Tertiary amine compound as well as preparation method and medical application thereof

Abstract

The invention belongs to the technical field of chemical drugs, and provides a tertiary amine compound and a preparation method and medical application thereof. # imgabs0 #

Description

A tertiary amine compound, its preparation method and medical use Technical Field

[0001] The invention belongs to the technical field of chemical medicines and provides a tertiary amine compound, a preparation method thereof and medical use thereof. Background Art

[0002] LPA is the name of the gene encoding apolipoprotein (a) (apo(a)). It is primarily expressed in the liver, with expression restricted to humans and non-primates. Apolipoprotein (a) is attached to apo(B)-100 via a disulfide bond, forming a lipid core that forms the lipoprotein (a) (Lp(a)) particle. Lp(a) particles are specialized, large cholesterol-rich lipoproteins with a surface coated with cholesterol and phospholipids and embedded with the hydrophilic apolipoprotein components apolipoprotein (a) and apo(B)-100. Lp(a) can enter and deposit on blood vessel walls, promoting atherosclerosis. Lp(a) shares structural homology with plasminogen (PLG) and can compete with plasminogen for binding sites on fibrin, thereby inhibiting fibrinogen hydrolysis and promoting thrombosis. Therefore, Lp(a) is closely associated with atherosclerosis and thrombosis. Studies have shown that Lp(a) levels in the blood are an independent risk factor for cardiovascular disease, stroke, and atherosclerotic stenosis. Human Lp(a) levels are genetically determined and do not change significantly with diet, exercise, or other lifestyle changes.

[0003] Currently reported LP(a) inhibitors are primarily macromolecular or small nucleic acid drugs. For example, WO2023046093A1 discloses a bispecific fusion polypeptide; CN111465694A discloses a nucleic acid for inhibiting LPA expression in cells; CN108368506A also discloses compositions and methods for inhibiting LPA gene expression; and CN113166759A discloses a chemically modified RNAi construct and its use. Currently, there are no marketed small-molecule LP(a) drugs. Among the projects under development, the only one, CN114008021A, reports a pyrrolidine compound as a small-molecule LP(a) drug. Therefore, there is an urgent need to provide more small-molecule LP(a) drugs.

[0004] Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a small molecule LP(a) drug to solve the problem of the shortage of small molecule LP(a) drugs in the prior art.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a tertiary amine compound, or an isomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, characterized in that the structure of the tertiary amine compound is as shown in general formula I:

[0008] Among them, X 1～ X 15 Each is independently selected from -N- or -CR1, wherein R1 is selected from H, halogen, -OH, -NH2, -CN, -NO2, -COOH, -SO3H, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl, substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C5-C 10 Heterocyclic group or

[0009] The X 1～ X 15 At least one of them is -N-;

[0010] Or when X 1～ X 15 are each independently selected from -CR1, wherein at least one R1 is not hydrogen and And the X 1～ At least one R1 in X5 is independently selected from

[0011] And the X 11～ X 15 At least one R1 is independently selected from

[0012] Or when X 1～ X 15 When each of the X 1～ X 15 Any two adjacent R1 and the connected carbon atom together form a 5-8 membered aryl or heterocyclic group;

[0013] Said R2 is selected from -H, halogen, substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 alkoxy;

[0014] Said R3 is selected from -(CH2) q -(5-8 membered heterocyclyl);

[0015] The substituted C1-C6 alkyl, substituted C1-C6 alkoxy, substituted C6-C 10 Aryl or substituted C5-C 10 The substituents in the heterocyclic group are independently selected from halogen, hydroxy, carboxyl, nitro, cyano, C1-C6 alkyl, C 1-C6 haloalkyl, C 1- C6 haloalkoxy, C 1- C6 alkoxy, C 3- C6 cycloalkyl, 3 to 6 membered heterocycloalkyl, C 3- C6 cycloalkoxy, C 6- C 10 one or more of aryl or 5- to 6-membered heteroaryl;

[0016] m, n, and p are independently selected from integers of 1 or 2, and q is selected from integers of 0, 1, or 2;

[0017] The tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt is not

[0018] As a preferred technical solution of the present invention, the R1 is selected from H, halogen, -OH, -NH2, -CN, -NO2, -COOH, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C 10 Cycloalkyl, substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C5-C 10 Heterocyclic group or The R2 is selected from -H, substituted or unsubstituted C1-C6 alkyl; the R3 is selected from 5-8 membered heterocyclic group; the substituted C1-C6 alkyl, substituted C6-C 10 Aryl or substituted C5-C 10 The substituents in the heteroaryl group are independently selected from one or more of halogen, hydroxy, carboxyl, nitro or cyano.

[0019] As a preferred technical solution of the present invention, the Selected from quinolinyl; More preferably:

[0020] As a preferred technical solution of the present invention, the structure of the tertiary amine compound is shown in Formula Ia or Ib:

[0021] Among them, X 1～ X 15 Each independently selected from -N- or -CR1, wherein R1 is selected from H, halogen, substituted C1-C6 alkyl, C1-C6 alkoxy or The R2 is selected from H, substituted or unsubstituted C1-C6 alkyl; the X 1～ X 15 At least one of them is -N-, or when X 1～ X15 are each independently selected from -CR1, wherein at least one R1 is not hydrogen and Substitution; the R3 is selected from a 5-8 membered heterocyclic group; the substituents in the substituted C1-C6 alkyl are independently selected from one or more of halogen, carboxyl, cyano, and C1-C6 alkyl.

[0022] As a preferred technical solution of the present invention, the structure of the tertiary amine compound is shown in the general formula IIa or IIb:

[0023] Among them, X 1～ X 15 Each independently selected from -N- or -CR1, wherein R1 is selected from H, halogen, substituted C1-C6 alkyl, C1-C6 alkoxy or The R2 is selected from H, substituted or unsubstituted C1-C6 alkyl; the X 1～ X 15 At least one of them is -N-, or when X 1～ X 15 are each independently selected from -CR1, wherein at least one R1 is not hydrogen and Substitution; the R3 is selected from a 5-8 membered heterocyclic group; the substituents in the substituted C1-C6 alkyl are independently selected from one or more of halogen, carboxyl, cyano, and C1-C6 alkyl.

[0024] As a preferred technical solution of the present invention, the structure of the tertiary amine compound is as shown in the general formula IIIa or IIIb:

[0025] Among them, X 1～ X 15 Each independently selected from -N- or -CR1, wherein R1 is selected from H, halogen, substituted C1-C6 alkyl, C1-C6 alkoxy or The R2 is selected from H, substituted or unsubstituted C1-C6 alkyl; the X 1～ X 15 At least one of them is -N-, or when X 1～ X 15 are each independently selected from -CR1, wherein at least one R1 is not hydrogen and Substitution; the R3 is selected from a 5-8 membered heterocyclic group; the substituents in the substituted C1-C6 alkyl are independently selected from one or more of halogen, carboxyl, cyano, and C1-C6 alkyl.

[0026] As a preferred technical solution of the present invention, the R1 is selected from H, fluorine, chlorine, bromine, iodine, trifluoromethyl, methyl, ethyl, propyl, methoxy, ethoxy, piperazinyl, -CH2COOH, -CH2CH2COOH, -CH2CH(CH3)COOH, The R3 is selected from pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyridinyl, pyrimidinyl or pyrazinyl.

[0027] As a preferred technical solution of the present invention, the Selected from:

[0028] As a preferred technical solution of the present invention, the Selected from:

[0029] As a preferred technical solution of the present invention, the Selected from:

[0030] As a preferred technical solution of the present invention, the C 1- C6 alkyl is preferably C 1- C2, C 1- C3, C 1- C4 or C 1- C5 alkyl; examples of the alkyl group include: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 1-ethylpropyl, 2-methylbutyl, tert-pentyl, 1,2-dimethylpropyl, isopentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, tert-hexyl, neohexyl, 2-methylpentyl, 1,2-dimethylbutyl, 1-ethylbutyl.

[0031] As a preferred technical solution of the present invention, the C 1- C6 alkoxy is preferably C 1- C2, C 1- C3, C 1- C4 or C 1- C5 alkoxy, further, the alkoxy is specifically selected from methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy.

[0032] As a preferred technical solution of the present invention, the C 3- C 10 Cycloalkyl is preferably selected from: C 3- C8 cycloalkyl, C 3- C6 cycloalkyl or C 3- C5 cycloalkyl, preferably C 3-C8 cycloalkyl, wherein the cycloalkyl is specifically selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0033] As a preferred embodiment of the present invention, the substituted or unsubstituted C6-C 10 The aryl group is preferably a substituted or unsubstituted C6-C8 aryl group, a substituted or unsubstituted C6-C7 aryl group; when the aryl group is substituted, the substituent is preferably selected from H, halogen, -OH, -NH2, -CN, -NO2, -COOH, -SO3H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C 10 Cycloalkyl, C6-C 10 Substituted or unsubstituted aryl, C5-C 10 Substituted or unsubstituted heteroaryl, the aryl is specifically selected from phenyl, naphthyl, anthracenyl, phenanthrenyl, etc.; the substituted or unsubstituted C5-C 10 Heteroaryl is preferably a substituted or unsubstituted C5-C8 aromatic heteroyl, a substituted or unsubstituted C5-C7 heteroaryl or a substituted or unsubstituted C5-C6 heteroaryl; when the heteroaryl is substituted, the substituent is preferably selected from H, halogen, -OH, -NH2, -CN, -NO2, -COOH, -SO3H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C 10 Cycloalkyl, substituted or unsubstituted C5-C 10 Aryl, substituted or unsubstituted C5-C 10 Heteroaryl, wherein the heteroatom in the heteroaryl is one or more of N, O, and S. The heteroaryl is preferably selected from: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazine, and the like.

[0034] As a preferred embodiment of the present invention, the 3-8 membered heterocyclic group or C5-C 10 The heterocyclic group is selected from saturated or unsaturated 3-8 membered heterocyclic group or C5-C 10Heterocyclic group, preferably 3-8 membered heteroaryl, C5-C8 heteroaryl or 3-8 membered heterocycloalkyl, more preferably 3-6 membered heteroaryl, C5-C6 heteroaryl or 3-6 membered heterocycloalkyl, more preferably 5-6 membered heteroaryl, C5-C6 heteroaryl or 5-6 membered heterocycloalkyl, the heteroatom in the heterocyclic group is one or more of N, O and S. Examples of such heterocycloalkyl groups include, but are not limited to, azetidinyl, morpholinyl, piperidinyl (e.g., 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, and 4-piperidinyl), pyrrolidinyl (e.g., 1-pyrrolidinyl, 2-pyrrolidinyl, and 3-pyrrolidinyl), pyrrolidonyl, pyranyl, tetrahydrofuranyl, tetrahydrothiophenyl, dioxanyl, tetrahydropyranyl (e.g., 4-tetrahydropyranyl), thiomorpholinyl, piperazinyl, N-alkylpiperazinyl (e.g., N-methylpiperazinyl), and N-alkylpiperidinyl (e.g., N-methylpiperidinyl). Examples of such heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepine, diazepine, isoxazolyl, benzofuranyl, isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, quinolinyl, isoquinolinyl, quinazolinyl, and quinoxalinyl. Particular heteroaryl groups include pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, isoxazolyl and isothiazolyl. More particular heteroaryl groups include imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, isoxazolyl and isothiazolyl.

[0035] As a preferred technical solution of the present invention, the tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt is selected from the structures shown in Table 1A:

[0036] Table 1A

[0037] As a preferred technical solution of the present invention, the tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt is selected from the structures shown in Table 1B:

[0038] Table 1B

[0039] The present invention further provides a method for preparing a tertiary amine compound, or an isomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, which is prepared by referring to the method of patent CN114008021A.

[0040] The present invention further provides a pharmaceutical composition, characterized in that it comprises a tertiary amine compound described in general formula I, general formula II or general formula III, or its isomer, or its racemate or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable excipients and / or carriers.

[0041] The present invention further provides a use of a tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt in the preparation of a compound for preventing or treating diseases associated with LP(a).

[0042] Furthermore, the Lp(a)-related disease is selected from cardiovascular disease;

[0043] Furthermore, the cardiovascular disease is selected from stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and any other disease associated with elevated Lp(a) levels.

[0044] The beneficial effects of the present invention over the prior art include but are not limited to:

[0045] Compared with the prior art, the tertiary amine compounds of the present invention have lower IC 90 value.

[0046] For the sake of clarity, general terms used in the description of the compounds are defined herein.

[0047] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered as undefined 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 its corresponding commercial product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0048] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared by reacting a compound having a specific substituent discovered in the present invention with a pharmaceutically acceptable acid or base.

[0049] In addition to the form of salts, the compounds provided by the present invention also exist in prodrug form. The prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to be converted into the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

[0050] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrates. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0051] 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 such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0052] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, separation of enantiomers and diastereomers is typically accomplished by using chromatography using a chiral stationary phase, optionally combined with chemical derivatization (e.g., formation of carbamates from amines).

[0053] "Alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups having 1 to 20 carbon atoms. Alkyl groups containing 1 to 6 carbon atoms are preferred. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and various branched chain isomers thereof. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from halogen, deuterium, hydroxy, oxo, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyloxy, C2-C6 alkynyloxy, C3-C6 cycloalkyl, 3 to 6 membered heterocycloalkyl, C5-C8 cycloalkenyl, C3-C6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C5-C8 cycloalkenyloxy, C6-C 10 aryl or 5 to 6 membered heteroaryl, the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyloxy, C2-C6 alkynyloxy, C3-C6 cycloalkyl, 3 to 6 membered heterocycloalkyl, C5-C8 cycloalkenyl, C3-C6 cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C5-C8 cycloalkenyloxy, C6-C 10 The aryl or 5- to 6-membered heteroaryl is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0054] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy.

[0055] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0056] The term "cycloalkyl" or "carbocycle" refers to a saturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0057] The term "aryl" or "aromatic ring" refers to a 6- to 10-membered all-carbon monocyclic or fused polycyclic (ie, rings sharing adjacent pairs of carbon atoms) group having a conjugated pi electron system, preferably 6- to 8-membered, such as phenyl and naphthyl.

[0058] The term "heteroaryl" or "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 3 heteroatoms and 5 to 10 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 8-membered, more preferably 5-membered or 6-membered. For example, non-limiting examples include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazine, etc.

[0059] The term "heterocyclyl" refers to a saturated or unsaturated heterocycloalkyl or heteroaryl group, wherein the heteroatom in the heterocyclyl group is one or more of N, O, and S. The heterocycloalkyl group is preferably a 3-8 membered heterocycloalkyl group, more preferably a 3-6 membered heterocycloalkyl group, and most preferably a 5-6 membered heterocycloalkyl group; the heteroaryl group is preferably a 3-8 membered heteroaryl group or a C5-C8 heteroaryl group, more preferably a 3-6 membered heteroaryl group or a C5-C6 heteroaryl group, and most preferably a 5-6 membered heteroaryl group or a C5-C6 heteroaryl group.

[0060] The term "heterocycloalkyl" refers to a monovalent saturated monocyclic or bicyclic ring system of 3 to 8 ring atoms, containing 1, 2, or 3 ring heteroatoms selected from N, O and S, with the remaining ring atoms being carbon. In a particular embodiment, heterocycloalkyl is a monovalent saturated monocyclic ring system of 4 to 7 ring atoms, containing 1, 2, or 3 ring heteroatoms selected from N, O and S, with the remaining ring atoms being carbon. Examples of monocyclic saturated heterocycloalkyl groups are aziridinyl, oxiranyl, azetidinyl, oxetanyl, pyrrolidinyl (e.g., 1-pyrrolidinyl, 2-pyrrolidinyl, and 3-pyrrolidinyl), tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, N-alkylpiperidinyl (e.g., N-methylpiperidinyl), tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl (e.g., 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, and 4-piperidinyl), morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, oxazepanyl, and thiazinyl. Examples of bicyclic saturated heterocycloalkyl groups are 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, 3-thia-9-aza-bicyclo[3.3.1]nonyl and 2,6-diaza-spiro[3.3]heptanyl.

[0061] The atoms of the molecules of the compounds of the present invention are isotopes, and isotope derivatization can generally extend half-life, reduce clearance, stabilize metabolism, and increase in vivo activity. In addition, an embodiment is included in which at least one atom is replaced by an atom having the same atomic number (number of protons) and a different mass number (protons and neutrons). Examples of isotopes included in the compounds of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, which respectively include 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 In particular, radioactive isotopes that emit radiation as they decay, such as 3 H or 14 C can be used for local anatomy testing of pharmaceutical preparations or compounds in vivo. Stable isotopes neither decay nor change with their amount nor are they radioactive, so they can be used safely. When the atoms constituting the molecules of the compounds of the present invention are isotopes, the isotopes can be converted according to general methods by replacing the reagents used in the synthesis with reagents containing the corresponding isotopes.

[0062] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen, such as trifluoromethyl, trifluoroethyl, etc.;

[0063] The term "haloalkoxy" refers to an alkoxy group in which one or more hydrogen atoms are replaced by halogen, for example, trifluoromethyloxy, trifluoroethyloxy, and the like.

[0064] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as deuterium ( 2 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0065] Furthermore, one or more hydrogen atoms of the compounds of the present invention are replaced by the isotope deuterium (2H). After deuteration, the compounds of the present invention have the effects of extending half-life, reducing clearance rate, metabolic stability and improving in vivo activity.

[0066] The preparation method of the isotopic derivative generally includes a phase transfer catalytic method. For example, a preferred deuteration method uses a phase transfer catalyst (e.g., a tetraalkylammonium salt, NBu4HSO4). The use of a phase transfer catalyst to exchange the methylene protons of the diphenylmethane compound results in a higher deuterium incorporation than reduction with a deuterated silane (e.g., triethyldeuterated monosilane) in the presence of an acid (e.g., methanesulfonic acid) or with a Lewis acid such as aluminum trichloride using sodium deuterated borate.

[0067] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical medicine. For additional information about carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0068] The term "excipient" generally refers to a carrier, diluent and / or vehicle required to formulate an effective pharmaceutical composition.

[0069] With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For the oral dosage forms of the present invention, an "effective amount" of an active substance in the composition means the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0070] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0071] "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 it does not. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below with reference to the embodiments, but the content of the invention is not limited to the embodiments.

[0073] Preparation of intermediates:

[0074] The preparation of intermediate 1-6 is as follows:

[0075] Intermediate 1: Synthesis of (R)-tert-butyl 3-(2-(S)-4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)pyrrolidine-1-carboxylate

[0076] Under ice-water bath, (R)-2-(1-tert-Butyloxycarbonyl)pyrrolidine-3-acetic acid (50.0 g, 218.34 mmol) was dissolved in tetrahydrofuran (500.0 ml), and triethylamine (72.1 ml) was added. After 5 minutes, pivaloyl chloride (33.2 ml) was added. After 15 minutes, lithium chloride (10.9 g, 259.5 mmol) and a solution of (S)-4-benzyloxazolidin-2-one (36.8 g, 207.91 mmol) in tetrahydrofuran (250.0 ml) were added in sequence, and the mixture was reacted at room temperature for 3 hours.

[0077] After the reaction was completed, the mixture was adjusted to weak acidity with 1N hydrochloric acid and diluted with water. The mixture was extracted with ethyl acetate (200 ml x 3 times). The organic phases were combined, washed with saturated brine (200 ml x 2 times), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 1 / 1) to obtain 35.0 g of (R)-tert-butyl 3-(2-(S)-4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)pyrrolidine-1-carboxylate. LCMS: RT = 2.02 min, [M+H] + =389.43.

[0078] Intermediate 2: Synthesis of (R)-3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(2-bromopyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0079] To a solution of tert-butyl (R)-3-(2-(S)-4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)pyrrolidine-1-carboxylate (1.0 g, 2.58 mmol) in tetrahydrofuran (4.0 ml) was slowly added a solution of lithium bistrimethylsilylamide in tetrahydrofuran (1.0 mol / L, 3.0 ml) at -78°C. After stirring at -78°C for 30 minutes, a solution of 2-bromo-4-bromomethylpyridine (776.0 mg, 3.10 mmol) in tetrahydrofuran (2.0 ml) was slowly added. The temperature was slowly raised to room temperature and the reaction was carried out at room temperature for 4 hours.

[0080] After the reaction was complete, an excess of saturated ammonium chloride solution was slowly added to the reaction solution, and the mixture was extracted with ethyl acetate (20 ml x 2). The organic phases were combined, washed with saturated brine (20 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 2 / 1) to obtain 1.1 g of tert-butyl (R)-3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(2-bromopyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.16 min, [M+H-Boc] + =458.15.

[0081] Intermediate 3: Synthesis of (S)-3-(2-bromopyridin-4-yl)-2-(R)-1-tert-butyloxycarbonylpyrrolidin-3-yl)propanoic acid

[0082] To a solution of tert-butyl (R)-3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(2-bromopyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (1.1 g, 1.97 mmol) in tetrahydrofuran (15.0 ml) was added hydrogen peroxide (300.0 μl) under ice-water bath. After 30 minutes, an aqueous solution (3.0 ml) of lithium hydroxide monohydrate (124.0 mg, 2.96 mmol) was added, and the mixture was heated to room temperature and reacted for 3 hours. After the reaction, an excess of saturated sodium bisulfate solution was slowly added to the reaction mixture to quench the reaction. The mixture was stirred for 10 minutes, and then a saturated aqueous solution of citric acid was added to adjust the pH to 3. The mixture was extracted with ethyl acetate (20 ml x 2). The organic phases were combined, washed with saturated brine (20 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 2 / 1) to obtain 750.0 mg of (S)-3-(2-bromopyridin-4-yl)-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-yl)propanoic acid. LCMS: RT = 1.90 min, [MH] - =397.11.

[0083] Intermediate 4: Synthesis of (R)-3-(S)-3-(2-bromopyridin-4-yl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0084] To a solution of (S)-3-(2-bromopyridin-4-yl)-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-yl)propanoic acid (750.0 mg, 1.88 mmol) in 2-methyltetrahydrofuran (10.0 ml) was added tert-butyl (Z)-N,N'-diisopropylcarbamate (2.03 ml) at room temperature. The atmosphere was replaced with nitrogen three times and then heated to 65°C for 7 hours.

[0085] After the reaction was complete, the mixture was filtered and the filter cake was washed with ethyl acetate. The filtrate was diluted with ethyl acetate, and the combined organic phases were washed with saturated brine (20 ml x 2) and then dried over anhydrous sodium sulfate. The residue was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 1) to obtain 540.0 mg of tert-butyl (R)-3-(S)-3-(2-bromopyridin-4-yl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.20 min, [M+H-tert-butyl] + =399.15.

[0086] Intermediate 5: Synthesis of (R)-3-(S)-1-tert-butoxy-1-oxo-3-(2-vinylpyridin-4-yl)propan-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0087] To a solution of tert-butyl (R)-3-(S)-3-(2-bromopyridin-4-yl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (540.0 mg, 1.19 mmol) in toluene (6.0 ml) at room temperature were added a solution of vinylboronic acid pinacol ester (262.3 μL, 1.55 mmol) and potassium carbonate (328.0 mg, 2.38 mmol) in water (2.0 ml). Under nitrogen protection, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (87.7 mg, 0.12 mmol) was added, and the reaction was carried out at 95°C for 4 hours.

[0088] After the reaction was complete, water (10 ml) was added to the reaction mixture, which was then filtered through a pad of Celite. The filter cake was washed with ethyl acetate, and the filtrate was extracted with ethyl acetate (20 ml x 2). The organic phases were combined, washed with saturated brine (20 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 1) to obtain 380.0 mg of tert-butyl (R)-3-(S)-1-tert-butoxy-1-oxo-3-(2-vinylpyridin-4-yl)propan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 1.98 min, [M+H] + =403.31.

[0089] Intermediate 6: Synthesis of (R)-3-(S)-1-tert-butyl-3-(2-formylpyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0090] To a solution of tert-butyl (R)-3-(S)-1-tert-butoxy-1-oxo-3-(2-vinylpyridin-4-yl)propan-2-yl)pyrrolidine-1-carboxylate (200.0 mg, 0.50 mmol) in tetrahydrofuran (4.0 ml) was added a solution of potassium osmate (1.5 mg, 0.005 mmol) in water (0.2 ml) at room temperature. The mixture was stirred at room temperature for 15 minutes. A solution of sodium periodate (214.0 mg, 1.0 mmol) in water (1.0 ml) was added and the mixture was reacted at room temperature. After completion of the reaction, the reaction was monitored by TLC. An excess of aqueous sodium bisulfate solution was slowly poured into the system to quench the reaction. The mixture was stirred for 30 minutes, and then extracted with ethyl acetate (20 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 5) to obtain 150.0 mg of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-formylpyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.11 min, [M+H-tert-butyl] + =349.10.

[0091] The preparation of intermediate 7-11 is as follows:

[0092] Intermediate 7: Synthesis of (R)-3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(6-bromopyridin-2-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0093] Intermediate 7 was prepared by referring to the method of Intermediate 2. LCMS: RT = 2.23 min, [M + H-tert-butyl] + =502.43.

[0094] Intermediate 8: Synthesis of (S)-3-(6-bromopyridin-2-yl)-2-(R)-1-tert-butyloxycarbonylpyrrolidin-3-yl)propanoic acid

[0095] Intermediate 8 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.08 min, [MH] - =397.03.

[0096] Intermediate 9: Synthesis of (R)-3-(S)-3-(6-bromopyridin-2-yl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0097] Intermediate 9 was prepared by referring to the method of Intermediate 4. LCMS: RT = 2.26 min, [M + H-tert-butyl] - =399.37.

[0098] Intermediate 10: Synthesis of (R)-3-(S)-1-tert-butyl-1-oxo-3-(6-vinylpyridin-2-yl)propyl-2-yl)pyrrolidine-1-carboxylate

[0099] Intermediate 10 was prepared by referring to the method of Intermediate 5. LCMS: RT = 2.16 min, [M+H] + =403.27.

[0100] Intermediate 11: Synthesis of (R)-3-(S)-1-tert-butyl-3-(6-formylpyridin-2-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0101] Intermediate 11 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.01 min, [M+H] + =405.28.

[0102] The preparation of intermediate 12-14, the specific steps are:

[0103] Intermediate 12: Synthesis of tert-butyl (3R)-3-(3-(4-bromopyridin-2-yl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0104] To a solution of tert-butyl (R)-3-(2-(tert-butoxy)-2-oxoethyl)pyrrolidine-1-carboxylate (1.64 g, 5.73 mmol) in THF (20 ml) was slowly added lithium bistrimethylsilylamide (7.8 ml, 1.0 mol per liter) at -78°C. After stirring at -78°C for 30 minutes, a solution of 4-bromo-2-(bromomethyl)pyridine (1.3 g, 5.20 mmol) in tetrahydrofuran (10 ml) was slowly added to the system, and the temperature was then naturally raised to room temperature for reaction overnight. After completion of the reaction, the mixture was quenched with saturated aqueous ammonium chloride solution, followed by extraction with ethyl acetate (20 mL x 2). The combined organic phases were washed with saturated brine (20 mL x 2), then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to obtain 1.0 g of tert-butyl (3R)-3-(3-(4-bromopyridin-2-yl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.20 min, [M+H] + =455.21.

[0105] Intermediate 13: Synthesis of tert-butyl (3R)-3-(1-(tert-butoxy)-1-oxo-3-(4-vinylpyridin-2-yl)propan-2-yl)pyrrolidine-1-carboxylate

[0106] Intermediate 13 was prepared by referring to the method of Intermediate 5. LCMS: RT = 2.00 min, [M+H] + =403.29.

[0107] Intermediate 14: Synthesis of tert-butyl (3R)-3-(1-(tert-butoxy)-3-(4-formylpyridin-2-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0108] Intermediate 14 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.11 min, [M+H] + =405.41.

[0109] The preparation of intermediate 15-19, the specific steps are:

[0110] Intermediate 15: Synthesis of (R)-3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(5-bromopyridin-3-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester Intermediate 15 was prepared according to the method of Intermediate 2. LCMS: RT = 2.25 min, [M-tert-butyl + H] + =502.17.

[0111] Intermediate 16: (S)-3-(5-bromopyridin-3-yl)-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-yl)propanoic acid

[0112] Intermediate 16 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.13 min, [MH] - =397.15.

[0113] Intermediate 17: Synthesis of tert-butyl (R)-3-(S)-3-(6-bromopyridin-2-yl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0114] Intermediate 17 was prepared by referring to the method of Intermediate 4. LCMS: RT = 2.26 min, [M-tert-butyl + H] - =399.17.

[0115] Intermediate 18: Synthesis of (R)-tert-butyl 3-(S)-1-tert-butoxy-1-oxo-3-(5-vinylpyridin-3-yl)propan-2-yl)pyrrolidine-1-carboxylate

[0116] Intermediate 18 was prepared by referring to the method of Intermediate 5. LCMS: RT = 2.16 min, [M+H] + =403.24.

[0117] Intermediate 19: Synthesis of (R)-3-(S)-1-tert-butyl-3-(5-formylpyridin-3-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0118] Intermediate 19 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.07 min, [M+H] + =405.33.

[0119] The preparation of intermediates 22-24 is as follows:

[0120] Intermediate 20: Synthesis of (R)-tert-butyl 3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(3-bromo-5-fluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate Intermediate 20 was prepared according to the method of Intermediate 2. LCMS: RT = 2.30 min, [M+H] + =475.18.

[0121] Intermediate 21: Synthesis of 3-(3-bromo-5-fluorophenyl)-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-ylpropionic acid

[0122] Intermediate 21 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.08 min, [MH] - =414.27.

[0123] Intermediate 22: Synthesis of tert-butyl (R)-3-(S)-3-(3-bromo-5-fluorophenyl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0124] Intermediate 22 was prepared by following the procedure of Intermediate 4.

[0125] Intermediate 23: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(3-fluoro-5-vinylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0126] Intermediate 23 was prepared by referring to the method of Intermediate 5. LCMS: RT = 2.37 min, [M + H-tert-butyl-tert-butyl] + =308.25.

[0127] Intermediate 24: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(3-fluoro-5-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0128] Intermediate 24 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.23 min, [M + H-Boc-tert-butyl] + =266.33.

[0129] The preparation of intermediates 25-29 is as follows:

[0130] Intermediate 25: Synthesis of tert-butyl (R)-3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(5-bromo-2-fluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0131] Intermediate 25 was prepared by referring to the method of Intermediate 2. LCMS: RT = 2.24 min, [M+H-Boc] + =475.32.

[0132] Intermediate 26: Synthesis of (S)-3-(5-bromo-2-fluorophenyl)-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-yl)propanoic acid

[0133] Intermediate 26 was prepared by following the procedure of Intermediate 3.

[0134] Intermediate 27: Synthesis of tert-butyl (R)-3-(S)-3-(5-bromo-2-fluorophenyl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0135] Intermediate 27 was prepared by referring to the method of Intermediate 4. LCMS: RT = 2.26 min, [M+H-Boc] + =372.14.

[0136] Intermediate 28: Synthesis of (R)-tert-butyl 3-(S)-1-tert-butoxy-3-(2-fluoro-5-vinylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0137] Intermediate 28 was prepared by following the procedure of Intermediate 5.

[0138] Intermediate 29: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-fluoro-5-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0139] Intermediate 29 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.45 min, [M+H-Boc] + =322.22.

[0140] The preparation of intermediates 30-34 is as follows:

[0141] Intermediate 30: Synthesis of (R)-tert-butyl 3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(3-bromo-2-fluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate Intermediate 30 was prepared according to the method of Intermediate 2. LCMS: RT = 2.27 min, [M+H-Boc] + =475.24.

[0142] Intermediate 31: Synthesis of (S)-3-(3-bromo-2-fluorophenyl)-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-yl)propanoic acid

[0143] Intermediate 31 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.08 min, [MH] - =414.10.

[0144] Intermediate 32: Synthesis of tert-butyl (R)-3-(S)-3-(3-bromo-2-fluorophenyl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0145] Intermediate 32 was prepared by following the procedure of Intermediate 4.

[0146] Intermediate 33: Synthesis of (R)-tert-butyl 3-(S)-1-tert-butoxy-3-(2-fluoro-3-vinylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0147] Intermediate 33 was prepared by following the procedure of Intermediate 5.

[0148] Intermediate 34: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-fluoro-3-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0149] Intermediate 34 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.21 min, [M+H-Boc] + =322.24.

[0150] The preparation of intermediates 35-39 is as follows:

[0151] Intermediate 35: Synthesis of (R)-tert-butyl 3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(5-bromo-2-chlorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate Intermediate 35 was prepared according to the method of Intermediate 2. LCMS: RT = 2.25 min, [M+H-Boc] + =491.12.

[0152] Intermediate 36: Synthesis of (S)-3-(5-bromo-2-chlorophenyl)-2-(R)-1-tert-butyloxycarbonylpyrrolidin-3-yl)propanoic acid

[0153] Intermediate 36 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.11 min, [M+H-Boc] + =332.01.

[0154] Intermediate 37: Synthesis of tert-butyl (R)-3-(S)-3-(5-bromo-2-chlorophenyl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0155] Intermediate 37 was prepared by referring to the procedure of Intermediate 4.

[0156] Intermediate 38: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-chloro-5-vinylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0157] Intermediate 38 was prepared by following the procedure of Intermediate 5.

[0158] Intermediate 39: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-chloro-5-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0159] Intermediate 39 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.23 min, [M + H-Boc-tert-butyl] + =282.14.

[0160] The preparation of intermediates 40-44 is as follows:

[0161] Intermediate 40: Synthesis of ethyl 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoate

[0162] Ethyl 2-(pyridin-3-yl)acetate (3.80 g, 23.0 mmol) was dissolved in anhydrous THF (50 mL) at room temperature and stirred at -30°C under nitrogen. Lithium bistrimethylsilylamide (30 mL, 1.0 mol / L) and 1-bromo-3-(bromomethyl)benzene (7.48 g, 29.9 mmol) were then added. Stirring was continued for 6 hours, as determined by LCMS and TLC. After the reaction was complete, aqueous ammonium chloride (50 mL) was added to quench the reaction at -30°C. The mixture was extracted with EA (50 mL x 3). The collected organic phases were dried over colorless sodium sulfate and evaporated. The crude product was purified by column chromatography using n-hexane:ethyl acetate = 4:1 to yield 6.55 g of ethyl 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoate. LCMS: RT = 1.93 min, [M+H]+ = 334.08.

[0163] Intermediate 41: Synthesis of 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoic acid

[0164] At room temperature, ethyl 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoate (6.55 g, 19.67 mmol) was dissolved in methanol (60 ml) and water (12 ml) and stirred. Then, lithium hydroxide monohydrate (1.65 g, 39.29 mmol) was added. After stirring for 2 hours, TLC plate detection showed that the raw materials had reacted completely. The reaction was terminated, and the methanol was removed by evaporation. The pH was adjusted to 3-4 with 1N hydrochloric acid solution, and the mixture was extracted with ethyl acetate (50 ml × 3 times) to obtain 3.87 g of 3-(3-bromophenyl)-2-(pyridin-3-yl)propionic acid.

[0165] Intermediate 42: Synthesis of tert-butyl 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoate

[0166] At room temperature, 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoic acid (2.0 g, 6.54 mmol) was dissolved in tert-butanol (25 mL) and stirred. Di-tert-butyl dicarbonate (2.14 g, 9.81 mmol) and 4-dimethylaminopyridine (79.3 mg, 0.65 mmol) were then added. The mixture was stirred at 50°C for two hours, and LCMS analysis indicated that the starting material had reacted completely. The reaction was terminated, and the tert-butanol was removed by distillation. The mixture was quenched by the addition of water (40 mL), and extracted with ethyl acetate (40 mL x 3). The collected organic phases were dried over colorless sodium sulfate and distilled to obtain the crude product, which was then purified by column chromatography using a 3:1 ratio of n-hexane:ethyl acetate to yield 1.08 g of tert-butyl 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoate. LCMS: RT = 2.11 min, [M+H] + =362.42.

[0167] Intermediate 43: Synthesis of tert-butyl 2-(pyridin-3-yl)-3-(3-vinylphenyl)propionate

[0168] At room temperature, tert-butyl 3-(3-bromophenyl)-2-(pyridin-3-yl)propanoate (1.08 g, 2.97 mmol) was dissolved in tetrahydrofuran (18 ml) and water (2 ml) and stirred. Potassium trifluoro(vinyl)borate (0.60 g, 4.46 mmol), cesium carbonate (3.90 g, 12 mmol) and triphenylphosphine (78 mg, 0.30 mmol) were then added. Palladium dichloride (27 mg, 0.5 mmol) was then added to the mixture. The reaction was terminated after the reaction mixture was stirred at 85°C overnight (15 hours) and analyzed by LCMS. The tetrahydrofuran was removed by distillation and quenched with sodium chloride solution (20 mL). The mixture was extracted with ethyl acetate (30 mL x 3). The collected organic phases were dried over colorless sodium sulfate and distilled. The crude product was purified by column chromatography using n-hexane:ethyl acetate = 3:1 to obtain 800 mg of tert-butyl 2-(pyridin-3-yl)-3-(3-vinylphenyl)propanoate. LCMS: RT = 2.09 min, [M+H] + =310.23.

[0169] Intermediate 44: Synthesis of tert-butyl 3-(3-formylphenyl)-2-(pyridin-3-yl)propanoate

[0170] At room temperature, tert-butyl 2-(pyridin-3-yl)-3-(3-vinylphenyl)propanoate (800 mg, 2.6 mmol) was dissolved in tetrahydrofuran (10 mL) and water (4 mL) and stirred at room temperature. Potassium osmate (10 mg, 0.26 mmol) was added, followed by sodium periodate (1.11 g, 5.19 mmol). The reaction was continued at room temperature with stirring for 9 hours. LCMS indicated complete reaction of the starting material. The tetrahydrofuran was removed by distillation at low temperature, and the mixture was quenched by the addition of aqueous sodium thiosulfate (15 mL). The mixture was extracted with ethyl acetate (15 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 1) to yield 318 mg of tert-butyl 3-(3-formylphenyl)-2-(pyridin-3-yl)propanoate. LCMS: RT = 1.86 min, [M+H] + =312.20.

[0171] The preparation of intermediates 45-49 is as follows:

[0172] Intermediate 45: Synthesis of tert-butyl (R)-2-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(3-bromophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0173] Intermediate 45 was prepared by referring to the method of Intermediate 2. LCMS: RT = 2.17 min, [M + H-tert-butyl] + =501.13.

[0174] Intermediate 46: Synthesis of (S)-3-(3-bromophenyl)-2-(R)-1-tert-butyloxycarbonyl)pyrrolidin-2-ylpropanoic acid

[0175] Intermediate 46 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.05 min, [MH] - =396.03.

[0176] Intermediate 47: Synthesis of (R)-2-(S)-3-(3-bromophenyl)-1-tert-butoxy-1-oxopropan-2-ylpyrrolidine-1-carboxylic acid tert-butyl ester Intermediate 47 was prepared according to the method of Intermediate 4. LCMS: RT = 2.24 min, [MH-tert-butyl] + =396.07.

[0177] Intermediate 48: Synthesis of (R)-2-(S)-1-tert-butoxy-1-oxo-3-(3-vinylphenyl)propyl-2-ylpyrrolidine-1-carboxylic acid tert-butyl ester

[0178] Intermediate 48 was prepared by referring to the method of Intermediate 5. LCMS: RT = 2.23 min, [M+H-Boc] + =302.13.

[0179] Intermediate 49: Synthesis of tert-butyl (R)-2-(S)-1-tert-butoxy-3-(3-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0180] Intermediate 49 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.07 min, [M+H] + =404.28.

[0181] The preparation of intermediates 50-53 is as follows:

[0182] Intermediate 50: Synthesis of tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-1-carboxylate

[0183] At room temperature, tert-butyl piperazine-1-carboxylate (5.0 g, 26.9 mmol) was dissolved in dichloromethane (40 mL) and stirred. Tert-butyl bromoacetate (5.51 g, 28.2 mmol) and triethylamine (5.43 g, 53.8 mmol) were then added. After stirring at room temperature for 6 hours, LCMS monitoring indicated that the starting material had reacted completely. The reaction was terminated and quenched with water (35 mL). The mixture was extracted with ethyl acetate (50 mL x 3 times), and the collected organic phases were dried over anhydrous sodium sulfate to yield 7.61 g of tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-1-carboxylate. LCMS: RT = 1.99 min, [M+H] + =301.21.

[0184] Intermediate 51: Synthesis of tert-butyl 4-(3-(3-bromophenyl)-1-tert-butoxy-1-oxopropan-2-yl)piperazine-1-carboxylate

[0185] At room temperature, tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-1-carboxylate (3.79 g, 12.6 mmol) was dissolved in anhydrous THF (35 ml) and stirred at -78°C under nitrogen protection. Then, lithium bistrimethylsilylamide (16.8 ml, 1.0 mol per liter) and 1-bromo-3-(bromomethyl)benzene (3.78 g, 15.1 mmol) were added. After stirring for 6.0 hours, LCMS and TLC plate detection showed that the raw material had reacted completely. The mixture was quenched by the addition of aqueous ammonium chloride (50 ml) at -78°C and extracted with EA (50 ml × 3 times). The collected organic phases were dried over colorless sodium sulfate and evaporated. The crude product was purified by column chromatography using n-hexane:ethyl acetate = 3:1 to obtain 4.63 g of tert-butyl 4-(3-(3-bromophenyl)-1-tert-butoxy-1-oxopropan-2-yl)piperazine-1-carboxylate. LCMS: RT = 2.34 min, [M+H] + =469.08.

[0186] Intermediate 52: Synthesis of tert-butyl 4-(1-tert-butoxy)-1-oxo-3-(3-vinylphenyl)propan-2-yl)piperazine-1-carboxylate

[0187] At room temperature, tert-butyl 4-(3-(3-bromophenyl)-1-tert-butoxy-1-oxopropan-2-yl)piperazine-1-carboxylate (4.63 g, 9.9 mmol) was dissolved in tetrahydrofuran (50 ml) and water (5.5 ml) and stirred. Potassium trifluoro(vinyl)borate (1.99 g, 14.9 mmol), cesium carbonate (9.65 g, 29.7 mmol) and triphenylphosphine (260 g, 0.99 mmol) were then added. Under nitrogen protection, palladium dichloride (90 mg, The reaction mixture was stirred at 85°C overnight (0.51 mmol) for 15 hours, followed by LCMS analysis. The reaction was complete, and the tetrahydrofuran was removed by distillation. The mixture was quenched by the addition of sodium chloride solution (60 ml), and extracted with ethyl acetate (60 ml x 3). The collected organic phases were dried over colorless sodium sulfate and distilled. The crude product was purified by column chromatography using n-hexane:ethyl acetate = 3:1 to obtain 3.61 g of tert-butyl 4-(1-tert-butoxy)-1-oxo-3-(3-vinylphenyl)propan-2-yl)piperazine-1-carboxylate. LCMS: RT = 2.29 min, [M+H] + =417.22.

[0188] Intermediate 53: Synthesis of tert-butyl 4-(1-tert-butoxy)-3-(3-formylphenyl)-1-oxopropan-2-yl)piperazine-1-carboxylate

[0189] At room temperature, tert-butyl 4-(1-tert-butoxy)-1-oxo-3-(3-vinylphenyl)propan-2-yl)piperazine-1-carboxylate (3.61 g, 8.65 mmol) was dissolved in tetrahydrofuran (40 ml) and water (20 ml) and stirred at room temperature. Potassium osmate (32 mg, 0.87 mmol) was added, followed by sodium periodate (3.68 g, 17.3 mmol). The reaction was continued with stirring at room temperature for 1 hour. LCMS analysis showed that the starting material had completely reacted, and the reaction was terminated. Most of the tetrahydrofuran was removed by distillation at low temperature, and the mixture was quenched with aqueous sodium sulfite (15 ml) and water (30 ml). The mixture was extracted with ethyl acetate (50 ml x 3 times), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 4 / 1) to obtain 3.06 g of tert-butyl 4-(1-tert-butoxy)-3-(3-formylphenyl)-1-oxopropan-2-yl)piperazine-1-carboxylate. LCMS: RT = 2.14 min, [M+H] + =419.15.

[0190] The preparation of intermediates 54-56 is as follows:

[0191] Intermediate 54: Synthesis of tert-butyl 3-(3-bromophenyl)propionate

[0192] 3-(3-Bromophenyl)propionic acid (343 mg, 1.5 mmol), tert-butanol (166 mg, 2.2 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (343 mg, 1.8 mmol) and 4-dimethylaminopyridine (18 mg, 0.15 mmol) were dissolved in dichloromethane (5 ml) at room temperature, replaced with nitrogen, and reacted for 6 hours.

[0193] After the reaction was completed, saturated sodium bicarbonate solution (10 ml) was added to quench the reaction, and the mixture was extracted with dichloromethane (20 ml × 2 times) and concentrated to dryness. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 20) to give 399 mg of tert-butyl 3-(3-bromophenyl)propionate.

[0194] Intermediate 55: Synthesis of tert-butyl 3-(3-vinylphenyl)propionate

[0195] At room temperature, tetrahydrofuran (9 ml), tert-butyl 3-(3-bromophenyl)propionate (255 mg, 1.1 mmol), triphenylphosphine (17.3 mg, 0.06 mmol), cesium carbonate (536 mg, 1.6 mmol), palladium dichloride (3.9 mg, 0.02 mmol), potassium ethylenetrifluoroborate (230 mg, 1.6 mmol), and purified water (1 ml) were added sequentially. The atmosphere was purged with nitrogen and the reaction was carried out at 85°C for 18 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with tap water (10 ml) and ethyl acetate (30 ml), and concentrated to dryness. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to obtain 164 mg of tert-butyl 3-(3-vinylphenyl)propionate.

[0196] Intermediate 56: Synthesis of tert-butyl 3-(3-formylphenyl)propionate

[0197] At room temperature, tert-butyl 3-(3-vinylphenyl)propionate (164 mg, 0.7 mmol) was dissolved in tetrahydrofuran (8 mL) and purified water (2 mL). The mixture was cooled with ice water, and then potassium osmate dihydrate (1.3 mL, 0.1 mmol, 40 mg) and sodium periodate (301 mg, 1.4 mmol) were added sequentially. The mixture was allowed to react in an ice-water bath for 1 hour. After completion of the reaction, the mixture was quenched with saturated aqueous sodium sulfite (10 mL), extracted with tap water (10 mL) and ethyl acetate (30 mL x 2), and concentrated to dryness. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 5) to yield 135 mg of tert-butyl 3-(3-formylphenyl)propionate. LCMS: RT = 2.23 min, [M+H] + =235.17.

[0198] The preparation of intermediates 57-58 is as follows:

[0199] Intermediate 57: Synthesis of tert-butyl 3-(2-vinylphenyl)propionate

[0200] Intermediate 57 was prepared by following the procedure of Intermediate 55.

[0201] Intermediate 58: Synthesis of tert-butyl 3-(2-formylphenyl)propionate

[0202] Intermediate 58 was prepared by following the procedure of Intermediate 56.

[0203] The preparation of intermediate 59 is as follows:

[0204] Intermediate 59: Synthesis of 5-ethylnicotinaldehyde

[0205] Tetrahydrofuran (10 ml) and 3-bromo-5-ethylpyridine (300 mg, 1.6 mmol) were added at room temperature, the atmosphere was replaced with nitrogen, and the dry ice / ethanol system was cooled to below -60°C. n-Butyl lithium (0.76 ml, 1.9 mmol, 2.5 mol / L) was added dropwise, and the reaction was kept warm for 1 hour. Subsequently, N,N-dimethylformamide (139 mg, 1.9 mmol) was added, and the temperature was gradually raised to room temperature for 2 hours.

[0206] After the reaction was completed, the mixture was quenched with saturated aqueous ammonium chloride (10 ml), extracted with ethyl acetate (20 ml), and concentrated to dryness. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to obtain 82 mg of 5-ethylnicotinaldehyde. LCMS: RT = 1.79 min, [M+H] + =136.06.

[0207] The preparation of intermediate 60-65 is as follows:

[0208] Intermediate 60: Synthesis of (R)-4-benzyl-3-(3-bromophenyl)propionyl)oxazolidin-2-one

[0209] Under ice bath, to a solution of 3-(3-bromophenyl)propionic acid (15.0 g, 65.5 mmol) in tetrahydrofuran (200 ml) were added triethylamine (16.5 g, 163.8 mmol) and pivaloyl chloride (9.4 g, 78.6 mmol) in sequence. After stirring for 30 minutes, lithium chloride (3.3 g, 78.6 mmol) was added to the system. Stirring was continued for 30 minutes, and (R)-4-benzyloxazolidin-2-one (12.7 g, 72.0 mmol) was added. The temperature was raised to room temperature and the reaction was carried out for 2 hours. After completion of the reaction, the reaction was quenched by addition of saturated aqueous ammonium chloride solution, followed by extraction with ethyl acetate (100 ml x 2). The combined organic phases were washed with saturated brine (100 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to yield 22.8 g of (R)-4-benzyl-3-(3-bromophenyl)propionyl)oxazolidin-2-one. LCMS: RT = 2.18 min, [M+H] + =388.02.

[0210] Intermediate 61: Synthesis of (R)-4-benzyl-3-(R)-3-(3-bromophenyl)-2-methylpropionyl)oxazolidin-2-one

[0211] To a solution of (R)-4-benzyl-3-(3-bromophenyl)propionyl)oxazolidin-2-one (8.0 g, 20.7 mmol) in tetrahydrofuran (120 ml) was added iodomethane (5.9 g, 41.4 mmol) at -45°C, followed by the slow dropwise addition of sodium bistrimethylsilylamide (31.1 ml, 31.1 mmol). The mixture was stirred at -45°C for 1 hour. After completion of the reaction, as monitored by TLC, saturated aqueous ammonium chloride was added to quench the reaction. The mixture was then extracted with ethyl acetate (100 ml x 2). The combined organic phases were washed with saturated brine (100 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a residue which was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to yield 4.5 g of (R)-4-benzyl-3-(R)-3-(3-bromophenyl)-2-methylpropionyl)oxazolidin-2-one. LCMS: RT = 2.23 min, [M+H] + =402.11.

[0212] Intermediate 62: Synthesis of (R)-3-(3-bromophenyl)-2-methylpropanoic acid

[0213] To a solution of (R)-4-benzyl-3-(R)-3-(3-bromophenyl)-2-methylpropanoyl)oxazolidin-2-one (4.5 g, 11.2 mmol) in tetrahydrofuran (90 ml) was added 30% aqueous hydrogen peroxide solution (0.6 g, 16.8 mmol) under ice-water bath, and the mixture was reacted for 30 minutes. Subsequently, an aqueous solution (10 ml) of lithium hydroxide (0.4 g, 16.8 mmol) was added, and the temperature was raised to room temperature and the reaction was carried out for 3 hours. After the reaction was completed, an excess of saturated sodium bisulfate solution was slowly added to the reaction mixture to quench the reaction. The mixture was stirred for 10 minutes, and then a saturated aqueous solution of citric acid was added to adjust the pH to 3. The mixture was extracted with ethyl acetate (50 ml x 2). The combined organic phases were washed with saturated brine (50 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to obtain 2.5 g of (R)-3-(3-bromophenyl)-2-methylpropanoic acid. LCMS: RT = 1.94 min, [MH] - =241.22.

[0214] Intermediate 63: Synthesis of (R)-tert-butyl 3-(3-bromophenyl)-2-methylpropionate

[0215] To a solution of (R)-3-(3-bromophenyl)-2-methylpropanoic acid (2.5 g, 10.7 mmol) in 2-methyltetrahydrofuran (50 mL) at room temperature was added O-tert-butyl-N,N'-diisopropylisourea (6.6 g, 32.2 mmol). The atmosphere was purged with nitrogen three times and the temperature was raised to 65°C for overnight reaction. After completion of the reaction, the mixture was filtered, the filter cake washed with ethyl acetate, and the filtrate diluted with ethyl acetate. The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to yield 2.2 g of (R)-tert-butyl 3-(3-bromophenyl)-2-methylpropanoate. LCMS: RT = 2.24 min, [MH-tert-butyl] - =240.87.

[0216] Intermediate 64: Synthesis of (R)-tert-butyl 2-methyl-3-(3-vinylphenyl)propionate

[0217] At room temperature, to the reaction flask were added tert-butyl (R)-3-(3-bromophenyl)-2-methylpropanoate (2.2 g, 7.4 mmol), triphenylphosphine (194 mg, 0.74 mmol), cesium carbonate (7.2 g, 22.2 mmol), palladium dichloride (66 mg, 0.37 mmol) and potassium ethylene trifluoroborate (1.5 g, 11.1 mmol), and the atmosphere was replaced with nitrogen three times. Then, tetrahydrofuran (40 ml) and purified water (5 ml) were added. The temperature was raised to 85°C and the reaction was allowed to proceed overnight. After completion of the reaction, the mixture was cooled to room temperature and diluted with water (20 mL). The mixture was then extracted with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a residue which was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to yield 1.4 g of (R)-tert-butyl 2-methyl-3-(3-vinylphenyl)propanoate. LCMS: RT = 2.14 min, [M+H] + =247.23.

[0218] Intermediate 65: Synthesis of (R)-tert-butyl 3-(3-formylphenyl)-2-methylpropionate

[0219] To a solution of tert-butyl (R)-2-methyl-3-(3-vinylphenyl)propanoate (1.4 g, 5.7 mmol) in tetrahydrofuran (28 ml) was added aqueous potassium osmate (1.0 ml, 0.1 mmol, 40 mg / ml) under ice-water bath. The mixture was stirred for 15 minutes, followed by the addition of an aqueous solution (12 ml) of sodium periodate (2.4 g, 11.4 mmol). The reaction was allowed to warm to room temperature. After completion of the reaction, as monitored by TLC, an excess of aqueous sodium bisulfate was slowly poured into the system to quench the reaction. The mixture was stirred for 30 minutes, and the mixture was extracted with ethyl acetate (20 ml × 2 times). The combined organic phases were washed with saturated brine (20 ml × 2 times), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue which was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 5) to give 0.8 g of tert-butyl (R)-3-(3-formylphenyl)-2-methylpropanoate. LCMS: RT = 2.04 min, [M+H] + =249.28.

[0220] Preparation of Intermediate 66, specifically the following steps: Intermediate 66: Synthesis of tert-butyl 4-(3-formylphenyl)piperazine-1-carboxylate

[0221] Toluene (3 ml), 3-bromobenzaldehyde (200 mg, 1.1 mmol), tert-butyl piperazine-1-carboxylate (241 mg, 1.3 mmol), sodium tert-butoxide (311 mg, 3.3 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (67 mg, 0.1 mmol), tris(dibenzylideneacetone)dipalladium (49 mg, 0.05 mmol) were added at room temperature, the atmosphere was replaced with nitrogen, and the temperature was raised to 100°C for reaction for 4 hours.

[0222] After the reaction, the mixture was cooled to room temperature, diluted with water (10 ml), and then adjusted to pH 5 with aqueous citric acid. The mixture was extracted with ethyl acetate (20 ml x 2) and concentrated to dryness. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to obtain 110 mg of tert-butyl 4-(3-formylphenyl)piperazine-1-carboxylate. LCMS: RT = 2.04 min, [M+H-tert-butyl] + =235.14.

[0223] The preparation of intermediate 67-70 is as follows:

[0224] Intermediate 67: Synthesis of dimethyl (S)-2-(3-bromobenzyl)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)methyl)malonate

[0225] To a solution of dimethyl (S)-2-(2-(1-(tert-butoxycarbonyl)pyrrolidine-3-ethyl)ethyl)malonate (3.1 g, 9.8 mmol) in tetrahydrofuran (60 ml) was slowly added sodium hydride (600 mg, 15 mmol) under ice-water bath. After stirring under ice-water bath for 30 min, a solution of m-bromobenzyl bromide (2.73 g, 11 mmol) in tetrahydrofuran (20 ml) was slowly added to the system, and the temperature was then naturally raised to room temperature for 3 hours. After completion of the reaction, the reaction was quenched by addition of saturated aqueous ammonium chloride solution, followed by extraction with ethyl acetate (20 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to yield 3.41 g of dimethyl (S)-2-(3-bromobenzyl)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)methyl)malonate. LCMS: RT = 2.33 min, [M+H] + =484.17.

[0226] Intermediate 68: Synthesis of (S)-2-(1-(tert-Butyloxycarbonyl)pyrrolidin-3-yl)methyl)-2-(3-vinylbenzyl)malonate

[0227] At room temperature, to the reaction flask were added dimethyl (S)-2-(3-bromobenzyl)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)methyl)malonate (490 mg, 1.01 mmol), triphenylphosphine (26.0 mg, 0.1 mmol), cesium carbonate (0.97 g, 3 mmol), palladium dichloride (9.2 mg, 0.05 mmol), potassium ethylene trifluoroborate (201 mg, 1.5 mmol), and the atmosphere was replaced with nitrogen three times. Then, tetrahydrofuran (18 ml) and water (2 ml) were added. The temperature was raised to 85°C and the reaction was allowed to proceed overnight. After completion of the reaction, the mixture was cooled to room temperature and diluted with water (20 mL). The mixture was then extracted with ethyl acetate (30 mL twice). The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to obtain 332 mg of (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)methyl)-2-(3-vinylbenzyl)malonate. LCMS: RT = 2.33 min, [M+H-Boc] + =332.23.

[0228] Intermediate 69: Synthesis of tert-butyl (3S)-3-(3-methoxy-3-oxo-2-(3-vinylbenzyl)propyl)pyrrolidine-1-carboxylate

[0229] To a solution of (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)methyl)-2-(3-vinylbenzyl)malonate (1.8 g, 4.17 mmol) in dimethyl sulfoxide (20 ml) was added lithium chloride (800 mg, 20 mmol) at room temperature. The temperature was raised to 140°C and the reaction was allowed to proceed for 4 hours. After completion of the reaction, the mixture was cooled to room temperature and diluted with water. The mixture was extracted with ethyl acetate (20 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 5) to give 1.2 g of tert-butyl (3S)-3-(3-methoxy-3-oxo-2-(3-vinylbenzyl)propyl)pyrrolidine-1-carboxylate. LCMS: RT = 2.55 min, [M+H-Boc] + =274.23.

[0230] Intermediate 70: Synthesis of tert-butyl (3S)-3-(2-(3-formylbenzyl)-3-methoxy-3-oxopropyl)pyrrolidine-1-carboxylate

[0231] To a solution of tert-butyl (3S)-3-(3-methoxy-3-oxo-2-(3-vinylbenzyl)propyl)pyrrolidine-1-carboxylate (411 mg, 1.1 mmol) in tetrahydrofuran (10 ml) under ice-water bath was added aqueous potassium osmate solution (0.7 ml, 0.07 mmol, 40 mg / ml) and stirred for 15 minutes. Subsequently, an aqueous solution (5.0 ml) of sodium periodate (426 mg, 2.0 mmol) was added and the mixture was allowed to warm to room temperature for reaction. After completion of the reaction, the reaction was monitored by TLC. An excess of aqueous sodium bisulfate solution was slowly poured into the system to quench the reaction. The mixture was stirred for 30 minutes, and then extracted with ethyl acetate (20 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 5) to obtain 342 mg of tert-butyl (3S)-3-(2-(3-formylbenzyl)-3-methoxy-3-oxopropyl)pyrrolidine-1-carboxylate. LCMS: RT = 2.09 min, [M+H] + =276.22.

[0232] The preparation of intermediates 71-75 is as follows:

[0233] Intermediate 71: Synthesis of tert-butyl (R)-3-((S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(4-bromophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0234] At room temperature, tert-butyl (R)-3-(2-((S)-4-benzyl-2-oxooxazolidin-3-yl)-2-oxoethyl)pyrrolidine-1-carboxylate (1.5 g, 3.9 mmol) was dissolved in anhydrous THF (20 ml) and stirred at -30°C under nitrogen protection. Then, lithium bistrimethylsilylamide (5.07 ml, 1.0 mol per liter) and 1-bromo-4-(bromomethyl)benzene (1.07 g, 4.3 mmol) were added. After stirring for 14.0 hours, the mixture was stirred for 14.0 hours. After completion of the reaction of the starting material, the reaction was quenched by adding aqueous ammonium chloride solution (30 ml) at -30°C and extracted with EA (40 ml x 3 times). The collected organic phases were dried over colorless sodium sulfate and evaporated. The crude product was purified by column chromatography using n-hexane:ethyl acetate = 4:1 to obtain 1.63 g of tert-butyl (R)-3-((S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(4-bromophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate.

[0235] Intermediate 72: Synthesis of (S)-3-(4-bromophenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoic acid

[0236] Intermediate 72 was prepared by following the procedure of Intermediate 3.

[0237] Intermediate 73: Synthesis of tert-butyl (S)-3-(4-bromophenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoate

[0238] Intermediate 73 was prepared by following the procedure of Intermediate 4.

[0239] Intermediate 74: Synthesis of tert-butyl (S)-3-(4-vinylphenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoate

[0240] At room temperature, tert-butyl (S)-3-(4-bromophenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoate (1.04 g, 2.29 mmol) was dissolved in tetrahydrofuran (18 ml) and water (2 ml) and stirred. Potassium trifluoro(vinyl)borate (0.46 g, 3.44 mmol), cesium carbonate (2.23 g, 6.86 mmol) and triphenylphosphine (60 mg, 0.23 mmol) were then added. Palladium dichloride (21 ml) was then added to the mixture. The reaction mixture was stirred at 85°C overnight (15 hours) and analyzed by LCMS. The reaction was complete and the tetrahydrofuran was removed by distillation. Water (25 mL) was added to quench the mixture and the mixture was extracted with ethyl acetate (30 mL x 3). The collected organic phases were dried over colorless sodium sulfate and evaporated. The crude product was purified by column chromatography using n-hexane:ethyl acetate = 3:1 to obtain 652 mg of tert-butyl (S)-3-(4-vinylphenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoate. LCMS: RT = 2.32 min, [M+H] + =402.47.

[0241] Intermediate 75: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(4-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0242] Intermediate 75 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.20 min, [M+H] + =404.28.

[0243] The preparation of intermediate 76-80 is as follows:

[0244] Intermediate 76: Synthesis of tert-butyl ((R)-3-((S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(2-bromophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0245] Intermediate 76 was prepared by following the procedure of Intermediate 2.

[0246] Intermediate 77: Synthesis of (S)-3-(2-bromophenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoic acid

[0247] Intermediate 77 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.06 min, [MH] - =396.03.

[0248] Intermediate 78: Synthesis of tert-butyl (S)-3-(2-bromophenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoate

[0249] Intermediate 78 was prepared by following the procedure of Intermediate 4.

[0250] Intermediate 79: Synthesis of tert-butyl (R)-3-((S)-1-(tert-butoxy)-1-oxo-3-(2-vinylphenyl)propan-2-yl)pyrrolidine-1-carboxylate

[0251] Intermediate 79 was prepared by following the procedure of Intermediate 5.

[0252] Intermediate 80: Synthesis of tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0253] Intermediate 80 was prepared by following the procedure of Intermediate 6.

[0254] The preparation of intermediate 81-82 is as follows:

[0255] Intermediate 81: Synthesis of tert-butyl (R)-3-((S)-3-(3-allylphenyl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0256] At room temperature, (R)-3-((S)-3-(3-bromophenyl)-1-(tert-butoxy)-1-oxoprop-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester (4.0 g, 8.83 mmol) was dissolved in toluene (60 ml) and stirred. Then tri-n-butyl allyltin (3.51 g, 10.6 mmol) was added. Under nitrogen protection, tetrakistriphenylphosphine palladium (1.02 g, 0.88 mmol) was added and the mixture was stirred at 100°C overnight ( After 15 hours (TLC detection), the starting material was completely reacted, and the reaction was terminated. 1N sodium chloride solution (40 ml) was added to quench the reaction, and the mixture was extracted with ethyl acetate (60 ml × 3 times). The collected organic phases were dried over colorless sodium sulfate and evaporated. The crude product was purified by column chromatography using n-hexane:ethyl acetate = 3:1 to obtain 2.1 g of tert-butyl (R)-3-((S)-3-(3-allylphenyl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate.

[0257] Intermediate 82: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-1-oxo-3-(3-(2-oxoethyl)phenyl)propan-2-yl)pyrrolidine-1-carboxylate

[0258] Intermediate 82 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.21 min, [M+H] + =418.27.

[0259] The preparation of intermediates 83-86 is as follows:

[0260] Intermediate 83: Synthesis of dimethyl (R)-2-(3-bromophenethyl)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate

[0261] To a solution of (R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate (3.0 g, 10 mmol) in tetrahydrofuran (60 ml) was slowly added sodium hydride (600 mg, 15 mmol) under ice-water bath. After stirring for 30 min under ice-water bath, a solution of 1-bromo-3-(2-iodoethyl)benzene (3.3 g, 10.6 mmol) in tetrahydrofuran (20 ml) was slowly added to the system, and then the temperature was naturally raised to room temperature for reaction for 3 hours. After completion of the reaction, the reaction was quenched by addition of saturated aqueous ammonium chloride solution, followed by extraction with ethyl acetate (20 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to obtain 3.13 g of dimethyl (R)-2-(3-bromophenethyl)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)malonate. LCMS: RT = 2.33 min, [M+H] +=484.16.

[0262] Intermediate 84: Synthesis of dimethyl (R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-2-(3-vinylphenethyl)malonate

[0263] Intermediate 84 was prepared by referring to the method of Intermediate 74. LCMS: RT = 2.36 min, [M+H-Boc] + =332.23.

[0264] Intermediate 85: Synthesis of tert-butyl (3R)-3-(1-methoxy-1-oxo-4-(3-vinylphenyl)butan-2-yl)pyrrolidine-1-carboxylate

[0265] To a solution of dimethyl (R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-2-(3-vinylphenylethyl)malonate (430 mg, 1.0 mmol) in dimethyl sulfoxide (10 mL) was added lithium chloride (200 mg, 4 mmol) at room temperature. The temperature was raised to 140°C and the reaction was allowed to react for 4 hours. After completion of the reaction, the mixture was cooled to room temperature and diluted with water. The mixture was extracted with ethyl acetate (20 mL x 2). The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 5) to give 257 mg of tert-butyl (3R)-3-(1-methoxy-1-oxo-4-(3-vinylphenyl)butan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.57 min, [M+H-Boc] + =274.13.

[0266] Intermediate 86: Synthesis of tert-butyl (3R)-3-(4-(3-formylphenyl)-1-methoxy-1-oxobutan-2-yl)pyrrolidine-1-carboxylate

[0267] Intermediate 86 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.13 min, [M+H] + =276.25.

[0268] The preparation of intermediates 87-91 is as follows:

[0269] Intermediate 87: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-hydroxyethyl)phenyl-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0270] To a solution of tert-butyl (R)-3-(S)-1-tert-butoxy-1-oxo-3-(2-vinylphenyl)propyl-2-ylpyrrolidine-1-carboxylate (2.7 g, 6.7 mmol) in tetrahydrofuran (25 ml) was added dropwise a solution of 9-borabicyclo[3.3.1]nonane in tetrahydrofuran (26 ml, 0.5 mol) under ice-water bath. After stirring at room temperature for 18 hours, methanol (10 ml), aqueous sodium hydroxide solution (20 ml, 3 mol) and 30% hydrogen peroxide (4 ml) were added to the reaction solution under ice-water bath, and the reaction was allowed to react at room temperature for 3 hours. After the reaction was completed, as monitored by TLC, an excess of a saturated sodium bisulfate solution was added to quench the reaction, followed by stirring for 10 minutes, dilution with water (30 ml), and extraction with ethyl acetate (50 ml x 2). The combined organic phases were washed with saturated brine (50 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 2) to obtain 2.5 g of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-hydroxyethyl)phenyl-1-oxopropan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.20 min, [M+H-tert-butyl-tert-butoxycarbonyl] + =264.17.

[0271] Intermediate 88: Synthesis of tert-butyl (R)-3-(S)-1-tert-butoxy-1-oxo-3-(2-oxoethyl)phenyl)propan-2-ylpyrrolidine-1-carboxylate

[0272] To a solution of tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-hydroxyethyl)phenyl-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (2.5 g, 6.0 mmol) in ethyl acetate (30 ml) was added 2-iodobenzoic acid (1.7 g, 6.0 mmol) at room temperature. The reaction temperature was raised to 70°C and the mixture was allowed to react for 8 hours. After completion of the reaction, the mixture was filtered with suction, the filter cake was washed with ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain a residue which was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to obtain 1.8 g of tert-butyl (R)-3-(S)-1-tert-butoxy-1-oxo-3-(2-oxoethyl)phenyl)propan-2-ylpyrrolidine-1-carboxylate. LCMS: RT = 2.22 min, [M+H-tert-butyl-tert-butoxycarbonyl] + =262.97.

[0273] Intermediate 89: (R)-tert-Butyl 3-(S)-1-oxo-3-(3-vinylphenyl)propyl-2-yl)pyrrolidine-1-carboxylate

[0274] To a solution of tert-butyl (R)-3-(S)-1-tert-butoxy-1-oxo-3-(3-vinylphenyl)propyl-2-yl)pyrrolidine-1-carboxylate (1.9 g, 4.74 mmol) in dichloromethane (80 ml) was slowly added dropwise DIBAL-H (5.6 ml, 1 M solution in toluene) at -78°C. The reaction was allowed to proceed at -78°C for 3 hours. After completion of the reaction, the mixture was quenched with water and extracted with dichloromethane (50 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2) and then dried over anhydrous sodium sulfate. The residue was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to give 1.3 g of tert-butyl (R)-3-(S)-1-oxo-3-(3-vinylphenyl)propyl-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.07 min, [M+H] + =330.31.

[0275] Intermediate 90: Synthesis of tert-butyl (R)-3-(R)-1-cyano-3-(3-vinylphenyl)propyl-2-yl)pyrrolidine-1-carboxylate

[0276] Under ice-water bath, to a solution of (R)-3-(S)-1-oxo-3-(3-vinylphenyl)propyl-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester (450 mg, 1.31 mmol) in tetrahydrofuran (15 ml) was added potassium tert-butoxide (183 mg, 1.5 mmol). After reacting for 30 minutes, p-toluenesulfonylmethyl isocyanide (292 mg, 1.5 mmol) was added to the system. After warming to room temperature and reacting for 1 hour, methanol (2 ml) was added to the system, followed by addition of The reaction was heated to 60°C for 1 hour. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate (50 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a residue which was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 10) to obtain 270 mg of tert-butyl (R)-3-(R)-1-cyano-3-(3-vinylphenyl)propyl-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.17 min, [M+H] + =341.23.

[0277] Intermediate 91: Synthesis of tert-butyl (R)-3-(R)-1-cyano-3-(3-formylphenyl)propan-2-yl)pyrrolidine-1-carboxylate

[0278] Intermediate 91 was prepared by following the procedure of Intermediate 6.

[0279] The preparation of intermediate 92-96 is as follows:

[0280] Intermediate 92: Synthesis of tert-butyl (R)-3-((S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(5-bromo-2,4-difluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0281] Intermediate 92 was prepared by following the procedure of Intermediate 2.

[0282] Intermediate 93: Synthesis of (S)-3-(5-bromo-2,4-difluorophenyl)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)propanoic acid

[0283] Intermediate 93 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.03 min, [MH] - =432.01.

[0284] Intermediate 94: Synthesis of tert-butyl (R)-3-((S)-3-(5-bromo-2,4-difluorophenyl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0285] Intermediate 94 was prepared by following the procedure of Intermediate 4.

[0286] Intermediate 95: Synthesis of tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2,4-difluoro-5-vinylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0287] Intermediate 95 was prepared by following the procedure of Intermediate 10.

[0288] Intermediate 96: Synthesis of tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2,4-difluoro-5-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0289] Intermediate 96 was prepared by referring to the method of Intermediate 6. LCMS: RT = 2.56 min, [M-tert-butyl-tert-butyloxycarbonyl + H] + =284.0.

[0290] The preparation of intermediate 97-106 is as follows:

[0291] Intermediate 97: Synthesis of tert-butyl (R)-3-(S)-1-(S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(2-bromo-5-fluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0292] Intermediate 97 was prepared by referring to the method of Intermediate 2. LCMS: RT = 2.27 min, [M + H-tert-butyl]+ =477.14.

[0293] Intermediate 98: Synthesis of (S)-3-(2-bromo-5-fluorophenyl)-2-(R)-1-tert-butyloxycarbonyl)pyrrolidin-3-yl)propanoic acid

[0294] Intermediate 98 was prepared by referring to the method of Intermediate 3. LCMS: RT = 2.08 min, [MH] - =414.06.

[0295] Intermediate 99: Synthesis of tert-butyl (R)-3-(S)-3-(2-bromo-5-fluorophenyl)-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0296] Intermediate 99 was prepared by referring to the method of Intermediate 4. LCMS: RT = 2.37 min, [M+H-Boc] + =372.12.

[0297] Intermediate 100: Synthesis of (R)-3-(S)-1-tert-butoxy-1-oxo-3-(2-vinyl-5-fluorophenyl)propyl-2-ylpyrrolidine-1-carboxylic acid tert-butyl ester

[0298] Intermediate 100 was prepared by referring to the method of Intermediate 75. LCMS: RT = 2.30 min, [M+H] + =420.21.

[0299] Intermediate 101: Synthesis of (R)-tert-Butyl 3-(S)-1-tert-Butoxy-3-(2-hydroxyethyl-5-fluoro)phenyl-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0300] Intermediate 101 was prepared by referring to the method of Intermediate 87. LCMS: RT = 2.14 min, [M+H-tert-butyl-tert-butoxycarbonyl] + =282.21.

[0301] Intermediate 102: Synthesis of (R)-3-(S)-1-tert-butyl-1-oxo-3-(2-oxoethyl-5-fluoro)phenyl)propan-2-ylpyrrolidine-1-carboxylate

[0302] Intermediate 102 was prepared by referring to the method of Intermediate 88. LCMS: RT = 2.19 min, [M+H] + =436.24.

[0303] Intermediate 103: Synthesis of tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2-fluoro-5-(hydroxymethyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0304] At room temperature, tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2-fluoro-5-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (500 mg, 1.18 mmol) and anhydrous isopropanol (10 ml) were added. The mixture was cooled with ice water, and then sodium borohydride (45 mg, 1.18 mmol) was added in three batches. The mixture was reacted in an ice-water bath for 1 hour.

[0305] After the reaction was completed, saturated aqueous ammonium chloride solution (10 ml) was added to quench the reaction, and the mixture was extracted with ethyl acetate (10 ml × 2 times), washed with saturated aqueous sodium chloride solution (10 ml × 1 time), dried over sodium sulfate, and concentrated to dryness to obtain 405 mg of yellow oily product (R)-3-((S)-1-(tert-butoxy)-3-(2-fluoro-5-(hydroxymethyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester.

[0306] Intermediate 104: (R)-tert-Butyl 3-((S)-1-(tert-butoxy)-3-(5-(((1,3-dioxoisoindolin-2-yl)methyl)-2-fluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0307] At room temperature, tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2-fluoro-5-(hydroxymethyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (400 mg, 0.94 mmol), phthalimide (180 mg, 1.22 mmol), triphenylphosphine (492 mg, 1.88 mmol), and tetrahydrofuran (8 ml) were added in sequence, followed by the slow dropwise addition of diisopropyl azodicarboxylate (380 mg, 1.88 mmol), and the reaction was carried out at room temperature for 2 hours.

[0308] After the reaction was completed, the mixture was concentrated to dryness, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 4) to obtain 770 mg of tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(5-(((1,3-dioxoisoindolin-2-yl)methyl)-2-fluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate.

[0309] Intermediate 105: (R)-tert-Butyl 3-((S)-3-(5-(aminomethyl)-2-fluorophenyl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0310] At room temperature, tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(5-(((1,3-dioxoisoindolin-2-yl)methyl)-2-fluorophenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (770 mg, 1.39 mmol) was added and dissolved in ethanol (15 ml). Hydrazine hydrate (218 mg, 3.49 mmol, 80%) was then added, and the temperature was raised to 85°C for 3 hours.

[0311] After the reaction was complete, the white solid was removed by filtration. The filter cake was washed with ethyl acetate (20 ml x 2). The combined filtrates were concentrated to dryness, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate (containing 0.5% v / v triethylamine) / n-hexane = 9 / 1) to obtain 340 mg of tert-butyl (R)-3-((S)-3-(5-(aminomethyl)-2-fluorophenyl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate as a yellow oil. LCMS: RT = 2.11 min, [M+H] + =423.20.

[0312] Intermediate 106: (R)-tert-Butyl 3-((S)-1-(tert-Butoxy)-3-(2-fluoro-6-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0313] Intermediate 106 was prepared by following the procedure of Intermediate 6.

[0314] The intermediates used in the examples were all obtained according to the preparation methods of the aforementioned intermediates.

[0315] Example 1

[0316] Step A: Synthesis of (R)-3-(S)-1-tert-butoxy)-3-(2-((3-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl(3-fluoro-5-methoxybenzyl)amino)methyl)pyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0317] At room temperature, tert-butyl (R)-3-(S)-1-tert-butoxy)-3-(3-((3-fluoro-5-methoxybenzyl)amino)methyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (50.0 mg, 0.09 mmol) (obtained with reference to patent CN202080041924.X) was dissolved in 1,2-dichloroethane (2.0 ml). tert-butyl (R)-3-(R)-3-tert-butoxy-2-(2-formylpyridin-4-yl)methyl)-3-oxopropyl)pyrrolidine-1-carboxylate (46.0 mg, 0.11 mmol) was added. After stirring at room temperature for 40 minutes, sodium triacetoxyborohydride (47.7 mg, 0.23 mmol) was added and the reaction was carried out at 45°C overnight.

[0318] After the reaction was completed, the reaction solution was slowly poured into 10.0 ml of water, and the mixture was extracted with dichloromethane (20 ml x 2). The organic phases were combined, washed with saturated brine (20 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 1 / 2) to obtain 50.0 mg of (R)-3-(S)-1-tert-butoxy)-3-(2-((3-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl(3-fluoro-5-methoxybenzyl)amino)methyl)pyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate. LCMS: RT = 2.69 min, [M+H] + =931.63.

[0319] Step B: Synthesis of (S)-3-(3-(S)-2-carboxy-2-(R)-pyrrolidin-3-ethyl)benzyl)(3-fluoro-5-methoxybenzyl)amino)methyl)pyridin-4-yl)-2-((R)-pyrrolidin-3-yl)propanoic acid At room temperature, (R)-3-(S)-1-tert-butoxy)-3-(2-((3-(S)-3-tert-butoxy)-2-(R)-1-( (tert-Butyloxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl (3-fluoro-5-methoxybenzyl)amino)methyl)pyridin-4-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (50.0 mg, 0.05 mmol) was dissolved in a mixture of dioxane / water (2 / 1) (1.5 ml), and concentrated hydrochloric acid (223.0 μl) was added, and the mixture was reacted at 45°C for 3 hours.

[0320] After the reaction was completed, the product was concentrated under reduced pressure to obtain a residue which was separated by high performance liquid chromatography to yield 20.0 mg of (S)-3-(3-(S)-2-carboxy-2-(R)-pyrrolidin-3-ethyl)benzyl)(3-fluoro-5-methoxybenzyl)amino)methyl)pyridin-4-yl)-2-((R)-pyrrolidin-3-yl)propanoic acid. LCMS: RT = 1.49 min, [M+H] + =619.41; 1 H NMR(400MHz,DeuteRium Oxide)δ8.16(d,J=5.2Hz,1H),7.19(dd,J=16.8,8.5Hz,1H),7.11(d,J=7.8Hz,2H),7.06–6.9 8(m,3H),6.69–6.55(m,1H),6.53(d,J=7.3Hz,1H),6.46(dt,J=10.9,2.4Hz,1H),3.77–3.68( m,2H),3.64(S,3H),3.54(S,2H),3.49(S,2H),3.39(S,2H),3.35–3.22(m,4H),3.19–3.05(m, 2H),2.89–2.66(m,4H),2.66–2.52(m,2H),2.45–2.19(m,4H),2.05–1.97(m,2H),1.64(m,2H).

[0321] Example 2

[0322] Synthesis of (S)-3-((4-(S)-2-carboxy-2-(R)-pyrrolidin-3-ethyl)benzyl)(4-(S)-2-carboxy-2-(R)-pyrrolidin-3-yl)ethyl)pyridin-2-yl)methyl)amino)methyl)phenyl)-2-((R)-pyrrolidin-3-yl)propanoic acid (Compound 2) was performed by the following synthetic route:

[0323] Step A: Synthesis of 3,3'-(4-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)pyridin-2-yl)methyl)azepinediyl)bismethylene(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0324] At room temperature, 3,3'-((2S,2'S)-(azadiylbismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate) (50.0 mg, 0.06 mmol) (obtained with reference to patent CN202080041924.X) was dissolved in 1,2-dichloroethane (2.0 ml), and tert-butyl (R)-3-(R)-3-tert-butoxy-2-(2-formylpyridin-4-yl)methyl)-3-oxopropyl)pyrrolidine-1-carboxylate (29.0 mg, 0.07 mmol) was added. After stirring at room temperature for 40 minutes, sodium triacetoxyborohydride (31.8 mg, 0.15 mmol) was added and the reaction was carried out at 45°C overnight.

[0325] After the reaction, 10.0 ml of water was added for dilution, and the mixture was extracted with dichloromethane (20 ml x 2). The combined organic phases were washed with saturated brine (20 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 1 / 3) to obtain 48.0 mg of 3,3'-(4-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)pyridin-2-yl)methyl)azepinediyl)bis(methylene(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate). LCMS: RT = 2.64 min.

[0326] Step B: Synthesis of (S)-3-((4-(S)-2-carboxy-2-(R)-pyrrolidin-3-ethyl)benzyl)(4-(S)-2-carboxy-2-(R)-pyrrolidin-3-yl)ethyl)pyridin-2-yl)methyl)amino)methyl)phenyl)-2-((R)-pyrrolidin-3-yl)propanoic acid

[0327] At room temperature, 3,3'-(4-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)pyridin-2-yl)methyl)azadiyl)bismethylene(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate) (48.0 mg, 0.04 mmol) was dissolved in a mixture of dioxane / water (2 / 1) (1.5 ml), and concentrated hydrochloric acid (271.0 μl) was added, and the mixture was reacted at 45°C for 3 hours.

[0328] After the reaction was completed, the product was concentrated under reduced pressure to obtain a residue which was separated by high performance liquid chromatography to yield 17.0 mg of (S)-3-((4-(S)-2-carboxy-2-(R)-pyrrolidin-3-ethyl)benzyl)(4-(S)-2-carboxy-2-(R)-pyrrolidin-3-yl)ethyl)pyridin-2-yl)methyl)amino)methyl)phenyl)-2-((R)-pyrrolidin-3-yl)propanoic acid. LCMS: RT = 1.41 min, [MH] - =710.45.

[0329] Example 3

[0330] Step A: Synthesis of (R)-3-(S)-1-tert-butoxy)-3-(3-(6-(S)-3-tert-butoxy-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-yl)-3-oxopropyl)pyridin-2-yl)methyl(3-fluoro-5-methoxybenzyl)amino)methyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate

[0331] At room temperature, tert-butyl ((R)-3-(S)-1-tert-butoxy-3-(6-formylpyridin-2-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (100 mg, 0.25 mmol), tert-butyl (R)-3-(S)-1-tert-butoxy)-3-(3-((3-fluoro-5-methoxybenzyl)amino)methyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (162 mg, 0.29 mmol) and glacial acetic acid (12 mg, 0.2 mmol) were dissolved in dry isopropanol (3 ml). The atmosphere was replaced with nitrogen three times and the temperature was raised to 45° C. for 5 hours. Then, sodium cyanoborohydride (38 mg, 0.95 mmol) was added and the mixture was stirred for 1 h. The reaction was continued at 45°C for 18 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate (20 ml x 2 times). The combined organic phases were washed with saturated brine (20 ml x 2 times), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a residue which was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to give 130 mg of (R)-3-(S)-1-tert-butoxy)-3-(3-(6-(S)-3-tert-butoxy-2-(R)-1-tert-butoxycarbonylpyrrolidin-3-yl)-3-oxopropyl)pyridin-2-yl)methyl(3-fluoro-5-methoxybenzyl)amino)methyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate.

[0332] Step B: Synthesis of (2S,2'S)-3,3'-((6-((S)-2-carboxy-2-(R)-pyrrolidin-3-yl)ethyl)pyridin-2-yl)methyl)azadiyl)bis(methylene)bis(methylene)bis(3,1-phenylene))bis(2-(R)-pyrrolidin-3-yl)propanoic acid)

[0333] Compound 3 was synthesized according to the method of Example 1. LCMS: RT = 1.54 min, [MH] - =617.41.

[0334] Example 4

[0335] Step A: Synthesis of 3,3'-(6-(S)-3-tert-butoxy-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)pyridin-2-yl)methyl)azepinediyl)bismethylene(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0336] At room temperature, tert-butyl ((R)-3-(S)-1-tert-butoxy-3-(6-formylpyridin-2-yl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (100 mg, 0.25 mmol), 3,3'-((2S,2'S)-(azadiylbismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate) (234 mg, 0.29 mmol) and glacial acetic acid (12 mg, 0.2 mmol) were dissolved in dry isopropanol (3 ml). The atmosphere was replaced with nitrogen three times, and the temperature was raised to 45° C. for reaction for 5 hours. Subsequently, sodium cyanoborohydride (38 mg, 0.95 mmol) was added and the reaction was carried out at 45° C. for 18 hours. After the reaction, the mixture was diluted with water and extracted with ethyl acetate (20 ml × 2 times). The combined organic phases were washed with saturated brine (20 ml × 2 times), then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to give 110 mg of 3,3'-(6-(S)-3-tert-butoxy-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)pyridin-2-yl)methyl)azepinediyl)bismethylene(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate).

[0337] Step B: Synthesis of (2S,2'S)-3,3'-((6-((S)-2-carboxy-2-(R)-pyrrolidin-3-yl)ethyl)pyridin-2-yl)methyl)azadiyl)bis(methylene)bis(methylene)bis(3,1-phenylene))bis(2-(R)-pyrrolidin-3-yl)propanoic acid)

[0338] Compound 4 was synthesized according to the method of Example 2. LCMS: RT = 1.38 min, [MH] -=710.19.

[0339] Example 12

[0340] Step A: Synthesis of 3,3'-((2S,2'S)di-tert-butyl(3-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)-2-fluorobenzyl)azepinediyl)bismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0341] At room temperature, tert-butyl (R)-3-(S)-1-tert-butoxy-3-(2-fluoro-3-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (150 mg, 0.36 mmol) and 3,3'-((2S,2'S)-(azadiylbismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate) (225 mg, 0.29 mmol) were dissolved in isopropanol (5 ml), glacial acetic acid (43 mg, 0.72 mmol) was added, and the reaction solution was stirred at 45°C overnight. Then, sodium cyanoborohydride (46 mg, 0.72 mmol) was added thereto and the reaction was continued at 45°C for 1 minute. After 8 hours, the reaction was completed and diluted with water. The mixture was extracted with ethyl acetate (20 ml × 2 times). The combined organic phases were washed with saturated brine (20 ml × 2 times), then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 1) to obtain 200 mg of 3,3'-((2S,2'S)di-tert-butyl(3-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)-2-fluorobenzyl)azepinediyl)bis(methylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate).

[0342] Step B: Synthesis of (2S,2'S)-3,3'(3-((S)-2-carboxy-2-(R)-pyrrolidin-3-yl)ethyl)-2-fluorobenzyl)azadiyl)bismethylene)bis(3,1-phenylene)bis(2-(R)-pyrrolidin-3-yl)propanoic acid)

[0343] At room temperature, 3,3'-((2S,2'S)di-tert-butyl(3-(S)-3-tert-butoxy)-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)-2-fluorobenzyl)azadiyl)bismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate) (200 mg, 0.17 mmol) was dissolved in dioxane (2.5 ml), concentrated hydrochloric acid (0.5 ml) was added, and the reaction was carried out at 45°C for 18 hours. After completion of the reaction, the product was concentrated under reduced pressure to obtain a residue which was separated and purified by high performance liquid chromatography to yield 24 mg of (2S,2'S)-3,3'-(3-((S)-2-carboxy-2-(R)-pyrrolidin-3-yl)ethyl)-2-fluorobenzyl)azadiyl)bismethylene)bis(3,1-phenylene)bis(2-(R)-pyrrolidin-3-yl)propanoic acid. LCMS: RT = 1.41 min, [MH] - =727.45, HPLC: 93.17%.

[0344] 1 H NMR(400MHz,D2O)δ7.38-7.23(m,5H),7.24-7.06(m,6H),4.27(s,6H),3.56-3.49(m,3H),3.38-3.32(m,3H),3. 22-3.15(m,3H),3.01-2.94(m,3H),2.87-2.66(m,6H),2.53-2.37(m,6H),2.10-2.04(m,3H),1.72-1.61(m,3H).

[0345] Example 13

[0346] Step A: Synthesis of 3,3'-di-tert-butyl((2S,2'S)-(((3-((S)-3-(tert-butoxy)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl)nitrogendiyl)bis(methylene)bis(6-fluoro-3,1-phenylene))bis(3-(tert-butoxy)-3-oxypropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0347] At room temperature, tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2-fluoro-5-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (312 mg, 0.74 mmol), tert-butyl (R)-3-((S)-3-(3-(aminomethyl)phenyl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (120 mg, 0.29 mmol) and glacial acetic acid (36 mg, 0.59 mmol) were dissolved in anhydrous isopropanol (3 ml), the atmosphere was replaced with nitrogen, and the mixture was reacted at 45°C for 5 hours. Subsequently, sodium cyanoborohydride (38 mg, 0.95 mmol) was added and the mixture was reacted at 45°C for 18 hours. After the reaction, the mixture was diluted with water and extracted with ethyl acetate (20 ml × 2 times). The combined organic phases were washed with saturated brine (20 ml × 2 times), then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to obtain 130 mg of 3,3'-di-tert-butyl((2S,2'S)-(((3-((S)-3-(tert-butoxy)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl)azinediyl)bis(methylene)bis(6-fluoro-3,1-phenylene))bis(3-(tert-butoxy)-3-oxypropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate).

[0348] Step B: Synthesis of (2S,2'S)-3,3'-(((3-((S)-2-carboxy-2-((R)-pyrrolidin-3-yl)ethyl)benzyl)azepinediyl)bis(methylene)bis(6-fluoro-3,1-phenylene))bis(2-(((R)-pyrrolidin-3-yl)propanoic acid)

[0349] Compound 13 was synthesized by the method of Reference Example 4. LCMS: RT = 1.44 min, [MH] - =745.41.

[0350] Example 24

[0351] Synthesis of (2S,2'S)-3,3'-(((((5-methoxypyridin-3-yl)methyl)azadiyl)bis(methylene))bis(3,1-phenylene))bis(2-((R)-pyrrolidin-3-yl)propanoic acid)

[0352] The specific synthetic route is as follows:

[0353] Step A: Synthesis of 3,3'-((2S,2'S)-(((((5-methoxypyridin-3-yl)methyl)azepinediyl)bis(methylene))bis(3,1-phenylene))bis(3-(tert-butoxy)-3-oxopropane-1,2-diyl))(3R,3'R)-bis(tert-butyl pyrrolidine-1-carboxylate)

[0354] At room temperature, tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-((((5-methoxypyridin-3-yl)methyl)amino)methyl)phenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (50.0 mg, 0.09 mmol) (obtained with reference to patent CN202080041924.X) was dissolved in 1,2-dichloroethane (2.0 ml). tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(3-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (44.3 mg, 0.11 mmol) (obtained with reference to Example 1) was added. After stirring at room temperature for 40 minutes, sodium triacetoxyborohydride (47.7 mg, 0.23 mmol) was added and the mixture was reacted at 45°C overnight. After the reaction was completed, the reaction solution was slowly poured into 10.0 ml of water, and the mixture was extracted with dichloromethane (20 ml x 2). The organic phases were combined, washed with saturated brine (20 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 1 / 3) to obtain 34.0 mg of 3,3'-((2S,2'S)-(((((5-methoxypyridin-3-yl)methyl)azepinediyl)bis(methylene))bis(3,1-phenylene))bis(3-(tert-butoxy)-3-oxopropane-1,2-diyl))(3R,3'R)-bis(tert-butyl pyrrolidine-1-carboxylate). LCMS: RT = 2.87 min, [M+H] + =913.67.

[0355] Step B: Synthesis of (2S,2'S)-3,3'-(((((5-methoxypyridin-3-yl)methyl)azepinediyl)bis(methylene))bis(3,1-phenylene))bis(2-((R)-pyrrolidin-3-yl)propanoic acid)

[0356] Compound 24 was prepared by the method of Example 3. LCMS: RT = 1.48 min, [MH] - =599.37.

[0357] Example 30

[0358] Synthesis of (2S,2'S)-3,3'-(5-ethylpyridin-3-yl)methyl)nitrogendiyl)bis(methylene)bis(3,1-phenylene))bis(2-((R)-pyrrolidin-3-yl)propionic acid) by the following synthetic route:

[0359] Step A: Synthesis of 3,3'-((2S,2'S)-(5-ethylpyridin-3-yl)methyl)azepinediyl)bis(methylene)bis(3,1-phenylene)bis(3-(tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0360] At room temperature, 3,3'-((2S,2'S)-((azadiylbis(methylene))bis(3,1-phenylene))-(3-(tert-butoxy)-3-oxopropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate) (469 mg, 0.6 mmol), 5-ethylnicotinaldehyde (80 mg, 0.6 mmol), anhydrous isopropanol (3 ml), and glacial acetic acid (72 mg, 1.2 mmol) were added in sequence. The atmosphere was replaced with nitrogen and the reaction was carried out at 45°C for 5 hours. Subsequently, sodium cyanoborohydride (75 mg, 1.2 mmol) was added and the reaction was carried out at 45°C for 18 hours.

[0361] After the reaction was completed, saturated aqueous ammonium chloride solution (10 ml) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 ml × 2 times) and concentrated to dryness. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to obtain 98 mg of 3,3'-((2S,2'S)-(5-ethylpyridin-3-yl)methyl)azepinediyl)bis(methylene)bis(3,1-phenylene)bis(3-(tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate).

[0362] Step B: Synthesis of (2S,2'S)-3,3'-(5-ethylpyridin-3-yl)methyl)nitrogendiyl)bis(methylene)bis(3,1-phenylene))bis(2-((R)-pyrrolidin-3-yl)propanoic acid)

[0363] Compound 30 was prepared by the method of Example 4. LCMS: RT = 1.49 min, [MH] - =597.37.

[0364] Example 39

[0365] Step A: Synthesis of 3,3'-(4-(S)-3-tert-butoxy-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl)azepinediyl)bismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0366] Tert-butyl (R)-3-(S)-1-tert-butoxy-3-(4-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (38 mg, 0.094 mmol) was dissolved in isopropanol (2.5 ml), and di-tert-butyl 3,3'-((2S,2'S)-((nitrogendiylbis(methylene))bis(3,1-phenylene))bis-(3-(tert-butoxy)-3-oxopropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate) (100 mg, 0.126 mmol) and sodium triacetoxyborohydride (55 mg, 0.26 mmol) were added and reacted at 45° C. for 14 hours. No starting material spot was detected by TLC spot plate, indicating the completion of the reaction. The reaction solution was evaporated to remove isopropanol, and the mixture was quenched by the addition of 1N sodium bicarbonate solution (10 ml). The mixture was washed with ethyl acetate (10 ml x 3 times), and the organic phase was collected and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure to obtain the crude target product. The product was purified by column chromatography using n-hexane:ethyl acetate in a ratio of 3:1 to obtain 100 mg of 3,3'-(4-(S)-3-tert-butoxy-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl)azepinediyl)bismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate).

[0367] Step B: Synthesis of (2S,2'S)-3,3'-((((4-((S)-2-carboxy-2-((R)-pyrrolidin-3-yl)ethyl)benzyl)azepinediyl)bis(methylene))bis(3,1-phenylene))bis-(2-(R)pyrrolidin-3-yl)propanoic acid)

[0368] 3,3'-(4-(S)-3-tert-butoxy-2-(R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl)azadiyl)bismethylene)bis(3,1-phenylene)bis(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R)-bis(pyrrolidine-1-carboxylate) (100 mg, 0.085 mmol) was dissolved in 1,4-dioxane (2.0 ml), concentrated hydrochloric acid (0.5 ml) was added and the mixture was reacted at room temperature for 2 hours. The reaction was completed after LCMS monitoring. The residue was concentrated under reduced pressure to obtain a crude product of the desired product, which was then purified by preparative high performance liquid chromatography to yield 42.8 mg of (2S,2'S)-3,3'-((((4-((S)-2-carboxy-2-((R)-pyrrolidin-3-yl)ethyl)benzyl)azepinediyl)bis(methylene))bis(3,1-phenylene))bis-(2-(R)pyrrolidin-3-yl)propanoic acid. LCMS: RT = 1.37 min, [MH] - =709.47, HPLC: 92.03%. 1 H NMR(400MHz,Deuterium Oxide)δ7.23-7.16(m,4H),7.10(dd,J=17.5,8.0Hz,8H),3.48(s,6H),3.35-3.23(m,6H),3. 15-3.04(m,3H),2.81-2.61(m,9H),2.41-2.25(m,6H),2.05-1.95(m,3H),1.68-1.55(m,3H).

[0369] Example 40

[0370] The specific synthetic route is as follows:

[0371] Step A: Synthesis of 3,3'-di-tert-butyl((2S,2'S)-(((3-((S)-3-(tert-butoxy)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl)nitrogendiyl)bis(methylene)bis(2,1-phenylene)bis(3-(tert-butoxy)-3-oxypropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate)

[0372] At room temperature, tert-butyl (R)-3-((S)-1-(tert-butoxy)-3-(2-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (299 mg, 0.74 mmol), tert-butyl (R)-3-((S)-3-(3-(aminomethyl)phenyl)-1-(tert-butoxy)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (120 mg, 0.29 mmol), anhydrous isopropanol (3 ml), and glacial acetic acid (36 mg, 0.59 mmol) were added in sequence. The atmosphere was replaced with nitrogen, and the reaction was carried out at 45°C for 5 hours. Subsequently, sodium cyanoborohydride (38 mg, 0.59 mmol) was added, and the reaction was carried out at 45°C for 18 hours.

[0373] After the reaction was completed, saturated aqueous ammonium chloride solution (10 ml) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 ml × 2 times). The mixture was concentrated to dryness, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane = 1 / 3) to obtain 210 mg of 3,3'-di-tert-butyl((2S,2'S)-(((3-((S)-3-(tert-butoxy)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-oxopropyl)benzyl)nitrogendiyl)bis(methylene)bis(2,1-phenylene)bis(3-(tert-butoxy)-3-oxypropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate).

[0374] Step B: (2S,2'S)-3,3'-(((3-((S)-2-carboxy-2-((R)-pyrrolidin-3-yl)ethyl)benzyl)azepinediyl)bis(methylene)bis(2,1-phenylene))bis(2-(((R)-pyrrolidin-3-yl)propanoic acid)

[0375] Compound 40 was prepared by the method of Example 4. LCMS: RT = 0.32 min, [MH] - =709.51.

[0376] Example 60

[0377] The specific synthetic route is as follows:

[0378] Step A: Synthesis of 3,3,3,3-tri-tert-butyl((2S,2,2"S)-((methylene))tris(4-fluorobenzene-3,1-diyl))tris(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R,3"R)-tris(pyrrolidine-1-carboxylate)

[0379] Tert-butyl (R)-3-(S)-3-(3-aminomethyl)-4-fluorophenyl-1-tert-butoxy-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (310 mg, 0.73 mmol) and tert-butyl (R)-3-(S)-1-tert-butoxy-3-(4-fluoro-3-formylphenyl)-1-oxopropan-2-yl)pyrrolidine-1-carboxylate (773 mg, 1.83 mmol) were dissolved in 1,2-dichloroethane (10 ml), and sodium triacetoxyborohydride (425 mg, 2.57 mmol) was added portionwise, and the reaction was carried out at 45° C. overnight. After the reaction, the solvent was evaporated under reduced pressure and the crude product was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 1) to obtain 200 mg of 3,3,3,3-tri-tert-butyl((2S,2,2"S)-((methylene))tris(4-fluorobenzene-3,1-diyl))tris(3-tert-butoxy)-3-oxopropane-1,2-diyl)(3R,3'R,3"R)-tris(pyrrolidine-1-carboxylate).

[0380] Step B: Synthesis of (2S,2'S,2"S)-3,3',3"-(((methylene))tris(4-fluorobenzene-3,1-diyl))tris(2-(R)-pyrrolidin-3-yl)propanoic acid)

[0381] Compound 60 was prepared by referring to the method of compound 12. LCMS: RT = 1.49 min, [MH] - =763.20. 1 HNMR(400MHz,Deuterium Oxide)δ7.04(d,J=6.7Hz,6H),6.90(t,J=9.2Hz,3H),3.69(s,6H),3.38–3.20(m,6H),3.08(ddd,J=11.7,9.9,7.3Hz,3H),2. 78(dd,J=11.5,9.0Hz,3H),2.62(qd,J=13.9,7.0Hz,6H),2.36–2.23(m,6H),2.06–1.92(m,3H),1.58(dq,J=12.9,9.0Hz,3H).

[0382] Examples 5-11, 14-23, 25-29, 31-38, 41-48, 50-59, 64-73

[0383] Compounds 5-11, 14-23, 25-29, 31-38, 41-48, 50-59, and 64-73 were prepared by referring to the methods of the aforementioned examples. The specific preparation and characterization data are as follows:

[0384] Example 74

[0385] In vitro Lp(a) assembly assay

[0386] Experimental procedures

[0387] The ability of compounds to inhibit Lp(a) particle formation in vitro was assessed using a cell-free assembly assay. Conditioned medium (DMEM supplemented with 10% FBS, 20 mM HEPES, and 1× penicillin / streptomycin) was collected from confluent wild-type HepG2 cells (a source of endogenously expressed ApoB) and from a HEK293 stable cell line expressing a human Apo(a) protein containing 17 Kringle repeats (selected on 1 μg / ml puromycin) after 96 hours of incubation at 37°C and 5% CO2. The in vitro assembly assay was performed by combining aliquots of HepG2 and HEK293 conditioned medium with test compounds added in a dilution series (final concentrations 0.03 to 1000 nM). The reaction was incubated at 37°C for 2 hours and terminated by the addition of 6-aminoacetic acid (EACA) to a final concentration of 150 mM. Lp(a) was detected using a sandwich ELISA with an anti-Apo(a) capture antibody (ab242565) and an HRP-conjugated anti-ApoB detection antibody (ab27622). The ELISA was developed using TMB, terminated with 1N sulfuric acid, and read at 450 nm on an Envision 2104 plate reader. The % inhibition of Lp(a) formed under each test condition was determined by setting the assembly reaction in the absence of inhibitor to 0% inhibition and the assembly reaction in the presence of minimal HepG2 conditioned medium (50-fold dilution) to 100% inhibition. The data were fitted to a 4-parameter curve to determine the IC 90 The results are shown in Table 1.

[0388] Table 1 IC of tertiary amine compounds 90 value

[0389] According to the results in Table 1, it can be seen that the tertiary amine compounds of the present invention all have good LP(a) inhibitory activity.

[0390] Example 75

[0391] Pharmacokinetic experiments in SD rats

[0392] (1) Experimental materials

[0393] SD rats: male, 180-250 g, purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0394] Reagents: Normal saline, EDTA-K2 (anticoagulant), TCA (trichloroacetic acid), and propranolol (internal standard) are all commercially available.

[0395] Instruments: Nexera LC-40; AB SCIEX QTRAP 5500+.

[0396] (2) Experimental methods

[0397] Weigh the compound and dissolve it in saline. After oral administration to rats, collect 200 μL of venous blood at 15, 30, 1, 2, 5, 7, and 24 hours (5 minutes additionally for the IV group) into EDTA-K2 anticoagulant EP tubes. Centrifuge at 12,000 rpm for 2 minutes, and freeze the plasma at -80°C for testing. Accurately weigh a certain amount of the test compound and dissolve it in ultrapure water to 2 mg / mL to prepare a stock solution. Accurately pipette an appropriate amount of the compound stock solution and dilute it with 50% acetonitrile-water to prepare a series of standard solutions. Accurately pipette 10 μL of each standard solution and add 90 μL of blank plasma. Vortex to mix thoroughly. Prepare plasma samples equivalent to plasma concentrations of 1, 3, 5, 10, 30, 100, 300, 1000, and 3000 ng / mL. Quality control samples were prepared at concentrations of 9, 240, and 2400 ng / mL. Duplicate samples were analyzed at each concentration to construct a standard curve. 30 μL of plasma (5, 15, and 30 minutes after intravenous administration, diluted 5-fold) was collected and 200 μL of a 5% trichloroacetic acid-water solution containing the internal standard propranolol (50 ng / mL) was added. After vortex mixing, the mixture was centrifuged at 4000 rpm for 10 minutes. The supernatant was collected, 150 μL of purified water was added, and the mixture was vortexed again for LC-MS / MS analysis. LC-MS / MS detection conditions were as follows:

[0398] Chromatographic column: YMC Triart C18, 50*3.0mm, 2.1μm.

[0399] Mobile phase: water (0.1% formic acid)-acetonitrile with gradient elution as shown in the table below.

[0400] Table 2 Gradient elution table

[0401] (3) Data processing

[0402] After LC-MS / MS detection of blood drug concentrations, pharmacokinetic parameters were calculated using WinNonlin 6.1 software and the non-compartmental model method. The results are shown in Table 2.

[0403] Table 3: Pharmacokinetic parameters of the compounds of the present invention in SD rats

[0404] According to the results in Table 3, it can be seen that the compound of the present invention has a higher exposure amount.

[0405] Example 76

[0406] Monkey pharmacokinetic experiments

[0407] (1) Experimental materials

[0408] Beagle dogs: male, 8-10 kg, purchased from Beijing Mas Biotechnology Co., Ltd.

[0409] Cynomolgus macaques: male, 3-6 kg, purchased from Guangzhou Huazhen Biotechnology Co., Ltd. and Hainan Jingang Biotechnology Co., Ltd.

[0410] Reagents: Normal saline, EDTA-K2 (anticoagulant), TCA (trichloroacetic acid), and propranolol (internal standard) are all commercially available.

[0411] Instruments: Nexera LC-40; AB SCIEX QTRAP 5500+.

[0412] (2) Experimental methods

[0413] Weigh the compound and dissolve it in physiological saline. After oral administration, collect 200 μL of venous blood into EDTA-K2 anticoagulant EP tubes at 15, 30, 1, 2, 4, 8, and 24 hours (5 minutes additionally for the IV group). The blood was centrifuged at 12,000 rpm for 2 minutes, and plasma was frozen at -80°C for testing. Accurately weigh a certain amount of the test compound and dissolve it in ultrapure water to 2 mg / mL to prepare a stock solution. Accurately pipette an appropriate amount of the compound stock solution and dilute it with 50% acetonitrile-water to prepare a series of standard solutions. Accurately pipette 10 μL of each standard solution and add 90 μL of blank plasma. Vortex to mix thoroughly. Plasma samples equivalent to plasma concentrations of 1, 3, 5, 10, 30, 100, 300, 1000, and 3000 ng / mL were prepared. Quality control samples were prepared at concentrations of 9, 240, and 2400 ng / mL. Duplicate samples were analyzed at each concentration to construct a standard curve. 30 μL of plasma (5, 15, and 30 minutes after intravenous administration, diluted 5-fold) was collected and 200 μL of a 5% trichloroacetic acid-water solution containing the internal standard propranolol (50 ng / mL) was added. After vortex mixing, the mixture was centrifuged at 4000 rpm for 10 minutes. The supernatant was collected, 150 μL of purified water was added, and the mixture was vortexed again for LC-MS / MS analysis. LC-MS / MS detection conditions were as follows:

[0414] Chromatographic column: YMC Triart C18, 50*3.0mm, 2.1μm.

[0415] Mobile phase: water (0.1% formic acid)-acetonitrile with gradient elution as shown in the table below.

[0416] Table 4 Gradient elution table

[0417] (3) Data processing

[0418] After LC-MS / MS detection of blood drug concentrations, WinNonlin 6.1 software was used to calculate the pharmacokinetic parameters using the non-compartmental model method. The results are shown in Table 5.

[0419] Table 5: Pharmacokinetic parameters of the compounds of the present invention in cynomolgus monkeys

[0420] According to the results in Table 5, it can be seen that the compound of the present invention has a higher exposure amount and a higher bioavailability.

[0421] Example 77

[0422] Plasma protein binding (PPB) test

[0423] 1. Experimental Materials

[0424] HT dialysis plates and dialysis membranes were purchased from Apricot Designs.

[0425] Reagents: NaH2PO4 (sodium dihydrogen phosphate), Na2HPO4 (disodium hydrogen phosphate), TCA (trichloroacetic acid), and propranolol (internal standard) are all commercially available.

[0426] Instruments: Nexera LC-40; AB SCIEX QTRAP 5500+.

[0427] 2. Solution Preparation

[0428] 2.1 Dialysis buffer (50 mM sodium phosphate buffer, pH 7.4 ± 0.1):

[0429] Dissolve 780.05g of AR-grade NaH2PO4 in 100mL of ultrapure water as Solution A, and 3581.4g of AR-grade Na2HPO4 in 200mL of ultrapure water as Solution B. Add Solution A to Solution B until the pH reaches 7.4 ± 0.1. Store the final solution in a refrigerator at 2-8°C for one month after preparation.

[0430] 2.2 Reaction termination solution

[0431] 5% trichloroacetic acid-water solution (containing 50 ng / mL propranolol internal standard)

[0432] 3. Experimental Procedure

[0433] 3.1 Prepare W1 (1000 μM) by diluting 10 μL of the 10 mM stock solution with 90 μL of ACN:H2O (1:1).

[0434] 3.2 Take 2 μL of W1 (1000 μM) and add it to 198 μL of blank plasma of the corresponding species, mix well, and prepare a 10 μM drug-containing plasma solution.

[0435] 3.3 Assemble the dialyzer according to the manufacturer's instructions.

[0436] 3.4 Add 100 μL of drug-containing plasma (in duplicate) to the red membrane side of the dialysis plate and 300 μL of blank dialysis buffer to the other white membrane side. Seal the dialysis plate and place it in a 37°C shaker at 100 rpm for 5 hours.

[0437] 3.5 Take 20 μL aliquots of drug-containing plasma and dialysis buffer into a 96-well plate in duplicate as T0 samples. Place the T0 sample plate at room temperature.

[0438] 3.6 After dialysis, aliquots of dialyzed plasma (20 μL) and buffer (20 μL) were transferred to a new 96-well plate.

[0439] 3.7 All samples in each well were mixed with an equal volume of the opposite blank matrix to obtain a final concentration ratio of plasma to dialysis buffer of 20:20 in each well.

[0440] 3.8 Dilute the sample with 150 μL of internal standard stop solution. Centrifuge at 4000 rpm for 10 minutes. Transfer 100 μL of the supernatant to the sample analysis plate. Mix the sample with 100 μL of ultrapure water and perform LC-MS / MS analysis.

[0441] LC-MS detection conditions are as follows:

[0442] Chromatographic column: YMC Triart C18, 50*3.0mm, 2.1μm.

[0443] Mobile phase: water (0.1% formic acid)-acetonitrile with gradient elution as shown in the table below.

[0444] Table 6 Gradient elution table

[0445] 4. Data Processing

[0446] The binding rate and recovery rate were calculated using the following formula:

[0447] %Bound=(DR) / D*100

[0448] %Recovery=(D+R) / T0*100

[0449] D is the donor (plasma side) peak area / internal standard peak area.

[0450] R is the receiving (buffer side) peak area / internal standard peak area.

[0451] T0 is the peak area of ​​the sample at T0 / the peak area of ​​the internal standard.

[0452] Table 6: Plasma protein binding parameters of the compounds of the present invention

[0453] According to the results in Table 6, it can be seen that the compound of the present invention has higher binding rates to human and monkey plasma proteins.

[0454] Example 78

[0455] Tissue distribution experiment in SD rats

[0456] (1) Experimental materials

[0457] SD rats: male, 180-250 g, purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0458] Reagents: MeOH (methanol), EDTA-K2 (anticoagulant), normal saline, TCA (trichloroacetic acid), and propranolol (internal standard) are all commercially available.

[0459] Instruments: Nexera LC-40; AB SCIEX QTRAP 5500+; fully automatic cryo-grinder.

[0460] (2) Experimental methods

[0461] The compound was weighed and dissolved in saline. After oral administration to rats, blood was collected from the abdominal aorta at 30 minutes, 3 hours, and 8 hours after administration into EDTA-K2 anticoagulant tubes. The blood was centrifuged at 4000 rpm for 5 minutes, and the plasma was frozen at -80°C for testing. Tissues collected included the liver, stomach, duodenum, eyeball, skin, heart, spleen, lung, kidney, testis, epididymis, brain, adipose tissue, and skeletal muscle. Each tissue sample was accurately weighed and added to 5 volumes of a 50% methanol-water solution (w:v = 1:5). The sample was placed in a cryo-grinder and ground at -50°C at 60 Hz for 60 seconds. The sample was then centrifuged at 10,000 rpm for 10 minutes. The supernatant was used as the tissue sample solution.

[0462] Accurately weigh a certain amount of test sample and dissolve it in ultrapure water to 2 mg / mL to prepare a stock solution. Accurately pipette an appropriate amount of the compound stock solution and dilute it with 50% acetonitrile-water to prepare a series of standard solutions. Accurately pipette 10 μL of each standard solution and add 90 μL of blank plasma and blank liver tissue homogenate, respectively. Vortex to mix thoroughly. Matrix standard curves are prepared at concentrations of 1, 3, 5, 10, 30, 100, 300, 1000, and 3000 ng / mL. Quality control samples are prepared at concentrations of 9, 240, and 2400 ng / mL. Duplicate samples are analyzed at each concentration to establish a standard curve. 30 μL of plasma and tissue samples are added to 200 μL of a 5% trichloroacetic acid-water solution containing the internal standard propranolol (50 ng / mL). Vortex to mix thoroughly, centrifuge at 4000 rpm for 10 minutes, remove 150 μL of the supernatant, add 150 μL of purified water, and vortex again for LC-MS / MS analysis. LC-MS / MS detection conditions are as follows:

[0463] Chromatographic column: YMC Triart C18, 50*3.0mm, 2.1μm.

[0464] Mobile phase: water (0.1% formic acid)-acetonitrile with gradient elution as shown in the table below.

[0465] Table 7 Gradient elution table

[0466] (3) Data processing

[0467] After LC-MS / MS detection of blood drug concentration, WinNonlin 6.1 software was used to calculate the pharmacokinetic parameters using the non-compartmental model method. The results are shown in Table 8.

[0468] Table 8: Tissue distribution results of the compounds of the present invention

[0469] According to the results in Table 8, it can be seen that the compounds of the present invention have higher liver tissue distribution.

[0470] It should be understood that the above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. For ordinary technicians in this field, they can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A tertiary amine compound, or an isomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the tertiary amine compound is shown in general formula I: Among them, X1~X 15 Each is independently selected from -N- or -CR1, wherein R1 is selected from H, halogen, -OH, -NH2, -CN, -NO2, -COOH, -SO3H, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C3-C 10 Cycloalkyl, substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C5-C 10 Heterocyclic group or X1~X 15 At least one of them is -N-; Or when X1~X 15 are each independently selected from -CR1, wherein at least one R1 is not hydrogen and And at least one R1 among X1 to X5 is independently selected from And the X 11 ~X 15 At least one R1 is independently selected from Or when X1~X 15 When all of X1 to X 15 Any two adjacent R1 and the connected carbon atom together form a 5-8 membered aromatic or heterocyclic group; The R2 is selected from -H, halogen, substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 alkoxy; The R3 is selected from -(CH2) q -(5-8 membered heterocyclyl); The substituted C1-C6 alkyl, substituted C1-C6 alkoxy, substituted C6-C 10 Aryl or substituted C5-C 10 The substituents in the heterocyclic group are independently selected from halogen, hydroxy, carboxyl, nitro, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 6 membered heterocycloalkyl, C3-C6 cycloalkoxy, C6-C 10 one or more of aryl or 5- to 6-membered heteroaryl; m, n, p are independently selected from integers of 1 or 2, and q is selected from integers of 0, 1 or 2; The tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt is not 2. The tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt according to claim 1, characterized in that: R1 is selected from H, halogen, -OH, -NH2, -CN, -NO2, -COOH, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C 10 Cycloalkyl, substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C5-C 10 Heterocyclic group or The R2 is selected from -H, substituted or unsubstituted C1-C6 alkyl; the R3 is selected from 5-8 membered heterocyclic group; the substituted C1-C6 alkyl, substituted C6-C 10 Aryl or substituted C5-C 10 Substituents in the heteroaryl group are independently selected from one or more of halogen, hydroxy, carboxyl, nitro or cyano.

3. The tertiary amine compound according to claim 1 or 2, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: The structure of the tertiary amine compound is shown in the general formula IIa or IIb: Among them, X1~X 15 Each is independently selected from -N- or -CR1, wherein R1 is selected from H, halogen, substituted C1-C6 alkyl, C1-C6 alkoxy or The R2 is selected from H, substituted or unsubstituted C1-C6 alkyl; the X1 to X 15 At least one of them is -N-, or when X1~X 15 are each independently selected from -CR1, wherein at least one R1 is not hydrogen and Substitution; the R3 is selected from a 5-8 membered heterocyclic group; the substituents in the substituted C1-C6 alkyl are independently selected from one or more of halogen, carboxyl, cyano, and C1-C6 alkyl.

4. The tertiary amine compound according to claim 1 or 2, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: The structure of the tertiary amine compound is shown in the general formula IIIa or IIIb: Among them, X1~X 15 Each is independently selected from -N- or -CR1, wherein R1 is selected from H, halogen, substituted C1-C6 alkyl, C1-C6 alkoxy or The R2 is selected from H, substituted or unsubstituted C1-C6 alkyl; the X1 to X 15 At least one of them is -N-, or when X1~X 15 are each independently selected from -CR1, wherein at least one R1 is not hydrogen and Substitution; the R3 is selected from a 5-8 membered heterocyclic group; the substituents in the substituted C1-C6 alkyl are independently selected from one or more of halogen, carboxyl, cyano, and C1-C6 alkyl.

5. The tertiary amine compound according to any one of claims 1 to 4, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: The R1 is selected from H, fluorine, chlorine, bromine, iodine, trifluoromethyl, methyl, ethyl, propyl, methoxy, ethoxy, piperazinyl, -CH2COOH, -CH2CH2COOH, -CH2CH(CH3)COOH, The R3 is selected from pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyridinyl, pyrimidinyl or pyrazinyl.

6. The tertiary amine compound according to any one of claims 1 to 5, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: Said Selected from quinolinyl.

7. The tertiary amine compound according to any one of claims 1 to 5, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: Said Selected from:

8. The tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt according to claim 7, characterized in that: Said Selected from:

9. The tertiary amine compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt according to claim 8, characterized in that: Said Selected from:

10. The tertiary amine compound according to any one of claims 1 to 9, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: The chemical structure of the tertiary amine compound is selected from the structures shown in Table 1A.

11. The tertiary amine compound according to any one of claims 1 to 10, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: The chemical structure of the tertiary amine compound is selected from the structures shown in Table 1B.

12. A pharmaceutical composition, characterized in that The invention comprises the tertiary amine compound according to any one of claims 1 to 11, or its isomer, or its racemate, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable excipients and / or carriers.

13. Use of the tertiary amine compound according to any one of claims 1 to 11, or its isomer, or its racemate, or its pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 12 in the preparation of a pharmaceutical composition for preventing or treating diseases associated with LP(a).

14. The use according to claim 13, characterized in that The Lp(a)-related disease is selected from cardiovascular disease.

15. The use according to claim 14, characterized in that The cardiovascular disease is selected from the group consisting of stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and any other disease associated with elevated Lp(a) levels.

Images