WO2016139292A1

WO2016139292A1 - Pyridinone compound, pharmaceutical composition containing the same and use

Info

Publication number: WO2016139292A1
Application number: PCT/EP2016/054516
Authority: WO
Inventors: John Alexander Brown; Philip G HUMPHREYS; Katherine Louise Jones; Christopher Roland Wellaway
Original assignee: Glaxosmithkline Intellectual Property (No.2) Limited
Priority date: 2015-03-05
Filing date: 2016-03-03
Publication date: 2016-09-09
Also published as: TW201704230A; GB201503720D0; AR103844A1; UY36574A

Abstract

The present invention is directed to the compound of formula (I), which is 5-[1-(1,3-dimethoxypropan-2-yl)-5-(morpholin-4-yl)-1H-1,3-benzodiazol-2-yl]-1,3-dimethyl-1,2-dihydropyridin-2-one or a salt thereof, in particular pharmaceutically acceptable salts thereof. The compound of formula (I) has been identified as a BET inhibitor and thus has potential for use in therapy, for example in the treatment of autoimmune and inflammatory diseases, such as rheumatoid arthritis; and cancers.

Description

,

THE SAME AND USE

FIELD OF THE INVENTION

The present invention relates to a compound, a process for its preparation, pharmaceutical compositions containing it, and to its use in the treatment of various disorders, in particular inflammatory and autoimmune diseases and cancers.

BACKGROUND TO THE INVENTION

The genomes of eukaryotic organisms are highly organised within the nucleus of the cell. The long strands of duplex DNA are wrapped around an octomer of histone proteins (most usually comprising two copies of histones H2A, H2B, H3 and H4) to form a nucleosome. This basic unit is then further compressed by the aggregation and folding of nucleosomes to form a highly condensed chromatin structure. A range of different states of condensation are possible, and the tightness of this structure varies during the cell cycle, being most compact during the process of cell division. Chromatin structure plays a critical role in regulating gene transcription, which cannot occur efficiently from highly condensed chromatin. The chromatin structure is controlled by a series of post translational modifications to histone proteins, notably histones H3 and H4, and most commonly within the histone tails which extend beyond the core nucleosome structure. These modifications include acetylation, methylation, phosphorylation, ubiquitinylation, SUMOylation. These epigenetic marks are written and erased by specific enzymes, which place tags on specific residues within the histone tail, thereby forming an epigenetic code, which is then interpreted by the cell to allow regulation of gene expression.

Histone acetylation is most usually associated with the activation of gene transcription, as the modification relaxes the interaction of the DNA and the histone octomer by changing the electrostatics. In addition to this physical change, specific proteins recognise and bind to acetylated lysine residues within histones to read the epigenetic code. Bromodomains are small (~110 amino acid) distinct domains within proteins that bind to acetylated lysine resides commonly but not exclusively in the context of histones. There is a family of around 50 proteins known to contain bromodomains, and they have a range of functions within the cell.

The BET family of bromodomain containing proteins comprises 4 proteins (BRD2,

BRD3, BRD4 and BRDT) which contain tandem bromodomains capable of binding to two acetylated lysine residues in close proximity, increasing the specificity of the interaction. Numbering from the N-terminal end of each BET protein the tandem bromodomains are typically labelled Binding Domain 1 (BDl) and Binding Domain 2 (BD2) (Chung et a\, J Med. Chem,. 2011, 54, 3827-3838j.

Inhibiting the binding of a BET protein to acetylated lysine residues has the potential to ameliorate progression of several diseases, including but not limited to, cancer (Dawson M.A. et a I, Nature, 2011: 478(7370): 529-33; Wyce, A. et a I, Oncotarget. 2013: 4(12):2419-29), sepsis (Nicodeme E. et al, Nature, 2010: 468(7327): 1119-23), autoimmune and inflammatory diseases such as rheumatoid arthritis and multiple sclerosis (Mele D.A. et al, Journal of Experimental Medicine, 2013: 210(ll):2181-90), heart failure (Anand P. et al, Cell, 2013: 154(3): 569-82), and lung fibrosis (Tang X. et al, Molecular Pharmacology, 2013: 83(1):.283-293).

There exists a need in the art for chemical compounds which inhibit the activity of bromodomains, in particular compounds that inhibit the binding of BET family bromodomain containing proteins to acetylated lysine residues. In particular, there is a need for compounds that possess an improved profile over known BET inhibitors.

SUMMARY OF THE INVENTION

The present invention provides, in a first aspect, 5-[l-(l,3-dimethoxypropan-2- yl)-5-(morpholin-4-yl)-lH-l,3-benzodiazol-2-yl]-l,3-dimethyl-l,2-dihydropyridin-2-one, of formula (I):

or a salt thereof.

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

In a third aspect of the present 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 BET inhibitor is indicated.

In a fourth aspect of the present invention, there is provided a method of treating diseases or conditions for which a BET inhibitor is indicated, which method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In a fifth aspect of the present invention, there is provided the use of 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 BET inhibitor is indicated.

In a further aspect, the present invention is directed to the use of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of an autoimmune and/or inflammatory disease.

In a further aspect, the present invention is directed to the use of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of rheumatoid arthritis.

Diseases and conditions for which a BET inhibitor may be indicated may include autoimmune and/or inflammatory diseases (such as rheumatoid arthritis), or cancers. In one embodiment, the diseases and conditions for which a BET inhibitor may be indicated may include autoimmune and/or inflammatory diseases (such as rheumatoid arthritis).

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

As used herein, the term "bromodomain" refers to evolutionary and structurally conserved modules (approximately 110 amino acids in length) that bind acetylated lysine residues, such as those on the N-terminal tails of histones. They are protein domains that are found as part of much larger bromodomain containing proteins (BCPs), many of which have roles in regulating gene transcription and/or chromatin remodelling. The human genome encodes for at least 57 bromodomains.

As used herein, the term "BET" refers to the bromodomain and extraterminal domain family of bromodomain containing proteins which include BRD2, BRD3, BRD4 and BRDT.

As used herein, the term "BET inhibitor" refers to a compound that is capable of inhibiting the binding of one or more BET family bromodomain containing proteins (e.g. BRD2, BRD3, BRD4 or BRDT) to, for example, acetylated lysine residues.

As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the compound of formula (I) and exhibit minimal undesired toxicological effects. These pharmaceutically-acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively. Furthermore, pharmaceutically-acceptable salts of the compound of formula (I) may be prepared during further processing of the free acid or base form, for example in situ during manufacture into a pharmaceutical formulation.

As used herein, the term "treatment" refers to prophylaxis of the condition, ameliorating or stabilising the specified condition, reducing or eliminating the symptoms of the condition, slowing or eliminating the progression of the condition, and preventing or delaying reoccurrence of the condition in a previously afflicted patient or subject.

As used herein, the term "therapeutically effective amount" refers to the quantity of a compound of formula (I), or a pharmaceutically acceptable salt thereof, which will elicit the desired biological response in an animal or human body.

As used herein, the term "subject" refers to an animal or human body.

It is to be understood that references herein to "compounds of the invention" mean a compound of formula (I) as the free base, or as a salt, for example a pharmaceutically acceptable salt.

STATEMENT OF THE INVENTION

The present invention provides a compound, or a salt thereof, which is a BET inhibitor and thus may have utility in the treatment of, for example, autoimmune and inflammatory diseases, and cancers. Further, the compound or a salt thereof may show an improved profile over known BET inhibitors in that it may possess, for example, one or more of the following properties:

(i) potent BET inhibitory activity;

(ii) selectivity over other known bromodomain containing proteins outside of the BET family of proteins;

(iii) selectivity for a particular BET family member over one or more other BET family members;

(iv) selectivity for one Binding Domain (i.e. BD1 over BD2) for any given BET family member;

(v) improved developability (e.g. desirable solubility profile, pharmacokinetics and pharmacodynamics); or

(vi) a reduced side-effect profile.

or a salt thereof. It will be appreciated that the present invention covers the compound of formula (I) as the free base and as salts thereof, for example as a pharmaceutically acceptable salt thereof. In one embodiment of the present invention, the compound of formula (I) is in the form of a free base.

Salts of the compound of formula (I) include both pharmaceutically and non- pharmaceutical ly acceptable salts. Non-pharmaceutically acceptable salts may have use in the preparation of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

Because of their potential use in medicine, salts of the compounds of formula (I) are desirably pharmaceutically acceptable. For a review on suitable salts see Berge et ai, J. Pharm. Sci., 66:1-19 (1977). Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base as appropriate. The resultant salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. In one embodiment of the present invention, the compound of formula (I) is in the form of a pharmaceutically acceptable salt.

A pharmaceutically acceptable acid addition salt may be formed by treatment of the compound of formula (I) with a suitable inorganic or organic acid. Representative pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methyl nitrate, sulfate, bisulfate, sulfamate, phosphate, acetate, hydroxyacetate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicyclate, glycollate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, naphthoate, hydroxynaphthoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate), and napthalene-2-sulfonate.

Pharmaceutically acceptable salts of the compound of formula (I) may possess, for example, improved stability or solubility, facilitating development as a medicine.

The invention includes within its scope all possible stoichiometric and non- stoichiometric forms of the salts of the compound of formula (I).

It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallised. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvents with high boiling points and/or solvents with a high propensity to form hydrogen bonds such as water, ethanol, /so-propyl alcohol, and /V-methyl pyrrolidinone may be used to form solvates. Methods for the identification of solvated include, but are not limited to, NMR and microanalysis. The compound of formula (I), or salt thereof, may form one or more solvates.

The compound of formula (I), or a salt thereof, may be in crystalline or amorphous form, each of which are included within the scope of the present invention. The most thermodynamically stable crystalline form of the compound of formula (I), or a salt thereof, is of particular interest.

Crystalline forms of the compound of formula (I), or a salt thereof, may be characterised and differentiated using a number of conventional analytical techniques, including, but not limited to, X-ray powder diffraction (XRPD), infrared spectroscopy (IR), Raman spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and solid-state nuclear magnetic resonance (ssNMR).

In one embodiment, there is provided a crystalline solid state form of 5-(l-(l,3- dimethoxypropan-2-yl)-5-morpholino-lH-benzo[<i]imidazol-2-yl)-l,3-dimethylpyridin- 2(lH)-one.

In a further embodiment, there is provided a crystalline solid state form of 5-(l- (l,3-dimethoxypropan-2-yl)-5-morpholino-lH-benzo[<i]imidazol-2-yl)-l,3-dimethylpyridin- 2(lH)-one characterised by an X-ray powder diffraction (XRPD) pattern having significant diffraction peaks at 2Θ values shown in Table 1.

In a further embodiment, the present invention is directed to a crystalline solid state form of 5-(l-(l,3-dimethoxypropan-2-yl)-5-morpholino-lH-benzo[<i]imidazol-2-yl)- l,3-dimethylpyridin-2(lH)-one, characterised by an X-ray powder diffraction pattern having diffraction peaks at 2Θ values, ± 0.10° 20 experimental error, of 8.2, 8.6, 11.0, 12.5, 13.0, 14.0, 16.3, 17.1, 18.5, 22.0, 23.7, and 26.7 (Form 1).

The present invention also includes all suitable isotopic variations of the compound of formula (I) or a salt thereof. An isotopic variation of the compound of formula (I), or a salt thereof, is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷0, ¹⁸0, ¹⁸F and ³⁶CI, respectively. Certain isotopic variations of the compound of formula (I) or a salt thereof, for example, those in which a radioactive isotope such as ³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the compound of formula (I), or a salt thereof, can generally be prepared by conventional procedures such as by the illustrative methods or by the preparations described in the Examples hereafter using appropriate isotopic variations of suitable reagents.

STATEMENT OF USE

The compound of formula (I) is known to be a BET inhibitor and thus this compound, or a pharmaceutically acceptable salt thereof, may have therapeutic utility in the treatment of a variety of diseases or conditions related to systemic or tissue inflammation, inflammatory responses to infection or hypoxia, cellular activation and proliferation, lipid metabolism, fibrosis and in the prevention and treatment of viral infections. In one embodiment, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is capable of inhibiting the binding of each BET family bromodomain containing protein (e.g. BRD2, BRD3, BRD4 and BRDT) to acetylated lysine residues. In a further embodiment, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is capable of inhibiting the binding of BRD4 to its cognate acetylated lysine residue.

The compound of formula (I), or a pharmaceutically acceptable salt thereof, may possess a profile that makes it particularly suitable for progression into development as a new therapy, as it possesses one or more of the following features:

i. potent BET family (BRD2, BRD3, BRD4 and BRDT) inhibitory activity; ii. selectivity over other known bromodomain containing proteins outside of the BET family of proteins;

iii. potent anti-inflammatory effect as evidenced by in vivo efficacy data generated in, for example, pre-clinical mouse models of TNP-KLH induced IgGl production and LPS induced IL-6 production;

iv. negative result in the Ames test;

v. high solubility in biologically relevant FaSSIF media (i.e. greater than 1 mg/mL)

vi. pharmacokinetic profile in pre-clinical species predictive of a high oral bioavailability in human (on the basis of the preclinical data for oral bioavailability (rat 94% and dog 101%) the oral bioavailability in human is expected to be high);

vii. pharmacokinetic and pharmacodynamic profile predictive of a low, once daily, oral dose, i.e. less than 50mg per day, for example 5, 10, 15, 20, 25, 30, 35, 40, 45 mg per day.

BET inhibitors may be useful in the treatment of a wide variety of acute or chronic autoimmune and/or inflammatory conditions such as rheumatoid arthritis, osteoarthritis, acute gout, psoriasis, systemic lupus erythematosus, pulmonary arterial hypertension (PAH), multiple sclerosis, inflammatory bowel disease (Crohn's disease and Ulcerative colitis), asthma, chronic obstructive airways disease, pneumonitis, myocarditis, pericarditis, myositis, eczema, dermatitis (including atopic dermatitis), alopecia, vitiligo, bullous skin diseases, nephritis, vasculitis, hypercholesterolemia, atherosclerosis, Alzheimer's disease, depression, Sjogren's syndrome, sialoadenitis, central retinal vein occlusion, branched retinal vein occlusion, Irvine-Gass syndrome (post cataract and postsurgical), retinitis pigmentosa, pars planitis, birdshot retinochoroidopathy, epiretinal membrane, cystic macular edema, parafoveal telengiectasis, tractional maculopathies, vitreomacular traction syndromes, retinal detachment, neuroretinitis, idiopathic macular edema, retinitis, dry eye (keratoconjunctivitis Sicca), vernal keratoconjunctivitis, atopic keratoconjunctivitis, uveitis (such as anterior uveitis, pan uveitis, posterior uveitis, uveitis-associated macular edema), scleritis, diabetic retinopathy, diabetic macula edema, age-related macular dystrophy, hepatitis, pancreatitis, primary biliary cirrhosis, sclerosing cholangitis, Addison's disease, hypophysitis, thyroiditis, type I diabetes, 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, Takayasu's Arteritis, pyoderma gangrenosum, vasculitis with organ involvement and acute rejection of transplanted organs. The use of BET inhibitors for the treatment of rheumatoid arthritis is of particular interest.

In one embodiment, the acute or chronic autoimmune and/or inflammatory condition is a disorder of lipid metabolism via the regulation of APO-A1 such as hypercholesterolemia, atherosclerosis and Alzheimer's disease.

In another embodiment, the acute or chronic autoimmune and/or inflammatory condition is a respiratory disorder such as asthma or chronic obstructive airways disease.

In another embodiment, the acute or chronic autoimmune and/or inflammatory condition is a systemic inflammatory disorder such as rheumatoid arthritis, osteoarthritis, acute gout, psoriasis, systemic lupus erythematosus, multiple sclerosis or inflammatory bowel disease (Crohn's disease and ulcerative colitis).

In another embodiment, the acute or chronic autoimmune and/or inflammatory condition is multiple sclerosis.

In a further embodiment, the acute or chronic autoimmune and/or inflammatory condition is Type I diabetes.

BET inhibitors may be useful in the treatment of diseases or conditions which involve inflammatory responses to infections with bacteria, viruses, fungi, parasites or their toxins, such as sepsis, acute sepsis, sepsis syndrome, septic shock, endotoxaemia, systemic inflammatory response syndrome (SIRS), multi-organ dysfunction syndrome, toxic shock syndrome, acute lung injury, ARDS (adult respiratory distress syndrome), acute renal failure, fulminant hepatitis, burns, acute pancreatitis, 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. In one embodiment, the disease or condition which involves an inflammatory response to an infection with bacteria, a virus, fungi, a parasite or their toxins is acute sepsis.

BET inhibitors may be useful in the treatment of conditions associated with ischaemia-reperfusion injury such as myocardial infarction, cerebro-vascular ischaemia (stroke), acute coronary syndromes, renal reperfusion injury, organ transplantation, coronary artery bypass grafting, cardio-pulmonary bypass procedures, pulmonary, renal, hepatic, gastro-intestinal or peripheral limb embolism.

BET inhibitors may be useful in the treatment of fibrotic conditions such as idiopathic pulmonary fibrosis, renal fibrosis, post-operative stricture, keloid scar formation, scleroderma (including morphea), cardiac fibrosis and cystic fibrosis.

BET inhibitors may be useful in the treatment of viral infections such as herpes simplex infections and reactivations, cold sores, herpes zoster infections and reactivations, chickenpox, shingles, human papilloma virus (HPV), human immunodeficiency virus (HIV), cervical neoplasia, adenovirus infections, including acute respiratory disease, poxvirus infections such as cowpox and smallpox and African swine fever virus. In one embodiment, the viral infection is a HPV infection of skin or cervical epithelia. In another embodiment, the viral infection is a latent HIV infection.

BET inhibitors may be useful in the treatment of cancer, including hematological (such as leukaemia, lymphoma and multiple myeloma), epithelial including lung, breast and colon carcinomas, midline carcinomas, mesenchymal, hepatic, renal and neurological tumours.

BET inhibitors may be useful in the treatment of one or more cancers selected from brain cancer (gliomas), glioblastomas, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, colorectal cancer, Wilm's tumor, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma cancer, osteosarcoma, giant cell tumor of bone, thyroid cancer, lymphoblastic T-cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma, megakaryoblastic leukemia, acute megakaryocyte leukemia, promyelocyte leukemia, mixed lineage leukaemia, erythroleukemia, malignant lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, lymphoblastic T-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulval cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumor), NUT- midline carcinoma and testicular cancer.

In one embodiment, the cancer is a leukaemia, for example a leukaemia selected from acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and mixed lineage leukaemia (MLL). In another embodiment, the cancer is NUT-midline carcinoma. In another embodiment, the cancer is multiple myeloma. In another embodiment, the cancer is a lung cancer such as small cell lung cancer (SCLC). In another embodimnet, the cancer is a neuroblastoma. In another embodiment, the cancer is Burkitt's lymphoma. In another embodiment, the cancer is cervical cancer. In another embodiment, the cancer is esophageal cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is breast cancer. In another embodiment, the cancer is colorectal cancer.

In one embodiment, the disease or condition for which a BET inhibitor is indicated is selected from diseases associated with systemic inflammatory response syndrome, such as sepsis, burns, pancreatitis, major trauma, haemorrhage and ischaemia. In this embodiment, the BET inhibitor would be administered at the point of diagnosis to reduce the incidence of SIRS, the onset of shock, multi-organ dysfunction syndrome, which includes the onset of acute lung injury, ARDS, acute renal, hepatic, cardiac or gastro-intestinal injury and mortality. In another embodiment, the BET inhibitor would be administered prior to surgical or other procedures associated with a high risk of sepsis, haemorrhage, extensive tissue damage, SIRS or MODS (multiple organ dysfunction syndrome). In a particular embodiment, the disease or condition for which a BET inhibitor is indicated is sepsis, sepsis syndrome, septic shock and endotoxaemia. In another embodment, the BET inhibitor is indicated for the treatment of acute or chronic pancreatitis. In another embodiment, the BET inhibitor is indicated for the treatment of burns.

In a further aspect, the present invention provides the compound of formula (I) , or a pharmaceutically acceptable salt thereof, for use in therapy.

In a further aspect, the present invention provides the compound of fomula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of diseases or conditions for which a BET inhibitor is indicated.

In a further aspect, the present invention also provides the compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of autoimmune and/or inflammatory diseases, and cancer.

In a further aspect, the present invention provides the compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of rheumatoid arthritis.

In a further aspect of the present invention, there is provided a method of treating a disease or condition for which a BET inhibitor is indicated, which method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In a further aspect, the present invention is directed to a method of treatment of an autoimmune and/or inflammatory disease, which comprises administering to a subject in need thereof, a safe and therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In one embodiment, the subject is a human subject.

In yet a further aspect, the present invention is directed to a method of treating rheumatoid arthritis, which comprises administering to a subject in need thereof, a safe and therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof.

PHARMACEUTICAL COMPOSITIONS/ROUTES OF ADMINISTRATION/DOSAGES

While it is possible that for use in therapy, the compound of formula (I) as well as pharmaceutically acceptable salts thereof may be administered as the raw chemical, it is common to present the active ingredient as a pharmaceutical composition.

In a further aspect, there is provided a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. In a further aspect, there is provided a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

The excipient(s) must be pharmaceutically acceptable and be compatible with the other ingredients of the composition. In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical composition including admixing a compound of formula (I), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable excipients. The pharmaceutical composition can be used in the treatment of any of the diseases described herein.

Since the compound of formula (I), or a pharmaceutically acceptable salt thereof, is intended for use in pharmaceutical compositions it will be readily understood that they are each preferably provided in substantially pure form, for example, at least 85% pure, especially at least 98% pure (% in a weight for weight basis).

Pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Preferred unit dosage compositions are those containing a daily dose or sub-dose, or an appropriate fraction thereof, of an active ingredient. Such unit doses may therefore be administered more than once a day.

Pharmaceutical compositions may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, inhaled, intranasal, topical (including buccal, sublingual or transdermal), ocular (including topical, intraocular, subconjunctival, episcleral, sub-Tenon), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the excipient(s).

In one aspect, the pharmaceutical composition is adapted for oral administration. Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as tablets or capsules; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Powders suitable for incorporating into tablets or capsules may be prepared by reducing the compound of formula (I), or a pharmaceutically acceptable salt thereof, to a suitable fine size (e.g. by micronisation) and mixing with a similarly prepared pharmaceutical excipient such as an edible carbohydrate, for example, starch or mannitol. Flavoring, preservative, dispersing and coloring agents, for example, may also be present.

Capsules may be made by preparing a powder mixture, as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested. Moreover, when desired or necessary, suitable binders, glidants, lubricants, sweetening agents, flavours, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compound of formula (I) or a pharmaceutically acceptable salt thereof can also be combined with a free flowing inert excipient and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be added.

Compositions for oral administration may be designed to provide a modified release profile so as to sustain or otherwise control the release of the therapeutically active agent.

Where appropriate, dosage unit compositions for oral administration can be microencapsulated. The composition may be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like.

Pharmaceutical compositions for nasal or inhaled administration may conveniently be formulated as aerosols, solutions, suspensions, gels or dry powders. For compositions suitable for and/or adapted for inhaled administration, it is preferred that a compound of formula (I) or a pharmaceutically acceptable salt thereof, is in a particle-size-reduced form e.g. obtained by micronisation. The preferable particle size of the size-reduced (e.g. micronised) compound or salt is defined by a D50 value of about 0.5 to about 10 microns (for example as measured using laser diffraction).

The pharmaceutical composition for inhaled administration may be a dry powder composition or an aerosol formulation, comprising a solution or fine suspension of the active substance in a pharmaceutically acceptable aqueous or non-aqueous solvent. Dry powder compositions can comprise a powder base such as lactose, glucose, trehalose, mannitol or starch, the compounds of formulae (I) or a pharmaceutically acceptable salt thereof (preferably in particle-size-reduced form, e.g. in micronised form), and optionally a performance modifier such as L-leucine or another amino acid and/or metal salt of stearic acid such as magnesium or calcium stearate. Preferably, the dry powder inhalable composition comprises a dry powder blend of lactose e.g. lactose monohydrate and the compound of formula (I) or a pharmaceutically acceptable salt thereof.

In one embodiment, a dry powder composition suitable for inhaled administration may be incorporated into a plurality of sealed dose containers provided on medicament pack(s) mounted inside a suitable inhalation device. The containers may be rupturable, peelable or otherwise openable one-at-a-time and the doses of the dry powder composition administered by inhalation on a mouthpiece of the inhalation device, as known in the art. The medicament pack may take a number of different forms, for instance a disk-shape or an elongate strip. Representative inhalation devices are the DISKHALER™ inhaler device, the DISKUS™ inhalation device, and the ELLIPTA™ inhalation device, marketed by GlaxoSmithKline. The DISKUS™ inhalation device is, for example, described in GB 2242134A, and the ELLIPTA™ inhalation device is, for example, described in WO 03/061743 Al WO 2007/012871 Al and/or WO2007/068896.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, emulsions, lotions, powders, solutions, pastes, gels, foams, sprays, aerosols or oils. Such pharmaceutical compositions may include conventional additives which include, but are not limited to, preservatives, solvents to assist drug penetration, co-solvents, emollients, propellants, viscosity modifying agents (gelling agents), surfactants and carriers.

For treatments of the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment, cream, gel, spray or foam. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

A therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, will depend upon a number of factors including, for example, the age and weight of the subject, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. In the pharmaceutical composition, each dosage unit may contain from 0.01 to 1000 mg, more preferably 0.5 to 100 mg, of a compound of formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. In a further embodiment, each dosage unit may contain 0.5 to 50 mg, for example 5 mg to 20 mg, such as 5 mg, 10 mg, 15 mg or 20 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. In a further embodiment, the dosage is administered more than once-daily (e.g. twice-daily), once-daily, or less frequently, for example once-weekly or once-monthly. In a further embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once-daily. In yet a further embodiment, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered once-daily at a dose of 10 mg. In yet a further embodiment, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered once-daily at a dose of 20 mg. The compound of formula (I) or a pharmaceutically acceptable salt thereof may be employed alone or in combination with other therapeutic agents. Combination therapies according to the present invention thus comprise the administration of at least one compound of formula (I) or a pharmaceutically acceptable salt thereof, and the use of at least one other therapeutically active agent. A compound of formula (I) or pharmaceutically acceptable salt thereof, and the other therapeutically active agent(s) may be administered together in a single pharmaceutical composition or separately and, when administered separately this may occur simultaneously or sequentially in any order.

In a further aspect, there is provided a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with one or more other therapeutically active agents, and optionally one or more pharmaceutically acceptable carriers, diluents or excipients.

In a further aspect, there is provided a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with one or more other therapeutically active agents, and optionally one or more pharmaceutically acceptable excipients.

It will be clear to a person skilled in the art that, where appropriate, the other therapeutic ingredient(s) may be used in the form of salts, for example as alkali metal or amine salts or as acid addition salts, or as solvates, for example hydrates, to optimise the activity and/or stability and/or physical characteristics, such as solubility, of the therapeutic ingredient. It will be clear also that, where appropriate, the therapeutic ingredients may be used in optically pure form.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical composition and thus pharmaceutical compositions comprising a combination as defined above together with one or more pharmaceutically acceptable excipients represent a further aspect of the invention.

EXAMPLE PREPARATION

Abbreviations

CV Column volumes

DCM Dichloromethane

DMSO Dimethylsulfoxide

g Grammes

h Hour(s)

EtOAc Ethyl acetate

HPLC High-performance liquid chromatography

L Litre

LCMS Liquid chromatography-mass spectrometry MeOH Methanol

min Minutes

mg Milligrammes

MHz Megahertz

ml_ Millilitre

mM Millimolar

nm Nanometre

nl_ Nanolitre

ppm Parts per million

THF Tetrahydrofuran

tRET Retention time

μΙΤΊ Micrometre

Experimental Details

LCMS

System A:

Column: Acquity BEH C18 (50 mm x 2.1 mm, 1.7 μηη)

Flow Rate: 0.6 ml_/min

Temperature: 35 °C

UV detection range: 190 to 400 nm

Mass spectrum: Recorded on a mass spectrometer using alternative-scan positive and negative mode electrospray ionisation

Mobile Phases: A: 0.1% formic acid in water

B: 0.1% formic acid in acetonitrile

Gradient: Time (min) A% B%

0 97 3

0.4 97 3

3.2 2 98

3.8 2 98

4.2 97 3

4.5 97 3

System B:

Column: Acquity UPLC CSH C18 (50 mm x 2.1 mm, 1.7 μηη)

Flow Rate: 1 ml_/min

Temperature: 40 °C

UV detection range: 210 to 350 nm Mass spectrum: Recorded on a mass spectrometer using alternative-scan positive and negative mode electrospray ionisation

Mobile Phases: A: 10 mM ammonium bicarbonate in water adjusted to pHIO with ammonia solution

B: acetonitrile

Gradient: Time Qnin) A% B%

0 97 3

0.05 97 3

1.5 5 95

1.9 5 95

2.0 97 3

H NMR

The ^ NMR spectrum was recorded in DMSO-c/₆ on a Bruker AVII+ 600 MHz with cryo- probe, and referenced to TMS at 0.00 ppm.

XRPD

The data were acquired on a PANalytical X'Pert Pro powder diffractometer, model PW3040/60 using an X'Celerator detector. The acquisition conditions were: radiation: Cu Ka, generator tension: 40 kV, generator current: 45 mA, start angle: 2.0° 2Θ, end angle: 40.0° 2Θ, step size: 0.0167° 2Θ, time per step: 31.75 seconds. The sample was prepared by mounting a few milligrams of sample on a silicon wafer (zero background plate), resulting in a thin layer of powder. Characteristic XRPD angles and d-spacings for Example 1 (5-(l-(l,3-Dimethoxypropan-2-yl)-5-morpholino-lH-benzo[<i]imidazol-2-yl)- l,3-dimethylpyridin-2(lH)-one) are recorded in Table 1 . The margin of error is approximately ± 0.1 ° 29 for each of the peak assignments. Peak intensities may vary from sample to sample due to preferred orientation. Peak positions were measured using PANalytical Highscore Plus software.

Table 1: Characteristic XRPD peak positions and d-spacinqs for Example 1

13.0 6.8

14.0 6.3

16.3 5.4

17.1 5.2

18.5 4.8

22.0 4.0

23.7 3.8

26.7 3.3

Intermediate Preparation

Intermediate 1: l,5-dimethyl-6-oxo-l,6-dihydropyridine-3-carbaldehyde

To a stirred mixture of 5-methyl-6-oxo-l,6-dihydropyridine-3-carbaldehyde (commercially available from, for example, Matrix Scientific or Milestone Pharmtech)(50 g, 365 mmol) in acetone (2000 mL) was added potassium carbonate (151 g, 1094 mmol) followed by iodomethane (68.4 mL, 1094 mmol) and stirred at room temperature for 72 h. The reaction mixture was filtered and the residue was washed with acetone (500 mL) and filtrate was evaporated under reduced pressure to afford Batch 1 (35 g) as a yellow solid.

A second batch was prepared as follows. To a stirred mixture of 5-methyl-6-oxo- l,6-dihydropyridine-3-carbaldehyde (50 g, 365 mmol) in acetone (2000 mL) was added potassium carbonate (151 g, 1094 mmol) followed by iodomethane (68.4 mL, 1094 mmol) and stirred at room temperature for 16 h. The reaction mixture was filtered and residue was washed with 10% MeOH in DCM (2 L) and the filtrate was evaporated under reduced pressure to afford a yellow solid (65 g). The crude product was purified by column chromatography using 60-120 silica gel, compound was eluted at 5% MeOH in DCM and evaporated under reduced pressure to afford Batch 2 (40 g) as a pale yellow solid. Batch 1 and Batch 2 were combined and purified by column chromatography by using 60-120 silica gel and eluted at 5% MeOH in DCM. The fractions were evaporated under reduced pressure to get solid material which was suspended in diethyl ether (500 mL) and stirred for 30 min then filtered and dried to afford the title compound (52 g, 44% total yield) as a pale yellow solid. LCMS (System A): t_RET = 1.15 min; MH⁺ 152.

Intermediate 2: 4-Bromo-/V-(l,3-dimethoxypropan-2-yl)-2-nitroaniline

4-Bromo-l-fluoro-2-nitrobenzene (commcerciaHy available from, for example, Aldrich) (50 g, 227 mmol) and l,3-dimethoxypropan-2-amine (commcerciaHy available from, for example, Aldrich) (32.5 g, 273 mmol) were dissolved in acetonitrile (300 mL) and potassium carbonate (47.1 g, 341 mmol) was added, then the mixture was stirred at 80 °C for 6 h, then the mixture was allowed to cool and stood over the weekend at room temperature. The mixture was diluted with water (500 mL) and extracted with EtOAc (2 x 500 mL). The organic layer was washed with water (300 mL) and brine (300 mL), dried and evaporated in vacuo to give the title compound (70 g, 220 mmol, 97% yield) as an orange solid. LCMS (System B): t_RET = 1.25 min; MH⁺ 319, 321.

Intermediate 3: 5-(5-Bromo-l-(l,3-dimethoxypropan-2-yl)-l y-benzorQlimidazol- 2-yl)-l,3-dimethylpyridin-2(lffl-one

4-Bromo-/V-(l,3-dimethoxypropan-2-yl)-2-nitroaniline (Intermediate 2, 69 g, 216 mmol) was dissolved in ethanol (400 mL) with heating, and on cooling the starting material crystallised out. l,5-Dimethyl-6-oxo-l,6-dihydropyridine-3-carbaldehyde (for an example preparation see Intermediate 1, 35.9 g, 238 mmol) was added to the suspension, followed by water (200 mL) and sodium dithionite (94 g, 540 mmol) and the mixture was heated at 90 °C for 18 h. The mixture was evaporated to approximately half its original volume, then diluted with water (200 mL) and extracted with DCM (2 x 300 mL). The combined organics were washed with brine, then dried and evaporated in vacuo and the resulting yellow solid triturated with EtOAc (200 mL) and the solid collected by filtration and washed with ether (200 mL) and dried under vacuum to give the title compound (Batch 1, 31 g, 34% yield). LCMS (System B): t_RET = 1.00 min; MH⁺ 420, 422.

The filtrate was evaporated in vacuo and the residue dissolved in DCM (100 mL) and loaded onto a 750 g silica column, then eluted with 0-10% MeOH/DCM and product- containing fractions evaporated in vacuo to give a yellow solid. This was dissolved in hot EtOAc (150 mL) and then allowed to cool and stand overnight. The resulting solid was collected by filtration and the product washed with ether (2 x 100 mL), stirring the product/ether mixture to ensure good trituration, and the product was then dried to give the title compound (Batch 2, 20 g, 47.6 mmol, 22% yield) as a very pale yellow solid. LCMS (System B): t_RET = 1.00 min; MH⁺ 420, 422.

Preparation of the compound of formula (I)

Example 1 : 5-(l-(l,3-Dimethoxypropan-2-yl)-5-morpholino-l y-benzorQlimidazol- 2-yl)-l,3-dimethylpyridin-2(lffl-one

5-(5-Bromo-l-(l,3-dimethoxypropan-2-yl)-lH-benzo[<i]imidazol-2-yl)-l,3- dimethylpyridin-2(lH)-one (for an example preparation see Intermediate 3, 29.5 g, 70.2 mmol), 2'-(dicyclohexylphosphino)-/V,/V-dimethyl-[l, -biphenyl]-2-amine (commercially available from, for example, Aldrich) (1.381 g, 3.51 mmol), tris(dibenzylideneacetone)dipalladium(0) (commercially available from, for example, Aldrich) (1.285 g, 1.404 mmol), morpholine (12.23 mL, 140 mmol) and 2- methyltetrahydrofuran (commercially available from, for example, Aldrich) (150 mL) were combined in a round bottom flask under nitrogen, then sodium tert-butoxide (2 M in THF, 105 mL, 211 mmol) was added and the mixture was heated at 80 °C for 2 h. The reaction mixture was diluted with brine (200 mL) and extracted with EtOAc (2 x 200 mL). The combined organics were dried and evaporated in vacuo to give a brown gum, which was then triturated with EtOAc (200 mL) and then diluted with ether (100 mL), giving a fine, beige precipitate. This was collected by filtration and washed with ether to give the title compound (Batch 1, 21.2 g, 71% yield). LCMS (System B): t_RET = 0.80 min; MH⁺ 427.

The fitrate was evaporated in vacuo to give a brown gum. This was dissolved in DCM (30 mL) and loaded onto a 340 g silica column, then eluted with 0-10% MeOH/DCM and product-containing fractions were evaporated in vacuo. The resulting solid was triturated with ether (100 mL) and collected by filtration to give the title compound (Batch 2, 6.5 g, 22% yield). LCMS (System B): t_RET = 0.80 min; MH⁺ 427.

5-(5-Bromo-l-(l,3-dimethoxypropan-2-yl)-lH-benzo[<i]imidazol-2-yl)-l,3- dimethylpyridin-2(lH)-one (Intermediate 3, 33.2 g, 79 mmol), 2'- (dicyclohexylphosphino)-/V,/V-dimethyl-[l, -biphenyl]-2-amine (Aldrich) (1.554 g, 3.95 mmol), tris(dibenzylideneacetone)dipalladium(0) (commercially available from, for example, Aldrich) (1.447 g, 1.580 mmol), morpholine (13.76 mL, 158 mmol) and 2- methyltetrahydrofuran (150 mL) were combined in a round bottom flask under nitrogen, then sodium tert-butoxide (2 M in THF, 118 mL, 237 mmol) was added and the mixture was heated at 80 °C for 2 h. The reaction mixture was diluted with brine (200 mL) and extracted with EtOAc (2 x 200 mL). The combined organics were dried and evaporated in vacuo to give a brown gum, which was then triturated with EtOAc (200 mL) and then diluted with ether (100 mL), giving a fine, beige precipitate. This was collected by filtration and washed with ether to give the title compound (Batch 3, 31.4 g, 93% yield). LCMS (System B): t_RET = 0.80 min; MH⁺ 427.

5-(5-Bromo-l-(l,3-dimethoxypropan-2-yl)-lH-benzo[<i]imidazol-2-yl)-l,3- dimethylpyridin-2(lH)-one (Intermediate 3, 20 g, 47.6 mmol), 2'- (dicyclohexylphosphino)-/V,/V-dimethyl-[l, -biphenyl]-2-amine (Aldrich) (0.936 g, 2.379 mmol), tris(dibenzylideneacetone)dipalladium(0) (commercially available from, for example, Aldrich) (0.871 g, 0.952 mmol), morpholine (8.29 mL, 95 mmol) and 2- methyltetrahydrofuran (commercially available from, for example, Aldrich) (150 mL) were combined in a round bottom flask under nitrogen, then sodium tert-butoxide (2 M in THF, 71.4 ml_, 143 mmol) was added and the mixture was heated at 80 °C for 2 h. The reaction mixture was diluted with brine (200 ml.) and extracted with EtOAc (2 x 300 ml_). The combined organics were dried and evaporated in vacuo to give a brown gum. This was combined with batches 1, 2, and 3 and dissolved in DCM (200 ml_), then loaded onto a 750 g silica column and eluted with EtOAc (5 CV), then a gradient of 0-25% EtOH/EtOAc (20 CV). Product-containing fractions were evaporated in vacuo to give a pale yellow solid. This was dissolved in DCM (500 ml.) and Silicycle thiourea silica resin (40 g) was added, then the mixture was stirred at room temperature for 1 h. The suspension was filtered and the filtrate evaporated in vacuo, then the resulting foam was triturated with ether (300 ml.) and the resulting solid collected by filtration to give (60 g) pale yellow solid. This was suspended in EtOAc (400 ml.) and heated to reflux with stirring for 1 h, then the mixture was allowed to cool over 2 h, then further cooled using an ice bath and stirred for 1 h. The suspension was filtered and the solid washed with ether (200 ml.) to give the title compound (49.8 g, 59% total yield) as an almost colourless solid. LCMS (System B): t_RET = 0.81 min; MH⁺ 427.

*H NMR (DMSO-de) δ: 8.00 (d, J=2.5 Hz, 1H), 7.65 (m, 1H), 7.63 (d, J=9.0 Hz, 1H), 7.10 (d, J=2.5 Hz, 1H), 6.96 (dd, J=9.0, 2.5 Hz, 1H), 4.77 (tt, J=9.0, 4.5 Hz, 1H), 3.97 (dd, J=10.5, 9.0 Hz, 2H), 3.76-3.78 (m, 4H), 3.75 (dd, J = 10.5, 4.5 Hz, 2H), 3.53 (s, 3H), 3.16 (s, 6H), 3.07-3.10 (m, 4H), 2.08 (s, 3H).

BIOLOGICAL DATA

Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay

Binding was assessed using a time resolved fluorescent resonance energy transfer binding assay. This utilises a 6 His purification tag at the N-terminal of the proteins as an epitope for an anti-6 His antibody labeled with Europium chelate (PerkinElmer AD0111) allowing binding of the Europium to the proteins which acts as the donor fluorophore. A small molecule, high affinity binder of the bromodomains BRD2, BRD3, BRD4 and BRDT has been labeled with Alexa Fluor647 (Reference Compound X) and this acts as the acceptor in the FRET pair.

Reference Compound X: 4-((Z)-3-(6-((5-(2-((45)-6-(4-chlorophenvn-8-methoxy-l- methyl-4H-benzorfiri,2,41triazolor4,3-airi,41diazepin-4-yl)acetamido)pentyl)amino)-6- oxohexyn-2-((2E,4E)-5-(3,3-dimethyl-5-sulfo-l-(4-sulfobutvn-3H-indol-l-ium-2-vnpenta- 2,4-dien-l-ylidene)-3-methyl-5-sulfoindolin-l-yl)butane-l-sulphonate)

To a solution of A/-(5-aminopentyl)-2-((4S)-6-(4-chlorophenyl)-8-methoxy-l-methyl-4H- benzo[f][l,2,4]triazolo[4,3-a][l,4]diazepin-4-yl)acetamide (for a preparation see Reference Compound J, WO2011/054848A1, 1.7 mg, 3.53 μιτιοΙ) in DMF (40μΙ) was added a solution of AlexaFluor647-ONSu (2.16 mg, 1.966 μιτιοΙ) also in DMF (ΙΟΟμΙ). The mixture was basified with DIPEA (1 μΙ, 5.73 μιτιοΙ) and agitated overnight on a vortex mixer. The reaction mixture was evaporated to dryness. The solid was dissolved in acetonitrile/water/acetic acid (5/4/1, < lml) filtered and was applied to a Phenomenex Jupiter C18 preparative column and eluted with the following gradient (A = 0.1% trifluoroacetic acid in water, B= 0.1% TFA/90% acetonitrile/10% water): Flow rate = lOml/min., AU = 20/10 (214nm):

5-35%, t=0min: B = 5%; t=10min: B = 5%; t=100min: B = 35%; t=115min: B = 100% (Sep. grad: 0.33%/min)

The major component was eluted over the range 26-28%B but appeared to be composed of two peaks. The middle fraction (F1.26) which should contain "both" components was analysed by analytical HPLC (Spherisorb ODS2, 1 to 35% over 60min): single component eluting at 28%B.

Fractions F1.25/26&27 were combined and evaporated to dryness. Transfered with DMF, evaporated to dryness, triturated with dry ether and the blue solid dried overnight at<0.2mbar: 1.54mg.

Analytical HPLC (Sphersisorb ODS2, 1 to 35%B over 60min): MSM10520-1:

[M+H]⁺ (obs): 661.8/- corresponding with M-29. This equates to [(M+2H)/2]⁺ for a calculated mass of 1320.984 which is M-29. This is a standard occurence with the Alexa Fluor 647 dye and represents a theoretical loss of two methylene groups under the conditions of the mass spectrometer.

Assay Principle: In the absence of a competing compound, excitation of the

Europium causes the donor to emit at λ618ηηη which excites the Alexa labelled bromodomain binding compound leading to an increased energy transfer that is measurable at λ647ηΜ. In the presence of a sufficient concentration of a compound that can bind these proteins, the interaction is disrupted leading to a quantifiable drop in fluorescent resonance energy transfer. The binding of the compound of formula (I) to Bromodomains BRD2, BRD3, BRD4 and BRDT was assessed using mutated proteins to detect differential binding to either Binding Domain 1 (BD1) or Binding Domain 2 (BD2) on the bromodomain. These single residue mutations in the acetyl lysine binding pocket greatly lower the affinity of the fluoroligand (Reference Compound X) for the mutated domain (>1000 fold selective for the non-mutated domain). Therefore in the final assay conditions, binding of the fluoroligand to the mutated domain cannot be detected and subsequently the assay is suitable to determine the binding of compounds to the single non-mutated bromodomain.

Protein production: Recombinant Human Bromodomains [(BRD2 (1-473) (Y113A) and (Y386A), BRD3 (1-435) (Y73A) and (Y348A) BRD4 (1-477) (Y97A) and (Y390A) and BRDT (1-397) (Y66A) and (Y309A)] were expressed in E. coli cells (in pET15b vector for BRD2/3/4 and in pET28a vector for BRDT) with a 6-His tag at the N-terminal. The His- tagged Bromodomain pellet was resuspended in 50mM HEPES (pH7.5), 300mM NaCI, lOmM imidazole & ΙμΙ/ml protease inhibitor cocktail and extracted from the E. coli cells using sonication and purified using a nickel sepharose high performance column, the proteins were washed and then eluted with a linear gradient of 0-500mM imidazole with buffer 50mM HEPES (pH7.5), 150mM NaCI, 500mM imidazole, over 20 column volumes. Final purification was completed by Superdex 200 prep grade size exclusion column. Purified protein was stored at -80°C in 20mM HEPES pH 7.5 and lOOmM NaCI. Protein identity was confirmed by peptide mass fingerprinting and predicted molecular weight confirmed by mass spectrometry.

Protocol for Bromodomain BRD2, 3, 4 and T, BD1 + BD2 mutant assays: All assay components were dissolved in buffer composition of 50 mM HEPES pH7.4, 150mM NaCI, 5% Glycerol, lmM DTT and lmM CHAPS. The final concentration of bromodomain proteins were ΙΟηΜ and the Alexa Fluor647 ligand was at Kd. These components were premixed and 5μΙ of this reaction mixture was added to all wells containing 50nl of various concentrations of test compound or DMSO vehicle (0.5% DMSO final) in Greiner 384 well black low volume microtitre plates and incubated in dark for 30 minutes at rt. 5μΙ of detection mixture containing 1.5nM final concentration anti-6His Europium chelate was added to all wells and a further dark incubation of at least 30 minutes was performed. Plates were then read on the Envision platereader, (λεχ = 317nm, donor λειτι = 615nm; acceptor λειτι = 665nm; Dichroic LANCE dual). Time resolved fluorescent intensity measurements were made at both emission wavelengths and the ratio of acceptor/donor was calculated and used for data analysis. All data was normalized to the mean of 16 high (inhibitor control - Example 11 of WO 2011/054846A1) and 16 low (DMSO) control wells on each plate. A four parameter curve fit of the following form was then applied:

y = a + (( b - a) / ( l + ( 10 x / 10 c ) d )

Where 'a' is the minimum, ^{^}ti is the Hill slope, is the pIC₅o and is the maximum.

Results: Example 1 was found to have a mean pIC₅o of 7.9 (n = 21) in the BRD4 BD1 assay and a mean pIC₅o of 6.6 (n = 23) in the BRD4 BD2 assay. Example 1 was found to have a mean pIC₅o of 7.6 (n = 4) in the BRD2 BD1 assay and a mean pIC₅o of 6.3 (n = 4) in the BRD2 BD2 assay. Example 1 was found to have a mean pIC₅o of 7.9 (n = 4) in the BRD3 BD1 assay and a mean pIC₅o of 7.0 (n = 4) in the BRD3 BD2 assay. Example 1 was found to have a mean pIC₅o of 6.9 (n = 8) in the BRDT BD1 assay and a mean p!C₅o of 6.1 (n = 8) in the BRDT BD2 assay.

Measurement of LPS induced MCP-1 production from human whole blood

Activation of monocytic cells by agonists of toll-like receptors such as bacterial lipopolysaccharide (LPS) results in production of key inflammatory mediators including MCP-1. Such pathways are widely considered to be central to the pathophysiology of a range of auto-immune and inflammatory disorders.

Blood was collected in a tube containing Sodium heparin (Leo Pharmaceuticals) (10 units of heparin/mL of blood). 96-well compound plates containing 0.5μΙ_ test sample (compound) in 100% DMSO were prepared (two replicates on account of donor variability). 130 μΙ_ of whole blood was dispensed into each well of the 96-well compound plates and incubated for 30 min at 37°C, 5% C0₂. 10 μΙ_ of lipopolysaccharide (from Salmonella typhosa; L6386) made up in PBS (200 ng/mL final assay concentration) was added to each well of the compound plates. The plates were then placed in the humidified primary cell incubator for 18-24 hours at 37°C, 5% C0₂. 140 μΙ_ of PBS was added to all wells of the compound plates containing blood. The plates were then sealed and centrifuged for 10 mins at 2500 rpm (r.t). 20 μΙ_ of cell supernatant was placed in a 96-well MSD plate pre-coated with human MCP-1 capture antibody. The plates were sealed and placed on a shaker at 600 rpm for 1.5 hour (r.t). 20 μΙ_ of Anti-human MCP-1 antibody labelled with MSD SULFO-TAG™ reagent was added to each well of the MSD plate (stock 50X was diluted 1:50 with Diluent 100, final assay concentration is 1 μg/mL). The plates were then re-sealed and shaken for 1 hour (r.t) before washing with 3x with PBS Tween 0.05%. 150 μΙ_ of 2X MSD Read Buffer T (stock 4X MSD Read Buffer T was diluted 50:50 with de-ionised water) was then added to each well and the plates read on the MSD Sector Imager 6000. Concentration response curves for each compound were generated from the data and an IC₅o value was calculated. Results: Example 1 was found to have a mean pIC₅o of 7.6 (n = 12), demonstrating that Example 1 inhibited the production of key inflammatory mediator MCP-1.

Measurement of LPS induced IL-6 production from human whole blood

Activation of monocytic cells by agonists of toll-like receptors such as bacterial lipopolysaccharide (LPS) results in production of key inflammatory mediators including IL-6. Such pathways are widely considered to be central to the pathophysiology of a range of auto-immune and inflammatory disorders.

Blood was collected in a tube containing Sodium heparin (Leo Pharmaceuticals) (10 units of heparin/mL of blood). 96-well compound plates containing Ιμί test sample (compound) in 100% DMSO were prepared (two replicates on account of donor variability). 130 μί of whole blood was dispensed into each well of the 96-well compound plates and incubated for 30 min at 37°C, 5% C0₂. 10 μί of lipopolysaccharide (from Salmonella typhosa; L6386) made up in PBS with 1% BSA (200 ng/mL final assay concentration) was added to each well of the compound plates. The plates were then placed in the humidified primary cell incubator for 22 (+/- 2hrs) hours at 37°C, 5% C0₂. 140 μί of PBS was added to all wells of the compound plates containing blood and the plates were sealed and shaken on a plate shaker at 600 rpm for 2 minutes. The plates were then centrifuged for 10 mins at 2500 rpm (r.t). 100 μί of cell supernatant was removed using a Bomec NX robot. MSD plates pre-coated with human IL-6 capture antibody were blocked with MSD Diluent2 for 30minutes on a plate shaker (600rpm r.t). The supernatants were diluted 1 in 40 in PBS and 25ul added to 96-well MSD IL-6 plates. The plates were sealed and placed on a shaker at 600 rpm for 1.5 hour (r.t). 20 μί of Anti-human IL-6 antibody labelled with MSD SULFO-TAG™ reagent is added to each well of the MSD plate (stock 50X was diluted 1:50 with Diluent 3, final assay concentration is 1 μg/mL). The plates were then re-sealed and shaken for 1 hour (r.t) before washing with 3x with PBS Tween 0.05%. 150 μί of 2X MSD Read Buffer T (stock 4X MSD Read Buffer T was diluted 50:50 with de-ionised water) was then added to each well and the plates read on the MSD Sector Imager 6000. Concentration response curves for each compound were generated from the data and an IC₅o value was calculated.

Results: Example 1 was found to have a mean pIC₅o of 7.7 (n = 6), demonstrating that Example 1 inhibited the production of key inflammatory mediator IL-6.

Solubility

The solubility of a compound in aqueous buffers and in physiological media is an important developability consideration. Example 1 was tested in a variety of well-known relevant media at 0.5 h, 4 h and 24 h timepoints and demonstrated high solubility, i.e. >0.82 mg/mL solubility under all conditions investigated (Table 2).

Table 2. Solubility of Example 1 in aqueous buffers and physiological media

Ames test

The Ames test is a widely used and well-known biological assay used to assess the mutagenic potential of a particular chemical compound. It is, thus, a simple established technique employed to evaluate the carcinogenic potential of a compound. A positive test in the assay indicates that a compound may be mutagenic and therefore may act as a carcinogen. A negative result is thus one of the properties required for a compound to be selected for further development as a potential therapy. Example 1 (tsted at concentrations of 50, 150, 500, 1500, 2500 and 5000 g/mL) was found to be Ames negative with four strains of Salmonella (Salmonella typhimurium TA1535, TA1537, TA98, TA100) and one strain of Escherichia coli (Escherichia coliWP2uvrA(pKM101)) in the presence and absence of S9-mix (commercially available from, for example, Moltox).

Selectivity

Example 1 possesses selectivity over other known bromodomain containing proteins outside of the BET family. Selectivity was evaluated by BROMOScan™

(DiscoverX™). BROMOScan™ is based on the KINOMEscan™ technology and is a robust and highly sensitive quantitative binding platform that can be applied to selectivity profiling to aid the identification of potent and selective small molecule bromodomain inhibitors.

Assay Process

1. Assemble Assay Components

• E. coli expressed bromodomain labeled with DNA tag for qPCR readout

• Known binding ligand immobilized on a solid support

· Test compound or solvent control

2. Equilibrate

3. Wash solid support to remove unbound bromodomain

4. Quantify bromodomain captured on solid support (qPCR)

5. Compare captured bromodomain levels in test compound & solvent control samples Assay Principle

Compounds that bind the bromodomain active site and directly (sterically) or indirectly (allosterically) prevent bromodomain binding to the immobilized ligand, will reduce the amount of protein captured on the solid support (Panels A & B). Conversely, test molecules that do not bind the bromodomain have no effect on the amount of bromodomain captured on the solid support (Panel C). Screening "hits" are identified by measuring the amount of bromodomain captured in test versus control samples by using a quantitative, precise and ultra-sensitive qPCR method that detects the associated DNA label. In a similar manner, dissociation constants (Kds) for test compound-bromodomain interactions are calculated by measuring the amount of bromodomain captured on the solid support as a function of the test compound concentration. KINOMEscan™ and BROMOscan™ use the same assay technology. For a detailed description of this assay technology see Fabian, M.A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329-336 (2005).

Results: Example 1 showed selectivity over all bromodomain containing proteins evaluated (ATAD2A, ATAD2B, BAZ2A, BAZ2B, BRD1, BRD2(1), BRD2(2), BRD3(1), BRD3(2), BRD4(1), BRD4(2), BRD7, BRD8(1), BRD8(2), BRD9, BRDT(l), BRDT(2), BRPF1, BRPF3, CECR2, CREBBP, EP300, FALZ, GCN5L2, PBRM1(2), PBRM1(5), PCAF, SMARCA2, SMARCA4, TAF1(2), TAF1L(2), TRIM24(PHD,Bromo.), TRIM33(PHD,Bromo.), and WDR9(2)). Example 1 was >3150 fold selectivity over all bromodomain containing proteins tested with the exception of CREBBP (19 fold) and EP300 (25 fold). Further, Example 1 was 4000 fold selective over CECR2 and 10000 fold selective over TAF1 BD2.

Lipopolysaccharide (LPS) induced interleukin-6 (IL-6) production mouse assay

Example 1 was assayed for its ability to inhibit lipopolysaccharide (LPS) induced interleukin-6 (IL-6) production in mice. Male CD1 mice (Charles River Laboratories, 5 per group) received an intravenous challenge of LPS (100 g/kg, L3192 E coli 0127:B8) 0.5 hours after oral administration of compound (in 1% (w/v) methylcellulose, aq 400). Serial blood samples were collected via tail vein up to 3 hours or via cardiac puncture at

5 hours (terminal sample) post LPS administration and the serum harvested from the blood samples was frozen at -80°C. On the day of analysis, the serum was thawed to room temperature, diluted 1:50 with assay diluent and levels of IL-6 were measured using single-spot cytokine assay plates (K152QXD) from Meso Scale Discovery (MSD, Gaithersburg, Maryland). The levels of IL-6 were detected according to the manufacturer's protocol (MSD) and read on a SECTOR imager 6000 (MSD). The mean IL-

6 Cmax and AUQH values were generated using WinNonlin Phoenix version 6.3 and the mean percent Cmax and AUQM IL-6 reduction following treatment with compound was calculated compared to the corresponding vehicle treated group. Levels of significance were calculated by analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test using Graphpad Prism version 5.04 (Graphpad Software, San Diego, CA). Statistical differences were determined as *P < 0.05, **P < 0.01. Results are shown in Table 3.

Table 3. Efficacy of Example 1 in the LPS-induced IL-6 assay

Trinitrophenol-keyhole limpet hemocyanin (TNP-KLH) induced Immunoqlobulin-1 (IgGl) production mouse assay

Example 1 was assayed for its ability to inhibit trinitrophenol-keyhole limpet hemocyanin (TNP-KLH) induced Immunoglobulin-1 (IgGl) production in mice. Male CD1 mice (Charles River Laboratories, 8 per group) received a single oral administration of compound (in 1% (w/v) methylcellulose, aq 400) either once every day (QD) or once every 24 hours (QOD) over a 14 day dosing period. On day 1 of the study, each mouse received a single bolus intraperitoneal (ip) administration of TNP-KLH (100 ug/kg, T- 5060-25, Lot # 021562-06) 0.5 hours after oral administration of compound. Serial blood samples were collected at 0.5 hour post oral compound administration via tail veil on days 1, 4, 7, 9 and 11 or via cardiac puncture (terminal sample) on day 14 and the serum harvested from the blood samples was frozen at -80°C. On the day of analysis, the serum was thawed to room temperature and levels of IgGl were measured using a TNP ELISA (developed in-house) and read on a SpectraMax 190 spectrophotometer (Molecular Devices, CA). The mean IgGl values were generated and the mean percent IgGl reduction on day 14 following treatment with compound was calculated compared to the corresponding vehicle treated group. Levels of significance were calculated by analysis of variance (ANOVA) followed by Dunnett's multiple comparison t-test using Graphpad Prism version 5.04 (Graphpad Software, San Diego, CA). Statistical differences were determined as **P < 0.01. Results are shown in Table 4.

Table 4. Efficacy of Example 1 in the TNP-KLH-induced IgGl production mouse assay

In vitro metabolic stability

The in vitro metabolic stability of Example 1 (0.5 μΜ) was determined in pooled cryopreserved primary hepatocytes for each species (CD-I mouse, Wistar Han rat, Beagle dog, and Human; supplied by BioreclamationlVT). The metabolic stability of Example 1 (0.5 μΜ) was determined in pooled cryopreserved primary hepatocytes for each species (CD-I mouse, Han Wistar rat, Beagle dog, and Human; supplied by BioreclamationlVT). Cryopreserved hepatocytes were stored in liquid nitrogen prior to use.

Williams E media (Sigma Aldrich), supplemented with 2 mM L-glutamine and 25 mM HEPES and test compound (final substrate concentration 3 μΜ; final DMSO concentration 0.25 %) were pre-incubated at 37 °C prior to the addition of a suspension of cryopreserved hepatocytes (final cell density 0.5 x 10⁶ viable cells/mL in Williams E media supplemented with 2 mM L-glutamine and 25 mM HEPES) to initiate the reaction. The final incubation volume was 500 μΙ_. Two control compounds were included for each species. The reaction was stopped by transferring 50 μΙ_ of incubate to 100 μΙ_ methanol containing internal standard at the appropriate time points. The termination plates are centrifuged at 2500 rpm at 4 °C for 30 min to precipitate the protein. Following protein precipitation, the sample supernatants were combined in cassettes of up to 4 compounds and analysed using Cyprotex generic LC-MS/MS conditions. The data for the two positive control compounds were determined to be within the accepted range enabling the assay to fulfill the Cyprotex acceptance criteria. Intrinsic clearance was calculated by non-linear regression analysis of peak area ratio vs time using the LINEST function in Excel to determine the first order elimination rate constant (k), from which the half life (ti ₂; minutes), intrinsic clearance (mL/min/g of liver) and scaled intrinsic clearance (scaled CLint; mL/min/kg) were then derived. To scale the hepatocyte clearance, a hepatocyte yield of 1.2 x 10⁸ cells per gram liver in human, rat and mouse and 1.7 x 10⁸ cells per gram liver in the dog were used.

Results: Example 1 was metabolically stable (below the assay lower limit of quantification) in mouse, rat, dog and human hepatocytes in the presence of NADPH, indicating low turnover across species (Table 5). Table 5. Hepatocyte intrinsic clearance for Example 1 in multiple species

All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. For all studies, the temperature and humidity were nominally maintained at 21°C ± 2°C and 55% ± 10%, respectively. For intravenous administration, Example 1 was formulated in DMSO and 10% (w/v) Kleptose™ in saline (2:98) at a concentration of 0.2 mg/mL (rat) and 0.1 mg/mL (dog). The dose was filtered using a ca. 0.2 μιτι syringe filter unit. Example 1 was administered as a 1 h iv infusion at 5 mL/kg/h to achieve a target dose of 1 and 0.5 mg/kg in the rat and dog respectively. For oral administration, Example 1 was formulated in 1% (w/v) methylcellulose (400 cps) (aq) at a concentration of 0.6 mg/mL (mouse and rat) and 0.3 mg/mL (dog). Example 1 was administered at 5 mL/kg to achieve a target dose of 3 mg/kg (mouse and rat) and 1.5 mg/kg (dog). The diet for rodents was 5LF2 Eurodent Diet 14% (PMI Labdiet, Richmond, Indiana) and for dogs was Harlan Teklad 2021C (HarlanTeklad, Madison, WI). There were no known contaminants in the diet or water at concentrations that could interfere with the outcome of the studies.

Rat pharmacokinetic study

Male Wistar Han rats (247-271 g, supplied by Charles River UK Ltd.) were surgically prepared under anaesthesia at GSK with implanted cannulae in the femoral vein (for drug administration) and jugular vein (for blood sampling). Each rat received Duphacillin (100 mg/kg s.c.) and Carprofen (7.5 mg/kg s.c.) as a pre-operative antibiotic and analgesic respectively. Each rat was allowed to recover from surgery for at least 2 days prior to dosing. Rats had free access to food and water throughout. Rat PK studies were conducted as a crossover design over 3 dosing occasions, with a minimum of 4 days between dose administrations. On dosing day 1, n=3 male rats each received a 1 h intravenous infusion of the test compound. On dosing day 2, the same three rats each received an oral administration of test compound suspended in 1% (w/v) methylcellulose aq. at a concentration of 0.6 mg/mL administered by gavage at 5 mL/kg to achieve a target dose of 3 mg/kg. On dosing day 3, the same three rats each received an oral administration of test compound suspended in 1% (w/v) methylcellulose aq. at a concentration of 6.0 mg/mL administered by gavage at 5 mL/kg to achieve a target dose of 30 mg/kg. Following dose administration, serial blood samples (ca. 60 μί) were collected up to 26 h after the start of dosing, via the jugular vein cannula.

Dog pharmacokinetic study

One healthy, laboratory-bred male Beagle dog (12.71 kg, supplied by Harlan Laboratories, UK) was fasted overnight prior to each dose administration and fed approximately 4 h after the start of dosing. The dog had free access to water throughout. The dog study was conducted as a crossover design on two dosing occasions with 6 days between dose administrations. On dosing day 1, n=l male dog received a 1 h intravenous infusion of Example 1 via temporary cannulation of the saphenous vein. On dosing day 2, the same dog received an oral administration of Example 1 suspended in 1% (w/v) methylcellulose aq. at a concentration of 0.3 mg/mL and administered by gavage at 5 mL/kg to achieve a target dose of 1.5 mg/kg. A temporary cannula was inserted into the cephalic vein from which serial blood samples (ca. 150 μΙ_) were collected from predose up to 2 h after the start of dosing. After the 2 h sample had been taken remaining samples were taken via direct venepuncture of the jugular vein. Mouse pharmacokinetic study

Three male CD1 mice (26.3-28.9 g, supplied by Charles River UK Ltd.) were used. Each mouse received an oral administration of Example 1 suspended in 1% (w/v) methylcellulose 400 aq. at a concentration of 0.6 mg/mL and administered by gavage at 5 mL/kg to achieve a target dose of 3 mg/kg. Following dose administration, serial blood samples (ca. 20 μΙ_) were collected up to 24 h after the start of dosing, via direct venepuncture of a tail vein.

For all PK studies serial blood samples were collected over an appropriate time course post administration, into tubes containing potassium EDTA as anti coagulant (rat and dog) and subaliquoted into a micronics tube or directly into a micronic tube (mouse). The aliquoted blood sample was then diluted with an equal volume of purified water. Samples were stored at -20°C prior to analysis by LC-MS/MS.

Blood Sample Analysis

Diluted blood samples were extracted using protein precipitation with 300 μΙ_ of acetonitrile containing an analytical internal standard. An aliquot of the supernatant was analysed by reverse phase LC MS/MS using a heat assisted electrospray interface in positive ion mode. Samples were assayed against calibration standards prepared in control blood and the assay fulfilled the in house good scientific practice acceptance criteria.

Rat and Dog Urine Sample Analysis

Urine samples (Rat; 25 μΙ_, Dog; 50 μΙ_) were mixed with an equal volume of 50:50 acetonitrile:water followed by 300 μΙ_ of acetonitrile containing an analytical internal standard. Samples were assayed against calibration standards prepared in control urine and the assay fulfilled the in house good scientific practice acceptance criteria. Pharmacokinetic data analysis from pharmacokinetic studies

Pharmacokinetic parameters were obtained from the blood concentration time profiles using non-compartmental analysis with WinNonlin Phoenix 6.3 (Pharsight, Mountain View, CA). The extrapolated AUCt-oovalues were < 10% of the AUCoo.

Results: The pharmacokinetics of Example 1 were assessed in the male CD-I mouse, male Wistar Han rat, and male Beagle dog following intravenous (iv infusion) and/or oral administration of Example 1. Following iv administration, Example 1 had low total blood clearance in the rat and dog (18 and 2 mL/min/kg respectively) and a moderate volume of distribution (2.3 and 0.9 L/kg respectively). Renal clearance of Example 1 was observed in both rat and dog, with 27% and 26% of the administered dose recovered as parent drug in the urine, respectively. The calculated terminal half-life was moderate at 3.8 and 5.3 h in rat and dog respectively. Following oral administration of Example 1 as a suspension to rat and dog, absorption was rapid with a Tmax of 0.5 - 1 h coupled with a high oral bioavailability of 95% in rat (3 mg/kg) and 101% in dog (1.5 mg/kg). Oral administration at 30 mg/kg in the rat resulted in a linear increase in exposure and a high bioavailability of 93%. Following oral administration of Example 1 as a suspension to the mouse at 3 mg/kg, systemic exposure was observed with the area under the curve (AUC) similar to that observed in the rat at the same dose level (3 mg/kg).

Claims

1. A compound of formula (I) which is 5-[l-(l,3-dimethoxypropan-2-yl)-5- (morpholin-4-yl)-lH-l,3-benzodiazol-2-yl]-l,3-dimethyl-l,2-dihydropyridin-2-one, of formula (I):

or a salt thereof.

2. A compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1, which is in the form of a free base.

4. A compound according to claim 3, which is in crystalline solid state form characterised by an X-ray powder diffraction pattern having diffraction peaks at 2Θ values of 8.2, 8.6, 11.0, 12.5, 13.0, 14.0, 16.3, 17.1, 18.5, 22.0, 23.7, and 26.7 (± 0.10° 2Θ experimental error).

5. A pharmaceutical composition which comprises a compound or a pharmaceutically acceptable salt thereof as defined in any of claims 2 to 4, and one or more pharmaceutically acceptable excipients.

6. A pharmaceutical composition according to claim 5, further comprising one or more other therapeutically active agents.

7. A compound or a pharmaceutically acceptable salt thereof as defined in any of claims 2 to 4 for use in therapy.

8. A compound or a pharmaceutically acceptable salt thereof as defined in any of claims 2 to 4 for use in the treatment of a disease or condition for which a BET inhibitor is indicated.

9. A compound or a pharmaceutically acceptable salt thereof for use according to claim 8, wherein the disease or condition is an autoimmune and/or inflammatory disease.

10. A compound or a pharmaceutically acceptable salt thereof for use according to claim 8, wherein the disease or condition is rheumatoid arthritis.

11. A method of treatment of a disease or condition for which a BET inhibitor is indicated, which method comprises administering to a subject in need thereof a therapeutically effective amount of a compound, or pharmaceutically acceptable salt thereof, as defined in any of claims 2 to 4.

12. A method of treatment according to claim 11, wherein the disease or condition for which a BET inhibitor is indicated is an autoimmune and/or inflammatory disease.

13. A method of treatment according to claim 11, wherein the disease or condition for which a BET inhibitor is indicated in rheumatoid arthritis.

14. The use of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of an autoimmune and/or inflammatory disease.

15. The use according to claim 14, wherein the disease is rheumatoid arthritis.