MXPA99005570A - Links to modulate the expression of exogenous genes through an ecdis receptor complex - Google Patents

Abstract

The present invention relates to an improved method for modulating expression of the exogenous gene, in which an ecdysone receptor complex comprises: a DNA binding domain, a ligand binding domain, a transactivation domain, and a ligand is contacted with a DNA construct comprising: the exogenous gene and a response element, wherein the exogenous gene is under the control of the response element, and the binding of the DNA binding domain to the response element, in the presence of the ligand, results in the activation or repression of the gene. The improvement resides in a selected group of non-steroidal ligands that show improved activity compared to known ligands.

Description

Ligands to Modulate the Expression of Exogenous Genes Through an Ecdysone Receptor Complex.

This application is a continuation in part of the US application no. 09 / 210,010, filed December 11, 1998. The present invention relates to ligands without steroids which are useful for inducing or repressing the expression of an exogenous gene in plant or animal cells.

In the field of genetic engineering, the precise temporal control of gene expression, that is, the ability to activate or repress the gene, is a valuable tool in the development of the study, manipulation and control, and other physiological processes (see , for example, Evans and No, PCT international application No. PCT / US97 / 05330 and references cited therein). In mammalian systems, applications include directing the induced gene, over-expression of toxic and teratogenic genes, anti -perception RNA expression and gene therapy. In plant systems, applications include control of plant characteristics, male or female fertility, overexpression of plant protection agents and production? modification of desired products from plants, including natural or non-natural materials. For both animals and plants, the stimulus can be valuable for the production of foreign proteins, for example, therapeutic proteins, industrial enzymes, polymers and the like. It is important that the agent used to control the expression of the gene, which is usually referred to as a "gene switch", is one that is not normally found in the organism in which the gene that is to be controlled resides. This is to avoid the unexpected expression or repression of the gene. For example, a system regulated with inducible tetracycline and used in transgenic mice has been conceived, from which the activity of the gene is induced in the absence of the antibiotic and repressed in its presence. In this case, unfortunately the pharmacokinetics of tetracycline can interfere with its use as an efficient switch of genes in an "on-off" state. The international patent application no. PCT / GB96 / 01195 discloses an insect steroid receptor isolated from Heliothis virescens ("HeCR") that is capable of acting as a gene switch sensitive both to the steroid (e.g., 20-hydroxyecdysone and Muristerone A) and to certain inducers without steroids Non-steroid inducers have a distinct advantage compared to steroids, in this and many other systems that are sensitive to both steroids and non-steroid inducers, for a number of reasons including, for example: low manufacturing cost, metabolic stability , absence of insects, plants or mammals, and environmental acceptance. The PCT application describes the utility of two dibenzoylhydrazines, 1,2-dibenzoyl-l-tert-butylhydrazine and (N- (4-ethylbenzoyl) -N '- (3,5-dimethylbenzoyl) -N' -tert-butyl -hydrazine) of tebufenozide, as gene switches for the HEcR system and suggests that other dibenzoyl hydrazines, such as those disclosed in U.S. Pat. 5,117,057, they can also function as gene switches in the system. Although this may be true, the activity of these dibenzoylhydrazines is uncertain. Specifically, 5,117,057 shows a very extensive class of dibenzoylhydrazines, many of which appear to be ineffective as gene switches. GB96 / 01195 indicates that when twenty of said dibenzoylhydrazines are tested, only seven show any activity. The international patent application no. PCT / EP9600686 discloses the use of tebufenozide as a chemical ligand for the ecdysone receptor from Drosophila melanogaster. This receptor is used to control the expression of the gene in transgenic plants that result in the control of several important characteristics in agronomy.

Unfortunately, although the ligands described in the aforementioned references show reporter gene induction activity in isolated cells, no consideration was made with respect to their use in all organisms, such as intact plants and animals. Therefore, there is still a continuing need to develop non-steroidal ligands with increased or consistent activity compared to known ligands, and which demonstrate activity in intact animals and plants. A limited group of dibenzoylhydrazine derivatives has been discovered which not only shows reporter gene induction activity in the isolated cells, but also have advantages compared to the known diacylhydrazines, 1,2-dibenzoyl-l-tert-butyl- hydrazine and tebufenozide, when used in intact plants and animals, due to their improved distribution and transport properties, metabolic stability, residual activity, receptor affinity, and without adverse effects. This invention relates to an improvement in a method for modulating - the expression of the exogenous gene, comprising contacting an ecdysone receptor complex comprising: a) a DNA binding domain; b) a ligand binding domain; c) a transactivation domain; and d) a ligand; with a DNA construct comprising: a) the exogenous gene; and b) a response element; wherein: a) the exogenous gene is under the control of the response element; and b) the binding of the DNA binding domain to the response element, in the presence of the ligand, results in the activation or repression of the gene; the improvement comprises: selecting the ligand from a compound of the formula I: wherein: E is an alkyl (C -Cd) containing a tertiary carbon or a cyano (C3-C5) alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, ÓEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2; R2 is H, Me Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi-Pr, SCN, SCHF2, SOMe, NH-CN or linked to R3 and the phenyl carbons to which R2 and R3 are linked to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R3 is H, Et or, together with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl-carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; R4, R5 and Rs are, independently, H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , OMe, OEt, SMe or SEt; provided that: a) when R1 is Me and R2 is OMe, then R3 will be H, and the combination of R4, R5 and R6 will be 3,5-di-Me, 3, 5-di-OMe, 4-Me, 3 , 5-di-Cl, or 3,5-di-F; b) when R1 is Me and R2 is OEt, then R3 will be H, and the combination of R4, R5 and Rd will be 3,5-di-Me, 3, 5-di-OMe-4-Me, 3,5- di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl; c) when R1 is Et and R2 is OMe or Oet, then R3 will be H, and the combination of R4, R5 and R6 will be: i) 3,5-di-OMe-4-Me, 3,5-di-Cl , 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br -5-Me, 2-C1, 2-Br, or 3 -Me; or ii) Rs is H, R4 is Me and R5 is Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , OMe, OEt, SMe, or SEt; d) when R1 is i-Pr, then R2 will be OMe or Oet; R3 will be H; and the combination of R4, R5 and Rs will be 3, 5-di-Me; e) when R3 is Et, then R2 will be H, R1 will be F or Cl, and the combination of R4, R5 and R6 will be 3,5-di-Me; f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring, then R1 will be Me or Et, and the combination of R4, R5 and R6 is 3,5-di-Me; g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring, then R1 will be Et and the combination of R4, R5 and Rs will be 3,5-di-Me; h) when R1 is formyl, CF3, CHF2, CHC12, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2, then R2 will be OMe or Oet, R3 will be H, and the combination of R4, R5 and Rs will be 3, 5-di-Me; and i) when R2 is Me, Et, n-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, Ac, F , Cl, OH, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi-Pr, SCN, SCHF2, SOMe , or NH-CN, then R1 will be Et, R3 will be H, the combination of R4, R5 and R6 will be 3,5-di-Me. This invention also relates to a method for modulating expression of the exogenous gene, comprising contacting an ecdysone receptor complex comprising: a) a DNA binding domain; b) a ligand binding domain; c) a transactivation domain; and d) a ligand consisting of a compound of formula I: with a DNA construct comprising: a) the exogenous gene; and b) a response element; wherein: a) the exogenous gene is under the control of the response element; and b) the binding of the DNA binding domain to the response element, in the presence of the ligand, results in the activation or repression of the gene. To achieve an optimal balance between a) ligand binding and the resulting activity of the gene switch, and b) transport, system, toxicity and metabolic stability in intact plants and animals, the position and size of the compound of the substituent groups of the formula I are important. The optimum balance of properties seems to occur when E is t-butyl, R1 is ethyl, R2 is ethoxy, R3 is hydrogen, and the combination of R4, R5 and R6 is 3,5-dimethyl. The composition of each of said "R" groups can be varied considerably. However, variations that lead to significant changes in the size, configuration and all polarity of the compound of formula I will tend to reduce the optimum balance and, consequently, the improved properties of the compound. For this reason, when the composition of any particular group R is changed from the optimum, the variations in the composition of the remaining R groups should be limited.

Preferably, E is (C4-C5) alkyl. More preferably, E is t-butyl. Preferably, R1 is Me, Et, i-Pr, F, CF3 / CHF2, CHC12, CH2F, CH2C1, CH2OH, CH20Me, CH2CN, CN, C = CH or CF2CF3. More preferably, R1 is Me, Et, i-Pr, F, CF3, CHF2, CH2F, CH2OMe, CH2CN, C = CH, or CF2CF3. Still more preferably, R1 is Me, Et, i-Pr or F. More preferably, R1 is Me or Et. Preferably, R2 is Et, CF3, CHF2, CHC12, CH2OH, CH2OMe, CH2CN, CN, OH, OMe, OEt, On-Pr, CF2CF3, azido, OCF3 or, together with R3 and the phenyl carbons to which R2 and R3 are linked to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon. More preferably, R2 is Et, CF3, CHF2, CHCl2, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, OH, OMe, OEt, CF2CF3 or, together with R3 and the phenyl carbons to which R2 and R3 are they join, to form an ethylenedioxy or dihydrofuryl ring with oxygen adjacent to a phenyl carbon. Still more preferably, R2 is OH, OMe, OEt or, together with R3 and the phenyl carbons to which R2 and R3 are attached, forms an ethylenedioxy or dihydrofuryl ring with oxygen adjacent to a phenyl carbon. More preferably, R2 is OMe or OEt.

Preferably, R3 is H, Et or, together with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon. More preferably, R3 is H, Et or is linked with R2 and the phenyl carbons, to which R2 and R3 are attached, to form an ethylenedioxy or dihydrofuryl ring with oxygen adjacent to a phenyl carbon. Still more preferably, R3 is linked with R2 and the phenyl carbons, to which R2 and R3 are attached, to form an ethylenedioxy or dihydrofuryl ring with oxygen adjacent to a phenyl carbon. Preferably, R4, R5 and R6 are, independently, Me, F, Cl, CH2OH or OMe. More preferably, R4, R5 and R6 are, independently, Me, F, Cl, CH2OH, or OMe. Even more preferable, R4, R5 and R6 are, independently. Me, F or Cl. It is still more preferred that the combination of R 4, R 5 and R 6 is 3,5-di-Me, 3,5-di-CL or 3,5-di-F. And it is better preferred, that the combination of R4, Rs and Rs is 3, 5-di-Me. The terms "Me", "Et", "n-Pr", "i-Pr" and "Ac" mean methyl, normal propyl ethyl, isopropyl and acetyl, respectively. When R4, R5 and R6 are mentioned, the term "2.4", "2.5", "3.5" and the like refers to the relative positions on the phenyl ring to which the groups are attached. The term "halo" means fluorine, chlorine, bromine or iodine. The term "modular" means the ability of a given receptor / ligand complex to induce or repress transactivation of an exogenous gene. The term "exogenous gene" means a gene foreign to the subject, that is, a gene that is introduced into the subject through a transformation process or an untransformed version of an exogenous transformed gene. The transformation method is not critical in this invention, and can be any suitable method known to the person skilled in the art. For example, transgenic plants are obtained by regeneration of transformed cells. Numerous transformation methods are known from documents, such as agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, and transformation by microprojectile. Complementary techniques are known for the transformation of animal cells, and the regeneration of said transformed cells in transgenic animals. The exogenous genes can be either natural or synthetic genes or therapeutic genes that are introduced into the substrate in the form of DNA or RNA that can function through a DNA intermediate as by reverse transcriptase. These genes can be introduced into target cells, directly introduced into the subject or indirectly introduced by the transfer of transformed cells within the subject. The term "therapeutic gene" means a gene that imparts a beneficial function to the host cell in which said gene is expressed. Therapeutic genes are not found naturally in host cells. The term "ecdysone receptor complex" generally refers to a heterodimeric protein complex consisting of two members of the steroid receptor family, the ecdysone receptor ("EcR") and the ultra-breathing proteins ("USP") ( see Yao, TP, et al. (1993) Nature 366, 476-479; Yao, T.-P., et. al , (1992) Cell 71, 63-72). The ecdysteroid receptor functional complex may also include additional protein (s), such as immunofilins. Additional members of the steroid receptor family of proteins, known as transcriptional factors (eg, DHR38, betaFTZ-1 or other insect homologs), may also be dependent or independent partners of the ligand for EcR and / or USP. The ecdysone receptor complex can also be a heterodimer of the ecdysone receptor protein and the vertebrate homologue of the ultra-breathing protein, retinoic acid receptor X protein ("RXR"). The homodimer complexes of the ecdysone receptor protein or USP may also be functional under some circumstances. A receptor complex of ecdysteroids can be activated by means of a ligand linkage without steroids or ecdysteroid for one of the complex proteins, including EcR, but without excluding other proteins of the complex. The ecdysone receptor complex includes proteins that are members of the large steroid receptor family, wherein all members are characterized by the presence of an amino terminal transactivation domain, a DNA binding domain ("DEA") and a domain ligand binding ("DEL") separated by a hinge region. Some members of the family may also have another transactivation domain on the carboxy terminal side of the LED. The DEA is characterized by the presence of two zinc fingers of cysteine, among which there are two amino acid motifs, the P-box and the D-box, which confer specificity for the ecdysone response elements. These domains can be natural, modified or chimeras of different domains of heterologous receptor proteins.

The term "response element" ("ER") means one or more cis-acting DNA elements that confer response on a promoter mediated through the interaction of the DNA binding domains of the ecdysone receptor complex. This DNA element can be palindromic (perfect or imperfect) in its sequence or be composed of sequence motifs or middle sites separated by a variable number of nucleotides. The average sites can be similar or identical, and be arranged as direct or inverted repetitions. The ecdysone receptor complex binds, in the presence or absence of a ligand, to the DNA sequence of an ER to initiate or repress transcription downstream of a gene or genes under the regulation of said response element. Examples of DNA sequences for the ER of the natural ecdysone receptor include: RRGG / TTCANTGAC / ACYY (see Cherbas L., et. Al., 81991), Genes Dev. 5, 120-131); AGGTCAN (n) AGGTCA, wherein N (n) can be one or more spacer nucleotides (see Dávino PP., Et al., (1995), Mol. Cell, Endocrinol, 113, 1-9); and GGTTGAATGAATTT (see Antoniewski C., et al. (1994), Mol Cell. Biol. 14, 4465-4474). The DNA sequences that construct the exogenous gene, the response element and the ecdysone receptor complex can be incorporated into prokaryotic cells such as Escherichia Coli, Bacillus subtilis or another enterobacteria. However, because many of the proteins expressed by the gene are incorrectly processed in the bacterium, eukaryotic cells are preferred. The nucleotide sequences for the exogenous gene, the response element and the receptor complex can also be incorporated as RNA molecules, preferably in the form of functional viral RNAs, such as tobacco mosaic virus. Of eukaryotic cells, vertebrate cells are preferred because they naturally lack molecules that confer response to the ligands for the ecdysone receptor. As a result, they are insensitive to the ligands of this invention. Thus, the ligands of this invention will have negligible, physiological or other effects on transformed cells or the whole organism. Therefore, the cells can grow and express the desired product, substantially unaffected by the presence of the ligand itself. The term "subject" means an intact plant or animal or a cell of a plant or animal. It is also envisaged that the ligands will work equally well when the subject is a fungus or yeast. When the subject is an intact animal, preferably the animal is a vertebrate, and more preferably a mammal.

The ligands of the present invention, when used with the ecdysone receptor complex which in turn is linked to the response element bound to an exogenous gene, provide means for the external temporal regulation of the expression of the exogenous gene. The order in which the various components are linked to each other, that is, the ligand to the receptor complex and the receptor complex to the response element, is not critical. Typically, expression modulation of the exogenous gene is in response to binding of the ecdysone receptor complex to a specific or regulatory control DNA element. The ecdysone receptor protein, like other members of the steroid receptor family, possesses at least three domains, a transactivation domain, a DNA binding domain and a ligand binding domain. This receptor, as a subset of the steroid receptor family, also has fewer well-defined regions responsible for the heterodimerization properties. The binding of the ligand to the ligand binding domain of the ecdysone receptor protein, after heterodimerization with the USP or RXR protein, allows the DNA binding domains of the heterodimeric proteins to bind to the response element in an activated form, thus resulting in the expression or repression of the exogenous gene. This mechanism does not exclude the potential of the binding of the ligand to the EcR or USP, and the resulting formation of active homodimeric complexes (eg, EcR + EcR or USP + USP). Preferably, one or more of the receptor domains can be changed by producing a chimeric gene switch. Typically, one or more of the three domains can be chosen from a source different from the source of the other domain, so that the chimeric receptor is optimized in the cell or host organism chosen for the transactivation activity, the complementary link of the ligand and the recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains, such as the GAL-4 protein of the yeast (see Sadowski, et al. (1988) Nature, 335, 563-564) or the LexA protein of E. coli (see Brent and Ptashne (1985), Cell, 43, 729-736) to accommodate chimeric ecdysone receptor complexes. Another advantage of the chimeric systems is that they allow the choice of a promoter used to drive the exogenous gene, according to a desired final result. Such double control can be particularly important in areas of gene therapy, especially when cyto-toxic proteins are produced, because both the expression time and the cells in which the expression occurs can be controlled.

The term "promoter" means a specific nucleotide sequence recognized by the RNA polymerase. The sequence is the site in which transcription can be initiated specifically under appropriate conditions. When exogenous genes operably linked to a suitable promoter are introduced into the cells into the subject, the expression of the exogenous genes is controlled by the presence of a ligand of this invention. The promoters can be constitutively or inductively regulated or they can be tissue specific (that is, expressed only in a particular type of cells) or specific for certain stages of organism development. Another aspect of the present invention is a method for modulating the expression of one or more exogenous genes in an organism, which comprises administering to the organism an effective amount, that is, the amount required to obtain the desired expression or repression of the gene, of a ligand comprising a compound of formula I, and wherein the cells of the organism contain: a) an ecdysone receptor complex, comprising: 1) a DNA binding domain 2) a binding domain for the ligand; and 3) a transactivation domain; and b) a DNA construct, comprising: 1) the exogenous gene; and 2) a response element; and wherein: a) the exogenous gene is under the control of the response element; and b) the binding of the DNA binding domain to the response element, in the presence of the ligand, results in the activation or repression of the gene. A related aspect of the invention is a method for regulating the expression of the endogenous or heterologous gene in a transgenic organism, comprising contacting a ligand comprising a compound of the formula I with an ecdysone receptor within the cells of the organism in wherein the cells contain a DNA-binding sequence for the ecdysone receptor, and wherein the formation of a DNA-ligand-receptor binding sequence complex induces the expression of the gene. A fourth aspect of the present invention is a method for producing a polypeptide, comprising the following steps: a) selecting a cell that is substantially insensitive to the exposure of a ligand comprising a compound of the formula I; b) introducing into the cell: 1) an A? 3N construct, which comprises: a) an exogenous gene encoding the polypeptide; and b) a response element; wherein the gene is under the control of the response element; and 2) an ecdysone receptor complex, comprising: a) a DNA binding domain; b) a binding domain for the ligand; and c) a transactivation domain; and c) exposing the cell to the ligand. In addition to the advantage of temporarily controlling the production of the polypeptide by means of the cell, this aspect of the invention provides another advantage, in those cases where the accumulation of said polypeptide can damage the cell, in which the expression of the polypeptide may be limited. to short periods. Such control is particularly important when the exogenous gene is a therapeutic gene. The therapeutic genes can be used to produce polypeptides that control necessary functions, such as the production of insulin in diabetic patients. They can also be used to produce harmful and even lethal proteins, such as those that are lethal to cancer cells. Such control can also be important when the levels of protein produced can constitute a metabolic drain on growth or reproduction, such as in transgenic plants. Numerous cDNA and genomic nucleic acid sequences encoding a variety of polypeptides are well known in the art. Exogenous genetic material useful with the ligands of this invention includes genes that encode biologically active proteins of interest, such as secretory proteins that can be released from a cell; enzymes that can metabolize a substrate from a toxic substance to a non-toxic substance or from an inactive substance to an active substance; regulatory proteins; cell surface receptors; and similar. Useful genes also include genes encoding blood coagulation factors, hormones such as insulin, parathyroid hormone, lutein hormone releasing factor, alpha and beta seminal inhibitors, and human growth hormone; the genes that encode proteins such as enzymes, in the absence of which they would be taken to the event of an abnormal state; genes encoding cytokines or lymphokines such as interferons, the granulocyte macrophage colony stimulating factor, factor 1 colony stimulator, tumor necrosis factor, and erythrospoietin; genes encoding inhibitory substances such as alpha-antitrypsin, genes encoding substances that function as drugs, for example cholera and diphtheria toxins; and similar. Useful genes also include those that are useful in cancer therapies, and to treat genetic diseases. Those skilled in the art have access to information on the nucleic acid sequence for virtually all known genes, and can also obtain the nucleic acid molecule directly in a public store, the institution that has published the sequence or by routine methods used to prepare the molecule. For use in gene therapy, the ligands described herein can be taken in pharmaceutically acceptable carriers, such as solutions, suspensions, tablets, capsules, ointments, elixirs and injectable compositions. The pharmaceutical preparations may contain from 0.01% to 99% by weight of the ligand. The preparations may be in single or double dose form. The amount of the ligand in any particular pharmaceutical preparation will depend on the effective dose, that is, the dose required to obtain expression or repression of the gene. Suitable routes of administration of pharmaceutical preparations include oral, rectal, topical (including dermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and by nasogastric tube. Those skilled in the art will understand that the preferred route of administration will depend on the condition being treated, and may vary with factors such as the condition of the recipient. The ligands described herein can also be administered together with other pharmaceutically active compounds. It will be understood by those skilled in the art that the pharmaceutically active compounds to be used in combination with the ligands described herein will be selected so as to avoid adverse effects on the receptor or undesirable interactions between the compounds. Examples of other pharmaceutically active compounds that can be used in combination with the ligands include, for example, chemotherapeutic agents for AIDS, amino acid derivatives, analgesics, anesthetics, anorectal products, antacids and antiflatulents, antibiotics, anticoagulants, antidotes, antifibrinolytic agents, antistamines, anti-inflammatory agents, antineoplastic, antiparasitic, antiprotozoal, antipyretic, antiseptic, antipasmodic and anticholinergic, antiviral, appetite suppressant, arthritis medication, biological response modifiers, bone metabolism regulators, bowel evacuators, cardiovascular agents, stimulants central nervous system, cerebral metabolic enhancers, cerumenolytics, cholinesterase inhibitors, preparations for the flu and cough, colony stimulation factors, contraceptives, cytoprotective agents, dental preparations, desodorant is, dermatological agents, detoxifying agents, diabetes agents, diagnostics, medicines for diarrhea, dopamine receptor agonists, electrolytes, enzymes and digestives, ergot preparations, fertility agents, fiber supplements, antifungal agents, galactorrhea inhibitors, gastric acid secretion inhibitors, gastrointestinal prokinetic agents, gonadotropin inhibitors, hair growth stimulants, haematinics, haemorheological agents, hemostats, H2 histamine receptor antagonists, hormones, hyperglycemic agents, hypolipidemic, immunosuppressive, laxative, leprostatic, adjuvants of leukapheresis, surfactants for the lung, migraine preparations, mucolytics, muscle relaxant antagonists, muscle relaxants, narcotic antagonists, nasal sprays, drugs for nausea, nucleoside analogues, food supplements, preparations for the teoporosis, oxytocics, parasympatholytics, parasympathomimetics, drugs for Parkinson's disease, penicillin auxiliaries, phospholipids, blood plate inhibitors, porphyria agents, prostaglandin analogues, prostaglandins, proton pump inhibitors, psychotropic pruritus drugs, quinolones, respiratory stimulants, saliva stimulants, salt substitutes, sclerosis agents, preparations for skin wounds, cessation aids, sulfonamides, sympatholytics, thrombolytics, agents for Tourette's syndrome, anti-tremor preparations, preparations for tuberculosis , uricosuric agents, agents for the urinary tract, agents for uterine contraction, uterine relaxants, vaginal preparations, dizziness agents, vitamin D analogues, vitamins, and medical imaging contrast media. In some cases, the ligands may be useful as an adjunct to drug therapy, for example, to "turn off" a gene that produces an enzyme that metabolizes a particular drug. For agricultural applications, in addition to the aforementioned applications, the ligands of this invention can also be used to control the expression of pesticidal proteins such as the toxin Bacillus thuringiensis (Bt). Said expression may be specific for tissues or plants. further, particularly when plague control in plants is also needed, one or more pesticides can be combined with the ligands described herein, thereby providing additional benefits and effectiveness, including less total applications, than if the pesticides are applied separately. When mixtures with pesticides are used, the relative proportions of each component in the composition will depend on the relative efficacy and the desired rate of application of each pesticide with respect to the crops, pests and / or herbs to be treated. Those skilled in the art will recognize that mixtures of pesticides can provide advantages such as a broader spectrum of activity, as compared to a pesticide used alone. Examples of pesticides that can be combined in the compositions with the ligands described herein include fungicides, herbicides, insecticides, miticides and biocides. The ligands described herein can be applied to the foliage of plants as aqueous sprays by means of commonly used methods, such as hydraulic sprinklers of many liters, sprinklers of a few liters, air injection and air sprinklers. The dilution and application rate will depend on the type of equipment used, the method and frequency of application desired, and the rate of application of the ligand. It may be convenient to include additional auxiliaries in the spray tank. Such auxiliaries include surfactants, dispersants, spreaders, adhesives, antifoaming agents, emulsifiers and other similar materials described in McCutcheon's Emulsifiers and Detergents, McCutcheon's Emulsifiers and Detergents / Functional Materials and McCutcheon's Functional Materials, McCutcheon's Emulsifiers and Detergents, McCutcheon Emulsifiers and Detergents / Functional Materials, and McCutcheon Functional Materials) all published annually by McCutcheon Division of MC Publishing Company (New Jersey). The ligands can also be mixed with fertilizers or fertilizer materials before application. The ligands and solid fertilizer material can also be mixed in the mixing or incorporation equipment, or they can be incorporated with fertilizers into granular formulations. Any relative proportion of fertilizer can be used as long as it is suitable for the crop or herbs that are to be treated. The ligands described herein will usually comprise from 5% to 50% of the fertilizer composition. These compositions provide fertilizer materials that promote the rapid growth of desired plants, and at the same time control the expression of the gene. The ligands of this invention are compounds known or readily prepared by one skilled in the art which adapts the processes described in US Pat. 5,117,057, 5,530,028 and 5,378,726. Typically a two-stage process is used. In the first step, a substituted benzoic acid chloride of the formula II is combined with a substituted hydrazine of the formula III, to obtain a monoacyl hydrazine of the formula IV. The monoacylhydrazine of formula IV is combined in a second step with another benzoic acid chloride of formula V, resulting in the formation of a compound of formula I.

II III The following examples demonstrate the activity of the ligands of this invention.

The following ligands and comparative ligands were evaluated: 3- 5- 4- Ligand Rl R2 R3 R4 R5 R6 CE-1 HH Et Me Me CE-2 HHHHH CE-3 1 Et OMe H OMe H 2 Me -0CH2CH20- Me Me 3 Me OMe H Me Me 4 Et OMe HFF 5 Et OMe H Me Me 6 Me OEt H Me Me 7 Et -0CH2CH20- Me Me 8 Et -CH2CH2? - Me Me 9 Me OMe H Cl Cl 10 Et OMe H Cl Me 11 i-Pr OMe H Me Me 12 Et OEt H Me Me 13 Et OMe H Cl Cl 14 Me OH H Me Me 15 Me OH H Me CH2? H 17 Et Me Me 18 Me OMe OMe OMe Me CE-1 = tebufenozide CE-2 = 1,2-dibenzoyl-l-tert-butyl-hydrazine CE-3 = muristerone A Gene constructions. PVgRXR (Invitrogen Corp., Carlsbad, Calif.) Is a 8728 kb plasmid that expresses both VgEcR and RXR to form a modified heterodimeric nuclear receptor (see No. D., et al., (1996) Proc. Nati. Acad. Sci. USA, 93, 3346-3351). The ecdysone receptor (VgEcR) is derived from the natural Drosophila ecdysone receptor, and is modified to contain the transactivation domain VP16 (see Cress, .D., And Triezenberg, SJ (1991), Science 251, 87-90; Sadowski, I., et al. (1988) Nature 335, 563-564, Triezenberg, SJ et al., (1988), Genes Dev 2, 718-729; Triezenberg, SJ, et. Al., (1988) ), Genes Dev 2, 730-742). RXR is the mammalian homolog of USP (ultraespiráculo), the natural partner of the ecdysone receptor of Drosophila (see Yao, T.-P., et. Al., (1993), Nature 366, 476-479; Yao, T.-P., et. al., (1992), Cell 71, 63-72). The P-box region of the link domain of VgEcR DNA was modified to recognize the hybrid response element of ecdysone which consists of a middle site of the glucuronide response element (see Umesono, K., and Evans, RM (1989), Cell 57, 1139-1146) and a middle site of the natural ecdysone response element. The hybrid response element reduces any possible interaction with the farsenoid X receptor that can bind the natural ecdysone response element (EcRE). The cytomegalovirus promoter-enhancer drives the expression of VgEcR, and the Rous sarcoma virus promoter drives the expression of RXR. The pIND vector (Invitrogen Corp.) is a 5024 bp vector based on pcDNA3.1. It contains five hybrid E / GREs recognized by the modified ecdysone receptor expressed by pVgRXR and a minimal heat shock promoter.

(See Yao, T.P., et al., (1993), Nature, 366, 476-479).

PIND / lacZ (Invitrogen Corp.) is a 8170 bp plasmid that contains the β-galactosidase gene in the manner of a reporter enzyme. The contrafection of pIND / lacZ and pVgRXR results in the induction of β-galactosidase expression with the addition of ecdysone agonists, such as muristerone A. The pIND / luc reporter plasmid was constructed by subcloning the firefly luciferase gene from pGL3 (Promega / E1741) as a Nhe I-BamHI fragment within pIND, also digested with Nhe I and BamHI.

Maintenance of mammalian cell lines, and transfection. CHO cells (ATCC # CCL-61) were maintained in a mixture of nutrients F-12 (Ham media) (Gibco / BRL, 11765-054) supplemented with 10% cerium fetal bovine (FBS, complete medium; / BRL, 16000-036). These cells were maintained at 37 ° C. in 5% C02 in 95% atmospheric air. The CHO cells were seeded in orifice tissue culture plates 12 at a concentration of 0.5 X 10 5 cells per ml. by hole. The lipid Pfx-8 (Invitrogen, T930-18) was used to transiently transfect the induced expression system of ecdysone (Invitrogen catalog No. K1000-01). The ecdysone induced expression system consists of pVgRXR, which encodes the subunits of the receptor, and pIND / lacZ or pIND / luc, containing the response element and a reporter gene encoding the β-galactosidase or luciferase, respectively. Twenty-four hours after seeding the cells, the lipid / DNA solution was prepared with 0.5mg./ml. of VgRXR and 0.5mg./ml. of pIND / lacZ or pIND / luc DNAs in sterile water. Thirty-six μl of Pfx-8 were diluted in a sterile polystyrene tube 17 x lOOmm. that contained 1.5ml. of the Opti-MEM medium (Gibco / BRL, 31985). Six μl of the plasmid DNA pool were mixed with 1.5 ml. of the Opti-MEM medium in another polystyrene tube. DNA and lipid solutions were combined to obtain 3ml. of transfection solution (enough for a set of samples in triplicate). The cells were rinsed three times with phosphate buffered solution (PBS; Gibco / BRL, 14190-144) by aspirating the medium from the cells. One ml was added per hole. of transfection solution. The cells were incubated for four hours, and the transfection medium replaced with the same volume of the complete medium. The cells were incubated for an additional twenty hours.

Line of mammalian cells transformed stably. CHO cells stably transformed with pVgRXR and pIND / LacZ (SPI) (Invitrogen Corp.) were maintained in Hams F12 media containing 10% FBS, 2mM glutamine, 300μg / ml. from Zeocin (Invitrogen Corp.) and 300μg / ml. Hygromycin B. The pIND (SPI), which is an alternative to the pIND vector, which can be induced at absolute expression levels five times larger than those of the pIND due to the presence of SPI elements 3 cis actors. The basic expression levels are correspondingly higher.

Treatment with ligands.

The stock solutions (10 ~ 2 M) were prepared for muristerone A and the non-steroidal ligands of the present invention in ethanol and acetone, respectively, and stored at -20 ° C. Twenty-four hours after transfection, it was added a test compound in a final concentration of μM (10 ~ 5 M) to each cell culture orifice of lml .. Muristerone A (Sigma, M7888), at a concentration of 10 μM, was used as a positive control. The acetone alone was added as a negative control.

Testing of the report gene. The expression of the reporter gene was evaluated 24-48 hours after the treatment of transiently transfected cells or 24 hours after treatment with the test ligands the line of stably transformed cells. The β-galactosidase was tested by staining cells stained or assayed for enzymatic activity in cell lysates. Β-galactosidase catalyses the hydrolysis of β-galactoside, X-gal (5-bromo-4-chloro-3-indolyl-β-galactopyranoside) produces a blue color within fixed cells (Invitrogen, K1465-01) which can be visualized with a microscope. Alternatively, the report lysis regulator (RLR; Promega / E397 A) was used to lyse the cells for the detection of β-galactosidase activity using a luminescent chemical substrate, Galacto-Star (Tropix / BMIOOS). The cells within each orifice plate 12 were Used with 250μl of RLR. Twenty μl of each extract was tested with lOOμl of substrate. To detect the luciferase activity, the cells were rinsed twice with PBS and lysed with 250μl of the luciferase assay reagent (Promega, E1500). For the galactosidase and luciferase assays, the luminescence was detected at room temperature using a DYNEX MLX microtiter plate luminometer equipped with a suuntoinjector for substrate distribution.

Preparation of cytosolic ecdysone receptor extract from Kc Drosophila cells. The Kcl67 line of dipteran cells, originally derived from Drosophila embryos (see Echalier, G. and Ohanessian, A. (1969) CR Acad. Sci., 268, 1771), was provided by Dr. Peter Cherbas (University of Indiana), and remained as described (Cherbas, L., et al., (1994), Methods in Cell Biology) 44, 161-179). A culture of 400ml. of Kc cells (3 x 10 7 cells per ml.) was centrifuged at 300 x g. for 10 minutes at room temperature to bind the cells. The top cream was aspirated and the pellet resuspended in 70ml. of cold TM regulator (10mM Tris, 5mM MgCl2, 1mM DTT, pH 7.2). After a 10 minute incubation on ice, the cells were centrifuged at 2300 xg. The cream was discarded. The cell pellet was frozen at -20 ° C. for an hour. The frozen cell pellet was slowly thawed on ice, and homogenized in a cold Potter-Elvehjem homogenizer using a Teflon mortar on a Caframo homogenizer motor, at a setting of 500 with 10 times from top to bottom at 4 ° C. This slurry was centrifuged at 100,000 x g. in an oscillating piston rotor for 60 minutes. The top cream, which contained the cytosolic protein extract, was diluted with the T-regulator (lOmM of Tris, lmM of DTT, pH 7.2) at a protein concentration of 5mg./ml. This extract was used immediately for the ligand binding assays.

Preparation of the nuclear ecdysone receptor from Plodia interpunctella cells. The IAL-PID2 cell line, derived from the imaginal wing discs of the Plodia interpunctella of the lepidopeteran, was provided by H. Oberlander and maintained as described (Lynn, EE and Oberlander, H., (1983), J. Insect Physiology , 29, 591-96 (Journal on the Physiology of Insects)). A stationary phase culture of 300ml. of P. interpunctella cells was centrifuged at 700 x gr. for 10 minutes at room temperature to bind the cells. The top cream was aspirated and the cell pellet resuspended in 35ml. of TMT (TM regulator with 0.1% Triton X-100). The suspension was homogenized with twenty top-down times of a dounce homogenizer on ice. The homogenized suspension was incubated on ice for 10 minutes, and then centrifuged for 15 minutes at 900 xg. The pellet was resuspended in 15 ml. of TM regulator and centrifuged at 2,300 x g .. Said pellet was extracted in the TMK regulator (regulator TM with 800 M KCl) using a glass bar to break the pellet until a gelatinous slurry formed, and incubated on ice during 15 minutes. The extract was centrifuged at 100,000 x g. in an oscillating piston rotor for 60 minutes. To the top cream, which constituted the nuclear extract, the salt was removed in a 10 DG salt elimination column (Bio-Rad, 732-2010) equilibrated with regulator T. The total protein concentration in the nuclear extract was adjusted at 5mg. per ml. with the regulator T that contained 1 mM of DTT.

Preparation of the bacterial glutathione-S-transferase fusion protein. The bacterial glutathione-S-transferase (GST) fusion proteins of the Spruce earthworm (Choristoneura fumif frog) EcR (CfEcR), which only contained CDEF domains, and ultraspiracle protein (CfUSP) were also used in the radioligand displacement assays. The cDNA encoding the CfEcR CDER domains was constructed following the PCR amplification using primers containing BamHl and EcoRI sites (Perera SC, Sundaram M, Krell PJ, Retnakaran A, Dhadialla TS, SR Palli, 1999 Arch. Insect Biochem, Physiol 41, In Press). The PCR product was digested with BamHI and EcoRI, and cloned into the vector pGEX-3X obtained from Pharmacia Biotech. The coding region of the CfUSP was amplified using primers containing BamHl and EcoRI sites, and cloned into the vector pGEX-2T obtained from Pharmacia Biotech. E. coli transformed with each of the two vectors were cultured and induced to produce the fusion proteins, as indicated in the GST technical bulletin of Pharmacia Biotech.

Competitive binding assay of the ecdysone receptor. 3H Ponasterone A, a potent phytoecdisteroid (660,000dpm, specific activity of 170 Ci / mmole, NEN Life Science Products, Boston, Massachusetts) was mixed with lOOμl of nuclear extracts of Plodia ecdysone receptor or cytosolic Kc in test tubes of 0.8 x 50mm glass in the absence or presence of 10 μM of unlabeled hydroxyecdysone 20 (20E), to obtain an estimate of the total or non-specific binding, respectively, of ponasterone A with tritium. The tubes were introduced into the vortex and incubated overnight at 4 ° C. for the cytosolic extracts Kc or 1.5 hours for the Plodia nuclear extracts, so that the binding reactions achieve equilibrium. At the end of equilibrium binding times, ponasterone A with bound tritium was separated from the unlinked one by adding 600 μl of ice or 300 μl of charcoal solution covered with dextran (500 mg of activated charcoal rinsed with Sigma HCL, 50 mg of Pharmacia Dextran T70, 50ml of regulator T) for Kc or Plodia extracts, respectively. The tubes were briefly vortexed, and centrifuged at 7,000 x g. to bind the charcoal. The top cream, 600μl or 300μl for the Kc or Plodia extract reactions, respectively, containing ponasterone A with bound tritium for the proteins, was aspirated into liquid scintillation flasks each containing 5ml. of scintillation mixture (ReadySafe®, Beckmann). The mixture was vortexed, and the amount of total or non-specifically bound radioactivity was measured in a Beckman LS500 liquid scintillation counter with 60% counting efficiency for tritium.

Determination of Kd values for competitive 3H-ponasterone A inhibitors that bind to ecdysteroid receptor complexes in Kc or Plodie cell extracts and bacterial fusion proteins. The concentrations of the competitors that inhibited 50% of the binding of ponasterone A with tritium (IC50) were determined by incubating nuclear extracts of Kc or Plodia or extracts of bacteria producing fusion proteins CfEcR (CDEF) -GST and CfUSP-GST with 3H -ponasterone A (66, OOOdpm per reaction) in the presence of a range of concentrations (O.lnM to lOμM) of test compounds. In the case of the binding reactions using bacterial fusion proteins, they were included by binding reaction 20 or Iμl of extract of bacteria that produced fusion proteins CfEcR (CDEF) -GST or CfUSP-GST, respectively. The conditions of assay and determination of the total and non-specific binding were as described above. The volume of the competitor in solvent, such as that of 20E or the solvent alone, was maintained at 1% of the total reaction volume using a 100-fold concentrated stock solution (ie, a 100-fold dilution of the stock solutions). The rest of the trial was as described previously. Each reaction was carried out in duplicate by concentration per test compound. The specific binding of 3H-ponasterone A was determined by subtracting the non-specific binding (that obtained in the presence of lOμM of 20E) from the competing or total radioactivity binding (non-competing). The data for each test compound was analyzed using the IGOR Pro (WaveMetrics, Lake Oswego, OR) computer program to calculate IC50 values. The linking constant (Kd in μM) for the test compounds was calculated by incorporating the Cheng-Prusoff equation (see Munson PJ and Rodbard D. (1980) Anal. Biochem. 107, 220-239) in the computer program IGOR Pro.

Tables 1 to 3 summarize the data obtained: Table 1 Ligand Kc Plodia CfECR bacterial Log (l / EC50) log (l / IC50) log (l / lC50) CE-1 6.55 8.7 CE-2 5.52 6.48 CE-3 7.96 1 7.36 8.52 2 7.16 9.14 3 6.73 9.04 8.76 4 7.41 8.51 5 7.70 9.49 6 8.82 7 8.78 8 8.85 9 9.00 10 8.80 11 8.93 12 8.10 9.63 13 7.44 9.06 14 8.61 15 8.49 17 9.24 18 8.82 ? abla 2 Activation of the luciferase gene in transiently transfected CHO cells.

CE-4 = ponasterone A 1 In units of relative light (ULR) - Average of the samples in duplicate 2 ULR index in the presence and absence of the ligand.

Table 3 Activation of the β-galactosidase gene in stably transformed CHO cells.

Ligand Kd index "1" activity for the lOmMs the ß-galactosidase induction Kc cells (nM) none 200 CE-1 323 2 192 CE-2 154 1 2000 CE-3 9830 49 2 CE-4 10386 52 0.7 1 1123 6 40 2 536 3 47 3 681 3 124 4 1963 10 26 5 11599 58 13 12 9830 49 5 13 6667 33 24 1 In units of relative light (ULR) - Average of the samples in triplicate 2 ULR index in the presence or absence of the ligand These data indicate that the ligands of this invention can induce the expression of the gene in concentrations comparable or lower than those of the known compounds CE-1 and CE-2.

In addition to the enhanced modulation capacity of gene expression, ligands are expected to have greater utility in intact animals and plants due to their superior metabolic and transport properties. The following sample demonstrates the improved transport of a ligand of this invention in a plant (Ligand 3) compared to a known ligand (CE-1) in plants.

Translaminar movement. A study was conducted to evaluate the translaminar movement of two compounds within cotton leaves using as a model system neonate beetworm larvae, Spodoptera exigua. Two treatments were evaluated: emulsifiable concentrates of CE-1 and ligand 3 (5% and 19%, respectively) at a concentration of 100μg / ml. to simulate concentrations in spray tank. These treatments were then compared with plants treated with a chemical standard known to have excellent translaminar movement (emamectin benzoate, 0.16% EC [20μg / ml.], Merck &Co.), and with untreated plants. Cotton plants with four weeks old, Gossypium hirsutum L. cv. Stoneville, were treated with each treatment by painting the upper surface of a single leaf / replica plant with a corresponding treatment. The plants were kept in a glasshouse with controlled environment maintained at 27 ° C. until it was needed. The residual effectiveness was evaluated by exposing treated and untreated foliage to neonatal beetworm larvae, Spodoptera exigua (Hübner), during three dates: 1, 7 and 14 days after treatment (DDT). At each sample date, the treated single leaf / plant was removed from each of the five replicate plants per treatment. The leaves were then attached to the top cover of a plastic Petri dish (100 x 20mm.) And then infested with ten first instar larvae. The first instar larvae of the beetworm are usually fed on the underside of the leaves, and usually do not feed on the upper surface. Therefore, the translaminar movement of the material would necessarily have to affect the larvae since the compounds were applied only to the upper surface of the leaf. The mortality of the larvae was recorded four days after the infestation. In addition, live larvae were observed to have characteristic symptoms of the shedding accelerator compounds (new cuticle formation and / or shed head capsule). Each treatment was repeated five times.

The data on the mortality percentage were transformed into square root, and then analyzed by means of ANOVA using JMP (Ver. 3.2.1). The averages were separated by means of the Tukey-Kramer test (P = 0.05). The results showed that ligand 3 was significantly more effective than CE-1 in killing S. exigua larvae from translaminar movement and, therefore, it would be expected to have a higher systematization in plants.

Table 4 of mortality Conc. Day after application Treatment (μg / ml, ./ 1 7 14 CE-1, 5% EC 100 2.5 0.0 22.7 3, 19% EC 100 100.0 93.8 100.0 emamectin benzoate 0.16 EC + AG-98 20 100.0 100.0 100.0 untreated mark 11.6 6.5 8.1 AG-98 = 0.12% of the surfactant Latron AG-98 nonionic surfactant (Rohm and Haas Company).

Claims (5)

Claims 1. In a method for modulating exogenous gene expression, comprising contacting an ecdysone receptor complex comprising: a) a DNA binding domain; b) a ligand binding domain; c) a transactivation domain; and d) a ligand; with a DNA construct comprising: a) the exogenous gene; and b) a response element; wherein: a) the exogenous gene is under the control of the response element; and b) the binding of the DNA binding domain to the response element, in the presence of the ligand, results in the activation or repression of the gene; the improvement comprises: selecting the ligand of a compound of the formula: wherein: E is an alkyl (C -C6) containing a tertiary carbon or a cyano (C3-C5) alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3CHF2, CHC12, CH2F, CH2C1, CH2OH, CH20Me, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe , OEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH20Me, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi Pr, SCN, SCHF2, SOMe, NH-CN or, linked to R3 and the phenyl carbons to which R2 and R3 are linked to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R3 is H, Et or, attached to R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R4, R5 and R6 are, independently, H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, 'CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propinyl, 2-propinyl, vinyl, OMe, OEt, SMe or SEt; provided that: a) when R1 is Me and R2 is OMe then R3 will be H; and the combination of R4, R? and Rs will be 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl or 3,5-di-F. b) when R1 is Me and R2 is OEt then R3 will be H and the combination of R4, R5 and R6 will be 3,5-di-Me, 3, 5-di-OMe-4-Me, 3,5-di- Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl; c) when R1 is Et and R2 is OMe or OEt then R3 will be H and the combination of R4, Rs and Rs will be: i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3 , 5-di-Cl, 3,5-di-F, 2,4-or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5 -Me, 2-Br-5-Me, 2-Cl, 2-Br or 3-Me; or ii) R6 will be H, R4 will be Me and R5 will be Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2Cl, CH2Cl, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , OMe, OEt, SMe or SEt; d) when R1 is i-Pr then R2 will be OMe or OEt; R3 will be H, and the combination of R4, R5 and R6 will be 3, 5-di-Me; e) when R3 is Et then R2 will be H, R1 will be F or Cl, and the combination of R4, R5 and Rs will be 3, 5-di-Me; f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring then R1 will be Me or Et and the combination of R4, R5 and R6 will be 3,5-di-Me; g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring then R1 will be Et and the combination of R4, R5 and Rd will be 3.5-di-Me; H) when R1 is formyl, CF3, CHF2, CHCl2, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2; then R2 will be OMe or Oet, R3 will be H, and the combination of R4, R5 and R6 will be 3,5-di-Me; ei) when R2 is Me, Et, n-Pr, i-Pr, formyl CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl 2-propynyl, vinyl, Ac , F, Cl, OH, On-Pr, OAc, NMe2, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3 / OCHF2 / Oi-Pr, SCN, SCHF2, SOMe or NH -CN; then R1 will be Et, R3 will be H, and the combination of R4, R5 and R6 will be 3, 5-di-Me.
1. 2. A method for modulating exogenous gene expression, comprising contacting an ecDisone receptor complex comprising: a) a DNA binding domain; b) a ligand binding domain; c) a transactivation domain; and d) a ligand of the formula: Wherein: E is an alkyl (C-C3) containing a tertiary carbon or a cyano (C3-C5) alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH20H, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH20H, CH20Me, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi Pr, SCN, SCHF2, SOMe, NH-CN or, together with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R3 is H, Et or, linked with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R4, R5 and R6 are, independently, H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH20H, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , OMe, OEt, SMe, or SEt; provided that: a) when R1 is Me and R2 is OMe then R3 will be H, and the combination of R4, R5 and R6 will be 3,5-di-Me, 3,5-di-OMe-4-Me, 3, 5-di-Cl or 3,5-di-F; b) when R1 is Me and R2 is OEt then R3 will be H, and the combination of R4, R5 and R6 will be 3,5-di-, 3,5-di-OMe-4-Me, 3,5-di -Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl; c) when R1 is Et and R2 is OMe or OEt then R3 will be H, and the combination of R4, R5 and Rs will be: i) 3,5-di-0Me-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F,
2. 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br-5-Me, 2-C1, 2-Br or
3. 3 - . 3 -Me; or ii) R6 will be H, R4 will be Me, and R5 will be Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt; d) when R1 is i-Pr then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and R6 will be 3.5-di-Me; e) when R3 is Et then R2 will be H, R1 will be F or Cl, and the combination of R4, R5 and R6 will be 3.5-di-Me; f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring then R1 will be Me or Et, and the combination of R4, R5 and R6 will be 3.5-di-Me; g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring then R1 will be Et, and the combination of R4, R5 and R6 will be 3.5-du-Me; h) when R1 is formyl, CF3, CHF, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH = CHCN, allyl , azido, SCN or SCHF2; then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and R6 will be 3, 5-di-Me; yi) when R2 is Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , Ac, F, Cl, OH, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi-Pr, SCN , SCHF2, SOMe, or NH-CN; then R 1 will be Et, R 3 will be H, and the combination of R 4, R 5 and R 6 will be 3, 5 -di-Me; with a DNA construct comprising: a) an exogenous gene; and b) a response element; wherein: a) the exogenous gene is under the control of the response element; and b) the binding of the DNA binding domain to the response element, in the presence of the ligand, results in the activation or repression of the gene. 3. A method for modulating the expression of one or more exogenous genes in a subject, comprising administering to the subject an effective amount of a ligand of the formula: I wherein: E is a (C
4. 4-C6) alkyl containing a tertiary carbon or a cyano (C3-C5) alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi Pr, SCN, SCHF2, SOMe, NH-CN or, together with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R3 is H, Et or, together with R2 and the phenyl carbons to which R2 and R3 are unidps to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R4, R5 and Rs are, independently, H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , OMe, OEt, SMe, or SEt; provided that: a) when R1 is Me and R2 is OMe then R3 will be H, and the combination of R4, R5 and R6 will be 3,
5. 5-di-Me, 3,5-di-OMe-4-Me, 3, 5-di-Cl or 3,5-di-F; b) when R1 is Me and R2 is OEt then R3 will be H, and the combination of R4, Rs and R6 will be 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di -Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl; c) when R1 is Et and R2 is OMe or OEt then R3 will be H, and the combination of R4, R5 and R6 will be: i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di- F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br- 5-Me, 2-Cl, 2-Br or 3-Me; or ii) R6 will be H, R4 will be Me, and R5 will be Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt; d) when R1 is i-Pr then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and R6 will be 3.5-di-Me; e) when R3 is Et then R2 will be H, R1 will be F or Cl, and the combination of R4, R5 and R6 will be 3.5-di-Me; f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring then R1 will be Me or Et, and the combination of R4, R5 and R6 will be 3.5-di-Me; g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring then R1 will be Et, and the combination of R4, R5 and R6 will be 3.5-di-Me; h} when R1 is formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido , SCN or SCHF2; then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and R6 will be 3, 5-di-Me; yi) when R2 is Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH20Me, CH20Me, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , Ac, F, Cl, OH, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi-Pr, SCN , SCHF2, SOMe, or NH-CN; then R1 will be Et, R3 will be H, and the combination of R4, R5 and R6 will be 3, 5-di-Me; wherein the cells of the subject contain: a) an ecdysone receptor complex comprising 1) a DNA binding domain; 2) a binding domain for the ligand; and 3) a transactivation domain; and b) the DNA construct comprises: 1) an exogenous gene; and 2) a response element; and wherein: a) the exogenous gene is under the control of the response element; and b) the binding of the DNA binding domain to the response element, in the presence of the ligand, results in the activation or repression of the gene. 4. A method for producing a polypeptide comprising the following steps: a) selecting a cell that is substantially not sensitive to the exposure of a ligand of the formula: wherein: E is a (C4-C6) alkyl containing a tertiary carbon or a cyano (C3-C5) alkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH20H, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, On-Pr, OAc, NMe2, Net2, SMe, SEt, S0CF3, 0CF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, 0CF3, OCHF2, Oi Pr, SCN, SCHF2, SOMe, NH-CN or, together with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R3 is H, Et or, together with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R4, R5 and Rs are, independently, H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , OMe, OEt, SMe, or SEt; provided that: a) when R1 is Me and R2 is OMe then R3 will be H, and the combination of R4, R5 and Rs will be 3,5-di-Me, 3,5-di-OMe-4-Me, 3, 5-di-Cl or 3,5-di-F; b) when R1 is Me and R2 is OEt then R3 will be H, and the combination of R4, R? and Rs will be 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di- F, 2,4- or 2,5-di-Cl; c) when R1 is Et and R2 is OMe or OEt then R3 will be H, and the combination of R4, R5 and R6 will be: i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br- 5-Me, 2-C1, 2-Br or 3-Me; or ii) R6 will be H, R4 will be Me, and R5 will be Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt; d) when R1 is i-Pr then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and R6 will be 3.5-di-Me; e) when R3 is Et then R2 will be H, R1 will be F or Cl, and the combination of R4, R5 and R6 will be 3.5-di-Me; f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring then R1 will be Me or Et, and the combination of R4, R5 and R6 will be 3.5-di-Me; g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring then R1 will be Et, and the combination of R4, R5 and R6 will be 3.5-di-Me; h} when R1 is formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido , SCN or SCHF2; then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and Rs will be 3, 5-di-Me; yi) when R2 is Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH20Me, CH20Me, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , Ac, F, Cl, OH, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi-Pr, SCN , SCHF2, SOMe, or NH-CN; then R1 will be Et, R3 will be H, and the combination of R4, R5 and R6 will be 3.5 -di-Me; b) introducing into the cell: 1) a DNA construct comprising: a) an exogenous gene encoding the polypeptide; and b) a response element; wherein the gene is under the control of the response element; and 2) an ecdysone receptor complex comprising: a) a DNA binding domain; b) a binding domain for the ligand; and c) a transactivation domain; and c) exposing the cell to a ligand. 5. A method for regulating the expression of the endogenous or heterologous gene in a transgenic organism, comprising contacting a ligand of the formula: Wherein: E is a (C4-C6) alkyl containing a tertiary carbon or a cyano (C3-C3) cyanoalkyl containing a tertiary carbon; R1 is H, Me, Et, i-Pr, F, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH20H, CH20Me, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, SCN or SCHF2; R2 is H, Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2CH, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi Pr, SCN, SCHF2, SOMe, NH-CN or, together with R3 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R3 is H, Et or, together with R2 and the phenyl carbons to which R2 and R3 are attached to form an ethylenedioxy, a dihydrofuryl ring with oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with oxygen adjacent to a phenyl carbon; R4, R5 and R6 are, independently, H, Me, Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2-propynyl, vinyl , OMe, OEt, SMe, or SEt; provided that: a) when R1 is Me and R2 is OMe then R3 will be H, and the combination of R4, R5 and R6 will be 3,5-di-Me / 3, 5-di-OMe-4-Me, 3, 5-di-Cl or 3,5-di-F; b) when R1 is Me and R2 is OEt then R3 will be H, and the combination of R4, R5 and R6 will be 3,5-di-Me, 3,5-di-OMe-4-Me, 3,5-di -Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl; c) when R1 is Et and R2 is OMe or OEt then R3 will be H, and the combination of R4, R5 and R6 will be: i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4- or 2,5-di-F, 2,4- or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br- 5-Me, 2-C1, 2-Br or 3-Me; or ii) Rs will be H, R4 will be Me, and R5 will be Et, F, Cl, Br, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CN, C = CH, 1-propynyl, 2 -propynyl, vinyl, OMe, OEt, SMe, or SEt; d) when R1 is i-Pr then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and R6 will be 3.5 -di-Me; e) when R3 is Et then R2 will be H, R1 will be F or Cl, and the combination of R4, R5 and Rs will be 3, 5-di-Me; f) when R2 and R3, together with the phenyl carbons to which they are attached, form an ethylenedioxy ring then R1 will be Me or Et, and the combination of R4, R5 and R6 will be 3.5-di-Me; g) when R2 and R3, together with the phenyl carbons to which they are attached, form a dihydrofuryl or dihydropyryl ring then R1 will be Et, and the combination of R4, R5 and R6 will be 3.5-di-Me; h} when R1 is formyl, CF3, CHF2, CHC12, CH2F, CH2Cl, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido , SCN or SCHF2; then R2 will be OMe or OEt, R3 will be H, and the combination of R4, R5 and R6 will be 3, 5-di-Me; and i) when R2 is Me, Et, n-Pr, i-Pr, formyl, CF3, CHF2, CHC12, CH2F, CH2C1, CH2OH, CH2OMe, CH2CN, CN, C = CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, On-Pr, OAc, NMe2, NEt2, SMe, SEt, SOCF3, OCF2CF2H, COEt, cyclopropyl, CF2CF3, CH = CHCN, allyl, azido, OCF3, OCHF2, Oi-Pr, SCN, SCHF2, SOMe, or NH-CN; then R1 will be Et, R3 will be H, and the combination of R4, R5 and R6 will be 3.5 -di-Me; with an ecdysone receptor complex within the cells of the organism, wherein the cells further contain a DNA-binding sequence for the ecdysone receptor complex when in combination with the ligand, and wherein the formation of a sequence complex of The complex-ligand-DNA link of the ecdysone receptor induces gene expression. 6. The method according to claim 2, 3, 4 or 5, wherein the ligand is of the specified formula, and E is t-butyl; R1 is Me, Et, i-Pr or F; R2 is OH, OMe, OEt or, together with R3 and the phenyl carbons to which R2 and R3 are attached, forms an ethylenedioxy or dihydrofuryl ring with oxygen adjacent to a phenyl carbon; R3 is H, Et or is attached to R2 and the phenyl carbons, to which R2 and R3 are attached, to form an ethylenedioxy or dihydrofuryl ring with oxygen adjacent to a phenyl carbon; and R4, R5 and R6 are, independently, Me, F, Cl, CH2OH or OMe. The method according to claim 2, 3, 4 or 5, wherein the ligand is of the specified formula, and E is t-butyl, R1 is Et, R2 is Oet, R3 is H, and the combination of R4, R5 and R6 is 3, 5 -di-Me. The method according to claim 2, 3, 4 or 5, wherein the ecdysone receptor complex is a chimeric ecdysone receptor complex, and the DNA construct further comprises a promoter. 9. The method according to claim 3, wherein the subject is a plant. 10. The method according to claim 3, wherein the subject is a mammal.