Use of a nitrogen-containing saturated heterocyclic compound

Abstract

The present application provides an application of a nitrogen-containing saturated heterocyclic compound. Specifically, the present application provides an application of a nitrogen-containing saturated heterocyclic compound represented by the following formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing chronic kidney disease:

Description

Technical Field

[0001] This invention relates to the use of a nitrogen-containing saturated heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and / or prevention of chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) is characterized by high prevalence, low awareness, poor prognosis, and high medical costs. It is another serious threat to human health after cardiovascular disease, diabetes, and malignant tumors. In recent years, the prevalence of CKD has been rising annually, reaching as high as 14.3% in the global general population. Cross-sectional epidemiological studies in my country show that the prevalence of CKD in people aged 18 and above is 10.8%. CKD is a progressive disease; if not treated promptly and effectively, it will worsen and progress, eventually leading to kidney failure. Without dialysis or kidney transplantation, survival becomes difficult. Currently, there is no particularly effective treatment for CKD; therefore, actively searching for effective drugs with minimal side effects is of great clinical value.

[0003] CN103562191B discloses a nitrogen-containing saturated heterocyclic compound or a pharmaceutically acceptable salt thereof with the following structural formula, which exhibits an inhibitory effect on renin and can be used to treat hypertension.

[0004]

[0005] In the formula, R 1 Indicates cycloalkane groups, etc., R 22 Indicates that aryl groups can be substituted, R indicates lower alkane groups, T indicates carbonyl groups, Z indicates -O-, etc. 3 R 4 R 5 and R 6 They can be the same or different, and they can represent hydrogen atoms, etc.

[0006] CN106928218A discloses novel pharmaceutically usable salts of morpholine derivatives (nitrogen-containing saturated heterocycles), including their malate, tartrate, hydrochloride, acetate, and naphthalene diphosphate. The tartrate salt has three crystalline salt forms: crystal form A, crystal form B, and dihydrate. The malate, hydrochloride, and acetate each have one crystalline salt form, and the naphthalene diphosphate is amorphous. Compared to known free bases of morpholine derivatives, the salts of morpholine derivatives possess one or more improved properties, such as better crystallinity, significantly improved water solubility, photostability, and thermal stability. The aforementioned morpholine derivative salts or their crystalline forms can be used to treat and / or prevent hypertension. Summary of the Invention

[0007] The technical problem to be solved by the present invention is that there are no particularly effective drugs for treating chronic kidney disease in the prior art, and the present invention provides the use of a nitrogen-containing saturated heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing chronic kidney disease.

[0008] The entire contents of patent CN103562191B and patent application CN106928218A described in the background art are incorporated herein by reference.

[0009] In one embodiment of the present invention, the use of a nitrogen-containing saturated heterocyclic compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing chronic kidney disease is provided:

[0010]

[0011] In a further embodiment of the invention, the pharmaceutically acceptable salt is a hydrochloride, sulfate, phosphate, hydrobromide, acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, maleate, malate, tartrate, acetate, or naphthalenedisulfonate.

[0012] In a further embodiment of the invention, the pharmaceutically acceptable salt is malate, tartrate, hydrochloride, acetate, or naphthalene disulfonate.

[0013] In one embodiment of the present invention, the pharmaceutically acceptable salt is a malate of compound I, which is a compound formed by compound I and malic acid in a 1:1 molar ratio, and its structural formula is as follows:

[0014]

[0015] In one embodiment of the present invention, the malate is a crystalline substance, and its X-ray powder diffraction pattern has characteristic peaks at 2θ of 7.767°±0.2°, 13.897°±0.2°, 14.775°±0.2°, 17.098°±0.2°, 18.999°±0.2°, 20.153±0.2°, 20.960°±0.2°, 21.423°±0.2°, 26.348°±0.2°, and 27.892°±0.2°. Specifically, the X-ray powder diffraction pattern of the malate crystals also exhibits characteristic peaks at 2θ values ​​of 5.598°±0.2°, 7.357°±0.2°, 10.395°±0.2°, 11.108°±0.2°, 16.037°±0.2°, 16.523°±0.2°, 19.410°±0.2°, 22.645°±0.2°, 26.630°±0.2°, 26.891°±0.2°, 27.380°±0.2°, 31.056°±0.2°, 33.306°±0.2°, 33.775°±0.2°, and 39.231°±0.2°. More specifically, the malate crystals of the compound of formula I have the following characteristics: Figure 1 The X-ray powder diffraction pattern shown is shown.

[0016] In one embodiment of the present invention, the chronic kidney disease includes hypertension complicated with kidney disease, hypertension complicated with kidney disease accompanied by abnormal glucose metabolism, chronic renal insufficiency complicated with chronic heart failure, or chronic kidney disease accompanied by abnormal glucose metabolism.

[0017] In one embodiment of the present invention, the chronic kidney disease refers to stages G1, G2, G3a, G3b, and G4 of chronic kidney disease, preferably stages G2, G3a, or G3b.

[0018] In one embodiment of the present invention, the hypertension in hypertension complicated with nephropathy is grade 1 hypertension, grade 2 hypertension or grade 3 hypertension, preferably grade 1 hypertension or grade 2 hypertension.

[0019] In one embodiment of the present invention, the dosage form of the drug includes tablets, capsules, intravenous injections, inhalers, nebulizers, lyophilized agents, patches, gels, sprays, or suppositories, preferably tablets.

[0020] In one embodiment of the present invention, the drug is a unit dose.

[0021] In one embodiment of the invention, the unit dose of the drug contains 25 mg to 200 mg of a nitrogen-containing saturated heterocyclic compound of Formula I or a pharmaceutically acceptable salt thereof, for example, 25 mg, 50 mg, 100 mg, 150 mg, or 200 mg of a nitrogen-containing saturated heterocyclic compound of Formula I or a pharmaceutically acceptable salt thereof.

[0022] The Kidney Disease Outcome Quality Initiative (KDOQI) working group of the Kidney Foundation (NKF) developed the definition and staging criteria for CKD in 2002.

[0023] Chronic kidney disease is defined as a structural or functional abnormality of the kidneys lasting for more than 3 months. The diagnostic criteria for chronic kidney disease include any one of the indicators in Table 1 below, lasting for more than 3 months.

[0024] Table 1: Diagnostic criteria for chronic kidney disease

[0025]

[0026] Note: At least one of the following must be met: AER: urinary albumin excretion rate; ACR: urinary albumin-to-creatinine ratio; GFR: glomerular filtration rate. Chronic kidney disease staging: Chronic kidney disease is divided into 5 stages according to glomerular filtration rate (GFR), as shown in Table 2 below.

[0027] Table 2: Chronic Kidney Disease Staging Based on GFR

[0028]

[0029] Hypertension is defined as: in the absence of antihypertensive medication, three separate measurements of office blood pressure on different days, with systolic blood pressure (SBP) ≥140 mmHg (1 mmHg = 0.133 kPa) and / or diastolic blood pressure (DBP) ≥90 mmHg.

[0030] Isolated systolic hypertension is defined as systolic blood pressure (SBP) ≥ 140 mmHg and DBP < 90 mmHg. Even if a patient has a history of hypertension and is currently using antihypertensive medication, hypertension should still be diagnosed if their blood pressure is below 140 / 90 mmHg.

[0031] Based on the level of blood pressure elevation, hypertension is further divided into grades 1, 2, and 3. The classification and definitions of blood pressure levels are shown in Table 3 below.

[0032] Table 3: Classification and Definition of Blood Pressure Levels

[0033]

[0034] Heart failure (HF) is a clinical syndrome defined as a complex group of clinical syndromes caused by any structural or functional abnormality of the heart that impairs the ability of the ventricles to fill or eject blood. Its main clinical manifestations include dyspnea and fatigue (limited exercise tolerance), as well as fluid retention (pulmonary congestion and peripheral edema).

[0035] Based on left ventricular ejection fraction (LVEF), heart failure is classified into three types: heart failure with reduced ejection fraction (HFrEF), heart failure with preserved ejection fraction (HFpEF), and heart failure with mid-range ejection fraction (HFmrEF). The definitions of these three types of heart failure are shown in Table 4 below.

[0036] Table 4: Classification and Definition of Heart Failure

[0037]

[0038]

[0039] Note: Heart failure with decreased HFrEF ejection fraction; heart failure with intermediate HFmrEF ejection fraction; heart failure with preserved HFpEF ejection fraction; a B-type natriuretic peptide (BNP) >35 ng / L and / or N-terminal pro-BNP (NT-proBNP) >125 ng / L; bE / e′≥13, mean e′ (interventricular septum and free wall) <9 cm / s; - None

[0040] Abnormal glucose metabolism includes prediabetes and diabetes.

[0041] Prediabetes, also known as impaired glucose regulation, refers to a condition where blood glucose levels exceed the normal range but have not yet met the diagnostic criteria for diabetes; it is an intermediate state between normal individuals and diabetic patients.

[0042] Diabetes mellitus is a group of common metabolic diseases characterized by hyperglycemia, glycosuria, impaired glucose tolerance, and abnormal insulin release tests.

[0043] The significant advantages of this invention are as follows: Studies on the efficacy and safety of nitrogen-containing saturated heterocyclic compound salts administered continuously for 8 weeks in rhesus monkeys (middle-aged / elderly) with chronic renal insufficiency and chronic heart failure demonstrate that the nitrogen-containing saturated heterocyclic compounds or their pharmaceutically acceptable salts provided by this invention can effectively prevent or treat chronic kidney disease, including hypertension with nephropathy, hypertension with nephropathy accompanied by abnormal glucose and lipid metabolism, chronic renal insufficiency with chronic heart failure, or chronic kidney disease accompanied by abnormal glucose and lipid metabolism. Furthermore, the nitrogen-containing saturated heterocyclic compounds or their pharmaceutically acceptable salts provided by this invention exhibit excellent safety, with no administration-related adverse events observed. Attached Figure Description

[0044] Figure 1 This is the XRPD spectrum of the formula I compound malate used in the embodiments of the present invention.

[0045] Figure 2 This is a concentration-time graph of compound I in monkeys after intravenous (iv) and oral (po) administration in Example 3. The intravenous dose in monkeys was 1 mg / kg, and the oral doses were 1, 3, and 9 mg / kg. Detailed Implementation

[0046] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0047] The formula I compound malate used in the following examples was prepared according to the preparation method described in Example 1 of patent application publication CN106928218A and its structure was confirmed; valsartan was purchased commercially; and the rhesus monkeys used were purchased from Sichuan Pumei Xingzhi Biotechnology Co., Ltd. (Plumi Primate Research Center).

[0048] abbreviations

[0049]

[0050]

[0051] Example 1: Efficacy and safety study of compound I malate administered continuously for 8 weeks in middle-aged / elderly rhesus monkeys with chronic renal insufficiency and chronic heart failure.

[0052] 1. Research Objectives

[0053] To evaluate the improvement of renal function after 8 weeks of continuous administration of the compound I malate, as well as the effective dose, intensity of action and risk of CVD events that can delay or improve renal function, providing a basis for clinical trial dose design, patient inclusion criteria and safety. Primary efficacy endpoint (ENDPOINT): 1. Improvement of renal function: estimated glomerular filtration rate eGFR creat-cys , cystatin C (CysC), creatinine and UACR; 2. Improvement of cardiac function: analysis of echocardiogram indexes for changes in cardiac structure and systolic and diastolic functions before and after drug administration; 3. Safety indexes: blood pressure, blood potassium, indexes related to glucose and lipid metabolism, etc.

[0054] 2. Test system

[0055] 2.1 Spontaneously chronically renal insufficiency rhesus monkeys

[0056] Animal species: Macaca mulatta (Rhesus Macaque)

[0057] Grade: ordinary grade. Quarantine qualified before the test, including physical examination, 2 tuberculin tests, parasites, Salmonella, Shigella and B virus examinations.

[0058] Animal identification: Wear stainless steel number plates engraved with Arabic numerals on the neck ring and tattoo on the chest.

[0059] Supplier: Ya'an Prime Bio-Tech Co., Ltd.

[0060] Production license number: SCXK(Sichuan)2019-027.

[0061] Animal certificate numbers: 0016710, 0016944, 0016966.

[0062] 2.2 Inclusion criteria

[0063] 23 male / female animals, aged 14 - 24 years (equivalent to 40 - 70-year-old adults), body weight 8.45 - 14.93 kg for males and 1 female: 6.43 kg;

[0064] Long-term abnormal glucose and lipid metabolism for more than 3 years; abnormal glucose metabolism is defined as fasting plasma glucose (FPG) > 4.8 mmol / l, vs age-matched control group 4.1 ± 0.3 mmol / l;

[0065] CKD-EPI rating G3a - G3b, estimated glomerular filtration rate eGFR: 30 - 59 ml / min / 1.73m 2Or moderate to severe proteinuria (A2-A3): urine albumin / creatinine ratio (UACR) ≥15mg / g-350mg / g (4-6 hours urine collection), vs age-matched control group 3±2mg / g.

[0066] Normal blood pressure, grade 1 or 2 hypertension (consistent with clinical patient standards);

[0067] HFrEF: LVEF 30%-49% (normal value: 50%-70% Simpson's two-plane method) or HFpEF (moderate to severe impairment): Ea<8 or E / Ea>10 (based on clinical diagnostic criteria).

[0068] 2.3 Exclusion Criteria

[0069] 1) Chronic liver disease (including known active hepatitis) and / or screening for alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >3x the upper limit of normal (ULN).

[0070] 2) Exclusion criteria related to dyslipidemia: TC>7mmol / l; TG>3.5mmol / l.

[0071] 3) Any other medical history that may affect the evaluation of drug efficacy.

[0072] Table 5 below shows the underlying etiology and characteristics of chronic DKD renal insufficiency in rhesus monkeys in each dose group at baseline.

[0073] Table 5: Baseline period - Underlying etiologies and characteristics of chronic DKD renal insufficiency in rhesus monkeys in each dose group

[0074]

[0075]

[0076] Note: #257 Animals with chest deformities cannot have their cardiac ultrasound images acquired.

[0077] Table 5: Baseline period - Underlying etiologies and characteristics of chronic DKD renal insufficiency in rhesus monkeys in each dose group (continued)

[0078]

[0079]

[0080] 3. Experimental Design

[0081] 3.1 Grouping and Dosage Design

[0082] This trial enrolled animals in three phases. Phase 1 consisted of two groups: a 5 mg / kg malate group (3 animals, #257, #4721, #287) and a placebo group (2 animals, #1029, #4713). Phase 2 consisted of a 5 mg / kg malate group (3 animals, #144, #5073, #6651), a Valsartan group (3 animals, #2091, #4861, #6645), and a placebo group (2 animals, #6653, #231). Phase 3 consisted of a 2 mg / kg malate group (5 animals, #4829, #6501, #287, #4721, #2091) and a 1 mg / kg malate group (5 animals, #1567, #4861, #5453, #6653, #5073).

[0083] In the first phase, two animals (#287 and #4721) were washed off after drug withdrawal for about 9 months. In the second phase, four animals (#5073, #2091, #4861 and #6653) were washed off after drug withdrawal for about 5 months before entering the third phase, the baseline period (which lasted for 1 month).

[0084] See Table 6 below for details.

[0085] Table 6: Group Design Information

[0086]

[0087] Note: In the Valsartan group, the medication was administered once daily during weeks 1-2 and twice daily during weeks 3-8.

[0088] 3.2 Dosage Information

[0089] Route of administration: Oral administration.

[0090] Dosage frequency: once daily for compound I malate, once daily for the Valsartan group during weeks 1-2, and twice daily for weeks 3-8.

[0091] Dosage calculation: Calculate the dosage for the following week based on the body weight measured at each weighing.

[0092] Administration time: 08:00-09:00.

[0093] 3.3 Main efficacy indicators

[0094] Glomerular filtration rate (eGFR) creat-cys The study measured creatinine (Cr-P), blood urea nitrogen (BUN), and cystatin (CysC) to calculate the eGFR value. Measurements were taken once before administration and every two weeks thereafter.

[0095] eGFR male=135×min(Cr / 0.9,1) -0.207 ×max(Cr / 0.9,1) -0.601 ×min(CysC / 0.8,1) -0.375 ×max(CysC / 0.8,1) -0.711 ×0.995 Age×3

[0096] eGFR female =135×min(Cr / 0.7,1) -0.284 ×max(Cr / 0.7,1) -0.601 ×min(CysC / 0.8,1) -0.375 ×max(CysC / 0.8,1) -0.711 ×0.995 Age×3 ×0.969

[0097] UACR: Urine samples were collected at 4h and 6h to measure urinary microalbumin (MALB) and urinary creatinine (Cr-U), and the UACR value was calculated. One dose was administered before administration and another at the end of administration. UACR = MALB / Cr-U.

[0098] Blood pressure: Blood pressure, including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and heart rate (HR), was measured after anesthesia. Once before administration, and once each at 4 and 8 weeks after administration.

[0099] Cardiac GE Doppler ultrasound: one echocardiogram within 1 year (for enrolled animals), one before administration (within the baseline period D-30 days), and one 8 weeks after administration.

[0100] 3.4 Secondary efficacy indicators

[0101] Serum potassium levels: twice before administration, and once every two weeks during the administration period;

[0102] Glucose and lipid metabolism and liver function: FPG, FRA, LDL-c, HDL-c, TG, TC, NT-proBNP, ALT, AST, TBIL, etc., twice before administration, and once each at 4 weeks and 8 weeks of administration.

[0103] Other biochemical and hematological indicators were measured once before and once after administration.

[0104] Weight: Once before administration, once a week during the administration period;

[0105] After administration, observe changes in food intake and behavior for 24 hours each day.

[0106] 3.5 Testing methods and equipment

[0107] Detection methods: see Table 7 and Table 8 below.

[0108] Hematological testing instrument: Siemens ADVIA 2120i Hematolagy Systems.

[0109] Blood biochemistry and urine indicators were detected using a Roche cobas 6000 analyzer series C501 module, and NT-proBNP was detected using an ELISA kit.

[0110] Table 7: Biochemical Testing Items

[0111]

[0112] Table 8: Hematological Test Items

[0113]

[0114] 3.6 Cardiac Doppler Ultrasound

[0115] Anesthesia method: Ketamine hydrochloride 15mg / kg was injected intramuscularly for anesthesia. Depending on the animal's anesthesia status, the veterinarian would determine whether to administer supplementary anesthesia. The dose of supplementary anesthesia was 1 / 2 of the initial dose each time.

[0116] Detection method: The animal was placed in the left lateral decubitus position, and images were acquired using a 6S-RS probe (frequency 2.7-8.0MHz).

[0117] Analysis method: After image acquisition and saving, the accompanying analysis workstation EchoPAC Software was used to repeatedly detect various indicators of cardiac systolic and diastolic function over three consecutive cardiac cycles.

[0118] Detection indicators and ultrasound techniques: see Table 9 below.

[0119] Testing instrument: GE Vivid S5 color Doppler ultrasound diagnostic instrument.

[0120] 3.7 Blood Pressure Measurement

[0121] Testing Method: Animals were anesthetized by intramuscular injection of 15 mg / kg ketamine hydrochloride. After anesthesia, the animals were placed in a supine position. The hair on the left upper arm was shaved clean, and a cuff of appropriate size was applied according to standard procedures. The pulse oximeter probe was clipped onto the animal's finger or toe (except for the left hand), with the red photosensitive side facing the fingertip. The animal's blood pressure was measured three times consecutively using automatic mode, with a 1-minute interval between each measurement. If the differences in DBP, SBP, and MBP between the three measurements were not significant (the difference between the highest and lowest values ​​was less than 15 mmHg), the measurement was considered complete.

[0122] Monitoring indicators: These include systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and heart rate (HR).

[0123] Testing instrument: GE B40i electrophysiological monitor.

[0124] Table 9: Echocardiography Detection Indicators

[0125]

[0126]

[0127] 3.8 Clinical observation

[0128] Observation frequency: once a day.

[0129] Observation method: Observe through the cage.

[0130] Observation content: injection site, skin, hair, eyes, ears, nose, mouth, chest, abdomen, urogenital area, limbs, etc., as well as changes in respiration, movement, urination, defecation and behavior.

[0131] 3.9 Food Intake Measurement

[0132] Feeding method: Feed once each at 8:00-9:00 AM, 10:00 AM, 2:00 PM, and 4:00 PM, ensuring there is food in the feeding box for free access. Remove any remaining feed at 7:40-8:00 AM the next day.

[0133] Feeding amount: approximately 250-500g / animal / day. During the experiment, feed was provided in the feeding box 24 hours a day, and the animals had free access to food.

[0134] Feed intake measurement method: Feed intake is estimated using a semi-quantitative method. The daily feed intake, waste, and remaining feed in the feed box are recorded. Feed intake = Feed intake - Waste - Remaining feed in the feed box.

[0135] 3.10 Weight Measurement

[0136] Weighing time: Before feeding on the same day.

[0137] Measurement method: The animal was fasted for 14-16 hours before weighing. The animal was placed in the transfer cage while conscious and weighed using a large animal scale.

[0138] Measuring instrument: METTLER TOLEDO electronic platform balance.

[0139] 4.0 Termination of Test Criteria

[0140] a. A serious adverse event occurs.

[0141] b. A serious infection occurs.

[0142] 5. Results and Analysis

[0143] 5.1 Impact on UACR

[0144] The effects of each group of drug administration on proteinuria (UACR) are shown in Table 10 below. CKD rhesus monkeys with moderate to severe proteinuria, characterized by a baseline urinary albumin / creatinine ratio (UACR) ≥15 mg / g-350 mg / g (urine collected over 4-6 hours), were selected from each group. The changes in albumin / creatinine ratio at baseline and 8 weeks (W8) after drug administration were analyzed.

[0145] In the placebo group (n=4), the mean UACR increased by 30.64±34.50% at the end of the trial period (W8) compared with baseline; the UACR of the four animals with proteinuria fluctuated steadily within a certain range and did not show rapid progression.

[0146] In the Valsartan group (n=3, including animals with moderate to severe proteinuria): compared with baseline, the UACR of the two animals (#2091, #4861) treated with W8 decreased by 40%-70%, both showing benefit; the baseline UACR of one animal was 15 mg / g, and the UACR of W8 after treatment was 20.4%, with no significant benefit. Due to the small number of animals enrolled, there was no statistically significant difference compared with the placebo group.

[0147] In the group treated with formula I malate (5 mg / kg once daily (qd)) (n=6, 4 / 6 animals with moderate to severe proteinuria were included in the statistics): compared with baseline, the mean UACR of W8 decreased by 46.87±36.93% after administration, which was significantly lower than that of the placebo group (p<0.05). Among them, 3 animals showed a 60%-70% decrease in UACR of W8 compared with baseline after administration, all of which showed benefits; 1 animal did not show any benefit.

[0148] In the group treated with formula I malate (2 mg / kg qd) (n=5, 3 / 5 animals with moderate to severe proteinuria were included in the statistics): compared with baseline, the mean UACR of W8 decreased by 66.00±17.42% after administration, which was significantly lower than that of the placebo group (p<0.01). Among them, 3 animals treated with this method showed a UACR decrease of approximately 50%-80% compared with baseline, all of which showed benefits.

[0149] In the formula I compound malate group (1 mg / kg qd) (n=5, 2 / 5 animals with moderate to severe proteinuria were included in the statistics): compared with baseline, the UACR of W8 decreased by 39% in 1 animal after administration, and the benefit was not obvious in 1 animal.

[0150] In summary, the administration of 2-5 mg / kg malate of Formula I compound once daily for 8 weeks can significantly improve proteinuria (UACR).

[0151] Table 10: Effects of Compound I malate administration for 8 weeks on UACR in rhesus monkeys with spontaneous chronic kidney disease (CKD).

[0152]

[0153]

[0154] Note 1. UACR = MALB / Cr-U

[0155] 2. Rate of change = (time point after administration - baseline value) / baseline value * 100%, negative values ​​indicate a decrease; "#" indicates a p < 0.05 compared to the placebo group.

[0156] 3. NA and UACR normal animals were not included in the statistics.

[0157] Table 10: Effects of Formula I compound malate administration for 8 weeks on UACR in rhesus monkeys with spontaneous chronic kidney disease (CKD) (continued)

[0158]

[0159]

[0160] Note 1. UACR = MALB / Cr-U

[0161] 2. Rate of change = (time point after administration - baseline value) / baseline value * 100%, negative values ​​indicate a decrease; "#" indicates p < 0.05 compared to the placebo group, "##" indicates p < 0.01 compared to the placebo group.

[0162] 3. NA and UACR normal animals were not included in the statistics.

[0163] 5.2 Glomerular filtration rate (eGFR) creat-cys Influence

[0164] The effects of each group on eGFR are shown in Tables 11 and 12 below. Each group included in the eGFR... creat-cys 30-59 ml / min / 1.73 m 2 The changes in glomerular filtration rate of W8 at baseline and after drug administration were analyzed.

[0165] Placebo group (n=4): compared with baseline, the glomerular filtration rate (eGFR) of the 4 animals was [value missing]. creat-cys It fluctuated steadily within a certain range without showing rapid progress.

[0166] Valsartan group (n=3): compared with baseline, the glomerular filtration rate (eGFR) of the Valsartan group after administration was 8%. creat-cys The average increase was 6 ± 2 ml / min / 1.73 m 2 The change value was significantly increased compared with that of the placebo group (p<0.01), which significantly improved glomerular deterioration, and the efficacy was time-dependent.

[0167] Formula I compound malate (5 mg / kg qd) group (n=6): W8 glomerular filtration rate (eGFR) after administration compared with baseline. creat-cys The average increase was 6 ± 6 ml / min / 1.73 m 2 The change value was significantly increased compared with that of the placebo group (p<0.05), indicating that the therapeutic effect was time-dependent.

[0168] Formula I compound malate (2 mg / kg qd) group (n=5): W8 glomerular filtration rate (eGFR) after administration compared with baseline. creat-cys The average increase was 3 ± 3 ml / min / 1.73 m 2 The change was significantly increased compared to the placebo group (p < 0.05).

[0169] Formula I compound malate (1 mg / kg qd) group (n=5): W8 glomerular filtration rate (eGFR) after administration compared with baseline. creat-cys The average increase was 3±4 ml / min / 1.73 m 2 The change was not statistically significant compared to the placebo group.

[0170] In summary, administration of 2-5 mg / kg malate of Formula I compound once daily for 8 weeks significantly improves glomerular filtration rate (eGFR). creat-cys .

[0171] Table 11: Effects of 8 weeks of administration of formula I compound malate on eGFRcreat-cys in rhesus monkeys with spontaneous chronic kidney disease.

[0172]

[0173]

[0174]

[0175] Note: Change value = 8 weeks - Baseline, negative values ​​indicate a decrease. "#" indicates a change value compared to the placebo group (p < 0.05), "##" indicates a change value compared to the placebo group (p < 0.01).

[0176] Table 12: Changes in eGFRcreat-cys values ​​in rhesus monkeys with spontaneous chronic kidney disease after 8 weeks of administration of formula I compound malate.

[0177]

[0178]

[0179] Note: Change value = current value - Baseline, negative value indicates a decrease.

[0180] 5.3 Effects on CysC, Cr-P and BUN The effects of each group on CysC, Cr-P and BUN after administration are shown in Tables 13 to 15 below.

[0181] Table 13: Effects of Compound I malate administration for 8 weeks on Cr-P in rhesus monkeys with spontaneous chronic kidney disease.

[0182]

[0183]

[0184] Note: "*" indicates p < 0.05 compared to the baseline value, and "**" indicates p < 0.01 compared to the baseline value.

[0185] Table 14: Effects of Compound I malate administration for 8 weeks on CysC in rhesus monkeys with spontaneous chronic kidney disease.

[0186]

[0187]

[0188]

[0189] Note: "**" is p < 0.01 compared to the baseline.

[0190] Table 15: Effects of Compound I malate administration for 8 weeks on BUN in rhesus monkeys with spontaneous chronic kidney disease.

[0191]

[0192]

[0193] 5.4 Effects on blood pressure

[0194] The effects of each drug administration group on blood pressure are shown in Table 16. Since blood pressure testing in primates requires anesthesia, it is necessary to control for heart rate variability at various time points in order to perform statistical analysis.

[0195] Placebo group (n=4, 2 / 4 hypertension): Compared with baseline, the blood pressure of the 4 animals fluctuated steadily within a certain range.

[0196] Valsartan group (n=3, 1 / 3 of which were hypertensive): Compared with baseline, W8, SBP and DBP decreased by an average of 14±4 mmHg and 7±0 mmHg, respectively, which were extremely significant compared with the placebo group (p<0.01).

[0197] In the formula I compound malate (5 mg / kg qd) group (n=6, 4 / 6 patients with hypertension): compared with baseline, W8, SBP and DBP decreased by an average of 21±8 mmHg and 10±7 mmHg respectively after administration, which were extremely significant compared with the placebo group (p<0.01), and the efficacy showed time-dependent effects.

[0198] In the formula I compound malate (2 mg / kg qd) group (n=5, 2 / 5 cases of hypertension): compared with baseline, W8, SBP and DBP decreased by an average of 19±9 mmHg and 9±8 mmHg respectively after administration, which was extremely significant compared with the placebo group (p<0.01), and the efficacy showed time-dependent effects.

[0199] In the formula I compound malate group (1 mg / kg qd) (n=5): compared with baseline, W8, SBP and DBP decreased by an average of 11±4 mmHg and 5±4 mmHg, respectively, which were significantly lower than the changes in the placebo group (p<0.05).

[0200] In summary, the malate of Formula I, administered at 1-5 mg / kg qd for 8 weeks, exhibited hypotensive activity.

[0201] Table 16: Effects of Compound I Malate Administration for 8 Weeks on Blood Pressure in Rhesus Monkeys with Spontaneous Chronic Kidney Disease

[0202]

[0203]

[0204] Note: "*" indicates p < 0.05 compared to baseline, "**" indicates p < 0.01 compared to baseline. "#" indicates p < 0.05 compared to placebo, "##" indicates p < 0.01 compared to placebo. NA (no heart rate) and unstable heart rates are not included in the statistical analysis.

[0205] 5.5 Effects on cardiac function

[0206] The effects of each group on echocardiographic parameters are shown in Table 17.

[0207] No significant adverse effects on cardiac systolic and diastolic function were observed after administration of the malate of Formula I at various dosage groups, the Valsartan group, and the placebo group.

[0208] In addition, two rhesus monkeys (#5073 and #287) in the Formula I compound malate 5 mg / kg group showed increased LVEF% at 8 weeks post-administration compared to baseline, indicating low ejection fraction (SD). However, whether this product can treat chronic heart failure requires further cohort data from subsequent studies.

[0209] Table 17: Effects of Compound I Malate Administration for 8 Weeks on Key Cardiac Function Indicators in Rhesus Monkeys with Spontaneous Chronic Kidney Disease and Evaluation of Efficacy

[0210]

[0211]

[0212] Note: The level of diastolic dysfunction is determined after comprehensively considering Ea, E / Ea, Ea / Aa and other functional indicators; when the level of diastolic dysfunction in the test animal improves from the baseline, it is considered "benefit", otherwise it is considered "ineffective"; #257 Animals with chest wall deformities could not have cardiac ultrasound images acquired.

[0213] 5.6 Effects of blood biomarkers on cardiac function impairment

[0214] 5.6.1 Effect on NT-proBNP

[0215] The effect of compound I malate on NT-proBNP is shown in Table 18.

[0216] Compared with the baseline period, no significant changes were observed in NT-proBNP levels in animals in each treatment group after 8 weeks of administration.

[0217] Table 18: Effects of Compound I malate administration for 8 weeks on NT-proBNP in rhesus monkeys with spontaneous chronic kidney disease.

[0218]

[0219]

[0220] 5.6.2 K + Impact

[0221] Formula I compound malate for K + The impact is shown in Table 19.

[0222] Compared with baseline, serum K levels in animals in each treatment group were significantly higher after 8 weeks of treatment. + The level has not changed significantly.

[0223] Table 19: Effects of 8-week administration of formula I compound malate on spontaneous chronic kidney disease in rhesus monkeys with K... + Impact

[0224]

[0225]

[0226]

[0227] 5.7 Safety and Tolerance Studies

[0228] No drug-related adverse events were observed in the formula I malate group during the administration period, and no significant changes were observed in liver function, kidney function, food intake, body weight, or hematological parameters.

[0229] 6. Conclusion

[0230] Compound I, malate, at 2-5 mg / kg once daily for 8 weeks, significantly improved uptake of proteinuria (UACR) and glomerular filtration rate (eGFR). creat-cys The efficacy was comparable to that of the Valsartan group.

[0231] Compound I malate, administered at 1-5 mg / kg once daily for 8 weeks, consistently exhibited hypotensive activity. Furthermore, no risk of hyperkalemia was observed under the conditions of this study.

[0232] Example 2: Preclinical safety assessment of compound I

[0233] 1. Materials and Methods

[0234] 1.1. Test Substances

[0235] Compound I was prepared and its structure confirmed according to the method described in CN103562191B. The solvent was 0.5% sodium carboxymethyl cellulose (CMC-Na). Compound I was stored at room temperature, away from light and moisture. In the safety study in cynomolgus monkeys, the test substance was suspended in 0.5% CMC-Na at the relevant concentration. The homogeneity, concentration, and one-week stability of the prepared suspension were analyzed to ensure correct administration of the reported dose. Accelerated stability of the active pharmaceutical ingredient (API) was tested over six months.

[0236] 1.2. Animal husbandry

[0237] Healthy male and female cynomolgus macaques aged 3-4 years were provided by Hainan Xinyuan Biotechnology Co., Ltd. (Hainan, China). At the start of treatment, the males weighed 3.2–5.8 kg and the females weighed 2.5–3.7 kg. They were placed in a 36-day acclimatization period prior to the study. The animals were given free access to food and water once a day, once in the morning.

[0238] This non-clinical study was conducted in accordance with the guidelines of the China Food and Drug Administration (CFDA). The experiment was carried out in a facility approved by the Association for Assessment and Evaluation of Laboratory Animal Care (AAA LAC), and the animals were housed according to the Guidelines for Laboratory Animal Care and Use (Nussberger et al., 2008).

[0239] 1.3. Dosage and Treatment Schedule

[0240] Cynomolgus monkeys (5 per sex per group) were selected using a weight-based computer randomization program and fed compound formula I via nasogastric tube (dose levels of 0 (control group), 20, 100, and 450 mg / kg / day, volume 5 ml / kg). Individual doses were adjusted weekly based on animal weight. For all groups, two-thirds of the animals were randomly selected and euthanized on day 28. The remaining animals were euthanized 28 days after drug withdrawal.

[0241] 1.4. Clinical observation

[0242] Assess mortality and clinical signs daily from the start of the quarantine period. Examine each animal's behavior, response to medication, or the occurrence of disease at least twice daily.

[0243] Observations included, but were not limited to, the following: changes in skin and fur; eyes and mucous membranes; respiration, circulation, autonomic nervous system, central nervous system, and behavioral patterns. Rectal temperature was measured before administration (twice), 1 and 24 hours after the first administration, and before and 1 and 24 hours after administration on day 26. Body weight was measured weekly. Daily food consumption was estimated. Ophthalmoscopy was performed on the monkeys using a portable slit lamp (YZ2) and a direct ophthalmoscope (GFJY-01B).

[0244] 1.5. Laboratory Testing

[0245] For the monkey study, the following parameters were measured during the quarantine period (twice, day 01 and day 02), day 14, day 28, and day 56, as shown in Table 20 below. (1) Blood and coagulation index tests were performed using a Bayer ADVIA2120 (Germany) and a Sysmex CA-1500 (Japan). (2) Serum biochemical tests were evaluated using a HITACHI 7080 automated analyzer (Japan) and an Easylyte PLUS electrolyte analyzer (MEDICA, USA). (3) 24-hour pooled urine was collected from each animal via a tray under each cage. Urine analysis was performed using a Uritest-300 (China). (4) 24-hour pooled feces were collected from each monkey via a tray under each cage. Fecal occult blood was detected. (5) ACCESS 2 chemiluminescent immunoassay was used for cardiac troponin I (non-GLP) testing.

[0246] Table 20: Parameters assessed in hematological, serological, and urinary analyses

[0247]

[0248]

[0249] 1.6. Routine Electrocardiogram Analysis

[0250] Routine ECG analysis was performed at each planned time (day 1, day 01, day 02, hours 1 and 24 of day 1, before administration, and hours 1 and 24 of days 26 and 56). The ECG system used a derived DII (ECG-6951E, Shanghai, China) to record P waves, R waves, T waves, PR intervals, QT intervals, QRS duration, and heart rate.

[0251] 1.7. Autopsy and Histopathology

[0252] At the end of the exposure phase (day 28) and recovery phase (day 56), a full necropsy was performed on all animals by visual inspection. The animals were trimmed, weighed, and assessed for the absolute weight and final body weight or brain weight as a percentage of the following selected organs: brain, heart, kidneys, liver, spleen, thymus, testes, epididymis, uterus, ovaries, adrenal glands, and thyroid gland (including parathyroid glands). The following tissues were preserved in 10% neutral formalin: brain, pituitary gland, thyroid gland (including parathyroid glands), trachea, heart, pancreas, spleen, adrenal glands, prostate, ovaries, uterus (including cervix and fallopian tubes), vagina, testes, epididymis, seminal vesicles, esophagus, duodenum, jejunum, ileum, cecum, colon, rectum, mesenteric lymph nodes, lymph nodes, submandibular lymph nodes, aorta, eyes, skeletal muscles, sciatic nerve, femur (including metaphysis), mammary glands, sternum, salivary glands, spinal cord, bladder, lungs (including bronchi), liver, kidneys, stomach, bone marrow (sternum), thymus, gallbladder, and any obvious lesions or masses. Lung tissue was infected with a fixative during autopsy. All preserved tissues were embedded, sectioned, stained with hematoxylin and eosin (HE), and examined under a microscope. Both the smears (monkey sternum) and the paired embedded sternum sections underwent bone marrow cell morphology examination.

[0253] 1.8. Toxicokinetics

[0254] Blood samples were collected at 0, 0.5, 1.5, 3, 5, 7, and 24 hours post-administration on days 1 and 26. Approximately 1.0 mL of blood was drawn from a vein each time and collected in EDTA-K2 anticoagulant tubes. The blood samples were placed on ice and then centrifuged at 4°C (3500 rpm, 5 min) to obtain plasma samples, which were then frozen at approximately -80°C until analysis. The toxicokinetics of compound I were determined at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0255] 1.9. Statistical Analysis

[0256] For each sex, weight, rectal temperature, ECG, organ weight, hematological parameters, and serum biochemical data were analyzed using the statistical software SAS 9.3. First, the uniformity of variance was analyzed using the Levene test. If P > 0.05, one-way ANOVA was performed. If P < 0.05, the Kruskal–Wallis test was performed. If the obtained ANOVA p-value < 0.05, the Dunnett T test was used for comparisons between groups. If the obtained Kruskal–Wallis test p-value < 0.05, the Dunnett T test was performed after rank transformation of the data.

[0257] 2. Results

[0258] 2.1. Clinical observation

[0259] Compared with the control group, the weight gain in the high-dose group was slightly lower (2.7±0.4 vs. 3.2±0.2 in females; 4.1±0.5 vs. 4.6±0.5 in males), which was not statistically significant.

[0260] Early drug-related death occurred on day 23 in one high-dose female, with clinical signs including lethargy, kyphosis, hypothermia, diarrhea, mild tympanic membrane and thymic atrophy. No obvious signs of treatment-related clinical toxicity were observed in other animals.

[0261] 2.2. Hematology and Coagulation

[0262] Increased NEUT% and decreased LYMPH% were observed in all treated females and high-dose males. Increased Fbg was also observed in medium- and high-dose animals, and increased APTT was observed in high-dose females (Table 21).

[0263] Table 21: Hematological data of monkeys treated with compound I for 4 weeks.

[0264]

[0265]

[0266] *p<0.05, compared with the control group; **p<0.01, compared with the control group; values ​​are mean ± SD.

[0267] 2.3. Serum Biochemistry

[0268] Serum chemical parameters CPK, BUN, and CREA were significantly increased in both male and female cynomolgus monkeys after high doses, especially in monkeys that died early on day 23 (day 14 data, BUN: 34.99; CREA: 526). CHOL was significantly decreased in high-dose males. Serum Na levels were slightly decreased in high-dose males, but this was statistically significant at day 26. Other statistically significant findings were isolated events and considered to have no toxicological significance (Table 22).

[0269] Table 22: Serum chemical composition of monkeys treated with compound I for 4 weeks

[0270]

[0271]

[0272] *p<0.05, compared with the control group; **p<0.01, compared with the control group; values ​​are mean ± SD.

[0273] 2.4. Urine Analysis

[0274] No significant changes were reported in any of the urine analysis parameters examined in either male or female monkeys.

[0275] 2.5. ECG parameters

[0276] A medium-dose female was observed to have a prolonged QTc interval at 1 hour on day 26 post-administration (296 ms in animal #303). A high-dose male was observed to have sinus arrhythmia and a prolonged QRS interval at 1 hour on day 26 post-administration (60 ms in animal #411). Prolonged QRS intervals were also observed at 1 hour on both day 1 and day 26 post-administration in high-dose females (Table 23).

[0277] Table 23: Electrocardiogram data of monkeys treated with compound I for 4 weeks

[0278]

[0279]

[0280] *p<0.05, compared with the control group; **p<0.01, compared with the control group; values ​​are mean ± SD.

[0281] 2.6. Autopsy

[0282] 2.6.1. Overall Pathology and Organ Weight

[0283] Macroscopic findings in no monkeys were thought to be associated with administration of the test substance, except for mild atrophy of the tympanic membrane and thymus in monkeys that died earlier. High-dose monkeys had higher relative liver and kidney weights, lower relative thymus weights, and lower absolute or relative uterine weights (Table 24).

[0284] Table 24: Organ weights of cynomolgus monkeys treated with compound I for 4 weeks.

[0285]

[0286]

[0287]

[0288]

[0289] *p<0.05, compared with the control group; **p<0.01, compared with the control group; values ​​are mean ± SD.

[0290] 2.7. Toxicokinetics

[0291] Systemic exposure (derived from plasma AUC of the original compound and major metabolite) in the dose range of 20-450 mg / kg 0–24h and C max The increase in (indicated by) was much greater than the increase in dosage. No accumulation was observed after 28 consecutive days of administration.

[0292] Example 3: Non-clinical pharmacokinetics

[0293] 1. Materials and Methods

[0294] 1.1. Materials

[0295] The compound of formula I was prepared and its structure was confirmed according to the method in CN103562191B.

[0296] 1.2. Animals

[0297] Crab-eating macaques (3 / sex / group: weight 3-5 kg, age 5-6 years) were obtained from Suzhou Xishan Zhongke Laboratory Animal Center (Shanghai, China). The animals were acclimatized for 7 days prior to the study. The monkeys were kept in individual cages in an animal room with controlled temperature (18–26℃) and humidity (50±20%), with an airflow of 10 rpm and fresh air replacement every hour. The light cycle was 12 hours on and 12 hours off. The monkeys were fed and watered once daily in the morning. All animal experiments were conducted in accordance with the Animal Care and Use Guidelines developed by the AMMS Animal Care and Use Committee.

[0298] 2. Intravenous and oral administration of compounds of formula I

[0299] Compound of Formula I was administered intravenously or orally to cynomolgus monkeys that had been fasted overnight. Doses were 1 mg / kg (intravenous), 1 mg / kg, 3 mg / kg, and 9 mg / kg (oral). Blood samples (0.2 mL each) were collected intravenously at 0, 5, and 15 minutes after administration, and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration. Immediately after collection, all blood samples were centrifuged at 10,000 × g for 5 minutes, and plasma was stored at -75 ± 10 °C until analysis. Plasma samples were prepared by protein precipitation. The concentration of compound of Formula I in plasma was determined by a validated liquid chromatography-tandem mass spectrometry method.

[0300] Pharmacokinetic parameters, such as the area under the plasma concentration-time curve (AUC) and the maximum plasma concentration (C). max ), reaching C max Time (T) max Half-life (T) 1 / 2 ), systemic plasma clearance (CL), mean residence time (MRT), and steady-state volume of distribution (V). ss The pharmacokinetic data were calculated using WinNonlin v1.3 (Pharsight, Mountain View, California, USA) via non-compartmental analysis. For the allometric growth model, animal pharmacokinetic data were simulated using a two-compartment model and the Kinetica 5.1 software package (v.3.0; InnaPhase, Philadelphia, PA, USA).

[0301] 3. Results

[0302] Plasma pharmacokinetics and bioavailability

[0303] Table 25 and Figure 2 Pharmacokinetic data after intravenous and oral administration in cynomolgus monkeys are presented. Following intravenous injection, drug concentrations decreased exponentially, with a clearance of 1.96 L / h / kg and a volume of distribution of 1.86 L / kg in monkeys. Following oral administration, compound I reached its C60 concentration in 1.0–1.30 h. max T in plasma 1 / 2 Disappearance time was 2.73–5.99 h. AUC after intravenous and oral administration. 0-24h The calculated oral bioavailability of compound I in monkeys was 3.3-11.3%, indicating the involvement of efflux transport proteins.

[0304] Table 25:

[0305]

[0306] CL clearance rate after IV, V at steady state after IV ss Distribution volume, average residence time of MRT, T1 / 2 Half-life, C max Maximum plasma concentration, reaching C max T max Time, AUC 总 Total area under the plasma concentration-time curve, CL / F clearance after oral administration, V ss / volume of distribution at steady state after oral administration of F, absolute bioavailability of F after oral administration, intravenous injection (iv), oral administration (po).

Claims

1. The use of nitrogen-containing saturated heterocyclic compounds of formula I or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment and / or prevention of chronic renal insufficiency complicated with chronic heart failure: 。 2. The application according to claim 1, wherein, The pharmaceutically acceptable salts are hydrochloride, sulfate, phosphate, hydrobromide, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, maleate, malate, tartrate, acetate, or naphthalenedisulfonate.

3. The application according to claim 1 or 2, wherein, The pharmaceutically acceptable salt is the malate of compound I, which is a compound formed by compound I and malic acid in a 1:1 molar ratio, and its structural formula is as follows: 。 4. The application according to claim 3, wherein, The malate is a crystalline substance, and its X-ray powder diffraction pattern has characteristic peaks at 2θ of 7.767°±0.2°, 13.897°±0.2°, 14.775°±0.2°, 17.098°±0.2°, 18.999°±0.2°, 20.153±0.2°, 20.960°±0.2°, 21.423°±0.2°, 26.348°±0.2°, and 27.892°±0.2°.

5. The application according to claim 4, wherein, The X-ray powder diffraction pattern of the malate crystals also shows characteristic peaks at 2θ of 5.598°±0.2°, 7.357°±0.2°, 10.395°±0.2°, 11.108°±0.2°, 16.037°±0.2°, 16.523°±0.2°, 19.410°±0.2°, 22.645°±0.2°, 26.630°±0.2°, 26.891°±0.2°, 27.380°±0.2°, 31.056°±0.2°, 33.306°±0.2°, 33.775°±0.2°, and 39.231°±0.2°.

6. The application according to claim 5, wherein, The malate crystals of the compound of formula I have the X-ray powder diffraction pattern shown in Figure 1.

7. The application according to claim 1 or 2, wherein, The dosage form of the drug is selected from tablets, capsules, intravenous injections, inhalers, lyophilized preparations, patches, gels, sprays, or suppositories.

8. The application according to claim 1 or 2, wherein, The drug is in the form of an nebulizer.

9. The application according to claim 1 or 2, wherein, The drug is a unit dose, wherein the unit dose contains 25 mg to 200 mg of a nitrogen-containing saturated heterocyclic compound of Formula I or a pharmaceutically acceptable salt thereof.

10. The application according to claim 9, wherein, The unit dose contains 25 mg, 50 mg, 100 mg, 150 mg or 200 mg of a nitrogen-containing saturated heterocyclic compound of Formula I or a pharmaceutically acceptable salt thereof.

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