Application of tanshinol ester derivative in preparation of medicine for treating acute lung injury and acute respiratory distress syndrome

Abstract

The invention discloses application of tanshinol methyl ester and tanshinol ethyl ester in preparation of drugs for treating acute lung injury and acute respiratory distress syndrome. The tanshinol methyl ester and the tanshinol ethyl ester have a good protection effect on LPS (Lipopolysaccharide)-induced acute lung injury C57BL / 6 mice; molecular docking analysis results show that the two compounds can be strongly and specifically combined with NLRP3 protein to inhibit overexpression of inflammatory factors, so that the effect of resisting acute lung injury and acute respiratory distress syndrome is achieved. The salvianic acid A methyl ester and the salvianic acid A ethyl ester can be prepared into dosage forms such as dripping pills, tablets and emulsion for injection, and are used for treating acute lung injury and acute respiratory distress syndrome.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of danshensu ester derivatives in the preparation of drugs for treating acute lung injury and acute respiratory distress syndrome. Background Art

[0002] Acute lung injury (ALI) refers to pathological phenomena such as diffuse interstitial and alveolar edema, atelectasis, and alveolar hyaline membrane formation caused by viral infection, inhalation of toxic gases or particles, severe trauma, and other factors that lead to damage to alveolar epithelial cells and capillary endothelial cells, increased vascular permeability, and thus cause persistent hypoxemia and non-cardiogenic pulmonary edema. If lung function continues to deteriorate, it will progress to acute respiratory distress syndrome (ARDS), with a mortality rate as high as 25%-40%. Therefore, drugs for the treatment of ALI are also used to treat ARDS.

[0003] At present, the clinical treatment for ALI and ARDS mainly includes fluid resuscitation, the use of broad-spectrum antibiotics, and basic life support measures, but it is still difficult to delay the deterioration of the disease and the resulting high mortality rate. Therefore, how to effectively treat ALI and ARDS is still an important issue that needs to be solved in the fields of respiratory and critical care medicine and public health.

[0004] The NLRP3 inflammasome is a multiprotein complex composed of the innate immune sensor NLRP3, the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD), and the effector protein caspase-1. The innate immune sensor NLRP3 (i.e., NLRP3 protein) contains a NACHT domain consisting of three parts: helical domain 1, winged helix domain, and helical domain 2. This domain has the ability to regulate the nucleotide binding and self-aggregation of the NLRP3 protein, and plays an important role in the initiation and assembly of the NLRP3 inflammasome.

[0005] NLRP3 inflammasome has been shown to play a key role in the development and pathogenesis of ALI. When the body is stimulated by pathogenic factors such as viruses and chemicals (such as LPS), under the action of the NACHT domain, the NLRP3 protein begins to recruit molecules such as ASC, initiates the assembly and activation of the NLRP3 inflammasome, and leads to excessive production of inflammatory factors such as IL-1β, which in turn triggers a severe inflammatory response and aggravates the severity of ALI and ARDS. Therefore, NLRP3-binding specific anti-ALI and ARDS drugs have good clinical application prospects and are a hot spot for new drug screening and development research.

[0006] Danshensu is produced by the degradation of tanshinolic acid B in Salvia miltiorrhiza Bunge, a plant of the Labiatae family. It is one of the main active ingredients of tanshensu and has a carboxyl group in its molecular structure, so it can undergo esterification. In order to explore more active anti-acute lung injury drugs, the inventors screened the activity of tanshensu and its multiple ester derivatives in an animal model of acute lung injury induced by LPS, and found that tanshensu methyl ester and tanshensu ethyl ester, especially tanshensu ethyl ester, had good anti-acute lung injury activity; the results of computer-simulated molecular docking analysis showed that tanshensu methyl ester and tanshensu ethyl ester had strong binding specificity with NLRP3 protein, and this characteristic was one of the main mechanisms for their anti-acute lung injury and acute respiratory distress syndrome activity. Summary of the invention

[0007] The present invention provides a technical solution for preparing a medicine for treating acute lung injury and acute respiratory distress syndrome, and the solution is achieved through a tanshinone ester derivative with a structure of formula I.

[0008] Wherein, R is: C1~C2 alkyl.

[0009]

[0010] The compound of formula I provided by the present invention can be combined with a pharmaceutically acceptable excipient or carrier to form a pharmaceutical composition, and can be made into oral preparations such as capsules, soft capsules, pills, tablets, granules, or injectable preparations such as emulsions for injection, lyophilized powders for injection, microspheres for injection, or nanoformulations for injection.

[0011] The present invention provides a compound represented by formula I, or use of a pharmaceutical composition comprising the compound in preparing a drug for treating acute lung injury and acute respiratory distress syndrome.

[0012] The dosage of the pharmaceutical composition containing the compound of formula I of the present invention varies depending on the condition and individual differences such as patient gender, age, weight, as well as the route of administration, number of administrations, etc. Preferably, based on the content of the compound of formula I, the daily oral dose of the drug for adults is 5 to 5000 mg, and the daily injection dose for adults is 5 to 500 mg.

[0013] Compared with the prior art, the present invention has the following beneficial effects: it provides two tanshinone ester derivatives that can specifically bind to NLRP3 protein to inhibit the assembly and activation of NLRP3 inflammasomes, and these two tanshinone ester derivatives can be used to prepare drugs for treating acute lung injury and acute respiratory distress syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Effects of danshensu and its ester derivatives on lung pathological changes in ALI C57BL / 6 mice (HE staining)

[0015] Figure 2 Effects of danshensu and its ester derivatives on lung pathological changes in ALI C57BL / 6 mice (pathological scores)

[0016] Figure 3 Molecular docking diagram of danshensu methyl ester and NLRP3 inflammasome

[0017] Figure 4 Molecular docking diagram of tanshinone ethyl ester and NLRP3 inflammasome DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples without specifying specific conditions are usually operated under normal conditions or according to the conditions and methods recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0019] Example 1: Preparation of Danshensu

[0020] Take 10 kg of Danshen medicinal material, crush it into the coarsest powder, add 12 times of 0.4% sodium hydroxide solution and boil it twice, each time for 2 hours, combine the decoctions, adjust the pH value to 2-3 with hydrochloric acid, let it stand, filter, concentrate to 5L, add the sample to AB-8 type macroporous resin column at a ratio of 4 mg Danshensu / ml resin, first elute with 1 column volume of water to remove impurities, then elute with 30% ethanol for 3 column volumes, collect this part of the eluate, concentrate it to 1L, add activated carbon, stir, filter, add NaOH to adjust the pH value to 6.0-6.5, refrigerate at 0℃-5℃ for crystallization, wash and decolorize the crystals with a small amount of 80% ethanol solution, recrystallize in the same way, and dry under reduced pressure to obtain Danshensu sodium with a purity of 99% (HPLC).

[0021] The obtained sodium danshensu was dissolved in 1L 0.1mol / L hydrochloric acid solution and extracted three times with an equal volume of ethyl acetate. The extracts were combined and dried under reduced pressure at 50°C to obtain 126g of colorless oil, which turned into colorless crystals after being placed in a refrigerator for one week. The purity was 99.6% (HPLC).

[0022] 1H NMR (600MHz, DMSO) δ12.37 (s, 1H), 8.68 (s, 1H), 8.60 (s, 1H), 6.62 (d, J = 2.1Hz, 1H), 6.60 (d, J = 8.0Hz, 1H), 6.45 (dd, J = 8.0, 2.1Hz, 1H), 5.17 (s, 1H), 4.03 (dd, J = 7.9, 4.8Hz, 1H), 2.76 (dd, J = 13.8, 4.7Hz, 1H), 2.60 (dd, J = 13.8, 4.7Hz, 1H); 13 C NMR (151 MHz, DMSO) δ 175.72, 145.17, 144.06, 129.24, 120.51, 117.32, 115.63, 71.93, 40.25. Its structure was confirmed to be danshensu.

[0023] Example 2: Preparation of Danshensu Methyl Ester

[0024] Take 10 g of Danshensu, dissolve it in 100 mL of anhydrous methanol, add 2 mL of hydrochloric acid, react at room temperature for 4 h, remove methanol under reduced pressure at 45 ° C, dissolve it in an appropriate amount of ethyl acetate, wash three times with water, collect the ethyl acetate phase, remove the organic solvent under reduced pressure to obtain a colorless oily liquid.

[0025] 1 H NMR (600MHz, DMSO) δ8.71 (s, 1H), 8.62 (s, 1H), 6.626.57 (m, 2H), 6.42 (dd, J=8.0, 2.1Hz, 1H), 5.45 (d, J =6.0Hz, 1H), 4.12 (dt, J=7.4, 5.5Hz, 1H), 3.59 (s, 3H), 2.74 (dd, J=13.7, 5.4Hz, 1H), 2.67-2.60 (m, 1H); 13 CNMR (151 MHz, DMSO) δ 174.50, 145.24, 144.16, 128.70, 120.44, 117.22, 115.70, 72.14, 51.76. Its structure was confirmed to be danshensu methyl ester.

[0026] Example 3: Preparation of Danshensu Ethyl Ester

[0027] Take 50g of Danshensu, dissolve it in 250mL of anhydrous ethanol, add 2.5mL of hydrochloric acid, react at 60℃ for 4h, remove ethanol under reduced pressure at 45℃, dissolve in appropriate amount of ethyl acetate, wash three times with water, collect the ethyl acetate phase, remove the organic solvent under reduced pressure to obtain a colorless oily liquid.

[0028] 1H NMR (600MHz, DMSO) δ8.70 (s, 1H), 8.62 (s, 1H), 6.62-6.57 (m, 2H), 6.42 (dd, J=8.0, 2.1Hz, 1H), 5.41 (d, J=6.1Hz, 1H), 4. 10 (dt, J=7.2, 5.7Hz, 1H), 4.04 (q, J=7.1Hz, 2H), 2.73 (dd, J=13.7, 5.5Hz, 1H), 2.682.61 (m, 1H), 1.14 (t, J=7.1Hz, 3H); 13 C NMR (151 MHz, DMSO) δ 174.03, 145.22, 144.15, 128.66, 120.48, 117.27, 115.67, 72.16, 60.37, 40.63, 14.55. Its structure was confirmed to be tanshinone ethyl ester.

[0029] Example 4: Preparation of Danshensu Isopropyl Ester

[0030] Take 10g of Danshen, dissolve it in 500mL of isopropanol, add 2.5mL of hydrochloric acid, react at 60℃ for 6h, remove ethanol under reduced pressure at 45℃, dissolve in appropriate amount of ethyl acetate, wash three times with water, collect the ethyl acetate phase, remove the organic solvent under reduced pressure to obtain a colorless oily liquid. Chromatography on a silica gel column, eluting with ethyl acetate-methanol (4:1), TLC detection, collect the part where the main spot is concentrated, remove the organic solvent under reduced pressure to obtain a colorless oily liquid.

[0031] 1 H NMR (600MHz, DMSO) δ8.71 (s, 1H), 8.62 (s, 1H), 6.626.57 (m, 2H), 6.42 (dd, J=8.0, 2.1Hz, 1H), 5.45 (d, J=6.0Hz , 1H), 4.12 (dt, J=7.4, 5.5Hz, 1H), 4.49 (m, 1H), 2.74 (dd, J=13.7, 5.4Hz, 1H), 2.67-2.60 (m, 1H), 1.15 (d, 6H); 13 C NMR (151 MHz, DMSO) δ 174.50, 145.24, 144.16, 128.70, 120.44, 117.22, 115.70, 72.14, 60.63, 40.58, 21.90. Its structure was confirmed to be danshensu isopropyl ester.

[0032] Example 5: Preparation of Danshensu Ethyl Ester Dropping Pills

[0033] prescription:

[0034]

[0035] The specific operation is: weigh the above-mentioned prescribed amount of PEG400, PEG4000, sodium carboxymethyl starch, and povidone K30, add them into a pill machine at 95°C to melt, add the prescribed amount of danshensu ethyl ester while stirring, mix thoroughly, the temperature of the medicine is 85°C, and the medicine is dripped into liquid paraffin at a drop distance of 20 cm and a drop rate of 28 drops / min. Condensate and solidify at 10°C to form pills, then take out and dry to obtain danshensu ethyl ester pills.

[0036] Example 6: Preparation of Danshensu Ethyl Ester Injection Emulsion

[0037] prescription:

[0038]

[0039] Operation: Weigh the prescribed amount of glycerol, add an appropriate amount of water for injection, dissolve it, prepare a glycerol aqueous solution, keep it warm at 50-60℃, as the water phase; weigh the prescribed amount of phospholipids, poloxamer and soybean oil, mix them, stir and dissolve them at 50-70℃, as the oil phase; add oleic acid and danshensu ethyl ester to the oil phase, stir to mix evenly, add them to the water phase while shearing, high-speed shearing to form uniform colostrum, dilute to 5000mL with water for injection, pass through a homogenizer, and obtain lipid microspheres with uniform particle size and an average particle size of 160-280nm; filter the obtained liquid with a 0.22μm membrane, fill it with nitrogen, fill it, and sterilize it with circulating steam at 125℃ for 5 minutes. 5mL / bottle, each bottle contains 25mg of danshensu ethyl ester.

[0040] Example 7: Therapeutic effects of danshensu and its esters on LPS-induced acute lung injury in mice

[0041] 1. Materials

[0042] 1.1 Experimental animals: 6-8 week old female C57BL / 6 mice, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., animal certificate number: SCXK(Lu)20140007, were kept in a clean animal observation room and fed adaptively for 3 days. During the administration period, they were allowed to drink water and eat freely.

[0043] 1.2 Drugs and reagents: Lipopolysaccharide (LPS, L8880) and dexamethasone (DEX, D6950) were purchased from Solebao Biotechnology Co., Ltd. (Beijing, China). TNF-a (abs520010), IL-6 (abs520004), and IL-1β (abs520001) mouse enzyme-linked immunosorbent assay (ELISA) kits were purchased from Aibixin Biotechnology Co., Ltd. (Shanghai, China). Anti-NLRP3 (A5652) antibody was purchased from ABclonal (San Mateo, CA USA) and anti-mouse caspase-1 (645101) antibody was purchased from Biolegend (San Diego, CA, USA). Anti-ASC (67824), anti-NF-κB p65 (3033S) and anti-NF-κB p-p65 (8242S) antibodies were from CST (Danvers MA, USA), and anti-β-actin (AP0060) antibody was purchased from Bioworld (Nanjing, China). Danshensu (DSS), danshensu methyl ester (DME), danshensu ethyl ester (DEE), and danshensu isopropyl ester (DIE) were all homemade in the laboratory (see Examples 1-4), and were prepared into sterile emulsions with 0.5% Tween 80 for injection.

[0044] 2. Methods

[0045] 2.1 Animal modeling and grouping

[0046] 42 (C57BL / 6) mice were randomly divided into 7 groups: control group (Control), model group (ALI), DSS (30mg / kg) group, DME (30mg / kg) group, DEE (30mg / kg) group, DIE (30mg / kg) group and DEX (5mg / kg) group. During modeling, except for the control group, the mice in other groups were intratracheally instilled with LPS (0.5mg / kg). 12 hours after modeling, except for the control group and the model group, which were intraperitoneally injected with an equal volume of normal saline, the mice in other groups were intraperitoneally injected with DSS, DME, DEE, DIE and DEX for three consecutive days, once a day in the morning and evening. 12 hours after the last administration, the mice in each group were intraperitoneally injected with 0.12-0.14ml of 1% sodium pentobarbital solution, and the mice were killed under anesthesia, and then lung tissue samples were collected.

[0047] 2.2 Pathological observation and analysis

[0048] Single lobe right lung tissue of mice was collected and fixed in 4% paraformaldehyde (Biosharp) for 24 hours, followed by 8 hours of water washing to remove excess fixatives. The tissue was then dehydrated with a series of alcohol concentration gradients, embedded after 6 hours of paraffin immersion, and cut into 5 μm thick slices, HE stained, and pathologically scored and analyzed using Image J software. The lung injury score was based on a 5-point scale of 0 to 4, where 0 means no injury, 1 means mild injury, 2 means moderate injury, 3 means severe injury, and 4 means extremely severe injury.

[0049] 3. Experimental results

[0050] The experimental results are only part of the research content in this embodiment.

[0051] Representative results are shown in the Appendix Figure 1 With attached Figure 2 :Effects of tanshinone and its ester derivatives on lung pathological changes in ALI C57BL / 6 mice (HE staining and Image J pathological scoring analysis).

[0052] HE staining results of lung tissue sections of mice in each group showed that compared with the control group, the alveolar wall of ALI mice was thickened, the alveolar cavity was narrowed, the number of inflammatory infiltrating cells increased, and the pulmonary hemorrhage increased, indicating that the ALI model was successful. Compared with the ALI model group, the alveolar structure of mice in the DME (30 mg / kg) group and the DEE (30 mg / kg) group was restored, and the alveolar wall damage was significantly improved, indicating that DME and DEE can effectively reduce LPS-induced lung injury, and even the effect of DEE (30 mg / kg) is equivalent to that of the hormone dexamethasone (DEX, 5 mg / kg). Figure 2 The Image J analysis results clearly showed this trend.

[0053] Example 8: Molecular docking analysis

[0054] In view of the key role of NLRP3 inflammasome in the course of ALI, the inventors used molecular docking to investigate the binding specificity of tanshinone and its ester derivatives to NLRP3 protein. Molecular docking is a method of drug design based on the characteristics of receptors and the interaction between receptors and drug molecules. It mainly studies the interaction between molecules (such as ligands and receptors) and predicts a theoretical simulation method of their binding mode and affinity. In recent years, molecular docking has become an important technology in the field of computer-assisted drug research, which can provide an important reference for the screening of lead drugs.

[0055] 1. Methods

[0056] Molecular docking was performed using Discovery Studio 2019 (Dassault Systèmes, France). The NLRP3 crystal structure (PDB: 7PZC) was obtained from the Protein Data Bank (PDB). Protein preparation was performed by removing water molecules, adding missing atoms, and optimizing the receptor structure. The ligand three-dimensional conformation was then generated and the ligand geometry was optimized. Molecular docking (LibDock) was run under default settings. The docking results were evaluated to determine the most reasonable docking pose. PyMOL ( Inc., USA) for visual analysis.

[0057] 2. Results

[0058] The molecular docking scores of danshensu, danshensu methyl ester, danshensu ethyl ester and danshensu isopropyl ester are shown in Table 1.

[0059] Table 1. Molecular docking scoring results

[0060]

[0061] It is generally believed that when the docking energy value is less than -4.25 kcal / mol, it indicates a certain binding activity between the two, less than -5.0 kcal / mol indicates good binding activity, and less than -7.0 kcal / mol indicates strong binding activity. The results of molecular docking analysis showed that the docking scores of DME, DEE and NLRP3 were -7.10 (kcal / mo1) and -7.34 (kcal / mol), respectively, indicating that there is a strong interaction between these two compounds and the NLRP3 protein. However, DSS and DIE do not have this strong binding property with the NLRP3 inflammasome.

[0062] Binding model of DME or DEE and NLRP3 protein (see Appendix Figure 3 and attached Figure 4). The analysis found that the hydrophobic pocket occupied by DME or DEE after binding to NLRP3 protein is located in the NACHT domain, where the specific binding site of DME is: GLN180PHE 683CYS 706PRO 709HIS 713ASP 750ASN 751SER 752ARG 779CVS 780ASP 807; the specific binding site of DEE is: ARG 172LEU 371PHE 373SER 374GLU 375ARG 378PHE 410SER 658THR659ARG 660HIS 724LEU 725THR 726SER 728PHE 729. This hydrogen bond binding between small molecules and specific protein sites has the characteristics of targeted specificity. It can be inferred that DME and DEE should inhibit the assembly and activation of NLRP3 inflammasome after binding to the NACHT domain of NLRP3.

[0063] The above animal-level pharmacodynamic study results show that danshensu methyl ester (DME) and danshensu ethyl ester (DEE) have a good protective effect on LPS-induced acute lung injury C57BL / 6 mice; the molecular docking analysis results show that the mechanism is that DME and DEE can bind to the NACHT domain in the NLRP3 protein, thereby blocking the initiation and assembly of the NLRP3 inflammasome, reducing the expression of inflammatory factors, and achieving the effect of anti-acute lung injury. The pathological manifestations of danshensu and danshensu isopropyl ester in Example 7 prove that this strong binding specificity with the NLRP3 inflammasome is not universally possessed by danshensu and its ester derivatives. Since the continued deterioration of acute lung injury will progress to acute respiratory distress syndrome, the above examples also show that danshensu methyl ester (DME) and danshensu ethyl ester (DEE) involved in the present invention can also be used to prepare drugs for treating acute respiratory distress syndrome.

[0064] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a danshensu derivative represented by formula I in the preparation of a drug for treating acute lung injury and acute respiratory distress syndrome, 2. The use according to claim 2, characterized in that: In the compound of formula I, R is an alkyl group having 1 to 2 carbon atoms.

3. The use according to any one of claims 1 to 3, characterized in that: When the compound of formula I is used to prepare drugs for treating acute lung injury and acute respiratory distress syndrome, the daily oral dose for adults is 5 to 5000 mg, and the daily injection dose for adults is 5 to 500 mg.

4. The use according to claim 5, characterized in that: When used to prepare drugs for treating acute lung injury and acute respiratory distress syndrome, the compound of formula I can be combined with a pharmaceutically acceptable excipient or carrier to form a pharmaceutical composition, prepared into oral preparations such as capsules, soft capsules, pills, tablets, granules, or injectable preparations such as emulsions for injection, lyophilized powders for injection, microspheres for injection, or nanoformulations for injection.

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