WO2018135918A1 - Composition pharmaceutique comprenant un composé ayant une activité inhibitrice de blt en tant que substance active pour la prévention ou le traitement de la bronchopneumopathie chronique obstructive - Google Patents

Composition pharmaceutique comprenant un composé ayant une activité inhibitrice de blt en tant que substance active pour la prévention ou le traitement de la bronchopneumopathie chronique obstructive Download PDF

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WO2018135918A1
WO2018135918A1 PCT/KR2018/000957 KR2018000957W WO2018135918A1 WO 2018135918 A1 WO2018135918 A1 WO 2018135918A1 KR 2018000957 W KR2018000957 W KR 2018000957W WO 2018135918 A1 WO2018135918 A1 WO 2018135918A1
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prop
methyl
phenyl
ynyl
fluorophenyl
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PCT/KR2018/000957
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English (en)
Korean (ko)
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최용석
김재홍
이경
한효경
위준동
권진선
구자일
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동국대학교 산학협력단
고려대학교 산학협력단
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Priority claimed from KR1020180006634A external-priority patent/KR20180087157A/ko
Application filed by 동국대학교 산학협력단, 고려대학교 산학협력단 filed Critical 동국대학교 산학협력단
Publication of WO2018135918A1 publication Critical patent/WO2018135918A1/fr

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    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease, comprising as an active ingredient a compound exhibiting inhibitory activity of Leukotriene B4 receptor 2. It relates to a composition.
  • Chronic obstructive pulmonary disease is a disease in which airflow restriction or obstruction is caused by chronic bronchitis or emphysema, and it is mainly a lung disease in which the airway is narrowed as a result of abnormal inflammatory reaction of the lung.
  • Chronic obstructive pulmonary disease is mainly caused by inhalation of harmful particles or gases, and smoking is known as a major cause.
  • Smoking acts as a powerful stimulant in lung tissue, increasing the production of various proinflammatory, growth, oxidative and chemotactic factors, and activating the inflammatory signaling system to induce inflammatory cells, including neutrophils and macrophages. To promote lung aggravation. Especially.
  • Chronic obstructive pulmonary disease is referred to collectively as chronic bronchitis with cough with chronic sputum and emphysema with abnormal increase in alveoli below terminal bronchiole and destruction of alveolar septum, which is difficult to distinguish between them.
  • COPD chronic obstructive pulmonary disease
  • COPD causes symptoms of airway disease such as dyspnea, cough, and sputum, which worsen lung function and lead to death. It is known that more than 90% of the causes are due to smoking, and pollution, congenital diseases and respiratory infections. Therefore, chronic obstructive pulmonary disease is a disease that causes the limitation of daily life and lowers the quality of life by gradually progressing the respiratory distress and eventually depends on others to the daily life, thereby lowering the productivity and quality of life of the people around.
  • leukotriene B4 is a group of inflammatory lipid mediators synthesized from arachidonic acid (AA) by the 5-lipoxygenase pathway that mediates acute and chronic inflammation.
  • LTB 4 is known to have a biological effect by binding to two types of receptors, BLT1 and BLT2.
  • Leukotriene B4 receptor 2 (BLT2) is a group of G protein-coupled receptors (GPCRs) that has low affinity for LTB 4 and is a lipid mediator of arachidonic acid (AA) induced through a 5-lipoxygenase dependent pathway. to be.
  • the present inventors while continuing to research to develop a more effective treatment for chronic obstructive pulmonary disease in order to solve the above-mentioned problems, the inventors prepared a compound that exhibits a BLT2 inhibitory activity, including the compound The first drug to treat obstructive pulmonary disease was designed.
  • the present invention has been made to solve the above problems, the present inventors have confirmed the effects of the treatment of chronic obstructive pulmonary disease of the compound showing the BLT2 inhibitory activity and based on this, to complete the present invention.
  • an object of the present invention is a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease (COPD), comprising as an active ingredient a compound, an isomer thereof, or a pharmaceutically acceptable salt thereof exhibiting BLT2 inhibitory activity.
  • COPD chronic obstructive pulmonary disease
  • the present invention comprises a compound exhibiting BLT2 inhibitory activity, an isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient, chronic obstructive pulmonary disease (COPD) ) Provides a pharmaceutical composition for prophylaxis or treatment.
  • COPD chronic obstructive pulmonary disease
  • the compound is tert -butyl 4- (4- (3- ( N -phenylpentaneamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate; N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide; N- (3- (4-methylpiperazin-1-carbonyl) phenyl) prop-2-ynyl) -N -phenylpentanamide; N- (3- (4- (4-ethylpiperazin-1-carbonyl) phenyl) prop-2-ynyl) -N -phenylpentanamide; N- (3- (4- (4-isopropylpiperazin-1-carbonyl) phenyl) prop-2-ynyl) -N -phenylpentaneamide; N- (3- (4- (4- (2-hydroxy
  • the composition may inhibit Leukotriene B4 receptor 2 (BLT2) activity.
  • BLT2 Leukotriene B4 receptor 2
  • It provides a method of treating chronic obstructive pulmonary disease comprising administering the pharmaceutical composition to a subject.
  • the present invention provides a therapeutic use for chronic obstructive pulmonary disease of a composition comprising the compound, an isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical for the prevention or treatment of chronic obstructive pulmonary disease (COPD), comprising as an active ingredient a compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, which exhibits Leukotriene B4 receptor 2 (BLT2) inhibitory activity.
  • COPD chronic obstructive pulmonary disease
  • BLT2 Leukotriene B4 receptor 2
  • the present inventors have experimentally confirmed the excellent chemotactic inhibitory effect and the treatment effect of chronic obstructive pulmonary disease of the compound showing the BTL2 inhibitory activity, the compound of the present invention can be usefully used as a pharmaceutical composition for treating chronic obstructive pulmonary disease It is expected to be able.
  • 1A to 1E and 2A to 2D are results of confirming the growth inhibitory effect of the compound treatment of the present invention in cells expressing BLT2 (CHO-BLT2).
  • 3A and 3B show the results of confirming chemotaxis inhibitory effect and IC 50 (50% inhibitory concentration) of cells by the compound treatment of the present invention in cells expressing BLT2 (CHO-BLT2 cells).
  • 4A and 4B show the results of confirming the chemotaxis inhibitory effect of cells treated with the compound of the present invention in cells expressing BLT2 (CHO-BLT2 cells) or cells expressing BLT1 (CHO-BLT1).
  • 5A to 5D show results of confirming chemotaxis inhibitory effect and IC 50 (50% inhibitory concentration) of cells by treatment with a compound of the present invention in cells expressing BLT2 (CHO-BLT2 cells).
  • 6A to 6C show the results of confirming the chemotaxis inhibitory effect of cells treated with the compound of the present invention in cells expressing BLT2 (CHO-BLT2 cells) or cells expressing BLT1 (CHO-BLT1).
  • 7A to 7C show the results of confirming the effect of inhibiting the binding of LTB4 and BLT2 by the compound treatment of the present invention in BLT2-expressing cells (CHO-BLT2 cells).
  • Figure 8a is a result confirming the inhibitory effect of TNF- ⁇ production by the compound treatment of the present invention in an animal model of chronic obstructive pulmonary disease.
  • Figure 8c is a result confirming the inhibitory effect of ROS production by the compound treatment of the present invention in an animal model of chronic obstructive pulmonary disease.
  • Figure 9 is a result of confirming the degree of infiltration of inflammatory cells by treatment of LMT-886 and LMT-1013 compounds of the present invention in animal models of chronic obstructive pulmonary disease induced by tobacco smoke and lipopolysaccharide.
  • FIG. 13 shows the results of measuring IL-6 by treatment with the compounds LMT-886 and LMT-1013 of the present invention in an animal model of chronic obstructive pulmonary disease induced by tobacco smoke and lipopolysaccharide.
  • Figure 14 shows the results confirmed by H & E staining lung tissue animal models of chronic obstructive pulmonary disease induced by tobacco smoke and lipopolysaccharide.
  • the present inventors based on the fact that the treatment of the compound prepared in Example can significantly inhibit the growth of BLT2 expressing cells, specific effects of BLT2-dependent chemotaxis and the effect of treating chronic obstructive pulmonary disease, etc. It confirmed with and based on this, this invention was completed.
  • the present invention provides a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease (COPD), comprising a compound represented by the following formula (1), an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • COPD chronic obstructive pulmonary disease
  • R 1 is C 1 -C 10 is alkyl
  • R 2 is , , , , or Is
  • R a is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Or hydroxy,
  • R b is , , , or ego
  • R C is , , or ego
  • R d is hydrogen or ego
  • R e is or ego
  • R 3 is hydrogen or fluorine.
  • Preferred examples of the compound represented by Formula 1 according to the present invention are as follows:
  • the present invention also provides a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease (COPD) comprising a compound represented by the following formula (2), an isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient. do.
  • COPD chronic obstructive pulmonary disease
  • R 1 is C 1 -C 10 alkyl, , or ego,
  • R 2 is hydrogen, , or ego
  • R 3 is hydrogen, , or ego
  • R 4 is hydrogen, , or ego
  • R a is hydrogen, C 1 -C 10 alkyl, C 1 -C 5 carboxyl,
  • R 5 , R 6 , and R 7 are each independently hydrogen, halogen, nitro, methyl, trifluoromethyl or methoxy,
  • R 1 is C 1 -C 10 alkyl and R a is hydrogen or C 1 -C 10 alkyl; And when R 2 , R 3 , and R 4 are all hydrogen at the same time.
  • Preferred examples of the compound represented by Formula 2 according to the present invention are as follows:
  • the term "pharmaceutically acceptable” is suitable for use in contact with the tissues of a subject (eg, a human being) because the benefit / risk ratio is reasonable without excessive toxicity, irritation, allergic reactions or other problems or complications.
  • a compound or composition is within the scope of sound medical judgment.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids.
  • Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide.
  • the acid addition salt according to the present invention is dissolved in a conventional method, for example, the compound represented by the formula (1) or (2) in an excess of aqueous acid solution, and the salt is water-miscible organic solvent such as methanol, ethanol, acetone Or by precipitation with acetonitrile. It may also be prepared by evaporating the solvent or excess acid from the mixture and then drying or by suction filtration of the precipitated salt.
  • a conventional method for example, the compound represented by the formula (1) or (2) in an excess of aqueous acid solution
  • the salt is water-miscible organic solvent such as methanol, ethanol, acetone Or by precipitation with acetonitrile. It may also be prepared by evaporating the solvent or excess acid from the mixture and then drying or by suction filtration of the precipitated salt.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).
  • a compound showing BLT2 inhibitory activity was prepared (see Example 1), and the growth inhibition of BLT2 expressing cells by the compound treatment was confirmed (see Experimental Example 2).
  • the chemotaxis of the BLT2 expressing cells can be inhibited (see Experimental Example 3)
  • using the compound of the present invention confirmed the inhibitory effect of LTB4 and BLT2 binding (see Experimental Example 4), chronic obstructive pulmonary disease
  • TNF- ⁇ , IL-6 and ROS production in bronchoalveolar lavage fluid see Experimental Example 5
  • the animal models of chronic obstructive pulmonary disease induced by tobacco smoke and lipopolysaccharide were prepared to confirm the efficacy of the compounds LMT-886 and LMT-1013 exhibiting BLT2 inhibitory activity (see Experimental Example 6). .
  • the inventors of the present invention to determine the effect of reducing the production of TNF- ⁇ and IL-6 to activate the inflammatory signaling system as an inflammatory cytokine in bronchoalveolar lavage fluid for chronic obstructive pulmonary disease-induced mice of the present invention, Changes in the production of TNF- ⁇ 2 and IL-6 in the bronchoalveolar lavage fluid of the experimental group to which the normal control group, the negative control group, the compound of the present invention and the comparative drug (ROF; Roflumilast PDE4 inhibitor) were administered were measured.
  • ROF Roflumilast PDE4 inhibitor
  • the tumor necrosis factor (TNF- ⁇ ) of the present invention is a kind of cytokinin produced by macrophages and the like, and interleukin-6 of the present invention produces B-cell antibody.
  • B-cell stimulating factor 2 (BSF-2) which induces final differentiation into cells, wherein the glycoprotein has an isolated molecular weight of 210,000. It is a cytokine produced by several cells such as T lymphocytes, B lymphocytes, macrophages and fibroblasts.
  • the reactive oxygen species (ROS) of the present invention are oxygen-containing chemical reaction molecules, which are formed as natural by-products of normal metabolism of oxygen and play an important role in cellular signaling and homeostasis. However, during environmental stress, ROS levels can increase significantly, which can lead to significant damage to cellular structure.
  • the compound of the present invention effectively inhibits the inflammatory response in the lung tissue through the inhibition of the inflammatory mediators, and thus can be usefully used as an active ingredient in the pharmaceutical composition for the prevention and treatment of chronic obstructive pulmonary disease.
  • prevention means any action that inhibits or delays the development of chronic obstructive pulmonary disease by administration of a pharmaceutical composition according to the present invention.
  • treatment means any action that improves or advantageously changes the symptoms for chronic obstructive pulmonary disease by administration of the pharmaceutical composition according to the present invention.
  • chronic obstructive pulmonary disease is an abnormal inflammatory reaction occurs in the lungs by inhalation of harmful particles such as smoking or industrial gases, thereby gradually limiting airflow and thus lowering lung function.
  • a respiratory disorder that causes respiratory distress More specifically, if a substance that stimulates the airways, such as cigarette smoke, repeatedly stimulates the airways and bronchus, the secretion of bronchial mucus secreted to remove foreign substances in the bronchus increases. Causes partial or complete closure. When the bronchus is closed, the alveoli expand and become damaged, and the ability to exchange oxygen and carbon dioxide is impaired. The closed alveoli are easily infected with bacteria.
  • chronic obstructive pulmonary disease When bacteria are infected by patients with chronic obstructive pulmonary disease, arterial oxygen pressure is a serious disorder of gas exchange. This rapidly decreasing situation may occur, and carbon dioxide coma may occur when the arterial blood carbon dioxide pressure increases due to respiratory failure.
  • chronic obstructive pulmonary disease may include, but is not limited to, emphysema, chronic bronchitis, and the like.
  • the chronic obstructive pulmonary disease of the present invention may be a disease due to overexpression of Leukotriene B4 receptor 2 (BLT2). More specifically, leukotriene is an immune mediator that causes bronchial contraction, and overexpression of Leukotriene B4 receptor 2 (BLT2) may cause chronic obstructive pulmonary disease.
  • BLT2 Leukotriene B4 receptor 2
  • BLT2 Leukotriene B4 receptor 2 is one of the group of G protein-coupled receptors (GPCR), which has a low affinity for LTB 4 ( Leukotriene B4; LTB 4 ), and the composition of the present invention is a cell growth by BLT2.
  • GPCR G protein-coupled receptors
  • the term “inhibition” means inhibiting any step of transcription, mRNA processing, translation, translocation, and maturation of a gene, or inhibition of protein-to-protein binding, activation of a protein, or signaling through it. .
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient.
  • the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • it may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug, It may be determined according to the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field.
  • the pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day.
  • the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
  • the present invention also provides a method of treating chronic obstructive pulmonary disease comprising administering the pharmaceutical composition to a subject.
  • subject means a subject in need of treatment for a disease, and more specifically, a mammal, such as a primate, mouse, dog, cat, horse and cow, which is human or non-human. .
  • non-limiting compounds to be used as active ingredients of the pharmaceutical composition according to the present invention include the following compounds, isomers thereof and pharmaceutically acceptable salts thereof.
  • the following compounds according to the invention were synthesized and the yield and 1 H NMR measurement results are described below.
  • N -Phenyl- N -(3- (4- (4- ( Prof -2- Inil Piperazine-1- Carbonyl ) Phenyl) Prof -2-ynyl) pentaneamide N -phenyl- N -(3- (4- (4- (prop-2- ynyl ) piperazine -1-carbonyl) phenyl) prop-2-ynyl) pentanamide) ( LMT -833)
  • N -(3- Fluorophenyl )- N -(3- (4- (4- Isopropyl piperazine -One- Carbonyl ) Phenyl) prop-2-ynyl) pentaneamide N -(3- fluorophenyl )- N -(3- (4- (4-isopropylpiperazine-1-carbonyl) phenyl) prop-2-ynyl) pentanamide
  • N -(4- Fluorophenyl )- N -(3- (4- (4- Isopropyl piperazine -One- Carbonyl ) Phenyl) prop-2-ynyl) pentaneamide N -(4- fluorophenyl )- N -(3- (4- (4-isopropylpiperazine-1-carbonyl) phenyl) prop-2-ynyl) pentanamide
  • Benzamide N , N -diethyl-4- (3- ( N -phenylpentanamido) prop-1-ynyl) benzamide) ( LMT -883)
  • N -(3- (3- (4-methylpiperazin-1-carbonyl) phenyl) prop-2-ynyl)- N -Phenylpentaneamide N -(3- (3- (4-methylpiperazine-1-carbonyl) phenyl) prop-2-ynyl)- N -phenylpentanamide
  • N -(4- Fluorophenyl )- N -(3- (3- (4- Isopropyl piperazine -One- Carbonyl ) Phenyl) prop-2-ynyl) pentaneamide N -(4- fluorophenyl )- N -(3- (3- (4-isopropylpiperazine-1-carbonyl) phenyl) prop-2-ynyl) pentanamide
  • tert -Butyl 4- (5- (3-(( N - Phenylpentaneamido ) Prof -1-yn-1-yl) Picolinoyl Piperazine-1-carboxylate ( tert -butyl 4- (5- (3- ( N - phenylpentanamido prop-1- yn -1-yl) picolinoyl) piperazine-1-carboxylate) ( LMT -834)
  • N -(3- (6- (4- Isopropyl piperazine -One- Carbonyl Pyridin-3-yl) Prof -2-yn-1-yl)- N -Phenylpentaneamide N -(3- (6- (4- isopropylpiperazine -1-carbonyl) pyridin -3-yl) prop-2-yn-1-yl)- N -phenylpentanamide
  • N, N -Diethyl-4- (3- ( N -(4-fluorophenyl) pentaneamido) prop-1-yn-1-yl) benzamide N, N -diethyl-4- (3- ( N -(4-fluorophenyl) pentanamido) prop-1-yn-1-yl) benzamide) ( LMT -927)
  • N -Ethyl-4- (3- ( N - Phenylpentaneamido ) Prof -1-yn-1-yl) Benzamide
  • N -ethyl-4- (3- ( N -phenylpentanamido) prop-1-yn-1-yl) benzamide
  • BLT2 is Expressed cells or BLT2 is Preparation of Unexpressed Cells
  • cells without BLT2 expression and cells with BLT2 expression were prepared in the following manner.
  • CHO cells were obtained from the Korea Cell Line Bank (KCLB, 10061), which contained 10% FBS (fetal bovine serum; Life technologies, Inc.), penicillin (50 units / ml), and antibiotic antimycotic solution (Life technologies, Inc.). It was incubated at 37 °C, 5% CO 2 conditions in RPMI 1640 medium (Invitrogen) containing the. The cells were maintained in the growth phase by splitting with Trypsin-EDTA for 3 days, respectively, with PBS (phosphate-buffered saline; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2m MKH 2 PO 4 ) And then added to fresh medium to prepare cells without BLT2 expression.
  • FBS fetal bovine serum
  • penicillin 50 units / ml
  • antibiotic antimycotic solution Life technologies, Inc.
  • CHO-K1 cells were transformed with pcDNA3-long form BLT2 encoding HA-tagged human BLT2, and 0.4 mg / ml of G418 (Invitrogen). , Carlsbad, CA, USA).
  • G418 Invitrogen
  • the selected clones were analyzed by RT-PCR using human-specific BLT2 primers, and representative clones were used in the experiments with cells expressing BLT2 (CHO-BLT2).
  • LTB 4 which is a ligand of BLT2 to BLT2-expressing cells (CHO-BLT2 cells) (300 nM) treated (DMSO +) and ethanol treated (DMSO-) cell growth increased from 20% to 35%, and in BLT2 expressed cells (CHO-BLT2), positive control Phosphorus LY255283 pre-treated, showed about 90% cell growth compared to the control group treated with DMSO, confirming the effect of inhibiting cell growth according to the compound treatment of the Example.
  • LTB 4 which is a ligand of BLT2 to BLT2-expressing cells (CHO-BLT2 cells) (300 nM) treated (DMSO +) and ethanol treated (DMSO-) cell growth increased from 20% to 35%, and in BLT2 expressed cells (CHO-BLT2), positive control Phosphorus LY255283 pre-treated, showed about 90% cell growth compared to the control group treated with DMSO, confirming the effect of inhibiting cell growth according to the compound treatment of the Example. Specifically, the growth inhibitory effect of the compounds of the present invention AC-1632 (78.7%), AC-1635 (71.6%), AC-1646 (72.1%) and AC-1650 (82.2%) was confirmed.
  • the compounds of the present invention can inhibit BLT2-induced cell proliferation with excellent efficiency, and the compound is used as a therapeutic agent for BLT2-related chronic obstructive pulmonary disease. It can be used as a possible pharmaceutical ingredient (BLT2-blocking pharmacological molecules).
  • Chemotactic motility was analyzed using a Transwell chamber equipped with a 6.5-mm diameter polycarbonate filter (8- ⁇ m pore size, Corning Costar). Specifically, the bottom surface of the filter was coated with 10 ⁇ g / ml fibronectin in serum free RPMI 1640 medium at 37 ° C. for 1 hour. A dry, coated filter with RPMI 1640 medium containing varying amounts of LTB 4 is placed in the bottom well of the Transwell chamber and finally 2 ⁇ 10 4 cells of CHO cells stably expressing BLT1 and BLT2 in serum-free RPMI 1640 medium. Experiments were performed by loading the upper wells with / 100 ⁇ l.
  • the cells were pretreated with each inhibitor for 30 minutes before dispensing. After 3 hours of incubation at 37 ° C., 5% CO 2 , the filters were fixed with methanol for 3 minutes and stained with hematoxylin and eosin for 10 minutes.
  • the cells used BLT2-expressing cells (CHO-BLT2 cells) and BLT1-expressing cells (CHO-BLT1), LY255283 and U75302 as the positive control, respectively, and LTB 4 , the ligand of BLT2 as the control. , (300 nM), LTB 4 , the ligand of BLT1 (10nM) and LPA (lysophosphatidic acid; 100nM) were used. Chemotaxis was quantified by counting cells on the lower side of the filter under an optical microscope (magnification, x 200). Six fields were counted in each assay, each sample analyzed twice, and the assay was repeated three times.
  • LTB 4 which is a ligand of BLT2 to BLT2-expressing cells (CHO-BLT2 cells)
  • DMSO + DMSO +
  • DMSO- ethanol treated
  • cell chemotaxis was 2.4-fold increased
  • LY255283 used as a positive control pretreated (10 ⁇ M)
  • the ligand LTB Compared to the case of 4 treated, it was confirmed that the coinability of 90%.
  • the ligand LTB 4 (10 nM) treated (DMSO +), ethanol treated compared to (DMSO-), the cell chemotaxis increased 2.2 times, the pretreatment (10 ⁇ M) of U75302 used as a positive control, the ligand LTB It was confirmed that 90% of chemotaxis was shown in comparison with the case where 4 was treated.
  • LTB 4 is a ligand Compared with treatment (DMSO +), chemotaxis was 66%, 90%, and 70.3% inhibited, whereas LTB 4 , a ligand, was expressed in BLT1-expressing cells (CHO-BLT1). It was confirmed that chemotaxis was not suppressed compared to treatment (DMSO +).
  • LTB 4 which is a ligand of BLT2 in BLT2-expressing cells (CHO-BLT2 cells) (300 nM) treated (DMSO +) and ethanol treated (DMSO-), compared to 2.9-fold increase in cell chemotaxis, 10 ⁇ M pretreated LY255283 used as a positive control, the ligand LTB 4 To It showed 90% chemotaxis compared to the treatment, and when the compound of the present invention (AC-1074) was pretreated with 10 ⁇ M of cells expressing BLT2, the ligand was treated with LTB 4 (DMSO +). It was confirmed that 53% inhibition of chemotaxis.
  • LTB 4 which is a ligand of BLT1 in BLT1-expressing cells (CHO-BLT1 cells)
  • 10 nM DMSO +
  • DMSO- DMSO-
  • the cell chemotaxis was increased by 2.8-fold
  • the compound (AC-1074) of the present invention was added to cells expressing BLT2 10.
  • pretreatment with M it was confirmed that there was no change in chemotaxis compared to (DMSO +) when the ligand LTB 4 was treated.
  • chemotactic activity was LTB 4 in cells stably expressing BLT2 (CHO-BLT2).
  • the compounds of the present invention LMT-692, LMT-696, LMT-1013, and AC-1074
  • LMT-692, LMT-696, LMT-1013, and AC-1074 can significantly inhibit this chemotaxis, as the BLT2-dependent strain induced by LTB 4 It can be used as a pharmaceutical component to inhibit Mars.
  • LTB 4 and BLT2 binding (ligand binding affinity) inhibition was analyzed using the isotope tritium (H3) label LTB 4 ([3H] LTB 4, ARC) (specific activity 160.0 Ci / mmol).
  • Experimental method is to put 2 ⁇ 10 6 CHO-BLT2 cells in a 100 mm culture dish and incubate for 48 hours before proceeding. The harvested cells are used five times for one minute in a homogenizer to separate the proteins of the cell membrane. Thereafter, centrifugation was performed for 40 minutes at 45,000 RPM at 4 ° C. to harvest only protein of the cell membrane and quantify it at a concentration of 40 ⁇ g / 45 ⁇ l.
  • the concentration of the compound (AC-1074) of the present invention increases (10 -9 , 10 -8 , 10 -7 , 10 -6 and 10 -5 ) the binding of LTB 4 and BLT2 was inhibited, the IC 50 (50% binding inhibition concentration) of the AC-1074 compound was 140.35 nM.
  • COPD Chronic obstructive pulmonary disease
  • Toxic smoke-induced chronic obstructive pulmonary disease (COPD) animal model experiments were conducted by the Korea Institute of Bioscience and Biotechnology (KRIBB). More specifically, standard tobacco extract (Cigarette smoking, CS) was prepared as follows to cause chronic obstructive pulmonary disease caused by tobacco extract in experimental mice. In accordance with ISO 3402, 60 cigarette smoke condensate collections were carried out in a smoking room maintained at a temperature of 22 ⁇ 2 ° C and a relative humidity of 60 ⁇ 5%, according to the ISO 3402 standard, Coresta Monitering Cigarette 7, Heinr Borgwaldt, Germany.
  • ISO 3402 60 cigarette smoke condensate collections were carried out in a smoking room maintained at a temperature of 22 ⁇ 2 ° C and a relative humidity of 60 ⁇ 5%, according to the ISO 3402 standard, Coresta Monitering Cigarette 7, Heinr Borgwaldt, Germany.
  • Cigarette cigarettes were burned using an automatic smoking machine (Automatic smoking machine, RM20, Heinr Borgwaldt, ISO 3308 standard) with a smoking volume of 35.0 ⁇ 0.3 ml, a smoking cycle of 60 ⁇ 0.5 seconds and a smoking time of 2.00 ⁇ 0.02 seconds.
  • the length of the butts was set to tip paper length + 3 mm (overwrap + 3 mm), and the smoke smoke condensate was collected by a 92 mm cambridge filter (ISO 3308 standard product).
  • the filter cartridge which collected the smoke smoke condensate, was separated from the cigaratte holder and placed in a 100 ml Erlenmeyer flask, and then shaken well by adding 50 ml of extraction solvent isopropanol, followed by extraction at room temperature for at least 8 hours. . After extraction, the resultant was filtered, concentrated with a vacuum filter concentrator, and the concentrates contained in three Erlenmeyer flasks were collected in scintillation vials and completely concentrated with nitrogen gas.
  • mice Male 8-week-old mice (BALB / c, SPF, 18-20 g) supplied by Orient Biotech Co., Ltd. (Korea) were thoroughly fed with solid feed (no antibiotics, Samyang Feed Co.) and water until the day of the experiment. It was supplied and used for experiments after 1 week of adaptation in a temperature of 22 ⁇ 2 ° C., a humidity of 55 ⁇ 15% and a light-dark cycle of 12 hours. After 8-week-old BALB / c mice were anesthetized with 7% chloral hydrate, the mouse's incisors were fixed with rubber bands when there was no movement, and then standard tobacco extract was added to 100 ⁇ g / ml of lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • the COPD-induced negative control group significantly increased the production of TNF- ⁇ in bronchoalveolar lavage fluid compared to the normal control group, the experimental group administered the compound of the present invention AC-1013 negative control group It was confirmed that the production of TNF- ⁇ is inhibited as compared with that of.
  • COPD Chronic obstructive pulmonary disease
  • the COPD-induced negative control group significantly increased the production of IL-6 in bronchoalveolar lavage fluid compared with the normal control group
  • the experimental group administered the compound of the present invention AC-1013 negative control group It was confirmed that the production of IL-6 is inhibited compared with that of.
  • ROS reactive oxygen species
  • mice were exposed to a total of 8 cigarette smokes for 1 hour per day, and LPS was intranasally administered at 5 ⁇ g / 50 ⁇ l / mouse.
  • Compounds of the invention (LMT-886 and LMT-1013) were administered orally 1 hour before exposure to tobacco smoke.
  • Tobacco smoke acts as a powerful stimulant in the lung tissue and activates the inflammatory signaling system, which further exacerbates pulmonary inflammation. Therefore, suppressing inflammation can be used as an index for improving chronic obstructive pulmonary disease.
  • Infiltration of inflammatory cells (Neutrophils, Macrophages, etc.) was confirmed. Therefore, in order to confirm the number of inflammatory cells in the bronchial alveolar lavage fluid of the COPD mouse described in Example 6-1, 700ul (total volume 1.4 ml) of saline (PBS: phosphate buffered saline) was added to the lungs of the mouse using bronchial implantation.
  • PBS phosphate buffered saline
  • centrifugation (14,000 rpm, 4 ° C, 5 min) was carried out by tapping 1 ml of PBS into a 1.5 ml tube from which cells were separated, and then 100 ⁇ l of each sample was obtained for cytospin (Hanil). Science, Korea) was attached to the slide by rotating for 5 minutes at 1000rpm. Thereafter, the nucleus and cytoplasm of the cells were stained using a Diff-Quik staining kit to distinguish inflammatory cells, and the number of cells was calculated by microscopic examination.
  • the compounds of the present invention LMT-886 and LMT-1013 significantly reduced the infiltration of inflammatory cells (neutrophils) of COPD mice.
  • Toxic agents that cause emphysema include neutrophil elastase (NE) and reactive oxygen species (ROS).
  • NE neutrophil elastase
  • ROS reactive oxygen species
  • DCFDA Dichlorofluorescin diacetate
  • NE see FIG. 10
  • ROS see FIG. 11
  • saline PBS: phosphate buffered
  • saline 700ul total volume 1.4 ml
  • ELISA kit (BD Bioscience, San Diego, Calif.) was used to measure the levels of TNF- ⁇ and IL-6 in the supernatant, and was performed according to the protocol of each manufacturer.
  • the supernatant was dispensed on a 96 well plate to which TNF- ⁇ or IL-6 antibody was attached and attached for 2 hours, followed by washing with a washing solution, and the detection antibody was attached with HRP for 1 hour and then washed again.
  • the reaction was stopped with sulfuric acid solution, and the absorbance was measured at 450 nm using a SPARK TM 10 M multimode microplate reader (Tecan system inc., CA, USA).
  • TNF- ⁇ (see FIG. 12) and IL-6 (see FIG. 13) were significantly increased in COPD mouse bronchial alveolar lavage fluid, but the compounds LMT-886 and LMT of the present invention.
  • Oral administration of -1013 was found to significantly reduce the production of cytokines such as TNF- ⁇ and IL-6.
  • lungs were removed, immediately fixed in 10% formaldehyde solution, rinsed and washed with running water for 8 hours, embedded in epoxy, and sections were made of microtome.
  • staining was performed with hematoxylin and eosin (H & E), and then fixed with Histomount solution (invitrogen, USA), and pathological changes of lung tissue were examined using an optical microscope. .
  • the compounds LMT-886 and LMT-1013 of the present invention is shown to improve the inflammation of the lungs in animal models of chronic obstructive pulmonary disease caused by tobacco smoke and lipopolysaccharide, and may be able to restore the reduced lung function. Judging.
  • Compounds exhibiting BTL2 inhibitory activity of the present invention exhibits an excellent chemotactic inhibitory effect and a treatment effect for chronic obstructive pulmonary disease, whereby the compounds of the present invention are for preventing or treating chronic obstructive pulmonary disease (COPD). It is expected that it may be usefully used as an active ingredient of a pharmaceutical composition.
  • COPD chronic obstructive pulmonary disease

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Abstract

La présente invention concerne un nouveau composé présentant une activité inhibitrice sur le récepteur 2 de leucotriène B4 (BLT2) et une composition comprenant celui-ci en tant que substance active pour la prévention ou le traitement de maladies inflammatoires. Dans la présente invention, un nouveau composé présentant une activité d'inhibition de BTL2 a été étudié. Le composé a été identifié de façon expérimentale comme ayant d'excellents effets tels que l'augmentation de la mort des cellules cancéreuses, la suppression des métastases, la suppression de la chimiotaxie, l'activité antiasthmatique, etc., et il est attendu de ce composé qu'il approche et traite une cible plus fondamentalement, en vue de prévenir ou traiter des maladies inflammatoires.
PCT/KR2018/000957 2017-01-23 2018-01-22 Composition pharmaceutique comprenant un composé ayant une activité inhibitrice de blt en tant que substance active pour la prévention ou le traitement de la bronchopneumopathie chronique obstructive WO2018135918A1 (fr)

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KR20170010613 2017-01-23
KR10-2017-0010613 2017-01-23
KR10-2018-0006634 2018-01-18
KR1020180006634A KR20180087157A (ko) 2017-01-23 2018-01-18 Blt 저해 활성을 갖는 화합물을 유효성분으로 포함하는 만성 폐쇄성 폐질환 예방 또는 치료용 약학적 조성물

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Citations (6)

* Cited by examiner, † Cited by third party
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KR20030068547A (ko) * 2000-10-27 2003-08-21 오르토-맥네일 파마슈티칼, 인코퍼레이티드 신경계 질환의 치료에 유용한 신규한 아미도알킬-피페리딘및 아미도알킬-피페라진 유도체
WO2008073929A1 (fr) * 2006-12-11 2008-06-19 Wyeth Modulateurs de canal ionique
US20110136877A1 (en) * 2008-01-07 2011-06-09 Ligand Pharmaceuticals Inc. 2-phenyl phenoxyacetic acids useful for treating inflammatory disorders
KR20130017073A (ko) * 2011-08-05 2013-02-19 동국대학교 산학협력단 신규한 바이페닐 유도체 또는 이의 약학적으로 허용가능한 염, 및 이를 유효성분으로 포함하는 염증성 질환 또는 자가면역질환의 예방 또는 치료용 약학적 조성물
CN104045552A (zh) * 2013-03-13 2014-09-17 上海先声药物研究有限公司 作为神经保护剂的药用化合物
KR20170012151A (ko) * 2015-07-24 2017-02-02 동국대학교 산학협력단 Blt 저해 활성을 갖는 신규 화합물 및 이를 유효성분으로 포함하는 염증성 질환 예방 또는 치료용 조성물

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030068547A (ko) * 2000-10-27 2003-08-21 오르토-맥네일 파마슈티칼, 인코퍼레이티드 신경계 질환의 치료에 유용한 신규한 아미도알킬-피페리딘및 아미도알킬-피페라진 유도체
WO2008073929A1 (fr) * 2006-12-11 2008-06-19 Wyeth Modulateurs de canal ionique
US20110136877A1 (en) * 2008-01-07 2011-06-09 Ligand Pharmaceuticals Inc. 2-phenyl phenoxyacetic acids useful for treating inflammatory disorders
KR20130017073A (ko) * 2011-08-05 2013-02-19 동국대학교 산학협력단 신규한 바이페닐 유도체 또는 이의 약학적으로 허용가능한 염, 및 이를 유효성분으로 포함하는 염증성 질환 또는 자가면역질환의 예방 또는 치료용 약학적 조성물
CN104045552A (zh) * 2013-03-13 2014-09-17 上海先声药物研究有限公司 作为神经保护剂的药用化合物
KR20170012151A (ko) * 2015-07-24 2017-02-02 동국대학교 산학협력단 Blt 저해 활성을 갖는 신규 화합물 및 이를 유효성분으로 포함하는 염증성 질환 예방 또는 치료용 조성물

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