EA045908B1 - COMBINATIONS FOR TREATMENT OF NASH/NAFLD AND RELATED DISEASES - Google Patents

Description

Field of invention

The invention relates to new pharmaceutical compositions containing (S)-2-(5-((3ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or its a pharmaceutically acceptable salt and in the same or a separate composition containing 4-(4-(1isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)- 6-methoxypyridin-2yl)benzoic acid or a pharmaceutically acceptable salt thereof, for the treatment of non-alcoholic fatty liver disease (NAFLD) and related diseases, such as non-alcoholic steatohepatitis (NASH) and metabolic diseases.

Prior Art

Non-alcoholic steatohepatitis (NASH) is a clinical and histological subset of non-alcoholic fatty liver disease (NAFLD, defined by the presence of >5% hepatic steatosis) that is associated with increased overall mortality from cirrhosis and end-stage liver disease, increased mortality from cardiovascular disease and increasing incidence of liver-related and non-liver cancers (Sanyal et al, Hepatology 2015;61(4):1392-1405). NAFLD is the hepatic manifestation of metabolic syndrome and represents a spectrum of liver conditions spanning steatosis, NASH, fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. NAFLD and NASH are considered the primary fatty liver diseases because they account for the largest proportion of individuals with elevated hepatic lipids. The severity of NAFLD/NASH depends on the presence of lipid, inflammatory cell infiltrate, hepatocyte ballooning, and degree of fibrosis. Currently, therapeutic options are limited to the treatment of associated conditions (EASL-EASD-EASO Clinical Practice Guidelines, J. Hepatol. 2016;64(6):1388-1402).

Alterations in lipid metabolism have been hypothesized to contribute to the molecular pathogenesis of NAFLD and NASH. Steatosis is a necessary but not sufficient component of the pathogenesis of NASH (Day C, and James O, Hepatology. 1998; 27(6): 1463-6). Consistent with this, numerous studies have demonstrated that the severity of steatosis predicts the risk of developing concomitant steatohepatitis, as well as the risk of progression to cirrhosis (Sorensen et al, Lancet. 1984; 2(8397): 241-4; Wanless I and Lentz J, Hepatology 1990; 12 (5): 1106-10; Reeves H, et al, J Hepatol 1996; 25(5): 677-83). Hepatic steatosis results from an imbalance in hepatic TG production/reuptake and clearance/removal (triglycerides) (Cohen JC, et al, Science. 2011; 332(6037):1519-1523). Reduction of steatosis, a metabolic driver supporting the development of NAFLD/NASH, is hypothesized to lead to subsequent improvements in liver inflammation and fibrosis.

Acetyl-CoA carboxylase (ACC) and diacylglycerol acyltransferase 2 (DGAT2) are two key enzymes regulating lipid metabolism. ACC catalyzes a critical and rate-limiting step in the process of de novo lipogenesis (DNL) (Saggerson D, Annu. Rev. Nutr. 2008;28:253-72.). In addition, ACC also regulates mitochondrial fatty acid beta-oxidation through allosteric regulation of the enzyme carnitine palmitoyltransferase 1 (CPT1) (Saggerson, 2008; Waite M, and Wakil SJ. J. Biol. Chem. 1962;237:2750-2757.). Emerging evidence also suggests that suppressing DNL through ACC inhibition may directly reduce inflammation by inhibiting the generation of inflammatory interleukin-17 (IL-17)-secreting T helper 17 cells (Th17 cells) and promoting the development of anti-inflammatory FoxP3(+) regulatory T cells. (Treg) cells (Berod L, et al. Nat. Med. 2014; 20(11): 1327-33).

Inhibition of ACC activity is hypothesized to benefit patients with NASH through at least two independent mechanisms. As summarized above, individuals with NAFLD demonstrate a marked increase in hepatic DNL, and normalization of this increased influx through pharmacologic inhibition of hepatic ACC is hypothesized to reduce steatosis. In addition, the effect of ACC inhibitors to increase fatty acid oxidation may also contribute to the reduction of liver fat. Consistent with this, ACC inhibitors have been shown to inhibit DNL (see Griffith DA, et al. J. Med. Chem. 2014;57(24):10512-10526; Kim CW, et al. CellMetab. 2017;26 , 394-406; Stiede K, et al. Hepatology. 2017;66(2):324-334; Lawitz EJ, et al. Clin Gastroenterol Hepatol. 2018 (https://doi.org/10.1016/j.cgh. 2018.04.042)). In addition, inhibition of DNL in IL-17-secreting T cells is expected to suppress liver inflammation by curbing the formation of inflammatory Th17 cells (Berod et al., 2014), a pathway that may be important in the pathogenesis of NASH (Rau M, et al. J. Immunol. 2016;196(1):97-105), and favoring the development of anti-inflammatory Treg cells. Additionally, ACC inhibition may reduce stellate cell activation and fibrosis (Ross et al., 2019).

Triglycerides or triacylglycerols (TG) are the main form of energy storage and storage in mammals. TGs are formed by the sequential esterification of glycerol with three fatty acids of varying chain lengths and varying degrees of saturation (Coleman, R. A., and Mashek, D. G. 2011. Chem. Rev. 111: 6359-6386). TGs synthesized in the intestine or liver are packaged into chylomicrons or very low density lipoprotein (VLDL) respectively and exported to peripheral tissues where they are hydrolyzed to their constituent fatty acids and

- 1 045908 glycerol under the action of lipoprotein lipase (LPL). The resulting non-esterified fatty acids (NEFA) can either be further metabolized to produce energy or can be re-esterified and stored.

Under normal physiological conditions, high-energy TG remains sequestered in various fat depots until there is a demand for its release, at which point it is hydrolyzed to glycerol and free fatty acids, which are then released into the bloodstream. This process is tightly regulated by the opposing actions of insulin and hormones such as catecholamines, which promote the deposition and mobilization of TG stores under various physiological conditions. In the postprandial setting, insulin acts by inhibiting lipolysis, thereby limiting the release of energy in the form of NEFA and ensuring adequate storage of dietary lipids in fat depots. However, in patients with type 2 diabetes, the ability of insulin to suppress lipolysis is improved and the influx of NEFA from adipocytes is inappropriately increased. In turn, this leads to increased lipid delivery to tissues such as muscle and liver. In the absence of energy demand, TG and other lipid metabolites such as diacylglycerol (DAG) can accumulate and cause loss of insulin sensitivity (Erion, D. M., and Shulman, G. I. 2010. Nat Med 16: 400402). Insulin resistance in muscle is characterized by decreased glucose reuptake and glycogen accumulation, while in the liver, loss of insulin signaling leads to dysregulated glucose production and excessive production of TG-rich VLDL, a hallmark of type 2 diabetes (Choi, S. H., and Ginsberg, H. N. 2011. Trends Endocrinol. Metab. 22: 353-363). Increased secretion of TG-rich VLDL, so-called VLDL1 particles, is thought to stimulate the production of small dense low-density lipoprotein (sdLDL), a proatherogenic subfraction of LDL that is associated with an increased risk of coronary heart disease (St-Pierre, A.C.et.al. 2005. Arterioscler. Thromb. Vasc. Biol. 25: 553-559).

In mammals, two diacylglycerol transferase (DGAT) enzymes (DGAT1 and DGAT2) have been characterized. Although these enzymes catalyze the same enzymatic reaction, their respective sequences are unrelated and occupy different gene families. Mice with a disruption of the gene encoding DGAT1 are resistant to diet-induced obesity and have increased energy intake and increased activity (Smith, S. J. et. al., 2000. Nat Genet 25: 87-90). Dgat1-/- mice exhibit dysregulated postabsorptive chylomicron release and accumulate lipids in enterocytes (Buhman, K.K. et.al. 2002. J. Biol. Chem. 277: 25474-25479). The metabolically favorable phenotype observed in these mice is hypothesized to be driven by loss of DGAT1 expression in the intestine (Lee, V., et.al. 2010. J. Lipid Res. 51: 1770-1780). Importantly, despite the defect in lactation in female Dgat1-/- mice, these animals retain the ability to synthesize TG, indicating the existence of additional DGAT enzymes. This observation and the isolation of a second DGAT from the fungus Mortierella rammaniana led to the identification and characterization of DGAT2 (Yen, S. L. et.al. 2008. J. Lipid Res. 49: 2283-2301).

DGAT2 is highly expressed in liver and fat and, unlike DGAT1, exhibits superior substrate specificity for DAG (Yen, C.L., 2008). Deletion of the DGAT2 gene in rodents results in defective intrauterine growth, severe lipemia, impaired skin barrier function, and early postnatal death (Stone, S. J. et.al. 2004. J. Biol. Chem. 279: 11767-11776). Because of the lethality caused by loss of DGAT2, much of our understanding of the physiological role of DGAT2 comes from studies performed with antisense oligonucleotides (ASOs) in rodent models of metabolic disease. In this setting, inhibition of hepatic DGAT2 led to an improvement in plasma lipoprotein profile (reduction of total cholesterol and TG) and a decrease in hepatic lipid burden, which was accompanied by an increase in insulin sensitivity and body glucose control (Liu, Y. et.al. 2008. Biochim. Biophys. Acta 1781: 97-104; Choi, S. S. et. al. 2007. J. Biol. Chem. 282: 22678-22688; Yu, X. X. et. al. 2005. Hepatology 42: 362-371 ). Although the molecular mechanisms underlying these observations are not fully understood, it is clear that DGAT2 downregulation results in down-regulation of the expression of many genes encoding proteins involved in lipogenesis, including sterol regulatory element-binding protein 1c (SREBP1c) and stearoyl-CoAdesaturase 1 (SCD1) (Choi, 2007; Yu, 2005). In parallel, oxidative pathways are induced, as evidenced by increased expression of genes such as carnitine palmitoyltransferase 1 (CPT1) (Choi, 2007). The net result of these changes is a decrease in hepatic DAG and TG lipid levels, which in turn leads to increased insulin sensitivity in the liver. In addition, inhibition of DGAT2 suppresses hepatic secretion of VLDL TG and reduces circulating cholesterol levels. Finally, plasma apolipoprotein B (APB) levels were suppressed, possibly due to reduced TG supply for lipidation of newly synthesized APOB protein (Liu, 2008; Yu, 2005). The beneficial effects of DGAT2 inhibition on glycemic control and plasma cholesterol profile suggest that this target may be valuable in the treatment of metabolic disease (Choi, 2007). Additionally, the observation that inhibition of DGAT2 activity leads to decreased hepatic lipid accumulation suggests that inhibitors of this enzyme may have utility in the treatment of NASH.

- 2 045908

In light of the above, there is a need for drugs, for example oral drugs, containing the combination of (b)-2-(5-((3-ethoxypyridin-2yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3- yl)pyrimidin-5-carboxamide (DGAT2 inhibitor) or a pharmaceutically acceptable salt thereof and in the same composition or in a separate composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[ indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid (ACCI inhibitor) or a pharmaceutically acceptable salt thereof. The specific combinations described herein address an existing need.

Summary of the invention

The invention relates to a pharmaceutical composition containing (b)-2-(5-((3-ethoxypyridin2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or its pharmaceutical an acceptable salt in combination with at least 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, each present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient.

The invention also relates to a pharmaceutical composition containing (S)-2-(5-((3ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or its a pharmaceutically acceptable salt and 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl )benzoic acid or a pharmaceutically acceptable salt thereof mixed with a pharmaceutically acceptable excipient.

The invention also relates to a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a metabolic disease comprising administering to a patient in need of such treatment a therapeutically effective amount of ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3yl)pyrimidin-5 -carboxamide or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

The invention also relates to a method for reducing the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis by at least one point, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing disease activity in non-alcoholic steatohepatitis or reducing the medical consequences of non-alcoholic steatohepatitis in humans, including the stage administering to a patient in need of such a reduction a therapeutically effective amount of ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H(tetrahydrofuran-3-yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

The invention also relates to a method for treating fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, comprising the step of administering to a person in need of such treatment, a therapeutically effective amount of the first composition and the second composition and optionally the third composition, wherein

1) the first composition contains ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient;

2) the second composition contains 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'piperidine]-1'-carbonyl)-6-methoxypyridine-2- yl)benzoic acid or a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient thereof; And

3) the third composition contains a pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an anti-diabetic agent, an anti-fibrotic agent, an anti-steatosis agent, a cholesterol/lipid-modulating agent and an anti-diabetic agent, and a pharmaceutically acceptable excipient.

The invention also relates to a method for treating fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, comprising the step of administering to a person in need of such treatment, a therapeutically effective amount of two separate pharmaceutical compositions containing

1) the first composition, which contains ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H-

- 3 045908 (tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof, present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient;

2) a second composition, which contains 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[inda zol-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient; and, perhaps

3) a third composition containing at least one additional pharmaceutical agent selected from the group consisting of a GLP-1R agonist, a KNK inhibitor or an FXR agonist, and a pharmaceutically acceptable excipient.

The invention also relates to a method of treating heart failure, congestive heart failure, coronary heart disease, peripheral vascular disease, renovascular disease, pulmonary hypertension, vasculitis, acute coronary syndromes and cardiovascular risk modification, comprising administering to a person in need of such treatment a therapeutically effective amount of ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2 -yl)benzoic acid or a pharmaceutically acceptable salt thereof.

The invention also relates to a method for treating obesity, type I diabetes, type II diabetes, idiopathic type I diabetes (type Ib), latent autoimmune diabetes in adults (LADA), early-onset type 2 diabetes (EOD), atypical early-onset diabetes young onset (YOAD), midlife diabetes of the young (MODY), malnutrition associated diabetes, gestational diabetes, coronary heart disease, ischemic stroke, post-angioplasty restenosis, peripheral vascular disease, intermittent claudication, myocardial infarction, dyslipidemia, postprandial lipemia , impaired glucose tolerance (IGT) conditions, impaired fasting plasma glucose conditions, metabolic acidosis, ketosis, arthritis, diabetic retinopathy, macular degeneration, cataracts, diabetic nephropathy, glomerulosclerosis, chronic renal failure, diabetic neuropathy, metabolic syndrome, syndrome X, hyperglycemia, hyperinsulinemia, hyperglyceridemia, insulin resistance, impaired glucose metabolism, skin and connective tissue disorders, foot ulcerations and ulcerative colitis, endothelial dysfunction and vascular elasticity, hyperapolipoprotein B-lipoproteinemia and leucinosis, including administration to a person in need of such treatment, a therapeutically effective amount of (b)-2-(5-((3-ethoxypyridin-2yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable compound thereof salt in combination with at least a therapeutically effective amount of 4-(4-(1isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2yl)benzoic acid or a pharmaceutically acceptable salt thereof.

The invention also relates to a method for treating hepatocellular carcinoma, clear cell renal carcinoma, squamous cell carcinoma of the head and neck, colorectal adenocarcinoma, mesothelioma, gastric adenocarcinoma, adenocortical carcinoma, papillary cell carcinoma of the kidney, cervical and endocervical carcinoma, urothelial carcinoma of the bladder, adeno lung carcinoma, including administering to a person in need of such treatment a therapeutically effective amount of N)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

It should be understood that both the foregoing general description and the following detailed description are illustrative and explanatory only and do not limit the invention as claimed.

Brief description of graphic materials

Fig. 1 is a representative X-ray powder diffraction pattern showing crystalline Form 1 of DGAT2i Compound (DGAT2 Inhibiting Compound) of Example 1 (vertical axis: intensity (counts); horizontal axis: two-theta (degrees)).

Fig. 2 is a representative X-ray powder diffraction pattern showing crystalline Form 2 DGAT2i of Compound Example 1 (vertical axis: intensity (counts); horizontal axis: two-theta (degrees)).

Fig. 3 shows an illustrative XRD pattern of Form 1 of Compound A obtained on a Bruker AXS D4 Endeavor diffractometer equipped with a Cu radiation source.

Fig. 4 shows an exemplary Raman spectrum of Form 1 of Compound A obtained with

- 4 045908 using a Nicolet NXR FT-Raman module attached to a FT-IR spectroscopy unit.

Fig. 5 shows an exemplary ¹³ C ssNMR spectrum of Form 1 of Compound A, performed on a Bruker-BioSpin CPMAS probe located in a Bruker-BioSpin Avance III 500 MHz NMR spectrometer ('H frequency).

Fig. 6 shows an illustrative XRD pattern of Form 2 of Compound A obtained on a Bruker AXS D4 Endeavor diffractometer equipped with a Cu radiation source.

Fig. 7 shows an exemplary Raman spectrum of Form 2 of Compound A obtained using a Nicolet NXR FT-Raman module coupled to a FT-IR spectroscopy unit.

Fig. 8 shows an exemplary ^13C tNMR spectrum of Form 2 of Compound A performed on a Bruker-BioSpin CPMAS sensor located in a Bruker-BioSpin Avance III 500 MHz NMR spectrometer ('H frequency).

Fig. 9 shows an exemplary single crystal structure of Form 2 of Compound A.

In fig. 10 summarizes the effects of oral administration, as monotherapy and in combination, of Compound A and Compound D on plasma triglyceride levels in Sprague Dawley rats fed a Western diet, measured postprandially.

In fig. 11 summarizes the effects of oral administration, as monotherapy and in combination, of Compound A and Compound D on plasma triglyceride levels in Sprague Dawley rats fed a Western diet, measured in the fasted state.

In fig. 12 summarizes the effects of administration of Compound A and Compound D as monotherapy and in combination on the nuclear localization of SREBP-1 in rats fed a Western diet.

In fig. 13 summarizes the effects of administration of Compound A and Compound D, as monotherapy and in combination, on the expression of lipogenic genes in the liver of rats fed a Western diet, specifically acetyl-CoA carboxylase (ACC1).

In fig. 14 summarizes the effects of administration of Compound A and Compound D, as monotherapy and in combination, on the expression of lipogenic genes in the liver of rats fed a Western diet, specifically fatty acid synthase (FASN).

In fig. 15 summarizes the effects of administration of Compound A and Compound D as monotherapy and in combination on the expression of lipogenic genes in the liver in rats fed a Western diet, specifically sterol-CoA desaturase (SCD1).

In fig. 16 summarizes the effects of administration of Compound A and Compound D as monotherapy and in combination on the expression of lipogenic genes in the liver in rats fed a Western diet, specifically sterol regulatory element-binding protein 1c (SREBP-1c).

In fig. 17 summarizes the effects of administration of Compound A and Compound D, alone and in combination, on the expression of lipogenic genes in the liver of rats fed a Western diet, specifically proprotein convertase subtilisin/kexin type 9 (PCSK9).

In fig. 18 summarizes the effects of administration of Compound A and Compound D as monotherapy and in combination on hepatic triglyceride levels in Sprague Dawley rats fed a Western diet.

In fig. 19 summarizes the effects of oral administration of Compound A and Compound D, alone and in combination, on liver elasticity, a marker of liver inflammation and fibrosis, in male Wistar Hann rats fed a choline-deficient high-fat diet (CDAHFD).

In fig. 20 summarizes the effects of oral administration of Compound A and Compound D, as monotherapy and in combination, on the immunohistochemistry of hepatic alpha-smooth muscle actin (aSMA), a marker of myofibroblast activation and fibrogenesis, in male Wistar Hann rats fed CDAHFD.

In fig. 21 summarizes the effects of oral administration of Compound A and Compound D, alone and in combination, on Picosirius red liver staining in CDAHFD-fed male Wistar Hann rats.

In fig. 22 summarizes the effects of oral administration of Compound A and Compound D, as monotherapy and in combination, on hepatic alpha-smooth muscle actin (aSMA) gene expression in CDAHFD-fed male Wistar Hann rats.

In fig. 23 summarizes the effects of oral administration of Compound A and Compound D, alone and in combination, on hepatic collagen 1A1 gene expression in CDAHFD-fed male Wistar Hann rats.

In fig. 24 summarizes the effects of oral administration of Compound A and Compound D, alone and in combination, on ionized calcium-binding adapter molecule 1 staining in CDAHFD-fed male Wistar Hann rats.

Detailed Description of the Invention

The invention can be better understood by reference to the following detailed description of the illustration.

- 5 045908 active embodiments of the invention and Examples included therein.

It should be understood that this invention is not limited to specific synthetic methods of preparation, which, of course, may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not limiting. In this specification and in the claims that follow, a number of terms are mentioned which are to be defined as having the following meanings.

As used herein, the singular may mean one or more. In claims used in combination with the word containing, the singular words may mean one or more than one. As used herein, other may mean at least second or more.

The term roughly refers to a relative term meaning an approximation of plus or minus 10% of the nominal value to which it refers, in one embodiment plus or minus 5%, in another embodiment plus or minus 2%. For the scope of this invention, this level of approximation is appropriate unless a value is specifically stated that requires a tight range.

The compounds herein include any pharmaceutically acceptable derivative or variant, including conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), racemic, diastereomeric and other mixtures of such isomers, as well as solvates, hydrates, isomorphs, polymorphs, tautomers, esters, salt forms and prodrugs. The term prodrug refers to compounds that are precursors to drugs and which, after administration, release the drug in vivo as a result of a chemical or physiological process (for example, the prodrug is converted to the target dosage form at physiological pH or by an enzyme).

Exemplary prodrugs upon cleavage release the corresponding free acid, and such hydrolyzable, ester-forming moieties of the compounds of the invention include, but are not limited to, those having a carboxyl moiety where the free hydrogen is replaced by a (C _g C ₄ )alkyl, (C ₂ - C7)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1 -(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-( N(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidiyl, 4-crotonolactonyl, gammabutyrolacton-4-yl, di-H.H-(CAC7)alkylamino(C|-C3)alkyl (such as β-dimethylaminoethyl), carbamoyl-(C ₁ -C ₂ )alkyl, ^Ndi(C1-C ₂ )alkylcarbamoyl-(C1-C ₂ )alkyl and piperidino-, pyrrolidino-or morpholino(C ₂ -C ₃ )alkyl.

In this document the arrow

or wavy line

denotes the point of attachment of a substituent to another group.

The patient is a warm-blooded animal such as, for example, guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, cattle, goats, sheep, horses, monkeys, chimpanzees and humans. The mammal is the patient.

Pharmaceutically acceptable means that the substance or composition must be compatible chemically and/or toxicologically with the other ingredients constituting the composition and/or with the mammal to be treated.

As used herein, the following terms have their usual meaning for the administration of pharmaceutical agents: QD means once daily, and BID means twice daily.

As used herein, the expressions reactive inert solvent and inert solvent refer to a solvent or mixture of solvents that does not react with the starting materials, reagents, intermediates or products in a manner that adversely affects the yield of the desired product.

As used herein, the term selectivity or selective refers to the greater effect of a compound in a first assay compared to the effect of the same compound in a second assay. For example, in enteric-selective compounds, a first assay determines the intestinal half-life of the compound, and a second assay determines the liver half-life of the compound.

The therapeutically effective amount means the amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl )benzoic acid (Compound A) in combination with an amount of ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidin-5- carboxamide (Compound D), possibly in combination with a quantity

- 6 045908 other compound(s) that treat the specific disease, condition or disorder described herein.

4-(4-(1-Isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxy pyridin-2-yl)benzoic the acid is a selective ACC inhibitor and is obtained as the free acid in Example 9 in US Pat. No. 8,859,577, which is US National Phase International Application No. PCT/1B2O11/O54119, all of which are incorporated herein by reference in their entirety. goals. Crystalline forms of the compound are described in International Patent Application No. PCT/1B2O18/O58966, published as WO 2019/102311 on May 31, 2019.

^)-2-(5-((3-Ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidin-5carboxamide is a DGAT2 inhibitor and is the compound of Example 1 in the published application US Patent No. 2018-0051012A1, which is incorporated herein by reference in its entirety for all purposes.

The term treating, treating or treating as used herein covers preventive, i.e. preventive treatment; palliative treatment, i.e. eliminating, reducing or slowing the progression of a patient's disease (or condition) or any tissue damage associated with that disease (or condition); and reversal, where the patient's disease (or condition) is not only alleviated, but any tissue damage associated with the disease (or condition) is in a better state than when treatment began. This latter may occur, for example, and without limitation, as a result of one or more of the following: demonstration of elimination of NASH and/or as a result of improved fibrosis assessment based on liver biopsy; lower incidence of progression to cirrhosis, hepatocellular carcinoma and/or other liver-related outcomes; reduction or improvement in serum or image-based markers of nonalcoholic steatohepatitis activity; decrease or positive dynamics of the activity of non-alcoholic steatohepatitis; or reducing the medical consequences of non-alcoholic steatohepatitis.

It appears that administration of an ACC inhibitor may have a beneficial effect in reducing hepatic TG and potentially other beneficial effects in the treatment of NASH. Increases in circulating TG levels have been reported to be a mechanistic consequence of hepatic ACC inhibition (Kim et al, 2017), although doses of ACC inhibitors that only partially inhibit DNL may not produce increases in circulating TGs (Bergman et al., 2018 ) J. of Hepatology, Volume 68, S582). WO2016/112305 provides methods for treating, stabilizing or reducing the severity or progression of non-alcoholic fatty liver disease using an ACC inhibitor, alone or with one or more additional therapeutic agents.

It was found that the introduction of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'piperidine]-1'-carbonyl)-6-methoxypyridine-2- yl)benzoic acid, possibly administered as a pharmaceutically acceptable salt, may result in elevated circulating levels of TG (usually measured in plasma) in Sprague Dawley rats fed a Western diet, as observed in human subjects.

The compounds of the invention may contain asymmetric or chiral centers and, therefore, may exist in different stereoisomeric forms. Unless specifically stated otherwise, all stereoisomeric forms of the compounds of the invention, as well as mixtures thereof, including racemic mixtures, are intended to form part of the invention. Moreover, the invention covers all geometric and positional isomers. For example, if a compound of the invention includes a double bond or a fused ring, then both the cis and trans forms, as well as mixtures, are within the scope of the invention.

The chiral compounds of the invention (and their chiral precursors) can be obtained in enantiomerically enriched form using chromatography, typically high pressure liquid chromatography (HPLC) or supercritical fluid chromatography (SFC) resin with an asymmetric stationary phase and a mobile phase consisting of a hydrocarbon , typically heptane or hexane, containing from 0 to 50% isopropanol, typically from 2 to 20%, and from 0 to 5% alkylamine, typically 0.1% diethylamine (DEA) or isopropylamine. Concentrating the eluent produces a rich mixture.

Diastereomeric mixtures can be separated into their individual diastereoisomers based on their physicochemical differences by methods well known to those skilled in the art, such as chromatography and/or fractional crystallization. Enantiomers can be separated by converting an enantiomeric mixture into a diastereomeric mixture by reaction with a suitable optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher acid chloride), separating the diastereoisomers, and converting (e.g., hydrolyzing) the individual diastereoisomers to the corresponding pure enantiomers. Enantiomers can also be separated using a chiral HPLC column. Alternatively, specific stereoisomers can be synthesized using an optically active starting material through asymmetric synthesis using optically active reagents, substrates,

- 7 045908 catalysts or solvents or by converting one stereoisomer into another through an asymmetric transformation.

If the compounds of the invention have two or more stereogenic centers, and the absolute or relative stereochemistry is given in the name, then the designations R and S refer respectively to each stereogenic center in increasing numerical order (1, 2, 3, etc.) according to standard schemes IUPAC numbers for each molecule. If the compounds of the invention have two or more stereogenic centers, and the absolute or relative stereochemistry is given in the name or structure, then it will be understood that the name or structure covers all forms of the compound, including the racemic form.

It is also possible that the intermediates and compounds of the invention may exist in different tautomeric forms, and all such forms are included within the scope of the invention. The term tautomer or tautomeric form refers to structural isomers of different energies that are capable of interconversion through a low energy barrier. For example, protic tautomers (also known as prototropic tautomers) involve interconversions resulting from proton migration, such as keto-enol isomerization and imino-enamine isomerization.

Valence tautomers involve interconversions resulting from the rearrangement of some bond electrons.

Included within the scope of the claimed compounds are all stereoisomers, geometric isomers and tautomeric forms of the compounds of the invention, including compounds exhibiting more than one type of isomerism and mixtures of one or more of them. Also included are acid or base addition salts where the counterion is optically active, such as D-lactate or L-lysine, or racemic, such as DL-tartrate or DL-arginine.

The invention covers all pharmaceutically acceptable isotopically labeled compounds of the invention wherein one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number typically found in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen such as ^2H and ^3H , carbon such as 11C, ^13C and ^14C , chlorine such as ^36Cl , fluorine such as ^18F , iodine , such as ¹²³ I, ¹²⁴ I and ¹²⁵ I, nitrogen, such as ¹³ N and ¹⁵ N, oxygen, such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus, such as ³² P, and sulfur, such as ³⁵ S.

Certain isotope-labeled compounds of the invention, eg compounds into which radioactive isotopes are introduced, are useful in drug and/or substrate tissue distribution studies. Radioactive isotopes tritium, i.e. H, and carbon-14, i.e. C are especially useful for these purposes due to the ease of their inclusion and the availability of ready-made detection tools.

Substitution with heavier isotopes such as deuterium, i.e. ^2H may provide some therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

Substitution with positron-emitting isotopes such as C, F, O, and N can be useful in positron emission tomography (PET) studies to study receptor occupancy by substrate.

Isotopically labeled compounds of formula I can generally be prepared by standard methods known to those skilled in the art, or by methods similar to those described in the accompanying Examples and Preparations, using an isotope-labeled reagent in place of the unlabeled reagent used previously.

The compounds of the invention can be isolated and used per se, or, when possible, in the form of a pharmaceutically acceptable salt. The term salts refers to inorganic and organic salts of the compound of the invention. These salts can be prepared in situ during the final isolation and purification of the compound or separately by treating the compound with a suitable organic or inorganic acid or a suitable organic or inorganic base and isolating the resulting salt. The acids that are used to prepare pharmaceutically acceptable acid addition salts of the above basic compounds of this invention are acids that form non-toxic acid addition salts (i.e., salts containing pharmacologically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, naphthylate, mesylate, glucoheptanoate, lactobionate, laurylsulfonate, hexafluorophosphate, benzenesulfonate, tosylate, formate, trifluoroacetate, oxalate, besylate, palmitiate, pamoate, malonate, stearate, laurate, malate, borate, p-toluenesulfonate and pamoate (i.e. 1,1'-methylene-bis-(2- hydroxy-3-naphthoate)).

The invention also relates to base addition salts of the compounds of the invention. Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base addition salts of those compounds of the invention that are acidic in nature are bases that form non-toxic base salts with

- 8 045908 such connections. Such non-toxic salts include, but are not limited to, salts derived from pharmacologically acceptable cations such as alkali metal cations (eg, lithium, potassium, and sodium) and alkaline earth metal cations (eg, calcium and magnesium), ammonium salts, or water-soluble addition salts amine, such as N-methylglucamine (meglumine), tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and lower alkanol ammonium addition salts and other basic salts of pharmaceutically acceptable organic amines (see, for example, Berge, et al. J. Pharm Sci 66, 1-19 (1977).

Some compounds of the invention may exist in more than one crystalline form (and these are commonly referred to as polymorphs). Polymorphs can be obtained by crystallization under different conditions, for example using different solvents or different mixtures of solvents for recrystallization; crystallization at different temperatures; and/or under different cooling conditions, ranging from very fast to very slow cooling during crystallization. Polymorphs can also be prepared by heating or melting the compound of the invention followed by gradual or rapid cooling. The presence of polymorphs can be determined by solid-state NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffraction, or such other methods.

In one embodiment, the present invention relates to a pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with at least 4-(4-(1isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6 -methoxypyridin-2yl)benzoic acid or a pharmaceutically acceptable salt thereof, each of which is present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient.

In a further embodiment, the composition further comprises at least one additional pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an anti-diabetic agent and a cholesterol/lipid-modulating agent.

In a further embodiment, the composition further comprises at least one additional pharmaceutical agent selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2methylazetidin-1-yl]-6-(trifluoromethyl )pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2. 1]octan-8-yl]-4-fluoro-1,3-benzothiazol-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl]-1H -benzimidazole-6carboxylic acid, or pharmaceutically acceptable salts thereof.

In a further embodiment, the pharmaceutical composition contains (S)-2-(5-((3ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide as a crystalline solid substance structure:

or a pharmaceutically acceptable salt thereof.

In a further embodiment, the crystalline solid has an X-ray powder diffraction pattern containing 2-theta (CuKa radiation, wavelength 1.54056 A) values of 5.3±0.2, 7.7±0.2 and 15.4± 0.2.

In a further embodiment, the crystalline solid has a powder x-ray diffraction pattern comprising 2-theta (CuKa radiation, wavelength 1.54056 A) values of 6.5±0.2, 9.3±0.2 and 13.6± 0.2.

In a further embodiment, the pharmaceutical composition contains 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine-2- yl)benzoic acid or a pharmaceutically acceptable salt thereof in the form of a crystalline solid with the structure:

or a pharmaceutically acceptable salt thereof.

In a further embodiment, the crystalline solid is 2-amino-2(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole5, 4'-piperidin]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid.

In a further embodiment, the pharmaceutical composition further comprises at least

- 9 045908 at least one additional pharmaceutical agent selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidine -4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol4-yl}methoxy)-8-azabicyclo[3.2.1]octane -8-yl]-4-fluoro-1,3-benzothiazol-6-carboxylic acid; or 2-[(4{6-[(4-cyano-2-fluorobenzyl)oxy]pyrigin-2-yl}piperigin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl]- 1Hbenzimidazole-6-carboxylic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease associated with metabolism, involving the administration to a patient in need of such treatment of a therapeutically effective amount of ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl) )pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with at least a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine ]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

In a further embodiment, the disease or condition is fatty liver disease. In another embodiment, the disease or condition is non-alcoholic fatty liver disease. In another embodiment, the disease or condition is non-alcoholic steatohepatitis. In another embodiment, the disease or condition is non-alcoholic steatohepatitis with liver fibrosis. In another embodiment, the disease or condition is non-alcoholic steatohepatitis with cirrhosis. In another embodiment, the disease or condition is non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma. In another embodiment, the disease or condition is non-alcoholic steatohepatitis with cirrhosis and a metabolic disease.

In a further embodiment, the method includes at least one other pharmaceutical agent selected from the group consisting of an acetyl-CoA carboxylase (ACC) inhibitor, a diacylglycerol O-acyltransferase 1 (DGAT-1) inhibitor, monoacylglycerol Oacyltransferase inhibitors, an phosphodiesterase (PDE)-10, AMPK activator, sulfonylurea, meglitinide, α-amylase inhibitor, α-glucosylhydrolase inhibitor, α-glucosidase inhibitor, PPARy agonist, PPAR α/γ agonist, biguanide, glucagon-like peptide 1 (GLP-1) modulator, liraglutide, albiglutide, exenatide, albiglutide, lixisenatide, dulaglutide, semaglutide, protein tyrosine phosphatase-1B (PTP-1B) inhibitor, SIRT-1 activator, dipeptidyl peptidase IV (DPP-IV) inhibitor, insulin secretagogue, fatty acid oxidation inhibitor, A2 antagonist, inhibitor c-jun amino terminal kinase (JNK), glucokinase activators (GKa), insulin, insulin mimetic, glycogen phosphorylase inhibitor, VPAC2 receptor agonist, SGLT2 inhibitors, glucagon receptor modulator, GPR119 modulators, FGF21 derivatives or analogues, TGR5 receptor modulators, GPBAR1 receptor modulators, GPR40 agonists, GPR120 modulators, high-affinity nicotinic acid receptor activators (HM74A), SGLT1 inhibitors, palmitoyltransferase enzyme inhibitors or modulators, fructose-1,6-diphosphatase inhibitors, aldose reductase inhibitors, mineralocorticoid receptor inhibitors, TORC2 inhibitors, CCR2 and/or CCR5 inhibitors, PKC isoform inhibitors (eg, PKCa, PKCe, PKOy), fatty acid synthetase inhibitors, serine palmitoyltransferase inhibitors, GPR81, GPR39, GPR43, GPR41, GPR105, Kv1.3 modulators, retinol binding protein 4, glucocorticoid receptor, somatostatin receptors, PDHK2 inhibitors or modulators, or PDHK4, MAP4K4 inhibitors, IL1 family modulators, including HLbeta, HMG-CoA reductase inhibitors, squalene synthetase inhibitors, fibrates, bile acid sequestrants, ACAT inhibitors, MTP inhibitors, lipoxygenase inhibitors, cholesterol absorption inhibitors, PCSK9 modulators, cholesteryl ester transfer protein inhibitors and RXRalpha modulators.

In a further embodiment, the method includes at least one other pharmaceutical agent selected from the group consisting of cysteamine or a pharmaceutically acceptable salt thereof, cystamine or a pharmaceutically acceptable salt thereof, an antioxidant compound, lecithin, vitamin B complex, bile salt preparations, antagonists cannabinoid receptor 1 (CB1), inverse agonists of cannabinoid receptor 1 (CB1), regulators of peroxisome proliferator-activated receptor activity, benzodiazepine or benzothiepine compound, antisense RNA construct for inhibiting protein tyrosine phosphatase PTPRU, heteroatom-linked substituted piperidine and its derivatives, azacyclopentane derivative, capable of inhibiting stearoyl coenzyme alpha delta-9 desaturase, acylamide compound having the activity of a secretagogue or inducer of adiponectin, quaternary ammonium compound, glatiramer acetate, pentraxin proteins, HMG-CoA reductase inhibitor, N-acetylcysteine, isoflavone compound, macrolide antibiotic, inhibitor galectin, antibody or any combination thereof.

- 10 045908

In a further embodiment, the method includes at least one other pharmaceutical agent selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6 (trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octane -8-yl]-4fluoro-1,3-benzothiazol-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl]-1H -benzimidazole-6-carboxylic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, the present invention relates to a method of reducing by at least one point in scoring systems for non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing disease activity in non-alcoholic steatohepatitis or reducing the medical consequences of non-alcoholic steatohepatitis in humans, comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of (b)-2-(5-((3-ethoxypyridin-2yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3-yl)pyrimidin- 5-carboxamide or a pharmaceutically acceptable salt thereof in combination with at least a therapeutically effective amount of 4-(4-(1isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine] - 1 '-carbonyl)-6-methoxypyridin-2yl)benzoic acid or a pharmaceutically acceptable salt thereof.

In a further embodiment, the method includes at least one other pharmaceutical agent selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6 (trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octane -8-yl]-4fluoro-1,3-benzothiazol-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl]-1H -benzimidazole-6-carboxylic acid, or pharmaceutically acceptable salts thereof.

In another embodiment, the present invention relates to a method of treating fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, comprising the step of administering to a person in need in such treatment, a therapeutically effective amount of two separate pharmaceutical compositions containing

1) the first composition, which contains ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or its pharmaceutical an acceptable salt, present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient;

2) a second composition which contains 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine -2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient; and, perhaps

3) a third composition containing at least one additional pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an anti-diabetic agent, an anti-fibrotic agent, an anti-steatosis agent and a cholesterol/lipid-modulating agent and an anti-diabetic agent, and a pharmaceutically acceptable excipient.

In a further embodiment, said first composition and said second composition are administered simultaneously. In another embodiment, the composition comprises a first composition, a second composition and a third composition.

In a further embodiment, the method includes a third composition, wherein at least one other pharmaceutical agent is selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2methylazetidin-1-yl]- 6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(N^^)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1] octan-8-yl]-4-fluoro-1,3-benzothiazol-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2fluorobenzyl)oxy]pyridin-2-yl} piperidin-1-yl)methyl]-1-[(2 S)-oxetan-2-ylmethyl] - 1H-benzimidazole-6carboxylic acid, or pharmaceutically acceptable salts thereof.

In another embodiment of the present invention, ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof in combinations with 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient.

In another embodiment of the present invention, ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof in combinations with at least 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'piperidine]-1'-carbonyl)-6-methoxypyridine-2- yl)benzoic acid or its pharmaceutical administration

- 11 045908 lem salt is present in the pharmaceutical composition in a therapeutically effective amount in admixture with a pharmaceutically acceptable excipient.

In another embodiment of the present invention, the composition further comprises at least one additional pharmaceutical agent selected from the group consisting of a GLP1R agonist, a KHK inhibitor, an FXR agonist, an anti-inflammatory agent, an anti-diabetic agent, an anti-fibrotic agent, an anti-steatosis agent and a cholesterol/lipid modulating agent.

In another embodiment, a method of treating a metabolic or metabolic-related disease, condition or disorder includes the step of administering to a patient a therapeutically effective amount of ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H -(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof in combination with 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine] -1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof.

In another embodiment, a method of treating a condition selected from the group consisting of hyperlipidemia, type I diabetes, type II diabetes mellitus, idiopathic type I diabetes (type Ib), latent autoimmune diabetes in adults (LADA), early onset diabetes type 2 (EOD ), atypical young-onset diabetes (YOAD), mature-onset diabetes of the young (MODY), malnutrition-related diabetes, gestational diabetes, coronary heart disease, ischemic stroke, restenosis after angioplasty, peripheral vascular disease, intermittent claudication, myocardial infarction (eg, necrosis and apoptosis), dyslipidemia, postprandial lipimia, impaired glucose tolerance (IGT) states, impaired fasting plasma glucose states, metabolic acidosis, ketosis, arthritis, obesity, osteoporosis, hypertension, congestive heart failure, left ventricular hypertrophy, peripheral arterial disease, diabetic retinopathy, macular degeneration, cataracts, diabetic nephropathy, glomerulosclerosis, chronic renal failure, diabetic neuropathy, metabolic syndrome, syndrome X, premenstrual syndrome, coronary heart disease, angina pectoris, thrombosis, atherosclerosis, myocardial infarction , transient ischemic attack, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia, hyperglyceridemia, insulin resistance, impaired glucose metabolism, conditions of impaired glucose tolerance, conditions of impaired fasting plasma glucose, obesity, erectile dysfunction, skin and connective tissue disorders tissue, foot ulcerations and ulcerative colitis, endothelial dysfunction and vascular elasticity, hyper-apolipoprotein Blipoproteinemia, Alzheimer's disease, schizophrenia, cognitive impairment, inflammatory bowel disease, ulcerative colitis, Crohn's disease and irritable bowel syndrome, non-alcoholic steatohepatitis (NASH) , non-alcoholic fatty liver disease (NAFLD), involves administering a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of Compound D or a pharmaceutically acceptable salt thereof.

In a further embodiment, a method of treating a metabolic or metabolic-related disease, condition or disorder includes the step of administering to a patient in need of such treatment at least two separate pharmaceutical compositions comprising

1) the first composition, which contains ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or its pharmaceutical an acceptable salt, present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient;

2) a second composition which contains 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine -2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient; and, perhaps

3) a third composition containing at least one additional pharmaceutical agent selected from the group consisting of a GLP-1R agonist, a KHK inhibitor, an FXR agonist, an anti-inflammatory agent, an anti-diabetic agent, an anti-fibrotic agent, an anti-steatosis agent and a cholesterol/lipid-modulating agent and an antidiabetic agent, and a pharmaceutically acceptable excipient.

In yet another embodiment, the method of the invention is carried out when said first composition, said second composition and said third composition are administered simultaneously.

In yet another embodiment, the method of the invention is carried out when the first composition, said second composition and said third composition are administered sequentially and in any order.

In one embodiment, when three agents are administered, then the first agent and the second agent are administered simultaneously, and the third agent is administered subsequently. In another embodiment, three separate agents are administered sequentially in any order.

In one embodiment, when three agents are administered, then the third agent contains a GLP-1R agonist. Ago

- 12 045908 nist GLP-1R 2-[(4-{6-[(4-cyano-2-(|)torbenzyl)oxy|pyridin-2-yl}piperidin-1-yl)methyl|-1- [(^)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or a pharmaceutical salt thereof [such as its 2-amino-2-(hydroxymethyl)propane-1,3-diol salt, also known as its tris -salt], as well as other GLP-1R agonists and methods for preparing these compounds, are described in US Pat. No. 10208019, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

In some embodiments, the GLP-1R agonist is selected from the group consisting of liraglutide, albiglutide, exenatide, albiglutide, lixisenatide, dulaglutide, semaglutide, HM15211, LY3298176, Medi0382, NN-9924, TTP-054, TTP-273, efpeglenatide, those described in WO2018109607, and DIAST-X2.

GLP-1 is a 30 amino acid long incretin hormone secreted by L cells in the intestine in response to food intake. GLP-1 has been shown to stimulate insulin secretion in a physiological and glucose-dependent manner, reduce glucagon secretion, inhibit gastric emptying, reduce appetite, and stimulate beta cell proliferation. In nonclinical experiments, GLP-1 promotes ongoing beta cell competence by stimulating the transcription of genes important for glucose-dependent insulin secretion and promoting beta cell neogenesis (Meier, et al. Biodrugs. 2003; 17(2):93-102).

In healthy individuals, GLP-1 plays an important role in the regulation of sprandial blood glucose levels by stimulating glucose-dependent insulin secretion from the pancreas, leading to increased peripheral glucose absorption. GLP-1 also inhibits glucagon secretion, resulting in decreased hepatic glucose output. In addition, GLP-1 delays gastric emptying and slows small intestinal motility, delaying the absorption of food. In people with T2DM, the normal postprandial rise of GLP-1 is absent and reduced (Vilsboll T, et al. Diabetes. 2001.50;609-613).

Hoist (Physiol. Rev. 2007, 87, 1409) and Meier (Nat. Rev. Endocrinol. 2012, 8, 728) describe that GLP-1 receptor agonists such as GLP-1, liraglutide and exendin-4 have 3 Key activities to improve glycemic control in patients such as those with T2DM by reducing fasting and postprandial glucose (FPG and PPG) levels are: (1) increasing glucose-dependent insulin secretion (phase 1 and 2 improvement), (2 ) suppression of glucagon activity under hyperglycemic conditions, (3) slowing the rate of gastric emptying, leading to slower absorption of glucose from food.

In another embodiment, when three agents are administered, then the third agent contains a KHK inhibitor.

In some embodiments, the KHK inhibitor is [(1R,5S,6R)-3-{2-[(2S)-2methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1 .0]hex-6-yl]acetic acid or a pharmaceutically acceptable salt thereof. [(1R,5S,6R)-3-{2-[(2S)-2-Methylazetidin-1-yl]-6(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6 -yl]acetic acid (including in crystalline free acid form) is a ketohexoxykinase inhibitor and is described in Example 4 in US Pat. No. 9,809,579, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

In some embodiments, the KHK inhibitor is the free acid crystalline form of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl} -3-azabicyclo[3.1.0|hex-6-yl|acetic acid.

Ketohexokinase (KHK) is an essential enzyme in fructose metabolism and catalyzes the conversion of fructose to fructose-1-phosphate (F1P). KHK is expressed as two alternative mRNA splice variants, designated KHKa and KHKc, resulting from alternative splicing of the third exon. The affinity and ability of KHKc to phosphorylate fructose is much stronger than that of KHKa, as evidenced by the much lower Km (Ishimoto, Lanaspa et al., PNAS 109, 4320-4325, 2012). While KHKa is expressed ubiquitously, KHKc expression is highest in the liver, kidney, and intestine, the primary sites of fructose metabolism in the body (Diggle CP, et al. (2009) J Histochem Cytochem 57:763–774; Ishimoto, Lanaspa, et al. , PNAS 109, 4320-4325, 2012). Additionally, loss-of-function mutations have been reported in humans, and the term Essential Fructosuria (OMIM #229800) has been applied, with no adverse effects other than the appearance of fructose in the urine after consuming sugar.

A more severe condition involved in fructose metabolism is hereditary fructose intolerance (HFI, OMIM #229600), which is caused by defects in aldolase B (GENE: ALDOB), which is the enzyme responsible for breaking down F1P and is located immediately after the KHK stage in this way (Bouteldja N, et. al, J. Inherit. Metab. Dis. 2010 Apr;33(2):105-12; Tolan, DR, Hum Mutat. 1995;6(3):210-8; http ://www.omim.org/entry/229600). It is a rare disorder that affects an estimated 1 in 20,000 people, and mutations lead to F1P accumulation, ATP depletion, and increased uric acid, the combination of which causes hypoglycemia, hyperuricemia, and lactic acidosis, among other metabolic disorders. HFI impairs the body's ability to metabolize dietary fructose, resulting in acute symptoms such as vomiting, severe hypoglycemia, diarrhea, and abdominal distress, leading to long-term growth defects, liver and kidney damage, and potential death (Ali M et al, J. Med. Genet. 1998 May:35(5):353-65). Typically, patients suffer during the first years of life before diagnosis, and the only course of treatment is from

- 13 045908 kaz from fructose in the diet. This becomes relevant due to the presence of this macronutrient in most foods. In addition to physical symptoms, many patients experience emotional and social isolation due to their unusual diets and constantly struggle to adhere to strict dietary restrictions (HFI-INFO Discussion Board, http://hfiinfo.proboards.com. Accessed December 14, 2015). Even when they appear asymptomatic, some patients develop NAFLD and kidney disease, highlighting the inadequacy of self-imposed dietary restrictions as the only treatment option as well as the high unmet medical need for this condition.

Under hyperglycemic conditions, endogenous fructose production occurs through the polyol pathway, a pathway that converts glucose to fructose with sorbitol as an intermediate. The activity of this pathway increases with hyperglycemia. In these studies, the authors demonstrated that KHK null mice were protected from glucose-induced weight gain, insulin resistance, and hepatic steatosis, suggesting that under hyperglycemic conditions, endogenously produced fructose may contribute to the development of insulin resistance and hepatic steatosis ( Lanaspa, M.A., et al., Nature Comm. 4, 2434, 2013). Thus, KHK inhibition is expected to be beneficial in many diseases that involve changes in both endogenous and ingested fructose.

In another embodiment, when three agents are administered, then the third agent contains an FXR agonist. In some embodiments, the FXR agonist is selected from the group consisting of tropifexor (2-[(1R,3R,5S)-3-({5cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl }methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4fluoro-1,3-benzothiazole-6-carboxylic acid) (Tropifexor); cilofexor (GS-9674); obeticholic acid; LY2562175; Met409; TERN-101; and EDP-305 and pharmaceutically acceptable salts thereof. The FXR agonist tropifexor or a pharmaceutically acceptable salt thereof is described, for example, in Example 1-1B in US Pat. No. 9,150,568, the disclosure of which is incorporated herein by reference in its entirety and for all purposes. Chemical name of tropifexor: 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo [ 3.2.1]octan-8-yl]-4-fluoro-1,3benzothiazol-6-carboxylic acid.

The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and is primarily expressed in the liver, kidney and intestine (see, for example, Seol et al. (1995) Mol. Endocrinol. 9:72-85 and Forman et al. ( 1995) Cell 81:687-693). It functions as a heterodimer with the retinoid X receptor (RXR) and binds to response elements in target gene promoters to regulate gene transcription. The FXR-RXR heterodimer binds with highest affinity to the inverted repeat response element-1 (IR-1), in which consensus receptor-binding hexamers are separated by a single nucleotide. FXR is part of an interrelated process in which FXR is activated by bile acids (an end product of cholesterol metabolism) (see, for example, Makishima et al. (1999) Science 284: 1362-1365, Parks et al. (1999) Science 284:1365- 1368, Wang et al. (1999) Mol. Cell. 3:543-553), which serve to inhibit cholesterol catabolism (see also Urizar et al. (2000) J. Bid. Chem. 275:39313-39317).

FXR is a key regulator of cholesterol homeostasis, triglyceride synthesis and lipogenesis (Crawley, Expert Opinion Ther. Patents (2010), 20(8): 1047-1057). In addition to the treatment of dyslipidemia, numerous indications for FXR have been described, including the treatment of liver disease, diabetes, vitamin D-related diseases, drug-induced side effects, and hepatitis (Crawley, supra). Despite advances in the development of new FXR agonists, there remains significant room for improvement.

In some other embodiments, when three agents are administered, then the third agent comprises an SGLT2 inhibitor, metformin, incretin analogues, an incretin receptor modulator, a DPP-4 inhibitor, a PPAR agonist.

In some other embodiments, when three agents are administered, then the third agent is an anti-heart failure agent selected from an ACE inhibitor, an angiotensin receptor blocker, a calcium channel blocker, or a vasodilator.

The present invention further covers kits that are suitable for use in carrying out the treatment methods described above. In one embodiment, the kit contains a first dosage form containing one or more agents (compounds) or pharmaceutically acceptable salts thereof of the present invention, and a container for the dosage form, in quantities sufficient to carry out the methods of the present invention.

In another embodiment, the kit contains a first dosage form containing one or more agents (compounds) or pharmaceutically acceptable salts thereof of the present invention, and a container for the first dosage form, and a second dosage form containing another agent (compound) or a pharmaceutically acceptable salt thereof according to the present invention and a container for the second dosage form, where both dosage forms are in quantities sufficient to carry out the methods of the present invention.

In another embodiment, the kit contains a first dosage form containing one of the agents

- 14 045908 (compounds) or pharmaceutically acceptable salts thereof of the present invention, and a container for a first dosage form, a second dosage form containing another agent (compound) or a pharmaceutically acceptable salt thereof of the present invention, and a container for a second dosage form, and a third dosage form, where all three dosage forms are in quantities sufficient to carry out the methods of the present invention.

In some embodiments of the present invention, a kit is provided that contains a first dosage form containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a container for a first dosage form, and a second dosage form containing 4-(4-(1-isopropyl7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4 '-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, and a container for a second dosage form, where both dosage forms are in quantities sufficient to carry out the methods of the present invention .

In another embodiment of the present invention, there is provided a kit that contains a first dosage form containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrophane-3- yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a container for a first dosage form, a second dosage form containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5, 4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, and a container for a second dosage form, a third dosage form containing another agent (compound) or a pharmaceutically acceptable salt thereof , and a container for a third dosage form, where all three dosage forms are in quantities sufficient to carry out the methods of the present invention.

In some embodiments, the dosage form(s) from the kit can be administered simultaneously or staggered in time, that is, at different times and at equal or different intervals for any of the dosage forms from the kit.

Any of the above-mentioned kits may provide means for separately storing each dosage form, such as a container, a compartmentalized vial, or a compartmentalized foil package. An example of such a set is the familiar blister packaging used for tablets, capsules, etc. Such a kit is particularly suitable for administering different dosage forms, for example oral and parenteral, for administering individual agents (compounds) at different intervals, or for titrating individual agents (compounds) against each other. To facilitate compliance with the administration regimen, the kit may contain instructions for administration and may be provided with a so-called reminder sheet.

The present invention also provides various other kits known to be used for packaging, dispensing and administering the agents of the present invention.

In a further embodiment, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, an HMG-CoA reductase inhibitor and a pharmaceutically acceptable excipient.

In some embodiments, the present invention provides a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, an HMGCoA reductase inhibitor selected from the group consisting of pravastatin, pitavastatin, lovastatin, atorvastatin, simvastatin, fluvastatin, itavastatin, nisvastatin, nisbastatin, rosuvastatin, atavastatin and visastatin, and a pharmaceutically acceptable excipient.

In some embodiments, the present invention provides a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, atorvastatin and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease metabolically involving the administration to a person in need of such treatment of a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1' -carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is atorvastatin.

In a further embodiment, the present invention relates to a method for reducing the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis by at least one point, reducing the level of serum markers of non-alcoholic fatty liver disease activity.

- 15 045908 atohepatitis, reducing disease activity in non-alcoholic steatohepatitis or reducing the medical consequences of non-alcoholic steatohepatitis in humans, including the step of administering to a patient in need of such a reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo1,4,6 ,7-te1rahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is atorvastatin.

In a further embodiment, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a fibrate agent and a pharmaceutically acceptable excipient.

In some embodiments, the present invention provides a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a fibrate agent selected from the group consisting of gemfibrozil, fenofibrate and clofibrate, and a pharmaceutically acceptable excipient.

In some embodiments, the present invention provides a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, fenofibrate and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease metabolically involving the administration to a person in need of such treatment of a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1' -carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt and fibrate agent thereof. In some embodiments, the fibrate agent is fenofibrate.

In a further embodiment, the present invention provides a method for reducing the severity of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by at least one point, reducing the level of serum markers of nonalcoholic steatohepatitis activity, reducing disease activity in nonalcoholic steatohepatitis, or reducing the medical consequences of nonalcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt and fibrate agent thereof. In some embodiments, the fibrate agent is fenofibrate.

In a further embodiment, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a bile acid sequestrant and a pharmaceutically acceptable excipient.

In some embodiments, the present invention provides a pharmaceutical composition comprising 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl) -6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a bile acid sequestrant selected from the group consisting of questran, colestipol and colesevelam, and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease associated with with metabolism comprising administering to a person in need of such treatment a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1'- carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a bile acid sequestrant.

In a further embodiment, the present invention provides a method for reducing the severity of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by at least one point, reducing the level of serum markers of nonalcoholic steatohepatitis activity, reducing disease activity in nonalcoholic steatohepatitis, or reducing the medical consequences of nonalcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a bile acid sequestrant.

- 16 045908

In a further embodiment, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a cholesterol absorption inhibitor and a pharmaceutically acceptable excipient. In some embodiments, the cholesterol absorption inhibitor is ezetimibe.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease metabolically involving the administration to a person in need of such treatment of a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1' -carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a cholesterol absorption inhibitor.

In a further embodiment, the present invention provides a method for reducing the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis by at least one point, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing disease activity in non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a cholesterol absorption inhibitor.

In a further embodiment, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a nicotinic acid agent and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a niacin agent selected from the group consisting of niacin, niacor and slo-niacin, and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease metabolically involving the administration to a person in need of such treatment of a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1' -carbonyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a nicotinic acid agent.

In a further embodiment, the present invention provides a method for reducing the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis by at least one point, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing disease activity in non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a nicotinic acid agent.

In a further embodiment, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'carbonyl)- 6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a PCSK9 modulator and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, a PCSK9 modulator selected from the group consisting of alirotsumab and evolutsumab, and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease metabolically involving the administration to a person in need of such treatment of a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1' -carbonyl)-6-methoxypyridin-2-yl)benzoic acid or

- 17 045908 its pharmaceutically acceptable salt and PCSK9 modulator.

In a further embodiment, the present invention provides a method for reducing the severity of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by at least one point, reducing the level of serum markers of nonalcoholic steatohepatitis activity, reducing disease activity in nonalcoholic steatohepatitis, or reducing the medical consequences of nonalcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl )-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof and a PCSK9 modulator.

In any of the previous embodiments, 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl )benzoic acid is a crystalline solid. In some embodiments, the crystalline solid is 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5 ,4'-piperidin]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid.

In a further embodiment, the present invention relates to a pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, an HMG-CoA reductase inhibitor and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof, an HMG-CoA reductase inhibitor selected from the group consisting of pravastatin, pitavastatin, lovastatin, atorvastatin, simvastatin, fluvastatin, itavastatin, nisvastatin, nisbastatin, rosuvastatin, atavastatin and visastatin, and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin- 5carboxamide or a pharmaceutically acceptable salt thereof, atorvastatin and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease associated with metabolism, involving the administration to a person in need of such treatment of a therapeutically effective amount of ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)N(tetrahydrofuran-3-yl) pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is atorvastatin.

In a further embodiment, the present invention provides a method for reducing the severity of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by at least one point, reducing the level of serum markers of nonalcoholic steatohepatitis activity, reducing disease activity in nonalcoholic steatohepatitis, or reducing the medical consequences of nonalcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such a reduction a therapeutically effective amount of N)-2-(5-((3-ethoxypyridin2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin- 5-carboxamide or a pharmaceutically acceptable salt thereof and an HMG-CoA reductase inhibitor. In some embodiments, the HMGCoA reductase inhibitor is atorvastatin.

In a further embodiment, the present invention relates to a pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt, a fibrate agent and a pharmaceutically acceptable excipient thereof.

In some embodiments, the present invention relates to a pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof, a fibrate agent selected from the group consisting of gemfibrozil, fenofibrate, and clofibrate, and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin- 5carboxamide or a pharmaceutically acceptable salt thereof, fenofibrate and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or with

- 18 045908 a metabolic disease comprising administering to a person in need of such treatment a therapeutically effective amount of (b)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)H- (tetrahydro(|)uran-3-yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt and fibrate agent thereof. In some embodiments, the fibrate agent is fenofibrate.

In a further embodiment, the present invention provides a method for reducing the severity of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by at least one point, reducing the level of serum markers of nonalcoholic steatohepatitis activity, reducing disease activity in nonalcoholic steatohepatitis, or reducing the medical consequences of nonalcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of f)-2-(5-((3-ethoxypyridin2-yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3-yl)pyrimidin- 5-carboxamide or a pharmaceutically acceptable salt and fibrate agent thereof. In some embodiments, the fibrate agent is fenofibrate.

In a further embodiment, the present invention relates to a pharmaceutical composition containing (S)-2-(5-((3-ethoxaccrdin-2-yl)oxy)ptrcdin-3-yl)-N-(tetracyclofuran-3yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof, a bile acid sequestrant and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing (S)-2-(5-((3-ethoxypcrdin-2-yl)oxy)pcirtsdin-3-yl)-N-(tetragsdrofuran-3-yl)pcirtsdin- 5carboxamide or a pharmaceutically acceptable salt thereof, a bile acid sequestrant selected from the group consisting of questran, colestipol and colesevelam, and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease metabolically involving the administration to a person in need of such treatment of a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)N-(tetragcdrofuran-3- yl)pcrimidin-5-carboxamcd or a pharmaceutically acceptable salt thereof and a bile acid sequestrant.

In a further embodiment, the present invention provides a method for reducing the severity of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by at least one point, reducing the level of serum markers of nonalcoholic steatohepatitis activity, reducing disease activity in nonalcoholic steatohepatitis, or reducing the medical consequences of nonalcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of f)-2-(5-((3-ethoxypyridin2-yl)oxy)pcrdin-3-yl)-N-(tetragcdrofuran-3-yl)pcricdin- 5-carboxamide or a pharmaceutically acceptable salt thereof and a bile acid sequestrant.

In a further embodiment, the present invention relates to a pharmaceutical composition containing (S)-2-(5-((3-ethoxypcrdin-2-yl)oxy)pcrdin-3-yl)-N-(tetragcdrofuran-3yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof, a cholesterol absorption inhibitor and a pharmaceutically acceptable excipient. In some embodiments, the cholesterol absorption inhibitor is ezetimibe.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease associated with metabolism, involving the administration to a person in need of such treatment of a therapeutically effective amount of f)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)N-(tetragcdrofuran-3-yl )pcrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof and a cholesterol absorption inhibitor.

In a further embodiment, the present invention provides a method for reducing the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis by at least one point, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing disease activity in non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of f)-2-(5-((3-ethoxypyridin2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin- 5-carboxamide or a pharmaceutically acceptable cholesterol absorption inhibitor salt thereof.

In a further embodiment, the present invention relates to a pharmaceutical composition containing f)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a nicotine-based agent

- 19 045908 acids and pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing (^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H-(tetrahydro(|)uranium-3- yl)pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof, a nicotinic acid agent selected from the group consisting of niacin, niacor and slo-niacin, and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease associated with metabolism, involving the administration to a person in need of such treatment of a therapeutically effective amount of (b)-2-(5-((.3-ethoxypyridin-2-yl)oxy)pyridin-.3-yl)N(tetrahydrofuran-3 -yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt and nicotinic acid agent thereof.

In a further embodiment, the present invention provides a method for reducing the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis by at least one point, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing disease activity in non-alcoholic steatohepatitis, or reducing the medical consequences of non-alcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of (b)-2-(5-((3-ethoxypyridin2-yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3-yl)pyrimidine -5-carboxamide or a pharmaceutically acceptable salt thereof and a nicotinic acid agent.

In a further embodiment, the present invention relates to a pharmaceutical composition containing (S)-2-(5-((3-ethoxunuridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof, a PCSK9 modulator and a pharmaceutically acceptable excipient.

In some embodiments, the present invention relates to a pharmaceutical composition containing (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin- 5carboxamide or a pharmaceutically acceptable salt thereof, a PCSK9 modulator selected from the group consisting of alirotsumab and evolutsumab, and a pharmaceutically acceptable excipient.

In a further embodiment, the present invention provides a method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a disease metabolically involving the administration to a person in need of such treatment of a therapeutically effective amount of (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)N(tetrahydrofuran-3-yl )pyrimidine-5-carboxamide or a pharmaceutically acceptable salt thereof and a PCSK9 modulator.

In a further embodiment, the present invention provides a method for reducing the severity of nonalcoholic fatty liver disease or nonalcoholic steatohepatitis by at least one point, reducing the level of serum markers of nonalcoholic steatohepatitis activity, reducing disease activity in nonalcoholic steatohepatitis, or reducing the medical consequences of nonalcoholic steatohepatitis in humans. comprising the step of administering to a patient in need of such reduction a therapeutically effective amount of ^)-2-(5-((3-ethoxypyridin2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin- 5-carboxamide or a pharmaceutically acceptable salt thereof and a PCSK9 modulator.

In any of the previous embodiments, (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide is a crystalline solid substance. In some embodiments, the crystalline solid has an X-ray powder diffraction pattern containing 2-theta (CuKa radiation, wavelength 1.54056 A) values of 5.3 ± 0.2, 7.7 ± 0.2, and 15.4 ± 0.2. In some embodiments, the crystalline solid has an X-ray powder diffraction pattern containing 2-theta (CuKa radiation, wavelength 1.54056 A) values of 6.5 ± 0.2, 9.3 ± 0.2, and 13.6 ± 0.2.

The compounds of the invention can be synthesized by synthetic routes including methods similar to those generally known in the chemical field, particularly in light of the description contained herein. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, WI) or can be readily prepared by methods known to those skilled in the art (for example, can be prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York (1967-1999 ed.) or in Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including appendices (also available via the Beilstein online database). Preparation of ^)-2-(5-((3-ethoxypyridine-2

- 20 045908 yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide is presented in Example 1 in application US 2018-0051012A1, incorporated herein in its entirety and for all purposes by reference. Preparation of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid set forth in Example 9 of US Pat. No. 8,859,577, which is incorporated herein by reference in its entirety and for all purposes. Preparation of [(1R,5S,6R)-3 -{2-[(2S)-2-methylazetigin-1 -yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3 azabicyclo[3.1.0]hex- 6-yl]acetic acid (including the free acid crystalline form) is described in Example 4 in US Patent No. 9809579. The preparation of GLP-1R agonists is described in US Patent No. 10208019.

Combination agents

The compounds of the invention may be administered alone or together as single agents, or in fixed dose combinations, or in combination with one or more additional therapeutic agents. Administration in combination or combination therapy means that Compound A and Compound D are administered together as two therapeutic agents alone or in combination with one or more additional therapeutic agents administered in parallel to the mammal being treated. When administered in combination, each component may be administered at the same time or sequentially in any order at different times. Thus, each component can be administered separately, but close enough in time to provide the desired therapeutic effect. Thus, the methods of treatment described herein include the use of combination agents to administer three or more agents in combination.

The combination agents are administered to the mammal in a therapeutically effective amount. A therapeutically effective amount means an amount of a compound of the invention that, when administered alone or in combination with an additional therapeutic agent to a mammal, is effective for treating the target disease/condition, for example NASH.

Preferred agents for the treatment of nonalcoholic steatohepatitis (NASH) and/or nonalcoholic fatty liver disease (NAFLD) (i.e., anti-NASH and anti-NAFLD agents) are an acetyl-CoA carboxylase (ACC) inhibitor, a ketohexokinase inhibitor (KNK), GLP-1 receptor agonist, FXRt agonist, CB1 antagonist, ASK1 inhibitor, CCR2 and/or CCR5 inhibitor, PNPLA3 inhibitor, hydroxysteroid 17-β-dehydrogenase inhibitor (HSD17B13), DGAT1 inhibitor, FGF21 analog, FGF19 analog, SGLT2 inhibitor, PPARt agonist , AMPK activator, SCD1 inhibitor or MPO inhibitor. This applicant's patent application PCT/IB2017/057577, filed on January 12, 2017, relates to GLP-1 receptor agonists. Most preferred are an FXR agonist, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, a PPAR agonist, a GLP-1 receptor agonist, an SGLT inhibitor, an ACC inhibitor, and a KHK inhibitor.

Given the activity of the compounds of the invention against NASH/NAFLD, they can be administered together with other agents for the treatment of non-alcoholic steatohepatitis (NASH) and/or non-alcoholic fatty liver disease (NAFLD) and associated diseases/conditions, such as orlistat, TZD and other insulin- sensitizing agents, FGF21 analogues, metformin, omega-3 ethyl ester (e.g. Lovasa), fibrates, HMG-CoA reductase inhibitors, ezetimibe, probucol, ursodeoxycholic acid, TGR5 agonists, FXR agonists, vitamin E, betaine, pentoxifylline, CB1 antagonists , carnitine, N-acetylcysteine, reduced glutathione, lorcaserin, nalrexone-buproprion combination, SGLT2 inhibitors (including dapagliflozin, canagliflozin, empagliflozin, tofogliflozin, ertugliflozin, ASP-1941, THR1474, TS-071, ISIS388626 and LX4211, as well as those who described in WO2010023594), phentermine, topiramate, GLP-1 receptor agonists, GIP receptor agonists, dual GLP-1 receptor/glucagon receptor agonists (i.e. OPK88003, MEDI0382, JNJ-64565111, NN9277, BI 456906), dual agonists receptor GLP-1/GIP receptor (i.e. tirzepatide (LY3298176), NN9423), angiotensin receptor blockers, acetyl-CoA carboxylase (ACC) inhibitor, BCKDK inhibitor, ketohexokinase (KHK) inhibitor, ASK1 inhibitors, branched chain alpha-keto acid dehydrogenase kinase inhibitors (BCBK inhibitors), CCR2 inhibitors and/or CCR5, PNPLA3 inhibitors, DGAT1 inhibitors, FGF21 analogue, FGF19 analogues, PPAR agonists, FXR agonists, AMPK activator, SCD1 inhibitors or MPO inhibitors.

Exemplary ACC inhibitors include 4-(4-[(1-isopropyl-7-oxo-1,4,6,7-tetrahydro-1'Hspiro[indazol-5,4'-piperidin]-1'-yl)carbonyl] -6-methoxypyridin-2-yl)benzoic acid and firsocostat (GS-0976) and pharmaceutically acceptable salts thereof.

Exemplary DGAT2 inhibitors include ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide;

2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4S)-4-fluoropiperidin-3yl)pyrimidin-5-carboxamide;

2-(5-((3-ethoxy-5-fluoropyridin-2-yl)oxy)pyridin-3-yl)-N-((3S,5S)-5-fluoropiperidin-3 yl)pyrimidin-5-carboxamide;

2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4S)-4-fluoropiperidin-3-yl)pyrimidin-5carboxamide;

- 21 045908

2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3R,4R)-4-fluoropiperidin-3-yl)pyrimidin-5carboxamide;

2-(5-((3-ethoxy-5-(|)torpyridin-2-yl)oxy)pyridin-3-yl)-H-((3P,4P)-4-(|)torpiperidin-3yl)pyrimidine -5-carboxamide; and

2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-((3S,5S)-5-fluoropiperidin-3-yl)pyrimidin-5carboxamide.

Examples of suitable antidiabetic agents include (eg, insulins, metformin, DPPIV inhibitors, GLP-1 receptor agonists, analogs and mimetics, SGLT1 and SGLT2 inhibitors). Suitable antidiabetic agents include an acetyl-CoA carboxylase (ACC) inhibitor such as those described in WO2009144554, WO2003072197, WO2009144555 and WO2008065508, a diacylglycerol O-acyltransferase 1 (DGAT-1) inhibitor such as those described in WO09 016462 or WO2010086820, AZD7687 or LCQ908, monoacylglycerol O-acyltransferase inhibitors, phosphodiesterase (PDE)-10 inhibitor, AMPK activator, sulfonylurea (eg, acetohexamide, chlorpropamide, diabenese, glibenslamide, glipizide, glyburide, glimepiride, gliclase ide, glipentide, gliquidone, glizolamide , tolazamide and tolbutamide), meglitinide, α-amylase inhibitor (eg, tendamistat, trestatin, and AL-3688), α-glucosylhydrolase inhibitor (eg, acarbose), α-glucosidase inhibitor (eg, adiposine, camiglibose, emiglitate, miglitol, voglibose, pradimycin -Q and salbostatin), PPARy agonist (eg, balaglitazone, ciglitazone, darglitazone, englitazone, isaglitazone, pioglitazone, and rosiglitazone), PPARα/γ agonist (eg, CLX-0940, GW-1536, GW-1929, GW-2433, KRP297, L-796449, LR-90, MK-0767 and SB-219994), biguanide (eg, metformin), glucagon-like peptide 1 (GLP-1) modulator such as agonist (eg, exendin-3 and exendin-4) , liraglutide, albiglutide, exenatide (Byetta®), albiglutide, lixisenatide, dulaglutide, semaglutide, NN-9924, TTP-054, protein tyrosine phosphatase-1B (PTP-1B) inhibitor (e.g., trodusquemin, hyrthiosal extract, and compounds described in Zhang, S., et al., Drug Discovery Today, 12(9/10), 373-381 (2007)), SIRT-1 activator (e.g. resveratrol, GSK2245840 or GSK184072), dipeptidyl peptidase IV (DPP-IV) inhibitor (e.g. , those described in WO2005116014, sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin and saxagliptin), insulin secretagogue, fatty acid oxidation inhibitor, A2 antagonist, c-jun amino terminal kinase (JNK) inhibitor, glucokinase (GKa) activators such as those described in WO2010103437, WO2010103438, WO2010013161, WO2007122482, TTR-399, TTR-355, TTR-547, AZD1656, ARRY403, MK-0599, TAK-329, AZD5658 or GKM-001 , insulin, insulin mimetic, glycogen phosphorylase inhibitor (eg, GSK1362885), VPAC2 receptor agonist, SGLT2 inhibitors such as those described in E.S. Chao et al. Nature Reviews Drug Discovery 9, 551-559 (July 2010), including dapagliflozin, canagliflozin, empagliflozin, tofogliflozin (CSG452), ertugliflozin, ASP-1941, THR1474, TS-071, ISIS388626 and LX4211, and those described in VP WO20100 23594 , a glucagon receptor modulator such as those described in Demong, D.E. et al. Annual Reports in Medicinal Chemistry 2008, 43, 119-137, GPR119 modulators, particularly agonists such as those described in WO2010140092, WO2010128425, WO2010128414, WO2010106457, Jones, R.M. et al. in Medicinal Chemistry 2009, 44, 149-170 (eg, MBX-2982, GSK1292263, APD597 and PSN821), derivatives or analogs of FGF21, such as those described in Kharitonenkov, A. et al. et al., Current Opinion in Investigational Drugs 2009, 10(4)359-364, modulators of the TGR5 receptor (also called GPBAR1), particularly agonists such as those described in Zhong, M., Current Topics in Medicinal Chemistry, 2010, 10(4), 386-396 and INT777, GPR40 agonists such as those described in Medina, J.C., Annual Reports in Medicinal Chemistry, 2008, 43, 75-85, including but not limited to TAK-875, GPR120 modulators, in particular agonists, high affinity nicotinic acid receptor activators (HM74A) and SGLT1 inhibitors such as GSK1614235. An additional representative list of antidiabetic agents that can be combined with the compounds of the invention can be found, for example, in WO2011005611 from line 35 on page. 28 to line 19 on p. 30. Preferred antidiabetic agents are metformin and DPP-IV inhibitors (eg, sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin, and saxagliptin). Other antidiabetic agents may include palmitoyltransferase enzyme inhibitors or modulators, fructose 1,6-biphosphatase inhibitors, aldose reductase inhibitors, mineralocorticoid receptor inhibitors, TORC2 inhibitors, CCR2 and/or CCR5 inhibitors, PKC isoform inhibitors (e.g., PKCa, PKCe, PKCy), fatty acid synthetase inhibitors, serine palmitoyltransferase inhibitors, modulators of GPR81, GPR39, GPR43, GPR41, GPR105, Kv1.3, retinol binding protein 4, glucocorticoid receptor, somatostatin receptors (eg, SSTR1, SSTR2, SSTR3, and SSTR5), PDHK2 or PDHK4 inhibitors or modulators , MAP41<4 inhibitors. modulators of the IL1 family, including Ibeta. RHRalfa modulators. In addition, suitable antidiabetic agents include the mechanisms listed in Carpino, P.A., Goodwin, B. Expert Opin. Ther. Pat, 2010, 20(12), 1627-51.

Suitable antiobesity agents include 11β-hydroxysteroid dehydrogenase-1 (11P-HSD type 1) inhibitors, stearoyl-CoA desaturase-1 (SCD-1) inhibitor, MCR-4 agonists, cholecystokinin-A (CCK-A) agonists, reverse monoamine uptake (such as sibutramine), sympathomy

- 22 045908 methic agents, β ₃ adrenergic agonists, dopamine agonists (such as bromocriptine), melanocyte-stimulating hormone analogs, 5HT2c agonists, melanin-concentrating hormone antagonists, leptin (OB protein), leptin analogs, leptin agonists, galanin antagonists, lipase inhibitors ( such as tetrahydrolipstatin, i.e. orlistat), anorectic agents (such as bombesin agonist), neuropeptide-Y antagonists (for example, NPY Y5 antagonists), PYY _3-36 (including its analogues), thyromimetic agents, dehydroepiandrosterone or its analogue, glucocorticoid agonists or antagonists, orexin antagonists, glucagon-like peptide-1 agonists, ciliary neurotrophic factors (such as Axokine™, available from Regeneron Pharmaceuticals, Inc., Tarrytown, NY, and Procter & Gamble Company, Cincinnati, ON), human protein inhibitors, agouti-related (AGRP), ghrelin antagonists, histamine 3 antagonists or inverse agonists, neuromedin U agonists, MTP/ApoB inhibitors (e.g., gut-selective MTP inhibitors such as dirlotapide), opioid antagonist, orexin antagonist, combination of nalrexone with buproprion, etc. .

Preferred anti-obesity agents for use in the combinations of the invention include gut-selective MTP inhibitors (eg, dirlotapide, mitratapide and implitapide, R56918 (CAS No. 403987) and CAS No. 913541-47-6), CCKa agonists (eg, benzyl -2-[4-(III-indol3-ylmethyl)-5-oxo-1-phenyl-4,5-dihydro-2,3,6,10b-tetraaza-benzo[e]azulen-6-yl]-N -isopropyl-acetamide described in PCT Application Publication No. WO2005/116034 or US Application Publication No. 2005-0267100 A1), 5HT2c agonists (eg, lorcaserin), MCR4 agonist (eg, compounds described in US Pat. No. 6,818,658), lipase inhibitor (eg , cetilistat), PYY _3-36 (as used herein, PYY _3-36 includes analogs such as pegylated PYY _3-36 , such as those described in US Application Publication 2006/0178501), opioid antagonists (eg, nalrexone) , combination of nalrexone with buproprion, oleoyl-estrone (CAS No. 180003-17-2), obinepitide (TM30338), pramlintide (Symlin®), tesofensine (NS2330), leptin, liraglutide, bromocriptine, orlistat, exenatide (Byetta®), AOD-9604 (CAS No. 221231-10-3), phentermine and topiramate (trade name: Qsymia) and sibutramine. Preferably, the compounds of the invention and combination therapeutic agents are administered in combination with exercise and a reasonable diet.

The compounds of the invention can be used in combination with cholesterol-modulating agents (including cholesterol-lowering agents), such as a lipase inhibitor, an HMG-CoA reductase inhibitor, an HMG-CoA synthase inhibitor, an HMG-CoA reductase gene expression inhibitor, an HMG gene expression inhibitor -CoA synthase, MTP/Apo B secretion inhibitor, CETP inhibitor, bile acid absorption inhibitor, cholesterol absorption inhibitor, cholesterol synthesis inhibitor, squalene synthetase inhibitor, squalene epoxidase inhibitor, squalene cyclase inhibitor, combined squalene epoxidase/squalene cyclase inhibitor, fibrate, niacin, ion exchange resin, an antioxidant, an ACAT inhibitor or a bile acid sequestrant or such agent, such as mipomersen.

Examples of suitable cholesterol/lipid-lowering agents and lipid profile therapies include: HMG-CoA reductase inhibitors (eg, pravastatin, pitavastatin, lovastatin, atorvastatin, simvastatin, fluvastatin, NK-104 (also known as itavastatin or nisvastatin or nisbastatin) and ZD-4522 (also known as rosuvastatin or atavastatin or visastatin); squalene synthetase inhibitors; fibrates (eg, gemfibrozil, fenofibrate, clofibrate); bile acid sequestrants (such as questran, colestipol, colesevelam); ACAT inhibitors; MTP inhibitors; liposkigenase inhibitors; cholesterol absorption inhibitors (eg, ezetimibe); niacin-based agents (eg, niacin, niacor, slo-niacin); omega-3 fatty acids; and cholesteryl ester transfer protein inhibitors. Other anti-sclerotic agents include PCSK9 modulators (eg , alirotsumab and evolosumab).

In another embodiment, the compounds of the invention can be co-administered with agents for the treatment of non-alcoholic steatohepatitis (NASH) and/or non-alcoholic fatty liver disease (NAFLD), such as orlistat, TZD and other insulin-sensitizing agents, FGF21 analogues, metformin, omega-ethyl esters 3-acids (eg, Lovaza), fibrates, HMG-CoA reductase inhibitors, esithimble, probucol, ursodeoxycholic acid, TGR5 agonists, FXR agonists, vitamin E, betaine, pentoxifylline, CB1 antagonists, carnitine, N-acetylcysteine, reduced glutathione, lorcaserin, nalrexone plus buproprion, SGLT2 inhibitors, phentermine, topiramate, incretin analogues (GLP and GIP) and angiotensin receptor blockers.

In another embodiment, the additional pharmaceutical agent is selected from the group consisting of cysteamine or a pharmaceutically acceptable salt thereof, cystamine or a pharmaceutically acceptable salt thereof, an antioxidant compound, lecithin, vitamin B complex, bile salt preparations, cannabinoid receptor 1 (CB1) antagonists , cannabinoid receptor 1 (CB1) inverse agonists, peroxisome proliferator-activated receptor activity regulator, benzodiazepine or benzothiepine compound, antisense RNA construct to inhibit protein tyrosine phosphatase PTPRU, heteroatom-linked substituted piperidine and its derivatives, azacyclopentane derivative capable of inhibiting stearoyl-coenzyme-alpha del ta9 -desaturase, an acylamide compound with secretagogue or adiponectin inducer activity

- 23 045908 on, quaternary ammonium compound, glatiramer acetate, pentraxin proteins, HMG-CoA reductase inhibitor, N-acetylcysteine, isoflaphone compound, macrolide antibiotic, galectin inhibitor, antibody or any combination thereof.

Additional therapeutic agents include anticoagulants or coagulation inhibitory agents, antiplatelet or platelet inhibitory agents, thrombin inhibitors, thrombolytic or fibrinolytic agents, antiarrhythmic agents, antihypertensive agents, calcium channel blockers (L-type and T-type), cardiac glycosides, diuretics , mineralocorticoid receptor antagonists, NO donor agents such as organonirates, NO-stimulating agents such as phosphodiesterase inhibitors, cholesterol/lipid-lowering agents and lipid profile therapies, antidiabetic agents, antidepressants, anti-inflammatory agents (steroidal and non-steroidal), anti-inflammatory agents osteoporosis, hormone replacement therapies, oral contraceptives, antiobesity agents, antianxiety agents, antiproliferative agents, antineoplastic agents, antiulcer agents and agents against gastroesophageal reflux disease, growth hormone and/or growth hormone secretagogues, thyroid mimetics (including thyroid hormone receptor antagonist) , anti-infective agents, antiviral agents, antibacterial agents and antifungal agents.

Agents used in the ICU (intensive care unit) setting include, for example, dobutamine, dopamine, epinephrine, nitroglycerin, nitroprusside, etc.

Combination agents useful for treating vasculitis include, for example, azathioprine, cyclophosphamide, mycophenolate, mofetil, rituximab, etc.

In another embodiment of the invention, a combination is provided wherein the third agent is at least one agent selected from a factor Xa inhibitor, an anticoagulant, an antiplatelet agent, a thrombin inhibitory agent, a thrombolytic agent, and a fibrinolytic agent. Exemplary factor Xa inhibitors include apixaban and rivaroxaban. Examples of suitable anticoagulants for use in combination with the compounds of the invention include heparins (eg, unfractionated and low molecular weight heparins such as enoxaparin and dalteparin).

In another preferred embodiment, the third agent is at least one selected from warfarin, dabigatran, unfractionated heparin, low molecular weight heparin, synthetic pentasaccharide, hirudin, argatrobanas, aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone , piroxicam, ticlopidine, clopidogrel, tirofiban, eptifibatide, abciximab, melagatran, disulfatohirudin, tissue plasminogen activator, modified tissue plasminogen activator, antistreplase, urokinase and streptokinase.

A preferred third agent is at least one antiplatelet agent. Particularly preferred antiplatelet agents are aspirin and clopidogrel.

The term antiplatelet agents (or platelet inhibitory agents) as used herein means agents that inhibit platelet function, for example by inhibiting platelet aggregation, adhesion or granular secretion of platelets. Agents include, without limitation, various known nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone, piroxicam, and pharmaceutically acceptable salts or prodrugs thereof. Of the NSAIDs, aspirin (acetylsalicylic acid or ASA) and COX-2 inhibitors such as CELEBREX or piroxicam are preferred. Other suitable platelet inhibitory agents include IIb/IIIa antagonists (e.g., tirofiban, eptifibatide, and abciximab), thromboxane A2 receptor antagonists (e.g., ifetroban), thromboxane A2 synthetase inhibitors, PDE-III inhibitors (e.g., Pletal, dipyridamole) and their pharmaceutically acceptable salts or prodrugs.

_The term antiplatelet agents (platelet inhibitory agents) as used herein also includes ADP (adenosine diphosphate ₎ receptor antagonists, preferably P2Y1 and _P2Y12 purinergic receptor antagonists, with _P2Y12 being even more preferred. Preferred P ₂ Y ₁₂ receptor antagonists include ticagrelor, prasugrel, ticlopidine and clopidogrel, including pharmaceutically acceptable salts or prodrugs thereof. Clopidogrel is an even more preferred agent. Ticlopidine and clopidogrel are also preferred compounds because they are known to be gentle on the gastrointestinal tract when administered.

The term thrombin inhibitors (or antithrombin agents) as used herein means inhibitors of thrombin serine protease. When thrombin is inhibited, various thrombin-mediated processes, such as thrombin-mediated platelet activation (ie, for example, platelet aggregation, and/or granular secretion of plasminogen activator inhibitor 1 and/or serotonin), and/or fibrin formation are impaired. A number of thrombin inhibitors are known to those skilled in the art, and these inhibitors are contemplated for use in combination with the present compounds. Such inhibitors include, but are not limited to, boroarginine derivatives, boropeptides,

- 24 045908 dabigatran, heparins, hirudin, argatroban and melagatran, including their pharmaceutically acceptable salts and prodrugs. Boroarginine derivatives and boropeptides include N-acetyl and peptide boronic acid derivatives, such as the C-terminal alpha-aminoboronic acid derivatives of lysine, ornithine, arginine, homoarginine and their corresponding isothiouronium analogues. The term hirudin as used herein includes suitable derivatives or analogs of hirudin, referred to herein as hirulogs, such as disulfatohirudin. The term thrombolytic or fibrinolytic agents (or thrombolytics or fibrinolytics) as used herein means agents that lyse blood clots (thrombi). Such agents include tissue plasminogen activator (natural or recombinant) and its modified forms, antistreplase, urokinase, streptokinase, tenesteplase (TNK), lanoteplase (nPA), factor VIIa inhibitors, PAI-1 inhibitors (i.e. tissue plasminogen activator inhibitor inactivators ), inhibitors of alpha2-antiplasmin and anisoylated plasminogenstreptokinase activator complex, including pharmaceutically acceptable salts or prodrugs thereof. The term antistreplase as used herein refers to an anisoylated plasminogenstreptokinase activator complex as described, for example, in EP 028,489, the disclosure of which is incorporated herein by reference. The term urokinase as used herein refers to both double- and single-chain urokinase, the latter also being referred to as pro-urokinase.

Examples of suitable antiarrhythmic agents include: Class I agents (such as propafenone); Class II agents (such as metoprolol, atenolol, carvediol and propanolol); Class III agents (such as sotalol, dofetilide, aminodarone, azimilide and ibutilide); Class IV agents (such as diltiazem and verapamil); K+ channel openers such as I _Ach inhibitors and I _Kur inhibitors (eg, the compounds that are disclosed in WOOI/40231).

The compounds of the present invention can be co-administered with anti-heart failure agents such as ACE inhibitors (eg, captopril, enalapril, fosinopril, lisinopril, perindpril, quinapril, ramipril, trandolapril), angiotensin II receptor blockers (eg, candesartan, losartan, valsartan) , angiotensin receptor neprilysin inhibitors (sacubitril/valsartan), If channel blocker ivabradine, beta-adrenergic blockers (eg, bisoprolol, metoprolol succinate, carvedilol), aldosterone antagonists (eg, spironolactone, eplerenone), hydralazine and isosorbide dinitrate, diuretics (eg , furosemide, bumetanide, torsemide, chlorothiazide, amiloride, hydrochlorothiazide, indaramide, metolazone, triamterene) or digoxin.

The compounds of the invention can be used in combination with antihypertensive agents, and such antihypertensive activity is readily determined by those skilled in the art according to standard assays (eg, blood pressure measurements). Examples of suitable antihypertensive agents include: alpha-adrenergic blockers; beta-adrenergic blockers; calcium channel blockers (eg, diltiazem, verapamil, nifedipine and amlodipine); vasodilators (eg, hydralazine), diuretics (eg, chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendroflumethiazide, methylchlorothiazide, trichlorothiazide, polythiazide, benzthiazide, ethacrynic acid, tricrinafen, chlorthalidone, torsemide, furosemide, indapamide, metho zolone, musolimin, bumetanide, triamterene, amiloride, spironolactone); renin inhibitors; ACE inhibitors (eg, captopril, zofenopril, fosinopril, enalapril, ceranopril, cilazopril, delapril, pentopril, perindopril, quinapril, ramipril, trandolapril, lisinopril); AT-1 receptor antagonists (for example, losartan, irbesartan, valsartan); angiotensin receptor neprilysin inhibitors (sacubitril/valsartan); beta-adrenergic blockers (for example, bisoprolol, metoprolol succinate, carvedilol); ET receptor antagonists (eg, sitaxentan, atresentan and the compounds disclosed in US Pat. Nos. 5,612,359 and 6,043,265); dual ET/AII antagonist (eg the compounds disclosed in WO 00/01389); neutral endopeptidase (NEP) inhibitors; Vasopeptidase inhibitors (dual NEP-ACE inhibitors) (eg hemopatrylate and nitrates). An exemplary antianginal agent is ivabradine.

Examples of suitable calcium channel blockers (L-type or T-type) include diltiazem, verapamil, nifedipine and amlodipine and mibefradil.

Examples of suitable cardiac glycosides include digitalis and ouabain.

In one embodiment, the compounds of the invention can be administered in conjunction with one or more diuretics. Examples of suitable diuretics include (a) loop diuretics such as furosemide (such as LASIX™), torsemide (such as DEMADEX™), bemetanide (such as BUMEX™) and ethacrynic acid (such as EDECRIN™); (b) thiazide-type diuretics, such as chlorothiazide (such as DIURIL™, ESIDRIX™ or HYDRODIURIL™), hydrochlorothiazide (such as MICROZIDE™ or ORETIC™), benzothiazide, hydroflumethiazide (such as SALURON™), bendroflumethiazide, methylchlorothiazide, poly azide, trichloromethiazide and indapamide (such as LOZOL™); (c) phthalimidine-type diuretics such as chlorthalidone (such as HYGROTON™) and metlazone (such as ZAROXOLYN™); (d) quinazoline-type diuretics such as quinetasone; and (e) potassium-sparing diuretics such as triamterene (such as DYRENIUM™) and amiloride (such as MIDAMOR™ or MODURETIC™).

In another embodiment, the compounds of the invention can be administered in conjunction with a loop diuretic

- 25 045908 com. In yet another embodiment, the loop diuretic is selected from furosemide and torsemide. In yet another embodiment, the compounds of the invention can be co-administered with furosemide. In yet another embodiment, the compounds of the invention may be co-administered with torsemide, which may optionally be torsemide in a controlled or modified release form.

In another embodiment, the compounds of the invention may be co-administered with a thiazide-type diuretic. In yet another embodiment, the thiazide-type diuretic is selected from the group consisting of chlorothiazide and hydrochlorothiazide. In yet another embodiment, the compounds of the invention can be co-administered with chlorothiazide. In yet another embodiment, the compounds of the invention can be co-administered with hydrochlorothiazide.

In another embodiment, the compounds of the invention may be co-administered with a phthalimidine-type diuretic. In yet another embodiment, the phthalimidine type diuretic is chlorthalidone. Examples of suitable mineralocorticoid receptor antagonists include spironolactone and eplerenone. Examples of suitable phosphodiesterase inhibitors: PDE III inhibitors (such as cilostazol); and PDE V inhibitors (such as sildenafil).

Those skilled in the art will appreciate that the compounds of this invention may also be used in combination with other cardiovascular and cerebrovascular therapies including PCI (percutaneous coronary angioplasty, stenting, drug-eluting stents, stem cell therapy, and medical devices such as implanted pacemakers, defibrillators or cardiac resynchronization therapy.

In particular, when provided in unit dosage form, there is the possibility of chemical interaction between the combined active ingredients. For this reason, when the first therapeutic agent and the second therapeutic agent are combined in a unit dosage form, then they can be formulated such that, although the active ingredients are combined in the unit dosage form, physical contact between the active ingredients is minimized (that is, there is reduced). For example, one active ingredient may be enteric coated. By coating one of the active ingredients with an enteric coating, it is possible not only to minimize contact between the combined active ingredients, but also to control the release of one of these components in the gastrointestinal tract so that one of these components is released in the intestine rather than in the stomach. One of the active ingredients may be coated with a substance that provides sustained release throughout the gastrointestinal tract and also serves to minimize physical contact between the combined active ingredients. In addition, the sustained release component may be further coated so that release of the component occurs only in the intestine. Yet another approach may involve preparing a combination product in which one component is coated with a polymer providing sustained and/or enteral release and the other component is also coated with a polymer such as low viscosity hydroxypropyl methylcellulose (HPMC) or other substance known in the art. in this area in order to further separate the active components from each other. The polymer coating serves to create an additional barrier that prevents interaction with another component.

These and other ways of minimizing contact between the components of the combination products of the present invention, whether administered in a single dosage form or administered in separate forms but at the same time in the same manner, will be apparent to those skilled in the art armed with the present description of the invention.

When treated with a combination therapeutic agent, both compounds of this invention and other drugs are administered to a mammal (eg, humans, male or female) by standard methods.

The dosage of each therapeutic agent, e.g., Compound A, Compound D, and any additional therapeutic agent, will generally depend on a number of factors, including the health status of the subject being treated, the extent of treatment desired, the nature and type of concurrent therapy, if any, and the frequency of treatment and the nature of the desired effect. Typically, the dosage range of each therapeutic agent is from about 0.001 mg to about 100 mg per kilogram of body weight of the individual per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight of the individual per day. However, some variability in the overall dosage may be required depending on the age and body weight of the subject being treated, the route of administration prescribed, the particular anti-obesity agent being administered, and the like. Determination of dosage ranges and optimal dosages for a particular patient is also within the purview of one skilled in the art having the benefit of the current description of the invention.

According to the methods of treatment of the invention, a compound of the invention or a combination of a compound of the invention and at least one additional pharmaceutical agent (referred to herein as a combination) is administered to a subject in need of such treatment, preferably

- 26 045908 specifically in the form of a pharmaceutical composition. In an aspect of the combination of the invention, the compound of the invention and at least one other pharmaceutical agent (eg, another anti-obesity agent) can be administered either separately or in a pharmaceutical composition containing both of them. It is generally preferred that such administration be oral.

When a combination of a compound of the invention and at least one other pharmaceutical agent is administered together, then such administration may be sequential in time or simultaneous. Concomitant administration of drug combinations is usually preferred. For sequential administration, the compound of the invention and the additional pharmaceutical agent can be administered in any order. It is generally preferred that such administration be oral. It is particularly preferred that such administration be oral and simultaneous. When a compound of the invention and an additional pharmaceutical agent are administered sequentially, then each may be administered in the same manner or in different manners.

According to the methods of the invention, the compound of the invention or combination is preferably administered in the form of a pharmaceutical composition. Accordingly, a compound of the invention or combination may be administered to a patient individually or together by any conventional oral, rectal, transdermal, parenteral (e.g., intravenous, intramuscular, or subcutaneous), intracisternal, intravaginal, intraperitoneal, topical (e.g., powder, ointment, cream, spray or lotion), buccal or nasal dosage form (eg, spray, drops or inhaled form).

The compounds of the invention or combinations may be administered alone, but will typically be administered in admixtures with one or more suitable pharmaceutical excipients, excipients, diluents or carriers known in the art and selected taking into account the intended route of administration and standard pharmaceutical practice. The compound of the invention or combination may be formulated into immediate, delayed, modified, sustained, pulsed or controlled release dosage forms depending on the desired route of administration and the specificity of the release profile according to therapeutic needs.

The pharmaceutical composition contains a compound of the invention or combination in an amount typically ranging from about 1% to about 75%, 80%, 85%, 90%, or even 95% by weight of the composition, typically ranging from about 1%, 2% or 3% to about 50%, 60% or 70%, more often in the range from about 1%, 2% or 3% to less than 50%, such as about 25%, 30% or 35%.

Methods for preparing various pharmaceutical compositions with specific amounts of active compound are known to those skilled in the art. For examples, see Remington: The Practice of Pharmacy, Lippincott Williams and Wilkins, Baltimore Md. 20.sup.th ed. 2000.

Compositions suitable for parenteral injection generally contain pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution in sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers or diluents (including solvents and carriers) include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerin, etc.), suitable mixtures thereof, triglycerides including vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The preferred carrier is the caprylic/capric acid glycerin or propylene glycol ester of the Miglyol® brand (e.g., Miglyol.RTM. 812, Miglyol.RTM. 829, Miglyol.RTM. 840), available from Condea Vista Co., Cranford, N.J. Proper flow can be maintained, for example, through the use of a coating such as lecithin, maintaining the required particle size in the case of dispersions, and the use of surfactants.

These parenteral injection compositions may also contain excipients such as preservatives, wetting agents, emulsifiers and dispersing agents. Prevention of microorganism contamination of compositions can be achieved by various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid and the like. It may be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of injectable pharmaceutical compositions can be achieved through the use of agents capable of delaying absorption, such as aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, chewing gums, wafers, pills, powders, and multiparticulate preparations (granules). In such solid dosage forms, the compound of the invention or combination is admixed with at least one inert excipient, diluent or carrier. Suitable excipients, diluents or carriers include substances such as sodium citrate or dicalcium phosphate and/or (a) one or more fillers or bulking agents (for example, microcrystalline cellulose (available as Avicel.TM. from FMC Corp.), starches, lactose, sucrose, mannitol, silicic acid, xylitol, sorbitol, dextrose, calcium hydrogen phosphate, dextrin, alpha-cyclodextrin, beta-cyclodextrin,

- 27 045908 liethylene glycol, medium chain fatty acids, titanium oxide, magnesium oxide, aluminum oxide, etc.); (b) one or more binding agents (e.g., carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gelatin, gum arabic, ethylcellulose, polyvinyl alcohol, pullulan, pregelatinized starch, agar, tragacanth, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, etc.) P.); (c) one or more humectants (eg, glycerin, etc.); (d) one or more disintegrants (e.g., agar agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, sodium carbonate, sodium lauryl sulfate, sodium starch glycolate (available as Explotab™ from Edward Mendell Co.), cross-linked polyvinylpyrrolidone, cross-carmellose sodium A-type (available as Ac-disol.TM.), potassium polyacryline (ion exchange resin), etc.); (e) one or more dissolution retardants (eg, paraffin, etc.); (f) one or more absorption accelerators (eg, quaternary ammonium compounds, etc.); (g) one or more wetting agents (eg, cetyl alcohol, glycerol monostearate, etc.); (h) one or more adsorbents (eg, kaolin, bentonite, etc.); and/or/and one or more lubricants (for example, talc, calcium stearate, magnesium stearate, stearic acid, polyoxyl stearate, cetanol, talc, hydrogenated castor oil, sucrose esters of fatty acids, dimethylpolysiloxane, microcrystalline wax, yellow beeswax, white beeswax, solid polyethylene glycols, sodium lauryl sulfate, etc.). In the case of capsules and tablets, dosage forms may also contain buffering agents.

Solid compositions of this type can also be used as fillers in soft or hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms such as tablets, dragees, capsules and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may also contain opacifying agents and may also be compositions that release a compound of the invention and/or an additional pharmaceutical agent in a sustained manner. Examples of potting compositions that can be used are polymeric substances and waxes. The drug may also be in microencapsulated form, if appropriate, with one or more of the above excipients.

In a tablet, the active agent will typically comprise less than 50% by weight of the composition, such as less than about 10%, such as 5% or 2.5% by weight. The majority of the composition contains fillers, diluents, disintegrants, lubricants and optionally flavoring agents . The composition of these excipients is well known in the art. Often, fillers/diluents will contain mixtures of two or more of the following: microcrystalline cellulose, mannitol, lactose (all types), starch and dicalcium phosphate. The filler/diluent mixtures typically constitute less than 98% of the composition and preferably less than 95%, such as 93.5%. Preferred disintegrants include Ac-di-sol.TM., Explotab.TM., starch and sodium lauryl sulfate. Disintegrant, when present, will typically constitute less than 10% of the composition or less than 5%, such as about 3%. The preferred lubricant is magnesium stearate. The lubricant, when present, will typically constitute less than 5% of the composition or less than 3%, such as about 1%.

Tablets can be produced by standard tableting processes, such as direct compression or wet granulation, dry or melt granulation, melt solidification and extrusion. Tablet cores may be single or multilayer and may be coated with suitable outer coatings known in the art.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the compound of the invention or combination, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (eg, cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil, etc.), Miglyole™ (available from CONDEA Vista Co., Cranford, N.J.), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters or mixtures of these substances, etc.

In addition to such inert diluents, the composition may also contain excipients such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and flavoring agents.

Oral liquid forms of the compounds or combinations of the invention include solutions where the active compound is completely dissolved. Examples of solvents include all pharmaceutically conventional solvents suitable for oral administration, in particular those in which the compounds of the invention have good solubility, for example polyethylene glycol, polypropylene glycol, edible oils and glyceryl and glyceride systems. Glyceryl and glyceride systems may include, for example, the following trademarked products (and associated

- 28 045908 tent products): Captex™ 355 EP (glyceryl tricaprylate/caprate from Abitec, Columbus Ohio), Crodamol™ GTC/C (medium chain triglyceride from Croda, Cowick Hall, UK) or Labrafac™ CC (medium chain triglyceride from Gattefosse), Captex ™ 500P (glyceryl triacetate, i.e. triacetin, from Abitec), Capmul™ MCM (medium chain mono- and diglycerides from Abitec), Migyol™ 812 (caprylic/capric triglyceride from Condea, Cranford NJ), Migyol™ 829 (caprylic/capric /succinic triglyceride from Condea), Migyol™ 840 (propylene glycol dicaprylate/dicaprate from Condea), Labrafil™ M1944CS (oleoyl macrogol-6 glycerides from Gattefosse), Peceol™ (glyceryl monooleate from Gattefosse) and Maisine™ 35-1 (glyceryl monooleate from Gattefosse ) . Of particular interest are medium chain (approximately C8- _C10 ) triglyceride oils. These solvents often form the predominant part of the composition, i.e. greater than about 50%, typically greater than about 80%, such as about 95% or 99%. Excipients and additives may also be included with the solvents, primarily as taste-masking agents, flavoring and flavoring agents, antioxidants, stabilizers, texture and viscosity modifiers, and solubilizers.

Suspensions, in addition to the compound of the invention or combination, may further contain carriers such as suspending agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures of these substances, etc. P.

Compositions for rectal or vaginal administration preferably include suppositories, which can be prepared by mixing a compound of the invention or combination with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax, which is solid at normal room temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity, releasing the active component(s).

Dosage forms for topical administration of compounds of the invention or combinations include ointments, creams, lotions, powders and sprays. The drugs are mixed with a pharmaceutically acceptable excipient, diluent or carrier and any preservatives, buffers and propellants that may be necessary.

When compounds are poorly soluble in water, eg, less than about 1 μg/ml, then liquid compositions in solubilizing, non-aqueous solvents, such as the medium chain triglyceride oils discussed above, are the preferred dosage form for these compounds.

Solid amorphous dispersions, including dispersions formed by spray drying, are the preferred dosage form for the poorly soluble compounds of the invention. Solid amorphous dispersion means a solid in which at least a portion of the poorly soluble compound is in amorphous form and dispersed in a water-soluble polymer. Amorphous means that the poorly soluble compound is not crystalline. Crystalline means that the compound exhibits long-range order in three dimensions with at least 100 repeating units in each dimension. Thus, the term amorphous covers not only matter that is essentially without order, but also matter that may have some small degree of order, but order in less than three dimensions and/or only over short distances. The amorphous material can be characterized by methods known in the art, such as X-ray powder diffraction (XRD), crystallography, solid-state NMR, or thermal methods such as differential scanning colorimetry (DSC).

Preferably, at least the majority (ie, at least about 60% by weight) of the poorly soluble compound in the solid amorphous dispersion is amorphous. The compound may exist in a solid amorphous dispersion in relatively pure amorphous domains or regions, as a solid solution of the compound homogeneously distributed throughout the polymer, or any combination of these states or those states intermediate between them. Preferably, the solid amorphous dispersion is substantially homogeneous such that the amorphous compound is distributed as uniformly as possible throughout the polymer. As used herein, substantially homogeneous means that the fraction of the compound that is found in relatively pure amorphous domains or regions in the solid amorphous dispersion is relatively small, an order of magnitude less than 20 wt.% and preferably less than 10 wt.% of the total amount of drug.

Water-soluble polymers suitable for use in solid amorphous dispersions must be inert in the sense that they do not react chemically with the poorly soluble compound in an unfavorable manner, are pharmaceutically acceptable, and have at least some solubility in aqueous solution at a physiologically relevant pH (e.g. , 1-8). The polymer may be neutral or ionizable and should have a solubility in aqueous solvents of at least 0.1 mg/ml within at least part of the pH range 1-8.

Water-soluble polymers suitable for use with the invention may be cellulosic or non-cellulosic. Polymers can be neutral or ionizable in water.

- 29 045908 creation. Of course, ionizable and cellulosic polymers are preferred, with ionizable cellulose polymers being more preferred.

Exemplary water-soluble polymers include hydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl methyl cellulose phthalate (HPMCP), carboxymethyl ethyl cellulose (CMEC), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), polyvinyl pyrrolidone (PVP), hydroxypropyl cellulose (HPC). ), methylcellulose (MC), block copolymers of ethylene oxide and propylene oxide (PEO/PPO, also known as polaxamers) and mixtures thereof. Particularly preferred polymers include HPMCAS, HPMC, HPMCCP, CMEC, CAP, CAT, PVP, poloxamers, and mixtures thereof. The most preferred is HPMCAS (see European Patent Application Publication No. 0901786 A2, the description of which is incorporated herein by reference.

Solid amorphous dispersions can be prepared by any method of forming solid amorphous dispersions that results in at least a major portion (at least 60%) of the poorly soluble compound being in an amorphous state. Such methods include mechanical, thermal and solvent methods. Exemplary mechanical methods include grinding and extrusion; melt processes including high temperature melting, solvent modified melting, and melt freezing methods; and solvent methods including precipitant deposition, spray coating and spray drying. See, for example, the following US patents, the respective disclosures of which are incorporated herein by reference: Nos. 5,456,923 and 5,939,099, which describe the formation of dispersions by extrusion processes; Nos. 5340591 and 4673564, which describe the formation of dispersions by grinding methods; and Nos. 5707646 and 4894235, which describe the formation of dispersions by melt-freeze methods. In a preferred method, the solid amorphous dispersion is formed by spray drying, as disclosed in European Patent Application Publication No. 0901786 A2. In this method, the compound and polymer are dissolved in a solvent such as acetone or methanol, and the solvent is then quickly removed from solution by spray drying to form a solid amorphous dispersion. Solid amorphous dispersions can be prepared such that they contain up to about 99 wt.% of the compound, for example 1 wt.%, 5 wt.%, 10 wt.%, 25 wt.%, 50 wt.%, 75 wt. %, 95 wt.% or 98 wt.%, as required.

The solid dispersion may be used as the dosage form itself, or it may serve as a manufacturing use product (MUP) in the preparation of other dosage forms such as capsules, tablets, solutions or suspensions. An example of an aqueous suspension is a 1:1 (w/w) aqueous suspension of compound/spray-dried HPMCAS-HF dispersion containing 2.5 mg/ml of compound in 2% polysorbate-80. Solid dispersions for use in a tablet or capsule will typically be mixed with other excipients or excipients typically found in such dosage forms. For example, an exemplary capsule fill contains a 2:1 (w/w) compound/spray-dried dispersion of HPMCAS-MF (60%), lactose (Fast Flow) (15%), microcrystalline cellulose (e.g., Avicel ^{(R0-102 )} (15.8%), sodium starch (7%), sodium lauryl sulfate (2%) and magnesium stearate (1%).

HPMCAS polymers are available in low, medium and high grades as Aqoa ^(R)-LF , Aqoat ^(R)-MF and Aqoat ^(R)-HF respectively from Shin-Etsu Chemical Co., LTD, Tokyo, Japan. Higher MF and HF grades are generally preferred.

The following paragraphs describe illustrative compositions, dosages, etc. useful in non-human animals. Administration of Compound A or a pharmaceutically acceptable salt thereof in combination with Compound D or a pharmaceutically acceptable salt thereof as two agents or in combinations with another agent may be orally or non-orally.

The amount of Compound A or a pharmaceutically acceptable salt thereof with Compound D or a pharmaceutically acceptable salt thereof together or in combination with another agent is administered so that an effective dose is obtained. Typically, the daily dose administered orally to an animal is from about 0.01 to about 1000 mg/kg body weight, such as from about 0.01 to about 300 mg/kg, or from about 0.01 to about 100 mg/kg. kg, or from about 0.01 to about 50 mg/kg body weight, or from about 0.01 to about 25 mg/kg, or from about 0.01 to about 10 mg/kg, or from about 0.01 to approximately 5 mg/kg. The daily dose of Compound A administered may be 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg or 50 mg. The daily dosage may be divided into multiple doses, such as BID/Q with dosing administered at 12-hour intervals. For example, in some cases, the daily dose of Compound A can be administered as 15 mg q12 hours. The daily dose of Compound D administered may be 50 mg, 100 mg, 200 mg or 300 mg. The daily dosage may be divided into multiple doses, such as BID/Q with dosing administered at 12-hour intervals. For example, in some cases, the daily dose of Compound D can be administered as 300 mg q12 hours.

Conveniently, the compound of the invention (or combination) can be added to drinking water so that a therapeutic dosage of the compound is ingested with daily water intake. The compound can be measured directly into drinking water, preferably in liquid form, water

- 30 045908 Roman concentrate (such as an aqueous solution of water-soluble salt).

For convenience, the compound of the invention (or combination) may also be added directly to food, either as such or in the form of an animal feed supplement, also referred to as a premix or concentrate. A premix or concentrate of the compound in an excipient, diluent or vehicle is more often used to incorporate the agent into the feed. Suitable excipients, diluents or carriers are liquid or solid as desired, such as water, various flours such as alfalfa meal, soybean meal, cottonseed oil meal, flaxseed meal, corn cob meal and corn meal, molasses , urea, bone meal and mineral mixtures such as those commonly used in poultry feed. A particularly effective excipient, diluent or carrier is the respective animal feed itself, a small portion of such feed. The carrier facilitates uniform distribution of the compound in the final feed with which the premix is mixed. Preferably, the compound is thoroughly mixed into the premix and then into the feed. In this regard, the compound can be dispersed or dissolved in a suitable oily carrier such as soybean oil, corn oil, cottonseed oil and the like, or in a volatile organic solvent and then mixed with the carrier. It should be understood that the proportion of compound in the concentrate can be varied within wide limits since the amount of compound in the finished feed can be adjusted by mixing a suitable proportion of the premix into the feed to obtain the desired level of compound.

High performance concentrates can be mixed by the feed manufacturer with a protein carrier such as soybean meal and other meal types as described above to produce concentrated supplements that are suitable for direct feeding to animals. In such cases, the animals are allowed to consume their normal diet. Alternatively, such concentrated supplements may be added directly to the feed to produce a nutritionally balanced finished feed containing a therapeutically effective level of the compound of the invention. The mixtures are thoroughly mixed using standard methods, such as in a V-blender, to ensure homogeneity.

If the additive is used as a feed additive, it also helps ensure that the compound is evenly distributed throughout the fed feed.

Drinking water and feed effective in increasing lean meat deposition and improving the lean to fat ratio are typically prepared by mixing a compound of the invention with an amount of animal feed sufficient to provide from about 10 ^-3 to about 500 ppm. (ppm) compounds in feed or water.

A preferred medicated feed for swine, cattle, sheep and goats typically contains from about 1 to about 400 grams of a compound of the invention (or combination) per ton of feed, with the optimal amount for these animals typically being from about 50 to about 300 grams per ton stern.

Preferred poultry and pet foods typically contain from about 1 to about 400 grams, and preferably from about 10 to about 400 grams, of a compound of the invention (or combination) per ton of feed.

For parenteral administration to animals, a compound of the invention (or combination) can be formulated as a paste or granule and administered as an implant, typically under the scalp or ear of an animal in which it is sought to increase lean deposition and improve the lean to fat ratio.

Paste formulations can be prepared by dispersing the drug in a pharmaceutically acceptable oil such as peanut oil, sesame oil, corn oil or the like.

Granules containing a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof in combination with Compound D or a pharmaceutically acceptable salt thereof, pharmaceutical composition or combination, can be prepared by mixing Compound A or a pharmaceutically acceptable salt thereof with Compound D or a pharmaceutically acceptable salt thereof, wherein a diluent such as carbowax, carnauba wax and the like, and a lubricant such as magnesium or calcium stearate may be added to improve the granulation process.

Of course, it is recognized that more than one pellet can be administered to an animal to achieve a desired dose level that will provide the desired increase in lean meat deposition and improvement in the lean to fat ratio. Moreover, implants can also be placed periodically during treatment of the animal to maintain proper levels of drug in the animal's body.

The invention has several advantageous veterinary features. For a pet owner or veterinarian who wishes to increase leanness and/or remove unwanted fat from pets, the present invention provides a means by which this can be accomplished. For poultry farmers, beef cattle breeders and pig farmers, using the method of the invention produces leaner animals for which they command higher selling prices from the meat industry.

- 31 045908

Examples

Unless specifically stated otherwise, starting materials are generally available from commercial sources such as Aldrich Chemicals Co. (Milwaukee, WI), Lancaster Synthesis, Inc. (Windham, NH), Acros Organics (Fairlawn, NJ), Maybridge Chemical Company, Ltd. (Cornwall, England) and Tyger Scientific (Princeton, NJ). Some common abbreviations and acronyms are used and may include: AcOH (acetic acid), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), CDI (1,1'carbonyldiimidazole), DCM (dichloromethane), DEA (diethylamine), DIPEA (NN-diisopropylethylamine). DMAP (4-dimethylaminopyridine), DMF (dimethylformamide), DMSO (dimethyl sulfoxide), EDCI (N(3-dimethylaminopropyl)-N'-ethylcarbodiimide), Et2O (diethyl ether), EtOAc (ethyl acetate), EtOH (ethanol) , G or g (gram), HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanamine), HBTU (O-benzotriazol-1-yl-H,^ MN-tetramethyluronium hexafluorophosphate), HOBT (1-hydroxybenzotriazole), H or h (hour), IPA (isopropyl alcohol), KHMDS (potassium hexamethyldisilazane), MeOH (methanol), L or l (liter), ml (milliliter) MTBE (tert-butyl methyl ether), mg (milligram), NaBH(OAc) ₃ (sodium triacetoxyborohydride), NaHMDS (sodium hexamethyldisilazane), NMP (N-methylpyrrolidone), RH (relative humidity), RT or rt. (room temperature, which is the same as ambient temperature (about 20 to 25°C)), SEM ([2(trimethylsilyl)ethoxy]methyl), TEA (triethylamine), TFA (trifluoroacetic acid), THF (tetrahydrofuran ) and T ₃ P (propanephosphonic acid anhydride).

The 1H nuclear magnetic resonance (NMR) spectra were in all cases consistent with the proposed structures. Characteristic chemical shifts (8) are given in parts per million (ppm) relative to the residual proton signal in the deuterated solvent (CHCl ₃ at 7.27 ppm; CD2HOD at 3.31 ppm) and are given using standard abbreviations for designations of the main peaks: for example, s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broadened.

ssNMR stands for solid-state NMR.

XRD stands for X-ray powder diffraction.

The term is essentially the same when used to describe powder x-ray diffraction patterns to include patterns in which the peaks are within a standard deviation of +/-0.2° 2θ.

As used herein, the term substantially pure, with reference to a particular crystalline form, means that the crystalline form includes less than 10%, preferably less than 5%, preferably less than 3%, preferably less than 1% by weight of any other physical form of Compound A or Compound D .

Reactions were carried out in air or, when oxygen- or moisture-sensitive reagents or intermediates were used, in an inert atmosphere (nitrogen or argon). When practical, the reactors were dried under dynamic vacuum using a heat gun and anhydrous solvents were used (Sure-Seal™ products from Aldrich Chemical Company, Milwaukee, Wisconsin or DriSolv™ products from EMD Chemicals, Gibbstown, NJ). Commercial solvents and reagents were used without further purification. When indicated, reaction mixtures were microwave heated using Biotage Initiator or Personal Chemistry Emrys Optimizer microwave reactors. The reactions were monitored using thin layer chromatography (TLC), liquid chromatography/mass spectrometry (LCMS), high performance liquid chromatography (HPLC) and/or gas chromatography/mass spectrometry (GCMS) analyses. TLC was performed on silica gel plates precoated with a fluorescence indicator (excitation wavelength 254 nm) and visualized under UV light and/or using I ₂ , KMnO ₄ , CoCl ₂ , phosphomolybdic acid and/or cerium ammonium molybdate dyes. LCMS data were acquired on an Agilent 1100 Series instrument with a Leap Technologies autosampler, Gemini C18 columns, MeCN/water gradients, and modifiers of either TFA, formic acid, or ammonium hydroxide. The column eluent was analyzed using a Waters ZQ mass spectrometer scanning in both positive and negative ion modes from 100 to 1200 Da (Dalton). Other similar devices have also been used. HPLC data were acquired on an Agilent 1100 Series instrument using Gemini or XBridge C18 columns, MeCN/water gradients, and either TFA or ammonium hydroxide modifiers. GCMS data were acquired using a Hewlett Packard 6890 oven with an HP 6890 injector, an HP-1 column (12 mx0.2 mmx0.33 μm) and helium carrier gas. The sample was analyzed on an HP 5973 mass selective detector scanning from 50 to 550 Da using electron ionization. Purifications were performed by medium-pressure liquid chromatography (MPLC) using an Isco CombiFlash Companion, AnaLogix IntelliFlash 280, Biotage SP1, or Biotage Isolera One instrument and prepackaged Isco RediSep or Biotage Snap silica cartridges. Chiral purifications were carried out by chiral supercritical fluid chromatography (SFC) using Berger or Thar instruments; ChiralPAK-AD, -AS, -IC, Chiralcel-OD or -OJ columns; and mixtures of CO ₂ with MeOH, EtOH, iPrOH or MeCN, pure or modified with TFA or iPrNH ₂ . UV detection was used to trigger fraction collection.

- 32 045908

Mass spectrometric data are from LCMS analyses. Mass spectrometry (MS) was performed using atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), electron impact ionization (EI), or electron scattering (ES) sources. Proton nuclear magnetic spectroscopy (1H NMR) chemical shifts are given in parts per million with a downfield shift from tetramethylsilane and were recorded on Varian 300, 400, 500 or 600 MHz spectrometers. Chemical shifts are expressed in parts per million (ppm, δ) referred to the residual peaks of the deuterated solvent. Peak shapes are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br s, broadened singlet; arr, apparent. Analytical SFC data were recorded on a Berger analytical instrument as described above. Optical rotation data were collected on a PerkinElmer model 343 polarimeter using a 1 dm cell. Silica gel chromatography was performed using Biotage or ISCO medium pressure systems using columns prepackaged by various commercial suppliers including Biotage and ISCO. Microanalyses were carried out by Quantitative Technologies Inc. and were within 0.4% of the calculated values.

Unless otherwise stated, chemical reactions were performed at room temperature (approximately 23 degrees Celsius).

The compounds and intermediates described below were named using the naming convention provided by ChemBioDraw Ultra, Version 12.0 (CambridgeSoft Corp., Cambridge, Massachusetts). The naming convention provided by ChemBioDraw Ultra, Version 12.0 is well known to those skilled in the art, and it is believed that the naming convention provided by ChemBioDraw Ultra, Version 12.0 is generally consistent with IUPAC (International Union of Pure and Applied Chemistry) recommendations. according to the nomenclature of organic chemistry and with the rules of the CAS Index. Unless otherwise stated, all reagents were obtained commercially without further purification or were prepared using methods known in the literature.

The terms concentrated, evaporated and concentrated in vacuo refer to the removal of solvent under reduced pressure on a rotary evaporator with a bath temperature of less than 60°C. The abbreviations min and h stand for minutes and hours, respectively. The term TLC refers to thin layer chromatography, room temperature or ambient temperature means a temperature of 18 to 25°C, GCMS refers to gas chromatography/mass spectrometry, LCMS refers to liquid chromatography/mass spectrometry, UPLC refers to ultra performance liquid chromatography, and HPLC refers to high pressure liquid chromatography, SFC refers to supercritical fluid chromatography.

Hydrogenation can be carried out in a Parr shaker under hydrogen gas pressure or in a Thales-nano H-Cube flow hydrogenation apparatus at full load of hydrogen and a flow rate of 1-2 ml/min at a specified temperature.

HPLC, UPLC, LCMS, GCMS, and SFC retention times were measured using the methods specified in the procedures.

Obtaining Intermediates and Compound Examples

Example 1 (DGAT2i Compound/Compound D): ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide

Stage 1: 3-Ethoxypyridine

Cesium carbonate (12 mol, 1.5 eq.) and ethyl iodide (9.7 mol, 1.2 eq.) were added to a solution of 3hydroxypyridine (8.10 mol, 1.0 eq.) in acetone (12 L) at 15 °C. This reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was filtered and the organic layer was concentrated to obtain the crude product. Ethyl acetate (20 L) was added and washed with water (3x5 L). The organic layer was dried over sodium sulfate, filtered and concentrated to give 3-ethoxypyridine (620 g, 62%) as an oil. 1H NMR (400 MHz, CDCl ₃ ) δ 1.44 (t, 3H), 4.07 (q, 2H), 7.15-7.23 (m, 2H), 8.20 (dd, 1H), 8.30 (d, 1H).

Step 2: 3-Ethoxypyridine-1-oxide meta-chloroperoxybenzoic acid (6.5 mol, 1.3 eq.) was added to a solution of 3-ethoxypyridine (5.0 mol, 1.0 eq.) in dichloromethane (12 L) at 10°C. This reaction mixture was stirred at room temperature for 24 hours. Sodium thiosulfate (4 kg, in 5 L of water) was added. The reaction mixture was stirred at 15°C for 2 hours. Another portion of sodium thiosulfate (1.5 kg, in 5 liters of water) was added. The reaction mixture was stirred at 15°C for 1 hour. The mixture was extracted with dichloromethane (16 x 10 L). The combined organic layers were concentrated to give the crude product. The crude product was purified by column chromatography on silica gel (dichloromethane

- 33 045908 tan:methanol 100:1-10:1) to obtain the title compound (680 g, 97%) as a brown oil. This oil was further purified by trituration with petroleum ether (4 L) at room temperature for 24 hours to give 3-ethoxypyridine-1-oxide (580 g, 83%) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 1.41 (t, 3H), 4.02 (q, 2H), 6.84 (dd, 1H), 7.12 (dd, 1H), 7.85 (d, 1H), 7.91-7.95 ( m, 1H).

Step 3: 2-((5-Bromopyridin-3-yl)oxy)-3-ethoxypyridine

This reaction was carried out in five parallel batches.

Diisopropylethylamine (2.69 mol, 3.7 eq.) and bromotripyrrolidinophosphonium hexafluorophosphate (0.93 mol, 1.3 eq.) were added to a stirred solution of 3-ethoxypyridine-1-oxide (0.72 mol, 1.0 eq. ) and 3bromo-5-hydroxypyridine (0.72 mol, 1.0 eq.) in tetrahydrofuran (2500 ml) at room temperature. This reaction mixture was stirred at room temperature for 2 days, then the individual batches were combined into one batch. The resulting suspension was concentrated to dryness and dissolved in dichloromethane (25 L). The organic layer was washed with 1 N. sodium hydroxide (15 l), water (3 x 20 l) and brine (20 l). The organic layer was dried over sodium sulfate, filtered and concentrated to give an oil. This crude oil was purified by silica gel column chromatography (petroleum ether:ethyl acetate 10:1-1:1) to obtain the crude product as a brown solid. This solid was triturated with methyl tert-butyl ether:petroleum ether (1:10; 11 L) to give 2-((5-bromopyridin-3-yl)oxy)-3-ethoxypyridine (730 g, 69%) as a yellowish solid. ^1H NMR (400 MHz, CDCl ₃ ) δ 1.49 (t, 3H), 4.16 (q, 2H), 7.04 (dd, 1H), 7.25 (dd, 1H), 7.68-7.73 (m, 2H), 8.44 ( d, 1H), 8.49 (d, 1H). MS (ERI+) 297.1 (M+N).

Step 4: Ethyl 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidin-5-carboxylate

A solution of 2-((5-bromopyridin-3-yl)oxy)-3-ethoxypyridine (300 mmol, 1.0 eq.) in tetrahydrofuran (1.3 L) was degassed with nitrogen for 30 minutes. Turbo Grignard reagent (RMgCl-LiCl) (390 mmol, 1.3 eq, 1.3 M in tetrahydrofuran) was added at room temperature at a rate such that the internal temperature was maintained below 30°C. The reaction mixture was left to cool to room temperature and stirred for 3 hours. The reaction mixture was cooled to 10°C and zinc chloride (390 mmol, 1.3 eq., 1.9 M in 2-methyltetrahydrofuran) was added at a rate to maintain the temperature below 15°C. The resulting suspension was warmed to room temperature until all the precipitate had dissolved and then cooled again to 10°C. Ethyl 2chloropyrimidine-5-carboxylate (360 mmol, 1.2 eq.) and palladium(II) dichloro[bis(2-(diphenylphosphino)phenyl)ether] (6.00 mmol, 0.02 eq.) were added as solids. The resulting suspension was degassed with nitrogen for 30 minutes, then heated to 50°C for 16 hours. The reaction mixture was treated under aqueous conditions, then treated sequentially with disodium ethylenediaminetetraacetic acid, thiosilica and charcoal to remove metal impurities. The crude compound was recrystallized from methanol (450 ml) to give ethyl 2-(5((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidin-5-carboxylate (77 g, 70%) as pale yellow solid. 1H NMR (400 MHz, CDCl ₃ ) δ 1.44 (t, 3H), 1.50 (t, 3H), 4.19 (q, 2H), 4.46 (q, 2H), 7.00-7.04 (m, 1H), 7.25 (s , 1H), 7.71 (d, 1H), 8.59 (s, 1H), 8.66 (d, 1H), 9.32 (s, 2H), 9.55 (s, 1H).

Step 5: 2-(5-((3-Ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidine-5-carboxylic acid (Intermediate 1)

Sodium hydroxide (307 mmol, 1.5 eq., 4 M aq.) and methanol (50 ml) were added to a suspension of 2-(5((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)pyrimidin-5- carboxylate (205 mmol, 1.0 eq.) in tetrahydrofuran (300 ml). The resulting solution was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (400 ml) and extracted with a mixture of 2:1 diethyl ether:heptanes (2 x 300 ml). The aqueous layer was acidified to pH 4 with 4 M hydrochloric acid. The resulting suspension was stirred at room temperature for 1 hour. The solid was filtered, washed with water and dried to give 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)pyrimidine-5-carboxylic acid (69 g, 100%) as a pale yellow solid substances. 1H NMR (400 MHz, DMSO-d ₆ ) δ 1.37 (t, 3H), 4.18 (q, 2H), 7.19 (dd, 1H), 7.58 (dd, 1H), 7.70 (dd, 1H), 8.35-8.40 (m, 1H), 8.66 (d, 1H), 9.33 (s, 2H), 9.41 (d, 1H), 13.9 (br. s, 1H).

Step 6: (S)-2-(5-((3-Ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide (Example 1 ( DGAT2i Connection))

Oxalyl chloride (13.8 mL, 160 mmol, 1.2 eq.) and dimethylformamide (0.510 mL, 6.65 mmol, 0.05 eq.) were added to the 2-(5-((3-ethoxypyridin-2-yl) suspension )oxy)pyridin-3-yl)pyrimidin-5-carboxylic acid (45.0 g, 133 mmol, 1.0 eq.) in dichloromethane (500 ml). The suspension was stirred for 2 hours until a solution formed. The reaction mixture was concentrated to give the crude acid chloride as a red solid. A solution of N-tetrahydrofuran-3-amine (12.2 g, 140 mmol, 1.05 eq.) and diisopropylethylamine (51.0 mL, 293 mmol, 2.2 eq.) in tetrahydrofuran (100 mL) was added dropwise into a solution of crude acid chloride in dichloromethane (200 ml) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. Water (1.0 L) and ethyl acetate (600 ml) were added and the organic layer was separated, washed with saturated sodium bicarbonate solution, dried over magnesium sulfate and filtered. The filtrate was treated with activated carbon (20 g) and stirred at 65°C for 20 minutes. The warm suspension was filtered and the filtrate was concentrated to a pale yellow solid, which was recrystallized from methanol in ethyl acetate (1:4, 1 L) to give (b)-2-(5-((3-ethoxypyridin-2yl)oxy) pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide (43.5 g, 81%) as a colorless solid. The title compound was combined with previous batches (108.7 g, 266.8 mmol) prepared in the same manner and suspended with ethyl acetate (1.0 L) at 80°C for 4 hours. The suspension was left to cool to room temperature and stirred for 4 days. The solid was filtered, washed with ethyl acetate (3 x 200 ml) and dried in high vacuum at 50°C for 24 hours to give ^)-2-(5-((3-ethoxypyridin-2yl)oxy)pyridin-3-yl )-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide (100.5 g, 92%) as a colorless solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.38 (t, 3H), 1.89-1.98 (m, 1H), 2.15-2.26 (m, 1H), 3.65 (dd, 1H), 3.70-3.78 (m , 1H), 3.85-3.92 (m, 2H), 4.18 (q, 2H), 4.46-4.55 (m, 1H), 7.18 (dd, 1H), 7.58 (dd, 1H), 7.69 (dd, 1H), 8.37 (dd, 1H), 8.64 (d, 1H), 8.95 (d, 1H), 9.28 (s, 2H), 9.39 (d, 1H). MS (ERI+) 408.4 (M+N). Melting point 177.5°C. Elemental analysis for C ₂₁ H ₂₁ N ₅ O ₄ : calculated C, 61.91; N, 5.20; N, 17.19; found C, 61.86; N, 5.18; N, 17.30.

The solid form obtained by this procedure was characterized by X-ray powder diffraction (XRD) analysis and was identified as Form 1 of Compound D.

Alternative Step 6 to prepare N)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide (Example 1 (Compound D))

A 100 ml reactor was charged with acetonitrile (35 ml), 2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)pyrimidine-5-carboxylic acid (5.0 g, 15 mmol) and ^ )-tetrahydrofuran-3-amine hydrochloride (2.2 g, 18 mmol, 1.2 eq.). Diisopropylethylamine (18 mL, 103 mmol, 7.0 eq) was charged while maintaining the temperature at 20°C to 30°C. A solution of propanephosphonic anhydride (T3P) in acetonitrile (21 mL, 30 mmol, 2.0 eq.) was charged at a rate such that the temperature was maintained below 45°C. The reactor was heated to 40 ± 5°C for 1 hour, then a sample was taken to complete the reaction. The reaction mixture was cooled to 20-25°C and tetrahydrofuran (25 ml) was added. Sodium bicarbonate solution (0.5M, 40 ml) was charged and the mixture was stirred for 1 hour. The pH was checked and the measured value was 8.5. Ethyl acetate (40 ml) was added and the mixture was stirred for 15 minutes. The mixture was allowed to settle and the phases were separated. The aqueous layer was transferred to a separatory funnel and extracted with ethyl acetate (100 ml). The organic phases were combined and washed with water (40 ml). The organic layer was transferred to a 100 ml reactor in portions and concentrated in vacuo to a small volume. Methyl ethyl ketone (100 ml) was added and the mixture was concentrated to a final volume of approximately 60 ml. The vacuum was removed and the slurry was heated to reflux and held until the solid was washed off the reactor walls. The suspension was cooled to 15°C over 2 hours and granulated overnight. The solid was isolated by filtration, washing the reactor and filter cake twice with methyl ethyl ketone (10 ml each time). The solid was dried in a vacuum oven at 50°C to obtain 4.86 g (81%) of the desired product. The solid form obtained by this procedure was characterized by XRD analysis and was recognized as Form 2 of Compound D.

Conversion of Form 2 to Form 1 of Compound D

Form 2^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide was loaded into a 100 ml reactor (Example 1) (10.0 g, 24.6 mmol, 1.00 eq.), methyl ethyl ketone (8.8 ml/g, 88.0 ml) and water (1.2 ml/g, 12.0 ml). The reactor was heated to 50°C for 30 minutes. Complete dissolution occurred at approximately 44°C. The reactor was cooled to 40°C for 30 minutes, then the seed was added Form 1 ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3 -yl)pyrimidin-5-carboxamide (Example 1, Compound D) (0.050 g, 0.123 mmol, 0.0050 eq.). After seeding, the cloudy suspension was stirred for 1 hour, then cooled to 5°C for 2 hours and then stirred at 5°C for 12 hours. A process control sample was collected and characterized by XRD analysis to confirm that the solid was Form 1 of Compound D. The suspension was filtered and the reactor and filter cake were washed with 0° C. methyl ethyl ketone (2.5 mL/g, 25 mL). The solid was dried in a vacuum oven at 50°C to obtain 8.15 g (81.5%) of the desired product. The DRLP patterns of the target product corresponded to Form 1 of Compound D.

X-ray diffraction by powder:

X-ray powder diffraction analysis was carried out using a Bruker AXS D8 Advance diffractometer equipped with a Cu radiation source (Κα-average wavelength 1.54056A). equipped with TWIN primary optics using a Goebel mirror. Diffracted radiation was detected using a PSD-Lynx Eye detector. Both primary and secondary, equipped with 2.5 Soller slits. The X-ray tube voltage and current were set to 40 kV and 40 mA, respectively. Data were collected in a theta-theta goniometer using a locked couple scan ranging from 3.0

- 35 045908 to 40.0 degrees 2-theta in steps of 1000 using a scan rate of 6 seconds per step. Samples were prepared by placing them in a low background silicon sample holder (C79298A3244B261). Data were collected using Bruker DIFFRAC Plus software. The analysis was carried out using EVA diffract plus software. The DRLP data file was not processed until peaks were identified. Using the peak identification algorithm in EVA software, peaks were selected with a threshold value of 5 and a width value of 0.2. The output of automated interpretations was visually checked to ensure accuracy, and adjustments were made manually when necessary. Typically, peaks with relative intensities >3% were selected. Peaks that were not resolved or inconsistent with noise were discarded. The typical USP specification for peak position error is +/- 0.2° (USP-941).

Table 1

Key XRD peaks characterizing the crystalline substance

EForm 1 of the compound of Example 1 Form 2 of the compound of Example 1

Angle 2Θ (°) Angle 2Θ (°)

5.3; 7.7; 15.4 6.5; 9.3; 13.6

Example 1 (Compound D)

Fig. 1 is a representative X-ray powder diffraction pattern showing crystalline Form 1 of the compound of Example 1 (Compound D) (vertical axis: intensity (pulses); horizontal axis: two-theta (degrees)).

Fig. 2 is a representative X-ray powder diffraction pattern showing crystalline Form 2 of the compound of Example 1 (Compound D) (vertical axis: intensity (pulses); horizontal axis: two-theta (degrees)).

Example 2: Preparation of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]1'-carbonyl)-6-methoxypyridin-2-yl )benzoic acid, Compound A (ACC-inhibiting Compound):

The preparation of Compound A notes that some preparation methods described herein may require protection of functional groups (eg, primary amine, secondary amine, carboxyl in Formula I precursors). The need for such protection will depend on the nature of the functional group and the conditions of the preparation methods. The need for such protection will be readily determined by one skilled in the art. The use of methods for such protection/deprotection is also within the competence of one skilled in the art. For a general description of protecting groups and their use, see T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991. Moreover, the present invention is not limited to the specific synthesis methods described herein, which may vary.

Intermediate A1: 1-Isopropyl-4,6-dihydrospiro[indazole-5,4'-piperidine]-7(III)-one, hydrochloride salt 'L ^/0

N-xJk

I^NH HCI

Step 1. tert-Butyl-9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate

Tert-butyl-4-formylpiperidine-1-carboxylate (108 kg), cyclohexane (1080 L) and pyrrolidine (64.8 kg) were charged into a dry reactor at 25-30°C. This mixture was stirred for 5-10 minutes and then heated to reflux for 12-16 hours while collecting water using a Dean-Stark trap. The reaction mixture was then cooled to 50-60°C, and at this temperature a vacuum was applied to distill off excess pyrrolidine and cyclohexane. The reaction mixture was then cooled to 25-30°C and cyclohexane (648 L) was charged, followed by methyl vinyl ketone (49.63 kg). The mixture was stirred for 12-16 hours, then filtered, and the filtrate was charged into a clean and dry reactor. The solution was cooled to 10-15°C, then a solution of acetic acid (54.75 kg) in water (54 L) was slowly added, maintaining the temperature below 15°C. After addition was complete, the mixture was heated to 25-30°C and stirred for 12-16 hours. The layers were separated and the aqueous was extracted with ethyl acetate (324 L). The combined organic layers were washed with a solution of sodium bicarbonate (32.34 kg) in water (324 L), then dried over sodium sulfate. The solid was washed with ethyl acetate (54 L) and the combined filtrates were concentrated under reduced pressure at a temperature below 40°C. n-Heptane (216 L) was charged into the reactor and distillation was carried out under reduced pressure at a temperature below 40° C. to dryness. The mixture was cooled to 25-30°C and n-heptane (216 L) was charged into the reactor. The mixture was stirred for 1-2 hours after mixing

- 36 045908 names of solids. The solid was then filtered, washed with n-heptane (54 L) and dried at 40-50°C for 10-12 hours to obtain the target substance (90.1 kg, 67% yield).

Step 2. (E)-tert-Butyl-10-((dimethylamino)methylene)-9-oxo-3-azaspiro[5.5]undec-7-ene-3 carboxylate

A clean and dry reactor was charged with tert-butyl-9-oxo-3-azaspiro[5.5]undec-7-ene-3-carboxylate (50 kg), N,N-dimethylformamide (500 L) and N,N-dimethylformamide dimethyl acetal (135 kg) at 25-30°C in a nitrogen atmosphere. This reaction mixture was stirred for 5-10 minutes, then heated to 120-130°C for 20 hours. The mixture was then cooled to 50-60°C, and the solvent was distilled off under high vacuum at a temperature below 60°C. Mixed xylenes (200 L) were loaded at a temperature below 45°C, and the solvent was distilled off under high vacuum at a temperature below 60°C. This operation was repeated with another batch of mixed xylenes (200 L). Then toluene (200 L) was loaded into the reactor, and the solvent was distilled off in high vacuum at a temperature below 60°C. This operation was repeated with another portion of toluene (200 l). Methyl tert-butyl ether (100 L) was then charged at a temperature below 30°C, and the solvent was distilled off under high vacuum at a temperature below 40°C. The mixture was cooled to 15-20°C and methyl tert-butyl ether (100 L) was charged at a temperature below 20°C. The mixture was stirred for 20-30 minutes and the solid was filtered, washed with methyl tert-butyl ether (50 L) and dried without vacuum at 50-55°C for 10 hours to obtain the title compound (52.1 kg, yield 87%). 1H NMR (400 MHz, CDCI3) δ ppm 7.48 (s, 1H), 6.57 (d, J=9.97 Hz, 1H), 5.99 (d, J=10.16 Hz, 1H), 3.32-3.51 (m, 4H), 3.06 (s, 6H), 2.72 (s , 2H), 1.57-1.66 (m, 2H), 1.41-1.53 (m, 11H).

Step 3. tert-Butyl-1-isopropyl-1,4-dihydrospiro[indazole-5,4'-piperidine]-1'-carboxylate o ¹

(E)-tert-butyl-10-((dimethylamino)methylene)-9-oxo-3azaspiro[5.5]undec-7-ene-3-carboxylate (80 kg), toluene (704 l) were loaded into a clean and dry reactor. and trimethylamine (16 l) at 25-30°C. This reaction mixture was heated to 70-80°C and a solution of isopropylhyrazine hydrochloride salt in methanol (1.25 eq., total 141 kg) was added over 4-5 hours. The reaction mixture was then stirred for 8-10 hours at 70 -80°C, after which it was cooled to 15-25°C. A solution of citric acid (48 kg) in water (480 L) was then slowly added, maintaining the internal temperature below 25°C. Ethyl acetate (208 L) was added and the mixture was stirred for 10 minutes. The layers were separated and the organic layer was washed successively with a solution of citric acid (48 kg) in water (480 L), then with water only (320 L). The combined aqueous layers were extracted with ethyl acetate (320 L). The combined organic layers were then dried over sodium sulfate (8 kg) and the solvents were evaporated to dryness under reduced pressure and at a temperature below 40°C. Dichloromethane (240 L) was charged into the reactor and the mixture was stirred at 2530°C until it became clear. Activated carbon (1.84 kg), magnesium silicate (1.84 kg) and silica gel (32 kg, 100-200 mesh) were sequentially charged at 25-30°C, and the heterogeneous mixture was stirred for 1 hour. The suspension was then filtered through Hyflow layer prepared by mixing Hyflow supercellular material (8 kg) and dichloromethane (40 l). The filter cake was washed with dichloromethane (three times, 120 L each). The combined filtrates were charged back into the reactor and the solvent was evaporated under reduced pressure at a temperature below 40°C. n-Heptane (160 L) was then charged and distilled under reduced pressure at a temperature below 40°C. n-Heptane (200 L) was loaded into the reactor and the mixture was cooled to 0-5°C. After stirring for 12-15 hours, the solid was filtered at 0°C, washed with cooled (0-5°C) n-heptane (160 L) and dried in vacuo at 40-50°C to give the title compound (82 .4 kg, 75%). 1H NMR (400 MHz, CDCl ₃ ) δ ppm 7.25 (s, 1H), 6.42 (dd, J=10.05, 0.49 Hz, 1H) 5.84 (d, J=9.95 Hz, 1H), 4.42-4.52 (m, 1H), 3.36-3.53 (m, 4H), 2.62 (s, 2H) 1.56-1.68 (m, 2H) 1.45-1.55 (m, 17H).

Step 4. 1-Isopropyl-4,6-dihydrospiro[indazole-5,4'-piperidine]-7(III)-one, hydrochloride salt 'G ⁰

X^NH HCI

A clean and dry reactor was charged with tert-butyl-1-isopropyl-1,4-dihydrospiro[indazole-5,4'piperidine]-1'-carboxylate (60 kg) and methanol (600 L) at 25-30°C. N-Bromosuccinimide (32.4 kg) was added in 5 portions over 30-40 minutes at 25-30°C and stirring was continued for 30-60 minutes. Slowly add a solution of sodium thiosulfate pentahydrate (5.4 kg) in water (102 L), maintaining

- 37 045908 internal temperature below 30°C. The mixture was stirred for 20-30 minutes, then the solvent was evaporated under reduced pressure at a temperature below 45 °C. The residue was cooled to 25-30°C and 2methyltetrahydrofuran (420 L) was charged into the reactor along with water (90 L). The mixture was stirred for 15-20 minutes, then the layers were separated, the aqueous layer was further extracted with 2methyltetrahydrofuran (120 L). The combined organic extracts were treated for 15-20 min at 25-30°C with a solution of sodium hydroxide (4.8 kg) in water (120 l). The layers were separated and the organic layer was washed with water (120 L), then a solution of sodium chloride (12 kg) in water (120 L) and then dried over sodium sulfate (6 kg). After filtration, the filter cake was washed with 2-methyltetrahydrofuran (30 L) and the combined filtrates were charged back to the reactor. The solvent was completely distilled off below 45° C. under reduced pressure, and the residue was solubilized in tetrahydrofuran (201 L). Another clean and dry reactor was charged with potassium tert-butoxide (60.6 kg) and tetrahydrofuran (360 L) at 25-30°C. A solution of the residue in tetrahydrofuran was slowly added to them, maintaining the temperature below 30°C. The reaction mixture was then heated to 60-65°C and maintained at this temperature for 1-2 hours. Once complete, the mixture was cooled to 0-10°C and slowly quenched with hydrochloric acid solution (1 N, 196 L), maintaining the internal temperature below 10°C. The reaction mixture was left to warm to 25-30°C and ethyl acetate (798 L) was charged. After stirring for 15-20 minutes, the layers were separated and the aqueous layer was further extracted with ethyl acetate (160 L). The combined organic layers were washed with water (160 L), dried over sodium sulfate (8 kg), filtered, and the filter cake washed with ethyl acetate (300 L). The solvents were completely distilled off under reduced pressure at a temperature below 45°C, and ethyl acetate (540 L) was charged into the reactor at 25-30°C, then methanol (156 L) was added. The mixture was cooled to 0-5°C, at which point acetyl chloride (79.8 kg) was slowly added, maintaining the temperature within the specified range. The mixture was then left to warm up to 20-25°C and kept at this temperature for 4-5 hours with stirring. The resulting suspension was filtered and the solid was washed with ethyl acetate (120 L), then dried at 40-45°C for 8-10 hours to obtain the desired crude product (33.5 kg, 65%).

The final purification step was carried out by solubilizing this crude solid (56.8 kg) in methanol (454.4 L) in a clean and dried reactor at 25-30°C. The solution was stirred for 30-45 minutes, then passed through a 0.2 micron cartridge filter into a clean and dry reactor at 25-30°C. Methanol was distilled off under reduced pressure at temperatures below 50°C until approximately 1 volume of solvent remained. The reaction mixture was cooled to 25-30°C and fresh acetonitrile (113.6 L) was charged through a 0.2 micron cartridge filter. The solvents were distilled off under reduced pressure at temperatures below 50°C until approximately 1 volume of solvent remained. The reaction mixture was cooled to 25-30°C and fresh acetonitrile (190 L) was charged into the reactor through a 0.2 micron cartridge filter. The mixture was heated to 65-70°C and stirred for 45 minutes, then cooled to 25-30°C and stirred for 1 hour. The resulting suspension was filtered and the filter residue was washed with cooled (15°C) acetonitrile (56 .8 l). The solid was dried under reduced pressure at 40-50°C for 8 hours to obtain Intermediate A1 (36.4 kg, 64%). ¹ H NMR (400 MHz, CD3OD) δ ppm 7.43 (s, 1H), 5.32-5.42 (w, 1H), 3.15-3.25 (m, 4H), 2.89 (s, 2H), 2.64 (s, 2H), 1.69-1.90 (m, 4H), 1.37- 1.45 (m, 6H); ERI [M+N]+ = 248.

Intermediate A2: 2-(4-(tert-Butoxycarbonyl)phenyl)-6-methoxyisonicotinic acid

ABOUT'

2,6-dichloroisonicotinic acid (30 kg) and methanol (120 l) were loaded into a clean and dried reactor at 20-25°C. The suspension was stirred for 5 minutes, then heated up to 65°C (reflux). A solution of sodium methoxide in methanol (30%, 87.2 kg) was then slowly charged over at least 4 hours through a dropping funnel. The funnel was rinsed with methanol (15 L) and stirring was carried out at 65°C for at least 15 hours. The mixture was then cooled to 45°C and distilled under reduced pressure to a residual volume of approximately 90 L. A solution of potassium bicarbonate (28.2 kg) and potassium carbonate (21.6 kg) in water (180 L) was then charged into the reactor at 40-45°C. The reactor containing the aqueous solution was washed with water (21 L), and the washing liquid was loaded into the reaction mixture. The mixture was distilled under reduced pressure at temperatures below 80°C to a residual volume of approximately 240 L, then cooled to 20-25°C.

Another clean and dry reactor was charged with tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoborolan2-yl)benzoate (52.3 kg) and dioxane (340 kg) and stirred at 2-25°C until completely dissolved. The contents of the first reactor were then heated at 40°C to ensure complete solubility and transferred to this new reactor. The reaction mixture was cooled to 20-25°C, and the deoxygenation stage was carried out

- 38 045908 were subjected to vacuum/nitrogen filling cycles. The mixture was further cooled to 0-10° C. and palladium acetate (0.65 kg) was charged into the reactor, followed by triphenylphosphine (2.46 kg) under a stream of nitrogen. The mixture was heated to 20-25°C and another deoxygenation step was carried out with vacuum/nitrogen filling cycles. The mixture was then heated to 80°C and maintained at this temperature for at least 18 hours. The mixture was cooled to 20-25°C, then methyl tert-butyl ether (133.2 kg) and water (30 l) were sequentially charged into the reactor. . The layers were separated and the aqueous layer was diluted with water (110 L), then extracted with methyl tert-butyl ether (110 L). The combined organic extracts were washed with a solution of citric acid (52 kg) in water (84 L) and the layers were separated. The aqueous layer was further extracted with methyl tert-butyl ether (88.8 kg) and the organic layers were combined, then washed three times with a solution of sodium chloride (43 kg) in water (80 L). After final separation of the layers, the organic layer was filtered through a Pall filter containing a charcoal cartridge and the filter cake was washed with methyl t-butyl ether (11.2 kg). The filtrate was distilled under reduced pressure at a temperature below 50°C to about 90 L and then successively co-distilled with heptane (120 L) at a temperature below 50°C to about 120 L. The mixture was then cooled to 20-25°C over 1 hour, then stirred at this temperature for a further 1 hour. The suspension was filtered and the filter cake was washed three times with heptane (3 x 18 L), then three times with acetonitrile (3 x 18 l). The resulting wet solid was dried under vacuum and a stream of nitrogen at a temperature below 45°C for at least 15 hours to give Intermediate A2 (44.6 kg, 87% yield). ¹ H NMR (400 MHz, CDCh) δ ppm 8.13 (s, 2H), 8.09 (s, 2H), 7.97 (d, J=1.17 Hz, 1H), 7.34 (d, J=0.98 Hz, 1H), 4.08 (s, 3H), 1.61 (s, 9H ); ERI [M+N]+ = 330.

Intermediate A3: tert-Butyl-4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridinium- 2-yl)benzoate

A round bottom flask was charged with 2-(4-(tert-butoxycarbonyl)phenyl)-6-methoxyisonicotinic acid (Intermediate A2, 15.2 g, 46.2 mmol) and ethyl acetate (140 ml). 1,1'Carbonyldiimidazole (8.98 g, 55.4 mmol) was added in one portion and stirred for 1 hour at RT. Add 1-isopropyl-4,6-dihydrospiro[indazole-5,4'-piperidine]-7(1H)-one hydrochloride (Intermediate A1, 14.8 g, 52.2 mmol), then add N-diisopropylethylamine (9.1 ml, 52.2 ml), and the reaction mixture was stirred for 18 hours at room temperature. An aqueous 2M HCl solution (40 ml) was added, followed by 1M potassium hydrogen sulfate (40 ml) and 50 ml of heptane. The resulting mixture was stirred for 1 hour at room temperature. The mixture was transferred to a separatory funnel. The organic phase was separated, washed successively with water (20 ml), saturated sodium bicarbonate solution (30 ml), water (20 ml), brine (20 ml), dried over 20 g of magnesium sulfate and 10 g of silica gel, filtered and concentrated in vacuo. Towards the end of concentration, a solid began to form. The residue was stirred in 40 ml of ethyl acetate at 80°C and heptane (120 ml) was added slowly dropwise. The mixture was stirred at 80°C for 1 hour, then slowly cooled to room temperature while stirring for 1 hour and stirred for 18 hours at room temperature. The solid was collected by filtration, washed with water and ethyl acetate-heptane (1:3) and dried in vacuo at 50°C for 18 hours to give Intermediate A3 (19.64 g, 76% yield).

Alternative Preparation of Intermediate A3:

A clean and dry reactor was charged with acetonitrile (219 kg) and 2-(4-(tert-butoxycarbonyl)phenyl)-6methoxyisonicotinic acid (Intermediate A2, 34.8 kg) at 20-25°C. The mixture was stirred for 5 minutes, then 1,1-carbodiimidazole (18.9 kg) was charged in three successive portions. The suspension was further stirred at 20-25°C for at least 1 hour, then 1-isopropyl-4,6-dihydrospiro[indazole-5,4'-piperidin]-7(III)-one hydrochloride salt ( Intermediate A1, 33.0 kg) was then charged with N-diisopropylethylamine (20.5 kg) via pump. The reagent pump and reactor walls were washed with acetonitrile (13.7 kg) and stirring was carried out at 20-25°C for at least 2 hours. Once complete, the mixture was seeded with tert-butyl-4-(4-(1- isopropyl 7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carboxyl)-6-methoxypyridin-2-yl)benzoate (Intermediate A3, 209 g) and stirred for at least 30 minutes. After confirmation of the onset of crystallization, a solution of citric acid monohydrate (58.5 kg) in water (257 L) was charged over 1 hour. The resulting suspension was further stirred at 20-25°C for at least 2 hours, then filtered, and the filter cake was washed with a mixture of acetonitrile (68.4 kg) and water (87 l). This wash was also used to wash the reactor. The solid was dried under reduced pressure at a temperature below 55°C to obtain Intermediate A3 (43.44 kg, 73% yield).

- 39 045908

Compound A (as free acid): 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6 -methoxypyridin-2-yl)benzoic acid

-W o

A round bottom flask was loaded with tert-butyl-4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6- methoxypyridin-2-yl)benzoate (3.7 g, 6.6 mmol) and toluene (25 ml). 85% phosphoric acid (3.0 ml) was added dropwise with stirring, and the reaction mixture was heated to 60°C for 4 hours. A colorless thick resin formed. The reaction mixture was cooled to room temperature. and added water. A white solid was observed. The toluene organic layer was discarded, leaving the aqueous layer and solid. Ethyl acetate (60 ml) was added and 4N was added. NaOH solution until the pH was adjusted to about 7. The layers were separated and the aqueous layer was extracted with ethyl acetate (50 ml). The combined ethyl acetate organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to give a white solid. This was dissolved in ethyl acetate (80 ml) at 50°C and heptane (90 ml) was added slowly. The heat was removed and the mixture was cooled to room temperature. and stirred for 16 hours. The resulting solid was collected by filtration, washed with mother liquor and dried to obtain the title compound (Compound A free form, 2.15 g, 65% yield) as a white solid.

Alternative preparation of Compound A (as free acid): A clear dry reactor was charged with acetonitrile (130.4 kg) and tert-butyl-4-(4-(1-isopropyl-7-oxo-1,4,6,7- tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoate (Intermediate A3, 20.72 kg) at 20-25°C. This mixture was stirred for 5 minutes, then p-toluenesulfonic acid (8.5 kg) was charged under low nitrogen pressure. The reaction mixture was heated to 70°C and maintained at this temperature for at least 6.5 hours. Once complete, the mixture was cooled to 40°C and slowly seeded with Compound A (104 g) and water (83 L) over for at least 1 hour. The mixture was further stirred at 40°C for at least 4 hours, then cooled to 20-25°C over 2 hours. After additional stirring for at least 2 hours, filtered and the filter cake was washed with solution acetonitrile (33 kg) and water (41 l). This wash was also used to wash the reactor. The resulting solid was dried under reduced pressure at a temperature below 55°C to obtain Compound A (16.5 kg, 89% yield).

Preparation of Form 1 of Compound A - Anhydrous Mono-Tris Salt of Compound A:

4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]1'-carbonyl)-6-methoxypyridin-2-yl) was loaded into the test tube benzoic acid (151 mg, 0.300 mmol) and 3 ml of ethanol. This mixture was heated to 80°C for 5 minutes until the solid dissolved and then cooled to room temperature. Tris(hydroxymethyl)aminomethane (39 mg, 0.32 mmol) was added and the mixture was stirred overnight at room temperature. Heptane (2.25 ml) was added dropwise to obtain a suspension, which was heated to 50°C until a clear solution was obtained. The mixture was cooled to room temperature. overnight with stirring. A white solid was observed and the mixture was stirred for another 3 days. The material was filtered and dried in a vacuum oven at 50°C overnight to obtain Form 1 (151 mg, 0.242 mmol, 81% yield).

Alternative Preparation of Form 1 of Compound A: Anhydrous Mono-Tris Salt of Compound A:

A clean and dry reactor was charged with ethanol (83 L), then Compound A (9.43 kg) and Tris (2.55 kg) were added while maintaining the temperature of the mixture at 20-25°C. The walls of the tank were washed with ethanol (2 L) and the resulting mixture was heated to 65-70°C, maintained at this temperature for at least 30 minutes until all solids had dissolved, then cooled to 45-50 °C. Warm filtration was performed through an inline 10 μm polypropylene filter, and the reactor as well as the filter were washed with ethanol (9 L). n-heptane (24 l) was loaded into the warm solution through the same built-in filter, and the mixture was seeded with 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydro spiro[indazole-5 ,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid anhydrous tris salt (100 g) in ethanol (0.5 l) at 45-50°C. The temperature was held for at least 2 hours, after which it was cooled to 20-25°C for at least 2 hours. Stirring was continued for at least 5 days. The suspension was then filtered and the filter cake was washed with an ethano mixture

- 40 045908 la (13 l) and n-heptane (6 l). The solid was dried under reduced pressure at a temperature below 45°C for at least 12 hours to obtain the compound of Example 1 (11.7 kg, 77%).

Preparation of Form 2 of Compound A - Compound A Mono-Tris Salt Trihydrate:

Form 2 of Compound A was prepared by conversion from Form 1 of Compound A. To a 50 mL EasyMax reactor was added Form 1 (1.7214 g, 2.760 mmol), isopropanol (16.50 mL, 215.8 mmol) and water (688 µl, 38.190 mmol). This mixture was stirred (300 rpm) for approximately 72 hours at a jacket temperature of 25°C. The reaction mixture was then heated to 40°C for 15 min and held at 40°C for approximately 24 hours, immediately cooled to 20°C to remove the sample for testing. A mixture of forms was determined by XRD, so additional water was added (688 μL, 38.190 mmol). The stirrer speed was increased to 400 rpm and the suspension was stirred for 6 hours and then cooled to 15°C. The solid was isolated on a 60 ml/40 M filter and washed with 96/4 isopropanol/water. The resulting substance corresponded to Form 2 of Compound A according to the results of XRD.

Alternative Preparation of Form 2 of Compound A - Compound A Mono-Tris Salt Trihydrate:

A clean and dry reactor was charged with isopropanol (60.4 kg) and Compound A (16.68 kg) and Tris (4.42 kg) were added while maintaining the mixture at a temperature of 20-25°C. The mixture was stirred for 5 min, then water (6.7 kg) was added and the suspension was heated to 55 °C. The current clear solution was filtered into a preheated, clean and dry reactor (50-55°C) through an inline 10 µm polypropylene filter. The solution was then seeded with the mono-tris salt of Compound A as trihydrate (167 g). After checking that the seed was retained, the mixture was cooled to 15°C for at least 2 hours, then kept at 15°C for a minimum of 16 hours. The suspension was filtered and the filter cake was washed with chilled isopropanol (13.1 kg). The solid was then dried under reduced pressure at a temperature below 25°C to obtain Compound A Form 2 alone (22.1 kg, 98% yield).

Form 1 of Compound A is anhydrous and thermodynamically stable below a water activity of about 0.2 (20% RH) at ambient temperature. Form 1 of Compound A has a DRLP pattern essentially the same as shown in FIG. 3 for Compound A. The characteristic XRD peaks of Form 1 of Compound A, expressed in 2Θ ± 0.2° 2θ, are as follows: 9.6, 10.7 and 11.3. The locations and intensities of the peaks for the DRLP pattern in Fig. 3 are given in table. 2.

table 2

XRD peaks and relative intensities of Form 1 of Compound A

Degrees 2Θ ± 0.2° 2Θ Relative intensity (%) Degrees 2Θ ± 0.2° 2Θ Relative intensity (%) Degrees 2Θ ± 0.2° 2Θ Relative intensity (%) 5.2 62 17.8 86 25.4 25 9.6 13 18.9 100 26.4 9 10.7 14 19.5 77 26.6 AND from AND 20.1 AND 27.2 9 12.1 6 21.1 29 28.3 9 13.3 60 22.2 15 29.3 10 13.9 9 22.4 19 29.9 9 14.0 10 22.6 21 30.4 13 15.5 AND 23.6 53 31.7 13 16.2 58 24.1 54 33.4 15 17.0 27 24.5 16

Form 1 of Compound A has a Raman spectrum substantially the same as the Raman spectrum shown in FIG. 4. Form 1 of Compound A has characteristic Raman peak shifts, expressed in cm ^-1 , at 568, 698, 989, 1218, 1511, 1561 and 1615 ± 2 cm ^-1 . Peak positions (± 2 cm ^-1 ) and normalized intensity (W = weak, M = medium, S = strong) of Form 1 of Compound A in FIG. 4 are listed in table. 3.

- 41 045908

Table 3

Raman peaks and normalized intensity for Form 1 of Compound A

Raman peak position (cm') Normalized intensity Raman peak position (cm’) Normalized intensity Raman peak position (cm’) Normalized intensity 115 M 794 W 1363 W 156 W 842 W 1388 W 170 W 885 W 1435 W 241 W 929 W 1466 W 274 W 989 W 1478 W 311 W 1011 W 1511 W 334 W 1047 W 1561 W 350 W 1071 W 1615 S 417 W 1090 W 1671 m 456 W 1119 W 2840 W 476 W 1143 W 2885 W 568 W 1169 W 2914 W 608 W 1187 W 2945 W 628 W 1196 W 2998 W 653 W 1218 W 3027 W 678 W 1244 W 3066 W 698 W 1265 W 3129 W 755 W 1315 W 779 W 1345 m

Form 1 of Compound A has ^{a 13} C mtNMR spectrum essentially the same as the spectrum shown in FIG. 5. Form 1 of Compound A has characteristic ¹³ C tNMR chemical shifts, expressed in ppm, at 22.9, 146.2, 157.9, 161.9 and 172.9 ± 0.2 ppm. ¹³ C chemical shifts (±0.2 ppm) for Form 1 of Compound A, which are shown in FIG. 5 are listed in table. 4.

Table 4

C chemical shifts and intensities for Form 1 of Compound A

ⁱⁿ c chemical shifts (ppm) Intensity ^B C chemical shifts (ppm) Intensity ^B C chemical shifts (ppm) Intensity 20.1 95 56.4 84 137.5 62 22.9 90 59.1 27 137.9 59 28.4 66 61.2 55 144.9 44 34.3 68 107.1 65 146.2 48 37.7 100 118.9 42 157.9 36 40.8 63 126.6 59 161.9 44 42.5 76 127.3 70 168.6 36 44.3 66 130.2 47 172.9 38 51.6 87 131.7 92 187.7 56 53.6 96 132.3 56

Form 2 of Compound A is a trihydrate and is thermodynamically stable above a water activity of about 0.2 at ambient temperature and 20% RH. Form 2 of Compound A has a DRLP pattern substantially the same as that shown in FIG. 6. The characteristic XRD peaks for Form 2 of Compound A, expressed in 2Θ ± 0.2° 2θ, are as follows: 8.4, 9.0, 10.5, 15.0 and 24.7. The locations and intensities of the peaks for the DRLP pattern in Fig. 6 are given in table. 5.

- 42 045908

Table 5

XRD peaks and relative intensities for Form 2 of Compound A

Degrees 2Θ ± 0.2° 2Θ Relative intensity (%) Degrees 2Θ ± 0.2° 2Θ Relative intensity (%) Degrees 2Θ ± 0.2° 2Θ Relative intensity (%) 5.0 AND 16.1 10 26.9 71 8.4 18 16.7 59 28.2 52 9.0 12 17.4 74 29.0 15 10.0 6 17.8 13 29.4 18 10.5 62 18.6 thirty 29.9 13 12.1 9 18.9 45 31.4 15 13.3 46 19.9 93 31.7 16 13.7 45 20.1 50 32.4 14 13.9 46 21.2 46 33.6 5 14.6 37 21.5 21 34.5 7 15.0 80 24.7 100 37.0 12 15.4 15 25.2 97

Form 2 of Compound A has a Raman spectrum essentially the same as the spectrum shown in FIG. 7. Form 2 of Compound A has characteristic Raman peak shifts, expressed in cm ^-1 , at 562, 692, 984, 1225, 1507, 1557 and 1610 ± 2 cm ^-1 . Peak positions (±2 cm ^-1 ) and normalized intensity (W = weak, M = medium, S = strong) for Form 2 of Compound A in FIG. 7 are listed in table. 6.

Table 6

Raman peaks and normalized intensity for Form 2 of Compound A

Position Normalized Position Normalized Position Normalized Raman and I Raman and I Raman and I th peak (cm' intensity th peak (cm' intensity th peak (cm' intensity 9 b 9 b 9 b 123 W 864 W 1344 W 179 W 884 W 1369 W 232 W 931 W 1387 W 284 W 984 W 1410 W 405 W 1019 W 1433 W 441 W 1048 W 1460 W 481 W 1077 W 1480 W 562 W 1097 W 1507 W 620 W 1109 W 1557 m 628 W 1118 W 1610 S 639 W 1140 W 1670 W 650 W 1194 W 2884 W 667 W 1225 W 2916 W 692 W 1246 W 2946 W 710 W 1261 W 2995 W 758 W 1277 W 3073 W 790 W 1305 W 3108 W 839 W 1321 W

Form 2 of Compound A has ^{a 13} C mtNMR spectrum essentially the same as the spectrum shown in FIG. 8. Form 2 of Compound A has characteristic ¹³ C ttNMR chemical shifts, expressed in ppm, at 19.2, 149.5, 155.6, 163.8 and 188.3 ± 0.2 ppm. ^13C chemical shifts (±0.2 ppm) for Form 2 of Compound A, which are shown in FIG. 8 are listed in table. 7.

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Table 7 ¹³ C chemical shifts and intensities for Form 2 of Compound A

¹³ s chemical shifts (ppm) Intensity ¹³ C chemical shifts (ppm) Intensity ¹³ C chemical shifts (ppm) Intensity 19.2 60 63.1 44 139.1 33 25.7 87 107.0 40 149.5 33 32.0 40 108.7 35 155.6 thirty 38.0 92 125.1 56 163.8 36 38.5 94 128.0 44 169.5 26 44.2 41 130.0 70 174.0 29 53.2 100 132.3 33 188.3 39 55.5 53 135.9 37 59.4 76 137.4 35

Based on the description given herein, one skilled in the art will understand that

Form 1 and Form 2 Compound A can each be uniquely identified by several different spectral peaks or patterns in varying combinations. Described below are exemplary combinations of characteristic peak values that may be used to separately identify Form 1 and Form 2 of Compound A, but these exemplary combinations should in no way be construed as limiting the other combinations of peak values disclosed herein.

To confirm the presence of three water molecules in Form 2 of Compound A, data was collected using a Bruker D8 Venture diffractometer at room temperature (see FIG. 9). The structure was determined by internal phasing using the SHELX software package in the monoclinic space group P21/c (version 5.1, Bruker AXS, 1997). The structure was then refined using the full-matrix least squares method. All non-hydrogen atoms were identified and refined using anisotropic substitution parameters.

Hydrogen atoms located on nitrogen and oxygen were identified from the Fourier difference map and refined with limited distances. The remaining hydrogen atoms were placed in the calculated positions and left to reside on the atoms that carried them.

The final R-index was 7.2%. The final difference Fourier map revealed no missing (lost) or misplaced electron density.

In table 8 shows the data collected regarding Form 2 of Compound A:

Table 8

[1=

Empirical formula SgvNzoad-SShchtsKhoz-ZNgO Mass according to formula 677.74 Temperature CT Wavelength 1.54178 A Crystal system Monoclinic Space group P2i/c Unit cell dimensions a = 17.6927(9) A a = 90° b = 13.2753(7) A 3 = 92.451(3) ^s c = 14.6480(8) A a = 90° Volume 3437.3(3) A ³ Z 4 Density (calculated) 1.310 mg/ ^m3 Goodness-of-fit criterion according to F ² 1.053 Final R indices RI = 0.0723, wR2 = 0.1835 gmats)] R indices (all data) RI = 0.1244, wR2 = 0.2110

Crystalline 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]- 1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid. This crystalline salt is commonly called the tris salt of Compound A.

Crystalline Tris salt of Compound A, wherein the ratio is 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine -2-yl)benzoic acid and salt are 1:1.

Crystalline tris salt of Compound A, wherein the crystalline salt is an anhydrous crystalline salt.

- 44 045908

Anhydrous crystalline tris salt of Compound A, wherein said anhydrous crystalline salt has a DRLP pattern containing peaks at diffraction angles of 9.6, 10.7 and 11.3 2θ ±0.2° 2θ.

Anhydrous crystalline tris salt of Compound A, wherein said anhydrous crystalline salt has a Raman spectrum containing Raman peak shifts at 1511, 1561 and 1615 cm ^-1 ±2 cm ^-1 .

Anhydrous crystalline tris salt of Compound A, wherein said anhydrous crystalline salt has ^{a 13} C tNMR spectrum containing chemical shifts at 22.9, 146.2 and 161.9 ppm. ± 0.2 ppm

Anhydrous crystalline tris salt of Compound A, wherein said anhydrous crystalline salt has an analytical parameter selected from the group consisting of a Raman spectrum containing peak shifts at 1511 and 1615 cm ^-1 ± 2 cm ^-1 , and ^{a 13} C tNMR spectrum containing at least one chemical shift at 22.9, 146.2 or 161.9 ppm. ± 0.2 ppm

Anhydrous crystalline tris salt of Compound A, wherein said anhydrous crystalline salt is substantially pure.

Crystalline tris salt of Compound A, wherein the crystalline salt is a trihydrate crystalline salt.

The trihydrate crystalline tris salt of Compound A, wherein said trihydrate crystalline salt has a DRLP pattern containing peaks at diffraction angles of 8.4, 9.0 and 10.5 29 ± 0.2° 2θ.

The trihydrate crystalline tris salt of Compound A, wherein said trihydrate crystalline salt has a Raman spectrum containing peak shifts at 1507, 1557 and 1610 cm ^-1 ± 2 cm ^-1 .

The trihydrate crystalline tris salt of Compound A, wherein said trihydrate crystalline salt has ^{a 13} C ttNMR spectrum containing chemical shifts at 19.2, 149.5 and 163.8 ppm. ± 0.2 ppm

A trihydrate crystalline tris salt of Compound A, wherein said trihydrate crystalline salt has an analytical parameter selected from the group consisting of an XRD pattern containing peaks at diffraction angles of 8.4 and 9.0 2θ ± 0.2° 2θ, a Raman spectrum containing peak shifts at 1557 and 1610 cm ^-1 ± 2 cm ^-1 , and ¹³ C ttNMR spectrum containing at least one chemical shift at 19.2, 149.5 or 163.8 ppm. ± 0.2 ppm

A trihydrate crystalline tris salt of Compound A, wherein said trihydrate crystalline salt has an analytical parameter selected from the group consisting of an XRD pattern containing peaks at diffraction angles of 8.4 and 9.0 2θ ± 0.2° 2θ, and a Raman spectrum, containing at least one peak shift at 1507, 1557 or 1610 cm ^-1 ± 2 cm ^-1 .

A trihydrate crystalline tris salt of Compound A, wherein said trihydrate crystalline salt has an analytical parameter selected from the group consisting of an XRD pattern containing peaks at diffraction angles of 8.4 and 9.0 2θ ± 0.2° 2θ, and ¹³ C ttNMR spectrum containing at least one chemical shift at 19.2, 149.5 or 163.8 ppm. ± 0.2 ppm

Pharmacological data

The following protocols are, of course, subject to modification by those skilled in the art.

A randomized, vehicle-controlled, 8 parallel group study was conducted in male Sprague-Dawley rats (Charles River (Boston, MA)) to obtain blood and liver TG levels. Standard laboratory conditions were used to house 96 rats (approximately 200 g); they were housed in pairs and maintained on a 12:12-hour light-dark cycle (lights turned off at 8 a.m.). On arrival, rats were randomized to either the balanced chow diet or the Western diet and dose groups and were given a 14-day run-in period on either the balanced chow diet or the Western diet before the start of the study. Standard laboratory rodent balanced diet, 5053, was from LabDiet (PMI, St Louis, Missouri). The Western diet, D12079Bi, was from Research Diets (New Brunswick, NJ).

The vehicle prepared for these studies was 0.5% (w/v) methylcellulose (MC, Sigma Aldrich; 274429) in deionized water. Stock solutions of either Compound A (derived from Tris salt) or Compound D were prepared with the appropriate compound so as to provide such a concentration that 10 ml of solution would deliver the desired dosage amount in mg/kg, where the average weight of the rats used was approximately 200 g.

Beginning on Day 1 of the study, rats were orally administered (10 mL/kg) with either vehicle control (0.5% MS (w/v%) in deionized water), a low dose, or a high dose of Compound A (1 mg/kg or 10 mg /kg QD, respectively), low or high dose of Compound D (5 mg/kg or 30 mg/kg BID, respectively), or co-administered low doses (Compound A 1 mg/kg QD and Compound D 5 mg/kg BID), or co-administered high doses (Compound A 10 mg/kg QD and Compound D 30 mg/kg BID)

Postmeal Plasma Analytes: Blood for dietary plasma TG concentrations was collected 2 hours post-dose (2 hours in the dark cycle) via the lateral tail vein and transferred to BD Microtainer tubes coated with ethylenediaminetetraacetic acid dipotassium (K2EDTA) ( PN365974), and centrifuged at 4°C. The resulting plasma samples were then analyzed on a Siemens Chemistry XPT clinical analyzer (Malven, PA) using Siemens triglyceride_2 reagents (ref 10335892).

Fasting plasma analytes: Blood for determination of TG concentrations in fasting plasma of individuals

- 45 045908 after a 4-hour fast, 2 hours after dosing (2 hours in the dark cycle) through the lateral tail vein, transferred into BD Microtainer tubes coated with dipotassium ethylenediaminetetraacetic acid (K ₂ EDTA) (PN365974), and centrifuged at 4°C. The resulting plasma samples were then analyzed on a Siemens Chemistry XPT clinical analyzer (Malven, PA) using Siemens triglyceride_2 reagents (ref 10335892).

On the last day of the study (Day 28), rats were sacrificed for tissue collection after a 4-hour fast 2 hours post-dose: two hours post-dose, blood for plasma analyte analysis was collected via the lateral tail vein, and the animals were then sacrificed by CO asphyxiation ₂ . Blood was transferred to BD Microtainer tubes coated with K2EDTA (PN365974), centrifuged at 4°C, and plasma was transferred to a 96-well microtiter plate and stored at -20°C. The livers were quickly removed, frozen in a Wollenberg clamp pre-cooled in liquid N ₂ , individually wrapped in aluminum foil and then stored at -80°C.

Tissue Grinding: Frozen livers were quickly crushed on an aluminum block cooled in liquid N2, ensuring that the tissue remained frozen throughout the grinding. Minced tissues were transferred and stored in 7 ml polypropylene conical tubes at -80°C until analysis.

Extraction for hepatic triglyceride: Approximately 50 to 100 mg of minced tissue was added to a 2 ml Lysing Matrix D tube (MP Bio) containing 800 μl of an ice-cold 1:1 CHCl3:MEON mixture. Samples were immediately extracted at 4°C using Qiagen Tissue Lyser II (Qiagen Cat No. 85300) for 4 minutes at 30 Hz. The homogenate was then transferred into 13 x 100 mm glass tubes and placed on ice. Lysis tubes were then washed with 800 μl of a 1:1 mixture _of CHCl:McOH, vortexed for 30 seconds and added to 13 x 100 mm glass tubes. On ice, 2.4 ml of 100% CHCl ₃ was added to all glass tubes to bring the CHCl ₃ : McOH ratio to 4:2. The samples were then placed in a freezer at -20°C overnight. The next day, 1.75 ml of 1 M KCl-H ₂ O was added, bringing the ratio to 4: 2: 1.75 CHCkMcO^HO. Samples were then vortexed for 30 seconds and centrifuged at 1500 rpm x 15 min at 4°C. After centrifugation, the organic phase was transferred to a fresh 13 x 100 mm extraction tube, dried at 37°C under N2 and resuspended in 750 µl CHCl ₃ . Aminopropyl solid phase extraction (SPE) cartridges (Waters Cat No. 054560, 6 mL, 500 mg) were wetted and washed with 5 mL hexane. After washing, 200 µl of sample extract in CHCl ₃ was applied to the cartridge and removed under vacuum without drying the column. Neutral lipids were then eluted with 5 ml of a 2:1 mixture of ΟΗΟ^κβοπρο^ηολΑΟ μM butylated hydroxytoluene. The samples were then dried at 37°C under N2 and resuspended with 1.75 ml of 98:2 isooctane:isopropanol. Samples were filtered through a 0.2 μM syringe filter and then injected onto a cyanopropyl phase HPLC column (particle size 3.5 μm, 4.6 x 150 mm Agilent Zorbax Eclipse XBD-CN column). The method performed was as follows: 4 μl injection with a run time of 27 minutes using solvent A (1000:1:2 isooctane:isopropanol:acetic acid) and solvent B (50:50 isopropanol:methyl tert-butyl ether). From minute 0 to minute 3, the solvent composition was maintained at 100% solvent A. From minute 3 to minute 8, the solvent composition was changed from 100% solvent A to 95% solvent A and 5% solvent B. From minute 8 to minute 18, the solvent composition was changed to the ratio 50:50. From minute 18 to minute 19, the solvent composition was changed back to 100% solvent A and maintained at this composition from minute 19 to minute 27.

Nuclear and membrane fractions were obtained by ultracentrifugation using standard methods from a portion of minced liver samples that were pooled by treatment group. Samples from nuclear extract and membrane fractions were analyzed by Western blotting for SREBP1. Western blots for calnexin were used as a marker for the membrane fraction, actin as a marker for total sample loading, and histone 2B as a marker for the nuclear fraction. Nuclear levels of SREBP1 were quantified using relative units and normalized to histone 2B to control for sample loss during nuclear fractionation and gel loading.

Another portion of minced liver was processed and analyzed for lipogenic gene expression. Rat taqman probes (linear degradable probes) against ACC1, FASN, SCD1, PCSK9 and SREBP-1c were all assessed using Actb as the housekeeping gene by qPCR.

Administration of f)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide (Compound D) or its pharmaceutical form acceptable salt in combination with 4-(4-(1isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2yl)benzoic acid (Compound A) or a pharmaceutically acceptable salt thereof resulted in a significant reduction in plasma (FIGS. 10, 11) and liver (FIG. 18) TG levels compared to plasma and liver TG levels when Compound A was administered as monotherapy.

Feeding a Western diet resulted in a 2.2-fold increase in plasma TG levels in

- 46 045908 state after eating relative to rats receiving a balanced one (Fig. 10). Oral administration of either a low dose (1 mg/kg QD) or a high dose (10 mg/kg QD) of Compound A alone (monotherapy) resulted in a 1.7-fold and 1.3-fold increase in plasma TG in the condition, respectively. after meals relative to rats fed a Western diet. Conversely, oral administration of either a low dose (5 mg/kg BID) or a high dose (30 mg/kg BID) of Compound D alone (monotherapy) reduced fasted plasma TG by 55% and 63%, respectively, relative to rats. , fed a Western diet and administered a vehicle. Co-administration of Compound A and Compound D resulted in complete blockade of Compound A-mediated increases in plasma TG in the postprandial state. Oral coadministration of Compound A and Compound D, either both at a low dose or both at a high dose, reduced postprandial plasma TG levels by 37% and 64% relative to vehicle-treated Western diet rats.

Feeding a Western diet resulted in a 1.6-fold increase in fasting plasma TG relative to rats fed a balanced diet (Figure 11). Oral administration of low and high doses of Compound A as monotherapy resulted in 2.4-fold and 1.7-fold increases, respectively, in fasting plasma TG relative to Western diet-fed vehicle-treated rats. Conversely, oral administration of low and high doses of Compound D as monotherapy reduced fasting plasma TG by 20% and 35%, respectively, relative to Western diet-fed vehicle-treated rats. Oral coadministration of Compound A and Compound D, each at both a low dose and a high dose, completely reduced the Compound A-mediated increase in fasting plasma TG observed with Compound A alone. Fasting plasma TG levels for both the low group dose (109 mg/dL) and high dose (81 mg/dL) groups of coadministered Compound A and Compound D were similar to levels in vehicle-treated Western diet-fed rats (96 mg/dL).

Nuclear localization of SREBP-1 was compared in samples from rats on a Western diet that were treated with vehicle, high dose Compound A as monotherapy, high dose Compound D as monotherapy, or co-administered high dose Compound A and high dose Compound D (Fig. 12 ). Relative to vehicle-treated Western diet-fed rats, administration of Compound A caused an increase in the nuclear localization of SREBP-1, an indicator of increased SREBP-1 activation. In contrast, administration of Compound D decreased SREBP-1 nuclear localization and SREBP-1 activation. Co-administration of Compound A and Compound D blocked the Compound A-mediated increase in SREBP-1 nuclear localization, producing a 50% reduction compared to Compound A alone.

Relative to vehicle-treated, balanced-fed rats, Western diet-fed and vehicle-treated animals tended to have decreased lipogenic gene expression: ACC1 (Figure 13), FASN (Figure 14), SCD1 (Figure 15) and SREBP1 (Figure 16), but not PCSK9 (Figure 17), which was lower in rats on the Western diet. Compound A administration tended to further increase, relative to Western diet-fed and vehicle-treated animals, the expression of ACC1, FASN (high dose Compound A only), SCD, but not PCSK9 and SREBP1. In contrast, administration of Compound D decreased the expression of all lipogenic genes. Co-administration of Compound A and Compound D resulted in expression levels comparable to or lower than those observed in vehicle-fed rats on a Western diet.

Relative to rats fed a balanced diet, rats on a Western diet fed vehicle showed an approximately 2.7-fold increase in hepatic triglyceride accumulation (Figure 18). Oral administration of low dose and high dose of Compound A caused a 36% and 53% reduction in steatosis, respectively, relative to vehicle-treated Western diet rats. Similarly, oral administration of a low dose and a high dose of Compound D reduced steatosis by 25% and 30%, respectively, relative to vehicle-treated Western diet rats. Oral coadministration of either low or high dose of both Compound A and Compound D reduced steatosis by 50% and 73%, respectively, relative to vehicle-treated Western diet rats. Oral administration of either a low dose or a high dose combination of Compound A and Compound D resulted in a greater reduction in steatosis than either Compound A or Compound D administered as monotherapy at the same dose levels.

A randomized, vehicle-controlled, 5 parallel group study was conducted in male Wistar-Han rats (Charles River (Boston, MA)) fed a choline-deficient high-fat diet (CDAHFD) (Research diets; A16092003) to identify differences in improvement in markers of hepatic inflammation and fibrosis when administered with either Compound A alone or Compound D alone, either as monotherapy or in combination. Standard laboratory conditions were used to house 60 rats (approximately 200 g); they were housed in pairs and maintained on a 12:12-hour light-dark cycle (lights turned off at 8 a.m.). Rats were fed a choline-deficient, high-fat diet (CDAHFD) starting 6 weeks before the start of the study. Rats randomized into 4 dose groups (n=12 per group) were administered vehicle, Co

- 47 045908 Compound A (5 mg/kg) as monotherapy, Compound D (30 mg/kg) as monotherapy or co-administered Compound A (5 mg/kg) and Compound D (30 mg/kg) for a period of time 6 weeks. Animals (n=12) that remained on a balanced diet throughout the study and were administered vehicle twice daily were used as a control group. Blood samples were collected before the start of compound administration and 3 and 6 weeks after compound administration to assess circulating markers in the blood. Shear wave electrography measurements (Aixplorer Ultimate imager, Supersoinc imagine) were taken at Week -3, Week 0 (before ^1st dose), Week 3 and Week 6 to assess progression of inflammation and fibrosis over time. Histology was assessed after 6 weeks of drug administration, which corresponded to 12 weeks on the CDAHFD diet. Results are given as mean values for animals in each dose group.

After 12 weeks on the CDAHFD diet, animals were sacrificed by CO ₂ asphyxiation. The right lateral, middle, and left lateral lobes of the liver were collected. Sections were taken from the left lateral, right middle and right lateral lobes and fixed in formalin and embedded in paraffin blocks for each animal. One section of the left lateral lobe per animal was cryopreserved at optimal cutting temperature (OCT). The remaining liver from each animal was frozen and quickly minced on an aluminum block cooled in liquid N2, ensuring that the tissue remained frozen throughout the mincing. The minced tissue was transferred and stored at -80°C until analysis. A portion of minced liver sample from each animal was processed and analyzed for markers of fibrogenesis gene expression. Rat taqman probes against aSMA and COL1A1 were all assessed using Actb as the housekeeping gene by qPCR.

The following endpoints were assessed by qualitative histological evaluation by a board-certified veterinary pathologist and quantitative histomorphometry: hepatic stellate cell activation and differentiation into myofibroblasts by aSMA immunohistochemistry (IHC); collagen as a correlate of fibrosis by Picrosirius Red staining. Images were analyzed using Visiopharm software. Visiopharm applications with threshold parameters were used uniformly to identify tissue sections and to quantify targets on each IHC (DAB (3,3'-diaminobenzidine) positive) or histochemically stained slides as a percentage of area: spot area of interest/total tissue ROI (area interest) - empty area) x 100%. Nonparametric statistics were used to analyze the data in this study. Group values are given as mean +/- standard error of the mean.

Relative to control animals that received a balanced diet and were administered vehicle, animals that received CDAHFD and were administered vehicle showed a marked increase in liver stiffness during the study (assessed using shear wave elastography (SWE), measured in kilopascals (kPa)). indicator of progressive inflammation and fibrosis of the liver (Fig. 19). Administration of Compound A or Compound D as monotherapy each reduced liver stiffness, indicating a reduction in liver inflammation and/or fibrosis. Coadministration of Compound A and Compound D resulted in a greater reduction in liver stiffness than administration of either agent as monotherapy (Figure 19).

Relative to control animals fed a balanced diet and treated with vehicle, animals fed CDAHFD and treated with vehicle showed a marked increase in hepatic alpha smooth muscle actin (aSMA) staining, an indicator of myofibroblast activation and fibrogenesis (Figure 20). Administration of Compound A or Compound D each as monotherapy reduced aSMA staining by 41% and 23%, respectively, indicating a reduction in fibroblast activation and liver fibrogenesis. Co-administration of Compound A and Compound D resulted in a greater reduction in aSMA staining than either agent administered as monotherapy, reducing staining by 72% (Figure 20).

Relative to the vehicle-fed control animals, the CDAHFD-fed and vehicle-fed animals showed a marked increase in Picosrius red (PSR) staining, an indicator of collagen deposition and fibrosis (FIG. 21). Administration of Compound A or Compound D as monotherapy each reduced PSR staining by 26% and 20%, respectively, indicating a reduction in collagen deposition and fibrosis. Co-administration of Compound A and Compound D produced a greater reduction in PSR staining than administration of either agent as monotherapy, reducing staining by 56% (Figure 21).

Relative to control animals that were fed a balanced diet and administered vehicle, animals that received CDAHFD and were administered vehicle showed a marked increase in ionized calcium-binding adapter molecule 1 (Iba1) staining, an indicator of hepatic macrophage activation (Figure 24). Administration of Compound A as monotherapy reduced Ibal staining by 15%, indicating a reduction in inflammation

-

Claims (27)

low liver tone. Although administration of D as monotherapy did not change Ibal staining, co-administration of A and D produced a greater reduction in Iba1 staining than Compound A administered as monotherapy, reducing staining by 33% (FIG. 24). Relative to control animals fed a balanced diet and treated with vehicle, animals fed CDAHFD and treated with vehicle showed a marked increase in the expression of the liver alpha smooth muscle actin (aSMA) (Figure 22) and collagen A1A (COL1A1) genes ( Fig. 23), an indicator of myofibroblast activation and fibrogenesis. Administration of Compound A or Compound D as monotherapy each reduced hepatic aSMA and COL1A1 gene expression, indicating a reduction in hepatic fibroblast activation and fibrogenesis. Coadministration of Compound A and Compound D produced greater reductions in hepatic aSMA (Figure 22) and COLIAI (Figure 23) gene expression than either agent alone. Various publications are referred to throughout this application. The descriptions of these publications are incorporated herein by reference in their entirety for all purposes. It will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from the scope or intent of the invention. Other embodiments of the invention will be apparent to those skilled in the art from the description and practice of the invention disclosed herein. It is intended that the Description and Examples be illustrative only and that the scope and spirit of the invention are defined by the following claims. CLAIM 1) the first agent contains ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-M-(tetrahydrofran-3-yl)pyrimidin-5-carboxamide or its pharmaceutical acceptable salt; And 1. A pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-H-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or its a pharmaceutically acceptable salt in combination with 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine -2-yl)benzoic acid or a pharmaceutically acceptable salt thereof, each of which is present in a therapeutically effective amount, in admixture with a pharmaceutically acceptable excipient. 2) the second agent contains 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridine- 2-yl)benzoic acid or a pharmaceutically acceptable salt thereof. 2. Pharmaceutical composition containing ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide or a pharmaceutically acceptable salt thereof , 4(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic an acid or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient. 3. The composition according to claim 1 or 2, further containing at least one additional pharmaceutical agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent and a cholesterol/lipid-modulating agent. 4. A method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a metabolic disease, comprising: administering to a patient in need of such treatment a therapeutically effective amount of (S)-2-(5-((3ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of 4-(4-(1isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)- 6-methoxypyridin-2yl)benzoic acid or a pharmaceutically acceptable salt thereof. 5. A method of treating a disease or condition selected from fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis and non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma or a metabolic disease, comprising: administering to a person in need of such treatment a therapeutically effective amount of (S)-2-(5-((3ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N-(tetrahydrofuran-3-yl)pyrimidin-5- carboxamide or a pharmaceutically acceptable salt thereof and 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'piperidine]-1'-carbonyl)-6-methoxypyridine- 2-yl)benzoic acid or a pharmaceutically acceptable salt thereof. 6. The method of claim 4 or 5, wherein the disease or condition is fatty liver disease. 7. The method of claim 4 or 5, wherein the disease or condition is non-alcoholic fatty liver disease. 8. The method according to claim 4 or 5, where the disease or condition is non-alcoholic steatohepatitis. 9. The method according to claim 4 or 5, where the disease or condition is non-alcoholic steatohepatitis with liver fibrosis. - 49 045908 10. The method according to claim 4 or 5, where the disease or condition is non-alcoholic steatohepatitis with cirrhosis. 11. The method according to claim 10, where the disease or condition is non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma. 12. The method of claim 10, wherein the disease or condition is non-alcoholic steatohepatitis with cirrhosis and a metabolic disease. 13. The method of claim 4 or 5, further comprising administering a therapeutically effective amount of at least one other pharmaceutical agent selected from the group consisting of an acetyl-CoA carboxylase (ACC) inhibitor, a diacylglycerol O-acyltransferase 1 inhibitor ( DGAT-1), monoacylglycerol O-acyltransferase inhibitors, phosphodiesterase (PDE)-10 inhibitor, AMPK activator (adenosine monophosphate-activated protein kinase), sulfonylurea, meglitinide, α-amylase inhibitor, α-glucoside hydrolase inhibitor, α-glucosidase inhibitor, PPARY agonist (peroxisome proliferator-activated receptor gamma), PPAR α/γ agonist, biguanide, glucagon-like peptide 1 (GLP-1) modulator, liraglutide, albiglutide, exenatide, albiglutide, lixisenatide, dulaglutide, semaglutide, protein tyrosine phosphatase-1B (PTP-1B) inhibitor, SIRT-1 activator (sirtuin 1), dipeptidyl peptidase IV inhibitor (DPP-IV), insulin secretagogue, fatty acid oxidation inhibitor, A2 antagonist, c-jun amino terminal kinase (JNK) inhibitor, glucokinase activators (GKa), insulin, insulin mimetic, glycogen phosphorylase inhibitor, VPAC2 receptor agonist, SGLT2 (sodium glucose cotransporter type 2) inhibitors, glucagon receptor modulator, GPR119 modulators (G protein coupled receptor 119), FGF21 derivatives or analogues (fibroblast growth factor 21), TGR5 receptor modulators, modulators GPBAR1 receptor (G protein coupled bile acid receptor 1), GPR40 agonists (G protein coupled receptor 40), GPR120 modulators (G protein coupled receptor 120), high affinity nicotinic acid receptor activators (HM74A), SGLT1 inhibitors (sodium glucose cotransporter type 1), carnitine palmitoyltransferase enzyme inhibitors or modulators, fructose 1,6-biphosphatase inhibitors, aldose reductase inhibitors, mineralocorticoid receptor inhibitors, TORC2 inhibitors, CCR2 (CC chemokine receptor 2) and/or CCR5 (CC chemokine receptor 5) inhibitors ), PKC (protein kinase C) isoform inhibitors (e.g. PKCa, PKCe, PKCy), fatty acid synthetase inhibitors, serine palmitoyltransferase inhibitors, GPR81, GPR39, GPR43, GPR41, GPR105, Kv1.3 modulators, retinol binding protein 4, glucocorticoid receptor, receptors somatostatin, inhibitors or modulators of PDHK2 (pyruvate dehydrogenase kinase 2) or PDHK4 (pyruvate dehydrogenase kinase 4), MAP4K4 inhibitors, IL1 (interleukin 1) family modulators, including HMG-CoA reductase inhibitors, squalene synthetase inhibitors, fibrates, bile acid sequestrants, inhibitors ACAT, MTP (microsomal triglyceride transfer protein) inhibitors, lipoxygenase inhibitors, cholesterol absorption inhibitors, PCSK9 (proprotein convertase subtilisin-kexin type 9) modulators, cholesteryl ester transport protein inhibitors and RXRalpha (retinoid X receptor alpha) modulators. 14. A method of reducing by at least one point in scoring systems the severity of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, reducing the level of serum markers of non-alcoholic steatohepatitis activity, reducing disease activity in non-alcoholic steatohepatitis or reducing the medical consequences of non-alcoholic steatohepatitis in humans, including a stage administering to a patient in need of such a reduction a therapeutically effective amount of N)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-M-(tetrahydrofuran-3-yl)pyrimidin- 5-carboxamide or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of 4-(4-( 1 -isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine] - 1' -carboxyl)-6-methoxypyridin-2-yl)benzoic acid or a pharmaceutically acceptable salt thereof. 15. A method of treating fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, comprising the step of administering to a person in need of such treatment a therapeutically effective quantities of the first and second agent, where: 16. Method according to any one of claims 4, 5, 14 or 15, where ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3yl)-N-(tetrahydrofuran-3-yl )pyrimidine-5-carboxamide is a crystalline solid with the structure: - 50 045908 or a pharmaceutically acceptable salt thereof. 17. The method of claim 16, wherein the crystalline solid has a powder X-ray diffraction pattern containing 2-theta values (CuKa radiation, wavelength 1.54056 A) of 5.3 ± 0.2, 7.7 ± 0, 2 and 15.4 ± 0.2. 18. The method of claim 16, wherein the crystalline solid has a powder X-ray diffraction pattern containing 2-theta values (CuKa radiation, wavelength 1.54056 A) of 6.5 ± 0.2, 9.3 ± 0, 2 and 13.6 ± 0.2. 19. Method according to any one of claims 4, 5, 14 or 15, where 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine ]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid is a crystalline solid with the structure: or a pharmaceutically acceptable salt thereof. 20. The method of claim 19, wherein the crystalline solid is 2-amino-2(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4,6, 7-tetrahydrospiro[indazol5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid. 21. Pharmaceutical composition according to claim 1 or 2, where ^)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin3-yl)^-(tetrahydrofuran-3-yl)pyrimidin-5-carboxamide is a crystalline solid with the structure: or a pharmaceutically acceptable salt thereof. 22. The pharmaceutical composition according to claim 21, wherein the crystalline solid has a powder x-ray diffraction pattern containing 2-theta values (CuKa radiation, wavelength 1.54056 A) of 5.3 ± 0.2, 7.7 ± 0 .2 and 15.4 ± 0.2. 23. The pharmaceutical composition according to claim 21, wherein the crystalline solid has a powder x-ray diffraction pattern containing 2-theta values (CuKa radiation, wavelength 1.54056 A) of 6.5 ± 0.2, 9.3 ± 0 .2 and 13.6 ± 0.2. 24. Pharmaceutical composition according to claim 1 or 2, where 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydro spiro[indazole-5,4'-piperidine]-1'- Carbonyl)-6-methoxypyridin-2-yl)benzoic acid is a crystalline solid with the structure: or a pharmaceutically acceptable salt thereof. 25. The pharmaceutical composition according to claim 24, where the crystalline solid is 2-amino-2-(hydroxymethyl)propane-1,3-diol salt 4-(4-(1-isopropyl-7-oxo-1,4, 6,7-tetrahydro spiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid. 26. The pharmaceutical composition according to claim 2, wherein the additional pharmaceutical agent is selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)-2-methylazetidin-1-yl]-6-( trifluoromethyl)pyrimidin-4-yl}3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1 ]octan-8-yl]-4-fluoro-1,3-benzothiazol-6-carboxylic acid; or 2-[(4-{6-[(4-cyano-2-fluorobenzyl)oxy]pyridin-2-yl}piperidin-1-yl)methyl]-1-[(2S)-oxetan-2-ylmethyl ]-1H-benzimidazole-6-carboxylic acid; or a pharmaceutically acceptable salt thereof. 27. The method according to any one of claims 14, 15, in which at least one other pharmaceutical agent selected from the group consisting of [(1R,5S,6R)-3-{2-[(2S)- 2-methylazetidin-1-yl]-6(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6-yl]acetic acid; 2-[(1R,3R,5S)-3-({5cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octane -8-il]-4 -