Synthesis method of histone deacetylase 4 small molecule inhibitor and application in anti-vascular endothelial senescence

Abstract

The present application relates to a kind of histone deacetylase 4 small molecule inhibitor synthesis method and the application of anti-vascular endothelial senescence.The present application is synthesized with simple and efficient new method a new HDAC4 small molecule inhibitor, its structural formula is as follows.The HDAC4 small molecule inhibitor has strong affinity with HDAC4, can significantly reduce the expression amount of HADC4 and P21 protein and mRNA of vascular endothelial cell and the mRNA expression amount of inflammation-related protein Il6, CCL2 and Il-1 beta, has the effect of significantly anti-vascular endothelial cell senescence, and has good drug property and safety, can be used for preparing the drug for treating vascular endothelial senescence and its related diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a synthetic method of a histone deacetylase 4 small molecule inhibitor and application of the histone deacetylase 4 small molecule inhibitor in anti-vascular endothelial senescence. BACKGROUND

[0002] Histone deacetylase 4 (HDAC4) is a kind of protease, which plays an important role in the structural modification of chromosomes and the regulation of gene expression. In the previous research (Di Yang, Cardiovascular Research (2018) 114, 1016-1028) of the research team of the present application, it is confirmed that HDAC4 is up-regulated in the model induced by angiotensin II (Ang II), and the inhibition of HDAC4 can significantly reduce the expression of senescence-associated secretory phenotype (SASP) factors, such as pro-inflammatory cytokines (COX2), interleukin 6 (IL6) and fibronectin (VCAM-1). Inflammatory senescence is gradually widely recognized in the art. Therefore, HDAC4 is an effective epigenetic target for inhibiting inflammatory senescence.

[0003] Studies have shown that HDAC4 has the following relationships with vascular endothelial cell senescence. First, it affects the senescence marker: HDAC4 may regulate the expression of senescence-related genes in vascular endothelial cells. For example, it may affect cell cycle regulators, thereby leading to decreased proliferation capacity of endothelial cells and promoting senescence. Second, oxidative stress: the senescence of endothelial cells is closely related to the increase of oxidative stress level. HDAC4 may be involved in regulating the signal pathways related to oxidative stress, thereby affecting the health status of endothelial cells. Third, pro-inflammatory response: HDAC4 may also be involved in regulating the expression of pro-inflammatory factors, and inflammation is another important promoting factor for endothelial cell senescence. By regulating the inflammatory response, HDAC4 may indirectly affect the senescence process of endothelial cells. Therefore, the intervention of HDAC4 can help to delay the senescence (especially inflammatory senescence) of endothelial cells, thereby improving vascular health. At present, the research on HDAC4 molecular inhibitors reported in the literature is still very insufficient, and most of the reported HDAC4 inhibitors have anti-tumor activity and high toxicity. SUMMARY

[0004] Based on this, the purpose of the present application is to provide a new HDAC4 small molecule inhibitor, which can be used for treating vascular endothelial senescence and other inflammatory senescence.

[0005] The technical scheme for achieving the above-mentioned purpose of the application is as follows.

[0006] The first aspect of the present application provides an application of a cyclic dipeptide compound or a pharmaceutically acceptable salt thereof in the preparation of a histone deacetylase 4 inhibitor, wherein the cyclic dipeptide compound has a structural formula of:

[0007]

[0008] The second aspect of the present application provides an application of the cyclic dipeptide compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting inflammatory aging.

[0009] The third aspect of the present application provides an application of the cyclic dipeptide compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for resisting vascular endothelial aging.

[0010] The fourth aspect of the present application provides an application of the cyclic dipeptide compound or the pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating diseases related to vascular endothelial aging.

[0011] The fifth aspect of the present application provides a preparation method of the cyclic dipeptide compound, comprising the following steps:

[0012] (1) reacting N-tert-butoxy carbonyl-L-phenylalanine with L-tyrosine methyl ester hydrochloride under the action of a condensing agent and a base to obtain a linear dipeptide protected by tert-butoxy carbonyl;

[0013] (2) removing the tert-butoxy carbonyl protecting group from the linear dipeptide protected by tert-butoxy carbonyl to obtain a linear dipeptide;

[0014] (3) reacting the linear dipeptide at a temperature of 80-100 DEG C to form a ring to obtain the cyclic dipeptide compound;

[0015] The reaction formula is as follows:

[0016]

[0017] The present application has the following beneficial effects:

[0018] The present application synthesizes a new HDAC4 small molecule inhibitor (i.e. the cyclic dipeptide compound of the present application) by a simple and efficient new method, which has strong affinity with HDAC4, can significantly reduce the expression amounts of HADC4 and P21 protein and mRNA of vascular endothelial cells and the mRNA expression amounts of inflammation-related proteins Il6, CCL2 and Il-1β, has a significant effect of resisting vascular endothelial cell aging, and has good drugability and safety, and can be used for preparing a drug for treating vascular endothelial aging and diseases related thereto.

[0019] The cyclic dipeptide small molecule inhibitor of the application has simple structure, is relatively easy to synthesize, is easy to store and transport, has the advantage of being orally taken, and is expected to become a promising small molecule drug for treating vascular endothelial aging and diseases related thereto. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0021] Figure 2 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0022] Figure 3 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0023] Figure 4 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0024] Figure 5 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0025] Figure 6 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0026] Figure 7 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0027] Figure 8 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0028] Figure 9 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0029] Figure 10 The structure of the HDAC4 small molecule inhibitor is confirmed by nuclear magnetic resonance hydrogen spectrum.

[0030] Figure 11 Schematic diagram of CPT animal toxicity experiment.

[0031] Figure 12 Schematic diagram of CPT, an HDAC4 small molecule inhibitor, inhibiting the vascular thickness of an Ang II-induced aging model mouse.

[0032] Figure 13 Schematic diagram of CPT, an HDAC4 small molecule inhibitor, inhibiting the expression of HDAC4 and aging-related protein P21 in an Ang II-induced aging model mouse.

[0033] Figure 14 Schematic diagram of CPT, an HDAC4 small molecule inhibitor, inhibiting the mRNA expression of aging-related protein P21, inflammation-related protein Il6, CCL2 and Il-1β in an Ang II-induced aging model mouse. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0035] The experimental methods in the following examples not specifically noted are generally carried out according to the conventional conditions or according to the conditions recommended by the manufacturers. The various common chemical reagents used in the examples are commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used in the present application includes any and all combinations of one or more related listed items.

[0037] In addition, as used in the present application, the term "or" is an inclusive "or" and is equivalent to the term "and / or", unless the context clearly indicates otherwise. The term "based on" is not exclusive and allows for other factors not described, unless the context clearly indicates otherwise. In addition, throughout the specification, the meanings of "one", "a", and "said / the" include plural referents. The meaning in "in" includes "in" and "on".

[0038] In some embodiments of the present application, a cyclic dipeptide compound or a pharmaceutically acceptable salt thereof is involved, and the structural formula of the cyclic dipeptide compound is:

[0039]

[0040] The present application also relates to a method for preparing the cyclic dipeptide compound, comprising the following steps:

[0041] (1) reacting N-tert-butyloxycarbonyl-L-phenylalanine with L-tyrosine methyl ester hydrochloride in the presence of a condensing agent and a base to obtain a tert-butyloxycarbonyl-protected linear dipeptide;

[0042] (2) removing the tert-butyloxycarbonyl protecting group from the tert-butyloxycarbonyl-protected linear dipeptide to obtain a linear dipeptide;

[0043] (3) reacting the linear dipeptide at a temperature of 80-100°C to obtain the cyclic dipeptide compound;

[0044] The reaction formula is as follows:

[0045]

[0046] In some embodiments, the molar ratio of N-tert-butyloxycarbonyl-L-phenylalanine to L-tyrosine methyl ester hydrochloride is 1:1-1.5.

[0047] In some embodiments, the condensing agent is 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0048] In some embodiments, the molar ratio of N-tert-butyloxycarbonyl-L-phenylalanine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-2:1-2.

[0049] In some embodiments, the base is N,N-diisopropylethylamine.

[0050] In some embodiments, the molar ratio of N-tert-butyloxycarbonyl-L-phenylalanine to the base is 1:2-4.

[0051] In some embodiments, the reaction in step (1) is carried out in an organic solvent, which is preferably dichloromethane.

[0052] In some embodiments, the reaction in step (1) is carried out under the protection of an inert gas.

[0053] In some embodiments, the reaction in step (1) is carried out at a temperature of 0-40°C for 1-4 hours.

[0054] In some embodiments, the reaction for removing the tert-butyloxycarbonyl protecting group in step (2) is carried out in a mixed solvent of dichloromethane and trifluoroacetic acid.

[0055] In some embodiments, the volume ratio of dichloromethane and trifluoroacetic acid is 1:0.8-1.2.

[0056] In some embodiments, the reaction for removing the tert-butyloxycarbonyl protecting group in step (2) is carried out in the presence of a catalyst, which is preferably triisopropylsilane.

[0057] In some embodiments, the reaction for removing the tert-butyloxycarbonyl protecting group in step (2) is carried out at a temperature of 0-40°C for 20 minutes to 2 hours.

[0058] In some embodiments, the reaction in step (3) is carried out in an organic solvent, which is preferably a mixture of isobutanol and toluene.

[0059] In some embodiments, the volume ratio of isobutanol and toluene is 1:0.8-1.2.

[0060] In some embodiments, the reaction in step (3) is carried out at a temperature of 85-95°C for 2-6 hours.

[0061] In some embodiments, the reaction in step (3) is carried out under the protection of an inert gas.

[0062] In some embodiments, the method for preparing the cyclic dipeptide compound comprises the following steps:

[0063] (1) dissolving the N-tert-butyloxycarbonyl-L-phenylalanine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in dichloromethane, cooling to 0-5°C, then adding the L-tyrosine methyl ester hydrochloride and N,N-diisopropylethylamine, stirring under the protection of an inert gas at 0-5°C for 20-40 minutes, then stirring at 20-35°C for 40-80 minutes to obtain a linear dipeptide protected by tert-butyloxycarbonyl;

[0064] (2) dissolving the linear dipeptide protected by tert-butyloxycarbonyl in a mixture of dichloromethane and trifluoroacetic acid at 0-5°C, and adding a catalytic amount of triisopropylsilane, stirring the resulting reaction mixture at 20-35°C for 20-40 minutes to obtain a linear dipeptide;

[0065] (3) dissolving the linear dipeptide in a mixture of isobutanol and toluene, refluxing the reaction under the protection of an inert gas at 85-95°C for 3-5 hours to obtain the cyclic dipeptide compound.

[0066] The present application also relates to a medicine for preventing and / or treating vascular endothelial senescence, which is prepared from the active ingredient comprising the cyclic dipeptide compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant.

[0067] The present application uses bio-membrane layer interferometry (BLI) to test the affinity of the cyclic dipeptide compound to HDAC4 protein, and uses rat aortic endothelial cells (RaECs) isolated from the thoracic aorta of 2-3-month-old SD rats to test the cytotoxicity of the cyclic dipeptide compound to RaECs. The drugability of the HDAC4 small-molecule inhibitor cyclic dipeptide compound is analyzed by computer, and the safety of the HDAC4 small-molecule inhibitor cyclic dipeptide compound is tested by using C57 mice. An AngII-induced senescence model is established, and the aorta is collected after 4 weeks of intragastrical administration, and then observed, counted and analyzed. The test results show that the HDAC4 small-molecule inhibitor cyclic dipeptide compound is dose-dependently combined with the HDAC4 protein; the HDAC4 small-molecule inhibitor cyclic dipeptide compound has no toxicity to the rat aortic endothelial cells within a dose of 250 uM, can effectively inhibit the protein expression of HADC4 and the senescence-related protein P21, can effectively reduce the expression of ROS and the cell senescence β-galactosidase SA-β-Gal, and can significantly reduce the mRNA expression amount of the senescence- and inflammation-related proteins P21, Il6, CCL2 and Il-1β, and has a significant anti-vascular endothelial cell senescence effect; the C57 mice are given a maximum dose of the HDAC4 small-molecule inhibitor cyclic dipeptide compound by intragastrical administration, and no mice die within 14 days after observation for 14 days; the HDAC4 small-molecule inhibitor cyclic dipeptide compound can inhibit the vascular thickness of the AngII-induced senescence model mice, and can reduce the protein expression amount of HDAC4 and P21 and the mRNA expression amount of the inflammation-related proteins Il6, CCL2 and Il-1β in the aorta in vivo. The above results prove that the HDAC4 small-molecule inhibitor cyclic dipeptide compound provided by the present application has a significant therapeutic effect on vascular endothelial senescence, is safe and effective, and can be used for preparing a medicine for treating vascular endothelial senescence and related diseases.

[0068] The present application is further described in detail below in combination with specific examples.

[0069] Example 1 Synthesis of histone deacetylase 4 (HDAC4) small-molecule inhibitor (CPT)

[0070]

[0071] N-tert-butoxycarbonyl-L-phenylalanine (Boc-L-Phe-OH, 530 mg, 2 mmol), 1- hydroxybenzotriazole (HOBT, 405 mg, 3 mmol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl, 575 mg, 3 mmol) were weighed into a 100 mL flask, dichloromethane (40 mL) was added, stirred to dissolve and cooled to 0 °C, then L-tyrosine methyl ester hydrochloride (510 mg, 2.2 mmol) and N,N-diisopropylethylamine (DIPEA, 775 mg, 6 mmol) were added slowly under stirring, nitrogen was purged into the reaction system, stirred for 30 min at 0 °C, then stirred for 1 h at room temperature, the reaction progress was monitored by TLC, after the reaction was completed, the reaction mixture was concentrated under reduced pressure and evaporated to dryness, extracted with dichloromethane, washed with water, the organic layer was collected, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a Boc-protected linear dipeptide (Boc-L-Phe-L-Tyr-OCH3-HCl), (815 mg, 85%), which was used in the next step without further purification.

[0072] Boc-protected linear dipeptide (Boc-L-Phe-L-Tyr-OCH3-HCl, 480 mg, 1 mmol) was weighed into a 50 mL flask, dissolved in dichloromethane / trifluoroacetic acid (DCM / TFA, 1:1 v / v, 20 mL) at 0 °C, and a catalytic amount of triisopropylsilane (TIPS, 0.5 mL) was added. The resulting reaction mixture was stirred at room temperature for 30 min. The reaction progress was monitored by TLC, after the reaction was completed, the reaction mixture was evaporated to dryness under reduced pressure and co-evaporated with toluene three times to obtain a linear dipeptide (H-L-Phe-L-Tyr-OCH3-HCl), (340 mg, 90%), which was used for cyclization without further purification.

[0073] Linear dipeptide (H-L-Phe-L-Tyr-OCH3-HCl, 308 mg, 0.9 mmol) was weighed into a 50 mL flask, isobutanol / toluene (1:1 v / v, 20 mL) was added, stirred to dissolve. The mixture was heated under reflux at 90 °C for 4 h under nitrogen protection. The reaction mixture was cooled to room temperature, the precipitate was filtered, washed with ice-cold ethanol (3 x 30 mL), and dried under vacuum to obtain a histone deacetylase 4 (HDAC4) small molecule inhibitor (named as CPT in the present application) 223 mg, with a yield of 80%. The nuclear magnetic resonance spectrum (as shown in Figure 1 and Figure 2 1 ​H NMR (600 MHz, DMSO) δ 9.24 (s, 1H), 7.87-7.82 (m, 2H), 7.27 (t, J = 7.4 Hz, 2H), 7.20 (t, J = 7.4 Hz, 1H), 7.03 (d, J = 6.9 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.6 Hz, 2H), 3.96 - 3.92 (m, 1H), 3.91-3.87 (m, 1H), 2.57 (dd, J = 13.6, 4.8 Hz, 1H), 2.52 (d, J = 4.6 Hz, 1H), 2.18 (ddd, J = 13.7, 9.7, 6.4 Hz, 2H). 13 C NMR (151 MHz, DMSO) δ 166.36, 156.15, 136.68, 130.89, 129.80, 128.25, 126.52, 126.46, 115.08, 55.77, 55.45, 38.58. The molecular weight was calculated as Figure 3 C 18 H 18 N2O3.

[0074] Example 2 Experiment of CPT binding to HDAC4 protein

[0075] Method: HDAC4-HIS tagged protein was immobilized on Ni-NTA biosensor (Fortebio), CPT was diluted into different concentrations (from 6.25 μΜ to 200 μΜ). After baseline setting with PBS, the biosensor tip was immersed into the well containing the serial dilutions of compound CPT, binding for 300 seconds, then dissociation step for 180 seconds, data was saved, KD value was calculated using data analysis software 9.0 (Fortebio), binding and dissociation curves were monitored in real-time mode.

[0076] Results please refer to Figure 4 , curve a is the binding and dissociation curve of 200 μΜ, curve b is the binding and dissociation curve of 100 μΜ, curve c is the binding and dissociation curve of 50 μΜ, curve d is the binding and dissociation curve of 25 μΜ, curve e is the binding and dissociation curve of 12.25 μΜ, curve f is the binding and dissociation curve of 6.25 μΜ; the linear regression determination coefficient R 2 = 0.8279 was calculated according to the binding and dissociation curve analysis, the equilibrium dissociation constant (KD) of the interaction between HDAC4 and CPT was 8.26 μΜ, the binding and dissociation of CPT to HDAC4 was dose-dependent and had strong affinity.

[0077] Example 3 Interaction site of CPT with HDAC4 protein

[0078] Methods: The best binding mode and interaction of CPT with HDAC4 were determined by Software virtual screening results were exported and visualized using Pymol (Version 2.5.2) software. The binding energy of CPT with HDAC4 was 53 kcal / mol, and the binding pocket was reasonable, which was conducive to maintaining the stable conformation of CPT and HDAC4, as shown in Figure 5 The active binding region includes multiple amino acid residues such as HIS-802, GLY-811, PHE-812, HIS-842, and RPO-942, which can form hydrogen bonds, van der Waals forces, and Pi-Pi conjugation and other interactions. Among them, GLY-811, PHE-812, HIS-842, and RPO-942 are of great significance to maintain the stability of the CPT and HDAC4 complex, and PHE-812 and HIS-842 are likely to be key sites for CPT to produce pharmacological effects.

[0079] Example 4 CPT cytotoxicity experiment

[0080] Methods: Rat aortic endothelial cells (RaECs) were isolated from the thoracic aorta of 2-3 month old SD rats, and the first three generations of RaECs were cultured in endothelial cell medium (ECM), which was composed of culture medium, 5% fetal bovine serum (FBS) and 1% endothelial cell growth supplement (ECGS). After the third generation, they were cultured in mixed culture medium composed of culture medium DMEM, 20% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The cells were cultured in a humidified incubator at 37°C and 5% CO2 for standby use. Twenty-four hours before the experiment, RaECs (1 x 10 4 cells / well) were seeded into 96-well plates and replaced with different concentrations of 100 μL CPT solution, with 5 replicates. The cells were then incubated for another 24 hours. Each well was cleaned with PBS, followed by replacement with freshly prepared serum-free medium containing 10% CCK-8 (NCM Biotech, China), and then the cells were incubated for another 4 hours. The optical density reading was performed at a wavelength of 450 nm using a multi-mode plate reader (Bioreader, Germany). The absorbance relative to the blank well was read. The cell survival rate (%) in each well was calculated by (OD450test-OD450blank) / (OD450control-OD450blank)*100%. The effect of CPT on RaECs was evaluated using a non-toxic dose.

[0081] As shown in Figure 6 CPT had no significant proliferation inhibition effect on rat aortic endothelial cells at a dose of 250 uM.

[0082] Example 5: Establishment and Use of an In Vitro Aging Model: Detection of Protein Level Changes Using Western Blotting

[0083] Methods: Twelve hours before modeling, RaECs cells in each group (including control group, model group, and drug treatment group) were starved in serum-free culture medium. Subsequently, the model group and drug treatment group were stimulated with angiotensin II at a final concentration of 2 μM / L for at least 48 hours. Fresh serum-free culture medium was added every 24 hours. The concentrations of CPT in the drug treatment group were 12.5 μM, 25 μM, and 50 μM, respectively. After modeling and drug administration, cells were lysed in ice-cold RIPA buffer containing 1 mM benzyl sulfonyl fluoride; proteins were obtained by centrifugation at 12,000 rpm for 15 minutes at 4°C; an equal volume of 10 μg protein was loaded onto a 10-15% SDS-PAGE unit and transferred to a nitrocellulose NC membrane by electroblotting; the NC membrane was blocked with 5% skim milk powder, stained with a 1:1000 diluted primary antibody, and then incubated overnight at 4°C; the cell membrane was then detected using a peroxidase-conjugated secondary antibody at a 1:10,000 dilution; the antigen-antibody complex was then detected using an enhanced chemiluminescence reagent, visualized using an AMERSHAmersham Image Quant 800 system, and analyzed using ImageJ software.

[0084] Analysis results as follows Figure 7 As shown, CPT can effectively reduce the protein expression levels of HADC4 and aging-related protein P21 in RaECs.

[0085] Example 6: Detection of Intracellular Reactive Oxygen Species (ROS) and Cellular Senescence β-Galactosidase SA-β-Gal

[0086] Methods: ROS detection kits were purchased from Beyotime. RaEC cells were cultured at 5 × 10⁶ cells / year. 4 The cells were seeded at a density of 1 / 2 well in 96-well plates. One day after seeding, the wells were treated with different doses of Ang II (2 μM) and CPT solution for 24 hours. To obtain dissociated microglia for ROS assay, the culture medium was first removed and the cells were washed three times with PBS. DCFH-DA was diluted to a final concentration of 10 μM with serum-free medium and added to the culture, and incubated at 37°C for 20 minutes. Fluorescence was read at 488 nm using an IN CellAnalyzer 6000 plate reader (Life, USA). Fluorescence intensity indicates intracellular ROS.

[0087] like Figure 8 As shown, CPT can significantly reduce the expression levels of ROS and cellular senescence β-galactosidase SA-β-Gal.

[0088] Establishment of in vitro aging model and mRNA level detection of Example 7

[0089] Method: 12 hours before modeling, the cells of each group (including the control group, the model group, and the drug group) were starved with serum-free medium, and then the model group and the drug group were stimulated with angiotensin II at a final concentration of 2 μM / L for at least 48 hours. The serum-free medium was changed every 24 hours. The CPT concentrations of the drug group were 12.5 μM, 25 μM, and 50 μM, respectively. β-nicotinamide mononucleotide (NMN) was used as a positive control drug at a concentration of 50 μM. The RaECs cells were stimulated with angiotensin II for at least 48 hours. After the stimulation, total RNA was obtained by TRIzol reagent. About 1 μg of total RNA of each sample was reversely transcribed into cDNA by a transgene kit, and then polymerase chain reaction (PCR) was performed using UltraSYBR mixture to detect in vitro aging markers and inflammatory indicators.

[0090] As shown in Figure 9 , CPT can significantly reduce the mRNA expression of aging and inflammation-related proteins P21, Il6, CCL2, and Il-1β.

[0091] Example 8: ADMET prediction of HDAC4 inhibitor CPT

[0092] Method: The Discovery Studio software was used to calculate the ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of the drug, i.e., aqueous solubility at 25 degrees Celsius, blood brain barrier penetration (BBB), cytochrome P4502D6 inhibition, hepatotoxicity, human intestinal absorption (HIA), and plasma protein binding. The ADMET properties of CPT were calculated by Small Molecules | Calculate Molecular Properties | ADMET Descriptors in Discovery Studio, with the default settings for parameter selection.

[0093] The results are shown in Figure 10As shown, the ADMET plot is a two-dimensional graph of ADMET_PSA_2D vs ADMET_AlogP98, representing the 95% and 99% confidence intervals of the blood-brain barrier permeability (BBB) ​​model and the human intestinal absorption (HIA) model, respectively. The experimental results show that CPT has good ADMET properties and good drug-likeness.

[0094] Example 9: CPT Animal Toxicity Experiment

[0095] C57BL / 6J mouse grouping: Sixteen 6-week-old, 22-26g, and healthy C57BL / 6J mice were randomly selected and randomly divided into a blank control group and a drug treatment group (n=10). The maximum dose of 2g / kg was administered by gavage once, and the mice were observed for 14 consecutive days.

[0096] like Figure 11 As shown in the results, no mice died within 14 days after administration, and no abnormalities were observed in their food and water intake, spontaneous activity, etc. The LD50 of CPT was >2 g / kg.

[0097] Example 10: Therapeutic effect of CPT on Ang II-induced aging mouse model

[0098] Mouse grouping: Forty-eight 6-week-old, 22-26g, and healthy C57BL / 6J mice were randomly selected and randomly divided into 6 groups (n=8): control group, angiotensin II infusion group, CPT (100, 50, and 25 mg / kg / day via gastric tube) group, and positive control (β-nicotinamide mononucleotide (NMN), 100 mg / kg / day via gastric tube).

[0099] Experimental method: After isoflurane anesthesia of mice, the hair on the back was removed, and a 1-2 cm incision was made with a scalpel, and an Alzet osmotic mini-pump (model 2004; ALZA Scientific Products, Mountain View, CA, USA) was implanted subcutaneously and sutured. The osmotic mini-pump of the control group only contained normal saline, and the osmotic mini-pump of the model group, the administration group and the positive control contained angiotensin II dissolved in normal saline. The administration group was given CPT at a drug concentration of 25-100 mg / Kg / day by gavage, and the positive control was given NMN positive control drug at a drug concentration of 100 mg / Kg / day by gavage. The administration time started from 7 days after modeling, that is, the administration lasted for 3 weeks, a total of 21 days. After the mice were continuously infused with normal saline or angiotensin II (1.5 mg / kg / d) for 4 weeks, the mice were euthanized by cervical dislocation, and the aorta of each mouse was cut into two sections. One section was immersed in liquid nitrogen and then stored at -80°C, and total RNA was extracted by Trizol method for gene expression detection. The other section was fixed with 4% paraformaldehyde overnight, then embedded in paraffin for immunofluorescence staining, and in this example, HDAC4 and p21 immunofluorescence double staining experiments were performed.

[0100] As shown in Figure 12 and Figure 13 , CPT can reduce the thickness of the aorta of the aging model mice induced by Ang II, reduce the expression of HDAC4, aging-related protein P21 protein and inflammatory-related protein Il6, CCL2 and Il-1β mRNA in the aorta of mice Figure 14 .

[0101] The above-described embodiments only express several embodiments of the present application, which are described in more detail and in more detail, but should not be understood as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims.

Claims

1. Use of a cyclic dipeptide compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of vascular endothelial senescence, characterized in that, A structural formula of the cyclic dipeptide compound is: 。 2. Use according to claim 1, characterized in that, The cyclic dipeptide compound can reduce the protein expression amount of the aging-related protein P21 in vascular endothelial cells; and / or, The cyclic dipeptide compound can reduce the mRNA expression amount of the aging-related protein P21 in vascular endothelial cells; and / or, The cyclic dipeptide compound can reduce the expression amount of ROS and cell aging beta-galactosidase SA-beta-Gal in vascular endothelial cells; and / or, The cyclic dipeptide compound can reduce the protein expression amount of the inflammation-related proteins Il6, CCL2 and Il-1beta in vascular endothelial cells; and / or, The cyclic dipeptide compound can reduce the mRNA expression amount of the inflammation-related proteins Il6, CCL2 and Il-1beta in vascular endothelial cells.

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