Medical use of combination of endothelin a (ETA) receptor antagonist and SGLT-2 inhibitor

Abstract

A pharmaceutical composition comprising an endothelin A (ETA) receptor antagonist and an SGLT-2 inhibitor and use thereof in the preparation of a drug for preventing and / or treating a disease selected from nephropathy and hypertension, wherein the ETA receptor antagonist is a compound of formula (I) or a salt thereof, and the SGLT-2 inhibitor is selected from dapagliflozin, empagliflozin, canagliflozin, and ertugliflozin, and salts thereof. (I)

Description

Pharmaceutical uses of endothelin A (ETA) receptor antagonists in combination with SGLT-2 inhibitors Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology. In particular, this invention provides a pharmaceutical composition of an endothelin A (ETA) receptor antagonist and an SGLT-2 inhibitor, and their combined pharmaceutical uses, including for hypertension, nephropathy, etc. The nephropathy includes acute nephropathy, chronic nephropathy, and further includes diabetic nephropathy, IgA, FSGS, and Alport. The hypertension includes refractory hypertension, portal hypertension due to cirrhosis, and elevated portal pressure due to cirrhosis. Background Technology

[0002] Chronic kidney disease (CKD) is a global challenge. It is a group of diseases, including: ① Primary glomerular diseases: chronic glomerulonephritis, nephrotic syndrome, IgA nephropathy, etc. (2) Secondary kidney diseases: allergic purpuric nephritis, diabetic nephropathy, hypertensive nephropathy, lupus nephropathy, etc. ③ Hereditary kidney diseases: polycystic kidney disease, etc. ④ Infectious diseases: urinary tract infection, etc. ⑤ Drug-induced kidney damage. ⑥ Chronic renal failure of unknown cause. Its fundamental pathology is damage to the glomeruli and renal tubules. This leads to inflammatory cell infiltration, resulting in mesangial cell proliferation and increased mesangial matrix (ECM). Clinical manifestations: proteinuria, hematuria, oliguria, edema, fatigue, etc. Currently, the treatment of these diseases is not ideal, with low cure rates and high relapse rates. Most of them gradually lead to renal fibrosis and develop into uremia.

[0003] Endothelin (ET) is currently the most potent vasoconstrictor known in the human body, exhibiting strong vasoconstrictive effects and promoting smooth muscle cell migration and proliferation. It also plays a crucial role in renal tubular water and electrolyte transport, acting extensively on multiple systems within the body. ET receptor antagonists can slow the progression of proteinuria-related nephropathy, such as diabetic nephropathy, and have a protective effect on the kidneys. ET-1 is involved in the pathophysiological processes of various diseases, including hypertension, atherosclerosis, restenosis, pulmonary hypertension, idiopathic cardiomyopathy, heart failure, and renal failure. Current clinical studies indicate that selective ETA receptor antagonists can reduce proteinuria, slow the progression of chronic kidney disease, increase renal blood flow, inhibit glomerular mesangial proliferation, reduce extracellular matrix accumulation, alleviate renal inflammation, delay renal fibrosis, and improve the prognosis of diabetic nephropathy. However, its main side effect is dose-dependent sodium and water retention, causing edema and thus worsening the symptoms of heart failure.

[0004] Sodium-glucose cotransporter 2 inhibitors (SGLT-2 inhibitors) are a new type of oral hypoglycemic drug that promotes the excretion of urinary glucose and sodium by blocking the SGLT2 transporter in the proximal tubules of the kidney, thereby effectively reducing glycated hemoglobin (HbA1c). They also have a mild diuretic effect, as well as the effects of weight loss, lowering blood pressure, and cardiovascular and renal protection.

[0005] However, the treatment of diabetic nephropathy still suffers from poor efficacy and poor durability. This invention, through research, has accidentally discovered the pharmaceutical use of an endothelin A (ETA) receptor antagonist in combination with an SGLT-2 inhibitor, including for the treatment of nephropathy, specifically, in the prevention and / or treatment of acute and chronic nephropathy, which is beneficial for the treatment of diabetic nephropathy, IgA, FSGS, alport, and other diseases. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide an application of endothelin A (ETA) receptor antagonist combined with SGLT-2 inhibitor in the prevention and / or treatment of diseases, the combination of which can achieve synergistic effects, wherein the diseases are selected from kidney disease, hypertension, etc., preferably, the kidney disease includes acute kidney disease and chronic kidney disease, and the hypertension includes refractory hypertension, portal hypertension due to cirrhosis, and elevated portal pressure due to cirrhosis.

[0007] The present invention is achieved through the following technical solution:

[0008] This invention first provides the use of an endothelin A (ETA) receptor antagonist in combination with an SGLT-2 inhibitor in the preparation of a medicament for the prevention and / or treatment of diseases, wherein the endothelin A (ETA) receptor antagonist is a compound of formula I or a salt thereof:

[0009] As a preferred embodiment of the present invention, the kidney disease includes acute kidney disease and chronic kidney disease (CKD).

[0010] As a preferred embodiment of the present invention, the kidney disease includes diabetic nephropathy (DKD), IgA, FSGS (focal segmental glomerulosclerosis), and Alport nephropathy.

[0011] The definition and classification of chronic kidney disease can be found in the "Guidelines for Screening, Diagnosis and Prevention of Chronic Kidney Disease", Chinese Journal of Practical Internal Medicine, January 2017, Vol. 37, No. 1.

[0012] For the definition and classification of IgA, please refer to the "Clinical Diagnosis and Treatment Guidelines for IgA+ Nephropathy (Interpretation)", Journal of Clinical Internal Medicine, May 2015, Vol. 32, No. 5.

[0013] The definition and classification of hypertension can be found in the "Guidelines for the Prevention and Treatment of Hypertension in China", which include refractory hypertension, portal hypertension due to cirrhosis, and elevated portal pressure due to cirrhosis.

[0014] As a preferred embodiment of the present invention, the SGLT-2 inhibitor is selected from dapagliflozin, empagliflozin, canagliflozin, and eltoggliflozin, as well as SGLT-2 inhibitors existing in their respective salt forms. Preferably, the SGLT-2 inhibitor is dapagliflozin or a salt thereof, or empagliflozin or a salt thereof.

[0015] As a preferred embodiment of the present invention, the mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to dapagliflozin (in free form) is 0.88:10-52.8:10; the preferred range of the endothelin A (ETA) receptor antagonist (in oxalate form) and dapagliflozin (in free form) is 1:10-60:10, for example 60:10, 50:10, 45:10, 40:10, 35:10, 30:10, 25:10, 15:10, 10:10, 8:10, 6:10, 5:10, 4:10, 2:10, 1:10.

[0016] As a preferred embodiment of the present invention, the mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to empagliflozin is selected from 0.88:10 to 52.8:10; the preferred range for the endothelin A (ETA) receptor antagonist (calculated as oxalate) and empagliflozin (calculated as empagliflozin) is 1:10 to 60:10, for example 60:10, 50:10, 45:10, 40:10, 35:10, 30:10, 25:10, 15:10, 10:10, 8:10, 6:10, 5:10, 4:10, 2:10, 1:10.

[0017] Unless otherwise specified, the aforementioned mass ratios are all in free form, i.e., the portion excluding salts or solvates.

[0018] The present invention further provides a pharmaceutical composition for the prevention and / or treatment of diseases, wherein the active ingredient comprises a compound of Formula I or a salt thereof and dapagliflozin or a salt thereof; or a compound of Formula I or a salt thereof and empagliflozin or a salt thereof.

[0019] As a preferred embodiment of the present invention, in the pharmaceutical composition, the mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to dapagliflozin (in free form) is 0.88:10-52.8:10, and the preferred range of the endothelin A (ETA) receptor antagonist (in oxalate form) and dapagliflozin (in free form) is 1:10-60:10, for example 60:10, 50:10, 45:10, 40:10, 35:10, 30:10, 25:10, 15:10, 10:10, 8:10, 6:10, 5:10, 4:10, 2:10, 1:10.

[0020] As a preferred embodiment of the present invention, in the pharmaceutical composition, the mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to empagliflozin is selected from 0.88:10 to 52.8:10. The preferred range for the endothelin A (ETA) receptor antagonist (calculated as oxalate) and empagliflozin (calculated as empagliflozin) is 1:10 to 60:10, for example 60:10, 50:10, 45:10, 40:10, 35:10, 30:10, 25:10, 15:10, 10:10, 8:10, 6:10, 5:10, 4:10, 2:10, and 1:10.

[0021] Unless otherwise specified, the aforementioned mass ratios are all in free form, i.e., the portion excluding salts or solvates.

[0022] As a preferred embodiment of the present invention, the salt of the compound shown in Formula I is selected from organic acid salts or inorganic acid salts. Specifically, the inorganic acid is selected from sulfuric acid, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, carbonic acid, or nitric acid.

[0023] The organic acid is selected from benzoic acid, 2,5-dihydroxybenzoic acid, 4-acetaminobenzoic acid, 4-aminobenzoic acid, oxalic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, hexanoic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, octanoic acid, capric acid, cinnamic acid, citric acid, aspartic acid, gluconic acid, glutamic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, succinic acid, formic acid, fumaric acid, gentian acid, glutamate, valeric acid, valeric acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, nicotinic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, palmitic acid, dihydroxynaphthyl acid, trifluoroacetic acid, thiocyanate, p-methylbenzenesulfonic acid, and L-malic acid.

[0024] As a preferred embodiment of the present invention, the salt is selected from compounds with the following structural formula (compound Ib):

[0025] Where M is an inorganic acid or an organic acid, and n:x = 1:1-2.

[0026] As a preferred embodiment of the present invention, the inorganic acid is selected from phosphoric acid.

[0027] As a preferred embodiment of the present invention, the inorganic acid is selected from phosphoric acid, wherein n = 3 and x = 3-6.

[0028] As a preferred embodiment of the present invention, the inorganic acid is selected from phosphoric acid, wherein n = 3, x = 3; or n = 3, x = 4; or n = 3, x = 5; or n = 3, x = 6.

[0029] As a preferred embodiment of the present invention, the organic acid is selected from oxalic acid.

[0030] In a preferred embodiment of the present invention, the organic acid is selected from oxalic acid, where n = 1 and x = 1. That is, a compound with the following structural formula:

[0031] Wherein, when the oxalate (compound Ic) of compound I is 1 mg, it is equivalent to 0.88 mg of compound I.

[0032] As a preferred embodiment of the present invention, dapagliflozin is selected from dapagliflozin propanediol hydrate and dapagliflozin di-L-proline, with the following structural formulas respectively. The dosage is based on the free form of dapagliflozin (in C...). 21 H 25 ClO6 (calculated):

[0033] As a preferred embodiment of the present invention, the structural formula of empagliflozin is as follows, and its dosage is based on empagliflozin (C 23 H 2v ClO7) calculation:

[0034] In this invention, the dosage of the endothelin A (ETA) receptor antagonist (in free form) ranges from 1 to 100 mg, the dosage of dapagliflozin (in free form) ranges from 1 to 100 mg, and the dosage of empagliflozin ranges from 1 to 100 mg. Specifically, it includes: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, and 42 mg. , 43mg, 44mg, 45mg, 46mg, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57 mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86m g, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg, 100mg.

[0035] The present invention has the following advantages and beneficial effects compared with the prior art:

[0036] This invention provides an application of an endothelin A (ETA) receptor antagonist combined with an SGLT-2 inhibitor in the prevention and / or treatment of diseases, exhibiting a synergistic effect. It can be used to prevent and / or treat kidney disease, hypertension, etc. Preferably, the kidney disease includes acute kidney disease and chronic kidney disease, and the hypertension includes refractory hypertension, portal hypertension due to cirrhosis, and elevated portal pressure due to cirrhosis.

[0037] This invention provides a pharmaceutical composition of an endothelin A (ETA) receptor antagonist and an SGLT-2 inhibitor, and its application in the prevention and / or treatment of diseases, including hypertension, nephropathy, etc., wherein the nephropathy includes acute nephropathy, chronic nephropathy, and further includes diabetic nephropathy, IgA, FSGS, Alport, etc., and the hypertension includes refractory hypertension, portal hypertension due to cirrhosis, and elevated portal pressure due to cirrhosis, etc. Attached Figure Description

[0038] Figure 1. Portal vein blood flow measured by ultrasound using compound A+Empagliflozin in portal hypertension caused by cirrhosis.

[0039] Figure 2 shows the mean internal diameter of the portal vein measured by HE staining of compound A+Empagliflozin in the histopathology of portal hypertension caused by cirrhosis. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the invention is not limited thereto.

[0041] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ increments. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-III NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3), with tetramethylsilane (TMS) as the internal standard.

[0042] MS measurements were performed using an ISQ EC mass spectrometer (manufacturer: Thermo, model: ISQ EC).

[0043] High-performance liquid chromatography (HPLC) analysis was performed using a Thermo U3000 HPLC DAD high-performance liquid chromatograph.

[0044] The CombiFlash rapid preparation system uses CombiFlash Rf+LUMEN (TELEDYNE ISCO).

[0045] Thin-layer chromatography silica gel plates used are from Yantai Yinlong HSGF. 254 or GF 254 Silica gel plates: The silica gel plates used in thin-layer chromatography (TLC) have a size of 0.17 mm to 0.23 mm, while those used for TLC separation and purification of products have a size of 0.4 mm to 0.5 mm.

[0046] Silica gel column chromatography generally uses 100-200 mesh silica gel from Rushan Shangbang as the carrier.

[0047] DMFN, N-dimethylformamide, chloromethylethyl carbonate, potassium iodide, cesium carbonate, DCM dichloromethane, n-hexane, ethyl acetate.

[0048] Example 1

[0049] 1-[(isopropoxycarbonyl)oxy]ethyl-(2R,3R,4S)-4-(benzo[d][1,3]dioxacyclopentan-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid ester

[0050] Step A: Synthesis of 1-[(isopropoxycarbonyl)oxy]ethyl-(2R,3R,4S)-4-(benzo[d][1,3]dioxacyclopentan-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid ester

[0051] At room temperature, atrasentan (2R,3R,4S)-4-(benzo[d][1,3]dioxolane-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (500 mg, 0.98 mmol), 1-chloroethyl isopropyl carbonate (325 mg, 1.96 mmol), cesium carbonate (640 mg, 1.96 mmol), and potassium iodide (325 mg, 1.96 mmol) were added to 10 mL of dry DMF, and the mixture was heated to 65°C and reacted for 2 hours.

[0052] After the reaction was complete, the mixture was cooled to room temperature and poured into 40 mL of ice-water solution. It was extracted with dichloromethane (20 mL × 3), and the organic phases were combined. The mixture was washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure to dryness, and the crude compound was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 1 / 1) to give 482 mg of colorless oily product 1-[(isopropoxycarbonyl)oxy]ethyl-(2R,3R,4S)-4-(benzo[d][1,3]dioxacyclopentan-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid ester (yield: 76.9%).

[0053] LC-MS: RT = 2.29 min, [M+H] + =641.48. 1H NMR (400MHz, DMSO) δ7.25 (t, J=9.0Hz, 2H), 7.07-7.04 (m, 1H), 6.93-6.89 (m, 2H), 6.85-6.78 (m, 2H), 6.59-6.54 (m, 1H), 5.99 (d, J=6.2Hz, 2H), 4.79-4.69 (m, 1H), 3.76-3.70 (m, 4H), 3.55-3.44 (m, 1H), 3.33-3.23 (m, 4H), 3.21 -3.13 (m, 1H), 3.02-2.90 (m, 3H), 2.80-2.75 (m, 1H) 2.71 (d, J = 13.9Hz, 1H), 1.39-1.33 (m, 1H), 1.32 (d, J = 5.4Hz , 2H), 1.26 (d, J=5.4Hz, 2H), 1.22-1.14 (m, 9H), 1.00-0.94 (m, 2H), 0.82 (t, J=7.3Hz, 3H), 0.72 (t, J=7.3Hz, 3H).

[0054] Example 2 Preparation of oxalate

[0055] The free base A (3.5 g) prepared according to the method of Example 1 was dissolved in isopropanol (10 ml), and oxalic acid dihydrate (689 mg) was added. The mixture was stirred at room temperature for 1 h, concentrated, and an oily substance was obtained. Isopropyl ether (50 ml) was added under ice bath and stirred to precipitate a solid. The mixture was stirred under ice bath for 1 day and filtered to obtain solid oxalate.

[0056] NMR data for oxalate:

[0057] Belonging to:

[0058] Analysis:

[0059] The peak with δ = 0.93 is the methyl hydrogen atom at C atom 34 and 37, which has 6 protons and consists of two sets of triplet peaks.

[0060] The peak with δ = 1.28 is the methyl hydrogen atom at C atom number 45, which has 3 protons and is a doublet.

[0061] The peak with δ = 1.34 is the methyl hydrogen on C atom 46, and the methylene hydrogen on C atoms 33 and 36, with 7 protons, which are a triplet and two doublets respectively.

[0062] The peak with δ = 1.52 is the methylene hydrogen atom at C atom 32 and 35, which has 4 protons and consists of two sets of triplet peaks.

[0063] The peak with δ = 1.59 is the methyl hydrogen atom on C atom number 39, which has 3 protons and is a doublet.

[0064] The methylene hydrogen atoms at C atoms 30, 31, 26, 11, 9, and 10, with δ = 3.24, 3.29, 3.31, 3.35, 3.41, 3.59, and 3.68, respectively, have 12 protons and represent three sets of multiplets.

[0065] The peak with δ = 3.78 is the methyl hydrogen atom at C atom number 25, which has 3 protons and forms a singlet.

[0066] The peak with δ = 4.39 is the methylene hydrogen atom on the 7th carbon atom, which has 1 proton and is represented by two doublets.

[0067] The peak with δ = 5.08 is the methylene hydrogen atom on C atom 44, which has 1 proton and is a multiplet.

[0068] The peak with δ = 5.93 is the methylene hydrogen atom on C atom number 2, which has 2 protons and is a triplet.

[0069] The peaks at δ = 6.63, 6.72, and 6.82 are the methylene hydrogen atoms at C atoms 18, 15, and 14, respectively, with 3 protons each, and are three sets of doublets.

[0070] The peak with δ = 6.91 is the methylene hydrogen atom on the C atom of the benzene ring at position 4 and 6, which has 2 protons and is represented by two sets of triplet peaks.

[0071] The peak with δ = 7.20 is the methyl hydrogen atom on the C atom of the benzene ring at position 3, which has 2 protons and is represented by two sets of triplet peaks.

[0072] The peak with δ = 7.51 is the methylene hydrogen atom on C atom 38, which has 1 proton and is a multiplet.

[0073] The peak at δ = 11.05 is the carboxyl hydrogen atom on the C atom of oxalic acid, with 2 protons, forming a singlet.

[0074] Example 3: Preparation of Phosphate

[0075] The free base A (4.1 g) prepared according to the method of Example 1 was dissolved in isopropanol (12 ml), phosphoric acid (800 mg) was added and reacted for 1 h, and isopropyl ether (80 ml) was added under ice bath and stirred for 4 h to obtain solid phosphate.

[0076] NMR data for phosphate:

[0077] Belonging to:

[0078] Analysis:

[0079] The peaks with δ = 0.73 and 0.83 are the methyl hydrogen atoms at C atoms 34 and 37, which have 6 protons and are two sets of triplet peaks.

[0080] The peaks with δ = 1.25 are the methyl hydrogen atoms at C atoms 45, 46, and 39, the methylene hydrogen atoms at C atoms 33 and 36, and the methylene hydrogen atoms at C atoms 32 and 35, with a proton number of 14, and are multiplets.

[0081] The methylene hydrogen atoms at C atoms 30, 31, 26, 11, 9, and 10, with δ = 2.78, 2.99, 3.19, 3.31, and 3.50, respectively, have 12 protons and form three sets of multiplets.

[0082] The peak at δ = 4.75 is the methylene hydrogen atom on carbon atom number 7, with 1 proton, and consists of two doublets.

[0083] The peak with δ = 6.00 is the methylene hydrogen atom on C atom number 20, which has 2 protons and is a triplet.

[0084] The peaks at δ = 6.57 and 6.84 are the methylene hydrogen atoms at C atoms 18, 15, and 14, respectively, with 3 protons each, and are three sets of doublets.

[0085] The peak with δ = 6.92 is the methylene hydrogen atom on the C atom of the benzene ring at position 4 and 6, which has 2 protons and is represented by two sets of triplet peaks.

[0086] The peaks at δ = 7.06 and 7.27 are the methylene hydrogen on the C atom of the benzene ring at position 1 and 3, and the methylene hydrogen on the C atom at position 38, with a proton number of 3, and are two sets of triplet peaks.

[0087] Elemental analysis showed that the phosphorus content (%) was 6.46 and the relative standard deviation (%) was 0.2.

[0088] Detection instrument: Full-spectrum direct-reading plasma atomic emission spectrometer, iCAP6500Duo (Thermo, USA);

[0089] Detection conditions: Incident power: 1150W; Plasma gas flow rate: 14L / min; Atomizer flow rate: 0.5L / min.

[0090] Example 4: Preparation of hydrochloride

[0091] Weigh 202.5 mg of the free base A obtained in Example 1 into a 5 mL vial, add 1.0 mL of MeOH and an equimolar ratio of hydrochloric acid to methanol solution. Suspend and stir at -20°C for 1 day, remove methanol by rotary evaporation; the sample is gel-like and remains gel-like even after vacuum drying. Add 2.0 mL of isopropyl ether, suspend and stir at -20°C for 1 day, precipitating a solid. Centrifuge and vacuum dry to obtain the solid.

[0092] Example 5: Preparation of Sulfate

[0093] Weigh 203.1 mg of the free base A obtained in Example 1 into a 5 mL vial, add 1.0 mL of IPA solvent and an equimolar ratio of sulfuric acid, suspend and stir at -20°C for 1 day, then transfer to vacuum drying to remove IPA, the sample is gel-like. Add 2.0 mL of isopropyl ether, suspend and stir at -20°C for 1 day, a solid precipitates, centrifuge and vacuum dry to obtain the solid.

[0094] Pharmacological research

[0095] Unless otherwise stated, in pharmacological studies, the compounds of this invention refer to the oxalate of Example 2, and compound A refers to the free form of Example 1.

[0096] Example 6: Study on the effect of the composition of the present invention on urinary sodium excretion in rats

[0097] (1) Test drug

[0098] Drug Name: Compound of this invention, Atrasentan Hydrochloride, Source: Shenzhen Sinopharm Pharmaceutical Co., Ltd.; Dapagliflozin, Source: Commercially available.

[0099] Preparation method: The compounds of the present invention and atrasentan hydrochloride were prepared using 60% PEG400 + 40% TPGS (14.3% aqueous solution, w / v), and dapagliflozin was prepared using 0.5% CMC-Na (w / v) aqueous solution. Dapagliflozin and atrasentan hydrochloride were calculated as free drug, and the amount of the compounds of the present invention was calculated as the oxalate of the compound of formula I (see Example 2).

[0100] (2) Laboratory animals

[0101] Male Wistar rats, weighing 200–220 g at reception, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0102] (3) Experimental steps

[0103] (3.1) Grouping

[0104] After 5 days of acclimatization, Wistar rats were divided into 8 groups according to body weight and administered solvent, single drug, or combination of two drugs, as detailed in the table below:

[0105] Table 1 Animal grouping and drug dosage

[0106] (3.2) Sample collection

[0107] After administering the medication in the morning, the animals were placed in a metabolic cage, fasted, and allowed free access to water. Urine was collected over 12 hours.

[0108] (3.3) Indicator Testing

[0109] 3.3.1 Rat urine volume

[0110] Urine from rats was collected in 50 ml centrifuge tubes, and the total urine volume was recorded.

[0111] 3.3.2 Rat urinary sodium detection

[0112] Take 1 ml of urine in a 1.5 ml centrifuge tube and centrifuge at 4000 rpm for 10 min. Collect the supernatant and use an automated biochemical analyzer (ion-selective electrode method) to detect the urinary sodium concentration. Calculate the urinary sodium excretion.

[0113] Urinary sodium excretion (mmol) = urinary sodium concentration (mmol / L) * urine volume (ml) * 0.001.

[0114] (4) Experimental Results

[0115] Based on the results of urinary sodium excretion in rats, at the experimental doses, atrasentan alone and the compound of this invention reduced urinary sodium excretion in rats, while dapagliflozin at the doses used in this study increased urinary sodium excretion in rats. When atrasentan or the compound of this invention was used in combination with dapagliflozin, the amount of urinary sodium excretion in rats increased compared to the single-use group, indicating improved sodium retention.

[0116] Compared with the group treated with atrasentan in combination with dapagliflozin, the animals treated with the compound of this invention showed a more significant increase in urinary sodium excretion.

[0117] The test results are as follows:

[0118] Table 2

[0119] Example 7: Study on the effect of the composition of the present invention on urinary sodium excretion in rats

[0120] (1) Test drug

[0121] Drug Name: Compound of this invention, Source: Shenzhen Sinopharm Pharmaceutical Co., Ltd.; Empagliflozin, Source: Commercially available; Atrasentan Hydrochloride, Source: Commercially available.

[0122] Preparation method: The compounds of the present invention and atrasentan hydrochloride were prepared using 60% PEG400 + 40% TPGS (14.3% aqueous solution, w / v), and empagliflozin was prepared using 0.5% CMC-Na (w / v) aqueous solution; empagliflozin was calculated as empagliflozin, and atrasentan hydrochloride was calculated as free drug. The amount of compound used in Example 1 of the present invention was calculated as oxalate of compound of formula I (see Example 2).

[0123] (2) Laboratory animals

[0124] Male Wistar rats, weighing 200–220 g at reception, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0125] (3) Experimental steps

[0126] (3.1) Grouping

[0127] After 5 days of acclimatization, Wistar rats were divided into 8 groups according to body weight and administered solvent, single drug, or combination of two drugs, as detailed in the table below:

[0128] Table 3 Animal grouping and drug dosage

[0129] (3.2) Sample collection

[0130] After administering the medication in the morning, the animals were placed in a metabolic cage, fasted, and allowed free access to water. Urine was collected over 12 hours.

[0131] (3.3) Indicator Testing

[0132] 3.3.1 Rat urine volume

[0133] Urine from rats was collected in 50 ml centrifuge tubes, and the total urine volume was recorded.

[0134] 3.3.2 Rat urinary sodium detection

[0135] Take 1 ml of urine in a 1.5 ml centrifuge tube and centrifuge at 4000 rpm for 10 min. Collect the supernatant and use an automated biochemical analyzer (ion-selective electrode method) to detect the urinary sodium concentration. Calculate the urinary sodium excretion.

[0136] Urinary sodium excretion (mmol) = urinary sodium concentration (mmol / L) * urine volume (ml) * 0.001.

[0137] (4) Experimental Results

[0138] Table 4

[0139] The results showed that the increase in urinary sodium excretion was more significant in animals treated with the compound of this invention in combination with empagliflozin compared to the group treated with atrasentan in combination with empagliflozin.

[0140] Example 8: Study on the effect of Zibotentan + Dapagliflozin on urinary sodium excretion in rats

[0141] (1) Test drug

[0142] Drug name: Zibotentan (dapagliflozin); Source: Commercially available.

[0143] Preparation method: Zibotentan is prepared using 60% PEG400 + 40% TPGS (14.3% aqueous solution, w / v), and dapagliflozin is prepared using 0.5% CMC-Na (w / v) aqueous solution; dapagliflozin is calculated as free drug, and Zibotentan is calculated as Zibotentan.

[0144] (2) Laboratory animals

[0145] Male Wistar rats, weighing 200–220 g at reception, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0146] (3) Experimental steps

[0147] (3.1) Grouping

[0148] After 5 days of acclimatization, Wistar rats were divided into four groups according to body weight and administered solvent, single drug, or a combination of two drugs, as detailed in the table below:

[0149] Table 5 Animal grouping and drug dosage

[0150] (3.2) Sample collection

[0151] After administering the medication in the morning, the animals were placed in a metabolic cage, fasted, and allowed free access to water. Urine was collected over 12 hours.

[0152] (3.3) Indicator Testing

[0153] 3.3.1 Rat urine volume

[0154] Urine from rats was collected in 50 ml centrifuge tubes, and the total urine volume was recorded.

[0155] 3.3.2 Rat urinary sodium detection

[0156] Take 1 ml of urine in a 1.5 ml centrifuge tube and centrifuge at 4000 rpm for 10 min. Collect the supernatant and use an automated biochemical analyzer (ion-selective electrode method) to detect the urinary sodium concentration. Calculate the urinary sodium excretion.

[0157] Urinary sodium excretion (mmol) = urinary sodium concentration (mmol / L) * urine volume (ml) * 0.001.

[0158] (4) Experimental Results

[0159] Table 6

[0160] Example 9: Inhibitory effect of ETA receptor antagonists on high glucose-induced vascular smooth muscle cell proliferation

[0161] Vascular disease is a common complication of diabetes and a significant factor contributing to death or disability. Adult vascular smooth muscle cells (VSMCs) primarily maintain vasoconstriction and regulate vascular diameter, thereby modulating blood flow distribution and blood pressure. Under sustained high glucose levels, cell proliferation and migration are promoted. VSMCs migrating to the vascular intima cause intimal thickening and luminal narrowing, leading to atherosclerotic lesions. Current research indicates that endothelin and high glucose levels play important roles in the proliferation and migration of vascular smooth muscle cells.

[0162] 1. Experimental materials:

[0163] (1) Cells: Human aortic smooth muscle cells (Sciencell);

[0164] (2) Drugs and reagents: The compound of this invention (Shenzhen Xinlitai Pharmaceutical Co., Ltd.); dapagliflozin and empagliflozin (Huainan Lianke Biomedical Co., Ltd.); CellTiter-Glo Luminescent Cell Viability Assay kit (Promega), smooth muscle cell culture medium (SMCM, Sciencell), smooth muscle cell growth factor (SMCGS, Sciencell); fetal bovine serum (FBS, Sciencell); penicillin and streptomycin (Sciencell).

[0165] (3) Main instruments: Multifunctional microplate reader (BMG PHERAstar FSX), CO2 incubator (Thermo), Olympus inverted microscope (CKX41SF);

[0166] 2. Experimental Grouping

[0167] Table 7

[0168] 3. Test Methods

[0169] (1) Culture medium preparation: complete culture medium SMCM + 2% FBS + 1% SMCGS + 1% penicillin and streptomycin; add glucose to the complete culture medium to make the final glucose concentration 25mM, which is the high glucose culture medium.

[0170] (2) Drug preparation: The compound of the present invention was prepared into a 100mM stock solution using DMSO, and empagliflozin and dapagliflozin were prepared into a 200mM stock solution. The DMSO stock solution of the corresponding drug was diluted into working solutions of different concentration gradients using incomplete culture medium so that the final concentration ratio of the drug was consistent with the grouping table.

[0171] (3) Cell preparation: Take cells that are growing well and have a confluence of more than 75%, digest them with trypsin, resuspend them in complete culture medium, and count them. Add 5×10⁻⁶ cells to each well of a 96-well black transparent plate. 3 Cells were cultured overnight at a volume of 100 μL per well.

[0172] (3) Cell drug administration and high glucose treatment: The original culture medium in the 96 plate was aspirated and discarded. 100 μL of high glucose culture medium was added to each well for drug treatment. Then, 0.5 μL of compound was added to each well. High glucose and low glucose control wells were set up at the same time. Cells were incubated under drug and high glucose conditions for 72 h.

[0173] (4) Microplate reader readings and data processing: ATP was measured according to the CellTiter-Glo kit instructions. Cell viability was calculated after reading the values ​​on the microplate reader. The viability of the low glucose control wells was taken as 100%, and the viability of the high glucose control wells and drug-treated wells was calculated. The percentage by which the drug-treated group inhibited the proliferation-promoting effect of the high glucose medium was also calculated.

[0174] 4. Results

[0175] Table 8

[0176] As shown in the table, high glucose conditions can promote the proliferation of vascular smooth muscle cells. The inhibitory effect on cell proliferation is weak when using the compound of this invention alone, dapagliflozin, or empagliflozin. Compound A, when used in combination with dapagliflozin or empagliflozin, exhibits a synergistic effect within a mass ratio range of 0.88:10 to 52.8:10.

[0177] Example 10: Compound A + Empagliflozin for portal hypertension caused by cirrhosis.

[0178] 1. Test Methods

[0179] Modeling: Eight-week-old male SD rats were used, and modeling was initiated after one week of acclimatization. Rats were randomly divided into a control group (n=10), a model group (n=10), and a drug treatment group (n=10) according to their body weight. The modeling group was injected with CCl4 / olive oil solution twice a week for 16 weeks; the control group was injected with olive oil solution.

[0180] Starting from week 9 of modeling, patients were administered solvent or therapeutic drugs (compound A + Empagliflozin combination dose group) according to their assigned groups, and continued for 8 weeks. During the trial, body weight was measured twice weekly, and serum liver function indicators were measured.

[0181] (1) Ultrasound examination

[0182] In the 7th week after drug administration, all animals underwent abdominal hair removal after anesthesia, and the portal vein was examined by ultrasound to determine blood flow and diameter.

[0183] (2) Histopathological examination

[0184] The liver and portal vein tissues were subjected to histopathological examination using HE staining.

[0185] 2. Results

[0186] (1) The portal vein blood flow measured by B-ultrasound is shown in Figure 1. Compared with the model group, the portal vein blood flow of the drug-treated group was reduced, which is beneficial to reduce portal vein pressure.

[0187] (2) The average inner diameter of the portal vein was measured by HE staining in histopathology, as shown in Figure 2. The average inner diameter of the portal vein in the liver of the model group animals was increased. The inner diameter of the portal vein was significantly reduced in the group using compound A in combination with empagliflozin, suggesting that it can improve portal hypertension.

[0188] In summary, in animal models of CCl4-induced cirrhosis and portal hypertension, compound A combined with empagliflozin can reduce portal vein blood flow and improve intrahepatic portal vein vessels.

[0189] Example 11

[0190] A multicenter, randomized, double-blind, placebo-controlled phase II clinical trial (Registration No.: CTR20243967) evaluating the efficacy and safety of compound A oxalate tablets (prepared according to the formulation and method described in CN118304295A) in the treatment of patients with chronic kidney disease (CKD).

[0191] Table 9

[0192] Conclusion: Compound A and its salts have a further proteinuria-reducing effect on the basis of current standard treatment, with a proteinuria-reducing effect of 15% to 30% in a subset of patients receiving standard treatment containing SGLT2.

[0193] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of an endothelin A (ETA) receptor antagonist in combination with an SGLT-2 inhibitor in the preparation of a medicament for the prevention and / or treatment of a disease, characterized in that, The endothelin A (ETA) receptor antagonist is selected from compounds of formula (I) or salts thereof: The diseases mentioned are selected from kidney disease, hypertension, etc.

2. The application according to claim 1, characterized in that, The kidney diseases mentioned include acute kidney disease and chronic kidney disease, and the hypertension mentioned includes refractory hypertension, portal hypertension due to cirrhosis, and elevated portal pressure due to cirrhosis.

3. The application according to claim 1 or 2, characterized in that, The kidney diseases mentioned include diabetic nephropathy, IgA, FSGS, and Alport nephropathy.

4. The application according to claim 1 or 4, characterized in that, The SGLT-2 inhibitors are selected from dapagliflozin, empagliflozin, canagliflozin, and eltoggliflozin, as well as SGLT-2 inhibitors that exist in their respective salt forms.

5. The application according to claim 1 or 4, characterized in that, The SGLT-2 inhibitor is dapagliflozin or a salt thereof, or empagliflozin or a salt thereof.

6. The application according to claim 1 or 5, characterized in that, The mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to dapagliflozin (in free form) is 0.88:10 to 52.8:

10.

7. The application according to claim 1 or 5, characterized in that, The mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to empagliflozin is selected from 0.88:10 to 52.8:

10.

8. The application according to any one of claims 1-7, characterized in that, The salts of the compounds of formula (I) are selected from their oxalates or phosphates; the salts of dapagliflozin are selected from dapagliflozin propylene glycol hydrate and dapagliflozin di-L-proline.

9. A pharmaceutical composition for the prevention and / or treatment of a disease, characterized in that, Its active ingredient includes compound (I) Or its salts and dapagliflozin or its salts.

10. A pharmaceutical composition for the prevention and / or treatment of a disease, characterized in that, Its active ingredients include or (I) compounds Or its salts and empagliflozin or its salts.

11. The pharmaceutical composition according to claim 9, characterized in that, The mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to dapagliflozin (in free form) is 0.88:10 to 52.8:

10.

12. The pharmaceutical composition according to claim 10, characterized in that, The mass ratio of the endothelin A (ETA) receptor antagonist (in free form) to empagliflozin is selected from 0.88:10 to 52.8:

10.

13. The pharmaceutical composition according to claim 9 or 10, wherein the disease is selected from kidney disease, hypertension, etc., preferably, the kidney disease includes acute kidney disease, chronic kidney disease, more preferably, the kidney disease includes diabetic nephropathy, IgA, FSGS, Alport nephropathy; the hypertension includes refractory hypertension, portal hypertension due to cirrhosis, and elevated portal pressure due to cirrhosis.

14. The pharmaceutical composition according to claim 9 or 10, characterized in that, The salts of the compounds of formula (I) are selected from their oxalates and phosphates; the salts of dapagliflozin are selected from dapagliflozin propylene glycol hydrate and dapagliflozin di-L-proline.

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