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Title:
INHIBITORS OF SUPPRESSION OF TUMORIGENCITY 2 (ST2) AND METHODS USING THE SAME
Document Type and Number:
WIPO Patent Application WO/2017/083242
Kind Code:
A1
Abstract:
Compounds capable of inhibiting suppression of tumorgenicity 2 (ST2) are disclosed. The compounds are useful in the treatment of a variety of diseases and conditions, such as alleviation of graft-versus-host disease (GVHD), cardiovascular diseases, asthma, lupus, and other Thl/Th2 imbalanced inflammatory diseases.

Inventors:
YANG CHAO-YIE (US)
PACZESNY SOPHIE (US)
Application Number:
PCT/US2016/060890
Publication Date:
May 18, 2017
Filing Date:
November 08, 2016
Export Citation:
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Assignee:
UNIV MICHIGAN REGENTS (US)
UNIV INDIANA RES & TECH CORP (US)
International Classes:
C07D405/14; A61P9/00; A61P11/06; A61P29/00; C07D209/08; C07D209/86; C07D209/88; C07D211/16; C07D307/52; C07D401/06; C07D403/06; C07D405/06; C07D453/02; C07D471/04
Domestic Patent References:
WO2013173761A22013-11-21
WO2008144610A12008-11-27
Other References:
DANIEL CHAN ET AL: "Structure-Based Discovery of Natural-Product-like TNF-[alpha] Inhibitors", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 49, no. 16, 6 April 2010 (2010-04-06), DE, pages 2860 - 2864, XP055338057, ISSN: 1433-7851, DOI: 10.1002/anie.200907360
FUNG-YI CHAN ET AL: "Identification of a New Class of FtsZ Inhibitors by Structure-Based Design and in Vitro Screening", JOURNAL OF CHEMICAL INFORMATION AND MODELING, vol. 53, no. 8, 26 August 2013 (2013-08-26), US, pages 2131 - 2140, XP055338059, ISSN: 1549-9596, DOI: 10.1021/ci400203f
BANERJEE MONIMOY ET AL: "Differential regulation of CYP3A4 promoter activity by a new class of natural product derivatives binding to pregnane X receptor", BIOCHEMICAL PHARMACOLOGY, vol. 86, no. 6, 2013, pages 824 - 835, XP028707033, ISSN: 0006-2952, DOI: 10.1016/J.BCP.2013.07.023
VANDER LUGT ET AL., N. ENGL. J. MED., vol. 396, no. 6, 2013, pages 529 - 539
BAELL, J. B.; HOLLOWAY, G. A.: "New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays", J MED CHERN, vol. 53, 2010, pages 2719 - 2740
J. ZHANG ET AL., SCI TRANSL MED, vol. 7, no. 308, 2015
Attorney, Agent or Firm:
NAPOLI, James, J. (US)
Download PDF:
Claims:
WHAT IS CLAIMED:

1. A method of inhibiting an interaction between ST2 and IL-33 comprising contacting the ST2 with one or more compound selected from the group consisting of

— CH2N

wherein R1 is selected from the group consisting of Rb , -Ci_3alkyl , substituted or unsubstituted heteroaryl, substituted and unsubstituted aryl,

and -CH2heterocycloalkyl;

R is selected from the group consisting

of -H, -Ci_3alkyl, -C(=0)Rc, -S02Ra, -C(=S)NHRd, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;

Ra is H, substituted or unsubstituted aryl, or Ci_4alkyl;

Rb is H, substituted or unsubstituted aryl, optionally

substituted -C(=S)NHaryl, -C(=0)Ci_3alkyl, optionally substituted heterocycloalkyl, optionally substituted -S02aryl, -C(=0)(CH2)i_ C02H, -C(=0)NHCi_3alkyl, or Ci_4alkyl;

Rc is Ci_3alkyl, -(CH2)i_3OCi_3alkyl, -(CH2)i_3C02H, substituted or unsubstituted aryl,

Rd is substituted or unsubstituted aryl, -(CH2)i_3OCi_4alkyl, -(CH2)i_3heteroaryl,

-(CH2)i_3heterocycloalkyl; and

n is 0 or 1 ;

or a pharmaceutically acceptable salt thereof;

wherein R is -C(=S)heterocycloalkyl (substituted), -(CH2)i_3heterocycloalkyl (substituted), -C(=0)NHaryl (substituted), heterocycloalkyl (substituted), or -(CH2)i_3N(Re);

R° is -N02, halo, or -CN; and

Re is H or -Ci_4alkyl;

or a pharmaceutically acceptable salt thereof;

wherein Z is null, NR or S;

\

C=0

D is -CH2- or /

R4 is phenyl, heteroaryl, -(CH2)i_2heteroaryl, cycloalkyl,

heterocycloalkyl, -C6H5-0-Rh, -(CH2)i_2-C6H5, or -(CH2)i_3ORh;

R5 is null, H, or -(CH2)i_3ORh, or

R4 and R5 are taken together with the nitrogen atom to which they are attached to form a heteroaryl group or a substituted or unsubstituted heterocycloalkyl group;

Rf is Ci_3alkyl or

Rg is H or halo; and

Rh is H or Ci_3alkyl, or

a pharmaceutically acceptable salt thereof; , or a pharmaceutically acceptable salt thereof.

The method of claim 1 wherein the ST2 is soluble ST2 (sST2).

3. The method of claim 1 wherein the ST2 is full-length membrane-spanning ST2 (FLST2).

4. A method of treating a disease or condition mediated by an interaction between ST2 and IL-33 comprising administering a therapeutically effective amount of an ST2 inhibitor to an individual in need thereof, wherein the ST2 inhibitor is a compound selected from the group consisting of

H

(i)

— CH2N

wherein R1 is selected from the group consisting of Rb , -Ci_3alkyl , substituted or unsubstituted heteroaryl, substituted and unsubstituted aryl,

and -CH2heterocycloalkyl;

R is selected from the group consisting

of -H, -Ci_3alkyl, -C(=0)Rc, -S02Ra, -C(=S)NHRd, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;

Ra is H, substituted or unsubstituted aryl, or Ci_4alkyl;

Rb is H, substituted or unsubstituted aryl, optionally

substituted -C(=S)NHaryl, -C(=0)Ci_3alkyl, optionally substituted heterocycloalkyl, optionally substituted -S02aryl, -C(=0)(CH2)i_ C02H, -C(=0)NHCi_3alkyl, or Ci_4alkyl;

Rc is Ci_3alkyl, -(CH2)i_3OCi_3alkyl, -(CH2)i_3C02H, substituted or unsubstituted aryl, ,Ra

— Nx

or Rb ;

Rd is substituted or unsubstituted aryl, -(CH2)i_3OCi_4alkyl, -(CH2)i_3heteroaryl, and -(CH2)i_3heterocycloalkyl; and

n is 0 or 1 ;

or a pharmaceutically acceptable salt thereof;

wherein R is -C(=S)heterocycloalkyl (substituted), -(CH2)i_3heterocycloalkyl (substituted), -C(=0)NHaryl (substituted), heterocycloalkyl (substituted), or -(CH2)i_3N(Re);

R° is -N02, halo, or -CN; and

Re is H or -Ci_4alkyl; or a pharmaceutically acceptable salt thereof;

(iii)

wherein Z is null, NR5 or S;

\

C=0

D is -CH2- or / ;

R4 is phenyl, heteroaryl, -(CH2)i-2heteroaryl, cycloalkyl,

heterocycloalkyl, -C6H5-0-Rh, -(CH2)i_2-C6H5, or -(CH2)i_3ORh;

R5 is null, H, or -(CH2)i_3ORh, or

R4 and R5 are taken together with the nitrogen atom to which they are attached to form a heteroaryl group or a substituted or unsubstituted heterocycloalkyl group;

Rf is Ci_3alkyl or

Rg is H or halo; and

Rh is H or Ci_3alkyl, or

a pharmaceutically acceptable salt thereof; , or a pharmaceutically acceptable salt thereof.

The method of claim 4 wherein the ST2 is soluble ST2 (sST2). The method of claim 4 wherein the ST2 is full-length membrane-spanning ST2 — CH2NH2 s optionally substituted with C6H4N CH3)2,

-CH2NHC(=0)(CH2)2C02H — CH2NHC(=0)NHC2H5

O

— CH2NH— < /— \ N- °C-CH3 2

8. The method of any precedin claim wherein R is -H, -C(=0)CH3, -C2H5,

-SO2CH3, -C(=0)NH-iPr, -C(=S)NH(CH2)2OCH3, ,

9. The method of any preceding claim wherein R° is N02, CN, CI, or F.

10. The method of any preceding claim wherein R is

1. The method of any preceding c

4 and R5 are taken together to form , or

or Z is NCH2CH2OH and R4 is -CH2CH2OH;

or Z is null and R4 is H or C6¾

12. The method of any of claims 3 through 11 wherein the disease or condition is graft-versus-host disease (GVHD), a cardiovascular disease, asthma, lupus, a Thl/Th2 imbalanced inflammatory disease, inflammatory bowel disease, rheumatoid arthritis, psoriasis, systemic sclerosis, linear fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, sepsis, trauma, HIV infection, systemic lupus erythematosus, Wegener's granulomatosis, Behchet disease, rhinosinusitis, nasal polyposis, eosinophilic bronchitis, atopic dermatitis, nasal polyposis, and a hematological cancer.

13. The method of any of claims 3 through 12 further comprising administering a therapeutically effective amount of a second therapeutic agent useful in the treatment of the disease or condition.

14. The method of any preceding claim wherein the compound is selected from the group consisting of

-46-

pharmaceutically acceptable salts thereof.

15. The method of claims 1 through 13 wherein the compound is selected from the group consisting of

and pharmaceutically acceptable salts thereof.

Description:
INHIBITORS OF SUPRESSION OF TUMORIGENCITY 2 (ST2) AND METHODS

USING THE SAME

STATEMENT OF GOVERNMENTAL INTEREST

[0001] This invention was made with government support under CA174667 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62/254,780, filed November 13, 2015, incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to inhibitors of Suppression of Tumorigenicity 2 (ST2) and to therapeutic methods of treating conditions and diseases wherein inhibition of ST2 provides a benefit.

BACKGROUND OF THE INVENTION

[0004] ST2, also known as IL1RL1, DER4 ,and Tl, is a binding receptor for interleukin-33 (IL-33), a cytokine related to IL-1 and IL-18. ST2 is expressed both as a soluble non- signaling variant (soluble ST2 or sST2) and a full-length membrane- spanning form (FL ST2, ST2 or ST2L) that mediates cellular responses to IL-33. The latter form is expressed on a wide range of cell types implicated in pathologic inflammation in a number of disease settings. These include lymphocytes, particularly IL-5 and IL-13 -expressing T helper cells, natural killer (NK), and natural killer-T (NKT) cells, as well as many innate immune cells, such as mast cells, basophils, eosinophils, macrophages, and innate helper cells (also known as nuocytes).

[0005] The presence of two ST2 isoforms, ST2L and sST2, creates an additional layer of complexity in the biology of IL-33 (Yanagisawaet al., 1993). ST2L or membrane ST2 (mST2) is the full-length protein and includes the extracellular immunoglobulin (Ig)-like domains, a short extracellular spacer, the transmembrane domain, and an intracellular TIR domain. Soluble ST2 (sST2) is a short form that lacks the final three exons, resulting in a soluble secreted protein consisting of the extracellular cytokine-binding domains. sST2 is present constitutively in human serum/plasma, where it acts as a decoy receptor by binding free IL-33. sST2 is increased by diverse inflammatory stimuli and in cardiovascular, rheumatologic, allergic diseases, and graft-versus-host disease (GVHD) potentially restricting the effects of systemic IL-33 (either deleterious or positive. In humans, genome-wide association studies (GWASs) have identified IL1RL1 variants associated with altered levels of serum sST2, which could influence susceptibility to IL-33-mediated responses.

[0006] IL-33 binding to ST2 on these cells leads to the recruitment of a broadly-expressed co-receptor known as the IL-1R Accessory Protein (AcP) and the activation of proinflammatory signaling, similar to IL-1 and IL-18. IL-33 thus is able to directly activate ST2- expressing cells or enhance their activation when in the presence of other activating stimuli. Examples of IL-33-induced cellular responses include the production of inflammatory cytokines, such as IL-5, IL-6, IL-13, TNF, IFN-g, and GM-CSF, as well as the production of chemokines, such as CXCL8, CCL17, and CCL24. IL-33 has also been shown to enhance acute allergic responses by augmenting mast cell and basophil activation triggered by IgE receptor signaling or other mast cell and basophil activators. IL-33 also enhances recruitment, survival, and adhesive properties of ST2 expressing immune cells, and thus is important in provoking and sustaining cellular inflammation in local tissues.

[0007] The pro-inflammatory actions of IL-33 on innate and adaptive immune cells culminate to promote a number of pathologic processes. In the lungs, these include increased airway inflammation, mucus production, airway hyper responsiveness, and fibrotic remodeling. IL-33 can also contribute to localized inflammation in the joints, as well as cutaneous and articular hypernociception, by promoting the production of proinflammatory cytokines. Excessive IL-33 has been linked to pathologic collagen deposition and fibrosis and also contributes to epithelial damage in the setting of inflammatory bowel disease.

Through its potent effects on basophils and IgE-sensitized mast cells, IL-33 also can trigger anaphylactic shock and may play a contributing role in allergic disease. Many of these diseases are chronic and progressive in nature and are difficult to treat.

[0008] There are several lines of evidence that the IL-33/ST2 pathway contributes to human disease. For example, abnormally high expression of IL-33 is found in diseases involving inflammation in mucosal tissues and articular inflammation. These include asthma, inflammatory bowel disease, and rheumatoid arthritis. IL-33 expression is elevated in psoriatic skin and the skin of atopic dermatitis patients and also is increased in pathologic settings of fibrosis, such as systemic sclerosis and liver fibrosis.

[0009] The concentration of circulating soluble ST2 also is elevated in numerous disease situations, further indicating a link between this cytokine pathway and these diseases.

Examples include asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, sepsis and trauma, HIV infection, systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, sclerosis, Wegener's granulomatosis, Behcet's disease, and cardiovascular disease. IL-33 potentiates eosinophillic inflammation, and evidence exists that this pathway is involved in eosinophil-associated disease, such as rhinosinusitis, nasal polyposis, and eosinophilic bronchitis.

[0010] Additional evidence linking the IL-33/ST2 pathway to human disease is provided by genetic studies, which have identified IL-33 and/or ST2 gene polymorphisms in the general population that are significantly associated with increased risk of disease or parameters of disease severity. Several large genome-wide association studies have linked genetic variation in ST2 (ILIRLI) or IL-33 with increased risk of asthma and other studies have genetically linked this pathway to increased asthma severity and bronchial hyper responsiveness. Similar findings have genetically implicated this pathway in allergic disorders such as atopic dermatitis as well as nasal polyposis. For additional diseases relating to the IL-33/ST2 pathway, see WO 2013/173761.

[0011] In addition, allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant diseases, but its clinical utility has been impeded by graft-versus-host disease (GVHD). Currently, no therapies exist to target proteins that are elevated in the blood of patients presenting with GVHD, and no effort has been made to develop drugs specific for GVHD. This is an important limitation of current therapies focused on targeting nonspecific effector cells. sST2 acts as a decoy receptor for IL-33, the only known ligand for ST2, which drives Th2 cells toward a Thl phenotype. This phenotypic change leads to GVHD, and the use of compounds inhibiting the binding association between ST2 and IL-33 could alleviate GVHD (Vander Lugt et al. N. Engl. J. Med. 396(6):529-539 (2013)).

[0012] Accordingly, a need still exists in the art for ST2 inhibitors having physical and pharmacological properties that permit use of the inhibitors in therapeutic applications, for example the treatment of GVHD and Thl/Th2 imbalanced inflammatory diseases. The present invention provides small molecule compounds that inhibit the ST2 and IL-33 interaction.

SUMMARY OF THE INVENTION

[0013] The present invention is directed to small molecule inhibitors of ST2, to compositions comprising the inhibitors, and to methods of using the inhibitors in a therapeutic treatment of conditions and diseases wherein inhibition of ST2 activity provides a benefit. [0014] The soluble form of ST2 is the most significant biomarker to predict a failure to respond to GVHD therapy. Therefore, one aspect of the invention is to identify small molecule compounds that target ST2 and that can be used to alleviate GVHD.

[0015] Another aspect of the present invention is to identify small molecule compounds that inhibit ST2, and therefore are useful in the treatment of hematological cancers by alleviating GVHD associated with cancer treatments.

[0016] Yet another aspect to the present invention is to provide a method of treating other diseases and conditions mediated by ST2, both the soluble form of ST2 and the full length form, by administering a therapeutically effective amount of a small molecule compound capable of inhibiting the ST2-IL33 interaction in an individual in need thereof. Nonlimiting examples of such diseases and conditions are GVHD, cardiovascular diseases, asthma, lupus, and other Thl/Th2 imbalanced inflammatory diseases.

[0017] Another aspect of the present invention is to identify small molecules capable of inhibiting ST2. Still another aspect of the invention is to utilize these compounds, or a composition containing one or more of these compounds, in the treatment of a disease or condition by the administration of a therapeutically effective amount of one or more of these compounds to an individual in need thereof.

[0018] By screening a chemical library, classes of ST2 inhibitors were identified. In one embodiment, a class of compounds capable of inhibiting ST2 has a structural formula (I)

H

[0019]

,R a

— CH 2 N

[0020] wherein R 1 is selected from the group consisting of R b , -Ci_ 3 alkyl , substituted or unsubstituted heteroaryl, substituted and unsubstituted aryl,

and -CH 2 heterocycloalkyl;

[0021] R is selected from the group consisting

of -H, -Ci_ 3 alkyl, -C(=0)R c , -S0 2 R a , -C(=S)NHR d , substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;

[0022] R a is H, substituted or unsubstituted aryl, or Ci_ 4 alkyl; [0023] R b is H, substituted or unsubstituted aryl, optionally

substituted -C(=S)NHaryl, -C(=0)Ci_ 3 alkyl, optionally substituted heterocycloalkyl, optionally substituted -S0 2 aryl, -C(=0)(CH 2 )i_ 3 C0 2 H, -C(=0)NHCi_ 3 alkyl, or Ci_ 4 alkyl;

[0024] R c is Ci_ 3 alkyl, -(CH 2 )i_ 3 OCi_ 3 alkyl, -(CH 2 )i_ 3 C0 2 H, substituted or unsubstituted

- b

aryl, or R ;

[0025] R d is substituted or unsubstituted aryl, -(CH 2 )i_ 3 OCi_ 4 alkyl, -(CH 2 )i_ 3 heteroaryl, and -(CH 2 )i_ 3 heterocycloalkyl; and

[0026] n is O or l;

[0027] or a pharmaceutically acceptable salt thereof.

[0028] In another embodiment, a second class of compounds capable of inhibiting ST2 has a structural formula (II):

[0030] wherein R is -C(=S)heterocycloalkyl (substituted), -(CH 2 )i_ 3 heterocycloalkyl (substituted), -C(=0)NHaryl (substituted), heterocycloalkyl (substituted), or -(CH 2 )i_ 3 N(R e );

[0031] R° is -N0 2 , halo, or -CN; and

[0032] R e is H or -Ci_ 4 alkyl;

[0033] or a pharmaceutically acceptable salt thereof.

[0034] In yet another embodiment, a third class of compounds capable of inhibiting ST2 has a structural formula (III):

[0036] wherein Z is null, NR 5 or S;

C=0

[0038] D is -CH 2 - or /

[0039] R 4 is phenyl, heteroaryl, -(CH 2 )i_ 2 heteroaryl, cycloalkyl,

heterocycloalkyl, -C 6 H 5 -0-R h , -(CH 2 )i_ 2 -C 6 H 5, or -(CH 2 )i_ 3 OR h ;

[0040] R 5 is null, H, or -(CH 2 )i_ 3 OR h , or

[0041] R 4 and R 5 are taken together with the nitrogen atom to which they are attached to form a heteroaryl group or a substituted or unsubstituted heterocycloalkyl group;

[0042] R f is Ci_ 3 alkyl or ; [0043] R g is H or halo; and [0044] R h is H or Ci_ 3 alkyl, or [0045] a pharmaceutically acceptable salt thereof.

[0046] Additional compounds capable of inhibiting ST2 include the following compounds of embodiment (IV):

, or a p armaceut ca y accepta e sa t t ereo .

[0048] Another embodiment of the present invention is to provide a composition comprising (a) an ST2 inhibitor of embodiments (I)-(IV) and (b) an excipient and/or pharmaceutically acceptable carrier useful in treating diseases or conditions wherein inhibition of ST2 provides a benefit.

[0049] Another embodiment of the present invention is to utilize a composition comprising a compound of embodiments (I)-(IV) and a second therapeutically active agent in a method of treating an individual for a disease or condition wherein inhibition of ST2 provides a benefit.

[0050] In a further embodiment, the invention provides for use of a composition comprising an ST2 inhibitor of embodiments (I)-(IV) and an optional second therapeutic agent for the manufacture of a medicament for treating a disease or condition of interest, e.g., asthma or lupus.

[0051] Still another embodiment of the present invention is to provide a kit for human pharmaceutical use comprising (a) a container, (bl) a packaged composition comprising an ST2 inhibitor of embodiments (I)-(IV), and, optionally, (b2) a packaged composition comprising a second therapeutic agent useful in the treatment of a disease or condition of interest, and (c) a package insert containing directions for use of the composition or compositions, administered simultaneously or sequentially, in the treatment of the disease or condition.

[0052] An ST2 inhibitor of embodiments (I)-(IV) and the second therapeutic agent can be administered together as a single-unit dose or separately as multi-unit doses, wherein the ST2 inhibitor of embodiments (I)-(IV) is administered before the second therapeutic agent or vice versa. It is envisioned that one or more dose of an ST2 inhibitor of embodiments (I)-(IV) and/or one or more dose of a second therapeutic agent can be administered.

[0053] In one embodiment, an ST2 inhibitor of embodiments (I)-(IV) and a second therapeutic agent are administered simultaneously. In related embodiments, an ST2 inhibitor of embodiments (I)-(IV) and a second therapeutic agent are administered from a single composition or from separate compositions. In a further embodiment, the ST2 inhibitor of embodiments (I)-(IV) and second therapeutic agent are administered sequentially. An ST2 inhibitor of embodiments (I)-(IV), as used in the present invention, can be administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose.

[0054] These and other embodiments and features of the present invention will become apparent from the following detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG 1 contains plots showing CD4 and CD8 T cell proliferation measured by CFSE dilution and IFN-γ production measured by intracellular staining in mixed leukocyte reactions with DMSO or an ST2 inhibitor; [0056] FIG 2 contains plots showing preservation of Treg cells in human mixed lymphocyte reactor with DMSO or an ST2 inhibitor;

[0057] FIG 3 contains plots for GVHD score and survival for NSG mice transplanted with human T cells after 300 cGy total body irradiation on day-1 and treated with DMSO or ST2 inhibitors administered via intra-peritoneal injection daily from day -1 to day 20 after HCT;

[0058] FIG 4 contains plots showing the percentage population of human IFNg+ and IL- 17+ CD4+ T cells and human Foxp3+ CD4+ Treg cells in live human CD45+CD4+ T cells collected from the gut after DMSO and ST2 inhibitors treatment at day 14 in the NSG mice;

[0059] FIG 5 contains plots showing the percentage population of human IFNg+ CD4+ T cells and human Foxp3+ CD4+ Treg cells in live human CD45+CD4+ T cells collected from the gut after DMSO and ST2 inhibitor treatment at day 21 in the NSG mice.

[0060] FIG 6 contains plots showing systemic human plasma ST2 and IFN-γ in a DMSO or ST2 inhibitor treated groups at day 7, 14, 21 and day 28 after HCT;

[0061] FIG 7 contains plots showing GVHD score and survival for C3H.SW mice transplanted with B57B/6 bone marrow and T cells after 1100 cGy total body irradiation on day 1 with DMSO or an ST2 inhibitor administered via intra-peritoneal injection daily from day -1 to day 20 after HCT.

[0062] FIGS 8-13 contain plots of an ex vivo analysis of the C3H.SW mice CD4+T cells from the gut at Day 14 and 21 after HCT;

[0063] FIG 14 contains bar graphs summarizing the percentage (expressed in mean + standard error of the mean) population of mouse IFN-yt, T-bet+, RORgt+ T cells and Foxp3+ CD4+ Treg cells in CD4+ T cells collected from the gut after DMSO and ST2 inhibitor treatment at day 14 and on day 21 in the C3H.SW mice and

[0064] FIG 15 contains bar graphs showing systemic mouse plasma (expressed in mean + standard error of the mean) ST2 and IFN-γ in a DMSO or ST2 inhibitor treated group at day 7 and 14 after HCT.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The present invention is described in connection with preferred embodiments. However, it should be appreciated that the invention is not limited to the disclosed embodiments. It is understood that, given the description of the embodiments of the invention herein, various modifications can be made by a person skilled in the art. Such modifications are encompassed by the claims below. [0066] The term "a disease or condition wherein inhibition of ST2 provides a benefit" pertains to a condition in which ST2, either the soluble form or the full length form, is important or necessary, e.g., for the onset, progress, expression of that disease or condition, or a disease or a condition which is known to be treated by an inhibition of ST2. Examples of such conditions include, but are not limited to, GVHD, cardiovascular disease, asthma, lupus, and other Thl/Th2 imbalanced inflammatory diseases. One of ordinary skill in the art is readily able to determine whether a compound treats a disease or condition mediated by ST2, for example, by assays which conveniently can be used to assess the activity of particular compounds.

[0067] The term "second therapeutic agent" refers to a therapeutic agent different from an ST2 inhibitor of embodiments (I)-(IV) and that is known to treat the disease or condition of interest. For example when a hematological cancer is the disease or condition of interest, the second therapeutic agent can be a known chemotherapeutic drug or radiation, for example. Second therapeutic agents useful to treat an autoimmune disease, like lupus, are disclosed in WO 2008/144610, designating the U.S., and incorporated herein by reference.

[0068] The term "disease" or "condition" denotes disturbances and/or anomalies that as a rule are regarded as being pathological conditions or functions, and that can manifest themselves in the form of particular signs, symptoms, and/or malfunctions. As demonstrated below, a compound of embodiments (I)-(IV) is a potent inhibitor of ST2 and can be used in treating diseases and conditions wherein inhibition of ST2 provides a benefit. In addition to the diseases and conditions disclosed herein, further conditions and diseases modified by IL- 33/SC-2 binding are disclosed in WO 2008/144610, designating the U.S. and incorporated herein by reference, specifically pages 51 and 52.

[0069] As used herein, the terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, or ameliorating a disease or condition, and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. As used herein, the terms "treat," "treating," "treatment," and the like may include "prophylactic treatment," which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously-controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition, as is the case with GVHD. The term "treat" and synonyms contemplate administering a therapeutically effective amount of a compound of the invention to an individual in need of such treatment. [0070] Within the meaning of the invention, "treatment" also includes relapse prophylaxis or phase prophylaxis, as well as the treatment of acute or chronic signs, symptoms and/or malfunctions. The treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy.

[0071] The term "therapeutically effective amount" or "effective dose" as used herein refers to an amount of the active ingredient(s) that is(are) sufficient, when administered by a method of the invention, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to an individual in need thereof.

[0072] The term "container" means any receptacle and closure therefor suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.

[0073] The term "insert" means information accompanying a pharmaceutical product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product. The package insert generally is regarded as the "label" for a pharmaceutical product.

[0074] "Concurrent administration," "administered in combination," "simultaneous administration," and similar phrases mean that two or more agents are administered concurrently to the subject being treated. By "concurrently," it is meant that each agent is administered either simultaneously or sequentially in any order at different points in time. However, if not administered simultaneously, it is meant that they are administered to an individual in a sequence and sufficiently close in time so as to provide the desired therapeutic effect and can act in concert. For example, an ST2 inhibitor of embodiments (I)-(IV) can be administered at the same time or sequentially in any order at different points in time as a second therapeutic agent. A present ST2 inhibitor and the second therapeutic agent can be administered separately, in any appropriate form and by any suitable route. When a present ST2 inhibitor and the second therapeutic agent are not administered concurrently, it is understood that they can be administered in any order to a subject in need thereof. For example, a present ST2 inhibitor can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent treatment modality (e.g., chemotherapy), to an individual in need thereof. In various embodiments, an ST2 inhibitor of embodiments (I)-(IV) and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In one embodiment, the components of the combination therapies are administered at 1 minute to 24 hours apart.

[0075] The use of the terms "a", "an", "the", and similar referents in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein merely are intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to better illustrate the invention and is not a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0076] As used herein, the term "alkyl" refers to straight chained and branched saturated Ci_io hydrocarbon groups , including but not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, and t-butyl. The term C n means the alkyl group has "n" carbon atoms. An alkyl, e.g., methyl, group can be substituted with one or more, and typically one to three, of independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for example.

[0077] As used herein, the term "halo" is defined as fluoro, chloro, bromo, and iodo.

[0078] The term "hydroxy" is defined as—OH.

[0079] The term "alkoxy" is defined as— OR, wherein R is alkyl.

[0080] The term "amino" is defined as— NH 2 , and the term "alkylamino" is defined as — NR 2 , wherein at least one R is alkyl and the second R is alkyl or hydrogen.

[0081] The term "carbamoyl" is defined as -C(=0)NR 2 .

[0082] The term "carboxy" is defined as -C(=0)OH or a salt thereof. [0083] The term "nitro" is defined as— N0 2 .

[0084] The term "cyano" is defined as— CN.

[0085] The term "trifluoromethyl" is defined as— CF 3 .

[0086] The term "trifluoromethoxy" is defined as— OCF 3 .

[0087] As used herein, groups such as is an abbreviation for .

[0088] As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to bicyclic and tricyclic carbon rings, where one ring is aromatic and the others are saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise indicated, an aryl group can be unsubstituted or substituted with one or more, and in particular one to four, groups independently selected from, for example, halo, alkyl, alkenyl, — OCF 3 ,— N0 2 ,— CN,— NC,—OH, alkoxy, amino, alkylamino,— C0 2 H,—

C0 2 alkyl, -OCOalkyl, aryl, and heteroaryl.

[0089] As used herein, the term "heterocyclic" refers to a heteroaryl and heterocycloalkyl ring systems.

[0090] As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. Each ring of a heteroaryl group can contain one or two O atoms, one or two S atoms, and/or one to four N atoms, provided that the total number of

heteroatoms in each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl group has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and triazolyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quiazolinyl, thiadiazolopyrimidyl, and thienopyridyl. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted with one or more, and in particular one to four, substituents selected from, for example, halo, alkyl, alkenyl,— OCF 3 ,— N0 2 ,— CN,— NC,— OH, alkoxy, amino, alkylamino,— C0 2 H,— C0 2 alkyl, -OCOalkyl, aryl, and heteroaryl.

[0091] As used herein, the term "cycloalkyl" means a monocyclic aliphatic ring containing three to eight carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, optionally substituted with one or more, and typically one to three, of independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for example.

[0092] As used herein, the term "heterocycloalkyl" means a monocyclic or a bicyclic aliphatic ring containing 4 to 12 total atoms, of which one to five of the atoms are

independently selected from nitrogen, oxygen, and sulfur and the remaining atoms are carbon. Nonlimiting examples of heterocycloalkyl groups are azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, dihydropyrrolyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, oxacycloheptyl, dioxacycloheptyl, thiacycloheptyl, diazacycloheptyl, each optionally substituted with one or more, and typically one to three, of independently selected halo, Ci_ 6 alkyl, C 1-6 alkoxy, cyano, amino, carbamoyl, nitro, carboxy, C 2 _ 7 alkenyl, C 2 _ 7 alkynyl, or the like on an atom of the ring.

[0093] An AlphaScreen detection assay was used to search for compounds that inhibit the ST2/IL33 interaction. The assay condition gave z'-factors > 0.7. Three specific studies were performed: 1) HTS implementation to discover compounds that inhibit the ST2/IL33 interaction; 2) enrich hits using focus compound libraries generated from pharmacophore models based on confirmed hits from HTS and identification of potential ligand binding sites in ST2; and 3) hits validation with in vitro human assays and in vivo murine models of GVHD. The assay integrated data for HTS technology of 87,000 small molecules with in silico enrichment of hits, and target binding site evaluation to discover small molecule inhibitors against biomarkers of GVHD refractoriness to therapy. The assay resulted in the discovery of small molecule compounds that can inhibit the binding between ST2 and IL-33. Toxicities evaluation of six compounds selected from these different classes of compounds showed that they are not toxic up to 40 mg/kg in healthy murine models.

[0094] The present invention therefore is directed to classes of inhibitors ST2. The compounds bind to ST2 and function as potent antagonists of the ST2/IL33 interaction. The ST2 inhibitors of the present invention therefore are useful in the treatment of a variety of diseases and conditions, including GVHD and autoimmune diseases, in subjects in need of such treatment, comprising administering a therapeutically effective amount of a compound of embodiments (I)-(IV).

[0095] In one aspect, the present invention is directed to the following embodiments: [0096] (I) A compound having a structural formula (I)

[0097]

,R a

— CH 2 N V

[0098] wherein R 1 is selected from the group consisting of R b , -Ci_ 3 alkyl , substituted or unsubstituted heteroaryl, substituted and unsubstituted aryl,

and -CH 2 heterocycloalkyl;

[0099] R is selected from the group consisting

of -H, -Ci_ 3 alkyl, -C(=0)R c , -S0 2 R a , -C(=S)NHR d , substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;

[0100] R a is H, substituted or unsubstituted aryl, or Ci_ 4 alkyl;

[0101] R b is H, substituted or unsubstituted aryl, optionally

substituted -C(=S)NHaryl, -C(=0)Ci_ 3 alkyl, optionally substituted heterocycloalkyl, optionally substituted -S0 2 aryl, -C(=0)(CH 2 )i_ 3 C0 2 H, -C(=0)NHCi_ 3 alkyl, or Ci_ 4 alkyl;

[0102] R c is Ci_ 3 alkyl, -(CH 2 )i_ 3 OCi_ 3 alkyl, -(CH 2 )i_ 3 C0 2 H, substituted or unsubstituted

,R a

- b

aryl, or R ;

[0103] R d is substituted or unsubstituted aryl, -(CH 2 )i_ 3 OCi_ 4 alkyl, -(CH 2 )i_ 3 heteroaryl, and -(CH 2 )i_ 3 heterocycloalkyl; and

[0104] n is O or l;

[0105] or a pharmaceutically acceptable salt thereof. [0106] (II) A compound having a structural formula (II)

[0108] wherein R 3 is -C(=S)heterocycloalkyl (substituted), -(CH 2 )i- 3 heterocycloalkyl (substituted), -C(=0)NHaryl (substituted), heterocycloalkyl (substituted), or -(CH 2 )i_ 3 N(R e );

[0109] R° is -N0 2 , halo, or -CN; and

[0110] R e is H or -C M alkyl;

[0111] or a pharmaceutically acceptable salt thereof.

[0112] A compound having a structural formula (III)

[0114] wherein Z is null, NR 5 or S ;

\

C=0

[0116] D is -CH 2 - or /

[0117] R 4 is phenyl, heteroaryl, -(CH 2 )i_ 2 heteroaryl, cycloalkyl,

heterocycloalkyl, -C 6 H 5 -0-R h , -(CH 2 )i- 2 -C 6 H5, or -(CH 2 ) ! _ 3 OR h ;

[0118] R 5 is null, H, or -(CH 2 )i- 3 OR h , or

[0119] R 4 and R 5 are taken together with the nitrogen atom to which they are attached to form a heteroaryl group or a substituted or unsubstituted heterocycloalkyl group;

[0121] R g is H or halo; and

[0122] R h is H or Ci_ 3 alkyl, or [0123] a pharmaceutically acceptable salt thereof.

[0124] A compound (IV) having a following structural formula:

, or a pharmaceutically acceptable salt thereof.

CH 3 me preferred embodiments of compounds (I), R' is ethyl, optionally substituted with

-CH 2 NHC(=0)(CH 2 ) 2 C0 2 H — CH 2 NHC(=0)NHC 2 H 5

— CH 2 NH — ( N-C-CH 3

[0127] In other preferred embodiments of compound (I), R is -H, -C(=0)CH 3 , -C 2 Hs,

— C(=S)NHCH

-S0 2 CH 3 , -C(=0)NH-iPr, -C(=S)NH(CH 2 ) 2 OCH 3 , 2 - 3 0

[0128] In some preferred embodiment of compounds (II), R° is N0 2 , CN, CI, or F.

[0129] In some preferred embodiments of compounds (II), R 3 is

- 18- [0131] or R 4 and R 5 are taken together to form or

N N— CH

[0132] or Z is NCH 2 CH 2 OH and R 4 is -CH 2 CH 2 OH; [0133] or Z is null and R 4 is H or C 6 H 5 .

[0134] Compounds of the invention can exist as salts. Pharmaceutically acceptable salts of the compounds of the invention often are preferred in the methods of the invention. As used herein, the term "pharmaceutically acceptable salts" refers to salts or zwitterionic forms of the compounds of structural formula (I). Salts of compounds of formula (I) can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of compounds of structural formula (I) can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.

Nonlimiting examples of salts of compounds of the invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hy-droxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylene-sulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methane- sulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present invention appearing herein is intended to include compounds of embodiments (I)-(VI) as well as pharmaceutically acceptable salts, hydrates, or solvates thereof.

[0135] Specific compounds found to inhibit ST2 and the IC 50 values for ST2 inhibitors are:

[0136] Compounds of Formula (I):

-21 -

-22- Compounds of Formula (III):

[0139] A combined library of small molecules was used in the high throughput screening experiment adopting the AlphaLISA assay. The AlphaLISA assay was developed to detect the inhibition between IL-33(with donor beads) and human ST2-Fc chimera (with acceptor beads). From the primary screening, compounds yielding greater than 30% of inhibition at 17 μΜ were selected for structural analysis. Molecular ACCess System (MACCS) fingerprints of compounds were employed in the structural cluster analysis using a 70% similarity threshold to separate groups. Each group was manually inspected to eliminate false positives including reported frequent binders and structures belonging to pan-assay interference compounds (PAINS) (Baell, J. B. & Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 53, 2719-2740, doi: 10.1021/jm901137j (2010)). For the remaining groups, each compound was inspected and the compounds with the highest inhibitory activity or synthetic feasibility were identified. Compounds then were selected and subject to the secondary dose dependent validation. Compound activity was confirmed in the dose response titration and various compounds gave does response curves. Various compounds then were evaluated by a second HEK-BLUE assay. In the HEK-BLUE assay, the activities of the compounds was observed by their inhibition to the binding between IL-33 in media and ST2 over-expressed on the HEK-BLUE cells monitored by the intracellular activation of NF-kB to release the SEAP reporter proteins. Compounds then were selected for a hit- enrichment study.

[0140] In the hit-enrichment, structural analogs of the compounds were identified first. A search based on topological similarity calculations was also performed to find candidates. Both methods gave a selection of additional 213 compounds. A number of compounds gave IC50 values of less than 50 μΜ in the AlphaLISA assay. Eleven compounds yielded dose dependent titration in the HEK-BLUE assays. The values for the HEK-BLUE assays are the HB values in the above list of compounds. Toxicity of these compounds to peripheral blood mononuclear cells (PBMC) were evaluated for a selected sets of compounds to determine dosages for consideration in in vivo mouse models. Six compounds were advanced to the toxicity study using healthy mouse models and no toxicities were observed up to 40 mg/kg up to 13 days.

[0141] The protocols for the AlphaLISA Assay and HEK-BLUE are as follows: [0142] AlphaLISA Assay

[0143] Dose Response for 45 Compounds HTS Hits [0144] (1 x 384 OptiPlate)

[0145] MATERIALS:

[0146] Recombinant biotinylated IL-33 (US Biological) - 55.25 uM in PBS (stored at -80)

[0147] Recombinant HuST2 Fc Chimera (R&D Systems) - 14.1 uM in PBS (stored at -80)

[0148] Human ST2 Antibody (R&D Systems) - SOOng/μΙ in PBS (stored at -80)

[0149] 20X Sappanone A DimethylEther (microSource Discovery Systems, Inc.) - lOmM stock at -80C. Dilute to 5mM with DMSO for assay.

[0150] 5% BSA (Gemini) in PBS

[0151] DMSO

[0152] PBS IX reconstitute from 10X with milliQ water

[0153] Tween 20

[0154] PE Acceptor Beads (5mg/ml stock)

[0155] PE Donor Beads (5mg/ml stock)

[0156] HTS Hits Compounds - 5mM working stocks (stored at -80)

[0157] White OptiPlate-384 (cat# 6007290)

[0158] Black Plate Sealers

[0159] En Vision Alpha Reader

[0160] PROCEDURE:

[0161] Reagent Preparation:

[0162] Thaw IL-33, ST2, ST2Ab and compounds (including Sappanone A) overnight

[0163] 5% BSA

[0164] 0.5 g BSA (Gemini) + 10 ml PBS

[0165] Assay Buffer: (prepare fresh each day)

[0166] For 384 wells : 90 ml PBS IX + 187 μΐ Tween20 (i.e. 0.2%) + 3.75 ml of 5% BSA (i.e. 0.2% total)

[0167] Compounds Preparation

[0168] Remove compounds (200mM working stock) from -80C and thaw O/N

[0169] Prepare 8 aliquots for further dilution [0170] Compound serial dilutions with 3 fold dilution factor:

[0171] 1. Vortex compound stock before harvest.

[0172] 2. Add Ιμΐ in the first aliquot and add 19μ1 of DMSO

[0173] 3. Then add ΙΟμΙ of DMSO in all the next 7 aliquots

[0174] 4. Perform serial dilutions by adding 5μ1 of compound to the next aliquots, mix well and vortex

[0175] ST2 without IL-33 (S I), working solution preparation:

[0176] 2.55 μΐ ST2 + 12 ml Assay Buffer. Keep 0.5 ml of this solution to make S 1, and use remaining 11 ml left for ST2 + IL-33 solution (see next step)

0177] ST2 DF = 4700, concentration = 3 nM

0178] ST2 + IL-33 (S2) , working solution preparation for one whole plate (11 ml):

0179] In 11 ml left of previous S 1 (ST2), add 2.39 μΐ IL-33

0180] ST2 DF = 4700, concentration = 3 nM

0181] IL-33 DF = 4604.16, concentration = 12 nM

0182] Procedure:

0183] 1. Blocking:

0184] Add 100 ul of assay buffer to each well

0185] Seal plate and incubate for 1 hour at RT (not on shaker)

0186] 2. Compounds and Controls:

0187] Load Samples (see plate map for well designation)

0188] Compounds and Sappanone A:

0189] Add 0.8μ1 of each dilution into designated wells

0190] Add 0.8μ1 DMSO into wells for compound positive and negative control

0191] ST2 and IL-33:

0192] ST2 + IL-33: 20μ1Λνε11 into all wells with compounds and ST2 Ab

0193] Controls : [0194] ST2 + IL-33 : 20μ1Λνε11 without compound (8 wells) = positive controls

[0195] ST2 without IL-33 : 20μ1Λνε11 without compound (8 wells) = negative control

[0196] Centrifuge plate at 1000 rpm for 15 seconds

[0197] Seal plate and incubate for 60 minutes at RT on shaker

[0198] 3. Acceptor B eads : (WORKING WITH LIGHTS OFF)

[0199] For 384 wells : 238 μΐ Beads + 4562 ul Assay Buffer (DF = 20.1)

[0200] Add 10 μΐ bead mixture to each assay well and MIX

[0201] Centrifuge plate at 1000 rpm for 15seconds

[0202] Seal plate and incubate for 60 minutes hour at RT

[0203] 4. Donor Beads: (WORKING WITH LIGHTS OFF)

[0204] For 384 wells : 238 μΐ Beads + 4562 ul Assay Buffer (DF = 20.1)

[0205] Add 10 μΐ donor bead mixture to each assay well and mix

[0206] Centrifuge plate at 1000 rpm for 15 seconds

[0207] Seal plate and incubate 30 minutes at RT

[0208] Read using EnVision-Alpha Reader.

[0209] 5. Reading AlphaLISA assay :

[0210] Instrument Setup :

[0211] Make certain plate reader is turned on, and open up Wallac En Vision® Manager software on computer.

[0212] Select "AlphaScreen 384" in the "Navigation Tree" menu. This protocol is located into the folder "Protocol/users/CGCF"

[0213] Check the "Protocol General Settings" in the PredictorMK window (just beside the Navigation Tree).

[0214] Output settings : rename ALWAYS the file by clicking on

"Browse Folder button" to select <date> - <time> and type file as ".csv" [0215] Then click on Well selection - Group 1 (into Plate 1). A plate map will appear and use the mouse to define your wells as needed. By using right click on the designated well, you can inactivate it.

[0216] Then click on "RUN"

[0217] To save on the hard drive/flash drive :

[0218] Browse to data (EnVision Data/Users/Sophie Paczesny) and save the

Excel file in the folder.

[0219] %Inh = [(Ave. signal of no-compound-controls - Ave. signal of no protein controls) - signal of compound)] / (Ave. signal of no-compound-controls - Ave. signal of no protein controls)

[0220] HTS Validation using HEK-Blue IL-33/IL-1B Cells - Dose Response [0221] 45 Compounds - Hits from HTS Screen [0222] MATERIALS:

[0223] HEK-Blue IL-33/IL-1B Cells in culture [0224] Zeocin ( 1 OOmg/ml) : stored at -20C [0225] Blasticidin (lOmg/ml): stored at -20C [0226] HygroGold: stored at -20C

[0227] Normocin (50mg/ml): formulation of three antibiotics active against mycoplasmas, bacteria and fungi. Stored at -20°C.

[0228] Quanti-Blue:

[0229] DMEM

[0230] FBS

[0231] Penicillin/Streptomycin

[0232] L-glutamine

[0233] Spectrophotometer

[0234] 37°C incubator

[0235] 96 well flat bottom plate for assay (costar cat#3599)

[0236] T-75 flask for culturing cells [0237] Compounds to be tested: 200 mM stock of each, except 4073-0156 and C226-3822 which are stocked at 100 mM

[0238] huIL-33: 10 ng/ul stock stored at -80°C

[0239] huTNFa: 10 ug/ml stock stored at -80°C

[0240] PROCEDURE:

[0241] Reagent Preparation:

[0242] Growth Medium:

[0243] DMEM, 4.5 g/1 glucose, 10% (v/v) fetal bovine serum, 50U/ml penicillin, 50ug/ml streptomycin, lOOug/ml Normocin, 2mM L-glutamine

[0244] Test Medium:

[0245] DMEM, 4.5 g/1 glucose, 10% (v/v) heat-inactivated fetal bovine serum, 50U/ml penicillin, 50ug/ml streptomycin, lOOug/ml Normocin, 2mM L-glutamine

[0246] QUANTI-Blue:

[0247] Pour contents of one pouch into clean 250ml glass bottle or flask.

[0248] Add 100ml of endotoxin-free water.

[0249] Swirl gently until powder is completely dissolved.

[0250] Warm lOminutes in water bath at 37°C.

[0251] Filter reconstituted QUANTI-Blue on a 0.2um membrane in a sterile 250ml bottle.

[0252] Use reconstituted detection medium immediately or store at 4°C. Reconstituted QUANTI-Blue is stable for at least 2weeks when properly stored.

[0253] huIL-33:

[0254] Prepare lOOng/ml working concentration from lOng/ul stock (DF=100).

[0255] lOul huIL-33 + 990ul media

[0256] huTNFa:

[0257] Prepare lOOng/ml working concentration from lOug/ml stock (DF=100)

[0258] lOul huTNFa + 990ul media

[0259] Compound preparation:

[0260] 1) Prepare 2 type of media : [0261] Growth medium (DMEM, 4.5 g/1 glucose, 10% (v/v) fetal bovine serum, 50U/ml penicillin, 50ug/ml streptomycin, lOOug/ml Normocin, 2mM L-glutamine) = 5 ml

[0262] Growth medium + DMSO 2% = 15 ml (14,7 media + 0.3 DMSO)

[0263] Procedure:

[0264] Dayl:

[0265] 1. Plate 50 ul of each compound into the designated wells. (IOOOUM final concentration for all compounds).

[0266] 2. Plate 1.5ul of DMSO into "IL-33 + 0.75% DMSO" and "TNFa + 0.75% DMSO" control wells as designated in plate map. Plate 3.0ul of DMSO into "IL-33 + 1.5% DMSO" and "TNFa + 1.5% DMSO" control wells as designated in plate map.

[0267] 3. Prepare cell suspension and plate cells.

[0268] Remove cells from flask wall by repeated pipetting. Cells adhere, but not tightly and should come off without addition of trypsin. (In general, one confluent flask of cells is sufficient to see one 96 well plate for assay.)

[0269] Move cell suspension to 50ml conical tube. Wash flask with 10ml fresh media and add wash to conical containing cells. (Pool cell suspension from multiple flaskes.)

[0270] Centrifuge cells at 300 x g for 5 minutes. Discard supernatant.

[0271] Resuspend cells in 5ml of test media (per flask of harvested eels) and resuspend well. Pool cell suspensions. Cells like to clump, so pipet vigorously and vortex to prepare single cell suspension for counting.

[0272] Count cells and calculate cell density.

[0273] Using test media, prepare final cell suspension at 265,000 cells/ml.

[0274] Plate 190ul of cell suspension (-50,000 cells) per well.

[0275] 4. Add lOul of IL-33 at 100 ng/ml to each compound, IL-33, and IL33 +DMSO control well. IL-33 and IL-33 + DMSO are your positive control wells

[0276] 5. Add lOul of TNFa at 100 ng/ml into TNFa and TNFa +IL-33 wells as negative control.

[0277] 6. Incubate at 37°C in C0 2 incubator for 20 hours. [0278] Day 2: [0279] 1. Prepare QUANTI-Blue media by warming in 37°C water bath for 10-15 minutes.

[0280] 2. Add 160 ul of resuspended QUANTI-Blue per well of flat bottom 96-well plate.

[0281] 3. Centrifuge culture plate at 2000 rpm for 2 minutes.

[0282] 4. Add 40 ul of induced HEK-Blue IL-33/IL- IB Cells supernatant. Carefully remove supernatant from top of wells using multichannel pipet. Mix supernatant with test media in wells by repeated pipetting with multichannel.

[0283] 5. Incubate at 37°C incubator for 1-3 hours

[0284] 6. Determine SEAP levels using spectrophotometer at 620-655 nm. [0285] The following are preferred compounds of the present invention.

[0286] Four ST2 inhibitors shown below were tested in vitro and in vivo to evaluate their efficacy in the ST2 Inhibition in cells and mouse models. The in vitro human mixed leukocytes allogeneic reaction data showed that ST2 Inhibitor 1 and 3 are effective in reducing the production of IFN-g in CD4+ and CD8+ T cells while maintaining the regulatory T cells population, compared to the DMSO treated control. ST2 Inhibitor 2 did not show efficacy in these tests (Figs. 1-2). Next, NSG mice transplanted with human T cells to establish xenogeneic GVHD were treated with 828 μg of ST2 Inhibitor 1 per dose in 200 μΐ daily, via intraperitoneal injection, from day-1 to day 20, an improved GVHD score and survival was observed compared to the mice treated with the DMSO control (Fig. 3). ST2 Inhibitor 2 (374 μg per dose in 200 μΐ daily) also showed efficacy in these experimental models whereas ST2 inhibitor 4 (408 μg per dose in 200 μΐ daily) did not (Fig. 3). In the ex vivo analysis of the live human CD45+CD4+ T cells collected from the gut, the main GVHD target organ, collected at Day 14 and 21 after HCT. Further a decrease in the population of human CD4+ T cells expressing IFN-γ-ι- as well as expressing IL-17, and significant increased population of human FoxP3+ T cells for mice treated with ST2 Inhibitor 1, was observed (Figs. 4-5). In addition, the plasma human ST2 levels of the mice decrease by 6 fold at Day 21 and Day 28 after ST2 Inhibitor 1 treatment in reference to the DMSO control (Fig. 6 top panel). The plasma human IFN-γ also decreased when mice were treated with ST2 Inhibitor 1 as compared to mice receiving DMSO (Fig. 6 bottom panel). Thus, the efficacy of ST2 Inhibitor 1 to the human-to-mouse xenogeneic model in NSG mice is associated with reduced IFN-g production and inhibition of soluble ST2 reflected in the reduced plasma ST2 levels. The compounds were tested according to modified protocols published in J. Zhang et al., Sci Transl Med 2015, 7(308):308ral60. Here, mice were irradiated with 300 cGy and ST2 inhibitors were administered at 2xIC50 concentrations in the experiments. [0287] In the second B57BL/6 to C3H.SW mouse GVHD model, same concentrations of the ST2 inhibitors were administered. Two independent experiments (experiment 1 and 2) were performed and the total number of mice is 13. Improved GVHD score and survival was also observed in the ST2 inhibitor 1 and 2 treatment when compared to the mice treated with the DMSO control (Fig. 7). In the ex vivo analysis of the mouse CD4+ T cells collected from the gut at Day 14 and 21 after HCT, the decreased population of IFN-y+, T-bet+, RORgt+ T cells and increased population of FoxP3+ T cells for mice treated with ST2 Inhibitor 1 was again confirmed (Figs. 8-13). At Day 21, greater increased population of IL-4 producing and Gata3+ T cells were found in the ST2 inhibitor 1 treatment group (Fig. 13). Summarized bar graphs are provided in Fig. 14. The plasma mouse ST2 levels of the mice decrease by 4 fold at Day 14 in the ST2 Inhibitor 1 treatment group in reference to the DMSO control whereas the plasma IFNg concentration decreases by 3 fold as compared to the DMSO control (Fig. 15). The efficacy of ST2 Inhibitor 1 to the B6→C3H.SW GVHD model is associated with the reduced IFN-g production and inhibition of soluble ST2 reflected in the reduced plasma ST2 levels.

ST2 Inhibitor 1 ST2 Inhibitor 2 ST2 Inhibitor 3 ST2 Inhibitor 4

[0288] In one test, ST2 Inhibitor 1, ST2 Inhibitor 2 and ST2 inhibitor 3 were used in an ex vivo analysis. The FIGS, show the efficacy of the present ST2 inhibitors in in vitro and in vivo GVHD models. FIG. 1 summarizes CD4 and CD8 T cell proliferation measured by CFSE dilution and IFN-γ production measured by intracellular staining in mixed leukocyte reactions with DMSO or ST2 inhibitors. FIG. 1 shows that ST2 inhibitors reduce type 1 cells proliferation in human mixed lymphocyte reaction. FIG. 2 shows that ST2 inhibitors preserve Treg cells in human mixed lymphocyte reaction.

[0289] In FIG. 3, NSG mice were transplanted with human T cells (10 6 ) after 300 cGy total body irradiation on day-1. DMSO or an ST2 inhibitor was administered via intraperitoneal injection daily from day -1 to day 20 after HCT. GVHD score and survival are shown. n=13 per group. **p<0.01, ***p<0.001.

[0290] Fig. 4 shows the percentage population of human IFNg+ CD4+ T cells and human Foxp3+ CD4+ Treg cells in live human CD45+CD4+ T cells collected from the gut after DMSO and ST2 inhibitors treatment at day 14 in the NSG mice.

[0291] Fig. 5 shows the percentage population of human IFNg+ CD4+ T cells and human Foxp3+ CD4+ Treg cells in live human CD45+CD4+ T cells collected from the gut after DMSO and ST2 inhibitors treatment at day 21 in the NSG mice.

[0292] FIG. 6 shows systemic human plasma ST2 and IFN-γ from plasma of NSG mice (produced by human T cells) in a DMSO or ST2 inhibitor treated group at day 7, 14, 21 and day 28 after HCT. n=3 per group. *p<0.05, **p<0.01.

[0293] In FIGS. 7-13, C3H.SW mice were transplanted with B57BL/6 bone marrow (5 xlO 6 ) and T cells (2 x 10 6 ) after 1100 cGy total body irradiation on day-1. DMSO or an ST2 inhibitor was administered via intra-peritoneal injection daily from day -1 to day 20 after HCT. GVHD score and survival are shown. n=13 per group from two experiments. **p<0.01, ***p<0.001.

[0294] Fig. 14 shows the percentage (expressed in mean + standard error of the mean) population of C3H.SW mouse IFN-y+, T-bet+, RORgt+ T cells and Foxp3+ CD4+ Treg cells in CD4+ T cells collected from the gut after DMSO or ST2 inhibitor treatment at day 14 in the C3H.SW mice. On day 21, IL-4+ and Gata3+ CD4+ T cell population are also analyzed.

[0295] FIG. 15 shows systemic mouse plasma (expressed in mean + standard error of the mean) ST2 and IFN-γ in DMSO or an ST2 inhibitor treated group at day 7 and 14 after HCT. n=3 per group. *p<0.05, **p<0.01.

[0296] The compounds of embodiments (I)-(IV) inhibit ST2 and are useful in the treatment of a variety of diseases and conditions. In particular, the compounds of embodiments (I)-(IV) are used in methods of treating a disease or condition wherein inhibition of ST2 provides a benefit, for example, GVHD and asthma. The method comprises administering a

therapeutically effective amount of a compound of embodiments (I)-(IV), or a mixture thereof, to an individual in need thereof. The present methods also encompass administering a second therapeutic agent to the individual in addition to the compound of embodiments (I)- (IV). The second therapeutic agent is selected from drugs known as useful in treating the disease or condition afflicting the individual in need thereof, e.g., a chemotherapeutic agent and/or radiation known as useful in treating a hematological cancer.

[0297] The compounds of the present invention typically are administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present invention are formulated in a conventional manner using one or more

physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of compounds of embodiments (I)-(IV). A second therapeutically active agents, one or more of which can be used in combination with an ST2 inhibitor of embodiments (I)- (IV), are prepared and administered as described in the art.

[0298] The pharmaceutical compositions can be manufactured, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of the compound of embodiments (I)-(IV) is administered orally, the composition typically is in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the

composition additionally can contain a solid carrier, such as a gelatin or an adjuvant. The tablet, capsule, and powder contain about 0.01% to about 95%, and preferably from about 1% to about 50%, of a compound of embodiments (I)-(IV). When administered in liquid form, a liquid carrier, such as water, petroleum, or oils of animal or plant origin, can be added. The liquid form of the composition can further contain physiological saline solution, dextrose or other saccharide solutions, or glycols. When administered in liquid form, the composition contains about 0.1% to about 90%, and preferably about 1% to about 50%, by weight, of a compound of embodiments (I)-(IV).

[0299] When a therapeutically effective amount of a compound of embodiments (I)-(IV) is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains, an isotonic vehicle. [0300] Compounds of embodiments (I)-(IV) can be readily combined with pharmaceutically acceptable carriers well-known in the art. Such carriers enable the active agents to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding the compound of embodiments (I)-(IV) to a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.

Suitable excipients include, for example, fillers and cellulose preparations. If desired, disintegrating agents can be added.

[0301] A compound of embodiments (I)-(IV) can be formulated for parenteral

administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.

[0302] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. Additionally, suspensions of a compound of embodiments (I)-(IV) can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, a present composition can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0303] A compound of embodiments (I)-(IV) also can be formulated in rectal

compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases. In addition to the formulations described previously, the compound of embodiments (I)-(IV) also can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds of embodiments (I)-(IV) can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins.

[0304] In particular, the compounds of embodiments (I)-(IV) can be administered orally, buccally, or sublingually in the form of tablets containing excipients, such as starch or lactose, or in capsules or ovules, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. Such liquid preparations can be prepared with pharmaceutically acceptable additives, such as suspending agents. The compounds of embodiments (I)-(IV) also can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the present ST2 inhibitors are best used in the form of a sterile aqueous solution which can contain other substances, for example, salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.

[0305] As an additional embodiment, the present invention includes kits which comprise one or more compounds or compositions packaged in a manner that facilitates their use to practice methods of the invention. In one simple embodiment, the kit includes a compound or composition described herein as useful for practice of a method (e.g., a composition comprising a compound of embodiments (I)-(IV) and an optional second therapeutic agent), packaged in a container, such as a sealed bottle or vessel, with a label affixed to the container or included in the kit that describes use of the compound or composition to practice the method of the invention. Preferably, the compound or composition is packaged in a unit dosage form. The kit further can include a device suitable for administering the composition according to the intended route of administration.

[0306] Prior ST2 inhibitors possessed properties that hindered their development as therapeutic agents. In accordance with an important feature of the present invention, compounds of embodiments (I)-(IV) were evaluated as inhibitors for ST2. For example, compounds of the present invention typically have a bonding affinity (IC 50 ) to ST2 of less than 500 μΜ, less than 100 μΜ, less than 50 μΜ, and less than 25 μΜ.