TWI838437B - Compounds for immunomodulation, manufacturing method and use thereof - Google Patents

Abstract

The presemt invention discloses a compound represented by formula (II) and a pharmaceutically acceptable salt thereof, and an application of the compound as an S1P1 agonist.

Description

Compounds as immunomodulators and preparation methods and applications thereof

The present invention relates to a compound represented by formula (II) and a pharmaceutically acceptable salt thereof, and the use of the compound as an S1P1 agonist.

This application claims the priority of Chinese patent application CN201811488085.7 filed on December 6, 2018, the priority of Chinese patent application CN201910414361.3 filed on May 17, 2019, and the priority of Chinese patent application CN201911161765.2 filed on November 22, 2019. This application cites the full text of the above Chinese patent applications.

Sphingosine-1-phosphate (S1P) is an amphipathic biosignaling molecule belonging to the lysophospholipid (LP). S1P can regulate important physiological and biochemical functions by acting on five G protein-coupled receptor subtypes, sphingosine-1-phosphate receptors (S1PR _1-5 ). S1P can regulate different physiological functions by binding to different S1P receptors, and plays an important role in maintaining the health of the body and the occurrence of diseases.

S1P1 receptor agonists interfere with lymphocyte trafficking, sequestering them in lymph nodes and other secondary lymphoid tissues. This results in a reduction in peripheral circulating lymphocytes. The clinical value of lymphocyte sequestration is to exclude them from the sphere of inflammatory and/or autoimmune responses in peripheral tissues. This sequestration of lymphocytes (e.g., in lymph nodes) is thought to be the result of a combination of agonist-driven functional antagonism of S1P1 receptors on T cells (thus reducing the ability of S1P to mobilize T cells to egress from the lymph nodes) and sustained agonism of S1P1 receptors on the lymph node endothelium (thus increasing the barrier function against lymphocyte migration). Thus, _S1P1 receptor agonists reduce the body's autoimmunity by preventing lymphocyte trafficking and can be used as immunosuppressants to treat a variety of autoimmune diseases.

Among them, the S1P1 agonist Fingolimod (FTY720) was approved by the FDA for the treatment of recurrent multiple sclerosis (MS), opening up a new therapeutic area for the treatment of immune diseases. Although FTY720 has clinical efficacy, it is a non-selective S1P receptor agonist. The binding of FTY720 to S1P3 in the body often leads to a series of important side effects, such as bradycardia, which greatly limits its application in the treatment of immune diseases. Therefore, the discovery of the second-generation highly selective S1P1 agonist, making it a drug for the treatment of immune diseases with better efficacy, fewer side effects and a wider range of applications, has become one of the hot spots in drug research.

In addition to improving target selectivity, shortening the half-life of drugs, namely S1P1 receptor agonists, in the body is also a goal of discovering second-generation S1P agonists. As an immunosuppressant drug, a longer half-life will lead to continuous inhibition of lymphocyte transport and a decrease in the number of peripheral blood lymphocytes, which will cause the user's immune function to be impaired and increase the risk of viral infection. In the event of an infection, it is often necessary to stop the drug so that the number of peripheral blood lymphocytes can return to normal levels as soon as possible so that the body's immune function can be quickly restored. Among them, the half-life of S1P1 receptor agonists such as FTY720 in the human body is as long as 6 to 9 days, so even if the drug is stopped, it will take a long time for the number of lymphocytes to return to normal.

Therefore, there is still a need in this field to develop novel S1P1 receptor agonists with S1P1 receptor selectivity and a shorter half-life to overcome the defects of existing therapies.

The present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof, wherein m is selected from 1, 2 and 3; n is selected from 1 and 2; y is selected from 1 and 2; _R1 is selected from H, halogen, OH, _NH2 , CN, _C1-6 alkyl, _C3-6 cycloalkyl, 3-7 membered heterocycloalkyl and _C1-6 heteroalkyl, wherein the _C1-6 alkyl, _C3-6 cycloalkyl, 3-7 membered heterocycloalkyl or _C1-6 heteroalkyl is optionally substituted by 1, 2 or 3 R; _R2 is selected from H, halogen, OH, _NH2 , CN and _C1-6 alkyl, wherein the _C1-6 alkyl is optionally substituted by 1, 2 or 3 R; Ring A is selected from _C3-7 cycloalkyl, 3-7 membered heterocycloalkyl and _C3-7 cycloalkenyl, wherein the _C3-7 cycloalkyl, 3-7 membered heterocycloalkyl or C1-6 heteroalkyl is optionally substituted by 1, 2 or 3 R; _3-7 cycloalkenyl is optionally substituted by 1, 2 or 3 R; L ₁ is selected from and ; L ₂ is selected from a single bond, O and S; T ₁ is selected from N and CH; T ₂ is selected from N and CH; T ₃ is selected from N and CH; R is independently selected from H, F, Cl, Br, I, OH, NH ₂ , C _1-3 alkyl and CF ₃ ; the C _1-6 heteroalkyl and 3-7 membered heterocycloalkyl contain 1, 2 or 3 heteroatoms or heteroatomic groups independently selected from NH, O and S.

In some embodiments of the present invention, the above R is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CH ₃ , CH ₂ CH ₃ and CF ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ is selected from H, F, Cl, Br, OH, NH ₂ , CN, C _1-3 alkyl, C _3-6 cycloalkyl, 3-7 membered heterocycloalkyl and C _1-3 alkoxy, and the C _1-3 alkyl, C _3-6 cycloalkyl, 3-7 membered heterocycloalkyl or C _1-3 alkoxy is optionally substituted by 1, 2 or 3 R, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ is selected from H, F, Cl, Br, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ , CF ₃ , , , , , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from H, F, Cl, Br, OH, NH ₂ , CN, CH ₃ and CH ₂ CH ₃ , and the CH ₃ or CH ₂ CH ₃ is optionally substituted by 1, 2 or 3 Rs, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from H, F, Cl, Br, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ and CF ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above _L1 is selected from , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is selected from , , , , , and , , , , , , or Optionally substituted with 1, 2 or 3 R, and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is selected from , , , , , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the structural unit Selected from , , , , , , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the structural unit Selected from , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from wherein m, n, T ₁ , T ₂ , T ₃ , R, L ₂ , R ₁ , R ₂ and Ring A are as defined above.

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein m is selected from 1, 2 and 3; n is selected from 1 and 2; _R1 is selected from H, halogen, OH, _NH2 , CN, _C1-6 alkyl, _C3-6 cycloalkyl, 3-7 membered heterocycloalkyl and _C1-6 heteroalkyl, wherein the _C1-6 alkyl, _C3-6 cycloalkyl, 3-7 membered heterocycloalkyl or _C1-6 heteroalkyl is optionally substituted by 1, 2 or 3 R; _R2 is selected from H, halogen, OH, _NH2 , CN and _C1-6 alkyl, wherein the _C1-6 alkyl is optionally substituted by 1, 2 or 3 R; Ring A is selected from _C3-7 cycloalkyl, 3-7 membered heterocycloalkyl and _C3-7 cycloalkenyl, wherein the C3-7 cycloalkyl, _3-7 membered heterocycloalkyl or C1-6 heteroalkyl is optionally substituted by 1, 2 or 3 R; _3-7 cycloalkenyl is optionally substituted by 1, 2 or 3 R; L ₁ is selected from and ; L ₂ is selected from a single bond, O and S; T ₁ is selected from N and CH; T ₂ is selected from N and CH; T ₃ is selected from N and CH; R is independently selected from H, F, Cl, Br, I, OH, NH ₂ , C _1-3 alkyl and CF ₃ ; the C _1-6 heteroalkyl and 3-7 membered heterocycloalkyl contain 1, 2 or 3 heteroatoms or heteroatomic groups independently selected from NH, O and S.

In some embodiments of the present invention, the above R is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CH ₃ , CH ₂ CH ₃ and CF ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ is selected from H, F, Cl, Br, OH, NH ₂ , CN, C _1-3 alkyl, C _3-6 cycloalkyl, 3-7 membered heterocycloalkyl and C _1-3 alkoxy, and the C _1-3 alkyl, C _3-6 cycloalkyl, 3-7 membered heterocycloalkyl or C _1-3 alkoxy is optionally substituted by 1, 2 or 3 R, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ is selected from H, F, Cl, Br, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ , CF ₃ , , , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from H, F, Cl, Br, OH, NH ₂ , CN, CH ₃ and CH ₂ CH ₃ , and the CH ₃ or CH ₂ CH ₃ is optionally substituted by 1, 2 or 3 Rs, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from H, F, Cl, Br, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ and CF ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above _L1 is selected from , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is selected from , , , , , and , , , , , , or Optionally substituted with 1, 2 or 3 R, and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is selected from , , , , , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the structural unit Selected from , , , , , , , and , other variables are as defined in the present invention.

In some embodiments of the present invention, the structural unit Selected from , , and , other variables are as defined in the present invention.

The present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention also provides use of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in preparing a drug for preventing and/or treating S1P1 receptor-related diseases.

In some embodiments of the present invention, the above-mentioned use, wherein the S1P1 receptor-related disease is selected from ulcerative colitis, Crohn’s disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, primary biliary cholangitis, atopic dermatitis, intracerebral hemorrhage, graft versus host disease, psoriasis, type I diabetes, acne, microbial infection or microbial disease and viral infection or viral disease.

Definition and description

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 ambiguous or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to the corresponding trade name or its active ingredient.

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

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from the compounds having specific substituents discovered in 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 form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amines 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 a solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrosulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, trifluoroacetic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, etc.; and 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 basic and acidic functional groups, and can be converted into any base or acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or alkaline groups by conventional chemical methods. Generally, such salts are prepared by reacting free acid or alkaline forms of these compounds with a stoichiometric amount of an 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, ( D )-isomers, ( L )-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present invention.

The compounds of the present invention may exist in specific forms. Unless otherwise specified, the term "tautomers" or "tautomeric forms" means that at room temperature, isomers with different functional groups are in dynamic equilibrium and can quickly convert to each other. If tautomers are possible (such as in solution), chemical equilibrium of tautomers can be reached. For example, proton tautomers (also called proton transfer tautomers (prototropic tautomers)) include interconversions carried out by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valence tautomers) include interconversions carried out by the reorganization of some bonding electrons. A specific example of keto-enol tautomerism is the tautomerism between pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ^3H ), iodine-125 ( ^125I ) or C-14 ( ^14C ). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic efficacy, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention. "Optional" or "optionally" means 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 where said event or circumstance does not occur.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. The term "optionally substituted" means that it may be substituted or unsubstituted, and unless otherwise specified, the type and number of substituents can be any on a chemically feasible basis.

When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence 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, and each occurrence of R is an independent choice. Furthermore, combinations of substituents and/or variants thereof are permitted only if such combinations result in stable compounds.

When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected, such as When _L2 represents a single bond, it means that the structure is actually .

When a substituent is listed without specifying the atom through which it is bonded to the substituted group, the substituent may be bonded through any atom thereof. For example, a pyridyl substituent may be bonded to the substituted group through any carbon atom on the pyridine ring.

When a linker group is listed without specifying its direction of attachment, the direction of attachment is arbitrary, e.g. The connecting group L is -CH ₂ O-. In this case, -CH ₂ O- can be connected to the phenyl group and the cyclopentyl group in the same direction as the reading order from left to right. , or by connecting phenyl and cyclopentyl groups in the opposite direction of the reading order from left to right. Combinations of linking groups, substituents and/or variants thereof are permitted only if such combinations result in stable compounds.

Unless otherwise specified, the number of atoms in a ring is generally defined as the ring member number, e.g., "3-6 membered ring" refers to a "ring" with 3-6 atoms arranged around the ring.

Unless otherwise specified, the term "C _1-6 alkyl" is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C _1-6 alkyl group includes C _1-5 , C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-4 , C ₆ and C ₅ alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-6 alkyl groups include but are not limited to methyl (Me), ethyl (Et), propyl (including n -propyl and isopropyl), butyl (including n -butyl, isobutyl, s -butyl and t -butyl), pentyl (including n -pentyl, isopentyl and neopentyl), hexyl, etc.

Unless otherwise specified, the term "C _1-3 alkyl" is used to represent a linear or branched 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 (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-3 alkyl groups include but are not limited to methyl (Me), ethyl (Et), propyl (including n -propyl and isopropyl), etc.

The term "heteroalkyl" by itself or in combination with another term refers to a stable straight or branched alkyl radical or combination thereof consisting of a certain number of carbon atoms and at least one heteroatom or heteroatom group. In some embodiments, the heteroatom is selected from B, O, N and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. In other embodiments, the heteroatom group is selected from -C(=O)O-, -C(=O)-, -C(=S)-, -S(=O), -S(=O) ₂ -, -C(=O)N(H)-, -N(H)-, -C(=NH)-, -S(=O) ₂ N(H)- and -S(=O)N(H)-. In some embodiments, the heteroalkyl group is a C _1-6 heteroalkyl group; in other embodiments, the heteroalkyl group is a C _1-3 heteroalkyl group. The heteroatom or heteroatom group may be located at any interior position of the heteroalkyl group, including the position where the alkyl group is attached to the rest of the molecule, but the term "alkoxy" is a conventional expression and refers to those alkyl groups that are attached to the rest of the molecule through an oxygen atom. Examples of heteroalkyl groups include, but are not limited to _{, -OCH3} , _-OCH2CH3 , _-OCH2CH2CH3 , _-OCH2 ( _{CH3 ) 2} , _-CH2 - _CH2 - _O - _CH3 , -NHCH3 _, -N(CH3) ₂ , _-NHCH2CH3 , -N( _CH3 )( _CH2CH3 ), _{-CH2 -CH2- NH - CH3} , -CH2- _CH2 - _N ( _CH3 )-CH3 _{, -SCH3 , -SCH2CH3} , _{-SCH2CH2CH3 , -SCH2} ( _CH3 ) ₂ , _-CH2- S- _CH2 -CH3, -CH2 _{- CH2 ,} -S(=O) _{-CH3 , -CH2 - CH2} -S(=O) ₂ -CH ₃ , and up to two impurity atoms may be consecutive, for example -CH ₂ -NH-OCH ₃ .

Unless otherwise specified, the term "C _1-3 alkoxy" refers to an alkyl group containing 1 to 3 carbon atoms that is attached to the rest of the molecule via an oxygen atom. The C _1-3 alkoxy group includes C _1-2 , C _2-3 , C ₃ and C ₂ alkoxy groups, etc. 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, "C _3-7 cycloalkyl" means a saturated cyclic hydrocarbon group consisting of 3 to 7 carbon atoms, which is a monocyclic and bicyclic system, and the C _3-7 cycloalkyl includes C _4-7 , C _5-7 , C _3-5 , C _4-5 and C _5-6 cycloalkyl, etc.; it can be monovalent, divalent or polyvalent. Examples of C _3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

Unless otherwise specified, the term "3-7 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 3 to 7 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) _p , p is 1 or 2). It includes monocyclic and bicyclic systems, wherein the bicyclic system includes spiro, cyclic and bridged rings. In addition, with respect to the "3-6 membered heterocycloalkyl", the heteroatom may occupy the position of attachment of the heterocycloalkyl to the rest of the molecule. The 3-7 membered heterocycloalkyl group includes 3-6 membered, 4-7 membered, 4 membered, 5 membered, 6 membered and 7 membered heterocycloalkyl groups. Examples of 3-7 membered heterocycloalkyl groups include, but are not limited to, azacyclobutyl groups, oxacyclobutyl groups, thiocyclobutyl groups, pyrrolidinyl groups, pyrazolidinyl groups, imidazolidinyl groups, tetrahydrothienyl groups (including tetrahydrothien-2-yl and tetrahydrothien-3-yl groups, etc.), tetrahydrofuranyl groups (including tetrahydrofuran-2-yl groups, etc.), tetrahydropyranyl groups, piperidinyl groups (including 1-piperidinyl groups, 2-piperidinyl groups and 3-piperidinyl groups, etc.), ... 1-piperidin 2-piperidin yl, etc.), phenothrin (including 3-phenothrin and 4-phenothrin, etc.), diphenothrin Alkyl, dithianyl, iso Oxazolidinyl, isothiazolidinyl, 1,2- 1,2-Thiazyl Hexahydrobenzoate Base, high Base or homopiperidinyl, etc.

Unless otherwise specified, "C _3-7 cycloalkenyl" means a partially unsaturated cyclic hydrocarbon group consisting of 3 to 7 carbon atoms containing at least one carbon-carbon double bond, which includes monocyclic and bicyclic systems, wherein the bicyclic system includes spirocyclic, cyclic and bridged rings, and any ring of this system is non-aromatic. The C _3-7 cycloalkenyl includes C _3-6 , C _3-5 , C _4-7 , C _4-8 , C _4-6 , C _4-5 , C _5-7 or C _5-6 cycloalkenyl, etc.; it can be monovalent, divalent or polyvalent. Examples of C _3-8 cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl and the like.

Unless otherwise specified, _Cn-n+m or _Cn - _Cn+m includes any specific case of n to n+m carbon atoms, for example, _C1-12 includes _C1 , _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , and _C12 , and also includes any range from n to n+m, for example, _C1-12 includes _C1-3 , _C1-6 , C1-9, _C3-6 , _C3-9 , _C3-12 , _C6-9 , _C6-12 , and C13 _{. Similarly} , n-membered to n+m-membered means that the number of atoms on the ring is 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, and also includes any 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, 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 (e.g., an affinity substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, iodine; sulfonate groups, such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate, etc.; acyloxy groups, such as acetyloxy, trifluoroacetyloxy, etc.

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxyl protecting group" or "alkyl 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, such as alkyl acyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butyloxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions of the hydroxy group. Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tertiary butyl; acyl groups such as alkyl acyl groups (such as acetyl); aryl methyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tertiary butyldimethylsilyl (TBS), and the like.

The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitution methods known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

The solvent used in the present invention can be obtained commercially.

The present invention uses the following abbreviations: DIAD represents diisopropyl azodicarboxylate; Xphos represents 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl; LiHMDS represents lithium hexamethyldisilamide; NIS represents N-iodosuccinimide; _Pd2 (dba) ₃ represents dipalladium tris(dibenzylideneacetone); THF represents tetrahydrofuran; DIAD represents diisopropyl azodicarboxylate; PPh ₃ represents triphenylphosphine; AntPhos represents 4-(9-anthryl)-3-(tert-butyl)-2,3-dihydrobenzo[d][1,3]oxyphosphine; DIEA represents N,N-diisopropylethylamine; HOAc represents acetic acid; DMF represents N,N-dimethylformamide; DIBAL-H represents diisobutylaluminum hydroxide; TBAF represents tetrabutylammonium fluoride.

The compounds were named according to the conventional naming principles in this field or using ChemDraw® software. Commercially available compounds were named according to the supplier's catalog name.

The present application is described in detail below through embodiments, but it does not mean that there are any adverse limitations on the present application. The present application has been described in detail herein, and its specific embodiments are also disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present application without departing from the spirit and scope of the present application.

Preparation of intermediates

Reference Example 1: Preparation of Intermediate I-1 Dissolve methyl 4-bromo-3-trifluoromethylbenzoate (6.32 g, 22.32 mmol) in dioxane/water (4:1, 30 mL), add cyclopentyl-1-ene-1-boronic acid (3.0 g, 26.79 mmol), tetrakistriphenylphosphine palladium (1.3 g, 1.12 mmol) and potassium carbonate (9.2 g, 67 mmol). Stir the reaction solution at 110°C under argon protection for 15 hours. After cooling to room temperature, the solvent was concentrated and evaporated to dryness to obtain a residue, which was purified by silica gel chromatography to obtain intermediate I-1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.32 (d, J = 1.5 Hz, 1H), 8.11 (dd, J = 8.0, 1.5 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 5.83 – 5.77 (m, 1H), 3.95 (s, 3H), 2.74 – 2.63 (m, 2H), 2.58 – 2.51 (m, 2H), 2.08 – 1.99 (m, 2H).

Reference Example 2: Preparation of Intermediate I-2 Dissolve the intermediate I-1 (1.0 g, 3.7 mmol) in 20 mL of methanol and add palladium carbon (50 mg, w/w=10%). Stir the reaction mixture at room temperature for 24 hours under a hydrogen atmosphere. Filter the reaction solution, and concentrate the filtrate to obtain the crude intermediate I-2. The crude product is used directly in the next reaction without purification.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.27 (d, J = 1.3 Hz, 1H), 8.13 (dd, J = 8.3, 1.3 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 3.93 (s, 3H), 3.47 – 3.36 (m, 1H), 2.16 – 2.07 (m, 2H), 1.93 – 1.82 (m, 2H), 1.82 – 1.70 (m, 2H), 1.69 – 1.55 (m, 2H).

Reference Example 3: Preparation of Intermediate I-3 Dissolve the intermediate I-2 (950 mg, 3.5 mmol) in 15 mL of tetrahydrofuran, and add a solution of tetrahydrogen lithium aluminum in tetrahydrofuran (1.5 M, 7 mL, 10.5 mmol) dropwise at -20°C under argon protection. After the addition is complete, stir the reaction solution at -20°C for 1 hour. After the temperature of the reaction solution is raised to 0°C, add a saturated aqueous ammonium chloride solution (4 mL) and ethyl acetate (5 mL). Filter the reaction solution, and concentrate the filtrate to obtain the intermediate I-3. The crude product is used directly in the next reaction without purification.

Reference Example 4: Preparation of Intermediate I-4 Intermediate I-3 (300 mg, 1.2 mmol) and 2-fluoro-4-hydroxybenzaldehyde (201 mg, 1.44 mmol) were dissolved in tetrahydrofuran (55 mL). Triphenylphosphine (644 mg, 2.4 mmol) and DIAD (484 mg, 2.4 mmol) were added under ice-water cooling, and stirred at room temperature overnight. Water (20 mL) and ethyl acetate (60 mL) were added to the reaction solution, and the organic phase was separated. The aqueous layer was extracted with ethyl acetate (60 mL × 2). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-4.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.22 (s, 1H), 7.84 (t, J = 8.4 Hz, 1H), 7.64 (s, 1H), 7.57 – 7.47 (m, 2H), 6.90 – 6.81 (m, 1H), 6.72 (dd, J = 12.2, 2.1 Hz, 1H), 5.11 (s, 2H), 3.44 – 3.33 (m, 1H), 2.17 – 2.05 (m, 2H), 1.92 – 1.80 (m, 2H), 1.79 – 1.69 (m, 2H), 1.65 – 1.58 (m, 2H).

Reference Example 5: Preparation of Intermediate I-5 Methyl 2,4-dihydroxybenzoate (5.0 g, 29.8 mmol) was dissolved in acetone (100 mL), potassium carbonate (8.2 g, 59.5 mmol) was added, the reaction solution was stirred at room temperature for 2 hours, benzyl bromide (5.6 g, 32.7 mmol) was added, and the reaction solution was stirred at 60 °C for 16 hours. After cooling to room temperature, the reaction solution was filtered, and the filtrate was concentrated to obtain a residue, which was purified by silica gel chromatography to obtain intermediate I-5.

LC-MS (ESI) [M+H] ⁺ 259.1.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.78 (s, 1H), 7.76 – 7.67 (m, 1H), 7.47 – 7.28 (m, 5H), 6.66 – 6.55 (m, 2H), 5.17 (s, 2H), 3.86 (s, 3H).

Reference Example 6: Preparation of Intermediate I-6 The intermediate I-5 (6.28 g, 24.3 mmol) was dissolved in tetrahydrofuran (100 mL), and LiHMDS (31.6 mL, 31.6 mmol, 1M tetrahydrofuran solution) was added dropwise at -78 °C. After the addition was completed, the reaction temperature was slowly raised to -40 °C, and after stirring for 2 hours, N,N-bistrifluoromethanesulfonylaniline (9.54 g, 26.7 mmol) was added. The reaction temperature was raised to room temperature and stirred for 16 hours. The reaction solution was quenched with 200 mL of water, 150 mL of ethyl acetate was added, and the organic layer was separated by extraction. The aqueous layer was further extracted with ethyl acetate (150 mL × 2). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-6.

Reference Example 7: Preparation of Intermediate I-7 To a tetrahydrofuran solution (100 mL) of intermediate I-6 (7.5 g, 19.2 mmol), potassium vinyl fluoroborate (2.83 g, 21.2 mmol), potassium phosphate (12.2 g, 57.7 mmol), sodium acetate (86.3 mg, 0.39 mmol) and Xphos (275 mg, 0.58 mmol) were added, and the reaction solution was stirred at 75°C for 4 hours under argon protection. After cooling to room temperature, the reaction solution was diluted with 200 mL of water and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-7.

LC-MS (ESI) [M+H] ⁺ 269.1.

Reference Example 8: Preparation of Intermediate I-8 Intermediate I-7 (500 mg, 1.87 mmol) was dissolved in methanol (20 mL), palladium carbon (100 mg, 10% w/w) was added, and the reaction solution was stirred at room temperature for 16 hours under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated to obtain a residue, which was purified by silica gel chromatography to obtain intermediate I-8.

LC-MS (ESI) [M+H] ⁺ 181.1.

Reference Example 9: Preparation of Intermediate I-9 The intermediate I-3 (2.5 g, 10.25 mmol) was dissolved in 10 mL of dichlorosulfonic acid and heated at 50 °C for 2 hours. After cooling to room temperature, the reaction solution was concentrated to obtain a residue. The residue was dissolved in ethyl acetate (10 mL) and washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (10 mL) in sequence. After drying over anhydrous sodium sulfate, the intermediate I-9 was obtained by filtration and concentration.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.53 (s, 1H), 7.49 – 7.36 (m, 2H), 4.50 (s, 2H), 3.35 – 3.23 (m, 1H), 2.07 – 1.95 (m, 2H), 1.84 – 1.72 (m, 2H), 1.71 – 1.59 (m, 2H), 1.57 – 1.44 (m, 2H).

Reference Example 10: Preparation of Intermediate I-10 Intermediate I-8 (310 mg, 1.72 mmol) was dissolved in N,N-dimethylformamide (20 mL), and intermediate I-9 (589.1 mg, 2.24 mmol) and cesium carbonate (1.12 g, 3.44 mmol) were added. The mixture was stirred at room temperature for 16 hours. The reaction solution was quenched with 50 mL of water and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-10.

Reference Example 11: Preparation of Intermediate I-11 The intermediate I-10 (670 mg, 1.65 mmol) was dissolved in tetrahydrofuran (20 mL), and diisobutylaluminum hydroxide (1.5M toluene solution, 3.3 ml, 4.95 mmol) was added dropwise at -40°C. After the addition, the reaction solution was stirred at room temperature for 16 hours. The reaction solution was quenched with 50 ml of water and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain the intermediate I-11.

LC-MS (ESI) [M-17] ⁺ 361.1.

Reference Example 12: Preparation of Intermediate I-12 The intermediate I-11 (400 mg, 1.06 mmol) was dissolved in 1,2-dichloroethane (20 mL), and manganese dioxide (921.6 mg, 10.6 mmol) was added, and the reaction solution was stirred at 70 °C for 16 hours. After cooling to room temperature, the reaction solution was filtered and the filtrate was evaporated to dryness to obtain a residue. The residue was purified by silica gel chromatography to obtain the intermediate I-12.

LC-MS (ESI) [M+H] ⁺ 377.1.

Reference Example 13: Preparation of Intermediate I-13 Intermediate I-3 (5.0 g, 20.47 mmol) was dissolved in 10 mL of 40% HBr/H ₂ O solution and heated at 100°C for 3 hours. After cooling to room temperature, the reaction solution was concentrated to obtain a residue. 100 mL of water was added to the residue and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-13.

¹ H NMR (400 MHz, MeOH- d ₄ ) δ 7.65 (d, J = 1.7 Hz, 1H), 7.60 (dd, J = 8.2, 1.6 Hz, 1H), 7.53 (d, J = 8.2 Hz, 1H), 4.58 (s, 2H), 3.41 – 3.32 (m, 1H), 2.10 – 2.01 (m, 2H), 1.94 – 1.83 (m, 2H), 1.78 – 1.67 (m, 2H), 1.67 – 1.57 (m, 2H).

Reference Example 14: Preparation of Intermediate I-14 Dissolve 4-hydroxybenzaldehyde (122 mg, 1 mmol) in acetonitrile (3 mL), add intermediate I-13 (306 mg, 1 mmol) and cesium carbonate (650 mg, 2 mmol). Stir the mixture at room temperature for 3 hours, and then filter the reaction solution. Concentrate the filtrate to obtain a residue. Purify the residue by silica gel chromatography to obtain intermediate I-14.

Reference Example 15: Preparation of Intermediate I-15 2-Chloro-4-hydroxybenzaldehyde (200 mg, 1.28 mmol) and intermediate I-13 (473 mg, 1.54 mmol) were dissolved in acetonitrile (4 mL), cesium carbonate (1.25 g, 3.84 mmol) was added, and the reaction solution was stirred at room temperature overnight. Water (10 mL) and ethyl acetate (30 mL) were added to the reaction solution and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (30 mL × 2). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-15.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.34 (s, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.65 (s, 1H), 7.58 – 7.48 (m, 2H), 7.02 (d, J = 2.4 Hz, 1H), 6.98 – 6.93 (m, 1H), 5.11 (s, 2H), 3.44 – 3.33 (m, 1H), 2.15 – 2.06 (m, 2H), 1.92 – 1.80 (m, 2H), 1.79 – 1.69 (m, 2H), 1.66 – 1.57 (m, 2H).

Reference Example 16: Preparation of Intermediate I-16 Intermediate I-13 (200 mg, 0.65 mmol) and 4-hydroxy-2-methylbenzaldehyde (80.2 mg, 0.59 mmol) were dissolved in acetonitrile (10 mL), cesium carbonate (423.8 mg, 1.30 mmol) was added, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was filtered and the filtrate was concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-16.

LC-MS (ESI) [MH] ^- 361.2.

Reference Example 17: Preparation of Intermediate I-17 Intermediate I-13 (460.0 mg, 1.50 mmol) and 4-hydroxy-2-bromobenzaldehyde (201.0 mg, 1.00 mmol) were dissolved in acetonitrile (10 mL), potassium carbonate (414.1 mg, 3.00 mmol) was added, and the reaction solution was stirred at 50°C for 16 hours. After the reaction solution was cooled to room temperature, water (10 mL) was added to the reaction solution, and it was extracted with ethyl acetate (15 mL × 3). The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, and filtered and concentrated to obtain the residue. The residue was purified by silica gel chromatography to obtain intermediate I-17.

LC-MS (ESI) [MH] ^- 425.1

Reference Example 18: Preparation of Intermediate I-18 Zinc cyanide (65.3 mg, 0.56 mmol) and palladium tetratriphenylphosphine (50 mg, 0.04 mmol) were added to a solution of intermediate I-17 (120.0 mg, 0.28 mmol) in N,N-dimethylformamide (10 mL), and the reaction solution was stirred at 140°C for 3 hours under argon protection. After cooling to room temperature, the reaction solution was diluted with 25 mL of water and extracted with ethyl acetate (20 mL × 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and filtered and concentrated to obtain the residue. The residue was purified by silica gel chromatography to obtain intermediate I-18.

LC-MS (ESI) [MH] ^– 372.2.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.23 (s, 1H), 8.01 (d, J = 8.7 Hz, 1H), 7.65 (s, 1H), 7.57 – 7.49 (m, 2H), 7.34 (d, J = 2.5 Hz, 1H), 7.30 (dd, J = 8.7, 2.5 Hz, 1H), 5.18 (s, 2H), 3.45 – 3.31 (m, 1H), 2.16 – 2.07 (m, 2H), 1.93 – 1.82 (m, 2H), 1.80 – 1.69 (m, 2H), 1.65 – 1.58 (m, 2H).

Reference Example 19: Preparation of Intermediate I-19 2-Hydroxy-3-trifluoromethylpyridine (5.00 g, 30.66 mmol) and NIS (6.90 g, 30.66 mmol) were dissolved in a mixed solvent of N,N-dimethylformamide and acetonitrile (60.0 mL, 1:1). The reaction mixture was heated to 80 °C and stirred at this temperature for 3 hours. The reaction mixture was cooled to 25 °C, sodium bicarbonate solution (35.0 mL, 1 M) was added and stirred for five minutes, and extracted with dichloromethane (500 mLx2). The organic phases were combined and concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was added with water (25.0 mL), stirred and filtered, and the filter cake was vacuum dried to obtain the crude intermediate I-19 product, which was directly used in the next reaction without further purification.

Reference Example 20: Preparation of Intermediate I-20 The intermediate I-19 (6.3 g, 21.80 mmol) was dissolved in phosphorus oxychloride (15.0 mL). The reaction mixture was heated to 100 °C and stirred at this temperature for 10 hours. The reaction solution was cooled to room temperature and slowly added to ice water (200 mL). The pH of the solution was adjusted to neutral with sodium carbonate and then extracted with ethyl acetate (400 mL × 2). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel column (PE:EA=100:1 to 10:1) to obtain intermediate I-20.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.78 (s, 1H), 8.28 (s, 1H).

Reference Example 21: Preparation of Intermediate I-21 Intermediate I-20 (2.10 g, 6.83 mmol), sodium acetate (1.12 g, 13.66 mmol), sodium acetate (76.78 mg, 0.342 mmol) and 1,1′-bis(diphenylphosphino)ferrocene (114 mg, 0.205 mmol) were dissolved in anhydrous methanol (25.0 mL). The reaction mixture was heated to 80 °C and reacted at this temperature under a CO atmosphere for 10 hours. The reaction solution was cooled to 25 °C and filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-21.

LC-MS (ESI) [M+H] ⁺ 240.1, [M+H+41] ⁺ 281.1.

¹ H NMR (400 MHz, CD ₃ OD) δ 9.13 (d, J = 1.8 Hz, 1H), 8.64 (d, J = 1.8 Hz, 1H), 3.99 (s, 3H).

Reference Example 22: Preparation of Intermediate I-22 The intermediate I-21 (500 mg, 2.09 mmol), cyclopentene-1-boronic acid (281 mg, 2.51 mmol), tricyclohexylphosphine (58.6 mg, 0.209 mmol), cesium carbonate (2.04 g, 6.27 mmol) and Pd ₂ (dba) ₃ were dissolved in a mixed solution of dioxane/hexanenitrile/water (12.00 mL, 2.5:2.5:1). The reaction solution was heated to 100 °C under nitrogen and reacted at this temperature for 8 hours. The reaction solution was cooled to room temperature and filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to remove the organic solvent. The residue was diluted with ethyl acetate (30 mL), washed with water (15 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by silica gel chromatography to obtain the target compound I-22.

LC-MS (ESI) [M+H+41] ⁺ 299.2

Reference Example 23: Preparation of Intermediate I-23 Intermediate I-22 (300 mg, 1.17 mmol) and PtO ₂ (53.1 mg) were dissolved in ethyl acetate (10.0 mL). The reaction mixture was reacted at 25 °C in a hydrogen atmosphere for 10 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain intermediate I-23.

LC-MS (ESI) [M+H+41] ⁺ 301.2.

Reference Example 24: Preparation of Intermediate I-24 The intermediate I-23 (270 mg, 1.04 mmol) was dissolved in tetrahydrofuran (4.00 mL). The mixture was cooled to 0 °C and BH ₃ (3.12 mL, 1 M in THF, 3.12 mmol) was slowly added dropwise under nitrogen. The temperature was then raised to 25 °C and the reaction was continued for 2 hours. The reaction solution was cooled to 0 °C again and quenched with methanol (4.00 mL). The reaction solution was concentrated under reduced pressure to remove the organic solvent, and the residue was diluted with ethyl acetate (30.0 mL) and washed with water (15.0 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product of intermediate I-24, which was directly used in the next reaction without purification.

LC-MS (ESI) [M+H] ⁺ 246.1

Reference Example 25: Preparation of Intermediate I-25 Intermediate I-24 (130 mg, crude product), PPh ₃ (170 mg, 0.649 mmol) and 2-fluoro-4-hydroxybenzaldehyde (53.5 mg, 0.382 mmol) were dissolved in tetrahydrofuran (3.00 mL), then cooled to 0 °C and DIAD (116 mg, 0.573 mmol) was slowly added dropwise under nitrogen protection. The reaction mixture was heated to 25 °C and the reaction was continued for 10 hours. Water (10.0 mL) was added to the reaction solution and extracted with ethyl acetate (15.0 mL× 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-25.

LC-MS (ESI) [M+H+41] ⁺ 409.2.

Reference Example 26: Preparation of Intermediate I-26 Intermediate I-25 (25.0 mg, 68.06 μmol) and 3-azacyclobutanecarboxylic acid methyl ester hydrochloride (10.32 mg, 68.06 μmol) were dissolved in methanol (1.00 mL), and then DIEA (8.80 mg, 68.06 μmol) and acetic acid (8.17 mg, 136.12 μmol) were added. The reaction mixture was reacted at 25 °C for 3 hours under nitrogen protection. Then NaBH ₃ CN (4.28 mg, 68.06 μmol) was added, and the reaction mixture was stirred at 25 °C for 10 hours. The reaction solution was added to water (10.0 mL) and extracted with ethyl acetate (15.0 mL). The organic phase was concentrated under reduced pressure to remove the organic solvent to obtain a crude intermediate I-26, which was directly used in the next reaction without purification.

LC-MS (ESI) [M+H] ⁺ 467.2

Reference Example 27: Preparation of Intermediate I-27 The intermediate I-1 (1.0 g, 3.70 mmol) was dissolved in tetrahydrofuran (10 mL) at -45 °C, and a toluene solution of diisobutylaluminum hydroxide (9.9 mL, 1.5 M, 14.85 mmol) was added. The reaction mixture was stirred at -45 ^° C for 2 hours. At 0 °C, a saturated aqueous ammonium chloride solution was added to quench the reaction (20 mL), and filtered. The filtrate was added with ethyl acetate (60 mL) for separation and extraction, and the aqueous phase was extracted with ethyl acetate (60 mL × 2) to obtain an organic phase. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-27.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.65 (s, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 5.72 (br.s, 1H), 4.73 (s, 2H), 2.69 – 2.59 (m, 2H), 2.55 – 2.46 (m, 2H), 2.09 – 1.97 (m, 2H).

Reference Example 28: Preparation of Intermediate I-28 At 0 °C, triphenylphosphine (1.13 g, 4.31 mmol) was dissolved in tetrahydrofuran (6 mL), and diisopropyl azodicarboxylate (578 mg, 2.86 mmol), intermediate I-27 (421 mg, 1.74 mmol) and 2-fluoro-4-hydroxybenzaldehyde (200 mg, 1.43 mmol) were added in sequence. The reaction mixture was stirred at room temperature overnight under argon protection. Water (20 mL) was added to the reaction mixture and the mixture was extracted with ethyl acetate (60 mL). The aqueous phase was extracted with ethyl acetate (60 mL × 2), and the organic phases were combined. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-28.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.22 (s, 1H), 7.85 (t, J = 8.8 Hz,1H), 7.70 (s, 1H), 7.54 – 7.49 (m, 1H), 7.33 (d, J = 8.8 Hz, 1H), 6.86 (dd, J = 8.8, 2.2 Hz, 1H), 6.73 (dd, J = 12.2, 2.3 Hz, 1H), 5.79 – 5.71 (br.s, 1H), 5.14 (s, 2H), 2.72 – 2.61 (m, 2H), 2.56 – 2.47 (m, 2H), 2.10 – 1.98 (m, 2H).

Reference Example 29: Preparation of Intermediate I-29 At room temperature, methyl-4-bromo-3-(trifluoromethyl)benzoate (2 g, 7.07 mmol), cyclohexene-1-ylboronic acid (1.07 g, 8.48 mmol) and potassium carbonate (2.93 g, 21.2 mmol) were dissolved in dioxane (20.0 mL) and water (4.00 mL), and then Pd(PPh ₃ ) ₄ (409 mg, 0.354 mmol) was added. Under nitrogen protection, the reaction mixture was heated to 110 °C and stirred at this temperature for 5 hours. The reaction solution was cooled to room temperature, water (200 mL) was added, and extracted with ethyl acetate (300 mL × 2). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-29.

¹ H NMR (400 MHz, CD ₃ OD) δ 8.25 (d, J = 1.5 Hz, 1H), 8.15 (dd, J = 8.0, 1.4 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 5.60 (s, 1H), 3.94 (s, 3H), 2.28 – 2.14 (m, 4H), 1.82 – 1.67 (m, 4H).

Reference Example 30: Preparation of Intermediate I-30 At room temperature, the intermediate I-29 (900 mg, 3.17 mmol) and Pd/C (10% w/w, 90 mg) were added to a 100 mL stainless steel reactor, and then methanol (10.0 mL) was added. The gas in the steel bottle was replaced with hydrogen, and the hydrogen was pressurized to 40 atm. The reaction mixture was heated to 50 °C under this pressure and stirred at this temperature for 10 hours. After the reaction solution was cooled to room temperature, it was filtered with diatomaceous earth, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the target compound I-30, which was directly used in the next reaction without purification.

¹ H NMR (400 MHz, CD ₃ OD) δ 8.22 (d, J = 1.4 Hz, 1H), 8.16 (dd, J = 8.3, 1.5 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 3.93 (s, 3H), 2.97 (dd, J = 11.6, 10.8 Hz, 1H), 1.84 (ddd, J = 23.6, 11.9, 3.3 Hz, 5H), 1.61 – 1.36 (m, 5H).

Reference Example 31: Preparation of Intermediate I-31 At room temperature, the intermediate I-30 (700 mg, 2.45 mmol) was dissolved in anhydrous tetrahydrofuran (10.0 mL). After the reaction solution was cooled to 0 °C under nitrogen protection, lithium aluminum tetrahydride (1M THF solution) (6.13 mL, 6.13 mmol) was slowly added dropwise. After the addition was completed, the reaction mixture was warmed to room temperature and stirred at room temperature for 10 hours. Ether (10.0 mL) was added to the reaction mixture and cooled to 0 °C, water (4.00 mL) was added, and then sodium hydroxide aqueous solution (6.00 mL, 10% w/w, aq.) was added. Water (18.0 mL) was added and stirred for 10 minutes. Anhydrous sodium sulfate was added to the system and stirring was continued for 10 minutes. Then the crude product was filtered, concentrated under reduced pressure to remove the organic solvent, and separated and purified by silica gel chromatography to obtain the intermediate I-31.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.55 – 7.47 (m, 2H), 4.60 (d, J = 6.1 Hz, 2H), 2.97 – 2.85 (m, 1H), 1.90 – 1.72 (m, 5H), 1.58 – 1.33 (m, 5H).

Reference Example 32: Preparation of Intermediate I-32 At room temperature, the intermediate I-31 (400 mg, 1.55 mmol), 2-fluoro-4-hydroxybenzaldehyde (217 mg, 1.55mmol) and PPh ₃ (1.22 g, 4.65 mmol) were dissolved in anhydrous tetrahydrofuran (10.0 mL), and the reaction solution was cooled to 0 °C under nitrogen protection. DIAD (471 mg, 2.33 mmol) was slowly added dropwise. After the addition was completed, the reaction solution was raised to room temperature and then reacted at room temperature for 10 hours. Water (100 mL) was added to the reaction mixture and extracted with ethyl acetate (150 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-32.

¹ H NMR (400 MHz, CD ₃ OD) δ 10.12 (s, 1H), 7.88 – 7.76 (m, 1H), 7.73 – 7.62 (m, 2H), 7.61 – 7.54 (m, 1H), 7.00 – 6.88 (m, 2H), 5.19 (d, J = 8.4 Hz, 2H), 3.02 – 2.86 (m , 1H), 1.92 – 1.72 (m, 5H), 1.59 – 1.33 (m, 5H).

Reference Example 33: Preparation of Intermediate I-33 At room temperature, methyl 4-bromo-3-(trifluoromethyl)benzoate (100 mg, 0.35 mmol) and cyclobutylboronic acid (70 mg, 0.70 mmol) were dissolved in xylene (4 mL), and then sodium acetate (4.5 mg, 0.02 mmol), AntPhos (11.1 mg, 0.03 mmol) and potassium phosphate (297 mg, 1.40 mmol) were added to the above system. The reaction system was replaced with argon three times and reacted at 140 °C for 16 hours under argon protection. The reaction solution was cooled to room temperature and water (15 mL) was added. Extracted with ethyl acetate (15 mL × 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate is concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product is purified by silica gel chromatography to obtain intermediate I-33.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.26 (s, 1H), 8.18 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 4.11 – 3.81 (m, 4H), 2.46 – 2.32 (m, 2H), 2.28 – 2.15 (m, 2H), 2.12 – 1.98 (m, 1H), 1.93 – 1.84 (m, 1H).

Reference Example 34: Preparation of Intermediate I-34 At 0°C, lithium aluminum tetrahydride (291.4 mg, 7.68 mmol) was added in batches to a solution of intermediate I-33 (660 mgl) in tetrahydrofuran (15 mL). The reaction mixture was reacted at room temperature for 16 hours. The reaction solution was cooled to 0°C and water (2 mL) was added to quench the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by reverse phase preparation to obtain intermediate I-34.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.64 – 7.49 (m, 3H), 4.72 (s, 2H), 3.94 – 3.81 (m, 1H), 2.41 – 2.29 (m, 2H), 2.25 – 2.12 (m, 2H), 2.09 – 1.96 (m, 1H), 1.92 – 1.80 (m, 1H).

Reference Example 35: Preparation of Intermediate I-35 At room temperature, triphenylphosphine (115 mg, 0.44 mmol) was added to a tetrahydrofuran solution (5 mL) of intermediate I-34 (50 mg, 0.22 mmol) and 2-fluoro-4-hydroxybenzaldehyde (46 mg, 0.33 mmol). The reaction system was replaced with argon three times. Under argon protection, diisopropyl azodicarboxylate (90 mg, 0.44 mmol) was slowly added dropwise to the above reaction solution at 0 °C and reacted at this temperature for 30 minutes. Then the reaction system was heated to room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-35.

LC-MS (ESI) [MH] ^- 351.1.

Reference Example 36: Preparation of Intermediate I-36 At room temperature, the intermediate I-35 (55.0 mg, 0.156 mmol) and methyl azocyclobutane-3-carboxylate hydrochloride (23.6 mg, 0.156 mmol) were dissolved in methanol (3 mL), and N,N-diisopropylethylamine (20.2 mg, 0.156 mmol) and acetic acid (18.7 mg, 0.311 mmol) were added to the above system in sequence. The reaction mixture was reacted at room temperature for 3 hours, and then sodium cyanoborohydride (14.7 mg, 0.234 mmol) was added to the reaction mixture, and the reaction was continued at room temperature for 16 hours. The reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain intermediate I-36, which was used directly in the next reaction without purification.

LC-MS (ESI) [M+H] ⁺ 452.3.

Reference Example 37: Preparation of Intermediate I-37 Dissolve methyl 4-hydroxy-3-(trifluoromethyl)benzoate (950 mg, 4.32 mmol) in N,N-dimethylformamide (15 mL), and add iodocyclopentane (1270 mg, 6.48 mmol) and cesium carbonate (4223 mg, 12.96 mmol) to the reaction system in sequence. Heat the reaction mixture to 45 °C and stir the reaction at this temperature for 16 hours. Cool the reaction solution to room temperature, add water (50 mL) and ethyl acetate (50 mL) in sequence, extract and separate the organic phase, extract the aqueous phase with ethyl acetate (50 mL × 2), combine the organic phases, wash with saturated brine (20 mL), dry over anhydrous sodium sulfate, and filter. The filtrate is concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-37.

LC-MS (ESI) [M+H] ⁺ 289.1.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.24 (d, J = 2.0 Hz, 1H), 8.15 (dd, J = 8.7, 2.1 Hz, 1H), 7.01 (d, J = 8.9 Hz, 1H), 4.96 – 4.92 (m, 1H), 3.91 (s, 3H), 1.95 – 1.92 (m, 2H), 1.92 – 1.88 (m, 2H),1.87 – 1.79 (m, 2H), 1.70 – 1.63 (m, 2H).

Reference Example 38: Preparation of Intermediate I-38 The intermediate I-37 (1.19 g, 4.13 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and DIBAL (5.51 mL, 8.26 mmol, 1.5M toluene solution) was added dropwise to the reaction mixture at -40°C. After the addition was completed, the reaction mixture was heated to room temperature and stirred at this temperature for 16 hours. Water (100 mL) and ethyl acetate (50 mL) were added in sequence, and the layers were extracted to separate the organic phase. The aqueous phase was extracted with ethyl acetate (50 mL × 2), and the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain the intermediate I-38.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.55 (d, J = 1.8 Hz, 1H), 7.45 (dd, J = 8.5, 1.8 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 4.89 – 4.85 (m, 1H), 4.64 (s, 2H), 1.92 – 1.89 (m, 2H), 1.88 – 1.84 (m, 2H), 1.84 – 1.76 (m, 2H), 1.65 – 1.61 (m, 2H).

Reference Example 39: Preparation of Intermediate I-39 Dissolve triphenylphosphine (606 mg, 2.31 mmol) in anhydrous tetrahydrofuran (15 mL) and add DIAD (467 mg, 2.31 mmol). The mixture is cooled to 0 °C and stirred at this temperature for 5 minutes, and then intermediate I-38 (400 mg, 1.54 mmol) and 2-fluoro-4-hydroxybenzaldehyde (216 mg, 1.54 mmol) are added in sequence. The reaction mixture is slowly raised to room temperature and stirred at this temperature for 16 hours. The reaction mixture is concentrated under reduced pressure to remove the organic solvent, and the residue is separated and purified by column chromatography (silica gel, ethyl acetate/petroleum ether = 0-30%) to obtain compound I-39.

LC-MS (ESI) [MH] ^- 381.0.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.21 (s, 1H), 7.84 (t, J = 8.4 Hz, 1H), 7.61 (d, J = 1.9 Hz, 1H), 7.50 (dd, J = 8.6, 2.0 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 6.85 (dd, J = 8.8, 2.2 Hz, 1H), 6.71 (dd, J = 12.3, 2.3 Hz, 1H),5.05 (s, 2H), 4.92 – 4.86 (m, 1H), 1.94 – 1.90 (m, 2H), 1.90 – 1.86 (m, 2H), 1.86 – 1.86 (m, 2H), 1.68 – 1.61 (m, 2H).

Reference Example 40: Preparation of Intermediate I-40 The intermediate I-39 (155 mg, 0.405 mmol) was dissolved in methanol (10.0 mL), and 3-methylformate azocyclobutane hydrochloride (61.4 mg, 0.405 mmol), DIEA (52.3 mg, 0.405 mmol), and HOAc (48.6 mg, 0.810 mmol) were added in sequence under stirring. The reaction mixture was stirred at room temperature for 4 hours, and then NaBH ₃ CN (38.2 mg, 0.608 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. Water (50 mL) and ethyl acetate (20 mL) were added in sequence, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (20 mL × 2) to obtain the product. The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-40.

LC-MS (ESI) [M+H] ⁺ 482.2.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.59 (d, J = 1.9 Hz, 1H), 7.50 (dd, J = 8.6, 1.9 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.02 – 6.98 (m, 1H), 6.75 (dd, J = 8.5, 2.4 Hz, 1H), 6.68 (dd, J = 11.6, 2.4 Hz, 1H), 4.96 (s, 2H), 4.90 – 4.86 (m, 1H), 3.77 – 3.73 (m, 2H), 3.73 (s, 3H), 3.72 – 3.68 (m, 2H), 3.52 (t, J = 7.9 Hz, 2H), 3.42 (dd, J = 15.4, 7.8 Hz, 1H), 1.94 – 1.91 (m, 2H), 1.90 – 1.88 (m, 2H), 1.84 – 1.81 (m, 2H), 1.66 – 1.61 (m, 2H).

Reference Example 41: Preparation of Intermediate I-41 Dissolve 2-fluoro-4-cyanobenzaldehyde (2.00 g, 13.41 mmol) and methyl azocyclobutane-3-carboxylate hydrochloride (4.07 g, 26.82 mmol) in methanol (30 mL). The temperature was lowered to 10°C, and then DIPEA (3.47 g, 26.82 mmol) and acetic acid (2.42 g, 40.23 mmol) were added in sequence. The reaction solution was stirred at 10°C for 2 hours, sodium cyanoborohydride (1.69 g, 26.82 mmol) was added and continued to stir for 1 hour, the reaction solution was poured into saturated sodium bicarbonate (100 mL), extracted with dichloromethane (50 mLx3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel chromatography to obtain intermediate I-41.

LC-MS (ESI) [M+H] ⁺ 249.2

Reference Example 42: Preparation of Intermediate I-42 The intermediate I-41 (1.00 g, 4.03 mmol) and Ni (100 mg) were added to acetic acid/water (3:2, 10 mL). Under argon protection, the reaction mixture was stirred at 50 °C for 72 hours. After the reaction solution was cooled to room temperature, it was filtered and the filtrate was extracted with ethyl acetate (30 mLx3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain the intermediate I-42.

LC-MS (ESI) [M+H] ⁺ 254.2.

Reference Example 43: Preparation of Intermediate I-43 3-Trifluoromethyl-4-bromophenol (1.0 g, 4.1 mmol) was dissolved in acetonitrile (20 mL), and benzyl bromide (1.05 g, 6.15 mmol), potassium carbonate (0.8 g, 5.79 mmol) and potassium iodide (66 mg, 0.4 mmol) were added in sequence. The reaction solution was stirred at 90°C for 10 hours. The reaction solution was cooled to room temperature, poured into 100 mL of water, and extracted with petroleum ether (30 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-43.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.78 (d, J = 8.8 Hz, 1H), 7.52 – 7.31 (m, 6H), 7.26 (dd, J = 8.8, 3.0 Hz, 1H), 5.21 (s, 2H).

Reference Example 44: Preparation of Intermediate I-44 Dissolve the intermediate I-43 (500 mg, 1.5 mmol) in a mixture of dioxane/water (4:1, 10 mL), add cyclopenteneboronic acid (203 mg, 1.8 mmol), tetrakistriphenylphosphine palladium (87 mg, 0.075 mmol) and potassium carbonate (621 mg, 4.5 mmol). Heat the reaction solution at 80 °C and stir for 10 hours under an argon atmosphere. Cool the reaction solution to room temperature, add 100 mL of water, and extract with ethyl acetate (20 mL×3). Combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, and filter. Concentrate the filtrate under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-44.

¹ HNMR (400 MHz, DMSO- d ₆ )7.64 – 7.13 (m, 8H), 5.75 – 5.57 (br.s, 1H), 5.20 (s, 2H), 2.63 – 2.54 (m, 2H), 2.49 – 2.42 (m, 2H), 2.00 – 1.90 (m, 2H).

Reference Example 45: Preparation of Intermediate I-45 Palladium carbon (20 mg) was added to an ethanol solution (10 mL) of intermediate I-44 (450 mg, 1.4 mmol), and heated and stirred at 80 °C for 10 hours under a hydrogen atmosphere. After cooling to room temperature, the reaction solution was filtered, and platinum dioxide (20 mg) was added to the filtrate. The reaction solution was heated and stirred at 80 °C for 10 hours under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was separated and purified by silica gel chromatography to obtain intermediate I-45.

¹ HNMR (400 MHz, DMSO- d ₆ ) δ 9.86 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.05 – 6.94 (m, 2H), 3.19 – 3.06 (m, 1H), 2.01 – 1.73 (m, 4H), 1.69 – 1.44 (m, 4H).

Reference Example 46: Preparation of Intermediate I-46 Intermediate I-42 (150 mg, 0.592 mmol), intermediate I-45 (136 mg, 0.592 mmol) and triphenylphosphine (186 mg, 0.710 mmol) were dissolved in anhydrous tetrahydrofuran (3 mL). After the reaction solution was cooled to 0 °C, it was stirred for 20 minutes under argon protection, and then DIAD (144 mg, 0.710 mmol) was slowly added dropwise. After the addition was completed, the reaction mixture was heated to room temperature and stirred for 2 hours. The mixture was concentrated to dryness, and the residue was separated and purified by silica gel chromatography to obtain intermediate I-46.

LC-MS (ESI) [M+H] ⁺ 466.1.

Reference Example 47: Preparation of Intermediate I-47 2-Furanboric acid (1.48 g, 13.25 mmol), methyl 3-trifluoromethyl-4-bromobenzoate (2.50 g, 8.83 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (644 mg, 0.88 mmol) and triethylamine (1.79 g, 17.66 mmol) were mixed in N,N-dimethylformamide (50 mL), and the reaction solution was stirred at 85 °C overnight under nitrogen protection. The reaction solution was cooled to room temperature and poured into water (500 mL), and extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-47.

¹ H NMR (400 MHz, Chloroform- d ) δ 8.42 (d, J = 1.7 Hz, 1H), 8.21 (dd, J = 8.3, 1.7 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 3.4 Hz, 1H), 6.55 (dd, J = 3.5, 1.8 Hz, 1H), 3.96 (s, 3H).

Reference Example 48: Preparation of Intermediate I-48 Intermediate I-47 (2.00 g, 7.40 mmol) and platinum dioxide (84 mg, 0.37 mmol) were mixed in ethyl acetate (20 mL). The reaction solution was stirred at 35 °C for 20 hours under an atmospheric pressure of hydrogen. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-48.

¹ H NMR (400 MHz, Chloroform- d ) δ 8.28 (s, 1H), 8.18 (dd, J = 8.2, 1.8 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 5.25 (t, J = 7.4 Hz, 1H), 4.22 – 4.16 (m, 1H), 4.00 – 3.94 (m, 1H), 3.93 (s, 3H), 2.43 (dq, J = 13.3, 6.8 Hz, 1H), 2.07 – 1.99 (m, 2H), 1.71 – 1.60 (m, 1H).

Reference Example 49: Preparation of Intermediate I-49 Dissolve the intermediate I-48 (380 mg, 1.39 mmol) in tetrahydrofuran (5 mL), cool the reaction system to -40 °C, and add a toluene solution of diisobutylaluminum hydroxide (1.5 M, 3.71 mL, 5.56 mmol) dropwise under argon protection. After the addition, stir the reaction solution at -40 °C for half an hour, and then stir at room temperature overnight. Cool the reaction solution to 0 °C, slowly quench with water (1 mL), pour the mixture into water (50 mL), and extract with ethyl acetate (20 mL× 3). Combine the organic phases, wash with saturated brine (30 mL), dry over anhydrous sodium sulfate, and filter. Concentrate the filtrate under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-49.

¹ H NMR (400 MHz, Chloroform- d ) δ 7.69 (d, J = 8.1 Hz, 1H), 7.62 (s, 1H), 7.54 (d, J = 8.1 Hz, 1H), 5.26 – 5.18 (m, 1H), 4.73 (s, 2H), 4.22 – 4.15 (m, 1H), 4.00 – 3.93 (m, 1H), 2.46 – 2.35 (m, 1H), 2.08 – 1.99 (m, 2H), 1.78 (s, 1H), 1.70 – 1.61 (m, 1H).

Reference Example 50: Preparation of Intermediate I-50 Intermediate I-49 (230 mg, 0.93 mmol), 2-fluoro-4-hydroxybenzaldehyde (157 mg, 1.12 mmol) and triphenylphosphine (341 mg, 1.30 mmol) were mixed in tetrahydrofuran (5 mL). After the reaction system was cooled to 0 °C, diisopropyl azodicarboxylate (263 mg, 1.30 mmol) was added. The reaction solution was stirred at 0 °C for half an hour, then raised to room temperature and stirred overnight at room temperature. The organic solvent was removed by decompression and concentration to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-50.

LC-MS (ESI) [M+H+41] ⁺ 410.2.

Reference Example 51: Preparation of Intermediate I-51 Dissolve the compound 3-furanboronic acid (1.97 g, 17.61 mmol) in DMF (30.0 mL), and add methyl 3-trifluoromethyl-4-bromobenzoate (3.84 g, 13.57 mmol), cesium carbonate (8.83 g, 27.10 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane complex (335 mg, 0.410 mmol) in sequence. The reaction solution was heated to 90 °C under argon protection and stirred for 16 hours. Water (60 mL) and ethyl acetate (60 mL) were added to the reaction solution, and the liquid was separated. The aqueous phase was extracted with ethyl acetate (60 mL x 2), and the organic phases were combined and washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness, and the crude product was separated and purified by silica gel chromatography to obtain intermediate I-51.

¹ H NMR (400 MHz, Chloroform- d ) δ 8.42 (d, J = 1.7 Hz, 1H), 8.19 (dd, J = 8.0, 1.7 Hz, 1H), 7.60 (d, J = 1.5 Hz, 1H), 7.54 – 7.48 (m, 2H), 6.62 – 6.56 (m, 1H), 3.97 (s, 3H).

Reference Example 52: Preparation of Intermediate I-52 Intermediate I-51 (3.11 g, 11.51 mmol) was dissolved in methanol (100 mL), Pd/C (500 mg, 10% w/w) was added, and the reaction mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere. The reaction solution was filtered and the filtrate was concentrated to dryness. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-52.

¹ H NMR (400 MHz, Chloroform- d ) δ 8.30 (d, J = 1.8 Hz, 1H), 8.20 – 8.15 (m, 1H), 7.62 (d, J = 8.3 Hz, 1H), 4.18 – 4.04 (m, 2H), 3.94 (s, 3H), 3.92 – 3.82 (m, 3H), 2.53 – 2.40 (m, 1H), 2.02 – 1.91 (m, 1H).

Reference Example 53: Preparation of Intermediate I-53 The intermediate I-52 (1.00 g, 3.65 mmol) was dissolved in anhydrous tetrahydrofuran (25 mL), cooled to -40 °C, and a toluene solution of diisobutylaluminum hydroxide (1.5 M, 9.73 mL, 14.60 mmol) was slowly added dropwise under the protection of argon. After the addition, the mixture was heated to room temperature and stirred for 2 hours. After the reaction was completed, the mixture was cooled to 0 °C, water (100 mL) was added to quench the reaction, ethyl acetate (50 mL) was added, and the liquids were separated. The aqueous phase was extracted with ethyl acetate (50 mL x 2), and the organic phases were combined, washed with brine (20 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent, and the residue was separated and purified by silica gel chromatography to obtain the intermediate I-53.

LC-MS (ESI) [MH] ^- 245.0.

Reference Example 54: Preparation of Intermediate I-54 The intermediate I-53 (123 mg, 0.500 mmol) was dissolved in dry tetrahydrofuran (2.00 mL), and 2-fluoro-4-hydroxybenzaldehyde (70.1 mg, 0.500 mmol) and triphenylphosphine (157 mg, 0.600 mmol) were added under argon protection. After stirring at room temperature for 20 minutes, the temperature was lowered to 0 °C, and diisopropyl azodicarboxylate (121 mg, 0.600 mmol) was slowly added dropwise. After the addition, the reaction solution was heated to room temperature and stirred for 3 hours. The reaction solution was reduced in pressure and concentrated to dryness. Ethyl acetate (10 mL) was added to the concentrate, and the mixture was washed with water (10 mLx3) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and dried, and then separated and purified by silica gel chromatography to obtain intermediate I-54.

LC-MS (ESI) [MH] ^- ：367.0

Reference Example 55: Preparation of Intermediate I-55 At 10 °C, methyl 4-bromo-3-(trifluoromethyl)benzoate (3.5 g, 12.37 mmol) and 3,6-dihydro- 2H -pyran-4-boronic acid pinacol ester (3.11 g, 14.8 mmol) were dissolved in dioxane/water (50 mL, 4/1), and then tetrakistriphenylphosphine palladium (710 mg, 0.614 mmol) and potassium carbonate (5.1 g, 36.9 mmol) were added. The reaction mixture was stirred at 110 °C for 3 hours under argon protection. Water (200 mL) was added to the reaction solution, and it was extracted with petroleum ether (50 mL × 4). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude intermediate I-55 product. The crude product was directly used in the next reaction without purification.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J = 1.4 Hz, 1H), 8.16 (dd, J = 8.0, 1.4 Hz, 1H), 7.35 (t, J = 6.6 Hz, 1H), 5.69 (s, 1H), 4.28 (q, J = 2.7 Hz, 2H), 3.95 (s, 3H), 3.92 (t, J = 5.4 Hz, 2H), 2.36 (dt, J = 7.2, 2.5 Hz, 2H).

Reference Example 56: Preparation of Intermediate I-56 At 18 °C, the intermediate I-55 (3.5 g, 12.23 mmol) was dissolved in ethanol (80 mL), and palladium carbon (100 mg, 10% w/w) was slowly added. The reaction mixture was stirred at 60 °C and 10 atmospheres for 72 hours. After the reaction solution was cooled to room temperature, the palladium carbon in the mixture was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain the target intermediate I-56.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.31 (s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 4.10 (dd, J = 11.5, 4.1 Hz, 2H), 3.94 (s, 3H), 3.56 (td, J = 11.8, 1.7 Hz, 2H), 3.25 (t, J = 11.8 Hz, 1H), 1.88 (dd, J = 12.6, 4.2 Hz, 2H), 1.75 – 1.67 (m, 2H).

Reference Example 57: Preparation of Intermediate I-57 The intermediate I-56 (2.0 g, 6.94 mmol) was dissolved in tetrahydrofuran, and diisobutylaluminum hydroxide solution (13.9 mL, 1.5 M toluene solution) was slowly added dropwise to the above mixture at -60 °C. After the reaction mixture was stirred at -30 °C for 3 hours, the reaction mixture was poured into dilute hydrochloric acid (100 mL, 2 N) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium bicarbonate (30 mL), then washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-57.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.64 (s, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 4.71 (s, 2H), 4.07 (dd, J = 11.5, 4.2 Hz, 2H), 3.54 (td, J = 11.9, 1.6 Hz, 2H), 3.18 (t, J = 11.8 Hz, 1H), 2.03 (d, J = 4.8 Hz, 1H), 1.85 (qd, J = 12.4, 4.3 Hz, 2H), 1.69 (d, J = 10.6 Hz, 2H).

Reference Example 58: Preparation of Intermediate I-58 At 10 °C, the intermediate I-57 (200 mg, 0.768 mmol) and 2-fluoro-4-hydroxybenzaldehyde (161 mg, 1.15 mmol) were dissolved in tetrahydrofuran (3 mL), and triphenylphosphine (404 mg, 1.54 mmol) and diisopropyl azodicarboxylate (311 mg, 1.54 mmol) were added in sequence. The reaction mixture was stirred at 30 °C for 5 hours. The reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (5 mL × 4). The organic phases were combined and washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-58.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.22 (s, 1H), 7.85 (t, J = 8.4 Hz, 1H), 7.69 (s, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 6.86 (dd, J = 8.8, 2.3 Hz, 1H), 6.72 (dd, J = 12.2, 2.3 Hz, 1H), 5.13 (s, 2H), 4.09 (dd, J = 11.6, 4.3 Hz, 2H), 3.56 (td, J = 11.9, 1.9 Hz, 2H), 3.21 (t, J = 11.9 Hz, 1H), 1.87 (dd, J = 12.6, 4.1 Hz, 2H), 1.72 (dd, J = 12.9, 1.8 Hz, 2H).

Reference Example 59: Preparation of Intermediate I-59 At 10 °C, the intermediate I-58 (90.0 mg, 0.235 mmol) was dissolved in methanol (3 mL), and 3-methylformate azocyclobutane hydrochloride (71.2 mg, 0.470 mmol), diisopropylethylamine (60.7 mg, 0.470 mmol) and glacial acetic acid (42.3 mg, 0.705 mmol) were added in sequence. The reaction mixture was stirred at 10 °C for 2 hours. Sodium cyanoborohydride (29.5 mg, 0.470 mmol) was added to the reaction system. The reaction mixture was stirred at 10 °C for 1 hour. The reaction mixture was poured into a saturated sodium bicarbonate solution (20 mL) and extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-59.

LC-MS (ESI) [M+H] ⁺ 482.2.

Reference Example 60: Preparation of Intermediate I-60 3,5-Difluoropicolinic acid (3.10 g, 19.5 mmol) was dissolved in aqueous lithium hydroxide solution (2N, 97.5 mL, 195 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 16 hours. After the reaction solution was cooled to room temperature, concentrated hydrochloric acid (12 M) was added to adjust the pH value of the reaction mixture to 4. The reaction mixture was concentrated under reduced pressure to remove the organic solvent to obtain a crude product.

At room temperature, the crude product was dissolved in methanol (60 mL), and concentrated sulfuric acid (5.00 mL) was added dropwise. After the addition was completed, the reaction mixture was stirred at 70 °C for 16 hours. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure to remove the organic solvent to obtain a concentrate. Water (100 mL) was added to the concentrate, and it was extracted with ethyl acetate (80 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-60.

LC-MS (ESI) [M+H] ⁺ 171.9.

Reference Example 61: Preparation of Intermediate I-61 At room temperature, the intermediate I-60 (500 mg, 2.92 mmol) was dissolved in acetonitrile (20 mL). The intermediate I-13 (1.08 g, 3.52 mmol) and cesium carbonate (2.85 g, 8.75 mmol) were added in sequence. After the addition was completed, the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain the intermediate I-61.

LC-MS (ESI) [M+H] ⁺ 398.1.

Reference Example 62: Preparation of Intermediate I-62 At 0 °C, a solution of intermediate I-61 (998 mg, 2.51 mmol) in anhydrous tetrahydrofuran (10 mL) was added dropwise to a suspension of lithium aluminum hydroxide in tetrahydrofuran (5.00 mL, 5.00 mmol, 1M). After the addition was complete, the reaction mixture was stirred at 0 °C for 1 hour. The reaction solution was quenched with saturated ammonium chloride (40 mL) at 0 °C and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-62.

LC-MS (ESI) [M+H] ⁺ 370.0.

Reference Example 63: Preparation of Intermediate I-63 At room temperature, the intermediate I-62 (200 mg, 0.541 mmol) was dissolved in dichloromethane (10 mL), and Dess-Martin oxidant (413 mg, 0.974 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-63.

LC-MS (ESI) [M+H] ⁺ 368.0.

Reference Example 64: Preparation of Intermediate I-64 At room temperature, the intermediate I-63 (158 mg, 0.430 mmol) was dissolved in methanol (3 mL) and dichloromethane (3 mL), and methyl 3-nitroaniline hydrochloride (65.2 mg, 0.430 mmol), triethylamine (43.5 mg, 0.430 mmol) and acetic acid (51.6 mg, 0.859 mmol) were added in sequence. The reaction mixture was stirred at 40 °C for 5 hours, and then sodium cyanoborohydride (54.0 mg, 0.859 mmol) was added and continued to stir at 40 °C for 2 hours. After the reaction mixture was cooled to room temperature, the organic solvent was removed and concentrated under reduced pressure to obtain a concentrate. Add water (20 mL) to the concentrate and extract with dichloromethane (10 mL × 3). Combine the organic phases, wash with saturated brine (20 mL), dry over anhydrous sodium sulfate, and filter. The filtrate is concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product is separated and purified by silica gel chromatography to obtain intermediate I-64.

LC-MS (ESI) [M+H] ⁺ 467.1.

Reference Example 65: Preparation of Intermediate I-65 Intermediate I-13 (200 mg, 0.65 mmol), 4-hydroxy-2-methoxybenzaldehyde (99 mg, 0.65 mmol) and potassium carbonate (180 mg, 1.30 mmol) were mixed in acetonitrile (5 mL), and the reaction solution was stirred at 50 °C overnight. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-65.

LC-MS (ESI) [M+H] ⁺ 379.2.

Reference Example 66: Preparation of Intermediate I-66 At room temperature, methyl 2,4-dihydroxybenzoate (4.50 g, 26.8 mmol) and intermediate I-13 (8.23 g, 26.8 mmol) were dissolved in acetone (50 mL), and potassium carbonate (7.41 g, 53.6 mmol) was added to the above system. The reaction system was stirred at 50 °C for 4 hours. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with water (60 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-66.

LC-MS (ESI) [MH] ^- 393.0.

Reference Example 67: Preparation of Intermediate I-67 At 0 °C, triethylamine (1.02 g, 10.1 mmol) was added to a dichloromethane (30 mL) solution of the intermediate I-66 (2.00 g, 5.07 mmol), and then trifluoromethanesulfonic anhydride (2.15 g, 7.62 mmol) was slowly added to the above reaction system. The reaction mixture was warmed to room temperature and stirred at room temperature for 3 hours. The reaction solution was diluted with dichloromethane (30 mL), and washed with water (30 mL) and saturated brine (30 mL) in sequence, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by silica gel chromatography to obtain the intermediate I-67.

LC-MS (ESI) [MH] ^- 525.2.

Reference Example 68: Preparation of Intermediate I-68 At room temperature, tetrakis(triphenylphosphine)palladium (164 mg, 0.142 mmol) and sodium carbonate (453 mg, 4.27 mmol) were added to a mixed solution of intermediate I-67 (750 mg, 1.42 mmol) and isopropenylboronic acid pinacol ester (958 mg, 5.70 mmol) in dioxane and water (24 mL, 5:1). The reaction system was replaced with argon three times. The reaction was carried out at 90 °C for 3 hours under argon protection. The reaction solution was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate is concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product is purified by silica gel chromatography to obtain intermediate I-68.

LC-MS (ESI) [M+H] ⁺ 419.2.

Reference Example 69: Preparation of Intermediate I-69 At room temperature, intermediate I-68 (594 mg, 1.42 mmol) was dissolved in methanol (10 mL) and platinum dioxide (60.0 mg) was added. The reaction mixture was replaced with hydrogen three times and stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-69.

LC-MS (ESI) [M+H] ⁺ 421.2.

Reference Example 70: Preparation of Intermediate I-70 At -30 °C, DIBAL-H (3.78 mL, 1 M in hexane, 3.78 mmol) was added to a tetrahydrofuran (15 mL) solution of intermediate I-69 (530 mg, 1.26 mmol). The reaction solution was continued to react at this temperature for 2 hours. After the reaction was completed, it was quenched with water (10 mL) and sodium hydroxide aqueous solution (10 mL, 1 M) at 0 °C. Filter, and extract the filtrate with ethyl acetate (10 mL × 3). Combine the organic phases, wash the organic phases with saturated brine (20 mL), dry over anhydrous sodium sulfate, and filter. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain intermediate I-70.

LC-MS (ESI) [MH] ^- 391.3.

Reference Example 71: Preparation of Intermediate I-71 Under argon protection, a mixed solution of intermediate I-70 (494 mg, 1.26 mmol) and manganese dioxide (548 mg, 6.30 mmol) in 1,2-dichloroethane (20 mL) was reacted at 70°C for 5 hours. The mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-71.

LC-MS (ESI) [M+H] ⁺ 391.2.

Reference Example 72: Preparation of Intermediate I-72 At room temperature, the intermediate I-71 (120 mg, 0.307 mmol) and methyl azocyclobutane-3-carboxylate hydrochloride (93.1 mg, 0.614 mmol) were dissolved in methanol (10 mL), and N,N-diisopropylethylamine (79.4 mg, 0.614 mmol) and acetic acid (55.3 mg, 0.921 mmol) were added to the above system in sequence. The reaction mixture was reacted at room temperature for 8 hours. Then sodium cyanoborohydride (38.6 mg, 0.614 mmol) was added to the reaction mixture, and the reaction was continued at 50 ° C for 16 hours. The reaction solution was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (15 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel chromatography to obtain intermediate I-72.

LC-MS (ESI) [M+H] ⁺ 490.4.

Reference Example 73: Preparation of Intermediate I-73 At room temperature, tetrakis(triphenylphosphine)palladium (49.2 mg, 0.0426 mmol) and sodium carbonate (452 mg, 4.26 mmol) were added to a mixed solution of intermediate I-67 (750 mg, 1.42 mmol) and cyclopropylboronic acid (366 mg, 4.26 mmol) in dioxane and water (36 mL, 5:1). The reaction system was replaced with argon three times. The reaction was carried out at 100 °C for 5 hours under argon protection. The reaction solution was cooled to room temperature. Water (20 mL) was added to dilute and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate is concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product is purified by silica gel chromatography to obtain intermediate I-73.

LC-MS (ESI) [MH] ^- 417.2.

Reference Example 74: Preparation of Intermediate I-74 At -30 °C, DIBAL-H (3.72 mL, 1 M in hexane, 3.72 mmol) was added to a tetrahydrofuran (10 mL) solution of intermediate I-73 (520 mg, 1.24 mmol). The reaction mixture was allowed to react at this temperature for 1 hour. After the reaction was completed, the mixture was quenched with water (5 mL) and aqueous sodium hydroxide solution (5 mL, 1 M) at 0 °C. The filtrate was filtered and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the intermediate I-74, which was directly used in the next reaction without further purification.

LC-MS (ESI) [M+H-18] ⁺ 373.2.

Reference Example 75: Preparation of Intermediate I-75 Under argon protection, a mixed solution of intermediate I-74 (484 mg, 1.24 mmol) and manganese dioxide (539 mg, 6.20 mmol) in 1,2-dichloroethane (15 mL) was reacted at 70°C for 3 hours. The mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-75.

LC-MS (ESI) [M+H] ⁺ 389.2.

Reference Example 76: Preparation of Intermediate I-76 At room temperature, the intermediate I-75 (200 mg, 0.515 mmol) and methyl azocyclobutane-3-carboxylate hydrochloride (156 mg, 1.03 mmol) were dissolved in methanol (10 mL), and N,N-diisopropylethylamine (133 mg, 1.03 mmol) and acetic acid (93.1 mg, 1.55 mmol) were added to the above system in sequence. The reaction mixture was reacted at room temperature for 5 hours. Then sodium cyanoborohydride (64.7 mg, 1.03 mmol) was added to the reaction mixture, and the reaction was continued at 50°C for 16 hours. The reaction solution was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (15 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel chromatography to obtain intermediate I-76.

LC-MS (ESI) [M+H] ⁺ 488.3.

Reference Example 77: Preparation of Intermediate I-77 At room temperature, the intermediate I-66 (700 mg, 1.77 mmol) was dissolved in acetonitrile (10 mL), and 2-iodopropane (3.0 g, 17.6 mmol) and cesium carbonate (1.73 g, 5.31 mmol) were added in sequence. The reaction mixture was stirred at 45 °C overnight. The organic solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain the intermediate I-77.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.82 (d, J = 9.1 Hz, 1H), 7.65 (s, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 6.58 – 6.53 (m, 2H), 5.07 (s, 2H), 4.56 – 4.47 (m, 1H), 3.84 (s, 3H), 3.42 – 3.32 (m, 1H), 2.14 – 2.05 (m, 2H), 1.89 – 1.58 (m, 6H), 1.36 (d, J = 6.1 Hz, 6H).

Reference Example 78: Preparation of Intermediate I-78 At -45 °C, the intermediate I-77 (770 mg, 1.76 mmol) was dissolved in tetrahydrofuran (5 mL), and diisobutylaluminum hydroxide (7 mL, 1 M in hexane, 7.00 mmol) was added. The reaction mixture was stirred at -45 °C for 1.5 hours. At 0 °C, the reaction was quenched by adding a saturated aqueous ammonium chloride solution (20 mL). Ethyl acetate (60 mL) was added for separation and extraction, and the aqueous phase was extracted with ethyl acetate (60 mL × 2) to obtain an organic phase. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-78.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.66 (s, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 6.55 – 6.52 (m, 1H), 6.49 (dd, J = 8.2, 2.3 Hz, 1H), 5.03 (s, 2H), 4.60 (s, 2H), 4.58 – 4.50 (m, 1H), 3.42 – 3.32 (m, 1H), 2.14 – 2.06 (m, 2H), 1.90 – 1.59 (m, 6H), 1.35 (d, J = 6.1 Hz, 6H).

Reference Example 79: Preparation of Intermediate I-79 At room temperature, the intermediate I-78 was dissolved in 1,2-dichloroethane (6 mL), and manganese dioxide (426 mg, 4.9 mmol) was added. The reaction mixture was stirred at 70 °C overnight under argon protection, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product I-79. The crude product was used directly in the next reaction without further purification.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.32 (s, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.66 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 6.61 – 6.57 (m, 1H), 6.51 (s, 1H), 5.10 (s, 2H), 4.64 – 4.57 (m, 1H), 3.42 – 3.34 (m, 1H), 2.15 – 2.07 (m, 2H), 1.92 – 1.61 (m, 6H), 1.38 (d, J = 6.0 Hz, 6H).

Reference Example 80: Preparation of Intermediate I-80 At room temperature, the intermediate I-79 and methyl cyclobutane-3-carboxylate hydrochloride (59 mg, 0.39 mmol) were dissolved in methanol (3 mL), and N,N-diisopropylethylamine (50 mg, 0.39 mmol) and glacial acetic acid (47 mg, 0.78 mmol) were added in sequence. After the reaction mixture was stirred at room temperature for 6 hours, sodium cyanoborohydride (49 mg, 0.78 mmol) was added to the reaction system and continued to stir at room temperature overnight. Water (10 mL) and ethyl acetate (30 mL) were added to the reaction system for separation and extraction, and the aqueous phase was extracted with ethyl acetate (30 mL × 2) to obtain an organic phase. The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product I-80. The crude product was directly used in the next reaction without further purification.

LC-MS (ESI) [M+H] ⁺ 506.6

Reference Example 81: Preparation of Intermediate I-81 4-Bromo-2-chlorophenol (5.00 g, 24.1 mmol), imidazole (4.11 g, 60.3 mmol) and triisopropylchlorosilane (5.57 g, 28.9 mmol) were dissolved in DCM (40.0 mL). The reaction mixture was stirred at 25 °C for 10 hours. The reaction solution was diluted with dichloromethane (200 mL) and washed with saturated brine (300 mL). The organic phase was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-81.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.51 (d, J = 2.4 Hz, 1H), 7.30 (dd, J = 8.8, 2.4 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 1.33 (m, 3H), 1.13 (d, J = 7.4 Hz, 18H).

Reference Example 82: Preparation of Intermediate I-82 Dissolve diisopropylamine (612 mg, 6.05 mmol) in anhydrous tetrahydrofuran (20.0 mL), cool to -78 °C under nitrogen, then slowly add n-BuLi (3.61 mL, 1.6 M in hexane, 5.78 mmol). Stir and react at this temperature for 0.75 hours. Then, slowly add intermediate I-81 (2.00 g, 5.50 mmol) to the system at -78 °C, and stir and react at this temperature for 0.5 hours. Finally, slowly add iodomethane (937 mg, 6.60 mmol) to the system at -78 °C. The reaction solution slowly rises to room temperature and stirs overnight. The reaction mixture was quenched with saturated ammonium chloride solution (15.0 mL), and then saturated brine (150 mL) was added and extracted with ethyl acetate (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-82.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.28 (d, J = 8.8 Hz, 1H), 6.65 (d, J = 8.8 Hz, 1H), 2.50 (s, 3H), 1.30 (m, 3H), 1.12 (d, J = 7.4 Hz, 18H).

Reference Example 83: Preparation of Intermediate I-83 Intermediate I-82 (1.00 g, 2.65 mmol) was dissolved in anhydrous tetrahydrofuran (15.0 mL). Under nitrogen protection, it was cooled to -78 °C, and then n-butyl lithium (1.99 mL, 1.6 M n-hexane solution, 3.18 mmol) was slowly added dropwise. The reaction was stirred at this temperature for 1 hour. Anhydrous N, N-dimethylformamide (290.6 mg, 3.975 mmol) was slowly added dropwise to the solution at -78 °C, and the reaction was stirred at this temperature for 2 hours before quenching the reaction with saturated ammonium chloride solution (10.0 mL). Water (40 mL) was added, and extracted with ethyl acetate (60 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-83.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.14 (s, 1H), 7.62 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 2.74 (s, 3H), 1.35 (m, 3H), 1.13 (d, J = 7.4 Hz, 18H).

Reference Example 84: Preparation of Intermediate I-84 Intermediate I-83 (650 mg, 1.99 mmol) and TBAF (1.04 g, 3.98 mmol) were dissolved in anhydrous tetrahydrofuran (15.0 mL) and reacted at 25 °C for 10 hours. The reaction solution was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-84.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.12 (s, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 2.74 (s, 3H).

Reference Example 85: Preparation of Intermediate I-85 Intermediate I-84 (80.0 mg, 0.469 mmol), intermediate I-13 (173 mg, 0.563 mmol) and potassium carbonate (195 mg, 1.41 mmol) were dissolved in acetonitrile (5.00 mL), the temperature was raised to 80°C, and the reaction was carried out at this temperature for 10 hours. The reaction solution was cooled to room temperature, water (20.0 mL) was added and extracted with ethyl acetate (15 mL × 2). The organic phases were combined, washed with saturated brine (10.0 mL), dried with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-85.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.15 (s, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.69 (s, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 5.22 (s, 2H), 3.38 (m, 1H), 2.76 (s, 3H), 2.10 (m, 2H), 1.86 (m, 2H), 1.74 (m, 2H), 1.62 (m, 2H).

Reference Example 86: Preparation of Intermediate I-86 Intermediate I-85 (80.0 mg, 0.202 mmol), 3-azacyclobutanecarboxylic acid methyl ester hydrochloride (30.6 mg, 0.202 mmol) and DIEA (26.1 mg, 0.202 mmol) were dissolved in methanol (3.00 mL), and then acetic acid (24.26 mg, 0.404 mmol) was added. The atmosphere was replaced with nitrogen and the reaction was carried out at 25°C for 3 hours. Then sodium cyanoborohydride (12.7 mg, 0.202 mmol) was added and the reaction was continued at 25°C for 10 hours. The reaction solution was added to water (10.0 mL) and extracted with ethyl acetate (15.0 mL × 2). The organic phases were combined and dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the intermediate I-86. The crude product was used directly in the next reaction without purification.

LC-MS (ESI) [M+H] ⁺ 496.2

Reference Example 87: Preparation of Intermediate I-87 Dissolve 2-methyl-4-bromobenzoic acid (5.00 g, 23.3 mmol) in N,N-dimethylformamide (50.0 mL), and add chlorosuccinimide (3.11 g, 23.3 mmol) and sodium acetate (523 mg, 2.33 mmol) in sequence. Heat the reaction solution to 100 °C and stir for 24 hours. After cooling the reaction solution to room temperature, add water (200 mL), and extract the mixture with ethyl acetate (40 mL*3). Combine the organic phases, wash with saturated brine (40 mL), dry over anhydrous sodium sulfate, and filter. The filtrate is concentrated under reduced pressure to dryness, and the residue is separated and purified by silica gel chromatography to obtain intermediate I-87.

¹ H NMR (400 MHz, Methanol- d ₄ ) δ 7.49 (s, 1H), 7.43 – 7.41 (s, 1H), 2.34 (s, 3H).

Reference Example 88: Preparation of Intermediate I-88 Dissolve the intermediate I-87 (4.50 g) in N,N-dimethylformamide (40.0 mL), and add iodomethane (5.11 g, 36.0 mmol) and potassium carbonate (7.46 g, 54.0 mmol) in sequence. Heat the reaction solution to 40 °C and stir for 2 hours. Add water (200 mL) at 40 °C, and extract the mixture with ethyl acetate (40 mL*3). Combine the organic phases, wash with saturated brine (40 mL), dry over anhydrous sodium sulfate, and filter. Concentrate the filtrate under reduced pressure to dryness, and separate and purify the residue by silica gel chromatography to obtain the intermediate I-88.

¹ H NMR (400 MHz, Chloroform- d ) δ 7.41 (s, 1H), 7.29 – 7.27 (s, 1H), 3.94 (s, 3H), 2.30 (s, 3H).

Reference Example 89: Preparation of Intermediate I-89 The intermediate I-88 (3.30 g, 12.5 mmol) was dissolved in dimethyl sulfoxide (40.0 mL), and pinacol diboron (4.14 g, 16.3 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.915 g, 1.25 mmol) and potassium acetate (3.68 g, 37.5 mmol) were added in sequence. The reaction solution was heated to 100 °C and stirred for 8 hours, then cooled to room temperature, water (50 mL) was added, and extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was separated and purified by silica gel chromatography to obtain intermediate I-89.

¹ H NMR (400 MHz, Chloroform- d ) δ 7.64 (s, 1H), 7.52 (s, 1H), 3.95 – 3.93 (s, 3H), 2.31 (s, 3H), 1.26 (s, 12H).

Reference Example 90: Preparation of Intermediate I-90 Dissolve the intermediate I-89 (3.31 g, 10.7 mmol) in tetrahydrofuran (30 mL), add sodium hydroxide solution (1 M, 10.7 mL, 10.7 mmol), and slowly add hydrogen peroxide (30% w/w, 2.41 g, 21.2 mmol) while stirring. Heat the reaction solution to 40 °C and stir for 1 hour, then add water (50 mL) at 40 °C, extract with ethyl acetate (50 mL*3), combine the organic phases, wash with saturated brine (30 mL), dry over anhydrous sodium sulfate, filter, reduce the pressure and concentrate the filtrate to dryness, and separate the residue by silica gel chromatography to obtain the intermediate I-90.

LC-MS (ESI) [MH] ^- 199.0.

Reference Example 91: Preparation of Intermediate I-91 The intermediate I-90 (602 mg, 3.00 mmol) was dissolved in acetonitrile (10.0 mL), and the intermediate I-13 (1.38 g, 4.50 mmol) and cesium carbonate (1.95 g, 6.00 mmol) were added in sequence. The reaction solution was heated to 40 °C and stirred for 4 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to dryness. The residue was separated and purified by silica gel chromatography to obtain the intermediate I-91.

LC-MS (ESI) [MH] ^- 425.1 .

Reference Example 92: Preparation of Intermediate I-92 Dissolve the intermediate I-91 (996 mg, 2.33 mmol) in tetrahydrofuran (10.0 mL), cool to -40 °C, slowly add DIBAL-H in n-hexane (1 M, 6.99 mL) under argon protection, warm to room temperature and continue stirring for 4 hours. Pour the reaction solution into 1 N glacial hydrochloric acid solution (50 mL), extract with ethyl acetate (40 mL*3), combine the organic phase, wash with saturated brine (40 mL), dry with anhydrous sodium sulfate, filter, reduce the pressure and concentrate the filtrate to dryness to obtain the intermediate I-92, which is directly used in the next reaction.

LC-MS (ESI) [MH] ^- 397.1.

Reference Example 93: Preparation of Intermediate I-93 Dissolve the intermediate I-92 (720 mg, 1.81 mmol) in dichloroethane (5.00 mL) and add manganese dioxide (785 mg, 9.03 mmol). Heat the reaction solution to 60 °C and stir for 5 hours, then filter under hot conditions, reduce pressure and concentrate the filtrate to dryness. Separate and purify the residue by silica gel chromatography to obtain the intermediate I-93.

LC-MS (ESI) [MH] ^- 395.1.

Reference Example 94: Preparation of Intermediate I-94 The intermediate I-93 (140 mg, 0.353 mmol) and acrilic acid methyl ester hydrochloride (161 mg, 1.06 mmol) were dissolved in methanol (3.00 mL), glacial acetic acid (0.50 mL) was added, the reaction solution was heated to 50 °C, stirred for 20 hours under argon protection, sodium cyanoborohydride (88.7 mg, 1.41 mmol) was added, and continued to stir at 50 °C for 2 hours. The reaction solution was cooled to room temperature, water (5 mL) was added to the mixture, extracted with ethyl acetate (20 mL x 3), the organic phases were combined, concentrated under vacuum, and separated and purified by silica gel chromatography to obtain the intermediate I-94.

LC-MS (ESI) [M+H] ⁺ 496.2.

Reference Example 95: Preparation of Intermediate I-95 At 0 °C, add 60% sodium hydroxide (2.17 g, 54.2 mmol) to a solution of methyl 4-methylsalicylate (6.00 g, 36.1 mmol) in tetrahydrofuran (100 mL). The reaction mixture was stirred at 10 °C for 30 minutes. At 10 °C, iodomethane (7.69 g, 54.2 mmol) was added to the reaction mixture. After the addition was completed, the reaction mixture was heated to 20 °C and stirred for 8 hours. The reaction mixture was quenched with saturated ammonium chloride (400 mL) and extracted with ethyl acetate (100 mL × 4). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate is concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product is separated and purified by silica gel chromatography to obtain intermediate I-95.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.72 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 6.8 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 2.38 (s, 3H).

Reference Example 96: Preparation of Intermediate I-96 At room temperature, N-bromosuccinimide (2.95 g, 16.6 mmol) and benzoyl peroxide (40.2 mg, 1.66 mmol) were added to a solution of intermediate I-95 (3.00 g, 16.6 mmol) in carbon tetrachloride (30.0 mL). The reaction mixture was stirred at 80 °C for 8 hours. The reaction system was cooled to room temperature, poured into water (100 mL), and extracted with ethyl acetate (50 mL × 4). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-96.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.77 (d, J = 8.3 Hz, 1H), 7.00 (d, J = 1.6 Hz, 2H), 4.46 (s, 2H), 3.93 (s, 3H), 3.89 (s, 3H).

Reference Example 97: Preparation of Intermediate I-97 At room temperature, intermediate I-96 (900 mg, 3.47 mmol) and intermediate I-45 (879 mg, 3.82 mmol) were dissolved in N,N -dimethylformamide (15.0 mL), and then cesium carbonate (3.39 g, 10.4 mmol) was added. The reaction mixture was stirred at 50 °C for 3 hours. The reaction system was cooled to room temperature, poured into water (50 mL), and extracted with ethyl acetate (20 mL × 4). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-97.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.82 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 7.20 (d, J = 2.8 Hz, 1H), 7.13 – 6.98 (m, 3H), 5.09 (s, 2H), 3.92 (s, 3H), 3.89 (s, 3H), 3.38 – 3.24 (m, 1H), 2.13 – 1.98 (m, 2H), 1.89 – 1.64 (m, 4H), 1.53 (m, 2H).

Reference Example 98: Preparation of Intermediate I-98 At 0 °C, the intermediate I-97 (700 mg, 1.71 mmol) was dissolved in tetrahydrofuran (15.0 mL), and then lithium aluminum tetrahydrate (71.3 mg, 1.88 mmol) was added at 0 °C. The reaction mixture was heated to 10 °C and stirred for 2 hours. Water/tetrahydrofuran (71 mg/20 mL) was added to the reaction system, the system was stirred at room temperature for 10 minutes, sodium hydroxide solution (71 mg, 10% w/w) was added, the mixture was stirred at room temperature for 30 minutes, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-98.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.36 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 2.8 Hz, 1H), 7.08 (dd, J = 8.7, 2.7 Hz, 1H), 7.02 – 6.94 (m, 2H), 5.05 (s, 2H), 4.69 (d, J = 6.4 Hz, 2H), 3.89 (s, 3H), 3.37 – 3.23 (m, 1H), 2.11 – 2.01 (m, 2H), 1.83 (m, 2H), 1.71 (m, 2H), 1.51 (m, 2H).

Reference Example 99: Preparation of Intermediate I-99 At room temperature, intermediate I-98 (520 mg, 1.37 mmol) was dissolved in dichloroethane (15.0 mL), and then manganese dioxide (956 mg, 11.0 mmol) was added. The reaction mixture was stirred at 80 °C for 3 hours. The reaction system was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-99.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.45 (s, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.20 (d, J = 2.6 Hz, 1H), 7.08 (m, 3H), 5.11 (s, 2H), 3.95 (s, 3H), 3.38 – 3.23 (m, 1H), 2.05 (s, 2H), 1.83 (s, 2H), 1.76 – 1.66 (m, 2H), 1.55 – 1.47 (m, 2H).

Reference Example 100: Preparation of Intermediate I-100 To a methanol solution (2.0 mL) of intermediate I-99 (150 mg, 0.396 mmol), 3-acridinic acid formate methyl hydrochloride (120 mg, 0.792 mmol), diisopropylethylamine (102 mg, 0.792 mmol) and glacial acetic acid (71.5 mg, 1.19 mmol) were added respectively. After stirring at room temperature for 1 hour, sodium triacetoxyborohydride (168 mg, 0.792 mmol) was added to the above reaction system, and then stirring was continued for 8 hours. Water (10 mL) was poured into the above reaction system, and extracted with ethyl acetate (5 mL × 4). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate is concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product is separated and purified by silica gel chromatography to obtain intermediate I-100.

LC-MS (ESI) [M+H] ⁺ 478.2.

Reference Example 101: Preparation of Intermediate I-101 The intermediate I-38 (200 mg, 0.768 mmol) was dissolved in tetrahydrofuran (10.0 mL). 4-Hydroxy-2-methoxy-benzaldehyde (140 mg, 0.922 mmol), triphenylphosphine (302 mg, 1.15 mmol) and DIAD (233 mg, 1.15 mmol) were added to the reaction system at 0 °C. The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel chromatography to obtain the intermediate I-101.

LC-MS (ESI) [M+H] ⁺ 395.2.

Reference Example 102: Preparation of Intermediate I-102 The intermediate I-31 (1.00 g, 3.87 mmol) was mixed in a 40% aqueous solution of hydrobromic acid (10 mL), and the reaction mixture was stirred at 100 °C for 2 hours. The reaction mixture was cooled to room temperature and extracted with dichloromethane (10 mL × 2). The organic phases were combined, washed with a saturated aqueous sodium bicarbonate solution (20 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain the intermediate I-102.

¹ H NMR (400 MHz, Chloroform- d ) δ 7.61 (d, J = 1.9 Hz, 1H), 7.51 (dd, J = 8.1, 2.0 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 4.48 (s, 2H), 2.98 – 2.86 (m, 1H), 1.90 – 1.73 (m, 5H), 1.49 – 1.25 (m, 5H).

Reference Example 103: Preparation of Intermediate I-103 4-Hydroxy-2-methoxy-benzaldehyde (109 mg, 0.716 mmol) was dissolved in acetonitrile (10 mL). Intermediate I-102 (230 mg, 0.716 mmol) and potassium carbonate (198 mg, 1.43 mmol) were added to the reaction system in sequence. The reaction mixture was stirred at 45 °C for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel chromatography to obtain intermediate I-103.

LC-MS (ESI) [M+H] ⁺ 393.6.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.18 (s, 1H), 7.77 (s, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 5.8 Hz, 1H), 7.66 (d, J = 5.2 Hz, 1H), 6.81 (d, J = 2.2 Hz, 1H), 6.75 (dd, J = 8.6, 1.8 Hz, 1H), 5.28 (s, 2H), 3.90 (s, 3H), 2.82 (t, J = 11.4 Hz, 1H), 1.85 – 1.78 (m, 2H), 1.74 – 1.66 (m, 3H), 1.59 – 1.48 (m, 2H), 1.41 – 1.28 (m, 3H).

Preparation of the embodiment:

Example 1: Preparation of Compound 1 Intermediate I-4 (93 mg, 0.25 mmol) and 3-carboxylic acid cyclobutylamine (51 mg, 0.5 mmol) were suspended in a mixture of tetrahydrofuran and methanol (5 mL, 1:1), sodium cyanoborohydride (47 mg, 0.75 mmol) and acetic acid (0.2 mL) were added, and the reaction solution was stirred at room temperature overnight. Most of the reaction solution was evaporated, and water (10 mL) and ethyl acetate (30 mL) were added to the residue to separate the organic phase, and the aqueous layer was extracted with ethyl acetate (30 mL × 2). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by preparative HPLC to obtain compound 1.

LC-MS (ESI) [M+H] ⁺ 452.1.

¹ H NMR (400 MHz, MeOH- d ₄ ) δ 7.69 – 7.57 (m, 3H), 7.46 – 7.41 (m, 1H), 6.98 – 6.90 (m, 2H), 5.17 (s, 2H), 4.42 (s, 2H), 4.36 – 4.23 (m, 4H), 3.66 – 3.59 (m, 1H), 3.39 – 3.34 (m, 1H), 2.10 – 2.02 (m, 2H), 1.95 – 1.85 (m, 2H), 1.79 – 1.70 (m, 2H), 1.68 – 1.59 (m, 2H).

Example 2: Preparation of Compound 2 Intermediate I-12 (110 mg, 0.293 mmol) and 3-carboxylic acid cyclobutylamine (44.3 mg, 0.439 mmol) were suspended in a mixture of tetrahydrofuran (18 mL) and methanol (18 mL), acetic acid (0.01 mL) was added and the reaction solution was stirred at room temperature for 16 hours. Sodium cyanoborohydride (55.4 mg, 0.882 mmol) was added and the stirring was continued at room temperature for 3 hours. 50 mL of water was added to the reaction solution and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by preparative HPLC to obtain compound 2.

LC-MS (ESI) [M+H] ⁺ 462.2.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.75 – 7.67 (m, 2H), 7.67 – 7.61 (m, 1H), 7.14 (d, J = 8.4 Hz, 1H), 6.85 – 6.80 (m, 1H), 6.79 – 6.73 (m, 1H), 5.11 (s, 2H), 3.46 (s, 2H), 3.28 – 3.22 (m, 3H), 3.18 – 3.11 (m, 3H), 2.61 (q, J = 7.2 Hz, 2H), 2.04 – 1.95 (m, 2H), 1.88 – 1.79 (m, 2H), 1.71 – 1.56 (m, 4H), 1.13 (t, J = 7.5 Hz, 3H).

Example 3: Preparation of Compound 3 Intermediate I-14 (210 mg, 0.60 mmol) and 3-carboxylic acid cyclobutylamine (60 mg, 0.60 mmol) were suspended in a mixture of tetrahydrofuran and methanol (1:4, 20 mL), acetic acid (1 mL) was added and the reaction solution was stirred at room temperature for 2 hours. Sodium cyanoborohydride (226 mg, 3.6 mmol) was added and stirring was continued for 16 hours. The reaction solution was evaporated to dryness, ethyl acetate (10 mL) and water (10 mL) were added to the residue, the organic layer was separated, and the residue was concentrated. The residue was purified by preparative HPLC to obtain compound 3.

LC-MS (ESI) [M+H] ⁺ 434.3.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.62 (s, 1H), 7.55 – 7.44 (m, 2H), 7.36 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 5.00 (s, 2H), 4.30 – 3.90 (m, 6H), 3.48 – 3.30 (m, 2H), 2.15 – 1.99 (m, 2H), 1.92 – 1.77 (m, 2H), 1.78 – 1.65 (m, 2H), 1.64 – 1.52 (m, 2H).

Example 4: Preparation of Compound 4 Intermediate I-15 (100 mg, 0.26 mmol) and 3-cyclobutylamine carboxylate (53 mg, 0.52 mmol) were suspended in a mixture of tetrahydrofuran and methanol (9 mL, 1:3). Sodium cyanoborohydride (193 mg, 3.12 mmol) and acetic acid (0.5 mL) were added, and the reaction solution was stirred at room temperature for two days. 0.5 mL of hydrochloric acid (2N) was added to the reaction solution, and compound 4 was obtained after preparative HPLC purification.

LC-MS (ESI) [M+H] ⁺ 468.1.

¹ H NMR (400 MHz, MeOH- d ₄ ) δ 7.68 (s, 1H), 7.66 – 7.62 (m, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.21 (d, J = 2.5 Hz, 1H), 7.06 (dd, J = 8.6, 2.6 Hz, 1H), 5.16 (s, 2H), 4.41 (s, 2H), 4.24 – 4.12 (m, 4H), 3.44 – 3.35 (m, 2H), 2.10 – 2.02 (m, 2H), 1.94 – 1.86 (m, 2H), 1.78 – 1.70 (m, 2H), 1.68 – 1.59 (m, 2H).

Example 5: Preparation of Compound 5 Intermediate I-16 (180 mg, 0.50 mmol) and 3-carboxylic acid cyclobutylamine (50.5 mg, 0.50 mmol) were suspended in a mixture of tetrahydrofuran and methanol (1:1, 10 mL), and acetic acid (0.5 mL) was added. After stirring at room temperature for 2 hours, sodium cyanoborohydride (94.5 mg, 1.50 mmol) was added, and the reaction solution was stirred at room temperature for 16 hours. 20 mL of water and 20 mL of ethyl acetate were added, and the organic layer was separated by extraction, and the aqueous layer was further extracted with ethyl acetate (20 mL × 2). The combined organic layers were washed with saturated brine and concentrated to obtain a residue. The residue was purified by preparative HPLC to obtain compound 5.

LC-MS (ESI) [M+H] ⁺ 448.3.

H NMR (400 MHz, MeOH- d ₄ ) δ 7.66 (s, 1H), 7.65 - 7.55 (m, 2H), 7.32 (d, J = 8.5 Hz, 1H), 6.96 (d, J = 2.5 Hz, 1H), 6.91 (dd, J = 8.4, 2.6 Hz, 1H), 5.13 (s, 2H), 4.35 (s, 2H), 4.25 – 4.13 (m, 4H), 3.45 – 3.33 (m, 2H), 2.40 (s, 3H), 2.11 – 2.02 (m, 2H), 1.95 – 1.85 (m, 2H), 1.79 – 1.59 (m, 4H).

Example 6: Preparation of Compound 6 Intermediate I-18 (55.0 mg, 0.15 mmol) and 3-carboxylic acid cyclobutylamine (22.2 mg, 0.22 mmol) were suspended in a mixture of tetrahydrofuran and methanol (5 mL/5 mL). Sodium cyanoborohydride (27.7 mg, 0.44 mmol) and acetic acid (3 drops) were added, and the reaction solution was stirred at room temperature for 16 hours. The pH of the reaction solution was adjusted to 3 with hydrochloric acid (1N), and the reaction solution was evaporated to dryness. The residue was purified by preparative HPLC to obtain compound 6.

LC-MS (ESI) [M+H] ⁺ 459.2.

¹ H NMR (400 MHz, MeOH- d ₄ ) δ 7.69 (s, 1H), 7.67 – 7.63 (m, 1H), 7.62 – 7.55 (m, 2H), 7.49 (d, J = 2.7 Hz, 1H), 7.38 (dd, J = 8.7, 2.7 Hz, 1H), 5.20 (s, 2H), 4.31 (s, 2H), 4.14 – 4.00 (m, 4H), 3.45 – 3.35 (m, 2H), 2.12 – 2.03 (m, 2H), 1.95 – 1.86 (m, 2H), 1.79 – 1.70 (m, 2H), 1.69 – 1.59 (m, 2H).

Example 7: Preparation of Compound 7 The intermediate I-26 (30 mg, crude product) and lithium hydroxide monohydrate (5.34 mg, 127.35 μmol) were dissolved in tetrahydrofuran (0.500 mL), methanol (0.500 mL) and water (0.200 mL). The reaction mixture was stirred at 25 °C for 10 hours, and then the reaction solution was directly reduced in pressure and concentrated to remove the organic solvent and water to obtain a crude product. The crude product was purified by preparative HPLC to obtain compound 7

LC-MS (ESI) [M+H] ⁺ 453.2.

¹ H NMR (400 MHz, CD ₃ OD) δ 8.82 (s, 1H), 8.10 (s, 1H), 7.46 (m, 1H), 7.09 – 6.88 (m, 2H), 5.23 (s, 2H), 4.34 (s, 2H), 4.16 (m, 4H), 3.57-3.46 (m, 1H), 3.44-3.37 (m,1H), 2.11 - 1.96 (m, 2H), 1.96 -1.82 (m, 4H),1.81-1.65(m, 2H).

Example 8: Preparation of Compound 8 At room temperature, the intermediate I-28 (140 mg, 0.38 mmol) and azocyclobutane-3-carboxylic acid methyl ester hydrochloride (58 mg, 0.38 mmol) were dissolved in methanol (3 mL), and N,N-diisopropylethylamine (49 mg, 0.38 mmol) and glacial acetic acid (46 mg, 0.766 mmol) were added in sequence. After the reaction mixture was stirred at room temperature for 6 hours, sodium cyanoborohydride (48 mg, 0.76 mmol) was added to the reaction system, and stirring was continued at room temperature overnight. Tetrahydrofuran/water (2 mL, 4:1) was added to the reaction solution, and lithium hydroxide monohydrate (32.0 mg, 0.76 mmol) was added. After the reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure to obtain a crude product, which was purified by preparative HPLC to obtain compound 8.

LC-MS (ESI) [M+H] ⁺ 450.2

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.66 (s, 1H), 7.59 – 7.39 (m, 2H), 7.32 – 7.27 (m, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.72 (d, J = 11.0 Hz, 1H), 5.71 (s, 1H), 5.01 (s, 2H), 4.51 – 3.94 (m, 6H), 3.62 – 3.49 (m, 1H), 2.68 – 2.57 (m, 2H), 2.55 – 2.44 (m, 2H), 2.08 – 1.93 (m, 2H).

Example 9: Preparation of Compound 9 At room temperature, the intermediate I-32 (90.0 mg, 0.24 mmol), 3-azacarboxylic acid (121 mg, 1.20 mmol) and glacial acetic acid (18.7 mg, 0.312 mmol) were dissolved in tetrahydrofuran (2.00 mL) and methanol (2.00 mL). Under nitrogen protection, the reaction mixture was stirred at 50°C for 10 hours. NaBH ₃ CN (60 mg, 0.96 mmol) was added, and then the reaction was continued to stir at 25°C for 5 hours. The reaction solution was directly adjusted to pH 5-6 with 1N hydrochloric acid solution, and then concentrated under reduced pressure to remove organic solvents and water to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 9.

LC-MS (ESI) [M+H] ⁺ 466.2.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.69 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.46 (t, J = 8.5 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 5.17 (s, 2H), 4.53 – 4.22 (m, 6H), 3.74 – 3.63 (m, 1H), 2.93 (m, 1H), 1.92 – 1.82 (m, 2H), 1.81 – 1.74 (m, 2H), 1.60 – 1.49 (m, 2H), 1.46 – 1.30 (m, 4H).

Example 10: Preparation of Compound 10 At room temperature, a solution of lithium hydroxide monohydrate (16.7 mg, 0.399 mmol) in water (1 mL) was added to a solution of intermediate I-36 (60.0 mg, 0.133 mmol) in tetrahydrofuran (3 mL). The reaction mixture was stirred at room temperature for 3 hours, and then the pH was adjusted to 4-6 with dilute hydrochloric acid (1 M). The reaction mixture was filtered through a filter membrane and purified by preparative HPLC to obtain compound 10.

LC-MS (ESI) [M+H] ⁺ 438.2.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.75 – 7.66 (m, 3H), 7.44 (t, J = 8.4 Hz, 1H), 6.97 – 6.93 (m, 2H), 5.18 (s, 2H), 4.34 (s, 2H), 4.17 (d, J = 8.4 Hz, 4H), 3.95 – 3.86 (m, 1H), 3.44 – 3.35 (m, 1H), 2.40 – 2.29 (m, 2H), 2.29 – 2.19 (m, 2H), 2.12 – 2.00 (m, 1H), 1.94 – 1.87 (m, 1H).

Example 11: Preparation of Compound 11 The intermediate I-40 (137 mg, 0.285 mmol) was dissolved in tetrahydrofuran (1.00 mL) and methanol (1.00 mL), and a solution of lithium hydroxide monohydrate (23.9 mg, 0.570 mmol) in water (1.00 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, and the pH value of the reaction mixture was adjusted to 6.0 with dilute hydrochloric acid (1 N). Ethyl acetate (10.0 mL) was added to extract the liquid, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (10.0 mL × 2), and the organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 11.

LC-MS (ESI) [M+H] ⁺ 468.2.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.66 – 7.57 (m, 2H), 7.42 (t, J = 8.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.98 – 6.88 (m, 2H), 5.09 (s, 2H), 4.99 – 4.95 (m, 1H), 4.36 (s, 2H), 4.19 (d, J = 8.4 Hz, 4H), 3.46 – 3.38 (m, 1H), 1.99 – 1.74 (m, 6H), 1.74 – 1.60 (m, 2H).

Example 12: Preparation of Compound 12 The intermediate I-46 (150 mg, 0.322 mmol) was dissolved in tetrahydrofuran (3.00 mL), and a solution of lithium hydroxide monohydrate (40.5 mg, 0.966 mmol) in water (0.5 mL) was added. The reaction mixture was stirred at room temperature for 2 hours, and then dilute hydrochloric acid (1 M) was added to adjust the pH value of the reaction mixture to 5. The reaction mixture was extracted with ethyl acetate (5 mL × 2), and the organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and filtered. The organic solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 12.

LC-MS (ESI) [M+H] ⁺ 452.2.

¹ H NMR (400 MHz, Methanol- d ₄ ) δ 7.57 – 7.44 (m, 2H), 7.40 – 7.29 (m, 2H), 7.24 – 7.16 (m, 2H), 5.18 (s, 2H), 4.40 (s, 2H), 4.17 (d, J = 8.3 Hz, 4H), 3.41 (m, 1H), 3.28 (m, 1H), 2.09 – 1.95 (m, 2H), 1.94 – 1.79 (m, 2H), 1.77 – 1.64 (m, 2H), 1.66 – 1.49 (m, 2H).

Example 13: Preparation of Compound 13 At room temperature, intermediate I-4 (100 mg, 0.27 mmol) and piperidine-4-carboxylic acid (70 mg, 0.54 mmol) were added to a mixed solvent of 1,2-dichloroethane/tetrahydrofuran (2.5 mL, 4:1), and glacial acetic acid (0.5 mL) was added. The reaction mixture was stirred at 50 °C for 2 hours and then cooled to room temperature. Sodium acetate borohydride (170 mg, 0.81 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature overnight. The organic solvent was removed by concentration under reduced pressure to obtain a crude product, and dilute hydrochloric acid (2N, 1 mL) was added. Compound 13 was obtained by separation and purification using preparative HPLC.

LC-MS (ESI) [M+H] ⁺ 480.6.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.63 (s, 1H), 7.60 – 7.44 (m, 3H), 6.80 (dd, J = 8.6, 2.3 Hz, 1H), 6.70 (dd, J = 11.5, 2.4 Hz, 1H), 5.03 (s, 2H), 4.00 (s, 2H), 3.42 – 3.33 (m, 1H), 3.33 – 3.19 (m, 2H), 2.70 – 2.46 (m, 2H), 2.40 – 2.29 (m, 1H), 2.19 – 2.04 (m, 4H), 2.04 - 1.91 (m, 2H), 1.91 – 1.81 (m, 2H), 1.80 – 1.68 (m, 2H), 1.66 – 1.54 (m, 2H).

Example 14: Preparation of Compound 14 The intermediate I-4 (183 mg, 0.50 mmol) was dissolved in 1,2-dichloroethane (2.50 mL), and 3-piperidine carboxylate (65.0 mg, 0.50 mmol) and acetic acid (0.100 mL) were added in sequence. The reaction solution was heated to 50 °C and stirred for 16 h. Sodium cyanoborohydride (158 mg, 2.50 mmol) was added and stirred for 4 h. The reaction solution was concentrated to dryness under reduced pressure, and methanol (5 mL) was added to dissolve it and purified by preparative HPLC to obtain compound 14.

LC-MS (ESI) [M+H] ⁺ 480.3.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.68 (s, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.45 (t, J = 8.7 Hz, 1H), 6.97 – 6.89 (m, 2H), 5.16 (s, 2H), 4.18 (s, 2H), 3.41 – 3.34 (m, 1H), 3.26 – 2.94 (br.s, 4H), 2.67 – 2.55 (m, 1H), 2.11 – 2.01 (m, 2H), 1.98 – 1.84 (m, 4H), 1.84 – 1.68 (m, 4H), 1.68 – 1.59 (m, 2H).

Example 15: Preparation of Compound 15 Intermediate I-50 (100 mg, 0.271 mmol), 3-acridinecarboxylic acid (82.5 mg, 0.816 mmol) and acetic acid (0.5 mL) were mixed in a mixed solvent of tetrahydrofuran (15 mL) and methanol (15 mL), and the reaction solution was stirred at 40 °C for 3 hours. Then sodium cyanoborohydride (51.3 mg, 0.816 mmol) was added, and the reaction solution was stirred at 40 °C overnight. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 15.

LC-MS (ESI) [M+H] ⁺ 454.1.

¹ H NMR (400 MHz, MeOH- d ₄ ) δ 7.81 – 7.68 (m, 3H), 7.45 (t, J = 8.4 Hz, 1H), 7.01 – 6.92 (m, 2H), 5.26 – 5.15 (m, 3H), 4.36 (s, 2H), 4.27 – 4.11 (m, 5H), 3.99 – 3.93 (m, 1H), 3.45 – 3.38 (m, 1H), 2.46 – 2.37 (m, 1H), 2.15 – 2.01 (m, 2H), 1.74 – 1.65 (m, 1H).

Example 16: Preparation of Compound 16 The intermediate I-54 (100 mg, 0.271 mmol) was dissolved in methanol/tetrahydrofuran (1:1, 10 mL), 3-azacarboxylic acid (82.2 mg, 0.813 mmol) and glacial acetic acid (0.500 mL) were added, the reaction solution was heated to 40 °C and stirred overnight, then sodium cyanoborohydride (51.1 mg, 0.813 mmol) was added and stirred at 40 °C for 2 hours, and glacial acetic acid (1 mL) was added to quench the excess sodium borohydride. The reaction solution was filtered and purified by preparative HPLC to obtain compound 16.

LC-MS (ESI) [M+H] ⁺ 454.2.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.73 (s, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 8.4 Hz, 1H), 6.98 – 6.91 (m, 2H), 5.18 (s, 2H), 4.35 (s, 2H), 4.18 (d, J = 8.4 Hz, 4H), 4.14 – 4.09 (m, 1H), 4.08 – 4.02 (m, 1H), 3.93 – 3.87 (m, 1H), 3.83 – 3.73 (m, 2H), 3.43 – 3.36 (m, 1H), 2.48 – 2.39 (m, 1H), 2.05 – 1.96 (m, 1H).

Example 17: Preparation of Compound 17 At 10 °C, the intermediate I-59 (60.0 mg, 0.125 mmol) was dissolved in ethanol/water (1 mL/1 mL), and lithium hydroxide monohydrate (15.7 mg, 0.375 mmol) was added. The reaction mixture was stirred at 10 °C for 2 hours. The reaction solution was directly separated and purified by preparative HPLC to obtain compound 17.

LC-MS (ESI) [M+H] ⁺ 468.2.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.80 – 7.66 (m, 3H), 7.25 (t, J = 8.6 Hz, 1H), 6.88 (dd, J = 12.0, 2.3 Hz, 1H), 6.82 (dd, J = 8.4, 2.3 Hz, 1H), 5.16 (s, 2H), 3.97 (dd, J = 11.2, 3.7 Hz, 2H), 3.48 – 3.40 (m, 4H), 3.33 (t, J = 6.7 Hz, 2H), 3.17 – 3.04 (m, 4H), 1.88 – 1.76 (m, 2H), 1.62 – 1.54 (m, 2H).

Example 18: Preparation of Compound 18 At room temperature, the intermediate I-64 (70.0 mg, 0.150 mmol) was dissolved in tetrahydrofuran (2 mL) and methanol (0.5 mL), and a solution of lithium hydroxide monohydrate (12.6 mg, 0.300 mmol) in water (0.5 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, and then dilute hydrochloric acid (1 M) was added to adjust the pH value of the reaction mixture to 5. The reaction mixture was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 18.

LC-MS (ESI) [M+H] ⁺ 453.2.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.19 (d, J = 1.8 Hz, 1H), 7.76 – 7.69 (m, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.51 (dd, J = 11.6, 2.3 Hz, 1H), 5.24 (s, 2H), 3.60 (d, J = 2.1 Hz, 2H), 3.39 (t, J = 7.5 Hz, 2H), 3.26 (m, 3H), 3.19 – 3.09 (m, 1H), 2.06 – 1.94 (m, 2H), 1.90 – 1.78 (m, 2H), 1.73 – 1.53 (m, 4H).

Example 19: Preparation of Compound 19 Intermediate I-65 (250 mg, 0.66 mmol), cyclobutane-3-carboxylic acid (200 mg, 1.98 mmol) and acetic acid (1 mL) were mixed in a mixed solvent of methanol and tetrahydrofuran (60 mL, 1:1). The reaction mixture was stirred at 40 °C for 3 hours. Then, a methanol (2 mL) solution of sodium cyanoborohydride (124 mg, 1.98 mmol) was added to the reaction system, and stirring was continued at 40 °C overnight. The organic solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 19.

LC-MS (ESI) [M+H] ⁺ 464.2.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.70 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H), 6.67 (dd, J = 8.4, 2.2 Hz, 1H), 5.17 (s, 2H), 4.38 -4.19 (m, 6H), 3.91 (s, 3H), 3.44-3.33 (m, 2H), 2.13-2.03 (m, 2H), 1.97-1.86 (m, 2H), 1.81-1.56 (m, 4H).

Example 20: Preparation of Compound 20 At room temperature, intermediate I-4 (100 mg, 0.27 mmol) and pyrrolidine-3-carboxylic acid (62 mg, 0.54 mmol) were added to a mixed solvent of 1,2-dichloroethane/tetrahydrofuran (2 mL, 4:1), and glacial acetic acid (0.5 mL) was added. The reaction mixture was stirred at 50°C for 7 hours and then cooled to room temperature. Sodium acetate borohydride (170 mg, 0.81 mmol) was added at 0°C. The reaction mixture was stirred at room temperature overnight, and the organic solvent was removed by concentration under reduced pressure to obtain a crude product. 2N dilute hydrochloric acid (1 mL) was added to the crude product, and compound 20 was obtained by separation and purification by preparative HPLC.

LC-MS (ESI) [M+H] ⁺ 466.3.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.73 – 7.67 (m, 2H), 7.65 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 8.6 Hz, 1H), 6.88 (dd, J = 11.9, 2.4 Hz, 1H), 6.83 (dd, J = 8.4, 2.4 Hz, 1H), 5.15 (s, 2H), 3.53 (s, 2H), 3.29 – 3.21 (m, 1H), 2.90 – 2.84 (m, 1H), 2.64 – 2.72 (m, 1H), 2.60 – 2.54 (m, 1H), 2.49 – 2.39 (m, 2H), 2.04 – 1.96 (m, 2H), 1.95 – 1.88 (m, 2H), 1.88 – 1.78 (m, 2H), 1.72 – 1.56 (m, 4H).

Example 21: Preparation of Compound 21 Under ice bath, lithium hydroxide monohydrate (12.8 mg, 0.305 mmol) in water (1 mL) was added to a tetrahydrofuran solution (3 mL) of intermediate I-72 (50.0 mg, 0.102 mmol). The reaction mixture was stirred at room temperature for 3 hours, and then the pH was adjusted to 5-6 with dilute hydrochloric acid (1 M). The reaction mixture was filtered through a filter membrane and purified by preparative HPLC to obtain compound 21.

LC-MS (ESI) [M+H] ⁺ 476.3.

¹ H NMR (400 MHz, CD ₃ OD) δ 7.70 (s, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 2.6 Hz, 1H), 6.92 (dd, J = 8.5, 2.7 Hz, 1H), 5.17 (s, 2H), 4.38 (s, 2H), 4.16 (d, J = 8.4 Hz, 4H), 3.43 – 3.35 (m, 2H), 3.23 – 3.16 (m, 1H), 2.12 – 2.04 (m, 2H), 1.96 – 1.86 (m, 2H), 1.80 – 1.69 (m, 2H), 1.69 -1.56 (m, 2H), 1.26 (d, J = 6.8 Hz, 6H).

Example 22: Preparation of Compound 22 Under an ice-water bath, a solution of lithium hydroxide monohydrate (11.6 mg, 0.276 mmol) in water (1 mL) was added to a solution of intermediate I-76 (45.0 mg, 0.0923 mmol) in tetrahydrofuran (3 mL). The reaction mixture was stirred at room temperature for 3 hours, and then the pH was adjusted to 5-6 with dilute hydrochloric acid (1 M). The reaction mixture was filtered through a filter membrane and purified by preparative HPLC to obtain compound 22.

LC-MS (ESI) [M+H] ⁺ 474.3.

¹ H NMR (400 MHz, CD ₃ OD) δ7.67 (s, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 6.90 (dd, J = 8.5, 2.6 Hz, 1H), 6.69 (d, J = 2.6 Hz, 1H), 5.13 (s, 2H), 4.52 (s, 2H), 4.21 (d, J = 8.3 Hz, 4H), 3.46 – 3.35 (m, 2H), 2.16 – 2.00 (m, 3H), 1.98 – 1.86 (m, 2H), 1.81 – 1.58 (m, 4H), 1.14 – 1.02 (m, 2H), 0.77 – 0.65 (m, 2H).

Example 23: Preparation of Compound 23 At room temperature, the crude intermediate I-80 (190 mg, 0.38 mmol) was dissolved in tetrahydrofuran/water (2 mL, 4:1), and lithium hydroxide monohydrate (32.0 mg, 0.76 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure to obtain a crude product. Compound 23 was obtained by separation and purification using preparative HPLC.

LC-MS (ESI) [M+H] ⁺ 492.3.

¹ H NMR (400 MHz,CD ₃ OD) δ7.69 (s, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 2.2 Hz, 1H), 6.65 (dd, J = 8.4, 2.3 Hz, 1H), 5.17 (s, 2H), 4.75 – 4.66 (m, 1H), 4.27 (s, 2H), 4.25 – 4.15 (m, 4H), 3.43 – 3.34 (m, 2H), 2.13 – 2.03 (m, 2H), 1.97 – 1.85 (m, 2H), 1.80 – 1.69 (m, 2H),1.69 - 1.57 (m, 2H), 1.37 (d, J = 6.0 Hz, 6H).

Example 24: Preparation of Compound 24 Intermediate I-86 (90.0 mg, 181.46 μmol) and lithium hydroxide monohydrate (22.84 mg, 554.38 μmol) were dissolved in tetrahydrofuran (3.00 mL) and water (0.500 mL). The reaction mixture was stirred at 25°C for 10 hours. The reaction solution was directly concentrated under reduced pressure to remove the organic solvent and water to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 24.

LC-MS (ESI) [M+H] ⁺ 482.2.

¹ H NMR (400 MHz, CD ₃ OD) δ7.74 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 5.24 (s, 2H), 4.40 (s, 2H), 4.16 (d, J = 8.3 Hz, 4H), 3.46 – 3.34 (m, 2H), 2.47 (s, 3H), 2.15 – 2.00 (m, 2H), 1.97 – 1.83 (m, 2H), 1.81 – 1.55 (m, 4H).

Example 25: Preparation of Compound 25 Intermediate I-94 (55.7 mg, 0.112 mmol) was dissolved in methanol (2.00 mL), and lithium hydroxide monohydrate (14.1 mg, 0.336 mmol) and water (0.500 mL) were added. The reaction mixture was stirred at 30 °C for 2 hours. The reaction solution was separated and purified by preparative HPLC to obtain compound 25.

LC-MS (ESI) [M+H] ⁺ 482.2.

¹ H NMR (400 MHz, Methanol- d ₄ ) δ 7.67 (s, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 2.7 Hz, 1H), 6.99 (d, J = 2.5 Hz, 1H), 5.15 (s, 2H), 4.63 (s, 2H), 4.42 (d, J = 8.6 Hz, 4H), 3.76 – 3.65 (m, 1H), 3.41 – 3.33 (m, 1H), 2.48 (s, 3H), 2.14 – 2.00 (m, 2H), 1.97 – 1.83 (m, 2H), 1.82 – 1.67 (m, 2H), 1.70 – 1.56 (m, 2H)

Example 26: Preparation of Compound 26 At room temperature, the intermediate I-100 (100 mg, 0.209 mmol) was dissolved in methanol/water (1 mL/1 mL), and lithium hydroxide monohydrate (17.5 mg, 0.418 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The product was separated and purified by preparative HPLC to obtain Example 26.

LC-MS (ESI) [M+H] ⁺ 464.2.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.52 (d, J = 8.7 Hz, 1H), 7.27 (dd, J = 8.7, 2.4 Hz, 1H), 7.24 – 7.17 (m, 2H), 7.05 (s, 1H), 6.98 (d, J = 7.6 Hz, 1H), 5.11 (s, 2H), 3.78 (s, 3H), 3.50 (s, 2H), 3.42 – 3.37 (m, 2H), 3.22 – 3.12 (m, 4H), 2.00 – 1.90 (m, 2H), 1.87 – 1.76 (m, 2H), 1.70 – 1.49 (m, 4H).

Example 27: Preparation of Compound 27 The intermediate I-101 (150 mg, 0.380 mmol) was dissolved in tetrahydrofuran/anhydrous methanol (1:1, 40.0 mL), and 3-azacarboxylic acid (154 mg, 1.52 mmol) and glacial acetic acid (0.500 mL) were added in sequence. The reaction mixture was stirred at 40 °C for 16 hours. Sodium cyanoborohydride (71.6 mg, 1.14 mmol) was added, and the reaction mixture was stirred at 40 °C for 16 hours. Water (80 mL) was added to quench the reaction and then extracted with ethyl acetate (30.0 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain compound 27.

LC-MS (ESI) [M+H] ⁺ 480.3.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.70 – 7.65 (m, 2H), 7.27 (d, J = 9.2 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.60 (d, J = 2.2 Hz, 1H), 6.55 (dd, J = 8.3, 2.2 Hz, 1H), 5.05 (s, 2H), 5.04 – 5.00 (m, 1H), 3.74 (s, 3H), 3.43 (s, 2H), 3.38 – 3.33 (m, 2H), 3.20 – 3.10 (m, 3H), 1.96 – 1.85 (m, 2H), 1.80 – 1.60 (m, 6H).

Example 28: Preparation of Compound 28 The intermediate I-103 (120 mg, 0.306 mmol) was dissolved in tetrahydrofuran/anhydrous methanol (1:1, 40.0 mL), and 3-azacarboxylic acid (92.8 mg, 0.918 mmol) and glacial acetic acid (0.500 mL) were added in sequence. The reaction mixture was stirred at 40 °C for 16 hours. Sodium cyanoborohydride (57.7 mg, 0.918 mmol) was added, and the reaction mixture was stirred at 40 °C for 16 hours. Water (80 mL) was added to quench the reaction and then extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by preparative HPLC to give compound 28.

LC-MS (ESI) [M+H] ⁺ 478.3.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.73 (s, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.62 (d, J = 2.1 Hz, 1H), 6.55 (dd, J = 8.3, 2.1 Hz, 1H), 5.12 (s, 2H), 3.75 (s, 3H), 3.44 (s, 2H), 3.39 – 3.33 (m, 2H), 3.21 – 3.11 (m, 3H), 2.82 (m, 1H), 1.86 – 1.78 (m, 2H), 1.75 – 1.66 (m, 3H), 1.59 – 1.48 (m, 2H), 1.40 – 1.27 (m, 3H).

Experimental Example 1: S1P1-mediated cAMP inhibition

The cell line used in the test is PathHunter® CHO-K1 EDG1 β-Arrestin Cell Line, supplier: DiscoverX, catalog number: 93-0207C2. The test evaluates the inhibitory effect of compounds on S1P1-mediated forskolin-induced cAMP activity.

For each test, different concentrations of compounds and a final concentration of 0.6 μM forskolin were added to the test plate wells and centrifuged at 1000 rpm for 10 seconds. Take a tube of frozen cells, wash twice with HBSS buffer, and resuspend. Add 5000 cells per well to the test plate, shake for 20 seconds, centrifuge at 1000 rpm for 10 seconds, and incubate at room temperature for 60 minutes. Add anti-cAMP-Eu2+-Cryptate and cAMP-d2, shake for 20 seconds, centrifuge at 1000 rpm for 10 seconds, incubate at room temperature for 60 minutes, and read the plate with Envision. The data were analyzed by nonlinear regression to determine the EC ₅₀ of the compound's inhibition of forskolin-induced cAMP. The experimental results are shown in Table 1. Table 1: Activation of S1P1 receptor-mediated cAMP inhibition by compounds Experimental data show that the compounds of the present invention exhibit good activation properties on the S1P1-mediated cAMP inhibition.

Experimental Example 2: S1P1-mediated activation of β-Arrestin reporter gene

The cell line used in the test is PathHunter® CHO-K1 EDG1 β-Arrestin Cell Line, supplier: DiscoverX, catalog number: 93-0207C2. The experimental operation was carried out according to the supplier's instructions. The cells were added to the test plate with 25μL of cell suspension containing 5000 cells per well and incubated at 37°C for 20 hours. 10 concentrations of 4-fold dilutions of the compound were added to the cell culture medium and incubated at 37°C for 90 minutes. The test solution was prepared, 12μL per well, incubated at room temperature for 60 minutes, and the plate was read by Envision. The data was analyzed by nonlinear regression to determine the EC ₅₀ of β-arrestin activity. The experimental results are shown in Table 2. Table 2: Activation effect of compounds on S1P1 receptor-mediated β-arrestin activity Experimental data show that the compound of the present invention exhibits a good activation effect on S1P1-mediated β-arrestin.

Experimental Example 3: Internalization effect experiment of the compound of the present invention on S1P1 receptor

1. CHO-K1 DEG1 cells (PathHunter® CHO-K1 EDG1 β-Arrestin Cell Line, supplier: DiscoverX, catalog number: 93-0207C2), remove the culture medium (F12 medium 1000 mL, 10% FBS, 800 μg/mL G418, 300 μg/mL Hygromycin, 1% GlutaMax and 1% Pen/Strep), rinse the cells with 2 ml DPBS, add 5 mL cell dispersion solution (Invitrogen-13151014) to disperse the cells, incubate in a 37 ℃ incubator for 1~2 minutes, tap the culture flask to detach the cells, add 5 mL growth medium, and gently pipette to fully suspend the cells. Use Vi-Cell to count the cells. 1. Centrifuge at 1000 rpm for 5 minutes at room temperature, gently pour off the supernatant, and resuspend the cells in FACS buffer to a concentration of 1.5e6 cells per ml. 2. Dilute S1P and compounds in DMSO in a 384-well plate and transfer 500 nL volume to a 96 V-well plate (Cat#Axygen-WIPP02280). 3. Add 50 μL of cells to the 96-well plate; 4. Incubate the 96-well plate in a 37 ℃ 5% CO2 incubator for 2 hours. 5. Centrifuge the cells at 1500 rpm for 5 minutes at room temperature and remove the supernatant. 6. Add 100 μl FACS buffer to the resuspended cells, centrifuge at 1500 rpm for 5 min, and remove the supernatant. 7. Dilute the antibodies of anti-human S1P1/EDG-1-Alex647 (R&D-FAB1864R) and anti-IgG2B-Alex647 (R&D-IC0041R) 200-fold with FACS buffer. 8. Add 50 μL of antibodies to the 96-well plate and transfer the plate to 4°C for 30 min. 9. Centrifuge the cells at 1500 rpm for 5 min at 4°C and remove the supernatant. 10. Add 100 μL FACS buffer to the resuspended cells. 11. Centrifuge the cells at 1500 rpm for 5 minutes at 4°C and remove the supernatant. 12. After washing, resuspend the cells in 50 μL FACS buffer per well. 13. Read the cell samples using iQue Screener PLUS-VBR. The experimental results are shown in Table 3. Table 3: Experiment on the internalization effect of compounds on S1P1 receptor Experimental data show that the compound of the present invention exhibits a good internalization activation effect on the S1P1 receptor.

Experimental Example 4: Determination of S1P3 receptor agonist activity

The cells, culture conditions and cell collection conditions used in this experiment are the same as those in Experimental Example 3. 1) Add 25 μL (5000 cells) of cell suspension to each well of the assay plate and incubate at 37°C for 20 hours. (2) Serially dilute the compound 4-fold to obtain 10 doses and incubate at 37°C for 90 minutes. (3) Add 12 μL of the test reagent to each well of the assay plate and incubate at 23°C for 60 minutes. (4) Read the Envision. The experimental results are shown in Table 4. Table 4: Assay for S1P3 receptor agonist activity Experimental data show that the compounds of the present invention exhibit good selectivity for the S1P3 receptor.

Experimental Example 5: In vivo pharmacokinetics experiment of the compound of the present invention

In this study, the pharmacokinetic properties of the drug were evaluated in rats by intravenous injection and oral administration.

Experimental methods and conditions: Male Sprague Dawley rats were given a single intravenous injection of the test compound at 1 mg/Kg (solvent 5% DMSO/15% Solutol/80% Saline) and oral gavage at 1 mg/Kg (solvent 0.5% MC). Blood was collected from the submandibular vein 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 24 hr after administration. Approximately 0.20 mL of blood was collected from each sample and anticoagulated with sodium heparin. The collected blood was placed on ice and the plasma was separated by centrifugation within 1 hour for testing. The blood drug concentration in plasma was measured by liquid phase tandem mass spectrometry (LC/MS/MS), and the pharmacokinetic parameters were calculated using Phoenix WinNonlin software. The experimental results are shown in Tables 5 and 6. Table 5: Pharmacokinetic parameters of oral administration (1 mg/kg) Table 6: Pharmacokinetics of intravenous injection (1 mg/kg) Experimental data show that the compound of the present invention exhibits good metabolic stability in rats.

Experimental Example 6: Effect of the compounds of the present invention on the peripheral lymphocyte lowering (PLL) assay in rats.

Male Sprague-Dawley rats, weighing 200-220g. Housing environment: temperature 23±2℃, relative humidity 40-70%, lighting time 7:00 am on, 7:00 pm off; animals are fed with ordinary feed and sterilized drinking water freely. All animal experiments were approved by the Animal Ethics Committee; all animal experimental operations complied with the relevant SOP requirements of the animal house. Animals were adaptively housed for one week before the experiment.

Animals were orally administered with a volume of 10 mL/kg. The administration solvent was 0.5% DMSO + 0.5% MC. Five hours after administration, the animals were anesthetized with isoflurane, and 100-150 μl of peripheral blood was collected through the eye socket in an EP tube, placed on ice, and blood analysis and lymphocyte count were performed within 30 minutes using an XT-2000i fully automatic blood analyzer; another 20 μl of whole blood was diluted with 40 μl DDW and quickly frozen in liquid nitrogen for blood compound concentration testing.

The results showed that compound 2 decreased the peripheral blood lymphocyte (PBL) counts of rats after 5 hours of testing, as shown in Figure 1. The IC50 was 94.6 nM (Compound 2).

Although the specific implementations of the present invention are described above, those skilled in the art should understand that these are only examples, and that various changes or modifications may be made to these implementations without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is limited by the scope of the attached patent application.

without

Figure 1 shows the results of peripheral blood lymphocyte (PBL) counts in rats after 5 hours of testing compound 2.

Claims (15)

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

wherein m is selected from 1, 2 and 3; n is selected from 1 and 2; y is selected from 1 and 2; R ₁ is selected from F, Cl, CH ₃ or CH ₂ CH ₃ ; R ₂ is selected from C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by 1, 2 or 3 R; Ring A is selected from

or

; L ₁ selected from

; _L2 is selected from a single bond; _T1 is selected from CH; _T2 is selected from CH; _T3 is selected from CH; R is independently selected from H and F. According to claim 1, the compound or a pharmaceutically acceptable salt thereof, wherein R ₁ is selected from F and Cl. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein R ₁ is selected from F. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R ₂ is CH ₃ or CH ₂ CH ₃ , and said CH ₃ or CH ₂ CH ₃ is optionally substituted by 1, 2 or 3 Rs. The compound or a pharmaceutically acceptable salt thereof according to claim 4, wherein R ₂ is selected from CH ₃ , CH ₂ CH ₃ and CF ₃ . The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein _L1 is selected from

,

. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein Ring A is selected from

. According to claim 1, the compound or a pharmaceutically acceptable salt thereof, wherein the structural unit

Selected from

. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the structural unit

Selected from

and

. According to claim 1, the compound or a pharmaceutically acceptable salt thereof is selected from

wherein m, n, T ₁ , T ₂ , T ₃ , R, and L ₂ are as defined in claim item 1; R ₁ is as defined in any one of claim items 1 to 3; R ₂ is as defined in claim items 1, 4, and 5; and ring A is as defined in claim items 1 and 7. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from any one of the following compounds:

A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from any one of the following compounds:

A drug composition, comprising a therapeutically effective amount of a compound as described in any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. A drug for preventing and/or treating diseases related to the S1P1 receptor, which is prepared using the compound described in any one of claims 1 to 12 or its pharmaceutically acceptable salt or the drug composition described in claim 13. The drug according to claim 14, wherein the disease associated with the S1P1 receptor is selected from ulcerative colitis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, primary cholangitis, allergic dermatitis, cerebral hemorrhage, graft-versus-host disease, psoriasis, type 1 diabetes, acne, microbial infection or microbial disease and viral infection or viral disease.