WO2011115892A1 - Modulators of the retinoic acid receptor-related orphan receptors - Google Patents

Abstract

The invention provides small molecule modulators of retinoic acid receptor-related orphan receptors such as RORα RORβ, or RORγ. Compounds of the invention can be effective modulators at concentrations ineffective to act on LXR receptors, or on other nuclear receptors, or other biological targets. Methods of modulation the RORs and methods of treating metabolic disorders, immune disorders, cancer, and CNS disorders wherein modulation of an ROR is medically indicated are also provided.

Description

MODULATORS OF THE RETINOIC ACID RECEPTOR-RELATED

ORPHAN RECEPTORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application serial number 61/313,933, filed March 15, 2010, which is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. U54- MH084512, awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

BACKGROUND

Retinoic acid receptor-related orphan receptors (RORs) are nuclear receptors that are constitutively active and can modulate gene transcription in the absence of binding endogenous ligand. Crystal structures of the ligand binding domain of RORs have found cholesterol and cholesterol sulfate in the ligand binding pocket. It is not clear if these ligands act as modulators or if this finding is an artifact of the purification and crystallization process. More recently, we have shown the oxygenated derivatives of cholesterol, 7-oc hydroxycholesterol as an example, are capable of modulating the activity of the RORs. However, it is still unclear if the oxysterols are endogenous ligands for the RORs. It is important to note that this subfamily of nuclear receptors was named not from any known propensity to interact with retinoids, but from sequence homology with retinoic acid receptors (RARs). Recently, it has been found that a high affinity ligand of RORa and RORy, compound T1317, which was developed previously as a LXR modulator, upon binding to the receptors modulates the receptor's ability to interact with transcriptional cofactor proteins and results in repression of ROR target genes.

Binding of the ligand T1317 was found to repress ROR α/γ dependent transactivation of ROR-responsive reporter genes, and in HepG2 cells reduced the recruitment of steroid receptor coactivator-2 (SRC-2) by RORa at an endogenous ROR target gene. This ligand exhibited a degree of selectivity among this class of nuclear receptors, as it was reported to be inactive versus RORp. Unlike RORa and RORy, ROR appears to not be constitutively active thus antagonists and inverse agonists have not effect on the receptors basal activity. However, we have

demonstrated that radiolabled T1317 does in fact bind to RORp. Thus, in the presence of a yet to be discovered endogenous agonist of RORb, T1317 and analogs of it, may prove effective at repression this receptor. Likewise, analogs of T1317 that are agonists, or agonists derived from other chemical scaffolds, are likely to be effective at modulating the activity of RORp. Each of the three major ROR isoforms has multiple variants. See N. Kumar, et al., Mol. Pharm., 77:228-236, 2010.

Accordingly, RORs are an attractive target for small molecule drugs useful for therapeutic intervention for metabolic and immune disorders, cancer, and CNS disorders as well as other diseases where the RORs play a role.

SUMMARY

In various embodiments, the invention is directed to compounds having retinoic acid receptor-related orphan receptor (ROR) modulating bioactivities and methods of modulating ROR comprising contacting the receptor with an effective amount of a compound. In various embodiments, the compounds are sulfonamides and carboxamide derivatives of substituted anilines, which are small molecule modulators of one or more isoforms of ROR. In various embodiments, the compounds are agonists of an ROR. In other embodiments, the compounds are repressors or inverse agonists, or antagonists of an ROR. In various embodiments the compounds are selective modulators of an ROR with little or no effect on the NR1H nuclear receptor subfamily, specifically LXRcc and LXRp, FXR.

In various embodiments, the invention provides a method of modulating the bioactivity of an ROR, comprising contacting the ROR with an effective amount of a compound of formula (I), wherein the compound is an agonist or an activator, or is a repressor, inverse agonist, or antagonist, of a receptor comprising any sequence variant of any isoform of the ROR subfamily, including RORa, RORp, or RORy; wherein the compound of formula (I) comprises

R^{1 ' X ^}N^{' R3}

r² (I)

wherein X is C(O) or S(0)₂;

R¹ is alkyl, aryl, or heteroaryl wherein any group is optionally mono- or independently multi- sub stituted with J ¹ ;

R is H, alkyl, haloalkyl, aryl, aroyl, heteroaryl, or heteroaroyl, wherein any non-hydrogen group is optionally mono- or independently multi- sub stituted with J ;

R is aryl or heteroaryl, wherein any group is optionally mono- or

independently multi- sub stituted with J ;

J¹ when present is halo, cyano, nitro, alkoxy, haloalkoxy, unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido alkoxycarbonyl, unsubstituted or substituted aryl, unsubstituted or substituted arylsulfonyl, unsubstituted or substituted heteroaryl, unsubstituted or substituted

heteroarylsulfonyl, or unsubstituted or substituted arylsulfonamido;

J when present is halo, cyano, nitro, alkoxy, haloalkoxy, unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido alkoxycarbonyl, unsubstituted or substituted aryl, unsubstituted or substituted arylsulfonyl, unsubstituted or substituted heteroaryl, unsubstituted or substituted

heteroarylsulfonyl, or unsubstituted or substituted arylsulfonamido;

J when present is alkyl, haloalkyl, hydroxyalkyl, or hydroxyhaloalkyl; or is an ester of hydroxyalkyl or hydroxyhaloalkyl;

including any stereoisomer thereof, or any salt, solvate, hydrate, metabolite, or prodrug thereof.

In various embodiments X can be CO, providing a carboxamide. In various other embodiments, X can be S0₂, providing a sulfonamide.

In various embodiments, pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable excipient are provided. The composition can be adapted for administration to a patient as a dosage form of the invention, such as a orally or parentally administered dosage form.

In various embodiments, pharmaceutical combinations comprising a compound of the invention and a second medicament are provided. In various embodiments, a method of treating a metabolic or immune disorder wherein modulation of an ROR is medically indicated, is provided. In various embodiments of an inventive method of treatment of a patient, such as a human, the ROR is modulated by a compound of the invention at a dose ineffective to modulate any other nuclear receptor, such as LXRa or LXRp, in the patient, providing an effect free of side effects resulting from modulation of nuclear receptors other than ROR. In various embodiments, the effective amount of the compound of the invention does not affect any other nuclear receptor, any G-protein coupled receptor (GPCR), any kinase, protease, or other enzyme, or any other cellular component or system at a

concentration effective to modulate the effect of at least one of RORa, RORp, or RORy.

In various embodiments, the invention provides novel compounds for carrying out the methods of the invention.

In various embodiments, the invention provides a dosage form adapted for administration to a patient afflicted with a malcondition comprising a metabolic or an immune disorder, cancer, or a CNS disorder, wherein the dosage form comprises a capsule, a tablet, a liquid or dispersed oral formulation, or a formulation adapted for parenteral administration comprising a novel compound of the invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows evidence that SRI 001 is a selective RORa and RORy inverse agonist. A) Structure of SR1001 and T1317. B) GAL4-LXRa, GAL4-RORa, and GAL4-RORy cotransfection assays in HEK293 cells comparing T1317 (left) to SR1001 (right) (n=8). C) Competition radioligand binding assays illustrating the direct binding of SRI 001 to the LBD of RORa (top) and RORy (bottom) relative to [ H]25-hydroxycholesterol (n=4). D) SR1001 dose-dependently inhibits an IL17 promoter-driven luciferase construct in the presence of RORa (left) or RORyt (right) in HEK293 cells. Results are normalized to vehicle (DMSO) control (n=4). E) AlphaScreen assay indicating SRI 001 dose-dependently inhibits the recruitment of a TRAP220 NR box 2 peptide to the LBD of RORy (n=3).

Figure 2 shows evidence that SR1001 modulates the expression of ROR target genes by decreasing coactivator recruitment: A) IL17a, Rora, and Rorc mRNA expression in EL4 cells treated with control (C), or mouse RORa/y siRNA, vehicle (DMSO), or SR1001 (10μΜ, 24 hours) (n=3). Protein expression of RORa and RORy is demonstrated by western blot. *P<0.05; ***P<0.005. B) ChlP-reCHIP assay in EL4 cells illustrating that SR1001 reduces RORa- (B) , RORy- (C) dependent recruitment of SRC-2 to the III 7 promoter.

Figure 3 shows: A) Comparison of the chemical structure of T0901317 (T1317) to SR1078. B) Scheme illustrating the Synthesis of SR1078. C) Biochemical coactivator interaction assay examining the ability of the RORy LBD to interact with the LXXLL domain peptide derived from the TRAP220 coactivator protein. ALPHA Screen technology was used for this assay. Increasing levels of SRI 078 result in a conformational change that results in a dose-dependent decrease in recruitment of the peptide. D) Cotransfection assays in HEK293 cells demonstrate RORa/RORy selectivity. Gal4 DBD-NR LBD chimeric receptors were transfected into cells along with a luciferase reporter responsive to Gal4. RORa, RORy, LXRa, LXR and FXR chimeric receptors were examined. SRI 078 (10 μΜ) resulted in reduced activity of RORa and RORy, but had no effect on LXRa, LXR or FXR activity. *, indicates p<0.05.

Figure 4 shows evidence that SR1078 is a RORa/y Agonist: A) Cotransfection of HEK293 cells with RORa and a reporter consisting of the G6Pase promoter upstream of a luciferase reporter gene. Addition of 10 μΜ SR1078 results in stimulation of transcription. This effect is dependent on the RORE since no activity was noted in a reporter that is identical except for the deletion of the RORE

(G6Pase^mt::luc). B) Cotransfection of HEK293 cells with RORy and a reporter consisting of the G6Pase promoter upstream of a luciferase reporter gene. Addition of 10 μΜ SR1078 results in stimulation of transcription. This effect is dependent on the RORE since no activity was noted in a reporter that is identical except for the deletion of the RORE (G6Pase^mt: :luc) . C) Cotransfection of HEK293 cells with RORa or RORy and a reporter consisting of the FGF21 promoter upstream of a luciferase reporter gene. Addition of 10 μΜ SR1078 results in stimulation of transcription. *, indicates p<0.05.

Figure 5 shows evidence that SR1078 dose-dependently activates RORa and RORy directed transcription. HEK293 cells were cotransfected with full length

RORa (A and C) or RORy (B and D) along with the FGF21 (A and B) and G6Pase (C and D) reporter as described in Figure 4. In all cases, SR1078 induced expression of the target gene reporter dose-dependently with significant activation occurring in the range of 2-5 μΜ. *, indicates p<0.05.

Figure 6 shows evidence that SRI 078 activates ROR target gene transcription both in vitro and in vivo. HepG2 cells expressing natural levels of RORa and RORy were treated with 10 μΜ SR1078 for 24h followed by assessment of either FGF21 (A) or G6Pase (B) gene expression. The expression of both of these ROR target genes was stimulated by the ROR agonist. C) Analysis of plasma levels of SRI 078 following i.p. injection of the compound at a dose of 10 mg/kg in mice. D and E) Levels of expression of FGF21 (D) and G6Pase (E) mRNA 2h following injection (i.p. 10 mg/kg) of SR1078.

Figure 7 shows the identification of a selective RORa synthetic ligand, SR3335. A) Comparison of the chemical structure of T0901317 to SR3335 and SR1078. B) Scheme illustrating the synthesis of SR3335. C) Competition radioligand binding assay illustrating the ability of SR3335 to displace radiolabeled 25- hydroxycholesterol from RORa LBD. D) Competition radioligand binding assay illustrating the inability of SR3335 to displace radiolabeled 25-hydroxycholesterol from RORy LBD.

Figure 8 shows evidence that SR3335 is a selective RORa partial inverse agonist. Cotransfection of HEK293 cells with RORa, RORy or LXRa LBD fused to a GAL4 DNA binding domain and a reporter containing 5 copies of the GAL4 UAS upstream of a luciferase reporter. The effect of T1317 is compared to SR3335 in each assay.

Figure 9 shows evidence that SR3335 suppresses the expression of RORa target genes. A) Treatment of HepG2 cells with 5 μΜ SR3335 results in suppression of transcription in a full-length RORa, G6Pase promoter-luciferase reporter cotransfection assay. G6Pase expression was normalized to cyclophilin. B)

Treatment of HEK293 cells with 5 μΜ SR3335 results in suppression of G6Pase and PEPCK mRNA expression. *, indicates p<0.05.

Figure 10 shows evidence that SR3335 suppresses gluconeogenesis in vivo. A) Pharmacokinetic profile SR3335 following a single injection of 10 mg/kg i.p. B) Pyruvate tolerance test in diet induced obese (DIO) mice (C57B1/6) following 1 week of b.i.d. dosing (i.p.) 15 mg/kg. C) Gene expression in mice following administration of SR3335 as inidicated in 9B. Gene expression was normalized to cyclophilin. *, indicates p<0.05.

DETAILED DESCRIPTION

Definitions

References in the specification to "an embodiment" indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

By "chemically feasible" is meant a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim. The structures disclosed herein, in all of their embodiments are intended to include only "chemically feasible" structures, and any recited structures that are not chemically feasible, for example in a structure shown with variable atoms or groups, are not intended to be disclosed or claimed herein.

When a substituent is specified to be an atom or atoms of specified identity, "or a bond", a configuration is referred to when the substituent is "a bond" that the groups that are immediately adjacent to the specified substituent are directly connected to each other in a chemically feasible bonding configuration.

All chiral, diastereomeric, racemic forms of a structure are intended, unless a particular stereochemistry or isomeric form is specifically indicated. Compounds used in the present invention can include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions, at any degree of enrichment. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention. As used herein, the terms "stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.

A "small molecule" refers to an organic compound, including an

organometallic compound, of a molecular weight less than about 2 kDa, that is not a polynucleotide, a polypeptide, a polysaccharide, or a synthetic polymer composed of a plurality of repeating units.

As to any of the groups described herein, which contain one or more substituents, it is understood that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non- feasible. In addition, the compounds of this disclosed subject matter include all stereochemical isomers arising from the substitution of these compounds.

Selected substituents within the compounds described herein are present to a recursive degree. In this context, "recursive substituent" means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target, and practical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the disclosed subject matter. One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents. To the degree that recursive substituents are present in a claim of the disclosed subject matter, the total number should be determined as set forth above.

The inclusion of an isotopic form of one or more atoms in a molecule that is different from the naturally occurring isotopic distribution of the atom in nature is referred to as an "isotopically labeled form" of the molecule. All isotopic forms of atoms are included as options in the composition of any molecule, unless a specific isotopic form of an atom is indicated. For example, any hydrogen atom or set thereof in a molecule can be any of the isotopic forms of hydrogen, i.e., protium (1H), deuterium ( 2 H), or tritium ( 3 H) in any combination. Similarly, any carbon atom or set thereof in a molecule can be any of the isotopic form of carbons, such as 11 C, 12 C, 13 C, or ¹⁴C, or any nitrogen atom or set thereof in a molecule can be any of the isotopic forms of nitrogen, such as ¹³N, ¹⁴N, or ¹⁵N. A molecule can include any combination of isotopic forms in the component atoms making up the molecule, the isotopic form of every atom forming the molecule being independently selected. In a multi- molecular sample of a compound, not every individual molecule necessarily has the same isotopic composition. For example, a sample of a compound can include molecules containing various different isotopic compositions, such as in a tritium or ¹⁴C radiolabeled sample where only some fraction of the set of molecules making up the macroscopic sample contains a radioactive atom. It is also understood that many elements that are not artificially isotopically enriched themselves are mixtures of naturally occurring isotopic forms, such as ¹⁴N and ¹⁵N, ³²S and ³⁴S, and so forth. A molecule as recited herein is defined as including isotopic forms of all its constituent elements at each position in the molecule. As is well known in the art, isotopically labeled compounds can be prepared by the usual methods of chemical synthesis, except substituting an isotopically labeled precursor molecule. The isotopes, radiolabeled or stable, can be obtained by any method known in the art, such as generation by neutron absorption of a precursor nuclide in a nuclear reactor, by cyclotron reactions, or by isotopic separation such as by mass spectrometry. The isotopic forms are incorporated into precursors as required for use in any particular synthetic route. For example, ¹⁴C and ³H can be prepared using neutrons generated in a nuclear reactor. Following nuclear transformation, ¹⁴C and ³H are incorporated into precursor molecules, followed by further elaboration as needed.

In general, "substituted" refers to an organic group as defined herein in which one or more bonds to a hydrogen atom contained therein are replaced by one or more bonds to a non-hydrogen atom such as, but not limited to, a halogen (i.e., F, CI, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, CI, Br, I, OR', OC(0)N(R')₂, CN, NO, N0₂, ON0₂, azido, CF₃, OCF₃, R', O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R')₂, SR', SOR', S0₂R', S0₂N(R')₂, S0₃R', C(0)R', C(0)C(0)R', C(0)CH₂C(0)R', C(S)R', C(0)OR', OC(0)R', C(0)N(R')₂, OC(0)N(R')₂, C(S)N(R')₂, (CH₂)₀-₂N(R')C(O)R', (CH₂)₀_ ₂N(R')N(R')₂, N(R')N(R')C(0)R', N(R')N(R')C(0)OR', N(R')N(R')CON(R')₂, N(R')S0₂R', N(R')S0₂N(R')₂, N(R')C(0)OR', N(R')C(0)R', N(R')C(S)R',

N(R')C(0)N(R')₂, N(R')C(S)N(R')₂, N(COR')COR', N(OR')R', C(=NH)N(R')₂, C(0)N(OR')R', or C(=NOR')R' wherein R' can be hydrogen or a carbon-based moiety, and wherein the carbon-based moiety can itself be further substituted.

When a substituent is monovalent, such as, for example, F or CI, it is bonded to the atom it is substituting by a single bond. When a substituent is more than monovalent, such as O, which is divalent, it can be bonded to the atom it is substituting by more than one bond, i.e., a divalent substituent is bonded by a double bond; for example, a C substituted with O forms a carbonyl group, C=0, which can also be written as "CO", "C(O)", or "C(=0)", wherein the C and the O are double bonded. When a carbon atom is substituted with a double-bonded oxygen (=0) group, the oxygen substituent is termed an "oxo" group. When a divalent substituent such as NR is double-bonded to a carbon atom, the resulting C(=NR) group is termed an "imino" group. When a divalent substituent such as S is double-bonded to a carbon atom, the results C(=S) group is termed a "thiocarbonyl" group.

Alternatively, a divalent substituent such as O, S, C(O), S(O), or S(0)₂ can be connected by two single bonds to two different carbon atoms. For example, O, a divalent substituent, can be bonded to each of two adjacent carbon atoms to provide an epoxide group, or the O can form a bridging ether group, termed an "oxy" group, between adjacent or non-adjacent carbon atoms, for example bridging the 1,4-carbons of a cyclohexyl group to form a [2.2.1]-oxabicyclo system. Further, any substituent can be bonded to a carbon or other atom by a linker, such as (CH₂)_n or (CR'₂)_n wherein n is 1, 2, 3, or more, and each R' is independently selected.

C(O) and S(0)₂ groups can be bound to one or two heteroatoms, such as nitrogen, rather than to a carbon atom. For example, when a C(O) group is bound to one carbon and one nitrogen atom, the resulting group is called an "amide" or "carboxamide." When a C(O) group is bound to two nitrogen atoms, the functional group is termed a urea. When a S(0)₂ group is bound to one carbon and one nitrogen atom, the resulting unit is termed a "sulfonamide." When a S(0)₂ group is bound to two nitrogen atoms, the resulting unit is termed a "sulfamate."

Substituted alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups as well as other substituted groups also include groups in which one or more bonds to a hydrogen atom are replaced by one or more bonds, including double or triple bonds, to a carbon atom, or to a heteroatom such as, but not limited to, oxygen in carbonyl (oxo), carboxyl, ester, amide, imide, urethane, and urea groups; and nitrogen in imines, hydroxyimines, oximes, hydrazones, amidines, guanidines, and nitriles.

Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and fused ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups can also be substituted with alkyl, alkenyl, and alkynyl groups as defined herein.

By a "ring system" as the term is used herein is meant a moiety comprising one, two, three or more rings, which can be substituted with non-ring groups or with other ring systems, or both, which can be fully saturated, partially unsaturated, fully unsaturated, or aromatic, and when the ring system includes more than a single ring, the rings can be fused, bridging, or spirocyclic. By "spirocyclic" is meant the class of structures wherein two rings are fused at a single tetrahedral carbon atom, as is well known in the art.

Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed above, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

Cycloalkyl groups are cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups can be mono- substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri- substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination denotes a cyclic alkenyl group.

The terms "carbocyclic," "carbocyclyl," and "carbocycle" denote a ring structure wherein the atoms of the ring are carbon, such as a cycloalkyl group or an aryl group. In some embodiments, the carbocycle has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms is 4, 5, 6, or 7. Unless specifically indicated to the contrary, the carbocyclic ring can be substituted with as many as N-l substituents wherein N is the size of the carbocyclic ring with, for example, alkyl, alkenyl, alkynyl, amino, aryl, hydroxy, cyano, carboxy, heteroaryl, heterocyclyl, nitro, thio, alkoxy, and halogen groups, or other groups as are listed above. A carbocyclyl ring can be a cycloalkyl ring, a cycloalkenyl ring, or an aryl ring. A carbocyclyl can be monocyclic or polycyclic, and if polycyclic each ring can be independently be a cycloalkyl ring, a cycloalkenyl ring, or an aryl ring.

(Cycloalkyl) alkyl groups, also denoted cycloalkylalkyl, are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkyl group as defined above.

Alkenyl groups include straight and branched chain and cyclic alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to about 20 carbon atoms, and typically from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to

vinyl, -CH=CH(CH₃), -CH=C(CH₃)₂, -C(CH₃)=CH₂, -C(CH₃)=CH(CH₃),

-C(CH₂CH )=CH₂, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

Cycloalkenyl groups include cycloalkyl groups having at least one double bond between 2 carbons. Thus for example, cycloalkenyl groups include but are not limited to cyclohexenyl, cyclopentenyl, and cyclohexadienyl groups. Cycloalkenyl groups can have from 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like, provided they include at least one double bond within a ring. Cycloalkenyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above.

(Cycloalkenyl)alkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above.

Alkynyl groups include straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to about 20 carbon atoms, and typically from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - C≡CH, -C≡C(CH₃), -C≡C(CH₂CH₃), -CH₂C≡CH, -CH₂C≡C(CH₃),

and -CH₂C≡C(CH₂CH₃) among others.

The term "heteroalkyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -0-CH₂-CH₂-CH₃, -CH₂-CH₂CH₂-OH, -CH₂-CH₂-NH-CH₃, -CH₂-S-CH₂-CH 3, -CH₂CH₂-S(=0)-CH₃, and -CH₂CH₂-0-CH₂CH₂-0-CH₃. Up to two heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃, or -CH₂-CH₂-S-S-CH₃.

A "cyclohetero alkyl" ring is a cycloalkyl ring containing at least one heteroatom. A cycloheteroalkyl ring can also be termed a "heterocyclyl," described below.

The term "heteroalkenyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain monounsaturated or di-unsaturated hydrocarbon group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Up to two heteroatoms may be placed consecutively. Examples

include -CH=CH-0-CH₃, -CH=CH-CH₂-OH, -CH₂-CH=N-OCH₃,

-CH=CH-N(CH₃)-CH₃, -CH₂-CH=CH-CH₂-SH, and and -CH=CH-0-CH₂CH₂-0- CH₃.

Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined above.

Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl group are alkenyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above.

Heterocyclyl groups or the term "heterocyclyl" includes aromatic and non- aromatic ring compounds containing 3 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Thus a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C₂-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C₄-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused ring species including those comprising fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a

benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both

heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl.

Heterocyclyl groups can be unsubstituted, or can be substituted as discussed above. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,

imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono- substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with groups such as those listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C₂-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C₄-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,

imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed above. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed above.

Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl,

3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1- imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-l-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl, l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4- oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2- pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5- pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4-pyridazinyl, 5- pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7- quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2- benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl,

6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro- benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro- benzo[b]furanyl), benzo[b] thiophenyl (2-benzo[b] thiophenyl, 3-benzo[b]thiophenyl,

4- benzo[b]thiophenyl, 5 -benzo[b] thiophenyl, 6-benzo[b] thiophenyl, 7- benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro- benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl,

2- indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl,

3- indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1 -benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6- benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4- benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1- carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H- dibenz[b,f]azepin-l-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H- dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,1 l-dihydro-5H- dibenz[b,f]azepine (10,1 l-dihydro-5H-dibenz[b,f]azepine-l-yl, 10,1 l-dihydro-5H- dibenz[b,f]azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro- 5H-dibenz[b,f]azepine-4-yl, 10,l l-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.

Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group as defined above is replaced with a bond to a heterocyclyl group as defined above. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.

Heteroarylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above.

The term "alkoxy" refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined above. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like.

Examples of cyclic alkoxy include but are not limited to cyclopropyloxy,

cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include one to about 12-20 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structures are substituted therewith.

The terms "halo" or "halogen" or "halide" by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.

A "haloalkyl" group includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3- dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.

A "haloalkoxy" group includes mono-halo alkoxy groups, poly-halo alkoxy groups wherein all halo atoms can be the same or different, and per-halo alkoxy groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkoxy include trifluoromethoxy, 1,1-dichloroethoxy, 1,2- dichloroethoxy, l,3-dibromo-3,3-difluoropropoxy, perfluorobutoxy, and the like.

A "hydroxyhaloalkyl" as the term is used herein refers to an alkyl group bearing at least one hydroxy group and at least one halo group. For example, a 1- hydroxy-l-trifluoromethyl-2,2,2-trifluoroethyl group is a hydroxyhaloalkyl group within the meaning herein.

The term "(C_x-C_y)perfluoroalkyl," wherein x < y, means an alkyl group with a minimum of x carbon atoms and a maximum of y carbon atoms, wherein all hydrogen atoms are replaced by fluorine atoms. Preferred is -(C₁-C₆)perfluoroalkyl, more preferred is -(C₁-C3)perfluoroalkyl, most preferred is -CF₃.

The term "(C_x-C_y)perfluoroalkylene," wherein x < y, means an alkyl group with a minimum of x carbon atoms and a maximum of y carbon atoms, wherein all hydrogen atoms are replaced by fluorine atoms. Preferred

is -(C₁-C₆)perfluoroalkylene, more preferred is -(C₁-C₃)perfluoroalkylene, most preferred is -CF₂-.

The terms "aryloxy" and "arylalkoxy" refer to, respectively, an aryl group bonded to an oxygen atom and an aralkyl group bonded to the oxygen atom at the alkyl moiety. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy.

An "acyl" group as the term is used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a "formyl" group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-20 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl-3-carbonyl) group is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group.

The term "amine" includes primary, secondary, and tertiary amines having, e.g., the formula N(group)₃ wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH₂, for example, alkylamines, arylamines, alkylarylamines; R₂NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R₃N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein.

An "amino" group is a substituent of the form -NH₂, -NHR, -NR₂, -NR ⁺, wherein each R is independently selected, and protonated forms of each, except for - NR₃ ⁺, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary or quaternary amino group. An

"alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group.

An "ammonium" ion includes the unsubstituted ammonium ion NH₄ ⁺, but unless otherwise specified, it also includes any protonated or quaternarized forms of amines. Thus, trimethylammonium hydrochloride and tetramethylammonium chloride are both ammonium ions, and amines, within the meaning herein.

The term "amide" (or "amido") includes C- and N-amide groups,

i.e., -C(0)NR₂, and -NRC(0)R groups, respectively. Amide groups therefore include but are not limited to primary carboxamide groups (-C(0)NH₂) and formamide groups (-NHC(O)H). A "carboxamido" group is a group of the formula C(0)NR₂, wherein R can be H, alkyl, aryl, etc.

The term "azido" refers to an N₃ group. An "azide" can be an organic azide or can be a salt of the azide (N₃ ^~) anion. The term "nitro" refers to an N0₂ group bonded to an organic moiety. The term "nitroso" refers to an NO group bonded to an organic moiety. The term nitrate refers to an ON0₂ group bonded to an organic moiety or to a salt of the nitrate (N0₃ ^~) anion.

The term "urethane" ("carbamoyl" or "carbamyl") includes N- and O-urethane groups, i.e., -NRC(0)OR and -OC(0)NR₂ groups, respectively. The term "sulfonamide" (or "sulfonamido") includes S- and N-sulfonamide groups, i.e., -SO₂NR₂ and -NRS0₂R groups, respectively. Sulfonamide groups therefore include but are not limited to sulfamoyl groups (-S0₂NH₂). An

organosulfur structure represented by the formula -S(0)(NR)- is understood to refer to a sulfoximine, wherein both the oxygen and the nitrogen atoms are bonded to the sulfur atom, which is also bonded to two carbon atoms.

The term "amidine" or "amidino" includes groups of the formula -C(NR)NR₂. Typically, an amidino group is -C(NH)NH₂.

The term "guanidine" or "guanidino" includes groups of the

formula -NRC(NR)NR₂. Typically, a guanidino group is -NHC(NH)NH₂.

A "salt" as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion. For example, acids in their anionic form can form salts with cations such as metal cations, for example sodium, potassium, and the like; with ammonium salts such as NH₄ ⁺ or the cations of various amines, including tetraalkyl ammonium salts such as tetramethylammonium, or other cations such as trimethylsulfonium, and the like. A "pharmaceutically acceptable" or "pharmacologically acceptable" salt is a salt formed from an ion that has been approved for human consumption and is generally non-toxic, such as a chloride salt or a sodium salt. A "zwitterion" is an internal salt such as can be formed in a molecule that has at least two ionizable groups, one forming an anion and the other a cation, which serve to balance each other. For example, amino acids such as glycine can exist in a zwitterionic form. A "zwitterion" is a salt within the meaning herein. The compounds of the present invention may take the form of salts. The term "salts" embraces addition salts of free acids or free bases which are compounds of the invention. Salts can be "pharmaceutically-acceptable salts." The term "pharmaceutically-acceptable salt" refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compounds of the invention.

Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,

trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts of compounds of the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example,

N,A^-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,

ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. Although pharmaceutically unacceptable salts are not generally useful as medicaments, such salts may be useful, for example as intermediates in the synthesis of Formula (I) compounds, for example in their purification by recrystallization. All of these salts may be prepared by conventional means from the corresponding compound according to Formula (I) by reacting, for example, the appropriate acid or base with the compound according to Formula (I). The term "pharmaceutically acceptable salts" refers to nontoxic inorganic or organic acid and/or base addition salts, see, for example, Lit et al., Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217, incorporated by reference herein. Lists of many suitable salts are also found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, (1985), 1418, and the disclosure of which is incorporated herein by reference.

A "hydrate" is a compound that exists in a composition with water molecules. The composition can include water in stoichiometic quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. As the term is used herein a "hydrate" refers to a solid form, i.e., a compound in water solution, while it may be hydrated, is not a hydrate as the term is used herein.

A "solvate" is a similar composition except that a solvent other that water replaces the water. For example, methanol or ethanol can form an "alcoholate", which can again be stoichiometic or non- stoichiometric. As the term is used herein a "solvate" refers to a solid form, i.e., a compound in solution in a solvent, while it may be solvated, is not a solvate as the term is used herein.

A "prodrug" as is well known in the art is a substance that can be administered to a patient where the substance is converted in vivo by the action of biochemical agents within the patient's body, such as enzymes, to the active pharmaceutical ingredient. Examples of prodrugs include esters of carboxylic acid groups, which can be hydrolyzed by endogenous esterases as are found in the bloodstream of humans and other mammals. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985. As used herein, the term "prodrug" refers to any pharmaceutically acceptable form of compound of the invention which, upon administration to a patient, provides a compound of the invention . Pharmaceutically acceptable prodrugs refer to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form a compound of the invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound. The compounds of the invention herein possess antiviral activity against HCV, or are metabolized to a compound that exhibits such activity.

As used herein, the term "metabolite" refers to any compound produced in vivo or in vitro from the parent drug of the invention, or any of its prodrugs that are converted biologically to a parent drug of the invention and then to a further biotransformation product of the parent drug.

"Treating" or "treatment" within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, or inhibition of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder. As used herein, the terms "treating" or "treat" includes (i) preventing a pathologic condition from occurring (e.g., prophylaxis); (ii) inhibiting the pathologic condition or arresting its development; (iii) relieving the pathologic condition; and/or (iv) diminishing symptoms associated with the pathologic condition

Similarly, as used herein, an "effective amount" or a "therapeutically effective amount" of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition. In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects.

An "effective amount" of a compound of the invention is an amount or concentration of the compound which is sufficient to modulate the effect of an ROR, e.g., RORa, RORp, or RORy, but does not affect a nuclear receptor of another type, e.g., LXRa or LXRp. In various embodiments, an effective amount of a compound of the invention does not affect any nuclear receptor other than an ROR. In various embodiments, an effective amount of a compound of the invention does not affect any G-protein coupled receptor (GPCR), kinase, protease, ion channel, enzyme, or any other biological component or system other than an ROR.

As used herein, the term "therapeutically effective amount" is intended to include an amount of a compound described herein, or an amount of the combination of compounds described herein, e.g., to treat or prevent the disease or disorder, or to treat the symptoms of the disease or disorder, in a host. The combination of compounds is preferably a synergistic combination. Synergy, as described for example by Chou and Talalay, Adv. Enzyme ReguL, 22:27 (1984), occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at suboptimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, increased activity, or some other beneficial effect of the combination compared with the individual components.

A "retinoic acid receptor-related orphan receptor" refers to nuclear receptors such as the sequence variants of RORa (NR1F1), RORp (NR1F2), and RORy (NR1F3), all having sequence homology to the retinoic acid receptor subfamily of nuclear receptors as are described in N. Kumar, et al., Mol. Pharm., 77:228-236, 2010, and documents cited therein.

A "modulator" as the term is used herein refers to a molecule that alters the basal activity of the ROR either positively (activates) or negatively (represses).

"Modulating" refers to the action of a modulator, either activating or repressing a receptor, such as an ROR or another nuclear receptor such as LXR, or as an agonist or antagonist of a receptor, such as a G-protein coupled receptor (GPCR), or as an inhibitor or activator of an enzyme, for example a kinase or a protease. A compound of the invention can be a modulator of an ROR, for example at an effective concentration or in an effective amount, but not be a modulator of any nuclear receptor other than an ROR, e.g., not a modulator, or not a modulator at some particular concentration or in some particular amount of LXRa or LXRP or another type of nuclear receptor, and not an agonist or antagonist of a GPCR or an inhibitor or activator of an enzyme. This can provide selectivity of effect of a compound of the invention when administered in a quantity to a patient for treatment of a malcondition such as a immune or metabolic disorder, cancer, or a central nervous system (CNS) disorder.

The term "medically indicated" refers to a course of treatment or a use of a medicinal compound or procedure wherein the treatment or use is recommended by competent medical authority, e.g., a physician treating a patient, wherein the physician based upon factors such as the physician's knowledge, experience, analysis and intuition recommends the treatment or use as potentially beneficial to the patient.

As used herein, the term "patient" refers to a warm-blooded animal, and preferably a mammal, such as, for example, a cat, dog, horse, cow, pig, mouse, rat, or primate, including a human.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

One diastereomer or one enantiomer of a compound of the invention may display superior biological activity compared with the other. When required, separation of the diastereomeric mixture or the racemic material can be achieved by HPLC, optionally using a chiral column or by a resolution using a resolving agent such as camphonic chloride as in Tucker et al., J. Med. Chem., 37, 2437 (1994), for the resolution of enantiomers. A chiral compound described herein may also be directly synthesized using a chiral catalyst or a chiral ligand, e.g., Huffman et al., L Org. Chem., 60: 1590 (1995).

As used herein, "μβ" denotes microgram, "mg" denotes milligram, "g" denotes gram, "μΙ_/' denotes microliter, "mL" denotes milliliter, "L" denotes liter, "nM" denotes nanomolar, "μΜ" denotes micromolar, "mM" denotes millimolar, "M" denotes molar, and "nm" denotes nanometer.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. Moreover, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any combination of individual members or subgroups of members of Markush groups. Thus, for example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, and Y is described as selected from the group consisting of methyl, ethyl, and propyl, claims for X being bromine and Y being methyl are fully described.

In various embodiments, the compound or set of compounds, such as are used in the inventive methods, can be any one of any of the combinations and/or subcombinations of the above-listed embodiments.

The present invention further embraces isolated compounds according to the formulas specified herein. The expression "isolated compound" refers to a

preparation of a compound of a specified formula, or a mixture of compounds, wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds. "Isolated" does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to compound in a form in which it can be used therapeutically. Preferably an "isolated compound" refers to a preparation of a compound or a mixture of compounds, which contains the named compound or mixture of compounds in an amount of at least 10 percent by weight of the total weight. Preferably the preparation contains the named compound or mixture of compounds in an amount of at least 50 percent by weight of the total weight; more preferably at least 80 percent by weight of the total weight; and most preferably at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation.

The compounds of the invention and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystallization or chromatography, including flash column chromatography, or HPLC.

Tautomerism

Within the present invention it is to be understood that a compound or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same compound. It is to be understood that the formulae drawings within this specification can represent only one of the possible tautomeric forms. However, it is also to be understood that the invention

encompasses any tautomeric form, and is not to be limited merely to any one tautomeric form utilized within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been convenient to show graphically herein. For example, tautomerism may be exhibited by a pyrazolyl group bonded as indicated by the wavy line. While both substituents would be termed a 4- pyrazolyl group, it is evident that a different nitrogen atom bears the hydrogen atom in each structure.

Such tautomerism can also occur with substituted pyrazoles such as 3-methyl, 5-methyl, or 3,5-dimethylpyrazoles, and the like. Another example of tautomerism is amido-imido (lactam-lactim when cyclic) tautomerism, such as is seen in heterocyclic compounds bearing a ring oxygen atom adjacent to a ring nitrogen atom. For example, the equilibrium:

is an example of tautomerism. Accordingly, a structure depicted herein as one tautomer is intended to also include the other tautomer.

Optical Isomerism

It will be understood that when compounds of the present invention contain one or more chiral centers, the compounds may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures. The present invention therefore includes any possible enantiomers, diastereomers, racemates or mixtures thereof of the compounds of the invention.

The isomers resulting from the presence of a chiral center comprise a pair of non-superimposable isomers that are called "enantiomers." Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the

Cahn-Ingold-Prelog system. The priority of substituents is ranked based on atomic weights, a higher atomic weight, as determined by the systematic procedure, having a higher priority ranking. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated (R) and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated (S). The Cahn-Ingold-Prelog ranking is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.

(R) configuration (S) configuration

The present invention is meant to encompass diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof. Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.

"Isolated optical isomer" means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula. Preferably, the isolated isomer is at least about 80%, more preferably at least 90% pure, even more preferably at least 98% pure, most preferably at least about 99% pure, by weight.

Isolated optical isomers may be purified from racemic mixtures by

well-known chiral separation techniques. According to one such method, a racemic mixture of a compound of the invention, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of DAICEL^® CHIRALPAK^® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.

Rotational Isomerism

It is understood that due to chemical properties {i.e., resonance lending some double bond character to the C-N bond) of restricted rotation about the amide bond linkage (as illustrated below) it is possible to observe separate rotamer species and even, under some circumstances, to isolate such species (see below). It is further understood that certain structural elements, including steric bulk or substituents on the amide nitrogen, may enhance the stability of a rotamer to the extent that a compound may be isolated as, and exist indefinitely, as a single stable rotamer. The present invention therefore includes any possible stable rotamers of formula (I) which are biologically active in the treatment of cancer or other proliferative disease states.

Regioisomerism

The preferred compounds of the present invention have a particular spatial arrangement of substituents on the aromatic rings, which is related to the structure activity relationship demonstrated by the compound class. Often such substitution arrangement is denoted by a numbering system; however, numbering systems are often not consistent between different ring systems. In six-membered aromatic systems, the spatial arrangements are specified by the common nomenclature "para" for 1,4-substitution, "meta" for 1,3 -substitution and "ortho" for 1,2- substitution as shown below.

"para-" "meta-" "ortho-"

Detailed Description

In various embodiments, the invention provides a method of modulating the bioactivity of an ROR, comprising contacting the ROR with an effective amount of a compound of formula (I), wherein the compound is an agonist or an activator, or is a repressor, inverse agonist, or antagonist, of a receptor comprising any sequence variant of any isoform of the ROR subfamily, including RORa, RORp, or RORy; wherein the compound of formula I) comprises

(I)

wherein X is C(O) or S(0)₂;

R¹ is alkyl, aryl, or heteroaryl wherein any group is optionally mono- or independently multi- substituted with J¹;

R is H, alkyl, haloalkyl, aryl, aroyl, heteroaryl, or heteroaroyl, wherein any non-hydrogen group is optionally mono- or independently multi- substituted with J ;

R is aryl or heteroaryl, wherein any group is optionally mono- or independently multi- substituted with J ;

J¹ when present is halo, cyano, nitro, alkoxy, or haloalkoxy; unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido, or alkoxycarbonyl; unsubstituted or substituted aryl; unsubstituted or substituted arylsulfonyl;

unsubstituted or substituted heteroaryl; unsubstituted or substituted

heteroarylsulfonyl; or unsubstituted or substituted arylsulfonamido;

J when present is halo, cyano, nitro, alkoxy, or haloalkoxy; unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido or alkoxycarbonyl; unsubstituted or substituted aryl; unsubstituted or substituted arylsulfonyl;

unsubstituted or substituted heteroaryl; unsubstituted or substituted

heteroarylsulfonyl; or unsubstituted or substituted arylsulfonamido; including any stereoisomer thereof, or any salt, solvate, hydrate, metabolite, or prodrug thereof.

The ROR-modulatory compound suitable for modulating an ROR can be an agonist or an activator, or a repressor, inverse agonist, or antagonist, of a receptor comprising any sequence variant of any isoforms of ROR, including RORa, RORp, or RORy [NR1F1, NR1F2, and NR1F3], thereby affecting the bioactivity of one or more of the ROR NRIF subfamily of nuclear receptors at concentrations of the compound accessible in vivo upon administration of the compound to a human patient.

In various embodiments of the compounds, X is C(O), providing

carboxamides. In other embodiments, X is S(0)₂, providing sulfonamides.

In various embodiments, R¹ can be unsubstituted or substituted phenyl, thiophenyl, quinolinyl, naphthyl, coumaryl, biphenyl, benzoxadiazolyl, thiazolyl, aroyloxymethyl, or trifluoromethyl. In various embodiments, J¹ can be fluoro, chloro, bromo, iodo, cyano, nitro, methoxy, methoxycarbonyl, trifluoromethoxy,

trifluoromethyl, methyl, t-butyl, n-butyl, substituted pyrimidinyl, isoxazolyl, pyridinyl, phenyl, phenylsulfonyl, substituted pyrazolyl, benzoylamidomethyl, halobenzoylamidomethyl,

In various embodiments, R 2 substituted with J 2 can comprise a 2,2,2- trifluoroethyl, benzoyl, toluoyl, or dinitrobenzoyl group.

In various embodiments, R can be unsubstituted or substituted phenyl. In various embodiments, J can be halo or hydroxyhaloalkyl, or an ester thereof. For example, R 3 and J 3 together can compri

or an ester thereof, wherein a wavy line indicates a point of attachment of J 3 -substituted R 3 to the nitrogen atom bearing R .

More specifically, the ester can comprise a substituted or unsubstituted aroyl or heteroaroyl ester o

, wherein a wavy line indicates a point of attachment of J 3 -substituted R 3 to the nitrogen atom bearing R 3.

For example, the aroyl ester can be an unsubstituted benzoyl or benzoyl substituted with halo, nitro, or alkyl, or any combination thereof. For example, the heteroaroyl ester can be an unsubstituted or substituted picolinoyl, thiophenoyl, furoyl, wherein any heteroaroyl can be substituted with halo, nitro, or alkyl, or any combination thereof.

In various embodiments, a method of the invention can use a compound of formula (I) which can be any of the following carboxamides:

or any salt, solvate, hydrate, metabolite, or prodrug thereof.

In various embodiments, a method of the invention can use a compound of formula (I) which can be any of the following sulfonamides:

or any salt, solvate, hydrate, metabolite, or prodrug thereof.

In various embodiments of the invention, a compound of the invention is inactive with respect to modulation of a nuclear receptor other than an ROR or with respect to modulation of a G-protein coupled receptor, an ion channel, or an enzyme; or the modulation of the ROR takes place at a concentration ineffective for modulation of a nuclear receptor other than an ROR at the concentration, or ineffective for modulation of a G-protein coupled receptor, an ion channel, or an enzyme at the concentration.

A compound of the invention that is an effective modulator (repressor or activator) of an ROR can be inactive with respect to modulation of another nuclear receptor, such as LXRa or LXRp, or the modulation of an ROR can be selective at some concentration with respect to modulation of another nuclear receptor, such as an LXR, providing an effect free of side effects resulting from modulation of a non- target nuclear receptor. In various embodiments of an inventive method of treatment of a patient, such as a human, the ROR is modulated by a compound of the invention at a dose ineffective to modulate any other nuclear receptor, such as LXRa or LXRp, in the patient, providing an effect free of side effects resulting from modulation of nuclear receptors other than ROR. In various embodiments, the effective amount of the compound of the invention does not affect any nuclear receptor other than an ROR, or does not affect any G-protein coupled receptor (GPCR), or any ion channel, or any kinase, protease, or other enzyme, or any other cellular component or system at a concentration effective to modulate the effect of an ROR such as RORa, RORp, or RORy.

In various embodiments, the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically effective excipient.

In various embodiments, the invention provides a pharmaceutical combination comprising a compound of the invention and a second medicament. The second medicament can comprise, for treatment of a metabolic disorder, an anti-diabetic or anti-insulin resistance agent, such as a glitazone, a sulfonylurea, metformin, insulin, an insulin mimetic, a DPP4 inhibitor, a GLP1 receptor agonist, a glucagon receptor antagonist, or an anti-obesity agent. For treatment of an immune disorder, the second medicament can comprise an anti-TNF agent or an immune- suppresive

glucocorticoid. For treatment of cancer, the second medicament can comprise an anticancer agent such as a platinum compound, a Vinca alkaloid or analog thereof, a taxane, a nitrogen mustard, or the like.

In various embodiments, the invention provides a use of a compound of a compound of the invention in the preparation of a medicament. More specifically, the medicament can be adapted for the treatment of metabolic and immune disorders, cancer, or CNS disorders.

In various embodiments, the invention provides a method of modulating the bioactivity of an ROR, comprising contacting the ROR with an effective amount of a compound of the invention. More specifically, the modulation can take place in vivo in a mammal. The mammal can be a human or a non-human primate.

In various embodiments, the invention provides a method of modulating the bioactivity of an ROR, wherein the bioactivity of an LXR is substantially unaffected by a concentration of the compound in a tissue effective for modulation of an ROR, providing an effect free of side effects resulting from LXR modulation. Modulation of LXR can result in an increase in blood triglycerides, which is undesirable therapeutically.

In various embodiments, the invention provides a method of treating a metabolic or immune disorder, cancer, or a CNS disorder in a patient for which modulation of an ROR is medically indicated, comprising administering to the patient an effective amount of a compound of the invention at a frequency and for a duration of time to provide a beneficial result to the patient. More specifically, the metabolic disorder can comprise insulin resistance, type 2 diabetes, diabetes, and obesity.

More specifically, the immune disorder can comprise an auto immune disorder such as Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, Type I diabetes, Rheumatoid arthritis, Systemic lupus erythematosus, Dermatomyositis, Sjogren syndrome, Lupus erythematosus, Multiple sclerosis, Myasthenia gravis, Reactive arthritis, Grave's disease, Crohn's disease, Lupus, etc.

More specifically, cancer can comprise prostate cancer, colon cancer, breast cancer, lung cancer, etc.

More specifically, a CNS disorder can comprise sleep disorder, anxiety, neurodegenerative disease such as Parkinson's or Alzheimer's, etc.

In various embodiments, the invention provides a compound of the invention as a novel compound per se, comprising any of the compounds disclosed as suitable for carrying out a method of the invention, with the exception of compounds of the following formulas:

Pharmaceutical Use

Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Compounds of the invention have been found to be high affinity ligands, agonists or repressors (antagonists), of at least

RORa and/or RORy classes of receptors. Binding of a radiolabeled compound of the invention to RORP has also been demonstrated. Modulation of one or more of these ROR receptors can be effective in controlling these and other physiological processes.

The role for RORa in regulation of metabolic pathways has been revealed by studies of a mutant mouse strain termed staggerer (RORa^sg/sg), wherein the RORa is rendered inactive. Such mice are less susceptible to hepatic steatosis and have a reduced body fat index relative to wild-type mice despite higher food consumption. RORa has also been implicated in regulation of glucose metabolism. RORy has been implicated in the regulation of immune function, such as in the development of TH17 cells that are believed to play an important role in autoimmunity. Accordingly, repressors of RORy may be able to block Thl7 cell proliferation and IL-17

production. Recently, RORa has been shown to attenuate Wnt/b-catenin signaling in colon cancer. Modulation of ROR may be able to stop cancer cell growth or induce cancer cell death. In addition, ROR plays a critical role in regulation of the core clock which controls circadian rhythms. Thus, modulation of RORs can be useful in the treatment of sleep dysfunction and other CNS disorders.

In various embodiments, a compound of the invention can be present in vivo in a patient in an amount or concentration of the compound which is sufficient to modulate the effect of an ROR, e.g., RORa, RORp, or RORy, but does not affect a nuclear receptor of another type, e.g., LXRa or LXRP in a living organism. In various embodiments, an effective amount of a compound of the invention does not affect any nuclear receptor other than an ROR. In various embodiments, an effective amount of a compound of the invention does not affect any G-protein coupled receptor (GPCR), kinase, protease, ion channel, enzyme, or any other biological component or system other than an ROR.

Compositions and Combinations

Another aspect of an embodiment of the invention provides compositions of the compounds of the invention, alone or in combination with another medicament. As set forth herein, compounds of the invention include stereoisomers, tautomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts and mixtures thereof. Compositions containing a compound of the invention can be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy, 19th Ed., 1995, incorporated by reference herein. The compositions can appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.

Typical compositions include a compound of the invention and a

pharmaceutically acceptable excipient which can be a carrier or a diluent. For example, the active compound will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which can be in the form of an ampoule, capsule, sachet, paper, or other container. When the active compound is mixed with a carrier, or when the carrier serves as a diluent, it can be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active compound. The active compound can be adsorbed on a granular solid carrier, for example contained in a sachet. Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The formulations can be mixed with auxiliary agents which do not deleteriously react with the active compounds. Such additives can include wetting agents, emulsifying and suspending agents, salt for influencing osmotic pressure, buffers and/or coloring substances preserving agents, sweetening agents or flavoring agents. The compositions can also be sterilized if desired.

In various embodiments, the invention provides a dosage form adapted for administration to a patient afflicted with a malcondition comprising a metabolic or an immune disorder, cancer, or a CNS disorder, wherein the dosage form comprises a capsule, a tablet, a liquid or dispersed oral formulation, or a formulation adapted for parenteral administration. The route of administration can be any route which effectively transports the active compound of the invention to the appropriate or desired site of action, such as oral, nasal, pulmonary, buccal, subdermal, intradermal, transdermal or parenteral, e.g., rectal, depot, subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solution or an ointment, the oral route being preferred.

If a solid carrier is used for oral administration, the preparation can be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. If a liquid carrier is used, the preparation can be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

Injectable dosage forms generally include aqueous suspensions or oil suspensions which can be prepared using a suitable dispersant or wetting agent and a suspending agent Injectable forms can be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution. Alternatively, sterile oils can be employed as solvents or suspending agents. Preferably, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the formulation can also be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations can optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and

combinations of these. The compounds can be formulated for parenteral

administration by injection such as by bolus injection or continuous infusion. A unit dosage form for injection can be in ampoules or in multi-dose containers.

The formulations of the invention can be designed to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. Thus, the formulations can also be formulated for controlled release or for slow release.

Compositions contemplated by the present invention can include, for example, micelles or liposomes, or some other encapsulated form, or can be administered in an extended release form to provide a prolonged storage and/or delivery effect.

Therefore, the formulations can be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections. Such implants can employ known inert materials such as silicones and biodegradable polymers, e.g., polylactide- polyglycolide. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).

For nasal administration, the preparation can contain a compound of the invention, dissolved or suspended in a liquid carrier, preferably an aqueous carrier, for aerosol application. The carrier can contain additives such as solubilizing agents, e.g., propylene glycol, surfactants, absorption enhancers such as lecithin

(phosphatidylcholine) or cyclodextrin, or preservatives such as parabens.

For parenteral application, particularly suitable are injectable solutions or suspensions, preferably aqueous solutions with the active compound dissolved in polyhydroxylated castor oil.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrier or binder or the like are particularly suitable for oral application. Preferable carriers for tablets, dragees, or capsules include lactose, corn starch, and/or potato starch. A syrup or elixir can be used in cases where a sweetened vehicle can be employed.

A typical tablet that can be prepared by conventional tabletting techniques can contain: Core:

Active compound (as free compound or salt thereof) 250 mg

Colloidal silicon dioxide (Aerosil)® 1.5 mg

Cellulose, microcryst. (Avicel)® 70 mg

Modified cellulose gum (Ac-Di-Sol)® 7.5 mg

Magnesium stearate Ad.

Coating:

HPMC approx. 9 mg

*Mywacett 9-40 T approx. 0.9 mg

*Acylated monoglyceride used as plasticizer for film coating.

A typical capsule for oral administration contains compounds of the invention (250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture is passed through a 60 mesh sieve and packed into a No. 1 gelatin capsule. A typical injectable preparation is produced by aseptically placing 250 mg of compounds of the invention into a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 2 mL of sterile physiological saline, to produce an injectable preparation.

The compounds of the invention can be administered to a mammal, especially a human in need of such treatment, prevention, elimination, alleviation or

amelioration of a malcondition. Such mammals include also animals, both domestic animals, e.g. household pets, farm animals, and non-domestic animals such as wildlife.

The compounds of the invention are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from about 0.05 to about 5000 mg, preferably from about 1 to about 2000 mg, and more preferably between about 2 and about 2000 mg per day can be used. A typical dosage is about 10 mg to about 1000 mg per day. In choosing a regimen for patients it can frequently be necessary to begin with a higher dosage and when the condition is under control to reduce the dosage. The exact dosage will depend upon the activity of the compound, mode of administration, on the therapy desired, form in which administered, the subject to be treated and the body weight of the subject to be treated, and the preference and experience of the physician or veterinarian in charge.

Generally, the compounds of the invention are dispensed in unit dosage form including from about 0.05 mg to about 1000 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.

Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal administration include from about 125 μg to about 1250 mg, preferably from about 250 μg to about 500 mg, and more preferably from about 2.5 mg to about 250 mg, of the compounds admixed with a pharmaceutically acceptable carrier or diluent.

Dosage forms can be administered daily, or more than once a day, such as twice or thrice daily. Alternatively dosage forms can be administered less frequently than daily, such as every other day, or weekly, if found to be advisable by a prescribing physician.

Evaluation of Bioactivity

It is within ordinary skill to evaluate any compound disclosed and claimed herein for effectiveness in modulation of an ROR in various cellular and biochemical assays using the procedures described above or found in the scientific literature. Accordingly, the person of ordinary skill can prepare and evaluate any of the claimed compounds without undue experimentation. It is also within ordinary skill to evaluate any compound disclosed and claimed herein for effectiveness in modulation of the nuclear receptors LXRcc and LXR in various cellular and biochemical assays using the procedures described above or found in the scientific literature, and in evaluating the results in light of the ROR modulatory results, evaluate the selectivity of the tested compound(s) for ROR selectivity over the LXRs, other nuclear receptors, and other biological targets and for further evaluation as a medicinal compound.

Any compound found to be an effective and selective inhibitor of an ROR can likewise be tested in animal models and in human clinical studies using the skill and experience of the investigator to guide the selection of dosages and treatment regimens.

EXAMPLES

Synthetic Procedures

Compounds of the present invention, containing a sulfonyl linker ("X") in structural formula I were synthesized as summarized in Scheme 1, below. An aniline of type A, which can be prepared as described in the literature (Farah, B. S.; Gilbert, E. E.; Sibilia, J. P. /. Org. Chem. 1965, 30, 1001) is treated with a sulfonyl chloride, numerous examples of which are known in the art, to give sulfonamides of type B. (See also, Xue, Y.; Chao, E.; Zuercher, W. J.; Willson T. M.; Collins, J. M.;

Redinboa, M. R. Bioorg. Med. Chem. 2007, 15, 2156-2166.). Alternatively, acylation of A with an acid chloride, or with a carboxylic acid using an appropriate dehydrating agent, numerous examples of which are well known in the art, will give amides C. Reduction of C under conventional conditions well known to a person skilled in the art with a reagent such as lithium aluminum hydride, in an inert solvent, provides an N-alkyl aniline of type D. Finally, treatment of D with a sulfonyl chloride, as discussed above, will give N-alkyl sulfonamide E. This example specifically illustrates the synthesis of a compound of structural formula I wherein X = S0₂ and R² = CH₂CF₃.

Scheme 1 : Synthetic Routes to Sulfonamide Compounds

Procedure A : Synthesis of Sulfonamides B

To a solution of 4-(l-hydroxy-l-trifluoromethyl-2,2,2-trifluoroethyl)aniline (A) (1.5M in THF, 128 μΐ,, 0.193 mmol) in acetone (643 μΙ_) under argon were successively added at room temperature 2,6-lutidine (29 uL, 0.251 mmol) and the corresponding arylsulfonylchloride (0.193 mmol). The mixture was heated at 80°C for 1 day, then cooled to room temperature and diluted with ethyl acetate (EtOAc) and saturated NaHC0₃ solution. The aqueous phase was extracted two times with EtOAc and combined organic phases were dried over Na₂S0₄, filtrated and evaporated. The crude residue was purified by column chromatography on silica gel to give sulfonamides B.

Procedure B : Synthesis of Sulfonamides E

To a solution of 4-(l-hydroxy-l-trifluoromethyl-2,2,2-trifluoroethyl)-N-2,2,2- trifluoroethylaniline (1.5M in THF, 128 μί, 0.193 mmol) under argon in pyridine (321 μ > was added at room temperature the arylsulfonylchloride (0.193 mmol). The mixture was heated at 100°C for 1 day, then diluted with toluene and concentrated under reduce pressure. The crude product was directly purified by column

chromatography on silica gel to give sulfonamides E.

Procedure C : Synthesis of Amides C.

To a solution of 4-(l-hydroxy-l-trifluoromethyl-2,2,2-trifluoroethyl)aniline

(1.5M in THF, 128 μΐ,, 0.193 mmol) under argon in CH₂C1₂ (275 μΐ.) was successively added at room temperature N,N-diisopropylethylamine (37 uL, 0.212 mmol) and the acyl chloride (0.193 mmol). The mixture was stirred for 8 hours and concentrated under reduce pressure. The crude residue was directly purified by column chromatography on silica gel to give amides C. Synthetic Examples— Sulfonamides

SR3335

SR3335 was prepared following procedure A and was purified by

hexane/EtOAc (7/3) to obtain 48 mg (62%) as a white powder. This compound known in literature and commercially available (CAS 2937-53-05-6).

R990

SR990 was prepared following procedure A and was purified by

hexane/EtOAc (7/3) to obtain 56 mg (67%) as a white powder.

FTIR: 3415, 3245, 1597, 1517, 1481, 1470, 1308, 1291, 1231, 1151, 1139, 1115, 972, 949, 924, 827, 681, 773 cm^"1.

1H NMR (400 MHz, (CD₃)₂SO): 3.75 (s, 3H), 7.19 (ddd, J = 8.2, 2.7, 1.0 Hz, 1H) , 7.23 (d, J = 9.0 Hz, 2H), 7.27 (t, J = 2.0 Hz, 1H), 7.38 (ddd, J = 7.8, 1.8, 0.9 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.54 (d, J = 8.6 Hz, 2H), 8.59 (s, 1H), 10.61 (s, 1H).

¹³C NMR (100 MHz, (CD₃)₂SO): 55.5, 1 1 1.47, 118.7, 1 19.0, 119.1 (2C), 125.7, 127.9 (2C), 130.6, 139.4, 140.7, 159.4. (Note: three carbons are missing in the ¹³C data reported for every compound with a 1 -hydroxy- l-trifluoromefhyl-2,2,2- trifluoroethyl group. The missing carbon resonances correspond to the three carbons of the 1 -hydroxy- l-trifluoromethyl-2,2,2-trifluoroethyl group. The fluorine coupling

13 with these carbons gives rise to a septuplet which is difficult to observe in the C spectrum.)

MS (ES-) m/z = 428 (found for Ci₆H₁₃F₆N0₄S-H⁺).

Mp = 125°C.

SR998

SR998 was prepared following procedure A starting from 36.79 μιηοΐ of 4-(l- Hydroxy- l-trifluoromethyl-2,2,2-trifluoroethyl)aniline and was purified by

Hexane/EtOAc (7/3) to obtain 3 mg (15%) as a white powder.

1H NMR (400 MHz, (CD₃)₂SO): 6.61 (d, J = 9.6 Hz, 1H), 7.22 (d, J = 9.0 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 7.57 (d, J = 8.8 Hz, 1H), 7.97 (dd, J = 8.7, 2.2 Hz, 1H), 8.16 (d, J = 9.7 Hz, 1H), 8.28 (d, J = 2.1 Hz, 1H), 8.59 (s, 1H), 10.80 (s, 1H).

MS (ES-) m/z = 466 (found for Ci₈H_uF₆N0₅S-H⁺).

SRI 000 was prepared following procedure A and was purified by

hexane/EtOAc (7/3) to obtain 42 mg (46%) as a yellow powder.

FTIR: 3436, 3243, 1614, 1517, 1402, 1337, 1266, 1242, 1216, 1192, 1153, 1105, 954, 926, 891, 858, 837, 704 cm^"1.

1H NMR (400 MHz, (CD₃)₂SO): 7.23 (d, J = 9.0 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 7.5 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.61 (s, 1H), 10.35 (s, 1H).

¹³C NMR (100 MHz, (CD₃)₂SO): 119.3 (2C), 126.0, 126.3, 126.7, 127.8 (2C), 130.6, 135.8, 138.5, 144.9, 148.9.

MS (ES-) m/z = 474 (found for Ci₅H₈ClF₆N₃0₄S-H⁺).

Mp = 142°C.

SR994 was prepared following procedure A and was purified by

Hexane/EtOAc (7/3) to obtain 52 mg (60%) as a white powder. FTIR: 3447, 3280, 1614, 1515, 1455, 1396, 1327, 1306, 1266, 1225, 1191, 1161, 1152, 1134, 1101, 977, 965, 925, 912, 847, 804, 769, 706 cm^"1.

¹H NMR (400 MHz, (CD₃)₂SO): 7.16 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 9.0 Hz, 2H), 7.63-7.69 (m, 2H), 7.73 (t, J = 7.6 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 8.30 (d, J = 7.3 Hz, 1H), 8.52 (s, 1H), 8.69 (d, J = 8.5 Hz, 1H), 11.05 (s, 1H).

¹³C NMR (100 MHz, (CD₃)₂SO): 117.9 (2C), 124.1, 124.5, 125.1, 127.0, 127.3, 127.8 (2C), 128.2, 129.2, 129.9, 133.8, 134.3, 134.6, 139.3.

MS (ES-) m/z = 448 (found for Ci₉H₁₃F₆N0₃S-H⁺).

Mp = 196°C.

SR992 was prepared following procedure A and was purified by

Hexane/EtOAc (5/5) to obtain 41 mg (47%) as a white powder.

FTIR: 3254 (broad), 1713, 1614, 1516, 1494, 1470, 1265, 1202, 1163, 1142,

1103, 964, 922, 831, 788, 679 cm^"1.

1H NMR (400 MHz, (CD₃)₂SO): 7.19 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 7.71 (dd, J = 8.4, 4.2 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 8.29 (d, J = 8.1 Hz, 1H), 8.43 (dd, J = 7.2, 1.2 Hz, 1H), 8.47 (s, 1H), 8.52 (dd, J = 8.2, 1.6 Hz, 1H), 9.11 (dd, J = 4.2, 1.8 Hz, 1H), 10.50 (s, 1H).

¹³C NMR (100 MHz, (CD₃)₂SO): 118.7 (2C), 122.7, 125.1, 125.7, 127.5 (2C), 128.4, 132.1, 134.4, 135.3, 137.0, 139.6, 142.7, 151.5.

MS (ES-) m/z = 449 (found for Ci₈H₁₂F₆N₂0₃S-H⁺).

Mp = 210°C.

RI 105

SRI 105 was prepared following procedure B starting from 0.6 mmol of 4-(l- Hydroxy-l-trifluoromethyl-2,2,2-trifluoroethyl)aniline and was purified by hexane/EtOAc (5/5) to obtain 240 mg (74%) as a white foam.

FTIR: 3257 (broad), 1706, 1642, 1614, 1537, 1515, 1309, 1265, 1210, 1188, 1147, 1107, 967, 923, 826, 705 cm^"1.

1H NMR (400 MHz, (CD₃)₂SO): 4.61 (d, J = 5.8 Hz, 2H), 7.03 (d, J = 3.8 Hz, 1H), 7.26 (d, J = 9.0 Hz, 2H), 7.44-7.50 (m, 2H), 7.51 (d, J = 3.8 Hz, 1H), 7.53-7.59 (m, 3H), 7.82-7.86 (m, 2H), 8.60 (s, 1H), 9.23 (t, J = 5.9 Hz, 1H), 10.78 (s, 1H).

¹³C NMR (100 MHz, (CD₃)₂SO): 38.1, 118.7 (2C), 125.5, 125.7, 127.2 (2C), 127.9 (2C), 128.4 (2C), 131.5, 132.5, 133.6, 138.2, 139.2, 151.0, 166.35.

MS (ES-) m/z = 537 (found for C₂₁Hi₆F₆N₂0₄S₂-H⁺).

SRI 106 was prepared following procedure B and was purified by

hexane/EtOAc (7/3) to obtain 62 mg (68%) as a white powder.

FTIR: 3462, 6231, 1694, 1516, 1448, 1311, 1261, 1242, 1213, 1175, 1151, 1107, 1092, 1055, 971, 925, 826, 756, 707 cm^"1.

1H NMR (400 MHz, (CD₃)₂SO): 2.30 (s, 3H), 3.81 (s, 3H), 7.26 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 7.68 (s, 1H), 8.65 (s, 1H), 10.08 (s, 1H).

1³C NMR (100 MHz, (CD₃)₂SO): 14.2, 52.8, 119.8 (2C), 126.6, 128.0 (2C),

134.9, 137.1, 138.4, 139.4, 142.4, 160.9.

MS (ES-) m/z = 476 (found for Ci₆H₁₃F₆N0₅S₂-H⁺).

Mp = 130°C. Compounds of the present invention, containing a carbonyl linker ("X") in structural formula I were synthesized as summarized in Scheme 2, below. An aniline of type A, which can be prepared as described in the literature (Farah, B. S.; Gilbert, E. E.; Sibilia, J. P. /. Org. Chem. 1965, 30, 1001) is treated with an acyl chloride, analogously to the conversion described above for Scheme 1, A to C.

Procedure C : Synthesis of Amides C. To a solution of 4-(l-hydroxy-l-trifluoromethyl-2,2,2-trifluoroethyl)aniline (1.5M in THF, 128 μΐ,, 0.193 mmol) under argon in CH₂C1₂ (275 yL) was successively added at room temperature N,N-diisopropylethylamine (37 μί, 0.212 mmol) and the acyl chloride (0.193 mmol). The mixture was stirred for 8 hours and concentrated under reduce pressure. The crude residue was directly purified by column chromatography on silica gel to give amides C.

Scheme 2: Synthesis of Carboxamido Compounds

Synthetic Examples— Carboxamides

SR987 was prepared following procedure C and was purified by hexane/EtOAc (8/2) to obtain 37 mg (48%) as a white powder. This compound is known in literature and commercially available (CAS 303126-97-8).

SR659 was prepared following procedure C and was purified by hexane/EtOAc (8/2) to obtain 52 mg (71%) as a white powder. This compound is known in literature and commercially available (CAS 301234-76-4). Table 1: Exemplary compounds

52

55

ag = agonist, effector; ant = antagonist, repressor.

Compounds of the invention are found to have bioactivity versus at least one ROR as an agonist or as a repressor (inverse agonist or antagonist). Compounds of the invention are found to have selective modulatory activity versus an ROR with respect to an LXR and may or may not be selective over any other nuclear receptor or any other protein target. See Table 2, below, showing inhibitory concentrations as determined by the below-described methods.

Cell culture and transcriptional Assays.

Luciferase reporter assays were conducted using a pBind Gal4-tagged RORoc/γ LBD construct and UAS luciferase reporter cotransfected into HEK293T cells (Kumar N, Solt LA, Conkright JJ, Wang Y, Istrate MA, Busby SA, Garcia- Ordonez RD, Bums TP, Griffin PR. Mol Pharmacol. 2010 Feb;77(2):228-36.).

Reverse transfections were performed in bulk using lxlO⁶ cells in 10 cm plates, 9 μg of total DNA in a 1: 1: 1 ratio of receptor, reporter and empty vector respectively, and FuGene6 (Roche) in a 1:3 DNA: lipid ratio. Following 24 hour bulk transfection, cells were counted and plated in 384 well plates at a density of 10,000 cells/well. The cells were treated with either DMSO or various compounds as indicated four hours after replating. Following additional 20 hour incubation, luciferase levels were assayed by one- step addition of 20μL BriteLite (Perkin Elmer) and read using an Envision multilabel plate reader (Perkin Elmer). Data was normalized to luciferase signal from UAS luciferase reporter/pBind control empty vector and displayed as fold change over UAS luciferase reporter.

RORa modulation of glucose 6-phosphatase wild-type promoter

For the glucose 6-phosphatase promoter, wild type promoter was used to transfect HEK293T cells with SRC-2 as co-activator in the presence or absence of full length RORa ( Chopra AR, Louet JF, Saha P, An J, Demayo F, Xu J, York B, Karpen S, Finegold M, Moore D, Chan L, Newgard CB, O'Malley BW. Science. 2008 Nov 28; 322(5906): 1395). The cells were replated and treated as above followed by luciferase measurements.

Radioligand Receptor Binding Assay.

Ninty nanograms of purified GST- RORa or GST-RORy was incubated with various concentrations of [ H]-25-hydroxycholesterol in assay buffer (50 mM

HEPES, pH 7.4, 0.01% bovine serum albumin, 150 mM NaCl and 5 mM MgCl₂) to determine the ¾ value (Kumar et al., 2010; Wang Y, Kumar N, Solt LA, Richardson TI, Helvering LM, Crumbley C, Garcia- Ordonez RD, Stayrook KR, Zhang X, Novick S, Chalmers MJ, Griffin PR, Burris TP. J Biol Chem. 2010 Feb 12; 285(7):5013-25).

Non-specific binding was defined in the absence of protein as well as excess of cold 25-hydroxycholesterol and were shown to be identical. The assays were terminated by rapid filtration through pre-soaked Whatman GF/B filters (0.5% PEI in PBS) in Multiscreen plates (Millipore) and were washed (3 x 0.1 ml) with ice-cold assay buffer. The radioligand binding results were analyzed using GraphPad Prism software. For the competition assay, various concentration of compounds were incubated with receptor in the presence of 3 nM of [ H] -25-hydroxycholesterol.

Reduction of Endogenous Gene Expression by Small Interference RNAs

To reduce endogenous ROR expression, HepG2 cells were seeded into a 12- well plate (2.5 x 10⁵/well) and transfected the next day with small interference RNAs (siRNAs) against human RORa (#L-003440-00-0005) and RORy (#L-003442-00- 0005; Dharmacon RNA Technologies, Lafayette, CO) at 50 nM following the instructions for Dharma-FECT 1 transfection reagent (Kumar et al., 2010). Forty-two hours post transfection, cells were treated with vehicle (DMSO) or T0901319 (10 μΜ) for 6 hours. Cells were harvested and total RNA was isolated. Quantitative RT- PCR was performed to analyze mRNA levels of human RORa, RORy, GAPDH, and G6Pase using SYBR Green technology. The primers used for quantitative PCR analysis are:

human RORa:

GTAGAAACCGCTGCCAACA (Forward) and ATCACCTCCCGCTGCTT (Reverse); human RORy::

CCCCTGACCGATGTGGACT (Forward) and CAGGATGCTTTGGCGATGA (Reverse);

human G6Pase TCATCTTGGTGTCCGTGATCG (Forward) and

TTTATCAGGGGCACGGAAGTG (Reverse);

GAPDH:

TGCACCACCAACTGCTTAGC (Forward) and GGCATGGACTGTGGTCATGAG (Reverse).

Table 2: Bioactivity of Compounds of the Invention versus RORa, RORy, and LXR

MFC = maximum fold change; IC₅₀ and EC₅₀ concentrations are molar.

Studies with Specific Exemplary Compounds of the Invention

SR1001

Compound SR1001 (compound 26, above; Figure la) was found to be devoid of all LXR activity, yet retained its ability to suppress the activity of RORcc and RORy. We found that SRI 001 repressed both GAL4-RORa and GAL4-RORy transcriptional activity in a dose dependent manner (Fig. lb). In contrast to T1317 (Figure la), SR1001 had no effect on LXRa activity (Fig. lb). Due to the

promiscuous nature of T1317, we assessed the specificity of SR1001 in a panel of all 48 human nuclear receptors in a cell-based cotransfection assay and did not observe activity on receptors other than RORcc or RORy (data not shown). We examined the direct binding of SRI 001 to RORa and RORy using competitive radioligand binding assays. SR1001 dose dependency displaced [ H]25-hydroxycholesterol binding to RORa and RORy (K; = 172 and 111 nM, respectively) (Fig. lc) but demonstrated no binding to ROR .

We assessed whether SRI 001 would affect RORa- and RORy-dependent

12

regulation of an III 7 promoter driven luciferase reporter . HEK 293 cells were transfected with the III 7 reporter and either full-length RORa or RORy and treated with SR1001 or vehicle. As illustrated in Fig. Id, SR1001 dose-dependently suppressed the 1117 promoter driven activity by either of the receptors. Since SRI 001 bound RORa and RORy, resulting in suppression of each receptors' transcriptional activity, we expected that SRI 001 would inhibit coactivator binding to the receptors. In a ligand- induced cofactor protein interaction assay, SRI 001 reduced the interaction of a coactivator TRAP220 NR box 2 peptide with RORy in a dose dependent manner (Fig. le) (IC₅₀ value -117 nM). Collectively, these data demonstrate that SRI 001 is a RORa/RORy ligand that functions as an inverse agonist by inhibiting coactivator which resulted in reduction of the transcriptional activity of each receptor.

Based on our 1117 reporter data, we examined whether SRI 001 affected endogenous 1117a gene expression. The EL4 murine tumor cell line constitutively expresses RORa (Rora), RORyt (Rorc), and IL- 17A (III 7a)¹³. EL4 cells were treated with either control siRNA or a mixture of RORa/y siRNA followed by treatment with either vehicle or SRI 001. Reduction in the expression of RORa (Rora) and RORyt (Rorc) significantly reduced the expression of IL- 17A (1117a) mRNA as measured by quantitative PCR (Fig. 2a). More importantly, treatment of the cells with SRIOOI suppressed III 7a mRNA expression whereas treatment of RORa/γ depleted cells displayed a significantly blunted response indicating that SRIOOI suppression of 1117 a mRNA expression is RORa/RORy dependent (Fig. 2a). Furthermore, SRIOOI suppressed the expression of the RORa and RORy target gene G6Pase in HepG2 cells, a human hepatocellular carcinoma cell line, providing further proof that the effect of SRIOOI is mediated by RORa and RORy¹⁴' ¹⁵.

We hypothesized that SRIOOI would inhibit binding of the coactivator SRC2 to either RORa or RORy when these receptors are occupying the III 7 promoter. We performed a sequential chromatin immunoprecipitation assay (ChlP-reChIP) assessing the relative amount of SRC2 associated with either RORa or RORy resident at the 1117 promoter in EL4 cells. SRIOOI completely blocked the ability of SRC2 to bind to RORa at the III 7 promoter (Fig. 2b, lanes 3 and 4). Similar to RORa, SRIOOI suppressed the ability of SRC2 to bind to RORy at the 1117 (Fig. 2c, lanes 3 and 4). Thus, SRIOOI suppresses 1117a expression by directly inhibiting coactivator binding to RORa and RORy.

We also explored whether SRIOOI would inhibit IL-17 protein production and secretion. Splenocytes were cultured under T_H17 polarizing conditions for 4, 5, and 6 days, and analyzed for IL-17 expression by intracellular flow cytometry. Treatment with SRIOOI inhibited the expression of IL-17 from CD4⁺ T cells at Day 4, 5, and 6. Similar to splenocyte cultures, intracellular flow cytometry demonstrated that SRIOOI significantly repressed IL-17 expression in purified differentiated murine CD4⁺ T cells (CD4⁺CD25^"CD62L^hiCD44^l0). Next we assessed the effect of SRIOOI on IL-17 secretion from splenocyte cultures by ELISA. Treatment with SRIOOI inhibited IL- 17 secretion over a three-day time course, when SRIOOI was added at either the initiation of ¾17 cell differentiation (initiation) or 48 hours post initiation of differentiation (post). SRIOOI was also effective at inhibiting intracellular IL-17 expression in human peripheral blood mononuclear cells (hPBMCs). Finally, we examined the effects of SRIOOI on other T helper cell lineages.

Given that RORa and RORyt are required for development of T_H17-mediated autoimmune diseases⁴' ⁵ and SRIOOI inhibits the activity of both of these receptors leading to suppressed T_H17 cell development in vitro, we evaluated the effects of SRIOOI treatment in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), a well characterized model of T_H17 cell-mediated autoimmune disease 17 ' 18. After myelin oligodendrocyte glycoprotein (MOG₃₅_5s) immunization at day 0, mice were treated with 25 mg kg^"1 of SRIOOI b .i.d. i.p. for the duration of the study. SRIOOI treatment delayed the onset and clinical severity of EAE. Further analysis of spinal cords from mice harvested at day 18 post- immunization revealed that SRIOOI repressed 1117 a mRNA expression by -60%, as well as reduced 1121, and 1122 mRNA expression. Intracellular cytokine analysis of splenocytes indicated significant reduction in IL-17 expression and reduced total CD4⁺ T cells with no effect on CD8⁺ T cells. mRNA expression of IL-14 and IFNy was unaffected in both spleen and spinal cords. These data are consistent with our interpretation that SRIOOI suppresses T_H17 cell function in vivo. Further

optimization of SRIOOI may yield compounds with greater activity.

While RORa and RORyt expression and activity are essential for full T_H17 cell development, it is important to note that RORa and RORy have roles outside of the immune system and are critical regulators of hepatic metabolism. Administration of SRIOOI to C57BL/6 mice, suppressed the expression of hepatic ROR target genes, Cyp7bl, Rev-erba, and Serpine 1 (Pai-1) ^19~21. One could hypothesize that the metabolic effects of SRIOOI would be positive and result in a phenotype similar to RORa/γ^"7" mice which present with reduced blood glucose levels and are resistant to weight gain when fed a high fat diet.

In summary, we describe a novel, first-in-class, highly selective drug targeting the orphan NRs RORa and RORy that effectively suppresses T_H17 cell differentiation and cytokine expression and reduces the severity of disease in an animal model of multiple sclerosis. Our data indicates that the targeting of this cell type, which mediates the pathology of many autoimmune disorders, by blocking RORoc/γ function with a synthetic ligand, is a tractable approach for potential therapeutic intervention. Current treatments for T_Hl7-mediated autoimmune diseases, including multiple sclerosis, utilize agents that are general immunosuppressant's and thus the side effect profile is significant. Clearly, our data demonstrates that by targeting RORa and RORy one can specifically inhibit T_H17 cells without affecting other T helper cell lineages thereby providing a more focused therapy that will not be a general immunosuppressant. Therefore, SRIOOI and derivatives of this compound may represent a novel class of superior drugs to not only treat T_H17-mediated autoimmune disorders, but ROR-mediated metabolic disorders as well.

Cited documents for SRI 001:

1. McGeachy, M.J. & Cua, D.J. Thl7 cell differentiation: The long and winding road. Immunity 28, 445-453 (2008).

2. Bettelli, E., Korn, T., Oukka, M. & Kuchroo, V.K. Induction and effector functions of T(H)17 cells. Nature 453, 1051-1057 (2008).

3. Littman, D.R. & Rudensky, A.Y. Thl7 and Regulatory T Cells in Mediating and Restraining Inflammation. Cell 140, 845-858.

4. Yang, X.X.O. et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28, 29-39 (2008). 5. Ivanov, II et al. The orphan nuclear receptor ROR gamma t directs the differentiation program of proinflammatory IL-17(+) T helper cells. Cell 126, 1121- 1133 (2006).

6. Ivanov, II, Zhou, L. & Littman, D.R. Transcriptional regulation of Thl7 cell differentiation. Semin. Immunol. 19, 409-417 (2007).

7. Manel, N., Unutmaz, D. & Littman, D.R. The differentiation of human T-H-17 cells requires transforming growth factor-beta and induction of the nuclear receptor ROR gamma t. Nat. Immunol. 9, 641-649 (2008).

8. Kumar, N. et al. The benzenesulfonamide T0901317 is a novel

ROR{ alpha }/{ gamma} Inverse Agonist. Molecular Pharmacology 77, 228-236 (2010).

9. Mitro, N., Vargas, L., Romeo, R., Koder, A. & Saez, E. T0901317 is a potent PXR ligand: Implications for the biology ascribed to LXR. FEBS Letters 581, 1721- 1726 (2007).

10. Houck, K.A. et al. T0901317 is a dual LXR/FXR agonist. Mol Genet Metab 83, 184-187 (2004).

11. Schultz, J.R. et al. Role of LXRs in control of lipogenesis. Genes &

Development 14, 2831-2838 (2000).

12. Zhang, F.P., Meng, G.X. & Strober, W. Interactions among the transcription factors Runxl, ROR gamma t and Foxp3 regulate the differentiation of interleukin 17- producing T cells. Nat. Immunol. 9, 1297-1306 (2008). 13. Ichiyama, K. et al. Foxp3 inhibits ROR gamma t-mediated IL-17A mRNA transcription through direct interaction with ROR gamma t. Journal of Biological Chemistry 283, 17003-17008 (2008).

14. Wang, Y. et al. Modulation of retinoic acid receptor-related orphan receptor alpha and gamma activity by 7-oxygenated sterol ligands. J Biol Chem 285, 5013-

5025.

15. Chopra, A.R. et al. Absence of the SRC-2 coactivator results in a

glycogenopathy resembling Von Gierke's disease. Science 322, 1395-1399 (2008).

16. Jin, L.H. et al. Structural Basis for Hydroxycholesterols as Natural Ligands of Orphan Nuclear Receptor ROR gamma. Molecular Endocrinology 24, 923-929

(2010).

17. Xu, J., Wagoner, G., Douglas, J.C. & Drew, P.D. Liver X receptor agonist regulation of Thl7 lympocyte function in autoimmunity. J Leukoc Biol 86, 401-409

(2009).

18. Xu, J.H., Racke, M.K. & Drew, P.D. Peroxisome proliferator- activated receptor- alpha agonist fenofibrate regulates IL-12 family cytokine expression in the CNS: relevance to multiple sclerosis. Journal of Neurochemistry 103, 1801-1810 (2007).

19. Delerive, P., Chin, W.W. & Suen, C.S. Identification of Reverb(alpha) as a novel ROR(alpha) target gene. J Biol Chem 277, 35013-35018 (2002).

20. Wada, T. et al. Identification of oxysterol 7alpha-hydroxylase (Cyp7bl) as a novel retinoid-related orphan receptor alpha (RORalpha) (NR1F1) target gene and a functional cross-talk between RORalpha and liver X receptor (NR1H3). Mol Pharmacol 73, 891-899 (2008).

21. Wang, J., Yin, L. & Lazar, M.A. The orphan nuclear receptor Rev-erb alpha regulates circadian expression of plasminogen activator inhibitor type 1. J Biol Chem 281, 33842-33848 (2006).

Methods for SR1001

A solution of 2-(4-aminophenyl)- 1,1, 1,3,3, 3-hexafluoropropan-2-ol (0.88 g, 3.4 mmol), 2-acetamido-4-methylthiazole-5-sulfonyl chloride (0.79 g, 3.1 mmol) in acetone (15 mL) and 2,6-lutidine (0.73 mL, 6.2 mmol) was warmed to 60°C for 18 h. The reaction was judged complete by analytical HPLC (starting materials consumed). The solvent was removed in vacuo, and the crude residue was diluted with EtOAc and aq 1M HC1. The layers were separated, and the organic layer was washed with 1M HC1, sat aq NaHC0₃, brine, dried (MgS0₄), and concentrated to give a solid.

Trituration with warm Et₂0/hexanes afforded N-(5-(N-(4-(l,l,l,3,3,3-hexafluoro-2- hydroxypropan-2-yl)phenyl)sulfamoyl)-4-methylthiazol-2-yl)acetamide (1.2 g, 86% yield) as a light tan solid, >95% pure as judged by analytical HPLC. A small amount of this was further purified by reverse-phase preparative HPLC to >99% purity to give a colorless solid. 1H NMR (DMSO-J₆, 400MHz) δ 12.5 (s, 1H); 10.8 (s, 1H); 8.6 (s, 1H); 7.60 (d, 2H); 7.25 (d, 2H); 2.30 (s, 3H); 2.15 (s, 3H); ¹³C NMR (DMSO-J₆, 100MHz) δ 170.0, 159.6, 153.0, 139.4, 128.4, 126.8, 124.8, 121.9, 121.7, 120.3, 22.8, 16.3; ¹⁹F NMR (DMSO-J₆, 376MHz) δ -74.1; HRMS (ESI-orbitrap) Calcd for C₁₅Hi₄F₆N₃0₄S₂, 478.0330; Found, 478.0319.

Mice.

All mice were maintained in specific pathogen free environment in accordance with institutional protocol. C57BL/6J mice purchased from Jackson laboratories (Bar Harbor, ME) were used for all in vitro experiments unless otherwise noted. EAE was induced in 8 week-old male wild-type C57BL/6J mice purchased from Harlan (Indianapolis, IN). Male DIO mice, 22 weeks of age, were purchased from Jackson Laboratories and fed a high fat diet (HFD) (60%kCal % fat) (Research Diets) for the duration of the study.

Induction and clinical evaluation of EAE.

EAE was induced in C57BL/6 wild-type mice by s.c. injection over four sites in the flank with 200 μg per mouse MOG 5 55 peptide (C S Bio Co., Menlo Park, CA, USA) in an emulsion with IFA supplemented with 2.25 mg ml^"1 Mycobacterium tuberculosis, strain H37Ra (Difco, Detroit, MI, USA). Pertussis toxin (List Biological Laboratories, Campbell, CA, USA) dissolved in PBS was injected i.p. at 200 ng pre mouse at the time of immunization (Day 0) and 48 h later. Mice were scored daily on a scale of 0-6, as described previously ^l: 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind-limb weakness; 3, severe hind-limb weakness; 4, complete hind-limb paralysis; 5, quadriplegia or pre-moribund state; 6, death. All mice were 7-10 weeks of age when experiments were performed. The SRI 001 was dissolved in DMSO at 25 mg ml^"1 and the mice were treated (i.p.) with 25 mg kg^"1 SR1001 (1 μΐ g^"1 body weight of mouse) or vehicle (DMSO, 1 μΐ g^"1 body weight of mouse) twice per day. The treatment was started 2 days before immunization and continued until the end of experiment. Where indicated in the figure legends, mice were anesthetized with halothane and transcardially perfused with PBS, and spinal cords were removed for RNA and protein isolation.

Cell lines.

HEK293 cells and EL4 cells (American Type Culture Collection) were maintained in Dulbecco' s modified Eagle's medium supplemented with 10% FBS and antibiotics (penicillin and streptomycin; Invitrogen). HepG2 cells were maintained in minimal essential medium supplemented with 10% FBS and antibiotics. PBMCs were obtained from Astarte Biologies and maintained in RPMI-1640 with 10% FBS and antibiotics.

T-cell differentiation in vitro.

The conditions for the different T_H cell subsets were: 20ug ml^"1 anti-IL-4 (clone 30340, R&D Systems) and 20ug ml^"1 anti-IFNy (clone H2, R&D Systems) for THO (neutral conditions); 20ug ml^"1 anti-IL-4, 20ng ml^"1 IL-12 (R&D Systems), and lOng ml^"1 IFNy (R&D Systems) for T_H1 conditions; 20ug ml^"1 anti-IFNy and lOng ml^{" 1} IL-4 (R&D systems) for T_H2 conditions; ΙΟμξ ml^"1 anti-IFNy, ΙΟμξ ml^"1 anti-IL-4, and 2ng ml^"1 TGF (R&D Systems) for iT_reg conditions; 20ug ml^"1 anti-IFNy, 20ug ml^{" 1} anti-IL-4, lng ml^"1 TGFp, and lOng ml^"1 IL-6 (R&D Systems) for T_H17 conditions. All cultures were stimulated with ^g ml^"1 anti-CD3 (eBiosciences) and ^g ml^"1 anti- CD28 (eBiosciences). For naive T cell differentiation, CD4⁺CD25^"CD62L^hiCD44^l0 cells were FACS sorted on a BD FACSAriall. Naive CD4⁺ T cells were activated with 5μg ml^"1 plate bound anti-CD3 and ^g ml^"1 anti-CD28 in the presence of 20ug ml^"1 anti-IFNy, 20ug ml^"1 anti-IL-4, lng ml^"1 TGFp, and lOng ml^"1 IL-6, similar to splenocyte activation. Four to five days after activation, all cells were restimulated with 5ng ml^"1 phorbol- 12-myristate- 13-acetate (PMA) (Sigma) and 500ng ml^"1 ionomycin (Sigma) for 2 hours with the addition of GolgiStop (BD Bioscience) for an additional 2 hours before intracellular staining. Similar restimulation with PMA and ionomycin occurred for ELISA. For hPBMC analysis, cells were restimulated with 5ng ml^"1 phorbol-12-myristate- 13-acetate (PMA) (Sigma) and 500ng ml^"1 ionomycin (Sigma) for 2 hours with the addition of GolgiStop (BD Bioscience) for an additional 2 hours before intracellular staining. Cells were cultured in RPMI 1640 medium (Invitrogen) with 10% FBS and antibiotics.

Reporter constructs.

Gal4-RORaLBD and Gal4-RORyLBD were gifts from Phenex

Pharmaceuticals AG (Ludwigshafen, Germany). The IL-17 reporter construct was purchased from ATCC and previously described .

Reporter gene assays.

4 h prior to transfection, HEK293 cells were plated in 96-well plates at a density of 15 x 10 3 cells/well. Transfections were performed using Lipofectamine XM 2000 (Invitrogen). 24 h post-transfection, the cells were treated with vehicle or compound. 24 h post-treatment, the luciferase activity was measured using the Dual- Glo™ luciferase assay system (Promega). Results were analyzed using GraphPad Prism software.

Radioligand binding assay.

Radioligand binding assays were performed as previously described . For the competition assay, various concentrations of SRI 001 were incubated with receptor in the presence of 3 nM [ H]-25-hydroxycholesterol. Results were analyzed using GraphPad Prism software and the Kj was determined using the Cheng-Prusoff equation.

Alpha Screen.

The ALPHA screen assays were performed as previously described ⁴. Assays were performed in triplicate in white opaque 384-well plates (Greiner Bio-One) under green light conditions (<100 lux) at room temperature. The final assay volume was 20 μL·. All dilutions were made in assay buffer (100 mM NaCl, 25 mM Hepes, 0.1% BSA, pH 7.4). The final DMSO concentration was 0.25%. A mix of 12 μΐ. of GST-RORy-LBD (10 nM), beads (12.5 μg ml^"1 of each donor and acceptor), and 4 μΐ. of increasing concentrations (210 nM - 50 μΜ) of compound SR-1001 were added to the wells, the plates were sealed and incubated for lh. After this preincubation step, 4 μΐ_^ of Biotin-TRAP220-2 peptide (50 nM) was added, the plates were sealed and further incubated for 2h. The plates were read on PerkinElmer Envision 2104 and data analyzed using GraphPad Prism software (La Jolla, CA).

RNA-mediated interference. EL4 cells were first electroporated with ΙΟΟηΜ total siRNA with the

GenePulserXcell Electroporator using siRNA against mouse RORa and RORy (Dharmacon RNA Technologies) followed by reverse using 50nM siRNA according to the instructions for Dharma-FECT 1 transfection reagent and seeded onto a 12 well plate. 24 hours post transfection, cells were treated with vehicle (DMSO) or SRI 001 (ΙΟμΜ) for 24 hours. Cells were harvested and total RNA was isolated. Quantitative reverse transcriptase PCR was performed to analyze mRNA levels of mouse RORa, RORyt, Gapdh, and III 7a using S YBR green technology. Primers sequences to mouse RORa, RORyt, III 7a, and Gapdh and have previously been described⁵'⁶'⁷. HepG2 cells were treated similarly to EL4 cells with the following exceptions: HepG2 cells were transfected with siRNA against human RORa and RORy (Dharmacon RNA Technologies) at 50nM according to the instructions for Dharma-FECT 1 transfection reagent. Quantitative reverse transcriptase PCR was performed to analyze mRNA levels of human RORa, RORA, RORy, RORC, CYCLOPHILIN, and G6Pase using SYBR green technology. The primer sequences have previously been described⁴. Quantitative RT-PCR.

Splenocyte total RNA was extracted using a RNeasy Plus Micro Kit (Qiagen) and reverse transcribed using iScript cDNA biosynthesis kit (Bio-Rad). Total RNA from spinal cord from MOG-immunized mice and livers from DIO mice was isolated using TRIzol reagent (Invitrogen) followed by DNasel treatment (In vitro gen) and reverse transcribed using iScript cDNA biosynthesis kit. Quantitative RT-PCR was performed with a 7900HT Fast Real Time PCR System (Applied Biosystems) using SYBR Green (Roche) as previously described . Primer sequences for mouse III 7a and RORyt, RORa, 1117 f, 1121, 1122, T-bet, Gata3, and Foxp3 have been previously described⁵'⁷'⁹'¹⁰. The level of mRNA expression was normalized to that of GAPDH mRNA expression. The following are the primer sequences for Cyp7bl:

Forward 5' - GGGAAGAAGCTGAAGACTTACG - 3';

Reverse, 5' - CCCTATAGGCTTCCTGTCGAT - 3' ;

Rev-erba:

Forward 5 ' - ACCTTTGAGGTGCTGATGGT - 3 ' :

Reverse, 5' - CTCGCTGAAGTCAAACATGG - 3';

Serpine 1:

Forward 5' - CTCGCTGAAGTCAAACATGG - 3';

Reverse, 5' - TTTTGCAGTGCCTGTGCTAC - 3' . Western Analysis.

HepG2 and EL4 cells were washed once with phosphate-buffered saline and then incubated for 10 min at 4 °C in 100 μΐ of TNT lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 1% Triton X-100) and a complete miniprotease inhibitor mixture (Roche Applied Science). Samples were then harvested into 1.5-ml microcentrifuge tubes, vortexed for 30 s, and then centrifuged (425 ⁰— g for 10 min). Protein levels in the supernatants were determined using a Coomassie protein assay kit (Bio-Rad), and 10 μg of protein from each sample was separated by SDSPAGE (BioRad - 10%) and then transferred to a polyvinylidene difluoride membrane (Millipore, Milford, MA) and immunoblotted with primary antibodies: mouse RORa (BioLegend), mouse RORy (BioLegend), human RORa (Perseus Proteomics), human RORy (IMGENEX), or a- tubulin (Sigma) and horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch). Detection of the bound antibody by enhanced chemiluminescence was performed according to the manufacturer's instructions (Santa Cruz).

Hydro en/deuterium exchange mass spectrometry.

Differential, solution phase HDX experiments were performed with a LEAP Technologies Twin HTS PAL liquid handling robot interfaced with an Orbitrap mass spectrometer (Exactive, ThermoFisher Scientific)¹¹. Each exchange reaction was initiated by incubating 4 μL· of 10μΜ protein complex (with or without SRI 001 and SRC2 RID) with 20 μΐ_^ of D₂0 protein buffer for a predetermined time (Is, 30s, 60s, 900s, and 3600s) at 25°C. The exchange reaction was quenched by mixing with 50 μΐ_^ of 3 M Urea, 0.1% TFA at 1 °C. The mixture was passed across at an in-house packed pepsin column (2mm x 2cm) at 50 μΐ min^"1 and digested peptides were captured onto a 2mm x 1cm Cg trap column (Agilent) and desalted (total time for digestion and desalting was 2.5 min). Peptides were then separated across a 2.1mm x 5cm Cn column (1.9μ Hypersil Gold, Thermo Scientific) with linear gradient of 4%-40% CH₃CN, 0.3 % formic acid, over 5 min. Protein digestion and peptide separation were performed within a thermal chamber (Mecour) held at 2°C to reduce D/H back exchange. Mass spectrometric analyses were carried out with capillary temperature at 225 °C and data were acquired with a measured resolving power of 65,000 at m/z 400. Three replicates were performed for each on-exchange time point.

Peptide Identification and HDX data processing. MS/MS experiments were performed with a linear ion trap mass spectrometer (LTQ, Thermo Fisher). Product ion spectra were acquired in a data-dependent mode and the five most abundant ions were selected for the product ion analysis. The MS/MS *.raw data files were converted to *.mgf files and then submitted to Mascot (Matrix Science, London, UK) for peptide identification. Peptides included in the peptide set used for HDX had a MASCOT score of 20 or greater. The MS/MS MASCOT search was also performed against a decoy (reverse) sequence and ambiguous identifications were ruled out. The MS/MS spectra of all of the peptide ions from the MASCOT search were further manually inspected and only those verifiable are used in the coverage. The intensity weighted average m/z value

(centroid) of each peptide isotopic envelopes were calculated with a new version of our in-house developed software; HD Desktop 12. The deuterium level was calculated as described previously¹³. Deuterium level (%)= { [m(P)-m(N)]/[m(F)-m(N)] }xl00%, where m(P), m(N) and m(F) are the centroid value of partly deuterated peptide, non- deuterated peptide and fully deuterated peptide, respectively. The corrections for back-exchange were made based on an estimated 70% deuterium recovery and accounting for the known 80% deuterium content of the on-exchange buffer.

ChJP/ReChJP.

EL4 cells were treated with plate bound anti-CD3 (5μg ml^"1) and soluble anti- CD28 (^g ml^"1) for 24 h and then treated with vehicle (DMSO) or SRIOOI (10 μΜ) for another 24 h. Re-ChIP assays were performed by using the kit from Active Motif Inc. (Carlsbad, CA). Anti-RORa (BioLegend) or anti-RORy (BioLegend) anti-body was used to do the first immunoprecipitation for all of the samples. The second immunoprecipitation was performed by using anti-rabbit IgG (Jackson

Immunoresearch) for RORa, anti-hamster IgG (Jackson Immunoresearch) for RORy, anti-RNA Pol II (Millipore), anti-SRC2 (Bethyl Laboratories, Montgomery, TX), or anti-NCoR (Santa Cruz). The IL-17 primers used in PCR have been previously described¹⁴.

Flow cytometry and antibodies.

For the analysis of all T cell subsets, single cell suspensions prepared from spleen were stained with fluorophore-conjugated monoclonal antibodies: FITC anti- CD4 (GK1.5, eBioscience) and Alexafluor 647 anti-Foxp3 (FJK-16s, eBioscience) along with the Foxp3 staining buffer set (eBioscience). For intracellular cytokine staining, phycoerythrin-conjugated anti-mouse IL-17A (eBiol7B7, eBioscience), phycoerythrin-conjugated anti-mouse IL-4 (11B11, eBioscience), and Alexafluor 647 anti-mouse IFNy (XMG1.2, eBioscience), in conjunction with the BD Bioscience Intracellular Cytokine staining kit were used. For the intracellular cytokine analysis of human PBMCs, FITC anti-CD4 (RPA-T4, eBioscience) and phycoerythrin- conjugated anti-human IL-17A (eBio64DEC17, eBioscience), were used. For the sorting of naive CD4⁺ T cells, CD4⁺CD25^~CD62L^hiCD44^l0 cells were gated on using the following fluorophore-conjugated antibodies: FITC anti-CD4 (GK1.5, eBioscience), phycoerythrin-conjugated anti-CD25 (PC61.5, eBioscience), APC- conjugated anti-CD62L (MEL- 14, eBioscience), and Alexa Fluor 700 anti-CD44 (IM7, eBioscience). Flow cytometric analysis was performed on a BD LSRII (BD Biosciences) instrument and analyzed using FlowJo software (TreeStar).

ELISA.

Concentration of IL-17 in the culture supernatant was determined by an ELISA kit according to the manufacturers protocol (R & D Systems).

Statistical analysis.

All data are expressed as the mean + s.e.m. (n=3 or more). Statistical analysis was performed using unpaired one-tailed Student's i-test.

Documents cited for SRI 001 methods

¹ Xu, J. H., Racke, M. K. & Drew, P. D. Peroxisome proliferator-activated receptor- alpha agonist fenofibrate regulates IL-12 family cytokine expression in the CNS: relevance to multiple sclerosis. Journal of Neurochemistry 103, 1801-1810, doi: 10.1111/j.l471-4159.2007.04875.x (2007).

Zhang, F. P., Meng, G. X. & Strober, W. Interactions among the transcription factors Runxl, ROR gamma t and Foxp3 regulate the differentiation of interleukin 17-producing T cells. Nat. Immunol. 9, 1297-1306,

doi: 10.1038/ni. l663 (2008).

Wang, Y. et al. Modulation of RORalpha and RORgamma activity by 7- oxygenated sterol ligands. Journal of Biological Chemistry 285, 5013-5025, doi: 10.1074/jbc.M109.080614 (2010).

4 Kumar, N. et al. The benzenesulfonamide T0901317 is a novel

ROR{ alpha }/{ gamma} Inverse Agonist. Molecular Pharmacology 77, 228- 236 (2010). Ivanov, II et al. The orphan nuclear receptor ROR gamma t directs the differentiation program of proinflammatory IL-17(+) T helper cells. Cell 126, 1121-1133, doi: 10.1016/j.cell.2006.07.035 (2006).

Ivanov, II, Zhou, L. & Littman, D. R. Transcriptional regulation of Thl7 cell differentiation. Semin. Immunol. 19, 409-417,

doi: 10.1016/j.smim.2007.10.011 (2007).

Okamoto, K. et al. I kappa B zeta regulates T(H)17 development by cooperating with ROR nuclear receptors. Nature 464, 1381-U1313, doi: 10.1038/nature08922.

Raghuram, S. et al. Identification of heme as the ligand for the orphan nuclear receptors REV-ERB [alpha] and REV-ERB[beta]. Nat Struct Mol Biol 14, 1207-1213 (2007).

Lighvani, A. A. et al. T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells. Proceedings of the National Academy of Sciences of the United States of America 98, 15137-15142 (2001).

Tamauchi, H. et al. Evidence of GATA-3-dependent T(h)2 commitment during the in vivo immune response. International Immunology 16, 179-187, doi: 10.1093/intimm/dxh026 (2004).

Chalmers, M. J. et al. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Analytical Chemistry 78, 1005-1014 (2006).

Pascal, B. D., Chalmers, M. J., Busby, S. A. & Griffin, P. R. HD Desktop: An Integrated Platform for the Analysis and Visualization of H/D Exchange Data. Journal of the American Society for Mass Spectrometry 20, 601-610, doi: 10.1016/j.jasms.2008.11.019 (2009).

Zhang, Z. Q. & Smith, D. L. DETERMINATION OF AMIDE HYDROGEN- EXCHANGE BY MASS-SPECTROMETRY - A NEW TOOL FOR

PROTEIN-STRUCTURE ELUCIDATION. Protein Science 2, 522-531 (1993).

Yang, X. X. O. et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28, 29-39, doi: 10.1016/j.immuni.2007.11.016 (2008). SR1078

Using the T1317 (Fig. la) as an initial scaffold, we synthesized an array of compounds and assessed their activity at RORa, RORy, FXR, LXRa, and LXR . One compound of particular interest was the amide SR1078 (Fig. 3A) that displayed a unique pharmacological profile indicating a high potential to be used as a chemical probe for assessment of ROR function. The synthetic scheme for SR1078 is shown in Fig. 3B (15). This compounds was initially identified based on its ability to inhibit the constitutive activity of RORoc/γ. In a biochemical coactivator interaction assay using Alpha screen technology, increasing doses of SR1078 resulted in a

concentration dependent reduction in the ability of RORy to recruit the TRAP220 coactivator LXXLL NR box (Fig. 3B). In a cell-based chimeric receptor Gal4 DNA- binding domain - NR ligand binding domain cotransfection assay, SR1078 significantly inhibited the constitutive transactivation activity of RORa and RORy, but had no effect on the activity of FXR, LXRa and LXR (Fig. 3C). These data clearly demonstrate that we developed a compound that selectively targeted RORa and RORy and no longer functioned as a LXR/FXR agonist.

In order to examine the activity of SRI 078 in more detail, we performed additional cotransfection assays where we transfected cells with full-length RORa or RORy and luciferase reporter genes driven by promoters derived from known ROR target genes. Two distinct reporter constructs were utilized: one driven by the glucose-6-phosphatase (G6Pase) promoter and one driven by the fibroblast growth factor-21 (FGF21) promoter. Both of these genes have been shown to be direct target genes of ROR and have characterized ROR response elements within their promoters (5, 16, 17). Unexpectedly, we noted that SRI 078 functioned as a ROR agonist, not inverse agonist, when used in the context of the full-length receptors. As shown in

Fig. 4A, in a RORa cotransfection assay, treatment of cells with SRI 078 resulted in a significant increase in transcription. Similarly, in the RORy cotransfection assay, SRI 078 treatment resulted in a stimulation of RORy-dependent transcription activity (Fig. 4B). In both cases, these effects were clearly mediated by ROR since the effect was lost when the RORE was mutated in the G6Pase promoter. Consistent with the G6Pase data, when the FGF21 promoter was used in the cotransfection assay, SRI 078 behaved as a RORo/y agonist stimulating ROR activity (Fig. 4C). With both RORcc and RORy using either promoter, we noted that the effects of SR1078 were dose-dependent as shown in Figs 5A-5D. Transcription was stimulated at concentrations of 2 to 5 μΜ and above. The lack of correlation between recruitment of a cofactor peptide and agonist vs. antagonist activity in cell-based assays has been observed previously with REV-ERBcc where the natural ligand heme causes displacement of the cofactor peptide but the ligand acts like an agonist in cells (18, 19).

In order to confirm that SR1078 is indeed an agonist in a more "physiological" context, we tested its activity in an assay that detects its effect on the expression of actual target genes in a target cell line expressing endogenous levels of RORcc and RORy. HepG2 cells were treated with SRI 078 for 24h followed by assessment of G6Pase and FGF21 gene expression. As shown in Fig. 6A, SR1078 treatment resulted in a significant 3-fold increase in FGF21 mRNA expression. G6Pase mRNA expression was also significantly stimulated ~2-fold by SR1078 treatment (Fig. 6B). These data support the results from the cotransfection data indicating that SRI 078 functions as a ROR agonist unlike its precursor T1317 which functions as an inverse agonist is these same assays.

We examined the pharmacokinetic properties of SRI 078 in mice and noted significant in vivo exposure. Plasma concentrations reached 3.6 μΜ lh after a 10 mg/kg i.p. injection of SR1078 and sustained levels of above 800 nM even 8h after the single injection (Fig. 6C). These levels were sufficient to perform a proof-of- principle experiment to determine if SRI 078 treatment would stimulate ROR target gene expression in an animal model. Mice were treated with SR1078 (10 mg/kg i.p.) and 2h after the injection the livers were harvested and mRNA purified for assessment of G6Pase and FGF21 gene expression. As shown in Fig. 6D and 6E the expression of both FGF21 and G6Pase was significantly stimulated by SR1078 treatment vs. vehicle control.

In summary, we report the identification of a synthetic ligand for RORcc and RORy that functions as an agonist in the context of the full-length receptors. Thus, SR1078 represents the first synthetic ligand that is able to function as an ROR agonist. In cotransfection assays, SRI 078 activates the transcription driven by ROR target gene promoters in a RORE-dependent manner. Furthermore, treatment of cells that express RORcc and RORy endogenously with SR1078 results in stimulation of expression of ROR target genes. It is worth noting that this compound activates the receptor beyond the level of its constitutive activity that is normally observed. The fact that the initial lead compound, T1317, functions as a RORoc/γ inverse agonist and SR1078 functions as a RORoc/γ agonist indicates that it is possible to develop synthetic ligands that will either suppress the constitutive activity of the receptors or further activate the receptors. Our work leading to the identification of SR1078 also demonstrates that it is possible to develop ROR selective synthetic ligands that lack activity at related receptors such as LXR and FXR. This degree of promiscuity that is displayed by T1317 limits the ability to utilize this particular compound as a chemical tool to probe the function of the RORs. Additionally, we show that SRI 078 displays pharmacokinetic properties that allow it to be used in vivo and as would be expected for a RORoc/γ agonist, administration of this compound to mice results in an increase in expression of ROR target genes in the liver. This proof-of-principle study demonstrates that additional experiments are warranted to examine the

pharmacological profile of this compound in vivo.

Methods for SR1078

Synthesis and Physical Characterization of SRI 078

Under argon, to a solution of 4-(l -Hydroxy- l-trifluoromethyl-2,2,2- trifluoroethyl)aniline (1.5M in THF, 128 μΐ,, 0.193 mmol) in CH2C12 (275 μΐ.) were successively added at room temperature N,N-Diisopropylethylamine (37 uL, 0.212 mmol) and 4-(Trifluoromethyl)benzoyl chloride (30 μί, 0.193 mmol). The mixture was stirred for 8 hours and concentrated under reduce pressure. The crude residue was directly purified by column chromatography on silica gel without any work-up by Hexane/AcOEt (8/2) to obtain 59 mg (71%) of SR1078 as a white powder.

FTIR: 3404, 3214, 1671, 1602, 1529, 1417, 1322, 1272, 1206, 1190, 1176, 1138, 1117, 1065, 1016, 973, 964, 948, 902, 857, 830, 765, 752, 737, 704, 692 cm^"1.

1H NMR (400 MHz, (CD₃)₂SO): 7.68 (d, J = 8.2 Hz, 2H), 7.89-7.96 (m, 4H), 8.16 (d, J = 8.2 Hz, 2H), 8.66 (s, 1H), 10.67 (s, 1H). ^1JC NMR (100 MHz, (CD₃)₂SO): 120.12 (2C), 125.4 (q, J = 4.0 Hz, 2C), 125.8, 127.3 (2C), 128.7 (2C), 131.5 (q, J = 31.9 Hz, 1C), 138.4, 140.4, 164.7.There are four carbons missing for the description of SRI 078. They correspond to the three carbons of the (1 -Hydroxy- l-trifluoromethyl-2,2,2-trifluoroethyl) moiety and the (trifluoromethyl)benzene. The fluorine coupling with these carbons give multiplets

13

that are difficult to see on the C spectrum even with a prolonged number of scans.

MS (ES-) m/z = 430 (found for C₁₇H₁₀F₉NO₂-H⁺).

Mp = 169°C.

Cell Culture and Cotransfections

HEK293 cells were maintained in Dulbecco's modified Eagle's medium

(DMEM) supplemented with 10% (v/v) fetal bovine serum at 37 °C under 5% C02. HepG2 cells were maintained and routinely propagated in minimum essential medium supplemented with 10% (v/v) fetal bovine serum at 37 °C under 5% C02. In experiments where lipids and sterols were depleted, cells were maintained on charcoal treated serum (10% (v/v) fetal bovine serum) and treated with 7.5 μΜ lovastatin and 100 μΜ mevalonic acid. 24 h prior to transfection, HepG2 or HEK293 cells were plated in 96-well plates at a density of 15 x 10 cells/well. Transfections were performed using LipofectamineTM 2000 (Invitrogen). 16 h post-transfection, the cells were treated with vehicle or compound. 24 h post-treatment, the luciferase activity was measured using the Dual-GloTM luciferase assay system (Promega). The values indicated represent the means + S.E. from four independently transfected wells. The experiments were repeated at least three times. The ROR and reporter constructs have been previously described (5, 12).

cDNA Synthesis and Quantitative PCR

Total RNA extraction and cDNA synthesis as well as the QPCR were performed as previously described (18, 20).

Coactivator Interaction Assay

The ALPHA screen assays were performed as previously described(72, 21- 23). Assays were performed in triplicate in white opaque 384- well plates (Greiner Bio-One) under green light conditions (<100 lux) at room temperature. The final assay volume was 20

. All dilutions were made in assay buffer (100 mM NaCl, 25 mM Hepes, 0.1% (w/v) BSA, pH 7.4). The final DMSO concentration was 0.25% (v/v). A mix of 12 of GST-RORy-LBD (10 nM), beads (12.5 μg/ml of each donor and acceptor), and 4 μL· of increasing concentrations (210 nM - 50 μΜ) of compound SR1078 were added to the wells, the plates were sealed and incubated for lh. After this preincubation step, 4 μL· of Biotin-TRAP220-2 peptide (50 nM) was added, the plates were sealed and further incubated for 2h. The plates were read on PerkinElmer Envision 2104 and data analyzed using GraphPad Prism software (GraphPad software).

Animal Studies

Plasma levels of SR1078 were evaluated in C57BL6 mice (n = 3 per time point) administered by i.p. injection. After 1, 2, 4, and 8h blood was taken. In the 2h time point liver was taken for target gene analysis. Plasma was generated using standard centrifugation techniques, and the plasma and tissues were frozen at -80°C. Plasma and tissues were mixed with acetonitrile (1:5 (v/v) or 1:5 (w/v), respectively), sonicated with a probe tip sonicator, and analyzed for drug levels by liquid

chromatography/tandem mass spectrometry. All the procedures were conducted in the Scripps vivarium, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and were approved by the Scripps Florida Institutional Animal Care and Use Committee.

SRI 078 Documents Cited

1. Jetten, A. M. (2009) Retinoid-related orphan receptors (RORs): critical roles in development,immunity, circadian rhythm, and cellular metabolism, Nucl Recept Signal 7, e003.

2. Solt, L. A., Griffin, P. R., and Burris, T. P. Ligand regulation of retinoic acid receptor-related orphan receptors: implications for development of novel therapeutics, Current Opinion in Lipidology 21, 204-211.

3. Kallen, J., Schlaeppi, J. M., Bitsch, F., Delhon, I., and Fournier, B. (2004) Crystal structure of the human ROR alpha ligand binding domain in complex with cholesterol sulfate at 2.2 angstrom, Journal of Biological Chemistry 279, 14033-14038.

4. Kallen, J. A., Schlaeppi, J. M., Bitsch, F., Geisse, S., Geiser, M., Delhon, I., and Fournier, B. (2002) X-ray structure of the hROR alpha LBD at 1.63 angstrom: Structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of ROR alpha, Structure 10, 1697-1707.

5. Wang, Y., Kumar, N., Solt, L. A., Richardson, T. I., Helvering, L. M., Crumbley, C, Garcia-Ordonez, R. A., Stayrook, K. R., Zhang, X., Novick, S., Chalmers, M. J., Griffin, P. R., and Burris, T. P. (2010) Modulation of RORalpha and RORgamma activity by 7-oxygenated sterol ligands, Journal of Biological Chemistry 285, 5013-5025.

6. Wang, Y., Kumar, N., Crumbley, C, Griffin, P. R., and Burris, T. P. (2010) A second class of nuclear receptors for oxysterols: Regulation of RORalpha and RORgamma activity by 24S-hydroxycholesterol (cerebro sterol), Biochim Biophys Acta 1801, 917-923.

. Jin, L. H., Martynowski, D., Zheng, S. Y., Wada, T., Xie, W., and Li, Y.

Structural Basis for Hydroxycholesterols as Natural Ligands of Orphan Nuclear Receptor ROR gamma, Molecular Endocrinology 24, 923-929.

8. Lau, P., Fitzsimmons, R. L., Raichur, S., Wang, S. C. M., Lechtken, A., and Muscat, G. E. O. (2008) The orphan nuclear receptor, ROR alpha, regulates gene expression that controls lipid metabolism - Staggerer (sg/sg) mice are resistant to diet- induced obesity, Journal of Biological Chemistry 283, 18411- 18421.

9. Yang, X. X. O., Pappu, B. P., Nurieva, R., Akimzhanov, A., Kang, H. S., Chung, Y., Ma, L., Shah, B., Panopoulos, A. D., Schluns, K. S., Watowich, S. S., Tian, Q., Jetten, A. M., and Dong, C. (2008) T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma, Immunity 28, 29-39.

10. Ivanov, II, McKenzie, B. S., Zhou, L., Tadokoro, C. E., Lepelley, A., Lafaille, J. J., Cua, D. J., and Littman, D. R. (2006) The orphan nuclear receptor ROR gamma t directs the differentiation program of proinflammatory IL-17(+) T helper cells, Cell 126, 1121-1133.

11. Meyer, T., Kneissel, M., Mariani, J., and Fournier, B. (2000) In vitro and in vivo evidence for orphan nuclear receptor ROR alpha function in bone metabolism, Proceedings of the National Academy of Sciences of the United States of America 97, 9197-+.

12. Kumar, N., Solt, L. A., Conkright, J. J., Wang, Y., Istrate, M. A., Busby, S. A., Garcia-Ordonez, R., Burris, T. P., and Griffin, P. R. (2010) The benzenesulfonamide T0901317 is a novel ROR{ alpha }/{ gamma} Inverse Agonist, Molecular Pharmacology 77, 228-236.

13. Schultz, J. R., Tu, H., Luk, A., Repa, J. J., Medina, J. C, Li, L. P., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D. J., Lustig, K. D., and Shan, B. (2000) Role of LXRs in control of lipogenesis, Genes &

Development 14, 2831-2838.

14. Houck, K. A., Borchert, K. M., Hepler, C. D., Thomas, J. S., Bramlett, K. S., Michael, L. F., and Burris, T. P. (2004) T0901317 is a dual LXR/FXR agonist, Molecular Genetics and Metabolism 83, 184-187.

15. Farah, B. S., Gilbert, E. E., and Sibilia, J. P. (1965) PERHALO KETONES .V.

REACTION OF PERHALOACETONES WITH AROMATIC HYDROCARBONS, Journal of Organic Chemistry 30, 99S-&.

16. Wang, Y. J., Solt, L. A., and Burris, T. P. (2010) Regulation of FGF21 Expression and Secretion by Retinoic Acid Receptor-related Orphan Receptor alpha, Journal of Biological Chemistry 285, 15668-15673.

17. Chopra, A. R., Louet, J. F., Saha, P., An, J., DeMayo, F., Xu, J. M., York, B., Karpen, S., Finegold, M., Moore, D., Chan, L., Newgard, C. B., and O'Malley, B. W. (2008) Absence of the SRC-2 Coactivator Results in a Glycogenopathy Resembling Von Gierke's Disease, Science 322, 1395-1399.

18. Raghuram, S., Stayrook, K. R., Huang, P., Rogers, P. M., Nosie, A. K., McClure, D. B., Burris, L. L., Khorasanizadeh, S., Burris, T. P., and Rastinejad, F. (2007) Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERBbeta, Nat Struct Mol Biol 14, 1207-1213. 19. Yin, L., Wu, N., Curtin, J. C, Qatanani, M., Szwergold, N. R., Reid, R. A., Waitt, G. M., Parks, D. J., Pearce, K. H., Wisely, G. B., and Lazar, M. A. (2007) Rev-erb{ alpha}, a Heme Sensor That Coordinates Metabolic and Circadian Pathways, Science 318, 1786-1789.

20. Kumar, N., Solt, L. A., Wang, Y., Rogers, P. M., Bhattacharyya, G., Kamenecka, T. M., Stayrook, K. R., Crumbley, C, Floyd, Z. E., Gimble, J. M., Griffin, P. R., and Burris, T. P. (2010) Regulation of Adipogenesis by Natural and Synthetic REV-ERB Ligands, Endocrinology, en.2009-0800.

21. Bettoun, D. J., Burris, T. P., Houck, K. A., Buck, D. W., Stayrook, K. R., Khalifa, B., Lu, J. F., Chin, W. W., and Nagpal, S. (2003) Retinoid X receptor is a nonsilent major contributor to vitamin D receptor-mediated transcriptional activation, Molecular Endocrinology 17, 2320-2328.

22. Houck, K. A., Borchert, K. M., Hepler, C. D., Thomas, J. S., Bramlett, K. S., Michael, L. F., and Burris, T. P. (2004) T0901317 is a dual LXR/FXR agonist, Mol Genet Metab 83, 184-187.

23. Bramlett, K. S., Houck, K. A., Borchert, K. M., Dowless, M. S., Kulanthaivel, P., Zhang, Y., Beyer, T. P., Schmidt, R., Thomas, J. S., Michael, L. F., Barr, R., Montrose, C, Eacho, P. I., Cao, G., and Burris, T. P. (2003) A natural product ligand of the oxysterol receptor, liver X receptor, / Pharmacol Exp Ther 307, 291-296.

SR3335

We recently identified the first synthetic ligand that binds to and regulates the activity of RORa and RORy, T0901317 (T1317) (Fig. 1A) (10). T1317 was originally identified as a liver X receptor agonist (LXR) (11), an NR that serves as a

physiological receptor for oxysterols and plays key roles in regulation of lipogenesis and reverse cholesterol transport (12). Our group demonstrated that T1317 displays a degree of promiscuity and also activated another NR that serves as a receptor for bile acids, FXR (13). Interestingly, T1317 acts as a LXR agonist, but a ROR inverse agonist. We utilized the benzenesulfonamide scaffold as an initiation point for development of the first selective ROR ligand, SRI 078 (see above) that behaves as a dual RORa/γ agonist (14).

Continued evaluation of the T1317 scaffold led to the identification of a RORa selective inverse agonist that is characterized in this study, SR3335 (ML- 176) (Fig. 7A). The synthetic scheme for SR3335 is shown in Fig. 7B. This compound was initially identified based on its ability to inhibit the constitutive activity of RORa in a GAL4-RORa ligand binding domain (LBD) cotransfection assay. In a biochemical radioligand binding assay using [ H]25-hydroxycholesterol as a label (5, 6, 10) it is clear that unlabeled SR3335 dose-dependently competes for binding to the RORa LBD (Fig. 7C). The K; was calculated as 220 nM using the Cheng-Prusoff equation. As shown in Fig. ID, SR3335 did not compete well for binding when the LBD of RORy was utilized. In a cell-based chimeric receptor Gal4 DNA-binding domain - NR ligand binding domain cotransfection assay, SR3335 significantly inhibited the constitutive transactivation activity of RORa (ICso=480 nM)(partial inverse agonist activity), but had no effect on the activity of LXRcc and RORy (Fig. 8). Although T1317 shows considerably more efficacy than SR3335 in terms of suppression of RORa activity, the RORa selectivity of SR3335 is clear. SR3335 also displays no activity on ROR (radioligand binding or cotransfection assays), FXR (cotransfection assays) or any other receptors in a selectivity panel for human nuclear receptors (10) (data not shown). We also observed no effect on the enzymatic activity of kinases (JNK or MAPK). These data clearly demonstrate that we developed a compound that selectively targets RORa.

In order to examine the activity of SR3335 in more detail, we performed additional cotransfection assays where we transfected cells with full-length RORa and a luciferase reporter gene driven by a promoter derived from a known ROR target gene, glucose-6-phosphatase (G6Pase). G6Pase is a well-characterized RORa target gene that plays a critical role in the gluconeogenesis pathway (5, 9, 15). As shown in Fig. 9A, in a RORa cotransfection assay, treatment of cells with SR3335 resulted in a significant suppression of transcription driven by the G6Pase promoter. Consistent with these cotransfection data, treatment of HepG2 cells with SR3335 lead to suppression of expression of both G6Pase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression (Fig. 9B). Given the critical roles that these two enzymes play in regulation of gluconeogenesis, we hypothesized that SR3335 may offer utility in suppression of hepatic glucose output, which is elevated in type 2 diabetics and contributes to the hyperglycemic state. In order to investigate this, we pursued additional studies in mice.

We examined the pharmacokinetic properties of SR3335 in mice and noted significant in vivo exposure. Plasma concentrations reached nearly 9 μΜ 0.5h after a 10 mg/kg i.p. injection of SR3335 and levels were sustained above 360 nM even 4h after the single injection (Fig. 10A). These levels were sufficient to perform a proof- of-principle experiment to determine if SR3335 treatment could suppress

gluconeogenesis in vivo. Diet induced obese mice were treated with SR3335 (15 mg/kg b.i.d., i.p.) for 6-days and a pyruvate tolerance test was performed on day 6 to estimate gluconeogenesis. As shown in Fig. 10B plasma glucose levels were slightly lower in SR3335 treated animals at time 0, but after injection of the pyruvate the SR3335 treated animals displayed significantly lower plasma glucose levels at each time point vs. vehicle treated animals (15, 30, and 60 min) indicating suppression of hepatic gluconeogenesis and an improvement in glucose homeostasis by the RORcc inverse agonist. Expression of hepatic pepck and g6pase expression in the mice revealed -50% decrease in pepck expression (the enzyme that catalyzes the rate limiting step in gluconeogenesis); however, g6pase expression was not significantly affected (Fig. IOC). It is unclear why only pepck expression was suppressed in vivo and not g6pase since both were repressed in cell culture experiments, but the suppression of only pepck in vivo may be responsible for the moderate effects on pyruvate stimulated gluconeogenesis. Clearly SR3335 is effectively targeting RORcc since a well characterized RORcc target gene (nrldl) (16, 17) is also repressed in the livers (Fig. IOC). Importantly, mice treated with SR3335 displayed no difference in body weight or food intake after 7-days of treatment with the compound (data not shown). The effects on glucose homeostasis are thus not secondary to weight loss and represent a metabolic response to the compound.

Several crystal structures of the LBD of both RORcc and RORy bound to sterol ligands have been solved (3, 4, 7). In all of these cases the LBD appears to be in an active conformation with helix 12 positioned in such a manner to allow for coactivator protein recruitment. It is unclear whether binding of a sterol is required for the transcriptional activity of these receptors since we have clearly observed that RORcc and RORy expressed in a sterol free environment retain constitutive ability to recruit coactivators (5, 10). We do observe that SR3335 is able to displace 25- hydroxycholesterol in a radioligand binding assay, thus whether it be by displacing an endogenous agonist or by binding to a receptor that has a basal conformation that is already active, SR3335 appears to limit the receptor's ability to activate transcription. This is most likely due to SR3335 inducing a conformation that reduces the affinity of the LBD for coactivators.

In summary, we report the identification of the first selective synthetic RORcc ligand that functions as an inverse agonist. In cotransfection assays, SR3335 suppresses transcription in both GAL4-RORCC LBD and full-length RORcc contexts. Furthermore, treatment of HepG2 cells with SR3335 results in suppression of RORcc target gene expression. Suppression of the expression of G6Pase and PEPCK mRNA suggested that SR3335 might be able to suppress gluconeogenesis. After determining that SR3335 displayed reasonable pharmacokinetics in mice, we tested this hypothesis and in vivo using a DIO mouse model and showed that SR3335 did indeed suppress gluconeogenesis. These data clearly define SR3335 as a valid chemical tool to evaluate the in vitro and in vivo actions of RORcc and suggest that compounds like SR3335 may hold utility in treatment of type 2 diabetes. Methods

Synthesis of SR3335

To a solution of 2-(4-aminophenyl)- 1,1,1, 3,3, 3-hexafluoropropan-2-ol (18) (1.5M in THF, 2.90 mL, 4.35 mmol) in acetone (4.3 mL) were successively added at room temperature 2,6-lutidine (658 μί, 5.65 mmol) and 2-thiophenesulfonyl chloride (910 mg, 4.78 mmol). The mixture was heated overnight at 60°C, then diluted by ethyl acetate (EtOAc) and quenched at room temperature by the addition of saturated NaHC0₃ solution. The aqueous phase was extracted two times with EtOAc. The combined organic phases were dried over Na₂S0₄, filtrated and concentrated on a rotary evaporator. The residue was purified by silica gel column and eluted with hexane-EtOAc (70/30) to obtain 1.1 g of SR3335 (62%, purity >98%) as a white powder: 1H NMR (400 MHz, (CD₃)₂SO) d 6.64 (dd, J = 5.0, 3.8 Hz, 1H), 6.77 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 7.14 (dd, J = 3.7, 1.4 Hz, 1H), 7.43 (d, J = 5.0, 1.4 Hz, 1H), 8.11 (s, 1H), 10.30 (s, 1H); ¹³C NMR (100 MHz, (CD₃)₂SO) d 119.1 (2C), 125.9, 127.7, 127.9 (2C), 132.7, 133.7, 139.2, 139.9; the three carbon resonances of the hexafluoropropanol unit are not observed in the 13 C spectrum of SR3335. The fluorine coupling with these carbons gives multiplets which were difficult to detect even with increased number of scans; FTIR 3362, 3228, 1614, 1519, 1472, 1404, 1341, 1308, 1280, 1257, 1230, 1186, 1146, 1098, 1067, 1027, 963, 946, 928, 832, 821, 730, 708 cm^"1; MS (ES-) m/z = 404 (found for Ci₃H₉F₆N0₃S₂H⁺). Cell Culture and Cotransfections

HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% C02. HepG2 cells were maintained and routinely propagated in minimum essential medium supplemented with 10% fetal bovine serum at 37 °C under 5% C0₂. 24 h prior to transfection, cells were plated in 96-well plates at a density of 15 x 103 cells/well. Transfections were performed using LipofectamineTM 2000 (Invitrogen). 16 h post- transfection, the cells were treated with vehicle or compound. 24 h post-treatment, the luciferase activity was measured using the Dual-GloTM luciferase assay system (Promega). The values indicated represent the means + S.E. from four independently transfected wells. The experiments were repeated at least three times. The ROR and reporter constructs have been previously described (5, 10).

cDNA Synthesis and Quantitative PCR

Total RNA extraction and cDNA synthesis as well as the QPCR were performed as previously described (19, 20).

Radioligand Binding Assay

The radioligand binding assay for RORcc and RORy using [Ή]25- hydroxycholesterol has been previously described (5, 6, 10).

Pharmacokinetic Studies

Plasma levels of SR3335 were evaluated in C57BL6 mice (n = 3 per time point) administered by i.p. injection. After 0.25, 0.5, 1, 2, 4, and 8h blood was taken. Plasma was generated using standard centrifugation techniques, and the plasma and tissues were frozen at -80°C. Plasma and tissues were mixed with acetonitrile (1 :5 v/v or 1 :5 w/v, respectively), sonicated with a probe tip sonicator, and analyzed for drug levels by liquid chromatography/tandem mass spectrometry. All the procedures were conducted in the Scripps vivarium, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and were approved by the Scripps Florida Institutional Animal Care and Use Committee.

Diet Induced Obesity Model

30 week old Diet induced obese (DIO) C57BL/6 male mice were purchased from Jackson Laboratories that were maintained on a 65% Kcal high-fat diet from weaning.. DIO mice were treated twice per day (07:00h and 18:00h) with 15 mg/kg SR3335 or vehicle for 6 days i.p. Pyruvate tolerance test was conducted on day 6 of the treatment. Food was removed from mice in the morning after SR3335 injection, fasted for 6 hours and the pyruvate tolerance test was conducted at 13:00h. Time 0 blood glucose was measured taken from the tail nip and the pyruvate challenge was initiated by injection of 2g/kg of pyruvate i.p. followed by measuring blood glucose at 15, 30 and 60 min following the injection. Blood glucose was measured by one touch ultra glucose-meter.

Documents cited for SR3335

1. Jetten, A. M. (2009) Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism, Nucl Recept Signal 7, e003.

2. Solt, L. A., Griffin, P. R., and Burris, T. P. Ligand regulation of retinoic acid receptor-related orphan receptors: implications for development of novel therapeutics, Current Opinion in Lipidology 21, 204-211.

3. Kallen, J., Schlaeppi, J. M., Bitsch, F., Delhon, I., and Fournier, B. (2004) Crystal structure of the human ROR alpha ligand binding domain in complex with cholesterol sulfate at 2.2 angstrom, Journal of Biological Chemistry 279, 14033-14038.

4. Kallen, J. A., Schlaeppi, J. M., Bitsch, F., Geisse, S., Geiser, M., Delhon, I., and Fournier, B. (2002) X-ray structure of the hROR alpha LBD at 1.63 angstrom: Structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of ROR alpha, Structure 10, 1697-1707.

5. Wang, Y., Kumar, N., Solt, L. A., Richardson, T. I., Helvering, L. M., Crumbley, C, Garcia-Ordonez, R. A., Stayrook, K. R., Zhang, X., Novick, S., Chalmers, M. J., Griffin, P. R., and Burris, T. P. (2010) Modulation of RORalpha and RORgamma activity by 7-oxygenated sterol ligands, Journal of

Biological Chemistry 285, 5013-5025.

6. Wang, Y., Kumar, N., Crumbley, C, Griffin, P. R., and Burris, T. P. (2010) A second class of nuclear receptors for oxysterols: Regulation of RORalpha and RORgamma activity by 24S-hydroxycholesterol (cerebro sterol), Biochim Biophys Acta 1801, 917-923.

7. Jin, L. H., Martynowski, D., Zheng, S. Y., Wada, T., Xie, W., and Li, Y.

(2010) Structural Basis for Hydroxycholesterols as Natural Ligands of Orphan Nuclear Receptor ROR gamma, Molecular Endocrinology 24, 923-929.

8. Lau, P., Fitzsimmons, R. L., Raichur, S., Wang, S. C. M., Lechtken, A., and Muscat, G. E. O. (2008) The orphan nuclear receptor, ROR alpha, regulates gene expression that controls lipid metabolism - Staggerer (sg/sg) mice are resistant to diet- induced obesity, Journal of Biological Chemistry 283, 18411- 18421.

9. Chopra, A. R., Louet, J. F., Saha, P., An, J., DeMayo, F., Xu, J. M., York, B., Karpen, S., Finegold, M., Moore, D., Chan, L., Newgard, C. B., and O'Malley,

B. W. (2008) Absence of the SRC-2 Coactivator Results in a Glycogenopathy Resembling Von Gierke's Disease, Science 322, 1395-1399.

10. Kumar, N., Solt, L. A., Conkright, J. J., Wang, Y., Istrate, M. A., Busby, S. A., Garcia-Ordonez, R., Burris, T. P., and Griffin, P. R. (2010) The benzenesulfonamide T0901317 is a novel ROR{ alpha }/{ gamma} Inverse

Agonist, Molecular Pharmacology 77, 228-236. 11. Schultz, J. R., Tu, H., Luk, A., Repa, J. J., Medina, J. C, Li, L. P., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D. J., Lustig, K. D., and Shan, B. (2000) Role of LXRs in control of lipogenesis, Genes & Development 14, 2831-2838.

12. Michael, L. F., Schkeryantz, J. M., and Burris, T. P. (2005) The pharmacology of LXR, Mini Rev Med Chem 5, 729-740.

13. Houck, K. A., Borchert, K. M., Hepler, C. D., Thomas, J. S., Bramlett, K. S., Michael, L. F., and Burris, T. P. (2004) T0901317 is a dual LXR/FXR agonist, Molecular Genetics and Metabolism 83, 184-187.

14. Wang, Y., Kumar, N., Nuhant, P., Cameron, M. D., Istrate, M. A., Roush, W.

R., Griffin, P. R., and Burris, T. P. (2010) Identification of SR1078, a Synthetic Agonist for the Orphan Nuclear Receptors RORA and RORG, ACS Chemical Biology, null-null.

15. Wang, Y. J., Solt, L. A., and Burris, T. P. (2010) Regulation of FGF21 Expression and Secretion by Retinoic Acid Receptor-related Orphan Receptor alpha, Journal of Biological Chemistry 285, 15668-15673.

16. Delerive, P., Chin, W. W., and Suen, C. S. (2002) Identification of Reverb alpha as a novel ROR alpha target gene, Journal of Biological Chemistry 277, 35013-35018.

17. Raspe, E., Mautino, G., Duval, C, Fontaine, C, Duez, H., Barbier, O., Monte, D., Fruchart, J., Fruchart, J. C, and Staels, B. (2002) Transcriptional regulation of human Rev-erb alpha gene expression by the orphan nuclear receptor retinoic acid-related orphan receptor alpha, Journal of Biological Chemistry 277, 49275-49281.

18. Farah, B. S., Gilbert, E. E., and Sibilia, J. P. (1965) Perhalo ketones. V.

Reaction of perhaloacetones with aromatic hydrocarbons, Journal of Organic Chemistry 30, 998-1001.

19. Kumar, N., Solt, L. A., Wang, Y., Rogers, P. M., Bhattacharyya, G., Kamenecka, T. M., Stayrook, K. R., Crumbley, C, Floyd, Z. E., Gimble, J. M., Griffin, P. R., and Burris, T. P. (2010) Regulation of Adipogenesis by

Natural and Synthetic REV-ERB Ligands, Endocrinology 151, 3015-3025. 0. Raghuram, S., Stayrook, K. R., Huang, P., Rogers, P. M., Nosie, A. K., McClure, D. B., Burris, L. L., Khorasanizadeh, S., Burris, T. P., and Rastinejad, F. (2007) Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERBbeta, Nat Struct Mol Biol 14,

1207-1213.

Reference will now be made in detail to certain claims of the disclosed subject matter, examples of which are illustrated in the accompanying structures and formulas. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that they are not intended to limit the disclosed subject matter to those claims. On the contrary, the disclosed subject matter is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the presently disclosed subject matter as defined by the claims.

All patents and publications referred to herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

What is claimed is:

1. A method of modulating the bioactivity of an ROR, comprising contacting the ROR with an effective amount of a compound of formula (I), wherein the compound is an agonist or an activator, or is a repressor, inverse agonist, or antagonist, of a receptor comprising any sequence variant of any isoform of the ROR subfamily, including RORa, RORp, or RORy;

wherein the compound of formula (I) comprises

Rⁱ - ½- ^R3

R²

(I)

wherein X is C(O) or S(0)₂;

R¹ is alkyl, aryl, or heteroaryl wherein any group is optionally mono- or independently multi- substituted with J¹;

R is H, alkyl, haloalkyl, aryl, aroyl, heteroaryl, or heteroaroyl, wherein any non-hydrogen group is optionally mono- or independently multi- substituted with J ;

R is aryl or heteroaryl, wherein any group is optionally mono- or

independently multi- substituted with J ;

J¹ when present is halo, cyano, nitro, alkoxy, or haloalkoxy; unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido, or alkoxycarbonyl; unsubstituted or substituted aryl; unsubstituted or substituted arylsulfonyl;

unsubstituted or substituted heteroaryl; unsubstituted or substituted

heteroarylsulfonyl; or unsubstituted or substituted arylsulfonamido;

J when present is halo, cyano, nitro, alkoxy, or haloalkoxy; unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido or alkoxycarbonyl; unsubstituted or substituted aryl; unsubstituted or substituted arylsulfonyl;

unsubstituted or substituted heteroaryl; unsubstituted or substituted

heteroarylsulfonyl; or unsubstituted or substituted arylsulfonamido;

J when present is alkyl, haloalkyl, hydroxyalkyl, or hydroxyhaloalkyl; or is an ester of hydroxyalkyl or hydroxyhaloalkyl; including any stereoisomer thereof, or any salt, solvate, hydrate, metabolite, or prodrug thereof.

2. The method of claim 1 wherein X is C(O).

3. The method of claim 1 wherein X is S(0)₂.

4. The method of claim 1 wherein R¹ is unsubstituted or substituted phenyl, thiophenyl, quinolinyl, naphthyl, coumaryl, biphenyl, benzoxadiazolyl, thiazolyl, aroyloxymethyl, or trifluoromethyl.

5. The method of claim 1 wherein J¹ is fluoro, chloro, bromo, iodo, cyano, nitro, methoxy, methoxycarbonyl, trifluoromethoxy, trifluoromethyl, methyl, t-butyl, n- butyl, substituted pyrimidinyl, isoxazolyl, pyridinyl, phenyl, phenylsulfonyl, substituted pyrazolyl, benzoylamidomethyl, or halobenzoylamidomethyl.

6. The method of claim 1 wherein R 2 substituted with J 2 comprises 2,2,2- trifluoroethyl, benzoyl, toluoyl, or dinitrobenzoyl.

The method of claim 1 wherein R is unsubstituted or substituted phi

The method of claim 1 wherein J is halo or hydroxyhaloalkyl, or an ester

9. The method of claim 1 wherein R 3 substituted with J 3 comprises

or an ester thereof, wherein a wavy line indicates a point of attachment of J 3 -substituted R 3 to the nitrogen atom bearing R 3.

10. The method of claim 9 wherein the ester comprises a substituted or unsubstituted aroyl or heteroaroyl ester of

wherein a wavy line indicates a point of attachment of J 3 -substituted R 3 to the nitrogen atom bearing R 3..

11. The method of claim 10 wherein the aroyl comprises unsubstituted benzoyl or benzoyl substituted with halo, nitro, or alkyl, or any combination thereof.

12. The method of claim 10 wherein the heteroaroyl comprises unsubstituted or substituted picolinoyl, thiophenoyl, furoyl, wherein any heteroaroyl can be substituted with halo, nitro, or alkyl, or any combination thereof.

The method of claim 2 comprising any of the following compounds of formula

alt, solvate, hydrate, metabolite, or prodrug thereof.

The method of claim 3 comprising any of the following compounds of formula

104

or any salt, solvate, hydrate, metabolite, or prodrug thereof.

15. The method of claim 1, wherein the compound of formula (I) is inactive with respect to modulation of a nuclear receptor other than an ROR or with respect to modulation of a G-protein coupled receptor, an ion channel, or an enzyme; or wherein the modulation of the ROR takes place at a concentration ineffective for modulation of a nuclear receptor other than an ROR at the concentration, or ineffective for modulation of a G-protein coupled receptor, an ion channel, or an enzyme at the concentration.

16. The method of claim 15 wherein the nuclear receptor comprises LXRa or LXRp, or both.

17. The method of claim 1 wherein the modulation takes place in vivo in a mammal.

18. The method of claim 1 wherein the bioactivity of a nuclear receptor other than an ROR is substantially unaffected by a concentration of the compound in a tissue effective to modulate an ROR.

19. A method of treating a disorder in a patient for which modulation of an ROR is medically indicated, comprising modulating an ROR receptor according to the method of claim 1 by administering to the patient an effective amount of a compound of formula (I) at a frequency and for a duration of time to provide a beneficial result to the patient.

20. The method of claim 19 wherein the disorder is a metabolic or immune disorder, cancer, or a CNS disorder.

21. The method of claim 20 wherein the metabolic disorder comprises insulin resistance, type 2 diabetes, diabetes, or obesity.

22. The method of claim 20 wherein the immune disorder comprises an auto immune disorder such as Hashimoto's thyroiditis, Pernicious anemia, Addison's disease, Type I diabetes, Rheumatoid arthritis, Systemic lupus erythematosus, Dermatomyositis, Sjogren syndrome, Lupus erythematosus, Multiple sclerosis, Myasthenia gravis, Reactive arthritis, Grave's disease, Crohn's disease, or Lupus.

23. The method of claim 20 wherein the cancer comprises prostate cancer, colon cancer, breast cancer, ovarian cancer, or lung cancer.

24. The method of claim 20 wherein the CNS disorder comprises a sleep disorder, anxiety, or a neurodegenerative disease such as Parkinson's or Alzheimer's.

25. The method of claim 1 further comprising administration of a second medicament.

26. The method of claim 25 wherein the second medicament comprises, for treatment of a metabolic disorder, an anti-diabetic or anti-insulin resistance agent, such as a glitazone, a sulfonylurea, metformin, insulin, an insulin mimetic, a DPP4 inhibitor, a GLPl receptor agonist, a glucagon receptor antagonist, or an anti-obesity agent.

27. The method of claim 25 wherein the second medicament comprises, for treatment of an immune disorder, an anti-TNF agent or an immune-suppresive glucocorticoid.

28. The method of claim 25 wherein the second medicament comprises, for treatment of cancer, an anticancer agent such as a platinum compound, a Vinca alkaloid or analog thereof, a taxane, or a nitrogen mustard.

29. A compound of formula (I) com rising

(I)

wherein X is C(O) or S(0)₂;

R¹ is alkyl, aryl, or heteroaryl wherein any group is optionally mono- or independently multi- substituted with J¹;

R is H, alkyl, haloalkyl, aryl, aroyl, heteroaryl, or heteroaroyl, wherein any non-hydrogen group is optionally mono- or independently multi- substituted with J ; R is aryl or heteroaryl, wherein any group is optionally mono- or

independently multi- substituted with J ;

J¹ when present is halo, cyano, nitro, alkoxy, or haloalkoxy; unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido, or alkoxycarbonyl; unsubstituted or substituted aryl; unsubstituted or substituted arylsulfonyl;

unsubstituted or substituted heteroaryl; unsubstituted or substituted

heteroarylsulfonyl; or unsubstituted or substituted arylsulfonamido;

J when present is halo, cyano, nitro, alkoxy, or haloalkoxy; unsubstituted or substituted alkyl, haloalkyl, alkylcarboxamido, arylcarboxamido or alkoxycarbonyl; unsubstituted or substituted aryl; unsubstituted or substituted arylsulfonyl;

unsubstituted or substituted heteroaryl; unsubstituted or substituted

heteroarylsulfonyl; or unsubstituted or substituted arylsulfonamido;

J when present is alkyl, haloalkyl, hydroxyalkyl, or hydroxyhaloalkyl; or is an ester of hydroxyalkyl or hydroxyhaloalkyl;

including any stereoisomer thereof, or any salt, solvate, hydrate, metabolite, or prodrug thereof;

provided the compound is not any of

30. The compound of claim 29 wherein X is C(O).

31. The compound of claim 29 wherein X is S(0)₂.

32. The compound of claim 29 wherein R¹ is unsubstituted or substituted phenyl, thiophenyl, quinolinyl, naphthyl, coumaryl, biphenyl, benzoxadiazolyl, thiazolyl, aroyloxymethyl, or trifluoromethyl.

33. The compound of claim 29 wherein J is fluoro, chloro, bromo, iodo, cyano, nitro, methoxy, methoxycarbonyl, trifluoromethoxy, trifluoromethyl, methyl, t-butyl, n-butyl, substituted pyrimidinyl, isoxazolyl, pyridinyl, phenyl, phenylsulfonyl, substituted pyrazolyl, benzoylamidomethyl, or halobenzoylamidomethyl.

34. The compound of claim 29 wherein R 2 substituted with J 2 comprises 2,2,2- trifluoroethyl, benzoyl, toluoyl, or dinitrobenzoyl.

35. The compound of claim 29 wherein R is unsubstituted or substituted phenyl.

36. The compound of claim 29 wherein J is halo or hydroxyhaloalkyl, or an ester thereof. ound of claim 29 wherein R 3 substituted with J 3 comprises

or an ester thereof, wherein a wavy line indicates a point of attachment of J 3 -substituted R 3 to the nitrogen atom bearing R 3.

38. The compound of claim 37 wherein the ester comprises a substituted or unsubstituted aroyl or heteroaro

, wherein a wavy line indicates a point of attachment of J 3 -substituted R 3 to the nitrogen atom bearing R 3.

39. The compound of claim 38 wherein the aroyl comprises unsubstituted benzoyl or benzoyl substituted with halo, nitro, or alkyl, or any combination thereof.

40. The compound of claim 38 wherein the heteroaroyl comprises unsubstituted or substituted picolinoyl, thiophenoyl, furoyl, wherein any heteroaroyl can be substituted with halo, nitro, or alkyl, or any combination thereof.

41. The compound of claim 30 comprising any of the following

alt, solvate, hydrate, metabolite, or prodrug thereof.

The compound of claim 31 comprising

or any salt, solvate, hydrate, metabolite, or prodrug thereof.

43. A dosage form comprising a compound of any one of claims 29-42 adapted for administration to a patient afflicted with a malcondition comprising a metabolic or an immune disorder, cancer, or a CNS disorder, wherein the dosage form comprises a capsule, a tablet, a liquid or dispersed oral formulation, or a formulation adapted for parenteral administration.

44. A pharmaceutical composition comprising a compound of any one of claims 29-42 and a pharmaceutically effective excipient.

45. A compound as recited in the method of any one of claims 1-14 for use as a medicament in treatment of a malcondition.

46. The compound of claim 45 wherein the malcondition comprises a metabolic or immune disorder, cancer, or a CNS disorder.

47. Use of the compound as recited in the method of any one of claims 1-14 for manufacture of a medicament.

48. The use of claim 47 wherein the medicament is for treatment of a

malcondition comprising a metabolic or immune disorder, cancer, or a CNS disorder.