalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido, or amidoalkyl. In some embodiments, R.sup.1 is alkyl, alkoxy, cyano, or halo.
[0194] In some embodiments, R.sup.1 is independently selected from alkyl, fluoroalkyl, alkoxy, fluoroalkoxy, or cyano. In some embodiments, at least one R.sup.1 is alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, or isobutyl). In some embodiments, at least one R.sup.1 is fluoroalkyl (e.g., trifluoromethyl, perfluoroethyl, or 2,2,2-trifluoroethyl). In some embodiments, at least one R.sup.1 is alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, or isobutoxy). In some embodiments, at least one R.sup.1 is fluoroalkoxy (e.g., trifluoromethoxy or 2,2,2-trifluoroethoxy). In some embodiments, at least one R.sup.1 is cyano (e.g., 4-cyano). In some embodiments, at least one R.sup.1 is selected from cyano, halo, or nitro.
[0195] In some embodiments, R.sup.1 is a para-substituent (e.g., 4-cyano). In some embodiments, R.sup.1 is a meta-substituent. In some embodiments, R.sup.1 is an ortho-substituent.
[0196] In some embodiments, two R.sup.1 substituents (e.g., adjacent R.sup.1 substituents) join to form an additional R.sup.1 ring, where the additional R.sup.1 ring is selected from aryl, heteroaryl, cycloalkyl, or heterocyclyl; and where the additional R.sup.1 ring is additionally substituted with 0, 1, 2, or 3 R.sup.6. In some embodiments, the additional R.sup.1 ring is cyclohexyl or cyclopentyl (e.g., cyclohexyl). In some embodiments, the additional R.sup.1 ring is heteroaryl (e.g., pyridyl, as in 62). In some embodiments, the additional R.sup.1 ring is substituted with 0 or 1 R.sup.6 (e.g., alkyl, such as methyl; halo, such as chloro).
[0197] In some embodiments, an R.sup.1 and a R.sup.2 substituent join to form an R.sup.1,2 ring, where the R.sup.1,2 ring is selected from cycloalkyl or heterocyclyl; and where the R.sup.1,2 ring is additionally substituted with 0, 1, 2, or 3 R.sup.6. In some embodiments, the R.sup.1,2 ring is cyclohexyl or cyclopentyl (e.g., cyclohexyl). In some embodiments, the R.sup.1,2 ring is substituted with 0 or 1 R.sup.6 (e.g., alkyl, such as methyl).
[0198] In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.
[0199] In some embodiments, R.sup.2 is alkyl. In some embodiments, R.sup.2 and R.sup.2′ are selected from the group including methyl, ethyl, and propyl.
[0200] In some embodiments, R.sup.2 and R.sup.2′ join to form the geminal R.sup.2 ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal cyclopropyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal cyclobutyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal cyclopenyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal cyclohexyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal 4-methylcyclohexyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal 4,4-dimethylcyclohexyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal piperidinyl, N-acylpiperidinyl, or N-alkylpiperidinyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal tetrahydro-2H-pyranyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal cycloheptyl ring.
[0201] In some embodiments, R.sup.2 is cycloalkyl. In some embodiments, R.sup.2 is cyclohexyl.
[0202] In some embodiments, R.sup.2 is aryl. In some embodiments, R.sup.2 is 4-fluorophenyl.
[0203] In some embodiments, R.sup.2 and R.sup.2′ are each a substituent independently selected from the group hydrogen, alkyl, or fluoroalkyl. In some embodiments, R.sup.2 is hydrogen. In some embodiments, R.sup.2 and R.sup.2′ are hydrogen. In some embodiments, R.sup.2 is alkyl. In a further aspect, R.sup.2 and R.sup.2′ are each a substituent independently selected from methyl, ethyl, or propyl. In some embodiments, R.sup.2 is fluoroalkyl (e.g., trifluoromethyl).
[0204] In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal R.sup.2 ring, where the geminal R.sup.2 ring is selected from the group cycloalkyl, cycloalkenyl, or heterocyclyl; and where the geminal R.sup.2 ring is additionally substituted with from 0 to 3 R.sup.6. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal R.sup.2 ring. In some further aspects, R.sup.2 and R.sup.2′ join to form a geminal cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl ring (e.g., cyclopropyl). In an alternative further aspect, R.sup.2 and R.sup.2′ join to form a geminal piperidinyl, N-acylpiperidinyl, or N-alkylpiperidinyl ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal ring, where the geminal R.sup.2 ring is additionally substituted with from 0, 1, 2, or 3 R.sup.6 (e.g., alkyl, such as methyl).
[0205] In some embodiments, R.sup.2 is joined into the R.sup.1,2′ ring.
[0206] In some embodiments, R.sup.2 is alkyl. In some embodiments, R.sup.2 and R.sup.2′ are each a substituent independently selected from methyl, ethyl, or propyl.
[0207] In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal R.sup.2 ring. In some embodiments, R.sup.2 and R.sup.2′ join to form a geminal cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl ring. In some alternative embodiments, R.sup.2 and R.sup.2′ join to form a geminal piperidinyl, N-acylpiperidinyl, or N-alkylpiperidinyl ring. In some alternative embodiments, R.sup.2 and R.sup.2′ join to form a geminal tetrahydro-2H-pyranyl ring.
[0208] In some embodiments, the compound is achiral (e.g., when R.sup.2 and R.sup.2′ are both hydrogen, and the compound contains no other enantiomeric centers). In some embodiments, the compound is racemic at the carbon substituted with R.sup.2 and R.sup.2′. In some embodiments, the compound is enantiomerically enriched with the R-stereocenter (i.e., at least 1% enantiomeric excess of the R-stereoisomer at this site, and in some embodiments, at least 10%, 20% 30%, 40%, 50%, 60% 70%, 80%, 90%, 93%, 95%, 97%, 98%, or 99% enantiomeric excess). In some embodiments, the steroecenter is enantiomerically enriched with the S-stereocenter (i.e., at least 1% enantiomeric excess of the S-stereoisomer at this site, and in some embodiments, at least 10%, 20% 30%, 40%, 50%, 60% 70%, 80%, 90%, 93%, 95%, 97%, 98%, or 99% enantiomeric excess).
[0209] In some embodiments, R.sup.3 is hydrogen.
[0210] In some embodiments, R.sup.3 is a substituent selected from the group hydrogen, alkyl, or fluoroalkyl. In some embodiments, R.sup.3 is hydrogen. In some embodiments, R.sup.3 is alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, or isobutyl). In some embodiments, R.sup.3 is fluoroalkyl (e.g., trifluoromethyl, perfluoroethyl, or 2,2,2-trifluoroethyl).
[0211] In some embodiments, R.sup.4 is hydrogen or alkyl. In some embodiments, R.sup.4 and R.sup.4′ are methyl.
[0212] In some embodiments, R.sup.4 and R.sup.4′ are each a substituent independently selected from hydrogen, alkyl, chloro, fluoro, or fluoroalkyl. In some embodiments, R.sup.4 and R.sup.4′ are each independently hydrogen or alkyl. In some embodiments, R.sup.4 is alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, or isobutyl). In some embodiments, R.sup.4 is fluoroalkyl (e.g., trifluoromethyl, perfluoroethyl, or 2,2,2-trifluoroethyl).
[0213] In some embodiments, the compound is achiral (e.g., when R.sup.4 and R.sup.4′ are both hydrogen, and the compound contains no other enantiomeric centers). In some embodiments, the compound is racemic at the carbon substituted with R.sup.4 and R.sup.4′. In some embodiments, the compound is enantiomerically enriched with the R-stereocenter (i.e., at least 1% enantiomeric excess of the R-stereoisomer at this site, and in some embodiments, at least 10%, 20% 30%, 40%, 50%, 60% 70%, 80%, 90%, 93%, 95%, 97%, 98%, or 99% enantiomeric excess). In some embodiments, the steroecenter is enantiomerically enriched with the S-stereocenter (i.e., at least 1% enantiomeric excess of the S-stereoisomer at this site, and in some embodiments, at least 10%, 20% 30%, 40%, 50%, 60% 70%, 80%, 90%, 93%, 95%, 97%, 98%, or 99% enantiomeric excess).
[0214] In some embodiments, the compound is racemic at a site other than the R.sup.2/R.sup.2′ carbon or the R.sup.4/R.sup.4′ carbon. In some embodiments, the compound is enantiomerically enriched with the R-stereocenter (i.e., at least 1% enantiomeric excess of the R-stereoisomer at this site, and in some embodiments, at least 10%, 20% 30%, 40%, 50%, 60% 70%, 80%, 90%, 93%, 95%, 97%, 98%, or 99% enantiomeric excess). In some embodiments, the steroecenter is enantiomerically enriched with the S-stereocenter (i.e., at least 1% enantiomeric excess of the S-stereoisomer at this site, and in some embodiments, at least 10%, 20% 30%, 40%, 50%, 60% 70%, 80%, 90%, 93%, 95%, 97%, 98%, or 99% enantiomeric excess).
[0215] In some embodiments, B is aryl. In some embodiments, B is phenyl.
[0216] In some embodiments, B is heteroaryl.
[0217] In some embodiments, B is a species or Markush that is set forth herein as an embodiment of A.
[0218] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2 (e.g., independently selected para- and meta-substituents). In some embodiments, n is 3.
[0219] In some embodiments, R.sup.5 is a para-substituent. In some embodiments, R.sup.5 is independently selected from the group including cyano, fluoro, halo, and nitro.
[0220] In some embodiments, R.sup.5 is a substituent independently selected from alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, heteroaryl (e.g., pyridyl), halo (e.g., fluoro, chloro), fluoroalkyl, hydroxyl, hydroxyalkyl, alkoxy, fluoroalkoxy, alkoxyalkyl, amino, aminoalkyl, cyano, carboxyl, carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, amido, or amidoalkyl. In some embodiments, R.sup.5 is alkyl, alkoxy, cyano, or halo. In some embodiments, R.sup.5 is selected from cyano, halo, or nitro.
[0221] In some embodiments, R.sup.5 is independently selected from alkyl, fluoroalkyl, alkoxy, fluoroalkoxy, or cyano. In some embodiments, at least one R.sup.5 is alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, or isobutyl). In some embodiments, at least one R.sup.5 is fluoroalkyl (e.g., trifluoromethyl, perfluoroethyl, or 2,2,2-trifluoroethyl). In some embodiments, at least one R.sup.5 is alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, or isobutoxy). In some embodiments, at least one R.sup.5 is fluoroalkoxy (e.g., trifluoromethoxy or 2,2,2-trifluoroethoxy). In some embodiments, at least one R.sup.5 is cyano (e.g., 4-cyano).
[0222] In some embodiments, R.sup.5 is selected from cyano, halo, or nitro. In some embodiments, R.sup.5 is halo. In some embodiments, R.sup.5 is cyano. In some embodiments, R.sup.5 is nitro. In some embodiments, R.sup.5 is para-.
[0223] In some embodiments, R.sup.5 is a hydrogen bond acceptor (i.e., a group that includes an electron pair on a electronegative heteroatom, such as N, O, S, or a halogen). In some embodiments, R.sup.5 is hydroxyl. In some embodiments, R.sup.5 is lower alkoxy or cyclopropyloxy.
[0224] In some embodiments, R.sup.5 is amino. In some embodiments, R.sup.5 is lower alkylamino. In some embodiments, R.sup.5 is carboxy. In some embodiments, R.sup.5 is lower alkoxycarbonyl. In some embodiments, R.sup.5 is amido. In some embodiments, R.sup.5 is lower alkylamido. In some embodiments, R.sup.5 is thio. In some embodiments, R.sup.5 is lower alkylthio. In some embodiments, the R.sup.5 acceptor is para-.
[0225] In some embodiments, R.sup.5 is a para-substituent (e.g., 4-cyano). In some embodiments, R.sup.5 is a meta-substituent (e.g., 3-halo). In some embodiments, R.sup.5 is an ortho-substituent.
[0226] In some embodiments, the ring includes para- and meta- R.sup.5 substituents. In some embodiments, one of the R.sup.5 substituents is a cyano group (e.g., 4-cyano). In some embodiments, one of the R.sup.5 substituents is a halo group (e.g., 3-chloro).
[0227] In some embodiments, two R.sup.5 substituents (e.g., adjacent R.sup.5 substituents) join to form an additional R.sup.5 ring, where the additional R.sup.5 ring is selected from aryl, heteroaryl, cycloalkyl, or heterocyclyl; and where the additional R.sup.5 ring is additionally substituted with 0, 1, 2, or 3 R.sup.6. In some embodiments, the additional R.sup.5 ring is cyclohexyl or cyclopentyl (e.g., cyclohexyl). In some embodiments, the additional R.sup.5 ring is heteroaryl (e.g., pyridyl). In some embodiments, the additional R.sup.5 ring is substituted with 0 or 1 R.sup.6 (e.g., alkyl, such as methyl; halo, such as chloro).
[0228] In some embodiments, each R.sup.6 is a substituent independently selected from alkyl, aryl, halo, fluoroalkyl, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl, or amido. In some embodiments, each R.sup.6 is a substituent independently selected from alkyl, halo, fluoroalkyl, cyano, or carboxyl. In some embodiments, each R.sup.6 is a substituent independently selected from the group alkyl, halo, or fluoroalkyl. In some embodiments, each R.sup.6 is a substituent independently selected from alkyl or halo.
[0229] In some embodiments, the sulfonamide agonist compound is any compound or set of compounds set forth in the specification and drawings of the instant application.
[0230] In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot.
[0231] In some embodiments, the abiotic stress tolerance comprises drought tolerance.
[0232] In some embodiments, the contacting step comprises delivering the formulation to the plant by aircraft or irrigation.
[0233] In some aspects, the present invention provides a method of inhibiting seed germination in a plant, the method comprising contacting a seed with a sufficient amount of the sulfonamide agonist compound set forth herein to inhibit germination. In some embodiments, the method comprises contacting a plant with a sufficient amount of the agricultural formulation set forth herein to inhibit germination.
[0234] In some aspects, the present invention provides a method of reducing transpiration in a plant, the method comprising contacting a plant with a sufficient amount of the sulfonamide agonist compound set forth herein to reduce transpiration. In some embodiments, the method comprises contacting a plant with a sufficient amount of the agricultural formulation set forth herein to reduce transpiration.
[0235] In some aspects, the present invention provides a method of activating a PYR/PYL protein, the method comprising contacting the PYR/PYL protein with the sulfonamide agonist compound set forth herein.
[0236] In some embodiments, the PYR/PYL protein is selectively activated.
[0237] In some embodiments, the PYR/PYL protein is expressed by a cell. In some aspects, the cell is a plant cell.
[0238] In some embodiments, the PYR/PYL protein is PYL-5. In some embodiments, the PYR/PYL protein is PYL-8. In some embodiments, the PYR/PYL protein is PYL-9.
[0239] In some aspects, the method further comprises using a second active compound.
[0240] In some embodiments, the second active compound is a PYR/PYL receptor full agonist. In some embodiments, the second active compound is a PYR/PYL receptor partial agonist.
[0241] In some embodiments, the second active compound is a PYR/PYL receptor superagonist.
[0242] In some embodiments, the second active compound is selected from the group including quinabactin, racemic ABA, R-ABA, and S-ABA.
[0243] In some embodiments, the second active compound is selected from the group including benoxacor, benzothiadiazole, dichlorobenil, fludioxonil, and mandipropamid.
[0244] In some aspects, the present invention provides the sulfonamide agonist compound as disclosed herein, with the proviso that the compound is not selected from the group including:
##STR00011##
[0245] In some aspects, the present invention provides a method of increasing abiotic stress tolerance in a plant, the method comprising: contacting a plant with a sufficient amount of the sulfonamide agonist compound as disclosed herein or the agricultural formulation as disclosed herein, thereby increasing abiotic stress tolerance in the plant.
[0246] In some aspects, the abiotic stress tolerance comprises drought tolerance.
[0247] In some aspects, the method comprises: delivering the compound or the formulation to the plant by aircraft or irrigation.
[0248] In some aspects, the present invention provides a method of inhibiting seed germination in a plant, the method comprising: contacting a seed with a sufficient amount of the sulfonamide agonist composition as disclosed herein.
[0249] In some aspects, the present invention provides a plant in contact with the sulfonamide agonist compound as disclosed herein or the agricultural formulation as disclosed herein. In some aspects, the plant is a seed.
[0250] In some aspects, the present invention provides a method of activating a PYR/PYL protein, the method comprising: contacting the PYR/PYL protein with the sulfonamide agonist compound as disclosed herein or the agricultural formulation as disclosed herein. In some embodiments, the PYR/PYL protein is expressed by a cell. In some embodiments, the cell is a plant cell.
[0251] Exemplary compounds according to the formulas above are shown below in Tables I to V, X, and XI. In some embodiments, each of the substituents included in Tables Ito V, X, and XI can be combined with the other substituents described. For example, certain embodiments include compounds of Formula I with the R.sup.1, R.sup.2, and R.sup.2′ substitution pattern set forth in exemplary compound 24 (i.e., R.sup.1=4-fluoro; R.sup.2 and R.sup.2′ join to form a geminal cyclopentyl ring).
[0252] In some embodiments, the contacting step comprises delivering the formulation to the plant by aircraft or irrigation.
[0253] In some embodiments, the abiotic stress tolerance comprises drought tolerance.
[0254] In some aspects, the present invention provides a plant in contact with a compound or formulation as set forth herein.
II. ABA Agonist Formulations
[0255] The present invention provides agricultural chemical formulations formulated for contacting to plants, wherein the formulation comprises an ABA agonist of the present invention. In some embodiments, the plants that are contacted with the agonists comprise or express an endogenous PYR/PYL polypeptide. In some embodiments, the plants that are contacted with the agonists do not comprise or express a heterologous PYR/PYL polypeptide (e.g., the plants are not transgenic or are transgenic but express heterologous proteins other than heterologous PYR/PYL proteins). In some embodiments, the plants that are contacted with the agonists do comprise or express a heterologous PYR/PYL polypeptide.
[0256] The formulations can be suitable for treating plants or plant propagation material, such as seeds, in accordance with the present invention, e.g., in a carrier. Suitable additives include buffering agents, wetting agents, coating agents, polysaccharides, and abrading agents. In some embodiments, the formulation further comprises a carrier. Exemplary carriers include water, aqueous solutions, slurries, solids and dry powders (e.g., peat, wheat, bran, vermiculite, clay, pasteurized soil, many forms of calcium carbonate, dolomite, various grades of gypsum, bentonite and other clay minerals, rock phosphates and other phosphorous compounds, titanium dioxide, humus, talc, alginate and activated charcoal. Any agriculturally suitable carrier known to one skilled in the art would be acceptable and is contemplated for use in the present invention). Optionally, the formulations can also include at least one surfactant, herbicide, fungicide, pesticide, or fertilizer.
[0257] In some aspects, the present invention provides an agricultural formulation consisting of, consisting essentially of, or comprising a compound as set forth herein.
[0258] In some aspects, the present invention provides an agricultural formulation comprising the sulfonamide agonist compound as disclosed herein and an agriculturally acceptable adjuvant.
[0259] In some embodiments, the formulation further comprises at least one of a fungicide, an herbicide, a pesticide, a nematicide, an insecticide, a plant activator, a synergist, an herbicide safener, a plant growth regulator, an insect repellant, an acaricide, a molluscicide, or a fertilizer.
[0260] In some aspects, the agricultural formulation further comprises a surfactant.
[0261] In some aspects, the agricultural formulation further comprises a carrier.
[0262] In some embodiments, the agricultural chemical formulation comprises at least one of a surfactant, an herbicide, a pesticide, such as but not limited to a fungicide, a bactericide, an insecticide, an acaricide, and a nematicide, a plant activator, a synergist, an herbicide safener, a plant growth regulator, an insect repellant, or a fertilizer. In some embodiments, the formulation further comprises a surfactant.
[0263] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more herbicides selected from paraquat (592), mesotrione (500), sulcotrione (710), clomazone (159), fentrazamide (340), mefenacet (491), oxaziclomefone (583), indanofan (450), glyphosate (407), prosulfocarb (656), molinate (542), triasulfuron (773), halosulfuron-methyl (414), or pretilachlor (632). The above herbicidal active ingredients are described, for example, in “The Pesticide Manual”, Editor C. D. S. Tomlin, 12th Edition, British Crop Protection Council, 2000, under the entry numbers added in parentheses; for example, mesotrione (500) is described therein under entry number 500. The above compounds are described, for example, in U.S. Pat. No. 7,338,920, which is incorporated by reference herein in its entirety.
[0264] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more fungicides selected from sedaxane, fludioxonil, penthiopyrad, prothioconazole, flutriafol, difenoconazole, azoxystrobin, captan, cyproconazole, cyprodinil, boscalid, diniconazole, epoxiconazole, fluoxastrobin, trifloxystrobin, metalaxyl, metalaxyl-M (mefenoxam), fluquinconazole, fenarimol, nuarimol, pyrifenox, pyraclostrobin, thiabendazole, tebuconazole, triadimenol, benalaxyl, benalaxyl-M, benomyl, carbendazim, carboxin, flutolanil, fuberizadole, guazatine, myclobutanil, tetraconazole, imazalil, metconazole, bitertanol, cymoxanil, ipconazole, iprodione, prochloraz, pencycuron, propamocarb, silthiofam, thiram, triazoxide, triticonazole, tolylfluanid, or a manganese compound (such as mancozeb, maneb). In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more of an insecticide, an acaricide, or a nematcide selected from thiamethoxam, imidacloprid, clothianidin, lamda-cyhalothrin, tefluthrin, beta-cyfluthrin, permethrin, abamectin, fipronil, or spinosad. Details (e.g., structure, chemical name, commercial names, etc) of each of the above pesticides with a common name can be found in the e-Pesticide Manual, version 3.1, 13th Edition, Ed. CDC Tomlin, British Crop Protection Council, 2004-05. The above compounds are described, for example, in U.S. Pat. No. 8,124,565, which is incorporated by reference herein in its entirety.
[0265] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more fungicides selected from cyprodinil ((4-cyclopropyl-6-methyl-pyrimidin-2-yl)-phenyl-amine) (208), dodine (289); chlorothalonil (142); folpet (400); prothioconazole (685); boscalid (88); proquinazid (682); dithianon (279); fluazinam (363); ipconazole (468); or metrafenone. Some of the above compounds are described, for example, in “The Pesticide Manual” [The Pesticide Manual—A World Compendium; Thirteenth Edition; Editor: C. D. S. Tomlin; The British Crop Protection Council, 2003], under the entry numbers added in parentheses. The above compounds also are described, for example, in U.S. Pat. No. 8,349,345, which is incorporated by reference herein in its entirety.
[0266] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more fungicides selected from fludioxonil, metalaxyl, or a strobilurin fungicide, or a mixture thereof. In some embodiments, the strobilurin fungicide is azoxystrobin, picoxystrobin, kresoxim-methyl, or trifloxystorbin. In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more of an insecticide selected from a phenylpyrazole or a neonicotinoid. In some embodiments, the phenylpyrazole is fipronil and the neonicotinoid is selected from thiamethoxam, imidacloprid, thiacloprid, clothianidin, nitenpyram or acetamiprid. The above compounds are described, for example, in U.S. Pat. No. 7,071,188, which is incorporated by reference herein in its entirety. In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more biological pesticide, including but not limited to, Pasteuria spp., Paeciliomyces, Pochonia chlamydosporia, Myrothecium metabolites, Muscodor volatiles, Tagetes spp., bacillus firmus, including bacillus firmus CNCM 1-1582.
[0267] In some aspects, the invention presents a formulation or method as set forth herein that further comprises using a second active compound. In some embodiments, the second active compound is a PYR/PYL receptor agonist. In some embodiments, the second active compound is a PYR/PYL receptor partial agonist. In some embodiments, the second active compound is a PYR/PYL receptor partial agonist.
[0268] In some embodiments, the second active compound is selected from the group quinabactin, racemic ABA, R-ABA, or S-ABA. In some embodiments, the second active compound is selected from the group benoxacor, benzothiadiazole, dichlorobenil, fludioxonil, or mandipropamid. In some embodiments, the second active compound is set forth in U.S. Pat. Publ. No. 2010/0216643 or 2013/0324409, which are incorporated by reference herein in their entirety.
III. Application to Plants
[0269] In some embodiments, the agricultural chemical formulations contemplated are formulated for contacting to plants. The formulations can be suitable for treating plants or plant propagation material, such as seeds, in accordance with the present invention, e.g., in a carrier. Suitable additives include buffering agents, wetting agents, coating agents, polysaccharides, and abrading agents. Exemplary carriers include water, aqueous solutions, slurries, solids and dry powders (e.g., peat, wheat, bran, vermiculite, clay, pasteurized soil, many forms of calcium carbonate, dolomite, various grades of gypsum, bentonite and other clay minerals, rock phosphates and other phosphorous compounds, titanium dioxide, humus, talc, alginate and activated charcoal). Any agriculturally suitable carrier known to one skilled in the art would be acceptable and is contemplated for use in the present invention. Optionally, the formulations can also include at least one surfactant, herbicide, fungicide, pesticide, or fertilizer.
[0270] In some aspects, the present invention provides a method of reducing transpiration in a plant, the method comprising contacting a plant with a sufficient amount of the compound of compound of Formula I, IB, II, III, IIIB, or IV as set forth herein, thereby reducing transpiration.
[0271] In some embodiments, a seed, flower, leaf, fruit, processed food, or food ingredient from a plant as described herein is provided. In some embodiments, the plant is a seed.
[0272] In some aspects, the present invention provides a method of activating a PYR/PYL protein, the method comprising contacting the PYR/PYL protein with a compound as set forth herein (e.g., a compound of Formula I). In some embodiments, the method comprises contacting the PYR/PYL protein with a formulation comprising a compound of Formula I, IB, II, III, IIIB, or IV. In some embodiments, the PYR/PYL protein is selectively activated. In some embodiments, the PYR/PYL protein is expressed by a cell. In a further embodiment, the cell is a plant cell. In some embodiments, the PYR/PYL protein is PYL-5. In some alternative embodiments, the PRY/PYL protein is PYL-8. In some alternative embodiments, the PYR/PYL protein is PYL-9.
[0273] Contacting the agricultural chemical formulation to the PYR/PYL receptor polypeptide can be performed in vitro (e.g., wherein the PYR/PYL receptor polypeptide exists in a purified form or is expressed in yeast cells) or in vivo (e.g., wherein the PYR/PYL receptor polypeptide is expressed by a plant). Contacting the agricultural chemical formulation to the PYR/PYL receptor polypeptide in vitro can be performed using a variety of known methods, e.g., by applying the formulation to protein binding assays, mammalian or yeast two-hybrid assays, competition assays, or cell-based assays using other organisms.
[0274] Contacting the agricultural chemical formulation to the PYR/PYL receptor polypeptide in vivo (e.g., to a plant) can be performed using a variety of known methods, e.g., by spraying, atomizing, dusting or scattering the formulations over the propagation material or brushing or pouring or otherwise contacting the formulations over the plant or, in the event of seed, by coating, encapsulating, or otherwise treating the seed. In an alternative to directly treating a plant or seed before planting, the formulations of the invention can also be introduced into the soil or other media into which the seed is to be planted. In some embodiments, a carrier is also used in this embodiment. The carrier can be solid or liquid, as noted above. In some embodiments peat is suspended in water as a carrier of the chemical agonist, and this mixture is sprayed into the soil or planting media or over the seed as it is planted.
[0275] The ABA agonist compounds or formulations can be applied to plants using a variety of known methods, e.g., by spraying, atomizing, dipping, pouring, irrigating, dusting or scattering the formulations over the propagation material, or brushing or pouring or otherwise contacting the formulations over the plant or, in the event of seed, by coating, encapsulating, spraying, dipping, immersing the seed in a liquid formulation, or otherwise treating the seed. In an alternative to directly treating a plant or seed before planting, the formulations of the invention can also be introduced into the soil or other media into which the seed is to be planted. For example, the formulations can be introduced into the soil by spraying, scattering, pouring, irrigating or otherwise treating the soil. In some embodiments, a carrier is also used in this embodiment. The carrier can be solid or liquid, as noted above.
[0276] In some embodiments peat is suspended in water as a carrier of the ABA agonist, and this mixture is sprayed into the soil or planting media or over the seed as it is planted.
[0277] The types of plant that can be treated with the ABA agonists described herein include both monocotyledonous (i.e., monocot) and dicotyledonous (i.e., dicot) plant species including cereals such as barley, rye, sorghum, tritcale, oats, rice, wheat, soybean and corn; beets (for example sugar beet and fodder beet); cucurbits including cucumber, muskmelon, cantaloupe, squash and watermelon; cole crops including broccoli, cabbage, cauliflower, bok choi, and other leafy greens, other vegetables including tomato, pepper, lettuce, beans, pea, onion, garlic and peanut; oil crops including canola, peanut, sunflower, rape, and soybean; solanaceous plants including tobacco; tuber and root crops including potato, yam, radish, beets, carrots and sweet potatoes; fruits including strawberry; fiber crops including cotton and hemp; other plants including coffee, bedding plants, perennials, woody ornamentals, turf and cut flowers including carnation and roses; sugar cane; containerized tree crops; evergreen trees including fir and pine; deciduous trees including maple and oak; and fruit and nut trees including cherry, apple, pear, almond, peach, walnut and citrus.
[0278] It will be understood that the ABA agonists described herein mimic the function of ABA on cells. Thus, it is expected that one or more cellular responses triggered by contacting the cell with ABA will also be triggered be contacting the cell with the ABA agonists described herein. The ABA agonists described herein mimic the function of ABA and are provided in a useful formulation.
[0279] In some embodiments, application of the ABA agonists described herein increases the abiotic stress resistance of a plant.
[0280] In some embodiments, application of the ABA agonists described herein to seeds inhibits germination of the seeds.
[0281] The present invention also provides plants in contact with the ABA formulations described herein. The plant in contact with the ABA formulation can include a plant part or a seed.
IV. Testing ABA Agonists and Antagonists
[0282] Embodiments of the present invention also provide for methods of screening putative chemical agonists to determine whether the putative agonist agonizes a PYR/PYL receptor polypeptide, when the putative agonist is contacted to the PYR/PYL receptor polypeptide. As used herein, an agent “agonizes” a PYR/PYL receptor protein if the presence of the agent results in activation or up-regulation of activity of the receptor, e.g., to increase downstream signaling from the PYR/PYL receptor. For the present invention, an agent agonizes a PYR/PYL receptor if, when the agent is present at a concentration no greater than 200 μM, contacting the agent to the PYR/PYL receptor results in activation or up-regulation of the activity of the PYR/PYL receptor as indicated by a substantial decrease in PP2C activity if measured in vitro, or induction of an ABA-regulated marker gene, or other physiological response (e.g., guard cell closure), if measured in vivo. If an agent does not activate a PYR/PYL receptor protein's activity when the agent is present at a concentration no greater than 200 μM, then the agent does not significantly agonize the PYR/PYL receptor. As used herein, “activation” requires a minimum threshold of activity to be induced by the agent. Determining whether this minimum threshold of activity has been met can be accomplished, e.g., by using an enzymatic phosphatase assay that sets a minimum value for the level of enzymatic activity that must be induced, or by using an enzymatic phosphatase assay in the presence of a colorimetric detection reagent (e.g., para-nitrophenylphosphate) wherein the minimum threshold of activity has been met if a color change is observed.
[0283] The present invention also provides methods of screening for ABA agonists and antagonists by screening for a molecule's ability to induce PYR/PYL-PP2C binding in the case of agonists, or to disrupt the ability of ABA and other agonists to promote PYR/PYL-PP2C binding in the case of antagonists. A number of different screening protocols can be utilized to identify agents that agonize or antagonize a PYR/PYL polypeptide.
[0284] Screening can take place using isolated, purified or partially purified reagents. In some embodiments, purified or partially purified PYR/PYL polypeptide can be used.
[0285] Alternatively, cell-based methods of screening can be used. For example, cells that naturally-express a PYR/PYL polypeptide or that recombinantly express a PYR/PYL polypeptide can be used. In some embodiments, the cells used are plant cells, animal cells, bacterial cells, fungal cells, including but not limited to yeast cells, insect cells, or mammalian cells. In general terms, the screening methods involve screening a plurality of agents to identify an agent that modulates the activity of a PYR/PYL polypeptide by, e.g., binding to PYR/PYL polypeptide, or activating a PYR/PYL polypeptide or increasing expression of a PYR/PYL polypeptide, or a transcript encoding a PYR/PYL polypeptide.
[0286] 1. PYR/PYL Polypeptide Binding Assays
[0287] Optionally, preliminary screens can be conducted by screening for agents capable of binding to a PYR/PYL polypeptide, as at least some of the agents so identified are likely PYR/PYL polypeptide modulators.
[0288] Binding assays can involve contacting a PYR/PYL polypeptide with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation or co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet, J. P. and Yamamura, H. I. (1985) “Neurotransmitter, Hormone or Drug Receptor Binding Methods,” in Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61-89). Other binding assays involve the use of mass spectrometry or NMR techniques to identify molecules bound to PYR/PYL polypeptide or displacement of labeled substrates (e.g., labeled ABA). The PYR/PYL polypeptide protein utilized in such assays can be naturally expressed, cloned or synthesized.
[0289] 2. Activity
[0290] PYR/PYL polypeptide agonists can be identified by screening for agents that activate or increase activity of a PYR/PYL polypeptide. Antagonists can be identified by their reducing activity.
[0291] One activity assay involves testing whether a candidate agonist can induce binding of a PYR/PYL protein to a type 2 protein phosphatase (PP2C) polypeptide in an agonist-specific fashion. Mammalian or yeast two-hybrid approaches (see, e.g., Bartel, P. L. et. al. Methods Enzymol, 254:241 (1995)) can be used to identify polypeptides or other molecules that interact or bind when expressed together in a cell. In some embodiments, agents that agonize a PYR/PYL polypeptide are identified in a two-hybrid assay between a PYR/PYL polypeptide and a type 2 protein phosphatase (PP2C) polypeptide (e.g., ABI1 or 2 or homologs thereof, e.g., from the group A subfamily of PP2Cs), wherein an ABA agonist is identified as an agent that activates or enables binding of the PYR/PYL polypeptide and the PP2C polypeptide. Thus, the two polypeptides bind in the presence, but not in the absence of the agent. In some embodiments, a chemical compound or agent is identified as an agonist of a PYR/PYL protein if the yeast cell turns blue in the yeast two hybrid assay.
[0292] The biochemical function of PYR1, and PYR/PYL proteins in general, is to inhibit PP2C activity. This can be measured in live cells using the yeast two hybrid or other cell-based methods. It can also be measured in vitro using enzymatic phosphatase assays in the presence of a colorimetric detection reagent (for example, para-nitrophenylphosphate). The yeast-based assay used above provides an indirect indicator of ligand binding. To address this potential limitation, one can use in vitro receptor-mediated phosphatase inhibition assays, or cell-based assays using other organisms, as alternative approaches for identifying weak binding target compounds.
[0293] 3. Expression Assays
[0294] Screening for a compound that increases the expression of a PYR/PYL polypeptide is also provided. Screening methods generally involve conducting cell-based or plant-based assays in which test compounds are contacted with one or more cells expressing PYR/PYL polypeptide, and then detecting an increase in PYR/PYL expression (either transcript or translation product). Assays can be performed with cells that naturally express PYR/PYL or in cells recombinantly altered to express PYR/PYL, or in cells recombinantly altered to express a reporter gene under the control of the PYR/PYL promoter.
[0295] Various controls can be conducted to ensure that an observed activity is authentic, including running parallel reactions with cells that lack the reporter construct or by not contacting a cell harboring the reporter construct with test compound.
[0296] 4. Validation
[0297] Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity or determine other biological effects of the agent. In some cases, the identified agent is tested for the ability to effect plant stress (e.g., drought tolerance), seed germination, or another phenotype affected by ABA. A number of such assays and phenotypes are known in the art and can be employed according to the methods of the invention.
[0298] 5. Solid Phase and Soluble High-Throughput Assays
[0299] In the high-throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 or more different compounds are possible using the integrated systems of the invention. In addition, microfluidic approaches to reagent manipulation can be used.
[0300] The molecule of interest (e.g., PYR/PYL or a cell expressing a PYR/PYL polypeptide) can be bound to the solid-state component, directly or indirectly, via covalent or non-covalent linkage.
[0301] The invention provides in vitro assays for identifying, in a high-throughput format, compounds that can modulate the expression or activity of PYR/PYL.
[0302] Abiotic stress resistance can be assayed according to any of a number of well-known techniques. For example, for drought tolerance, plants can be grown under conditions in which less than optimum water is provided to the plant. Drought resistance can be determined by any of a number of standard measures including turgor pressure, growth, yield, and the like.
V. Methods of Increasing Abiotic Stress Tolerance in Plants
[0303] The present invention also provides methods of increasing abiotic stress tolerance in a plant. Thus, in some embodiments, a plant is contacted with an ABA agonist compound as set forth herein, or an ABA agonist formulation as set forth herein, in sufficient amount to increase the abiotic stress tolerance in the plant. The amount of the ABA agonist compound or formulation applied to the plant can be sufficient to increase the abiotic stress tolerance compared to not contacting the plant with the ABA agonist compound or formulation. The plant can be contacted with the ABA agonist compound or formulation using any of the methods described herein. The increase in abiotic stress tolerance can improve the plants growth or survival to abiotic stress conditions that adversely affect the plant's growth or survival. Abiotic stress includes physical or chemical conditions described herein.
[0304] In some embodiments, the plant is a monocot. In some alternative embodiments, the plant is a dicot. In some embodiments, the abiotic stress tolerance comprises drought tolerance.
[0305] In some embodiments, the contacting step comprises delivering the formulation to the plant by aircraft or irrigation.
VI. Methods of Inhibiting Seed Germination in a Plant
[0306] The present invention also provides methods of inhibiting seed germination. Thus, in some embodiments, a plant, plant part, or a seed is contacted with an ABA agonist formulation in an amount sufficient to inhibit seed germination. The seed can be contacted with the ABA formulation using any of the methods described herein. In some embodiments, the seed is directly contacted with the ABA agonist formulation. In some embodiments, the ground or soil is contacted with the ABA agonist formulation either prior to or after planting or sowing the seeds. In some embodiments, a plant is contacted with sufficient ABA agonist formulation to inhibit germination of seeds that later develop from the plant. In some aspects, the present invention provides a method of inhibiting seed germination in a plant, the method comprising contacting a seed with a sufficient amount of the compound or formulation as set forth herein, thereby inhibiting germination.
VII. Methods of Activating a PYR/PYL Receptor Protein
[0307] The present invention also provides methods of activating a PYR/PYL receptor protein. In some embodiments, a PYR/PYL protein is contacted with a compound or formulation set forth herein. In some embodiments, the activated PYR/PYL protein binds to a PP2C polypeptide. In some embodiments, the PYR/PYL protein that is activated is substantially identical to any one of SEQ ID NOs:1-119. Examples of sequences of ABA receptors from various plants are provided in U.S. Patent Publication 2011/0271408, which is incorporated by reference herein in its entirety.
[0308] In some embodiments, the PYR/PYL protein is an endogenous protein. In some embodiments, the PYR/PYL protein is a heterologous protein. In some embodiments, the cell further expresses a type 2 protein phosphatase (PP2C). In some embodiments, the type 2 protein phosphatase is HAB1 (Homology to ABI1), ABI1 (Abscisic acid insensitive 1), or ABI2 (Abscisic acid insensitive 2).
[0309] In some embodiments, the PYR/PYL protein is expressed by a cell. In some embodiments, the cell is a plant cell. In some alternative embodiments, the cell is a plant, animal, mammalian, or fungal cell.
[0310] In some embodiments, the method activates a PYR/PYL receptor in a cell free in vitro assay. In some embodiments, the method activates a PYR/PYL receptor expressed in a cell. In some embodiments, the cell also expresses a PP2C polypeptide. In some embodiments, the cell is a plant cell. In some embodiments, the cell is an animal or mammalian cell. In some embodiments, the cell expresses an endogenous PYR/PYL protein. In some embodiments, the cell is engineered to express a heterologous PYR/PYL polypeptide. In some embodiments, the cell expresses a heterologous PP2C polypeptide. In some embodiments, the cell expresses a PP2C polypeptide selected from HAB1 (homology to ABI1), ABI1, or ABI2.
[0311] In some embodiments, the activated PYR/PYL polypeptide induces expression of heterologous genes. In some embodiments, the heterologous genes are ABA responsive genes. In some embodiments, the induced gene expression occurs in cells that express an endogenous PYR/PYL polypeptide. In some embodiments, the induced gene expression occurs in cells that express a heterologous PYR/PYL polypeptide.
[0312] It is understood that the examples and embodiments described herein are for illustrative purposes only. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES
Example 1
ABA Activity of N-Acylsulfonamide Derivatives
[0313] 1. N-Acylsulfonamide agonists of Monomeric Receptor Activity
[0314] A family of 62 structurally related N-acyl sulfonamides (Tables I-V) were tested for ABA agonist activity using multiple receptor-mediated PP2C inhibition in vitro assays (Park et al., 2009). Recombinant proteins for all Arabidopsis receptors were prepared, with the exceptions of PYL7 and PYL12, which failed to yield active proteins. The constructs utilized for protein expression have been previously described (Okamoto et al. 2013). For constructs encoding 6X-His-fusion proteins (all receptors except PYL11), the coding sequences were of the receptors were cloned in the vector pET28, expressed in BL21[DE3]pLysS E. coli host cells at 18° C. overnight, and subsequently purified from sonicated lysates using Ni-NTA agarose (Qiagen, USA), according to the manufacturer's instructions. PYL11 was constructed as a maltose binding protein fusion in the vector pMAL-c, expressed in BL21┌DE3┐pLysS host cells and purified using amylose resin (New England Biolabs) as described by the manufacturer. Recombinant GST-HAB1 was expressed and purified as described previously (Park et al., 2009). PP2C activity assays were conducted using the fluorogenic phosphatase substrate 4-methylumbelliferyl-phosphate. Recombinant receptors and HAB1 were used to examine ligand-induced PP2C inactivation in response to multiple test compounds. Enzyme inhibition assays were conducted using the following assay conditions: 50 nM GST-PP2C, 100 mM Tris-OAc (pH 7.9), 100 mM NaCl, 1 mM MnCl.sub.2, 1% β-mercaptoethanol. The activity of recombinant ABA receptors dropped rapidly in the 24-48 hours after purification and then stabilized, suggesting inactivation of a subset of the receptors after purification. Because of this, the ratio of each receptor used in the PP2C-inhibitions assays was established empirically as the minimum fold-excess of receptor required to elicit maximal PP2C inhibition at a saturating ABA concentration (10 04).
[0315] Based on this criterion, we used a ratio of 2:1 receptor in the assays described herein.
[0316] Using the above assay conditions, compounds were tested at 25 μM for agonist activity on PYL4, PYLS, PYL8 and PYL9. As shown in Table VI, most of the compounds tested showed activity on at least one of the receptors tested. Consequently, a subset of the compounds was tested at multiple concentrations against 11 receptors so that IC.sub.50 values and receptor selectivity profiles could be examined. As shown in Table VII, these assays revealed many compounds with nanomolar IC.sub.50 values (shown in bold in Table VII); thus, the N-acylsulfonamide agonists disclosed include potent activators of monomeric ABA receptors. Importantly, none of the compounds tested appreciably activated dimeric ABA receptors (i.e., none possessed IC.sub.50 values less than 50 μM). Thus, the N-acylsulfonamide agonists disclosed are selective for monomeric receptors.
[0317] The tricyclic N-acylsulfonaimdes shown in Table II incorporated rings into the alpha carbon of their phenacyl substructures (position n in Table II). Compounds with cyclopropyl rings (n=3) provided potent and relatively selective PYLS agonists (i.e., compounds 30, 50 and 44). Moreover, compounds incorporating five- and six-membered rings into the same position provided potent and relatively selective PYL9 agonists (i.e., compounds 51 and 24). These data define the alpha carbon as a valuable site for controlling both agonist selectivity and potency.
[0318] 2. N-Acylsulfonamides Regulate ABA Signaling in Planta
[0319] To examine if the agonists identified possess bioactivity in vivo, we examined the effects of several compounds on Arabidopsis seed germination and hypocotyl growth. We germinated seeds from wild type on ½×strength Murashige and Skoog salts growth medium containing either 10 or 50 μM for all 62 compounds as well as 1 μM (+)-ABA and mock controls. As shown in Table VIII, 41 of the 62 compounds tested inhibit seed germination or hypocotyl growth, as expected of ABA agonists. To investigate if the any of the compounds are sufficient to control vegetative ABA responses, we treated three week old Arabidopsis plants with aqueous solutions containing 0.02% Silwet and either 50 μM of selected N-acylsulfonamides, 50 μM ABA or 0.1% DMSO (the carrier solvent for the compounds tested). The leaf temperatures were subsequently examined using a thermal imaging camera 24 hours after treatment. It is well known that ABA-induced guard cell closure reduces transpiration, which leads to increases in leaf temperatures. It has been estimated that a 1° C. increase in leaf temperature correlates with a decrease in transpiration rates by approximately 50% (Sirault et al., 2009). Since guard cell aperture is the primary determinant of transpiration rates, thermal imaging is a useful way to indirectly infer relative transpiration rates and, indirectly, relative degrees of guard cell opening at the whole plant level. As shown in Table IX, several N-acylsulfonamides altered leaf temperature within 24 hours of treatment, indicating they reduced transpiration by inducing guard cell closure, as expected for ABA agonists. These biological data indicate broad potency of the N-acylsulfonamide agonist family in vivo.
TABLE-US-00001 TABLE I Exemplary Structures
TABLE-US-00002 TABLE II Exemplary Structures
TABLE-US-00003 TABLE III Exemplary Structures
TABLE-US-00004 TABLE IV Exemplary Structures
TABLE-US-00005 TABLE V Exemplary Structures Cmpd. No. Structure 61
TABLE-US-00006 TABLE VI PP2C Activity (25 μm) Cmpd. No. PYL4 PYL5 PYL8 PYL9 1 96 106 89 83 24 92 99 21 10 25 94 102 37 21 26 99 105 33 18 27 97 39 77 63 56 94 105 106 108 28 96 65 55 37 29 96 90 28 14 30 90 12 82 69 31 97 77 80 74 32 92 74 24 12 33 98 81 39 24 34 95 99 103 107 35 94 20 62 49 2 89 23 69 47 36 91 34 98 94 37 84 30 78 60 3 90 47 35 18 4 97 106 59 36 5 89 86 83 61 6 90 21 52 32 57 91 100 56 39 38 92 41 77 66 58 96 83 84 83 59 92 65 97 98 39 98 110 71 66 53 89 95 99 98 7 90 33 54 38 40 92 101 106 103 41 93 27 89 81 42 88 45 65 42 8 91 41 77 69 61 91 16 75 68 9 91 43 58 47 43 95 95 41 46 10 93 75 83 73 44 89 18 63 50 11 93 44 62 58 45 93 30 74 53 12 92 43 62 47 13 98 37 32 17 14 97 103 108 108 46 95 24 73 55 47 95 28 86 75 15 96 51 57 46 16 93 47 43 30 17 85 96 95 94 18 91 12 52 34 54 97 68 106 109 48 93 76 60 59 19 86 85 36 15 20 95 88 104 98 49 92 77 93 81 21 89 89 98 102 22 95 85 91 92 50 67 7 36 18 51 93 97 16 8 52 94 99 91 98 62 98 103 100 100 55 96 95 72 49 60 90 70 101 101 23 ND ND ND ND
TABLE-US-00007 TABLE VII PP2C Dose Curves Cmpd. No. PYR1 PYL1 PYL2 PYL3 PYL4 PYL5 PYL6 PYL8 PYL9 PYL10 PYL11 24 >50 >50 — — >50 >50 >50 6.38 0.60 14.77 4.09 25 — — — — — — — 12.56 6.69 >50 12.71 26 — — — — — — — 11.51 7.11 41.60 7.52 29 — — — — — >50 >50 3.36 1.27 >50 1.77 30 — — >50 >50 — 0.86 >50 >50 >50 — 4.63 32 — — — — — >50 >50 3.23 1.40 >50 2.25 35 — — — — — 3.91 34.70 >50 27.18 >50 3.29 3 — — — — — 29.51 45.53 8.58 3.00 — 1.94 6 — — >50 — — >50 >50 26.28 8.96 >50 4.16 7 — — >50 — — 10.71 >50 40.04 18.45 — 13.95 42 — — — — — 29.40 >50 52.41 17.39 17.89 14.70 43 — — — — — — — 22.69 28.24 >50 >50 44 — — >50 — — 2.48 12.17 49.47 21.15 79.12 13.26 13 — — >50 — — 11.09 18.92 4.45 1.62 >50 2.48 46 — — — — — 7.06 15.27 111.47 39.62 — 18.51 18 — — >50 >50 >50 1.95 12.60 18.61 8.29 31.48 14.92 19 >50 >50 >50 >50 >50 >50 57.13 9.65 3.90 50.83 2.44 50 — — 54.71 20.84 >50 0.39 2.43 8.39 5.76 >50 2.52 51 — — — — — >50 >50 1.11 0.04 4.46 5.33 23 — — — — — 39.63 >50 48.92 1.88 >50 7.84
TABLE-US-00008 TABLE VIII Germination Effects Compound Germination Hypocotyl Growth No. Inhibition Inhibition Mock − − ABA 1 1 − 50 24 10 25 50 10 26 50 10 27 − 50 56 − − 28 − 10 29 50 10 30 − 50 31 − − 32 50 10 33 − 10 34 50 35 − 10 2 50 10 36 − − 37 − 50 3 50 10 4 − 50 5 − 50 6 50 10 57 − − 38 − 50 58 − − 59 − − 39 50 10 53 − 50 7 50 10 40 − 41 − 10 42 50 10 8 50 10 61 − 50 9 − 10 43 50 10 10 − 50 44 − 50 11 − − 45 − − 12 − 50 13 10 10 14 − − 46 − − 47 − − 15 − 10 16 50 10 17 − 50 18 − − 54 − 50 48 50 19 10 20 − − 49 − − 21 − − 22 − 10 50 − 51 10 52 − − 62 − − 55 − − 60 − − 23 50 10
TABLE-US-00009 TABLE IX Thermal Response Compound Thermal No. Response Mock − ABA ++++ 29 +++ 30 + 32 +++ 35 − 13 +++ 18 ++ 50 + 51 +
Example 2
Synthesis of N-Acylsulfonamide Derivatives and N-Sulfonylcarbamate Derivatives
[0320] Schemes 1 and 2 below show the general route for the preparation of N-acylsulfonamide and N-sulfonyl carbamate derivatives. A series of exemplary derivatives are set forth in Tables X and XI.
##STR00020## ##STR00021##
##STR00022## ##STR00023##
General Methods:
[0321] Unless stated otherwise, all reactions were carried out under an atmosphere of nitrogen in oven-dried glassware. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (rt) is noted as 25° C. All other solvents were of anhydrous quality purchased from Aldrich Chemical Co. and used as received. Pure reaction products were typically dried under high vacuum. Commercially available starting materials and reagents were purchased from Aldrich, TCI and Fisher Scientific and were used as received unless specified otherwise. Analytical thin layer chromatography (TLC) was performed with (5 ×20 cm, 60 Å, 250 μm). Visualization was accomplished using a 254 nm UV lamp. .sup.1H NMR spectra were recorded on Inova 400 MHz spectrophotometer. Chemical shifts are reported in ppm with the solvent resonance as internal standard ([CDCl.sub.3 7.27 ppm, 77.23 ppm] [DMSO-d.sub.6 2.5 ppm, 39.51 ppm] and [MeODd.sub.4 4.78, 49.0] for .sup.1H, .sup.13C respectively). Data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, dd=doublet of doublet, t=triplet, q=quartet, br=broad, m=multiplet, abq=ab quartet), number of protons, and coupling constants. High resolution mass spectral data was collected using a Agilent 6224 LC-TOF. All compounds submitted for biological testing were found to be ≧95% pure.
[0322] 1-(4-Fluorophenyl)-4-methylcyclohexanecarbonitrile (4a). To a solution of 2a (4.8 g, 0.019 mol) and 1a (2.5 g, 0.019 mol) in anhydrous DMF was added NaH (1.85 g, 0.046 mol) portion wise at 0° C. and the reaction allowed to attain room temperature and stirred for 12 h. The reaction was quenched with water and extracted with EtOAc (3×30 mL). The combined organic extracts were washed with brine, dried (Na.sub.2SO.sub.4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (100:0.fwdarw.80:20, hexanes: EtOAc) to a give compound 4a as a pale yellow oil (3.2 g, 80%). .sup.1H NMR (400 MHz, CDCl3): δ 7.40-7.48 (m, 2H), 7.01-7.10 (m, 2H), 2.07-2.17 (m, 2H), 1.92-2.02 (m, 2H), 1.77-1.88 (m, 3H), 1.39-1.59 (m, 3H), 0.95-0.93 (m, 3H).
[0323] 1-(4-Fluorophenyl)-4,4-dimethylcyclohexanecarbonitrile (5a). The synthesis of intermediate 5a is similar to that reported for intermediate 4a, and 5a is obtained as a white solid in 70% yield. .sup.1H NMR (400 MHz, CDCl3): δ 7.39-7.51 (m, 2H), 7.02-7.11 (m, 2H), 2.01-1.47 (m, 8H), 1.03 (s, 3H), 0.97 (s, 3H).
[0324] 1-(Thiophen-2-yl)-1-cyclohexane-1-carbonitrile (15a) The synthesis of intermediate 15a is similar to that reported for intermediate 4a, and 15a is obtained as a yellow oil in 75% yield. .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ 7.48-7.54 (m, 1H), 7.13-7.19 (m, 1H), 6.99-7.04 (m, 1H), 2.22 (d, J=12.87Hz, 2H), 1.8-1.54 (m, 8H).
[0325] 1-(Thiophen-3-yl)-1-cyclohexane-1-carbonitrile (16a) The synthesis of intermediate 16a is similar to that reported for intermediate 4a, and 16a is obtained as a yellow oil in 88% yield. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.54-7.60 (m, 1H), 7.49 (dd, J=2.73, 1.56 Hz, 1H), 7.19-7.26 (m, 1H), 2.07-2.16 (m, 2H), 1.63-1.80 (m, 6H), 1.48-1.63(m, 2H).
[0326] 1-(4-Fluorophenyl)-4-methylcyclohexanecarboxylic acid (6a). To a solution of 4a (2 g, 9.2 mmole) in 20 mL of 1,3 propanediol was added freshly powdered NaOH (3.7 g, 92 mmol) and the mixture stirred in a pressure vessel for 3 days at 110° C. Upon completion, the reaction is cooled to room temperature, quenched with concentrated hydrochloric and cooled for an hour at 0° C. to a give compound 6a as a white solid (1.5 g, 68%) which is filtered off and used without further purification.
[0327] 1-(4-Fluorophenyl)-4,4-dimethylcyclohexanecarboxylic acid (7a). The synthesis of intermediate 7a is similar to that reported for intermediate 6a, and 7a is obtained as a white solid in 55% yield.
[0328] 1-(Thiophen-2-yl)-1-cyclohexane-1-carboxylic acid (17a) The synthesis of intermediate 17a is similar to that reported for intermediate 6a, and 17a is obtained as a white solid in 75% yield.
[0329] 1-(Thiophen-3-yl)-1-cyclohexane-1-carboxylic acid (18a) The synthesis of intermediate 18a is similar to that reported for intermediate 6a, and 18a is obtained as a white solid in 60% yield.
[0330] 1-(3-Bromo-4-cyanophenyl)methanesulfonamide. To a suspension of 1-(4-cyanophenyl)-methanesulfonamide (2 g, 0.01 mol) in anhydrous dichloroethane was added N-bromosuccinmide (3.26 g, 0.0183 mol), Pd(OAc).sub.2 (0.228 g, 0.00102 mol) and p-toluenesulfonic acid (1.938 g, 0.0102). The suspension was heated in a sealed pressure vessel at 70′C for 12 h. After cooling to room temperature, the volatiles were removed under pressure, and the residue was purified by flash chromatography chromatography (100:0.fwdarw.10:90, hexanes: EtOAc) to give the brominated sulfonamide as a white solid (2.5 g, 89% yield.) . .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.98-7.91 (m, 1H), 7.79-7.86 (m, 1H), 7.58-7.50 (m, 1H), 6.93 (s, 2H), 4.34 (s, 2H).
General Procedure for Synthesis of N-Acylsulfonamide Derivatives.
[0331] To a solution of acid (1 equiv) was added EDCI (1.5 equiv) and DMAP (2 equiv) in anhydrous dichloroethane (10 mL/mmol of acid) and stirred at room temperature for an hour. The 4-cyanobenzyl sulfonamide (1.2 equiv) is added to the reaction mixture and the reaction heated at 65° C. overnight. The reaction quenched by adding brine and extracted with EtOAc (30 mL/mmole of acid). The organic extracts were washed with brine, dried (Na.sub.2SO.sub.4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (100:0.fwdarw.50:50, hexanes:EtOAc) to give compounds 67, 68, 69, 71, 73, 75, and 78 as white foamy solids in 65-70% yields. The compound 72 was obtained in 20% yield.
[0332] N-[(4-Cyanophenyl)methanesulfonyl]-1-(4-fluorophenyl)-4-methylcyclohexane-1-carboxamide (67) .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.93 (bs, 1H), 7.60-7.70 (m, 2H), 7.35-7.42 (m, 2H), 7.14-7.24 (m, 4H), 4.74 (s, 2H), 1.76-1.35 (m, 4H), 0.99-0.73 (m, 6H). HRMS (ESI) m/z [M+H].sup.+ for C.sub.22H.sub.23FN.sub.2O.sub.3S, found 415.15.
[0333] N-[(4-Cyanophenyl)methanesulfonyl]-1-(4-fluorophenyl)-4,4-dimethylcyclohexane-1-carboxamide (68) .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.2 (bs, 1H), 7.66 (d, J=8.19 Hz, 2H), 7.36-7.27 (m, 4H), 7.23-7.16 (m, 2H), 4.81 (s, 2H), 2.32 (d, J=13.26 Hz, 2H), 1.67-1.81 (m, 2H), 1.20-1.27 (m, 4H), 0.87 (s, 3H), 0.82 (s, 3H). HRMS (ESI) m/z [M+H].sup.+ for C.sub.23H.sub.25FN.sub.2O.sub.3S, found 429.16.
[0334] N-[(4-Cyanophenyl)methanesulfonyl]-1-phenylcyclohexane-1-carboxamide (69) .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.1 (bs, 1H), 7.64 (d, J=8.19 Hz, 2H), 7.28-7.37 (m, 5H), 7.20-7.26 (m, 2H), 4.80 (s, 2H), 2.41 (d, J=13.26 Hz, 2H), 1.44-1.11 (m, 10H). HRMS (ESI) m/z [M+H].sup.+ for C.sub.21H.sub.22N.sub.2O.sub.3S, found 383.14.
[0335] N-[(4-Cyanophenyl)methanesulfonyl]-2,2-diphenylacetamide (71).sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 12.1 (bs, 1H), 7.64-7.72 (m, 2H), 7.32-7.39 (m, 4H), 7.24-7.31 (m, 8H) 4.98 (s, 1H), 4.82 (s, 1H). HRMS (ESI) m/z [M+H].sup.+ for C.sub.22H.sub.18N.sub.2O.sub.3S, found 391.11.
[0336] N-[(4-Cyanophenyl)methanesulfonyl]-2,2-bis(4-fluorophenyl)acetamide (72).sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 12.15 (bs, 1H), 7.71-7.76 (m, 2H), 7.34-7.07 (m, 10H), 5.00 (s, 1H), 4.82 (s, 1H). HRMS (ESI) m/z [M+H].sup.+ for C.sub.22H.sub.16F.sub.2N.sub.2O.sub.3S, found 427.09.
[0337] N-[(4-Cyanophenyl)methanesulfonyl]-2,2-cyclohexylacetamide (73).sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.64 (bs, 1H), 7.98-7.74 (m, 2H), 7.67-7.63 (m, 2H), 4.95 (s, 2H), 2.09 (m, 1H), 1.66-1.42 (m, 22H). HRMS (ESI) m/z [M+H].sup.+ for C.sub.22H.sub.30N.sub.2O.sub.3S, found 403.20.
[0338] N-[(4-Cyanophenyl)methanesulfonyl]-1-(thiophen-2-yl)cyclohexane-1-carboxamide (75).sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.20 (bs, 1H), 7.65-7.69 (m, 2H), 7.52-7.50 (m, 1H), 7.17-7.22 (m, 2H), 7.04-7.02 (m, 1H), 6.97-7.00 (m, 1H), 4.79 (s, 2H), 2.42 (d, J=13.26 Hz, 2H), 1.75-1.70 (m, 2H), 1.55-1.45 (m, 3H), 1.34-1.22 (m, 3H). HRMS m/z [M+H].sup.+ for C.sub.19H.sub.20N.sub.2O.sub.3S.sub.2, found 389.09.
[0339] N-[(4-Cyanophenyl)methanesulfonyl]-1-(thiophen-3-yl)cyclohexane-1-carboxamide (78) .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.06 (bs, 1H), 7.65-7.70 (m, 2H), 7.54-7.58 (m, 1H), 7.27-7.32 (m, 2H), 7.19-7.24 (m, 2H), 7.10-7.08 (m, 1H), 4.79 (s, 2H), 2.40 (d, J=13.65 Hz, 2H), 1.68-1.63 (m, 2H), 1.55-1.45 (m, 3H), 1.34-1.22 (m, 3H). HRMS m/z [M+H].sup.+ for C.sub.19H.sub.20N.sub.2O.sub.3S.sub.2, found 389.09.
[0340] N-[(4-Cyano-3-bromophenyl)methanesulfonyl]-1-(thiophen-3-yl)cyclohexane-1-carboxamide (106).sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.06 (bs, 1H), 7.79 (d, J=7.80 Hz, 1H 2H), 7.75-7.59 (m, 1H), 7.58-7.34 (m, 1H), 7.32-7.13 (m, 2H), 7.03 (dd, J=5.07 Hz, 1.17 Hz, 1H), 4.79 (bs, 2H), 2.36 (d, J=11.70 Hz, 2H), 1.70-1.53 (m, 2H), 1.34-1.19 (m, 3H), 1.19-1.08 (m, 3H). HRMS m/z [M+H].sup.+ for C.sub.19H.sub.19N.sub.2O.sub.3S.sub.2Br, found 467.01.
General Procedure for Synthesis of N-Sulfonylcarbamate Derivatives.
[0341] To an ice cold solutionp-cyanobenzyl sulfonamide (1 equiv) was added NaH (1.5 equiv) portionwise and stirred at the same temperature for 1 hr. A solution of chloroformate 11a or 12a (1.2 equiv) was then added dropwise, and the reaction stirred at room temperature for 12 hr. The reaction was quenched by adding brine and extracted with EtOAc (30 mL/mmol of p-cyanobenzylsulfonamide). The organic extracts were washed with brine, dried (Na.sub.2SO.sub.4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (100:0.fwdarw.50:50, hexanes:EtOAc) to a give compound 65 as a white solid in 60% yield.
[0342] 2-Methylpropyl N-[(4-cyanophenyl)methanesulfonyl]carbamate (65) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.70-7.66 (m, 2H), 7.52-7.48 (m, 2H), 7.08 (s, 1H), 4.01 (d,J=6.63, 2H), 1.93-2.03 (m, 1H), 0.93-0.97 (m, 6H). HRMS (ESI) m/z [M+H].sup.| for C.sub.13H.sub.16N.sub.2O.sub.4S, found 297.09.
TABLE-US-00010 TABLE X Exemplary Structures Compound Structure No. 63
General Procedure for Synthesis of Additional Derivatives
[0343] The following compounds are made by similar procedures to those above.
TABLE-US-00011 TABLE XI Exemplary Structures
Example 3
Biological Activity of Additional N-Acylsulfonamide and N-Sulfonylcarbamate Derivatives
[0344] Additional compounds were tested for biological activity following the procedures of Example 1 with minor modifications. The results are indicated in Tables XII to XV.
TABLE-US-00012 TABLE XII PP2C Activity (50 μm) Cmpd. No. PYR1 PYL1 PYL2 PYL3 PYL4 PYL5 PYL6 PYL8 PYL9 PYL10 PYL11 63 101.4 101.1 115.2 126.6 103.4 107.3 108.5 96.6 75.5 88.9 92.6 64 98.4 101.7 106.8 97.4 105.1 103.2 108.8 101.1 91.1 89.8 97.5 65 96.2 91.7 95.2 92.9 95.5 23.6 58.9 35.8 18.6 39.2 42.1 67 99.6 98.3 93.0 101.0 91.9 43.0 46.6 25.8 8.0 48.2 14.7 68 94.4 96.0 94.8 98.3 94.0 102.7 46.6 57.5 22.6 83.3 16.2 69 96.0 98.4 96.2 102.8 95.0 118.9 72.6 14.7 5.8 27.3 13.5 71 97.9 93.8 102.5 101.6 93.2 102.0 96.9 96.8 81.8 86.5 43.2 72 93.9 94.2 100.5 100.0 90.1 101.6 95.1 98.5 85.8 98.0 37.4 73 91.6 95.5 93.8 92.7 98.2 109.7 91.0 66.1 56.3 85.7 26.9 75 96.1 95.5 89.5 95.4 98.0 81.8 57.2 14.9 6.9 27.0 9.8 78 90.9 95.6 81.2 102.8 101.6 68.5 44.9 14.3 6.1 25.6 9.9 106 98.9 94.3 29.6 96.9 89.2 11.9 17.6 13.6 5.4 22.5 7.8 ABA 6.0 5.9 6.1 5.4 10.2 4.9 10.9 7.0 6.3 13.8 5.7 (10 μM)
TABLE-US-00013 TABLE XII-B PP2C Activity (IC.sub.50 in uM) Cmpd. No. PYR1 PYL1 PYL2 PYL3 PYL4 PYL5 PYL6 PYL8 PYL9 PYL10 PYL11 63 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 64 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 65 >50 >50 >50 >50 >50 14.2 >50 20.4 6.5 19.2 50 67 >50 >50 >50 >50 >50 25.3 50 9.6 2.131 50.0 3.130 68 >50 >50 >50 >50 >50 >50 50 >50 17.70 >50 2.220 69 >50 >50 >50 >50 >50 >50 >50 0.750 0.135 1.690 2.530 71 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 25 72 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 25 73 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 22.40 75 >50 >50 >50 >50 >50 >50 >50 0.134 0.083 0.380 0.390 78 >50 >50 >50 >50 >50 >50 25 0.073 0.038 0.250 0.190 106 >50 >50 32.96 >50 >50 7.261 7.841 0.037 0.014 0.022 0.065
[0345] For these in vitro receptor assays, the compounds listed were tested in in vitro PP2C inhibition assays with 50 uM of the test compound except for ABA (10 uM). IC.sub.50s were calculated from dose curves, but only for selected compounds.
TABLE-US-00014 TABLE XIII Germination Inhibition II Compound Germination Hypocotyl Growth No. Inhibition Inhibition Mock − − ABA 1 1 − 50 24 10 25 50 10 26 50 10 27 − 50 56 − − 28 − 10 29 50 10 30 − 50 31 − − 32 50 10 33 − 10 34 50 35 − 10 2 50 10 36 − − 37 − 50 3 50 10 4 − 50 5 − 50 6 50 10 57 − − 38 − 50 58 − − 59 − − 39 50 10 53 − 50 7 50 10 40 − 41 − 10 42 50 10 8 50 10 61 − 50 9 − 10 43 50 10 10 − 50 44 − 50 11 − − 45 − − 12 − 50 13 10 14 − − 46 − − 47 − − 15 − 10 16 50 10 17 − 50 18 − − 54 − 50 48 50 19 10 20 − − 49 − − 21 − − 22 − 10 50 − 51 10 52 − − 62 − − 55 − − 60 − − 23 50 10
[0346] For the germination inhibition experiment, the compounds were tested at 10 and 50 uM. The amounts shown are the lowest of the two concentrations that either inhibited germination or hypocotyl growth in wells where seeds germinated.
TABLE-US-00015 TABLE XIV Thermal Response II Compound No. Response 63 nd 64 nd 65 nd 67 — 68 + 69 +++ 71 nd 72 nd 73 nd 75 +++ 78 +++ 106 +++ 13 ++ ABA +++
[0347] For the thermal response experiment, the compounds were tested by spraying Arabidopsis plants with 50 uM solution of test compound. Thermal imaging was then conducted after 24 hours.
TABLE-US-00016 TABLE XIII Germination Effects III Compound Germination Growth No. Inhibition(uM) Inhibition(uM) 63 — — 64 — — 65 — 50 67 25 10 68 25 10 69 5 1 71 — — 72 — — 73 — — 75 5 5 78 2.5 2.5 106 1 1 13 10 5
[0348] For the germination test, the compounds were tested at 1, 5, 10, 25 and 50 uM. The amounts shown are the lowest of the concentrations that either inhibited germination or hypocotyl growth in the wells where seeds germinated.
[0349] For the thermal response test, the compounds were tested by spraying Arabidopsis plants with 50 uM solution of test compound. Thermal imaging was conducted after 24 hours.