Use of 2-((((2,6-di(E)-benzylidenecyclohexylidene)amino)oxy)acetic acid in the preparation of a drug for preventing and treating heart failure related diseases

Abstract

This invention provides the application of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in the preparation of drugs for the prevention and treatment of heart failure-related diseases, belonging to the field of biomedical technology. The application of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in the preparation of drugs for the prevention and treatment of heart failure-related diseases, the structural formula of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid (CG9a) is: [Insert structural formula here]. The CG9a described in this invention exhibits significant cardioprotective effects in various heart failure-related disease models, including diabetic cardiomyopathy, obesity-induced cardiomyopathy, TAC-induced heart failure, and ISO-induced chronic heart failure. It can also improve myocardial hypertrophy and fibrosis, ultimately achieving the effect of preventing and treating heart failure.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in the preparation of drugs for the prevention and treatment of heart failure-related diseases. Background Technology

[0002] Heart failure (HF) is a major disease posing a serious threat to human health worldwide. Epidemiological studies show that the incidence rate increases exponentially with age in people over 65 years of age. With the increasing number of survivors of hypertension, diabetes, and acute myocardial infarction, the hospitalization and readmission rates for HF patients continue to rise. Its causes are diverse, including myocardial infarction, cardiomyopathy, hemodynamic overload, and inflammation, leading to progressive changes in myocardial structure and function, ultimately manifesting as impaired ventricular pumping or filling function. Chronic heart failure is a persistent end-stage state and a common outcome of many heart diseases. Current treatment centers on a "golden triangle" or "quadruple therapy" consisting of diuretics, ACE inhibitors / ARBs, beta-blockers, aldosterone antagonists, and emerging ARNI and SGLT2 inhibitors. However, it cannot reverse the already occurred myocardial cell loss and fibrotic scarring, and the 5-year mortality rate remains as high as approximately 50%.

[0003] Therefore, developing novel and effective drugs to combat heart failure has become an urgent problem to be solved in the field of cardiovascular disease. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art by providing the application of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in the preparation of drugs for the prevention and treatment of heart failure-related diseases.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in the preparation of drugs for the prevention and treatment of heart failure-related diseases; The structural formula of the 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid is: .

[0006] Preferably, the heart failure-related disease is a disease characterized by decreased cardiac function and myocardial pathological damage.

[0007] Preferably, the heart failure-related diseases include diabetic cardiomyopathy, obesity-induced cardiomyopathy, aortic arch constriction-induced heart failure, and isoproterenol-induced chronic heart failure.

[0008] Preferably, the myocardial pathological damage includes myocardial hypertrophy and myocardial fibrosis; the myocardial hypertrophy and myocardial fibrosis are caused by elevated expression of profibrotic and pathological hypertrophy-related molecules.

[0009] Preferably, the use of 2-((((2,6-di(E)-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of heart failure-related diseases.

[0010] Preferably, the drug contains an effective dose of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid or a pharmaceutically acceptable salt thereof.

[0011] Preferably, the drug formulation includes tablets, capsules, injections, and sustained-release formulations.

[0012] The beneficial effects of this invention include: The CG9a described in this invention exhibits significant cardioprotective effects in various heart failure-related disease models, including diabetic cardiomyopathy, obesity-induced cardiomyopathy, aortic arch coarctation (TAC)-induced heart failure, and isoproterenol (ISO)-induced chronic heart failure. It also significantly increases left ventricular ejection fraction and left ventricular fractional shortening in model mice, reduces serum ANP levels, and effectively improves cardiac function. Simultaneously, it reduces myocardial fibrosis area, improves cardiomyocyte hypertrophy, downregulates the mRNA expression of pro-fibrotic and pathological hypertrophy molecules in myocardial tissue, inhibits the progression of myocardial damage, and ultimately achieves the effect of preventing and treating heart failure. This invention provides a new potential drug option for the treatment of heart failure-related diseases, possessing significant application value and clinical translational prospects. Attached Figure Description

[0013] Figure 1 Echocardiography (A), quantitative analysis of ejection fraction (EF) and left ventricular shortening fraction (FS) in mice with diabetic cardiomyopathy (B-C), and quantitative analysis of the myocardial injury marker ANP (D) are shown in Example 1. Figure 2 The images show the heart morphology and size (A), heart weight / tibia length ratio (HW / TL) quantitative analysis (B), HE staining of heart tissue (C), wheat germ lectin (WGA) staining (D), and quantitative analysis of cardiomyocyte cross-sectional area (E) in diabetic cardiomyopathy mice in Example 1, as well as genes related to myocardial hypertrophy. Nppa, Nppb In vivo transcriptional level (F~G); Figure 3The images show Masson's trichrome staining (A) and its quantitative analysis (B) and Sirius red staining (C) of cardiac tissue from diabetic cardiomyopathy mice in Example 1, along with their quantitative analysis (D). The images also show fibrosis-related genes. Col1a1、Tgfb1 In vivo transcriptional level (E); Figure 4 Echocardiography (A), quantitative analysis of ejection fraction (EF) and left ventricular shortening fraction (FS) (B-C), and quantitative analysis of the myocardial injury marker ANP (D) in mice with high-fat diet-induced obesity, as shown by compound CG9a in Example 2. Figure 5 The images shown in Example 2 include: heart morphology and size (A), heart weight / tibia length ratio (HW / TL) quantitative analysis (B), HE staining of heart tissue (C), wheat germ lectin (WGA) staining (D), quantitative analysis of cardiomyocyte cross-sectional area (E), and myocardial hypertrophy-related genes. Nppa, Nppb In vivo transcriptional level (F); Figure 6 The images show Masson's trichrome staining (A) and its quantitative analysis (B), Sirius red staining (C) and its quantitative analysis (D) of heart tissue from obese mice in Example 2, and fibrosis-related genes. Col1a1、Tgfb1 In vivo transcriptional level (E); Figure 7 Echocardiography (A), quantitative analysis of ejection fraction (EF) and left ventricular shortening fraction (FS) (B-C), and quantitative analysis of the myocardial injury marker ANP (D) in mice with TAC-induced heart failure relieved by compound CG9a in Example 3; Figure 8 The images shown in Example 3 are: heart morphology and size (A), heart weight / tibia length ratio (HW / TL) quantitative analysis (B), HE staining of heart tissue (C), wheat germ lectin (WGA) staining (D), and quantitative analysis of cardiomyocyte cross-sectional area (E) in mice with heart failure. Also included are images of genes related to myocardial hypertrophy. Nppa, Nppb In vivo transcriptional level (F~G); Figure 9 The images show Masson's trichrome staining (A) and its quantitative analysis (B) and Sirius red staining (C) of cardiac tissue from mice with myocardial infarction in Example 3, along with their quantitative analysis (D). Fibrosis-related genes are also included. Col1a1、Tgfb1 In vivo transcriptional level (E~F); Figure 10 Echocardiography (A), quantitative analysis of ejection fraction (EF) and left ventricular shortening fraction (FS) (B-C), and quantitative analysis of the myocardial injury marker ANP (D) in mice with ISO-induced chronic heart failure alleviated by compound CG9a in Example 4. Figure 11The images shown in Example 4 are: heart morphology and size (A), heart weight / tibia length ratio (HW / TL) quantitative analysis (B), heart tissue HE staining (C), wheat germ lectin (WGA) staining (D), and cardiomyocyte cross-sectional area quantitative analysis (E), as well as myocardial hypertrophy-related genes. Nppa, Nppb In vivo transcriptional level (F); Figure 12 The images show Masson's trichrome staining (A) and its quantitative analysis (B) and Sirius red staining (C) of the heart tissue from the ISO model experimental animals in Example 4, along with their quantitative analysis (D), and fibrosis-related genes. Col1a1、Tgfb1 At the in vivo transcriptional level (E). Detailed Implementation

[0014] This invention provides the application of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in the preparation of drugs for the prevention and treatment of heart failure-related diseases; The structural formula of the 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid is: .

[0015] The 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid of the present invention is the same as the 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in Chinese invention patent CN202510422015.5, and the preparation method is also the same.

[0016] In this invention, the heart failure-related disease is preferably a disease characterized by decreased cardiac function and myocardial pathological damage.

[0017] In this invention, the heart failure-related diseases preferably include diabetic cardiomyopathy, obesity-induced cardiomyopathy, aortic arch coarctation-induced heart failure, and isoproterenol-induced chronic heart failure.

[0018] In this invention, the pathological damage to the myocardium preferably includes myocardial hypertrophy and myocardial fibrosis; the myocardial hypertrophy and myocardial fibrosis are preferably caused by elevated expression of pro-fibrotic and pathological hypertrophy-related molecules.

[0019] In this invention, the use of 2-((((2,6-di(E)-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of heart failure-related diseases is discussed.

[0020] In this invention, the drug contains an effective dose of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid or a pharmaceutically acceptable salt thereof.

[0021] In this invention, the preferred formulations of the drug include tablets, capsules, injections, and sustained-release formulations.

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] In the following examples, the synthetic route and preparation method of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid (CG9a) are as follows:

[0024] Step a): 20 mL of ethanol solution containing 6 g of o-trifluoromethylbenzaldehyde and 20 mL of ethanol solution containing 1.77 g of cyclohexanone were added to 50 mL of sodium hydroxide aqueous solution (sodium hydroxide aqueous solution mass fraction was 1%). The reaction was carried out at 25 °C for 3 h. The reaction was monitored by TLC. Water was added to induce crystallization. The mixture was filtered, and the filter cake was dissolved and extracted with ethyl acetate to obtain the crude product. Recrystallization from n-heptane gave 2.8 g of compound 2 as a white solid.

[0025] Step b): Add 10 g (1 eq) of compound 2, 3.3 g (2 eq) of hydroxylamine hydrochloride, 3.03 g (3 eq) of potassium carbonate, 2 g of anhydrous magnesium sulfate and 50 mL of anhydrous ethanol to the reaction flask. Reflux at 80 °C for 5 h under anaerobic conditions. After the reaction is complete, add water to the reaction flask to 250 mL. The solid precipitates and is filtered. The filter cake is dried at 40 °C and transferred to a 500 mL beaker. Add 200 mL of n-hexane and stir for 0.5 h. Filter to obtain compound 3, and dry at 40 °C to obtain 7 g of white powder.

[0026] Step c): Mix 1g of compound 3, 270mg of tert-butyl bromoacetate, 2g of potassium carbonate and 25mL of acetonitrile thoroughly, reflux at 85℃ for 4h, monitor the reaction by TLC, the solution color changes from colorless transparent liquid to milky white liquid, evaporate the acetonitrile to dryness, add 100mL of water, extract three times with 40mL of ethyl acetate, wash the organic phase with saturated brine, collect the organic phase liquid, add anhydrous magnesium sulfate to dry and remove water, filter under reduced pressure, evaporate the filtrate to dryness to obtain 1.13g of yellow oily liquid (compound 4).

[0027] Step d): Add 3 mL of dichloromethane and 1 mL of trifluoroacetic acid to 500 mg of compound 4, heat at 42 °C for 8 h, monitor the reaction by TLC. After the reactants have reacted completely, evaporate the dichloromethane, add 100 mL of water, extract three times with 40 mL of ethyl acetate, wash the organic phase with saturated brine, collect the organic phase liquid, add anhydrous magnesium sulfate to dry and remove water, filter under reduced pressure, evaporate the filtrate, add 10 mL of petroleum ether and heat in a water bath. After the precipitate color becomes uniformly light, filter under reduced pressure to obtain a light yellow powder (compound 5), dry at 37 °C and weigh 170 mg. Compound 5 is 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid (CG9a), with a purity > 99.90%.

[0028] Laboratory animal housing: Male C57BL / 6J mice aged 6-8 weeks with an initial weight of 23-25g were used as experimental subjects and were purchased and housed at the Laboratory Animal Center of Hangzhou Medical College. All animals were housed in a specific pathogen-free (SPF) facility.

[0029] Example 1: Compound CG9a was used to alleviate diabetic cardiomyopathy in mice.

[0030] (1) Establishment of a mouse model of T1DM-induced diabetic cardiomyopathy and CG9a treatment

[0031] For three consecutive days, 6-8 week old C57BL / 6J mice were intraperitoneally injected with streptozotocin (STZ) (60 mg / kg, dissolved in sodium citrate buffer; Sigma-Aldrich, St. Louis, Missouri, USA). Fasting blood glucose (FBG) was monitored every two weeks using a glucometer. Diabetes was considered successfully induced when the average fasting blood glucose level was ≥12 mmol / L over two consecutive measurements. Control group (Ctrl group) mice received an equivalent dose of sodium citrate buffer. Mice treated with STZ were divided into five groups: T1DM group, compound treatment group (T1DM+CG9a 2.5 mg / kg, T1DM+CG9a 5 mg / kg, T1DM+CG9a 10 mg / kg), positive control group (T1DM+Noxintu LCZ696 60 mg / kg). T1DM refers to three consecutive days of injection of the above STZ at 60 mg / kg per day.

[0032] (2) Echocardiographic assessment of cardiac function in mice

[0033] Echocardiography was used to assess cardiac function and confirm the establishment of a diabetic cardiomyopathy model. After drug administration, echocardiography was performed on mice in each group using a Vevo 1100 high-resolution small animal ultrasound system. Mice were placed in a supine position on a physiological information monitoring platform and anesthetized with isoflurane inhalation. Long-axis B-mode and M-mode images of the heart were acquired parasternally on the left side. Left ventricular parameters were then measured, and the average value over three cardiac cycles was taken. The main parameters included ejection fraction (EF) and fractional shortening (FS). Mice were weighed after ultrasound, and blood samples were collected and centrifuged to collect serum for serum biochemical assays. The levels of ANP, a myocardial injury marker, in mouse serum were detected using an ELISA quantitative assay kit.

[0034] Figure 1 Echocardiography (A), quantitative analysis of ejection fraction and left ventricular shortening fraction (B-C), and quantitative analysis of the myocardial injury marker ANP (D) in mice with diabetic cardiomyopathy induced by compound CG9a in Example 1. Figure 1 The results of A showed that, compared with the model group, CG9a treatment significantly improved cardiac dysfunction in diabetic cardiomyopathy mice, manifested by increasing left ventricular ejection fraction and shortening the ejection fraction. Figure 1 B~ Figure 1 C). Figure 1 The results showed that, compared with the Ctrl group, the ANP content in the T1DM group was significantly increased; compared with the T1DM group, CG9a reduced the ANP content in a dose-dependent manner, and the high-dose CG9a group was comparable to the positive control group treated with LCZ696. These results suggest that CG9a alleviates cardiac function impairment in diabetic cardiomyopathy mice.

[0035] (3) Take left ventricular heart tissue from mice and detect the degree of myocardial hypertrophy and fibrosis at the histological level.

[0036] At the end of the experiment, mice that had undergone ultrasound were euthanized by cervical dislocation for tissue sampling. The heart was dissected by cutting along the left ribcage using surgical scissors for pathological sectioning. After extraction, the heart tissue was quickly placed in a 10% KCl aqueous solution to stop beating in diastole. Blood was squeezed out of the heart chambers, and the tissue was trimmed and weighed (mg). The heart tissue was then soaked in formalin. Alternatively, after extraction, blood could be squeezed out of the heart chambers, trimmed, and weighed (mg). One tibia was exposed and its length (cm) was measured. The weighed mouse heart was fixed in formalin for 36 hours, then trimmed, placed in an embedding frame for dehydration, clearing, and embedding to prepare pathological sections for subsequent experiments.

[0037] Hematoxylin-eosin (HE) staining for cardiomyocyte hypertrophy: Pathological sections were baked at 55℃ for 35 min. The sections were then dewaxed by immersing them in xylene twice (5 min each time). Subsequently, the sections were sequentially hydrated in a gradient of 100% anhydrous ethanol, an aqueous solution containing 90% anhydrous ethanol, an aqueous solution containing 80% anhydrous ethanol, and an aqueous solution containing 70% anhydrous ethanol (3 min each time). Finally, the sections were immersed in pure water for 10 min. Next, the cell nuclei were stained with hematoxylin. After differentiation and blue reversion treatment, the cytoplasm was stained with eosin. Finally, the sections were dehydrated, cleared, and mounted. The morphology and structure of the cardiac tissue were observed under a microscope.

[0038] Wheat germ lectin (WGA) staining for cardiomyocyte cross-sectional area detection: Pathological sections were baked at 55℃ for 35 min, then dewaxed by immersing them in xylene twice (5 min each time). The sections were then sequentially hydrated in a gradient of 100% anhydrous ethanol, 90% anhydrous ethanol aqueous solution, 80% anhydrous ethanol aqueous solution, and 70% anhydrous ethanol aqueous solution (3 min each time), and finally immersed in pure water for 10 min. Antigen retrieval sections were prepared using trypsin method (50 μL / heart tissue incubated at 37℃ for 15 min), washed with PBS, and 100 μL of prepared WGA-AF488 working solution was added to the heart tissue. Incubation was carried out for 2 h, followed by thorough rinsing with PBS. Finally, the sections were mounted with 4',6-diamidinyl-2-phenylindole (DAPI) and photographed under a fluorescence microscope. WGA staining and photography: Each image should contain at least 20 cells with clear boundaries and nuclei located in the center of the cell. After taking the photos, the cross-sectional area of ​​the cardiomyocytes should be measured using Image-Pro Plus 6.0 image analysis software.

[0039] Masson staining: The pathological sections were baked at 55℃ for 35 min. The sections were then dewaxed by immersing them in xylene twice (5 min each time). The sections were then hydrated in a gradient of 100% anhydrous ethanol, 90% anhydrous ethanol, 80% anhydrous ethanol, and 70% anhydrous ethanol (3 min each time). Finally, the sections were soaked in pure water for 10 min. The nuclei were stained with Weigert iron hematoxylin and differentiated by hydrochloric acid-ethanol. The cytoplasm was then stained with Ponceau S and acid fuchsin. The cytoplasm was then differentiated with phosphomolybdic acid and stained with aniline blue. Finally, the sections were rapidly rinsed with 1% glacial acetic acid, dehydrated by gradient ethanol, cleared with xylene, and mounted with neutral resin. The contrast between the red myocardial fibers and the blue collagen fibers was observed under a microscope.

[0040] Sirius Red staining for myocardial collagen deposition: Pathological sections were baked at 55°C for 35 min, then dewaxed by immersing twice in xylene (5 min each time). The sections were then sequentially hydrated in a gradient of 100% anhydrous ethanol, 90% anhydrous ethanol, 80% anhydrous ethanol, and 70% anhydrous ethanol (3 min each time), and finally soaked in pure water for 10 min. The sections were then incubated in Sirius Red staining solution (0.1% Direct Red 80 dissolved in picric acid saturated aqueous solution) at room temperature for 60 min. They were then quickly rinsed twice with 0.5% glacial acetic acid aqueous solution for 30 s each time to remove unbound dye. After gradient ethanol dehydration, xylene clearing, and mounting with neutral resin were performed. Under a regular optical microscope, collagen fibers appeared red, and cardiomyocytes appeared yellow. Under a polarized light microscope, type I collagen showed strong red / yellow birefringence, and type III collagen showed weak green birefringence. Five to ten non-overlapping fields of view were randomly selected from each slice, and the collagen volume fraction (collagen-positive area / total myocardial tissue area × 100%) was calculated using Image Pro Plus 6.0 image analysis software.

[0041] Figure 2 The images show the heart morphology and size (A), heart weight / tibia length ratio quantitative analysis (B), HE staining of heart tissue (C), wheat germ lectin staining (D), and quantitative analysis of cardiomyocyte cross-sectional area (E) of diabetic cardiomyopathy mice in Example 1, as well as genes related to myocardial hypertrophy. Nppa, Nppb At the in vivo transcriptional level (F~G). Figure 2 The results of A showed that the heart appearance of the T1DM group was significantly enlarged, and different concentrations of compound CG9a could improve cardiac hypertrophy in diabetic mice; Figure 2 B shows that the heart weight / tibia length ratio (HW / TL) was significantly increased in the T1DM group, and treatment with different concentrations of compound CG9a improved this abnormal increase. Furthermore, the high-dose CG9a group showed comparable effects to the positive control drug LCZ696 treatment group. Figure 2 As shown in C~E, the T1DM group exhibited disordered cell arrangement and increased cardiomyocyte cross-sectional area. Treatment with different concentrations of compound CG9a significantly improved the pathological state of mouse cardiac tissue and decreased the cardiomyocyte cross-sectional area. The high-dose CG9a group showed comparable effects to the positive control drug LCZ696 treatment group.

[0042] Figure 3 The images show Masson's trichrome staining (A) and its quantitative analysis (B) and Sirius red staining (C) of cardiac tissue from diabetic cardiomyopathy mice in Example 1, along with their quantitative analysis (D). The images also show fibrosis-related genes. Col1a1、Tgfb1 The in vivo transcriptional level (E).

[0043] Depend on Figure 3 As seen in A~3D, the T1DM group mice showed increased cardiac tissue fibrosis and abnormal collagen fiber deposition. Treatment with different concentrations of compound CG9a significantly improved the degree of left ventricular myocardial fibrosis and collagen deposition in diabetic cardiomyopathy mice, and the high-dose CG9a group had comparable effects to the positive control drug LCZ696 treatment group.

[0044] (4) RT-qPCR was used to assess the transcriptional levels of markers of myocardial hypertrophy and fibrosis in mice in vivo.

[0045] At the end of the experiment, mice were sacrificed after echocardiography, and the hearts were cleaned of blood and trimmed, leaving left ventricular heart tissue which was then placed in cryovials. A certain amount of left ventricular heart tissue was excised and total RNA was extracted. To detect mRNA expression, total RNA was extracted from each heart tissue sample using TRIzol reagent. cDNA synthesis was performed using Hifair® Ⅲ 1st Strand cDNASynthesis SuperMix for qPCR (Shanghai, China), followed by real-time quantitative PCR using Hieff UNICON® qPCR SYBR Green Master Mix. Gene expression was amplified and measured using a 3-step amplification program in a real-time quantitative PCR instrument. The expression of target genes in each group was compared with β-actin levels based on changes in CT cycle values, using a 2- ΔΔCT Methods and normalized statistical analysis. Detection of hypertrophy markers in mouse heart tissue. Nppa and Nppb and fibrosis markers Col1a1、Tgfb1 The transcription level. Primer sequences are shown in Table 1.

[0046] Table 1 Primer Sequences

[0047] Figure 2 F~G and Figure 3 The results of E showed that, compared with the T1DM group mice, CG9a treatment significantly reduced markers of myocardial hypertrophy. Nppa, Nppb and markers of myocardial fibrosis Col1a1、Tgfb1 The levels were similar, and the high-dose CG9a group was comparable to the positive control group treated with LCZ696.

[0048] Depend on Figures 2-3 The results show that compound CG9a can significantly alleviate myocardial hypertrophy and fibrosis in diabetic cardiomyopathy mice, and the high-dose CG9a group was comparable to the positive control group treated with LCZ696. These results indicate that CG9a can alleviate T1DM-induced diabetic cardiomyopathy to some extent.

[0049] Example 2: Compound CG9a was used to alleviate cardiac damage in high-fat-fed obese mice.

[0050] (1) Establishment of a high-fat feeding obese mouse model and CG9a treatment

[0051] C57BL / 6J mice were randomly divided into 4 groups (n=6 per group): control group (Ctrl group): fed with the standard diet of healthy mice; obese mouse model group (HFD group): fed with a high-fat diet (HFD, fat accounts for 60.3% of total calories, Research Diets, D12492) for 10 weeks; compound treatment group (HFD+CG9a 10mg / kg or HFD+CG9a 20mg / kg): after the obese mouse model was established, the mice were administered compound CG9a by gavage at the corresponding dose for each group after week 10, with 0.5% CMC-Na as the solvent, once every two days for 8 consecutive weeks.

[0052] (2) Echocardiographic assessment of cardiac function in mice

[0053] The steps are the same as those in Example 1 (2) for evaluating cardiac function in mice by echocardiography.

[0054] Figure 4 The images show the echocardiogram (A), quantitative analysis of ejection fraction and left ventricular shortening fraction (B-C), and quantitative analysis of the myocardial injury marker ANP in mice induced by high-fat feeding, all obtained by compound CG9a in Example 2.

[0055] Mouse echocardiography results are as follows Figure 4 As shown in A~C, compared with the HFD group, CG9a treatment significantly improved cardiac dysfunction in HFD-induced obese mice, manifested as increased left ventricular ejection fraction and shortened ejection fraction. Figure 4 The results showed that serum ANP levels were significantly increased in the HFD group compared to the control group; and CG9a reduced ANP levels in a dose-dependent manner compared to the HFD group. These results indicate that CG9a alleviated obesity-induced cardiac damage in mice.

[0056] (3) Take left ventricular heart tissue from mice and detect the degree of myocardial hypertrophy and fibrosis at the histological level.

[0057] The steps were the same as in Example 1 (3), where left ventricular heart tissue was taken from mice and the degree of myocardial hypertrophy and fibrosis was detected at the histological level.

[0058] Figure 5The images shown in Example 2 include: heart morphology and size (A), heart weight / tibia length ratio quantitative analysis (B), HE staining of heart tissue (C), wheat germ lectin staining (D), and quantitative analysis of cardiomyocyte cross-sectional area (E), as well as genes related to myocardial hypertrophy. Nppa, Nppb At the in vivo transcriptional level (F).

[0059] Figure 5 The results of A showed that, compared with the control group, the heart size in the HFD group was significantly larger, and different concentrations of compound CG9a could improve the cardiac hypertrophy induced by HFD; Figure 5 As shown in Figure B, the heart weight / tibia length ratio (HW / TL) was significantly increased in the HFD group, and treatment with different concentrations of compound CG9a could improve this abnormally high ratio. Figure 5 As seen in C~E, the HFD group showed disordered cell arrangement and increased cardiomyocyte cross-sectional area. Treatment with different concentrations of compound CG9a significantly improved the pathological state of mouse heart tissue and reduced the cross-sectional area of ​​cardiomyocytes.

[0060] Figure 6 The images show Masson's trichrome staining (A) and its quantitative analysis (B), Sirius red staining (C) and its quantitative analysis (D) of heart tissue from obese mice in Example 2, and fibrosis-related genes. Col1a1、Tgfb1 At the in vivo transcriptional level (E).

[0061] Depend on Figure 6 As shown in A~D, the HFD group mice showed increased cardiac tissue fibrosis and abnormal collagen fiber deposition. Treatment with different concentrations of compound CG9a significantly improved the degree of left ventricular myocardial fibrosis and collagen deposition in diabetic cardiomyopathy mice.

[0062] (4) RT-qPCR was used to assess the transcriptional levels of markers of myocardial hypertrophy and fibrosis in mice in vivo.

[0063] The steps were the same as those in Example 1 (4) for assessing the transcriptional levels of in vivo myocardial hypertrophy and fibrosis markers in mice using RT-qPCR.

[0064] Figure 5 F and Figure 6 The results of E showed that, compared with the HFD group mice, CG9a treatment significantly reduced markers of myocardial hypertrophy. Nppa, Nppb and markers of myocardial fibrosis Col1a1、Tgfb1 The level.

[0065] Depend on Figures 5-6 The compound CG9a was found to significantly alleviate myocardial hypertrophy and fibrosis in obese mice. These results indicate that CG9a can alleviate cardiac damage induced by high-fat feeding in obese mice to some extent.

[0066] Example 3: Compound CG9a was used to alleviate TAC-induced heart failure in mice.

[0067] (1) Establishment of TAC-induced mouse myocardial infarction model and CG9a treatment

[0068] Male C57BL / 6J mice aged 6-8 weeks were randomly divided into 4 groups (n=6 per group) after one week of acclimatization: Sham group: Mice in the sham group underwent the same surgical procedure as the TAC group, until the aortic arch was exposed and the sutures were bypassed, but no ligation or narrowing was performed. Model group (TAC): After isoflurane induction anesthesia, 1% sodium pentobarbital (50 mg / kg) was injected intraperitoneally to maintain anesthesia. Mice were fixed in a supine position, endotracheally intubated, and connected to a small animal ventilator (tidal volume 6-8 mL / kg, respiratory rate 120 breaths / min, respiratory ratio 1:1.5). An 8 mm longitudinal incision was made in the skin from the upper sternal border to the second intercostal space. The thymus was bluntly dissected and pushed towards the head to expose the aortic arch. The aortic arch was dissected between the brachiocephalic artery and the left common carotid artery. A 27G suture was placed parallel to the arch, and the arch was ligated with 8-0 atraumatic sutures. The suture was then quickly withdrawn to form a fixed stenosis. After confirming the absence of active bleeding, the fascia and skin were sutured layer by layer with 6-0 absorbable sutures. Before closing the chest, the lungs were briefly inflated to expel air from the pleural cavity. Penicillin was administered intramuscularly for three consecutive days post-surgery to prevent infection. Animals were kept individually in cages and kept warm until they regained consciousness. Compound treatment group (TAC + CG9a 5mg / kg or TAC + CG9a 10mg / kg): After successful model establishment, mice were administered compound CG9a by gavage at the corresponding dose for each group, using 0.5% CMC-Na as the solvent, once every two days for eight consecutive weeks.

[0069] (2) Echocardiographic assessment of cardiac function in mice

[0070] The steps are the same as those in Example 1 (2) for evaluating cardiac function in mice by echocardiography.

[0071] Figure 7 Echocardiography (A), quantitative analysis of ejection fraction and left ventricular shortening fraction (B-C), and quantitative analysis of the myocardial injury marker ANP (D) in mice with TAC-induced heart failure relieved by compound CG9a in Example 3.

[0072] Figure 7 The results of A~C showed that, compared with the model group, CG9a treatment significantly improved TAC-induced cardiac dysfunction in mice, manifested as an increase in left ventricular ejection fraction and a decrease in ejection fraction. Figure 7 The results showed that, compared with the Sham group, the ANP content in the TAC group was significantly increased; compared with the TAC group, CG9a reduced the ANP content in a dose-dependent manner. These results indicate that CG9a alleviated cardiac injury in TAC mice.

[0073] (3) Take left ventricular heart tissue from mice and detect the degree of myocardial hypertrophy and fibrosis at the histological level.

[0074] The steps were the same as in Example 1 (3), where left ventricular heart tissue was taken from mice and the degree of myocardial hypertrophy and fibrosis was detected at the histological level.

[0075] Figure 8 The images shown in Example 3 are: heart morphology and size (A), heart weight / tibia length ratio quantitative analysis (B), HE staining of heart tissue (C), wheat germ lectin staining (D), and quantitative analysis of cardiomyocyte cross-sectional area (E), and myocardial hypertrophy-related genes in mice with heart failure. Nppa, Nppb At the in vivo transcriptional level (F~G).

[0076] Figure 8 The results of A showed that the hearts of the TAC group were significantly enlarged, and different concentrations of compound CG9a could improve cardiac hypertrophy in TAC group mice; Figure 8 As shown in Figure B, the heart weight / tibia length ratio was significantly increased in the TAC group, and treatment with different concentrations of compound CG9a could improve this abnormally high ratio. Figure 8 As seen in C~E, the TAC group showed disordered cell arrangement, increased cardiomyocyte cross-sectional area, and treatment with different concentrations of compound CG9a significantly improved the pathological state of mouse heart tissue and decreased cardiomyocyte cross-sectional area.

[0077] Figure 9 The images show Masson's trichrome staining (A) and its quantitative analysis (B) and Sirius red staining (C) of cardiac tissue from mice with myocardial infarction in Example 3, along with their quantitative analysis (D). Fibrosis-related genes are also included. Col1a1、Tgfb1 At the in vivo transcriptional level (E~F).

[0078] Depend on Figure 9 As shown in A~D, the TAC group mice showed increased cardiac tissue fibrosis and abnormal collagen fiber deposition. Treatment with different concentrations of compound CG9a significantly improved the degree of left ventricular myocardial fibrosis and collagen deposition in diabetic cardiomyopathy mice.

[0079] (4) RT-qPCR was used to assess the transcriptional levels of markers of myocardial hypertrophy and fibrosis in mice in vivo.

[0080] The steps were the same as those in Example 1 (4) for assessing the transcriptional levels of in vivo myocardial hypertrophy and fibrosis markers in mice using RT-qPCR.

[0081] Figure 8 F~G and Figure 9 The results from E to F showed that, compared with the TAC group mice, CG9a treatment significantly reduced markers of myocardial hypertrophy. Nppa, Nppb and markers of myocardial fibrosis Object Col1a1, Tgfb1 The level.

[0082] Depend on Figures 8-9 It was found that compound CG9a significantly alleviated myocardial hypertrophy and fibrosis in TAC mice. These results indicate that CG9a can alleviate TAC-induced heart failure in mice to some extent.

[0083] Example 4: Compound CG9a was used to alleviate ISO-induced chronic heart failure in mice.

[0084] (1) Establishment of ISO-induced chronic heart failure mouse model and CG9a treatment

[0085] Thirty male C57BL / 6 mice aged 6-8 weeks were randomly divided into 5 groups of 6 mice each: control group (Ctrl), model group (ISO), 2.5 mg / kg compound group (ISO+2.5 mg / kg), 5 mg / kg compound group (ISO+5 mg / kg), and 10 mg / kg compound group (ISO+10 mg / kg). Compound CG9a was administered intraperitoneally for 7 days before model induction. Both the model group and the drug group received subcutaneous injections of isoproterenol (ISO, 5 mg / kg) once daily for 10 days to simulate a chronic heart failure animal model. The control group received an equal volume of physiological saline. Twenty-eight days after administration, echocardiography was performed on all mice. After echocardiography, the mice were weighed, sacrificed, and blood was collected. Serum samples were obtained by centrifugation at 3000 rpm for 15 minutes after standing at room temperature for 4 hours. Simultaneously, heart tissue was fixed or cryopreserved for subsequent experiments.

[0086] (2) Echocardiographic assessment of cardiac function in mice

[0087] The steps are the same as those in Example 1 (2) for evaluating cardiac function in mice by echocardiography.

[0088] Figure 10 Echocardiography (A), quantitative analysis of ejection fraction and left ventricular shortening fraction (B-C), and quantitative analysis of the myocardial injury marker ANP (D) in mice with ISO-induced chronic heart failure, as shown by compound CG9a in Example 4.

[0089] Mouse echocardiography results are as follows Figure 10 As shown in Figure A, compared with the control group, the echocardiographic left ventricular ejection fraction (EF) and fulminant suture (FS) of mice in the ISO group were significantly decreased; compared with the ISO group, the EF and FS of each dose group of CG9a were significantly increased. Figure 10 (B~C) indicates that CG9a can improve myocardial contractile function in mice with heart failure. For example... Figure 10As shown in Figure D, compared with the control group, the ANP content in the ISO group was significantly increased; compared with the ISO group, CG9a decreased the ANP content in a dose-dependent manner. These results indicate that CG9a has a protective effect on the heart of mice with heart failure.

[0090] (3) Take left ventricular heart tissue from mice and detect the degree of myocardial hypertrophy and fibrosis at the histological level.

[0091] The steps were the same as in Example 1 (3), where left ventricular heart tissue was taken from mice and the degree of myocardial hypertrophy and fibrosis was detected at the histological level.

[0092] Figure 11 The images shown in Example 4 are: heart morphology and size (A), heart weight / tibia length ratio quantitative analysis (B), HE staining of heart tissue (C), wheat germ lectin staining (D), and quantitative analysis of cardiomyocyte cross-sectional area (E), as well as genes related to myocardial hypertrophy. Nppa, Nppb At the in vivo transcriptional level (F).

[0093] Figure 11 The results of A showed that, compared with the control group, the heart size in the ISO group was significantly larger, and different concentrations of compound CG9a could improve the cardiomegaly induced by ISO; Figure 11 As shown in Figure B, the heart weight / tibia length ratio was significantly increased in the ISO group, and treatment with different concentrations of compound CG9a could improve this abnormally high ratio. Figure 11 As seen in C~E, the ISO group showed disordered cell arrangement and increased cardiomyocyte cross-sectional area. Treatment with different concentrations of compound CG9a significantly improved the pathological state of mouse heart tissue and decreased the cross-sectional area of ​​cardiomyocytes.

[0094] Figure 12 The images show Masson's trichrome staining (A) and its quantitative analysis (B) and Sirius red staining (C) of the heart tissue from the ISO model experimental animals in Example 4, along with their quantitative analysis (D), and fibrosis-related genes. Col1a1、Tgfb1 At the in vivo transcriptional level (E).

[0095] Depend on Figure 12 As shown in A~D, the ISO group mice showed increased cardiac tissue fibrosis and abnormal collagen fiber deposition. Treatment with different concentrations of compound CG9a significantly improved the degree of left ventricular myocardial fibrosis and collagen deposition in diabetic cardiomyopathy mice.

[0096] (4) RT-qPCR was used to assess the transcriptional levels of markers of myocardial hypertrophy and fibrosis in mice in vivo.

[0097] The steps were the same as those in Example 1 (4) for assessing the transcriptional levels of in vivo myocardial hypertrophy and fibrosis markers in mice using RT-qPCR.

[0098] Figure 11 F and Figure 12 The results of E showed that, compared with the ISO group mice, CG9a treatment significantly reduced markers of myocardial hypertrophy. Nppa, Nppb and markers of myocardial fibrosis Object Col1a1, Tgfb1 The level.

[0099] Depend on Figures 11-12 It was found that compound CG9a significantly alleviated myocardial hypertrophy and fibrosis in mice. These results indicate that CG9a can improve ISO-induced chronic heart failure in mice to some extent.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid in the preparation of drugs for the prevention and treatment of heart failure-related diseases; The structural formula of the 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid is: 。 2. The application according to claim 1, characterized in that, The heart failure-related diseases are those characterized by decreased cardiac function and myocardial pathological damage.

3. The application according to claim 2, characterized in that, The heart failure-related diseases include diabetic cardiomyopathy, obesity-induced cardiomyopathy, aortic arch coarctation-induced heart failure, and isoproterenol-induced chronic heart failure.

4. The application according to claim 2, characterized in that, The myocardial pathological damage includes myocardial hypertrophy and myocardial fibrosis; the myocardial hypertrophy and myocardial fibrosis are caused by elevated expression of profibrotic and pathological hypertrophy-related molecules.

5. The application according to any one of claims 2 to 4, characterized in that, The use of the 2-((((2,6-di(E)-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of heart failure-related diseases.

6. The application according to claim 5, characterized in that, The drug contains an effective dose of 2-((((2,6-di(E)-o-trifluoromethylbenzyl)cyclohexyl)amino)oxy)formic acid or a pharmaceutically acceptable salt thereof.

7. The application according to claim 1 or 6, characterized in that, Drug formulations include tablets, capsules, injections, and sustained-release formulations.

Images