WO2017018750A1 - Nouveau composé ayant une activité inhibitrice de blt et composition pour prévenir ou traiter des maladies inflammatoires, le contenant en tant que principe actif - Google Patents

Nouveau composé ayant une activité inhibitrice de blt et composition pour prévenir ou traiter des maladies inflammatoires, le contenant en tant que principe actif Download PDF

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WO2017018750A1
WO2017018750A1 PCT/KR2016/008069 KR2016008069W WO2017018750A1 WO 2017018750 A1 WO2017018750 A1 WO 2017018750A1 KR 2016008069 W KR2016008069 W KR 2016008069W WO 2017018750 A1 WO2017018750 A1 WO 2017018750A1
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Prior art keywords
prop
ynyl
phenyl
carbonyl
fluorophenyl
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PCT/KR2016/008069
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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 KR1020160093762A external-priority patent/KR101796391B1/ko
Application filed by 동국대학교 산학협력단, 고려대학교 산학협력단 filed Critical 동국대학교 산학협력단
Priority to US15/745,337 priority Critical patent/US10179764B2/en
Priority to JP2018503553A priority patent/JP6574517B2/ja
Priority to ES16830783T priority patent/ES2834549T3/es
Priority to CN201680055514.4A priority patent/CN108349874B/zh
Priority to EP16830783.3A priority patent/EP3327000B1/fr
Publication of WO2017018750A1 publication Critical patent/WO2017018750A1/fr
Priority to US16/181,953 priority patent/US10766857B2/en

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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
    • A61K31/33—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
    • 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
    • A61K31/4965—Non-condensed pyrazines
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/02—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
    • C07C233/04—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C233/07—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/04—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/30—1,4-Oxazines; Hydrogenated 1,4-oxazines not condensed with other rings

Definitions

  • the present invention relates to a novel compound and its use, and more particularly, to a novel compound exhibiting inhibitory activity of Leukotriene B4 receptor 2 (BLT2) and a pharmaceutical composition for preventing or treating inflammatory diseases comprising the same as an active ingredient.
  • BLT2 Leukotriene B4 receptor 2
  • Inflammatory reactions are one of the body's immune systems that are activated through various mechanisms of action to protect against physical or chemical agents, bacterial infections, and immunological stimuli. However, if such an inflammatory response persists, it promotes mucosal damage, thereby causing inflammatory diseases such as rheumatoid arthritis, arteriosclerosis, gastritis, and asthma due to redness, fever, swelling, pain, and dysfunction. have. These inflammatory reactions are classified into acute and chronic inflammations over time, and acute inflammations can cause symptoms such as erythema, fever, pain, and edema, while inflammatory reactions last several days to several weeks. Is a prolonged inflammatory state, sometimes over several years to decades, and is accompanied by histological changes such as invasion of monocytes, proliferation of fibroblasts and capillaries, fibrosis due to increased connective tissue, and tissue destruction. .
  • pro-inflammatory cytokines pro-inflammatory cytokines
  • interferon-gamma INF- ⁇
  • tumor necrosis factor- ⁇ TNF- ⁇
  • interleukin-1 interleukin-1, IL-1
  • interleukin during inflammatory processes in the body INF- ⁇
  • cytokines such as -6 (interleukin-6, IL-6), nitric oxide (NO) and prostaglandin E2 (PGE2)
  • cytokines such as -6 (interleukin-6, IL-6), nitric oxide (NO) and prostaglandin E2 (PGE2) are well known as major inflammatory agents.
  • 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 substance that induces more effective termination of inflammation, in order to solve the conventional problems as described above, has produced a novel compound showing a BLT2 inhibitory activity, including the compound It was the first to develop an inflammatory disease treatment.
  • the present invention has been made to solve the above problems, the present inventors have confirmed the therapeutic effect of the inflammatory disease of the novel compound showing the BLT2 inhibitory activity and completed the present invention based on this.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, comprising the novel compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a novel compound or a pharmaceutically acceptable salt thereof exhibiting BLT2 inhibitory activity.
  • 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 present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the novel compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the inflammatory disease may be selected from the group consisting of asthma, atherosclerosis, cancer, itching of the skin, rheumatoid arthritis and inflammatory bowel disease.
  • the composition may inhibit Leukotriene B4 receptor 2 (BLT2) activity.
  • BLT2 Leukotriene B4 receptor 2
  • It provides a method of treating an inflammatory disease comprising administering the pharmaceutical composition to a subject.
  • the present invention provides a therapeutic use of an inflammatory disease of a composition comprising the novel compound or a pharmaceutically acceptable salt thereof.
  • the present invention relates to a novel compound that exhibits Leukotriene B4 receptor 2 (BLT2) inhibitory activity and a pharmaceutical composition for preventing or treating an inflammatory disease comprising the same.
  • BLT2 Leukotriene B4 receptor 2
  • the present inventors have identified a novel compound that exhibits BTL2 inhibitory activity in order to solve in vivo instability and difficulty in mass production, which are problems of conventional inflammatory disease treatment substances, and has excellent cancer cell death enhancement and metastasis suppression effect and chemotaxis inhibition of the compound.
  • the effects, and anti-asthma effects have been confirmed experimentally, it is expected that it can be usefully used as a pharmaceutical composition for treating inflammatory diseases.
  • 1A to 1E show the results of confirming the growth inhibitory effect of the compound of the present invention in cells expressing BLT2 (CHO-BLT2).
  • Figures 2a and 2b is a result of confirming the chemotaxis inhibitory effect and IC 50 (50% inhibitory concentration) of the cells by the compound treatment of the present invention in CLT-expressing cells (CHO-BLT2 cells).
  • 3A and 3B 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).
  • Figures 4a and 4b is a result of confirming the effect of inhibiting the binding of LTB 4 and BLT2 by the compound treatment of the present invention in BLT2-expressing cells (CHO-BLT2 cells).
  • Figures 5a and 5b is the result of confirming the inhibitory effect of the production of active oxygen by the compound treatment of the present invention in MDA-MB-231 cells or MDA-MB-435 cells.
  • Figures 6a and 6b is a result of confirming the inhibitory effect of IL-8 (interleukin-8) expression by the compound treatment of the present invention in MDA-MB-231 cells or MDA-MB-435 cells.
  • Figures 7a and 7b is a result of confirming the cancer cell infiltration inhibition effect by the compound treatment of the present invention in MDA-MB-231 cells or MDA-MB-435 cells.
  • 13A to 13C show that asthma-induced mice reduced total cells and neutrophils introduced into the abdominal cavity of the mouse by the compound treatment of the present invention.
  • 14A and 14B are results confirming that the total cells and neutrophils introduced into the abdominal cavity of the mouse by the compound treatment of the present invention are reduced in asthma-induced mice.
  • the present inventors when treated with the novel compound prepared in Example, can significantly inhibit the growth of BLT2 expressing cells, thereby promoting cancer cell death, inhibiting cancer cell metastasis, inhibiting BLT2-dependent chemotaxis and The asthma effect, etc. were specifically confirmed and the present invention was completed based on this.
  • the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
  • 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 may be hydrogen or fluorine.
  • Preferred examples of the compound represented by Formula 1 according to the present invention are as follows:
  • the term "pharmaceutically acceptable” is suitable for use in contact with the tissue 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.
  • Acid addition salts according to the invention are dissolved in conventional methods, for example, the compounds represented by the formulas (1) to (4) in an excess of aqueous acid solution, and the salts are water miscible organic solvents such as methanol, ethanol, acetone or It can be prepared 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.
  • water miscible organic solvents such as methanol, ethanol, acetone or It can be prepared 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 novel compound exhibiting BLT2 inhibitory activity was prepared a novel compound exhibiting BLT2 inhibitory activity (see Examples 1 to 46), and confirmed the growth inhibition of BLT2 expressing cells by the novel compound treatment (see Experimental Example 2). In addition, it was confirmed that chemotaxis of BLT2 expressing cells can be suppressed (see Experimental Example 3).
  • the compound was used to confirm the inhibitory effect of LTB 4 and BLT2 binding (see Experimental Example 4), inhibited intracellular free radicals, inhibited IL-8 expression, inhibited cancer cell infiltration and inhibited metastasis of cancer cells (Experimental Example). 5), the effect of reducing airway hypersensitivity, inhibiting IL-4 production, and inhibiting immune cell influx into the mouse abdominal cavity was specifically confirmed in asthma-induced mice (see Experimental Example 6). It was confirmed that it can be used.
  • the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • prevention means any action that inhibits or delays the development of an inflammatory disease by administration of a pharmaceutical composition according to the present invention.
  • treatment means any action that improves or advantageously alters the symptoms of an inflammatory disease by administration of a pharmaceutical composition according to the present invention.
  • the inflammatory disease is due to overexpression of Leukotriene B4 receptor 2 (BLT2), and may be at least one selected from asthma, atherosclerosis, cancer, skin itch, rheumatoid arthritis and inflammatory bowel disease, but is limited thereto. It is not.
  • BLT2 Leukotriene B4 receptor 2
  • all BLT2-associated inflammatory diseases known in the art are considered to be included in inflammatory diseases that can be prevented or treated with a compound having the structure of Formula 1 of the present invention.
  • the cancer may be any cancer caused by overexpression of BLT2 or the oncogene Ras.
  • the cancer may be selected from the group consisting of bladder cancer, prostate cancer, pancreatic cancer, breast cancer, brain tumor, skin cancer, and liver cancer, but is not limited thereto.
  • BLT2 Leukotriene B4 receptor 2
  • LTB 4 is one of the group GPCR (G protein-coupled receptor)
  • GPCR G protein-coupled receptor
  • the composition of the present invention can prevent or treat inflammatory diseases by inhibiting cell growth by BLT2. More specifically, the inhibition of the production of ROS induced by BLT2 activity may inhibit LTB 4 -induced chemotaxis.
  • 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 kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day.
  • the dose may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.
  • the present invention also provides a method of treating an inflammatory 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. .
  • Step 1 N -Phenylpentaneamide ( N -phenylpentanamide)
  • Step 2 tert -Butyl 4- (4- Bromobenzoyl Piperazine-1- Carboxylate ( tert -butyl Preparation of 4- (4-bromobenzoyl) piperazine-1-carboxylate)
  • Isopropylethylamine (DIPEA) (2.34 ml, 13.44 mmol) was added and stirred at room temperature for 15 hours.
  • the reaction solution was concentrated under reduced pressure and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated under reduced pressure.
  • Step 3 tert -Butyl 4- (4- (3- Hydroxyprop -One- Inil ) Benzoyl Piperazine-1- Carboxylate ( tert -butyl 4- (4- (3- hydroxyprop -One- ynyl ) benzoyl ) piperazine -1-carboxylate)
  • Triethyl tert -butyl 4- (4-bromobenzoyl) piperazine-1-carboxylate (1.00 g, 2.71 mmol) and propagyl alcohol (0.32 ml, 5.42 mmol) obtained in step 2 were obtained. Dissolved in amine (12 ml) and stirred for 5 minutes. Bis (triphenylphosphine) palladium (II) dichloride (190 mg, 0.271 mmol) and copper iodide (I) (52 mg, 0.271 mmol) were added to the mixture, which was heated and refluxed at 60 ° C. for 17 hours. .
  • the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated under reduced pressure.
  • Step 4 tert -Butyl 4- (4- (3- ( Methylsulfonyloxy ) Prof -One- Inil ) Benzoyl Piperazine-1-carboxylate ( tert -butyl 4- (4- (3- ( methylsulfonyloxy ) prop-1-ynyl) benzoyl) piperazine-1-carboxylate)
  • Step 5 tert - Butyl 4 -(4- (3- ( N - Phenylpentaneamido ) Prof -One- Inil ) Benzoyl Piperazine-1-carboxylate ( tert -butyl 4- (4- (3- ( N - phenylpentanamido ) prop-1-ynyl) benzoyl) piperazine-1-carboxylate)
  • the reaction solution was concentrated under reduced pressure and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated under reduced pressure.
  • Piperazine-1-carboxylate ( tert- butyl 4- (4- (3- ( N- phenylpentanamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate) was obtained (382 mg, 73% yield).
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (33.3 mg, 0.072 mmol) obtained in Example 2 and potassium hydroxide ( KOH) (9.09 mg, 0.108 mmol) was dissolved in N , N -dimethylformamide (DMF) (1 ml) and stirred at room temperature for 5 minutes. Iodomethane (9 ⁇ l, 0.144 mmol) was added to the mixture, followed by stirring at the same temperature for 17 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was diluted with dichloromethane and washed with water and brine.
  • KOH potassium hydroxide
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (24.8 mg, 0.061 mmol) obtained in Example 3 and potassium hydroxide ( 8.62 mg, 0.154 mmol) was dissolved in N , N -dimethylformamide (DMF) (1 ml) and stirred at room temperature for 5 minutes. Iodoethane (20 ⁇ l, 0.246 mmol) was added to the mixture and stirred for 17 hours at the same temperature. The reaction solution was concentrated under reduced pressure, and the obtained residue was diluted with dichloromethane and washed with water and brine.
  • DMF N -dimethylformamide
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (106 mg, 0.263 mmol) and sodium hydrogencarbonate obtained in Example 2
  • N , N -dimethylformamide (DMF) (2 ml) was added thereto and stirred for 1 hour.
  • 2-iodopropane (30 ⁇ l, 0.316 mmol) was added to the mixture, ice was removed, and the mixture was heated and refluxed at 60 ° C. for 24 hours.
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (56 mg, 0.139 mmol) obtained in Example 2 and potassium carbonate ( 77 mg, 0.556 mmol) was dissolved in acetonitrile (3 ml) and stirred at room temperature for 5 minutes. 2-bromoethanol (99 ⁇ l, 1.39 mmol) was added to the mixture, and the mixture was heated and refluxed at 60 ° C. for 17 hours. The reaction solution was cooled to room temperature, filtered to remove solids, and then concentrated under reduced pressure.
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (50 mg, 0.124 mmol) obtained in Example 2 and potassium carbonate ( 51 mg, 0.372 mmol) was dissolved in N , N -dimethylformamide (DMF) (2 ml) and then stirred at room temperature for 5 minutes.
  • Cyclopropylmethyl bromide (15 ⁇ l, 0.145 mmol) was added to the mixture, heated at 80 ° C., refluxed and stirred for 4 hours. The reaction solution was cooled to room temperature, filtered to remove solids, and then concentrated under reduced pressure.
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (50 mg, 0.124 mmol) obtained in Example 2 and iodocyclo
  • N - (3- (4- ( 4-cyclohexylpiperazine-1-carbonyl) phenyl) prop-2-ynyl) - N -phenylpentanamide was obtained (20 mg, 33% yield ).
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (50 mg, 0.124 mmol) and bromomethyl obtained in Example 2 above Using cyclohexane (20 ⁇ l, 0.145 mmol) in the same manner as in Example 7, the final product, N- (3- (4- (4- (cyclohexylmethyl) piperazin-1-carbonyl) phenyl) prop profile-2-ynyl) - N - phenyl pentane amide (N - (3- (4- ( 4- (cyclohexylmethyl) piperazine-1-carbonyl) phenyl) prop-2-ynyl) - N -phenylpentanamide a) to give ( 35 mg, 56% yield).
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (50 mg, 0.124 mmol) obtained in Example 2 and potassium carbonate ( 51 mg, 0.372 mmol) was dissolved in N , N -dimethylformamide (DMF) (2 ml) and then stirred at room temperature for 5 minutes. Propargyl bromide (12 ⁇ l, 0.145 mmol) was added to the mixture and stirred at room temperature for 17 hours.
  • N -phenyl- N- (3- (4- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide 50 mg, 0.124 mmol
  • trimethylsilyl cya obtained in Example 2 above
  • Nide 49 ⁇ l, 0.372 mmol
  • acetonitrile 2 ml
  • Sodium hypochlorite 43 ⁇ l, 0.620 mmol
  • the reaction solution was cooled to room temperature, filtered to remove solids, and then concentrated under reduced pressure.
  • Step 1 N -(3- Fluorophenyl ) Pentaneamide ( N -(3- fluorophenyl ) pentanamide Manufacturing
  • Step 2 tert - Butyl 4- (4- (3- ( N -(3- Fluorophenyl ) Pentanamido ) Prof -One- Inil ) Benzoyl) piperazine-1-carboxylate ( tert -butyl 4- (4- (3- ( N Preparation of-(3-fluorophenyl) pentanamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate)
  • N- (3-fluorophenyl) pentaneamide (101 mg, 0.519 mmol) obtained in step 1 and tert -butyl 4- (4- (3- (methylsulfonyl) obtained in step 4 of Example 1 Oxy) prop-1-ynyl) benzoyl) piperazine-1-carboxylate (329 mg, 0.779 mmol) in the same manner as in step 5 of Example 1 above, the final product tert - butyl 4- (4- (3- ( N- (3-fluorophenyl) pentaneamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate ( tert -butyl 4- (4- (3- ( N- (3 -fluorophenyl) pentanamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate) was obtained (197 mg, 73% yield).
  • Example 14 A N obtained in Example 14 - (3-fluorophenyl) - N - (3- (4- (piperazine-1-carbonyl) phenyl) prop-2-ynyl), pentane amide (46 mg, 0.109 mmol) and 2-iodopropane (0.375 ml, 3.77 mmol) to give the final product N- (3-fluorophenyl) -N- (3- (4- (4-isopropylpipepe) piperazine-1-carbonyl) phenyl) prop-2-ynyl) pentane amide (N - (3-fluorophenyl) - N - (3- (4- (4-isopropylpiperazine-1-carbonyl) prop-2- phenyl) ynyl) pentanamide) was obtained (20 mg, 40% yield).
  • Step 1 N - (4-fluorophenyl) pentaneamide ( N -(4-fluorophenyl) pentanamide Manufacturing
  • Step 2 tert -Butyl 4- (4- (3- ( N -(4- Fluorophenyl ) Pentanamido ) Prof -One- Inil ) Benzoyl) piperazine-1-carboxylate ( tert -butyl 4- (4- (3- ( N Preparation of-(4-fluorophenyl) pentanamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate)
  • Example 17 The embodiment one N obtained in Example 17 - (4-fluorophenyl) - N - (3- (4- (piperazine-1-carbonyl) phenyl) prop-2-ynyl) pentane amide (1.06 g, 2.51 mmol) and 2-iodopropane (0.375 ml, 3.77 mmol) in the same manner as in Example 15 above, where the final product was N- (4-fluorophenyl) -N- (3- ( 4- (4-isopropyl-piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentane amide (N - (4-fluorophenyl) - N - (3- (4- (4-isopropylpiperazine-1- carbonyl) phenyl) prop-2-ynyl) pentanamide) was obtained (465 mg, 40% yield).
  • Step 1 (4-bromophenyl) (morpholino) methanone ((4-bromophenyl) (morpholino) methanone)
  • Step 2 (4- (3-hydroxyprop-1-ynyl) phenyl) (morpholino) methanone)
  • Example 4 was prepared using (4-bromophenyl) (morpholino) methanone (455 mg, 1.68 mmol) and propagyl alcohol (0.196 ml, 3.36 mmol) obtained in step 1. (4- (3-hydroxyprop-1-ynyl) phenyl) (morpholino) methanone ((4- (3-hydroxyprop-1-ynyl) phenyl) (morpholino) methanone) was obtained (371 mg, yield 90%).
  • step 3 3 -(4-( Morpholine -4- Carbonyl ) Phenyl) Prof -2- Inil Methanesulfonate Preparation of (3- (4- (morpholine-4-carbonyl) phenyl) prop-2-ynyl methanesulfonate)
  • Step 4 N -(3- (4- ( Morpholine -4- Carbonyl ) Phenyl) Prof -2- Inil )- N - Phenylpentane amy De ( N -(3- (4- (morpholine-4-carbonyl) phenyl) prop-2-ynyl)- N -phenylpentanamide)
  • N -phenylpentaneamide (143 mg, 0.81 mmol) obtained in step 1 of Example 1 and 3- (4- (morpholine-4-carbonyl) phenyl) prop-2- obtained in step 3 above.
  • Example 20 N -Phenyl- N -(3- (4- (piperidine-1- Carbonyl ) Phenyl) Prof -2- Inil ) Pentaneamide ( N -phenyl- N -(3- (4- ( piperidine Preparation of -1-carbonyl) phenyl) prop-2-ynyl) pentanamide) (LMT-683)
  • Step 1 (4-bromophenyl) (piperidin-1-yl) methanone ((4- bromophenyl ) ( piperidin -1-yl) methanone))
  • Step 2 (4- (3- Hydroxyprop -One- Inil ) Phenyl) pyridin-1-yl) Metanon Preparation of ((4- (3-hydroxyprop-1-ynyl) phenyl) (piperidin-1-yl) methanone)
  • step 3 3 -(4- (pyridine-1- Carbonyl ) Phenyl) Prof -2- Inil Methanesulfonate Preparation of (3- (4- (piperidine-1-carbonyl) phenyl) prop-2-ynyl methanesulfonate)
  • Step 4 N -Phenyl- N -(3- (4- (piperidine-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide ( N -phenyl- N Preparation of-(3- (4- (piperidine-1-carbonyl) phenyl) prop-2-ynyl) pentanamide)
  • N -phenylpentanamide (146 mg, 0.82 mmol) obtained in step 1 of Example 1 and 3- (4- (pyridine-1-carbonyl) phenyl) prop-2-ynyl obtained in step 3 above.
  • Prop-2-ynyl) pentaneamide N- phenyl- N- (3- (4- (piperidine-1-carbonyl) phenyl) prop-2-ynyl) pentanamide
  • step 1 4 - Bromo - N , N - Diethylbenzamide (4- bromo - N , N -diethylbenzamide)
  • Step 2 N , N - Diethyl -4- (3-hydroxyprop-1-ynyl) benzamide ( N , N Preparation of -diethyl-4- (3-hydroxyprop-1-ynyl) benzamide)
  • N , N -diethyl-4- (3-hydroxyprop-1-ynyl) benzamide was obtained.
  • N , N- diethyl-4- (3-hydroxyprop-1-ynyl) benzamide (425 mg, 67% yield).
  • step 3 3 -(4-( Diethylcarbamoyl ) Phenyl) Prof -2- Inil Methanesulfonate Preparation of (3- (4- (diethylcarbamoyl) phenyl) prop-2-ynyl methanesulfonate)
  • N -diethyl-4- (3-hydroxyprop-1-ynyl) benzamide (425 mg, 1.84 mmol) and methanesulfonyl chloride (0.16 ml, 2.02 mmol) obtained in step 2 above.
  • 3- (4- (diethylcarbamoyl) phenyl) prop-2-ynyl methanesulfonate (3- (4- (diethylcarbamoyl) phenyl) prop-2-ynyl in the same manner as in Step 4 of Example 1 above methanesulfonate) was obtained (483 mg, 85% yield).
  • Step 4 N , N - Diethyl -4- (3- ( N -Phenylpentaneamido) prop-1-ynyl) benzamy De ( N , N -diethyl-4- (3- ( N -phenylpentanamido) prop-1-ynyl) benzamide)
  • N -phenylpentanamide (94 mg, 0.530 mmol) obtained in step 1 of Example 1 and 3- (4- (diethylcarbamoyl) phenyl) prop-2-ynyl obtained in step 3 above.
  • methanesulfonate (246 mg, 0.795 mmol) in the same manner as in Step 5 of Example 1, the final product, N , N -diethyl-4- (3- ( N -phenylpentaneamido) prop- 1-ynyl) benzamide ( N , N- diethyl-4- (3- ( N- phenylpentanamido) prop-1-ynyl) benzamide) was obtained (81 mg, 39% yield).
  • Example 22 N -Phenyl- N -(3- (3- (piperazin-1- Carbonyl ) Phenyl) Prof -2- Inil ) Pentaneamide ( N -phenyl- N -(3- (3- ( piperazine Preparation of -1-carbonyl) phenyl) prop-2-ynyl) pentanamide) (LMT-837)
  • Step 1 tert -Butyl 4- (3- Bromobenzoyl Piperazine-1- Carboxylate ( tert -butyl Preparation of 4- (3-bromobenzoyl) piperazine-1-carboxylate)
  • Step 2 tert -Butyl 4- (3- (3- Hydroxyprop -One- Inil ) Benzoyl Piperazine-1- Carboxylate ( tert -butyl 4- (3- (3- hydroxyprop -One- ynyl ) benzoyl ) piperazine -1-carboxylate)
  • Step 3 tert -Butyl 4- (3- (3- ( Methylsulfonyloxy ) Prof -One- Inil ) Benzoyl Piperazine-1-carboxylate ( tert -butyl 4- (3- (3- ( methylsulfonyloxy ) prop-1-ynyl) benzoyl) piperazine-1-carboxylate)
  • Step 4 tert -Butyl 4- (3- (3- ( N - Phenylpentaneamido ) Prof -One- Inil ) Benzoyl Piperazine-1-carboxylate ( tert -butyl 4- (3- (3- ( N - phenylpentanamido ) prop-1-ynyl) benzoyl) piperazine-1-carboxylate)
  • N -phenylpentaneamide (275 mg, 1.55 mmol) obtained in step 1 of Example 1 and tert -butyl 4- (3- (3- (methylsulfonyloxy) prop-1 obtained in step 3) -Inyl) benzoyl) piperazine-1-carboxylate (982 mg, 2.33 mmol) in the same manner as in Step 5 of Example 1, tert -butyl 4- (3- (3- ( N- Phenylpentaneamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate ( tert -butyl 4- (3- (3- ( N -phenylpentanamido) prop-1-ynyl) benzoyl) piperazine-1- carboxylate) (323 mg, 70% yield).
  • Step 5 N -Phenyl- N -(3- (3- (piperazin-1- Carbonyl ) Phenyl) Prof -2- Inil ) Pentaneamide ( N -phenyl- N -(3- (3- ( piperazine -1-carbonyl) phenyl) prop-2- ynyl ) pentanamide ) (LMT-837)
  • N -phenyl- N- (3- (3- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (50 mg, 0.124 mmol) obtained in Example 22 and formaldehyde ( 37% in H 2 O) (1.5 ml) was dissolved in formic acid (2.0 ml), heated at 100 ° C., refluxed and stirred for 4 hours. The reaction solution was concentrated under reduced pressure, and then titrated by addition of aqueous sodium hydroxide solution (2.0 M). Thereafter, the mixture was diluted with dichloromethane and washed with water and brine.
  • Example 24 N -(3- (3- (4- Isopropyl piperazine -One- Carbonyl ) Phenyl) Prof -2- Inil )- N -Phenylpentaneamide ( N -(3- (3- (4- isopropylpiperazine -1-carbonyl) phenyl) prop-2-ynyl)- N -phenylpentanamide) (LMT-842)
  • N -phenyl- N- (3- (3- (piperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide (152 mg, 0.36 mmol) obtained in Example 22 and 2-Io
  • Example 25 tert -Butyl-4- (3- (3- ( N -(4- Fluorophenyl ) Pentanamido ) Prof -One- Inil ) Benzoyl) piperazine-1-carboxylate ( tert -butyl 4- (3- (3- ( N -(4-fluorophenyl) pentanamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate) ( LMT -887)
  • N- (4-fluorophenyl) pentaneamide (275 mg, 1.41 mmol) obtained in Step 1 of Example 16 and tert -butyl 4- (3- (3- (Methylsulfonyloxy) prop-1-ynyl) benzoyl) piperazine-1-carboxylate (894 mg, 2.12 mmol) in the same manner as in Step 5 of Example 1 above, the final product tert -butyl- 4- (3- (3- ( N- (4-fluorophenyl) pentaneamido) prop-1-ynyl) benzoyl) piperazin-1-carboxylate ( tert -butyl 4- (3- (3- ( N- (4-fluorophenyl) pentanamido) prop-1-ynyl) benzoyl) piperazine-1-carboxylate) was obtained (625 mg, 85% yield).
  • Example 26 N -(4- Fluorophenyl )- N -(3- (3- (piperazin-1- Carbonyl ) Phenyl) Prof -2-ynyl) pentaneamide ( N -(4- fluorophenyl )- N -(3- (3- ( piperazine Preparation of -1-carbonyl) phenyl) prop-2-ynyl) pentanamide) (LMT-888)
  • Example 27 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) ( LMT - 889) Produce
  • Example 26 A N obtained in Example 26 - (4-fluorophenyl) - N - (3- (3- (piperazine-1-carbonyl) phenyl) prop-2-ynyl) pentane amide (150 mg, 0.356 mmol) and 2-iodopropane (53 ⁇ l, 0.534 mmol) in the same manner as in Example 5, where the final product, N- (4-fluorophenyl) -N- (3- ( 3- (4-isopropylpiperazin-1-carbonyl) phenyl) prop-2-ynyl) pentaneamide was obtained (66 mg, 40% yield).
  • Step 1 N -(Prop-2-ynyl) aniline ( N Preparation of-(prop-2-ynyl) aniline)
  • Step 2 N -Phenyl- N - (Prop-2-ynyl) pentaneamide ( N -phenyl- N Preparation of-(prop-2-ynyl) pentanamide)
  • Step 3 N -(3- (4- Hydroxyphenyl ) Prof -2- Inil )- N - Phenylpentaneamide ( N -(3- (4-hydroxyphenyl) prop-2-ynyl)- N -phenylpentanamide)
  • Triethyl N -phenyl- N- (prop-2-ynyl) pentaneamide (550 mg, 2.55 mmol) and 4-iodophenol (422 mg, 1.92 mmol) obtained in step 2 were obtained. Dissolved in amine (15 ml) and stirred for 5 minutes. Bis (triphenylphosphine) palladium (II) dichloride (89 mg, 0128 mmol) and copper iodide (I) (49 mg, 0.255 mmol) were added to the mixture, heated at 50 ° C., refluxed and stirred for 5 hours. It was.
  • the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated under reduced pressure.
  • Step 1 ethyl 2- (4- (3- ( N - Phenylpentaneamido ) Prof -One- Inil ) Phenoxy Acetate (ethyl (2- (4- (3- ( N -phenylpentanamido) prop-1-ynyl) phenoxy) acetate)
  • N- (3- (4-hydroxyphenyl) prop-2-ynyl) -N -phenylpentanamide (590 mg, 1.92 mmol) and potassium carbonate (796 mg, 5.76 mmol) obtained in Example 28 were prepared. It was dissolved in acetonitrile (15 ml) and stirred for 30 minutes. Ethyl bromoacetate (0.23 ml, 2.11 mmol) was added to the mixture, followed by stirring at room temperature for 17 hours. The reaction solution was filtered to remove solids, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine.
  • step 2 2 -(4- (3- ( N - Phenylpentaneamido ) Prof -One- Inil ) Phenoxy Acetic acid (2- (4- (3- ( N -phenylpentanamido) prop-1-ynyl) phenoxy) acetic acid)
  • step 1 The ethyl 2- (4- (3- ( N -phenylpentaneamido) prop-1-ynyl) phenoxy) acetate (530 mg, 1.35 mmol) obtained in step 1 was dissolved in ethanol (15 ml). , And stirred at room temperature for 5 minutes. To the mixture was added 2N sodium hydroxide (NaOH) (0.50 ml), heated at 80 ° C., refluxed and stirred for 3 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine.
  • NaOH sodium hydroxide
  • Example 30 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 -phenyl- N- (prop-2-ynyl) pentaneamide and tert -butyl 4- (5-bromopicolinoyl) piperazine-1-carboxylate (86 mg) obtained in step 2 of Example 28 above , 0.4 mmol) using the same method as in Step 3 of Example 28, the final product tert -butyl 4- (5- (3-(( N -phenylpentaneamido) prop-1-yn-1 -Yl) picolinoyl) piperazine-1-carboxylate (tert-butyl 4- (5- (3- (N-phenylpentanamido) prop-1-yn-1-yl) picolinoyl) piperazine-1-carboxylate) Obtained (36 mg, 35% yield).
  • Example 31 N -Phenyl- N -(3- (6- (piperazin-1- Carbonyl Pyridin-3-yl) Prof -2-yn-1-yl) pentaneamide ( N -phenyl-N- (3- (6- ( piperazine -1-carbonyl) pyridin -3- yl ) prop-2-yn-1-yl) pentanamide) (LMT-835)
  • Example 32 N -(3- (6- Isopropyl piperazine -One- Carbonyl Pyridin-3-yl) Prof -2-yn-1 yl) pentaneamide (N- (3- (6- (4- isopropylpiperazine -1-carbonyl) pyridin Preparation of -3-yl) prop-2-yn-1-yl) -N-phenylpentanamide) (LMT-836)
  • 0.14 mmol) and sodium hydrogencarbonate (27 mg, 0.316 mmol) were cooled in ice, and N , N -dimethylformamide (DMF) (2 ml) was added thereto, followed by stirring for 1 hour.
  • 2-iodopropane (30 ⁇ l, 0.316 mmol) was added to the mixture, ice was removed, and the mixture was heated and refluxed at 60 ° C. for 24 hours.
  • the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated under reduced pressure.
  • step 1 4 - Bromo - N, N - Diethylbenzamide (4- bromo - N, N - diethylbenzamide Manufacturing
  • 4-Bromobenzoic acid (4-gromobenzoic acid) (5.00 g, 24.9 mmol) was dissolved in N, N -dimethylformamide (100.00 ml) and then diisopropylamine (13 ml, 74.6 mmol) was added. To the mixture was added 1-hydroxybenzotriazole hydrate (7.15 mg, 37.30 mmol) and 1-ethyl-3- (3-dibutylaminopropyl) calvoimide hydrochloride (1-Ethyl -3- (3-dimethylaminopropyl) carbodiimide hydrochloride) (5.04 mg, 37.30 mmol) was added and stirred for 5 minutes.
  • Step 2 N, N - Diethyl -4- (3- Hydroxyprop -One- Inil ) Benzamide ( N, N Preparation of -diethyl-4- (3-hydroxyprop-1-ynyl) benzamide)
  • the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated under reduced pressure.
  • step 3 3 -(4-( N, N - Diethylcarbamoyl ) Phenyl) Prof -2- Inil Methanesulfonate Preparation of (3- (4- (diethylcarbamoyl) phenyl) prop-2-yn-1-yl methanesulfonate)
  • N, N -diethyl-4- (3-hydroxyprop-1-ynyl) benzamide (5.16 mg, 22.3 mmol) was dissolved in dichloromethane (100 ml) and cooled on ice. Triethylamine (4.80 ml, 34.4 mmol) was added to the mixture, which was then stirred for 5 minutes. Methanesulfonyl chloride (1.95 ml, 25.2 mmol) was added at the same temperature, ice was removed, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure and the obtained residue was diluted with ethyl acetate and washed with water and brine.
  • Step 4 N, N - Diethyl -4- (3- (N- (3- Fluorophenyl Pentaamido) Prof -1-yn-1-yl) benzamide ( N, N -diethyl-4- (3- ( N -(3- fluorophenyl ) pentanamido prop-1- yn -1-yl) benzamide)
  • N- (3-fluorophenyl) pentaneamide 200 mg, 1.02 mmol was dissolved in tetrahydrofuran (10.00 ml) and cooled on ice. Sodium hydride (73 mg, 3.06 mmol) was added to the mixture, followed by stirring for 1 hour. At the same temperature, 3- (4- ( N, N -diethylcarbamoyl) phenyl) prop-2-ynyl methanesulfonate (475 mg, 1.54 mmol) obtained in step 3 was added thereto, and the mixture was allowed to stand at room temperature. Stir for 4 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was diluted with dichloromethane and washed with water and brine.
  • Example 34 N, N -Diethyl-4- (3- ( N -(4-fluorophenyl) pentamido) prop-1-yn-1-yl) benzamide ( N, N -diethyl-4- (3- ( N -(4-fluorophenyl) pentanamido) prop-1-yn-1-yl) benzamide) (LMT-927)
  • N- (4-fluorophenyl) pentaneamide 200 mg, 1.02 mmol
  • 3- (4- ( N, N -diethylcarbamoyl) phenyl) prop-2-ynyl methanesulfonate (3- (4 -(diethylcarbamoyl) phenyl) prop-2-yn-1-yl methanesulfonate) using N, N-diethyl-4- (3- (N- (4- Fluophenyl) pentaamido) prop-1-yn-1-yl) benzamide
  • N, N -diethyl-4- (3- ( N- (4-fluorophenyl) pentanamido) prop-1-yn-1 -yl) benzamide was obtained (291.7 mg, 70% yield).
  • Example 35 N -(3- (4- ( N, N - Diethylsulfamoyl ) Phenyl) Prof -2- Inil )- N - Phenylpentamide ( N -(3- (4- ( N, N - diethylsulfamoyl ) phenyl) prop-2- ynyl )- N -phenylpentanamide) (LMT-946)
  • step 1 4 - Bromo - N, N - Diethylbenzenesulfonamide (4- bromo - N, N -diethylbenzenesulfonamide)
  • Step 2 N, N - Diethyl -4- (3- Hydroxyprop -One- Inil ) Benzenesulfonamide ( N, N Preparation of -diethyl-4- (3-hydroxyprop-1-ynyl) benzenesulfonamide)
  • the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered and concentrated under reduced pressure.
  • step 3 3 -(4-( N, N - Diethylsulfamoyl ) Phenyl) Prof -2- Inil Methanesulfonate (3- (4- ( N, N -diethylsulfamoyl) phenyl) prop-2-ynyl methanesulfonate)
  • N, N -diethyl-4- (3-hydroxyprop-1-ynyl) benzenesulfonamide (273.00 mg, 1.02 mmol) obtained in step 2 was dissolved in dichloromethane (10 ml), and then dried on ice. Cooled. Triethylamine (0.21 ml, 1.53 mmol) was added to the mixture, which was then stirred for 5 minutes. Methanesulfonyl chloride (0.09 ml, 1.12 mmol) was added at the same temperature, ice was removed, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure and the obtained residue was diluted with ethyl acetate and washed with water and brine.
  • step 4 4 -(3- Bromoprop -One- Inil )- N, N - Diethylbenzenesulfonamide (4- (3-bromoprop-1-ynyl)- N, N -diethylbenzenesulfonamide)
  • step 3 3- (4- ( N, N -diethylsulfamoyl) phenyl) prop-2-ynyl methanesulfonate (260.00 mg, 0.75 mmol) obtained in step 3 was added to tetrahydrofuran (20.00 ml). It was dissolved in and cooled on ice. Lithium bromide (196.28 mg, 2.26 mmol) was added to the mixture at the same temperature, followed by stirring for 4 hours. The reaction solution was concentrated under reduced pressure and the obtained residue was diluted with ethyl acetate and washed with water and brine. The organic solvent layers were combined, the water was removed with anhydrous magnesium sulfate (MgSO 4 ), filtered, and concentrated under reduced pressure.
  • MgSO 4 anhydrous magnesium sulfate
  • the concentrate was purified by a filter to afford 4- (3-bromoprop-1-ynyl) -N, N -diethylbenzenesulfonamide (4- (3-bromoprop-1-ynyl) -N, N -diethylbenzenesulfonamide). Obtained (240.00 mg, 97%)
  • Step 5 N, N - Diethyl -4- (3- ( Phenylamino ) Prof -One- Inil ) Benzenesulfonamide ( N, N Preparation of -diethyl-4- (3- (phenylamino) prop-1-ynyl) benzenesulfonamide)
  • Step 6 N -(3- (4- ( N, N - Diethylsulfamoyl ) Phenyl) Prof -2- Inil )- N - Phenylpentamide ( N -(3- (4- ( N, N - diethylsulfamoyl ) phenyl) prop-2- ynyl )- N -phenylpentanamide)
  • N, N -diethyl-4- (3- (phenylamino) prop-1-ynyl) benzenesulfonamide (174.00 mg, 0.51 mmol) obtained in step 5 was dissolved in dichloromethane (15.00 ml). Cooled on ice. Triethylamine (0.14 ml, 1.02 mmol) was added to the mixture, which was then stirred for 5 minutes. Valeroyl chloride (0.06 ml, 0.53 mmol) was added at the same temperature, ice was removed, and the mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was diluted with dichloromethane and washed with water and brine.
  • Example 36 N -(3- (4- ( N - Isopropyl sulfamoyl ) Phenyl) Prof -2- Inil )- N - Phenylpentamide ( N -(3- (4- ( N - isopropylsulfamoyl ) phenyl) prop-2- ynyl )- N -phenylpentanamide) (LMT-947)
  • step 1 4 - Bromo - N - Isopropylbenzenesulfonamide (4- bromo - N -isopropylbenzenesulfonamide)
  • step 2 4 -(3- Hydroxyprop -One- Inil )- N - Isopropylbenzenesulfonamide (4- (3-hydroxyprop-1-ynyl)- N -isopropylbenzenesulfonamide)
  • 4- (3-hydroxyprop-1-ynyl) -N -isopropylbenzenesulfonamide (4- (3-hydroxyprop-1-ynyl) -N- isopropylbenzenesulfonamide) was obtained (420.00 mg, 92% yield).
  • step 3 3 -(4-( N - Isopropyl sulfamoyl ) Phenyl) Prof -2- Inil Methanesulfonate (3- (4- ( N -isopropylsulfamoyl) phenyl) prop-2-ynyl methanesulfonate)
  • step 2 4- (3-hydroxyprop-1-ynyl) -N -isopropylbenzenesulfonamide (410.00 mg, 1.62 mmol) and methanesulfonyl chloride (0.14 ml, 1.78 mmol) obtained in step 2;
  • step 3 of Example 35 using 3- (4- ( N -isopropylsulfamoyl) phenyl) prop-2-ynyl methanesulfonate (3- (4- ( N- isopropylsulfamoyl) phenyl) prop-2-ynyl methanesulfonate) was obtained (330.00 mg, 61% yield).
  • step 4 4 -(3- Bromoprop -One- Inil )- N - Isopropylbenzenesulfonamide (4- (3-bromoprop-1-ynyl)- N -isopropylbenzenesulfonamide)
  • Step 5 N - Isopropyl -4- (3- ( Phenylamino ) Prof -One- Inil ) Benzenesulfonamide ( N Preparation of -isopropyl-4- (3- (phenylamino) prop-1-ynyl) benzenesulfonamide)
  • Step 6 N -(3- (4- ( N - Isopropyl sulfamoyl ) Phenyl) Prof -2- Inil )- N - Phenylpentaneamide ( N -(3- (4- ( N - isopropylsulfamoyl ) phenyl) prop-2- ynyl )- N -phenylpentanamide)
  • N -isopropyl-4- (3- (phenylamino) prop-1-ynyl) benzenesulfonamide (150.00 mg, 0.46 mmol) and valeroyl chloride (0.06 ml, obtained in step 2) 0.48 mmol) to the same method as in step 6 of example 35 using the N - (3- (4- (N - isopropyl-sulfamoyl) phenyl) prop-2-ynyl) - N - phenyl pentane amide (N -(3- (4- ( N- isopropylsulfamoyl) phenyl) prop-2-ynyl) -N- phenylpentanamide) was obtained (94.00 mg, 49.5% yield).
  • Step 2 N -Phenyl- N - (Pro-2yn-1-yl) pentaneamide ( N -phenyl- N -(prop-2- yn -1-yl) pentanamide)
  • N -phenylpentaneamide (19.10 g, 107.40 mmol) obtained in step 1 was dissolved in N, N -dimethylformamide (DMF) (100 ml), the reaction system was nitrogen-substituted, and sodium hydride (5.20). g, 214.80 mmol) was added below freezing and then stirred for 2 hours. Propagyl bromide (18.10 ml, 214.80 mmol) was added to the mixture, followed by stirring for 2 hours at -40 ° C. Water was added to the reaction solution below freezing, diluted with ethyl acetate, and washed with water and brine.
  • DMF N, N -dimethylformamide
  • Step 4 tert -Butyl 4- (3- ( N Phenylpentane amido) prop -1-yn-1-yl) Benzoa ( tert -butyl 4- (3- ( N -phenylpentanamido) prop-1-yn-1-yl) benzoate)
  • Example 38 4- (3- ( N -Phenylpentaneamido) prop-1-pin-1-yl) benzoic acid Preparation of (4- (3- (N-phenylpentanamido) prop-1-yn-1-yl) benzoic acid) (LMT-1013)
  • Example 38 4- (3- (N-phenylpentaneamido) prop-1-pin-1-yl) benzoic acid obtained in Example 38 (4- (3- (N-phenylpentanamido) prop-1-yn-1 -yl) benzoic acid) (80.00 mg, 0.24 mmol) was dissolved in N, N-dimethylformamide (DMF) (0.70 ml) and stirred in triethylamine (0.70 ml) for 1 hour.
  • DMF N, N-dimethylformamide
  • Example 38 4- (3- (N-phenylpentaneamido) prop-1-pin-1-yl) benzoic acid obtained in Example 38 (4- (3- (N-phenylpentanamido) prop-1-yn-1 -yl) benzoic acid) (80.00 mg, 0.24 mmol) using N, N-dimethylethane-1,2-diamine (0.04 ml, 0.36 mmol) N- (2- (diethylamino) ethyl) -4- (3- (N-phenylpentaneamido) prop-1-yn-1-yl) benzamide (N- (2- (dimethylamino) ethyl) -4- (3- (N-phenylpentanamido) prop-1-yn-1-yl) benzamide) was obtained (71.72 mg, 74% yield).
  • Example 41 Ethyl 2- (4- (3- (N- Phenylpentaneamido ) Prof -1-yn-1-yl) Benzamido Acetate (ethyl 2- (4- (3- (N-) phenylpentanamido prop-1- yn -1-yl) benzamido) acetate) (LMT-1014)
  • Example 38 4- (3- (N-phenylpentaneamido) prop-1-pin-1-yl) benzoic acid obtained in Example 38 (4- (3- (N-phenylpentanamido) prop-1-yn-1 -yl) benzoic acid) (150.00 mg, 0.45 mmol) using glycineethylester hydrochloride (93.70 mg, 0.67 mmol) in the same manner as in Example 39 to obtain ethyl 2- (4- (3- ( N-phenylpentaneamido) prop-1-yn-1-yl) benzamido) acetate (ethyl 2- (4- (3- (N-phenylpentanamido) prop-1-yn-1-yl) benzamido) acetate) was obtained (102.18 mg, 54% yield).
  • Ethyl 2- (4- (3- (N-phenylpentaneamido) prop-1-yn-1-yl) benzamido) acetate obtained in Example 41 (ethyl 2- (4- (3- (N-phenylpentanamido) prop-1-yn-1-yl) benzamido) acetate) (46.20 mg, 0.10 mmol) and a 2-mol aqueous solution of sodium hydroxide (0.07 ml, 0.14 mmol) were dissolved in methanol (0.1 ml). Stirred at room temperature for 30 minutes. The acidity of the reaction solution was increased using hydrochloric acid, diluted with ethyl acetate, and washed with water and brine.
  • Example 43 methyl 2- (4- (3- (N- Phenylpentaneamido ) Prof -1-yn-1-yl) Benzamido Propanoate (methyl 2- (4- (3- (N-) phenylpentanamido prop-1- yn -1-yl) benzamido) propanoate) (LMT-1018)
  • Example 38 4- (3- (N-phenylpentaneamido) prop-1-pin-1-yl) benzoic acid obtained in Example 38 (4- (3- (N-phenylpentanamido) prop-1-yn-1 L-alaninemethylester hydrochloride (83.70 mg, 0.60 mmol) in -yl) benzoic acid) (100.00 mg, 0.30 mmol) in the same manner as in Example 39.
  • Step 1 N- (3- Fluorophenyl ) Pentaneamide (N- (3- fluorophenyl ) pentanamide Manufacturing
  • N- (3-fluorophenyl) pentanamide (N- (3-fluorophenyl) pentanamide in the same manner as in Step 1 of Example 37 using 3-fluoroaniline (200.00 mg, 1.79 mmol) ) was obtained (349.00 mg, 99% yield).
  • Step 2 N- (3- FluoroPetyl ) -N- ( Prof -2- Inil ) Pentaneamide Preparation of (N- (3-fluorophenyl) -N- (prop-2-ynyl) pentanamide)
  • N- (3-fluorophenyl) pentaneamide 400.00 mg, 2.05 mmol
  • Potassium hydroxide 230.61 mg, 4.11 mmol
  • tetrabutylammonium iodide tetrabutyl ammonium Iodide
  • Tetrahydrofuran 20.00 ml
  • propargyl bromide (0.19 ml, 2.30 mmol) was added to the mixture, followed by stirring for 20 hours.
  • the reaction solution was concentrated under reduced pressure and the obtained residue was diluted with ethyl acetate and washed with water and brine.
  • Step 3 N- (3- Fluorophenyl ) -N- (3- (4- Hydroxyphenyl ) Prof -2- Inil ) Pentanah Amide (N- (3- fluorophenyl ) -N- (3- (4- hydroxyphenyl prop-2- ynyl ) pentanamide Manufacturing
  • N- (3-fluorofetyl) -N- (prop-2-ynyl) pentaneamide (270.00 mg, 1.16 mmol) and 4-iodophenol (127.60 mg, obtained in step 2) 0.58 mmol) with N- (3-fluorophenyl) -N- (3- (4-hydroxyphenyl) prop-2-ynyl) pentaneamide (N- (3-fluorophenyl) -N- (3 -(4-hydroxyphenyl) prop-2-ynyl) pentanamide) was obtained (128.00 mg, 67.8% yield).
  • Step 4 ethyl 2- (4- (3- (N- (3- Fluorophenyl ) Pentanamido ) Prof -One- Inil ) Phenoxy Acetate (ethyl 2- (4- (3- (N- (3- fluorophenyl ) pentanamido ) prop-1-ynyl) phenoxy) acetate)
  • step 5 2 -(4- (3- (N- (3- Fluorophenyl ) Pentanamido ) Prof -One- Inil ) Phenoxy ) Acetic acid Preparation of (2- (4- (3- (N- (3-fluorophenyl) pentanamido) prop-1-ynyl) phenoxy) acetic acid)
  • Sodium hydroxide 2M (0.30 ml) was added to the mixture, and the mixture was heated and refluxed at 80 ° C. for 3 hours.
  • the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate and washed with water and brine.
  • Step 1 N- (4- Fluorophenyl ) Pentaneamide (N- (4- fluorophenyl ) pentanamide Manufacturing
  • Step 2 N- (4- Fluorophenyl ) -N- ( Prof -2- Inil ) Pentaneamide Preparation of (N- (4-fluorophenyl) -N- (prop-2-ynyl) pentanamide)
  • Step 3 N- (4- Fluorophenyl ) -N- (3- (4- Hydroxyphenyl ) Prof -2- Inil ) Pentanah Amide (N- (4- fluorophenyl ) -N- (3- (4- hydroxyphenyl prop-2- ynyl ) pentanamide Manufacturing
  • N- (4-fluorophenyl) -N- (prop-2-ynyl) pentaneamide (300.00 mg, 1.29 mmol) and 4-iodophenol (140.80 mg, obtained in step 3)
  • N- (4-fluorophenyl) -N- (3- (4-hydroxyphenyl) prop-2-ynyl) pentaneamide N- (4-fluorophenyl) -N- (3 -(4-hydroxyphenyl) prop-2-ynyl) pentanamide
  • Step 4 ethyl 2- (4- (3- (N- (4- Fluorophenyl ) Pentanamido ) Prof -One- Inil ) Phenoxy Acetate (ethyl 2- (4- (3- (N- (4- fluorophenyl ) pentanamido ) prop-1-ynyl) phenoxy) acetate)
  • step 5 2 -(4- (3- (N- (4- Fluorophenyl ) Pentanamido ) Prof -One- Inil ) Phenoxy ) Acetic acid Preparation of (2- (4- (3- (N- (4-fluorophenyl) pentanamido) prop-1-ynyl) phenoxy) acetic acid)
  • 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.
  • the compounds of the present invention can inhibit BLT2-induced cell proliferation with very good efficiency, and the compound can be used as a pharmaceutical ingredient (BLT2-) as a therapeutic agent for the inhibition of anticancer, anti-asthma or other forms of BLT2-related inflammatory diseases. 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 into the upper wells at / 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 respectively as a positive control, LTB 4 ligand of BLT2 as a comparative control , (300 nM), LTB 4 , the ligand of BLT1 (10 nM) and LPA (lysophosphatidic acid; 100 nM) were used. Chemotaxis was quantified by counting cells on the lower side of the filter under an optical microscope (magnification, x200). Six fields were counted in each assay, each sample analyzed twice, and the assay was repeated three times.
  • IC 50 50% inhibitory concentration
  • LTB 4 which is a ligand of BLT2 in BLT2-expressing cells (CHO-BLT2 cells)
  • DMSO + DMSO +
  • DMSO- ethanol treated
  • the cell chemotaxis was increased by 2.4 times
  • LY255283 used as a positive control (10 ⁇ M) the ligand LTB Compared to the case of 4 treated, it was confirmed that the coinability of 90%.
  • 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 the treatment (DMSO +).
  • LTB 4 and BLT2 binding (ligand binding affinity) inhibition was analyzed by isotope tritium (H3) using the 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 proteins of the cell membrane and quantitate them at a concentration of 40 ⁇ g / 45 ⁇ L.
  • MDA-MB-231 and MDA-MB-453 were intended to confirm the inhibition of the production of free radicals and IL-8 expression in the cells according to the compound treatment of the present invention.
  • MDA-MB-231 cells breast cancer cells, were obtained from Korea Cell Line Bank (Seoul, Korea), and MDA-MB-435 cells were obtained from J.-H. Obtained from Lee (Asan Medical Center, Seoul, Korea). This was 37 ° C. in RPMI 1640 medium (Invitrogen) containing 10% FBS (fetal bovine serum; Life technologies, Inc.), 1% penicillin (50 units / mL), and antibiotic antimycotic solution (Life technologies, Inc.). , And cultured under the condition of 5% CO 2 .
  • FBS fetal bovine serum
  • penicillin 50 units / mL
  • antibiotic antimycotic solution Life technologies, Inc.
  • Intracellular reactive oxygen (H 2 O 2 ) following compound (LMT-696) treatment of the present invention was measured as a function of DCF fluorescence. Specifically, before the measurement of free radicals (ROS) 2 ⁇ 10 5 Cells were grown in 60-mm wells and incubated for 24 hours in RPMI 1640 medium supplemented with FBS. To assess the effect of the compounds of the invention, the cells were pretreated with compound (LMT-696) for 30 minutes each. To measure intracellular free radicals, cells were incubated for 20 minutes in a dark, humid CO 2 incubator at 37 ° C. with H 2 O 2 -sensitive fluorescent substance H 2 DCFDA [Molecular Probes (Eugene, OR)] (20 ⁇ M). It was.
  • H 2 DCFDA is hydrolyzed to DCF in the cell and is present in the cell, and oxidized to DCF showing high fluorescence in the presence of H 2 O 2 , and thus the amount of active oxygen was measured using this property.
  • cells were harvested using trypsin-EDTA and resuspended in serum-free RPMI 1640 without phenol red for confirmation of free radical production in the detector.
  • DCF fluorescence was measured with excitation and emission wavelengths of 488 and 530 nm, respectively, using a FACS Calibur flow cytometer (Becton Dickinson, NJ).
  • RNAs were isolated from cells by Easy Blue (Intron, Sungnam, Korea) and quantified at 260 nm absorbance. RNA (1.25 ⁇ g) was synthesized as cDNA by reverse transcription by polymerase chain reaction (PCR) technique. Expression levels were confirmed using primers that specifically bind to IL-8 and glyceraldehydes-3-phosphate dehydrogenase (GAPDH).
  • PCR polymerase chain reaction
  • BioCoat Matrigel Invasion Chambers (BD Biosciences, Bedford, Mass.) was used to measure the penetration of breast cancer cells following the compound treatment of the present invention.
  • 5 ⁇ 10 4 breast cancer cells were harvested with trypsin-EDTA, then resuspended in RPMI 1640 included in 0.5% serum and transferred to Matrigel inserts.
  • RPMI 1640 containing 5% serum was placed in the lower chamber and the cells were incubated at 37 ° C. for 36 hours.
  • Each filter was fixed with methanol for 3 minutes and stained with hematoxylin and eosin for 10 minutes.
  • Permeability of cancer cells was quantified by the number of cells on the lower side of the filter under an optical microscope (magnification x 200). Six fields were quantified for each assay. Each sample was analyzed twice, and the analysis was repeated three times.
  • the metastasis experiment of breast cancer cells according to the compound treatment of the present invention was approved by Korea University Ethics Committee, and all experimental animals used in this experiment were performed according to the approved guidelines of Korea University Animal Care and Use Committee. Cancer cells were injected into 6-week-old female nude mice (Charles River, Wilmington, Mass.) To confirm cancer cell metastasis. After 24 hours pretreatment of the compound of the present invention (LMT-696, 10 ⁇ M), LY255283, U75302 and DMSO in breast cancer cells, harvested with trypsin-EDTA, resuspended in PBS, and anesthetized with zoletil (50 mg / kg) in mice. 2 x 10 6 breast cancer cells were injected by intraperitoneal injection.
  • metastasis of cancer cells was inhibited 40% by treatment of the compound of the present invention (LMT-696) compared to the control, positive
  • the control group LY255283 was suppressed 36% by treatment, while the U75302 treatment did not inhibit the metastasis of cancer cells MDA-MB-231.
  • the compound of the present invention (LMT-696) can inhibit the production of free radicals and IL-8 in the cells of the cancer cells and consequently inhibit the penetration and metastasis of the cancer cells, a pharmaceutical component having excellent anticancer efficacy It can be used as.
  • mast cells play an important role.
  • mast cells are activated to release various cytokines (interlukin-4 and interlukin-13).
  • the cytokine causes phenomena such as influx of inflammatory cells, mucus production, and airway contraction.
  • the present inventors received female BALB / c mice that were 7 weeks old (18-20 g) from Orient (Seoungnam, Korea) and used them in the experiments to induce asthma.
  • Airway hypersensitivity was measured 24 days after initial sensitization, and mice were dissected on day 25 to observe the asthma phenotype of inflammatory cytokine IL-4 expression and the influx of inflammatory cells (neutrophils).
  • LPS Lipopolysaccharide
  • SVA egg white albumin
  • OVA egg white albumin
  • Airway constrictor administration was sprayed through the inlet of the chamber for 3 minutes using an ultrasonic nebulizer. Airway hypersensitivity was analyzed using enhanced pause as an indicator of asthma. Airway wash cell numbers were quantified by counting cells under an optical microscope (magnification, x 200). Four fields were counted in each assay, each sample analyzed twice, and the assay was repeated three times.
  • airway resistance was increased by about 13 times compared to asthma-induced mouse (OVA + LPS) compared to non-asthma-induced mouse (Normal), and the compound of the present invention (LMT-1013)
  • OVA + LPS asthma-induced mouse
  • Normal non-asthma-induced mouse
  • LMT-1013 the compound of the present invention
  • the present invention relates to a novel compound that exhibits Leukotriene B4 receptor 2 (BLT2) inhibitory activity and a pharmaceutical composition for preventing or treating an inflammatory disease comprising the same.
  • BLT2 Leukotriene B4 receptor 2
  • the present inventors have identified a novel compound that exhibits BTL2 inhibitory activity in order to solve in vivo instability and difficulty in mass production, which are problems of conventional inflammatory disease treatment substances, and has excellent cancer cell death enhancement and metastasis suppression effect and chemotaxis inhibition of the compound.
  • the effects, and anti-asthma effects have been confirmed experimentally, it is expected that it can be usefully used as a pharmaceutical composition for treating inflammatory diseases.

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Abstract

La présente invention concerne un nouveau composé présentant une activité inhibitrice du récepteur 2 du leukotriène B4 (BLT2) et une composition pharmaceutique permettant de prévenir ou traiter des maladies inflammatoires, contenant ledit composé comme principe actif. Dans la présente invention, un nouveau composé présentant une activité inhibitrice de BLT2 est étudié et la mort améliorée des cellules cancéreuses, la suppression des métastases, l'inhibition de la chimiotaxie, l'effet anti-asthmatique et analogue présentés dans le composé ont été expérimentalement confirmés. Par conséquent, la présente invention permet une approche plus fondamentale et un traitement cible pour le traitement ou la prévention de maladies inflammatoires.
PCT/KR2016/008069 2015-07-24 2016-07-23 Nouveau composé ayant une activité inhibitrice de blt et composition pour prévenir ou traiter des maladies inflammatoires, le contenant en tant que principe actif WO2017018750A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US15/745,337 US10179764B2 (en) 2015-07-24 2016-07-23 Compound having BLT inhibitory activity and composition, for preventing or treating inflammatory diseases, comprising same as active ingredient
JP2018503553A JP6574517B2 (ja) 2015-07-24 2016-07-23 Ｂｌｔ阻害活性を有する新規化合物およびこれを有効成分として含む炎症性疾患の予防または治療用組成物
ES16830783T ES2834549T3 (es) 2015-07-24 2016-07-23 Compuesto novedoso que tiene actividad inhibidora de BLT y composición, para prevenir o tratar enfermedades inflamatorias, que comprende el mismo como principio activo
CN201680055514.4A CN108349874B (zh) 2015-07-24 2016-07-23 具有blt抑制活性的化合物和包含其作为活性成分的用于预防或治疗炎症性疾病的组合物
EP16830783.3A EP3327000B1 (fr) 2015-07-24 2016-07-23 Nouveau composé ayant une activité inhibitrice de blt et composition pour prévenir ou traiter des maladies inflammatoires, le contenant en tant que principe actif
US16/181,953 US10766857B2 (en) 2015-07-24 2018-11-06 Compound having BLT inhibitory activity and composition, for preventing or treating inflammatory diseases, comprising same as active ingredient

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KR20150105097 2015-07-24
KR10-2015-0105097 2015-07-24
KR10-2016-0093762 2016-07-22
KR1020160093762A KR101796391B1 (ko) 2015-07-24 2016-07-22 Blt 저해 활성을 갖는 신규 화합물 및 이를 유효성분으로 포함하는 염증성 질환 예방 또는 치료용 조성물

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040466A2 (fr) * 2000-10-27 2002-05-23 Ortho-Mcneil Pharmaceutical, Inc. Derives d'amidoalkyl-piperidine et d'amidoalkyl-piperazine utilises dans le traitement de troubles du systeme nerveux
US20060264415A1 (en) * 2005-04-01 2006-11-23 Methylgene Inc. Inhibitors of histone deacetylase
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
KR20150080428A (ko) * 2013-12-30 2015-07-09 이화여자대학교 산학협력단 신규한 아미드 유도체 또는 이의 약학적으로 허용가능한 염, 이의 제조방법 및 이를 포함하는 통증의 예방 또는 치료용 약학적 조성물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040466A2 (fr) * 2000-10-27 2002-05-23 Ortho-Mcneil Pharmaceutical, Inc. Derives d'amidoalkyl-piperidine et d'amidoalkyl-piperazine utilises dans le traitement de troubles du systeme nerveux
US20060264415A1 (en) * 2005-04-01 2006-11-23 Methylgene Inc. Inhibitors of histone deacetylase
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
KR20150080428A (ko) * 2013-12-30 2015-07-09 이화여자대학교 산학협력단 신규한 아미드 유도체 또는 이의 약학적으로 허용가능한 염, 이의 제조방법 및 이를 포함하는 통증의 예방 또는 치료용 약학적 조성물

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