WO2023104051A1 - 吲哚取代四氢异喹啉类化合物与他汀类化合物的联合应用 - Google Patents

吲哚取代四氢异喹啉类化合物与他汀类化合物的联合应用 Download PDF

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WO2023104051A1
WO2023104051A1 PCT/CN2022/137011 CN2022137011W WO2023104051A1 WO 2023104051 A1 WO2023104051 A1 WO 2023104051A1 CN 2022137011 W CN2022137011 W CN 2022137011W WO 2023104051 A1 WO2023104051 A1 WO 2023104051A1
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substituted
unsubstituted
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pharmaceutical composition
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PCT/CN2022/137011
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French (fr)
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柳红
王江
蒋华良
李永国
戈震
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广州嘉越医药科技有限公司
中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings

Definitions

  • the invention relates to the research field of combined drug treatment of hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, fatty liver deformation, atherosclerosis, obesity and other metabolic diseases.
  • Cardiovascular and cerebrovascular diseases generally refer to ischemic or hemorrhagic diseases in the heart, brain, and systemic tissues caused by hyperlipidemia, atherosclerosis, and hypertension. CVD ranks first in the global incidence of disease, mortality and disability, and seriously endangers people's life and health. Since 1990, the incidence and mortality of cardiovascular diseases have continued to increase globally. The number of CVD patients has increased from 271 million to 523 million in 2019, and the number of deaths has increased from 12.1 million to 17.9 million in 2020, accounting for the total number of global disease deaths. About 1/3 of the patients aged 30-70 died of CVD. With the development of social economy and the improvement of living standards, population growth, aging, changes in dietary structure and lifestyle changes have caused the prevalence of CVD to continue to rise. The number of CVD patients in my country is about 290 million. It is the leading cardiovascular disease in the world. countries with the highest incidence and mortality rates.
  • the Chinese market is the world's most potential lipid-lowering drug market, and it is also one of the fastest-growing markets in the world.
  • the market size of China's blood lipid-lowering drugs will be 36.30 billion yuan, a year-on-year increase of 9.1%.
  • the product composition of blood lipid-lowering drugs is mainly based on statins, supplemented by other types of drugs.
  • Statins lipid-lowering drugs can significantly reduce the morbidity and mortality of AS and coronary heart disease caused by hypercholesterolemia, and have the characteristics of good selectivity and high efficacy, so they occupy the main market share of lipid-lowering drugs.
  • Statins are the first-line drugs for the treatment of hyperlipidemia. Although statins are well tolerated, there are still some patients (such as those with familial hypercholesterolemia) who fail to significantly reduce LDL-C levels even with the highest dose of potent statin therapy. In addition, statins can easily cause liver damage, leading to elevated liver transaminases and side effects such as myalgia, myopathy and even rhabdomyolysis. High-dose statin therapy is associated with increased incidence of diabetes, pancreatitis, and renal failure, further limiting statin use.
  • the object of the present invention is to provide a combination of compositions and active ingredients for the treatment of hyperlipidemia, and its use in the treatment of hyperlipidemia, using the synergistic effect of two drugs for the treatment of hyperlipidemia with different mechanisms of action, To enhance the effect of treating hyperlipidemia.
  • the first aspect of the present invention provides a pharmaceutical composition or a pharmaceutical combination, the pharmaceutical composition or pharmaceutical combination comprising:
  • a therapeutically effective amount of the second active ingredient which is an HMG-CoA reductase inhibitor statin drug for treating hyperlipidemia.
  • the pharmaceutical composition or pharmaceutical combination consists of (A) a therapeutically effective amount of the first active ingredient, and (B) a therapeutically effective amount of the second active ingredient;
  • the first active ingredient is an indole-substituted tetrahydroisoquinoline compound or a pharmaceutically acceptable salt thereof;
  • the second active ingredient is an HMG-CoA reductase inhibitor statin drug for treating hyperlipidemia.
  • the (A) therapeutically effective amount of the first active ingredient and (B) therapeutically effective amount of the second active ingredient can be administered simultaneously or separately.
  • Said “separate administration” such as "a separate pharmaceutical composition comprising (A) a therapeutically effective amount of a first active ingredient” and "a separate pharmaceutical composition comprising (B) a therapeutically effective amount of a second active ingredient” are separated at different times Administration, for example: one of “a separate pharmaceutical composition comprising (A) a therapeutically effective amount of a first active ingredient” and "a separate pharmaceutical composition comprising (B) a therapeutically effective amount of a second active ingredient” is administered first, and the other One is administered subsequently.
  • the separate administrations may be close in time or distant in time.
  • the administration regimen (including administration route, administration dose, administration interval, etc.) of (A) a therapeutically effective amount of the first active ingredient and (B) a therapeutically effective amount of the second active ingredient may be the same or Different, it can be adjusted as needed by those skilled in the art to provide the optimal therapeutic effect.
  • the (A) therapeutically effective amount of the first active ingredient can be administered by any suitable route in the art, including oral administration.
  • the (A) therapeutically effective amount of the first active ingredient may be administered to the patient in a fixed dose, ie a fixed or predetermined amount of dose is given to the patient.
  • a fixed dose meaning a single dose
  • the (B) therapeutically effective amount of the second active ingredient can be administered by any suitable route in the art, including oral administration.
  • the (B) therapeutically effective amount of the second active ingredient may be administered to the patient in a fixed dose, ie a fixed or predetermined amount of dose is given to the patient.
  • a fixed dose meaning a single dose
  • the content of the first active ingredient ranges from 0.01% to 99.99%, based on the total weight of the active ingredients in the composition; preferably 0.1% to 99.9%, more preferably 1% to 99% more preferably 10% to 99%; more preferably 20% to 99%; more preferably 30% to 99%; more preferably 40% to 99%; more preferably 50% to 99%.
  • the content of the second active ingredient ranges from 0.01% to 99.99%, based on the total weight of the active ingredients in the composition; preferably 0.1% to 99.9%, more preferably 1% to 99%; more preferably 10% to 99%; more preferably 20% to 99%; more preferably 30% to 99%; more preferably 40% to 99%; more preferably 50% to 99%.
  • the first active ingredient is a compound represented by formula (I), or a pharmaceutically acceptable salt thereof:
  • R 1 and R 2 are each independently selected from the following group: hydrogen, deuterium, tritium, halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, Substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted 5-7 membered heterocycle, substituted or unsubstituted C1 -C6 alkylphenyl, substituted or unsubstituted C 1 -C 6 alkane 5-7 membered heteroaryl, substituted or unsubstituted C 3 -C 12 cycloalkyl, substituted or unsubstituted C 2 -C 10 acyl, substituted or unsubstituted C 2 -C 10 ester, amino, Substituted or unsubstituted C1-C6 amido, -SO 2 R 9 , -OSO 2 R 9 ,
  • R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of hydrogen, deuterium, tritium, halogen, hydroxyl, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 - C 6 alkoxy, substituted or unsubstituted C 3 -C 12 cycloalkyl, cyano, nitro or carboxyl;
  • n 0 or 1
  • R 1 and R 2 are each independently selected from the following group: hydrogen, deuterium, tritium, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted Substituted 5-7 membered heterocycle, substituted or unsubstituted C 1 -C 6 alkylphenyl, substituted or unsubstituted C 1 -C 6 alkyl 5-7 membered heteroaryl;
  • R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of hydrogen, deuterium, tritium, halogen, hydroxyl, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 - C 6 alkoxy, substituted or unsubstituted C 3 -C 12 cycloalkyl, cyano, nitro or carboxyl;
  • n 0 or 1.
  • the first active ingredient is a compound selected from the following group, or a pharmaceutically acceptable salt thereof:
  • the first active ingredient compound comes from: chemical synthesis or semichemical synthesis.
  • the second active ingredient is an HMG-CoA reductase inhibitor statin therapeutic drug.
  • the second active ingredient is selected from the group consisting of fluvastatin, lovastatin, atorvastatin, cerivastatin, Pravastatin, Simvastatin, Rosuvastatin, Pitavastatin, or a pharmaceutically acceptable salt thereof.
  • the second active ingredient is lovastatin, atorvastatin, pravastatin, rosuvastatin, and pitavastatin.
  • the second active ingredient is lovastatin, atorvastatin, rosuvastatin.
  • the second active ingredient is atorvastatin.
  • the mass ratio of the first active ingredient to the second active ingredient ranges from 1:10000 to 10000:1; preferably 1:1000 to 1000:1; more preferably, is 1:500 to 500:1; more preferably, 1:100 to 100:1; more preferably, 1:10 to 10:1.
  • the pharmaceutical composition or drug combination is used to prevent or treat PCSK9-related diseases, including the prevention or treatment of hyperlipidemia, hypercholesterolemia, hypertriglyceridemia Esteremia, fatty liver deformation, atherosclerosis, obesity and other metabolic diseases.
  • Fig. 1 is the effect of compound 6 combined with atorvastatin calcium (Ator-Ca) on the HepG2 cell PCSK9 protein expression level induced by atorvastatin calcium;
  • Figure 2 is the effect of compound 6 combined with atorvastatin calcium (Ator-Ca) on the LDLR protein expression level of HepG2 cells induced by atorvastatin calcium;
  • Figure 3 is the effect of compound 6 combined with atorvastatin calcium (Ator-Ca) on the LDL uptake activity of HepG2 cells induced by atorvastatin calcium;
  • Figure 4 is a schematic diagram of the influence (mmol/L) of compound 6 combined with atorvastatin calcium (Ator-Ca) on rat serum triglyceride (TG) levels;
  • Figure 5 is a schematic diagram of the influence (mmol/L) of compound 6 combined with atorvastatin calcium (Ator-Ca) on rat serum total cholesterol (TC) levels;
  • Figure 6 is a schematic diagram of the effect (mmol/L) of compound 6 combined with atorvastatin calcium (Ator-Ca) on rat serum high-density lipoprotein cholesterol (HDL-C) levels;
  • Figure 7 is a schematic diagram of the influence (mmol/L) of compound 6 combined with atorvastatin calcium (Ator-Ca) on rat serum low-density lipoprotein cholesterol (LDL-C) levels;
  • Figure 8 is a schematic diagram of the effect (g) of compound 6 combined with atorvastatin calcium (Ator-Ca) on the body weight of rats;
  • Figure 9 is a schematic diagram of the effect (U/L) of compound 6 combined with atorvastatin calcium (Ator-Ca) on rat serum ALT levels;
  • Figure 10 is a schematic diagram of the effect (U/L) of compound 6 combined with atorvastatin calcium (Ator-Ca) on rat serum AST levels;
  • Fig. 11 is a schematic diagram showing the effect of compound 6 combined with atorvastatin calcium (Ator-Ca) on the mRNA levels of PCSK9 and LDLR in rat liver.
  • indole-substituted tetrahydroisoquinoline compounds or pharmaceutically acceptable salts thereof can reduce total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG), and increased hepatic low-density lipoprotein receptor (LDLR) expression.
  • TC total cholesterol
  • LDL-C low-density lipoprotein cholesterol
  • TG triglycerides
  • LDLR hepatic low-density lipoprotein receptor
  • the combined drug regimen and pharmaceutical composition can be used for the prevention and treatment of metabolic diseases such as hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, fatty liver deformation, atherosclerosis, and obesity. Based on the above findings, the inventors have accomplished the present invention.
  • substituent selected from the following group: halogen, amino, hydroxyl, nitro, cyano, trifluoromethane C 1 -C 12 alky
  • C 1 -C 6 alkyl refers to a straight chain or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl group, tert-butyl group, or similar groups.
  • C 3 -C 12 cycloalkyl refers to a cycloalkyl group having 3 to 12 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or the like.
  • C 1 -C 6 alkoxy refers to a straight-chain or branched alkoxy group having 1-12 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy , isobutoxy, sec-butoxy, tert-butoxy, or similar groups.
  • halogen refers to F, Cl, Br and I.
  • C 2 -C 10 ester group refers to a substituent having a structure of "straight chain or branched chain alkyl/cycloalkyl/aryl/heteroaryl-carbonyl-oxy-" having 1-9 carbon atoms groups, such as ethyl, propyl, butyl, or similar groups.
  • C 1 -C 6 acyl refers to a substituent in the form of a "straight chain or branched chain alkyl/cycloalkyl/aryl/heteroaryl-carbonyl-" structure with 0-5 carbon atoms, such as acetyl group, propionyl, butyryl, or similar groups.
  • C 6 -C 10 aryl refers to an aryl group having 1-12 (preferably 6-10, ie C 6-10 ) carbon atoms, such as phenyl, naphthyl, etc., and the aryl group can be be substituted or not.
  • C 1 -C 12 heteroaryl refers to a heteroaryl group having 1-12 carbon atoms and one or more (preferably 1-3) heteroatoms selected from O, S and/or N, preferably C5 -C8 heteroaryl.
  • the heteroaryl group can be substituted or unsubstituted.
  • 5-7 membered heterocycle refers to a 5-7 membered cyclic saturated, partially unsaturated or aromatic group, wherein the heterocycle has at least one ring atom selected from the following group: O , S and/or N.
  • heteroaryl with 5-7 members refers to a cyclic aromatic group with 5-7 members, wherein the heterocycle has at least one ring atom selected from the group consisting of O, S and/or N.
  • C1-Cn means that the group has 1-n carbon atoms
  • C1-C12 means that the group has 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11 or 12 carbon atoms
  • C6-C10 means that the group has 6, 7, 8, 9 or 10 carbon atoms.
  • the term "pharmaceutically acceptable” ingredient refers to a substance that is suitable for human and/or animal without undue adverse side effects (such as toxicity, irritation and allergic reaction), ie, has a reasonable benefit/risk ratio.
  • the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or exhibits a detectable therapeutic or preventive effect.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agents and/or combination of therapeutic agents chosen for administration. Therefore, it is not useful to prespecify an exact effective amount. However, the effective amount can be determined by routine experimentation, within the judgment of the clinician, for a given situation.
  • each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.
  • the term "compound of the present invention” refers to a compound represented by formula I.
  • the term also includes the various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.
  • the term "pharmaceutically acceptable salt” refers to a salt of a compound of the present invention with an acid or a base which is suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • composition refers to a composition containing the specified active ingredients that can be prepared in the same dosage form.
  • indole-substituted tetrahydroisoquinoline compounds and their derivatives have good lipid-lowering activity in vivo and in vitro.
  • this type of compound can effectively inhibit the expression of PCSK9 protein, increase the level of LDLR protein, and promote the uptake of LDL in HepG2 cells, and has good lipid-lowering activity in vitro, which is better than the positive drug pravastatin.
  • this type of compound has good lipid-lowering activity in animals, and can effectively reduce the TC level, LDL-C level and TG level of the golden hamster hyperlipidemia animal model. It has significant lipid-lowering activity in spontaneous rhesus monkeys, and can reduce TC levels and LDL-C levels in spontaneous rhesus monkeys.
  • the present invention relates to the research field of drug combination therapy for hyperlipidemia, in particular to a drug comprising indole-substituted tetrahydroisoquinoline compounds or pharmaceutically acceptable salts thereof and statins for lowering blood lipids Composition, combination of active ingredients and its application in the treatment of hyperlipidemia.
  • the indole-substituted tetrahydroisoquinoline compounds and statin hypolipidemic drugs can be prepared for use as a pharmaceutical composition, or can be used in a separate dosage form or in the form of a drug combination, such as being administered simultaneously or successively for the treatment of object.
  • the dosage of the indole-substituted tetrahydroisoquinoline compounds and the statin hypolipidemic drugs can be determined according to actual needs, for example, the content of the first active ingredient ranges from 0.01% to 99.99% %, preferably 0.1% to 99.9%, more preferably 1% to 99%; more preferably 10% to 99%; more preferably 20% to 99%; more preferably 30% to 99%; more preferably 40% % to 99%; more preferably 50% to 99%, based on the total weight of the active ingredients of the composition.
  • the content range of the second active ingredient is 0.01% to 99.99%, preferably 0.1% to 99.9%, more preferably 1% to 99%; more preferably 10% to 99%; more preferably 20% to 99%; more preferably Preferably it is from 30% to 99%; more preferably from 40% to 99%; more preferably from 50% to 99%, based on the total weight of the active ingredients of the composition.
  • the mass ratio of the first active ingredient to the second active ingredient ranges from 1:10000 to 10000:1; preferably 1:1000 to 1000:1; more preferably, is 1:500 to 500:1; more preferably, 1:100 to 100:1; more preferably, 1:10 to 10:1.
  • the indole-substituted tetrahydroisoquinoline compound is preferably a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
  • R 1 and R 2 are each independently selected from the following group: hydrogen, deuterium, tritium, halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, Substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted 5-7 membered heterocycle, substituted or unsubstituted C1-C6 alkylphenyl, substituted or unsubstituted C 1 -C 6 alkane 5-7 membered heteroaryl, substituted or unsubstituted C 3 -C 12 cycloalkyl, substituted or unsubstituted C 2 -C 10 acyl, substituted or unsubstituted C 2 -C 10 ester, amino, Substituted or unsubstituted C1-C6 amido, -SO 2 R 9 , -OSO 2 R 9 ,
  • R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of hydrogen, deuterium, tritium, halogen, hydroxyl, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 - C 6 alkoxy, substituted or unsubstituted C 3 -C 12 cycloalkyl, cyano, nitro or carboxyl;
  • n 0 or 1
  • statin hypolipidemic drugs are preferably selected from the group consisting of fluvastatin, lovastatin, atorvastatin, cerivastatin, pravastatin, simvastatin, rosuvastatin, pitavastatin or a pharmaceutically acceptable salt thereof; optimally, the second active ingredient is atorvastatin.
  • the pharmaceutical composition or pharmaceutical combination of the present invention can be used to prevent or treat a series of cardiovascular diseases or metabolic diseases, such as (but not limited to) diseases selected from the group consisting of hyperlipidemia, hypercholesterolemia, hyperlipidemia Triglyceridemia, fatty liver deformation, atherosclerosis, obesity and other metabolic diseases.
  • cardiovascular diseases or metabolic diseases such as (but not limited to) diseases selected from the group consisting of hyperlipidemia, hypercholesterolemia, hyperlipidemia Triglyceridemia, fatty liver deformation, atherosclerosis, obesity and other metabolic diseases.
  • the main advantages of the present invention include:
  • the present invention relates to a pharmaceutical composition or pharmaceutical combination, the composition or pharmaceutical combination contains indole-substituted tetrahydroisoquinoline compounds or pharmaceutically acceptable salts thereof, and the composition or pharmaceutical combination also contains a drug , this drug is an HMG-CoA reductase inhibitor statin.
  • a pharmaceutical composition or pharmaceutical combination contains indole-substituted tetrahydroisoquinoline compounds or pharmaceutically acceptable salts thereof, and the composition or pharmaceutical combination also contains a drug , this drug is an HMG-CoA reductase inhibitor statin.
  • a pharmaceutical composition or pharmaceutical combination comprising: (A) a therapeutically effective amount of the first active ingredient, the first active ingredient being an indole-substituted tetrahydroisoquinoline compound or a pharmaceutically acceptable Accepted salt; (B) a therapeutically effective amount of the second active ingredient, the second active ingredient is an HMG-CoA reductase inhibitor statin lipid-lowering drug, and the quality of the first active ingredient and the second active ingredient The ratio is 1:10000 to 10000:1.
  • the invention also discloses an active component combination, a pharmaceutical composition and its application in treating hyperlipidemia.
  • the pharmaceutical composition or pharmaceutical combination of the present invention has an excellent therapeutic effect on hyperlipidemia.
  • a pharmaceutical composition or pharmaceutical combination which contains an indole-substituted tetrahydroisoquinoline compound or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition or pharmaceutical combination also contains Contains a medicine, this medicine is an HMG-CoA reductase inhibitor statin.
  • statin lipid-lowering drugs are the first-line lipid-lowering drugs for hyperlipidemia, but during clinical use, statin intolerance exists received phenomenon.
  • indole-substituted tetrahydroisoquinoline compounds or pharmaceutically acceptable salts thereof are used in combination with statins, which can effectively down-regulate the expression level of PCSK9 protein in patients taking statins, and can further improve LDLR in patients protein expression, further improving its therapeutic activity for hyperlipidemia.
  • the pharmaceutical composition or drug combination and combined drug regimen can be used for the prevention and treatment of metabolic diseases such as hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, fatty liver deformation, atherosclerosis, and obesity. .
  • the indole-substituted tetrahydroisoquinoline compounds or their pharmaceutically acceptable salts involved in the present invention are not the substrates of the main transporters OATP1B1 and OATP1B3 of atorvastatin.
  • the indoles involved in the present invention The IC 50 values of the azole-substituted tetrahydroisoquinoline compounds or their pharmaceutically acceptable salts for inhibiting the uptake and transport of atorvastatin by OATP1B1 and OATP1B3 are all greater than 50 ⁇ M. It is suggested that indole-substituted tetrahydroisoquinoline compounds or their pharmaceutically acceptable salts affect the metabolites of statins.
  • the reagents and raw materials used in the present invention are all commercially available.
  • Example 1 In vitro study of compound 6 combined with atorvastatin
  • HepG2 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), incubated at 37°C in 5% CO 2 ; after 24 hours, the medium was changed to DMEM high-glucose medium containing 2% delipidated serum (LPDS) The culture medium was continued for 24 hours; the old culture medium was discarded, and the medium was changed to a new DMEM high-glucose medium containing 2% LPDS, and the compound to be tested was added for co-cultivation for 24 hours.
  • FBS fetal bovine serum
  • LPDS delipidated serum
  • Protein sample loading and electrophoresis According to the difference of the electrophoresis comb, take 15-30 ⁇ g protein sample for loading, 80-120V constant voltage electrophoresis, and stop the electrophoresis after the band of the target protein in the separation gel is clearly distinguished;
  • Membrane transfer (semi-dry transfer method): Activate the filter paper and PVDF membrane with methanol and infiltrate the membrane transfer buffer respectively, cut out the part containing the target protein in the separation gel, and place the filter paper, PVDF membrane and gel on the membrane transfer apparatus in sequence. Gel, and finally cover the gel with filter paper, drive out the air bubbles and install the membrane transfer instrument, adjust the transfer current and transfer time according to the size of the target protein and the concentration of the gel;
  • Secondary antibody incubation After the primary antibody incubation, take out the target protein band and wash it in TBST solution for 3 times for 5, 10, and 15 min respectively, and then transfer the protein band to the corresponding secondary antibody solution ( Prepared according to the instructions), after incubating on a shaker at room temperature for 2 hours in the dark, wash with TBST solution 3 times for 5, 10, and 15 minutes respectively;
  • HepG2 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS), 5% CO 2 , 37°C.
  • FBS fetal bovine serum
  • HepG2 cells were seeded in a 24-well plate and cultured in DMEM containing 10% FBS for 24 hours;
  • Compound 6 is used in combination with atorvastatin calcium, and the detection results of the influence of atorvastatin calcium on the expression of PCSK9 protein in hepatocytes are as follows:
  • Compound 6 combined with atorvastatin calcium can down-regulate the expression level of PCSK9 protein in HepG2 cells induced by atorvastatin calcium.
  • the results are shown in Figure 1.
  • Compound 6 (5 ⁇ M) and atorvastatin calcium were co-incubated with HepG2 cells for 24h, and then the changes in the expression of PCSK9 protein in cells were detected.
  • Compound 6 combined with atorvastatin calcium can increase the LDL uptake activity of HepG2 cells induced by atorvastatin calcium.
  • the results are shown in Figure 3, the combined use of compound 6 can further enhance the uptake of LDL and enhance the lipid-lowering activity in vitro.
  • the main transporters of atorvastatin are OATP1B1 and OATP1B3.
  • a study on the transporter of compound 6 was carried out.
  • Test method LC-MS/MS method was used to determine the concentration of compound 6 and positive compound inhibitor rifampicin.
  • the uptake rate of Compound 6 at 1.00 and 20.0 ⁇ M on HEK293-OATP1B1 cell lines was no more than twice that of Mock cells, and the uptake rate of Compound 6 at 5.00 ⁇ M on HEK293-OATP1B1 cell lines was more than twice that of Mock cells, but After adding the inhibitor rifampicin, the uptake rate did not decrease, indicating that compound 6 is not a substrate of the OATP1B1 transporter; the uptake rates of compound 6 at 1.00, 5.00 and 20.0 ⁇ M on HEK293-OATP1B3 cell lines were not higher than that of Mock cells 2 times, indicating that compound 6 is not a substrate of the OATP1B3 transporter, the results are shown in Table 1 and Table 2.
  • compound 6 is not a substrate of OATP1B1 transporter and OATP1B3 transporter, and compound 6 does not affect the transport of atorvastatin, that is, it does not affect the metabolic behavior of atorvastatin.
  • Example 3 Compound 6 inhibits the activity of OATP1B1 and OATP1B3 uptake and transport atorvastatin
  • the main transporters of atorvastatin are OATP1B1 and OATP1B3.
  • a study on the transporter of compound 6 was carried out.
  • Test method LC-MS/MS method was used to determine the concentration of compound 6 and positive compounds.
  • Compound 6 was co-incubated with the corresponding transporter probe substrates on HEK293-OATP1B1 and HEK293-OATP1B3 cell lines, and whether compound 6 was an inhibitor of each transporter was evaluated by measuring the changes in the substrate uptake rate of each transporter.
  • HEK293-Mock cell group, probe substrate control group and probe substrate + compound 6 treatment group were set up.
  • Example 4 In vivo pharmacodynamics study of compound 6 combined with atorvastatin in rats
  • the blank control group was fed with common feed
  • the high-fat control group was fed with high-fat feed and fed with equal volume of 0.5% CMC-Na;
  • Ator low-dose group fed with high-fat feed, atorvastatin calcium: 3mg/kg/day;
  • Ator high-dose group fed with high-fat feed, atorvastatin calcium: 10mg/kg/day;
  • the compound 6 administration group was fed with high-fat feed, compound 6: 10 mg/kg/day;
  • Combined administration group (1) fed with high-fat feed, atorvastatin calcium: 3mg/kg/day, compound 6: 10mg/kg/day;
  • Combined administration group (2) was fed with high-fat feed, atorvastatin calcium: 10 mg/kg/day, compound 6: 10 mg/kg/day.
  • Each drug group was intragastrically administered the corresponding dose of drugs once a day.
  • Wistar rats Male, weighing about 150g were adaptively fed for 1 week, and the basic blood lipid level was detected. Three rats were randomly selected as the blank control group, fed with common feed, and the remaining rats were fed with high-fat feed. After 10 days, detection Blood lipid level, according to the level of serum TG, the rats were evenly divided into 6 groups, 8 in each group, the animals in the treatment group continued to be given high-fat feed, and the administration was continued for 28 days, the high-fat model group continued to be given high-fat feed, and the blank control group continued to be given high-fat feed. The group was given normal chow. At the end of the experiment, the blood lipid level, ALT and AST were detected, the animals were euthanized, and the liver samples were weighed and serum samples were reserved.
  • the changes of physiological indexes such as blood fat and body weight were observed in animals before and after administration.
  • TG triglyceride
  • TC total cholesterol
  • HDL-C high-density lipoprotein cholesterol
  • LDL-C low-density lipoprotein cholesterol
  • the TG levels Further decrease, among them, the decrease rate of Ator high-dose group, compound 6 administration group and combined administration group (2) were 67.29%, 45.79% and 114.02%, respectively, and the decline rates of the three administration groups for TC were 2.99%, 2.99% and 34.33%, the reductions against LDL-C were 0, 4.55% and 50%, respectively.
  • the serum triglyceride and cholesterol of the rats in the high-fat control group showed different degrees of increase, in which the level of triglyceride increased by 2.09 times, with a very significant difference, successfully established Rat hyperlipidemia model; after 28 days of administration of atorvastatin calcium and compound 6, the levels of triglycerides in the serum of rats decreased to varying degrees, and the levels of triglycerides further decreased after the combination of the two; atorvastatin calcium Or compound 6 administered alone failed to change the serum cholesterol level of rats, but the cholesterol level decreased significantly after the two were used in combination, indicating that the combination of atorvastatin calcium and compound 6 has a significant blood lipid-lowering effect on hyperlipidemia rats, and the effect Superior to atorvastatin calcium or compound 6 alone.

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Abstract

一种吲哚取代四氢异喹啉类化合物与他汀类化合物的联合应用，具体地，一种安全的、非相互作用的药物组合物或药物组合，该组合物含有吲哚取代四氢异喹啉类化合物或其药用上可接受的盐，该组合物还含有一种药物，这种药物是HMG-CoA还原酶抑制剂他汀类降脂药物。所述组合的联合应用可用于高血脂症、高胆固醇血症、高甘油三酯血症、脂肪肝变形、动脉粥样硬化、肥胖等代谢性疾病的治疗。

Description

吲哚取代四氢异喹啉类化合物与他汀类化合物的联合应用 技术领域
本发明涉及联合用药治疗高血脂症、高胆固醇血症、高甘油三酯血症、脂肪肝变形、动脉粥样硬化、肥胖等代谢性疾病的研究领域，具体涉及一种包括吲哚取代四氢异喹啉类化合物或其药用上可接受的盐和他汀类降血脂药物的组合物、活性成分组合及其在高血脂症治疗中的应用。
背景技术
心脑血管疾病(CVD)泛指由于高脂血症、动脉粥样硬化、高血压等所导致的心脏、大脑及全身组织发生的缺血性或出血性疾病。CVD位列全球疾病发生率、死亡率和致残率首位，严重危害到人们的生命健康。自1990年以来，全球心血管疾病发生率和死亡率持续增长，CVD患病人数从2.71亿增加到2019年的5.23亿，死亡人数从1210万增加到2020年的1790万，占全球疾病死亡人数的1/3，30-70岁的患病人群中就有约610万患者死于CVD。随着社会经济的发展和生活水平的提高，人口增长、老龄化、膳食结构改变和生活方式转变等使得CVD患病率处于持续上升阶段，我国CVD患者人数约有2.9亿，是全球心血管疾病发生率和死亡率最高的国家。
目前，中国市场是全球最具潜力的降血脂药市场，也是全球发展最快的市场之一。2020年中国降血脂药市场规模为363.0亿元，同比增长9.1％。降血脂药产品构成中主要以他汀类药物为主，其他药物种类为辅。他汀类降脂药物可明显降低由高胆固醇血症引起的AS和冠心病的发病率和死亡率，具有选择性好和疗效高的特点，因此占据了主要的降脂药物市场份额。
他汀类药物是高血脂症治疗的一线药物。尽管他汀类药物具有良好的耐受性，但仍存在一些患者(如家族性高胆固醇血症患者)即使接受最大剂量的强效他汀类药物治疗，也无法显著降低LDL-C水平。此外，他汀类药物易引起肝功能损害，导致肝脏转氨酶的升高以及引起肌痛、肌病甚至横纹肌溶解症等副作用。高剂量的他汀类药物治疗与糖尿病、胰腺炎及肾衰竭的发病率增加有关，进一步限制了他汀类药物的使用。
因此，本领域迫切需要提供一种安全有效，副作用少的心脑血管疾病药物组合物或药物组合。
发明内容
本发明的目的在于提供一种用于高血脂症治疗的组合物以及活性成分的组合，及其在高血脂症治疗中的用途，利用两种不同作用机制的高血脂症治疗药物的协同作用，以增强治疗高血脂症的效果。
本发明的第一方面，提供了一种药物组合物或药物组合，所述药物组合物或药物组合包括：
(A)治疗有效量的第一活性成分，所述第一活性成分为吲哚取代四氢异喹啉类化合物，或其药学上可接受的盐；
(B)治疗有效量的第二活性成分，所述第二活性成分为HMG-CoA还原酶抑制剂他汀类高血脂症治疗药物。
在另一优选例中，所述的药物组合物或药物组合，由(A)治疗有效量的第一活性成分， 和(B)治疗有效量的第二活性成分组成；
其中，所述第一活性成分为吲哚取代四氢异喹啉类化合物或其药学上可接受的盐；所述第二活性成分为HMG-CoA还原酶抑制剂他汀类高血脂症治疗药物。
所述的药物组合物或药物组合中，所述(A)治疗有效量的第一活性成分和(B)治疗有效量的第二活性成分可以同时施用或分开施用。
所述“同时施用”例如所述(A)治疗有效量的第一活性成分和(B)治疗有效量的第二活性成分包含在单独药物组合物中同时施用；或者，“包含(A)治疗有效量的第一活性成分的单独药物组合物”与“包含(B)治疗有效量的第二活性成分的单独药物组合物”同时施用。
所述“分开施用”例如“包含(A)治疗有效量的第一活性成分的单独药物组合物”与“包含(B)治疗有效量的第二活性成分的单独药物组合物”在不同时间分开施用，例如：“包含(A)治疗有效量的第一活性成分的单独药物组合物”和“包含(B)治疗有效量的第二活性成分的单独药物组合物”其中之一首先施用，另一个随后施用。所述的分开施用可在时间上距离接近或时间上距离较远。
无论同时施用还是分开施用，所述(A)治疗有效量的第一活性成分和(B)治疗有效量的第二活性成分的施用方案(包括施用途径、施用剂量、施用间隔等)可以相同或不同，其可以由本领域技术人员根据需要进行调整，以提供最优的治疗效果。
在另一优选例中，所述(A)治疗有效量的第一活性成分可以采用本领域中任何合适的途径施用，包括口服等。
所述(A)治疗有效量的第一活性成分可以固定剂量施用于患者，即给予患者固定或预定量的剂量。固定剂量(指单次剂量)的非限制性实例范围可以为10-600mg，例如10mg、20mg、30mg、40mg、50mg、60mg、70mg、80mg、90mg、100mg、110mg、120mg、130mg、140mg、150mg、170mg、180mg、190mg、200mg、300mg、400mg、500mg或600mg。
在另一优选例中，所述(B)治疗有效量的第二活性成分可以采用本领域中任何合适的途径施用，包括口服等。
所述(B)治疗有效量的第二活性成分可以固定剂量施用于患者，即给予患者固定或预定量的剂量。固定剂量(指单次剂量)的非限制性实例范围可以为10-80mg，例如10mg、20mg、30mg、40mg、50mg、60mg、70mg或80mg。
在另一优选例中，所述的第一活性成分的含量范围为0.01％至99.99％，以组合物活性成分的总重量计；优选为0.1％至99.9％，更优选为1％至99％；更优选为10％至99％；更优选为20％至99％；更优选为30％至99％；更优选为40％至99％；更优选为50％至99％。
在另一优选例中，第二活性成分的含量范围为0.01％至99.99％，以组合物活性成分的总重量计；优选为0.1％至99.9％，更优选为1％至99％；更优选为10％至99％；更优选为20％至99％；更优选为30％至99％；更优选为40％至99％；更优选为50％至99％。
在另一优选例中，所述第一活性成分是式(I)所示的化合物，或其药学上可接受的盐：
Figure PCTCN2022137011-appb-000001
在式(I)中：
其中，R 1和R 2各自独立地选自下组：氢、氘、氚、卤素、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 6～C 10芳基、取代或未取代的5-7元的杂环、取代或未取代的C1～C6烷基苯基、取代或未取代的C 1～C 6烷基5-7元杂芳基、取代或未取代的C 3～C 12环烷基、取代或未取代的C 2～C 10酰基、取代或未取代的C 2～C 10酯基、氨基、取代或未取代的C1-C6酰胺基、-SO 2R 9、-OSO 2R 9、-OCOR 9；且所述的R 1和R 2不同时为氢；
或所述的R 1和R 2和相邻的-(CH 2) n-O以及C＝C共同构成取代或未取代的5-7元的杂环，其中，所述的杂环为全部饱和的杂环、部分不饱和的杂环或芳杂环；
R 3、R 4、R 5和R 6各自独立地选自下组：氢、氘、氚、卤素、羟基、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 3～C 12环烷基、氰基、硝基或羧基；
n为0或1；
所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、C 1-C 4烷基、三氟甲基、C 1～C 4烷氧基、C 3-C 6环烷基、C 1～C 4直链或支链烷基取代的胺基、羟基、氰基、硝基、氧原子(＝O)、羟基-C 1～C 6烷基、羧基或巯基。
在另一优选例中，所述的式(I)中：
其中，R 1和R 2各自独立地选自下组：氢、氘、氚、取代或未取代的C 1～C 6烷基、取代或未取代的C 6～C 10芳基、取代或未取代的5-7元的杂环、取代或未取代的C 1～C 6烷基苯基、取代或未取代的C 1～C 6烷基5-7元杂芳基；
或所述的R 1和R 2和相邻的-(CH 2) n-O以及C＝C共同构成取代或未取代的5-7元的杂环，其中，所述的杂环为全部饱和的杂环、部分不饱和的杂环或芳杂环；
R 3、R 4、R 5和R 6各自独立地选自下组：氢、氘、氚、卤素、羟基、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 3～C 12环烷基、氰基、硝基或羧基；
n为0或1。
在另一优选例中，所述第一活性成分为选自下组的化合物，或其药学上可接受的盐：
Figure PCTCN2022137011-appb-000002
Figure PCTCN2022137011-appb-000003
在另一优选例中，所述第一活性成分化合物来自：化学合成或半化学合成。
在另一优选例中，所述第二活性成分为HMG-CoA还原酶抑制剂他汀类治疗药物。
在另一优选例中，所述的第二活性成分选自下组：氟伐他汀(Fluvastatin)、洛伐他汀(Lovastatin)、阿托伐他汀(Atorvastatin)、西立伐他汀(Cerivastatin)、普伐他汀(Pravastatin)、辛伐他汀(Simvastatin)、瑞舒伐他汀(Rosuvastatin)、匹伐他汀(Pitavastatin)，或其药用上可接受的盐。
在另一优选例中，所述的第二活性成分为洛伐他汀、阿托伐他汀、普伐他汀、瑞舒伐他汀、匹伐他汀。
更佳地，所述的第二活性成分为洛伐他汀、阿托伐他汀、瑞舒伐他汀。
更佳地，所述的第二活性成分为阿托伐他汀。
在另一优选例中，所述第一活性成分与所述第二活性成分的质量比范围为1：10000至10000：1；较佳地为1:1000至1000:1；更佳地，为1:500至500:1；更佳地，为1:100至100:1；更佳地，为1:10至10:1。
在另一优选例中，所述的药物组合物或药物组合用于预防或治疗与PCSK9相关的疾病，所述PCSK9相关的疾病包括预防或治疗高脂血症、高胆固醇血症、高甘油三酯血症、脂肪肝变形、动脉粥样硬化、肥胖等代谢性疾病。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为化合物6与阿托伐他汀钙(Ator-Ca)联用对于由阿托伐他汀钙诱导增加的HepG2细胞PCSK9蛋白表达水平的影响；
图2为化合物6与阿托伐他汀钙(Ator-Ca)联用对于由阿托伐他汀钙诱导增加的HepG2细胞LDLR蛋白表达水平的影响；
图3为化合物6与阿托伐他汀钙(Ator-Ca)联用对于由阿托伐他汀钙诱导增加的HepG2细胞对LDL的摄取活性的影响；
图4为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠血清甘油三酯(TG)水平的影响(mmol/L)示意图；
图5为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠血清总胆固醇(TC)水平的影响(mmol/L)示意图；
图6为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠血清高密度脂蛋白胆固醇(HDL-C)水平的影响(mmol/L)示意图；
图7为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠血清低密度脂蛋白胆固醇(LDL-C)水平的影响(mmol/L)示意图；
图8为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠体重的影响(g)示意图；
图9为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠血清ALT水平的影响(U/L)示意图；
图10为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠血清AST水平的影响(U/L)示意图；
图11为化合物6与阿托伐他汀钙(Ator-Ca)联用对大鼠肝脏中PCSK9和LDLR的mRNA水平的影响示意图。
具体实施方式
本发明经过长期而深入的研究，意外地发现，吲哚取代四氢异喹啉类化合物或其药用上可接受的盐能降低总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)和甘油三酯(TG)，并且增加肝低密度脂蛋白受体(LDLR)表达。与他汀类药物联合使用，能够有效下调他汀类药物服用患者体内的PCSK9蛋白表达水平，进一步提升患者体内 LDLR的蛋白表达，进一步提高其高血脂症的治疗活性。降脂作用机制研究结果表明，将PCSK9调节剂与他汀类药物联合用药，可有效改善他汀不耐受患者的血脂水平。因此，该联合用药方案和药物组合物可用于高脂血症、高胆固醇血症、高甘油三酯血症、脂肪肝变形、动脉粥样硬化、肥胖等代谢性疾病的预防和治疗。基于上述发现，发明人完成了本发明。
术语
在本文中，除特别说明之处，术语“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、氨基、羟基、硝基、氰基、三氟甲基、C 1-C 12烷基或环烷基、C 1-C 12烷氧基、氧原子(即＝O)、未取代或被C 1-4烷胺基取代的C 1-C 12烷胺基、C 2-C 6酯基、C 2-C 6酰基、C 2-C 6酰胺基、硫代C 1-C 12烷基、羧基、C 5-C 12芳基或杂芳基、C 5-C 12杂环基(含有1-5个，优选1-3个选自N、O或S的杂原子)。
术语“C 1-C 6烷基”指具有1～6个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
术语“C 3-C 12环烷基”指具有3～12个碳原子的环烷基，例如环丙基、环丁基、环戊基、环庚基、或类似基团。
术语“C 1-C 6烷氧基”指具有1-12个碳原子的直链或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、或类似基团。
术语“卤素”指F、Cl、Br和I。
术语“C 2-C 10酯基”指形如“具有1-9个碳原子的直链或支链烷基/环烷基/芳基/杂芳基-羰基-氧基-”结构的取代基，如乙酯基、丙酯基、丁酯基，或类似基团。
术语“C 1-C 6酰基”指形如“具有0-5个碳原子的直链或支链烷基/环烷基/芳基/杂芳基-羰基-”结构的取代基，如乙酰基、丙酰基、丁酰基，或类似基团。
术语“C 6-C 10芳基”指具有1-12个(优选为6-10个，即C 6-10)碳原子的芳基，例如苯基、萘基等，所述的芳基可以是取代或未取代的。
术语“C 1-C 12杂芳基”指具有1-12个碳原子和一个或多个(优选1-3个)选自O、S和/或N的杂原子的杂芳基，优选C5-C8杂芳基。所述的杂芳基可以是取代或未取代的。
术语“5-7元的杂环”指具有5-7元的环状饱和、部分不饱和或芳香性基团，其中，所述的杂环具有至少1个选自下组的环原子：O、S和/或N。
术语“5-7元的杂芳基”指具有5-7元的环状芳香性基团，其中，所述的杂环具有至少1个选自下组的环原子：O、S和/或N。
特别地，形如“C1-Cn”的表述指基团具有1-n个碳原子，例如，“C1-C12”的表述指基团具有1个，2个，3个，4个，5个，6个，7个，8个，9个，10个，11个或12个碳原子；“C6～C10”指基团具有6个，7个，8个，9个或10个碳原子。
本发明中，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比的物质。
本发明中，术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。
除非特别说明，本发明中，所有出现的化合物均意在包括所有可能的光学异构体，如单一手性的化合物，或各种不同手性化合物的混合物(即外消旋体)。本发明的所有化合物之 中，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。
如本文所用，术语“本发明化合物”指式I所示的化合物。该术语还包括式I化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。
如本文所用，术语“药物组合物”是指含有指定的活性成分、可被制备成同一剂型的组合物。
吲哚取代四氢异喹啉类化合物的药物联用
在申请人的前期研究结果中表明，吲哚取代四氢异喹啉类化合物及衍生物具有良好的体内外降脂活性。一方面，该类化合物可以有效地抑制PCSK9蛋白的表达，提高LDLR蛋白水平，促进对HepG2细胞中LDL的摄取，具有良好的体外降脂活性，降脂活性优于阳性药物普伐他汀。另一方面，该类化合物在动物体内具有良好的降脂活性，能够有效降低金黄地鼠高脂动物模型的TC水平、LDL-C水平和TG水平。在自发恒河猴体内具有显著的降脂活性，可以降低自发恒河猴体内的TC水平和LDL-C水平。
基于前期发现的研究结果，本发明涉及联合用药治疗高血脂症的研究领域，具体涉及一种包括吲哚取代四氢异喹啉类化合物或其药用上可接受的盐和他汀类降血脂药物的组合物、活性成分组合及其在高血脂症治疗中的应用。所述的吲哚取代四氢异喹啉类化合物和他汀类降血脂药物可以被制备为药物组合物使用，也可以以单独的剂型，以药物组合的形式使用，例如同时或先后被施用于治疗对象。
所述的药物组合物或药物组合中，吲哚取代四氢异喹啉类化合物和他汀类降血脂药物的用量可以根据实际需要来确定，例如，第一活性成分的含量范围为0.01％至99.99％，优选为0.1％至99.9％，更优选为1％至99％；更优选为10％至99％；更优选为20％至99％；更优选为30％至99％；更优选为40％至99％；更优选为50％至99％，以组合物活性成分的总重量计。第二活性成分的含量范围为0.01％至99.99％，优选为0.1％至99.9％，更优选为1％至99％；更优选为10％至99％；更优选为20％至99％；更优选为30％至99％；更优选为40％至99％；更优选为50％至99％，以组合物活性成分的总重量计。
在另一优选例中，所述第一活性成分与所述第二活性成分的质量比范围为1：10000至10000：1；较佳地为1:1000至1000:1；更佳地，为1:500至500:1；更佳地，为1:100至100:1；更佳地，为1:10至10:1。
所述的吲哚取代四氢异喹啉类化合物优选地是式(I)所示的化合物或其药学上可接受的盐：
Figure PCTCN2022137011-appb-000004
在式(I)中：
其中，R 1和R 2各自独立地选自下组：氢、氘、氚、卤素、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 6～C 10芳基、取代或未取代的5-7元的杂环、取代或未取代的C1～C6烷基苯基、取代或未取代的C 1～C 6烷基5-7元杂芳基、取代或未取代的C 3～C 12环烷基、取代或未取代的C 2～C 10酰基、取代或未取代的C 2～C 10酯基、氨基、取代或未取代的C1-C6酰胺基、-SO 2R 9、-OSO 2R 9、-OCOR 9；且所述的R 1和R 2不同时为氢；
或所述的R 1和R 2和相邻的-(CH 2) n-O以及C＝C共同构成取代或未取代的5-7元的杂环，其中，所述的杂环为全部饱和的杂环、部分不饱和的杂环或芳杂环；
R 3、R 4、R 5和R 6各自独立地选自下组：氢、氘、氚、卤素、羟基、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 3～C 12环烷基、氰基、硝基或羧基；
n为0或1；
所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、C 1-C 4烷基、三氟甲基、C 1～C 4烷氧基、C 3-C 6环烷基、C 1～C 4直链或支链烷基取代的胺基、羟基、氰基、硝基、氧原子(＝O)、羟基-C 1～C 6烷基、羧基或巯基。
所述的他汀类降血脂药物较佳地选自下组：氟伐他汀、洛伐他汀、阿托伐他汀、西立伐他汀、普伐他汀、辛伐他汀、瑞舒伐他汀、匹伐他汀或其药用上可接受的盐；最佳地，所述的第二活性成分为阿托伐他汀。
本发明的药物组合物或药物组合可以用于预防或治疗一系列心血管疾病或代谢性疾病，例如(但并不限于)选自下组的疾病：高脂血症、高胆固醇血症、高甘油三酯血症、脂肪肝变形、动脉粥样硬化、肥胖等代谢性疾病。
与现有技术相比，本发明的主要优点包括：
本发明涉及一种药物组合物或药物组合，该组合物或药物组合含有吲哚取代四氢异喹啉类化合物或其药用上可接受的盐，该组合物或药物组合还含有一种药物，这种药物是HMG-CoA还原酶抑制剂他汀类化合物。特定药物组合物或药物组合的联合应用可用于高血脂症的治疗。具体地公开了一种药物组合物或药物组合，包括：(A)治疗有效量的第一活性成分，所述第一活性成分为吲哚取代四氢异喹啉类化合物或其药用上可接受的盐；(B)治疗有效量的第二活性成分，所述第二活性成分为HMG-CoA还原酶抑制剂他汀类降脂药物，并且所述第一活性成分与第二活性成分的质量比为1:10000至10000:1。本发明还公开了一种活性成分组合、药物组合物及其在高血脂症治疗中的用途。本发明的药物组合物或药物组合具有优异的高血脂症治疗作用。
(1)提供了一种药物组合物或药物组合，该药物组合物或药物组合含有吲哚取代四氢异喹啉类化合物或其药用上可接受的盐，该药物组合物或药物组合还含有一种药物，这种药物是HMG-CoA还原酶抑制剂他汀类化合物。
(2)提供的药物组合物或药物组合为他汀不耐受患者提供了新的治疗方案，他汀类降脂药物是高血脂症的一线降脂药物，但是在临床使用过程中，存在他汀不耐受的现象。本发明将吲哚取代四氢异喹啉类化合物或其药用上可接受的盐与他汀类药物联合使用，能够有效下调他汀类药物服用患者体内的PCSK9蛋白表达水平，能够进一步提升患者体内LDLR的蛋白表达，进一步提高其高血脂症的治疗活性。降脂作用机制研究结果表明，将吲哚取代四氢异喹啉类化合物或其药用上可接受的盐与他汀类药物联合用药，可有效改善他汀不耐受患者的血脂水平。
因此该药物组合物或药物组合和联合用药方案可以用于高脂血症、高胆固醇血 症、高甘油三酯血症、脂肪肝变形、动脉粥样硬化、肥胖等代谢性疾病的预防和治疗。
另外，本发明涉及的吲哚取代四氢异喹啉类化合物或其药用上可接受的盐不是阿托伐他汀的主要转运体OATP1B1和OATP1B3的底物，另一方面，本发明涉及的吲哚取代四氢异喹啉类化合物或其药用上可接受的盐抑制OATP1B1和OATP1B3摄取转运阿托伐他汀的IC 50值均大于50μM。提示吲哚取代四氢异喹啉类化合物或其药用上可接受的盐对他汀类药物的代谢物影响。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：化合物6与阿托伐他汀联合用药体外研究
1、实验方法与步骤：
(1)化合物6对于HepG2肝细胞PCSK9和LDLR蛋白表达影响的检测
细胞培养：
HepG2细胞(ATCC)培养于含10％胎牛血清(FBS)DMEM高糖培养基中，5％CO 2，37℃孵育；24h后，换液至含2％脱脂血清(LPDS)的DMEM高糖培养基继续培养24h；吸弃旧培养液，换液至新的含2％LPDS DMEM高糖培养基，同时加入待测化合物共培养24h。
蛋白免疫印迹方法如下：
a)SDS-PAGE凝胶配制：取浓缩胶浓度为4％，分离胶浓度依据目的蛋白大小选取8％；
b)蛋白样品上样与电泳：依据电泳梳的差异，取15～30μg蛋白样品进行上样，80～120V恒压电泳，待分离胶中目的蛋白条带显著区分开后，结束电泳；
c)转膜(半干转法)：将滤纸与PVDF膜分别用甲醇活化及转膜缓冲液浸润，切出分离胶中含目的蛋白部分，依次在转膜仪上放置滤纸、PVDF膜及凝胶，最后于凝胶上覆以滤纸，赶去其中气泡并安装好转膜仪，依据目的蛋白大小及胶的浓度调整转膜电流和转膜时间；
d)膜封闭：转膜结束后，取出PVDF膜置于TBST溶液中稍洗涤后，转至含5％脱脂奶粉的TBST溶液中室温摇床封闭2h；
e)一抗孵育：封闭结束后，取出PVDF膜，依据目的蛋白分子量大小切割出相应目的蛋白条带，加入对应的一抗溶液中(一抗按说明书要求进行稀释)，置于4℃冰箱中摇床孵育过夜；
f)二抗孵育：一抗孵育结束后，取出目的蛋白条带置于TBST溶液中分3次洗涤，时间分别为5、10、15min，之后将蛋白条带转移至相对应的二抗溶液(按说明书要求配制)中，室温避光摇床孵育2h后，TBST溶液洗涤3次，时间分别为5、10、15min；
g)显影：按说明书要求配制ECL发色液，均匀覆于目的蛋白条带，覆以自封 袋，置于凝胶成像仪中显色拍摄；
h)实验结果计算：imageJ定量。
(2)化合物6对于肝细胞摄取DiI-LDL作用影响的检测
细胞培养：
HepG2细胞培养于含10％胎牛血清(FBS)DMEM高糖培养基中，5％CO 2，37℃。
DiI-LDL细胞摄取实验：
a)HepG2细胞接种于24孔板，利用含10％FBS的DMEM中培养24h；
b)换液至含2％脱脂血清(LPDS)的DMEM高糖培养基(500μl/每24孔单孔)，饥饿24h以促进低密度脂蛋白受体(LDLR)表达；
c)吸弃旧培养液，换液至新的含2％LPDS DMEM高糖培养基(500μl/每24孔单孔)，同时加入待测化合物(化合物利用DMSO配置，工作浓度为5μM，每份样品设3个平行孔)，孵育24h；
d)吸弃旧培养液，加入含DiI-LDL DMEM(300μl/每24孔单孔)，孵育3～4h；
e)吸弃含DiI-LDL DMEM，用含0.4％BSA的PBS小心冲洗2次，PBS冲洗3次，以去除多余DiI-LDL；
f)向24孔板中加入异丙醇(500μl/每24孔板单孔)，避光，摇床20min后，吸取上清200μl至96孔黑色酶标板，于激发光520nm、发射光578nm处检测荧光强度(FLU)；
g)实验结果计算：LDL摄取率＝待测样品组FLU值/空白(DMSO)组FLU值。
化合物6与阿托伐他汀钙联用，对阿托伐他汀钙诱导增加的肝细胞PCSK9蛋白表达作用影响的检测结果如下：
(1)化合物6与阿托伐他汀钙(Ator-Ca)联用，可下调由阿托伐他汀钙诱导增加的HepG2细胞PCSK9蛋白表达水平。结果如图1中所示，化合物6(5μM)和阿托伐他汀钙(Ator-Ca，2.5μM)与HepG2细胞共孵育24h，之后检测细胞PCSK9蛋白表达变化，实验结果表明，Ator-Ca可将细胞PCSK9蛋白表达水平上调1.76( **)倍，在此基础上，加入化合物6可将PCSK9蛋白表达水平降至Ator-Ca组的0.48( ##)倍(数据来自3次独立重复实验，实验结果为均数±SEM表示。 **：P<0.01，与DMSO组相比。 ##：P<0.01，与Ator-Ca组相比)。
(2)化合物6与阿托伐他汀钙(Ator-Ca)联用，可增加由阿托伐他汀钙诱导增加的HepG2细胞LDLR蛋白表达水平。结果如图2中所示，针对细胞LDLR蛋白表达变化的检测结果表明，化合物6可将细胞LDLR蛋白表达水平上调至DMSO组2.16( *)倍，Ator-Ca可将细胞LDLR蛋白表达水平上调至DMSO组2.93( **)倍，化合物6与Ator-Ca联用后，可将LDLR蛋白表达水平上调至DMSO组的5.36( *)倍(数据来自3次独立重复实验，实验结果为均数±SEM表示， **：P<0.01，与DMSO组相比； ##：P<0.01，与Ator-Ca组相比)。
(3)化合物6与阿托伐他汀钙(Ator-Ca)联用，可增加由阿托伐他汀钙诱导增加的HepG2细胞对LDL的摄取活性。结果如图3中所示，化合物6的联用可进一步增强对LDL的摄取作用，提升体外降脂活性。
实施例2：化合物6与转运体OATP1B1和OATP1B3的关系
阿托伐他汀的主要转运体是OATP1B1和OATP1B3，为了考察化合物6与阿托伐他汀联合用药时，是否影响阿托伐他汀的代谢行为，开展了化合物6的转运体相关研究。
具体实验操作和实验结果如下：
细胞模型：HEK293细胞模型
测试方法：采用LC-MS/MS法测定化合物6及阳性化合物抑制剂利福平的浓度。
底物筛查实验操作：
1)37℃预热的HBSS溶液清洗细胞三次后，向每孔中加入300μL 37℃预热的HBSS溶液平衡10min。
2)吸去孔中HBSS溶液，向每孔加入300μL含1.00μM、5.0μM、20.0μM待测物化合物6或5.0μM待测物化合物6和抑制剂利福平的HBSS溶液，摄取时间为10min，实验设置三样本。
3)摄取过程结束后，快速移去孔中HBSS溶液，以终止摄取反应；随后用冰冷的HBSS溶液(不含药物)清洗细胞三次，去除细胞表面的药物。
4)检测化合物6的孔中加入200μL双蒸水，反复冻融3次，取150μL样品，存于－80℃待测，取20μL样品用于蛋白浓度测定。
底物选择性筛查结果
1.00和20.0μM的化合物6在HEK293-OATP1B1细胞株上的摄取速率均不超过Mock细胞的2倍，5.00μM的化合物6在HEK293-OATP1B1细胞株上的摄取速率为Mock细胞的2倍以上，但加入抑制剂利福平后，摄取速率未出现降低，表明化合物6不是OATP1B1转运体的底物；1.00、5.00和20.0μM的化合物6在HEK293-OATP1B3细胞株上的摄取速率均不超过Mock细胞的2倍，表明化合物6不是OATP1B3转运体的底物，结果见表1和表2。
表1、化合物6在OATP1B1摄取转运体细胞株上的摄取
Figure PCTCN2022137011-appb-000005
Figure PCTCN2022137011-appb-000006
表2、化合物6在OATP1B3摄取转运体细胞株上的摄取
Figure PCTCN2022137011-appb-000007
实验结论：化合物6不是OATP1B1转运体和OATP1B3转运体的底物，化合物6不影响阿托伐他汀的转运，即不影响阿托伐他汀的代谢行为。
实施例3：化合物6抑制OATP1B1、OATP1B3摄取转运阿托伐他汀的活性
阿托伐他汀的主要转运体是OATP1B1和OATP1B3，为了考察化合物6与阿托伐他汀联合用药时，是否影响阿托伐他汀的代谢行为，开展了化合物6的转运体相关研究。
具体实验操作和实验结果如下：
细胞模型：HEK293细胞模型
测试方法：采用LC-MS/MS法测定化合物6及阳性化合物的浓度。
抑制实验操作：
1)37℃预热的HBSS溶液清洗细胞三次后，向每孔中加入300μL 37℃预热的阳性抑制剂阿托伐他汀或待测物化合物6溶液平衡30min。
2)吸去孔中HBSS溶液，向每孔加入300μL相应的含有一定浓度的阳性抑制剂或待测物化合物6和阳性底物的HBSS溶液。
3)摄取过程结束后，快速移去孔中HBSS溶液，以终止摄取反应；随后用冰冷的HBSS溶液(不含药物)清洗细胞三次，去除细胞表面的药物。
4)每孔中加入200μL超纯水，反复冻融3次，取150μL样品，存于－80℃待测,取20μL样品用于蛋白浓度测定。
抑制结果:
化合物6与相应的转运体探针底物在HEK293-OATP1B1和HEK293-OATP1B3细胞株上共同孵育，通过测定各个转运体底物摄取速率的变化，评价化合物6是否是各个转运体的抑制剂。同时设立HEK293-Mock细胞组、探针底物对照组和探针底物+化合物6处理组。结果显示，在0.1、0.3、1、3、10和30μM的化合物6作用下，探针底物阿托伐他汀在两种转运体细胞株(HEK293-OATP1B1)上的摄取速率相对于底物对照组分别为94.6％、87.3％、85.9％、84.9％、84.1％和92.3％；在0.1、0.3、1、3、10和30μM的化合物6作用下，探针底物阿托伐他汀在两种转运体细胞株(HEK293-OATP1B3)上的摄取速率相对于底物对照组分别为73.3％、78.0％、72.1％、75.5％、76.9％和80.6％。结果见表3和表4。
表3阿托伐他汀联用不同剂量的化合物6在OATP1B1摄取转运体细胞株上的摄取
Figure PCTCN2022137011-appb-000008
Figure PCTCN2022137011-appb-000009
NA:不适用
表4、阿托伐他汀联用不同剂量的化合物6在OATP1B3摄取转运体细胞株上的摄取
Figure PCTCN2022137011-appb-000010
Figure PCTCN2022137011-appb-000011
实验结论：上述结果表明，化合物6不干预阿托伐他汀的转运，提示两者具有良 好的联合作用生物学基础。
实施例4：化合物6与阿托伐他汀联合用药大鼠体内药效学研究
剂量与分组设置
空白对照组，给予普通饲料喂养；
高脂对照组，给予高脂饲料喂养，等体积0.5％CMC-Na灌胃；
Ator低剂量组，给予高脂饲料喂养，阿托伐他汀钙：3mg/kg/day；
Ator高剂量组，给予高脂饲料喂养，阿托伐他汀钙：10mg/kg/day；
化合物6给药组，给予高脂饲料喂养，化合物6：10mg/kg/day；
联合给药组(1)，给予高脂饲料喂养，阿托伐他汀钙：3mg/kg/day，化合物6：10mg/kg/day；
联合给药组(2)，给予高脂饲料喂养，阿托伐他汀钙：10mg/kg/day，化合物6：10mg/kg/day。
样品给药途径和频率
各给药组每天一次灌胃给予相应剂量的药物。
实验过程
Wistar大鼠(雄性，体重约150g)适应性预饲养1周后，检测基础血脂水平，随机取3只作为空白对照组，给予普通饲料喂养，其余大鼠给予高脂饲料喂养，10天后，检测血脂水平，根据血清TG水平将大鼠均匀分为6组，每组8只，给药组动物继续给予高脂饲料，并连续给药28天，高脂模型组继续给予高脂饲料，空白对照组给予普通饲料。实验终点时检测血脂水平、ALT和AST，动物安乐死，肝脏称重留样，血清留样。
血脂检测
异氟烷麻醉动物，眼眶后静脉丛采血，静置30分钟，4000rpm离心10分钟，分离血清，检测血清中甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)水平。
观察指标
观察动物给药前后血脂、体重等生理指标的变化。
统计学方法
实验数据用
Figure PCTCN2022137011-appb-000012
表示，用one-way ANOVA进行显著性检验。
实验结果
1)对大鼠血脂水平的影响
高脂饲料饲喂大鼠10天后，与空白对照组比较，大鼠血清中甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)及低密度脂蛋白胆固醇(LDL-C)水平显著上升，给药28天后，与模型对照组比较，阿托伐他汀钙和化合物6多次给药使大鼠血清中TG水平呈现不同程度下降，二者联用后TG水平进一步下降，其中，Ator高剂量组、化合物6给药组及联合给药组(2)的降幅分别为67.29％，45.79％和114.02％，三个给药组针对TC的降幅分别为2.99％，2.99％和34.33％，针对LDL-C的降幅分别为0,4.55％和50％。
表5.化合物6对大鼠血脂水平的影响(mmol/L，
Figure PCTCN2022137011-appb-000013
)
Figure PCTCN2022137011-appb-000014
Figure PCTCN2022137011-appb-000015
2)对大鼠血清甘油三酯水平的影响
结果如图4中所示，高脂饲料喂养10天后，与空白对照组比较，大鼠血清TG水平显著升高(P＜0.001)。给药28天后，与高脂对照组比较，各给药组大鼠的TG水平显著下降(P＜0.01)。与单独给药组比较，联用组的TG水平进一步下降。
3)对大鼠血清总胆固醇水平的影响
结果如图5中所示，高脂饲料喂养10天后，与空白对照组比较，大鼠血清TC水平显著升高(P＜0.001)。给药28天后，与高脂对照组比较，阿托伐他汀钙10mg/kg和化合物6 10mg/kg联用组大鼠血清中的TC水平显著下降。
4)对大鼠血清高密度脂蛋白水平的影响
结果如图6中所示，高脂饲料喂养10天后，与空白对照组比较，大鼠血清高密度脂蛋白(HDL-C)水平显著升高(P＜0.05)。给药28天后，与高脂对照组相比较，各给药组大鼠血清HDL-C水平无明显变化。
5)对大鼠血清LDL-C水平的影响
结果如图7中所示，高脂饲料喂养10天后，与空白对照组比较，大鼠血清低密度脂蛋白(LDL-C)水平显著升高(P＜0.001)。给药28天后，与高脂对照组比较，阿托伐他汀钙10mg/kg和化合物6 10mg/kg联用组大鼠血清LDL-C显著下降(P＜0.01)，其余各给药组大鼠的LDL-C水平则无显著变化。
6)对大鼠体重的影响
结果如图8中所示，与空白对照组比较，高脂对照组及各给药组大鼠的体重明显下降；与高脂对照组比较，各给药组大鼠体重无明显变化。
7)对大鼠血清ALT和AST的影响
结果如图9、图10中所示，高脂饲料喂养、阿托伐他汀钙多次给药和化合物6多次给药对大鼠血清中代表肝功能的ALT和AST水平均无明显影响。
8)对大鼠肝脏中PCSK9和LDLR mRNA水平的影响
结果如图11所示，给药28天后，与高脂对照组比较，阿托伐他汀钙10mg/kg显著上调大鼠肝脏中PCSK9mRNA水平(P＜0.001)，化合物6 10mg/kg显著下调大鼠肝脏中PCSK9mRNA水平(P＜0.05)，阿托伐他汀钙10mg/kg和化合物6 10mg/kg联用组大鼠肝脏中LDLR mRNA水平显著上升(P＜0.01)；与阿托伐他汀钙10mg/kg组比较，阿托伐他汀钙10mg/kg和化合物6 10mg/kg联用组大鼠肝脏中PCSK9 mRNA水平显著下降(P＜0.001)。
结论
给予高脂饲料喂养10天后，与空白对照组比较，高脂对照组大鼠血清中甘油三酯和胆固醇呈现不同程度升高，其中甘油三酯水平升高2.09倍，具有极显著差异，成功建立大鼠高血脂模型；阿托伐他汀钙及化合物6给药28天后，大鼠血清中甘油三酯水平呈现不同程度的下降，二者联用后甘油三酯水平进一步下降；阿托伐他汀钙或化合 物6单独给药未能改变大鼠血清胆固醇水平，但二者联用后胆固醇水平显著下降，表明阿托伐他汀钙和化合物6联合用药对高血脂大鼠有显著的降血脂作用，效果优于阿托伐他汀钙或化合物6单独用药。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考一样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种药物组合物或药物组合，其特征在于，所述药物组合物或药物组合包括：
    (A)治疗有效量的第一活性成分，所述第一活性成分为吲哚取代四氢异喹啉类化合物，或其药学上可接受的盐；
    (B)治疗有效量的第二活性成分，所述第二活性成分为HMG-CoA还原酶抑制剂他汀类高血脂症治疗药物。
  2. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述的药物组合物或药物组合，由(A)治疗有效量的第一活性成分，和(B)治疗有效量的第二活性成分组成；
    其中，所述第一活性成分为吲哚取代四氢异喹啉类化合物或其药学上可接受的盐；所述第二活性成分为HMG-CoA还原酶抑制剂他汀类高血脂症治疗药物。
  3. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述的第一活性成分的含量范围为0.01％至99.99％，以组合物活性成分的总重量计；优选为0.1％至99.9％，更优选为1％至99％；更优选为10％至99％；更优选为20％至99％；更优选为30％至99％；更优选为40％至99％；更优选为50％至99％。
  4. 如权利要求1所述的药物组合物或药物组合，其特征在于，第二活性成分的含量范围为0.01％至99.99％，以组合物活性成分的总重量计；优选为0.1％至99.9％，更优选为1％至99％；更优选为10％至99％；更优选为20％至99％；更优选为30％至99％；更优选为40％至99％；更优选为50％至99％。
  5. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述第一活性成分是式(I)所示的化合物，或其药学上可接受的盐：
    Figure PCTCN2022137011-appb-100001
    在式(I)中：
    其中，R 1和R 2各自独立地选自下组：氢、氘、氚、卤素、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 6～C 10芳基、取代或未取代的5-7元的杂环、取代或未取代的C1～C6烷基苯基、取代或未取代的C 1～C 6烷基5-7元杂芳基、取代或未取代的C 3～C 12环烷基、取代或未取代的C 2～C 10酰基、取代或未取代的C 2～C 10酯基、氨基、取代或未取代的C1-C6酰胺基、-SO 2R 9、-OSO 2R 9、-OCOR 9；且所述的R 1和R 2不同时为氢；
    或所述的R 1和R 2和相邻的-(CH 2) n-O以及C＝C共同构成取代或未取代的5-7元的杂环，其中，所述的杂环为全部饱和的杂环、部分不饱和的杂环或芳杂环；
    R 3、R 4、R 5和R 6各自独立地选自下组：氢、氘、氚、卤素、羟基、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 3～C 12环烷基、氰基、硝基或羧基；
    n为0或1；
    所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、C 1-C 4烷基、三氟甲基、C 1～C 4烷氧基、C 3-C 6环烷基、C 1～C 4直链或支链烷基取代的胺基、羟基、氰基、硝基、氧原子(＝O)、羟基-C 1～C 6烷基、羧基或巯基。
  6. 如权利要求5所述的药物组合物或药物组合，其特征在于，所述的式(I)中：
    其中，R 1和R 2各自独立地选自下组：氢、氘、氚、取代或未取代的C 1～C 6烷基、取代或未取代的C 6～C 10芳基、取代或未取代的5-7元的杂环、取代或未取代的C 1～C 6烷基苯基、取代或未取代的C 1～C 6烷基5-7元杂芳基；
    或所述的R 1和R 2和相邻的-(CH 2) n-O以及C＝C共同构成取代或未取代的5-7元的杂环，其中，所述的杂环为全部饱和的杂环、部分不饱和的杂环或芳杂环；
    R 3、R 4、R 5和R 6各自独立地选自下组：氢、氘、氚、卤素、羟基、取代或未取代的C 1～C 6烷基、取代或未取代的C 1～C 6烷氧基、取代或未取代的C 3～C 12环烷基、氰基、硝基或羧基；
    n为0或1。
  7. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述第一活性成分为选自下组的化合物，或其药学上可接受的盐：
    Figure PCTCN2022137011-appb-100002
    Figure PCTCN2022137011-appb-100003
  8. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述第二活性成分为HMG-CoA还原酶抑制剂他汀类治疗药物。
  9. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述的第二活性成分选自下组：氟伐他汀(Fluvastatin)、洛伐他汀(Lovastatin)、阿托伐他汀(Atorvastatin)、西立伐他汀(Cerivastatin)、普伐他汀(Pravastatin)、辛伐他汀(Simvastatin)、瑞舒伐他汀(Rosuvastatin)、匹伐他汀(Pitavastatin)，或其药用上可接受的盐。
  10. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述的第二活性成分为洛伐他汀、阿托伐他汀、普伐他汀、瑞舒伐他汀、匹伐他汀；更佳地，所述的第二活性成分为洛伐他汀、阿托伐他汀、瑞舒伐他汀；更佳地，所述的第二活性成分为阿托伐他汀。
  11. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述第一活性成分与所述第二活性成分的质量比范围为1：10000至10000：1。
  12. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述第一活性成分与所述第二活性成分的质量比范围为1:1000至1000:1。
  13. 如权利要求1所述的药物组合物或药物组合，其特征在于，所述第一活性成分与所述第二活性成分的质量比范围为1:500至500:1；更佳地，所述第一活性成分与所述第二活性成分的质量比范围为1:100至100:1；更佳地，为1:10至10:1。
  14. 如权利要求1所述的药物组合物或药物组合的应用，其特征在于，所述的药物组合物或药物组合用于预防或治疗与PCSK9相关的疾病。
  15. 如权利要求14所述的应用，其特征在于，所述PCSK9相关的疾病选自下组：代谢性疾病；较佳地，所述的代谢性疾病选自下组：高脂血症、高胆固醇血症、高甘油三酯血症、脂肪肝变形、动脉粥样硬化、肥胖。
PCT/CN2022/137011 2021-12-07 2022-12-06 吲哚取代四氢异喹啉类化合物与他汀类化合物的联合应用 WO2023104051A1 (zh)

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WO2017167202A1 (zh) * 2016-03-30 2017-10-05 中国科学院上海药物研究所 苯基[a]吲哚[2,3-g]并喹嗪类化合物、其制备方法、药物组合物及其应用

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