Use of an ester group-containing aromatic propanamide compound and its metabolite in the preparation of a drug for treating heart failure

Abstract

Use of an ester group-containing aromatic propanamide compound and its metabolite in the preparation of a drug for treating heart failure, wherein the ester group-containing aromatic propanamide compound is C<subgt;25< / subgt;H<subgt;17< / subgt;F<subgt;3< / subgt>N<subgt;4< / subgt>O<subgt;4< / subgt>. The ester group-containing aromatic propanamide compound can improve the reduction of ejection fraction, fractional shortening and cardiac output in rats with chronic heart failure, and increase the LVSP of heart failure rats.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of an ester group-containing aromatic propanamide compound and its metabolite in the preparation of a drug for treating heart failure. Background Art

[0002] Heart failure, abbreviated as HF, refers to a syndrome in which the heart's systolic function and / or diastolic function are impaired, and the venous return blood volume cannot be fully discharged from the heart, resulting in blood stasis in the venous system and insufficient blood perfusion in the arterial system, thereby causing a cardiac circulatory disorder syndrome. This disorder syndrome is mainly manifested as pulmonary congestion and vena cava congestion. The occurrence of heart failure is generally due to the apoptosis of cardiomyocytes after myocardial infarction, resulting in the progressive loss of effective contractile functional units - cardiomyocytes. The compensatory function of surviving cardiomyocytes is enhanced, the cardiomyocytes adaptively hypertrophy, the deposition of extracellular matrix and reactive interstitial fibrosis occur, and the left ventricular wall thickness decreases. As the degree of hypertrophy progresses, the increase in cardiomyocyte apoptosis further reduces the number of cardiomyocytes, and the overall myocardial contractility decreases. At the same time, the apoptotic cardiomyocytes after myocardial infarction will release a large number of factors to promote collagen production to repair the injured tissue. The overexpressed collagen deposition is further manifested as myocardial fibrosis, resulting in abnormal cardiac systolic and diastolic functions. At the same time, this repair effect will extend to the area around the infarct zone. The increase in fibrosis also reduces the compliance of the left ventricle, and the myocardial contractility cannot exert its due ejection effect, thus forming a vicious cycle, resulting in myocardial decompensation and ultimately leading to heart failure. Summary of the Invention

[0003] In view of the above-mentioned defects in the prior art, the purpose of the present invention is to provide the application of an ester group-containing aromatic propanamide compound and its metabolite in the preparation of a drug for treating heart failure.

[0004] The purpose of the present invention will be achieved through the following technical solutions:

[0005] The application of an ester group-containing aromatic propanamide compound in the preparation of a drug for treating heart failure.

[0006] Preferably, the ester group-containing aromatic propanamide compound is C 25 H 17 F3N4O4.

[0007] Preferably, the dosage of the ester group-containing aromatic propanamide compound in the preparation of a drug for treating heart failure is 2 mg / kg - 5 mg / kg.

[0008] Preferably, the dosage of the ester group-containing aromatic propanamide compound in the preparation of a drug for treating heart failure is 3 mg / kg.

[0009] The outstanding effect of the present invention is that the ester group-containing aromatic propanamide compounds can improve the reduction of ejection fraction, fractional shortening, and cardiac output in rats with chronic heart failure, and increase the LVSP in heart failure rats. It can play a very positive role in the application of preparing drugs for treating heart failure.

[0010] The following will further elaborate on the specific implementation manners of the present invention in conjunction with the accompanying drawings of the embodiments, so that the technical solutions of the present invention are easier to understand and master. Description of the Drawings

[0011] Figure 1 : Bar chart of the change in body weight gain of rats with different compounds in the present invention.

[0012] Figure 2 : Bar chart of the heart weight index of rats with different compounds in the present invention.

[0013] Figure 3 : Bar chart of the brain-heart weight index of rats with different compounds in the present invention.

[0014] Figure 4 : Bar chart of the effect of different compounds in the present invention on the LVEF of heart failure rats.

[0015] Figure 5 : Bar chart of the effect of different compounds in the present invention on the FS of heart failure rats.

[0016] ] Figure 6 : Bar chart of the effect of different compounds in the present invention on the SV of heart failure rats.

[0017] Figure 7 : Bar chart of the effect of different compounds in the present invention on the CO of heart failure rats.

[0018] Figure 8 : Bar chart of the effect of different compounds in the present invention on the ESV of heart failure rats.

[0019] Figure 9 : Bar chart of the effect of different compounds in the present invention on the EDV of heart failure rats.

[0020] Figure 10 : Bar chart of the effect of different compounds in the present invention on the LVPWTs of heart failure rats.

[0021] Figure 11 : Bar chart of the effect of different compounds in the present invention on the LV mass of heart failure rats.

[0022] [[ID=5,2]] Figure 12 : Bar chart of the effect of different compounds in the present invention on the heart rate of heart failure rats.

[0023] Figure 13: Bar graph showing the effects of different compounds in the present invention on the systolic blood pressure (SBP) of heart failure rats.

[0024] Figure 14 : Bar graph showing the effects of different compounds in the present invention on the diastolic blood pressure (DBP) of heart failure rats.

[0025] Figure 15 : Bar graph showing the effects of different compounds in the present invention on the mean blood pressure (MBP) of heart failure rats.

[0026] Figure 16 : Bar graph showing the effects of different compounds in the present invention on the left ventricular systolic pressure (LVSP) of heart failure rats. Detailed implementation manners

[0027] The present invention discloses the application of an ester group-containing aromatic propanamide compound in the preparation of a drug for treating heart failure. The following specific experiments are used to confirm the application of the present invention. EG017 mentioned in the text is C 25 H 17 F3N4O4. The preparation method and structural formula of the EGO17 refer to the content recorded in the patent document with the patent number CN201410033958.0. In all the drawings of the present invention, the bar order from left to right is arranged in the Sham, Model, and EG017 groups in sequence.

[0028] Instruments used in the experiments of the present invention

[0029] Small animal ventilator, model: V-200046; manufacturer: Harvard Apparatus.

[0030] Vevo small animal ultrasonic imaging system, model: 1100, manufacturer: Visualsonics.

[0031] Powerlab 8 / 35 signal acquisition system, model: 8 / 35, manufacturer: ADinstrunents.

[0032] Animal experiment treadmill, model: DB030, manufacturer: Beijing Zhishu Biotechnology Co., Ltd.

[0033] Analytical balance, model: SQP, manufacturer: sartorius.

[0034] Electronic balance, model: FA-2204, manufacturer: Bangxi Instrument Technology (Shanghai) Co., Ltd.

[0035] Electronic balance, model: MP5002, manufacturer: Changzhou Tianzhiping Instrument Equipment Co., Ltd.

[0036] Centrifuge, model: 5424R, manufacturer: Eppendorf.

[0037] Thermostatic magnetic stirrer, model: 85-2, manufacturer: Shanghai Sile Instrument Co., Ltd.

[0038] Micro vortex mixer, model: XW-80A, manufacturer: Shanghai Huxi Analytical Instrument Co., Ltd.

[0039] The test compounds used in the experiments of the present invention are as follows:

[0040] Test compound Production batch number Supplier Storage conditions EG017 Q18-078 MedChemExpress (Shanghai) Co., Ltd. Keep in a sealed container at room temperature

[0041] Reagents:

[0042] CMC Na, manufacturer: Sigma, Lot#: SLBV9664, storage condition: room temperature.

[0043] Isoflurane, manufacturer: Jiangsu Hengfengqiang Biotechnology Co., Ltd., batch number: 20191223, storage condition: sealed at room temperature.

[0044] Pentobarbital sodium injection, manufacturer: AlfaMedic Ltd, batch number: 1709296-02, physical state: pink liquid, 0.2 g / ml, expiration date: September 2020, storage condition: sealed at room temperature.

[0045] Meloxicam injection, manufacturer: Qilu Animal Health Products Co., Ltd., batch number: 1710002, storage condition: sealed at room temperature.

[0046] Gentamicin sulfate injection, manufacturer: Huazhong Pharmaceutical Co., Ltd., batch number: 20180622 storage condition: protected from light at room temperature.

[0047] Normal saline, manufacturer: Chenxin Pharmaceutical Co., Ltd., batch number: 18101907, storage condition: sealed at room temperature.

[0048] Solvent and compound preparation

[0049] 1. Solvent (0.5% CMC Na): Preparation: Weigh 5 g of CMC Na, make up the volume to 1000 ml with ddwater, and stir with a stirrer until dissolved.

[0050] 2. EG017: Preparation: Weigh an appropriate amount of the sample, add the prescribed amount of 0.5% CMCNa, and prepare solutions of 1 mg / kg, 3 mg / kg, and 10 mg / kg, and stir with a stirrer until dissolved.

[0051] Drug administration plan: One week after animal model establishment, the sham operation / myocardial infarction rats were grouped and administered orally once a day for 28 consecutive days, and the administration volume was 10 ml / kg.

[0052] Use of experimental animals

[0053] Sprague Dawley rats (SD rats), provided by Vital River Laboratory Animal Technology Co., Ltd., female, 120 rats, license number: SCXK (Jing) 2016-0006, experimental animal certificate number: 1100112011006510. Female, 50 rats, license number: SCXK (Zhe) 2019-0001, experimental animal certificate number: 2003050021.

[0054] Feeding of animals

[0055] All animals used in the following experiments were SD rats. After the animals arrived at the facilities of Shanghai Wuxi AppTec New Drug Development Co., Ltd., they were housed in an animal breeding room with strictly controlled environmental conditions. The temperature in the breeding room was maintained at 20-24°C, and the humidity was maintained at 30-70%. The temperature and humidity in the breeding room were monitored in real time by a thermometer and hygrometer, and the temperature and humidity were recorded twice a day (once in the morning and once in the afternoon). The lighting in the animal breeding room was controlled by an electronic timing light system, with the light on for 12 hours and off for 12 hours every day (on at 6:00 AM and off at 6:00 PM). The animals had free access to feed and were ensured free drinking water.

[0056] Specific experimental methods

[0057] After the animals were acclimated, on the day of the experiment, the animals were anesthetized by intraperitoneal injection of sodium pentobarbital injection (60 mpk), and atropine (0.5 mpk) was intraperitoneally injected to relieve phlegm, and then the trachea was connected to a ventilator for assisted respiration; thoracotomy was performed between the third and fourth ribs, and the chest was spread with a rib retractor; the pericardium was torn open, and the left anterior descending coronary artery was ligated with 5-0 silk thread, and the ribs and skin were sutured, and the animals were placed in a warming blanket for recovery after the operation. The sham operation group (Sham group) also underwent the same surgical procedure, except that the silk thread ligation was not performed. After the operation, all animals were intramuscularly injected with meloxicam (1 mpk) and gentamicin injection (8 mpk) for analgesia and anti-infection. One day before drug administration, the rats were anesthetized by inhaling isoflurane (1.5%-5% v / v in oxygen) and then underwent echocardiography. There was no obvious contraction of the anterior wall of the left ventricle, the left ventricular cavity became larger, and the left ventricular ejection fraction decreased by 30% compared with the normal control group, indicating successful ligation. The rats with successful models were selected for subsequent experiments (Note: Ensure that there are 7 rats in each group of animals after modeling, and 7 rats in the sham operation group). Each group of rats was given the treatment drug once a day by gavage for four consecutive weeks. During the experiment, the living status of the animals was observed, and abnormal conditions were recorded; endpoint index detection and sample collection were performed one day after the last drug administration.

[0058] One week after animal model establishment, rats were anesthetized with isoflurane, and the left ventricular function of the model rats was examined using a Vevo small animal ultrasound imaging system. A 30% reduction in LVFE% was defined as a successful model.

[0059] Except for the Sham group, the rats were divided into 9 groups according to LVEF% and body weight, with 7 rats in each group. The details of each group are as follows in the table:

[0060]

[0061] Endpoint detection indicators

[0062] A. Echocardiogram examination (one day after the last administration): The animals were anesthetized by inhaling isoflurane (1.5%-5% v / v in oxygen), and the left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular end-systolic volume (ESV), left ventricular end-diastolic volume (EDV), stroke volume (SV), cardiac output (CO), left ventricular mass (LV mass), left ventricular posterior wall end-systolic thickness (LVPWTs), and left ventricular posterior wall end-diastolic thickness (LVPWTd) were measured.

[0063] B. Hemodynamics (one day after the last administration): The rats were anesthetized with sodium pentobarbital (~60 mpk, ip), fixed in the supine position on the operating table, the common carotid artery was isolated, and a catheter was inserted to measure the rat carotid artery blood pressure (SP / DP) through the Powerlab system. Then the catheter was advanced into the left ventricle to measure the left ventricular systolic / diastolic pressure (LVSP / LVDP); the maximum rate of rise / fall of the left ventricular pressure (±dp / dtmax) and heart rate (HR). All procedures were completed within 30 minutes after anesthesia.

[0064] C. Organ weight index: Heart weight / 100 g body weight; Heart-brain ratio: Heart weight / brain weight.

[0065] D. Myocardial staining: Sirius red staining was performed on the myocardium to observe the level of myocardial fibrosis.

[0066] Data statistics

[0067] The data were expressed as Mean±S.E.M, and statistical plots were made using Graphpad Prism 5.0. One-way ANOVA Dunntt's test and t-test were used. A P value < 0.05 indicated a statistically significant difference.

[0068] Preliminary observation

[0069] Studies have shown that the weight of the test group given EG017 alone increased significantly. At the end-point autopsy, the hearts of the Sham group were ruddy and plump, while the left ventricles of the model rats were shriveled, and the infarcted area was grayish-white.

[0070] Effect of the compound on the cardiac weight index of heart failure rats

[0071] At the end-point of the rats, the hearts were collected by autopsy, washed with normal saline, weighed after absorbing the moisture, and the cardiac weight index was calculated according to the body weight. After weighing the brain, the brain-heart weight ratio was calculated. From Figure 2 - Figure 3 As can be seen from Table 1, compared with the Sham group, the cardiac weight index and brain-heart weight ratio of the Model group were significantly increased. There was no significant statistical difference in the cardiac weight index and brain-heart weight ratio of the test group rats compared with the Model group (P>0.05).

[0072] Table 1 Cardiac weight index and brain-heart weight ratio of rats (Mean±SEM) N = 7

[0073]

[0074] ΔP < 0.05 vs sham, &P < 0.05 vs model, by t-test; #P < 0.05 vs sham, by one-way ANOVA Dunnett’s test.

[0075] Effect of the compound on the cardiac function of rats

[0076] Effect of the compound on the cardiac systolic function of rats

[0077] An important manifestation of heart failure is the reduction of left ventricular systolic function. Therefore, at the end-point of the experiment, the hearts of rats in each group were examined by longitudinal echocardiography, and the changes in left ventricular ejection fraction (LVEF), fractional shortening (FS), cardiac output (CO) and stroke volume (SV) were analyzed and compared. From Figure 4 - 7 As can be seen from Table 2, after myocardial infarction, the rats in the Model group showed severe left ventricular dysfunction. The LVEF, FS, CO and SV were 36.3±3.83%, 18.5±2.22%, 55.7±4.90 ml / min and 159±8.62 μl respectively, which were significantly lower than those of the Sham group (84.8±2.83%, 56.1±3.14%, 77.2±5.10 ml / min and 187±9.46 μl), and the statistical differences were significant (P<0.001, P<0.001, P<0.05, P<0.05). Compared with the Model group, the EG017 group had a significant improvement in the reduction of LVEF and FS in heart failure rats, and the statistical difference was significant (P<0.05). The EG017 group had a significant improvement in the reduction of CO and SV (P<0.05).

[0078] Table 2 Effects of compounds on LVEF, FS, CO and SV in heart failure rats (Mean±SEM) N = 7

[0079]

[0080] *** # < 0.001 vs sham, *P < 0.05, ** P < 0.01 vs Model, by one-way ANOVA Dunnett’s test; ΔP < 0.05 vs sham, &P < 0.05, &&P < 0.01 vs Model by t-test.

[0081] Effects of compounds on left ventricular volume in rats

[0082] After heart failure develops, due to compensatory effects, the left ventricular cavity becomes enlarged and the left ventricular posterior wall becomes thinner. As can be seen from Figure 8 - Figure 9 and Table 3, compared with the Sham group, the EDV and ESV of the hearts of heart failure rats were significantly increased, with extremely significant statistical differences (P < 0.001). Compared with the Model group, the EG017 group had a significant improvement in the increase in the systolic volume (ESV) of the heart caused by heart failure (P < 0.05).

[0083] Table 3 Effects of compounds on left ventricular volume in systole and diastole in heart failure rats (Mean±SEM) N = 7

[0084] Group dose ESV (ul) EDV (ul) Sham Vehicle 36.0±9.05 223±16.1 Model Vehicle 294±32.6### 453±28.5### EG017 3mpk 238±52.0 477±48.2

[0085] P < 0.001 vs Sham, by one-way ANOVA Dunnett’s test; &P < 0.05 vs model, by t-test.

[0086] Effects of compounds on left ventricular posterior wall thickness and heart mass in rats

[0087] As can be seen from Figure 10 - Figure 11 and Table 4, compared with the Sham group, the LVPWTs and LVPWTd of the rats in the Model group were slightly decreased (P > 0.05), while the heart mass was significantly increased, with significant statistical differences (P < 0.05). Compared with the Model group, the effects of each drug administration group on LVPWTs, LVPWTd and LVmass were not obvious (P > 0.05).

[0088] Table 4 Effects of compounds on left ventricular posterior wall thickness and left ventricular mass in heart failure rats (Mean±SEM) N = 7

[0089]

[0090] &P < 0.05 vs model by t - test.

[0091] Effect of the compound on hemodynamics in heart - failure rats

[0092] After myocardial infarction in rats, left ventricular myocardial infarction occurred, resulting in a decrease in left ventricular systolic function, manifested as a decrease in ventricular systolic pressure and a decrease in the rate of rise / fall of intra - ventricular pressure (±dp / dtmax).

[0093] From Figure 12 - Figure 15 , and Table 5, it can be seen that there was no significant effect on the heart rate in each experimental group (P > 0.05). The arterial blood pressure (SBP, DBP, MBP) in the Model group was significantly lower compared with that in the Sham group, with significant statistical differences (P < 0.001, P < 0.05, P < 0.01). Compared with the Model group, the EG017 group had no significant effect on the arterial blood pressure in rats after heart failure.

[0094] Table 5 Effect of the compound on heart rate and arterial blood pressure in heart - failure rats (Mean ± SEM) N = 7

[0095]

[0096] #P < 0.05, ##P < 0.01, P < 0.001 vs sham, *P < 0.05 vs model, by one - way ANOVA Dunnett′s test; &P < 0.05 vs model, by t - test.

[0097] Effect of the compound on ventricular pressure and the rate of change of ventricular pressure

[0098] As shown in Figure 16 and Table 6, compared with the Sham group, the left ventricular systolic pressure (LVSP) in the Model group of rats decreased significantly, with significant statistical differences (P < 0.01). Compared with the Model group, EG017 had a significant alleviating effect on the decrease in LVSP caused by heart failure, with significant statistical differences (P < 0.05).

[0099] Compared with the Sham group, the +dp / dt max of rats in the Model group was 5164 ± 352 mmHg / s and the - dp / dt max was - 3789 ± 220 mmHg / s, which were significantly lower than 6210 ± 429 mmHg / s and 5091 ± 456 mmHg / s in the Sham group, with significant statistical differences (P < 0.05, P < 0.01).

[0100] Table 6 Effects of compounds on left ventricular blood pressure in rats with heart failure (Mean±SEM) N=7

[0101]

[0102] ##P<0.01, P<0.001 vs sham, *P<0.05 vs model, by one-way ANOVA Dunnett′s test; &P<0.05, &&P<0.01 vs model, by t-test.

[0103] The following examples are used to prove that the metabolites of EG017 have a positive promoting effect in the preparation of drugs for the treatment of heart failure.

[0104] 2.1. Test articles and solvents

[0105] 2.1.1. Test articles

[0106] Name: EG017; Physical state: Off-white powder;

[0107] Main component: (S)-1-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-(4-cyanophenoxy)-2-methyl-1-oxopropan-2-yl nicotinate; Content: 99.3%;

[0108] 2.1.2. Solvents

[0109] 2.1.2.1. CMC-Na

[0110] Name / code: Sodium carboxymethyl cellulose / CMC-Na; Character: White or slightly yellow fibrous powder;

[0111] 2.1.2.2. DMA

[0112] Name / code: N,N-Dimethylacetamide / DMA

[0113] Character: Colorless or nearly colorless clear liquid;

[0114] 2.1.2.3. solutol

[0115] Name / code: Polyethylene glycol 15-hydroxystearate / solutol; Character: Light yellow paste at room temperature, becomes liquid at about 30 °C. Dissolves in

[0116] water or ethanol to form a clear solution;

[0117] Storage condition: 2 - 8 °C;

[0118] 2.1.2.4. Sodium chloride injection

[0119] Name: Sodium Chloride Injection;

[0120] Batch number: 1804172726; Appearance: Colorless transparent liquid;

[0121] Preparation of Test Sample

[0122] Suspension of Test Sample

[0123] Take a certain amount of CMC-Na (allowing a weighing error of ±1%), dissolve it in pure water to prepare 0.5% CMC-Na. Preparation method of the suspension of test sample: Weigh a certain amount of EG017 according to the following table, place it in a mortar, add a small amount of 0.5% CMC-Na and grind it thoroughly, transfer it to a container, then wash the mortar 4 times, transfer the washing liquid to the container, and dilute it with 0.5% CMC-Na to the required volume to prepare the suspension of test sample with the required concentration. Stir with a magnetic stirrer at a speed of 1200 rpm for at least 15 min before analysis sampling and administration.

[0124] Thirty-two Beagle dogs were used in the experiment, randomly divided into 4 groups with 8 dogs in each group, half male and half female, and were respectively set as the intravenous injection group (1 mg / kg) and the kinetic low (1 mg / kg), medium (3 mg / kg), and high (10 mg / kg) dose groups by gavage. Blood samples were collected at the corresponding time points after administration, anticoagulated with EDTA-K2. After the animals in the medium dose group completed sample collection, they were used for continuous gavage administration for 7 days and blood samples were collected at the corresponding time points. The concentrations of EG017 and its main metabolite EG-2 in plasma were detected by LC-MS / MS method, and pharmacokinetic parameters were calculated using WinNonlin 6.4 software.

[0125] There are still various implementation manners of the present invention. All technical solutions formed by using equivalent transformation or equivalent substitution fall within the protection scope of the present invention.

Claims

1. Use of an ester group-containing aromatic propanamide compound in the preparation of an oral drug for treating heart failure, characterized in that, The chemical formula of the ester group-containing aromatic propanamide compound is C 25 H 17 F3N4O4, and the ester group-containing aromatic propanamide compound is (S)-1-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-(4-cyanophenoxy)-2-methyl-1-oxopropan-2-yl nicotinate.

2. The application according to claim 1, wherein: The dosage of the ester group-containing aromatic propanamide compound in the preparation of an oral drug for treating heart failure is 1 mg / kg - 10 mg / kg.

3. The application according to claim 1 or 2, characterized in that: The dosage of the ester group-containing aromatic propanamide compound in the preparation of an oral drug for treating heart failure is 3 mg / kg.

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