CXCR7 receptor modulator and use thereof

Abstract

The present disclosure relates to a compound of formula (I), a stereoisomer, isotopically labeled compound, hydrate, solvate, or pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising same, and a use thereof in the treatment of CXCR7-mediated diseases.

Description

CXCR7 receptor modulators and uses thereof

[0001] The present disclosure relates to novel CXCR7 (CXC chemokine receptor 7) receptor modulators, pharmaceutical compositions comprising the same, and uses thereof for the treatment of CXCR7-mediated diseases.

[0002] Chemokine receptors, a subset of G protein-coupled receptors (GPCRs), play a pivotal role in coordinating cellular responses to chemotactic signals in diverse biological contexts. Among these receptors, CXCR7 (CXC chemokine receptor type 7), also known as ACKR3, RDC1, CMKOR1, and GPR159, is known to be involved in regulating cell migration, proliferation, and tissue regeneration.

[0003] CXCR7 has two known ligands: CXCL12 (stromal cell-derived factor 1 (SDF-1), also known as Pre-B cell growth stimulating factor (PBSF)) and CXCL11 (interferon-inducible T cell alpha chemoattractant (I-TAC)).

[0004] CXCL12 participates in the regulation of immune surveillance and inflammatory responses. CXCL12 is secreted by bone marrow stromal cells, endothelial cells, heart, skeletal muscle, liver, brain, kidney, and parenchymal cells, and plays an essential role in stem cell proliferation, survival, and the homing of hematopoietic / progenitor cells to the bone marrow. CXCL12 also recruits bone marrow-derived progenitor cells to sites of vasculature. It also plays a crucial role in carcinogenesis. CXCL12 promotes the recruitment of endothelial progenitor cells and myeloid-derived suppressor cells to tumor sites, as well as other bone marrow-derived cells. CXCL12 also regulates angiogenesis / angiogenesis associated with tumor progression and plays a crucial role in the seeding of circulating tumor cells to metastatic sites. In addition to its chemotactic function, CXCL12 has been shown to regulate tumor cell proliferation, motility, and survival. In addition to CXCR7, CXCL12 binds to and activates CXCR4 (also known as Fusin, Leukocyte-derived seven-transmembrane-domain receptor (LESTR), D2S201E, seven-transmembrane-segment receptor, HM89, lipopolysaccharide-associated protein 3, LPS-associated protein 3), and CXCL11 binds to and activates CXCR3 (also known as GPR9, CD183).

[0005] Therefore, dysfunction of a series of pathways consisting of CXCR7, its ligands CXCL12 and CXCL11, and CXCR4 that binds CXCL12 is known to be associated with various diseases related to inflammation, immune damage, immune dysfunction, tissue damage, and cell growth abnormalities.

[0006] For example, indirect regulation of CXCL12 and CXCR4 through CXCR7 agonists is known to have therapeutic utility in hepatitis and liver fibrosis (Stromal cell-derived factor-1 (SDF-1) as a target in liver diseases TC-14012, Am J Physiol Gastrointest Liver Physiol 2016).

[0007] In addition, recent studies have shown that CXCR7 participates in the Wnt / β-catenin pathway and exhibits an inhibitory effect on fibrosis (CXCR7 Inhibits Fibrosis via Wnt / β-Catenin Pathways during the Process of Angiogenesis in Human Umbilical Vein Endothelial Cells, BioMed Research International, 2020), and it has also been reported that CXCR7 agonists exhibit a protective effect on isoproterenol-induced cardiac injury (Discovery of a Novel Small-Molecule Modulator of CXC Chemokine Receptor Type 7 as a Treatment for Cardiac Fibrosis, J Med Chem 2018).

[0008] CXCR7 agonists have been reported to reduce the effects of LPS-induced cytokines related to RANKL and TNF-α, reduce osteoclastogenesis and bone resorption induced by these, and have therapeutic effects on acute ischemic injury and thrombosis (CXC receptor 7 agonist acts as a CXC motif chemokine ligand 12 inhibitor to ameliorate osteoclastogenesis and bone resorption, Molecular Medicine REPORTS 2022; ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury Nature Communications 2022; Discovery and Development of First-in-Class ACKR3 / CXCR7 Superagonists for Platelet Degranulation Modulation, J Med Chem 2022).

[0009] Therefore, based on its potential as a therapeutic target for various diseases, there is a need in the art for the development of CXCR7 modulators that can selectively regulate CXCR7.

[0010] Accordingly, the present inventors have unexpectedly discovered that novel compounds having a specific substituent combination among a group of compounds previously known as adenosine receptor antagonists (compounds disclosed in International Publication Nos. WO 2021 / 099837 and WO 2022 / 107044) exhibit excellent activity as CXCR7 modulators, thereby completing the present invention.

[0011] An object of the present invention is to provide a compound of the following formula I, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, which is useful as a CXCR7 modulator.

[0012] [Chemical Formula I]

[0013]

[0014] In the above formula,

[0015] X and Y are each independently CR a or N;

[0016] R 1 is selected from the group consisting of hydrogen, halogen, C1-C6 haloalkyl, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl;

[0017] R 2 is -CN or -CONR b R c and;

[0018] R a , R b and R c are each independently H or C1-C6 alkyl;

[0019] n is an integer from 0 to 2.

[0020] An object of the present invention is to provide a pharmaceutical composition for the treatment of CXCR7-mediated diseases, such as fibrosis, cholestatic liver disease, inflammatory diseases, angiogenic diseases, cancer, autoimmune disorders, osteoporosis, pulmonary hypertension, acute ischemic injury and thrombosis.

[0021] An object of the present invention is to provide a method for treating a disease mediated by CXCR7, comprising administering to a subject a compound of the above formula I, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof.

[0022] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0023] One aspect of the present invention provides a compound of formula I, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof:

[0024] [Chemical Formula I]

[0025]

[0026] In the above formula, X and Y are each independently CR a Or N.

[0027] In one specific example, X is CR a , and Y can be N. In one specific example, X is CR a and Y is CR a may be. In one specific example, X is N and Y is CR a may be. In one specific example, X may be N and Y may be N. In the above definitions and specific examples, R a is H or C1-C6 alkyl. For example, R a is H or methyl.

[0028] Accordingly, the compound of the above formula I can be represented by any one of the following formulae IA to ID:

[0029]

[0030]

[0031] (In the above chemical formulas IA to ID, R 1 , R 2 And the definition of n is as described below for chemical formula I, and R a The definition of is as described above for chemical formula I.)

[0032] In the above chemical formula I and chemical formulas IA to ID, R 1 is selected from the group consisting of hydrogen, halogen, C1-C6 haloalkyl, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl.

[0033] In one specific example, R 1 may be H, halogen, C1-C3 haloalkyl (e.g., C1-C3 alkyl substituted with F or Cl), C1-C3 alkyl, C1-C3 alkoxy, or C2-C3 alkynyl. R 1 can be H, F, Cl, Br, I, CF3, -CH3, -OCH3 or -C≡C. For example, R 1 can be H, F, Cl, Br or CF3.

[0034] In the above chemical formulas I and IA to ID, n is an integer from 0 to 2. In one specific example, n may be 0 or 1.

[0035] In the above chemical formula I and chemical formulas IA to ID, R 2 is -CN or -CONR b R c It is. R b and R c are each independently H or C1-C6 alkyl.

[0036] In one specific example, R 2 is -CN or -CONH2. In one specific example, R 2 is -CN. In one specific example, R 2 is -CONH2.

[0037] In one specific example, the compound of formula I may be represented by the following formulae I-1 to I-3.

[0038]

[0039] In the above chemical formulas I-1 to I-3, X, Y, R 1 and R 2 The definition is as described above for Chemical Formula I and Chemical Formulas IA to ID.

[0040] In one specific example, R of the above chemical formulas I-1 to I-3 1may be H, halogen or C1-C3 haloalkyl (e.g., C1-C3 alkyl substituted with F or Cl). For example, in the above formulae I-1 to I-3, R 1 can be H, F or Cl. In formula I-3, two R 1 may be identical or different.

[0041] In one specific example, R of the above chemical formulas I-1 to I-3 2 is CN or -CONH2.

[0042] In one specific example, X in the above formulas I-1 to I-3 is N, and Y is CR a It can be. In this case, R a is H or C1-C6 alkyl. For example, R a is H or methyl.

[0043] Meanwhile, International Publication Nos. WO 2021 / 099837 and WO 2022 / 107044 disclose that compounds having a pyrimidine core fused to a 5-membered heterocycle are useful for treating neurodegenerative diseases such as Parkinson's disease through adenosine receptor antagonist activity. Based on the core disclosed in the International Publication Nos., the present inventors surprisingly discovered that when 3-CF3 is introduced to a benzyl group bonded to a 5-membered ring and a CN or amide group at the 5-position together with 3-CN is introduced to a phenyl group bonded to a 6-membered ring, it does not act on adenosine receptors but selectively acts on CXCR7 receptors, exhibiting excellent CXCR7 modulating activity.

[0044] Therefore, the compound of formula (I) according to the present invention comprises 3-CF3 of the benzyl group bonded to the core and 3-CN of the phenyl group bonded to the core, and 5-CN or 5-CONR b R c It has a characteristic structure.

[0045]

[0046] The compound of the above formula I may be a compound selected from Table 1 below:

[0047]

[0048]

[0049]

[0050]

[0051] Compounds of the present invention, including stereoisomers, isotopically labeled compounds, hydrates, solvates and salts, can be prepared by known organic synthetic methods and can be synthesized via a number of synthetic routes.

[0052] The reaction for preparing the compound of the present invention can be performed in a suitable solvent that can be appropriately selected by those skilled in the art of organic synthesis. Suitable solvents are those that are substantially non-reactive with the starting materials (reactants), intermediates, or target products at the temperature at which the reaction occurs. Those skilled in the art will be able to appropriately select the appropriate solvent for each specific reaction step.

[0053] During the synthesis of the compound of the present invention, protection and deprotection of various functional groups can be achieved. Those skilled in the art will readily be able to determine the necessity of protection and deprotection and select appropriate protecting groups.

[0054] Each reaction can be monitored by any suitable method known in the art. For example, the synthesis of the target compound can be monitored by spectroscopic means, such as NMR (e.g., 1H or 13C), mass spectroscopy, or chromatography (HPLC or TLC).

[0055] If necessary, the compounds of the present invention can be purified by, for example, chromatography, crystallization from a solvent or solvent mixture, distillation, extraction, etc. Chromatography can be, but is not limited to, reverse-phase, normal phase, size exclusion, ion exchange, preparative, or flash chromatography. Those skilled in the art will readily be able to select the optimal technique for purifying the target compound.

[0056] If the compounds of the present invention are stereoisomers, they can be isolated from the racemic mixture by any suitable method, if necessary. For example, separation by formation of ionic or diastereomeric salts using chiral compounds and fractional crystallization, formation and separation of diastereoisomers using chiral derivatizing reagents and subsequent conversion to pure stereoisomers, and methods for directly separating substantially pure stereoisomers under chiral conditions can be used.

[0057] The compound of the present invention can be synthesized according to the synthetic process described in the examples below, and based on this, the target compound can be manufactured by appropriately changing the reactants and reaction conditions according to the structure of the target compound.

[0058] Specifically, the compound of formula I according to the present invention can be prepared by introducing an appropriate substituent corresponding to the structure of the target compound into an intermediate having a pyrazolopyrimidine core (e.g., intermediate S1, intermediate S2, intermediate S3), a pyrrolopyrimidine core (e.g., intermediate S4), a purine core (e.g., intermediate S5), or a triazolopyrimidine core (e.g., intermediate S6).

[0059] The preparation method of the intermediate having each of the above cores and the exemplary compounds according to the present invention in which appropriate substituents are introduced therein are described in detail in the following Preparation Examples. As an example, the compound of the present invention having a pyrazolopyrimidine core can be prepared according to the following Reaction Scheme I.

[0060] [Reaction Formula I]

[0061]

[0062] According to the above reaction scheme I, the compound of the present invention having a pyrazolopyrimidine core can be prepared through the following steps 1 to 4:

[0063] Step 1: Dissolving 2-amino-4,6-dichloropyrimidine-5-carbaldehyde in a solvent of tetrahydrofuran and H2O, and subjecting the solution to a chain reaction with hydrazine monohydrate at 50°C for 3 hours to prepare intermediate S1.

[0064] Step 2a: Dissolve intermediate S1 in N,N dimethylformamide solvent and add potassium carbonate or cesium carbonate at room temperature for 4 hours to obtain R 1 and a step of nucleophilic substitution reaction with a benzene bromide compound substituted with CF3

[0065] Step 3a: A step of preparing a compound of formula I in which 3,5-CN is substituted on the phenyl group by dissolving the product of step 2a in 1,4-dioxane and distilled water and then subjecting the solution to a Suzuki coupling reaction with 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile at 110°C for 12 hours.

[0066] Step 4: If necessary, a step of preparing a compound of formula I in which 3-CN and 5-amide are substituted on the phenyl group by reacting at 70°C for 40 minutes under the conditions of InCl3 and acetaldoxime through additional monocyano hydrolysis.

[0067] Alternatively, steps 2a and 3a may be performed in reverse order, similar to steps 2b and 3b of the above reaction scheme. The above reaction conditions, reaction times, reagents, etc. are merely examples, and those skilled in the art will be able to appropriately modify and prepare the compounds of the present invention based on the disclosures of the specification, including the examples herein.

[0068] definition

[0069] All technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art, and unless otherwise stated, conventional measuring methods, manufacturing methods, conventional ingredients or materials based on conventional techniques of pharmacology, pharmaceutical manufacturing, mass spectrometry, NMR, HPLC, biochemistry, etc. are used.

[0070] The individual features and components of each embodiment described and illustrated in this specification may be combined with the features and components of any other embodiment without departing from the scope or spirit of the present disclosure.

[0071] Unless otherwise specified, in this specification and any attached claims, "and" and "or" mean "and / or." The terms "comprises" and "comprised" are open-ended, meaning that the compound, composition, or method may include additional features or components in addition to the specific features or components listed.

[0072] In this specification, a numerical range indicated using the term “to” refers to a range that includes the numerical values ​​described before and after the term “to” as the lower and upper limits, respectively.

[0073] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the term "optionally substituted" means that the occurrences include instances where the occurrences are either substituted or unsubstituted with the specified substituent.

[0074] compound

[0075] The term "halogen" refers to an atom belonging to Group 17 of the periodic table. Halogen atoms include fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). The term "halogen" may be used interchangeably with the term "halo," which refers to a monovalent functional group composed of a halogen.

[0076] The term "hydroxy" refers to the -OH functional group (hydroxyl group).

[0077] The term "-CN" or "cyano" refers to a functional group consisting of a triple bond between a carbon atom and a nitrogen atom.

[0078] The term "amino" refers to a nitrogen atom with hydrogen attached to it, i.e. -NH2.

[0079] The term "alkyl" refers to a fully saturated branched or unbranched (or straight-chain or linear) hydrocarbon. The alkyl may be a substituted or unsubstituted alkyl group. The alkyl may be an alkyl group having a carbon number of C1 to C6, C1 to C5, C1 to C4, C1 to C3, or C1 to C2. Non-limiting examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, iso-amyl, or n-hexyl.

[0080] The term "haloalkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group having a specified number of carbon atoms, substituted with one or more halogen atoms. Haloalkyl groups include perhaloalkyl groups, wherein all hydrogens of the alkyl group are replaced with halogens (e.g., -CF3, -CF2CF3). The halogens may be the same (e.g., CHF2, -CF3) or different (e.g., CF2Cl). Where specified, a haloalkyl group may be optionally substituted with one or more substituents other than halogen. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, dichloroethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl groups.

[0081] As used herein, the term "alkylene" refers to a divalent, fully saturated, branched or unbranched (or straight-chain or linear) hydrocarbon having the formula -C n H 2n - refers to a functional group expressed as a. For example, C 1- C6 alkylene may include ethylene, propylene, butylene, and hexylene.

[0082] The term "alkenyl" denotes a linear or branched hydrocarbyl group having 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms, and at least one site of vinyl unsaturation (>C=C<). For example, (C x -C y )Alkenyl represents an alkenyl group having x to y carbon atoms, and may include, for example, ethenyl, propenyl, isopropylene, 1,3-butadienyl, etc.

[0083] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" may also include hydrocarbyl groups having one triple bond and one double bond. For example, (C2-C6)alkynyl may include ethynyl, propynyl, and the like.

[0084] The term "alkoxy" refers to a substituent in which a substituted or unsubstituted straight or branched chain alkyl moiety is linked to another chemical structure by an oxygen atom. The alkoxy may include, without limitation, all possible isomers thereof, such as methoxy, ethoxy, propoxy, and butoxy, or isopropoxy, isobutoxy, and t-butoxy.

[0085] The term "substitution" in the above "optionally substituted" refers to introducing a substituted hydrogen atom in the case where one or more hydrogen atoms in an organic compound are replaced with another atomic group to form a derivative, and "substituent" refers to the introduced atomic group. As used herein, a "substituted" group is one in which one or more hydrogen atoms are replaced with one or more non-hydrogen atomic groups, provided that the valence requirement is satisfied and a chemically stable compound is generated from the substitution. As used herein, unless explicitly described as "unsubstituted," all substituents should be interpreted as being either substituted or unsubstituted.

[0086] In this specification, when a combination of substituents is referred to as one group, for example, haloalkyl, hydroxyalkyl, etc., it generally contains the atom attached to the end of the molecule as the last group mentioned.

[0087] In this specification " ", "*" or "-" are used to indicate the position at which the substituent is bonded to the residue of the compound. For example, when - is displayed at the end of a substituent, it means that the end is bonded to the remaining residue of the compound. Also, when two or more substituents are connected with "-", it means that the substituent immediately before "-" is bonded to the substitutable atom of the substituent immediately after "-".

[0088] The term "isotope" as used herein means an element of the same element but with different mass numbers, i.e., an element having the same number of protons but a different number of neutrons. For example, 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl, 125 I may include, but is not limited to, compounds labeled with isotopes may have advantages in terms of improved stability in the body and longer half-lives.

[0089] The term "solvate" as used herein may refer to a compound of the present invention or a salt thereof that contains a stoichiometric or non-stoichiometric solvent bound by non-covalent intermolecular forces. Preferred solvents include those that are volatile, non-toxic, and / or suitable for human administration. Those skilled in the art will readily be able to prepare solvates, such as hydrates, of the compounds disclosed herein using appropriate techniques known in the art.

[0090] The term "stereoisomer" as used herein may mean a compound of the present invention or a salt thereof having the same chemical or molecular formula but different optically or sterically, and specifically may be a diastereomer, an enantiomer, or a geometric isomer.

[0091] In some embodiments, the compounds of the present invention may contain one or more asymmetric centers, and may be in the form of racemates, single enantiomers, mixtures of enantiomers, single diastereomers, mixtures of diastereomers, etc. In one embodiment, due to the nature or restricted rotation of the asymmetric center, the compounds of the present invention may exist in the form of enantiomers or diastereomers.

[0092] When two or more asymmetric centers are present in the compounds of the present invention, multiple diastereoisomers and enantiomers of the chemical structures disclosed herein may exist, and all such pure isomers, isolated isomers, partially pure isomers, or racemic mixtures are intended to fall within the scope of the present invention.

[0093] Purification of the above isomers and separation of the isomer mixture can be achieved by standard techniques known in the art. For example, a diastereomeric mixture can be separated into individual diastereoisomers by chromatographic processes or crystallization, and racemates can be separated into individual enantiomers by chiral phase chromatographic processes or resolution.

[0094] The term "salt" refers to inorganic and organic acid addition salts of a compound. The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. The pharmaceutically acceptable salts may be salts that do not cause serious irritation to the organism to which the compound is administered and do not impair the biological activity and physical properties of the compound. The inorganic salts may be hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salt may be formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl, trichloroacetic acid, trifluoroacetate, benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspartate. In addition, the metal salt may be calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

[0095] A pharmaceutically acceptable salt of a compound according to the present invention can be prepared by dissolving the compound of formula I in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, adding an excess of an organic acid or an aqueous solution of an inorganic acid, and then precipitating or crystallizing the mixture. Subsequently, the solvent or the excess of acid is evaporated from the mixture, followed by drying to obtain an addition salt, or the precipitated salt can be prepared by suction filtration.

[0096] Medicinal uses, pharmaceutical compositions and methods of administration

[0097] Another aspect provides a pharmaceutical composition comprising a compound according to one aspect, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof. The compound, stereoisomer, isotopically labeled compound, solvate, and salt are as described above.

[0098] A compound according to one aspect of the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, selectively acts on the CXCR7 receptor and is useful for treating CXCR7-mediated diseases.

[0099] The compounds, stereoisomers, isotopically labeled compounds, hydrates, solvates, or pharmaceutically acceptable salts thereof according to the present invention selectively activate CXCR7 and activate its downstream signaling pathways, and provide high specificity for CXCR7, improved pharmacokinetic properties, reduced off-target effects, and improved efficacy in modulating cellular responses associated with CXCR7 activation. As used herein, a "CXCR7-mediated disease" may be a disease caused by a dysfunction of a series of pathways consisting of CXCR7, its ligands CXCL12 and CXCL11, and CXCR4 that binds to CXCL12. For example, the compounds according to the present invention may exhibit therapeutic effects on various diseases by counteracting the effects of CXCL12 through the activation of CXCR7.

[0100] The above CXCR7-mediated diseases include fibrosis, cholestatic liver disease, inflammatory diseases, angiogenic diseases, cancer, autoimmune disorders, osteoporosis, pulmonary hypertension, acute ischemic injury, and thrombosis.

[0101] In one embodiment, the compound according to the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, may be useful in the treatment of fibrosis through selective modulatory activity on CXCR7.

[0102] In one embodiment, the fibrosis comprises fibrosis of the liver, lungs, skin, kidneys, heart, joints, or intestines.

[0103] In one specific example, the fibrosis may be selected from the group consisting of liver fibrosis, liver cirrhosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, renal fibrosis, cardiac fibrosis, and arthrofibrosis. For example, the fibrosis may be liver fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, or cardiac fibrosis.

[0104] Fibrosis is a pathological process characterized by the excessive accumulation of fibrous connective tissue, forming scar tissue within an organ or tissue. This abnormal tissue formation disrupts the normal structure and function of the affected organ, affecting various organs, including the lungs (pulmonary fibrosis), liver (cirrhosis), heart (cardiac fibrosis), kidneys, and skin.

[0105] CXCR7 has been reported to have an anti-fibrotic effect by participating in the Wnt / β-catenin pathway, and this effect is particularly evident in the process of angiogenesis (CXCR7 Inhibits Fibrosis via Wnt / β-Catenin Pathways during the Process of Angiogenesis in Human Umbilical Vein Endothelial Cells, BioMed Research International, 2020).

[0106] Overexpression of CXCR7 was observed to suppress fibrosis through the Wnt / β-catenin pathway during angiogenesis in human umbilical vein endothelial cells (HUVECs), implying that CXCR7 effectively influences the vascular fibrosis switch in the pathophysiology of angiogenesis.

[0107] A significant antifibrotic effect of CXCR7 agonist has been reported in a carbon tetrachloride (CCl4)-induced liver fibrosis model (Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis, Nature 2013; Stimulation of the atypical chemokine receptor 3 (ACKR3) by a small-molecule agonist attenuates fibrosis in a preclinical liver but not lung injury model, Cell Mol Life Sci 2022).

[0108] Additionally, a study has reported that a selective agonist of CXCR7 significantly reduces isoproterenol-induced cardiac fibrosis (Discovery of a Novel Small-Molecule Modulator of CXC Chemokine Receptor Type 7 as a Treatment for Cardiac Fibrosis, J Med Chem 2018).

[0109] Without being bound by theory, CXCR7 is thought to play a role in fibrosis, including:

[0110] (1) Fibroblast activation and proliferation: CXCR7 signaling may affect the activation and proliferation of fibroblasts, which are the main cause of excessive deposition of extracellular matrix proteins observed in fibrotic tissues.

[0111] (2) Inflammation and immune response: CXCR7 plays a role in regulating inflammatory responses and immune cell migration. In fibrosis, chronic inflammation is often accompanied by tissue scarring, and CXCR7 may contribute to this.

[0112] (3) Angiogenesis and tissue remodeling: CXCR7 influences angiogenesis, which may significantly impact tissue scarring during fibrosis.

[0113] The present inventors experimentally demonstrated that the compound according to the present invention significantly reduces fibrosis in an idiopathic pulmonary fibrosis model and a liver fibrosis model.

[0114] In one embodiment, the compound, stereoisomer, isotopically labeled compound, hydrate, solvate, or pharmaceutically acceptable salt thereof according to the present invention may be useful for treating cholestatic liver disease through selective modulatory activity on CXCR7. Cholestatic liver disease is a general term for biochemical, physiological, and clinical changes due to circulatory disorders of bile produced in the liver and circulating through the biliary tract and intestine. For example, the cholestatic liver disease may include, but is not limited to, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), cholesterol gallstones, gestational intrahepatic cholestasis, progressive familial intrahepatic cholestasis (PFIC), or Alagille syndrome.

[0115] CXCR7 can exert significant therapeutic effects in various pathologies, primarily through mechanisms such as suppression of liver inflammation, inhibition of fibrosis, cytoprotection, and regulation of cholesterol metabolism. For example, CXCR7 activation can reduce the expression of inflammatory cytokines and suppress immune cell overactivation in primary biliary cholangitis (PBC), an autoimmune liver disease, thereby preventing hepatocellular damage (CXCR7's anti-inflammatory effects in autoimmune liver diseases, Zhang et al., 2022, Autoimmunity Reviews, 21(8), 125-134). In addition, CXCR7 may contribute to inhibiting the progression of fibrosis and protecting bile duct epithelial cells by regulating the TGF-β signaling pathway in primary sclerosing cholangitis (PSC), which is characterized by chronic inflammation and fibrosis of the bile duct (CXCR7 as a therapeutic target in chronic cholangitis, Lin et al., 2021,Liver International, 41(2), 210-222). In terms of preventing cholesterol gallstones, CXCR7 agonists have also been reported to regulate cholesterol metabolism and excretion in the liver and affect bile acid metabolism, thereby inhibiting gallstone formation (Role of CXCR7 in cholesterol metabolism and gallstone prevention, Huang et al., 2020,Journal of Hepatology, 74(5), 1120-1130). In addition, in cases of intrahepatic cholestasis of pregnancy, CXCR7 activation may protect hepatocytes and improve bile flow, thereby reducing liver damage due to bile acid accumulation (The hepatoprotective role of CXCR7 in intrahepatic cholestasis of pregnancy, Wang et al., 2021, Placenta, 105, 150-160). In the genetic cholestatic disease progressive familial intrahepatic cholestasis (PFIC), liver function protection can be expected by suppressing overactivation of inflammatory chemokine pathways and promoting hepatocyte survival through CXCR7 modulation (Modulation of CXCR7 in genetic cholestasis disorders, Lee et al., 2023, Hepatology Communications, 6(3), 330-340). In this respect, CXCR7 agonists may provide an important therapeutic approach in cholestatic liver diseases.

[0116] In particular, the inventors experimentally confirmed that the compound of the present invention reduces bile duct proliferation, portal inflammation and confluent necrosis and significantly inhibits liver tissue fibrosis in a cholestatic liver disease model induced by bile duct ligation.

[0117] In one embodiment, the compounds of the present invention, stereoisomers, isotopically labeled compounds, hydrates, solvates, or pharmaceutically acceptable salts thereof may be useful for the treatment of inflammatory diseases through their selective modulatory activity against CXCR7. For example, the inflammatory diseases may include, but are not limited to, arthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, myocarditis, sarcoidosis, or sinusitis.

[0118] It has been reported that CXCR7 agonist reduces the effects of RANKL and TNF-α-related cytokines induced by LPS (CXC receptor 7 agonist acts as a CXC motif chemokine ligand 12 inhibitor to ameliorate osteoclastogenesis and bone resorption, Molecular Medicine REPORTS 2022, 25(3), 78), so the compound of the present invention exhibiting selective regulatory activity against CXCR7 will be useful for the treatment of diseases caused by persistent inflammatory responses such as arthritis.

[0119] In one embodiment, the compound according to the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, may be useful in the treatment of angiogenic diseases through selective modulatory activity toward CXCR7. For example, angiogenic diseases may include, but are not limited to, rheumatoid arthritis, psoriasis, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, posterior lens fibroplasia, rosacea, Osler-Weber syndrome, myocardial neovascularization, plaque neovascularization, telangiectasias, hemophilic joints, angiofibromas, intestinal adhesions, Crohn's disease, eczema, scleroderma, diabetes, atherosclerosis, wound granulation, or keloids.

[0120] Previous studies have shown that CXCR7 activation inhibits CXCL12-induced HUVEC tube formation and reduces CXCR4 protein levels. These findings suggest that when CXCR7 expression is increased by inflammation, CXCR7 dimerizes with CXCR4, inducing its internalization and degradation, thereby acting as a negative regulator of CXCR4. Therefore, it has been proposed that CXCR7 activation may have therapeutic effects against CXCL12-induced diseases by counteracting the effects of CXCL12 (CXCR7 agonists inhibit the function of CXCL12 by down-regulating CXCR4, Biochem Biophys Res Commun 2013).

[0121] In this regard, CXCL12 promotes tumor growth, angiogenesis, and HCC metastasis in hepatocellular carcinoma (HCC). High CXCR4 expression has been previously known to indicate an unfavorable prognosis in HCC patients. Tumor-infiltrating myeloid-derived suppressor cells (MDSCs) also express CXCR4 and migrate toward CXCL12. Therefore, CXCL12 inhibition not only directly blocks HCC growth but also alters the tumor environment (angiogenesis, MDSCs), making HCC patients more sensitive to existing treatments. Therefore, indirect regulation of CXCL12 and CXCR4 by CXCR7 agonists has therapeutic utility in hepatitis, liver cancer, and liver fibrosis. (Stromal cell-derived factor-1 (SDF-1) as a target in liver diseases TC-14012, Am J Physiol Gastrointest Liver Physiol 2016).

[0122] In one embodiment, a compound according to the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof may be useful in the treatment of cancer through selective modulatory activity against CXCR7.

[0123] In this application, “cancer” is a general term for a disease caused by cells that have aggressive characteristics in which cells divide and grow while ignoring normal growth limits, invasive characteristics in which cells invade surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body. For example, the cancer includes, but is not limited to, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, large intestine cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, stomach cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, genital cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, glioblastoma, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. In one specific embodiment, the cancer for which the compound according to the present invention can exhibit a therapeutic effect through selective modulating activity for CXCR7 is hepatobiliary cancer. In one specific example, the cancer for which the compounds of the present invention, etc., can exhibit therapeutic effects through selective modulatory activity against CXCR7 is breast cancer. In particular, the inventors of the present invention confirmed that the compounds of the present invention, etc., exhibit a cancer metastasis-inhibiting effect by inhibiting CXCL12-mediated breast cancer cell migration.

[0124] CXCR7 agonists can inhibit tumor progression by binding to CXCL12 and blocking the interaction with CXCR4 in an environment where the CXCL12 / CXCR4 pathway is activated and cancer cell migration and metastasis are promoted. Furthermore, CXCR7 agonists can prevent cancer metastasis and suppress tumor spread by reducing the ability of cancer cells to migrate and invade surrounding tissues in the tumor microenvironment. CXCR7 agonists have also been reported to inhibit angiogenesis around tumors, preventing the delivery of oxygen and nutrients to cancer cells, and modulating the recruitment of immune cells, thereby altering the tumor microenvironment. Specifically, CXCR7 acts as a competitive receptor for CXCL12 in breast cancer cells, blocking CXCR4 signaling and thereby inhibiting tumor metastasis (CXCR7 antagonizes CXCR4 in breast cancer cell migration and invasion, Miao et al., 2019, Cancer Letters). In addition, it has been reported that treatment of breast cancer cell lines with VUF11207, a CXCR7 agonist, effectively reduces the motility and invasiveness of cancer cells by binding to both receptors, internalizing both CXCR4 and CXCR7 into the cells and inhibiting their cell surface expression (VUF11207, a Biased Ligand for CXCR4 and ACKR3, Induces Internalization and Degradation of Both Receptors in Breast Cancer Cell Lines; Int J Mol Sci. 2018 Nov 14;19(11):3592).CXCR7 activation is known to contribute to the inhibition of cancer growth by inhibiting tumor angiogenesis in densely vascularized cancers such as hepatocellular carcinoma, thereby blocking oxygen and nutrient supply (Inhibition of angiogenesis and hepatocellular carcinoma growth by targeting CXCR7 signaling pathway, Liu et al., 2021, Journal of Experimental & Clinical Cancer Research). In ovarian and prostate cancers, CXCR7 activation can prevent cancer metastasis by inhibiting tumor cell motility (The CXCR7 / CXCL12 axis in ovarian cancer: implications for tumor progression and therapy, Wang et al., 2020, Journal of Cancer Research and Clinical Oncology). In addition, CXCR7 has been reported to play an important role in regulating the recruitment of immune cells, suppressing immune evasion of cancer cells, and promoting anticancer immune responses (CXCR7 promotes tumor immune evasion by altering the recruitment of immune cells, Miao et al., 2020, Cancer Immunology Research). Recently, it has been reported that CXCR7 activation in neuronal cancers such as glioblastoma can suppress tumor progression by inhibiting tumor motility and invasiveness and enhancing neural tissue protection mechanisms (CXCR7-mediated regulation of glioma cell migration and proliferation, Li et al., 2022, Frontiers in Oncology). In a glioblastoma mouse model, knockdown of CXCR7 worsens survival outcomes and increases PD-L1 expression compared to controls.In vitro T cell experiments showed that VUF11207, a selective CXCR7 agonist, enhanced tumor cell cytotoxicity and reversed immunosuppression in glioblastoma cell-macrophage-T cell cocultures. The CXCR7 agonist VUF11207 inhibits PD-L1 expression and restores the function of intratumoral CD8+ T cells, demonstrating a synergistic effect that improves survival when combined with anti-PD-L1 antibodies, suggesting a novel therapeutic approach to enhance the efficacy of immune checkpoint inhibitors in glioblastoma (CXCR7 activation evokes the anti-PD-L1 antibody against glioblastoma by remodeling CXCL12-mediated immunity; Liu et al, Cell Death & Disease volume 15,434,2024). Therapeutic strategies targeting CXCR7 offer a promising approach to inhibit cancer progression and overcome the limitations of existing anticancer treatments through these diverse mechanisms.

[0125] In one specific embodiment, the compound according to the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof may be useful for the treatment of an autoimmune disorder through selective modulatory activity against CXCR7. For example, the autoimmune disorder includes, but is not limited to, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, lupus nephritis, interstitial cystitis, celiac disease, autoimmune encephalomyelitis, a dehydrating disease, osteoarthritis, or type I diabetes. For example, an autoimmune disorder that can be treated with the compound of the present invention may be multiple sclerosis. The inventors of the present invention confirmed that the compound of the present invention exhibits an excellent autoimmune disease therapeutic effect in an autoimmune disease disease model, particularly an experimental autoimmune encephalomyelitis (EAE) mouse model, which is widely known as a multiple sclerosis model.

[0126] In one specific embodiment, the compound according to the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, may be useful in the treatment of osteoporosis through selective modulatory activity on CXCR7.

[0127] It has been reported that a CXCR7 agonist can reduce the effects of LPS-induced RANKL and TNF-α-related cytokines, and at the same time, reduce osteoclastogenesis and bone resorption induced by these (CXC receptor 7 agonist acts as a CXC motif chemokine ligand 12 inhibitor to ameliorate osteoclastogenesis and bone resorption, Molecular Medicine REPORTS 2022, 25(3), 78). Without being bound by theory, the compound of the present invention having selective modulatory activity against CXCR7 may be useful for the treatment of osteoporosis caused by an imbalance between bone formation and bone resorption.

[0128] In one specific embodiment, a compound according to the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, may be useful for the treatment of pulmonary hypertension through selective modulatory activity against CXCR7. Pulmonary hypertension includes, but is not limited to, pulmonary arterial hypertension or chronic thromboembolic pulmonary hypertension.

[0129] Agonism / activation of CXCR7 is a promising mechanism for the treatment of pulmonary arterial hypertension (PAH), which can inhibit disease progression through various mechanisms. CXCR7 activation is known to alleviate pulmonary endothelial cell damage caused by PAH by promoting the survival and function of vascular endothelial cells and inhibiting apoptosis (Chen, L., & Hu, X. (2020). "CXCR7-mediated signaling pathways in cardiovascular disease." International Journal of Molecular Sciences, 21(7), 2524). It suppresses inflammation in the lungs through anti-inflammatory effects that reduce the inflammatory response that promotes PAH progression by inhibiting the activation of inflammatory cells, and regulates the CXCL12 signaling pathway together with CXCR4 to balance the angiogenesis and regeneration process, and reduces the increase in pulmonary blood pressure by inhibiting the CXCR4-dependent pathway (Wang, Y., He, H., Chen, L., & Hao, D. (2021). "Potential role of CXCR7 in the progression of pulmonary arterial hypertension." Frontiers in Pharmacology, 12, 623795). CXCR7 agonism is known to play an important role in alleviating vascular occlusion, one of the major pathological findings of PAH, by inhibiting smooth muscle cell proliferation during the vascular remodeling process (Rajagopal, S., Kim, J., Ahn, S., Craig, S., Lam, CM, Gerard, NP, ... & Lefkowitz, RJ (2010). "Beta-arrestin-but not G protein-mediated signaling by the 'decoy' receptor CXCR7."Proceedings of the National Academy of Sciences, 107(2), 628-632). Based on this, a novel therapeutic approach aimed at controlling the pathological progression of PAH through CXCR7 activation and ultimately improving the clinical condition can be proposed.

[0130] In one embodiment, the compound according to the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof may be useful in the treatment of acute ischemic injury and thrombosis through selective modulatory activity on CXCR7.

[0131] Single-cell RNA sequencing analysis has shown that CXCR7 is the most abundantly expressed gene in cardiomyocytes and fibroblasts among 510 7TMR genes, and cardiomyocyte-specific CXCR7 null mice have been reported to exhibit significant cardiac enlargement and functional decline compared to control mice 4 weeks after myocardial infarction. These results suggest that CXCR7, abundantly expressed in cardiomyocytes, may have a protective function in the heart as a β-arrestin-biased receptor, and that CXCR7 agonists have a therapeutic effect on myocardial infarction (CXCR7 ameliorates myocardial infraction as a β-arrestin-biased receptor, Scientific reports 2021).

[0132] Inhibition of platelet activation plays a pivotal role in the treatment of acute organ ischemia. CXCR7 expression in platelets of patients with coronary artery disease (CAD) is associated with clinical prognosis, and genetic deficiency of platelet CXCR7 is known to cause platelet activation and damage to ischemic myocardium and brain tissue, thereby aggravating tissue inflammation and systemic thrombotic inflammation. In this regard, activation of platelet-CXCR7 through a CXCR7 agonist (VUF11207) has been reported to have an inhibitory effect on platelet activation and thrombus formation, and can alleviate ischemic myocardium and brain tissue damage (ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury, Nature Communications, 2022; Discovery and Development of First-in-Class ACKR3 / CXCR7 Superagonists for Platelet Degranulation Modulation, J Med Chem, 2022). Without being bound by theory, compounds of the present invention having selective modulatory activity toward CXCR7 would be useful in the treatment of acute organ ischemic injury, such as ischemic heart injury, and thrombosis.

[0133] As used herein, the term "treating" or "treatment" means inhibiting a disease, condition or disorder, e.g., preventing further development of the pathology and / or symptoms in a subject experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder, or ameliorating a disease, condition or disorder, e.g., ameliorating a disease, condition or disorder, e.g., reversing the pathology and / or symptoms, e.g., reducing disease severity.

[0134] The pharmaceutical composition may comprise, in addition to the compound of the present invention, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, an additional therapeutically active agent. In this case, the compound of the present invention and the additional therapeutically active agent may be a single composition or separate compositions. For example, the compound of the present invention may be provided as a composition in an oral dosage form, and the additional therapeutically active agent may be provided in a parenteral dosage form, or the compound of the present invention may be provided as a parenteral dosage form, and the additional therapeutically active agent may be provided in an oral dosage form. When the compound of the present invention is used for the treatment of fibrosis, it may be administered simultaneously, sequentially, or separately with, for example, nintedanib, pirfenidone, etc. When the compound of the present invention is used for the treatment of pulmonary arterial hypertension, it may be administered simultaneously, sequentially, or separately with, for example, sildenafil, etc. When the compound of the present invention is used for the treatment of multiple sclerosis, it may be administered simultaneously, sequentially, or separately with, for example, fingolimod (FTY720).

[0135] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used to mean an excipient, diluent, or auxiliary. The carrier may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, saline, a buffer such as PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0136] The pharmaceutical composition described above may be prepared in any dosage form according to conventional methods. For example, the composition may be formulated as an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral dosage form (e.g., injection). Furthermore, the composition may be prepared as a systemic dosage form or a topical dosage form.

[0137] In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid preparation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the liquid preparation for oral administration may be a suspension, an oral solution, an emulsion, or a syrup. The liquid preparation may include water or liquid paraffin. The liquid preparation may include an excipient such as a wetting agent, a sweetener, a flavoring agent, or a preservative. In the above pharmaceutical composition, the preparation for parenteral administration may be a sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized product, or suppository. The non-aqueous solvent or suspension may contain a vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, Tween 61, cocoa butter, laurin butter, or glycerogelatin.

[0138] The pharmaceutical composition comprises a compound according to one aspect, a stereoisomer thereof, an isotopically labeled compound, a solvate, or a pharmaceutically acceptable salt thereof as an active ingredient of the pharmaceutical composition. The term "active ingredient" refers to a physiologically active substance used to achieve pharmacological activity (e.g., an antifibrotic effect).

[0139] The pharmaceutical composition may comprise an effective amount of a compound according to one aspect, a stereoisomer thereof, an isotopically labeled compound, a solvate, or a pharmaceutically acceptable salt thereof. The term "effective amount" refers to an amount sufficient to exhibit the effect of preventing or treating a disease when administered to a subject in need of prevention or treatment. The effective amount can be appropriately selected by those skilled in the art depending on the selected cell or subject. The preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the subject, the degree of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, the compound, its stereoisomer, isotopically labeled compound, solvate, or pharmaceutically acceptable salt may be administered in divided doses of, for example, about 0.0001 mg / kg to about 100 mg / kg, or about 0.001 mg / kg to about 100 mg / kg, once to 24 times a day, once to 7 times every 2 days to 1 week, or once to 24 times every 1 month to 12 months. In the pharmaceutical composition, the compound, its stereoisomer, isotopically labeled compound, solvate, or pharmaceutically acceptable salt may be included in an amount of about 0.0001 wt% to about 10 wt%, or about 0.001 wt% to about 1 wt%, based on the total weight of the entire composition.

[0140] Administration may be oral or parenteral. For example, the route of administration may be oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal. The composition may be administered systemically or locally, and may be administered alone or in combination with other therapeutically active agents.

[0141] Another aspect provides a method for treating a disease mediated by CXCR7, comprising administering to a subject a compound according to one aspect, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof. The compound, stereoisomer, isotopically labeled compound, a hydrate, a solvate, a pharmaceutically acceptable salt, and the disease treatable therefor are as described above.

[0142] Another aspect provides the use of a compound according to one aspect, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of a disease mediated by CXCR7. The compound, stereoisomer, isotopically labeled compound, hydrate, solvate, pharmaceutically acceptable salt, and the disease treatable therefor are as described above.

[0143] According to one aspect, the compound, its use as a CXCR7 receptor modulator, a pharmaceutical composition comprising the same, and its use for the treatment of CXCR7-mediated diseases, the compound has excellent CXCR7 modulating activity and may be useful in the treatment of fibrosis, cholestatic liver disease, inflammatory diseases, angiogenic diseases, cancer, autoimmune disorders, osteoporosis, pulmonary hypertension, acute ischemic injury, and thrombosis.

[0144] Figure 1 shows the results of BALF absolute differential cell counts in a bleomycin-induced IPF model. Data are presented as mean ± standard deviation; #p<0.05, ##p<0.01, ###p<0.001 (vs. normal control); *p<0.05, **p<0.01, ***p<0.001 (vs. bleomycin + vehicle); One-way or Two-way ANOVA followed by Dunnett's multiple comparisons test; n=10-12.

[0145] Figure 2 shows the results of measuring lung hydroxyproline content in a bleomycin-induced IPF model. Data are presented as mean ± standard deviation; ###p <0.001 (vs. normal control); ***p <0.001 (vs. bleomycin + vehicle); One-way ANOVA followed by Dunnett's multiple comparisons test; n=10-12.

[0146] Figure 3 illustrates the results of evaluating the Ashcroft Fibrosis Score in a bleomycin-induced IPF model. Data are presented as mean ± standard deviation; ###p <0.001 (vs. normal control); **p <0.01, ***p <0.001 (vs. bleomycin + vehicle); one-way ANOVA followed by Dunnett's multiple comparisons test; n = 10-12.

[0147] Figure 4 illustrates the results of measuring hepatic hydroxyproline in a carbon tetrachloride-induced liver fibrosis model. Data are presented as mean ± standard deviation; ###p <0.001 (vs. normal control); ***p <0.001 (vs. CCl4 + vehicle); One-way ANOVA followed by Dunnett's multiple comparisons test; n=10.

[0148] Figure 5 shows the results of Ishak's fibrosis score measurements in a carbon tetrachloride-induced liver fibrosis model. Data are presented as mean ± standard deviation; ###p <0.001 (vs. normal control); **p<0.01, ***p<0.001 (vs. CCl4 + vehicle); One-way ANOVA followed by Dunnett's multiple comparisons test; n=10.

[0149] Figure 6 shows the results of measuring ALT in a carbon tetrachloride-induced liver fibrosis model. Data are expressed as mean ± standard deviation; ###p<0.001 (vs. normal control); *p<0.05 (vs. CCl4+vehicle); One-way ANOVA followed by Dunnett's multiple comparisons test; n=10.

[0150] Figure 7 shows the results of clinical scores recorded in an autoimmune neuroinflammation model induced by MOG 35-55.

[0151] Figure 8 shows the results of measuring right ventricular systolic pressure (RVSP) in a model of pulmonary arterial hypertension induced by monocrotaline administration. Data are expressed as mean ± standard deviation; ****P<0.0001 (vs. normal control group); # P<0.05 (vs. pulmonary hypertension model)

[0152] Figure 9 shows the results of measuring the ratio of right ventricular weight to left ventricular and interventricular septal weights (RV / LV+S) in a model of pulmonary arterial hypertension induced by monocrotaline administration. Data are expressed as mean ± standard deviation; ****P<0.0001 (vs. normal control group); # P<0.05 (vs. pulmonary hypertension model)

[0153] Figure 10 shows the results of measuring right ventricular wall thickness in a model of pulmonary arterial hypertension induced by monocrotaline administration. Data are expressed as mean ± standard deviation; ****P<0.0001 (vs. normal control group);# P<0.05, ### P<0.001 (vs. pulmonary hypertension model)

[0154] Figure 11 shows the results of measuring the inhibitory effect on cell migration of MDA-MB-231 breast cancer cells induced by CXCL12. **P<0.01 (compared to the CXCL12 treatment group).

[0155] Figure 12 shows the results of measuring AST, ALT, ALP, and total bilirubin (TBIL) in a cholestatic liver disease model induced by bile duct ligation. *p<0.05, **p<0.01, ***p<0.001 (compared to the bile duct ligation model).

[0156] Figure 13 shows the results of the HE Score and a-SMA immunohistochemistry (IHC) quantitative morphometry in a cholestatic liver disease model induced by bile duct ligation, as evaluated by the criteria in Table 6 of this study after H&E staining. *p<0.05, **p<0.01, ***p<0.001 (compared to the bile duct ligation model).

[0157] The following examples are provided to illustrate the present disclosure and should not be construed as limiting the scope of the present technology in any way. All functionally equivalent methods are within the scope of the present technology. Various modifications of the present technology, beyond those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications fall within the scope of the appended claims.

[0158] The meanings of the abbreviations used in the examples below are as follows, and abbreviations not listed below have the meanings commonly used in the relevant fields.

[0159] THF: Tetrahydrofuran

[0160] TEA: Triethanolamine

[0161] DMF: N,N-dimethylformamide

[0162] DCM: Dichloromethane

[0163] TFA: Trifluoroacetic acid

[0164] NaOAc: sodium acetate

[0165] EA: Ethyl Acetate

[0166] TPP: Triphenylphosphine

[0167] TMEDA: N,N,N',N'-tetramethylethylenediamine

[0168] Preparation Example S1: 4-chloro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (Intermediate S1)

[0169]

[0170] To a stirred solution of 2-amino-4,6-dichloropyrimidine-5-carbaldehyde (100 g, 521 mmol) in a mixture of THF (700 mL) and water (300 mL) were added TEA (87.7 mL, 625 mmol) and hydrazine monohydrate (30.7 mL, 625 mmol) at 0 °C, and the resulting mixture was stirred at 50 °C for 3 h. After completion of the reaction, the solvent was distilled off, and the resulting residue was filtered, washed with diethyl ether, and dried in vacuo to give intermediate S1 (80 g, 90.58% yield) as a pale yellow solid. LCMS: m / z = 170 (M+1, ESI+).

[0171] Manufacturing Example S2: 5-(6-amino-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S2)

[0172]

[0173] To a stirred solution of intermediate S1 (5 g, 29.5 mmol) in a mixture of THF (144 mL) and H2O (36 mL) in a sealed tube were added 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (8.99 g, 35.4 mmol) and K2CO3 (8.15 g, 59 mmol) at 25 °C and degassed with argon for 10 min. Then, Pd(PPh3)4 (1.7 g, 1.47 mmol) was added and the resulting mixture was stirred at 110 °C for 12 h. After completion of the reaction, the mixture was cooled to room temperature and filtered through a Celite bed, washed with ethyl acetate (250 mL x 2) and 10% MeOH in DCM (500 mL x 6). The filtrate was concentrated under reduced pressure. The residue was triturated with 10% acetonitrile in diethyl ether to give intermediate S2 (2.4 g, yield 86%) as a pale yellow solid. LCMS: m / z = 262 (M+1, ESI+).

[0174] Preparation Example S3: 4-chloro-1-(3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (Intermediate S3)

[0175]

[0176] To a stirred solution of intermediate S1 (5 g, 29.5 mmol) in dry DMF was added K2CO3 (8.15 g, 59 mmol) at 0°C. After 10 min, 1-(bromomethyl)-3-(trifluoromethyl)benzene (4.52 mL, 29.5 mmol) was added, and the resulting mixture was stirred at 25°C for 4 h. After completion of the reaction, the reaction mixture was quenched with ice water (150 mL), and the resulting precipitate was filtered, washed with ice water (100 mL x 2), and dried in vacuo. The residue was purified by column chromatography to give intermediate S3 (1.7 g, 17.59% yield) as an off-white solid. LCMS: m / z = 328 (M+1, ESI+).

[0177] Preparation Example S4: 5-(2-amino-7H-pyrrolo[2,3-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S4)

[0178]

[0179] To a stirred solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine (4 g, 23.7 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (7.23 g, 28.5 mmol) in a mixture of THF (80 mL) and water (20 mL) was added K2CO3 (6.56 g, 47.5 mmol) at 25 °C, and the mixture was degassed with argon for 10 min. Then, Pd(PPh3)4 (1.37 g, 1.19 mmol) was added, and the resulting mixture was stirred at 100 °C for 16 h. After completion of the reaction, the reaction mixture was filtered through a Celite bed, and the bed was washed with EA (600 mL) and MeOH (200 mL). The filtrate was collected and concentrated to obtain a residue. The residue was triturated with 5% MeOH in DCM to obtain intermediate S4 (2.5 g, 62% yield) as a pale yellow solid. LCMS: m / z = 261 (M+1, ESI+).

[0180] Manufacturing Example S5: 5-(2-amino-9H-purin-6-yl)isophthalonitrile (Intermediate S5)

[0181]

[0182] To a stirred solution of 6-chloro-9H-purin-2-amine (6 g, 22.98 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (7.59 g, 29.88 mmol) in a mixture of 1,4-dioxane (80 mL) and water (20 mL) was added K2CO3 (9.51 g, 68.96 mmol) at 25 °C, and the mixture was degassed with argon for 10 min. Then, Pd(dppf)Cl2.DCM (1.87 g, 2.2 mmol) was added, and the resulting mixture was stirred at 100 °C for 16 h. After the reaction was completed, the reaction mixture was filtered through a Celite bed, and the bed was washed with EA (600 mL) and MeOH (200 mL). The filtrate was collected and concentrated. The residue was purified by column chromatography to give intermediate S5 (3 g, crude) as a pale yellow solid. LCMS: m / z = 262 (M+1, ESI+).

[0183] Manufacturing Example S6: 5-(5-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)isophthalonitrile trifluoroacetate (Intermediate S6)

[0184]

[0185] Step 1: 6-chloro-N4-(4-methoxybenzyl)pyrimidine-2,4,5-triamine

[0186] To a stirred solution of 4,6-dichloropyrimidine-2,5-diamine (5 g, 27.9 mmol) in ethanol (100 mL) were added triethylamine (9.75 mL, 69.8 mmol) and (4-methoxyphenyl)methanamine (7.3 mL, 55.9 mmol) at 0 °C, and the resulting mixture was heated to reflux at 80 °C for 16 h. After the reaction was complete, the reaction mixture was cooled to 25 °C, and the solvent was evaporated. The crude product was dissolved in DCM (100 mL), washed with water (25 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography to give 6-chloro-N4-(4-methoxybenzyl)pyrimidine-2,4,5-triamine (6.0 g, 76.82% yield) as an orange solid. LCMS: m / z = 280 (M+1, ESI+).

[0187] Step 2: 7-chloro-3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine

[0188] Conc. To a stirred solution of 6-chloro-N4-(4-methoxybenzyl)pyrimidine-2,4,5-triamine (2.5 g, 8.94 mmol) in HCl (30 mL) was added dropwise a solution of NaNO2 (925 mg, 13.4 mmol) in water (3 mL) at 0 °C for 20 min. The resulting mixture was then stirred at 0 °C for 30 min. After the reaction was completed, the reaction mixture was poured into a saturated NaHCO3 solution with vigorous stirring. The resulting precipitate was filtered and dried in vacuo to give 7-chloro-3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine (1.8 g, 69.49%) as an off-white solid. LCMS: m / z = 291 (M+1, ESI+).

[0189] Step 3: 5-(5-amino-3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)isophthalonitrile

[0190] To a stirred solution of 7-chloro-3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine (2 g, 6.89 mmol) in 1,4-dioxane (25 mL) and water (8 mL) were added 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (2.1 g, 8.27 mmol) and K2CO3 (1.90 g, 13.79 mmol) at 25 °C, and the mixture was degassed with argon for 10 min. Pd(dppf)Cl2.DCM (0.281 g, 0.344 mmol) was then added, and the resulting mixture was stirred at 100 °C for 12 h. After the reaction was completed, the reaction mixture was diluted with EA (100 mL), washed with water (30 mL) and brine (30 mL), and concentrated. The residue was purified by column chromatography to give 5-(5-amino-3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)isophthalonitrile (1.5 g, 57.03% yield) as an orange solid. LCMS: m / z = 383 (M+1, ESI+).

[0191] Step 4: 5-(5-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)isophthalonitrile trifluoroacetate (Intermediate S6)

[0192] To a stirred solution of 5-(5-amino-3-(4-methoxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)isophthalonitrile (1.3 g, 3.40 mmol) in TFA (20 mL) was added trifluoromethanesulfonic acid (3.46 mL, 39.2 mmol) at 0 °C, and the resulting mixture was stirred at 25 °C for 4 h. After completion of the reaction, the volatiles were evaporated, and the residue was triturated with diethyl ether to give intermediate S6 (1.2 g TFA salt, quantitative yield) as an orange solid. LCMS: m / z = 263 (M+1, ESI+).

[0193] Preparation Example S7: 5-(2-amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S7)

[0194]

[0195]

[0196] Step 1a: 2-chloro-1,1-dimethoxypropane

[0197] To a stirred solution of sulfuryl chloride in DCM (1 M, 100 mL) was added propionaldehyde (70 mL, 976 mmol) at 0°C over 10 minutes. After stirring at 25°C for 1 hour, methanol (240 mL) was added to the reaction mixture over 15 minutes. Vigorous gas evolution was observed during the addition. After stirring at 25°C for 2.5 hours, the DCM was distilled off. The reaction mixture was added saturated NaHCO3 solution (15 mL), extracted with ether, dried over Na2SO4, and concentrated under reduced pressure to give 2-chloro-1,1-dimethoxypropane (40 g crude, quantitative) as a colorless semisolid. The residue was used as is in the next step based on TLC.

[0198] Step 1: 2-Amino-5-methyl-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

[0199] A mixture of 2-chloro-1,1-dimethoxypropane (7.5 mL, 39.6 mmol) in 1 N HCl (22 mL) and ethanol (7 mL) was stirred at 70 °C for 2 h. Cooled to 25 °C. A mixture of 2,6-diaminopyrimidin-4(1H)-one (5 g, 39.6 mmol) and NaHCO3 in water (50 mL) was stirred at 50 °C for 15 min. The hydrolyzed acetal mixture was then slowly added and stirred for 15 min. Cooled to 0 °C, and the reaction mixture was quenched with saturated NH4Cl solution. The residue was treated with ice, and the resulting precipitate was filtered to obtain 2-amino-5-methyl-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (5 g, crude, quantitative). LCMS: m / z = 165 (M+1, ESI+).

[0200] Step 2: N-(5-methyl-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide

[0201] A stirred mixture of 2-amino-5-methyl-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (15 g, 91.46 mmol) and N,N-dimethyl-4-pyridylamine (558 mg, 4.57 mmol) in pivalic anhydride (82 mL) was stirred at 120 °C for 1.5 h. Cooled to 0 °C, cold ether was added to the reaction mixture, and the resulting precipitate was filtered to give N-(5-methyl-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide (14 g crude, quantitative) as a pale yellow solid. LCMS: m / z = 249 (M+1, ESI+).

[0202] Step 3: N-(5-methyl-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide

[0203] A mixture of N-(5-methyl-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide (14 g, 56.4 mmol) in POCl3 (113 mL, 141 mmol) was heated at 110°C for 1 h. After the reaction was completed, the excess POCl3 was removed from the reaction mixture under vacuum, and the residue was added to ice and 25% ammonia solution. The mixture was stirred vigorously for 30 min, and the resulting precipitate was filtered to obtain crude N-(5-methyl-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide (15 g, quantitative). LCMS: m / z = 267 (M+1, ESI+).

[0204] Step 4: 4-Chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine

[0205] A mixture of N-(5-methyl-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide (15 g, 56.2 mmol) in 2N NaOH (200 mL) was stirred at 120°C for 2.5 h. The reaction mixture was cooled to room temperature, and the resulting precipitate was filtered to obtain 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (7 g, quantitative) as a pale yellow solid. LCMS: m / z = 183 (M+1, ESI+).

[0206] Step 5: 5-(2-Amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S7)

[0207] To a stirred solution of 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (1.2 g, 6.59 mmol) in 1,4-dioxane (8 mL) and water (2 mL) was added 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (2.01 g, 7.91 mmol) and Cs2CO3 (6.44 g, 19.78 mmol) at 25 °C, and the mixture was degassed with argon for 10 min. Pd(PPh3)4 (761 mg, 0.659 mmol) was then added, and the mixture was stirred at 100 °C for 16 h. After the reaction was completed, the reaction mixture was filtered through a Celite bed, and the bed was washed with EA (100 mL x 5) and MeOH (100 mL). The filtrate was collected and concentrated. The residue was triturated with 5% MeOH in DCM to give intermediate S7 (1.2 g, crude, 66.44% yield) as a pale yellow solid. The crude product was used as the intermediate. LCMS: m / z = 275 (M+1, ESI+).

[0208] Preparation Example S8: 5-(2-amino-6-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S8)

[0209]

[0210] Step 1: 2-Amino-6-methyl-1, 7-dihydro-4H-pyrrolo[2, 3-d]pyrimidin-4-one

[0211] A suspension of 2,6-diamino-4(1H)-pyrimidinone (10 g, 79.3 mmol) and NaOAc (6.5 g, 79.3 mmol) in water (100 mL) was stirred at 100 °C. After 10 min, 1-chloro-2-propanone (7.34 mL, 79.3 mmol) was slowly added, and the mixture was stirred at 100 °C for 3 h. After the reaction was completed, the reaction mixture was filtered, washed with water (200 mL), and dried in vacuo to give 2-amino-6-methyl-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (10.8 g, 83.01% yield) as an off-white solid. LCMS: m / z = 165 (M+1, ESI+).

[0212] Step 2: 4-Chloro-6-methyl-7H-pyrrolo[2, 3-d]pyrimidin-2-amine

[0213] A mixture of 2-amino-6-methyl-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (10.8 g, 65.8 mmol) in POCl3 (110 mL, 660 mmol) and N,N-dimethylaniline (0.3 mL, 2.37 mmol) was stirred at 110 °C for 3 h. After the reaction was completed, the reaction mixture was evaporated under reduced pressure. The residue was poured onto ice, basified with 25% aqueous ammonia solution (120 mL), and the precipitate was filtered and dried under vacuum to give 4-chloro-6-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (4.4 g, 36.63% yield) as an off-white solid. LCMS: m / z = 183 (M+1, ESI+).

[0214] Step 3: 5-(2-amino-6-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S8)

[0215] To a stirred solution of 4-chloro-6-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (1.5 g, 8.24 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (2.51 g, 9.89 mmol) in a mixture of 1,4-dioxane (36 mL) and water (4 mL) was added K2CO3 (2.27 g, 16.48 mmol) and degassed with argon for 10 min. Then, Pd(dppf)Cl2.DCM (0.33 g, 0.41 mmol) was added and the mixture was stirred at 110 °C for 16 h. After completion of the reaction, the reaction mixture was filtered through a Celite bed and washed with EA (600 mL). The organic layer was washed with water (100 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give intermediate S8 (1.1 g crude, 30% yield) as a yellow solid. LCMS: m / z = 275 (M+1, ESI+).

[0216] Manufacturing Example S9: 5-(2-amino-8-methyl-9H-purin-6-yl)isophthalonitrile (Intermediate S9)

[0217]

[0218] Step 1: 5-(2,5-diamino-6-((4-methoxybenzyl)amino)pyrimidin-4-yl)isophthalonitrile

[0219] 6-Chloro-N obtained in step 1 of Preparation Example S6 in a mixture of 1,4-dioxane (36 mL) and water (4 mL) 4To a stirred solution of -(4-methoxybenzyl)pyrimidine-2,4,5-triamine (4 g, 14.30 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (4.3 g, 17.16 mmol) was added Cs2CO3 (13.9 g, 42.90 mmol) and degassed with argon for 10 min. Then, Pd(PPh3)4 (1.65 g, 1.43 mmol) was added, and the mixture was stirred at 100 °C for 16 h. After completion of the reaction, the reaction mixture was filtered through a Celite bed and washed with EA (600 mL). The organic phase was washed with water (100 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give 5-(2,5-diamino-6-((4-methoxybenzyl)amino)pyrimidin-4-yl)isophthalonitrile (4.8 g, crude, 67% yield) as a yellow solid. LCMS: m / z = 372 (M+1, ESI+).

[0220] Step 2: 5-(2-amino-9-(4-methoxybenzyl)-8-methyl-9H-purin-6-yl)isophthalonitrile

[0221] To a stirred solution of 5-(2,5-diamino-6-((4-methoxybenzyl)amino)pyrimidin-4-yl)isophthalonitrile (4.8 g, crude, 8.65 mmol) in dry DCM was added 1,1,1-triethoxyethane (2.1 g, 12.98 mmol) and BF3.OEt2 (1.2 g, 8.65 mmol) at 0 °C, and the mixture was stirred at 25 °C for 16 h. After the reaction was completed, the reaction mixture was basified (pH ~ 7-8) with saturated NaHCO3 solution and extracted with 10% MeOH in DCM (400 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give 5-(2-amino-9-(4-methoxybenzyl)-8-methyl-9H-purin-6-yl)isophthalonitrile (2.28 g, 66.58% yield) as a yellow solid. LCMS: m / z = 396 (M+1, ESI+).

[0222] Step 3: 5-(2-amino-8-methyl-9H-purin-6-yl)isophthalonitrile (intermediate S9)

[0223] To a stirred solution of 5-(2-amino-9-(4-methoxybenzyl)-8-methyl-9H-purin-6-yl)isophthalonitrile (1.8 g, 4.55 mmol) in TFA (20 ml) was added trifluoromethanesulfonic acid (6.8 g, 45.52 mmol), and the mixture was stirred at 75°C for 1 h. After completion of the reaction, the reaction mixture was quenched with ice and basified with saturated NaHCO3 solution (pH ~ 7–8). The precipitated solid was filtered and collected to give intermediate S9 (1.2 g, 95.77% yield) as a yellow solid. LCMS: m / z = 276 (M+1, ESI+).

[0224] Manufacturing Example S10: 5-(6-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S10)

[0225]

[0226] Step 1: 1-(2-amino-4, 6-dichloropyrimidin-5-yl)ethan-1-ol

[0227] To a solution of 2-amino-4,6-dichloropyrimidine-5-carbaldehyde (20 g, 104 mmol) in THF (50 mL) was added 2 M MeMgBr (185 mL, 871 mmol) at -78 °C, and the mixture was stirred at -78 °C for 4 h. After the reaction was completed, the reaction mixture was quenched with saturated NH4Cl solution and extracted with EA (350 mL x 2). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography to give 1-(2-amino-4,6-dichloropyrimidin-5-yl)ethan-1-ol (13 g, 60.01% yield) as an off-white solid. LCMS: m / z = 208 (M+1, ESI+).

[0228] Step 2: 1-(2-amino-4, 6-dichloropyrimidin-5-yl)ethan-1-one

[0229] To a solution of 1-(2-amino-4,6-dichloropyrimidin-5-yl)ethan-1-ol (13 g, 62.5 mmol) in DCM (130 mL) was added Dessmartin periodinane (53 g, 125 mmol) at 0 °C, and the mixture was stirred at 0 °C for 2 h. After the reaction was completed, the reaction mixture was quenched with water (100 mL) and extracted with EA (500 mL x 2). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography to give 1-(2-amino-4,6-dichloropyrimidin-5-yl)ethan-1-one (8.4 g, 65.24% yield) as an off-white solid. LCMS: m / z = 207 (M+1, ESI+).

[0230] Step 3: 4-chloro-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine

[0231] To a solution of 1-(2-amino-4,6-dichloropyrimidin-5-yl)ethan-1-one (8.4 g, 40.8 mmol) in DCM (90 mL) was added hydrazine hydrate (99%) (1.92 mL, 61.2 mmol) at 25 °C, and the mixture was stirred at 25 °C for 16 h. After completion of the reaction, the reaction mixture was concentrated and diluted with ice-cold water (100 mL), and the precipitated solid was filtered, washed with water (200 mL), and dried in vacuo to give 4-chloro-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine (6.7 g, 89.81% yield) as an off-white solid. LCMS: m / z = 184 (M+1, ESI+).

[0232] Step 4: 5-(6-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile (Intermediate S10)

[0233] To a stirred solution of 4-chloro-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine (4 g, 21.7 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (6.6 g, 26.1 mmol) in a mixture of 1,4-dioxane (45 mL) and water (5 mL) was added Cs2CO3 (21.2 g, 65.1 mmol) and degassed with argon for 10 min. Then, Pd(PPh3)2Cl2 (2.29 g, 3.26 mmol) was added, and the mixture was stirred at 100 °C for 16 h. After completion of the reaction, the reaction mixture was filtered through a Celite bed and washed with EA (400 mL). The organic layer was washed with water (100 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give intermediate S10 (1.8 g, 30.05%) as a yellow solid. LCMS: m / z = 276 (M+1, ESI+).

[0234] Manufacturing Example A1: 1-(Bromomethyl)-3-ethynyl-5-(trifluoromethyl)benzene (Intermediate A1)

[0235]

[0236] Step 1: 3-(trifluoromethyl)-5-((trimethylsilyl)ethynyl)benzaldehyde

[0237] To a stirred solution of 3-bromo-5-(trifluoromethyl)benzaldehyde (5 g, 19.7 mmol) in TEA (40 mL) were added CuI (376 mg, 1.97 mmol) and Pd(PPh3)2Cl2 (416 mg, 0.59 mmol) at 0 °C. After 10 min, ethynyltrimethylsilane (3.37 mL, 23.7 mmol) was added dropwise, and the mixture was stirred at 50 °C for 4 h. After completion of the reaction, the reaction mixture was quenched with water (100 mL) and extracted with EA (400 mL x 2). The combined organic phases were washed with brine (50 mL x 2), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to obtain 3-(trifluoromethyl)-5-((trimethylsilyl)ethynyl)benzaldehyde (3.66 g, 68.66% yield) as a yellow liquid. 1 H NMR (400 MHz, CDCl3) δ 10.03 (s, 1H), 8.12 (s, 1H), 8.06 (s, 1H), 7.94 (s, 1H), 0.28 (s, 9H).

[0238] Step 2: 3-Ethynyl-5-(trifluoromethyl)benzaldehyde

[0239] To a stirred solution of 3-(trifluoromethyl)-5-((trimethylsilyl)ethynyl)benzaldehyde (2.3 g, 8.51 mmol) in MeOH (30 mL) was added K2CO3 (1.17 g, 8.51 mmol) at 0°C, and the resulting mixture was stirred at 25°C for 0.5 h. After completion of the reaction, the reaction mixture was quenched with water (50 mL) and extracted with EA (350 mL x 2). The combined organic phases were washed with brine (50 mL), dried over Na2SO4, and concentrated to give 3-ethynyl-5-(trifluoromethyl)benzaldehyde (1.98 g crude) as a yellow liquid. The crude product was used as is in the next step without further purification. 1 H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 8.15 (m, 1H), 8.10 (m, 1H), 7.97 (m, 1H), 4.12 (d,J=7.2 Hz, 1H).

[0240] Step 3: (3-ethynyl-5-(trifluoromethyl)phenyl)methanol

[0241] To a stirred solution of 3-ethynyl-5-(trifluoromethyl)benzaldehyde (1.98 g, 9.94 mmol) in EtOH (20 mL) was added NaBH4 (188 mg, 4.97 mmol) at 0°C, and the mixture was stirred at 25°C for 0.5 h. After the reaction was completed, the reaction mixture was quenched with ice and extracted with EA (250 mL x 2). The combined organic layers were washed with brine solution (40 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give (3-ethynyl-5-(trifluoromethyl)phenyl)methanol (1.14 g, 57.00% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.69 (m, 3H), 5.48 (t,J=6.0 Hz, 1H), 4.58 (d,J=5.6 Hz, 2H), 4.38 (s, 1H).

[0242] Step 4: 1-(Bromomethyl)-3-ethynyl-5-(trifluoromethyl)benzene (Intermediate A1)

[0243] To a stirred solution of (3-ethynyl-5-(trifluoromethyl)phenyl)methanol (1.0 g, 5.0 mmol) in dry DCM (15 mL) were added CBr4 (3.54 g, 10.0 mmol) and TPP (2.80 g, 10.0 mmol) at 0 °C, and the mixture was stirred at 25 °C for 3 h. After the reaction was completed, the reaction mixture was quenched with ice and extracted with EA (200 mL x 2). The combined organic phases were washed with brine (40 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give intermediate A1 (1.0 g, 76.04% yield) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.68 (d,J=6.4 Hz, 2H), 7.61 (s, 1H), 4.46 (s, 2H), 3.18 (s, 1H).

[0244] Example 1: 5-(6-amino-1-(3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0245]

[0246] To a stirred solution of intermediate S3 (48 g, 146.4 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (44.6 g, 175.7 mmol) in a mixture of 1,4-dioxane (430 mL) and water (50 mL) was added K2CO3 (40.5 g, 293 mmol) at 25 °C, and the mixture was degassed with argon for 10 min. Then, Pd(PPh3)4 (33 g, 29.3 mmol) was added, and the mixture was stirred at 110 °C for 12 h. After completion of the reaction, the solvent was evaporated, water (100 mL) was added, and extracted with EA (500 ml x 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography to obtain the compound of Example 1 (22 g, 35.81% yield) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 2H), 8.69 (s, 1H), 8.60 (s, 1H), 7.67 - 7.56 (m, 3H), 7.47 (d, 1H), 7.22 (s, 2H), 5.58 (s, 2H); LCMS: m / z = 420 (M+1, ESI+); HRMS: 420.1185 (M+1, ESI+); MR: 196℃-200℃

[0247] Example 2: 5-(6-amino-1-(3-fluoro-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0248]

[0249] To a stirred solution of intermediate S2 (0.6 g, crude, 1.98 mmol) in DMF (10 mL) was added K2CO3 (545 mg, 3.96 mmol) at 0°C and stirred for 10 min. Then, 1-(bromomethyl)-3-fluoro-5-(trifluoromethyl)benzene (508 mg, 1.98 mmol) was added and the resulting mixture was stirred at 25°C for 4 h. After completion of the reaction, the reaction mixture was quenched with ice, and the resulting precipitate was filtered and washed with water. The residue was purified by column chromatography to obtain the compound of Example 2 (140 mg, 16.2%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 2H), 8.69 (s, 1H), 8.61 (s, 1H), 7.63 (d, 1H), 7.47 (s, 1H), 7.33 (d, 1H), 7.23 (s, 2H), 5.59 (s, 2H); LCMS: m / z = 438 (M+1, ESI+); HRMS: 438.0567; MR: 204℃-208℃.

[0250] Example 3: 3-(6-amino-1-(3-fluoro-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0251]

[0252] To a stirred solution of the compound of Example 2 (0.3 g, 0.686 mmol) in dry toluene (10 mL) were added InCl3 (7.6 mg, 0.034 mmol) and acetaldoxime (203 mg, 3.43 mmol) at room temperature, and the resulting mixture was stirred in a preheated oil bath at 70°C for 40 min. After completion of the reaction, it was cooled to room temperature and the solvent was evaporated to obtain a residue. The residue was purified by column chromatography to obtain the compound of Example 3 (93 mg, 27.3%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6)δ 8.84 (s, 1H), 8.65 (s, 1H), 8.49 (d, 2H), 8.33 (s, 1H), 7.78 (s, 1H), 7.63 (d, 1H), 7.48 (s, 1H), 7.33 (d, 1H), 7.20 (s, 2H), 5.59 (s, 2H); LCMS:m / z = 456 (M+1, ESI+); MR:248℃-252℃.

[0253] Example 4: 5-(6-amino-1-(3-chloro-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0254]

[0255] The compound of Example 4 was obtained as an off-white solid in the same manner as in Example 2 using intermediate S2 (0.3 g, crude, 0.988 mmol) and 1-(bromomethyl)-3-chloro-5-(trifluoromethyl)benzene (270 mg, 0.988 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 2H), 8.69 (s, 1H), 8.61 (s, 1H), 7.82 (s, 1H), 7.60 (s, 1H), 7.55 (s, 1H), 7.24 (s, 2H), 5.58 (s, 2H); LCMS: m / z = 454 (M+1, ESI+); HRMS: 454.0257; MR: 214℃-219℃.

[0256] Example 5: 3-(6-amino-1-(3-chloro-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0257]

[0258] Using the compound of Example 4 (0.4 g, 0.881 mmol) in the same manner as in Example 3, the compound of Example 5 (95 mg, 22.8%) was obtained as an off-white solid. 1H NMR (400 MHz, DMSO-d6)δ 8.84 (t, 1H), 8.65 (t, 1H), 8.49 (s, 2H), 8.33 (s, 1H), 7.80 (d, 2H), 7.58 (s, 2H,), 7.22 (s, 2H), 5.59 (s, 2H); LCMS: m / z = 472 (M+1, ESI+).

[0259] Example 6: 5-(6-amino-1-(3-bromo-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0260]

[0261] To a stirred solution of intermediate S2 (0.6 g, crude, 1.97 mmol) in DMF (10 mL) was added K2CO3 (0.545 g, 3.95 mmol) at 0°C and stirred for 10 min. Then, 1-bromo-3-(bromomethyl)-5-(trifluoromethyl)benzene (0.626 g, 1.97 mmol) was added and stirred at 25°C for 2 h. After the reaction was completed, the reaction mixture was quenched with ice and extracted with EA (75 mL x 2). The combined organic layers were washed with cold water (20 mL x 2) and brine (20 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give the compound of Example 6 (160 mg, 16.29% yield) as an off-white solid. 1 NMR (400 MHz, DMSO-d6) δ 8.81 (s, 2H), 8.68 (s, 1H), 8.60 (s, 1H), 7.91 (s, 1H), 7.70 (s, 1H), 7.63 (s, 1H), 7.23 (s, 2H), 5.58 (s, 2H); LCMS: m / z = 498 (M+1, ESI+); MR: 214℃-219℃.

[0262] Example 7: 3-(6-amino-1-(3-bromo-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0263]

[0264] Using the compound of Example 6 (0.350 g, 0.70 mmol) in the same manner as in Example 3, the compound of Example 7 (75 mg, 20.71% yield) was obtained as an off-white solid. 1 NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.65 (s, 1H), 8.49 (d, 2H), 8.33 (s, 1H), 7.92 (s, 1H), 7.79 (s, 1H), 7.71 (s, 1H), 7.64 (s, 1H), 7.22 (s, 2H), 5.58 (s, 2H); LCMS: m / z = 516 (M+1, ESI+).

[0265] Example 8: 5-(6-amino-1-(3-iodo-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0266]

[0267] The compound of Example 8 (55 mg, 5%) was obtained as an off-white solid in the same manner as in Example 2 using intermediate S2 (0.6 g, crude, 1.97 mmol) and 1-(bromomethyl)-3-iodo-5-(trifluoromethyl)benzene (717 mg, 1.97 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 2H), 8.69 (s, 1H), 8.60 (s, 1H), 8.02 (s, 1H), 7.88 (s, 1H), 7.61 (s, 1H), 7.24 (s, 2H), 5.54 (s, 2H); LCMS: m / z = 546 (M+1, ESI+); MR: 200℃-206℃.

[0268] Example 9: 3-(6-amino-1-(3-iodo-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0269]

[0270] Using the compound of Example 8 (0.2 g, 0.366 mmol) in the same manner as in Example 3, the compound of Example 9 (50 mg, 24%) was obtained as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.66 (s, 1H), 8.49 (d, 2H), 8.34 (s, 1H), 8.02 (s, 1H), 7.89 (s, 1H), 7.79 (s, 1H), 7.62 (s, 1H), 7.22 (s, 2H), 5.55 (s, 2H); LCMS: m / z = 562

[0271] Example 10: 5-(6-amino-1-(3,5-bis(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0272]

[0273] The compound of Example 10 (170 mg, 17.6%) was obtained as an off-white solid in the same manner as in Example 2 using intermediate S2 (0.6 g, crude, 1.98 mmol) and 1-(bromomethyl)-3,5-bis(trifluoromethyl)benzene (608 mg, 1.98 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 2H), 8.68 (s, 1H), 8.62 (s, 1H), 8.06 (s, 1H), 7.92 (s, 2H), 7.23 (s, 2H), 5.68 (s, 2H); LCMS: m / z = 488 (M+1, ESI+); MR: 220℃-226℃.

[0274] Example 11: 3-(6-amino-1-(3,5-bis(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0275]

[0276] Using the compound of Example 10 (0.3 g, 0.6 mmol) in the same manner as in Example 3, the compound of Example 11 (85 mg, 27.33% yield) was obtained as an off-white solid. 1 HNMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.66 (s, 1H), 8.50 (d, 2H), 8.33 (s, 1H), 8.08 (s, 1H), 7.94 (s, 2H), 7.79 (s, 1H), 7.22 (s, 2H), 5.68 (s, 2H); LCMS: m / z = 506 (M+1, ESI+).

[0277] Examples 12 to 40

[0278] Compounds of Examples 12 to 40 were obtained in the same manner as in Example 6 using the starting materials listed in Table A below. In Examples 20, 21, 22, 23, 28, 30, and 32 to 40, Cs2CO3 was used instead of K2CO3. Stirring time, purification method, etc. were appropriately changed.

[0279] [Table A]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285] [Table B]

[0286]

[0287]

[0288]

[0289] Example 41: 5-(6-amino-1-(4-chloro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0290]

[0291] Step 1: 4-chloro-1-(4-chloro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine

[0292] The same procedure as in Example 6 was followed using intermediate S1 (300 mg, 2.96 mmol) and 4-(bromomethyl)-1-chloro-2-(trifluoromethyl)benzene (533 mg, 1.95 mmol) and using CS2CO3 instead of K2CO3 to obtain 4-chloro-1-(4-chloro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (201 mg, 31.37% yield) as an off-white solid. LCMS: m / z = 362 (M+1, ESI+).

[0293] Step 2: 5-(6-amino-1-(4-chloro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0294] To a stirred solution of 4-chloro-1-(4-chloro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (185 mg, 0.51 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (168 mg, 0.66 mmol) in a mixture of THF (15 mL) and water (5 mL) was added K2CO3 (140 mg, 1.02 mmol) at 25 °C, and the mixture was degassed with argon for 10 min. Pd(PPh3)4 (29.45 mg, 0.025 mmol) was then added, and the mixture was stirred at 110 °C for 12 h. After the reaction was completed, the reaction mixture was quenched with ice and extracted with EA (100 mL x 2). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to obtain the compound of Example 41 (98 mg, 42.24% yield) as an off-white solid. 1 H NMR (DMSO-d6) 8.81 (s, 2H), 8.70 (s, 1H), 8.61 (s, 1H), 7.80 (s, 1H), 7.70 (d, 1H), 7.44 (d, 1H), 7.23 (s, 2H), 5.56 (s, 2H); LCMS: m / z = 454 (M+1, ESI+); MR: 228℃-234℃.

[0295] Example 42: 5-(6-amino-1-(2-chloro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0296]

[0297] The compound of Example 42 (60 mg, 15.71% yield) was obtained as an off-white solid in the same manner as in Example 6 using intermediate S2 (500 mg, crude, 0.842 mmol) and 1-(bromomethyl)-2-chloro-3-(trifluoromethyl)benzene (228 mg, 0.842 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.82 (d, 2H), 8.71 (s, 1H), 8.64 (s, 1H), 7.84 (d, 1H), 7.50 (t, 1H), 7.24 (s, 2H), 7.15 (d, 1H), 5.64 (s, 2H); LCMS: m / z = 454 (M+1, ESI+); MR: 220℃-226℃.

[0298] Example 43: 5-(6-amino-1-(3-ethynyl-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0299]

[0300] The compound of Example 43 (90 mg, 35.85% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (500 mg, 2.96 mmol) and intermediate A1 (856 mg, 3.25 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 2H), 8.69 (s, 1H), 8.60 (s, 1H), 7.77 (s, 1H), 7.67 (s, 1H), 7.53 (s, 1H), 7.24 (s, 2H), 5.57 (s, 2H), 4.43 (s, 1H); LCMS: m / z = 444 (M+1, ESI+); MR: 176℃-182℃.

[0301] Example 44: 5-(6-amino-1-(2,5-bis(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0302]

[0303] The compound of Example 44 (107 mg, 54.25% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (300 mg, 1.77 mmol) and 2-(bromomethyl)-1,4-bis(trifluoromethyl)benzene (543 mg, 1.77 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.84 (d, 2H), 8.70 (s, 1H), 8.66 (s, 1H), 8.08 (d, 1H), 7.96 (d, 1H), 7.33 (s, 1H), 7.26 (s, 2H), 5.72 (s, 2H); LCMS: m / z = 488 (M+1, ESI+); MR: 244℃-250℃.

[0304] Example 45: 5-(6-amino-1-(2-methoxy-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0305]

[0306] The compound of Example 45 (85 mg, 33.86% yield) was obtained as a pale yellow solid in the same manner as in Example 41 using intermediate S1 (430 mg, 2.54 mmol) and 2-(bromomethyl)-1-methoxy-4-(trifluoromethyl)benzene (684 mg, 2.54 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (d, 2H), 8.68 (s, 1H), 8.59 (s, 1H), 7.66 (d, 1H), 7.24 (d, 1H), 7.19 (s, 2H), 7.07 (s, 1H), 5.46 (s, 2H), 3.90 (s, 3H); LCMS: m / z = 450 (M+1, ESI+); MR: 248℃-254℃.

[0307] Example 46: 5-(6-amino-1-(4-methoxy-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0308]

[0309] The compound of Example 46 (70 mg, 26.77% yield) was obtained as a pale yellow solid in the same manner as in Example 41 using intermediate S1 (430 mg, 2.54 mmol) and 4-(bromomethyl)-1-methoxy-2-(trifluoromethyl) benzene (684 mg, 2.54 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 2H), 8.69 (s, 1H), 8.56 (s, 1H), 7.56 (s, 1H), 7.49 (d, 1H), 7.24 - 7.21 (m, 3H), 5.47 (s, 2H), 3.86 (s, 3H); LCMS: m / z = 450 (M+1, ESI+); MR: 202℃-212℃.

[0310] Example 47: 5-(6-amino-1-(2-methoxy-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0311]

[0312] The compound of Example 47 (57 mg, 37.82% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (300 mg, 1.76 mmol) and 1-(bromomethyl)-2-methoxy-3-(trifluoromethyl)benzene (520 mg, 1.93 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (d, 2H), 8.70 (s, 1H), 8.61 (s, 1H), 7.62 (d, 1H), 7.27 - 7.18 (m, 4H), 5.59 (s, 2H), 3.94 (s, 3H); LCMS: m / z = 450 (M+1, ESI+); MR: 204℃-212℃.

[0313] Example 48: 5-(6-amino-1-(2-methyl-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0314]

[0315] The compound of Example 48 (121 mg, 47.71% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (250 mg, 1.47 mmol) and 2-(bromomethyl)-1-methyl-4-(trifluoromethyl)benzene (410 mg, 1.62 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (d, 2H), 8.69 (s, 1H), 8.59 (s, 1H), 7.57 (d, 1H), 7.46 (d, 1H), 7.22 (s, 3H), 5.53 (s, 2H), 2.43 (s, 3H); LCMS: m / z = 434 (M+1, ESI+); MR: 204℃-210℃.

[0316] Example 49: 5-(6-amino-1-(4-methyl-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0317]

[0318] The compound of Example 49 (60 mg, 23.65% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (300 mg, 1.76 mmol) and 4-(bromomethyl)-1-methyl-2-(trifluoromethyl)benzene (489 mg, 1.93 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.81 (d, 2H), 8.69 (t, 1H), 8.58 (s, 1H), 7.59 (s, 1H), 7.41 - 7.34 (m, 2H), 7.21 (s, 2H), 5.51 (s, 2H), 2.40 (s, 3H); LCMS: m / z = 434 (M+1, ESI+); MR: 176℃-182℃.

[0319] Example 50: 5-(6-amino-1-(2-methyl-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0320]

[0321] The compound of Example 50 (110 mg, 43.37% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (300 mg, 1.76 mmol) and 4-(bromomethyl)-1-methyl-2-(trifluoromethyl)benzene (445 mg, 1.76 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.81 (d, 2H), 8.69 (s, 1H), 8.59 (s, 1H), 7.62 (d, 1H), 7.31 (t, 1H), 7.19 (s, 2H), 7.05 (d, 1H), 5.56 (s, 2H), 2.50 - 2.48 (m, 3H); LCMS: m / z = 434 (M+1, ESI+).

[0322] Example 51: 5-(6-amino-1-(2-fluoro-5-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0323]

[0324] The compound of Example 51 (110 mg, 57.95% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (300 mg, 1.76 mmol) and 2-(bromomethyl)-1-fluoro-4-(trifluoromethyl)benzene (497 mg, 1.93 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.81 (d, 2H), 8.69 (d, 1H), 8.57 (s, 1H), 7.80 - 7.79 (m, 1H), 7.63 (d, 1H), 7.50 (t, 1H), 7.24 (s, 2H), 5.58 (s, 2H); LCMS: m / z = 438 (M+1, ESI+); MR: 206℃-212℃.

[0325] Example 52: 5-(6-amino-1-(4-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0326]

[0327] The compound of Example 52 (98 mg, 38.68% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (300 mg, 1.76 mmol) and 4-(bromomethyl)-1-fluoro-2-(trifluoromethyl)benzene (496 mg, 1.93 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 2H), 8.68 (s, 1H), 8.58 (s, 1H), 7.71 (d, 1H), 7.53 - 7.45 (m, 2H), 7.22 (s, 2H), 5.54 (s, 2H); LCMS: m / z = 438 (M+1, ESI+).

[0328] Example 53: 5-(6-amino-1-(2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0329]

[0330] The compound of Example 53 (119 mg, 46.94% yield) was obtained as an off-white solid in the same manner as in Example 41 using intermediate S1 (300 mg, 1.76 mmol) and 1-(bromomethyl)-2-fluoro-3-(trifluoromethyl)benzene (497 mg, 1.93 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.80 (d, 2H), 8.69 (d, 1H), 8.58 (s, 1H), 7.75 (t, 1H), 7.47 (t, 1H), 7.38 (t, 1H), 7.23 (s, 2H), 5.59 (s, 2H); LCMS: m / z = 438 (M+1, ESI+); MR: 188℃-194℃.

[0331] Example 54: 5-(5-amino-3-(2-fluoro-3-(trifluoromethyl)benzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)isophthalonitrile

[0332]

[0333] In Example 20, 1-(bromomethyl)-2-fluoro-3-(trifluoromethyl)benzene was used instead of 1-(bromomethyl)-3-(trifluoromethyl)benzene, and the compound of Example 54 (110 mg, 6.26% yield) was obtained as a gray-white solid in the same manner as in Example 20. 1 H NMR (400 MHz, DMSO-d6) δ 9.25 (d, 2H), 8.77 (t, 1H), 7.80 (t, 1H), 7.64-7.62 (m, 3H), 7.42 (t, 1H), 5.86 (s, 2H); LCMS: m / z = 439 (M+1, ESI+); MR: 244℃-248℃.

[0334] Example 55: 5-(2-amino-9-(2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-6-yl)isophthalonitrile

[0335]

[0336] The compound of Example 55 (290 mg, 57.30% yield) was obtained as an off-white solid in the same manner as in Example 41 using 6-chloro-9H-purin-2-amine (0.4 g, 2.36 mmol) and 1-(bromomethyl)-2-fluoro-3-(trifluoromethyl)benzene (0.6 g, 2.36 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 9.31 (s, 2H), 8.65-8.64 (m, 1H), 8.39 (s, 1H), 7.76 (t, 1H), 7.46 (t, 1H), 7.38 (t, 1H), 6.84 (brs, 2H), 5.52 (s, 2H); LCMS: m / z = 438 (M+1, ESI+); MR: 247℃-251℃.

[0337] Example 56: 5-(2-amino-9-(2-fluoro-3-(trifluoromethyl)benzyl)-8-methyl-9H-purin-6-yl)isophthalonitrile

[0338]

[0339] Intermediate S9 (210 mg, 0.76 mmol) was dissolved in DMF (8 mL), and Cs2CO3 (494 mg, 1.52 mmol) was added at 0°C. After 10 min, 1-(bromomethyl)-2-fluoro-3-(trifluoromethyl)benzene (214 mg, 0.83 mmol) was added at 0°C, and the mixture was stirred at 0°C for 2 h. The reaction was stopped by quenching with ice. The precipitate formed in the ice-cooled reaction mixture was filtered and washed with water (30 mL), and the residue was purified by column chromatography to obtain the compound of Example 56 (72 mg, 20.93% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.31 (d, 2H), 8.63 (t, 1H), 7.75 (t, 1H), 7.38-7.28 (m, 2H), 6.73 (s, 2H), 5.49 (s, 2H), 2.53 (s, 3H); LCMS: m / z = 452 (M+1, ESI+); MR: Not melted up to 300°C.

[0340] Example 57: 5-(6-amino-1-(2-fluoro-3-(trifluoromethyl)benzyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0341]

[0342] To a stirred solution of intermediate S10 (0.15 g, 0.54 mmol) in DMF (10 mL) was added Cs2CO3 (0.26 g, 0.81 mmol) at 0°C. After 10 min, 1-(bromomethyl)-2-fluoro-3-(trifluoromethyl)benzene (0.14 g, 0.54 mmol) was added and stirred at 25°C for 2 h. After completion of the reaction, the reaction mixture was quenched with ice water (30 mL) and extracted with EA (2 x 50 mL). The organic phase was washed with ice water (20 mL x 2) and brine (20 mL), dried over Na2SO4, and concentrated to obtain a residue. The residue was purified by column chromatography to obtain the compound of Example 57 (0.15 g, 60.98% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.53 (s, 2H), 7.50 (t, 1H), 7.49 (t, 1H), 7.39 (t, 1H), 7.16 (brs, 2H), 5.50 (s, 2H), 2.13 (s, 3H); LCMS: m / z = 452 (M+1, ESI+); MR:190℃-196℃

[0343] Example 58: 3-(6-amino-1-(2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0344]

[0345] The compound of Example 53 (230 mg, 0.526 mmol) was added to toluene (10 mL), and indium(III) chloride (5.82 mg, 0.026 mmol) and (Z)-acetaldehyde oxime (0.224 mL, 3.15 mmol) were added, and the mixture was stirred at 70°C for 20 minutes. After the reaction was completed, the solvent was evaporated, and the residue was diluted with water (30 mL) and extracted with EA (100 mL x 2). The combined organic layers were dried over Na2SO4 and concentrated, and the residue was purified by column chromatography to obtain the compound of Example 58 (96 mg, 40.16% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.83 (s, 1H), 8.63 (s, 1H), 8.47 (d, 2H), 8.33 (s, 1H), 7.78-7.73 (m, 2H), 7.47 (t, 1H), 7.38 (t, 1H), 7.20 (s, 2H), 5.59 (s, 2H); LCMS: m / z = 456 (M+1, ESI+); MR: 258℃-261℃.

[0346] Example 59: 3-(2-amino-9-(2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-6-yl)-5-cyanobenzamide

[0347]

[0348] Using the compound of Example 55 (0.21 g, 0.48 mmol) as a starting material, the compound of Example 59 (46 mg, 21.04% yield) was obtained as a gray-white solid in the same manner as in Example 58.

[0349] 1H NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H), 9.26 (s, 1H), 8.45 (s, 1H), 8.37 (s, 1H), 8.27 (s, 1H), 7.79-7.77 (m, 2H), 7.46-7.38 (m, 2H), 6.80 (s, 2H), 5.53 (s, 2H); LCMS: m / z = 456 (M+1, ESI+); MR: 244℃-250℃.

[0350] Example 60: 3-(5-amino-3-(2-fluoro-3-(trifluoromethyl)benzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)-5-cyanobenzamide

[0351]

[0352] Using the compound of Example 54 (100 mg, 0.22 mmol) as a starting material, the compound of Example 60 (32 mg, 30.76% yield) was obtained as a gray-white solid in the same manner as in Example 58. 1 H NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H), 9.18 (s, 1H), 8.56 (s, 1H), 8.31 (s, 1H), 7.81-7.78 (m, 2H), 7.64-7.60 (m, 3H), 7.42 (t, 1H), 5.85 (s, 2H); LCMS: m / z = 457 (M+1, ESI+); MR: Not melted up to 300°C.

[0353] Example 61: 5-(6-amino-1-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0354]

[0355] Step 1: 5-chloro-2-fluoro-3-(trifluoromethyl)benzaldehyde

[0356] 4-Chloro-1-fluoro-2-(trifluoromethyl)benzene (0.5 g, 2.52 mmol) was dissolved in THF (30 mL) and stirred at room temperature for 10 min. n-BuLi (1.89 mL, 3.0 mmol) was added to the reaction mixture at -78°C and stirred at the same temperature for 1 h. DMF (0.24 mL, 3.0 mmol) was added to the reaction mixture and stirred for an additional 1 h at 25°C. After the reaction was completed, the reaction mixture was quenched with aqueous ammonium chloride solution and extracted with EA (50 mL x 2). The combined organic layers were washed with water (10 mL x 2) and brine (10 mL), dried over Na2SO4, and concentrated to obtain 5-chloro-2-fluoro-3-(trifluoromethyl)benzaldehyde (0.5 g, crude) as a sticky black liquid. 1 H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.25 (dd, 1H), 8.16 (dd, 1H).

[0357] Step 2: (5-chloro-2-fluoro-3-(trifluoromethyl)phenyl)methanol

[0358] To a solution of 5-chloro-2-fluoro-3-(trifluoromethyl)benzaldehyde (2 g, crude) at 0°C was added MeOH (30 mL), NaBH4 (0.33 g, 8.8 mmol) and stirred at 25°C for 1 h. After completion of the reaction, the solvent was concentrated, and the residue was diluted with ice water (20 mL) and extracted with DCM (50 mL x 2). The combined organic layers were washed with water (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated. The residue was purified by column chromatography to obtain (5-chloro-2-fluoro-3-(trifluoromethyl)phenyl)methanol (1 g quantitative) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (t, 2H), 5.60 (t, 1H), 4.61 (d, 2H).

[0359] Step 3: 1-(Bromomethyl)-5-chloro-2-fluoro-3-(trifluoromethyl)benzene

[0360] To a solution of 5-chloro-2-fluoro-3-(trifluoromethyl)phenyl)methanol (1 g, 4.37 mmol) at 0°C was added DCM (30 mL), TPP (2.3 g, 8.7 mmol) and CBr4 (2.9 g, 8.7 mmol) and the mixture was stirred at 25°C for 1 h. After completion, the reaction mixture was cooled with ice and extracted with DCM (50 mL x 2). The combined organic layers were washed with water (10 mL) and brine (20 mL), dried over Na2SO4 and concentrated, and the residue was purified by column chromatography to give 1-(bromomethyl)-5-chloro-2-fluoro-3-(trifluoromethyl)benzene (0.9 g, 70.86% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.06 (dd, 1H), 7.91 (dd, 1H), 4.74 (s, 2H).

[0361] Step 4: 4-chloro-1-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine

[0362] To a solution of 4-chloro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (1 g, 5.9 mmol) at 0°C was added DMF (20 mL) and stirred at the same temperature for 10 min. 1-(bromomethyl)-5-chloro-2-fluoro-3-(trifluoromethyl)benzene was added and stirred at 25°C for 2 h. The reaction mixture was cooled with ice and extracted with EA (50 mL x 2). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give 4-chloro-1-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (350 mg, 26.90% yield) as a pale yellow solid. LCMS: m / z = 380 (M+1, ESI+).

[0363] Step 5: 5-(6-amino-1-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0364] 4-Chloro-1-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (350 mg, 0.9 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (297 mg, 1.17 mmol) were added to a mixture of THF (15 mL) and water (5 mL) at 25 °C. K2CO3 (254 mg, 1.8 mmol) was added, the mixture was filled with argon for 10 min, and stirred. Pd(Ph3)4 (53 mg, 0.01 mmol) was added, and the mixture was stirred at 100 °C for 12 h. After completion of the reaction, the reaction mixture was filtered through a Celite bed and washed with EA (600 mL) and MeOH (200 mL). The filtrate was concentrated, and the residue was purified by column chromatography to obtain the compound of Example 61 (80 mg, 18.43% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 2H), 8.69 (s, 1H), 8.58 (s, 1H), 7.89 (m, 1H), 7.62-7.61 (m, 1H), 7.25 (s, 2H), 5.57 (s, 2H); LCMS: m / z = 472 (M+1, ESI+); MR: 230℃-236℃.

[0365] Example 62: 3-(6-amino-1-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0366]

[0367] Using the compound of Example 61 (40 mg, 0.08 mmol) as a starting material, the compound of Example 62 (9 mg, 21.95% yield) was obtained as a gray-white solid in the same manner as in Example 58. 1 H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H), 8.64 (s, 1H), 8.48-8.47 (m, 2H), 8.33 (s, 1H), 7.91-7.89 (m, 1H), 7.79 (s, 1H), 7.64-7.62 (m, 1H), 7.23 (s, 2H), 5.58 (s, 2H); LCMS: m / z = 490 (M+1, ESI+).

[0368] Example 63: 5-(6-amino-1-(2,5-difluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0369]

[0370] Step 1: (2,5-difluoro-3-(trifluoromethyl)phenyl)methanol

[0371] 1,4-Difluoro-2-(trifluoromethyl)benzene (5.00 g, 27.31 mmol) and tetramethylethylenediamine (TMEDA, 4.76 g, 40.96 mmol) were dissolved in THF (50 mL). n-BuLi (1.6 M in hexane) (22 mL, 35.50 mmol) was added dropwise at -78°C and stirred at the same temperature for 90 min. DMF (2.59 g, 35.50 mmol) was added and stirred at the same temperature for 30 min. After the mixture was maintained at 0°C in an ice bath, sodium borate (2.07 g, 54.62 mmol) was slowly added. The resulting mixture was stirred at room temperature for 2 hours, then the reaction mixture was diluted with water (500 mL), extracted with EtOAc (500 mL × 3), and the organic layer was dried over anhydrous Na2SO4(s) and concentrated. The resulting residue was purified by silica gel column chromatography (PE: EtOAc = 5:1) to obtain (2,5-difluoro-3-(trifluoromethyl)phenyl)methanol (3.00 g, 51.55% yield) as a yellow oil. 1 HNMR (400 MHz, DMSO-d6) δ: 7.66-7.51 (m, 2H), 5.67-5.62 (m, 2H), 4.63 (d, 2H).

[0372] Step 2: 2,5-Difluoro-3-(trifluoromethyl)benzyl 4-methylbenzenesulfonate

[0373] (2,5-Difluoro-3-(trifluoromethyl)phenyl)methanol (3.00 g, 14.07 mmol) and triethylamine (2.85 g, 28.15 mmol) were dissolved in DCM (30 mL), and TsCl (3.49 g, 18.30 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 1 h, and the reaction mixture was diluted with water (100 mL), extracted with DCM (100 mL × 3), and the organic layer was dried over anhydrous Na2SO4 and concentrated to give 2,5-difluoro-3-(trifluoromethyl)benzyl 4-methylbenzenesulfonate (6.0 g, 100% yield) as a white oil without further purification.

[0374] Step 3: 4-chloro-1-(2,5-difluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine

[0375] Intermediate S1 (500 mg, 2.96 mmol), 2,5-difluoro-3-(trifluoromethyl)benzyl 4-methylbenzenesulfonate (1.3 g, 3.55 mmol), and K2CO3 (817 mg, 5.92 mmol) were added to DMSO (10 mL) and stirred at 0°C for 1 h. The reaction mixture was diluted with water (20 mL) and extracted with EA (20 mL * 2). The combined organic phases were washed with brine (30 mL), dried over Na2SO4, concentrated and purified by silica gel column chromatography (PE: EtOAc = 3:1) to give 4-chloro-1-(2,5-difluoro-3-(trifluoromethyl)benzyl)-1H-pyrazol[3,4-d]pyrimidin-6-amine (100 mg, 9.34% yield) as a white solid. MS: m / z = 364.0 (M+1, ESI+)

[0376] Step 4: 5-(6-amino-1-(2,5-difluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)isophthalonitrile

[0377] 4-Chloro-1-(2,5-difluoro-3-(trifluoromethyl)benzyl)-1H-pyrazole[3,4-d]pyrimidin-6-amine (100 mg, 0. 27 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (105 mg, 0.41 mmol), Pd(dppf)Cl2 (22 mg, 0.03 mmol), and K2CO3 (74 mg, 0. 54 mmol) were added to 1,4-dioxane:H2O (4:1) (5 mL) and stirred at 100°C for 2 h. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (PE:EtOAc= 1:1) to obtain the compound of Example 63 (28 mg, 22.76% yield) as a white solid. 1 HNMR (400 MHz, DMSO-d6) δ: 8.80 (s, 2H), 8.69 (s, 1H), 8.59 (s, 1H), 7.75-7.73 (m, 1H), 7.39-7.38 (m, 1H), 7.24 (s, 2H), 5.58 (s, 2H); MS: m / z = 455.9 (M+1, ESI+).

[0378] Example 64: 3-(6-amino-1-(2,5-difluoro-3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-5-cyanobenzamide

[0379]

[0380] The compound of Example 63 (150 mg, 0.33 mmol) was dissolved in 1,4-dioxane (1 mL), and acetaldoxime (97 mg, 1.64 mmol) and InCl3 (7 mg, 0.03 mmol) in toluene (1 mL) were added, and the mixture was stirred at 90°C for 50 minutes. The mixture was concentrated to obtain a residue, which was purified by Pre-HPLC (ACN / H2O = 40 / 60) to obtain the compound of Example 64 (10 mg, 6.41% yield) as a white solid. 1HNMR (400 MHz, DMSO-d6) δ: 8.84-8.83 (m, 1H), 8.64-8.63 (m, 1H), 8.48-8.47 (m, 2H), 8.33 (s, 1H), 7.78-7.72 (m, 2H), 7.42-7.38 (m, 1H), 7.22 (s, 2H), 5.58 (s, 2H); MS: m / z = 474.0 (M+1, ESI+).

[0381] Example 65: 5-(2-amino-9-(2,5-difluoro-3-(trifluoromethyl)benzyl)-9H-purin-6-yl)isophthalonitrile

[0382]

[0383] Using (2,5-difluoro-3-(trifluoromethyl)phenyl)methanol (3.00 g, 14.07 mmol) as a starting material, the compound of Example 65 (300 mg, 12.89% yield) was obtained as a yellow solid in the same manner as in Example 63. 1 HNMR (400 MHz, DMSO-d6) δ: 9.31 (s, 2H), 8.65 (s, 1H), 8.38 (s, 1H), 7.78-7.74 (m, 1H), 7.49-7.45 (m, 1H), 6.84 (s, 2H), 5.50 (s, 2H); MS: m / z = 456.0 (M+1, ESI+).

[0384] Example 66: 3-(2-amino-9-(2,5-difluoro-3-(trifluoromethyl)benzyl)-9H-purin-6-yl)-5-cyanobenzamide

[0385]

[0386] The compound of Example 65 (80 mg, 0.18 mmol) was dissolved in toluene (1 mL), and acetaldoxime (52 mg, 0.88 mmol) and InCl3 (4 mg, 0.02 mmol) were added. The mixture was stirred at 90°C for 50 minutes. The mixture was concentrated, and the residue obtained was purified by Pre-HPLC (ACN / H2O = 40 / 60) to obtain the compound of Example 66 (20 mg, 23.47%) as a white solid. 1 HNMR (400 MHz, DMSO-d6) δ: 9.43-9.42 (m, 1H), 9.26-9.25 (m, 1H), 8.44-8.43 (m, 1H), 8.35 (s, 1H), 8.25 (s, 1H), 7.78-7.74 (m, 2H), 7.48-7.44 (m, 1H), 6.79 (s, 2H), 5.50 (s, 2H); MS: m / z = 474.0 (M+1, ESI+).

[0387] Example 67: 5-(2-amino-9-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-6-yl)isophthalonitrile

[0388]

[0389] Step 1: (5-chloro-2-fluoro-3-(trifluoromethyl)phenyl)methanol

[0390] 4-Chloro-1-fluoro-2-(trifluoromethyl)benzene (2100 mg, 10.57 mmol) was dissolved in THF (15 mL) under nitrogen atmosphere, and TMEDA (1843 mg, 15.86 mmol) and n-BuLi (5.5 mL, 13.75 mmol) were slowly added at -78 °C, and the mixture was stirred for 1 h. Then, DMF (1082 mg, 14.80 mmol) was added, and the mixture was stirred at 25 °C for 0.5 h, and then NaBH4 (799 mg, 21.15 mmol) was added, and the mixture was further stirred at 25 °C for 0.5 h. Water (200 mL) was poured into the resulting mixture, and the mixture was extracted three times with EtOAc (400 mL). The collected organic layer was dried over anhydrous Na2SO4 and concentrated to obtain (5-chloro-2-fluoro-3-(trifluoromethyl)phenyl)methanol (2400 mg, 99.27% ​​yield) as a yellow oil. 1 H NMR (300 MHz, DMSO-d6) δ: 7.84-7.79 (m, 2H), 5.67-5.63 (m, 1H), 4.64 (d, 2H).

[0391] Step 2: 6-chloro-9-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-2-amine

[0392] Under nitrogen atmosphere, (5-chloro-2-fluoro-3-(trifluoromethyl)phenyl)methanol (2.4 g, 10.50 mmol) was dissolved in DCM (30 mL) at 0 °C, and TsCl (2.16 g, 11.34 mmol) and TEA (3.28 g, 32.4 mmol) were added. The reaction mixture was stirred at 0 °C for 1 h, after which 6-chloro-9H-purin-2-amine (3.63 g, 21.4 mmol) and K2CO3 (4.44 g, 32.1 mmol) were added. After stirring for 1 hour at 40°C, the solvent was removed, and the residue was purified by silica gel column chromatography (DCM:MeOH = 25:1~10:1) to obtain 6-chloro-9-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-2-amine (1.6 g, yield 39.25%) as a white solid. MS: m / z = 379.80 (M+1, ESI+).

[0393] Step 3: 5-(2-amino-9-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-6-yl)isophthalonitrile

[0394] 6-Chloro-9-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-2-amine (300 mg, 0.78 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalonitrile (970 mg, 1.18 mmol), K2CO3 (327 mg, 2.36 mmol), and Pd(PPh3)4 (136 mg, 0.11 mmol) were dissolved in 1,4-dioxane / H2O(4 / 1) (5 mL), and stirred at 100 °C for 2 h under nitrogen. The mixture was filtered, concentrated in vacuo, washed with DCM (20 mL x 2), and concentrated in vacuo to give the compound of Example 67 (60 mg, 16.11% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 9.31 (d, 2H), 8.64 (s, 1H), 8.39 (s, 1H), 7.92-7.90 (m, 1H), 7.66-7.64 (m, 1H), 6.85 (s, 2H), 5.50 (s, 2H). MS: m / z = 471.9 (M+1, ESI+).

[0395] Example 68: 3-(2-amino-9-(5-chloro-2-fluoro-3-(trifluoromethyl)benzyl)-9H-purin-6-yl)-5-cyanobenzamide

[0396]

[0397] The compound of Example 67 (300 mg, 0.63 mmol), acetaldoxime (247 mg, 4.19 mmol), and InCl3 (18 mg, 0.08 mmol) were dissolved in 1,4-dioxane / toluene (1 / 1) (5 mL), and stirred at 90°C for 1 h under nitrogen (argon or air). The mixture was then filtered, concentrated in vacuo, washed with DCM (20 mLx2), and concentrated to obtain the compound of Example 68 (80 mg, 25.72% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ: 9.43 (s, 1H), 9.25 (s, 1H), 8.44 (s, 1H), 8.36 (s, 1H), 8.26 (s, 1H), 7.91 (d, 1H), 7.75 (s, 1H), 7.65 (d, 1H), 6.80 (s, 2H), 5.50 (s, 2H). MS: m / z = 490.0 (M+1, ESI+).

[0398]

[0399] Experimental Example 1: In vitro testing for the human chemokine GPCR cell-based agonist Arrestin assay.

[0400] The agonist activity of the compounds of the present invention against the human ACKR3 (CXCR7) receptor was measured in stably transfected CHO-K1 cells (PathHunter® CHO-K1 CXCR7 (CMKOR1) β-Arrestin Cell Line, Cat no. 93-0248C2) using a GPCR cell-based assay. Cells were seeded in a total volume of 20 μL in a 384-well microplate and incubated overnight at 37°C prior to testing. For agonist determinations, cells were incubated with the sample to induce the response. Intermediate dilutions of the sample stock were performed to generate a 5X sample in assay buffer. 5 μL of the 5X sample was added to the cells and incubated at 37°C for 120 minutes. The final assay vehicle concentration was 1%.

[0401] The results are presented in Table 2 as efficacy percentage ratings for the maximum response to the control ligand (CXCL12). The efficacy percentage ratings presented in Table 2 are as follows:

[0402] A: Efficacy percentage ≥ 50%, B: 20 ​​≤ Efficacy percentage < 50%, C: Efficacy percentage < 20%

[0403]

[0404] Through the experimental results in Table 2, it was confirmed that the compounds of the present disclosure act as CXCR7 agonists, and in particular, it was confirmed that Examples 1, 2, 4, 5, 6, 10, 17, 18, 20, 21, 22, 23, 33, 53, 54, 55, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 exhibited excellent effects.

[0405] Experimental Example 2: Bleomycin-induced idiopathic pulmonary fibrosis test

[0406] Male C57BL / 6N Tac mice were administered the compound of Example 1 and pirfenidone on day 0 and the experiment was conducted for 21 days. One hour after drug administration, the animals were anesthetized with ketamine + xylazine (70 + 5 mg / kg, intravenous injection). An endotracheal catheter was inserted through the mouse's mouth, and a solution of bleomycin (1 mg, 1.5–2 U / kg based on a 50 μL mouse dose) was infused using a micropipette, and the solution was allowed to be absorbed into the lungs spontaneously. Afterwards, 300 μL of air was additionally injected to ensure uniform distribution of the BLM solution.

[0407] Example 1 Compounds (10, 30, and 60 mg / kg, orally once daily) and Pirfenidone (100 mg / kg, orally twice daily) were administered for 21 days. After euthanizing the mice, bronchoalveolar lavage fluid (BALF) was collected, slides were prepared, and the number of each cell was evaluated. Lung tissues from euthanized mice were collected, immediately frozen in liquid nitrogen, and stored at -80°C. A portion of the lung lobe was used for hydroxyproline analysis. Hydroxyproline levels were evaluated by hydrolyzing the lung tissues of each mouse with 12 N HCl (1:20) in an oven at 110°C, oxidizing with 0.2 M chloramine-T, reacting with Ehrlich's reagent, and measuring the color change using a spectrophotometer at 550 nm. For histological examination, lung tissue (left lobe) was collected, fixed in 10% NBF for 24 hours, embedded in paraffin wax, and sectioned at 5-micrometer thickness. Lung tissue sections were stained with H&E for light microscopic analysis, and fibrosis grade was assessed using the Ashcroft Fibrosis Score Scale (Hubner et al., 2008).

[0408]

[0409] Figure 1 shows the effect of the compound of the present invention on BALF absolute differential cell count in a bleomycin-induced mouse IPF model. As shown in Figure 1, macrophages, lymphocytes, and neutrophils significantly increased in the BALF of the bleomycin-treated disease control group compared to the normal control group. On the other hand, the number of lymphocytes decreased in the groups administered with the compound of Example 1 (10, 30, and 60 mg / kg), and a significant decrease was observed particularly in the 60 mg / kg group.

[0410] Figure 2 illustrates the effect of Example 1 compound on pulmonary hydroxyproline in a bleomycin-induced IPF model. Hydroxyproline content is known to be an indicator of collagen deposition in fibrotic tissue. As shown in Figure 2, pulmonary hydroxyproline content was significantly increased in the bleomycin-treated diseased control group compared to the normal control group. In contrast, hydroxyproline content was significantly decreased in the Example 1 compound (10, 30, and 60 mg / kg) administration groups.

[0411] Figure 3 illustrates the effect of Example 1 compound on the Ashcroft fibrosis score in the bleomycin-induced IPF model. As shown in Figure 3, the Ashcroft score was significantly increased in the bleomycin-treated disease control group compared to the normal control group, confirming that fibrosis had significantly progressed 21 days after bleomycin administration. Meanwhile, the fibrosis score was reduced in the Example 1 compound (10, 30, and 60 mg / kg) administration groups compared to the disease control group, and in particular, the 30 mg / kg and 60 mg / kg administration groups showed statistically significant reductions.

[0412] Experimental Example 3: Carbon tetrachloride (CCl4)-induced liver fibrosis test

[0413] Male C57BL / 6N Tac mice were injected intraperitoneally twice weekly for 6 weeks with a mixture of carbon tetrachloride (CCl4) and olive oil (3:1 ratio, CCl4 25% in olive oil). Compound 1 (10, 30, 60 mg / kg, orally once daily) and elafibranor (30 mg / kg, orally once daily) were administered for 6 weeks, starting on day 0 before CCl4 administration. After the end of the test, the mice were euthanized, and serum and liver samples were collected for further analysis. The collected serum was separated and stored at -80°C for liver enzyme analysis, including ALT levels. For hydroxyproline analysis, liver sample pieces were flash-frozen in liquid nitrogen and stored at -80°C. Each ~50 mg of liver tissue was hydrolyzed with 12 N HCl (1:20) at 111°C for 20 h. The hydrolyzate was centrifuged (14,000 rpm, 15 min) and the supernatant was separated. After incubating 15 μL of the supernatant + 100 μL of chloramine T in a 96-well plate for 20 min, 100 μL of Ehrlich's solution was added and incubated at 75°C for 30 min in the dark. The final absorbance of the plate was measured at 550 nm to assess the hydroxyproline concentration.

[0414] Liver tissue was collected, preserved in 10% neutral buffered formalin for 24 hours, embedded in paraffin wax, and sectioned into 5 μm-thick sections. After H&E staining, the degree of fibrosis was assessed according to the Ishak fibrosis grade (see Table 4 below).

[0415]

[0416] The results of measuring hepatic hydroxyproline in a carbon tetrachloride-induced hepatic fibrosis model are shown in Figure 4. Hydroxyproline content is known to be an indicator of collagen deposition in fibrotic tissue. As shown in Figure 4, hepatic hydroxyproline content was significantly increased in the CCl4+ vehicle-treated diseased control group compared to the normal control group. In contrast, the groups administered with Example 1 compound (10, 30, and 60 mg / kg) showed a significant decrease in hepatic hydroxyproline content compared to the diseased control group, demonstrating an effect equivalent to elafibranor.

[0417] In addition, the results of measuring the Ishak fibrosis score in the carbon tetrachloride-induced liver fibrosis model are shown in Fig. 5. As can be seen in Fig. 5, the Ishak fibrosis score in the H&E-stained liver sections of the disease control group increased rapidly after 6 weeks of CCl4 administration compared to the normal control group, indicating that fibrosis had progressed significantly. However, it was confirmed that the fibrosis score was significantly reduced in the groups administered with the compound of Example 1 (10, 30, 60 mg / kg) compared to the disease control group.

[0418] Furthermore, from the results of measuring ALT in a carbon tetrachloride-induced liver fibrosis model (Fig. 6), it was confirmed that the ALT level, which increased rapidly in the disease control group compared to the normal control group, was significantly reduced by administration of the compound of Example 1 (10, 30, 60 mg / kg).

[0419] Experimental Example 4: EAE model test induced by MOG35-55

[0420] Myelin oligodendrocyte glycoprotein (MOG 35-55) was dissolved in saline at a concentration of 2 mg / mL, and heat-killed tuberculosis bacilli were added to prepare a complete Freund's adjuvant at a final concentration of 4 mg / mL. The 2 mg / mL MOG 35-55 solution and an equal volume of 4 mg / mL CFA were emulsified on ice at 30,000 rpm for 1.5 h using a high-speed homogenizer. Male C57BL / 6N Tac mice were anesthetized with 1-4% isoflurane, and 100 μL of the emulsion was injected subcutaneously at three sites: one site along the midline of the back between the shoulders and two sites on each side of the midline of the waist. Forty-eight hours later, disease was induced in all mice by intraperitoneal injection of pertussis toxin (200 ng in 200 μL of PBS). Compounds of Examples 1, 2, and 4 at a dose of 30 mg / kg were administered orally for 21 days.

[0421] The results of scoring EAE clinical signs according to the EAE clinical scoring system described in Table 5 below are shown in Figure 7.

[0422]

[0423] The EAE (Experimental Autoimmune Encephalomyelitis) mouse model is widely known as a model for autoimmune diseases, particularly multiple sclerosis. The therapeutic effects of the compound of the present invention in the EAE model were confirmed. As shown in Figure 7, the groups administered compounds of Examples 1, 2, and 4 showed a significant reduction in clinical signs of neuroinflammation compared to the disease control group (Model-Vehicle).

[0424] Experimental Example 5: MCT (monocrotaline)-induced pulmonary arterial hypertension model test in male SD rats

[0425] On Day 1, male SD rats were administered 60 mg / kg of MCT (monocrotaline) via intraperitoneal injection, and the test substances were administered orally once a day from D1 to D28. The test substances were the compound of Example 5 (10, 30, 60 mg / kg, po), Sildenafil (30 mg / kg, po) was used as a positive control, and AMD3100 octahydrochloride (CAS155148-31-5) (5 mg / kg, po) was used as a comparison substance. On Day 29, after anesthetizing the rats, a PE-50 catheter was inserted into the right jugular vein and slowly advanced into the subclavian vein, right atrium, and right ventricle, and when the right ventricular waveform stabilized, the right ventricular systolic pressure (RVSP) was measured.

[0426] Right ventricular hypertrophy index (RVH Index) was measured by perfusing PBS through the abdominal aorta of rats, isolating the heart, separating the right ventricle from the interventricular septum along the pulmonary artery orifice, and resecting and washing the left ventricle and interventricular septum. The right ventricle (RV) and the combined weight of the left ventricle and interventricular septum (LV+S) were then measured. The ratio of right ventricle weight to the weight of the left ventricle and interventricular septum (RV / LV+S) and right ventricle weight to body weight (RV / BW) were calculated.

[0427] For histopathological analysis, the left lung and right ventricle of rats were fixed by perfusion with 10% neutral buffered formalin, sectioned in paraffin, and stained with H&E. Right ventricular wall thickness was measured at three locations using an optical microscope, and the average of the measurements was used as the right ventricular wall thickness.

[0428] As can be seen in Figures 8 to 10, pulmonary artery pressure, i.e., right ventricular systolic pressure (RVSP), increased in the MCT administration model. However, in the group administered with the compound of Example 5, RVSP decreased in a dose-dependent manner, and a significant decrease was confirmed in the group administered with the compound of Example 5 (60 mg / kg). No significant changes were observed in the groups administered with AMD3100 and the positive control, sildenafil. An increase in pulmonary artery pressure increases the load on the right ventricle, leading to right ventricular hypertrophy. As a result, the RV / LV+S ratio significantly increased in the MCT administration model compared to the control group, and when the compound of Example 5 was administered at doses of 10, 30, and 60 mg / kg, the RV / LV+S ratio decreased in a dose-dependent manner. In particular, the RV / LV+S ratio was significantly lower in the group administered with the compound of Example 5 at a dose of 60 mg / kg compared to the control group. No significant changes were observed in the groups administered with AMD3100 and the positive control, sildenafil. Right ventricular wall thickness was significantly thickened in the MCT model, and the groups administered Example 5 compound at doses of 30 mg / kg and 60 mg / kg showed a significant decrease in right ventricular wall thickness. On the other hand, AMD3100 showed no significant decrease, and the positive control sildenafil significantly reduced right ventricular wall thickness.

[0429] Experimental Example 6: CXCL12-induced breast cancer (MDA-MB-231, TNBC) metastasis model test

[0430] Mount 24 Oris™ cell seeding stoppers in a 96-well plate, and suspend MDA-MB-231 cells in DMEM to obtain a cell count of 5x10 5After adjusting to cells / mL, 100 μL of cells were pipetted into each test well through one of the side ports of the Oris™ cell seeding stopper. The seeding plate containing the Oris™ cell seeding stopper was incubated in a humidified chamber (37°C, 5% CO2) for 24 h. After 24 h, the stopper was removed, and 100 μL of fresh medium with or without CXCL12 (100 ng / mL) was added. Compounds of Examples 1, 2, and 4 were each added at a concentration of 1 μM and incubated in a humidified chamber (37°C, 5% CO2) for 28 h to determine cell migration. After incubation, migrated cells were stained with Calcein-AM (1 μM) for 30 min, and cell migration was photographed using a Leica DMI3000 B. Images were calculated using ImageJ, and data were analyzed using GraphPad Prism.

[0431] As illustrated in Figure 11, cell migration was increased in breast cancer cells treated with CXCL12. However, cell migration was inhibited in the group administered with the compounds of Examples 1, 2, and 4 at a concentration of 1 μM. The results demonstrate that the compounds of the present invention can inhibit cancer metastasis by inhibiting cell migration.

[0432] Experimental Example 7: Bile duct ligation-induced cholestatic liver disease model in male C57BL / 6J mice.

[0433] On Day 0, serum was collected from male C57BL / 6J mice fasted for one day to assess AST, ALT, ALP, and total bilirubin. On Day 1, male C57BL / 6J mice were anesthetized with isoflurane, and a 1-cm common bile duct was exposed through a midline incision in the upper abdomen. The common bile duct was ligated twice with 5 / 0 silk sutures, and an incision was made between the ligatures. After surgery, the muscles and skin were separately sutured with 4 / 0 silk. The normal group underwent only exploratory laparotomy after midline incision and sutured. From Day 2 to Day 15, the compound of Example 2 (30 mg / kg, 60 mg / kg) was administered orally once daily for two weeks. On Day 15, the mice were euthanized with CO2, and blood was collected from the heart to isolate the serum. AST, ALT, ALP, and total bilirubin in the serum were assessed. Liver tissues were isolated, weighed, and the mesenchymal portion was fixed in 10% NBF and evaluated by hematoxylin and eosin (H&E) staining and α-SMA immunohistochemistry (IHC). For H&E staining, liver sections were cut into 4 μm thick, dried in an oven for 1 h, and then stained with an integrated workstation Leica SPECTRA ST (HistoCore SPECTRA H&E Staining System, S1#3801654, Lot#050223). For histological evaluation, H&E stained images were scanned using a LEICA Aperio GT450, and ductal proliferation, portal inflammation, and confluent necrosis scores were determined based on the criteria in Table 6 below.

[0434]

[0435] Immunohistochemical staining was performed by incubating primary rabbit polyclonal anti-a-SMA antibody (Abcam#ab5694) at a 1:400 dilution for 1 hour, adding anti-rabbit IgG(H+L) (Jackson immuno #111-035-045) at a 1:500 dilution, and scanning the a-SMA-stained sections using a Leica Aperio GT450 scanner for analysis.

[0436] As shown in Figure 12, the blood analysis results on Day 15 showed that ALT, AST, ALP, and total bilirubin levels were significantly higher in the bile duct ligation model compared to normal animals. However, in the group administered the Example 2 compound, the levels of ALT, AST, ALP, and total bilirubin decreased in a dose-dependent manner. In particular, a significant decrease in these levels was observed in the group administered the Example 2 compound at a dose of 60 mg / kg.

[0437] As shown in Figure 13, liver tissue fibrosis scores increased in the bile duct ligation model compared to normal animals in liver tissue stained with H&E solution. However, liver tissue fibrosis was significantly suppressed in the Example 2 compound administration group (60 mg / kg). Furthermore, the expression of a-SMA, a fibrogenic protein, significantly increased in the bile duct ligation model. However, a-SMA expression was significantly reduced in the Example 2 compound administration group (60 mg / kg).

Claims

1. A compound of the following formula I, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above formula, X and Y are each independently CR a or N; R 1 is selected from the group consisting of hydrogen, halogen, C1-C6 haloalkyl, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl; R 2 is -CN or -CONR b R c and; R a , R b and R c are each independently H or C1-C6 alkyl; n is an integer from 0 to 2.

2. In paragraph 1, R 1 A compound, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein said compound is H, F, Cl, Br, I, CF3, -CH3, -OCH3 or -C≡C.

3. In paragraph 1, R 2 A compound, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is -CN or -CONH2.

4. In paragraph 1, A compound, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is represented by any one of the following formulae IA to ID. In the above formula, R 1 , R 2 , R a And the definition of n is as described in paragraph 1.

5. In paragraph 1, A compound, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following chemical formula I-1: In the above formula, X, Y, R 1 and R 2 The definition is as described in paragraph 1.

6. In paragraph 1, A compound of formula I selected from the following, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof:

7. A pharmaceutical composition comprising a compound of any one of claims 1 to 6, a stereoisomer thereof, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof as an active ingredient.

8. In paragraph 7, A pharmaceutical composition for the treatment of a disease mediated by CXCR7.

9. In paragraph 8, A pharmaceutical composition, wherein the disease is selected from fibrosis, cholestatic liver disease, inflammatory disease, angiogenic disease, cancer, autoimmune disorder, osteoporosis, pulmonary hypertension, acute ischemic injury and thrombosis.

10. In paragraph 9, A pharmaceutical composition, wherein the above fibrosis is fibrosis of the liver, lungs, skin, kidneys, heart, joints or intestines.

11. In paragraph 9, A pharmaceutical composition, wherein the fibrosis is selected from the group consisting of liver fibrosis, liver cirrhosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, renal fibrosis, myocardial fibrosis, and arthrofibrosis.

12. In paragraph 9, A pharmaceutical composition, wherein the above cholestatic liver disease is selected from primary biliary cholangitis, primary sclerosing cholangitis, cholesterol gallstones, pregnancy-related intrahepatic cholestasis, progressive familial intrahepatic cholestasis, and Alagille syndrome.

13. In paragraph 9, The above inflammatory diseases are arthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, myocarditis, sarcoidosis or sinusitis; A pharmaceutical composition, wherein the angiogenic disease is rheumatoid arthritis, psoriasis, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, posterior lens fibroplasia, rosacea, Osler-Weber syndrome, myocardial neovascularization, plaque neovascularization, telangiectasia, hemophilic joint, angiofibroma, intestinal adhesions, Crohn's disease, eczema, scleroderma, diabetes, atherosclerosis, wound granulation or keloid.

14. In paragraph 9, A pharmaceutical composition wherein the cancer is selected from glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, leukemia, lymphoma, prostate cancer, Burkitt lymphoma, head and neck cancer, colon cancer, large intestine cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, stomach cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, genital cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, glioblastoma, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma.

15. In paragraph 9, A pharmaceutical composition, wherein the autoimmune disorder is multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, lupus nephritis, interstitial cystitis, celiac disease, autoimmune encephalomyelitis, a dehydrating disease, osteoarthritis, or type I diabetes.

16. In paragraph 9, A pharmaceutical composition wherein the above pulmonary hypertension is pulmonary arterial hypertension or chronic thromboembolic pulmonary hypertension.

17. A method for treating a disease mediated by CXCR7, comprising administering to a subject a compound of any one of claims 1 to 6, a stereoisomer, an isotopically labeled compound, a hydrate, a solvate or a pharmaceutically acceptable salt thereof.

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