Pharmaceutical composition targeting cancer-associated fibroblasts

Abstract

The present invention relates to a pharmaceutical composition targeting cancer-associated fibroblasts and, more specifically, to a pharmaceutical composition comprising a homoharringtonine analog as an active ingredient, wherein the pharmaceutical composition targets cancer-associated fibroblasts, and has the ability to inhibit the proliferation or activity thereof.

Description

Pharmaceutical composition targeting cancer-associated fibroblasts

[0001] The present invention relates to a pharmaceutical composition targeting cancer associated fibroblasts, and more specifically, to a pharmaceutical composition comprising a homoharringtonine analogue as an active ingredient and having the ability to target cancer associated fibroblasts and inhibit their proliferation or activity.

[0002]

[0003] Cancer cells exist in a cancer microenvironment, a protective and supportive environment, and through this, normal cells surrounding cancer cells have the ability to change their properties to protect and support the growth of cancer cells. The cancer microenvironment includes proteins or extracellular support tissues that are signaling substances secreted by cells, such as cytokines or chemokines. Cancer cells or surrounding cells also produce cytokines or chemokines, and the signaling substances secreted by cancer cells are known to vary among cancer cells (Erik Sahai et al., 2020, Nature Reviews Cancer 20, 174-186).

[0004] The cancer microenvironment includes fibroblasts, blood vessels, lymphatic vessels, immune cells, extracellular matrix, and adipocytes, and through various interactions, regulates the growth and proliferation of cancer cells, significantly affecting cancer progression or metastasis, as well as the response and effectiveness of cancer treatment. For example, normal fibroblasts suppress tumor development, whereas cancer-associated fibroblasts (CAFs) induce tumor metastasis and invasion through extensive tissue remodeling through the secretion of proteolytic enzymes, deposition of extracellular matrix, and development of vascular networks. As cancer grows, the cells that make up the cancer microenvironment change. Fibroblasts transform into cancer-associated fibroblasts, secreting substances and growth factors such as TGF-beta that promote cancer growth. They also create blood vessels around the tumor to help supply nutrients to cancer cells and promote metastasis (Raghu Kalluri, 2016, Nature Reviews Cancer 16, 582-598).

[0005] Meanwhile, homoharringtonine (HHT), a natural single component extracted from the Cephalotaxus harringtonii tree, is known to inhibit protein production by binding to 80S ribosomes in eukaryotic cells and inhibiting chain elongation. Homoharringtonine is used to treat certain types of chronic myeloid leukemia (CML), which does not improve following other anticancer treatments, and research is ongoing to determine whether it can be used to treat other cancers as well (Chenying Li et al., haematologica, Vol. 105, No. 1 (2020)). Additionally, it has been recently reported that homoheringtonin inhibits the expression of Prrx1 (Paired related homeobox 1), which is overexpressed in CAF and known to act as a master transcription factor (mTF) (Keun-Woo Lee et al., Nature Communications 13, 2793 (2022)).

[0006] Accordingly, the inventors of the present invention have conducted extensive research efforts to develop a drug having cancer cell inhibitory activity in a cancer microenvironment where CAFs exist, and as a result, have confirmed that homoheringtonine analogs can reduce CAF contractility, decrease cancer cell migration and invasion ability, and inhibit aggregation of cancer cells and CAFs, thereby confirming that excellent therapeutic effects can be exhibited against various cancers such as solid cancers, thereby completing the present invention.

[0007] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0008]

[0009] Summary of the invention

[0010] The purpose of the present invention is to provide a homoheringtonine analog that exhibits an effect of inhibiting the proliferation and activity of CAF.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the homoheringtonine analogue as an active ingredient.

[0012] Another object of the present invention is to provide a method for preventing or treating cancer, comprising a step of administering to a subject the homoheringtonine analogue or a pharmaceutical composition containing the same as an active ingredient.

[0013] Another object of the present invention is to provide a use of the homoheringtonine analogue or a pharmaceutical composition containing the same as an active ingredient for preventing or treating cancer.

[0014] Another object of the present invention is to provide the use of the homoheringtonine analogue or a pharmaceutical composition containing the same as an active ingredient for the manufacture of a drug for preventing or treating cancer.

[0015] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the homoheringtonine analogue as an active ingredient and is to be administered in combination with an anticancer agent.

[0016] Another object of the present invention is to provide a method for preventing or treating cancer, characterized in that the homoheringtonine analogue or a pharmaceutical composition containing the same as an active ingredient is administered to a subject in combination with an anticancer agent or a pharmaceutical composition containing the same.

[0017] Another object of the present invention is to provide a use of the homoheringtonine analogue or a pharmaceutical composition containing the same as an active ingredient for preventing or treating cancer in combination with an anticancer agent or a pharmaceutical composition containing the same.

[0018] Another object of the present invention is to provide the use of the homoheringtonine analogue or a pharmaceutical composition containing it as an active ingredient for the manufacture of a medicament for preventing or treating cancer in combination with an anticancer agent or a pharmaceutical composition containing it.

[0019]

[0020] In order to achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a compound represented by the following chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof:

[0021] [Chemical Formula 1]

[0022]

[0023] The present invention also provides a method for preventing or treating cancer, comprising administering to a subject the homoheringtonine analogue or a pharmaceutical composition containing the same as an active ingredient.

[0024] The present invention also provides a use of the homoheringtonine analogue or a pharmaceutical composition containing the same as an active ingredient for preventing or treating cancer.

[0025] The present invention also provides the use of the homoheringtonine analogue or a pharmaceutical composition comprising the same as an active ingredient for the manufacture of a medicament for preventing or treating cancer.

[0026] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the homoheringtonine analogue as an active ingredient and for co-administration with an anticancer agent.

[0027] The present invention also provides a method for preventing or treating cancer, characterized in that the homoheringtonine analog or a pharmaceutical composition containing the same as an active ingredient is administered to a subject in combination with an anticancer agent or a pharmaceutical composition containing the same.

[0028] The present invention also provides a use of the homoheringtonine analogue or a pharmaceutical composition comprising the same as an active ingredient for preventing or treating cancer in combination with an anticancer agent or a pharmaceutical composition comprising the same.

[0029] The present invention also provides the use of the homoheringtonine analogue or a pharmaceutical composition comprising it as an active ingredient for the manufacture of a medicament for preventing or treating cancer in combination with an anticancer agent or a pharmaceutical composition comprising it.

[0030] The present invention also provides a compound represented by the following chemical formula 10 or chemical formula 11, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof:

[0031]

[0032] Figures 1a to 1k show the EC of 11 homoheringtonine analogs and homoheringtonine (HHT) against HCT116 cancer cells, DLD1 cancer cells, CAF1, and CAF2. 50 This is a graph that measures the values.

[0033] Figure 2 shows the results of measuring the CAF contraction ability after culturing CAF for one week with 33 homoheringtonine analogues, collecting conditioned medium (CM) (serum-free), inoculating HCT116 cells, adding the collected CM, and culturing for 2-3 days.

[0034] Figure 3 shows the results of a migration analysis performed using cancer cells cultured for 1 week after treating CAF with 33 homoheringtonine analogues, collecting conditioned medium (CM) (serum-free), inoculating HCT116 cells, adding the collected CM, and culturing for 2-3 days.

[0035] Figure 4 shows the results of sphere formation analysis performed after culturing CAF for one week with 33 homoheringtonine analogues and mixing them with HCT116 cells.

[0036] Figures 5a to 5c show the results of spheroid formation assays performed after treating HCT116 cancer cells alone, HCT116 cancer cells and CAF1, and HCT116 cancer cells and CAF2 with 33 homoherin analogues, respectively. Lighter and darker colors indicate smaller and larger spheroid sizes, respectively, and "No treatment," "Treatment 1," "Treatment 2," and "Treatment 3" indicate the following (Figure 5a): No treatment: Spheroid size increases when CAFs are present; Treatment 1: Spheroid size decreases when CAFs are present only; Treatment 2: Spheroid size decreases regardless of the presence of CAFs, but the effect is greater when cancer cells are present alone; Treatment 3: Spheroid size decreases regardless of the presence of CAFs, but the effect is less when cancer cells are present alone.

[0037] Figure 6 is a table summarizing the screening results of 33 homoherin analogues. Analogues with excellent anticancer effects are highlighted in green. *Spheroid growth was tested only in one type of CAF cell line. **EC50 was measured, but the data significantly deviated from the log-logistic model.

[0038] Figure 7 shows the EC of homoheringtonine and 11 analogues for CCD-18Co cells. 50 This is a graph that measures the values.

[0039] Figure 8 is a table summarizing the final screening results of 11 homoheringtonine analogs. Analogs with excellent anticancer effects are highlighted in green. *Spheroid growth was tested only in one type of CAF cell line.

[0040]

[0041] Detailed description of the invention and preferred embodiments

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0043]

[0044] The present invention aims to develop a drug having cancer cell inhibitory activity in a cancer microenvironment where CAFs exist, and selected 33 homoheringtonine analogs (ARC-12102 to ARC-12134) as candidate substances, and among these, 11 homoheringtonine analogs (ARC-12104, ARC-12110, ARC-12112, ARC-12113, ARC-12116, ARC-12117, ARC-12121, ARC-12127, ARC-12128, ARC-12129, and ARC-12131) were excellent in reducing CAF contraction, reducing cancer cell migration and invasion ability, and reducing aggregation of cancer cells and CAFs in a cancer microenvironment where CAFs exist, and thus confirmed to have excellent cancer cell inhibitory activity in a cancer microenvironment where CAFs exist.

[0045] Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a compound represented by the following chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof:

[0046] [Chemical Formula 1]

[0047]

[0048] In the present invention, the chemical formulas of 11 homoheringtonine analogues included as active ingredients in the pharmaceutical composition are as shown in Chemical Formulas 2 to 12 of Table 1 below.

[0049]

[0050]

[0051] From another perspective, the present invention relates to a method for preventing or treating cancer, characterized in that it comprises administering in combination a compound represented by the above chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a pharmaceutical composition containing the same as an active ingredient, and an anticancer agent or a pharmaceutical composition containing the same.

[0052] In another aspect, the present invention relates to the use of a compound represented by the above chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a pharmaceutical composition comprising the same as an active ingredient, for preventing or treating cancer in combination with an anticancer agent or a pharmaceutical composition comprising the same.

[0053] In another aspect, the present invention relates to the use of a compound represented by the above chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a pharmaceutical composition comprising the same as an active ingredient for the manufacture of a medicament for preventing or treating cancer in combination with an anticancer agent or a pharmaceutical composition comprising the same.

[0054]

[0055] The present invention also identified two novel homoheringtonine analogs (ARC-12128 and ARC-12129) with excellent cancer cell inhibitory activity in a cancer microenvironment where CAFs exist.

[0056] Accordingly, the present invention relates to a compound represented by the following chemical formula 10 or 11, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof from another aspect:

[0057]

[0058] In the present invention, 33 homoheringtonine analogues have the structure of chemical formula 1 and the residues of R as shown in Table 2 below.

[0059]

[0060]

[0061]

[0062] In the present invention, cancer may be characterized by including cancer-associated fibroblasts in the surrounding cancer microenvironment.

[0063] In the present invention, the pharmaceutical composition may be characterized by further comprising a culture containing cancer-associated fibroblasts.

[0064] In the present invention, the cancer may be characterized by being at least one selected from the group consisting of lung cancer, thyroid cancer, breast cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, and liver cancer, but is not limited thereto.

[0065] In the present invention, the pharmaceutical composition may be characterized in that it is administered in combination with an anticancer agent.

[0066] The above anticancer drugs are gossypol, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tyusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, Pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, It may be at least one selected from the group consisting of cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, navelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine, but is not limited thereto.

[0067] In the present invention, the pharmaceutical composition may be characterized by being formulated into any one dosage form selected from the group consisting of injections, oral administration preparations, patches, liquids, capsules, granules, tablets, powders, sprays, ointments, gels, mucosal administration preparations, and suppositories, but is not limited thereto.

[0068] The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. The pharmaceutical composition of the present invention is preferably administered parenterally, and can be administered by, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration, but is not limited thereto. In the case of intraperitoneal administration for ovarian cancer and portal vein administration for liver cancer, it can be administered by injection, and in the case of breast cancer, it can be administered by direct injection into the tumor mass, in the case of colon cancer, it can be administered by direct injection with an enema, and in the case of bladder cancer, it can be administered by direct injection into a catheter.

[0069] In the present invention, the pharmaceutical composition may be characterized by further comprising a pharmaceutically acceptable carrier.

[0070] The carrier is one commonly used in the formulation and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0071] The pharmaceutical composition of the present invention can be manufactured in a unit dosage form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. A typical dosage form refers to, for example, an oral (tablet, capsule, powder), intrabuccal, sublingual, intrarectal, intravaginal, intranasal, topical or parenteral (including intravenous, intracavernous, intramuscular, subcutaneous and intraductal) administration dosage form. For example, the compound according to the present invention can be administered orally, intrabuccally or sublingually in the form of a tablet containing starch or lactose, or in the form of a capsule containing alone or with an excipient, or in the form of an elixir or suspension containing a chemical agent for flavoring or coloring. Liquid preparations are prepared with pharmaceutically acceptable excipients such as suspending agents (e.g., methylcellulose, semi-synthetic glycerides such as witepsol, or glyceride mixtures such as a mixture of apricot kernel oil and PEG-6 esters or a mixture of PEG-8 and caprylic / capric glycerides). Furthermore, when injected parenterally, for example, intravenously, intracavernosally, intramuscularly, subcutaneously and intraductally, they are most preferably used in the form of sterile aqueous solutions, which may contain other substances (e.g., salts or monosaccharides such as mannitol or glucose) to render them isotonic with blood.

[0072] The homoheringtonine analogue according to the present invention can be used in the form of a pharmaceutically acceptable salt, and as a salt, an acid addition salt formed by a pharmaceutically acceptable free acid can be used. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid and fumaric acid. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, Including, but not limited to, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

[0073] Acid addition salts can be prepared by a conventional method, for example, by dissolving the active ingredient included in the composition of the present invention in an organic solvent, for example, methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, filtering and drying the resulting precipitate, or by distilling the solvent and an excess of acid under reduced pressure and then drying or crystallizing the same in an organic solvent.

[0074] In addition, pharmaceutically acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are pharmaceutically suitable as metal salts. In addition, the corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate). In addition, the present invention includes not only the thienopyrimidine derivative represented by the above chemical formula 1 and its pharmaceutically acceptable salts, but also all possible solvates, hydrates, stereoisomers, etc. that can be prepared therefrom.

[0075] In the present invention, the pharmaceutical composition may further include, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0076] In the present invention, the daily dosage of the compound, its stereoisomer, its pharmaceutically acceptable salt or its solvate is 1 μg / kg to 100 mg / kg, and may be characterized by administration once a day or 2-3 times a week, but is not limited thereto.

[0077] The appropriate dosage may vary depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity.

[0078]

[0079] Hereinafter, the present invention will be described in detail with examples and the like to aid understanding. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples.

[0080]

[0081] Example 1. Experimental preparation and experimental method

[0082] 1-1. Isolation and culture of human fibroblasts

[0083] Fresh tissue from a patient with colorectal cancer undergoing surgery was cut into small pieces in a culture dish and minced with a scalpel. The sample was placed in a 15-ml tube with 10 ml of D / F12 complete medium (10% FBS + 1% antibiotics) containing collagenase type I and collagenase / dispase. Digestion was performed for 12 hours on an orbital shaker in a 37°C CO2 incubator. After 12 hours, the sample was centrifuged at 700 rpm for 5 minutes, washed twice with PBS, and cultured in D / F12 complete medium to culture cancer-associated fibroblasts.

[0084]

[0085] 1-2. Culture of colon cancer cell lines and colon fibroblast lines

[0086] Colon cancer cell lines HCT116 and DLD-1 were subcultured in DMEM complete medium (10% FBS + 1% antibiotics), and the cells were cultured in a CO2 incubator at 37℃ for less than 6 months. CCD18co normal type colon normal fibroblasts (KCLB Korea Cell Line Bank) were also subcultured using DMEM complete medium, and the subcultivation ratio was 1:3 or less during subculture.

[0087]

[0088] 1-3. EC 50 value concentration measurement

[0089] EC of each analogue 50 To determine the concentration value, 5x10 of cancer-associated fibroblasts and colon cancer cell lines HCT116 and DLD1 were prepared in a 96-well plate. 3The following day, replace the medium with 100 μl of DMEM complete medium and treat each analog compound at concentrations ranging from 0 nM to 2 μM. After culturing for 3 days, proceed according to the instructions of the EZ-cytox plus cell viability assay kit (Cat: EZ-3000P, DaeilLab, Seoul, Korea) dedicated to measuring cell viability and cytotoxicity. Add 10 μl of EZ-Cytox to each well and incubate for about 1 hour. Before measuring the absorbance, gently shake for about 1 minute and measure the absorbance at 450 nm using a plate reader (Mithras LB-943, microplate reader (luminometer), Berthold technologies). Each experiment is performed in triplicate. In addition, CCD18co (colon fibroblast cell line, Korea Cell Line Bank KCLB NO. 21459) was used to confirm toxicity in normal conditions. EC 50 The dose-response curve was calculated using the R package drc, and the EC 50 is determined as the concentration opposite to the mid-distance between the upper and lower ends of the four-parameter logistic curve (Ritz C, Baty F, Streibig JC, Gerhard D (2015) Dose-Response Analysis Using R. PLoS ONE 10(12): e0146021).

[0090]

[0091] 1-4. Preparation of Conditioned Medium

[0092] 1x10 prepared cancer-associated fibroblasts (CAFs) 6Seed in a 100mm cell culture dish. The next day, when the cells are all attached and growing healthily, wash with PBS and measure the EC of each analog compound. 50 Treat with the value concentration (the CAF control is DMSO treatment, and at this time, DMSO-treated CM of HCT116, which is not treated with colon cancer cell analogues, is also prepared). After culturing for 3 days, wash twice using PBS, add 8 ml of serum-free DMEM medium, and culture for 2-3 days (cells reached 80% confluence). Collect this, transfer it to a 15 ml tube, and spin it at 1300 rpm, 4 ° C for 5 minutes to obtain clean conditioned medium without pellet debris. Conditioned medium used in the experiment is replaced daily.

[0093]

[0094] 1-5. Gel contraction assay

[0095] Gel contraction assay was performed using a cell contraction assay kit (Cat: CBA-201, Cell Biolabs, Inc., San Diego, CA) according to the instructions. Prepared cancer-associated fibroblasts were detached using trypsin and washed twice with PBS. 1x10 5After resuspending cancer-associated fibroblasts in 20ul of DMEM complete medium, mix well with a mixture of 5XDMEM and collagen, and add a total of 500ul of cell-collagen mixture to a 24-well plate. Incubate in a CO2 incubator at 37℃ for 2 hours for collagen polymerization. Replace the DMEM complete medium every 2 days, and measure the EC of each analog compound at this time. 50 Treat with value concentration. Observe the collagen gel size during culture.

[0096]

[0097] 1-6. Migration assay

[0098] 2x10 colon cancer cells HCT116 5 Seed in a 60mm cell culture dish. When the cells reach 70-80% confluence, wash twice with PBS. Replace the conditioned medium (CM) of each prepared analog compound cancer-associated fibroblast (CAF). At this time, not only the DMSO-treated CM of CAF but also the DMSO-treated CM of colon cancer cells HCT116 are tested as a control. After culturing for 3 days with the CM, 1x10 5Resuspend the cells in 100 μl of DMEM medium supplemented with 1% FBS and 0.1% BSA and place them in the upper chamber of a 24-well Transwell insert (Cat: 354234, Corning, Corning City, NY). Add 400 μl of DMEM complete medium to the 24-well below the insert well. After 24 hours, fix the cells with methanol for 5 minutes and stain with hematoxylin & eosin. Remove the cells in the upper chamber and observe the migrated cells by taking images using a microscope.

[0099]

[0100] 1-7. Sphere forming assay

[0101] 2x10 colon cancer cells HCT116 5 Seed in a 60mm cell culture dish. When the cells reach 70-80% confluence, wash twice with PBS. Replace the conditioned medium (CM) of each prepared analog compound cancer-associated fibroblast (CAF). At this time, not only the DMSO-treated CM of CAF but also the DMSO-treated CM of colon cancer cells HCT116 are tested as a control. After culturing for 3 days with the CM, 1x10 3Cells are seeded in ultra-low-attachment 6-well plates. At this time, the cells are used DMEM / F-12 medium supplemented with B27 (Invitrogen, Carlsbad, CA, USA), 10 ng / mL EGF, and bFGF (Invitrogen, Carlsbad, CA, USA). The next day, 500 μl of each CAF and cancer cell CM is added. Thereafter, the sphere forming medium containing 500 μl of CM is replaced every two days. During the two-week culture, the spheres are imaged under a microscope and the number is observed.

[0102]

[0103] 1-8. Hanging drop 3D spheroid coculture system

[0104] To perform this experiment, 15 ml of PBS was placed in a 100 mm petri dish to serve as a hydration chamber. Colon cancer cells HCT116 and cancer-associated fibroblasts (CAFs) were detached with trypsin and counted. 1 x 10 3 3x10 cancer cells and 3CAF (ratio 1:3) was resuspended in DMEM complete medium and 10 ul was added to a 100 mm petri dish by turning the lid over. The dish was then covered with a 15 ml petri dish and cultured in a 37°C CO2 incubator. After 24 hours, the shape of the aggregation was observed and the aggregated spheroids were collected and placed in a 100 mm petri dish. 10 ml of DMEM complete medium was added and culture was started on a shaker in a 37°C 5% CO2 incubator. After 24 hours, the medium was replaced with new DMEM complete medium and each analog compound was measured for its EC. 50 Treat with the value concentration and culture again in a shaker. Replace with new medium treated with each analog compound every two days and culture, and image the spheroids using a microscope every day and observe the size. After 7 days, collect the spheroids, transfer to a 15 ml tube, wash twice with PBS, and fix with 4% PFA (paraformaldehyde) at 4℃ for 12 hours. After fixation, place the spheroids in a tissue staining cassette to make a paraffin block, perform H&E staining, and scan with Leica Biosystems Aperio - Pathology Slide Scanners to observe cell changes within the spheroids and measure the size using the Aperio image scope program.

[0105]

[0106] 1-9. Reagents

[0107] All reagents and related research kits used in this study are as follows:

[0108] -DMEM : welgene LM 001-05

[0109] -DMEM / F12 : biowest, L0092-500

[0110] -FBS : welgene, S001-07

[0111] -Trypsin / EDTA solution : welgene, LS 015-01

[0112] -Antibiotic-Antimycotic solution, 100X : welgene, LS203-01

[0113] -24well plate : SPL, 30024

[0114] -96well plate : SPL, 30096

[0115] -15ml tube : NEST, 601002

[0116] -100mm petri dish : SPL, 10091

[0117] -6well low attachment plate : Corning, 3471

[0118] -24well transwell insert : Corning, 354234

[0119] -FGF : Peprotech, cat# AF-100-18B

[0120] -EGF : Merck, cat# E4127

[0121] -B27 : Thermo Fisher Scientific, cat# 17504044

[0122] -collagenase type I : sigma aldrich, SCR-103

[0123] -collagenase / Dispase : Roche, 10269638001

[0124] -EZ-cytox plus cell viability assay kit (Cat: EZ-3000P, DaeilLab)

[0125] -Contraction kit: (Cat: CBA-201, Cell Biolabs, Inc, San Diego, CA)

[0126]

[0127] Example 2. EC of HHT analogues against cancer cells and CAFs 50 measurement

[0128] EC of 33 homoheringtonine analogs against cancer cells and CAFs 50 To measure the value, an experiment was conducted as in Example 1-3.

[0129] As a result, 18 analogues of homoheringtonine (ARC-12102, ARC-12104, ARC-12110, ARC-12112, ARC-12113, ARC-12114, ARC-12116, ARC-12117, ARC-12121, ARC-12122, ARC-12127, ARC-12128, ARC-12129, ARC-12130, ARC-12131, ARC-12132, ARC-12133, and ARC-12134) showed EC values ​​for at least one of HCT116, DLD1, CAF1, and CAF2. 50 It was confirmed that the value was excellent at less than 40 nM (Fig. 1a to Fig. 1k).

[0130]

[0131] Example 3. CAF contraction reduction effect of HHT analogues

[0132] A fibroblast contraction assay was performed as in Example 1-5.

[0133] As a result, it was confirmed that 21 analogues of homoheringtonine (ARC-12104, ARC-12105, ARC-12107, ARC-12109, ARC-12110, ARC-12112, ARC-12113, ARC-12114, ARC-12116, ARC-12117, ARC-12118, ARC-12119, ARC-12120, ARC-12121, ARC-12122, ARC-12123, ARC-12124, ARC-12127, ARC-12128, ARC-12129, and ARC-12131) reduced CAF contractile ability by more than 50% (Fig. 2).

[0134]

[0135] Example 4. Migration-reducing effect of HHT analogues

[0136] To confirm the change in the migration ability of cancer cells increased by CAF, CAF CM was collected, HCT116 was cultured, and the process was carried out as in Example 1-6.

[0137] As a result, it was confirmed that 30 analogs of homoheringtonine (all analogs except ARC-12107, ARC-12124, and ARC-12125) reduced the migration ability of cancer cells increased by CAF by more than 50% (Fig. 3).

[0138]

[0139] Example 5. Effect of HHT analogues on sphere formation reduction

[0140] To confirm the change in the sphere-forming ability of cancer cells, CAF CM was collected, HCT 116 was cultured, and the procedure was carried out as in Example 1-7.

[0141] As a result, it was confirmed that 27 analogs of homoheringtonine (all analogs except ARC-12107, ARC-12108, ARC-12109, ARC-12124, ARC-12125, and ARC-12126) reduced the sphere-forming ability of cancer cells by more than 50% (Fig. 4).

[0142]

[0143] Example 6. Effect of HHT analogues on spheroid size reduction

[0144] To confirm the effect of reducing the spheroid size of cancer cells, the procedure was carried out as in Example 1-8.

[0145] As a result, it was confirmed that 28 analogs of homoheringtonine (all analogs except ARC-12107, ARC-12108, ARC-12123, ARC-12124, and ARC-12126) excellently reduced the spheroid size of at least one of two cancer cells, HCT 116 and DLD1, in the presence of CAF (Figs. 5a to 5c).

[0146] Based on the results of Examples 2 to 5, it was confirmed that 11 HHT analogs represented by Chemical Formulas 2 to 12 (ARC-12104, ARC-12110, ARC-12112, ARC-12113, ARC-12116, ARC-12117, ARC-12121, ARC-12127, ARC-12128, ARC-12129, and ARC-12131) had excellent cancer cell inhibitory activity in the tumor microenvironment where CAFs exist (Fig. 6).

[0147]

[0148] Example 7. Toxicity evaluation of HHT analogues

[0149] In order to measure the normal toxicity of homoheringtonine analogues with excellent efficacy, a toxicity evaluation was conducted using CCD18co normal type colonic fibroblasts as in Example 1-3.

[0150] As a result, six analogues of homoheringtonine (ARC-12104, ARC-12116, ARC-12127, ARC-12128, ARC-12129, and ARC-12131) showed excellent cancer cell inhibition activity in the cancer microenvironment where CAFs exist, and EC of normal fibroblast-associated CCD-18Co cells. 50 It can be confirmed that the value is excellent at 40 nM or more and the predicted toxicity is low (Fig. 7 and Fig. 8).

[0151]

[0152] A pharmaceutical composition comprising a homoheringtonine analogue according to the present invention as an active ingredient can reduce CAF contractility, decrease the migration and invasion capabilities of cancer cells, and inhibit the aggregation of cancer cells and CAF, and thus has a preventive and therapeutic effect on various cancers such as solid cancer.

[0153]

[0154] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a compound represented by the following chemical formula 1, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient: [Chemical Formula 1] 2. A pharmaceutical composition according to claim 1, wherein the compound is a compound represented by the following chemical formula 10 or chemical formula 11:

3. A pharmaceutical composition according to claim 1 or 2, characterized in that the cancer comprises cancer associated fibroblasts in the surrounding cancer microenvironment.

4. A pharmaceutical composition according to claim 1 or 2, characterized in that it further comprises a culture containing cancer-associated fibroblasts.

5. A pharmaceutical composition according to claim 1 or 2, characterized in that the cancer is at least one selected from the group consisting of lung cancer, thyroid cancer, breast cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, and liver cancer.

6. A pharmaceutical composition according to claim 1 or 2, characterized in that it is administered in combination with an anticancer agent.

7. A pharmaceutical composition according to claim 1 or 2, characterized in that it is formulated into any one dosage form selected from the group consisting of injections, oral administration preparations, patches, liquids, capsules, granules, tablets, powders, sprays, ointments, gels, mucosal administration preparations, and suppositories.

8. A pharmaceutical composition according to claim 1 or 2, characterized in that it further comprises a pharmaceutically acceptable carrier.

9. A pharmaceutical composition according to claim 1 or 2, wherein the daily dosage of the compound, its stereoisomer, its pharmaceutically acceptable salt or its solvate is 1 μg / kg to 100 mg / kg, and is administered once a day or 2-3 times a week.

10. A compound represented by the following chemical formula 10 or 11, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof:

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