WO2018164549A1 - Nouveau composé ayant une activité inhibitrice de malate déshydrogénase et composition pharmaceutique destinée à prévenir ou à traiter le cancer le contenant à titre de principe actif - Google Patents

Nouveau composé ayant une activité inhibitrice de malate déshydrogénase et composition pharmaceutique destinée à prévenir ou à traiter le cancer le contenant à titre de principe actif Download PDF

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Publication number
WO2018164549A1
WO2018164549A1 PCT/KR2018/002861 KR2018002861W WO2018164549A1 WO 2018164549 A1 WO2018164549 A1 WO 2018164549A1 KR 2018002861 W KR2018002861 W KR 2018002861W WO 2018164549 A1 WO2018164549 A1 WO 2018164549A1
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WIPO (PCT)
Prior art keywords
methyl
phenoxy
benzoate
trimethylpentan
acetamido
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PCT/KR2018/002861
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English (en)
Korean (ko)
Inventor
이경
원미선
반현승
김민경
김보경
Original Assignee
동국대학교 산학협력단
한국생명공학연구원
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Priority claimed from KR1020180027397A external-priority patent/KR102082504B1/ko
Application filed by 동국대학교 산학협력단, 한국생명공학연구원 filed Critical 동국대학교 산학협력단
Priority to US16/491,237 priority Critical patent/US20200031764A1/en
Publication of WO2018164549A1 publication Critical patent/WO2018164549A1/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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/38—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring

Definitions

  • the present invention relates to a novel compound having inhibitory activity of MDH1 (malate dehydrogenases 1) or MDH2 (malate dehydrogenases 2) and a pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient.
  • the malic acid-asphalic acid shuttle is a mechanism that plays an important role in delivering NADH to the mitochondria in the cytoplasm generated during glycolysis.
  • the malic acid-asphalic acid shuttle is made by malic dehydrogenases (MDHs) and glutamate oxaloacetate transaminases (GOTs) present in the cytoplasm and mitochondria.
  • Aminooxyacetic acid an inhibitor of the maleic acid-asphalic acid shuttle, inhibits the proliferation of breast cancer cells by preventing glucose from becoming a product of the tricarboxylic acid cycle.
  • GOT2 Mitochondrial GOT2
  • MDH1 and MDH2 isozymes produced by different MDH genes are present in the cytoplasm and mitochondrial matrix.
  • MDH1 and MDH2 reversibly serve to convert malate and oxaloacetate (OAA) using the NAD / NADH cofactor system.
  • OAA oxaloacetate
  • MDH1 in the cytoplasm oxidizes NADH to NAD + by reducing oxalic acid to malic acid.
  • malic acid-asphalic acid shuttle malic acid is transported into the mitochondria and oxidized back to oxalic acid by mitochondrial MDH2 to produce NADH.
  • the generated NADH generates ATP through an electron transport system.
  • MDH2 involved in the TCA cycle is involved in ATP production through respiration.
  • the present inventors while continuing to research to develop a more effective cancer treatment material, in order to solve the conventional problems as described above, the substrate binding site and active site structure of MDH1 and MDH2 is very similar to that of MDH1 and MDH2
  • a compound was prepared that simultaneously inhibits the activities of MDH1 and MDH2, and a cancer therapeutic agent containing the compound was first devised to complete the present invention.
  • the present invention has been made to solve the above problems, the present inventors have confirmed the cancer preventive or therapeutic effect of the compound that inhibits the activity of MDH1 and / or MDH2 to complete the present invention based on this.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, which exhibits MDH1 and / or MDH2 inhibitory activity.
  • the present invention provides a compound represented by the following formula (1), an isomer thereof or a pharmaceutically acceptable salt thereof.
  • X is methylene group, ethane group, ethylene group, n -propylene group or isopropylene;
  • R 1 is a nitro group, trifluoromethyl group, C 1 -C 20 alkyl or C 1 -C 20 cycloalkyl
  • R 2 is , , , , , , ,
  • R 2 is when,
  • R 3 is methyl or 2-propynyl
  • R 4 is methyl, hydrogen, hydroxy, methoxy, 2-propynyl, , , , , or ego;
  • R 2 is when,
  • R 5 is , or ego
  • R 2 is when,
  • R 6 is methyl or hydroxy
  • R 2 is when,
  • R 7 may be C or N.
  • R 1 may be substituted with adamantyl, tert -butyl, pentyl, cyclopentyl, cyclohexyl, or 2,4,4-trimethylpentin-2-yl.
  • R 1 is adamantyl
  • X is methylene group, ethane group, ethylene group, n -propylene group or isopropylene
  • R 2 is ego
  • R 4 is methyl, hydrogen, hydroxy, , , or And
  • R 3 may be methyl.
  • the compound may be a compound represented by the following formula (2).
  • R 1 is adamantyl or tert -butyl
  • R 2 is , or ego
  • R 2 is when,
  • R 3 is methyl or 2-propynyl
  • R 4 is methyl, hydrogen or hydroxy
  • R 2 is when,
  • R 6 is methyl or hydroxy
  • R 2 is when,
  • R 7 may be C or N.
  • the compound may be a compound represented by the following formula (3).
  • R 1 is a nitro group, trifluoromethyl group, adamantyl, tert -butyl, pentyl, cyclopentyl, cyclohexyl, or 2,4,4-trimethylpentin-2-yl;
  • R 2 is , , , , , or ego
  • R 2 is when,
  • R 3 is methyl or 2-propynyl
  • R 4 is methyl, hydrogen, hydroxy, methoxy, 2-propynyl, , , , , or Can be.
  • the compound may be any one or more selected from the group consisting of:
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the compound represented by Formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the composition may inhibit the activity of malate dehydrogenases 1 (MDH1) and / or malate dehydrogenases 2 (MDH2).
  • MDH1 malate dehydrogenases 1
  • MDH2 malate dehydrogenases 2
  • the composition may simultaneously inhibit the activity of MDH1 (malate dehydrogenases 1) and MDH2 (malate dehydrogenases 2).
  • the present invention provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a subject.
  • the present invention provides a cancer prevention or treatment of the composition comprising the compound, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention relates to a compound exhibiting inhibitory activity of MDH1 (malate dehydrogenases 1) and / or MDH2 (malate dehydrogenases 2) and a pharmaceutical composition for preventing or treating cancer, comprising the same as an active ingredient.
  • MDH1 malate dehydrogenases 1
  • MDH2 malate dehydrogenases 2
  • a pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient.
  • the present inventors have experimentally confirmed the mitochondrial respiratory inhibitory effect and excellent cancer cell growth inhibitory effect in cancer cells of the compound showing MDH1 and / or MDH2 inhibitory activity, the compound of the present invention is useful as a pharmaceutical composition for treating cancer It is expected to be used.
  • 1 is a schematic diagram illustrating the synthesis of 55 compounds of the present invention (compounds 3-1 to 3-34, compounds 6-1 to 6-10 and compounds 9-1 to 9-11).
  • Figure 2 shows the overexpressed recombinant MDH2 protein isolated and purified in the present invention.
  • Figure 3 is the result of measuring the enzyme kinetics (enzyme kinetics) of MDH1 and MDH2 according to the change of NADH concentration of the compound of the present invention.
  • OCR oxygen consumption rate
  • Figures 7a and 7b is a result of confirming that the tumor size or volume after administration of the compound of the present invention.
  • Figure 7c is a result confirming that the weight of the tumor after administration of the compound of the present invention.
  • the present inventors when treated with the compound prepared in Example, exhibits the inhibitory activity of MDH1 / 2 expression, HIF-1 ⁇ expression inhibitory activity, mitochondrial respiratory inhibitory effect in cancer cells and excellent cancer cell growth
  • the inhibitory effect etc. were confirmed specifically, and this invention was completed based on this.
  • the present invention provides a compound represented by Formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.
  • X is methylene group, ethane group, ethylene group, n -propylene group or isopropylene;
  • R 1 is a nitro group, trifluoromethyl group, C 1 -C 20 alkyl or C 1 -C 20 cycloalkyl
  • R 2 is , , , , , , ,
  • R 2 is when,
  • R 3 is methyl or 2-propynyl
  • R 4 is methyl, hydrogen, hydroxy, methoxy, 2-propynyl, , , , , or Can be.
  • R 2 is when,
  • R 5 is , or Can be.
  • R 2 is when,
  • R 6 may be methyl or hydroxy.
  • R 2 is when,
  • R 7 may be C or N.
  • R 1 is adamantyl, tert -butyl, pentyl, cyclopentyl, cyclohexyl, or 2,4,4-trimethylpentin-2-yl.
  • R 1 is adamantyl
  • X is methylene group, ethane group, ethylene group, n -propylene group or isopropylene
  • R 2 is ego
  • R 4 is methyl, hydrogen, hydroxy, , , or And
  • R 3 may be methyl.
  • the compound may be a compound represented by the following formula (2).
  • R 1 is adamantyl or tert -butyl
  • R 2 is , or ego
  • R 2 is when,
  • R 3 is methyl or 2-propynyl
  • R 4 is methyl, hydrogen or hydroxy
  • R 2 is when,
  • R 6 is methyl or hydroxy
  • R 2 is when,
  • R 7 is C or N.
  • the compound may be a compound represented by the following formula (3).
  • R 1 is a nitro group, trifluoromethyl group, adamantyl, tert -butyl, pentyl, cyclopentyl, cyclohexyl, or 2,4,4-trimethylpentin-2-yl;
  • R 2 is , , , , , or ego
  • R 2 is when,
  • R 3 is methyl or 2-propynyl
  • R 4 is methyl, hydrogen, hydroxy, methoxy, 2-propynyl, , , , , or Can be.
  • C 1 -C 20 alkyl refers to a monovalent alkyl group having 1 to 20 carbon atoms.
  • the term is exemplified by functional groups such as methyl, ethyl, n -propyl, i -propyl, n -butyl, i -butyl, tert -butyl, n -hexyl and the like.
  • C 1 -C 20 cycloalkyl refers to a saturated hydrocarbon ring compound having a single ring (eg cyclohexyl) or multiple condensed ring (eg norbornyl), cyclopentyl, Cyclohexyl, norbornyl, adamantane and the like.
  • methylene group used in the present invention means a bond in the form of-(CH 2 )-, and means a form when one carbon is bonded to X in the general formula (1) of the present invention.
  • N is 1 or 2.
  • ethane group used in the present invention means a bond in the form of-(C 2 H 4 )-, and refers to a form when two carbons are bonded to X in Formula 1 of the present invention.
  • ethylene group used in the present invention means a bond in the form of-(C 2 H 2 )-, and means the form when two carbons are bonded to X in the general formula (1) of the present invention.
  • n -propylene group used in the present invention means a bond in the form of-(C 3 H 6 )-, and in the formula (1) of the present invention means a form when three carbons are bonded to the X in a straight chain form. do.
  • isopropylene group refers to a bond in the form of-(C 3 H 6 )-, wherein in the formula (1) of the present invention, when X is combined with three carbons in a crushed or branched form, it means.
  • 2-propynyl refers to -CH 2 C ⁇ CH, and means a linear hydrocarbon group of three carbon atoms, including unsaturated carbon bonded to the triple bond at the end.
  • Substituents comprising alkyl and other alkyl moieties described in the present invention include both straight and pulverized forms.
  • 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 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 Formula 1 of the present invention in an excess of an aqueous acid solution, and the salt is mixed with a water miscible organic solvent such as methanol, ethanol, It can be prepared by precipitation with acetone or 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 water miscible organic solvent such as methanol, ethanol
  • 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).
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the compound represented by Formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. .
  • composition of the present invention includes a pharmaceutical and nutraceutical composition.
  • prevention means any action that inhibits cancer or delays the onset by administration of a pharmaceutical composition according to the invention.
  • treatment means any action that improves or beneficially alters the symptoms of cancer by administration of a pharmaceutical composition according to the present invention.
  • Cancer a disease to be prevented and treated by the composition of the present invention, is classified into a disease in which normal tissue cells proliferate unlimitedly for any cause and continue to develop rapidly regardless of the living phenomenon of the living body or the surrounding tissue state.
  • Cancer includes, but is not limited to, dysplasia, hyperplasia, solid tumors, and hematopoietic stem cell cancer, and includes various cancer types known in the art.
  • Other cancers may include, but are not limited to, cancers of the following organs or organs: brain, heart, lung, stomach, large intestine, genitourinary tract, liver, bone, nervous system, gynecology, blood, skin, breast and adrenal gland. It doesn't work.
  • cancer cells include glioma (Schwannoma, glioblastoma, astrocytoma), neuroblastoma, pheochromocytoma, adrenal ganglia, meningioma, adrenal cortex, medulloblastoma, rhabdomyosarcoma, kidney cancer, various types of cancer Vascular cancer, osteoblastic osteocarcinoma, prostate cancer, ovarian cancer, uterine myoma, salivary gland cancer, choroid plexus cancer, breast cancer, pancreatic cancer, colon cancer, colon cancer and megakaryocyte leukemia; And sarcomas, such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, hemangioma, cutaneous fibroma, keloid, fibrosarcoma or angiosarcoma, and melanoma Includes skin cancer, including.
  • MDH1 or MDH2 of the present invention are isoenzymes produced by different MDH genes, where MDH is present in the cytoplasm and mitochondrial matrix. More specifically, MDH1 and MDH2 reversibly serve to convert malate and oxaloacetate (OAA) using a NAD / NADH cofactor system. MDH1 in the cytoplasm oxidizes NADH to NAD + by reducing oxalic acid to malic acid. In the malic acid-asphalic acid shuttle, malic acid is transported into the mitochondria and oxidized back to oxalic acid by mitochondrial MDH2 to produce NADH. The generated NADH generates ATP through an electron transport system. In addition, MDH2 involved in the TCA cycle is involved in ATP production through respiration.
  • OOA oxaloacetate
  • composition of the present invention can inhibit the activity of MDH1 (malate dehydrogenases 1) and / or MDH2 (malate dehydrogenases 2), and in particular can inhibit the activity of MDH1 and MDH2 simultaneously.
  • 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 inhibitory activity of MDH1 and MDH2 by the compound treatment synthesized according to the preparation method of the present invention was confirmed (see Experimental Example 1).
  • MDH inhibition mechanism was confirmed using the compound of the present invention (see Experimental Example 2)
  • HIF-1 ⁇ inhibitory activity was confirmed using the compound of the present invention (see Experimental Example 3)
  • mitochondria in cancer cells The respiratory inhibitory effect was confirmed (see Experimental Example 4), and cancer cell transplantation mouse model was used to specifically confirm the effect of reducing tumor weight and size (see Experimental Example 5) as a pharmaceutical composition for cancer prevention or treatment. It was confirmed that it can be used very usefully.
  • the compound represented by Formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof according to the present invention may be usefully used as a pharmaceutical composition for preventing, ameliorating or treating cancer including the same as an active ingredient.
  • 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 for preventing, controlling or treating cancer 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. .
  • examples of the non-limiting compound used as an active ingredient of the pharmaceutical composition include the following compounds, isomers thereof and pharmaceutically acceptable salts thereof.
  • the reactants and / or starting materials of known methods were appropriately modified to synthesize the following compounds according to the present invention.
  • Reagents and conditions a) (i) K 2 CO 3 , methyl-4-bromobutanoate or methyl-2-bromo-2-methylpropanoate (Methyl 2-bromo-2 -methylpropanoate), DMF; (ii) LiOH, THF / H 2 O; b) EDC.HCl, HOBT, DIPEA, DMF, NH 2 -R 2 ; c) PPh 3 , Methylpropiolate, Toluene; d, g) LiOH, THF / H 2 O; e) EDC.HCl, HOBT, DIPEA, DMF, NH 2 -R 2 or HATU, DIPEA, DMF, NH 2 -R 2 ; f) Pd / C, H 2 , MeOH; h) EDC.HCl, HOBT, DIPEA, DMF, NH 2 -R 2 or HATU, DIPEA, DMF /
  • the concentrate was purified by silica gel column chromatography to afford methyl 3- (2- (4- (2,4,4-trimethylpentane-2-yl) phenoxy) acetamido) benzoate (white solid, 0.10 g, 64.1% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 4-hydroxy-3- (2- (4- (2,4,4-trimethylpenten-2-yl) phenoxy) acetamido) benzoate (White solid, 1.00 g, 64.1% yield).
  • the concentrate was purified by silica gel column chromatography to obtain N- (4- (trifluoromethyl) phenyl) -2- (4- (2,4,4-trimethylpenten-2-yl) phenoxy) acetamide. Obtained (white solid, 0.11 g, 71.5% yield).
  • the concentrate was purified by silica gel column chromatography to obtain N- (4- (4-methylpiperazin-1-yl) phenyl) -2- (4- (2,4,4-trimethylpentane-2-yl) phenoxy Acetamide was obtained (white solid, 0.12 g, 72.7% yield).
  • the concentrate was purified by silica gel column chromatography to obtain 1- (4- (4- (trifluoromethyl) benzyl) piperazin-1-yl) -2- (4- (2,4,4-trimethylpentane- 2-yl) phenoxy) ethanone was obtained (white solid, 0.15 g, 81.0% yield).
  • the concentrate was purified by silica gel column chromatography to obtain 1- (4- (prop-2-ynyl) piperazin-1-yl) -2- (4- (2,4,4-trimethylpentane-2-yl Phenoxy) ethanone was obtained (white solid, 0.11 g, 78.5% yield).
  • the concentrate was purified by silica gel column chromatography to obtain N- (3-hydroxy-adamantane-1-yl) -2- (4- (2,4,4-trimethylpentane-2-yl) phenoxy) Acetamide was obtained (white solid, 0.12 g, 76.9% yield).
  • Methyl 3- (2- (4-adamantan-1-yl-phenoxy) acetamido) -4-hydroxybenzoate (1.0 equiv), anhydrous potassium carbonate (3.0 equiv) and ethyl chloroacetate (2.0 equiv) are mixed in DMF and at room temperature After stirring overnight, the mixture was concentrated under reduced pressure, and the obtained residue was diluted with EtOAc and washed with sodium bicarbonate, water and brine. The organic layers were combined, dried over anhydrous magnesium sulfate (anhydrous MgSO 4 ), and then concentrated under reduced pressure.
  • the concentrate was purified by silica gel column chromatography to methyl 3- (2- (4-adamantane-1-yl-phenoxy) acetamido) -4- (2- (pyrrolidin-1-yl) Oxy) benzoate was obtained (white solid, 0.09 g, 75.0% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 3- (2- (4-adamantane-1-yl-phenoxy) acetamido) -4- (3-morpholinopropoxy) benzoate. (White solid, 0.09 g, 69.7% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 4-methoxy-3- (2- (4- (2,4,4-trimethylpenten-2-yl) phenoxy) acetamido) benzoate (White solid, 0.08 g, 77.6% yield).
  • the methoxyethane (2.0 equiv) was mixed with DMF, stirred at room temperature overnight, concentrated under reduced pressure, and the obtained residue was diluted with EtOAc, washed with sodium bicarbonate, water and brine. After collection of the organic layer was removed, the dried over anhydrous magnesium sulfate (anhydrous MgSO 4), and concentrated under reduced pressure.
  • the concentrate was purified by silica gel column chromatography to give methyl 3- (2- (4-adamantane-1-yl-phenoxy) -2-methylpropaneamido) benzoate (white solid, 0.12 g, 84.5% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 3- (2-methyl-2- (4- (2,4,4-trimethylpenten-2-yl) phenoxy) propaneamido) benzoate. (White solid, 0.11 g, 75.8% yield).
  • the concentrate was purified by silica gel column chromatography to prepare methyl-4-hydroxy-3- (2-methyl-2- (4- (2,4,4-trimethylpentan-2-yl) phenoxy) propaneamido) Obtained benzoate (white solid, 0.03 g, 83.2% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 4-hydroxy-3- (4- (4- (4- (2,4,4-trimethylpenten-2-yl) phenoxy) butanamido) benzoate (White solid, 0.10 g, 66.2% yield).
  • the concentrate was purified by silica gel column chromatography ( E ) -3H- [1,2,3] triazolo [4,5-b] pyridin-3-yl 3- (4- (2,4,4-trimethyl Pentan-2-yl) phenoxy) acrylate was obtained (white solid, 0.05 g, 44.6% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 3- (3- (4- (2,4,4-trimethylpenten-2-yl) phenoxy) propaneamido) benzoate (white solid, 0.13 g, 88.4% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 4-hydroxy-3- (3- (4- (2,4,4-trimethylpentan-2-yl) phenoxy) propaneamido) benzoate. (White solid, 0.01 g, 75.0% yield).
  • the concentrate was purified by silica gel column chromatography to afford methyl 2-methyl-5- (3- (4- (2,4,4-trimethylpentan-2-yl) phenoxy) propaneamido) benzoate ( Yellow solid, 0.05 g, 61.0% yield).
  • the concentrate was purified by silica gel column chromatography to give isopropyl 3- (3- (4- (2,4,4-trimethylpentan-2-yl) phenoxy) propaneamido) benzoate (clear liquid, 0.06 g, 26.5% yield).
  • the concentrate was purified by silica gel column chromatography to give methyl 5- (3- (4- (2,4,4-trimethylpentan-2-yl) phenoxy) propaneamido) nicotinate (white solid, 0.06 g, 40.3% yield).
  • the concentrate was purified by silica gel column chromatography to obtain N- (3- (morpholine-4-carbonyl) phenyl) -3- (4- (2,4,4-trimethylpentan-2-yl) phenoxy) propane Amide was obtained (white solid, 0.08 g, 53.0% yield).
  • the concentrate was purified by silica gel column chromatography to give ethyl 2- (3- (3- (4- (2,4,4-trimethylpentan-2-yl) phenoxy) propaneamido) benzamido) acetate (White solid, 0.14 g, 64.4% yield).
  • an MDH2 recombinant protein was prepared.
  • the MDH2 gene was purchased from the Korea Human Genetics Bank (Korean Human GeneBank, KUGI, NM_005918), and amplified by PCR and cloned into the pET28a vector (Merck, Germany). Thereafter, the plasmid vector was introduced into E. coli Rosetta 2 (DE3), followed by IPTG treatment to overexpress MDH2 recombinant protein.
  • the overexpressed recombinant MDH2 protein was purified by Ni-NTA affinity chromatography, TEV enzyme cleavage, and size-exclusion chromatography.
  • the inhibitory activity of the compounds against MDH1 and MDH2 recombinant proteins produced in Experimental Examples 1-1, 1-2, 1-3 and 1-4 were measured. More specifically, 0.25 nM MDH1 recombinant protein (Biovision) or MDH2 recombinant protein was prepared using 200 ⁇ M oxaloacetate, nicotinamide adenine dinucleotide (NADH), the compound prepared in Example 1, and MDH assay buffer (100 mM potassium phosphate, pH 7.4) for 30 minutes. Thereafter, the change in NADH concentration in the solution by oxidation of NADH (NAD + ) by MDH1 or MDH2 enzyme was measured at absorbance 340 nm.
  • NADH nicotinamide adenine dinucleotide
  • HIF-1 ⁇ hyperoxia-inducible factor-1 ⁇
  • HIF-1 ⁇ hypoxia-inducible factor-1 ⁇
  • FBS fetal calf serum
  • the cells were incubated for 24 hours in a cell incubator. Cells were incubated for 6 hours at 1% oxygen, 94% nitrogen and 5% carbon dioxide conditions to induce accumulation of HIF-1 ⁇ protein by hypoxia.
  • the change in oxygen consumption rate was measured. More specifically, the oxygen consumption rate (OCR) of the mitochondria was measured using an XF24 extracellular flux analyzer (Seaholes). After HCT-116 cells (1 ⁇ 10 5 cells) were incubated for 24 hours in a measuring plate (XF24 cell cultureplate), the culture medium was exchanged with XF measuring medium and incubated for 1 hour in a cell incubator without carbon dioxide.
  • OCR oxygen consumption rate
  • the oxygen consumption rate of mitochondria was measured three times in non-drug cells, three times after administration of the ATP synthesis inhibitor oligomycin (1 ⁇ M), and three times after administration of the chemical decoupling agent carbonyl cyanide p -trifluoromethoxyphenylhydrazone (0.5 ⁇ M). Oxygen consumption was measured three times after administration of rotenone (1 ⁇ M) and antimycin A (1 ⁇ M).
  • HCT-116 cells (1 ⁇ 10 5 cells) were treated with compound 9-2 and hypoxic specific sensitive fluorescent probe MAR (Gray, 0.5 ⁇ M), and then incubated for 6 hours in a cell incubator under hypoxic conditions. Thereafter, changes in intracellular fluorescence intensity due to hypoxia were measured and quantified by a real-time cell observation analysis system (Incusite, Essen).
  • compound 9-2 was orally administered once daily at a concentration of 20 mg / kg, and 3, 5, 7, 10, 12, 14, 17, 19, 21, and 24 days Body weight and tumor size of the nude mouse were measured using Equation 1 below. Mice were sacrificed 24 days after Compound 9-2 administration to determine the weight and size of the extracted tumor.
  • the cell proliferation inhibitory activity of the compound 9-2 prepared according to the above Example was confirmed.
  • the cell lines used in the experiment were lung cancer cell lines A549 and H1703, colorectal cancer cell lines HCT116 and HT29, liver cancer (liver).
  • cancer cell lines Hep3B and HepG2 gastric or stomach cancer cell lines NUGC-3 and AGS, kidney and renal cancer cell lines 786-O and Caki-1, breast cancer cell lines MCF-7 and MDA -MB-231, prostate cancer cell line PC3, pancreatic cancer cell line MIA-PaCa-2, cervical cancer cell line HeLa, normal cell WI-38 and CCD-32Lu cell lines.
  • the cell line was suspended in DMEM medium containing 5% fetal bovine serum (FBS), and then transferred to a 96 well plate (3 ⁇ 10 3 cells / well) for 24 hours in a 37 ° C. cell incubator maintaining 5% carbon dioxide. It was.
  • Compound 9-2 was treated at various concentrations, incubated for 72 hours, and the cells were fixed with an aqueous 10% formalin solution, followed by staining with 0.5% methylene blue solution. Thereafter, the concentration change of methylene blue extracted with an aqueous 0.5% hydrochloric acid solution was measured at an absorbance of 600 nm.
  • Compound 9-2 inhibited the proliferation of various cancer cell lines that are normal cell lines, and showed a particularly strong inhibitory effect in A549, HCT116, HepG2 cells. Compound 9-2 was found to have no effect on the proliferation of normal cell lines in a concentration range that inhibits cancer cell proliferation.

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Abstract

La présente invention concerne un composé présentant une activité inhibitrice d'au moins une des malate déshydrogénases 1 (MDH1) et/ou malate déshydrogénases 2 (MDH2), et une composition pharmaceutique destinée à prévenir ou à traiter le cancer le contenant à titre de principe actif. Les inventeurs de la présente invention ont confirmé expérimentalement que le composé présentant l'activité inhibitrice de MDH1 et/ou MDH2 a un effet inhibiteur sur la respiration mitochondriale dans les cellules cancéreuses, un excellent effet inhibiteur sur la croissance des cellules cancéreuses, etc. Par conséquent, le composé selon la présente invention devrait être utilisé de manière efficace sous forme de composition pharmaceutique pour traiter le cancer.
PCT/KR2018/002861 2017-03-09 2018-03-09 Nouveau composé ayant une activité inhibitrice de malate déshydrogénase et composition pharmaceutique destinée à prévenir ou à traiter le cancer le contenant à titre de principe actif WO2018164549A1 (fr)

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KR20220139513A (ko) * 2021-04-07 2022-10-17 동국대학교 산학협력단 자가유화형 나노에멀전을 이용한 경구 투여 제제

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Cited By (3)

* Cited by examiner, † Cited by third party
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EP3610866A4 (fr) * 2017-04-14 2020-12-23 National Cancer Center Composition pharmaceutique permettant de prévenir et de traiter le cancer, contenant un inhibiteur de navette malate-aspartate et un médicament anticancéreux en tant que principes actifs
KR20220139513A (ko) * 2021-04-07 2022-10-17 동국대학교 산학협력단 자가유화형 나노에멀전을 이용한 경구 투여 제제
KR102566464B1 (ko) 2021-04-07 2023-08-14 동국대학교 산학협력단 자가유화형 나노에멀전을 이용한 경구 투여 제제

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