RU2846904C1 - Novel pyrazole derivative - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to organic chemistry and includes a compound of chemical formula I-1, 6-benzyl-3-methoxy-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine, their pharmaceutically acceptable salts and pharmaceutical composition based thereon. In formula I-1, X is NR3; R3 is selected from Ra-L-, Ra-L-CO-, Ra-L-NHCO-, Ra-L-OCO- and Ra-L-SO₂-; Ra is selected from cyano, halogen, substituted or unsubstituted C1-C5 straight or branched alkyl, C2-C6alkynyl, C2-C6alkenyl, unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C6-C20aryl, or substituted or unsubstituted C5-C20 heteroaryl, and heteroaryl contains 1, 2 or 3 heteroatoms from O, N and S; L is selected from a direct bond, and C1-C3 alkylene and substituted or unsubstituted phenylene; and R4 is unsubstituted C1-C5 alkyl; A is CR5 or N, B is CR6, D is CR7, E is CR8, G is CR9 or N, where A or G is N; radicals R5 to R9 are identical or different and each is independently selected from hydrogen, cyano, halogen, carbonyl, nitro, carboxyl, C1-C5alkoxy, substituted or unsubstituted C1-C5 straight or branched alkyl, an unsubstituted amino group, and two or more adjacent groups, can be combined with each other to form an unsubstituted C5-C10 heteroaryl ring containing 1, 2 or 3 heteroatoms selected from N and S, the rest of the substituents are as indicated in the formula of invention.

EFFECT: pyrazole derivatives of formula I-1, having oxidative stress inhibitory activity.

6 cl, 6 dwg, 2 tbl, 9 ex

Description

AREA OF TECHNOLOGY

This application claims priority from Korean Patent Application No. 10-2020-0004471 filed on January 13, 2020, and the entire contents disclosed in the corresponding Korean Patent Application are incorporated as part of this specification.

The present invention relates to a novel pyrazole derivative with excellent oxidative stress inhibitory activity, a method for producing such a derivative and a pharmaceutical composition thereof.

PRIOR ART

Oxidative stress refers to the phenomenon in which the balance between the production of reactive oxygen species or reactive nitrogen species and the antioxidant defense mechanism in biomolecules, cells and tissues in vivo is disturbed, and the production of reactive oxygen species or reactive nitrogen species becomes relatively excessive, and tissue damage usually occurs in this case.

Reactive oxygen species (ROS) collectively refer to the oxygen free radicals formed due to the chemical properties of oxygen and _the oxygen compounds derived from them, such as superoxide anion ( _O2 ^- '), hydrogen peroxide ( _H2O2 ), hydroxyl group (OH'), alkoxyl group (RO'), peroxyl group (ROO'), and the like. Reactive nitrogen species (RNS) collectively refer to the highly reactive peroxynitrite (ONOO ^- ), formed by NOS (nitric oxide synthase) in vivo as a result of the interaction of nitric oxide (NO') and superoxide anion ( _O2 ^- '), and nitrogen dioxide (' _NO2 ), nitrosoperoxycarbonate ( _ONOOCO2 ^- ), carbonate radical (O=C(O')O ^- ), and the like, derived from peroxynitrite.

Because reactive oxygen species (ROS) or reactive nitrogen species (RNS) are chemically very unstable and highly reactive, they trigger inflammation and act as a major factor in tissue fibrosis, causing extensive oxidative damage to enzymatic catalysis reactions, mitochondrial electron transfer, cell signaling and gene expression, transcription factor activation, and biomolecules, cells, tissues, and other in vivo processes. This oxidative damage causes various diseases in all tissues of the human body. In particular, it is known to be involved in the development of cancer in tissues such as the skin, kidneys, heart, joints, lungs, brain, blood vessels, gastrointestinal tract, and eyes, and in the progression of existing cancer. It is also known to play a significant role in almost all diseases, including cardiovascular disease, inflammation, fibrosis, diabetes, and the like.

It has recently been established that the interaction between the tumor microenvironment and cancer cells plays a key role in tumor growth, metastasis, and resistance to antitumor agents, with tumor-associated fibroblasts (CAFs) playing a key role in this regard. CAFs are known to be activated by ROS, which promotes tumor evasion from the immune response and impacts cancer malignancy and metastasis. Therefore, CAFs have recently emerged as a new target for cancer treatment.

In liver tissue from patients with liver fibrosis, increased ROS production in hepatocytes has been reported to be caused by stimulation of TGF-β1 (transforming growth factor beta-1), which increases the expression of collagen and α-smooth muscle actin (α-SMA), which are important for fibrosis. ROS has been reported to exacerbate pulmonary fibrosis in lung tissue from patients with idiopathic pulmonary fibrosis.

The brain is a highly metabolic tissue and therefore highly vulnerable to oxidative damage. Therefore, numerous studies have been conducted on effectively scavenging ROS for the treatment of various neurodegenerative diseases (Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, ischemic or traumatic brain injury, and so on), but to no avail. Recently, when ROS were discovered to be a significant factor in these brain diseases, the possibility of treatment through ROS inhibition was reported.

Keloid is a type of benign tumor in which connective tissue in the skin abnormally grows, forming hard nodules, and the epidermis becomes thin, shiny, and reddish. Overexpression of connective tissue growth factor (CTGF), mediated by ROS produced by TGF-β1 stimulation, has been reported as one of the causes of keloid development and recurrence.

In diabetes, complications such as diabetic nephropathy and diabetic retinopathy can accompany prolonged hyperglycemia, and ROS are known to play a significant role in the development of these complications. In other words, high blood sugar levels cause increased ROS production, and the resulting ROS can cause diabetic nephropathy, diabetic retinopathy, and other conditions through a series of processes that cause kidney and retinal cell death.

In addition, according to observations in keloid diseases, overexpression of the CTGF gene in the retina has been reported to cause retinal fibrosis and macular degeneration.

Therefore, the inventors of the present invention conducted a study focusing on the fact that a therapeutic agent for the treatment of cancer, an inflammatory disease, a fibrotic disease, a neurodegenerative disease, a liver disease, a skin disease or a retinal disease caused by oxidative stress can be developed using a molecular mechanism that inhibits the occurrence of oxidative stress, and as a result, they found that the novel pyrazole derivative of the present invention has an excellent inhibitory activity against the formation of ROS, and confirmed that it can be used for the treatment of various diseases associated with oxidative stress, which completed the present invention.

Prior art documents

Patent Document 1: WO 2016-207785.

Patent Document 2: WO 2015-144801.

Non-patent document 1: Mengjia Song, Xinfeng Chen, et al., Chinese Journal of Cancer Research, 2018, 30(2): 157–172.

Non-patent document 2: Lan T, Kisseleva T, Brenner DA, PLoS One 2015; 10: e0129743.

Non-patent document 3: Merry W. Ma, Jin Wang, et al., Molecular neurodegeneration, 12:7, 2017.

Non-Patent Document 4: Alessandro G. Fois, Panagiotis Paligiannis et al. Respir Res. 2018, 19:51.

Non-patent document 5: Rohan Samarakoon, Jessica M. Overstreet et al., Cell Signal, 2013, Jan, 25(1), 264-268.

DISCLOSURE OF THE ESSENCE OF THE INVENTION

TECHNICAL TASK

The aim of the present invention is to provide a new pyrazole derivative which is a compound of chemical formula I, a solvate, stereoisomer or a pharmaceutically acceptable salt thereof.

The aim of the present invention is to provide a pharmaceutical composition comprising a compound of chemical formula I, a solvate, stereoisomer or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The purpose of the present invention is to provide a method for regulating ROS production using a compound of chemical formula I, a solvate, stereoisomer or pharmaceutically acceptable salt thereof.

The purpose of the present invention is to provide a method for treating a disease associated with oxidative stress by administering to a subject a compound of chemical formula I, a solvate, stereoisomer or pharmaceutically acceptable salt thereof.

The purpose of the present invention is to provide a method for preparing a compound of chemical formula I.

The aim of the present invention is to provide the use of a compound of chemical formula I, a solvate, stereoisomer or pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of a disease associated with oxidative stress.

TECHNICAL SOLUTION

According to the present invention, to achieve the above objectives, a new pyrazole derivative is provided, which is a compound of the following chemical formula I, a solvate, stereoisomer or pharmaceutically acceptable salt thereof.

chemical formula I

in which

X, Y represent CR1R2 or NR3; and

Rl, R2 or R3 are the same or different from each other and each is independently selected from Ra-L-, Ra-L-CO-, Ra-L-NHCO-, Ra-L-OCO-, Ra-L-SO ₂ -; and

Ra is selected from hydrogen, heavy hydrogen, cyanogen, halogen, nitro, haloalkyl, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C5-C20 aryl, or substituted or unsubstituted C5-C20 heteroaryl, and the heteroaryl contains 1, 2 or 3 heteroatoms from O, N and S; and

L is selected from a direct bond, C1-C5 alkylene, and substituted or unsubstituted C5-C10 arylene; and

R4 is selected from hydrogen or substituted or unsubstituted C1-C10 alkyl; and

A is CR5 or N, B is CR6 or N, D is CR7 or N, and E is CR8 or N, and G is CR9 or N; and

radicals R5 through R9 are the same or different and are each independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5alkoxy, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20aryl, or substituted or unsubstituted C5-C20 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

substituted C1-C10 straight or branched alkyl may be optionally partially unsaturated and may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20 aryl or C5-C20 heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl; and

substituted C3-C8cycloalkyl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20aryl or C5-C20 heteroaryl, C1-C5alkoxy and C1-C3haloalkyl, or the same or different 2 or more substituents are linked; and

substituted C5-C20 aryl or substituted C5-C20 heteroaryl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, C1-C5 alkoxy, C1-C5 haloalkyl, C1-C10 alkyl, C3-C8 cycloalkylC1-C10 alkyl and C5-C20 arylC1-C10 alkyl, or the same or different 2 or more substituents are linked; and

the substituted amine may be substituted with one or two C1-C5 alkyl groups; and

substituted C5-C10arylene may be substituted with one or more substituents selected from C1-C3 alkyl, halogen, cyano, amine, nitro and heavy chain, or the same or different 2 or more substituents are linked.

Furthermore, according to the present invention, there is provided a compound of chemical formula I, a solvate, a stereoisomer or a pharmaceutically acceptable salt used for the treatment of a disease associated with oxidative stress.

Furthermore, according to the present invention, a pharmaceutical composition for the treatment of a disease associated with oxidative stress is provided, containing a compound of chemical formula I, its solvate, stereoisomer or pharmaceutically acceptable salt and an acceptable carrier as an active ingredient.

Furthermore, according to the present invention, there is provided a method for treating a disease associated with oxidative stress, comprising administering an effective dose of a compound of chemical formula I, a solvate, stereoisomer or pharmaceutically acceptable salt thereof to a subject in need of treatment.

Furthermore, according to the present invention, there is provided the use of a compound of chemical formula I, a solvate, stereoisomer or pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of a disease associated with oxidative stress.

BENEFICIAL EFFECTS OF THE INVENTION

The novel pyrazole derivative and the pharmaceutical composition containing it according to the present invention have an excellent effect on a disease associated with oxidative stress.

In addition, the new pyrazole derivative effectively inhibits oxidative stress and the production of αSMA, extracellular matrix or IL-1β (interleukin 1 beta) in various cells, and in particular, effectively improves the condition of cancer, inflammatory disease, fibrous disease, neurodegenerative disease, liver disease, skin disease or retinal disease and the like caused by oxidative stress.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 shows the effect of the compounds from the synthesis examples on the inhibition of ROS production induced by PMA (phorbol 12-myristate 13-acetate) in LX-2 (human liver stellate cells) and NHLF (normal human lung fibroblasts) cells.

Fig. 2 shows the effect of the compounds from the synthesis examples on the inhibition of αSMA expression induced by TGF-β1 in ARPE19 cells (human retinal pigment epithelium).

Fig. 3a and Fig. 3b show the effect of the compound of Synthesis Example 3 on inhibiting the expression of αSMA induced by TGF-1β in various cells.

Fig. 4 shows the effect of the compound from Synthesis Example 3 on inhibiting MPP+ (1-methyl-4-phenylpyridinium)-induced ROS production in N27 cells (rat dopaminergic neurons).

Fig. 5 shows the effect of the compound from Synthesis Example 3 on inhibiting the expression of IL-1β induced by LPS (lipopolysaccharide) in LX-2 and NHLF cells.

Fig. 6 shows the effect of inhibition of αSMA expression by compounds from the synthesis examples in lung tissue in a pulmonary fibrosis model.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described in detail using embodiments. However, these are provided as examples, and the present invention is not limited to them, but is defined only by the scope of the claims described below. Furthermore, even if a configuration is necessary for implementing the present invention, a detailed description of such a configuration, which can be easily implemented by those skilled in the art using known technology, will be omitted.

Unless otherwise specified below, the term "a compound of the present invention" or "a compound of chemical formula I" is used as a concept that includes all of the compound as such, its solvates, stereoisomers and salts.

In the present specification, the term "straight or branched alkyl" means a straight, branched monovalent saturated hydrocarbon group. Unless otherwise specified, an alkyl group usually contains 1~10, 1~8, 1~6, 1~4 or 1~3 carbon atoms. An example of an alkyl group includes methyl, ethyl, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl and mpem-butyl), pentyl (e.g., n-pentyl, isopentyl and neopentyl), n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. In the present specification, the alkyl group may optionally be partially unsaturated and may be alkenyl or alkynyl as defined below. Furthermore, the alkyl group may be further substituted with other substituents.

In the present description, the term "alkenyl" means a straight or branched monovalent unsaturated hydrocarbon group having one or more carbon-carbon double bonds. Unless otherwise specified, an alkenyl group typically contains 2~10, 2~8, 2~6, 2~4, or 2~3 carbon atoms. An alkenyl group includes, for example, ethenyl, n-propenyl, isopropenyl, cyclohexenyl, and the like, and may be further substituted with other substituents.

In the present description, the term "alkynyl" means a straight or branched monovalent unsaturated hydrocarbon group having one or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group typically contains 2~10, 2~8, 2~6, 2~4, or 2~3 carbon atoms. An alkynyl group includes, for example, ethynyl, n-propynyl, and the like, and may be further substituted with other substituents.

In the present description, the term "alkoxy" may be a straight chain, a branched chain, or a ring chain. The number of carbon atoms of the alkoxy group is not particularly limited; however, the number of carbon atoms is preferably from 1 to 5. Specifically, it may be methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentyloxy, neopentyl, isopentyl, and the like, but is not limited thereto.

As used herein, the term "haloalkyl" means an alkyl group having one or more halogen substituents. Haloalkyl includes _-CF3 , _-C2F5 , _-CHF2 , _-CCl3 , _-CHCl2 and _-C2Cl5 . Unless otherwise specified, a haloalkyl group typically contains 1~6, 1~ ₅ , 1~4 or 1~ ₃ carbon atoms and may be further substituted with other substituents.

As used herein, the term "cycloalkyl" represents a non-aromatic carbon ring comprising cyclized alkyl, alkenyl, and alkynyl groups. A cycloalkyl group may comprise a monocyclic or polycyclic ring. A polycyclic ring has, for example, 2, 3, or 4 fused rings. Unless otherwise specified, a cycloalkyl group typically contains from 3 to 10 or from 3 to 7 carbon atoms in the ring. A cycloalkyl group includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexadienyl, cycloheptatrienyl, and the like, and may be further substituted with other substituents.

In the present description, the term "aryl" means an aromatic hydrocarbon group having a monocyclic or polycyclic ring and may be further substituted with other substituents. Here, "polycyclic" means a group in which the aryl group is directly bonded by condensation to another ring group. Here, the "other ring group" may be an aryl group, but may be another kind of ring group, for example, a cycloalkyl group, a heteroaryl group, and the like. A polycyclic ring may have, for example, 2, 3, or 4 rings. Unless otherwise specified, an aryl group typically has 5 to 20, 6 to 15, 6 to 12, or 6 to 10 carbon atoms in a ring. An aryl group includes, for example, phenyl, naphthyl (e.g., naphthyl-1-yl and naphthyl-2-yl), a group other than phenyl, anthracenyl, phenanthreneyl, and the like.

As used herein, the term "heteroaryl" means a monovalent aromatic group having one or more heteroatoms selected from N, O, and S as a ring member. A heteroaryl group has a monocyclic or heterocyclic structure. A polycyclic ring may have, for example, 2, 3, or 4 fused rings. Unless otherwise specified, a heteroaryl group typically contains 3 to 10, 3 to 7, or 3 to 5 ring atoms. A heteroaryl group may have 1, 2, or 3 heteroatoms. The heteroaryl group includes, for example, furanyl, pyridyl, N-oxopyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, furanyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isooxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, furinyl, benzimidazolyl, indolinyl and the like, and may be further substituted with other substituents.

In this description, the term "halogen" represents fluorine, chlorine, bromine or iodine.

As used herein, the term "arylalkyl" represents an alkyl group substituted with an aryl group. "Aryl" and "alkyl" are as defined above.

As used herein, the term "heteroarylalkyl" represents an alkyl group substituted with a heteroaryl group. "Heteroaryl" and "alkyl" are as defined above.

In the present specification, the "substituted or unsubstituted amino group" may be selected from the group consisting of a monoalkylamino group, -NH ₂ and a dialkylamino group, wherein "alkyl" is as defined above. A specific example of a substituted or unsubstituted amino group includes, but is not limited to, NH ₂ , methylamino group, dimethylamino group, ethylamino group, diethylamino group and the like.

In this description, "arylene group" means an aryl group with two binding sites, i.e., a divalent group. Except for the definition of each divalent group, the above-mentioned aryl group may be used.

In the present description, the term "substitution" means that a hydrogen atom bonded to a carbon atom of a compound is replaced by another substituent, and the position to be substituted is not limited as long as the position at which the hydrogen atom is located, that is, the position at which the substituent is replaceable, and when two or more substituents are substituted, the two or more substituents may be the same or different from each other, and two or more same or different substituents may be bonded and substituted.

In the present description, the term "substituted straight or branched alkyl" is a form in which a carbon atom of _C1 - _C10 straight or branched alkyl is substituted with the following substituent. In particular, it is a form in which one or more substituents are selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5- _C20 aryl or C5- _C20 heteroaryl, C1 _-C5 alkoxy and _C1 -C5 haloalkyl, or the same or different 2 or more substituents are linked and substituted. As a typical substituent, trifluoromethyl, halogen, cyano, methoxy, carboxyl, methyl, amino, nitro, dimethylamino, cyclopropyl, substituted imidazole and the like can be used, without limitation.

In the present description, the term "substituted cycloalkyl" is a form in which one or more substituents are selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5- _C20 aryl or C5- _C20 heteroaryl, C1 _- C5 alkoxy and _C1 -C5 haloalkyl, or the same or different 2 or more substituents are linked and substituted on _C3 - _C8 cycloalkyl. As a typical substituent, trifluoromethyl, halogen, cyano, methoxy, carboxyl, methyl, amino, nitro, dimethylamino, cyclopropyl, substituted imidazole and the like can be used, without limitation.

In the present description, the term "substituted aryl" or "substituted heteroaryl" is a form in which one or more substituents are selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, _C1- C5alkoxy, _{C1- C5haloalkyl} , _C1 - _C10alkyl , _C3 - _C8cycloalkyl , _C1 - _C10alkyl , C5-C20aryl, _C1 - _C10alkyl , or the same or different 2 or more substituents are bonded and substituted on C5- _C20aryl or _C5 -C5heteroaryl. Typical substituents that may be used include, but are not limited to, trifluoromethyl, halogen, cyanogen, methoxy, carboxyl, methyl, amino, nitro, dimethylamino, cyclopropyl, substituted imidazole, and the like.

As used herein, an "adjacent" group may mean a substituent used as a substituent at an atom directly bonded to an atom substituted by the corresponding substituent, a substituent spatially closest to the corresponding substituent, or another substituent used as a substituent at an atom substituted by the corresponding substituent. For example, two substituents at the ortho position on a benzene ring and two substituents at the same carbon atom on an aliphatic ring may be defined as an "adjacent" group with respect to each other.

According to the present invention, there is provided a compound of the following chemical formula I, a solvate, a stereoisomer or a pharmaceutically acceptable salt of chemical formula I

in which

X, Y represent CR1R2 or NR3; and

R1, R2 or R3 are the same or different from each other and each is independently selected from Ra-L-, Ra-L-CO-, Ra-L-NHCO, Ra-L-OCO, Ra-L-SO ₂ -; and

Ra is selected from hydrogen, heavy hydrogen, cyanogen, halogen, nitro, haloalkyl, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C5-C20 aryl, or substituted or unsubstituted C5-C20 heteroaryl, and the heteroaryl contains 1, 2 or 3 heteroatoms from O, N and S; and

L is selected from a direct bond, C1-C5 alkylene, and substituted or unsubstituted C5-C10 arylene; and

R4 is selected from hydrogen or substituted or unsubstituted C1-C10 alkyl; and A is CR5 or N, B is CR6 or N, D is CR7 or N, and E is CR8 or N, and G is CR9 or N; and

radicals R5 through R9 are the same or different and are each independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5alkoxy, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20aryl, or substituted or unsubstituted C5-C20 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

substituted C1-C10 straight or branched alkyl may be optionally partially unsaturated and may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20 aryl or C5-C20 heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl; and

substituted C3-C8 cycloalkyl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20 aryl or C5-C20 heteroaryl, C1-C5 alkoxy and C1-C5 haloalkyl, or the same or different 2 or more substituents are linked; and

substituted C5-C20 aryl or substituted C5-C20 heteroaryl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, C1-C5 alkoxy, C1-C5 haloalkyl, C1-C10 alkyl, C3-C8 cycloalkylC1-C10 alkyl and C5-C20 arylC1-C10 alkyl, or the same or different 2 or more substituents are linked;

the substituted amine may be substituted with one or two C1-C5 alkyl groups; and

substituted C5-C10arylene may be substituted with one or more substituents selected from C1-C3 alkyl, halogen, cyano, amine, nitro and heavy chain, or the same or different 2 or more substituents are linked.

Additionally, the chemical formula of I is the chemical formula I-1 given below.

chemical formula I-1

in which

X is NR3; and

R3 is selected from Ra-L-, Ra-L-CO-, Ra-L-NHCO, Ra-L-OCO- and Ra-L-SO ₂ -; and

Ra is selected from hydrogen, heavy hydrogen, cyanogen, halogen, nitro, haloalkyl, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C5-C20 aryl, or substituted or unsubstituted C5-C20 heteroaryl, and the heteroaryl contains 1, 2 or 3 heteroatoms from O, N and S; and

L is selected from a direct bond, C1-C5 alkylene, or substituted or unsubstituted C5-C10 arylene; and

R4 is selected from hydrogen or substituted or unsubstituted C1-C10 alkyl; and

A is CR5 or N, and B is CR6 or N, and D is CR7 or N, and E is CR8 or N, and G is CR9 or N; and

radicals R5 through R9 are the same or different and are each independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5alkoxy, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20aryl, or substituted or unsubstituted C5-C20 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

substituted C1-C10 straight or branched alkyl may be optionally partially unsaturated and may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20 aryl or C5-C20 heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl; and

substituted C3-C8 cycloalkyl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20 aryl or C5-C20 heteroaryl, C1-C5 alkoxy and C1-C5 haloalkyl, or the same or different 2 or more substituents are linked; and

substituted C5-C20 aryl or substituted C5-C20 heteroaryl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, C1-C5 alkoxy, C1-C5 haloalkyl, C1-C10 alkyl, C3-C8 cycloalkylC1-C10 alkyl and C5-C20 arylC1-C10 alkyl, or the same or different 2 or more substituents are linked;

the substituted amine may be substituted with one or two C1-C5 alkyl groups; and

substituted C5-C10arylene may be substituted with one or more substituents selected from C1-C3 alkyl, halogen, cyano, amine, nitro and heavy chain, or the same or different 2 or more substituents are linked.

Additionally, the chemical formula of I is the chemical formula I-2 below.

chemical formula I-2

in which

Y is NR3, and

R3 is selected from Ra-L-, Ra-L-CO-, Ra-L-NHCO-, Ra-L-OCO- and Ra-L-SO ₂ -; and

Ra is selected from hydrogen, heavy hydrogen, cyanogen, halogen, nitro, haloalkyl, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C5-C20 aryl, or substituted or unsubstituted C5-C20 heteroaryl, and the heteroaryl contains 1, 2 or 3 heteroatoms from O, N and S; and

L is selected from a direct bond, C1-C5 alkylene, or substituted or unsubstituted C5-C10 arylene; and

R4 is selected from hydrogen or substituted or unsubstituted C1-C10 alkyl; and A is CR5 or N, and B is CR6 or N, and D

represents CR7 or N, and E represents CR8 or N, and G represents

is CR9 or N; and

radicals R5 through R9 are the same or different and are each independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5alkoxy, substituted or unsubstituted C1-C10 straight or branched alkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20aryl, or substituted or unsubstituted C5-C20 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

substituted C1-C10 straight or branched alkyl may be optionally partially unsaturated and may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20 aryl or C5-C20 heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl; and

substituted C3-C8 cycloalkyl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C20 aryl or C5-C20 heteroaryl, C1-C5 alkoxy and C1-C5 haloalkyl, or the same or different 2 or more substituents are linked; and

substituted C5-C20 aryl or substituted C5-C20 heteroaryl may be independently substituted with one or more substituents selected from heavy hydrogen, halogen, cyano, nitro, carboxyl, substituted or unsubstituted amine, C1-C5 alkoxy, C1-C5 haloalkyl, C1-C10 alkyl, C3-C8 cycloalkylC1-C10 alkyl and C5-C20 arylC1-C10 alkyl, or the same or different 2 or more substituents are linked;

the substituted amine may be substituted with one or two C1-C5 alkyl groups; and

substituted C5-C10arylene may be substituted with one or more substituents selected from C1-C3 alkyl, halogen, cyano, amine, nitro and heavy chain, or the same or different 2 or more substituents are linked.

In addition, in a compound of chemical formula I-1 or 1-2

R3 is Ra-L; and

L is selected from a direct bond and C1-C3 alkylene; and

N in A, B, D, E, G is 0 or 1; And

radicals R5 to R9 are the same or different and each is independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 haloalkyl in which one or more halogens are substituted, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C10 aryl, or substituted or unsubstituted C5-C10 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

R4 may be selected from hydrogen or substituted or unsubstituted C1-C3 alkyl.

In addition, in a compound of chemical formula I-1 or I-2

R3 is Ra-L-CO-; and

L is selected from a direct bond, C1-C3 alkylene and substituted phenylene, and N in A, B, D, E, G is 0 or 1; and

radicals R5 to R9 are the same or different and are each independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 haloalkyl in which one or more halogens are substituted, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C10 aryl, or substituted or unsubstituted C5-C10 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

R4 may be selected from hydrogen or substituted or unsubstituted C1-C3 alkyl.

In addition, in a compound of chemical formula 1-1 or 1-2

R3 is Ra-L-NHCO-; and

L is selected from a direct bond and C1-C3 alkylene; and

N in A, B, D, E, G is 0 or 1; And

radicals R5 to R9 are the same or different and each is independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 haloalkyl in which one or more halogens are substituted, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C10 aryl, or substituted or unsubstituted C5-C10 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

R4 may be selected from hydrogen or substituted or unsubstituted C1-C5 alkyl.

Additionally, in a compound of chemical formula 1-1 or 1-2

R3 is Ra-L-OCO-; and

L is selected from a direct bond and C1-C3 alkylene, and

N in A, B, D, E, G is 0 or 1; And

radicals R5 to R9 are the same or different and each is independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 haloalkyl in which one or more halogens are substituted, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C10 aryl, or substituted or unsubstituted C5-C10 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

R4 may be selected from hydrogen or substituted or unsubstituted C1-C5 alkyl.

Additionally, in a compound of chemical formula 1-1 or 1-2

R3 is Ra-L-SO ₃ -; and

L is selected from a direct bond and C1-C3 alkylene, and

N in A, B, D, E, G is 0 or 1; And

radicals R5 to R9 are the same or different and each is independently selected from hydrogen, heavy hydrogen, cyano, halogen, haloalkyl, carbonyl, nitro, carboxyl, C1-C5 alkoxy, C1-C5 alkyl, C1-C5 haloalkyl in which one or more halogens are substituted, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted amine, substituted or unsubstituted C5-C10 aryl, or substituted or unsubstituted C5-C10 heteroaryl, and two or more groups adjacent to each other can be combined with each other to form a substituted or unsubstituted aromatic hydrocarbon ring, where the aromatic hydrocarbon ring can form a C5-C10 heteroaryl ring or an aryl ring containing 0, 1, 2 or 3 heteroatoms selected from N, O and S; and

R4 may be selected from hydrogen or substituted or unsubstituted C1-C5 alkyl.

The compound of chemical formula I may be any of the following compounds:

2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol;

2-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol;

6-benzyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-benzyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-benzyl-2-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(4-fluorobenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(4-methylbenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

4-((3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methyl)benzonitrile;

6-(3,5-difluorobenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

2-(pyridin-2-yl)-6-(3-(trifluoromethyl)benzyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(naphthalen-2-ylmethyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

1-(4,5-dihydro-3-hydroxy-2-(pyridin-2-yl)-2H-pyrazolo[3,4-c]pyridin-6(7H)-yl)butan-1-one;

1-(3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)ethan-1-one;

6-(4-methoxybenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-ethyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(prop-2-yn-1-yl)-2(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-propyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-allyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

(3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)(phenyl)methanone;

benzyl 3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate;

(4-chlorophenyl)(3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-5-yl)methanone;

ethyl 3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate;

N-cyclohexyl-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide;

furan-2-yl(3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone;

6-(methylsulfonyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(phenylsulfonyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

N-(4-fluorophenyl)-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide;

N-(4-fluorobenzyl)-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide;

N-(furan-2-ylmethyl)-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide;

N-(4-chlorophenethyl)-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide;

6-(naphthalene-2-ylsulfonyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

4-nitrobenzyl-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate;

(5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylphenyl)(3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone;

5-benzyl-2-(pyridin-2-yl)-4D6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

6-benzyl-2-(5-chloropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

4-(6-benzyl-3-hydroxy-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-2-yl)benzoic acid;

6-benzyl-2-(4-bromophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-benzyl-2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

5-benzyl-2-(5-trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-(4-bromophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-(2-chlorophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

6-benzyl-2-(2-chlorophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-benzyl-2-(2-methoxyphenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

5-benzyl-2-(5-chloropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-(thieno[3,2-c]pyridin-4-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(naphthalen-2-ylmethyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(naphthalen-2-ylmethyl)-2-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(3-(trifluoromethyl)benzyl)-2-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(3,5-difluorobenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(3,5-difluorobenzyl)-2-(5-trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

6-benzyl-3-methoxy-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine;

6-(6-benzyl-3-hydroxy-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-2-yl)nicotinonitrile;

6-benzyl-2-(6-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-benzyl-2-(3-fluoropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-benzyl-2-(6-methylpyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-benzyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

5-benzyl-2-(6-methylpyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-(6-trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

6-(5-benzyl-3-hydroxy-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)nicotinonitrile;

5-benzyl-2-(3-fluoropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-(thieno[3,2-c]pyridin-4-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-benzyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

6-benzyl-2-(thieno[3,2-c]pyridin-4-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

2-(pyridin-2-yl)-6-(pyridin-2-ylmethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

2-(pyridin-2-yl)-6-(pyridin-4-ylmethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(4-(dimethylamino)benzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(3-(dimethylamino)benzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

2-(6-aminopyridin-2-yl)-6-benzyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

5-(4-(dimethylamino)benzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(3-(dimethylamino)benzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

2-(pyridin-2-yl)-6-(quinolin-6-ylmethyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

6-(isoquinolin-3-ylmethyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol;

4-((3-hydroxy-2-(pyridin-2-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile;

4-((3-hydroxy-2-(5-(trifluoromethyl)pyridin-2-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methyl)benzonitrile;

2-(pyridin-2-yl)-5-(3-(trifluoromethyl)benzyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(3-(trifluoromethyl)benzyl)-2-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(isoquinolin-3-ylmethyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

5-(isoquinolin-3-ylmethyl)-2-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol;

4-(5-benzyl-3-hydroxy-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-2-yl)benzoic acid.

The compound of the present invention not only effectively reduced the level of ROS generated by stimulation with PMA, TGF-β1, palmitate, or glucose at a high concentration, or the like in all NHLF (normal human lung fibroblasts), LX-2 (human liver stellate cells), ARPE19 (human retinal pigment epithelium), and KEL-FIB (keloid fibroblasts) cells, but also showed an inhibitory effect on ROS generation of 30% to 70% or more when differentiated HL-60 cells (human leukemia cells) were treated with each of the compounds of the synthesis examples at a concentration of 0.5 μM. Accordingly, the compound of the present invention can be used for the prevention or treatment of a disease associated with oxidative stress.

It has been reported that tumor-associated fibroblasts (CAFs) are formed or fibrosis is induced in various tissues when fibroblasts differentiate into myofibroblasts. Treatment of HFF-1 (human foreskin fibroblasts), LX-2, NHLF, ARPE19, and KEL-FIB cells, in which fibrosis is induced by treatment with TGF-β1, with the compounds of the present invention exhibits an inhibitory effect on the expression of αSMA, an indicator of fibrosis. Therefore, the compound of the present invention can be used to prevent or treat fibrosis-related diseases and can be used to treat cancer by regulating tumor-associated fibroblasts, which are important indicators of interaction with the tumor microenvironment.

When ROS levels in nerve cells are elevated, lipid oxidation is induced, which is known to be a key factor in nerve cell death. Simultaneous application of MPP+ (1-methyl-4-phenylpyridinium), an ROS-inducing compound, and the compounds from the synthesis examples to N27 cells (rat dopaminergic neurons) demonstrated effective inhibition of ROS production. Therefore, the compound of the present invention can be used to prevent or treat a disease associated with nerve cell death caused by oxidative stress.

One of the main causes of fibrous skin disease is the production of extracellular matrix, such as type I collagen and the like, by CTGF from keloid fibroblasts. When KELIB cells (keloid fibroblasts) were treated with the compound from the synthesis examples and then treated with TGF-β1 to induce fibrosis, the expression of the CTGF gene and type I collagen gene was effectively inhibited. Therefore, the compound of the present invention can be used to prevent or treat keloid skin disease.

IL-1β is a typical cytokine associated with inflammatory disease. When LX-2 and NHLF cells were treated with the compound from the synthesis examples following LPS treatment, IL-1β expression was effectively inhibited. Therefore, the compound of the present invention can be used to prevent or treat inflammatory disease.

The compound of the present invention not only inhibited the expression of αSMA and collagen I, indicators of pulmonary fibrosis, in a mouse model of pulmonary fibrosis, but also demonstrated an ameliorating effect on pulmonary fibrosis, such as reducing the infiltration of inflammatory cells into the alveoli and bronchioles. Specifically, the compound of the present invention demonstrated effective amelioration of pulmonary fibrosis compared with nintedanib, a conventional therapeutic agent for idiopathic pulmonary fibrosis. Therefore, the compound of the present invention can be used for the prevention or treatment of fibrotic diseases, including pulmonary fibrosis.

The compound of the present invention demonstrated the effect of alleviating the symptoms of liver fibrosis and hepatitis in STAM mice in which non-alcoholic steatohepatitis and fibrosis were induced. Therefore, the compound from the synthetic examples of the present invention can be used to prevent or treat liver disease, including non-alcoholic hepatitis or liver fibrosis.

The compound of the present invention alleviated behavioral symptoms in a mouse model of Parkinsonism and improved α-synuclein aggregation and accumulation by 50% or more in brain tissue. Therefore, the compound of the present invention can be used to prevent or treat neurodegenerative diseases such as Parkinson's disease and the like.

The pharmaceutical composition of the present invention can be used to treat a disease associated with oxidative stress. The disease can be cancer, an inflammatory disease, a fibrotic disease, a neurodegenerative disease, a liver disease, a skin disease, or a retinal disease.

The cancer may be selected from the group consisting of liver cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, neurofibromatosis-associated meningioma, pancreatic cancer, leukemia, myeloproliferative/myelodysplastic disease, skin fibrosarcoma, breast cancer, lung cancer, thyroid cancer, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, brain tumor, head and neck cancer, glioblastoma, and the like. In addition, the cancer may be a secondary cancer that has metastasized to another organ from among the various cancer types listed above.

Inflammatory diseases may be rheumatoid arthritis, osteoarthritis, pneumonia, inflammatory bowel disease, colitis, glomerulonephritis, gastritis, angiitis, pancreatitis, peritonitis, bronchitis, cardiac myositis, encephalitis, inflammation in post-ischemic reperfusion injury and inflammation as a result of immune rejection after tissue and organ transplantation, various inflammatory processes on the skin such as burns, allergic contact dermatitis and the like, inflammation occurring in multiple organ disorders, inflammation in diabetes including diabetic nephropathy, infectious inflammation caused by a viral or bacterial infection, or an autoimmune disease such as lupus, psoriasis, atherosclerosis and the like.

Fibrotic disease may be liver fibrosis or cirrhosis caused by metabolic disorders, liver fibrosis or cirrhosis caused by NAFLD (non-alcoholic fatty liver disease), liver fibrosis or cirrhosis caused by NASH (non-alcoholic steatohepatitis), liver fibrosis or cirrhosis caused by alcohol, liver fibrosis or cirrhosis caused by drugs, liver fibrosis or cirrhosis caused by infectious agents, liver fibrosis or cirrhosis caused by parasitic infection, liver fibrosis or cirrhosis caused by bacterial infection, liver fibrosis or cirrhosis caused by viral infection, liver fibrosis or cirrhosis caused by HBV (hepatitis B virus), liver fibrosis or cirrhosis caused by HCV (hepatitis C virus), liver fibrosis or Liver cirrhosis due to HIV (human immunodeficiency virus) infection, liver fibrosis or cirrhosis due to HCV/HIV co-infection, liver fibrosis or cirrhosis due to radiation or chemotherapy, biliary fibrosis, liver fibrosis or cirrhosis due to any chronic cholestatic disease, gastrointestinal fibrosis of any etiology, fibrosis due to Crohn's disease, fibrosis due to ulcerative colitis, intestinal fibrosis, colon fibrosis, gastric fibrosis, pulmonary fibrosis, skin fibrosis, epidermal fibrosis, epithelial fibrosis, skin fibrosis due to dermatosclerosis/systemic sclerosis, chronic obstructive pulmonary disease (COPD), asthma, pulmonary emphysema, smoker's lung, tuberculosis, pulmonary fibrosis, pulmonary fibrosis with subsequent Idiopathic pulmonary fibrosis, cardiac fibrosis, renal fibrosis, nephrogenic systemic fibrosis, muscle fibrosis, soft tissue fibrosis, bone marrow fibrosis, joint fibrosis, dry fibrosis, cartilage fibrosis, pancreatic fibrosis, uterine fibrosis, nerve fibrosis, testicular fibrosis, ovarian fibrosis, adrenal fibrosis, arterial fibrosis, venous fibrosis, ocular fibrosis, endomyocardial fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, progressive massive fibrosis which is a complication of pneumoconiosis, proliferative fibrosis, fibrosis after antitumor therapy, peritransplant fibrosis, asbestosis, joint fibrosis or adhesive capsulitis.

The neurodegenerative disease may be Alzheimer's disease (including mild or early Alzheimer's disease, mild to moderate Alzheimer's disease, moderate to moderate Alzheimer's disease, moderate to severe Alzheimer's disease, moderately severe Alzheimer's disease, severe Alzheimer's disease, and Alzheimer's disease (AD) with Lewy bodies), Parkinson's disease (including chemically induced Parkinson's disease by exposure to an environmental agent such as a pesticide, insecticide, or herbicide and/or a metal such as manganese, aluminum, cadmium, copper, or zinc, SNCA-associated Parkinson's disease, sporadic or idiopathic Parkinson's disease, or Parkinson- or LRRK2-associated Parkinson's disease (PD)), autosomal dominant Parkinson's disease, diffuse Lewy body disease (DLBD) (also known as dementia with Lewy bodies (DLB)), true autonomic dysfunction, Lewy body dysphagia (LBD), incidental LBD, genetic LBD (e.g., alpha-synuclein, PARK3, and PARK4 gene mutations), polyphyletic kraurosis (including olivopontocerebellar atrophy, striatonigral degeneration, Shy-Drager syndrome (MSA (multisystem atrophy))), a disorder or condition characterized by Alzheimer's disease-Parkinson complex and/or MSA, Huntington's disease, synucleinopathy, or the presence of Lewy bodies, multiple sclerosis, amyotrophic lateral sclerosis (ALS), dementia (including vascular dementia, dementia with Lewy bodies, dementia in Parkinson's disease, and frontotemporal dementia), psychosis (including anxiety due to a neurodegenerative disease or associated with dopamine therapy, such as psychosis in Parkinson's disease, psychosis in Alzheimer's disease, psychosis in dementia with Lewy bodies (but not limited to)), dyskinesia (including anxiety due to a neurodegenerative disease or associated with dopamine therapy), anxiety (anxiety due to a neurodegenerative disease or associated with dopamine therapy), dopamine therapy-associated condition (including dysmyotonia, myoclonus, or fibrillation), synucleinopathy, or a disorder associated with abnormal expression of alpha-synuclein.

Liver disease may include metabolic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis, drug-induced liver disease, alcoholic liver disease, infectious liver disease, inflammatory liver disease, immune dysfunction-mediated liver disease, or dyslipidemia.

Skin disease may be keloid disease, psoriasis, or leukoplakia, and retinal disease may be retinal fibrosis, macular degeneration, diabetic retinopathy, cataract, or neovascular glaucoma.

The compound of the present invention may be in the form of its pharmaceutically acceptable salt. This salt means a salt widely used in the medical field to which the present invention pertains, and, for example, it may be an inorganic salt obtained with hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid and sulfuric acid, and the like. In addition, it may be an organic salt obtained with acetic acid, perfluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, aspartic acid, ascorbic acid, carbonic acid, and the like. In addition, it may be a sulfonic acid salt obtained using methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid and the like, and the type of salt of the present invention is not limited to these listed salts.

The compound of the present invention may also exist in the form of its solvate. "Solvate" means a complex or aggregate formed by one or more molecules of a solute, i.e., a compound of chemical formula I or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. A solvate may be a complex or aggregate formed by molecules of various solvents, such as, for example, water, methanol, ethanol, isopropyl alcohol, or acetic acid, and the like.

The compound of the present invention may also exist in the form of its stereoisomer. A stereoisomer includes all stereoisomers, such as an enantiomer and a diastereomer. The compound may be a stereoisomerically pure form of a stereoisomer or a mixture of one or more stereoisomers, such as a racemic mixture. Separation of a specific stereoisomer can be accomplished by any of the conventional methods known in the art. Some examples of the compound of the present invention may have a more potent inhibitory effect on oxidative stress when present as a specific stereoisomer compared to their racemic mixture. In this case, using a specific stereoisomer can reduce the dosage. Thus, by separating a specific stereoisomer, such as an enantiomer and a diastereomer, with a potent inhibitory effect on oxidative stress, it is possible to effectively treat a disease associated with oxidative stress.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective dose of a compound of chemical formula I as defined above, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, and a pharmaceutically acceptable carrier.

In the composition of the present invention, the compound or pharmaceutically acceptable salt or solvate or stereoisomer thereof is as defined above.

In the composition of the present invention, a "pharmaceutically acceptable carrier" is a substance, typically an inactive substance, used in combination with an active ingredient to facilitate the use of the active ingredient. The carrier includes conventional pharmaceutically acceptable excipients, additives, or diluents. The carrier may include one or more substances selected from, for example, fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, flavoring agents, thickeners, colorants, emulsifiers, suspending agents, stabilizers, pH adjusting agents, and tonicity adjusting agents.

Sugar, starch, microcrystalline cellulose, lactose (lactose hydrate), glucose, dimannitol, alginate, alkaline earth metal salts, clay, polyethylene glycol, anhydrous calcium hydrogen phosphate or a mixture thereof, or the like can be used as a diluent; and starch, microcrystalline cellulose, highly dispersed silicon dioxide, mannitol, dimannitol, sucrose, lactose hydrate, polyethylene glycol, polyvinylpyrrolidone (povidone), copolymer can be used as a binder.

polyvinylpyrrolidone (copovidone), hypromellose, hydroxypropyl cellulose, natural gum, synthetic gum, gelatin or a mixture thereof, or the like.

As a disintegrating agent, there can be used starch or modified starch such as sodium starch gluconic acid, corn starch, potato starch, pregelatinized starch and the like; clay such as bentonite, montmorillonite or veegum or the like; celluloses such as microcrystalline cellulose, hydroxypropyl cellulose or carboxymethyl cellulose or the like; algins such as sodium alginate or alginic acid or the like; cross-linked celluloses such as sodium croscarmellose or the like; gums such as guar gum, xanthan gum or the like; cross-linked polymers such as cross-linked polyvinylpyrrolidone (crospovidone) or the like; effervescent agents such as sodium bicarbonate, citric acid or the like; or a mixture thereof.

Talc, stearic acid, magnesium stearate, calcium stearate, sodium lauryl sulfate, hydrogenated vegetable oil, sodium benzoate, sodium stearyl fumarate, glyceryl monostearate, glyceryl monostearate, glyceryl palmitostearate, colloidal silicon dioxide or a mixture thereof, or the like can be used as a lubricant.

As the pH adjusting agent, an acidifier such as acetic acid, ascorbic acid, sodium ascorbate, sodium ester acid, malic acid, succinic acid, tartaric acid, fumaric acid and citric acid, and an alkalizing agent such as precipitated calcium carbonate, aqueous ammonia, meglumine, sodium carbonate, magnesium oxide, magnesium carbonate, sodium citrate and tribasic calcium phosphate and the like can be used.

Dibutylhydroxytoluene, butylated hydroxyanisole, tocopherol acetate, tocopherol, propyl gallate, sodium bisulfite, sodium pyrosulfite, and the like can be used as antioxidants. Polyoxyethylene sorbitan fatty acid esters, such as sodium lauryl sulfate, polysorbate, and the like, sodium docusate, poloxamer, and the like, can be used as solubilizers in the pre-release cell.

In addition, to obtain a sustained-release agent, it may include an enteric polymer, a water-insoluble polymer, a hydrophobic compound, and a hydrophilic polymer.

An enteric polymer refers to a polymer that is soluble or degradable under certain pH conditions at a pH of 5 or greater, which is insoluble or stable under acidic conditions at a pH of less than 5, and it includes, for example, enteric cellulose derivatives such as hypromellose acetate succinate, hypromellose phthalate (hydroxypropyl methylcellulose phthalate), hydroxymethylethylcellulose phthalate, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate malate, cellulose benzoate phthalate, cellulose propionate phthalate, methylcellulose phthalate, carboxymethylethylcellulose, and ethyl hydroxyethyl cellulose phthalate and methyl hydroxyethyl cellulose; enteric-coated acrylate-based copolymers such as styrene-acrylate copolymer, acrylate methyl acrylate copolymer, acrylate methyl methacrylate copolymer (e.g. acrylic), acrylate butyl-styrene-acrylate copolymer and acrylate methyl methacrylate-acrylate octyl copolymer; enteric-coated polymethacrylate copolymers such as poly(methacrylate-methyl methacrylate) copolymer (e.g. Eudragit L, Eudragit S, Evonik, Germany) and poly(methacrylate-ethyl acrylate) copolymer (e.g. Eudragit L100-55); Enteric-coated maleate-based copolymers such as vinyl acetate-maleate anhydride copolymer, styrene-maleate anhydride copolymer, styrene-maleate monoether copolymer, vinyl methyl ether-maleate anhydride copolymer, ethylene-maleate anhydride copolymer, vinyl butyl ether-maleate anhydride copolymer, acrylonitrile-methyl acrylate maleate anhydride copolymer and acrylate-butyl styrene-maleate anhydride copolymer; and enteric-coated polyvinyl derivatives such as polyvinyl alcohol phthalate, ivory acetal phthalate, polyvinyl butylate phthalate and ivory acetal phthalate.

The water-insoluble polymer refers to a pharmaceutically acceptable polymer insoluble in water that controls the release of a drug. For example, the water-insoluble polymer includes polyvinyl acetate (e.g., Kollicoat SR30D), water-insoluble polymethacrylate copolymer [e.g., poly(ethyl acrylate-methyl methacrylate) copolymer (e.g., Eudragit NE30D), poly(ethyl acrylate-methyl methacrylate-trimethylaminoethyl methacrylate) copolymer (e.g., Eudragit RSPO), etc.], ethyl cellulose, cellulose ester, cellulose ether, cellulose acylate, cellulose diacylate, cellulose triacylate, cellulose acetate, cellulose diacetate, and cellulose triacetate, and the like.

A hydrophobic compound refers to a pharmaceutically acceptable substance that is insoluble in water and regulates the release of a drug. For example, it includes fatty acids and fatty acid esters such as glyceryl palmitostearate, glyceryl stearate, glyceryl behenate, cetyl palmitate, glyceryl monooleate, and stearic acid; waxes such as carnauba wax, sega wax, and microcrystalline wax; and mineral substances such as talc, precipitated calcium carbonate, calcium monohydrogen phosphate, zinc oxide, titanium oxide, kaolin, bentonite, montmorillonite, and veegum, and the like.

The hydrophilic polymer refers to a pharmaceutically acceptable substance soluble in water that controls the release of a drug. For example, it includes saccharides such as dextrin, polydextrin, dextran, pectin and pectin derivatives, alginate salts, polygalacturonic acid, xylan, arabinoxylan, arabinogalactan, starch, hydroxypropyl starch, amylose and amylopectin; cellulose derivatives such as hypromellose, hydroxypropyl cellulose, hydroxymethylcellulose, hydroxyethylcellulose, methyl cellulose A and sodium carboxymethylcellulose; gums such as guar gum, locust bean gum, tragacanth, carrageenan, gum arabic, gum acacia, gellan gum and xanthan gum; proteins such as gelatin, casein and zein; polyvinyl derivatives such as polyvinyl alcohol, polyvinylpyrrolidone and polyvinyl acetal diethylaminoacetate; hydrophilic copolymers based on polymethacrylate such as poly(butyl methacrylate-(2-dimethylaminoethyl) methacrylate-methyl methacrylate) copolymer (e.g. Eudragit El00, Evonik, Germany), poly(ethyl acrylate-methyl methacrylate-triethylaminoethyl methacrylate chloride) copolymer (e.g. Eudragit RL, RS, Evonik, Germany); polyethylene derivatives such as polyethylene glycol and polyethylene oxide; and carbomers and the like.

In addition, the preparation of the present invention can be manufactured by selectively using pharmaceutically acceptable additives in the form of various additives selected from dyes and flavorings.

In the present description, the amount of the additive is not limited when using the above additives, and it can be introduced into the composition of the preparation by including a dose within the general range by selecting the above additives.

The pharmaceutical composition of the present invention can be used in the form of an oral preparation such as powder, granules, tablets, capsules, suspension, emulsion, syrup and aerosol and the like, for external use, suppository or sterile injection solution according to a conventional technique.

The composition of the present invention can be administered orally or parenterally, including intravenous, intraperitoneal, subcutaneous, rectal and topical administration.

According to another aspect of the present invention, there is provided a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective dose of a compound of chemical formula I or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

With this method, those skilled in the art can appropriately select the route of administration based on the patient's condition. Administration can be oral or parenteral. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

In this method, the dosage can be varied in various ways according to various factors, such as the patient's condition, the route of administration, the opinion of the family physician, and the like, as indicated above. The effective dose can be determined from a dose-response curve obtained as a result of an in vitro experiment or testing on an animal model. The ratio and concentration of the compound of the present invention present in the composition for administration can be determined depending on the chemical properties, the route of administration, the therapeutic dose, and the like. The dose can be administered to the subject at an effective dose of from about 1 μg/kg to about 1 g/kg/day or from about 0.1 mg/kg to about 500 mg/kg/day. The dose can be varied depending on the age, body weight, sensitivity, or symptoms present in the subject.

Furthermore, a pharmaceutical composition comprising a compound or a solvate, stereoisomer or pharmaceutically acceptable salt thereof according to the present invention can be used in a method for preventing or treating a disease selected from cancer, keloidosis, liver fibrosis, liver cirrhosis, pulmonary fibrosis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, ischemic or traumatic brain injury, retinal fibrosis, macular degeneration and inflammatory disease, comprising administering the pharmaceutical composition to a subject in need thereof.

METHOD FOR CARRYING OUT THE INVENTION

The present invention will now be described in more detail using examples. These examples are intended to more specifically illustrate the present invention, and it will be clear to those skilled in the art that the scope of the present invention is not limited to these examples in accordance with the essence of the present invention.

The compound of chemical formula I of the present invention can be prepared by the method illustrated in the following reaction schemes, but not limited thereto.

General synthesis method 1 (synthetic examples 1-4)

The compounds of Synthesis Examples 1-4 were synthesized by the following method, and specific synthetic examples are described.

The 2-hydrazinopyridine derivative (100 mmol) and the beta-ketoester (100 mmol) dissolved in ethanol (30~50 mL) are placed in a 100 mL round-bottomed flask and heated to 140°C for 24 hours. Ethanol is then slowly distilled at atmospheric pressure for approximately 3 hours. The reaction progress and consumption of the intermediate (hydrazinimine) are monitored by LC-MS (liquid chromatography-mass spectrometry). The product is dissolved in hot ethanol, cooled, and purified by recrystallization to afford a white solid (yield 40-70%).

Synthesis Example 1: 2-(Pyridin-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 8.23-8.55 (m, 1H), 7.71-8.04 (m, 2H), 7.10 (m, 1H), 2.44-1.95-2.77 (m, 4H), 1.50-1.80 (m, 3H), 1.07 (2d, 2H), mass [M+H] ⁺ : 216.0, mixture of conformational isomers.

Synthesis example 2. 2-(5-(Trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 11.80 (s, 0.5H), 8.81 (s, 0.5H), 8.67 (d, J=9.0 Hz, 0.5H), 8.53 (d, J=11.0 Hz, 1H), 8.02 (dd, J=9.0, 2.1 Hz, 0.5H), 7.94-7.96 (m, 1H), 2.59-2.65 (dt, J=64.0, 6.2 Hz, 2H), 2.35-2.45 (dt, J=59.0, 6.0 Hz, 2H), 1.72-1.87 (m, 4H), mass [M+H] ⁺ : 284.0, mixture of conformational isomers.

Synthesis example 3. 6-Benzyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazol[3,4-c]pyridin-3-ol

1H NMR (400 MHz, DMSO (dimethyl sulfoxide)-d ₆ ) δ 11.75 (s, 1H), 8.39 (s, 1H), 7.94 (s, 1H), 7.65 (s, 2H), 7.45 (s, 3H), 7.35-7.15 (m, 2H), 4.42 (bs, 2H), 4.11 (bs, 2H), 3.56 (bs, 1H), 3.21 (bs, 1H), 2.8-2.6 (m, 2H), mass [M+H] ⁺ : 307.1; HPLC (high performance liquid chromatography) retention time: 3.4 min, λ _max : 304 nm, purity 97.8%.

Synthesis example 4. 6-Benzyl-2-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 8.52 (s, 1H), 8.00 (s, 2H), 7.20-7.37 (m, 5H), 3.72 (d, J=11.0 Hz, 2H), 3.53 (s, 2H), 2.74 (bs, 2H), 2.56 (bs, 2H), mass [M+H] ⁺ : 375.2.

General synthesis method 2 (alkyl derivatives among compounds from the synthesis examples)

The general method for synthesizing alkyl derivatives among the compounds from the synthesis examples is as follows.

Interaction at stage 1

A mixture of ethyl 1-benzyl-3-oxopiperidine-4-carboxylate (1.0 g, 3.35 mmol), ZnCl (0.46 g, 3.36 mmol), and NaBH ₄ (0.13 g, 3.36 mmol) was dissolved in methanol (20 mL) and stirred at 70 °C overnight. The solvent was removed under reduced pressure, and the residues were diluted with water (50 mL). The aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated to give the desired product as a pale yellow oil. Reaction in step 2

A mixture of ethyl 1-benzyl-3-hydroxypiperidine-4-carboxylate (0.5 g, 2.01 mmol) and Pd/C (10%, 50 mg) in methanol (20 mL) was stirred under hydrogen balloon pressure at room temperature overnight. The solvent was removed under reduced pressure to yield the desired product as a pale yellow oil.

Interaction at stage 3

Secondary amine (0.5 g; 1 mol equivalent), N,N-diisopropylethylamine (1.5 mol equivalent), alkyl halide (1.1 mol equivalent), and 10 mL of DMF (dimethylformamide) were added to a round-bottomed flask and stirred at room temperature under nitrogen atmosphere. After completion of the reaction (TLC monitoring), the reaction mixture was evaporated and dried under reduced pressure. The residue was dissolved in 5 mL of methylene chloride and washed with 5 mL of distilled water. The aqueous layer was washed with 3×5 mL portions of methylene chloride. The collected organic fractions were dried with MgSO ₄ and filtered, and then the solvent was removed under reduced pressure to yield the product.

Interaction at stage 4

To 40 mL of acetonitrile was added H ₅ IO ₆ (1.19 g, 5.15 mmol) and stirred vigorously at room temperature for 15 min. After adding the alcohol intermediate (under ice cooling), PCC dissolved in 10 mL of acetonitrile was added twice successively and stirred for 2 h. Then, the reaction mixture was diluted with 100 mL of ethyl acetate and washed with a 1:1 mixture of brine:water, saturated aqueous Na ₂ SO ₃ solution and brine, respectively, and dried over anhydrous Na ₂ SO ₄ and concentrated to give the product.

Interaction at stage 5

The 2-hydrazinopyridine derivative (1.1 mol equivalent) and the beta-ketoester (1 mol equivalent), dissolved in ethanol (0.5~5 ml), were placed in a 10 ml round-bottomed flask and heated to 140°C for 12 hours. After the reaction began, ethanol was distilled off at atmospheric pressure for 3 hours. The product was purified on silica gel (yield 5-30%).

General synthesis method 3 (synthetic examples 5-33, 35-38, 43, 44, 53-58, 65-70, 73,

74)

The compounds of Synthesis Examples 5-33, 35-38, 43, 44, 53-58, 65-70, 73, 74 are synthesized according to the general reaction scheme given above, and an example of the synthetic method that applies to a specific compound is given below.

Interaction at stage 1

2-Hydrazinopyridine derivative (2 g, 1.1 mol equivalent) and beta-ketoester (5 g, 1 mol equivalent) dissolved in ethanol (40 mL) were placed in a 100 mL round-bottomed flask and heated to 140 °C for 24 h. After the reaction had started, ethanol was slowly distilled at atmospheric pressure over a period of approximately 3 h. The extent of the reaction and the consumption of the intermediate (hydrazinimine) were monitored by LC-MS. The product was dissolved in hot ethanol (50 mL), cooled, and purified by recrystallization to give a white solid (yield 3.7 g, 85%).

Interaction at stage 2

6-Benzyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol hydrochloride (0.5 g, 2.01 mmol) dissolved in methanol (20 mL) and Pd/C (wet, 10%, 250 mg) was stirred under hydrogen balloon pressure at room temperature for 2 days. The solvent was removed under reduced pressure to give the product, the hydrochloride salt, as a pale yellow solid.

Interaction at stage 3-1 (alkyl derivatives)

2-(Pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol (50 mg; 0.12 mmol) was dissolved in 5 mL of dimethylacetamide. Triethylamine (97 μL; 3 equivalents) and alkyl halide (1.2 equivalents) were added to the reaction solution at room temperature. The reaction mixture was stirred at room temperature for 2 h. It was diluted with ethyl acetate (50 mL) and mixed with brine/saturated aqueous _NaHCO3 solution for washing. The product was purified by recrystallization, and the yield was in the range of 10-50%.

Interaction at stage 3-2 (method for the synthesis of derivatives of amides, carbonates, urea, sulfonamides)

2-(Pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol (20 mg, 0.08 mmol) was dissolved in a mixed solution of 5 mL of acetonitrile and 1 mL of water, and saturated NaHCO ₃ solution (200 μL) was added. For the synthesis of each derivative, one compound was selected from carboxyl chloride, chloroformate, isocyanate, and sulfonyl chloride and added at room temperature. The reaction mixture as a solution was stirred at room temperature for 1 h, the completion of the reaction was confirmed by LC-MS, and then 50 mL of ethyl acetate was added for dilution and washed with brine and admixed saturated NaHCO ₃ solution. The product was isolated and purified by TLC, and the yield was in the range of 10-70%. In addition, data on compounds from the synthesis examples obtained according to the general synthesis method and the synthesis method using a specific substance are described.

Synthesis example 5. 2-(Pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H-NMR (600 MHz, DMSO-D6) δ 9.74 (bs, 1H), 8.45 (d, J=4.8 Hz, 1H), 8.05-7.92 (1H), 7.37-7.24 (1H), 4.14 (s, 2H), 3.31 (t, J=5.9 Hz, 2H), 2.50 (s, 2H), mass [M+H] ⁺ : 217.0.

Synthesis example 6. 6-(4-Fluorobenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H-NMR (600 MHz, CDCl ₃ ) δ 8.24 (d, J=4.8 Hz, 1H), 7.80 (m, 2H), 7.35 (dd, J=8.3, 5.5 Hz, 2H), 7.00-7.12 (m, 3H), 3.68 (d, J=4.8 Hz, 2H), 3.54 (s, 2H), 2.74 (t, J=5.9 Hz, 2H), 2.58 (t, J=5.9 Hz, 2H), mass [M+H] ⁺ : 325.0.

Synthesis example 7. 6-(4-Methylbenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H-NMR (600 MHz, CDCl ₃ ) δ 8.23 (m, 1H), 7.80 (m, 2H), 7.00-7.12 (m, 5H), 3.68 (s, 2H), 3.54 (s, 2H), 2.72 (m, 2H), 2.56 (m, 2H), 2.34 (s, 3H), mass [M+H] ⁺ : 321.0.

Synthesis example 8. 4-((3-Hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methyl)benzonitrile

1H NMR (600 MHz, CDCl ₃ ) δ 8.25 (d, J=4.8 Hz, 1H), 7.81 (m, 2H), 7.64 (m, 3H), 7.52 (d, J=7.6 Hz, 2H), 3.77 (d, J=4.8 Hz, 2H), 3.55 (s, 2H), 2.74-2.78 (m, 2H), 2.59 (t, J=5.9 Hz, 2H), mass [M+H] ⁺ : 332.0.

Synthesis example 9. 6-(3,5-Difluorobenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 8.22 (d, J=4.8 Hz, 1H), 7.79 (t, J=7.9 Hz, 2H), 7.09 (s, 1H), 6.92 (d, J=6.2 Hz, 1H), 6.67-6.69 (m, 2H), 3.67 (s, 3H), 3.53 (s, 2H), 2.73 (m, 2H), 2.57 (t, J=5.5 Hz, 2H), mass [M+H] ⁺ : 343.0.

Synthesis Example 10 2-(Pyridin-2-yl)-6-(3-(trifluoromethyl)benzyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H-NMR (600 MHz, CDCl ₃ ) δ 8.24 (d, J=4.8 Hz, 1H), 7.80 (m, 2H), 7.35 (m, 2H), 7.00-7.12 (m, 3H), 3.68 (d, J=4.8 Hz, 2H), 3.54 (s, 2H), 2.74 (t, J=5.9 Hz, 2H), 2.58 (t, J=5.9 Hz, 2H), mass [M+H] ⁺ : 374.9.

Synthesis example 11. 6-(Naphthalen-2-ylmethyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H-NMR (600 MHz, CDCl ₃ ) δ 8.56 (s, 1H), 7.77-7.88 (m, 6H), 7.43-7.47 (m, 3H), 7.18 (m, 1H), 3.92 (s, 2H), 3.73 (s, 2H), 3.01 (t, J=5.9 Hz, 2H), 2.80 (m, 2H), mass [M+H] ⁺ : 356.9.

Synthesis example 12. 1-(4,5-Dihydro-3-hydroxy-2-(pyridin-2-yl)-2H-pyrazolo[3,4-c]pyridin-6(7H)-yl)butan-1-one

1H NMR (600 MHz, CDCl ₃ ) δ 8.27 (q, J=5.3 Hz, 1H), 7.81-7.89 (m, 2H), 7.13-7.17 (m, 1H), 4.65 (ss, 2H), 3.76 (mixture of rotational isomers dt, J=5.7 Hz, 2H), 2.58 (dt, J=29.4, 5.9 Hz, 2H), 2.41 (m, 2H), 1.64-1.74 (m, 2H), 0.96-1.01 (m, 3H), mass [M+H] ⁺ : 286.9.

Synthesis example 13. 1-(3-Hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)etan-1-one

1H NMR (600 MHz, CDCl ₃ ) δ 8.27 (q, J=5.7 Hz, 1H), 7.82-7.90 (m, 2H), 7.14-7.18 (m, 1H), 4.64 (2s, 2H), 3.74 (2t, J=5.9 Hz, 2H), 2.59 (2t, J=6.0 Hz, 2H), 2.18-2.20 (2s, 3H), mass [M+H] ⁺ : 258.1: mixture of conformational isomers.

Synthesis example 14. 6-(4-Methoxybenzyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 8.24 (d, J = 4.8 Hz, 1H), 7.81 (bs, 2H), 7.29 (d, J = 8.3 Hz, 2H), 7.10 (m, 1H), 6.86 (d, J = 8.6 Hz, 2H), 3.80 (s, 3H), 3.67 (s, 2H), 3.55 (s, 2H), 2.74 (t, J = 5.9 Hz, 2H), 2.57 (t, J = 5.9 Hz, 2H), mass [M+H] ⁺ : 337.0.

Synthesis Example 15: 6-Ethyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, DMSO-D6) δ 8.34 (bs, 1H), 7.85-7.96 (m, 2H), 7.26-7.28 (bs, 1H), 4.31 (2bs, 2H), 3.60 (bs, 2H), 3.22 (q, J=7.3 Hz, 1H), 2.86-2.96 (m, 2H), 1.47-1.56 (m, 3H), mass [M+H] ⁺ : 244.9.

Synthesis example 16. 6-(Prop-2-yn-1-yl)-2(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, _CDCl3 ) δ 8.25 (bd, J=4.8 Hz, 1H), 7.84 (bs, 2H), 7.12 (dd, J=8.3, 5.5 Hz, 1H), 3.72 (s, 2H), 3.55 (d, J=2.1 Hz, 2H), 2.83 (t, J=5.9 Hz, 2H), 2.63 (t, J=5.5 Hz, 2H), 2.30 (t, J=2.4 Hz, 1H), mass [M+H] ⁺ : 254.9.

Synthesis example 17. 6-Propyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, DMSO-D6) δ 8.24 (s, 1H), 7.79 (b, 2H), 7.16 (s, 1H), 4.16 (b, 2H), 3.33-3.48 (m, 2H), 3.13 (bs, 2H), 2.77-2.92 (m, 2H), 1.7-2.0 (m, 5H), mass [M+H] ⁺ : 258.9.

Synthesis example 18. 6-Allyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 8.24 (bs, 1H), 7.82 b (s, 2H), 7.11 (bs, 2H), 5.19-5.27 (m, 3H), 3.72 (bs, 2H), 3.22 (d, J=6.2 Hz, 3H), 2.75 (bs, 2H), 2.59 (bs, 2H), mass [M+H] ⁺ : 257.0.

Synthesis Example 19. (3-Hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)(phenyl)methanone

1H NMR (600 MHz, CDCl ₃ ) δ 8.19-8.20 (m, 1H), 7.66-7.85 (m, 2H), 7.39-7.49 (m, 5H), 7.11 (broad, 1H), 4.76 (m, 2H), 3.58-4.13 (m, 2H), 2.59-2.76 (m, 2H), mass [M+H] ⁺ : 320.9: mixture of conformational isomers.

Synthesis example 20. Benzyl 3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate

1H NMR (600 MHz, CDCl ₃ ) δ 8.24 (d, J=4.8 Hz, 1H), 7.80-7.85 (m, 2H), 7.34 (m, 5H), 7.13 (t, J=6.5 Hz, 1H), 5.16 (s, 2H), 4.61 (s, 2H), 3.72 (bs, 2H), 2.57 (bs, 2H), mass [M+H] ⁺ : 350.9.

Synthesis Example 21. (4-Chlorophenyl)(3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-5-yl)methanone

1H NMR (600 MHz, CDCl ₃ ) δ 8.13 (d, J=8.3 Hz, 1H), 7.71-7.86 (m, 2H), 7.41-7.44 (m, 4H), 7.09-7.15 (m, 1H), 4.54-4.92 (m, 2H), 3.59-4.01 (m, 2H), 2.64 (m, 2H), mass [M+H] ⁺ : 354.9, mixture of conformational isomers.

Synthesis example 22. Ethyl 3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate

1H NMR (600 MHz, CDCl ₃ ) δ 8.24-8.39 (m, 1H), 7.70-7.86 (m, 2H), 7.12-7.18 (m, 1H), 4.58-4.80 (m, 2H), 4.17-4.23 (m, 2H), 3.67 (m, 2H), 2.44-2.60 (m, 2H), 1.21-1.30 (m, 3H), mass [M+H] ⁺ : 288.9, mixture of conformational isomers.

Synthesis example 23. N-Cyclohexyl-3-hydroxy-2-(pyrid1 sh-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide

1H-NMR (600 MHz, CDCl ₃ ) δ 8.28 (d, J=4.8 Hz, 1H), 7.84-7.89 (m, 2H), 7.17 (t, J=6.2 Hz, 1H), 4.49 (s, 2H), 3.67-3.71 (m, 1H), 3.65 (t, J=5.5 Hz, 2H), 2.59 (t, J=5.9 Hz, 2H), 1.35-1.99 (m, 10H), mass [M+H] ⁺ : 341.9.

Synthesis Example 24. Furan-2-yl(3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone

1H NMR (600 MHz, CDCl ₃ ) δ 8.31 (d, J=5.2 Hz, 1H), 7.90 (m, 2H), 7.56 (s, 1H), 7.19 (t, J=6.5 Hz, 1H), 7.10 (s, 1H), 6.54 (s, 1H), 5.08-4.76 (m, 2H), 4.00 (m, 2H), 2.76 (bs, 2H), mass [M+H] ⁺ : 310.9.

Synthesis example 25. 6-(Methylsulfonyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H-NMR (600 MHz, CDCl ₃ ) δ 8.28 (d, J=4.8 Hz, 1H), 7.83-7.90 (m, 2H), 7.18 (t, J=6.2 Hz, 1H), 4.45 (s, 2H), 3.59-3.62 (m, 2H), 3.38 (s, 2H), 3.11 (s, 3H), mass [M+H] ⁺ : 294.8.

Synthesis example 26. 6-(Phenylsulfonyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 8.25 (d, J = 4.8 Hz, 1H), 7.83-7.85 (m, 2H), 7.51-7.60 (m, 5H), 7.15 (t, J = 6.2 Hz, 1H), 4.26 (s, 2H), 3.39 (t, J = 5.9 Hz, 2H), 2.60 (t, J = 5.9 Hz, 2H), mass [M+H] ⁺ : 356.9.

Synthesis example 27. N-(4-Fluorophenyl)-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide

1H NMR (600 MHz, CDCl ₃ ) δ 8.26 (d, J=5.5 Hz, 1H), 7.82-7.87 (m, 2H), 7.29-7.31 (m, 2H), 7.15 (t, J=6.5 Hz, 1H), 6.97 (t, J=9.0 Hz, 2H), 6.56 (bs, 1H), 4.60 (s, 2H), 3.72 (t, J=5.9 Hz, 2H), 2.63 (t, J=5.9 Hz, 2H), mass [M+H] ⁺ : 353.9.

Synthesis example 28. N-(4-Fluorobenzyl)-3-hydroxy-2-(1-sridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide

1H NMR (600 MHz, CDCl ₃ ) δ 8.26 (d, J = 4.8 Hz, 1H), 7.78-7.86 (m, 2H), 7.26 (dd, J = 8.6, 5.9 Hz, 2H), 7.14 (t, J = 6.2 Hz, 1H), 6.99 (t, J = 8.6 Hz, 2H), 4.49 (s, 2H), 4.38 (d, J = 5.5 Hz, 2H), 3.63 (t, J = 5.5 Hz, 2H), 2.56 (t, J = 5.2 Hz, 2H), mass [M+H] ⁺ : 367.9.

Synthesis example 29. N-(Furan-2-ylmethyl)-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide

1H-NMR (600 MHz, CDCl ₃ ) δ 8.26 (d, J=4.8 Hz, 1H), 7.79-7.86 (m, 2H), 7.35 (s, 1H), 7.14 (t, J=6.2 Hz, 1H), 6.31 (t, J=2.4 Hz, 1H), 6.23 (d, J=3.4 Hz, 1H), 4.49 (s, 2H), 4.43 (d, J=5.5 Hz, 2H), 3.62-3.65 (m, 2H), 2.56 (m, 2H), mass [M+H] ⁺ : 339.9.

Synthesis example 30. N-(4-Chlorophenethyl)-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxamide

1H NMR (600 MHz, CDCl ₃ ) δ 8.26 (bs, 1H), 7.83 (d, J=30.3 Hz, 2H), 7.25-7.27 (m, 2H), 7.07-7.15 (m, 3H), 4.42 (s, 2H), 3.58 (s, 2H), 3.47 (m, 2H), 2.77-2.81 (m, 2H), 2.54 (s, 2H), mass [M+H] ⁺ : 397.9.

Synthesis example 31. 6-(Naphthalen-2-ylsulfonyl)-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

1H NMR (600 MHz, CDCl ₃ ) δ 8.38 (s, 1H), 7.94 (d, J=6.9 Hz, 2H), 7.52-7.60 (m, 7H), 7.11 (bs, 1H), 4.29 (s, 2H), 3.54 (m, 2H), 2.57 (bs, 2H), mass [M+H] ⁺ : 406.8.

Synthesis example 32. 4-Nitrobenzyl-3-hydroxy-2-(pyridin-2-yl)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate

1H NMR (600 MHz, CDCl ₃ ) δ 8.27 (bs, 1H), 8.23 (m, 2H), 7.82-7.87 (m, 2H), 7.54 (d, J=8.3 Hz, 2H), 7.16 (bs, 1H), 5.28 (d, J=10.3 Hz, 2H), 4.66 (s, 2H), 3.75 (t, J=5.9 Hz, 2H), 2.61 (bs, 2H), mass [M+H] ⁺ : 395.9.

Synthesis example 35. Preparation of 6-benzyl-2-(5-chloropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol

Ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate hydrochloride (0.50 g, 1.68 mmol) and 5-chloro-2-hydrazinopyridine (0.24 g, 1.68 mmol) were dissolved in 10 ml of ethanol and 2 drops of acetate were added. The reaction mixture was stirred at room temperature for 1 h, sodium triacetoxyborohydride (0.53 g, 2.52 mmol) was added and then stirred for 30 h. The reaction mixture was concentrated under reduced pressure and then extracted with water and dichloromethane. The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The reaction mixture was recrystallized from ethyl acetate to give the title compound (0.12 g, 22%).

¹ H-NMR (600 MHz, DMSO-de) δ 11.54 (brs, 1H), 8.40 (d, G=2.4 Hz, 1H), 8.35 (s, 1H), 7.97 (dd, J=8.4, 2.4 Hz, 1H), 7.34-7.28 (m, 4H), 7.26-7.20 (m, 1H), 3.64 (s, 2H), 3.33 (s, 2H), 2.63 (t, J=5.8 Hz, 2H), 2.23 (t, J=5.3 Hz, 2H). MS: calculated: 340.1; MS: found: 341.1 ([M+H] ⁺ ).

Synthesis example 36. Preparation of 4-(6-benzyl-3-hydroxy-4,5,6,7-tetrahydropyrazolo[3,4-c]pyridin-2-yl)-benzoic acid hydrochloride

Using ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate hydrochloride (0.50 g, 1.68 mmol) and 4-hydrazinobenzoic acid hydrochloride (0.31 g, 1.68 mmol), the title compound (0.28 g, 43%) was obtained according to the preparation method of Synthesis Example 38 below.

¹ H-NMR (600 MHz, DMSO-de) δ 12.96 (brs, 1H), 10.61 (brs, 1H), 7.97 (d, J=8.8 Hz, 2H), 7.84 (d, J=8.8 Hz, 2H), 7.61-7.52 (m, 2H), 7.51-7.43 (m, 3H), 4.52-4.36 (m, 2H), 4.28-4.12 (m, 2H), 3.75-3.51 (m, 1H), 3.30-3.15 (m, 1H), 2.90-2.65 (m, 2H). MS: calculated: 349.1; MS: found: 350.1 ([M+H] ⁺ ).

Synthesis example 37. Preparation of 6-benzyl-2-(4-bromophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol hydrochloride

Using ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate hydrochloride (0.50 g, 1.68 mmol) and 4-bromophenylhydrazine hydrochloride (0.35 g, 1.68 mmol), the title compound (0.29 g, 41%) was obtained according to the preparation method of Synthesis Example 38 below.

¹ H-NMR (600 MHz, DMSO-de) δ 10.65 (brs, 1H), 7.65-7.59 (m, 4H), 7.59-7.53 (m, 2H), 7.51-7.41 (m, 3H), 4.50-4.38 (m, 2H), 4.22-4.07 (m, 2H), 3.65-3.50 (m, 1H), 3.27-3.10 (m, 1H), 2.85-2.65 (m, 2H). MS: calculated: 383.1; MS: found: 384.1 ([M+H] ⁺ ).

Synthesis example 38. Preparation of 6-benzyl-2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-3-ol hydrochloride

Ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate hydrochloride (0.50 g, 1.68 mmol) and phenylhydrazine hydrochloride (0.24 g, 1.68 mmol) were dissolved in 10 ml of ethanol, and then 2 drops of acetate were added. The reaction mixture was stirred at 120°C for 1 hour and then concentrated under reduced pressure. The reaction mixture was recrystallized from ethanol and diethyl ether to give the title compound (0.25 g, 44%).

¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 10.30 (brs, 1H), 7.63 (d, J=8.0 Hz, 2H), 7.60-7.51 (m, 2H), 7.50-7.44 (m, 3H), 7.41 (t, J=7.7 Hz, 2H), 7.31-7.20 (m, 1H), 4.44 (s, 2H), 4.30-4.07 (m, 2H), 3.63 (dd, 1H), 3.25-3.19 (m, 1H), 2.84-2.60 (m, 2H). MS: calculated: 305.1; MS: found: 306.1 ([M+H] ⁺ ).

General synthesis method 4 (synthetic examples 34, 39-42, 45-52, 59-64, 71, 72, 75-81)

The compounds from Synthesis Examples 34, 39-42, 45-52, 59-64, 71, 72, 75-81 are synthesized according to the general reaction scheme given above, and an example of the synthetic method that relates to a specific compound is given below. In addition, these compounds from the Synthesis Examples are described according to the synthetic method.

Synthesis example 39. Preparation of 5-benzyl-2-(5-trifluoromethyl-pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol hydrochloride

Ethyl 1-benzyl-4-oxo-3-piperidinecarboxylate hydrochloride (0.50 g, 1.68 mmol) and 2-hydrazinyl-5-(trifluoromethyl)pyridine (0.29 g, 1.68 mmol) were dissolved in 10 ml of acetate and stirred at 65°C for 12 h. The reaction mixture was concentrated under reduced pressure and then recrystallized from ethanol to give the title compound (0.14 g, 21%).

¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 10.99 (brs, 1H), 8.90-8.78 (m, 1H), 8.60-8.45 (m, 1H), 8.30 (d, J=9.,8 Hz, 1H), 7.65-7.55 (m, 2H), 7.48-7.43 (m, 3H), 4.60-4.30 (m, 2H), 3.90-3.60 (m, 3H), 3.50-3.20 (m, 1H), 3.10-2.80 (m, 2H). MC: calculated: 374.1; MC: found: 375.1 ([M+H] ⁺ ).

Synthesis example 40. Preparation of 5-benzyl-2-phenyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol hydrochloride

Using ethyl 1-benzyl-4-oxo-3-piperidinecarboxylate hydrochloride (1.02 g, 3.46 mmol) and phenylhydrazine hydrochloride (0.50 g, 3.46 mmol), the title compound (0.48 g, 41%) was obtained according to the preparation method of Synthesis Example 38 above.

¹ H-NMR (600 MHz, DMSO-d ₆ ) δ 10.94 (brs, 1H), 7.68-7.60 (m, 4H), 7.50-7.44 (m, 3H), 7.40 (t, J=7.7 Hz, 2H), 7.22 (t, G=7.3 Hz, 1H), 4.54-4.46 (m, 1H), 4.36 (dd, J=12.6, 5.9 Hz, 1H), 4.06-3.87 (m, 2H), 3.72-3.66 (m, 1H), 3.38-3.28 (m, 1H), 3.08-2.96 (m, 1H), 2.90-2.81 (m, 1H). MS: counted: 305.1; MS: found: 306.2 ([M+H] ⁺ ).

Synthesis example 41. Preparation of 5-benzyl-2-(4-bromophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol hydrochloride

Using ethyl 1-benzyl-4-oxo-3-piperidinecarboxylate hydrochloride (0.50 g, 1.68 mmol) and 4-bromophenylhydrazine hydrochloride (0.38 g, 1.68 mmol), the title compound (0.17 g, 24%) was obtained according to the preparation method of Synthesis Example 38 above.

¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.10 (brs, 1H), 7.66-7.61 (m, 4H), 7.67-7.61 (m, 2H), 7.47-7.43 (m, 3H), 4.54-4.42 (m, 1H), 4.40-4.30 (m, 1H), 4.05-3.85 (m, 2H), 3.70-3.60 (m, 1H), 3.40-3.21 (m, 1H), 3.10-2.95 (m, 1H), 2.90-2.80 (m, 1H). MS: calculated: 383.1; MC: found: 384.1 ([M+H] ⁺ ).

Synthesis example 42. Preparation of 5-benzyl-2-(2-chlorophenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3-ol hydrochloride

Using ethyl 1-benzyl-4-oxo-3-piperidinecarboxylate hydrochloride (0.50 g, 1.68 mmol) and 2-chlorophenylhydrazine hydrochloride (0.30 g, 1.68 mmol), the title compound (0.23 g, 36%) was obtained according to the preparation method of Synthesis Example 38 above.

¹ H-NMR (600 MHz, DMSO-de) δ 11.19 (brs, 1H), 7.71-7.67 (m, 2H), 7.65-7.60 (m, 1H), 7.52-7.45 (m, 5H), 7.41 (d, J=7.3 Hz, 1H), 4.54 (dd, J=12.6, 3.5 Hz, 1H), 4.39 (dd, J=12.6, 6.3 Hz, 1H), 4.09-3.91 (m, 2H), 3.68 (t, J=13.6 Hz, 1H), 3.45-3.31 (m, 1H), 3.14-3.00 (m, 1H), 2.93-2.79 (m, 1H). MC: Calculated: 339.1; MC: Found: 340.2 ([M+H] ⁺ ).

Example 1

In order to confirm the effect of the compounds of the Synthesis Examples on the generation of ROS from fibroblasts and specific substrate cells, ROS generation was induced by stimulation with PMA (phorbol 12-myristate 13-acetate), TGF-β1, palmitate or glucose at a high concentration or the like in EL-60 (Korean Cell Line Bank), NHLF (normal human lung fibroblasts, Lonza), LX-2 (human liver stellate cells, Sigma), ARPE19 (human retinal pigment epithelial cells, ATCC (American Type Culture Collection)), KEL-FIB (keloid fibroblasts, ATCC) cells differentiated into neutrophils by treatment with DMSO, and the inhibitory effect of the compounds of the Synthesis Examples on ROS generation was observed under these conditions.

HL-60 cells (human leukemia cells, ATCC) were suspended in a medium containing 1.25% DMSO (RPMI plus 10% FBS (fetal bovine serum)) and cultured under 5% _CO2 , 37°C for 6 days to induce differentiation into neutrophils. HL-60 cells differentiated into neutrophils were aliquoted into a 96-well plate at a concentration of 2 × 104 cells/well and treated with the compound from the synthesis example at a concentration of 0.5 mM for 30 min. Each well containing cells and drug was then treated with 200 mM PMA to induce ROS production, and this ROS production was measured by monitoring the luminescence level in each well using 100 μM L-012 (chemiluminescent probe).

NHLF (normal human lung fibroblasts, Lonza), LX-2 (human liver stellate cells, Sigma), ARPE19 (human retinal pigment epithelial cells, ATCC), and KEL-FIB (keloid fibroblasts, ATCC) cells were suspended in a culture medium containing 10% FBS and aliquoted into a 96-well plate and then cultured under 5% _CO2 , 37°C for 24 hours. They were pretreated with the compound from the synthesis example for 30 minutes to 1 hour, and then each well containing the cells and the drug was treated with a ROS production-stimulating substance. After further culturing for 48 h, the ROS production level was confirmed using L-012 or DCF-DA (fluorescent dye dichlorofluorescein diacetate).

As a result of the experiment, the compounds from the synthesis examples inhibited the ROS production achieved by ROS stimulation in all NHLF, LX-2, ARPE19, and KEL-FIB cells. The results presented in Table 2 show that the compounds from the synthesis examples (0.5 μM) inhibited the ROS production induced by PMA in HL-60 cells differentiated into neutrophils, and Fig. 1 shows the result according to which both the compound from synthesis example 3 and the compound from synthesis example 34 inhibited the ROS production induced by PMA in LX-2 and NHLF cells.

Therefore, it was confirmed that ROS production was effectively inhibited when the compound of the synthesis example was treated with differentiated HL-60 cells, or various fibroblasts, or cells of specific substrates in which ROS production was induced.

Table 2

Inhibitory effect on ROS production in differentiated HL-60 cells

Example 2. Analysis of changes in αSMA expression induced by TGF-β1

Fibroblasts have been reported to differentiate into myofibroblasts to form tumor-associated fibroblasts (CAFs) or cause fibrosis in various tissues.

In order to confirm whether the compound of the synthesis example of the present invention affects the differentiation of fibroblasts into myofibroblasts, the inhibitory effect of the compound of the synthesis example on the increase in the expression of αSMA (a biomarker for confirming differentiation into myofibroblasts) induced by TGF-β1 was examined for HL-60 cells (Korean Cell Line Bank), HFF-1 (human foreskin fibroblasts, ATCC), NHLF (normal human lung fibroblasts, Lonza), LX-2 (human liver stellate cells, Sigma), ARPE19 (human retinal pigment epithelial cells, ATCC), KEL-FIB cells (keloid fibroblasts, ATCC).

Each cell type was suspended in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS, aliquoted into a 4-well slide chamber (Nunc) at a concentration of 1× ¹⁰⁴ cells/well, and treated with the compound from the synthesis example and TGF-β1 (TGF-β1, TGF-β have the same meaning) to prepare the cells for use in confirming αSMA expression. Confirmation of αSMA expression in the prepared cells was performed by immunocytochemistry as follows. After fixation with 4% paraformaldehyde for 10 minutes and permeabilization of the cells with 0.1% Triton X-100, the primary antibody (anti-αSMA antibody, 1:200, at 4°C for 16 hours) and secondary antibody (A1exa-594-conjugated antibody, 1:800, at 4°C for 1 hour) against αSMA were successively treated, and then the expression level of αSMA was observed using a fluorescence microscope.

Fig. 2 shows the effect of treating ARPE19 cells with various compounds from Synthesis Examples (5 μM) after inducing αSMA expression by treatment with TGF-β1 (5 μM). All the compounds from Synthesis Examples 3, 8, 9, 26 showed an effect of inhibiting αSMA expression by 35%~70% or more, although there was a difference in the degree.

In addition, as confirmed in Fig. 3a and Fig. 3b, also in HFF-1, LX-2, NHLF, ARPE19, KEL-FIB cells, it was noted that the increase in αSMA expression induced by TGF-β1 (10 ng/ml) was significantly inhibited by treatment with 5 μM of the compound from Synthesis Example 3.

Example 3. Analysis of changes in ROS production by dopaminergic neurons

High levels of ROS in neurons induce lipid oxidation, which is known to be a key factor in neuronal death. To confirm the effect of the compound from the synthesis example on ROS production by dopaminergic neurons, ROS production was induced by stimulation with MPP+ (1-methyl-4-phenylpyridinium) in N27 cells (rat dopaminergic neurons, Sigma). Under these conditions, the inhibitory effect of the compound from the synthesis example on ROS production was observed.

N27 cells were cultured in RPMI medium containing 10% FBS, 1 μM angiotensin II, and the cultured cells were aliquoted into 6-well plates at a concentration of 5 × 10 ³ ~ 6 × 10 ³ cells/well. The cells were treated with a ROS production stimulator, MPP+ (1-methyl-4-phenylptridinium), and the compound from Synthesis Example 3 (1 μM). Twenty-four hours after treatment with the compound from Synthesis Example, the level of ROS production was confirmed by measuring fluorescence in each well using DCF-DA.

As a result of the experiment, as confirmed in Fig. 4, it was found that the ROS production induced by MPP+ was effectively inhibited by treatment with the compound from the synthesis example.

Example 4. Analysis of changes in the expression of the CTGF gene and the COL1A1 gene (collagen type I) by keloid fibroblasts

To confirm the effect of the compound from the synthesis example on the process of extracellular matrix formation from keloid fibroblasts, the change in the expression of the factor that promotes the formation of extracellular matrix, CTGF, and the typical extracellular matrix, collagen type I, was analyzed at the gene level when KEL-FIB cells (keloid fibroblasts) were treated with the compound from the synthesis example 3.

The KEL-FIB cell line was adapted in DMEM containing 2% FBS for 24 hours and then pretreated with the compound from Synthesis Example 3 at a concentration of 5 μM or 10 μM for 1 hour. To induce the expression of the CTGF and type I collagen genes, treatment with 2 ng/ml TGF-β1 was performed, and mRNA (messenger ribonucleic acid) was isolated from the cells after 24 or 48 hours. The presence of mRNA and the expression level of the CTGF and type I collagen genes were confirmed by qRT-PCR (quantitative real-time PCR).

As a result of the experiment, it was confirmed that the expression of the CTGF gene and the type I collagen gene was increased by 26 times and 2.3 times, respectively, when treated with 2 ng/ml TGF-β1, and then the expression of the two genes was inhibited by 76% and 75% when treated with 5 μM or 10 μM of the compound of Synthesis Example 3.

Example 5. Analysis of changes in IL-1β expression induced by LPS

In order to confirm the effect of the compounds from the synthesis examples of the present invention on the inflammatory response in human-derived fibroblasts, it was observed whether the expression of IL-1β, increased by LPS treatment, was inhibited in fibroblasts obtained from various tissues.

After inoculating the cells at 2× ¹⁰⁵ in a 6-well plate and culturing for 24 hours, and then further culturing in a serum-free culture solution for 16 hours, pretreatment with the compound of Synthesis Example 3 was performed according to each condition for 30 minutes, and an inflammatory response was induced with LPS for 6 hours. IL-1β expression was confirmed using RT-PCR (reverse transcription PCR). Total RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen), and cDNA (complementary deoxyribonucleic acid) was synthesized from the isolated 2 μg of RNA using the PrimeScript™ II Single-Strand cDNA Synthesis Kit (TaKaRa), followed by PCR using the AccuPower® PCR PreMix Kit (Bioneer) for gene amplification. The sequence of target primers is as follows. IL-1β (forward: 5'-CCACAGACCTTCCAGGAGAATG-3', reverse: 5'-GTGCAGTTCAGTGATCGTACAGG-3'); GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (forward: 5'-GTGGCTGGCTCAGAAAAAGG-3', reverse: 5'-GGTGGTCCAGGGGTCTTACT-3'); β-actin (forward: 5'-CACCATTGGCAATGAGCGGTTC-3', reverse: 5'-AGGTCTTTGCGGATGTCCACGT-3'). The PCR product was confirmed by electrophoresis in 1.5% agarose gel.

As a result of the experiment, as confirmed in Fig. 5, it was found that IL-1β induced by LPS was effectively inhibited in a concentration-dependent manner when treated with the compound of Synthesis Example 3.

Example 7. Confirmation of the inhibitory effect on pulmonary fibrosis in a mouse model of bleomycin-induced pulmonary fibrosis

As a mouse model of bleomycin-induced pulmonary fibrosis, 5-week-old male C57BL/6J mice weighing 20 g before and after were used. During the experiment, the experimental animals were divided into a control group in which distilled water was orally administered (control), a group in which pulmonary fibrosis was induced by bleomycin (BLM), an experimental group in which bleomycin was administered, and after 3 weeks, nintedanib was orally administered every day at a dose of 100 mg/kg as a positive control (BLM+NIN), and an experimental group in which bleomycin was administered, and after 3 weeks, the compound from the synthesis example of the present invention was orally administered every day at a dose of 3 mg/kg (BLM+compound from the synthesis example), 7 mice per group.

Nintedanib and the compound from the synthesis example were orally administered once daily for 28 days. On the day following the final administration of nintedanib or the compound from the synthesis example, each animal was anesthetized, blood was collected via cardiac puncture, and then sacrificed. BAL (bronchoalveolar lavage fluid) and lung tissue were collected and used for further analysis. Hematoxylin and eosin (H&E), a cellular stain, and Masson's trichrome (MT) staining were performed to confirm fibrosis, and the expression rate of αSMA and type I collagen in lung tissue was confirmed.

In general, based on the above histological and immunological results, the severity of pulmonary fibrosis in each of the groups receiving the compound from the synthesis example (BLM + compound from the synthesis example) was evaluated compared with the negative control group (control), the experimental group (BLM), and the positive control group by quantifying it using the improved Ashcroft score (modified Ashcroft scale), which is widely used in the field of lung fibrotic tissue analysis. The histopathological examination of the prepared tissue preparations was confirmed using an optical microscope (Carl Zeiss, Oberkochen, Germany). The mean and standard deviation of the experimental result were calculated using SPSS version 22.0 statistical software (SPSS Inc., Chicago, IL, USA), and the significance of the difference between the experimental groups was tested at p<0.05 using Student's t-test.

As a result of the experiment, as confirmed in Fig. 6, according to the histological analysis using H&E staining and MT staining, it was found that the degree of inflammatory cell infiltration into the alveoli and bronchioles was significantly lower in the experimental group in which pulmonary fibrosis was induced (BLM+vehicle), the positive control group (BLM+NIN), and the group receiving the compound of the synthesis example (BLM+compound of the synthesis example) than in the experimental group and the positive control group, and it was confirmed that the hypertrophy of epithelial cells was reduced, and the structural deformation of the lungs was reduced, and the areas of accumulation of pathological tissue were reduced.

Furthermore, it was confirmed that the size of fibrotic areas and collagen accumulation due to fibrosis were significantly reduced in the group receiving the compound from the synthesis example (BLM + compound from the synthesis example) compared with the experimental group and the positive control group, and it was confirmed that αSMA expression was significantly inhibited to the level of the normal group. Thus, it was confirmed that the compound from the synthesis example effectively reduced pulmonary fibrosis.

Overall, based on the above histological and immunological results, according to the evaluation of the severity of pulmonary fibrosis in each of the groups receiving the compound of the synthesis example (BLM+compound of the synthesis example), compared with the negative control group (control), the experimental group (BLM), the positive control group by quantification using the improved Ashcroft score (modified Ashcroft scale) which is widely used in the field of lung fibrotic tissue analysis, the score in the negative control group (PBS (phosphate-buffered saline)) showing normal values was 0.4±0.55, and in the experimental group in which pulmonary fibrosis was induced by bleomycin (BLM) was 5.4±0.5. The improved Ashcroft score in the positive control group (BLM+NIN) was reduced to 5.0±0, and the improved Ashcroft score in the experimental group administered with the compound of the synthesis example (BLM+compound of the synthesis example) was 3.44±0.88, and thus it was confirmed that the compound of the present invention effectively improves pulmonary fibrosis compared with nintedanib used as a conventional therapeutic agent for idiopathic pulmonary fibrosis.

Example 8. Confirmation of the therapeutic effect in non-alcoholic steatohepatitis and liver fibrosis in the STAM mouse model

To confirm the therapeutic effect of the compound from the synthesis example of the present invention on non-alcoholic steatohepatitis and liver fibrosis, STAM mice (6-week-old, female, SMC Laboratories Inc, Japan) in which non-alcoholic steatohepatitis and liver fibrosis were induced were used. All mice were maintained under standard conditions. During the experiment, the experimental animals were divided into a normal control group in which distilled water was orally administered (negative control), a STAM group in which non-alcoholic steatohepatitis and fibrosis were induced (vehicle control group), an experimental group in which telmisartan was orally administered daily at a dose of 100 mg/kg as a STAM positive control group (STAM+TEL), and an experimental group in which the compound from the synthesis example was orally administered daily at a dose of 3 or 10 mg/kg (STAM+compound from the synthesis example), 7 mice per group. Telmisartan and the compound from the synthesis example were orally administered once a day for 28 days.

After completion of the study, the animals were anesthetized, and liver tissue was removed from each animal, and blood was collected for analysis. Liver tissue weight was measured and photographed. The tissue was fixed in 10% neutral buffered formalin. After sectioning the fixed tissue to a specified thickness, it was embedded in paraffin, following the general tissue processing method, to prepare 4~5 μm tissue sections. Hematoxylin and eosin (H&E) staining, a general staining method, was then used to establish histopathological characteristics. Sirius red staining was used to confirm fibrosis, and Oil Red O working solution was used to confirm the degree of lipid formation.

Overall, based on the above histological and immunological results, according to the evaluation of the severity of hepatitis and liver fibrosis in each of the groups receiving the compound of the synthesis example (STAM+compound of the synthesis example), compared with the negative control group (control), the experimental group (STAM), the positive control group (STAM+TEL) by quantification using the improved NAS (Non-alcoholic Fatty Liver Disease Activity Scale) scale, which is widely used in the field of hepatitis and fibrotic tissue analysis, the score in the normal control group showing normal values (normal control) was 0.0±0.0, and that in the STAM experimental group (STAM+vehicle) was 4.8±0.7. The improved NAS score in the positive control group (STAM+TEL) was reduced to 2.8±0.7, and the improved NAS score in the experimental group administered with the compound from the synthesis example (STAM+compound from the synthesis example) was confirmed at 2.6±0.7.

In addition, according to the results of Sirius red staining to confirm the degree of liver fibrosis, the liver fibrosis area was 0.29±0.06 in the normal control group showing normal parameters (normal control) and 0.93±0.36 in the experimental STAM group (STAM+vehicle). It was confirmed that the fibrosis area in the positive control group (STAM+TEL) was reduced to 0.44±0.15, and the fibrosis area in the experimental group administered with the compound from the synthesis example (STAM+compound from the synthesis example) was reduced to 0.49±0.16.

It was confirmed that the compound from the synthesis example of the present invention effectively improves the course of non-alcoholic hepatitis and liver fibrosis.

Example 9. Confirmation of the effect in the treatment of traumatic brain injury

To study the effect of compounds from the synthesis examples in the prevention or treatment of brain disease or illness, a mouse model of Parkinson's disease (transgenic mice with the A53T mutation in the alpha-synuclein gene) was created.

All mice were maintained under standard conditions. During the experiment, the experimental animals were divided into a normal control group, which received distilled water orally (negative control), a Parkinson's disease-induced group (vehicle control group), and an experimental group, which received the compound from the synthesis example orally at a dose of 25 mg/kg daily (compound from the synthesis example), with 8 mice per group. The normal control group received saline, while the compound from the synthesis example was administered orally once daily for 2 weeks. Subsequently, behavioral changes and histopathological examination were performed.

Behavioural changes were analysed using the rotarod test, vertical pole test and tucked hind limb test, and the average of three experiments was measured for each animal.

After completing the study, the animals were anesthetized for brain tissue extraction, and the tissues were fixed in 4% buffered neutral formalin. After sectioning the fixed tissues to a specified thickness, they were embedded in paraffin, following the standard tissue processing protocol, to prepare 4-5 μm tissue sections. To confirm whether the Parkinson's disease pathology index, based on the level of protein aggregation in the striatum, was improved, alpha-synuclein was first labeled by phosphorylation at serine 129, and then stained with thioflavin-T to establish histopathological markers.

As a result of the experiment, according to the behavioral change analysis data using the rotarod test, the vertical pole test, and the tucked hind limb test, in the group receiving the compound of the synthesis example, an improvement in Parkinsonian-like behavioral symptoms by 50% or more was confirmed, and the aggregation and accumulation of α-synuclein, an important indicator of the pathological process, were reduced by 50% or more.

In conclusion, it was confirmed that the compounds of the synthesis examples of the present invention effectively improved the course of Parkinson's disease.

Claims (22)

1. A compound of the following chemical formula I-1 or a pharmaceutically acceptable salt thereof chemical formula I-1, in which X is NR3; and R3 is selected from Ra-L-, Ra-L-CO-, Ra-L-NHCO-, Ra-L-OCO- and Ra-L-SO ₂ -; and Ra is selected from hydrogen, cyano, halogen, substituted or unsubstituted C1-C5 straight or branched alkyl, C2-C6 alkynyl, C2-C6 alkenyl, unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C6-C20 aryl, or substituted or unsubstituted C5-C20 heteroaryl, and the heteroaryl contains 1, 2, or 3 heteroatoms from O, N, and S; and L is selected from a direct bond, C1-C3 alkylene, and substituted or unsubstituted phenylene; and R4 is unsubstituted C1-C5 alkyl; and A is CR5 or N, and B is CR6, and D is CR7, and E is CR8, and G is CR9 or N, where A or G is N; and radicals R5 to R9 are the same or different and each is independently selected from hydrogen, cyano, halogen, carbonyl, nitro, carboxyl, C1-C5 alkoxy, substituted or unsubstituted C1-C5 straight or branched alkyl, unsubstituted amino group, and two or more groups adjacent to each other can be combined with each other to form an unsubstituted C5-C10 heteroaryl ring containing 1, 2 or 3 heteroatoms selected from N and S; and substituted C1-C5 straight or branched alkyl may be independently substituted with one or more substituents selected from halogen, substituted or unsubstituted C6-C20 aryl, or C5-C20 heteroaryl containing 1 heteroatom selected from N and O; and substituted C6-C20 aryl or substituted C5-C20 heteroaryl may be independently substituted with one or more substituents selected from halogen, cyano, nitro, substituted amino, C1-C5 alkoxy, C1-C5 haloalkyl, C1-C10 alkyl, C3-C8 cycloalkyl; and the substituted amino group may be substituted by one or two C1-C5 alkyl groups; and substituted C6-C10arylene may be substituted with one or more substituents selected from C1-C3 alkyl, halogen. 2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R3 is Ra-L-; L is C1-C3 alkylene; and A is CR5 or N, and B is CR6, and D is CR7, and E is CR8, and G is CR9 or N, where A or G is N. 3. A compound or a salt thereof, wherein said compound is 6-benzyl-3-methoxy-2-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine. 4. A pharmaceutical composition for the prevention or treatment of a disease associated with oxidative stress, comprising an effective dose of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier. 5. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable carrier is one or more selected from the group consisting of excipients, diluents, disintegrants, binders, lubricants, surfactants, emulsifiers, suspending agents, and extenders. 6. The pharmaceutical composition of claim 4, wherein the disease associated with oxidative stress is selected from cancer, non-alcoholic steatohepatitis, liver fibrosis, pulmonary fibrosis, and Parkinson's disease, wherein the cancer is selected from the group consisting of liver cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, meningioma associated with neurofibromatosis, pancreatic cancer, leukemia, myeloproliferative/myelodysplastic disease, skin fibrosarcoma, breast cancer, lung cancer, thyroid cancer, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, brain tumor, head and neck cancer, and glioblastoma.