Sodium-hydrogen exchanger 3 inhibitor compounds

Abstract

The present invention relates to sodium-hydrogen exchanger 3 (NHE3) inhibitor compounds of the following formula: to pharmaceutical compositions comprising the compounds and to the use of the compounds for treating certain diseases associated with elevated sodium and / or phosphate levels.

Description

[0001] The present invention relates to novel sodium-hydrogen exchanger 3 (NHE3) inhibitor compounds, to pharmaceutical compositions comprising said compounds, and to the use of said compounds for treating certain diseases associated with elevated sodium and / or phosphate levels. The present invention further relates to the use of novel NHE3 compounds in combination with certain sodium-dependent phosphate cotransporter (NPT2b) inhibitors.

[0002] NHE3 is an epithelial sodium-hydrogen exchanger present in the thick and thin limbs of the small intestine, colon, gallbladder, renal proximal duct and Henle's loop in mammals. In the intestine, NHE3 regulation occurs sharply as part of digestion and is inhibited immediately after a meal. NHE3-deficient mice show sodium-fluid balance disorders, highlighting the role of NHE3 in maintaining volume homeostasis and indicating that NHE3 is a major contributor to intestinal sodium absorption. In addition, recent in vivo studies have shown that NHE3 inhibition leads to reduced intestinal phosphate absorption (Orlowski, J. et al., Pflugers Arch.-Eur. J. Physiol., 2004, 447: 549-565, King, AJ et al., Sci. Transl. Med., 2018, 10, 1-17).

[0003] Excessive sodium intake is ubiquitous in the Western diet and is closely associated with the development of hypertension, which in turn is associated with an increased risk of cardiovascular and renal disease. Maintaining an optimal level of sodium intake can help manage blood pressure and prevent cardiovascular and renal disease (Weintraub, WS et al., J. Am. Coll. Cardiol., 2015, 65(10), 1042-1050).

[0004] The reduced renal clearance of chronic kidney disease (CKD) and end-stage renal disease (ESRD) leads to an increased phosphate load, which can promote secondary hyperparathyroidism of the kidneys and ultimately lead to hyperphosphatemia (Wolf M., J. Am. Soc. Nephrol., 2010, 21, 1427-1435). CKD is not only the most common cause of hyperphosphatemia in humans, but also in adult cats and dogs (Kidder, A. et al., J. Feline Med. Sur., 2009, 11, 913-924). A well-documented consequence of hyperphosphatemia is cardiovascular disease (CVD), which can manifest as vascular and heart valve calcification, left ventricular hypertrophy, heart failure, arrhythmias and sudden cardiac death (Fujii, H. et al., Clin. Exp. Nephrol., 2017, 21 (S1), S53-S63). Secondary hyperparathyroidism caused by increased phosphate load can also lead to bone disease (Yuen, NK et al., Perm. J., 2016, 20(3), 15-127).

[0005] Effective reduction of intestinal phosphate absorption through dietary phosphate restriction and use of phosphate absorption inhibitors will reduce the phosphate load in CKD and help reduce the sequelae that lead to further deterioration of CVD, bone disease, and renal function.

[0006] WO2010 / 078449 discloses compounds and methods for inhibiting NHE-mediated antiport in the treatment of conditions associated with fluid retention or salt overload and gastrointestinal conditions. WO2014 / 169094 discloses NHE3 binding compounds and methods for inhibiting phosphate transport.

[0007] In summary, it is recognized that excessive sodium and phosphate absorption in the intestine leads to increased morbidity and associated mortality of diseases such as hypertension, CKD, CVD, and bone disease. Therefore, alternative treatments for these conditions are needed, especially treatments that inhibit NHE3. Specifically, compounds that effectively reduce sodium and phosphate absorption in the body and are highly selective for NHE3 inhibition are needed. In addition, it is also desirable to have NHE3 inhibitor compounds that are effective in combination with NPT2b inhibitor compounds.

[0008] Therefore, the present invention provides compounds of formula I:

[0009]

[0010] wherein both R are CN or both R are C(O)NH2, or a pharmaceutically acceptable salt thereof.

[0011] In a particular embodiment, both R are C(O)NH2.

[0012] In a particular embodiment, the compound is of the formula:

[0013]

[0014] or a pharmaceutically acceptable salt thereof.

[0015] In another embodiment, the compound is of the formula:

[0016]

[0017] In another embodiment, the compound is the dihydrochloride salt of:

[0018]

[0019] In a particular embodiment, the compound is of the formula:

[0020]

[0021] In a particular embodiment, the compound is the dihydrochloride salt of:

[0022]

[0023] The present invention also provides a method for treating a disease selected from CKD, hyperphosphatemia, secondary hyperparathyroidism, heart failure, hypertension and CVD, comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to a mammal in need thereof. In one embodiment, the present invention provides a method for treating CKD in a cat, comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to a cat in need thereof.

[0024] The present invention also provides a method for treating a disease selected from CKD, hyperphosphatemia, secondary hyperparathyroidism, heart failure and CVD, comprising administering to a mammal in need thereof a therapeutically effective combination of a compound of formula I or a pharmaceutically acceptable salt thereof and a compound of formula II or a pharmaceutically acceptable salt thereof:

[0025]

[0026] In one embodiment, the present invention provides a method of treating CKD in a cat, comprising administering to a cat in need thereof a therapeutically effective combination of a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0027] In addition, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in therapy. Specifically, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in treating CKD, hyperphosphatemia, secondary hyperparathyroidism, heart failure, hypertension or CVD. In one embodiment, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in treating CKD in cats.

[0028] In addition, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof, which is used for simultaneous, separate or sequential combination with a compound of formula II or a pharmaceutically acceptable salt thereof for treating CKD, hyperphosphatemia, secondary hyperparathyroidism, heart failure or cardiovascular disease. In one embodiment, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof, which is used for simultaneous, separate or sequential combination with a compound of formula II or a pharmaceutically acceptable salt thereof for treating CKD in cats.

[0029] In addition, the present invention provides a compound of formula II or a pharmaceutically acceptable salt thereof, which is used for simultaneous, separate or sequential combination with a compound of formula I or a pharmaceutically acceptable salt thereof for treating CKD, hyperphosphatemia, secondary hyperparathyroidism, heart failure or cardiovascular disease. In one embodiment, the present invention provides a compound of formula II or a pharmaceutically acceptable salt thereof, which is used for simultaneous, separate or sequential combination with a compound of formula I or a pharmaceutically acceptable salt thereof for treating CKD in cats.

[0030] In addition, the present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent.

[0031] In addition, the present invention provides a pharmaceutical composition, which comprises a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent, and further comprises a compound of formula II or a pharmaceutically acceptable salt thereof.

[0032] In addition, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating CKD, hyperphosphatemia, secondary hyperparathyroidism, heart failure, hypertension or CVD. In one embodiment, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating CKD in cats.

[0033] The compounds and combinations of the present invention are useful for treating diseases and conditions associated with sodium and / or phosphate imbalance, including: hyperphosphatemia, particularly hyperphosphatemia in ESRD or CKD or familial hyperphosphatemia; hyperparathyroidism, particularly secondary hyperparathyroidism associated with CKD; calcium phosphate kidney stones; heart valve calcification associated with CKD or ESRD; bone fractures associated with CKD; calciphylaxis; tumoral calcification; and acute kidney disease.

[0034] In a particular embodiment, the compound of formula II is a free acid. In a particular embodiment, the compound of formula II is a disodium salt. The free acid compound of formula II is referred to herein as "Compound A".

[0035] As used herein, the term "treating" includes inhibiting, slowing, stopping or reversing the progression or severity of existing symptoms or conditions.

[0036] In one embodiment, the mammal to be treated is a human. In another embodiment, the mammal is a cat or a dog, preferably a cat.

[0037] As used herein, the term "effective amount" refers to the amount or dosage of a compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof that provides the desired effect in a patient being diagnosed or treated after single or multiple dose administration to a mammal.

[0038] The effective amount can be determined by one skilled in the art by using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for a patient, many factors are considered, including, but not limited to: the species of the patient; the size, age, diet and general health of the patient; the specific disease or condition involved; the extent or involvement or severity of the disease or condition; the response of the individual patient; the specific compound administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosage regimen selected; the use of concomitant drugs; and other relevant circumstances. The compounds of Formula I and Formula II are effective at daily doses ranging from about 0.01 to about 15 mg / kg body weight.

[0039] The compound of formula I or formula II is formulated as a pharmaceutical composition administered by any route that makes the compound bioavailable. Preferably, the pharmaceutical composition is for oral administration. The pharmaceutical composition and its preparation method are known in the art (see, for example, Remington, JP, Remington: The Science and Practice of Pharmacy, LV Allen, ed., 22nd edition, Pharmaceutical Press, 2012).

[0040] The compounds of formula I and their pharmaceutically acceptable salts can be used for the therapeutic purposes of the present invention, wherein certain configurations are preferred. The following list of compounds of the present invention describes such configurations. It should be understood that these preferred contents apply to the therapeutic purposes and compounds of the present invention.

[0041] The skilled artisan will appreciate that for compounds where both R are C(O)NH2, the guanylurea moiety can exist in either the E or Z configuration and can be readily converted between the two. The compounds of the present invention include both E and Z isomers, and mixtures thereof.

[0042] Compounds of formula I include:

[0043]

[0044]

[0045] or a pharmaceutically acceptable salt thereof.

[0046] Although the present invention contemplates all individual enantiomers and diastereomers, as well as mixtures of enantiomers of the compounds, including racemates, compounds of Formula Ia' and Formula Ia" and pharmaceutically acceptable salts thereof are particularly preferred. In particular, the dihydrochloride salt of the compound of Formula Ia" is preferred.

[0047] One of ordinary skill in the art can separate or resolve individual enantiomers at any convenient point in the synthesis of the compounds of the invention by methods such as selective crystallization techniques, chiral chromatography (see, e.g., J. Jacques et al., Enantiomers, Racemates, and Resolutions, John Wiley and Sons, Inc., 1981, and ELEiel and SH Wilen, Stereochemistry of Organic Compounds, Wiley-Interscience, 1994), or supercritical fluid chromatography (SFC) (see, e.g., TABerger; Supercritical Fluid Chromatography Primer, Agilent Technologies, July 2015).

[0048] Pharmaceutically acceptable salts of compounds of formula I can be formed, for example, by reacting a suitable free base form of a compound of formula I with a suitable pharmaceutically acceptable acid in a suitable solvent under standard conditions known in the art (see, for example, Bastin, RJ et al.; Org. Process. Res. Dev., 4, 427-435, 2000 and Berge, SM et al.; J. Pharm. Sci., 66, 1-19, 1977). Specifically, a preferred salt of a compound of formula I is a dihydrochloride.

[0049] The pharmaceutically acceptable salt of the compound of formula II can be formed, for example, by reacting the compound of formula II in the form of an appropriate free acid with an appropriate pharmaceutically acceptable base in a suitable solvent under standard conditions known in the art (see, for example, Bastin, RJ et al.; Org. Process. Res. Dev., 4, 427-435, 2000 and Berge, SM et al.; J. Pharm. Sci., 66, 1-19, 1977). Specifically, the preferred salt of the compound of formula II is the disodium salt.

[0050] WO2018 / 034883 describes a class of compounds, including compounds of formula II, which are inhibitors of sodium-dependent phosphate co-transporter 2b (NPT2b or NaPiIIb). NPT2b is present on the luminal surface of the small intestine, where it promotes phosphate absorption by active transport. The compounds of formula II can be prepared by the method described in WO2018 / 034883.

[0051] Formula I compound or its salt can be prepared by various methods known to those of ordinary skill in the art, some of which are described in the following preparation examples and examples. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration and crystallization. Reagents and starting materials are readily available to those of ordinary skill in the art. Without limiting the scope of the invention, the following preparation examples and examples are provided to further illustrate the present invention. In addition, it is understood by those of ordinary skill in the art that Formula I compound can be prepared by using starting materials or intermediates with corresponding desired stereochemical configurations that can be prepared by those skilled in the art.

[0052] Certain abbreviations are defined as follows: “ACN” refers to acetonitrile; “BCECF-AM” refers to 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; “BSA” refers to bovine serum albumin; “DCM” refers to dichloromethane; “DMSO” refers to dimethyl sulfoxide; “EtOAc” refers to ethyl acetate; “ES / MS” refers to electrospray mass spectrometry; “EMS” refers to ethyl methanesulfonate; “EtOH” refers to ethanol; “FBS” refers to fetal bovine serum; and “h” refers to hour. ; “HEPES” refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; “HPLC” refers to high performance liquid chromatography; “IC50” refers to the inhibitor concentration at half-maximal inhibition; “LC-MS” refers to liquid chromatography-mass spectrometry; “MeOH” refers to methanol; “min” refers to minutes; “MTBE” refers to methyl tert-butyl ether; “m / z” refers to mass-to-charge ratio; “Pd(dba)2” refers to bis(dibenzylideneacetone)palladium(0); “RT” refers to room temperature; and “TLC” refers to thin layer chromatography.

[0053] LC-MS HP1260 LC system. Mass spectrometry was done by ES / MS (acquired in positive and / or negative mode) and performed on a mass selective detector quadrupole mass spectrometer connected to an HPLC which may or may not have an ELSD. LC-MS conditions (low pH): Column: NX C18 2.0×50 mm 3.0 μm, 110 Angstroms; Gradient: 5-95% B in 1.5 minutes, then 95% B for 0.5 minutes; Column temperature: 50°C + / - 10°C; Flow rate: 1.2 ml / min; 1 μl injection volume; Solvent A: 0.1% HCOOH in deionized water; Solvent B: 0.1% formic acid in ACN; Wavelengths 200-400 nm and 212-216 nm. If the HPLC is equipped with an ELSD, set to 45°C evaporator temperature, 40°C nebulizer temperature, and 1.6 SLM gas flow rate. Alternative LC-MS conditions (high pH): Column: Waters C18 column 2.1×50 mm, 3.5 μm; Gradient: 5-95% B in 1.5 minutes, then 95% B for 0.50 minutes; Column temperature: 50℃+ / -10℃; Flow rate: 1.2 ml / min; 1 μL injection volume; Solvent A: 10 mM NH4HCO3 pH 9; Solvent B: ACN; Wavelengths: 200-400 nm and 212-216 nm; If ELSD is available: 45℃ evaporator temperature, 40℃ nebulizer temperature and 1.60 SLM gas flow rate.

[0054] Preparation Example 1

[0055] 4-(3-Benzylthiophenyl)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinoline

[0056]

[0057] Three reactions were set up in parallel at the same scale. For each reaction, two solutions, solution 1 and solution 2, were prepared. Solution 1: To a solution of 4-(3-bromophenyl)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinoline (54.0 g, 145.5 mmol) in xylene (1.08 L) was added Pd(dba)2 (3.35 g, 5.82 mmol) and Xantphos (3.37 g, 0.04 eq. 5.82 mmol) at 15°C under nitrogen. The mixture was stirred for 1 hour. Solution 2: To a mixture of K2CO3 (10.1 g, 72.7 mmol) in xylene (1.08 L) was added phenylmethylmercaptan (27.1 g, 218.3 mmol) dropwise at 0°C under nitrogen. The mixture was stirred for 1 hour at 15°C. Solution 1 was added dropwise to solution 2 at 15°C, and the mixture was stirred at 140°C for 12 hours.

[0058] The three reaction mixtures were combined and washed with water (500 ml).The organic layer was concentrated and purified by silica gel chromatography using a gradient of 1% to 17% EtOAc in petroleum ether to give the title compound (92 g, 50%). 1 H-NMR (400MHz, CDCl3) δ7.28-7.17(m,8H),7.05-7.04(m,1H),7.00-6.95(m,1H),6.74-6.69(m,1H),4.15-4. 04(m,3H),3.76(d,J=16.0Hz,1H),3.47(d,J=16.0Hz,1H),2.90(dd,J=11.2,7.2MHz,1H),2.50-2.42(m,4H).

[0059] Preparation Example 2

[0060] 3-(6,8-Dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl)benzenesulfonyl chloride; hydrochloride

[0061]

[0062] Two reactions were set up in parallel on the same scale. For each reaction, 1,3-dichloro-5,5-dimethyl-imidazolidine-2,4-dione (47.5 g, 241 mmol) was added portionwise to a stirred mixture of 4-(3-benzylthiophenyl)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinoline (50.0 g, 121 mmol), acetic acid (52.17 g, 868.8 mmol) and water (62 ml) in CAN (690 ml) at 0 ° C. The mixture was stirred for 2 hours at 0 to 5 ° C, and the reaction mixture was then concentrated in vacuo. MTBE (200 ml) and HCl (4M solution in 1,4-dioxane, 10 ml) were added to each reaction, and the reaction mixtures were combined. Stir for 30 minutes, and filter to collect the solid. The solid was washed with MTBE (100 ml) to obtain the title compound (112 g), which can be used without further purification. Assuming quantitative yield, the purity was estimated to be 92% w / w based on a theoretical yield of 103 g. TLC (1:6 EtOAc: petroleum ether) R f =0.05.

[0063] Preparation Example 3

[0064] tert-Butyl N-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]carbamate

[0065]

[0066] To a solution of tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (5.22 g, 21.0 mmol) and trimethylamine (6.38 g, 8.79 ml, 63.0 mmol, 3.00 equiv) in anhydrous DCM (120 ml) was added racemic 3-(6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl)benzenesulfonyl chloride (Abacipharm, 8.21 g, 21.0 mmol) and stirred overnight at room temperature under nitrogen. The reaction mixture was concentrated in vacuo. EtOAc (50 ml) was added to the oily residue, the solids were filtered off and the filtrate was concentrated in vacuo to give an orange oil. Purification by silica gel column chromatography eluting with EtOAc gave the racemic compound (6.92 g, 55%) as a colorless oil. 1H-NMR(400MHz, CDCl3)δ7.75(d,J=8.0Hz,1H),7.67(s,1H),7.44(t,J=7.6Hz,1H), 7.36(d,J=7.2Hz,1H),7.23(d,J=2.0Hz,1H),6.71(d,J=1.2Hz,1H),5.42-5.05(br m,1H),4.29(t,J=6.0Hz,1H),3.73(d,J=16.2Hz,1H),3.57(d,J=15.8Hz,1H),3.55-3.46(m,8H),3.34-3.25(m, 2H), 3.14-3.08 (m, 2H), 2.97 (dd, J=11.5, 5.29Hz, 1H), 2.59 (dd, J=11.5, 7.2Hz, 1H), 2.46 (s, 3H), 1.41 (s, 9H).

[0067] exist The enantiomers were separated on an AD (8×35 cm) column (flow rate 400 ml / min, detection at 260 nm), 11 injections of 629 mg, eluted with 95:5 EtOH:ACN to obtain the title compound (2.94 g, 23%), which was the second eluting isomer. Chiral HPLC (column: AD-H 4.6×150 mm, flow rate: 0.6 ml / min, detection: 250 nm, eluent: 95:5 EtOH:ACN) showed >99% ee.

[0068] Alternative synthesis

[0069] To a solution of tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (57.1 g, 230 mmol) in DCM (1.70 L) was added triethylamine (65.2 g, 645 mmol) at 0°C. 3-(6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl)benzenesulfonyl chloride; hydrochloride (starting material prepared according to Preparation Example 2; 84.0 g, 181 mmol) was added portionwise to the mixture at 0°C. The mixture was heated to 25°C and stirred for 4 hours. The reaction mixture was combined with two reaction mixtures prepared essentially as described above starting with 3-(6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl)benzenesulfonyl chloride; hydrochloride (starting material prepared according to Preparation Example 2; 52 g each, 112 mmol, 104 g, 224 mmol in total). The resulting mixture was poured into ice water (1.8 L), then the organic layer was washed with water (2 x 1.5 L) and concentrated to give a yellow residue. Purification by silica gel chromatography using a gradient of 1% to 2% MeOH in DCM gave the racemic compound (157 g, 64% yield) as a yellow oil.

[0070] This racemate (157 g, 261 mmol) was combined with additional racemate (23 g, 38 mmol) prepared as above. The enantiomers were separated by SFC on an AD-H (5 μm, 3×25 cm) column (flow rate 70 g / min, UV detection at 220 nM) eluting with 70:30 CO2:(0.1% NH4OH) in MeOH to afford the title compound (50 g, 28%) as a yellow gum, the second eluting isomer, >99% ee. ES / MS m / z ( 35 Cl / 37 Cl)602 / 604[M+H] + .

[0071] Preparation Example 4

[0072] N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide dihydrochloride

[0073]

[0074] To tert-butyl N-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]carbamate (2.94 g, 4.88 mmol) was added 4M hydrogen chloride in 1,4-dioxane (10 ml, 40 mmol) and the resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give a white foam (2.85 g, >100%). It was dried under high vacuum overnight and used without further manipulation. ES / MS m / z ( 35 Cl / 37 Cl)502 / 504[M+H] +

[0075] Preparation Example 5

[0076] N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide

[0077]

[0078] Using 10g N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide dihydrochloride (992 mg, 1.84 mmol) was purified by SCX column to obtain 740 mg of free base (740 mg, 80%) as a clear colorless semisolid.

[0079] Preparation Example 6

[0080] N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide

[0081]

[0082] To tert-butyl N-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]carbamate (48 g, 80 mmol) in MeOH (190 ml) at 15°C was added 4M hydrochloric acid in 1,4-dioxane (100 ml, 400 mmol) and the resulting solution was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was dissolved in water (150 ml). To this solution was added 5N NaOH (30 ml) and 2N NaOH (50 ml) until pH 9-10, then 20% aqueous Na2CO3 (75 ml) was added to bring the solution to pH 11. The aqueous mixture was extracted with EtOAc (2×200 ml, then 1×100 ml). The organics were combined and washed with a mixture of saturated aqueous sodium chloride (120 ml) and 20% Na2CO3 (30 ml). The organics were dried over sodium sulfate, filtered and concentrated in vacuo. The residue was dissolved in toluene and concentrated in vacuo, and this process was repeated twice more. The residue was dissolved in DCM and concentrated in vacuo, and this process was repeated twice more to give a dark yellow oil (42 g, 94%) based on anisole as an internal standard. 1 H-NMR comparison showed that the purity was about 90 wt %. 1 H-NMR (400MHz, CDCl3) δ7.76-7.72(m,1H),7.68-7.66(m,1H),7.43(t,J=7.6Hz,1 H),7.36-7.33(m,1H),7.23(d,J=2.1Hz,1H),6.74-6.72(m,1H),4.28-4.22(m,1H ),3.67(d,J=16.2Hz,1H),3.62-3.49(m,9H),3.11(t,J=4.6Hz,2H),2.91(dd,J=1 1.8, 5.5Hz, 1H), 2.87 (t, J = 5.1Hz, 2H), 2.58 (dd, J = 11.7, 7.4Hz, 1H), 2.44 (s, 3H). ES / MS m / z( 35 Cl / 37 Cl)502 / 504[M+H] + .

[0083] Example 1

[0084] 2-Cyano-1-[4-[[N'-cyano-N-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinoline

[0085] [(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]amidino]amino]butyl]-3-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]guanidine

[0086]

[0087] To a solution of N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide (prepared according to Preparation Example 5; 740 mg, 1.47 mmol) in 1,4-dioxane (10 ml) and pyridine (10 ml) was added N-cyanocarbonyl diphenyl imide (351 mg, 1.47 mmol) and stirred overnight at room temperature under nitrogen. Butane-1,4-diamine (65 mg, 0.74 mmol) was added and heated at 60° C. for 24 hours and then at 90° C. overnight. The reaction mixture was cooled to room temperature. The reaction mixture was diluted with EtOAc, washed twice with 5% aqueous potassium carbonate solution, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to give an orange oil. The crude mixture was purified by silica gel column chromatography eluting with a gradient of 0 to 10% MeOH in DCM to afford the title compound as a yellow foam (591 mg, 67%). ES / MS m / z ( 35 Cl)1193[M+H] +

[0088] Alternative synthesis

[0089] To a solution of N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide (prepared according to Preparation Example 6; purity 90 wt%, 24.5 g, 44 mmol) in 1,4-dioxane (240 ml) was added pyridine (17.7 ml, 219 mmol) and N-cyanocarbonyl imide diphenyl ester (10.5 g, 44 mmol) and stirred overnight at room temperature under nitrogen. Butane-1,4-diamine (1.93 g, 21.9 mmol) was added to the reaction mixture, the reactant was divided into four equal portions, and each portion was heated to 90° C. for 21 hours. Butane-1,4-diamine (102mg, 1.16mmol) and 1,4-dioxane (5ml) were added to each portion of the reactants and continued to heat at 90°C for 23 hours. Butane-1,4-diamine (42mg, 0.48mmol) and 1,4-dioxane (5ml) were added to the portion of the reaction mixture that showed a cyanoisourea intermediate:title compound ratio greater than 1:5 by LC-MS determination. All portions of the reaction mixture were heated at 90°C overnight and then cooled to room temperature. The reaction portions were combined. The mixture was combined with two additional batches prepared in essentially the same manner using N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]benzenesulfonamide (90 wt% purity) (10.05 g, 18.00 mmol and 5.20 g, 9.32 mmol, respectively). The solvent was removed under reduced pressure and the residue was dissolved in DCM (250 ml) and the organics were washed with 5% aqueous potassium carbonate solution (2 x 80 ml). The aqueous washes were combined and extracted with DCM (2 x 40 ml). All organic washes were combined together and washed with saturated aqueous sodium chloride solution (80 ml). The organics were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a yellow-brown oil. The crude material was purified by silica gel column chromatography eluting with a gradient of 8 to 16% EtOH in DCM. The material was then re-purified twice by silica gel chromatography, eluting with gradients of 5 to 12% and 2 to 12% MeOH in DCM. Impure fractions were re-purified by silica gel chromatography, eluting with a gradient of 2% to 12% MeOH in DCM, and the pure material was combined to give the title compound as an off-white foamy solid (15.3 g, 36%). ES / MS m / z ( 35 Cl)1193[M+H] + .

[0090] Example 2

[0091] [[4-[[N'-Carbamoyl-N-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethyl]amidino]amino]butylamino]-[2-[2-[2-[[3-[(4S)-6,8-dichloro-2-methyl]-3,4-dihydro-1H-isoquinolin-4-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethylamino]methylene]urea dihydrochloride

[0092]

[0093] To Example 1 (670 mg, 0.562 mmol) was added trifluoroacetic acid (10 ml) and water (1 ml) and stirred at room temperature overnight. The volatiles were removed under reduced pressure. The residue was dissolved in a minimum amount of MeOH and passed through 10 g Purification by SCX column gave yellow foam (629 mg). EVO C 18 100 x 30 mm, 5 μm particle size, in-line heater at 50°C; eluent: 51 to 86% (5% MeOH in 10 mM NH4HCO3) in ACN] to give the title compound as the free base as an orange oil (253 mg, 37%). ES / MS m / z ( 35 Cl)1229[M+H] + In a vial, the free base compound (223 mg, 0.181 mmol) was dissolved in DCM (0.5 ml) and hydrochloric acid (1 M solution in ether, 1 ml) was added dropwise under shaking. A white solid precipitated from the solution. The mixture was shaken for 5 minutes and then concentrated in vacuo to give the title compound as a fine white powder (236 mg, 100%). ES / MS m / z ( 35 Cl)1229[free base M+H] + ,1227[Free base MH] +

[0094] Alternative synthesis

[0095] Prepare two equal portions of the mixture in the following manner: For each mixture, the compound of Example 1 (5.1 g, 4.3 mmol), a dioxane solution of 4M hydrochloric acid (50 ml, 200 mmol) and water (25 ml) were mixed. Each solution was heated to 65 ° C for 2 hours. The mixture was cooled to room temperature and stirred at room temperature overnight, and then they were combined with another batch of materials prepared in substantially the same manner as the compound of Example 1 (3.1 g, 2.6 mmol) as raw material. The mixture was concentrated in vacuo. Water / ethanol (1:2, 100 ml) was added to the residue and concentrated in vacuo. This operation was repeated three times. EtOH was added to the residue and stirred at 45 ° C for 10 minutes, then concentrated in vacuo. This operation was repeated three times. The residue was dried in vacuo at room temperature for 22 hours. Another batch of products (1 g, 0.8 mmol) prepared in substantially the same manner as described above were added to the residue. Dry under vacuum at room temperature for 23 hours, 50°C for 16 hours, 55°C for 6 hours, and then at room temperature for 72 hours to give the title compound (14.7 g, 94%). ES / MS m / z ( 35 Cl)1229[free base M+H] + .

[0096] Determination

[0097] In vitro NHE3 inhibitory activity

[0098] To create the NHE3 assay, cells selectively expressing NHE3 were generated by overexpressing human NHE3 in an endogenous NHE-deficient cell line. The NHE-deficient cell line was generated by chemically induced mutagenesis. After mutagenesis, NHE-deficient cells were selected by placing lithium (Li)-loaded cells in an acidic extracellular environment. Under these conditions, NHE converts intracellular Li + Exchanged for extracellular protons, only NHE-deficient cells can avoid toxic intracellular acidosis and survive.

[0099] The NHE-deficient cell line NHD C8 was derived from the cell line Dede ( CCL-39TM) was produced. Mutagenesis was induced by treating Dede CCL-39 cells with EMS (Sigma-Aldrich) for 20 hours in a growth medium at a final concentration of 500 μg / ml. NHE-deficient cells were selected by culturing in a LiCl solution (130 mM LiCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM glucose and 20 mM Hepes-Tris, pH 7.4) for 2 hours, and then in a choline chloride solution (130 mM choline chloride, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2 and 20 mM 2-(N-morpholino)ethanesulfonic acid-Tris, pH 5.5) for 30 minutes. Surviving cells were grown to 90% confluence in standard culture medium and then a second round of selection was performed. Monoclonal colonies were isolated, grown to complete confluence, and a third round of selection was performed. Surviving clones were expanded and tested for sodium-hydrogen exchanger activity (see assay below).

[0100] The myc-tagged cDNA encoding human NHE3 was subcloned into plasmid pcDNA3.1 and a stable cell line was generated in the NHE-deficient NHD C8 cells mentioned above. Cells stably overexpressing NHE3 were maintained in McCoy's 5A medium (Hyclone-GE Healthcare Bio science) containing 10% FBS, 400 mg / ml G418 (Gibco-ThermoFisher Scientific) and antibiotic / antimycotic solution.

[0101] To measure NHE activity, the intracellular pH of the cells was lowered by adding NH4Cl. The sodium-dependent intracellular pH changes were then measured by the intracellular pH-sensitive fluorescein dye BCECF-AM (Molecular Probes-ThermoFisher Scientific). NHD8 / NHE3 cells were spread into 96-well poly-D-lysine plates (Corning) at 30,000 per well using a multichannel pipette. The cells were cultured at 37°C plus 5% CO2 for 24 hours. The cell culture medium was aspirated, and the cells were washed twice with 100 μl NaCl-HEPES solution (100 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% BSA, 50 mM HEPES, pH 7), and then incubated with 100 μl of 5 μM BCECF-AM in NH4Cl / pluronic F-127 / probenecid solution (130 mM NH4Cl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% BSA, 0.0625% pluronic F-127, 2 mM probenecid, 20 mM HEPES, pH 7) at room temperature for 60 minutes. After incubation, wash cells twice with 100 μl of HEPES / 0.1% BSA solution (100 mM choline chloride, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% BSA, 50 mM HEPES, pH 7) without ammonium and sodium. Then add 86 μl of HEPES / 0.1% BSA containing 30 μM amiloride and compound or control to the appropriate wells. NHE activity is initiated by adding 14 μl 1M NaCl to a final concentration of 140 mM. Immediately read the fluorescence intensity of the plate at 505 nm excitation wavelength and 550 nm emission wavelength. The percentage inhibition at each test concentration is calculated relative to the NHE activity (0% inhibition) at 1% DMSO and the NHE activity (100% inhibition) at the saturation concentration of the standard inhibitor. IC was determined by fitting nine concentration response curves from 1000 nM to 0.152 nM plus no compound to a 4-parameter model using Prism. 50 .

[0102] Example 2 Inhibition of human NHE3 in a concentration-dependent manner, IC 50 5.76nM (IC 50 Example 2 is selective for NHE3 because it is not an effective NPT2b (IC 50 =58.4 μM) or SGLT1 (IC 50=6.6 μM) inhibitor. Example 1 Inhibits human NHE3 in a concentration-dependent manner, IC 50 37.9nM (IC 50 The geometric mean, n = 2, the standard error of the geometric mean is 6.2).

[0103] Example 2 Effect on Blood Pressure

[0104] Male spontaneously hypertensive (SHR) rats weighing 250-300g at 7 weeks of age were adapted to the reverse light cycle (lights off at 8:00 am and lights on at 8:00 pm) and were free to eat regular food and water. An implantable telemetry device (model TA11PA-C40, DataSciences International) was implanted in the abdominal aorta. After recovery from surgery, rats were placed in a separate cage in a quiet telemetry room for measuring blood pressure (mean arterial pressure, MAP) and heart rate (HR). DSI DataquestIV 4.0 software was used to collect 20 seconds of digital pressure signals every 5 minutes.

[0105] Thirty-one SHR rats were randomized into two groups by MAP and treated twice daily for 14 days with vehicle (n=15) (10% gum arabic, 0.05% antifoam solution in purified water) or 0.15 mg / kg / day Example 2. The first dose was given between 6:00 and 6:30 in the morning (2 hours before lights out) and the second dose was given between 4:00 and 4:30 in the afternoon. Daily food consumption and body weight gain were also monitored.

[0106] MAP, HR, and body weight (BW) data were analyzed by repeated measures analysis of covariance (ANCOVA) with baseline as a covariate. Food consumption (FC) data were analyzed by repeated measures analysis of variance (ANOVA). For the group treated with 0.15 mg / kg / day Example 2, the mean blood pressure over the entire study time course (Days 1-14) was 4.83 mm Hg lower than the control group with a p value of 0.0007 (Table 1). No significant effects were observed on HR, BW, or FC.

[0107] Table 1: Effect of Example 2 on mean arterial pressure (MAP) in male spontaneously hypertensive rats (SHR)

[0108]

[0109] Example 1 Effect on Sodium Absorption

[0110] Urinary sodium excretion after oral administration of large doses of sodium chloride is an indirect measure of sodium absorption in the intestine.

[0111] Male Sprague-Dawley rats with a body weight range of 200 to 250 g were randomized into groups based on equal mean body weight. Hereinafter, the following formulation is referred to as "1% HEC": 1% hydroxyethylcellulose (HEC) and 0.25% 80 with defoamer vehicle. 0.1 mg / ml of Example 1 (final dose of 1 mg / kg, administered in 10 ml / kg volume) was prepared in a glass vial containing a pre-weighed amount of compound with 1% HEC and then probe sonicated until it appeared as a uniform suspension. To ensure that the compound did not adhere to the sides or bottom of the vial, a stirring bar was added to the bottle and the suspension was stirred throughout the preparation and administration process. Subsequent dosing solutions of Example 1 were prepared by serial dilution with 1% HEC. 10 mg / ml of NaCl was prepared by adding sterile water (final dose of 200 mg / kg, administered in 20 ml / kg volume).

[0112] On the day before the study, the animals were placed in clean cages without food but with access to water, fasting overnight. On the day of the study, the rats were orally administered vehicle or different doses of Example 1. 30 minutes later, the animals were administered NaCl and then immediately transferred to metabolic cages. Urine samples were collected for 2 hours. Net urine volume was recorded. Urine sodium and creatinine were assessed using a clinical biochemical analyzer.

[0113] The values ​​expressed as the ratio of urine sodium to creatinine (mM / mM) were calculated and expressed as mean ± SEM. These curves were fitted with a 4-parameter logarithmic curve fitting tool, GraphPad Prism 6, to calculate ED50. For the purpose of curve fitting, the dose of Example 1 was artificially set to 0.001 mg / kg in the software for the vehicle alone group.

[0114] After oral administration of Example 1 and NaCl, urinary sodium excretion decreased in a dose-dependent manner (Table 2). ED of urinary sodium excretion of Example 1 50 The dose-dependent reduction in urinary sodium excretion is consistent with the dose-dependent inhibition of intestinal sodium absorption in Example 1.

[0115] Table 2: Effect of Example 1 on urinary sodium in rats

[0116]

[0117] Example 2 Effect on Sodium and Phosphate Absorption

[0118] Urinary sodium and phosphate excretion after oral administration of large doses of sodium phosphate is an indirect measure of sodium and phosphate absorption in the intestine.

[0119] Based on equal average body weight, male Sprague-Dawley rats of about 7 weeks of age with a body weight range of 195 to 221 g were randomized. Example 2 (final dose of 3 mg / kg, administered in 10 ml / kg volume) was prepared at 0.3 mg / ml by adding 1% HEC to a glass vial containing a pre-weighed compound and then probe sonicated until it appeared as a uniform suspension. To ensure that the compound does not adhere to the side or bottom of the vial, a stirring bar was added to the bottle and the suspension was stirred throughout the preparation and administration process. The subsequent dosing solution of Example 2 was prepared by serial dilution with 1% HEC. 34.5 mg / ml of NaH2PO4 (final dose of 690 mg / kg, administered in 20 ml / kg volume) was prepared by adding sterile water.

[0120] On the day of the study, the animals were placed in clean cages without food but with access to water, and fasted for 4 hours before the study. Rats were orally administered vehicle or different doses of Example 2. After 15 minutes, the animals were administered sterile water or NaH2PO4 and then immediately transferred to a metabolic cage. Urine samples were collected for 4 hours. Net urine volume was recorded. Urine sodium, creatinine, and phosphate were assessed using a clinical biochemical analyzer. The values ​​expressed as the ratio of urine to dietary phosphorus or sodium were calculated and expressed as mean ± SEM. These curves were fitted with a 4-parameter logarithmic curve fitting tool, GraphPod Prism 6, to calculate ED50. For the purpose of curve fitting, for the vehicle group alone, the dosage of Example 2 was artificially set to 0.00001 mg / kg in the software.

[0121] After oral administration of Example 2 and phosphate, urinary sodium and phosphate excretion decreased in a dose-dependent manner (Table 3). ED for urinary phosphate excretion of Example 2 50 is 0.041 mg / kg, and the ED for sodium excretion in Example 2 50 The dose-dependent reduction in urinary sodium and phosphate excretion is consistent with the dose-dependent inhibition of intestinal sodium and phosphate absorption in Example 2.

[0122] Table 3: Effect of Example 2 on urinary phosphorus and sodium in rats

[0123]

[0124] Example 2 Effect of Combination with NPT2b Inhibitor (Compound A) on Phosphate Absorption in Rats

[0125] NPT2b inhibitor dosing solution: Compound A and poly-1-vinylpyrrolidone-co-vinyl acetate (PVP-VA, Sigma-Aldrich) were weighed into a bottle at a weight ratio of 30% Compound A and 70% PVP-VA. A clear yellow solution was prepared by diluting the mixture in MeOH followed by 5N NaOH with the addition of 2 mol NaOH / mol Compound A. The solution was spray dried by a stream of hot nitrogen and the solid dispersion powder was collected and then further dried in a vacuum oven. The dose of the spray dried solid dispersion (SDD) was always expressed as the active pharmaceutical ingredient (API).

[0126] To prepare the Compound A dosing solution, an appropriate amount of Compound ASDD was weighed into a vial and dissolved in water (10 ml / kg dosing volume). To ensure that the compound does not adhere to the sides or bottom of the vial, a stirring bar was added to the bottle and the suspension was stirred throughout the preparation and dosing process. Subsequent solutions for lower doses were prepared by serial dilution with water. 7 mg / ml of PVP-VA was used as a vehicle control.

[0127] NHE3 inhibitor dosing solution: To prepare the Example 2 dosing solution, the appropriate amount of compound was weighed into a vial and dissolved in 1% HEC (10 ml / kg dosing volume). To ensure that the compound did not adhere to the sides or bottom of the vial, a stir bar was added to the bottle and the suspension was stirred throughout the formulation and dosing process. Subsequent solutions for lower doses were prepared by serial dilution with HEC.

[0128] To prepare the radiolabeled phosphate dosing solution, a 16.25 mM Na2HPO4, 0.9% saline, pH 7.4 solution was prepared and filtered using a sterile 0.22 μm polyethersulfone Millex-GP syringe filter unit (EMD Millipore). Radioactive phosphate (H3 33 PO4, Perkin Elmer) and filtered again.

[0129] Three independent studies were conducted. In these studies, male Sprague-Dawley rats were randomized into groups with approximately equal mean body weights. After an overnight fast, all animals were administered vehicle at 10 ml / kg, different doses of Compound A (Study 1), different doses of Example 2 (Study 2), or 1.2 mg / kg Compound A and different doses of Example 2 (Study 3). After 15 minutes, radiolabeled phosphate was orally administered in a volume of 2 ml. After 15 minutes, blood was collected and plasma was prepared. The radioactivity (dpm) in 50 μL plasma was measured by scintillation counting and used to calculate phosphate absorption. The results were normalized with a vehicle control representing 100% absorption. The curves were fitted using GraphPad Prism 6 with nonlinear regression with a variable slope to calculate the ED values ​​given in Table 7. 50 and E max . Because of the ED 50 For the purpose of curve fitting in the calculations, the dose of Example 2 was artificially set in the software to 0.00001 mg / kg for the vehicle alone group in Study 2 and the vehicle + 1.2 mg / kg Compound A group in Study 3. For Compound A alone in Study 1, no curve fitting was performed.

[0130] The results of three independent studies conducted in rats, i.e., a dose response study of Compound A (Study 1), a dose response study of Example 2 (Study 2), and a dose response study of Example 2 in the presence of 1.2 mg / kg Compound A (Study 3), are summarized in Tables 4-7 below. Both Example 2 and Compound A inhibited phosphate absorption in a dose-dependent manner, with percentage inhibition of 37% and 18%, respectively, at the highest dose tested. However, when given in combination, 70% inhibition was achieved at the highest dose of Example 2 combined with 1.2 mg / kg Compound A. Thus, the two compounds were more effective when given together than when either compound was given alone, consistent with each compound inhibiting a different pathway that contributes to phosphate absorption in the rat intestine.

[0131] Table 4: Effects of Compound A on Phosphate Absorption in Rats (Study 1)

[0132] deal with Absorption (% relative to vehicle control, mean ± SEM) Medium 100±7.23 Compound A, 0.01 mg / kg 94.39±7.88 Compound A, 0.1 mg / kg 87.61±6.08 Compound A, 1 mg / kg 87.64±7.26 Compound A, 10 mg / kg 81.53±5.50

[0133] Table 5: Effect of Example 2 on phosphate absorption in rats (Study 2)

[0134] deal with Absorption (% relative to vehicle control, mean ± SEM) Medium 100.00±14.22 Example 2, 0.001 mg / kg 97.16±20.76 Example 2, 0.003 mg / kg 100.81±20.74 Example 2, 0.01 mg / kg 75.15±15.52 Example 2, 0.03 mg / kg 83.17±17.99 Example 2, 0.1 mg / kg 57.29±11.20 Example 2, 0.3 mg / kg 71.09±13.98 Example 2, 1 mg / kg 44.29±9.09 Example 2, 3 mg / kg 60.04±12.00 Example 2, 10 mg / kg 63.47±8.39

[0135] Table 6: Effect of the combination of Compound A and Example 2 on phosphate absorption in rats (Study 3)

[0136]

[0137]

[0138] Table 7:

[0139] Compound A Example 2 Example 2 + 1.2 mg / kg Compound A <![CDATA[ED 50 (mg / kg)]]> No curve fitting 0.041 0.056 % inhibition at the highest dose 18％ 37％ 70％

[0140] Example 2 Effect of Combination with an NPT2b Inhibitor (Compound A) on Phosphate Absorption and Intestinal Phosphate Retention in Rats Influence

[0141] The purpose of this study was to investigate in rats the effects of Example 2, Compound A and the combination of Example 2 / Compound A on phosphate absorption 15 minutes after compound administration and phosphate retained in the intestine 4.25 hours after compound administration. The latter is a measure of the oral phosphate load absorbed over a long period of time.

[0142] Two vehicle carriers were used in this study: 1) 1% HEC and 2) 0.46% poly-1-vinylpyrrolidone-co-vinyl acetate (PVP-VA vehicle) in water. The vehicle control group in this study received a 1:1 combination of the two vehicles.

[0143] Example 2, Compound A and phosphate dosing solutions were prepared similarly to the above, except that the dose of Example 2 was 0.4 mg / kg, the dose of Compound A was 10 mg / kg, and they were prepared at a concentration that would be administered in a volume of 5 ml / kg. Example 2 and the Compound A group alone were mixed with the vehicle of the other compound at 1:1, while Compound A and Example 2 were mixed at 1:1 before administration. In all cases, the final dosing volume was 10 ml / kg.

[0144] Male Sprague-Dawley rats were fasted for 4 hours and then administered the appropriate vehicle, 10 mg / kg Compound A, 0.4 mg / kg Example 2, or a combination of the two compounds. Fifteen minutes later, the animals were administered a radiolabeled phosphate solution. Fifteen minutes later, blood was collected and plasma was prepared. The radioactivity in the plasma was measured by scintillation counting and used to calculate the inhibition of phosphate absorption. Example 2 (0.4 mg / kg) inhibited phosphate absorption by 29% (P=0.046, relative to the vehicle control), Compound A (10 mg / kg) inhibited phosphate absorption by 35% (P=0.013, relative to the vehicle control), and the combination of the two compounds inhibited phosphate absorption by 63% (P=0.0001, relative to the vehicle control). The 63% inhibition of the Example 2 / Compound A combination exceeded the inhibition of either compound alone. Data are expressed as mean ± SEM, and the number of animals in each group is equal to 8. Statistical significance was determined by ANOVA with Dunnett's comparison of the Example 2 / Compound A combination using JMP 12.1.

[0145] Four hours after administration of the radiolabeled phosphate solution, the stomach, small intestine, large intestine, and feces were collected, weighed, and digested with 1N NaOH overnight at 37° C. The radioactivity in each fraction was measured by scintillation counting.

[0146] The percentage of dose recovered in the gastrointestinal tract was defined as the radioactivity recovered in the stomach, small intestine, large intestine and feces compared to the dose administered. Data are presented as mean ± SEM, and the number of animals in each group was equal to 8. Statistical significance was determined by ANOVA with Dunnett's comparison of the Example 2 / Compound A combination using JMP 12.1.

[0147] Table 8: Percent of dose recovered in various parts of the gastrointestinal tract.

[0148]

[0149] Percentage of radiolabeled phosphate dose recovered from each segment of the gastrointestinal tract, mean ± SEM

[0150] The total radioactivity recovered in animals dosed with both Example 2 and Compound A (46%) was greater than either compound alone (31% and 32%, respectively) (Table 8). Thus, the combination was more potent than either compound alone.

[0151] The percentage of dose recovered in the gastrointestinal tract was further analyzed by two-way ANOVA in JMP 12.1 to test the additivity of the effects of Example 2 and Compound A on the inhibition of phosphate absorption. The detection of the Example 2 / Compound A interaction was significant (p=0.0187), indicating that there was a synergistic relationship between the compounds at the doses tested in this study. That is, the inhibitory effect of the combination was greater than the sum of the effects of the individual compounds.

Claims

1. A compound of the formula: wherein both R are CN or both R are C(O)NH2, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein both R are C(O)NH2, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 or claim 2, wherein the compound is: or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, wherein the compound is:

5. The compound according to claim 3, wherein the compound is the dihydrochloride salt of:

6. A compound, which is:

7. A compound which is the dihydrochloride salt of:

8. Use of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease selected from hyperphosphatemia and hypertension in a mammal in need thereof.

9. Use of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof in combination with a compound of the following formula or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hyperphosphatemia in a mammal in need thereof:

10. The use according to claim 8 or 9, wherein the mammal is a human.

11. The use according to claim 8 or 9, wherein the mammal is a dog.

12. The use according to claim 8 or 9, wherein the mammal is a cat.

13. A compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof for simultaneous, separate or sequential combination with a compound of the following formula or a pharmaceutically acceptable salt thereof: Use in medicines for treating hyperphosphatemia.

14. A compound of the formula: or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hyperphosphatemia in simultaneous, separate or sequential combination with a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent.

16. The pharmaceutical composition according to claim 15, further comprising a compound of the formula: or a pharmaceutically acceptable salt thereof.

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