Novel compounds that bind to MDM2 protein and composition for preventing or treating hyper proliferative disorder containing the same

Abstract

The present invention relates to a novel compound that binds to MDM2 (Mouse Double Minute 2 homolog) protein, a composition for inhibiting or degrading MDM2 protein activity using the same, and a composition for preventing or treating hyperproliferative disorders. The novel compound according to the present invention is a low-molecular-weight compound that effectively binds to the MDM2 protein and induces the degradation of the MDM2 protein, thereby having the advantage of exhibiting excellent inhibition and regulation of the activity of the MDM2 protein. In addition, the present invention can be utilized as a pharmaceutical composition for the prevention or treatment of abnormal proliferative diseases using the novel compound, and the pharmaceutical composition has the advantage of exhibiting excellent preventive or therapeutic efficacy, particularly for cancer diseases including solid tumors.

Description

Technology Field

[0001] The present invention relates to a novel compound having therapeutic efficacy for hyperproliferative disorders and a pharmaceutical composition using the same. Specifically, the present invention relates to a novel compound that binds to MDM2 (Mouse Double Minute 2 homolog) protein, a composition for inhibiting or degrading MDM2 protein activity using the same, and a composition for preventing or treating hyperproliferative disorders. Background Technology

[0002] MDM2 (Mouse Double Minute 2 homolog) is a type of oncoprotein as an E3 ubiquitin-protein ligase involved in the intracellular protein degradation process. MDM2 acts as a negative regulatory protein that degrades p53 using p53 as a substrate and inhibits its activity. p53 is a tumor suppressor factor that plays a central role in preventing tumor formation and is known to play a decisive role in tumor suppression through various mechanisms that regulate apoptosis, DNA repair, maintenance of normal stem cells, and self-renewal. Accordingly, research has been conducted on anticancer treatment and the development of anticancer drugs using protein-protein interaction inhibitors (PPIs) to treat cancer by inhibiting the interaction between MDM2 and p53, thereby inhibiting MDM2's p53 degradation activity.

[0003] In addition, research on compounds that bind to E3 ligases and degrade target proteins, such as molecular glue and PROTAC (Proteolysis-targeting chimera), has recently been actively conducted, and MDM2 is also being utilized as an E3 ligase to induce the degradation of target proteins. In other words, technologies utilizing compounds that bind to MDM2 to induce ubiquitination by MDM2 on disease-causing target proteins, and ultimately to induce the degradation of the target proteins, are being actively researched. Dual-acting proteolytic agents using MDM2-binding compounds studied to date include proteolytic agents that induce the degradation of MDM2 by linking a Cereblon ligand to an MDM2-binding compound, and proteolytic agents that degrade other targets by using an MDM2-binding compound as an E3 ligase ligand.

[0004] However, existing known MDM2-binding compounds such as Nutlin-3 and RG-7388 have large molecular weights, making them difficult to use in the development of dual-acting proteolytic agents such as PROTAC. It remains an important task to improve the pharmacological properties of dual-acting proteolytic agents by developing MDM2-binding compounds with small molecular weights, such as thalidomide-based compounds that bind to Cereblon. Prior art literature

[0005] Non-patent Document 1: Mengyang Chang, et al. MDM2-BCL-XL PROTACs enable degradation of BCL-XL and stabilization of p53. Acta Materia Medica 2022, Volume 1, Issue 2, p. 244-259. The problem to be solved

[0006] The main purpose of the present invention is to provide a novel compound that binds to the MDM2 (Mouse Double Minute 2 homolog) protein in order to solve the aforementioned conventional problems.

[0007] In addition, another objective of the present invention is to provide a composition for inhibiting or degrading the activity of an MDM2 protein, comprising a novel compound that binds to the MDM2 protein as an active ingredient.

[0008] In addition, another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a hyperproliferative disorder comprising a novel compound that binds to the MDM2 protein as an active ingredient.

[0009] The purpose of the present invention is not limited to the description above and is provided for all cases in which appropriate effects can be obtained by utilizing the present invention. means of solving the problem

[0010] The inventors conducted research on small molecule compounds that bind to the MDM2 (Mouse Double Minute 2 homolog) protein in order to develop a therapeutic agent for hyperproliferative disorders that exhibits efficacy in inhibiting the activity of the MDM2 protein. As a result, novel compounds that bind to the MDM2 protein were identified using proteomic techniques with LC-MS / MS (Liquid chromatography-mass spectrometry), and the binding of the novel compounds to the MDM2 protein was confirmed through 2D NMR spectroscopic analysis. Furthermore, the excellent MDM2 protein degradation efficacy of the novel compounds was confirmed through Western blot analysis. Accordingly, novel small molecule compounds that bind to the MDM2 protein and degrade it to effectively inhibit its activity were finally identified, and the present invention was completed.

[0012] Specifically, the present invention provides a compound represented by the following [Chemical Formula a] that binds to the MDM2 (Mouse Double Minute 2 homolog) protein.

[0013] [Chemical formula a]

[0014]

[0015] In the above [chemical formula a],

[0016] The above R 1 and R 2 are identical or different from each other, and each independently -H, -CH 3, , , , , and It is any one selected from the group consisting of;

[0017] The above R 1 and R 2They can be connected to each other to form a loop together, and in this case, the above NR 1 R 2 Is , , , and It is any one selected from the group consisting of;

[0018] The above R 4 , R 5 , R 6 and R 7 They are identical or different from each other, and each independently , , , , , , , , , , and It is any one selected from the group consisting of;

[0019] The above R 3 -C2H5, , , and It is any one selected from the group consisting of;

[0020] The above R 8 is -H or -CH3;

[0021] The above R 9 is -CH2- or And;

[0022] The above R 10 is -NH- or -O- and;

[0023] The above n is an integer from 1 to 4.

[0025] In addition, the present invention provides a compound wherein the compound represented by [Chemical Formula a] is selected from any one of the compounds represented by [Chemical Formula 1] to [Chemical Formula 28b].

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0044] In addition, the present invention provides a compound having the efficacy of inhibiting the activity of MDM2 protein.

[0045] In addition, the present invention provides a compound having the efficacy to degrade MDM2 protein.

[0046] In addition, the present invention provides a composition for inhibiting or degrading the activity of MDM2 (Mouse Double Minute 2 homolog) protein, comprising the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0047] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of a hyperproliferative disorder comprising the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0048] In addition, the present invention provides a pharmaceutical composition in which the abnormal proliferative disease is cancer.

[0049] In addition, the present invention provides a pharmaceutical composition wherein the cancer is one or more selected from the group consisting of lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, and brain tumor.

[0051] The present invention will be described in more detail below.

[0053] In a specific embodiment, the present invention provides a compound represented by the following [Chemical Formula a] that binds to the MDM2 (Mouse Double Minute 2 homolog) protein.

[0054] [Chemical formula a]

[0055]

[0056] In the above [chemical formula a],

[0057] The above R 1 and R 2 are identical or different from each other, and each independently -H, -CH 3, , , , , and It is any one selected from the group consisting of;

[0058] The above R 1 and R 2 They can be connected to each other to form a loop together, and in this case, the above NR 1 R 2 Is , , , and It is any one selected from the group consisting of;

[0059] The above R 4 , R 5 , R 6 and R 7 They are identical or different from each other, and each independently , , , , , , , , , , and It is any one selected from the group consisting of;

[0060] The above R 3 -C2H5, , , and It is any one selected from the group consisting of;

[0061] The above R 8 is -H or -CH3;

[0062] The above R 9 is -CH2- or And;

[0063] The above R 10 is -NH- or -O- and;

[0064] The above n is an integer from 1 to 4.

[0066] In the present invention, the compound represented by [Chemical Formula a] may be any one of the compounds selected from [Chemical Formula 1] to [Chemical Formula 28b] below.

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0085] The specific names of the novel compounds 1 to 28b represented by [Chemical Formula 1] to [Chemical Formula 28b] in the present invention are as follows.

[0086] Compound 1: 1-(4-(4-fluorophenethyl)piperazin-1-yl)propan-1-one [Formula 1]

[0087] Compound 2: 1-(4-(3-fluoro-4-methoxybenzyl)piperazin-1-yl)propan-1-one [Formula 2]

[0088] Compound 3: 1-(4-(5-ethylthiazol-2-yl)piperazin-1-yl)propan-1-one [Formula 3]

[0089] Compound 4: 1-(2-(phenoxymethyl)morpholino)propan-1-one [Formula 4]

[0090] Compound 5: 1-(4-(1-(4-methylthiazol-2-yl)ethyl)piperazin-1-yl)propan-1-one [화학식 5]

[0091] Compound 6: N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropionamide [화학식 6]

[0092] Compound 7: 1-(4-(4,5-dimethylthiazol-2-yl)piperidin-1-yl)propan-1-one [화학식 7]

[0093] Compound 8: 1-(4-(thiazol-2-yl)piperazin-1-yl)propan-1-one [화학식 8]

[0094] Compound 9: 1-(4-(3-fluorobenzyl)piperazin-1-yl)propan-1-one [화학식 9]

[0095] Compound 10: 1-(4-(thiophene-3-carbonyl)piperazin-1-yl)propan-1-one [화학식 10]

[0096] Compound 11: 1-(2-(3-(difluoromethoxy)phenyl)morpholino)propan-1-one [화학식 11]

[0097] Compound 12: N-(1-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)-1H-pyrazol-4-yl)propionamide [화학식 12]

[0098] Compound 13: 1-(4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propan-1-one [화학식 13]

[0099] Compound 14:1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)propan-1-one [화학식 14]

[0100] Compound 15: 5-phenyl-2-propionamidothiophene-3-carboxamide [화학식 15]

[0101] Compound 16: 1-(3-(5-(trifluoromethyl)-1H-imidazol-2-yl)piperidin-1-yl)propan-1-one [화학식 16]

[0102] Compound 17: N-methyl-N-(quinolin-4-ylmethyl)propionamide [화학식 17]

[0103] Compound 18: N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methyl-4-nitrobenzamide [화학식 18]

[0104] Compound 19: 4-amino-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide [화학식 19]

[0105] Compound 20a: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 20a]

[0106] Compound 20b:3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 20b]

[0107] Compound 20c: 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 20c]

[0108] Compound 20d: 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 20d]

[0109] Compound 20e: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-inden-5-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 20e]

[0110] Compound 20f: N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methyl-3-(2-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-inden-4-yl)amino)ethoxy)propenamide [화학식 20f]

[0111] Compound 21a:4-((2-(2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione [화학식 21a]

[0112] Compound 21b: 5-((2-(2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione [화학식 21b]

[0113] Compound 21c: 4-((2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione [화학식 21c]

[0114] Compound 22: 2-(2,6-dioxopiperidin-3-yl)-5-((2-(2-(3-oxo-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione [화학식 22]

[0115] Compound 23a: 4-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide [화학식 23a]

[0116] Compound 23b:4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide [화학식 23b]

[0117] Compound 23c: 5-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide [화학식 23c]

[0118] Compound 24a: 1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpiperidine-4-carboxamide [화학식 24a]

[0119] Compound 24b: 1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)propanoyl)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpiperidine-4-carboxamide [화학식 24b]

[0120] Compound 25:(2S,4R)-1-(2-(4-(2-(3-(difluoromethoxy)phenyl)morpholino)-4-oxobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide [화학식 25]

[0121] Compound 26: 3-(2-((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 26]

[0122] Compound 27: 3-(2-((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 27]

[0123] Compound 28a: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 28a]

[0124] Compound 28b: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide [화학식 28b]

[0126] Novel compounds 1 to 17 represented by [Chemical Formula 1] to [Chemical Formula 17] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 1] below.

[0127] [Reaction Equation 1]

[0128]

[0130] The novel compounds 18 and 19 represented by [Chemical Formula 18] and [Chemical Formula 19] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Scheme 2] below.

[0131] [Reaction Equation 2]

[0132]

[0134] The novel compounds 20a to 24b represented by [Chemical Formula 20a] to [Chemical Formula 24b] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Scheme 3] below.

[0135] [Reaction Equation 3]

[0136]

[0138] The novel compound 25 represented by [Chemical Formula 25] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 4] below.

[0139] [Reaction Equation 4]

[0140]

[0142] The novel compound 26 represented by [Chemical Formula 26] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 5] below.

[0143] [Reaction Equation 5]

[0144]

[0146] The novel compound 27 represented by [Chemical Formula 27] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 6] below.

[0147] [Reaction Equation 6]

[0148]

[0150] The novel compounds 28a and 28b represented by [Chemical Formula 28a] and [Chemical Formula 28b] according to the present invention can be prepared through a synthesis process schematically illustrated in [Reaction Scheme 7] below.

[0151] [Reaction Equation 7]

[0152]

[0154] The novel compound according to the present invention effectively binds to the MDM2 protein.

[0155] In the present invention, the MDM2 (Mouse Double Minute 2 homolog) protein is a type of tumor protein as an E3 ubiquitin-protein ligase involved in the intracellular protein degradation process. MDM2 is known to act as a negative regulatory protein that degrades p53 using p53 as a substrate protein and inhibits the activity of p53.

[0156] The novel compound of the present invention binds to the MDM2 protein and exhibits the efficacy of effectively inhibiting the activity of the MDM2 protein. More specifically, the novel compound of the present invention binds to the MDM2 protein and induces the degradation of the MDM2 protein, thereby exhibiting the efficacy of excellently inhibiting the activity of the MDM2 protein.

[0157] In another specific embodiment, the present invention provides a composition for inhibiting or degrading the activity of MDM2 (Mouse Double Minute 2 homolog) protein, comprising a compound represented by [Chemical Formula a] or a pharmaceutically acceptable salt thereof as an active ingredient.

[0158] In another specific embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of a hyperproliferative disorder comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0159] In the present invention, the compound represented by [Chemical Formula a] may be a novel compound 1 to 28b represented by [Chemical Formula 1] to [Chemical Formula 28b].

[0160] In the present invention, the hyperproliferative disorder generally refers to a pathological condition caused by excessive cell growth, division, and migration that are not controlled by general limiting means within the body. Non-limiting examples of the hyperproliferative disorder may include cancer, diabetic retinopathy, retinopathy of prematurity, corneal transplant rejection, neovascular glaucoma, erythematosus, proliferative retinopathy, psoriasis, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, recurrent stenosis, atherosclerosis, intestinal stenosis, ulcer, liver cirrhosis, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy, organ transplant rejection, renal glomerulopathy, etc.

[0161] In addition, the above-mentioned abnormal proliferative disease may particularly include cancer, and more specifically, the cancer may include solid tumors including lung cancer, stomach cancer, pancreatic cancer, colorectal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, brain tumor, etc.

[0162] The novel compound of the present invention has the advantage of exhibiting excellent preventive or therapeutic efficacy against abnormal proliferative diseases, particularly cancer, by effectively binding to MDM2 protein, a type of tumor protein, and inducing the degradation of MDM2 protein to inhibit its activity.

[0163] In the present invention, the efficacy for prevention or treatment of the abnormal proliferative disease may include not only the prevention or treatment of the abnormal proliferative disease itself, but also the efficacy for prevention or treatment of symptoms or complications that may appear due to the abnormal proliferative disease.

[0164] In the present invention, the above-mentioned pharmaceutically acceptable salt refers to a salt that can be prepared by conventional methods in the relevant technical field and is a salt in which a cation and an anion are bonded by electrostatic attraction, and is a form of salt that can be used pharmaceutically. Typically, it may be a metal salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, etc. For example, it means forming an acid salt with an inorganic acid such as hydrochloric acid, hydrogen bromide, sulfuric acid, sodium bisulfate, phosphoric acid, carbonic acid, or an acid salt with an organic acid such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestisic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin), or forming a metal salt of these by reacting with alkali metal ions such as sodium or potassium, or forming another form of pharmaceutically acceptable salt by reacting with ammonium ions. Non-limiting examples of the above pharmaceutically acceptable salts may include hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartrate, maleate, fumarate, gluconate, methanesulfonate, glyconate, succinate, 4-toluenesulfonate, gluturonic acid, emvonate, glutamate, aspartate, etc.

[0165] In the present invention, "included as an active ingredient" means that the corresponding ingredient is included in an amount necessary or sufficient to realize the desired biological effect. In actual application, the amount included as an active ingredient is determined as an amount for treating the target disease, taking into account factors that do not cause other toxicities, and may vary depending on various factors such as the disease or condition being treated, the form of the administered composition, the size of the subject, or the severity of the disease or condition. A person skilled in the art to which the present invention pertains can empirically determine the effective amount of individual compositions without resorting to excessive experimentation.

[0166] In the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and may be formulated together with said carrier and provided as food, medicine, etc. In the present invention, said pharmaceutically acceptable carrier generally refers to a carrier or diluent that does not stimulate living organisms and does not impair the biological activity and properties of the administered compound. The types of said carriers usable in the present invention are not particularly limited, and any carrier that is commonly used in the relevant technical field or similar fields and is pharmaceutically acceptable may be used. Non-limiting examples of said carriers include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in a mixture of two or more. In addition, if necessary, other conventional additives such as antioxidants, buffers and / or bacteriostatic agents may be added, and diluents, dispersants, surfactants, binders and / or lubricants may be additionally added to formulate the product into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0167] The method of administration of the pharmaceutical composition of the present invention is not particularly limited and may follow methods commonly used in the relevant technical field or similar fields. As a non-limiting example of the method of administration, the composition may be administered orally or parenterally.

[0168] The pharmaceutical composition of the present invention may be prepared in various formulations depending on the intended mode of administration. Non-limiting examples of formulations for oral administration include troches, lozenges, tablets, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. In order to formulate the pharmaceutical composition of the present invention into an oral administration formulation such as a tablet or capsule, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; an excipient such as dicalcium phosphate; and a disintegrant such as corn starch or sweet potato starch; It may include lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. Furthermore, in the case of capsule formulations, in addition to the aforementioned substances, it may additionally contain liquid carriers such as fatty oils.

[0169] Methods for parenterally administering the pharmaceutical composition of the present invention may include, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration, and methods of applying or spraying the composition to a diseased area may also be used, but are not limited thereto. Formulations for parenteral administration may include, for example, injectable forms such as subcutaneous injection, intravenous injection, or intramuscular injection; suppository administration; or formulations for sprays such as aerosols that allow inhalation through the respiratory tract, but are not limited thereto. To formulate the composition of the present invention into an injectable form, the composition of the present invention may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, and this may be formulated for unit administration in ampoules or vials. When formulating for a spray such as an aerosol, a propellant or the like may be combined with an additive to disperse the water-dispersed concentrate or wet powder.

[0170] The suitable dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the method of formulation, method of administration, time of administration and / or route of administration, as well as the age, weight, gender, severity of disease symptoms, food consumed, and excretion rate of the subject to administration, and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment.

[0171] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. In the present invention, "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and this can be easily determined by a person skilled in the art. Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, condition, body weight, absorption rate, inactivation rate, and elimination rate of the active ingredient in the body, type of disease, and concurrently used drugs.

[0173] Terms not otherwise defined in the present invention shall be interpreted as having the meanings commonly used in the relevant technical field. Additionally, the expression "or" as used in the present invention may be interpreted as a concept including "and" unless otherwise noted.

[0174] The scope of the present invention is not limited by the specific descriptions disclosed herein, and each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all possible combinations of the various elements disclosed herein are to be interpreted as falling within the scope of the present invention. Furthermore, a person skilled in the art may recognize or identify a number of equivalents to specific embodiments of the present invention through ordinary experimentation, and such equivalents are to be interpreted as falling within the scope of the present invention. Effects of the invention

[0175] The present invention relates to a novel compound that binds to MDM2 (Mouse Double Minute 2 homolog) protein, a composition for inhibiting or degrading MDM2 protein activity using the same, and a composition for preventing or treating hyperproliferative disorders.

[0176] The novel compound according to the present invention is a low-molecular-weight compound that effectively binds to the MDM2 protein and induces the degradation of the MDM2 protein, thereby having the advantage of exhibiting excellent inhibition and regulation of the activity of the MDM2 protein.

[0177] In addition, the present invention can be utilized as a pharmaceutical composition for the prevention or treatment of abnormal proliferative diseases using the novel compound, and the pharmaceutical composition has the advantage of exhibiting excellent preventive or therapeutic efficacy, particularly for cancer diseases including solid tumors. Brief explanation of the drawing

[0178] Figure 1 is a graph comparing the average chemical shift perturbation of major amino acids using 2D NMR spectroscopic analysis of novel compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17 and 19 of the present invention in one embodiment of the present invention. FIG. 2 is a figure showing the average chemical shift perturbation of major amino acids using 2D NMR spectroscopic analysis of novel compounds 6 and 11 of the present invention in one embodiment of the present invention. Figure 3 shows the results of evaluating the MDM2 protein degradation efficacy of the novel compound of the present invention in one embodiment of the present invention. Figure 4 shows the results of evaluating the MDM2 protein degradation efficacy of the novel compound of the present invention through Western blot in one embodiment of the present invention. Figure 5 shows the results of evaluating the MDM2 protein degradation efficacy of the novel compound 20a [Chemical Formula 20a] of the present invention at different concentrations through Western blot in one embodiment of the present invention. Figure 6 shows the results of evaluating the MDM2 protein degradation efficacy of the novel compound 20a [Chemical Formula 20a] of the present invention over time using Western blot in one embodiment of the present invention. Figure 7 shows the results of a Western blot performed to confirm the mechanism of action (MoA) of a novel compound of the present invention in one embodiment of the present invention. Specific details for implementing the invention

[0179] The present invention will be described in more detail below through specific embodiments. However, these embodiments are merely examples for explaining the present invention and should not be interpreted as limiting the scope of the present invention in any way.

[0181] [Synthetic Example]

[0182] Novel compounds 1 to 28b represented by [Chemical Formula 1] to [Chemical Formula 28b] according to the present invention are as follows.

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0201] Compound 1: 1-(4-(4-fluorophenethyl)piperazin-1-yl)propan-1-one

[0202] Compound 2: 1-(4-(3-fluoro-4-methoxybenzyl)piperazin-1-yl)propan-1-one

[0203] Compound 3:1-(4-(5-ethylthiazol-2-yl)piperazin-1-yl)propan-1-one

[0204] Compound 4: 1-(2-(phenoxymethyl)morpholino)propan-1-one

[0205] Compound 5: 1-(4-(1-(4-methylthiazol-2-yl)ethyl)piperazin-1-yl)propan-1-one

[0206] Compound 6: N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropionamide

[0207] Compound 7: 1-(4-(4,5-dimethylthiazol-2-yl)piperidin-1-yl)propan-1-one

[0208] Compound 8: 1-(4-(thiazol-2-yl)piperazin-1-yl)propan-1-one

[0209] Compound 9: 1-(4-(3-fluorobenzyl)piperazin-1-yl)propan-1-one

[0210] Compound 10: 1-(4-(thiophene-3-carbonyl)piperazin-1-yl)propan-1-one

[0211] Compound 11: 1-(2-(3-(difluoromethoxy)phenyl)morpholino)propan-1-one

[0212] Compound 12: N-(1-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)-1H-pyrazol-4-yl)propionamide

[0213] Compound 13:1-(4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propan-1-one

[0214] Compound 14: 1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)propan-1-one

[0215] Compound 15: 5-phenyl-2-propionamidothiophene-3-carboxamide

[0216] Compound 16: 1-(3-(5-(trifluoromethyl)-1H-imidazol-2-yl)piperidin-1-yl)propan-1-one

[0217] Compound 17: N-methyl-N-(quinolin-4-ylmethyl)propionamide

[0218] Compound 18: N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methyl-4-nitrobenzamide

[0219] Compound 19: 4-amino-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0220] Compound 20a: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0221] Compound 20b:3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0222] Compound 20c: 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0223] Compound 20d: 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0224] Compound 20e: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-inden-5-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0225] Compound 20f: N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methyl-3-(2-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-inden-4-yl)amino)ethoxy)propenamide

[0226] Compound 21a:4-((2-(2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0227] Compound 21b: 5-((2-(2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0228] Compound 21c: 4-((2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0229] Compound 22: 2-(2,6-dioxopiperidin-3-yl)-5-((2-(2-(3-oxo-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione

[0230] Compound 23a: 4-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0231] Compound 23b:4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0232] Compound 23c: 5-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0233] Compound 24a: 1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpiperidine-4-carboxamide

[0234] Compound 24b: 1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)propanoyl)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpiperidine-4-carboxamide

[0235] Compound 25: (2S,4R)-1-(2-(4-(2-(3-(difluoromethoxy)phenyl)morpholino)-4-oxobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0236] Compound 26:3-(2-((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0237] Compound 27: 3-(2-((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0238] Compound 28a: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0239] Compound 28b: 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0241] Novel compounds 1 to 17 represented by [Chemical Formula 1] to [Chemical Formula 17] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 1] below.

[0242] [Reaction Equation 1]

[0243]

[0245] <Synthetic Example 1> Novel Compound 1 [Chemical Formula 1]

[0246] 1-(4-(4-fluorophenethyl)piperazin-1-yl)propan-1-one

[0247] 1-(4-(4-fluorophenethyl)piperazine (20 mg, 0.10 mmol) was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 1 of [Formula 1] (18 mg, 68%).

[0249] H NMR (600 MHz, CDCl3) δ ppm 7.07 (dd, J = 12.0, 1.8 Hz, 1H), 6.96 (t, J = 8.7 Hz, 2H), 3.65 - 3.63 (m, 2H), 3.48 - 3.47 (m, 2H), 2.78 - 2.75 (m, 2H), 2.59 - 2.56 (m, 2H), 2.49 - 2.46 (m, 4H), 2.33 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.4, 3H).

[0251] <Synthesizing Example 2> Novel Compound 2 [Chemical Formula 2]

[0252] 1-(4-(3-fluoro-4-methoxybenzyl)piperazin-1-yl)propan-1-one

[0253] 1-(3-fluoro-4 methoxybenzyl)piperazine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 2 of [Formula 2].

[0255] H NMR (600 MHz, CDCl3) δ ppm 7.14 (dd, J = 10.7, 5.5 Hz, 2H), 6.98 (d, J = 8.3 Hz, 1H), 6.89 (t, J = 8.4 Hz, 1H), 3.87 (s, 3H), 3.62 - 3.61 (m, 2H), 3.45 - 3.43 (m, 4H), 2.40 - 2.38 (m, 4H), 2.32 (q, J = 7.4 Hz, 2H), 1.13 (t, J = 7.4, 3H).

[0257] <Synthesizing Example 3> Novel Compound 3 [Chemical Formula 3]

[0258] 1-(4-(5-ethylthiazol-2-yl)piperazin-1-yl)propan-1-one

[0259] 5-ethyl-2-(piperazine-1-yl)thiazole was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 3 of [Formula 3].

[0261] H NMR (600 MHz, DMSO) δ ppm 7.07 (s, 1H), 3.61 (s, 4H), 3.50 - 3.43 (m, 4H), 2.69 - 2.65 (m, 2H), 2.36 (q, J = 7.4 Hz, 2H), 1.18 (t, J = 7.4) Hz, 3H), 1.00 (t, J = 7.4, 3H).

[0263] <Synthesizing Example 4> Novel Compound 4 [Chemical Formula 41]

[0264] 1-(2-(phenoxymethyl)morpholino)propan-1-one

[0265] 2-(phenoxymethyl)morpholine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 4 of [Formula 4].

[0267] H NMR (600 MHz, CDCl3) δ ppm 7.31 - 7.28 (m, 2H), 6.97 (qui, J = 7.6 Hz, 1H), 6.91 (d, J = 7.9 Hz, 2H), 4.62 (d J = 13.3 Hz, 0.5H), 4.42 (d, J = 13.5 Hz, 0.5H), 4.10 - 3.94 (m, 3H), 3.88 (d, J = 13.1 Hz, 0.5H), 3.79 - 3.78 (m, 1H), 3.66 (d, J = 13.3, 0.5H), 3.62 - 3.56 (m, 1H), 3.31 - 3.27 (m, 0.5H), 3.17 - 3.13 (m, 0.5H), 2.90 - 2.86 (m, 0.5H), 2.72 (t, J = 11.6, 0.5H), 2.43 - 2.31 (m, 2H), 1.17 (t, J = 7.4 Hz, 3H).

[0269] <Synthesizing Example 5> Novel Compound 5 [Chemical Formula 5]

[0270] 1-(4-(1-(4-methylthiazol-2-yl)ethyl)piperazin-1-yl)propan-1-one

[0271] 4-methyl-2-(1-(piperazine-1-yl)ethyl)thiazole was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 5 of [Formula 5].

[0273] H NMR (600 MHz, CDCl3) δ ppm 7.31 (s, 1H), 3.97 - 3.93 (m, 1H), 3.63 (br s, 2H), 3.46 (br s, 2H), 2.57 - 2.51 (m, 4H), 2.42 (s, 3H), 2.31 (q, J = 7.5 Hz, 2H), 1.44 (d, J = 6.8 Hz, 3H), 1.12 (t, J = 7.4 Hz, 3H).

[0275] <Synthesizing Example 6> Novel Compound 6 [Chemical Formula 6]

[0276] N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropionamide

[0277] 1-(4-fluorophenyl)-N-methyl-1-(pyridine-3-yl)methaneamine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 6 of [Formula 6].

[0279] H NMR (600 MHz, CDCl3) δ ppm 8.56 - 8.55 (m, 1H), 8.46 (s, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.20 (s, 1H), 7.14 (t, J = 5.3 Hz, 2H), 7.05 (t, J = 8.4 Hz, 2H), 2.80 (s, 1H), 2.45 (q, J = 7.3, 2H), 1.20 (t, J = 7.3 Hz, 3H).

[0281] <Synthesizing Example 7> Novel Compound 7 [Chemical Formula 7]

[0282] 1-(4-(4,5-dimethylthiazol-2-yl)piperidin-1-yl)propan-1-one

[0283] 4,5-dimethyl-2-(piperidin-4-yl)thiazole was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 7 of [Formula 7].

[0285] H NMR (600 MHz, CDCl3) δ ppm 4.66 (d, J = 13.3, Hz 1H), 3.91 (d, J = 13.5 Hz, 1H), 3.14 - 3.07 (m, 2H), 2.71 - 2.67 (m, 1H), 2.34 (q, J = 7.4) Hz, 2H), 2.29 (s, 3H), 2.26 (s, 3H), 2.08 (dd, J = 34.1, 13.1 Hz, 2H), 1.71 - 1.61 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H).

[0287] <Synthetic Example 8> Novel Compound 8 [Chemical Formula 8]

[0288] 1-(4-(thiazol-2-yl)piperazin-1-yl)propan-1-one

[0289] 2-(piperazine-1-yl)thiazole was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 8 of [Formula 8].

[0291] H NMR (600 MHz, CDCl3) δ ppm 7.19 (d, J = 3.6 Hz, 1H), 6.60 (d, J = 3.6 Hz, 1H), 3.76 - 3.74 (m, 2H), 3.59 - 3.57 (m, 2H), 3.54 - 3.52, (m, 2H), 3.44 - 3.42 (m, 2H), 2.37 (q, J = 7.4 Hz, 2H), 1.15 (t, J = 7.4 Hz, 3H).

[0293] <Synthesizing Example 9> Novel Compound 9 [Chemical Formula 9]

[0294] 1-(4-(3-fluorobenzyl)piperazin-1-yl)propan-1-one

[0295] 1-(3-fluorobenzyl)piperazine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 9 of [Formula 9].

[0297] H NMR (600 MHz, CDCl3) δ ppm 7.28 (q, J = 7.7 Hz, 1H), 7.08 (t, J = 8.6 Hz, 2H), 6.96 (t, J = 8.0 Hz, 1H), 3.65 - 3.63 (m, 2H), 3.51 (s, 2H), 3.47 - 3.46 (m, 2H), 2.43 - 2.41 (m, 4H), 2.34 (q, J = 7.5 2H), 1.15 (t, J = 7.5 Hz, 3H).

[0299] <Synthetic Example 10> Novel Compound 10 [Chemical Formula 10]

[0300] 1-(4-(thiophene-3-carbonyl)piperazin-1-yl)propan-1-one

[0301] Piperazine-1-yl(thiophene-3-yl)methanone was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 10 of [Formula 10].

[0303] H NMR (600 MHz, CDCl3) δ ppm 7.53 (dd, J = 2.4, 1.1 Hz, 1H), 7.35 (dd, J = 5.4, 3 Hz, 1H), 7.17 (dd, J = 4.8, 1.1 Hz, 1H), 3.63 (br s, 6H), 3.50 (br s, 2H), 2.36 (q, J = 7.1, 2H), 1.15 (t, J = 7.5 Hz, 3H).

[0305] <Synthetic Example 11> Novel Compound 11 [Chemical Formula 11]

[0306] 1-(2-(3-(difluoromethoxy)phenyl)morpholino)propan-1-one

[0307] 2-(3-(difluoromethoxy)phenyl)morpholine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 11 of [Formula 11].

[0309] H NMR (600 MHz, CDCl3) δ ppm 7.36 (qui, J = 7.9 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.17 (s, 1H), 7.07 (dd, J = 19.1, 8 Hz, 1H), 6.51 (td, J = 73.7, 8.8 Hz, 1H), 4.60 (dd, J = 84, 13.5 Hz, 1H), 4.52 (d, J = 10.6 Hz, 1H), 4.09 - 4.07 (m, 1H), 3.76 (dd, J = 53.7, 13.3 Hz, 1H) 3.70 - 3.64 (m, 1H), 3.37 - 3.32 (m, 0.5H), 3.10 - 3.06 (m, 0.5H), 2.90 - 2.85 (m, 0,5H), 2.66 - 2.62 (m, 0.5H), 2.45 - 2.29 (m, 2H), 1.19 - 1.16 (m, 3H).

[0311] <Synthetic Example 12> Novel Compound 12 [Chemical Formula 12]

[0312] N-(1-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)-1H-pyrazol-4-yl)propionamide

[0313] 1-((1-methyl-1H-benzo[d]imidazole-2-yl)methyl)-1H-pyrazole-4-amine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 12 of [Formula 12].

[0315] H NMR (600 MHz, CDCl3) δ ppm 7.91 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.52 (s, 1H), 7.31 - 7.27 (m, 3H), 7.10 (br s, 1H), 5.58 (s, 2H), 3.75 (s, 3H), 2.32 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H).

[0317] <Synthetic Example 13> Novel Compound 13 [Chemical Formula 13]

[0318] 1-(4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propan-1-one

[0319] 2-(piperazine-1-yl)-4-(trifluoromethyl)pyrimidine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 13 of [Formula 13].

[0321] H NMR (600 MHz, CDCl3) δ ppm 8.51 (d, J = 4.9 Hz, 1H), 6.80 (d, J = 4.9 Hz, 1H), 3.90 - 3.85 (m, 4H), 3.72 - 3.70 (m, 2H), 3.55 - 3.53 (m, 2H), 5.58 (s, 2H), 2.39 (q, J = 7.4 Hz, 2H), 1.18 (t, J = 7.4 Hz, 3H).

[0323] <Synthetic Example 14> Novel Compound 14 [Chemical Formula 14]

[0324] 1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)propan-1-one

[0325] 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 14 of [Formula 14].

[0327] H NMR (600 MHz, CDCl3) δ ppm 5.00 - 4.90 (m, 2H), 4.20 - 4.00 (m, 4H), 2.43 (q, J = 7.3 Hz, 2H), 1.15 (t, J = 7.4 Hz, 3H).

[0329] <Synthetic Example 15> Novel Compound 15 [Chemical Formula 15]

[0330] 5-phenyl-2-propionamidothiophene-3-carboxamide

[0331] 2-amino-5-phenylthiophene-3-carboxamide was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 15 of [Formula 15].

[0333] H NMR (600 MHz, CDCl3) δ ppm 11.81 (s, 1H), 7.58 - 7.56 (m, 2H), 7.39 - 7.37 (m, 2H), 7.30 - 7.28 (m, 1H) 7.10 (s, 1H), 5.63 (br s, 2H), 2.55 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H).

[0335] <Synthetic Example 16> Novel Compound 16 [Chemical Formula 16]

[0336] 1-(3-(5-(trifluoromethyl)-1H-imidazol-2-yl)piperidin-1-yl)propan-1-one

[0337] 3-(5-(trifluoromethyl)-1H-imidazole-2-yl)piperidine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 16 of [Formula 16].

[0339] H NMR (600 MHz, CDCl3) δ ppm 7.21 (s, 1H), 4.45 - 4.42 (m, 1H), 3.68 - 3.65 (m, 1H), 3.49 - 3.46 (m, 1H), 3.35 - 3.30 (m, 1H), 3.19 - 3.17 (m, 1H), 2.56 - 2.53 (m, 1H), 2.41 - 2.34 (m, 2H), 2.05 - 2.00 (m, 1H), 1.64 - 1.60 (m, 1H), 1.45 - 1.38 (m, 1H), 1.13 (t, J = 7.5 Hz, 3H).

[0341] <Synthetic Example 17> Novel Compound 17 [Chemical Formula 17]

[0342] N-methyl-N-(quinolin-4-ylmethyl)propionamide

[0343] N-methyl-1-(quinoline-4-yl)methaneamine was dissolved in a saturated aqueous solution of tetrahydrofuran / sodium bicarbonate (THF / NaHCO3(aq)) (1:1, 2 ml), and then acryloyl chloride was added dropwise at 0°C. After stirring the reaction mixture for 5 minutes, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 17 of [Formula 17].

[0345] H NMR (600 MHz, CDCl3) δ ppm 8.86 (d, J = 4.4, 1H), 8.13 (d, J = 8.4 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.73 (t, J = 7.1, 1H), 7.58 (t, J = 7.3, 1H), 7.18 (d, J = 4.3, 1H), 5.09 (s, 2H), 2.96 (s, 3H), 2.47 (q, J = 7.4 Hz, 2H), 1.23 (t, J = 7.4 Hz, 3H).

[0347] The novel compounds 18 and 19 represented by [Chemical Formula 18] and [Chemical Formula 19] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Scheme 2] below.

[0348] [Reaction Equation 2]

[0349]

[0351] <Synthetic Example 18> Novel Compound 18 [Chemical Formula 18]

[0352] N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methyl-4-nitrobenzamide

[0353] 1-(4-fluorophenyl)-N-methyl-1-(pyridin-3-yl)methaneamine (200 mg, 0.93 mmol), 4-nitrobenzoyl chloride (185 mg, 1.19 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (527 mg, 1.38 mmol), and N,N-diisopropylethylamine (327 μl, 1.85 mmol) were dissolved in dimethylformamide (5 ml). After stirring the reaction mixture for 4 hours, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 18 (236 mg, 70%) of [Formula 18].

[0355] H NMR (600 MHz, CDCl3) δ ppm 8.60 (s, 1H), 8.49 (br s, 1H), 8.47 - 8.44 (m, 2H), 7.53 (br s, 1H), 7.33 - 7.26 (m, 5H), 7.17 (br s, 2H), 7.10 - 7.08 (m, 2H), 2.82 - 2.81 (m, 3H).

[0357] <Synthetic Example 19> Novel Compound 19 [Chemical Formula 19]

[0358] 4-amino-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0359] N-((4-fluorophenyl)(phenyl)methyl)-N-methyl-4-nitrobenzamide (236 mg, 0.65 mmol) was dissolved in methanol (6 ml), and then palladium / carbon (3.5 mg, 5 mol%) was added at room temperature under a hydrogen atmosphere. After stirring the reaction mixture for 30 minutes, the palladium / carbon was filtered out using diatomite and concentrated under vacuum to obtain the novel compound 19 of [Formula 19] as a yellow solid (173 mg, 80%).

[0361] H NMR (600 MHz, CDCl3) δ ppm 8.60 (s, 1H), 8.49 (br s, 1H), 7.53 (br s, 1H), 7.33 - 7.26 (m, 5H), 7.17 (br s, 2H), 7.10 - 7.08 (m, 2H), 6.66 - 6.65 (m, 2H), 3.90 (br s, 2H), 2.82 - 2.81 (m, 3H).

[0363] The novel compounds 20a to 24b represented by [Chemical Formula 20a] to [Chemical Formula 24b] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Scheme 3] below.

[0364] [Reaction Equation 3]

[0365]

[0367] <Synthesizing Example 20-1> Novel Compound 20a [Chemical Formula 20a]

[0368] 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0369] 1-(4-fluorophenyl)-N-methyl-1-phenylmethaneamine (100 mg 0.46 mmol), 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxosoidolindolin-4-yl)amino)ethoxy)propanoic acid (215 mg, 0.55 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (527 mg, 1.38 mmol), and N,N-diisopropylethylamine (327 μl, 1.85 mmol) were reacted with dimethylformamide (5 ml) and stirred for 4 hours. After stirring, the mixture was diluted with ethyl acetate, washed with water and brine, dried (MgSO4), and then concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 20a of [Formula 20a] (81 mg, 30%).

[0371] H NMR (600 MHz, CDCl3) δ ppm 8.57 (s, 1H), 8.49 (s, 1H), 7.52 - 7.47 (m, 2H), 7.31 - 7.29 (m, 1H), 7.17 (s, 1H), 7.15 - 7.13 (m, 2H), 7.10 (d, J = 7.1 Hz, 1H), 7.04 (t, J = 8.5 Hz, 2H) 6.90 (d, J = 8.5 Hz, 1H), 6.50 - 6.45 (m, 1H), 4.91 - 4.88 (m, 1H), 3.86 (t, J = 6.5) Hz, 2H), 3.72 (t, J = 5.3 Hz, 2H), 3.69 - 3.65 (m, 4H), 3.47 - 3.45 (m, 2H), 2.88 - 2.85 (m, 1H), 2.81 - 2.68 (m, 8H), 2.13 - 2.09 (m, 1H).

[0373] <Synthesizing Example 20-2> Novel Compound 20b [Chemical Formula 20b]

[0374] 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0375] A novel compound 20b of [Chemical Formula 20b] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0377] H NMR (600 MHz, CDCl3) δ ppm 8.57 (s, 1H), 8.49 (s, 1H), 7.52 - 7.47 (m, 2H), 7.31 - 7.29 (m, 1H), 7.17 (s, 1H), 7.15 - 7.13 (m, 2H), 7.10 (d, J = 7.1 Hz, 1H), 7.04 (t, J = 8.5 Hz, 2H) 6.90 (d, J = 8.5 Hz, 1H), 6.50 - 6.45 (m, 1H), 4.91 - 4.88 (m, 1H), 3.86 (t, J = 6.5) Hz, 2H), 3.72 (t, J = 5.3 Hz, 2H), 3.69 - 3.65 (m, 4H), 3.47 - 3.45 (m, 2H), 2.88 - 2.85 (m, 1H), 2.81 - 2.68 (m, 8H), 2.13 - 2.09 (m, 1H).

[0379] <Synthesizing Example 20-3> Novel Compound 20c [Chemical Formula 20c]

[0380] 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0381] A novel compound 20c of [Chemical Formula 20c] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0383] H NMR (600 MHz, CDCl3) δ ppm 8.91 - 8.86 (m, 1H), 8.60 - 8.57 (m, 1H), 8.48 (s, 1H), 7.57 - 7.42 (m, 2H), 7.32 - 7.30 (m, 1H), 7.18 (s, 1H), 7.16 - 7.03 (m, 5H), 6.91 (d, J = 8.5 Hz, 2H), 6.50 - 6.45 (m, 1H), 5.00 - 4.88 (m, 1H), 3.86 (t, J = 6.5 Hz, 2H), 3.71 (t, J = 5.3 Hz, 2H), 3.66 - 3.65 (m, 8H), 3.46 - 3.45 (m, 2H), 2.94 - 2.69 (m, 8H), 2.12 - 2.09 (m, 1H).

[0385] <Synthesizing Example 20-4> Novel Compound 20d [Chemical Formula 20d]

[0386] 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0387] A novel compound 20d of [Chemical Formula 20d] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0389] H NMR (600 MHz, CDCl3) δ ppm 8.80 - 8.72 (m, 1H), 8.60 - 8.57 (m, 1H), 8.48 (s, 1H), 7.53 - 7.47 (m, 2H), 7.32 - 7.30 (m, 1H), 7.18 (s, 1H), 7.16 - 7.03 (m, 5H), 6.91 (d, J = 8.5 Hz, 2H), 6.50 - 6.46 (m, 1H), 4.91 - 4.88 (m, 1H), 3.86 (t, J = 6.5 Hz, 2H), 3.71 (t, J = 5.3 Hz, 2H), 3.66 - 3.64 (m, 12H), 3.47 - 3.44 (m, 2H), 2.88 - 2.68 (m, 8H), 2.13 - 2.09 (m, 1H).

[0391] <Synthesizing Example 20-5> Novel Compound 20e [Chemical Formula 20e]

[0392] 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-inden-5-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0393] A novel compound 20e of [Chemical Formula 20e] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0395] 1H NMR (600 MHz, DMSO) δ 11.03 (s, 1H), 8.51 - 8.50 (m, 1H), 8.38 - 8.36 (m, 1H), 7.53 - 7.51 (m, 2H), 7.38 - 7.36 (m, 1H), 7.19 - 7.14 (m, 1H), 7.19 - 7.14 (m, 4H), 7.11 - 7.09 (m, 1H), 6.95 (s, 1H), 6.86 (dd, J = 8.34 Hz, 1H), 5.01 (dd, J = 12.9 Hz, 1H), 3.72 - 3.68 (m, 2H), 3.61 - 3.54 (m, 2H), 3.35 - 3.32 (m, 2H), 2.88 - 2.82 (m, 1H), 2.75 - 2.74 (m, 3H), 2.73 - 2.70 (m, 2H), 2.59 - 2.51 (m, 2H), 1.98 - 1.95 (m, 1H)

[0397] <합성예 20-6> 신규 화합물 20f [화학식 20f]

[0398] N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methyl-3-(2-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-inden-4-yl)amino)ethoxy)propenamide

[0399] A novel compound 20f of [Chemical Formula 20f] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0401] H NMR (600 MHz, CDCl3) δ ppm 8.49 (s, 1H), 7.52 - 7.47 (m, 2H), 7.31 - 7.29 (m, 1H), 7.17 (s, 1H), 7.15 - 7.13 (m, 2H), 7.10 (d, J = 7.1 Hz, 1H), 7.04 (t, J = 8.5 Hz, 2H) 6.90 (d, J = 8.5 Hz, 1H), 6.50 - 6.45 (m, 1H), 4.91 - 4.88 (m, 1H), 3.86 (t, J = 6.5 Hz, 2H), 3.76 (s, 3H), 3.72 (t, J = 5.3 Hz, 2H), 3.69 - 3.65 (m, 4H), 3.47 - 3.45 (m, 2H), 2.88 - 2.85 (m, 1H), 2.81 - 2.68 (m, 8H), 2.13 - 2.09 (m, 1H).

[0403] <합성예 21-1> 신규 화합물 21a [화학식 21a]

[0404] 4-((2-(2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0405] A novel compound 21a of [Chemical Formula 21a] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0407] H NMR (600 MHz, CDCl3) δ ppm 7.45 (q, J = 7.4 Hz, 1H), 7.34 - 7.31 (m, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.15 (s, 1H), 7.08 - 7.04 (m, 2H), 6.89 - 6.85 (m, 1H), 6.64 - 6.39 (m, 2H), 4.87 (br s, 1H), 4.65 - 4.38 (m, 2H), 4.06 - 4.01 (m, 1H), 3.93 - 3.76 (m, 3H), 3.71 - 3.60 (m, 7H), 3.47 - 3.40 (m, 2H), 3.47 - 3.44 (m, 2H), 3.33 - 3.01 (m, 1H), 2.86 - 2.52 (m, 6H), 2.07 (br s, 1H).

[0409] <합성예 21-2> 신규 화합물 21b [화학식 21b]

[0410] 5-((2-(2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0411] A novel compound 21b of [Chemical Formula 21b] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0413] H NMR (600 MHz, CDCl3) δ ppm 7.55 (t, J = 7.3 Hz, 1H), 7.32 (dt, J = 22.4, 7.9 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.16 - 7.14 (m, 1H), 7.05 (dd, J = 24.0, 8.0 Hz, 1H), 6.98 (br s, 1H), 6.79 - 6.75 (m, 1H), 6.51 (td, J = 73.9, 12.5 Hz, 1H), 5.45 (d, J = 35.4 Hz, 1H), 4.89 - 4.88 (m, 1H), 4.67 - 4.53 (m, 1H), 4.48 - 4.40 (m, 1H), 4.07 (t, J = 9.5 Hz, 1H), 3.91 - 3.76 (m, 3H), 3.71 - 3.65 (m, 7H), 3.37 - 3.35 (m, 2H), 3.10 - 2.53 (m, 7H), 2.09 (br s, 1H).

[0415] <합성예 21-3> 신규 화합물 21c [화학식 21c]

[0416] 4-((2-(3-(2-(2-(difluoromethoxy)phenyl)morpholino)-3-oxopropoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0417] A novel compound 21c of [Chemical Formula 21c] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0419] 1H NMR (600 MHz, DMSO) δ 11.07 (s, 1H), 7.58 - 7.53 (m, 1H), 7.41 - 7.34 (m, 1H), 7.22 - 7.21 (m, 1H), 7.15 - 7.10 (m, 1H), 7.03 - 7.00 (m, 1H), 6.58 - 6.54 (m, 1H), 5.02 (dd, J = 12.8 Hz), ), 4.45 (d, J = 10.4 Hz, 1H), 4.37 (t, J = 10.5 Hz, 1H), 4.26 (d, J = 13.2 Hz, 0.5H), 3.98 (d, J = 9.2 Hz, 0.5H), 3.94 (d, J = 12.6 Hz, 1H), 3.82 (d, J = 13.3 Hz, 0.5H), 3.69 (t, J = 6.3 Hz, 2H), 3.59 (q, J = 5.4 Hz, 2H), 3.46 - 3.44 (m, 2H), 3.34 - 3.33 (s, 0.5H), 3.20 (t, J = 12.0 Hz, 0.5H), 2.97 (t, J = 11.9 Hz, 0.5H), 2.90 - 2.68 (m, 3.5H), 2.60 - 2.50 (m, 4H), 2.01 - 1.93 (m, 1H).

[0421] <합성예 22> 신규 화합물 22 [화학식 22]

[0422] 2-(2,6-dioxopiperidin-3-yl)-5-((2-(2-(3-oxo-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione

[0423] A novel compound 22 of [Chemical Formula 22] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0425] H NMR (600 MHz, CDCl3) δ ppm 8.50 - 8.48 (m, 1H), 7.45 (q, J = 7.6 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 4.7) Hz, 1H), 6.55 - 6.48 (m, 1H), 4.85 - 4.77 (m, 1H), 3.88 - 3.79 (m, 6H), 3.70 - 3.64 (m, 8H), 3.57 - 3.55 (m, 2H), 3.45 - 3.43 (m, 2H), 4.48 - 4.40 (m, 1H), 2.82 - 2.80 (m, 1H), 2.71 - 2.46 (m, 4H), 2.07 (br s, 1H).

[0427] <Synthesizing Example 23-1> Novel Compound 23a [Chemical Formula 23a]

[0428] 4-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0429] A novel compound 23a of [Chemical Formula 23a] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0431] H NMR (600 MHz, CDCl3) δ ppm 8.60 (s, 1H), 8.40 - 8.33 (m, 1H), 7.49 - 7.30 (m, 7H), 7.21 (br s, 1H), 7.09 - 7.05 (m, 3H), 6.98 (br s, 1H), 6.55 (br s, 1H), 4.90 - 4.87 (m, 1H), 3.84 - 3.83 (m, 2H), 3.77 (br s, 2H), 3.52 (m, 2H), 2.86 - 2.61 (m, 8H), 2.07 (br s, 1H).

[0433] <Synthesizing Example 23-2> Novel Compound 23b [Chemical Formula 23b]

[0434] 4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0435] A novel compound 23b of [Chemical Formula 23b] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0437] H NMR (600 MHz, CDCl3) δ ppm 8.76 (d, J = 2.6 Hz, 1H), 8.59 (d, J = 4.7 Hz, 1H), 8.48 (br s, 1H), 7.30 (d, J = 7.9 Hz, 2H), 7.50 (br s, 1H), 7.46 (t, J = 8.0 1H), 7.32 - 7.31 (m, 3H), 7.15 (br s, 1H), 7.08 - 7.06 (m, 3H), 6.82 (d, J = 8.5 1H), 6.51 (t, J = 5.0, 1H), 4.87 - 4.84 (m, 1H), 3.83 (t, J = 5.2 Hz, 1H), 3.75 - 3.73 (m, 6H), 3.41 - 3.40 (m, 2H), 2.84 - 2.65 (m, 8H), 2.10 - 2.07 (m, 1H).

[0439] <Synthesizing Example 23-3> Novel Compound 23c [Chemical Formula 23c]

[0440] 5-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanamido)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylbenzamide

[0441] A novel compound 23c of [Chemical Formula 23c] was obtained by synthesizing in the same manner as in <Synthesizing Example 20-1> above.

[0443] H NMR (600 MHz, MeOD) δ ppm 8.53 (d, J = 4.4 Hz, 1H), 8.40 (br s, 1H), 7.70 (br s, 1H), 7.49 - 7.48 (m, 2H), 7.40 (br s, 2H), 7.24 (br s, 2H), 7.16 (t, J = 8.5 Hz, 2H), 6.96 - 6.96 (m, 1H), 6.80 - 6.78 (m, 1H), 5.03 - 5.00 (m, 1H), 3.82 (t, J = 5.8, 2H), 3.67 - 3.65 (m, 6H), 3.30 - 3.29 (m, 2H), 2.87 - 2.62 (m, 8H), 2.08 - 2.06 (m, 1H).

[0445] <Synthesizing Example 24-1> Novel Compound 24a [Chemical Formula 24a]

[0446] 1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpiperidine-4-carboxamide

[0447] A novel compound 24a of [Chemical Formula 24a] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0449] 1H NMR (600 MHz, CDCl3) δ 8.56 (dd, J = 23.6, 14.7 Hz, 2H), 7.54 - 7.42 (m, 2H), 7.32 - 7.27 (m, 1H), 7.18 - 7.01 (m, 6H), 6.90 (d, J = 8.5 Hz, 1H), 6.54 (dd, J = 39.5, 12.9 Hz, 1H), 4.91 (ddd, J = 17.2, 12.4, 5.2 Hz, 1H), 4.61 (d, J = 11.7 Hz, 1H), 4.01 (ddd, J = 17.3, 9.4, 9.0 Hz, 1H), 3.90 - 3.79 (m, 2H), 3.71 (s, 2H), 3.47 - 3.39 (m, 2H), 2.88 - 2.52 (m, 10H), 2.14 - 2.07 (m, 1H), 1.91 - 1.72 (m, 4H), 1.58 (d, J = 29.3 Hz, 2H)

[0451] <Synthesizing Example 24-2> Novel Compound 24b [Chemical Formula 24b]

[0452] 1-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)propanoyl)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpiperidine-4-carboxamide

[0453] A novel compound 24b of [Chemical Formula 24b] was obtained by synthesizing in the same manner as in <Synthesization Example 20-1> above.

[0455] 1H NMR (600 MHz, CDCl3) δ 8.60 - 8.46 (m, 2H), 7.57 (t, J = 7.0 Hz, 1H), 7.46 (dd, J = 18.8, 9.2 Hz, 1H), 7.30 (d, J = 4.5 Hz, 1H), 7.10 (ddd, J = 34.7, 27.6, 15.4 Hz, 6H), 6.77 (dd, J = 15.0, 7.7 Hz, 1H), 6.11 - 5.90 (m, 1H), 4.91 (dd, J = 12.2, 5.1 Hz, 1H), 4.70 (dd, J = 50.3, 15.2 Hz, 1H), 3.96 (t, J = 14.6 Hz, 1H), 3.84 - 3.69 (m, 4H), 3.42 - 3.30 (m, 2H), 2.92 - 2.56 (m, 10H), 2.12 (dd, J = 8.9, 4.0 Hz, 1H), 1.93 - 1.70 (m, 4H), 1.63 (s, 2H).

[0457] The novel compound 25 represented by [Chemical Formula 25] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 4] below.

[0458] [Reaction Equation 4]

[0459]

[0461] <Synthetic Example 25> Novel Compound 25 [Chemical Formula 25]

[0462] (2S,4R)-1-(2-(4-(2-(3-(difluoromethoxy)phenyl)morpholino)-4-oxobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

[0463] 2-(3-(difluoromethoxy)phenyl)morpholine (30 mg, 0.13 mmol), 4-((1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazole-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-4-oxobutanoic acid, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (527 mg, 1.38 mmol), and N,N-diisopropylethylamine (327 μl, 1.85 mmol) were reacted with dimethylformamide (5 ml) and the mixture was stirred for 4 hours, after which the mixture was diluted with ethyl acetate, washed with water and brine, and then After drying (MgSO4), the product was concentrated under vacuum. The product was purified by preparative chromatography (MPLC) to obtain the novel compound 25 of [Formula 25] (15 mg, 15%).

[0465] H NMR (600 MHz, DMSO-d6) δ ppm 8.99 (s, 1H), 8.58 - 8.56 (m, 1H), 7.96 - 7.92 (m, 1H), 7.45 - 7.38 (m, 6H), 7.35 - 7.18 (m, 3H), 7.13 - 7.12 (m, 1H), 5.12 (br s, 1H), 4.55 - 4.50 (m, 2H), 4.45 - 4.36 (m, 5H), 4.28 - 4.21 (m, 2H), 4.02 - 3.95 (m, 2H), 3.69 - 3.62 (m, 3H), 2.79 - 2.71 (m, 1H), 2.68 - 2.61 (m, 1H), 2.47 - 2.41 (m, 6H), 2.09 - 2.03 (m, 1H), 1.93 - 1.88 (m, 1H), 0.95 (s, 9H).

[0467] The novel compound 26 represented by [Chemical Formula 26] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 5] below.

[0468] [Reaction Equation 5]

[0469]

[0471] <Synthetic Example 26> Novel Compound 26 [Chemical Formula 26]

[0472] 3-(2-((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0473] 1-(4-fluorophenyl)-N-methyl-1-(pyridine-3-yl)methaneamine (41 mg 0.19 mmol), (S)-3-(2-((2-(2,6-dioxopiperidine-3-yl)-1-oxoisoindolin-4-yl)amino)ethoxy)propanoic acid (71 mg 0.19 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (87 mg, 0.23 mmol), and N,N-diisopropylethylamine (100 μl, 0.3 mmol) were reacted with dimethylformamide (1.9 ml) and stirred for 24 hours. Afterward, the mixture was diluted with ethyl acetate, washed with water and brine, dried (Na2SO4), and then concentrated under vacuum. The product was purified by high-performance liquid chromatography (HPLC) to obtain the novel compound 26 of [Chemical Formula 26].

[0475] 1H NMR (600 MHz, CDCl3) δ 8.89 (d, J = 82.7 Hz, 1H), 8.54 (d, J = 50.8 Hz, 2H), 7.45 (dt, J = 24.8, 12.5 Hz, 1H), 7.38 - 7.28 (m, 1H), 7.23 (t, J = 9.3 Hz, 2H), 7.17 (s, 1H), 7.13 - 6.96 (m, 4H), 6.80 - 6.71 (m, 1H), 5.15 - 5.07 (m, 1H), 4.40 - 4.31 (m, 1H), 4.24 - 4.12 (m, 1H), 3.88 - 3.73 (m, 4H), 3.34 (dd, J = 15.4, 9.4 Hz, 2H), 2.79 (s, 3H), 2.76 - 2.65 (m, 5H), 2.16 - 2.08 (m, 1H), 2.04 (dd, J = 8.5, 3.7 Hz, 1H).

[0477] The novel compound 27 represented by [Chemical Formula 27] according to the present invention can be produced through a synthesis process schematically illustrated in [Reaction Formula 6] below.

[0478] [Reaction Equation 6]

[0479]

[0481] <Synthetic Example 27> Novel Compound 27 [Chemical Formula 27]

[0482] 3-(2-((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0483] 1-(4-fluorophenyl)-N-methyl-1-(pyridine-3-yl)methaneamine (28 mg 0.13 mmol), (S)-3-(2-((2-(2,6-dioxopiperidine-3-yl)-1-oxoisoindolin-4-yl)oxy)ethoxy)propanoic acid (49 mg 0.13 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (59 mg, 0.16 mmol), and N,N-diisopropylethylamine (70 μl, 0.39 mmol) were reacted with dimethylformamide (1.3 ml) and stirred for 24 hours. Afterward, the mixture was diluted with ethyl acetate, washed with water and brine, dried (Na2SO4), and then concentrated under vacuum. The product was purified by high-performance liquid chromatography (HPLC) to obtain the novel compound 27 of [Chemical Formula 27].

[0485] 1H NMR (600 MHz, CDCl3) δ 8.61 - 8.41 (m, 2H), 7.52 - 7.45 (m, 2H), 7.41 (dd, J = 12.1, 5.8 Hz, 1H), 7.28 - 7.26 (m, 1H), 7.11 (ddd, J = 29.1, 12.8, 8.2 Hz, 3H), 7.06 - 6.97 (m, 3H), 5.20 (ddd, J = 21.7, 13.3, 5.1 Hz, 1H), 4.55 - 4.34 (m, 1H), 4.35 - 4.25 (m, 1H), 4.26 - 4.16 (m, 2H), 3.96 - 3.88 (m, 2H), 3.88 - 3.80 (m, 2H), 2.97 - 2.62 (m, 8H), 2.40 - 2.26 (m, 1H), 2.23 - 2.12 (m, 1H).

[0487] The novel compounds 28a and 28b represented by [Chemical Formula 28a] and [Chemical Formula 28b] according to the present invention can be prepared through a synthesis process schematically illustrated in [Reaction Scheme 7] below.

[0488] [Reaction Equation 7]

[0489]

[0491] <Synthesizing Example 28-1> Novel Compound 28a [Chemical Formula 28a]

[0492] 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0493] 1-(4-fluorophenyl)-N-methyl-1-(pyridine-3-yl)methaneamine (13 mg 0.06 mmol), 3-(2-((2-(2,6-dioxopiperidine-3-yl)-1,3-dioxosisoindolin-4-yl)oxy)ethoxy)propanoic acid (23 mg 0.06 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (27 mg, 0.072 mmol), N,N-diisopropylethylamine 30 μl, 0.18 mmol) were reacted with dimethylformamide (0.6 ml) and stirred for 18 hours. Afterward, the mixture was diluted with ethyl acetate, washed with water and brine, dried (Na2SO4), and then [under] vacuum The product was concentrated. The product was purified by high-performance liquid chromatography (HPLC) to obtain the novel compound 28a of [Chemical Formula 28a].

[0495] 1H NMR (600 MHz, CDCl3) δ 8.65 - 8.45 (m, 2H), 7.63 - 7.39 (m, 2H), 7.37 - 7.26 (m, 2H), 7.13 (dt, J = 24.4, 13.9 Hz, 4H), 7.10 - 6.94 (m, 2H), 5.01 - 4.87 (m, 1H), 4.26 - 3.88 (m, 2H), 3.82 (t, J = 5.9 Hz, 2H), 3.71 - 3.56 (m, 2H), 2.86 - 2.52 (m, 8H), 2.09 - 2.02 (m, 2H).

[0497] <Synthesizing Example 28-2> Novel Compound 28b [Chemical Formula 28b]

[0498] 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)ethoxy)-N-((4-fluorophenyl)(pyridin-3-yl)methyl)-N-methylpropanamide

[0499] 1-(4-fluorophenyl)-N-methyl-1-(pyridine-3-yl)methaneamine (22 mg 0.1 mmol), 3-(2-((2,6-dioxopiperidine-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)ethoxy)propanoic acid (39 mg 0.1 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (46 mg, 0.12 mmol), N,N-diisopropylethylamine 50 μl, 0.30 mmol) were reacted with dimethylformamide (1.0 ml) and stirred for 18 hours. Afterward, the mixture was diluted with ethyl acetate, washed with water and brine, dried (Na2SO4), and then concentrated under vacuum. The product was purified by high-performance liquid chromatography (HPLC) to obtain the novel compound 28b of [Chemical Formula 28b].

[0501] 1H NMR (600 MHz, CDCl3) δ 8.53 (dd, J = 24.8, 20.5 Hz, 2H), 7.62 (ddd, J = 31.3, 23.8, 7.9 Hz, 2H), 7.45 - 7.32 (m, 1H), 7.23 - 7.12 (m, 4H), 7.12 - 7.00 (m, 3H), 5.00 - 4.87 (m, 1H), 4.31 - 3.94 (m, 2H), 3.93 - 3.75 (m, 2H), 3.74 - 3.52 (m, 2H), 3.12 - 2.63 (m, 8H), 2.05 (tt, J = 84.8, 42.4 Hz, 2H).

[0503] [Example]

[0504] <Example 1> Discovery of Novel Compounds Binding to MDM2 Protein through Evaluation of Binding Ability Using LC-MS / MS

[0505] Proteomic techniques utilizing LC-MS / MS (Liquid chromatography-mass spectrometry) were applied to rapidly identify compounds that bind to the cysteine ​​(Cys, C)-introduced MDM2 (Mouse double minute 2) protein. The cysteine-introduced MDM2 protein was synthesized and prepared by exchanging the buffer four times with a composition of 25 mM Tris pH 7.4, 150 mM NaCl, 30 µM TCEP (tris(2-carboxyethyl)phosphine), and 1 mM PMSF. Fifty compound groups were prepared by mixing 10 non-overlapping compounds from 500 compounds selected via in-silico methods from the FBDD (Fragment-Based Drug Discovery) library, ensuring that each compound had a molecular weight within 1 Da, resulting in a concentration of 100 µM for each compound. MDM2 protein was added to 100 µl of each compound group solution to achieve a protein concentration of 20 µM, and the mixture was reacted at 4°C for 24 hours. Subsequently, IAA (indolacetic acid) was added and the mixture was reacted at 25°C for 4 hours to alkylate the remaining unreacted free cysteine, thereby terminating the reaction.

[0506] LC-MS / MS analysis was performed to confirm the covalent bond between the compound and the MDM2 protein. Prior to the analysis, the protein samples from which the reaction had concluded were solvent-exchanged and denatured to enable protein hydrolysis using trypsin. The peptides generated by hydrolysis were separated by reverse-phase chromatography and detected using a mass spectrometer. To confirm the covalent bond between the MDM2 protein and the compound and to evaluate the binding ability, the MS / MS spectra of the following four peptides were examined, and the quantitative values ​​of the peptides were determined from the peak area (or mass intensity).

[0507] 1) Target peptide: A trypsin hydrolyzed peptide containing target cysteine ​​introduced into a protein

[0508] 2) Target peptide bound to the compound

[0509] 3) Target peptide containing cysteine ​​alkylated by IAA

[0510] 4) Reference peptide: The representative MDM2 peptide generated by enzymatic protein degradation excluding the target peptide.

[0512] For each of the above peptides, the RBA (Relative binding affinity) (RBA = Quantity of target peptide bound to the compound / Quantity of reference peptide) was calculated by comparing it with the quantitative value of the reference peptide, and after arranging them in descending order, the compound corresponding to 80% of the total RBA was selected as a compound with high reactivity (binding strength) to the MDM2 protein.

[0514] <Example 2> 2D NMR Spectroscopic Analysis

[0515] In order to determine whether the novel compounds selected in Example 1 above interact with the protein action site, the MDM2 protein 1 H- 15 N HSQC spectra were measured using a Bruker 600 MHz NMR spectrometer (triple-resonance, pulse field gradient probe (Bruker, Germany)). Each spectrum was measured at a protein:compound molar concentration ratio of 1:5, and the data were processed with the NMRfx program and analyzed using the NMRview program.

[0516] The chemical shift perturbation (CSP) of the peak depending on the presence or absence of the compound was calculated using the following [Equation 1] (δ H and δ N are respectively the 1H-axis and 15(Represents the amount of change along the N-axis). For the bond strength between compounds, the average CSP was compared by selecting three major residues (G58, V93, Y100) among the MDM2 residues involved in bonding.

[0517] [Formula 1]

[0518]

[0520] As shown in Figure 1, the experimental results showed that when the average CSP of the three major amino acid sites (G58, V93, Y100) of the MDM2 protein was calculated, the average CSP for novel compounds 10 and 17 ranged from 0.045 to 0.055 ppm, while for novel compounds 2, 4, 5, 7, 8, 9, 12, 15, and 16, the average CSP ranged from 0.007 to 0.025 ppm, indicating that there was not a significant change in the major amino acids. The top three compounds were novel compounds 6, 11, and 13, which showed the largest change in average CSP, ranging from 0.07 to 0.09 ppm, while compound 19, the result of the optimization process for 6, showed the largest change at 0.116 ppm. This indicates that the affinity between the compounds and the MDM2 protein increased compared to the existing compounds due to the optimization process.

[0521] In addition, as shown in Figure 2, for each major amino acid of novel compounds 6 and 11 representing the average CSP of the top two major amino acids 1 H- 15 When comparing the N HSQC spectra, the novel compound 11 (gray) showed a large movement in Y100 compared to the control (black), while the changes in G58 and V93 were not significant. In the case of the novel compound 6, balanced changes in the major residues were observed overall, which means that the compound is responding well at the target site in the binding of the protein.

[0523] <Example 3> Expression and Purification of MDM2 Protein

[0524] MDM2 is E. coliIt was cloned from BL21 (DE3) RIPL by the expression vector pET-28a-ySUMO, an N-terminal His-tag fusion protein. Cell density (OD) at 37°C incubator. 600 After growing to 0.8, the temperature was lowered to 18°C ​​to induce protein expression with 0.5 mM IPTG (isopropyl-bD-thiogalactoside), and the samples were harvested after 16 hours. Uniformly labeled 15 To obtain N MDM2, bacterial cells 15Cells were grown in M9 minimal medium containing N NH4Cl. After obtaining the cells, they were resuspended in elution buffer (50 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, 7 mM β-ME (β-mercaptoethanol), and 1 mM PMSF (phenylmethylsulfonyl fluoride)). Cell lysis was performed by sonication while maintaining a low temperature on ice. Subsequently, the cell lysate was centrifuged at 3,800 rpm at 4°C for 30 minutes, and only the supernatant was collected and filtered through a 0.8 μM filter. The supernatant was run through a 10 mL nickel column (Sepharose 6FF, GE healthcare) and elution buffer (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole, 7 mM β-ME, and 1 mM PMSF) to obtain the His-SUMO-MDM2 fusion protein. ULP-1 protease was added to the solution containing the protein inside a 3.5 K membrane, and the fusion protein was cleaved overnight in dialysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 7 mM β-ME, and 1 mM PMSF). Subsequently, the solution inside the membrane was recovered and run through a 15 mL nickel column to obtain the MDM2 protein with the His-SUMO tag cleaved. Pure MDM2 protein was obtained by concentrating to 5 mL using 10K amicon (Merck, USA) and loading onto a Superdex S75 16 / 60 gel filtration column (16 / 60 GE Healthcare).

[0526] <Example 4> Evaluation of the MDM2 protein degradation efficacy of a novel compound

[0527] MDM2 is E.coliThe C-terminal GFP-P2A-RFP fusion protein was cloned from DH5α using the expression vector pcDNA5 / FRT and transfected into the human embryonic kidney cell line HEK293. During the translation of the MDM2-GFP-P2A-RFP protein, self-cleavage was induced at the P2A initiator, resulting in the production of MDM2-GFP and RFP proteins. Expression in protein-stable cell lines was observed by fluorescence microscopy (Zeiss, Germany) and flow cytometry (FACS, BD bioscience, USA) to monitor the luminescence of GFP and RFP.

[0528] HEK293 expressing MDM2 was cultured, and an appropriate amount of cells were maintained in a 96-well plate. Compounds according to the present invention (20a, 21a, 22) at predetermined concentrations (1, 10, 100 μM) were mixed and treated to the cells. After incubation for 24 hours at 37°C and 5% CO2, the cells were harvested, and GFP and RFP luminescence were analyzed using a flow cytometer. As a positive control, MD224, currently undergoing clinical trials, was co-treated at 10 nM for verification. The proteolytic efficacy was calculated using the following [Formula 2].

[0529] [Formula 2]

[0530] Protein degradation efficacy (Normalized GFP / RFP, 1) = GFP / RFP of sample χ GFP / RFP of control

[0532] As shown in Fig. 3a, the experimental results indicate that the normalized GFP / mCherry value of the negative control (DMSO) serves as the reference point (1.0), and values ​​approaching 0 signify that the MDM2 protein has been degraded. MD224, used as a positive control, exhibited a normalized GFP / mCherry value of approximately 0.2, demonstrating high protein degradation. Concentration-dependent reductions in MDM2 were observed for all of the novel compounds 20a, 21a, and 22. Notably, 20a showed protein degradation similar to that observed at 3 μM and 10 μM.

[0534] In addition, 30 μM of the compounds according to the present invention (20a, 20b, 20c, 20d, 21a, 21b, 22, 23a, 23b, 23c) were treated to cells in the same manner as above, and GFP and RFP luminescence were analyzed using a flow cytometer. As in the experiment above, 10 nM of MD224 was used as a positive control. Protein degradation efficacy was calculated using the following [Formula 3].

[0535] [Formula 3]

[0536] Degradation efficacy = [1 - Normalized GFP / RFP] x 100 %

[0538] As a result of the experiment, as shown in Figure 3b, MD224, used as a positive control, showed a protein degradation efficiency of 83.7% compared to the negative control (DMSO). Novel compounds 20a, 21a, and 22 showed a protein degradation efficiency of 32.6 to 37.2%, while 20b, 20c, 20d, 21b, 23a, 23b, and 23c showed a protein degradation efficiency of 5.5 to 31.1%. Through this experiment, it was confirmed that novel compounds based on the top three compounds obtained from 2D NMR spectroscopic analysis can degrade MDM2 protein.

[0540] <Example 5> Confirmation of MDM2 proteolytic efficacy of novel compound via Western blot

[0541] HEK293 expressing MDM2 according to Example 4 above was cultured to maintain an appropriate amount of cells in a cell culture plate. Then, the novel compounds 20a [Chemical Formula 20a], 21a [Chemical Formula 21a], and 22 [Chemical Formula 22] of the present invention were each treated to the cells under different concentration conditions (3, 10, 30 μM) or treatment time conditions (2, 6, 24 hours), cultured at 37°C and 5% CO2, and then Western blot was performed.

[0542] Experimental results confirmed that the novel compounds 20a and 21a exhibited excellent MDM2 protein degradation efficacy in a concentration-dependent manner (Fig. 4a), and in particular, the novel compound 20a also showed excellent MDM2 protein degradation efficacy as the treatment time increased (Fig. 4b).

[0543] Western blot analysis was performed after treatment with the novel compound 20a at more detailed concentration conditions (0.3, 1, 3, 10, 30, 100 μM), and it was confirmed that the compound exhibited excellent MDM2 protein degradation efficacy in a concentration-dependent manner, and a slight hook effect was observed in the case of treatment at 100 μM (Fig. 5).

[0544] In addition, Western blot was performed after treatment under more detailed treatment time conditions (2, 6, 12, 24, 48 hours) using the novel compound 20a, and it was confirmed that the longer the treatment time, the more excellent the MDM2 protein degradation efficacy was up to 24 hours, and there was no significant difference in degradation efficacy after 24 hours (Fig. 6).

[0546] <Example 6> Confirmation of the Mechanism of Action (MoA) of a Novel Compound via Western Blot

[0547] Western blot was performed to confirm the mechanism of action of the novel compound of the present invention using the novel compound 20f of the present invention, which has a methyl group added to the E3 ligase binder portion compared to the novel compound 20a of the present invention, the proteasome inhibitor MG-132, and the neddylation inhibitor MLN4924.

[0548] Specifically, HEK293 expressing MDM2 according to Example 4 above was cultured, and an appropriate amount of cells were maintained in a cell culture plate. Then, the cells were treated with the novel compounds 20a [Formula 20a] and 20f [Formula 20f] of the present invention, respectively, at different concentrations (3, 10 μM), cultured at 37°C and 5% CO2, and then Western blot was performed (Fig. 7a). The same cells were treated with MG-132 and MLN4924 at a non-dead cell concentration (0.4 μM), and after 4 hours, treated with the novel compound 20a [Formula 20a] of the present invention and cultured at 37°C and 5% CO2, and then Western blot was performed (Figs. 7b, 7c).

[0549] Experimental results confirmed that MDM2 was not degraded in all cases: when treated with the novel compound 20f (Fig. 7a), when treated with MG-132 at a concentration of 0.4 uM 4 hours prior to treatment with 20a (Fig. 7b), and when treated with MLN4924 at a concentration of 0.4 uM 4 hours prior to treatment with 20a (Fig. 7c). This indicates that the novel compound 20a degrades MDM2 according to the mechanism of PROTAC, which utilizes the ubiquitin proteasome system to cause protein degradation, which is the mechanism of the novel compound.

[0551] This specification omits detailed descriptions of matters that can be sufficiently recognized and inferred by those skilled in the art, and various modifications are possible within the scope of not altering the technical concept or essential configurations of the invention, in addition to the specific examples described herein. Accordingly, the invention may be implemented in a manner different from that specifically described and exemplified in this specification, and this is a matter that can be understood by those skilled in the art.

Claims

Claim 1 delete Claim 2 A compound characterized by binding to MDM2 (Mouse Double Minute 2 homolog) protein, and selected from any one of the compounds represented by [Chemical Formula 1] to [Chemical Formula 28b] below. Claim 3 In claim 2, the compound is a compound having the efficacy of inhibiting the activity of MDM2 protein. Claim 4 In claim 2, the compound is a compound having the efficacy to degrade MDM2 protein. Claim 5 delete Claim 6 A pharmaceutical composition for the prevention or treatment of a hyperproliferative disorder, comprising, as an active ingredient, the compound according to any one of claims 2 to 4 or a pharmaceutically acceptable salt thereof. Claim 7 A pharmaceutical composition according to claim 6, wherein the above-mentioned abnormal proliferative disease is cancer. Claim 8 A pharmaceutical composition according to claim 7, wherein the cancer is one or more selected from the group consisting of lung cancer, stomach cancer, pancreatic cancer, colorectal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, and brain tumor.

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