Left-handed bicyclic morpholine and salt thereof, preparation method therefor, pharmaceutical composition, and application

Abstract

To provide a left-handed bicyclic morpholine and a pharmaceutically acceptable salt thereof, a preparation method therefor, and use thereof in the preparation of medicaments for preventing and / or treating liver diseases and the like.SOLUTION: A left-handed bicyclic morpholine and a pharmaceutically acceptable salt thereof are provided, wherein the structure of the left-handed bicyclic morpholine is represented by Compound 5, and the pharmaceutically acceptable salt has structural formula I, where X is selected from inorganic acids and organic acids.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular, to levorotatory bicyclic morpholine and its pharmaceutically acceptable salts, methods for their preparation, pharmaceutical compositions, and their use in the preparation of agents for preventing and / or treating liver diseases and the like.

Background Art

[0002] Liver diseases are a kind of global diseases, and China is a major country with liver diseases. Currently, in our country, there are many patients suffering from liver damage and inflammation caused by various causes, mainly due to viral hepatitis. Statistically, the direct economic losses caused by chronic hepatitis (including liver cirrhosis and liver cancer caused by late-onset chronic hepatitis) in our country reach 900 billion yuan per year. In recent years, the incidences of drug-induced liver diseases, alcoholic and non-alcoholic fatty liver diseases, and autoimmune liver diseases have also shown an increasing trend year by year. Searching for safe and effective drugs for preventing and treating liver diseases has always been a research hot spot for various research institutes and pharmaceutical companies around the world.

[0003] Bicyclol is a kind of Category 1 chemical drug for treating hepatitis in China with independent intellectual property rights developed by the Institute of Materia Medica, Chinese Academy of Medical Sciences (MMA). In foreign clinics, they have the advantages of good effects in liver protection and enzyme decomposition, specific activity against hepatitis virus, convenient administration without any significant adverse reactions, and broad pharmacological activities. The research achievements related to bicyclol are 9 thIt has won multiple awards, including the Five-year National Science and Technology Key Task Achievement Award (2002), the Top Ten News in the Chinese Pharmaceuticals Industry in 2002, the First Prize of Beijing Scientific and Technological Progress (2005), and the Second Prize of National Scientific and Technological Progress (2007). Bicyclol is protected by compound invention patent rights in 16 countries / regions, such as the United States, the European Union, Japan, South Korea, etc., and the Taiwan region, and is sold in countries such as Ukraine. Since it was announced at a press conference held in the Great Hall of the People in November 2001 that Bicyclol would be commercially available, it has achieved great social and economic benefits in our country.

[0004] However, Bicyclol has low water solubility in vivo because of its low bioavailability. Researchers from the Institute of Materia Medica, Chinese Academy of Medical Sciences optimized the structure of Bicyclol and found that bicyclic morpholine and its salts have good pharmacological activities and pharmacokinetic properties (Wu Song, Sun Hua, et al., Bicyclol Derivative and Preparations and Applications Thereof, 201610922563.5). Based on previous findings, the researchers resolved the racemate of bicyclic morpholine and found that levorotatory bicyclic morpholine and its salts have better pharmacological activities and pharmacokinetic properties in terms of anti-inflammatory and liver protection than the racemate, as well as dextromorpholine and its salts.

Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a levorotatory bicyclic morpholine and a pharmaceutically acceptable salt thereof, a method for preparing the same, and its use in the preparation of a medicament for preventing and / or treating liver diseases and the like.

[0006] To solve the technical problem of the present invention, the present invention provides the following technical solution: The first aspect of the technical solution according to the present invention is Compound 5:

Chemical formula

[0007] Therefore, pharmaceutically acceptable acid addition salts can be prepared from inorganic acids and organic acids. Therefore, X is selected from inorganic acids and organic acids; the inorganic acids are selected from hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, sulfuric acid, and phosphoric acid; the organic acids are acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, quinic acid, borneolic acid, camphorsulfonic acid, aspartic acid, glutamic acid, pyroglutamic acid, L-tartaric acid, L-dibenzoyl tartaric acid, L-di-p-methylbenzoyl tartaric acid, diethyl L-tartrate, L-malic acid, L-borneolic acid, L-10-camphorsulfonic acid, R-(-)-mandelic acid, L-quinic acid, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, D-tartaric acid, D-dibenzoyl tartaric acid, D-di-p-methylbenzoyl tartaric acid, diethyl D-tartrate, D-malic acid, D-borneolic acid, D-10-camphorsulfonic acid, S-(-)-mandelic acid, D-quinic acid, D-aspartic acid, D-glutamic acid, and D-pyroglutamic acid, where the ratio of free base to acid is optionally 1:1, 2:1, or 3:1. Therefore, the preferred structures are as follows:

Chemical formula

Brief Description of the Drawings

[0008]

Figure 1

Modes for Carrying Out the Invention

[0009] The second aspect of the technical solution according to the present invention is to provide a method for preparing a levorotatory bicyclic morpholine and a pharmaceutically acceptable salt thereof according to the first aspect, and it is as follows:

Chemical Formula

[0010] The third aspect of the technical solution according to the present invention is to provide a pharmaceutical composition comprising a therapeutically and prophylactically effective amount of the levorotatory bicyclic morpholine according to the first aspect of the present invention and its pharmaceutically acceptable salts, and optionally one or more pharmaceutically acceptable carriers or excipients.

[0011] The pharmaceutical composition can be prepared by methods well known in the art. The compounds of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants and formulated into any dosage form suitable for human or animal use. The compounds of the present invention are typically provided in the state of their pharmaceutical compositions in an amount of 0.1 to 95% by weight.

[0012] The compound of the present invention or a pharmaceutical composition containing the same can be administered in unit dosage forms, and the administration routes can be, for example, enteral or parenteral, such as oral administration, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0013] The dosage form may be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form may be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type, and multiple emulsion), a suspension, an injection solution (including aqueous injection solution, powder injection solution, and infusion solution), eye drops, nasal drops, lotion, and application agent; the solid dosage form may be a tablet (including general tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, and orally disintegrating tablet), a capsule (including hard capsule, soft capsule, and enteric-coated capsule), granule, powder, pellet, pill, suppository, film, patch, aerosol (powder), spray, etc.; the semi-solid dosage form may be an ointment, gel, paste, etc. The compound of the present invention can also be formulated into general preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle administration systems.

[0014] To prepare the compound of the present invention into tablets, various excipients known in the art may be used, including diluents, binders, wetting agents, disintegrants, lubricants, and co-solvents. The diluents may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents may be water, ethanol, isopropanol, etc.; the binders may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, etc.; the lubricants and co-solvents may be talc powder, silica, stearate, tartaric acid, liquid paraffin, and polyethylene glycol.

[0015] The tablets may further be prepared into coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or two-layer and multi-layer tablets. To prepare dosage units in capsules, the compound of the present invention as the active ingredient is mixed with diluents and co-solvents, and the resulting mixture is directly filled into hard capsules or soft capsules. Also, the compound of the present invention as the active ingredient is mixed with diluents, binders, and disintegrants to prepare granules or pellets, which are then filled into hard capsules or soft capsules. Various types of diluents, binders, wetting agents, disintegrants, and co-solvents for preparing the tablets of the compound of the present invention can also be used to prepare the capsules of the compound of the present invention.

[0016] To prepare the compound of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent, and an appropriate amount of solubilizer, cosolvent, pH adjuster and osmotic pressure adjuster generally used in the art may be added; the solubilizer or cosolvent may be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster may be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjuster may be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When a lyophilized powder is prepared for an injection, mannitol or glucose may also be added as a proponent.

[0017] In addition, if desired, coloring agents, preservatives, flavoring agents, or other additives may also be added to the pharmaceutical preparation. In order to achieve the administration purpose and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method. The administration dosage of the pharmaceutical composition containing the compound of the present invention can vary within a wide range depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the administration route, and the dosage form. Generally, the preferred range of the daily dosage of the compound of the present invention is 0.001 - 5 mg / kg body weight. The above dosage may be administered in one dosage unit or divided into several dosage units according to the administration regimen including the clinical experience of the doctor and the use of other treatment means.

[0018] The compound or composition of the present invention may be administered alone or in combination with other therapeutic or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage must be adjusted according to the actual situation. The fourth aspect of the technical solution according to the present invention is to provide the use of the levorotatory bicyclic morpholine according to the first aspect and its pharmaceutically acceptable salts, and the pharmaceutical composition according to the third aspect in the preparation of a medicament for preventing and / or treating liver-related diseases. Therefore, liver-related diseases refer to liver disorder-related diseases, and in particular, hepatitis-related diseases selected from hepatitis A, hepatitis B, hepatitis C, drug-induced liver diseases, alcoholic liver diseases, non-alcoholic liver diseases, autoimmune liver diseases, liver fibrosis caused by the progression of liver diseases, cirrhosis, and liver failure.

[0019] Advantageous technical effects of the present invention The levorotatory bicyclic morpholine according to the present invention and its pharmaceutically acceptable salts exhibit better pharmacological activities than the dextrorotatory enantiomer and the racemate in various liver injury animal models, and there are statistical significant differences (P < 0.05). Moreover, the levorotatory bicyclic morpholine and its pharmaceutically acceptable salts have better pharmacokinetic properties than the dextrorotatory enantiomer and the racemate.

[0020] Best mode for carrying out the present invention The present invention provides a levorotatory bicyclic morpholine and its pharmaceutically acceptable salts for treating liver diseases, a method for preparing the same, a pharmaceutical composition, and uses thereof. The following examples are listed for further illustrating the present invention and are not intended to limit the present invention in any way. It can be understood by those skilled in the art that various changes and modifications can be made to the present invention without departing from the gist and scope thereof. The nuclear magnetic resonance spectra of the levorotatory bicyclic morpholine and its salts provided by the present invention are measured by using a Varain Mercury-500 nuclear magnetic resonance spectrometer together with TMS as an internal standard, and the mass spectra are measured by using a ZAB-2F mass spectrometer.

Example

[0021] Example 1 Preparation of Levo(-)-IMM-H014

Chemical formula

[0022] Compound 1 (i.e., bicyclol, 5.1 g, 13.1 mmol) was placed into a 100 mL three-necked flask equipped with a magnetic stirrer and a thermometer, to which 50 mL of dry DMF was added, and the solid was completely dissolved. After cooling the reaction system to 0 °C in an ice bath, SOCl2 (4.5 mL, 61.8 mmol) was slowly added dropwise, and at the same time, the temperature of the system was controlled not to exceed 5 °C. After the dropwise addition, the reaction was continued in the ice bath for 30 minutes until TLC indicated that the raw material had completely reacted. The reaction system was poured into about 100 g of crushed ice, and stirred well to precipitate a large amount of white solid, which was filtered, and the filter cake was washed with a small amount of distilled water and ether, and dried by removing moisture. The product was naturally dried in air and weighed to produce 4.9 g of a white solid (Compound 2) in total, yield: 91.7%.

[0023] Morpholine (1.28 g, 14.7 mmol) was placed into a 50 mL round-bottom flask to which 25 mL of acetone and 2.2 mL of triethylamine were added, and while stirring at room temperature, Compound 2 (2.76 g, 6.8 mmol) was added thereto. The mixture was reacted at room temperature for 5 hours and then left to stand overnight. TLC indicated that the raw material had completely reacted, and a pink insoluble solid formed in the system. By filtration, the filtrate was distilled under reduced pressure to remove the solvent therein. The obtained yellow oil was separated by a vacuum column (petroleum ether: ethyl acetate = 2:1), and the product component was recovered to produce 3.1 g of a colorless oil (Compound 3), yield: 92.3%. MS-FAB [M+H] + = 460.1.

[0024] Compound 3 (3.0 g, 6.54 mmol) was weighed, dissolved in 45 ml of ethyl acetate, and L-dibenzoyl tartaric acid (L-DBTA, 1.2 g, 3.27 mmol) was added thereto. At room temperature, the mixture was stirred to precipitate a white solid, and after 30 minutes, it was filtered to recover the solid, which was further dried at 60 °C for 1 hour, then weighed to obtain 1.25 g of a white solid (levorotatory bicyclic morpholine: L-dibenzoyl tartaric acid = 2:1), yield: 56.8%. By chiral HPLC analysis, the enantiomeric excess (%e.e.) of Intermediate 4 was 98.0%, [α] 25 = -130.4 (CH2Cl2).

[0025] Intermediate 4 (1.25 g) was dispersed with 25 mL of ethyl acetate, washed twice with 25 mL of saturated sodium bicarbonate solution, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to produce a colorless oil. The oil was dissolved in 10 ml of ethyl acetate, and 120 μL of methanesulfonic acid was added thereto. Crystallization was carried out with stirring at room temperature, and the obtained solid was recrystallized with 10 ml of methanol by filtration to produce 0.7 g of (-)-IMM-H014, a white solid, HPLC purity > 98.0%, [α] 25 = -65.6 (CH2Cl2). 1 H NMR (500 MHz, chloroform-d) δ 7.45 (s, 1H), 7.38 (s, 1H), 6.14 (s, 1H), 6.02 (s, 1H), 5.96 (d, J = 8.9 Hz, 2H), 4.37 (dd, J = 13.3, 4.0 Hz, 1H), 4.16 (t, J = 12.0 Hz, 1H), 4.05 (d, J = 19.7 Hz, 8H), 3.96 - 3.81 (m, 3H), 3.76 (s, 3H), 3.57 (d, J = 11.8 Hz, 1H), 3.41 (d, J = 12.5 Hz, 1H), 2.91 (s, 3H), 2.79 - 2.68 (m, 1H), 2.50 (d, J = 11.3 Hz, 1H).

[0026] Comparative Example 1 Preparation of Dextrorotatory Enantiomer (+) IMM-H014 [Chemical formula]

[0027] Compound 3 (3.0 g, 6.54 mmol) was further obtained, dissolved in 45 ml of ethyl acetate, and D-dibenzoyl tartaric acid (1.2 g, 3.27 mmol) was added thereto. The mixture was stirred at room temperature to precipitate a white solid, and after 30 minutes, the solid was recovered, dried at 60 °C for 1 hour, weighed, and 1.2 g of a white solid (dextrorotatory bicyclic morphine:D-dibenzoyl tartaric acid = 2:1) was produced, yield: 54.5%. By chiral HPLC analysis, the enantiomeric excess (% e.e.) of Intermediate 4 was 97.3%, [α] 25 = +133.2 (CH2Cl2).

[0028] The intermediate (1.2 g) was dispersed with 25 mL of ethyl acetate, washed twice with 25 mL of saturated sodium bicarbonate solution, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oil. The oil was dissolved in 10 ml of ethyl acetate, and 120 μL of methanesulfonic acid was added thereto. Crystallization was carried out with stirring at room temperature, and the obtained solid was recrystallized with 10 mL of methanol by filtration to obtain 0.6 g of (+) IMM-H014 as a white solid, HPLC purity > 98.0%, [α] 25 = -78.2 (CH2Cl2). 11H NMR (500 MHz, chloroform-d) δ 7.45 (s, 1H), 7.38 (s, 1H), 6.14 (s, 1H), 6.02 (s, 1H), 5.96 (d, J = 8.9 Hz, 2H), 4.37 (dd, J = 13.3, 4.0 Hz, 1H), 4.16 (t, J = 12.0 Hz, 1H), 4.05 (d, J = 19.7 Hz, 8H), 3.96 - 3.81 (m, 3H), 3.76 (s, 3H), 3.57 (d, J = 11.8 Hz, 1H), 3.41 (d, J = 12.5 Hz, 1H), 2.91 (s, 3H), 2.79 - 2.68 (m, 1H), 2.50 (d, J = 11.3 Hz, 1H).

[0029] Pharmacological Test Example 1 Effect of IMM-H014 Optical Isomers on ConA-Induced Acute Immune Liver Injury

[0030] 1. Method and Administration Method for Establishing a ConA-Induced Mouse Acute Immune Liver Disease Model After acclimation, SPF-grade male ICR mice (20 - 22 g) were randomly assigned to five groups: a blank control group, a ConA-induced model group, a 200 mg / kg (+) IMM-H014 group, a 200 mg / kg (-) IMM-H014 group, and a 200 mg / kg (±) IMM-H014 group, with 10 mice in each group. In each administration group, it was administered intragastrically once a day and a total of 3 times. In the blank control group and the model group, the same dose of physiological saline was used for intragastric administration. Two hours after the last administration, mice in each group except the blank control group were injected with 20 mg / kg of ConA via the tail vein, and each administration dose was 10 ml / kg. The mice were fasted for 16 hours but not water-restricted, and then the animals waited for further treatment.

[0031] 2. Measurement of Biochemical Indexes The mice were decapitated, blood was collected, and the blood samples were allowed to stand at room temperature for 2 hours and then centrifuged at 4000 rmp for 10 minutes to separate the serum. The ALT, AST, and LDH contents in the serum were detected using an automatic biochemical analyzer.

[0032] 3. Statistical analysis Each data was expressed as the numerical mean ± standard deviation (x ± SD). Inter-group comparison was performed by t-test, and P < 0.05 indicated a significant difference.

[0033] 4. Experimental results 4.1 Effect of IMM-H014 enantiomers on ConA-induced increase in ALT, a biomarker of serum liver injury Since the serum ALT level shows a direct and positive correlation with the degree of liver injury, it is a generally recognized serum biomarker of liver injury. The results in Table 1 show that 20 mg / kg of ConA will cause significant liver injury in mice and significantly enhanced the serum ALT level compared with the blank control group (P < 0.001). Both (+)IMM-H014 and (-)IMM-H014 can significantly reduce the ConA-induced increase in serum ALT (P < 0.001). The percentage reduction of ALT by the IMM-H014 enantiomers was 95.3% and 97.3% respectively, and the ALT content was reduced to the level of the blank control group. Both (+)IMM-H014 and (-)IMM-H014 showed a significant protective effect against ConA-induced immune liver injury, and the activity of (-)IMM-H014 was slightly better than that of (+)IMM-H014. When compared with (±)IMM-H014 at the same dose, the reduction effects of (+)IMM-H014 and (-)IMM-H014 on the ConA-induced increase in mouse serum ALT level were superior to those of (±)IMM-H014 (and it also showed significant efficacy, with the percentage reduction of ALT being 88.6%), where the reduction effect of (-)IMM-H014 on ALT had a statistical difference (P < 0.05) when compared with that of (±)IMM-H014.

[0034] Table 1 Reduction effect of IMM-H014 enantiomers on ConA-induced increase in mouse serum ALT (n = 10)

Table 1

[0035] 4.2 Effect of IMM-H014 enantiomers on ConA-induced increase in serum AST, a biomarker of liver injury An increase in serum AST level is also one of the important markers of hepatocyte injury, especially mitochondrial injury of hepatocytes. When mitochondria are damaged, the serum AST level increases significantly, reflecting the severity of hepatocytes. The results are shown in Table 2. 20 mg / kg of ConA caused significant damage to mouse hepatocyte mitochondria and significantly increased the serum AST level compared with the blank control group (P < 0.001). All of (+)IMM-H014, (-)IMM-H014 and (±)IMM-H014 could reduce the level of ConA-induced increase in serum AST. Compared with the model group, the percentage reduction of AST by (+)IMM-H014, (-)IMM-H014 and (±)IMM-H014 was 44.8%, 72.9% and 22.2% respectively. Here, the (-)IMM-H014 group had a statistical difference compared with the model group (P < 0.01). The activities of (+)IMM-H014 and (-)IMM-H014 for reducing AST were superior to those of (±)IMM-H014. The activity of the (-)IMM-H014 was optimal and had a statistical difference compared with the (±)IMM-H014 group at the same dose (P < 0.05).

[0036] Table 2 Reduction effect of IMM-H014 enantiomers on ConA-induced increase in mouse serum AST (n = 10)

Table 2

[0037] 4.3 Effect of IMM-H014 enantiomers on ConA-induced increase in serum LDH When the liver is damaged, the serum LDH level can also reflect the status and degree of hepatocyte damage. The results are shown in Table 3. Intravenous injection of 20 mg / kg of ConA caused severe hepatocyte damage and significantly increased the serum LDH level compared with the blank control group (P<0.001). (-)IMM-H014 could significantly reduce the level of ConA-induced serum LDH increase, and the percentage reduction of LDH was up to 54.4%, and there was a statistical difference compared with the model group (P<0.01). (+)IMM-H014 also had a reducing effect on the increase in serum LDH, and the percentage reduction was 27.6%, but there was no statistical difference compared with the model group. (±)IMM-H014 only had a weak reducing effect on the increase in LDH at the given dose, and the percentage reduction was 6.4%. In terms of the mode of reducing the LDH level, the activity of (-)IMM-H014 was still superior to that of (+)IMM-H014 and (±)IMM-H014, and there was a statistical difference between (-)IMM-H014 and (±)IMM-H014 at the same dose (P<0.05).

[0038] Table 3 Reduction effect of IMM-H014 enantiomers on ConA-induced increase in mouse serum LDH (n = 10)

Table 3

[0039] Example 2 Effect of IMM-H014 optical enantiomers on ethionine-induced non-alcoholic fatty liver disease 1. Method and administration method for establishing an ethionine-induced mouse non-alcoholic fatty liver model After acclimation, male ICR mice (20 - 22 g) of SPF grade were randomly assigned to 5 groups: blank control group, ethionine-induced model group, 200 mg / kg (+) IMM-H014 group, 200 mg / kg (-) IMM-H014 group, and 200 mg / kg (±) IMM-H014 group, with 5 mice in each group. Three days before model establishment, each administration group was given intragastric administration once a day, and a total of 3 administrations were given. In the animals of the blank control group and the model group, the same dose of physiological saline was used for intragastric administration. Each administration dose was 10 mL / kg. One hour after the last administration, 250 mg / kg of ethionine was given intragastrically to the mice in each group once. The administration dose was 20 ml / kg. The mice were fasted for 24 hours, but not in a water-restricted state, and then the animals waited for further treatment.

[0040] 2. Measurement of the contents of neutral fat and cholesterol in liver tissue The mice were subjected to thoracotomy, the liver was excised, and it was washed with physiological saline at 4°C. A part of the liver tissue was prepared into a 10% liver tissue homogenate using a lysate and an electric homogenizer; here, a part of it was measured according to the measurement methods of the neutral fat kit and the cholesterol kit to measure the triglyceride and cholesterol in it, and the other part was measured using a BCA protein quantification kit to measure the protein content in it. The triglyceride and cholesterol in the liver tissue were subjected to protein correction.

[0041] 3. Statistical analysis By adopting SPSS software, each data was shown as numerical mean ± standard deviation (x ± SD). The comparison between groups was carried out by t-test, and P < 0.05 indicated a significant difference.

[0042] 4. Experimental results 4.1 Effect of IMM-H014 enantiomers on the ethionine-induced increase in the cholesterol (TC) content of mouse liver tissue By interfering with methionine metabolism so as to further affect the synthesis of apolipoproteins, ethionine results in preventing cholesterol, triglycerides, etc. synthesized in hepatocytes from being transported into the blood, and this causes the accumulation of hepatocyte lipids, thereby forming a drug-induced non-alcoholic fatty liver model. The results are shown in Table 4. Ethionine at 250 mg / kg can cause a significant increase in the cholesterol content in liver tissue compared with the blank control group (P<0.05). Both (+)IMM-H014 and (-)IMM-H014 can reduce the ethionine-induced accumulation of TC in liver tissue. The percentage reduction of TC by the IMM-H014 enantiomers (i.e., (+)IMM-H014 and (-)IMM-H014) is 27.2% and 32.4% respectively. Here, there was a statistical difference in the (-)IMM-H014 group compared with the model group (P<0.05). (±)IMM-H014 also has a significant inhibitory effect on the ethionine-induced increase in TC in liver tissue, and there was a statistical difference compared with the model group (P<0.05). The activity of (-)IMM-H014 was slightly better than that of (±)IMM-H014.

[0043] Table 4 Reduction effect of IMM-H014 enantiomers on ethionine-induced TC accumulation in mouse liver tissue (n = 10)

Table 4

[0044] 4.2 Effect of IMM-H014 enantiomers on ethionine-induced increase in triglyceride (TG) content in mouse liver tissue The results are shown in Table 5. Ethionine at 250 mg / kg caused a significant increase in the TG content in liver tissue (P<0.05) compared with that in the blank control group, indicating the symptoms of non-alcoholic fatty liver. (-)IMM-H014 could significantly reduce the ethionine-induced TG accumulation in liver tissue, with a TG reduction percentage of 39.0%, and there was a statistical difference compared with the model group (P<0.05). (+)IMM-H014 showed a weak reducing effect on the increase in ethionine-induced triglycerides in liver tissue, with only a 7.1% reduction rate.

[0045] Table 5 Reduction effect of IMM-H014 enantiomers on ethionine-induced TG accumulation in liver tissue (n = 10)

Table 5

[0046] Example 3 Effect of IMM-H014 enantiomers on 4-acetamidophenol-induced hepatocyte injury 1. Cell culture Human liver cancer HepG2 cells well retain the characteristics of normal human hepatocytes and were cultured in DMEM medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin) under culture conditions of 37°C, 5% CO2, and saturated humidity. A solution containing 0.25% trypsin and 0.02% EDTA was used for digestion and passage.

[0047] 2. Protective effect of IMM-H014 enantiomers on 4-acetamidophenol-induced in vitro hepatocyte injury The MTT method was adopted. HepG2 cells were inoculated into 96-well cell culture plates. After culturing for 24 hours, (+)IMM-H014, (-)IMM-H014, and (±)IMM-H014 were added at non-toxic concentrations, and furthermore, 4-acetamidophenol (APAP, final concentration of 8 mM) was added thereto. A bicyclol positive control group, a solvent control group, and a model group were set up for the experiment. The culturing was continued for 24 hours. 100 μL of the culture solution was collected, centrifuged, and the LDH level was detected there using a fully automatic biochemical analyzer with an LDH detection kit. The remaining culture solution was discarded, and 100 μL of MTT (0.5 mg / mL) solution was added to each well to continue culturing for 4 hours. The MTT solution was discarded, and 150 μL of DMSO was added to each well. The mixture was shaken by a mixing shaker, and the absorbance value was measured at a wavelength of 570 nm using a microplate reader. Cell viability (%) = (average OD of the administration group / average OD of the solvent control group) × 100%.

[0048] 3. Statistical analysis The data were shown as numerical mean ± standard deviation (x ± SD). The comparison between groups was performed by t-test, and P < 0.05 indicated a significant difference.

[0049] 4. Experimental results 10 μM of (+)-IMM-H014, 10 μM of (-)-IMM-H014, and 10 μM of (±)-IMM-H014 affected HepG2 cells over 48 hours, and they were not significantly toxic to the cells, with cell viabilities of 90% each. These concentrations were used to investigate the protective effect against APAP-impaired hepatocytes. The results are shown in Table 6. 8 mM of APAP could significantly impair HepG2 cells, and its cell viability was only 38.33% compared to the blank control group (P < 0.001). (+)-IMM-H014, (-)-IMM-H014, and (±)-IMM-H014 at a dose of 10 μM each had a significant protective effect against APAP-induced in vitro human hepatocyte injury (P < 0.05, P < 0.001, P < 0.01), and the increases in the proportion of cell survival were 41.4%, 74.8%, and 32.1% respectively. The activity of (-)-IMM-H014 was relatively optimal, and there was a statistical difference compared to the (±)-IMM-H014 group at the same dose (P < 0.05). Bicyclol could also significantly improve APAP-induced hepatocyte injury (P < 0.05).

[0050] Table 6 Effects of IMM-H014 enantiomers on 4-acetamidophenol-induced decrease in hepatocyte viability (n = 3 - 4)

Table 6

[0051] LDH (lactate dehydrogenase) is one of the important enzymes for the energy metabolism of cells. When cells die, the inner cell membrane ruptures, and LDH is released from the cytoplasm, so the LDH level is proportional to the degree of cell damage. The results are shown in Figure 1. 8 mM of APAP affected HepG2 cells over 24 hours, and the LDH level in the cell culture supernatant increased significantly (P<0.01) compared with that of the blank control group, further indicating significant hepatocyte injury. (+)IMM-H014, (-)IMM-H014, and (±)IMM-H014 at a dose of 10 μM each were able to reduce the LDH level. Here, the LDH levels of (-)IMM-H014 and (±)IMM-H014 were statistically different (P<0.05, P<0.05) compared with those of the model group, and the reduction effect of the (-)IMM-H014 group was slightly better than that of the (±)IMM-H014 group at the same dose.

[0052] Example 4 Effect of optical isomers of IMM-H014 on carbon tetrachloride-induced hepatocyte injury 1. Cell culture Human liver cancer HepG2 cells well retain the characteristics of normal human hepatocytes and were cultured in DMEM medium containing 10% fetal bovine serum (containing 100 U / mL of penicillin and 100 μg / mL of streptomycin) under culture conditions: 37 °C, 5% CO2, and saturated humidity. A solution containing 0.25% trypsin and 0.02% EDTA was used for digestion and passage.

[0053] 2. Protective effect of IMM-H014 isomers on carbon tetrachloride-induced in vitro hepatocyte injury The MTT method was adopted. HepG2 cells were inoculated into a 96-well cell culture plate. After culturing for 24 hours, (+) IMM-H014, (-) IMM-H014, and (±) IMM-H014 were added at non-toxic concentrations, and furthermore, carbon tetrachloride (CCl4, final concentration of 0.6%) was added thereto. A bicyclol positive control group, a solvent control group, and a model group were set up in the experiment. They continuously affected the cells for 24 hours. The culture medium was discarded, and 100 μL of MTT (0.5 mg / mL) solution was added to each well and cultured for 4 hours. The MTT solution was discarded, and 150 μL of DMSO was added to each well. The mixture was shaken by a mixing shaker, and the absorbance value was measured at a wavelength of 570 nm using a microplate reader. Cell viability (%) = (average OD of the administration group / average OD of the solvent control group) × 100%.

[0054] 3. Statistical analysis The data were shown as numerical mean ± standard deviation (x ± SD). The comparison between groups was performed by t-test, and P < 0.05 indicated a significant difference.

[0055] 4. Experimental results 10 μM (+)IMM-H014, 10 μM (-)IMM-H014, and 10 μM (±)IMM-H014 affected HepG2 cells for 48 hours, and each concentration was not significantly toxic to the cells, with cell viability of 90%. These concentrations were used to investigate the protective effect on CCl4-injured hepatocytes. The results are shown in Table 7. 0.6% CCl4 significantly injured HepG2 cells, and the cell viability was 77.50% compared with the blank control group (P<0.001). (+)IMM-H014, (-)IMM-H014, and (±)IMM-H014 at a dose of 10 μM each had a significant ameliorative effect on CCl4-induced in vitro human hepatocyte injury (P<0.05, P<0.01, P<0.01), and the increases in cell survival rates were 12.59%, 34.66%, and 18.74%, respectively. Compared with (±)IMM-H014 at the same dose, (-)IMM-H014 had better protective activity against CCl4-induced hepatocyte injury. Bicyclol could also significantly ameliorate APAP-induced hepatocyte injury.

[0056] Table 7. Effect of IMM-H014 enantiomers on the decrease in hepatocyte viability caused by 4-acetamidophenol (n=3-4) [Table 7]

[0057] Pharmacokinetic studies Example 5 1. Purpose of the experiment The single enantiomers and the racemate of IMM-H014 were intragastrically administered to male SD rats to compare their pharmacokinetic properties in the rat body.

[0058] 2. Experimental equipment and materials 2.1 Experimental equipment Agilent 6470 triple tandem quadrupole LC-MS (Agilent Technologies Inc.), Mettler AG135-model electronic analytical balance, pipette gun, TDL-5-A centrifuge, Sigma mini centrifuge, nitrogen blower, and animal weighing scale.

[0059] 2.2 Experimental materials (+)IMM-H014, (-)IMM-H014, (±)IMM-H014; methanol (MS Grade, product of Fisher Scientific Inc., catalog number 179097); acetonitrile (MS Grade, product of Fisher Scientific Inc., catalog number 177802); deionized water (Hangzhou Wahaha Co.); formic acid (HPLC Grade, ROE SCIENTIFIC INC, catalog number 6F2941); ethyl acetate (MS Grade, product of Fisher Scientific Inc., catalog number 166828); 1.5 ml EP tubes.

[0060] 2.3 Experimental animals Thirty-six male SD rats (200 ± 10 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed in a clean room of the animal house in the Institute of Materia Medica, Chinese Academy of Medical Sciences, at a temperature of 23 ± 2 °C and a relative humidity of 55 ± 5%, with 12 hours of light irradiation per day. The experimental animals had free access to water and food, and after 2 weeks of acclimation, the experiment was started. The study complied with the rules established by the Animal Experiment Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

[0061] 3. Animal experiments 3.1 Animal allocation and administration Thirty-six male SD rats were randomly divided into six groups of six rats each, including the (+) IMM-H014 intragastric administration group, (-) IMM-H014 intragastric administration group, and (±) IMM-H014 intragastric administration group. The dosing amount was 50 mg / kg. The rats were fasted for 12 hours before administration but could drink water freely.

[0062] 3.2 Preparation of the administration solution 100 mg of the IMM-H014 raw drug was weighed and dissolved in 20 ml of purified water to prepare a 5 mg / ml administration solution, which was administered as a single dose according to body weight (1 ml / 100 g).

[0063] 3.3 Collection of plasma samples At 0 hours before administration and at 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 12 hours, and 24 hours after administration, 250 μL of blood was collected from the orbital venous plexus, placed in an EP tube containing 10 μL of sodium heparin, centrifuged at 4000 rpm for 10 minutes, and then plasma was obtained and stored in a refrigerator at -80°C.

[0064] 4. Analytical method 4.1 Preparation of solutions 4.1.1 Preparation of the stock solution 5 mg of the IMM-H014 control sample was accurately weighed, placed in a 5 ml volumetric flask, dissolved in methanol, and diluted to the degree scale to prepare a 1.0 mg / ml control stock solution.

[0065] 4.1.2 Preparation of the internal standard dilution standard solution A 5.0 mg carbamazepine control sample was accurately weighed, placed into a 5 mL volumetric flask, and dissolved by adding methanol. It was adjusted to a constant volume stepwise to prepare an internal standard stock solution with a concentration of 1.0 mg / mL. 50 μL of the stock solution was accurately pipetted, placed into a 10 mL volumetric flask, and diluted by adding methanol. It was adjusted to a constant volume stepwise to prepare an internal standard concentrated stock solution with a concentration of 5.0 μg / mL. 5.0 mL of the internal standard concentrated stock solution was accurately pipetted, placed into a 50 mL volumetric flask, and adjusted to a constant volume stepwise by adding methanol to prepare an internal standard diluted standard solution with a concentration of 500 ng / mL.

[0066] 4.1.3 Preparation of calibration dilution standard solutions and quality control standard solutions with a series of concentrations 100 μL of 1.0 mg / mL IMM-H014 stock solution was accurately pipetted, placed into a 10 mL volumetric flask, and diluted with methanol to prepare a 10 μg / mL IMM-H014 solution. The solution was serially diluted with methanol to prepare IMM-H014 series standard solutions with concentrations of 2, 5, 10, 50, 100, 500, 1000, 2000, 4000, and 5000 ng / ml. 1.0 mg / ml of IMM-H014 stock solution was accurately pipetted and serially diluted with methanol to prepare quantitative control standard solutions with concentrations of 5, 100, and 4000 ng / ml.

[0067] 4.1.4 Preparation of matrix calibration samples and quality control samples 50 μl of the standard solution at 2, 5, 10, 50, 100, 500, 1000, 2000, 4000, and 5000 ng / ml respectively was accurately pipetted, dried by blowing nitrogen gas at 30 °C, and 50 μl of rat blank plasma was added thereto. After stirring the mixture for 3 minutes, matrix calibration samples were prepared.

[0068] Accurately pipette 50 μl of standard solutions at 5, 100, and 4,000 ng / ml respectively, dry them by blowing nitrogen gas at 30 °C, and then add 50 μl of rat blank plasma thereto. After stirring the mixture for 3 minutes, quality control samples were prepared.

[0069] 4.2 Pretreatment of Samples Accurately pipette 50 μL of plasma samples, add 10 μL of internal standard dilution standard solution thereto, and then put them into 1.5 ml EP tubes. Stir them for 30 seconds, add 500 μl of ethyl acetate. Next, stir and shake them for 5 minutes, maintain them at 4 °C for 10 minutes, and then centrifuge at 13,000 r for 5 minutes. Collect the supernatant and dry it by blowing N2 at 30 °C. The residue was redissolved by adding 100 μL of the first mobile phase, centrifuged at 13,000 r for 5 minutes, and the supernatant was transferred into a sampling bottle for measurement.

[0070] 4.3 Chromatography Conditions Chromatography column: Agilent ZORBAX SB C18 (2.1×100 mm, 3.5 μm) Mobile phase A: water (0.1% formic acid, 1 mM ammonium acetate); Mobile phase B: acetonitrile (0.1% formic acid) Column temperature: 35 °C; Sampling volume: 3 μL Elution: 0 - 2 minutes: from 40% to 53% of B; 2 - 3 minutes: from 53% to 40% of B

[0071] 4.4 Mass Spectrometry Conditions Ion source: ESI; Detection mode: positive ion; Temperature of drying gas: 300 °C; Flow rate of drying gas: nitrogen gas, 11 L / min; Spray gas: nitrogen, 15 psi; Capillary tube voltage: 4,000 V; Scanning mode: multiple reaction monitoring (MRM); Ion pairs and related voltage parameters are shown below:

Table 8

[0072] 5. Data processing The original data obtained after sampling the samples was processed by adopting MassHunter QQQ data processing software to obtain blood drug concentration data; next, pharmacokinetic parameters were calculated by DAS software; finally, by adopting SPSS software, a t-test was performed to compare whether there were statistical differences between drugs of the same optical activity but different batches and drugs with different optical activities. Here, P < 0.05 was regarded as having a significant difference.

[0073] In the test, the pharmacokinetic properties of IMM-H014 single enantiomer or racemate in rats were tested by intragastric administration to three groups of SD rats respectively. The results of the test suggested that the in vivo exposure levels of intragastric administration of (-)IMM-H014 and (±)IMM-H014 were superior to those of (+)IMM-H014.

[0074] Table 8 Average pharmacokinetic parameters (excluding outliers) of each rat group

Table 9

Claims

1. Levorotatory bicyclic morpholine and pharmaceutically acceptable salts thereof, wherein the structure of the levorotatory bicyclic morpholine is represented by Compound 5, and the pharmaceutically acceptable salts thereof have Structural Formula (I): 【Chemical 1】 {wherein X is selected from inorganic acids and organic acids}, characterized by levorotatory bicyclic morpholine and pharmaceutically acceptable salts thereof.

2. The inorganic acids include hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, sulfuric acid, and phosphoric acid; the organic acids include acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, quinic acid, borneolic acid, camphorsulfonic acid, aspartic acid, glutamic acid, and pyroglutamic acid, characterized by the levorotatory bicyclic morpholine and pharmaceutically acceptable salts thereof according to Claim 1.

3. The organic acids are selected from L-tartaric acid, L-dibenzoyl tartaric acid, L-di-p-methylbenzoyl tartaric acid, diethyl L-tartrate, L-malic acid, L-borneolic acid, L-10-camphorsulfonic acid, R-(−)-mandelic acid, L-quinic acid, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, D-tartaric acid, D-dibenzoyl tartaric acid, D-di-p-methylbenzoyl tartaric acid, diethyl D-tartrate, D-malic acid, D-borneolic acid, D-10-camphorsulfonic acid, S-(−)-mandelic acid, D-quinic acid, D-aspartic acid, D-glutamic acid, and D-pyroglutamic acid, characterized by the levorotatory bicyclic morpholine and pharmaceutically acceptable salts thereof according to Claim 1.

4. The levorotatory bicyclic morpholine and pharmaceutically acceptable salts thereof are selected from (−)-bicyclic morpholine methanesulfonate having the following structure: [Chemical 2] characterized by the levorotatory bicyclic morpholine and pharmaceutically acceptable salts thereof according to Claim 1.

5. A method for preparing the levorotatory bicyclic morpholine and pharmaceutically acceptable salts thereof according to any one of Claims 1 to 4, comprising the following steps: [Chemical Formula 3] a) Chlorinating the hydroxyl group of the bicyclic compound to obtain Compound 2; b) Reacting Compound 2 with morpholine to obtain Compound 3; c) By reacting compound 3 with chiral acid Y to form a salt and performing resolution in an organic solvent by utilizing the difference in salt solubility to obtain the levo salt 4, wherein the chiral acid Y is selected from L-tartaric acid, L-dibenzoyl tartaric acid, L-di-p-methylbenzoyl tartaric acid, diethyl L-tartrate, L-malic acid, L-camphoric acid, L-10-camphorsulfonic acid, R-(−)-mandelic acid, L-quinic acid, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, D-tartaric acid, D-dibenzoyl tartaric acid, D-di-p-methylbenzoyl tartaric acid, diethyl D-tartrate, D-malic acid, D-camphoric acid, D-10-camphorsulfonic acid, S-(−)-mandelic acid, D-quinic acid, D-aspartic acid, D-glutamic acid, and D-pyroglutamic acid; The organic solvent is selected from ethyl acetate, acetone, methanol, ethanol, and isopropanol, and solvents obtained by mixing the above solvents in different ratios; the %e.e. values of the levo enantiomer and the dextro enantiomer are each greater than 95%; d) Enabling salt 4 to become the free amine under the action of a base; e) Optionally, forming a salt by reacting the free amine 5 with acid X to obtain the compound of formula (I); including wherein the definition of X is the same as that described in any one of claims 1 to 4, Method.

6. A pharmaceutical composition comprising a therapeutically effective amount and / or a prophylactically effective amount of the levo bicyclic morpholine according to any one of claims 1 to 4 and a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable carriers or excipients.

7. Use of the levo bicyclic morpholine according to any one of claims 1 to 4 and a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 5 in the preparation of a medicament for preventing and / or treating liver-related diseases.

8. The use according to claim 7, characterized in that the liver-related diseases include liver injury-related diseases and hepatitis-related diseases.

9. The use according to claim 7, characterized in that the liver-related diseases are selected from hepatitis A, hepatitis B, hepatitis C, drug-induced liver disease, alcoholic liver disease, non-alcoholic liver disease, autoimmune liver disease, liver fibrosis caused by the progression of liver disease, cirrhosis, and liver failure.

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