WO2023068835A1 - Pak4 inhibitor and uses thereof - Google Patents

Abstract

The present disclosure relates to a novel PAK4 inhibitor and uses thereof. In addition, the present disclosure newly reveals the role of PAK4 in the process of tissue damage caused by ischemia-reperfusion or hypoxia-reoxygenation, and provides new pharmaceutical uses of a PAK4 inhibitor on the basis of the revelation.

Description

PAK4 inhibitors and uses thereof

This application claims priority to Korean Patent Application No. 10-2021-0140107 filed on October 20, 2021, and the entire specification is a reference in this application.

The present disclosure relates to PAK4 inhibitors and their pharmaceutical uses.

PAK4 (p21 activated kinase 4) is a serine/threonine kinase and a subprotein of Rho GTPases. The main functions of PAK4 reported so far are known to be involved in a wide range of cell functions such as cell proliferation, invasion, migration, and anchorage independent growth through cytoskeletal remodeling. Dysregulation of PAK4 expression and activity results in various pathological conditions.

PAK4 plays a central role in cancer progression by promoting epithelial-mesenchymal transition, invasion, and metastasis, and its expression is increased in various types of cancer tissues, including prostate, breast, lung, brain, gastric, ovarian, and gallbladder cancers ( Won et al., Experimental & Molecular Medicine (2019) 51:11). Therefore, PAK4 is regarded as a therapeutic target for various cancers, and PAK4 inhibitors are being developed as anticancer agents. The first PAK4 inhibitor reported to have anticancer effects in animal experiments is PF-3758309 (IC ₅₀ = 1.3 nM) developed by Pfizer. It has no selectivity for PAK1, so the clinical trial was terminated early, and the drug has been successfully developed. there is no Therefore, it is necessary to develop novel PAK4-selective inhibitors.

In addition, PAK4 is known to interact with many effectors such as Cdc42 and CREB. PAK4 has been reported to protect lung epithelial cells from oxidative stress by directly binding to keratinocyte growth factor receptors (Lu Y et al., J Biol Chem (2003) 278: 10374), Korea Patent Publication No. In No. 2016-0112181, based on the fact that PAK4 plays a role as an accelerator in the melanin production process induced by α-MSH and UVB, a pharmaceutical for the treatment of melanin hyperpigmentation disease containing a PAK4 activity or expression inhibitor as an active ingredient composition was provided. The discovery of additional effectors and mechanisms of action may lead to the discovery of new medicinal uses of PAK4. Therefore, further efforts are needed to identify the effectors and mechanism of action of PAK4.

One technical problem to be solved by the present disclosure is to provide a novel inhibitor of PAK4 and to provide a pharmaceutical composition thereof.

Another technical problem to be solved in the present disclosure is to identify a novel effector and mechanism of action of PAK4, and to provide a new therapeutic agent based thereon.

However, the technical task to be achieved by the present disclosure is not limited to the tasks mentioned above, and other tasks not mentioned above will be clearly understood by those skilled in the art from the description below.

Pyrrolopyrimidine derivative compounds

In order to solve the above problems, the present disclosure provides, in one aspect, a compound represented by Formula 1 below, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, X is -C ₁ haloalkyl,

Y is aryl or 5-6 membered heteroaryl {wherein at least one H of the aryl or 5-6 membered heteroaryl is -C _1-6 alkyl, -C _1-6 aminoalkyl, -C _1-6 hydroxy Alkyl, -C _1-6 Haloalkyl, -C _1-6 Alkyl-OC _1-6 Alkyl, -CN, -NR ₁ R ₂ , -OR ₃ , -Halo, -Cycloalkyl, -C(=O)- may be substituted with cycloalkyl, -heterocycloalkyl, or -C(=O)-heterocycloalkyl [wherein -cycloalkyl, -C(=O)-cycloalkyl, -heterocycloalkyl, or -C( At least one H of the =O)-heterocycloalkyl ring may be substituted with -C _1-6 alkyl, -C _1-6 aminoalkyl, -C _1-6 hydroxyalkyl, -cycloalkyl, or -heterocycloalkyl has exist]}

L ₁ is -C(=O)-NR ₄ -, -C(=O)-O-, -NR ₅ -C(=O)-, or -C(=O)-(heterocycloalkyl)-; {wherein the heterocycloalkyl ring of -C(=O)-(heterocycloalkyl)- contains at least one N and N is connected to -C(=O)-}

L ₂ is -NH- or -O-;

n is O, 1, 2, 3, 4, or 5 {wherein, when n is 0, L ₁ and L ₂ are directly connected};

R ₁ and R ₂ are each independently -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), or -C _1-6 alkyl- OC _1-6 alkyl;

R ₃ is -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), -C _1-6 alkyl-OC _1-6 alkyl, or -C _1-6 alkyl-heterocycloalkyl;

R ₄ and R ₅ are each independently -H or -C _1-6 alkyl.

According to specific examples of the present disclosure, the compound represented by Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof may be within the following ranges.

X is -CF ₃ ;

Y is phenyl or 5-6 membered heteroaryl {wherein, at least one H of the phenyl or 5-6 membered heteroaryl is -C _1-6 alkyl, -C _1-6 alkyl-OC _1-6 alkyl, -NR May be substituted with ₁ R ₂ , -OR ₃ , -heterocycloalkyl, or -C(=O)-heterocycloalkyl [wherein, -heterocycloalkyl, or -C(=O)-heterocycloalkyl ring one or more H may be substituted with -C _1-6 alkyl or -heterocycloalkyl]},

L ₁ is -C(=O)-NR ₄ -, -C(=O)-O-, -NR ₅ -C(=O)-,

,

,

,

, or

ego;

L ₂ is -NH- or -O-;

n is O, 1, 2, 3, or 4 {wherein, when n is 0, L ₁ and L ₂ are directly connected};

R ₁ and R ₂ are each independently -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), or -C _1-6 alkyl- OC _1-6 alkyl;

R ₃ is -H, -C _1-6 alkyl, or -C _1-6 alkyl-heterocycloalkyl;

R ₄ and R ₅ are each independently -H or -C _1-6 alkyl.

In the present disclosure, "alkyl", unless otherwise indicated, may mean a straight-chain or branched-chain, acyclic, cyclic, or saturated hydrocarbon to which they are bonded. For example, "C _1-6 alkyl" may mean an alkyl containing 1 to 6 carbon atoms. Non-cyclic alkyl may include, for example, methyl, ethyl, n -propyl, n -butyl, isopropyl, sec -butyl, isobutyl, or tert -butyl, etc. Not limited to this. Cyclic alkyl may be used interchangeably with "cycloalkyl" herein, and may include, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, as examples. It doesn't work.

For purposes of this disclosure, “alkoxy” can mean -(O-alkyl) as an alkyl ether group, where alkyl is as defined above. For example, "C _1-6 alkoxy" may mean C _1-6 alkyl-containing alkoxy, that is, -(OC _1-6 alkyl), and as an example, alkoxy is methoxy. ), ethoxy, n -propoxy, isopropoxy , n -butoxy, isobutoxy, sec - butoxy ), or tert -butoxy ( tert -butoxy), etc., but is not limited thereto.

In the context of this disclosure, “halo” can be F, Cl, Br, or I.

For purposes of this disclosure, “haloalkyl” may mean a straight or branched chain alkyl (hydrocarbon) having carbon atoms substituted with one or more halo as defined herein. Examples of such haloalkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl or n -butyl independently substituted with one or more halogens such as F, Cl, Br, or I .

For purposes of this disclosure, “hydroxyalkyl” can mean a straight or branched chain alkyl (hydrocarbon) having carbon atoms substituted with hydroxy (OH).

For purposes of this disclosure, "aminoalkyl" can mean a straight or branched chain alkyl (hydrocarbon) having carbon atoms substituted with amino (NR'R"), wherein R' and R" are each independently It may be selected from the group consisting of hydrogen and C _1-6 alkyl, and the selected R' and R" may each independently be substituted or unsubstituted.

For purposes of this disclosure, “cyanoalkyl” may refer to a straight or branched chain alkyl (hydrocarbon) having carbon atoms substituted with cyano (CN).

For purposes of this disclosure, “heterocycloalkyl” may refer to a ring containing at least one selected from N, O, P, P(=O), and S in the ring, and may be saturated or partially unsaturated. . Here, when unsaturated, it may be referred to as a heterocycloalkene. Unless otherwise stated, a heterocycloalkyl can be a single ring or multiple rings such as spiro rings, bridged rings or fused rings. In addition, "3 to 12 membered heterocycloalkyl" may mean a heterocycloalkyl containing 3 to 12 atoms forming a ring, and as an example, heterocycloalkyl is pyrrolidine, piperidine, Imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidine-2,4 (1 H ,3 H ) -Dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine- S -oxide, thiomorpholine- S , S -oxide , piperazine, pyran, pyridone, 3-pyrroline , thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, tropane, 2-azaspiro[3.3]heptane, (1 R ,5 S )-3-azabicyclo[3.2.1 ]octane, ( 1s , 4s )-2-azabicyclo[2.2.2]octane, or ( 1R , 4R )-2-oxa-5-azabicyclo[2.2.2]octane, etc. It can be done, but is not limited thereto.

In the context of this disclosure, “arene” can mean an aromatic hydrocarbon ring. Arenes can be monocyclic arenes or polycyclic arenes. The number of ring carbon atoms of the arene may be 5 or more and 30 or less, 5 or more and 20 or less, or 5 or more and 15 or less. Examples of arenes include benzene, naphthalene, fluorene, anthracene, phenanthrene, bibenzene, terbenzene, quarterbenzene, quinquebenzene, sexybenzene, triphenylene, pyrene, benzofluoranthene, chrysene, etc. , but not limited to these. In the present specification, a residue obtained by removing one hydrogen atom from the above "arene" is referred to as "aryl".

In the present disclosure, "heteroarene" may be a ring containing one or more of O, N, P, Si, and S as heterogeneous elements. The number of ring carbon atoms of the heteroarene may be 2 or more and 30 or less, or 2 or more and 20 or less. The hetero arene may be a monocyclic hetero arene or a polycyclic hetero arene. Polycyclic heteroarenes may have, for example, a bicyclic or tricyclic structure. Examples of heteroarenes include thiophene, purine, pyrrole, pyrazole, imidazole, thiazole, oxazole, isothiazole, oxadiazole, triazole, pyridine, bipyridyl, triazine, acridyl, pyridazine , pyrazine, quinoline, quinazoline, quinoxaline, phenoxazine, phthalazine, pyrimidine, pyrido pyrimidine, pyrido pyrazine, pyrazino pyrazine, isoquinoline, indole, carbazole, imidazopyridazine, imidazopyridine , imidazopyrimidine, pyrazolopyrimidine, imidazopyrazine or pyrazolopyridine, N -arylcarbazole, N -heteroarylcarbazole, N -alkylcarbazole, benzooxazole, benzoimidazole, benzothiazole , benzocarbazole, benzothiophene, dibenzothiophene, thienothiophene, benzofuran, phenanthroline, isoxazole, oxadiazole, thiadiazole, benzothiazole, tetrazole, phenothiazine , dibenzosilol and dibenzofuran, but are not limited thereto. In one embodiment of the present disclosure, a heteroarene may also include a bicyclic heterocyclo-arene including an arene ring fused to a heterocycloalkyl ring or a heteroarene fused to a cycloalkyl ring. In the present specification, a residue obtained by removing one hydrogen atom from the "heteroarene" is referred to as "heteroaryl".

In one embodiment of the present disclosure, the compound represented by Formula 1 may be selected from the group consisting of compounds listed in Table 1 described in 'Specific details for carrying out the invention' below.

For purposes of this disclosure, the term “enantiomer” means a compound of this disclosure or a salt thereof that has the same chemical formula or molecular formula but is sterically different. Each of these optical isomers and mixtures thereof are also included within the scope of this disclosure. Unless otherwise specified, the solid bond (-) connecting an asymmetric carbon atom is a wedge-shaped solid bond representing the absolute configuration of the stereogenic center.

or Wedge Dotted Combination

can include

Compounds of Formula 1 of the present disclosure may exist in the form of “pharmaceutically acceptable salts”. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The term "pharmaceutically acceptable salt" in the present disclosure is a concentration that has a relatively non-toxic and harmless effective effect on patients, and the side effects caused by the salt do not reduce the beneficial efficacy of the compound represented by Formula 1. means any and all organic or inorganic acid addition salts.

Acid addition salts are prepared by conventional methods, for example, by dissolving a compound in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol in water may be heated, and then the mixture may be evaporated to dryness, or the precipitated salt may be suction filtered.

At this time, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid may be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, glue Conic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, or hydroiodic acid, etc. Can be used. However, it is not limited to these.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable for preparing a sodium, potassium, or calcium salt, but is not limited thereto. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of the present disclosure include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of Formula 1 above. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of a hydroxy group, and other pharmaceutically acceptable salts of an amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate), and p-toluenesulfonate (tosylate) salts; It can be prepared through a method for preparing a salt known to.

As confirmed in <Experimental Example 1>, the compound of Formula 1 according to the present disclosure was found to inhibit the enzymatic activity of PAK4. In addition, as confirmed in the following <Experimental Example 8>, the compound according to the present disclosure has distinct inhibitory effects on hepatocellular necrosis, apoptosis, oxidative stress, and cytokine release in ischemia-reperfusion injury model mice compared to the vehicle control group. It has been shown to have a therapeutic effect on liver damage.

Use of pyrrolopyrimidine derivative compounds

In one aspect, the present disclosure provides a pharmaceutical composition comprising a compound represented by Formula 1 below, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

X, Y, L1 and L2 of the compound of Formula 1 are as described in the 'pyrrolopyrimidine derivative compound' section.

The compound represented by Formula 1 of the present disclosure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof exhibits inhibitory activity against the enzymatic activity of PAK4. Accordingly, the present disclosure can be used to prevent or treat diseases caused by overexpression or overactivity of PAK4.

In one aspect, the present disclosure provides a compound represented by Formula 1, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof provided in the present disclosure as an active ingredient for inflammatory diseases, ischemia, -Diseases caused by reperfusion, diseases caused by hypoxia-reoxygenation, melanin hyperpigmentation diseases, muscle regeneration (Mao Y et al., J Cachexia Sarcopenia Muscle (2021), in press), Parkinson's disease (Won SY et al., Sci Transl Med ( 2016) 8: 367), neurological diseases such as amyotrophic lateral sclerosis (Cong C et al., Cell Prolif (2021) 54: e13003), hypertension (Wu Z et al., Pulm Circ (2020) 10: 2015894020974919), AIDS (Vargas B et al., Antimicrob Agents Chemother (2019) 63: e01744), polycystic nephropathy (Hwang VJ et al., Kidney Int (2017) 92: 922-933), osteoporosis (Choi SW et al., J Bone Miner Res (2015) 30: 1494-1507 ) Provides a pharmaceutical composition for the prevention or treatment of a disease selected from.

In one aspect of the present disclosure, the compound of Formula 1 included in the pharmaceutical composition has at least one activity selected from the group consisting of inhibiting PAK4, inhibiting phosphorylation of Nrf2, and increasing the stability and transcriptional activity of Nrf2 protein. can be characterized.

In one aspect of the present disclosure, the inflammatory disease may be selected from the group consisting of viral infection, bacterial infection, fungal infection, autoimmune disease and chronic inflammatory disease, but is not limited thereto.

In one aspect of the present disclosure, the ischemia-reperfusion or hypoxia-reoxygenation-related disease consists of liver damage, brain damage, lung damage, kidney damage, myocardial damage, and skeletal muscle damage due to ischemia-reperfusion or hypoxia-reoxygenation. It may be selected from the group, but is not limited thereto.

In one aspect of the present disclosure, the hypermelanin pigmentation disease may be selected from the group consisting of melanin hyperpigmentation diseases including melasma, freckles, senile pigment spots, solar melanosis, and skin whitening, but is not limited thereto .

Composition for preventing and treating tissue damage caused by ischemia-reperfusion

Liver damage due to ischemia-reperfusion occurs in surgical situations such as liver transplantation, hepatectomy, and trauma, and in medical situations such as hypovolemic shock and blood transfusion after excessive bleeding. From a pathophysiological point of view, ischemia-reperfusion liver injury consists of an early ischemic phase and a late reperfusion injury phase. Reactive oxygen species (ROS) in the ischemic phase and Kupffer cells or inflammatory cells infiltrated from the outside (eg macrophages, neutrophil cells) in the reperfusion phase are major liver damage factors. Therefore, production of reactive oxygen species and suppression of inflammatory responses are key mechanisms to inhibit liver damage caused by ischemia-reperfusion.

In order to suppress the damage caused by reactive oxygen species, hepatocytes activate defense mechanisms. A typical example is Nrf2. Normally, Nrf2 binds to a protein called Keap1 and exists in the cytoplasm. When reactive oxygen species are generated, Nrf2 detaches from Keap1 and migrates to the nucleus, binds to antioxidant response elements (ARE), and promotes the expression of various antioxidant enzymes. Various intracellular kinases regulate the stability and transcriptional activity of Nrf2 protein through phosphorylation.

The inventors of the present disclosure found that PAK4 expression increased during ischemia-reperfusion-induced liver damage, and hypothesized that PAK4 is a key mechanism for ischemia-reperfusion-induced liver damage. And to prove this, the inventors of the present disclosure constructed a hepatocyte-specific PAK4 knockout (KO) mouse, synthesized a PAK4-selective inhibitor, and conducted an experiment. Therefore, the inventors of the present disclosure found that PAK4 expression is increased in hepatocytes by ischemia-reperfusion to induce phosphorylation at T369 of Nrf2, and phosphorylated Nrf2 is transported from the nucleus to the cytoplasm, proteolysis occurs through ubiquitination and proteasome, and in the nucleus It was confirmed that the expression of antioxidant enzymes regulated by Nrf2 was reduced by the reduction of Nrf2. In addition, the inventors of the present disclosure completed the present disclosure by confirming the inhibitory effect of PAK4 inhibitors on liver damage as a result of studying agents for inhibiting liver damage caused by ischemia-reperfusion.

Accordingly, in one aspect, the present disclosure provides a pharmaceutical composition for preventing or treating tissue or cell damage caused by ischemia-reperfusion or hypoxia-reoxygenation, comprising a PAK4 activity or expression inhibitor as an active ingredient.

In one aspect of the present disclosure, the PAK4 activity or expression inhibitor may be selected from compounds, peptides, peptidomimetics, antibodies, aptamers, or protein kinase A inhibitors that specifically bind to PAK4. can

An antibody capable of specifically binding to and inhibiting the activity of PAK4 protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies to the PAK4 protein can be prepared by methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U.S. Patent No. 4,816 , 56) or by the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). It can be. General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991, which are incorporated herein by reference. Antibodies against the PAK4 protein may be purchased and used commercially. The antibody can specifically and directly bind to the PAK4 protein to effectively inhibit the activity of the PAK4 protein. In the present invention, a peptide capable of specifically binding to and inhibiting the activity of PAK4 protein can be obtained by a conventional method known in the art, for example, a phage display method (Smith GP, "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface". Science 228 (4705):13151317(1985); Smith GP, Petrenko VA, "Phage display". Chem. Rev. 97(2):391410(1997)).

In the present disclosure, compounds capable of specifically binding to and inhibiting the activity of PAK4 protein are preferably PAK4 activity specific inhibitors. For example, a compound of Formula 1 provided in the present disclosure may be used, and is a known PAK4 activity inhibitory compound, PF-3758309 [(S)-N-(2-(dimethylamino)-1-phenylethyl)- 6,6-dimethyl-3-((2-methylthieno[3,2-d]pyrimidin-4-yl)amino)-4,6-dihydropyrrolo[3,4-c]pyrazole- 5(1H)-carboxamide)], or LCH-7749944 [N2-(3-methoxyphenyl)-N4-((tetrahydrofuran-2-yl)methyl) quinazoline-2,4-diamine] (Cancer Lett. 2012 Apr 1;317(1);24-32), but is not limited thereto.

In the present disclosure, peptide mimetics that specifically bind to the PAK4 protein inhibit the activity of the protein by binding to a specific domain of the PAK4 protein. Peptidomimetics can be peptides or non-peptides, with amino acids bound by non-peptide linkages, such as psi linkages (Benkirane, N., et al. J. Biol. Chem., 271:33218-33224, 1996). can be configured. Also included are "conformationally constrained" peptides, cyclic mimetics, cyclic mimetics comprising at least one exocyclic domain, a binding moiety (binding amino acid) and an active site. can be chained Peptide mimics are structured similarly to the secondary structural characteristics of UBAP2 (Ubiquitin-Associated Protein 2) protein and can be used as antibodies (Park, B. W. et al. Nat Biotechnol 18, 194-198, 2000) or water-soluble receptors (Takasaki, W. et al. Nat Biotechnol 15; , 1261-1265, 1997).

In the present disclosure, an aptamer that specifically binds to the PAK4 protein is a single-stranded DNA or RNA molecule, and is a specific chemical molecule by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). It can be obtained by isolating oligomers that bind to molecules or biological molecules with high affinity and selectivity (C. Tuerand L. Gold, Science 249, 505 - 510, 2005; A. D. Ellington and J. W. Szostak, Nature 346, 818 - 822 , 1990; M. Famulok, et. al., Acc. Chem. Res. 33, 591 - 599, 2000; D. S. Wilson and Szostak, Annu. Rev. Biochem. 68, 611 - 647, 1999). An aptamer can specifically bind to a target and modulate the activity of the target, such as blocking the function of the target through binding.

In the present disclosure, the PAK protein activity inhibitor is preferably a protein kinase A inhibitor, more preferably the H89 compound N-[2-[[3-(4-bromophenyl)-2-pro phenyl]amino]ethyl]-5-isoquinolinesulfonamide. The H89 compound competitively binds to the ATP binding site of the catalytic domain of PAK and inhibits the activity of PAK.

In one aspect of the present disclosure, the PAK4 activity or expression inhibitor may be an antisense nucleotide, siRNA, shRNA, or ribozyme that complementarily binds to the mRNA of the PAK4 gene.

As used herein, the term "antisense nucleotide" refers to DNA or RNA or derivatives thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and binds to a complementary sequence in mRNA to inhibit translation of mRNA into protein. It works. Antisense sequence refers to a DNA or RNA sequence that is complementary to and capable of binding to PAK4 mRNA and is essential for translation, translocation, maturation, or all other overall biological functions of PAK4 mRNA. activity may be impaired. The length of the antisense nucleotide is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases. The antisense nucleotide may be modified at one or more bases, sugars or backbone positions to enhance its activity (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55 (1995 )). The backbone of antisense nucleic acids can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomic, heterocyclic intersugar linkages, and the like. In addition, antisense nucleic acids may contain one or more substituted sugar moieties. Antisense nucleic acids may contain modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-mepyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 2,6-diaminopurine etc. Antisense nucleic acids of the present disclosure may also be chemically linked with one or more moieties or conjugates that enhance the antisense nucleic acid's activity and cell adhesion. Oligonucleotides comprising various moieties and methods for their preparation are known in the art of this disclosure (U.S. Pat. Nos. 5,138,045, 5,218,105, and 5,459,255). The modified nucleic acid may increase stability to nucleases and increase binding affinity between the antisense nucleic acid and the target mRNA. The design of antisense oligonucleotides that can be used in the present disclosure can be readily prepared according to methods known in the art (Weiss, B. (ed.): Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents, CRC Press , Boca Raton, FL, 1997; Weiss, B., et al., Antisense RNA gene therapy for studying and modulating biological processes.Cell. Mol. Life Sci., 55:334-358 (1999).

In the present disclosure, the PAK4 expression inhibitor may be shRNA or siRNA comprising a sequence complementary to the nucleotide sequence of the PAK4 gene. As used herein, the term "small hairpin RNA or short hairpin RNA" (shRNA) refers to a sequence of RNA that makes a robust hairpin turn, which can be used to silence gene expression through RNA interference. For shRNA, a vector for cell introduction is used, and a U6 promoter capable of expressing shRNA is mainly used. These vectors are always passed on to the daughter cells so that gene silencing can be inherited. The shRNA hairpin structure is degraded into siRNA, which is an intracellular mechanism, and is bound to the RNA-induced silencing complex. The complex described above binds to and degrades mRNA matched to the siRNA bound thereto. shRNAs are transcribed by RNA polymerase III, and shRNA production in mammalian cells may trigger an interferon response, as cells recognize shRNAs as viral attack and seek defense.

As used herein, the term "small interference RNA (siRNA)" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (WO 00/44895, WO 01/36646, WO 99/32619 , WO 01/29058, WO 99/07409 and WO 00/44914). Since siRNA can suppress the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method. siRNA was first discovered in plants, worms, fruit flies and parasites, but has recently been developed/used and applied to mammalian cell studies (Degot S, et al. 2002; Degot S, et al. 2004; Ballut L, et al. 2005). [0029] The siRNA molecule of the present invention may have a double-stranded structure in which the sense strand and the antisense strand are positioned opposite to each other. In addition, the siRNA molecules of the present invention may have a single-stranded structure having self-complementary sense and antisense strands.

As used herein, the term complementary means not only 100% complementarity but also incomplete complementarity, preferably 90% complementarity, more preferably 98% complementarity, and most preferably 100% complementarity. do. Where 100% complementarity is expressed herein, it is specifically described as completely complementary. siRNA is not limited to complete pairing of double-stranded RNA parts that pair with each other, but is not paired by mismatch (corresponding bases are not complementary), bulge (no base corresponding to one chain), etc. parts may be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, and even more preferably 20 to 70 bases. The siRNA may be composed of a 15 to 30 mer sense sequence selected from the nucleotide sequence of the mRNA of a gene encoding the PAK4 protein and an antisense sequence complementary to the sense sequence. As for the siRNA end structure, either a blunt end or a cohesive end can be used as long as the expression of the PAK4 gene can be suppressed by the RNAi effect. The sticky end structure can be both a 3' end protruding structure and a 5' end protruding structure. The siRNA molecule of the present disclosure may have a form in which a short nucleotide sequence (eg, about 5-15 nt) is inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by expression of the nucleotide sequence is A hairpin structure is formed by intramolecular hybridization, and a stem-and-loop structure is formed as a whole. This stem-and-loop structure can be processed in vitro or in animals to generate active siRNA molecules capable of mediating RNAi.

An RNA interference (RNAi) molecule that specifically binds to the PAK4 gene of the present disclosure can be inserted into a gene carrier. The gene carrier is a plasmid, recombinant adenovirus (Thimmappaya, B. et al., Cell, 31:543-551 (1982); and Riordan, J. R. et al., Science, 245:1066-1073 (1989) ), Adeno-associated viruses (AAV) (LaFace et al, Viology, 162:483486 (1988), Zhou et al., Exp. Hematol. (NY), 21:928-933 (1993), Walsh et al, J. Clin. Invest, 94:1440-1448 (1994) and Flotte et al. Gene Therapy, 2:29-37 (1995)), retroviruses (Mann et al., Cell, 33:153- 159 (1983)), lentivirus (Wang G. et al., J. Clin. Invest. 104(11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc Natl.

In one aspect of the present disclosure, the pharmaceutical composition may be administered before, concurrently with, or after ischemia-reperfusion or hypoxia-reoxygenation.

In one aspect of the present disclosure, the pharmaceutical composition may inhibit the ubiquitination of Nrf2 in the cytoplasm and the proteolysis of Nrf2 through the proteasome.

In one aspect of the present disclosure, the pharmaceutical composition inhibits tissue infiltration of inflammatory cells or inhibits the production of one or more cytokines selected from the group consisting of TNF-α, IL-1β, IL-6 and CCL-2. can do.

In one aspect of the present disclosure, the pharmaceutical composition can inhibit NF-κB transcriptional activity.

In one aspect of the present disclosure, the pharmaceutical composition can inhibit generation of reactive oxygen species in tissues or cells or reduce blood reactive oxygen species markers.

In one aspect of the present disclosure, the pharmaceutical composition may inhibit apoptosis or necrosis of cells.

In one aspect of the present disclosure, the ischemia-reperfusion or hypoxia-reoxygenation-related disease consists of liver damage, brain damage, lung damage, kidney damage, myocardial damage, and skeletal muscle damage due to ischemia-reperfusion or hypoxia-reoxygenation. It may be selected from the group, but is not limited thereto.

The pharmaceutical composition of the present disclosure includes at least one active ingredient exhibiting the same or similar efficacy in addition to the compound represented by Formula 1, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof. can include more.

The pharmaceutical composition of the present disclosure can be used for clinical administration and can be formulated to be administered in various oral and parenteral dosage forms.

In addition, according to one embodiment of the present disclosure, a therapeutically effective amount of the compound represented by Formula 1, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof ( It provides a method for treating or preventing a PAK4-associated disease, comprising the step of administering to a subject). The subject may be a mammal including a human.

The term "therapeutically effective amount" used in the present disclosure refers to an amount of the compound represented by Formula 1 effective for the treatment or prevention of PAK4-related diseases. Specifically, "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, type of disease, It may be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition of the present disclosure may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a commercially available therapeutic agent. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present disclosure may be determined by an expert according to various factors such as the patient's condition, age, sex, and complications. The dosage of the pharmaceutical composition of the present disclosure may be, for example, 0.0001-100 mg/kg body weight per day.

In addition, according to one embodiment of the present disclosure, the present disclosure provides a compound represented by Formula 1, an optical isomer thereof, and a compound thereof for use in the preparation of a medicament for use in the treatment or prevention of PAK4-related diseases. A hydrate or solvate, or a pharmaceutically acceptable salt thereof, is provided. The compound represented by Formula 1 for the preparation of a drug may be mixed with an acceptable adjuvant, diluent, carrier, etc., and may be prepared as a combined preparation with other active agents to have a synergistic action of the active ingredients.

In the present disclosure, the administration method of the "pharmaceutical composition" is determined according to the degree of symptoms, and a topical administration method is generally recommended. In addition, the dosage of the active ingredient in the pharmaceutical composition may vary depending on conditions such as the severity of the disease, the age, sex, weight, and route of administration of the patient, and may be administered from once to several times a day. Appropriate carriers, excipients, and diluents commonly used in the preparation of the pharmaceutical composition may further be included.

In the present disclosure, vehicle refers to a compound that facilitates the addition of proteins or peptides into cells or tissues, for example, dimethyl sulfoxide (DMSO) is the input of many organic substances into cells or tissues of organisms. It is a commonly used carrier that facilitates.

A “diluent” is defined in this disclosure as a compound that is diluted in water which will dissolve the protein or peptide as well as stabilize the biologically active form of the protein or peptide of interest. A salt dissolved in a buffer is used as a diluent in the art. A commonly used buffer is phosphate buffered saline (PBS), as it mimics the salt state of human solutions. Because buffers can control the pH of a solution at low concentrations, buffering diluents rarely modify the biological activity of a compound. Compounds containing azelaic acid as used herein may be administered to human patients by themselves or as pharmaceutical compositions mixed with other ingredients, as in combination therapy, or with suitable carriers or excipients.

In addition, the PAK4 inhibitor composition may be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injection solutions according to conventional methods, respectively, and may be included in the composition. Examples of carriers, excipients and diluents include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, decomposers, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid formulations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous agents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition of the present disclosure may be administered orally or parenterally, and in the case of parenteral administration, intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, and the like. The appropriate dosage of the pharmaceutical composition of the present disclosure varies depending on factors such as formulation method, administration mode, patient's age, weight, sex, medical condition, food, administration time, route of administration, excretion rate and response sensitivity. may be prescribed. The pharmaceutical composition of the present disclosure is prepared in unit dosage form by formulating using pharmaceutically acceptable carriers and excipients according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by putting it in a large-capacity container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

In one aspect, the present disclosure provides ischemia-reperfusion-induced ischemia-reperfusion or hypoxia-reperfusion-induced hepatic injury model animals or hypoxia-reperfusion-damaged hepatocytes, including the step of administering a candidate substance or a candidate gene through adenovirus to hepatocytes damaged by hypoxia-reperfusion. A screening method for a drug that inhibits tissue or cell damage caused by reoxygenation is provided.

Matters mentioned in the uses, compositions, and treatment methods of the present disclosure apply equally unless contradictory to each other.

Since the compound represented by Formula 1 according to the present disclosure inhibits the enzymatic activity of PAK4, it can be used for preventing and treating diseases caused by excessive activity of PAK4.

In addition, the present disclosure newly identified the role and mechanism of PAK4 in tissue or cell damage caused by ischemia-reperfusion or hypoxia-reoxygenation. Accordingly, PAK4 inhibitors can be used for prevention or treatment of tissue or cell damage caused by ischemia-reperfusion or hypoxia-reoxygenation.

1 is a liver injury model (A) by ischemia-reperfusion (I/R) in mice and hypoxia-reoxygenation (H/R) of hepatocytes in primary hepatocytes A schematic diagram showing the fabrication of the damage model (B) and the experimental protocol using it. A. Mice were subjected to partial ischemia for 1 hour followed by reperfusion. For overexpression of adenoviral PAK4, mice were intravenously injected with Ad-LacZ, Ad-PAK4, or Ad-PAK4 ^S474A 2 days prior to ischemic injury. B. The primary cultured stem cells were cultured for 1 hour in an anoxic culture tank, and oxygen was then supplied for 12 hours. For PSK4 overexpression using adenovirus, primary cultured hepatocytes were infected with Ad-LacZ, Ad-PAK4, or Ad-PAK4 ^S474A in mice 12 hours before hypoxic injury.

Figure 2 shows the expression of PAK4 protein (A) and mRNA expression (B) in liver tissue after ischemia-reperfusion in mice, and immunohistochemical staining for PAK4 protein 6 hours or 24 hours after ischemia-reperfusion (C) and immunofluorescence staining (D) results are shown. E and F show the expression of PAK4 protein (E) and mRNA (F) in the primary cultured hepatocyte injury model caused by hypoxia-reoxygenation. ** indicates p<0.01 for time 0. HNF4: hepatocyte nuclear factor 4

Figure 3 shows the effect on various indicators when hepatocyte-specific PAK4 KO mice are induced liver damage by ischemia-reperfusion. A: PAK4 levels in the livers of wild-type mice and PAK4 KO mice, B and C: micrographs and necrotic areas of liver cuts. D: Blood levels of AST and AKT, which are markers of liver cell damage, E: CD68 ⁺ (macrophages), F4/80 ⁺ (macrophages), Ly6G ⁺ (neutrophils) and TUNEL ⁺ (apoptotic) cells in liver tissue Photographs of immunofluorescence staining results and graphs quantifying them, F and G: protein and mRNA levels of pro-inflammatory cytokines/chemokines in blood and liver tissue. **, p<0.01.

Figure 4 shows the effect on various indicators when liver damage by ischemia-reperfusion is induced in mice overexpressing PAK using adenovirus. A: Expression of PAK4 in liver tissue when mice were injected with PAK4 adenovirus (Ad-PAK4) and PAK4 mutant adenovirus (Ad-PAK4 ^S474A ) with phosphorylation removed,

B and C: Micrographs and necrotic area of liver cuts (**, p<0.01). D: Blood levels of AST and AKT, which are markers of hepatocellular damage (**, p<0.01 against sham, ##, p<0.01 against Ad-LacZ, $, p<0.05 against Ad-PAK4, $$, p<0.01 for Ad-PAK4), E: Immunofluorescence of CD68 ⁺ (macrophages), F4/80 ⁺ (macrophages), Ly6G ⁺ (neutrophils neutrophils) and TUNEL ⁺ (apoptotic) cells in liver tissue Photographs of staining results and graphs quantifying them (**, p<0.01), F and G: Protein and mRNA levels of pro-inflammatory cytokines/chemokines in blood and liver tissue (**, p<0.01) .

Figure 5 shows the effects on various indicators when liver damage is induced by ischemia-reperfusion in PAK4 KO mice. A: RNA sequencing analysis results of liver tissue, B: Gene set enrichment analysis (GSEA) results, C: GeneMANIA analysis results, D: 4-HNE staining results, E: blood levels of GSH and MDA. F and G: Results of 4-HNE staining and blood levels of GSH and MDA in liver tissue after ischemia-reperfusion with PAK4 overexpression. **, p<0.01

Figure 6 shows the experimental results showing the effect of PAK4 on the regulation of stability and transcriptional activity of Nrf2. A: Results of ARE-luciferase assay after overexpressing Nrf2 and PAK4 in HEK293T cells, B: Nrf2 and cage in total protein extract (total), cytoplasmic extract (CE) and nuclear extract (NE) of ischemia-reperfusion-damaged liver tissue Level of gene, C: results of Nrf2 immunostaining of primary cultured hepatocytes after hypoxia-reoxygenation challenge, D: primary cultured hepatocytes isolated from wild-type and PAK4 KO mice and primary cultured hepatocytes overexpressing PAK4 after hypoxia-reoxygenation ( 12 hours), Western blot analysis of Nrf2 protein in whole cell lysates (Total), nuclear extracts (NE) and cytoplasmic extracts (CE) showed that E: In PAK4 overexpressing hepatocytes, Nrf2 and PAK4 were found in subcellular units. F: relative protein level of Nrf2 in primary cultured hepatocytes after treatment with cyclohexamid (CHX, 20 μg/ml, n=5), G: expression of Nrf2 in HEK293T cells in the presence of MG132 (2 μM) Analysis of protein levels of signal regulatory molecules for ubiquitination and degradation through proteasome, H:Keap 1 and Nrf2 regulation by Western blot.

7 is a result of observing that PAK4 directly phosphorylates Nrf2. A: Coimmunoprecipitation in HEK293 T cells, B: Radiographs and Coomassie Brilliant Blue staining results after incubation of full-length recombinant Nrf2 protein with [γ- ³² P] ATP in the presence or absence of PAK4. , C: Effect of synthetic oligopeptides on Nrf2 phosphorylation by PAK4. In vitro kinase assay performed with or without 10 μg of each oligopeptide, D: ARE luciferase activity in HEK293T cells, E: D coimmunoprecipitation in HEK293T cells, F: hypoxia-reoxygenation-induced impairment Subcellular localization of Nrf2 in hepatocytes, G: ubiquitination of Nrf2 (experiment similar to G in Fig. 6c), H: protein and mRNA levels of pro-inflammatory cytokines, I Nrf2 target genes and apoptosis-related pathways. **,p < 0.01

8 shows the indicators when wild-type and PAK4 KO mice were intravenously injected with Ad-shLacZ or Ad-shNrf2 and subjected to hepatic ischemia-reperfusion injury (A, C-H). Or scrambled siRNA (siCtrl) or siRNA against Nrf2dp (siNrf2) In primary cultured hepatocytes isolated from wild-type and PAK4 KO mice infected with hypoxia-reoxygenation damage (B, I, J) A: Liver Protein level of Nrf2 in tissues, B: protein level of Nrf2, C: serum AST and ALT levels (**, p < 0.01 versus WT-Ad-shLacZ; ##, p < 0.01 versus KO-Ad-shLacZ) , D: hepatocyte necrosis, apoptosis and lipid peroxidation in liver tissue (**, p < 0.01), E and F: apoptosis-related proteins and oxidative stress markers in liver tissue (**, for WT-Ad-shLacZ p <0.01;##, p < 0.01 for KO-Ad-shLacZ), G and H: levels of pro-inflammatory cytokines in blood and liver (**, p < 0.01), I: in primary cultured hepatocytes Apoptosis-related proteins and oxidative stress markers, J: levels of pro-inflammatory cytokines in primary cultured hepatocytes (**, p < 0.01).

9 shows experimental results evaluating the effect of ND201651, a novel PAK4 inhibitor provided through the present disclosure, on liver damaged by ischemia-reperfusion. A: Chemical structure of ND201651, B: Experimental protocol after treatment of ND20165 in C57B/6 mice, C: Liver necrosis, apoptosis and oxidative stress in liver tissue damaged by ischemia-reperfusion, D: AST in serum and ALT level, E: level of MDA and GSH in liver, F: cytokine level in serum, G: mRNA expression of cytokine in liver, H level of Nrf2 in nucleus and cytoplasm and target protein of NrF2 in liver (**, p <0.01; *, p < 0.05).

10 schematically illustrates the mechanism of action of PAK4 and PAK4 inhibitors on ischemia-reperfusion-induced damage.

Hereinafter, the present disclosure will be described in detail by examples and experimental examples. The present disclosure may apply various transformations and may have various embodiments. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present disclosure to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present disclosure. In describing the present disclosure, if it is determined that a detailed description of related known technologies may obscure the gist of the present disclosure, the detailed description will be omitted.

I. Pyrrolopyrimidine derivative compounds

<Analysis and purification conditions>

Compounds synthesized in Examples of the present disclosure were purified or structurally analyzed by the following HPLC conditions.

1. HPLC conditions I

Analytical HPLC conditions (ACQUITY UPLC H-Class Core System)

A UPLC system (ACQUITY UPLC PDA Detector) manufactured by Waters, equipped with a mass QDA detector manufactured by Waters, was used. The column used is ACQUITY UPLC from Waters.BEH C18 (1.7 μm, 2.1 X 50 mm), and the column temperature was run at 30 °C.

Mobile phase A was water containing 0.1% formic acid, mobile phase B used acetonitrile containing 0.1% formic acid.

Gradient condition (10-100% B for 3 minutes, moving speed = 0.6 ml/min)

Prep-LCMS (Preparative-Liquid chromatography mass spectrometry)

Autopurification HPLC system manufactured by Waters (2767 sample manger, 2545 binary gradient module, 2998 Photodiode Array Detector) equipped with mass QDA detector manufactured by Waters was used. The column used is Waters' SunFire.Prep C18 OBD ^TM (5 μm, 19 X 50 mm) and the column temperature was carried out at room temperature.

Mobile phase A was water containing 0.035% trifluoroacetic acid, and mobile phase B was methanol containing 0.035% trifluoroacetic acid.

Gradient condition (10 minutes at 15-100% B, moving speed = 25 ml/min)

Prep-150 LC System for Preparative-Liquid chromatography UV spectrometry

A Prep 150 LC system (2545 Quaternary gradient module, 2998 Photodiode Array Detector, Fraction collector III) equipment manufactured by Waters was used. The column used is Waters' XTERRAPrep RP18 OBD ^TM (10 μm, 30 X 300 mm) and the column temperature was carried out at room temperature.

Mobile phase A was water containing 0.035% trifluoroacetic acid, and mobile phase B was methanol containing 0.035% trifluoroacetic acid.

Gradient condition (120 min at 3-100% B, moving speed = 40 ml/min)

Preparative HPLC System for purification (Preparative-Liquid chromatography UV spectrometry)

ACCQPrep HP150 equipment manufactured by Teledyne was used. The column used is Waters' XTERRAPrep RP18 OBD ^TM (10 μm, 30 X 300 mm) and the column temperature was carried out at room temperature.

Mobile phase A was water containing 0.035% trifluoroacetic acid, and mobile phase B was methanol containing 0.035% trifluoroacetic acid.

Gradient condition (10-100% B for 120 minutes, moving speed = 42 ml/min)

2. NMR analysis

NMR analysis was performed using an AVANCE III 400 or AVANCE III 400 HD manufactured by Bruker, and data were expressed in ppm (parts per milion (δ)).

Commercial reagents used were used without further purification. In the present disclosure, room temperature or normal temperature refers to a temperature of about 5 ° C to 40 ° C, one example, 10 ° C to 30 ° C, and another example, 20 ° C to 27 ° C, and is not strictly limited within the above range. Concentration under reduced pressure or solvent distillation was performed using a rotary evaporator.

<Preparation Example 1> Preparation of ethyl 4-amino-1,3-dimethyl-1H-pyrazole-5-carboxylate

Step 1: Preparation of ethyl 1,3-dimethyl-4-nitro-1H-pyrazole-5-carboxylate

Nitric acid (60%, 4 mL, 3.34 eq) and sulfuric acid (13 mL, 14.14 eq) were mixed at 0 °C.

After stirring the mixture to room temperature, ethyl 1,3-dimethyl-1H-pyrazole-5-carboxylate (2.9 g, 1 eq) was added. The reaction mixture was then stirred at room temperature for 6 hours. As a result of LCMS analysis, all of the starting materials disappeared and the target compound was detected. The reaction mixture was poured into ice water, and the precipitated solid was filtered and dried to obtain the desired compound (2 g, 54% yield).

MS m/z: 214.1 [M+1].

Step 2: Preparation of ethyl 4-amino-1,3-dimethyl-1H-pyrazole-5-carboxylate

After dissolving ethyl 1,3-dimethyl-4-nitro-1H-pyrazole-5-carboxylate (2 g, 1 eq) obtained in step 1 in ethanol (50 mL), palladium/carbon (Pd/ C; 10%, 0.2 g, 0.02 eq) was added. The reaction mixture was stirred at room temperature under hydrogen gas for 16 hours. As a result of LCMS analysis, all of the starting materials disappeared and the target compound was detected. The reaction mixture was filtered through diatomaceous earth. The filtrate was evaporated to dryness under reduced pressure to obtain the desired compound (1.7 g, 100% yield).

MS m/z: 184.2 [M+1].

<Example 1> 3 ^{1,3 3} -dimethyl-1 ^5- (trifluoromethyl)-1 ⁷ H,3 ¹ H-2,5,9-triaza-1(2,4)-pyrrolo Preparation of [2,3-d]pyrimidina-3(4,5)-pyrazolacyclononapan-4-one

Step 1: tert-butyl (3-((2-chloro-5-(trifluoromethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- Preparation of d]pyrimidin-4-yl)amino)propyl)carbamate

Starting material 2,4-dichloro-5-(trifluoromethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine ( After dissolving 1.2 g, 1 eq) in acetonitrile (ACN; 10 mL), triethylamine (TEA; 0.65 mL, 1.5 eq) was added. Thereafter, tert-butyl (3-aminopropyl) carbamate (0.541 g, 1 eq) was added to the reaction mixture and stirred at room temperature for 16 hours. As a result of LCMS analysis, all of the starting materials disappeared and the target compound was detected. The organic layer was extracted using ethyl acetate (EA; 200mL) and water (100mL), and the organic layer was washed several times with saturated brine (100ml * 2). Thereafter, the organic layer was dried over magnesium sulfate and then concentrated under reduced pressure. The concentrated compound was purified by chromatography to obtain the target compound (1.6 g, 98% yield).

MS m/z: 524.3 [M+1].

Step 2: Ethyl 4-((4-((3-((tert-butoxycarbonyl)amino)propyl)amino)-5-(trifluoromethyl)-7-((2-(trimethylsilyl) Preparation of ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1,3-dimethyl-1H-pyrazole-5-carboxylate

tert-butyl (3-((2-chloro-5-(trifluoromethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2, After dissolving 3-d] pyrimidin-4-yl) amino) propyl) carbamate (0.542 g, 1 eq) in sec-butanol (8 mL), ethyl 4-amino-1 obtained in <Preparation Example 1> ,3-Dimethyl-1H-pyrazole-5-carboxylate (0.227 g, 1.2 eq) and potassium carbonate (0.715 g, 5 eq) were added. After degassing the reaction mixture, it was reacted at 60 °C for 5 minutes. Tris(dibenzylideneacetone)dipalladium(0) (0.095 g, 0.1 eq) and dicyclohexyl(2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl ) Phosphane (Xphos; 0.049 g, 0.1 eq) was added. Then, the reaction mixture was stirred at 100° C. for 1 hour under a nitrogen atmosphere. As a result of LCMS analysis, all of the starting materials disappeared and the target compound was detected. The reaction mixture was filtered through diatomaceous earth. The filtrate was evaporated to dryness under reduced pressure and purified by chromatography (30-40% ethyl acetate in hexane) to give the desired compound (367 mg, 68%).

MS m/z: 671.4 [M+1].

Step 3: 4-((4-((3-((tert-butoxycarbonyl)amino)propyl)amino)-5-(trifluoromethyl)-7-((2-(trimethylsilyl) Preparation of Toxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1,3-dimethyl-1H-pyrazole-5-carboxylic acid

Ethyl 4-((4-((3-((tert-butoxycarbonyl)amino)propyl)amino)-5-(trifluoromethyl)-7-((2-(trimethyl) obtained in step 2 above silyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1,3-dimethyl-1H-pyrazole-5-carboxylate (367 mg, 1 eq) was dissolved in methanol (8 mL), and sodium hydroxide (6M; 1.824 mL, 20 eq) was added. The reaction mixture was stirred at room temperature for 2 hours. As a result of LCMS analysis, all of the starting materials disappeared and the target compound was detected. The organic layer was extracted using ethyl acetate (EA; 200mL), water (100mL), and hydrochloric acid (3N; 100mL), dried over magnesium sulfate, and then concentrated under reduced pressure to obtain the target compound (150 mg, 42%).

MS m/z: 643.4 [M+1].

Step 4: 4-((4-((3-aminopropyl)amino)-5-(trifluoromethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[ Preparation of 2,3-d] pyrimidin-2-yl) amino) -1,3-dimethyl-1H-pyrazole-5-carboxylic acid

* 4-((4-((3-((tert-butoxycarbonyl)amino)propyl)amino)-5-(trifluoromethyl)-7-((2-(trimethyl) obtained in step 3 above silyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-1,3-dimethyl-1H-pyrazole-5-carboxylic acid (150 mg, 1 eq ) was dissolved in methanol (5 mL), and hydrochloric acid/dioxane (4N, 0.46 mL, 8 eq) was added. The reaction mixture was then stirred at room temperature for 3 hours. As a result of LCMS analysis, all starting materials disappeared, and the reaction mixture was concentrated under reduced pressure to obtain the target compound (76 mg, 60%).

MS m/z: 543.3 [M+1].

Step 5: 3 ^One ,3 ³ -Dimethyl-1 ⁵ -(trifluoromethyl)-1 ⁷ -((2-(trimethylsilyl)ethoxy)methyl)-1 ⁷ H, 3 ^One H -2,5,9-triaza-1(2,4)-pyrrolo[2,3- d Preparation of ]pyrimidina-3(4,5)-pyrazolacyclononapan-4-one

4-((4-((3-aminopropyl)amino)-5-(trifluoromethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-p obtained in step 4 above Rollo[2,3- d ]pyrimidin-2-yl)amino)-1,3-dimethyl-1H-pyrazole-5-carboxylic acid (76 mg, 1 eq) was mixed with N,N-dimethylformamide ( 10 mL) and tetrahydrofuran (10 mL), and diisopropylethylamine (0.249 mL, 10 eq) was added. The reaction mixture was stirred at room temperature for 5 minutes, then 1-[bis(dimethylamino)methylene] -1H -1,2,3-triazolo[4,5- b ]pyridinium 3-oxide hexafluoro Phosphate (HATU; 75 mg, 1.4 eq) was added and stirred at room temperature for 1 hour. As a result of LCMS analysis, all of the starting materials disappeared and the target compound was detected. The organic layer was extracted using ethyl acetate (EA; 200mL) and water (100mL), and the organic layer was washed with saturated sodium chloride (100ml * 2). Thereafter, the organic layer was dried over magnesium sulfate and then concentrated under reduced pressure. The concentrated compound was purified by chromatography to obtain the target compound (50 mg, 68%).

MS m/z: 525.3 [M+1].

Step 6: 3 ¹ ,3 ³ -dimethyl-1 ⁵ -(trifluoromethyl)-1 ⁷ H ,3 ¹ H -2,5,9-triaza-1(2,4)-pyrrolo[2 Preparation of ,3- d ]pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one

3 ^{1,3 3} -dimethyl-1 ⁵ -(trifluoromethyl)-1 ⁷ -((2-(trimethylsilyl)ethoxy)methyl)-1 ⁷ H ,3 ¹ H - obtained in step 5 above 2,5,9-triaza-1 (2,4)-pyrrolo [2,3- d ] pyrimidina-3 (4,5) -pyrazolacyclononapan-4-one (50 mg, 1 eq) was dissolved in dichloromethane (2 mL), and then trifluoroacetic acid (1 mL) was added. The reaction mixture was stirred at room temperature for 1 hour. LCMS analysis showed that all of the starting materials had disappeared and the reaction mixture was concentrated under reduced pressure. After dissolving the concentrated compound in 1,4-dioxane (2 mL), ammonium hydroxide (2 mL) was added. The reaction mixture was then stirred at room temperature for 1 hour. As a result of LCMS analysis, the target compound was detected and the reaction mixture was concentrated under reduced pressure. The concentrated compound was purified by chromatography to obtain the target compound (18 mg, 48%).

MS m/z: 395.3 [M+1].

^1H NMR (400 MHz, DMSO-d6) δ = 11.99 (s, 1H), 8.55 (s, 1H), 8.24 (t, J = 4.8 Hz, 1H), 7.51 (s, 1H), 6.06 (s, 1H), 4.13 (s, 1H), 3.78 (s, 3H), 3.52 (s, 1H), 3.10 (s, 1H), 2.85 (s, 2H), 2.13 (s, 3H), 1.80 (d, J = 19.7 Hz, 2H)

<Examples 2 to 30>

The compounds of Examples 2 to 30 were prepared in a similar manner to Example 1.

The compound name, chemical structure, NMR and LCMS analysis results of each Example compound are summarized in [Table 1].

Example compound	structure		compound name		1 H NMR; MS[M+H] +
One			3 1 ,3 3 -Dimethyl-1 5 -(trifluoromethyl)-1 7 H,3 1 H-2,5,9-triaza-1(2,4)-pyrrolo[2,3-d] Pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ=11.99 (s, 1H), 8.55 (s, 1H), 8.24 (t, J = 4.8 Hz, 1H), 7 .51 (s, 1H), 6.06 ( s, 1H), 4.13 (s, 1H), 3.78 (s, 3H), 3.52 (s, 1H), 3.10 (s, 1H), 2.85 (s, 2H), 2.1 3 (s, 3H), 1.80 ( d, J = 19.7 Hz, 2H); 395.3[M+H] +
2			3 1 , 3 3 -Dimethyl-1 5- (trifluoromethyl)-1 7 H,3 1 H-2,5,10-triaza-1(2,4)-pyrrolo[2,3-d]pyryl midina-3(4,5)-pyrazolacyclodecapan-4-one		1H NMR (400 MHz, DMSO-d6) δ = 12.23 (d, J = 20.5 Hz, 1H), 9.82 (s, 1H), 8.60 (d, J = 38.9 Hz, 1H), 7.59 (s, 1H) , 6.21 (s, 1H), 3.88 (s, 3H), 2.10 (d, J = 0.9 Hz, 3H), 1.70 (d, J = 7.9 H z, 4H), 1.41 (d, J = 6.8 Hz, 4H) ); 409.2 [M+H] +
3			3 1 -(2-methoxyethyl)-3 3 -methyl-1 5- (trifluoromethyl)-1 7 H,3 1 H-2,5,9-triaza-1(2,4)-pyrrolo [2,3-d]pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one		1 H NMR (400 MHz, DMSO-d6) δ = 11.90 (s, 1H), 8.41 (s, 1H), 8.01 (s, 1H), 7.48 (s, 1H) , 6.00 (s, 1H), 4.25 ( s, 2H), 4.17 (s, 2H), 4.06 (s, 1H), 3.59 (d, J = 12.4 Hz, 3H), 3.45 (s, 1H), 3.06 (s, 1H), 2.83 (s , 1H), 2.10 (s, 3H), 1.80 (s, 1H), 1.68 (d, J = 6.7 Hz, 1H); 439.2 [M+H] +
4			3 1 ,3 3 -Dimethyl-1 5 -(trifluoromethyl)-1 7 H,3 1 H-5-oxa-2,9-diaza-1(2,4)-pyrrolo[2, 3-d] pyrimidina-3 (4,5) -pyrazolacyclononaphan -4-one		1 H NMR (400 MHz, DMSO-d6) δ = 11.87 (s, 1H), 8.63 (s, 1H), 7.46 (s, 1H), 5.94 (s, 1H), 4.54 (t, J = 10.8 Hz, 1H), 4.00 (s, 2H), 3.77 (d, J = 1.5 Hz, 3H), 3.07 (d, J = 12.9 Hz, 1H), 2.09 (d, J = 1.5 Hz, 3H), 1.85 (s, 2H); 396.2 [M+H] +
5			3 3 -Methyl-3 1- (1-methylpiperidin-4-yl)-1 5- (trifluoromethyl)-1 7 H,3 1 H-2,5,9-triaza-1( 2,4)-pyrrolo[2,3-d]pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ = 11.88 (s, 1H), 9.47 (s, 1H), 8.40 (s, 1H), 8.25 (t, J = 4.8 Hz, 1H), 7.49 (s, 1H), 5.90 (s, 1H), 4.45 (td, J = 11.5, 10.3, 4.9 Hz, 2H), 4.03 (d, J = 7.1 Hz, 2H), 3.66 - 3.34 (m, 4H), 3.18 (s , 2H), 2.88 - 2.81 (m, 1H), 2.79 (d, J = 4.3 Hz, 3H), 2.16 (s, 2H), 1.97 (d, J = 11.8 Hz, 2H), 1.82 (t, J = 19.1 Hz, 2H); 478.3 [M+H] +
6			3 3 -methyl-3 1 -(morpholine-4-carbonyl)-1 5 -(trifluoromethyl)- 1 7 H,3 1 H-2,5,9-triaza-1(2,4 )-pyrrolo[2,3-d]pyrimidina-3(4,5)-pyrazola cyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ=11.74 (d, J = 2.7 Hz, 1H), 8.29 (s, 1H), 8.03 - 7.82 (m, 1H), 7.48 (t, J = 2.1 Hz, 1H), 5.73 (s, 1H), 3.69 (t, J = 4.6 Hz, 8H), 3.08 (s, 2H), 2.85 - 2.65 (m, 2H), 2.20 (s, 3H), 1.76 ( s, 2H) ); 494.3 [M+H] +
7			3 1 -Methyl-1 5- (trifluoromethyl)-1 7 H,3 1 H- 2,5,9-triaza-1(2,4)-pyrrolo[2,3-d]pyrimidi Na-3(3,4)-pyrazolacyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ=11.80 (d, J = 2.9 Hz, 1H), 8.95 (d, J = 2.5 Hz, 1H), 7.6 5 - 7.57 (m, 1H), 7.52 (d , J = 2.5 Hz, 1H), 7.36 (d, J = 2.3 Hz, 1H), 5.61 (d, J = 7.2 Hz, 1H), 3.70 (d, J = 2.3 Hz, 3H), 2.98 (s, 2H) ), 2.89 (s, 1H), 2.71 (s, 1H), 1.76 (s, 2H); 381.2 [M+H] +
8			3 4 -((2-(dimethylamino)ethyl)(methyl)amino)-1 5- (trifluoromethyl)-1 7 H-2,5,9-triaza-1(2,4)- Pyrrolo[2,3-d]pyrimidina-3(1,3)-benzenecyclononaphan-4-one		477.3 [M+H] +
9			3 5 -morpholino-1 5 -(trifluoromethyl)-1 7 H-2,5,9-triaza-1(2,4)-pyrrolo[2,3-d]pyrimidina -3(1,3)-benzenecyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ=11.88 (d, J = 2.7 Hz, 1H), 9.06 (s, 1H), 7.71 (s, 1H), 7 .64 (t, J = 7.3 Hz, 1H), 7.52 (t, J = 2.1 Hz, 1H), 6.71 (t, J = 2.1 Hz, 1H), 6.59 (d, J = 2.3 Hz, 1H), 6.24 (d, J = 6.1 Hz, 1H) , 3.75 (d, J = 9.8 Hz, 8H), 3.18 (q, J = 11.3, 8.8 Hz, 2H), 3.09 (t, J = 4.9 Hz, 2H), 1.94 (d, J = 22.6 Hz, 2H) ; 462.2 [M+H] +
10			3 4 -Morpholino-1 5- (trifluoromethyl)-1 7 H-2,5,9-triaza-1(2,4)-pyrrolo[2,3-d]pyrimidina- 3(1,3)-benzenecyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ=12.01 - 11.81 (m, 1H), 9.10 (s, 1H), 7.83 (q, J = 7.3 Hz, 1H), 7.57 (d, J = 2.5 Hz, 1H), 7.50 (t, J = 2.1 Hz, 1H), 7.15 - 7.09 (m, 1H), 7.04 (dd, J = 8.6, 2.5 Hz, 1H), 6.31 (s, 1H), 3.67 (t , J = 4.6 Hz, 4H), 3.08 (s, 2H), 3.01 (s, 4H), 2.96 - 2.80 (m, 2H), 1.98 - 1.82 (m, 2H); 462.0 [M+H] +
11			3 6 -Methoxy-3 4 -morpholino-1 5 -(trifluoromethyl)-1 7 H-2,4,10-triaza-1(2,4)-pyrrolo[2,3- d]pyrimidina-3(1,3)-benzenecyclodecapan-5-one		1H NMR (400 MHz, DMSO-d6) δ=12.38 (s, 1H), 8.77 (s, 1H), 8.45 (d, J = 24.1 Hz, 1H), 7.65 (s, 1H), 7.63 - 7.56 ( m, 1H), 6.71 (s, 2H), 3.89 (s, 3H), 3.75 - 3.67 (m, 4H), 3.40 (q, J = 7.2 Hz, 2H), 2.96 (t, J = 4.5 Hz, 3H) ), 2.86 (t, J = 4.5 Hz, 1H), 2.24 - 2.16 (m, 2H), 1.68 (s, 2H), 1.60 - 1.50 (m, 2H); 506.3 [M+H] +
12			3 4 -((2-methoxyethyl)amino)-1 5- (trifluoromethyl)-1 7 H-2,5,9-triaza-1(2,4)-pyrrolo[2,3 -d] pyrimidina-3(1,3)-benzenecyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ=12.27 (s, 1H), 9.51 (s, 1H), 7.99 - 7.82 (m, 2H), 7.56 (s, 2H), 7.06 (dd, J = 8.7 , 2.4 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.71 (s, 1H), 3.53 (t, J = 5.2 Hz, 2H), 3.29 - 3.24 (m, 3H), 3.21 (d , J = 7.9 Hz, 2H), 3.11 (q, J = 7.9 Hz, 2H), 2.85 (s, 2H), 2.04 - 1.92 (m, 2H); 450.3 [M+H] +
13			3 6 -methoxy-3 4- (4-morpholinopiperidin-1-yl)-1 5- (trifluoromethyl)-1 7 H-2,4,10-triaza-1(2, 4)-pyrrolo[2,3-d]pyrimidina-3(1,3)-benzenecyclodecapan-5-one		589.4 [M+H] +
14			1 5 -(trifluoromethyl)-1 7 H-2,5,10-triaza-1(2,4)-pyrrolo[2,3-d]pyrimidina-3(1,3)- Benzenecyclodecapan-4-one		391.3 [M+H] +
15			3 6,5 -dimethyl-3 4 -morpholino-1 5- (trifluoromethyl)-1 7 H-2,5,9-triaza-1(2,4)-pyrrolo[2, 3-d] pyrimidina-3(1,3)-benzenecyclononaphan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 8.25 (s, 1H), 7.43 (d, J = 1.8 Hz, 1H), 7.21 (s, 1H), 6.90 (s , 1H), 6.06 (t, J = 5.0 Hz, 1H), 3.62 (t, J = 4.6 Hz, 4H), 3.17 (d, J = 4.8 Hz, 1H), 3.15 - 3.04 (m, 4H), 2.98 - 2.87 (m, 5H), 2.77 (dq, J = 9.6, 4.6 Hz, 2H), 2.25 (s, 3H), 1.81 - 1.72 (m, 1H); 490.3 [M+H] +
16			3 6,4 -dimethyl-3 4 -morpholino-1 5- (trifluoromethyl)-1 7 H-2,4,10-triaza-1(2,4)-pyrrolo[2, 3-d] pyrimidina-3 (1,3) -benzenecyclodecapan-5-one		1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 8.08 (s, 1H), 7.45 (d, J = 1.8 Hz, 1H), 7.24 (s, 1H), 6.90 (s , 1H), 5.84 - 5.77 (m, 1H), 4.15 - 4.05 (m, 2H), 3.88 - 3.77 (m, 1H), 3.67 (ddt, J = 14.0, 10.8, 5.5 Hz, 4H), 3.09 (s , 3H), 2.99 - 2.91 (m, 2H), 2.88 - 2.82 (m, 1H), 2.79 - 2.73 (m, 2H), 2.26 (s, 3H), 1.98 - 1.88 (m, 1H), 1.87 - 1.80 (m, 1H), 1.47 - 1.41 (m, 1H), 1.35 - 1.29 (m, 1H); 504.4 [M+H] +
17			3 6 -methyl-3 4 -morpholino-1 5 -(trifluoromethyl)-1 7 H-2,5,9-triaza-1(2,4)-pyrrolo[2,3-d ]pyrimidina-3(1,3)-benzenecyclononaphan-4-one		1 H NMR (400 MHz, DMSO-d6) δ = 12.48 (s, 1H), 9.44 (s, 1H), 8.23 (s, 1H), 7.84 (s, 1H) , 7.45 (s, 1H), 7.19 ( s, 1H), 7.06 (s, 1H), 6.28 - 6.22 (m, 1H), 3.61 (d, J = 6.2 Hz, 5H), 3.34 (t, J = 6.3 Hz, 3H), 2.96 (s, 2H) ), 2.20 (s, 3H), 1.90 - 1.77 (m, 3H); 476.3 [M+H] +
18			3 6 -methoxy-3 4 -(4-(oxetan-3-yl)piperazin-1-yl)-1 5- (trifluoromethyl)-1 7 H-2, 4,10-triaza -1(2,4)-pyrrolo[2,3-d]pyrimidina-3(1,3)-benzenecyclodecapan-5-one		1H NMR (400 MHz, DMSO-d6) δ=12.19 (s, 1H), 8.78 (d, J = 2.9 Hz, 1H), 8.09 (s, 1H), 7 .71 (d, J = 14.3 Hz, 1H), 7.63 - 7.48 (m, 1H), 6.69 (d, J = 4.9 Hz, 1H), 6.43 (s, 1H), 4.83 - 4.72 (m, 4H), 3.89 (s, 3H), 3.43 - 3.34 (m, 4H), 3.14 (s, 4H), 3.09 (s, 2H), 2.25 (dd, J = 10.4, 5.1 Hz, 3 H), 1.68 (s, 2H), 1.55 (s, 2H); 561.4 [M+H] +
19			(3 3 S)-5 6- (4-methylpiperazin-1-yl)-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-p Rolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO) δ 11.95 (s, 1H), 8.68 (s, 1H), 8.20 (d, J = 2.4 Hz, 1H), 7.56 (d, J = 1.6 Hz, 1H), 7 09 (dd, J = 8.6, 2.4 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 5.20 (s, 1H), 4.60 (d, J = 9.5 Hz, 1H), 4.39 (d, J = 11.9 Hz, 1H), 3.76 (s, 1H), 3.24 - 3.12 (m, 2H), 2.73 - 2.62 (m, 3H), 2.58 (dd, J = 21.6, 9.5 Hz, 1H), 2.36 (s, 4H), 2.18 (s, 3H), 1.96 (s, 1H), 1.85 (d, J = 12.6 Hz, 2H), 1.71 (t, J = 13.0 Hz, 1H); 501.3 [M+H]+
20			(3 3 R)-5 6- (4-methylpiperazin-1-yl)-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-p Rolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.69 (s, 1H), 8.20 (d, J = 2.5 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H) , 7.09 (dd, J = 8.6, 2.5 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 5.20 (dd, J = 3.8, 1.7 Hz, 1H), 4.60 (d, J = 12.7 Hz, 1H), 4.40 (d, J = 11.8 Hz, 1H), 3.81 - 3.70 (m, 2H), 3.23 - 3.18 (m, 2H), 2.71 - 2.61 (m, 3H), 2.6 1 - 2.53 (m, 1H) ), 2.36 (s, 4H), 2.18 (s, 3H), 2.01 - 1.94 (m, 1H), 1.87 - 1.79 (m, 2H), 1.76 - 1.62 (m, 1H); 501.3 [M+H] +
21			(3 3 S)-5 6 -morpholino-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-pyrrolo[2,3-d] Pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.92 (br s, 1H), 8.72 (s, 1H), 8.24 (d, J = 2.5 Hz, 1H) , 7.57 (d, J = 1.8 Hz, 1H) , 7.12 (dd, J = 8.5, 2.4 Hz, 1H), 6.97 (d, J = 8.6 Hz, 1H), 5.21 (d, J = 3.7 Hz, 1H), 4.61 (d, J = 12.7 Hz, 1H), 4.42 (d, J = 12.3 Hz, 1H), 3.78 (d, J = 8.7 Hz, 1H), 3.62 (t, J = 4.7 Hz, 4H), 3.26 - 3.19 (m, 2H), 2.73 - 2.62 (m, 3H), 2.58 (dd, J = 12.4, 10.0 Hz, 1H), 2.00 - 1.92 (m, 1H), 1.87 - 1.80 (m, 2H), 1.78 - 1.63 (m, 1H); 488.3 [M+H] +
22			(3 3 R)-5 6 -morpholino-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-pyrrolo[2,3-d] Pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.92 (br s, 1H), 8.72 (s, 1H), 8.24 (d, J = 2.5 Hz, 1H) , 7.57 (d, J = 1.8 Hz, 1H) , 7.12 (dd, J = 8.5, 2.4 Hz, 1H), 6.97 (d, J = 8.6 Hz, 1H), 5.21 (d, J = 3.7 Hz, 1H), 4.61 (d, J = 12.7 Hz, 1H), 4.42 (d, J = 12.3 Hz, 1H), 3.78 (d, J = 8.7 Hz, 1H), 3.62 (t, J = 4.7 Hz, 4H), 3.22 (dt, J = 11.5, 4.6 Hz, 2H), 2.66 (dt, J = 11.8, 4.8 Hz, 3H), 2.58 (dd, J = 12.4, 10.0 Hz, 1H), 2.00 - 1.92 (m, 1H), 1.87 - 1.80 (m, 2H) , 1.78 - 1.63 (m, 1H); 488.3 [M+H] +
23			(3 3 S)-5 4 -methyl-5 6 -morpholino-1 5 -(trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-pyrrolo[ 2,3-d] pyrimidina-3 (3,1) - piperidina-5 (1,3) - benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.92 (br s, 1H), 8.24 (s, 1H), 8.11 (s, 1H), 7.56 (d, J = 1.7 Hz, 1H), 6.85 ( s, 1H), 5.11 (d, 1H), 4.59 (d, J = 12.4 Hz, 1H), 4.36 (d, J = 11.3 Hz, 1H), 3.73 - 3.66 (m, 1H), 3.6 2 (ddd, J = 5.5, 3.6, 1.5 Hz, 4H), 3.25 (dt, J = 11.9, 4.6 Hz, 2H), 2.70 - 2.60 (m, 3H), 2.55 (dd, J = 12.4, 10.1 Hz, 1H), 2.28 (s , 3H), 1.99 - 1.90 (m, 1H), 1.88 - 1.78 (m, 2H), 1.74 - 1.62 (m, 1H); 5 02.3 [M+H] +
24		(3 3 S)-5 4 -methoxy-5 6 -morpholino-1 5 -(trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-pyrrolo[ 2,3-d] pyrimidina-3 (3,1) -piperidina-5 (1,3 ) -benzenecyclohexapan-4-one		1 H NM R (400 MHz, DMSO-d6) δ 11.86 (s, 1H), 8.21 (s, 1H), 7.60 - 7.57 (m, 1H), 7.48 (s, 1H), 6.68 (s, 1H) , 5.25 (d, J = 2.1 Hz, 1H), 4.60 (d, J = 13.3 Hz, 1H), 4.36 (d, J = 11.8 Hz, 1H), 4.09 - 3.96 (m, 1H), 3.85 (s, 3H) ), 3.82 - 3.72 (m, 2H), 3.68 - 3.60 (m, 5H), 3.38 - 3.31 (m, 3H), 2.74 - 2.67 (m, 2H), 2.66 - 2.54 (m, 2H); 518.3 [M+H] +
25		(3 3 S)-5 6 -morpholino-1 5 -(trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-pyrrolo[2,3-d] Pyrimidina-3(3,1)-azefana-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 8.60 (s, 1H), 8.11 (d, J = 2.5 Hz, 1H), 7.57 - 7.54 (m, 1H), 7.08 (dd, J = 8.5, 2.4 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 4.85 - 4.81 (m, 1H), 4.22 (d, J = 13.6 Hz, 1H), 3.95 - ( m, 3H), 1.80 - 1.72 (m, 2H); 502.3 [M+H] +
26		(3 3 R)-5 6 -morpholino-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-pyrrolo[2,3-d] Pyrimidina-3(3,1)-azefana-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 8.61 (s, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.57 - 7.55 (m, 1H), 7.08 (dd, J = 8.5, 2.5 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 4.86 - 4.81 (m, 1H), 4.22 (d, J = 13.7 Hz, 1H), 3.95 - ( m, 3H), 1.80 - 1.72 (m, 2H); 502.3 [M+H] +
27		(3 3 S)-5 5 -morpholino-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4,2)-pyrrolo[2,3-d] Pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 8.78 (s, 1H), 8.21 (s, 1H), 7.59 (d, J = 1.9 Hz, 1H), 6.86 - 6.79 (m , 1H), 6.61 - 6.54 (m, 1H), 5.26 (d, J = 3.6 Hz, 1H), 4.83 - 4.72 (m, 1H) , 4.53 - 4.42 (m, 1H), 3.88 - 3.79 (m, 1H) ), 3.73 (t, J = 4.9 Hz, 4H), 3.10 - 3.00 (m, 4H), 2.72 - 2.63 (m, 1H), 2.57 - 2.51 (m, 1H), 2.07 - 1.98 (m, 1H) ), 1.92 - 1.83 (m, 2H), 1.78 - 1.64 (m, 1H); 488.3 [M+H] +
28		(3 3 S)-5 6- (2-(pyrrolidin-1-yl)ethoxy)-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4, 2)-pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 8.68 (s, 1H), 8.25 (d, J = 2.6 Hz, 1H), 7.59 - 7.54 (m, 1H), 7.10 (dd , J = 8.7, 2.6 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 5.22 - 5.18 (m, 1H), 4.56 (d, J = 13.0 Hz, 1H), 4.44 - 4.38 (m, 1H), 4.07 (dt, J = 9.7, 6.0 Hz, 2H), 3.97 (dt, J = 10.0, 6.1 Hz, 2H), 3.81 - 3.72 (m, 4H), 2.73 - 2.67 (m, 2H), 2.65 - 2.53 (m, 2H), 2.50 - 2.43 (m, 4H), 2.01 - 1.95 (m, 1H), 1.88 - 1.80 (m, 2H); 516.3 [M+H] +
29		(3 3 R)-5 4 -methyl-5 6- (4-methylpiperazin-1-yl)-1 5- (trifluoromethyl)-1 7 H-2,6-diaza-1(4 ,2)-pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one		1H NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 8.22 (s, 1H), 8.07 (s, 1H), 7.56 (q, J = 1.7 Hz, 1H), 6.8 3 (s, 1H), 5.12 - 5.08 (m, 1H), 4.63 - 4.55 (m, 2H), 4.34 (d, J = 12.7 Hz, 2H), 3.72 - 3.63 (m, 2H), 3.27 - 3.19 (m, 2H) , 2.71 - 2.62 (m, 3H), 2.55 (s, 3H), 2.37 - 2.34 (m, 3H), 2.26 (s, 3H), 1.98 - 1.90 (m, 1H), 1.78 - 1.59 (m, 2H); 515.2 [M+H] +
30		3 1 ,3 3 -Dimethyl-1 5 -(trifluoromethyl)-1 7 H,3 1 H-9-oxa-2,5-diaza-1(2,4)-pyrrolo[2, 3-d] pyrimidina-3 (4,5) -pyrazolacyclononaphan -4-one		1H NMR (400 MHz, DMSO-d6) δ = 11.84 (d, J = 2.7 Hz, 1H), 8.51 (s, 1H), 8.35 (t, J = 5.2 Hz, 1H), 7.54 (dt, J = 3.1, 1.7 Hz, 1 H), 3.78 (s, 3H), 3.57 (s, 4H), 2.13 (s, 3H), 1.99 - 1.85 (m, 2H); 396.2 [M+H] +

<Experimental Example 1> Evaluation of PAK4 enzyme inhibitory activity

In order to evaluate the inhibition of PAK4 kinase activity of the compounds according to the present disclosure, the following method was performed.

The example compounds were reacted with the purified human PAK4 (full-length, SignalChem) enzyme and the CDC42 enzyme, and the enzyme inhibitory ability was evaluated by the following method. The reaction buffer was used in the composition of 40 mM Tris-HCl pH 7.4, 20 mM MgCl ₂ , 0.5 mg/ml BSA, and 50 μM DTT, and all the test materials were reacted on the reaction buffer. The compound was diluted in 12 steps from a 10 mM DMSO stock by serial dilution method, and the enzyme activity was measured at concentrations of 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0003, 0.0001, and 0 μM of the final compound. .

In the test, after reacting human PAK4 (0.8 ng)/human CDC42 (1.6 ng) mixed enzyme with purified ATP (5 μM) and enzyme substrate (0.2 μg) at 25 ° C for 1 hour, enzyme activity was measured by in vitro ADP-GloTM It was confirmed using a kinase assay (Promega). Enzyme activation reaction solution, ADP-Glo reaction solution, and enzyme activity detection solution were reacted at a ratio of 2:2:1, and the degree of inhibition of enzyme activity was measured by luminescence. Based on the luminescence of the enzyme activity of the solvent control group that was not treated with the compound, the degree of enzyme activity inhibition according to the treatment concentration of each compound _was calculated. was determined and obtained using GraphPad Prism 8.3.0 (GraphPad software Inc., San Diego)). The results are shown in [Table 2] below.

Example compound	PAK4 enzyme activity	Example compound	PAK4 enzyme activity	Example compound	PAK4 enzyme activity
19	A	21	A	28	A
20	B	27	A

A: IC ₅₀ < 100 nM; B: 100 nM ≤ IC ₅₀ < 1,000 nM; C: 1,000 nM ≤ IC ₅₀ ;

II. Effect of PAK4 on tissue damage caused by ischemia-reperfusion/hypoxia-reoxygenation

The present disclosure identifies the role and mechanism of action of PAK4 in tissue damage caused by ischemia-reperfusion or hypoxia-reoxygenation, and based on this, provides new uses of PAK4 inhibitors.

Liver damage due to ischemia-reperfusion occurs in various morbid situations such as liver transplantation, hepatectomy, trauma, hypovolemic shock, and blood transfusion after excessive bleeding. During this process, the expression of PAK4 increases in hepatocytes, and the expression of Nrf2 in the nucleus decreases, resulting in a decrease in antioxidant capacity. Therefore, maintenance of Nrf2 expression and transcriptional activity in the nucleus by treatment with PAK4 inhibitors maintains antioxidant capacity in the liver and body, ultimately protecting the liver from ischemia-reperfusion damage.

The experimental protocol used in the examples below was as follows.

laboratory animals

Experimental animals were allowed free access to a standard diet and water in a controlled barrier facility (12-hour light/dark cycle, 23+1 ^o C, 60-70% humidity). All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised in 2011) and approved by the Laboratory Animal Ethics Committee of Chonbuk National University (approval number: JBNU -2019-00122).

Construction of hepatocyte-specific PAK4 knockout (KO) mice

Hepatocyte-specific Pak4 KO mice were obtained by mating Pak4 ^flox/flox mice (B6.129S2-Pak4 ^tm2.1Amin /J) and Albumin-cre (B6.Cg-Speer6-ps1 ^{Tg(Alb-cre)21Mgn} /J) mice. (Pak4 ^flox/flox ; Albumin-cre) was produced.

Construction of a partial liver injury model by ischemia-reperfusion (I/R)

C57BL/6 mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 m/kg). A midline incision was made and an atraumatic clip was placed across the portal vein, hepatic artery, and bile duct just above the right branch to block blood flow to the left lateral and median lobe, which It accounts for about 70% of the total blood supply to the liver. The liver was moistened with saline-soaked gauze, and the body temperature was maintained at 37 °C with a warm blanket during the ischemic period. After 60 minutes of partial hepatic ischemia, the clip was removed and reperfusion was started. Sham mice underwent the same procedure without vascular occlusion.

After reperfusion for the desired time period, mice were sacrificed by exsanguination under anesthesia, and serum samples were collected. The left lateral and median lobes of the liver were obtained and stored at -80 °C until further analysis, or immediately fixed in 10% formalin.

Isolation of primary hepatocytes

Primary hepatocytes were prepared by perfusion from 8 to 10-week-old mice of littermates. After cannulation into the inferior vena cava, calcium-free HEPES buffer (10 mM, pH 7.4) for 5 min at a flow rate of 0.35 ml/min, followed by 5 μg of collagenase IV (Sigma-Aldrich, St. Louis) with 5 mM calcium chloride. , MO, USA) for 5 min. Hepatocytes were resuspended in DMEM/F12 supplemented with 10% FBS, 10 units/ml penicillin, 10 μg/ml streptomycin and 10 nM dexamethasone mixed with 42% Percoll and centrifuged at 1300 rpm for 5 minutes. Cells were plated in a 6-well culture dish at 1x10 ⁶ cells/well.

Hypoxia-reoxygenation (H/R) protocol

Primary stem cells were cultured at 37 °C in an anoxic culture tank (Oxoid, Basingstoke, Hampshire, England) with an oxygen absorption pack (AnaeroGen, Oxoid). This method achieved an oxygen level in the culture vessel of less than 1%. After varying periods of hypoxia, the chamber was opened and the hypoxic medium was replaced with an oxygen-containing medium to initiate reoxygenation of the cells.

Cell culture and transient infection

The human embryonic kidney cell line HEK239T was purchased from ATCC (Manassas, VA, USA). For Nrf2 reporter gene analysis, 1 μg of a plasmid containing a promoter together with an antioxidant responsive element (ARE) driving luciferase expression was used. HEK293T cells were infected with 0.1-1 μg of NrF2, PAK4, or PAK4 ^S474A together with Lipofectamine 3000 (Invitroge, Carlsbad, CA, USA) to express foreign proteins. Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega, Madson, WI, USA) with a Lumat LB 9570 (Berthold, Bad Wildbad, Germany).

chemical analysis

Alanine aminotransferase (ALT), aspartic acid aminotransferase (AST) (Asan Pharmaceutical, Seoul, Korea) and levels of glutathione in the liver (GSH, Arbor Assays, AnnArbor, Michiganm, USA) and malondialdehyde (MDA, ab118970, Abcam, Cambridge, UK) was assayed using a commercial kit. Blood levels of IL-1β, IL-6, TNF-α and CCL-2 were all measured using ELISA kits purchased from Invitrogen.

Flow cytometry analysis of nonparenchymal cells

Liver non-parenchymal cells were isolated by enzymatic digestion with collagenase IV. Cells were incubated with anti-CD16/CD32 antibody (1:100, #14-0161-86) for 30 minutes to block non-specific binding to Fcγ receptors. FITC-conjugated anti-F4/80 antibody (1:200, #123108), PE-conjugated anti-Ly6G antibody (1:200, #108408) and APC-conjugated anti-CD11b antibody (1: 200, #101212) at 4°C for 30 minutes. All antibodies were purchased from Invitrogen. After washing three times with FACS buffer (3% FBS in phosphate buffer), the cells were analyzed with an Accur ^™ flow cytometer (BD Biosciences, San Jose, CA, USA).

RNA isolation and qPCR

Total RNA was extracted from frozen liver tissue or primary cultured hepatocytes using TRIzol reagent (Invitrogen). RNA was precipitated with isopropanol dried using 70% ethanol and dissolved in diethylpyrocarbonate-treated distilled water. Fist-strand cDNA was generated using random hexamer primers provided by the First-strand cDNA Synthesis Kit (Applied Biosystems, Foster City, CA, USA). Specific primers were designed using PrimerBank ( https://pga.mgh.harvard.edu/primerbank ). The table below shows the sequences of the primers used for qPCR.

qPCR reactions were run in 384-well plates using the ABI Prism ^TM 7900 sequence Detection System (applied Biosystems) in a final volume of 10 μl containing 10 ng reverse transcribed total RNA, 200 nM forward and reverse primers and PCR master mixture. performed.

Subcellular fractionation, western blot, co-immunoprecipitation (Co-IP)

Nuclear and cytoplasmic fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, Waltham, MA, USA). Tissue homogenates or cell lysates (15 μg) were separated by 6-14% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, blots were collected for PAK4, p-PAK4 (S474A), p-IKKα/β, p-IKBα, p-p65, cleaved caspase-3, Bax (Cell Signaling Technology, Beverly, MA, USA), GAPDH, lamin B1, Bcl2, NQO1 (Bioworld Technology, St Louis Park, MN, USA), p65, IKBα, Ub (Santa Cruz Biotechnology, Dallas, TX, USA), Nrf2 (Proteintech, Rosemont, IL, USA) ), HO-1 (Enzo Life Sciences, Farmingdale, New York, USA), p-Thr, and p-Ser (Merck KGaA, Darmstadt, Germany).

For co-immunoprecipitation, 600 μg of protein was incubated overnight at 4° C. with anti-Nrf2 antibody (Proteintech, Sankt Leon-Rot, Germany). Next, incubation was performed with protein G agarose at 4° C. for 2 hours. Blots were validated with antibodies against PAK4, Nrf2, p-Ser, or p-Thr.

* Histology

For light microscopic observation, paraffin sections (5 μm) of liver tissue were stained with hematoxylin and eosin (H&E). TUNEL staining was performed using a commercial kit (Promega, Madison, WI, USA). Five to six random sections per slide were examined to determine the area of necrosis and the percentage of apoptotic cells.

To determine the area of necrosis, sections were viewed with an Axiovert 40 CFL microscope (CARl Zeiss, Oberkochen, Germany) and measured using iSolution DT 36 software (Carl Zeiss). Immunohistochemical staining was performed using the DAKO Envision system (DAKO, Carpinteria, CA, USA). After deparaffinization and hydration, tissue sections were subjected to a microwave antigen-retrieval procedure in 0.01 M sodium citrate buffer. For immunofluorescence staining, the paraffin was removed and sections were collected with antibodies to PAK4 (ab173315, Abcam), antibodies to hepatocyte nuclear factor 4α (HNF4α, ab41898), antibodies to F4/80 (ab6640), and antibodies to Ly6G (ab25377). ), 650 CD68 antibody (ab125212), and 4-hydroxynonenal antibody (HNE, ab46545) (all Abcam products) were immunostained. After washing with phosphate buffer, secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG1 and Alexa fluor 594-conjugated goat anti-earth IgM, Thermo Fisher Scientific) were reacted at 37°C for 1 hour. Sections were counterstained with DAPI.

RNA sequencing (RNA-seq) analysis

RNA from liver tissue of littermates and hepatocyte-specific PAK4 KO mice subjected to liver ischemia-reperfusion (i.e., 1 hour partial ischemia followed by 6 hours reperfusion) was used. Total RNA concentration was calculated with Quant-IT RiboGreen (Invitrogen, #R11490). Samples were run on a TapeStation RNA screentape (Agilent, #5067-5576) to assess the integrity of total RNA. Only high quality RNA preparations with RIN greater than 7.0 were used for RNA library construction. Libraries were independently prepared with Illumina's TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina Inc, San Diego, CA, USA, #20020599) with 0.5 μg of total RNA per sample. The first step in the workflow involved removing rRNA from total RNA. After this step, the remaining mRNA was cleaved into small pieces using divalent cations under elevated temperature. The cut RNA fragment was cloned into first-strand cDNA using SuperScript II reverse transcriptase (Invtrogen, #18064014) and random primers. Next, second-strand cDNA synthesis was performed using DNA Polymerase I, RNase H and dUTP. Next, the end repair process of adding one '' base to these cDNA fragments, and then, the adapter was ligated. Next, the product was purified and enriched by PCR to make the final cDNA library. Libraries were quantified using the KAPA Library Quantification Kit for Illumina Sequencing platforms according to the qPCR quantification protocol guide (KAPA BIOSYSTEMS, #KK4854) and screened using a TapeStation D1000 ScreenTape (agilent Technologies, #5067-5582). Next, the indexed library was submitted to Illumina NovaSeq (Illumina, Inc., San Diego, CA, USA), and paired-end (2 x 100 bp) sequencing was performed by Macrogen Inc.

Relative abundances of genes were measured by Read Count using StringTie. In order to find differentially expressed genes (DEGs), statistical analysis was performed using the estimated frequency for each gene in the sample. Genes greater than 1 greater than the zeroed Read Count value in the sample were excluded. For Log2-transformation, 1 was added to each Read Count value of the filtered gene. The filtered data was log2 transformed and TMM normalized. Statistical significance of differential expression data was determined using exactTest with edgeR and fold change. The false discovery rate (FDR) was adjusted by correcting the p value using the Benjamini-Hochberg algorithm. For the DEG set, hierarchical clustering analysis was performed using Euclidean distance as a measure of complete linkage and similarity. Meaning based on gProfiler ( https://biit.cs.ut.ee/gprofiler/gost ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ( https://www.genome.jp/keff/pathway.html ) Gene-enrichment and functional annotation analysis and pathway analysis were performed on the gene list.

Gene set enrichment analysis (GSEA)

RNA-seq data were analyzed using GSEA 4.1.0 software. For analysis, hallmark gene set of Molecular Signatures Database ( https://software.broadinstitute.org/gsea/msigdb ) v7.4 was used. FDR was used to evaluate the statistical significance of NES. Gene sets with FDR<0.25 were considered statistically significant. Gene interaction networks were evaluated by GeneMANIA (https://genemania.org/) and visualized using Cytoscape 3.8.2 software ( https://www.cytoscape.org ).

In vitro kinase assay

Recombinant Nrf2 ( _0.6 μg, ab132356 , Abcam) was incubated with active PAK4 (0.2 μg, ab96405, Abcam) at 30 °C. The reaction was then SDS-PAGEd and 32 P-labeled protein was detected by autoradiography. For Coomassie blue staining, the gel was stained in Coomassie protein staining buffer (ab119211, Abcam) for 1 hour. For peptide competition analysis, synthetic 15-residue oligopeptides corresponding to each region including Ser168, Thr211 or Thr369 were used.

Mutagenesis of Nrf2 mutants

The human Nrf2 plasmid vector (clone ID, OHu26812) was supplied by GenScript (Tokyo, Japan). Mutants of Nrf2 were generated by site-directed mutagenesis (Stratagene, La Jolla, CA, USA). Nrf2-S168A, Nrf2-T211A and Nrf2-T360A mutants were generated by introducing point mutations of serine or threonine residues of Nrf2 to alanine as follows: Nrf2-S168A (TCT→ GCT), Nrf2-T211A (ACC→ GCC) and Nrf2-T360A (ACA→GCA).

statistical processing

Data are presented as mean±SD. For statistical comparison, Fixher'post hoc one-way ANOVA was used. Significance of differences between groups was determined using the Student'unpaired t-test. A p-value of less than 0.05 was considered significant.

Experimental Example 2: Expression of PAK4 in ischemia-reperfusion-damaged liver and hypoxia-reoxygenated primary cultured hepatocytes

Expression of PAK4 was evaluated in livers of wild-type mice (A) after ischemia for 1 hour and then reperfused according to the protocol of FIG. 1 and in primary cultured hepatocytes (B) after hypoxia and reoxygenation for 1 hour.

Both protein and mRNA levels of PAK4 were low in normal liver (sham, prior to ischemia), but increased significantly after ischemia/reperfusion, reaching a peak at 24 hours postoperatively (FIGS. 2A-2D). (A) shows an increase in PAK4 protein expression with time after ischemia-reperfusion, and (B) shows an increase in PAK4 mRNA expression. (C, D) are observations of PAK4 protein increase 6 hours or 24 hours after ischemia-reperfusion through immunohistochemical staining (C) and immunofluorescence staining (D).

In addition, the protein and mRNA expression of PAK4 also increased in primary cultured hepatocytes after hypoxia-reoxygenation (FIG. 2E, 2F).

This upregulation in hepatocytes suggests that PAK4 may be involved in the pathogenesis of liver damage due to ischemia/reperfusion.

Experimental Example 3: Effect of genetic deletion of PAK4 on liver damage caused by ischemia-reperfusion

Hepatocyte-specific PAK4 KO (knockout) mice were used to examine whether PAK4 genetic overexpression or deficiency affects liver damage caused by ischemia-reperfusion.

Figure 3A shows PAK4 levels in the liver of wild-type mice and PAK4 KO mice. It shows that PAK is missing in PAK4 KO mice.

3B and 3C are hematoxylin-eosin staining and analysis of necrotic area after ischemia-reperfusion, respectively. When ischemia-reperfusion was performed on wild-type (WT) and KO mice, necrosis of liver tissue was significantly reduced in KO mice compared to wild-type mice. This result was confirmed by the decrease in blood levels of AST and AKT, which are markers of hepatocyte damage (FIG. 3D).

Figure 3E is a photograph of immunofluorescence staining results for CD68 ⁺ (macrophages), F4/80 ⁺ (macrophages), Ly6G ⁺ (neutrophils) and TUNEL ⁺ (apoptotic) cells in liver tissue and a graph quantifying them. . After ischemia-reperfusion, infiltration of macrophages and neutrophils, which are inflammatory cells, was reduced in the liver tissue of KO mice compared to wild-type mice.

3F and 3G are graphs showing protein and mRNA levels of pro-inflammatory cytokines/chemokines in blood and liver tissue, respectively. It shows that the levels of various cytokines in blood and liver tissue are reduced in KO mice.

As such, in the case of PAK4 KO mice, necrosis of liver tissue caused by ischemia-reperfusion (FIG. 3B, 3C), hepatocyte damage (FIG. 3D), inflammatory cell infiltration (FIG. 3E), and apoptosis ( Figure 3E), cytokine production (Figures 3F and 3G) was significantly reduced compared to normal control mice. This shows that hepatocyte-specific PAK4 KO mice are protected from ischemia-reperfusion-induced liver damage compared to controls (wild type, WI).

Experimental Example 4: Effect of genetic overexpression of PAK4 on liver damage and inflammation caused by ischemia-reperfusion

To overexpress PAK4, C57BL/6 mice were injected with 1 x 10 ⁹ pfu of PAK4-expressing adenovirus (Ad-PAK4), a PAK4 mutant adenovirus devoid of kinase function (Ad-PAK4 ^S474A ), and a control virus (Ad-LacZ ) was injected through the tail vein. After virus injection, ischemia for 1 hour and reperfusion for 6 or 24 hours were performed (Fig. 1A).

4 shows that liver damage caused by ischemia-reperfusion is aggravated when PAK is overexpressed using adenovirus.

Figure 4A is the result of detecting the PAK4 protein in the liver tissue homogenate. When mice were injected with PAK4 adenovirus (Ad-PAK4) and PAK4 mutant adenovirus (Ad-PAK4 ^S474A ) with phosphorylation removed, PAK4 expression increased in liver tissue.

4B and 4C are micrographs of liver sections and graphs quantifying the area of necrosis, respectively. This shows that necrosis and hepatocellular damage increased in the Ad-PAK4-administered group compared to the control adenovirus (Ad-LacZ)-administered group. However, the Ad-PAK4 ^S474A administration group did not cause necrosis or damage. This result was confirmed by the decrease in blood levels of AST and ALT, which are markers of liver cell damage (FIG. 4D).

Figure 4E is a photograph of immunofluorescence staining results for CD68 ⁺ (macrophages), F4/80 ⁺ (macrophages), Ly6G ⁺ (neutrophils, neutrophils) and TUNEL ⁺ (apoptotic) cells in liver tissue and quantification thereof. it's a graph After ischemia-reperfusion, infiltration of inflammatory cells such as macrophages and neutrophils was increased in the Ad-PAK4-administered group compared to the control group or the mutant adenovirus-administered group.

4F and 4G are graphs showing protein and mRNA levels of pro-inflammatory cytokines/chemokines in blood and liver tissue, respectively. It shows that cytokine expression is increased in liver tissue and blood by PAK4.

As such, when PAK4 was overexpressed (Ad-PAK4 group), necrosis of liver tissue due to ischemia-reperfusion (FIG. 4B and 4C), hepatocyte damage (FIG. 4D), inflammatory cell infiltration (FIG. 4E), and apoptosis (FIG. 4B) 4E), and cytokine generation (Fig. 4F, 4G) was significantly increased compared to the control virus (Ad-LacZ group) mice. However, when PAK4 mutant adenovirus with PAK4 phosphorylation removed (Ad-PAK4 ^S474A group) was administered, liver damage did not worsen, suggesting that PAK4 kinase function is a key factor in liver damage.

Experimental Example 5: Increased generation of active oxygen by PAK4

In order to find the mechanism by which PAK4 genetic deficiency or inhibition of enzyme activity suppresses ischemia-reperfusion-induced liver damage, wild-type and PAK4 KO mice were subjected to ischemia-reperfusion injury, followed by RNA sequencing.

Wild-type and PAK4 KO mice (Figs. 5A-5E) or PAK4 overexpressing C57BKL/6 mice (Figs. 5F, 5G) were subjected to hepatic ischemia-reperfusion injury.

Figure 5A shows the results of RNA sequencing of liver tissue after ischemia-reperfusion injury to wild-type and PAK4 KO mice. The KEGG pathway and gene ontology biological process were performed for genes differentially expressed in the liver. Expression of previously known genes related to cytoskeleton, proliferation, and growth as well as genes related to inflammation or stress were reduced in PAK4 KO mice. Gene set enrichment analysis (GSEA) confirmed that the generation of reactive oxygen species was reduced in KO mice (FIG. 5B). As a result of GeneMANIA analysis, Nrf2 was related to the gene group related to the generation of reactive oxygen species in the ROS pathway identified in B above (FIG. 5C).

As a result of these genetic changes, PAK4 expression and reactive oxygen species are positively correlated in liver tissue and blood after ischemia-reperfusion. Next, oxidative stress markers, 4-hydroxy-2-nonenal (4-HNE) and malonaldehyde (MDA), which are lipid peroxidation markers, and GSH levels were evaluated in the livers of these mice.

These oxidative stress markers correlated with the expression of PAK4. In PAK4-deficient liver tissue of PAK4 KO mice, the number of 4-HNE-positive hepatocytes, which mark reactive oxygen generation after ischemia-reperfusion, was significantly reduced compared to wild-type (Fig. 5D), and on the contrary, when PAK4 was overexpressed by adenovirus After hepatic ischemia-reperfusion, the number of 4-HNE-positive hepatocytes increased. However, this was not the case for PAK4 ^S474A (FIG. 5a F). These histological results are supported by the levels of MDA and GSH in the liver. Defective PAK4 increased the level of GSH and decreased the level of MDA (Fig. 5E), whereas overexpression of PAK4 decreased blood GSH. and blood MDA increased (FIG. 5G).

Experimental Example 6: Regulation of stability and transcriptional activity of Nrf2 by PAK4

Through the Genemania analysis of Example 34, it was confirmed that Nrf2 was closely related to the generation of reactive oxygen species in PAK4 KO mice (FIG. 5C), and Nrf2 was expected to be a potential sub-target of PAK4. Therefore, we investigated whether PAK4 directly phosphorylates Nrf2 to regulate the expression of Nrf2 antioxidant enzymes.

After overexpressing Nrf2 and PAK4 in HEK293T cells, ARE-luciferase assay was performed, and the results showed that PAK4 significantly suppressed the transcriptional activity of Nrf2. However, PAK4 ^S474A did not (Fig. 6A).

Consistent with the above experimental results, when PAK4 was overexpressed and subjected to ischemia-reperfusion, the levels of total Nrf2 and nuclear Nrf2 were reduced compared to wild-type mice, whereas the amount of Nrf2 in the nucleus after ischemia-reperfusion was decreased in PAK4-deficient KO mice. It increased compared to mice (FIG. 6B). Considering that the amount of Nrf2 in the nucleus is essential for the expression of Nrf2-regulated antioxidant enzymes (eg, NQO1, HO-1, etc.), it was confirmed that the transcriptional activity of Nrf2 was also inhibited by PAK4 in mouse liver tissue.

This effect was also confirmed in hypoxia-reoxygenation injury experiments of primary cultured hepatocytes. By hypoxia-reoxygenation, PAK4 transported Nrf2 from the nucleus to the cytoplasm (Fig. 6C, 6D, 6E).

Nrf2 activity is primarily determined by nuclear accumulation and protein stability. According to the results of the CHX tracking experiment, removal of PAK4 increased the protein stability of Nrf2 (Fig. 6F). Consistent with this, PAK4 overexpression markedly increased ubiquitination of Nrf2 in the presence of MG132 (Fig. 6G). Keap1 expression was increased by PAK4 deficiency, thus excluding the possibility of Keap 1 dependent regulation of Nrf2 (FIG. 6H).

The above results show that generation of reactive oxygen species by PAK4 occurs through transcriptional activity of Nrf2 protein and regulation of protein stability.

Experimental Example 7: T369 phosphorylation of Nrf2 by PAK4

To elucidate the mechanism of regulation of Nrf2 protein stability and transcriptional activity by PAK4, phosphorylation of Nrf2 was measured, as it is an important factor determining changes in Nrf2's intracellular distribution and protein stability.

After overexpressing PAK4 in HEK293T cells, co-immunoprecipitation (Co-IP) and in vitro kinase assays revealed a physical interaction between PAK4 and Nrf2 and that PAK4 directly phosphorylated Nrf2 (Fig. 7A, 7B) . However, phosphorylation of Ser40 of Nrf2 mediated by PKC or GSK-3β was not changed in the liver of PAK4 KO mice (see Fig. 6H).

To identify the specific site of Nrf2 phosphorylated by PAK4, a bioinformatic approach using PhosphoNET was used to predict potential phosphorylation sites. Three residues (ie, S168, T211 and T369) have been identified in the human Nrf2 protein.

Through peptide competition in vitro kinase assay, among the three candidates for phosphorylation of Nrf2 protein, the peptide containing the T369 region efficiently inhibited Nrf2 phosphorylation by PAK4, and the peptide containing S168 or T211 effectively inhibited Nrf2 phosphorylation by PAK4. slightly inhibited (Fig. 7C). This suggests that in the presence of PAK4, all three sites contribute to phosphorylation of Nrf2.

In order to further confirm this, a phosphorylation-inactivated mutant in which serine or threonine was substituted with alanine was prepared and subjected to Co-IP and Western blot analysis. In the case of the T369A mutant in which the T369 site of Nrf2 was substituted with alanine, Nrf2 phosphorylation by PAK4 was effectively inhibited (FIG. 7D). In the ARE reporter assay, only the Nrf2 ^T369A mutant significantly blocked the inhibition of Nrf2 transcriptional activity by PAK4 (Fig. 7E), indicating that PAK4-induced Nrf2 T369 phosphorylation is a key process in reducing Nrf2 transcriptional activity.

Furthermore, PAK4-induced nuclear export and ubiquitination of Nrf2 were not observed in the Nrf2 ^T369A mutant (Fig. 7F, 7G). The Nrf2 ^T369A mutant evaded the inhibition of Nrf2 transcriptional activity by PAK4 (Fig. 7E), increased Nrf2 expression in the nucleus (Fig. 7F), inhibited Nrf2 protein degradation through the proteasome (Fig. 7G), and produced cytokines. (FIG. 7H), inhibited apoptosis, and increased antioxidant enzyme expression (FIG. 7I).

From the above experimental results, it can be seen that PAK4 phosphorylates Nrf2 at T369 and destabilizes Nrf2 to regulate oxidative stress in hepatocytes during liver ischemia-reperfusion.

Experimental Example 8: Effect of Nrf2 gene knockdown on the protective effect of PAK4 deficiency against hepatic ischemia-reperfusion

To demonstrate a direct causal relationship between Nrf2 inhibition and PAK4-dependent liver ischemia-reperfusion injury, Nrf2 knockdown experiments were performed. Ad-shNrf2 or Ad-shLacZ was injected into wild-type and PAK4 KO mice (Fig. 8A), and Nrf2-targeting siRNA was introduced into primary cultured hepatocytes isolated from wild-type and PAK4 KO mice (Fig. 8B) to inhibit Nrf2 expression. did Knockdown of Nrf2 increased ischemia-reperfusion-induced liver damage (serum AST and ALT levels, increased liver damage and apoptosis, oxidative stress and inflammation) and abrogated the enhancement of these markers by PAK4 deficiency (Fig. 8E-8H).

The specificity of Nrf2 related to PAK4 effect was also reproduced in primary cultured hepatocytes. There were no differences in the levels of apoptotic markers and pro-inflammatory cytokines between wild-type and PAK4 KO mouse hepatocytes infected with Nrf2-siRNA (Fig. 8I, 8J).

Experimental Example 9: Effect of PAK4 inhibitors on liver damage

The compound of Example 19, which is a novel small molecule PAK4 inhibitor that can be administered orally provided by the present disclosure ((3 ³ S)-5 ^6- (4-methylpiperazin-1-yl)-1 ⁵ -(trifluoromethyl )-1 ⁷ H-2,6-diaza-1(4,2)-pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3) The effect of -benzenecyclohexapan-4-one) (FIG. 9A) on liver damage caused by ischemia-reperfusion was evaluated. The compound of Example 19 (50 mg/kg) was injected orally 48 hours, 12 hours, and 6 hours before ischemia-reperfusion, as shown in B of FIG. 9a. After 6 hours and 24 hours after reperfusion, serum and liver tissues were collected, histologically analyzed, and mRNA and protein expression were compared.

The compound of Example 19 was found to be a potent ATP-competitive PAK4 inhibitor and to have excellent PAK4 selectivity over PAK1. PAK4 IC ₅₀ was 4.22 nM and PAK1 IC ₅₀ was 1200 nM.

At 72 hours after the first oral administration of the compound of Example 19 (50 mg/kg, 3 consecutive days), ischemia-reperfusion-induced liver damage to the mouse liver was almost completely blocked. Compared to the vehicle control group, this was shown by significant inhibition of hepatocyte necrosis, apoptosis, oxidative stress, and cytokine release (FIG. 9D and FIG. 9E-9G), and Nrf2 accumulation in the nucleus and induction of target genes under ischemia-reperfusion conditions. It is related to (Fig. 9C).

These results are consistent with the results in PAK4 KO mice (Fig. 2, Fig. 5B).

As above, specific parts of the present disclosure have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present disclosure is not limited thereby. The point will be clear. Accordingly, the substantial scope of the present disclosure will be defined by the appended claims and their equivalents.

The present invention is to proceed with an application with the support of the tasks described below.

The present invention relates to a PAK4 inhibitor and its pharmaceutical use, and can be used for preventing and treating diseases caused by excessive activity of PAK4. More specifically, PAK4 inhibitors can be used for prevention or treatment of tissue or cell damage caused by ischemia-reperfusion or hypoxia-reoxygenation.

Claims (23)

1. A compound represented by Formula 1, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, including inflammatory diseases, diseases caused by ischemia-reperfusion, and diseases caused by hypoxia-reoxygenation A pharmaceutical composition for preventing or treating a disease selected from diseases, melanin hyperpigmentation disease, muscle regeneration, Parkinson's disease, neurological diseases such as amyotrophic lateral sclerosis, hypertension, AIDS, polycystic nephropathy, and osteoporosis.

   [Formula 1]

   

   In Formula 1,

   X is -C ₁ haloalkyl;

   Y is aryl or 5-6 membered heteroaryl {wherein at least one H of the aryl or 5-6 membered heteroaryl is -C _1-6 alkyl, -C _1-6 aminoalkyl, -C _1-6 hydroxy Alkyl, -C _1-6 Haloalkyl, -C _1-6 Alkyl-OC _1-6 Alkyl, -CN, -NR ₁ R ₂ , -OR ₃ , -Halo, -Cycloalkyl, -C(=O)- may be substituted with cycloalkyl, -heterocycloalkyl, or -C(=O)-heterocycloalkyl [wherein -cycloalkyl, -C(=O)-cycloalkyl, -heterocycloalkyl, or -C( At least one H of the =O)-heterocycloalkyl ring may be substituted with -C _1-6 alkyl, -C _1-6 aminoalkyl, -C _1-6 hydroxyalkyl, -cycloalkyl, or -heterocycloalkyl has exist]}

   L ₁ is -C(=O)-NR ₄ -, -C(=O)-O-, -NR ₅ -C(=O)-, or -C(=O)-(heterocycloalkyl)-; {wherein the heterocycloalkyl ring of -C(=O)-(heterocycloalkyl)- contains at least one N and N is connected to -C(=O)-}

   L ₂ is -NH- or -O-;

   n is O, 1, 2, 3, 4, or 5 {wherein, when n is 0, L ₁ and L ₂ are directly connected};

   R ₁ and R ₂ are each independently -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), or -C _1-6 alkyl- OC _1-6 alkyl;

   R ₃ is -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), -C _1-6 alkyl-OC _1-6 alkyl, or -C _1-6 alkyl-heterocycloalkyl;

   R ₄ and R ₅ are each independently -H or -C _1-6 alkyl.
2. According to claim 1,

   X is -CF ₃ ;

   Y is phenyl or 5-6 membered heteroaryl {wherein, at least one H of the phenyl or 5-6 membered heteroaryl is -C _1-6 alkyl, -C _1-6 alkyl-OC _1-6 alkyl, -NR May be substituted with ₁ R ₂ , -OR ₃ , -heterocycloalkyl, or -C(=O)-heterocycloalkyl [wherein, -heterocycloalkyl, or -C(=O)-heterocycloalkyl ring one or more Hs may be substituted with -C _1-6 alkyl or -heterocycloalkyl]}

   L ₁ is -C(=O)-NR ₄ -, -C(=O)-O-, -NR ₅ -C(=O)-,

   

   ,

   

   ,

   

   ,

   

   , or

   

   ego;

   L ₂ is -NH- or -O-;

   n is O, 1, 2, 3, or 4 {wherein, when n is 0, L ₁ and L ₂ are directly connected};

   R ₁ and R ₂ are each independently -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), or -C _1-6 alkyl- OC _1-6 alkyl;

   R ₃ is -H, -C _1-6 alkyl, or -C _1-6 alkyl-heterocycloalkyl;

   R ₄ and R ₅ are each independently -H or -C _1-6 alkyl, characterized in that, the pharmaceutical composition.
3. According to claim 1,

   A pharmaceutical composition wherein the compound represented by Formula 1 is selected from the group consisting of the following compounds:

   (1) 3 ^1,3 3 -dimethyl- ^{1 5 -} (trifluoromethyl)-1 ⁷ H,3 ¹ H-2,5,9-triaza-1(2,4)-pyrrolo[2 ,3-d] pyrimidina-3 (4,5) -pyrazolacyclononapan-4-one

   (2) 3 ^1,3 3 -dimethyl- ^{1 5 -} (trifluoromethyl)-1 ⁷ H,3 ¹ H-2,5,10-triaza-1(2,4)-pyrrolo[2 ,3-d] pyrimidina-3 (4,5) -pyrazolacyclodecapan-4-one

   (3) 3 ^1- (2-methoxyethyl)-3 ³ -methyl-1 ^5- (trifluoromethyl)-1 ⁷ H,3 ¹ H-2,5,9-triaza-1(2, 4)-pyrrolo[2,3-d]pyrimidina-3(4,5)-pyrazolacyclononapan-4-one

   (4) 3 ^1,3 3 -dimethyl- ^{1 5 -} (trifluoromethyl)-1 ⁷ H,3 ¹ H-5-oxa-2,9-diaza-1(2,4)-pyrrolo [2,3-d]pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one

   (5) 3 ³ -Methyl-3 ^1- (1-methylpiperidin-4-yl)-1 ^5- (trifluoromethyl)-1 ⁷ H,3 ¹ H-2,5,9-triaza -1(2,4)-pyrrolo[2,3-d]pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one

   (6) 3 ³ -methyl-3 ¹ -(morpholine-4-carbonyl)-1 ⁵ -(trifluoromethyl)-1 ⁷ H,3 ¹ H-2,5,9-triaza-1( 2,4)-pyrrolo[2,3-d]pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one

   (7) 3 ¹ -Methyl-1 ^5- (trifluoromethyl)-1 ⁷ H,3 ¹ H-2,5,9-triaza-1(2,4)-pyrrolo[2,3-d ]pyrimidina-3(3,4)-pyrazolacyclononaphan-4-one

   (8) 3 ⁴ -((2-(dimethylamino)ethyl)(methyl)amino)-1 ^5- (trifluoromethyl)-1 ⁷ H-2,5,9-triaza-1(2, 4)-pyrrolo[2,3-d]pyrimidina-3(1,3)-benzenecyclononaphan-4-one

   (9) 3 ⁵ -morpholino-1 ⁵ -(trifluoromethyl)-1 ⁷ H-2,5,9-triaza-1(2,4)-pyrrolo[2,3-d]pyridine midina-3(1,3)-benzenecyclononaphan-4-one

   (10) 3 ⁴ -morpholino-1 ⁵ -(trifluoromethyl)-1 ⁷ H-2,5,9-triaza-1(2,4)-pyrrolo[2,3-d]pyryl midina-3(1,3)-benzenecyclononaphan-4-one

   (11) 3 ⁶ -Methoxy-3 ⁴ -morpholino-1 ⁵ -(trifluoromethyl)-1 ⁷ H-2,4,10-triaza-1(2,4)-pyrrolo[2 ,3-d] pyrimidina-3 (1,3) -benzenecyclodecapan-5-one

   (12) 3 ⁴ -((2-methoxyethyl)amino)-1 ⁵ -(trifluoromethyl)-1 ⁷ H-2,5,9-triaza-1(2,4)-pyrrolo[ 2,3-d] pyrimidina-3 (1,3) -benzenecyclononaphan-4-one

   (13) 3 ⁶ -methoxy-3 ^4- (4-morpholinopiperidin-1-yl)-1 ^5- (trifluoromethyl)-1 ⁷ H-2,4,10-triaza-1 (2,4)-pyrrolo[2,3-d]pyrimidina-3(1,3)-benzenecyclodecapan-5-one

   (14) 1 ⁵ -(trifluoromethyl)-1 ⁷ H-2,5,10-triaza-1(2,4)-pyrrolo[2,3-d]pyrimidina-3(1, 3)-benzenecyclodecapan-4-one

   (15) 3 ^6,5 -dimethyl-3 ⁴ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,5,9-triaza-1(2,4)-pyrrolo [2,3-d]pyrimidina-3(1,3)-benzenecyclononaphan-4-one

   (16) 3 ^6,4 -dimethyl-3 ⁴ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,4,10-triaza-1(2,4)-pyrrolo [2,3-d]pyrimidina-3(1,3)-benzenecyclodecapan-5-one

   (17) 3 ⁶ -methyl-3 ⁴ -morpholino-1 ⁵ -(trifluoromethyl)-1 ⁷ H-2,5,9-triaza-1(2,4)-pyrrolo[2, 3-d] pyrimidina-3(1,3)-benzenecyclononaphan-4-one

   (18) 3 ⁶ -Methoxy-3 ⁴ -(4-(oxetan-3-yl)piperazin-1-yl)-1 ⁵ -(trifluoromethyl)-1 ⁷ H-2,4,10 -Triaza-1(2,4)-pyrrolo[2,3-d]pyrimidina-3(1,3)-benzenecyclodecapan-5-one

   (19) (3 ³ S)-5 ^6- (4-methylpiperazin-1-yl)-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2 )-pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (20) (3 ³ R)-5 ^6- (4-methylpiperazin-1-yl)-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2 )-pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (21) (3 ³ S)-5 ⁶ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2)-pyrrolo[2,3 -d] pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (22) (3 ³ R)-5 ⁶ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2)-pyrrolo[2,3 -d] pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (23) (3 ³ S)-5 ⁴ -methyl-5 ⁶ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2)-p Rolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (24) (3 ³ S)-5 ⁴ -methoxy-5 ⁶ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2)- Pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (25) (3 ³ S)-5 ⁶ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2)-pyrrolo[2,3 -d] pyrimidina-3(3,1)-azefana-5(1,3)-benzenecyclohexapan-4-one

   (26) (3 ³ R)-5 ⁶ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2)-pyrrolo[2,3 -d] pyrimidina-3(3,1)-azefana-5(1,3)-benzenecyclohexapan-4-one

   (27) (3 ³ S)-5 ⁵ -morpholino-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1(4,2)-pyrrolo[2,3 -d] pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (28) (3 ³ S)-5 ^6- (2-(pyrrolidin-1-yl)ethoxy)-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza-1 (4,2)-pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one

   (29) (3 ³ R)-5 ⁴ -methyl-5 ^6- (4-methylpiperazin-1-yl)-1 ^5- (trifluoromethyl)-1 ⁷ H-2,6-diaza- 1(4,2)-pyrrolo[2,3-d]pyrimidina-3(3,1)-piperidina-5(1,3)-benzenecyclohexapan-4-one; and

   (30) 3 ^1,3 3 -dimethyl- ^{1 5 -} (trifluoromethyl)-1 ⁷ H,3 ¹ H-9-oxa-2,5-diaza-1(2,4)-pyrrolo [2,3-d]pyrimidina-3(4,5)-pyrazolacyclononaphan-4-one.
4. The pharmaceutical composition according to claim 1 or 2, wherein the compound of Formula 1 has at least one activity selected from the group consisting of PAK4 inhibition, Nrf2 phosphorylation inhibition, and Nrf2 protein stability and transcriptional activity increase. .
5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the inflammatory disease is selected from the group consisting of viral infection, bacterial infection, fungal infection, autoimmune disease and chronic inflammatory disease.
6. The method according to any one of claims 1 to 3, wherein the ischemia-reperfusion or hypoxia-reoxygenation-related disease is liver damage, brain damage, lung damage, kidney damage, myocardium due to ischemia-reperfusion or hypoxia-reoxygenation Damage, a pharmaceutical composition characterized in that selected from the group consisting of skeletal muscle damage.
7. The method according to any one of claims 1 to 3, wherein the melanin hyperpigmentation disease is selected from the group consisting of melanin hyperpigmentation diseases including melasma, freckles, senile pigment spots, solar melanosis, and skin whitening. pharmaceutical composition.
8. A pharmaceutical composition for preventing or treating tissue or cell damage caused by ischemia-reperfusion or hypoxia-reoxygenation, comprising a PAK4 activity or expression inhibitor as an active ingredient,

   The PAK4 activity or expression inhibitor is a pharmaceutical composition, characterized in that a compound, peptide, peptidomimetic, aptamer, antibody, or protein kinase A (protein kinase A) inhibitor that specifically binds to PAK4.
9. According to claim 8,

   The PAK4 activity or expression inhibitor comprises as an active ingredient a compound represented by Formula 1, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for preventing or treating tissue or cell damage caused by hypoxia-reoxygenation.

   [Formula 1]

   

   In Formula 1,

   X is -C ₁ haloalkyl;

   Y is aryl or 5-6 membered heteroaryl {wherein at least one H of the aryl or 5-6 membered heteroaryl is -C _1-6 alkyl, -C _1-6 aminoalkyl, -C _1-6 hydroxy Alkyl, -C _1-6 Haloalkyl, -C _1-6 Alkyl-OC _1-6 Alkyl, -CN, -NR ₁ R ₂ , -OR ₃ , -Halo, -Cycloalkyl, -C(=O)- may be substituted with cycloalkyl, -heterocycloalkyl, or -C(=O)-heterocycloalkyl [wherein -cycloalkyl, -C(=O)-cycloalkyl, -heterocycloalkyl, or -C( At least one H of the =O)-heterocycloalkyl ring may be substituted with -C _1-6 alkyl, -C _1-6 aminoalkyl, -C _1-6 hydroxyalkyl, -cycloalkyl, or -heterocycloalkyl has exist]}

   L ₁ is -C(=O)-NR ₄ -, -C(=O)-O-, -NR ₅ -C(=O)-, or -C(=O)-(heterocycloalkyl)-; {wherein the heterocycloalkyl ring of -C(=O)-(heterocycloalkyl)- contains at least one N and N is connected to -C(=O)-}

   L ₂ is -NH- or -O-;

   n is O, 1, 2, 3, 4, or 5 {wherein, when n is 0, L ₁ and L ₂ are directly connected};

   R ₁ and R ₂ are each independently -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), or -C _1-6 alkyl- OC _1-6 alkyl, R ₃ is -H, -C _1-6 alkyl, -C _1-6 alkyl-N(C _1-6 alkyl)(C _1-6 alkyl), -C _1-6 alkyl- OC _1-6 alkyl, or -C _1-6 alkyl-heterocycloalkyl, and R ₄ and R ₅ are each independently -H or -C _1-6 alkyl.
10. The pharmaceutical composition according to claim 8, wherein the compound that specifically binds to PAK4 is one or more of the compounds according to claims 1 to 3.
11. [Claim 9] The pharmaceutical composition according to claim 8, wherein the PAK4 activity or expression inhibitor is an antisense nucleotide, siRNA, shRNA, or ribozyme complementary to PAK4 gene mRNA.
12. 12. The pharmaceutical composition according to any one of claims 8 to 11, wherein the composition is administered before, concurrently with, or after ischemia-reperfusion or hypoxia-reoxygenation.
13. The pharmaceutical composition according to any one of claims 8 to 11, wherein the composition can inhibit the ubiquitination of Nrf2 in the cytoplasm and the proteolysis of Nrf2 through the proteasome.
14. The method according to any one of claims 8 to 11, wherein the composition inhibits tissue infiltration of inflammatory cells or is selected from the group consisting of inflammation-related TNF-α, IL-1β, IL-6 and CCL-2. A pharmaceutical composition characterized by inhibiting the production of more than one cytokine.
15. Claims 8 to 11 The pharmaceutical composition according to any one of the preceding, wherein the composition inhibits NF-κB transcriptional activity.
16. The pharmaceutical composition according to any one of claims 8 to 11 , wherein the composition suppresses generation of reactive oxygen species related to inflammation in tissues or cells or reduces active oxygen markers in the blood.
17. The pharmaceutical composition according to any one of claims 8 to 11, wherein the composition inhibits cell apoptosis (apoptosis) or cell necrosis (necrosis).
18. The method according to any one of claims 8 to 11, wherein the ischemia-reperfusion or hypoxia-reoxygenation-related disease is liver damage, brain damage, lung damage, kidney damage, myocardium due to ischemia-reperfusion or hypoxia-reoxygenation Damage, a pharmaceutical composition characterized in that selected from the group consisting of skeletal muscle damage.
19. Ischemia-reperfusion-induced liver injury model animals or hypoxia-reoxygenation-damaged hepatocytes, including the step of administering a candidate material or a candidate gene through adenovirus, to tissue or cell damage caused by ischemia-reperfusion or hypoxia-reoxygenation A method for screening drugs that inhibit damage.
20. Inflammatory diseases, ischemia, including the step of administering to a subject a composition comprising the compound represented by Formula 1 of claim 1, its optical isomer, its hydrate or solvate, or its pharmaceutically acceptable salt as an active ingredient. -Prevention of diseases caused by reperfusion, diseases caused by hypoxia-reoxygenation, melanin hyperpigmentation diseases, muscle regeneration, Parkinson's disease, diseases of the nervous system such as amyotrophic lateral sclerosis, hypertension, AIDS, polycystic nephropathy, osteoporosis, or treatment method.
21. A composition comprising the compound represented by Formula 1 of claim 1, its optical isomer, its hydrate or solvate, or its pharmaceutically acceptable salt as an active ingredient for inflammatory diseases, diseases caused by ischemia-reperfusion, hypoxia-property Use for prevention or treatment of diseases caused by digestion, melanin hyperpigmentation diseases, muscle regeneration, Parkinson's disease, nervous system diseases such as amyotrophic lateral sclerosis, hypertension, AIDS, polycystic nephropathy, and osteoporosis.
22. The step of administering to a subject a composition comprising the compound represented by Formula 1 of claim 9, its optical isomer, its hydrate or solvate, or its pharmaceutically acceptable salt as an active ingredient of a PAK4 activity or expression inhibitor. A method for preventing or treating tissue or cell damage caused by ischemia-reperfusion or hypoxia-reoxygenation, comprising:
23. A composition comprising the compound represented by Formula 1 of claim 9, an optical isomer thereof, a hydrate or solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient of an activity or expression inhibitor of PAK4, ischemia-reperfusion or hypoxia- Use to prevent or treat tissue or cell damage caused by reoxygenation.

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