Inhibitor for promoting collagen expression

Abstract

The present disclosure relates to an inhibitor for promoting collagen expression, the inhibitor having a structural formula as shown in the following formula (I). The azacycloalkane compound and a pharmaceutical composition thereof provided in the present disclosure have significant Rho kinase inhibitory activity, have better enzymatic activity and cell activity than existing ROCK inhibitors such as Ripasudil, Netarsudil and belumosudil, and have JAK inhibitory activity. Therefore, the compound and composition have great application potential.

Description

An inhibitor for promoting collagen expression TECHNICAL FIELD

[0001] The present disclosure provides an azacycloalkane compound, a stereoisomer comprising the compound, a pharmaceutical composition, and the use of the compound and the pharmaceutical composition, which is an inhibitor for promoting collagen expression and targeting ROCK, and can be used for regulating Rho kinase-mediated diseases. BACKGROUND

[0002] The Rho kinase / ROCK (Rho-associated kinase) signaling pathway induces cytoskeleton reorganization, cell migration, adhesion and stress fiber formation, and is related to various physiological functions. The ROCK family includes ROCK1 and ROCK2. ROCK1 is highly expressed in the lung, liver, spleen, kidney and testis, and ROCK2 is highly expressed in the brain and heart. ROCK1 indirectly acts on the epithelial-cadherin complex by binding to the scaffold protein P120-catenin of E-cadherin. ROCK1 can be concentrated in the microtubule organizing center and the edge of the pseudopod of the motile cell, and is related to cell migration. ROCK2 is mainly concentrated in the cytoplasm, and it is localized to the plasma membrane through the C-terminal region, and is related to vimentin and actin stress fibers.

[0003] ROCK mediates many pathophysiological signals, and is related to various physiological functions such as endothelial permeability, tissue contraction and growth. ROCK inhibitors have potential application value in diseases including asthma, cancer, glaucoma, insulin resistance, renal failure, neuronal degeneration and osteoporosis. It is very urgent to develop a drug targeting ROCK to solve the unmet clinical needs.

[0004] Type XVII collagen (COL17), also known as BP180 or BPAG2, is a transmembrane collagen protein widely distributed in the epidermis, basal layer of the skin, hair follicle stem cells, and epithelial cells of the eye. As a transmembrane protein on the hemidesmosome, Type XVII collagen plays a bridging role between the epidermis and the dermis, mainly responsible for tightly binding the epidermis to the dermis. This binding not only helps maintain the integrity of the skin structure and the tightness of the skin, but also plays an important role in stabilizing the basement membrane, skin aging mechanism, and maintenance of hair follicle cells. Studies have shown that COL17 expression deficiency and acquired dysfunction are associated with bullous skin diseases (Wataru NISHIE, Acta Derm Venereol, 2020), and lack of BP180 and / or its functional disorder leads to keratinocyte separation from the basal layer, resulting in separation of the epidermis and dermis, leading to skin blistering (Jussi Tuusa, Int. J. Mol. Sci., 2021). Bullous pemphigoid (BP) is an autoimmune skin disease characterized by tense bullous disease, erythema, leprosy-like skin lesions or eczema, and intense itching. Autoantibody degradation and depletion of COL17 are considered to be the main mechanism of BP occurrence (Nishie W, J Dermato Sci, 2014). Loss-of-function mutations in the COL17 gene can cause junctional epidermolysis bullosa (JEB) (McGrath JA et al., Nat Genet, 1995). In addition, mucous membrane pemphigoid (MMP), pemphigoid gestationis (PG), acquired epidermolysis bullosa (EBP), lichen planus pemphigoid (LPP), linear IgA bullous dermatosis (LABD), dermatitis herpetiformis (DH), cicatricial pemphigoid (CP), and other autoimmune subepidermal bullous diseases are also associated with COL17 (BP180) (Schmidt E et al., Lancet, 2013; Wang G, Jin HZ, Zhonghua Pingfen Zazhi, 2022). In addition, Type XVII collagen promotes hair growth in androgenetic alopecia (AGA) (Cheng HR et al., Zhonghua Zhongliu Waike Zazhi, 2024). Emi K. Nishimura et al. found that the chemical small molecule Y27632 can promote the expression of COL17 in human keratinocytes (Nan Liu et al., Nature, 2019), and it is a ROCK inhibitor. The present study found a batch of new skeletal ROCK small molecule inhibitors that can promote the expression of COL17 with better effects. SUMMARY

[0005] The present disclosure develops a novel skeleton of high-activity azacycloalkane compounds and pharmaceutical compositions thereof, which can promote the expression of collagen type 17 and target ROCK, for use in the regulation of COL17 or Rho kinase-mediated diseases.

[0006] The present disclosure provides an azacycloalkane compound represented by Formula I or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof:

[0007] One aspect of the present disclosure is to provide a compound, isotopologue, or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof represented by Formula I and its use in treating ROCK-mediated diseases or diseases associated with COL17.

[0008] One aspect of the present disclosure is to provide a use of a compound, isotopologue, or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof represented by Formula I in the preparation of a medicament for treating Rho kinase-mediated diseases or a medicament for treating diseases associated with COL17.

[0009] Another aspect of the present disclosure is to provide a method for treating Rho kinase-mediated diseases or a method for treating diseases associated with COL17, comprising administering to a patient in need thereof a therapeutically effective amount of a compound, isotopologue, or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof represented by Formula I. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 shows the immunoblotting test of Y-27632 on the promotion of collagen type 17 expression;

[0011] Figure 2 shows the immunoblotting test of the compound of Example 6 on the promotion of collagen type 17 expression;

[0012] Figure 3 shows the immunoblotting test of the compound of Example 33 on the promotion of collagen type 17 expression;

[0013] Figure 4A shows the data of the measured basal intraocular pressure each day, and Figure 4B shows the change value of the basal intraocular pressure of each day relative to D1. DETAILED DESCRIPTION

[0014] The present disclosure is further explained by the accompanying drawings and examples. The features and advantages of the present disclosure will become more apparent from the following description in conjunction with the accompanying drawings.

[0015] The term "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations. Unless specifically stated otherwise, the drawings are not drawn to scale and are merely intended to conceptually illustrate the features described herein.

[0016] Moreover, the technical features involved in the different embodiments of the present disclosure described below can be combined with each other as long as there is no conflict.

[0017] Definitions

[0018] When a substituent is described by a conventional chemical formula that is written from left to right, the substituent is also meant to include an alternative chemical formula that is the reverse of the first formula, i.e., read from right to left. For example, -CH2O is equivalent to OCH2.

[0019] The term "optionally" or "optional" means that the subsequently described event or circumstance can or can not occur, and thus the description includes instances where the event or circumstance occurs and instances where it does not. For example, an ethyl group "optionally" substituted with a halogen means that the ethyl group can be unsubstituted (-CH2CH3), mono-substituted (e.g., -CH2CH2F), poly-substituted (e.g., -CHFCH2F, -CH2CHF2, etc.), or fully substituted (-CF2CF3). It will be understood by those skilled in the art that, for any group containing one or more substituents, such group does not include any substitution or substitution pattern that is not

[0020] The term "substituted" means that any one or more hydrogen atoms on the particular atom is replaced with a substituent, as long as the valency of the particular atom is not normally exceeded and the resulting compound is stable. When the substituent is oxo (i.e., =0), it is meant that two hydrogen atoms are replaced by the oxo group. Oxos are not intended to be on aromatic groups.

[0021] When any variable (e.g., R) occurs more than one time in a constituent or in a combination of constituents, its definition in each instance is independent of its definition at every other location. Thus, for example, if a group is substituted with 0-2 R groups, then such group can optionally be substituted with one R or two R groups, and at each occurrence R is selected independently. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0022] As used herein, C m～n means that the moiety has m to n carbon atoms. For example, the group "C 1～8 " means that the moiety has 1 to 8 carbon atoms, i.e., the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms,... 8 carbon atoms. Thus, for example, the group "C 1～8"Alkyl" refers to an alkyl group of from 1 to 8 carbon atoms, i.e., the alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and the like. The numerical ranges given herein are used to define the various embodiments, for example, "1 to 8 carbon atoms" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, or 8 carbon atoms. It will be appreciated that the upper and lower limits of the ranges of atoms can be combined to form "ranges of ranges," e.g., "1-8, or 1-6, or 1-4, or 1-3 carbon atoms."

[0023] The term "alkyl" refers to an optionally substituted straight-chain or optionally substituted branched-chain saturated aliphatic hydrocarbon group, which is attached to the rest of the molecule by a single bond. An "alkyl" group herein can have from 1 to about 8 carbon atoms, for example, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms, or from 1 to 3 carbon atoms. Examples of "alkyl" groups herein include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-l-propyl, 2-methyl-2-propyl, 2-methyl-l-butyl, 3-methyl-l-butyl, 2-methyl-3-butyl, 2,2-dimethyl-l- propyl, 2-methyl- 1-pentyl, 3-methyl- 1-pentyl, 4-methyl- 1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l-butyl, 3,3-dimethyl-l-butyl, 2-ethyl-l-butyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, t-pentyl, hexyl, and the like, as well as longer alkyl groups such as heptyl, octyl, and the like. Where a group defined herein, such as "alkyl," appears more than once in a formula, each occurrence of the group is independent of the others unless otherwise specified. For example, a "dialkyl" group is defined as two alkyl groups attached to the rest of the molecule by two separate carbon atoms. 1-8 "Alkyl" refers to an alkyl group of from 1 to 8 carbon atoms, i.e., the alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and the like. The numerical ranges given herein are used to define the various embodiments, for example, "1 to 8 carbon atoms" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, or 8 carbon atoms. It will be appreciated that the upper and lower limits of the ranges of atoms can be combined to form "ranges of ranges," e.g., "1-8, or 1-6, or 1-4, or 1-3 carbon atoms." 1-4 "Alkyl" refers to an alkyl group of from 1 to 8 carbon atoms, i.e., the alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and the like. The numerical ranges given herein are used to define the various embodiments, for example, "1 to 8 carbon atoms" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, or 8 carbon atoms. It will be appreciated that the upper and lower limits of the ranges of atoms can be combined to form "ranges of ranges," e.g., "1-8, or 1-6, or 1-4, or 1-3 carbon atoms."

[0024] The term "alkenyl" refers to an optionally substituted straight-chain or optionally substituted branched-chain monovalent hydrocarbon group having at least one C=C double bond. The alkenyl group has, without limitation, from 2 to about 8 carbon atoms, such as from 2 to about 6 carbon atoms, or from 2 to about 4 carbon atoms. The double bond in these groups can be in the cis or trans configuration, and shall be understood to encompass both isomers. Examples of alkenyl groups include, but are not limited to, ethenyl (CH=CH2), 1-propenyl (CH2CH=CH2), isopropenyl (C(CH3)=CH2), butenyl, and 1,3-buten-2-yl, and the like. Where a group defined herein, such as "alkenyl," appears more than once in a formula, each occurrence of the group is independent of the others unless otherwise specified. For example, a "dialkenyl" group is defined as two alkenyl groups attached to the rest of the molecule by two separate carbon atoms. 2-8"Alkenyl" refers to an alkenyl group that can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, and the like. Alkenyl groups herein also encompass instances where no number range is specified.

[0025] The term "alkynyl" refers to an optionally substituted straight or branched chain monovalent hydrocarbon radical having at least one C≡C triple bond. The alkynyl group has, without limitation, 2 to about 8 carbon atoms, such as 2 to about 6 carbon atoms, or 2 to about 4 carbon atoms. Examples of alkynyl groups herein include, but are not limited to, ethynyl, 2-propynyl, 2-butynyl, and 1,3- butadiynyl, and the like. Where a number range is specified for an alkynyl group defined herein, such as "C2-C6alkynyl," the range is inclusive of the numbers specified as the lower and upper number of the range. 2-8 "Alkynyl" refers to an alkynyl group that can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, and the like. Alkynyl groups herein also encompass instances where no number range is specified.

[0026] The term "cycloalkyl" refers to a non-aromatic carbon-containing ring, including saturated carbon rings (such as cycloalkyl) or unsaturated carbon rings (such as cycloalkenyl). The carbon ring can include a single carbon ring (having one ring), such as a monocyclic cycloalkyl; a double carbon ring (having two rings), such as a bicyclic cycloalkyl; a multiple carbon ring (having more than two rings). The rings can be fused or spirocyclic. The carbon ring (such as cycloalkyl or cycloalkenyl) can have 3 to 8 carbon atoms, such as 3 to about 6 ring-forming carbon atoms or 3 to about 5 ring-forming carbon atoms.

[0027] The term "aryl" refers to an optionally substituted aromatic hydrocarbon group having 6 to about 20, such as 6 to 12 or 6 to 10 ring-forming carbon atoms, which can be a monocyclic aryl, bicyclic aryl, or polycyclic aryl. The bicyclic aryl or polycyclic aryl can be a monocyclic aryl fused to other independent rings, such as aliphatic, heterocyclic, aromatic, aromatic heterocyclic rings. Non-limiting examples of monocyclic aryl groups include 6 to about 12, 6 to about 10, or 6 to about 8 ring-forming carbon atoms, such as phenyl; bicyclic aryl groups such as naphthyl; polycyclic aryl groups such as phenanthryl, anthryl, azulenyl.

[0028] The term "heteroaryl" refers to any substituted heteroaryl group comprising from about 5 to about 20, such as 5 to 12 or 5 to 10 ring-forming atoms, at least one (such as 1-4, 1-3, 1-2) of which is a heteroatom independently selected from oxygen, nitrogen, sulfur, phosphorus, silicon, selenium, and tin, without limitation. Heteroaryl groups include monocyclic heteroaryl groups (having one ring), bicyclic heteroaryl groups (having two rings), or polycyclic heteroaryl groups (having more than two rings). In embodiments where two or more heteroatoms occur in the ring, the two or more heteroatoms can be the same as one another, or some or all of the two or more heteroatoms can be different from one another. Bicyclic or more ring heteroaryl groups can be one monocyclic heteroaryl group fused to other independent rings, such as aliphatic, heterocyclic, aromatic, and aromatic heterocyclic rings (which can be collectively referred to as fused ring heteroaryl groups). Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

[0029] The term "heterocyclyl" refers to non-aromatic heterocycle, which includes saturated heterocycles or unsaturated heterocycles (containing unsaturated bonds) that do not have a fully conjugated pi-electron system, and can be classified as acyclic, fused, bridged, or spiro systems, without limitation. One or more (such as 1-4, 1-3, 1-2) of the ring-forming atoms is a heteroatom, such as an oxygen, nitrogen, or sulfur atom. Heterocyclyl groups can include single heterocyclic rings (having one ring) or double heterocyclic rings (having two fused rings) or multiple heterocyclic rings (having more than two fused rings); and also include spiro rings. Heterocyclyl groups can have from 3 to about 20, such as 3 to about 10, 3 to about 8, 4 to 8, 4 to 7, 5 to about 8, or 5 to about 6 ring-forming atoms. Non-limiting examples of heterocyclyl groups include oxiranyl, thiiranyl, aziridinyl, oxetanyl, thietanyl, tetrahydrofuranyl, pyrrolidinyl, oxazolidinyl, tetrahydropyrazolyl, pyrrolinyl, dihydrofuranyl, dihydrothienyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyranyl, tetrahydropyranyl, dihydrothiopyranyl, azepinyl, oxepinyl, thiepinyl, oxazepinyl, diazepinyl, and the like.

[0030] The term "halo" or "halogen" refers to an optionally substituted group (such as alkyl, alkenyl, alkynyl, alkoxy, and the like) in which at least one hydrogen atom is replaced with a halogen (such as fluorine, chlorine, bromine, iodine, or combinations thereof). In some embodiments, two or more hydrogens are replaced with the same halogen as one another (e.g., difluoromethyl, trifluoromethyl); in other embodiments, two or more hydrogens are replaced with different halogens from one another (e.g., 1-chloro-1-fluoro-1-iodoethyl).

[0031] The term "alkoxy" refers to an alkyl ether group (0-alkyl), non-limiting examples of which include methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, sec-butyloxy, t-butyloxy, and the like.

[0032] The term "alkylcarbonyl" refers to a group in which an alkyl group is attached to a -CO- group, non-limiting examples of which include formyl, acetyl, propionyl, butyryl, and the like. For example, the term "C 1-6 alkylcarbonyl" refers to a group in which a C 1-6 alkyl group is attached to a -CO- group. For another example, the term "C 1-4 alkylcarbonyl" refers to a group in which a C 1-4 alkyl group is attached to a -CO- group.

[0033] The term "alkylsulfonyl" refers to a group in which an alkyl group is attached to a -SO2- group, non-limiting examples of which include methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, and the like. For example, the term "C 1-6 alkylsulfonyl" refers to a group in which a C 1-6 alkyl group is attached to a -SO2- group. For another example, the term "C 1-4 alkylsulfonyl" refers to a group in which a C 1-4 alkyl group is attached to a -SO2- group.

[0034] The term "amino" refers to a -NH2 group, a -NH(C 1～6 alkyl) group, or a -N(C 1～6 alkyl)2 group. Specific examples of amino groups include, but are not limited to, -NH2, -NHCH3, -N(CH3)2, -NHC2H5, -N(C2H5)2, -N(C3H7)2, -N(CH3)C2H5, and the like.

[0035] Other group terms herein also include: "hydroxy" refers to an -OH group, "mercapto" refers to an -SH group, "cyano" refers to a -CN group, and "carboxy" refers to a -COOH group.

[0036] The term "membered" refers to the number of skeletal atoms that make up a ring. For example, pyridine is a six-membered ring, and pyrrole is a five-membered ring.

[0037] The term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0038] The term "pharmaceutical composition" refers to a biologically active compound optionally admixed with at least one pharmaceutically acceptable chemical constituent or agent, i.e., a "carrier," which facilitates the introduction of the compound into a cell or tissue, including but not limited to stabilizers, diluents, suspending agents, thickening agents, and / or excipients.

[0039] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acids and bases of the designated compound and that is not biologically or otherwise undesirable. Unless otherwise specified, the salts in the present disclosure can refer to metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Non-limiting examples of metal salts include, but are not limited to, alkali metal salts such as sodium salts, potassium salts, and the like; alkaline earth metal salts such as calcium salts, magnesium salts, barium salts, and the like; aluminum salts, and the like. Non-limiting examples of salts with organic bases include, but are not limited to, salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and the like. Non-limiting examples of salts with inorganic acids include, but are not limited to, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Non-limiting examples of salts with organic acids include, but are not limited to, salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Non-limiting examples of salts with basic amino acids include, but are not limited to, salts with arginine, lysine, ornithine, and the like. Non-limiting examples of salts with acidic amino acids include, but are not limited to, salts with aspartic acid, glutamic acid, and the like.

[0040] Pharmaceutically acceptable salts can be synthesized from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, such salts are prepared either by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, and the like, are preferred.

[0041] The term "solvent" refers to a physical aggregate formed by a compound of this disclosure with one or more solvent molecules, including varying degrees of ions and covalent bonds, such as hydrogen bonds. It has been demonstrated that this solvate can be separated, for example, when one or more solvent molecules are mixed in the crystal lattice. "Solvent" comprises both a solvent phase and a separable solvate component. Numerous examples of solvates exist, including ethanol solvates, methanol solvates, etc. "Hydrate" is a solvate that uses water (H₂O) molecules as a solvent. One or more compounds of this disclosure can be prepared as solvates at will. The preparation of solvates is well known. A typical, non-limiting preparation process involves dissolving the compound of the invention in a desired amount of an ideal solvent (organic solvent, water, or a mixture thereof) at a temperature above room temperature, cooling, allowing crystals to crystallize, and then separating and picking out the crystals using standard methods. The presence of the solvent (water) in the crystallization can be confirmed using IR spectroscopy.

[0042] The term "active metabolite" refers to an active derivative of a compound that is formed during its metabolism.

[0043] The term "polymorph" refers to the compounds of this disclosure that exist in different lattice forms.

[0044] The term "isotope-labeled" refers to compounds of this disclosure that are labeled with isotopes. For example, the isotopes in the compounds of this disclosure may include various isotopes of elements such as H, C, N, O, P, F, and S, such as... 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 S.

[0045] The term "pharmaceuticalally acceptable prodrug" or "prodrug" refers to any pharmaceutically acceptable salt, ester, salt of ester, or other derivative of the compounds disclosed herein, which, upon administration to a receptor, can directly or indirectly provide the compound disclosed herein or its pharmaceutically active metabolites or residues. Particularly preferred derivatives or prodrugs are those compounds that, when administered to a patient, can improve the bioavailability of the compounds disclosed herein (e.g., make orally administered compounds more readily absorbed into the bloodstream), or those compounds that facilitate the delivery of the parent compound to a biological organ or site of action (e.g., the brain or lymphatic system). Prodrugs can be prepared by modifying functional groups present in the compound in a manner that allows them to be broken down into the parent compound, either through conventional procedures or in vivo. Various forms of prodrugs are well known in the art.

[0046] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule. The compounds disclosed herein contain asymmetric or chiral centers, double bonds, and other structures; therefore, the compounds disclosed herein may include various isomer forms such as optical isomers, geometric isomers, tautomers, and transisomers. These isomers, their single isomers, racemates, etc., are all included within the scope of this disclosure. For example, optical isomers can be prepared by chiral resolution, chiral synthesis, or chiral reagents or other conventional techniques to produce optically active (R)- and (S)- isomers, as well as D- and L isomers. For example, they can be converted to diastereomers by reacting with suitable optically active substances (e.g., chiral alcohols or Mosher's chloride), and then separated and converted (e.g., hydrolyzed) to the corresponding single isomers. Furthermore, separation can also be achieved by chromatographic column chromatography.

[0047] The “pharmaceutical compositions” described herein may be prepared in a manner well known in the pharmaceutical field and may be administered or applied via a variety of routes, depending on whether local or systemic treatment is required and the area to be treated. They may be administered topically (e.g., transdermal, skin, eye, and mucous membrane delivery, including intranasal, vaginal, and rectal delivery), pulmonaryly (e.g., by inhalation or blowing of powders or aerosols, including via nebulizers; intratracheal, intranasal), or orally or parenterally. Parenterally administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Parenterally administration may be in the form of a single large dose or via, for example, a continuous infusion pump. The pharmaceutical compositions described herein include, but are not limited to, the following forms: tablets, pills, powders, lozenges, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or soluble in liquid solvents); ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders containing, for example, up to 10% by weight of an active compound.

[0048] The pharmaceutical compositions herein can be formulated in unit dosage form for administration once or more than once per day. Each dosage unit contains a predetermined amount, calculated to produce the desired therapeutic effect, of the active agent, and the unit dosage can range from about 0.1 to 1000 mg, usually about 5 to 1000 mg, more usually about 100 to 500 mg, of the active agent. The term "unit dosage form" refers to physically discrete units suitable for single administration for human patients and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier.

[0049] The term "individual" refers to an individual having a disease, disorder, or condition, and the like, including mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (such as chimpanzees and other apes and monkey species); farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.

[0050] The terms "treat," "treatment," and other similar terms include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a symptom caused by the disease or condition, or stopping the symptoms of the disease or condition, and in addition, the term can encompass the purpose of preventing. The terms also include achieving a therapeutic effect and / or a prophylactic effect. The therapeutic effect refers to curing or ameliorating the underlying disorder being treated. In addition, a cure or amelioration of one or more symptoms associated with the underlying disorder is a therapeutic effect, e.g., a patient can be observed to have an improvement in condition even though the patient can still be affected by the underlying disorder. In terms of prophylactic effects, the compositions or compounds can be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis can not have been made.

[0051] The term "amount effective to obtain the desired therapeutic effect" or "therapeutically effective amount" refers to that amount of at least one pharmaceutical agent or compound that, when administered, is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological

[0052] The proportion or concentration of the compounds of the present disclosure in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical properties (e.g., hydrophobicity), route of administration, and the like. For example, a composition containing about 0.1 to 10% w / v of the compound can be used for parenteral administration. Certain typical dose ranges are about 1 pg / kg to about 1 g / kg body weight / day. In certain embodiments, the dose range is about 0.01 mg / kg to about 100 mg / kg body weight / day. The dosage will likely depend on such variables as the type and extent of progression of the disease or disorder, the general health of the particular patient, the preferred mode of administration, the relative biological efficacy of the compound selected, and the like.

[0053] The term "administering" refers to methods allowing delivery of a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral routes, transduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injections or infusions), topical, and rectal administration. Those skilled in the art are familiar with the techniques of administration that can be used for the compounds and methods described herein.

[0054] In one aspect, the present disclosure provides a new use of the pyrrolopyrimidine compound represented by general formula (I) or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically labeled, isomer or prodrug thereof, particularly in the preparation of a medicament for treating or preventing Rho kinase-mediated diseases or diseases associated with COL17.

[0055] The present study unexpectedly found that the compound of the example has the effect of promoting the expression of collagen 17, which is significantly better than that of a single ROCK inhibitor (such as Y-27632 and KD-025, etc.), and a single JAK inhibitor (such as Baricitinib, Itacitinib, etc.) has no such effect.

[0056] The pyrrolopyrimidine compound of the present disclosure has a structural formula represented by general formula (I)

[0057] wherein,

[0058] L is selected from -C(=O)-, -S(=O)2- or -CONH-;

[0059] X is selected from CR4 or N;

[0060] Ar is selected from substituted or unsubstituted single bond, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl or C 5～20 heteroaryl, the substituent is selected from halogen, C 1～8alkyl, C 1～8 haloalkyl or C 1～8 alkoxy;

[0061] R1is selected from the group consisting of substituted or unsubstituted: halo, NR 71 R 72 , C 1～8 alkyl, C 1～8 alkoxy, C 2-8 alkenyl, C 2-8 alkynyl, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl or C 5～20 heteroaryl; substituents are selected from the group consisting of halo, C 1～8 alkyl, C 1～8 haloalkyl, C 1～8 alkoxy, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, C 5～20 heteroaryl, cyano, NR 71 R 72 , hydroxyl, carboxyl or thiol;

[0062] R2is selected from -NHR7, wherein R7is selected from the group consisting of substituted or unsubstituted: hydrogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 alkylacyl, C 1～6 alkylsulfonyl, C 1～6 haloalkyl or C 1～6 alkoxy; substituents are selected from the group consisting of cyano, NR 71 R 72 , hydroxyl, carboxyl, thiol, C 3～6 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl or C 5～20 heteroaryl;

[0063] R3is selected from hydrogen, halo, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 haloalkyl, C 1～6 alkoxy, cyano, amino, hydroxyl, carboxyl or thiol;

[0064] R4is selected from hydrogen, halo or C 1～6 alkyl;

[0065] R5is selected from cyano, -CONH2or carboxyl;

[0066] R6is selected from hydrogen, halo or C1～6 alkyl;

[0067] R 71 and R 72 are each independently selected from hydrogen or C 1～6 alkyl.

[0068] For the pyrrolopyrimidine compounds of Formula (I), in one embodiment, R2is selected from -NHR7, wherein R7is selected from substituted or unsubstituted hydrogen, C 1～4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1～4 alkylacyl, C 1～4 alkylsulfonyl, C 1～4 haloalkyl, or C 1～4 alkoxy; the substituents are selected from cyano, NR 71 R 72 , hydroxy, carboxy, mercapto, C 3～5 cycloalkyl, C 3～6 heterocyclyl, C 6～12 aryl, or C 5～12 heteroaryl. In one embodiment, R7may be hydrogen, i.e., R2is -NH2. In one embodiment, R7may be C 1～4 alkyl (e.g., methyl, ethyl, etc.); or, R7may be amino-substituted C 1～4 alkyl, e.g., dimethylamino-substituted C 1～4 alkyl (e.g., methyl, ethyl, etc.). In one embodiment, R7may be C 1～4 alkylacyl, e.g., acetyl, etc. In one embodiment, R2is -NH2. In one embodiment, R2is -NHCOC 1-4 alkyl, e.g., -NHCOCH3. In one embodiment, R2is -NHC 1-4 alkyl, e.g., -NHCH2CH3. In one embodiment, R2is amino-substituted -NHC 1-4 alkyl, e.g., -NHCH2CH2N(CH3)2.

[0069] For the pyrrolopyrimidine compounds of Formula (I), in one embodiment, X is selected from CH.

[0070] For the pyrrolopyrimidine compounds of Formula (I), in one embodiment, R1is selected from substituted or unsubstituted C 1～6 alkyl, C 1～6 alkoxy, C 3～6 cycloalkyl, C 6～12 aryl, C 3～6 heterocyclyl, or C5～12 Heteroaryl, wherein the heteroatom is selected from N, O, or S; preferably selected from the following substituted or unsubstituted groups: C 1～4 Alkyl, C 1～4 Alkoxy, phenyl, biphenyl, naphthyl, and C atoms containing 1 to 3 nitrogen atoms 5～6 Heterocyclic group or C 5～6 Heteroaryl groups; substituents selected from halogens, C 1～4 Alkyl or C 1～4 Halogenated alkyl groups.

[0071] For the pyrrolopyrimidine compound represented by general formula (Ⅰ), in one embodiment, R3 is selected from hydrogen, halogen, C 1～4 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 1～4 Haloalkyl, C 1～4 Alkoxy, cyano, NR 71 R 72 , hydroxyl, carboxyl or thiol group. Preferably, R3 is hydrogen.

[0072] For the pyrrolopyrimidine compound represented by general formula (I), in one embodiment, Ar is selected from the following substituted or unsubstituted groups: C 5～7 cycloalkyl, C 6～12 aryl or C containing at least one heteroatom 5～12 Heterocyclic group or C 5～12 Heteroaryl groups, wherein the heteroatoms are selected from N, O, or S; preferably selected from substituted or unsubstituted C groups containing 1 to 3 N atoms. 5～6 Heterocyclic group or C 5～6 heteroaryl; substituents are selected from halogens.

[0073] Preferably, in one embodiment, Ar is one of the following groups, substituted or unsubstituted:

[0074] in, Indicates the position connected to L. Indicates the position where it is attached to the nitrogen-containing heterocyclic butyl group.

[0075] The substituents are halogens, particularly F; the number of substituents can be 1 to 3, for example, 1, 2, or 3. In a further embodiment, Ar is one of the following groups:

[0076] in, Indicates the position connected to L. Indicates the position where it is attached to the nitrogen-containing heterocyclic butyl group;

[0077] In particular, Ar is

[0078] For the pyrrolopyrimidine compounds of the general formula (I), in an embodiment, Ar is a single bond, i.e. the pyrrolopyrimidine compounds of the following formula IA

[0079] The above limitations for the groups in the general formula (I) apply equally to formula IA. Further, for the pyrrolopyrimidine compounds of the general formula (I) or formula IA, in an embodiment, R6is hydrogen.

[0080] For the pyrrolopyrimidine compounds of the above general formula (I) or formula IA, R 71 and R 72 may both be hydrogen; or, R 71 and R 72 may both be C 1-4 alkyl, such as methyl, and the like.

[0081] For the pyrrolopyrimidine compounds of the general formula (I), in an embodiment, the pyrrolopyrimidine compounds are selected from the following structures:

[0082] Pharmaceutical compositions and uses

[0083] The present disclosure also provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof of any of the above technical solutions, and a pharmaceutically acceptable carrier.

[0084] The pharmaceutical composition includes, but is not limited to, oral dosage forms, parenteral dosage forms, topical dosage forms, rectal dosage forms, and the like. For example, the pharmaceutical composition can be oral tablets, capsules, pills, powders, sustained-release preparations, solutions and suspensions, sterile solutions, suspensions or emulsions for parenteral injection, ointments, creams, gels and the like for external use, eye drops for external use, inhalants for external use, or suppositories for rectal administration.

[0085] The pharmaceutical composition can also include other active ingredients or drugs, which are used in combination with the compound or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof.

[0086] The present disclosure also provides the use of the above compound or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof, and the above pharmaceutical composition in the preparation of a medicament for treating Rho kinase-mediated diseases or a medicament for treating diseases related to COL17.

[0087] The present disclosure also relates to a method for treating a Rho kinase-mediated disease, comprising administering to a patient in need thereof a therapeutically effective amount of the above-mentioned azacycloalkane compound or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof, or the above-mentioned pharmaceutical composition. The Rho kinase can include ROCK1 and ROCK2, etc.

[0088] In one embodiment, the Rho kinase-mediated disease is graft-versus-host disease (GVHD), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, cancer, glaucoma, insulin resistance, kidney failure, neuronal degeneration or osteoporosis.

[0089] In one embodiment, the Rho kinase-mediated disease is asthma, cancer, glaucoma, insulin resistance, kidney failure, neuronal degeneration or osteoporosis.

[0090] The azacycloalkane compound and the pharmaceutical composition thereof provided by the present disclosure have significant Rho kinase inhibitory activity, and the enzymatic activity and cellular activity thereof are superior to those of the existing ROCK inhibitors such as Ripasudil, Netarsudil and belumosudil, and thus have great application potential.

[0091] In another aspect, the present disclosure also provides use of the above-mentioned compound or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof, and the above-mentioned pharmaceutical composition in the preparation of a medicament for treating a disease associated with COL17. The azacycloalkane compound and the pharmaceutical composition thereof provided by the present disclosure can significantly promote the expression of COL17 (Collagen XVII), and can be used for treating a disease associated with COL17 (in particular, a disease whose treatment requires promotion of the expression of COL17).

[0092] The present disclosure also provides a method for treating a disease associated with COL17, comprising administering to a patient in need thereof a therapeutically effective amount of the pyrrolopyrimidine compound represented by general formula (I) or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer or prodrug thereof.

[0093] In one embodiment, the disease associated with COL17 is a bullous or blistering disease; preferably, it is a pemphigoid, an autoimmune blistering disease. In particular, the disease associated with COL17 is junctional bullous epidermolysis, bullous pemphigoid, mucous membrane pemphigoid, pemphigoid of pregnancy, acquired bullous epidermolysis, lichen planus pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis and cicatricial pemphigoid.

[0094] In an embodiment, the disease related to COL17 is a skin healing related disease. In an embodiment, the skin healing related disease is diabetic foot ulcer, pressure ulcer, decubitus, burn, scald, sunburn, abrasion, incised wound.

[0095] In an embodiment, the disease related to COL17 is androgenetic alopecia, skin aging.

[0096] In order to make the purposes, technical solutions and advantages of the present disclosure clearer, the technical solutions of the exemplary embodiments of the present disclosure will be further described below.

[0097] Embodiments

[0098] I. Compound synthesis

[0099] Example 1: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (benzoyl)azetidin-3-yl)acetonitrile

[0100] Step A: 2-((1-benzoyl)azetidin-3-ylidene)acetonitrile

[0101] 300 mg (2.3 mmol, 1.0 eq) of 2-(azetidin-3-ylidene)acetonitrile hydrochloride and 700 mg (6.9 mmol, 3.0 eq) of triethylamine were dissolved in 20 mL of dichloromethane, and 387 mg (2.7 mmol, 1.2 eq) of benzoyl chloride was slowly added dropwise to the above reaction solution at room temperature. The reaction system was left overnight at room temperature. The reaction was quenched with 30 mL of saturated aqueous sodium bicarbonate solution, and the aqueous phase was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was obtained by column chromatography (PE:EA = 2:1 ~ 1:1) to obtain the target compound (397 mg, yield = 87%).

[0102] Step B: 4-chloro-7-([2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine

[0103] To a solution of 38.4 g (250.4 mmol, 1.0 eq.) of 4-chloro-7H-pyrrolo[2,3- d]pyrimidine in 200 mL of dry DMF was added 13 g (305 mmol, 1.2 eq) of 57% NaH under ice-bath stirring. The reaction was stirred at room temperature for 1 h and then 50.9 g of SEMCl (305 mmol, 1.2 eq.) was added dropwise under ice-bath cooling. After the addition was completed, the reaction was stirred at ice-bath for 1 h, quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The target compound was isolated by column chromatography on silica gel (71 g, yield = 100%).

[0104] Step C: 2-cyano-2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)acetic acid ethyl ester

[0105] To a solution of 33.5 g (118 mmol, 1.0 eq.) of 4-chloro-7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine in a mixture of 40.1 g (354.0 mmol, 3.0 eq.) of cyanoacetic acid ethyl ester and 33.0 g (238 mmol, 2.0 eq.) of potassium carbonate was stirred at room temperature. The reaction was heated to 60 °C for 0.5 h and then heated to 130 °C for 1.0 h. After cooling to room temperature, the reaction was quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The target compound was isolated by column chromatography on silica gel (30.6 g, yield = 72%).

[0106] Step D: 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)acetonitrile

[0107] To a solution of 50 g (855.6 mmol, 10.0 eq.) of sodium chloride in a mixture of DMSO and water was added 30.6 g (84.9 mmol, 1.0 eq.) of 2-cyano-2-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)acetic acid ethyl ester under stirring at room temperature. The reaction was heated to 150 °C under nitrogen protection for 6 days. After cooling to room temperature, the reaction was quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The target compound was isolated by column chromatography on silica gel (18.1 g, yield = 74%).

[0108] Step E: 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3- amino-1H-pyrazole

[0109] To a solution of 18.1 g (62.8 mmol, 1.0 eq) of 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)acetonitrile and 22.4 g (188 mmol, 3 eq) of DMF-DMA in 80 mL of DMF was added 37 g of 85% hydrazine hydrate (628 mmol, 10.0 eq.) at room temperature with stirring. The reaction was stirred at 90 °C under nitrogen protection for 3 hours. After cooling to room temperature, 100 mL of water was added and stirred, filtered and dried to obtain the target compound (11.0 g, yield = 53%). 1 HNMR (400 MHz, DMSO-d6) δ 12.13 (br s, 1H), 8.65 (s, 1H), 8.18 (br s, 1H), 7.61 (d, J = 3.2 Hz, 1H), 7.01 (d, J = 3.6 Hz, 1H), 6.55 (br s, 2H), 5.60 (s, 2H), 3.52 (t, J = 8.0 Hz, 2H), 0.83 (t, J = 8.0 Hz, 2H), -0.10 (s, 9H). LC-MS: m / z = 331 [M+1] + .

[0110] Step F: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(benzoyl)azetidin-3-yl)acetonitrile

[0111] 335 mg (1.0 mmol, 1.0 eq) of 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3-amine and 200 mg (1.0 mmol, 1.0 eq) of 2-((1-benzoyl)azetidin-3-ylidene)acetonitrile were dissolved in 10 mL of acetonitrile, and 310 mg (2.0 mmol, 2.0 eq) of 1,8-diazabicyclo[5.4.0]undec-7-ene was slowly added dropwise to the reaction system at room temperature, and the reaction was stirred at room temperature overnight. The reaction was quenched with 30 mL of saturated aqueous sodium bicarbonate solution, and the aqueous phase was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure, and the residue was purified by column chromatography (PE:EA = 2:1 ~ EA) to obtain the target compound (405 mg, yield = 76%).

[0112] Step G: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (benzoyl)azetidin-3-yl)acetonitrile

[0113] Step G: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (benzoyl)azetidin-3-yl)acetonitrile 1 H-NMR (400 MHz, DMSO-d6): δ 12.04 (s, 1H), 8.64 (d, J = 12.9 Hz, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.59-7.46 (m, 4H), 7.10 (s, 1H), 6.38 (s, 2H), 4.98 (d, J = 9.3 Hz, 1H), 4.65 (d, J = 10.4 Hz, 1H), 4.57 (d, J = 8.7 Hz, 1H), 4.35 (d, J = 10.1 Hz, 1H), 3.63 (s, 2H). LC-MS: m / z = 399 [M+H] + .

[0114] Example 2: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (p-tolylsulfonyl)azetidin-3-yl)acetonitrile

[0115] Step A: 2-(1-(p-tolylsulfonyl)azetidin-3-ylidene)acetonitrile

[0116] The compound was prepared according to the procedure described in Step A of Example 1 (495 mg, yield = 87%).

[0117] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1-yl)-1-(p-tolylsulfonyl)azetidin-3-yl)acetonitrile

[0118] The compound was prepared according to the procedure described in Step F of Example 1 (243 mg, yield = 52%).

[0119] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(p- toluenesulfonyl)azetidin-3-yl)acetonitrile

[0120] The compound was prepared according to the procedure described in Step G of Example 1 (70 mg, yield = 37%). 1 H-NMR (400 MHz, DMSO-d6): δ 12.05 (s, 1H), 8.64 (s, 1H), 8.25 (s, 1H), 7.71 (d, J = 7.5 Hz, 2H), 7.58 (s, 1H), 7.32 (d, J = 7.7 Hz, 2H), 7.01 (s, 1H), 6.25 (s, 2H), 4.31 (d, J = 9.7 Hz, 2H), 4.13 (d, J = 9.8 Hz, 2H), 3.42 (s, 2H), 2.03 (s, 3H). LC-MS: m / z = 449 [M+H] + .

[0121] Example 3: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(n- propylsulfonyl)azetidin-3-yl)acetonitrile

[0122] Step A: 2-(1-(n-propylsulfonyl)azetidin-3-ylidene)acetonitrile

[0123] The compound was prepared according to the procedure described in Step A of Example 1 (383 mg, yield = 76%).

[0124] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(n-propylsulfonyl)azetidin-3-yl)acetonitrile

[0125] The compound was prepared according to the procedure described in Step F of Example 1 (374 mg, yield = 72%).

[0126] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(n- propylsulfonyl)azetidin-3-yl)acetonitrile

[0127] The compound was prepared according to the procedure described in Step G in Example 1 (140 mg, yield = 50%). 1 H-NMR (400 MHz, DMSO-d6): δ 12.06 (s, 1H), 8.67 (s, 1H), 8.62 (s, 1H), 7.56 (s, 1H), 7.10 (s, 1H), 6.39 (s, 2H), 4.55 (d, J = 8.9 Hz, 2H), 4.14 (d, J = 8.8 Hz, 2H), 3.58 (s, 2H), 3.23-3.16 (m, 2H), 1.79-1.68 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H). LC-MS: m / z = 401 [M+H] + .

[0128] Example 4: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (cyclopropylsulfonyl)azetidin-3-yl)acetonitrile

[0129] Step A: 2-(1-(cyclopropylsulfonyl)azomethine)acetonitrile

[0130] The compound was prepared according to the procedure described in Step A in Example 1 (396 mg, yield = 79%).

[0131] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1-yl)-1-(cyclopropylsulfonyl)azetidin-3-yl)acetonitrile

[0132] The compound was prepared according to the procedure described in Step F in Example 1 (368 mg, yield = 69%).

[0133] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (cyclopropylsulfonyl)azetidin-3-yl)acetonitrile

[0134] The compound was prepared according to the procedure described in Step G in Example 1 (165 mg, yield = 59%). 1H-NMR (400 MHz, DMSO-d6): δ 12.06 (s, 1H), 8.67 (s, 1H), 8.63 (s, 1H), 7.56 (s, 1H), 7.11 (s, 1H), 6.40 (s, 2H), 4.62 (d, J = 9.1 Hz, 2H), 4.18 (d, J = 9.0 Hz, 2H), 3.60 (s, 2H), 2.90-2.78 (m, 1H), 1.09-0.97 (m, 4H). LC-MS: m / z = 399 [M+H] + .

[0135] Example 5: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (ethanesulfonyl)azetidin-3-yl)acetonitrile

[0136] Step A: 2-(1-(ethanesulfonyl)azomethine)acetonitrile

[0137] The compound was prepared according to the procedure described in US2009 / 233903A1 Page 63-64.

[0138] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1-yl)-1-(ethanesulfonyl)azetidin-3-yl)acetonitrile

[0139] The compound was prepared according to the procedure described in Step F in Example 1 (1.05 g, yield = 85%).

[0140] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (ethanesulfonyl)azetidin-3-yl)acetonitrile

[0141] The compound was prepared according to the procedure described in Step G in Example 1 (384 mg, yield = 49%). 1H-NMR (400 MHz, DMSO-d6): δ 12.06 (s, 1H), 8.67 (s, 1H), 8.61 (s, 1H), 7.56 (s, 1H), 7.10 (s, 1H), 6.39 (s, 2H), 4.56 (d, J = 8.9 Hz, 2H), 4.13 (d, J = 9.0 Hz, 2H), 3.59 (s, 2H), 3.25-3.16 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H). LC-MS: m / z = 387 [M+H] + .

[0142] Example 6: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (isopropylsulfonyl)azetidin-3-yl)acetonitrile

[0143] Step A: 2-(1-(isopropylsulfonyl)azomethine)acetonitrile

[0144] The compound was prepared according to the procedure described in Step A of Example 1 (506 mg, yield = 100%).

[0145] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1-yl)-1-(isopropylsulfonyl)azetidin-3-yl)acetonitrile

[0146] The compound was prepared according to the procedure described in Step F of Example 1 (316 mg, yield = 60%).

[0147] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (isopropylsulfonyl)azetidin-3-yl)acetonitrile

[0148] The compound was prepared according to the procedure described in Step G of Example 1 (100 mg, yield = 43%). 1H-NMR (400 MHz, DMSO-d6): δ 12.05 (s, 1H), 8.67 (s, 1H), 8.61 (s, 1H), 7.55 (s, 1H), 7.09 (s, 1H), 6.40 (s, 2H), 4.56 (d, J = 8.7 Hz, 2H), 4.10 (d, J = 8.7 Hz, 2H), 3.60 (s, 2H), 3.38-3.33 (m, 1H), 1.28 (s, 3H), 1.26 (s, 3H). LC-MS: m / z = 401 [M+H] + .

[0149] Example 7: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (n-butylsulfonyl)azetidin-3-yl)acetonitrile

[0150] Step A: 2-(1-(n-butylsulfonyl)azomethine)acetonitrile

[0151] The compound was prepared according to the procedure described in Step A of Example 1 (517 mg, yield = 95%).

[0152] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1-yl)-1-(n-butylsulfonyl)azetidin-3-yl)acetonitrile

[0153] The compound was prepared according to the procedure described in Step F of Example 1 (321 mg, yield = 59%).

[0154] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(n- butylsulfonyl)azetidin-3-yl)acetonitrile

[0155] The compound was prepared according to the procedure described in Step G of Example 1 (108 mg, yield = 45%). 1H-NMR (400 MHz, DMSO-d6): δ 12.06 (s, 1H), 8.67 (s, 1H), 8.62 (s, 1H), 7.56 (s, 1H), 7.10 (s, 1H), 6.39 (s, 2H), 4.56 (d, J = 9.0 Hz, 2H), 4.14 (d, J = 9.0 Hz, 2H), 3.58 (s, 2H), 3.25-3.19 (m, 2H), 1.71-1.65 (m, 2H), 1.48-1.34 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). LC-MS: m / z = 415 [M+H] + .

[0156] Example 8: 2-(l-acetyl-3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-lH-pyrazol-l- yl)azetidin-3-yl)acetonitrile

[0157] Step A: 2-(l-(acetyl)azetidin-3-ylidene)acetonitrile

[0158] The compound was prepared according to the procedure described in Step A in Example 1 (327 mg, yield = 95%).

[0159] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)-lH-pyrazol-l-yl)-l-(acetyl)azetidin-3-yl)acetonitrile

[0160] The compound was prepared according to the procedure described in Step F in Example 1 (328 mg, yield = 70%).

[0161] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-lH-pyrazol-l-yl)-l- (acetyl)azetidin-3-yl)acetonitrile

[0162] The compound was prepared according to the procedure described in Step G in Example 1 (36 mg, yield = 16%). 1H-NMR (400 MHz, DMSO-d6): δ 8.84 (s, 1H), 8.73 (s, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.30 (d, J = 2.8 Hz, 1H), 6.50 (s, 2H), 4.83 (d, J = 9.3 Hz, 1H), 4.56 (d, J = 10.3 Hz, 1H), 4.49 (d, J = 9.4 Hz, 1H), 4.21 (d, J = 10.3 Hz, 1H), 3.70 (s, 2H), 1.94 (s, 3H). LC-MS: m / z = 337 [M+H] + .

[0163] Example 9: 2-(3-(3,5-diamino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (ethylsulfonyl)azetidin-3-yl)acetonitrile

[0164] Step A: 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)propanedinitrile

[0165] To a mixture of 3.5 g (53 mmol, 1.5 eq.) of propanedinitrile and 7.2 g (53 mmol, 1.5 eq.) of potassium carbonate, 10.0 g (35.2 mmol, 1.0 eq.) of 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine was added under stirring at room temperature. The reaction was warmed to 60 °C for 6 h. Cooled to room temperature, quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo, column chromatography on silica gel column to isolate the target compound (9.3 g, yield = 84%).

[0166] Step B: 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,5- diamino-1H-pyrazole

[0167] To a solution of 4.0 g (12.8 mmol, 1.0 eq.) of 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)propanedinitrile in 100 mL of absolute ethanol, 7.5 g of 85% hydrazine hydrate (127 mmol, 10.0 eq.) was added under stirring at room temperature. The reaction was stirred at 90 °C under nitrogen protection overnight. Cooled to room temperature, concentrated under reduced pressure, suction filtered and dried to give the target compound (1.98 g, yield = 45%). LC-MS: m / z = 346 [M+1] +.

[0168] Step C: 2-(3-(3,5-diamino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (ethylsulfonyl)azetidin-3-yl)acetonitrile

[0169] The compound was prepared according to the procedure described in Step F in Reference Example 1 (130 mg, yield = 75%).

[0170] Step D: 2-(3-(3,5-diamino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (ethylsulfonyl)azetidin-3-yl)acetonitrile

[0171] The compound was prepared according to the procedure described in Step G in Reference Example 1 (54 mg, yield = 61%). LC-MS: m / z = 402 [M+H] + .

[0172] Examples 10-16: The following compounds were synthesized according to the above examples using similar procedures.

[0173] Example 17: 2-(3-(3-amino-4-(9H-purin-6-yl)-1H-pyrazol-1-yl)-1- (ethylsulfonyl)azetidin-3-yl)acetonitrile

[0174] The compound was prepared according to the procedure described in Steps in Reference Example 1 and Example 5, replacing 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with 6-chloropurine, to produce the target compound (64 mg). LC-MS: m / z = 388 [M+H] + .

[0175] Examples 18-24: The following compounds were synthesized according to the above examples using similar procedures.

[0176] Example 25: 2-(3-(3,5-diamino-4-(9H-purin-6-yl)-1H-pyrazol-1-yl)-1- (ethylsulfonyl)azetidin-3-yl)acetonitrile

[0177] The compound was prepared according to the procedure described in Steps in Reference Example 1 and Example 9, replacing 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with 6-chloropurine, to produce the target compound (73 mg). LC-MS: m / z = 403 [M+H] + .

[0178] Examples 26-32: The following compounds were synthesized according to the above examples using similar procedures.

[0179] Example 33: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-lH-pyrazol-l- yl)-l-(l-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3- yl)acetonitrile

[0180] Step A: tert-butyl 4-(3-(cyanomethyl)azetidin-l-yl)piperidine-l-carboxylate

[0181] To a solution of 40 g (0.3 mol, 1.0 eq) of 2-(azetidin-3-ylidene)acetonitrile hydrochloride (compound prepared according to US2014 / 256941 Al Paragraph 0145) and 61 g (0.3 mol, 1.0 eq) of N-tert-butoxycarbonyl-4-piperidinone in 500 mL of dichloromethane, under nitrogen protection, ice water bath was used to cool the solution to 0 °C, 130 g (0.6 mol, 2.0 eq) of sodium borohydride in acetic acid was added slowly in batches, after the addition was completed, the ice bath was maintained for reaction, TLC was used to monitor the completion of the reaction. The reaction solution was slowly poured into water, the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with water and saturated sodium bicarbonate in turn, the organic phase was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the residue was purified by column chromatography to obtain the product (40 g, yield = 85%).

[0182] Step B: 2-(l-(piperidin-4-yl)azetidin-3-ylidene)acetonitrile hydrochloride

[0183] To a solution of 40 g (0.14 mol, 1.0 eq) of tert-butyl 4-(3-(cyanomethyl)azetidin-l- yl)piperidine-l-carboxylate in 250 mL of anhydrous ethanol, under nitrogen protection, ice water bath was used to cool the solution to 0 °C, 40 g of concentrated hydrochloric acid was added slowly dropwise, after the addition was completed, the solution was slowly warmed to room temperature for reaction, TLC was used to monitor the completion of the reaction. The suspension was filtered, the filter cake was washed with anhydrous ethanol, and dried under vacuum to obtain the product (24 g, yield = 81%).

[0184] Step C: 2-(l-(l-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3- yl)acetonitrile

[0185] Step B: 2-(l-(piperidin-4-yl)azetidin-3-yl)acetonitrile hydrochloride 650 mg (3.0 mmol, 1.0 eq) of 2-(l-(piperidin-4-yl)azetidin-3-yl)acetonitrile hydrochloride, 630 mg (3.0 mmol, 1.0 eq) of 2-fluoro-3-trifluoromethylpicolinic acid and 2 g (15 mmol, 5.0 eq) of N,N-diisopropylethylamine were dissolved in 30 mL of dichloromethane, 1.4 g (3.6 mmol, 1.2 eq) of HATU was added and the reaction was stirred at room temperature overnight. The reaction was quenched by the addition of 100 mL of water and the aqueous phase was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography (PE:EA = 1:1 to 0:1) to give the product (1.1 g, yield = 98%). 1 H-NMR (400 MHz, DMSO-D6): δ 8.67 (d, J = 4.5 Hz, 1H), 7.91 (t, J = 4.5 Hz, 1H), 5.69-5.71 (m, 1H), 3.90-4.15 (m, 5H), 3.25-3.50 (m, 2H), 3.07-3.20 (m, 1H), 2.51-2.64 (m, 1H), 1.55-1.85 (m, 2H), 1.10-1.30 (m, 2H).

[0186] Step D: 4-chloro-7-([2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine

[0187] A solution of 38.4 g (250.4 mmol, 1.0 eq.) of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine in 200 mL of dry DMF was stirred in an ice bath and 13 g (305 mmol, 1.2 eq) of 57% NaH was added. The reaction was stirred at room temperature for 1 hour and then 50.9 g of SEMCl (305 mmol, 1.2 eq.) was added dropwise while the reaction was cooled in an ice bath. After the addition was complete, the reaction was stirred in an ice bath for 1 hour, quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated under vacuum. The product was isolated by column chromatography on silica gel (71 g, yield = 100%).

[0188] Step E: 2-cyano-2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)acetic acid ethyl ester

[0189] To a mixture of 40.1 g (354.0 mmol, 3.0 eq.) ethyl cyanoacetate and 33.0 g (238 mmol, 2.0 eq.) potassium carbonate was added 33.5 g (118 mmol, 1.0 eq.) 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine at room temperature under stirring. The reaction was warmed to 60 °C for 0.5 h and then warmed to 130 °C for 1.0 h. Cooled to room temperature, quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo, column chromatography on silica gel column to give the target compound (30.6 g, yield = 72%).

[0190] Step F: 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)acetonitrile

[0191] To a mixture of 40.1 g (354.0 mmol, 3.0 eq.) ethyl cyanoacetate and 33.0 g (238 mmol, 2.0 eq.) potassium carbonate was added 33.5 g (118 mmol, 1.0 eq.) 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine at room temperature under stirring. The reaction was warmed to 60 °C for 0.5 h and then warmed to 130 °C for 1.0 h. Cooled to room temperature, quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo, column chromatography on silica gel column to give the target compound (30.6 g, yield = 72%).

[0192] Step G: 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3- amino-1H-pyrazole

[0193] To a mixture of 40.1 g (354.0 mmol, 3.0 eq.) ethyl cyanoacetate and 33.0 g (238 mmol, 2.0 eq.) potassium carbonate was added 33.5 g (118 mmol, 1.0 eq.) 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine at room temperature under stirring. The reaction was warmed to 60 °C for 0.5 h and then warmed to 130 °C for 1.0 h. Cooled to room temperature, quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo, column chromatography on silica gel column to give the target compound (30.6 g, yield = 72%). 1HNMR (400 MHz, DMSO-d6): δ 12.13 (br s, 1H), 8.65 (s, 1H), 8.18 (br s, 1H), 7.61 (d, J = 3.2 Hz, 1H), 7.01 (d, J = 3.6 Hz, 1H), 6.55 (br s, 2H), 5.60 (s, 2H), 3.52 (t, J = 8.0 Hz, 2H), 0.83 (t, J = 8.0 Hz, 2H), -0.10 (s, 9H). LC-MS: m / z = 331 [M+1] + .

[0194] Step H: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3- fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0195] Step H: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3- fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0196] Step I: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3- fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0197] Method 1: 260 mg (0.37 mmol, 1.0 eq) of 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile was dissolved in 8 mL of dichloromethane and 1 mL of trifluoroacetic acid at room temperature, and the reaction was allowed to proceed overnight at room temperature. The solvent was evaporated under reduced pressure, and the residue was dissolved in 10 mL of methanol. 0.5 mL of ethylenediamine was added at room temperature, and the reaction was stirred at room temperature for 30 minutes. The reaction solution was poured into 50 mL of water, and the aqueous phase was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the residue was purified by column chromatography (EA:MeOH = 1:0 ~ 25:1) to obtain the product (150 mg, yield = 70%).

[0198] Method 2: 478 mg (0.7 mmol, 1.0 eq) of 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile was dissolved in 15 mL of acetonitrile at room temperature, and 500 mg (3.5 mmol, 5.0 eq) of boron trifluoride etherate was slowly added dropwise while being cooled in an ice bath. After the addition was completed, the reaction was allowed to proceed at room temperature for 4 hours. The reaction system was cooled to below 5°C in an ice bath, and 8 mL of 25% aqueous ammonia solution was slowly added dropwise. The reaction was stirred at room temperature for 60 minutes. The reaction solution was poured into 80 mL of water, and the aqueous phase was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the residue was purified by column chromatography (EA:MeOH = 1:0 ~ 25:1) to obtain the product (195 mg, yield = 50%).

[0199] 1 H-NMR (400 MHz, CDCl3): δ 10.15 (s, 1H), 8.82 (s, 1H), 8.60 (d, J = 4.5 Hz, 1H), 8.06 (s, 1H), 7.56 (t, J = 4.4 Hz, 1H), 7.37 (dd, 1H), 6.69 (dd, 1H), 5.74 (s, 2H), 4.17-4.29 (m, 1H), 3.76-3.69 (m, 2H), 3.57-3.64 (m, 2H), 3.42-3.54 (s, 2H), 3.34 (s, 2H), 3.07-3.20 (m, 1H), 2.54-2.64 (m, 1H), 1.60-1.92 (m, 2H), 1.37-1.57 (m, 2H). LC-MS: m / z = 569 [M+1] + .

[0200] Example 34: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-fluoro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0201] Step A: 2-(1-(1-(2-fluoro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)azetidin-3- ylidene)acetonitrile

[0202] The compound was prepared according to the procedure described in Step C in Example 33 (550 mg, yield = 98%). 1 H NMR (400 MHz, CDC13): δ 7.71 (t, J = 7.0 Hz, 1H), 7.62 (t, J = 6.2 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 5.29-5.36 (m, 1H), 4.20-4.30 (m, 1H), 4.10 (s, 2H), 4.01 (s, 2H), 3.48-3.59 (m, 1H), 3.37-3.48 (m, 1H), 3.10-3.25 (m, 1H), 2.52-2.63 (m, 1H), 1.65-1.95 (m, 2H), 1.25-1.59 (m, 2H).

[0203] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-fluoro-3-(trifluoromethyl)benzoyl)piperidin- 4-yl)azetidin-3-yl)acetonitrile

[0204] The compound was prepared according to the procedure described in Step H in Example 33 (720 mg, yield = 71%).

[0205] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2-fluoro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0206] The compound was prepared according to the procedure described in Step E in Example 33, Method 1 (210 mg, yield = 36%). 1H NMR (400 MHz, DMSO-D6): δ 12.09 (s, 1H), 8.68 (s, 1H), 8.54 (s, 1H), 7.90 (t, J = 7.1 Hz, 1H), 7.79 (t, J = 6.6 Hz, 1H), 7.54 (dd, J = 16.7, 8.9 Hz, 2H), 7.09 (d, J = 1.7 Hz, 1H), 6.36 (s, 2H), 4.02 - 4.22 (m, 1H), 3.65 - 3.78 (m, 2H), 3.46 - 3.57 (m, 4H), 3.40 - 3.45 (m, 1H), 3.22 - 3.33 (m, 1H), 3.03 - 3.15 (m, 1H), 2.50 - 2.60 (m, 1H), 1.74 - 1.84 (m, 1H), 1.61 - 1.73 (m, 1H), 1.16 - 1.37 (m, 2H). LC-MS: m / z = 568 [M+1] + .

[0207] Example 35: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(pyrimidine-4- carboxamido)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0208] Step A: 2-(1-(1-(pyrimidine-4-carboxamido)piperidin-4-yl)azetidin-3-ylidene)acetonitrile

[0209] The compound was prepared according to the procedure described in Step C of Example 33 (570 mg, yield = 99%). 1 H NMR (400 MHz, CDCl3): δ 9.28 (s, 1H), 8.93 (d, J = 5.0 Hz, 1H), 7.63 (d, J = 4.8 Hz, 1H), 5.33 (s, 1H), 4.17 - 4.26 (m, 1H), 4.11 (s, 2H), 4.01 (s, 2H), 3.70 - 3.87 (m, 1H), 3.40 - 3.50 (m, 1H), 3.24 - 3.37 (m, 1H), 2.54 - 2.65 (m, 1H), 1.70 - 1.92 (m, 2H), 1.41 - 1.59 (m, 2H).

[0210] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(pyrimidine-4-carboxamido)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0211] The compound was prepared according to the procedure described in Step H in Example 33 (980 mg, yield = 82%).

[0212] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(pyrimidine-4- carboxamido)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0213] The compound was prepared according to the procedure described in Step E in Example 33, Method 1 (150 mg, yield = 20%). 1 H NMR (400 MHz, DMSO): δ 12.06 (s, 1H), 9.24 (s, 1H), 8.95 (d, J = 5.1 Hz, 1H), 8.65 (s, 1H), 8.50 (s, 1H), 7.65 (d, J = 5.1 Hz, 1H), 7.54 (s, 1H), 7.06 (s, 1H), 6.33 (s, 2H), 4.02-4.13 (m, 1H), 3.68 (d, J = 7.7 Hz, 2H), 3.38-3.54 (m, 5H), 3.16-3.26 (m, 1H), 3.03-3.14 (m, 1H), 2.50-2.57 (m, 1H), 1.72-1.82 (m, 1H), 1.59-1.70 (m, 1H), 1.18-1.33 (m, 2H). LC-MS: m / z = 484 [M+1] + .

[0214] Example 36: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2- trifluoromethylpyrimidine-4-carboxamido)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0215] Step A: 2-(1-(1-(2-trifluoromethylpyrimidine-4-carboxamido)piperidin-4-yl)azetidin-3- ylidene)acetonitrile

[0216] The compound was prepared according to the procedure described in Step C in Example 33 (480 mg, yield = 95%).

[0217] Step B: 2-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 1H-pyrazol-1-yl)-1-(1-(2-trifluoromethylpyrimidine-4-carboxamido)piperidin-4-yl)azetidin-3- yl)acetonitrile

[0218] The compound was prepared according to the procedure described in Step H in Example 33 (780 mg, yield = 78%).

[0219] Step C: 2-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(2- trifluoromethylpyrimidine-4-carbonyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0220] The compound was prepared according to the procedure described in Step E in Example 33, Method 1 (185 mg, yield = 32%). LC-MS: m / z = 552 [M+1] + .

[0221] Example 37: 4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidine-1-carboxamide

[0222] Step A: 4-(3-(cyanomethyliden)azetidin-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidine- 1-carboxamide

[0223] To 325 mg (1.8 mmol, 1.2 eq) of 4-fluoro-2-trifluoromethylaniline and 760 mg (7.5 mmol, 5.0 eq) of triethylamine in 20 mL of dichloromethane, 225 mg (0.76 mmol, 0.5 eq) of triphosgene was added under ice bath, the reaction was stirred for 5 minutes under ice bath, 320 mg (1.5 mmol, 1.0 eq) of 2-(1-(piperidin-4-yl)azetidin-3-ylidene)acetonitrile hydrochloride was added at once, the reaction was slowly warmed to room temperature overnight. To the reaction system, 50 mL of water was added to quench the reaction, the aqueous phase was extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, evaporated under reduced pressure, the residue was purified by column chromatography (PE:EA = 1:1 ~ 0:1) to give the product (324 mg, yield = 57%). 1H NMR (400 MHz, CDC13): δ 7.98 (dd, J = 8.9, 5.0 Hz, 1H), 7.25-7.32 (m, 1H), 7.19-7.24 (m, 1H), 6.66 (s, 1H), 5.28 (s, 1H), 4.02-4.09 (m, 2H), 3.92-4.01 (m, 2H), 3.77-3.85 (m, 2H), 3.15-3.20 (m, 2H), 2.42-2.53 (m, 1H), 1.71-1.82 (m, 2H), 1.44-1.37 (m, 2H).

[0224] Step B: 4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidine-1- carboxamide

[0225] The compound was prepared according to the procedure described in Step H in Reference Example 33 (500 mg, yield = 84%).

[0226] Step C: 4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-N-(4-fluoro-2-(trifluoromethyl)phenyl)piperidine-1- carboxamide

[0227] The compound was prepared according to the procedure described in Step E, Method 1 in Reference Example 33 (90 mg, yield = 22%). 1 H NMR (400 MHz, DMSO): δ 12.09 (s, 1H), 8.68 (s, 1H), 8.53 (s, 1H), 8.25 (s, 1H), 7.62-7.44 (m, 4H), 7.09 (s, 1H), 6.36 (s, 2H), 3.80-3.93 (m, 2H), 3.66-3.75 (m, 2H), 3.55-3.46 (m, 4H), 2.94-3.09 (m, 2H), 2.37-2.49 (m, 1H), 1.63-1.75 (m, 2H), 1.10-1.24 (m, 2H). LC-MS: m / z = 583 [M+1] + .

[0228] Example 38: Methyl 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)pyrazine-2-carboxylate

[0229] Step A: 5-(3-(cyanomethyl)azetidin-l-yl)pyrazine-2-carboxylic acid methyl ester

[0230] To a solution of 520 mg (4.0 mmol, 1.0 eq) of 5-chloropyrazine-2-carboxylic acid methyl ester, 690 mg (4.0 mmol, 1.0 eq) of 2-(l-(piperidin-4-yl)azetidin-3-ylidene)acetonitrile hydrochloride and 1.6 g (12 mmol, 3.0 eq) of N,N-diisopropylethylamine in 20 mL of dioxane, the reaction was heated to reflux with stirring for 2 hours. The reaction was monitored by TLC and upon completion, the reaction was cooled to room temperature and poured into 150 mL of water. The suspension was filtered and the filter cake was washed with water and diethyl ether successively. The filter cake was collected and dried under vacuum to give the product (840 mg, yield = 91%). 1 H NMR (400 MHz, CDC13): δ 8.85 (s, 1H), 7.89 (s, 1H), 5.53 (s, 1H), 5.01 (s, 2H), 4.93 (s, 2H), 3.97 (s, 3H).

[0231] Step B: 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-lH-pyrazol-l-yl)-3- (cyanomethyl)azetidin-l-yl)pyrazine-2-carboxylic acid methyl ester

[0232] The compound was prepared according to the procedure described in Step H in Reference Example 33 (820 mg, yield = 86%).

[0233] Step C: 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-lH-pyrazol-l-yl)-3- (cyanomethyl)azetidin-l-yl)pyrazine-2-carboxylic acid methyl ester

[0234] The compound was prepared according to the procedure described in Step E, Method 2 in Reference Example 33 (40 mg, yield = 48%). 1H NMR (400 MHz, DMSO-D6): δ 12.09 (s, 1H), 8.71 (d, J = 1.3 Hz, 1H), 8.67 (d, J = 2.6 Hz, 2H), 8.07 (d, J = 1.3 Hz, 1H), 7.59-7.54 (m, 1H), 7.11 (dd, J = 3.5, 1.6 Hz, 1H), 6.43 (s, 2H), 4.81 (d, J = 10.0 Hz, 2H), 4.48 (d, J = 9.9 Hz, 2H), 3.82 (s, 3H), 3.73 (s, 2H). LC-MS: m / z = 431 [M+1] + .

[0235] Example 39: (S)-4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide

[0236] Step A: (S)-4-chloro-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide

[0237] Step A: (S)-4-chloro-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide

[0238] Step B: (S)-4-(3-(cyanomethyl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2- yl)benzamide

[0239] The compound was prepared according to the method described in US20150246046A1 Paragraph 0166 (350 mg, yield = 56%).

[0240] Step C: (S)-4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide

[0241] The compound was prepared according to the procedure described in Step H in Reference Example 33 (260 mg, yield = 82%).

[0242] Step D: (S)-4-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-2,5-difluoro-N-(1,1,1-trifluoropropan-2-yl)benzamide

[0243] The compound was prepared according to the procedure described in Step E, Method 2 in Reference Example 33 (46 mg, yield = 53%). LC-MS: m / z = 546 [M+1] + .

[0244] Example 40: 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-N-isopropylpyrazine-2-carboxamide

[0245] Step A: 5-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azetidin-1-yl)pyrazine-2-carboxylic acid

[0246] To a solution of 200 mg (0.37 mmol, 1.0 eq) of methyl 5-(3-(3-amino-4-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)pyrazine-2-carboxylate in 10 mL of methanol and 3 mL of water, was added 52 mg (1.1 mmol, 3.0 eq) of lithium hydroxide monohydrate and stirred at room temperature overnight. The reaction was monitored by TLC and the solvent was evaporated. The pH was adjusted to 2-3 with 0.5 N hydrochloric acid solution and the suspension was filtered. The filter cake was washed with water, collected and dried under vacuum to give the product (195 mg, quant).

[0247] Step B: 5-(3-(3-amino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azetidin-1-yl)-N- isopropylpyrazine-2-carboxamide

[0248] The compound was prepared according to the procedure described in Step C in Reference Example 33 (100 mg, yield = 49%).

[0249] Step C: 5-(3-(3-amino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3- (cyanomethyl)azetidin-1-yl)-N-isopropylpyrazine-2-carboxamide

[0250] The compound was prepared according to the procedure described in Step E, Method 2 in Reference Example 33 (60 mg, yield = 77%). 1 H NMR (400 MHz, DMSO-D6): δ 12.09 (s, 1H), 8.71-8.62 (m, 3H), 8.13 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 1.4 Hz, 1H), 7.57 (dd, J = 3.5, 2.5 Hz, 1H), 7.11 (dd, J = 3.6, 1.8 Hz, 1H), 6.43 (s, 2H), 4.78 (d, J = 9.7 Hz, 2H), 4.45 (d, J = 9.6 Hz, 2H), 4.15-4.06 (m, 1H), 3.73 (s, 2H), 1.17 (d, J = 6.6 Hz, 6H). LC-MS: m / z = 458 [M+1] + .

[0251] Example 41: 2-(3-(3,5-diamino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1- (1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0252] Step A: 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)propanedinitrile

[0253] To a mixture of 3.5 g (53 mmol, 1.5 eq.) of malononitrile and 7.2 g (53 mmol, 1.5 eq.) of potassium carbonate was added 10.0 g (35.2 mmol, 1.0 eq.) of 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine at room temperature under stirring. The reaction was warmed to 60 °C for 6 hours. Cooled to room temperature, quenched with water, extracted with ethyl acetate, the combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo, column chromatography on silica gel column to isolate the target compound (9.3 g, yield = 84%).

[0254] Step B: 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,5- diamino-1H-pyrazole

[0255] To a solution of 4.0 g (12.8 mmol, 1.0 eq.) of 2-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)propanedinitrile in 100 mL of anhydrous ethanol was added 7.5 g of 85% hydrazine hydrate (127 mmol, 10.0 eq.) at room temperature under stirring. The reaction was stirred at 90 °C under nitrogen overnight. Cooled to room temperature, concentrated under reduced pressure, suction filtered and dried to give the target compound (1.98 g, yield = 45%). LC-MS: m / z = 346 [M+1] + .

[0256] Step C: 2-(3-(3,5-diamino-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0257] The compound was prepared according to the procedure described in Step H in Example 33 (110 mg, yield = 60%).

[0258] Step D: 2-(3-(3,5-diamino-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3- fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile

[0259] The compound was prepared according to the procedure described in Step I in Example 33 (54 mg, yield = 51%). LC-MS: m / z = 584 [M+H] + .

[0260] Examples 42-48

[0261] The following compounds were synthesized using a similar method as described in the examples above.

[0262] Example 49: 2-(3-(3-amino-4-(9H-purin-6-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0263] Following the steps described in Example 33, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was replaced with 6-chloropurine to prepare the target compound (67 mg). LC-MS: m / z = 570 [M+H] + .

[0264] Examples 50-56

[0265] The following compounds were synthesized using a similar method as described in the examples above.

[0266] Example 57: 2-(3-(3,5-diamino-4-(9H-purin-6-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinyl)piperidin-4-yl)azacyclobut-3-yl)acetonitrile

[0267] Following the steps described in Examples 33 and 41, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was replaced with 6-chloropurine to prepare the target compound (68 mg). LC-MS: m / z = 585 [M+H] + .

[0268] Examples 58-64

[0269] The following compounds were synthesized using a similar method as described in the examples above.

[0270] The following compounds were synthesized according to the steps in Example 5.

[0271] Comparison of compound synthesis methods:

[0272] Synthetic route for reference 1:

[0273] Step A: Ethyl 2-cyano-2-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3- d]pyrimidin-4-yl)acetate

[0274] To a solution of ethyl 2-cyano-2-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3- d]pyrimidin-4-yl)acetate (1000 mg, 2.8 mmol) and triethylamine (310 mg, 3.0 mmol) in dry tetrahydrofuran (20 mL) was added diisobutylaluminum hydride (1 M in THF, 8.0 mL) dropwise at 0 °C and the solution was stirred at 25 °C for 3 h. After the reaction was completed, it was diluted with water (50 mL) and extracted with ethyl acetate (30 mL*3). The organic phase was dried, concentrated and purified by silica gel column (petroleum ether: ethyl acetate = 1:1) to give ethyl (2E)-3-amino-2-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3- d]pyrimidin-4-yl)prop-2-enoate (700 mg, yield = 70.0%).

[0275] LC-MS (ESI), m / z: [M+H] + = 361.0

[0276] Step B: Ethyl (2E)-3-amino-2-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3- d]pyrimidin-4-yl)prop-2-enoate

[0277] To a solution of ethyl 2-cyano-2-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3- d]pyrimidin-4-yl)acetate (1000 mg, 2.8 mmol) and triethylamine (310 mg, 3.0 mmol) in dry tetrahydrofuran (20 mL) was added diisobutylaluminum hydride (1 M in THF, 8.0 mL) dropwise at 0 °C and the solution was stirred at 25 °C for 3 h. After the reaction was completed, it was diluted with water (50 mL) and extracted with ethyl acetate (30 mL*3). The organic phase was dried, concentrated and purified by silica gel column (petroleum ether: ethyl acetate = 1:1) to give ethyl (2E)-3-amino-2-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3- d]pyrimidin-4-yl)prop-2-enoate (700 mg, yield = 70.0%).

[0278] LC-MS (ESI), m / z: [M+H] + = 361.0

[0279] Step C: 4-{7-[(2-(trimethylsilyl)ethoxy)methyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}- 1 H-pyrazol-3 -ol

[0280] (2E)-3-amino-2-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3-d]pyrimidin-4- yl)prop-2-enoate (700 mg, 1.9 mmol) and water and hydrazine (193 mg, 3.86 mmol) were added into dioxane (10 mL) and stirred at 80 °C for 2 h. The reaction was concentrated to dryness and purified by silica gel column (dichloromethane:methanol = 20:1) to give the product pyrrolo[2,3-d] (600 mg, yield = 75.79%).

[0281] Step D: 2-[3-(3-hydroxy-4-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}-1H-pyrazol-1-yl)-1- (isopropylsulfonyl)azetidin-3-yl]acetonitrile

[0282] 4-{7-[(2-(trimethylsilyl)ethoxy)methyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}-1H-pyrazol- 3-ol pyrrolo[2,3-d] (300 mg, 0.9 mmol), 2-[1-(propan-2-sulfonyl)azetidin-3-ylidene]acetonitrile (181 mg, 0.9 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (206 mg, 1.35 mmol) were added into N,N-dimethylformamide (5 mL) and stirred at 50 °C for 2 h. After the reaction was completed, it was diluted with water (50 mL) and extracted with ethyl acetate (30 mL*30), the organic phase was dried, concentrated to dryness and purified by silica gel column (petroleum ether: ethyl acetate = 2:1) to give the product azetidin-3 (80 mg, yield = 16.67%).

[0283] LC-MS (ESI), m / z: [M+H] + = 532.1

[0284] Step E: 2-[3-(3-hydroxy-4-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}-1H-pyrazol-1-yl)-1- (isopropylsulfonyl)azetidin-3-yl]acetonitrile

[0285] To 2-[3-(3-hydroxy-4-{7-[(2-(trimethylsilyl)ethoxy)methyl]-7H-pyrrolo[2,3- d]pyrimidin-4-yl}-1H-pyrazol-1-yl)-1-(isopropanesulfonyl)azetidin-3-yl]acetonitrile azetidin-3 (50 mg, 0.09 mmol) and boron trifluoride etherate (40 mg, 0.28 mmol) were added into dichloromethane (2 mL) and stirred at 25 °C for 3 hours. After completion, concentrated under vacuum and dissolved with methanol (2 mL). Ethylenediamine (17 mg, 0.28 mmol) was added and the solution was stirred at 25 °C for 0.5 hours. The reaction was concentrated and the product azetidin-3 (5 mg, yield = 13.1%) was obtained by Prep-HPLC (column type: gemini-C18 150 x 21.2 mm, 5 um, mobile phase: ACN-H2O (0.1% TFA), gradient 5-10%, flow rate: (20 mL / min).

[0286] LC-MS (ESI), m / z: [M+H] + = 402.0

[0287] 1 H-NMR (400 MHz, MeOD) δ 8.67 (s, 1H), 8.63 (s, 1H), 8.10 (s, 2H), 7.49 (d, J = 3.6 Hz, 1H), 7.06 (d, J = 3.6 Hz, 1H), 4.61 (d, J = 9.3 Hz, 2H), 4.18 (d, J = 9.4 Hz, 2H), 3.48 (s, 2H), 3.25 (d, J = 1.8 Hz, 1H), 1.35 (s, 3H), 1.33 (s, 3H).

[0288] Synthetic route for Control 2:

[0289] Step A: 2-[3-(3-methoxy-4-{7-[(2-methoxyethyl)trimethyl-{5}-silyl]pyrrolo[2,3- d]pyrimidin-4-yl}pyrazol-1-yl)-1-(propan-2-sulfonyl)azetidin-3-yl]acetonitrile

[0290] To a stirred solution of 2-[3-(3-hydroxy-4-{7-[(2-methoxyethyl)trimethyl-{5}-silyl]pyrrolo[2,3-d]pyrimidin-4-yl}pyrazol-l-yl)-l-(propan-2-sulfonyl)azetidin-3-yl]acetonitrile (50 mg, 0.09 mmol) in N,N-dimethylformamide (2 mL) was added iodomethane (15 mg, 0.1 mmol) and potassium carbonate (39 mg, 0.28 mmol) at 25 °C and stirred for 3 h. After completion of the reaction, diluted with water (20 mL) and extracted with ethyl acetate (10 mL*3), dried the organic phase, concentrated and evaporated to get the product 2-[3-(3-methoxy-4-{7-[(2-methoxyethyl)trimethyl-{5}-silyl]pyrrolo[2,3-d]pyrimidin-4-yl}pyrazol-l-yl)-l-(propan-2-sulfonyl)azetidin-3-yl]acetonitrile (50 mg, yield = 77.96%) as crude which was used as such for the next step.

[0291] LC-MS (ESI), m / z: [M+H] = 546.1 + = 546.1

[0292] Step B: 2-[3-(3-methoxy-4-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}pyrazol-l-yl)-l-(propan-2- sulfonyl)azetidin-3-yl]acetonitrile

[0293] To a stirred solution of 2-[3-(3-methoxy-4-{7-[(2-methoxyethyl)trimethyl-{5}-silyl]pyrrolo[2,3-d]pyrimidin-4-yl}pyrazol-l-yl)-l-(propan-2-sulfonyl)azetidin-3-yl]acetonitrile (50 mg, 0.09 mmol) in dichloromethane (2 mL) was added boron trifluoride etherate (40 mg, 0.28 mmol) after dissolution, stirred for 2 h. The reaction was concentrated and evaporated to get dissolved in methanol (2 mL), added ethylenediamine (17 mg, 0.28 mmol) to adjust the pH to 8 and stirred for 0.5 h at 25 °C. The reaction was concentrated and purified by Prep-HPLC (column type: gemini-C18 150 x 21.2 mm, 5 um, mobile phase: ACN-H20 (0.1% TFA), gradient 5-10%, flow rate: 20 mL / min) to get the product 2-[3-(3-methoxy-4-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}pyrazol-l-yl)-l-(propan-2-sulfonyl)azetidin-3-yl]acetonitrile (3 mg, yield = 7.76%).

[0294] LC-MS (ESI), m / z: [M+H] = 416.1 + = 416.1

[0295] 1 H-NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.68 (s, 1H), 8.65 (s, 1H), 7.55-7.51 (m, 1H), 6.94 (dd, J = 3.5, 1.7 Hz, 1H), 4.54 (d, J = 9.0 Hz, 2H), 4.15 (d, J = 9.0 Hz, 2H), 4.00 (s, 3H), 3.63 (s, 2H), 3.36 (d, J = 6.8 Hz, 1H), 1.28 (s, 3H), 1.26 (s, 3H).

[0296] Synthesis route of Control 3:

[0297] Step A: 4-bromo-1-(4-methoxybenzyl)-1H-pyrazole-3-carboxylic acid methyl ester

[0298] To a solution of 4-bromo-1H-pyrazole-3-carboxylic acid methyl ester (5 g, 0.0025 mol) in acetonitrile (50 mL) was added potassium carbonate (6.78 g, 0.005 mol) and p-methoxybenzyl chloride (7.69 g, 0.0025 mmol). The reaction was stirred at 25 °C for 2 h. After the reaction was completed, the reaction was concentrated under reduced pressure to give the product 4-bromo-1-(4-methoxybenzyl)-1H-pyrazole-3-carboxylic acid methyl ester (5.88 g, crude product was used directly in the next step) as a yellow solid.

[0299] LC-MS (ESI), m / z: [M+H]+: 325.0 + = 325.0

[0300] Step B: 1-(4-methoxybenzyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-3-carboxylic acid methyl ester

[0301] A mixture of 4-bromo-l-(4-methoxybenzyl)-lH-pyrazole-3-carboxylic acid methyl ester (5.88 g, 0.018 mol), bis(pinacolato)diboron (4.58 g, 0.018 mol) and potassium acetate (5.292 g, 0.054 mol) in 1,4-dioxane (200 mL) was added [l,l'-bis(diphenylphosphino)ferrocene]dichloropalladium(ll) (1.316 g, 0.0018 mol). The reaction was stirred at 90 °C under nitrogen for 16 h. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by Combiflash column (petroleum ether: ethyl acetate = 2: 1) to give the crude product (3.74 g, which was used directly in the next step).

[0302] LC-MS (ESI), m / z: [M+H] + = 373.1

[0303] Step C: 1-(4-methoxybenzyl)-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-lH-pyrazole-3-carboxylic acid methyl ester

[0304] A mixture of 1-(4-methoxybenzyl)-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-lH- pyrazole-3-carboxylic acid methyl ester (3.74 g, 0.01 mol) and 4-chloro-7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (2.83 g, 0.01 mol) and potassium carbonate (4.14 g, 0.03 mol) in 1,4-dioxane was added dichlorobis(triphenylphosphine)palladium (0.7 g, 0.001 mol). The reaction was stirred at 90 °C under nitrogen for 16 h. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by Combiflash column (dichloromethane:methanol = 20: 1) to give the crude product (1.85 g, which was used directly in the next step).

[0305] LC-MS (ESI), m / z: [M+H] + = 494.1

[0306] Step D: 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 1 H-pyrazole-3 -carboxylic acid methyl ester

[0307] To a solution of methyl 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazole-3-carboxylate (1.85 g, 0.0037 mol) in methanol (20 mL) was added cerium ammonium nitrate (9.8 g, 0.0185 mol). The reaction was stirred at 25 °C overnight. The reaction was diluted with water (20 mL) and then extracted with ethyl acetate (10 mL*3), the combined organic phase was washed with saturated brine (20 mL), the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was obtained, the crude product was purified by Combiflash column (dichloromethane:methanol = 15:1) to give the crude product (900 mg, yield = 65%) as a yellow solid.

[0308] LC-MS (ESI), m / z: [M+H]+= 374.1

[0309] Step E: 4-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazole-3-carboxamide

[0310] Methyl 4-(7-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazole-3-carboxylate (200 mg, 0.54 mmol) was added to a solution of ammonia in methanol (5 mL) at room temperature and stirred at 25 °C for 16 hours. After the reaction was completed, it was concentrated and evaporated to dryness to give 4-(7-{[2- (trimethylsilyl)ethoxy]methyl}pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide (110 mg, crude), which was used directly in the next step.

[0311] LC-MS (ESI), m / z: [M+H] + = 359.2

[0312] Step F: 1-(3-(cyanomethyl)-1-(ethylsulfonyl)azetidin-3-yl)-4-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3- carboxamide

[0313] To a solution of 4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide (170 mg, 0.48 mmol), 1,8- diazobicyclo[5.4.0]undec-7-ene (144 mg, 0.96 mmol) in N,N-dimethylformamide (5 mL) was added 2-(1-ethylsulfonyl)azetidin-3-ylideneacetonitrile (85 mg, 0.48 mmol) and the reaction was stirred at 25 °C overnight. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (20 mL*3), the combined organic phase was washed with saturated brine (20 mL), the combined organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give the crude product. The crude product was purified by Combiflash column (dichloromethane:methanol = 25:1) to give the product (75 mg, yield = 29%).

[0314] LC-MS (ESI), m / z: [M+H] + = 545.1

[0315] Step G: 1-(3-(cyanomethyl)-1-(ethylsulfonyl)azetidin-3-yl)-4-(7-(hydroxymethyl)- 7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide

[0316] To a solution of 1-(3-(cyanomethyl)-1-(ethylsulfonyl)azetidin-3-yl)-4-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3- carboxamide (75 mg, 0.14 mmol) in dichloromethane (5 mL) was added boron trifluoride-ether solution (0.5 mL). The reaction was stirred at 25 °C for 2 h. After the reaction was completed, the reaction was concentrated under reduced pressure to give the product (43 mg, crude) as a yellow solid.

[0317] LC-MS (ESI), m / z: [M+H] + = 445.1

[0318] Step H: 1-(3-(cyanomethyl)-1-(ethylsulfonyl)azetidin-3-yl)-4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide

[0319] To a solution of 1-(3-(cyanomethyl)-1-(ethylsulfonyl)azetidin-3-yl)-4-(7- (hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide (43 mg, 0.097 mmol) in methanol (2 mL) was added ethylenediamine (0.2 mL). The reaction was stirred at 25 °C for 2 h. The solution was concentrated under vacuum, the reaction was stirred at 25 °C for 2 h. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (10 mL*3), the combined organic phase was washed with saturated brine (20 mL), the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was obtained, the combined organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure, the residue was purified by Prep-HPLC (column type: gemini-C18 150 x 21.2 mm, 5um, mobile phase: ACN-H2O (0.1% TFA), gradient 25%-30%, flow rate: 20 mL / min) to give the product (14.4 mg, 36%). LC-MS (ESI), m / z: [M+H]=415.0 +

[0320] 1 H-NMR (400 MHz, CD3OD) d 8.81 (m, 1H), 8.14 (s, 1H), 7.60 (dd, J = 3.7, 0.8 Hz, 1H), 6.91 (m, 1H), 4.70 (d, J = 9.3 Hz, 2H), 4.32 (d, J = 9.1 Hz, 2H), 3.63 (s, 2H), 3.16 (m, 2H), 1.35 (m, 3H).

[0321] Synthetic route for Control 4:

[0322] Step A refers to Step E of Control 3, except that methylamine is used instead of ammonia gas.

[0323] Step B: 1-(3-(cyanomethyl)-1-(isopropylsulfonyl)azetidin-3-yl)-N-methyl-4-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3- carboxamide

[0324] ​To a solution of N-methyl-4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide (72 mg, 0.19 mmol), 1,8-diazobicyclo[5.4.0]undec-7- ene (59 mg, 0.38 mmol) in N,N-dimethylformamide (5 mL) was added 2-(1- isopropylsulfonyl)azetidin-3-ylideneacetonitrile (38 mg, 0.19 mmol) and the reaction was stirred at 25 °C overnight. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (20 mL*3), the combined organic phase was washed with saturated brine (20 mL), the combined organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give the crude product. The crude product was purified by Combiflash column (dichloromethane:methanol = 25:1) to give the product (94 mg, yield = 86%).

[0325] LC-MS (ESI), m / z: [M+H] + = 573.1

[0326] Step C: 1-(3-(cyanomethyl)-1-(isopropylsulfonyl)azetidin-3-yl)-4-(7-(hydroxymethyl)-7H- pyrrolo[2,3-d]pyrimidin-4-yl)-N-methyl-1H-pyrazole-3-carboxamide

[0327] To a solution of 1-(3-(cyanomethyl)-1-(isopropylsulfonyl)azetidin-3-yl)-N-methyl-4-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide (94 mg, 0.17 mmol) in dichloromethane (5 mL) was added boron trifluoride-etherate solution (0.5 mL). The reaction was stirred at 25 °C for 2 h. After the reaction was completed, the reaction was concentrated under reduced pressure to give the product as a yellow solid (55 mg, crude).

[0328] LC-MS (ESI), m / z: [M+H] + = 473.1

[0329] Step D: 1-(3-(cyanomethyl)-1-(isopropylsulfonyl)azetidin-3-yl)-N-methyl-4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazole-3-carboxamide

[0330] To a solution of 1-(3-(cyanomethyl)-1-(isopropylsulfonyl)azetidin-3-yl)-4-(7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-methyl-1H-pyrazole-3-carboxamide (55 mg, 0.11 mmol) in methanol (2 mL) was added ethylenediamine (0.2 mL). The reaction was stirred at 25 °C for 2 h. The solution was concentrated under vacuum, the reaction was stirred at 25 °C for 2 h. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (10 mL*3), the combined organic phase was washed with saturated brine (20 mL), the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was obtained, the combined organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure, the residue was purified by Prep-HPLC (column type: gemini-C18 150 x 21.2 mm, 5um, mobile phase: ACN-H2O (0.1% TFA), gradient 25%-30%, flow rate: 20 mL / min) to give the product (18.7 mg, yield = 36%).

[0331] LC-MS (ESI), m / z: [M+H] + = 443.0

[0332] 1 H-NMR (400MHz, MeOD) δ 9.33 (s, 1H), 9.11 (s, 1H), 7.96 (d, J = 3.8 Hz, 1H), 7.48 (d, 1H), 4.79 (d, J = 9.8 Hz, 2H), 4.33 (m, 2H), 3.68 (d, J = 6.3 Hz, 2H), 3.04 (s, 3H), 1.35 (m, 6H).

[0333] Control 5: 3-(cyanomethyl)-3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide

[0334] Step A: 3-cyanomethyl-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide

[0335] To a solution of 2-(azetidin-3-ylidene)acetonitrile hydrochloride (200 mg, 1.53 mmol, 1.0 eq) in dichloromethane (8 ml) was added N,N-diisopropylethylamine (593 mg, 4.59 mmol, 3.0 eq) dropwise at 0 °C. After the reaction mixture was stirred at 25 °C for 15 min, phenyl (2,2,2-trifluoroethyl)carbamate (337 mg, 1.53 mmol, 1.0 eq) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 18 h. After the reaction was completed, the reaction mixture was evaporated under reduced pressure. The residue was concentrated and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to give 3-cyanomethyl-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide (200 mg, yield = 95%) as a white solid.

[0336] LC-MS (ESI), m / z: [M+H] + = 262.0.

[0337] Step B: 3-cyanomethyl-3-hydroxy-4-(7-(2-trimethylsilylethoxymethyl)-7H-pyrrolizin-4-ylpyrimidin-1-ylpyrazol-1-yl-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide

[0338] To a solution of 4-(2-trimethylsilylethoxymethyl)pyrrolopyrazin-4-ylpyrazole (302 mg, 0.91 mmol, 1.0 eq) and 3-cyanomethyl-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide (200 mg, 0.91 mmol, 1.0 eq) in N,N-dimethylformamide (8 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (92 mg, 0.91 mmol, 1.0 eq). The reaction mixture was stirred at 25 °C for 18 h. After the reaction was completed, water (20 mL) was added to the reaction mixture. The mixture was extracted with dichloromethane (20 mL*3). The combined organic phase was washed with saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate. The residue was concentrated and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to give 3-cyanomethyl-3-hydroxy-4-(7-(2-trimethylsilylethoxymethyl)-7H-pyrrolizin-4-ylpyrimidin-1-ylpyrazol-1-yl-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide (100 mg, yield = 30%) as a yellow oil.

[0339] LC-MS (ESI), m / z: [M+H] + = 551.0.

[0340] Step C: 3-cyanomethyl-3-hydroxy-4-(7-(2-trimethylsilylethoxymethyl)-7H- pyrrolizin-4-ylpyrrolo[2,3-d]pyrimidin-1-yl)-N-(2,2,2-trifluoroethyl)azetidine-1- carboxamide

[0341] 3-cyanomethyl-3-hydroxy-4-(7-(2-trimethylsilylethoxymethyl)-7H- pyrrolizin-4-ylpyrrolo[2,3-d]pyrimidin-1-yl)-N-(2,2,2-trifluoroethyl)azetidine-1- carboxamide (150 mg, 0.27 mmol, 1.0 eq) was dissolved in dichloromethane (6 ml). Boron trifluoride-ether solution (2 mL) was added dropwise under ice bath. The reaction was stirred at 25 °C for 2 h. After the reaction was completed, it was evaporated under reduced pressure. The crude product 3-cyanomethyl-3-(3-hydroxy-4-hydroxymethyl-7H-pyrrolizin-4-ylpyrrolo[2,3-d]pyrimidin-1-yl)-2,3-d-pyrrolidin-4-yl-1-pyrazolyl-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide (82 mg, crude) was obtained as a yellow oil.

[0342] LC-MS (ESI), m / z: [M+H] + = 451.0.

[0343] Step D: 3-cyanomethyl-3-hydroxy-4-pyrrolidin-4-ylpyrrolo[2,3-d]pyrimidine-2,3- dimethylpyrazol-1-yl-N-(2,2,2-trifluoroethyl)azetidine-1-carboxamide

[0344] 3-cyanomethyl-3-hydroxy-4-(7-(2-trimethylsilylethoxymethyl)-7H- pyrrolizin-4-ylpyrrolo[2,3-d]pyrimidin-1-yl)-N-(2,2,2-trifluoroethyl)azetidine-1- carboxamide (82 mg, 0.18 mmol, 10.0 eq) was dissolved in methanol (5 mL), ethylenediamine (0.5 ml) was added under ice bath. After the reaction was completed, water (20 ml) was added to the reaction solution, extracted with dichloromethane (10 mL*3), the combined organic phase was washed with saturated brine (20 mL), the organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was purified by Prep-HPLC (column type: gemini-C18 150 x 21.2 mm, 5 um, mobile phase: ACN-H2O (0.1% FA), gradient 10%-40%, flow rate: 20 mL / min) to give the product (5.3 mg, yield = 7%).

[0345] LC-MS (ESI), m / z: [M+H] + = 421.0.

[0346] 1H-NMR (400 MHz, CD3OD) δ 8.67 (s, 1H), 8.60 (s, 1H), 7.49 (d, J = 3.6 Hz, 1H), 7.05 (d, J = 3.6 Hz, 1H), 4.56 (d, J = 9.3 Hz, 2H), 4.28 (d, J = 9.3 Hz, 2H), 3.82 (d, J = 9.3 Hz, 2H), 3.48 (s, 2H).

[0347] II. Testing

[0348] 1. Enzymatic activity (IC50) of compounds 50 detection

[0349] (1) ROCK1 / 2 detection experiment

[0350] (a) Transfer 50 nL of diluted compound working solution to each well of the reaction plate (784075, Greiner) using Echo 655.

[0351] (b) Seal the reaction plate with a plate sealer and centrifuge at 1000g for 1 minute.

[0352] (c) Prepare the kinase solution.

[0353] (d) Add 5 μL of the kinase solution to each well of the reaction plate. Centrifuge the plate at 1000g for 30 seconds and let it stand at room temperature for 10 minutes.

[0354] (e) Prepare the STK2-substrate-biotin kinase substrate and ATP mix.

[0355] (f) Add 5 μL of the STK2-substrate-biotin and ATP mix to the reaction plate, centrifuge at 1000g for 30 seconds and start the reaction.

[0356] (g) Let the ROCK1 kinase reaction proceed for 20 minutes at room temperature and the ROCK2 kinase reaction proceed for 30 minutes at room temperature.

[0357] (h) Prepare the Sa-XL 665 (125 nM) and STK-antibody-Cryptate mix using the HTRF detection buffer.

[0358] (i) Add 10 μL of the Sa-XL 665 and STK-antibody-Cryptate mix to each well, centrifuge at 1000g for 30 seconds and let it react for 1 hour at room temperature.

[0359] (j) Read the 615 nm (Cryptate) and 665 nm (XL665) signals using Envision 2104 and use the signal intensity to characterize the degree of kinase activity.

[0360] Kinase activity data is expressed as the ratio of kinase activity with test compound to kinase activity of the blank (containing only DMSO), and IC 50 values at different ATP concentrations are shown in Table 1-1 and Table 1-2, respectively. 50 values at different ATP concentrations are shown in Table 1-1 and Table 1-2, respectively.

[0361] (2) JAK1 / 2 Assay

[0362] The JAK1 / 2 kinase activity detection platform is established based on the principle of Lance Ultra, and the activity of the compound is determined. At the same time, known Ruxolitinib and the like are determined as controls. In the detection plate, the enzyme, Ulight-labeled polypeptide substrate, ATP and detection compound are mixed and incubated. After the reaction, EDTA is added to terminate the reaction, and Eu-labeled antibody is added at the same time for detection. The detection plate is analyzed by Envision of PE company, and the analysis mode is TR-FRET. The data are represented by the readings of fluorescence signals at 665 nm and 615 nm, respectively. A high ratio of 665 nm / 615 nm indicates high enzyme activity, and a low ratio of 665 nm / 615 nm indicates inhibited enzyme activity.

[0363] Reagents: kinase (JAK1 / 2), substrate (ULight-JAK-1 peptide and ATP), detection reagent (Eu-W1024 Anti-phosphotyrosine and EDTA).

[0364] Instrument: Echo, Envision.

[0365] Dissolve the test compound into a 10 mM DMSO solution and store it in a nitrogen cabinet for long-term storage. Take 10 μL of 10 mM test compound solution and dilute it into a 1 mM working solution. Dilute it 3 times with Echo, a total of 11 concentrations, and the final concentration of the compound in the reaction system is 10 μM to 0.17 nM. Add 5 μL of enzyme and polypeptide substrate mixture to the detection plate with an electric pipette, centrifuge the detection plate, and incubate the detection plate at room temperature (23°C) for 15 minutes. Add 5 μL of kinase buffer containing ATP to the detection plate with an electric pipette, centrifuge the detection plate, and use aluminum foil to seal the plate. Incubate the detection plate at room temperature (23°C) for 90 minutes. Terminate the reaction by adding detection reagent to the detection plate with an electric pipette, centrifuge the detection plate, and use aluminum foil to seal the plate. Incubate the detection plate at room temperature (23°C) for half an hour, and detect the signal value of the reaction plate with Envision instrument. The results are shown in Table 1-1 and Table 1-2.

[0366] Enzymatic activity (IC 50 )

[0367] ROCK1 / ROCK2 ~250 mM ATP, JAK1 38 mM ATP, JAK2 12 mM ATP

[0368] Enzymatic activity (IC 50 )

[0369] The structures of the control compounds are as follows:

[0370] As can be seen from the data in the table, the enzymatic activity of the compounds in the embodiments of the present disclosure has better enzymatic activity than the marketed ROCK inhibitors Ripasudil, Netarsudil and Belumosudil and the control compounds, and some of the compounds have strong activity on ROCK1 / 2 kinase and can be used for the treatment of diseases mediated by ROCK kinase.

[0371] Among them, the marketed ROCK inhibitors have the following structural formula:

[0372] Cell activity (IC 50 ) detection (In-Cell Western detection of myosin light chain phosphorylation)

[0373] ROCK causes changes in the cytoskeleton by phosphorylating the myosin light chain T18 / S19 two amino acid sites. Using the rat smooth muscle cell line A7r5, the culture conditions of A7r5 cells are DMEM containing 10% FBS, and the level of myosin light chain phosphorylation is detected by In-Cell Western using phspho-MLC-T18 / S19 specific antibody and secondary detection antibody. Cells treated with positive compounds are used as positive controls, and cells without compounds only with compound vehicle are used as negative controls. DRAQ5 is used to stain the nucleus as an internal reference. Using GraphPad Priism 7.0 software, a nonlinear regression curve fitting with a changing slope is used to determine the absolute IC 50 value.

[0374] On day 1, A7r5 cells were resuspended in serum-free medium and seeded at a density of 5000 cells per well in 384-well clear-bottom black plates coated with PDL. Cells were serum-starved for 4 hours, followed by incubation with the compound in serum-free medium for 1 hour. 50 μL of 8% PFA (paraformaldehyde) was added to each well for fixation at room temperature for 1 hour. The liquid in the wells was discarded, and 90 μL of ice-cold methanol was added to each well for permeation at 4°C for 1 hour. The plates were then washed three times with PBST (0.1% Tween 20-PBS) using an automated separatory funnel. After drying the plates, 50 μL of blocking buffer was added to each well for blocking at room temperature for 1 hour. Phspho-MLC-T18 / S19 specific antibody was diluted 1:200 with blocking buffer, and 20 μL was added to each well. The plates were then sealed and incubated overnight at 4°C.

[0375] On the second day, the liquid in the wells was discarded, and the plate was washed 5 times with PBST using an automated separatory plunger. After drying the plate, the second detection antibody was diluted 1:800 with blocking buffer and DRAQ5 was diluted 1:1000. 20 μL of each antibody was added to each well and incubated at room temperature for 1 hour. Then, the liquid in the wells was discarded, and the plate was washed 3 times with PBST using an automated separatory plunger, followed by 3 washes with ddH2O. After drying the plate, it was scanned using a LICOR Odyssey near-infrared imaging scanner. The specific detection results are shown in Table 2 below.

[0376] Table 2 Cell activity (IC50) of compounds 50 )

[0377] The compounds in this disclosure exhibit better cellular activity compared to marketed ROCK inhibitors Ripasudil, Netarsudil, and Belumosudil, as well as control compounds. They demonstrate a stronger ability to alter the cytoskeleton by phosphorylating two amino acid sites on the myosin light chain, T18 and S19. Therefore, the compounds disclosed herein have broader application potential.

[0378] 3. Immunoblotting assay

[0379] Human immortalized keratinocytes (HaCat, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) in logarithmic growth phase were harvested and cell counts were performed. Cell viability was assessed using the trypan blue exclusion assay to ensure that the viability of each cell line was above 90%. The cell concentration was adjusted to 5.55 × 10⁻⁶ cells using MEM (HyClone, Cat#SH30024.01) medium supplemented with 10% FBS (Gibco, Cat#10099-141). 5 / mL; 1.8 mL cell suspension was added to 6-well plates (Corning, Cat# 3516) respectively. 10x concentration of compounds was prepared, and 200 μL 10x compound was added to the 6-well plates with cells respectively. The cells in 6-well plates were incubated at 37°C, 5% CO2 for 24 hours.

[0380] The 6-well plates were taken out, and the cells were washed with PBS, then lysed with RIPA Lysis and Extraction Buffer (Thermo, Cat# 89901) on ice to extract total protein, and stored at -80°C overnight. The samples were thawed on ice, centrifuged at 13,000 rpm for 15 min, and the supernatant was collected. The total protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo, Cat# 23225). 20 μg sample protein was mixed with a certain volume of Sample Reducing Agent (10x) (Thermo, Cat# NP0009) and LDS Sample Buffer (4x) (Thermo, Cat# NP0008), and boiled in boiling water for 5 min. TM The total protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo, Cat# 23225). 20 μg sample protein was mixed with a certain volume of Sample Reducing Agent (10x) (Thermo, Cat# NP0009) and LDS Sample Buffer (4x) (Thermo, Cat# NP0008), and boiled in boiling water for 5 min.

[0381] 20 μg sample protein was subjected to SDS-4-12% PAGE gel (Thermo, Cat# NW04120BOX) electrophoresis, and transferred to membrane using iBlot 2 (Thermo, Cat# IB24001). After transfer, the PVDF membrane was blocked with 5% BSA in TBS-T solution at room temperature for 1 h. The corresponding primary antibody was added according to the table below, and incubated at 4°C overnight. The membrane was washed with TBS-T, and secondary antibody (Anti-rabbit IgG, HRP-linked Antibody) was added, and incubated at room temperature for 1 h. After washing the membrane, chemiluminescence was performed, and exposed.

[0382] The main reagents used are as follows in Table 3

[0383] Table 3 Main reagents

[0384] The results are shown in Figures 1-3. As can be seen from the figures, the inhibitors of Examples 6 and 33 can significantly promote the expression of collagen XVII in keratinocytes, which helps to promote skin repair (Figures 2 and 3); while the JAK inhibitors Baricitinib and Itacitinib have no such effect; the control drug ROCK1 / 2 inhibitor Y-27632 has a certain promoting effect on the expression of collagen XVII (Figure 1), but the effect is not as good as the compounds of the examples (Figure 2); the effect of the ROCK2 inhibitor Belumosudil (KD-025) is not obvious (Figure 3). The abnormally prominent effect of the compounds of the examples in promoting the expression of collagen XVII may be related to their high activity in inhibiting ROCK1 / 2, or may be related to their dual-target inhibition characteristics of acting on ROCK and JAK at the same time.

[0385] The structural formula of the control drug ROCK1 / 2 inhibitor Y-27632 is as follows:

[0386] 4. Effect of compounds in animal models of glaucoma

[0387] Test method: The frozen GFP-SD rat Tenon's capsule fibroblasts (referred to as Tenon's cells) were quickly placed in a 37°C water bath for recovery, then cultured in a 37°C, 5% CO2 incubator with 10% FBS·DMEM complete culture medium. When the cells were 70-80% confluent, they were passaged, and after 2-3 stable passages, they were digested with 0.25% trypsin-0.02% EDTA mixed solution, neutralized with complete culture medium (1:4), resuspended by blowing the culture medium, and centrifuged to remove the supernatant. The cells were diluted to a concentration of 4.5×10 7 cells / mL for standby use.

[0388] After quarantine, male SD rats were anesthetized with isoflurane, and the left eye (OS) was surface anesthetized with propoxycaine hydrochloride eye drops. 10 μL of allogeneic Tenon's capsule fibroblasts at a concentration of 4.5×10 7 cells / mL (i.e. the amount of cells injected was 4.5×10 5 cells / eye) was injected into the anterior chamber of the left eye, and tobramycin eye ointment was applied to the needle hole. The sham-operated SD rats were injected with the same volume of PBS into the anterior chamber of the left eye, and the rest of the operations were the same as for the model animals. After the rats woke up, they were put back in the cage.

[0389] On the 7th day after modeling, according to the intraocular pressure and body weight, relatively uniform animals were selected for random grouping. Randomly divided into normal control group, model control group, test substance group, 8 in each group. After modeling, the test substance was given by conjunctival sac eye drops, once a day, one drop each time, for 20 days (a total of 20 times), and the first administration day was defined as the first day of the test (D1). Test substance eye drops preparation: 0.02% / 0.05% test substance + 5% ELP35 + 0.60% xanthan gum + 0.02% benzalkonium chloride + 94.36% / 94.33% pH 7.4 buffer solution.

[0390] During the test period, the general state of the animals in each group was observed; after the start of administration, the baseline intraocular pressure before administration each day was measured; after the end of administration, the animals were dissected according to the plan, and ophthalmic examination was performed, and general autopsy observation was performed after euthanasia. The results are shown in Figures 4A and 4B (intraocular pressure data), wherein Figure 4A shows the baseline intraocular pressure data measured each day in each group, and Figure 4B shows the change value of the baseline intraocular pressure each day in each group relative to D1. The results show that: compared with the glaucoma model group of animals, the administration group of Example 6 can significantly reduce the intraocular pressure of the glaucoma model rats; the pure JAK inhibitor Ruxolitinib has no obvious reduction in intraocular pressure.

[0391] The above describes the present disclosure in combination with preferred embodiments, but these embodiments are only exemplary and are only for illustrative purposes. On this basis, various substitutions and improvements can be made to the present disclosure, which all fall within the protection scope of the present disclosure.

Claims

1. Use of a pyrrolopyrimidine compound of general formula (Ⅰ) or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotope label, isomer or prodrug thereof in the preparation of a medicament for treating Rho kinase-mediated diseases; wherein L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CR4or N; Ar is selected from the group consisting of a substituted or unsubstituted single bond, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl, substituted with a substituent selected from the group consisting of halogen, C 1～8 alkyl, C 1～8 haloalkyl, or C 1～8 alkoxy; R1 is selected from the following groups, whether substituted or unsubstituted: halogen, NR 71 R 72 C 1～8 Alkyl, C 1～8 Alkoxy, C 2-8 alkenyl, C 2-8 alkynyl group, C 3～8 cycloalkyl, C 3～8 Heterocyclic group, C 6～20 Aryl or C 5～20 Heteroaryl groups; substituents selected from halogens, C 1～8 Alkyl, C 1～8 Haloalkyl, C 1～8 Alkoxy, C 3～8 cycloalkyl, C 3～8 Heterocyclic group, C 6～20 Aryl, C 5～20 heteroaryl, cyano, NR 71 R 72 hydroxyl, carboxyl, or thiol groups; R2is selected from -NHR7, wherein R7is selected from substituted or unsubstituted: hydrogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 alkylacyl, C 1～6 alkylsulfonyl, C 1～6 haloalkyl, C 1～6 alkoxy; the substituents are selected from cyano, NR 71 R 72 , hydroxy, carboxy, mercapto, C 3～6 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl; R3is selected from hydrogen, halogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 haloalkyl, C 1～6 alkoxy, cyano, NR 71 R 72 , hydroxyl, carboxyl, or thiol; R4is selected from hydrogen, halo or C 1～6 alkyl; R5is selected from cyano, -CONH2or carboxyl; R6is selected from hydrogen, halo or C 1～6 alkyl; R 71 and R 72 are each independently selected from hydrogen or C 1～6 alkyl.

2. Use according to claim 1, wherein, R2is selected from -NHR7, wherein R7is selected from substituted or unsubstituted hydrogen, C 1～4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1～4 alkylacyl, C 1～4 alkylsulfonyl, C 1～4 haloalkyl or C 1～4 alkoxy; the substituents are selected from cyano, NR 71 R 72 , hydroxy, carboxy, mercapto, C 3～5 cycloalkyl, C 3～6 heterocyclyl, C 6～12 aryl or C 5～12 heteroaryl; preferably, R7is hydrogen.

3. Use according to claim 1 or 2, wherein, said X is selected from CH.

4. Use according to any one of claims 1 to 3, wherein, R1is selected from the group consisting of substituted or unsubstituted C 1～6 alkyl, C 1～6 alkoxy, C 3～6 cycloalkyl, C 6～12 aryl, C 3～6 heterocyclyl or C 5～12 heteroaryl, said heteroatom being selected from N, O or S; preferably from the group consisting of substituted or unsubstituted C 1～4 alkyl, C 1～4 alkoxy, phenyl, biphenyl, naphthyl, C 5～6 heterocyclyl or C 5～6 heteroaryl; the substituents being selected from halogen, C 1～4 alkyl or C 1～4 haloalkyl.

5. Use according to any one of claims 1 to 4, wherein, said R3is selected from hydrogen, halogen, C 1～4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 1～4 haloalkyl, C 1～4 alkoxy, cyano, NR 71 R 72 , hydroxyl, carboxyl or thiol; preferably, R3is hydrogen.

6. Use according to any one of claims 1 to 5, wherein, Ar is selected from the group consisting of substituted or unsubstituted C 5～7 cycloalkyl, C 6～12 aryl or C 5～12 heterocyclyl or C 5～12 heteroaryl, the heteroatom being selected from N, O or S; preferably from the group consisting of substituted or unsubstituted C 5～6 heterocyclyl or C 5～6 heteroaryl; the substituents being selected from halogen; Preferably, Ar is a substituted or unsubstituted group: wherein denotes the position of attachment to L, denotes the position of attachment to the azetidinyl group, the substituents are halogen, in particular F; the number of substituents can be 1-3, for example 1, 2 or 3.

7. Use according to any one of claims 1 to 6, wherein, Ar is a single bond.

8. Use according to any one of claims 1 to 7, wherein, R6is selected from hydrogen.

9. Use according to any one of claims 1 to 8, wherein, The compound is selected from one of the following structures:

10. The use according to any one of claims 1 to 9, wherein, The Rho kinase mediated disease is graft versus host disease (GVHD), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, cancer, glaucoma, insulin resistance, renal failure, neuronal degeneration or osteoporosis.

11. Use of a pyrrolopyrimidine compound of Formula (I) or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotopically-labeled, isomer, or prodrug thereof, in the manufacture of a medicament for treating a COL17 -associated disease. wherein L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CR4or N; Ar is selected from the group consisting of a substituted or unsubstituted single bond, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl, substituted with a substituent selected from the group consisting of halogen, C 1～8 alkyl, C 1～8 haloalkyl, or C 1～8 alkoxy; R1is selected from substituted or unsubstituted: halo, NR 71 R 72 , C 1～8 alkyl, C 1～8 alkoxy, C 2-8 alkenyl, C 2-8 alkynyl, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl; substituents are selected from halo, C 1～8 alkyl, C 1～8 haloalkyl, C 1～8 alkoxy, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, C 5～20 heteroaryl, cyano, NR 71 R 72 , hydroxyl, carboxyl, or thiol; R2is selected from -NHR7, hydroxy or thiol, wherein R7is selected from substituted or unsubstituted hydrogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 alkylacyl, C 1～6 alkylsulfonyl, C 1～6 haloalkyl or C 1～6 alkoxy; the substituents are selected from cyano, NR 71 R 72 , hydroxy, carboxy, thiol, C 3～6 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl or C 5～20 heteroaryl; R3is selected from hydrogen, halogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 haloalkyl, C 1～6 alkoxy, cyano, NR 71 R 72 , hydroxyl, carboxyl, or thiol; R4is selected from hydrogen, halo or C 1～6 alkyl; R5is selected from cyano, -CONH2or carboxyl; R6is selected from hydrogen, halo or C 1～6 alkyl; R 71 and R 72 are each independently selected from hydrogen or C 1～6 alkyl.

12. The use according to claim 11, wherein, The disease associated with COL17 is a bullous or blistering disease; preferably, it is a pemphigoid, an autoimmune blistering disease; preferably, the disease associated with COL17 is junctional bullous epidermolysis, bullous pemphigoid, mucous membrane pemphigoid, pemphigoid gestationis, acquired bullous epidermolysis, lichen planus pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis and cicatricial pemphigoid.

13. The use according to claim 11, wherein, The disease associated with COL17 is a skin healing related disease or disorder.

14. The use according to claim 13, wherein, The skin healing related disease or disorder is a diabetic foot ulcer, a pressure ulcer, a decubitus ulcer, a burn, a scald, a sunburn, a graze or a cut.

15. The use of claim 11, wherein, The disease associated with COL17 is androgenetic alopecia, skin aging.

16. A method of treating a Rho kinase mediated disease comprising administering to a patient in need thereof a therapeutically effective amount of a ROCK inhibitor, wherein, The ROCK inhibitor is selected from the group consisting of pyrrolopyrimidine compounds represented by general formula (I); wherein L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CR4or N; Ar is selected from the group consisting of a substituted or unsubstituted single bond, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl, substituted with a substituent selected from the group consisting of halogen, C 1～8 alkyl, C 1～8 haloalkyl, or C 1～8 alkoxy; R1 is selected from the following groups, whether substituted or unsubstituted: halogen, NR 71 R 72 C 1～8 Alkyl, C 1～8 Alkoxy, C 2-8 alkenyl, C 2-8 alkynyl group, C 3～8 cycloalkyl, C 3～8 Heterocyclic group, C 6～20 Aryl or C 5～20 Heteroaryl groups; substituents selected from halogens, C 1～8 Alkyl, C 1～8 Haloalkyl, C 1～8 Alkoxy, C 3～8 cycloalkyl, C 3～8 Heterocyclic group, C 6～20 Aryl, C 5～20 heteroaryl, cyano, NR 71 R 72 hydroxyl, carboxyl, or thiol groups; R2is selected from -NHR7, wherein R7is selected from substituted or unsubstituted hydrogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 alkylacyl, C 1～6 alkylsulfonyl, C 1～6 haloalkyl or C 1～6 alkoxy; the substituents are selected from cyano, NR 71 R 72 , hydroxy, carboxy, mercapto, C 3～6 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl or C 5～20 heteroaryl; R3is selected from hydrogen, halogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 haloalkyl, C 1～6 alkoxy, cyano, NR 71 R 72 , hydroxyl, carboxyl, or thiol; R4is selected from hydrogen, halo, or C 1～6 alkyl; R5is selected from cyano, -CONH2or carboxyl; R6is selected from hydrogen, halo or C 1～6 alkyl; R 71 and R 72 are each independently selected from hydrogen or C 1～6 alkyl.

17. A method of treating a disease associated with COL17, comprising administering to a patient in need a therapeutically effective amount of a pyrrolopyrimidine compound of general formula (I); wherein L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CR4or N; Ar is selected from the group consisting of a substituted or unsubstituted single bond, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl, substituted with a substituent selected from the group consisting of halogen, C 1～8 alkyl, C 1～8 haloalkyl, or C 1～8 alkoxy; R1is selected from substituted or unsubstituted: halo, NR 71 R 72 , C 1～8 alkyl, C 1～8 alkoxy, C 2-8 alkenyl, C 2-8 alkynyl, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl; substituents are selected from halo, C 1～8 alkyl, C 1～8 haloalkyl, C 1～8 alkoxy, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, C 5～20 heteroaryl, cyano, NR 71 R 72 , hydroxyl, carboxyl, or thiol; R2is selected from -NHR7, wherein R7is selected from substituted or unsubstituted: hydrogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 alkylacyl, C 1～6 alkylsulfonyl, C 1～6 haloalkyl or C 1～6 alkoxy; the substituents are selected from cyano, NR 71 R 72 , hydroxy, carboxy, mercapto, C 3～6 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl or C 5～20 heteroaryl; R3is selected from hydrogen, halogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 haloalkyl, C 1～6 alkoxy, cyano, NR 71 R 72 , hydroxyl, carboxyl, or thiol; R4is selected from hydrogen, halo or C 1～6 alkyl; R5is selected from cyano, -CONH2or carboxyl; R6is selected from hydrogen, halo or C 1～6 alkyl; R 71 and R 72 are each independently selected from hydrogen or C 1～6 alkyl.

18. A compound, or a pharmaceutically acceptable salt, solvate, active metabolite, polymorph, isotope label, isomer, or prodrug thereof, for the treatment of Rho kinase-mediated diseases or diseases associated with COL17, having the structural formula shown in general formula (I). wherein L is selected from -C(=O)-, -S(=O)2- or -CONH-; X is selected from CR4or N; Ar is selected from the group consisting of a substituted or unsubstituted single bond, C 3～8 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl, or C 5～20 heteroaryl, substituted with a substituent selected from the group consisting of halogen, C 1～8 alkyl, C 1～8 haloalkyl, or C 1～8 alkoxy; R1 is selected from the following groups, whether substituted or unsubstituted: halogen, NR 71 R 72 C 1～8 Alkyl, C 1～8 Alkoxy, C 2-8 alkenyl, C 2-8 alkynyl group, C 3～8 cycloalkyl, C 3～8 Heterocyclic group, C 6～20 Aryl or C 5～20 Heteroaryl groups; substituents selected from halogens, C 1～8 Alkyl, C 1～8 Haloalkyl, C 1～8 Alkoxy, C 3～8 cycloalkyl, C 3～8 Heterocyclic group, C 6～20 Aryl, C 5～20 heteroaryl, cyano, NR 71 R 72 hydroxyl, carboxyl, or thiol groups; R2is selected from -NHR7, wherein R7is selected from substituted or unsubstituted: hydrogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 alkylacyl, C 1～6 alkylsulfonyl, C 1～6 haloalkyl or C 1～6 alkoxy; the substituents are selected from cyano, NR 71 R 72 , hydroxy, carboxy, mercapto, C 3～6 cycloalkyl, C 3～8 heterocyclyl, C 6～20 aryl or C 5～20 heteroaryl; R3is selected from hydrogen, halogen, C 1～6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1～6 haloalkyl, C 1～6 alkoxy, cyano, NR 71 R 72 , hydroxyl, carboxyl, or thiol; R4is selected from hydrogen, halo, or C 1～6 alkyl; R5is selected from cyano, -CONH2or carboxyl; R6is selected from hydrogen, halo or C 1～6 alkyl; R 71 and R 72 are each independently selected from hydrogen or C 1～6 alkyl.

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