MET Protein Degradation Inducing Compounds

Abstract

The present invention relates to a compound for degrading a target protein on the basis of a chaperone-mediated protein degradation (CMPD) technology, and a preparation method and use thereof, and more particularly, to a compound for degrading a target protein on the basis of a chaperone-mediated protein degradation (CMPD) technology. The present invention provides a target protein degrading compound comprising a first moiety (CB) capable of binding to a chaperonin or protein component of a chaperonin complex and a second moiety (TB) capable of binding to a target protein or protein, said compound being a compound that is formed by a chaperonin binding moiety-linker (linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker, linker L)-target protein binding moieties or stereoisomers thereof. The compound provided by the invention shows the effect of inducing the degradation of MET protein based on a CMPD technology. Therefore, the compound of the present invention is expected to exert an excellent anti-cancer effect that can overcome the drug resistance and mutation of a targeted anti-cancer agent through complete degradation of a target protein (MET).

Description

Technical Field

[0001] The present invention relates to a compound for degrading a target protein based on chaperone-mediated protein degradation (CMPD) technology, a preparation method thereof, and uses thereof. Background Art

[0002] Cancer is one of the diseases that has not been conquered since the development of the first therapeutic agent in the 1940s. With the technological development of cancer treatment, the form of anti-cancer agents has been constantly changing. Approximately 40% of the R&D investment in new drug development is concentrated on anti-cancer agents, so many new anti-cancer agents have been marketed, but the unmet needs of cancer patients are increasing day by day. Currently, the direction of anti-cancer agent development is to develop new anti-cancer agents that overcome drug resistance or increase the treatment response rate. Existing anti-cancer agents rely on existing technologies such as small molecule compounds and antibodies. Only 400 out of approximately 3,000 disease-causing genes have drug approvals (there are 85% of undruggable targets), and drugs developed as anti-cancer agents also have problems such as side effects, drug resistance, and response rate. Therefore, there is an urgent need to introduce next-generation drugs / technologies.

[0003] Existing drug development technologies focus on inhibiting the functions of these disease-related proteins, thus having limitations. Therefore, "protein degradation technology (PDT)" is considered a new strategy for overcoming the difficulties of drug development. Protein degradation technology is a technology for selectively degrading proteins and is a new concept technology for removing disease-related proteins themselves. Chemically induced target protein degradation is a new method for developing small molecule compound drugs. Small molecule compounds are used to promote the interaction between components of various cellular protein degradation pathways and the target protein or protein, thereby inducing the degradation of the target protein or protein as a way to treat diseases. It is expected that it can also be applied to diseases with proteins that cannot be conquered by existing drug development technologies (undruggable) as drug targets. Not only can the pathogenic protein be fundamentally removed by degrading the protein, but also the side effects and drug resistance of the developed drugs can be overcome.

[0004] In the field of PDT, proteolysis-targeting chimera (PROTAC) that utilizes E3 ligase is a platform for small molecules that induce the degradation of specific proteins. PROTAC molecules are bifunctional small molecules that can simultaneously bind to the target protein and the E3 ubiquitin ligase. Therefore, PROTAC molecules induce the polyubiquitination of the target protein, followed by the degradation of the target protein by the proteasome. PROTAC molecules have good tissue distribution and the ability to act on intracellular proteins. However, there is a problem that PROTAC molecules are resistant to PROTAC due to mutations in the E3 ligase. Therefore, considering the substrate specificity of new target proteins, PROTAC technology needs to explore suitable new E3 ligases.

[0005] In contrast, the CMPD technology of the present invention does not require the direct participation of E3 ligase, but mediates the interaction with various E3 ligases in vivo through the chaperone complex, and then promotes and induces protein degradation through the Ubiquitin Proteasome System (UPS pathway). Therefore, the limitations of the existing PROTAC technology can be overcome.

[0006] Moreover, when the disease-related target protein is a client protein of the chaperone complex, it can be selectively targeted for degradation. Therefore, compared with PROTAC, it can have a wider range of target proteins and high scalability. Therefore, various diseases such as cancer, neurodegenerative brain diseases, and rare diseases can be studied, and it is easy to expand the product line.

[0007] On the other hand, lung cancer is the most common cancer globally and a major cause of cancer-related deaths. Although the 5-year survival rate has improved since the 2000s, the 5-year relative survival rate remains at 28.2%. In cases of medical records with distant metastases, the observed survival rate in South Korea is as low as 6.1%. Non-small cell lung cancer is a cancer with various gene mutations. Although many therapeutic agents have been developed, there are still many unmet needs. Therefore, various studies are underway, such as the development of therapeutic agents for drug-resistant mutations and the development of combination therapy agents. Among all non-small cell lung cancer patients, 30% are caused by mutations in the epidermal growth factor receptor (EGFR). Therefore, the development focus of the therapeutic agent market lies in the first-generation therapeutic agents as EGFR inhibitors and the second-generation therapeutic agents for the EGFR T790M mutation that confers resistance to the first-generation therapeutic agents. Among all non-small cell lung cancer patients, 6% are caused by mutations in the hepatocyte growth factor receptor (c-MET), which is a rare type. However, approximately 250,000 patients are caused by MET mutations, which are the main cause of drug resistance after the first-generation and second-generation EGFR treatments. Therefore, the expectation and demand for MET-targeted therapeutic agents in the non-small cell lung cancer therapeutic agent market are increasing continuously.

[0008] The present invention can provide a new solution through target protein degradation in non-small cell lung cancer with MET mutations by developing CMPD-based drugs targeting the MET protein.

[0009] The inventors of the present invention combined a CMPD-inducing compound, which is a molecular chaperone complex-related protein-binding part, with existing targeted drugs (such as crizotinib, capmatinib, tepotinib, etc.) used as target-binding substances using various linkers to design the CMPD drug structure. As a result, it was confirmed that by inducing the degradation and complete removal of the key target protein MET in non-small cell lung cancer through CMPD, it is expected to exert a more excellent anti-cancer effect than existing anti-cancer agents through the complete degradation of the target protein MET, thus completing the present invention. Summary of the Invention

[0010] Technical Problem

[0011] An object of the present invention is to provide a compound that degrades proteins through CMPD.

[0012] Another object of the present invention is to provide a method for preparing a compound that degrades proteins through CMPD.

[0013] Another object of the present invention is to provide the use of a compound that degrades proteins through CMPD.

[0014] Technical solution

[0015] The present invention provides a compound for degrading a target protein based on CMPD technology. Specifically, the present invention provides a target protein degrading compound, which comprises a first part (chaperone binding, CB) capable of binding to a chaperone protein or a protein component of a chaperone complex and a second part (target binding, TB) capable of binding to a target protein or a protein.

[0016] The compound of the present invention is a compound represented by the following chemical formula I or a stereoisomer thereof, which is composed in the form of a chaperone binding part (hereinafter referred to as "CB") - linker (L) - target protein binding part (hereinafter referred to as "TB").

[0017] Chemical formula I:

[0018] CB-L-TB

[0019] CB is the first part capable of binding to a chaperone protein or a protein component of a chaperone complex, and it is and exists in the meta or para form.

[0020] The chaperone complex of the present invention can be selected from heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), inhibitor of apoptosis protein (IAP), E3 ligase (for example, Carboxyl Terminus of HSC70-interacting Protein (CHIP), Homologous to E6AP C-Terminus domain-containing 3 (HECTD3), cullin 5 (CUL 5)) and other cofactors or cochaperones.

[0021] TB is the second part capable of binding to a target protein or a protein, and is selected from and any one of them, preferably

[0022] The target protein of the present invention can be selected from targeted drugs such as crizotinib, savolitinib, cabozantinib, capmatinib, tepotinib, etc.

[0023] L is a linker that binds a first moiety (CB) capable of binding to a chaperone protein or a protein component of a chaperone complex and a second moiety (TB) capable of binding to a target protein or a protein, and is selected from

[0024] any one of them, preferably

[0025] In the present invention, the linker is non-degradable in vivo and does not interfere with the ability of the chaperone binding substance and the target binding substance that are respectively bound thereto.

[0026] The present invention can prepare a compound represented by Chemical Formula I having a structure as shown in Table 1 below.

[0027] Table 1

[0028]

[0029]

[0030] Moreover, the present invention provides a method for preparing the compound represented by the above Chemical Formula I.

[0031] In the present invention, the method for preparing the compound represented by Chemical Formula I includes: Step i), preparing a first moiety (CB) capable of binding to a chaperone protein or a protein component of a chaperone complex, especially an acidic intermediate CB in the meta or para form; Step ii), preparing an L-TB intermediate by binding a second moiety (TB) capable of binding to a target protein or a protein to a linker; and Step iii), coupling the CB intermediate prepared in Step i) with the L-TB intermediate prepared in Step ii) to prepare a compound CB-L-TB represented by Chemical Formula I.

[0032] In the step (i), the compound (c) of Formula 3 is prepared by reacting the compound (a) of the following Formula I with the compound (b) of Formula 2, and then hydration is carried out to prepare the compound (d) of Formula 4 as an intermediate, or the compound (h) of Formula 6 is prepared by reacting the compound (g) of the following Formula 5 with the compound (b) of Formula 2, and then hydration is carried out to prepare the compound (i) of Formula 7 as an intermediate.

[0033] Formula I:

[0034]

[0035] Formula 2:

[0036]

[0037] Formula 3:

[0038]

[0039] Formula 4:

[0040]

[0041] Formula 5:

[0042]

[0043] Formula 6:

[0044]

[0045] Formula 7:

[0046]

[0047] In the step (ii), by using the

[0048] as the targeted drug and

[0049] as the linker to react to prepare L-TB as an intermediate of the combination of the targeted drug and the linker.

[0050] Step (iii) includes the step of preparing the compound CB-L-TB shown in Chemical Formula I by coupling the compound (d) of Chemical Formula 4 as an intermediate prepared in step (i) or the compound (i) of Chemical Formula 7 as an intermediate with L-TB prepared in step (ii).

[0051] Moreover, the present invention provides a use of the compound shown in the Chemical Formula I.

[0052] The compounds of the present invention represented by Chemical Formula I are generally used in anti-cancer therapies, and thus the diseases treated by the compounds of the present invention are cancers. The terms "cancer" or "tumor" are well-known in the art and, for example, indicate the presence in a subject of cells having typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, reduced apoptosis / death, and specific characteristic morphological features. Cancer cells generally exist in the form of solid tumors. However, cancer also includes non-solid tumors, such as hematological tumors, such as leukemia, in which the cancer cells originate from the bone marrow. The term "cancer" as used in the present invention includes pre-cancerous lesions and malignant tumors. Cancers include acoustic neuroma, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelogenous (granulocytic) leukemia, chronic myeloid leukemia, colorectal cancer, colon cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, dysplastic changes (dysplasia and metaplasias), embryonal carcinoma, endometrial cancer, endothelial sarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, liver tumor, hepatocellular carcinoma, hormone-independent prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin and non-Hodgkin), malignant and hyperplastic diseases of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin and uterus, lymphatic system malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, solid tumors (cancers and sarcomas), small cell lung cancer, stomach cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, Waldenström's macroglobulinemia, testicular tumor, uterine cancer and Wilms' tumor, but are not limited thereto.Other cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, small intestine cancer, urinary tract cancer, kidney cancer, urothelial cancer, female genital cancer, uterine cancer, gestational trophoblastic disease, male genital cancer, seminal vesicle cancer, testicular cancer, germ cell tumor, endocrine tumor, thyroid cancer, adrenal cancer, pituitary cancer, hemangioma, bone and soft tissue sarcoma, Kaposi's sarcoma, nerve cancer, eye cancer, meningioma, glioblastoma, neuroma, neuroblastoma, Schwannomas, solid tumors derived from hematopoietic malignancies such as leukemia, metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumor, colon cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small cell lung cancer, malignant glioma, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine cancer, refractory malignancy, triple-negative breast cancer, HER2-amplified breast cancer, nasopharyngeal cancer, oral cancer, biliary tract cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN), non-medullary thyroid cancer, recurrent glioblastoma multiforme, neurofibroma type 1, central nervous system (CNS) cancer, liposarcoma, leiomyosarcoma, salivary gland cancer, mucosal melanoma, acral / lentiginous melanoma, paraganglioma, pheochromocytoma, advanced metastatic cancer, solid tumor, triple-negative breast cancer, colon cancer, sarcoma, melanoma, kidney cancer, endometrial cancer, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumor, mantle cell lymphoma, and refractory malignancy.

[0053] The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods. When formulating, it is prepared by using common diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the compound with one or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. And, in addition to simple excipients, lubricants such as magnesium stearate, talc powder, etc. are also used. Liquid preparations for oral administration include suspensions, internal liquids, emulsions, syrups, etc., and in addition to common simple diluents such as water or liquid paraffin, various excipients can be included, for example, wetting agents, sweeteners, flavoring agents, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate can be used as non-aqueous solvents, suspensions. Semi-synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, glycerogelatin, etc. can be used as the matrix of suppositories. All modes of administration can be envisaged, for example, it can be injected orally, rectally or intravenously, intramuscularly, subcutaneously, by bronchial inhalation, intrauterinely, intrathecally or intracerebrally.

[0054] Moreover, the dosage of the pharmaceutical composition of the present invention can be increased or decreased according to the administration route, the severity of the disease, gender, body weight, age. The specific dosage and treatment regimen for a particular patient will vary depending on various factors including the activity of the specific compound used, age, body weight, general health status, gender, diet therapy, administration time, excretion rate, drug combination and the judgment of the attending physician and the severity of the specific disease to be treated. Therefore, the dosage does not limit the scope of the present invention in any way.

[0055] Effects of the Invention

[0056] The compounds of the present invention exhibit the effect of inducing the degradation of MET protein based on CMPD technology. Therefore, it can be expected that the compounds of the present invention have excellent anti-cancer effects that can overcome the drug resistance and mutations of targeted anti-cancer agents through the complete degradation of the target protein (MET). Description of the Drawings

[0057] Figure 1 Shows the trend of protein degradation of the compound OZD-MET 01 of the present invention over time in H596 cells.

[0058] Figure 2Shows the dose-dependent trend of protein degradation by the compound OZD-MET 01 of the present invention in H596 cells.

[0059] Figure 3 Shows the time-dependent trend of protein degradation by the compound OZD-MET 01 of the present invention in H1437 cells.

[0060] Figure 4 Shows the dose-dependent trend of protein degradation by the compound OZD-MET 01 of the present invention in H1437 cells.

[0061] Figure 5 Shows the trend of target protein degradation after 72 hours by the compounds OZD-MET 01 to 11 of the present invention at a dose of 5 μM in H596 cells.

[0062] Figure 6 Shows the trend of target protein degradation after 72 hours by the compounds OZD-MET 01 to 11 of the present invention at a dose of 5 μM in H1437 cells.

[0063] Figure 7 Shows the reduction and recovery of the expression of the target protein by the compound OZD-MET 01 of the present invention in H596 cells.

[0064] Figure 8 Shows the reduction and recovery of the expression of the target protein by the compound OZD-MET 01 of the present invention in H1437 cells.

[0065] Figure 9 Shows the reduction and recovery of the expression of the target protein by the compound OZD-MET 02 of the present invention in H596 cells.

[0066] Figure 10 Shows the reduction and recovery of the expression of the target protein by the compound OZD-MET 02 of the present invention in H1437 cells.

[0067] Figure 11 Shows the reduction and recovery of the expression of the target protein by the compound OZD-MET 03 of the present invention in H596 cells.

[0068] Figure 12 Shows the reduction and recovery of the expression of the target protein by the compound OZD-MET 03 of the present invention in H1437 cells. Detailed implementation mode

[0069] Hereinafter, the present invention will be described in more detail by way of examples and experimental examples. However, these examples and experimental examples are used to illustrate the present invention exemplarily, and the scope of the present invention is not limited to these examples and experimental examples.

[0070] Example 1: Preparation of 2-{2-[(3-{[1-(2,4-dihydroxy-5-isopropylphenyl)-N-methylformamido]methyl}phenyl)formamido]ethoxy}ethyl 4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}pyrazol-1-yl)piperidine-1-carboxylate (hereinafter referred to as "OZD-MET 01")

[0071]

[0072] 1) Preparation of Intermediate Compound (d) (meta)

[0073] Dissolve methyl 3-formylbenzoate in methanol, add methylamine, then add sodium borohydride at 0 °C and stir. After all the starting materials have reacted, remove methanol under reduced pressure. Dissolve the residue in methylene chloride, wash with a small amount of brine (saturated sodium chloride solution), dry over MgSO4, filter, and remove the solvent under reduced pressure to obtain the following compound (a), which is immediately used in the next reaction.

[0074] Dissolve 2,4-dihydroxy-5-isopropylbenzoate in methanol:tetrahydrofuran (THF):H2O (1:1:1), then add lithium hydroxide and react for 12 hours. After all the reactions are completed, filter through celite 545 and remove the solvent from the filtrate under reduced pressure. Add H2O to dissolve the residue, then acidify with hydrochloric acid and extract twice with ethyl acetate (EA), wash with brine, dry over MgSO4, filter, and remove the solvent under reduced pressure to obtain the following compound (b) with a yield of 97%.

[0075] Dissolve compound (a) and compound (b) in dimethylformamide, and add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIPEA). Stir at room temperature for 12 hours to terminate the reaction. Add sodium bicarbonate solution (NaHCO3(aq.)) to the solution and extract twice with EA. Wash with brine, dry over MgSO4, filter, remove the solvent under reduced pressure, and separate by silica gel column chromatography to obtain a transparent liquid compound (c) with a yield of 86%.

[0076] Dissolve compound (c) in methanol:THF:H2O (1:1:1), then add lithium hydroxide and react at room temperature for 15 hours. Remove the solvent, add H2O to dissolve the residue, acidify with hydrochloric acid, extract with EA, wash with brine, add MgSO4 for drying, and obtain a solid compound (d) with a yield of 99%.

[0077] 1 H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 2.81 (s, 3H), 3.19 (m, 1H), 4.39 (t, 2H), 6.41 (s, 1H), 7.40 - 7.61 (m, 3H), 8.19 (m, 1H), 8.46 (m, 1H), 10.07 (s, 1H)

[0078]

[0079] 2) Preparation of Crizotinib-Linker Intermediate Compound (f)

[0080] Dissolve carbonyldiimidazole (CDI) in dichloromethane. After the temperature reaches 0 °C, add (R)-crizotinib and 2-[2-(hydroxyethoxy)ethyl]-1H-isoindole-1,3(2H)-dione, and then react for 10 hours. After all the starting materials have reacted, remove dichloromethane to obtain the following compound (e).

[0081] Dissolve compound (e) in ethanol, then add hydrazine hydrate and reflux at 110 °C for 1 hour. Then stir at room temperature and filter the resulting white solid to obtain the following compound (f) with a yield of 66%.

[0082] 11H NMR (500 MHz, DMSO) δ 1.51 - 1.53 (m, 5H), 2.00 (m, 2H), 2.47 (m, 2H) 2.66 (t, 1H), 3.06 - 3.17 (m, 3H), 3.37 (t, 1H), 3.56 - 3.70 (m, 4H), 3.98 (t, 1H), 4.14 - 4.27 (m, 2H), 4.42 (t, 1H), 4.82 (q, 1H), 5.46 (s, 2H), 7.05 (q, 1H), 7.29 (q, 1H), 7.52 - 7.69 (m, 2H), 7.83 (d, 1H), 8.13 (d, 1H)

[0083]

[0084] 3) Preparation of Compound OZD-MET 01

[0085] Compound (f) and compound (d) were dissolved in DMF, and EDC, HOBT, and DIPEA were added for reaction. NaHCO3(aq.) was added to the solution, and it was extracted twice with EA, washed with brine, dried over MgSO4, filtered, the solvent was removed under reduced pressure, and separated by silica gel column to obtain the white solid compound OZD-MET 01 with a yield of 75%.

[0086] 1 1H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 1.51 (d, 3H), 1.99 (m, 2H), 2.45 (m, 2H), 2.81 (s, 3H), 3.15 - 3.24 (m, 4H), 3.45 - 3.59 (m, 3H), 3.64 - 3.70 (m, 3H), 3.98 (t, 1H), 4.14 (t, 1H), 4.26 - 4.42 (m, 3H), 4.73 - 4.74 (m, 2H), 5.46 (s, 2H), 6.41 (s, 1H), 7.05 - 7.20 (m, 2H), 7.40 - 7.47 (m, 4H), 7.65 - 7.69 (m, 2H), 7.83 - 7.97 (m, 2H), 8.51 - 8.53 (m, 2H), 10.07 (s, 1H)

[0087]

[0088] Example 2: 2-{2-[(4-{[1-(2,4-Dihydroxy-5-isopropylphenyl)-N-methylformamido]methyl}benz yl)formamido]ethoxy}ethyl-4-(4-{6-Amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3- yl}pyrazol-1-yl)piperidine-1-carboxylate (hereinafter referred to as "Compound OZD-MET 02") Preparation

[0089]

[0090] 1) Preparation of Intermediate Compound (i) (para)

[0091] Methyl 4-formylbenzoate was dissolved in methanol and methylamine was added. Then, sodium borohydride was added at a temperature of 0 °C and the mixture was stirred. After all the starting materials had reacted, methanol was removed under reduced pressure. The residue was dissolved in dichloromethane, washed with H2O (brine), dried over MgSO4, filtered, and the solvent was removed under reduced pressure to obtain the following compound (g).

[0092]

[0093] Compound (g) and compound (b) of Example 1 were dissolved in dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 1-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIPEA) were added, and the mixture was stirred at room temperature for 12 hours to terminate the reaction. NaHCO3(aq.) was added to the solution, and the mixture was extracted twice with EA, washed with brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure to obtain compound (e). The compound was separated by silica gel column chromatography to obtain a transparent liquid compound (h) with a yield of 52%.

[0094]

[0095] Compound (h) was dissolved in methanol:THF:H2O (1:1:1), then lithium hydroxide was added and the mixture was reacted at room temperature for 15 hours. The solvent was removed, H2O was added to dissolve the residue, and the solution was acidified with HCl, extracted with EA, washed with brine, and dried over MgSO4 to obtain a foam solid compound (i) with a yield close to 100%.

[0096] 1 H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 2.81 (s, 3H), 3.19 (m, 1H), 4.42 (t, 2H), 6.41 (s, 1H), 7.42 - 7.61 (m, 3H), 8.02 (d, 2H), 10.07 (s, 1H)

[0097]

[0098] 2) Preparation of Compound OZD-MET 02

[0099] Dissolve the compound (i) and the compound (f) prepared in 2) of Example 1 in DMF, add EDC, EBOT, and DIPEA for reaction. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, separate it by silica gel column to obtain a light yellow solid compound OZD-MET 02 with a yield of 44%.

[0100] 1 H NMR(500MHz,DMSO)δ1.24(d,6H),1.51(d,3H),1.99(m,2H),2.45(m,2H),2.81(s,3H),3.16-3.24(m,4H),3.45-3.59(m,3H),3.64-3.70(m,3H),3.98(t,1H),4.14(t,1H),4.26-4.42(m,3H),4.71-4.74(m,2H),5.46(s,2H),6.41(s,1H),7.05-7.20(m,2H),7.42-7.47(m,4H),7.58-7.65(m,2H),7.83(d,2H),8.05(d,2H),8.51(d,1H),10.07(s,1H)

[0101] Example 3: N-{[4-({5-[4-(4-{6-Amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin- 3-yl}pyrazol-1-yl)piperidin-1-yl]-5-oxopentyl}aminocarbonyl)phenyl]methyl}-2,4-dihydroxy-5-isoprop yl-N-methylbenzamide (hereinafter referred to as "Compound OZD-MET 03") Preparation

[0102]

[0103] 1) Preparation of Crizotinib-Linker Intermediate Compound (j)

[0104] Dissolve (R)-crizotinib and 5-aminovaleric acid in DMF, add EDC and HOB T for reaction. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a crude compound (j) with a yield of 28%.

[0105] 11H NMR (500 MHz, DMSO) δ 1.36 - 1.39 (m, 3H), 1.51 - 1.61 (m, 5H), 1.84 (m, 1H), 1.98 - 1.99 (m, 3H), 2.40 - 2.53 (m, 4H), 3.09 (q, 2H), 3.53 (0t, 2H), 4.29 (m, 1H), 4.77 (q, 1H), 5.46 (s, 2H), 7.05 (q, 1H), 7.30 (q, 1H), 7.51 - 7.61 (m, 2H), 7.83 (d, 1H), 8.13 (d, 1H)

[0106]

[0107] 2) Preparation of Compound OZD-MET 03

[0108] Dissolve the compound (j) and the compound (i) prepared in 1) of Example 2 in DMF, add EDC and HOBT for reaction. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with Brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, separate it by a Silica gel column to obtain a yellow solid compound OZD - MET 03 with a yield of 63%.

[0109] 1 1H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 1.40 (m, 1H), 1.51 (d, 3H), 1.68 (m, 2H), 1.90 - 1.98 (m, 4H), 2.44 - 2.53 (m, 3H), 2.81 (s, 3H), 3.08 (m, 2H), 3.13 - 3.19 (m, 2H), 3.47 - 3.52 (m, 3H), 4.28 - 4.32 (m, 2H), 4.68 - 4.75 (m, 2H), 5.46 (s, 2H), 6.41 (s, 1H), 6.77 (s, 1H), 7.05 (q, 1H), 7.21 (q, 1H), 7.39 - 7.44 (m, 4H), 7.65 - 7.83 (m, 2H), 8.05 (d, 2H), 8.51 (d, 1H), 10.07 (s, 1H)

[0110] Example 4: 2-{2-[(3-{[1-[2,4-Dihydroxy-5-isopropylphenyl]-N-methylformamido]methyl}benz yl)formamido]ethoxy}ethyl-4-[3-(1-{Imidazo[1,2-a]pyridin-6-yl}ethyl)-[1,2,3]triazolo[4, 5-b]pyrazin-5-yl]pyrazole-1-carboxylate (hereinafter referred to as "Compound OZD-MET 04") Preparation

[0111]

[0112] 1) Preparation of Savolitinib-Linker Intermediate Compound (k)

[0113] Use (R)-savolitinib instead of (R)-crizotinib, and prepare the savolitinib-linker intermediate compound (k) with the following structure by the same method as in 2) of Example 1 (yield: 33%).

[0114] 1 H NMR (500 MHz, DMSO) δ 1.53 (s, 2H), 2.11 (d, 3H), 2.66 (t, 1H) 3.06 (t, 1H), 3.37 (t, 1H), 3.56 (t, 1H), 3.70 (t, 1H), 3.98 - 4.14 (m, 2H), 4.42 (t, 1H), 6.01 (m, 1H), 7.10 (d, 1H), 8.00 - 8.25 (m, 5H), 9.37 (m, 2H)

[0115]

[0116] 2) Preparation of Compound OZD-MET 04

[0117] Dissolve compound (k) and compound (d) prepared in 1) of Example 1 in DMF, add EDC, HOBT, and DIPEA for reaction. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, and separate it by silica gel column to obtain the solid compound OZD-MET 04 with a yield of 37%.

[0118] 1 H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 2.10 (d, 3H), 2.81 (s, 3H), 3.19 (m, 1H), 3.41 - 3.45 (m, 2H), 3.64 - 3.70 (m, 2H), 3.98 - 4.14 (m, 2H), 4.42 - 4.46 (m, 2H), 4.89 (t, 1H), 6.00 (m, 1H), 6.41 (s, 1H), 7.40 - 7.42 (m, 3H), 7.69 - 7.85 (m, 2H), 7.97 - 8.17 (m, 5H), 8.57 (m, 2H), 9.37 - 9.44 (m, 2H), 10.07 (s, 1H)

[0119] Example 5: 2,4-Dihydroxy-N-({4-[(5-{4-[3-(1-{Imidazo[1,2-a]pyridin-6-yl}ethyl)-[1, 2,3]triazolo[4,5-b]pyrazin-5-yl]pyrazol-1-yl}-5-oxopentyl)aminocarbonyl]phenyl}methyl)-5-isoprop yl-N-methylbenzamide (hereinafter referred to as "Compound OZD-MET 05") Preparation

[0120]

[0121] 1)Preparation of Savolitinib-Linker Intermediate Compound (l)

[0122] Use (R)-savolitinib instead of (R)-crizotinib and prepare the savolitinib-linker intermediate compound (l) with the following structure by the same method as in 1) of Example 3 (yield: 39%).

[0123] 1 H NMR (500 MHz, DMSO) δ 1.36 - 1.39 (m, 3H), 1.59 (m, 1H), 1.91 (m, 1H), 2.14 - 2.15 (m, 4H), 2.40 (t, 1H), 2.71 - 2.79 (m, 2H), 3.27 (t, 1H), 6.05 (m, 1H), 7.11 (d, 1H), 7.96 (d, 1H), 8.03 - 8.16 (m, 4H), 9.33 - 9.37 (m, 2H)

[0124]

[0125] 2) Preparation of Compound OZD-MET 05

[0126] Dissolve compound (l) and compound (i) prepared in 1) of Example 2 in DMF, add EDC and HOBT and react. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, and separate it by silica gel column to obtain the white solid compound OZD-MET05 with a yield of 29%.

[0127] 1 H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 1.39 (m, 1H), 1.70 (m, 1H), 1.97 - 2.20 (m, 5H), 2.71 (t, 1H), 2.81 (s, 3H), 3.13 - 3.27 (m, 3H), 3.47 (t, 1H), 4.48 (t, 1H), 4.91 (t, 1H), 6.03 (m, 1H), 6.41 (s, 1H), 6.77 (s, 1H), 7.42 - 7.68 (m, 4H), 8.02 - 8.17 (m, 6H), 8.54 (d, 1H), 9.37 - 9.39 (m, 2H), 10.01 (s, 1H)

[0128] Example 6: 2-{2-[(3-{[1-(2,4-Dihydroxy-5-isopropylphenyl)-N-methylformamido]methyl}benz yl)formamido]ethoxy}ethyl-N-[4-(4-{1-[(4-Fluorophenyl)aminocarbonyl]cyclopropanecarboxamido}phenoxy)quin olin-6-yl)carbamate (hereinafter referred to as "Compound OZ D-MET 06") Preparation

[0129]

[0130] 1) Preparation of Cabozantinib-Linker Intermediate Compound (m)

[0131] Use cabozantinib instead of (R)-crizotinib, and prepare the cabozantinib-linker intermediate compound (m) with the following structure by the same method as in 2) of Example 1 (yield: 56%).

[0132] 1 H NMR (500 MHz, DMSO) δ 1.49 - 1.53 (m, 4H), 1.72 (q, 2H), 2.86 (t, 2H), 3.46 (t, 2H), 3.84 (t, 2H), 4.28 (t, 2H), 6.65 (d, 1H), 6.95 (d, 2H), 7.20 - 7.45 (m, 5H), 7.71 (d, 1H), 8.20 (m, 3H), 8.59 (s, 1H), 8.82 (d, 1H), 9.83 (s, 1H), 9.92 (s, 1H)

[0133]

[0134] 2) Preparation of Compound OZD-MET 06

[0135] Dissolve compound (m) and compound (d) prepared in 1) of Example 1 in DMF, add EDC and HOBT and react. Add NaHCO3(aq.) to the solution, extract 3 times with EA, wash with brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, and separate it by silica gel column to obtain a dark yellow solid compound OZD-MET 06 with a yield of 42%.

[0136] 1 H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 1.48 (q, 2H), 1.71 (1, 2H), 2.81 (s, 3H), 3.19 - 3.25 (m, 3H), 3.55 (5, 2H), 3.84 (t, 2H), 4.28 - 4.32 (m, 4H), 6.41 (s, 1H), 6.65 (d, 1H), 6.95 (d, 2H), 7.20 - 7.33 (m, 4H), 7.40 - 7.45 (m, 4H), 7.64 - 7.71 (m, 2H), 7.97 (m, 1H), 8.20 - 8.27 (m, 3H), 8.44 - 8.59 (m, 2H), 8.82 (d, 1H), 9.43 - 9.52 (m, 2H), 10.07 (s, 1H)

[0137] Example 7: N′1-{4-[(6-{5-[(4-{[1-(2,4-Dihydroxy-5-isopropylphenyl)-N-methylformamide{[1-(2,4-dihydroxy-5-isopropylphenyl)-N-methylformamido]methyl}phenyl)-N1-(4-fluorophenyl)cyclopropane-1,1- Preparation of 2-{2-[(3-{[1-(2,4-dihydroxy-5-isopropylphenyl)-N-methylformamido]methyl}phenyl)formamido]ethoxy}ethyl-N-{2-fluoro-4-[7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl]phenyl}carbamate (hereinafter referred to as "Compound OZD-MET07")

[0138]

[0139] 1) Preparation of cabozantinib-linker intermediate compound (n)

[0140] Use cabozantinib instead of (R)-crizotinib, and prepare the cabozantinib-linker intermediate compound (n) with the following structure by the same method as in 1) of Example 3 (yield: 63%).

[0141] 1 H NMR (500 MHz, DMSO) δ 1.39 (s, 2H), 1.47 - 1.57 (m, 6H), 1.76 (q, 2H), 2.29 (t, 2H), 2.59 (t, 2H), 6.65 (d, 1H), 6.95 (d, 2H), 7.20 - 7.45 (m, 5H), 7.71 (d, 1H), 8.04 - 8.20 (m, 3H), 8.49 (s, 1H), 8.82 (d, 1H), 10.05 (s, 1H), 10.14 (s, 1H)

[0142]

[0143] 5) Preparation of Compound OZD-MET 07

[0144] Dissolve compound (n) and compound (i) prepared in 1) of Example 2 in DMF, add EDC and HOBT and react. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, and separate it by silica gel column to obtain the solid compound OZD-MET 07 with a yield of 54%.

[0145] 1 H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 1.51 - 1.54 (m, 4H), 1.63 - 1.74 (m, 4H), 2.29 (t, 2H), 2.81 (s, 3H), 3.19 - 3.30 (m, 3H), 4.34 (t, 2H), 6.41 (s, 1H), 6.65 - 6.77 (m, 2H), 6.95 (d, 2H), 7.20 (q, 2H), 7.33 - 7.45 (m, 5H), 7.63 - 7.71 (m, 2H), 8.05 - 8.20 (m, 6H), 8.82 (d, 1H), 9.65 (s, 1H), 9.75 (s, 1H), 10.07 (s, 1H)

[0146] Example 8: 2-{2-[(3-{[1-(2,4-dihydroxy-5-isopropylphenyl)-N-methylformamido]methyl} phenyl)formamido]ethoxy}ethyl-N-{2-fluoro-4-[7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin- 2-yl]phenyl}carbamate (hereinafter referred to as "Compound OZ D-MET 08")

[0147]

[0148] 1) Preparation of capmatinib-linker intermediate compound (o)

[0149] Use capmatinib instead of (R)-crizotinib, and prepare the capmatinib-linker intermediate compound (o) with the following structure in the same method as 2) of Example 1 (yield: 58%).

[0150] 1 H NMR (500 MHz, DMSO) δ 1.53 (s, 2H), 2.86 (t, 2H), 3.46 (t, 2H), 3.84 (t, 2H), 4.24 - 4.28 (m, 4H), 7.19 (m, 1H), 7.39 - 7.62 (m, 4H), 7.91 - 8.04 (m, 2H), 8.20 - 8.39 (m, 3H), 8.87 (q, 1H), 9.19 (s, 1H)

[0151]

[0152] 2) Preparation of Compound OZD-MET 08

[0153] Dissolve compound (o) and compound (d) prepared in 1) of Example 1 in DMF, add EDC and HOBT for reaction. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with brine, dry with MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, and separate it by silica gel column to obtain the solid compound OZD-MET 08 with a yield of 55%.

[0154] 11H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 2.81 (s, 3H), 3.19 (m, 1H), 3.46 - 3.55 (m, 4H), 3.84 (t, 2H), 4.22 - 4.28 (m, 4H), 4.53 (t, 2H), 6.41 (s, 1H), 7.19 (m, 1H), 7.40 - 7.42 (m, 3H), 7.52 - 7.57 (m, 2H), 7.62 - 7.72 (m, 4H), 7.97 (m, 1H), 8.12 - 8.21 (m, 2H), 8.34 (t, 1H), 8.44 - 8.46 (m, 2H), 8.87 (q, 1H), 9.01 (s, 1H), 10.01 (s, 1H)

[0155] Example 9: Preparation of N-[(4-{[4-({2-fluoro-4-[7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]tri azin-2-yl]phenyl}carbamoyl)butyl]carbamoyl}phenyl)methyl]-2,4-dihydroxy-5-isopropyl-N-methyl benzamide (hereinafter referred to as "Compound OZD-MET 09")

[0156]

[0157] 1) Preparation of capmatinib-linker intermediate compound (p)

[0158] Camatinib was used instead of (R)-crizotinib, and the camatinib-linker intermediate compound (p) with the following structure was prepared by the same method as in 1) of Example 3 (yield 60%).

[0159] 1 1H NMR (500 MHz, DMSO) δ 1.39 (s, 2H), 1.47 (m, 2H), 1.77 (m, 2H), 2.29 (t, 2H), 2.59 (t, 2H), 4.27 (q, 2H), 7.19 (m, 1H), 7.39 - 7.44 (m, 3H), 7.62 (d, 1H), 7.91 (q, 1H), 8.21 - 8.40 (m, 3H), 8.64 (s, 1H), 8.87 (q, 1H), 9.19 (s, 1H)

[0160]

[0161] 2) Preparation of Compound OZD-MET 09

[0162] Compound (p) and compound (i) prepared in 1) of Example 2 were dissolved in DMF, and EDC and HOBT were added for reaction. NaHCO3(aq.) was added to the solution, and it was extracted 3 times with EA, washed with brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure to obtain a compound, which was separated by a silica gel column to obtain the white solid compound OZD-MET09 with a yield of 63%.

[0163] 1 1H NMR (500 MHz, DMSO) δ 1.24 (d, 6H), 1.54 (m, 2H), 1.84 (m, 2H), 2.29 (t, 2H), 2.81 (s, 3H), 3.19 - 3.30 (m, 3H), 4.26 (q, 2H), 4.55 (t, 2H), 6.41 (s, 1H), 6.77 (s, 1H), 7.19 (m, 1H), 7.42 - 7.44 (m, 4H), 7.62 - 7.63 (m, 2H), 8.05 - 8.12 (m, 3H), 8.21 - 8.25 (m, 2H), 8.41 - 8.54 (m, 2H), 8.87 (q, 1H), 9.01 (s, 1H), 10.07 (s, 1H)

[0164] Example 10: 2-{2-[(3-{[1-(2,4-dihydroxy-5-isopropylphenyl)-N-methylformamido]methyl} phenyl)formamido]ethoxy}ethyl-4-({[2-(3-{[3-(3-cyanophenyl)-6-oxopyridazin-1-yl]methyl}benz yl)pyrimidin-5-yl]oxy}methyl)piperidine-1-carboxylate (hereinafter referred to as "Compound OZD-MET 10")

[0165]

[0166] 1) Preparation of tepotinib-linker intermediate compound (q)

[0167] Use tepotinib instead of (R)-crizotinib, and prepare the tepotinib-linker intermediate compound (q) with the following structure in the same method as in 2) of Example 1 (yield: 76%).

[0168] 1 1H NMR (500 MHz, DMSO) δ 1.53 (s, 2H), 1.65 (m, 2H), 2.04 (m, 3H), 2.86 (t, 2H), 3.05 (q, 2H), 3.46 - 3.49 (m, 4H), 3.84 (t, 2H), 4.07 (d, 2H), 4.28 (t, 2H), 5.64 (t, 2H), 7.29 - 7.42 (m, 4H), 7.70 - 7.77 (m, 2H), 8.13 - 8.20 (m, 3H), 8.63 (s, 2H), 8.93 (d, 1H)

[0169]

[0170] 2) Preparation of Compound OZD-MET 10

[0171] Dissolve compound (q) and compound (d) prepared in 1) of Example 1 in DMF, add EDC and HOBT and react. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with Brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, and separate it by a silica gel column to obtain a white compound OZD-MET 10 with a yield of 78%.

[0172] 1 H NMR(500MHz,DMSO)δ1.24(d,6H),1.63(m,2H),2.03(m,3H),2.81(s,3H),3.03 - 3.19(m,3H),3.45 - 3.47(m,4H),3.55(t,2H),3.84(t,2H),4.08 - 4.28(m,4H),4.53(t,2H),5.63(t,2H),6.41(s,1H),7.01(m,1H),7.29 - 7.42(m,5H),7.65 - 7.77(m,4H),7.97(m,1H),8.20(m,1H),8.34 - 8.45(m,3H),8.63(s,2H),10.07(s,1H)

[0173] Example 11: N-{[4-({5-[4-({[2-(3-{[3-(3-cyanophenyl)-6-oxopyridazin-1-yl]methyl} phenyl)pyrimidin-5-yl]oxy}methyl)piperidin-1-yl]-5-oxopentyl}carbamoyl)phenyl]methyl}-2,4-dihydro xy-5-isopropyl-N-methylbenzamide (hereinafter referred to as "Compound OZD-MET 11")

[0174]

[0175] 1) Preparation of tepotinib-linker intermediate compound (r)

[0176] Use tepotinib instead of (R)-crizotinib, and prepare the tepotinib-linker intermediate compound (r) with the following structure in the same method as in 1) of Example 3 (yield: 74%).

[0177] 1 H NMR(500MHz,DMSO)δ1.39(s,2H),1.47(m,2H),1.63 - 1.74(m,4H),2.03 - 2.26(m,5H),2.59(t,2H),2.97(q,2H),3.41(t,2H),4.07(d,2H),5.68(t,2H),7.29 - 7.42(m,4H),7.70 - 7.77(m,2H),8.13

[0178] -8.20(m,3H),8.63(s,2H),8.93(d,1H)

[0179]

[0180] 2) Preparation of Compound OZD-MET 11

[0181] Dissolve compound (r) and compound (i) prepared in 1) of Example 2 in DMF, add EDC and HOBT and react. Add NaHCO3(aq.) to the solution, extract twice with EA, wash with Brine, dry over MgSO4, filter, remove the solvent under reduced pressure to obtain a compound, separate it by silica gel column to obtain the yellow solid compound OZD-MET11 with a yield of 79%.

[0182] 1 H NMR(500MHz,DMSO)δ1.24(d,6H),1.54 - 1.62(m,4H),1.80(m,2H),2.01 - 2.05(m,3H),2.26(t,2H),2.96(q,2H),3.19(m,1H),3.30 - 3.40(m,4H),4.08(d,2H),4.55(t,2H),5.67(t,2H),6.41(s,1H),6.77(s,1H),7.01(m,1H),7.29 - 7.42(m,5H),7.65 - 7.77(m,3H),8.05(d,2H),8.20 - 8.40(m,3H),9.43(d,1H),10.07(s,1H)

[0183] Experimental Example 1: Cell viability effect

[0184] In this experiment, after treating cells with each corresponding compound prepared in Examples 1 to 11 respectively, the changes in cytotoxicity and cell number were confirmed. By confirming the change in cell number, the IC 50 value can be obtained, and the inhibitory effect of the compounds of the present invention on cancer cell growth and the anti-cancer effect can be evaluated.

[0185] Experimental method: Cell viability confirmation test

[0186] Seed cells (H596 or H1437 cells) at 3X10 per well 3Cells were seeded in a 96-well plate and then cultured for more than 12 hours. After removing the medium, 200 μl of each compound prepared in Examples 1 to 11 was added to each well. After 3 days, the medium was removed, 10 μl of CCK8 reagent + 90 μl of medium was dispensed into each well, and after reacting for 3 to 4 hours, the absorbance at a wavelength of 450 nM was measured using an ELISA microplate reader. The IC 50 value was obtained using the Graphpad prism program and is shown in Table 2.

[0187] As shown in Table 2, each compound prepared in Examples 1 to 11 exhibited a cell viability that was approximately 10 times higher than that of crizotinib as a control drug, the intermediate compound (d) of the first part bound to the molecular chaperone complex, and compound (i).

[0188] Therefore, compared with the compounds of the first part and the second part, the compounds of the present invention have very excellent anti-cancer efficacy.

[0189] Table 2

[0190]

[0191] Experimental Example 2: Confirmation of c-MET degradation effect

[0192] This experiment evaluated the degradation trend and degree of the target protein c-MET (total form) itself in cells treated with each compound prepared in Examples 1 to 11.

[0193] Experimental method: Target protein degradation confirmation test

[0194] Cells (H596 or H1437 cells) were seeded at 5x10 5 or 1x10 6Cells were seeded in 60-mm or 100-mm culture dishes and cultured for more than 12 hours. After removing the media, each compound prepared in Examples 1 to 11 was used to treat the cells under different conditions (different concentrations (0, 2.5, 5, 10 μM), different times (0, 6, 8, 24, 48, 72 hours), and the specified concentration and time of all compounds (5 μM, 72 hours)). Then, the cells were harvested and lysed using RIPA buffer, followed by centrifugation (4°C, 13,000 rpm, 15 min), and the supernatant was transferred to a new tube. Protein quantification was performed using the Bradford Assay, and then the samples for loading were prepared by boiling at 95°C, and Western blot was carried out. The samples were loaded onto an SDS polyacrylamide gel (8% concentration), electrophoresed at 80 V, and then switched to 100 - 120 V (when the loaded samples were transferred from the stacking gel to the running gel) for electrophoresis. The completed polyacrylamide gel was transferred to a membrane (after diluting the transfer buffer in a ratio of 10X transfer buffer:methanol:D.W = 1:2:7 and performing transfer at 100 V for 1 hour or 80 V for 2 hours in the cold state), the membrane was blocked with 5% skim milk for 30 minutes, and washed with phosphate-buffered saline with Tween 20 (PBS-T) buffer. The primary antibody was diluted in 5% BSA + NaN3 solution and reacted with the membrane for more than 12 hours, then washed with PBS-T, and reacted with 5% skim milk + secondary antibody for 1 hour 30 minutes. After washing with PBS-T, the ECL solution was reacted with the membrane, and the bands were detected using an imaging device to confirm the degradation of the bands corresponding to the target protein c-MET, which are shown in Table 3 and Figures 1 to 6 in.

[0195] As shown in Table 3 below, crizotinib, compound (d) of the first part that binds to the molecular chaperone complex, and compound (i) did not show degradation of the target protein MET. In contrast, each compound prepared in Examples 1 to 11 showed significant degradation of the target protein.

[0196] Therefore, the present invention has a very excellent degradation effect on the target protein.

[0197] Table 3

[0198]

[0199] Experimental Example 3: Degradation and recovery of target protein through the UPS pathway

[0200] In this experiment, each compound prepared in Examples 1 to 11 and bortezomib, a proteasome inhibitor, were used to simultaneously treat cells to evaluate the degradation and recovery of the target protein through the ubiquitin-proteasome system (UPS) pathway.

[0201] Experimental method: Target protein degradation and recovery confirmation test

[0202] H596 or H1437 cells were seeded at 3x10 5After inoculating cells into a 6-well culture plate, they were cultured for more than 12 hours. After changing the culture medium, the cells were treated with 100 nM bortezomib and 5 μM of each compound prepared in Examples 1 to 11. The control group was treated with an equal amount of dimethyl sulfoxide (DMSO) as the drug solvent. After 16 hours, the culture medium was removed, the cells were washed with phosphate buffered saline (PBS), and then the cells were harvested using trypsin-EDTA. Cell lysis was performed using radio-immuno precipitation assay buffer (RIPA buffer), followed by centrifugation (4 °C, 13,000 rpm, 15 min), and the supernatant was transferred to a new tube. Protein quantification was performed using the Bradford Assay, and then the samples were boiled at 95 °C for 10 minutes. The samples were loaded onto an 8% SDS (sodium dodecyl sulfate) polyacrylamide gel, and electrophoresis was performed at 80 V. When the protein migrated from the stacking gel to the running gel, the voltage was switched to 100 V in the cold state for Western blot for 60 minutes. The membrane was blocked with a 5% skim milk solution for more than 30 minutes and washed with phosphate-buffered saline with Tween 20 (PBS-T) buffer. The membrane was reacted with the primary antibody diluted with 5% bovine serum albumin (5% BSA) and NaN3 solution for more than 12 hours. After washing with PBS-T, the membrane was reacted with the secondary antibody diluted with a 5% skim milk solution for 1 hour and 30 minutes. After washing with PBS-T, the membrane was reacted with ECL solution, and the bands were confirmed using an imaging device. When treated with bortezomib, it was confirmed whether the reduced MET expression due to each compound prepared in Examples 1 to 11 was restored, and it is shown in Table 4 and Figures 7 to 12 in.

[0203] As shown in Table 4, crizotinib, intermediate compound (d) of the first part bound to the molecular chaperone complex, and compound (i) did not show degradation and restoration of the MET protein. In contrast, significant degradation of the target protein and restoration of the target protein upon treatment with bortezomib, a proteasome inhibitor, were exhibited in each of the compounds prepared in Examples 1 to 11.

[0204] Table 4

[0205]

Claims

1. A compound of the following chemical formula I or its stereoisomer, characterized in that, Chemical formula I: CB-L-TB, In the said formula, CB is T B is selected from and any one of them, L is selected from and any one of them 2. The compound or its stereoisomer shown by Chemical Formula I according to Claim 1, characterized in that, CB exists in the meta or para form.

3. The compound or its stereoisomer shown by Chemical Formula I according to Claim 1, characterized in that, TB is selected from and any one of them.

4. The compound or its stereoisomer shown in Chemical Formula I according to Claim 1, characterized in that, L is 5. A compound of Formula I, characterized in that, Selected from the group consisting of the following compounds:

6. A pharmaceutical composition for treating cancer, characterized in that, Comprising the compound of chemical formula I according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition for treating cancer according to claim 6, characterized in that, Cancers include acoustic neuroma, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia including monocytes, myeloblasts, adenocarcinoma, angiosarcoma, astrocytoma, acute myelomonocytic and promyelocytic leukemia, acute T-cell leukemia, basal cell carcinoma, cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia including granulocytes, chronic myelogenous leukemia, colorectal cancer, colon cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt lymphoma, dysplastic changes including dysplasia and metaplasia, embryonal carcinoma, endometrial cancer, endothelial sarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, liver tumor, hepatocellular carcinoma, hormone-independent prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphomas including Hodgkin and non-Hodgkin, malignant and hyperplastic diseases of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin and uterus, lymphoid system malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, solid tumors including cancer and sarcoma, small cell lung cancer, stomach cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, Waldenström macroglobulinemia, testicular tumor, uterine cancer and Wilms tumor, but are not limited thereto, and other cancers are selected from primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, small intestine cancer, urinary tract cancer, kidney cancer, urothelial cancer, female genital cancer, uterine cancer, gestational trophoblastic disease, male genital cancer, seminal vesicle cancer, testicular cancer, germ cell tumor, endocrine tumor, thyroid cancer, adrenal cancer, pituitary cancer, hemangioma, bone and soft tissue sarcoma, Kaposi sarcoma, nerve cancer, eye cancer, meningeal cancer, glioblastoma, neuroma, neuroblastoma, schwannoma, solid tumors derived from hematopoietic system malignancies including leukemia,Metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumor, colon cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small cell lung cancer, malignant glioma, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine cancer, refractory malignancies, triple-negative breast cancer, HER2-amplified breast cancer, nasopharyngeal carcinoma, oral cancer, biliary tract cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, non-medullary thyroid cancer, recurrent glioblastoma multiforme, neurofibroma type 1, central nervous system cancer, liposarcoma, leiomyosarcoma, salivary gland cancer, mucosal melanoma, acral / lentiginous melanoma, paraganglioma, pheochromocytoma, advanced metastatic cancer, solid tumors, triple-negative breast cancer, colon cancer, sarcoma, melanoma, renal cancer, endometrial cancer, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumor, mantle cell lymphoma, and refractory malignancies.

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