WO2022059996A1 - Blood circulation micro in-vitro corpuscle-mediated cancer treatment composition - Google Patents

Blood circulation micro in-vitro corpuscle-mediated cancer treatment composition Download PDF

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WO2022059996A1
WO2022059996A1 PCT/KR2021/012192 KR2021012192W WO2022059996A1 WO 2022059996 A1 WO2022059996 A1 WO 2022059996A1 KR 2021012192 W KR2021012192 W KR 2021012192W WO 2022059996 A1 WO2022059996 A1 WO 2022059996A1
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substituted
unsubstituted
group
alkyl
cancer
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PCT/KR2021/012192
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French (fr)
Korean (ko)
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강건욱
박미소
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서울대학교 기술지주 주식회사
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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
    • A61K31/495—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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/48—Two nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to a composition for the treatment of blood circulation microexosome-mediated cancer, and more particularly, to a composition for preventing or treating cancer comprising the compound represented by Formula 1 or a salt thereof as an active ingredient.
  • Cancer is a generic term for a group of diseases that start from the uncontrolled proliferation of cells, invade and destroy surrounding normal tissues or organs, and create a new growth site that can take away the life of an individual.
  • new targets including oncogenes and cancer suppressor genes, the regulation of the cell cycle or apoptosis to conquer cancer, the incidence of cancer has increased with the development of civilization. is increasing as
  • Chemotherapy, surgery, radiation therapy, etc. are used as a treatment method for cancer.
  • chemotherapy is the most used for the treatment of cancer as a method of using an anticancer agent.
  • Today about 60 kinds of various anticancer drugs are being used in clinical practice, and new anticancer drugs are continuously being developed as knowledge about cancer occurrence and characteristics of cancer cells is widely known.
  • mitomycin C mitomycin-C
  • adriamycin adriamycin myelosuppressive action
  • cisplatin the most useful anticancer drug developed so far, is widely used in the treatment of testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer and cervical cancer, but hematopoietic toxicity such as anemia, vomiting, and nausea
  • hematopoietic toxicity such as anemia, vomiting, and nausea
  • side effects such as digestive toxicity such as digestive toxicity, kidney toxicity such as kidney tubular damage, hearing loss, electrolyte abnormality in the body, shock, and peripheral nerve abnormality is a big problem (RT Skeel, Handbook of Cancer Chemotherapy, pp.89-91, 1999).
  • gefitinib gefitinib; Some drugs, such as the trade name, Iressa
  • Iressa Some drugs, such as the trade name, Iressa
  • these molecular targeted therapeutics that selectively inhibit only a specific target have a problem in that resistance occurs due to long-term administration, and there is a disadvantage in showing a selective therapeutic effect according to the mutation status of a specific gene.
  • the present invention has been devised to solve the prior art needs as described above, and the present inventors confirmed that the compound represented by Formula 1 inhibits cancer cell proliferation as well as metastasis and mobility, thereby preventing cancer
  • the preventive or therapeutic effect and the cancer metastasis inhibitory effect were confirmed, and the present invention was completed based on this.
  • a pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the cancer is a circulating microextrabody (csEV)
  • csEV circulating microextrabody
  • Another object of the present invention is to provide a pharmaceutical composition for inhibiting cancer metastasis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the cancer is It provides a pharmaceutical composition, characterized in that it is a blood circulating microextrabody (csEV) mediated cancer.
  • csEV blood circulating microextrabody
  • R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
  • substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
  • R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
  • substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted
  • R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.
  • the R 3 and R 4 may each independently be one or more hydrogen, Cl, F, or methyl, but is not limited thereto.
  • the compound represented by Formula 1 may be at least one selected from the group consisting of the compounds shown in Table 1, but is not limited thereto.
  • the composition may further include gefitinib or a pharmaceutically acceptable salt thereof, but is not limited thereto.
  • the blood circulation micro-extrabody (csEV)-mediated cancer is a cancer with migratory ability, that is, if it is a metastatic cancer, but is not limited thereto, for example, prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer , thyroid cancer, ovarian cancer, bladder cancer, kidney cancer, stomach cancer, uterine cancer, rectal cancer, may be at least one selected from the group consisting of pancreatic cancer and melanoma.
  • the compound represented by Formula 1 may have one or more effects selected from the group consisting of cancer cell growth inhibition, invasion inhibition, and metastasis inhibition, but limited thereto it is not going to be
  • the pharmaceutical composition may be an immuno-cancer agent, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for enhancing the anticancer efficacy of gefitinib comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for inhibiting cancer metastasis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the cancer may be a blood circulation microextrabody (csEV) mediated cancer, but is not limited thereto.
  • csEV blood circulation microextrabody
  • the compound represented by Formula 1 may control the M2 immune response of cancer cells, but is not limited thereto.
  • the present invention provides a method for preventing cancer or cancer metastasis, comprising administering a pharmaceutical composition comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient to an individual in need of treatment for cancer or a method of treatment.
  • the present invention provides a use for preventing or treating cancer or cancer metastasis of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for producing a medicament for preventing or treating cancer or cancer metastasis.
  • the compounds of the present invention have excellent anti-proliferative activity of cancer cells such as lung cancer, colon cancer, prostate cancer, breast cancer, and liver cancer mediated by blood circulating microextrabody (csEV), and also have excellent anti-proliferative activity against anticancer drug-resistant cancer cell lines, and for invasive cancer Since it not only exhibits a proliferation inhibitory effect, but also significantly inhibits cancer growth in xenograft and allograft animal models of cancer cells, it can be usefully used as a drug for inhibiting cancer metastasis as well as preventing or treating cancer.
  • cancer cells such as lung cancer, colon cancer, prostate cancer, breast cancer, and liver cancer mediated by blood circulating microextrabody (csEV)
  • csEV blood circulating microextrabody
  • FIG. 1A illustrates a process for isolating blood circulating microexosomes (csEV) according to an embodiment of the present invention.
  • Figure 1b shows the result of checking the size of the separated csEV by DLS.
  • Figure 1c shows the results of confirming the incorporation of isolated csEV into LNCaP-SL prostate cancer cells when exposed to the cells with a confocal microscope.
  • Figure 1d shows the effect of csEV to promote migration of LNCaP-SL prostate cancer cells.
  • Figure 1e shows the migration promoting effect of csEV cancer cells (HCT116, SW480, GR-HCC827, H1975, primary BCC).
  • Fig. 1f shows a csEV separation method according to the difference in surface charge with electric field stimulation of csEV.
  • Figure 1g shows the migration effect according to the surface charge of csEV.
  • Figure 2 shows the inhibitory effect of KRCT-6j on the migration of LNCaP-SL cells.
  • Figure 3a shows the rate of cancer cell proliferation in the presence or absence of csEV in FBS.
  • Figure 3b shows the rate of cancer cell proliferation by csEV treatment in FBS depletion condition.
  • Figure 3c shows the activity of AKT and ERK by csEV treatment.
  • 3D shows the results of reduced YAP expression and nuclear localization by KRCT-6j treatment in csEV-mediated cancer cells.
  • Figure 4a shows the results of confirming the proliferation of metastatic prostate cancer cells by docetaxel treatment in the presence or absence of csEV in FBS.
  • Figure 4b shows the results of confirming the proliferation of H1993 and GR-H1993 cells by csEV and KRCT-6j treatment.
  • Figure 4c shows the results of confirming the proliferation of parental H1993 cells, GR-H1993 cells and ER-H1993 cells by csEV and KRCT-6j treatment.
  • Figure 4d shows the results of confirming the YAP expression level in H1993 and GR-H1993 cells.
  • Figure 4e shows the results of confirming the nuclear localization of YAP by KRCT-6j treatment in GR-H1993 cells.
  • Figure 5a shows the synergistic effect of the combined treatment of KRCT-6j and gefitinib.
  • Figure 5b shows the tumor growth inhibitory effect in the GR-H1993 xenograft mouse model according to the combined treatment of KRCT-6j and gefitinib.
  • Figure 5c shows the inhibitory effect of tumor volume increase according to the combination treatment of KRCT-6j and gefitinib.
  • Figure 5d shows the results of confirming the expression of the tumor growth marker Ki67 according to the combined treatment of KRCT-6j and gefitinib.
  • FIG 6 shows the inhibitory effect of LNCaP-SL and HCT116 cancer cell proliferation according to the treatment of the compound of the present invention.
  • HCC827 and GR-HCC827 according to compounds No. 87 and No. 203 treatment.
  • H1975, H1993, ER-H1993, and GR-H1993 shows the effect of inhibiting the proliferation of cancer cells.
  • Figure 8 shows the inhibitory effect on the proliferation of HT29, OR-HT29, HCT116, OR-HCT116 colon cancer cells according to the treatment of compounds 87 and 203.
  • Figure 9a shows the inhibitory effect on the proliferation of LNCaP-O, and LNCaP-SL prostate cancer cells according to the treatment of compounds 87 and 203.
  • Figure 9b shows the inhibitory effect on the proliferation of Huh7, and HepG2 hepatocellular carcinoma cells according to the treatment of compounds No. 87 and No. 203.
  • FIG. 10 shows the inhibitory effect on cancer cell proliferation in 4T1 mammary gland cancer cells and MC38 colorectal cancer cell allograft mouse models according to compounds 87 and 203 treatment.
  • HCT116 HCT116, OR-HCT116 (abnormal colorectal cancer cells), MDA-MB-231, 4T1, tamoxifen-resistant MCF-7 (TAMR-MCF-7) [abnormal human and mouse breast (adenocarcinoma) cells], LNCaP-SL (prostate cancer cells), erlotinib-resistant H292 cells (ER-H292), GR-H1993, GR-HCC-827, H1975 [abnormal EGFR-TKI-resistant lung cancer cells] showed the effect of inhibiting the migration of cancer cells.
  • TAMR-MCF-7 tamoxifen-resistant MCF-7
  • Figure 12 shows the spheroid formation inhibitory activity of 4T1 and GR-H1993 cells according to the treatment of compounds No. 87 and No. 203.
  • Figure 13 shows the spheroid formation inhibitory activity of HCT116 and OR-HCT116 cells according to compound 87 and 203 treatment.
  • Figure 14 shows the spheroid formation inhibitory activity of HT29 and OR-HT29 cells according to compound 87 and 203 treatment.
  • Figure 15a shows the results of confirming the expression of TGF-beta in macrophages exposed to IL-4 treatment according to compounds 87 and 203 treatment.
  • 15B shows the results of confirming the expression of arg1 and ciita, which are representative markers of M2 macrophages, in macrophages exposed to IL-4 treatment according to compound 87 treatment.
  • 16A shows an experimental protocol for administering compounds No. 87 and No. 203 to an allograft model transplanted with 4T1 mouse mammary cancer cells.
  • Figure 16b shows the results of measuring the tumor volume as a result of administering the compounds No. 87 and No. 203 to the allograft model transplanted with 4T1 mouse mammary cancer cells.
  • Figure 16c shows the body weight as a result of administering the compounds No. 87 and No. 203 to the allograft model transplanted with 4T1 mouse mammary cancer cells.
  • 17A shows an experimental protocol for administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • Figure 17b shows the results of measuring the tumor volume as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • Figure 17c shows the results of measuring the number of CD45+ cells compared to the tumor weight as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • FIG. 17d shows that the tumor-associated macrophages were defined as CD45+Ly6G-Ly6C-F4/80+CD11b+ as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • 17e shows the results of measuring the I-A/I-E ratio expressed by TAM as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • 17f shows the results of measuring the number of cytotoxic CD8+ T cells as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • 17g shows the results of measuring the ratio of FOXP3+CD4+ T cells, which are markers of regulatory T cells, as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • Figure 17h shows an experimental protocol for confirming the phenotypic change of T cells by administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
  • Figure 17i shows the secretion of interferon- ⁇ (IFN- ⁇ ) and tumor necrosis factor- ⁇ (TNF- ⁇ ) in CD4+ and CD8+ T cells following administration of compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells. shows the results of checking .
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the present invention provides a pharmaceutical composition for preventing, treating, and inhibiting cancer metastasis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the compound of the present invention may be one represented by the following formula (1):
  • R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
  • substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
  • R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
  • substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted
  • R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.
  • C 1-5 alkyl refers to a monovalent alkyl group having 1 to 5 carbon atoms
  • C 1-3 alkyl refers to a monovalent alkyl group having 1 to 3 carbon atoms.
  • the term includes functional groups such as methyl, ethyl, n -propyl, i -propyl, n -butyl, i -butyl, tert -butyl and the like.
  • Alkyl, and other substituents containing alkyl moieties described herein include both straight-chain and comminuted forms.
  • C 1-5 alkoxy refers to an -OR group, where R stands for "C 1 -C 5 alkyl".
  • Preferred alkoxy groups include, for example, methoxy, ethoxy, phenoxy, and the like.
  • the substituents containing alkyl, alkoxy and other alkyl moieties described in the present invention include both straight chain and comminuted forms.
  • halogen may include fluoro (F), chloro (Cl), and bromo (Br) and iodine (I).
  • C 6 -C 10 aryl refers to an unsaturated aromatic ring compound having 6 to 10 carbon atoms having a single ring (eg phenyl) or a plurality of condensed rings (eg naphthyl). .
  • the aryl may be selected from the group consisting of phenyl, naphthyl, anthryl and biaryl.
  • C 6 -C 10 heteroaryl is an aryl group containing 1 to 3 heteroatoms selected from S, O and N, dioxolyl, pyridyl, pyrimidyl, thiophenyl, p It may be selected from the group consisting of rollyl, furanyl and triazolyl, but is not limited thereto.
  • C 6 -C 10 arylalkyl used in the present invention is represented by -(CH 2 ) n -R, where R means an aryl group, having an aryl substituent including benzyl, phenethyl, etc.
  • R means an aryl group, having an aryl substituent including benzyl, phenethyl, etc.
  • C 6 -C 10 aryl refers to an unsaturated aromatic ring compound having 6 to 20 carbon atoms having a single ring (eg phenyl) or a plurality of condensed rings (eg naphthyl). .
  • the aryl includes phenyl, naphthyl, and the like.
  • the R 3 and R 4 may each independently be one or more hydrogen, Cl, F, or methyl, but is not limited thereto.
  • the compound represented by Formula 1 may be one or more selected from the group consisting of the compounds shown in Table 1, but is not limited thereto.
  • composition may further include gefitinib or a pharmaceutically acceptable salt thereof, but is not limited thereto.
  • the compound represented by Formula 1 may have one or more effects selected from the group consisting of cancer cell growth inhibition, invasion inhibition, and metastasis inhibition, but is not limited thereto.
  • the blood circulating microextrabody promotes the migration and proliferation of cancer cells (see Example 1), csEV-mediated cancer migration, and expression of YAP in cancer cells and the compound of the present invention inhibits nuclear localization (see Examples 2 to 4), and the compound of the present invention exhibits a synergistic effect on gefitinib-resistant cancer cells when used in combination with gefitinib (see Example 5),
  • the compound of the present invention not only inhibits the proliferation of colon cancer, prostate cancer, metastatic prostate cancer, pesso-cell lung cancer, anticancer drug-resistant colorectal cancer, and liver cancer cells, but also has excellent cancer cell proliferation inhibitory effect in mammary and colon cancer allograft mouse models.
  • the compound of Formula 1 of the present invention can be used for cancer treatment and cancer metastasis inhibition, as it was confirmed that there was an effect of inhibiting proliferation as well as migration inhibition (see Example 6).
  • cancer is a disease related to the regulation of cell death, and refers to a disease caused by excessive cell proliferation when the balance of normal apoptosis is disrupted. In some cases, these abnormal hyperproliferative cells may invade surrounding tissues and organs to form a mass, and may cause destruction or transformation of normal structures in the body. This condition is collectively called cancer.
  • a tumor refers to an abnormally grown mass due to the autonomous overgrowth of body tissues, and tumors can be divided into benign tumors and malignant tumors. Malignant tumors grow very rapidly compared to benign tumors, and metastasis occurs while infiltrating the surrounding tissues, threatening life. Such malignant tumors are commonly referred to as 'cancer'.
  • Cancer metastasis is the spread of cancer cells from primary cancer to other organs to form new cancer. Since metastasis is a major life-threatening phenomenon in various cancer patients, preventing or controlling metastasis is an important goal in cancer research. Surgery, chemotherapy, or radiation therapy are effective in the case of early diagnosis without metastasis, but the effectiveness of these treatments is reduced if metastasis has occurred at the time of diagnosis. In addition, although metastasis could not be confirmed at the time of diagnosis, metastasis is often identified during or after treatment. Although metastasis of cancer is clinically important, the metastasis process is still not fully understood.
  • Metastasis consists of successive stages such as invasion, intravasation, arrest, extravasation, and colonization, and through this process, Eventually, cancer will form in other organs.
  • the first stage, invasion is the starting stage of metastasis, and includes changes in the interaction of cancer cells with cells or extracellular matrix, degradation of surrounding tissues, and migration of cancer cells into tissues.
  • the second step, intravascular entry is when cancer cells pass through the endothelial cells of blood vessels or lymphatic vessels and are included in the systemic circulation. It has been confirmed that only a small fraction of the introduced cancer cells survive the cycle. A part of the cancer cells that survived succeed in extravasation through the capillary endothelial cells in other areas, adapt to the new environment, and proliferate to form metastatic cancer.
  • the cancer may be a blood circulation microextrabody (csEV) mediated cancer, but is not limited thereto.
  • the blood circulation microextrabody (csEV) mediated cancer is a cancer with mobility, that is, metastatic cancer, but is not limited thereto, for example, prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, thyroid cancer, ovarian cancer, bladder cancer, kidney It may be at least one selected from the group consisting of cancer, stomach cancer, uterine cancer, rectal cancer, pancreatic cancer and melanoma.
  • the compound represented by Formula 1 may control the M2 immune response of cancer cells, but is not limited thereto.
  • M2-type macrophages are cells that have an absolute effect on the survival of cancer by inhibiting the activity of other immune cells and helping with angiogenesis.
  • the compound of the present invention was shown to significantly inhibit the increase in TGF-beta expression of M2-type macrophages by IL-4 treatment. was confirmed to be controlled.
  • the present invention provides an immunotherapy agent comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the immuno-oncology agent refers to a therapeutic agent that induces immune cells to selectively attack only cancer cells by stimulating the immune system. It is known that CD8+ T cells infiltrating the tumor, CD4+ T cells differentiated into T helper 1 (TH1), and CD103+ DCs play a central role in the anticancer immune response, and in one embodiment of the present invention, a colorectal cancer allograft mouse model As a result of administering the compound of the present invention, CD8+ T cells, which can directly induce cancer cell death by recognizing the antigen of cancer cells, significantly increase, and are known to decrease the immune function of helper T cells in the vicinity of cancer cells. It was confirmed that the ratio of FOXP3 + CD4 + T cells, which is a regulatory T cell marker, was rather decreased. Therefore, it is expected that the compound of the present invention can be usefully used as an immuno-cancer agent.
  • the present invention provides a pharmaceutical composition for inhibiting cancer metastasis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a method for treating cancer comprising administering to an individual in need of cancer treatment a pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient do.
  • the present invention provides a cancer treatment use of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for producing a cancer therapeutic agent.
  • the present invention provides a treatment for cancer metastasis comprising administering a pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient to an individual in need of treatment for cancer metastasis provide a way
  • the present invention provides a use for inhibiting cancer metastasis of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for producing a cancer metastasis inhibitor.
  • the present invention may also include the pharmaceutically acceptable salt as an active ingredient.
  • pharmaceutically acceptable salt includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
  • acids examples include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like.
  • Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an aqueous solution of an excess of acid, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can also be prepared by heating an equimolar amount of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or by suction filtration of the precipitated salt.
  • a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile.
  • Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • the alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate.
  • the metal salt it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).
  • the content of the compound in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition.
  • the content ratio is a value based on the dry amount from which the solvent is removed.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.
  • the excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.
  • the pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, alcohols, troches, fragrances, and limonaade.
  • tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • formulation it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone,
  • sucrose solution other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifying agent, thickening agent, etc. may be used.
  • Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.
  • a suspending agent such as acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910 may be used. and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.
  • Injectables according to the present invention include distilled water for injection, 0.9% sodium chloride injection, ring gel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated ring gel injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin, pepton
  • the suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neos
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • excipients for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • composition according to 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 is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.
  • the pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration can be contemplated, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.
  • “individual” means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. It may be a mammal of, but is not limited thereto.
  • administration means providing a given composition of the present invention to a subject by any suitable method.
  • prevention means any action that inhibits or delays the onset of a desired disease
  • treatment means that the desired disease and metabolic abnormalities are improved or It means all actions that are advantageously changed
  • improvement means all actions that reduce the parameters related to the desired disease, for example, the degree of symptoms by administration of the composition according to the present invention.
  • Example 1 Identification of the role of negatively charged blood circulating microextrabody (csEV) in cancer cell migration
  • csEV was isolated from human, bovine and porcine serum using five-step successive centrifugation.
  • csEV derived from bovine serum of the same lot number was used in a similar experiment, and the main data were confirmed using csEV from human serum.
  • Porcine serum and bovine serum were purchased from Invitrogen (Karlsruhe, Germany), and the serum obtained from a healthy adult male was centrifuged step by step to remove dead cells and residues. First, the cells were centrifuged at 300 g for 10 minutes and at 2500 g for 20 minutes to remove residual cells and residues, and the supernatant was taken. The obtained supernatant was centrifuged at 10000 g for 30 minutes to remove microvesicles. 200nm filtration was performed to remove vesicles and contaminants larger than the EV size from the supernatant obtained therefrom.
  • the supernatant finally obtained through this process was ultracentrifuged at 120000 g, 120 minutes (Optima XE-100, Type 32Ti rotor, Beckman Coulter). Next, discard all of the supernatant to remove EV-contaminated proteins, fill the tube with DPBS, perform ultracentrifugation once more at 120000 g, 90 minutes, discard the supernatant, minimize contamination and separate pure EVs For this purpose, 200 nm filtration was performed again. After dissolving in a small amount of PBS or solution for the experiment, it was used for the experiment, and Bradford assay was performed for EV quantification. Approval from the Seoul National University Ethics Committee was obtained for the acquisition of adult male serum (#SNU 19-2-040), and the isolated csEV was stored at 4°C and used within 5 days.
  • the size of the EVs isolated from the direct light scattering (DLS) data was less than 100 nm, and they were classified as microex vivos.
  • Transaswell migration experiments were performed using IncuCyte ClearView 96-well chemotaxis plates (Essen bioscience, MI, USA). The lower part of the transwell unit was coated with Type I Collagen (Collaborative Research, KY, USA) and dried at room temperature for 1 hour. After preparing at 13 ⁇ 10 3 cells/well in RPMI medium without FBS, the experimental group or control group treated with the drug was seeded at the upper end of the transwell unit by 60 ⁇ L. A total of 200 ⁇ L of medium containing 10% FBS, 10% EV-free FBS, or csEV in serum-free RPMI media was used for the lower well containing the attracting chemical. Cell migration was monitored at 4-hour intervals using Incucyte Chemotaxis live-cell analysis (ESSEN bioscience).
  • csEV derived from bovine, porcine or human serum promoted migration of LNCaP-SL cancer cells, an aggressive subline of LNCaP prostate cancer cells.
  • csEV derived from human serum exhibited the strongest migratory capacity (38.1 fold).
  • LNCaP-SL prostate cancer
  • tamoxifen-resistant-MCF-7 breast cancer
  • gefitinib-resistant-HCC-827 lung cancer
  • HCT116 colon cancer
  • SW480 colon cancer
  • the cell migration of five cancer cells was increased in a concentration-dependent manner with csEV.
  • NTA Nanoparticle Tracking Analysis
  • csEVs having a charge distribution were distinguished by electron stimulation, and the differential migration ability according to the 'surface charge' of csEVs was observed.
  • the relatively negatively charged csEV showed the strongest migration effect than other csEVs. It was determined that the lipid component of EV is involved because the lipid having a negative charge is a surface component of EV.
  • a transwell migration experiment was performed using an IncuCyte ClearView 96-well chemotactic plate (Essen bioscience, MI, USA). The lower part of the transwell unit was coated with Type I collagen (Collaborative Research, KY, USA) and dried at room temperature for 1 hour. After preparing 3 ⁇ 10 3 cells/well in RPMI medium without FBS, 60 ⁇ L of the experimental group or control group treated with the drug was dispensed at the upper end of the transwell unit. A total of 200 ⁇ L of medium supplemented with csEV or PBS as a control was used for the lower wells containing the attracting chemical. Cell migration was monitored at 4-hour intervals using Incucyte Chemotaxis live-cell analysis (ESSEN bioscience).
  • the proliferation of LNCaP-SL was not affected by csEV under FBS-containing medium, but csEV treatment significantly promoted cell proliferation in the serum-free state (7.8-fold VS 12.8-fold at 96 h). ).
  • Example 4 Identification of a target for overcoming chemical resistance in invasive cancer
  • YAP activity is closely related to chemical resistance in various cancer types, particularly epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Therefore, we measured whether the Tyro3-YAP axis could affect the chemosensitivity to anticancer drugs.
  • proliferation of LNCaP-SL cells was measured using EV-containing normal FBS or EV-free FBS and 50 nM docetaxel, a representative chemotherapeutic agent for metastatic prostate cancer.
  • LNCaP-SL cells were found to be much more resistant to docetaxel under the EV-containing normal 10% FBS compared to the FBS 10% condition without EVs.
  • csEV treatment increased cell proliferation of GR-H1993 (gefitinib-resistant-H1993), but showed low levels of Tyro3 expression in H1993 or ER-H1993 (erlotinib-resistant-H1993). ), not in cells.
  • KRCT-6j was shown to inhibit only the proliferative effect of csEV-mediated GR-H1993.
  • a cell lysis buffer in which a dephosphorylation enzyme inhibitor (phosphatase inhibitor, Sigma-Aldrich, St.Louis, MO, USA) and a proteinase inhibitor (proteinase inhibitor, Roche, Basel, Switzerland) were added to the cells (50mM Tris-Cl, pH7.6, 120mM NaCl, 1mM EDTA, pH8.0, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 50mM NaF, 200 ⁇ M sodium orthovanadate, 1mM phenylmethylsulfonyl fluoride (PMSF) ) was added and the cells were lysed on ice for 1 hour.
  • phosphatase inhibitor phosphatase inhibitor, Sigma-Aldrich, St.Louis, MO, USA
  • a proteinase inhibitor proteinase inhibitor, Roche, Basel, Switzerland
  • each protein sample was separated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) using a gel between 8-12%. After electrophoresis, the gel was transferred to a nitrocellulose membrane 0.45 ⁇ m filter (GE healthcare Life Sciences, Chalfont, Buckinghamshire, UK) with a transfer buffer solution (25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3). Transfer was carried out at 40V for 190 minutes.
  • CI Combination Index
  • 3X10 3 GR-H1993 cells were aliquoted in a 96-well plate in a volume of 100 ⁇ L, and the next day, the medium was changed to a medium containing csEV, gefitinib or KRCT-6j. Then, the phase confluence of the cells was measured by IncuCyte. Monitoring was performed every 4 hours using an S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI).
  • GR-H1993 cells (5 ⁇ 10 6 cells) were dissolved in 50 ⁇ L Cultrex pathclear basement membrane extract (PBS, R&D systems) and subcutaneously injected into 5-week-old male Athymic BALB/c-nu mice.
  • PBS Cultrex pathclear basement membrane extract
  • GR-H1993 tumor model when tumors became visible, mice were grouped based on pre-treatment tumor volume (>100 mm 3 ).
  • Gefitinib 25 mg/kg, oral administration (PO)
  • KRCT-6j (20 mg/kg, intraperitoneal injection (IP) was administered daily for 28 days and tumor growth was measured every 3 days. Tumor length and width were measured with calipers, and tumor volume was calculated using the formula [(length ⁇ width 2 ) ⁇ ⁇ /6].
  • the mice were humanely euthanized.
  • the volume of the isolated tumor tissue was measured and further stained with Ki67, a representative proliferation marker. As shown in FIGS. 5c and 5d , it was confirmed that the combined administration of KRCT-6j and gefitinib effectively inhibited the growth of aggressive cancer induced as a result of YAP inactivation.
  • N-cyclohexylpyrimidin-4-amine derivatives 14 compounds were tested in HCT116 colon cancer cells and metastatic LNCaP-SL prostate cancer cells that responded to csEV. The proliferation inhibitory response was evaluated.
  • the compounds exhibiting both cell inhibitory responses were KRCT-5, 31, 62, 77, 87 and 203 compounds.
  • Example 6-1 Based on the results of Example 6-1, the proliferation inhibitory effects of various cancer cells, including anticancer drug-resistant cell lines, were evaluated for compounds No. 87 and No. 203, which had the best effects.
  • HCC827 cells a non-small cell lung cancer (NSCLC) cell line
  • NSCLC non-small cell lung cancer
  • the antiproliferative IC 50 values of compounds 87 and 203 were 0.95 and 0.97 ⁇ M, respectively
  • GR-HCC827 gefitinib-resistant HCC827 cells, 2.1 and 1.5 ⁇ M.
  • the IC 50 was 1.4 and 1.2 ⁇ M.
  • erlotinib-resistant H1993 (ER-H1993) cells showed 1.8 and 1.9 ⁇ M
  • gefitinib-resistant H1993 (GR-H1993) cells showed 2.8 and 0.9 ⁇ M.
  • ER-H1993 cells showed 1.8 and 1.9 ⁇ M
  • GR-H1993 cells showed 2.8 and 0.9 ⁇ M.
  • a similar antiproliferative effect was observed for both EGFR-TKI-resistant lung cancer and reactive lung cancer.
  • the proliferation inhibitory IC 50 values of compounds 87 and 203 were 0.90 and 0.52 ⁇ M, respectively, and in the case of oxaliplatin-resistant HT297 (OR-HT29) cells, 0.64 and 0.44 ⁇ M.
  • HCT116 cells showed 1.13 and 0.83 ⁇ M.
  • oxaliplatin-resistant HCT116 (OR-HCT116) cells showed 1.15 and 0.99 ⁇ M, and similar antiproliferative effects were observed for both platinum complex-resistant colorectal cancer and reactive lung cancer.
  • LNCaP androgen receptor-positive, non-metastatic cells
  • metastatic LNCaP-SL cells which are human prostate cancer cell lines.
  • the antiproliferative IC 50 values of compounds 87 and 203 were 2.3 and 1.7 ⁇ M, respectively, and in the case of LNCaP-SL cells, a metastatic sub-line with high cancer cell mobility. , 0.9 and 2.2 ⁇ M.
  • Huh7 metalastatic
  • HepG2 non-metastatic
  • HCT116 In the case of cancer cells, not only proliferation but also mobility is an important factor in determining cancer metastasis, which is a problem in chemotherapy.
  • the mobility inhibitory activity of KRCT-0087 or KRCT-0203 was evaluated.
  • Transwell migration experiments were performed using IncuCyte ClearView 96-well chemotaxis plates (Essen bioscience, MI, USA). The lower part of the transwell unit was coated with Type I collagen (Collaborative Research, KY, USA) and dried at room temperature for 1 hour. After preparing 3 to 5 ⁇ 10 3 cells/well in RPMI or DMEM medium without FBS, the experimental group or control group treated with the drug at the specified concentration (1 ⁇ M) was seeded at the upper end of the transwell unit by 60 ⁇ L. . KRCT-0087 or KRCT-0203 was treated at the top of the transwell unit. A total of 200 ⁇ L of 10% FBS medium was used for the bottom wells containing the attractant chemicals. Cell migration was monitored at 4-hour intervals using Incucyte Chemotaxis live-cell analysis (ESSEN bioscience).
  • GR-H1993, 4T1, HCT116, OR-HCT116, HT-29 and OR-HT-29 cells were cultured on a ULA plate, and three-dimensional sphericity was observed, and in the case of compound 87, , showed a strong spheroid formation inhibitory ability.
  • the spheroid formation inhibitory activity did not appear in GR-H1993, OR-HCT116 cells, and in the case of other cancer cells, the inhibitory activity was less than that of compound 87.
  • Bone marrow cells isolated from mice were treated with macrophage-colony stimulating factor (M-CSF) to differentiate them into macrophages.
  • M-CSF macrophage-colony stimulating factor
  • IL-4 was exposed for M2-type differentiation mediating an environment favorable for the survival of cancer cells, and in this case, mRNA expression of M2-type markers TGF- ⁇ , Arg1 (Arginase 1), and CIITA (Class II Major Histocompatibility Complex Transactivator) was reduced. increased.
  • Both femurs and shinbones were separated from 8-week-old C57BL/6J mice, washed three times with 1X PBS, and then both leg bones were cut with scissors. Then, RMPI1640 medium (2.5% HEPES, 10% FBS, 1% Penicillin/streptomycin) was flowed through a 1mL syringe. After filtering using a 70 ⁇ M cell strainer, it was centrifuged (400 g, 5 min). And red blood cells were removed with ACK lysis buffer (Life Technologies, Grand island, NY). After centrifugation again (400 g, 5 min) to release the cells in a new medium, the cells were filtered using a 70 ⁇ M cell strainer. After treatment with M-CSF 30ng/mL, and after 3 days of change to a new medium, after differentiation for a total of 7 days, primary cultured bone marrow-derived immune cells were obtained, which were used in the experiment.
  • compounds 87 and 203 significantly inhibited both the increase in TGF- ⁇ expression in macrophages by IL-4 treatment at a concentration of 1 ⁇ M.
  • compound 87 significantly inhibited the increase in Arg1 and CIITA expression in macrophages by IL-4 treatment at a concentration of 0.5 ⁇ M.
  • 4T1 cells (1 ⁇ 10 6 cells) were diluted in 100 ⁇ L PBS and injected subcutaneously into 5-week-old female Balb/c mice. Administration was started when the average tumor size reached 100 mm 3 or more, and KRCT-0087 5 mg/kg and KRCT-0203 10 mg/kg were administered intraperitoneally. Tumor growth was measured every 3 days. Tumor length and width were measured with calipers, and tumor volume was calculated using the formula [(length ⁇ width 2 ) ⁇ ⁇ /6]. When the tumor volume of the control group reached about 1,000 mm 3 , the mice were humanely euthanized.
  • MC38 cells (2 ⁇ 10 5 cells) were diluted in 100 ⁇ L PBS and injected subcutaneously into 5-week-old male Balb/c mice. Administration was started when the average tumor size reached 100 mm 3 or more, and 5 mg/kg of KRCT-0087 was administered intraperitoneally for 9 days (see FIG. 17a ). After administration, tumor growth was measured every 3 days. Tumor length and width were measured with calipers, and tumor volume was calculated using the formula [(length ⁇ width 2 ) ⁇ ⁇ /6]. When the tumor volume of the control group reached about 1,000 mm 3 , the mice were humanely euthanized.
  • CD is an essential cell membrane protein required when acting as an antigen-presenting cell that promotes the division of activated T cells, and is used as an important indicator in relation to cell activation.
  • the T cell differentiation group is expressed as CD3, and is mainly classified into CD4, which plays a role in cytokine production, and CD8, which is a cytotoxic T cell.
  • lymphocytes in the tumor were isolated from the collected tumor tissue using the Tumor dissassociation kit ((Miltenyi Biotec, Auburn, CA) and Percoll density gradient centrifugation technique. Macrophages and T cells were used using FACS (fluorescence analysis cells sorting).
  • ⁇ -CD45-APC/Cy7 ⁇ -BV785-IA/IE, ⁇ -CD11b-PerCP/Cy5.5, ⁇ -Ly6C-APC, ⁇ -Ly6G-FITC, ⁇ -F4/80- PE/Cy7, ⁇ -CD4-FITC, and ⁇ -CD8-APC were purchased from biolegend (San Diego, CA, USA)
  • ⁇ -FOXP3-EF450 was purchased from eBioscience (San Diego, CA, USA).
  • the tumor-associated macrophage (TAM) population was defined as CD45 + Ly6G - Ly6C - F4/80 + CD11b + .
  • compound 87 of the present invention acts on macrophages and T cells to cause an immune synergistic effect, thereby exhibiting an anticancer effect, in particular a therapeutic effect for colorectal cancer.
  • Interferon- ⁇ (IFN- ⁇ ) and tumor necrosis factor- ⁇ (TNF- ⁇ ) are known as essential cytokines for inducing apoptosis in T cells and exhibiting strong anticancer effects. Also, CD4+ T cells that secrete IFN- ⁇ and TNF- ⁇ are defined as Th1 CD4+ T cells.
  • the cytokines IFN- ⁇ and TNF- ⁇ secreted from CD4+ and CD8+ T cells were significantly increased by administration of compound 87.
  • the present inventors confirmed that the compound of the present invention not only inhibited csEV-mediated cancer growth, but also had a very excellent metastasis inhibitory effect.
  • the compound of the present invention induces the activity of innate immunity by increasing the fraction of M1/M2, a differentiated type of macrophages that inhibit immune cell attack on cancer cells, and increases Th1 CD4+ T cells and cytotoxic CD8+ T cells that are acquired immunity was confirmed to be activated.
  • the compound of the present invention can be used as a variety of cancer preventive or therapeutic agents, cancer metastasis inhibitors, and immuno-cancer agents.
  • the compounds of the present invention have excellent anti-proliferative activity of cancer cells such as lung cancer, colon cancer, prostate cancer, breast cancer, and liver cancer mediated by blood circulating microextrabody (csEV), and also have excellent anti-proliferative activity against anticancer drug-resistant cancer cell lines, and for invasive cancer Not only does it exhibit a proliferation inhibitory effect, but it also significantly inhibits cancer growth in xenograft and allograft animal models of cancer cells. there is.
  • cancer cells such as lung cancer, colon cancer, prostate cancer, breast cancer, and liver cancer mediated by blood circulating microextrabody (csEV)
  • csEV blood circulating microextrabody

Abstract

The present invention relates to a blood circulation micro in-vitro corpuscle-mediated cancer treatment composition and, more specifically, to a composition for preventing or treating cancer comprising a compound represented by chemical formula 1 or a salt thereof as an active ingredient.

Description

혈액 순환 미세체외소체 매개 암 치료용 조성물Composition for the treatment of blood circulation microextrabody-mediated cancer
본 발명은 혈액 순환 미세체외소체 매개 암 치료용 조성물에 관한 것으로, 구체적으로 화학식 1로 표시되는 화합물 또는 그 염을 유효성분으로 포함하는 암 예방 또는 치료용 조성물 등에 관한 것이다.The present invention relates to a composition for the treatment of blood circulation microexosome-mediated cancer, and more particularly, to a composition for preventing or treating cancer comprising the compound represented by Formula 1 or a salt thereof as an active ingredient.
본 출원은 2020년 9월 15일에 출원된 한국특허출원 제10-2020-0118272호 및 2021년 9월 1일에 출원된 한국특허출원 제10-2021-0116558호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다. This application claims priority based on Korean Patent Application No. 10-2020-0118272, filed on September 15, 2020 and Korean Patent Application No. 10-2021-0116558, filed on September 1, 2021, All contents disclosed in the specification and drawings of the application are incorporated herein by reference.
암이란 주로 통제되지 않는 세포의 증식에서 시작되어 주위의 정상조직 또는 기관으로 침윤하여 파괴시키고 새로운 성장 장소를 만들 수 있어 개체의 생명을 빼앗아 갈 수 있는 질환 군을 총칭한다. 지난 10 여년 동안 암을 정복하기 위해 세포주기나 세포사멸(apoptosis)의 조절과 발암유전자나 암억제 유전자들을 포함한 새로운 표적을 모색함에 있어서 눈에 띄는 발전을 거듭해 왔음에도 불구하고 암의 발생률은 문명이 발달됨에 따라 증가되고 있다.Cancer is a generic term for a group of diseases that start from the uncontrolled proliferation of cells, invade and destroy surrounding normal tissues or organs, and create a new growth site that can take away the life of an individual. In the past decade or so, despite the remarkable progress made in the search for new targets including oncogenes and cancer suppressor genes, the regulation of the cell cycle or apoptosis to conquer cancer, the incidence of cancer has increased with the development of civilization. is increasing as
암의 치료방법으로는 화학 요법, 수술 요법 및 방사선 치료 요법 등이 사용되고 있다. 이중에서, 화학 요법은 항암제를 이용하는 방법으로서 암의 치료에 가장 많이 사용되고 있다. 오늘날에는 약 60여종의 다양한 항암제가 임상에 사용되고 있으며, 암 발생 및 암세포의 특성에 대한 지식이 많이 알려짐에 따라 새로운 항암제가 계속적으로 개발되고 있다. 그러나, 현재 임상에서 사용되고 있는 항암제의 대부분은 화학적으로 합성된 물질들로 오심, 구토, 구강 및 소장의 궤양, 설사, 탈모, 혈액의 유효성분의 생산이 저하되는 골수 억제 등과 같은 부작용을 수반하는 경우가 많다. 예를 들어, 마이토마이신 C(mitomycin-C)는 신부전증을, 아드리아마이신(adriamycin)은 골수억제작용 등의 부작용이 알려져 있다. 특히, 지금까지 개발된 항암제 중 가장 유용한 약제인 시스플라틴(cisplatin)은 고환암, 난소암, 폐암, 두경부암, 방광암, 위암 및 자궁경부암 등의 치료에 널리 사용되고 있으나, 빈혈 등의 조혈독성, 구토, 메스꺼움 등의 소화기 독성, 콩팥 세뇨관 손상 등의 신장독성, 난청, 체내 전해질 이상, 쇼크, 말초신경 이상 등과 같은 부작용의 발생이 큰 문제가 되고 있다(R.T. Skeel, Handbook of Cancer Chemotherapy, pp.89-91, 1999). 또한, 제피티닙[gefitinib; 상품명, 이레사 (Iressa)]과 같은 일부 약제는 비소세포폐암에서 분자표적치료제로서 가장 먼저 미국식품의약청에서 승인을 받아 이미 임상에 적용되어 환자들에게 도움을 주고 있다. 그러나 암은 매우 복잡한 경로로 발전하기 때문에 특정 표적인자만을 선택적으로 억제하는 이러한 분자표적치료제는 장기 투여에 의한 내성이 생기는 문제점이 있으며 특정 유전자의 돌연변이 상태에 따라 선택적인 치료효과를 나타내는 단점이 있다.Chemotherapy, surgery, radiation therapy, etc. are used as a treatment method for cancer. Among them, chemotherapy is the most used for the treatment of cancer as a method of using an anticancer agent. Today, about 60 kinds of various anticancer drugs are being used in clinical practice, and new anticancer drugs are continuously being developed as knowledge about cancer occurrence and characteristics of cancer cells is widely known. However, most of the anticancer drugs currently used in clinical practice are chemically synthesized substances, and when accompanied by side effects such as nausea, vomiting, ulcers of the mouth and small intestine, diarrhea, hair loss, and bone marrow suppression that reduces the production of active ingredients in blood there are many For example, mitomycin C (mitomycin-C) is known to have side effects, such as renal failure, and adriamycin (adriamycin) myelosuppressive action. In particular, cisplatin, the most useful anticancer drug developed so far, is widely used in the treatment of testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer and cervical cancer, but hematopoietic toxicity such as anemia, vomiting, and nausea The occurrence of side effects such as digestive toxicity such as digestive toxicity, kidney toxicity such as kidney tubular damage, hearing loss, electrolyte abnormality in the body, shock, and peripheral nerve abnormality is a big problem (RT Skeel, Handbook of Cancer Chemotherapy, pp.89-91, 1999). In addition, gefitinib [gefitinib; Some drugs, such as the trade name, Iressa], have been approved by the US Food and Drug Administration for the first time as molecular targeted therapy for non-small cell lung cancer, and have already been applied to clinical trials to help patients. However, since cancer develops in a very complex path, these molecular targeted therapeutics that selectively inhibit only a specific target have a problem in that resistance occurs due to long-term administration, and there is a disadvantage in showing a selective therapeutic effect according to the mutation status of a specific gene.
따라서, 종래 항암제가 가지고 있는 부작용 및 독성을 해결할 수 있는 안전성이 우수한 새로운 항암제의 개발이 절실히 요구되고 있다.Therefore, there is an urgent need to develop a new anticancer drug with excellent safety that can solve the side effects and toxicity of conventional anticancer drugs.
이에, 본 발명은 상기와 같은 종래 기술상의 필요성을 해결하기 위해 안출된 것으로서, 본 발명자들은 화학식 1로 표시되는 화합물이 암 세포 증식의 억제 뿐만 아니라, 전이성 및 이동성을 억제하는 것을 확인함으로써, 암에 대한 예방 또는 치료 효과 및 암 전이 억제 효과를 확인한바, 이에 기초하여 본 발명을 완성하게 되었다.Accordingly, the present invention has been devised to solve the prior art needs as described above, and the present inventors confirmed that the compound represented by Formula 1 inhibits cancer cell proliferation as well as metastasis and mobility, thereby preventing cancer The preventive or therapeutic effect and the cancer metastasis inhibitory effect were confirmed, and the present invention was completed based on this.
따라서, 본 발명의 목적은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물로서, 상기 암은 혈액 순환 미세체외소체(csEV, circulating small extracellular vesicles) 매개 암인 것을 특징으로 하는, 약학적 조성물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the cancer is a circulating microextrabody (csEV) It is to provide a pharmaceutical composition, characterized in that small extracellular vesicles) mediated cancer.
본 발명의 다른 목적은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암 전이 억제용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for inhibiting cancer metastasis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물로서, 상기 암은 혈액 순환 미세체외소체(csEV) 매개 암인 것을 특징으로 하는, 약학적 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the cancer is It provides a pharmaceutical composition, characterized in that it is a blood circulating microextrabody (csEV) mediated cancer.
[화학식 1][Formula 1]
Figure PCTKR2021012192-appb-img-000001
Figure PCTKR2021012192-appb-img-000001
(상기 화학식 1에 있어서,(In Formula 1,
상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
본 발명의 일 구현예에서, In one embodiment of the present invention,
Figure PCTKR2021012192-appb-img-000002
Figure PCTKR2021012192-appb-img-000002
상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, Cl, F 또는 메틸일 수 있으나, 이에 제한되는 것은 아니다.The R 3 and R 4 may each independently be one or more hydrogen, Cl, F, or methyl, but is not limited thereto.
본 발명의 다른 구현예에서, 상기 화학식 1로 표시되는 화합물은 표 1에 기재된 화합물로 이루어진 군으로부터 선택된 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the compound represented by Formula 1 may be at least one selected from the group consisting of the compounds shown in Table 1, but is not limited thereto.
[표 1][Table 1]
Figure PCTKR2021012192-appb-img-000003
Figure PCTKR2021012192-appb-img-000003
Figure PCTKR2021012192-appb-img-000004
Figure PCTKR2021012192-appb-img-000004
Figure PCTKR2021012192-appb-img-000005
Figure PCTKR2021012192-appb-img-000005
본 발명의 다른 구현예에서, 상기 조성물은 제피티닙(gefitinib) 또는 이의 약학적으로 허용 가능한 염을 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the composition may further include gefitinib or a pharmaceutically acceptable salt thereof, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 혈액 순환 미세체외소체(csEV) 매개 암은 이동능이 있는 암, 즉 전이성 암이라면 이에 제한되는 것은 아니나, 예를 들어 전립선암, 유방암, 폐암, 결장암, 결장직장암, 갑상선암, 난소암, 방광암, 신장암, 위암, 자궁암, 직장암, 췌장암 및 흑색종으로 이루어진 군으로부터 선택된 하나 이상일 수 있다.In another embodiment of the present invention, the blood circulation micro-extrabody (csEV)-mediated cancer is a cancer with migratory ability, that is, if it is a metastatic cancer, but is not limited thereto, for example, prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer , thyroid cancer, ovarian cancer, bladder cancer, kidney cancer, stomach cancer, uterine cancer, rectal cancer, may be at least one selected from the group consisting of pancreatic cancer and melanoma.
본 발명의 또 다른 구현예에서, 상기 화학식 1로 표시되는 화합물이 암 세포의 성장 억제, 침윤(invasion) 억제 및 전이(metastasis) 억제로 이루어진 군으로부터 선택된 하나 이상의 효과를 갖는 것일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the compound represented by Formula 1 may have one or more effects selected from the group consisting of cancer cell growth inhibition, invasion inhibition, and metastasis inhibition, but limited thereto it is not going to be
본 발명의 또 다른 구현예에서, 상기 약학적 조성물은 면역항암제일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the pharmaceutical composition may be an immuno-cancer agent, but is not limited thereto.
또한, 본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 제피티닙의 항암 효능 증진용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for enhancing the anticancer efficacy of gefitinib comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암 전이 억제용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 일 구현예에서, 상기 암은 혈액 순환 미세체외소체(csEV) 매개 암일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the cancer may be a blood circulation microextrabody (csEV) mediated cancer, but is not limited thereto.
본 발명의 다른 구현예에서, 상기 화학식 1로 표시되는 화합물은 암세포의 M2 면역반응을 제어할 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the compound represented by Formula 1 may control the M2 immune response of cancer cells, but is not limited thereto.
또한, 본 발명은 암의 치료를 필요로 하는 개체에 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 약학적 조성물을 투여하는 단계를 포함하는 암 또는 암 전이의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing cancer or cancer metastasis, comprising administering a pharmaceutical composition comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient to an individual in need of treatment for cancer or a method of treatment.
또한, 본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 조성물의 암 또는 암 전이의 예방 또는 치료 용도를 제공한다.In addition, the present invention provides a use for preventing or treating cancer or cancer metastasis of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 암 또는 암 전이 예방 또는 치료 약제를 생산하기 위한 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염의 용도를 제공한다.In addition, the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for producing a medicament for preventing or treating cancer or cancer metastasis.
본 발명의 화합물들은 혈액 순환 미세체외소체(csEV)가 매개하는 폐암, 결장암, 전립선암, 유방암, 간암과 같은 암세포 증식 억제 활성이 뛰어나고, 항암제 내성 암세포주에 대한 증식 억제 활성도 뛰어나며, 침습성 암에 대한 증식 억제 효과를 나타낼 뿐만 아니라, 암세포의 이종이식 및 동종이식 동물모델에서 암 성장을 유의하게 억제하므로, 암의 예방 또는 치료 뿐만 아니라 암 전이 억제용 약제로 유용하게 사용할 수 있다.The compounds of the present invention have excellent anti-proliferative activity of cancer cells such as lung cancer, colon cancer, prostate cancer, breast cancer, and liver cancer mediated by blood circulating microextrabody (csEV), and also have excellent anti-proliferative activity against anticancer drug-resistant cancer cell lines, and for invasive cancer Since it not only exhibits a proliferation inhibitory effect, but also significantly inhibits cancer growth in xenograft and allograft animal models of cancer cells, it can be usefully used as a drug for inhibiting cancer metastasis as well as preventing or treating cancer.
도 1a는 본 발명의 일 실시예에 따른 혈액 순환 미세체외소체(csEV)를 분리하는 과정을 나타낸 것이다.1A illustrates a process for isolating blood circulating microexosomes (csEV) according to an embodiment of the present invention.
도 1b는 분리된 csEV의 크기를 DLS로 확인한 결과를 나타낸 것이다.Figure 1b shows the result of checking the size of the separated csEV by DLS.
도 1c는 분리된 csEV를 LNCaP-SL 전립선 암세포에 노출 시 세포 내로 함입 됨을 공초점 현미경으로 확인한 결과를 나타낸 것이다.Figure 1c shows the results of confirming the incorporation of isolated csEV into LNCaP-SL prostate cancer cells when exposed to the cells with a confocal microscope.
도 1d는 csEV의 LNCaP-SL 전립선 암세포의 이동 촉진 효과를 나타낸 것이다.Figure 1d shows the effect of csEV to promote migration of LNCaP-SL prostate cancer cells.
도 1e는 csEV의 암세포(HCT116, SW480, GR-HCC827, H1975, primary BCC)의 이동 촉진 효과를 나타낸 것이다.Figure 1e shows the migration promoting effect of csEV cancer cells (HCT116, SW480, GR-HCC827, H1975, primary BCC).
도 1f는 csEV의 전기장 자극으로 표면 전하 차에 따른 csEV 분리 방법을 나타낸 것이다.Fig. 1f shows a csEV separation method according to the difference in surface charge with electric field stimulation of csEV.
도 1g는 csEV의 표면 전하에 따른 이동 효과를 나타낸 것이다.Figure 1g shows the migration effect according to the surface charge of csEV.
도 2는 KRCT-6j의 LNCaP-SL 세포의 이동 억제 효과를 나타낸 것이다.Figure 2 shows the inhibitory effect of KRCT-6j on the migration of LNCaP-SL cells.
도 3a는 FBS에서 csEV 유무에 의한 암세포 증식 비율을 나타낸 것이다.Figure 3a shows the rate of cancer cell proliferation in the presence or absence of csEV in FBS.
도 3b는 FBS 고갈조건에서 csEV 처리에 의한 암세포 증식 비율을 나타낸 것이다.Figure 3b shows the rate of cancer cell proliferation by csEV treatment in FBS depletion condition.
도 3c는 csEV 처리에 의한 AKT 및 ERK의 활성을 나타낸 것이다.Figure 3c shows the activity of AKT and ERK by csEV treatment.
도 3d는 csEV 매개 암세포에서 KRCT-6j 처리에 의하여 YAP 발현 및 핵 국소화가 감소된 결과를 나타낸 것이다.3D shows the results of reduced YAP expression and nuclear localization by KRCT-6j treatment in csEV-mediated cancer cells.
도 4a는 FBS의 csEV 유무 조건에서 도세탁셀 처리에 의한 전이성 전립선 암세포의 증식을 확인한 결과를 나타낸 것이다.Figure 4a shows the results of confirming the proliferation of metastatic prostate cancer cells by docetaxel treatment in the presence or absence of csEV in FBS.
도 4b는 csEV 및 KRCT-6j 처리에 의한 H1993 및 GR-H1993 세포의 증식을 확인한 결과를 나타낸 것이다.Figure 4b shows the results of confirming the proliferation of H1993 and GR-H1993 cells by csEV and KRCT-6j treatment.
도 4c는 csEV 및 KRCT-6j 처리에 의한 Parental H1993세포, GR-H1993 세포 및 ER-H1993 세포의 증식을 확인한 결과를 나타낸 것이다.Figure 4c shows the results of confirming the proliferation of parental H1993 cells, GR-H1993 cells and ER-H1993 cells by csEV and KRCT-6j treatment.
도 4d는 H1993 및 GR-H1993 세포에서 YAP 발현 수준을 확인한 결과를 나타낸 것이다.Figure 4d shows the results of confirming the YAP expression level in H1993 and GR-H1993 cells.
도 4e는 GR-H1993 세포에 KRCT-6j 처리하여 YAP의 핵 국소화를 확인한 결과를 나타낸 것이다.Figure 4e shows the results of confirming the nuclear localization of YAP by KRCT-6j treatment in GR-H1993 cells.
도 5a는 KRCT-6j 및 제피티닙의 병용 처리에 따른 시너지 효과를 나타낸 것이다.Figure 5a shows the synergistic effect of the combined treatment of KRCT-6j and gefitinib.
도 5b는 KRCT-6j 및 제피티닙의 병용 처리에 따른 GR-H1993 이종이식 마우스 모델에서의 종양 성장 억제 효과를 나타낸 것이다.Figure 5b shows the tumor growth inhibitory effect in the GR-H1993 xenograft mouse model according to the combined treatment of KRCT-6j and gefitinib.
도 5c는 KRCT-6j 및 제피티닙 병용 처리에 따른 종양 부피 증가 억제 효과를 나타낸 것이다.Figure 5c shows the inhibitory effect of tumor volume increase according to the combination treatment of KRCT-6j and gefitinib.
도 5d는 KRCT-6j 및 제피티닙 병용 처리에 따른 종양 증식 마커 Ki67의 발현을 확인한 결과를 나타낸 것이다.Figure 5d shows the results of confirming the expression of the tumor growth marker Ki67 according to the combined treatment of KRCT-6j and gefitinib.
도 6은 본 발명 화합물 처리에 따른 LNCaP-SL 및 HCT116 암세포 증식 억제 효과를 나타낸 것이다.6 shows the inhibitory effect of LNCaP-SL and HCT116 cancer cell proliferation according to the treatment of the compound of the present invention.
도 7a 및 도 7b는 화합물 87번 및 203번 처리에 따른 HCC827, GR-HCC827. H1975, H1993, ER-H1993, 및 GR-H1993 암세포의 증식 억제 효과를 나타낸 것이다.7a and 7b show HCC827 and GR-HCC827 according to compounds No. 87 and No. 203 treatment. H1975, H1993, ER-H1993, and GR-H1993 shows the effect of inhibiting the proliferation of cancer cells.
도 8은 화합물 87번 및 203번 처리에 따른 HT29, OR-HT29, HCT116, OR-HCT116 대장암세포의 증식 억제 효과를 나타낸 것이다.Figure 8 shows the inhibitory effect on the proliferation of HT29, OR-HT29, HCT116, OR-HCT116 colon cancer cells according to the treatment of compounds 87 and 203.
도 9a는 화합물 87번 및 203번 처리에 따른 LNCaP-O, 및 LNCaP-SL 전립선 암세포의 증식 억제 효과를 나타낸 것이다.Figure 9a shows the inhibitory effect on the proliferation of LNCaP-O, and LNCaP-SL prostate cancer cells according to the treatment of compounds 87 and 203.
도 9b는 화합물 87번 및 203번 처리에 따른 Huh7, 및 HepG2 간암세포의 증식 억제 효과를 나타낸 것이다.Figure 9b shows the inhibitory effect on the proliferation of Huh7, and HepG2 hepatocellular carcinoma cells according to the treatment of compounds No. 87 and No. 203.
도 10은 화합물 87번 및 203번 처리에 따른 4T1 유선암세포 및 MC38 대장암 세포 동종이식 마우스 모델에서의 암세포 증식 억제 효과를 나타낸 것이다.10 shows the inhibitory effect on cancer cell proliferation in 4T1 mammary gland cancer cells and MC38 colorectal cancer cell allograft mouse models according to compounds 87 and 203 treatment.
도 11은 화합물 87번 및 203번 처리에 따른 HCT116, OR-HCT116(이상 대장암세포), MDA-MB-231, 4T1, 타목시펜(tamoxifen)-저항성 MCF-7(TAMR-MCF-7)[이상 인간 및 마우스 유방(선)암 세포], LNCaP-SL(전립선암 세포), 엘로티닙 내성 H292세포(ER-H292), GR-H1993, GR-HCC-827, H1975[이상 EGFR-TKI 내성 폐암세포]의 암세포 이동 억제 효과를 나타낸 것이다.11 shows HCT116, OR-HCT116 (abnormal colorectal cancer cells), MDA-MB-231, 4T1, tamoxifen-resistant MCF-7 (TAMR-MCF-7) [abnormal human and mouse breast (adenocarcinoma) cells], LNCaP-SL (prostate cancer cells), erlotinib-resistant H292 cells (ER-H292), GR-H1993, GR-HCC-827, H1975 [abnormal EGFR-TKI-resistant lung cancer cells] showed the effect of inhibiting the migration of cancer cells.
도 12는 화합물 87번 및 203번 처리에 따른 4T1 및 GR-H1993 세포의 스페로이드 형성 억제 활성을 나타낸 것이다.Figure 12 shows the spheroid formation inhibitory activity of 4T1 and GR-H1993 cells according to the treatment of compounds No. 87 and No. 203.
도 13은 화합물 87번 및 203번 처리에 따른 HCT116 및 OR-HCT116 세포의 스페로이드 형성 억제 활성을 나타낸 것이다.Figure 13 shows the spheroid formation inhibitory activity of HCT116 and OR-HCT116 cells according to compound 87 and 203 treatment.
도 14는 화합물 87번 및 203번 처리에 따른 HT29 및 OR-HT29 세포의 스페로이드 형성 억제 활성을 나타낸 것이다.Figure 14 shows the spheroid formation inhibitory activity of HT29 and OR-HT29 cells according to compound 87 and 203 treatment.
도 15a는 화합물 87번 및 203번 처리에 따른 IL-4 처리에 노출된 대식세포에서의 TGF-beta 발현을 확인한 결과를 나타낸 것이다.Figure 15a shows the results of confirming the expression of TGF-beta in macrophages exposed to IL-4 treatment according to compounds 87 and 203 treatment.
도 15b는 화합물 87번 처리에 따른 IL-4 처리에 노출된 대식세포에서의 M2 대식세포의 대표 마커인 arg1 및 ciita의 발현을 확인한 결과를 나타낸 것이다.15B shows the results of confirming the expression of arg1 and ciita, which are representative markers of M2 macrophages, in macrophages exposed to IL-4 treatment according to compound 87 treatment.
도 16a는 4T1 마우스 유선암 세포를 이식한 동종이식모델에 화합물 87번 및 203번을 투여하는 실험 프로토콜을 나타낸 것이다.16A shows an experimental protocol for administering compounds No. 87 and No. 203 to an allograft model transplanted with 4T1 mouse mammary cancer cells.
도 16b는 4T1 마우스 유선암 세포를 이식한 동종이식모델에 화합물 87번 및 203번을 투여한 결과 종양 부피를 측정한 결과를 나타낸 것이다.Figure 16b shows the results of measuring the tumor volume as a result of administering the compounds No. 87 and No. 203 to the allograft model transplanted with 4T1 mouse mammary cancer cells.
도 16c는 4T1 마우스 유선암 세포를 이식한 동종이식모델에 화합물 87번 및 203번을 투여한 결과 체중을 나타낸 것이다.Figure 16c shows the body weight as a result of administering the compounds No. 87 and No. 203 to the allograft model transplanted with 4T1 mouse mammary cancer cells.
도 17a는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여하는 실험 프로토콜을 나타낸 것이다.17A shows an experimental protocol for administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17b는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여한 결과 종양 부피를 측정한 결과를 나타낸 것이다.Figure 17b shows the results of measuring the tumor volume as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17c는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여한 결과 종양 무게 대비 CD45+ 세포의 수를 측정한 결과를 나타낸 것이다. Figure 17c shows the results of measuring the number of CD45+ cells compared to the tumor weight as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17d는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여한 결과 종양 관련 대식세포를 CD45+Ly6G-Ly6C-F4/80+CD11b+로 정의한 것을 나타낸 것이다.FIG. 17d shows that the tumor-associated macrophages were defined as CD45+Ly6G-Ly6C-F4/80+CD11b+ as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17e는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여한 결과 TAM이 발현하는 I-A/I-E 비율을 측정한 결과를 나타낸 것이다.17e shows the results of measuring the I-A/I-E ratio expressed by TAM as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17f는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여한 결과 세포독성 CD8+ T 세포의 수를 측정한 결과를 나타낸 것이다.17f shows the results of measuring the number of cytotoxic CD8+ T cells as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17g는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여한 결과 조절 T 세포의 마커인 FOXP3+CD4+ T 세포의 비율을 측정한 결과를 나타낸 것이다.17g shows the results of measuring the ratio of FOXP3+CD4+ T cells, which are markers of regulatory T cells, as a result of administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17h는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여하여 T 세포의 표현형 변화를 확인하는 실험 프로토콜을 나타낸 것이다.Figure 17h shows an experimental protocol for confirming the phenotypic change of T cells by administering compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells.
도 17i는 MC38 마우스 대장암 세포를 이식한 동종이식모델에 화합물 87번을 투여에 따른 CD4+ 및 CD8+ T 세포에서의 인터페론-γ(IFN-γ) 및 종양 괴사 인자-α (TNF-α)의 분비를 확인한 결과를 나타낸 것이다. Figure 17i shows the secretion of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in CD4+ and CD8+ T cells following administration of compound 87 to an allograft model transplanted with MC38 mouse colorectal cancer cells. shows the results of checking .
따라서, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암의 예방, 치료 및 암 전이 억제용 약학적 조성물을 제공한다.Accordingly, the present invention provides a pharmaceutical composition for preventing, treating, and inhibiting cancer metastasis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 화합물은 하기 화학식 1로 표시되는 것일 수 있다 : The compound of the present invention may be one represented by the following formula (1):
[화학식 1][Formula 1]
Figure PCTKR2021012192-appb-img-000006
Figure PCTKR2021012192-appb-img-000006
(상기 화학식 1에 있어서,(In Formula 1,
상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
다음은 본 발명에 따른 화합물들을 제조하는 여러 가지 치환기의 정의를 설명한다.The following describes the definitions of various substituents making up the compounds according to the present invention.
본 발명에서 사용된 용어 "C1-5 알킬"은 탄소원자수 1 내지 5의 1가 알킬기를 의미하고, "C1-3 알킬"은 탄소원자수 1 내지 3의 1가 알킬기를 의미한다. 이 용어는 메틸, 에틸, n-프로필, i-프로필, n-부틸, i-부틸, tert-부틸 등과 같은 기능기를 예로 들 수 있다.As used herein, the term “C 1-5 alkyl” refers to a monovalent alkyl group having 1 to 5 carbon atoms, and “C 1-3 alkyl” refers to a monovalent alkyl group having 1 to 3 carbon atoms. The term includes functional groups such as methyl, ethyl, n -propyl, i -propyl, n -butyl, i -butyl, tert -butyl and the like.
본 발명에 기재된 알킬, 및 그 외 알킬 부분을 포함하는 치환체는 직쇄 또는 분쇄 형태를 모두 포함한다.Alkyl, and other substituents containing alkyl moieties described herein include both straight-chain and comminuted forms.
본 발명에서 사용된 용어 "C1-5 알콕시"는 -O-R기를 의미하며, 여기서 R은 "C1-C5 알킬"을 의미한다. 바람직한 알콕시기는 예를 들면, 메톡시, 에톡시, 페녹시 등을 포함한다.As used herein, the term "C 1-5 alkoxy" refers to an -OR group, where R stands for "C 1 -C 5 alkyl". Preferred alkoxy groups include, for example, methoxy, ethoxy, phenoxy, and the like.
본 발명에 기재된 알킬, 알콕시 및 그 외 알킬부분을 포함하는 치환체는 직쇄 또는 분쇄 형태를 모두 포함한다.The substituents containing alkyl, alkoxy and other alkyl moieties described in the present invention include both straight chain and comminuted forms.
본 발명에서 사용된 용어 "할로겐"은 플루오로(F), 클로로(Cl), 및 브로모(Br), 요오드(I) 를 포함할 수 있다.As used herein, the term “halogen” may include fluoro (F), chloro (Cl), and bromo (Br) and iodine (I).
본 발명에서 사용된 용어 "C6-C10 아릴"은 단일링(예를 들면 페닐) 또는 복수의 축합링(예를 들면 나프틸)을 갖는 탄소원자수 6 내지 10의 불포화 방향족 고리화합물을 의미한다. 상기 아릴은 페닐, 나프틸, 안트릴 및 바이아릴로 이루어지는 군으로부터 선택될 수 있다.As used herein, the term "C 6 -C 10 aryl" refers to an unsaturated aromatic ring compound having 6 to 10 carbon atoms having a single ring (eg phenyl) or a plurality of condensed rings (eg naphthyl). . The aryl may be selected from the group consisting of phenyl, naphthyl, anthryl and biaryl.
본 발명에서 사용된 용어 상기 "C6-C10 헤테로아릴"은 S, O 및 N으로부터 선택되는 1 내지 3개의 이종원자를 포함하는 아릴기로서, 다이옥솔일, 피리딜, 피리미딜, 티오페닐, 피롤릴, 퓨라닐 및 트리아졸릴로 이루어지는 군으로부터 선택될 수 있으나, 이에 제한되지 않는다.As used herein, the term "C 6 -C 10 heteroaryl" is an aryl group containing 1 to 3 heteroatoms selected from S, O and N, dioxolyl, pyridyl, pyrimidyl, thiophenyl, p It may be selected from the group consisting of rollyl, furanyl and triazolyl, but is not limited thereto.
본 발명에서 사용된 용어 "C6-C10 아릴알킬"은 -(CH2)n-R로 표시되는 것으로, 여기서 R은 아릴기를 의미하는 것이며, 벤질, 펜에틸 등을 포함하는 아릴 치환체를 갖는 알킬기를 의미하는 것으로, "C6-C10아릴"은 단일링(예를 들면 페닐) 또는 복수의 축합링(예를 들면 나프틸)을 갖는 탄소원자수 6 내지 20의 불포화 방향족 고리화합물을 의미한다. 상기 아릴은 페닐, 나프틸 등을 포함한다.The term "C 6 -C 10 arylalkyl" used in the present invention is represented by -(CH 2 ) n -R, where R means an aryl group, having an aryl substituent including benzyl, phenethyl, etc. By means of an alkyl group, "C 6 -C 10 aryl" refers to an unsaturated aromatic ring compound having 6 to 20 carbon atoms having a single ring (eg phenyl) or a plurality of condensed rings (eg naphthyl). . The aryl includes phenyl, naphthyl, and the like.
본 발명에서, In the present invention,
Figure PCTKR2021012192-appb-img-000007
Figure PCTKR2021012192-appb-img-000007
상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, Cl, F 또는 메틸일 수 있으나, 이에 제한되는 것은 아니다.The R 3 and R 4 may each independently be one or more hydrogen, Cl, F, or methyl, but is not limited thereto.
본 발명에서, 상기 화학식 1로 표시되는 화합물은 표 1에 기재된 화합물로 이루어진 군으로부터 선택된 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the compound represented by Formula 1 may be one or more selected from the group consisting of the compounds shown in Table 1, but is not limited thereto.
본 발명에서, 상기 조성물은 제피티닙 또는 이의 약학적으로 허용 가능한 염을 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the composition may further include gefitinib or a pharmaceutically acceptable salt thereof, but is not limited thereto.
본 발명에서, 상기 화학식 1로 표시되는 화합물이 암 세포의 성장 억제, 침윤(invasion) 억제 및 전이(metastasis) 억제로 이루어진 군으로부터 선택된 하나 이상의 효과를 갖는 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the compound represented by Formula 1 may have one or more effects selected from the group consisting of cancer cell growth inhibition, invasion inhibition, and metastasis inhibition, but is not limited thereto.
본 발명의 일 실시예에서는, 혈액 순환 미세체외소체(csEV)가 암세포의 이동 및 증식을 촉진함을 확인하고(실시예 1 참조), csEV에 의해 매개된 암의 이동, 암 세포 내 YAP의 발현 및 핵 국소화를 본 발명의 화합물이 억제하며(실시예 2 내지 4 참조), 본 발명의 화합물이 제피티닙 내성 암세포에 대하여 제피티닙과 병용시 시너지 효과를 나타내며(실시예 5 참조), 본 발명의 화합물이 대장암, 전립선암, 전이성 전립선암, 페소세포성 폐암, 항암제 내성 대장암, 간암 세포의 증식을 억제할 뿐만 아니라, 유선암 및 대장암 동종 이식 마우스 모델에서도 암세포 증식 억제 효과가 우수하며, 증식 억제 뿐만 아니라 이동 억제 효과가 있음을 확인하였는 바(실시예 6 참조), 본 발명의 화학식 1의 화합물을 암 치료 및 암 전이 억제에 사용할 수 있음을 확인하였다.In one embodiment of the present invention, it was confirmed that the blood circulating microextrabody (csEV) promotes the migration and proliferation of cancer cells (see Example 1), csEV-mediated cancer migration, and expression of YAP in cancer cells and the compound of the present invention inhibits nuclear localization (see Examples 2 to 4), and the compound of the present invention exhibits a synergistic effect on gefitinib-resistant cancer cells when used in combination with gefitinib (see Example 5), The compound of the present invention not only inhibits the proliferation of colon cancer, prostate cancer, metastatic prostate cancer, pesso-cell lung cancer, anticancer drug-resistant colorectal cancer, and liver cancer cells, but also has excellent cancer cell proliferation inhibitory effect in mammary and colon cancer allograft mouse models. , It was confirmed that the compound of Formula 1 of the present invention can be used for cancer treatment and cancer metastasis inhibition, as it was confirmed that there was an effect of inhibiting proliferation as well as migration inhibition (see Example 6).
본 발명에서 사용되는 용어 "암"은 세포의 사멸 조절과 관련된 질병으로서, 정상적인 세포 자살 (apoptosis)의 균형이 깨지는 경우 세포가 과다 증식하게 됨으로써 생기는 질병을 의미한다. 이러한 비정상적 과다 증식 세포들은 경우에 따라 주위 조직 및 장기에 침입하여 종괴 (腫塊)를 형성할 수 있으며 체내의 정상적인 구조의 파괴나 변형을 유발할 수 있는데, 이러한 상태를 통칭하여 암이라고 한다.As used herein, the term “cancer” is a disease related to the regulation of cell death, and refers to a disease caused by excessive cell proliferation when the balance of normal apoptosis is disrupted. In some cases, these abnormal hyperproliferative cells may invade surrounding tissues and organs to form a mass, and may cause destruction or transformation of normal structures in the body. This condition is collectively called cancer.
일반적으로 종양 (tumor)이라 하면 신체 조직의 자율적인 과잉 성장에 의해 비정상적으로 자란 덩어리를 의미하며, 종양은 양성 종양 (benign tumor)과 악성 종양 (malignant tumor)으로 구분할 수 있다. 악성 종양은 양성 종양에 비해 성장 속도가 매우 빠르며 주변 조직에 침윤하면서 전이 (metastasis)가 일어나 생명을 위협하게 된다. 이러한 악성 종양을 통상적으로 '암 (cancer)'이라 한다.In general, a tumor refers to an abnormally grown mass due to the autonomous overgrowth of body tissues, and tumors can be divided into benign tumors and malignant tumors. Malignant tumors grow very rapidly compared to benign tumors, and metastasis occurs while infiltrating the surrounding tissues, threatening life. Such malignant tumors are commonly referred to as 'cancer'.
암 전이는 원발성 암에서 암 세포가 타 장기로 퍼져 새로운 암을 형성하는 것이다. 전이는 다양한 암 환자에서 목숨을 위협하는 주요 현상이기에 전이를 예방하거나 조절하는 것은 암 연구분야의 중요 목표이다. 전이가 되지 않은 초기에 진단이 된 경우에는 수술, 항암치료, 또는 방사선 치료가 효과적이나 진단시에 전이가 되어 있는 경우에는 이들 치료의 효과는 감소된다. 이뿐 아니라 진단시 전이를 확인하지 못하였으나 전이가 치료 중이나 후에 확인되는 경우도 종종 있다. 임상적으로 암의 전이의 중요성이 높지만 전이 과정은 아직 완벽히 이해되고 있지 못한 상태이다.Cancer metastasis is the spread of cancer cells from primary cancer to other organs to form new cancer. Since metastasis is a major life-threatening phenomenon in various cancer patients, preventing or controlling metastasis is an important goal in cancer research. Surgery, chemotherapy, or radiation therapy are effective in the case of early diagnosis without metastasis, but the effectiveness of these treatments is reduced if metastasis has occurred at the time of diagnosis. In addition, although metastasis could not be confirmed at the time of diagnosis, metastasis is often identified during or after treatment. Although metastasis of cancer is clinically important, the metastasis process is still not fully understood.
전이는 침투 (invasion), 혈관내 유입 (intravasation), 멈춤 (arrest), 혈관외 배출 (extravasation), 그리고, 집락화 (colonization) 등의 연속적인 단계로 구성되어 있으며 이 과정을 통하여 원발성 장기에서 시작하여 최종적으로 다른 장기에 암을 형성하게 된다. 첫 번째 단계인 침투는 전이의 시작 단계로 암 세포의 세포간 또는 세포외 기질과의 상호작용 변화, 주변 조직의 분해, 그리고 조직내로의 암 세포의 이동 등을 포함한다. 두 번째 단계인 혈관내 유입은 암 세포가 혈관이나 림프관의 내피세포를 통과하여 전신적인 순환에 포함되는 것이다. 유입된 암 세포 중 극히 일부분만이 순환과정에서 살아남는 것으로 확인되고 있다. 살아 남은 일부분의 암 세포는 다른 부위의 모세관 내피세포를 투과하는 혈관외 배출에 성공하고 새로운 환경에 적응하여 증식하여 전이 암을 형성한다.Metastasis consists of successive stages such as invasion, intravasation, arrest, extravasation, and colonization, and through this process, Eventually, cancer will form in other organs. The first stage, invasion, is the starting stage of metastasis, and includes changes in the interaction of cancer cells with cells or extracellular matrix, degradation of surrounding tissues, and migration of cancer cells into tissues. The second step, intravascular entry, is when cancer cells pass through the endothelial cells of blood vessels or lymphatic vessels and are included in the systemic circulation. It has been confirmed that only a small fraction of the introduced cancer cells survive the cycle. A part of the cancer cells that survived succeed in extravasation through the capillary endothelial cells in other areas, adapt to the new environment, and proliferate to form metastatic cancer.
본 발명에서, 상기 암은 혈액 순환 미세체외소체(csEV) 매개 암일 수 있으나, 이에 제한되는 것은 아니다. 상기 혈액 순환 미세체외소체(csEV) 매개 암은 이동능이 있는 암, 즉 전이성 암이라면 이에 제한되는 것은 아니나, 예를 들어 전립선암, 유방암, 폐암, 결장암, 결장직장암, 갑상선암, 난소암, 방광암, 신장암, 위암, 자궁암, 직장암, 췌장암 및 흑색종으로 이루어진 군으로부터 선택된 하나 이상일 수 있다.In the present invention, the cancer may be a blood circulation microextrabody (csEV) mediated cancer, but is not limited thereto. The blood circulation microextrabody (csEV) mediated cancer is a cancer with mobility, that is, metastatic cancer, but is not limited thereto, for example, prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, thyroid cancer, ovarian cancer, bladder cancer, kidney It may be at least one selected from the group consisting of cancer, stomach cancer, uterine cancer, rectal cancer, pancreatic cancer and melanoma.
본 발명에서, 상기 화학식 1로 표시되는 화합물은 암세포의 M2 면역반응을 제어할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the compound represented by Formula 1 may control the M2 immune response of cancer cells, but is not limited thereto.
종양 내 면역세포 중 M2형 대식세포는 타 면역세포의 활성을 억제하고 혈관생성을 돕는 등 암의 생존에 절대적으로 영향을 미치는 세포로, 이를 억제하면 암세포의 성장 및 전이를 억제할 수 있다. 본 발명의 일 실시예에서는 본 발명의 화합물이 IL-4 처치에 의한 M2형 대식세포의 TGF-beta 발현 증가를 현저하게 억제하는 것으로 나타난 바, 본 발명의 화학식 1의 화합물이 암세포의 M2 면역반응을 제어함을 확인하였다.Among the immune cells in a tumor, M2-type macrophages are cells that have an absolute effect on the survival of cancer by inhibiting the activity of other immune cells and helping with angiogenesis. In one embodiment of the present invention, the compound of the present invention was shown to significantly inhibit the increase in TGF-beta expression of M2-type macrophages by IL-4 treatment. was confirmed to be controlled.
따라서, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 면역항암제를 제공한다.Accordingly, the present invention provides an immunotherapy agent comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명에서, 면역항암제란 면역체계를 자극함으로써 면역세포가 선택적으로 암세포만을 공격하도록 유도하는 치료약제를 의미한다. 종양으로 침투한 CD8+ T 세포, T helper 1 (TH1)으로 분화된 CD4+ T 세포 및 CD103+ DC가 항암 면역반응에 중추적인 역할을 하는 것으로 알려져 있으며, 본 발명의 일 실시예에서는 대장암 동종이식 마우스 모델에 본 발명의 화합물을 투여한 결과, 암세포의 항원을 인지하여 직접적으로 암세포의 사멸을 유도할 수 있는 CD8+ T 세포가 유의하게 증가하고, 암세포의 주변에서 도움 T 세포의 면역 기능을 저하시키는 것으로 알려진 조절 T 세포 마커인 FOXP3+CD4+ T 세포의 비율이 오히려 감소됨을 확인하였다. 따라서, 본 발명의 화합물을 면역항암제로서 유용하게 사용할 수 있을 것으로 기대된다.In the present invention, the immuno-oncology agent refers to a therapeutic agent that induces immune cells to selectively attack only cancer cells by stimulating the immune system. It is known that CD8+ T cells infiltrating the tumor, CD4+ T cells differentiated into T helper 1 (TH1), and CD103+ DCs play a central role in the anticancer immune response, and in one embodiment of the present invention, a colorectal cancer allograft mouse model As a result of administering the compound of the present invention, CD8+ T cells, which can directly induce cancer cell death by recognizing the antigen of cancer cells, significantly increase, and are known to decrease the immune function of helper T cells in the vicinity of cancer cells. It was confirmed that the ratio of FOXP3 + CD4 + T cells, which is a regulatory T cell marker, was rather decreased. Therefore, it is expected that the compound of the present invention can be usefully used as an immuno-cancer agent.
또한, 본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암 전이 억제용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 암의 치료를 필요로 하는 개체에 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 약학적 조성물을 투여하는 단계를 포함하는 암의 치료 방법을 제공한다.In addition, the present invention provides a method for treating cancer comprising administering to an individual in need of cancer treatment a pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient do.
또한, 본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 조성물의 암 치료 용도를 제공한다.In addition, the present invention provides a cancer treatment use of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 암 치료제를 생산하기 위한 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염의 용도를 제공한다.In addition, the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for producing a cancer therapeutic agent.
또한, 본 발명은 암 전이의 치료를 필요로 하는 개체에 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 약학적 조성물을 투여하는 단계를 포함하는 암 전이의 치료 방법을 제공한다.In addition, the present invention provides a treatment for cancer metastasis comprising administering a pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient to an individual in need of treatment for cancer metastasis provide a way
또한, 본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 조성물의 암 전이 억제 용도를 제공한다.In addition, the present invention provides a use for inhibiting cancer metastasis of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 암 전이 억제제를 생산하기 위한 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염의 용도를 제공한다.In addition, the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for producing a cancer metastasis inhibitor.
본 발명은 또한, 상기 약학적으로 허용가능한 염을 유효성분으로 포함할 수 있다. 본 발명에서 용어, "약학적으로 허용 가능한 염"이란 약학적으로 허용되는 무기산, 유기산, 또는 염기로부터 유도된 염을 포함한다. The present invention may also include the pharmaceutically acceptable salt as an active ingredient. As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
적합한 산의 예로는 염산, 브롬산, 황산, 질산, 과염소산, 푸마르산, 말레산, 인산, 글리콜산, 락트산, 살리실산, 숙신산, 톨루엔-p-설폰산, 타르타르산, 아세트산, 시트르산, 메탄설폰산, 포름산, 벤조산, 말론산, 글루콘산, 나프탈렌-2-설폰산, 벤젠설폰산 등을 들 수 있다. 산부가염은 통상의 방법, 예를 들면 화합물을 과량의 산 수용액에 용해시키고, 이 염을 메탄올, 에탄올, 아세톤 또는 아세토니트릴과 같은 수혼화성 유기 용매를 사용하여 침전시켜서 제조할 수 있다. 또한, 동몰량의 화합물 및 물 중의 산 또는 알코올을 가열하고 이어서 상기 혼합물을 증발시켜서 건조시키거나, 또는 석출된 염을 흡인 여과시켜 제조할 수 있다.Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an aqueous solution of an excess of acid, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can also be prepared by heating an equimolar amount of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or by suction filtration of the precipitated salt.
적합한 염기로부터 유도된 염은 나트륨, 칼륨 등의 알칼리 금속, 마그네슘 등의 알칼리 토금속, 및 암모늄 등을 포함할 수 있으나, 이에 제한되는 것은 아니다. 알칼리 금속 또는 알칼리 토금속염은, 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리토 금속 수산화물 용액 중에 용해하고, 비용해 화합물염을 여과한 후 여액을 증발, 건조시켜 얻을 수 있다. 이 때, 금속염으로서는 특히 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하며, 또한 이에 대응하는 은염은 알칼리 금속 또는 알칼리토 금속염을 적당한 은염(예, 질산은)과 반응시켜 얻을 수 있다.Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. In this case, as the metal salt, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).
본 발명의 조성물 내의 상기 화합물의 함량은 질환의 증상, 증상의 진행 정도, 환자의 상태 등에 따라서 적절히 조절 가능하며, 예컨대, 전체 조성물 중량을 기준으로 0.0001 내지 99.9중량%, 또는 0.001 내지 50중량%일 수 있으나, 이에 한정되는 것은 아니다. 상기 함량비는 용매를 제거한 건조량을 기준으로 한 값이다.The content of the compound in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition. However, the present invention is not limited thereto. The content ratio is a value based on the dry amount from which the solvent is removed.
본 발명에 따른 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 부형제는 예를 들어, 희석제, 결합제, 붕해제, 활택제, 흡착제, 보습제, 필름-코팅 물질, 및 제어방출첨가제로 이루어진 군으로부터 선택된 하나 이상일 수 있다. The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.
본 발명에 따른 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 서방형 과립제, 장용과립제, 액제, 점안제, 엘실릭제, 유제, 현탁액제, 주정제, 트로키제, 방향수제, 리모나아데제, 정제, 서방형정제, 장용정제, 설하정, 경질캅셀제, 연질캅셀제, 서방캅셀제, 장용캅셀제, 환제, 틴크제, 연조엑스제, 건조엑스제, 유동엑스제, 주사제, 캡슐제, 관류액, 경고제, 로션제, 파스타제, 분무제, 흡입제, 패취제, 멸균주사용액, 또는에어로졸 등의 외용제 등의 형태로 제형화하여 사용될 수 있으며, 상기 외용제는 크림, 젤, 패치, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 또는 카타플라스마제 등의 제형을 가질 수 있다. The pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, alcohols, troches, fragrances, and limonaade. , tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.
본 발명에 따른 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
본 발명에 따른 정제, 산제, 과립제, 캡슐제, 환제, 트로키제의 첨가제로 옥수수전분, 감자전분, 밀전분, 유당, 백당, 포도당, 과당, 디-만니톨, 침강탄산칼슘, 합성규산알루미늄, 인산일수소칼슘, 황산칼슘, 염화나트륨, 탄산수소나트륨, 정제 라놀린, 미결정셀룰로오스, 덱스트린, 알긴산나트륨, 메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 카올린, 요소, 콜로이드성실리카겔, 히드록시프로필스타치, 히드록시프로필메칠셀룰로오스(HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, 프로필렌글리콜, 카제인, 젖산칼슘, 프리모젤 등 부형제; 젤라틴, 아라비아고무, 에탄올, 한천가루, 초산프탈산셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스칼슘, 포도당, 정제수, 카제인나트륨, 글리세린, 스테아린산, 카르복시메칠셀룰로오스나트륨, 메칠셀룰로오스나트륨, 메칠셀룰로오스, 미결정셀룰로오스, 덱스트린, 히드록시셀룰로오스, 히드록시프로필스타치, 히드록시메칠셀룰로오스, 정제쉘락, 전분호, 히드록시프로필셀룰로오스, 히드록시프로필메칠셀룰로오스, 폴리비닐알코올, 폴리비닐피롤리돈 등의 결합제가 사용될 수 있으며, 히드록시프로필메칠셀룰로오스, 옥수수전분, 한천가루, 메칠셀룰로오스, 벤토나이트, 히드록시프로필스타치, 카르복시메칠셀룰로오스나트륨, 알긴산나트륨, 카르복시메칠셀룰로오스칼슘, 구연산칼슘, 라우릴황산나트륨, 무수규산, 1-히드록시프로필셀룰로오스, 덱스트란, 이온교환수지, 초산폴리비닐, 포름알데히드처리 카제인 및 젤라틴, 알긴산, 아밀로오스, 구아르고무(Guar gum), 중조, 폴리비닐피롤리돈, 인산칼슘, 겔화전분, 아라비아고무, 아밀로펙틴, 펙틴, 폴리인산나트륨, 에칠셀룰로오스, 백당, 규산마그네슘알루미늄, 디-소르비톨액, 경질무수규산 등 붕해제; 스테아린산칼슘, 스테아린산마그네슘, 스테아린산, 수소화식물유(Hydrogenated vegetable oil), 탈크, 석송자, 카올린, 바셀린, 스테아린산나트륨, 카카오지, 살리실산나트륨, 살리실산마그네슘, 폴리에칠렌글리콜(PEG) 4000, PEG 6000, 유동파라핀, 수소첨가대두유(Lubri wax), 스테아린산알루미늄, 스테아린산아연, 라우릴황산나트륨, 산화마그네슘, 마크로골(Macrogol), 합성규산알루미늄, 무수규산, 고급지방산, 고급알코올, 실리콘유, 파라핀유, 폴리에칠렌글리콜지방산에테르, 전분, 염화나트륨, 초산나트륨, 올레인산나트륨, dl-로이신, 경질무수규산 등의 활택제;가 사용될 수 있다.Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodite, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Additive soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, A lubricant such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.
본 발명에 따른 액제의 첨가제로는 물, 묽은 염산, 묽은 황산, 구연산나트륨, 모노스테아린산슈크로스류, 폴리옥시에칠렌소르비톨지방산에스텔류(트윈에스텔), 폴리옥시에칠렌모노알킬에텔류, 라놀린에텔류, 라놀린에스텔류, 초산, 염산, 암모니아수, 탄산암모늄, 수산화칼륨, 수산화나트륨, 프롤아민, 폴리비닐피롤리돈, 에칠셀룰로오스, 카르복시메칠셀룰로오스나트륨 등이 사용될 수 있다.As additives for the liquid formulation according to the present invention, water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.
본 발명에 따른 시럽제에는 백당의 용액, 다른 당류 혹은 감미제 등이 사용될 수 있으며, 필요에 따라 방향제, 착색제, 보존제, 안정제, 현탁화제, 유화제, 점조제 등이 사용될 수 있다.In the syrup according to the present invention, a sucrose solution, other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifying agent, thickening agent, etc. may be used.
본 발명에 따른 유제에는 정제수가 사용될 수 있으며, 필요에 따라 유화제, 보존제, 안정제, 방향제 등이 사용될 수 있다.Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.
본 발명에 따른 현탁제에는 아카시아, 트라가칸타, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 미결정셀룰로오스, 알긴산나트륨, 히드록시프로필메칠셀룰로오스, HPMC 1828, HPMC 2906, HPMC 2910 등 현탁화제가 사용될 수 있으며, 필요에 따라 계면활성제, 보존제, 안정제, 착색제, 방향제가 사용될 수 있다.In the suspending agent according to the present invention, a suspending agent such as acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910 may be used. and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.
본 발명에 따른 주사제에는 주사용 증류수, 0.9%염화나트륨주사액, 링겔주사액, 덱스트로스주사액, 덱스트로스+염화나트륨주사액, 피이지(PEG), 락테이티드 링겔주사액, 에탄올, 프로필렌글리콜, 비휘발성유-참기름, 면실유, 낙화생유, 콩기름, 옥수수기름, 올레인산에칠, 미리스트산 이소프로필, 안식향산벤젠과 같은 용제; 안식향산나트륨, 살리실산나트륨, 초산나트륨, 요소, 우레탄, 모노에칠아세트아마이드, 부타졸리딘, 프로필렌글리콜, 트윈류, 니정틴산아미드, 헥사민, 디메칠아세트아마이드와 같은 용해보조제; 약산 및 그 염(초산과 초산나트륨), 약염기 및 그 염(암모니아 및 초산암모니움), 유기화합물, 단백질, 알부민, 펩톤, 검류와 같은 완충제; 염화나트륨과 같은 등장화제; 중아황산나트륨(NaHSO3) 이산화탄소가스, 메타중아황산나트륨(Na2S2O5), 아황산나트륨(Na2SO3), 질소가스(N2), 에칠렌디아민테트라초산과 같은 안정제; 소디움비설파이드 0.1%, 소디움포름알데히드 설폭실레이트, 치오우레아, 에칠렌디아민테트라초산디나트륨, 아세톤소디움비설파이트와 같은 황산화제; 벤질알코올, 클로로부탄올, 염산프로카인, 포도당, 글루콘산칼슘과 같은 무통화제; 시엠시나트륨, 알긴산나트륨, 트윈 80, 모노스테아린산알루미늄과 같은 현탁화제를 포함할 수 있다.Injectables according to the present invention include distilled water for injection, 0.9% sodium chloride injection, ring gel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated ring gel injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin, peptone and gum; isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO 3 ) carbon dioxide gas, sodium metabisulfite (Na 2 S 2 O 5 ), sodium sulfite (Na 2 SO 3 ), nitrogen gas (N 2 ), ethylenediaminetetraacetic acid; sulphating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesic agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as SiMC sodium, sodium alginate, Tween 80, or aluminum monostearate.
본 발명에 따른 좌제에는 카카오지, 라놀린, 위텝솔, 폴리에틸렌글리콜, 글리세로젤라틴, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 스테아린산과 올레인산의 혼합물, 수바날(Subanal), 면실유, 낙화생유, 야자유, 카카오버터+콜레스테롤, 레시틴, 라네트왁스, 모노스테아린산글리세롤, 트윈 또는 스판, 임하우젠(Imhausen), 모놀렌(모노스테아린산프로필렌글리콜), 글리세린, 아뎁스솔리두스(Adeps solidus), 부티룸 태고-G(Buytyrum Tego-G), 세베스파마 16(Cebes Pharma 16), 헥사라이드베이스 95, 코토마(Cotomar), 히드록코테 SP, S-70-XXA, S-70-XX75(S-70-XX95), 히드록코테(Hydrokote) 25, 히드록코테 711, 이드로포스탈(Idropostal), 마사에스트라리움(Massa estrarium, A, AS, B, C, D, E, I, T), 마사-MF, 마수폴, 마수폴-15, 네오수포스탈-엔, 파라마운드-B, 수포시로(OSI, OSIX, A, B, C, D, H, L), 좌제기제 IV 타입(AB, B, A, BC, BBG, E, BGF, C, D, 299), 수포스탈(N, Es), 웨코비(W, R, S, M ,Fs), 테제스터 트리글리세라이드 기제(TG-95, MA, 57)와 같은 기제가 사용될 수 있다.The suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), supostal (N, Es), Wecobi (W, R, S, M, Fs), tester triglyceride base (TG-95, MA, 57) and The same mechanism may be used.
경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "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 is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.
본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 본 발명이 속하는 기술분야에 통상의 기술자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.
본 발명의 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구 복용, 피하 주사, 복강 투여, 정맥 주사, 근육 주사, 척수 주위 공간(경막내) 주사, 설하 투여, 볼점막 투여, 직장 내 삽입, 질 내 삽입, 안구 투여, 귀 투여, 비강 투여, 흡입, 입 또는 코를 통한 분무, 피부 투여, 경피 투여 등에 따라 투여될 수 있다.The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration can be contemplated, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.
본 발명의 약학적 조성물은 치료할 질환, 투여 경로, 환자의 연령, 성별, 체중 및 질환의 중등도 등의 여러 관련 인자와 함께 활성성분인 약물의 종류에 따라 결정된다.The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.
본 발명에서 "개체"란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐(mouse), 쥐(rat), 개, 고양이, 말, 및 소 등의 포유류일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. It may be a mammal of, but is not limited thereto.
본 발명에서 "투여"란 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다.In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.
본 발명에서 "예방"이란 목적하는 질환의 발병을 억제하거나 지연시키는 모든 행위를 의미하고, "치료"란 본 발명에 따른 약학적 조성물의 투여에 의해 목적하는 질환과 그에 따른 대사 이상 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미하며, "개선"이란 본 발명에 따른 조성물의 투여에 의해 목적하는 질환과 관련된 파라미터, 예를 들면 증상의 정도를 감소시키는 모든 행위를 의미한다. In the present invention, "prevention" means any action that inhibits or delays the onset of a desired disease, and "treatment" means that the desired disease and metabolic abnormalities are improved or It means all actions that are advantageously changed, and "improvement" means all actions that reduce the parameters related to the desired disease, for example, the degree of symptoms by administration of the composition according to the present invention.
본 명세서에서 언급된 모든 문헌은 그 내용이 본 명세서에 기재된 것처럼 본 명세서에 참조로 포함된다. 본 발명 또는 이의 바람직한 태양(들)의 요소를 도입할 때, 관사 "하나의(a)", "하나의(an)", "그(the)" 및 "상기(said)"는 하나 이상의 요소가 있는 것을 의미하는 것으로 의도된다. 용어 "포함하는(comprising)", "포함하는(including)" 및 "갖는(having)"은 포괄적인 것으로 의도되고, 나열된 요소 이외의 추가적인 요소가 있을 수 있다는 것을 의미한다. 비록 본 발명이 특정 양태 또는 태양에 관하여 설명되었지만, 이들 양태의 세부 사항을 한정하는 것으로 해석되어서는 안된다.All documents mentioned herein are incorporated herein by reference as if their contents were set forth herein. When introducing an element of the present invention or preferred aspect(s) thereof, the articles “a”, “an”, “the” and “said” refer to one or more of the elements. is intended to mean that there is The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Although the invention has been described with respect to specific aspects or aspects, it should not be construed as limiting the details of these aspects.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
실시예 1. 음전하를 띠는 혈액 순환 미세체외소체(csEV)의 암세포 이동에서의 역할 확인Example 1. Identification of the role of negatively charged blood circulating microextrabody (csEV) in cancer cell migration
1-1. 미세소체의 분리1-1. Isolation of microbody
먼저, 도 1a에 나타난 바와 같이, 5단계 연속적인 원심 분리를 사용하여 csEV를 인간, 소 및 돼지 혈청에서 분리하였다. First, as shown in Fig. 1a, csEV was isolated from human, bovine and porcine serum using five-step successive centrifugation.
종 내 분산을 최소화하기 위하여, 유사한 실험에서 동일한 로트 수의 소 혈청에서 유래된 csEV를 사용했으며, 주요 데이터는 인간 혈청의 csEV를 사용하여 확인하였다. To minimize intraspecies variance, csEV derived from bovine serum of the same lot number was used in a similar experiment, and the main data were confirmed using csEV from human serum.
돼지 혈청(Porcine serum), 소 혈청(bovine serum)은 Invitrogen(Karlsruhe, Germany)로부터 구입하였으며, 건강한 성인 남성에게서 얻은 혈청을 죽은 세포, 잔여물 제거를 위해 단계별로 원심 분리 하였다. 먼저 300g로 10분, 2500g로 20분 동안 원심 분리하여 잔여 세포와 잔여물을 제거하고 상층액을 취하였다. 얻은 상층액을 10000g로 30분 동안 원심 분리하여 microvesicle을 제거하였다. 이로부터 얻은 상층액에서 EV 크기보다 큰 vesicles, contaminants 제거를 위해 200nm filtration을 수행하였다. 이 과정을 통하여 최종적으로 얻은 상등액을 120000g, 120분으로 초원심분리하였다(Optima XE-100, Type 32Ti rotor, Beckman Coulter). 다음으로 EV에 오염된 단백질 등의 제거를 위해 상층액을 모두 버린 후 DPBS로 튜브를 채워 한 번 더 120000g, 90분 조건에서 초원심분리 한 후, 상층액을 버리고 오염 최소화 및 순수한 EV의 분리를 위하여 200 nm filteration을 재수행하였다. 소량의 PBS 혹은 실험을 위한 solution에 녹인 후 실험에 사용하였으며 EV 정량을 위하여 Bradford assay를 수행하였다. 성인 남성의 혈청 획득을 위하여 서울대학교 윤리위원회의 승인을 획득하였으며 (#SNU 19-2-040), 분리된 csEV는 4℃에 보관하고 5일 내로 사용되었다.Porcine serum and bovine serum were purchased from Invitrogen (Karlsruhe, Germany), and the serum obtained from a healthy adult male was centrifuged step by step to remove dead cells and residues. First, the cells were centrifuged at 300 g for 10 minutes and at 2500 g for 20 minutes to remove residual cells and residues, and the supernatant was taken. The obtained supernatant was centrifuged at 10000 g for 30 minutes to remove microvesicles. 200nm filtration was performed to remove vesicles and contaminants larger than the EV size from the supernatant obtained therefrom. The supernatant finally obtained through this process was ultracentrifuged at 120000 g, 120 minutes (Optima XE-100, Type 32Ti rotor, Beckman Coulter). Next, discard all of the supernatant to remove EV-contaminated proteins, fill the tube with DPBS, perform ultracentrifugation once more at 120000 g, 90 minutes, discard the supernatant, minimize contamination and separate pure EVs For this purpose, 200 nm filtration was performed again. After dissolving in a small amount of PBS or solution for the experiment, it was used for the experiment, and Bradford assay was performed for EV quantification. Approval from the Seoul National University Ethics Committee was obtained for the acquisition of adult male serum (#SNU 19-2-040), and the isolated csEV was stored at 4°C and used within 5 days.
도 1b에 나타난 바와 같이, 직접 광 산란(DLS) 데이터에서 분리된 EV의 크기는 100nm 미만으로, 미세체외소체로 분류하였다.As shown in Figure 1b, the size of the EVs isolated from the direct light scattering (DLS) data was less than 100 nm, and they were classified as microex vivos.
도 1c에 나타난 바와 같이, 공초점 현미경에 의한 Did-표지 csEV 흡수를 확인한 결과, 분리된 소포가 미세체외소체임을 확인하였다.As shown in Figure 1c, Did-labeled csEV absorption was confirmed by confocal microscopy, and it was confirmed that the separated vesicles were microex vivo.
1-2. csEV의 암세포 이동 촉진 효과 확인1-2. Confirmation of the effect of csEV to promote cancer cell migration
종양 세포에서 비롯된 EV가 전이를 증가시킨 것을 고려하여, csEV가 암세포의 이동을 촉진하는지 여부를 조사하였다. Considering that EVs derived from tumor cells increased metastasis, we investigated whether csEV promotes the migration of cancer cells.
IncuCyte ClearView 96-well chemotaxis plates(Essen bioscience, MI, USA)를 이용하여 tranaswell migration 실험을 수행하였다. 트랜스웰 유닛의 아랫부분에 Type Ⅰ Collagen(Collaborative Research, KY, USA)을 코팅한 후 1시간 동안 상온에서 말리는 과정을 수행하였다. 이후 FBS가 없는 조건의 RPMI 배지에 13×103 cells/well로 준비한 후, 약물을 함께 처리한 실험군 또는 대조군을 60 μL씩 트랜스웰 유닛 상단부에 시딩하였다. 유인 화학 물질이 들어가는 하단부 well에는 10% FBS, 10% EV-free FBS, 또는 serum free RPMI media에 csEV을 추가한 배지 총 200μL가 사용되었다. 세포의 이동은 Incucyte Chemotaxis live-cell analysis (ESSEN bioscience)을 사용하여 4시간 간격으로 모니터 하였다.Transaswell migration experiments were performed using IncuCyte ClearView 96-well chemotaxis plates (Essen bioscience, MI, USA). The lower part of the transwell unit was coated with Type I Collagen (Collaborative Research, KY, USA) and dried at room temperature for 1 hour. After preparing at 13×10 3 cells/well in RPMI medium without FBS, the experimental group or control group treated with the drug was seeded at the upper end of the transwell unit by 60 μL. A total of 200 μL of medium containing 10% FBS, 10% EV-free FBS, or csEV in serum-free RPMI media was used for the lower well containing the attracting chemical. Cell migration was monitored at 4-hour intervals using Incucyte Chemotaxis live-cell analysis (ESSEN bioscience).
도 1d에 나타난 바와 같이, 소, 돼지 또는 인간 혈청에서 유래한 csEV는 LNCaP 전립선 암세포의 공격적인 하위 라인인 LNCaP-SL 암세포의 이동을 촉진하였다. 특히, 인간 혈청에서 유래된 csEV는 가장 강력한 이동 능력(38.1 배)을 나타내었다.As shown in Fig. 1d, csEV derived from bovine, porcine or human serum promoted migration of LNCaP-SL cancer cells, an aggressive subline of LNCaP prostate cancer cells. In particular, csEV derived from human serum exhibited the strongest migratory capacity (38.1 fold).
또한, 상기와 동일한 방법으로, 다른 이동능이 높은 암 세포주(LNCaP-SL(전립선암), 타목시펜 내성-MCF-7(유방암), 제피티닙 내성-HCC-827(폐암), HCT116(결장암) 및 SW480(결장암)) 및 환자 유래 원발성 유방암에서 csEV의 이동 효과를 평가하였다. In addition, by the same method as above, other high-mobility cancer cell lines (LNCaP-SL (prostate cancer), tamoxifen-resistant-MCF-7 (breast cancer), gefitinib-resistant-HCC-827 (lung cancer), HCT116 (colon cancer) and SW480 (colon cancer)) and the migration effect of csEV in patient-derived primary breast cancer were evaluated.
도 1e에 나타난 바와 같이, 5개의 암세포의 세포 이동은 csEV에 농도 의존적으로 증가하였다.As shown in FIG. 1E , the cell migration of five cancer cells was increased in a concentration-dependent manner with csEV.
다음으로, csEV의 어떤 구성 요소가 암세포의 강력한 이동을 유발하는지 조사하였다. Next, we investigated which components of csEV induce robust migration of cancer cells.
먼저, csEV의 포획 및 방출을 위해 과산화된 폴리피롤(overoxidized Ppy) 표면을 500μL 세럼에 30분간 배양했다. csEV를 포획하기 위해 30초간 Ppy 표면에 +0.5, 0, -0.5V의 전기 자극을 가한 후 탈이온수로 Ppy 표면을 두 번 헹구었다. 포획된 엑소좀은 3분간 -1.3V에서 전위를 가하여 Ppy 표면에서 용출되었다. 포획된 EV의 총 수 및 이동능은 제조사의 프로토콜에 따라 나노입자 추적 분석(NTA) 소프트웨어(NanoSight NS300, Malvern Instruments, 영국 Malvern Instruments)를 사용하여 정량화되었다.First, for capture and release of csEV, the surface of overoxidized Ppy was incubated in 500 μL serum for 30 minutes. To capture csEV, electrical stimulation of +0.5, 0, -0.5V was applied to the Ppy surface for 30 seconds, and then the Ppy surface was rinsed twice with deionized water. The captured exosomes were eluted from the Ppy surface by applying a potential at -1.3V for 3 min. The total number and mobility of captured EVs were quantified using Nanoparticle Tracking Analysis (NTA) software (NanoSight NS300, Malvern Instruments, Malvern Instruments, UK) according to the manufacturer's protocol.
먼저, 도 1f에 나타난 바와 같이, 전자 자극에 의해 전하 분포를 갖는 csEV를 구분하고 csEV의 '표면 전하'에 따른 차등 이동 능력을 관찰하였다. First, as shown in FIG. 1f , csEVs having a charge distribution were distinguished by electron stimulation, and the differential migration ability according to the 'surface charge' of csEVs was observed.
도 1g에 나타난 바와 같이, 상대적으로 음전하를 띠는 csEV는 다른 csEV보다 가장 강력한 이동 효과를 나타냈다. 이는 음전하를 갖는 지질이 EV의 표면 성분이기 때문에, EV의 지질 성분이 관여하는 것으로 판단하였다. As shown in Fig. 1g, the relatively negatively charged csEV showed the strongest migration effect than other csEVs. It was determined that the lipid component of EV is involved because the lipid having a negative charge is a surface component of EV.
실시예 2. N-사이클로헥실피리미딘-4-아민(N-cyclohexylpyrimidin-4-amine) 유도체의 csEV 매개 암 이동 억제 확인Example 2. Confirmation of inhibition of csEV-mediated cancer migration by N-cyclohexylpyrimidin-4-amine derivatives
N-사이클로헥실피리미딘-4-아민(N-cyclohexylpyrimidin-4-amine) 유도체인 KRCT-6j의 csEV 매개 암 억제 활성을 확인하기 위하여, 실시예 1에서 확인된 csEV 매개 LNCaP-SL 세포를 사용하였다. In order to confirm the csEV-mediated cancer inhibition activity of KRCT-6j, an N-cyclohexylpyrimidin-4-amine derivative, the csEV-mediated LNCaP-SL cells identified in Example 1 were used. did
구체적으로, IncuCyte ClearView 96-웰 화학주성 플레이트(Essen bioscience, MI, USA)를 이용하여 트랜스웰 이동(tranaswell migration) 실험을 수행하였다. 트랜스웰 유닛의 아랫부분에 Type Ⅰ 콜라겐(Collaborative Research, KY, USA)을 코팅한 후 1시간 동안 상온에서 말리는 과정을 수행하였다. 이후 FBS가 없는 조건의 RPMI 배지에 3×103 cells/well을 준비한 후, 약물을 함께 처리한 실험군 또는 대조군을 60 μL씩 트랜스웰 유닛 상단부에 분주하였다. 유인 화학 물질이 들어가는 하단부 웰에는 csEV 혹은 대조군인 PBS를 추가한 배지 총 200μL가 사용되었다. 세포의 이동은 Incucyte Chemotaxis live-cell analysis (ESSEN bioscience)을 사용하여 4시간 간격으로 모니터링 하였다.Specifically, a transwell migration experiment was performed using an IncuCyte ClearView 96-well chemotactic plate (Essen bioscience, MI, USA). The lower part of the transwell unit was coated with Type I collagen (Collaborative Research, KY, USA) and dried at room temperature for 1 hour. After preparing 3×10 3 cells/well in RPMI medium without FBS, 60 μL of the experimental group or control group treated with the drug was dispensed at the upper end of the transwell unit. A total of 200 μL of medium supplemented with csEV or PBS as a control was used for the lower wells containing the attracting chemical. Cell migration was monitored at 4-hour intervals using Incucyte Chemotaxis live-cell analysis (ESSEN bioscience).
도 2에 나타난 바와 같이, csEV에 의해 촉진된 LNCaP-SL세포의 이동은 농도 의존적으로 KRCT-6j에 의해 억제되었다.As shown in FIG. 2 , the migration of LNCaP-SL cells promoted by csEV was inhibited by KRCT-6j in a concentration-dependent manner.
<KRCT-6j(KRCT-0005)><KRCT-6j(KRCT-0005)>
IUPAC name:IUPAC name:
N 4-Cyclohexyl-N 2-(3,5-dichlorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidine-2,4-diamine N 4 -Cyclohexyl- N 2 -(3,5-dichlorophenyl)-5-(1-methyl-1 H -pyrazol-4-yl)pyrimidine-2,4-diamine
Figure PCTKR2021012192-appb-img-000008
Figure PCTKR2021012192-appb-img-000008
실시예 3. YAP(Yes-Associated Protein) 활성화를 통한 csEV에 의한 암세포 생존 확인Example 3. Confirmation of cancer cell survival by csEV through YAP (Yes-Associated Protein) activation
암세포 증식에서 csEV의 역할을 더 확인하기 위해, LNCaP-SL 세포의 증식에 대한 csEV의 효과를 조사하였다. To further confirm the role of csEV in cancer cell proliferation, the effect of csEV on the proliferation of LNCaP-SL cells was investigated.
1X103의 LNCaP-SL cells을 96 웰 플레이트에 100μL 부피로 분주한 후 다음 날 csEV 혹은 PBS(대조군)가 포함된 배지로 배지를 바꾸었다. 그 후 세포의 phase confluence을 IIncuCyte ZOOM Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI)을 사용하여 4시간마다 모니터링하였다.After dispensing 1X10 3 LNCaP-SL cells in a volume of 100 μL in a 96-well plate, the medium was changed to a medium containing csEV or PBS (control) the next day. Thereafter, cell phase confluence was monitored every 4 hours using the IIncuCyte ZOOM Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI).
도 3a 및 도 3b에 나타난 바와 같이, LNCaP-SL의 증식은 FBS 함유 배지 하에서 csEV에 의해 영향을 받지 않았지만, csEV 처리는 무혈청 상태에서 세포 증식 을 상당히 촉진하였다(96 시간에서 7.8배 VS 12.8배).3a and 3b, the proliferation of LNCaP-SL was not affected by csEV under FBS-containing medium, but csEV treatment significantly promoted cell proliferation in the serum-free state (7.8-fold VS 12.8-fold at 96 h). ).
csEV 매개 증식을 매개하는 분자 기전을 밝히기 위하여, 대표적인 성장 인자 신호 전달 경로(PI3 kinase-AKT 및 MEK1/2-ERK)의 활성을 조사하였다.In order to elucidate the molecular mechanisms mediating csEV-mediated proliferation, the activities of representative growth factor signaling pathways (PI3 kinase-AKT and MEK1/2-ERK) were investigated.
도 3c에 나타난 바와 같이, csEV 처리 후 AKT(Protein kinase B) 또는 ERK(Extracellular-signal-regulated kinase)의 활성에는 뚜렷한 변화가 없는 것으로 나타났다.As shown in FIG. 3c , there was no significant change in the activity of AKT (Protein kinase B) or ERK (Extracellular-signal-regulated kinase) after csEV treatment.
또한, YAP/TAZ 반응성 TEAD 리포터 루시퍼라제 분석을 사용하여 csEV가 TEAD 리포터 유전자를 트랜스 활성화하는지 여부를 조사하였다.In addition, we investigated whether csEV trans-activates the TEAD reporter gene using a YAP/TAZ-responsive TEAD reporter luciferase assay.
도 3d에 나타난 바와 같이, 면역세포화학 분석으로, csEV와의 배양에 의해 YAP의 발현 및 핵 국소화가 증가하고 KRCT-6j 처리에 의해 다시 감소됨을 확인하였다.As shown in Fig. 3d, immunocytochemical analysis confirmed that YAP expression and nuclear localization were increased by incubation with csEV and decreased again by KRCT-6j treatment.
실시예 4. 침습성 암에서 화학내성 극복을 위한 타겟 확인Example 4. Identification of a target for overcoming chemical resistance in invasive cancer
최근 연구에 따르면 YAP 활성은 다양한 암 유형, 특히 표피 성장 인자 수용체 티로신 키나제 억제제(EGFR-TKI)에서 화학 내성과 밀접한 관련이 있는 것으로 확인되었다. 따라서 Tyro3-YAP 축이 항암제에 대한 화학 감수성에 영향을 미칠 수 있는지 여부를 측정하였다. 먼저, EV 함유 정상 FBS 또는 EV가 없는 FBS와 전이성 전립선 암에 대한 대표적인 화학 요법제인 50nM 도세탁셀(docetaxel)을 이용하여 LNCaP-SL 세포의 증식을 측정하였다. Recent studies have confirmed that YAP activity is closely related to chemical resistance in various cancer types, particularly epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Therefore, we measured whether the Tyro3-YAP axis could affect the chemosensitivity to anticancer drugs. First, proliferation of LNCaP-SL cells was measured using EV-containing normal FBS or EV-free FBS and 50 nM docetaxel, a representative chemotherapeutic agent for metastatic prostate cancer.
1X103의 LNCaP-SL cells을 96 웰 플레이트에 100 μL 부피로 분주한 후 다음 날 csEV 및 도세탁셀(docetaxel)이 포함된 배지로 배지를 바꾸었다. 그 후 세포의 phase confluence을 IIncuCyte ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI)을 사용하여 4시간마다 모니터링하였다.After dispensing 1X10 3 LNCaP-SL cells in a volume of 100 μL in a 96-well plate, the medium was changed to a medium containing csEV and docetaxel the next day. Then, cell phase confluence was monitored every 4 hours using IIncuCyte ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI).
도 4a에 나타난 바와 같이, LNCaP-SL 세포는 EV가 없는 FBS 10% 조건에 비해 EV 함유 정상 10% FBS 하에서 도세탁셀에 훨씬 더 내성을 보이는 것으로 나타났다.As shown in Figure 4a, LNCaP-SL cells were found to be much more resistant to docetaxel under the EV-containing normal 10% FBS compared to the FBS 10% condition without EVs.
또한, 도 4b 및 4c에 나타난 바와 같이, csEV 처리는 GR-H1993(제피티닙 내성-H1993)의 세포 증식을 증가시켰지만, 낮은 수준의 Tyro3 발현을 나타내는 H1993 또는 ER-H1993(엘로티닙 내성-H1993) 세포에서는 그렇지 않았다. 게다가, KRCT-6j는 csEV 매개 GR-H1993의 증식 효과만을 억제하는 것으로 나타났다. In addition, as shown in FIGS. 4B and 4C , csEV treatment increased cell proliferation of GR-H1993 (gefitinib-resistant-H1993), but showed low levels of Tyro3 expression in H1993 or ER-H1993 (erlotinib-resistant-H1993). ), not in cells. Moreover, KRCT-6j was shown to inhibit only the proliferative effect of csEV-mediated GR-H1993.
다음으로 H1993 및 GR-H1993 세포에서 YAP의 발현 수준과 인산화 강도를 모니터링하였다. Next, the expression level and phosphorylation intensity of YAP in H1993 and GR-H1993 cells were monitored.
세포의 배양 배지를 제거한 후 세포에 탈인산화효소 억제제(phosphatase inhibitor, Sigma-Aldrich, St.Louis, MO, USA)와 프로티네이즈 억제제(proteinase inhibitor, Roche, Basel, Switzerland)를 첨가한 세포 용해 완충액(50mM Tris-Cl, pH7.6, 120mM NaCl, 1mM EDTA, pH8.0, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 50mM NaF, 200μM sodium orthovanadate, 1mM phenylmethylsulfonyl fluoride(PMSF))을 가하여 1시간 동안 얼음 위에서 세포를 용해하였다. 이후 13000g에서 15분 동안 원심분리하여 얻은 상등액을 전세포 추출액으로 사용하여 Bradford 정량법을 이용하여 단백질 농도를 정량하였다. 이후 각각의 단백질 샘플을 8-12% 사이의 젤을 이용해서 SDS-PAGE(sodium dodecylsulfate-polyacrylamide gel electrophoresis)법으로 분리하였다. 이후 전기영동이 끝난 겔을 나이트로셀룰로오즈 막 0.45μm 필터(GE healthcare Life Sciences, Chalfont, Buckinghamshire, UK)로 전이완충용액(25mM Tris, 192mM glycine, 20% v/v methanol, pH 8.3)을 이용하여 40V, 190분간 transfer 시켰다. 이후 transfer시킨 나이트로셀룰로오즈 막을 5% skim milk(BD Biosciences, San Jose, CA)로 1시간 동안 인큐베이션 한 후, 1차 항체와 4℃에서 밤새 배양 후, HRP-결합된 항-IgG를 2차 항체로 사용하였다. 이를 enhanced chemiluminescence(ECL) system reagent(EMD Milipore, Billerica, MA, USA)로 발색하여 LAS3000-mini(Fujifilm, Tokyo, Japan)으로 측정하였다. p-YAP (#4911) 및 YAP (#8418) 항체는 Cell Signaling Technology (Beverly, MA)에서 구매하였다.After removing the cell culture medium, a cell lysis buffer in which a dephosphorylation enzyme inhibitor (phosphatase inhibitor, Sigma-Aldrich, St.Louis, MO, USA) and a proteinase inhibitor (proteinase inhibitor, Roche, Basel, Switzerland) were added to the cells (50mM Tris-Cl, pH7.6, 120mM NaCl, 1mM EDTA, pH8.0, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 50mM NaF, 200μM sodium orthovanadate, 1mM phenylmethylsulfonyl fluoride (PMSF) )) was added and the cells were lysed on ice for 1 hour. Then, the supernatant obtained by centrifugation at 13000 g for 15 minutes was used as a whole cell extract, and the protein concentration was quantified using the Bradford quantification method. Then, each protein sample was separated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) using a gel between 8-12%. After electrophoresis, the gel was transferred to a nitrocellulose membrane 0.45 μm filter (GE healthcare Life Sciences, Chalfont, Buckinghamshire, UK) with a transfer buffer solution (25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3). Transfer was carried out at 40V for 190 minutes. After incubating the transferred nitrocellulose membrane with 5% skim milk (BD Biosciences, San Jose, CA) for 1 hour, incubated with the primary antibody overnight at 4°C, HRP-conjugated anti-IgG secondary antibody was used as The color was developed with an enhanced chemiluminescence (ECL) system reagent (EMD Milipore, Billerica, MA, USA) and measured with LAS3000-mini (Fujifilm, Tokyo, Japan). p-YAP (#4911) and YAP (#8418) antibodies It was purchased from Signaling Technology (Beverly, MA).
도 4d에 나타난 바와 같이, YAP의 발현은 기원 H1993 세포보다 GR-H1993 세포에서 현저하게 증가했다.As shown in FIG. 4d , the expression of YAP was significantly increased in GR-H1993 cells than in origin H1993 cells.
또한, 도 4e에 나타난 바와 같이, GR-H1993 세포를 KRCT-6j로 처리하면 csEV로 유도된 YAP의 핵 국소화가 억제되었다.In addition, as shown in Fig. 4e, treatment of GR-H1993 cells with KRCT-6j inhibited csEV-induced nuclear localization of YAP.
실시예 5. 제피티닙과 시너지 효과 확인Example 5. Confirmation of synergistic effect with gefitinib
실시간 증식 분석을 수행하여 GR-H1993 세포에서 제피티닙과 KRCT-6j의 시너지 효과를 평가하였다. 시너지 효과는 조합 지수(CI)를 계산하는 Chou-Talalay 방법으로 평가되었다(Cancer Research, Volume 70, Issue 2, pp. 440-446). Compusyn에 의해 계산된 조합 지수는 상승 작용 (CI <1), 부가 효과 (CI = 1) 및 길항 작용 (CI> 1)을 계산하는 데 사용되었다. 두 가지 다른 농도의 제피티닙과 KRCT-6j를 사용하여 GR-H1993 세포에 대해 적절한 항 증식 효과를 관찰하였다. A real-time proliferation assay was performed to evaluate the synergistic effect of gefitinib and KRCT-6j in GR-H1993 cells. Synergy was assessed by the Chou-Talalay method for calculating the Combination Index (CI) (Cancer Research, Volume 70, Issue 2, pp. 440-446). Combination indices calculated by Compusyn were used to calculate synergism (CI < 1), additive effects (CI = 1) and antagonism (CI > 1). Appropriate antiproliferative effects on GR-H1993 cells were observed using two different concentrations of gefitinib and KRCT-6j.
3X103의 GR-H1993 cells을 96 웰 플레이트에 100 μL 부피로 분주한 후 다음 날 csEV, 제피티닙(gefitinib) 혹은 KRCT-6j가 포함된 배지로 배지를 바꾸었다. 그 후 세포의 phase confluence을 IncuCyte　 S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI)을 사용하여 4시간마다 모니터링하였다.3X10 3 GR-H1993 cells were aliquoted in a 96-well plate in a volume of 100 μL, and the next day, the medium was changed to a medium containing csEV, gefitinib or KRCT-6j. Then, the phase confluence of the cells was measured by IncuCyte.　Monitoring was performed every 4 hours using an S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI).
도 5a에 나타난 바와 같이, KRCT-6j와 제피티닙의 병용 처리에 대한 CI를 평가했을 때, 사용한 두 약제는 시너지 효과를 나타냈다(CI <1).As shown in Fig. 5a, when the CI for the combined treatment of KRCT-6j and gefitinib was evaluated, the two drugs used showed a synergistic effect (CI <1).
이에 더하여, 제피티닙과 KRCT-6j가 생체 내 종양 성장에 미치는 시너지 효과를 확인하기 위해 GR-H1993 세포를 이식하는 이종 이식(xenograft)을 채택하였다.In addition, in order to confirm the synergistic effect of gefitinib and KRCT-6j on tumor growth in vivo, a xenograft of GR-H1993 cells was adopted.
모든 실험과 방법은 관련 지침과 규정에 따라 수행되었다. 모든 동물 시술은 서울대 동물윤리위원회의 승인(SNU-190919-1)을 받았다. GR-H1993세포(5 × 106세포)는 50μL Cultrex pathclear 기저막 추출물(basement membrane extract)(PBS, R&D systems)에 용해하여 생후 5주 된 수컷 Athymic BALB/c-nu 마우스에 피하 주사 하였다. GR-H1993 종양 모델의 경우 종양이 가시화되면 치료 전 종양 부피를 기준으로 마우스를 그룹 지었다(>100mm3). 제피티닙(25mg/kg, 경구 투여(PO)) 또는 KRCT-6j(20mg/kg, 복강 내 주사(IP))를 매일 28일간 투여하였으며 3일마다 종양 성장을 측정했다. 종양 길이와 너비는 캘리퍼스로 측정하였으며 종양 부피는 공식[(길이 × 너비2) × π/6]을 사용하여 계산되었다. 대조군의 종양 부피가 약 1,000 mm3에 이르는 경우 쥐들을 인도적으로 안락사하였다.All experiments and methods were performed in accordance with relevant guidelines and regulations. All animal procedures were approved by the Animal Ethics Committee of Seoul National University (SNU-190919-1). GR-H1993 cells (5 × 10 6 cells) were dissolved in 50 μL Cultrex pathclear basement membrane extract (PBS, R&D systems) and subcutaneously injected into 5-week-old male Athymic BALB/c-nu mice. For the GR-H1993 tumor model, when tumors became visible, mice were grouped based on pre-treatment tumor volume (>100 mm 3 ). Gefitinib (25 mg/kg, oral administration (PO)) or KRCT-6j (20 mg/kg, intraperitoneal injection (IP)) was administered daily for 28 days and tumor growth was measured every 3 days. Tumor length and width were measured with calipers, and tumor volume was calculated using the formula [(length × width 2 ) × π/6]. When the tumor volume of the control group reached about 1,000 mm 3 , the mice were humanely euthanized.
도 5b에 나타난 바와 같이, 제피티닙 단독 투여는 종양 성장에 영향을 미치지 않았지만, KRCT-6j 및 제피티닙의 병용 투여는 GR-H1993 세포가 이식 된 이종이식 동물에서 종양 성장을 유의하게 억제했으며, 이는 시험관 내 데이터와 일치한다.As shown in Figure 5b, administration of gefitinib alone did not affect tumor growth, but combined administration of KRCT-6j and gefitinib significantly inhibited tumor growth in xenograft animals transplanted with GR-H1993 cells. , which is consistent with in vitro data.
또한, 분리된 종양 조직의 부피를 측정하고, 대표적인 증식 마커인 Ki67로 추가 염색하였다. 도 5c 및 도 5d에 나타난 바와 같이, KRCT-6j와 제피티닙의 병용 투여가 YAP 비활성화의 결과로 유도된 공격적인 암의 성장을 효과적으로 억제한다는 것을 확인하였다.In addition, the volume of the isolated tumor tissue was measured and further stained with Ki67, a representative proliferation marker. As shown in FIGS. 5c and 5d , it was confirmed that the combined administration of KRCT-6j and gefitinib effectively inhibited the growth of aggressive cancer induced as a result of YAP inactivation.
실시예 6. N-사이클로헥실피리미딘-4-아민(N-cyclohexylpyrimidin-4-amine) 유도체의 csEV 매개 암 세포 증식 억제 효과 확인Example 6. Confirmation of the inhibitory effect of N-cyclohexylpyrimidin-4-amine derivatives on csEV-mediated cancer cell proliferation
6-1. HCT116 대장암 세포와 전이성 LNCaP-SL 전립선암 세포에서 증식 억제 반응 평가6-1. Evaluation of antiproliferative responses in HCT116 colorectal cancer cells and metastatic LNCaP-SL prostate cancer cells
N-사이클로헥실피리미딘-4-아민(N-cyclohexylpyrimidin-4-amine) 유도체 14종의 화합물(표 1)을 대상으로 csEV에 반응을 보인 HCT116 대장암 세포와 전이성 LNCaP-SL 전립선암 세포에서 증식 억제 반응을 평가하였다.N-cyclohexylpyrimidin-4-amine derivatives 14 compounds (Table 1) were tested in HCT116 colon cancer cells and metastatic LNCaP-SL prostate cancer cells that responded to csEV. The proliferation inhibitory response was evaluated.
3X103 cells을 96 웰 플레이트에 100μL 부피로 분주한 후 다음 날 csEV 및 화합물이 포함된 배지로 배지를 바꾸었다. 그 후 세포의 phase confluence을 IncuCyte　 ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI)을 사용하여 4시간마다 모니터링하였다.After dispensing 3X10 3 cells in a volume of 100 μL in a 96-well plate, the medium was changed to a medium containing csEV and compound the next day. Then, cell phase confluence was monitored every 4 hours using IncuCyte ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI).
도 6에 나타난 바와 같이, 두 세포 공히 억제반응을 보인 화합물은 KRCT-5, 31, 62, 77, 87 및 203 화합물로 나타났다.As shown in FIG. 6 , the compounds exhibiting both cell inhibitory responses were KRCT-5, 31, 62, 77, 87 and 203 compounds.
Figure PCTKR2021012192-appb-img-000009
Figure PCTKR2021012192-appb-img-000009
Figure PCTKR2021012192-appb-img-000010
Figure PCTKR2021012192-appb-img-000010
Figure PCTKR2021012192-appb-img-000011
Figure PCTKR2021012192-appb-img-000011
6-2. 항암제 내성 세포주에서 증식 억제 반응 평가6-2. Evaluation of proliferation inhibition response in anticancer drug-resistant cell lines
실시예 6-1의 결과를 바탕으로, 효과가 가장 우수하였던 87번, 203번 화합물을 대상으로 항암제 내성 세포주를 포함하는 다양한 암세포의 증식억제효과를 평가하였다. Based on the results of Example 6-1, the proliferation inhibitory effects of various cancer cells, including anticancer drug-resistant cell lines, were evaluated for compounds No. 87 and No. 203, which had the best effects.
1 내지 3X103 cells을 96 웰 플레이트에 100 μL 부피로 분주한 후 다음 날 2X 약물 농도의 100μL 배지를 추가하였다. 그 후 세포의 phase confluence을 IncuCyte　 ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI)을 사용하여 4시간마다 모니터링하였다.After dispensing 1 to 3X10 3 cells in a volume of 100 μL in a 96-well plate, 100 μL medium of 2X drug concentration was added the next day. Then, cell phase confluence was monitored every 4 hours using IncuCyte ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI).
도 7a 및 도 7b에 나타난 바와 같이, 비소세포성 폐암(NSCLC) 세포주인 HCC827세포에서 87번과 203번 화합물의 증식억제 IC50 값은 각각 0.95 및 0.97 μM로 나타났으며, 제피티닙 내성 HCC827(GR-HCC827) 세포의 경우, 2.1 및 1.5 μM로 나타났다. 7A and 7B , in HCC827 cells, a non-small cell lung cancer (NSCLC) cell line, the antiproliferative IC 50 values of compounds 87 and 203 were 0.95 and 0.97 μM, respectively, and gefitinib-resistant HCC827 (GR-HCC827) cells, 2.1 and 1.5 μM.
T790M 돌연변이된 제피티닙 내성 H1975 폐암 세포의 경우, IC50이 1.4 및 1.2 μM으로 나타났다. For T790M mutated gefitinib-resistant H1975 lung cancer cells, the IC 50 was 1.4 and 1.2 μM.
또한, 기본 H1993 세포의 경우, 2.3 및 2.0 μM, 엘로티닙(erlotinib) 내성 H1993(ER-H1993) 세포는 1.8 및 1.9 μM, 제피티닙 내성 H1993(GR-H1993)세포는 2.8 및 0.9 μM로 나타나 EGFR-TKI 내성 폐암이나 반응성 폐암 모두에 대해 유사한 증식억제 효과가 관찰되었다.In addition, for primary H1993 cells, 2.3 and 2.0 μM, erlotinib-resistant H1993 (ER-H1993) cells showed 1.8 and 1.9 μM, and gefitinib-resistant H1993 (GR-H1993) cells showed 2.8 and 0.9 μM. A similar antiproliferative effect was observed for both EGFR-TKI-resistant lung cancer and reactive lung cancer.
6-3. 항암제 내성 대장암 세포주에서 증식 억제 반응 평가6-3. Evaluation of antiproliferative response in anticancer drug-resistant colorectal cancer cell lines
인간 대장암세포주인 HT29세포와 HCT116 세포의 경우에도 유사한 양상을 확인하였다. A similar pattern was confirmed in the case of human colorectal cancer cell lines, HT29 cells and HCT116 cells.
도 8에 나타난 바와 같이, HT29세포의 경우, 87번과 203번 화합물의 증식억제 IC50 값은 각각 0.90 및 0.52 μM으로 나타났으며, 옥살리플라틴(oxalliplatin) 내성 HT297(OR-HT29) 세포의 경우, 0.64 및 0.44 μM로 나타났다. HCT116 세포의 경우, 1.13 및 0.83 μM을 보였다. 또한, 옥살리플라틴 내성 HCT116(OR-HCT116) 세포는 1.15 및 0.99 μM으로 나타나 백금착화합물 내성 대장암이나 반응성 폐암 모두 유사한 증식억제효과가 관찰되었다. As shown in Figure 8, in the case of HT29 cells, the proliferation inhibitory IC 50 values of compounds 87 and 203 were 0.90 and 0.52 μM, respectively, and in the case of oxaliplatin-resistant HT297 (OR-HT29) cells, 0.64 and 0.44 μM. HCT116 cells showed 1.13 and 0.83 μM. In addition, oxaliplatin-resistant HCT116 (OR-HCT116) cells showed 1.15 and 0.99 μM, and similar antiproliferative effects were observed for both platinum complex-resistant colorectal cancer and reactive lung cancer.
6-4. 전립선암 세포주에서 증식 억제 반응 평가6-4. Evaluation of antiproliferative responses in prostate cancer cell lines
인간 전립선암세포주인 LNCaP(안드로겐 수용체 양성, 비전이성) 세포와 전이성 LNCaP-SL 세포의 경우에도 유사한 양상을 확인하였다. A similar pattern was confirmed in the case of LNCaP (androgen receptor-positive, non-metastatic) cells and metastatic LNCaP-SL cells, which are human prostate cancer cell lines.
도 9a에 나타난 바와 같이, LNCaP세포의 경우, 87번과 203번 화합물의 증식억제 IC50 값은 각각 2.3 및 1.7 μM으로 나타났으며, 암세포 이동능이 높은 전이성 sub-line인 LNCaP-SL 세포의 경우, 0.9 및 2.2 μM로 나타났다. As shown in FIG. 9a , in the case of LNCaP cells, the antiproliferative IC 50 values of compounds 87 and 203 were 2.3 and 1.7 μM, respectively, and in the case of LNCaP-SL cells, a metastatic sub-line with high cancer cell mobility. , 0.9 and 2.2 μM.
6-5. 간세포암 세포주에서 증식 억제 반응 평가6-5. Evaluation of antiproliferative responses in hepatocellular carcinoma cell lines
또한, 인간 간암 세포주인 Huh7(전이성)과 HepG2(비전이성) 세포의 경우에서도 평가를 진행하였다. In addition, in the case of human liver cancer cell lines Huh7 (metastatic) and HepG2 (non-metastatic) cells were evaluated.
도 9b에 나타난 바와 같이, Huh7 세포의 경우, 2.32 및 1.51 μM, HepG2 세포의 경우, 1.43 및 1.06 μM으로 나타나 대부분의 인간 고형암 세포주의 내성 및 전이성 유무와 상관없이 이들 화합물들의 암 증식 억제활성을 확인하였다.As shown in FIG. 9B , in the case of Huh7 cells, 2.32 and 1.51 μM, and in the case of HepG2 cells, 1.43 and 1.06 μM, the cancer proliferation inhibitory activity of these compounds was confirmed regardless of the resistance and metastasis of most human solid cancer cell lines. did
6-6. 동종이식 마우스 모델에서 암세포 증식 억제 반응 평가6-6. Evaluation of cancer cell proliferation inhibition in an allograft mouse model
항암면역활성 평가를 위한 동종이식 마우스 모델 활용을 위한 기초자료로 Tyro3 발현이 관찰된 마우스 기원 암세포주에도 증식 억제활성을 평가하였다.As basic data for the use of an allograft mouse model for the evaluation of anticancer immune activity, the proliferation inhibitory activity was also evaluated in a mouse-derived cancer cell line in which Tyro3 expression was observed.
1X103 개 세포를 96 웰 플레이트에 100μL 부피로 분주한 후 다음 날 2X 약물 농도의 100 μL 배지를 추가하였다. 그 후 세포의 phase confluence을 IncuCyte　 ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI)을 사용하여 4시간마다 모니터링하였다.After dispensing 1X10 3 cells in a volume of 100 μL in a 96-well plate, 100 μL medium of 2X drug concentration was added the next day. Then, cell phase confluence was monitored every 4 hours using IncuCyte ZOOM or S3 Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI).
도 10에 나타난 바와 같이, 4T1(마우스 유선암세포), MC38(마우스 대장암세포) 세포 이식 모델에서 두 화합물 모두 3 μM 농도에서 현저한 암세포 증식 억제 효과를 나타냈다.As shown in FIG. 10 , in the 4T1 (mouse mammary cancer cell) and MC38 (mouse colorectal cancer cell) cell transplantation models, both compounds showed a significant inhibitory effect on cancer cell proliferation at a concentration of 3 μM.
6-7. 암세포 이동능 억제 반응 평가6-7. Evaluation of cancer cell migration inhibition response
암세포의 경우, 증식 뿐 아니라 이동능이 항암치료에서 문제가 되는 암 전이를 결정하는 중요한 요소가 된다. Transwell migration assay로 암 이동능이 현저하게 관찰되는 HCT116, OR-HCT116(이상 대장암세포), MDA-MB-231, 4T1, 타목시펜(tamoxifen)-내성 MCF-7(TAMR-MCF-7)[이상 인간 및 마우스 유방(선)암 세포], LNCaP-SL(전립선암 세포), 엘로티닙 내성 H292세포(ER-H292), GR-H1993, GR-HCC-827, H1975[이상 EGFR-TKI 내성 폐암세포]에서 KRCT-0087 혹은 KRCT-0203의 이동능 억제 활성을 평가하였다. In the case of cancer cells, not only proliferation but also mobility is an important factor in determining cancer metastasis, which is a problem in chemotherapy. HCT116, OR-HCT116 (abnormal colorectal cancer cells), MDA-MB-231, 4T1, tamoxifen-resistant MCF-7 (TAMR-MCF-7) [abnormal human and In mouse breast (adenocarcinoma) cells], LNCaP-SL (prostate cancer cells), erlotinib-resistant H292 cells (ER-H292), GR-H1993, GR-HCC-827, H1975 [abnormal EGFR-TKI-resistant lung cancer cells] The mobility inhibitory activity of KRCT-0087 or KRCT-0203 was evaluated.
IncuCyte ClearView 96-well chemotaxis plates(Essen bioscience, MI, USA)를 이용하여 트랜스웰 이동 실험을 수행하였다. 트랜스웰 유닛의 아랫부분에 Type Ⅰ 콜라겐(Collaborative Research, KY, USA)을 코팅한 후 1시간 동안 상온에서 말리는 과정을 수행하였다. 이후 FBS가 없는 조건의 RPMI 혹은 DMEM 배지에 3 내지 5×103 cells/well로 준비한 후, 약물을 지정된 농도 (1 μM)로 함께 처리한 실험군 또는 대조군을 60 μL씩 트랜스웰 유닛 상단부에 시딩하였다. KRCT-0087 혹은 KRCT-0203은 트랜스웰 유닛 상단부에 처치하였다. 유인 화학 물질이 들어가는 하단부 웰에는 10% FBS 배지 총 200μL가 사용되었다. 세포의 이동은 Incucyte Chemotaxis live-cell analysis (ESSEN bioscience)을 사용하여 4시간 간격으로 모니터링하였다.Transwell migration experiments were performed using IncuCyte ClearView 96-well chemotaxis plates (Essen bioscience, MI, USA). The lower part of the transwell unit was coated with Type I collagen (Collaborative Research, KY, USA) and dried at room temperature for 1 hour. After preparing 3 to 5 × 10 3 cells/well in RPMI or DMEM medium without FBS, the experimental group or control group treated with the drug at the specified concentration (1 μM) was seeded at the upper end of the transwell unit by 60 μL. . KRCT-0087 or KRCT-0203 was treated at the top of the transwell unit. A total of 200 μL of 10% FBS medium was used for the bottom wells containing the attractant chemicals. Cell migration was monitored at 4-hour intervals using Incucyte Chemotaxis live-cell analysis (ESSEN bioscience).
도 11에 나타난 바와 같이, 사용한 암세포주에 따라서 약간의 활성 차이가 관찰되었으나 87번과 203번 화합물 모두 암세포의 이동능을 억제하는 활성을 관찰하였다.As shown in FIG. 11 , a slight difference in activity was observed depending on the cancer cell line used, but both compounds No. 87 and No. 203 were observed to inhibit the migration ability of cancer cells.
6-8. 스페로이드(Spheroid) 형성능에 미치는 영향 평가6-8. Evaluation of the effect on the ability to form spheroids
Ultra-low attachment(ULA) plate 상에서 암세포를 배양하면 암줄기세포의 특성을 갖는 세포만 3차원 구 형성을 보이는 스페로이드를 형성한다. 스페로이드 형성능에 미치는 두 화합물의 활성을 평가하였다. When cancer cells are cultured on an ultra-low attachment (ULA) plate, only cells with the characteristics of cancer stem cells form spheroids showing three-dimensional sphere formation. The activity of the two compounds on the ability to form spheroids was evaluated.
스페로이드 생성을 평가하기 위해서 Ultralow attachment(ULA) plate (corning, 7007)에 세포를 분주하고 200 rmp로 5분간 원심분리하였다. 다음 날 본 발명의 화합물을 지정된 농도 (3 μM)로 처리하였으며, 7-10일 동안 배양 후 형성된 스페로이드의 사진을 실시간 세포 영상 분석기 Incucyte S3를 이용하여 사진을 찍었다. 사진을 바탕으로 지름을 측정하고 [VOLUME=0.5X단경2X장경] 공식을 이용하여 스페로이드의 부피를 계산하였다.In order to evaluate spheroid production, cells were seeded on an Ultralow attachment (ULA) plate (corning, 7007) and centrifuged at 200 rpm for 5 minutes. The next day, the compound of the present invention was treated at a specified concentration (3 μM), and a picture of the spheroid formed after culture for 7-10 days was taken using a real-time cell image analyzer Incucyte S3. The diameter was measured based on the photo, and the volume of the spheroid was calculated using the [VOLUME=0.5X minor diameter 2X major diameter] formula.
도 12 내지 14에 나타난 바와 같이, GR-H1993, 4T1, HCT116, OR-HCT116, HT-29 및 OR-HT-29세포는 ULA plate에 배양 시 3차원 구형성이 관찰되었고, 87번 화합물의 경우, 강력한 스페로이드 형성 억제능을 보였다. 203번 화합물의 경우, 스페로이드 형성 억제 활성이 GR-H1993, OR-HCT116 세포에서는 나타나지 않았으며, 다른 암세포의 경우 그 억제 활성이 87번 화합물 보다는 적은 것으로 나타났다. As shown in FIGS. 12 to 14, GR-H1993, 4T1, HCT116, OR-HCT116, HT-29 and OR-HT-29 cells were cultured on a ULA plate, and three-dimensional sphericity was observed, and in the case of compound 87, , showed a strong spheroid formation inhibitory ability. In the case of compound 203, the spheroid formation inhibitory activity did not appear in GR-H1993, OR-HCT116 cells, and in the case of other cancer cells, the inhibitory activity was less than that of compound 87.
6-9. 면역세포에 미치는 영향 평가6-9. Assessment of the effect on immune cells
다음으로 87번 및 203번 화합물의 항암면역 활성에 관여할 가능성을 평가하기 위하여 면역세포에 미치는 영향을 평가하였다. 마우스에서 분리한 골수세포에 macrophage-colony stimulating factor(M-CSF)를 처치하여 대식세포로 분화시켰다. 암세포의 생존에 유리한 환경을 매개하는 M2형 분화를 위해 IL-4 노출시켰고, 이 경우 M2 형 마커인 TGF-β, Arg1(Arginase 1), 및 CIITA(Class II Major Histocompatibility Complex Transactivator)의 mRNA 발현이 증가하였다. Next, the effect on immune cells was evaluated in order to evaluate the possibility that the compounds No. 87 and No. 203 may be involved in the anticancer immune activity. Bone marrow cells isolated from mice were treated with macrophage-colony stimulating factor (M-CSF) to differentiate them into macrophages. IL-4 was exposed for M2-type differentiation mediating an environment favorable for the survival of cancer cells, and in this case, mRNA expression of M2-type markers TGF-β, Arg1 (Arginase 1), and CIITA (Class II Major Histocompatibility Complex Transactivator) was reduced. increased.
8주령 C57BL/6J 마우스에서 양쪽 대퇴골과 정강뼈를 분리하여 1X PBS로 세 번 세척한 후 다리뼈 양쪽을 가위로 잘라내었다. 그 후 1mL 주사기로 RMPI1640 배지(2.5% HEPES, 10% FBS, 1% Penicillin/streptomycin)를 흘려주었다. 70μM cell strainer를 사용하여 걸러낸 후 이를 원심분리(400g, 5min) 하였다. 그리고 ACK lysis buffer(Life Technologies, Grand island, NY)로 적혈구를 제거하였다. 다시 원심분리를 하여(400g, 5min) 새 배지에 세포를 푼 뒤, 70μM cell strainer를 사용하여 걸러내었다. M-CSF 30ng/mL를 처리하고, 3일 후 새로운 배지로 갈아 준 후 총 7일간 분화시킨 후 일차배양 골수 유래 면역세포를 획득하였으며, 이를 실험에 사용하였다.Both femurs and shinbones were separated from 8-week-old C57BL/6J mice, washed three times with 1X PBS, and then both leg bones were cut with scissors. Then, RMPI1640 medium (2.5% HEPES, 10% FBS, 1% Penicillin/streptomycin) was flowed through a 1mL syringe. After filtering using a 70 μM cell strainer, it was centrifuged (400 g, 5 min). And red blood cells were removed with ACK lysis buffer (Life Technologies, Grand island, NY). After centrifugation again (400 g, 5 min) to release the cells in a new medium, the cells were filtered using a 70 μM cell strainer. After treatment with M-CSF 30ng/mL, and after 3 days of change to a new medium, after differentiation for a total of 7 days, primary cultured bone marrow-derived immune cells were obtained, which were used in the experiment.
도 15a에 나타난 바와 같이, 87번과 203번 화합물은 1 μM 농도에서 IL-4 처치에 의한 대식세포의 TGF-β 발현 증가를 모두 현저하게 억제하였다.As shown in FIG. 15a , compounds 87 and 203 significantly inhibited both the increase in TGF-β expression in macrophages by IL-4 treatment at a concentration of 1 μM.
또한, 도 15b에 나타난 바와 같이, 87번 화합물은 0.5 μM 농도에서 IL-4 처치에 의한 대식세포의 Arg1, 및 CIITA 발현 증가를 현저하게 억제하였다In addition, as shown in FIG. 15b , compound 87 significantly inhibited the increase in Arg1 and CIITA expression in macrophages by IL-4 treatment at a concentration of 0.5 μM.
6-10. 유선암 동종이식 모델에서의 효과 평가6-10. Efficacy evaluation in mammary gland cancer allograft model
상기 암세포 증식, 이동능 억제, 3차원 구 형성 억제 및 항암면역능에 미치는 두 화합물들의 효능이 실제 항암활성으로 나타나는지를 4T1 마우스 유선암 세포를 이식한 동종이식 암 발생 모델에서 평가하였다(도 16a 참조). Whether the efficacy of the two compounds on the cancer cell proliferation, inhibition of migration, inhibition of three-dimensional sphere formation and anticancer immunity was actually shown as anticancer activity was evaluated in an allograft cancer development model transplanted with 4T1 mouse mammary gland cancer cells (see FIG. 16a ).
동물 실험 및 방법은 서울대학교 동물실험윤리위원회의 승인을 받아 진행 되었다 (SNU-200218-3-1). 4T1 cells(1×106 cells)을 100μL PBS에 희석하여 5주령 암컷 Balb/c 마우스의 피하로 주입하였다. 종양의 평균적 크기가 100 mm3 이상이 되었을 때 투여를 시작하였으며, 복강 내 투여로 KRCT-0087 5mg/kg, KRCT-0203 10mg/kg을 투여하였다. 3일마다 종양 성장을 측정하였다. 종양 길이와 너비는 캘리퍼스로 측정하였으며 종양 부피는 공식[(길이×너비2)×π/6]을 사용하여 계산되었다. 대조군의 종양 부피가 약 1,000 mm3에 이를 시 쥐들을 인도적으로 안락사하였다.Animal experiments and methods were carried out with the approval of the Animal Experimentation Ethics Committee of Seoul National University (SNU-200218-3-1). 4T1 cells (1×10 6 cells) were diluted in 100 μL PBS and injected subcutaneously into 5-week-old female Balb/c mice. Administration was started when the average tumor size reached 100 mm 3 or more, and KRCT-0087 5 mg/kg and KRCT-0203 10 mg/kg were administered intraperitoneally. Tumor growth was measured every 3 days. Tumor length and width were measured with calipers, and tumor volume was calculated using the formula [(length × width 2 ) × π/6]. When the tumor volume of the control group reached about 1,000 mm 3 , the mice were humanely euthanized.
도 16b에 나타난 바와 같이, 87번 화합물 5mg/kg, 203번 화합물 10mg/kg을 16일간 매일 복강주사한 결과, 4T1 암 성장이 유의적으로 억제됨을 확인하였다. As shown in FIG. 16b , as a result of intraperitoneal injection of compound 87 at 5 mg/kg and compound 203 at 10 mg/kg daily for 16 days, it was confirmed that 4T1 cancer growth was significantly inhibited.
더불어, 도 16c에 나타난 바와 같이, 체중감소와 같은 물질의 독성 또한 관찰되지 않았다. In addition, as shown in Fig. 16c, toxicity of the substance such as weight loss was also not observed.
6-11. 대장암 동종이식 모델에서의 효과 평가6-11. Effect evaluation in colorectal cancer allograft model
동물 실험 및 방법은 서울대학교 동물실험윤리위원회의 승인을 받아 진행 되었다 (SNU-200218-3-1). 대장암 유래 세포 MC38 세포(2×105 cells)을 100μL PBS에 희석하여 5주령 수컷 Balb/c 마우스의 피하로 주입하였다. 종양의 평균적 크기가 100 mm3 이상이 되었을 때 투여를 시작하였으며, 복강 내 투여로 KRCT-0087 5mg/kg를 9일간 매일 투여하였다 (도 17a 참조). 투여 후, 3일마다 종양 성장을 측정하였다. 종양 길이와 너비는 캘리퍼스로 측정하였으며 종양 부피는 공식[(길이×너비2)×π/6]을 사용하여 계산되었다. 대조군의 종양 부피가 약 1,000 mm3에 이를 시 쥐들을 인도적으로 안락사하였다.Animal experiments and methods were carried out with the approval of the Animal Experimentation Ethics Committee of Seoul National University (SNU-200218-3-1). Colorectal cancer-derived cells MC38 cells (2×10 5 cells) were diluted in 100 μL PBS and injected subcutaneously into 5-week-old male Balb/c mice. Administration was started when the average tumor size reached 100 mm 3 or more, and 5 mg/kg of KRCT-0087 was administered intraperitoneally for 9 days (see FIG. 17a ). After administration, tumor growth was measured every 3 days. Tumor length and width were measured with calipers, and tumor volume was calculated using the formula [(length × width 2 ) × π/6]. When the tumor volume of the control group reached about 1,000 mm 3 , the mice were humanely euthanized.
도 17b에 나타난 바와 같이, 87번 화합물 5mg/kg을 9일간 매일 복강주사한 결과, 대장암의 부피가 유의하게 감소함을 확인하였다.As shown in FIG. 17b , as a result of intraperitoneal injection of compound 87 at 5 mg/kg daily for 9 days, it was confirmed that the volume of colorectal cancer was significantly reduced.
이에 더하여, 87번 화합물이 대식세포 및 T 세포에 작용하여 면역상승효과를 야기함으로써 항암 효과를 나타내는지 확인하였다. CD는 활성화된 T 세포의 분열을 촉진시키는 항원제시 세포로서의 역할시 필수적으로 요구되는 세포막 단백질로 세포의 활성화와 관련하여 중요한 지표로 이용된다. 일반적으로 T 세포의 분화집단은 CD3로 표현되며, 주로 사이토카인을 생산하는 역할을 하는 CD4와 세포독성 T 세포인 CD8으로 분류된다.In addition, it was confirmed whether compound 87 exerts an anticancer effect by acting on macrophages and T cells to induce an immune synergistic effect. CD is an essential cell membrane protein required when acting as an antigen-presenting cell that promotes the division of activated T cells, and is used as an important indicator in relation to cell activation. In general, the T cell differentiation group is expressed as CD3, and is mainly classified into CD4, which plays a role in cytokine production, and CD8, which is a cytotoxic T cell.
먼저, 채취한 종양 조직으로부터 Tumor dissassociation kit ((Miltenyi Biotec, Auburn, CA) 및 Percoll density gradient centrifugation 기법을 사용하여 종양 내 림프구를 분리하였다. FACS(fluorescence analysis cells sorting)를 이용하여 대식세포 및 T 세포의 양상을 확인하였다. α-CD45-APC/Cy7, α-BV785-I-A/I-E, α-CD11b-PerCP/Cy5.5, α-Ly6C-APC, α-Ly6G-FITC, α-F4/80-PE/Cy7, α-CD4-FITC, α-CD8-APC는 biolegend (San Diego, CA, USA)에서 구입하였다. α-FOXP3-EF450은 eBioscience (San Diego, CA, USA)에서 구입하First, lymphocytes in the tumor were isolated from the collected tumor tissue using the Tumor dissassociation kit ((Miltenyi Biotec, Auburn, CA) and Percoll density gradient centrifugation technique. Macrophages and T cells were used using FACS (fluorescence analysis cells sorting). α-CD45-APC/Cy7, α-BV785-IA/IE, α-CD11b-PerCP/Cy5.5, α-Ly6C-APC, α-Ly6G-FITC, α-F4/80- PE/Cy7, α-CD4-FITC, and α-CD8-APC were purchased from biolegend (San Diego, CA, USA) α-FOXP3-EF450 was purchased from eBioscience (San Diego, CA, USA).
도 17c에 나타난 바와 같이, 87번 화합물의 투여에 따라, 림프구 공통항원인 CD45 양성 세포가 유의미하게 증가한 것을 확인하였다. As shown in FIG. 17c , it was confirmed that, according to the administration of compound 87, the number of CD45-positive lymphocyte common antigen cells was significantly increased.
또한, 도 17d에 나타난 바와 같이, 종양 관련 대식세포(TAM) 군집을 CD45+Ly6G-Ly6C-F4/80+CD11b+로 정의하였다.In addition, as shown in FIG. 17D , the tumor-associated macrophage (TAM) population was defined as CD45 + Ly6G - Ly6C - F4/80 + CD11b + .
또한, 도 17e에 나타난 바와 같이, TAM이 발현하는 I-A/I-E(MHC 제II 부류) 비율을 FACS로 확인하였을 때, 87번 화합물의 투여에 의해 I-A/I-E의 발현이 유의미하게 증가하였다.In addition, as shown in FIG. 17E , when the ratio of I-A/I-E (MHC class II) expressed by TAM was confirmed by FACS, the expression of I-A/I-E was significantly increased by administration of compound 87.
또한, 도 17f에 나타난 바와 같이, 87번 화합물의 투여에 의해 세포독성 CD8+ T 세포의 숫자 또한 증가하였다.In addition, as shown in FIG. 17f , the number of cytotoxic CD8+ T cells was also increased by administration of compound 87.
또한, 도 17g에 나타난 바와 같이, 87번 화합물의 투여에 의해 조절 T 세포의 마커인 FOXP3+CD4+ T 세포의 비율은 오히려 감소하였음을 확인하였다. In addition, as shown in FIG. 17G , it was confirmed that the ratio of FOXP3 + CD4 + T cells, a marker of regulatory T cells, was rather decreased by the administration of compound 87.
상기 결과는 본 발명의 87번 화합물이 대식세포 및 T 세포에 작용하여 면역상승효과를 야기하며, 그에 따라 항암 효과, 특히 대장암 치료 효과를 나타낼 수 있음을 나타낸다.The above results indicate that compound 87 of the present invention acts on macrophages and T cells to cause an immune synergistic effect, thereby exhibiting an anticancer effect, in particular a therapeutic effect for colorectal cancer.
이에 더하여, 선천적 면역 반응에 해당하는 대식세포의 M1/M2 표현형의 증가 및 후천적 면역에 해당하는 tumor infilterating lymphocyte가 증가함을 확인함에 따라, T 세포의 표현형 변화를 확인하였다. 동물 실험 및 방법은 서울대학교 동물실험윤리위원회의 승인을 받아 진행 되었으며(SNU-200827-2-3) 실험 방법은 위와 동일하다. α-IFNr-PE/Cy7, α-TNFα-PerCP/Cy5.5는 biolegend (San Diego, CA, USA)에서 구입하였다.In addition, as it was confirmed that the M1/M2 phenotype of macrophages corresponding to the innate immune response and tumor infiltering lymphocytes corresponding to the acquired immunity increased, the phenotypic change of T cells was confirmed. Animal experiments and methods were carried out with the approval of the Animal Experimentation Ethics Committee of Seoul National University (SNU-200827-2-3), and the experimental methods are the same as above. α-IFNr-PE/Cy7 and α-TNFα-PerCP/Cy5.5 were purchased from biolegend (San Diego, CA, USA).
인터페론-γ(IFN-γ) 및 종양 괴사 인자-α(TNF-α)는 T 세포에서 세포 사멸을 유도하고, 강력한 항암 효과 나타내기 위해 필수적인 사이토카인으로 알려져 있다. 또한 IFN-γ 및 TNF-α를 분비하는 CD4+ T 세포는 Th1 CD4+ T 세포로 정의된다.Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are known as essential cytokines for inducing apoptosis in T cells and exhibiting strong anticancer effects. Also, CD4+ T cells that secrete IFN-γ and TNF-α are defined as Th1 CD4+ T cells.
도 17i에 나타난 바와 같이, CD4+ 및 CD8+ T 세포에서 분비하는 사이토카인인 IFN-γ 및 TNF-α가 87번 화합물의 투여에 의하여 유의미하게 증가하였다. As shown in FIG. 17i , the cytokines IFN-γ and TNF-α secreted from CD4+ and CD8+ T cells were significantly increased by administration of compound 87.
이상의 실시예를 종합하여, 본 발명자들은 본 발명의 화합물이 csEV 매개 암 증식을 억제할 뿐만 아니라, 전이 억제 효과 또한 매우 우수함을 확인하였다. 더불어 본 발명의 화합물이 면역세포의 암세포 공격을 저해하는 대식세포의 분화형인 M1/M2의 분율을 증가시켜 선천적 면역의 활성을 유도하며, 후천적 면역인 Th1 CD4+ T 세포의 증가 및 세포독성 CD8+ T 세포가 활성화됨을 확인하였다. Summarizing the above examples, the present inventors confirmed that the compound of the present invention not only inhibited csEV-mediated cancer growth, but also had a very excellent metastasis inhibitory effect. In addition, the compound of the present invention induces the activity of innate immunity by increasing the fraction of M1/M2, a differentiated type of macrophages that inhibit immune cell attack on cancer cells, and increases Th1 CD4+ T cells and cytotoxic CD8+ T cells that are acquired immunity was confirmed to be activated.
따라서, 본 발명의 화합물을 다양한 암 예방 또는 치료제, 암 전이 억제제 및 면역항암제로 이용할 수 있을 것으로 기대된다.Therefore, it is expected that the compound of the present invention can be used as a variety of cancer preventive or therapeutic agents, cancer metastasis inhibitors, and immuno-cancer agents.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
본 발명의 화합물들은 혈액 순환 미세체외소체(csEV)가 매개하는 폐암, 결장암, 전립선암, 유방암, 간암과 같은 암세포 증식 억제 활성이 뛰어나고, 항암제 내성 암세포주에 대한 증식 억제 활성도 뛰어나며, 침습성 암에 대한 증식 억제 효과를 나타낼 뿐만 아니라, 암세포의 이종이식 및 동종이식 동물모델에서 암 성장을 유의하게 억제하므로, 암의 예방 또는 치료 뿐만 아니라 암 전이 억제용 약제로 유용하게 사용할 수 있어, 산업상 이용가능성이 있다.The compounds of the present invention have excellent anti-proliferative activity of cancer cells such as lung cancer, colon cancer, prostate cancer, breast cancer, and liver cancer mediated by blood circulating microextrabody (csEV), and also have excellent anti-proliferative activity against anticancer drug-resistant cancer cell lines, and for invasive cancer Not only does it exhibit a proliferation inhibitory effect, but it also significantly inhibits cancer growth in xenograft and allograft animal models of cancer cells. there is.

Claims (16)

  1. 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물로서,As a pharmaceutical composition for the prevention or treatment of cancer comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient,
    상기 암은 혈액 순환 미세체외소체(csEV) 매개 암인 것을 특징으로 하는, 약학적 조성물.The cancer is characterized in that the blood circulating microextrabody (csEV) mediated cancer, the pharmaceutical composition.
    [화학식 1][Formula 1]
    Figure PCTKR2021012192-appb-img-000012
    Figure PCTKR2021012192-appb-img-000012
    (상기 화학식 1에 있어서,(In Formula 1,
    상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
    여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
    다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
    상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
    또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
    여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
    다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
    또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
    상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
  2. 제1항에 있어서,According to claim 1,
    Figure PCTKR2021012192-appb-img-000013
    Figure PCTKR2021012192-appb-img-000013
    상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, Cl, F 또는 메틸인 것을 특징으로 하는, 약학적 조성물.The R 3 and R 4 are each independently one or more hydrogen, Cl, F or methyl, the pharmaceutical composition.
  3. 제1항에 있어서,The method of claim 1,
    상기 화학식 1로 표시되는 화합물은 하기 표 1에 기재된 화합물로 이루어진 군으로부터 선택된 하나 이상인 것을 특징으로 하는, 약학적 조성물.The compound represented by Formula 1 is a pharmaceutical composition, characterized in that at least one selected from the group consisting of the compounds shown in Table 1.
    [표 1][Table 1]
    Figure PCTKR2021012192-appb-img-000014
    Figure PCTKR2021012192-appb-img-000014
    Figure PCTKR2021012192-appb-img-000015
    Figure PCTKR2021012192-appb-img-000015
    Figure PCTKR2021012192-appb-img-000016
    Figure PCTKR2021012192-appb-img-000016
  4. 제1항에 있어서,According to claim 1,
    상기 조성물은 제피티닙 또는 이의 약학적으로 허용 가능한 염을 더 포함하는 것을 특징으로 하는, 약학적 조성물.The composition is characterized in that it further comprises gefitinib or a pharmaceutically acceptable salt thereof, a pharmaceutical composition.
  5. 제1항에 있어서,The method of claim 1,
    상기 혈액 순환 미세체외소체(csEV) 매개 암은 전이성 암인 것을 특징으로 하는, 약학적 조성물.The blood circulation microextrabody (csEV) mediated cancer is characterized in that the metastatic cancer, the pharmaceutical composition.
  6. 제1항에 있어서,According to claim 1,
    상기 화학식 1로 표시되는 화합물이 암 세포의 성장 억제, 침윤(invasion) 억제 및 전이(metastasis) 억제로 이루어진 군으로부터 선택된 하나 이상의 효과를 갖는 것을 특징으로 하는, 약학적 조성물.A pharmaceutical composition, characterized in that the compound represented by Formula 1 has at least one effect selected from the group consisting of growth inhibition, invasion inhibition, and metastasis inhibition of cancer cells.
  7. 제1항에 있어서,According to claim 1,
    상기 약학적 조성물은 면역항암제인 것을 특징으로 하는, 약학적 조성물.The pharmaceutical composition is characterized in that the immuno-cancer agent, the pharmaceutical composition.
  8. 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 제피티닙의 항암 효능 증진용 약학적 조성물.A pharmaceutical composition for enhancing the anticancer efficacy of gefitinib comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
    [화학식 1][Formula 1]
    Figure PCTKR2021012192-appb-img-000017
    Figure PCTKR2021012192-appb-img-000017
    (상기 화학식 1에 있어서,(In Formula 1,
    상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
    여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
    다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
    상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
    또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
    여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
    다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
    또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
    상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
  9. 제8항에 있어서,9. The method of claim 8,
    상기 조성물은 제피티닙 또는 이의 약학적으로 허용 가능한 염을 더 포함하는 것을 특징으로 하는, 약학적 조성물.The composition is characterized in that it further comprises gefitinib or a pharmaceutically acceptable salt thereof, a pharmaceutical composition.
  10. 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 암 전이 억제용 약학적 조성물.A pharmaceutical composition for inhibiting cancer metastasis comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
    [화학식 1][Formula 1]
    Figure PCTKR2021012192-appb-img-000018
    Figure PCTKR2021012192-appb-img-000018
    (상기 화학식 1에 있어서,(In Formula 1,
    상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
    여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
    다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
    상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
    또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
    여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
    다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
    또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
    상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
  11. 제10항에 있어서,11. The method of claim 10,
    상기 암은 혈액 순환 미세체외소체(csEV) 매개 암인 것을 특징으로 하는, 약학적 조성물.The cancer is characterized in that the blood circulating microextrabody (csEV) mediated cancer, the pharmaceutical composition.
  12. 제10항에 있어서,11. The method of claim 10,
    상기 조성물은 제피티닙 또는 이의 약학적으로 허용 가능한 염을 더 포함하는 것을 특징으로 하는, 약학적 조성물.The composition is characterized in that it further comprises gefitinib or a pharmaceutically acceptable salt thereof, a pharmaceutical composition.
  13. 제10항에 있어서,11. The method of claim 10,
    상기 화학식 1로 표시되는 화합물은 암세포의 M2 면역반응을 제어하는 것을 특징으로 하는, 약학적 조성물.The compound represented by Formula 1 is a pharmaceutical composition, characterized in that it controls the M2 immune response of cancer cells.
  14. 암의 치료를 필요로 하는 개체에 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 약학적 조성물을 투여하는 단계를 포함하는 암 또는 암 전이의 예방 또는 치료 방법.A method for preventing or treating cancer or cancer metastasis, comprising administering to an individual in need of cancer treatment a pharmaceutical composition comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
    [화학식 1][Formula 1]
    Figure PCTKR2021012192-appb-img-000019
    Figure PCTKR2021012192-appb-img-000019
    (상기 화학식 1에 있어서,(In Formula 1,
    상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
    여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
    다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
    상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
    또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
    여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
    다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
    또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
    상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
  15. 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 조성물의 암 또는 암 전이의 예방 또는 치료 용도.Use of a composition comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention or treatment of cancer or cancer metastasis.
    [화학식 1][Formula 1]
    Figure PCTKR2021012192-appb-img-000020
    Figure PCTKR2021012192-appb-img-000020
    (상기 화학식 1에 있어서,(In Formula 1,
    상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
    여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
    다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
    상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
    또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
    여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
    다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
    또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
    상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
  16. 암 또는 암 전이 예방 또는 치료 약제를 생산하기 위한, 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염의 용도.Use of a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof for producing a medicament for preventing or treating cancer or cancer metastasis.
    [화학식 1][Formula 1]
    Figure PCTKR2021012192-appb-img-000021
    Figure PCTKR2021012192-appb-img-000021
    (상기 화학식 1에 있어서,(In Formula 1,
    상기 R1은 치환 또는 비치환된 C6-10의 아릴 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이며, Wherein R 1 is a substituted or unsubstituted C 6-10 aryl or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S,
    여기서, 상기 치환된 아릴 또는 헤테로아릴은 할로젠, 히드록시, 시아노, 아미노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 및 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고, Here, the substituted aryl or heteroaryl is halogen, hydroxy, cyano, amino, nitro, substituted or unsubstituted C 1-5 straight or branched chain alkyl, and substituted or unsubstituted C 1-5 substituted with one or more substituents selected from the group consisting of straight-chain or branched alkoxy,
    다시 여기서, 상기 치환된 알킬 또는 치환된 알콕시는 할로젠, 옥소(=O) 및 히드록시로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고;again wherein said substituted alkyl or substituted alkoxy is substituted with one or more substituents selected from the group consisting of halogen, oxo (=O) and hydroxy;
    상기 R2는 치환 또는 비치환된 C3-10의 사이클로알킬, N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로사이클로알킬, 치환 또는 비치환된 C6-10의 아릴, 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 치환 또는 비치환된 5 내지 10각환의 헤테로아릴이거나,Wherein R 2 is a substituted or unsubstituted C 3-10 cycloalkyl, a substituted or unsubstituted 5- to 10-membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, substituted Or an unsubstituted C 6-10 aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O, and S;
    또는 C6-10의 아릴과 C3-10의 사이클로알킬 또는 N, O, 및 S로 이루어진 군으로부터 선택된 하나 이상의 헤테로 원자를 포함하는 5 내지 10각환의 헤테로사이클로알킬이 융합된(fused), 치환 또는 비치환된 융합 고리이되,Or C 6-10 aryl and C 3-10 cycloalkyl or 5 to 10-membered heterocycloalkyl containing at least one hetero atom selected from the group consisting of N, O, and S is fused (fused), substituted or an unsubstituted fused ring,
    여기서, 상기 치환된 사이클로알킬, 치환된 헤테로사이클로알킬, 치환된 아릴, 치환된 헤테로아릴, 또는 치환된 융합 고리는 치환 또는 비치환된 아미노, 할로젠, 히드록시, 시아노, 니트로, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알킬, 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄의 알콕시, 또는 치환 또는 비치환된 C6-10아릴C1-10알킬로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,wherein the substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, or substituted fused ring is substituted or unsubstituted amino, halogen, hydroxy, cyano, nitro, substituted or unsubstituted The group consisting of substituted or unsubstituted C 1-5 straight or branched chain alkyl, substituted or unsubstituted C 1-5 straight or branched chain alkoxy, or substituted or unsubstituted C 6-10 arylC 1-10 alkyl substituted with one or more substituents selected from
    다시 여기서, 상기 치환된 아미노, 치환된 알킬, 치환된 알콕시, 치환된 C6-10아릴C1-10알킬은 치환 또는 비치환된 C1-5의 직쇄 또는 분지쇄 알킬, 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고,Again, wherein said substituted amino, substituted alkyl, substituted alkoxy, substituted C 6-10 arylC 1-10 alkyl is a substituted or unsubstituted C 1-5 straight or branched chain alkyl, halogen, and oxo (=O) is substituted with one or more substituents selected from the group consisting of,
    또 다시 여기서, 상기 치환된 C1-5의 직쇄 또는 분지쇄 알킬은 할로젠, 및 옥소(=O)로 이루어진 군으로부터 선택된 하나 이상의 치환기로 치환되고; 및Again here, the substituted C 1-5 straight or branched chain alkyl is substituted with one or more substituents selected from the group consisting of halogen, and oxo (=O); and
    상기 R3 및 R4는 각각 독립적으로 하나 이상의 수소, 할로겐 또는 C1-5의 알킬이다.)R 3 and R 4 are each independently one or more hydrogen, halogen, or C 1-5 alkyl.)
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WO2006035067A2 (en) * 2004-09-30 2006-04-06 Tibotec Pharmaceuticals Ltd. Hiv inhibiting 5-heterocyclyl pyrimidines
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KR20190027765A (en) * 2017-09-07 2019-03-15 한국화학연구원 Pyrazole substituted pyrimidine derivative, optical isomer thereof, or pharmaceutically acceptable salts thereof, and composition comprising its same for preventing or treating of cancer
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KR20180045065A (en) * 2008-06-27 2018-05-03 셀젠 카르 엘엘씨 Heteroaryl compounds and uses thereof
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KR20190027765A (en) * 2017-09-07 2019-03-15 한국화학연구원 Pyrazole substituted pyrimidine derivative, optical isomer thereof, or pharmaceutically acceptable salts thereof, and composition comprising its same for preventing or treating of cancer
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