TREA

Novel cyanoenamines useful as ligands for modulating gene expression in plants or animals

Follow

Information

  • Patent Application
  • 20030109705
  • Publication Number
    20030109705
  • Date Filed
    February 27, 2002
    22 years ago
  • Date Published
    June 12, 2003
    21 years ago
Information
Abstract
Accordingly, the present invention provides a compound of Formula I, or tautomers or isomers thereof, 1
Description


TECHNICAL FIELD

[0002] The present invention relates, in general, to novel compounds that are useful as ligands for modulating gene expression in living organisms (plants and/or animals). More particularly, the present invention relates to compounds that are cyanoenamines that are useful as non-steroidal ligands for modulating exogenous gene expression in eukaryotic organisms (i.e., those where the cell has a nucleus), more particularly plants, especially chlorophyll-containing plants.
1Table of Abbreviationsn-BuLin-butyllithiumt-butyltert-butylCcentigradeDMAPN,N-dimethylaminopyridineDNAdeoxyribonucleic acidEcRecdysone receptorEC80effective concentration that producesan 80% effectGCgas chromatographyGRglucocorticoid receptorggramHvHeliothis virescenshhourL1first larval instar, namely the stagebetween the egg and the first moltLCliquid crystalLDAlithium diisopropylamideMSmass spectroscopympmelting pointμMmicromolemLmillilitermmmillimeterminminuteMmoleNMRnuclear magnetic resonance#numberppmparts per millionRNAribonucleic acidRXRretinoid X receptorSPODLISpodoptera littoralisTHFtetrahydrofuranUSPultraspiracle



BACKGROUND OF THE INVENTION

[0003] Precise temporal control of gene expression is a valuable tool in the field of genetic engineering. The ability to activate (i.e., to induce) or to suppress a gene is of vast importance in manipulating, controlling, and/or studying development and other physiological processes. Inducability is often valuable for foreign protein production, such as production of therapeutic proteins, industrial enzymes, and polymers, in both plants and animals.

[0004] Specifically in the case of plants, often desirable is the control of the timing and level of expression of a phenotypic trait in a plant, plant cell or plant tissue. Ideally, regulation of expression of such a trait can be achieved whenever desired by triggering gene expression with a chemical that is easily applied to field crops, ornamental shrubs and other plants of economic importance. This triggering mechanism for gene expression control is referred to as a gene switch. In order to avoid unexpected activation of the gene switch, the chemical should be one that is normally absent from the plant.

[0005] One such gene switch mechanism is the ecdysone receptor (EcR). EcR is a member of the nuclear hormone family of receptors. Members of this receptor family are multi-domain proteins, capable of regulating gene expression in response to a chemical ligand. The DNA binding domain (also known as the C domain) binds to a specific target DNA sequence. This specificity determines which target genes are activated by the receptor. The ligand binding domain (E domain) plays a critical role in the determination of ligand specificity as well as the ligand regulated activation property of the receptor. The hinge domain (domain D) resides between the DNA binding and ligand binding domains. The hinge domain modulates the receptor's response to ligand induction.

[0006] Ligands that are complementary to the ligand binding domain of the ecdysone receptor are known. Steroidal agonists such as 20-hydroxy ecdysone, muristerone, and ponasterone are capable of activating an ecdysone receptor gene switch. Non-steroidal agonists have advantages over steroidal agonists due to such factors as greater stability, cheaper cost, and environmental acceptance. One known non-steroidal agonist is the insecticide Tebufenozide (also known as the insecticide sold under the trademark MIMIC®).

[0007] Of interest is European Published Patent Application No. 0 965 644 A2 to Carlson et al., assignors to Rohm and Haas Company, which relates to a method of modulating exogenous gene expression in which an ecdysone receptor complex is contacted with a DNA construct having an exogenous gene under the control of a response element, and where the binding of the ecdysone receptor to the response element results in activation or suppression of the gene. The ligand is chosen from certain dibenzoyl-tert-butyl-hydrazine compounds.

[0008] As referred to herein, an “ecdysone receptor gene switch” means a gene switch comprising an ecdysone receptor. The ecdysone receptor gene switch may be a heterodimer of EcR and USP, or EcR and RXR. The heterodimerization partner may be native to the organism or cell type in which the gene switch is present, or the heterodimerization partner may be provided exogenously. The ecdysone receptor gene switch may be comprised only of EcR in the absence of a heterodimerization partner. EcR may be in its native form, as isolated from insects, comprising a DNA binding domain, hinge and ligand binding domain from an insect EcR. EcR may be a chimeric protein comprising a DNA binding domain from another EcR or another transcription factor such as Ga14. EcR may comprise its native activation domain or an activation domain of another protein. Furthermore, EcR may comprise a ligand binding domain of an insect ecdysone receptor or a ligand binding domain from a member of the nuclear hormone family of receptors.

[0009] Also of interest is International Publication No. WO 00/15791 to Albertsen et al., assignors to Pioneer Hi-Bred International, Inc. This Publication relates to novel ecdysone receptors from the insect species Ostrinia and the genus Pyrilidae and their use for gene regulation in plants.

[0010] Additionally of interest is International Publication No. WO 99/02683 to Gage et al., assignors to The Salk Institute for Biological Studies. This Publication relates to nuclear receptor proteins from the silk moth Bombyx mori, useful for the regulation of gene expression.

[0011] Also of interest is International Publication No. WO 96/37609 to Jepson et al., assignors to Zeneca, relating to the use of a chimeric ecdysone receptor gene switch in plants.

[0012] Of general background interest is each of the following describing examples of ecdysone receptor gene switches: No et al., Proc. Nat'l. Acad. Sci., 93: 3346-3351 (1996), describing EcR in mammalian cells; Godowski et al., International Publication No. WO 93/03162, describing EcR and chimeric EcR proteins and related gene switches; Evans et al., International Publication Nos. WO 99/58155 and WO 97/38117, describing EcR and chimeric EcR proteins and related gene switches; Martinez et al., Insect. Biochem. Mol. Biol., 29 (10):915-930 (October, 1999), describing a chimeric EcR gene switch in plants; Martinez et al., Plant J., 19(1):97-106 (July, 1999), describing a chimeric EcR gene switch in plants; Martinez et al., Mol. Gen. Genet., 261(3):546-552 (April, 1999), describing a chimeric EcR gene switch in plants; Suhret al., Proc. Nat'l. Acad. Sci. U.S.A., 95(14):7999-8004 (Jul. 7, 1998), describing a chimeric EcR switch in mammalian cells; and Hoppe et al., Mol. Ther., 1(2):159-164 (February, 2000), describing an adenovirus mediated EcR gene switch.

[0013] Especially of interest is U.S. Pat. No. 5,880,333 to Goff et al., assignors to Novartis Finance Corporation. This patent discloses a method of controlling gene expression in plants. Specifically, the method involves obtaining a transgenic plant that has at least 2 receptor expression cassettes and at least 1 target expression cassette. A first of the 2 receptor expression cassettes has a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence that encodes a first receptor polypeptide and a 3′ termination region. A second of the 2 receptor expression cassettes has a nucleotide sequence for a 5′ regulator region operably linked to a nucleotide sequence that encodes a second receptor polypeptide and a 3′ termination region. The target expression cassette has a nucleotide sequence operably linked to a nucleotide sequence that encodes a target polypeptide and a 3′ termination region, wherein the 5′ regulatory region of the target expression cassette is activated by the first and second receptor polypeptides in the presence of a certain chemical ligand that is complimentary to the ligand binding domain of the receptor polypeptides, as a result of which expression of the target polypeptide is accomplished. In a preferred embodiment, the method involves expressing in a plant an insect EcR and a second receptor as a heterodimerization partner and activating the expression of a target polypeptide by contacting the plant cells with a ligand that is complimentary to the ligand binding domain of one of the receptors. The method of U.S. Pat. No. 5,880,333 to Goff et al. is useful for controlling various traits of agronomic importance, such as plant fertility.

[0014] Lastly, of interest is U.S. Provisional Application No. 60/242,969, filed Oct. 24, 2000, describing novel ecdysone receptor gene switches and methods of use, the disclosure of which is incorporated in its entirety.

[0015] All of the patents and published patent applications mentioned here are incorporated by reference.

[0016] Despite the plethora of available ecdysone receptor gene switch systems, there still remain a continuing need to develop non-steroidal ligands with increased activity as compared to known ligands and a need to develop ligands that demonstrate improved consistent activity in intact plants and animals.


SUMMARY AND OBJECTS OF THE INVENTION

[0017] Accordingly, the present invention provides a compound comprising Formula I
2

[0018] and also, Formula I may be in its tautomeric form comprising Formula II
3

[0019] and also, Formula I may be in its isomeric form comprising Formula III
4

[0020] wherein:

[0021] R1

[0022] is a branched chain lower alkyl (C3 to C8), cycloalkyl (C3 to C8), alkyl-substituted alkyl (C4 to C8), bicycloalkyl, 1-adamantyl, polyhaloalkyl, trialkylsilyl, unsubstituted phenyl or optionally substituted phenyl;

[0023] R2 and R3

[0024] are independently unsubstituted or substituted aromatic rings, chosen from phenyl, pyridyl, pyrimidinyl, furyl, thiophenyl, pyrazinyl, pyrrolyl, pyrazolyl, 1,2,4-triazolyl, naphthyl, fluorenonyl, xanthenyl, 4-oxo-1,4-dihydro-(1,8)naphthyridinyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, benzo-1,2,3-thiadiazolyl, oxazolyl, imidazolyl, quinolinyl, or isoquinolinyl, where a substituent on the rings is one or more chosen independently from hydrogen, alkyl (C1 to C4), alkoxy, alkoxyalkyl, hydroxy, amino, alkylamino, dialkylamino, acylamino, halo, haloalkyl, hydroxyalkyl, dihydroxyalkyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, unsubstituted or substituted alkylphenyl, unsubstituted or substituted phenyl, unsubstituted or substituted phenoxy, nitro, cyano, alkylthio, alkylsulfonyl, aminoalkyl, carboxyalkyl, or sulfonylalkyl; and

[0025] R4

[0026] is hydrogen, alkylthio, alkylthioalkyl, alkyloxyalkyl, acyloxyalkyl, alkyl, acyl, trialkylsilyl, or cyclized together with R3 and the O in Formula II to form a lactone.

[0027] The compounds described in the above paragraph are useful for modulation of an exogenous gene in a living organism. The compounds are also useful for the control of pests, such as anthropods, parasites, and the like, by acting as agonists of 20-hydroxyecdysone, the molting hormone.

[0028] Therefore, it is an object of the present invention to provide a compound that has the ability to activate or to suppress an exogenous gene.

[0029] It is another object of the present invention to provide a compound that is useful as a pesticide.

[0030] Some of the objects of the invention having been stated, other objects will become evident as the description proceeds, when taken in connection with the laboratory examples described below.


DETAILED DESCRIPTION OF THE INVENTION

[0031] The inventive ligand is an addition to the ligands described in the above-noted U.S. Pat. No. 5,880,333 to Goff et. al.

[0032] A ligand according to the present invention is described by the below recited general Formula I and its below recited tautomer, Formula II, and its below recited isomer, Formula III:
5

[0033] wherein:

[0034] R1

[0035] is a branched chain lower alkyl (C3 to C8), cycloalkyl (C3 to C8), alkyl-substituted alkyl (C4 to C8), bicycloalkyl, 1-adamantyl, polyhaloalkyl, trialkylsilyl, or optionally substituted phenyl;

[0036] R2 and R3

[0037] are independently optionally substituted aromatic rings, such as phenyl, pyridyl, pyrimidinyl, furyl, thiophenyl, pyrazinyl, pyrrolyl, pyrazolyl, 1,2,4-triazolyl, naphthyl, fluorenonyl, xanthenyl, 4-oxo-1,4-dihydro-(1,8)naphthyridinyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, benzo-1,2,3-thiadiazolyl, oxazolyl, imidazolyl, quinolinyl, or isoquinolinyl. Substituents on these rings can be one or more chosen independently from hydrogen, alkyl (C1 to C4), alkoxy, alkoxyalkyl, hydroxy, amino, alkylamino, dialkylamino, acylamino, halo, haloalkyl, hydroxyalkyl, dihydroxyalkyl, alkoxycarbonyl, alkylaminocarbonyl, dialkyl-aminocarbonyl, (optionally substituted) alkylphenyl, (optionally substituted) phenyl, (optionally substituted) phenoxy, nitro, cyano, alkylthio, alkylsulfonyl, aminoalkyl, carboxyalkyl, and sulfonylalkyl; and

[0038] R4

[0039] is hydrogen, or a substituent that may be easily removed in planta, serving as an aid in absorption and/or translocation, such as alkylthio, alkylthioalkyl, alkyloxyalkyl, acyloxyalkyl, alkyl, acyl, trialkylsilyl, or cyclized together with R3 and the O in Formula II to form a lactone.

[0040] Halo may be selected from the group consisting of fluoro, chloro, bromo, iodo, and combinations thereof. The substituents on R2 and R3 may also be joined to form cyclic structures on adjacent atoms of the aromatic ring, such as 1,2-methylenedioxy and 1,2-difluoromethylenedioxy. The preferred R1 is tert-butyl. The preferred R2 is phenyl, 3,5-dimethylphenyl, 2,4-dimethylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylphenyl, or 3,4-methylenedioxyphenyl. The preferred R3 is phenyl, 3-pyridyl, 3-methoxy-2-methylphenyl, 3-ethoxy-2-methylphenyl, 3-methoxy-2-ethylphenyl, 4-ethylphenyl, 2,6-difluorophenyl, 2,3-dimethylphenyl, 3-chloro-2-methylphenyl, or 3-bromo-2-methylphenyl. When R3, R4, and O are cyclized to form the cyclic ester known as a lactone, the lactone may be:
6

[0041] The compounds described as per Formula I and its tautomer, Formula II, and its isomer, Formula III, are useful for modulation of an exogenous gene in a living organism. They have the ability to activate or to suppress an exogenous gene.

[0042] Additionally, the described compounds can be used as pesticides, i.e., for arthropod control (control of segmented invertebrates such as insects, arachnids, crustaceans, or myriapods) on plants in soil or water, in structures, and on parasites in or on vertebrate animals, acting as agonists of 20-hydroxyecdysone, the molting hormone.

[0043] The preparation of these compounds may be accomplished by the following general scheme:
7

[0044] and in this scheme, R1 is tert-butyl, but that is not a requirement.






LABORATORY EXAMPLES


Example 1

[0045] Preparation of starting material; (E)-3-amino-2-(3,5-dimethylphenyl)-4,4-dimethylpent-2-enenitrile
8

[0046] To 30 mL of n-butyllithium (2.5 M in hexane) at 5° C. was added 30 mL of dry tetrahydrofuran. The resulting solution was cooled back to 5° C. and a solution of 5 g of 3,5-dimethylphenylacetonitrile in 10 mL of tetrahydrofuran was added over 30 min., keeping the temperature between 5° C. and 10° C. The mixture was stirred at 5° C. for 1 h., and then a solution of 2.86 g of trimethylacetonitrile in 10 mL of tetrahydrofuran was added. The resulting mixture was stirred overnight at ambient temperature. The mixture was poured into ice water and extracted with 2 portions of ethyl acetate.

[0047] The combined ethyl acetate layers were washed with brine, dried over MgSO4, filtered, and evaporated in vacuo to afford the crude product as an oil, which was crystallized from petroleum ether to yield 3.2 g of a solid with a 1HNMR spectrum consistent with the expected product, namely (E)-3-amino-2-(3,5-dimethylphenyl)-4,4-dimethylpent-2-enenitrile.

[0048] Preparation of starting material; 3-methoxy-2-methylbenzoyl chloride
9

[0049] Thionyl chloride (5 mL) was gradually added to 0.95 g of 3-methoxy-2-methylbenzoic acid at room temperature. The resulting mixture was heated at 65° C. for 1 h. The excess thionyl chloride was evaporated in vacuo, and a small portion of carbon tetrachloride was added. Then, the mixture was again evaporated in vacuo to yield the desired product as an oil. This was used directly in the next reaction.

[0050] Preparation of N-[(E)-1-tert-butyl-2-cyano-2-(3,5-dimethylphenyl)-vinyl]-3-methoxy-2-methylbenzamide
10

[0051] To 3.8 mL of lithium diisopropylamide solution (1.5 M in cyclohexane) at −78° C., was added dropwise, a solution of 0.51 g of (E)-3-amino-2-(3,5-dimethylphenyl)-4,4-dimethylpent-2-enenitrile in 30 mL of dry tetrahydrofuran. The mixture was stirred for 30 min. at −78° C., and then 1.05 g of 3-methoxy-2-methylbenzoyl chloride was added in one portion. The resulting mixture was stirred overnight at ambient temperature. The reaction mixture was poured into ice water and extracted with ethyl acetate.

[0052] The ethyl acetate layer was washed with brine, dried over MgSO4, filtered, and evaporated in vacuo to yield 1.4 g of crude solid product. The crude product was partially purified using 3 plates, each being a 600 mm×20 mm silica gel preparative layer chromatography plate, eluted with 20% ethyl acetate in hexane to afford 0.17 g of slightly impure material. This was recrystallized from a 3 mL tetrahydrofuran and 15 mL hexane mixture to yield 0.15 g of white crystalline material (mp was 203 to 204° C.) with GC/MS and 1HNMR spectra consistent with the desired product, namely N-[(E)-1-tert-butyl-2-cyano-2-(3,5-dimethylphenyl)-vinyl]-3-methoxy-2-methylbenzamide.


Example 2

[0053] Using essentially the same procedure as described above, the following selected cyanoenamine compounds have also been prepared, as reported in Table A1 below.
2TABLE A1Melting point LC/MSCompounddegrees C. molecular ion11499Compound 112476Compound 213363Compound 314465Compound 415429Compound 516521Compound 617439Compound 718390Compound 819436Compound 920507Compound 1021516Compound 1122485Compound 1223457Compound 1324371Compound 1425473Compound 1526485Compound 1627437Compound 1728508Compound 1829477Compound 1930449Compound 2031530Compound 2132499Compound 2233471Compound 2334385Compound 2435487Compound 2536451Compound 2637459Compound 2738468Compound 2839437Compound 2940409Compound 3041323Compound 3142425Compound 3243377Compound 3344389Compound 3445475Compound 3546407Compound 3647463Compound 3748431Compound 3849429Compound 3950377Compound 4051433Compound 4152401Compound 4253459Compound 4354391Compound 4455473Compound 4556431Compound 4657447Compound 4758415Compound 4859413Compound 4960391Compound 5061401Compound 5162371Compound 5263491Compound 5364383Compound 5465439Compound 5566483Compound 5667424Compound 5768452Compound 5869416Compound 5970399Compound 6071521Compound 6172363Compound 6273483Compound 6374443Compound 6475375Compound 6576431Compound 6677475Compound 6778416Compound 6879408Compound 6980391Compound 7081513Compound 7182385Compound 7283505Compound 7384397Compound 7485453Compound 7586497Compound 7687438Compound 7788466Compound 7889430Compound 7990413Compound 8091464Compound 8192535Compound 8293323Compound 8394443Compound 8495390Compound 8596335Compound 8697391Compound 8798435Compound 8899376Compound 89100404Compound 90101368Compound 91102351Compound 92103431Compound 93104401Compound 94105120Compound 95106165Compound 96107170Compound 97108128-133Compound 98109165Compound 99110195Compound 100111145Compound 101112190Compound 102113150Compound 103114198Compound 104115153Compound 105116205Compound 106117130Compound 107118165Compound 108119165Compound 10912082.1Compound 110121125Compound 111122170Compound 112123145Compound 113124160Compound 114125125Compound 115126220Compound 116127391Compound 117128179-182Compound 118129175Compound 119130170Compound 120131225Compound 121132184.4Compound 122133125.9Compound 123134171-174Compound 124135113.6Compound 125136>300Compound 126137194.7Compound 127138190Compound 128139393Compound 129140>300Compound 130141178-180Compound 131142197-200Compound 132143203-205Compound 133144178-180Compound 134145173-174Compound 135146194-195Compound 136147194-195Compound 137148203-204Compound 138149146-147 Compound 139150142-144Compound 140151175-176Compound 141152138-140Compound 142153180-183Compound 143154219-222Compound 144


[0054] The various R1, R2, R3, and R4 moieties (from the compounds made as per Table A1 above) are summarized in Table A2 below.
3TABLE A2Compound#R1R2R3R41t-Bu155156H2t-Bu157158H3t-Bu159160H4t-Bu161162H5t-Bu163164H6t-Bu165166H7t-Bu167168H8t-Bu169170H9t-Bu171172H10t-Bu173174H11t-Bu175176H12t-Bu177178H13t-Bu179180H14t-Bu181182H15t-Bu183184H16t-Bu185186H17t-Bu187188H18t-Bu189190H19t-Bu191192H20t-Bu193194H21t-Bu195196H22t-Bu197198H23t-Bu199200H24t-Bu201202H25t-Bu203204H26t-Bu205206H27t-Bu207208H28t-Bu209210H29t-Bu211212H30t-Bu213214H31t-Bu215216H32t-Bu217218H33t-Bu219220H34t-Bu221222H35t-Bu223224H36t-Bu225226H37t-Bu227228H38t-Bu229230H39t-Bu231232H40t-Bu233234H41t-Bu235236H42t-Bu237238H43t-Bu239240H44t-Bu241242H45t-Bu243244H46t-Bu245246H47t-Bu247248H48t-Bu249250H49t-Bu251252H50t-Bu253254H51t-Bu255256H52t-Bu257258H53t-Bu259260H54t-Bu261262H55t-Bu263264H56t-Bu265266H57t-Bu267268H58t-Bu269270H59t-Bu271272H60t-Bu273274H61t-Bu275276H62t-Bu277278H63t-Bu279280H64t-Bu281282H65t-Bu283284H66t-Bu285286H67t-Bu287288H68t-Bu289290H69t-Bu291292H70t-Bu293294H71t-Bu295296H72t-Bu297298H73t-Bu299300H74t-Bu301302H75t-Bu303304H76t-Bu305306H77t-Bu307308H78t-Bu309310H79t-Bu311312H80t-Bu313314H81t-Bu315316H82t-Bu317318H83t-Bu319320H84t-Bu321322H85t-Bu323324H86t-Bu325326H87t-Bu327328H88t-Bu329330H89t-Bu331332H90t-Bu333334H91t-Bu335336H92t-Bu337338H93t-Bu339340H94t-Bu341342H95t-Bu343344H96t-Bu345346H97t-Bu347348H98t-Bu349350H99t-Bu351352H100t-Bu353354H101t-Bu355356H102t-Bu357358H103t-Bu359360H104t-Bu361362H105t-Bu363364H106t-Bu365366H107t-Bu367368H108t-Bu369370H109t-Bu371372H110t-Bu373374H111t-Bu375376H112t-Bu377378H113t-Bu379380H114t-Bu381382H115t-Bu383384H116t-Bu385386H117t-Bu387388H118t-Bu389390H119t-Bu391392H120t-Bu393394H121t-Bu395396H122t-Bu397398H123t-Bu399400H124t-Bu401402H125t-Bu403404H126t-Bu405406H127t-Bu407408H128t-Bu409410H129t-Bu411412H130t-Bu413414H131t-Bu415416H132t-Bu417418H133t-Bu419420H134t-Bu421422H135t-Bu423424H136t-Bu425426H137t-Bu427428H138t-Bu429430H139t-Bu431432H140t-Bu433434H141t-Bu435436H142t-Bu437438H143t-Bu439440H144t-Bu441442H



Example 3

[0055] The following cyanoenamine compounds have been tested for pesticidal activity, according to the following procedures.

[0056]

Spodoptera littoralis
(abbreviated as SPODLI) (commonly known as Egyptian cotton leafworm): larvicide, feeding/contact activity. Cotton leaf discs were placed on agar in petri dishes and individually sprayed with test solution of cyanoenamine in an application chamber. After drying, the leaf discs were infested with 20 to 25 L1 larvae. The samples were checked for mortality, repellent effect, feeding behavior, and growth regulation 2 and 6 days after treatment.

[0057]

Heliothis virescens
(abbreviated as Hv) (commonly known as tobacco budworm): ovo-larvicide, feeding/contact activity. 30 to 35 fresh eggs (0 to 24 hours old), deposited on filter paper, were placed in petri dishes on a layer of artificial diet and 0.8 mL of each test solution of cyanoenamine was individually pipetted onto them. After an incubation period of 6 days, samples were checked for egg mortality, larval mortality, and growth regulation.

[0058] Each of Spodopertera littoralis and Heliothis virescens is a larval form of an insect in the order Lepidoptera.

[0059] The results are summarized in Table B below.
4TABLE BInsecticidal activity(EC80 ppm)COMPOUND #SPODLIHv443505444200200445>50>50446>>100>1004475050448100100449200200450>>100>>100



Example 4

[0060] Construction of Reporter Plasmid

[0061] A minimal promoter vector was made by ligating a synthetic TATA box sequence oligonucleotide pair, 5′-agcttgagggtataatg-3′ (SEQ ID NO: 1) and 3′-actcccatattactcga-5′ (SEQ ID NO:2), into the HindIII site of vector pGL3-basic (Promega) so that the HindIII site was recreated 5′ to the inserted oligonucleotide and destroyed between the oligonucleotide and the downstream luciferase gene. This vector was designated TATA5.

[0062] The binding site from the hsp27 gene (Koelle et al., Cell 67(1): 59-77 (1991)) was made with the oligonucleotide pair, 5′-gatccgagacaagggttcaatgcacttgtccaatga-3′ (SEQ ID NO:3) and 3′-gctctgttcccaagttacgtgaacaggttactctag-5′ (SEQ ID NO:4). This site was multimerized and ligated into the BglII site of vector TATA-5. One isolate, pCGS154, contained the sequence below in the inserted region, having 2 pairs of sites in inverted orientations. One site had a deletion of a single base from the consensus sequence. The sequence of the inserted region in pCGS154 is shown below:
51gatccgagac aagggttcaa tgcacttgtc caatgagatc(SEQ ID NO:5)41cgagacaagg gttcaatgca cttgtccaat gagatctcat81tggacaagtg cattgaacct tgtctcggat ctcattggac121aagtgcattg aacccttgtc tcggatc


[0063] Cloning of EcR Receptor Plasmid

[0064] PCR primers were designed based on the published sequence for Manduca Sexta ecdysone receptor (EcR) (genbank accession number U19812 (SEQ ID Nos:6 and 7) to clone the gene in two halves. RNA was prepared from prepupae larva of Manduca sexta using the LiCl/phenol method (Current Protocols in Molecular Biology, Vol. 1, Unit 4.3, 1987, John Wiley and Sons, publishers) and 1 μg of total RNA was used to prepare cDNA using MMLV reverse transcriptase (Promega). The cDNA was used in a PCR reaction with the primers described above to generate two PCR products for the 5′ and 3′ halves of the gene. These were subcloned into the pGEM-TA vector (Promega) and sequenced. The two fragments were joined at a unique Ndel site within each fragment and ligated into pBS-KS (Stratagene) to create a full length Manduca sexta EcR clone named pBSFLMa. A HindIII site followed by an inframe stop codon and BamHI site was placed at the 3′ end of the E domain (ligand binding domain) of the Manduca EcR receptor using the oligonucleotide: 5′-ggatcctaaagcttcgtcgtcgacacttcg-3′ (SEQ ID NO:8).

[0065] A truncated Manduca EcR containing domains C, D and E of the receptor was constructed as follows. A BamHI site and in-frame ATG was engineered just 5′ to the C domain using the degenerate primers 5′-ggatccatgggycgagaagaattrtcaccr-3′ (SEQ ID NO:9) and 5′-ccacrtcccagatctcctcga-3′ (SEQ ID NO:10). This fragment was then joined using the Nde site to the 3′ end of Manduca EcR, which has an engineered HindIII site at the 3′ end as described above.

[0066] A fragment containing the herpes simplex VP16 transactivation domain was cloned from plasmid 35S/USP-VP16 (U.S. Pat. No. 5,880,333) using the PCR primers 5′-aagcttgcccccccgaccg-3′ (SEQ ID NO:11) (placing a HindIII site at the 5′ end of the domain) and 5′-tctagaggatcctacccaccgtact-3′ (SEQ ID NO:12) (placing an inframe stop codon followed by BamHI and Xbal sites at the 3′ end of the domain). The VP16 domain was fused in frame to the 3′ end of the E domain of the ecdysone receptor using the HindIII site 3′ to EcR clone and the HindIII site engineered at the 5′ end of VP16.

[0067] The plasmid pPacU (Courey A J and Tjian R (1988) Cell 55, 887-898) was used as the starting vector for expression constructs. The truncated Manduca EcR-VP16 was ligated into pPacU using the BamHI sites flanking the coding region to create the construct referred to as MMV.

[0068] Cell-Based Assay

[0069] An in vivo cell based assay was used to measure transcriptional activation by the EcR receptor plasmid in the presence of the chemical ligands as described above. S2 Drosophila cells (ATCC CRL-1963) (commonly known as cells from the fruit fly) were transiently transfected with luciferase reporter (pCGS154) and receptor expression plasmid (MMV) using the calcium phosphate precipitation procedure (Di Nocera and David (1983) PNAS 80, 7095-7098). S2 cells were plated in 96 well format at a density of 2×105 in 166.6 μl of Schneider's Drosophila media supplemented with antibiotics and 10% heat inactivated fetal bovine serum (GIBO-BRL). The following day, 33.4 μl of a calcium phosphate precipitate containing 3-6 ng of pCGS154 reporter plasmid, and 3-6 ng of EcR receptor plasmid MMV along with salmon sperm DNA, to a total of 400 ng DNA per well were added. Chemical ligands (cyanoenamine test compounds) were added 16-24 hours after DNA addition to the cells at a final concentration of 2 μM. Cells were then harvested and extracted 24 hours after chemistry addition following the procedures for the luciferase assay by centrifuging and resuspending the cell pellets in 100 μl of cell culture lysis reagent (Promega). Luciferase activity was quantitatively determined using chemiluminescence (Promega) using an analytical luminescence model 2001 luminometer. Results were normalized as a ratio of induction relative to the reporter construct without chemical ligand addition.

[0070] The results are summarized in Table C below.
6TABLE CGene switch activityCHEMISTRYfold induction45166452191453225454636455111245611454571012458112145929460893461239462314463364464394465913


[0071] It will be understood that various details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation—the invention being defined by the claims.

Claims
  • 1. A compound comprising Formula I
  • 2. The compound of claim 1, wherein R1 is tert-butyl.
  • 3. The compound of claim 1, wherein at least one of R2 and R3 is substituted with a substituent forming a cyclic structure on adjacent atoms of the aromatic ring.
  • 4. The compound of claim 3, wherein the substituent is selected from the group consisting of 1,2-methylenedioxy and 1,2-difluoromethylenedioxy.
  • 5. The compound of claim 1, wherein R2 is selected from the group consisting of phenyl, 3,5-dimethylphenyl, 2,4-dimethylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylphenyl, and 3,4-methoxydioxyphenyl.
  • 6. The compound of claim 1, wherein R3 is selected from the group consisting of phenyl, 3-pyridyl, 3-methoxy-2-methylphenyl, 3-ethoxy-2-methylphenyl, 3-methoxy-2-ethylphenyl, 4-ethylphenyl, 2,6-difluorophenyl, 2,3-dimethylphenyl, 3-chloro-2-methylphenyl, and 3-bromo-2-methylphenyl.
  • 7. The compound of claim 1, wherein halo is selected from the group consisting of fluoro, chloro, bromo, iodo, and combinations thereof.
  • 8. The compound of claim 1, wherein Formula I is in its tautomeric form as Formula II:
  • 9. The tautomeric compound of claim 8, wherein R3 and R4 and O together form a cyclic structure resulting in a lactone.
  • 10. The compound of claim 9, wherein the lactone is selected from the group consisting of:
  • 11. The compound of claim 1, wherein Formula I is in its isomeric form as Formula III:
  • 12. The isomeric compound of claim 11, wherein R1 is tert-butyl, R2 is 3,5-dimethylphenyl, and R3 is fluoromethylphenyl or 2-methyl-3-methoxyphenyl.
  • 13. The isomeric compound of claim 12, wherein the compound is selected from the group consisting of:
  • 14. The compound of claim 1, wherein the compound is selected from the group consisting of:
  • 15. A method of controlling gene expression comprising contacting an ecdysone receptor gene switch with a compound of Formula I
  • 16. The method of claim 15, wherein R1 is tert-butyl.
  • 17. The method of claim 15, wherein at least one of R2 and R3 is substituted with a substituent forming a cyclic structure on adjacent atoms of the aromatic ring.
  • 18. The method of claim 17, wherein the substituent is selected from the group consisting of 1,2-methylenedioxy and 1,2-difluoromethylenedioxy.
  • 19. The method of claim 15, wherein R2 is selected from the group consisting of phenyl, 3,5-dimethylphenyl, 2,4-dimethylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylphenyl, and 3,4-methoxydioxyphenyl.
  • 20. The method of claim 15, wherein R3 is selected from the group consisting of phenyl, 3-pyridyl, 3-methoxy-2-methylphenyl, 3-ethoxy-2-methylphenyl, 3-methoxy-2-ethylphenyl, 4-ethylphenyl, 2,6-difluorophenyl, 2,3-dimethylphenyl, 3-chloro-2-methylphenyl, and 3-bromo-2-methylphenyl.
  • 21. The method of claim 15, wherein halo is selected from the group consisting of fluoro, chloro, bromo, iodo, and combinations thereof.
  • 22. The method of claim 15, wherein Formula I is in its tautomeric form as Formula II:
  • 23. The method of claim 22, wherein in the tautomeric form, R3 and R4 and O together form a cyclic structure resulting in a lactone.
  • 24. The method of claim 23, wherein the lactone is selected from the group consisting of:
  • 25. The method of claim 15, wherein Formula I is in its isomeric form as Formula III:
  • 26. The isomeric method of claim 25, wherein R1 is tert-butyl, R2 is 3,5-dimethylphenyl, and R3 is 2-trifluoromethylphenyl or 2-methyl-3-methoxyphenyl.
  • 27. The isomeric method of claim 26, wherein the compound is selected from the group consisting of:
  • 28. The method of claim 15, wherein the compound is selected from the group consisting of:
CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application claims benefit of U.S. Provisional Patent Application Serial No. 60/272,905, filed Mar. 2, 2001 and is incorporated herein by reference.

Provisional Applications (1)
Number Date Country
60272905 Mar 2001 US