EP300 protein degrader and use thereof

Abstract

Provided are a compound defined by chemical formula 1, a stereoisomer thereof, a tautomer thereof, and a pharmaceutically acceptable salt thereof. In chemical formula 1, R is a site which binds to a histone acetyl transferase region of EP300. The compound of the present invention can reduce the intracellular level of EP300. [Chemical formula 1]

Description

EP300 Protein Degrading Agent and Uses thereof

[0001] The present invention relates to a novel heterofunctional group compound capable of inhibiting the activity of a specific protein in the body. Furthermore, the present invention relates to a small organic compound that induces the degradation of this specific protein.

[0002] Histone acetyltransferases (HATs) catalyze the acetylation of the ε-amino group of the target lysine side chain within the substrate histone protein, while histone deacetylases (HDACs) catalyze the removal of the acetyl group from the acetylated lysine residue. Histone acetyltransferases are broadly divided into four protein families, one of which is the lysine acetyltransferase type III (KAT3) protein family, which includes two proteins: EP300 (also known as p300) and CREB-binding protein (CBP), which are paralogous to each other. EP300 and CBP are proteins conserved in all animals within the metazoan subkingdom and share more than 90% sequence homology. EP300 and CBP contain several additional protein domains in addition to the enzymatically active histone acetyltransferase domain (HAT domain), including three protein domains (CH1, CH2, and CH3) rich in cysteine ​​and histidine, the KIX domain, the bromodomain, and the steroid receptor coactivator interaction domain (SRC-1 interaction domain). The HAT domain of EP300 and CBP is responsible for the acetylation of the majority of active histone H3s, lysine 18 and lysine 27, which are histone modifications associated with the activation of promoters and enhancers in the genome.

[0003] EP300 and CBP are involved in many cellular processes, including cell growth, proliferation, and differentiation. Mutations in EP300 and CBP have been observed in many human diseases, reaching a frequency of 30% in cancer. Mutations occur even more frequently within the HAT domain, suggesting that there is pressure in cancer that selectively modifies the acetyltransferase activity of EP300 or CBP.

[0004] In addition to its acetyltransferase function, EP300 activates transcription by acting as a scaffold for transcription factors or as a bridge between transcription factors and the basic components of transcription. High expression of EP300 has been observed in various cancers, which correlates with low survival rates and malignant phenotypes. Therefore, inhibiting the activity of EP300 or CBP holds promise as an anticancer treatment. In particular, in cancers lacking CBP, if only EP300 is selectively inhibited over CBP, the cancer cells lacking CBP die, while normal cells can avoid death because CBP can take over the function of EP300. Thus, treatment that selectively inhibits EP300 over CBP can be expected to reduce side effects because it can provide selective killing power against cancer cells.

[0005] However, there were several difficulties in finding small organic compound inhibitors with strong inhibitory effects on the acetyltransferase activity of EP300 (Cole, Nat. Chem. Biol. Vol. 4 (2008), pp. 590–597), and designing an inhibitor with high specificity for EP300 is not easy due to the sequence homology between CBP and EP300. Meanwhile, EP300 HAT inhibitors derived from natural compounds have moderate efficacy but lack specificity. There have also been attempts to block the efficacy of EP300 by inducing the in vivo degradation of target proteins called protein targeting chimeras (Protac), but there were difficulties in developing them as drugs because the design principles of heterogeneous functional group compounds used in protein targeting chimeras result in increased molecular weight and consequently poor physical properties.

[0006] For this reason, the demand in this field for small organic compounds with excellent efficacy and specificity that can inhibit the activity of EP300 or reduce the amount of EP300 has not yet been sufficiently met.

[0007] The technical problem of the present invention is to provide a novel compound capable of inhibiting the activity of EP300. More specifically, the technical problem of the present invention is to provide a compound capable of reducing the intracellular amount of EP300. Even more specifically, the technical problem of the present invention is to provide a compound with degradation-inducing activity that exhibits selectivity toward EP300 among CBP and EP300. Furthermore, the technical problem of the present invention is to provide a novel compound in the form of a small organic compound having these functions and a molecular weight lower than that of a heterogeneous functional group substance.

[0008] In one aspect of the present invention, a compound defined by Formula 1, its tautomers, its stereoisomers, or pharmaceutically acceptable salts thereof are provided.

[0009] [Chemical Formula 1]

[0010]

[0011] In this case, R in Chemical Formula 1 is the site that binds to the histone acetyltransferase domain (HAT domain) of EP300.

[0012] In one embodiment of the present invention, the aforementioned compound satisfies the structure of Formula 2.

[0013] [Chemical Formula 2]

[0014]

[0015] In this case, R' in Chemical Formula 2 is the site that binds to the histone acetyltransferase region of EP300. That is, compound R'-H can bind to the HAT region of EP300.

[0016] In a more specific embodiment of the present invention, the aforementioned R' is selected from Formula 3 or Formula 4.

[0017] [Chemical Formula 3]

[0018]

[0019] [Chemical Formula 4]

[0020]

[0021] In chemical formulas 3 and 4 The symbol indicates a position where it is covalently connected to the carbonyl carbon directly connected to R' of chemical formula 2.

[0022] X 1 and X 2 are independently CH or N,

[0023] L 5 is a chemical bond or

[0024] It is a 5-membered ring selected from among, where m is an integer from 0 to 2, and Is Indicates the connection in the display direction, Is Indicates a connection of directions.

[0025] Z 1 It is hydrogen or CH2-Z'. Z' is a 5-6-membered heteroaryl or phenyl.

[0026] (1) Z 1 When this is hydrogen, Z 2 ga is an 8- to 10-membered double-fused ring heteroaryl containing nitrogen, and

[0027] R 1 is methylene and R 2 is ethylene, and at this time R 1 and R 2 are connected to each other, and R 2 The nitrogen atom directly connected to and R 1 It forms an azacyclopentane ring with a carbon atom directly connected to it.

[0028] L 2 , L 3 and L 4 is non-existent;

[0029] L 1 It is a chemical bond or 1,1-cyclohexylene.

[0030] (2) Z 1 When this is CH2-Z', Z 2 α is a 4- to 7-membered cycloalkyl, and

[0031] L 1 is CH, and L 2 , L 3 and L 4 is methylene.

[0032] R 1 is hydrogen, and R 2 is C(=O)NH, where R 2 The nitrogen atom of L 1 It is directly connected to form an imidazolidin-2,4-dione ring.

[0033] R 3 and R 4Each is independently selected from the group consisting of hydrogen, halogen, OH, CN, NH2, and NO2. R 5 is a 5-6-membered heteroaryl or phenyl.

[0034] Only L in chemical formula 3 1 and L 5 Cases where all are chemical bonds are excluded, and

[0035] In Chemical Formulas 3 and 4, each of the azacyclopentane ring, 5-6 member heteroaryl or phenyl, 4-7 member cycloalkyl, 8-10 member difused ring heteroaryl 1,1-cyclohexylene or 4-7 member cycloalkyl may be unsubstituted or may be substituted with a functional group selected independently from each ring hydrogen atom from the group consisting of halogen, OH, CN, NH2 and NO2.

[0036] In another aspect of the present invention, a pharmaceutical composition for the treatment or prevention of cancer is provided, comprising a pharmaceutically acceptable excipient and containing the aforementioned compound, its tautomer, its stereoisomer, or its pharmaceutically acceptable salt in a therapeutically effective amount.

[0037] In yet another aspect of the present invention, a method for treating or preventing cancer is provided. The method comprises the step of administering a therapeutically effective amount of a compound of the present invention, such as a compound defined by Formula 1, or a pharmaceutical composition comprising such a compound of the present invention, to a subject requiring treatment or prevention.

[0038] The compound of the present invention can reduce the intracellular amount of EP300 in the form of a small organic compound. The compound of the present invention exhibits antitumor activity.

[0039] Embodiments of the present invention will be described in detail below. Prior to this, terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings. Instead, based on the principle that the inventor can appropriately define the concepts of terms to best describe his invention, they should be interpreted in a meaning and concept consistent with the technical spirit of the present invention.

[0040] Therefore, the embodiments described in this specification are merely examples presented for the purpose of helping to understand the invention and do not represent all technical ideas of the invention; thus, it should be understood that various equivalents and modifications that can replace them may exist at the time of filing this application.

[0041] Unless otherwise defined, all technical terms used in this invention are used in the sense generally understood by a person skilled in the art related to this invention. While preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. Numerical values ​​described herein are deemed to include the meaning of "approximately" unless explicitly stated otherwise. Numerical ranges indicated by the term "to" in this specification include ranges that include the values ​​described before and after the term "to" as lower and upper limits, respectively.

[0042] Definition of Terms

[0043] In this specification, EP300 is a term that encompasses E1A binding protein p300, whose amino acid sequence is the sequence described in GENBANK access number NP_001420.2 (human), the genetic sequence database of the National Institutes of Health (NIH), and all human or mammalian allelic variants and all isoforms of this polypeptide sequence.

[0044] In this specification, the term EP300 HAT region refers to a protein region in EP300 that possesses histone acetyltransferase activity, and is a polypeptide sequence corresponding to residues 1195 through 1673 of the sequence described in GENBANK access number NP_001420.2 (human), and encompasses all human or mammalian allelic variants of this sequence and all isomers thereof.

[0045] In this specification, the terms “binding” or “binding” of the entire compound or any part of the compound to a target indicate that the compound or the corresponding part of the compound specifically binds to the target substance. Specifically binding means that the binding strength of the compound or the corresponding part of the compound to the target is stronger than the binding strength to other substances other than the target. The dissociation constant (K) when the compound or a part of the compound binds to the first target. D When the value of ) is smaller than the dissociation constant value when binding to the second target, it can be said that it binds more strongly to the first target. Such targets may be proteins such as EP300 or E3 ligase, or specific sites or protein regions within a protein, such as the HAT domain of EP300. Methods for measuring the dissociation constant when a compound binds to proteins, including EP300 or E3 ligase, under in vivo conditions or environmental conditions close thereto are well known in the art. The value of the dissociation constant for the specifically bound target is, compared to the binding dissociation constant for a non-target substance, at least one-tenth or less, preferably one-twentieth or less, more preferably one-fiftieth or less, even more preferably one-hundredth or less, and even more preferably one-thousandth or less.

[0046] In this specification, to a specific atom of a substituent forming part of a compound, " " The presence of a mark indicates that the corresponding substituent is chemically bonded to the rest of the compound through that atom.

[0047] In this specification, if a connection line is marked with a dotted line such as "---", it indicates that the connection line is an optional element that may or may not exist. For example, Notations such as imply that this structural formula encompasses benzene and 1,3-cyclohexadiene. However, "---" or " The maximum possible number of covalent bonds between two adjacent ring atoms, implied or contained per dotted line mark such as ", does not necessarily have to be one, and if otherwise specified in this specification for the relevant chemical structural formula, it follows the number specified therein, e.g., the number designated as no bond, single bond, or double bond.

[0048] In this specification, the meaning of the statement that a functional group represented by a symbol may be a "chemical bond" when the symbols before and after the symbol are connected by chemical bonds means that there is no functional group at the position corresponding to the symbol.

[0049] In this specification, the term “pharmaceuticalally acceptable salt” for any compound refers to a type of salt that does not cause toxicity, irritation, or allergic reactions, etc., unsuitable for contact with human or animal tissues without impairing the desirable biological activity of the compound. Pharmaceutically acceptable salts are well known in the art. For example, reference may be made to prior art such as the article published by Berge et al. (1977), *Pharmaceutical Sciences*, Vol. 66, pp. 1–19. To give just a few examples of pharmaceutically acceptable salts, acid addition salts include hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, adipose, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrosulfide, etc., and base addition salts include alkali metal salts, alkaline earth metal salts, ammonium salts, and quaternary ammonium (N + (C 1~4 There are alkyls, and examples of alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. In this specification, "subject requiring treatment" or "subject" refers to a person diagnosed by a physician with a proliferative disease, e.g., cancer, or a mammal diagnosed by a veterinarian with a proliferative disease, e.g., cancer. Furthermore, "subject requiring treatment" or "subject" includes a person or mammal who has suffered from such a proliferative disease but whose symptoms have improved, and a subject suspected of having or likely to have such a proliferative disease, such as a person or mammal exhibiting one or more symptoms related to such a proliferative disease. Here, "proliferative disease" refers to a condition in which symptoms or progression can be alleviated by the compound of the present invention.

[0050] In this specification, "treatment" refers to a measure or action that alleviates, to some extent, the symptoms, markers, and / or all adverse effects of a condition in a subject currently suffering from such condition. In order to reduce the risk of progression to the said disease, disorder, and / or condition, depending on the embodiment, such treatment may be performed on a subject exhibiting only early signs of the said condition.

[0051] In this specification, "prevention" refers to any method of wholly or partially delaying or preventing the manifestation of one or more symptoms or signs of a related disease, condition, and / or pathology. Prevention may be performed on subjects that do not exhibit signs of the disease, condition, and / or pathology.

[0052] A person skilled in this field will be aware that some of the compounds of the present invention may exist in one or more tautomeric forms. Since a single chemical structural formula can only represent one tautomeric form, it will be well understood that when a compound is referred to by a single structural formula for convenience, that formula encompasses even the tautomeric variations of the compound. Depending on the compound, it may primarily exist in one of several tautomeric forms, exist as a mixture of multiple tautomerics at room temperature, or be able to isolate only a single tautomeric form. Examples of tautomeric forms include the pyridone form and the hydroxypyridine form, and the keto form and the enol form.

[0053] compound

[0054] In one aspect of the present invention, a compound defined by Formula 1, its tautomers, its stereoisomers, or pharmaceutically acceptable salts thereof are provided.

[0055]

[0056] In Chemical Formula 1, R is the site that binds to the histone acetyltransferase (HAT) region of EP300.

[0057] The compound of the present invention has the property of binding to E3 ligase.

[0058] In one embodiment, the compound of the present invention further takes the structure of Formula 2.

[0059]

[0060] In Chemical Formula 2, R' is the binding site to the histone acetyltransferase region of EP300. That is, compound R'-H can bind to the HAT region of EP300.

[0061] In one specific embodiment of the compound of the present invention, R' of Formula 2 is in the form of Formula 3.

[0062]

[0063] In chemical formula 3 The symbol indicates a position where it is covalently connected to the carbonyl carbon directly connected to R' of chemical formula 2.

[0064] X in Chemical Formula 3 1 and X 2 They are independently CH or N.

[0065] In chemical formula 3, L 5 It is a chemical bond or a five-membered ring selected from the following functional groups.

[0066] In this case, m is an integer from 0 to 2, and Is Indicates the connection in the direction of indication, Is Indicates a connection in the direction;

[0067] Z 1 It is hydrogen or CH2-Z', where Z' is a 5-6-membered heteroaryl or phenyl.

[0068] (1) Z in Chemical Formula 3 1When this is hydrogen, Z 2 It is an 8- to 10-membered double-fused ring heteroaryl containing nitrogen.

[0069] Z 1 When this is hydrogen, R 1 This is methylene and R 2 ga is ethylene, and at this time R 1 and R 2 are connected to each other, and R 2 The nitrogen atom directly connected to and R 1 It forms an azacyclopentane ring with a carbon atom directly connected to it.

[0070] Z 1 When this is hydrogen, L 2 , L 3 and L 4 is non-existent,

[0071] L 1 It is a chemical bond or 1,1-cyclohexylene.

[0072] (2) Z in Chemical Formula 3 1 When this is CH2-Z', Z 2 α is a 4- to 7-membered cycloalkyl.

[0073] L 1 is CH, and L 2 , L 3 and L 4 is methylene, and

[0074] R 1 It is hydrogen.

[0075] Z in Chemical Formula 3 1 When this is hydrogen, R 2 is C(=O)NH, and at this time R 2 The nitrogen atom of L 1 Directly connected to R 2 , L 1 This R 2 and L 1 It forms an imidazolidin-2,4-dione ring together with the interposed NC(=O).

[0076] However, in R' of the aforementioned chemical formula 3, L 1and L 5 Cases where all are chemical bonds are excluded.

[0077] R in Chemical Formula 3 1 , R 2 , L 1 , Z' and Z 2 It may be unsubstituted. Alternatively, each of the azacyclopentane ring, 5-6 member heteroaryl or phenyl, 4-7 member cycloalkyl, 8-10 member difused ring heteroaryl 1,1-cyclohexylene or 4-7 member cycloalkyl of the aforementioned chemical formula 3 may optionally be substituted with a functional group independently selected from the group consisting of halogen, OH, CN, NH2 and NO2 for each ring hydrogen atom.

[0078] In one specific embodiment of the compound of the present invention, R' of Formula 2 is in the form of Formula 4.

[0079]

[0080] In chemical formula 4 The notation indicates a position connected by a covalent bond to the carbonyl carbon directly connected to R' of Chemical Formula 2, and

[0081] X 1 and X 2 They are independently CH or N.

[0082] L 5 is a chemical bond or

[0083] It is a 5-membered ring selected from among, where m is an integer from 0 to 2, and Is Indicates the connection in the direction of indication, Is Indicates a connection in the direction.

[0084] In chemical formula 4, Z' is a 5-6-membered heteroaryl or phenyl.

[0085] R 3 and R 4Each is independently selected from the group consisting of hydrogen, halogen, OH, CN, NH2, and NO2. R 5 is a 5-6-membered heteroaryl or phenyl.

[0086] Z' and R in Chemical Formula 4 5 can be unsubstituted. Or the above Z' and R 5 Each of the 5-6-membered heteroaryls or phenyls may be substituted with one or more of its ring hydrogen atoms by functional groups selected independently from each ring hydrogen atom from the group consisting of halogens, OH, CN, NH2 and NO2.

[0087] In a more specific embodiment of the compound of the present invention, R' of Formula 2 takes the structure of any one of Formulas 5 to 8 below.

[0088]

[0089]

[0090]

[0091]

[0092] In chemical formulas 5 to 8 The notation indicates the position connected by a covalent bond to the carbonyl carbon leading to R' in chemical formula 2.

[0093] In one more specific embodiment of the present invention, the compound of Formula 7 takes the structure of Formula 9 below.

[0094]

[0095] In another more specific embodiment of the present invention, the compound of Formula 8 takes the structure of Formula 10 below.

[0096]

[0097] In chemical formulas 9 and 10 The notation indicates the position connected by a covalent bond to the carbonyl carbon leading to R' in Chemical Formula 2.

[0098] In the most specific embodiment of the compound of the present invention, the compound of the present invention is the following compound, its stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt:

[0099] (E)-N-Benzyl-N-Cyclohexyl-2-(6'-(1-(2-(4-(4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-1-yl)acetamide,

[0100] (R,E)-N-Benzyl-N-Cyclohexyl-2-(6'-(1-(2-(4-(4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-1-yl)acetamide,

[0101] (S,E)-N-Benzyl-N-Cyclohexyl-2-(6'-(1-(2-(4-(4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-1-yl)acetamide,

[0102] (E)-2-((4-cyanofenethyl)amino)-N-(5-(1-(2-(4-(4-methoxyphenyl)-4-oxobut-2-enoil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)pyridine-2-yl)-2-phenylacetamide,

[0103] (E)-4-fluoro-1-(1-(4-(4-(4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-carbonyl)phenyl)cyclohexanecarbonyl)-N-(1H-pyrazolo[4,3-b]pyridin-5-yl)pyrrolidin-2-carboxamide,

[0104] (2R,4R)-4-fluoro-1-(1-(4-(4-((E)-4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-carbonyl)phenyl)cyclohexanecarbonyl)-N-(1H-pyrazolo[4,3-b]pyridin-5-yl)pyrrolidin-2-carboxamide,

[0105] (E)-4-fluoro-1-(4-(1-(2-(4-(4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)benzoyl)-N-(1H-pyrazolo[4,3-b]pyridin-5-yl)pyrrolidin-2-carboxamide and

[0106] (2R,4R)-4-fluoro-1-(4-(1-(2-(4-((E)-4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)benzoyl)-N-(1H-pyrazolo[4,3-b]pyridin-5-yl)pyrrolidine-2-carboxamide.

[0107] The compound of the present invention can reduce the intracellular amount of EP300. The compound of the present invention can reduce the intracellular activity of EP300.

[0108] In one embodiment of the present invention, the compound of the present invention selectively reduces the amount of intracellular EP300 compared to CBP.

[0109] Synthesis of the compound of the present invention

[0110] The compound of Formula I of the present invention may be synthesized by a synthetic route comprising methods similar to those well known in the field of chemistry. The starting material may generally be available from commercial sources, such as Aldrich Chemicals (Milwaukee, Wisconsin, USA), or may be prepared using methods well known to those skilled in the art (e.g., Reagents for Organic Synthesis, v. 1-19, Wiley, NY (eds. 1967–1999) by Louis F. Fieser and Mary Fieser or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin; including appendix (also available via the Beilstein online database)).

[0111] Pharmaceutical composition

[0112] In another aspect of the present invention, a pharmaceutical composition is disclosed that contains the compound of the present invention described above in a therapeutically effective amount and further contains a pharmaceutically acceptable excipient. A pharmaceutical composition comprising one compound according to the present invention and a pharmaceutically acceptable excipient can be used to reduce the intracellular amount of EP300 in the body. For example, this pharmaceutical composition can be used for the treatment of cancer.

[0113] In the pharmaceutical composition of the present invention, the aforementioned compound may exist as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, for example, base addition salts and acid addition salts. Pharmaceutically acceptable base addition salts may be formed as addition salts of metals or amines, for example, alkali metal bases, alkaline earth metal bases, or organic amines. Pharmaceutically acceptable salts of the compound may also be prepared using pharmaceutically acceptable cations. Pharmaceutically acceptable acid addition salts include inorganic acid or organic acid salts.

[0114] In the present invention, a pharmaceutically acceptable excipient refers to an inactive ingredient approved by relevant administrative authorities (e.g., the Korea Food and Drug Administration, the U.S. Food and Drug Administration (FDA)) as suitable for use in combination with an active pharmaceutical ingredient for the manufacture of medicines for the treatment of diseases in humans or livestock. Such pharmaceutically acceptable excipients include, but are not limited to, carriers, lubricants, fluidizing agents, disintegrants, sweeteners, diluents, preservatives, coloring agents, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvent emulsifiers, and adjuvants.

[0115] The pharmaceutical composition of the present invention may be in a form suitable for oral administration, such as, for example, tablets, capsules, pills, powders, sustained-release formulations, solutions, and suspensions; in a form suitable for parenteral injection, such as sterile solutions, suspensions, or emulsions; in a form suitable for topical administration, such as ointments or creams; or in a form suitable for rectal administration, such as suppositories.

[0116] The pharmaceutical composition according to the present invention can be prepared in a conventional manner, for example by conventional mixing, dissolving, granulating, making a coated tablet, powdering, emulsifying, encapsulating, encapsulating, or freeze-drying processes. The appropriate formulation depends on the selected route of administration.

[0117] Pharmaceutical compositions suitable for oral administration can be easily formulated by combining the compounds disclosed herein with pharmaceutically acceptable excipients, such as carriers well known in the art. Using such excipients and carriers, the compounds disclosed herein can be formulated as tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by a target to be treated. Oral pharmaceutical formulations can be obtained by adding the compounds of the present invention together with solid excipients, grinding the resulting mixture as needed, adding suitable adjuvants if necessary, and then processing the granular mixture to form tablet or coated tablet cores. Suitable excipients include, for example, fillers and cellulose preparations. If necessary, disintegrants may be added.

[0118] In the case of a pharmaceutical composition for oral administration of a therapeutically effective amount of the compound of the present specification, the composition is generally in the form of a solid (e.g., tablet, capsule, pill, powder, or troche) or a liquid formulation (e.g., aqueous suspension, solution, elixir, or syrup).

[0119] When administered in the form of tablets, the composition may additionally contain a functional solid and / or solid carrier, such as gelatin or an adjuvant. Compositions in the form of tablets, capsules, and powders may contain the compound of the present invention in an amount of about 1 to about 95 weight% based on the total weight of the composition, preferably in an amount of about 15 to about 90 weight%. Examples of tablet compositions include, for instance, about 80 weight% or less of an active pharmaceutical ingredient, about 10 weight% to about 90 weight% of a binder, about 0 weight% to about 85 weight% of a diluent, about 2 weight% to about 10 weight% of a disintegrant, and about 0.25 weight% to about 10 weight% of a lubricant.

[0120] When administered in liquid or suspension form, a functional liquid and / or liquid carrier, such as water, petroleum, or animal or vegetable oil, may be added. The composition in liquid form may further contain physiological saline, a sugar alcohol solution, dextrose or other sugar solutions, or glycol. When administered in liquid or suspension form, the composition may contain about 0.5 to about 90 weight percent of the compound of the present invention, preferably about 1 to about 50 percent of the compound of the present invention. In one embodiment considered, the liquid carrier is non-aqueous or substantially non-aqueous. For administration in liquid form, the composition may be supplied as a rapidly dissolving solid formulation for dissolution or suspension immediately before administration.

[0121] When the pharmaceutical composition of the present invention is administered by intravenous, deslicating, or subcutaneous injection, it is in the form of a parenteral aqueous solution that does not contain a pyrogen. The preparation of such parenteral solutions, taking into account pH, isotonicity, stability, etc., falls within the art of the art. Preferred compositions for intravenous, deslicating, or subcutaneous injection generally contain an isotonic vehicle. Such compositions may be prepared for administration as a solution of a free base or a pharmacologically acceptable salt in water, suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as dispersions in oil, may also be prepared. Under normal storage and use conditions, such formulations may optionally contain a preservative to prevent microbial growth.

[0122] The injectable composition may comprise a sterile aqueous solution, suspension, or dispersion, and a sterile powder for the immediate preparation of the sterile injectable solution, suspension, or dispersion. The sterile injectable solution is prepared by mixing the required amount of the active compound, along with the various other components as needed, in a suitable solvent, and then filtering and sterilizing. Generally, the dispersion is prepared by mixing various sterile active ingredients in a sterile vehicle containing a base dispersion medium and other necessary components among those listed above. In an embodiment of the sterile powder for the preparation of the sterile injectable solution, a preferred manufacturing method is vacuum drying and freeze-drying techniques to produce a powder of the active ingredient and any additional necessary components from a pre-sterile filtered solution.

[0123] Sustained-release or sustained-release formulations may be prepared to achieve controlled release of an active compound in contact with body fluids in the gastrointestinal tract, and to provide substantially constant and effective levels of the active compound in plasma. For example, release may be controlled by one or more of dissolution, diffusion, and ion exchange. Additionally, sustained-release approaches may enhance absorption through saturation or restriction pathways within the gastrointestinal tract. For example, for this purpose, the compound may be embedded in a polymer matrix consisting of a biodegradable polymer, a water-soluble polymer, or a mixture thereof, and optionally a suitable surfactant. In this context, embedding may mean the incorporation of microparticles into a polymer matrix. Controlled-release formulations may also be obtained by encapsulating dispersed microparticles or emulsified microdroplets through known dispersion or emulsion coating techniques.

[0124] The pharmaceutical composition of the present invention may be formulated for parenteral administration by injection (e.g., bolus injection or continuous infusion). The injectable formulation may be provided in a unit dosage form (e.g., ampoule or multi-dose container) with added preservatives. The composition may take the form of a suspension, solution, or emulsion in an oil or aqueous vehicle and may contain formulation agents such as suspenders, stabilizers, and / or dispersants.

[0125] The pharmaceutical composition may exist in a unit dosage form suitable for a single administration of an accurate dosage.

[0126] A pharmaceutical composition containing the compound of the present invention can be used according to the method described below.

[0127] Treatment or prevention methods

[0128] In another aspect, the present invention provides a method for treating or preventing cancer. The method comprises the step of administering a therapeutically effective amount of a compound of the present invention, such as a compound of the present invention defined by Formula 1, to a subject requiring treatment or prevention. Alternatively, the present invention comprises the step of administering a pharmaceutical composition comprising the aforementioned compound of the present invention and a pharmaceutically acceptable excipient to a subject requiring treatment, in a therapeutically effective amount.

[0129] In a method for treating cancer, the compound of the present invention may be administered alone or in combination with at least one medicine. The compound of the present invention and such other medicine may be administered simultaneously or sequentially.

[0130] In this specification, unless specifically stated otherwise, the term “therapeutic effective dose” of any compound refers to an amount of the compound sufficient to delay or minimize one or more symptoms associated with any disease, condition, or state, or an amount of the compound sufficient to provide a therapeutic effect against any disease, condition, or state. The term “therapeutic effective dose” encompasses an amount that improves overall treatment, an amount that alleviates or avoids the cause of symptoms or the disease or state, and even an amount that enhances the therapeutic efficacy of other therapeutic agents.

[0131] The amount of compound administered may vary depending on the subject requiring treatment or prophylaxis, the subject's age, health, gender, and weight, the type of concurrent treatment (if any), the severity of pain, the nature of the desired effect, the mode and frequency of treatment, and the prescribing physician's judgment. The frequency of administration may also vary depending on the pharmacodynamic effect on arterial oxygen pressure. However, the most desirable dosage may be tailored to the individual subject. This generally involves adjusting the standard dose (e.g., reducing the dose if the subject's body weight is low).

[0132] When administered to humans for the therapeutic or prophylactic treatment of pathological conditions and disorders using the compounds of the present invention, for example, a typical dosage of the compounds of the present invention may be about 0.01 mg / kg / day to about 100 mg / kg / day, for example, about 5 mg / day to about 500 mg / day. These doses may be administered as a single dose or divided into multiple doses.

[0133] The present invention will be explained in more detail below through the following examples and experimental examples. However, these examples and experimental examples are intended only to aid in understanding the present invention and do not limit the scope of the present invention in any way. Various changes and modifications may be made to these examples, and such changes and modifications are also included within the scope of the appended claims.

[0134] The full names of the abbreviations listed in the synthesis method of the following manufacturing example are as follows.

[0135] Boc: tertiary butyloxycarbonyl

[0136] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate

[0137] DEIPA:N,N-Diethylisopropylamine DMF:N,N-Dimethylformamide

[0138] TEA: Triethylamine LiHMDS: Lithium bis(trimethylsilyl)amide

[0139] SPhos Pd G3: (2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl)[2-(2'-amino-1,1'-biphenyl)palladium(II)methanesulfonate

[0140] TFA: Trifluoroacetic acid THF: Tetrahydrofuran tBu: Tertiary butyl

[0141] DIPEA: N,N-Diisopropylethylamine DCM: Dichloromethane

[0142] EtOH: Ethanol

[0143] FmocCl: Fluorenylmethyloxycarbonyl chloride

[0144]

[0145] Preparation Example 1: Preparation of (E)-1-(4-methoxyphenyl)-4-(piperazine-1-yl)bu-2-en-1,4-dione 2,2,2-trifluoroacetate

[0146]

[0147] Step 1) Preparation of tert-butyl (E)-4-(4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-carboxylate

[0148] 1.0 g (4.66 mmol) of (E)-4-(4-methoxyphenyl)-4-oxo-but-2-enoic acid and 2.2 g (5.59 mmol) of HATU were diluted in 40 mL of N,N-dimethylformamide, and 2.5 mL (14.00 mmol) of N,N-diethylisopropylamine was added under a nitrogen atmosphere at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. 1.0 g (5.12 mmol) of tert-butylpiperazine-1-carboxylate was diluted in 40 mL of N,N-dimethylformamide and added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 5 hours. Once the reaction was complete, the mixture was washed with water and extracted with ethyl acetate. The organic layer thus separated was dried with anhydrous sodium sulfate, filtered under reduced pressure, and distilled under reduced pressure. The residue obtained in this way was separated by column chromatography (dichloromethane : methanol = 20 : 1 (volume ratio)) to obtain 1.6 g of the title compound (yield: 93%).

[0149] 1 H-NMR (300 MHz, CDCl3)δ8.04 (d, 2H), 7.96 (dd, 1H), 7.46 (d, 1H), 6.98 (d, 2H), 3.89 (s, 3H), 3.72 (t, 2H), 3.62 (t, 2H), 3.49 (t, 4H), 1.48 (s, 9H).

[0150] Step 2) Preparation of (E)-1-(4-methoxyphenyl)-4-(piperazine-1-yl)bu-2-en-1,4-dione 2,2,2-trifluoroacetate

[0151] 70 mg (0.19 mmol) of the compound prepared in Step 1) above was diluted in 0.46 mL (5.98 mmol) of trifluoroacetic acid. The resulting reaction mixture was stirred at room temperature for 1 hour. Once the reaction was complete, the reaction mixture was distilled under reduced pressure to obtain 73 mg of the title compound (yield: 94%).

[0152] 1H-NMR (300 MHz, CDCl3)δ9.17 (brs, 1H), 8.05~8.00 (m, 3H), 7.45~7.35 (m, 1H), 7.01~6.97 (m, 2H), 4.06~4.01 (d, 4H), 3.90 (s, 3H), 3.36~3.33 (m, 4H).

[0153] Preparation Example 2: Preparation of (S)-2-(4-(1-(2-(benzyl(cyclohexyl)amino)-2-oxoethyl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-6'-yl)-1H-pyrazole-1-yl)acetic acid

[0154]

[0155] Step 1) Preparation of 6'-bromo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione

[0156] 5.1 g (22.2 mmol) of 6-bromo-3,4-dihydronaphthalene-1(2H)-one was diluted in 25 mL of ethanol, and 2.3 g (33.3 mmol) of potassium cyanide, 7.5 g (77.8 mmol) of ammonium carbonate, and 2.8 g (22.2 mmol) of water ammonia were added. The reaction mixture was stirred at 60°C for 18 hours. Upon completion of the reaction, the mixture was cooled to room temperature, washed with distilled water, and extracted with a dichloromethane:methanol = 4:1 (volume ratio) solution. The organic layer thus separated was dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The resulting solid was diluted with ethyl acetate and stirred at room temperature for 30 minutes. The resulting mixture was washed with ethyl acetate and vacuum filtration to obtain 5.7 g of the title compound (yield: 88%).

[0157] 1H-NMR (300 MHz, DMSO-d6): δ10.86 (brs, 1H), 8.52 (s, 1H), 7.40~7.38 (m, 2H), 7.04~7.01 (d, 1H), 2.79~2.75 (t, 2H), 2.11~2.02 (m, 2H), 1.94~1.76 (m, 2H).

[0158] Step 2) Preparation of N-Benzyl-2-Bromo-N-Cyclohexylacetamide

[0159] 11 mL (52.8 mmol) of N-benzylcyclohexaneamine was diluted in 120 mL of dichloromethane, and 9.4 mL (106 mmol) of 2-bromoacetyl bromide was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 3 hours. Once the reaction was complete, water was added and the mixture was extracted with dichloromethane. The organic layer thus separated was dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained was diluted with ethyl acetate, stirred at room temperature for 30 minutes, and then vacuum filtration. The resulting filtrate was separated by column chromatography (hexane:ethyl acetate = 6:1 (volume ratio)) to obtain 10.1 g (yield: 62%) of the title compound.

[0160] 1 H-NMR (300 MHz, CDCl3): δ7.39~7.30 (m, 3H), 7.28~7.19 (m, 2H), 4.59 (s, 2H), 4.58~4.42 (m, 1H), 3.99~3.88 (m, 2H), 1.83~1.07 (m, 10H).

[0161] Step 3) Preparation of N-benzyl-2-(6'-bromo-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-1-yl)-N-cyclohexylacetamide

[0162] 4.2 g (14.30 mmol) of the compound prepared in Step 1) above was diluted in 80 mL of N,N-dimethylformamide, and 5.3 g (17.20 mmol) of the compound prepared in Step 2) above and 5.0 g (35.80 mmol) of potassium carbonate were added. This reaction mixture was stirred at room temperature for 3 hours. Once the reaction was complete, water was added and stirred for 30 minutes. The solid obtained was washed with water and filtered under reduced pressure. The solid obtained in this way was diluted in dichloromethane, dried with anhydrous sodium sulfate, and then subjected to reduced pressure filtration and reduced pressure distillation. The residue obtained in this way was separated by column chromatography (hexane:ethyl acetate = 2:1 (volume ratio)) to obtain 3.6 g (yield: 47%) of the title compound.

[0163] 1 H-NMR (300 MHz, CDCl3): δ7.42~7.17 (m, 8H), 5.81~5.76 (m, 1H), 4.62~4.50 (m, 3H), 4.42~4.23 (d, 1H), 2.95~2.82 (m, 2H), 2.38~2.23 (m, 2H), 2.10~2.00 (m, 2H), 1.85~0.95 (14 H).

[0164] Step 4) Preparation of (S)-N-Benzyl-2-(6'-Bromo-2,5-Dioxo-3',4'-Dihydro-2'H-Spiro[Imidazolidine-4,1'-Naphthalene]-1-yl)-N-Cyclohexylacetamide

[0165] 2.6 g (4.96 mmol) of the racemic mixture prepared in Step 3) above was separated by prep-HPLC (Chiral ART cellulose SZ; hexane:ethanol = 7:3 (volume ratio) + 0.2% diethylamine) to determine the lag time (R) among the enantiomers of the title compound. t 1.1 g of compound with a duration of 26.5 minutes was obtained.

[0166] 1H-NMR (300 MHz, CDCl3): δ7.42~7.17 (m, 8H), 5.81~5.76 (m, 1H), 4.62~4.50 (m, 3H), 4.42~4.23 (d, 1H), 2.95~2.82 (m, 2H), 2.38~2.23 (m, 2H), 2.10~2.00 (m, 2H), 1.85~0.95 (14 H).

[0167] Step 5) Preparation of (S)-2-(4-(1-(2-(benzyl(cyclohexyl)amino)-2-oxoethyl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-6'-yl)-1H-pyrazole-1-yl)acetic acid

[0168] 1.1 g (2.19 mmol) of the compound prepared in step 4) above was diluted with 20 mL of a mixed solution of water and dioxane (water:1,4-dioxane by volume ratio = 1:4), 875 mg (3.29 mmol) of methyl 2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)pyrazole-1-yl]acetate, 180 mg (0.22 mmol) of (2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate, and 2.14 g (6.58 mmol) of cesium carbonate were added sequentially, and the mixture was stirred at 100°C for 16 hours. When the reaction was complete, the reaction mixture was cooled to room temperature and 2 N hydrochloric acid was slowly added, after which the resulting solid was washed with water, filtered under reduced pressure, and dried at 50°C to obtain 1.2 g of the title compound (yield: 94%).

[0169] 1H-NMR (300 MHz, DMSO-d6): δ 8.87 (d, 1H), 8.14 (d, 1H), 7.89 (d, 1H), 7.41~7.36 (m, 5H), 7.28~7.19 (m, 3H), 4.95 (d, 2H), 4.66 (s, 1H), 4.53 (d, 2H), 2.89~2.75 (m, 2H), 2.13~1.89 (m, 4H), 1.70~1.13 (m, 10H), 1.11~1.01 (m, 2H).

[0170] Preparation Example 3: Preparation of (R)-2-(4-(1-(2-(benzyl(cyclohexyl)amino)-2-oxoethyl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-6'-yl)-1H-pyrazole-1-yl)acetic acid

[0171]

[0172] Step 1) Preparation of (R)-N-benzyl-2-(6'-bromo-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-1-yl)-N-cyclohexylacetamide

[0173] 2.6 g (4.96 mmol) of the racemic mixture prepared in Step 3) of Preparation Example 2 above was separated by preparative HPLC (Chiral ART cellulose SZ; hexane:ethanol = 7:3 (volume ratio) + 0.2% diethylamine), and the enantiomer and lag time (R) of the compound in Step 4) of Preparation Example 2 were obtained t 1.2 g of compound with a value of 28.0 min was obtained.

[0174] 1 H-NMR (300 MHz, CDCl3): δ7.42~7.17 (m, 8H), 5.81~5.76 (m, 1H), 4.62~4.50 (m, 3H), 4.42~4.23 (d, 1H), 2.95~2.82 (m, 2H), 2.38~2.23 (m, 2H), 2.10~2.00 (m, 2H), 1.85~0.95 (14 H).

[0175] Step 2) Preparation of (R)-2-(4-(1-(2-(benzyl(cyclohexyl)amino)-2-oxoethyl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-6'-yl)-1H-pyrazole-1-yl)acetic acid

[0176] 1.1 g (2.19 mmol) of the compound prepared in step 1) above was diluted with 20 mL of a mixed solution of water and 1,4-dioxane (water:1,4-dioxane = 1:4 (volume ratio)), 875 mg (3.29 mmol) of methyl 2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)pyrazole-1-yl]acetate, 180 mg (0.22 mmol) of (2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II)methanesulfonate, and 2.14 g (6.58 mmol) of cesium carbonate were added sequentially, and the mixture was stirred at 100°C for 16 hours. When the reaction was complete, the reaction mixture was cooled to room temperature and 2N hydrochloric acid was slowly added, after which the resulting solid was washed with water, filtered under reduced pressure, and dried at 50°C to obtain 1.1 g of the title compound (yield: 89%).

[0177] 1 H-NMR (300 MHz, DMSO-d6): δ8.87 (d, 1H), 8.14 (d, 1H), 7.89 (d, 1H), 7.41~7.36 (m, 5H), 7.28~7.19 (m, 3H), 4.95 (d, 2H), 4.66 (s, 1H), 4.53 (d, 2H), 2.89~2.75 (m, 2H), 2.13~1.89 (m, 4H), 1.70~1.13 (m, 10H), 1.11~1.01 (m, 2H).

[0178] Preparation Example 4: Preparation of 2-(4-(6-(2-((4-cyanopenetyl)amino)-2-phenylacetamido)pyridine-3-yl)-1H-pyrazole-1-yl)acetic acid

[0179]

[0180] Step 1) Preparation of ethyl 2-((4-cyanopenetyl)amino)-2-phenylacetate

[0181] 10 g (41.14 mmol) of ethyl 2-bromo-2-phenylacetate was diluted in 50 mL of N,N-dimethylformamide, and 9.7 g (53.48 mmol) of 4-(2-aminoethyl)benzonitrile hydrochloride and 17.2 mL (123.41 mmol) of triethylamine were added sequentially, followed by stirring at 60°C for 3 hours. Once the reaction was complete, the reaction mixture was diluted with ethyl acetate and washed with saturated brine. The organic layer thus separated was dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The resulting residue was separated by column chromatography (hexane:ethyl acetate = 2:1 (volume ratio)) to obtain 9.5 g of the title compound (yield: 75%).

[0182] 1 H-NMR (300 MHz, CDCl3): δ7.58 (m, 2H), 7.34~7.28 (m, 7H), 4.39 (m, 1H), 4.28~4.10 (q, 2H), 2.90~2.77 (m, 4H), 1.21 (t, 3H).

[0183] Step 2) Preparation of 2-((4-cyanofenethyl)amino)-N-(5-iodopyridine-2-yl)-2-phenylacetamide

[0184] 6 g (27.27 mmol) of 5-iodopyridine-2-amine and 8.4 g (27.27 mmol) of the compound prepared in Step 1) above were diluted in 50 mL of N,N-dimethylformamide, and 36.3 mL (54.54 mmol) of 1.0 M lithium bis(trimethylsilyl)amide tetrahydrofuran solution was slowly added at 0°C and stirred for 2 hours. Once the reaction was complete, the reaction mixture was diluted with ethyl acetate and washed with saturated brine. The organic layer thus separated was dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained was separated by column chromatography (hexane:ethyl acetate = 2:1 (volume ratio)) to obtain 9.0 g (yield: 68%) of the title compound.

[0185] 1 H-NMR (300 MHz, DMSO-d6): δ10.54 (s, 1H), 8.52 (s, 1H), 8.11 (m, 1H), 7.93 (m, 1H), 7.72 (m. 2H), 7.44 (m, 5H), 7.27 (m, 3H), 4.52 (m, 1H), 2.85 (m, 2H), 2.74 (m, 2H).

[0186] Step 3) Preparation of 2-(4-(6-(2-((4-cyanopenetyl)amino)-2-phenylacetamido)pyridine-3-yl)-1H-pyrazole-1-yl)acetic acid

[0187] 9 g (18.66 mmol) of the compound prepared in step 2) above was diluted in 90 mL of a mixed solution of water:1,4-dioxane = 1:5 (volume ratio), 7.8 g (27.99 mmol) of ethyl 2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)pyrazole-1-yl]acetate, 1.5 g (1.87 mmol) of (2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II)methanesulfonate, and 18.2 g (56.98 mmol) of cesium carbonate were added sequentially, and the mixture was stirred at 90°C for 16 hours. Once the reaction was complete, the reaction mixture was cooled to room temperature and washed with ethyl acetate. 4N hydrochloric acid was slowly added to the aqueous layer obtained in this way at 0°C, and the resulting solid was filtered under reduced pressure and dried under reduced pressure to obtain 6.5 g of the title compound (yield: 72%).

[0188] 1 H-NMR (300 MHz, DMSO-d6): δ10.75 (s, 1H), 8.55 (s, 1H), 8.17 (s, 1H), 7.98 (m, 3H), 7.71 (m. 2H), 7.44~7.38 (m, 7H), 4.94 (m, 2H),4.78 (s, 1H), 2.94 (m, 4H).

[0189] MS (ESI + ):m / z= 481.19 [M+H] + .

[0190] Preparation Example 5: Preparation of 4-(1-((2R,4R)-2-((1H-pyrazolo[4,3-b]pyridin-5-yl)carbamoyl-4-fluoropyrrolidine-1-carbonyl)cyclohexyl)benzoic acid

[0191]

[0192] Step 1) Preparation of 2-(4-methoxycarbonylphenyl)acetic acid

[0193] 40 g (188.27 mmol) of methyl 4-(2-methoxy-2-orthoethyl)benzoate was diluted in 360 mL of a tetrahydrofuran:methanol:water = 4:1:1 (volume ratio) solution, 8.7 g (197.69 mmol) of lithium hydroxide was added, and the mixture was stirred at room temperature for 22 hours. Once the reaction was complete, the reaction mixture was concentrated, diluted with distilled water, and acidified to pH 2 with 1N hydrochloric acid. After the reaction was complete, the reaction mixture was filtered under reduced pressure and washed with distilled water to obtain 30.8 g (yield: 84%) of the title compound.

[0194] 1 H-NMR (300 MHz, DMSO-d6): δ7.88 (d, 2H), 7.39 (d, 2H), 3.83 (s, 3H), 3.66 (s, 2H).

[0195] Step 2) Preparation of methyl 4-(2-tert-butoxy-2-oxo-ethyl)benzoate

[0196] 6.0 g (30.90 mmol) of the compound prepared in Step 1) above was diluted in 100 mL of dichloromethane, and 10.06 mL (92.69 mmol) of tertiary butanol, 12.9 g (33.99 mmol) of HATU, and 12.92 mL (92.69 mmol) of N,N-diisopropylethylamine were added at 0°C, followed by stirring at room temperature for 1 hour. Once the reaction was complete, the reaction mixture was diluted in dichloromethane and washed with distilled water. The organic layer thus separated was dried with anhydrous magnesium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained was separated by column chromatography (hexane:ethyl acetate = 9:1 (volume ratio)) to obtain 6.8 g of the title compound (yield: 88%).

[0197] 1 H-NMR (300 MHz, CDCl3): δ8.00 (d, 2H), 7.36 (d, 2H), 3.92 (s, 3H), 3.70 (s, 2H), 1.45 (s, 9H).

[0198] Step 3) Preparation of methyl 4-(1-tert-butoxycarbonylcyclohexyl)benzoate

[0199] 6.5 g (25.97 mmol) of the compound prepared in Step 2) above was diluted in 65 mL of N,N-dimethylformamide, and 3.1 g (77.91 mmol) of sodium hydride (60% dispersion in mineral oil) was added at 0°C, followed by the dropwise addition of 5.30 mL (38.95 mmol) of 1,5-dibromopentane at the same temperature. The reaction mixture was stirred at room temperature for 2 hours. Upon completion of the reaction, the mixture was washed with distilled water and extracted with ethyl acetate. The organic layer obtained by extraction was dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The resulting residue was separated by column chromatography (hexane:ethyl acetate = 4:1 (volume ratio)) to obtain 0.9 g (yield: 11%) of the title compound.

[0200] 1 H-NMR (300 MHz, CDCl3): δ8.02 (d, 2H), 7.47 (d, 2H), 3.93 (s, 3H), 2.60~2.41(m, 2H), 1.74~1.37 (m, 8H), 1.30 (s, 9H).

[0201] Step 4) Preparation of 1-(4-methoxycarbonylphenyl)cyclohexanecarboxylic acid

[0202] 1.2 g (3.76 mmol) of the compound prepared in Step 3) above was diluted in 10 mL of dichloromethane, and 4.8 mL (62.70 mmol) of trifluoroacetic acid was slowly added dropwise at 0°C. The reaction mixture was raised to room temperature and stirred for 3 hours. Once the reaction was complete, the reaction mixture was distilled under reduced pressure. The obtained solid was filtered under reduced pressure and dried under reduced pressure to obtain 0.6 g of the title compound (yield: 60%).

[0203] 1H-NMR (300 MHz, CDCl3): δ8.01 (d, 2H), 7.54 (d, 2H), 3.93 (s, 3H), 2.53-2.48 (m, 2H), 1.70-1.50 (m, 8H).

[0204] Step 5) Preparation of 4-(1-chlorocarbonylcyclohexyl)benzoate methyl

[0205] 0.28 g (1.06 mmol) of the compound prepared in Step 4) above was diluted in 4 mL of dichloromethane, and 0.77 mL (10.67 mmol) of thionyl chloride and 1.6 mg (0.0213 mmol) of N,N-dimethylformamide were added. This reaction mixture was stirred at 40°C for 30 minutes. Once the reaction was complete, the reaction mixture was cooled to room temperature, and then subjected to vacuum distillation to obtain 0.26 g of the title compound (yield: 87%).

[0206] Step 6) Preparation of (2R, 4R)-1-(((9H-fluorene-9-yl)methoxy)carbonyl)-4-fluoropyrrolidine-2-carboxylic acid

[0207] 10 g (42.87 mmol) of (2R, 4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidine-2-carboxylic acid was added to 107 mL of 4N hydrochloric acid and stirred at room temperature for 12 hours. When the reaction was complete, the resulting solid was filtered under reduced pressure. The resulting solid was diluted in 360 mL of a 1,4-dioxane:water = 1:1 (volume ratio) solution, and then 18.0 g (214 mmol) of sodium bicarbonate and 13.3 g (51.4 mmol) of fluorenylmethyloxycarbonyl chloride were added at 0°C and stirred at room temperature for 16 hours. When the reaction was complete, water was added, the mixture was washed twice with ethyl acetate, and the aqueous layer was acidified to pH 4 with 1N hydrochloric acid. After extracting the acidified aqueous layer twice with chloromethane, the separated organic layer was dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained in this way was separated by column chromatography (hexane : ethyl acetate = 1 : 1 (volume ratio)) to obtain 14.0 g (yield: 92%) of the title compound.

[0208] 1 H-NMR (300 MHz, CDCl3): δ7.90 (d, 2H), 7.55 (d, 2H), 7.38 (dd, 2H), 7.28 (dd, 2H), 4.70 (d, 2H), 4.46 (d, 1H), 4.32 (t, 1H), 3.73~3.62 (m, 2H), 3.53~3.48(m, 1H), 2.31~2.19(m, 1H), 2.10~1.95 (m, 1H).

[0209] Step 7) Preparation of (9H-fluorene-9-yl)methyl(2R,4R)-2-(chlorocarbonyl)-4-fluoropyrrolidine-1-carboxylate

[0210] 12.0 g (33.77 mmol) of the compound prepared in Step 6) above was diluted in 120 mL of dichloromethane, 24 mL (337.68 mmol) of thionyl chloride and 0.052 mL of N,N-dimethylformamide were added, and the reaction mixture was stirred at room temperature for 3 hours. Once the reaction was complete, the reaction mixture was concentrated, diluted in 15 mL of dichloromethane, and 150 mL of hexane was added. The solid thus obtained was dried under reduced pressure to obtain 11.44 g (yield: 90%) of the title compound.

[0211] Step 8) Preparation of tert-butyl 5-((2R, 4R)-1-((((9H-fluorene-9yl)methoxy)carbonyl)-4-fluoropyrrolidine-2-carboxamido)-1H-pyrazolo[4,3-b]pyridine-1-carboxylate

[0212] 6.58 g (28.10 mmol) of tert-butyl 5-aminopyrazolo[4,5-b]pyridine-1-carboxylate was diluted in 110 mL of dichloromethane, and 5.8 mL (36.52 mmol) of N,N-diisopropylethylamine was added at 0°C. To the reaction mixture, 10.5 g (28.09 mmol) of the compound prepared in step 7) was diluted in 110 mL of dichloromethane and slowly added dropwise, followed by stirring at room temperature for 12 hours. After washing the reaction with water, the organic layer thus separated was dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained was separated by column chromatography (hexane : ethyl acetate = 1 : 1 (volume ratio)) to obtain 10.18 g (yield: 64%) of the title compound.

[0213] 1H-NMR (300 MHz, CDCl3): δ10.62 (s, 1H), 8.53 (d, 1H), 8.30 (s, 1H), 7.91 (d, 2H), 7.55 (d, 2H), 7.38 (dd, 2H), 7.28 (dd, 2H), 4.15 (dd, 1H), 3.91 (dd, 1H), 3.55 (dd, 1H), 3.40 (dd, 1H), 2.82~2.67(m, 1H), 2.52~2.30(m, 2H), 2.12~1.93 (m, 1H).1.86 (s, 9H).

[0214] Step 9) Preparation of tert-butyl 5-((2R, 4R)-4-fluoropyrrolidin-2-carboxamido)-1H-pyrazolo[4,3-b]pyridine-1-carboxylate

[0215] 11.4 g (19.98 mmol) of the compound prepared in Step 8) above was diluted with 110 mL of N,N-dimethylformamide, and 9.9 mL (99.89 mmol) of piperidine was added at 0°C, followed by stirring at room temperature for 1 hour. Once the reaction was complete, the reaction mixture was diluted with ethyl acetate and washed with distilled water. The organic layer thus separated was dried with anhydrous magnesium sulfate, followed by vacuum filtration and vacuum distillation. The resulting residue was separated by column chromatography (hexane : ethyl acetate = 10 : 1 (volume ratio)) to obtain 4.27 g of the title compound (yield: 61%).

[0216] 1 H-NMR (300 MHz, CDCl3):δ10.32 (s, 1H) 8.58 (d, 1H), 8.50(d, 1H), 8.27 (s, 1H), 4.18 (dd, 1H), 3.92 (dd, 1H), 3.53 (dd, 1H), 3.35 (dd, 1H), 2.78~2.65 (m, 1H), 2.51~2.28(m, 2H), 1.82 (s, 9H).

[0217] Step 10) Preparation of tert-butyl 5-((2R,4R)-4-fluoro-1-(1-(4-(methoxycarbonyl)phenyl)cyclohexane-1-carbonyl)pyrrolidine-2-carboxamido)-1H-pyrazolo[4,3-b]pyridine-1-carboxylate

[0218] 324 mg (0.93 mmol) of the compound prepared in Step 9) was diluted with 10 mL of dichloromethane, and 9.9 mL (99.89 mmol) of N,N-diisopropylethylamine was added at 0°C. Subsequently, 260 mg (0.93 mmol) of the compound prepared in Step 5), diluted in 2 mL of dichloromethane, was slowly added dropwise. Once the addition was complete, the reaction mixture was stirred at room temperature for 2 hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane and washed with distilled water. The organic layer thus separated was dried with anhydrous magnesium sulfate, followed by vacuum filtration and vacuum distillation. The resulting residue was separated by column chromatography (hexane:ethyl acetate = 9:1 (volume ratio)) to obtain 230 mg of the title compound (yield: 42%).

[0219] 1 H-NMR (300 MHz, CDCl3):δ10.26 (s, 1H) 8.52 (dd, 2H), 8.50 (d, 1H), 8.28 (s, 1H), 8.12 (d, 1H) 7.47 (d, 2H), 4.18 (dd, 1H), 3.95 (s, 3H), 3.55 (dd, 1H), 3.38 (dd, 1H), 2.78~2.65 (m, 1H), 2.51~2.28(m, 4H), 2.10~1.95 (m, 1H). 1.82 (s, 9H), 1.70~1.50 (m, 8H).

[0220] Step 11) Preparation of 4-(1-((2R,4R)-2-((1H-pyrazolo[4,3-b]pyridin-5-yl)carbamoyl-4-fluoropyrrolidine-1-carbonyl)cyclohexyl)benzoic acid

[0221] 400 mg (0.67 mmol) of the compound prepared in Step 10) above was diluted in 6 mL of a solution of ethanol:tetrahydrofuran:distilled water = 1:1:1 (volume ratio), and then 85 mg (2.02 mmol) of lithium hydroxide was added and stirred at room temperature for 14 hours. Once the reaction was complete, the reaction mixture was concentrated and acidified to pH 4 with 1N hydrochloric acid. The organic layer thus separated was dried with anhydrous magnesium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained was separated by column chromatography (hexane:ethyl acetate = 9:1 (volume ratio)) to obtain 230 mg of the title compound (yield: 42%).

[0222] 1 H-NMR (300 MHz, DMSO-d6): δ10.31 (s, 1H) 8.12 (dd, 2H), 8.14~8,10 (m, 2H), 8.02 (d, 1H), 7.97 (d, 1H), 7.47 (t, 2H), 4.40 (dd, 1H), 3.55 (dd, 1H), 3.38 (dd, 1H), 2.78~2.65 (m, 1H), 2.45~2.22 (m, 4H), 2.10~1.95 (m, 1H). 1.70~1.50 (m, 8H).

[0223] MS (ESI + ):m / z= 479.20 [M+H] + .

[0224] Preparation Example 6: Preparation of 2-(4-(4-((2R,4R)-2-((1-(tert-butoxycarbonyl)-1H-pyrazolo[4,3-b]pyridin-5-yl)carbamoyl)-4-fluoropyrrolidin-1-carbonyl)phenyl-1H-pyrazolo-1-yl)acetic acid

[0225]

[0226] Step 1) Preparation of tert-butyl 5-((2R,4R)-4-fluoro-1-(4-iodobenzoyl)pyrrolidine-2-carboxamido)-1H-pyrazolo[4,3-b]pyridine-1-carboxylate

[0227] 2.7 g (7.73 mmol) of the compound prepared in Step 9) of Preparation Example 5 above was diluted in 100 mL of dichloromethane, and 5.29 g (13.91 mmol) of HATU, 2.49 g (10.05 mmol) of 4-iodobenzoic acid, and 4.1 mL (23.18 mmol) of N,N-diisopropylethylamine were added, followed by stirring at room temperature for 2 hours. Once the reaction was complete, the reaction mixture was diluted in dichloromethane and washed with distilled water. The organic layer thus separated was dried with anhydrous magnesium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained was separated by column chromatography (hexane:ethyl acetate = 1:1 (volume ratio)) to obtain 3.8 mg of the title compound (yield: 85%).

[0228] 1 H-NMR (300 MHz, CDCl3):δ9.28 (s, 1H) 8.47 (d, 2H) 8.27 (s, 1H), 7.82 (dd, 2H), 7.30 (dd, 2H) 4.18 (dd, 1H), 3.92 (dd, 1H), 3.35 (dd, 1H), 2.78~2.65 (m, 1H), 2.51~2.28(m, 2H), 1.90 (s, 9H).

[0229] Step 2) Preparation of 2-(4-(4-((2R,4R)-2-((1H-pyrazolo[4,3-b]pyridin-5-yl)carbamoyl)-4-fluoropyrrolidin-1-carbonyl)phenyl-1H-pyrazolo-1-yl)acetic acid

[0230] 2.7 g (4.46 mmol) of the compound prepared in Step 1) above, 1.3 g (4.66 mmol) of methyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-yl)acetate, 363 mg (0.46 mmol) of SPhos Pd G3, and 4.5 g (13.98 mmol) of cesium carbonate were diluted in 25 mL of a 1,4-dioxane : distilled water = 4 : 1 (volume ratio) mixed solution. This reaction mixture was stirred at 120°C for 18 hours. Once the reaction was complete, the reaction mixture was cooled to room temperature, and then 6N aqueous hydrochloric acid solution was added dropwise until the pH reached 4. The mixture was washed with distilled water and filtered under reduced pressure. The solid obtained in this way was dissolved in dichloromethane and dried with anhydrous sodium sulfate, then filtered under reduced pressure and distilled under reduced pressure to obtain 1.5 g of the title compound (final stage yield: 67%).

[0231] 1 H-NMR (300 MHz, DMSO-d6):δ10.32 (s, 1H) 8.32~8.08 (m, 5H), 7.91~7.65 (m, 5H), 5.02 (s, 2H), 4.15 (dd, 1H), 3.90 (dd, 1H), 3.38 (dd, 1H), 2.79~2.70 (m, 1H), 2.50~2.30(m, 2H).

[0232] MS (ESI+): m / z = 477.16 [M+H] + .

[0233] [Example 1]

[0234] N-Benzyl-N-Cyclohexyl-2-(6'-(1-(2-(4-(4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)-2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-1-yl)Preparation of Acetamide

[0235]

[0236]

[0237] Preparation of the first enantiomer

[0238] 50 mg (0.09 mmol) of the compound from Preparation Example 2) above was diluted in 1 mL of N,N-dimethylformamide, and 0.07 mL (0.11 mmol) of a 50% solution of propylphosphoic acid anhydride and 0.03 mL (0.18 mmol) of N,N-diethylisopropylamine were added. This reaction mixture was stirred at room temperature for 10 minutes. 36 mg (0.09 mmol) of the compound from Preparation Example 1) above was diluted in 1 mL of N,N-dimethylformamide, and 0.03 mL (1.18 mmol) of N,N-diethylisopropylamine was added at 0°C and stirred for 10 minutes, then dropwise added to the above reaction mixture at 0°C. This reaction mixture was stirred at 0°C for 2 hours. When the reaction was complete, water was added and stirred for 10 minutes. The solid obtained in this way was washed with water and filtered under reduced pressure. The solid obtained in this way was diluted with dichloromethane and dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained in this way was separated by column chromatography (dichloromethane : methanol = 15 : 1 (volume ratio)) to obtain 11 mg (yield: 14%) of the first enantiomer of the title compound.

[0239] 1 H-NMR (300 MHz, DMSO-d6): δ8.88 (d, 1H), 8.09~8.04 (m, 3H), 7.88 (d, 1H), 7.83~7.78 (m, 1H), 7.45~7.31 (m, 6H), 7.31~7.15 (m, 3H), 7.08 (d, 2H), 5.18 (d, 2H), 4.65 (s, 1H), 4.55 (d, 2H), 4.35~4.19 (m, 2H), 3.89 (s, 3H), 3.78~3.49 (m, 8H), 2.88~2.78 (m, 2H), 2.15~0.81 (m, 14H);

[0240] MS (ESI + ):m / z= 826.38 [M+H] +.

[0241] Preparation of second enantiomers

[0242] 150 mg (0.26 mmol) of the compound from Preparation Example 3) above was diluted in 1 mL of N,N-dimethylformamide, and 0.20 mL (0.34 mmol) of a 50% solution of propylphosphonic acid anhydride and 0.09 mL (0.53 mmol) of N,N-diethylisopropylamine were added. This reaction mixture was stirred at room temperature for 10 minutes. 107 mg (0.28 mmol) of the compound from Preparation Example 1) above was diluted in 1 mL of N,N-dimethylformamide, and 0.09 mL (0.53 mmol) of N,N-diethylisopropylamine was added at 0°C and stirred for 10 minutes, after which the mixture was added dropwise to the above reaction mixture at 0°C. This reaction mixture was stirred at 0°C for 2 hours. When the reaction was complete, water was added and stirred for 10 minutes. The solid obtained in this way was washed with water and filtered under reduced pressure. The solid obtained in this way was diluted with dichloromethane and dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained in this way was separated by column chromatography (ethyl acetate : dichloromethane : methanol = 7 : 7 : 1 (volume ratio)) to obtain 23 mg (yield: 11%) of the second enantiomer of the title compound.

[0243] 1 H-NMR (300 MHz, DMSO-d6): δ8.88 (d, 1H), 8.09~8.04 (m, 3H), 7.88 (d, 1H), 7.83~7.78 (m, 1H), 7.45~7.31 (m, 6H), 7.31~7.15 (m, 3H), 7.08 (d, 2H), 5.18 (d, 2H), 4.65 (s, 1H), 4.55 (d, 2H), 4.35~4.19 (m, 2H), 3.89 (s, 3H), 3.78~3.49 (m, 8H), 2.88~2.78 (m, 2H), 2.15~0.81 (m, 14H);

[0244] MS (ESI +):m / z= 826.38 [M+H] + .

[0245] [Example 2]

[0246] Preparation of (E)-2-((4-cyanopenethyl)amino)-N-(5-(1-(2-(4-(4-methoxyphenyl)-4-oxobut-2-enoil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazole-4-yl)pyridine-2-yl)-2-phenylacetamide

[0247]

[0248] 90 mg (0.19 mmol) of the compound from Preparation Example 4) above was diluted in 2 mL of N,N-dimethylformamide, and 0.14 mL (0.24 mmol) of a 50% solution of propylphosphonic acid anhydride and 0.06 mL (0.37 mmol) of N,N-diethylisopropylamine were added. This reaction mixture was stirred at room temperature for 10 minutes. 73 mg (0.19 mmol) of the compound from Preparation Example 1) above was diluted in 2 mL of N,N-dimethylformamide, and 0.06 mL (0.37 mmol) of N,N-diethylisopropylamine was added at 0°C and stirred for 10 minutes, then dropwise added to the above reaction mixture at 0°C. This reaction mixture was stirred at 0°C for 2 hours. When the reaction was complete, water was added and stirred for 10 minutes. The solid obtained in this way was washed with water and filtered under reduced pressure. The solid obtained in this way was diluted with dichloromethane and dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained in this way was separated by column chromatography (dichloromethane : methanol = 19 : 1 (volume ratio)) to obtain 3 mg of the title compound (yield: 2%).

[0249] 1H-NMR (300 MHz, DMSO-d6): δ10.44 (s, 1H), 8.57 (d, 1H), 8.13 (s, 1H), 8.09~8.01 (m, 3H), 7.99~7.91 (m, 2H), 7.85~7.71 (m, 3H), 7.50~7.41 (m, 6H), 7.39~7.25 (m, 3H), 7.11 (d, 2H), 5.21 (s, 2H), 4.52 (s, 1H), 3.87 (s, 3H), 3.78~3.49 (m, 8H), 2.95~2.70 (m, 4H);

[0250] MS (ESI + ):m / z= 737.31 [M+H] + .

[0251] [Example 3]

[0252] Preparation of (2R,4R)-4-fluoro-1-(1-(4-(4-((E)-4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-carbonyl)phenyl)cyclohexane-1-carbonyl)-N-(1H-pyrazolo[4,3-b]pyridine-5yl)pyrrolidine-2-carboxamide

[0253]

[0254] 180 mg (0.37 mmol) of the compound from Preparation Example 5) above was diluted in 1 mL of N,N-dimethylformamide, and 0.29 mL (0.48 mmol) of a 50% solution of propylphosphonic acid anhydride and 0.13 mL (0.75 mmol) of N,N-diethylisopropylamine were added. This reaction mixture was stirred at room temperature for 10 minutes. 153 mg (0.39 mmol) of the compound from Preparation Example 1) above was diluted in 1 mL of N,N-dimethylformamide, and 0.13 mL (0.75 mmol) of N,N-diethylisopropylamine was added at 0°C and stirred for 10 minutes, then dropwise added to the above reaction mixture at 0°C. This reaction mixture was stirred at 0°C for 2 hours. When the reaction was complete, water was added and stirred for 10 minutes. The solid obtained in this way was washed with water and filtered under reduced pressure. The solid obtained in this way was diluted with dichloromethane and dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained in this way was separated by column chromatography (dichloromethane:methanol = 19:1 (volume ratio)) to obtain 53 mg of the title compound (yield: 18%).

[0255] 1 H-NMR (300 MHz, DMSO-d6): δ10.63 (s, 1H), 8.16 (s, 2H), 8.08 (d, 3H) 7.91 (d, 1H), 7.44~7.33 (m, 5H), 7.02 (d, 2H), 3.96 (s, 3H), 3.77~3.62 (m, 4H), 3.40~3.23 (m, 2H), 2.32~2.25 (m, 2H), 2.12~2.08(m, 1H), 1.83~1.77(m, 2H), 1.59~1.23 (m, 5H).

[0256] MS (ESI + ):m / z= 735.32 [M+H] + .

[0257] [Example 4]

[0258] Preparation of (2R,4R)-4-fluoro-1-(4-(1-(2-(4-((E)-4-(4-methoxyphenyl)-4-oxobut-2-en-oil)piperazine-1-yl)-2-oxoethyl)-1H-pyrazol-4-yl)benzoyl)-N-(1H-pyrazolo[4,3-b]pyridine-5-yl)pyrrolidine-5-yl)pyrrolidine-2-carboxamide

[0259]

[0260] 145 mg (0.30 mmol) of the compound from Preparation Example 6) above was diluted in 2 mL of N,N-dimethylformamide, and 0.24 mL (0.40 mmol) of a 50% solution of propylphosphoic acid anhydride and 0.11 mL (0.60 mmol) of N,N-diethylisopropylamine were added. This reaction mixture was stirred at room temperature for 10 minutes. 118 mg (0.30 mmol) of the compound from Preparation Example 1) above was diluted in 1 mL of N,N-dimethylformamide, and 0.11 mL (0.60 mmol) of N,N-diethylisopropylamine was added at 0°C and stirred for 10 minutes, then dropwise added to the above reaction mixture at 0°C. This reaction mixture was stirred at 0°C for 2 hours. When the reaction was complete, water was added and stirred for 10 minutes. The solid obtained in this way was washed with water and filtered under reduced pressure. The solid obtained in this way was diluted with dichloromethane and dried with anhydrous sodium sulfate, followed by vacuum filtration and vacuum distillation. The residue obtained in this way was separated by column chromatography (dichloromethane : methanol = 19 : 1 (volume ratio)) to obtain 7 mg of the title compound (yield: 3%).

[0261] 1H-NMR (300 MHz, DMSO-d6): δ13.26(s, 1H), 10.54 (s, 1H), 8.18~8.09 (m, 7H), 7.97 (d, 1H), 7.67 (dd, 3H) 7.46~7.38 (m, 1H), 7.08 (d, 2H), 5.23 (s, 2H), 4.15 (dd, 1H), 3.96 (s, 3H), 3.75~3.60 (m, 4H), 3.40~3.13 (m, 7H), 2.30~2.27 (m, 1H), 2.14~2.09(m, 1H).

[0262] MS (ESI + ):m / z= 733.28 [M+H] + .

[0263] [Experimental Example] Test of EP300 and CBP reduction effects of a compound

[0264] 2 × 10⁶ HAP1 cells, a near-haploid human cell line derived from the KBM-7 chronic myelogenous leukemia cell line, were placed in each well of a 6-well plate. 5Each sample was cultured for 24 hours. Afterward, the growth medium was replaced, and while the total volume of the medium was fixed at 3 mL, the positive control drug or the example compound was added at the experimental concentration (100 nM or 1,000 nM), respectively. As the positive control drug, 2-((1'S,3'R)-5'-(1-(2-((7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxosisoindolin-5-yl)amino)heptyl)amino)-2-oxoethyl)-4,5-dihydro-1H-pyrazole-4-yl)-3'-fluoro-2,5-dioxo-2',3'-dihydrospiro[imidazolidin-4,1'-indene]-1-yl)-N-(4-fluorobenzyl)-N-((S)-1,1,1-trifluoropropane-2-yl)acetamide (BT-02C), a compound that selectively inhibits EP300, was used, and cells to which neither the positive control drug nor the example compound was administered were used as the negative control. After 24 hours of culture, the amounts of EP300 and CBP were quantified as follows. At the end of culture, a cell density of 60% to 80% was confirmed, and the cells were washed with DPBS. Subsequently, the cells were lysed using a cell lysis buffer containing PIC / PS (protease & phosphatase inhibitor) and EDTA to extract proteins. The total protein content of the cell lysate was measured by the biciconic acid (BCA) quantification method. Cell lysates containing the same amount of total protein were taken from each sample, subjected to electrophoresis, and visualized using an immunoblot method with rabbit anti-human EP300 antibody (D2X6N, Cell Signaling, USA Catalog No. 54062) and anti-human CBP antibody (D6C5, Cell Signaling, USA Catalog No. 7389). The density of the electrophoresis bands was measured. The density of the negative control was set to 100%, and the measured values ​​of each sample were calculated as relative values ​​based on this standard to compare the effects with the positive control.The intracellular EP300 or CBP protein band signal intensity was compared with the negative control signal to classify the degradation rate of the target protein as A when it was 80% or more, B when it was 45% or more but less than 80%, C when it was 10% or more but less than 45%, and D when it was less than 10%, and the results are shown in Table 1 below.

[0265]

[0266] Through this, it was confirmed that the compound of the present invention decomposes EP300 at a rate greater than the CBP decomposition rate. Although the present invention has been described above by limited embodiments, the technical concept and scope of the present invention are not limited to these embodiments. The invention of the claims described below, as well as various modifications or variations of the invention described in the claims that are obvious to those skilled in the art to which this invention belongs, are included within the scope of the present invention within the equivalent scope of the invention described in the claims.

Claims

Compounds of Formula 1, their tautomers, their stereoisomers, or pharmaceutically acceptable salts thereof: [Chemical Formula 1] In Chemical Formula 1, R is the site that binds to the histone acetyltransferase (HAT) region of EP300. A compound according to claim 1, characterized in that the compound can bind to an E3 ligase. A compound according to claim 1, characterized in that the compound has the structure of Formula 2: [Chemical Formula 2] In chemical formula 2, R' is the site that binds to the histone acetyltransferase domain of EP300. A compound according to claim 3, characterized in that R' has the structure of Chemical Formula 3: [Chemical Formula 3] In chemical formula 3 The symbol indicates a position connected by a covalent bond to the carbonyl carbon directly connected to R' of Chemical Formula 2; X 1 and X 2 are independently CH or N; L 5 is a chemical bond or It is a 5-membered ring selected from among, where m is an integer from 0 to 2, and Is Indicates the connection in the display direction, Is Indicates a connection of directions; Z 1 It is hydrogen or CH2-Z', where Z' is a 5-6-membered heteroaryl or phenyl; (1) Z 1 When this is hydrogen, Z 2 ga is an 8- to 10-membered double-fused ring heteroaryl containing nitrogen; R 1 is methylene and R 2 is ethylene, and at this time R 1 and R 2 are connected to each other, and R 2 The nitrogen atom directly connected to and R 1 Forming an azacyclopentane ring together with a carbon atom directly connected to it; L 2 , L 3 and L 4 is non-existent; L 1 is a chemical bond or 1,1-cyclohexylene; (2) Z 1 When this is CH2-Z', Z 2 α is a 4- to 7-membered cycloalkyl; L 1 is CH, and L 2 , L 3 and L 4 is methylene; R 1 It is hydrogen; R 2 is C(=O)NH, and at this time R 2 The nitrogen atom of L 1 Directly connected to form an imidazolidin-2,4-dione ring; Only L in chemical formula 3 1 and L 5 Cases where all are chemical bonds are excluded, and Each of the above azacyclopentane ring, 5-6 member heteroaryl or phenyl, 4-7 member cycloalkyl, 8-10 member difused ring heteroaryl 1,1-cyclohexylene or 4-7 member cycloalkyl may be unsubstituted or may be substituted with a functional group independently selected from the group consisting of halogen, OH, CN, NH2 and NO2 for each ring hydrogen atom. A compound according to claim 3, characterized in that R' has the structure of Chemical Formula 4: [Chemical Formula 4] In chemical formula 4 The symbol indicates a position connected by a covalent bond to the carbonyl carbon directly connected to R' of Chemical Formula 2; X 1 and X 2 are independently CH or N; L 5 is a chemical bond or It is a 5-membered ring selected from among, where m is an integer from 0 to 2, and Is Indicates the connection in the display direction, Is Indicates a connection of directions; Z' is a 5-6-membered heteroaryl or phenyl; R 3 and R 4 Each is independently selected from the group consisting of hydrogen, halogen, OH, CN, NH2, and NO2; R 5 is a 5-6-membered heteroaryl or phenyl; The above Z' and R 5 Each of the 5-6-membered heteroaryl or phenyl groups may be unsubstituted or one or more of the ring hydrogen atoms may be substituted with functional groups selected independently from each ring hydrogen atom from the group consisting of halogens, OH, CN, NH2 and NO2. A compound according to claim 3, characterized in that R' is any one of the structures of Chemical Formula 5 to Chemical Formula 8: [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] In chemical formulas 5 to 8 The notation indicates the position connected by a covalent bond to the carbonyl carbon leading to R' in chemical formula 2. In claim 6, the compound of chemical formula 7 is characterized by having the structure of chemical formula 9 below: [Chemical Formula 9] In claim 6, the compound of chemical formula 8 is characterized by having the structure of chemical formula 10 below: [Chemical Formula 10] A pharmaceutical composition for the treatment or prevention of cancer comprising a therapeutically effective amount of one compound according to claim 1 and a pharmaceutically acceptable excipient.

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