WO2023122325A2 - Meta anilide compounds and methods for use of the same to treat diseases and disorders - Google Patents

Abstract

Disclosed herein are meta anilide inhibitors of gene transcription mediated by myocardin-related transcription factor and serum response factor, and methods for their use in treating or preventing diseases, such as cancer and fibrosis. In particular, disclosed herein are compounds of Formula (I) and pharmaceutically acceptable salts thereof: (I) wherein the substituents are as described.

Description

META ANILIDE COMPOUNDS AND METHODS FOR USE OF THE SAME TO TREAT DISEASES AND DISORDERS

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under grant numbers R01 GM115459-01 A1 and R01 AR066049-01 , each awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

[0002] The present disclosure relates to meta anilide compounds that act as inhibitors of gene transcription mediated by myocardin-related transcription factor and/or serum response factor (“MRTF/SRF”), and methods of using the compounds to inhibit MRTF/SRF- mediated gene transcription and to treat diseases.

[0003] Cell growth, proliferation, migration, and invasion are dependent on many growth factors, mitogens, and chemotactic agents. The medium for growing cells in tissue culture generally contains serum (e.g., fetal bovine serum) and serum also stimulates migration and invasion of cancer cells and fibroblasts. Treatment of cells with serum results in robust activation of gene transcription via the serum response factor (“SRF”) (see Norman et aL, Cell 55:989-1003 (1988)). SRF is associated with cellular transformation and epithelial- mesenchymal transformation (see Iwahara et aL, Oncogene 22:5946-5957 (2003); Psichari et aL, J Biol Chem 277:29490-29495 (2002)).

[0004] One key mechanism of activation of serum response factor (“SRF”) by serum involves activation of the Rho GTPases (especially rhoA and rhoC) through G protein- coupled receptors and possibly other mechanisms. Activation of rhoA and rhoC induces actin polymerization and release of the transcriptional coactivator, myocardin-related transcription factor (“MRTF”) (see Cen et aL, Mol Cell Biol 23:6597-6608 (2003); Miralles et aL, Cell 113:329-342 (2003); Selvaraj and Prywes, J Biol Chem 278:41977-41987 (2003)). MRTF, which is also known as MKL, was first identified as a site of gene translocation in leukemia (megakaryoblastic leukemia), like the leukemia-associated rhoGEF (“LARG”, see Mercher et aL, Genes Chromosomes Cancer 33:22-28 (2002)). The protein product of the translocated gene is hyperactive compared to the wild-type protein. MRTF has also been called modified in acute leukemia (“MAL”) and BSAC (see Miralles et aL, Cell 113:329-342 (2003); Sasazuki et aL, J Biol Chem 277:28853-28860 (2002)). There are two MRTF genes (MRTFA and MRTFB, previously known as MKL1 and MKL2, respectively). Their actions are largely redundant on where both proteins are expressed (see Shaposhnikov, Cell Cycle 12:1762-72 (2013)). MRTF was identified in an antiapoptosis screen for genes that abrogate tumor necrosis factor-induced cell death (see Sasazuki et aL, J Biol Chem 277:28853-28860 (2002)).

[0005] As a consequence of Rho signaling, MRTF translocates to the nucleus and binds SRF leading to the expression of fibronectin, CXCL1 , and caveolin-2 (see Wu et aL, Breast Cancer Res Treat 84(1 ):3-12 (2004). Also, the MRTF/SRF transcription complex directly binds to induce expression of the WWTR1 gene and its gene product, TAZ protein (see Esnault et aL, Genes Dev. 28(9):943-58 (2014); Foster et aL, Genes Dev. 31 (23-24):2361 - 2375 (2017). YAP and TAZ are transcription factors that interact with MRTF/SRF and play critical roles in cancer proliferation, migration, metastasis, and drug resistance (see Foster et aL, Genes Dev. 31 (23-24):2361 -2375 (2017); Thompson Bioessays 42(5):e1900162 (2020)). Expression of these genes leads to cancer cell invasion, metastasis, and resistance to multiple cancer therapies. Thus, there is a link between Rho/MRTF-controlled biological processes and cancer metastasis.

[0006] In addition to metastasis, cancer cell resistance to chemotherapy, targeted therapies, and radiation has been shown to be driven by Rho GTPase signaling (see Lim et aL, J Biol Chem. 294(48):18192-18206 (2019); Mokady et aL, Drug Resist Update 19:22-32 (2015); Pranatharthi et aL, Stem Cells Int. 2016:5785786 (2016)) and MRTF/SRF-regulated gene expression (see Xu et aL, Oncotarget 8(5):8436-8446 (2017); Lionarons et aL, Cancer Cell 36(1 ):68-83 (2019); Misek et aL, Oncogene 39(7):1466-1483 (2020); and Girard et aL, Cancer Res. 80(10):1927-1941 (2020)). While most extensively documented in melanoma, Rho/MRTF/SRF signaling contribute to resistance in prostate, breast, ovarian, and other cancers.

[0007] Rho GTPase signaling and MRTF-regulated gene transcription have also been implicated in tissue fibrosis (see Small J Cardiovasc Transl Res. 5(6):794-804 (2012); Janmey et aL, Differentiation 86(3):112-20 (2013); Tsou et aL, Am J Physiol Cell Physiol. 307(1 ):C2-13 (2014); Lighthouse et aL, J Mol Cell Cardiol. 91 :52-60 (2016); Gasparics et aL, Dev Dyn. 247(3) :396-404 (2018); Miranda et aL, Int J Mol Sci 22(1 1 ) (2021 )). For example, Rho GTPase signaling and MRTF-regulated gene transcription has been implicated in tissues, such as lung tissue (see Sandbo et al, Am J Respir Cell Mol BioL 41 :332-8 (2009); Luchsinger J Biol Chem. 286:44116-25 (2011 )), skin (see Haak et al, J Pharmacol Exp Ther. 349:480-6 (2014)), intestinal tissue (see Johnson et al, Inflamm Bowel Dis. 20:154-65 (2014), kidney tissue (see 6 hAinmhire et aL, Am J Physil Renal Physiol. 316(1 ):F63-F75 (2019); Bialik et aL, Sci Rep. 9(1 ):4323 (2019); Huang et aL, J Diabetes Res 2021 :9570405 (2021 )), liver tissue (see Zheng et aL, Clin Res Hepatol Gastroenterol. 41 (3):303-310 (2017); Shi et aL, Am J Physiol Gastrointest Liver Physiol. 18(3):G504-G517 (2020)), and eye tissue (see Korol et aL, Mol Med. 22:713-723 (2016); Yu-Wai-Man Sci Rep. 7(1 ):518 (2017);

Ichikawa et a., Taiwan J Ophthalmol. 10(2):100-105 (2020)). Many genes involved in fibrosis (e.g., ACTA2, CTGF, and several collagen genes) are activated by Rho-regulated MRTF/SRF mechanisms (see Haak et al, J Pharmacol Exp Ther. 349:480-6, (2014)).

[0008] Cancer metastasis is a significant medical problem in the United States, where it is estimated that over 500,000 cancer-related deaths in 2003 resulted from metastatic tumors rather than primary tumors (approximately 90% of cancer deaths). Cancer metastasis requires malfunction in several tightly regulated cellular processes controlling cell movement from a primary site to a secondary site. These cellular processes include cell survival, adhesion, migration, and proteolysis resulting in extracellular matrix remodeling, immune escape, angiogenesis and lymphangiogenesis, and target ‘homing’. Most existing cancer treatments focus on killing tumor cells; however, such chemotherapeutic intervention leads to substantial toxicity to healthy cells and tissue. Since spread, or metastasis, of cancers is the primary cause of cancer-related mortalities, there is a need for agents that can specifically inhibit or prevent signals that trigger metastasis.

[0009] Rho proteins are overexpressed in various tumors, including colon, breast, lung, testicular germ cell, and head and neck squamous-cell carcinoma (see Sawyer, Expert Opin. Investig. Drugs., 13: 1-9 (2004)). The rho family of small GTP binding proteins plays important roles in many normal biological processes and in cancer (see Schmidt and Hall, Genes Dev., 16:1587-1609 (2002); Burridge and Wennerberg, Cell, 116:167-179 (2004)). This family includes three main groups: rho, rac, and cdc42. Rho is activated by numerous external stimuli including growth factor receptors, immune receptors, cell adhesion, and G protein coupled receptors (GPCRs) (see Schmidt and Hall, Genes Dev., 16:1587-1609 (2002), Sah et al., Annu. Rev. Pharmacol. Toxicol., 40:459-489 (2000)).

[0010] RhoA and rhoC play roles in metastasis (see Clark et aL, Nature 406:532-535 (2000); Ikoma et aL, Clin Cancer Res 10:1192-1200 (2004); Shikada et aL, Clin Cancer Res 9:5282-5286 (2003); Wu et aL, Breast Cancer Res Treat 84:3-12 (2004); Hakem et al, Genes Dev 19:1974-9 (2005). Both rhoA and rac1 can regulate the function of the extracellular matrix (ECM) proteins, ezrin, moesin, and radixin, by the phosphorylation of ezrin via the rhoA pathway and the phosphorylation of the ezrin antagonist, neurofibromatosis 2, by the rac1 pathway (see Shaw et aL, Dev Cell 1 :63-72 (2001); Matsui et aL, J Cell Biol 140:647-657 (1998)). These ECM proteins promote cell movement by utilizing the ECM receptor, CD44, to link the actin cytoskeleton with the plasma membrane. In addition, rhoA and rad regulate ECM remodeling by controlling the levels of matrix metalloproteinases (MMPs) or their antagonists, tissue inhibitors of metalloproteinases (TIMPs) (see Bartolome et aL, Cancer Res 64:2534-2543 (2004)). RhoA is also required for monocyte tail retraction during transendothelial migration, indicating a role in extravasation, which is a key process in metastasis (see Worthylake et aL, J Cell Biol 154:147-160 (2001). [0011] The relative contributions of rho and rac proteins in the metastatic phenotype has been studied (see Sahai and Marshall, Nat Rev Cancer 2:133-142 (2002); Whitehead et aL, Oncogene 20:1547-1555 (2001 )). Sahai and Marshall (see Nat Cell Biol 5:711 -719 (2003)) showed that different tumor cell lines exhibit different mechanisms of motility and invasion. In particular, 375m2 melanoma and LS174T colon carcinoma cell lines showed striking “rounded” and “blebbed” morphology during invasion into Matrigel matrices. This invasion was entirely rho-dependent and was blocked by C3 exotoxin, the N17rho dominant negative protein, and a ROCK kinase inhibitor. In contrast, two other cell lines were blocked instead by a rac dominant negative mutation, but not rho or ROCK inhibitors. These latter two cell lines (BE colon carcinoma and SW962 squamous cell carcinoma) had elongated morphologies. A third line showed a mixed morphology and was blocked partially by both rho and rac inhibitors. Additionally, mice lacking rhoC have greatly reduced metastasis of virally-induced breast tumors to lung (see Hakem et al, Genes Dev 19:1974-9 (2005)). Also, knock-down of SRF or its transcriptional co-activator MRTF reduced lung metastases from breast or melanoma xenografts (see Medjkane et al, Nat Cell Biol. 11 :257-68 (2009)). Even rac1 -driven melanomas, though, appear to depend on MRTF (see Lionarons et aL, Cancer Cell. 36(1):68-83 (2019)). Thus, there is important heterogeneity in mechanisms of tumor cell behavior that contributes to metastasis. It is widely recognized that cell growth and apoptosis mechanisms vary greatly among tumors, necessitating customized therapeutic approaches.

[0012] Diseases involving fibrosis or excess deposition of extracellular matrix, including collagen, are major medical problems. Nearly 40% of chronic diseases, such as cirrhosis, heart failure, and diabetic nephropathy (see 6 hAinmhire et aL, Am J Physil Renal Physiol. 316(1):F63-F75 (2019); Bialik et aL, Sci Rep. 9(1 ) :4323 (2019); Huang et aL, J Diabetes Res 2021 :9570405 (2021)) involve tissue fibrosis. The poor clinical outcome of several orphan diseases (scleroderma or systemic sclerosis (“SSc”), idiopathic pulmonary fibrosis (“IPF”) etc.) is primarily determined by tissue fibrosis; and despite their rapid and lethal clinical course, there are few effective treatments, and those that exist are not well-tolerated.

[0013] Systemic sclerosis is an orphan, multisystem autoimmune disorder that can cause fibrosis of the skin and internal organ systems (lungs, heart, kidneys, and gastrointestinal system). It has the highest case fatality rate of any rheumatic disease. SSc predominately affects women (see Beyer et aL, Arthritis Rheum 62: 2831-2844 (2010); Boukhalfa G, et aL, Exp Nephrol 4: 241-247 (1996); Buhl AM, et aL, J Biol Chem 270: 24631-24634 (1995); Chaqour et aL, FEBS J 273: 3639-3649 (2006); Charles et aL, Lancet 367: 1683-1691 (2006) and increases with age. The precise pathogenesis of SSc is yet to be defined but the major clinical features of SSc — collagen production, vascular damage and inflammation/autoimmunity — require environmental triggers and genetic effects which interact with the three cardinal features of the disease at several points (see Charles et aL, Lancet 367: 1683-1691 (2006)). Generally, there is initial inflammation but fibrosis persists even after the inflammation has resolved or has been suppressed by medications (see Beyer et aL, Curr Opin Rheumatol 24: 274-280 (2012); Wynn TA, and Ramalingam TR. Nat Med 18: 1028-1040 (2012)).

[0014] In addition to orphan diseases that result in lung fibrosis, lung fibrosis is also a major treatment-limiting toxicity for cancer patients treated with thoracic radiotherapy or certain chemotherapies, particularly bleomycin sulfate. See Skeoch et aL, J. Clin. Med. 7(10):356 (2018); Carter et aL, Curr. Treat. Options OncoL 21 :42 (2020); Palma et aL, Int J Radiat Oncol Biol Phys. 85(2), 444-450 (2013); and Bang et aL,TransL Lung Cancer Res. 8, S139-S146 (2019). The disease is initiated by pulmonary inflammation which develops into lung pneumonitis. Over 30% of the patient population is afflicted during treatment, and if it progresses to late-stage fibrosis, it proves fatal for 2-5% of individuals. See Palma et aL, Int J Radiat Oncol Biol Phys. 85(2), 444-450 (2013); Keffer et aL, Adv. Rad.OncoL 5(2), 238- 249 (2020); and Sleijfer et aL, Chest 120, 617-624 (2001). Those who survive can be afflicted with permanent scarring in the lung, drastically reducing quality of life and creating a high burden of follow-on healthcare costs. Patients with lung fibrosis are 80% more likely to be hospitalized in a given year, and have a life expectancy of only four to five years post diagnosis despite medical intervention. See Olson et aL, Adv. Ther. 37, 3292-3298 (2020); Wuyts et aL, Adv.Ther. 37, 3246-3264 (2020).

[0015] Like all fibrotic diseases, pulmonary fibrosis is caused by an excess of extracellular matrix (ECM) deposition, namely collagen, within the lung. Although initiated by diverse mechanisms, the process generally begins with inflammatory responses. See Guiranno et aL, Front. OncoL 9,877 (2019); Wynn et aL, Nat.Med. 18, 1028-1040 (2012). These drive the critical transition from a normal healthy lung fibroblast to a myofibroblast - a rapidly dividing cell responsible for increased collagen deposition in the lungs. See Hu et aL, Curr Opin Rheumatol 25, 71-77 (2013); Small et aL, J CardiovascTransL Res. 5, 794-804 (2012). Without the intervention of therapeutics, the build-up of ECM in the lungs impairs tissue elasticity, eventually leading to respiratory failure. See Raghu et aL, Lancet Respir. Med. 2, 566-572 (2014).

[0016] Currently, when lung fibrosis develops, cancer treatment is halted and patients are administered steroids, such as prednisolone. Steroid administration can only treat the symptoms of fibrosis, with no clinical evidence that it can halt the progression of fibrotic disease, and chronic steroid treatment is problematic for patient health. See Skeoch et aL, J. Clin. Med. 7(10):356 (2018). Approved anti-fibrotics treat idiopathic pulmonary fibrosis (IPF), but must be dosed two to three times daily, and carry toxicities which limit their use in cancer treatment. See Abdel- Rah man et aL, Future Oncol. 12(18), 2163-2172 (2016); Nobel et aL, Eur. Respir. J. 47, 243-253 (2016).

SUMMARY

[0017] One aspect of the disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein: R¹ is H, Me, F, Cl, CN, OH, or OMe; each of R² and R³ independently is H or F, provided that at least one of R² and R³ is H; R⁴ is Cl, C2-₃alkyl, C2-₃alkenyl, C2-₃alkynyl, cyclopropyl, CF₃, or OCF₃; R⁵ is H, Cl, F, Ci-₃alkyl, OCi-₃alkyl, Ci-₃alkenyl, Ci-₃alkynyl, cyclopropyl, CN, CF₃, or OCF₃; one of X¹ and X² is CH and the other of X¹ and X² is N; and Y is CH or N.

[0018] In some cases, R¹ is H. In some cases, R¹ is Me. In some cases, R¹ is F or Cl. In some cases, R¹ is OH or OMe. In some cases, R¹ is CN. In some cases, R² is H. In some cases, R² is F. In some cases, R³ is H. In some cases, R³ is F. In some cases, R² is F and R³ is H. In some cases, R² is H and R³ is F. In some cases, R² and R³ are each H. In some cases, R⁴ is Cl. In some cases, R⁴ is C2-₃alkyl, C2-₃alkenyl, or C2-₃alkynyl. In some cases, R⁴ is ethyl, propyl, or isopropyl. In some cases, R⁴ is ethenyl or propenyl. In some cases, R⁴ is ethynyl or propynyl. In some cases, R⁴ is cyclopropyl. In some cases, R⁴ is CF₃, or OCF₃. In some cases, R⁵ is H. In some cases, R⁵ is Cl, F, Ci-₃alkyl, OCi-₃alkyl, Ci-₃alkenyl, Ci- ₃alkynyl , cyclopropyl, CN, CF₃, or OCF₃. In some cases, R⁵ is Cl or F. In some cases, R⁵ is Cl. In some cases, R⁵ is F. In some cases, R⁵ is Ci-₃alkyl or OCi-₃alkyl. In some cases, R⁵ is Me or OMe. In some cases, R⁵ is Ci-₃alkenyl or Ci-₃alkynyl. In some cases, R⁵ is CF₃, or OCF₃. In some cases, R⁵ is Cl, F, Me, OMe, CF₃, OCF₃, or CN. In some cases, X¹ is CH and X² is N. In some cases, X¹ is N and X² is CH. In some cases, Y is CH. In some cases,

Y is N. In some cases, X¹ is N, X² is CH, and Y is N. In some cases, X¹ is CH, X² is N, and

Y is N. In some cases, X¹ is N, X² is CH, and Y is CH. In some cases, X¹ is CH, X² is N, and Y is CH.

[0019] In some cases, the compound of Formula (I) is:

, or a pharmaceutically acceptable salt of any of the foregoing. In some cases, the compound of Formula (I) is

pharmaceutically acceptable salt of any of the foregoing.

[0020] Another aspect of the disclosure relates to a pharmaceutical formulation comprising a compound disclosed herein, such as a compound of Formula (I), and a pharmaceutically acceptable excipient.

[0021] Still another aspect of the disclosure relates to a kit comprising the pharmaceutical formulation disclosed herein and instructions for administering the pharmaceutical formulation to a patient.

[0022] Yet another aspect of the disclosure relates to a method of inhibiting myocardin- related transcription factor and/or serum response factor (“MRTF/SRF”)-mediated gene transcription in a subject, comprising contacting the MRTF/SRF with a compound disclosed herein, such as a compound of Formula (I), in an amount to inhibit the gene transcription.

[0023] Another aspect of the disclosure relates to a method of treating a disease associated with dysfunction of MRTF/SRF- mediated gene transcription in a patient comprising administering to the patient a therapeutically effective amount of a compound disclosed herein or a pharmaceutical formulation disclosed herein. In some cases, the disease is cancer, fibrotic disease, diabetes, insulin insensitivity, hyperactive platelets, metabolic disease, diabetic complications, inflammation, inflammatory disease, pulmonary arterial hypertension, axon regeneration following nerve damage, Raynaud's phenomenon, cerebral vascular disease, cardiovascular disease, erectile dysfunction, or combinations thereof.

[0024] In some cases, the cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, Ewing's tumor, lymphangioendotheliosarcoma, synovioma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, glioblastoma, leukemia, megakaryoblastic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, or combinations thereof. For example, the cancer can be megakaryoblastic leukemia, melanoma, breast cancer, prostate cancer, pancreatic cancer, glioblastoma, or combinations thereof.

[0025] In some cases, the fibrotic disease wherein the fibrotic disease is systemic sclerosis, localized scleroderma, pulmonary fibrosis, cardiac fibrosis, liver fibrosis, liver cirrhosis, renal fibrosis, diabetic nephropathy, high-glucose-induced nephropathy, chronic renal failure, lung fibrosis, nephrogenic systemic fibrosis, graft versus host disease, Dupuytren's contracture, keloid, scarring, and wound healing abnormalities, inflammatory bowel disease, Crohn’s disease, ocular fibrosis, glaucoma, post-trabeculectomy fibrosis, postoperative capsular contraction, corneal fibrosis, pterygia, Graves opthmalopathy, diabetic retinopathy, age-related macular degeneration, degenerative disc disease, intervertebral disc degeneration, postoperative adhesions, reactive fibrosis, chronic heart failure, aortic dissection, or combinations thereof. For example, the fibrotic disease can be systemic sclerosis or idiopathic pulmonary fibrosis. In some cases, the pulmonary fibrosis is drug-induced, chemotherapy-induced, radiation-induced, environmental-induced, or toxin- induced.

[0026] In some cases, the metabolic disease is obesity, diabetes (e.g., type II diabetes), insulin resistance, or combinations thereof.

[0027] The disclosure also provides a compound, salt, or formulation as described herein for use in the treatment or prevention of a disease or disorder in a subject. The disclosure also provides use of a compound, salt, or formulation, as described herein, in a method for treating or preventing a disease or disorder in a subject.

[0028] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. The description hereafter includes specific embodiments with the understanding that the disclosure is illustrative, and is not intended to limit the invention to the specific embodiments described herein.

DETAILED DESCRIPTION

[0029] Disclosed herein are meta anilide compounds that can inhibit gene transcription mediated by myocardin-related transcription factor and/or serum response factor (“MRTF/SRF”) with superior potency (e.g., nanomolar potency) and/or aqueous solubility over known MRTF/SRF inhibitors, while also exhibiting decreased or minimal cytochrome P450 inhibition (“CYP inhibition”). The compounds disclosed herein are useful in the treatment and/or prevention of diseases, such as cancer or fibrotic disease, improving the quality of life for afflicted individuals. For example, the compounds of the disclosure are useful in the prevention of cancer treatment-induced lung fibrosis. By dosing patients prophylactically with the compound disclosed herein, at the start of their cancer treatment, lung fibrosis can be prevented from ever forming. With no fear of lung fibrosis, oncologists can be free to treat all cancer patients with the full number of cycles of chemotherapy or radiotherapy necessary to kill tumors and achieve more effective long-term survival. Such a therapy could change the standard of care for cancer patients and improve the quality of life for cancer survivors.

[0030] The compounds disclosed herein have a structure of Formula (I), wherein the anilide group is substituted at the meta position (R⁴) with Cl, Ca-salkyl, Cs salkenyl, C2- salkynyl, cyclopropyl, CF₃, or OCF₃, and the remaining substituents are as described herein.

The compounds of Formula (I) can be more potent, such as at least two times more potent (e.g., at least three, four, five, six, seven, eight and nine times more potent) and up to ten times more potent, than compounds that do not have a meta R⁴ group, as defined in Formula (I). Moreover, in some cases, the compounds of Formula (I) exhibit greater aqueous solubility than structurally similar compounds having the same cLogP value but without a meta R⁴ group. In some cases, the compounds of Formula (I) are up to five times more soluble than structurally similar compounds having the same cLogP value but without a meta R⁴ group. Tables 1 and 2 in the Examples section below, show the biological activity of the compounds of the disclosure (Table 1 ) and comparative compounds (Table 2) assessed using the serum responsive element firefly luciferase reporter assay (“SRE.L”), as well as their solubilities. For example, a compound of Formula (I):

exhibits a SRE.L IC₅o value of 0.08 pM and an aqueous solubility of 23 pM. However, comparative compounds having a para- or ortho-R⁴ group (E-041 and C2, respectively), a meta R⁴ group other than Cl, C2-₃alkyl, C2-₃alkenyl, C2-

₃alkynyl, cyclopropyl, CF₃, or OCF₃ (C1 and C7), or a methylene spacer (C3) exhibit significantly lower potencies and solubilities. See Table A, below. Further a compound of

Formula (I) wherein Y is N (a 2-pyridinyl group):

(B1) exhibits a SRE.L IC₅o value of 0.37 and an aqueous solubility of 327 pM. However, as shown in Table A, comparative compounds in which the pyridinyl group is a 4-, 5-, or 6- pyridinyl group (C4, C5, and C6) exhibit significantly lower potencies.

Table A. Comparative Compounds

[0031] In various cases, the compounds of Formula (I) inhibit the serum response element (SRE) with an IC₅o up to about 0.5 pM, or up to about 0.4 pM, or up to about 0.3 pM, or up to about 0.2 pM, or up to about 0.1 pM, or up to about 0.05 pM. In some embodiments, the compounds of Formula (I) have an IC₅o value for SRE of less than about 0.5 pM, or less than about 0.4 pM, or less than about 0.3 pM, or less than about 0.2 pM, or less than about 0.1 pM. In some cases, the compounds of Formula (I) have an IC₅o value for SRE between about or less than about 0.8 pM, or less than about 0.6 pM, or less than about 0.4 pM, or less than about 0.3 pM, or less than about 0.2 pM, or less than about 0.1 pM, or less than about 0.05 pM. In various cases, the IC₅o value of the compound of Formula (I) is about 0.05 pM to about 0.5 pM, or abut 0.1 pM to about 0.4 pM, or about 0.15 pM to about 0.4 pM, or about 0.1 pM to about 0.3 pM.

[0032] In some cases, the compounds of Formula (I) exhibit an aqueous solubility of greater than about 10 pM, or greater than about 25 pM, or greater than about 50 pM, or greater than about 75 pM, or greater than about 100 pM, or greater than about 125 pM, or greater than about 150 pM, or greater than about 175 pM, or greater than about 200 pM, or greater than about 225 pM, or greater than about 250 pM, or greater than about 275 pM, or greater than about 300 pM, or greater than about 325 pM, or greater than about 350 pM, or greater than about 375 pM, or greater than about 400 pM.

Compounds of the Disclosure

[0033] Provided herein are compounds of Formula (I), or pharmaceutically acceptable salts thereof:

wherein:

R¹ is H, Me, F, Cl, CN, OH, or OMe; each of R² and R³ independently is H or F, provided that at least one of R² and R³ is

yl, CN, CF₃, or OCF₃; one of X¹ and X² is CH and the other of X¹ and X² is N; and Y is CH or N.

[0034] In some cases, R¹ is H. In some cases, R¹ is Me. In some cases, R¹ is F or Cl. In some cases, R¹ is F. In some cases, R¹ is Cl. In some cases, R¹ is CN. In some cases, R¹ is OH or OMe. In some cases, R¹ is OH. In some cases, R¹ is OMe.

[0035] In some cases, R² is H. In some cases, R² is F. In some cases, R³ is H. In some cases, R³ is F. In some cases, R² is H and R³ is F. In some cases, R² is F and R³ is H. In some cases, each of R² and R³ is H.

[0036] In some cases, R⁴ is Cl. In some cases, R⁴ is C2-₃alkyl, C2-₃alkenyl, or C2-₃alkynyl. In some cases, the C2-₃alkyl, C2-₃alkenyl, and C2-₃alkynyl groups are unsubstituted. In some cases, R⁴ is ethyl, propyl, or isopropyl. In some cases, R⁴ is ethenyl or propenyl. In some cases, R⁴ is ethynyl or propynyl. In some cases, R⁴ is cyclopropyl. In some cases, R⁴ is CF₃, or OCF₃. In some cases, R⁴ is CF₃. In some cases, R⁴ is OCF₃.

[0037] In some cases, R⁵ is H. In some cases, R⁵ is Cl, F, Ci-₃alkyl, OCi-₃alkyl, Cisalkenyl, Ci-₃alkynyl, cyclopropyl, CN, CF₃, or OCF₃. In some cases, R⁵ is Cl or F. In some cases, R⁵ is Cl. In some cases, R⁵ is F. In some cases, R⁵ is Ci-₃alkyl or OCi-₃alkyl. In some cases, R⁵ is Me or OMe. In some cases, R⁵ is Me. In some cases, R⁵ is OMe. In some cases, R⁵ is Ci-₃alkenyl or Ci-₃alkynyl. In some cases, R⁵ is CF₃, or OCF₃. In some cases, R⁵ is CF₃. In some cases, R⁵ is OCF₃. In some cases, R⁵ is H, Cl, F, Me, OMe, CF₃, OCF₃, or CN.

[0038] In some cases, X¹ is CH and X² is N. In some cases, X¹ is N and X² is CH. In some cases, Y is CH. In some cases, Y is N. In some cases X¹ is N, X² is CH, and Y is N. In some cases, X¹ is CH, X² is N, and Y is N. In some cases, X¹ is N, X² is CH, and Y is CH. In some cases, X¹ is CH, X² is N, and Y is CH.

[0039] In some cases, X¹ is N, X² is CH, one of R² and R³ is F and the other of R² and R³ is H, and Y is N. In some cases, R⁴ is Cl.

[0040] Contemplated compounds of the disclosure include, but are not limited to

the foregoing.

[0041] Other contemplated compounds of Formula (I) include, but are not limited to:

foregoing. As shown in Table 1 in the Examples section, disubstituted anilide compounds exhibit excellent potency.

[0042] Unless otherwise indicated, and when applicable, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, cis-trans, conformational, and rotational) forms of the structure. For example, the F?and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this disclosure, unless only one of the isomers is specifically indicated. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, cis/trans, conformational, and rotational mixtures of the present compounds are within the scope of the disclosure. In some cases, the compounds disclosed herein are stereoisomers. "Stereoisomers" refer to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds disclosed herein can exist as a single stereoisomer, or as a mixture of stereoisomers. Stereochemistry of the compounds shown herein indicate a relative stereochemistry, not absolute, unless discussed otherwise. As indicated herein, a single stereoisomer, diastereomer, or enantiomer refers to a compound that is at least more than 50% of the indicated stereoisomer, diastereomer, or enantiomer, and in some cases, at least 90% or 95% of the indicated stereoisomer, diastereomer, or enantiomer.

[0043] Unless otherwise indicated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure. For example, compounds in which X¹ is N

[0044] Additionally, unless otherwise indicated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹¹C- ¹³C- or deenriched carbon are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays. Such compounds, especially deuterium analogs, can also be therapeutically useful. Thus, further disclosed herein are deuterated compounds or salts of Formula (I), in which one or more isotopes of hydrogen have been replaced with deuterium. Further disclosed herein are compounds of Formula (I) having one or more tritium atoms. Also disclosed herein are compounds of Formula (I) in which one or more carbon atoms have been replaced by one or more carbon isotopes, such as one or more C¹¹ atoms, one or more C¹³ atoms, one or more C¹⁴ atoms, or any combination of the foregoing.

[0045] The compounds of the disclosure are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

[0046] As used herein, the term “alkyl” refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms. The term C_n means the alkyl group has “n” carbon atoms. For example, C4 alkyl refers to an alkyl group that has 4 carbon atoms. C1-7 alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 7 carbon atoms), as well as all subgroups (e.g., 1-6, 2-7, 1-5, 3-6, 1 , 2, 3, 4, 5, 6, and 7 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), t-butyl (1 ,1 -dimethylethyl), 3,3- dimethylpentyl, and 2-ethylhexyL Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0047] As used herein, the term “alkenyl” is defined identically as “alkyl” except for containing at least one carbon-carbon double bond, and having two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms. The term C_n means the alkenyl group has “n” carbon atoms. For example, C4 alkenyl refers to an alkenyl group that has 4 carbon atoms. C2-7 alkenyl refers to an alkenyl group having a number of carbon atoms encompassing the entire range (i.e., 2 to 7 carbon atoms), as well as all subgroups (e.g., 2-6, 2-5, 3-6, 2, 3, 4, 5, 6, and 7 carbon atoms). Specifically contemplated alkenyl groups include ethenyl, 1 -propenyl, 2-propenyl, and butenyl. Unless otherwise indicated, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.

[0048] As used herein, the term “alkynyl” is defined identically as “alkyl” except for containing at least one carbon-carbon triple bond, and having two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms. The term C_n means the alkynyl group has “n” carbon atoms. For example, C4 alkynyl refers to an alkynyl group that has 4 carbon atoms. C2-7 alkynyl refers to an alkynyl group having a number of carbon atoms encompassing the entire range (i.e., 2 to 7 carbon atoms), as well as all subgroups (e.g., 2-6, 2-5, 3-6, 2, 3, 4, 5, 6, and 7 carbon atoms). Specifically contemplated alkynyl groups include ethynyl, 1 -propynyl, 2-propenyl, and butynyl. Unless otherwise indicated, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.

[0049] As used herein, the term “cycloalkyl” refers to an aliphatic cyclic hydrocarbon group containing three to eight carbon atoms (e.g., 3, 4, 5, 6, 7, or 8 carbon atoms). The term C_n means the cycloalkyl group has “n” carbon atoms. For example, C₅ cycloalkyl refers to a cycloalkyl group that has 5 carbon atoms in the ring. C₅-s cycloalkyl refers to cycloalkyl groups having a number of carbon atoms encompassing the entire range (i.e., 5 to 8 carbon atoms), as well as all subgroups (e.g., 5-6, 6-8, 7-8, 5-7, 5, 6, 7, and 8 carbon atoms). Nonlimiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be an unsubstituted cycloalkyl group or a substituted cycloalkyl group. The cycloalkyl groups described herein can be isolated, share a carbon atom with another cycloalkyl or heterocycloalkyl group, or fused to another cycloalkyl group, a heterocycloalkyl group, an aryl group and/or a heteroaryl group. Cycloalkyl groups can be saturated or partially unsaturated ring systems optionally substituted with, for example, one to three groups, independently selected alkyl, alkylene-OH, C(O)NH₂, NH₂, oxo (=0), aryl, haloalkyl, halo, and OH.

[0050] As used herein, the term “halo” refers to a fluoro, chloro, bromo, or iodo group, the term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen.

[0051] A used herein, the term “substituted," when used to modify a chemical functional group, refers to the replacement of at least one hydrogen radical on the functional group with a substituent. Substituents can include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycloalkyl, thioether, polythioether, aryl, heteroaryl, hydroxyl, oxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, ester, thioester, carboxy, cyano, nitro, amino, amido, acetamide, and halo (e.g., fluoro, chloro, bromo, or iodo). When a chemical functional group includes more than one substituent, the substituents can be bound to the same carbon atom or to two or more different carbon atoms. A substituted chemical functional group can itself include one or more substituents.

Pharmaceutically Acceptable Salts

[0052] The compounds described herein can exist in free form, or where appropriate, as salts. Those salts that are pharmaceutically acceptable are of particular interest because they are useful in administering the compounds described herein for medical purposes. Salts that are not pharmaceutically acceptable are useful in manufacturing processes, for isolation and purification purposes, and in some cases, for use in separating stereoisomeric forms of the compounds of the disclosure or intermediates thereof.

[0053] As used herein, the term "pharmaceutically acceptable salt" refers to salts of a compound which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue side effects, such as, toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.

[0054] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et aL, describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

[0055] Where the compound described herein contains a basic group, or a sufficiently basic bioisostere, acid addition salts can be prepared by 1 ) reacting the purified compound in its free-base form with a suitable organic or inorganic acid and 2) isolating the salt thus formed. In practice, acid addition salts might be a more convenient form for use and use of the salt amounts to use of the free basic form.

[0056] Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0057] Where the compound described herein contains a carboxyl group or a sufficiently acidic bioisostere, base addition salts can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) isolating the salt thus formed. In practice, use of the base addition salt might be more convenient and use of the salt form inherently amounts to use of the free acid form. Salts derived from appropriate bases include alkali metal (e.g., sodium, lithium, and potassium), alkaline earth metal (e.g., magnesium and calcium), ammonium and N⁺(Ci-4alkyl)₄ salts. This disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0058] Basic addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum. The sodium and potassium salts are usually preferred. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, dietanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)- aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, dicyclohexylamine and the like.

[0059] Other acids and bases, although not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

[0060] It should be understood that a compound disclosed herein can be present as a mixture/combination of different pharmaceutically acceptable salts. Also contemplated are mixtures/combinations of compounds in free form and pharmaceutically acceptable salts.

Pharmaceutical Formulations

[0061] Also provided herein are pharmaceutical formulations that include a compound described herein, such as a compound of Formula (I), and one or more pharmaceutically acceptable excipients. As used herein, the term “formulation” is used interchangeably with “composition.” As used herein, the term “excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API), suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

[0062] The compounds of the disclosure, such as compounds of Formula (I), can be administered alone or as part of a pharmaceutically acceptable composition or formulation. In addition, the compounds can be administered all at once, as for example, by a bolus injection, multiple times, e.g. by a series of tablets, or delivered substantially uniformly over a period of time, as for example, using transdermal delivery. It is also noted that the dose of the compound can be varied over time.

[0063] The compounds of the disclosure, such as compounds of Formula (I), can be administered in combination with one or more additional pharmaceutically active compounds/agents. The additional pharmaceutically active compounds/agents may be small molecules or can be macromolecules such as a proteins, antibodies, peptibodies, DNA, RNA or fragments of such macromolecules.

[0064] The compounds disclosed herein, such as compounds of Formula (I), and other pharmaceutically active compounds, if desired, can be administered to a subject or patient by any suitable route, e.g. orally, topically, rectally, parenterally, (for example, subcutaneous injections, intravenous, intramuscular, intrasternal, and intrathecal injection or infusion techniques), or as a buccal, inhalation, or nasal spray. The administration can be to provide a systemic effect (e.g. eneteral or parenteral). All methods that can be used by those skilled in the art to administer a pharmaceutically active agent are contemplated. In some cases, the disclosed formulations can be administered orally or topically.

[0065] Suitable oral compositions or formulations in accordance with the disclosure include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs. Compositions or formulations suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions.

[0066] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Microorganism contamination can be prevented by adding various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0067] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0068] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active compound can also be in micro- encapsulated form, optionally with one or more excipients. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

[0069] The active compounds can also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0070] The pharmaceutical compositions and formulations described herein may also be administered topically or transdermally, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract, e.g., can be effected in a rectal suppository formulation or in a suitable enema formulation. Dosage forms for topical or transdermal administration of a compound described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, suppositories, or patches.

[0071] For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment, cream, lotion, or gel, containing the active component suspended or dissolved in one or more carriers, and any needed preservatives or buffers as may be required. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

[0072] Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this disclosure. Additionally, the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0073] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanedioL Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0074] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suspensions, in addition to the active compound, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, or mixtures of these substances, and the like.

[0075] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0076] In order to prolong the effect of a compound described herein, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0077] Compositions for rectal or vaginal administration are specifically suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0078] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butanedioL Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0079] The pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

[0080] The compounds for use in the methods of the disclosure can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0081] The compounds of the disclosure, such as compound of Formula (I), can be administered to a subject or patient at dosage levels in the range of about 0.1 to about 3,000 mg per day. For a normal adult human having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kilogram body weight is typically sufficient. The specific dosage and dosage range that will be used can potentially depend on a number of factors, including the requirements of the subject or patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. The determination of dosage ranges and optimal dosages for a particular subject or patient is within the ordinary skill in the art.

[0082] When a patient or subject is to receive or is receiving multiple pharmaceutically active compounds, the compounds can be administered simultaneously, or sequentially. For example, in the case of tablets, the active compounds may be found in one tablet or in separate tablets, which can be administered at once or sequentially in any order. In addition, it should be recognized that the compositions may be different forms. For example, one or more compound may be delivered via a tablet, while another is administered via injection or orally as a syrup. All combinations, delivery methods and administration sequences are contemplated.

Methods

[0083] In some cases, the compounds disclosed herein can inhibit MRTF/SRF-mediated gene transcription, which is useful in preventing or treating diseases related to a dysfunction in MRTF/SRF-mediated gene transcription.

[0084] The rho family of GTPases regulates many aspects of intracellular actin dynamics. Rho signaling causes MRTF to translocate to the cell nucleus and bind to SRF. As used herein the term “rho” or “rho protein” refers to the rho subfamily that includes rhoA, rhoB, rhoC, and others, and is described in Sahai and Marshall Nat. Rev. Cancer 2:133-142 (2002). The binding of MRTF to SRF leads to expression of c-fos, which along with c-jun, forms the transcription factor AP-1 . The AP-1 transcription factor promotes the activity of various matrix metalloproteinases (“MMPs”) and other cell motility genes, the overexpression of which leads to cancer cell invasion and metastasis. Thus, dysfunction of MRTF/SRF- mediated gene transcription has been implicated in cancer metastasis. [0085] Thus, one aspect of the disclosure relates to a method of inhibiting MRTF/SRF- mediated gene transcription in a subject, comprising contacting the MRTF/SRF with a compound (e.g., a compound of Formula (I)) or formulation disclosed herein in an amount effective to inhibit gene transcription. The MRTF/SRF can be contacted in a subject by administering to the subject a compound disclosed herein, such as a compound of Formula (I), or a formulation disclosed herein.

[0086] Dysfunction of MRTF/SRF-mediated gene transcription also has been implicated in fibrosis. The hallmark of fibrotic disease is the transition of normal fibroblasts into myofibroblasts, which are characterized by the expression of alpha smooth muscle actin (“a- SMA”) and the production of extracellular matrix (“ECM”). Fibroblast activation to myofibroblasts results from gene transcription stimulated by a common Rho-mediated signaling pathway that originates from divergent extracellular profibrotic stimuli. Specifically, rho mediates the conversion of G-actin to F-actin, which releases G-actin-bound MRTF. The release of G-actin-bound MRTF results in accumulation of MRTF in the nucleus, where it binds to SRF on the serum response element (“SRE”) promoter. Thus, MRTF serves as a regulator of the fibrotic process used in wound healing, and dysregulation and/or overstimulation of it can lead to fibrosis.

[0087] Cancer patients suffer high morbidity and mortality, not only from the cancer itself, but from secondary toxicities associated with the cancer treatment. The patients at risk for developing treatment-induced lung fibrosis represent a diverse set of cancers: Hodgkin’s lymphoma, germ cell cancers (testicular and ovarian), lung cancer and breast cancer. Currently, when lung fibrosis develops, cancer treatment is halted and patients are administered steroids, such as prednisolone. By dosing patients prophylactically with MRTF/SRF inhibitors, at the start of their cancer treatment, lung fibrosis can be prevented from ever forming

[0088] As such, further provided are methods of treating or preventing a disease associated with dysfunction of MRTF/SRF-mediated gene transcription in a patient comprising administering to the patient an effective amount of a compound as disclosed herein, such as a compound of Formula (I), or a formulation disclosed herein. As used herein the terms “treating”, “treat” or “treatment” and the like include preventative (e.g., prophylactic) and palliative treatment. In some cases, the treating refers to treating a symptom of a disorder or disease as disclosed herein. As used herein, the “MRTF/SRF- mediated gene transcription” refers to gene transcription that is mediated by myocardin- related transcription factor, serum response factor, or both myocardin-related transcription factor and serum response factor. As used herein, the term “dysfunction of MRTF/SRF- mediated gene transcription” relates to an abnormality or impairment gene transcription that is mediated by MRTF, SRF, or both MRTF and SRF. As used herein, an "effective amount" includes a therapeutically effective amount and a prophylactically effective amount. In some cases, the compounds described herein, such as compounds of Formula (I), are administered in therapeutically effective amount. The term "therapeutically effective amount" refers to an amount effective in treating and/or ameliorating a disease or condition in a subject. In some cases, the compounds described herein, such as compounds of Formula (I), are administered in prophylactically effective amount. The term "prophylactically effective amount" refers to an amount effective in preventing and/or substantially lessening the chances of a disease or condition in a subject. As used herein, the terms “patient” and “subject” may be used interchangeably and can mean animals, such as dogs, cats, cows, horses, and sheep (i.e., non-human animals) and humans. Particular patients or subjects are mammals (e.g., humans). The terms “patient” and “subject” include males and females

[0089] In some cases, the disease is cancer, fibrotic disease, diabetes, insulin insensitivity, hyperactive platelets, metabolic disease, diabetic complications (e.g., diabetic nephropathy, insulin resistance, heart failure, and fibrosis), inflammation, inflammatory disease, pulmonary arterial hypertension, axon regeneration following nerve damage, Raynaud's phenomenon, cerebral vascular disease, cardiovascular disease, erectile dysfunction, or combinations thereof.

[0090] In some cases, the disease is a cancer. In some cases, the cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, Ewing's tumor, lymphangioendotheliosarcoma, synovioma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, glioblastoma, leukemia, megakaryoblastic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, or combinations thereof. In some cases, the cancer is megakaryoblastic leukemia, melanoma, breast cancer, prostate cancer, pancreatic cancer, glioblastoma, or combinations thereof.

[0091] In some cases, the disease is a fibrotic disease. As used herein, the term “fibrotic disease” relates to diseases involving fibrosis, which may, e.g., be due to chronic inflammation or repair and reorganization of tissues. Fibrosis may involve any organ of the human body (e.g. the skin, lung, pancreas, liver or kidney). In some cases, the fibrotic disease is systemic sclerosis, localized scleroderma, pulmonary fibrosis, cardiac fibrosis, liver fibrosis, liver cirrhosis, renal fibrosis, diabetic nephropathy, high-glucose-induced nephropathy, chronic renal failure, lung fibrosis, nephrogenic systemic fibrosis, graft versus host disease, Dupuytren's contracture, inflammatory bowel disease, Crohn’s disease, ocular fibrosis, glaucoma, post-trabeculectomy fibrosis, corneal fibrosis, pterygia, Graves opthmalopathy, diabetic retinopathy, age-related macular degeneration, degenerative disc disease, intervertebral disc degeneration, keloid and other scarring/wound healing abnormalities, postoperative adhesions, reactive fibrosis, chronic heart failure (e.g., aftger myocardial infarction) aortic dissection, or combinations thereof. In some cases, the disease is systemic sclerosis or idiopathic pulmonary fibrosis. In some cases, the disease is induced pulmonary fibrosis. In some cases, the pulmonary fibrosis is induced by drugs (drug- induced), chemotherapy (chemotherapy-induced), radiation (radiation-induced), environmental factors (environmental-induced), or toxins (toxin-induced).

[0092] In some embodiments, the disease is a metabolic disease. In some cases, the metabolic disease is obesity, diabetes, insulin resistance, or combinations thereof. In some cases, the metabolic disease is diabetes, such as type II diabetes.

[0093] Use of a compound disclosed herein, such as a compound of Formula (I), to treat a disease condition resulting from dysfunction of MRTF/SRF- mediated gene transcription in a subject, as well as use of the compound in the preparation of a medicament for treating the condition, also are contemplated.

[0094] Further guidance for using compounds disclosed herein for inhibiting MRTF/SRF- mediated gene transcription, such as a compound of Formula (I), can be found in the Examples section, below.

[0095] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities. Kits

[0096] Also provided herein are kits that include a pharmaceutical formulation comprising a compound described herein, such as a compound of Formula (I) and instructions for administering the pharmaceutical formulation to a patient. In some embodiments the kit is provided with a device for administering the formulation to a patient. The kit may further include a variety of containers, e.g., vials, tubes, bottles, and the like. In some cases, the device of the kit is an aerosol dispensing device, wherein the formulation is prepackaged within the aerosol device. In various embodiments, the kit comprises a syringe and a needle, wherein the formulation is optionally prepackaged within the syringe.

EXAMPLES

[0097] The following examples are provided for illustration and are not intended to limit the scope of the invention.

[0098] Synthetic Procedures.

[0099] The compounds of the disclosure can be synthesized by any method known to one skilled in the art. In some cases, the compounds of the disclosure can be synthesized according to Scheme A, below.

Scheme A

[00100] Method G: General Procedure for Basic Ester Hydrolysis of Methyl Esters

[00101] In a round-bottomed flask, the starting methyl ester (1 eq) was dissolved in THE (1-2 mL/1 mmol). 1 M NaOH (1 -2 mL/1 mmol) was added to the solution and the reaction was stirred at 25 °C for 3 h. The THE was evaporated in vacuo, and then the aqueous layer was acidified with 1 N HCI (7 mL). The product was extracted with EtOAc (3 x 15 mL), washed with brine (3 x 10 mL), dried with MgSO4, and concentrated in vacuo. The residue was dissolved in minimal EtOAc and the product was triturated with hexane (Hex), producing a precipitate that was filtered and dried under vacuum.

[00102] Method I: General Procedure for Amide Coupling Using HATU:

[00103] In a round bottomed flask, the carboxylic acid (1 eq) was dissolved in DMF (2-10 mL), and HATU (1 .5 eq), desired aniline (1.1 eq), and DIPEA (3-4 eq) were added, respectively. The reaction was stirred at 25 °C for 16 hr, and then quenched with brine (10 mL). The product was extracted with EtOAc (3 x 15 mL), dried with MgSC , and concentrated in vacuo. The subsequent oil was subjected to silica gel chromatography eluting with 15% EtOAc: 85% Hex. The fractions containing product were concentrated in vacuo.

[00104] Method J: General Procedure for Boc-Deprotection:

[00105] In a round-bottomed flask or scintillation vial, the Boc-amine (1 eq) was dissolved in 4 M HCI/Dioxanes (1.5-10 mL) and the reaction was stirred at 25 °C for 1 hr. The solvents were evaporated in vacuo, and the subsequent solid was taken into the next step without further purification.

[00106] Synthesis of tert-butyl 5-((6-chloropyridin-2-yl)carbamoyl)-3,3- difluoropiperidine-1 -carboxylate (A-2). Method I starting from 1 -(tert-butoxycarbonyl)-5,5- difluoropiperidine-3-carboxylic acid (A-1 , commercial, 0.06 g, 0.23 mmol) and 6- chloropyridin-2-amine (commercial, 0.03 g, 0.23 mmol) gave tert-butyl 5-((6-chloropyridin-2- yl)carbamoyl)-3,3-difluoropiperidine-1 -carboxylate; column: 10-15% EtOAc/Hex; reaction done at 85 °C; ¹H NMR (500 MHz, CDCI₃-d) 8 8.44 (br. s, 1 H) 8.1 1 -8.05 (m, 1 H) 7.72-7.65 (s, 1 H) 7.12-7.06 (m, 1 H) 4.45-4.18 (m, 2H) 3.09 (s, 2H) 2.80 (s, 1 H) 2.39 (s, 1 H) 2.32-2.22 (m, 1 H) 1 .48 (s, 9H); TOP ES+ MS: (M+Na) 398.10; HPLC Ret: 7.71 min.

[00107] Synthesis of N-(6-chloropyridin-2-yl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (B1). Method J followed by Method I starting from tert-butyl 5-((6-chloropyridin-2-yl)carbamoyl)-3,3-difluoropiperidine-1 -carboxylate (A-2, 0.04 g, 0.1 mmol) and 3-fluoro-5-(pyridin-4-yl)benzoic acid (commercial, 0.02 g, 0.1 mmol) gave N-(6-chloropyridin-2-yl)-5,5-difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3- carboxamide (2 steps); column: 75% to 100% EtOAc: Hex; H₂O wash post column needed; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.75 (br.s 1 H) 8.66 (d, J = 5.2 Hz, 2H) 7.96 (d, J = 8.2 Hz, 1 H) 7.83-7.68 (m, 4H) 7.62 (s, 1 H) 7.31 (d, J = 5.8 Hz, 1 H) 7.15 (d, J = 7.7 Hz, 1 H) 4.32-4.10 (m, 2H) 3.55 (dd, J = 29.2, 13.8 Hz, 1 H) 3.29 (t, J = 13.2 Hz, 1 H) 2.98 (br. s, 2H) 2.39-2.23 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -97.46 — 104.04 (m, 2H) -1 11 .16 (t, J = 9.5 Hz, 1 H); QTOF ES+ MS: (M+H) 475.11 ; HPLC Ret: 5.67 min; 98% pure.

[00108] Synthesis of tert-butyl 5-((3-chlorophenyl)carbamoyl)-3,3-difluoropiperidine- 1 -carboxylate (A-2). Method I starting from 1 -(tert-butoxycarbonyl)-5,5-difluoropiperidine-3- carboxylic acid (A-1 , 0.07 g, 0.26 mmol) and 3-chloroaniline (0.03 g, 0.26 mmol) gave tertbutyl 5-((3-chlorophenyl)carbamoyl)-3,3-difluoropiperidine-1 -carboxylate; column: 15% to 30% EtOAc/Hex; ¹H NMR (500 MHz, CDCI₃-d) 8 8.15 (br. s, 1 H) 7.71 (s, 1 H) 7.36 (d, J = 8.0 Hz, 1 H) 7.25 (t, J = 8.1 Hz, 1 H) 7.10 (d, J = 8.0 Hz, 1 H) 4.15-4.01 (m, 2H) 3.38 (br. s, 2H) 2.79 (s, 1 H) 2.50 (s, 1 H) 2.31 (s, 1 H) 1 .48 (s, 9H); TOF ES+ MS: (M+Na) 397.1 1 ; HPLC Ret: 8.01 min.

[00109] Synthesis of 1-(5-bromo-2-fluorobenzoyl)-N-(3-chlorophenyl)-5,5- difluoropiperidine-3-carboxamide. (A-4). Method J followed by Method I starting from tertbutyl 5-((3-chlorophenyl)carbamoyl)-3,3-difluoropiperidine-1 -carboxylate (A-2, 0.06 g, 0.2 mmol) and 5-bromo-2-fluorobenzoic acid (commercial, 0.04 g, 0.2 mmol) gave 1 -(5-bromo-2- fluorobenzoyl)-N-(3-chlorophenyl)-5,5-difluoropiperidine-3-carboxamide (2 steps); column: 20% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.07 (br. s, 1 H) 7.77-7.65 (m, 2H) 7.54 (br. s, 1 H) 7.42 (br. s, 1 H) 7.29 (q, J = 9.4, 9.0 Hz, 2H) 7.09 (d, J = 8.0 Hz, 1 H) 4.50-4.12 (m, 2H) 3.74-3.38 (m, 2H) 3.37-3.31 (m, 1 H) 2.90 (br. s, 1 H) 2.38-2.22 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -97.59 — 102.14 (m, 2H) -117.67 (s, 1 H); QTOF ES+ MS: (M+Na) 498.98; HPLC Ret: 7.78 min; 99% pure.

[00110] Synthesis of N-(3-chlorophenyl)-5,5-difluoro-1-(2-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (B2). In a 10 mL round-bottomed flask, 1 -(5-bromo- 2-fluorobenzoyl)-N-(3-chlorophenyl)-5,5-difluoropiperidine-3-carboxamide (A-4, 0.034 g, 0.071 mmol) and pyridin-4-ylboronic acid (commercial, 0.01 g, 0.075 mmol) were dissolved in a solution of toluene:EtOH (0.5 mL:0.5 mL). The solution was degassed and sodium carbonate (0.02 g, 0.21 mmol) and Tetrakis (0.003 g, 0.05 mmol) were added. The reaction was stirred under N₂ at 90 °C for 16 hr. The reaction was cooled to RT, and the solution was diluted with water (10 mL). The product was extracted with EtOAc (3 x 15 mL), washed with brine (20 mL), dried with MgSO4, and concentrated in vacuo. The yellow residue was subjected to silica gel chromatography eluting with 3% MeOH: 97% DCM. The fractions containing product were concentrated in vacuo. ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.05 (br. s, 1 H) 8.68-8.60 (m, 2H) 7.91 (br. s, 1 H) 7.79-7.69 (m, 2H) 7.70-7.50 (m, 4H) 7.43 (t, J = 8.8 Hz, 2H) 7.30 (t, J = 8.2 Hz, 1 H) 7.08 (d, J = 7.5 Hz, 1 H) 4.66-4.13 (m, 2H) 3.71 - 3.19 (m, 3H) 2.89 (br. s, 1 H) 2.41 -2.25 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -96.18 — 104.76 (m, 2H) -115.80 (s, 1 H); QTOF ES+ MS: (M+Na) 496.10; HPLC Ret: 5.85 min; 98% pure.

[00111] Synthesis of Methyl 5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxylate (A-6). Method J followed by Method I starting from 1 - tert-butyl 3-methyl 5,5-difluoropiperidine-1 ,3-dicarboxylate (commercial, A-5, 0.39 g, 1.79 mmol) and 3-fluoro-5-(pyridin-4-yl)benzoic acid (0.39 g, 1.79 mmol) gave methyl 5,5-difluoro- 1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylate (Intermediate A-6, (2 steps); column: 80% EtOAc: 20% Hex; ¹H NMR (500 MHz, DMSO-cfe) 8 ppm 8.80-8.66 (m, 2H) 7.95 (s, 1 H) 7.86 (d, J = 9.7 Hz, 1 H) 7.84-7.77 (m, 2H) 7.72 (br. s, 1 H) 7.45-7.36 (m, 1 H) 4.58 (br. s, 1 H) 4.26 (br. s, 1 H) 3.70 (br. s, 1 H) 3.56 (br. s, 2H) 3.02 (br. s, 1 H) 2.89 (s, 3H) 2.47-2.30 (m, 1 H); QT0F ES+ MS: (M+H) 379.13; HPLC Ret: 4.73 min.

[00112] Synthesis of 5,5-difluoro-1-(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3- carboxylic acid (A-7). Method G starting from methyl 5,5-difluoro-1 -(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxylate (A-6, 0.7 g, 1.72 mmol) gave 5,5-difluoro-1 -(3-fluoro-5- (pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid; filtered solid that formed after acidification; crystalized with EtOH; ¹H NMR (500 MHz, DMSO-cfe) 8 ppm 13.50 (br. s, 1 H) 8.80-8.66 (m, 2H) 7.95 (s, 1 H) 7.86 (d, J = 9.7 Hz, 1 H) 7.84-7.77 (m, 2H) 7.72 (br. s, 1 H) 7.45-7.36 (m, 1 H) 4.58 (br. s, 1 H) 4.26 (br. s, 1 H) 3.70 (br. s, 1 H) 3.56 (br. s, 2H) 3.02 (br. s, 1 H) 2.47-2.30 (m, 1 H); QTOF ES+ MS: (M+H) 365.1 ; HPLC Ret: 2.10 min.

[00113] Synthesis of 5,5-difluoro-1-(3-fluoro-5-(pyridin-4-yl)benzoyl)-N-(3- (trifluoromethyl)phenyl)piperidine-3-carboxamide. (B3). Method I starting from 5,5- difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.05 g, 0.12 mmol) and 3-(trifluoromethyl)aniline (0.02 g, 0.15 mmol) gave 5,5-difluoro-1 -(3-fluoro-5- (pyridin-4-yl)benzoyl)-N-(3-(trifluoromethyl)phenyl)piperidine-3-carboxamide; column: 70% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.22 (br. s, 1 H) 8.66 (d, J = 4.9 Hz, 2H) 8.01 (s, 1 H) 7.80-7.67 (m, 4H) 7.62 (s, 1 H) 7.52 (t, J = 8.0 Hz, 1 H) 7.40-7.28 (m, 2H) 4.40-4.12 (m, 2H) 3.59 (dd, J = 29.6, 14.0 Hz, 1 H) 3.28 (t, J = 12.5 Hz, 1 H) 2.98 (br. s, 2H) 2.42-2.22 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -61.54 (s, 3H) -96.90 — 101.35 (m, 2H) -11 1.10 (t, J = 9.4 Hz, 1 H); QTOF ES+ MS: (M+H) 508.15; HPLC Ret: 6.12 min; 97% pure.

[00114] Synthesis of N-(3-chlorophenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (B4). Method J followed by Method I starting from tert-butyl 5-((3-chlorophenyl)carbamoyl)-3,3-difluoropiperidine-1 -carboxylate (Intermediate A-2, 0.06 g, 0.16 mmol) and 3-fluoro-5-(pyridin-4-yl)benzoic acid (0.04 g, 0.16 mmol) gave N-(3-chlorophenyl)-5,5-difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide (2 steps); column: 90% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.07 (br.s 1 H) 8.66 (d, J = 5.7 Hz, 2H) 7.78-7.67 (m, 4H) 7.62 (d, J = 1 .6 Hz, 1 H) 7.43 (d, J = 8.3 Hz, 1 H) 7.35-7.26 (m, 2H) 7.09 (d, J = 8.1 Hz, 1 H) 4.44-4.00 (m, 2H) 3.59 (dd, J = 29.6, 14.1 Hz, 1 H) 3.27 (t, J = 12.1 Hz, 1 H) 2.96 (br. s, 1 H) 2.42-2.24 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO- cfe, 80 °C) 8 ppm -97.46 — 102.51 (m, 2H) -11 1.09 (s, 1 H); QTOF ES+ MS: (M+H) 474.1 1 ; HPLC Ret: 5.87 min; 99% pure.

[00115] Synthesis of N-(3-cyclopropylphenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (B5). Method I starting from 5,5-difluoro-1 -(3-fluoro- 5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.05 g, 0.12 mmol) and 3- cyclopropylaniline (0.02 g, 0.15 mmol) gave N-(3-cyclopropylphenyl)-5,5-difluoro-1 -(3-fluoro- 5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide; column: 70% to 80% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 9.78 (s, 1 H) 8.66 (d, J = 4.9 Hz, 2H) 7.77-7.67 (m, 3H)

7.61 (s, 1 H) 7.37-7.23 (m, 3H) 7.14 (t, J = 7.9 Hz, 1 H) 6.79 (d, J = 7.6 Hz, 1 H) 4.20 (br. s, 2H) 3.56 (dd, J = 30.0, 14.0 Hz, 1 H) 3.25 (t, J = 12.5 Hz, 1 H) 2.98 (br. s, 2H) 2.39-2.20 (m, 1 H) 1 .92-1 .81 (m, 1 H) 0.96-0.82 (m, 2H) 0.61 -0.58 (m, 2H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -96.90 — 101 .35 (m, 2H) -1 11 .09 (t, J = 9.4 Hz, 1 H); QTOF ES+ MS: (M+H) 480.19; HPLC Ret: 5.98 min; 99% pure.

[00116] Synthesis of 5,5-difluoro-1-(3-fluoro-5-(pyridin-4-yl)benzoyl)-N-(3- isopropylphenyl)piperidine-3-carboxamide. (B6). Method I starting from 5,5-difluoro-1 -(3- fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.05 g, 0.12 mmol) and 3- isopropylaniline (0.02 g, 0.15 mmol) gave 5,5-difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)-N- (3-isopropylphenyl)piperidine-3-carboxamide; column: 65% to 80% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 9.82 (s, 1 H) 8.66 (d, J = 4.9 Hz, 2H) 7.77-7.67 (m, 3H)

7.62 (s, 1 H) 7.45-7.28 (m, 3H) 7.18 (t, J = 7.9 Hz, 1 H) 6.93 (d, J = 7.7 Hz, 1 H) 4.20 (br. s, 2H) 3.56 (dd, J = 30.0, 13.9 Hz, 1 H) 3.26 (t, J = 12.1 Hz, 1 H) 2.98 (br. s, 2H) 2.84 (p, J = 6.9 Hz, 1 H) 2.37-2.19 (m, 1 H) 1.22-1.13 (m, 6H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm - 96.90 — 104.28 (m, 2H) -1 11 .09 (t, J = 9.4 Hz, 1 H); QTOF ES+ MS: (M+H) 482.21 ; HPLC Ret: 6.23 min; 99% pure.

[00117] Synthesis of N-(3-ethylphenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (B7). Method I starting from 5,5-difluoro-1 -(3-fluoro- 5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.05 g, 0.12 mmol) and 3- ethylaniline (0.02 g, 0.15 mmol) gave N-(3-ethylphenyl)-5,5-difluoro-1 -(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide; column: 65% to 80% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 9.83 (s, 1 H) 8.66 (d, J = 4.9 Hz, 2H) 7.78-7.67 (m, 3H) 7.62 (s, 1 H) 7.42-7.27 (m, 3H) 7.18 (t, J = 7.8 Hz, 1 H) 6.90 (d, J = 7.5 Hz, 1 H) 4.20 (br. s, 2H) 3.56 (dd, J = 29.9, 14.0 Hz, 1 H) 3.26 (t, J = 9.9 Hz, 1 H) 2.98 (br. s, 2H) 2.54 (q, J = 7.6 Hz, 2H) 2.30 (dd, J = 19.2, 13.2 Hz, 1 H) 1.17 (t, J = 7.6 Hz, 3H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -97.30 — 102.57 (m, 2H) -1 11.09 (t, J = 9.3 Hz, 1 H); QTOF ES+ MS: (M+H) 468.19; HPLC Ret: 5.99 min; 98% pure.

[00118] Synthesis of 5,5-difluoro-1-(3-fluoro-5-(pyridin-4-yl)benzoyl)-N-(3- (trifluoromethoxy)phenyl)piperidine-3-carboxamide. (B8). Method I starting from 5,5- difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.05 g, 0.12 mmol) and 3-(trifluoromethoxy)aniline (0.03 g, 0.15 mmol) gave 5,5-difluoro-1 -(3-fluoro-5- (pyridin-4-yl)benzoyl)-N-(3-(trifluoromethoxy)phenyl)piperidine-3-carboxamide; column: 65% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.17 (br. s, 1 H) 8.66 (d, J = 4.9 Hz, 2H) 7.78-7.59 (m, 5H) 7.48 (d, J = 8.1 Hz, 1 H) 7.40 (t, J = 8.1 Hz, 1 H) 7.28 (d, J = 8.4 Hz, 1 H) 7.00 (d, J = 8.2 Hz, 1 H) 4.38-4.08 (m, 2H) 3.58 (dd, J = 29.8, 13.9 Hz, 1 H) 3.27 (t, J = 12.5 Hz, 1 H) 2.98 (br. s, 2H) 2.42-2.25 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -56.61 (s, 3H) -96.90 — 101.35 (m, 2H) -1 11.10 (t, J = 9.4 Hz, 1 H); QTOF ES+ MS: (M+H) 524.14; HPLC Ret: 6.23 min; 99% pure.

[00119] Synthesis of N-(3-ethynylphenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (B9). Method I starting from 5,5-difluoro-1 -(3-fluoro- 5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.05 g, 0.14 mmol) and 3- ethynylaniline (0.02 g, 0.14 mmol) gave N-(3-ethynylphenyl)-5,5-difluoro-1 -(3-fluoro-5- (pyridin-4-yl)benzoyl)piperidine-3-carboxamide; column: 80% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 9.97 (br. s, 1 H) 8.70-8.62 (m, 2H) 7.77-7.67 (m, 4H) 7.62 (d, J = 1 .7 Hz, 1 H) 7.53 (d, J = 8.3 Hz, 1 H) 7.29 (td, J = 8.0, 2.0 Hz, 2H) 7.16 (d, J = 8.2 Hz, 1 H) 4.38-4.10 (m, 2H) 3.92 (s, 1 H) 3.58 (dd, J = 29.6, 13.9 Hz, 1 H) 3.27 (t, J = 12.2 Hz, 1 H) 2.98 (br. s, 2H) 2.42-2.22 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -98.91 — 101.74 (m, 2H) -1 11.08 (t, J = 9.4 Hz, 1 H); QTOF ES+ MS: (M+H) 464.16; HPLC Ret: 5.74 min; 99% pure.

[00120] Synthesis of ferf-butyl 5-((3,4-dichlorophenyl)carbamoyl)-3,3- difluoropiperidine-1 -carboxylate (A-2). Method I starting from 1 -(tert-butoxycarbonyl)-5,5- difluoropiperidine-3-carboxylic acid (A-1 , 0.07 g, 0.26 mmol) and 3,4-dichloroaniline (0.04 g, 0.26 mmol) gave tert-butyl 5-((3,4-dichlorophenyl)carbamoyl)-3,3-difluoropiperidine-1 - carboxylate; column: 15% to 30% EtOAc/Hex; ¹H NMR (500 MHz, CDCh- ) 8 8.39 (br. s, 1 H) 7.85 (s, 1 H) 7.39-7.34 (m, 1 H) 7.28-7.25 (m, 1 H) 4.00-3.94 (m, 2H) 3.51 (br. s, 2H) 2.80 (s, 1 H) 2.55 (s, 1 H) 2.28 (s, 1 H) 1 .48 (s, 9H); TOF ES+ MS: (M+Na) 431 .07; HPLC Ret: 8.42 min.

[00121] Synthesis of 1-(5-bromo-2-fluorobenzoyl)-N-(3,4-dichlorophenyl)-5,5- difluoropiperidine-3-carboxamide (A-4). Method J followed by Method I starting from tertbutyl 5-((3,4-dichlorophenyl)carbamoyl)-3,3-difluoropiperidine-1 -carboxylate (A-2, 0.06 g, 0.15 mmol) and 5-bromo-2-fluorobenzoic acid (0.03 g, 0.15 mmol) gave 1 -(5-bromo-2- fluorobenzoyl)-N-(3,4-dichlorophenyl)-5,5-difluoropiperidine-3-carboxamide (2 steps): 20% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.18 (br. s, 1 H) 7.90 (br. s, 1 H) 7.71 -7.67 (m, 1 H) 7.56-7.45 (m, 3H) 7.28 (t, J = 9.1 Hz, 1 H) 4.65-4.12 (m, 2H) 3.74-3.30 (m, 3H) 2.89 (br. s, 1 H) 2.37-2.21 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm - 95.46 — 104.76 (m, 2H) -1 17.69 (s, 1 H); QTOF ES+ MS: (M+Na) 532.94; HPLC Ret: 8.16 min; 99% pure.

[00122] Synthesis of N-(3,4-dichlorophenyl)-5,5-difluoro-1-(2-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (D1). As described for the synthesis of B2 starting with 1 -(5-bromo-2-fluorobenzoyl)-N-(3,4-dichlorophenyl)-5,5-difluoropiperidine-3- carboxamide. ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.19 (br. s, 1 H) 8.63 (dt, J = 4.9, 1.6 Hz, 2H) 7.91 (br. s, 1 H) 7.79-7.71 (m, 1 H) 7.69-7.55 (m, 3H) 7.56-7.39 (m, 3H) 4.70-4.15 (m, 2H) 3.75-3.18 (m, 3H) 2.96-2.90 (m, 1 H) 2.42-2.21 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO- cfe, 80 °C) 8 ppm -96.90—104.52 (m, 2H) -115.85 (s, 1 H); QTOF ES+ MS: (M+Na) 579.16; HPLC Ret: 6.22 min; 99% pure.

[00123] Synthesis of N-(3-chloro-5-fluorophenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-

4-yl)benzoyl)piperidine-3-carboxamide. (D2). 5,5-dif luoro- 1 -(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.03 g, 0.08 mmol) was dissolved in DMF (1 .0 mL), and EDC (0.02, 0.1 mmol) DMAP (0.01 g, 0.08 mmol), 3-chloro-5-fluoroaniline (0.02 g, 0.12 mmol), and DIPEA (0.03 g, 0.04 mL, 0.25 mmol) were added. The reaction was stirred at 25 °C for 16 hr and brine (10 mL) was added. The product was extracted with EtOAc (3 x 15 mL), dried with MgSC , and concentrated in vacuo. The subsequent oil was subjected to silica gel chromatography eluting with 70% to 80% EtOAc: Hex (2-steps). ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.24 (br. s, 1 H) 8.66 (d, J = 5.7 Hz, 2H) 7.77-7.67 (m, 3H) 7.62 (s, 1 H) 7.48 (s, 1 H) 7.40 (d, J = 1 1 .1 Hz, 1 H) 7.30 (d, J = 8.2 Hz, 1 H) 7.01 (d, J = 8.7 Hz, 1 H) 4.37-4.10 (m, 2H) 3.60 (dd, J = 29.2, 14.0 Hz, 1 H) 3.25 (s, 1 H) 2.98 (br. s, 2H) 2.41 - 2.23 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -98.93 — 101 .84 (m, 2H) - 109.91— -1 10.56 (m, 1 H) -11 1.09 (t, J = 9.4 Hz, 1 H); QTOF ES+ MS: (M+H) 492.1 ; HPLC Ret: 6.02 min; 97% pure.

[00124] Synthesis of N-(3-chloro-5-(trifluoromethyl)phenyl)-5,5-difluoro-1-(3-fluoro-

5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide. (D3). Method I starting from 5,5- difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.03 g, 0.08 mmol) and 3-chloro-5-(trifluoromethyl)aniline (0.02 g, 0.12 mmol) gave N-(3-chloro-5- (trifluoromethyl)phenyl)-5,5-difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3- carboxamide; reaction done at 85 °C; column: 60% EtOAc: Hex; washed with water postcolumn to remove remaining PFe salt; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.35 (br. s, 1 H) 8.65 (d, J = 5.5 Hz, 2H) 7.90 (d, J = 19.9 Hz, 2H) 7.77-7.66 (m, 3H) 7.61 (s, 1 H) 7.43 (s, 1 H) 7.30 (d, J = 8.6 Hz, 1 H) 4.38-4.09 (m, 2H) 3.60 (dd, J = 29.2, 13.8 Hz, 1 H) 3.27 (s, 1 H) 2.96 (br. s, 2H) 2.45-2.22 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -61.83 (s, 3H) -99.16 — 101.82 (m, 2H) -1 11.10 (t, J = 9.0 Hz, 1 H); QTOF ES+ MS: (M+H) 542.1 ; HPLC Ret: 6.48 min; 99% pure.

[00125] Synthesis of N-(3-chloro-5-cyanophenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin- 4-yl)benzoyl)piperidine-3-carboxamide. (D4). Method I starting from 5,5-difluoro-1 -(3- fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.03 g, 0.08 mmol) and 3- amino-5-chlorobenzonitrile (0.01 g, 0.09 mmol) gave N-(3-chloro-5-cyanophenyl)-5,5- difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide; reaction done at 75 °C; column: 65% to 75% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.37 (br. s, 1 H) 8.66 (d, J = 5.3 Hz, 2H) 7.94 (s, 1 H) 7.88 (s, 1 H) 7.78-7.67 (m, 3H) 7.61 (d, J = 7.0 Hz, 2H) 7.30 (d, J = 8.8 Hz, 1 H) 4.36-4.09 (m, 2H) 3.61 (dd, J = 29.5, 13.9 Hz, 1 H) 3.27 (s, 1 H) 2.96 (br. s, 2H) 2.47-2.21 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -97.22 — 101.90 (m, 2H) -11 1.08 (t, J = 9.3 Hz, 1 H); QTOF ES+ MS: (M+H) 499.1 ; HPLC Ret: 5.83 min; 96% pure.

[00126] Synthesis of N-(3-chloro-5-(trifluoromethoxy)phenyl)-5,5-difluoro-1-(3- fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide. (D5). Method I starting from 5,5-difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.03 g, 0.08 mmol) and 3-chloro-5-(trifluoromethoxy)aniline (0.03 g, 0.12 mmol) gave N-(3-chloro-5- (trifluoromethoxy)phenyl)-5,5-difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3- carboxamide; reaction done at 85 °C; column: 60% EtOAc: Hex; washed with water postcolumn to remove remaining PFe salt; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.31 (br. s, 1 H) 8.65 (d, J = 5.6 Hz, 2H) 7.78-7.67 (m, 3H) 7.69-7.55 (m, 3H) 7.30 (d, J = 8.5 Hz, 1 H) 7.12 (s, 1 H) 4.40-4.10 (m, 2H) 3.60 (dd, J = 29.4, 13.9 Hz, 1 H) 3.29 (s, 1 H) 2.97 (br. s, 2H) 2.45-2.14 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -56.82 (s, 3H) -99.08 — 101.85 (m, 2H) -11 1.10 (t, J = 9.3 Hz, 1 H); QTOF ES+ MS: (M+H) 558.1 ; HPLC Ret: 6.58 min; 99% pure.

[00127] Synthesis of N-(3,4-dichlorophenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (D6). Method J followed by Method I starting from tert-butyl 5-((3,4-dichlorophenyl)carbamoyl)-3,3-difluoropiperidine-1 -carboxylate (A-2, 0.06 g, 0.15 mmol) and 3-fluoro-5-(pyridin-4-yl)benzoic acid (0.03 g, 0.15 mmol) gave N-(3,4- dichlorophenyl)-5,5-difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide; column: 90% EtOAc: Hex; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.17 (br.s 1 H) 8.66 (d, J = 5.7 Hz, 2H) 7.90 (d, J = 2.3 Hz, 1 H) 7.78-7.67 (m, 3H) 7.61 (d, J = 1 .7 Hz, 1 H) 7.54- 7.42 (m, 2H) 7.30 (d, J = 8.1 Hz, 1 H) 4.39-4.02 (m, 2H) 3.59 (dd, J = 29.6, 14.0 Hz, 1 H) 3.27 (t, J = 12.3 Hz, 1 H) 2.96 (br. s, 2H) 2.42-2.21 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -97.46 — 103.79 (m, 2H) -1 11 .08 (s, 1 H); QTOF ES+ MS: (M+H) 508.08; HPLC Ret: 6.22 min; 96% pure.

[00128] Synthesis of N-(3-chloro-5-methylphenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin- 4-yl)benzoyl)piperidine-3-carboxamide. (D7). Method I starting from 5,5-difluoro-1 -(3- fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.03 g, 0.08 mmol) and 3- chloro-5-methylaniline (0.01 g, 0.08 mmol) gave N-(3-chloro-5-methylphenyl)-5,5-difluoro-1 - (3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide; reaction done at 80 °C; column: 65% to 75% EtOAc: Hex; washed with water post-column to remove remaining PF₆ salt; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 9.97 (br. s, 1 H) 8.66 (d, J = 8.0 Hz, 2H) 7.77-7.67 (m, 3H) 7.62 (s, 1 H) 7.52 (s, 1 H) 7.30 (ddt, J = 8.7, 2.4, 1.1 Hz, 1 H) 7.26 (s, 1 H) 6.93 (s, 1 H) 4.35-4.09 (m, 2H) 3.58 (dd, J = 29.7, 14.0 Hz, 1 H) 3.26 (t, J = 12.2 Hz, 1 H) 2.97 (br. s, 2H) 2.41 -2.29 (m, 1 H) 2.27 (s, 3H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -96.90 — 102.27 (m, 2H) -11 1.09 (t, J = 9.3 Hz, 1 H); QTOF ES+ MS: (M+H) 488.1 ; HPLC Ret: 6.20 min; 99% pure.

[00129] Synthesis of N-(3,5-dichlorophenyl)-5,5-difluoro-1-(3-fluoro-5-(pyridin-4- yl)benzoyl)piperidine-3-carboxamide. (D8). Method I starting from 5,5-difluoro-1 -(3-fluoro- 5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.03 g, 0.08 mmol) and 3,5- dichloroaniline (0.01 g, 0.08 mmol) gave N-(3,5-dichlorophenyl)-5,5-difluoro-1 -(3-fluoro-5- (pyridin-4-yl)benzoyl)piperidine-3-carboxamide; reaction done at 85 °C; column: 65% to 80% EtOAc: Hex; washed with water post-column to remove remaining PFe salt; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.22 (br. s, 1 H) 8.66 (d, J = 8.0 Hz, 2H) 7.78-7.67 (m, 3H) 7.67-7.60 (m, 3H) 7.30 (d, J = 8.5Hz, 1 H) 7.20 (s, 1 H) 4.38-4.10 (m, 2H) 3.60 (dd, J = 29.3, 14.0 Hz, 1 H) 3.26 (s, 1 H) 2.93 (br. s, 2H) 2.46-2.20 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -97.22 — 106.44 (m, 2H) -1 11 .08 (t, J = 9.5 Hz, 1 H); QTOF ES+ MS: (M+H) 508.1 ; HPLC Ret: 6.49 min; 95% pure.

[00130] Synthesis of N-(3-chloro-5-methoxyphenyl)-5,5-difluoro-1-(3-fluoro-5- (pyridin-4-yl)benzoyl)piperidine-3-carboxamide. (D9). Method I starting from 5,5-difluoro- 1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxylic acid (A-7, 0.03 g, 0.08 mmol) and 3-chloro-5-methoxyaniline (0.01 g, 0.08 mmol) gave N-(3-chloro-5-methoxyphenyl)-5,5- difluoro-1 -(3-fluoro-5-(pyridin-4-yl)benzoyl)piperidine-3-carboxamide; reaction done at 80 °C; column: 65% to 75% EtOAc: Hex; washed with water post-column to remove remaining PFe salt; ¹H NMR (400 MHz, DMSO-cfe, 80 °C) 8 ppm 10.01 (br. s, 1 H) 8.66 (d, J = 8.0 Hz, 2H) 7.78-7.67 (m, 3H) 7.61 (s, 1 H) 7.34-7.25 (m, 2H) 7.12 (s, 1 H) 6.69 (s, 1 H) 4.35-4.08 (m, 2H) 3.75 (s, 3H) 3.58 (dd, J = 29.6, 14.0 Hz, 1 H) 3.28 (t, J = 12.2 Hz, 1 H) 2.96 (br. s, 2H) 2.43- 2.19 (m, 1 H); ¹⁹F NMR (376 MHz, DMSO-cfe, 80 °C) 8 ppm -99.07 — 101 .76 (m, 2H) -11 1 .09 (t, J = 9.4 Hz, 1 H); QTOF ES+ MS: (M+H) 504.1 ; HPLC Ret: 6.06 min; 98% pure.

[00131 ] Assays.

[00132] Dual Luciferase Assay. Seed PC-3 prostate cancer cells (40,000 cells/well) were grown in 96-well plates in 10% FBS containing DMEM medium. Cells were transiently transfected with the Gal2QL activator of the Rho/MKLI pathway, along with the SRE.L- firefly luciferase reporter construct for 6 hours. Additionally, cells were co-transfected with the TK- Ren ilia luciferase reporter as an indicator of non-specific compound effects. Various concentrations of the compounds disclosed herein were added to the 96-well plates. Plates were incubated for 19 hours at 37°C and 5% C02 in 0.5% FBS containing DMEM medium. Cells were lysed with IX Passive Lysis Buffer (Promega). Plates were incubated for 30 minutes at room temperature. Luminescence counts were read with a Victor2 (Perkin-Elmer) plate reader.

[00133] SRE.L-Luciferase Reporter Assay. Biological activity of the compounds disclosed herein, as well as comparative compounds, were assessed in the SRE.L luciferase reporter assay. PC-3 prostate cancer cells were co-transfected with 2 ng of the Ga12Q231 L expression plasmid along with 50 ng of the SRE.L and 7 ng of the pRL-TK luciferase reporter plasmids, as described above. Cells were treated with 0 (vehicle, DMSO alone), 1 , 3, 10, 30, and 100 pM of a compound disclosed herein for 19 hrs after transfection before lysis. Luminescence was determined as described above. Just before cell lysis, the viability of the cells was measured using the WST-1 cell proliferation reagent as described in the Materials and Methods section. Data are expressed as percentage of inhibition (DMSO alone = 0%). The experiments were performed three separate times to achieve n = 3 in triplicate. Results of SRE.L-Luciferase assay are shown in Table 1 (compounds of the disclosure) and Table 2 (comparative compounds), below.

[00134] Assay for inhibition of TGF-p stimulated expression of a-smooth muscle actin (a- SMA). Dermal fibroblasts from healthy human donors were treated for 72 hours with the indicated concentration of the compounds of the disclosure (and 0.1% DMSO control), with or without stimulation by 10 ng/mL TGFpi . Cells were then probed with primary antibody for a-SMA, then fluorophore-conjugated secondary antibody +DAPL Cells were imaged using a fluorescent microscope at 40X magnification. For quantification, cells from three, random non-overlapping fields of view were scored as a-SMA positive or negative by an observer blinded to the treatment.

[00135] Table 1 . Assay Data for Exemplary Compounds of the Disclosure

[00136] Table 2. Assay Data for Comparative Compounds

[00137] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

[00138] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[00139] Throughout the specification, where compositions are described as including components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Likewise, where methods are described as including particular steps, it is contemplated that the methods can also consist essentially of, or consist of, any combination of the recited steps, unless described otherwise. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

[00140] The practice of a method disclosed herein, and individual steps thereof, can be performed manually and/or with the aid of or automation provided by electronic equipment. Although processes have been described with reference to particular embodiments, a person of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various of the steps may be changed without departing from the scope or spirit of the method, unless described otherwise. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[00141] All patents, publications and references cited herein are hereby fully incorporated by reference. In case of conflict between the present disclosure and incorporated patents, publications and references, the present disclosure should control.

Claims

We Claim:

1. A compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein:

R¹ is H, Me, F, Cl, CN, OH, or OMe; each of R² and R³ independently is H or F, provided that at least one of R² and R³ is

cyclopropyl, CF₃, or OCF₃; one of X¹ and X² is CH and the other of X¹ and X² is N;

R⁵ is H, Cl, F, Ci-₃alkyl, OCi-₃alkyl, Ci-₃alkenyl, Ci-₃alkynyl, cyclopropyl, CN, CF₃, or OCF₃; and

Y is CH or N.

2. The compound or salt of claim 1 , wherein R¹ is H.

3. The compound or salt of claim 1 , wherein R¹ is Me.

4. The compound or salt of claim 1 , wherein R¹ is F or Cl.

5. The compound or salt of claim 1 , wherein R¹ is CN.

6. The compound or salt of claim 1 , wherein R¹ is OH or OMe.

7. The compound or salt of any one of claims 1 -6, wherein R² is F and R³ is H.

8. The compound or salt of any one of claims 1 -6, wherein R² is H and R³ is F.

9. The compound or salt of any one of claims 1 -6, wherein each of R² and R³ is

H.

10. The compound or salt of any one of claims 1 -9, wherein R⁴ is Cl.

11. The compound or salt of any one of claims 1 -9, wherein R⁴ is Casalkyl, C2- 3alkenyl, or C2-₃alkynyl.

12. The compound or salt of claim 11 , wherein R⁴ is ethyl, propyl, or isopropyl.

13. The compound of salt of claim 11 , wherein R⁴ is ethenyl or propenyl.

14. The compound of salt of claim 11 , wherein R⁴ is ethynyl or propynyl.

15. The compound or salt of any one of claims 1-9, wherein R⁴ is cyclopropyl.

16. The compound or salt of any one of claims 1-9, wherein R⁴ is CF3 or OCF3.

17. The compound or salt of any one of claims 1-16, wherein R⁵ is H.

18. The compound or salt of any one of claims 1-16, wherein R⁵ is Cl, F, Ci-salkyl,

OCi-salkyl, Cvsalkenyl, Ci-3alkynyl, cyclopropyl, CN, CF₃, or OCF₃.

19. The compound or salt of claim 18, wherein R⁵ is Cl or F.

20. The compound or salt of claim 18, wherein R⁵ is Ci-salkyl or OCi-salkyl.

21. The compound or salt of claim 18, wherein R⁵ is Cvsalkenyl or Ci-salkynyL

22. The compound or salt of claim 18, wherein R⁵ is CF3, or OCF3.

23. The compound or salt of claim 18, wherein R⁵ is Cl, F, Me, OMe, CF3, OCF3, or CN.

24. The compound or salt of any one of claims 1 -23, wherein X¹ is CH and X² is N.

25. The compound or salt of any one of claims 1 -23, wherein X¹ is N and X² is CH.

26. The compound or salt of any one of claims 1 -23, wherein Y is CH.

27. The compound or salt of any one of claims 1 -23, wherein Y is N.

28. The compound or salt of any one of claims 1-23, wherein:

(a) X¹ is N, X² is CH, and Y is N; or

(b) X¹ is CH, X² is N, and Y is N; or

(c) X¹ is N, X² is CH, and Y is CH; or (d) X¹ is CH, X² is N, and Y is CH.

29. The compound or salt of any one of claims 1 -17 and 24-28, wherein the

pharmaceutically acceptable salt of any of the foregoing.

30. The compound or salt of any one of claims 1 -15 and 17-27, wherein the

pharmaceutically acceptable salt of any of the foregoing.

31. A pharmaceutical formulation comprising a compound of any one of claims 1 - 21 and a pharmaceutically acceptable excipient.

32. A kit comprising the pharmaceutical formulation of claim 22 and instructions for administering the pharmaceutical formulation to a patient.

33. A method of inhibiting MRTF/SRF- mediated gene transcription in a subject, comprising contacting the MRTF/SRF with a compound of any one of claims 1-21 or the formulation of claim 22 in an amount effective to inhibit the gene transcription.

34. A method of treating a disease associated with dysfunction of MRTF/SRF- mediated gene transcription in a patient comprising administering to the patient a therapeutically effective amount of the compound of any one of claims 1-21 or the pharmaceutical formulation of claim 22.

35. The method of claim 25, wherein the disease is cancer, fibrotic disease, diabetes, insulin insensitivity, hyperactive platelets, metabolic disease, diabetic complications, inflammation, inflammatory disease, pulmonary arterial hypertension, axon regeneration following nerve damage, Raynaud's phenomenon, cerebral vascular disease, cardiovascular disease, erectile dysfunction, or combinations thereof.

36. The method of claim 26, wherein the cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, Ewing's tumor, lymphangioendotheliosarcoma, synovioma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, glioblastoma, leukemia, megakaryoblastic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, or combinations thereof.

37. The method of claim 27, wherein the cancer is megakaryoblastic leukemia, melanoma, breast cancer, prostate cancer, pancreatic cancer, glioblastoma, or combinations thereof.

38. The method of claim 26, wherein the fibrotic disease is systemic sclerosis, localized scleroderma, pulmonary fibrosis, cardiac fibrosis, liver fibrosis, liver cirrhosis, renal fibrosis, diabetic nephropathy, high-glucose-induced nephropathy, chronic renal failure, lung fibrosis, nephrogenic systemic fibrosis, graft versus host disease, Dupuytren's contracture, keloid, scarring, and wound healing abnormalities, inflammatory bowel disease, Crohn’s disease, ocular fibrosis, glaucoma, post-trabeculectomy fibrosis, postoperative capsular contraction, corneal fibrosis, pterygia, Graves opthmalopathy, diabetic retinopathy, age- related macular degeneration, degenerative disc disease, intervertebral disc degeneration, postoperative adhesions, reactive fibrosis, chronic heart failure, aortic dissection, or combinations thereof.

39. The method of claim 29, wherein the fibrotic disease is systemic sclerosis or idiopathic pulmonary fibrosis.

40. The method of claim 29, wherein the pulmonary fibrosis is drug-induced, chemotherapy-induced, radiation-induced, environmental-induced, or toxin-induced.

41 . The method of claim 26, wherein the metabolic disease is obesity, diabetes, insulin resistance, or combinations thereof.

42. The method of claim 32, wherein the diabetes is type II diabetes.

43. A compound or salt of any one of claims 1 -21 , or a formulation claim 22 for use in the treatment or prevention of a disease or disorder in a subject.

44. Use of the compound or salt of any one of claims 1 -21 or the pharmaceutical composition of claim 22 in a method for treating or preventing a disease or disorder in a subject.