NR4A1 Ligands, Pharmaceutical Compositions, and Related Methods of Use

Abstract

To provide this summary to introduce a selection of concepts in a simplified form that are further described herein in the Detailed Description, where this summary is neither intended to identify key features of the claimed subject matter, nor intended to be used as an aid in determining the scope of the claimed subject matter.SOLUTION: NR4A1 ligands, pharmaceutical compositions including the NR4A1 ligands, and related methods of use are described. Provided are methods of treating a disease or condition in an individual treatable by modulation of NR4A1 activity, comprising administering to the individual a therapeutically effective amount of a compound or a pharmaceutical composition described herein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 62 / 543,761, filed August 10, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] Nuclear receptor subfamily 4 group A member 1 (NR4A1) is overexpressed in colon, pancreatic, breast (estrogen receptor-positive and -negative), and lung tumors, and high NR4A1 expression predicts decreased survival in patients with breast, colon, and lung tumors. The functional activity of NR4A1 in cancer has been extensively investigated in cancer cell lines by either knockdown or overexpression. Results have shown that NR4A1 regulates one or more of cancer cell proliferation, viability, cell cycle progression, migration, and invasion (Figure 1) in lung, melanoma, lymphoma, pancreatic, colon, breast, kidney, cervical, ovarian, and gastric cancer cell lines. Studies have identified β1 and other integrins as NR4A1-regulated genes. Studies have also demonstrated that NR4A1 is overexpressed in tumors from patients with rhabdomyosarcoma (RMS), and that this receptor regulates pro-oncogenic pathways in RMS cells, as shown in Figure 1. The pro-oncogenic functions of NR4A1 include regulation of several genes that are themselves individual drug targets, including integrins, survivin, EGFR, and other receptor tyrosine kinases.

[0003] This suggests that the development of NR4A1 antagonists represents a unique chemotherapy strategy that simultaneously targets multiple pro-oncogenic pathways associated with solid tumor growth, viability, and migration / invasion. Studies first demonstrated that several 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compounds inactivated NR4A1. Subsequently, studies identified DIM-C-pPhOH and other p-substituted phenyl analogs as NR4A1 ligands. DIM-C-pPhOH and related compounds block the pro-oncogenic pathways outlined in Figure 1 at concentrations of 10–20 μM in cell culture and partially inhibit tumor growth at doses of 30–40 mg / kg / day. DIM-C-pPhOH has a high binding affinity for NR4A1 (K 0–0.100 μM) but exhibits a short in vivo half-life.

[0004] As described above, research has focused on developing NR4A1 ligands that bind to NR4A1 and act as antagonists (inverse agonists). Studies by one or more of the present inventors have relied on RNA interference (RNAi) to knock down NR4A1 and molecular / biochemical studies to determine the effects of receptor knockdown on cellular function and related pathways required for cellular function. Initial studies have shown that NR4A1 knockdown results in reduced pancreatic cancer cell proliferation and induction of apoptosis, which has also been observed in rhabdomyosarcoma, lung cancer, breast cancer, kidney cancer, and colon cancer cell lines. Mechanistic studies demonstrate that NR4A1 acts as a cofactor to activate pro-survival (survivin and bcl2) and pro-growth (EGFR and other receptor tyrosine kinase) genes by interacting with Sp transcription factors bound to their proximal GC-rich regions. NR4A1, in conjunction with other cofactors (e.g., p300), activates and regulates the expression of these genes.

[0005] Among a series of C-DIM analogs, the p-hydroxyphenyl analog (DIM-C-pPhOH; CDIM8) was demonstrated to not only bind to NR4A1 but also act as an antagonist. Accordingly, DIM-C-pPhOH inhibits cancer cell proliferation and survival and inhibits the expression of survivin, EGFR, and other NR4A1 / Sp-regulated genes. Knockdown of NR4A1 by RNAi also inhibits mTOR signaling through p53-dependent and -independent activation of Sestrin, and similar results were observed with DIM-C-pPhOH and other NR4A1 / C-DIM antagonists in multiple cancer cell lines. NR4A1 also plays an important role in maintaining low oxidative stress in cancer cell lines by regulating the expression of isocitrate dehydrogenase 1 (IDH1) and thioredoxin domain-containing 5 (TXNDC5). Knockdown of NR4A1 or treatment with DIM-C-pPhOH and other NR4A1 antagonists reduced the expression of IDH1 and TXNDC6, resulting in the induction of reactive oxygen species (ROS), ROS-dependent endoplasmic reticulum stress, and cell death in many cancer cell lines (Figure 1). Recent studies have shown that NR4A1 plays a key role in cancer cell migration / invasion, which is due to NR4A1 / Sp-mediated regulation of the invasion-promoting gene β1-integrin. Knockdown of NR4A1 or treatment with DIM-C-pPhOH or other NR4A1 antagonists reduced cancer cell migration and downregulated β1-integrin and other integrins (Figure 1).

[0006] These studies established that NR4A1 is pro-tumorigenic in solid tumors, and DIM-C-pPhOH and other C-DIMs were characterized as NR4A1 antagonists in cancer cell lines. All first-generation C-DIM compounds contain a p-substituted phenyl moiety, and DIM-C-pPhOH (p-hydroxyphenyl) was characterized as a high-affinity ligand that primarily exhibited NR4A1 antagonist activity in cancer cell lines while exhibiting minimal receptor-independent activity such as mitochondrial toxicity. However, the in vivo tumor growth inhibitor activity of DIM-C-pPhOH and related compounds was in the range of approximately 30 mg / kg / day, and their in vivo half-lives were very short.

[0007] The present disclosure seeks to fulfill these needs and provides further related advantages. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,232,843 [Non-patent literature]

[0009] [Non-Patent Document 1] Remington's Pharmaceutical Sciences, 18th edition (Easton, Pa.: Mack Publishing Company, 1990) Summary of the Invention [Problem to be solved by the invention]

[0010] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. [Means for solving the problem]

[0011] In one aspect, the disclosure provides a nuclear receptor subfamily 4 group A member 1 (NR4A1) ligand. In one embodiment, the ligand is a compound of the formula:

[0012] [ka]

[0013] or a salt thereof, During the ceremony, R1, R2, R1', and R2' are each independently selected from the group consisting of hydrogen, a linear alkyl group containing from 1 to about 10 carbon atoms, and a branched alkyl group containing from 1 to about 10 carbon atoms; R3, R4, R5, R6, R3', R4', R5', and R6' are each independently selected from the group consisting of hydrogen, halogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, an alkoxy group containing 1 to about 10 carbon atoms, and a nitro group; R7 is selected from the group consisting of hydrogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, a cycloalkyl group containing 1 to about 10 carbon atoms, and an aryl group; R8, R9, R 10 , R 11 , and R 12 are independently selected from the group consisting of H, halogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, an alkoxy group containing 1 to about 10 carbon atoms, a haloalkyl group containing 1 to about 10 carbon atoms, a nitro group, a hydroxyl group, and a haloalkoxy group containing 1 to about 10 carbon atoms; R8, R9, R 10 , R 11 , and R 12 at least one of is OH, R 10 When is OH, R8, R9, R 10 , R 11 , and R 12 At least one of is not hydrogen.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.

[0015] In another aspect, the disclosure provides a method for treating a disease or condition in an individual treatable by modulation of NR4A1 activity, comprising administering to the individual a therapeutically effective amount of a compound or pharmaceutical composition described herein.

[0016] In another aspect, the disclosure provides a method of modulating NR4A1 activity in a cell, the method comprising administering to the cell a compound or pharmaceutical composition described herein.

[0017] The foregoing aspects and many of the attendant advantages of the claimed disclosure will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0018] [Figure 1] 10A-10C schematically illustrate the inhibition of nuclear receptor subfamily 4 group A member 1 (NR4A1) by 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methane (CDIM) / NR4A1 ligand by attenuating NR4A1-dependent proliferation and survival pathways. [Figure 2] 1 graphically depicts luciferase activity in Panc1 cells transfected with GAL4-NR4A1 and GAL4-NR4A2 and UAS-Luc (GAL4-linked luciferase construct) treated with 4-, 3-, and 2-hydroxy C-DIM analogs according to embodiments of the present disclosure. [Figure 3] 1 includes images of Western blots of Panc1 (pancreatic) and SKBR3 (breast) cancer cells treated with monohydroxy C-DIM compounds according to embodiments of the present disclosure, showing downregulation of expression of two NR4A1-dependent factors, β1-integrin and TXNDC5. [Figure 4] 1 includes images of Western blots of Rh30 (RMS) cells treated with 4-, 3-, and 2-hydroxy C-DIM compounds according to embodiments of the present disclosure, showing downregulation of PAX3-FOX01A and TXNDC5 expression. [Figure 5]1 includes images of Western blots of SKBR3 (breast) cancer cells treated with 4-, 3-, and 2-hydroxy C-DIM compounds according to embodiments of the present disclosure, showing upregulation of SERPINB5 expression. [Figure 6] 1 graphically depicts luciferase activity in Panc1 cells transfected with GAL4-NR4A1 and UAS-Luc treated with 2-hydroxy C-DIM analogs according to embodiments of the present disclosure. [Figure 7] 1 graphically depicts luciferase activity in Panc1 cells transfected with GAL4-NR4A1 and UAS-Luc treated with 3-hydroxy C-DIM analogs according to embodiments of the present disclosure. [Figure 8] 1 graphically depicts luciferase activity in Panc1 cells transfected with GAL4-NR4A1 and UAS-Luc treated with 4-hydroxy C-DIM analogs according to embodiments of the present disclosure. [Figure 9] 1 graphically depicts luciferase activity in Panc1 cells transfected with GAL4-NR4A1 and UAS-Luc treated with 4-hydroxy C-DIM analogs according to embodiments of the present disclosure. [Figure 10A] 1 includes images of Western blots of Panc1 cells treated with 4-hydroxyC-DIM analog (5 μM) and 4-OH (DIM-C-pPhOH parent compound) showing induction of SERPINB5 according to embodiments of the present disclosure. [Figure 10B] 1 includes images of Western blots of Rh30 cells treated with 4-hydroxyC-DIM analog (5 μM) and 4-OH (DIM-C-pPhOH parent compound) showing reduced induction of PAX3-FOX01A and TXNDC5 according to embodiments of the present disclosure. [Figure 11A] 1 graphically depicts the NR4A1-dependent induction of interleukin-24 (IL-24), guanine deaminase (GDA), and doublecortin domain-containing 2 (DCDC2) by DIM-C-pPhOH (4-OH compound) and substituted C-DIM analogs according to embodiments of the present disclosure in Rh30 cells. [Figure 11B]1 graphically depicts the NR4A1-dependent induction of interleukin-24 (IL-24), guanine deaminase (GDA), and doublecortin domain-containing 2 (DCDC2) by DIM-C-pPhOH (4-OH compound) and substituted C-DIM analogs according to embodiments of the present disclosure in Rh30 cells. [Figure 11C] 1 graphically depicts the NR4A1-dependent induction of interleukin-24 (IL-24), guanine deaminase (GDA), and doublecortin domain-containing 2 (DCDC2) by DIM-C-pPhOH (4-OH compound) and substituted C-DIM analogs according to embodiments of the present disclosure in Rh30 cells. [Figure 12] Figure 12A graphically illustrates mammary tumor volume in an athymic nude mouse xenograft model using human MDA-MB-231 breast cancer cells treated with a 4-hydroxy C-DIM analog (10 mg / kg / day) (black) versus a control (gray) according to an embodiment of the present disclosure. Figure 12B graphically illustrates mammary tumor volume in an athymic nude mouse xenograft model using human MDA-MB-231 breast cancer cells treated with a 4-hydroxy C-DIM analog (10 mg / kg / day) (black) versus a control (gray) according to an embodiment of the present disclosure. Figure 12C graphically illustrates mammary tumor volume in an athymic nude mouse xenograft model using human MDA-MB-231 breast cancer cells treated with a 4-hydroxy C-DIM analog (10 mg / kg / day) (black) versus a control (gray) according to an embodiment of the present disclosure. [Figure 13] 1 graphically depicts luciferase activity in Panc1 cells transfected with GAL4-NR4A2 and UAS-Luc treated with 3-hydroxy C-DIM analogs according to embodiments of the present disclosure. [Figure 14] 1 includes an image of a Western blot of SKBR3 cells treated with a 3-hydroxy C-DIM analog according to an embodiment of the present disclosure. [Figure 15] 1 graphically depicts luciferase activity in Panc1 cells transfected with GAL4-NR4A2 and UAS-Luc treated with 2-hydroxy C-DIM analogs according to embodiments of the present disclosure. [Figure 16] 1 includes an image of a Western blot of Panc1 cells treated with a 2-hydroxy C-DIM analog according to an embodiment of the present disclosure, showing reduced induction of TXNDC5 and β1-integrin. [Figure 17] 1 includes an image of a Western blot of Panc1 cells treated with a 2-hydroxy C-DIM analog according to an embodiment of the present disclosure, showing induction of SERPINB5 and GADD45α. [Figure 18] 1 includes an image of a Western blot of dissociated tumor cell lysates from an orthotopic breast cancer model treated with a 4-hydroxy C-DIM analog according to an embodiment of the present disclosure, showing inhibition of the mTOR pathway. [Figure 19] 10A-10C show downregulation of the NR4A1 gene product and upregulation of GADD45α, SERPINB5, and c-PARP. 10B includes images of Western blots of dissociated tumor cell lysates from an orthotopic breast cancer model treated with a 4-hydroxy C-DIM analog according to embodiments of the present disclosure. [Figure 20A] 1 graphically depicts luciferase activity in glucose-deprived HepG2 cells treated with C-DIM analogs according to embodiments of the present disclosure, demonstrating downregulation of G6Pase. [Figure 20B] 1 graphically depicts luciferase activity in glucose-deprived HepG2 cells treated with C-DIM analogs according to embodiments of the present disclosure, demonstrating downregulation of PEPCK. [Figure 21] 1 graphically depicts NR4A2-dependent induction of osteopontin (OPN) gene expression in Panc1 cells treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 22] 1 is a surface plasmon resonance (SPR) response curve of a C-DIM analog according to an embodiment of the present disclosure that binds to NR4A1. [Figure 23A] 1A-C graphically depict xenograft breast tumor volume (AC) and mass (D) in mice treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 23B]1A-C graphically depict xenograft breast tumor volume (AC) and mass (D) in mice treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 23C] 1A-C graphically depict xenograft breast tumor volume (AC) and mass (D) in mice treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 23D] 1A-C graphically depict xenograft breast tumor volume (AC) and mass (D) in mice treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 24A] 1 graphically depicts tumor volume of RMS xenograft tumors in mice treated with 3,5-dibromo-4-hydroxyC-DIM compound according to an embodiment of the present disclosure. [Figure 24B] 1 graphically depicts tumor volume of RMS xenograft tumors in mice treated with 3,5-dibromo-4-hydroxyC-DIM compound according to an embodiment of the present disclosure. [Figure 24C] 1 graphically depicts tumor volume of RMS xenograft tumors in mice treated with 3,5-dibromo-4-hydroxyC-DIM compound according to an embodiment of the present disclosure. [Figure 24D] 1 graphically depicts tumor volume of RMS xenograft tumors in mice treated with 3,5-dibromo-4-hydroxyC-DIM compound according to an embodiment of the present disclosure. [Figure 25A] 1 is an image of a Western blot of whole cell lysates of C2C12 cells treated with a C-DIM analog according to an embodiment of the present disclosure. [Figure 25B] 1 is an image of a Western blot of whole cell lysates of C2C12 cells treated with a C-DIM analog according to an embodiment of the present disclosure. [Figure 25C] 1 is an image of a Western blot of whole cell lysates of C2C12 cells treated with a C-DIM analog according to an embodiment of the present disclosure. [Figure 25D] 1 is an image of a Western blot of whole cell lysates of C2C12 cells treated with a C-DIM analog according to an embodiment of the present disclosure. [Figure 26A]1 graphically depicts relative NR4A1 / glucose transporter 4 (GLUT-4) mRNA expression in C2C12 cells treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 26B] 1 graphically depicts relative NR4A1 / glucose transporter 4 (GLUT-4) mRNA expression in C2C12 cells treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 26C] 1 graphically depicts relative NR4A1 / glucose transporter 4 (GLUT-4) mRNA expression in C2C12 cells treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 26D] 1 graphically depicts relative NR4A1 / glucose transporter 4 (GLUT-4) mRNA expression in C2C12 cells treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 27] 1 graphically illustrates glucose uptake in C2C12 cells treated with C-DIM analogs according to embodiments of the present disclosure. [Figure 28A] Figure 1 shows the relative mRNA expression of glycolytic genes in C2C12 cells treated with DIM-C-pPhOH (A), DIM-C-pPhOH-3,5-Br2 (B), DIM-C-pPhOH-3-Cl (C), and DIM-C-pPhOH-3-Cl-5-OCH3 (D) for 24 hours or transfected with an NR4A1 expression plasmid (E). [Figure 28B] Figure 1 shows the relative mRNA expression of glycolytic genes in C2C12 cells treated with DIM-C-pPhOH (A), DIM-C-pPhOH-3,5-Br2 (B), DIM-C-pPhOH-3-Cl (C), and DIM-C-pPhOH-3-Cl-5-OCH3 (D) for 24 hours or transfected with an NR4A1 expression plasmid (E). [Figure 28C]Figure 1 shows the relative mRNA expression of glycolytic genes in C2C12 cells treated with DIM-C-pPhOH (A), DIM-C-pPhOH-3,5-Br2 (B), DIM-C-pPhOH-3-Cl (C), and DIM-C-pPhOH-3-Cl-5-OCH3 (D) for 24 hours or transfected with an NR4A1 expression plasmid (E). [Figure 28D] Figure 1 shows the relative mRNA expression of glycolytic genes in C2C12 cells treated with DIM-C-pPhOH (A), DIM-C-pPhOH-3,5-Br2 (B), DIM-C-pPhOH-3-Cl (C), and DIM-C-pPhOH-3-Cl-5-OCH3 (D) for 24 hours or transfected with an NR4A1 expression plasmid (E). [Figure 28E] Figure 1 shows the relative mRNA expression of glycolytic genes in C2C12 cells treated with DIM-C-pPhOH (A), DIM-C-pPhOH-3,5-Br2 (B), DIM-C-pPhOH-3-Cl (C), and DIM-C-pPhOH-3-Cl-5-OCH3 (D) for 24 hours or transfected with an NR4A1 expression plasmid (E). [Figure 29A] 1 graphically depicts the relative rates of blood glucose in vehicle control and C-DIM8-3-Cl-5-OCH3 (10 mg / kg / d) treated mice according to an embodiment of the present disclosure. [Figure 29B] 1 graphically illustrates the reduction in blood glucose levels during glucose tolerance in HFD-fed C57BL / 6 mice treated with vehicle control and C-DIM8-3-Cl-5-OCH3 according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides nuclear receptor subfamily 4 group A member 1 (NR4A ligands), pharmaceutical compositions comprising a therapeutically effective amount of the NR4A1 ligand, and related methods of use.

[0020] NR4A1 ligand In one aspect, the present disclosure provides compounds that are NR4A1 ligands. As further described herein, in certain embodiments, 4-, 3-, and 2-hydroxyphenyl C-DIM analogs are used as scaffolds to explore the synthesis and eventual development of second-generation NR4A1 ligands that exhibit potent activity against cancer and other diseases, where NR4A1 is a potential therapeutic target, such as metabolic and neurological disorders.

[0021] Accordingly, in one embodiment, the ligand is a compound having the formula:

[0022] [ka]

[0023] or a salt thereof, During the ceremony, R1, R2, R1', and R2' are each independently selected from the group consisting of hydrogen, a linear alkyl group containing from 1 to about 10 carbon atoms, and a branched alkyl group containing from 1 to about 10 carbon atoms; R3, R4, R5, R6, R3', R4', R5', and R6' are each independently selected from the group consisting of hydrogen, halogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, an alkoxy group containing 1 to about 10 carbon atoms, and a nitro group; R7 is selected from the group consisting of hydrogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, a cycloalkyl group containing 1 to about 10 carbon atoms, and an aryl group; R8, R9, R 10 , R 11 , and R 12are independently selected from the group consisting of H, halogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, an alkoxy group containing 1 to about 10 carbon atoms, a haloalkyl group containing 1 to about 10 carbon atoms, a nitro group, a hydroxyl group, and a haloalkoxy group containing 1 to about 10 carbon atoms; R8, R9, R 10 , R 11 , and R 12 at least one of is OH, R 10 When is OH, R8, R9, R 10 , R 11 , and R 12 At least one of is not hydrogen.

[0024] As discussed further herein, the compounds of the present disclosure are NR4A1 ligands. In that regard, in some embodiments, antagonist ligands block the constitutive function of the receptor and its stimulatory ability to bind the cognate ligand to the NR4A1 protein and activate NR4A1-dependent genes.

[0025] Chemical moieties referred to as monovalent chemical moieties (e.g., alkyl, aryl, etc.) also encompass structurally permissible multivalent moieties as understood by those of skill in the art. For example, an "alkyl" moiety is commonly referred to as a monovalent group (e.g., CHCH-), but in appropriate circumstances, an "alkyl" moiety can also be referred to as a divalent group (e.g., -CHCH-, equivalent to an "alkylene" group). Similarly, in situations where a divalent moiety is required, those of skill in the art will understand that the term "aryl" refers to the corresponding divalent arylene group.

[0026] Terms used herein may be preceded or followed by a hyphen "-" or an equal sign "=" to indicate the bond order of the bond between the named substituents, where the term is the parent moiety, the hyphen indicates a single bond, and the equal sign indicates a double bond. In the absence of a hyphen or equal sign, a single bond is formed between the substituent and its parent moiety, and further, unless otherwise specified in the symbol, the substituents are intended to be read "left to right." For example, C1-C6 alkoxycarbonyloxy and -OC(O)C1-C6 alkyl indicate the same functionality, and similarly, arylalkyl and alkylaryl indicate the same functionality.

[0027] All atoms are understood to have the normal number of valencies for bond formation (e.g., 4 for carbon, 3 for N, 2 for O, 2, 4, or 6 for S, depending on the oxidation state of the atom). Sometimes a moiety can be defined as, for example, (A)aB, where a is 0 or 1. In such a case, when a is 0, the moiety is B, and when a is 1, the moiety is AB.

[0028] Where substituents can have different numbers of the same type of group or atom (e.g., an alkyl group can be C1, C2, C3, etc.), the number of repeating atoms or groups can be expressed by ranges (e.g., C1-C6 alkyl), including each and every number within any and all subranges. For example, C1-C3 alkyl means C1, C2, C3, C 1~2 , C 1~3 , and C 2~3 Contains alkyl.

[0029] The term "alkyl," as used herein, unless otherwise specified, means a straight or branched chain hydrocarbon containing 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. When an "alkyl" group is a linking group between two other moieties, it may also be straight or branched, and examples include, but are not limited to, -CH-, -CHCH-, -CHCHCHC(CH)-, and -CHCH(CHCH)CH-.

[0030] The term "cycloalkyl," as used herein, refers to a monocyclic or bicyclic cycloalkyl ring system. A monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, which may be saturated or unsaturated, but is not aromatic. In certain embodiments, a cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. A bicyclic cycloalkyl ring system is a bridged monocycle or a fused bicycle. A bridged monocycle includes a monocyclic cycloalkyl ring in which two non-adjacent carbon atoms of the monocycle are connected by an alkylene bridge between 1 to 3 additional carbon atoms (i.e., a bridging group of the type -(CH)-, where w is 1, 2, or 3).

[0031] "Alkoxy" refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0032] The term "aryl," as used herein, refers to phenyl (i.e., monocyclic aryl), or an aromatic bicycle containing only carbon atoms in a bicyclic ring system or aromatic bicyclic ring system containing at least one phenyl ring. The bicyclic aryl may be an azulenyl, naphthyl, or phenyl fused to a monocyclic cycloalkyl, monocyclic cycloalkenyl, or monocyclic heterocyclyl. The bicyclic aryl is bonded to the parent molecular moiety through any carbon atom contained within the phenyl portion of the bicyclic system or any carbon atom of the naphthyl or azulenyl ring. The monocyclic heterocyclyl portion of the fused monocyclic cycloalkyl or bicyclic aryl is optionally substituted with one or two oxo and / or oxo groups.

[0033] "Halogen" refers to a chloro, bromo, fluoro, or iodo atomic radical. The term "halogen" also contemplates "halo" or "halide."

[0034] The terms "haloalkyl," "haloalkenyl," and "haloalkoxy" refer to alkyl, alkenyl, or alkoxy groups, as the case may be, substituted with one or more halogen atoms.

[0035] The term "nitro" as used herein refers to the group --NO.sub.2.

[0036] The term "substituted," as used herein, means that a hydrogen radical of the specified moiety is replaced with a specified substituent group, provided that the substitution results in a stable or chemically feasible compound. The term "substitutable," when used in reference to a specified atom, means that attached to the atom is a hydrogen radical that can be replaced with a suitable substituent group.

[0037] 2-hydroxyligands In some embodiments, R is OH. In certain instances, such ligands are referred to herein as "2-hydroxy" and / or "2-OH" due to the substitution of the OH group on the central phenyl group. In certain such embodiments, R, R 10, R 11 , and R 12 are each H. In certain other embodiments, R, R 10 , R 11 , and R 12 is independently selected from the group consisting of halogen, CH, OCCl, CF, t-butyl, OCH, OH, CH, and CN. 10 is OCH3. In one embodiment, R 11 is selected from the group consisting of CH3, OCH3, and CF3. In one embodiment, R9 and R 11 is Br.

[0038] In one embodiment, the composition of the present disclosure has the following structure:

[0039] [ka]

[0040] and one of these salts.

[0041] 3-hydroxyligand In some embodiments, R is OH. In certain instances, such ligands are referred to herein as "3-hydroxy" and / or "3-OH" due to the substitution of the OH group on the central phenyl group. In certain such embodiments, R, R 10 , R 11 , and R 12 are each H. In certain other embodiments, R, R 10 , R 11 , and R 12 is independently selected from the group consisting of halogen, CH3, OCCl3, CF3, t-butyl, OCH3, OH, C6H5, and CN. In certain embodiments, R8 is halogen.

[0042] In one embodiment, the composition of the present disclosure has the following structure:

[0043] [ka]

[0044] and one of these salts.

[0045] 4-hydroxyligand In some embodiments, R 10 is OH. In certain instances, such ligands are referred to herein as "4-hydroxy" and / or "4-OH" due to the substitution of the OH group on the central phenyl group. In certain such embodiments, R, R, R 11 , and R 12 is independently selected from the group consisting of halogen, CH, OCCl, CF, t-butyl, OCH, OH, C6H5, and CN. In certain other embodiments, R is halogen and R 11 is selected from the group consisting of H, halogen, and OCH3.

[0046] In one embodiment, the composition of the present disclosure has the following structure:

[0047] [ka]

[0048] and one of these salts.

[0049] The C-DIM compounds of the present disclosure can be prepared by the condensation of substituted benzaldehyde with indole or substituted indole. The compounds can be synthesized by incubating two parts indole or substituted indole with one part benzaldehyde or substituted benzaldehyde in dilute acetic acid at 80-90°C for 24-48 hours. The solid is collected by filtration and crystallized from benzene or benzene / hexane to give C-DIM in 70-90% yield. The use of a single indole starting material results in a symmetrical product, while the use of two different indole starting materials results in an asymmetrical product.

[0050] The preparation and characterization of representative C-DIM compounds is described, for example, in US Pat. No. 7,232,843, the entire contents of which are incorporated herein by reference.

[0051] Pharmaceutical Composition In certain aspects, the present disclosure provides pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present disclosure, together with a pharmaceutically acceptable carrier and optionally other therapeutic and / or prophylactic ingredients.

[0052] The term "therapeutically effective amount," as used herein, refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, e.g., to ameliorate, alleviate, relieve, and / or delay one or more of its symptoms. With respect to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to affect a tumor so as to shrink and / or reduce the rate of tumor growth (inhibit tumor growth). In some embodiments, an effective amount is an amount sufficient to cause delay. In some embodiments, an effective amount is an amount sufficient to prevent onset and / or recurrence. An effective amount can be administered in one or more administrations.

[0053] "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical industry and are described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (Easton, Pa.: Mack Publishing Company, 1990). For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes, and even flavoring agents can be added to the pharmaceutical composition. For example, sodium benzoate, sortie acid, and p-hydroxybenzoic acid esters can be added as preservatives. In addition, antioxidants and suspending agents can be used.

[0054] Suitable excipients for non-flowing formulations are also known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients and salts can be found in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990).

[0055] Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffer substances, surfactants, etc., may be present in such vehicles. The biological buffer may be any solution that is pharmacologically acceptable and provides the formulation with a desired pH, i.e., a pH within the physiologically acceptable range. Examples of buffers include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered saline, etc.

[0056] Depending on the intended method of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions, etc., preferably in unit dosage forms suitable for single administration of precise dosage amounts. The compositions will contain an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier, and may, in addition, include other medicinal agents, adjuvants, diluents, buffers, etc.

[0057] The present disclosure includes pharmaceutical compositions comprising compounds of the present disclosure, including isomers, racemic or non-racemic mixtures of isomers, or pharmaceutically acceptable salts or solvates thereof, together with one or more pharmaceutically acceptable carriers, and optionally other therapeutic and / or prophylactic ingredients.

[0058] In general, the compounds of the present disclosure are administered in a therapeutically effective amount by any of the recognized administration methods. The range of suitable dosages depends on many factors, such as the severity of the disease being treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication of the subject being administered, and the preferences and experience of the physician involved. Those skilled in the art of treating such diseases can determine the therapeutically effective amount of the compounds of the present disclosure for a given disease without undue experimentation, relying on their own knowledge and the disclosures of this application.

[0059] Thus, the compounds of the present disclosure can be administered in pharmaceutical formulations, including those suitable for oral (including buccal and sublingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous, and intravenous) administration, or in a form suitable for administration by inhalation or insufflation. The preferred method of administration is intravenous or oral, using a convenient daily regimen that can be adjusted according to the level of discomfort.

[0060] For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc., an active compound described herein and optional adjuvants in an excipient, for example, water, saline, aqueous dextrose, glycerol, ethanol, or the like, thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of non-toxic auxiliary substances (wetting or emulsifying agents, pH buffering agents, and the like, e.g., sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like). Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences (supra).

[0061] In yet another embodiment, permeation enhancer excipients are used, including polymers such as polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin), polyanions (N-carboxymethylchitosan, polyacrylic acid), and thiolated polymers (carboxymethylcellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).

[0062] For oral administration, the compositions generally take the form of tablets, capsules, soft capsules, or may be aqueous or non-aqueous solutions, suspensions, or syrups. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use may contain one or more commonly used carriers (such as lactose and cornstarch). Lubricants such as magnesium stearate are also typically added. Typically, the compounds of the present disclosure are combined with a non-toxic, pharmaceutically acceptable inert carrier for oral use (such as lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc.). Furthermore, if desired or necessary, suitable binders, lubricants, disintegrants, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars (such as glucose or β-lactose), corn syrup, natural and synthetic gums (such as acacia, tragacanth, sodium alginate, etc.), carboxymethylcellulose, polyethylene glycol, waxes, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0063] For example, capsules can be prepared by conventional procedures, with a dosage unit of 100 mg of the compound of the present disclosure, 100 mg of cellulose, and 10 mg of magnesium stearate. Larger capsules can also be prepared by filling two standard hard gelatin capsules with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg of cellulose, and 10 mg of magnesium stearate. Alternatively, tablets can be prepared by conventional procedures, with a dosage unit of 100 mg of the compound of the present disclosure, 150 mg of lactose, 50 mg of cellulose, and 10 mg of magnesium stearate. Larger tablets can also be prepared by conventional procedures, with a dosage unit of 100 mg of the compound of the present disclosure, with other ingredients being 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 250 mg of microcrystalline cellulose, 10 mg of starch, and 100 mg of lactose. Suitable coatings can be applied to enhance palatability or delay absorption.

[0064] When a liquid suspension is used, the active agent can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water, etc.), as well as emulsifying and suspending agents. If desired, flavoring agents, coloring agents, and / or sweetening agents may also be added. Other optional ingredients for incorporation into oral formulations herein include, but are not limited to, preservatives, suspending agents, thickening agents, etc.

[0065] Parenteral preparations can be prepared in conventional forms, either as liquid solutions or suspensions, as solid forms that can be settled for solubilization or suspension in liquid prior to injection, or as emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents, and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Among acceptable vehicles and solvents, water, Ringer's solution, and isotonic saline solution can be used. In addition, sterile fixed oils, fatty esters, or polyols are conventionally used as solvents or suspending media. In addition, parenteral administration may require the use of a slow-release or sustained-release system to maintain a constant level of dosage.

[0066] Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and includes aqueous and non-aqueous sterile isotonic injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the intended recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Administration via certain parenteral routes may require the introduction of the formulations of the present disclosure into the patient's body through a needle or catheter propelled by a sterile syringe or some other mechanical device, such as a continuous infusion system. The formulations provided by the present disclosure can be administered using a syringe, infuser, pump, or any other device recognized in the art for parenteral administration.

[0067] Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents, and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Among acceptable vehicles and solvents, water, Ringer's solution, and isotonic saline solution can be used. In addition, sterile fixed oils, fatty esters, or polyols are conventionally used as solvents or suspending media. In addition, parenteral administration may require the use of a slow-release or sustained-release system to maintain a constant level of dosage.

[0068] Preparations according to the present disclosure for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and organic ester injectables such as ethyl oleate. Such dosage forms may also contain auxiliary substances such as preservatives, wetting agents, emulsifying agents, and dispersing agents. They can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition. They can also be prepared using sterile water or some other injectable sterile medium immediately before use.

[0069] The formulation can optionally contain an isotonicity agent.The formulation preferably contains an isotonicity agent, and glycerin is the most preferred isotonicity agent.The concentration of glycerin (if used) is within the range known in the art, for example, about 1 mg / mL to about 20 mg / mL.

[0070] The pH of the parenteral formulation can be controlled by a buffer such as phosphate, acetate, TRIS, or L-arginine. The concentration of the buffer is preferably sufficient to buffer the pH during storage so as to maintain the pH at the target pH ± 0.2 pH units. The preferred pH is about 7 to about 8 when measured at room temperature.

[0071] Other additives, such as pharmaceutically acceptable solubilizers such as Tween 20® (polyoxyethylene (20) sorbitan monolaurate), Tween 40® (polyoxyethylene (20) sorbitan monopalmitate), Tween 80® (polyoxyethylene (20) sorbitan monooleate), Pluronic F68® (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol), may optionally be added to the formulation and may be useful when the formulation comes into contact with plastic materials. In addition, parenteral formulations may contain various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0072] Sterile injectable solutions are prepared by incorporating one or more of the compounds of the present disclosure in the required amount in a suitable solvent, optionally with various other ingredients as described above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients described above. For sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of the active ingredient and any additional desired ingredients from the previously described sterile filtered solution. Thus, for example, a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of the active ingredient in 10% by volume of propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

[0073] Alternatively, the pharmaceutical composition of the present disclosure can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0074] The pharmaceutical compositions of the present disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques known in the art of pharmaceutical formulation and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, propellants such as fluorocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents.

[0075] Preferred formulations for topical drug delivery are ointments and creams. Ointments are semisolid preparations, typically based on petrolatum or other petroleum derivatives. Creams containing selected active agents are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil, as is known in the art. Cream bases are water-washable and contain an oily phase, an emulsifier, and an aqueous phase. The oily phase, sometimes referred to as the "internal" phase, is generally composed of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol, while the aqueous phase usually, although not necessarily, exceeds the oily phase in volume and generally contains a moisturizer. Emulsifiers in cream formulations are generally nonionic, anionic, cationic, or amphoteric surfactants. The particular ointment or cream base used will provide optimal drug delivery, as will be appreciated by those skilled in the art. As with other carriers or vehicles, ointment bases should be inert, stable, nonirritating, and nonsensitizing.

[0076] Formulations for oral administration include tablets, lozenges, gels, and the like. Alternatively, oral administration can be achieved using transmucosal delivery systems known to those skilled in the art. The compounds of the present disclosure can also be delivered through the skin or mucosal tissue using conventional transdermal drug delivery systems, i.e., transdermal "patches," in which the drug is typically contained in a laminated structure that functions as a drug delivery device affixed to the body surface. In such structures, the drug composition is typically contained in a layer or "reservoir" underlying an upper backing layer. The laminated device can contain a single reservoir or can contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylene, polysiloxane, polyisobutylene, polyacrylate, polyurethane, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive can be present as separate, distinct layers, with the adhesive underlying the reservoir, which can be either a polymeric matrix or a liquid or gel reservoir as described above, or can take another form. The backing layer in these laminates serves as the upper surface of the device, functions as the primary structural element of the laminate structure, and provides considerable flexibility to the device. The material chosen for the backing layer should be substantially impermeable to the active agent and any other materials present.

[0077] The compounds of the present disclosure can be formulated for aerosol administration, particularly to the respiratory tract (including intranasal administration). The compounds generally have a small particle size, for example, about 5 microns or less. Such a particle size can be obtained by means known in the art, for example, by micronization. The active ingredient is placed in a pressurized pack with a suitable propellant, such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol conveniently also contains a surfactant, such as lecithin. The dosage can be controlled by a metered valve. Alternatively, the active ingredient can be supplied in dry powder form, for example, a powder mix of the compound in a suitable powder base, such as lactose, starch, starch derivatives, such as hydroxypropylmethylcellulose, and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition can be presented in unit dose form, for example, in capsules or (e.g., gelatin) cartridges or blister packs from which the powder can be administered by an inhaler.

[0078] A pharmaceutically or therapeutically effective amount of the composition is delivered to the subject. The exact effective amount will vary from subject to subject and will depend on the species, age, size, and health of the subject, the nature and severity of the condition being treated, the advice of the treating physician, and the treatment or combination of treatments selected for administration. Therefore, the effective amount for a given situation can be determined by routine experimentation. For purposes of this disclosure, a therapeutic amount generally ranges from about 0.01 mg / kg (body weight) to about 250 mg / kg, more preferably from about 0.1 mg / kg to about 10 mg / kg, in at least one dose. For larger mammals, an indicated daily dosage may range from about 1 mg to 300 mg, more preferably from about 10 mg to 200 mg, in one or more doses per day. A subject may be administered as high a dose as necessary to reduce and / or alleviate the signs, symptoms, or pathogenesis of the disorder in question, or to effect any other desired alteration in a biological system. If desired, formulations can be prepared with enteric coatings suitable for sustained- or controlled-release administration of the active ingredient.

[0079] The pharmaceutical product is preferably in unit dosage form. In such dosage form, the preparation is subdivided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form may be a packaged preparation, a package containing discrete amounts of the preparation (such as packeted tablets, capsules, and powders in vials or ampoules). The unit dosage form may also be a capsule, tablet, cachet, or lozenge itself, or any of these in an appropriate packaged form.

[0080] Methods for modulating NR4A1 activity In another aspect, the disclosure provides a method of modulating NR4A1 activity in a cell, the method comprising administering to the cell a compound or pharmaceutical composition described herein.

[0081] In some embodiments, modulating NR4A1 activity involves binding of a compound described elsewhere herein to the NR4A1 protein. In some embodiments, the compound has antagonist activity, i.e., the compound reduces or eliminates the effect of stimulating the cognate function of the receptor (e.g., an antagonist ligand). In some embodiments, an antagonist ligand blocks the receptor's constitutive function and its stimulatory power, the ability of the cognate ligand to bind to the NR4A1 protein, and the ability to activate NR4A1-dependent genes. In some embodiments, the NR4A1 ligand may be a tissue-, response-, or gene-specific agonist.

[0082] The term "antagonist" refers to a compound that can bind to the NR4A1 receptor and reduce or inhibit molecular and cellular activity. An antagonist can be a ligand that directly binds to the receptor. Alternatively, an antagonist can indirectly bind to the receptor, for example, (a) by forming a complex with another molecule or protein that directly binds to the receptor, or (b) otherwise cause the modification of another compound so that the other compound directly binds to the NR4A1 receptor.

[0083] The term "agonist" refers to a compound that can bind to the NR4A1 receptor to produce or increase molecular and cellular activity. An agonist may be a ligand that binds directly to the receptor. Alternatively, an agonist may bind indirectly to the receptor, for example, (a) by forming a complex with another molecule or protein that binds directly to the receptor, or (b) otherwise cause the modification of another compound such that the other compound binds directly to the NR4A1 receptor.

[0084] The term "activation" and its variants refer to a measurable increase in molecular and cellular activity.

[0085] In one embodiment, the cell is a cancer cell.

[0086] In one embodiment, the cell is contacted with the compound or pharmaceutical composition in vitro. In one embodiment, the cell is contacted with the compound or pharmaceutical composition in vivo by administering an effective amount of the compound or pharmaceutical composition to a subject.

[0087] In one embodiment, modulation of NR4A1 activity induces downregulation of a protein selected from the group consisting of β1-integrin, TXNDC5, survivin, EFGR, PAX3-FOX01A, and combinations thereof. In one embodiment, modulation of NR4A1 activity induces upregulation of a protein selected from the group consisting of SERPINB5, GADD45α, and combinations thereof.

[0088] In one embodiment, the compound has the formula:

[0089] [ka]

[0090] or a salt thereof, During the ceremony, R1, R2, R1', and R2' are each independently selected from the group consisting of hydrogen, a linear alkyl group containing from 1 to about 10 carbon atoms, and a branched alkyl group containing from 1 to about 10 carbon atoms; R3, R4, R5, R6, R3', R4', R5', and R6' are each independently selected from the group consisting of hydrogen, halogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, an alkoxy group containing 1 to about 10 carbon atoms, and a nitro group; R7 is selected from the group consisting of hydrogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, a cycloalkyl group containing 1 to about 10 carbon atoms, and an aryl group; R8, R9, R 10 , R 11 , and R 12are independently selected from the group consisting of H, halogen, a linear alkyl group containing 1 to about 10 carbon atoms, a branched alkyl group containing 1 to about 10 carbon atoms, an alkoxy group containing 1 to about 10 carbon atoms, a haloalkyl group containing 1 to about 10 carbon atoms, a nitro group, a hydroxyl group, and a haloalkoxy group containing 1 to about 10 carbon atoms; R8, R9, R 10 , R 11 , and R 12 at least one of is OH, R 10 When is OH, R8, R9, R 11 , and R 12 At least one of is not hydrogen.

[0091] 4-hydroxyligand In one embodiment, R 10 is OH. In certain such embodiments, R, R, R 11 , and R 12 is independently selected from the group consisting of halogen, CH, OCCl, CF, t-butyl, OCH, OH, C6H5, and CN. In certain other embodiments, R is halogen and R 11 is selected from the group consisting of H, halogen, and OCH3.

[0092] In one embodiment, the composition of the present disclosure has the following structure:

[0093] [ka]

[0094] and one of these salts.

[0095] 2-hydroxyligands In one embodiment, R9 is OH. In certain such embodiments, R8, R 10 , R 11 , and R 12 are each H. In certain other embodiments, R, R10 , R 11 , and R 12 is independently selected from the group consisting of halogen, CH3, OCCl3, CF3, t-butyl, OCH3, OH, C6H5, and CN. In certain embodiments, R8 is halogen.

[0096] In one embodiment, the composition of the present disclosure has the following structure:

[0097] [ka]

[0098] and one of these salts.

[0099] 3-hydroxyligand In one embodiment, R8 is OH. In certain such embodiments, R9, R 10 , R 11 , and R 12 are each H. In certain other embodiments, R, R 10 , R 11 , and R 12 is independently selected from the group consisting of halogen, CH, OCCl, CF, t-butyl, OCH, OH, CH, and CN. 10 is OCH3. In one embodiment, R 11 is selected from the group consisting of CH3, OCH3, and CF3. In one embodiment, R9 and R 11 is Br.

[0100] In one embodiment, the composition of the present disclosure has the following structure:

[0101] [ka]

[0102] and one of these salts.

[0103] Methods of Treating a Disease or Condition In another aspect, the disclosure provides a method for treating a disease or condition in an individual treatable by modulation of NR4A1 activity, comprising administering to the individual a therapeutically effective amount of a compound or pharmaceutical composition described herein.

[0104] Those skilled in the art can determine the most effective dose and time for administering the composition, taking into account the delivery route, compound metabolism, and other pharmacokinetic parameters (volume of distribution, clearance, age of the subject, etc.). For example, the NR4A1 antagonist can be administered by any known method, such as topical administration, oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, intranasal administration, transdermal administration, rectal administration, or by any means that delivers an effective amount of the active agent to the tissue or site to be treated. A suitable dosage is one that achieves the desired endpoint. It should be understood that different dosages may be required for the treatment of different disorders. An effective amount of a drug is, for example, an amount that causes a cessation or significant reduction in neoplastic cell count, proliferation, size, cell migration, or cell invasion.

[0105] The composition can be administered with a pharmaceutical carrier and / or diluent. The agent may also be administered in combination with other agents, such as other chemotherapeutic or immunostimulant agents, or other agents associated with therapeutic agents, such as in the treatment of cancer. Examples of pharmaceutical carriers or diluents useful in the present invention include any physiological buffer medium (i.e., pH of about 7.0 to 7.4) containing a suitable water-soluble organic carrier. Suitable water-soluble organic carriers include, but are not limited to, corn oil, dimethyl sulfoxide, gelatin capsules, and the like.

[0106] The individual may be any animal, such as a mammal, bird, reptile, or fish. Exemplary mammal categories include rodents, primates, canines, felines, ungulates, lagomorphs, etc. For example, the individual may be a human, monkey, ape, or other primate, mouse, rat, or other rodent, dog, cat, pig, horse, cow, or rabbit, etc.

[0107] As used herein, the term "treatment" means providing an ameliorative, curative, or prophylactic effect to a disorder or condition. In some embodiments, treatment includes preventing the progression or progression of a condition, or slowing the rate of progression or progression (compared to no treatment or other treatment). In the context of cancer (described further below), treatment includes slowing or preventing the rate of cell proliferation or division, slowing or preventing cell migration, and / or slowing or preventing cell invasion.

[0108] Exemplary conditions include cancer, diabetes, thrombosis, colitis, Crohn's disease, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, immunosuppressive disorders, arthritis, asthma, stroke, restenosis, rhinitis, and osteoporosis. Exemplary cancers include pancreatic ring, kidney cancer, colon cancer, rhabdomyosarcoma, lung cancer, and breast cancer.

[0109] In one embodiment, the disease is cancer, hi one embodiment, the cancer is selected from the group consisting of pancreatic cancer, breast cancer, colon cancer, rhabdomyosarcoma, and lung cancer.

[0110] In one embodiment, modulation of NR4A1 activity induces downregulation of a protein selected from the group consisting of β1-integrin, TXNDC5, survivin, EFGR, PAX3-FOX01A, and combinations thereof. In one embodiment, modulation of NR4A1 activity induces upregulation of a protein selected from the group consisting of SERPINB5, GADD45α, and combinations thereof.

[0111] In one embodiment, the disease is diabetes and modulation of NR4A1 activity induces glucose uptake in the individual, hi one embodiment, modulation of NR4A1 activity induces upregulation of GLUT-4 and Rab4 and phosphorylation of AMPK. [Example]

[0112] NR4A1 binding to 4-hydroxy-substituted NR4A1 ligands Below is a description of compounds according to embodiments of the present disclosure that bind to NR4A1.

[0113] The 4-hydroxy compound (C-DIM8) was a potent NR4A1 antagonist that bound to NR4A1 with high affinity and inhibited the expression of several tumor-promoting NR4A1-regulated genes and pathways. The 4-hydroxy analog was a relatively potent tumor growth inhibitor in mouse xenograft models but had a relatively short serum half-life. Therefore, nine substituted 4-hydroxy analogs were synthesized, some of which reinforced the hydroxy group, to examine their relative potencies as NR4A1 ligands and their half-lives. Their effects on GAL4-NR4A1 and GAL4-NR4A2 activation / inactivation in Panc1 cells were determined (see Figures 9 and 13).

[0114] Significant inhibition of GAL4-NR4A1 transactivation by the 4-hydroxy reference compound was observed only at 22.5 μM but not at 15 μM (steep dose-response curve). The substituted 4-hydroxy analogs were tested at concentrations of 7.5 μM and 15 μM, and with the exception of the 3-fluoro-4-hydroxy analog, the remaining eight 4-hydroxy-substituted compounds were more potent than the 4-hydroxy reference compound as inhibitors of NR4A1-dependent transactivation. See Figure 9.

[0115] The effects of the 4-hydroxy reference compound and nine analogs were compared for downregulation of two NR4A1-regulated gene products, TXNDC5 and PAX3-FOX01A, in Rh30 rhabdomyosarcoma cells (see Figure 10B). At a high dose of 20 μM, the 4-hydroxy reference compound reduced expression of both gene products by 50–60%, while all nine analogs reduced expression of both gene products by 80–95% at a concentration of 5 μM. Thus, all analogs were more than four-fold more potent than the 4-hydroxy reference compound and therefore represent promising novel, second-generation NR4A1 ligands. The results also indicate that the NR4A1-dependent transactivation assay appears to be less predictive than NR4A1-regulated gene products for determining differences in NR4A1 antagonist potency.

[0116] Additionally, we examined C-DIM-mediated inhibition of NR4A1-regulated gene products in Panc1 and SKBR3 cells by the same compounds (see Figures 10A, 14, and 19). These results are similar to those observed in Rh30 cells.

[0117] Because ligands for nuclear receptors can both activate and repress gene expression and NR4A1 is pro-tumorigenic, we focused on NR4A1 ligands as antagonists or inhibitors of NR4A1-regulated genes.

[0118] Compared to the 4-bromo reference compound [i.e., 1,1-bis(3'-indolyl)-1-(p-bromophenyl)methane], the 4-hydroxy analogs showed minimal GAL4-NR4A2 activation, demonstrating that these compounds appear to be NR4A1 specific. See, e.g., Figure 13.

[0119] Structure-activity relationships of 4-hydroxyligands Below is a description of the structure-activity relationships of compounds according to embodiments of the present disclosure.

[0120] NR4A1 is thought to be a key player in metabolic diseases, and derivatives of DIM-C-pPhOH (C-DIM8; NR4A1 standard) have been identified that represent second-generation NR4A1 ligands that are potent in both in vivo and in vitro assays (described further herein).

[0121] Figures 11A-11C summarize the completed structure-activity relationship studies for the induction of interleukin-24 (IL-24), guanine deaminase (GDA), and doublecortin domain-containing 2 (DCDC2) mRNA by DIM-C-pPhOH and six substituted analogs in Rh30 cells. EC of induction 50 The values ​​clearly show a structure-dependent potency, which also reflects their efficacy (ie, maximal induction potential).

[0122] NR4A1 and NR4A2 binding to 4-hydroxyligands measured by surface plasmon resonance Below is a description of the binding of compounds according to embodiments of the present disclosure to NR4A1 using surface plasmon resonance (SPR).

[0123] Binding experiments were performed using SPR at 25°C on a Biacore 3000 system (GE Healthcare). Purified NR4A1 and NR4A2 LBD proteins were covalently immobilized onto a CM5 sensor chip (GE) using the amine coupling method. Alternatively, a more expensive His-tag-conjugated NTA sensor chip (GE) can be used to ensure accurate protein surface orientation. SPR response curves (sensorgrams) were generated after ligand injection using 5 or 10 μM DIM-C-pPhX analogs (where X = Br (#2), X = OH (#8), X = CN (#10), and X = CO2Me (#14)) (see Figure 22). While both proteins were successfully immobilized on the chip, NR4A2 appeared to aggregate / oligomerize in the test buffer conditions, and therefore, the buffer ionic strength required further optimization. DIM-C-pPhOH (X-DIM8) binds NR4A1 with high affinity, and DIM-C-pPhBr (C-DIM2) shows adhesive interactions with both proteins, which is consistent with K d This is problematic for decisions.

[0124] NR4A2-dependent osteopontin gene expression in cells treated with 4-hydroxyligand 1 shows the induction of osteopontin (OPN) expression by compounds according to embodiments of the present disclosure.

[0125] Panc1 cells were treated with DIM-C-pPhX (halogen-substituted) analogs containing 4-F, 4-Cl, and 4-I substituents at concentrations ranging from 2.5 to 20 μM (in increments of 2.5 μM), and NR4A2-dependent OPN gene expression was determined using qPCR analysis. Compound treatment affected the expression of GAPDH, which was originally intended to serve as an internal control. However, mRNA levels were determined, and equal amounts of mRNA were used for each qPCR reaction. The fold induction of OPN was used to generate dose-response curves and EC 50 Values ​​can be determined (Figure 21). In certain embodiments, other housekeeping genes such as β-actin and 18S ribosomal RNA are currently used for internal controls.

[0126] NR4A1 binds to 3-hydroxy and 2-hydroxy ligands Below is a description of NR4A1 binding by 3-hydroxy and 2-hydroxy compounds according to embodiments of the present disclosure.

[0127] The 4-hydroxy compound (C-DIM8) was a potent NR4A1 antagonist that bound to NR4A1 with high affinity and inhibited the expression of several tumor-promoting NR4A1-regulated genes / pathways. Above, we show that analogs of 4-hydroxy compounds represent a new generation of more potent NR4A1 ligands.

[0128] The results described herein demonstrate that, in certain embodiments, 3-hydroxy (3-OH) and 2-hydroxy (2-OH) compounds are more potent as NR4A1 antagonists / agonists than the 4-OH standard.

[0129] The transactivation results demonstrate that the 4-, 3-, and 2-hydroxy compounds all reduced transactivation in Panc1 cells transfected with GAL4-NR4A1, with a steep dose-response curve between 15 and 22.5 μM (see Figures 8, 7, and 6). Compared to the 4-bromo standard NR4A2 ligand, the 3- and 2-hydroxy isomers had minimal NR4A2 activity (see Figures 13 and 15).

[0130] Figure 3 shows that both the 2- and 3-hydroxy compounds were more potent than the 4-hydroxy compound in downregulating β1-integrin and TXNDC5 in Panc1 and SKBR3 cells. Note that β1-actin loading control was also reduced at high concentrations of the 3-hydroxy (SKBR cells) and 2-hydroxy (SKBR3 and Panc1 cells) compounds. Results correlate with the transactivation data.

[0131] Western blot analysis shows that the potency of these 3- and 2-hydroxy CDIM compounds to induce SERPINB5 (i.e., 2-OH / 3-OH > 4-OH) was similar to that observed in downregulating β1-integrin and TXNDC5 (see, e.g., Figures 3 and 5).

[0132] The efficacy of 2-, 3-, and 4-hydroxy compounds in downregulating TXNDC5 and β1-integrin in Rh30 rhabdomyosarcoma cells was similar to that observed in Panc1 and SKBR3 cells. See Figure 4.

[0133] NR4A1 binding and transactivation of substituted 2-hydroxy and 3-hydroxy ligands Results obtained with 2- and 3-hydroxy C-DIM analogs demonstrated that, in certain embodiments, they were more potent NR4A1 antagonists than the parent 4-hydroxy reference standard. Therefore, several 2- and 3-hydroxy DIM substitution analogs were synthesized and their activity in transactivation assays was examined. Furthermore, for the 2-hydroxy analogs, we also examined their functional effects on gene induction (SERPINB5 and GADD45α) (see Figure 17) and repression (β1-integrin and TXNDC5) (see Figure 16).

[0134] Transactivation assays of 10 2-hydroxy analogs and their effects on NR4A1 showed that the 4-methoxy, 5-methyl, and 5-methoxy derivatives reduced transactivation at concentrations similar to those observed with the parent 2-hydroxy compound (see Figure 6).

[0135] Within this same series of substituted 2-hydroxy analog compounds, certain compounds (e.g., 2-bromo, 5-trifluoromethyl, and 3,5-dibromo) activated NR4A2 with a maximum induction response greater than 33% of that observed with the 4-bromoDIM reference standard. See Figure 15.

[0136] The effects of 2-hydroxyDIM analogs on NR4A1-dependent reduction (β1-integrin and TXNDC5 (see Figure 16)) and NR4A1-dependent increase (SERPINB5 and GADD45α (see Figure 17)) gene product expression in Panc1 and SKBR3 cells were examined, and the results were compared to those observed with unsubstituted 2-hydroxyDIM compounds. Results suggested that the substituted 2-hydroxyDIM compounds analyzed were significantly less active than the parent compound, which contrasts with previous studies with 4-hydroxyDIM and substituted analogs.

[0137] The effects of five substituted 3-hydroxy DIM analog compounds on NR4A1-dependent transactivation were also examined in Panc1 cells. Both the 2-chloro and 2-bromo analogs were more potent than the unsubstituted 3- and 4-hydroxy DIM standards (Note: the 5-hydroxy analog is equivalent to the 3-hydroxy substitution). See Figure 7.

[0138] 4-Hydroxyligands Activate the Tumor-Suppressor SERPINB5 Ligands for nuclear receptors both activate and repress gene expression, and these effects are specific to the cell context. Although our initial studies focused on the NR4A1 antagonist C-DIM, which downregulates tumor-promoting genes such as β1-integrin and TXNDC5, C-DIM also induces tumor-suppressor gene expression in cancer cells.

[0139] In Panc1 cells, 20 μM of the 4-hydroxy positive control compound (C-DIM8) and 4 μM of the substituted analogs induced SERPINB5 (mapsin), a tumor suppressor gene that inhibits cell invasion and metastasis (see Figure 10A). However, only the disubstituted 3,5-Br2 and 3-Cl-5-methoxy and 3-Cl analogs were more than fourfold more potent than the 4-hydroxyC-DIM positive control in this response in Panc1 cells.

[0140] In SKBR3 breast cancer cells, 20 μM of the 4-hydroxy positive control compound minimally induced SERPINB5, whereas significant induction of SERPINB5 was observed in cells treated with 5 μM of the substituted 4-hydroxy analogs (see Figure 14). In this study, DMSO (control) values ​​were relatively high, resulting in low induction by the 4-hydroxy compounds, whereas induction was observed in other experiments due to low basal SERPINB5 levels. Results in SKBR3 and Panc1 cells demonstrated that the C-DIM analogs induced SERPINB5, albeit with some differences in potency, and these results support the idea that the 4-hydroxy-substituted C-DIM analogs represent a second generation of potent NR4A1 ligands.

[0141] In vitro and in vivo assays of 4-OH ligands Below is a description of three of the more active DIM-4-OH analogs according to embodiments of the present disclosure in both in vivo and in vitro assays.

[0142] Figures 12A-12C summarize the in vivo tumor growth inhibition by three analogs of DIMC-pPh-OH in athymic nude mice bearing triple-negative MDA-MD-231 cells in an orthotopic model (black lines) versus a control (gray lines). The 3-chloro, 3,5-dibromo-, and 3-chloro-5-methoxy analogs of DIM-C-pPh-OH inhibited tumor growth at a dose of 10 mg / kg / day, with EDTA of 10 mg / kg / day. 50 It was clear that the values ​​were in the low mg / kg / day or high mg / kg / day range (see Figures 12A-12C). It is clear that the new substituted analogs represent a second generation of NR4A1 ligands that are significantly more potent than DIM-C-pPh-OH, which only partially inhibited tumor growth at doses of 40-50 mg / kg / day.

[0143] Tumor lysates from control and treated mice were analyzed for their effects on NR4A1-dependent responses previously characterized in vitro. Lysates from individual tumors were analyzed by Western blot, and the three analogs (a) reduced the mTOP pathway, including phosphorylated mTOR, p70S6K, pS6RP, and p-EBP1 (see Figure 18), (b) reduced NR4A1 / Sp regulators, including survivin, EGFR, TXNDC5, and β1-integrin (see Figure 19), and (c) induced expression of the NR4A1-regulated GAD045a and SERPINB5 gene products, as well as PARP cleavage, a marker of apoptosis (see Figure 19).

[0144] The structure-dependent induction of three NR4A1-responsive genes by DIM-C-pPhOH (DIM-4-OH) used in the in vivo studies and two more potent DIM-C-pPhOH analogs (3-chloro- and 3,5-bibromo-) was examined in Rh30 cells. The three NR4A1-induced genes, IL-24, GDA, and DCDC2, were identified by RNA sequencing. Preliminary studies showed maximal mRNA induction by the C-DIM NR4A1 ligand after 12 hours. The results (Figures 11A-C) demonstrate differences in the structure-dependent potency of the three NR4A1 ligands as inducers of gene expression. EC 50Based on the values, the 3-chloro- or 3,5-dibromo analogs were up to 10-fold more potent than DIM-C-pPhOH, but these differences in potency were complex and gene-dependent.

[0145] As confirmation of the in vitro SAR, Figure 23 shows that three second-generation C-DIM / NR4A1 ligands completely inhibit mammalian tumor growth at a dose of 5 mg / kg / day, significantly lower than previous studies in which DIM-C-pPhOH (C-DIM8) at 30-40 mg / kg / day only partially (40-50%) inhibited tumor growth.

[0146] Figure 24 summarizes the results of mouse xenograft studies using the 3,5-dibromo second-generation C-DIM / NR4A1 ligand, which completely inhibited RMS tumor growth at doses of 10.0, 7.5, 5.0, and 2.5 mg / kg / day. No toxicity was observed in any in vivo studies, and in vivo studies showed inhibition of RMS tumor growth at a dose of 0.25 mg / kg / day. Thus, the in vitro GAL4-NR4A1 screening assay highly predicts NR4A1-dependent anticancer activity both in vitro and in vivo, and preliminary results in mouse muscle cells (C2C12) indicate that the SAR for cancer is comparable to that for modulation of NR4A1-mediated metabolic activity.

[0147] Glucose uptake in C2C12 cells treated with displacing NR4A1 ligands Below is a description of glucose uptake by cells treated with compounds according to embodiments of the present disclosure.

[0148] Previous studies have shown that NR4A1 regulates genes related to glucose metabolism and enhances GLUT-4 expression in C2C12 muscle cells. Furthermore, mice with GLUT-4 knockdown in muscle cells are insulin resistant, suggesting that drugs that increase GLUT-4 expression in muscle are potential antidiabetic drugs. NR4A1 also plays a role in enhancing glucose metabolism in muscle, consistent with the potential antidiabetic activity of NR4A1 ligands previously reported for cytosporone-derived NR4A1 ligands in mouse models. Initial studies using DIM-C-pPhOH and second-generation substituted analogs used C2C12 muscle cells as a model to examine the antidiabetic activity of these compounds. Figures 25A-25D illustrate that NR4A1 was expressed in C2C12 cells, and treatment with C-DIM8 (DIM-C-pPhOH) or the second-generation substituted C-DIM8 analogs 3,5-bromo- (C-DIM8-3,5-Br2), 3-chloro- (C-DIM8-3-Cl), and 3-chloro-5-methoxy- (C-DIM8-3-Cl-5-OCH3) increased NR4A1 expression in C2C12 cells. In addition, we observed enhanced expression of Rab4 and activation (phosphorylation) of AMPK, and similar results were observed with the antidiabetic drug metformin in C2C12 cells. The results illustrated in Figures 26A-26D show that both DIM-C-pPhOH (C-DIM8) and metformin induced NR4A1 and GLUT-4 gene expression in C2C12 cells, with maximal induction observed after treatment with 20 μM DIM-C-pPhOH. Second-generation replacement compounds induced similar responses at concentrations between 2.5 and 5.0 μM.

[0149] The effects of DIM-C-pPhOH and three substituted analogs on glucose uptake in C2C12 cells were examined (Figure 27). DIM-C-pPhOH (15 μM and 20 μM) and the substituted analogs (2.5–5.0 μM) significantly induced glucose uptake. The responses observed with C-DIM8-3,5-Br2 and C-DIM8-3-Cl-5-OCH3 were similar to those observed with 500 μM metformin.

[0150] Overexpression of NR4A1 in C2C12 cells induces several genes involved in glycolysis, including phosphofructokinase (PFKM), phosphoglycerate mutase 2 (PGAM2), bisphosphoglycerate mutase (BPGM), and glycogen phosphorylase M (PYGM). Treatment of C2C12 cells with NR4A1 ligands and metformin significantly induced the expression of all genes, and a similar induction response was observed after overexpression of NR4A1 in C2C12 cells (Figure 28E). These results (Figures 25-28) demonstrate that DIM-C-pPhOH and substituted analogs induce glucose uptake and glycolysis in C2C12 myocytes and represent a novel class of antidiabetic drugs acting through NR4A1.

[0151] Figure 20 summarizes the results in glucose-deprived HepG2 cells showing enhanced gluconeogenesis. DIM-C-pPhOH (C-DIM8) and the 3,5-dibromo analog inhibited G6Pase and PEPCK mRNA levels, and the results suggest that the mechanism involves NR4A1 / AMPK-dependent inhibition of mTOR, but the role of LKB / NR4A1 interaction may differ from that previously published.

[0152] These data demonstrate that C-DIM analogs are selective receptor modulators of NR4A1, acting in an agonistic manner in diabetic models, while acting in an antagonistic manner in tumor models.

[0153] Treatment of mice maintained on a high-fat diet with a substituted NR4A1 ligand Below is a description of mice maintained on a high-fat diet treated with compounds according to embodiments of the present disclosure.

[0154] C57BL / 6 mice were maintained on a high-fat diet for several weeks and then treated every other day with C-DIM8-3-Cl-5-OCH3 (10 mg / kg / d in corn oil) by oral gavage. Blood glucose was tested at several intervals throughout the treatment period, and a significant decrease in blood glucose levels was observed (Figures 29A and 29B). In addition, mice treated with C-DIM8-3-Cl-5-OCH3 (10 mg / kg / d) also exhibited reduced blood glucose levels compared with control animals in a glucose tolerance test; in the present study (25 mg / kg / d), we observed reduced blood glucose levels and increased blood insulin levels. These results are also typically observed with antidiabetic drugs, confirming that C-DIM8 analogs exhibit antidiabetic activity, and this in vivo data complements the results of in vitro studies, establishing that C-DIM8 and related substituted analogs represent a novel class of NR4A1-dependent antidiabetic drugs.

[0155] Any embodiment, feature, element, definition, or summary provided in any aspect of this disclosure can be applied to any other aspect of this disclosure without limitation, unless expressly stated otherwise. Thus, any embodiment described herein can be implemented with reference to any method, agent, or composition of the invention, and vice versa. Furthermore, the agents and compositions of the invention can be used to achieve the methods of the invention.

[0156] The use of the words "a" or "an," when used in conjunction with the term "comprising" herein, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0157] Although use of the term "or" is used to mean "and / or" unless otherwise clearly indicated to refer to alternatives only or that the alternatives are mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0158] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of the device and the method used to determine the value or variation present in the study subject.

[0159] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs is open-ended, such as "comprises," "comprising," "has," "having," "includes," and "including." For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only that one or more steps, but extends to other unlisted steps. As an alternative to or in addition to "comprising," any embodiment herein can include "consisting of." The transitional phrase "consisting of" excludes all elements, steps, or ingredients not specified in the claim. Words using the singular or plural also include the plural and singular, respectively. Additionally, the words "herein," "above," and "below," and words of similar meaning, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0160] Publications cited herein and the subject matter for which they are cited are specifically incorporated herein by reference in their entirety.

[0161] While the preferred embodiment of the invention has been illustrated and described, it will be understood that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

1. The following formula: 【Chemistry 1】 or a salt thereof (In the formula, R 1 , R 2 , R 1 ', R 2 ', R 3 , R 4 , R 5 , R 6 , R 3 ', R 4 ', R 5 ', R 6 ' and R 7 are hydrogen, R 8 is a halogen, R 9 is OH, R 10 , R 11 , and R 12 are independently H, halogen, CH 3 , O.C.Cl 3 , C.F. 3 , t-butyl, and OCH 3 is selected from the group consisting of R 8 , R 10 , R 11 , and R 12 At least one of these is not H) However, the following compounds 【Chemistry 2】 Not a compound. 【Request Item 2】 【Chemistry 3】 and salts thereof.

3. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 or 2, or a salt thereof, and a pharmaceutically acceptable carrier.

4. 4. The pharmaceutical composition of claim 3, for administration to an individual in a therapeutically effective amount to treat a disease or condition in the individual that can be treated by modulating Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1) activity.

5. 5. The pharmaceutical composition of claim 4, wherein the disease is selected from the group consisting of cancer, thrombosis, colitis, Crohn's disease, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis immunosuppressive disorder, arthritis, asthma, stroke, restenosis, rhinitis, diabetes, and osteoporosis.

6. 6. The pharmaceutical composition of claim 5, wherein the cancer is selected from the group consisting of pancreatic cancer, breast cancer, colon cancer, rhabdomyosarcoma, and lung cancer.

7. 5. The pharmaceutical composition of claim 4, wherein modulation of NR4A1 activity induces downregulation of a protein selected from the group consisting of β1-integrin, TXNDC5, survivin, EFGR, PAX3-FOX01A, and combinations thereof.

8. The pharmaceutical composition of claim 4, wherein modulation of NR4A1 activity induces upregulation of a protein selected from the group consisting of SERPINB5, GADD45α, and combinations thereof.

9. 6. The pharmaceutical composition of claim 5, wherein the disease is diabetes and modulation of NR4A1 activity induces glucose uptake in the individual.

10. The pharmaceutical composition of claim 9, wherein modulation of NR4A1 activity induces upregulation of GLUT-4 and Rab4 and phosphorylation of AMPK.

11. 5. The pharmaceutical composition of claim 4, wherein administration comprises topical administration, oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, intranasal administration, transdermal administration, rectal administration, or a combination thereof.

12. The pharmaceutical composition of claim 3 for administration to a cell to modulate NR4A1 activity in the cell.

13. The pharmaceutical composition of claim 12, wherein modulating NR4A1 activity in a cell comprises reducing the level of functional NR4A1 in the cell.

14. The pharmaceutical composition of claim 12, wherein the cell is a cancer cell.

15. 13. The pharmaceutical composition of claim 12, wherein the cell is contacted with the pharmaceutical composition in vitro.

16. 13. The pharmaceutical composition of claim 12, wherein the cells are contacted with the pharmaceutical composition in vivo by administering an effective amount of the pharmaceutical composition to a subject.

17. 13. The pharmaceutical composition of claim 12, wherein modulation of NR4A1 activity induces downregulation of a protein selected from the group consisting of β1-integrin, TXNDC5, survivin, EFGR, PAX3-FOX01A, and combinations thereof.

18. The pharmaceutical composition of claim 12, wherein modulation of NR4A1 activity induces upregulation of a protein selected from the group consisting of SERPINB5, GADD45α, and combinations thereof.

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