EA040490B1 - 2,6-DIAMINOPYRIDINE COMPOUNDS - Google Patents

Description

The present invention relates to novel ketohexokinase (KHK) inhibitor compounds, to pharmaceutical compositions containing these compounds, and to the use of the compounds for the treatment of certain metabolic conditions such as type 2 diabetes mellitus (T2DM), heart failure, diabetic kidney disease and non-alcoholic steatohepatitis (NASH).

Metabolic syndrome is commonly defined as a collection of conditions that reflect overnutrition and a sedentary lifestyle, and its manifestations include type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), obesity, dyslipidemia, heart failure, and kidney disease.

Type 2 diabetes is characterized by relative insulin deficiency caused by pancreatic P-cell dysfunction and target organ insulin resistance. It accounts for more than 90% of diabetic patients and leads to microvascular and macrovascular complications that cause severe psychological and physical suffering for patients and place a high burden on healthcare systems (Davies, M.J., et al., Lancet, 389, 2239-2251, 2017).

Heart failure is a syndrome caused by structural or functional abnormalities of the heart that result in increased intracardiac pressure or decreased cardiac output at rest or during stress. Heart failure is a major and growing cause of morbidity and mortality worldwide (Teerlink, J.R., et al., Lancet, 390, 1981-1995, 2017).

Diabetic kidney disease develops in almost half of patients with type 2 diabetes and is the leading cause of chronic kidney disease worldwide. Metabolic changes associated with diabetes lead to glomerular hyperfiltration, progressive albuminuria, reduced glomerular filtration rate, and eventually end-stage renal disease (Alicic, R.Z., et al., Clin. J. Am. Soc. Nephrol., 12:2032-2045, 2017).

NAFLD is a spectrum of liver diseases that can lead to progressive NASH, fibrosis, and eventually hepatocellular carcinoma and liver failure. In the next 20 years, NAFLD is expected to become the leading cause of liver-related morbidity and mortality, as well as the main indication for liver transplantation (Bertot, L.C., et al., Int. J. Mol. Sci., 17(5), 774 , 2016).

KHK, also called fructokinase, is a rate-limiting enzyme involved in fructose metabolism. It catalyzes the phosphorylation of fructose to fructose-1-phosphate (F1P), causing a concomitant depletion of cellular ATP levels. Unlike glucose, fructose metabolism lacks feedback inhibition and triggers the accumulation of subsequent intermediates involved, for example, in lipogenesis, gluconeogenesis, and oxidative phosphorylation (Hannou, S.A., et al., J. Clin. Invest., 128 (2), 544-555, 2018). This has negative consequences for metabolism, which are associated with a number of serious metabolic disorders.

KHK exists in two alternatively spliced isoforms, consisting of KHK-C and KHKA, differing in exon 3. KHK-C is expressed primarily in the liver, kidney, and intestine, while KHK-A is ubiquitous. Mice deficient in both isoforms are completely protected from the onset of fructose-induced metabolic syndrome. However, adverse metabolic effects are exacerbated in mice lacking only KHK-A (Ishimoto T., et al.; Proc. Natl. Acad. Sci. USA, 109(11), 4320-4325, 2012).

Several epidemiological and experimental studies have reported that increased fructose intake, and more specifically an increase in fructose metabolism, may play an important role in the development of the metabolic syndrome, in particular in the development of type 2 diabetes (Softie et al.; J. Clin. Invest, 127(11 ), 4059-4074, 2017), heart failure (Mirtschink, P., et al.; Eur. Heart J., 39, 2497-2505, 2018), diabetic kidney disease (Cirillo, P., et al.; J Am. Soc. Nephrol, 20, 545-553, 2009) and NAFLD/NASH (Vos, M.B., et a.l; Hepatology, 57, 2525-2531, 2013). Targeted inhibition of KHK is expected to limit fructose metabolism and provide effective treatment options for a number of metabolic disorders.

US 2017/0183328 A1 describes substituted 3-azabicyclo[3.1.0]hexanes as KHK inhibitors.

There is a need for alternative treatments for the metabolic syndrome and its associated indications, including type 2 diabetes, heart failure, diabetic kidney disease, and NASH. In particular, there is a need for compounds having KHK inhibitory activity to provide treatment options for these diseases. In addition, there is a need for potent KHK inhibitors with properties that are important for therapeutic use in humans, such as oral bioavailability and a half-life sufficient to support daily dosing, or a limited drug-drug interaction profile.

Accordingly, the present invention provides a compound of formula I

- 1 040490 where n is 1 or 2;

or a pharmaceutically acceptable salt thereof.

Formula I includes all of their individual enantiomers and diastereomers, as well as mixtures of enan-

thiomers and racemates.

In a particular embodiment, a compound of the present invention is a compound of the formula

or a pharmaceutically acceptable salt thereof.

In one implementation, n is 1.

In this embodiment, the compound of the present invention is a compound of the formula

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of the present invention is a compound of the formula

or a pharmaceutically acceptable salt thereof.

In another implementation, n is 2.

In this embodiment, the compound of the present invention is a compound of the formula

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of the present invention is a compound of the formula

or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the compound of the present invention is a compound of the formula

- 2 040490

or a pharmaceutically acceptable salt thereof.

In this embodiment, the compound of the present invention is 2-[(3R)-1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4-(trifluoromethyl)-2-pyridyl] pyrrolidin-3-yl]acetic acid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention also provides a method of treating type 2 diabetes in a patient in need of such treatment, which comprises administering to the patient an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the method comprises introducing 2-[(3K)-1-[5-cyano-6-[(28)-2-methylazetidin-1-yl]-4(trifluoromethyl)-2-pyridyl]pyrrolidin-3- yl]acetic acid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention also provides a method of treating heart failure in a patient in need of such treatment, which comprises administering to the patient an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the method comprises administering 2-[(3K)-1-[5-cyano-6-[(28)-2-methylazetidin-1yl]-4-(trifluoromethyl)-2-pyridyl]pyrrolidin-3-yl ]acetic acid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention also provides a method of treating diabetic kidney disease in a patient in need of such treatment, which comprises administering to the patient an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the method comprises administering 2-[(3K)-1-[5-cyano-6-[(28)-2-methylazetidin-1yl]-4-(trifluoromethyl)-2-pyridyl]pyrrolidin-3-yl ]acetic acid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention also provides a method of treating NASH in a patient in need of such treatment, which comprises administering to the patient an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the method comprises administering 2-[(3R)-1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4(trifluoromethyl)-2-pyridyl]pyrrolidin-3- yl]acetic acid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention also provides a method of treating chronic kidney disease in a patient in need of such treatment, which comprises administering to the patient an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the method comprises administering 2-[(3K)-1-[5-cyano-6-[(28)-2-methylazetidin-1yl]-4-(trifluoromethyl)-2-pyridyl]pyrrolidin-3-yl ]acetic acid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention further provides a method of treating a disease selected from the group consisting of metabolic syndrome, NAFLD, obesity, complications of diabetes, e.g., diabetic retinopathy, cardiovascular disease, coronary artery disease, and dyslipidemia, in a patient in need of such treatment comprising administering to a patient an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the method comprises administering 2-[(3R)-1-[5-cyano-6-[(28)-2methylazetidin-1-yl]-4-(trifluoromethyl)-2-pyridyl]pyrrolidin-3-yl ]acetic acid or a pharmaceutically acceptable salt thereof.

Moreover, in one embodiment, the present invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy. In one embodiment, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of type 2 diabetes. In one embodiment, the present invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of heart failure. In one embodiment, the present invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of diabetic kidney disease. In one embodiment, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of NASH. In one embodiment, the present invention also provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic kidney disease. In one embodiment, the present invention also provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of metabolic syndrome, NAFLD, obesity, complications of diabetes, such as diabetic retinopathy, cardiovascular disease, coronary artery disease, or lipid dysrhythmias. In a preferred embodiment, the compound of formula I in the therapeutic uses above is 2-[(3R)-1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4(trifluoromethyl) -2-pyridyl]pyrrolidin-3-yl]acetic acid or a pharmaceutically acceptable salt thereof.

Moreover, in one embodiment, the present invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of type 2 diabetes. In one embodiment, the present invention provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of heart failure. In one embodiment, the present invention provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of diabetic kidney disease. In one embodiment, the present invention provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of NASH. In one embodiment, the present invention provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of chronic kidney disease. In one embodiment, the present invention also provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of metabolic syndrome, NAFLD, obesity, complications of diabetes, such as diabetic retinopathy, cardiovascular disease, coronary artery disease, or dyslipidemia. In a preferred embodiment, the compound of formula I is 2-[(3R)-1-[5-cyano-6-[(2S)-2methylazetidin-1-yl]-4-(trifluoromethyl)-2-pyridyl]pyrrolidin-3 -yl]acetic acid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention further provides a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present invention further provides a method for preparing a pharmaceutical composition comprising a mixture of a compound of Formula I, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

As used herein, the term treatment or treat includes curbing, slowing, stopping or reversing the progression or severity of an existing symptom or disorder.

As used herein, the term patient refers to a mammal. Preferably the patient is a human.

As used herein, the term "effective amount" refers to an amount or dose of a compound of formula I, or a pharmaceutically acceptable salt thereof, which, when administered single or multiple times to a patient, provides the desired effect in the patient being diagnosed or treated.

The effective amount can be determined by a person skilled in the art using known techniques and observing results obtained under similar circumstances. In determining an effective amount for a patient, many factors are considered, including, but not limited to, the type of patient; its size, age and general health; the specific disease or disorder involved; the degree of involvement or severity of the disease or disorder; individual patient response; the particular compound being administered; method of administration; characteristics of the bioavailability of the administered drug; the chosen scheme of introduction; use of concomitant medicinal products and other relevant circumstances. The compounds of the present invention are effective at a daily dosage that is in the range of about 0.1 to about 15 mg/kg body weight.

The compounds of the present invention are formulated into pharmaceutical compositions that are administered by any route that ensures the bioavailability of the compound. Preferably, said compositions are for oral administration. Such pharmaceutical compositions and methods for preparing them are well known in the art (see, for example, Remington, J.P., Remington: The Science and Practice of Pharmacy, L.V. Allen, Editor, 22nd ed., Pharmaceutical Press, 2012).

The compounds of formula I or their pharmaceutically acceptable salts are particularly suitable for the methods of treatment according to the present invention, but certain configurations are preferred. The following list of compounds of the present invention describes such configurations. It should be understood that these preferences apply to the compounds of the present invention, as well as methods of treatment, therapeutic applications, and pharmaceutical compositions.

Compounds of the present invention include

- 4 040490

Formula 1b and their pharmaceutically acceptable salts.

Additional compounds according to the present invention include

Formula Pa

NC

F ₃ C

CO ₂ H

Formula II and their pharmaceutically acceptable salts.

Additional compounds according to the present invention include

Formula IIIa’

Formula Sha”

- 5 040490

Formula IIIb" and their pharmaceutically acceptable salts.

While the present invention embraces all individual enantiomers and diastereomers, as well as mixtures thereof, including racemates, compounds of formula Ia, formulas IIa and IIIa and their pharmaceutically acceptable salts are particularly preferred.

The individual enantiomers may be separated or separated by one of skill in the art at any convenient point in the synthesis of the compounds of the present invention by methods such as selective crystallization techniques, chiral chromatography (see, for example, J. Jacques, et al., Enantiomers, Racemates, and Resolutions , John Wiley and Sons, Inc., 1981, and E. L. Eliel and S. H. Wilen, Stereochemistry of Organic Compounds, Wiley-Interscience, 1994), or supercritical fluid chromatography (SFC) (see, for example, T. A. Berger; Supercritical Fluid Chromatography Primer (Agilent Technologies, July 2015).

A pharmaceutically acceptable salt of the compounds of the present invention can be prepared, for example, by reaction between a suitable neutral form of a compound of the present invention and a suitable pharmaceutically acceptable acid or base, in a suitable solvent, under standard conditions known in the art (see, for example, Bastin, R. J., et al., Org. Process. Res. Dev., 4, 427-435, 2000 and Berge, S. M., et al.; J. Pharm. Sci., 66, 1-19, 1977).

The compounds of the present invention, or salts thereof, are prepared by a variety of procedures known to those skilled in the art, some of which are presented in the Schemes, Preparations and Examples below. The products of each step in the schemes below can be isolated by conventional means well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the following schemes, all substituents, unless otherwise indicated, are as previously defined. Reagents and starting materials are generally available to those skilled in the art. Without limiting the scope of the invention, the following schemes, methods of preparation and examples are presented to further illustrate the invention. Additionally, a person skilled in the art will understand that a compound of formula I can be prepared using a starting material or an intermediate with the corresponding desired stereochemical configuration, which can be obtained by a person skilled in the art.

Some abbreviations are defined as follows: ABT refers to 1-aminobenzotriazole; ACN refers to acetonitrile; BSA refers to bovine serum albumin; CAS# refers to the reference service registry number; DXM refers to methylene chloride or dichloromethane; DIPEA refers to diisopropylethylamine; DMEM refers to Dulbecco's Modified Eagle's Medium; DMSO refers to dimethyl sulfoxide; ELSD refers to an evaporative light scattering detector; ER/MS refers to electrospray mass spectrometry; EtOAc refers to ethyl acetate; EtOH refers to ethanol or ethyl alcohol; FBS refers to fetal bovine serum; h refers to the hour or hours; HPLC refers to high performance liquid chromatography; Me refers to methyl; MeOH refers to methanol; MTBE refers to methyl tert-butyl ether; min refers to the minute or minutes; m/z refers to the ratio of mass to charge; PBS refers to phosphate buffered saline; Ph refers to phenyl; FBC refers to a round bottom flask; RT refers to room temperature; SCX refers to selective cation exchange; SFC refers to supercritical fluid chromatography; THF refers to tetrahydrofuran.

- 6 040490

Scheme

h 4

⁵ la

The scheme shows the general preparation of compounds of formula I. In step 1, 3-cyano-2,6-dichloro-4(trifluoromethyl)pyridine (1) and a cyclic amine (2) are reacted in the presence of a base such as NaHCO ₃ or DIPEA in EtOH or MeOH to form aminopyridine (3). Alternatively, the reaction solvent for this step may be DCM. In step 2, compound 3 is reacted with 2-methylazetidine (4) at elevated temperature and in the presence of a base such as NaHCO ₃ or DIPEA in EtOH or MeOH to give diaminopyridine (5). Alternatively, the reaction solvent for this step may be THF. In step 3, the ester moiety is hydrolysed using a base such as NaOH or LiOH in MeOH or THF at elevated temperature to give a compound of formula Ia. Alternatively, this step can be carried out using the same reagents in a microwave reactor.

Getting and examples.

The following preparations and examples further illustrate the present invention and demonstrate exemplary methods for synthesizing a compound of the present invention. Reagents and starting materials are readily available to those skilled in the art or can be easily synthesized by them. It should be understood that these preparations and examples are provided by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

LC-ESR/MS was performed on an AGILENT® HP1100 liquid chromatography system. Electrospray mass spectrometry measurements (recorded in positive and/or negative mode) were performed on a quadrupole mass spectrometer with a mass selective detector connected to an HPLC system, which may or may not be equipped with an ELSD. LC-MS conditions (low pH): column: PHENOMENEX® GEMINI® NX C18 2.0x50 mm 3.0 µm, 110 A; gradient: 5-95% B over 1.5 min followed by 95% B over 0.5 min; column temperature: 50±10°C; flow rate: 1.2 ml/min; injection volume 1 μl; solvent A: deionized water with 0.1% HCOOH; solvent B: ACN with 0.1% formic acid; wavelength 200-400 nm and 212-216 nm. If the HPLC is equipped with an ELSD, the settings are as follows: evaporator temperature 45°C, nebulizer temperature 40°C and gas flow 1.6 scl/min. Alternate LC-MS conditions (high pH): Column: Waters xBridge® C18 2.1x50 mm, 3.5 µm column; gradient: 5-95% B in 1.5 min, then 95% B in 0.50 min; column temperature: 50±10°C; flow rate: 1.2 ml/min; injection volume 1 μl; solvent A: 10 mM NH ₄ HCO ₃ , pH 9; solvent B: ACN; wavelength: 200-400 nm and 212-216 nm; with ELSD: evaporator temperature 45°C, nebulizer temperature 40°C and gas flow 1.60 scl/min.

Preparation 1. Salt of (2S)-1-benzhydryl-2-methylazetidine[(1R,4S)-7,7-dimethyl-2-oxonorbornan-1yl]methanesulfonic acid

A 2000 ml three neck round bottom flask was connected to an addition funnel, nitrogen inlet and thermometer adapter. The vessel was purged with nitrogen and (3R)-butane-1,3-diol (25 g, 277.4 mmol), DIPEA (127 ml, 731 mmol) and ACN (556 ml) were added. Cooled to -30°C. Trifluoromethanesulfonic acid anhydride (101 ml, 601 mmol) was added dropwise over 3 h so that the internal temperature was maintained at -35 to -30°C. After the addition was completed, the mixture was stirred for 10

- 7 040490 min at -35 to -30°C. Trifluoromethanesulfonic acid anhydride (1.9 ml, 11 mmol) was added dropwise over 5 minutes so that the internal temperature was maintained at -35 to -30°C. After completion of the addition was stirred for 10 min at a temperature of from -35 to -30°C. DIPEA (127 ml, 731 mmol) was added dropwise over 15 minutes so that the internal temperature was maintained at -35 to -30°C. After completion of the addition was stirred for 10 min at a temperature of from -35 to -30°C. In a separate flask under nitrogen atmosphere, aminodiphenylmethane (48.0 ml, 270 mmol) was dissolved in ACN (49 ml, 935 mmol) and the resulting solution was transferred to an addition funnel. The amine solution was added dropwise to the cold triflate over 40 minutes to keep the internal temperature between -20°C and -35°C. After completion of the addition was stirred for 30 min at a temperature of from -35 to -30°C. The reaction mixture was transferred to a water bath and allowed to warm slowly over 30 minutes. The bath was removed and the reaction mixture was allowed to warm to room temperature over 30 minutes. The vessel was transferred to a heating mantle and the reaction mixture was heated to 45°C for 30 min, then cooled to room temperature. The resulting mixture was poured into 1200 ml of water and extracted with toluene (400 mlx3). The extracts were combined, washed with water, sat. aq. NaCl solution, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated on a rotary evaporator. The material was dried under vacuum overnight. The residue was dissolved in DCM (400 ml). A silica pad was prepared on a sintered funnel and equilibrated with a 1:1 heptane/EtOAc mixture. The product solution was loaded onto a silica pad and washed with 1600 ml of heptane/EtOAc 1:1. The filtrate was concentrated to give a red oil. The oil was dissolved in MeOH (250 ml) and the flask was placed in a water bath (~10°C). Partially added L(-)-camphorsulfonic acid (61.6 g, 265 mmol), maintaining the internal temperature below 20°C. The resulting mixture was stirred for 15 minutes and then concentrated on a rotary evaporator to a brown foam. The foam was dried on a vacuum pump for 2 hours. The foam was dissolved in 130 ml DCM. The dropping funnel was attached to the flask. Using a funnel, 1100 ml of EtOAc was slowly added to the stirred solution. The resulting mixture was transferred to a 4000 ml beaker and stirred in air overnight. The beaker was cooled in an ice bath for 10 min. The precipitate was collected in a sintered-bed funnel by vacuum filtration, washing with a minimal amount of ice-cold EtOAc. The solid was dried on the sintered bed for 2 hours. The resulting white solid was dissolved in a minimum amount of DCM, transferred to a 2000 ml beaker and then slowly diluted with EtOAc until the clear solution turned cloudy. The suspension was stirred for 4 hours in the open air. The solids were collected by vacuum filtration using a sintered funnel and dried on the sintered bed overnight to give the title compound (111.8 g, 238.06 mmol, 86% yield) as a white solid.

¹ H NMR (400 MHz, (d6-DMSO): 10.54-10.47 (m, 1H), 7.61 (d, J=7.3 Hz, 5H), 7.47-7.37 ( m, 7H), 5.85 (d, J=10.3 Hz, 1H), 4.68-4.61 (m, 1H), 3.91-3.83 (m, 2H), 3.37 (s, 8H), 2.99 (d, J=14.6 Hz, 1H), 2.77-2.68 (m, 1H), 2.51-2.44 (m, 4H), 2, 30-2.16 (m, 2H), 1.91-1.81 (m, 2H), 1.42-1.28 (m, 3H), 1.08 (s, 3H), 1.01 ( d, J=6.6 Hz, 3H), 0.77 (s, 4H), >98% ee [HPLC: Chiralcel OJ (10 cm x 4.6 mm, 5 µm), 5 ml/min, 40°C isocratic 10% EtOH (0.2% ⁱ PrNH2)/CO ₂ ].

Preparation 2 [(1R,4S)-7,7-dimethyl-2-oxonorbornan-1-yl]methanesulfonate (2S)-2methylazetidin-1-ium salt

To a 2250 ml Parr flask was added 20 wt% Pd(OH) ₂ on charcoal (6.62 g). The bottle was purged with nitrogen and 250 ml MeOH was added. The salt of (2S)-1-benzhydryl2-methylazetidine[(1R,4S)-7,7-dimethyl-2-oxonorbornan-1-yl]methanesulfonic acid (111 g, 236 mmol) dissolved in 250 ml MeOH. The vessel was blocked. Purged with nitrogen, then hydrogen and pressurized to 60 PSI. Shake the reaction vessel vigorously in a Parr mixer for 15 hours at room temperature. The vessel was purged with nitrogen and then the reaction mixture was filtered through a pad of celite, rinsing with MeOH. The filtrate was concentrated to give a white solid and dried under vacuum. The solid was suspended in 780 ml of MTBE/EtOAc 1:1, the mixture was heated to 65° C. for 20 h, then cooled to room temperature and stirred overnight. Solids were collected by filtration. The solids were suspended in 380 ml of MTBE and stirred at room temperature for 24 hours. The white solid was collected by filtration to give the title compound (41.5 g, 136.78 mmol, 58% yield).

1H NMR (400 MHz, d ₆ -DMSO): 8.68-8.55 (m, 1H), 4.51-4.42 (m, 1H), 3.91-3.75 (m, 1H) , 3.36 (s, 3H), 2.91 (d, J=14.6 Hz, 1H), 2.69-2.61 (m, 1H), 2.52-2.46 (m, 2H ), 2.28-2.22 (m, 1H), 2.17-2.10 (m, 1H), 1.96 (t, J=4.5 Hz, 1H), 1.89-1, 79 (m, 1H), 1.43 (d, J=6.7 Hz, 2H), 1.36-1.26 (m, 1H), 1.05 (s, 2H), 0.75 (s , 2H).

- 8 040490

Preparation 3. Methyl 2-[(3R)-1-[6-chloro-5-cyano-4-(trifluoromethyl)-2-niridyl]n-pyrrolidin-3yl]acetate

3-cyano-2,6-dichloro-4-(trifluoromethyl)pyridine (123 mmol, 29.6 g) and EtOH (230 ml) were added to a round bottom flask. The mixture was cooled to 0°C. NaHCO ₃ (368 mmol, 31 g) was added, after which a solution of methyl (R)-pyrrolidine-3-acetate hydrochloride (23 g, 123 mmol) in EtOH (230 ml) was added. The resulting mixture was allowed to warm to room temperature overnight. The reaction mixture was evaporated to dryness on a rotary evaporator. Water (200 ml) was added and extracted with MTBE (2x200 ml). The extracts were combined and evaporated to dryness. Purify by silica gel chromatography using hexane/MTBE (20 to 70% gradient) to give the title compound (34.7 g, 99.89 mmol, 81% yield) as a white solid. ER/MS (m/z): 348.0, 350.0 [M+H]+.

Preparation 4. Methyl 2-[(3R)-1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4-(trifluoromethyl)-2pyridyl]pyrrolidin-3-yl]acetate

L ^CH 3 N

NC X

A 1 CO ₂ CH ₃

To a round bottom flask was added methyl 2-[(3R)-1-[6-chloro-5-cyano-4-(trifluoromethyl)-2pyridyl]pyrrolidin-3-yl]acetate (25.5 g, 73.3 mmol), [(1R,4S)-7,7-dimethyl-2-oxonorbornan-1-yl]methanesulfonate (2S)-2-methylazetidin-1-ium salt (88.0 mmol, 26.7 g), MeOH (255 ml ), and NaHCO ₃ (220 mmol, 18.5 g). The mixture was stirred at 65° C. for 16 hours. [(1R,4S)-7,7-dimethyl-2-oxonorbornan-1-yl]methanesulfonate (2S)-2-methylazetidin-1-ium salt (22.0 mmol, 6.68 g) and NaHCO ₃ (147 mmol, 12.3 g) continued stirring for 32 h. The solvent was removed on a rotary evaporator. Water (300 ml) was added to the residue and extracted with MTBE (2x200 ml). The extracts were combined and dried over anhydrous MgSO ₄ , filtered through silica gel and concentrated to dryness to give the title compound (28.0 g, 73.2 mmol, 99% yield) as a white solid. ER/MS (m/z): 383.2 [M+H] ⁺ .

Preparation 5 Methyl 2-[1-[6-chloro-5-cyano-4-(trifluoromethyl)-2-pyridyl]azetidin-3-yl]acetate

To a solution of 3-cyano-2,6-dichloro-4-(trifluoromethyl)pyridine (500 mg, 2.03 mmol) in EtOH (15 ml) was added NaHCO ₃ (0.549 g, 6.51 mmol) and methyl salt 2- trifluoroacetic acid (azetidin-3-yl)acetate (0.494 g, 2.03 mmol). The mixture was stirred at RT for 16 hours. The reaction mixture was diluted with saturated aqueous NaCl (30 ml). The mixture was extracted with EtOAc (20 ml x3). The extracts were combined and washed with saturated aqueous NaCl (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel column chromatography eluting with EtOAc in petroleum ether (0-30% gradient) to give the title compound (470 mg, 1.41 mmol, 66% yield) as a white solid. ER/MS (m/z) = 333.9 [M+H] ⁺ .

Preparation 6 Methyl 2-[1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4-(trifluoromethyl)-2-pyridyl]azetidin-3-yl]acetate

_NaHCO (0.173 g, 2.05 mmol) and [(1R,4S)-7,7-dimethyl-2-oxonorbornan-1-yl]methanesulfonate (2S)-2-methylazetidin-1-ium salt (0.194 g, 0.626 mmol ). The mixture was heated to 80° C. for 16 hours. The mixture was diluted with water (50 ml) and extracted with EtOAc (40 mlx3). The extracts were combined and washed with saturated aqueous NaCl (50 ml), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (0-10%,

EtOAc in petroleum ether) to give the title compound (176 mg, 0.46 mmol, 77% yield) as a white solid. ER/MS (m/z): 383.1 [M+H]+.

Example 1 2-[(3R)-1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4-(trifluoromethyl)-2-pyridyl]pyrrolidin-3-yl]acetic acid

ASN ₃

N ^{Z 3 ν} Αν

L II from ₂ n

Methyl 2-[(3R)-1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4(trifluoromethyl)-2-pyridyl]pyrrolidin-3-yl] was added to a round bottom flask. acetate (26.5 g, 69.3 mmol), MeOH (265 ml) and 2M aq. NaOH solution (416 mmol, 208 ml). The mixture was stirred at 60° C. for 3 hours. Cooled to room temperature and MeOH was removed on a rotary evaporator. The aqueous phase was acidified to pH 3-4 with concentrated aqueous HCl. Washed with EtOAc (400 ml). The organic layer was washed with water (200 ml), dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give the title compound (22.5 g, 61.11 mmol, 88% yield) as a white foam. ER/MS (m/z): 369.2 [M+H] ⁺ .

Example 2 2-[1-[5-Cyano-6-[(2S)-2-methylazetidin-1-yl]-4-(trifluoromethyl)-2-pyridyl]azetidin-3yl]acetic acid

To a mixture of methyl 2-[1-[5-cyano-6-[(2S)-2-methylazetidin-1-yl]-4-(trifluoromethyl)-2-pyridyl]azetidin3-yl]acetate (176 mg, 0.478 mmol ) in THF (5.00 ml) and water (1.00 ml) was added LiOH (0.04 g, 0.955 mmol). The resulting mixture was stirred at room temperature for 2 hours. It was diluted with EtOAc (4 ml) and the aqueous layer was extracted with EtOAc (2 mlx3). The organic extracts were combined and washed with saturated aqueous NaCl (3 mlx2), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated to give the title compound (128 mg, 0.36 mmol, 78% yield) as a white solid. ER/MS (m/z) 354.9 [M+H]+.

Analysis methods.

Assay of KHK enzyme activity for human KHK-C and human KHK-A.

The intrinsic activity of the compounds can be measured using an enzymatic assay that measures F1P production. Compounds were prepared in DMSO and tested using a 10-point concentration curve to give 3-fold serial dilutions of compounds in a 96-well plate ranging from 20 μM to 1.02 nM. The enzyme was prepared in assay buffer [50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 10 mM potassium chloride, 100 mM magnesium chloride, 2 mM tris(2-carboxyethyl)phosphine (TCEP), 0 01% n-octylglucoside] and incubated with compounds at room temperature for 15 min. The reaction was carried out in volumes of 100 μl containing concentrations of the substrate fructose (250 μM for the KHK-C assay and 1.25 mM for the KHK-A assay) and ATP (150 μM for both isoforms); which were additionally incubated at room temperature for 20 min. The reaction was then stopped by adding stop buffer; consisting of 0.2% formic acid and 1 µg/ml internal standard ¹³ C6-fructose-6-phosphate ( ¹³ C6-F6P). The plates were stored at -20° C. until RapidFire MS analysis.

RapidFire MS assay for F1P quantification.

An Agilent 300 RapidFire automated extraction system (Agilent, Santa Clara, CA) with three quaternary HPLC pumps is connected to an Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an electrospray ionization source (ESI). The RapidFire Mass Spec system is equipped with a reusable RapidFire C18 (type C) solid phase extraction (SPE) cartridge (G9205A).

Solvent A used to load and wash the sample is 6 mM octylamine (Acros Organics 129495000) adjusted to pH 5.0 with acetic acid. Solvent B used to elute the sample is 20% water in ACN containing 0.1% formic acid. Samples were analyzed sequentially by withdrawing 10 μl per suction loop under vacuum directly from the multiwell plates. 10 µl of sample was loaded into a Cl8 cartridge and washed with solvent A at a flow rate of 1.25 ml/min for 5000 ms. The retained analyte was eluted into the mass spectrometer using solvent B at a flow rate of 1.25 ml/min for 5000 ms. The system was re-equilibrated using a solvent

And at a flow rate of 1.25 ml/min for 2000 ms.

The tri-quadrupole mass spectrometer is equipped with an ESI source and analytes are monitored by Selected Reaction Monitoring (SRM) in negative mode [M-H]. F1P was monitored at m/z 259.02/96.9 and ¹³ C ₆ -fructose-6-phosphate was monitored at m/z 264.99/97. Area ratio values for F1P were calculated using ¹³ Cb-fructose-6-phosphate as an internal standard.

The compounds of examples 1 and 2 were tested as described above.

____________________________________________________________ Table 1

Example number hKNK-S 1C ₅₀ (nM) hKHK-A 1С ₅₀ (nM) 1 9 19 2 27 37

These results demonstrate that the compounds of examples 1 and 2 inhibit the enzymatic activity of both KNK-C and KNK-A.

Analysis of CNK cellular activity.

Efficacy can be measured using a cellular assay of inhibition of the conversion of fructose to F1P of cellular CNK. HepG2 cells were seeded in 96-well cell culture plates in growth medium [Dulbecco's Modified Eagle's Medium (DMEM) high glucose, 10% heat-inactivated fetal bovine serum (HFBS), 1x penicillin/streptomycin] and allowed to attach overnight in an incubator at 37°C. Growth medium was washed and replaced with an assay medium consisting of reduced whey Gibco OptiMEM 1 medium, 0.1% casein, 8.33 mM O-fructose- ^13Cb , and compound at concentrations ranging from 100 to 0.0051 µM (10- dotted concentration curve). The plates were incubated at 37°C for 3 hours, after which the assay medium was pumped out of the cell wells. A stopping solution consisting of 80% methanol, 2 mM _{ammonium acetate, and 50 ng/ml fructose-6phosphate-13C6} ^was then added to the cells. The plates were stored at -20° C. until RapidFire MS analysis (described above).

The compounds of examples 1 and 2 were tested as described above.

table 2

Example number HepG2 IC50 1 127 2 316

These results demonstrate that the compounds of examples 1 and 2 inhibit the metabolism of fructose to F1P.

Liquid chromatographic method with tandem mass spectrometry (LC-MS/MS) for pharmacokinetic analyses. Samples were extracted by protein precipitation by adding 180 µl MeOH:ACN (1:1, v/v) containing an internal standard and 25 µl (rodent samples) or 50 µl (non-rodent samples) plasma. Samples were then diluted with MeOH:water (1:1, v/v) to obtain concentrations within the range of the standard curve. Diluted samples were analyzed by LC-MS/MS using a Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS; Foster City, CA) equipped with a TurboIonSpray interface and operating in positive ion mode. Analyzes were separated chromatographically using a Javelin Thermo Betasil Cl8 5 µm 20x2.1 mm column (rodent samples) or an Advantage ECHELON Cl8 4 µm 20 mm x 2.1 mm column (non-rodent samples). LC conditions were as follows: water/1M ammonium bicarbonate (2000:10, v/v) (mobile phase A) and MeOH/1M ammonium bicarbonate, (2000:10, v/v) (mobile phase B) .

Pharmacokinetics in mice.

The in vivo pharmacokinetic properties and involvement of the OATP1A/1B transporter in the pharmacokinetics of Example 1 were demonstrated in wild-type FVB and OATP1A/1B knockout mice (Taconic # 10707) (fasted; η = 4/genotype). Example 1 was administered once intravenously (IV; 1 mg/kg; volume 1 ml/kg) in a vehicle. Blood was collected from each animal at 0, 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours post-dose. Liver, spleen, and pancreas were harvested, weighed, and perfused 24 hours post-dose. Plasma and tissue concentrations of Example 1 were determined by the LC-MS/MS method as described above.

In FVB mice, Example 1 had a half-life of 5.43 hours, a mean clearance of 6.91 ml/h/kg, a volume of distribution of 2.74 L/kg, with a mean hepatic partition coefficient of the unbound form (K _puu )

- 11 040490

67.5. In OATP1A/1B knockout mice, Example 1 has a half-life of 9.36 hours, a mean clearance of 1.34 ml/h/kg, a volume of distribution of 0.986 L/kg, with a mean hepatic partition coefficient of the unbound form (K _puu ) of 24, 2. These data show that OATP is involved in the hepatic uptake of Example 1 in mice and that the involvement of this transporter affects clearance.

Pharmacokinetics in dogs.

The in vivo pharmacokinetic properties of Example 1 were demonstrated in beagle dogs (post-fed, n=3-4). Example 1 was administered once orally (PO; 3 mg/kg; volume 2 ml/kg) or intravenously (IV; 1 mg/kg; volume 1 ml/kg) in a vehicle. Blood was taken from each animal at 0, 0.03 (group IV only), 0.08 (IV), 0.25, 0.5, 1, 2, 4, 8, 12, 24, 32 (IV), 48 (IV) and 72 (IV) hours after dosing. Plasma concentrations for Example 1 were determined by LC-MS/MS as described above.

For oral doses, the mean half-life of Example 1 was 6.9 hours and the bioavailability was ~93%. For intravenous doses, the mean half-life of Example 1 was 7.4 hours and the mean clearance was 2.52 ml/h/kg with a low volume of distribution (1.33 l/kg). These data show that Example 1 has low total clearance, low volume of distribution and high oral bioavailability in dogs.

In vivo characterization of major excretion pathways was demonstrated using cannulated bile ducts (BDC) in beagle dogs (post-fed; n = 3). Example 1 was administered once intravenously (IV; 1 mg/kg; volume 1 ml/kg) in a vehicle. Blood was collected from each animal at 0, 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 32, 48 and 72 hours post-dose. Bile was collected from each animal at 1, 2, 3, 4, 5, 6, 12, 18, 24, 32, 48 and 72 hours post-dose. Urine was collected at 12, 24, 48 and 72 hours and feces were collected at 24, 48 and 72 hours post-dose. Plasma, bile, urine and feces concentrations from Example 1 were determined by LC-MS/MS as described above.

The mean half-life of Example 1 was 2.9 h and the mean clearance was 4.69 ml/h/kg with a low volume of distribution (0.546 l/kg). Urinary levels of Example 1 are negligible and ~10% of the administered intravenous dose is excreted in the bile. These data show that Example 1 is excreted via the kidneys and bile in negligible amounts. Overall, the half-life in dogs with cannulated bile ducts, 2.9 hours, is faster than the half-life determined in intact dogs, 7.4 hours, suggesting enterohepatic recirculation.

Pharmacokinetics in cynomolgus monkeys.

The in vivo pharmacokinetic properties of Example 1 were demonstrated in cynomolgus monkeys. Compounds were administered once (orally; 10 mg/kg; volume 5 ml/kg) in vehicle. Blood was collected from each animal at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 32, 48 and 72 hours post-dose. Plasma concentrations for Example 1 were determined by LC-MS/MS as described above.

The mean half-life of Example 1 was 15.3 hours. These data indicate that Example 1 is orally bioavailable and is slowly cleared in monkeys.

Internal clearance in human hepatocytes (± AVT).

This method is designed to determine the metabolic clearance in vitro no substrate depletion in hepatocytes. Incubation in the presence and absence of ABT, an inhibitor of the panCYP450 enzyme, was used to evaluate the role in CYP-mediated metabolism. Cryopreserved human hepatocytes were thawed at 37°C, centrifuged and reconstituted in hepatocyte maintenance medium to a density of 1x106 viable cells/ml. In a pre-warmed 96-well plate, 196 μl of hepatocyte suspension was added to each well. Cells were preincubated with and without ABT as follows: for preincubation with ABT, 2 µl of 100 mM ABT solution was added (2 µl of medium without ABT was added to control samples), and the plate was incubated at 37°C for 30 min with constant shaking ( ~600 rpm). After that, 2 µl of a 30 µM stock solution of the test substance was added, and after 0, 15, 30, 60, 120, 240 min, 20 µl aliquots were taken and quenched by transfer into ACN containing an internal standard. After centrifugation at 4000 rpm for 30 min, the supernatant concentration was determined by LC-MS/MS. Clearance was calculated from the slope of the % remaining compound over time.

The clearance of Example 1 is inhibited by AWT by ~12% in human hepatocytes, suggesting a limited involvement of CYP enzymes in the hepatic clearance of Example 1.

Inhibition of OATP1B1 and OATP1B3.

OATP1B1, OATP1B3 cells and vector control (VC) were grown in 5% CO ₂ at 37°C in a humid atmosphere in DMEM medium supplemented with 10% FBS, 50 μg/ml gentamicin and 5 μg/ml blasticidin. Cells were seeded in 24-well BioCoat Poly-D-Lysine plates. Cells were treated with 5 mM sodium butyrate in DMEM supplemented 24 h before the experiment. Cell cultures were washed twice with pre-warmed PBS before the experiment. After washing, all cell types were incubated for 30 min at 37°C in 200 µl of buffer, buffer with varying concentration of the test substance or with a suitable positive control inhibitor. Based on preliminary data, concentrations were tested from 0.025 to 12.5 μM for OATP1B1 and from 0.20 to 100 μM for OATP1B3 (but

Claims (16)

minal). At the beginning of the pre-incubation, 50 μl of buffer was removed to determine the concentrations of test substances in the wells using LC-MS/MS. After the preincubation period, the buffer was removed. Experiments started with the addition of 200 µl of substrate solution (total 400 nM rosuvastatin, including 1.4 nM tritiated rosuvastatin). Incubation was carried out with and without inhibitor. The positive control inhibitor used for each cell line was 50 μM rifamycin SV. Experiments were run for 1 min at 37° C. at which the reaction was stopped and washed by adding 1000 μl of ice-cold PBS per well. Each well was then aspirated and washed twice with ice-cold PBS. The cells in each well were lysed in 400 μl of 1% Triton X 100 in PBS (v/v). Samples were taken from each well of the radioactivity scoring plates, and the protein concentration in each well was determined by the bicinchoninic acid method. IC ₅₀ values were determined by fitting data using GraphPad Prism. Before installation, the nominal concentrations were converted to measured ones. Example 1 inhibited OATP1B1 more than OATP1B3, with IC ₅₀ values of 0.12 and 5.5 μM, respectively. CLAIM 1. Compound formula where n is 1 or 2; or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, characterized in that n is 1, or a pharmaceutically acceptable salt thereof. 3. The compound according to claim 2, characterized in that the compound is or a pharmaceutically acceptable salt thereof. 4. A compound according to claim 1, characterized in that n is 2, or a pharmaceutically acceptable salt thereof. 5. The compound according to claim 4, characterized in that the compound is or a pharmaceutically acceptable salt thereof. 6. The compound according to claim 5, characterized in that the compound is or a pharmaceutically acceptable salt thereof. 7. A method of treating type 2 diabetes in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof. 8. A method for treating heart failure in a patient, comprising administering to a patient - 13 040490 given in such treatment, an effective amount of a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof. 9. A method of treating diabetic kidney disease in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof. 10. A method of treating nonalcoholic steatohepatitis in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof. 11. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 for the treatment of type 2 diabetes. 12. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 for the treatment of heart failure. 13. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 for the treatment of diabetic kidney disease. 14. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 for the treatment of non-alcoholic steatohepatitis. 15. A pharmaceutical composition containing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 with one or more pharmaceutically acceptable carriers, diluents or excipients. 16. A method for preparing a pharmaceutical composition, comprising mixing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 with one or more pharmaceutically acceptable carriers, diluents or excipients. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2