# Patent

## Title
Compounds as RORγt modulators and uses thereof

## Abstract

Heterocyclic compounds, and pharmaceutical compositions thereof, are disclosed as RORγt modulators that have a formula represented by the following:




and wherein n1, n2, R1, R2, R3, and R4 are as described herein. These compounds may be used for the prevention and treatment of a variety of conditions in mammals including humans, including by way of non-limiting example, inflammatory conditions, autoimmune disorders, cancer, and graft-versus-host disease.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a National Stage Application claiming the priority of co-pending PCT Application No. PCT/US2011/000460 filed Mar. 11, 2011, which in turn claims priority from U.S. Provisional Application No. 61/339,996, filed Mar. 11, 2010. Applicants claim the benefits of 35 U.S.C. Section 120 as to the PCT application and priority under 35 U.S.C. Section 119 as to the said U.S. Provisional application, and the entire disclosures of both applications are incorporated herein by reference in their entireties.


GOVERNMENTAL SUPPORT
The research leading to the present invention was supported, at least in part, by a grant from the National Institutes of Health Grant Number R01AI080885. Accordingly, the Government has certain rights in the invention.


FIELD OF THE INVENTION
This invention relates to compounds capable of modulating RORγt activity and uses of such compounds to treat diseases or conditions related to RORγt activity. More particularly, the compounds may be used to diminish inflammation associated with an inflammatory disease or condition or to reduce symptoms associated with an autoimmune disorder. Also encompassed herein, are assays and methods for using same to identify compounds capable of modulating RORγt activity.
BACKGROUND OF THE INVENTION
The retinoic acid receptor-related orphan nuclear receptor (ROR) RORγ and its isoform RORγt (collectively “RORγ/γt”) play a major role in regulation of a variety of biological systems. To illustrate, RORγt has a central role in immune system development, homeostasis, and responses to microbial pathogens. For example, RORγt is required for the differentiation of Th17 cells (Ivanov, I I et al. Cell, 2006, 126, 1121-33), a subset of T helper cells that protect the host from infection by secreting inflammatory cytokines such as IL-17, IL-17A, IL-17F, IL-22, and TNFα. These cytokines are signaling proteins that have been shown to be essential in regulating numerous immune responses, including inflammatory responses to antigens. Th17 cells have also recently been shown to have important roles in activating and directing immune responses in a variety of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA), inflammatory bowel disease (IBD), cancer (Weaver, C. et al. Ann. Rev. Immunol., 2007, 25, 821-52; Kryczek, I. et al. J. Immunol., 2007, 178, 6730-3; Cua, D. J. et al. Nature, 2003, 421, 744-8; Langrish, C. L. et al. J. Exp. Med., 2005, 201, 233-40; Yen, D. et al. J. Clin. Invest., 2006, 116, 1310-6), and graft-versus-host disease (Carlson, M. J. et al. Blood, 28 Oct. 2008. [Epub ahead of print]; Kappel, L. W. et al. Blood, 17 Oct. 2008. [Epub ahead of print]). Th17 cells have also been implicated in asthma, psoriasis, rheumatoid arthritis, multiple sclerosis (Tzartos, J. S., et al. Am. J. Pathology, 2008, 172, 146-55; Yu, J. J., and Gaffen, S. L. Front. Biosci., 2008, 13, 170-77; and Zheng, Y. et al. Nature, 2007, 445, 648-51), and Crohn's disease (Duerr, R. H., et al. Science, 2006, 314, 1461-63). Additionally, it has been shown that mice defective for expression of RORγt lack Th17 cells and are resistant to a variety of autoimmune diseases and that the absence of Th17-producing microbiota in the small intestine of mice alters the Th17: regulatory T (Treg) cell balance with implications for intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases (Ivanov, I. I. Cell Host & Microbe, 2008, 4, 337-49).
The formation of immune cell aggregates, such as cryptopatches (CP) and isolated lymphoid follicles (ILF), which contain RORγt expressing cells, is known to be a vital step in many immune responses. For example, CPs and ILFs are required for mucosal immunity and for production of the intestinal antibody IgA. Such immune responses can result in inflammation in various diseases, such as Crohn's disease. The ability to inhibit such immune responses by inhibiting the formation of immune cell aggregates may offer another way to treat diseases associated with such responses.
T-cells have also been demonstrated to play a role in diseases characterized by bone loss and degradation, such as osteoarthritis. For example, in autoimmune arthritis, activation of T cells results in bone destruction mediated by osteoclasts. Th17, whose differentiation is regulated by RORγt, has been shown to be osteoclastogenic, thus linking T cell activation and bone resorption (Sato, K. et al. J. Ex. Med., 2008, 203, 2673-82). Thus, the ability to regulate Th17 cell differentiation via RORγt modulation may offer a way to treat bone loss and degradation, such as that associated with autoimmune disease. Furthermore, interferon gamma (IFN-γ) suppresses osteoclast formation by rapidly degrading the RANK adaptor protein TRAF6 in the RANK-RANKL signaling pathway, and RORγt has been shown to down-regulate the production of IFN-γ (Ivanov, I. I. et al. Cell, 2006, 126, 1121-33). Thus, the ability to regulate osteoclast formation through modulation of RORγt-mediated IFN-γ osteoclast suppression may provide additional methods to treat bone loss and degradation, such as that associated with autoimmune disease (e.g., osteoarthritis).
Circadian rhythm relates to an approximately daily periodicity in the biochemical, physiological or behavioral processes of living beings, including plants, animals, fungi and some bacteria. Members of the ROR family of orphan nuclear receptors have been implicated in regulation of control of circadian clock function by regulation of clock genes (Ueda, H. R. et al. Nature, 2002, 418, 534-39; Sato, T. K. et al. Neuron, 2004, 43, 527-37), and RORγ/γt has been implicated in the regulation of genes that govern circadian metabolism (Kumaki, Y. et al. PNAS, 2008, 105, 14946-51; Liu, C. et al. Nature, 2007, 447, 477-81). Moreover, RORγ gene expression is known to oscillate in a circadian manner in metabolically active tissues such as liver and brown adipose tissue (Yang, X. et al., Cell, 2006, 126, 801-10), which further confirms that a role exists for RORγ in regulating circadian function. Hence, the ability to modulate RORγ/γt expression may also result in circadian rhythm regulation and treatment of disorders associated with disruption of circadian rhythm. Since circadian rhythm is integral in maintaining metabolic levels, whose imbalance is linked to obesity and diabetes, modulators of RORγ/γt may also be useful in treating obesity and diabetes through regulation of circadian rhythm.
In view of the above, a need exists for therapeutic agents, and corresponding pharmaceutical compositions and related methods of treatment that address conditions causally related RORγt activity, and it is toward the fulfillment and satisfaction of that need, that the present invention is directed.
SUMMARY OF THE INVENTION
The present invention provides a method for preventing, treating or ameliorating in a mammal a disease or condition that is causally related to the activity of the RORγ or RORγt activity in vivo, which comprises administering to the mammal an effective disease-treating or condition-treating amount of a compound according to formula I:




    
    
        wherein
        R1 is OH, or substituted or unsubstituted alkoxy;
        R2 is H, or Me;
        each R3 and R4 is independently selected from H, OH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted acyl, substituted or unsubstituted acylamino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylarylamino, substituted or unsubstituted amino, substituted or unsubstituted arylalkyl, sulfo, substituted sulfo, substituted sulfonyl, substituted sulfinyl, substituted sulfanyl, substituted or unsubstituted aminosulfonyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, azido, substituted or unsubstituted carbamoyl, carboxyl, cyano, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted dialkylamino, halo, nitro, and thiol; and
        each n1 and n2 is independently 1, 2, 3, 4 or 5;
        or a pharmaceutically acceptable salt, solvate or prodrug thereof; and stereoisomers, isotopic variants and tautomers thereof.
    
    


In another aspect, the present invention provides a compound according to formula II:




wherein

    
    
        R1 is OH, or substituted or unsubstituted alkoxy;
        each R3 and R4 is independently selected from H, OH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted acyl, substituted or unsubstituted acylamino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylarylamino, substituted or unsubstituted amino, substituted or unsubstituted arylalkyl, sulfo, substituted sulfo, substituted sulfonyl, substituted sulfonyl, substituted sulfanyl, substituted or unsubstituted aminosulfonyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, azido, substituted or unsubstituted carbamoyl, carboxyl, cyano, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted dialkylamino, halo, nitro, and thiol; and
        each n1 and n2 is independently 1, 2, 3, 4 or 5;
        or a pharmaceutically acceptable salt, solvate or prodrug thereof; and stereoisomers, isotopic variants and tautomers thereof.
    
    


In yet another aspect, the present invention provides a method for preventing, treating or ameliorating in a mammal a disease or condition that is causally related to RORγ or RORγt activity in vivo, which comprises administering to the mammal an effective disease-treating or condition-treating amount of a compound according to formula I:

    
    
        i) a steroid derivative selected from
    
    









    
    
        ii) a heterocyclic compound selected from
    
    









    
    
        iii) a fused heterocyclic compound
    
    









    
    
        iv) a thiourea compound
    
    






and

    
    
        v) a compound of formula:
    
    









    
    
        or a pharmaceutically acceptable salt, solvate or prodrug thereof;
        and stereoisomers, isotopic variants and tautomers thereof.
    
    


In a further aspect, the present invention provides pharmaceutical compositions comprising a compound of the invention, and a pharmaceutical carrier, excipient or diluent. In this aspect of the invention, the pharmaceutical composition can comprise one or more of the compounds described herein. Moreover, the compounds of the present invention useful in the pharmaceutical compositions and treatment methods disclosed herein, are all pharmaceutically acceptable as prepared and used.
In a further aspect, this invention provides a method of treating a mammal susceptible to or afflicted with a condition from among those listed herein, and particularly, such condition as may be associated with RORγt. Such conditions include, without limitation, multiple sclerosis (and the animal model thereof, EAE), rheumatoid arthritis (and the animal model thereof, CIA), inflammatory bowel disease (IBD), cancer, graft-versus-host disease, asthma, psoriasis, diabetes, uveitis, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, arteriosclerosis, and H. pylori infections and ulcers resulting from such infection.
In a further aspect, an assay for screening to identify modulators of RORγ/γt transcriptional activity is envisioned, the assay comprising a first insect cell line that expresses a fusion protein (SEQ ID NO: 2; encoded by SEQ ID NO: 1) comprising a RORγ/γt sequence, wherein the RORγ/γt sequence does not comprise the DNA binding domain (DBD) of full length RORγ/γt and a yeast GAL4 DBD, wherein expression of the fusion protein is transcriptionally regulated by an inducible promoter, and wherein the first insect cell line further comprises a reporter, whose expression is upregulated in the presence of the fusion protein. Accordingly, the component of the fusion protein representative of RORγ/γt sequences is a DBD-deleted RORγ/γt sequence. The fusion protein is essentially a chimeric protein wherein the RORγ/γt DBD is deleted from the RORγ/γt sequences and replaced with the yeast GAL4 DNA binding domain DBD. In a particular embodiment, the Gal4 DNA binding domain (G4 DBD) corresponds to amino acids 1 through 147 of Gal4 protein and the DBD-deleted RORγ/γt sequence is amino acids 79 to the carboxyl terminal end. Accordingly, the G4 DBD corresponds to amino acids 1-147 of SEQ ID NO: 2 and the DBD-deleted RORγ/γt sequence corresponds to amino acids 148-564 of SEQ ID NO: 2. In a particular embodiment, the inducible promoter is a copper inducible promoter.
In an embodiment of the assay, the reporter is transcriptionally regulated by a plurality of copies of the GAL4 binding site enhancer (UAS) operatively linked to nucleic acid sequences encoding the reporter. In a particular embodiment of the assay, the plurality of copies of the GAL4 binding site enhancer (UAS) is between 1 and 5 copies. In a more particular embodiment, the reporter is the firefly luciferase reporter.
In an aspect of the assay, the first insect cell line is the S2 cell line. In another aspect of the assay, the fusion protein is encoded by nucleic acids that are integrated into the genome of the first insect cell line or encoded by extrachromosomal nucleic acids incorporated into the first insect cell line. Accordingly, the assay may relate to stably transfected cell line or a transiently transfected cell line. The choice of stably or transiently transfected cell line depends, in part, on the number of compounds to be tested and availability and cost of assay reagents required.
As described herein, the assay may further comprise a second insect cell line that expresses a second fusion protein (SEQ ID NO: 4; encoded by SEQ ID NO: 3) comprising a RORα sequence, wherein the RORα sequence does not comprise the DBD of full length RORα sequence and a yeast GAL4 DBD; a third insect cell line that expresses a third fusion protein (SEQ ID NO: 6; encoded by SEQ ID NO: 5) comprising a DHR3 sequence, wherein the DHR3 sequence does not comprise the DNA binding domain (DBD) of full length DHR3 sequence and a yeast GAL4 DBD, and a fourth insect cell line that expresses a fourth fusion protein (SEQ ID NO: 8; encoded by SEQ ID NO: 7) comprising a transcriptionally active domain of general transcriptional activator VP16 and a yeast GAL4 DBD, wherein expression of the second, third and fourth fusion proteins is transcriptionally regulated by inducible promoters. In a particular embodiment, the DBD-deleted mouse RORα sequence is amino acids 142 to the carboxyl terminal end of mouse RORα and the DBD-deleted Drosophila DHR3 is amino acids 120 to the carboxyl terminal end of Drosophila DHR3. Accordingly, the G4 DBD corresponds to amino acids 1-147 of SEQ ID NOs: 4, 6, and 8 and the DBD-deleted RORα sequence corresponds to amino acids 148-529 of SEQ ID NO: 4, the DBD-deleted DHR3 sequence corresponds to amino acids 148-513 of SEQ ID NO: 6, and the VP16 sequence corresponds to amino acids 148-231 of SEQ ID NO: 8. In an embodiment, inducible promoters regulating expression of the first, second, third, and fourth fusion proteins are identical promoters. In yet another embodiment, the inducible promoters regulating expression of the first, second, third, and fourth fusion proteins are copper inducible promoters.
Methods for using the assay of the invention are also encompassed herein. Such methods include those involving use of the cell-based assay systems described herein to screen diverse compound libraries, such as those available from research institutions, federal agencies, and/or commercial vendors, to identify modulators of RORγ/γt transcriptional activity. Modulators of RORγ/γt transcriptional activity may be identified based on results determined using the first insect cell-based assay system described herein alone or in combination with at least one of the second, third, and fourth insect cell-based assay systems described herein to identify compounds that are specific modulators of RORγ/γt transcriptional activity. Modulators identified using assays described herein may be identified as inhibitors or agonists of RORγ/γt transcriptional activity. Compounds identified using the cell based systems described herein may be assessed in secondary screens, also described herein and understood in the art, to validate their identity as bona fide modulators of RORγ/γt transcriptional activity.
Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and B show the structures of RORγ specific compounds with inverse agonist activity and fluorescence activated cell sorter (FACS) plot analyses revealing the effects of these compounds on mouse CD4 T cells.
FIGS. 2A and B show the structure of a RORγ specific compound with inverse agonist activity and FACS plot analyses revealing the effects of this compound on mouse CD4 T cells.
FIG. 3 shows a graph depicting the effects of ursolic acid in the Drosophila S2 cell assay and its specificity for RORγ inhibition.
FIG. 4 shows a FACS plot analyses revealing the effects of ursolic acid on human CD4 T cell cytokine production.
FIGS. 5A, B, and C show the structures of RORγ specific compounds with inverse agonist activity and a graph depicting the effects of these compounds in the Drosophila S2 cell assay and their specificity for RORγ inhibition and FACS plot analyses revealing the effects of these compounds on mouse CD4 T cells.
FIGS. 6A and B show the structures of RORγ specific compounds with inverse agonist activity and FACS plot analyses revealing the effects of these compounds on human CD4 T cell cytokine production.
FIGS. 7A and B show the structure of a RORγ specific compound with inverse agonist activity and FACS plot analyses revealing the effects of this compound on mouse CD4 T cells.
FIGS. 8A and B show the structure of a RORγ specific compound with inverse agonist activity and a graph depicting the effects of this compound in the Drosophila S2 cell assay and its specificity for RORγ inhibition.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
When describing the compounds, pharmaceutical compositions containing such compounds and methods of using such compounds and compositions, the following terms have the following meanings unless otherwise indicated. It should also be understood that any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope. It should be further understood that the terms “groups” and “radicals” can be considered interchangeable when used herein.
“Acyl” refers to a radical —C(O)R20, where R20 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein. Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.
“Acylamino” refers to a radical —NR21C(O)R22, where R21 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl and R22 is hydrogen, alkyl, alkoxy, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl or heteroarylalkyl, as defined herein. Representative examples include, but are not limited to, formylamino, acetylamino, cyclohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino and the like.
“Acyloxy” refers to the group —OC(O)R23 where R23 is hydrogen, alkyl, aryl or cycloalkyl.
“Substituted alkenyl” includes those groups recited in the definition of “substituted” herein, and particularly refers to an alkenyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Alkoxy” refers to the group —OR24 where R24 is alkyl. Particular alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
“Substituted alkoxy” includes those groups recited in the definition of “substituted” herein, and particularly refers to an alkoxy group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, heteroaryl, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Alkoxycarbonylamino” refers to the group —NR25C(O)OR26, where R25 is hydrogen, alkyl, aryl or cycloalkyl, and R26 is alkyl or cycloalkyl.
“Alkyl” refers to monovalent saturated alkane radical groups particularly having up to about 11 carbon atoms, more particularly as a lower alkyl, from 1 to 8 carbon atoms and still more particularly, from 1 to 6 carbon atoms. The hydrocarbon chain may be either straight-chained or branched. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, n-octyl, tert-octyl and the like. The term “lower alkyl” refers to alkyl groups having 1 to 6 carbon atoms. The term “alkyl” also includes “cycloalkyls” as defined below.
“Substituted alkyl” includes those groups recited in the definition of “substituted” herein, and particularly refers to an alkyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, heteroaryl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2—, and aryl-S(O)2—.
“Alkylene” refers to divalent saturated alkene radical groups having 1 to 11 carbon atoms and more particularly 1 to 6 carbon atoms which can be straight-chained or branched. This term is exemplified by groups such as methylene (—CH2—), ethylene (—CH2CH2—), the propylene isomers (e.g., —CH2CH2CH2— and —CH(CH3)CH2—) and the like.
“Substituted alkylene” includes those groups recited in the definition of “substituted” herein, and particularly refers to an alkylene group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, amino-carbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbyl groups preferably having 2 to 11 carbon atoms, particularly, from 2 to 8 carbon atoms, and more particularly, from 2 to 6 carbon atoms, which can be straight-chained or branched and having at least 1 and particularly from 1 to 2 sites of olefinic unsaturation. Particular alkenyl groups include ethenyl (—CH═CH2), n-propenyl (—CH2CH═CH2), isopropenyl (—C(CH3)═CH2), vinyl and substituted vinyl, and the like.
“Alkenylene” refers to divalent olefinically unsaturated hydrocarbyl groups particularly having up to about 11 carbon atoms and more particularly 2 to 6 carbon atoms which can be straight-chained or branched and having at least 1 and particularly from 1 to 2 sites of olefinic unsaturation. This term is exemplified by groups such as ethenylene (—CH═CH—), the propenylene isomers (e.g., —CH═CHCH2— and —C(CH3)═CH— and —CH═C(CH3)—) and the like.
“Alkynyl” refers to acetylenically or alkynically unsaturated hydrocarbyl groups particularly having 2 to 11 carbon atoms, and more particularly 2 to 6 carbon atoms which can be straight-chained or branched and having at least 1 and particularly from 1 to 2 sites of alkynyl unsaturation. Particular non-limiting examples of alkynyl groups include acetylenic, ethynyl (—C≡CH), propargyl (—CH2C≡CH), and the like.
“Substituted alkynyl” includes those groups recited in the definition of “substituted” herein, and particularly refers to an alkynyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Alkanoyl” or “acyl” as used herein refers to the group R27—C(O)—, where R27 is hydrogen or alkyl as defined above.
“Aryl” refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like. Particularly, an aryl group comprises from 6 to 14 carbon atoms.
“Substituted Aryl” includes those groups recited in the definition of “substituted” herein, and particularly refers to an aryl group that may optionally be substituted with 1 or more substituents, for instance from 1 to 5 substituents, particularly 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, alkoxycarbonyl, alkyl, substituted alkyl, alkynyl, substituted alkynyl, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Fused Aryl” refers to an aryl having two of its ring carbon in common with a second aryl ring or with an aliphatic ring.
“Alkaryl” refers to an aryl group, as defined above, substituted with one or more alkyl groups, as defined above.
“Aralkyl” or “arylalkyl” refers to an alkyl group, as defined above, substituted with one or more aryl groups, as defined above.
“Aryloxy” refers to —O-aryl groups wherein “aryl” is as defined above.
“Alkylamino” refers to the group alkyl-NR28R29, wherein each of R28 and R29 are independently selected from hydrogen and alkyl.
“Arylamino” refers to the group aryl-NR30R31, wherein each of R30 and R3I are independently selected from hydrogen, aryl and heteroaryl.
“Alkoxyamino” refers to a radical —N(H)OR32 where R32 represents an alkyl or cycloalkyl group as defined herein.
“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is as defined herein.
“Alkylarylamino” refers to a radical —NR33R34 where R33 represents an alkyl or cycloalkyl group and R34 is an aryl as defined herein.
“Alkylsulfonyl” refers to a radical —S(O)2R35 where R35 is an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl and the like.
“Alkylsulfinyl” refers to a radical —S(O)R35 where R35 is an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfinyl and the like.
“Alkylthio” refers to a radical —SR35 where R35 is an alkyl or cycloalkyl group as defined herein that may be optionally substituted as defined herein. Representative examples include, but are not limited to, methylthio, ethylthio, propylthio, butylthio, and the like.
“Amino” refers to the radical —NH2.
“Substituted amino” includes those groups recited in the definition of “substituted” herein, and particularly refers to the group —N(R36)2 where each R36 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, and where both R groups are joined to form an alkylene group. When both R groups are hydrogen, —N(R36)2 is an amino group.
“Aminocarbonyl” refers to the group —C(O)NR37R37 where each R37 is independently hydrogen, alkyl, aryl and cycloalkyl, or where the R37 groups are joined to form an alkylene group.
“Aminocarbonylamino” refers to the group —NR38C(O)NR38R38 where each R38 is independently hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form an alkylene group.
“Aminocarbonyloxy” refers to the group —OC(O)NR39R39 where each R39 is independently hydrogen, alkyl, aryl or cycloalkyl, or where the R groups are joined to form an alkylene group.
“Arylalkyloxy” refers to an —O-arylalkyl radical where arylalkyl is as defined herein.
“Arylamino” means a radical —NHR40 where R40 represents an aryl group as defined herein.
“Aryloxycarbonyl” refers to a radical —C(O)—O-aryl where aryl is as defined herein.
“Arylsulfonyl” refers to a radical —S(O)2R41 where R41 is an aryl or heteroaryl group as defined herein.
“Azido” refers to the radical —N3.
“Bicycloaryl” refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent bicycloaromatic ring system. Typical bicycloaryl groups include, but are not limited to, groups derived from indane, indene, naphthalene, tetrahydronaphthalene, and the like. Particularly, an aryl group comprises from 8 to 11 carbon atoms.
“Bicycloheteroaryl” refers to a monovalent bicycloheteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent bicycloheteroaromatic ring system. Typical bicycloheteroaryl groups include, but are not limited to, groups derived from benzofuran, benzimidazole, benzindazole, benzdioxane, chromene, chromane, cinnoline, phthalazine, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, benzothiazole, benzoxazole, naphthyridine, benzoxadiazole, pteridine, purine, benzopyran, benzpyrazine, pyridopyrimidine, quinazoline, quinoline, quinolizine, quinoxaline, benzomorphan, tetrahydroisoquinoline, tetrahydroquinoline, and the like. Preferably, the bicycloheteroaryl group is between 9-11 membered bicycloheteroaryl, with 5-10 membered heteroaryl being particularly preferred. Particular bicycloheteroaryl groups are those derived from benzothiophene, benzofuran, benzothiazole, indole, quinoline, isoquinoline, benzimidazole, benzoxazole and benzdioxane.
“Carbamoyl” refers to the radical —C(O)N(R42)2 where each R42 group is independently hydrogen, alkyl, cycloalkyl or aryl, as defined herein, which may be optionally substituted as defined herein.
“Carboxy” refers to the radical —C(O)OH.
“Carboxyamino” refers to the radical —N(H)C(O)OH.
“Cycloalkyl” refers to cyclic hydrocarbyl groups having from 3 to about 10 carbon atoms and having a single cyclic ring or multiple condensed rings, including fused and bridged ring systems, which optionally can be substituted with from 1 to 3 alkyl groups. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, and multiple ring structures such as adamantanyl, and the like.
“Substituted cycloalkyl” includes those groups recited in the definition of “substituted” herein, and particularly refers to a cycloalkyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Cycloalkoxy” refers to the group —OR43 where R43 is cycloalkyl. Such cycloalkoxy groups include, by way of example, cyclopentoxy, cyclohexoxy and the like.
“Cycloalkenyl” refers to cyclic hydrocarbyl groups having from 3 to 10 carbon atoms and having a single cyclic ring or multiple condensed rings, including fused and bridged ring systems and having at least one and particularly from 1 to 2 sites of olefinic unsaturation. Such cycloalkenyl groups include, by way of example, single ring structures such as cyclohexenyl, cyclopentenyl, cyclopropenyl, and the like.
“Substituted cycloalkenyl” includes those groups recited in the definition of “substituted” herein, and particularly refers to a cycloalkenyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Fused Cycloalkenyl” refers to a cycloalkenyl having two of its ring carbon atoms in common with a second aliphatic or aromatic ring and having its olefinic unsaturation located to impart aromaticity to the cycloalkenyl ring.
“Cyanato” refers to the radical —OCN.
“Cyano” refers to the radical —CN.
“Dialkylamino” means a radical —NR44R45 where R44 and R45 independently represent an alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, or substituted heteroaryl group as defined herein.
“Ethenyl” refers to substituted or unsubstituted —(C═C)—.
“Ethylene” refers to substituted or unsubstituted —(C—C)—.
“Ethynyl” refers to —(C≡C)—.
“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo. Preferred halo groups are either fluoro or chloro.
“Hydroxy” refers to the radical —OH.
“Nitro” refers to the radical —NO2.
“Substituted” refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to —X, —R46, —O−, ═O, —OR46, —SR46, —S−, ═S, —NR46R47, ═NR46, —CX3, —CF3, —CN, —OCN, —SCN, —NO, —NO2, ═N2, —N3, —S(O)2O−, —S(O)2OH, —S(O)2R46, —OS(O2)O−, —OS(O)2R46, —P(O)(O−)2, —P(O)(OR46)(O−), —OP(O)(OR46)(OR47), —C(O)R46, —C(S)R46, —C(O)OR46, —C(O)NR46R47, —C(O)O−, —C(S)OR46, —NR48C(O)NR46R47, —NR48C(S)NR46R47, —NR49C(NR48)NR46R47 and —C(NR48)NR46R47, where each X is independently a halogen; each R46, R47, R48 and R49 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted alkyl, arylalkyl, substituted alkyl, cycloalkyl, substituted alkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, —NR50R51, —C(O)R50 or —S(O)2R50 or optionally R50 and R51 together with the atom to which they are both attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R50 and R51 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted alkyl, arylalkyl, substituted alkyl, cycloalkyl, substituted alkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.
Examples of representative substituted aryls include the following




In these formulae one of R52 and R53 may be hydrogen and at least one of R52 and R53 is each independently selected from alkyl, alkenyl, alkynyl, cycloheteroalkyl, alkanoyl, alkoxy, aryloxy, heteroaryloxy, alkylamino, arylamino, heteroarylamino, NR54COR55, NR54SOR55, NR54SO2R57, COOalkyl, COOaryl, CONR54R55, CONR54OR55, NR54R55, SO2NR54R55, S-alkyl, S-alkyl, SOalkyl, SO2alkyl, Saryl, SOaryl, SO2aryl; or R52 and R53 may be joined to form a cyclic ring (saturated or unsaturated) from 5 to 8 atoms, optionally containing one or more heteroatoms selected from the group N, O or S. R54, R55, and R56 are independently hydrogen, alkyl, alkenyl, alkynyl, perfluoroalkyl, cycloalkyl, cycloheteroalkyl, aryl, substituted aryl, heteroaryl, substituted or hetero alkyl or the like.
“Hetero” when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen, or sulfur heteroatom. Hetero may be applied to any of the hydrocarbyl groups described above such as alkyl, e.g. heteroalkyl, cycloalkyl, e.g. cycloheteroalkyl, aryl, e.g. heteroaryl, cycloalkenyl, cycloheteroalkenyl, and the like having from 1 to 5, and especially from 1 to 3 heteroatoms.
“Heteroaryl” refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. Preferably, the heteroaryl group is between 5-15 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred. Particular heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.
Examples of representative heteroaryls include the following:




wherein each Y is selected from carbonyl, N, NR58, O, and S; and R58 is independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, heteroalkyl or the like.

As used herein, the term “cycloheteroalkyl” refers to a stable heterocyclic non-aromatic ring and fused rings containing one or more heteroatoms independently selected from N, O and S. A fused heterocyclic ring system may include carbocyclic rings and need only include one heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, piperazinyl, homopiperazinyl, piperidinyl and morpholinyl, and are shown in the following illustrative examples:




wherein each X is selected from CR582, NR58, O and S; and each Y is selected from NR58, O and S; and R58 is independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, heteroalkyl or the like. These cycloheteroalkyl rings may be optionally substituted with one or more groups selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—. Substituting groups include carbonyl or thiocarbonyl which provide, for example, lactam and urea derivatives.

Examples of representative cycloheteroalkenyls include the following:




wherein each X is selected from CR582, NR58, O and S; and each Y is selected from carbonyl, N, NR58, O and S; and R58 is independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, heteroalkyl or the like.

Examples of representative aryl having hetero atoms containing substitution include the following:




wherein each X is selected from C—R582NR58, O and S; and each Y is selected from carbonyl, NR58, O and S; and R58 is independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, heteroalkyl or the like.

“Hetero substituent” refers to a halo, O, S or N atom-containing functionality that may be present as an R4 in a R4C group present as substituents directly on A, B, W, Y or Z of the compounds of this invention or may be present as a substituent in the “substituted” aryl and aliphatic groups present in the compounds.
Examples of hetero substituents include:
-halo,
—NO2, —NH2, —NHR59, —N(R59)2,
—NRCOR, —NR59SOR59, —NR59SO2R59, OH, CN,
—CO2H,
—R59—OH, —O—R59, —COOR59,
—CON(R59)2, —CONROR59,
—SO3H, —R59—S, —SO2N(R59)2,
—S(O)R59, —S(O)2R59 
wherein each R59 is independently an aryl or aliphatic, optionally with substitution. Among hetero substituents containing R59 groups, preference is given to those materials having aryl and alkyl R59 groups as defined herein. Preferred hetero substituents are those listed above.
“Hydrogen bond donor” group refers to a group containing O—H, or N—H functionality. Examples of “hydrogen bond donor” groups include —OH, —NH2, and —NH—R59a and wherein R59a is alkyl, cycloalkyl, aryl, or heteroaryl.
“Dihydroxyphosphoryl” refers to the radical —PO(OH)2.
“Substituted dihydroxyphosphoryl” includes those groups recited in the definition of “substituted” herein, and particularly refers to a dihydroxyphosphoryl radical wherein one or both of the hydroxyl groups are substituted. Suitable substituents are described in detail below.
“Aminohydroxyphosphoryl” refers to the radical —PO(OH)NH2.
“Substituted aminohydroxyphosphoryl” includes those groups recited in the definition of “substituted” herein, and particularly refers to an aminohydroxyphosphoryl wherein the amino group is substituted with one or two substituents. Suitable substituents are described in detail below. In certain embodiments, the hydroxyl group can also be substituted.
“Thioalkoxy” refers to the group —SR60 where R60 is alkyl.
“Substituted thioalkoxy” includes those groups recited in the definition of “substituted” herein, and particularly refers to a thioalkoxy group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, selected from the group consisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)2— and aryl-S(O)2—.
“Sulfanyl” refers to the radical HS—. “Substituted sulfanyl” refers to a radical such as RS— wherein R is any substituent described herein.
“Sulfonyl” refers to the divalent radical —S(O2)—. “Substituted sulfonyl” refers to a radical such as R61—(O2)S— wherein R61 is any substituent described herein. “Aminosulfonyl” or “Sulfonamide” refers to the radical H2N(O2)S—, and “substituted aminosulfonyl” “substituted sulfonamide” refers to a radical such as R622N(O2)S— wherein each R62 is independently any substituent described herein.
“Sulfone” refers to the group —SO2R63. In particular embodiments, R63 is selected from H, lower alkyl, alkyl, aryl and heteroaryl.
“Thioaryloxy” refers to the group —SR64 where R64 is aryl.
“Thioketo” refers to the group ═S.
“Thiol” refers to the group —SH.
One having ordinary skill in the art of organic synthesis will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring, whether it is aromatic or non aromatic, is determined by the size of the ring, the degree of unsaturation and the valence of the heteroatoms. In general, a heterocyclic ring may have one to four heteroatoms so long as the heteroaromatic ring is chemically feasible and stable.
“Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
“Pharmaceutically acceptable salt” refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of non toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. The term “pharmaceutically acceptable cation” refers to a non toxic, acceptable cationic counter-ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like.
“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
“Preventing” or “prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
“Prodrugs” refers to compounds, including derivatives of the compounds of the invention, which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
“Solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. Conventional solvents include water, ethanol, acetic acid and the like. The compounds of the invention may be prepared e.g. in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and further include both stoichiometric solvates and non-stoichiometric solvates.
“Subject” includes humans. The terms “human,” “patient” and “subject” are used interchangeably herein.
“Therapeutically effective amount” means the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.
“Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
As used herein, the term “operably linked” refers to a regulatory sequence capable of mediating the expression of a coding sequence and which is placed in a DNA molecule (e.g., an expression vector) in an appropriate position relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.
A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
An “expression vector” or “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
The terms “transform”, “transfect”, or “transduce”, shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.
The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism. In other applications, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but in the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Preferred are the C1 to C8 alkyl, C2-C8 alkenyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds of the invention.
As used herein, the term “isotopic variant” refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. For example, an “isotopic variant” of a compound can contain one or more non-radioactive isotopes, such as for example, deuterium (2H or D), carbon-13 (13C), nitrogen-15 (15N), or the like. It will be understood that, in a compound where such isotopic substitution is made, the following atoms, where present, may vary, so that for example, any hydrogen may be 2H/D, any carbon may be 13C, or any nitrogen may be 15N, and that the presence and placement of such atoms may be determined within the skill of the art. Likewise, the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Further, compounds may be prepared that are substituted with positron emitting isotopes, such as 11C, 18F, 15O and 13N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
All isotopic variants of the compounds provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.
It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”.
Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.
“Tautomers” refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of π electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro-forms of phenylnitromethane, that are likewise formed by treatment with acid or base.
Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.
The Compounds
The present invention provides a method for preventing, treating or ameliorating in a mammal a disease or condition that is causally related to the activity of the RORγ or RORγ activity in vivo, which comprises administering to the mammal an effective disease-treating or condition-treating amount of a compound according to formula I:




wherein

    
    
        R1 is OH, or substituted or unsubstituted alkoxy;
        R2 is H, or Me;
        each R3 and R4 is independently selected from H, OH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted acyl, substituted or unsubstituted acylamino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylarylamino, substituted or unsubstituted amino, substituted or unsubstituted arylalkyl, sulfo, substituted sulfo, substituted sulfonyl, substituted sulfinyl, substituted sulfanyl, substituted or unsubstituted aminosulfonyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, azido, substituted or unsubstituted carbamoyl, carboxyl, cyano, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted dialkylamino, halo, nitro, and thiol; and
        each n1 and n2 is independently 1, 2, 3, 4 or 5;
        or a pharmaceutically acceptable salt, solvate or prodrug thereof; and stereoisomers, isotopic variants and tautomers thereof.
    
    


In one embodiment, with respect to the compounds of formula I, R2 is H.
In one embodiment, with respect to the compounds of formula I, R2 is Me.
In one embodiment, with respect to the compounds of formula I, the compound is according to formula II




    
    
        and wherein R1, R3, R4, n1 and n2 are as in claim 1;
        or a pharmaceutically acceptable salt, solvate or prodrug thereof; and stereoisomers, isotopic variants and tautomers thereof.
    
    


In another aspect, the present invention provides a compound according to formula II:




wherein

    
    
        RI is OH, or substituted or unsubstituted alkoxy;
        each R3 and R4 is independently selected from H, OH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted acyl, substituted or unsubstituted acylamino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylarylamino, substituted or unsubstituted amino, substituted or unsubstituted arylalkyl, sulfo, substituted sulfo, substituted sulfonyl, substituted sulfonyl, substituted sulfanyl, substituted or unsubstituted aminosulfonyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, azido, substituted or unsubstituted carbamoyl, carboxyl, cyano, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted dialkylamino, halo, nitro, and thiol; and
        each n1 and n2 is independently 1, 2, 3, 4 or 5;
        or a pharmaceutically acceptable salt, solvate or prodrug thereof; and stereoisomers, isotopic variants and tautomers thereof.
    
    


In one embodiment, with respect to the compounds of formulae I-II, the compound is according to formula III:




wherein

    
    
        and wherein R1, R3, R4, n1 and n2 are as in claim 1 or claim 5;
        
            or a pharmaceutically acceptable salt, solvate or prodrug thereof; and stereoisomers, isotopic variants and tautomers thereof.
        
        
    
    


In one embodiment, with respect to the compounds of formulae I-III, R1 is OH.
In one embodiment, with respect to the compounds of formulae I-III, R1 is alkoxy.
In one embodiment, with respect to the compounds of formulae I-III, R1 is OMe or OEt.
In one embodiment, with respect to the compounds of formulae I-III, each R3 is H.
In one embodiment, with respect to the compounds of formulae I-III, n1 is 1, 2, or 3; and each R3 is independently halo, alkyl, OH, alkoxy, haloalkyl or CN.
In one embodiment, with respect to the compounds of formulae I-III, n1 is 1, 2, or 3; and each R3 is independently F, Cl, Br, Me, Et, OMe, OCF3, CN, or CF3.
In one embodiment, with respect to the compounds of formulae I-III, each R4 is H.
In one embodiment, with respect to the compounds of formulae I-III, n2 is 1, 2, or 3; and each R4 is independently halo, alkyl, OH, alkoxy, haloalkyl or CN.
In one embodiment, with respect to the compounds of formulae I-III, n2 is 1, 2, or 3; and each R4 is independently F, Cl, Br, Me, Et, OMe, OCF3, CN, or CF3.
In one embodiment, with respect to the compounds of formulae I-III, n1 is 1; R′ is H, Br, or OMe; n2 is 1; and R4 is H, F, Br, Cl, or CF3.
In one embodiment, with respect to the compounds of formulae I-III, the compound is according to formula IVa, IVb, IVc, or IVd:




wherein

    
    
        and wherein R1 is OMe or OEt; each of R3 and R4 are independently selected from H, OMe, F, Cl, Me, or CF3;
        
            or a pharmaceutically acceptable salt, solvate or prodrug thereof; and stereoisomers, isotopic variants and tautomers thereof.
        
        
    
    


In one embodiment, with respect to the method, the compound is any one of compounds listed in Table 3.
In one embodiment, with respect to the compound, the compound is any one of compounds listed in Table 1; provided that the compound is other than




In yet another aspect, the present invention provides a method for preventing, treating or ameliorating in a mammal a disease or condition that is causally related to the activity of the RORγ or RORγt activity in vivo, which comprises administering to the mammal an effective disease-treating or condition-treating amount of any one of compounds selected from
i) a steroid derivative selected from







ii) a heterocyclic compound selected from







iii) a fused heterocyclic compound







iv) a thiourea compound




and

v) a compound of formula:







    
    
        or a pharmaceutically acceptable salt, solvate or prodrug thereof;
        and stereoisomers, isotopic variants and tautomers thereof.
    
    


In one embodiment, with respect to the compound used in method of treatment, the compound is any one of the steroid derivatives listed herein.
In one embodiment, with respect to the compound used in method of treatment, the compound is any one of the monocyclic heterocyclic compound listed listed herein.
In one embodiment, with respect to the compound used in method of treatment, the compound is any one of the fused heterocyclic compound listed listed herein.
In one embodiment, with respect to the compound used in method of treatment, the compound is the thiourea compound listed listed herein.
In one embodiment, with respect to the compound used in method of treatment, the compound is the compound of formula:




In one embodiment, with respect to the compound used in method of treatment, the compound is the compound of formula:




In one embodiment, with respect to the method of treatment, the disease or condition is autoimmune disease.
In one embodiment, with respect to the method of treatment, the disease or condition is inflammatory disease.
In one embodiment, with respect to the method of treatment, the disease or condition is selected from arthritis, diabetes, multiple sclerosis (and the animal model thereof, EAE), uveitis, rheumatoid arthritis (and the animal model thereof, CIA), psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, cancer, graft-versus-host disease, H. pylori infections and ulcers resulting from such infection, and inflammatory bowel diseases.
In one embodiment, with respect to the method of treatment, the disease or condition is selected from Crohn's disease, ulcerative colitis, sprue and food allergies.
In certain aspects, the present invention provides prodrugs and derivatives of the compounds according to the formulae above. Prodrugs are derivatives of the compounds of the invention, which have metabolically cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention, which are pharmaceutically active, in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Preferred are the C1 to C8 alkyl, C2-C8 alkenyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds of the invention.
Pharmaceutical Compositions
When employed as pharmaceuticals, the compounds of this invention are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
Generally, the compounds of this invention are administered in a pharmaceutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound-administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
The pharmaceutical compositions of this invention can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, the compounds of this invention are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.
The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the furansulfonic acid compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. As before, the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight. When formulated as a ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base. Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope of this invention.
The compounds of this invention can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.
The above-described components for orally administrable, injectable or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.
The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences. 
The following formulation examples illustrate representative pharmaceutical compositions of this invention. The present invention, however, is not limited to the following pharmaceutical compositions.
Formulation 1
Tablets
A compound of the invention is admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 240-270 mg tablets (80-90 mg of active amide compound per tablet) in a tablet press.
Formulation 2
Capsules
A compound of the invention is admixed as a dry powder with a starch diluent in an approximate 1:1 weight ratio. The mixture is filled into 250 mg capsules (125 mg of active amide compound per capsule).
Formulation 3
Liquid
A compound of the invention (125 mg), sucrose (1.75 g) and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water is then added to produce a total volume of 5 mL.
Formulation 4
Tablets
A compound of the invention is admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active amide compound) in a tablet press.
Formulation 5
Injection
A compound of the invention is dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/ml.
Formulation 6
Topical
Stearyl alcohol (250 g) and a white petrolatum (250 g) are melted at about 75° C. and then a mixture of a compound of the invention (50 g) methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate (10 g), and propylene glycol (120 g) dissolved in water (about 370 g) is added and the resulting mixture is stirred until it congeals.
Methods of Treatment
The present compounds are used as therapeutic agents for the treatment of conditions in mammals that are causally related or attributable to RORγ or RORγt activity. Accordingly, the compounds and pharmaceutical compositions of this invention find use as therapeutics for preventing and/or treating a variety of inflammatory conditions and autoimmune disorders in mammals, including humans.
In a method of treatment aspect, this invention provides a method of treating a mammal susceptible to or afflicted with a condition associated with an inflammatory condition and/or an autoimmune disorder, which method comprises administering an effective amount of one or more of the pharmaceutical compositions just described.
In additional method of treatment aspects, this invention provides methods of treating a mammal susceptible to or afflicted with an inflammatory condition or autoimmune disorder causally related or attributable to RORγt activity. Such condition and disorders include, without limitation, arthritis, diabetes, multiple sclerosis (and the animal model thereof, EAE), uveitis, rheumatoid arthritis (and the animal model thereof, CIA), psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, cancer, graft-versus-host disease, H. pylori infections and ulcers resulting from such infection, and inflammatory bowel diseases. Such methods comprise administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions just described.
The present inventors have shown that treatment of wildtype cells with RORγt inhibitor digoxin resulted in changes in gene expression that were very similar to those observed in RORγt-deficient cells. See Huh et al. (2011) Digoxin and its derivatives suppress Th17 cell differentiation by antagonizing RORγt activity. Nature, in press, the entire contents of which is incorporated herein by reference. That being the case, RORγt inhibitors can be used to treat any diseases caused by RORγ or RORγt expressing cells, including Th17, NK22, and other innate lymphoid cells. The pro-atherogenic contribution of IL-17 to atherosclerotic lesions, for example, suggests that atherosclerosis can be treated efficaciously with the compounds and compositions described herein. See Chen et al. J Innate Immun. 2010; 2(4):325-33. Epub 2010 May 7, the entire contents of which is incorporated herein by reference.
As a further aspect of the invention there is provided the present compounds for use as a pharmaceutical especially in the treatment or prevention of the aforementioned conditions and diseases. Also provided herein is the use of the present compounds in the manufacture of a medicament for the treatment or prevention of one of the aforementioned conditions and diseases.
Injection dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to 96 hours. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient.
For the prevention and/or treatment of long-term conditions, such as, e.g., arthritis, diabetes, multiple sclerosis (and the animal model thereof, EAE), rheumatoid arthritis (and the animal model thereof, CIA), psoriasis, or asthma, the regimen for treatment usually stretches over many months or years, so oral dosing is preferred for patient convenience and tolerance. With oral dosing, one to five and especially two to four and typically three oral doses per day are representative regimens. Using these dosing patterns, each dose provides from about 0.01 to about 20 mg/kg of the compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg and especially about 1 to about 5 mg/kg.
Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses. Modes of administration suitable for mucosal sites are also envisioned herein and include without limitation: intra-anal swabs, enemas, intranasal sprays, and aerosolized or vaporized compounds and/or compositions for delivery to the lung mucosa. One of skill in the art would choose an appropriate delivery mode/s based on a variety of parameters, including the organ or tissue site in a patient with a disease or condition that is most severely affected by the disease or condition. A skilled practitioner could, for example, treat a patient afflicted with an inflammatory bowel disease (IBD) with a therapeutic regimen that included delivery of the compounds or compositions of the invention using an enema for direct delivery to the bowel.
When used to prevent the onset of an inflammatory condition or autoimmune disorder, the compounds of this invention will be administered to a patient at risk for developing the condition or disorder, typically on the advice and under the supervision of a physician, at the dosage levels described above. Patients at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.
The compounds of this invention can be administered as the sole active agent or they can be administered in combination with other agents, including other compounds that demonstrate the same or a similar therapeutic activity and are determined to safe and efficacious for such combined administration.
General Synthetic Procedures
The compounds of this invention may be purchased from various commercial sources or can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.
The compounds of the invention may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis. Compounds according to formula I can be prepared by the method reported by Lu, Y. et al, Chem. Commun., 2010, 7578-7580.
Exemplary Compounds of the Invention
The following compounds, as exemplified in Table 1, have been purchased or can be prepared according to the general synthetic methods known to one skilled in the art of organic synthesis. Catalog numbers listed below refer to the NCGC numbers.







TABLE 1







Exemplary Compounds of the Invention









NCGC #
ID
Structure












NCGC00168746
1






 


NCGC00185070
2






 


digoxin
3






 


b-acetyl-digoxin
4






 


NCGC00095652
5






 


NCGC00016442
6






 


MLS000728569
7






 


MLS000696943
8






 


NCGC00167761
9






 


MLS000402315
10






 


MLS001217081
11






 


MLS000526165
12






 


MLS000777984
13






 


MLS000772650
14






 


MLS000767819
15






 


MLS000778763
16






 


MLS001066232
17






 


MLS001066267
18






 


MLS000118457
19






 


MLS001048850
20






 


MLS000700815
21






 


MLS000566729
22






 


MLS000592121
23






 


MLS001034558
24






 


MLS000569401
25






 


MLS000324596
26






 


MLS001201644
27






 


MLS001157801
28






 


MLS001141034
29






 


MLS000394148-02
30













Assays
Development of a High-Throughput Screening Assay
Methods:
The Drosophila S2 cell line was originally purchased from Invitrogen and was maintained in Schneider's medium supplemented with 10% heat-inactivated bovine fetal calf serum and antibiotics (Invitrogen). Gal4 DNA binding domain (G4 DBD) corresponding to amino acids 1 through 147 of Gal4 protein was PCR amplified to make a fusion construct with mouse RORγt (amino acids 79 to the carboxyl terminal end) lacking its DNA binding domain. The resulting chimeric gene was subcloned into the copper inducible pMT/V5-His A vector (available from Invitrogen). In a similar manner, Gal4 fusion constructs were prepared with mouse RORα (amino acids 142 to end) or Drosophila DHR3 (amino acids 120 to end). Coding sequences for firefly luciferase (available from Promega) were PCR amplified and subcloned into pUAST vector containing Gal4 binding enhancer sequences. pUAST is a commonly used Drosophila vector used by ordinarily skilled practitioners and has been described by Brand et al. (Development 118(2):401-415, 1993), the entire contents of which is incorporated herein in its entirety.
Renilla luciferase construct under the polIII promoter was obtained from Dr. Dasgupta's laboratory (NYU medical center). In order to generate stable cell lines, cells were co-transfected with pMT-G4 DBD-RORγt, pMT-G4 DBD-RORα, pMT-G4 DBD-DHR3, or pMT-G4DBDVP16 and pHygro plasmids (Invitrogen) and screened for resistant colonies in the presence of hygromycin (0.3 mg/mL). More than 150 independent RORγ stable clones with luciferase reporters (pMt-RORγ_luc) were tested for their suitability for the high-throughput screening (HTS) based on the following criteria: high signal-to background ratio in 384- and 1,536-well plates, their high induction rate upon the addition of copper, and RORγ/γt specificity probed by the dsRORγ- or RORγ/γt antagonist-mediated suppression. Positive clones were further transfected with pUAST-firefly luciferase, polIII-Renilla luciferase, and pCoPuro (Iwaki, Figuera et al. 2003) (“Rapid selection of Drosophila S2 cells with the puromycin resistance gene.” Biotechniques 35(3): 482-4, 486.) and selected with puromycin (2.5 ug/ml). Seven clones were finally selected, and one of them (stable clone #25) was subsequently used for large-scale HTS. Using similar methods, stable clones with genomic integration of the luciferase reporters and other reporters were generated such as pMT-RORα_luc, pMT-DHR3_luc, or pMT-VP16_luc.
Results and Discussion:
An activity-based assay system that permits high-throughput screening (HTS) for chemical modulators of RORγt transcriptional activity was developed. Since RORγt is located exclusively in cell nuclei, even when mutations are introduced in the putative ligand binding pocket, RORγt-dependent transcriptional activation, rather than the often used ligand-induced cytoplasm to nucleus translocation of hormone receptor, served as a better read-out. A cell-based assay was used to eliminate cell-impermeable or toxic molecules and to perform screening in a biologically relevant setting. The system employed provided a high signal-to-noise ratio and was able to handle a large-scale screen, as well as be cost-effective. S2 cells were derived from late stage Drosophila melanogaster embryos with hemocyte-like morphology and gene expression profiles (Schneider, I. J Embryol Exp Morphol, 1972, 27, 353-65). They grew at room temperature as a semi-adherent monolayer with no requirement for CO2, making it easy to apply large sets of small chemical molecules and to transfer cells without trypsin treatment.
Like other hormone receptors, RORγt contains both a DNA binding domain (DBD) and a ligand binding domain (LBD). The DBD was replaced with the heterologous yeast GAL4 DBD, because the endogenous DNA binding sites for RORγt are less well characterized.
The GAL4-RORγt fusion construct was placed under the control of a copper inducible promoter, and stable S2 cell lines with genomic integration of this construct were generated. The copper inducible promoter ensures a tight regulation of GAL4-RORγt expression and allows small molecules to get into the cells prior to its induction, thus increasing their effects on GAL4-RORγt. The reporter cells also carried the firefly luciferase reporter, whose expression is regulated by five copies of the GAL4 binding site enhancer (UAS) and a constitutive heat shock promoter, along with a control plasmid, pol III-driven Renilla luciferase. Pol III-Renilla luciferase was previously shown to serve as an excellent transfection and cell viability control in the S2 cell system. Use of Pol III-Renilla luciferase permitted normalization of RORγt-driven firefly luciferase activity and reduced cytotoxic effects and corrected for potential imprecise dispensation of cells in culture medium (Armknecht, S. et al. Methods Enzymol, 2005, 392, 55-73). When Cu++ was added, the ratio of firefly to Renilla luciferase activity (FR ratio) in these cells increased more than 100-fold compared to control cells lacking GAL4-RORγt ( ̃34 fold increase compared to GAL4-RORγt cells treated with dsROR). RORγt induces robust transcriptional activation in Drosophila S2 cells in 384-well plates. For test with transient transfection, firefly reporter under GAL4 binding sites and Pol III-Renilla control plasmids were transiently transfected along with dsRNA (75 ng), targeting EYFP or RORγ, into both control or pMT-GAL4-RORγt stable S2 cell lines (10,000 cells/well). After three days, copper was added to induce GAL4-RORγt, and dual luciferase activity was measured following overnight incubation. The increase was also observed when the experiment was carried out in 384-well plates, demonstrating that it can be adopted as a high-throughput screen. Co-transfecting dsRNA that targets RORγt suppressed ROR-mediated firefly luciferase induction, demonstrating that this activity is ROR dependent.
In order to confirm that the RORγt function in S2 cells was physiologically relevant, it was first confirmed that Drosophila has a RORγt homologue. Mouse encodes three different ROR proteins, RORα, RORβ, and RORγ. RORγ and RORγt are two isoforms that differ in non-translated N-terminal mRNA sequences. Indeed, Drosophila has one ROR homologue, Drosophila hormone receptor 3(DHR3) (King-Jones, K. & Thummel, C. S, Nat Rev Genet, 2005, 6, 311-23). Structure-based alignment using BLAST revealed 48% amino acid identity between DHR3 and RORγt.
Next it was confirmed that RORγt ligands are likely present in Drosophila S2 cells. Since many ligands for nuclear hormone receptors are found to be sterols or their derivatives, growth of cells in medium lacking FBS (fetal bovine serum) or medium supplemented with fatty-acid stripped serum (charcoal treated) was attempted. Only a small decrease of the FR ratio was detected in cells grown in this condition, and the results were not conclusive. Previous studies have shown that the introduction of a single amino acid change inside the ligand binding pocket of RORβ abrogates its function as a transcriptional activator, suggesting that RORβ is a ligand-dependent hormone receptor. The crystal structure of the protein strongly suggested that replacement of alanine 269 with amino acids carrying bulkier side chains, such as valine and phenylalanine, would prevent binding of endogenous ligands without affecting the correct folding of the ligand binding domain (Stehlin, C. et al. Embo J, 2001, 20, 5822-31). When the corresponding alanine residue in the putative ligand binding pocket of RORγt was replaced with phenylalanine, the mutant protein was no longer sufficient to induce Th17 cell differentiation when transduced into naive mouse CD4+ T cells, consistent with the presence of cognate RORγt ligand(s) in these cells. The Ala to Phe mutation in RORγt also completely abrogated transcriptional activation of firefly luciferase expression without affecting the transcription of control Renilla luciferase, suggesting that RORγt ligand is present in the Drosophila assay system. Indeed, introduction of alanine to phenylalanine (F) mutation in a putative ligand binding pocket abolishes activity of RORγt in this system. As confirmed by immunoblot, the alanine to phenylalanine mutation did not, however, affect protein stability.
DHR3 is transcriptionally regulated by 20-hydroxyecdysone (20E) and is essential for fly larval development (King-Jones, supra). It has been shown that E75, another 20E-dependent fly nuclear hormone receptor, negatively regulates the function of DHR3 (White, K. P., Hurban, P., Watanabe, T. & Hogness, D. S. Science, 1997, 276, 114-17; Reinking, J. et al. Cell, 2005, 122, 195-207. Indeed, co-expression of E75a or E75b (two Drosophila E75 isoforms) decreased the level of DHR3-mediated transcriptional activation in a dosage-dependent manner. E75 is a hormone receptor having antagonizing activities of DHR3 and RORγt. Transfection of increased amount of E75a or E75b resulted in concomitant reduction of FR ratio. Each well received the same amount of DNA in transfection mix. These fly genes also function as dosage-dependent negative regulators for mouse RORγt activity in S2 cells, without affecting the functions of an irrelevant transcriptional activator VP16, strongly suggesting that the ROR/DHR3 core regulatory mechanism is conserved between mouse and fly systems. Collectively, these data confirm the accuracy and relevance of the above approach utilizing the heterologous S2 cell system in order to identify chemical agonists or antagonists of the mouse hormone receptor RORγt.
Measurement of Luciferase Activity.
Methods:
Promega Dual-Glo system is widely used for HTS as the luciferase substrates. Cell culture medium was reduced to the final volume of 10 μl or less and 10 μl of Dual-glo and 10 μl of Stop-glo luciferase substrates were added in a sequencial manner (Promega). Firefly and Renilla luciferase activity was determined by measuring luminescence signals using an automated 384-well plate reader equipped with multiple stack units (such as the Analyst GT or Envision plate readers).
Results and Discussion:
The Dual-Glo luciferase assay system from Promega facilitated measuring luciferase activity in HTS. First, it did not require a washing step, and the luciferase activities of both the firefly and the Renilla could be measured in the same well one after the other. Second, the signals that it produced remained stable for more than two hours, making it possible to measure activity from many different plates for a given experiment. Since the reagents are expensive, the volume of medium was reduced to one third prior to adding luciferase substrates, so that fewer substrates were used. However, when minimizing cost is less of a priority, the luciferase substrates used in the primary assay may be directly added to cells without going through this additional step.
HTS for Identification of RORγ/γt Antagonists using RORγ/γt-luc Stable Cell Lines
Methods:
600 G4 DBD-RORγ/γt-luc reporter (#25) or G4 DBD-VP16-luc reporter cells were distributed into each well of 1,536-well white bottom plates in 4 μl S2 cell culture volume containing hygromicin (300 μg/ml) and puromycin (2 μg/ml). Small compounds in seven different concentrations range from 46 μM to 1.7 nM were pin-transferred (23 nl) by a robot (Inglese et al., 2006, Proc. Natl. Acad. Sci. 103:11473-8). After one-hour incubation, 1 μl of culture medium solution containing copper sulfate (to final 0.7 mM) and 10 μM digoxin (or other RORγ/γt antagonists) were added to each well. After 20-hour incubation at ambient temperature, 1.5 μl firefly luciferase detection mix was added and the luciferase activities were measured in 10 min. ViewLux luminometers were used for measuring luciferase signal.
Results and Discussion:
Even though G4BD-RORγt stable cells with transient transfection of two luciferase constructs were successfully used for small-scale screening and led the present inventors to identify specific RORγ/γt antagonists such as digoxin and digitoxin, it became problematic to apply the same method to larger scale screens with chemical libraries covering more than 250,000 compounds. First, transient transfection method often produced well-to-well variation due to incomplete mixing and unequal distribution of transfection mix. There is also day-to-day variation when preparing the master transfection mix. Second, in order to save reagent and to handle large quantities of chemicals, it was necessary to do the screen with reduced numbers of cells in a smaller culture volume. Moreover, performing the screen with 1,536-well plates is more efficient than performing the screen with 384-well plates. Thus, new RORγ/γt-luc reporter stable cells were developed to eliminate the necessity of repeated transfection and to achieve increased well-to-well consistency. When tested in a 384-well plate, even small numbers of cells (400 cells/well) gave high firefly luciferase signal (thus high RORγ/γt activity), which is suppressed (22-fold reduction when 400 cells are used) by addition of antagonist digoxin. The new stable cell line systems also turned out to be suitable for HTS in 1,536-well plates, because z′ value is 0.75 when 600 cells are used per well with 10 μl total cell culture volume.
Using RORγ/γt-luc reporter lines, the pilot screen with the LOPAC (Sigma) library was performed through a NIH roadmap program to identify RORγ/γt antagonist compounds. LOPAC contains 1,280 pharmacologically active compounds. Among these, approximately 40 compounds were found as initial hits ( ̃0.3%). These hits were tested against validation screens to identify RORγ/γt specific compounds. To facilitate a large-scale validation, VP16-luc reporter cell lines with genomic integrations of G4BD-VP16, UASt-ffluc, and polIII-Rluc were developed as discussed earlier. These cells also exhibited robust and consistent VP16 activity in 1,536-well plates (data not shown), and thus can be used as a good control reporter to weed-out compounds inhibiting general transcription, the function of GAL4 DNA binding domain, and a nuclear import of GAL4 chimeric proteins. Indeed, many compounds were identified as initial hits to reduce RORγ/γt activity from the pilot LOPAC screen, but later were found to be non-specific inhibitors, because they reduced both RORγ/γt and VP16 activities. Intriguingly, both digoxin and β-acetyl-digoxin, previously identified RORγ/γt specific antagonists from small scale HTS, selectively inhibited RORγ/γt activity without affecting VP16, confirming their specificity on RORγ/γt. A large-scale screen covering more than 250,000 compounds was carried out against RORγ/γt-luc and VP16-luc reporter systems in parallel to identify RORγ/γt specific antagonists in a systematic manner.
Secondary assays directed toward evaluating the ability of such compounds to suppress mouse or human Th17 cell differentiation are also encompassed herein. Exemplary secondary assays that serve to confirm bona fide RORγ inhibitors include without limitation:
Secondary Screening Lists
S2 cell reporter system: Identified hits from HTS are further screened and their specificity confirmed by testing against RORγ-luc, VP16-luc, RORα-luc, and DHR3-luc reporter S2 cell lines. Compounds either having no activity on VP16, RORα, and DHR3 or having ten times higher IC50 values on such reporters are selected for further tests.
Cytokine induced mouse Th17 cells differentiation. Effects on mouse endogenous RORγt in a relevant physiological setting were determined by testing compounds in the Th17 cell differentiation assay. Compounds having RORγt inhibitory activity are predicted to suppress Th17 cell differentiation. Th1 or regulatory T cell differentiation was used as a counter-screen method to select specific compounds that only affect Th17 cell differentiation without having pleiotrophic effects on general T cell proliferation or cytokine production.
ROR dependent Th17 cell differentiation. Compounds were further tested, by examining their effects on T cells expressing RORα or RORγ. Compounds that directly inhibit RORγ are expected to inhibit RORγ- but not RORα-dependent Th17 cell differentiation. Compounds affecting IL17a production or ROR regulatory pathways, however, are expected to inhibit both.
Human Th17 cell differentiation. Compound effects on human RORγt will be tested by treating human cord blood CD4 T cells with select compounds to determine if such compounds alter differentiation into Th17 lineages.
With aspects of the claimed invention now being generally described, these will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain features and embodiments of the presently claimed invention and are not intended to be limiting.
EXAMPLES
As detailed above, in order to identify small molecules to antagonize RORγ/γt transcriptional activity, the present inventors developed insect cell line based reporter systems, expressing murine RORγ/γt or closely related transcriptional activators. Since their cognate DNA binding sites were not well characterized, the DNA binding domains (DBD) of RORγ/γt, RORα(mouse homolog for PORγ), and DHR3 (Drosophila orthologue for ROR family proteins) were replaced with the heterologous yeast GAL4 DBD. The transcriptionally active domain of general transcriptional activator VP16 was also fused with GAL4 DBD. The GAL4 fusion constructs were placed under the control of a copper inducible promoter, and stable S2 cell lines with genomic integration of these four reporter constructs were generated. The copper inducible promoter ensures tight regulation of GAL4-fusion protein expression and it allows small molecules to get into the cells prior to protein induction, thus increasing their effects on GAL4 reporters. The stable reporter cell lines also encode the firefly luciferase reporter, whose expression is regulated by five copies of the GAL4 binding site enhancer (UAS), along with the pol III-driven Renilla luciferase reporter. Pol III-Renilla luciferase was included to serve as cell viability control in the S2 cell system (Armknecht, S. et al. Methods Enzymol 392, 55-73 (2005).
Table 2 below presents a list of compounds, which were identified by the HTS and additional confirmatory and secondary assays to be inhibitors of RORγt. The table lists the RORγ/γt-inhibiting ability of these compounds as measured by FR ratio (indicated as percentile normalized to DMSO control) as well as the IC50 values:







TABLE 2







Screening Data of Exemplary Compounds









NCGC #
80% Inhib. @ μM
IC50 (μM) at S2 cell












MLS000566729-02
0.438944625
0.13395


NCGC00167761-02
0.775863419
0.28105


MLS001048850-02
1.645808001
0.543325


NCGC00185070-01
3.025993987
1.27125


MLS000324596-02
3.293296235
2.10825


MLS000728569-02
4.271924974
1.462925


NCGC00168746-02
6.270268372
1.9906


MLS000402315-02
7.292161088
2.610025


MLS000767819-02
7.561862037
2.7818


MLS001034558-02
8.843053355
1.6036


MLS001066267-01
10
5


NCGC00095652-02
10.59736565
3.971875


MLS000772650-02
12.38316505
8.671125


MLS000696943-02
35.5651686
13.3996


NCGC00016442-02
38.20138301
9.69525


MLS001201644-02
40.36349458
16.7467


MLS001157801-02
45.84869793
14.677325


MLS000592121-02
62.16624241
17.843


MLS001141034-02
161.1873652
38.732425









Table 3 below presents a list of additional heterocyclic compounds of the invention, which were identified by the HTS and additional confirmatory and secondary assays to be inhibitors of RORγt. The table lists the RORγ/γt AC50 and the curve class values. Concentration response curves (CRC) are placed into four curve class values. Class 1 contains complete CRCs showing both upper and lower asymptotes and r2 values >0.9. Class 2 contains incomplete CRCs having only one asymptote and shows r2 values >0.9. Class 3 curves are of the lowest confidence because they are poorly fit or based on activity at a single concentration point. Class 1 and 2 curves are divided further into subclasses to indicate efficacies 80% or greater (Class 1.1 and 2.1) or between 30% and 80% (Class 1.2 and 2.2). Class 4 compounds are poorly active having either no curve-fit or an efficacy below threshold activity (3 SD of the mean activity).







TABLE 3







Screening Data of Exemplary Compounds














Curve



Sample ID
ID
Structure
Class
AC50(uM)














MLS001066268-01
31




−2.1
8.9125


 


MLS001066231-01
32




−2.1
10


 


MLS001066267-01
33




−2.1
5.0119


 


MLS001066482-01
34




−2.1
12.5893


 


MLS001066318-01
35




−2.1
7.9433


 


MLS001066232-01
36




−2.1
10


 


MLS001066096-01
37




−2.2
8.9125


 


MLS001066483-01
38




−2.2
12.5893


 


MLS001066281-01
39




−2.2
11.2202


 


MLS001066026-01
40




−2.2
17.7828


 


MLS001066523-01
41




−3
15.8489


 


MLS001066270-01
42




−3
12.5893


 


MLS001066276-01
43




−3
17.7828


 


MLS001066319-01
44




−3
15.8489


 


MLS001066241-01
45




4
3.9811


 


MLS001066294-01
46




4
0.1122


 


MLS001066263-01
47




4
0.0045


 


MLS001066242-01
48




4
0.0224


 


MLS001066301-01
49




4
0.631


 


MLS001066305-01
50




4
0.8913


 


MLS0010660292-01
51




4
11.2202


 


MLS001066046-01
52




4
0.0126


 


MLS001066502-01
53




4
0.7943


 


MLS001066286-01
54




4
null


 


MLS001066291-01
55




4
0.1122


 


MLS001066230-01
56




4
35.4813


 


MLS001066259-01
57




4
35.4813


 


MLS001066306-01
58




4
7.0795


 


MLS001066238-01
59




4
35.4813


 


MLS001066025-01
60




4
35.4813


 


MLS001066443-01
61




4
3.9811


 


MLS001066302-01
62




4
null


 


MLS001066321-01
63




4
6.3096


 


MLS001066228-01
64




4
0.1413


 


MLS001066035-01
65




4
3.9811


 


MLS001066308-01
66




4
null


 


MLS001066279-01
67




4
null


 


MLS001066454-01
68




4
11.2202


 


MLS001066503-01
69




4
2.5119


 


MLS001066256-01
70




4
null


 


MLS001066275-01
71




4
5.0119


 


MLS001066253-01
72




4
null


 


MLS001066225-01
73




4
35.4813


 


MLS001066514-01
74




4
14.1254


 


MLS001066297-01
75




4
0.8913


 


MLS001066243-01
76




4
0.8913


 


MLS001066248-01
77




4
null


 


MLS001066453-01
78




4
0.2818


 


MLS001066492-01
79




4
0.1413


 


MLS001066313-01
80




4
0.0447


 


MLS001066244-01
81




4
0.0056


 


MLS001066250-01
82




4
4.4668


 


MLS001066311-01
83




4
10


 


MLS001066434-01
84




4
3.1623


 


MLS001066444-01
85




4
0.0071


 


MLS001066254-01
86




4
1.122









Tables 4 and 5 show the results from FACS plot analyses demonstrating that the indicated RORγ inhibitory compounds (tested at 5, 10, and 20 μm, as indicated) suppress mouse Th17 cell differentiation, as measured by IL17a production, when compared to DMSO treated cells. Naive CD4 T cells were cultured with CD3/CD28 stimulatory antibodies in presence of TGFβ and IL6. Compounds were added on Day 1 and cells were analyzed on Day 4. Numbers shown in Table 3 indicate the percentage of mouse Th17 cells.







TABLE 4







Compounds with RORγ and Th17 inhibitory activity











NCGC_ID
5 μM
20 μM















NCGC00168746-02
4.5
 1.2



NCGC00185070-01
7.4
 1.5



NCGC00016442-02
4.8
 2.6



MLS000696943-02
13.9
 3.1



NC0000167761-02
11.6
 3.6



MLS000402315-02

 4.8



NCGC00095652-02
11.1
 6.7



MLS001217081-02

 8.3



MLS000526165-02

 8.3



MLS000777984-02

 8.9



MLS000772650-02
16.9
 9.9



MLS000767819-02

10.2



MLS000778763-02

10.2



MLS001066232-01

10.4 (at 10 uM)



MLS000118457-02

10.7



MLS001048850-02

11.9



MLS000700815-02

11.9



MLS000566729-02
16.3
12.1



MLS001066267-01

13.5 (at 10 uM)



MLS000592121-02
27.7
27.7



MLS000728569-02
3.7
dead



MLS001034558-02
14.3
dead



MLS000569401-02
15




DMSO
25%
17%




Th17
Th17

















TABLE 5







Additional compounds with RORγ and Th17 


inhibitory activity tested at 10 μm











Th17




10 μM














digoxin
0.91



b-acetyl-digoxin
1.17



DMSO
26%










FIG. 1 depicts the RORγt specific inhibitory effects of digoxin and derivatives on mouse CD4 T cells. Briefly, FACS-sorted naive T cells were transduced with retroviral vectors encoding murine RORα or RORγt on Day 1 (16 hours after TCR stimulation) and intracellular staining was performed on Day5. DMSO or Digoxin was added 6 ̃8 hours after viral transduction.
FIG. 2 depicts the RORγt specific inhibitory effects of ursolic acid on mouse CD4 T cells. Briefly, the figure shows flow cytometry of intracellular staining for IL-17a and IFN-γ by sorted naive T cell populations activated and expanded in the presence of mouse Th17 cytokines (IL-6 and TGF-β). CD25−, CD62L+, and CD44int-high cells were sorted from B6 mice. DMSO or ursolic acid was added on 16 hours after cell plating and cells were analyzed on day 5.
FIG. 3 depicts the RORγt specific inhibitory effects of ursolic acid in the S2 cell assay. The results show that ursolic acid treatment at various concentrations (μM) as indicated on x-axis leads to more potent suppression of RORγ reporter activity compared to other closely related reporters. Relative luciferase activity is shown on the y-axis.
FIG. 4 illustrates the RORγt specific inhibitory effects of ursolic acid on human CD4 T cells. Briefly, the figure shows flow cytometry of the production of IL-17a and IFN-γ by human naive cord blood T cells (CD45RO−CD45RA+CD3+CD4+CD25−HLA-DR−), which were cultured for six days in presence of IL2, IL23, ILβ, with various concentrations of TGFβ. DMSO or ursolic acid was added 16 hours after cytokine addition.
FIG. 5 shows the RORγt specific inhibitory effects of the indicated compound on the S2 cell assay and murine CD4 T cells. For S2 cells, compound 1066232 leads to inhibition of RORγ reporter without affecting the activity of other closely related reporters. X-axis denotes compound concentrations and y-axis indicate relative luciferase activity. For mouse CD4 T cells, flow cytometry of intracellular staining for IL-17a and IFN-γ by sorted naive T cell populations activated and expanded in the presence of mouse Th17 cytokines (IL-6 and TGF-β). CD25−, CD62L+, and CD44int-high cells were sorted from B6 mice. DMSO or compound 1066232 or 1066267 were added 16 hours after plating cells. FACS analysis was done at day 5.
FIG. 6 reveals the RORγt specific inhibitory effects of the indicated compounds on human CD4 T cells. Compounds 1066232 and 1066267 inhibit RORγ overexpresison dependent, but not RORα dependent, IL17a production in human CD4 T cells, demonstrating their specificity as RORγ inhibitors. The results show flow cytometry of the production of IL-17a and IFN-γ by peripheral blood naive CD4+ T cells (CD45RA+CD3+CD4+) transduced with RORαd-IRES-GFP or RORγt-IRES-GFP on day 1 and analyzed on day 6. GFP expressing cells were gated for analysis. DMSO or compounds were added 6-8 hours after viral transduction.
FIG. 7 shows the RORγt specific inhibitory effects of the indicated compound on murine CD4 T cells. The figure shows flow cytometry of intracellular staining for IL-17a and IFN-γ by sorted naive T cell populations activated and expanded in the presence of mouse Th17 cytokines (IL-6 and TGF-β). CD25−, CD62L+, and CD44int-high cells were sorted from B6 mice. DMSO or compound MLS000777984 was added 16 hours after plating T cells. FACS analysis was done at day 5.
FIG. 8 depicts the RORγt specific inhibitory effects of the indicated compound in the S2 cell assay. Compound MLS000767819 treatment at various concentrations (μM) as indicated on x-axis leads to suppression of RORγ reporter activity without having effects on other closely related reporters. Relative luciferase activity is shown on the y-axis.
From the foregoing description, various modifications and changes in the compositions and methods of this invention will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
The chemical names of compounds of invention given in this application are generated using Open Eye Software's Lexichem naming tool, Symyx Renassance Software's Reaction Planner or MDL's ISIS Draw Autonom Software tool and not verified. Preferably, in the event of inconsistency, the depicted structure governs.

## Claims

1. A method for treating in a mammal an autoimmune or inflammatory disease or condition, which comprises administering to the mammal an effective disease-treating or condition-treating amount of a compound according to formula I:



wherein
R1 is OH, or substituted or unsubstituted alkoxy;
R2 is H, or Me;
each R3 and R4 is independently selected from H, OH, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted acyl, substituted or unsubstituted acylamino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylarylamino, substituted or unsubstituted amino, substituted or unsubstituted arylalkyl, sulfo, substituted sulfo, substituted sulfonyl, substituted sulfinyl, substituted sulfanyl, substituted or unsubstituted aminosulfonyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, azido, substituted or unsubstituted carbamoyl, carboxyl, cyano, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted dialkylamino, halo, nitro, and thiol; and
each n1 and n2 is independently 1, 2, 3, 4 or 5;
or a pharmaceutically acceptable salt or solvate thereof; and
stereoisomers, isotopic variants and tautomers thereof, wherein the autoimmune or inflammatory disease or condition is arthritis, diabetes, multiple sclerosis, uveitis, psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, H. pylori infection, ulcers resulting from H. pylori infection, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, sprue, food allergies, experimental autoimmune encephalomyelitis (EAE).

2. The method according to claim 1, wherein the compound is according to formula II



and wherein R1, R3, R4, n1 and n2 are as in claim 1;
or a pharmaceutically acceptable salt or solvate thereof; and
stereoisomers, isotopic variants and tautomers thereof.

3. The method according to claim 1, wherein the compound is according to formula III:



wherein
and wherein R1, R3, R4, n1 and n2 are as in claim 1;
or a pharmaceutically acceptable salt or solvate thereof; and stereoisomers, isotopic variants and tautomers thereof.

4. The method according to claim 1, wherein each R3 is H.

5. The method according to claim 1, wherein n1 is 1, 2, or 3; and each R3 is independently halo, alkyl, OH, alkoxy, haloalkyl, OCF3, or CN.

6. The method according to claim 1, wherein each R4 is H.

7. The method according to claim 1, wherein n2 is 1, 2, or 3; and each R4 is independently halo, alkyl, OH, alkoxy, haloalkyl, OCF3, or CN.

8. The method according to claim 1, wherein the compound is according to formula IVa, IVb, IVc, or IVd:



wherein
and wherein R1 is OMe or OEt; and each of R3 and R4 are independently selected from H, OMe, F, Cl, Me, or CF3;
or a pharmaceutically acceptable salt or solvate thereof; and stereoisomers, isotopic variants and tautomers thereof.

9. The method of claim 1, wherein the compound is










































or a pharmaceutically acceptable salt or solvate thereof; and stereoisomers, isotopic variants and tautomers thereof.

10. The method of claim 1, wherein the arthritis is collagen-induced arthritis (CIA).

11. The method of claim 1, wherein the arthritis is rheumatoid arthritis.