WO2015049667A1 - Novel compound as a diacylglycerol acyltransferase inhibitor - Google Patents

Abstract

This invention relates to a novel compound which is an inhibitor of acyl coenzyme A: diacylglycerol acyltransferase 1 (DGAT1), to pharmaceutical compositions containing it, to processes for its preparation, and to its use in therapy, alone or in combination with weight management therapies or other triglyceride lowering therapy, for the prevention or treatment of diseases related to DGAT1 dysfunction or where modulation of DGAT1 activity may have therapeutic benefit including but not limited to obesity, obesity related disorders, genetic (Type 1, Type 5 hyperlipidemia) and acquired forms of hypertriglyceridemia or hyperlipoproteinemia- related disorders, caused by but not limited to lipodystrophy, hypothyroidism, medications (beta blockers, thiazides, estrogen, glucocorticoids, transplant) and other factors (pregnancy, alcohol intake), hyperlipoproteinemia, chylomicronemia, dyslipidemia, non-alcoholic steatohepatitis, diabetes, insulin resistance, metabolic syndrome, cardiovascular outcomes, angina, excess hair growth (including syndromes associated with hirsutism), nephrotic syndrome, fibrosis such as myocardial, renal and liver fibrosis, hepatitis C virus infection and acne or other skin disorders.

Description

NOVEL COMPOUND AS A DIACYLGLYCEROL AC YL TRANSFERASE INHIBITOR

FIELD OF INVENTION

This invention relates to a novel compound which is an inhibitor of acyl coenzyme A: diacylglycerol acyltransferase 1 (DGATl), to pharmaceutical compositions containing it, to processes for its preparation, and to its use in therapy, alone or in combination with weight management therapies or other triglyceride lowering therapy, for the prevention or treatment of diseases related to DGATl dysfunction or where modulation of DGATl activity may have therapeutic benefit including but not limited to obesity, obesity related disorders, genetic (Type 1, Type 5 hyperlipidemia) and acquired forms of hypertriglyceridemia or hyperlipoproteinemia- related disorders, caused by but not limited to lipodystrophy, hypothyroidism, medications (beta blockers, thiazides, estrogen, glucocorticoids, transplant) and other factors (pregnancy, alcohol intake), hyperlipoproteinemia, chylomicronemia, dyslipidemia, non-alcoholic steatohepatitis, diabetes, insulin resistance, metabolic syndrome, cardiovascular outcomes, angina, excess hair growth (including syndromes associated with hirsutism), nephrotic syndrome, fibrosis such as myocardial, renal and liver fibrosis, hepatitis C virus infection and acne or other skin disorders.

BACKGROUND OF THE INVENTION

Obesity is a medical condition that is reaching epidemic proportions among humans in a number of countries throughout the world. It is a condition that is also associated with or induces other diseases or conditions that disrupt life activities and lifestyles. Obesity is recognized as a serious risk factor for other diseases and conditions such as diabetes, hypertension, and arteriosclerosis. It is also known that increased body weight due to obesity can place a burden on joints, such as knee joints, causing arthritis, pain, and stiffness.

Because overeating and obesity have become such a problem in the general population, many individuals are now interested in losing weight, reducing weight, and maintaining a healthy body weight and desirable lifestyle. One approach to treating obesity is to reduce food intake and/or hyperlipidemia. It has been suggested that molecules which are developed to prevent the accumulation of triglyceride would not only reduce obesity but also have the additional beneficial effect of reducing insulin resistance, a primary factor contributing to the development of diabetes.

Acyl coenzyme A: diacylglycerol acyltransferase 1 (DGATl) is one of two known DGAT enzymes that catalyze the final step in mammalian triglyceride synthesis. DGATl is an enzyme that is implicated in the development of both diabetes and insulin resistance. Studies of DGATl deficient mice show that DGATl deficiency protects against insulin resistance and obesity, see Chen, H.C. et al., J Clin Invest.. 109(8), 1049-1055 (2002). Therefore, inhibitors of DGATl should be useful for the treatment of metabolic disorders, e.g. obesity, Type 2 diabetes, and insulin resistance syndrome (or metabolic syndrome) and other associated or related diseases and conditions.

SUMMARY OF THE INVENTION

This invention relates to 4-amino-6-(l-(2-hydroxy-2-methylpropyl)indolin-5-yl)-7,8- dihydropyrimido[5,4- ][l,4]o la (I):

(I),

or pharmaceutically acceptable salts thereof.

This invention also relates to a pharmaceutical composition comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

This invention also relates to a method of treating obesity comprising administering to a human in need thereof an effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

DETAIL DESCRIPTION OF THE INVENTION

A person of ordinary skills in the art recognizes that the compound of the present invention may have alternative names when different naming software is used.

This invention also relates to the compound of Formula (I), or pharmaceutically acceptable salts thereof, for use in therapy. In particular, for use in the treatment of diseases mediated by Acyl coenzyme A: diacylglycerol acyltransferase 1 (DGATl), such as obesity, obesity related disorders, genetic (Type 1, Type 5 hyperlipidemia) and acquired forms of hypertriglyceridemia or hyperlipoproteinemia-related disorders, hyperlipoproteinemia, chylomicronemia, dyslipidemia, non-alcoholic steatohepatitis, diabetes, insulin resistance, metabolic syndrome, cardiovascular outcomes, angina, excess hair growth (including syndromes associated with hirsutism), nephrotic syndrome, fibrosis such as mycocardial, renal and liver fibrosis, hepatitis C virus infection and acne or other skin disorders. In particular, this invention relates to the compound of Formula (I), or pharmaceutically acceptable salts thereof, for use in the treatment of obesity.

This invention also relates to the compound of Formula (I), or pharmaceutically acceptable salts thereof, for use as a medicament. This invention also relates to the compound of Formula (I), or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for the treatment of obesity.

Typically, but not absolutely, the salts of the present invention are pharmaceutically acceptable salts. Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compound of this invention. Salts of the disclosed compound may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid,

methanesulfonic acid, ethanesulfonic acid or the like. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates,

dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, phenylacetates,

phenylpropionates, phenylbutrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates mandelates, and sulfonates, such as xylenesulfonates, methanesulfonates, propanesulfonates, naphthalene- 1-sulfonates and naphthalene-2-sulfonates.

Salts of the disclosed compound can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N^-dibenzylethylenediamine, 2- hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine,

dibenzylpiperidine, dehydroabietylamine, N,N-bisdehydroabietylamine, glucamine, N- methylglucamine, collidine, choline, quinine, quinoline, and basic amino acid such as lysine and arginine. Other salts, which are not pharmaceutically acceptable, may be useful in the preparation of compounds of this invention and these should be considered to form a further aspect of the invention. These salts, such as oxalic or trifluoroacetate, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts.

The compound of Formula (I) may exist in solid or liquid form. In the solid state, it may exist in crystalline or noncrystalline form, or as a mixture thereof. The skilled artisan will appreciate that pharmaceutically acceptable solvates may be formed for crystalline or noncrystalline compounds. In crystalline solvates, solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve non-aqueous solvents such as, but not limited to, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent incorporated into the crystalline lattice are typically referred to as "hydrates." Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The invention includes all such solvates.

The skilled artisan will further appreciate that crystalline forms of the compound of the invention, including the various solvates thereof, may exhibit polymorphism (i.e. the capacity to occur in different crystalline structures). These different crystalline forms are typically known as "polymorphs." The invention includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which may be used for identification. The skilled artisan will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents, used in making the compound. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, one polymorph may spontaneously convert to another polymorph under certain conditions.

The subject invention also includes isotopically-labelled compounds, which are identical to the compound of Formula (I), but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compound of the invention and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine, iodine, and chlorine, such as H, H, C, C, C, N, O, ¹⁸0, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶C1, ¹²³I, and ¹²⁵I. Compounds of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically -labelled compounds of the present invention, for example those into which radioactive isotopes such as ³H or ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. ^UC and ¹⁸F isotopes are particularly useful in PET (positron emission tomography), and ¹²⁵I isotopes are particularly useful in SPECT (single photon emission computerized tomography), all useful in brain imaging. Further, substitution with heavier isotopes such as deuterium, i.e., ²H, can 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. An isotopically labelled compound of Formula (I) can generally be prepared by carrying out the procedures disclosed in the Examples below, by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

PHARMACEUTICAL COMPOSITIONS

The invention further provides a pharmaceutical composition (also referred to as pharmaceutical formulation) comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more excipients (also referred to as carriers and/or diluents in the pharmaceutical arts). The excipients are pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof (i.e., the patient).

In accordance with another aspect of the invention there is provided a process for the preparation of a pharmaceutical composition comprising mixing (or admixing) the compound of Formula (I) or a pharmaceutically acceptable salt thereof, with at least one excipient.

Pharmaceutical compositions may be in unit dose form containing a predetermined amount of active ingredient per unit dose. Such a unit may contain a therapeutically effective dose of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, or a fraction of a therapeutically effective dose such that multiple unit dosage forms might be administered at a given time to achieve the desired therapeutically effective dose. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Furthermore, such pharmaceutical compositions may be prepared by any of the methods well-known in the pharmacy art.

Pharmaceutical compositions may be adapted for administration by any appropriate route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes. 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).

When adapted for oral administration, pharmaceutical compositions may be in discrete units such as tablets or capsules, powders or granules, solutions or suspensions in aqueous or nonaqueous liquids, edible foams or whips, oil-in-water liquid emulsions or water-in-oil liquid emulsions. The compound or salt thereof of the invention or the pharmaceutical composition of the invention may also be incorporated into a candy, a wafer, and/or tongue tape formulation for administration as a "quick-dissolve" medicine.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Powders or granules are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agents can also be present.

Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin or non-gelatinous sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, 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 medicine when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants, 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, 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, without limitation, starch, methylcellulose, 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, and 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 a binder such as a 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 a tablet machine, resulting in 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 or salt of the present invention can also be combined with a free-flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear 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 dosages.

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

Where appropriate, dosage unit formulations for oral administration can be

microencapsulated. The formulation can also be prepared to prolong or sustain the release as, for example, by coating or embedding particulate material in polymers, wax, or the like.

In the present invention, tablets and capsules are preferred for delivery of the

pharmaceutical composition.

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

The present invention provides a method of treatment in a mammal, especially a human, suffering from obesity, diabetes, hypertension, depression, anxiety, drug addiction, substance addiction, or a combination thereof. Such treatment comprises the step of administering a therapeutically effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, to said mammal, particularly a human. Treatment can also comprise the step of administering a therapeutically effective amount of a pharmaceutical composition containing the compound of Formula (I) or a pharmaceutically acceptable salt thereof, to said mammal, particularly a human.

As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician.

The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For use in therapy, therapeutically effective amounts of the compound of Formula (I), as well as salts thereof, may be administered as the raw chemical.

Additionally, the active ingredient may be presented as a pharmaceutical composition. While it is possible that, for use in therapy, a therapeutically effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, may be administered as the raw chemical, it is typically presented as the active ingredient of a pharmaceutical composition or formulation.

The precise therapeutically effective amount of the compound or salt thereof of the invention will depend on a number of factors, including, but not limited to, the age and weight of the subject (patient) being treated, the precise disorder requiring treatment and its severity, the nature of the pharmaceutical formulation/composition, and route of administration, and will ultimately be at the discretion of the attending physician or veterinarian. Typically, the compound of Formula (I) or a pharmaceutically acceptable salt thereof, will be given for the treatment in the range of about 0.1 to 100 mg/kg body weight of recipient (patient, mammal) per day and more usually in the range of 0.1 to 10 mg/kg body weight per day. Acceptable daily dosages may be from about 0.1 to about 1000 mg/day, and preferably from about 1 to about 100 mg/day. This amount may be given in a single dose per day or in a number (such as two, three, four, five, or more) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt thereof may be determined as a proportion of the effective amount of the compound of Formula (I) per se. Similar dosages should be appropriate for treatment of the other conditions referred herein for treatment. In general, determination of appropriate dosing can be readily arrived at by one skilled in medicine or the pharmacy art.

Additionally, the present invention provides the use of the compound of the invention in combination with weight management therapies or other triglyceride lowering therapy. In particular, the present invention provides a combination of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof with at least one other therapeutically active agent, including another anti-obesity drug and/or an anti-diabetes drug. Such other therapeutically active agent can include, for example, metformin (Glucophage^®), CB1 receptor antagonists, GLP-1 agonists, opioid antagonists, and neurotransmitter reuptake inhibitors. When the compound of the invention is employed in combination with another anti-obesity drug or anti -diabetes drug, it is to be appreciated by those skilled in the art that the dose of each compound or drug of the combination may differ from that when the drug or compound is used alone.

Appropriate doses will be readily appreciated and determined by those skilled in the art. The appropriate dose of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and the other therapeutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect, and are within the expertise and discretion of the attending doctor or clinician.

COMPOUNDS PREPARATION

Unless otherwise noted, reagents are commercially available or are prepared according to procedures in the literature. The symbols and conventions used in the descriptions of processes, schemes, and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry.

In the Examples:

Chemical shifts are expressed in parts per million (ppm) units. Coupling constants (J) are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), dt (double triplet), m (multiplet), br (broad).

Flash column chromatography was performed on silica gel.

The naming program used is ChemDraw^®.

Abbreviations:

CH₃CN acetonitrile

Cs₂C0₃ cesium carbonate

DCM dichloromethane

DMF dimethylformamide

DMSO dimethylsulfoxide

Et₃N triethylamine

g gram(s)

h hour(s) HC1 hydrochloric acid

in z mass to charge ratio

MeMgBr methyl magnesium bromide

MeOH methanol

mmol millimoles

NH₃ ammonia gas

NMR nuclear magnetic resonance

Pd(OAc)₂ palladium acetate

RT room temperature

SOCl₂ thionyl chloride

TBDMS (TBS) fert-butyldimethylsilyl

THF tetrahydrofuran

X-Phos 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl

Example 1 :

Reagents and conditions: a) Ethyl bromoacetate, CS₂CO₃, DMF, RT; b) MeMgBr, THF, 0 °C then RT; c) NH₂(CH₂)₂OTBDMS, Pd(OAc)₂, Cs₂C0₃, X-Phos, Toluene, 110 °C; d) 4,6- dichloropyrimidine-5-carboxylic acid, SOCl₂, reflux; then 1C, DCM, Et₃N, RT; e) HC1, MeOH, RT; f) CH₃CN, Et₃N, 80 °C; g) NH₃, Dioxane, RT.

Procedures

4-Amino-6-(l-(2-hvdroxy-2-methylpropynindolin-5-vn-7.,8-dihvdropyriniido[5.,4- f\ [l,41oxazepin-5(6H)-one

Ethyl 2-(5-bromoindolin-l-v0acetate (1A)

1A Ethyl bromoacetate (4.05 g, 24.24 mmol) was added to a solution of 5-bromo-lH-indole (4 g, 20.2 mmol) in DMF (30 mL) followed by cesium carbonate (13.16 g, 40.4 mmol), and the mixture was stirred at room temperature for 12 h. Insoluble solids were filtered, the filtrate was concentrated, and the residue was partitioned between diethyl ether and water. Separated organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the title compound (2.4 g, 41.8%) as a brown oil. ¾ NMR (400 MHz, DMSO- d₆y. 5 7.15 (s, 1H), 7.07 (dd, J_x = 2.0 Hz, J₂ = 8.4 Hz, 1H), 6.39 (d, J= 8.0 Hz, 1H), 4.1 (q, J= 6.8 Hz, 2H), 4.01 (s, 2H), 3.47 (t, J= 8.8 Hz, 2H), 2.94 (t, J= 8.4 Hz, 2H), 1.18 (t, J= 6.8 Hz, 3H). l-(5-Bromoindolin-l-vO-2-methylpropan-2-ol (IB)

1 B

Methyl magnesium bromide (3M diethyl ether solution) (11.26 mL, 33.8 mmol) was added to an ice-cold solution of ethyl 2-(5-bromoindolin-l-yl)acetate (2.4 g, 8.45 mmol) in THF (60 mL) dropwise, and the mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with aqueous 2N HC1 and partitioned between ethyl acetate and water. Separated organic layer was dried over sodium sulphate and filtered, and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography using 8% ethyl acetate in hexanes to afford the title compound (1.4 g, 61.4%) as a brown oil. Ti NMR (300 MHz, DMSO- ₆): δ 7.10 (s, 1H), 7.05 (m, 1H), 6.4 (d, J= 8.4 Hz, 1H), 4.38 (s, 1H), 3.5 (t, J= 8.7 Hz, 2H), 2.91 (m, 4 H), 1.18 (s, 6H). ESI- MS m/z = 270 (M+H)⁺. l-(5-(2-(terf-Butyldimethylsilyloxy)ethylamino)indolin-l-yl)-2-methylpropan-2-ol (1C)

Palladium acetate (0.116 g, 0.518 mmol) and X-Phos (0.247 g, 0.518 mmol) were added to a solution of l-(5-bromoindolin-l-yl)-2-methylpropan-2-ol (1.4 g, 5, 18 mmol), 2- tert- butyldimethylsilyloxy)ethanamine (1.09 g, 6.22 mmol) and cesium carbonate (3.38 g, 10.36 mmol) in toluene (30 mL) under argon atmosphere. The mixture was then degassed for 15 min and stirred at 110 °C for 4 h. The reaction mixture was filtered through a pad of celite, and the filtrate was concentrated in vacuo. The crude sample was purified by flash chromatography using 12% ethyl acetate in hexanes to afford the title compound (0.8 g, 42.3%) as a brown oil. ESI-MS m/z = 365 (M+H)⁺.

A^r-(2-(ter^Butyldimethylsilyloxy)ethvn-4.,6-dichloro-jV-(l-(2-hvdroxy-2-methylpropynindolin-

5-yl)pyrimidine-5-carboxamide (ID)

A solution of 4,6-dichloropyrimidine-5-carboxylic acid (0.508 g, 2.63 mmol) in thionyl chloride (12 mL, 164 mmol) was refluxed for 3 h and was then concentrated. 4,6- Dichloropyrimidine-5-carbonyl chloride thus obtained was dissolved in DCM (8 mL) and was then added dropwise to an ice-cold solution of l-(5-(2-(teri-butyldimethylsilyloxy)ethylamino)indolin- l-yl)-2-methylpropan-2-ol (0.8 g, 2.194 mmol) and triethylamine (1.223 mL, 8.78 mmol) in DCM (15 mL), and the mixture was stirred for 2 h. The reaction mixture was concentrated in vacuo, and the residue was partitioned between dichloromethane and water. The separated organic layer was dried over sodium sulphate and filtered. The filtrate was concentrated in vacuo and purified by flash chromatography using 16% ethyl acetate in hexanes to afford the title compound (0.64 g, 54.1%) as a brown oil. ¾ NMR (300 MHz, DMSO-t/₆): δ 8.8 (s, 1H), 7.03 (s, 1H), 6.95 (dd, J_x= 2.1 Hz, J₂= 8.4 Hz, 1H), 6.3 (d, J= 8.4 Hz, 1H), 4.34 (s, 1H), 3.83 (m, 2H), 3.56 (m, 2H), 3.48 (t, J = 8.7 Hz, 2H), 2.83(m, 4H), 1.07 (s, 6H), 0.85 (s, 9H), 0.01 (s, 6H). ESI-MS m/z = 539 (M+H)⁺.

4,6-Dichloro-jV-(l-(2-hvdroxy-2-methylpropyl)indolin-5-yl)-jV-(2-hvdroxyethyl)pyrimidine-5- carboxamide (IE)

A solution of N-(2-(teri-butyldimethylsilyloxy)ethyl)-4,6-dichloro-N-(l-(2-hydroxy-2- methylpropyl)indolin-5-yl)pyrimidine-5-carboxamide (0.62 g, 1.149 mmol) in methanolic solution of HQ (0.3 mL of HC1 in 10 mL of MeOH) was stirred at room temperature for 0.33 h. Methanol was removed in vacuo, the residue was dissolved in ethyl acetate, and washed with saturated aqueous sodium bicarbonate and brine. The separated organic layer was dried over sodium sulfate and filtered, and the filtrate was concentrated in vacuo to afford the title compound (0.34 g, 69.6%) as a pale yellow solid. ^lH NMR (300 MHz, DMSO- g): δ 8.79 (s, 1H), 7.03 (s, 1H), 6.94 (m, 1H), 6.31 (d, J= 8.4 Hz, 1H), 4.74 (t, J= 5.4 Hz, 1H), 4.34 (s, 1H), 3.78 (t, J= 6.3 Hz, 2H), 3.56 (q, J = 6.3 Hz, 2H), 3.48 (t, J= 8.4 Hz, 2H), 2.83 (m, 4H), 1.08 (s, 6H). ESI-MS m/z = 425 (M+H)⁺.

4-Chloro-6-(l-(2-hvdroxy-2-niethylpropynindolin-5-yl)-7.,8-dihvdropyrimido[5.,4- mi,41oxazepin-5(6H)-one (IF)

A solution of 4,6-dichloro-N-(l-(2-hydroxy-2-methylpropyl)indolin-5-yl)-N-(2- hydroxyethyl)pyrimidine-5-carboxamide (0.34 g, 0.799 mmol) and triethylamine (0.446 mL, 3.2 mmol) in acetonitrile (20 mL) was stirred at 80 °C for 6 h. The reaction mixture was cooled, concentrated in vacuo and partitioned between ethyl acetate and water. The separated organic layer was dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo to afford the title compound (0.23 g, 74%) as a pale yellow solid. ¾ NMR (400 MHz, DMSO-t/₆): 5 : 8.8 (s, 1H), 7.01 (s, 1H), 6.96 (dd, J_x = 1.5 Hz, J₂ = 6.3 Hz, 1H), 6.49 (d, J= 6.6 Hz, 1H), 4.68 (t, J= 1.8 Hz, 2H), 4.4 (s, 1H), 4.01 (t, J= 3.9 Hz, 2H), 3.54 (t, J= 6 Hz, 2H), 2.94 (m, 4H), 1.16 (s, 6H). ESI- MS m/z = 389 (M+H)⁺.

4-Amino-6-(l-(2-hvdroxy-2-methylpropyl)indolin-5-yl)-7,8-dihvdropyrimido[5,4- f] [l,41oxazepin-5(6H)-one

Ammonia gas was passed through a solution of 4-chloro-6-(l-(2-hydroxy-2- methylpropyl)indolin-5-yl)-7,8-dihydropyrimido[5,4- |[l,4]oxazepin-5(6H)-one (0.23 g, 0.59 mmol) in 1,4-dioxane (10 mL) at room temperature for 4 h. The reaction mixture was concentrated in vacuo and partitioned between chloroform and water. The separated organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated in reduced pressure, and the residue was triturated with ethyl acetate and «-pentane. The resulting solid was filtered through a Buchner funnel, washed with «-pentane, and dried to afford the title compound (0.13 g, 56%) as an off-white solid. i NMR ^OO MHz, DMSO-dg): δ 8.15 (s, 1H), 7.57 (bs, 2H), 6.95 (s, 1H), 6.89 (d, J= 8.4 Hz, 1H), 6.45 (d, J = 8.1 Hz, 1H), 4.57 (t, J= 4.8 Hz, 2H), 4.38 (s, 1H), 3.86 (t, J= 4.2 Hz, 2H), 3.52 (t, J= 8.4 Hz, 2H), 2.92 (m, 4H), 1.15 (s, 6H). ESI-MS m/z = 370 (M+H)⁺.

Biological Assays Inhibition of human DGAT1 activity in vitro

Human DGAT1 was expressed in Sf9 insect cells using a baculovirus expression system. Microsomes were prepared and used as enzyme for in vitro inhibition testing. Fluorescence based and radioactive based assay formats were developed to measure the formation of the co-product, thiolated coenzyme A or triacylglycerol product, respectively. All steps were performed at 21- 23 °C. All data for DGAT1 inhibition by test compounds were collected under conditions where product formation was linear with reaction time.

CPM assay: For inhibition of thiolated CoA co-product formation, test compounds were prepared in 100% DMSO, diluted into assay buffer, and 10 uL was added to 96-well half-area plates (Greiner 675076). An equal volume (10 uL) of 3X enzyme in buffer was added and the components incubated for 30 minutes pre-reaction incubation to allow enzyme and test compounds to attain binding equilibrium. As an experimental control, 30 uM of {4-[4-(4-amino-7,7-dimethyl- 7H-pyrimido[4,5-b][l,4]oxazin-6-yl)phenyl]cyclohexyl}acetic acid was added to determine the 100% inhibition. Some assays were performed with inclusion of didecanoylglycerol in the pre- reaction incubation of test compound and enzyme. DGAT reactions (30 uL) were initiated upon addition of 10 uL of 3X substrate solution. Final reaction conditions consisted of 20 mM HEPES pH 7.5, 2 mM MgCl₂, 1 mM CHAPS, 50 uM didecanoylglycerol, 3 uM decanoyl-CoA, 1 ug/mL microsomal protein, and 0.7% DMSO. Following a 60 minute reaction incubation, reactions were stopped and thiolated-CoA product derivatized with 30 uL of buffer containing 10 uM of {4-[4-(4- amino-7,7-dimethyl-7H-pyrimido[4,5-b][l,4]oxazin-6-yl)phenyl]cyclohexyl}acetic acid and 50 uM of 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Fluorescence was read using Envision reader at Ex 405 nm/Em 480 nm about 30 minutes after addition of final solution. Inhibition was normalized to controls containing 0.7% DMSO or 10 uM of {4-[4-(4-amino-7,7- dimethyl-7H-pyrimido[4,5-b][l,4]oxazin-6-yl)phenyl]cyclohexyl}acetic acid. IC₅₀s of compounds were determined using biostatistics software GraphPad prsim by analyzing the data using non- linear regression (curve fit), sigmoidal dose response analysis (variable slope). LE assay: For inhibition of triacylglycerol product formation, 11 uL reactions were run in white Polyplate-384 (PerkinElmer6007300) starting with a 30 minute pre-reaction incubation of 5 uL of 2.2X enzyme and 1 uL of 100% DMSO containing test compound or control compound, {4- [4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][l,4]oxazin-6-yl)phenyl]cyclohexyl}acetic acid. Some assays were performed with inclusion of didecanoylglycerol in the pre-reaction incubation of test compounds and enzyme. Reactions were initiated after 30 minute pre-reaction incubation via addition of 5 uL of 2.2X substrate. Final reaction conditions consisted of 50 mM HEPES pH 7.5, 2 mM MgCl₂, 1 mM CHAPS, 25 uM didecanoylglycerol, 0.5 uM decanoyl-CoA, 0.3 nCi/uL [14C]- decanoyl-CoA or 5.5 nCi/uL [3H]-decanoyl-CoA, 0.05-4 ug/mL microsomal protein, and 0.7% DMSO. Following 60 minute reaction incubation, reactions were stopped with 40 uL of 45% isopropanol and 50 mM sodium carbonate in water and mixed. Extraction of triacylglycerol product was accomplished via addition of 30 uL Microscint-E (Perkin Elmer) and 2 hours of incubation (sealed). Plates were read on a Microbeta Microplate reader. Inhibition was normalized to controls containing DMSO or 10 uM of {4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5- b][l,4]oxazin-6-yl)phenyl]cyclohexyl}acetic acid. IC50S of compounds were determined using biostatistics software GraphPad prsim by analyzing the data using non-linear regression (curve fit), sigmoidal dose response analysis (variable slope).

Pharmacokinetic assay in Rats (Rat IV PK)

Male Crl:CD (SD) rats were housed individually in cages with clean bedding in a controlled environment (22 to 25° C temperature, humidity of 30-70 % RH, and a 12 hour light/12 hour dark cycle). Certified rodent diet was provided ad libitum to the rats. Rats were fasted overnight 14-16 hours prior to the dosing on each study day and fed approximately 4 hours post dose (following collection of the 4 hour blood sample). Water was available ad libitum. Normal healthy rats certified by the attending veterinarian were selected and acclimatized for a minimum of three days prior to initiation of the study. Rats were randomized according to the body weight and were identified with body markings.

Intravenous formulation of 1 mg/mL concentration was prepared in DMA (N,N- Dimethylacetamide):TEG (Tetraethylene glycol): Water for Injection (20:40:40) freshly on study day. Animals were dosed through tail vein to achieve a target dose of 1 mg/kg. The actual dose volume was based on the body weight obtained on the study day, which was documented in the dosing sheet. Blood samples (approx. 300 μΕ/ΐηηε point) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after intravenous bolus dose and transferred to a pre-labeled K2EDTA coated tubes. Blood volume was replaced with administration of equal volume of saline through the jugular vein. After collection of blood samples at each time point, the blood samples were stored immediately on ice and centrifuged at 2320 g at 4 °C for 15 minutes. The plasma was separated and transferred to pre-labeled micro-centrifuge tubes and promptly frozen at -80 ± 10 °C until bioanalysis.

The plasma concentrations were determined by using a fit for purpose analytical method based on protein precipitation, followed by LC/MS/MS analysis. Analyst Version 1.5.1 software was used to acquire and quantify data. Pharmacokinetic analysis of plasma concentration-time data was performed by a non-compartmental method using Pheonix WinNonLin™ (WLN) Version 6.3. The area under the plasma concentration-time curve (AUC) from the time of dosing to the last quantifiable time point (AUCo-t) was determined by WLN using linear logarithmic trapezoidal rule. Terminal half life Tm), was determined by WLN using linear logarithmic trapezoidal rule for plasma concentrations following intravenous administration.

Biological Data

Example 1 was tested in human DGAT1 lipid extraction (LE) assay and demonstrated an average IC₅₀ of 37.6 nM (n=6).

Pharmacokinetics properties are one of the critical parameters in determining the success of drug discovery. (See Lin, J. H.; Lu, A. Y. H. "Role of Pharmacokinetics and Metabolism in Drug Discovery and Development". Pharmacological Reviews, 1997, 49, 403.) Most drugs are administrated at a given dose and duration to achieve a pharmacological benefit. In general, compounds with a long plasma half life (T_{1 2}) and high systemic exposure (AUC) will result in less frequent dosing (generally improves patient compliance) and lower doses (potentially reduces toxicity). Conversely, compounds with short plasma Ti_/2 and low AUC will result in more frequent dosing (generally worse patient compliance) and higher doses (potentially increased toxicity).

Comparison data of the compound of Example 1 with two Examples from International Patent Application Publication No. WO 12/ 162129 (Examples 49 and 51) in Rat IV PK (T_1/2 and AUC) are shown in Table 1.

Table 1

Claims

Claims:

1. A compound which is 4-amino-6-(l-(2-hydroxy-2-methylpropyl)indolin-5-yl)-7,8- dihydropyrimido[5,4- ][l,4]oxazepin-5(6H)-one, represented by Formula (I):

(I),

or a pharmaceutically acceptable salt thereof.

2. A compound which is 4-amino-6-(l-(2-hydroxy-2-methylpropyl)indolin-5-yl)-7,^ dihydropyrimido[5,4- |[l,4]oxazepin-5(6H)-one, represented by Formula (I):

(I).

3. A pharmaceutical composition comprising the compound or pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable excipient.

4. A pharmaceutical composition comprising the compound according to claim 2 and a pharmaceutically acceptable excipient.

5. A method of treating obesity comprising administering to a human in need thereof an effective amount of the compound or pharmaceutically acceptable salt according to claim 1.

6. A method of treating obesity comprising administering to a human in need thereof an effective amount of the compound according to claim 2.

7 The compound or pharmaceutically acceptable salt according to claim 1 for use in therapy.

8 The compound according to claim 2 for use in therapy.

9 The compound or pharmaceutically acceptable salt according to claim 1 for use in the treatment of obesity.

10. The compound according to claim 2 for use in the treatment of obesity.

11. The use of the compound or pharmaceutically acceptable salt according to claim 1 in the preparation of a medicament for use in the treatment of obesity.

12. The use of the compound according to claim 2 in the preparation of a medicament for use in the treatment of obesity.