WO2025009852A1 - Novel pyrazole derivative and use thereof - Google Patents

Abstract

The present invention provides, as a caspase-3 inhibitor, a novel pyrazole derivative compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof. The novel pyrazole derivative compound of the present invention has been confirmed to exhibit excellent caspase-3 inhibitory activity and thus may be used for the prevention or treatment of caspase-3-related disease, particularly pulmonary arterial hypertension. Thus, the novel pyrazole derivative compound of the present invention may be usefully employed for the prevention or treatment of caspase-3-related disease in the medical and pharmaceutical fields.

Description

Novel pyrazole derivatives and their uses

This application claims the benefit of priority from Korean Patent Application No. 10-2023-0085927, filed on July 3, 2023, the entire contents of which are incorporated herein by reference. The present invention relates to novel pyrazole derivative compounds, and more particularly to novel pyrazole derivative compounds as caspase-3 inhibitors.

Apoptosis is an essential function of cells that plays an important role in various physiological processes such as organism development, immune system regulation, and tissue formation, and is caused by the removal of unnecessary cells, the removal of self and damaged cells in immune responses, and the removal of tumor cells. Cells undergoing apoptosis exhibit unique morphological and biochemical changes, such as membrane remodeling, nuclear condensation, DNA fragmentation, and the activation of a family of intracellular cysteine-degrading enzymes called caspases. Caspases involved in apoptosis can be distinguished according to their substrate specificity. In particular, primary effector caspases, including caspase-3 or caspase-7, show a preference for substrates containing the four-amino acid recognition sequence DEVD, which are found in abundance in cytoplasmic or nuclear caspase substrates. Caspase-3 is known to play a key role in the execution of apoptosis because it cleaves chromosomes by nucleosomal fragmentation and promotes the activity of other effector caspases. In addition, activated caspase-3 cleaves a number of cellular substrates, such as poly-(ADP-ribose) polymerase (PARP), inhibitor of caspase-activated deoxyribonuclease (ICAD), and alpha-fodrin, which induces apoptosis.

Caspase-3, a member of the caspase enzyme family, is a protease that specifically reacts with Cys and Asp amino acid residues, and is known to play an important role in tissue differentiation, regeneration, and neural development (Biotechnol Appl Biochem. 2022 Aug;69(4):1633-1645.). This enzyme is a key enzyme in cell death, and is converted to an active form by cleavage by a signal pathway that induces cell death. Activated caspase-3 recognizes specific proteins inside the cell and degrades the protein at a specific amino acid binding site, thereby activating other caspase enzymes. This protein degradation process causes nuclear disintegration of the cell, cell membrane damage, and degradation of cell cycle control proteins, which are the execution stages of cell death. In addition, abnormal activation of caspase-3 is associated with various diseases. In cancer cells, abnormal activation of caspase-3 can inhibit apoptosis and promote tumor proliferation. On the other hand, lack of caspase-3 activity may be linked to neurological diseases, which may interfere with the normal progression of apoptosis and affect the regulation of cell survival signaling pathways.

As such, caspase-3 is known to play an important role in the biological signal transduction system. In addition, it has been reported that apoptosis, inflammation, and fibrosis contribute to vascular remodeling in hypertension, and that apoptosis and microvascular disease are related ( Hypertension . 2001;38[part 2]:581-587.), and a study has also reported that endothelial cell death in pulmonary arterial hypertension is controlled by the microRNA/programmed cell death 4/caspase-3 axis (Hypertension. 2014;64:185-194.). Accordingly, caspase-3 is considered as a new therapeutic strategy for treating apoptosis-related diseases in the field of medical research.

The problem to be solved by the present invention is to provide a novel structural caspase-3 inhibitory compound exhibiting excellent caspase-3 inhibitory activity.

In addition, the problem solved by the present invention is to provide a pharmaceutical composition for preventing or treating a caspase-3 related disease, comprising a caspase-3 inhibitory compound having the novel structure.

In addition, the problem to be solved by the present invention is to provide a method for preventing or treating a caspase-3 related disease using the caspase-3 inhibitory compound having the novel structure.

In addition, the problem to be solved by the present invention is to provide a use of the novel caspase-3 inhibitory compound of the above structure for preventing or treating a caspase-3 related disease.

The problems to be solved by the present invention are not limited to the problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

In order to solve the above problem, according to one aspect of the present invention, a pyrazole derivative compound represented by the following chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof is provided.

<Chemical formula (I)>

In the above formula,

R ¹ is -C(O)OR ³ or a 5- to 7-membered heteroaryl ring containing 1 to 4 N,

L is -N(R ⁴ )C(O)-, -N(R ⁴ )C(O)O-, -N(R ⁴ )C(O)CH ₂ N(R ⁵ )- or -C(O)N(R ⁴ )-,

R ² is a fused ring of two rings in which an aryl ring of 1 to 2 rings having C _5-12 , or a 5 to 7 membered heterocycloalkyl ring containing 1 to 2 N is fused with an aryl ring, and R ² is optionally substituted with one or more independent R ⁶ ,

R ^a and R ^b are independently C _1-3 alkyl,

R ³ , R ⁴ and R ⁵ are independently -H or C _1-3 alkyl,

R ⁶ is C _1-3 alkoxy, oxo, hydroxy, -C(O)OH, -C(O)C(O)OH, -S(O) ₂ R ⁷ or aryl,

R ⁷ is a 5- to 7-membered heterocycloalkyl ring containing 1 to 2 N, and R ⁷ is optionally substituted with R ⁸ ,

R ⁸ is C _1-3 alkoxyC _1-3 alkyl,

m and n are independently integers from 1 to 3,

p is an integer from 0 to 3.

According to another aspect of the present invention, a pharmaceutical composition for preventing or treating a caspase-3-related disease is provided, comprising a pyrazole derivative compound represented by the chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

According to another aspect of the present invention, a method for preventing or treating a caspase-3 related disease using a pyrazole derivative compound represented by the chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof is provided.

According to another aspect of the present invention, there is provided a use of a pyrazole derivative compound represented by the above formula (I), a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof for the prevention or treatment of a caspase-3 related disease.

According to another aspect of the present invention, a pharmaceutical composition comprising a pyrazole derivative compound represented by the above formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive is provided.

It was confirmed that the novel pyrazole derivative compound of the present invention exhibits excellent caspase-3 inhibitory activity, and thus it was found that it can be used for the prevention or treatment of caspase-3 related diseases, particularly pulmonary arterial hypertension.

Therefore, the novel pyrazole derivative compound of the present invention can be usefully used in the medical and pharmaceutical fields for the prevention or treatment of caspase-3 related diseases.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

The present invention provides a pyrazole derivative compound represented by the following chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.

<Chemical formula (I)>

In the above formula,

R ¹ is -C(O)OR ³ or a 5- to 7-membered heteroaryl ring containing 1 to 4 N,

L is -N(R ⁴ )C(O)-, -N(R ⁴ )C(O)O-, -N(R ⁴ )C(O)CH ₂ N(R ⁵ )- or -C(O)N(R ⁴ )-,

R ² is a fused ring of two rings in which an aryl ring of 1 to 2 rings having C _5-12 , or a 5 to 7 membered heterocycloalkyl ring containing 1 to 2 N is fused with an aryl ring, and R ² is optionally substituted with one or more independent R ⁶ ,

R ^a and R ^b are independently C _1-3 alkyl,

R ³ , R ⁴ and R ⁵ are independently -H or C _1-3 alkyl,

R ⁶ is C _1-3 alkoxy, oxo, hydroxy, -C(O)OH, -C(O)C(O)OH, -S(O) ₂ R ⁷ or aryl,

R ⁷ is a 5- to 7-membered heterocycloalkyl ring containing 1 to 2 N, and R ⁷ is optionally substituted with R ⁸ ,

R ⁸ is C _1-3 alkoxyC _1-3 alkyl,

m and n are independently integers from 1 to 3,

p is an integer from 0 to 3.

In one embodiment, R ¹ can be ethoxycarbonyl, hydroxycarbonyl, or tetrazolyl.

In one embodiment, L can be -NHC(O)-, -NHC(O)O-, -NHC(O)CH ₂ NH-, or -C(O)NH-.

In one embodiment, R ² can be phenyl, naphthalenyl, indolinyl, isoindolinyl, dihydroisoquinolinyl or tetrahydroisoquinolinyl.

In one embodiment, R ^a and R ^b can independently be methyl or ethyl.

In one embodiment, R ³ , R ⁴ and R ⁵ can independently be -H or ethyl.

In one embodiment, R ⁶ can be methoxy, oxo, hydroxy, -C(O)OH, -C(O)C(O)OH, pyrrolidinylsulfonyl or phenyl.

In one embodiment, R ⁸ can be methoxymethyl.

In one embodiment, m may be an integer 1, and n may be an integer 1.

In one embodiment, p can be 0, 1, or 2.

In defining the pyrazole derivative compound represented by the chemical formula (I) throughout this specification, the following definitions apply unless otherwise stated.

The term "alkyl" as used herein refers to a straight-chain or branched-chain saturated hydrocarbon, preferably C _1-10 alkyl. For example, the alkyl includes, but is not limited to, methyl, ethyl, n -propyl, iso -propyl, n -butyl, iso -butyl, tert -butyl, n -pentyl, iso -pentyl, n -hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n -heptyl, n -octyl, n -nonyl, and n -decyl.

The term "cycloalkyl" as used herein refers to a partially or fully saturated single or fused ring hydrocarbon, preferably C _3-10 cycloalkyl. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexynyl, and the like.

As used herein, the term "hydroxy" or "hydroxyl" is defined as -OH, and the term "alkoxy", unless otherwise defined, means alkyloxy, a radical in which the hydrogen atoms of the hydroxy group are replaced with 1 to 10 alkyl atoms.

The term "heteroatom" as used herein means N, O or S.

The term “heterocycloalkyl” as used herein refers to a non-aromatic alkyl ring, which is a non-aromatic carbocyclic ring containing one or more heteroatoms, such as N, O or S, within the ring. The ring may be 5, 6, 7 or 8 membered and/or may be fused to another ring, such as a cycloalkyl or an aromatic ring. Examples of such compounds include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, oxathiolanyl, dithiolanyl, piperidinyl, tetrahydropyranyl, thianyl, piperazinyl, morpholino, and dioxanyl.

The term "aryl" as used herein means an aromatic hydrocarbon, and includes a polycyclic aromatic ring system in which a carbocyclic aromatic ring or a heteroaryl ring is fused with one or more other rings. Preferably, it is C _5-12 aryl, and more preferably, C _5-10 aryl. For example, the aryl includes, but is not limited to, phenyl, naphthyl, tetrahydronaphthyl, and the like. Also included are groups in which a heteroaryl ring is fused to a cycloalkyl or a non-aromatic heterocyclic ring, for example, indanyl or tetrahydrobenzopyranyl.

The term "heteroaryl" or "aromatic heterocycle" as used herein means a _{3 to 12 membered, more preferably 5 to 10 membered aromatic hydrocarbon group containing one or more heteroatoms selected from N, O and S as a reducing group, and forming a single or fused ring which may be fused with benzo or C 3-8} cycloalkyl. For example, the heteroaryl includes, but is not limited to, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrazinyl, pyrimidyl, pyridazinyl, triazinyl, oxadiazolyl, isoxadiazolyl, tetrazolyl, indolyl, indazolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, furanyl, benzofuranyl, thiophenyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, quinolinyl, isoquinolinyl, and the like. Additionally, the heteroaryl includes groups in which the heteroaryl ring is fused to a cycloalkyl or a non-aromatic heterocyclic ring, for example, dihydrocyclopentapyrazinyl, and the like.

Specific examples of pyrazole derivative compounds according to the present invention are as follows.

[1] 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid,

[2] 2-(1-(4-(2-(2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid,

[3] Ethyl 2-(1-(4-(2-(2,3-dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate,

[4] 2-(2-((2-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)amino)-2-oxoethyl)amino)-5-(pyrrolidin-1-ylsulfonyl)phenyl)-2-oxoacetic acid,

[5] Ethyl (S)-2-(1-(4-(2-(5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)-2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate,

[6] Ethyl 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate,

[7] 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetic acid,

[8] Ethyl 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetate,

[9] 6-((4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid,

[10] 6-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid,

[11] Ethyl 2-(3,5-dimethyl-1-(4-(3-(1,3,4-trioxo-3,4-dihydroisoquinolin-2(1H)-yl)propanamido)benzyl)-1H-pyrazol-4-yl)acetate,

[12] 2-(3,5-dimethyl-1-(4-((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-1H-pyrazol-4-yl)acetic acid,

[13] 2-(3,5-dimethyl-1-(4-(((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-1H-pyrazol-4-yl)acetic acid,

[14] 2-(1-(4-(((2-methoxynaphthalen-1-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid,

[15] 2-(1-(4-((([1,1'-biphenyl]-4-ylmethoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid,

[16] N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide, and

[17] N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-3-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)propanamide.

The pyrazole derivative compound represented by the chemical formula (I) according to the present invention can be prepared and used in the form of a prodrug, a hydrate, a solvate, and a pharmaceutically acceptable salt to promote absorption in a body or increase solubility. Therefore, the prodrug, hydrate, solvate, and pharmaceutically acceptable salt are also within the scope of the present invention.

The term "prodrug" refers to a substance that is transformed in vivo into the parent drug. Prodrugs are often used because, in some cases, they are easier to administer than the parent drug. For example, they may be bioactive by oral administration, whereas the parent drug may not be. Prodrugs may also have improved solubility in pharmaceutical compositions than the parent drug. For example, prodrugs may be bio-hydrolyzable esters of compounds according to the invention and pharmaceutically acceptable salts thereof. Another example of a prodrug may be a short peptide (polyamino acid) that is linked to an acid group that is metabolized to reveal the active site of the peptide.

The term "hydrate" means a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

The term "solvate" means a compound of the present invention or a salt thereof which contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefor include those which are volatile, non-toxic, and/or suitable for administration to humans.

The term "isomer" means a compound of the present invention or a salt thereof having the same chemical formula or molecular formula but structurally or sterically different. Such isomers include structural isomers such as tautomers, and stereoisomers such as R or S isomers having an asymmetric carbon center, and geometric isomers (trans, cis). All of these isomers and mixtures thereof are also included in the scope of the present invention.

The term "pharmaceutically acceptable salt" means a salt form of a compound which does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutical salts include acid addition salts formed by acids which form non-toxic acid addition salts containing pharmaceutically acceptable anions, for example, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. For example, pharmaceutically acceptable carboxylic acid salts include metal salts or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, etc.; amino acid salts such as lysine, arginine, guanidine, etc.; organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline, and triethylamine. The compound of formula 1 according to the present invention can be converted into its salt by a conventional method.

In addition, the present invention provides a method for producing a pyrazole derivative compound represented by the chemical formula (I).

The manufacturing method of the pyrazole derivative compound represented by the chemical formula (I) of the present invention is exemplified in Examples 1 to 17, and the manufacturing method below does not limit the method of manufacturing the pyrazole derivative compound represented by the chemical formula (I) according to the present invention. The manufacturing methods of the following Examples 1 to 17 are merely illustrative, and it is obvious that they can be easily modified by those skilled in the art depending on a specific substituent.

The present invention also provides a pharmaceutical composition for preventing or treating a caspase-3-related disease, comprising a pyrazole derivative compound represented by the chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a method for treating or preventing a caspase-3-related disease by administering a pyrazole derivative compound represented by the above chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof to a patient in need thereof.

The present invention also provides a use of a pyrazole derivative compound represented by the above chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof for the prevention or treatment of a caspase-3-related disease.

In one embodiment, the caspase-3 associated disease may preferably be pulmonary arterial hypertension.

As a result of measuring the caspase-3 inhibitory activity of the pyrazole derivative compound of the present invention, it was confirmed that it exhibited excellent caspase-3 inhibitory activity.

The present invention also provides a pharmaceutical composition characterized by comprising a pyrazole derivative compound represented by the above chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive.

The above additives may include pharmaceutically acceptable carriers or diluents, and may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

The pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants are included. Oral solid preparations include tablets, pills, powders, granules, capsules, etc., and these solid preparations may contain at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may contain a lubricant, such as magnesium stearate, talc, etc. Oral liquid preparations include suspensions, solutions, emulsions, syrups, etc., and may contain diluents, such as water or liquid paraffin, wetting agents, sweeteners, flavoring agents, preservatives, etc. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, creams, lyophilized preparations, suppositories, and non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable esters, such as ethyl oleate. Suppository bases that can be used include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

The dosage of the active ingredient contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of the disease, the form of the active ingredient, the route and period of administration, and can be appropriately adjusted depending on the patient. For example, the active ingredient may be administered at a dosage of 0.0001 to 1000 mg/kg per day, preferably 0.001 to 100 mg/kg, and the administration may be administered once a day or divided into several times. In addition, the pharmaceutical composition of the present invention may contain the active ingredient at a weight percentage of 0.001 to 90% based on the total weight of the composition.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, for example, orally, cutaneously, intraperitoneally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

Hereinafter, the present invention will be described in more detail through manufacturing examples, examples, and experimental examples. However, the following examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

Manufacturing Example 1. Ethyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

Step 1: Ethyl 2-(3,5-dimethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)acetate

Ethyl 2-(3,5-dimethyl-1H-pyrazol-4-yl)acetate (67.8 g, 372 mmol) was dissolved in acetonitrile (750 mL), and 4-nitrobenzyl bromide (64.3 g, 298 mmol) and potassium carbonate (46.3 g, 335 mmol) were added at room temperature. The reaction mixture was stirred at 40 ℃ for 16 h. After cooling to room temperature, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure, and ethyl acetate (1.00 L) was added, and the residue was washed with water (2.00 L) and brine (1.00 L). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then diethyl ether/hexane (200 mL/500 mL) was added. The mixture was stirred at room temperature for 1 hour and filtered to obtain ethyl 2-(3,5-dimethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)acetate (91.3 g, 77%).

^1H NMR (400 MHz, CDCl ₃ ) δ 8.16 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 8.8 Hz, 2H), 5.31 (s, 2H), 4.13 (q, J = 7.1 Hz) , 2H), 3.36 (s, 2H), 2.23 (s, 3H), 2.12 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H).

Step 2: Ethyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

Ethyl 2-(3,5-dimethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)acetate (91.3 g, 288 mmol) obtained in Step 1 was dissolved in methanol (800 mL) and palladium charcoal (18.3 g, 20 wt%) was added. The mixture was stirred for 24 hours under hydrogen gas, and the reaction mixture was filtered using Celite. The filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and then diethyl ether/hexane (200 mL/800 mL) was added. The mixture was stirred at room temperature for 30 minutes and filtered to obtain the target compound ethyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (65.2 g, 79%).

¹ H NMR (400 MHz, CD ₃ OD) δ 6.84 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.4 Hz, 2H), 5.08 (s, 2H), 4.10 (q, J = 7.1 Hz, 2H), 3.38 (s, 2H), 2.16 (s, 3H), 2.12 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H).

Manufacturing Example 2. Ethyl 2-(1-(4-(3-chloropropanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

To a mixture of ice-cooled ethyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (200 mg, 0.696 mmol) and potassium carbonate (443 mg, 3.2 mmol) in dichloromethane (2.3 mL) was added 3-chloropropionyl chloride (100 μL, 1.044 mmol) dropwise, and the mixture was stirred at room temperature for 17 h. After concentrating the reaction mixture, the residue was diluted with water and dichloromethane, and the aqueous layer was extracted with dichloromethane. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure to obtain the target compound ethyl 2-(1-(4-(3-chloropropanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (256 mg, 97%) as a white solid.

^1H NMR (400 MHz, CDCl ₃ ) δ 7.85 (s, 1H), 7.38 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 5.16 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.86 (t, J = 6.4 Hz, 2H), 3.34 (s, 2H), 2.79 (t, J = 6.4 Hz, 2H), 2.22 (s, 3H), 2.13 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H).

Manufacturing Example 3. Ethyl 2-(1-(4-(2-chloroacetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

To a mixture of ice-cold ethyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (200 mg, 0.696 mmol) and potassium carbonate (443 mg, 3.2 mmol) in dichloromethane (5 mL) was added chloroacetyl chloride (84 μL, 1.044 mmol) dropwise, and the mixture was stirred at room temperature for 6 h. After concentrating the reaction mixture, the residue was diluted with water and dichloromethane, and the aqueous layer was extracted with dichloromethane. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure to obtain the target compound ethyl 2-(1-(4-(2-chloroacetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (204 mg, 80%) as a white solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 10.28 (s, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 5.13 (s, 2H) , 4.23 (s, 2H), 4.04 (d, J = 7.1 Hz, 2H), 2.09 (s, 3H), 2.04 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H).

Manufacturing Example 4. 1,3-Dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid

Step 1: Methyl 4-cyano-3-(cyanomethyl)benzoate

Methyl cyanoacetate (4.9 mL, 55.81 mmol) was slowly added dropwise to a DMSO (17 mL) suspension of ice-cold sodium hydride (60%, 2.23 g, 55.81 mmol), and the mixture was stirred at room temperature for 30 minutes. To the reaction mixture was added a DMSO (27 mL) solution of methyl 4-cyano-3-fluorobenzoate (5 g, 27.9 mmol), and the mixture was stirred at 90 °C for 12 hours. A small amount of aqueous hydrochloric acid (2 M) was added to the reaction solution, and the mixture was extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure to obtain methyl 4-cyano-3-(1-cyano-2-methoxy-2-oxoethyl)benzoate, which was dissolved in DMSO-H ₂ O (9:1, 30 mL) and stirred at 120 ℃ for 16 hours. The reaction mixture was diluted with EtOAc and brine, and the aqueous layer was extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure, and the resulting residue was purified by silica gel chromatography (Hx:EtOAc = 98:2 to 90:10) to obtain the target compound methyl 4-cyano-3-(cyanomethyl)benzoate (3.5 g, 63%) as a yellow solid.

^1H NMR (400 MHz, CDCl ₃ ) δ 8.31 (d, J = 0.8 Hz, 1H), 8.14 (dd, J = 8.0, 1.5 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 4.05 (s, 2H), 3.99 (s, 3H).

Step 2: 1,3-Dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid

Methyl 4-cyano-3-(cyanomethyl)benzoate (3.5 g, 17.48 mmol) obtained in Step 1 was stirred in concentrated hydrochloric acid (80 mL) at 110 ℃ for 15 hours. After gradually cooling to room temperature, the solid precipitated in the reaction solution was filtered under reduced pressure. The solid was washed with EtOAc, water, MeOH, and Et ₂ O in that order, and then dried to obtain the target compound 1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid (3.58 g, 100%) as a white solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 13.38 (s, 1H), 11.43 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.02-7.89 (m, 2H), 4.11 ( s, 2H).

Manufacturing Example 5. 4-((4-(2-Ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoic acid

Step 1: tert-butyl 4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoate

t-Butyl 4-bromomethyl benzoate (1.31 g, 4.833 mmol) and potassium carbonate (668 mg, 4.833 mmol) were added to a mixture of ethyl 2-(3,5-dimethyl-1H-pyrazol-4-yl) acetate (5.370 mmol) in CH3CN (5.5 mL), and the mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with EtOAc and brine, and the aqueous layer was extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (CH ₂ Cl ₂ :EtOAc = 95:5 to 90:10) to obtain the target compound tert-butyl 4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoate (1.4 g, 70%) as a yellow liquid.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.77 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 4.73 (s, 2H), 4.01 (q, J = 7.1 Hz, 2H), 3.19 (s, 3H), 2.06 (s, 3H), 1.46 (s, 9H), 1.11 (t, J = 7.1 Hz, 3H).

Step 2: 4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoic acid

Trifluoroacetic acid (7.5 mL) was added to a solution of tert-butyl 4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoate (1.38 g, 3.71 mmol) obtained in Step 1 in dichloromethane (2 mL), and the mixture was stirred at room temperature for 7 hours. After concentrating the reaction mixture under reduced pressure, _Et2O was added, and the mixture was stirred at room temperature for 30 minutes. The solid in the mixture was filtered under reduced pressure and dried to obtain the target compound 4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoic acid (978 mg, 83%) as a white solid.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.83 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 5.22 (s, 2H), 3.98 (q, J = 7.1 Hz, 2H), 3.16 (s, 3H), 2.06 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H).

Manufacturing Example 6. (1,3-Bis((tert-butyldiphenylsilyl)oxy)isoquinolin-6-yl)methanol

Step 1: tert-Butyldiphenylsilyl 1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinoline-6-carboxylate

To a mixture of 1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid (300 mg, 1.46 mmol) and imidazole (402 mg, 5.84 mmol) in dichloromethane (12 mL) was added tert-Butyldiphenylsilyl chloride (1.14 mL, 4.38 mmol), and the mixture was stirred at room temperature for 15 hours. The reaction mixture was filtered, and the filtrate was diluted with dichloromethane and water, and the aqueous layer was extracted with dichloromethane. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (Hx:EtOAc = 97:3) to obtain the target compound tert-butyldiphenylsilyl 1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinoline-6-carboxylate (850 mg, 63%).

^1H NMR (400 MHz, CDCl ₃ ) δ 8.33 (d, J = 8.6 Hz, 1H), 8.13 (d, J = 1.2 Hz, 1H), 7.96 (dd, J = 8.6, 1.6 Hz, 1H), 7.75 -7.68 (m, 8H), 7.51 (dd, J = 8.0, 1.2 Hz, 4H), 7.42-7.37 (m, 2H), 7.37-7.31 (m, 7H), 7.28 (dt, J = 14.6, 4.2 Hz, 5H), 7.23-7.16 (m, 5H), 1.17 (d, J = 3.6 Hz, 18H), 0.90 (s, 9H).

Step 2: (1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinolin-6-yl)methanol

Lithium aluminum hydride (15 mg, 3.69 mmol) was added in small portions several times to a THF (10 mL) solution of tert-butyldiphenylsilyl 1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinoline-6-carboxylate (850 mg, 0.923 mmol) obtained in Step 1, and the mixture was stirred at room temperature for 4 hours. EtOAc and a saturated aqueous potassium sodium tartrate solution were added to the reaction mixture, and the mixture was stirred at room temperature until the organic layer and the aqueous layer were separated. The aqueous layer was extracted with EtOAc, the organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (Hx:EtOAc = 80:20) to obtain the target compound ((1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinolin-6-yl)methanol (137 mg, 22%).

^1H NMR (400 MHz, CDCl ₃ ) δ 8.26 (d, J = 8.5 Hz, 1H), 7.76 (dd, J = 8.1, 1.3 Hz, 4H), 7.55 (dd, J = 8.0, 1.3 Hz, 4H) , 7.40-7.27 (m, 10H), 7.23 (t, J = 7.4 Hz, 4H), 6.25 (s, 1H), 4.76 (d, J = 5.7 Hz, 2H), 1.19 (s, 9H), 0.91 (s, 9H).

MS (ESI) m/z 668 [M+H] ⁺

Manufacturing Example 7. Ethyl 2-(1-(4-(azidocarbonyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

In Preparation Example 5, 4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoic acid (500 mg, 1.58 mmol) and triethylamine (0.28 mL, 2.05 mmol) obtained in THF (5 mL) were added diphenylphosphoryl azide (0.44 mL, 2.05 mmol), stirred at room temperature for 15 hours, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography (Hx:EtOAc = 1:1) to obtain the target compound ethyl 2-(1-(4-(azidocarbonyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (475 mg, 88%) as a white solid.

MS (ESI) m/z 364 [M+Na] ⁺

Manufacturing Example 8. 4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)aniline

Step 1: 2-(3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)acetonitrile

(4-nitrobenzyl)hydrazine (2.77 g, 16.57 mmol) in MeOH was slowly added dropwise to a MeOH solution of 4-oxo-3-propionylhexanitrile (2.5 mL 14.91 mmol), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with water and EtOAc, and the aqueous layer was extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure, and the residue was purified by silica gel chromatography (CHCl ₃ :EtOAc = 98:2 to 95:5) to obtain the target compound 2-(3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)acetonitrile (2.6 g, 58%).

^1H NMR (400 MHz, CDCl ₃ ) δ 8.16 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 5.33 (s, 2H), 3.46 (s, 2H), 2.61 (dd, J = 26.4, 7.6 Hz, 4H), 1.27 (t, J = 7.6 Hz, 3H), 1.07 (t, J = 7.7 Hz, 3H).

MS (ESI) m/z 321 [M+Na] ⁺

Step 2: 5-((3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)methyl)-1H-tetrazole

Sodium azide (1.81 g, 27.9 mmol) and zinc bromide (6.28 g, 27.9 mmol) were added to a solution of 2-(3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)acetonitrile (2.5 g, 8.38 mmol) obtained in Step 1 in ⁱ PrOH-H ₂ O (1:1, 30 mL), and the mixture was stirred at 100 ℃ for 25 hours. The reaction mixture was slowly cooled to room temperature, and the precipitated solid was filtered. The solid was dissolved in MeOH, and 6 M hydrochloric acid aqueous solution was added, and the mixture was stirred at room temperature for 1 hour.

The mixture was extracted with CH ₂ Cl ₂ :MeOH (9:1), and the organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure to obtain the target compound 5-((3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)methyl)-1H-tetrazole (2.46 g, 72%) as a white solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 8.20 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 5.41 (s, 2H), 4.03 (s, 2H) , 2.59 (q, J = 7.5 Hz, 2H), 2.45 (t, J = 7.5 Hz, 2H), 1.05 (t, J = 7.5 Hz, 3H), 0.85 (t, J = 7.5 Hz, 3H).

MS (ESI) m/z 364 [M+Na] ⁺

Step 3: 5-((3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)methyl)-1-trityl-1H-tetrazol

DCM of 5-((3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)methyl)-1H-tetrazole (380 mg, 1.11 mmol) obtained in step 2 To the solution were added trityl chloride (372 mg, 1.33 mmol), triethylamine (279 μL, 2.0 mmol), and DMAP (5 mg, 0.04 mmol), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water and DCM, and the aqueous layer was extracted with DCM. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure, and the residue was purified by silica gel chromatography (CHCl ₃ :MeOH = 98:2 to 95:5) to obtain the target compound 5-((3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)methyl)-1-trityl-1H-tetrazole (430 mg, 66%) as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.13 (d, J = 8.8 Hz, 2H), 7.39 - 7.31 (m, 9H), 7.23 (d, J = 8.7 Hz, 2H), 6.99 (dd, J = 7.8, 1.8 Hz, 6H), 5.38 (s, 2H), 4.00 (s, 2H), 2.54 (d, J = 7.6 Hz, 2H), 2.43 (t, J = 7.6 Hz, 2H), 1.02 (t, J = 7.6 Hz, 3H), 0.82 (t, J = 7.5 Hz, 3H).

MS (ESI) m/z 606 [M+Na] ⁺

Step 4: 4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)aniline

To a solution of 5-((3,5-diethyl-1-(4-nitrobenzyl)-1H-pyrazol-4-yl)methyl)-1-trityl-1H-tetrazole (430 mg, 0.737 mmol) obtained in Step 3 in MeOH (7 mL) was added Pd/C (10%, 75 mg), and the mixture was stirred at room temperature for 3 hours under a stream of hydrogen. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound 4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)aniline (400 mg, 98%) as a pale yellow solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 6.82 (d, J = 8.2 Hz, 2H), 6.48 (dd, J = 8.7, 2.1 Hz, 2H), 4.98 (s, 2H), 3.89 (s, 2H), 2.54 (d, J = 7.8 Hz, 2H), 2.44 (dd, J = 15.6, 7.8 Hz, 2H), 1.05 (t, J = 7.5 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H).

MS (ESI) m/z 334 [M+Na] ⁺

Manufacturing Example 9. 3-(4-cyano-3-(cyanomethyl)phenyl)propanoic acid

Step 1: 4-Bromo-2-(cyanomethyl)benzonitrile

2-Methyl cyanoacetate (0.44 mL, 5.00 mmol) was added dropwise to a suspension of ice-cold sodium hydride (60%, 200 mg, 5.00 mmol) in DMSO (1.5 mL), and the mixture was stirred at room temperature for 30 minutes. To the reaction mixture was added a solution of 4-bromo-2-fluorobenzonitrile (500 mg, 2.50 mmol) in DMSO (2.5 mL), and the mixture was stirred at 90 °C for 5 hours. A small amount of aqueous hydrochloric acid (2 M) was added to the reaction mixture, and the mixture was extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure to obtain methyl 2-(5-bromo-2-cyanophenyl)-2-cyanoacetate, which was dissolved in DMSO-H ₂ O (9:1, 3 mL) and stirred at 120 ℃ for 6 hours. The reaction mixture was diluted with EtOAc and brine, and the aqueous layer was extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure, and the resulting residue was purified by silica gel chromatography (Hx:EtOAc = 98:2 to 95:5) to obtain the target compound 4-bromo-2-(cyanomethyl)benzonitrile (895 mg, 81%) as a yellow solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 7.89 (dd, J = 5.0, 3.2 Hz, 2H), 7.82 (dd, J = 8.3, 1.9 Hz, 1H), 4.28 (s, 2H).

Step 2: Ethyl 3-(4-cyano-3-(cyanomethyl)phenyl)propanoate

To a DMF (36 mL) solution of 4-bromo-2-(cyanomethyl)benzonitrile (2.0 g, 9.05 mmol) obtained in Step 1 was added tetra-n-butylammonium bromide (2.9 g, 9.05 mmol), tributylamine (4.3 mL, 18.1 mmol), palladium(II) acetate (61 mg, 0.27 mmol), and acrolein diethylacetal (4.1 mL, 27.14 mmol), and the mixture was stirred at 100 ℃ for 5 hours. After adding aqueous hydrochloric acid solution (1 M) to the reaction solution, the solution was diluted with _Et2O and water, and the aqueous layer was extracted with _Et2O . The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (Hx:EtOAc = 90:10 to 80:20) to obtain the target compound ethyl 3-(4-cyano-3-(cyanomethyl)phenyl)propanoate (1.2 g, 50%) as an orange liquid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 7.83 (d, J = 7.9 Hz, 1H), 7.52 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 4.24 (s, 2H) , 4.04 (q, J = 7.1 Hz, 2H), 2.95 (t, J = 7.4 Hz, 2H), 2.67 (t, J = 7.4 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H).

MS (ESI) m/z 265 [M+Na] ⁺

Step 3: 3-(4-cyano-3-(cyanomethyl)phenyl)propanoic acid

In step 2, ethyl 3-(4-cyano-3-(cyanomethyl)phenyl)propanoate (1.0 g, 4.13 mmol) obtained was added to a THF (10 mL) solution, water (2 mL) and lithium hydroxide (198 mg, 8.26 mmol) were added, and the mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. The residue was acidified by adding aqueous hydrochloric acid solution (1 M), and then extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure to obtain the target compound 3-(4-cyano-3-(cyanomethyl)phenyl)propanoic acid (890 mg, 100%) as a yellow solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.20 (s, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.52 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H) , 4.24 (s, 2H), 2.92 (t, J = 7.4 Hz, 2H), 2.59 (t, J = 7.5 Hz, 2H).

MS (ESI) m/z 237 [M+Na] ⁺

Example 1. 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

Step 1: Ethyl 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

To a suspension of ice-cold sodium hydride (60%, 12 mg, 0.289 mmol) in DMF (1 mL) were added isatin (36 mg, 0.241 mmol), ethyl 2-(1-(4-(3-chloropropanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (100 mg, 0.265 mmol) obtained in Preparation Example 2, and a catalytic amount of potassium iodide, and the mixture was stirred at 120 °C for 20 hours. An aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (CHCl ₃ :EtOAc = 90:10 to 75:25 then CHCl ₃ :EtOAc:MeOH = 60:40:2) to obtain the target compound ethyl 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (25 mg, 21%) as an orange solid.

MS (ESI) m/z 511 [M+Na] ⁺

Step 2: 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

To a THF (0.5 mL) solution of ethyl 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (25 mg, 0.051 mmol) obtained in Step 1 was added water (0.25 mL) and lithium hydroxide (3 mg, 0.13 mmol). The reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was acidified with aqueous hydrochloric acid (1.0 M) and extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (CHCl ₃ :MeOH = 6:1) to obtain the target compound 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (10 mg, 42%) as an orange solid.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.63-7.36 (m, 4H), 7.16 (d, J = 8.2 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.02 (d, J = 8.6 Hz, 2H), 5.16 (s, 2H), 4.09 (t, J = 6.8 Hz, 2H), 3.21 (s, 2H), 2.74 (t, J = 6.9 Hz, 2H), 2.18 (s, 3H), 2.13 (s, 3H).

Example 2. 2-(1-(4-(2-(2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

Isatin (10 mg, 0.07 mmol) and ethyl 2-(1-(4-(2-chloroacetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (29 mg, 0.08 mmol) obtained in Preparation Example 3 were treated with the method of Example 1 to obtain the target compound 2-(1-(4-(2-(2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (12 mg, 35%) as an orange solid.

^1H NMR (400 MHz, CDCl ₃ ) δ 8.96 (s, 1H), 7.59 (t, J = 6.7 Hz, 2H), 7.26 (d, J = 2.4 Hz, 2H), 7.14 (t, J = 7.5 Hz) , 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.3 Hz, 2H), 5.07 (s, 2H), 4.48 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.32 (s, 2H), 2.13 ( d, J = 5.8 Hz, 6H), 1.23 (d, J = 7.1 Hz, 3H).

Example 3. Ethyl 2-(1-(4-(2-(2,3-dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

5-(Pyrrolidin-1-ylsulfonyl)indolin-2,3-dione (400 mg, 1.427 mmol) and ethyl 2-(1-(4-(2-chloroacetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (519 mg, 1.427 mmol) obtained in Preparation Example 3 were treated with the same solution as in Step 1 of Example 1 to obtain the target compound ethyl 2-(1-(4-(2-(2,3-dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (287 mg, 33%) as a yellow solid.

^1H NMR (400 MHz, CDCl ₃ ) δ 9.05 (s, 1H), 8.07-7.98 (m, 2H), 7.27 (s, 1H), 7.24 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 8.5 Hz, 2H), 5.14 (s, 2H), 4.52 (s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.35 (s, 2H), 3.23 (t, J = 6.7 Hz, 4H), 2.18 ( s, 3H), 2.15 (s, 3H), 1.84 - 1.78 (m, 4H), 1.25 (dd, J = 9.2, 5.0 Hz, 3H).

MS (ESI) m/z 630 [M+Na] ⁺

Example 4. 2-(2-((2-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)amino)-2-oxoethyl)amino)-5-(pyrrolidin-1-ylsulfonyl)phenyl)-2-oxoacetic acid

Ethyl 2-(1-(4-(2-(2,3-dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (100 mg, 0.165 mmol) obtained in Example 3 was applied to Step 2 of Example 1 to obtain the target compound 2-(2-((2-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)amino)-2-oxoethyl)amino)-5-(pyrrolidin-1-ylsulfonyl)phenyl)-2-oxoacetic acid (12 mg, 12%) as a white solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.12 (s, 1H), 10.30 (s, 1H), 9.31 (t, J = 5.2 Hz, 1H), 7.93 (d, J = 2.2 Hz, 1H) , 7.86 (dd, J = 9.1, 2.1 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 9.2 Hz, 1H), 5.13 (s, 2H), 4.29 (d, J = 5.2 Hz, 2H), 3.26 (s, 2H), 3.09 (t, J = 6.6 Hz, 4H), 2.09 (s, 3H), 2.05 (s, 3H), 1.68 (dd, J = 8.1, 5.3 Hz, 4H).

Example 5. Ethyl (S)-2-(1-(4-(2-(5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)-2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

(S)-5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)indoline-2,3-dione (100 mg, 0.308 mmol) and ethyl 2-(1-(4-(2-chloroacetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (112 mg, 0.308 mmol) obtained in Preparation Example 3 were applied to the method of Step 1 of Example 1 to obtain the target compound ethyl (S)-2-(1-(4-(2-(5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)-2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (150 mg, 61%). Obtained.

^1H NMR (400 MHz, CDCl ₃ ) δ 8.81 (s, 1H), 8.18-7.95 (m, 2H), 7.28 (d, J = 8.5 Hz, 2H), 7.11 (d, J = 8.1 Hz, 1H) , 6.89 (d, J = 8.2 Hz, 2H), 5.14 (s, 2H), 4.53 (s, 2H), 4.34 (d, J = 7.0 Hz, 1H), 4.13 (dt, J = 9.8, 5.8 Hz, 2H), 4.00-3.92 (m, 1H), 3.86 (dd, J = 9.2, 6.1 Hz, 1H), 3.79-3.64 (m, 2H), 3.57 (dd, J = 9.4, 3.8 Hz, 1H), 3.45 - 3.40 (m, 1H), 3.35 (d, J = 4.5 Hz, 3H), 3.11 (d, J = 8.0 Hz, 1H), 2.19 (s, 3H), 2.14 (s, 3H), 1.84 (d, J = 1.7 Hz, 2H), 1.68 (s, 2H), 1.26 (d, J = 3.5 Hz, 3H).

MS (ESI) m/z 674 [M+Na] ⁺

Example 6. Ethyl 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

To a pyridine (5 mL) solution of 1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid (500 mg, 2.437 mmol) obtained in Manufacturing Example 4 and ethyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (700 mg, 2.437 mmol) obtained in Manufacturing Example 1 was added N,N-diisopropylethylamine (0.42 mL, 2.437 mmol), followed by slow dropwise addition of phosphorus oxychloride (0.23 mL, 2.437 mmol). The reaction mixture was stirred at room temperature for 12 hours. After cooling the reaction mixture on ice, water was slowly added dropwise over 2 hours, and the precipitated solid was filtered under reduced pressure. The solid was washed successively with water and _Et2O , then dried to obtain the target compound ethyl 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (269 mg, 23%) as a yellow solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 13.39 (s, 1H), 11.43 (s, 2H), 10.45 (s, 1H), 7.94 (t, J = 9.5 Hz, 3H), 7.71 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 5.16 (s, 2H), 4.11 (s, 4H), 4.04 (q, J = 7.1 Hz, 2H), 2.11 (s, 3H), 2.06 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H).

Example 7. 2-(3,5-Dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetic acid

Step 1: 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

In Example 6, ethyl 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (20 mg, 0.042 mmol) obtained was added to a THF (1 mL) solution, water (1 mL) and lithium hydroxide (10 mg, 0.42 mmol). The reaction mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure. The residue was acidified with citric acid, and the solid was filtered under reduced pressure. The solid was dried to obtain 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (3 mg).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.09 (s, 1H), 11.43 (s, 1H), 10.45 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.01-7.82 ( m, 2H), 7.71 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 5.16 (s, 2H), 4.11 (s, 2H), 3.26 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H) ).

MS (ESI) m/z 445 [MH] ^-

During the above purification process, the residual solid after filtration was dissolved using MeOH, EtOAc and CHCl ₃ , and the solution was collected and concentrated to obtain 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetic acid (approximately 2 mg).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.08 (s, 1H), 10.69 (s, 1H), 8.61-7.86 (m, 3H), 7.74 (d, J = 8.5 Hz, 2H), 7.13 ( d, J = 8.5 Hz, 2H), 5.17 (s, 2H), 3.26 (s, 2H), 2.11 (s, 3H), 2.06 (s, 3H).

MS (ESI) m/z 459 [MH] ^-

Step 2: 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetic acid

2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (3 mg) obtained in Step 1 was dissolved in DMSO-d ₆ (0.75 mL) and left at room temperature. The progress of the reaction was monitored by ¹ H NMR measurement. After the oxidation was complete, DMSO was evaporated under reduced pressure to obtain the target compound 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetic acid (3 mg, 100%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.08 (s, 1H), 10.69 (s, 1H), 8.67-7.88 (m, 3H), 7.74 (d, J = 8.3 Hz, 2H), 7.13 ( d, J = 8.4 Hz, 2H), 5.17 (s, 2H), 3.26 (s, 2H), 2.11 (s, 3H), 2.06 (s, 3H).

Example 8. Ethyl 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetate

Ethyl 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (6 mg, 0.012 mmol) obtained in Example 6 was applied to the method of Step 2 of Example 7 to obtain the target compound ethyl 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetate (6 mg, 100%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.09 (s, 1H), 10.69 (s, 1H), 8.58 (d, J = 1.7 Hz, 1H), 8.42 (dd, J = 8.1, 1.8 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H), 5.18 (s, 2H), 4.04 (d, J = 7.1 Hz, 2H), 2.11 (s, 3H), 2.06 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H).

Example 9. 6-((4-((4-(2-Ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid

In Example 8, ethyl 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetate (50 mg, 0.102 mmol) obtained was added to a THF (1.5 mL) solution, water (0.5 mL) and lithium hydroxide (6 mg, 0.255 mmol). The reaction mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was acidified with aqueous hydrochloric acid (1 M), and then extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (CHCl ₃ :MeOH) to obtain the target compound 6-((4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid (3 mg, 6%) as an orange solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 10.42 (s, 1H), 8.75 (s, 1H), 7.98 (dd, J = 7.8, 1.4 Hz, 1H), 7.87 (s, 1H), 7.71 ( d, J = 8.5 Hz, 2H), 7.64 (d, J = 7.8 Hz, 1H), 7.09 (t, J = 8.1 Hz, 2H), 6.66 (s, 1H), 5.16 (s, 2H), 4.04 (q, J = 7.1 Hz, 2H), 3.37 (d, J = 3.6 Hz, 2H), 2.11 (s, 3H), 2.05 (d, J = 2.2 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H).

MS (ESI) m/z 505 [M-H]-

Example 10. 6-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid

In Example 8, ethyl 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetate (31 mg, 0.063 mmol) obtained was added to a THF (1 mL) solution, water (0.3 mL) and lithium hydroxide (4 mg, 0.159 mmol). The reaction mixture was stirred at room temperature for 12 hours and then concentrated under reduced pressure. The residue was acidified with 1 M hydrochloric acid solution and then extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (CHCl ₃ :MeOH) to obtain the target compound 6-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid (3 mg, 9%) as an orange solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 10.42 (s, 1H), 8.75 (s, 1H), 7.97 (dd, J = 7.8, 1.5 Hz, 1H), 7.87 (s, 1H), 7.70 ( d, J = 8.6 Hz, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.68 (s, 1H), 5.10 (s, 2H), 2.88 (s, 2H), 2.08 (s, 3H) , 2.05 (s, 3H).

Example 11. Ethyl 2-(3,5-dimethyl-1-(4-(3-(1,3,4-trioxo-3,4-dihydroisoquinolin-2(1H)-yl)propanamido)benzyl)-1H-pyrazol-4-yl)acetate

3-(1,3-dioxo-3,4-dihydroisoquinolin-2(1H)-yl)propanoic acid (50 mg, 0.214 mmol) in THF (0.2 mL) was added a DMF (0.4 mL) solution of EDCI (49 mg, 0.257 mmol) and DMAP (31 mg, 0.257 mmol) and ethyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (56 mg, 0.193 mmol) obtained in Preparation Example 1, and the mixture was stirred at room temperature for 45 hours. The reaction mixture was concentrated under reduced pressure, and a 1 M hydrochloric acid solution was added to the residue, followed by extraction with EtOAc. The organic layer was dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (Et ₂ O:CHCl ₃ :EtOAc = 1:1:1) to obtain the target compound ethyl 2-(3,5-dimethyl-1-(4-(3-(1,3,4-trioxo-3,4-dihydroisoquinolin-2(1H)-yl)propanamido)benzyl)-1H-pyrazol-4-yl)acetate (5 mg, 5%) as an orange solid.

^1H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J = 7.2 Hz, 1H), 8.20 (dd, J = 7.6, 1.0 Hz, 1H), 7.87 (dddd, J = 15.1, 8.7, 7.5, 1.2 Hz, 3H), 7.38 (d, J = 8.3 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 5.15 (s, 2H), 4.44 (t, J = 7.3 Hz, 2H), 4.10 (d, J = 7.1 Hz, 2H), 3.33 (s, 2H), 2.76 (t, J = 7.3 Hz, 2H), 2.21 (s, 3H), 2.11 (s, 3H), 1.22 (d, J = 7.1 Hz, 3H).

MS (ESI) m/z 539 [M+Na] ⁺

Example 12. 2-(3,5-Dimethyl-1-(4-((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-1H-pyrazol-4-yl)acetic acid

Step 1: Methyl 4-(4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzamido)-2-(2-methoxy-2-oxoethyl)benzoate

4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzoic acid (345 mg, 1.09 mmol) and methyl 4-amino-2-(2-methoxy-2-oxoethyl)benzoate (243 mg, 1.09 mmol) obtained in Preparation Example 5 were reacted with each other according to the method of Example 11 to obtain the target compound methyl 4-(4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzamido)-2-(2-methoxy-2-oxoethyl)benzoate (399 mg, 70%) as a white solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 10.49 (s, 1H), 7.94 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.85-7.81 (m, 1H), 7.77 (d, J = 2.1 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 5.29 (s, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.97 (s, 2H), 3.77 (s, 3H), 3.60 ( s, 3H), 3.37 (s, 2H), 2.11 (s, 3H), 2.06 (s, 3H), 1.17 (t, J = 7.1 Hz, 3 H).

Step 2: Methyl 2-(1-(4-((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

In step 1, methyl 4-(4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzamido)-2-(2-methoxy-2-oxoethyl)benzoate (300 mg, 0.575 mmol) obtained was added an aqueous solution of potassium hydroxide (322 mg, 5.75 mmol) in MeOH, stirred at room temperature for 48 hours, and then concentrated under reduced pressure. The residue was acidified by adding an aqueous hydrochloric acid solution (6 M), and concentrated under reduced pressure to obtain compound 2-(carboxymethyl)-4-(4-((4-(2-methoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzamido)benzoic acid as a yellow solid.

Then, a suspension of 2-(carboxymethyl)-4-(4-((4-(2-methoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzamido)benzoic acid in AcOH (1.2 mL) was dissolved by stirring at 100 °C, and urea (80 mg, 1.33 mmol) was added and stirred at 120 °C for 5 hours. After the reaction mixture was cooled to room temperature, water was added and stirred for 30 minutes. The precipitated solid was filtered and dried to obtain the target compound methyl 2-(1-(4-((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (200 mg, 75%) as an ivory solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 11.19 (s, 1H), 10.54 (s, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.93-7.84 (m, 3H), 7.79 ( dd, J = 8.6, 1.7 Hz, 1H), 7.23 (d, J = 8.3 Hz, 2H), 5.29 (s, 2H), 4.03 (s, 2H), 3.59 (s, 3H), 3.40 (s, 2H), 2.11 (s, 3H), 2.06 (s) , 3H).

Step 3: 2-(1-(4-((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

Methyl 2-(1-(4-((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (30 mg, 0.065 mmol) obtained in step 2 was applied to the method of step 2 of Example 1 to obtain the target compound 2-(1-(4-((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (3 mg, 11%) as a white solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.13 (s, 1H), 11.19 (s, 1H), 10.54 (s, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.92-7.85 ( m, 3H), 7.79 (dd, J = 8.7, 1.7 Hz, 1H), 7.24 (d, J = 8.2 Hz, 2H), 5.29 (s, 2H), 4.03 (s, 2H), 3.28 (s, 2H), 2.11 (s, 3H), 2.06 ( s, 3H).

Step 4: 2-(3,5-dimethyl-1-(4-((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-1H-pyrazol-4-yl)acetic acid

2-(1-(4-((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (3 mg) obtained in Step 3 was applied to the method of Step 2 of Example 7 to obtain the target compound 2-(3,5-dimethyl-1-(4-((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-1H-pyrazol-4-yl)acetic acid (3 mg, 100%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 11.86 (s, 1H), 10.77 (s, 1H), 8.51 (d, J = 2.2 Hz, 1H), 8.30-8.25 (m, 1H), 8.11 ( d, J = 8.6 Hz, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 5.30 (s, 2H), 3.28 (s, 2H), 2.09 (d, J = 17.1 Hz, 6H).

Example 13. 2-(3,5-Dimethyl-1-(4-(((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-1H-pyrazol-4-yl)acetic acid

Step 1: Ethyl 2-(1-(4-(((1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

To a mixture of ethyl 2-(1-(4-(azidocarbonyl)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (41 mg, 0.119 mmol) obtained in Manufacturing Example 7 and toluene (2 mL) was added (1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinolin-6-yl)methanol (88 mg, 0.131 mmol) obtained in Manufacturing Example 6, and the mixture was stirred at 100 °C for 8 hours. Triethylamine was added to the reaction mixture, stirred at room temperature for 1 hour, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (Hx:EtOAc=80:20) to obtain the target compound ethyl 2-(1-(4-(((1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (89 mg, 76%).

^1H NMR (400 MHz, CDCl ₃ ) δ 8.31 (d, J = 8.5 Hz, 1H), 7.78 (dd, J = 8.1, 1.3 Hz, 4H), 7.60-7.56 (m, 4H), 7.42-7.37 ( m, 4H), 7.37-7.31 (m, 7H), 7.28 (dt, J = 14.8, 4.1 Hz, 6H), 7.05 (d, J = 8.5 Hz, 2H), 6.80 (s, 1H), 6.30 (s, 1H), 5.27 (s, 2H), 5.19 (s, 2H), 4.15 (dt, J = 10.0, 5.7 Hz, 3H), 3.36 (s, 2H), 2.26 (s, 3H), 2.13 (s, 3H), 2.08 (s, 1H), 1.29 (dd, J = 9.1, 5.2 Hz, 2H), 1.22 (s, 9H), 0.95 (s, 9H).

MS (ESI) m/z 981 [M+H] ⁺

Step 2: Ethyl 2-(1-(4-(((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate

Aqueous hydrochloric acid solution (1 M) was added to a THF (1 mL) solution of ethyl 2-(1-(4-(((1,3-bis((tert-butyldiphenylsilyl)oxy)isoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (30 mg, 0.031 mmol) obtained in Step 1, and the mixture was stirred at room temperature for 9 hours. After THF in the reaction mixture was evaporated under reduced pressure, the precipitated solid was filtered and dried to obtain ethyl 2-(1-(4-(((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (15 mg, 96%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 11.30 (s, 1H), 9.84 (s, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H) , 7.44-7.35 (m, 3H), 7.03 (d, J = 8.6 Hz, 2H), 5.21 (s, 2H), 5.10 (s, 2H), 4.03 (q, J = 7.1 Hz, 4H), 2.09 (s, 3H), 2.04 (s, 3H) , 1.15 (t, J = 7.1 Hz, 3H).

MS (ESI) m/z 505 [M+H] ⁺

Step 3: 2-(1-(4-(((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

Ethyl 2-(1-(4-(((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (12 mg, 0.023 mmol) obtained in step 2 was applied to the method of step 2 of Example 1 to obtain the target compound 2-(1-(4-((((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (3 mg, 27%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.08 (s, 1H), 11.30 (s, 1H), 9.84 (s, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.53-7.30 ( m, 4H), 7.04 (d, J = 6.8 Hz, 2H), 5.21 (s, 2H), 5.10 (s, 2H), 4.04 (s, 2H), 3.24 (s, 2H), 2.06 (d, J = 17.8 Hz, 6H).

Step 4: 2-(3,5-dimethyl-1-(4-(((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-1H-pyrazol-4-yl)acetic acid

2-(1-(4-(((1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (3 mg) obtained in Step 3 was applied to the method of Step 2 of Example 7 to obtain the target compound 2-(3,5-dimethyl-1-(4-((((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-1H-pyrazol-4-yl)acetic acid (3 mg, 100%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.21-11.30 (m, 2H), 9.89 (d, J = 13.8 Hz, 1H), 8.40-7.51 (m, 3H), 7.40 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), 5.33 (s, 2H), 5.10 (s, 2H), 3.24 (s, 2H), 2.08 (s, 3H), 2.04 (s, 3H).

Example 14. 2-(1-(4-(((2-methoxynaphthalen-1-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

Triphosgene (0.054 g, 0.183 mmol) was dissolved in THF (0.37 mL), and (2-methoxy-1-naphthyl)methanol (0.034 g, 0.183 mmol) dissolved in DCM was slowly added dropwise. Then, triethylamine (0.056 mL, 0.402 mmol) diluted in DCM was slowly added dropwise. After stirring at room temperature for 30 minutes, the mixture was concentrated under reduced pressure. Then, the mixture was dissolved in THF (0.37 mL), and methyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (0.050 g, 0.183 mmol) dissolved in THF was added dropwise. The resulting residue was stirred at 70°C for 30 minutes and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography to obtain the target compound methyl 2-[1-[[4-[(2-methoxy-1-naphthyl)methoxycarbonylamino]phenyl]methyl]-3,5-dimethyl-pyrazol-4-yl]acetate (0.034 g).

Next, methyl 2-[1-[[4-[(2-methoxy-1-naphthyl)methoxycarbonylamino]phenyl]methyl]-3,5-dimethyl-pyrazol-4-yl]acetate (0.034 g, 0.0697 mmol) was dissolved in methanol (0.35 mL), and 4 N NaOH (0.061 mL, 0.244 mmol) was added dropwise. After stirring at room temperature for 12 h, the mixture was neutralized with 1 N HCl. The resulting residue was extracted with EtOAc, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain the target compound 2-(1-(4-((((2-methoxynaphthalen-1-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (31 mg, 35%).

^1H NMR (400 MHz, CDCl ₃ ) δ 8.03 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.54 (ddd , J = 8.4, 6.8, 1.4 Hz, 1H), 7.43 - 7.37 (m, 1H), 7.39 - 7.29 (m, 3H), 7.01 (d, J = 8.2 Hz, 2H), 6.78 (s, 1H), 5.76 (s, 2H), 5.17 (s) , 2H), 3.99 (s, 3H), 3.37 (s, 2H), 2.22 (s, 3H), 2.11 (s, 3H).

Example 15. 2-(1-(4-((([1,1'-biphenyl]-4-ylmethoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid

Methyl 2-(1-(4-aminobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate (0.050 g, 0.183 mmol) and biphenyl-4-methanol (0.034 g, 0.183 mmol) were reacted according to the method of Example 14 to obtain the target compound (0.032 g, 37%).

^1H NMR (400 MHz, CDCl3) δ 7.62 - 7.58 (m, 4H), 7.49 - 7.37 (m, 4H), 7.35 - 7.32 (m, 3H), 7.05 (d, J = 8.2 Hz, 2H), 6.92 (s, 1H), 5.24 (s, 2H), 5.21 (s, 2H), 3.38 (s, 2H), 2.24 (s, 3H), 2.13 (s, 3H).

Example 16. N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide

Step 1: 4-Cyano-3-(cyanomethyl)-N-(4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)phenyl)benzamide

To a solution of 4-cyano-3-(cyanomethyl)benzoic acid (135 mg, 0.722 mmol) in DMF (3 mL) were added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (412 mg, 1.083 mmol), N,N-diisopropylethylamine (0.38 mL, 2.166 mmol), and 4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)aniline (400 mg, 0.722 mmol) obtained in Preparation Example 8. The reaction mixture was stirred at 90 °C for 2 hours and then concentrated under reduced pressure. The residue was stirred for 1 h, then hydrochloric acid aqueous solution (1 M) was added and extracted with EtOAc. The organic layer was washed with NaHCO ₃ aqueous solution, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (CH ₂ Cl ₂ :EtOAc = 80:20 to 60:40) to obtain the target compound 4-cyano-3-(cyanomethyl)-N-(4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)phenyl)benzamide (200 mg, 38%) as a yellow solid.

^1H NMR (400 MHz, DMSO-d ₆ ) δ 10.55 (s, 1H), 8.14-8.05 (m, 3H), 7.67 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 7.2 Hz, 9H), 7.06 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 1.4 Hz, 3H), 6.98 (d, J = 2.0 Hz, 3H), 5.19 (d, J = 7.0 Hz, 2H), 4.38 (s, 2H), 3.98 (s, 2H), 2.55 (d, J = 7.7 Hz, 2H), 2.44 (d, J = 7.6 Hz, 2H), 1.01 (d, J = 7.6 Hz, 3H), 0.84 (d, J = 7.5 Hz, 3H).

MS (ESI) m/z 744 [M+Na] ⁺

Step 2: N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide

A solution of 4-cyano-3-(cyanomethyl)-N-(4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)phenyl)benzamide (45 mg, 0.062 mmol) obtained in Step 1 and concentrated hydrochloric acid (0.4 mL) was stirred at 50 °C for 1 hour. After gradually cooling to room temperature, the precipitated solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue obtained by concentration was purified by C18 reverse phase column chromatography (H ₂ O:MeOH = 4:6) to obtain the target compound N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (6 mg, 19%).

MS (ESI) m/z 521 [M+Na] ⁺

Step 3: N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide

N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (3 mg) obtained in step 2 was dissolved in DMSO-d ₆ (0.75 mL) and left at room temperature. The progress of the reaction was monitored by ¹ H NMR measurement. After the oxidation was completed, DMSO was evaporated under reduced pressure to obtain the target compound N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (3 mg, 100%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 12.08 (s, 1H), 10.69 (s, 1H), 8.59 (d, J = 1.7 Hz, 1H), 8.42 (dd, J = 8.1, 1.8 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 5.21 (s, 2H), 3.98 (s, 2H), 2.62-2.55 (m, 2H), 2.46 (t, J = 7.6 Hz, 2H), 1.07 (t, J = 7.5 Hz, 3H), 0.86 (t, J = 7.5 Hz, 3H).

MS (ESI) m/z 535 [M+Na] ⁺

Example 17. N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-3-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)propanamide

3-(4-cyano-3-(cyanomethyl)phenyl)propanoic acid (193 mg, 0.903 mmol) obtained in Manufacturing Example 9 and 4-((3,5-diethyl-4-((1-trityl-1H-tetrazol-5-yl)methyl)-1H-pyrazol-1-yl)methyl)aniline (500 mg, 0.903 mmol) obtained in Manufacturing Example 8 were reacted with each other according to the method of Example 16 to obtain the target compound N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-3-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)propanamide (6 mg, 1.2%).

^1H NMR (400 MHz, DMSO-d ₆ ) δ 11.89 (s, 1H), 9.96 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 1.5 Hz, 1H) , 7.81 (dd, J = 8.0, 1.7 Hz, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), 6.73 (dd, J = 79.5, 1.8 Hz, 1H), 5.14 (s, 2H), 3.99 ( s, 2H), 3.08 (t, J = 7.5 Hz, 2H), 2.70 (d, J = 7.5 Hz, 2H), 2.58 - 2.54 (m, 2H), 2.43 (d, J = 7.6 Hz, 2H), 1.04 (t, J = 7.5 Hz, 3H), 0.81 (d, J = 7.6 Hz, 3H).

Experimental Example 1. Evaluation of Caspase-3 Inhibitory Activity

In order to measure the Caspase-3 inhibitory activity of the compound of the present invention manufactured in the examples, Caspase-3 activity was measured using a substrate to which a fluorescent dye was attached. The fluorescent dye attached to the substrate is released from the substrate by Caspase and exhibits fluorescence.

The compounds were dissolved in DMSO and stored at -20 °C. The enzymatic reaction was performed in a buffer solution containing 25 mM HEPES, 10 mM DTT, 0.1% CHAPS (w/v), pH 7.5, and the enzyme activities were measured in the presence of various concentrations of the compounds using 50 μM of the fluorescent substrate N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin (Ac-DEVD-AFC) (Cayman) and 2 ng of recombinant human Caspase-3 (R&D System). Kinetics measurements were performed at 400/505 nm (Ex/Em) wavelengths using a CLARIOstar Plus spectrometer (BMG Labtech). The results were marked as A if the IC ₅₀ (concentration that reduces activity by 50%) was less than 1 μM, B if it was 1 μM or more but less than 10 μM, and C if it was more than that.

As a result, as shown in Table 1 below, it was confirmed that each compound inhibited Caspase-3 activity. Therefore, it was confirmed that the compound of the present invention is a direct Caspase-3 inhibitor.

IC50 (μM) compound Caspase-3 1 C 2 C 3 B 4 C 5 C 6 A 7 A 8 A 9 C 10 C 11 B 12 A 13 A 14 C 15 C 16 A 17 A

The above description of the present invention is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single component may be implemented in a distributed manner, and likewise, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (14)

A pyrazole derivative compound represented by the following chemical formula (I), a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof: <Chemical formula (I)>

In the above formula, R ¹ is -C(O)OR ³ or a 5- to 7-membered heteroaryl ring containing 1 to 4 N, L is -N(R ⁴ )C(O)-, -N(R ⁴ )C(O)O-, -N(R ⁴ )C(O)CH ₂ N(R ⁵ )- or -C(O)N(R ⁴ )-, R ² is a fused ring of two rings in which an aryl ring of 1 to 2 rings having C _5-12 , or a 5 to 7 membered heterocycloalkyl ring containing 1 to 2 N is fused with an aryl ring, and R ² is optionally substituted with one or more independent R ⁶ , R ^a and R ^b are independently C _1-3 alkyl, R ³ , R ⁴ and R ⁵ are independently -H or C _1-3 alkyl, R ⁶ is C _1-3 alkoxy, oxo, hydroxy, -C(O)OH, -C(O)C(O)OH, -S(O) ₂ R ⁷ or aryl, R ⁷ is a 5- to 7-membered heterocycloalkyl ring containing 1 to 2 N, and R ⁷ is optionally substituted with R ⁸ , R ⁸ is C _1-3 alkoxyC _1-3 alkyl, m and n are independently integers from 1 to 3, p is an integer from 0 to 3. A pyrazole derivative compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, in claim 1, wherein R ¹ is ethoxycarbonyl, hydroxycarbonyl, or tetrazolyl. A pyrazole derivative compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, in claim 1, wherein L is -NHC(O)-, -NHC(O)O-, -NHC(O)CH ₂ NH-, or -C(O)NH-. A pyrazole derivative compound, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, in claim 1, wherein R ² is phenyl, naphthalenyl, indolinyl, isoindolinyl, dihydroisoquinolinyl or tetrahydroisoquinolinyl. A pyrazole derivative compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, in claim 1, wherein R ^a and R ^b are independently methyl or ethyl. A pyrazole derivative compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, in claim 1, wherein R ³ , R ⁴ and R ⁵ are independently -H or ethyl. A pyrazole derivative compound, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, in claim 1, wherein R ⁶ is methoxy, oxo, hydroxy, -C(O)OH, -C(O)C(O)OH, pyrrolidinylsulfonyl or phenyl. A pyrazole derivative compound, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, in claim 1, wherein R ⁸ is methoxymethyl. A pyrazole derivative compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, in claim 1, wherein m is an integer 1 and n is an integer 1. A pyrazole derivative compound, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein p is 0, 1, or 2 in claim 1. In claim 1, a pyrazole derivative compound represented by the chemical formula (I) is any one selected from the group consisting of the following compounds, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof: [1] 2-(1-(4-(3-(2,3-dioxoindolin-1-yl)propanamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid, [2] 2-(1-(4-(2-(2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid, [3] Ethyl 2-(1-(4-(2-(2,3-dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate, [4] 2-(2-((2-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)amino)-2-oxoethyl)amino)-5-(pyrrolidin-1-ylsulfonyl)phenyl)-2-oxoacetic acid, [5] Ethyl (S)-2-(1-(4-(2-(5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)-2,3-dioxoindolin-1-yl)acetamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate, [6] Ethyl 2-(1-(4-(1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetate, [7] 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetic acid, [8] Ethyl 2-(3,5-dimethyl-1-(4-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)benzyl)-1H-pyrazol-4-yl)acetate, [9] 6-((4-((4-(2-ethoxy-2-oxoethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid, [10] 6-((4-((4-(carboxymethyl)-3,5-dimethyl-1H-pyrazol-1-yl)methyl)phenyl)carbamoyl)-1-hydroxy-3-oxoisoindoline-1-carboxylic acid, [11] Ethyl 2-(3,5-dimethyl-1-(4-(3-(1,3,4-trioxo-3,4-dihydroisoquinolin-2(1H)-yl)propanamido)benzyl)-1H-pyrazol-4-yl)acetate, [12] 2-(3,5-dimethyl-1-(4-((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)carbamoyl)benzyl)-1H-pyrazol-4-yl)acetic acid, [13] 2-(3,5-dimethyl-1-(4-(((1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)methoxy)carbonyl)amino)benzyl)-1H-pyrazol-4-yl)acetic acid, [14] 2-(1-(4-(((2-methoxynaphthalen-1-yl)methoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid, [15] 2-(1-(4-((([1,1'-biphenyl]-4-ylmethoxy)carbonyl)amino)benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid, [16] N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-1,3,4-trioxo-1,2,3,4-tetrahydroisoquinoline-6-carboxamide, and [17] N-(4-((4-((1H-tetrazol-5-yl)methyl)-3,5-diethyl-1H-pyrazol-1-yl)methyl)phenyl)-3-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)propanamide. A pharmaceutical composition for preventing or treating a caspase-3-related disease, comprising a pyrazole derivative compound of any one of claims 1 to 11, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition according to claim 12, characterized in that the caspase-3 related disease is pulmonary hypertension. A pharmaceutical composition comprising a pyrazole derivative compound according to any one of claims 1 to 11, a hydrate thereof, a solvate thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive.