IRF4 inhibitors and their use for treating cancer and autoimmune disease

Abstract

Compounds are provided that are useful for inhibiting interferon regulatory factor 4 (IRF4). Also provided are pharmaceutical compositions comprising such compounds and methods for treating diseases related to IRF4 activity, such as cancer, using such compounds.

Description

IRF4 INHIBITORS AND THEIR USE FOR TREATING CANCER AND AUTOIMMUNE DISEASE

[0001] Compounds useful for inhibiting interferon regulatory factor 4 (IRF4) are provided, as are pharmaceutical compositions comprising such compounds and methods for treating diseases related to IRF4 activity, such as cancer, using such compounds.BACKGROUND

[0002] Transcription factors are promising therapeutic targets in cancers and other disease states but are often mislabeled as ‘undruggable’ due to their dynamic structures, lack of small molecule binding pockets, non-globular tertiary structures, and lack of feasible paths for bioactive small molecules. Interferon regulatory factor 4 (IRF4) is a transcription factor in the interferon regulatory factor family encoded by the IRF4 gene. Distinct from other IRF family members, IRF4 is expressed upon activation of T-cell receptor signaling in T-cells, B-cell receptor, IL-4, CD40 signaling in B cells, and Toll-like receptor signaling by lipopolysaccharide in macrophages. It is noted that IRF4 has been referred to by different names in earlier studies including LSIRF, MUM1, ICSAT, Pipl, NF-EM5, and SHEP8.

[0003] IRF4 overexpression is known in melanoma, leukemia, myeloma, and lymphoma and is associated with a unique gene expression pattern in each. More specifically, IRF4 overexpression is shown in diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM), classical Hodgkin lymphoma (cHL), plasma cell myeloma, and primary effusion lymphoma (PEL). High levels of IRF4 also are noted in Epstein-Barr virus (EBV)-transformed cells, B- cell lymphomas with Type 3 latency, and Human T-cell Leukemia Virus-1 (HTLVl)-infected cell lines, acute lymphocytic leukemia (ALL), and adult T-cell leukemia / lymphoma (ATL). Additionally, translocation and genetic mutation of IRF4 have been shown in MM, peripheral T-cell lymphomas, and chronic lymphocytic leukemia (CLL).

[0004] Dysregulated IRF4 is observed in the pathogenesis of immune-inflammatory or autoimmune diseases such as ulcerative colitis, Crohn’s disease, type 1 diabetes, multiple sclerosis, and lupus. Therefore, targeting IRF4 may also have therapeutic potential in the treatment of immune disorders.

[0005] IRF4 is a well-validated clinical target with immunomodulatory drug (IMiD) based treatment regimens. Attempts at targeting IRF4 have relied on the clinically used IMiDs, suchas lenalidomide and pomalidomide, which target Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3) - upstream transcription factors of IRF4. Unfortunately, IMiDs are highly susceptible to resistance mechanisms in the clinic and fail to provide deep and sustained downregulation of IRF4 due to their indirect mechanism of action. IRF4 expression is driven by numerous transcriptional activators including STAT6, NF-kB, and c-Myc, which often counteract the effects of IMiD based treatments.

[0006] The effectiveness of direct IRF4 inhibition has been demonstrated using a selective antisense oligonucleotide (ASO), ION251, to treat MM. (Cell Stem Cell 28, 623-636, April 1, 2021). Pre-clinical models showed a reduction in myeloma regeneration and sensitized myeloma cells to standard of care treatments, specifically, lenalidomide. This is mechanistically achieved via disruption of cell cycle progression and downregulation of stem cell and cell adhesion transcript expression. However, antisense oligonucleotides often are limited by pharmacological effects including off-target toxicities, poor cellular delivery, biochemical attack, and poor target binding.

[0007] Given the validation of IRF4 as a clinical target with IMiD-based and ASO therapy, orally available small molecules that can directly target IRF4 are expected to have significant benefit for patients with MM. Small molecule therapies are generally more stable with predictable pharmacokinetics and pharmacodynamics and oral bioavailability. The predictability of chemical synthesis also provides manufacturing and development benefits for small molecule drugs.

[0008] In this regard, the present inventors reported in Blood 142 (2023) 3635-3636 (https: / / doi.org / 10.1182 / blood-2023-186864) the discovery of a first-in-class small molecule inhibitor of the IRF4-PU.1 interaction, with a TR-FRET IC50 of 2 pM. The structure of said inhibitor, was later disclosed as l-(4-methoxyphenyl)-N-[2-(methylamino)-2-oxo-l- phenylethyl] cyclopropanecarboxamide (referred to as Compound (X) herein) at the American Society of Hematology (ASH) Annual Meeting and Exposition on 10 December 2023.

[0009] Even so, inhibition of IRF4 is severely under-explored, and therefore presents the possibility for discovering new therapeutic strategies for cancers and autoimmune diseases. There is an urgent need, in particular, for novel MM therapeutics as over 100,000 MM patients die annually, and those newly diagnosed have a 5-year survival rate of only 53.9%.SUMMARY

[0010] Further investigations have led to the discovery of new compounds that are IRF4 inhibitors.

[0011] In a first aspect, there is provided a method of treating cancer or an autoimmune disease, comprising administering to a subject in need thereof, a therapeutically effective amount of a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: the mole fraction of the 5-enantiomer in said compound of formula (I) is greater than zero, except when R2 is CH2C(O)NHCH3 in which case the mole fraction of the A’-enantiomer in said compound of formula (I) is greater than zero;Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent, naphthyl, or pyridinyl;R21S C(O)NH2, C(O)NHCH3, CH2C(O)NHCH3, C(O)OH, C(O)OCH3, or tetrazolyl; each R3is independently methyl, hydroxy, methoxy, ethoxy, fluoro, chloro, bromo, phenyl, pyrazolyl optionally substituted with one methyl substituent, triazolyl optionally substituted with one methyl substituent, pyridinyl optionally substituted with one methyl or amino substituent, or OCH2CO2H; m is 1 or 2; and n is 1 or 2.

[0012] This first aspect includes: a compound of formula (I) as defined above for use in the treatment of cancer or an autoimmune disease; and the use of a compound of formula (I) as defined above in the manufacture of a medicament for the treatment of cancer or an autoimmune disease.

[0013] In a second aspect, there is provided a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: the mole fraction of the 5-enantiomer in said compound of formula (I) is greater than zero, except when R2 is CH2C(O)NHCH3 in which case the mole fraction of the A’-enantiomer in said compound of formula (I) is greater than zero;Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent, naphthyl, or pyridinyl;R21S C(O)NH2, C(O)NHCH3, CH2C(O)NHCH3, C(O)OH, C(O)OCH3or tetrazolyl; each R3is independently methyl, hydroxy, methoxy, ethoxy, fluoro, chloro, bromo, phenyl, pyrazolyl optionally substituted with one methyl substituent, triazolyl optionally substituted with one methyl substituent, pyridinyl optionally substituted with one methyl or amino substituent, or OCH2CO2H; m is 1 or 2; and n is 1 or 2; with the proviso that the compound of formula (X)is excluded when the mole fraction of the 5-enantiomer in said compound of formula (X) is 0.5.

[0014] In third aspect, there is provided a pharmaceutical composition, comprising a compound of formula (I) as defined in the first aspect, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.DETAILED DESCRIPTION

[0015] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the art to which this application belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. To facilitate ready understanding, certain terms used herein are first defined below.

[0016] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Likewise, the term "and / or" as used in a phrase such as “A, B, and / or C” is used interchangeably with “A and / or B and / or C” and is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0017] The word “comprise”, or variations such as “comprises”, or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is further understood that wherever embodiments are described herein with the language “comprising” or grammatical equivalents thereof, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. In other words, if a composition comprising A, B and C is recited, a composition consisting essentially of A, B and C is also contemplated as is a composition consisting of A, B and C.

[0018] All publications and other reference materials referenced herein are hereby incorporated by reference in their entirety. Although a number of documents are cited herein,this citation does not constitute an admission that any one of these documents forms part of the common general knowledge in the art.

[0019] The term “cancer” is used herein to describe a disease characterized by the uncontrolled, abnormal growth of cells.

[0020] The term “hematological cancer” (or “blood cancer”) is used herein to describe any type of cancer that affects blood cells. This definition encapsulates the five subtypes of hematological cancer: leukemia, lymphoma, myeloma, myelodysplastic syndrome (MDS), and myeloproliferative disorder (MPD).

[0021] “Leukemia” refers to a type of hematological cancer that affects blood cells in bone marrow - usually white blood cells. Examples of leukemia include: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), childhood leukemia and blastic plasmacytoid dendritic cell neoplasm (BPDCN) (although the last of these has features different than traditional leukemias).

[0022] "Lymphoma” refers to a type of hematological cancer that affects the immune system - specifically white blood cells called lymphocytes. Examples of lymphoma include: diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal B cell lymphoma, mantle cell lymphoma, post-transplant lymphoproliferative disorder, marginal zone lymphoma, Waldenstrom's macroglobulinemia, and Burkitt lymphoma.

[0023] “Myeloma” (or “multiple myeloma”) refers to a type of hematological cancer that affects plasma cells. Examples of myeloma include: light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma.

[0024] “Myelodysplastic syndrome (MDS)” refers to a type of hematological cancer characterized by the bone marrow producing faulty blood cells and insufficient healthy blood cells. Examples of MDS include: MDS with single lineage dysplasia (MDS-SLD), MDS with ring sideroblasts (MDS-RS), MDS with multilineage dysplasia (MDS-MLD), and MDS with excess blasts (MDS-EB).

[0025] “Myeloproliferative disorder (MPD)” refers to a type of hematological cancer whereby the bone marrow produces too many of a particular type of blood cell. Examples ofMPD include: polycythaemia vera (PV), essential thrombocythaemia (ET), and myelofibrosis (MF).

[0026] The term “autoimmune disease” refers to any condition that results from a subject’s immune system attacking their own tissue. Examples of autoimmune disease include: rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and type I diabetes.

[0027] The terms “treating” or “treatment” describe the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treating includes preventing the onset of symptoms or complications, alleviating or eliminating symptoms or complications, or eliminating the underlying disease, condition, or disorder. For example, treating cancer in a subject includes reducing, repressing, delaying or preventing the growth of cancerous cells as well as killing cancerous cells within the subject.

[0028] The terms “administering” and “administration” refer to any method of providing the active substance to a subject. Where the method involves administration of two or more active substances, it results in them exerting their desired pharmacodynamic effects at the same time, or for an overlapping period of time, within the patient's body. Thus, administration of the two or more active substances is not limited to simultaneous administration, nor administration via the same route. It encompasses separate, sequential and simultaneous administration via the same or different routes and in the same or different medicaments, provided that it results in them exerting their desired pharmacodynamic effects at the same time, or for an overlapping period of time, within the patient’s body.

[0029] Methods of administration are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intraaural administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable administration such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent. While the above means of administration may provide systemic exposure to the active substance(s), local administration / exposure is also contemplated, e.g., to the bone marrow of cells.

[0030] “Pharmaceutically acceptable salt” means a salt such as those described in standard texts on salt formation, see for example: S.M. Berge, el al., “Pharmaceutical Salts” (1977) Journal of Pharmaceutical Sciences, 66, 1-19. Suitable salts include those formed with organic or inorganic acids or bases.

[0031] “Pharmaceutical composition” refers to a formulation of the described compounds (or a pharmaceutical salt thereof) in association with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions suitable for the delivery of described compounds and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in “Remington’s Pharmaceutical Sciences”, 19th Edition (Mack Publishing Company, 1995).

[0032] A compound is considered to be an “IRF4 inhibitor” herein if it exhibits an IC50 less than or equal to 20pM in the described TR-FRET binding assay. Accordingly, IRF4 inhibitors are compounds which bind to the interferon accessory domain (IAD) of the IRF4 protein.

[0033] The term "subject” refers to mammals, preferably humans.

[0034] The cancer or the autoimmune disease may be one which is characterized by overexpression and / or dysregulation of interferon regulatory factor 4 (IRF4).

[0035] The cancer may be a hematological cancer, such as lymphoma, myeloma, or leukemia. Preferably, the hematological cancer is multiple myeloma.

[0036] The autoimmune disease may be rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, or type I diabetes.

[0037] In a first aspect, there is provided a method of treating cancer or an autoimmune disease, comprising administering to a subject in need thereof, a therapeutically effective amount of a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein:the mole fraction of the 5-enantiomer in said compound of formula (I) is greater than zero, except when R2 is CH2C(O)NHCH3 in which case the mole fraction of the A’-enantiomer in said compound of formula (I) is greater than zero;Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent, naphthyl, or pyridinyl;R21S C(O)NH2, C(O)NHCH3, CH2C(O)NHCH3, C(O)OH, C(O)OCH3, or tetrazolyl; each R3is independently methyl, hydroxy, methoxy, ethoxy, fluoro, chloro, bromo, phenyl, pyrazolyl optionally substituted with one methyl substituent, triazolyl optionally substituted with one methyl substituent, pyridinyl optionally substituted with one methyl or amino substituent, or OCH2CO2H; m is 1 or 2; and n is 1 or 2.

[0038] This first aspect includes: a compound of formula (I) as defined above for use in the treatment of cancer or an autoimmune disease; and the use of a compound of formula (I) as defined above in the manufacture of a medicament for the treatment of cancer or an autoimmune disease.

[0039] In a second aspect, there is provided a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: the mole fraction of the 5-enantiomer in said compound of formula (I) is greater than zero, except when R2 is CH2C(O)NHCH3in which case the mole fraction of the R-enantiomer in said compound of formula (I) is greater than zero;Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent, naphthyl, or pyridinyl;R21S C(O)NH2, C(O)NHCH3, CH2C(O)NHCH3, C(O)OH, C(O)OCH3or tetrazolyl; each R3 is independently methyl, hydroxy, methoxy, ethoxy, fluoro, chloro, bromo, phenyl, pyrazolyl optionally substituted with one methyl substituent, triazolyl optionally substituted with one methyl substituent, pyridinyl optionally substituted with one methyl or amino substituent, or OCH2CO2H; m is 1 or 2; and n is 1 or 2; with the proviso that the compound of formula (X)

[0040] is excluded when the mole fraction of the 5-enantiomer in said compound of formula (X) is 0.5.

[0041] The skilled artisan will appreciate that the described compounds, or pharmaceutically acceptable salts thereof, are comprised of a core that contain a chiral center, represented by * in formula (I) below:

[0042] All individual enantiomers, as well as mixtures of the enantiomers of said compounds including racemates, except for pure A’-enantiomers when R2 is C(O)NH2, C(O)NHCH3, C(O)OH, C(O)OCH3 or tetrazo lyl and pure 5-enantiomers when R2 is CH2C(O)NHCH3, are contemplated. Enantiomerically enriched samples of compounds of formula (I) may be prepared beginning with chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively, enantiomerically enriched samples ofcompounds of formula (I) may be prepared from enantiomeric mixtures by standard chiral chromatographic or crystallization techniques at any convenient point in the synthesis of compounds of the invention.

[0043] 5-enantiomers of described compounds are preferred, except when R2 is CH2C(O)NHCH3 in which case / / -enantiomers are preferred. When present, the mole fraction of the 5-enantiomer (or / / -enantiomer when R2 is CH2C(O)NHCH3) in said compound of formula (I) is greater than 0.5, preferably greater than 0.8, more preferably greater than 0.95, still more preferably greater than 0.99, and most preferably 1.00.

[0044] In some embodiments of the first and second aspects, Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent. Preferably, Ri is phenyl.

[0045] In some embodiments of the first and second aspects, R2 is C(O)NHCH3.

[0046] In some embodiments of the first and second aspects, each R3 is independently methyl, methoxy, phenyl, pyrazolyl, triazolyl optionally substituted with one methyl substituent, pyridinyl, or OCH2CO2H. Preferably, each R3 is methoxy, and more preferably p- methoxy.

[0047] In some embodiments of the first and second aspects, m is 1.

[0048] In some embodiments of the first and second aspects, n is 1.

[0049] In some embodiments of the first and second aspects, the compound is:

[0050] In a third aspect, there is provided a pharmaceutical composition, comprising i) a compound of formula (I) as defined in the first aspect, or a pharmaceutically acceptable salt thereof, or a compound of formula (I) as defined in the second aspect, or a pharmaceutically acceptable salt thereof, and ii) a pharmaceutically acceptable carrier.

[0051] The compound of formula (I) is generally effective over a wide dosage range. It will be understood that the amount of the compound actually administered will 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 or compounds administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.

[0052] It is well known in the art that agents for the treatment of cancer may be combined with other agents for the treatment of cancer. The compound of formula (I), or a pharmaceutically acceptable salt thereof, may be co-administered, simultaneously or sequentially, with other effective treatment(s) for cancer.

[0053] The compound of formula (I) may be prepared by a variety of procedures known in the art, some of which are illustrated in the Examples below.EXAMPLES

[0054] The following Examples further illustrate the invention and represent typical syntheses of the compounds of the invention. The reagents and starting materials are readily available, or may be readily synthesized by one of ordinary skill in the art. It should be understood that these Examples are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

[0055] The R or S configuration of the described compounds may be determined by standard techniques such as X-ray analysis and correlation with chiral-HPLC retention time. The naming of the compounds prepared in the following Examples is generally performed using the IUPAC naming feature in PerkinElmer CHEMDRAW® version 22.0.0.22.

[0056] Unless otherwise specified, percentages in the below Examples refer to percent yield.Procedure 1:

[0057] A flask was charged with 2-amino-A-methyl-2-phenylacetamide [1] (20 mg, 0.122 mmol) and carboxylic acid [2] (0.134 mmol, 1.1 eq) and dissolved in anhydrous DMF (2 mL). HATU (92 mg, 0.244 mmol, 2 equiv.), and DIPEA (52.8 pL, 0.366 mmol, 3 equiv.) were then added and the mixture was stirred at rt for 16 h. The solvent was then removed under flow of nitrogen. The resulting residue was then purified by silica gel column chromatography gradient (DCM / MeOH) 0-10%. Average yield = 60% (0.073 mmol).

[0058] The Examples in Table 1, below, were prepared in accordance with Procedure 1, by using the appropriately substituted carboxylic acid [2],Table 1Procedure 2:

[0059] (a) A flask was charged with 2-amino-N-methyl-2-phenylacetamide [1] (50 mg,0.305 mmol) and l-(3 / 4-bromophenyl)cyclopropane-carboxylic acid [3] (80.9 mg , 0.335 mmol, 1.1 equiv.) and dissolved in anhydrous DMF (2 mL). HATU (231.8 mg, 0.610 mmol), and DIPEA (132 pL, 0.915 mmol) were then added and the mixture was stirred at room temperature for 16 h. The solvent was then removed under flow of nitrogen and the resulting residue was dissolved in dichloromethane. The mixture was purified by silica gel column chromatography gradient (DCM / MeOH) 0-10%. Average yield = 60% (0.183 mmol).

[0060] (b) A dry flask was charged with l-(4 / 3-bromophenyl)-A-(2-(methylamino)-2-oxo- l-phenylethyl)cyclopropane-l -carboxamide [4] (10 mg, 0.026 mmol), Pd(dppf)Ch (0.9 mg, 0.001 mmol), aryl boronic acids (0.026 mmol, 1 equiv.), and potassium carbonate (10.8 mg, 0.078 mmol). The flask was then purged with nitrogen gas and a solution (3 mL) of dioxane:water (4: 1) mixture was added. The reaction was refluxed for 16 h under nitrogen gas. After cooling the reaction to room temperature, the solvent was removed under vacuum, and then purified by silica gel column chromatography gradient (DCM / MeOH) 0-10%. Average yield = 40% (0.01 mmol).

[0061] The Examples in Table 2, below, were prepared in accordance with Procedure 2, by using the appropriately substituted carboxylic acid [3], the appropriately substituted amide [4], and the appropriately substituted boronic acid of step (b).Table 2Procedure 3:

[0062] (a) A flask was charged with amine derivative [5] (0.700 mmol) and carboxylic acid derivative [6] (0.770 mmol, 1.1 equiv.) and then dissolved in anhydrous DMF (2 mL). To the same flask, HATU (532 mg, 1.4 mmol), and DIPEA (303 pL, 2.1 mmol) were then added and the mixture was stirred at room temperature for 16 h. The solvent was then removed under flow of nitrogen, and the resulting residue was dissolved in dichloromethane. The mixture was then purified by silica gel column chromatography gradient (DCM / MeOH) 0-10%. Average yield = 70% (0.539 mmol).

[0063] (b) A flask was charged with methyl-ester derivative [7] (0.539 mmol, 1 equiv.) and Li OH (38.8 mg, 1.62 mmol) and then dissolved in a solution of THF / H2O (1 :1) and stirred at room temperature for 24 h. The solution was then acidified with cone. HC1, diluted with water, and then extracted 3 x with EtOAc. The combined organic phases were then dried with sodium sulfate and concentrated under reduced pressure, and used in the next reaction without further purification.

[0064] (c) A flask was charged with carboxylic acid [8] (0.050 mmol, 1 equiv.), aminederivative (0.050 mmol, 1 equiv.), EDCI (15.5 mg, 0.10 mmol), and HOBt (13.5 mg, 0.10 mmol) and then dissolved in anhydrous DCM (2 mL). DIPEA (21.6 pL, 0.15 mmol) was then added and the reaction was stirred at room temperature overnight. The solvent was then removed under reduced pressure and the resulting residue was purified by silica gel column chromatography gradient (DCM / MeOH) 0-10%. Average yield = 80% (0.040 mmol).

[0065] The Examples in Table 3, below, were prepared in accordance with Procedure 3, by using the appropriately substituted ester [5], the appropriately substituted carboxylic acid [6], and the appropriately substituted amine of step (c).Table 3Procedure 4 - preparation of l-(4-methoxyphenyl)-N-(phenyl(lH-tetrazol-5- yl)methyl)cyclopropane-l-carboxamide (Example 34)

[0066] (a) A flask was charged with 2-amino-2-phenylacetonitrile [9] (50 mg, 0.380 mmol), 1 -(4-methoxyphenyl) cyclopropane- 1 -carboxylic acid

[0010] (72.6 mg, 0.380 mmol), and HATU (288.8 mg, 0.760 mmol) and dissolved in dry DMF (2 mL). To the same flask, DIPEA (165 pL, 1.14 mmol) was then added and the reaction was stirred for 16 h. The solvent was then removed under reduced pressure and purified by silica gel column chromatography (DCM / MeOH) 0-10%. Yield = 90% (0.342 mmol)

[0067] (b) A dry flask was charged with A-(cyano(phenyl)methyl)-l-(4- methoxyphenyl)cyclopropane-l -carboxamide

[0011] (10 mg, 0.033 mmol), TEA»HC1 (5.9 mg, 0.043 mmol), and sodium azide (2.8 mg, 0.043 mmol) and then dissolved in dry toluene (2 mL), and refluxed for 16 h. After cooling to room temperature, the solvent was removed underreduced pressure, and the resulting residue was purified by silica gel column chromatography (DCM / MeOH) 0-15%. Yield = 34% (5.1 mg, 0.015 mmol).Procedure 5

[0068] (a) A flask was charged with 2-amino-A-methyl-2-phenylacetamide [1] (50 mg,0.305 mmol) and 1 -(4-hydroxyphenyl) cyclopropane- 1 -carboxylic acid

[0012] (60 mg, 0.336 mmol, 1.1 equiv.) and dissolved in anhydrous DMF (2 mL). HATU (232 mg, 0.610 mmol, 2 equiv. ), and DIPEA (132 pL, 0.915 mmol, 3 equiv. ) were then added and the mixture was stirred at room temperature for 16 h. The solvent was then removed under flow of nitrogen. The resulting residue was then purified by silica gel column chromatography gradient (DCM / MeOH) 0-10%. Average yield = 60% (0.183 mmol).

[0069] (b) A dry flask was charged with 1 -(4-hydroxyphenyl)-A-(2-(methylamino)-2-oxo- l-phenylethyl)cyclopropane-l -carboxamide

[0013] (10 mg, 0.031 mmol), potassium carbonate (12.9 mg, 0.093 mmol), and Bromo-derivatives (0.093 mmol) and then dissolved in dry acetone (2 mL), and refluxed for 16 h. After cooling to room temperature, the solvent was removed under reduced pressure, and then resulting residue was purified by silica gel column chromatography (DCM / MeOH) 0-15%. Average yield = 72% (0.022 mmol).

[0070] The Examples in Table 4, below, were prepared in accordance with Procedure 5, by using the appropriately substituted alkyl bromine of step (b).Table 4Procedure 6 - preparation of N-(2-amino-2-oxo-l-phenylethyl)-l-(4- methoxyphenyl)cyclopropane-l-carboxamide (Example 37)

[0071] A flask was charged with phenylglycinamide

[0014] (0.038 mmol), l-(4- methoxyphenyl) cyclopropane- 1 -carboxylic acid

[0015] (7.26 mg, 0.038 mmol), and HATU (28.9 mg, 0.076 mmol) and then dissolved in dry DMF (2 mL). DIPEA (16.5 pL, 0.11 mmol) was then added and the reaction was stirred for 16 h. The solvent was then removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography (DCM / MeOH) 0-10%. Yield = 90% (0.034 mmol)Procedure 7 - Preparation of ( -l-(4-methoxyphenyl)- / V-(2-(methylamino)-2-oxo-l- phenylethyDcyclopropane-l-carboxamide (Example 38)

[0072] (a) A flask was charged with (5)-2-((tert-butoxycarbonyl)amino)-2-phenylacetic acid

[0016] (100 mg, 0.40 mmol) and dissolved in anhydrous DMF (2 mL). NMI (66 mg, 0.80 mmol) and TCFH (224 mg, 0.80 mmol) were then added and the mixture was stirred at room temperature for 2 h. The solvent was then removed under reduced pressure, and the resulting residue was dissolved in DCM. The mixture was then purified by silica gel column chromatography gradient (DCM / MeOH) 0-10%.

[0073] (b) A flask was charged with tert-butyl (5)-(2-(methylamino)-2-oxo-l- phenylethyl)carbamate

[0017] (20 mg, 0.075 mmol) and dissolved in a solution of 6M HCl / Dioxane (2mL) and stirred at room temperature for 24 h. The solvent was then removed under reduced pressure. The product was used in the next reaction without further purification.

[0074] (c) A flask was charged with (5)-2-amino-A-methyl-2-phenylacetamide

[0018] (10 mg, 0.037 mmol), 1 -(4-methoxyphenyl) cyclopropane- 1 -carboxylic acid

[0015] (7.3 mg, 0.037 mmol), NMI (6.6 mg, 0.074 mmol), and TCFH (20.7 mg, 0.074 mmol) and then was stirred at room temperature for 2 h. The solvent was then removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography gradient (DCM / MeOH) 0- 10%. Yield = 61% (7.8 mg, 0.032 mmol).

[0075] The Reference Example in Table 5 below was prepared in accordance with Procedure 7, by using the ( / ?) enantiomer of

[0016] ,Table 5Procedure 8 - Preparation of 1 -(4-methoxyphenyl)- V-(3 -(methylamino)-3 -oxo- 1 - phenylpropyl)cyclopropane- 1 -carboxamide (Example 39)O O / =\ MeCN, 0 °C-rt, 2 h / =\1 2To a solution of compound 1 (2.00 g, 7.50 mmol) in MeCN (20 mL) was added NMI (1.00 g, 15.0 mmol), l-methylimidazole(1.30 g, 15.7 mmol) and TCFH (2.30 g, 8.25 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water (10 mL), extracted with EA (20 mL x 3), washed with brine, dried over Na2SO_i, combined the organic layer and concentrated under reduced pressure to give a residue. The residue was purified by chromatographic column on silica gel, eluted with 50% to 100% EA in PE to give compound 2 (200 mg, 9.5% yield) as a white solid. LCMS: [M+H]+=279.1. 'H NMR (400 MHz, DMSO-^) 8 7.71 - 7.64 (m, 1H), 7.38 (d, J= 8.4 Hz, 1H), 7.31 - 7.25 (m, 4H), 7.22 - 7.16 (m, 1H), 4.96 - 4.83 (m, 1H), 2.50 - 2.47 (m, 3H), 2.47 - 2.42 (m, 2H), 1.35 (s, 9H).13C NMR (101 MHz, DMSO-r / s) 8 169.74, 154.68, 143.57, 128.09, 126.66, 126.23, 77.77, 51.42, 42.59, 28.23, 25.42.To a solution of compound 2 (200 mg, 0.720 mmol) in DCM (2 mL) was added 4M-HC1 in dioxane (2 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to afford compound 3 (150 mg, crude) as a white solid. LCMS: [M+H]+=179.1To a solution of compound 3 (100 mg, 0.560 mmol) in DMF (5 mL) was added compound 4 (1.07 g, 0.560 mmol), DIEA (220 mg, 1.68 mmol) and HATU (320 mg, 0.84 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered and the filtrate was purified by prep-HPLC to afford IRF4-int-R (32 mg, 16% yield) as a white solid. LCMS: [M+H]+=353.2. 'H NMR (400 MHz, DMSO-t / r,) 87.69 - 7.61 (m, 1H), 7.44 (d, J= 8.1 Hz, 1H), 7.33 - 7.24 (m, 4H), 7.22 - 7.12 (m, 3H), 6.97 - 6.90 (m, 2H), 5.15 - 5.07 (m, 1H), 3.76 (s, 3H), 2.51 - 2.48 (m, 1H), 2.48 - 2.44 (m, 1H), 2.44 - 2.41 (m, 3H), 1.38 - 1.18 (m, 2H), 0.97 - 0.86 (m, 2H).13C NMR (101 MHz, DMSO-^) 8 172.48, 170.62, 158.87, 143.11, 132.04, 131.85, 128.58, 127.10, 126.43, 114.56, 55.55, 50.80, 41.32, 29.89, 25.78, 15.28.AssaysProtein Production Protocol:

[0076] For IAD domain production and purification, double-stranded fragments containing the IAD domain of IRF4 (residues 240-420) were cloned into bacterial expressionvectors (addgene plasmid #29653). These vectors were transformed into BL21(DE3)pLysS (PROMEGA™ cat. LI 195), grown to an optical density of 0.6 at 37 °C, and ITPG (lOOmM final concentration) was added and the cells were allowed to shake at 16 °C overnight. The cells were pelleted and lysed at 18,000 psi in a LM10 microfluidizer (lysis buffer 25 mM Tris pH 8.0, 500 mM NaCl, 1 mM TCEP, 20 mM Imidazole, 0.1% IGEPAL, 10% glycerol, 1 mM PMSF) and the supernatant was passed to nickel column chromatography with 2 mL of nickel beads. The supernatant was incubated with beads for 45 - 60 min at 0 °C, and the beads were washed with >20 column volumes (CVs) of lysis buffer and >5 CV of high salt buffer (lysis buffer + 1500 mM NaCl). The protein was eluted using 0.1 CV of elution buffer (lysis buffer + 400 mM Imidazole) and loaded onto an AKTA pure 25M FPLC (GEHealth product no. 29018226). The mixture was purified by FPLC in running buffer (20 mM TRIS, pH 8, 200 mM NaCl, 1 mM TCEP) and the purity of protein fractions were assessed by SDS / Page gel. Pure fractions were pooled, and the concentration was assessed using nanodrop absorbances at A260 / A280.Protein Labeling Protocol:

[0077] Recombinant IRF4 (240-420) was first labeled with terbium by incubating IRF4 (50 pM) with Corafluor-1 (TOCRIS™ cat.7920) (2500 pM) in labeling buffer (100 mM sodium carbonate, pH 8.5, 0.05% tween-20) then labeled for 40 min at rt. Following labeling, the reaction was buffered exchanged using Zebra-spin desalting columns (THERMOFISHER™ cat. 89882) into storage buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM KC1, 20% glycerol).TR-FRET Assay General Protocol:

[0078] A master-mix solution of 10 nM Terbium-Labeled IRF4 (240-420) and 300 nM Probe (FITC-ahx-SPPLEV(pS)DG-NH2) was prepared using a IX TR-FRET Buffer (10 mM NaCl, 100 mM HEPES™ pH = 8.0, 0.5 mg / mL BSA, 0.1% Tween-20). 19 pL of the master mix is multi-channeled into a corning 384- well white plates. Compounds were serial diluted in DMSO (in a 1 :3 ratio) and added to the plate in 1 pL quantities to yield final drug concentrations of 0 - 100 pM. The plate was covered and incubated at rt for 30 min. TR-FRET values were measured using a CLARIOSTAR® plus plate reader at the 495 nm channel and the 520 nm channel. IC50 values were calculated using PRISM software and fit using non-linear regression four parameter inhibitor vs. response.TR-FRET Assay Probe:

[0079] FITC-ahx-SPPLEV(pS)DGEAD-NH2(Purchased from peptide2.0) **N-Term = FITC dye C-Term = AMIDE (-CONH2)BRET Assay General Protocol:

[0080] HEK293T (ATCC cat. CRL-3216) were stably transfected with plasmids expressing full-length IRF4-Nanoluc fusions which were cloned into a lentiviral GFP expression vector (addgene plasmid #21373). The cells stabling expressing IRF4-Nanoluc fusions were harvested and lysed using a IX NP-40%, supplemented with 100X proteasome and phosphatase inhibitor (THERMOFISHER™, cat. 78440). The lysate was cleared and diluted using IX BRET Buffer (10 mM NaCl, 100 mM HEPES pH = 8.0, 0.5 mg / mL BSA, 0.1% TWEEN® 20) to a concentration of IxlO4cell lysate / mL. To the master mix, TAMRA- ahx-PEPl was added to a final concentration of 1 pM (peptide2.0 custom peptide synthesis), and the NanoLuc substrate (PROMEGA™ cat no. N1661) was added to a IX final concentration (e.g. 20uL substrate in ImL of media). 49 pL of the master-mix is multichanneled into a low-volume CORNING® 384-well white plate (cat. CLS4511). Compounds were serial diluted (in a 1:3 ratio) in DMSO and added to the plate in 1 pL quantities to yield final drug concentrations of 0 - 100 pM. The plate was covered and incubated for 30 min at rt. BRET values were measured using a PHERASTAR® Fsx plate reader at the 495 nm channel and the 610 nm channel. IC50 values were calculated using PRISM software and fit using nonlinear regression four parameter inhibitor vs. response.BRET Assay Probe:

[0081] TAMRA-ahx-SPPLEV(pS)DGEAD-NH2(Purchased from peptide2.0) * *N-Term = TAMRA dye, C-Term = AMIDE (-CONH2)Table 6

Claims

What is claimed is:

1. A method of treating cancer or an autoimmune disease, comprising administering to a subject in need thereof, a therapeutically effective amount of a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: the mole fraction of the 5-enantiomer in said compound of formula (I) is greater than zero, except when R2 is CH2C(O)NHCH3 in which case the mole fraction of the A’-enantiomer in said compound of formula (I) is greater than zero;Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent, naphthyl, or pyridinyl;R2IS C(O)NH2, C(O)NHCH3, CH2C(O)NHCH3, C(O)OH, C(O)OCH3, or tetrazolyl; each R3is independently methyl, hydroxy, methoxy, ethoxy, fluoro, chloro, bromo, phenyl, pyrazolyl optionally substituted with one methyl substituent, triazolyl optionally substituted with one methyl substituent, pyridinyl optionally substituted with one methyl or amino substituent, or OCH2CO2H; m is 1 or 2; and n is 1 or 2.

2. A compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein:the mole fraction of the 5-enantiomer in said compound of formula (I) is greater than zero, except when R2is CH2C(O)NHCH3in which case the mole fraction of the A’-enantiomer in said compound of formula (I) is greater than zero;Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent, naphthyl, or pyridinyl;R2IS C(O)NH2, C(O)NHCH3, CH2C(O)NHCH3, C(O)OH, C(O)OCH3or tetrazolyl; each R3is independently methyl, hydroxy, methoxy, ethoxy, fluoro, chloro, bromo, phenyl, pyrazolyl optionally substituted with one methyl substituent, triazolyl optionally substituted with one methyl substituent, pyridinyl optionally substituted with one methyl or amino substituent, or OCH2CO2H; m is 1 or 2; and n is 1 or 2; with the proviso that the compound of formula (X)is excluded when the mole fraction of the 5-enantiomer in said compound of formula (X) is 0.5.

3. The method of claim 1 or the compound of claim 2, wherein the mole fraction of the S- enantiomer (when R2is C(O)NH2, C(O)NHCH3, C(O)OH, C(O)OCH3, or tetrazolyl) or R- enantiomer (when R2is CH2C(O)NHCH3) in said compound of formula (I) is greater than 0.5, preferably greater than 0.8, more preferably greater than 0.95, still more preferably greater than 0.99, and most preferably 1.00.

4. The method of claim 1 or 3, or the compound of claim 2 or 3, wherein Ri is phenyl optionally substituted with one methyl, hydroxy, methoxy, fluoro or chloro substituent.

5. The method or compound of claim 4, wherein Ri is phenyl.

6. The method of claims 1 or 3 to 5, or the compound of claims 2 to 5, wherein R2 is C(O)NHCH3.

7. The method of claims 1 or 3 to 6, or the compound of claims 2 to 6, wherein each R3 is independently methyl, methoxy, phenyl, pyrazolyl, triazolyl optionally substituted with one methyl substituent, pyridinyl, or OCH2CO2H.

8. The method or the compound of claim 7, wherein each R3 is methoxy, preferably p- methoxy.

9. The method of claims 1 or 3 to 8, or the compound of claims 2 to 8, wherein m is 1.

10. The method of claims 1 or 3 to 9, or the compound of claims 2 to 9, wherein n is 1.

11. The method of claims 1 or 3 to 10, or the compound of claims 2 to 10, wherein the compound is:

12. A pharmaceutical composition, comprising i) a compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, or a compound according to any one of claims 2 to 11, or a pharmaceutically acceptable salt thereof, and ii) a pharmaceutically acceptable carrier.

13. The method of claims 1 or 3 to 11, wherein the cancer or the autoimmune disease is mediated by interferon regulatory factor 4 (IRF4).

14. The method of claims 1 or 3 to 11 or 13, wherein the cancer is a hematological cancer.

15. The method of claim 14, wherein the hematological cancer is lymphoma, myeloma, or leukemia.

16. The method of claim 14, wherein the hematological cancer is multiple myeloma.

17. The method of claims 1 or 3 to 11 or 13, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis), or type I diabetes.

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