WO2022100614A1 - Benzourea ring derivative, and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are a benzourea ring derivative as represented by formula (I) or a pharmaceutically acceptable salt thereof, and a preparation method therefor and the use thereof.

Description

Benzourea ring derivative and its preparation method and application

This application claims the following priority:

CN202011259175.6, November 11, 2020.

technical field

The invention relates to the field of medicine, in particular to a class of benzourea ring derivatives and a preparation method and application thereof, in particular to a compound represented by formula (I) and a pharmaceutically acceptable salt thereof.

Background technique

RHO-associated protein kinase (ROCK), a serine/threonine protein kinase, is a downstream target effector molecule of RHO and is widely expressed in the human body. RHO-related protein kinase (ROCK) is involved in the regulation of myosin light chain (MLC) and is suitable for the treatment of vasodilation. New research supports that ROCK kinase is involved in the regulation of the immune response of TH17 cells and the activation of fibroblasts, which can expand adaptation It is used for pulmonary fibrosis, asthma and other lung diseases, and further indications include autoimmune diseases. The ROCK kinase family includes two isoforms, ROCK1 and ROCK2. ROCK2 kinase is associated with inflammation and fibrosis. Selective ROCK2 inhibitors do not induce vasodilation at high concentrations in isolated vasodilation experiments, which can reduce cardiovascular side effects. Although ROCK1 knockout mice have low embryonic mortality, most of them die after birth due to cytoskeletal variation caused by reduced MLC phosphorylation, while 90% of ROCK2 knockout mice die in the embryonic stage, but surviving mice and wild-type mice There was no difference, and selective inhibition of ROCK2 activity may have a higher safety profile. Therefore, selective ROCK2 protein kinase inhibitors can avoid the cardiovascular side effects of the drug.

KD025 (WO2006105081; WO2008054599; WO2010104851; WO2014055996) is an oral ROCK2 kinase selective inhibitor developed by Kadmon Company. Studies have shown that KD025 represents a novel mechanism for the treatment of idiopathic pulmonary fibrosis (IPF) by inhibiting fibrosis-regulating proteins such as RHO kinase. Idiopathic pulmonary fibrosis (IPF) may be caused by physical damage. The body's response to injury involves the reorganization of the actin cytoskeleton of various cells (such as epithelial cells, fibroblasts, endothelial cells, and macrophages), and the assembly of actin filaments and actomyosin The contraction of actomyosin is guided and regulated by RHO kinase family proteins (including ROCK1 and ROCK2). Previous studies have shown that RHO kinase family proteins are activated in the lungs of IPF patients and animal models of the disease, and RHO kinase inhibitors can prevent tissue fibrosis in these models and induce regression of established fibrosis . It has completed a Phase II clinical trial for the treatment of moderate to severe psoriasis and is in the Phase II clinical study phase for the treatment of idiopathic pulmonary fibrosis (IPF).

WO2014134388 and WO2016028971 also disclose a class of compounds whose general structural formulas are shown in formula (a) and (b), which can also be used for cardiovascular diseases, neuropathological diseases, tumors, autoimmune diseases, and pulmonary fibrosis. , treatment of inflammatory diseases, etc.

The compounds of the present invention are ROCK2 inhibitors, which have significant kinase inhibitory activity.

SUMMARY OF THE INVENTION

On the one hand, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

in,

T ₁ is N or CH; T ₃ is N or CR ₃ ;

为

When _T2 is O, _T3 is N, the structural unit

for

为

When _T2 is N, the structural unit

for

T ₄ is N or CH;

R ₁ , R ₂ , R ₃ and R ₄ are each independently H, F, Cl, Br, I, -OH, -CN, -NH ₂ , -NO ₂ , C _1-3 alkyl or C _1-3 alkoxy, wherein the C _1-3 alkyl and C _1-3 alkoxy groups are optionally 1, 2 or 3 independently selected from F, Cl, Br, I, -OH, -OCH ₃ , -CN , -NH ₂ or -NO ₂ substituents;

R ₆ and R ₇ are each independently H, F, Cl or C _1-3 alkyl, wherein said C _1-3 alkyl is optionally selected from 1, 2 or 3 independently selected from F, Cl, Br, I , -OH, -OCH ₃ , -CN, -NH ₂ or -NO ₂ substituent;

Each R ₈ is independently H, F, Cl, Br, I, -OH, -CN, -NH ₂ , -NO ₂ , C _1-3 alkyl or C _1-3 alkoxy, wherein said C _{1 -3} alkyl and C _1-3 alkoxy optionally by 1, 2 or 3 independently selected from F, Cl, Br, I, -OH, -OCH ₃ , -CN, -NH ₂ or -NO ₂ Substituent substituted; n is 1, 2, 3 or 4.

In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof has the structure represented by formula (I-1) or (I-2):

wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₆ , R ₇ , R ₈ , T ₁ , T ₃ , T ₄ and n are as defined in the present invention.

In some aspects of the present invention, the above R ₁ , R ₂ , R ₃ and R ₄ are each independently H, F, Cl, Br and -CH ₃ , and other variables are as defined herein.

In some aspects of the present invention, the above R ₆ and R ₇ are each independently H and -CH ₃ , wherein the -CH ₃ is optionally 1, 2 or 3 independently selected from F, Cl, Br, -OH or -NH ₂ substituents, other variables are as defined in the present invention.

_In some aspects of the present invention, R6 and _R7 above are each independently H and _-CH2NH2 , and other variables are as defined _herein .

In some aspects of the present invention, the above R ₈ is independently H, F, Cl, Br, -OH, -CN, -NH ₂ , -NO ₂ or -OCH ₃ , and other variables are as defined herein.

为

n、R ₈及其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned structural units

for

n, _R8 and other variables are as defined in the present invention.

为

R ₈及其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned structural units

for

_R8 and other variables are as defined herein.

为

其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned structural units

for

Other variables are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds or pharmaceutically acceptable salts thereof have the structures represented by formulae (I-3) to (I-7):

wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₆ , R ₇ , R ₈ and n are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds or pharmaceutically acceptable salts thereof have the structures represented by formulae (I-8) to (I-12):

wherein, R ₁ , R ₆ , R ₇ , R ₈ and n are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds or pharmaceutically acceptable salts thereof have the structures represented by formulae (I-13) to (I-17):

Wherein, R ₁ , R ₈ and n are as defined in the present invention; the carbon atom with "*" is a chiral carbon atom, in the form of (R) or (S) single enantiomer or enriched in one enantiomer form exist.

There are also some solutions of the present invention that are formed by any combination of the above variables.

In some aspects of the invention, the above compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

技术效果

technical effect

The compounds of the present invention have high kinase inhibitory activity against ROCK2.

Definition and Explanation

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without specific definitions, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue , without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, and methanesulfonic acids; also include salts of amino acids such as arginine, etc. , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized from the acid or base containing parent compound by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to this within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

Unless otherwise indicated, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

Unless otherwise specified, the terms "cis-trans isomer" or "geometric isomer" result from the inability to rotate freely due to double bonds or single bonds to ring carbon atoms.

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirror-image relationship.

Unless otherwise specified, "(D)" or "(+)" means dextrorotatory, "(L)" or "(-)" means levorotatory, and "(DL)" or "(±)" means racemic.

和楔形虚线键

表示一个立体中心的绝对构型，用直形实线键

和直形虚线键

表示立体中心的相对构型，用波浪线

表示楔形实线键

或楔形虚线键

或用波浪线

表示直形实线键

和直形虚线键

Use solid wedge keys unless otherwise specified

and wedge-dotted keys

Indicate the absolute configuration of a stereocenter, using a straight solid key

and straight dashed keys

Indicate the relative configuration of the stereocenter, with a wavy line

Represents a solid wedge key

or wedge-dotted key

or with wavy lines

Represents a straight solid key

and straight dashed keys

The compounds of the present invention may exist in particular. Unless otherwise specified, the term "tautomer" or "tautomeric form" refers to isomers of different functional groups that are in dynamic equilibrium and are rapidly interconverted at room temperature. A chemical equilibrium of tautomers can be achieved if tautomers are possible (eg, in solution). For example, proton tautomers (also called prototropic tautomers) include interconversions by migration of protons, such as keto-enol isomerization and imine-ene Amine isomerization. Valence tautomers include interconversions by recombination of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer" or "enriched in one enantiomer" refer to one of the isomers or pairs The enantiomer content is less than 100%, and the isomer or enantiomer content is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the terms "isomeric excess" or "enantiomeric excess" refer to the difference between two isomers or relative percentages of two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80% .

Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art The diastereoisomers were resolved and the pure enantiomers recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).

Compounds of the invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). For another example, deuterated drugs can be formed by replacing hydrogen with deuterium, and the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

The terms "optional" or "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. .

The term "substituted" means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable. When the substituent is oxygen (ie =O), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically achievable basis.

When any variable (eg, R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may optionally be substituted with up to two Rs, with independent options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

When the number of a linking group is 0, such as -(CRR) ₀ -, it means that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups connected to it are directly connected, for example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituents do not specify through which atom it is attached to the substituted group, such substituents may be bonded through any of its atoms, for example, pyridyl as a substituent may be through any one of the pyridine rings. The carbon atom is attached to the substituted group.

中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成

也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成

所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。 When the listed linking group does not indicate its direction of attachment, the direction of attachment is arbitrary, for example,

The linking group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right.

It is also possible to connect ring A and ring B in the opposite direction to the reading order from left to right.

Combinations of the linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

直形虚线键

或波浪线

表示。例如-OCH ₃中的直形实线键表示通过该基团中的氧原子与其他基团相连；

中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；

中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连。

表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括

这4种连接方式，即使-N-上画出了H原子，但是

仍包括

这种连接方式的基团，只是在连接1个化学键时，该位点的的H会对应减少1个变成相应的一价哌啶基。 Unless otherwise specified, when a group has one or more attachable sites, any one or more sites in the group can be linked to other groups by chemical bonds. When the connection method of the chemical bond is not located, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of the connected chemical bond. the group. The chemical bond connecting the site to other groups can be represented by straight solid line bonds

straight dotted key

or wavy lines

express. For example, a straight solid bond in -OCH ₃ indicates that it is connected to other groups through the oxygen atom in this group;

The straight dashed bond in the group indicates that it is connected to other groups through the two ends of the nitrogen atom in the group;

The wavy lines in the phenyl group indicate connections to other groups through the 1 and 2 carbon atoms in the phenyl group.

Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least

These 4 connection methods, even if the H atom is drawn on -N-, but

still includes

For the group in this connection mode, when one chemical bond is connected, the H at the site will be correspondingly reduced by one to become the corresponding monovalent piperidinyl group.

Unless otherwise specified, the term "C _1-3 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C _1-3 alkyl group includes C _1-2 and C _2-3 alkyl groups, etc.; it can be monovalent (eg methyl), divalent (eg methylene) or multivalent (eg methine) . Examples of _C1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, the term " _C1-3alkoxy " refers to those alkyl groups containing 1 to 3 carbon atoms attached to the remainder of the molecule through an oxygen atom. The C _1-3 alkoxy group includes C _1-2 , C _2-3 , C ₃ and C ₂ alkoxy and the like. Examples of C _1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

Unless otherwise specified, _Cn-n+m or _Cn - _Cn+m includes any particular instance of n to n+ _m carbons, eg _C1-12 includes _C1 , _C2 , C3, C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , and C ₁₂ , also including any range from n to n+ _m , eg C _1-12 includes C _1-3 , C _1-6 , C _1-9 , C _3-6 , C _3-9 , C _3-12 , C _6-9 , C _6-12 , and C _9-12 , etc.; in the same way, n yuan to n +m-membered means that the number of atoms in the ring is from n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring , 10-membered ring, 11-membered ring, and 12-membered ring, also including any one range from n to n+m, for example, 3-12-membered ring includes 3-6 membered ring, 3-9 membered ring, 5-6 membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 6-10 membered ring, etc.

The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, affinity substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy, such as acetoxy, trifluoroacetoxy, and the like.

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: formyl; acyl groups, such as alkanoyl groups (eg, acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS), 2-(trimethylsilyl)ethoxymethyl (SEM) and tert-butyldiphenyl Methylsilyl (TBS) and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing hydroxyl side reactions. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (eg acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and the like.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments enumerated below, embodiments formed in combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.

扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。 The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is:

After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.

The solvent used in the present invention is commercially available.

The following abbreviations are used in the present invention: g stands for grams; mg stands for milligrams; μL stands for microliters; mL stands for milliliters; mol stands for moles; mmol stands for millimoles; /L; μM stands for micromoles/liter; nM stands for nanomoles/liter. Me represents methyl; Boc represents tert-butoxycarbonyl; DMSO represents dimethyl sulfoxide; DMSO-d ₆ represents deuterated dimethyl sulfoxide.

软件命名，市售化合物采用供应商目录名称。 Compounds of the present invention are either used according to conventional nomenclature in the art

Software naming, commercially available compounds use supplier catalog names.

Detailed ways

The present invention will be described in detail by the following examples, but it does not mean any unfavorable limitation of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. For those skilled in the art, various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention. will be obvious.

Intermediate A

synthetic route:

first step

Compound A-1 (3.45 g, 16.4 mmol), compound A-2 (4.76 g, 24.5 mmol), tris(dibenzylideneacetone)dipalladium (1.50 g, 1.63 mmol), 2-dicyclohexylphosphine- 2',4',6'-Triisopropylbiphenyl (779mg, 1.63mmol), potassium carbonate (6.78g, 49.0mmol) were dissolved in dioxane (80mL) and water (20mL), the reaction solution was Stir at 95°C for 12 hours. After the reaction was completed, the reaction solution was directly filtered, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate, 15/1～1/1, V/V) to obtain Compound A-3. MS-ESI calculated [M+H] ⁺ 281, found 281.

second step

Compound A-3 (3 g, 9.55 mmol) was dissolved in tetrahydrofuran (32 mL), water (8 mL) and methanol (8 mL), lithium hydroxide monohydrate (1.20 g, 28.7 mmol) was added, and stirred at 28 degrees Celsius for 12 Hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove tetrahydrofuran and methanol, diluted with water (100 mL), and extracted with methyl tert-butyl ether (60 mL×1). The aqueous phase was adjusted to pH 3 with dilute aqueous hydrochloric acid (1M), filtered with suction, the filter cake was washed with water, and dried to obtain compound intermediate A. MS-ESI calculated [M+H] ⁺ 253, found 253.

Intermediate B

synthetic route:

first step

Urotropine (30.6 g, 218 mmol) was added to a solution of compound B-1 (50.0 g, 218 mmol) in dichloromethane (625 mL), and the reaction solution was stirred at 30 degrees Celsius for 1 hour. The reaction solution was filtered, and the filter cake was collected to obtain the crude compound B-2.

second step

Compound B-2 (65.0 g, 176 mmol) was dissolved in ethanol (429 mL), then 35% concentrated hydrochloric acid (52.0 mL) was added, and the reaction solution was stirred at 30 degrees Celsius for 72 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the hydrochloride of compound B-3.

third step

The hydrochloride salt of compound B-3 (30 g, 149 mmol) was dissolved in water (375 mL), then sodium bicarbonate (31.2 g, 372 mmol), methanol (375 mL) and di-tert-butyl dicarbonate (48.7 g, 223 mmol) were added , 51.3mL). The reaction solution was stirred at 30°C for 1 hour. The reaction solution was extracted with ethyl acetate (600 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product obtained by concentration under reduced pressure was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate, 1/0-5/1, V/V) to obtain compound B-4.

the fourth step

Ammonium acetate (116 g, 1.51 mol) and sodium cyanoborohydride (9.47 g, 151 mmol) were added to a solution of compound B-4 (40.0 g, 151 mmol) in methanol (400 mL). The reaction solution was stirred at 50°C for 18 hours. The reaction solution was concentrated under reduced pressure, dichloromethane (600 mL) was added to the residue, stirred and filtered, and the filtrate was concentrated under reduced pressure. The crude product obtained by concentration under reduced pressure was purified by reverse-phase preparative chromatography column (aqueous ammonia system) to obtain Intermediate B. MS-ESI calculated [M+H] ⁺ 267, found 267.

Intermediate C

synthetic route:

first step

Compound C-1 (8.00 g, 40.6 mmol) was dissolved in N,N-dimethylacetamide (80 mL), and 1-chloromethyl-4-fluoro-1,4 was added to the reaction solution under nitrogen atmosphere. - Diazabicyclo[2.2.2]octane bis(tetrafluoroborate) salt (28.8 g, 81.2 mmol), the reaction solution was stirred at 60 degrees Celsius for 14 hours. The reaction solution was concentrated under reduced pressure, and the obtained crude product was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate, 50/1～25/1, V/V) to obtain compound C-2. MS-ESI calculated [M+H] ⁺ 215 and 217, found 215 and 217.

second step

Compound C-2 (2.30 g, 10.7 mmol) and pinacol biboronate (5.43 g, 21.4 mmol) were dissolved in dioxane (50 mL), and [1,1′-bis(diphenylphosphine) was added ) ferrocene] palladium (II) dichloromethane complex (873 mg, 1.07 mmol) and potassium acetate (2.10 g, 21.4 mmol), the reaction solution was stirred at 95 degrees Celsius for 14 hours. The reaction solution was concentrated under reduced pressure, and the obtained crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate, 30/1～10/1, V/V) to obtain Intermediate C. MS-ESI calculated [M+H] ⁺ 263, found 263.

Example 1

synthetic route:

first step

Compound 1-1 (6.00 g, 39.7 mmol) was dissolved in dichloromethane (20 mL), N-bromosuccinimide (7.06 g, 39.7 mmol) and p-toluenesulfonic acid (1.37 g, 7.94 mmol) were added mmol), the reaction solution was stirred at 50 degrees Celsius for 12 hours. The reaction solution was diluted with water (150 mL), extracted with ethyl acetate (250 mL×2), and the organic phases were combined. The organic phase was washed once with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product concentrated under reduced pressure was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate, 100/1～20/1, V/V) to obtain compound 1-2.

second step

Urotropine (2.68 g, 19.1 mmol) was added to a solution of compound 1-2 (4.40 g, 19.1 mmol) in dichloromethane (60 mL), and the reaction solution was stirred at 20 degrees Celsius for 1 hour. The reaction solution was filtered, and the filter cake was collected to obtain the crude compound 1-3.

third step

Compound 1-3 (3.00 g, 8.17 mmol) was dissolved in ethanol (30 mL), then 35% concentrated hydrochloric acid (2.41 mL) was added, and the reaction solution was stirred at 80 degrees Celsius for 2 hours. The reaction solution was filtered, and the filtrate was concentrated and dried under reduced pressure to obtain the hydrochloride salt of compound 1-4.

the fourth step

The hydrochloride salt of compound 1-4 (2.80 g, 13.8 mmol) was dissolved in water (25 mL) and tetrahydrofuran (25 mL), then di-tert-butyl dicarbonate (2.41 g, 11.1 mmol, 2.54 mL) and hydrogen carbonate were added Sodium (3.58 g, 41.5 mmol). The reaction solution was stirred at 20°C for 1 hour. The reaction solution was diluted with water (60 mL) and extracted with ethyl acetate (100 mL x 2). The combined organic phases were washed once with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product concentrated under reduced pressure was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate, 30/1～10/1, V/V) to obtain compound 1-5.

the fifth step

Ammonium acetate (409 mg, 5.30 mol) and sodium cyanoborohydride (33.3 mg, 530 mmol) were added to a solution of compound 1-5 (300 mg, 530 mmol) in methanol (10 mL). The reaction solution was stirred at 50°C for 1 hour. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was separated and purified by preparative high performance liquid chromatography (chromatographic column: Waters Xbridge 150 x 25 mm x 5 μm; mobile phase: [10 mM aqueous ammonium bicarbonate solution-acetonitrile]; B%: 20%-50%, 10 minutes) to obtain the compound 1-6. MS-ESI calculated [M+H] ⁺ 268, found 268.

Step 6

Intermediate A (151 mg, 599 μmol) was dissolved in N,N-dimethylformamide (8 mL), followed by the addition of 2-(7-azobenzotriazole)-N,N,N',N '-Tetramethylurea hexafluorophosphate (296 mg, 778 μmol), N,N-diisopropylethylamine (232 mg, 1.80 mmol, 313 μL) and compound 1-6 (160 mg, 599 μmol). The reaction solution was stirred at 20°C for 12 hours. The reaction solution was diluted with water (80 mL), extracted with ethyl acetate (100 mL x 2), and the organic phases were combined. The organic phase was washed once with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by thin layer chromatography (developing solvent: ethyl acetate) to obtain compound 1-7.

Step 7

Compound 1-7 (220 mg, 420 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2 mL) was added, and the reaction solution was stirred at 20 degrees Celsius for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was subjected to preparative high performance liquid chromatography (chromatographic column: Phenomenex Synergi C18 150 x 30 mm x 4 μm; mobile phase: [0.05% aqueous hydrochloric acid-acetonitrile]; B%: 20%-40%, 10 minutes) separation and purification to obtain the hydrochloride of compound 1.

MS-ESI calculated [M+H] ⁺ 402, found 402. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.31(d, J=8.0Hz, 1H), 8.40(s, 1H), 8.32(s, 3H), 8.16(s, 1H), 7.99-7.88( m, 2H), 7.78 (dd, J=1.6, 8.8Hz, 1H), 7.72 (dd, J=7.6, 8.0Hz, 1H), 7.63-7.53 (m, 2H), 7.06 (d, J=7.3Hz) , 1H), 6.76 (d, J=8.0Hz, 1H), 5.48-5.37 (m, 1H), 3.90 (s, 3H), 3.61-3.24 (m, 2H), 2.56 (s, 3H) ppm.

Example 2

synthetic route:

first step

Compound 2-1 (1 g, 7.29 mmol) and tert-butylsulfinamide (928 mg, 7.66 mmol) were dissolved in dichloromethane (10 mL), cesium carbonate (4.75 g, 14.6 mmol) was added, and the reaction solution was heated at 20 degrees Celsius The reaction was stirred for 15 hours. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude compound 2-2. MS-ESI calculated [M+H] ⁺ 241, found 241.

second step

Compound nitromethane (3.55 mL, 65.8 mmol) was dissolved in dimethyl sulfoxide (15 mL), sodium tert-butoxide (160 mg, 1.66 mmol) was added, the reaction solution was stirred at 30 degrees Celsius for 1 hour, and compound 2 was added -2 (2 g, 8.32 mmol), the reaction solution was continuously stirred for 15 hours at 30 degrees Celsius. The reaction solution was added with saturated aqueous ammonium chloride solution (100 mL), extracted with ethyl acetate (30 mL x 4), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude compound 2-3. MS-ESI calculated [M+H] ⁺ 302, found 302.

the fourth step

Compound 2-3 (2 g, 6.64 mmol) was dissolved in ethyl acetate (3 mL), 4M hydrogen chloride ethyl acetate solution (13.3 mL) was added, and the reaction solution was stirred at 15 degrees Celsius for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product obtained by concentration was added to ethyl acetate (50 mL), stirred for 0.5 hour, and filtered to obtain the hydrochloride salt of compound 2-4.

the fifth step

The hydrochloride salt of compound 2-4 (250mg, 1.07mmol), Intermediate A (270mg, 1.07mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (410mg , 2.14 mmol) and 1-hydroxybenzotriazole (217 mg, 1.60 mmol) were dissolved in tetrahydrofuran (4 mL) and N,N-dimethylformamide (1 mL), to the reaction solution was added N,N-diiso Propylethylamine (553 mg, 4.28 mmol, 745 μL). The reaction solution was stirred at 30°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the obtained crude product was separated by thin layer chromatography (developing solvent: dichloromethane/methanol, 10/1, V/V) to obtain compound 2-5. MS-ESI calculated [M+H] ⁺ 432, found 432.

Step 6

Compound 2-5 (120 mg, 220 μmol) was dissolved in ethanol (2 mL) and water (0.5 mL), iron powder (77.7 mg, 1.39 mmol) and ammonium chloride (149 mg, 2.78 mmol) were added, and the reaction solution was heated at 95 degrees Celsius. The reaction was stirred for 2 hours. The reaction solution was concentrated under pressure, dichloromethane/methanol (10/1, V/V) solution (50 mL) was added to the residue, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was concentrated under reduced pressure, and subjected to preparative high performance liquid chromatography (chromatographic column: Xtimate C18 150 x 25 mm x 5 μm; mobile phase: [0.05% aqueous ammonium bicarbonate solution-acetonitrile]; B%: 21%-41%, 10 minutes ) was isolated and purified to obtain compound 2.

MS-ESI calculated [M+H] ⁺ 402, found 402. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.36(d, J=6.0Hz, 1H), 8.24(s, 1H), 8.05(s, 1H), 7.93-7.81(m, 2H), 7.74( dd,J=1.6,8.8Hz,1H),7.61-7.54(m,2H),7.04-6.94(m,1H),6.87(dd,J=2.4,5.6Hz,1H),5.14-4.93(m, 1H), 3.82 (s, 3H), 3.17-2.92 (m, 2H), 2.56 (s, 3H) ppm.

Example 3

synthetic route:

first step

In the first step of Reference Example 2, compound 3-2 was obtained. MS-ESI calculated [M+H] ⁺ 211, found 211.

second step

Compound 3-3 was obtained in the second step of Reference Example 2.

third step

Refer to the third step of Example 2 to obtain the hydrochloride of compound 3-4.

the fourth step

Compound 3-5 was obtained in the fourth step of Reference Example 2. MS-ESI calculated [M+H] ⁺ 402, found 402.

the fifth step

The fifth step of reference example 2 obtains the crude product, and the obtained crude product is subjected to preparative high performance liquid chromatography (chromatographic column: Phenomenex Synergi C18 150 x 30 mm x 4 μm; mobile phase: [0.05% aqueous hydrochloric acid solution-acetonitrile]; B%: 7%-27 %, 11 minutes) separation and purification to obtain the hydrochloride of compound 3.

MS-ESI calculated [M+H] ⁺ 372, found 372. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.92(d, J=7.6Hz, 1H), 9.17(s, 1H), 8.98-8.76(m, 2H), 8.60(s, 3H), 8.50( s,1H),8.28(s,1H),8.18-8.05(m,1H),7.93(t,J=8.4Hz,2H),7.83(d,J=8.8Hz,1H),7.66-7.47(m , 2H), 5.76-5.59 (m, 1H), 3.74-3.39 (m, 2H), 2.58 (s, 3H) ppm.

Example 4

synthetic route:

first step

In the first step of Reference Example 2, compound 4-2 was obtained. MS-ESI calculated [M+H] ⁺ 241, found 241.

second step

Compound 4-3 was obtained in the second step of Reference Example 2. MS-ESI calculated [M+H] ⁺ 302, found 302.

third step

Refer to the third step of Example 2 to obtain the hydrochloride salt of compound 4-4.

the fourth step

Compound 4-5 was obtained in the fourth step of Reference Example 2. MS-ESI calculated [M+H] ⁺ 432, found 432.

the fifth step

Compound 4 was obtained in the fifth step of Reference Example 2.

MS-ESI calculated [M+H] ⁺ 402, found 402. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.71(s, 1H), 8.85(d, J=8.0Hz, 1H), 8.28-8.15(m, 3H), 8.04(s, 1H), 7.93- 7.81(m, 2H), 7.72(dd, J=1.6, 8.4Hz, 1H), 7.61-7.49(m, 2H), 7.45-7.38(m, 1H), 5.08-4.91(m, 1H), 3.83( s, 3H), 3.09-2.81 (m, 2H), 2.55 (s, 3H) ppm.

Example 5

synthetic route:

first step

Compound 5-2 was obtained in the sixth step of Reference Example 1. MS-ESI calculated [M+H] ⁺ 451,453, found 451,453.

second step

Compound 5-2 (60.0 mg, 133 μmol) and compound 5-3 (41.2 mg, 160 μmol) potassium carbonate (36.8 mg, 266 μmol) were added to dioxane (4.5 mL) and water (0.5 mL), followed by [1,1'-bis(diphenylphosphino)ferrocene]dichloride palladium(II) (9.73 mg, 13.3 μmol) was added, and the reaction solution was stirred at 90 degrees Celsius for 16 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was separated and purified by silica gel column chromatography (eluent: dichloromethane/methanol, 200/1～10/1, V/V) to obtain compound 5-4. MS-ESI calculated [M+H] ⁺ 503, found 503.

third step

Referring to the seventh step of Example 1, the hydrochloride of compound 5 was obtained.

MS-ESI calculated [M+H] ⁺ 403, found 403. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.56(d,J=8.8Hz,1H),8.75(d,J=5.2Hz,1H), 8.56-8.42(m,1H), 8.38-8.27( m,1H),8.21-8.03(m,3H),7.85(d,J=8.4Hz,1H),7.74(t,J=7.6Hz,1H),7.62(d,J=8.8Hz,1H), 7.06(d,J=7.6Hz,1H),6.80(d,J=8.0Hz,1H),5.54-5.40(m,1H),3.92(s,3H),3.58-3.42(m,2H),2.64 -2.54(m,3H)ppm..

Example 6

synthetic route:

first step

Compound 6-1 (1 g, 3.80 mmol) was dissolved in N,N-dimethylformamide (8 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added (1.12 g, 5.85 mmol), 1-hydroxybenzotriazole (579 mg, 4.29 mmol) and ammonia (1.82 g, 13.0 mmol, 2.00 mL, 25% purity). The reaction solution was stirred at 20°C for 15 hours. Water (60 mL) was added to the reaction solution, the reaction solution was stirred at 0 degrees Celsius for 1 hour, filtered, and the filter cake was collected to obtain the crude compound 6-2.

second step

Compound 6-2 (470 mg, 1.79 mmol) was added to trimethoxymethane (4.84 g, 45.6 mmol, 5 mL) and N-methylpyrrolidone (1 mL), followed by p-toluenesulfonic acid (34.1 mg, 179 μmol) . The reaction solution was stirred at 110 degrees Celsius for 12 hours. Water (60 mL) was added to the reaction solution, and the reaction solution was further stirred at 0 degrees Celsius for 1 hour. The reaction solution was filtered, and the filter cake was collected to obtain the crude compound 6-3.

third step

Compound 6-3 (370 mg, 1.36 mmol) was added to N,N-dimethylformamide (0.1 mL), then thionyl chloride (4 mL) was added, and the reaction solution was stirred at 90 degrees Celsius for 14 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. Toluene (20 mL) was added to the residue obtained by concentration under reduced pressure, and the mixture was washed once with saturated sodium bicarbonate (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude compound 6-4.

MS-ESI calculated [M+H] ⁺ 291, found 291.

the fourth step

Compound 6-4 (220 mg, 757 μmol) and Intermediate B (205 mg, 757 μmol) were dissolved in isopropanol (5 mL) and sodium carbonate (161 mg, 1.51 mmol) was added. The reaction solution was stirred at 60 degrees Celsius for 15 hours. The reaction solution was concentrated under reduced pressure, and the obtained crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate, 10/1～4/1, V/V) to obtain compound 6-5.

MS-ESI calculated [M+H] ⁺ 521, found 521.

the fifth step

Compound 6-5 (200 mg, 384 μmol), Intermediate C (101 mg, 384 μmol), potassium phosphate (163 mg, 769 μmol) and [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride ( The dichloromethane complex of II) (62.8 mg, 76.9 μmol) was added to N,N-dimethylformamide (3 mL) and water (0.3 mL). The reaction solution was stirred at 85°C for 16 hours. The reaction solution was concentrated under reduced pressure, and the obtained crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate, 10/1-1/1, V/V) to obtain compound 6-6.

MS-ESI calculated [M+H] ⁺ 529, found 529.

Step 6

Compound 6-6 (75 mg, 142 μmol) was added to a 4M hydrogen chloride ethyl acetate solution (5 mL), and the reaction solution was stirred at 20 degrees Celsius for 40 minutes. The reaction solution was concentrated under reduced pressure, and the crude product obtained by concentration was subjected to preparative high performance liquid chromatography (chromatographic column: Phenomenex Synergi C18 150×25mm 10μm; mobile phase: [0.05% aqueous hydrochloric acid-acetonitrile]; B%: 8%-28%, 10 minutes ) was isolated and purified to obtain the hydrochloride of compound 6.

MS-ESI calculated [M+H] ⁺ 429, found 429. ¹ H NMR (400MHz, DMSO-d ₆ )δ13.04-12.67(m,1H), 9.42-9.06(m,1H), 8.68(s,1H), 8.59-8.44(m,3H), 8.01(d ,J=7.2Hz,1H),7.89-7.80(m,2H),7.67(d,J=8.8Hz,1H),7.58-7.49(m,1H),7.38-7.23(m,2H),7.19- 7.09(m,1H),6.96-6.85(m,1H),5.98(d,J=6.4Hz,1H),4.09-3.82(m,1H),3.79-3.70(m,3H),3.45-3.21( m,1H)ppm.

Example 7

synthetic route:

first step

Compound 7-1 was obtained in the fifth step of Reference Example 6.

MS-ESI calculated [M+H] ⁺ 525, found 525.

second step

Referring to the sixth step of Example 6, the hydrochloride of compound 7 was obtained.

MS-ESI calculated [M+H] ⁺ 425, found 425. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.42 (d, J=7.6 Hz, 1H), 9.29 (d, J=8.4 Hz, 1H), 8.69 (s, 1H), 8.58 (s, 3H) ,8.02(dd,J＝1.0,7.6Hz,1H),7.92-7.82(m,2H),7.65(d,J＝8.4Hz,1H),7.42(dd,J＝1.6,8.4Hz,1H), 7.34-7.27(m, 2H), 7.18(d, J=7.8Hz, 1H), 6.90(dd, J=2.0, 8.0Hz, 1H), 6.10-5.93(m, 1H), 4.10-3.87(m, 1H), 3.76 (s, 3H), 3.35 (dd, J=4.0, 7.2 Hz, 1H), 2.52 (s, 3H) ppm.

Example 8

synthetic route:

first step

Compound 8-1 (4.4 g, 17.3 mmol) was dissolved in dichloromethane (40 mL), the reaction solution was cooled to 0 degrees Celsius, and m-chloroperoxybenzoic acid (5.25 g, 25.9 mmol, 85% purity) was added in portions. The reaction solution was stirred at 20 degrees Celsius for 15 hours. The reaction solution was diluted with dichloromethane (100 mL), washed with 10% aqueous sodium bicarbonate solution (100 mL), 10% aqueous sodium thiosulfate solution (100 mL) and saturated brine (100 mL) in this order. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product obtained by concentration was separated and purified by silica gel column chromatography (eluent: dichloromethane/methanol, 100/1～10/1, V/V) to obtain compound 8-2.

MS-ESI calculated [M+H] ⁺ 272, found 272.

second step

Compound 8-2 (1 g, 3.69 mmol) was dissolved in dichloromethane (20 mL), triethylamine (747 mg, 7.38 mmol, 1.03 mL) was added, the reaction solution was cooled to minus 70 degrees Celsius, and oxalyl chloride ( 937 mg, 7.38 mmol, 646 μL). The reaction solution was stirred and reacted at minus 70 degrees Celsius for 0.5 hours, heated to 20 degrees Celsius, and continued to be stirred and reacted for 2 hours. Methanol (5 mL) was added to the reaction solution to dilute, and the obtained organic phase was washed with saturated sodium bicarbonate (50 mL) and water (50 mL) successively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product obtained by concentration was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate, 100/1～50/1, V/V) to obtain compound 8-3.

MS-ESI calculated [M+H] ⁺ 290, found 290.

third step

Compound 8-4 was obtained in the fifth step of Reference Example 6.

MS-ESI calculated [M+H] ⁺ 298, found 298.

the fourth step

Compound 8-5 was obtained in the fourth step of Reference Example 6.

MS-ESI calculated [M+H] ⁺ 528, found 528.

the fifth step

Referring to the sixth step of Example 6, the hydrochloride of compound 8 was obtained.

MS-ESI calculated [M+H] ⁺ 428, found 428. ¹ H NMR (400MHz, DMSO-d ₆ )δ10.33(s, 1H), 9.31(d, J=8.4Hz, 1H), 8.55(s, 3H), 8.03-7.96(m, 1H), 7.95- 7.88(m, 1H), 7.77(s, 1H), 7.62(dd, J=3.6, 7.6Hz, 2H), 7.45-7.26(m, 4H), 7.08(d, J=7.2Hz, 1H), 6.92 (d, J=7.6 Hz, 1H), 5.85-5.75 (m, 1H), 4.02-3.85 (m, 1H), 3.77 (s, 3H), 3.47-3.35 (m, 1H) ppm.

Example 9

synthetic route:

first step

Compound 9-1 was obtained in the fifth step of Reference Example 6.

MS-ESI calculated [M+H] ⁺ 294, found 294.

second step

Compound 9-2 was obtained in the fourth step of Reference Example 6.

MS-ESI calculated [M+H] ⁺ 524, found 524.

third step

Referring to the sixth step of Example 6, the hydrochloride of compound 9 was obtained.

MS-ESI calculated [M+H] ⁺ 424, found 424. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.81 (d, J=7.6Hz, 1H), 9.29-9.02 (m, 1H), 8.76-8.29 (m, 3H), 8.01-7.84 (m, 2H) ), 7.76(s, 1H), 7.71-7.61(m, 2H), 7.55-7.44(m, 1H), 7.39-7.20(m, 3H), 7.02(d, J＝5.6Hz, 1H), 6.94- 6.88 (m, 1H), 5.84-5.70 (m, 1H), 3.99-3.71 (m, 4H), 3.50-3.35 (m, 1H) ppm.

activity test

1. In vitro evaluation of ROCK protein kinase inhibitory activity

Objective: To detect the ROCK protein kinase inhibitory IC ₅₀ value of the compound.

Experimental Materials:

Assay buffer solution: 20 mM 4-hydroxyethylpiperazineethanesulfonic acid (pH 7.5), 10 mM magnesium chloride, 1 mM ethylene glycol diethyl ether diamine tetraacetic acid, 0.02% polyoxyethylene lauryl ether, 0.02 mg/mL bovine serum albumin , 0.1 mM sodium vanadate, 2 mM dithiothreitol, 1% DMSO. Experimental operation:

Add the ROCK2 protein kinase substrate Long S6 Kinase substrate peptide to the freshly prepared buffer solution at a concentration of 20 μM, then add 1 nM ROCK2 protein kinase, and stir evenly. Use Echo550 to add serial DMSO dilutions (starting at 10 μM, 3-fold serial dilution) containing the compounds to be tested, pre-incubate at room temperature for 20 minutes, add ³³ P-ATP (radiation intensity 10 μCi/μL) to initiate the reaction, and react at room temperature for two times. Hour. It was then filtered using P81 ion exchange paper (Whatman #3698-915) and washed with 0.75% phosphoric acid. Radiation intensity was detected using the Filter-Binding method.

The protein kinase inhibitory activity of the compounds was expressed as the residual protein kinase activity relative to the blank substrate (DMSO alone). _IC50 values and curves were calculated using the Prism software package (GraphPad Software, San Diego California, USA).

Experimental results:

Table 1 ROCK inhibitory activity test results

compound IC ₅₀ (nM) for ROCK2 1 (hydrochloride) 8 3 (hydrochloride) 45 4 54 5 (hydrochloride) 27 6 (hydrochloride) 56 7 (hydrochloride) 50 8 (hydrochloride) 63 9 (hydrochloride) 72

The compounds of the present invention have significant and even unexpected ROCK2 inhibitory activity.

Claims (13)

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

in, T ₁ is N or CH; T ₃ is N or CR ₃ ; When _T2 is O, _T3 is N, the structural unit

for

When _T2 is N, the structural unit

for

T ₄ is N or CH; R ₁ , R ₂ , R ₃ and R ₄ are each independently H, F, Cl, Br, I, -OH, -CN, -NH ₂ , -NO ₂ , C _1-3 alkyl or C _1-3 alkoxy, wherein the C _1-3 alkyl and C _1-3 alkoxy groups are optionally 1, 2 or 3 independently selected from F, Cl, Br, I, -OH, -OCH ₃ , -CN , -NH ₂ or -NO ₂ substituents; R ₆ and R ₇ are each independently H, F, Cl or C _1-3 alkyl, wherein said C _1-3 alkyl is optionally selected from 1, 2 or 3 independently selected from F, Cl, Br, I , -OH, -OCH ₃ , -CN, -NH ₂ or -NO ₂ substituent; Each R ₈ is independently H, F, Cl, Br, I, -OH, -CN, -NH ₂ , -NO ₂ , C _1-3 alkyl or C _1-3 alkoxy, wherein said C _{1 -3} alkyl and C _1-3 alkoxy optionally by 1, 2 or 3 independently selected from F, Cl, Br, I, -OH, -OCH ₃ , -CN, -NH ₂ or -NO ₂ Substituent substituted; n is 1, 2, 3 or 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (I-1) or (I-2):

in, R ₁ , R ₂ , R ₃ , R ₄ , R ₆ , R ₇ , R ₈ , T ₁ , T ₃ , T ₄ and n are as defined in claim 1 . The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently H, F, Cl, Br and -CH ₃ . The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ₆ and R ₇ are each independently H and -CH ₃ , wherein -CH ₃ is optionally separated by 1, 2 or 3 Substituents independently selected from F, Cl, Br, -OH or _-NH2 . The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein R ₆ and R ₇ are each independently H and -CH ₂ NH ₂ . The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein each _R8 is independently H, F, Cl, Br, -OH, -CN, _-NH2 , _-NO2 , or -OCH ₃ . The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein the structural unit

for

The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the structural unit

for

The compound according to claim 8 or a pharmaceutically acceptable salt thereof, wherein the structural unit

for

The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formulas (I-3) to (I-7):

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined in claim 1 , 2 or 3; R ₆ and R ₇ are as defined in claim 1 , 2, 4 or 5; _R8 is as defined in claim 1, 2 or 6; n is as defined in claim 1 . The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein the compound has the structures represented by formulae (I-8) to (I-12):

wherein R ₁ , R ₆ , R ₇ , R ₈ and n are as defined in claim 10 . The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the compound has the structures represented by the formulae (I-13) to (I-17):

wherein, R ₁ , R ₈ and n are as defined in claim 11; the carbon atom with "*" is a chiral carbon atom, in the form of (R) or (S) single enantiomer or enriched in one enantiomer form exists. A compound of the following formula or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: