WO2022059996A1 - Micro-composition de traitement du cancer à médiation par corpuscules in vitro à circulation sanguine - Google Patents

Micro-composition de traitement du cancer à médiation par corpuscules in vitro à circulation sanguine 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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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

La présente invention concerne une micro-composition de traitement du cancer à médiation par corpuscules in vitro à circulation sanguine et, plus particulièrement, une composition pour la prévention ou le traitement du cancer comprenant un composé représenté par la formule chimique 1 ou un sel de celui-ci en tant que principe actif.
PCT/KR2021/012192 2020-09-15 2021-09-08 Micro-composition de traitement du cancer à médiation par corpuscules in vitro à circulation sanguine WO2022059996A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
WO2006035067A2 (fr) * 2004-09-30 2006-04-06 Tibotec Pharmaceuticals Ltd. 5-heterocyclyl-pyrimidines inhibitrices du vih
US20150038481A1 (en) * 2012-05-22 2015-02-05 The University Of North Carolina At Chapel Hill Pyrimidine compounds for the treatment of cancer
KR20180045065A (ko) * 2008-06-27 2018-05-03 셀젠 카르 엘엘씨 헤테로아릴 화합물 및 이의 용도
KR20190027765A (ko) * 2017-09-07 2019-03-15 한국화학연구원 피라졸기로 치환된 피리미딘 유도체 화합물, 이의 광학이성질체, 또는 이의 약학적으로 허용 가능한 염, 및 이를 유효성분으로 포함하는 암 예방 또는 치료용 조성물
WO2019222538A1 (fr) * 2018-05-16 2019-11-21 The University Of North Carolina At Chapel Hill Aminopyrimidines et aminopyridines en tant qu'inhibiteurs de mertk et leur application dans le traitement du cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006035067A2 (fr) * 2004-09-30 2006-04-06 Tibotec Pharmaceuticals Ltd. 5-heterocyclyl-pyrimidines inhibitrices du vih
KR20180045065A (ko) * 2008-06-27 2018-05-03 셀젠 카르 엘엘씨 헤테로아릴 화합물 및 이의 용도
US20150038481A1 (en) * 2012-05-22 2015-02-05 The University Of North Carolina At Chapel Hill Pyrimidine compounds for the treatment of cancer
KR20190027765A (ko) * 2017-09-07 2019-03-15 한국화학연구원 피라졸기로 치환된 피리미딘 유도체 화합물, 이의 광학이성질체, 또는 이의 약학적으로 허용 가능한 염, 및 이를 유효성분으로 포함하는 암 예방 또는 치료용 조성물
WO2019222538A1 (fr) * 2018-05-16 2019-11-21 The University Of North Carolina At Chapel Hill Aminopyrimidines et aminopyridines en tant qu'inhibiteurs de mertk et leur application dans le traitement du cancer

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