WO2021103749A1 - 一类左旋双环吗啉及其盐，其制备方法、药物组合物和应用 - Google Patents

一类左旋双环吗啉及其盐，其制备方法、药物组合物和应用 Download PDF

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WO2021103749A1
WO2021103749A1 PCT/CN2020/115410 CN2020115410W WO2021103749A1 WO 2021103749 A1 WO2021103749 A1 WO 2021103749A1 CN 2020115410 W CN2020115410 W CN 2020115410W WO 2021103749 A1 WO2021103749 A1 WO 2021103749A1
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acid
imm
liver
bicyclomorpholine
pharmaceutically acceptable
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PCT/CN2020/115410
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English (en)
French (fr)
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吴松
孙华
张金兰
张文轩
王喆
杨庆云
姜琳
陈子涵
申静
张洁
张弛
韩遵圣
秦通
张媛媛
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中国医学科学院药物研究所
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Priority to JP2022532069A priority Critical patent/JP2023508844A/ja
Priority to AU2020390812A priority patent/AU2020390812B2/en
Priority to EP20892802.8A priority patent/EP4050004A4/en
Priority to US17/775,402 priority patent/US20230002343A1/en
Priority to KR1020227022153A priority patent/KR20220107036A/ko
Publication of WO2021103749A1 publication Critical patent/WO2021103749A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/64—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to a L-bicyclomorpholine and a pharmaceutically acceptable salt thereof, a preparation method thereof, and application thereof in the preparation of medicines for preventing and/or treating liver diseases.
  • Liver disease is a worldwide disease, and my country is a major country with liver disease. At present, there are a large number of patients with liver injury and liver inflammation caused by various reasons in my country. Viral hepatitis is the main cause. According to statistics, my country's annual direct economic loss due to chronic hepatitis (including late-stage liver cirrhosis and liver cancer) is 900 billion yuan. In recent years, the incidence of drug-induced liver disease, alcoholic and non-alcoholic fatty liver disease, and autoimmune liver disease has also been increasing year by year. Finding safe and effective drugs for the prevention and treatment of liver disease has always been a hot spot for research and development by major research institutes and pharmaceutical companies around the world.
  • Bicyclol is the first chemical class I hepatitis drug developed by the Institute of Materia Medica, Chinese Academy of Medical Sciences with independent intellectual property rights in my country. It has clinically good liver-protecting and enzyme-lowering effects and certain anti-hepatitis virus activity. It is convenient to take and has no obvious adverse effects. The reaction has a wide range of pharmacological activities.
  • bicyclic morpholine and its salt have good pharmacological activity and pharmacokinetic properties ( Wu Song, Sun Hua, et al., a bicyclic alcohol derivative and its preparation and application, 201610922563.5).
  • the researchers split the bicyclomorpholine racemate and found that L-bicyclomorpholine and its salt have better anti-inflammatory and hepatoprotective properties than the racemate and dextrorotary morpholine and its salt. Pharmacological activity and pharmacokinetic properties.
  • the technical problem solved by the present invention is to provide a class of L-bicyclomorpholine and its pharmaceutically acceptable salt, its preparation method and its application in the preparation of drugs for preventing and/or treating liver-related diseases.
  • the present invention provides the following technical solutions:
  • the first aspect of the technical solution of the present invention is to provide a levorotatory bicyclomorpholine with a structure as shown in compound 5, and a pharmaceutically acceptable salt thereof, with the structural formula shown in (I) below:
  • X is selected from inorganic acid and organic acid; wherein the inorganic acid is selected from hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, sulfuric acid, phosphoric acid; the organic acid is selected from 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, camphor acid, camphor sulfonic acid, aspartic acid, glutamic acid, pyroglutamic acid, L-tartaric acid, L-dibenzoyl tartaric acid,
  • the second aspect of the technical solution of the present invention is to provide the preparation method of the L-bicyclomorpholine and the pharmaceutically acceptable salt thereof as described in the first aspect, as shown below:
  • Y is selected from: L-tartaric acid, L-dibenzoyltartaric acid, L-dimethylbenzoyltartaric acid, L-diethyl tartaric acid, L-malic acid, L-camphoric acid, L-10-camphoric acid Acid, R-(-)-mandelic acid, L-quinic acid, L-aspartic acid, L-pyroglutamic acid, D-tartaric acid, D-dibenzoyltartaric acid, D-dimethylbenzene Formyltartaric acid, D-diethyl tartrate, D-malic acid, D-camphoric acid, D-10-camphorsulfonic acid, S-(-)-mandelic acid, D-
  • the third aspect of the technical solution of the present invention provides a pharmaceutical composition, which comprises a therapeutically and/or preventively effective amount of the L-bicyclomorpholine and pharmaceutically acceptable salts thereof according to the first aspect of the present invention, and optionally One or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition can be prepared according to methods well known in the art.
  • the compound of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants to prepare any dosage form suitable for human or animal use.
  • the content of the compound of the present invention in its pharmaceutical composition is usually 0.1-95% by weight.
  • the compound of the present invention or a pharmaceutical composition containing it can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosal, eye, lung, and Respiratory tract, skin, vagina, rectum, etc.
  • the dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w type, w/o type and double emulsion), suspensions, injections (including water injections, powder injections and infusions), eye drops
  • the solid dosage form can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, air (powder) sprays, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.
  • the compounds of the present invention can be made into ordinary preparations, and can also be made into slow-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.
  • the diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the humectant can be water, ethanol, iso Propanol, etc.
  • the binder can be starch syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.
  • the disintegrant can be dry starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the humectant can be water, ethanol, iso Propanol, etc.
  • the binder can be starch syrup
  • the tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • the active ingredient of the compound of the present invention can be mixed with a diluent and a co-solvent, and the mixture can be directly placed in a hard capsule or a soft capsule.
  • the compound of the present invention can also be used as the active ingredient to form granules or pellets with diluents, binders, and disintegrants, and then placed in hard or soft capsules.
  • the various diluents, binders, wetting agents, disintegrants, and solubilizers used to prepare the compound tablets of the present invention can also be used to prepare the compound capsules of the present invention.
  • solubilizers can be poloxamer, lecithin, hydroxypropyl- ⁇ -cyclodextrin, etc.
  • the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.
  • the osmotic pressure regulator can be It is sodium chloride, mannitol, glucose, phosphate, acetate, etc.
  • mannitol, glucose, etc. can also be added as proppants.
  • coloring agents can also be added to the pharmaceutical preparations.
  • the drug or pharmaceutical composition of the present invention can be administered by any known administration method.
  • the dosage of the compound pharmaceutical composition of the present invention can vary widely according to the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally speaking, the appropriate daily dose range of the compound of the present invention is 0.001-5 mg/Kg body weight.
  • the above-mentioned dosage can be administered in one dosage unit or divided into several dosage units, depending on the doctor's clinical experience and the dosage regimen including the use of other treatment methods.
  • the compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.
  • the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.
  • the fourth aspect of the technical solution of the present invention is to provide the L-bicyclomorpholine of the first aspect and the pharmaceutically acceptable salt thereof, and the pharmaceutical composition of the third aspect in the preparation of drugs for the prevention and/or treatment of liver-related diseases
  • the liver-related diseases are selected from liver injury-related diseases and hepatitis-related diseases, specifically including: hepatitis A, hepatitis B, hepatitis C, drug-induced liver disease, alcoholic liver disease, non-alcoholic liver disease, autoimmune liver disease, and liver disease progression Liver fibrosis, cirrhosis, liver failure.
  • L-bicyclomorpholine and its pharmaceutically acceptable salts involved in the present invention show better pharmacological activity than dextrorotate and racemate in a variety of animal models of liver injury, and the statistical difference is significant (P ⁇ 0.05) ).
  • levorotatory bicyclomorpholine and its pharmaceutically acceptable salts have better pharmacokinetic properties than dextrorotatory and racemates.
  • the invention provides a L-bicyclomorpholine for treating liver disease and a pharmaceutically acceptable salt thereof, a preparation method, a pharmaceutical composition and an application thereof.
  • the following examples further illustrate the present invention, but do not limit the present invention in any way. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.
  • the nuclear magnetic resonance spectrum of the L-bicyclomorpholine and its salt provided by the present invention is measured with a Varain Mercury-500 nuclear magnetic resonance instrument, TMS is an internal standard, and the mass spectrum is measured with a ZAB-2F mass spectrometer.
  • a.SOCl 2 /DMF b.TEA/CH 2 Cl 2 or CH 3 COCH 3 ; cL-DBTA/EA; d.NaHCO 3 /H 2 O/EA; e.CH 3 SO 3 H/EA.
  • 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 with anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oil.
  • SPF male ICR mice (20-22g) were randomly divided into 5 groups after adapting to the environment, blank control group, Con A model group, (+)IMM-H014 200mg/kg group, (-)IMM-H014 200mg/kg Group, ( ⁇ )IMM-H014 200mg/kg group, 10 animals in each group.
  • Each administration group was given intragastric administration once a day for 3 times in total.
  • the blank control group and the model group were given the same amount of normal saline via intragastric administration.
  • mice in each group were injected with 20 mg/kg Con A once through the tail vein. The dosage is 10ml/kg. After the mice were fasted with water for 16 hours, the animals were treated.
  • mice were decapitated to take blood, the blood samples were left at room temperature for 2 hours, and the blood samples were centrifuged at 4000 rpm for 10 minutes.
  • the level of serum ALT is directly and positively correlated with the degree of liver damage, and is a recognized serum biomarker of liver damage.
  • the results in Table 1 show that ConA 20mg/kg caused significant liver damage in mice, and the serum ALT content was significantly higher than that of the blank control group (P ⁇ 0.001).
  • (+)IMM-H014 and (-)IMM-H014 can significantly reduce the increase in serum ALT caused by ConA (P ⁇ 0.001), and the percentage of reduction in ALT is 95.3% and 97.3%, respectively, and the ALT content is reduced to the blank control group
  • (+)IMM-H014 and (-)IMM-H014 both showed significant protective effects on immune liver injury caused by ConA, and the activity of (-)IMM-H014 was slightly better than (+)IMM-H014.
  • (+)IMM-H014 and (-)IMM-H014 are better than ( ⁇ )IMM-H014 in reducing the increase in serum ALT levels induced by ConA in mice.
  • Significant efficacy, the percentage of ALT reduction is 88.6%), among which (-)IMM-H014 has a statistically different effect on ALT reduction compared with ( ⁇ )IMM-H014 (P ⁇ 0.05).
  • Elevated serum AST level is also one of the important markers of liver cell damage, especially liver cell mitochondrial damage. When mitochondrial damage, serum AST level is significantly increased, reflecting the severity of liver cell damage.
  • the results are shown in Table 2.
  • ConA 20mg/kg caused significant damage to the mitochondria of mouse liver cells, and the serum AST level was significantly higher than that of the blank control group (P ⁇ 0.001).
  • (+)IMM-H014, (-)IMM-H014 and ( ⁇ )IMM-H014 can reduce the level of serum AST increase caused by ConA.
  • the percentage reduction of AST is 44.8%, 72.9% and 22.2%, respectively , Among which (-)IMM-H014 group has statistical difference compared with model group (P ⁇ 0.01).
  • (+)IMM-H014, (-)IMM-H014 have better AST lowering activity than ( ⁇ )IMM-H014, (-)IMM-H014 has the best activity, compared with the same dose of ( ⁇ )IMM-H014 group There are statistical differences (P ⁇ 0.05).
  • the serum LDH level can also reflect the damage and degree of liver cell damage.
  • the results are shown in Table 3.
  • ConA 20mg/kg tail vein injection caused severe liver cell damage, and the serum LDH level was significantly higher than that of the blank control group (P ⁇ 0.001).
  • (-) IMM-H014 can significantly reduce the increase in serum LDH caused by ConA, and the percentage of LDH reduction is 54.4%, which is statistically different from the model group (P ⁇ 0.01).
  • (+) IMM-H014 can also reduce the increase in serum LDH, the reduction percentage is 27.6%, but there is no statistical difference compared with the model group.
  • ( ⁇ ) IMM-H014 has only a weak reduction effect on the increase of LDH at the current dose, and the reduction percentage is 6.4%.
  • SPF-grade male ICR mice (20-22g) were randomly divided into 5 groups after adapting to the environment, blank control group, ethionine model group, (+)IMM-H014 200mg/kg group, (-)IMM-H014 200mg /kg group, ( ⁇ )IMM-H014 200mg/kg group, 5 animals in each group.
  • Each administration group was given intragastric administration 3 days before modeling, once a day, 3 times in total.
  • the animals in the blank control group and the model group were given the same amount of normal saline.
  • the dosage is 10mL/kg.
  • mice in each group were given 250 mg/kg ethionine once by gavage.
  • the dosage is 20mL/kg. After the mice were fasted with water for 24 hours, the animals were treated.
  • liver tissue triglyceride kit Open the chest to take the liver, wash it with 4°C normal saline, take part of the liver tissue with lysate and electric homogenizer to prepare 10% liver tissue homogenate, part of the liver tissue is measured according to the detection method of tissue triglyceride kit and cholesterol kit The other part uses BCA protein quantification kit to detect protein content, and performs protein correction on liver tissue triglyceride and cholesterol.
  • Ethionine can interfere with the metabolism of methionine, thereby affecting the synthesis of apolipoproteins, etc., causing the cholesterol and triglycerides synthesized by liver cells to be unable to be transported into the blood, causing lipid accumulation in liver cells to form drug-induced Non-alcoholic fatty liver model.
  • the results are shown in Table 4. 250mg/kg ethionine caused a significant increase in liver cholesterol content compared with the blank control group (P ⁇ 0.05). Both (+)IMM-H014 and (-)IMM-H014 can reduce ethionine Acid-induced liver tissue TC accumulation, the percentage of TC reduction was 27.2% and 32.4%, respectively.
  • the (-)IMM-H014 group was significantly different from the model group (P ⁇ 0.05).
  • ( ⁇ ) IMM-H014 also has a significant inhibitory effect on the elevation of liver tissue TC caused by ethionine, which is statistically different from the model group (P ⁇ 0.05).
  • the activity of (-) IMM-H014 is slightly better than ( ⁇ ) IMM-H014.
  • Human liver cancer HepG 2 cells which retain the characteristics of normal human liver cells. Grow in DMEM culture medium (containing 100 U/mL penicillin and 100 ⁇ g/mL streptomycin) containing 10% fetal bovine serum, and the culture conditions are 37° C., 5% CO 2 , and saturated humidity. Digestion and passage with solution containing 0.25% trypsin and 0.02% EDTA.
  • Cell survival rate (%) (average OD of administration group/average OD of solvent control group) ⁇ 100%.
  • (+)IMM-H014, (-)IMM-H014 and ( ⁇ )IMM-H014 10 ⁇ M doses have a significant protective effect on APAP-induced in vitro human hepatocyte damage (P ⁇ 0.05, P ⁇ 0.001, P ⁇ 0.01), The percentage increase in cell survival was 41.4%, 74.8% and 32.1%, respectively.
  • the activity of (-) IMM-H014 is relatively best, and there is a statistical difference compared with the same dose of ( ⁇ ) IMM-H014 group (P ⁇ 0.05).
  • Bicyclic alcohol can also significantly improve liver cell damage caused by APAP (P ⁇ 0.05).
  • LDH lactate dehydrogenase
  • LDH lactate dehydrogenase
  • the level of LDH is directly proportional to the degree of cell damage.
  • the results are shown in Figure 1, 8mM APAP treated HepG2 cells for 24h, the LDH level in the cell culture supernatant was significantly higher than that in the blank control group (P ⁇ 0.01), further suggesting significant damage to liver cells. (+)IMM-H014, (-)IMM-H014, and ( ⁇ )IMM-H014 10 ⁇ M doses can all reduce LDH levels.
  • Human liver cancer HepG 2 cells which retain the characteristics of normal human liver cells. Grow in DMEM culture medium (containing penicillin 100 U/mL, streptomycin 100 ⁇ g/mL) containing 10% fetal bovine serum, and the culture conditions are 37° C., 5% CO 2 , and saturated humidity. Digestion and passage with solution containing 0.25% trypsin and 0.02% EDTA.
  • HepG 2 cells were seeded in 96-well cell culture plates. After 24 hours of culture, non-toxic concentrations of (+)IMM-H014, (-)IMM-H014 and ( ⁇ )IMM-H014 were added, and carbon tetrachloride (CCl) was added at the same time. 4. The final concentration is 0.6%).
  • the experiment has a bicyclic alcohol positive drug control group, a solvent control group and a model group. Continue to act on the cells for 24h.
  • Cell survival rate (%) (average OD of administration group/average OD of solvent control group) ⁇ 100%.
  • (+)IMM-H014, (-)IMM-H014 and ( ⁇ )IMM-H014 10 ⁇ M doses have a significant improvement effect on CCl 4 induced human liver cell injury in vitro (P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.01) , The percentages of cell survival improvement were 12.59%, 34.66% and 18.74%, respectively.
  • ( ⁇ )IMM-H014, (-)IMM-H014 has better protective activity against CCl 4 induced hepatocyte damage.
  • Bicyclic alcohol can also significantly improve liver cell damage caused by APAP.
  • Agilent 6470 triple quadrupole liquid-mass spectrometer (Agilent), Mettler AG135 electronic analytical balance, pipette, TDL-5-A centrifuge, SIGMA small centrifuge, nitrogen blowing instrument, animal weighing scale.
  • 36 male SD rats were randomly divided into 6 groups, each with 6 rats, including: (+)IMM-H014 intragastric administration group, (-)IMM-H014 intragastric administration group, ( ⁇ )IMM-H014 Gavage administration group.
  • the dosage is 50mg/kg, fasting 12h before administration, and drinking water freely.
  • IMM-H014 stock solution Precisely pipet 100 ⁇ L of 1.0mg/mL IMM-H014 stock solution into a 10mL volumetric flask and dilute with methanol to obtain a 10 ⁇ g/mL solution of IMM-H014.
  • the solution was gradually diluted with methanol to obtain IMM-H014 series standard solutions with concentrations of 2, 5, 10, 50, 100, 500, 1000, 2000, 4000 and 5000 ng/ml.
  • Mobile phase A water (0.1% formic acid, 1mM ammonium acetate)
  • Mobile phase B acetonitrile (0.1% formic acid)
  • Ion source ESI
  • detection mode positive ion
  • drying gas temperature 300°C
  • drying gas flow rate nitrogen, 11L/min
  • atomizing gas nitrogen, 15psi
  • capillary voltage 4000V
  • scanning mode multiple reaction detection (MRM)
  • Ion pairs and related voltage parameters are as follows:
  • the original data obtained after sample collection is processed by MassHunter QQQ data processing software to obtain blood drug concentration data; then the pharmacokinetic parameters are calculated by DAS software; finally, SPSS software is used for t-test to compare different batches of drugs with the same optical properties Whether the pharmacokinetic parameters of drugs with different optical properties are statistically different, P ⁇ 0.05 is considered to be a significant difference.

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Abstract

本发明属于药物化学领域，具体涉及一种左旋双环吗啉及其药学上可接受的盐、其制备方法、药物组合物及其在制备预防和/或治疗肝病等方面药物中的应用。

Description

一类左旋双环吗啉及其盐，其制备方法、药物组合物和应用 技术领域
本发明属于药物化学领域，具体涉及一种左旋双环吗啉及其药学上可接受的盐、其制备方法、及其在制备预防和/或治疗肝病等方面药物中的应用。
背景技术
肝病是世界性疾病，我国是肝病大国。当前我国各种原因引起的肝损伤及肝脏炎症患者数量众多，以病毒性肝炎为主，具统计，我国每年因慢性肝炎(包括后期导致的肝硬化、肝癌)直接经济损失达9000亿人民币。近年来，药物性肝病、酒精性及非酒精性脂肪性肝病、自身免疫性肝病发病率亦呈逐年增加趋势。寻找安全有效的防治肝病相关药物一直都是全世界各大研究院所及制药公司研发的热点。
双环醇是中国医学科学院药物研究所研发的第一个拥有我国自主知识产权的化学一类治疗肝炎药物，临床具有良好的保肝降酶作用和一定的抗肝炎病毒活性，服用方便，无明显不良反应，药理活性广泛。双环醇相关研究成果先后获得多个奖项，包括“九五”优秀科研攻关成果奖(2002年)，2002年中国医药十大新闻，北京市科技进步一等奖(2005年)和国家科技进步二等奖(2007年)等，已获得美国、欧盟、日本、韩国等16个国家和台湾地区的化合物发明专利保护，产品远销乌克兰等国。自2001年11月在人民大会堂召开新闻发布会宣布上市，双环醇已为国家创造了巨大的社会效益和经济效益。
但是双环醇生物水溶性很差，导致生物利用度不高，中国医学科学院药物研究所研究人员对其结构进行了优化，发现了双环吗啉及其盐具有良好药理活性和药代动力学性质(吴松，孙华，等，一种双环醇类衍生物及其制备和应用，201610922563.5)。在此前发现的基础上，研究人员对双环吗啉消旋体进行了拆分，发现左旋双环吗啉及其盐比消旋体和右旋体及其盐在抗炎保肝方面具有更好的药理活性和药代动力学性质。
发明内容
本发明解决的技术问题是提供一类左旋双环吗啉及其药学上可接受的盐、其制备方法以及在制备预防和/或治疗与肝脏有关疾病的药物中的应用。
为解决本发明的技术问题，本发明提供如下技术方案：
本发明技术方案的第一方面是提供了一种左旋双环吗啉，结构如化合物5所示，及其药学上可接受的盐，具有如下(I)所示的结构式：
Figure PCTCN2020115410-appb-000001
其中，药物上可接受的酸加合盐可从无机酸和有机酸制备。X选自无机酸、有机酸；其中无机酸选自氢氟酸、盐酸、氢溴酸、氢碘酸、醋酸、硫酸、磷酸；所述的有机酸选自乙酸、三氟乙酸、丙酸、草酸、苹果酸、丙二酸、琥珀酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸、奎宁酸、樟脑酸、樟脑磺酸、天冬氨酸、谷氨酸、焦谷氨酸、L-酒石酸、L-二苯甲酰酒石酸、L-二对甲基苯甲酰酒石酸、L-酒石酸二乙酯、L-苹果酸、L-樟脑酸、L-10-樟脑磺酸、R-(-)-扁桃酸、L-奎宁酸、L-天冬氨酸、L-谷氨酸、L-焦谷氨酸、D-酒石酸、D-二苯甲酰酒石酸、D-二对甲基苯甲酰酒石酸、D-酒石酸二乙酯、D-苹果酸、D-樟脑酸、D-10-樟脑磺酸、S-(-)-扁桃酸、D-奎宁酸、D-天冬氨酸、D-谷氨酸、D-焦谷氨酸，其中游离碱和酸的比例任选为1:1、2:1或3:1。其中，优选的结构为：
Figure PCTCN2020115410-appb-000002
本发明技术方案的第二方面是提供了第一方面所述左旋双环吗啉及其药学 上可接受盐的制备方法，如下所示：
Figure PCTCN2020115410-appb-000003
a)双环醇羟基进行氯代得到化合物2；
b)化合物2与吗啉反应得到化合物3；
c)化合物3与手性酸Y成盐，利用盐溶解度差异，在有机溶剂中进行拆分得到左旋体的盐4，左旋体对映体过量百分率(％e.e.)大于95.0％；其中所述的Y选自:L-酒石酸、L-二苯甲酰酒石酸、L-二对甲基苯甲酰酒石酸、L-酒石酸二乙酯、L-苹果酸、L-樟脑酸、L-10-樟脑磺酸、R-(-)-扁桃酸、L-奎宁酸、L-天冬氨酸、L-焦谷氨酸、D-酒石酸、D-二苯甲酰酒石酸、D-二对甲基苯甲酰酒石酸、D-酒石酸二乙酯、D-苹果酸、D-樟脑酸、D-10-樟脑磺酸、S-(-)-扁桃酸、D-奎宁酸、D-天冬氨酸、D-谷氨酸、D-焦谷氨酸及上述酸的衍生物，其中游离碱和酸的比例任选为1:1、2:1或3:1。所述有机溶剂为：乙酸乙酯、丙酮、甲醇、乙醇、异丙醇及混合溶剂；
d)盐4在碱的作用下游离；
e)游离胺和酸X成盐，得到化合物I。
其中，X的定义和本发明技术方案第一方面的定义项相同。
本发明技术方案的第三方面提供了一种药物组合物，其包含治疗和/或预防有效量的本发明第一方面所述左旋双环吗啉及其药学上可接受的盐，以及任选的一种或多种药学可接受的载体或赋形剂。
该药物组合物可根据本领域公知的方法制备。可通过将本发明化合物与一种或多种药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。本发明化合物在其药物组合物中的含量通常为0.1-95重量％。本发明化合物或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括o/w型、w/o型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明化合物可以制成普通制剂、也制成是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将本发明化合物制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助溶剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；湿润剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助溶剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分本发明化合物与稀释剂、助溶剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分本发明化合物先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制 备本发明化合物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助溶剂品种也可用于制备本发明化合物的胶囊剂。
为将本发明化合物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、pH调节剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调节剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其它添加剂。
为达到用药目的，增强治疗效果，本发明的药物或药物组合物可用任何公知的给药方法给药。
本发明化合物药物组合物的给药剂量依照所要预防或治疗疾病的性质和严重程度，患者或动物的个体情况，给药途径和剂型等可以有大范围的变化。一般来讲，本发明化合物的每天的合适剂量范围为0.001-5mg/Kg体重。上述剂量可以一个剂量单位或分成几个剂量单位给药，这取决于医生的临床经验以及包括运用其它治疗手段的给药方案。
本发明的化合物或组合物可单独服用，或与其他治疗药物或对症药物合并使用。当本发明的化合物与其它治疗药物存在协同作用时，应根据实际情况调整它的剂量。
本发明技术方案的第四方面是提供了第一方面所述左旋双环吗啉及其药学上可接受的盐以及第三方面所述药物组合物在制备预防和/或治疗与肝脏有关疾病的药物中的应用。其中所述的与肝脏有关疾病选自肝损伤相关疾病、肝炎相关疾病，具体而言包括：甲肝、乙肝、丙肝、药物性肝病、酒精性肝病、非酒精性肝病、自身免疫性肝病、肝病进展的肝纤维化、肝硬化、肝衰竭。
有益技术效果
本发明涉及的左旋双环吗啉及其药学上可接受的盐在多种肝损伤动物模型中都显示出比右旋体和消旋体更好的药理活性，而且统计学差异显著(P<0.05)。此外，左旋双环吗啉及其药学上可接受的盐的具有比右旋体和消旋体更好的药代动力学性质。
附图说明
图1 IMM-H014对映异构体对对乙酰氨基酚引起细胞培养上清LDH水平升高的降低作用(n＝3-4)。 **P<0.01，与空白对照组比较； #P<0.05，与模型组比较。
具体实施方式
本发明提供了一种治疗肝病的左旋双环吗啉及其药学上可接受的盐，其制备方法，药物组合物和应用。下面列举实施例进一步对本发明予以说明，但不以任何方式限制本发明。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
本发明提供的左旋双环吗啉及其盐核磁共振谱用Varain Mercury-500核磁共振仪测定，TMS为内标，质谱用ZAB-2F质谱仪测定。
实施例1左旋(-)IMM-H014的制备
Figure PCTCN2020115410-appb-000004
a.SOCl 2/DMF；b.TEA/CH 2Cl 2or CH 3COCH 3；c.L-DBTA/EA；d.NaHCO 3/H 2O/EA；e.CH 3SO 3H/EA.
将化合物1(即双环醇，5.1g，13.1mmol)置于100ml带有磁力搅拌和温度计的三口瓶中，加入50ml干燥DMF，固体完全溶解，冰浴冷却至0℃后，缓慢滴加SOCl 2(4.5ml，61.8mmol)，控制体系温度不超过5℃。滴加完毕后冰浴反 应30min至TLC显示原料反应完全。将反应体系倒在约100g碎冰中，充分搅拌，析出大量白色固体，过滤，滤饼依次用少量蒸馏水、乙醚洗，抽干。自然晾干，称重，得到白色固体(化合物2)共4.9g，收率：91.7％。
将吗啉(1.28g，14.7mmol)置于50ml的圆底烧瓶中，加入丙酮25ml和三乙胺2.2ml，室温搅拌下加入化合物2(2.76g，6.8mmol)，室温反应5h，静置过夜，TLC显示原料反应完全，体系有粉色不溶固体生成，过滤，滤液减压蒸去溶剂，所得黄色油状物减压柱分离(石油醚∶乙酸乙酯＝2∶1)，收集产物组分，共得到无色油状物(化合物3)3.1g，收率：92.3％。MS-FAB[M+H] +＝460.1
称取化合物3(3.0g，6.54mmol)，溶于45ml乙酸乙酯中，加入L-二苯甲酰酒石酸(L-DBTA,1.2g，3.27mmol)，室温下搅拌析出白色固体，30分钟后过滤收集固体，再用60℃干燥1h，称重，共得白色固体1.25g(左旋双环吗啉：L-二苯甲酰酒石酸＝2:1)，收率：56.8％。手性HPLC分析，中间体4对映体过量百分率(％e.e.)为98.0％，[α] 25＝-130.4(CH 2Cl 2)。
将中间体4(1.25g)用25mL乙酸乙酯分散，用25mL饱和碳酸氢钠溶液洗两次，有机相用无水硫酸钠干燥，过滤，浓缩得无色油状物。用10ml乙酸乙酯将油状物溶解，加入120μL甲磺酸，室温下搅拌析晶。过滤，所得固体用10mL甲醇重结晶，得到(-)IMM-H014为白色固体0.7g，HPLC纯度>98.0％,[α] 25＝-65.6(CH 2Cl 2)。 1H NMR(500MHz,Chloroform-d)δ7.45(s,1H),7.38(s,1H),6.14(s,1H),6.02(s,1H),5.96(d,J＝8.9Hz,2H),4.37(dd,J＝13.3,4.0Hz,1H),4.16(t,J＝12.0Hz,1H),4.05(d,J＝19.7Hz,8H),3.96–3.81(m,3H),3.76(s,3H),3.57(d,J＝11.8Hz,1H),3.41(d,J＝12.5Hz,1H),2.91(s,3H),2.79–2.68(m,1H),2.50(d,J＝11.3Hz,1H).
对比例1右旋体(+)IMM-H014的制备
Figure PCTCN2020115410-appb-000005
另取化合物3(3.0g，6.54mmol)，溶于45ml乙酸乙酯中，加入D-二苯甲酰酒石酸(1.2g，3.27mmol)，室温下搅拌析出白色固体，30分钟后过滤收集固体，60℃干燥1h，称重，共得白色固体1.2g(右旋双环吗啉：D-二苯甲酰酒石酸＝2:1)，收率：54.5％。手性HPLC分析，中间体4对映体过量百分率(％e.e.)为97.3％，[α] 25＝+133.2(CH 2Cl 2)。
将中间体(1.2g)用25mL乙酸乙酯分散，用25mL饱和碳酸氢钠溶液洗两次，有机相用无水硫酸钠干燥，过滤，浓缩得无色油状物。用10ml乙酸乙酯将油状物溶解，加入120μL甲磺酸，室温下搅拌析晶。过滤，所得固体用10mL甲醇重结晶，得到(+)IMM-H014为白色固体0.6g，HPLC纯度>98.0％,[α] 25＝-78.2(CH 2Cl 2)。 1H NMR(500MHz,Chloroform-d)δ7.45(s,1H),7.38(s,1H),6.14(s,1H),6.02(s,1H),5.96(d,J＝8.9Hz,2H),4.37(dd,J＝13.3,4.0Hz,1H),4.16(t,J＝12.0Hz,1H),4.05(d,J＝19.7Hz,8H),3.96–3.81(m,3H),3.76(s,3H),3.57(d,J＝11.8Hz,1H),3.41(d,J＝12.5Hz,1H),2.91(s,3H),2.79–2.68(m,1H),2.50(d,J＝11.3Hz,1H).
药理实验
实验例1 IMM-H014光学对映异构体对植物凝集素(ConA)诱导的急性免疫性肝损伤的作用
1.Con A诱导的小鼠急性免疫性肝损伤模型建立及给药方法
SPF级雄性ICR小鼠(20～22g)，适应环境后随机分为5组，空白对照组，Con A模型组，(+)IMM-H014 200mg/kg组，(-)IMM-H014 200mg/kg组，(±)IMM-H014 200mg/kg组，每组10只。各给药组灌胃给药，每天1次，共给药3次，空白对照组和模型组灌胃给予相同量生理盐水。于末次给药后2h，除空白对照组，各组小鼠尾静脉注射20mg/kg Con A一次。给药量均为10ml/kg。小鼠禁食不禁水16h后，处理动物。
2.生化指标测定
小鼠断头取血，血液样本室温静置2h，4000rmp离心10min，分离血清，全自动生化分析仪检测血清中ALT、AST、LDH含量。
3.统计学分析
数据以均数平均值±标准
Figure PCTCN2020115410-appb-000006
表示。组间比较用t检验，以p<0.05表 示有显著性差异。
4.实验结果
4.1 IMM-H014对映异构体对ConA引起血清肝损伤生物标记物ALT升高的作用
血清ALT的水平与肝损伤程度呈直接正相关，为公认的肝损伤血清生物标志物。表1的结果显示，ConA 20mg/kg引起小鼠显著肝损伤，血清ALT含量较空白对照组显著升高(P<0.001)。(+)IMM-H014，(-)IMM-H014均能显著降低ConA导致的血清ALT升高(P<0.001)，对ALT的降低百分率分别为95.3％和97.3％，ALT含量降低到空白对照组水平，(+)IMM-H014和(-)IMM-H014对ConA引起的免疫性肝损伤均表现显著保护作用，(-)IMM-H014活性略优于(+)IMM-H014。与同剂量的(±)IMM-H014比较，(+)IMM-H014和(-)IMM-H014对ConA诱导小鼠血清ALT水平升高的降低作用均优于(±)IMM-H014(亦显示显著药效，ALT降低百分率88.6％)，其中(-)IMM-H014对ALT的降低作用与(±)IMM-H014比较有统计学差异(P<0.05)。
表1 IMM-H014对映异构体对ConA引起小鼠血清ALT升高的降低作用(n＝10)
Figure PCTCN2020115410-appb-000007
***P<0.001，与空白对照组比较； ###P<0.001，与模型组比较； &P<0.05，与(±)IMM-H014组比较。
4.2 IMM-H014对映异构体对ConA引起血清肝损伤生物标记物AST升高的作用
血清AST水平升高亦为肝细胞损伤、特别是肝细胞线粒体损伤的重要标志物之一，线粒体损伤时，血清AST水平明显升高，反应肝细胞损伤的严重程度。结果见表2，ConA 20mg/kg引起小鼠肝细胞线粒体显著损伤，血清AST水平较空白对照组显著升高(P<0.001)。(+)IMM-H014，(-)IMM-H014及(±)IMM-H014均能够降低ConA引起的血清AST升高的水平，较模型组，AST降低百分率分别为44.8％，72.9％和22.2％，其中(-)IMM-H014组与模型组比较有统计学差异 (P<0.01)。(+)IMM-H014、(-)IMM-H014对AST的降低活性优于(±)IMM-H014，(-)IMM-H014的活性最佳，与同剂量的(±)IMM-H014组比较有统计学差异(P<0.05)。
表2 IMM-H014对映异构体对ConA引起小鼠血清AST升高的降低作用(n＝10)
Figure PCTCN2020115410-appb-000008
***P<0.001，与空白对照组比较； ##P<0.01，与模型组比较； &P<0.05，与(±)IMM-H014组比较。
4.3 IMM-H014对映异构体对ConA引起血清LDH升高的作用
当肝脏受损伤时，血清LDH水平亦能反映肝细胞受损情况和损伤程度。结果见表3，ConA 20mg/kg尾静脉注射给药引起严重肝细胞损伤，血清LDH水平较空白对照组显著升高(P<0.001)。(-)IMM-H014能显著降低ConA引起的血清LDH升高水平，LDH降低百分率达54.4％，与模型组比较有统计学差异(P<0.01)。(+)IMM-H014对血清LDH升高亦有降低作用，降低百分率为27.6％，但与模型组比较无统计学差异。(±)IMM-H014在目前所用剂量下，对LDH的升高仅有弱的降低作用，降低百分率为6.4％。在降低LDH水平方面，(-)IMM-H014活性仍优于(+)IMM-H014和(±)IMM-H014，(-)IMM-H014与同剂量的(±)IMM-H014比较，有统计学差异(P<0.05)。
表3 IMM-H014对映异构体对ConA引起小鼠血清LDH升高的降低作用(n＝10)
Figure PCTCN2020115410-appb-000009
Figure PCTCN2020115410-appb-000010
***P<0.001，与空白对照组比较； ##P<0.01，与模型组比较； &P<0.05，与(±)IMM-H014组比较。
实验例2 IMM-H014光学对映异构体对乙硫氨酸诱导的非酒精性脂肪肝的作用
1.乙硫氨酸诱导的小鼠非酒精性脂肪肝模型建立及给药方法
SPF级雄性ICR小鼠(20～22g)，适应环境后随机分为5组，空白对照组，乙硫氨酸模型组，(+)IMM-H014 200mg/kg组，(-)IMM-H014 200mg/kg组，(±)IMM-H014 200mg/kg组，每组5只。各给药组于造模前3天灌胃给药，每天1次，共3次，空白对照组及模型组动物给予相同量的生理盐水。给药量均为10mL/kg。于末次给药后1h，各组小鼠灌胃给予250mg/kg乙硫氨酸一次。给药量为20mL/kg。小鼠禁食不禁水24h后，处理动物。
2.肝组织甘油三酯及胆固醇含量测定
开胸取肝脏，用4℃生理盐水冲洗，取部分肝组织用裂解液和电动匀浆器制备10％的肝组织匀浆，一部分按组织甘油三酯试剂盒及胆固醇试剂盒检测方法测定肝组织中甘油三酯和胆固醇，另一部分用BCA蛋白定量试剂盒检测蛋白含量，对肝组织甘油三酯、胆固醇进行蛋白校正。
3.统计学分析
采用SPSS软件，数据以均数平均值±标准差(mean±SD)表示。组间比较用t检验，以P<0.05表示有显著性差异。
4.实验结果
4.1 IMM-H014对映异构体对乙硫氨酸引起小鼠肝组织胆固醇(TC)含量升高的作用
乙硫氨酸能干扰甲硫氨酸的代谢，进而影响载脂蛋白等的合成，导致肝细胞合成的胆固醇及甘油三酯等无法转运到血液中，引起肝细胞脂质堆积，形成药物诱导的非酒精性脂肪肝模型。结果见表4，250mg/kg乙硫氨酸引起肝组织内胆固醇含量较空白对照组显著升高(P<0.05)，(+)IMM-H014和(-)IMM-H014均能降低乙硫氨酸诱导的肝组织TC堆积，TC降低百分率分别为27.2％和32.4％，其中，(-)IMM-H014组与模型组比较有统计学差异(P<0.05)。(±)IMM-H014对乙硫氨酸引起的肝组织TC升高亦有显著抑制作用，与模型组比较有统计学差异 (P<0.05)。(-)IMM-H014活性略优于(±)IMM-H014。
表4 IMM-H014对映异构体对乙硫氨酸引起小鼠肝组织TC蓄积的降低作用(n＝10)
Figure PCTCN2020115410-appb-000011
*P<0.05，与空白对照组比较； #P<0.05，与模型组比较。
4.2 IMM-H014对映异构体对乙硫氨酸引起小鼠肝组织甘油三酯(TG)含量升高的作用
结果见表5，250mg/kg乙硫氨酸引起肝组织内TG含量较空白对照组显著升高(P<0.05)，表现非酒精性脂肪肝症状。(-)IMM-H014能显著降低乙硫氨酸诱导的肝组织TG堆积，TG降低百分率为39.0％，与模型组比较有统计学差异(P<0.05)。(+)IMM-H014对乙硫氨酸诱导的肝组织TG升高显示较弱的降低作用，降低百分率仅为7.1％。
表5 IMM-H014对映异构体对乙硫氨酸引起小鼠肝组织TG蓄积的降低作用(n＝10)
Figure PCTCN2020115410-appb-000012
*P<0.05，与空白对照组比较； #P<0.05， ##P<0.01，与模型组比较。
实验例3 IMM-H014光学对映异构体对对乙酰氨基酚诱导肝细胞损伤的作用
1.细胞培养
人肝癌HepG 2细胞，该细胞较好的保留了人正常肝细胞的特性。在含10％ 胎牛血清的DMEM培养液(含青霉素100U/mL，链霉素100μg/mL)中生长，培养条件为37℃、5％CO 2，饱和湿度。用含0.25％胰蛋白酶和0.02％EDTA液消化传代。
2.IMM-H014对映异构体对对乙酰氨基酚引起体外肝细胞损伤的保护作用
采用MTT方法。HepG 2细胞接种于96孔细胞培养板中，培养24h后，加入无毒浓度的(+)IMM-H014、(-)IMM-H014及(±)IMM-H014，同时加入对乙酰氨基酚(APAP，终浓度8mM)，实验设双环醇阳性对照药组、溶剂对照组及模型组。继续培养24h。取培养液100μL，离心，利用LDH检测试剂盒全自动生化分析仪测定LDH水平。弃去剩余培养液，每孔加入MTT(0.5mg/mL)液100μL，继续培养4h，弃去MTT液，每孔加入DMSO 150μL，混和振荡器振荡，于酶标仪570nm波长处测定吸光度值。细胞存活率(％)＝(给药组OD平均值/溶剂对照组OD平均值)×100％。
3.统计学分析
数据以均数平均值±标准差
Figure PCTCN2020115410-appb-000013
表示。组间比较用t检验，以p<0.05表示有显著性差异。
4.实验结果
10μM(+)IMM-H014、10μM(-)IMM-H014、10μM(±)IMM-H014作用HepG 2细胞48h对细胞无明显毒性，细胞存活率均90％。利用该浓度考察其对APAP损伤肝细胞的保护作用。结果见表6，8mM APAP可显著损伤HepG 2细胞，细胞存活率较空白对照组仅为38.33％(P<0.001)。(+)IMM-H014、(-)IMM-H014及(±)IMM-H014 10μM剂量对APAP引起的体外人肝细胞损伤均有显著保护作用(P<0.05，P<0.001，P<0.01)，细胞存活提高百分率分别为41.4％，74.8％和32.1％。(-)IMM-H014活性相对最佳，与同剂量的(±)IMM-H014组比较有统计学差异(P<0.05)。双环醇亦能显著改善APAP引起的肝细胞损伤(P<0.05)。
表6 IMM-H014对映异构体对对乙酰氨基酚降低肝细胞存活率的作用(n＝3-4)
Figure PCTCN2020115410-appb-000014
Figure PCTCN2020115410-appb-000015
***P<0.001，与空白对照组比较； #P<0.05， ##P<0.01， ###P<0.001，与模型组比较； &P<0.05，与(±)IMM-H014组比较。
LDH(乳酸脱氢酶)是细胞能量代谢的重要酶之一，当细胞死亡时，胞膜破裂，LDH从胞浆释放，LDH水平与细胞损伤程度呈正比。结果见附图1，8mM APAP作用HepG2细胞24h，细胞培养上清中LDH水平较空白对照组显著升高(P<0.01)，进一步提示肝细胞显著损伤。(+)IMM-H014、(-)IMM-H014及(±)IMM-H014 10μM剂量均能够降低LDH水平，其中(-)IMM-H014、(±)IMM-H014组LDH水平与模型组比较有统计学差异(P<0.05，P<0.05)，(-)IMM-H014的降低作用略优于同剂量的(±)IMM-H014。
实验例4 IMM-H014光学对映异构体对四氯化碳诱导肝细胞损伤的作用
1.细胞培养
人肝癌HepG 2细胞，该细胞较好的保留了人正常肝细胞的特性。在含10％胎牛血清的DMEM培养液(含青霉素100U/mL，链霉素100μg/mL)中生长，培养条件为37℃、5％CO 2，饱和湿度。用含0.25％胰蛋白酶和0.02％EDTA液消化传代。
2.IMM-H014对映异构体对四氯化碳引起体外肝细胞损伤的保护作用
采用MTT方法。HepG 2细胞接种于96孔细胞培养板中，培养24h后，加入无毒浓度的(+)IMM-H014、(-)IMM-H014及(±)IMM-H014，同时加入四氯化碳(CCl 4，终浓度0.6％)，实验设双环醇阳性药对照组、溶剂对照组及模型组。继续作用细胞24h。弃去培养液，每孔加入MTT(0.5mg/mL)液100μL，培养4h，弃去MTT液，每孔加入DMSO 150μL，混和振荡器振荡，于酶标仪570nm波长处测定吸光度值。细胞存活率(％)＝(给药组OD平均值/溶剂对照组OD平均值)×100％。
3.统计学分析
数据以均数平均值±标准差
Figure PCTCN2020115410-appb-000016
表示。组间比较用t检验，以p<0.05表示有显著性差异。
4.实验结果
10μM(+)IMM-H014、10μM(-)IMM-H014、10μM(±)IMM-H014作用HepG 2细胞48h对细胞无明显毒性，细胞存活率均90％。利用该浓度考察其对CCl 4损伤肝细胞的保护作用。结果见表7，0.6％CCl 4可显著损伤HepG 2细胞，细胞存活率较空白对照组为77.50％(P<0.01)。(+)IMM-H014、(-)IMM-H014及(±)IMM-H014 10μM剂量对CCl 4引起的体外人肝细胞损伤均有显著改善作用(P<0.05，P<0.01，P<0.01)，细胞存活提高百分率分别为12.59％，34.66％和18.74％。与同剂量的(±)IMM-H014比较，(-)IMM-H014对CCl 4引起肝细胞损伤的保护活性更优。双环醇亦能显著改善APAP引起的肝细胞损伤。
表7 IMM-H014对映异构体对对乙酰氨基酚降低肝细胞存活率的作用(n＝3-4)
Figure PCTCN2020115410-appb-000017
**P<0.01，与空白对照组比较； #P<0.05， ##P<0.01，与模型组比较。
药代动力学实验
实验例5
1实验目的
通过灌胃给药方式分别给予雄性SD大鼠的IMM-H014单一对映体和消旋体，比较它们在大鼠体内的药代动力学特点。
2实验仪器与材料
2.1实验仪器
Agilent 6470三重串联四极杆液质联用仪(安捷伦公司)、Mettler AG135型电子分析天平、移液枪、TDL-5-A离心机、SIGMA小型离心机、氮吹仪、动物体重秤。
2.2实验材料
(+)IMM-H014、(-)IMM-H014、(±)IMM-H014；甲醇(质谱级，Fisher Scientific公司产品，货号179097)；乙腈(质谱级，Fisher Scientific公司产品，货号177802)； 去离子水(杭州哇哈哈公司)；甲酸(HPLC级，ROE SCIENTIFIC INC，货号6F2941)；乙酸乙酯(质谱级，Fisher Scientific公司产品，货号166828)；1.5ml EP管。
2.3实验动物
雄性SD大鼠36只(200±10g)，购自北京维通利华实验动物技术有限公司。大鼠在中国医学科学院药物研究所动物房清洁间中饲养，每天12小时光照，温度为23±2℃，相对湿度为55±5％，实验动物可自由饮水进食，适应环境两周后开始实验。研究遵守中国医学科学院药物研究所的动物实验伦理委员会制定的规程。
3动物实验
3.1动物分组和给药
雄性SD大鼠36只，随机分为6组，每组6只，包括：(+)IMM-H014灌胃给药组，(-)IMM-H014灌胃给药组，(±)IMM-H014灌胃给药组。给药剂量为50mg/kg，给药前12h禁食，自由饮水。
3.2给药液的配制
称取IMM-H014原料药100mg用20ml纯净水溶解，制成5mg/ml的给药液，按体重进行单次给药(1ml/100g)。
3.3血浆样本采集
分别于给药前0h，给药后5min，10min，30min，1h，2h，3h，5h，8h，12h，24h通过眼底静脉丛取血250μL，置于含10μL肝素钠的EP管中，4000rpm离心10min，取血浆，置-80℃冰箱保存。
4分析方法
4.1溶液配制
4.1.1储备液的配制
精密称取IMM-H014对照品5mg置于5ml容量瓶中，用甲醇溶解并稀释至刻度，配置成1.0mg/ml的对照品储备液。
4.1.2内标工作液的配制
精密称取卡马西平对照品约5.0mg，置于5mL容量瓶中，加甲醇溶解并定容至刻度，得到浓度为1.0mg/mL的内标储备液，精密吸取该储备液50μL，置于10mL容量瓶，加甲醇稀释并定容至刻度，得到浓度为5.0μg/mL的内标浓储 备溶液。精密吸取内标浓储备溶液5.0mL，置50mL容量瓶，加甲醇稀释并定容至刻度，得到浓度为500ng/mL的内标工作溶液。
4.1.3系列浓度标准曲线工作溶液和质控标准溶液的配制
精密吸取1.0mg/mL IMM-H014储备液100μL置于10mL容量瓶，用甲醇稀释，得到IMM-H014为10μg/mL的溶液。用甲醇逐级稀释该溶液，得到浓度为2、5、10、50、100、500、1000、2000、4000和5000ng/ml的IMM-H014系列标准溶液。
精密吸取1.0mg/ml IMM-H014储备液，用甲醇逐级稀释，得到浓度为5、100、和4000ng/ml的质控标准溶液。
4.1.4基质标准曲线样品和质控样品的制备
精密吸取2、5、10、50、100、500、1000、2000、4000和5000ng/ml的标准溶液50μl，30℃下氮气吹干，加入50μl大鼠空白血浆，涡旋3min，制备基质标准曲线样品。
精密吸取5、100和4000ng/ml标准溶液50μl，30℃下氮气吹干，加入50μl大鼠空白血浆，涡旋3min，制备质控样品。
4.2样品前处理
精密吸取血浆样品50μl，加入10μL内标工作溶液至1.5ml EP管中，涡旋30s，加入500μl乙酸乙酯，涡旋振荡5min，4℃下静置10min，13000转离心5min，取上清液，30℃下N2吹干，残渣加入初始流动相100μL复溶，13000转离心5min，上清液转移至进样瓶中待测。
4.3色谱条件
色谱柱：Agilent ZORBAX SB-C18(2.1×100mm，3.5μm)
流动相A：水(0.1％甲酸，1mM乙酸铵)流动相B：乙腈(0.1％甲酸)
柱温：35℃ 进样量：3μL
洗脱：0-2min：40％-53％B 2-3min：53％-40％B
4.4质谱条件
离子源：ESI；检测模式：正离子；干燥气温度：300℃；干燥气流速：氮气，11L/min；雾化气：氮气，15psi；毛细管电压：4000V；扫描模式：多反应检测(MRM)；离子对及相关电压参数如下：
Figure PCTCN2020115410-appb-000018
Figure PCTCN2020115410-appb-000019
5数据处理
样本采集后得到的原始数据采用MassHunter QQQ数据处理软件处理后，得到血药浓度数据；再通过DAS软件进行药代参数的计算；最后采用SPSS软件进行t-检验，比较同一旋光性质不同批次药物之间以及不同旋光性质药物之间的药代参数是否有统计学差异，P<0.05认为具有显著性差异。
本研究通过对3组SD大鼠分别灌胃给予IMM-H014的单一对映体和消旋体后，研究了它们在大鼠体内的药代动力学特点。研究结果提示灌胃给药(-)IMM-H014和(±)IMM-H014的体内暴露水平优于(+)IMM-H014。
表8每组大鼠的平均药代参数(剔除离群值)
Figure PCTCN2020115410-appb-000020
*：(+)-IMM-H014与消旋IMM-H014相比，P<0.05。

Claims (9)

  1. 一种左旋双环吗啉以及药学上可接受的盐，其特征在于，该左旋双环吗啉的结构如化合物5所示，其药学上可接受的盐具有式I所述的结构式：
    Figure PCTCN2020115410-appb-100001
    其中，X选自无机酸、有机酸。
  2. 根据权利要求1的左旋双环吗啉以及药学上可接受的盐，其特征在于，所述的无机酸包括氢氟酸、盐酸、氢溴酸、氢碘酸、醋酸、硫酸、磷酸；所述的有机酸包括乙酸、三氟乙酸、丙酸、草酸、苹果酸、丙二酸、琥珀酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸、奎宁酸、樟脑酸、樟脑磺酸、天冬氨酸、谷氨酸、焦谷氨酸。
  3. 根据权利要求1的左旋双环吗啉以及药学上可接受的盐，其特征在于，所述的有机酸选自L-酒石酸、L-二苯甲酰酒石酸、L-二对甲基苯甲酰酒石酸、L-酒石酸二乙酯、L-苹果酸、L-樟脑酸、L-10-樟脑磺酸、R-(-)-扁桃酸、L-奎宁酸、L-天冬氨酸、L-谷氨酸、L-焦谷氨酸、D-酒石酸、D-二苯甲酰酒石酸、D-二对甲基苯甲酰酒石酸、D-酒石酸二乙酯、D-苹果酸、D-樟脑酸、D-10-樟脑磺酸、S-(-)-扁桃酸、D-奎宁酸、D-天冬氨酸、D-谷氨酸、D-焦谷氨酸。
  4. 根据权利要求1的左旋双环吗啉以及药学上可接受的盐，其特征在于，选自(-)-双环吗啉甲磺酸盐，具有如下所示结构：
    Figure PCTCN2020115410-appb-100002
  5. 权利要求1-4任一项所述的左旋双环吗啉以及药学上可接受的盐的制备方法，其特征在于，包括以下步骤：
    Figure PCTCN2020115410-appb-100003
    a)对双环醇羟基进行氯代得到化合物2；
    b)化合物2与吗啉反应得到化合物3；
    c)化合物3与手性酸Y成盐，利用盐溶解度差异，在有机溶剂中进行拆分得到左旋体的盐4，其中所述的Y选自:L-酒石酸、L-二苯甲酰酒石酸、L-二对甲基苯甲酰酒石酸、L-酒石酸二乙酯、L-苹果酸、L-樟脑酸、L-10-樟脑磺酸、R-(-)-扁桃酸、L-奎宁酸、L-天冬氨酸、L-谷氨酸、L-焦谷氨酸、D-酒石酸、D-二苯甲酰酒石酸、D-二对甲基苯甲酰酒石酸、D-酒石酸二乙酯、D-苹果酸、D-樟脑酸、D-10-樟脑磺酸、S-(-)-扁桃酸、D-奎宁酸、D-天冬氨酸、D-谷氨酸、D-焦谷氨酸；
    所述有机溶剂为：乙酸乙酯、丙酮、甲醇、乙醇、异丙醇以及上述溶剂按不同比例混合后的溶剂；左旋体和右旋体的ee值分别大于95％；
    d)盐4在碱的作用下游离；
    e)任选地，游离胺5和酸X成盐，得到化合物I；
    其中，X的定义和权利要求1-4任一项相同。
  6. 一种药物组合物，其特征在于，包含预防和/或治疗有效量的权利要求1-4任一项所述的左旋双环吗啉及其药学上可接受的盐，以及任选的一种或多种药学上可接受的载体或赋形剂。
  7. 权利要求1-4任一项所述左旋双环吗啉及其药学上可接受的盐或者权利要求5所述药物组合物在制备预防和/或治疗与肝脏有关疾病的药物中的应用。
  8. 根据权利要求7的应用，其特征在于，所述的与肝脏有关疾病包括肝损伤相关疾病、肝炎相关疾病。
  9. 根据权利要求7的应用，其特征在于，所述的与肝脏有关疾病选自甲肝、乙肝、丙肝、药物性肝病、酒精性肝病、非酒精性肝病、自身免疫性肝病、肝病进展的肝纤维化、肝硬化、肝衰竭。
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