Novel myricetin-colchicine hybrid and its synthesis method and application

Abstract

The present invention discloses a novel myricetin-colchicine hybrid and its synthesis method and application. Animal experimental studies have confirmed that this novel myricetin-colchicine hybrid (CMyrH) has a good therapeutic effect on acute lung injury and is a potential low-toxicity and highly effective anti-ALI / ARDS drug for the clinical prevention and treatment of ALI / ARDS. It can not only solve the problems of narrow clinical treatment window and severe side effects of colchicine in anti-ALI / ARDS, but also enhance the anti-inflammatory and anti-ALI / ARDS activities of colchicine, showing good medicinal prospects. The synthesis method of the present invention uses colchicine and myricitrin as raw materials. First, an amino group is introduced into the colchicine molecule under the condition of sodium hydroxide, and then the retained carboxyl group is dehydrated and esterified with pentamethylmyricetin to obtain the final product, the novel myricetin-colchicine hybrid. This synthesis method has the advantages of inexpensive and easily available raw materials, simple operation, low cost and high yield.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a new type of myricetin-colchicine hybrid and a synthesis method and application thereof. Background Art

[0002] Acute lung injury (ALI) is an inflammatory disease caused by lung inflammation, ischemia-reperfusion, sepsis, etc. The pathological characteristics of ALI include inflammatory cell infiltration, production of proinflammatory mediators, increased levels of oxidative stress, and disruption of vascular permeability in lung tissue, which ultimately lead to destruction of the alveolar capillary epithelial barrier and pulmonary edema. Acute Respiratory Distress Syndrome (ARDS) is the main cause of death in intensive care units (ICUs). It is reported that 10% of ICU patients and 23% of mechanically ventilated patients suffer from ARDS. 40% to 58% of patients with moderate to severe ARDS have diffuse alveolar damage, and the mortality rate of severe ARDS is as high as 45%. In addition, the COVID-19 pandemic has led to an increase in ARDS.

[0003] At present, in the treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), some drugs are listed as alternative intervention methods due to their characteristics: glucocorticoids can inhibit the generation and release of proinflammatory factors, improve the time of weaning from mechanical ventilation, respiratory compliance and oxygenation; surfactants can maintain alveolar opening and promote gas exchange, improve oxygenation and shorten mechanical ventilation time; antioxidants improve patients' blood oxygen saturation and oxygen partial pressure by removing excess oxygen free radicals in lung tissue; non-steroidal drugs play an anti-inflammatory role by causing platelet aggregation and reduce the protein and white blood cell content in alveolar lavage fluid; vasodilators improve oxygenation by improving tissue oxygen supply, reducing pulmonary hypertension; in addition, sivelestat sodium is used in the treatment of ALI, but the actual situation of the patient needs to be considered. In addition, although the respiratory support strategy can quickly improve the patient's hypoxemia and hypercapnia, the complications caused by respiratory support, such as pneumothorax, atelectasis, and human-machine confrontation, should still be considered. Therefore, there is currently no specific and effective therapeutic drug for ALI / ARDS, and it is very important to explore new drugs and targets that can effectively prevent and treat ALI / ARDS.

[0004] Colchicine is a natural alkaloid with potent anti-inflammatory ability. It was initially isolated from the seeds and bulbs of Colchicum autumnale, a plant in the Liliaceae family, and is the earliest known microtubule protein polymerization inhibitor. Colchicine has various biological activities, such as anti-tumor and anti-inflammatory effects, and is mostly used for the treatment of familial Mediterranean fever, acute gout, and as a prophylactic drug for gouty arthritis. With the in-depth progress of research, colchicine is currently also used as a second-line drug for the treatment of acute lung injury caused by novel coronavirus infection. The results of recent clinical randomized controlled trials (RCTs) reported in authoritative SCI journals show that colchicine can effectively reduce the mortality rate of severe patients with novel coronavirus infection (COVID-19) (the severe mortality rate decreased from 44% to 20%). Its mechanism is related to the fact that colchicine can inhibit ALI / ARDS and cytokine storm in patients with severe COVID-19 pneumonia. However, the toxic and side effects of colchicine affect the safety of drug use, severely limiting the clinical application of this toxic compound. After oral administration, colchicine is rapidly absorbed, reaching the peak plasma concentration within 0.5 - 3 hours, and there is a first-pass effect in the liver, with a bioavailability of 20% - 50%. After absorption, colchicine is rapidly distributed in various tissues and tightly binds to microtubulin in cells. Once a colchicine poisoning reaction occurs, there is no specific drug to dissociate it and excrete it from the body. Therefore, as a microtubulin inhibitor, colchicine has the disadvantages of high toxicity and many adverse reactions, which has a great impact on the safety of drug use. Colchicine has a prominent therapeutic effect in ALI / ARDS. It is necessary to modify its chemical structure to overcome its disadvantages of large toxic and side effects and improve its anti-ALI / ARDS activity. Myricetin is a class of natural polyhydroxyflavonol compounds widely present in vegetables, fruits, and medicinal plants. There are research reports that myricetin has pharmacological effects such as hypoglycemic, liver-protecting, antioxidant, antiviral, anti-cancer, protecting the nervous system, and anti-cardiovascular diseases, and also has pharmacological activities such as anti-inflammatory and immunomodulatory effects. Currently, myricetin is mainly used in the health product field. As a dietary flavonoid supplement, it has been marketed as a health product in Europe.

[0005] In view of the obvious toxic and side effects of colchicine, the present invention modifies the chemical structure of colchicine in order to reduce its toxic and side effects and provide a new strategy for the treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Summary of the Invention

[0006] The object of the present invention is to provide a novel myricetin-colchicine hybrid and its synthesis method and application. By using the ophiocoma synthetic method, a novel C-10 modified "multi-functional" myricetin-colchicine hybrid (Colchicine Myricetin Hybrid, CMyrH) is synthesized for the first time. Pharmacological experiments have proved that the novel myricetin-colchicine hybrid provided by the present invention is a potential low-toxicity and high-efficiency anti-ALI / ARDS drug for the clinical prevention and treatment of ALI / ARDS. It can not only solve the problems of narrow clinical treatment window and serious side effects of colchicine in anti-ALI / ARDS treatment, but also enhance the anti-inflammatory and anti-ALI / ARDS activities of colchicine.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A novel myricetin-colchicine hybrid is synthesized from colchicine and myricetin as raw materials.

[0009] Further, the molecular formula of the above-mentioned novel myricetin-colchicine hybrid is: C 44 H 46 N2O 14 , and the structure is:

[0010]

[0011] Furthermore, the above-mentioned novel myricetin-colchicine hybrid is a light yellow solid; melting point: 182 °C - 184 °C; 1H NMR: 1H NMR (400 MHz, CDCl3) δ 7.53–7.43 (m, 3H), 7.41 (d, J = 11.1 Hz, 1H), 7.05 (s, 2H), 6.63 (d, J = 11.3 Hz, 1H), 6.55 (d, J = 2.2 Hz, 1H), 6.51 (s, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.71–4.64 (m, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.88 (s, 6H), 3.62 (s, 3H), 3.08 (t, J = 7.4 Hz, 2H), 2.46 (dd, J = 13.3, 6.5 Hz, 1H), 2.36 (td, J = 12.9, 6.7 Hz, 1H), 2.28–2.18 (m, 1H), 1.98 (s, 3H), 1.94–1.80 (m, 3H); 13C NMR: 13C NMR (101 MHz, CDCl3) δ 175.47, 170.48, 170.07, 168.84, 164.74, 161.30, 159.34, 153.84, 153.40, 153.04, 151.45, 151.42, 151.24, 141.66, 140.70, 139.11, 134.68, 134.08, 130.99, 126.95, 124.81, 123.68, 108.75, 108.33, 107.32, 105.77, 104.98, 96.41, 92.88, 61.56, 61.45, 61.15, 60.56, 56.55, 56.48, 56.26, 56.04, 52.68, 38.51, 37.25, 33.31, 30.21, 23.01, 14.34; High resolution mass spectrometry: HRMS (m / z): calcd for C 44 H 46 N2NaO 14 [M+Na]+ 849.2841, found 849.2859.

[0012] The synthesis method of the novel myricetin-colchicine hybrid as described in claim 1 comprises the following steps:

[0013] (1) Synthesis of intermediate pentamethylmyricetin

[0014]

[0015] ① Take myricetin and potassium carbonate, mix them evenly, add acetonitrile, stir well, then add dimethyl sulfate, stir well to obtain a mixed solution;

[0016] ② The mixed solution is refluxed for 24 hours, filtered, and the filtrate is desolvated to obtain crude product 1;

[0017] ③ Take crude product 1, dissolve it in ethanol, then add concentrated hydrochloric acid, react at 70 °C for 12 hours, desolvate to remove ethanol to obtain crude product 2;

[0018] ④ Take crude product 2, dissolve it in 50 mL of dichloromethane, wash it with 50 mL of water, dry it with 50 g of anhydrous sodium sulfate, filter, and desolvate the filtrate to obtain the intermediate pentamethylmyricetin;

[0019] (2) Synthesis of novel myricetin-colchicine hybrid

[0020]

[0021] ① Take colchicine and amino acid, mix them evenly, add ethanol and mix well, then add sodium hydroxide solution, mix well, and react at 70 °C for 5 hours;

[0022] ② The reaction solution is placed in a rotary evaporator to remove the ethanol solvent, wash it once with 15 mL of ethyl acetate, separate the aqueous phase, adjust the pH value to 4 - 5 with hydrochloric acid, wait for white solid to precipitate, extract and wash it 3 times with dichloromethane, 30 mL each time, then dry it with 50 g of anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain a solid;

[0023] ③ Take the solid obtained in ②, dissolve it in dichloromethane, add the pentamethylmyricetin obtained in step (1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine respectively, mix well, react at room temperature for 12 hours, quench with water, wash the reaction solution with 30 mL of water, and separate the aqueous phase;

[0024] ④ Extract the aqueous phase with dichloromethane 3 times, 50 mL each time, combine the organic phases, dry with 50 g of anhydrous sodium sulfate, filter, and concentrate the filtrate until no more distillate is obtained to obtain a crude product;

[0025] ⑤ Take the crude product obtained in ④, perform silica gel column chromatography, elute, collect the eluate, and desolvate to obtain the novel myricetin-colchicine hybrid.

[0026] Further, in the synthesis method of the novel myricetin-colchicine hybrid described above, in step (1) item ①, the dosage of myricetin is 0.8 g, the dosage of potassium carbonate is 1.8 g, the dosage of acetonitrile is 40 mL, and the dosage of dimethyl sulfate is 1.2 mL.

[0027] Further, in the synthesis method of the novel myricetin-colchicine hybrid described above, in step (1) item ③, the dosage of ethanol is 40 mL, and the dosage of concentrated hydrochloric acid is 1.4 mL.

[0028] Further, in the synthesis method of the novel myricetin-colchicine hybrid described above, in step (2) item ①, the dosage of colchicine is 2.0 g, the dosage of amino acid is 10 mmol, the dosage of ethanol is 10 mL, and the concentration of the sodium hydroxide solution used is 1 mol / L.

[0029] Further, in the synthesis method of the novel myricetin-colchicine hybrid described above, in step (2) item ③, the dosage of dichloromethane is 50 mL, the dosage of pentamethylmyricetin is 1.9 g, the dosage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1.0 g, and the dosage of 4-dimethylaminopyridine is 1.22 mg.

[0030] Further, in the synthesis method of the novel myricetin-colchicine hybrid described above, in step (2) item ⑤, the eluent used for silica gel column chromatography is a petroleum ether-ethyl acetate solution with a volume ratio of 1:1, and the dosage is 500 mL.

[0031] The application of the novel myricetin-colchicine hybrid as described in claim 1 is for the application in the preparation of a drug for preventing and treating acute lung injury.

[0032] The beneficial effects of the present invention are as follows:

[0033] 1. It is confirmed by animal experimental studies that the novel myricetin-colchicine hybrid (CMyrH) obtained by the synthesis method of the present invention can reduce the toxic and side effects of colchicine, reduce the mortality of model rats, and significantly reduce the acute pulmonary edema (lung wet / dry weight ratio), lung tissue pathology score, and the expression levels of inflammatory factors (IL-1β, IL-6, IL-18, and TNF-α) in BLM-induced ALI model rats. Further molecular docking studies found that CMyrH also has good binding affinity for the key pathological target of acute lung injury (neutrophil elastase, NE). It shows that the novel myricetin-colchicine hybrid (CMyrH) provided by the present invention has a good therapeutic effect on acute lung injury, and is a potential low-toxicity and high-efficiency anti-ALI / ARDS drug for the clinical prevention and treatment of ALI / ARDS. It can not only solve the problems of narrow clinical treatment window and severe toxic and side effects of colchicine in anti-ALI / ARDS, but also enhance the anti-inflammatory and anti-ALI / ARDS activities of colchicine, and has good medicinal prospects.

[0034] 2. The present invention provides a synthesis method of a novel myricetin-colchicine hybrid. This synthesis method uses colchicine and myricitrin as raw materials, which are inexpensive and easily available. With an amino acid as the linker, the method is simple and easy to operate. First, an amino group is introduced into the colchicine molecule under the condition of sodium hydroxide, and then the retained carboxyl group is subjected to dehydration esterification with pentamethylmyricetin to obtain the final product, the novel myricetin-colchicine hybrid. The whole synthesis route has a low cost and a high yield. Description of the Drawings

[0035] Figure 1 It is a graph showing the comparison results of the survival rates of rats in each ALI model group. Among them, Ctrl: blank control group; Mod: model group; CMyrH 1.0 mg / kg i.p.: 1.0 mg / kg novel myricetin-colchicine hybrid group; CMyrH 3.0 mg / kg i.p.: 3.0 mg / kg novel myricetin-colchicine hybrid group; Col 1.0 mg / kg i.p.: 1.0 mg / kg colchicine group; Col 1.5 mg / kg i.p.: 1.0 mg / kg colchicine group.

[0036] Figure 2 It is a graph showing the comparison results of pulmonary edema (lung wet / dry ratio) of rats in each group. Among them, Ctrl: blank control group; Mod: model group; Col 1.0 mg / kg i.p.: 1.0 mg / kg colchicine group; CMyrH 1.0 mg / kg i.p.: 1.0 mg / kg novel myricetin-colchicine hybrid group; CMyrH 3.0 mg / kg i.p.: 3.0 mg / kg novel myricetin-colchicine hybrid group.

[0037] Figure 3 It is a graph showing the comparison results of the histopathology of the lung tissues of rats in each group. Among them, the left figure is an image of the histopathology of the rat lung tissue magnified 40×, and the right figure is an image of the histopathology of the rat lung tissue magnified 200×; Ctrl: blank control group; Mod: model group; Col 1.0 mg / kg i.p.: 1.0 mg / kg colchicine group; CMyrH 1.0 mg / kg i.p.: 1.0 mg / kg novel myricetin-colchicine hybrid group; CMyrH 3.0 mg / kg i.p.: 3.0 mg / kg novel myricetin-colchicine hybrid group.

[0038] Figure 4The figure shows the histopathological scoring results of the lung tissues of rats in each group. Among them, Ctrl: blank control group; Mod: model group; Col 1.0mg / kg i.p.: colchicine group at 1.0mg / kg; CMyrH 1.0mg / kg i.p.: novel myricetin-colchicine hybrid group at 1.0mg / kg; CMyrH 3.0mg / kg i.p.: novel myricetin-colchicine hybrid group at 3.0mg / kg.

[0039] Figure 5 The figure shows the comparison results of the expression levels of inflammatory factors in the lung tissues of rats in each group. Among them, A shows the comparison of the expression level of IL-1β in the lung tissues of rats in each group, B shows the comparison of the expression level of IL-6 in the lung tissues of rats in each group, C shows the comparison of the expression level of IL-18 in the lung tissues of rats in each group, and D shows the comparison of the expression level of TNF-α in the lung tissues of rats in each group; Ctrl: blank control group; Mod: model group; Col1.0mg / kg i.p.: colchicine group at 1.0mg / kg; CMyrH 1.0mg / kg i.p.: novel myricetin-colchicine hybrid group at 1.0mg / kg; CMyrH 3.0mg / kg i.p.: novel myricetin-colchicine hybrid group at 3.0mg / kg.

[0040] Figure 6 The figure shows the molecular docking diagrams of CMyrH, colchicine, and myricetin with neutrophil elastase (HNE), a key pathological target of acute lung injury. Among them, A is the molecular docking diagram of CMyrH with HNE enzyme, B is the molecular docking diagram of colchicine with HNE enzyme, and C is the molecular docking diagram of myricetin with HNE enzyme. Detailed implementation manners

[0041] The present invention will be further described below in combination with specific embodiments, but the protection scope and application scope of the present invention are not limited.

[0042] I. Synthesis of novel myricetin-colchicine hybrid (CMyrH)

[0043] Example 1 Synthesis of novel myricetin-colchicine hybrid (CMyrH)

[0044] (1) Synthesis of intermediate pentamethylmyricetin (3-hydroxy-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one)

[0045] Synthesis route:

[0046]

[0047] ① Take 0.8 g of myricetin and 1.8 g of potassium carbonate, mix them evenly, add 40 mL of acetonitrile, stir well, then add 1.2 mL of dimethyl sulfate, stir well to obtain a mixed solution;

[0048] ② The mixed solution is refluxed for 24 hours, filtered, and the filtrate is desolvated to obtain crude product 1;

[0049] ③ Take crude product 1, dissolve it in 40 mL of ethanol, then add 1.4 mL of concentrated hydrochloric acid, react at 70 °C for 12 hours, desolvate to remove ethanol to obtain crude product 2;

[0050] ④ Take crude product 2, dissolve it in 50 mL of dichloromethane, wash it with 50 mL of water, dry it with 50 g of anhydrous sodium sulfate, filter, and desolvate the filtrate to obtain the intermediate pentamethylmyricetin;

[0051] (2) Synthesis of novel myricetin-colchicine hybrid

[0052] Synthesis route:

[0053]

[0054] ① Take 2.0 g of colchicine and 10 mmol of amino acid, mix them evenly, add 10 mL of ethanol and mix well, then add sodium hydroxide solution (0.4 g of sodium hydroxide dissolved in 10 mL of water), mix well, and react at 70 °C for 5 hours;

[0055] ② The reaction solution is placed in a rotary evaporator to remove the ethanol solvent, washed once with 15 mL of ethyl acetate, the aqueous phase is separated, the pH value is adjusted to 4 - 5 with hydrochloric acid, wait for white solid to precipitate, extract and wash with dichloromethane 3 times, 30 mL each time, then dry with 50 g of anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain a solid;

[0056] ③ Take the solid obtained in ②, dissolve it in 50 mL of dichloromethane, add 1.9 g of the pentamethylmyricetin obtained in step (1), 1.0 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.22 mg of 4-dimethylaminopyridine, mix well, react at room temperature for 12 hours, quench with water, wash the reaction solution with 30 mL of water, and separate the aqueous phase;

[0057] ④ The aqueous phase is extracted with dichloromethane 3 times, 50 mL each time, the organic phases are combined, dried with 50 g of anhydrous sodium sulfate, filtered, and the filtrate is concentrated until no more distillate is obtained to obtain a crude product;

[0058] ⑤ Take the crude product obtained in ④, perform silica gel column chromatography, elute with 500 mL of a petroleum ether - ethyl acetate solution with a volume ratio of 1:1, collect the eluate, desolvate to obtain the novel myricetin - colchicine hybrid;

[0059] 1.8 g, light yellow solid, yield 45%; melting point: 182 °C - 184 °C; 1H NMR (400 MHz, CDCl3) δ 7.53–7.43 (m, 3H), 7.41 (d, J = 11.1 Hz, 1H), 7.05 (s, 2H), 6.63 (d, J = 11.3 Hz, 1H), 6.55 (d, J = 2.2 Hz, 1H), 6.51 (s, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.71–4.64 (m, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.88 (s, 6H), 3.62 (s, 3H), 3.08 (t, J = 7.4 Hz, 2H), 2.46 (dd, J = 13.3, 6.5 Hz, 1H), 2.36 (td, J = 12.9, 6.7 Hz, 1H), 2.28–2.18 (m, 1H), 1.98 (s, 3H), 1.94–1.80 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 175.47, 170.48, 170.07, 168.84, 164.74, 161.30, 159.34, 153.84, 153.40, 153.04, 151.45, 151.42, 151.24, 141.66, 140.70, 139.11, 134.68, 134.08, 130.99, 126.95, 124.81, 123.68, 108.75, 108.33, 107.32, 105.77, 104.98, 96.41, 92.88, 61.56, 61.45, 61.15, 60.56, 56.55, 56.48, 56.26, 56.04, 52.68, 38.51, 37.25, 33.31, 30.21, 23.01, 14.34; high resolution mass spectrometry: HRMS (m / z): calcd for C 44 H 46 N2NaO 14 [M+Na]+ 849.2841, found 849.2859.

[0060] II. Pharmacological research

[0061] Example 2 Effect of CMyrH on the survival rate of rats with ALI model

[0062] 1. Experimental method:

[0063] 1.1 Experimental animals

[0064] Healthy SPF-grade SD rats, 6-8 weeks old, male, with a body weight of 180±20 g, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (License: SCXK(Yue): 2022-0063). All experimental animals were adaptively raised in an SPF-grade environment, with free access to food and water during the period. The environmental temperature was controlled at 20℃-26℃, and the daily light exposure time reached 12 h. During the breeding process, the welfare of experimental animals was ensured, and all experiments were carried out strictly in accordance with the animal experiment regulations and systems. All experiments were approved by the local experimental animal ethics committee.

[0065] 1.2 Grouping, drug administration and model establishment

[0066] (1) Grouping and drug administration: 48 rats were randomly divided into 6 groups, with 8 rats in each group, and drug administration was carried out continuously for 3 days.

[0067] High-dose colchicine group: Colchicine was administered by gavage at a dose of 1.5 mg / kg (6 / 8 rats died);

[0068] Low-dose colchicine group: Colchicine was administered by gavage at a dose of 1.0 mg / kg;

[0069] High-dose CMyrH group: CMyrH was administered by gavage at a dose of 3.0 mg / kg;

[0070] Low-dose CMyrH group: CMyrH was administered by gavage at a dose of 1.0 mg / kg;

[0071] Blank control group: 0.9% Nacl of equal volume was administered by gavage;

[0072] Model group: 0.9% Nacl of equal volume was administered by gavage.

[0073] (2) Acute lung injury rat model: Under complete anesthesia, the rats were fixed in the supine position on a 30° slope operating table, and the skin of the anterior neck was incised to expose the trachea. Except for the blank control group, bleomycin (BLM dose was 5 mg / kg, and the infusion volume was 0.5 mL / kg) was slowly injected into the trachea of the remaining groups to induce the ALI model in rats; under the same conditions, an equal volume of 0.9% sodium chloride injection was instilled into the trachea of the control group.

[0074] 1.3 Specimen collection

[0075] Three days after drug administration, the rats were sacrificed and specimens were collected. Blood was taken from the abdominal aorta to collect serum and plasma. The whole lung was weighed and photographed to measure the lung index of the rats. The left lung lobe was placed in 4% paraformaldehyde solution and fixed at 4℃; the right upper and lower lobes were weighed, then dried to measure the wet / dry weight ratio; the middle and lower lobes of the right lung were quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for subsequent ELISA experiments.

[0076] 1.4 Observation of general conditions of rats

[0077] During the modeling period, the rats' diet, drinking water, mental state, and activity were closely observed, and the number of deaths was recorded.

[0078] 2. Experimental results

[0079] See Figure 1 The survival rate within 72 hours in the model group and CMyrH (1.0mg / kg and 3.0mg / kg) group was 100%, 6 / 8 mice died in the 1.5mg / kg colchicine group (P=0.0027, vs CMyrH 3.0mg / kg); 2 / 8 mice died in the 1.0mg / kg colchicine group (P=0.1435, vs CMyrH 3.0mg / kg). This result shows that CMyrH can reduce the toxicity of colchicine, thereby improving the survival rate.

[0080] Example 3 CMyrH can reduce the lung wet-to-dry weight ratio of ALI model rats

[0081] 1. Experimental Methods

[0082] After the rats were killed, the chest cavity was cut open, the upper lobe and the lateral lobe of the right lung were placed on filter paper, and the weight of the whole lung was measured after the surface blood was absorbed and recorded as the wet weight (Wet weight, W); then the whole lung was placed in an embedding frame and baked in a constant temperature oven at 60°C for 72 hours. Its mass no longer changed, and its weight was weighed again and recorded as the dry weight (D). The W / D ratio of each rat was calculated.

[0083] 2. Experimental results

[0084] like Figure 2 As shown in the figure, compared with the blank control group, the lung W / D in the model group was significantly increased (P=0.001); compared with the model group, the lung W / D in the CMyrH (1.0 mg / kg and 3.0 mg / kg) group and the 1.0 mg / kg colchicine group was significantly reduced (P=0.001), and there was no significant difference in lung W / D between the CMyrH (1.0 mg / kg and 3.0 mg / kg) group and the 1.0 mg / kg colchicine group (P>0.05). This indicates that CMyrH can alleviate BLM-induced acute pulmonary edema, and there is no difference in the effect of 1.0 mg / kg CMyrH and 1.0 mg / kg colchicine in alleviating pulmonary edema.

[0085] Example 4 CMyrH can improve the lung tissue pathology score of ALI model rats

[0086] 1. Experimental Methods

[0087] ① Observe the pathological changes of lung tissue by HE staining: Remove the left lung of the rats, soak it in 4% paraformaldehyde for fixation for 24 h, embed it in paraffin at room temperature for 5 μm sections, and after dewaxing, rehydration, blueing, and clearing operations, stain with hematoxylin and eosin, and then observe and collect images under an optical microscope.

[0088] ② Histologically score the lung tissue involvement: Score according to alveolar edema, intrapulmonary congestion, infiltration of inflammatory cells, and degree of interstitial pulmonary edema respectively. No change or slight change is 0 point, mild change is 1 point, moderate change is 2 points, severe change is 3 points, and extremely severe change is 4 points. Take the average value as the pathological score of lung injury for each sample, as shown in Table 1.

[0089] Table 1 Pathological scoring criteria for lung tissue of rats with acute lung injury

[0090]

[0091] 2. Experimental results

[0092] The results of histopathological examination found that in the model group, the lung tissue of the rats showed extensive pulmonary consolidation, thickening of the lung interstitium, infiltration of inflammatory cells such as neutrophils and mononuclear macrophages, and disorder or even disappearance of the alveolar structure; the pathological changes of the lung tissue in the 1.0 mg / kg CMyrH group were similar, but the lesions were alleviated, showing moderate pulmonary consolidation; the 3.0 mg / kg CMyrH group and the 1.0 mg / kg colchicine group showed mild to moderate interstitial lung lesions (see Figure 3 ). Histopathological scoring showed that compared with the model rats, the histopathological scores of the lung tissue in the 3.0 mg / kg CMyrH group and the 1.0 mg / kg colchicine group were significantly reduced (P < 0.01), and the histopathological score of the lung tissue in the 1.0 mg / kg CMyrH group also showed a downward trend, but there was no statistical significance (P > 0.05), see Figure 4 .

[0093] Example 5 CMyrH can reduce the expression level of inflammatory factors in the lung tissue of ALI model rats

[0094] 1. Experimental method

[0095] Approximately 30 mg of lung tissue was taken from each group of samples, and 300 μL of tissue homogenate was added to each for homogenization. The protein concentration of the samples was detected using a BCA kit. Using the Luminex detection technique, the expression levels of factors such as IL-1β, IL-6, IL-18, and TNF-α in the lung tissue samples were detected: The kit was first equilibrated at room temperature for 30 min. Meanwhile, the standards, washing solution, beads, detection antibodies, and PE-streptavidin were prepared. After the samples were diluted, the beads were resuspended, and 50 μL of the diluted beads was added to each well, and the plate was washed twice. According to the layout of the experimental design, 50 μL of the standards and samples were added to the corresponding wells respectively, and the plate was shaken on a shaker at 800 rpm at room temperature for 30 min; the microplate was placed on a magnetic rack for at least 1 min to ensure that the beads were adsorbed, and each well was washed 3 times with 100 μL of the washing solution; 25 μL of the diluted biotin-labeled detection antibody complex was added to each well, shaken at room temperature for 30 min, and 50 μL of the diluted streptavidin-labeled PE was added, shaken at room temperature for 10 min, and each well was washed 3 times with 100 μL of the washing solution; the beads were resuspended with 125 μL of Assay Buffer, shaken and incubated for 2 min, and detected on a Luminex instrument. The concentration was calculated based on the OD value of the samples and the standard curve.

[0096] 2. Experimental results

[0097] As Figure 5 shown, compared with the blank control group, the expression levels of the inflammatory factors IL-1β, IL-6, IL-18, and TNF-α in the lung tissue of the model group rats were significantly increased; compared with the model group, the expression levels of the inflammatory factors IL-1β, IL-6, IL-18, and TNF-α in the lung tissue of the 1.0 mg / kg and 3.0 mg / kg CMyrH groups of rats showed a concentration-dependent decrease, and the decrease in the expression levels of the inflammatory factors in the 3.0 mg / kg CMyrH group was more significant. There was no significant difference in the expression levels of the inflammatory factors between the 1.0 mg / kg CMyrH group and the 1.0 mg / kg colchicine group.

[0098] Example 6 CMyrH can inhibit the activity of the key pathological target (neutrophil elastase, HNE) of acute lung injury

[0099] 1. Experimental method

[0100] The crystal structure of neutrophil elastase (PDB ID: 6SMA) is from the RSCB PDB database (http: / / www.rcsb.org / ). The structure of the CMyrH ligand was drawn by ChemDraw 15.0 and then converted into a 3D structure after energy minimization by Chem3D 16.0. Molecular docking was performed using Autodock 4.2, and the binding energy was predicted to evaluate the affinities of HNE with CMyrH, colchicine, and myricetin. Based on the criteria of the lowest binding energy and root mean square deviation value (RMSD < 1), the best binding conformation was selected from 50 lowest-energy docking structures and displayed using PyMOL. The evaluation criteria are as follows: if the binding energy < 0, the ligand and receptor bind spontaneously; if the binding energy ≤ -5.0 kcal / mol, the docking of the ligand and receptor is stable and reliable.

[0101] 2. Experimental results

[0102] The molecular docking study showed (see Figure 6 ), that the α,β-unsaturated carbonyl on the colchicine fragment of CMyrH could form a covalent bond with Ser195, the amino group outside the heptacyclic ring on the colchicine fragment of CMyrH could form a hydrogen bond with Ser214, and the ketone carbonyl of the myricetin fragment could form a hydrogen bond with Gly216. In short, CMyrH could bind to the active site of HNE enzyme through binding modes such as covalent bonds and hydrogen bonds, thereby inhibiting the activity of HNE enzyme. In addition, the binding energy value of CMyrH with HNE enzyme was -13.54 kcal / mol (Ki value was 118.49 pM), and the binding energy values of colchicine and myricetin with HNE enzyme were -5.86 kcal / mol and -1.89 kcal / mol (Ki values were 51.03 μM and 40.93 mM), respectively. This indicates that CMyrH has a stronger binding force with HNE enzyme than colchicine and myricetin, and is more likely to inhibit the activity of HNE enzyme, which is a key pathological target for acute lung injury.

Claims

1. A novel myricetin-colchicine hybrid, characterized in that, The novel myricetin-colchicine hybrid is synthesized from colchicine and myricitrin as raw materials, and its molecular formula is: C 44 H 46 N2O 14 , and the structure is:

2. A method for synthesizing a novel myricetin-colchicine hybrid as described in claim 1, characterized in that, It includes the following steps: (1) Synthesis of intermediate pentamethylmyricetin ① Take myricitrin and potassium carbonate, mix them evenly, add acetonitrile, stir evenly, then add dimethyl sulfate, and stir evenly to obtain a mixed solution; ② The mixed solution is refluxed for 24 hours, filtered, and the filtrate is desolvated to obtain crude product 1; ③ Take crude product 1, dissolve it with ethanol, then add concentrated hydrochloric acid, react at 70 °C for 12 hours, and desolvate to remove ethanol to obtain crude product 2; ④ Take crude product 2, dissolve it in 50 mL of dichloromethane, wash it with 50 mL of water, dry it with 50 g of anhydrous sodium sulfate, filter, and evaporate the solvent from the filtrate to obtain the intermediate pentamethyl myricetin. (2) Synthesis of novel myricetin-colchicine hybrid ① Take colchicine and β-alanine, mix them evenly, add ethanol and mix well, then add sodium hydroxide solution, mix well, and react at 70 °C for 5 hours; ② The reaction solution is placed in a rotary evaporator to remove the ethanol solvent, washed once with 15 mL of ethyl acetate, the aqueous phase is separated, the pH value is adjusted to 4 - 5 with hydrochloric acid, wait for white solid to precipitate, extracted and washed 3 times with dichloromethane, 30 mL each time, then dried with 50 g of anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a solid; ③ Take the solid obtained in ②, dissolve it with dichloromethane, add the pentamethylmyricetin obtained in step (1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine respectively, mix well, react at room temperature for 12 hours, quench with water, the reaction solution is washed with 30 mL of water, and the aqueous phase is separated; ④ The aqueous phase is extracted 3 times with dichloromethane, 50 mL each time, the organic phases are combined, dried with 50 g of anhydrous sodium sulfate, filtered, and the filtrate is concentrated until no more distillate comes out to obtain a crude product; ⑤ Take the crude product obtained in ④, perform silica gel column chromatography, elute, collect the eluate, and desolvate to obtain the novel myricetin-colchicine hybrid.

3. The synthesis method of the novel myricetin-colchicine hybrid according to claim 2, wherein In ① under step (1), the dosage of myricitrin is 0.8 g, the dosage of potassium carbonate is 1.8 g, the dosage of acetonitrile is 40 mL, and the dosage of dimethyl sulfate is 1.2 mL.

4. The synthesis method of the novel myricetin-colchicine hybrid according to claim 2, characterized in that, In ③ under step (1), the dosage of ethanol is 40 mL, and the dosage of concentrated hydrochloric acid is 1.4 mL.

5. The synthesis method of the novel myricetin-colchicine hybrid according to claim 2, characterized in that, In ① under step (2), the dosage of colchicine is 2.0 g, the dosage of β-alanine is 10 mmol, the dosage of ethanol is 10 mL, and the concentration of the sodium hydroxide solution used is 1 mol / L.

6. The synthesis method of the novel myricetin-colchicine hybrid according to claim 2, characterized in that, In ③ under step (2), the dosage of dichloromethane is 50 mL, the dosage of pentamethylmyricetin is 1.9 g, the dosage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1.0 g, and the dosage of 4-dimethylaminopyridine is 1.22 mg.

7. The synthesis method of the novel myricetin-colchicine hybrid according to claim 2, characterized in that, In ⑤ under step (2), the eluent used for silica gel column chromatography is a petroleum ether - ethyl acetate solution with a volume ratio of 1:1, and the dosage is 500 mL.

8. Use of the novel myricetin-colchicine hybrid as described in claim 1, characterized in that, The said application is the application in the preparation of drugs for preventing and treating acute lung injury.

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