Heterocyclic compounds as bromodomain inhibitors

The present disclosure relates to compounds, which are useful for inhibition of BET protein function by binding to bromodomains, and their use in therapy.

The present disclosure relates to novel compounds, pharmaceutical compositions containing such compounds, and their use in prevention and treatment of diseases and conditions.

Post-translational modifications (PTMs) of histones are involved in regulation of gene expression and chromatin organization in eukaryotic cells. Histone acetylation at specific lysine residues is a PTM that is regulated by histone acetylases (HATs) and deacetylases (HDACs) [1]. Small molecule inhibitors of HDACs and HATs are being investigated as cancer therapy [2-5]. Histone acetylation controls gene expression by recruiting protein complexes that bind directly to acetylated lysine via bromodomains [6]. One such family, the bromodomain and extra terminal domain (BET) proteins, comprises Brd2, Brd3, Brd4, and BrdT, each of which contains two bromodomains in tandem that can independently bind to acetylated lysines, as reviewed in [7].

Interfering with BET protein interactions via bromodomain inhibition results in modulation of transcriptional programs that are often associated with diseases characterized by dysregulation of cell cycle control, inflammatory cytokine expression, viral transcription, hematopoietic differentiation, insulin transcription, and adipogenesis [8].

BET inhibitors are believed to be useful in the treatment of diseases or conditions related to systemic or tissue inflammation, inflammatory responses to infection or hypoxia, cellular activation and proliferation, lipid metabolism, fibrosis, and the prevention and treatment of viral infections [8, 9].

Autoimmune diseases, which are often chronic and debilitating, are a result of a dysregulated immune response, which leads the body to attack its own cells, tissues, and organs. Pro-inflammatory cytokines including IL-1β, TNF-α, IL-6, MCP-1, and IL-17 are overexpressed in autoimmune disease. IL-17 expression defines the T cell subset known as Th17 cells, which are differentiated, in part, by IL-6, and drive many of the pathogenic consequences of autoimmune disease. Thus, the IL-6/Th17 axis represents an important, potentially druggable target in autoimmune disease therapy [10].

BET inhibitors are expected to have anti-inflammatory and immunomodulatory properties [8, 9]. BET inhibitors have been shown to have a broad spectrum of anti-inflammatory effects in vitro including the ability to decrease expression of pro-inflammatory cytokines such as IL-1β, MCP-1, TNF-α, and IL-6 in activated immune cells [11-13]. The mechanism for these anti-inflammatory effects may involve BET inhibitor disruption of Brd4 co-activation of NF-κB-regulated pro-inflammatory cytokines and/or displacement of BET proteins from cytokine promoters, including IL-6 [12, 14, 15]. In addition, because Brd4 is involved in T-cell lineage differentiation, BET inhibitors may be useful in inflammatory disorders characterized by specific programs of T cell differentiation [16].

The anti-inflammatory and immunomodulatory effects of BET inhibition have also been confirmed in vivo. A BET inhibitor prevented endotoxin- or bacterial sepsis-induced death and cecal ligation puncture-induced death in mice, suggesting utility for BET inhibitors in sepsis and acute inflammatory disorders [12]. A BET inhibitor has been shown to ameliorate inflammation and kidney injury in HIV-1 transgenic mice, an animal model for HIV-associated nephropathy, in part through inhibition of Brd4 interaction with NF-κB [14]. The utility of BET inhibition in autoimmune disease was demonstrated in a mouse model of multiple sclerosis, where BET inhibition resulted in abrogation of clinical signs of disease, in part, through inhibition of IL-6 and IL-17 [17]. These results were supported in a similar mouse model where it was shown that treatment with a BET inhibitor inhibited T cell differentiation into pro-autoimmune Th1 and Th17 subsets in vitro, and further abrogated disease induction by pro-inflammatory Th1 cells [18].

BET inhibitors may be useful in the treatment of a wide variety of acute inflammatory conditions including but not limited to, acute gout, giant cell arteritis, nephritis including lupus nephritis, vasculitis with organ involvement, such as glomerulonephritis, vasculitis, including giant cell arteritis, Wegener's granulomatosis, polyarteritis nodosa, Behcet's disease, Kawasaki disease, and Takayasu's arteritis.

BET inhibitors may be useful in the prevention and treatment of diseases or conditions that involve inflammatory responses to infections with bacteria, viruses, fungi, parasites, and their toxins, such as, but not limited to sepsis, sepsis syndrome, septic shock [12], systemic inflammatory response syndrome (SIRS), multi-organ dysfunction syndrome, toxic shock syndrome, acute lung injury, adult respiratory distress syndrome (ARDS), acute renal failure, fulminant hepatitis, burns, post-surgical syndromes, sarcoidosis, Herxheimer reactions, encephalitis, myelitis, meningitis, malaria, and SIRS associated with viral infections, such as influenza, herpes zoster, herpes simplex, and coronavirus [8].

Cancer is a group of diseases caused by dysregulated cell proliferation. Therapeutic approaches aim to decrease the numbers of cancer cells by inhibiting cell replication or by inducing cancer cell differentiation or death, but there is still significant unmet medical need for more efficacious therapeutic agents. Cancer cells accumulate genetic and epigenetic changes that alter cell growth and metabolism, promoting cell proliferation and increasing resistance to programmed cell death, or apoptosis. Some of these changes include inactivation of tumor suppressor genes, activation of oncogenes, and modifications of the regulation of chromatin structure, including deregulation of histone PTMs [20, 21].

The present disclosure provides a method for treating human cancer, including, but not limited to, cancers that result from aberrant translocation or overexpression of BET proteins (e.g., NUT midline carcinoma (NMC) [22]) and B-cell lymphoma [23]). NMC tumor cell growth is driven by a translocation of the Brd4 or Brd3 gene to the nutlin 1 gene [24]. BET inhibition has demonstrated potent antitumor activity in murine xenograft models of NMC, a rare but lethal form of cancer [24].

The present disclosure provides a method for treating human cancers, including, but not limited to, cancers dependent on a member of the myc family of oncoproteins including c-myc, MYCN, and L-myc [25]. These cancers include Burkitt's lymphoma, acute myelogenous leukemia, multiple myeloma, and aggressive human medulloblastoma [25]. Cancers in which c-myc is overexpressed may be particularly susceptible to BET protein inhibition; it has been shown that treatment of tumors that have activation of c-myc with a BET inhibitor resulted in tumor regression through inactivation of c-myc transcription [26-30].

The present disclosure provides a method for treating human cancers including cancers that rely on BET proteins and pTEFb (Cdk9/CyclinT) to regulate oncogenes [31], and cancers that can be treated by inducing apoptosis or senescence by inhibiting Bcl2, cyclin-dependent kinase 6 (CDK6) [26], or human telomerase reverse transcriptase (hTERT) [27, 32].

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity in the United States [34]. Atherosclerosis, an underlying cause of CVD, is a multifactorial disease characterized by dyslipidemia and inflammation. BET inhibitors are expected to be efficacious in atherosclerosis and associated conditions because of aforementioned anti-inflammatory effects as well as ability to increase transcription of ApoA-I, the major constituent of HDL [11, 35].

Up-regulation of ApoA-I is considered to be a useful strategy in treatment of atherosclerosis and CVD [36]. BET inhibitors have been shown to increase ApoA-I transcription and protein expression [11, 35]. It has also been shown that BET inhibitors bind directly to BET proteins and inhibit their binding to acetylated histones at the ApoA-1 promoter, suggesting the presence of a BET protein repression complex on the ApoA-1 promoter, which can be functionally disrupted by BET inhibitors. It follows that, BET inhibitors may be useful in the treatment of disorders of lipid metabolism via the regulation of ApoA-I and HDL such as hypercholesterolemia, dyslipidemia, atherosclerosis [36], and Alzheimer's disease and other neurological disorders [37].

BET inhibitors may be useful in the prevention and treatment of conditions associated with ischemia-reperfusion injury such as, but not limited to, myocardial infarction, stroke, acute coronary syndromes [9], renal reperfusion injury, organ transplantation, coronary artery bypass grafting, cardio-pulmonary bypass procedures, hypertension, pulmonary, renal, hepatic, gastro-intestinal, or peripheral limb embolism.

Obesity-associated inflammation is a hallmark of type II diabetes, insulin resistance, and other metabolic disorders [8, 19]. Consistent with the ability of BET inhibitors to inhibit inflammation, gene disruption of Brd2 in mice ablates inflammation and protects animals from obesity-induced insulin resistance [38]. It has been shown that Brd2 interacts with PPARy and opposes its transcriptional function. Knockdown of Brd2 in vitro promotes transcription of PPARy-regulated networks, including those controlling adipogenesis [39]. In addition Brd2 is highly expressed in pancreatic β-cells and regulates proliferation and insulin transcription [38]. Taken together, the combined effects of BET inhibitors on inflammation and metabolism decrease insulin resistance and may be useful in the treatment of pre-diabetic and type II diabetic individuals as well as patients with other metabolic complications [8].

Host-encoded BET proteins have been shown to be important for transcriptional activation and repression of viral promoters. Brd4 interacts with the E2 protein of human papilloma virus (HPV) to enable E2 mediated transcription of E2-target genes [40]. Similarly, Brd2, Brd3, and Brd4 all bind to latent nuclear antigen 1 (LANA1), encoded by Kaposi's sarcoma-associated herpes virus (KSHV), promoting LANA1-dependent proliferation of KSHV-infected cells [41]. A BET inhibitor has been shown to inhibit the Brd4-mediated recruitment of the transcription elongation complex pTEFb to the Epstein-Barr virus (EBV) viral C promoter, suggesting therapeutic value for EBV-associated malignancies [42]. Also, a BET inhibitor reactivated HIV in models of latent T cell infection and latent monocyte infection, potentially allowing for viral eradication by complementary anti-retroviral therapy [43-46].

BET inhibitors may be useful in the prevention and treatment of episome-based DNA viruses including, but not limited to, human papillomavirus, herpes virus, Epstein-Barr virus, human immunodeficiency virus [8], adenovirus, poxvirus, hepatitis B virus, and hepatitis C virus.

Some central nervous system (CNS) diseases are characterized by disorders in epigenetic processes. Brd2 haplo-insufficiency has been linked to neuronal deficits and epilepsy [47]. SNPs in various bromodomain-containing proteins have also been linked to mental disorders including schizophrenia and bipolar disorders [9]. In addition, the ability of BET inhibitors to increase ApoA-I transcription may make BET inhibitors useful in Alzheimer's disease therapy considering the suggested relationship between increased ApoA-I and Alzheimer's disease and other neurological disorders [37].

BRDT is the testis-specific member of the BET protein family which is essential for chromatin remodeling during spermatogenesis [48, 49]. Genetic depletion of BRDT or inhibition of BRDT interaction with acetylated histones by a BET inhibitor resulted in a contraceptive effect in mice, which was reversible when small molecule BET inhibitors were used [50, 51]. These data suggest potential utility of BET inhibitors as a novel and efficacious approach to male contraception.

Monocyte chemotactic protein-1 (MCP-1, CCL2) plays an important role in cardiovascular disease [52]. MCP-1, by its chemotactic activity, regulates recruitment of monocytes from the arterial lumen to the subendothelial space, where they develop into macrophage foam cells, and initiate the formation of fatty streaks which can develop into atherosclerotic plaque [53]. The critical role of MCP-1 (and its cognate receptor CCR2) in the development of atherosclerosis has been examined in various transgenic and knockout mouse models on a hyperlipidemic background [54-57]. These reports demonstrate that abrogation of MCP-1 signaling results in decreased macrophage infiltration to the arterial wall and decreased atherosclerotic lesion development.

The association between MCP-1 and cardiovascular disease in humans is well-established [52]. MCP-1 and its receptor are overexpressed by endothelial cells, smooth muscle cells, and infiltrating monocytes/macrophages in human atherosclerotic plaque [58]. Moreover, elevated circulating levels of MCP-1 are positively correlated with most cardiovascular risk factors, measures of coronary atherosclerosis burden, and the incidence of coronary heart disease (CHD) [59]. CHD patients with among the highest levels of MCP-1 are those with acute coronary syndrome (ACS) [60]. In addition to playing a role in the underlying inflammation associated with CHD, MCP-1 has been shown to be involved in plaque rupture, ischemic/reperfusion injury, restenosis, and heart transplant rejection [52].

MCP-1 also promotes tissue inflammation associated with autoimmune diseases including rheumatoid arthritis (RA) and multiple sclerosis (MS). MCP-1 plays a role in the infiltration of macrophages and lymphocytes into the joint in RA, and is overexpressed in the synovial fluid of RA patients [61]. Blockade of MCP-1 and MCP-1 signaling in animal models of RA have also shown the importance of MCP-1 to macrophage accumulation and proinflammatory cytokine expression associated with RA [62-65].

Overexpression of MCP-1, in the brain, cerebrospinal fluid (CSF), and blood, has also been associated with chronic and acute MS in humans [66]. MCP-1 is overexpressed by a variety of cell types in the brain during disease progression and contributes to the infiltration of macrophages and lymphocytes which mediate the tissue damage associated with MS [66]. Genetic depletion of MCP-1 or CCR2 in the experimental autoimmune encephalomyelitis (EAE) mouse model, a model resembling human MS, results in resistance to disease, primarily because of decreased macrophage infiltration to the CNS [67, 68].

Preclinical data have suggested that small- and large-molecule inhibitors of MCP-1 and CCR2 have potential as therapeutic agents in inflammatory and autoimmune indications.

The present disclosure includes compounds that are useful for inhibition of BET protein function by binding to bromodomains, and their use in the treatment and prevention of diseases and conditions, including, but not limited to, cancer, autoimmune, and cardiovascular diseases.

The first aspect of the present disclosure includes compounds of Formula I and methods of administering a therapeutically effective amount of those compounds to a mammal (e.g., a human) in need thereof.

The present invention includes compounds that are useful for inhibition of BET protein function by binding to bromodomains, and their use in the treatment and prevention of diseases and conditions, including, but not limited to, cancer, autoimmune, and cardiovascular diseases.

The first aspect of the invention includes compounds of Formula I and methods of administering a therapeutically effective amount of those compounds to a mammal (e.g., a human) in need thereof:

or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof,
wherein:W1is selected from N and CR5;W2is selected from N and CR4;W3is selected from N and CR3;each W may be the same or different from each other;R1is selected from a carbocycles or heterocycles;R2is selected from a 5- or 6-membered monocyclic carbocycle or a 5- or 6-membered monocyclic heterocycle;R3, R4, and R5are each independently selected from hydrogen, alkyl, —OH, —NH2, thioalkyl, alkoxy, ketone, ester, carboxylic acid, urea, carbamate, carbonate, amino, amide, halogen, carbocycle, heterocycle, sulfone, sulfoxide, sulfide, sulfonamide, and —CN;R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle;R4may be connected to B or R2to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO2—, —CRxRyCRzRvO—, —CRxRyCRzRvN—, —CRxRyCRzRvS—, and —CRxRyCRzRvCRQRR—;Rx, Ry, Rz, Rv, RQ, and RRare each independently selected from hydrogen, alkyl(C1-C8), halogen, —OH, —CF3, amino, alkoxy (C1-C8), carboxyl, —CN, sulfone, and sulfoxide, carbocycle, heterocycle, or two substituents selected from Rx, Ry, Rz, Rv, RQand RRmay form an oxo or thio-oxo group, or two substituents selected from Rx, Ry, Rz, Rv, R5, and R1may be connected in a 5- or 6-membered ring to form a bicyclic carbocycle or bicyclic heterocycle;B is selected from —(CRaRb)n—, —(CRaRbCRcRd)—, —O—, —OCRaRb—, —CRaRbO—, —NH—, —NHCRaRb—, —CRaRbNH—, —S—, —SCRaRb—, —CRaRbS—, —S(O)—, —S(O)CRaRb—, —CRaRbS(O)—, —SO2—, —SO2CRaRb—, and —CRaRbSO2—;n is selected from 0 and 1, meaning if n=0 then B is absent and R2is connected directly to the center ring;Ra, Rb, Rc, and Rdare each independently selected from hydrogen, alkyl(C1-C3), and alkoxy(C1-C3).

In another aspect of the invention, a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients is provided.

In yet another aspect of the invention there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof for use in therapy, in particular in the treatment of diseases or conditions for which a bromodomain inhibitor is indicated.

In yet another aspect of the invention there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of diseases or conditions for which a bromodomain inhibitor is indicated.

DEFINITIONS

As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise. The following abbreviations and terms have the indicated meanings throughout.

“Subject” refers to an animal, such as a mammal, that has been or will be the object of treatment, observation, or experiment. The methods described herein may be useful for both human therapy and veterinary applications. In one embodiment, the subject is a human.

As used herein, “treatment” or “treating” refers to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, “treatment” or “treating” refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In yet another embodiment, “treatment” or “treating” refers to inhibiting the progression of a disease or disorder, either physically, e.g., stabilization of a discernible symptom, physiologically, e.g., stabilization of a physical parameter, or both. In yet another embodiment, “treatment” or “treating” refers to delaying the onset of a disease or disorder. For example, treating a cholesterol disorder may comprise decreasing blood cholesterol levels.

As used herein, “prevention” or “preventing” refers to a reduction of the risk of acquiring a given disease or disorder.

A dash (“-”) that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, —CONH2is attached through the carbon atom.

By “optional” or “optionally” is meant that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which is does not. For example, “optionally substituted aryl” encompasses both “aryl” and “substituted aryl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and/or inherently unstable.

As used herein, the term “hydrate” refers to a crystal form with either a stoichiometric or non-stoichiometric amount of water is incorporated into the crystal structure.

The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-8 carbon atoms, referred to herein as (C2-C8)alkenyl. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, and 4-(2-methyl-3-butene)-pentenyl.

The term “alkoxy” as used herein refers to an alkyl group attached to an oxygen (—O-alkyl-). “Alkoxy” groups also include an alkenyl group attached to an oxygen (“alkenyloxy”) or an alkynyl group attached to an oxygen (“alkynyloxy”) groups. Exemplary alkoxy groups include, but are not limited to, groups with an alkyl, alkenyl or alkynyl group of 1-8 carbon atoms, referred to herein as (C1-C8)alkoxy. Exemplary alkoxy groups include, but are not limited to methoxy and ethoxy.

The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-8 carbon atoms, referred to herein as (C2-C8)alkynyl. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl.

The term “amide” as used herein refers to the form —NRaC(O)(Rb)— or —C(O)NRbRc, wherein Ra, Rband Rcare each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. The amide can be attached to another group through the carbon, the nitrogen, Rb, or Rc. The amide also may be cyclic, for example Rband Rc, may be joined to form a 3- to 8-membered ring, such as 5- or 6-membered ring. The term “amide” encompasses groups such as sulfonamide, urea, ureido, carbamate, carbamic acid, and cyclic versions thereof. The term “amide” also encompasses an amide group attached to a carboxy group, e.g., -amide-COOH or salts such as -amide-COONa, an amino group attached to a carboxy group (e.g., -amino-COOH or salts such as -amino-COONa).

The term “amine” or “amino” as used herein refers to the form —NRdReor —N(Rd)Re—, where Rdand Reare independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, carbamate, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. The amino can be attached to the parent molecular group through the nitrogen. The amino also may be cyclic, for example any two of Rdand Remay be joined together or with the N to form a 3- to 12-membered ring (e.g., morpholino or piperidinyl). The term amino also includes the corresponding quaternary ammonium salt of any amino group. Exemplary amino groups include alkylamino groups, wherein at least one of Rdor Reis an alkyl group. In some embodiments Rd and Re each may be optionally substituted with hydroxyl, halogen, alkoxy, ester, or amino.

The term “aryl” as used herein refers to a mono-, bi-, or other multi-carbocyclic, aromatic ring system. The aryl group can optionally be fused to one or more rings selected from aryls, cycloalkyls, and heterocyclyls. The aryl groups of this present disclosure can be substituted with groups selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. Exemplary aryl groups also include, but are not limited to a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as “(C6)aryl.”

The term “arylalkyl” as used herein refers to an alkyl group having at least one aryl substituent (e.g., -aryl-alkyl-). Exemplary arylalkyl groups include, but are not limited to, arylalkyls having a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as “(C6)arylalkyl.”

The term “carbamate” as used herein refers to the form —RgOC(O)N(Rh)—, —RgOC(O)N(Rh)Ri—, or —OC(O)NRhRi, wherein Rg, Rhand Riare each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. Exemplary carbamates include, but are not limited to, arylcarbamates or heteroaryl carbamates (e.g., wherein at least one of Rg, Rhand Riare independently selected from aryl or heteroaryl, such as pyridine, pyridazine, pyrimidine, and pyrazine).

The term “carboxy” as used herein refers to —COON or its corresponding carboxylate salts (e.g., —COONa). The term carboxy also includes “carboxycarbonyl,” e.g. a carboxy group attached to a carbonyl group, e.g., —C(O)—COOH or salts, such as —C(O)—COONa.

The term “cyano” as used herein refers to —CN.

The term “cycloalkoxy” as used herein refers to a cycloalkyl group attached to an oxygen.

The term “ester” refers to the structure —C(O)O—, —C(O)O—Rj-, —RkC(O)O—Rj-, or —RkC(O)O—, where O is not bound to hydrogen, and Rjand Rkcan independently be selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, cycloalkyl, ether, haloalkyl, heteroaryl, and heterocyclyl. Rkcan be a hydrogen, but Rjcannot be hydrogen. The ester may be cyclic, for example the carbon atom and Rj, the oxygen atom and Rk, or Rjand Rkmay be joined to form a 3- to 12-membered ring. Exemplary esters include, but are not limited to, alkyl esters wherein at least one of Rj or Rk is alkyl, such as —O—C(O)-alkyl, —C(O)—O-alkyl-, and -alkyl-C(O)—O-alkyl-. Exemplary esters also include aryl or heteoraryl esters, e.g. wherein at least one of Rj or Rk is a heteroaryl group such as pyridine, pyridazine, pyrimidine and pyrazine, such as a nicotinate ester. Exemplary esters also include reverse esters having the structure —RkC(O)O—, where the oxygen is bound to the parent molecule. Exemplary reverse esters include succinate, D-argininate, L-argininate, L-lysinate and D-lysinate. Esters also include carboxylic acid anhydrides and acid halides.

The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I.

The term “haloalkyl” as used herein refers to an alkyl group substituted with one or more halogen atoms. “Haloalkyls” also encompass alkenyl or alkynyl groups substituted with one or more halogen atoms.

The terms “hydroxy” and “hydroxyl” as used herein refer to —OH.

The term “hydroxyalkyl” as used herein refers to a hydroxy attached to an alkyl group.

The term “hydroxyaryl” as used herein refers to a hydroxy attached to an aryl group.

The term “ketone” as used herein refers to the structure —C(O)—Rn (such as acetyl, —C(O)CH3) or —Rn-C(O)—Ro-. The ketone can be attached to another group through Rnor Ro. Rnor Rocan be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, or Rnor Rocan be joined to form a 3- to 12-membered ring.

The term “monoester” as used herein refers to an analogue of a dicarboxylic acid wherein one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is a free carboxylic acid or salt of a carboxylic acid. Examples of monoesters include, but are not limited to, to monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid.

The term “thioalkyl” as used herein refers to an alkyl group attached to a sulfur (—S-alkyl-).

As used herein, a suitable substitution on an optionally substituted substituent refers to a group that does not nullify the synthetic or pharmaceutical utility of the compounds of the present disclosure or the intermediates useful for preparing them. Examples of suitable substitutions include, but are not limited to: C1-8alkyl, alkenyl or alkynyl; C1-6aryl, C7-5heteroaryl; C3-7cycloalkyl; C1-8alkoxy; C6aryloxy; —CN; —OH; oxo; halo, carboxy; amino, such as —NH(C1-8alkyl), —N(C1-8alkyl)2, —NH((C6)aryl), or —N((C6)aryl)2; formyl; ketones, such as —CO(C1-8alkyl), —CO((C6aryl) esters, such as —CO2(C1-8alkyl) and —CO2(C6aryl). One of skill in art can readily choose a suitable substitution based on the stability and pharmacological and synthetic activity of the compound of the present disclosure.

The term “pharmaceutically acceptable carrier” as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

The term “pharmaceutically acceptable composition” as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

The term “pharmaceutically acceptable prodrugs” as used herein represents those prodrugs of the compounds of the present disclosure that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present disclosure. A discussion is provided in Higuchi et al., “Prodrugs as Novel Delivery Systems,”ACS Symposium Series, Vol. 14, and in Roche, E. B., ed.Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

The term “pharmaceutically acceptable salt(s)” refers to salts of acidic or basic groups that may be present in compounds used in the present compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, matate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.

Individual stereoisomers of compounds of the present disclosure can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Stereoisomers can also be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

Geometric isomers can also exist in the compounds of the present disclosure. The present disclosure encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the E and Z isomers.

Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangements of substituents around a carbocyclic ring are designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”

The compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the present disclosure, even though only one tautomeric structure is depicted.

EXEMPLARY EMBODIMENTS

In a preferred aspect of Formula I, the invention is directed to a compound according to Formula II:

or a stereoisomer, tautomer, pharmaceutical acceptable salt, or hydrate thereof,
wherein:W1is selected from N and CR5;W2is selected from N and CR4;W3is selected from N and CR3, with the proviso that if W3is N then neither R5nor R4is —OH;each W may be the same or different from each other;R1is a carbocycle or heterocycle;V is selected from a 5-membered monocyclic carbocycle or monocyclic heterocycle, where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond,with the proviso that V cannot be unsubstituted thiophene, cyclopentyl, cyclopentenyl, ribofuranosyl, or furan,and with the proviso that if W1═CR5and V is an optionally substituted

then at least one of R3and R4are different from hydrogen, or if W3═N, then R4is different from hydrogen,and with the proviso that if W1═CR5and V is

then R1is different from

and with the proviso that if W1═CR5and V is

then R1is not

and with the proviso that if W1═N and V is an optionally substituted

then at least one of R3and R4are different from hydrogen, or if W3→N, then R4is different from hydrogen,and with the proviso that if W1═N and V is an optionally substituted

then R1-A is different from

and with the proviso that if W1═N and V is

then R3and R4cannot be

and with the proviso that if B is present (meaning n is different from zero) then neither R4or R5can be hydroxyl;R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle;R4may be connected to B or V to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO3—, —CRxRyCRzRvO—, —CRxRyCRzRvN—, —CRxRyCRzRvS—, and —CRxRyCRzRvCRQRR—;with the proviso that Rxand Rycannot both be an unsubstituted phenyl ring,and with the proviso that if A is —CH2CH2CH2— and W3is N then R4is not —OH,and with the proviso that if A is —CH2CH2O— or —CH2C(O)NH— then V is not a substituted

or a substituted

and with the proviso that if A is —CH2CH2O— the R1is not

In some embodiments, according to Formula II, V is selected from an optionally substituted 5-membered monocyclic heterocycle, such as, but not limited to:

In some embodiments according to Formula II, V is selected from an optionally substituted 5-membered monocyclic heterocycle containing one oxygen and one or two nitrogens, where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond.

In some embodiments, according to Formula II, V is an optionally substituted isoxazole.

In some embodiments, according to Formula II, V is

In some embodiments, according to Formula II, W1is CR5.

In some embodiments, according to Formula II, W2is CR4.

In some embodiments, according to Formula II, X is oxygen.

In some embodiments, according to Formula II, n=0, meaning B is absent.

In some embodiments, according to Formula II, A is selected from C═O and —CRxRy—.

In some embodiments, according to Formula II, R1is selected from an optionally substituted 5- and 6-membered carbocycle and heterocycle (such as phenyl, pyridyl, thiophene, or cyclopentyl).

In some embodiments, according to Formula II, R1is selected from an optionally substituted phenyl or pyridyl ring.

In some embodiments, according to Formula II, R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle such as

In some embodiments, according to Formula II, Rxand Ryare independently selected from hydrogen, methyl, and —CF3.

In some embodiments, according to Formula II, Rxand Rvare independently selected from hydrogen, methyl, and —CF3.

In some embodiments, according to Formula II, Ra, Rb, Rc, and Rdare independently selected from hydrogen, methyl, methoxy, and —CF3.

In some embodiments, according to Formula II, B is selected from —(CRaRb)n—, —O—, —NH—, —S—, —S(O)—, and —SO2—, where n is 0 or 1, meaning if n=0 then B is absent.

In some embodiments, according to Formula II, B is selected from —(CRaRb)n—, —O—, —NH—, and —S—, where n is 0 or 1, meaning if n=0 then B is absent.

In certain embodiments of the disclosure, the compound of formula I is 1-(4-chlorobenzyl)-5-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)pyridin-2(1H)-one (Example 197).

In a second aspect of Formula I, the invention is directed to a compound according to Formula III:

or a stereoisomer, tautomer, pharmaceutical acceptable salt, or hydrate thereof,wherein:W2is selected from N and CR4,with the proviso that if W2is N and R2is

then R5is not hydrogen;W3is selected from N and CR3,with the proviso that if W3is N then neither R5or R4can be —OH;each W may be the same or different from each other;R1is a carbocycle or heterocycle,with the proviso R1-A is not

and with the proviso that if R1-A is

then at least one of Q1, Q2, Q3, or Q4is different from hydrogen,and with the proviso that if R1-A is

then at least one of R3and R4is not hydrogen,and with the proviso that if R1is

then R2is not

and with the proviso that if R1is

then R2is not

R2is selected from a 6-membered monocyclic carbocycle or monocyclic heterocycle, with the proviso that R2is not

or an optionally substituted

and with the proviso that if R2is

then at least one of R3and R4is not hydrogen,and with the proviso that if R3is —CN, then R2is not

and with the proviso that if R2is

then R1is not

and with the proviso that if R2is

then R5is not —COOMeand with the proviso that if R4is —NH2then R2is not

R3, R4, and R5are each independently selected from hydrogen, alkyl, —OH, —NH2, thioalkyl, alkoxy, ketone, ester, carboxylic acid, urea, carbamate, carbonate, amino, amide, halogen, carbocycle, heterocycle, sulfone, sulfoxide, sulfide, sulfonamide, and —CN,with the proviso that R4is not —OH and R5is not —COOH or -ester;R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle;R4may be connected to B or R2to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO2—, —CRxRyCRzRvO—, —CRxRyCRzRvN—, —CRxRyCRzRvS—, and —CRxRyCRzRvCRQRR—;with the proviso that Rxand Rycannot both be an unsubstituted phenyl ring,and with the proviso that if A is —CH2CH2CH2— and W3is N then R4is not —OH,and with the proviso that if A is —CH2CH2O— the R1is not

In some embodiments, according to Formula III, R2is selected from an optionally substituted 6-membered monocyclic carbocycle (such as phenyl) or heterocycle (such as pyridyl, pyrimidine, pyrazine, and triazine), where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond.

In some embodiments, according to Formula III, R2is selected from

In some embodiments, according to Formula III, R2is selected from

In some embodiments, according to Formula III, R1is selected from a 3-, 4-, 5-, or 6-membered carbocycle or heterocycle.

In some embodiments, according to Formula III, R1is selected from an optionally substituted phenyl.

In some embodiments, according to Formula III, R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle such as

In some embodiments, according to Formula III, R5is hydrogen.

In some embodiments, according to Formula III, R4is hydrogen.

In some embodiments, according to Formula III, X is oxygen.

In some embodiments, according to Formula III, n=0, meaning B is absent.

In some embodiments, according to Formula III, B is selected from —(CRaRb)n—, —O—, —NH—, —S—, where n is 0 or 1, meaning if n=0 then B is absent.

In some embodiments, according to Formula III, A is selected from C═O and —CRxRy—

In a third aspect of Formula I, the invention concerns a compound according to Formula IV:

or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof,wherein:W2is selected from N and CR4,with the proviso that if W2is N and R2is

then R5is not hydrogen;W3is selected from N and CR3,with the proviso that if W3is N then neither R5or R4can be —OH;each W may be the same or different from each other;R1is a carbocycle or heterocycle,with the proviso that R1is different from an amino group with nitrogen attached to A (such as

a substituted napthyl, or cyclohexyl,and with the proviso that R1-A is not

and with the proviso that if R1-A is

then R2is not an optionally substituted

where T is halogen,and with the proviso that if R1-A is

then R2is not

and with the proviso that if R1A is

then R2is not substituted with —OH or —NH2;R2is selected from a 6-membered monocyclic carbocycle or monocyclic heterocycle,with the proviso that R2is not unsubstituted thiophene, furane, cyclopentyl, cyclohexyl, or

where T can be any atom,and with the proviso that R2is not

where T is Cl, Br, —OMe, or Me,and with the proviso that R2is not

where T and Y are independently selected from Cl, F, -Me, —CN, and —OH,and with the proviso that R2is not

and with the proviso that if R2is

then R1-A is not

where T and Y are independently selected from hydrogen, F, Cl, Br, —CF3, and -Me, and R1is not unsubstituted pyridyl, substituted furane, or unsubstituted naphthyl,and with the proviso that if R2is

where T is an —OH, Alkoxy, —OAcyl, —NH2, amino, amide, carbamate, or urea, substituent, then at least one of R3and R4is different from hydrogen,and with the proviso that if R2is an unsubstituted pyridyl, then at least one of R3and R4is different from hydrogen, or R1-A is different from

or R3and R4are not connected to form an unsubstituted benzene ring,and with the proviso that if R2is

then R3is not methyl, at least one of R3and R4cannot be connected to

or R1-A cannot be

R4may be connected to B or R2to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO2—, —CRxRyCRzRvO—, —CRxRyCRzRvN—, —CRxRyCRzRvS—, and —CRxRyCzRvCRQRR—;with the proviso that if A is C═O, then R2is not an optionally substituted

where T is halogen,and with the proviso that Rxand Rycannot both be an unsubstituted phenyl ring,and with the proviso that if A is —CH2CH2CH2— and W3is N then R4is not —OH,and with the proviso that if A is —CH2CH2O— the R1is not

In some embodiments, according to Formula IV, R2is selected from an optionally substituted 6-membered monocyclic carbocycle (such as phenyl) or heterocycle (such as pyridyl, pyrimidine, pyrazine, and triazine), where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond.

In some embodiments, according to Formula IV, R2is selected from

In some embodiments, according to Formula IV, R2is selected from

In some embodiments, according to Formula IV, R1is selected from a 3-, 4-, 5-, or 6-membered carbocycle or heterocycle.

In some embodiments, according to Formula IV, R1is selected from an optionally substituted phenyl.

In some embodiments, according to Formula IV, R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle such as

In some embodiments, according to Formula IV, R4is hydrogen.

In some embodiments, according to Formula IV, X is oxygen.

In some embodiments, according to Formula IV, n=0, meaning B is absent.

In some embodiments, according to Formula IV, B is selected from —(CRaRb)n—, —O—, —NH—, —S—, where n is 0 or 1, meaning if n=0 then B is absent.

In some embodiments, according to Formula IV, A is selected from C═O and —CRxRy—

Another aspect of the invention provides a method for inhibition of BET protein function by binding to bromodomains, and their use in the treatment and prevention of diseases and conditions in a mammal (e.g., a human) comprising administering a therapeutically effective amount of a compound of Formulae I-IV.

In one embodiment, BET inhibitor compounds of Formulae I-IV may be used for treating rheumatoid arthritis (RA) and multiple sclerosis (MS). Strong proprietary data exist for the utility of BET inhibitors in preclinical models of RA and MS [17]. Both RA and MS are characterized by a dysregulation of the IL-6 and IL-17 inflammatory pathways [10] and thus would be especially sensitive to BET inhibition. In another embodiment, BET inhibitor compounds of Formulae I-IV may be used for treating sepsis and associated afflictions. BET inhibition has been shown to inhibit development of sepsis, in part, by inhibiting IL-6 expression, in preclinical models in both published [12] and proprietary data.

In one embodiment, BET inhibitor compounds of Formulae I-IV may be used to treat cancer. Cancers that have an overexpression, translocation, amplification, or rearrangement c-myc or other myc family oncoproteins (MYCN, L-myc) are particularly sensitive to BET inhibition [27, 28]. These cancers include, but are not limited to, B-acute lymphocytic leukemia, Burkitt's lymphoma, Diffuse large cell lymphoma, Multiple myeloma, Primary plasma cell leukemia, Atypical carcinoid lung cancer, Bladder cancer, Breast cancer, Cervix cancer, Colon cancer, Gastric cancer, Glioblastoma, Hepatocellular carcinoma, Large cell neuroendocrine carcinoma, Medulloblastoma, Melanoma, nodular, Melanoma, superficial spreading, Neuroblastoma, esophageal squamous cell carcinoma, Osteosarcoma, Ovarian cancer, Prostate cancer, Renal clear cell carcinoma, Retinoblastoma, Rhabdomyosarcoma, and Small cell lung carcinoma [25].

In one embodiment, BET inhibitor compounds of Formulae I-IV may be used to treat cancers that result from an aberrant regulation (overexpression, translocation, etc) of BET proteins. These include, but are not limited to, NUT midline carcinoma (Brd3 or Brd4 translocation to nutlin 1 gene) [22], B-cell lymphoma (Brd2 overexpression) [23], non-small cell lung cancer (BrdT overexpression) [147, 148], esophageal cancer and head and neck squamous cell carcinoma (BrdT overexpression) [147], and colon cancer (Brd4) [149].

In one embodiment, because BET inhibitors decrease Brd-dependent recruitment of pTEFb to genes involved in cell proliferation, BET inhibitor compounds of Formulae I-IV may be used to treat cancers that rely on pTEFb (Cdk9/cyclin T) and BET proteins to regulate oncogenes. These include, but are not limited to, chronic lymphocytic leukemia and multiple myeloma [150], follicular lymphoma, diffuse large B cell lymphoma with germinal center phenotype, Burkitt's lymphoma, Hodgkin's lymphoma, follicular lymphomas and activated, anaplastic large cell lymphoma [151], neuroblastoma and primary neuroectodermal tumor [152], rhabdomyosarcoma [153], prostate cancer [154], and breast cancer [45].

Published and proprietary data have shown direct effects of BET inhibition on cell proliferation in various cancers. In one embodiment, BET inhibitor compounds of Formulae I-IV may be used to treat cancers for which exist published and, for some, proprietary, in vivo and/or in vitro data showing a direct effect of BET inhibition on cell proliferation. These cancers include NMC(NUT-midline carcinoma), acute myeloid leukemia (AML), acute B lymphoblastic leukemia (B-ALL), Burkitt's Lymphoma, B-cell Lymphoma, Melanoma, mixed lineage leukemia, multiple myeloma, pro-myelocytic leukemia (PML), and non-Hodgkin's lymphoma [24, 26-30, 33]. Examples provided within this application have also shown a direct effect of BET inhibition on cell proliferation in vitro for the following cancers: Neuroblastoma, Medulloblastoma, lung carcinoma (NSCLC, SCLC), and colon carcinoma.

In one embodiment, because of their ability to up-regulate ApoA-1 transcription and protein expression [11, 35], BET inhibitor compounds of Formulae I-IV may be used to treat cardiovascular diseases that are generally associated with including dyslipidemia, atherosclerosis, hypercholesterolemia, and metabolic syndrome [8, 19]. In another embodiment, BET inhibitor compounds of Formulae I-IV may be used to treat non-cardiovascular disease characterized by deficits in ApoA-1, including Alzheimer's disease [37].

In one embodiment, BET inhibitor compounds of Formulae I-IV may be used in patients with insulin resistance and type II diabetes [8, 19, 38, 39]. The anti-inflammatory effects of BET inhibition would have additional value in decreasing inflammation associated with diabetes and metabolic disease [163].

In one embodiment, because of their ability to down-regulate viral promoters, BET inhibitor compounds of Formulae I-IV may be used as therapeutics for cancers that are associated with viruses including Epstein-Barr Virus (EBV), hepatitis virus (HBV, HCV), Kaposi's sarcoma associated virus (KSHV), human papilloma virus (HPV), Merkel cell polyomavirus, and human cytomegalovirus (CMV) [40-42, 164]. In another embodiment, because of their ability to reactivate HIV-1 in models of latent T cell infection and latent monocyte infection, BET inhibitors could be used in combination with anti-retroviral therapeutics for treating HIV [43-46].

In one embodiment, because of the role of epigenetic processes and bromodomain-containing proteins in neurological disorders, BET inhibitor compounds of Formulae I-IV may be used to treat diseases including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington disease, bipolar disorder, schizophrenia, Rubinstein-Taybi syndrome, and epilepsy [9, 165].

In one embodiment, because of the effect of BRDT depletion or inhibition on spermatid development, BET inhibitor compounds of Formulae I-IV may be used as reversible, male contraceptive agents [50, 51].

Pharmaceutical Compositions

Pharmaceutical compositions of the present disclosure comprise at least one compound of Formulae I-IV, or tautomer, stereoisomer, pharmaceutically acceptable salt or hydrate thereof formulated together with one or more pharmaceutically acceptable carriers. These formulations include those suitable for oral, rectal, topical, buccal and parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) administration. The most suitable form of administration in any given case will depend on the degree and severity of the condition being treated and on the nature of the particular compound being used.

Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of a compound of the present disclosure as powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. As indicated, such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association at least one compound of the present disclosure as the active compound and a carrier or excipient (which may constitute one or more accessory ingredients). The carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and must not be deleterious to the recipient. The carrier may be a solid or a liquid, or both, and may be formulated with at least one compound described herein as the active compound in a unit-dose formulation, for example, a tablet, which may contain from about 0.05% to about 95% by weight of the at least one active compound. Other pharmacologically active substances may also be present including other compounds. The formulations of the present disclosure may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components.

For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmacologically administrable compositions can, for example, be prepared by, for example, dissolving or dispersing, at least one active compound of the present disclosure as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. In general, suitable formulations may be prepared by uniformly and intimately admixing the at least one active compound of the present disclosure with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, a tablet may be prepared by compressing or molding a powder or granules of at least one compound of the present disclosure, which may be optionally combined with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, at least one compound of the present disclosure in a free-flowing form, such as a powder or granules, which may be optionally mixed with a binder, lubricant, inert diluent and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, where the powdered form of at least one compound of the present disclosure is moistened with an inert liquid diluent.

Formulations suitable for buccal (sub-lingual) administration include lozenges comprising at least one compound of the present disclosure in a flavored base, usually sucrose and acacia or tragacanth, and pastilles comprising the at least one compound in an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present disclosure suitable for parenteral administration comprise sterile aqueous preparations of at least one compound of Formulae I-IV or tautomers, stereoisomers, pharmaceutically acceptable salts, and hydrates thereof, which are approximately isotonic with the blood of the intended recipient. These preparations are administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing at least one compound described herein with water and rendering the resulting solution sterile and isotonic with the blood. Injectable compositions according to the present disclosure may contain from about 0.1 to about 5% w/w of the active compound.

Formulations suitable for rectal administration are presented as unit-dose suppositories. These may be prepared by admixing at least one compound as described herein with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin may take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers and excipients which may be used include Vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof. The active compound (i.e., at least one compound of Formulae I-IV or tautomers, stereoisomers, pharmaceutically acceptable salts, and hydrates thereof) is generally present at a concentration of from about 0.1% to about 15% w/w of the composition, for example, from about 0.5 to about 2%.

The amount of active compound administered may be dependent on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician. For example, a dosing schedule may involve the daily or semi-daily administration of the encapsulated compound at a perceived dosage of about 1 μg to about 1000 mg. In another embodiment, intermittent administration, such as on a monthly or yearly basis, of a dose of the encapsulated compound may be employed. Encapsulation facilitates access to the site of action and allows the administration of the active ingredients simultaneously, in theory producing a synergistic effect. In accordance with standard dosing regimens, physicians will readily determine optimum dosages and will be able to readily modify administration to achieve such dosages.

A therapeutically effective amount of a compound or composition disclosed herein can be measured by the therapeutic effectiveness of the compound. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being used. In one embodiment, the therapeutically effective amount of a disclosed compound is sufficient to establish a maximal plasma concentration. Preliminary doses as, for example, determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.

Data obtained from the cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. Therapeutically effective dosages achieved in one animal model may be converted for use in another animal, including humans, using conversion factors known in the art (see, e.g., Freireich et al.,Cancer Chemother. Reports50(4):219-244 (1966) and the following Table for Equivalent Surface Area Dosage Factors).

Equivalent Surface Area Dosage Factors:

The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Generally, a therapeutically effective amount may vary with the subject's age, condition, and gender, as well as the severity of the medical condition in the subject. The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

In one embodiment, a compound of Formulae I-IV or a tautomer, stereoisomer, pharmaceutically acceptable salt or hydrate thereof, is administered in combination with another therapeutic agent. The other therapeutic agent can provide additive or synergistic value relative to the administration of a compound of the present disclosure alone. The therapeutic agent can be, for example, a statin; a PPAR agonist, e.g., a thiazolidinedione or fibrate; a niacin, a RVX, FXR or LXR agonist; a bile-acid reuptake inhibitor; a cholesterol absorption inhibitor; a cholesterol synthesis inhibitor; a cholesteryl ester transfer protein (CETP), an ion-exchange resin; an antioxidant; an inhibitor of AcylCoA cholesterol acyltransferase (ACAT inhibitor); a tyrophostine; a sulfonylurea-based drug; a biguanide; an alpha-glucosidase inhibitor; an apolipoprotein E regulator; a HMG-CoA reductase inhibitor, a microsomal triglyceride transfer protein; an LDL-lowing drug; an HDL-raising drug; an HDL enhancer; a regulator of the apolipoprotein A-IV and/or apolipoprotein genes; or any cardiovascular drug.

REFERENCES

EXAMPLES

General Procedure A

Preparation of 2-Benzyl-6-(3,5-dimethylisoxazol-4-yl)pyridazin-3(2H)-one

General Procedure B

Preparation of 2-Benzyl-64(5,6-dimethoxypyridin-2-yl)-amino)pyridazin-3(2H)-one

General Procedure C

Preparation of 2-Benzyl-6-(3,5-dimethylisoxazol-4-yl)pyridazin-3(2H)-one

General Procedure D

General Procedure E

Preparation of 5-(3,5-Dimethylisoxazol-4-yl)-1-(1-(3-fluorophenyl)ethyl)pyridin-2(1H)-one

General Procedure F

Preparation of 1-Benzyl-5-(thiazol-5-yl)pyridin-2(1H)-one

General Procedure G

Preparation of 1-(3-(Difluoromethyl)benzyl)-5-(3,5-dimethylisoxazol-4-yl)pyridin-2(1H)-one

General Procedure H

Preparation of 5-(3,5-Dimethylisoxazol-4-yl)-1-(4-fluorobenzoyl)pyridin-2(1H)-one

General Procedure I

Preparation of 1-Benzyl-5-(3,5-dimethylisoxazol-4-yl)-3-(4-fluorophenyl)pyridin-2(1H)-one

General Procedure J

Preparation of 1-Benzyl-5-(3,5-dimethylisoxazol-4-yl)-3-(phenylamino)pyridin-2(1H)-one

General Procedure K

Chiral Separation of Example 110 and Example 111

General Procedure L

Preparation of 1-((1H-benzo[d]imidazol-5-yl)methyl)-5-(3,5-dimethylisoxazol-4-yl)pyridin-2(1H)-one

General Procedure M

Preparation of 1-Benzyl-5-(3,5-dimethylisoxazol-4-yl)-3-(piperazin-1-yl)pyridin-2(1H)-one Hydrochloride

General Procedure N

Preparation of 3-amino-5-(3,5-dimethylisoxazol-4-yl)-1-(4-fluorobenzyl)pyridin-2(1H)-one

General Procedure O

Preparation of 3-Amino-5-(3,5-dimethylisoxazol-4-yl)-1-((3-methyl-1H-indol-4-yl)methyl)pyridin-2(1H)-one

To a solution of 23 (1.52 g, 10.0 mmol) in ethanol (10 mL) was added propionaldehyde (696 mg, 12.0 mmol) at room temperature. The mixture was heated at 80° C. for 1 h and cooled at room temperature. Then concentrated hydrochloric acid (2.5 mL) was added and heated at 80° C. overnight. The mixture was concentrated under vacuum. The residue was dissolved in MeOH and basified with sodium carbonate (20% in water). The mixture was concentrated and purified by chromatography (silica gel, 0-50% ethyl acetate/hexanes) to give a mixture of 24 and 25 (650 mg, 32%) as an orange oil.

A mixture of 29 (4.00 g, 18.3 mmol), ethanol (15 mL), and 48% HBr (20 mL) was heated at 85° C. for 1 h under nitrogen. The reaction mixture was concentrated under vacuum to give crude 30 (6.40 g) that was used in the next step without further purification.

General Procedure P

Preparation of 3-Amino-5-(3,5-dimethylisoxazol-4-yl)-1-(4-isopropylbenzyl)pyridin-2(1H)-one

Preparation of 2-Benzyl-6-(3,4-dimethoxyphenoxy)pyridazin-3(2H)-one

Preparation of 5-(3-Amino-5-methylisoxazol-4-yl)-1-benzylpyridin-2(1H)-one

Preparation of 3-(Azetidin-1-yl)-1-benzyl-5-(3,5-dimethylisoxazol-4-yl)pyridin-2(1H)-one

Preparation of 3-Amino-1-benzyl-5-(3-(hydroxymethyl)-5-methylisoxazol-4-yl)pyridin-2(1H)-one

Preparation of 1-(4-Chlorobenzyl)-5-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)pyridin-2(1H)-one

Preparation of 1-Benzyl-5-(3,5-dimethylisoxazol-4-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile

Examples 229 and 230

Preparation of N-(1-Benzyl-5-(3,5-dimethylisoxazol-4-yl)-2-oxo-1,2-dihydropyridin-3-yl)methanesulfonamide

Preparation of 2-Benzyl-6-(((3,5-dimethylisoxazol-4-yl)methyl)amino)pyridazin-3(2H)-one

To a solution of 50 (439 mg, 1.8 mmol) in MeOH/water (15 mL/5 mL) was added NaOH (360 mg, 9.0 mmol). The reaction mixture was refluxed for 5 h and concentrated. The residue was partitioned between DCM and water, and extracted with DCM. The organic layer was dried over sodium sulfate, filtered and concentrated to give 51 (368 mg, 100%) as a yellow solid: ESI m/z 202 [M+H]+.

Preparation of 3-Amino-1-(4-chlorobenzyl)-5-(3-(hydroxymethyl)-5-methylisoxazol-4-yl)pyridin-2(1H)-one

Preparation of 5-(3,5-Dimethylisoxazol-4-yl)-1-(4-vinylbenzyl)pyridin-2(1H)-one

Preparation of 3-Amino-5-(3,5-dimethylisoxazol-4-yl)-1-methylpyridin-2(1H)-one

Preparation of 3-Amino-1-(4-(azetidin-1-yl)benzyl)-5-(3,5-dimethylisoxazol-4-yl)pyridin-2(1H)-one

Preparation of 3-Amino-5-(3,5-dimethylisoxazol-4-yl)-1-(4-morpholinobenzyl)pyridin-2(1H)-one

To a solution of 60 (450 mg, 2.33 mmol) in chloroform (5 mL) was added thionyl chloride (1.00 mL). The mixture was heated at 60° C. for 2 h and concentrated to afford crude 61 (624 mg, >99%) as a yellow solid.

Preparation of 3-Amino-1-(4-bromobenzyl)-5-(3,5-dimethylisoxazol-4-yl)pyridin-2(1H)-one

Preparation of 1-(4-Chlorobenzyl)-5-(3,5-dimethylisoxazol-4-yl)-3-((2,2,2-trifluoroethyl)amino)pyridin-2(1H)-one

Preparation of 1-((1H-Indol-4-yl)methyl)-3-amino-5-(3,5-dimethylisoxazol-4-yl)pyridin-2(1H)-one

Preparation of 3-(Aminomethyl)-1-benzyl-5-(3,5-dimethylisoxazol-4-yl)pyridin-2(1H)-one

A mixture of 5-bromo-2-oxo-1,2-dihydropyridine-3-carboxylic acid 60 (10 g, 45.9 mmol), H2SO4, and EtOH (225 mL) was heated at reflux for 1 hour. The solution was cooled to room temperature and concentrated. The residue was taken up in CH2Cl2(200 mL) and washed with saturated sodium bicarbonate solution, dried over sodium sulfate and filtered. The solvent was removed and purified by silica gel chromatography (0 to 5% methanol in CH2Cl2) to provide compound 61 (8 g, 71%).

To a mixture of LiAlH4(300 mg, 7.93 mmol) and THF (40 mL) at 0° C. under nitrogen was slowly added a solution of ethyl 5-bromo-2-oxo-1,2-dihydropyridine-3-carboxylate 61 (1.5 g, 6.09 mmol) and THF (20 mL). After 2.5 hours, the reaction was quenched by slow addition of water. The resultant solid was removed by filtration and the filtrate was extracted with CH2Cl2(2×100 mL). The combined extracts were dried over sodium sulfate and filtered. The solvent was removed under reduced pressure to provide compound 62 (380 mg, 28%).

To a suspension of 5-bromo-3-(hydroxymethyl)pyridin-2(1H)-one 62 (350 mg, 1.72 mmol), Et3N (0.71 mL, 5.16 mmol) and CH2Cl2(15 mL) was slowly added methanesulfonyl chloride (0.27 mL, 3.43 mmol) at 0° C. under nitrogen. The reaction mixture was allowed to warm to room temperature for 17 h and then water was added. The layers were separated and the aqueous was extracted with CH2Cl2. The organic phase was dried over sodium sulfate and filtered. The solvent was removed and the residue was purified by silica gel chromatography (eluting with 0 to 30% ethyl acetate in hexanes) to provide compound 63 (75 mg, 15%).

To a solution of (5-bromo-2-oxo-1,2-dihydropyridin-3-yl)methyl methanesulfonate 63 (75 mg, 0.27 mmol), and DMF (5 mL) was added sodium azide at room temperature. The mixture was stirred for 18 hours under nitrogen. The solvent was removed under reduced pressure and the residue was partitioned between water (20 mL) and ethyl acetate (20 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (20 mL). The organic layers were combined and dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and the product was purified by silica gel chromatography (eluting with 0 to 70% ethyl acetate in hexanes) to provide compound 64 (34 mg, 55%).

To a mixture of 3-(azidomethyl)-5-bromopyridin-2(1H)-one 64 (34 mg, 0.15 mmol), K2CO3(42 mg, 0.30 mmol) and CH3CN (5 mL) was added benzyl bromide (30 mg, 0.18 mmol) at room temperature. The reaction mixture was stirred for 18 hours and then diluted with CH2Cl2. The mixture was washed with water, dried over sodium sulfate and filtered. The solvent was removed under reduced pressure and the product was purified by silica gel chromatography (eluting with 0 to 50% ethyl acetate in hexanes) to provide compound 65 (30 mg, 63%).

Preparation of 1-Benzyl-5-(5-oxopyrrolidin-3-yl)pyridin-2(1H)-one

Sodium hydride (60% suspension in mineral oil, 0.92 g, 22.8 mmol) was added in one portion to a stirred suspension of 4-chloro-2H-phthalazin-1-one (3.74 g, 20.7 mmol) in anhydrous DMF (80 mL). The reaction was stirred for 15 min and then cooled to 10° C. Benzyl bromide (4.25 g, 24.8 mmol) was added drop wise and the reaction mixture was then stirred for 21 h at rt. After that time the reaction was diluted with ethyl acetate (200 mL), washed with water (5×80 mL) then brine (80 mL), dried over MgSO4and concentrated under reduced pressure. The resulting pale yellow solid was suspended in hexanes (80 mL) and stirred for 3 h. After that time, the precipitate was collected by filtration, washed with hexanes and dried to give 2-benzyl-4-chloro-2H-phthalazin-1-one (5.05 g, 90%) as white solid:1H NMR (400 MHz, CDCl3) δ 8.45 (d, J=7.6 Hz, 1H), 7.98 (d, J=8.8 Hz, 1H), 7.97-7.83 (m, 2H), 7.50 (d, J=6.8 Hz, 2H), 7.36-7.7.29 (m, 3H), 5.37 (s, 2H).

Inhibition of Tetra-Acetylated Histone H4 Binding Individual BET Bromodomains

Proteins were cloned and overexpressed with a N-terminal 6×His tag, then purified by nickel affinity followed by size exclusion chromatography. Briefly,E. coliBL21 (DE3) cells were transformed with a recombinant expression vector encoding N-terminally Nickel affinity tagged bromodomains from Brd2, Brd3, Brd4. Cell cultures were incubated at 37° C. with shaking to the appropriate density and induced overnight with IPTG. The supernatant of lysed cells was loaded onto Ni-IDA column for purification. Eluted protein is pooled, concentrated and further purified by size exclusion chromatography. Fractions representing monomeric protein were pooled, concentrated, aliquoted, and frozen at −80° C. for use in subsequent experiments.

Binding of tetra-acetylated histone H4 and BET bromodomains was confirmed by a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method. N-terminally His-tagged bromodomains (200 nM) and biotinylated tetra-acetylated histone H4 peptide (25-50 nM, Millipore) were incubated in the presence of Europium Cryptate-labeled streptavidin (Cisbio Cat. #610SAKLB) and XL665-labeled monoclonal anti-His antibody (Cisbio Cat. #61HISXLB) in a white 96 well microtiter plate (Greiner). For inhibition assays, serially diluted test compound was added to these reactions in a 0.2% final concentration of DMSO. Final buffer concentrations were 30 mM HEPES pH 7.4, 30 mM NaCl, 0.3 mM CHAPS, 20 mM phosphate pH 7.0, 320 mM KF, 0.08% BSA). After a 2-h incubation at room temperature, the fluorescence by FRET was measured at 665 and 620 nm by a SynergyH4 plate reader (Biotek). Illustrative results with the first bromodomain of Brd4 and the second bromodomain of Brd2 are shown below. The binding inhibitory activity was shown by a decrease in 665 nm fluorescence relative to 620 nm. IC50values were determined from a dose response curve.

Compounds with an IC50value less than 30 μM were deemed to be active.

Inhibition of c-Myc Expression in Cancer Cell Lines

MV4-11 cells (2.5×104cells) were plated in 96 well U-bottom plates with test compound or DMSO (0.1%), and incubated for 3 h at 37° C. Cells were then harvested by centrifugation, lysed, and mRNA was isolated using the mRNA catcher plus kit (Invitrogen). Reverse transcription of the mRNA and duplex amplification of the c-myc and cyclophilin cDNAs was performed using the RNA Ultrasense kit (Invitrogen) and a ViiA7 real-time PCR machine (Applied Biosystems). IC50values were determined from a dose response curve.

Compounds with an IC50value less than 30 μM were deemed to be active.

Inhibition of Cell Proliferation in Cancer Cell Lines

MV4-11 cells: 96-well plates were seeded with 5×104cells per well of exponentially growing human AML MV-4-11 (CRL-9591) cells and immediately treated with two-fold dilutions of test compounds, ranging from 30 μM to 0.2 μM. Triplicate wells were used for each concentration, as well as a media only and three DMSO control wells. The cells and compounds were incubated at 37° C., 5% CO2for 72 h before adding 20 μL of the CellTiter Aqueous One Solution (Promega) to each well and incubating at 37° C., 5% CO2for an additional 3-4 h. The absorbance was taken at 490 nm in a spectrophotometer and the percentage of proliferation relative to DMSO-treated cells was calculated after correction from the blank well. IC50were calculated using the GraphPad Prism software.

Compounds with an IC50value less than 30 μM were deemed to be active.

Inhibition of hIL-6 mRNA Transcription

In this example, hIL-6 mRNA in tissue culture cells was quantitated to measure the transcriptional inhibition of hIL-6 when treated with a compound of the present disclosure.

A human leukemic monocyte lymphoma cell line (U937) was plated (3.2×104cells per well) in a 96-well plate in 1004 RPMI-1640 containing 10% FBS and penicillin/streptomycin, and differentiated into macrophages for 3 days in 60 ng/mL PMA (phorbol-13-myristate-12-acetate) at 37° C. in 5% CO2prior to the addition of the compound of interest. The cells were pretreated for 1 h with the test compound prior to stimulation with 1 ug/mL lipopolysaccharide fromEscherichia coli. The cells were incubated at 37° C. for 3 h before the cells were harvested. At time of harvest, the spent media was removed from the cells and the cells were rinsed in 2004 PBS. Cell lysis solution (70 μL) was added the cells in each well and incubated for 5-10 min at room temperature, to allow for complete cell lysis and detachment. mRNA was then prepared using the “mRNA Catcher PLUS plate” (Invitrogen), according to the protocol supplied. After the last wash, as much wash buffer as possible was aspirated without allowing the wells to dry. Elution buffer (E3, 70 μL) was then added to each well. mRNA was then eluted by incubating the mRNA Catcher PLUS plate with Elution Buffer for 5 min at 68° C. and then immediately placing the plate on ice.

The eluted mRNA isolated was then used in a one-step quantitative real-time PCR reaction, using components of the Ultra Sense Kit together with Applied Biosystems primer-probe mixes. Real-time PCR data was analyzed, normalizing the Ct values for hIL-6 to an internal control, prior to determining the fold induction of each unknown sample, relative to the control.

Compounds with an IC50value less than 30 μM were deemed to be active.

Inhibition of IL-17 mRNA Transcription

In this example, hIL-17 mRNA in human peripheral blood mononuclear cells was quantitated to measure the transcriptional inhibition of hIL-17 when treated with a compound of the invention.

Human peripheral blood mononuclear cells were plated (2.0×105cells per well) in a 96-well plate in 45 μL OpTimizer T Cell expansion media containing 20 ng/ml IL-2 and penicillin/streptomycin. The cells were treated with the test compound (45 μL. at 2× concentration), and then the cells were incubated at 37° C. for 1 h before addition of 10× stock OKT3 antibody at 10 ug/ml in media. Cells were incubated at 37° C. for 3 h before the cells were harvested. At time of harvest, cells were centrifuged (800 rpm, 5 min). Spent media was removed and cell lysis solution (70 μL) was added the cells in each well and incubated for 5-10 min at room temperature, to allow for complete cell lysis and detachment. mRNA was then prepared using the “mRNA Catcher PLUS plate” (Invitrogen), according to the protocol supplied. After the last wash, as much wash buffer as possible was aspirated without allowing the wells to dry. Elution buffer (E3, 70 μL) was then added to each well. mRNA was then eluted by incubating the mRNA Catcher PLUS plate with Elution Buffer for 5 min at 68° C. and then immediately placing the plate on ice.

The eluted mRNA isolated was then used in a one-step quantitative real-time PCR reaction, using components of the Ultra Sense Kit together with Applied Biosystems primer-probe mixes. Real-time PCR data was analyzed, normalizing the Ct values for hIL-17 to an internal control, prior to determining the fold induction of each unknown sample, relative to the control.

Compounds with an IC50value less than 30 μM were deemed to be active.

Inhibition of hVCAM mRNA Transcription

In this example, hVCAMmRNA in tissue culture cells is quantitated to measure the transcriptional inhibition of hVCAM when treated with a compound of the present disclosure.

Human umbilical vein endothelial cells (HUVECs) are plated in a 96-well plate (4.0×103cells/well) in 100 μL EGM media and incubated for 24 h prior to the addition of the compound of interest. The cells are pretreated for 1 h with the test compound prior to stimulation with tumor necrosis factor-α. The cells are incubated for an additional 24 h before the cells are harvested. At time of harvest, the spent media is removed from the HUVECs and rinsed in 200 μL PBS. Cell lysis solution (70 μl) is then added the cells in each well and incubated for˜5-10 min at room temperature, to allow for complete cell lysis and detachment. mRNA is then prepared using the “mRNA Catcher PLUS plate” (Invitrogen), according to the protocol supplied. After the last wash, as much wash buffer as possible is aspirated without allowing the wells to dry. Elution buffer (E3, 70 μL) is then added to each well. mRNA is then eluted by incubating the mRNA Catcher PLUS plate with elution buffer for 5 min at 68° C. and then immediately placing the plate on ice.

The eluted mRNA so isolated is then used in a one-step quantitative real-time PCR reaction, using components of the Ultra Sense Kit together with Applied Biosystems primer-probe mixes. Real-time PCR data was analyzed, normalizing the Ct values for hVCAM to an internal control, prior to determining the fold induction of each unknown sample, relative to the control.

Compounds with an IC50value less than 30 μM are deemed to be active.

Inhibition of hMCP-1 mRNA Transcription

In this example, hMCP-1 mRNA in human peripheral blood mononuclear cells was quantitated to measure the transcriptional inhibition of hMCP-1 when treated with a compound of the present disclosure.

Human Peripheral Blood Mononuclear Cells were plated (1.0×105cells per well) in a 96-well plate in 45 μL RPMI-1640 containing 10% FBS and penicillin/streptomycin. The cells were treated with the test compound (45 μL at 2× concentration), and then the cells were incubated at 37° C. for 3 h before the cells were harvested. At time of harvest, cells were transferred to V-bottom plates and centrifuged (800 rpm, 5 min). Spent media was removed and cell lysis solution (70 μL) was added the cells in each well and incubated for 5-10 min at room temperature, to allow for complete cell lysis and detachment. mRNA was then prepared using the “mRNA Catcher PLUS plate” (Invitrogen), according to the protocol supplied. After the last wash, as much wash buffer as possible was aspirated without allowing the wells to dry. Elution buffer (E3, 70 μL) was then added to each well. mRNA was then eluted by incubating the mRNA Catcher PLUS plate with Elution Buffer for 5 min at 68° C. and then immediately placing the plate on ice.

The eluted mRNA isolated was then used in a one-step quantitative real-time PCR reaction, using components of the Ultra Sense Kit together with Applied Biosystems primer-probe mixes. Real-time PCR data was analyzed, normalizing the Ct values for hMCP-1 to an internal control, prior to determining the fold induction of each unknown sample, relative to the control.

Compounds with an IC50value less than 30 μM were deemed to be active.

Up-Regulation of hApoA-1 mRNA Transcription

In this example, ApoA-I mRNA in tissue culture cells was quantitated to measure the transcriptional up-regulation of ApoA-I when treated with a compound of the present disclosure.

Huh7 cells (2.5×105per well) were plated in a 96-well plate using 100 μL DMEM per well, (Gibco DMEM supplemented with penicillin/streptomycin and 10% FBS), 24 h before the addition of the compound of interest. After 48 h treatment, the spent media was removed from the Huh-7 cells and placed on ice (for immediate use) or at −80° C. (for future use) with the “LDH cytotoxicity assay Kit II” from Abcam. The cells remaining in the plate were rinsed with 100 μL PBS.

Then 85 μL of cell lysis solution was added to each well and incubated for 5-10 min at room temperature, to allow for complete cell lysis and detachment. mRNA was then prepared using the “mRNA Catcher PLUS plate” from Life Technologies, according to the protocol supplied. After the last wash, as much wash buffer as possible was aspirated without allowing the wells to dry. Elution Buffer (E3, 80 μL) was then added to each well. mRNA was then eluted by incubating the mRNA Catcher PLUS plate with Elution Buffer for 5 min at 68° C., and then 1 min at 4° C. Catcher plates with mRNA eluted were kept on ice for use or stored at −80° C.

The eluted mRNA isolated was then used in a one-step real-time PCR reaction, using components of the Ultra Sense Kit together with Life Technologies primer-probe mixes. Real-time PCR data was analyzed, using the Ct values, to determine the fold induction of each unknown sample, relative to the control (that is, relative to the control for each independent DMSO concentration).

Compounds with an EC170value less than 30 μM were deemed to be active.

In Vivo Efficacy in Athymic Nude Mouse Strain of an Acute Myeloid Leukemia Xenograft Model Using MV4-11 Cells

MV4-11 cells (ATCC) are grown under standard cell culture conditions and (NCr) nu/nu fisol strain of female mice age 6-7 weeks were injected with 5×106cells/animal in 100 μl PBS+100 μL Matrigel in the lower left abdominal flank. By approximately day 18-21 after MV4-11 cells injection, mice are randomized based on tumor volume (L×W×H)/2) of average˜100-300 mm3. Mice are dosed orally with compound at 5 to 120 mg/kg b.i.d and or 30 mg/kg q.d in EA006 formulation at 10 mL/kg body weight dose volume. Tumor measurements are taken with electronic micro calipers and body weights measured on alternate days beginning from dosing period. The average tumor volumes, percent Tumor Growth Inhibition (TGI) and % change in body weights are compared relative to Vehicle control animals. The means, statistical analysis and the comparison between groups are calculated using Student's t-test in Excel.

In Vivo Efficacy in Athymic Nude Mouse Strain of an Acute Myeloid Leukemia Xenograft Model Using OCI-3 AML Cells

OCl-3 AML cells (DMSZ) are grown under standard cell culture conditions and (NCr) nu/nu fisol strain of female mice age 6-7 weeks are injected with 10×106cells/animal in 100 μL PBS+100 μL Matrigel in the lower left abdominal flank. By approximately day 18-21 after OCl-3 AML cells injection, mice are randomized based on tumor volume (L×W×H)/2) of average˜300 mm3. Mice are dosed orally with compound at 30 mg/kg b.i.d in EA006 formulation at 10 mL/kg body weight dose volume. Tumor measurements are taken with electronic micro calipers and body weights measured on alternate days beginning from dosing period. The average tumor volumes, percent Tumor Growth Inhibition (TGI) and % change in body weights are compared relative to Vehicle control animals. The means, statistical analysis and the comparison between groups are calculated using Student's t-test in Excel.

In Vivo Efficacy in Athymic Nude Mouse Strain of Multiple Myeloma Xenograft Model Using MM1.s Cells

MM1.s cells (ATCC) were grown under standard cell culture conditions and SCID-Beige strain of female mice age 6-7 weeks were injected with 10×106cells/animal in 100 μl PBS+100 μL Matrigel in the lower left abdominal flank. By approximately day 21 after MM1.s cells injection, mice were randomized based on tumor volume (L×W×H)/2) of average˜120 mm3. Mice were dosed orally with compound at 25 to 90 mg/kg b.i.d in EA006 formulation at 10 mL/kg body weight dose volume. Tumor measurements were taken with electronic micro calipers and body weights measured on alternate days beginning from dosing period. The average tumor volumes, percent Tumor Growth Inhibition (TGI) and % change in body weights were compared relative to Vehicle control animals. The means, statistical analysis and the comparison between groups were calculated using Student's t-test in Excel.

TABLE 9In vivo efficacy in athymic nude mouse strain of multiplemyeloma xenograft model using MM1.s cellsExampleIn vivo activityExample 152Active

In Vivo Efficacy in Mouse Endotoxemia Model Assay

Sub lethal doses of Endotoxin (E. Colibacterial lipopolysaccharide) were administered to animals to produce a generalized inflammatory response which was monitored by increases in secreted cytokines. Compounds were administered to C57/B16 mice orally at 75 mg/kg dose to evaluate inhibition in IL-6 and IL-17 cytokines post 4-h challenge with lipopolysaccharide (LPS) at 0.5 mg/kg dose intraperitoneally.

In Vivo Efficacy in Rat Collagen-Induced Arthritis

Rat collagen-induced arthritis is an experimental model of polyarthritis that has been widely used for preclinical testing of numerous anti-arthritic agents. Following administration of collagen, this model establishes a measurable polyarticular inflammation, marked cartilage destruction in association with pannus formation and mild to moderate bone resorption and periosteal bone proliferation. In this model, collagen was administered to female Lewis strain of rats on Day 1 and 7 of study and dosed with compounds from Day 11 to Day 17. Test compounds were evaluated to assess the potential to inhibit the inflammation (including paw swelling), cartilage destruction and bone resorption in arthritic rats, using a model in which the treatment is administered after the disease has been established.

In Vivo Efficacy in Experimental autoimmune encephalomyelitis (EAE) Model of MS

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune disease of the CNS which shares many clinical and histopathological features with human multiple sclerosis (MS). EAE is the most commonly used animal model of MS. T cells of both Th1 and Th17 lineage have been shown to induce EAE. Cytokines IL-23, IL-6 and IL-17, which are either critical for Th1 and Th17 differentiation or produced by these T cells, play a critical and non-redundant role in EAE development. Therefore, drugs targeting production of these cytokines are likely to have therapeutic potential in treatment of MS.

This study may be conducted to assess the potential anti-inflammatory effect of test compounds to inhibit the inflammation and clinical EAE scores of a 28 day preventative mouse model. In this model, EAE is induced by MOG35-55/CFA immunization and pertussis toxin injection in female C57Bl/6 mice.

In view of the preceding disclosure and examples, the invention includes at least the following exemplary embodiments:

1. A compound of Formula I:

or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof,
wherein:W1is selected from N and CR5;W2is selected from N and CR4;W3is selected from N and CR3;each W may be the same or different from each other;R1is selected from a carbocycles or heterocycles;R2is selected from a 5- or 6-membered monocyclic carbocycle or a 5- or 6-membered monocyclic heterocycle;R3, R4, and R5are each independently selected from hydrogen, alkyl, —OH, —NH2, thioalkyl, alkoxy, ketone, ester, carboxylic acid, urea, carbamate, carbonate, amino, amide, halogen, carbocycle, heterocycle, sulfone, sulfoxide, sulfide, sulfonamide, and —CN;R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle;R4may be connected to B or R2to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO2—, —CRxRyCRzRvO—, —CRxRyCRzRvN—, —CRxRyCRzRvS—, and —CRxRyCRzRvCRQRR—;Rx, Ry, Rz, Rv, RQ, and RRare each independently selected from hydrogen, alkyl(C1-C8), halogen, —OH, —CF3, amino, alkoxy (C1-C8), carboxyl, —CN, sulfone, sulfoxide, carbocycle, and heterocycle, or two substituents selected from Rx, Ry, Rz, Rv, RQand RRmay form an oxo or thio-oxo group, or two substituents selected from Rx, Ry, Rz, Rv, RS, and R1may be connected in a 5- or 6-membered ring to form a bicyclic carbocycle or bicyclic heterocycle;B is selected from —(CRaRb)n—, —(CRaRbCRcRd)—, —O—, —OCRaRb—, —CRaRbO—, —NH—, —NHCRaRb—, —CRaRbNH—, —S—, —SCRaRb—, —CRaRbS—, —S(O)—, —S(O)CRaRb—, —CRaRbS(O)—, —SO2—, —SO2CRaRb—, and —CRaRbSO2—;n is selected from 0 and 1, meaning if n=0 then B is absent and R2is connected directly to the center ring;
Ra, Rb, Rc, and Rdare each independently selected from hydrogen, alkyl(C1-C3), and alkoxy(C1-C3).

2. A compound of Formula II:

or a stereoisomer, tautomer, pharmaceutical acceptable salt, or hydrate thereof,
wherein:W1is selected from N and CR5;W2is selected from N and CR4;W3is selected from N and CR3, with the proviso that if W3is N then neither R5nor R4is —OH;each W may be the same or different from each other;R1is a carbocycle or heterocycle;V is selected from a 5-membered monocyclic carbocycle or monocyclic heterocycle, where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond,with the proviso that V cannot be unsubstituted thiophene, cyclopentyl, cyclopentenyl, ribofuranosyl, or furan,R3, R4, and R5are each independently selected from hydrogen, alkyl, —OH, —NH2, thioalkyl, alkoxy, ketone, ester, carboxylic acid, urea, carbamate, carbonate, amino, amide, halogen, carbocycle, heterocycle, sulfone, sulfoxide, sulfide, sulfonamide, and —CN,R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle;R4may be connected to B or V to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO2—, —CRxRyCRzRvO—, —CRxRyCRzRvN—, —CRxRyCRzRvS—, and —CRxRyCRzRyCRQRR—,with the proviso that Rxand Rycannot both be an unsubstituted phenyl ring,and with the proviso that if A is —CH2CH2CH2— and W3is N then R4is not —OH,and with the proviso that if A is —CH2CH2O— or —CH2C(O)NH— then V is not a substituted

or a substituted

and with the proviso that if A is —CH2CH2O— then R1is not

3. The compound according to embodiment 2, wherein if W1═CR5and V is an optionally substituted

then at least one of R3and R4are not hydrogen.

4. The compound according to embodiment 2, wherein if W3═N, then R4is not hydrogen.

5. The compound according to embodiment 2, wherein if W1═CR5and V is

then R1is not

6. The compound according to embodiment 2, wherein if W1═CR5and V is

then R1is not

7. The compound according to embodiment 2, wherein if W1═N and V is an optionally substituted

then at least one of R3and R4are not hydrogen.

8. The compound according to embodiment 2, wherein if W3═N, then R4is not hydrogen.

9. The compound according to embodiment 2, wherein if W1═N and V is an optionally substituted

then R1-A is not

10. The compound according to embodiment 2, wherein if W1═N and V is

then R3and R4cannot be connected to form

11. The compound according to embodiment 2, wherein if R5is —COOMe then V is not a substituted thiophene.

12. The compound according to embodiment 2, wherein if R5is methyl then R2is not

13. The compound according to embodiment 2, wherein if B is present then neither R4nor R5is hydroxyl.

14. The compound according to embodiment 2, wherein Rxand Rycannot both be an unsubstituted phenyl ring.

15. The compound according to any one of embodiments 2 to 14, wherein V is selected from an optionally substituted 5-membered monocyclic heterocycle selected from

17. The compound according to any one of embodiments 2 to 14, wherein V is selected from an optionally substituted 5-membered monocyclic heterocycle containing one oxygen and one or two nitrogens, where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond.

18. The compound according to any one of embodiments 2 to 14, wherein V is an optionally substituted isoxazole.

19. The compound according to any one of embodiments 2 to 14, wherein V is

20. The compound according to any one of embodiments 2 to 14, wherein W1is CRs.

21. The compound according to any one of embodiments 2 to 14, wherein W2is CR4.

22. The compound according to any one of embodiments 2 to 14, wherein X is oxygen.

23. The compound according to any one of embodiments 2 to 14, wherein n=0, meaning B is absent.

24. The compound according to any one of embodiments 2 to 14, wherein A is selected from C═O and —CRxRy—.

25. The compound according to any one of embodiments 2 to 14, wherein R1is selected from an optionally substituted 3-, 4-, 5-, and 6-membered carbocycle or heterocycle.

27. The compound according to embodiment 25, wherein R1is selected from an optionally substituted 5- and 6-membered carbocycle or heterocycle.

28. The compound according to embodiment 27, wherein the carbocycle or heterocycle is selected from phenyl, pyridyl, thiophene, and cyclopentyl.

29. The compound according to embodiment 28, wherein R1is selected from an optionally substituted phenyl or pyridyl ring.

33. The compound according to any one of embodiments 2 to 14, wherein R3is selected from hydrogen, —NH2, amino (such as —NHMe, —NHEt, —NHcyclopropyl, —NHPh, —NHBn, —NMe2, —NHpyridyl, —NHcyclopentyl), and —NHheterocycle or heterocycle selected from

34. The compound according to embodiment 33, wherein R3is selected from hydrogen, —NH2, and amino.

35. The compound according to any one of embodiments 2 to 14, wherein R3and R4are connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle selected from

37. The compound according to any one of embodiments 2 to 14, wherein Ra, Rb, Rcand Rdare independently selected from hydrogen, methyl, methoxy, and —CF3.

38. The compound according to any one of embodiments 2 to 14, wherein B is selected from —(CRaRb)n—, —O—, —NH—, —S—, —S(O)—, —SO2—, where n is 0 or 1, meaning if n=0 then B is absent.

40. A compound of Formula III:

or a stereoisomer, tautomer, pharmaceutical acceptable salt, or hydrate thereof,wherein:W2is selected from N and CR4,W3is selected from N and CR3,each W may be the same or different from each other;R1is a carbocycle or heterocycle,R2is selected from a 6-membered monocyclic carbocycle or monocyclic heterocycle,R3, R4, and R5are each independently selected from hydrogen, alkyl, —OH, —NH2, thioalkyl, alkoxy, ketone, ester, carboxylic acid, urea, carbamate, carbonate, amino, amide, halogen, carbocycle, heterocycle, sulfone, sulfoxide, sulfide, sulfonamide, and —CN,with the proviso that R4is not —OH and R5is not —COOH or -ester;R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle;R4may be connected to B or R2to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO2—, —CRxRyCRzRvO—, —CRxRyCRzRyN—, —CRxRyCRzRyS—, and —CRxRyCRzRvCRQRR;with the proviso that Rxand Rycannot both be an unsubstituted phenyl ring,and with the proviso that if A is —CH2CH2CH2— and W3is N then R4is not —OH,and with the proviso that if A is —CH2CH2O— then R1is not

41. The compound according to embodiment 40, wherein if W2is N and R2is

then R5is not hydrogen.

42. The compound according to embodiment 40, wherein if W3is N then neither R5nor R4is —OH.

43. The compound according to embodiment 40, wherein R1-A is not

44. The compound according to embodiment 40, wherein if R1-A is

then at least one of Q1, O2, Q3, or Q4is not hydrogen.

45. The compound according to embodiment 40, wherein if R1-A is

then at least one of R3and R4is not hydrogen.

46. The compound according to embodiment 40, wherein if R1is

then R2is not

47. The compound according to embodiment 40, wherein if R1is

then R2is not

48. The compound according to embodiment 40, wherein R2is not

or an optionally
substituted

49. The compound according to embodiment 40, wherein if R2is

then at least one of R3and R4is not hydrogen

50. The compound according to embodiment 40, wherein if R3is —CN, then R2is not

51. The compound according to embodiment 40, wherein if R2is

then R1is not

or if R2is

then R5is not —COOMe; or if R4is —NH2then R2is not

52. The compound according to any one of embodiments 40 to 51, wherein R2is selected from an optionally substituted 6-membered monocyclic carbocycle (such as phenyl) or heterocycle (such as pyridyl, pyrimidine, pyrazine, and triazine), where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond.

53. The compound according to any one of embodiments 39 to 50, wherein R2is selected from

54. The compound according to any one of embodiments 40 to 51, wherein R2is selected from

55. The compound according to any one of embodiments 40 to 51, wherein R1is selected from a 3, 4-, 5-, or 6-membered carbocycle or heterocycle.

56. The compound according to embodiment 55, wherein R1is selected from an optionally substituted phenyl.

61. The compound according to any one of embodiments 40 to 51, wherein R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle such as

62. The compound according to any one of embodiments 40 to 51, wherein R5is hydrogen.

63. The compound according to any one of embodiments 40 to 51, wherein R4is hydrogen.

64. The compound according to any one of embodiments 40 to 51, wherein X is oxygen.

65. The compound according to any one of embodiments 40 to 51, wherein n=0, meaning B is absent.

66. The compound according to any one of embodiments 40 to 51, wherein B is selected from —(CRaRb)n—, —O—, —NH—, —S—, where n is 0 or 1, meaning if n=0 then B is absent.

67. The compound according to any one of embodiments 40 to 51, wherein A is selected from C═O and —CRxRy—.

69. A compound of Formula IV:

or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof,wherein:W2is selected from N and CR4,W3is selected from N and CR3,each W may be the same or different from each other;R1is a carbocycle or heterocycle,R2is selected from a 6-membered monocyclic carbocycle or monocyclic heterocycle,R3and R4are each independently selected from hydrogen, alkyl, —OH, —NH2, thioalkyl, alkoxy, ketone, ester, carboxylic acid, urea, carbamate, carbonate, amino, amide, halogen, carbocycle, heterocycle, sulfone, sulfoxide, sulfide, sulfonamide, and —CN,with the proviso that R4is not —OH;R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle,with the proviso that R3and R4are not connected to form

R4may be connected to B or R2to form a carbocycle or heterocycle;X is selected from O and S;A is selected from —CRxRy—, C═O, —C(O)CRxRy—, —CRxRyCRzRv—, —SO2—, —CRxRyCRzRvO—, —CRxRyCRzRvN—, —CRxRyCRzRvS—, and —CRxRyCRzRvCRQRR—;with the proviso that if A is C═O, then R2is not an optionally substituted

where T is halogen,and with the proviso that Rxand Rycannot both be an unsubstituted phenyl ring,and with the proviso that if A is —CH2CH2CH2— and W3is N then R4is not —OH,and with the proviso that if A is —CH2CH2O— then R1is not

70. The compound according to embodiment 69, wherein if W2is N and R2is

then R5is not hydrogen.

71. The compound according to embodiment 69, wherein if W3is N then neither R5nor R4is —OH.

72. The compound according to embodiment 69, wherein R1is not an amino group with nitrogen attached to A, a substituted napthyl, or cyclohexyl.

73. The compound according to embodiment 69, wherein R1-A is not

74. The compound according to embodiment 69, wherein if R1-A is

then R2is not an optionally substituted

where T is halogen.

75. The compound according to embodiment 69, wherein if R1-A is

then R2is not

76. The compound according to embodiment 69, wherein if R1A is

then R2is not substituted with —OH or —NH2.

77. The compound according to embodiment 68, wherein R2is not an unsubstituted thiophene, furan, cyclopentyl, cyclohexyl, or

where T is any atom.

78. The compound according to embodiment 69, wherein R2is not

where T is Cl, Br, —OMe, or Me.

79. The compound according to embodiment 69, wherein R2is not

where T and Y are independently selected from Cl, F, -Me, —CN, or —OH.

80. The compound according to embodiment 69, wherein R2is not

81. The compound according to embodiment 69, wherein if R2is

then R1-A is not

where T and Y are independently selected from hydrogen, F, Cl, Br, —CF3, and -Me, and R1is not unsubstituted pyridyl, substituted furan, or unsubstituted naphthyl.

82. The compound according to embodiment 69, wherein if R2is

where T is an —OH, Alkoxy, —OAcyl, —NH2, amino, amide, carbamate, or urea substituent, then at least one of R3and R4is not hydrogen.

83. The compound according to embodiment 69, wherein if R2is an unsubstituted pyridyl, then at least one of R3and R4is not hydrogen, or R1-A is not

or R3and R4are not connected to form an unsubstituted benzene ring.

84. The compound according to embodiment 69, wherein if R2is

then R3is not methyl, at least one of R3and R4cannot be connected to

or R1-A cannot be

85. The compound according to any one of embodiments 69 to 84, wherein R2is selected from an optionally substituted 6-membered monocyclic carbocycle (such as phenyl) or heterocycle (such as pyridyl, pyrimidine, pyrazine, and triazine), where the heterocycle is connected to the rest of the molecule via a carbon-carbon bond.

86. The compound according to any one of embodiments 69 to 84, wherein R2is selected from

87. The compound according to any one of embodiments 69 to 84, wherein R2is selected from

88. The compound according to any one of embodiments 69 to 84, wherein R1is selected from a 3, 4-, 5-, or 6-membered carbocycle or heterocycle.

89. The compound according to embodiment 87, wherein R1is an optionally substituted phenyl.

94. The compound according to any one of embodiments 69 to 84, wherein R3and R4may be connected to form an optionally substituted 5-, 6-, or 7-membered carbocycle or heterocycle such as

95. The compound according to any one of embodiments 69 to 84, wherein R4is hydrogen.

96. The compound according to any one of embodiments 69 to 84, wherein X is oxygen.

97. The compound according to any one of embodiments 69 to 84, wherein n=0, meaning B is absent.

98. The compound according to any one of embodiments 69 to 84, wherein B is selected from —(CRaRb)n—, —O—, —NH—, —S—, where n is 0 or 1, meaning if n=0 then B is absent.

99. The compound according to any one of embodiments 69 to 84, wherein A is selected from C═O and —CRxRy—.

101. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 99.

102. A compound according to any one of embodiments 1 to 100 for use as a medicament.

103. A method for inhibiting BET proteins in a mammal comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

104. A method for treating a disease that is sensitive to a BET inhibitor comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

105. A method for treating an autoimmune disease in a mammal comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

107. A method for treating inflammatory diseases or disorders in a mammal comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

109. A method for treating or preventing a cancer in a mammal comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

110. The method of embodiment 109 wherein the cancer is a midline carcinoma.

111. The method of embodiment 109 wherein the cancer exhibits overexpression, translocation, amplification, or rearrangement of a myc family oncoproteins.

112. The method of embodiment 109 wherein the cancer is characterized by overexpression of c-myc.

113. The method of embodiment 109 wherein the cancer is characterized by is characterized by overexpression n-myc.

114. The method of embodiment 109 wherein the cancer results from aberrant regulation of BET proteins.

115. The method of embodiment 109 wherein the cancer is characterized by recruitment of pTEFb to regulate oncogenes.

116. The method of embodiment 109 wherein the cancer is characterized by upregulation of CDK6, Bcl2, TYRO3, MYB and/or hTERT.

118. The method of any one of embodiments 109 to 117 wherein the compound of Formula I is administered in combination with another anticancer agent.

120. A method of treating a cardiovascular disease comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

121. The method of embodiment 120, wherein the cardiovascular disease is dyslipidemia, atherosclerosis, hypercholesterolemia, or metabolic syndrome.

122. A method of treating insulin resistance diabetes comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

123. A method of treating a neurological disorder comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

125. A method of male contraception comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

126. A method of treating HIV comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

127. A method of treating a cancer associated with a viral infection comprising administering a therapeutically effective amount of a compound according to any one of embodiments 1 to 100.

128. The method of embodiment 127 wherein the virus is selected from Epstein-Barr Virus, hepatitis B virus, hepatitis C virus, Kaposi's sarcoma associated virus, human papilloma virus, Merkel cell polyomavirus, and human cytomegalovirus.

129. The compound of embodiment 1, wherein the compound of formula I is 1-(4-chlorobenzyl)-5-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)pyridin-2(1H)-one (Example 197).