WO2020040582A1 - Nouveau dérivé de benzopyrane ou sel pharmaceutiquement acceptable de celui-ci et composition pharmaceutique le comprenant en tant que principe actif - Google Patents

Nouveau dérivé de benzopyrane ou sel pharmaceutiquement acceptable de celui-ci et composition pharmaceutique le comprenant en tant que principe actif Download PDF

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WO2020040582A1
WO2020040582A1 PCT/KR2019/010731 KR2019010731W WO2020040582A1 WO 2020040582 A1 WO2020040582 A1 WO 2020040582A1 KR 2019010731 W KR2019010731 W KR 2019010731W WO 2020040582 A1 WO2020040582 A1 WO 2020040582A1
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formula
group
cancer
nct
ethoxy
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서재홍
김지영
김윤재
오은혜
이지우
안지혜
콩트렁응웬
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Korea University Research and Business Foundation
SNU R&DB Foundation
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Seoul National University R&DB Foundation
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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4

Definitions

  • the present invention relates to a novel benzopyran derivative or a pharmaceutically acceptable salt thereof and a pharmaceutical composition containing the same as an active ingredient. More particularly, the present invention relates to a novel anti-cancer activity by inhibiting the activity of Hsp90 (Heat shock protein 90).
  • Hsp90 Heat shock protein 90.
  • One benzopa is related to a derivative or a pharmaceutically acceptable salt thereof and a pharmaceutical composition containing the same as an active ingredient.
  • HER2-positive breast cancer is a cancer that overexpresses HER2 (human epidermal growth factor receptor 2), a membrane protein receptor present in the cancer cell membrane, and appears in 20-30% of all breast cancer patients and has a poor prognosis than HER2-negative patients. It is inferior and has a very low reactivity to commonly used cytotoxic anticancer agents.
  • HER2 human epidermal growth factor receptor 2
  • Hsp90 inhibitors are potential chemotherapeutic agents for angiogenesis-related diseases (Eccles, S .; Massey, A .; Raynaud, F .; Sharp, S .; Box, G .; Valenti, M .; Patterson, L .; de Haven Brandon, A .; Gowan, S .; Boxall, F. NVP-AUY922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth , angiogenesis, and metastasis. Cancer Res . 2008, 68, 2850).
  • trastuzumab (trademark: ®Herceptin) and Pertuzumab (trade name: ®Perjeta) and T-DM1 (trade name: ®Kadcyla), which target HER2-positive breast cancers, have been developed, but at a high price. It creates an economic burden.
  • trastuzumab is used as a first-line treatment in patients with HER2-positive breast cancer, but initial resistance is seen in about 40-50% of patients and in 90% of patients with good initial response within one year. Secondary resistance. Such resistance is a major cause of relapse and metastasis and plays a decisive role in reducing the survival rate of patients.
  • HER2 is one of the major client proteins of Hsp, and the activity of kinase is directly regulated by Hsp. Therefore, Hsp inhibitors can directly inhibit the activation of p95HER2 and HER2 / HER3 dimerization, which are ultimately responsible for resistance, so Hsp inhibitors will be the top-targeted therapeutics for HER2 and are valuable as drug-resistant drugs. Since it was first reported that the expression and activity of HER2 was inhibited by an Hs inhibitor ansamycin, many derivatives of Hsp inhibitors have been developed until now as a drug for controlling the activation of HER2 in HER2-positive breast cancer.
  • Hsp90 inhibitors currently in clinical trials inhibit N-terminal Hsp90, and most of them failed or stopped due to serious side effects such as lack of pharmacological activity, toxicity and heat shock response.
  • the present inventors have diligently tried to solve the above problems, and as a result, the novel benzopyran derivatives of the present invention or pharmaceutically acceptable salts thereof, or solvates thereof are based on the C-terminal homology model structure. It was confirmed that inhibiting the activity of Hsp90 by imparting a strong binding force to the, to complete the present invention.
  • An object of the present invention is to provide a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, or solvate thereof exhibiting anticancer activity by inhibiting the activity of Hsp90 (Heat shock protein 90).
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.
  • the present invention provides a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, or solvate thereof.
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.
  • the compound represented by Formula 1 of the present invention does not affect the complex structure of HSP90 and HSF-1, it does not induce HSF-1 activation or expression of HSP genes. It can effectively promote the death of cancer cells, can be used as an active ingredient of an anticancer agent.
  • Figure 2 is a result of measuring the cell viability after treating the compound of the present invention by concentration in a breast cancer cell line.
  • Figure 3 shows the changes in cell morphology after treatment of the compounds of the present invention to breast cancer cell lines BT474, JIMT-1 and MDA-MB-231.
  • Figure 6 shows the results of measuring the expression of HSP90 clients after the treatment of the compound of the present invention.
  • FIG. 7 shows the results of measuring cell viability after treatment of the compound of the present invention to human prostate cancer cell line DU145, colon cancer cell line HCT116, liver cancer cell line HepG2 and ovarian cancer cell line SKOV-3.
  • the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, or a solvate thereof increases casapse-3 activity and expresses not only the expression of EGFR and STAT3, but also the active forms of phospho-EGFR and phospho-STAT3. And significantly reduced the expression of AKT, ERK, Cyclin D1, and Survivin, and effectively promoted the death of cancer cells.
  • the present invention relates to a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, or solvate thereof.
  • R 1 is a substituted or unsubstituted alkoxy group
  • R 2 , R 3 , R 4 and R 5 are each independently a hydrogen atom; Or a C 1 -C 2 alkyl group
  • the substituent is a nitrogen atom; Oxygen atom; Alkyl groups; Or a heterocyclic alkyl substituted with a C 1 -C 6 alkyl group (heteroatoms comprise at least one of nitrogen, oxygen, sulfur, and the heterocycle is a pentagonal, hexagonal ring).
  • the compound represented by Formula 1 is a derivative of benzopyran.
  • substitution in the present invention is a reaction that replaces an atom or a group of atoms contained in a molecule of a compound with another atom or group of atoms.
  • alkoxy group refers to an alkyl group (O-alkyl group) bonded to oxygen, and in the present invention, a methoxy group (C 1 ), an ethoxy group (C 2 ), a propoxy group ( C 3 ), a group consisting of C 1 to C 8 alkoxy groups including butoxy group (C 4 ), pentyloxy group (C 5 ), hexyloxy group (C 6 ), heptyloxy group (C 7 ) and octyloxy (C8) It may be selected from, but is not limited thereto.
  • C 1 -C 2 alkyl means a monovalent linear hydrocarbon residue consisting solely of carbon and hydrogen atoms, having from 1 to 2 carbon atoms.
  • C 1 -C 6 alkyl means a monovalent linear or branched or cyclic saturated hydrocarbon residue consisting solely of carbon and hydrogen atoms, having 1 to 6 carbon atoms. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary-butyl, tert-butyl, pentyl, n-hexyl and the like.
  • branched alkyl examples include isopropyl, isobutyl, tert-butyl and the like.
  • a “cyclic alkyl” is a cyclic alkyl group having 3 to 6 carbon atoms, and examples thereof include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group or a cyclohexyl group.
  • hetero ring refers to heterocyclyl and is saturated of 4 to 9 ring atoms, including 1, 2 or 3 ring heteroatoms selected from N, O and S in addition to carbon atoms; Partially unsaturated carbocyclic rings. It is meant that two rings consist of two rings having two common ring atoms, ie the bridge separating the two rings is a single bond or a chain of one or two ring atoms.
  • the heterocyclyl ring may be substituted or unsubstituted as defined herein.
  • heterocyclyl may be azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, pipepe Ridinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, arc Furperazinyl, oxazpanyl, dihydroindolyl, dihydrofuryl, dihydroimidazolinyl, dihydrooxazolyl, tetrahydropyridinyl, dihydropyranyl or benzodioxolyl.
  • cyclic refers to a structure in which both ends of the chain are connected in the skeleton of the organic compound to form a ring shape.
  • the heterocyclic alkyl group of R 1 may be characterized in that the pyridine, piperidine, morpholine or piperazine.
  • R 1 is a methoxy group (methoxy), 2-methoxy-ethoxy group (2-methoxy-ethoxy), 2-aminylethoxy group (2-aminylethoxy), 2-methyl aminyl 2-methylaminylethoxy, 2-dimethylaminylethoxy, 3-aminylpropoxy, 3-methyl aminylpropoxy, 3-methylaminylpropoxy, 3 -Dimethylaminylpropoxy, (1-methylpiperidin-4-yl) oxy group ((1-Methylpiperidin-4-yl) oxy), (piperidin-4-yl) methok Period ((1-Methylpiperidin-4-yl) methoxy), (1-methylpiperidin-4-yl) methoxy group ((1-Methylpiperidin-4-yl) methoxy), (2-piperidine-1- Yl) ethoxy group (2- (piperidin-1-yl) ethoxy), (2-piperidine-1
  • R 1 is ; ; ; ; ; ; ; ; ; ; ; And; It may be characterized in that the one selected from.
  • novel benzopyran derivatives or pharmaceutically acceptable salts thereof, or solvates thereof inhibit Hsp90 activity by imparting strong binding force to Hsp90 C-terminal based on the C-terminal homology model structure.
  • the compounds of the present invention may exist in the form of salts, in particular pharmaceutically acceptable salts.
  • salts conventionally used in the art may be used without limitation, such as acid addition salts formed by pharmaceutically acceptable free acid.
  • pharmaceutically acceptable salt of the present invention is any concentration of the compound which is relatively nontoxic to the patient and has a harmless effective action, in which the side effects caused by the salt do not degrade the beneficial efficacy of the compound represented by the formula (1).
  • the novel compounds represented by Formula 1 can be used in the form of pharmaceutically acceptable salts, and acid addition salts formed by pharmaceutically acceptable free acid are useful as salts.
  • 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 salts according to the present invention are dissolved in conventional methods, for example, a novel compound represented by the formula (1) 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.
  • a novel compound represented by the formula (1) in an excess of aqueous acid solution
  • the salts are water miscible organic solvents such as methanol, ethanol, acetone or It can be prepared by precipitation with acetonitrile.
  • the same amount of the novel compound represented by the formula (1) and the acid or alcohol in water may be heated and then the mixture is evaporated to dryness, or the precipitated salt may be produced by suction filtration.
  • 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.
  • Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable silver salt (e.g., silver nitrate).
  • the prepared new benzopyran derivatives can be separated and purified by silica gel column chromatography after preparation, and the molecular structure can be confirmed by nuclear magnetic resonance.
  • the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the compound or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.
  • the composition may be characterized by exhibiting anticancer activity by inhibiting Hsp90.
  • composition is considered to include not only products comprising specific components, but also any products made directly or indirectly by the combination of specific components.
  • molecular chaperones such as heat-shock protein families (HSPs) regulate the folding of client proteins through ATP-dependent structural changes to activate nascent proteins, refold the damaged proteins, A protein that helps to break down.
  • HSPs heat-shock protein families
  • client proteins avoid aggregation by binding to molecular chaperones, and their binding aids intracellular deposition through membrane translocation of client proteins.
  • Hsp90 a family of heat shock proteins
  • the molecular chaperone function of Hsp90 is known to be necessary for the stability and activation of various client proteins involved in cell signaling pathways. Under normal conditions without external stimulation, the amount of Hsp90 accounts for 1-2% of the intracellular protein, but under external stimulation, the amount of Hsp90 doubles.
  • Cancer-causing mutations in the client protein require higher levels of Hsp90 function and lead to overexpression of Hsp90, and overexpressed Hsp90 is a common feature of cancer cells compared to normal tissue (Bagatell, R .; Whitesell, L. Altered Hsp90 function in cancer: A unique therapeutic opportunity.Mol. Cancer Ther. 2004, 3, 1021-1030).
  • ErbB2 ErbB2
  • Src Met tyrosine kinase, MEK 1/2 (mitogen-activated protein kinase kinase), Akt, Raf-1, Cyclin-dependent serine kinases, steroid hormone receptors, telomerases, Hsp90 client proteins, including metalloprotein-2 (MMP-2) and hypoxia-inducible factor-1 ⁇ (HIF-1 ⁇ ), are available on a variety of signaling pathways, including cell survival, proliferation, invasion, metastasis, and neovascularization.
  • MMP-2 metalloprotein-2
  • HIF-1 ⁇ hypoxia-inducible factor-1 ⁇
  • the cancer is prostate cancer, ovarian cancer, lung cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, gallbladder and biliary tract cancer, breast cancer, leukemia, esophageal cancer, non-Hodgkin's lymphoma, thyroid cancer, cervical cancer, skin cancer It may be characterized in that it is one or more cancers selected from the group consisting of metastatic cancer caused by metastasis to other organs, and tumor cell disease produced by promoting abnormal hypercellular division.
  • the cancer may be characterized as breast cancer, the breast cancer may be characterized as triple negative breast cancer or HER2-positive breast cancer.
  • HSP90 increases casapse activity and is associated with cell infiltration, migration and cancer metastasis of triple negative breast cancer.
  • the representative clients of phospho-EGFR and phospho-STAT3 they significantly reduced the expression of AKT, ERK, Cyclin D1, and Survivin. It confirmed (FIG. 4, FIG. 5, and FIG. 6).
  • NCT 44, NCT 50 and NCT 58 is superior in solubility than deguelin.
  • compositions of the invention can be formulated in a variety of oral or parenteral dosage forms.
  • Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc.
  • These formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose). Rose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols.
  • Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally such as starch, agar, alginic acid or its sodium salt. Disintegrant or boiling mixtures and / or absorbents, colorants, flavors, and sweeteners.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally such as starch, agar, alginic acid or its sodium salt.
  • compositions comprising the compound of formula 1 according to the present invention as an active ingredient may be parenterally administered, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.
  • parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.
  • the compound of Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or a buffer to prepare a solution or suspension, which is prepared in ampule or vial unit dosage form. can do.
  • compositions may contain sterile and / or preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating It may be formulated according to the formulation or coating method.
  • the compound of formula (1) is orally or parenterally divided or administered once daily in an amount of 0.1 to 500 mg / kg body weight, preferably 0.5 to 100 mg / kg body weight, for mammals including humans. Administration can be via the old route.
  • compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
  • Reagents and conditions include (a) 3-methyl-but-2-enal, pyridine, 140 ° C., on; (b) MeI, K 2 CO 3 , DMF, 70 ° C., 2 h; (c) H 2 O 2 , NaH 2 PO 4 , NaClO 2 , AC,, rt, 2h; (d) K 2 CO 3 , DMF, 70 ° C., 3 h; (e) 10% Pd / C, H 2; (g) DCM, rt, 2h; (f) EDC-HCl, TEA, HOBt, DCM.
  • the synthesized nitro compound (1 eq) was dissolved in methanol, and then 10% Pd / C (0.1 eq) was added and stirred for 1 hour at room temperature under the condition of supplying hydrogen gas. After confirming that the reaction was completed using TLC, the resultant was concentrated under reduced pressure after the celite filter to obtain aniline.
  • the synthesized aniline compound and the carboxylic acid compound were dissolved in DCM, and then EDC.HCl (1.5 eq), HOBt (1.5eq), and TEA (2.5eq) were added.
  • the mixed solution was stirred for 15 hours under reflux conditions, and after completion of the reaction, the heat transfer plate was removed, cooled to room temperature, and water was added to terminate the reaction.
  • the mixed organic material was extracted with DCM, washed several times with brine, dried over MgSO 4 and concentrated under reduced pressure.
  • the synthesized compound was purified using silica gel column chromatography.
  • novel benzopyran derivatives were separated and purified by silica gel column chromatography, and the molecular structure was confirmed by nuclear magnetic resonance.
  • Example 2 benzopyran derivatives HSP90 inhibitor NCT1 Survival of breast cancer cell lines BT474, JIMT-1 and MDA-MB-231 of 13 synthetics
  • the human breast cancer cell lines BT474, JIMT-1 and MDA-MB-231 each contain 10% fetal bovine serum (FBS), streptomycin-penicillin (100 U / ml) and Fungizone (0.625 ⁇ g / ml).
  • FBS fetal bovine serum
  • streptomycin-penicillin 100 U / ml
  • Fungizone 0.625 ⁇ g / ml
  • DMEM was incubated in a 5% CO 2 , 37 °C environment.
  • Novel benzopyrani derivatives HSP90 inhibitor NCT1 13 in human breast cancer cell lines BT474, JIMT-1 and MDA-MB-231 (NCT-20, NCT-29, NCT-44, NCT-58, NCT-364, NCT- 375, NCT-377, NCT-383, NCT-385, NCT-394, and NCT-407) were treated at 10 ⁇ M for 72 hours, and then cell viability was measured by MTS assay.
  • NCT-58 and NCT-407 were treated with HSP90 inhibitors NCT-58 and NCT-407 at various concentrations of 0, 0.1, 0.5, 1, 5, 10 and 20 ⁇ M, respectively, for 72 hours. Then, cell viability was measured by the MTS assay technique (Cell seeding numbers: 2 (B), 1 (J), 0.7 (M) x 10 4 cells / wells (confluency ⁇ 25%)). It was confirmed that NCT-58 and NCT-407 drugs significantly inhibited cell viability in three breast cancer cell lines.
  • Example 4 Investigation of cell morphology of benzopyran derivatives NCT-58 and NCT-407 in breast cancer cell lines BT474, JIMT-1 and MDA-MB-231
  • Example 5 Determination of apoptosis rates of benzopyran derivatives NCT-58 and NCT-407 drugs in breast cancer cell lines BT474, JIMT-1 and MDA-MB-231
  • NCT-58 and NCT-407 drug-induced apoptosis were measured by DNA content analysis using flow cytometry.
  • BT474, JIMT-1 and MDA-MB-231, control (DMSO), NCT-58 and NCT-407 were treated for 72 hours at a concentration of 10 ⁇ M, respectively, and the cells were harvested, followed by 0.5 for 24 hours.
  • PI propidium iodide
  • RNase 50 ⁇ g / ml
  • the cell cycle is divided into G1 (cell growth phase) -S (cell replication phase) -G2 / M (cell division phase) according to the content of intracellular DNA, and DNA fragmentation (DNA) is induced when cell death is induced. With fragmentation, the content of DNA in each cell is significantly less than that of the G1 phase.
  • the result of such apoptosis is shown in the cell cycle as the Sub G1 site, and the ratio of Sub G1 is expressed in numerical values, respectively, in FIG. 4.
  • NCT-58 and NCT-407 drugs were found to significantly induce apoptosis (Sub-G1 population) in BT474, JIMT-1, MDA-MB-231 breast cancer cell lines. Apoptosis experiments were performed three times independently, the significance was verified by unpaired Students t-test (* p ⁇ 0.01; DMSO control vs NCT-58 or NCT-407), shown in Figure 4 graphically for each cell line It was.
  • Example 6 Expression of apoptosis-related factors by benzopyran derivatives NCT-58 and NCT-407
  • Example 5 in order to investigate the apoptosis mechanism by NCT-58 and NCT-407, activation of the apoptosis-related factor caspase family member was investigated by Western Blotting technique.
  • the membrane was prepared with primary antibodies diluted in 5% bovine serum albumin (BSA) [pro PARP (1: 1000), cleaved PARP (1: 2000), cleaved caspase-3 (1: 1000), cleaved caspase-7 (1: 1000) and ⁇ -actin (1: 5000)] at 4 ° C. for 24 hours, and then reacted with a secondary antibody, horseradish peroxidase (HRP) -conjugated rabbit IgG (1: 3000), at room temperature for 2 hours.
  • BSA bovine serum albumin
  • HRP horseradish peroxidase
  • Example 7 Expression of HSP90 clients by benzopyran derivatives NCT-58 and NCT-407
  • the BT474 and JIMT-1 cell lines are HER2-positive breast cancer cell lines and Western blotting whether HSP90 inhibitors NCT-58 and NCT-407 can downregulate the expression and activity of HER2 and HER1 (EGFR), the representative clients of HSP90.
  • the technique was investigated.
  • NCT-58 and NCT-407 were treated at 10 ⁇ M concentration for 72 hours, the expression of HER2 and EGFR of BT474 and JIMT-1 cell lines was decreased, and the expression of the active forms of phospho-HER2 and phospho-EGFR was also reduced. Significantly reduced.
  • both drugs inhibited the expression of cell growth factors, ERK, Cyclin D1 and Survivin.
  • the MDA-MB-231 cell line is a triple-negative breast cancer (TNBC) cell line and is a carcinoma having a high recurrence rate and metastasis rate of breast cancer.
  • TNBC triple-negative breast cancer
  • the expression and phosphorylation of EGFR and STAT3, the representative clients of HSP90, related to cell invasion, migration and cancer metastasis of triple negative breast cancer were measured, and expression of survival factors AKT, ERK, Cyclin D1 and Survivin was investigated.
  • Both NCT-58 and NCT-407 drugs significantly reduced the expression of the active forms of phospho-EGFR and phospho-STAT3 as well as the expression of EGFR and STAT3 in trinegative breast cancer MDA-MB-231 cell lines, and AKT, ERK, and Cyclin D1. And significantly inhibited the expression of Survivin. Expression results of the client by the drug is shown in FIG.
  • the primary antibodies used for Western blotting are [HER2 (1: 5000), phospho-HER2 (Tyr1221 / 1222, 1: 1000), EGFR (1: 2000), phospho-EGFR (Tyr1068, 1: 2000), Akt ( 1: 2000), ERK (1: 2000), phospho-ERK (Thr202 / Tyr204, 1: 2000), STAT3, phospho-STAT3 (Tyr705, 1: 1000), Cyclin D1 (1: 3000), Survivin (1: 1000), ⁇ -actin (1: 5000)] and diluted with 5% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Example 8 Investigation of the effect of benzopyran derivatives on cell viability in human prostate cancer cell line DU145, colon cancer cell line HCT116, liver cancer cell line HepG2 and ovarian cancer cell line SKOV-3
  • HSF-1 is a transcriptional regulator that is known to upregulate the expression of HSP70, a gene related to heat shock response, and is present in the cytoplasmic binding to HSP90.
  • HSF-1 is separated from HSP90 to form trimerization in the cytoplasm and phosphorylated to have activity.
  • HSF-1 enters the nucleus, binds to the DNA's heat shock response element (HSE), and increases the expression of HSP genes involved in thermal shock responses such as HSP70, HSP40, and HSP27. Inhibitory defense mechanisms are known to activate.
  • HSE heat shock response element
  • NCT-58 (N58), a benzopyran derivative of the present invention, confirmed that HSF-1 did not accumulate in the nucleus of SKBR3 cells as a C-terminal HSP90 inhibitor, and geldanamycin (Gelda), an N-terminal HSP90 inhibitor, was HSF-1. Strong expression in this nucleus was confirmed by immunocytochemistry. Expression of HSF-1 by the drug is shown in FIG. 8.
  • SKBR3 cell lines were dispensed into chamber slides and treated with NCT-58 or geldanamycin for 24 hours, and the cells were fixed with 4% paraformaldehyde. Immunofluorescence staining was performed using HSF-1 primary antibody and Alexa-488 goat anti-rabbit IgG secondary antibody. Cell nuclei were stained with DAPI. The image was taken at a magnification of 500 times with a confocal microscope.
  • HSP70 The expression of HSP70 was examined to confirm that NCT-58 (N58) compound did not induce a thermal shock response.
  • HSF-1-induced HSP70 binds to caspases, death receptors and bax, which are cell death inducing factors, and is known to activate defense mechanisms that ultimately inhibit cell death.
  • Geldanamycin (Gelda) an N-terminal HSP90 inhibitor, strongly confirmed the induction of HSP70 by immunocytochemistry.
  • the C-terminal HSP90 inhibitor NCT-58, a benzopyran derivative demonstrated that both SKBR3 and JIMT-1 cell lines did not induce the expression of HSP70. Expression of HSP70 by the drug is shown in FIG. 9.
  • SKBR3 and JIMT-1 cell lines were dispensed into chamber slides, treated with NCT-58 or geldanamycin for 24 hours, and the cells were fixed with 4% laformaldehyde. Immunofluorescence staining was performed using HSP70 primary antibody and Alexa-488 goat anti-rabbit IgG secondary antibody. Cell nuclei were stained with DAPI. The image was taken at a magnification of 500 times with a confocal microscope.
  • the standard curve was serial dilution of 1 mg / ml product solution to produce solutions of final concentrations of 100 ng / ml, 200 ng / ml and 500 ng / ml. Using this solution as a standard, the concentration of the sample was calculated, and MeOH was used as the mobile phase in serial dilution.
  • NCT 44 and NCT 58 had better solubility than deguelin.

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  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un nouveau dérivé de benzopyrane ou un sel pharmaceutiquement acceptable de celui-ci et une composition pharmaceutique le comprenant en tant que principe actif. Le composé représenté par la formule chimique 1, un sel pharmaceutiquement acceptable de celui-ci, ou un solvate du composé, selon la présente invention n'a aucune influence sur la structure complexe de HSP90 et de HSF-1, n'induit pas l'activation de HSF-1 ou l'expression de gènes de HSP, a une augmentation efficace conséquente de la mort des cellules cancéreuses, ce qui lui permet d'être utilisé en tant que principe actif d'un agent anticancéreux.
PCT/KR2019/010731 2018-08-23 2019-08-23 Nouveau dérivé de benzopyrane ou sel pharmaceutiquement acceptable de celui-ci et composition pharmaceutique le comprenant en tant que principe actif Ceased WO2020040582A1 (fr)

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KR1020180098788A KR102304532B1 (ko) 2018-08-23 2018-08-23 신규한 벤조파이란 유도체 또는 이의 약학적으로 허용가능한 염 및 이를 유효성분으로 함유하는 약학적 조성물
KR10-2018-0098788 2018-08-23

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Cited By (1)

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EP4603488A4 (fr) * 2022-10-12 2025-12-24 Univ Korea Res & Bus Found Nouveau dérivé d'indazole ou sel pharmaceutiquement acceptable de celui-ci et son utilisation

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KR102455529B1 (ko) * 2020-01-09 2022-10-20 연세대학교 산학협력단 새로운 벤조피란 유도체 제조방법 및 이를 포함하는 항암제 조성물
WO2022158624A1 (fr) * 2021-01-25 2022-07-28 연세대학교 산학협력단 Nouveau composé dérivé de benzopyrane, son procédé de préparation et composition anticancéreuse le comprenant
KR102634945B1 (ko) * 2021-11-16 2024-02-08 (주)케이메디켐 신규한 카나비크로멘산 유도체, 이의 제조방법 및 이를 포함하는 인지기능 개선용 조성물
WO2024144327A1 (fr) * 2022-12-29 2024-07-04 의료법인 성광의료재단 Nouvelle composition pour le traitement de maladies inflammatoires de la peau

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