Compounds for the treatment of cardiac hypertrophy diseases

Abstract

The present application relates to a kind of compound for treating myocardial hypertrophy disease, it is related to the field of biological medicine.The compound can effectively improve the left ventricular systolic function of myocardial hypertrophy mouse, reduce heart weight / body weight, lung weight / body weight, myocardial cell cross-sectional area and fibrosis;Meanwhile, the compound can relieve angiotensin II induced myocardial hypertrophy in vitro.Especially, the compound can improve the damage and dysfunction of mitochondria in myocardial tissue of myocardial hypertrophy mouse, while improving the oxidative phosphorylation level of myocardial cell after angiotensin II stimulation, has the potential of preparation treatment myocardial mitochondrial damage, inhibits myocardial cell hypertrophy, treatment myocardial hypertrophy and heart failure drug.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a compound for treating hypertrophic cardiomyopathy. Background Technology

[0002] Heart failure (HF) is an end-stage cardiovascular disease caused by various etiologies and is the leading cause of death. Clinically, it is mainly manifested as reduced left ventricular pump function, insufficient peripheral tissue perfusion, and decreased exercise tolerance. In particular, increased cardiac afterload due to aortic coarctation and hypertension is a significant contributing factor to cardiac structural remodeling changes such as myocardial hypertrophy and myocardial fibrosis, ultimately leading to chronic heart failure. For many years, scholars from various countries have made tireless efforts in treating myocardial remodeling and heart failure, achieving some progress. However, overall efficacy remains unsatisfactory, with no fundamental improvement observed. Treatment options remain limited, and patient prognosis remains poor. Cardiac dysfunction caused by systemic diseases such as hypertension and diabetes, as well as myocardial tissue lesions such as dilated cardiomyopathy, hypertrophic cardiomyopathy, and mitochondrial cardiomyopathy, all exhibit varying degrees of ventricular wall thickening and cardiomyocyte hypertrophy. Currently, drug treatment strategies targeting myocardial hypertrophy are not very effective. Summary of the Invention

[0003] The purpose of this invention is to provide a compound for treating myocardial hypertrophy, which has good therapeutic effects on stress overload-induced myocardial hypertrophy and heart failure.

[0004] The technical solution provided by this invention is as follows:

[0005] In a first aspect, the present invention provides a compound of formula (I) (hereinafter referred to as SRK3IN-1) and its pharmaceutically acceptable derivatives:

[0006]

[0007] This compound can enhance the function of mitochondria in cells, inhibit mitochondrial damage, improve the oxidative phosphorylation function of cardiomyocytes, effectively inhibit cardiomyocyte hypertrophy, improve energy metabolism during cell and myocardial tissue repair, significantly improve cardiac function in stress overload model mice, and has a good preventive effect on stress overload-induced myocardial hypertrophy and heart failure.

[0008] Based on the above compounds, the present invention also provides a pharmaceutical composition comprising the compound represented by formula (I) and its pharmaceutically acceptable derivatives, pharmaceutically acceptable salts, solvates, optical isomers or polymorphs and pharmaceutically acceptable carriers.

[0009] Based on the above technical solution, the carrier is one or more of the following: diluent, adjuvant, excipient and formulation medium.

[0010] The present invention has found that the above compounds can inhibit the expression of SRPK3. Based on this, the present invention provides the use of the compound of formula (I) and its pharmaceutically acceptable derivatives, pharmaceutically acceptable salts, solvates, optical isomers or polymorphs in the preparation of a drug for inhibiting the expression of SRPK3.

[0011] Based on the medicinal functions of the above compounds in treating and / or preventing diseases related to myocardial hypertrophy, the present invention provides the use of the compound of formula (I) and its pharmaceutically acceptable derivatives, pharmaceutically acceptable salts, solvates, optical isomers or polymorphs as active ingredients in the preparation of medicaments for treating and / or preventing diseases related to myocardial hypertrophy.

[0012] Based on the above technical solutions, the drug provided by the present invention for treating and / or preventing diseases related to myocardial hypertrophy targets SRPK3 and inhibits the production of SRPK3 protein.

[0013] Based on the above technical solutions, the drug dosage forms provided by the present invention for treating and / or preventing diseases related to myocardial hypertrophy are capsules, tablets, oral preparations, microcapsule preparations, injections, suppositories, sprays, or ointments.

[0014] Based on the above technical solutions, the drug provided by the present invention for treating and / or preventing diseases related to myocardial hypertrophy is a drug for preventing mitochondrial damage.

[0015] Based on the above technical solution, the diseases related to myocardial hypertrophy are selected from: hypertension, arrhythmia, sudden cardiac death, infective endocarditis, and heart failure.

[0016] Based on the above technical solution, the disease related to myocardial hypertrophy is hypertension.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a compound that can inhibit SRPK3 expression and, for the first time, applies it to the preparation of products for the treatment of myocardial hypertrophy and heart failure. This compound can enhance the function of mitochondria in cells, inhibit mitochondrial damage, and improve the oxidative phosphorylation function of cardiomyocytes, thereby effectively inhibiting cardiomyocyte hypertrophy, improving energy metabolism during cell and myocardial tissue repair, effectively improving mitochondrial function decline caused by various reasons and promoting cell and tissue repair. It can significantly improve cardiac function in stress overload model mice and has the potential to treat myocardial mitochondrial damage, inhibit cardiomyocyte hypertrophy, and treat myocardial hypertrophy and heart failure. Attached Figure Description

[0019] Figure 1 Figure A: Structural formula of SRK3IN-1; Figure B: IC50 values ​​of SRK3IN-1 and MSC-1186 (final concentration 0.01 nM to 100 μM) against various protein kinases, with SRK3IN-1 pre-stimulated before ATP addition; Figure C: SRK3IN-1 inhibits RPS3-p(S149) phosphorylation. HEK293T cells stably expressing Flag-SRPK3, HA-SRPK1, or Myc-SRPK2 were treated with the specified concentration of SRK3IN-1 for 1 hour, lysed, and immunoblotted with the specified antibody.

[0020] Figure 2 Protein kinase profiles were detected using 500 nM SRK3IN-1. https: / / www.medchemexpress.cn / ).

[0021] Figure 3 Figure A: Composite thumbnail showing the crystal structure of SRK3IN-1, with a grid outline; Figure B: Molecular splicing of human SRPK3 protein and SRK3IN-1 in a 3D image; Figure C: Total protein extracted from mouse hearts 48 hours after SRK3IN-1 (10 mg / kg / d) intervention, and immunoblotting detection of SRPK3 protein levels and quantitative analysis results (p-values ​​are shown in the figure).

[0022] Figure 4 Figure A: Flowchart of SRK3IN-1 treatment of Ang II-stimulated neonatal rat cardiomyocytes; Figure B: Protein collection and immunoblotting detection of SRPK3 protein expression level after SRK3IN-1 treatment of neonatal rat cardiomyocytes for 48 hours.

[0023] Figure 5 Figure A: SRK3IN-1 treatment of Ang II-stimulated neonatal rat cardiomyocytes, protein collection, and immunoblotting detection of BNP, β-MHC and PGC1α protein levels, with β-Tubulin as an internal control; Figure B: Protein quantification results in Figure A (p-values ​​are shown in the figure).

[0024] Figure 6 Figure A: SRK3IN-1 treated Ang II-stimulated neonatal rat cardiomyocytes undergoing α-Actinin immunofluorescence staining; Figure B: Image J software quantitative analysis of cardiomyocyte area (n=40-80), violin plot showing the cardiomyocyte area distribution results of each group (p-value shown in the figure).

[0025] Figure 7Figure A: Mitochondrial oxidative respiration rate curves of SRK3IN-1-treated Ang II-stimulated neonatal rat cardiomyocytes detected by Seahorse. Oligo represents oligomycin, FCCP is the mitochondrial oxidative phosphorylation uncoupling agent, and Antimycin A / R represents anti-enzyme A / R. Figure B: Quantitative results of basal respiration, ATP production, maximum oxygen consumption, and respiratory potential (p-values ​​are shown in the figure).

[0026] Figure 8 Figure A: Mouse cardiac function was assessed by echocardiography weekly before and after aortic coarctation (TAC). SRK3IN-1 (10 mg / kg / d) was administered intraperitoneally for two consecutive weeks starting in week 2. Mouse hearts were harvested four weeks post-surgery. Figure B: M-mode echocardiograms of mice in each group four weeks post-TAC (two weeks of SRK3IN-1 intervention). Figure C: Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd) before and for four consecutive weeks post-TAC.

[0027] Figure 9 Figure A: Gross images of the hearts of mice in each group 4 weeks after aortic coarctation (TAC); Figure B: Heart weight (HW) to tibia length (TL) ratio; Figure C: WGA staining of heart tissue in mice 4 weeks after sham surgery or TAC modeling; Figure D: Statistical quantitative results of cardiomyocyte cross-sectional area (p-values ​​are shown in the figure).

[0028] Figure 10 Figure A: Masson staining images of paraffin sections of mouse hearts 4 weeks after aortic coarctation (TAC); Figure B: Statistical quantitative results of collagen deposition in the left ventricle of mice; Figure C: Transmission electron micrographs of left ventricular tissue in mice of each group, with magnified portions mainly showing mitochondrial morphology; Figure D: Statistical quantitative graphs showing the number of mitochondria, the proportion of mitochondrial regions, and the proportion of mitochondria with cristae damage under electron microscopy, from left to right (p-values ​​are shown in the figure).

[0029] Figure 11 The chemical equation for the preparation of SRK3IN-1. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0031] Unless otherwise specified, this invention uses triethylamine-containing dichloromethane as the reaction solvent, compounds 1b, 2b-1, and 3b as raw materials, and a chiral ligand (S)-TF-BiphamPhos / AgOAc as a catalyst to synthesize compound 4b, namely compound SRK3IN-1 as shown in formula (I). See details... Figure 11The chemical equation for the preparation of SRK3IN-1 is shown.

[0032] In the following examples, this invention compared the effects of SRK3IN-1 and the pan-inhibitor of the SRPKs family, MSC-1186, on SRPK3 expression levels and kinase activity. Through IC50 assays and Western blotting in HeLa cells treated with both, it was found that SRK3IN-1 inhibited SRPK3 expression more strongly than MSC-1186, but had little effect on the expression of SRPK1 and SRPK2. Furthermore, SRK3IN-1 effectively inhibited the phosphorylation modification of RPS3 by SRPK3. Simultaneously, protein kinase profile analysis of 500 nmol / L SRK3IN-1 showed that SRK3IN-1's inhibitory effect on SRPK3 was significantly superior to other protein kinases.

[0033] This invention also used C57BL / 6J mice (8 to 10 weeks old, weighing 24.5 ± 1.0 g) as experimental subjects to study the function of SRK3IN-1 in an animal model of myocardial hypertrophy induced by transverse aortic constriction (TAC). The results showed that, compared with the placebo group, the SRK3IN-1 treatment group significantly improved cardiac contractile function in the mouse model of myocardial hypertrophy. Simultaneously, SRK3IN-1 treatment reduced heart weight / body weight, lung weight / body weight, cardiomyocyte cross-sectional area, and fibrosis level in the mice. Furthermore, transmission electron microscopy images showed that SRK3IN-1 could alleviate mitochondrial damage in the left ventricular myocardial tissue. These results indicate that inhibiting SRPK3 with SRK3IN-1 can alleviate the disease progression of pathological myocardial hypertrophy.

[0034] This invention also utilizes isolated neonatal rat cardiomyocytes to induce an in vitro cardiomyocyte hypertrophy model using angiotensin II. Results showed that, compared to the phosphate-buffered saline (PBS) treatment group, the SRK3IN-1 intervention group (1.0 μmol / L) exhibited significantly increased cardiomyocyte hypertrophy, with markedly decreased levels of hypertrophy markers such as BNP and β-MHC, and significantly increased levels of the mitochondrial biosynthesis-related factor PGC1α. Furthermore, mitochondrial oxidative respiration rate (OCR) curves revealed that, compared to the Ang II-only treatment group, the SRK3IN-1 treatment group significantly increased cardiomyocyte oxidative phosphorylation and ATP production.

[0035] This invention validates, through mouse models of myocardial hypertrophy and heart failure induced by stress overload and an in vitro model of angiotensin II-induced cardiomyocyte hypertrophy, that SRK3IN-1 intervention can reduce significant cardiomyocyte hypertrophy and alleviate the disease progression of pathological myocardial hypertrophy. Therefore, SRK3IN-1 and its pharmaceutically acceptable derivatives, pharmaceutically acceptable salts, solvates, optical isomers, or polymorphs can be used as active ingredients to prepare drugs for the treatment and / or prevention of diseases related to myocardial hypertrophy or drugs for enhancing mitochondrial function.

[0036] When using the above drugs, the dosage of SRK3IN-1 is 1-20 mg / kg, preferably 10 mg / kg, and the dosage is measured in the form of the above chemical formula.

[0037] Laboratory animals and their feeding

[0038] Experimental animals: 8-10 week old C57BL / 6J mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) weighing 24.5±1.0g were selected as experimental subjects.

[0039] Housing environment: All experimental mice were housed in the Specific Pathogen Free (SPF) Laboratory Animal Center of the Institute of Cardiovascular Diseases, Wuhan University. Housing conditions: room temperature between 22-24℃, humidity between 50-70%, alternating light and dark lighting for 12 hours, and free access to water and food.

[0040] Example 1: Verification of the inhibitory effect of SRK3IN-1 on SRPK3

[0041] (1) Cell culture: HEK 293T cells were purchased from the American Type Culture Collection (ATCC, USA) and cultured in DMEM-F12 (Invitrogen, USA) medium containing 10%-15% fetal bovine serum (FBS (Gibco, USA)) at 37°C in a 5% CO2 incubator (Thermo Electron Corporation, USA).

[0042] (2)IC 50 Assay: The purified kinases were detected using the ADP-Glo ​​kinase assay system (Promega, Madison, WI). In vitro, the kinases at final concentrations between 0.01 nM and 100 μM were analyzed by IC50 assays with SRK3IN-1 and MSC-1186. 50Measurements were taken (SRK3IN-1 or MSC-1186 was added to the kinase reaction before ATP was mixed in), and these values ​​were expressed as a percentage of the DMSO control. In 5 μL final volumes of peptide sequences of KKISGRLSPIMTEQ (SEQ ID NO:1), KKKVSRSGLYRSPSMPENLNRPR (SEQ ID NO:2), and ARTKQTARKSTGGKAPRKQLA (SEQ ID NO:3), containing 50 mM Tris pH 7.5, 150 μM substrate peptide, 5 mM MgCl2, and 10–50 μM ATP (CLK1, CLK3, SRPK2, DYRK2, DYRK3 at 10 μM, CLK2, DYRK1A, SRPK1 at 25 μM, and CLK4, DYRK1B, SRPK3 at 50 μM), DYRKs, CLKs, CLKs, and SRPKs (substrate peptide diluted in 50 mM Tris-HCl pH 7.5 and 2 mM DTT), DYRKs, CLKs, and SRPKs are determined, and incubated at room temperature for 60–120 minutes.

[0043] (3) After stimulating HEK 293T cells with SRK3IN-1 at different concentration gradients for 48 hours, the expression and activity of protein kinases were detected by Western blotting: 1) Prepare a 12% SDS-polyacrylamide gel according to the gel preparation kit instructions; 2) Electrophoresis: Dilute the 5× electrophoresis buffer with double-distilled water to make 1× electrophoresis buffer. Perform stacking gel electrophoresis at a constant voltage of 75V for 20 minutes. After all samples are stacked to a horizontal line, switch to 10V stacking gel electrophoresis. 5V. Constant voltage separation gel until electrophoresis is complete; 3) Transfer: Place the appropriately sized PVDF membrane in a bowl containing methanol and soak for half a minute to activate it. Remove the gel after electrophoresis and place it in the transfer solution. Then assemble it in the following order: sponge pad - 3 layers of filter paper - gel - PVD membrane - 3 layers of filter paper. Place it in the transfer tank with the gel facing the negative electrode and the membrane facing the positive electrode. Place the transfer tank in an ice box and press ice packs on top to maintain low temperature transfer. Adjust the transfer current to 200mA and the time to 1.5h; 4) Sealing: After the transfer is complete, remove the PVDF membrane and place it in 5% BSA-TBS. 5) Block the PVDF membrane on a shaker at room temperature for 1 hour; 6) Prepare the corresponding primary antibody dilution solution (1:1000) using 3% BSA-TBST according to the instructions, cut the PVDF membrane according to different molecular weights, add the corresponding primary antibody and incubate overnight on a shaker at 4°C; 7) After the primary antibody incubation, wash the membrane 3 times with TBST for 5 minutes each time, add the secondary antibody (1:2000) diluted with TBST and the HRP-labeled internal control antibody, incubate for 1 hour, and after the secondary antibody incubation, wash the membrane 3 times with TBST for 5 minutes each time; 8) Prepare the developing solution in the dark for development, and use the Bio-Rad Laboratories chemiluminescence imaging system. After development, use Quantity One software for quantitative analysis.

[0044] Example 2: Effect of SRK3IN-1 on cardiomyocyte hypertrophy following angiotensin II (Ang II) stimulation

[0045] 1. Primary culture of neonatal SD rat cardiomyocytes

[0046] (1) Ten Sprague-Dawley suckling mice aged 1-3 days were disinfected below the neck with 75% alcohol. The heart was removed with ophthalmic scissors and microforceps and placed in a glass petri dish containing 10 mL of DMEM / F12 solution. The same process was repeated for another mouse.

[0047] (2) Wash the heart with DMEM / F12 medium and cut the heart into 1-2 mm pieces. 3 The fragments were transferred to a serum bottle containing a rotor, DMEM / F12 was removed, and trypsin digestion solution was added. The rotor speed was 120 r / min, and digestion was carried out for 15 min. After standing for a few seconds, the supernatant was discarded.

[0048] (3) Add trypsin digestion solution, rotate at 120 r / min, and digest for 15 min. Let stand for a few seconds, aspirate the supernatant, terminate digestion with DMEM / F12 medium containing 20% ​​fetal bovine serum, and store at 4°C. Repeat this step several times. When collecting the supernatant, try to collect as much as possible. When the tissue block turns white and becomes significantly smaller, stop digestion.

[0049] (4) Centrifuge the collected myocardial cell suspension at 1500 rpm for 8 min and discard the supernatant. Add an appropriate amount of culture medium to the centrifuge tube, gently pipette to resuspend the cells, and concentrate them into a 50 mL centrifuge tube. Filter the cell suspension through a 40 μm cell filter.

[0050] (5) Seed the cells in a 100 mm culture dish and allow them to adhere for 90 min. Then, filter the unadhered cell suspension. Add BrdU (final concentration 0.1 mM) to the total amount of cell suspension, mix well, and then add the mixture to a dish coated with 0.1% gelatin.

[0051] (6) Gently shake to disperse the cells, do not vortex. Incubate at 37°C and 5% CO2 for 48 hours, wash once with PBS, and change the culture medium.

[0052] 2. After isolating and culturing neonatal Sprague-Dawley rat cardiomyocytes (1-3 days old), primary neonatal rat cardiomyocytes were cultured for 48 hours, followed by medium change. Serum-free DMEM / F12 starved cardiomyocytes were added for 12 hours to synchronize the cells. The cells were then incubated with SRK3IN-1 (1.0 μM) or an equal volume of DMSO (dimethyl sulfoxide) for 48 hours, followed by stimulation with PBS or angiotensin II (Ang II, 1 μM) for 24 hours. Proteins were extracted from the cardiomyocytes and subjected to SDS-PAGE-Western blot assay. The inhibitory effect of SRK3IN-1 was detected using an antibody specifically recognizing SRPK3 protein. Simultaneously, the expression of cardiomyocyte hypertrophy markers BNP and β-MHC, as well as the mitochondrial production regulator PGC1α, was measured in each group, with β-Tubulin used as an internal control. The results are as follows: Figure 4 and Figure 5 As shown, SRK3IN-1 significantly inhibited SRPK3 levels in primary cardiomyocytes and alleviated cardiomyocyte hypertrophy following Ang II stimulation. Furthermore, immunofluorescence assays indicated that the surface area of ​​cardiomyocytes treated with SRK3IN-1 was significantly smaller than that in the Ang II-positive control group. Figure 6 ).

[0053] Example 3: Effects of SRK3IN-1 on cardiac function in mice with myocardial hypertrophy after in vivo application

[0054] 1. The mouse model of myocardial hypertrophy was established using aortic arch constriction surgery (TAC). The model operation procedure is as follows:

[0055] 1.1 Preoperative preparation

[0056] (1) Anesthesia: First, weigh the mice and calculate the required amount of anesthetic (3% sodium pentobarbital) based on 90 mg / kg body weight. Administer the anesthetic via intraperitoneal injection and record the injection time. Successful anesthesia is defined as no obvious reaction to tail or toe clamping and the mouse being in good condition (generally, there is no obvious reaction about 10 minutes after injection; the optimal time for surgery is about 30 minutes after anesthesia, with the mouse showing a toe clamping reaction approximately 50 minutes after anesthesia).

[0057] (2) Surgical area preparation: Remove hair from the skin of the left chest, left lateral chest, and left forelimb axilla of the mouse. After shaving, wipe the surgical area with a damp gauze to remove the mouse hair, so as not to affect the surgical field.

[0058] (3) Endotracheal intubation: Fix the upper incisors of the mouse to the inclined surface of the V-shaped plate with a rubber band, and quickly and accurately insert the endotracheal tube into the trachea through the glottis. Then place the mouse in a right lateral decubitus position on a heating pad (the heating pad needs to be preheated). Then connect the endotracheal tube to the ventilator and fix the mouse. If the rise and fall of the mouse's chest is consistent with the ventilator frequency, it indicates that the endotracheal intubation was successful.

[0059] 2. Transaortic coarctation (TAC)

[0060] Myocardial hypertrophy model group: The mice were placed in a right lateral decubitus position with their left forelimb placed above their right forelimb, and both forelimbs were secured with medical tape. A cotton swab was placed under the right chest to elevate the thoracic cavity. The surgical area was disinfected with iodine and 75% alcohol. Using ophthalmic forceps in the left hand, the skin of the left chest was lifted, and with ophthalmic scissors in the right hand, the skin was cut approximately 1 cm. The muscles and soft tissues were then separated sequentially. The thoracic cavity was opened at the level of the 2nd-3rd rib. The left lung was slightly opened with a cotton swab, and the descending branch of the aortic arch was freed. The surgical suture was passed through the blood vessel, and a 26G (25.0-27.5g mouse) or 27G (23.5-25.0g) syringe needle was placed parallel above the blood vessel. The blood vessel and needle were ligated together, and the needle was then withdrawn to achieve the corresponding degree of vascular constriction. After ligation, the sutures were closed, and the thoracic cavity was closed. A syringe was inserted into the thoracic cavity through the suture opening, and 1cc of air was withdrawn to restore negative pressure within the thoracic cavity. The syringe was then removed, and the skin incision was quickly sutured.

[0061] Sham surgery group: After the descending branch of the aorta was freed, only the suture was threaded but not ligated. The rest of the steps were the same as the hypertrophic myocardial model group.

[0062] 3. Postoperative care

[0063] After aortic coarctation surgery, once the mice exhibited spontaneous breathing and a strong toe-clamping response, the endotracheal tube was removed, and the mice were placed in a rearing cage containing autoclaved bedding, feed, and drinking water for continued rearing and observation in the rearing room.

[0064] 4. SRK3IN-1 in vivo mouse intervention: Two weeks after TAC surgery, mice were intraperitoneally injected with either SRK3IN-1 (SRK3IN-1 treatment group) or placebo (placebo group; in this experiment, the placebo was physiological saline) at a dose of 10 mg / kg / day (determined through pre-experiment and baseline blood drug concentration). Administration continued for two weeks, starting before TAC surgery. Echocardiography was used weekly to assess cardiac function in mice (see...). Figure 7 A) The echocardiogram examination is as follows.

[0065] 5. Echocardiography to detect cardiac function in mice

[0066] 5.1. Preliminary Preparations

[0067] (1) Preparation of the anesthesia machine: First connect the oxygen cylinder to the air inlet on the anesthesia machine, then unscrew the sealing cap of the drug delivery port on the anesthesia machine, quickly add isoflurane to the safe mark and then tighten the sealing cap. Unscrew the main valve on the oxygen cylinder, adjust the knob of the flow control valve, and maintain the outlet pressure at 0.2-0.3 MPa.

[0068] (2) Preparation of test mice: After the test mice are rapidly anesthetized with isoflurane, the hair on the left chest area is shaved, and the head of the prepared mouse is inserted into the anesthetic catheter. The mouse is kept under stable anesthesia with 1.5-2.0% isoflurane.

[0069] 5.2. Cardiac Function Testing

[0070] Mice were placed in a left lateral decubitus or supine position, and ultrasound coupling agent was evenly applied to the shaved area. A high-frequency ultrasound diagnostic instrument with a frequency of 15 MHz was used, and a standard left ventricular papillary muscle short-axis section was selected to measure the left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction, and short-axis shortening rate.

[0071] 5.3 Figure 8 The main findings are the cardiac function test results of mice after TAC surgery treated with SRK3IN-1 or placebo. Compared with the sham-operated group, mice in the myocardial hypertrophy model group showed weakened cardiac function and myocardial hypertrophy at 3 and 4 weeks after TAC surgery. This was mainly manifested by increased left ventricular end-diastolic diameter and left ventricular end-systolic diameter, indicators of myocardial hypertrophy, while decreased ejection fraction and fractional shortening, indicators of cardiac function. Treatment with SRK3IN-1 alleviated both myocardial hypertrophy and heart failure compared to placebo-treated mice. Figure 8 ).

[0072] Example 4: Pathological examination of myocardial hypertrophy and fibrosis in mice in the myocardial hypertrophy model group

[0073] 1. Get materials

[0074] (1) Preliminary work: Prepare a urine cup containing 20 mL of 10% formaldehyde and label it (mouse number, group, surgery type, and collection date). Place a petri dish filled with 10% KCl solution at the collection point. Turn on the analytical balance and zero it for later use. Weigh and euthanize the mice.

[0075] (2) Sample collection: The vascular pedicle below the atrial appendage was grasped with ophthalmic curved forceps, the heart was cut off, and quickly placed in a 10% KCl solution. After the heart stopped beating in diastole, it was placed on sterile gauze, the fluid in the heart chamber was gently squeezed out, the surface fluid was dried, the weight was weighed and recorded, the heart was placed in the corresponding urine cup, fixed for 48 hours, and then used for pathological examination.

[0076] (3) Related measurements and calculations: Remove the mouse heart and lungs, trim them, blot them dry with filter paper, weigh them, and record the weight. Cut open the skin at the tibia of the mouse's hind limb, measure and record the tibia length. Calculate the ratio of heart weight to tibia length (HW / TL).

[0077] 2. Pathological examination

[0078] 2.1 Preparation of paraffin-embedded specimen sections

[0079] The main procedures include: trimming the heart → preparing the embedding frame → rinsing with running water → dehydration → clearing → wax infiltration → embedding → sectioning → spreading → drying or baking for later use.

[0080] 2.2 Wheat germ agglutinin (WGA) staining

[0081] Main steps: Paraffin-embedded specimens baked at 60℃ for 30 min are placed in xylene for 5 min × 3 times → 100% ethanol for 5 min × 2 times → 95% ethanol for 5 min → 70% ethanol for 5 min → rinsed with distilled water for 5 min × 2 times → rinsed with PBS for 5 min → rinsed with PBS for 10 min → discard PBS, add trypsin working solution (DIG-3008, Fuzhou Maixin) and incubate at 37℃ in the dark for 20 min → rinsed with PBS for 5 min × 3 times → remove the sections, wipe the liquid around the tissue with filter paper (do not dry the tissue), draw circles with a histochemistry pen and place flat in a humidified chamber → add WGA-Alexa Flour 488 working solution (10 μg / mL) and incubate at 37℃ in the dark for 2 h → discard the staining solution, rinse with PBS for 5 min × 3 times → mount with SlowFade Gold antifade reagent with DAPI → observe under a fluorescence microscope and take pictures under a microscope.

[0082] 2.3 Masson's trichrome staining

[0083] The main steps are as follows: baking at 55℃ for 30 min → xylene for 2 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → rinsing with running water for 10 min → double distilled water for 1 min → Weigert's iron hematoxylin staining for 5 min → rinsing with tap water for 5 min → removing residual liquid → differentiation with 1% hydrochloric acid alcohol for 4 s → rinsing with tap water for 5 min to return to blue → staining with Ponceau S and acid fuchsin solution for 10 min → rinsing with distilled water for 5 min → treatment with phosphomolybdic acid aqueous solution for about 5 min → counterstaining with aniline blue solution for 5 min → treatment with 1% ice CH3COOH for 1 min → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 s, 3 times → xylene for 2 min, 3 times → immediately cover with a glass slide while the xylene is still wet, and take pictures under a microscope.

[0084] 2.4 There were no significant differences in the gross phenotype of the hearts of mice in the sham-operated group. The hearts of mice in the myocardial hypertrophy model group were larger than those in the sham-operated group, while the hearts of mice in the SRK3IN-1 treatment group were significantly smaller compared to mice in the placebo group after TAC surgery. Furthermore, in the sham-operated group, regardless of whether SRK3IN-1 stimulation was administered, there were no significant differences in the heart weight / tibia length ratio (HW / TL), but the HW / TL of mice in the SRK3IN-1 treatment group was significantly lower than that of mice in the placebo group. Figure 9 AB). WGA staining sections revealed that compared with the placebo group, the SRK3IN-1 treatment group showed a significant reduction in cardiomyocyte hypertrophy, with statistically significant differences. Figure 9 CD). Masson staining revealed a significant increase in collagen content in the left ventricular tissue of mice in the myocardial hypertrophy model group, while intraperitoneal injection of SRK3IN-1 significantly alleviated left ventricular fibrosis. Figure 10 AB).

[0085] Example 5: Effects of SRK3IN-1 on mitochondrial structure and function

[0086] 1. Morphological analysis of mitochondria in mouse myocardial tissue:

[0087] (1) Preliminary work: Prepare EP tubes containing 100 μL of tissue electron microscopy fixative (G1102, Servicebio) and label them (mouse number, group, surgery type, and sampling date). Place the culture dish containing 10 mL of tissue electron microscopy fixative at the sampling site. Weigh and euthanize the mice.

[0088] (2) Sample collection: Using ophthalmic curved forceps, grasp the vascular pedicle below the auricle, cut off the heart, quickly place it on sterile gauze, gently squeeze out the fluid inside the heart chambers, pat dry the surface fluid, and then place the heart in tissue electron microscopy fixation solution. Use ophthalmic scissors to cut approximately 1.0 mm from the apex of the heart. 3Tissue blocks of the appropriate size, avoiding compression of the apical tissue, are placed in labeled EP tubes, and transmission electron microscopy tissue samples are prepared within 48 hours.

[0089] (3) Related measurements and calculations: Mitochondrial morphology was observed under a transmission electron microscope, and the morphology of mitochondria in myocardial tissue of each group was recorded by taking pictures. Image J software was used to count the number of mitochondria, the proportion of mitochondria in the tissue, and the proportion of mitochondria with cristae damage.

[0090] (4) The results of mitochondrial morphology analysis showed that compared with the sham-operated group mice, the number of mitochondria in the apical tissue of mice in the myocardial hypertrophy model group decreased and the proportion of mitochondria with crest damage increased 4 weeks after TAC surgery. In contrast, the number of mitochondria in mice treated with SRK3IN-1 increased and the proportion of mitochondria with crest damage decreased, suggesting that the application of SRK3IN-1 can significantly reduce mitochondrial damage in the myocardial tissue of stress-loaded mice.

[0091] 2. Mitochondrial Function Assay: Mitochondrial oxidative respiration rate (OCR) in neonatal rat cardiomyocytes was measured using a Seahorse XFe 24 cell energy analyzer. Working concentrations of the required mitochondrial stress assay drugs (oligomycin-A and FCCP) for primary cardiomyocytes were determined by dostotation. Cells were cultured in V-7 hippocampal plates at a density of ≥10⁻⁶ cells per well. 5 Primary cardiomyocytes were cultured for 1 hour at 37°C in a CO2-free incubator (1 mM pyruvate + glucose-free Seahorse assay medium). A syringe was then loaded with 1.0 μM oligomycin, 1.0 μM FCCP, and 2.0 μM antimycin A. Mitochondrial oxidative respiration parameters, such as basal respiration, ATP production, maximum oxygen consumption, and respiratory potential, were recorded as mitochondrial oxygen consumption rate (OCR). Results showed that Ang II stimulation significantly reduced mitochondrial function in primary cardiomyocytes, while SRK3IN-1 treatment alleviated mitochondrial respiration and energy production.

[0092] 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. Use of the compound represented by formula (I) in the preparation of drugs that inhibit SRPK3 expression: （I）。 2. Use of the compound represented by formula (I) as an active ingredient in the preparation of medicaments for the treatment and / or prevention of diseases associated with myocardial hypertrophy: （I）。 3. The use according to claim 2, characterized in that: The drugs used to treat and / or prevent diseases associated with myocardial hypertrophy are drugs for preventing mitochondrial damage.

4. The use according to claim 2, characterized in that: The drug used to treat and / or prevent diseases associated with myocardial hypertrophy is a drug that inhibits SRPK3 expression.

5. The use according to claim 2, characterized in that: The drug dosage forms for treating and / or preventing diseases related to myocardial hypertrophy are capsules, tablets, granules, oral preparations, microcapsules, injections, suppositories, sprays, or ointments.

6. The use according to claim 2, characterized in that: The diseases associated with myocardial hypertrophy are selected from: hypertension, arrhythmia, sudden cardiac death, infective endocarditis, and heart failure.

7. The use according to claim 6, characterized in that: The disease associated with myocardial hypertrophy is hypertension.

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