S1PR4 modulator compounds for autoimmune disease treatment and uses thereof

Abstract

The invention also discloses a compound shown in the formula I and application of the compound as an S1PR regulator, especially an S1PR4 agonist. The compound provided by the invention can be used for treating related diseases mediated by S1PR4 and mutants thereof, especially autoimmune diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and in particular to novel compounds and applications of S1PR4 modulators for preventing and treating autoimmune diseases. Background Art

[0002] Sphingosine-1-phosphate (S1P) can mediate the transmission of biological signals to G protein-coupled receptors on biological membranes or directly act on intracellular receptors, thereby playing a special role in mediating inflammatory responses, allergic reactions, and participating in the migration, proliferation, and differentiation of immune cells. Abnormal regulation of S1P signals is associated with autoimmune diseases, cardiovascular diseases, nervous system diseases, and cancer. As a member of the G protein-coupled receptor family, five different S1P receptor subtypes have been found in mammals, namely S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5 (formerly known as EDG1, EDG5, EDG3, EDG6, and EDG8). Among them, S1PR1, S1PR2, and S1PR3 are widely expressed in various body tissues, S1PR4 is mainly expressed in the lymphatic system and blood system, and S1PR5 is mainly expressed in the central nervous system. Current studies have confirmed that S1P can activate the β-arrestin signaling pathway to induce receptor invagination by binding to S1PR1 on the cell surface, thereby inducing lymphocyte homing and inhibiting lymphocyte migration from peripheral lymph nodes and secondary lymphoid organs to the outside, thereby preventing lymphocyte migration to inflammatory sites, and has a good immunosuppressive regulatory effect. Targeted regulation of the S1PR1 signaling pathway can achieve the regulation of a variety of autoimmune diseases, such as systemic lupus erythematosus, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, etc. However, the current marketed drugs targeting S1PR1 have good agonist activity against other subtypes of S1PR and lack selectivity, especially the activation of S1PR3 can lead to off-target side effects such as bradycardia, so the development of selective S1PRs agonists is of great significance.

[0003] However, there are few reports on S1PR4 selective drugs. Almost all the disclosed S1PR4 agonists / antagonists were developed by Edward Roberts of the Scripps Research Institute in the United States. However, these S1PR4 modulators are only explored as in vitro tool molecules, and there is no systematic mechanism research and drugability evaluation. Therefore, it is of great scientific value to develop orally effective selective S1PR4 agonists / antagonists for the treatment of autoimmune diseases. Summary of the invention

[0004] The object of the present invention is to provide relevant compounds as S1PR modulators, in particular S1PR4 agonists.

[0005] Another object of the present invention is to provide a pharmaceutical composition comprising the above compound.

[0006] Another object of the present invention is to provide the use of the above-mentioned compound or pharmaceutical composition in the preparation of drugs for treating S1PR4-related diseases or regulating S1P receptors.

[0007] In a first aspect, the present invention provides a compound represented by formula I, or an optical isomer or a pharmaceutically acceptable salt thereof:

[0008]

[0009] A is selected from substituted or unsubstituted C5-C8 aromatic ring (such as phenyl) or C8-C 14 A fused aromatic ring (e.g. naphthyl), a substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 3 heteroatoms selected from N or O;

[0010] R is independently selected from the following group: hydrogen, halogen, cyano, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl or cycloalkenyl, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C5-C8 aryl (e.g., phenyl), substituted or unsubstituted five-membered or six-membered heterocyclic or heteroaryl containing 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, p is an integer of 1-3;

[0011] R 1 Selected from the following group: hydrogen, halogen, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C3-C8 lactone, substituted or unsubstituted C1-C 10 An amide group, a substituted or unsubstituted C5-C8 aryl group, a substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O or S, or a substituted or unsubstituted C5-C8 aromatic heterocyclic group;

[0012] n is selected from an integer of 1 to 3;

[0013] R 2 Select from the following group:

[0014]

[0015] m is selected from an integer of 1 to 4;

[0016] q is selected from an integer of 1-2.

[0017] In a preferred embodiment, the compound is not the following:

[0018]

[0019] In a specific embodiment, the compound is shown in the following formula II:

[0020]

[0021] In the formula,

[0022] R 1 Selected from the following group: hydrogen, halogen (preferably F, Cl or Br), cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C 10 Alkyl (preferably substituted or unsubstituted C1-C6 alkyl, for example, methyl, ethyl, trifluoromethyl), substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O or S, substituted or unsubstituted C5-C8 aromatic heterocyclic group;

[0023] R 2 Select from the following group:

[0024]

[0025] X is selected from: C or N;

[0026] R 3 is absent or is a substituent selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S;

[0027] R 5 Selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S;

[0028] R 4 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl or cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic or heteroaryl containing 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C1-C 10 an alkylformyl group, or a substituted or unsubstituted arylformyl group.

[0029] In a specific embodiment, the compound is represented by the following formula III:

[0030]

[0031] In the formula,

[0032] R 1 Selected from the following group: hydrogen, halogen (preferably F, Cl or Br), cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C 10 Alkyl (preferably substituted or unsubstituted C1-C6 alkyl, for example, methyl, ethyl, trifluoromethyl), substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O or S, substituted or unsubstituted C5-C8 aromatic heterocyclic group;

[0033] R 2 Select from the following group:

[0034]

[0035] R 3 A substituent selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S;

[0036] R 5Selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S;

[0037] R 4 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl or cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic or heteroaryl containing 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C1-C 10 an alkylformyl group, or a substituted or unsubstituted arylformyl group.

[0038] In a specific embodiment, the present invention provides a compound selected from the following group, or an optical isomer or a pharmaceutically acceptable salt thereof:

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] In a specific embodiment, in Formula III, R 1Selected from the following group: hydrogen, halogen (preferably F, Cl or Br), cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C6 alkyl (preferably methyl, ethyl, propyl);

[0052] R 2 for:

[0053]

[0054] R 3 and R 5 Independently selected from the following group: hydrogen, substituted or unsubstituted C1-C6 alkyl (preferably methyl, ethyl or propyl), halogen (preferably F);

[0055] R 4 Selected from the following group: substituted or unsubstituted C5-C8 aryl (preferably phenyl or F-substituted phenyl), substituted or unsubstituted five-membered or six-membered heterocyclic group or heteroaryl group containing 1-2 heteroatoms selected from N, O or S.

[0056] In a specific embodiment, the present invention provides a compound selected from the following group, or an optical isomer or a pharmaceutically acceptable salt thereof:

[0057]

[0058]

[0059] In a specific embodiment, in Formula III, R 1 Selected from the following group: cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C6 alkyl (preferably methyl, ethyl, propyl);

[0060] R 2 for:

[0061]

[0062] R 3 and R 5 Independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C3 alkyl (preferably methyl, ethyl or propyl), halogen (preferably F);

[0063] R 4 Selected from the following group: substituted or unsubstituted C5-C8 aryl (preferably phenyl or F-substituted phenyl), substituted or unsubstituted five-membered or six-membered heteroaryl containing 1-2 heteroatoms selected from N, O or S.

[0064] In a specific embodiment, the present invention provides a compound selected from the following group, or an optical isomer or a pharmaceutically acceptable salt thereof:

[0065]

[0066] In a preferred embodiment, the compound is an S1PR modulator; preferably, the compound is an S1PR4 agonist; more preferably, the compound has an agonistic effect on S1PR4 but not on S1PR1.

[0067] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first aspect, or an optical isomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0068] In a preferred embodiment, the pharmaceutical composition is in a dosage form suitable for oral administration, including but not limited to tablets, solutions, suspensions, capsules, granules, and powders.

[0069] In a preferred embodiment, the pharmaceutical composition is used as a S1PR4 agonist or a S1PR4 mutant agonist.

[0070] In a preferred embodiment, the pharmaceutical composition is used to treat or prevent S1PR4-mediated diseases.

[0071] In a preferred embodiment, the S1PR4-mediated disease comprises an autoimmune disease.

[0072] In a preferred embodiment, the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, and the like.

[0073] In a third aspect, the present invention provides use of the compound described in the first aspect, or an optical isomer or a pharmaceutically acceptable salt thereof, in the preparation of an S1PR modulator. In a preferred embodiment, the S1PR modulator is an S1PR4 selective agonist or an S1PR4 mutant selective agonist.

[0074] In a preferred embodiment, the S1PR modulator is a S1PR4 agonist or a S1PR4 mutant agonist.

[0075] In a preferred embodiment, the S1PR4 agonist or S1PR4 mutant agonist is a drug for treating or preventing a disease mediated by S1PR4.

[0076] In a preferred embodiment, the S1PR4-mediated disease comprises an autoimmune disease.

[0077] In a preferred embodiment, the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, and the like.

[0078] In a fourth aspect, the present invention provides a method for regulating S1PR in a subject, the method comprising the step of administering an effective amount of the compound of the first aspect, or an optical isomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect to a subject in need thereof.

[0079] In a preferred embodiment, the method is a method for agonizing S1PR4 in a subject; more preferably, the method is a method for agonizing S1PR4 in a subject without agonizing S1PR1.

[0080] In a preferred embodiment, the method is used to treat or prevent a S1PR4-mediated disease.

[0081] In a preferred embodiment, the S1PR4-mediated disease comprises an autoimmune disease.

[0082] In a preferred embodiment, the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, and the like.

[0083] In a fifth aspect, the present invention provides the compound according to the first aspect, or an optical isomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9, for use in treating or preventing a disease mediated by S1PR4.

[0084] In a preferred embodiment, the S1PR4-mediated disease comprises an autoimmune disease.

[0085] In a preferred embodiment, the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, and the like.

[0086] In a sixth aspect, the present invention provides a drug for treating or preventing a disease mediated by S1PR4, comprising the compound of the first aspect, or an optical isomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect.

[0087] In a preferred embodiment, the S1PR4-mediated disease comprises an autoimmune disease.

[0088] In a preferred embodiment, the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, and the like.

[0089] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 The restoration effect after compound 30-induced S1PR4 internalization is shown;

[0091] Figure 2 Compound 30 was shown to reduce the mortality of lupus erythematosus animals;

[0092] Figure 3 Compound 30 was shown to dose-dependently reduce subcutaneous nodules and antinuclear antibody levels in SLE mice;

[0093] Figure 4 It shows that compound 30 regulates the number and ratio of T lymphocytes;

[0094] Figure 5 It was shown that compound 30 improved the renal injury pathological changes in SLE;

[0095] Figure 6 The survival curve of acute UC model mice administered with compound 30 is shown;

[0096] Figure 7 The effect of compound 30 on body weight in acute UC model mice is shown;

[0097] Figure 8 The effect of compound 30 on daily DAI scores in acute UC model mice is shown;

[0098] Fig. 9 The effect of compound 30 on the total DAI score of acute UC model mice is shown;

[0099] Fig.10 The colon length of each experimental group is shown;

[0100] Fig.11 Colon weights of each experimental group are shown;

[0101] Fig.12 The colon swelling rate of each experimental group is shown;

[0102] Fig.13 H&E staining was shown to detect pathological changes in mouse colon;

[0103] Fig.14 The colon pathological scores of mice in each group were shown by H&E staining;

[0104] Fig.15 Immunohistochemical detection of Occludin in the colon of acute UC mice is shown;

[0105] Fig.16 Immunohistochemical detection of Occludin in the colon of acute UC mice is shown;

[0106] Fig.17 The changes in body weight of each group during chronic UC modeling are shown;

[0107] Fig.18 The changes in body weight of each group after the start of drug administration are shown;

[0108] Fig.19 The weight change rate of each group before and after administration is shown;

[0109] Fig. 20 The daily DAI scores of the chronic UC model during the modeling period are shown;

[0110] Fig.21 The daily DAI scores of the chronic UC model during the drug administration period are shown;

[0111] Fig. 22 The sum of daily DAI scores of the chronic UC model in each group during the drug administration period is shown;

[0112] Fig.23 The colon length of each group of chronic UC is shown;

[0113] Fig.24 The colon weights of each group in chronic UC are shown;

[0114] Fig.25 The colon swelling rate of each group in chronic UC is shown;

[0115] Fig.26 H&E staining was shown to detect pathological changes in mouse colon;

[0116] Fig. 27 The colon pathological scores of mice in each group were shown by H&E staining;

[0117] Fig.28 Immunohistochemical detection of Occludin in the colon of chronic UC mice is shown;

[0118] Fig.29 Immunohistochemical detection of Occludin in the colon of chronic UC mice is shown;

[0119] Fig.30The efficacy of compounds 30 / 38 / 53 / 67 / 77 / 80 in the acute UC model of mice was shown;

[0120] Fig.31 The experimental process of osteoarthritis OA is shown;

[0121] Fig.32 The pain index statistics of each group in the osteoarthritis experiment of compound 30 are shown;

[0122] Fig.33 The five-minute walking distance statistics of each group in the osteoarthritis experiment of compound 30 are shown;

[0123] Fig.34 The statistics of the number of standing upright within five minutes for each group in the osteoarthritis experiment of compound 30 are shown;

[0124] Fig.35 The anatomical diagram of the knee joints of each group in the osteoarthritis experiment of compound 30 is shown;

[0125] Fig.36 The effects of compound 30 on rat knee joint safranin fast green staining and OARSI score in osteoarthritis experiment are shown;

[0126] Fig.37 The pain index statistics in the osteoarthritis experiment of compound 67 are shown;

[0127] Fig.38 The five-minute walking distance statistics of each group in the osteoarthritis experiment of compound 67 are shown;

[0128] Fig.39 The statistics of the number of standing upright within five minutes for each group in the osteoarthritis experiment of compound 67 are shown;

[0129] Fig.40 shows the anatomical diagram of the knee joints of each group in the osteoarthritis experiment of compound 67;

[0130] Fig.41 The effects of compound 67 on rat serum cytokines in osteoarthritis experiments are shown;

[0131] Fig.42 The effects of compound 67 on safranin fast green staining and OARSI scores in rat knee joints in osteoarthritis experiments are shown;

[0132] Fig.43 The weight and survival rate of lupus erythematosus MRL / Lpr model mice in the systemic lupus erythematosus experiment of compound 67 within 8 weeks are shown;

[0133] Fig.44 It was shown that in the systemic lupus erythematosus experiment of compound 67, compound 67 significantly reduced the level of urinary microalbumin in mice;

[0134] Fig.45 It was shown that in the systemic lupus erythematosus experiment of compound 67, compound 67 significantly reduced the level of anti-double-stranded DNA antibodies in mice;

[0135] Fig.46 It was shown that in the systemic lupus erythematosus experiment of compound 67, compound 67 significantly reduced the blood creatinine level of mice;

[0136] Fig.47 It shows that compound 67 regulates the number and ratio of T lymphocytes in the systemic lupus erythematosus experiment;

[0137] Fig.48 Compound 67 was shown in the systemic lupus erythematosus experiment;

[0138] Fig.49 The peripheral clinical symptom scores of each group of mice in the mouse ankylosing spondylitis model (8 weeks) are shown;

[0139] Fig.50 The peripheral clinical symptom scores of each group of mice in the mouse ankylosing spondylitis model (12 weeks) are shown;

[0140] Fig.51 Typical photos of inflammatory symptoms in the feet of mice in each group in the mouse ankylosing spondylitis model are shown;

[0141] Fig.52 The spinal CT imaging scores of each group of mice in the mouse ankylosing spondylitis model are shown;

[0142] Fig.53 The typical CT images of the spine of each group of mice in the mouse ankylosing spondylitis model are shown; and

[0143] Fig.54 The skin reactions of each group after IMQ modeling are shown. DETAILED DESCRIPTION

[0144] After extensive and in-depth research, the inventors found that S1PR4 agonists have therapeutic effects on certain autoimmune diseases at the animal level. Furthermore, the inventors synthesized candidate compounds with S1PR4 receptor modulating activity. The obtained candidate compounds were structurally optimized, and a series of selective S1PR4 compounds that have not been reported in the literature were designed and synthesized, and the structures were characterized. This series of compounds were tested for activity at the cellular level, and a batch of compounds with S1PR4 agonist activity were obtained. These compounds can modulate S1P receptor activity, among which the EC with S1PR4 agonist activity 50 The value reaches nM level, and has a good targeting effect on S1PR4. On this basis, the present invention is completed.

[0145] Definition of terms

[0146] Some of the groups involved in this article are defined as follows:

[0147] As used herein, "alkyl" refers to a saturated branched or straight chain or cycloalkyl group having a carbon chain length of 1 to 10 carbon atoms, and preferred alkyl groups include alkyl groups having 1 to 5, 1 to 2, 1 to 6, 1 to 4, 3 to 8 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl, and the like. Alkyl groups may be substituted with one or more substituents, such as halogen or haloalkyl. For example, an alkyl group may be an alkyl group substituted with 1 to 4 fluorine atoms, or an alkyl group may be an alkyl group substituted with a fluoroalkyl group.

[0148] Herein, "alkenyl" generally refers to a monovalent hydrocarbon group having at least one double bond, generally containing 2-8 carbon atoms, preferably containing 2-6 carbon atoms, and can be straight chain or branched. Examples of alkenyl include, but are not limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, etc.

[0149] Herein, "ester group" generally refers to a carboxylic acid derivative having at least one ester group, generally containing 3-8 carbon atoms, preferably containing 3-6 carbon atoms, and can be straight chain or branched. Examples of ester groups include, but are not limited to, methyl formate, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, and the like.

[0150] Herein, "hydroxyl" refers to a branched or straight chain alcohol having a carbon chain length of 1 to 10 carbon atoms, usually containing 1 to 10 carbon atoms, preferably containing 1 to 6 carbon atoms, and can be straight or branched. Examples of ester hydroxyl groups include, but are not limited to, 1-hydroxy-n-butyl, 1-hydroxy-isobutyl, and the like.

[0151] As used herein, "acylamino" refers to a group having the structural formula "-R'-NH-C(O)-R", wherein R' can be selected from hydrogen or alkyl, and R can be selected from alkyl, alkenyl, alkynyl, NR c R d Alkyl substituted, NR c R d Substituted alkenyl and NR c R d substituted alkynyl, alkyl substituted by halogen, alkenyl substituted by cyano, wherein R c and R d It may be selected from alkyl and alkenyl groups.

[0152] Herein, "aryl" refers to a monocyclic, bicyclic or tricyclic aromatic group containing 6 to 14 carbon atoms, including phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, fluorenyl, tetrahydronaphthyl, indanyl, etc. The aryl group may be optionally substituted with 1-5 (e.g., 1, 2, 3, 4 or 5) substituents selected from the following: halogen, C 1-4 Aldehyde, C 1-6 alkyl, cyano, nitro, amino, amide, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., trifluoromethoxy), carboxyl, C 1-4 Alkoxy, ethoxycarbonyl, N(CH3) and C 1-4 acyl group, heterocyclic group or heteroaryl group, etc.

[0153] Herein, "heterocyclic group" includes but is not limited to a 5-membered or 6-membered heterocyclic group containing 1-3 heteroatoms selected from O, S or N, including but not limited to furanyl, thienyl, pyrrolyl, pyrrolidinyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, pyranyl, pyridinyl, pyrimidinyl, pyrazinyl, piperidinyl, morpholinyl and the like.

[0154] Herein, "aromatic heterocyclic group" refers to a group containing 5-14 ring atoms and having 6, 10 or 14 electrons shared in the ring system. The ring atoms contained are carbon atoms and 1-3 heteroatoms selected from oxygen, nitrogen and sulfur. Useful aromatic heterocyclic groups include piperazinyl, morpholinyl, piperidinyl, pyrrolidinyl, thienyl, furanyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, including but not limited to pyrimidinyl, etc. The aromatic heterocyclic group may be optionally substituted by 1-5 (e.g., 1, 2, 3, 4 or 5) substituents selected from the following: halogen, C 1-4 Aldehyde, C 1-6 Straight-chain or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., trifluoromethoxy), carboxyl, C 1-4 Alkoxy, ethoxycarbonyl, N(CH3) and C 1-4 Acyl.

[0155] As used herein, "alkoxy" refers to an oxy group substituted by an alkyl group. Preferred alkoxy groups are alkoxy groups having 1 to 6 carbon atoms in length, and more preferably alkoxy groups having 1 to 3 carbon atoms in length. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and the like. Alkoxy groups may be substituted by one or more substituents, such as halogen or haloalkyl. For example, an alkoxy group may be an alkyl group substituted by 1 to 4 fluorine atoms, or an alkyl group may be an alkyl group substituted by a fluoroalkyl group.

[0156] As used herein, "halogen" refers to fluorine, chlorine, bromine or iodine.

[0157] Herein, "optionally substituted" means that the substituent it modifies can be optionally substituted by 1-5 (e.g., 1, 2, 3, 4 or 5) substituents selected from the following: halogen, C 1-4 Aldehyde, C 1-6 Straight-chain or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., trifluoromethoxy), carboxyl, C 1-4 Alkoxy, ethoxycarbonyl, N(CH3) and C 1-4 Acyl.

[0158] Compounds of the present invention

[0159] The inventors found that males and females in a family began to show muscular dystrophy phenotypes at the age of 50, and the phenotype worsened with age, showing strong family hereditary disease characteristics. Through whole-genome sequencing and other studies on this family, it was found that NDUFA10 and S1PR4 mutations were potential pathogenic gene mutations of muscular dystrophy.

[0160] In view of the fact that there are currently no drugs targeting S1PR4 in clinical practice, the S1P receptor modulators on the market and under development are mainly concentrated on S1PR1 and S1PR5 related targets. The inventors have designed and synthesized a series of compounds with new structures that are selective between different subtypes of S1PR. Therefore, the compounds of the present invention have different regulatory effects on different subtypes of S1PR; in other words, the compounds of the present invention are modulators of S1PR. In a specific embodiment, the compounds of the present invention are S1PR4 agonists. In a preferred embodiment, the compounds of the present invention are selective between S1PR4 and S1PR1; that is, the compounds of the present invention have an agonistic effect on S1PR4, but not on S1PR1.

[0161] In a specific embodiment, the compound of the present invention is a compound represented by the following formula I, or an optical isomer or a pharmaceutically acceptable salt thereof,

[0162]

[0163] In the formula, A, R, R 1 ,n,R 2 , m, and q are as described above.

[0164] In a preferred embodiment, the compounds of the present invention do not include the following compounds:

[0165]

[0166] In a preferred embodiment, the compound of the present invention is represented by the following formula II:

[0167]

[0168] In the formula, R 1 , R 2 ,X,R 3 , R 4 , R 5 As mentioned above.

[0169] In a preferred embodiment, the compound of the present invention is represented by the following formula III:

[0170]

[0171] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 As mentioned above.

[0172] Specifically, the present invention provides a compound selected from the following group, or an optical isomer or a pharmaceutically acceptable salt thereof:

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] The following compounds are preferred:

[0184]

[0185]

[0186] More preferred are the following compounds:

[0187]

[0188]

[0189] Based on the compound of the present invention, the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0190] Examples of pharmaceutically acceptable salts of the compounds of the invention include, but are not limited to, inorganic and organic acid salts, such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and inorganic and organic base salts formed with bases such as sodium hydroxy, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methylglucamine.

[0191] The pharmaceutical composition of the present invention can be formulated into a dosage form suitable for various routes of administration, including but not limited to being formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal or external administration, for the treatment of tumors and other diseases. The dosage is the amount of medicine that effectively improves or eliminates one or more symptoms. For the treatment of a specific disease, an effective amount is an amount of medicine sufficient to improve or alleviate in some way the symptoms associated with the disease. Such a dosage can be administered as a single dose, or it can be administered according to an effective treatment regimen. The dosage may cure the disease, but the administration is usually to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement. The dosage of the drug will be determined based on the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

[0192] The pharmaceutical preparation of the present invention can be administered to any mammal as long as they can obtain the therapeutic effect of the compound of the present invention. The most important of these mammals is human.

[0193] The compounds of the present invention or their pharmaceutical compositions can be used to prepare S1PR regulators. In a specific embodiment, the S1PR regulator is an S1PR4 agonist or an S1PR4 mutant agonist. Thus, the compounds of the present invention or their pharmaceutical compositions can be used to treat various diseases mediated by S1PR4 (including its mutants), including autoimmune diseases. In a specific embodiment, the autoimmune diseases mediated by S1PR4 (including its mutants) include but are not limited to multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, Crohn's disease, etc.

[0194] The pharmaceutical preparations of the present invention can be manufactured in a known manner. For example, they can be manufactured by conventional mixing, granulation, tableting, dissolution, or freeze-drying processes. When manufacturing oral preparations, solid excipients and active compounds can be combined and the mixture can be selectively ground. After adding appropriate amounts of auxiliary agents as needed or necessary, the granular mixture can be processed to obtain tablets or lozenge cores.

[0195] Suitable excipients are in particular fillers, for example sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate; and binders, for example starch paste, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, disintegrants such as the starches mentioned above, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, can be added. Auxiliary agents are in particular flow regulators and lubricants, for example, silica, talc, stearates, such as magnesium calcium stearate, stearic acid or polyethylene glycol. If necessary, the tablet core can be provided with a suitable coating that can resist gastric juice. For this purpose, a concentrated sugar solution can be used. This solution can contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, a lacquer solution and a suitable organic solvent or solvent mixture. To prepare coatings resistant to gastric juices, suitable cellulose solutions may be used, for example cellulose acetate phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the coatings of tablets or dragee cores, for example, for identification or to characterize the combination of active ingredient doses.

[0196] Based on the above compounds and pharmaceutical compositions, the present invention further provides a method for treating S1PR4-mediated diseases, which comprises administering the compounds or pharmaceutical compositions of the present invention to a subject in need thereof.

[0197] The administration method includes but is not limited to various administration methods known in the art, which can be determined according to the actual situation of the patient, including but not limited to parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal or topical administration.

[0198] In view of the fact that the compound of the present invention or its pharmaceutical composition can be used to prepare an S1PR modulator, the present invention also provides a method for regulating S1PR in a subject, comprising administering an effective amount of the compound of the present invention, or its optical isomer or pharmaceutically acceptable salt or pharmaceutical composition to a subject in need thereof. In a specific embodiment, the method is a method for exciting S1PR4 in a subject; more preferably, the method excites S1PR4 in a subject without exciting S1PR1.

[0199] Advantages of the present invention:

[0200] 1. The compound provided by the present invention is a S1PR modulator with a completely new structure, especially a S1PR4 selective receptor modulator;

[0201] 2. The compounds provided by the present invention have excellent biological activity against S1PR4 and S1PR4 mutant proteins;

[0202] 3. The compounds provided by the present invention lay the foundation for the development of drugs that can target S1PR4, have great industrialization and commercialization prospects and market value, and have significant economic benefits.

[0203] The technical scheme of the present invention is further described below in conjunction with specific implementation cases, but the following implementation cases do not constitute a limitation of the present invention, and all various application methods adopted according to the principles and technical means of the present invention belong to the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0204] Materials and Methods

[0205] The synthesis of the compound of the present invention is as follows:

[0206] Example 1

[0207] Synthesis of compounds

[0208] Synthesis of 4-(Bromomethyl)-2-methyl-1,1'-biphenyl

[0209]

[0210] Dissolve 3-methyl-4-phenylbenzoic acid (2.00 g, 9.40 mmol) in 10 mL of anhydrous tetrahydrofuran and stir under an ice bath. Add 1.0 M borane tetrahydrofuran solution (14 mL, 14.20 mmol) dropwise. After the addition, stir at room temperature. After 2 hours, the reaction is complete. Add water dropwise to quench the reaction, and then extract with dichloromethane. Wash with saturated brine. Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 1.50 g of a colorless oil with a yield of 80.6%. The product is used directly in the next step without further purification. LC-MS (ESI): m / z: 199.1 (M+H) + .

[0211] Dissolve (2-methyl-[1,1'-biphenyl]-4-yl)methanol (1.50 g, 7.60 mmol) in 10 mL of dichloromethane, add phosphorus tribromide (820 mg, 3.10 mmol) dropwise under ice bath, stir at room temperature after addition, and the reaction is complete after 0.5 hour. After quenching with water, extract with dichloromethane, and wash with saturated brine. Combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 1.80 g of colorless oil, with a yield of 91.0%.

[0212] 1H NMR (400MHz, DMSO-d6) δ7.45 (dd, J=14.9, 7.9Hz, 2H), 7.39-7.30 (m, 5H), 7.18 (d, J=7.8Hz, 1H), 4.72 (s, 2H), 2.21 (s, 3H). LC-MS (ESI): m / z: 261.3 (M+H) + .

[0213] Synthesis of Ethyl (E)-N-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)acetylcarbamate

[0214]

[0215] Ethyl acetohydroxamate (400 mg, 3.90 mmol) was dissolved in 10 mL of tetrahydrofuran solution and then placed in an ice bath. 1.0 M potassium tert-butoxide tetrahydrofuran solution (15.2 mL, 15.20 mmol) was added dropwise to the reaction system. After the addition was completed, stirring was continued in an ice bath for 0.5 hours. 4-(bromomethyl)-2-methyl-1,1'-biphenyl (1.01 g, 3.90 mmol) was dissolved in 5 mL of tetrahydrofuran and then added dropwise to the reaction system. After the addition was completed, the mixture was returned to room temperature and stirred, and the reaction progress was monitored by TLC. After the reaction was completed, water was added to quench the reaction, extracted with dichloromethane, and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated, and 821 mg of an oily colorless liquid was obtained through a silica gel column with a yield of 74.4%.

[0216] 1 H NMR(400MHz, DMSO-d6)δ7.43(t,J=7.3Hz,2H),7.38-7.30(m,3H),7.27-7.16(m,3H), 4.89(s,2H),3.95(q,J=7.5Hz,2H),2.22(s,3H),1.90(s,3H),1.20(t,J=7.5Hz,3H).

[0217] Synthesis of (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one O-((2-methyl-1,1'-biphenyl]-4-yl)methyl)oxime

[0218]

[0219] Dissolve (E)-N-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)acetylcarbamate (200mg, 0.70mmol) in 5mL methanol. Add 4.0M hydrogen chloride dioxane solution (0.4mL, 0.30mmol) to the reaction solution. After stirring for 0.5 hours, add triethylamine to adjust the pH to between 4-6. Then dissolve 1-(3-ethyl-4-(hydroxymethyl)phenyl)ethanone (125mg, 0.70mmol) in 2mL methanol and add it dropwise to the reaction system. Monitor the progress of the reaction by TLC. After the reaction is completed, add water to quench the reaction, extract with dichloromethane, and wash with saturated brine. Combine the organic phases, dry with anhydrous sodium sulfate, and concentrate through a silica gel column to obtain 140mg of an oily colorless liquid with a yield of 53.6%.

[0220] 1 H NMR (400MHz, CDCl3) δ7.46 (s, 1H), 7.42 (dd, J = 8.0, 1.6Hz, 1H), 7.36-7.27 (m, 4H), 7.29-7.18 (m, 3H), 7.18-7 .06(m,2H),5.18(s,2H),4.66(s,2H),2.65(q,J=7.5Hz,2H),2.21(s,3H),1.50(s,3H),1.18(t,J=7.5Hz,3H).

[0221] Synthesis of (E)-2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)iminoethyl)benzaldehyde

[0222]

[0223] (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one O-((2-methyl-[1,1'-biphenyl]-4-yl)methyl)oxime (112 mg, 0.30 mmol) was dissolved in 3 mL of DMSO solvent. 2-iodobenzoic acid (IBX) oxidant (84 mg, 0.30 mmol) was added. Then stirred at room temperature for about 2 hours. TLC was used to monitor the progress of the reaction. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with dichloromethane and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to obtain 99 mg of an oily colorless liquid with a yield of 89.1%.

[0224] 1H NMR (400MHz, CDCl3) δ10.29(s,1H),7.82(d,J=6.4Hz,1H),7.64(dd,J=12.9,5.5Hz,1H),7.60(d,J=9.7Hz,1H),7.40(dd,J=18.7,11. 4Hz,2H),7.36-7.30(m,5H),7.25-7.19(m,1H),5.29(s,2H),3.09(q,J=7.5Hz,2H),2.31(s,3H),2.30(s,3H),1.33(t,J=7.5Hz,3H).

[0225] Synthesis of (S,E)-1-(2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 30)

[0226]

[0227] Dissolve (E)-2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)iminoethyl)benzaldehyde (74 mg, 0.20 mmol) in 5 mL of methanol, add 1 drop of acetic acid, and then add S-pyrrolidine 3-carboxylic acid (35 mg, 0.30 mmol). After stirring at room temperature for 3 minutes, add sodium cyanoborohydride (19 mg, 0.30 mmol). Monitor the progress of the reaction by TLC. After the reaction is complete, the reaction system is distilled under reduced pressure, and water and dichloromethane are added for extraction. Wash with saturated brine, combine the organic phases, dry with anhydrous sodium sulfate, and concentrate on a silica gel column to obtain 29 mg of a white solid with a yield of 30.8%.

[0228] 1 H NMR (400MHz, CD3OD) δ7.57 (s, 1H), 7.51 (d, J = 8.0Hz, 1H), 7.44-7.35 (m, 3H), 7. 33-7.26(m,5H),7.17(d,J=7.7Hz,1H),5.20(s,2H),4.02(s,2H),3.19(t,J=8.6 Hz,1H),3.11-3.00(m,3H),2.90(dd,J=17.2,7.9Hz,1H),2.79(q,J=7.5Hz,2H) ,2.26(s,3H),2.24(s,3H),2.15(dd,J=14.6,7.3Hz,2H),1.23(t,J=7.5Hz,3H). 13C NMR (151MHz, CD3OD) δ179.95,154.70,143.25,141.79,141.48,137.08,136.62,134.91,130.01,129.84,129.31,128.79,127.77 ,126.49,126.28,125.37,123.57,75.60,57.40,55.54,53.76,44.56,28.06,25.20,19.22,14.48,11.50.HRMS(ESI)(m / z):[M+H] + Calculate for C 30 H 34 N2O3, 471.2648; found: 471.2650. HPLC Purity: 98.16%, retention time: 1.52min.

[0229] The synthetic route of representative compound 13 is as follows:

[0230] Synthesis of Ethyl (E)-N-((3-(Trifluoromethyl)benzyl)oxy)acetylcarbamate

[0231]

[0232] Ethyl acetohydroxamate (865mg, 8.40mmol) was dissolved in 10mL tetrahydrofuran solution and then placed in an ice bath. 1.0M potassium tert-butoxide tetrahydrofuran solution (17mL, 16.70mmol) was added dropwise to the reaction system. After the addition was complete, stirring was continued under an ice bath for 0.5 hours. 3-(trifluoromethyl)benzyl bromide (1.99g, 8.40mmol) was dissolved in 5mL tetrahydrofuran and then added dropwise to the reaction system. After the addition was complete, stirring was restored to room temperature and the reaction progress was monitored by TLC. After the reaction was completed, water was added to quench the reaction, extracted with dichloromethane, and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated over a silica gel column to obtain 1.81g of an oily colorless liquid with a yield of 82.6%.

[0233] 1 H NMR (400MHz, CDCl3) δ7.63 (s, 1H), 7.54 (d, J = 7.1Hz, 2H), 7.45 (t, J = 7.7Hz, 1H), 4.97 (s, 2H), 3.98 (q, J = 7.1Hz, 2H), 1.95 (s, 3H), 1.24 (t, J = 7.1Hz, 3H).

[0234] Synthesis of (E)-1-(3-ethyl-4-hydroxymethyl)phenyl)ethane-1-one O-(3-(trifluoromethyl)benzyl)oxime

[0235]

[0236] Dissolve (E)-N-((3-(trifluoromethyl)benzyl)oxy)acetylcarbamate (157mg, 0.60mmol) in 5mL of methanol. Add 0.4mL of 4.0M hydrogen chloride dioxane solution to the reaction solution. After stirring for 0.5 hours, add triethylamine to adjust the pH to between 4-6. Then dissolve 1-(3-ethyl-4-(hydroxymethyl)phenyl)ethanone (107mg, 0.60mmol) in 2mL of methanol and add it dropwise to the reaction system. Monitor the progress of the reaction by TLC. After the reaction is completed, add water to quench the reaction, extract with dichloromethane, and wash with saturated brine. The combined organic phases are dried over anhydrous sodium sulfate and concentrated through a silica gel column to obtain 150mg of an oily colorless liquid with a yield of 71.1%.

[0237] 1 H NMR(400MHz, CDCl3)δ7.69(s,1H),7.59(dd,J=12.7,7.9Hz,3H),7.51-7.46(m,2H),7.37(d,J=8.0Hz, 1H),5.28(s,2H),4.74(s,1H),4.72(s,2H),2.71(q,J=7.6Hz,2H),2.28(s,3H),1.24(t,J=7.5Hz,3H).

[0238] Synthesis of (E)-2-ethyl-4-(1-(((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzaldehyde

[0239]

[0240] Dissolve (E)-1-(3-ethyl-4-hydroxymethyl)phenyl)ethane-1-one O-(3-(trifluoromethyl)benzyl)oxime (211 mg, 0.60 mmol) in 3 mL of DMSO solvent. Add IBX oxidant (308 mg, 1.10 mmol). Then stir at room temperature for about 2 hours. Monitor the progress of the reaction by TLC. After the reaction is completed, add water to quench the reaction, extract with dichloromethane, and wash with saturated brine. Combine the organic phases, dry with anhydrous sodium sulfate, and concentrate on a silica gel column to obtain 126 mg of an oily colorless liquid with a yield of 60.0%.

[0241] 1H NMR (400MHz, CDCl3) δ10.29 (s, 1H), 7.82 (d, J = 8.1Hz, 1H), 7.69 (s, 1H), 7.64-7.53 (m, 4H), 7 .50(d,J=7.7Hz,1H),5.31(s,2H),3.08(d,J=7.5Hz,2H),2.30(s,3H),1.28(t,J=7.5Hz,3H).

[0242] Synthesis of (E)-1-(2-ethyl-4-(1-((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 13)

[0243]

[0244] (E)-2-ethyl-4-(1-(((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzaldehyde (105 mg, 0.30 mmol) was dissolved in 5 mL of methanol, 1 drop of acetic acid was added, and then 3-azetidinecarboxylic acid (61 mg, 0.60 mmol) was added. After stirring at room temperature for 3 minutes, sodium cyanoborohydride (38 mg, 0.60 mmol) was added. TLC was used to monitor the progress of the reaction. After the reaction was complete, the reaction system was distilled under reduced pressure, an appropriate amount of water was added, and the mixture was extracted with dichloromethane, washed with saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated over a silica gel column to obtain 78 mg of an oily substance with a yield of 60.0%.

[0245] 1 H NMR (400MHz, CD3OD) δ7.71(s,1H),7.67(d,J=7.4Hz,1H),7.62-7.51(m,3H),7.47(dd,J=8.0,1.6Hz,1H),7.31(d,J=8.0Hz,1H),5.28(s,2H),3. 98(s,2H),3.83(t,J=8.8Hz,2H),3.67(t,J=8.4Hz,2H),3.28(dd,J=15.2,6.9Hz,1H),2.73(q,J=7.5Hz,2H),2.26(s,3H),1.21(t,J=7.5Hz,3H). 13C NMR (151MHz, CD3OD) δ178.20,155.32,142.88,139.73,136.16,133.52,131.41,130.23(q,J=32.0Hz),128.87,128.82,12 6.17,124.32,124.31(q,J=272.0Hz),124.06,123.61,74.70,57.68,35.94,25.11,14.28,11.47.HRMS(ESI)(m / z):[M+H] + Calculate for C 23 H 25 F3N2O3, 435.1896; found: 435.1897. HPLC Purity: 99.79%, retention time: 1.17min.

[0246] The synthetic route of representative compound 16 is shown below:

[0247] Synthesis of 5-(Bromomethyl)-2-isopropoxybenzonitrile

[0248]

[0249] 2.0M oxalyl chloride dichloromethane solution (14.6mL, 29.20mmol) was added to 3-cyano-4-(isopropoxy)benzoic acid (2.01g, 9.80mmol) and one drop of DMF was added. The reaction mixture was stirred at room temperature for 0.5 hours and then concentrated under reduced pressure. 3-cyano-4-(isopropoxy)benzoyl chloride (923mg, 4.50mmol) was dissolved in 10mL tetrahydrofuran and cooled to 0°C. Sodium borohydride (414mg, 11.20mmol) was added, followed by 2mL methanol and the reaction mixture was stirred at 0°C for 20 minutes and then warmed to room temperature. After 2 hours, the reaction mixture was acidified to pH=3 with 1.0M hydrochloric acid. The aqueous layer was extracted with ethyl acetate, the organic layers were combined and dried over anhydrous sodium sulfate, and concentrated to give 1.12g of colorless oil, with a yield of 59.9%.

[0250] 1 H NMR (400MHz, CDCl3) δ7.48(s,1H),7.47(d,J=12Hz,1H),7.45(s,1H),6.92(d,J =8.6Hz,1H),4.61(dt,J=12.2,6.2Hz,1H),4.57(s,2H),1.36(d,J=6.1Hz,6H).

[0251] Dissolve 5-(Hydroxymethyl)-2-isopropoxybenzonitrile (993 mg, 5.20 mmol) in 10 mL of dichloromethane, add phosphorus tribromide (0.2 mL, 2.10 mmol) dropwise under ice bath, stir at room temperature after addition, and the reaction is complete after 0.5 hour. Quench with water, extract with dichloromethane, and wash with saturated brine. Combine the organic phases, dry with anhydrous sodium sulfate, and concentrate to obtain 1.19 g of colorless oil, with a yield of 90.5%.

[0252] 1 H NMR (400MHz, CDCl3) δ7.56 (d, J=2.1Hz, 1H), 7.51 (dd, J=8.7, 2.2Hz, 1H), 6.92 (d ,J=8.7Hz,1H),4.65(dt,J=12.0,6.0Hz,1H),4.42(s,2H),1.39(d,J=6.1Hz,6H).

[0253] Synthesis of Ethyl-N-((3-cyano-4-(isopropoxy)benzyl)oxy)acetylcarbamate

[0254]

[0255] Ethyl acetohydroxamate (402mg, 3.90mmol) was dissolved in 10mL tetrahydrofuran solution and then placed in an ice bath. 1.0M potassium tert-butoxide tetrahydrofuran solution (8.0mL, 8.00mmol) was added dropwise to the reaction system, and stirring continued for 0.5 hours under an ice bath after the addition was complete. 3-cyano-4-isopropoxybenzyl bromide (987mg, 3.90mmol) was dissolved in 5mL tetrahydrofuran and then added dropwise to the reaction system. After the addition was complete, stirring was restored to room temperature and the reaction progress was monitored by TLC. After the reaction was completed, water was added to quench the reaction, extracted with dichloromethane, and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated over a silica gel column to obtain 619mg of an oily colorless liquid with a yield of 57.5%.

[0256] 1 H NMR (400MHz, CDCl3) δ7.54(d,J=2.0Hz,1H),7.48(dd,J=8.7,2.1Hz,1H),6.92(d,J=8.7Hz,1H),4.82(s,2H),4 .64(dt,J=12.1,6.1Hz,1H),3.97(q,J=7.1Hz,2H),1.92(s,3H),1.39(d,J=6.1Hz,6H),1.24(t,J=7.1Hz,3H).

[0257] Synthesis of (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one-O-(3-cyano-4-(isopropoxy)benzyl)oxime

[0258]

[0259] Ethyl-N-((3-cyano-4-(isopropoxy)benzyl)oxy)acetylcarbamate (138 mg, 0.50 mmol) was dissolved in 5 mL of methanol. 4.0 M hydrogen chloride dioxane solution (0.4 mL, 0.30 mmol) was added dropwise to the reaction solution. After stirring for 0.5 hours, triethylamine was added dropwise to adjust the pH to between 4 and 6. Then 1-(3-ethyl-4-(hydroxymethyl)phenyl)ethanone (90 mg, 0.50 mmol) was dissolved in 2 mL of methanol and added dropwise to the reaction system. TLC was used to monitor the progress of the reaction. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with dichloromethane and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated over a silica gel column to obtain 127 mg of an oily colorless liquid with a yield of 69.3%.

[0260] 1 H NMR (400MHz, CDCl3) δ7.61(d,J=2.0Hz,1H),7.54(dd,J=8.7,2.1Hz,1H),7.45(dd,J=12.1,4.1Hz,2H),7.38(d,J=7.9Hz,1H),6.94(d,J=8.7Hz ,1H),5.13(s,2H),4.73(s,2H),4.64(dt,J=12.1,6.0Hz,1H),2.72(q,J =7.6Hz,2H),2.24(s,3H),1.40(d,J=6.1Hz,6H),1.24(t,J=7.6Hz,3H).

[0261] Synthesis of (E)-5-((((1-(3-ethyl-4-formylphenyl)ethylidene)amino)oxy)methyl)-2-isopropoxybenzonitrile

[0262]

[0263] Dissolve (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one-O-(3-cyano-4-(isopropoxy)benzyl)oxime (147 mg, 0.40 mmol) in 4 mL of DMSO solvent, and add IBX oxidant (225 mg, 0.80 mmol). Stir at room temperature for about 2 hours, and monitor the progress of the reaction by TLC. After the reaction is completed, water is added to quench the reaction, and the mixture is extracted with dichloromethane and washed with saturated brine. The combined organic phases are dried over anhydrous sodium sulfate and concentrated through a silica gel column to obtain 119 mg of an oily colorless liquid with a yield of 81.2%.

[0264] 1 H NMR (400MHz, CDCl3) δ10.28(s,1H),7.82(d,J=8.1Hz,1H),7.65-7.58(m,2H),7.58-7.50(m,2H),6.95(d,J=8.7Hz,1H),5. 16(s,2H),4.65(dt,J=12.1,6.1Hz,1H),3.08(q,J=7.5Hz,2H),2.26(s,3H),1.40(d,J=6.1Hz,6H),1.28(t,J=7.5Hz,3H).

[0265] Synthesis of (E)-1-(2-ethyl-4-(1-((3-cyano-4-(isopropoxy)benzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 16)

[0266]

[0267] (E)-5-((((1-(3-ethyl-4-formylphenyl)ethylidene)amino)oxy)methyl)-2-isopropoxybenzonitrile (109 mg, 0.30 mmol) was dissolved in 5 mL of methanol, 1 drop of acetic acid was added, and then 3-azetidinecarboxylic acid (61 mg, 0.60 mmol) was added. After stirring at room temperature for 3 minutes, sodium cyanoborohydride (19 mg, 0.30 mmol) was added. TLC was used to monitor the progress of the reaction. After the reaction was complete, the reaction system was distilled under reduced pressure, water and dichloromethane were added for extraction, and the mixture was washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated over a silica gel column to obtain 70 mg of a light-colored solid with a yield of 52.3%.

[0268] 1 H NMR (400MHz, CD3OD) δ7.64 (d, J=6.7Hz, 2H), 7.56 (s, 1H), 7.50 (dd, J=8.0, 1.6Hz, 1H),7.33(d,J=8.1Hz,1H),7.16(d,J=9.4Hz,1H),5.14(s,2H),4.78-4.73(m,1H) ,4.18(s,2H),3.99(t,J=9.3Hz,2H),3.90(t,J=8.6Hz,2H),3.40-3.33(m,1H),2. 76(q,J=7.5Hz,2H),2.23(s,3H),1.38(s,3H),1.37(s,3H),1.23(t,J=7.5Hz,3H). 13C NMR(151MHz,CD3OD)δ177.23,159.53,154.92,143.23,136.96,134.67,133.44,131.00,129.23,126.42,123.80, 116.06,113.66,101.88,74.22,71.68,57.49,56.20,35.40,25.12,20.71,14.38,11.39.HRMS(ESI)(m / z):[M+H] + Calculate for C 26 H 31 N3O4,450.2396; found:450.2395.HPLCPurity:96.46%,retention time:1.02min.

[0269] The synthetic route of compound 17 is as follows:

[0270] Synthesis of Ethyl (E)-N-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)acetylcarbamate

[0271]

[0272] Ethyl acetohydroxamate (196mg, 1.90mmol) was dissolved in 5mL tetrahydrofuran solution and then placed in an ice bath. 1.0M potassium tert-butoxide tetrahydrofuran solution (4mL, 3.90mmol) was added dropwise to the reaction system, and the addition was continued to stir for 0.5 hours under an ice bath. 4-Chloromethyl-1-cyclopentyl-2-trifluoromethylbenzene (498mg, 1.90mmol) was dissolved in 3mL tetrahydrofuran and then added dropwise to the reaction system. After the addition was completed, stirring was restored to room temperature, and TLC monitored the reaction progress. After the reaction was completed, water was added to quench the reaction, extracted with dichloromethane, and washed with saturated brine. The combined organic phase was dried over anhydrous sodium sulfate and concentrated over a silica gel column to obtain 532mg of an oily colorless liquid with a yield of 85.1%.

[0273] 1 H NMR (400MHz, CDCl3) δ7.59(s,1H),7.48(d,J=8.4Hz,1H),7.43(d,J=8.1Hz,1H),4.91(s,2H),3.99(q,J=7.1Hz,2H),3.41 -3.28(m,1H),2.07(m,2H),1.94(s,3H),1.89-1.80(m,2H),1.73-1.68(m,2H),1.63-1.56(m,2H),1.25(t,J=7.1Hz,3H).

[0274] Synthesis of (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one-O-(4-cyclopentyl-3-(trifluoromethyl)benzyl)oxime

[0275]

[0276] Dissolve (E)-N-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)acetylcarbamate (461 mg, 1.40 mmol) in 5 mL of methanol. Add 4.0 M hydrogen chloride dioxane solution (1.0 mL, 0.70 mmol) dropwise to the reaction solution. After stirring for 0.5 hours, triethylamine was added dropwise to adjust the pH to between 4 and 6. Then, 1-(3-ethyl-4-(hydroxymethyl)phenyl)ethanone (267 mg, 1.50 mmol) was dissolved in 2 mL of methanol and added dropwise to the reaction system. TLC was used to monitor the progress of the reaction. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with dichloromethane and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated over a silica gel column to obtain 489 mg of an oily colorless liquid with a yield of 83.4%.

[0277] 1 H NMR (400MHz, CDCl3) δ7.66 (s, 1H), 7.54 (d, J = 8.0Hz, 1H), 7.49 (d, J = 6.2Hz, 1 H),7.45(d,J=8.6Hz,2H),7.38(t,J=8.6Hz,1H),5.22(s,2H),4.72(s,2H),3. 36(p,J=8.5Hz,1H),2.71(q,J=7.6Hz,2H),2.22(s,3H),2.14-2.04(m,2H),1. 92-1.81(m,2H),1.77-1.69(m,2H),1.61-1.56(m,2H),1.25(t,J=7.1Hz,3H).

[0278] Synthesis of (E)-4-(1-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzaldehyde

[0279]

[0280] (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one-O-(4-cyclopentyl-3-(trifluoromethyl)benzyl)oxime (503 mg, 1.20 mmol) was dissolved in 3 mL of DMSO solvent. IBX oxidant (952 mg, 2.40 mmol) was added, and then stirred at room temperature for about 2 hours. TLC was used to monitor the progress of the reaction. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with dichloromethane and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and passed through a silica gel column to obtain 302 mg of an oily colorless liquid, 60.3%.

[0281] 1 H NMR (400MHz, CDCl3) δ10.29(s,1H),7.82(d,J=8.1Hz,1H),7.69-7.59(m,2H),7.59-7.52(m,2H),7.47(d,J=8.1Hz,1H),5.25(s,2H),3.45-3.30(m ,1H),3.08(q,J=7.5Hz,2H),2.28(s,3H),2.07(d,J=12.3Hz,2H),1.88-1 .80(m,2H),1.73-1.70(m,2H),1.64-1.54(m,2H),1.28(t,J=7.1Hz,3H).

[0282] Synthesis of (E)-1-(4-(1-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 17)

[0283]

[0284] (E)-4-(1-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzaldehyde (500 mg, 1.20 mmol) was dissolved in 5 mL of methanol, and 1 drop of acetic acid was added, followed by 3-azetidinecarboxylic acid (242 mg, 2.40 mmol). After stirring at room temperature for 3 minutes, sodium cyanoborohydride (113 mg, 1.80 mmol) was added. TLC was used to monitor the progress of the reaction. After the reaction was complete, the reaction system was distilled under reduced pressure, an appropriate amount of water was added, and the mixture was extracted with dichloromethane and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated over a silica gel column to obtain 182 mg of a light-colored solid with a yield of 30.2%.

[0285] 1H NMR (400MHz, CD3OD) δ7.64(s,1H),7.53(dt,J=9.8,5.1Hz,4H),7.38(t,J=11.5Hz,1H),5.22(s,2H),4.25(s,2H),4.02(d,J=8.3Hz,4H),3. 43-3.33(m,1H),2.75(dd,J=15.0,7.5Hz,2H),2.29-2.14(s,3H),2.03(m,2H),1.93-1.81(m,2H),1.78-1.57(m,4H),1.20(t,J=7.1Hz,3H). 13 C NMR(151MHz,CD3OD)δ177.79,154.79,145.36,143.28,137.08,136.13,131.80,130.92,129.27,127.96,127.80(q,J=28.7Hz),1 26.46,124.78(q,J=273.5Hz),124.76,123.84,74.79,57.14,40.91,35.57,25.62,25.20,14.36,11.42.HRMS(ESI)(m / z):[M+H] + Calculate for C 28 H 33 F3N2O3,503.2522; found:503.2523.HPLC Purity:96.46%,retention time:2.28min.

[0286] The reaction conditions of other compounds are similar to those of the above compounds.

[0287] (E)-1-(4-(1-((4-cyclohexylbenzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 1)

[0288]

[0289] 1H NMR (400MHz, Methanol-d4) δ7.60(d,J=8.3Hz,2H),7.30(dd,J=8.2,2.7Hz,4H),7.19(d,J=8.1Hz,2H),5.14(s,2H),3.64(d,J=2.4Hz,2H),3.53(t,J=8.1Hz ,2H),3.38–3.32(m,2H),3.20(dd,J=16.6,8.2Hz,1H),2.57–2.45(m,1H),2.2 2(s,3H),1.84(d,J=9.2Hz,4H),1.75(d,J=12.2Hz,1H),1.43(d,J=4.4Hz,6H). 13 C NMR(151MHz,Methanol-d4)δ154.60,147.52,138.04,135.64,135.45,128.59,128.01,126.36,125.82 ,75.68,62.35,57.58,44.40,36.48,34.30,29.46,26.62,25.86,11.45.LC-MS(ESI):m / z:421.1(M+H) + .

[0290] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 2)

[0291] 1 H NMR (400MHz, Methanol-d4) δ7.56(d,J=17.6Hz,1H),7.53(d,J=7.4Hz,1H),7.39(t,J=12.5Hz,1H),7.30(d,J=7.9Hz,2H),7.19(d,J=7.9Hz,2H),5.11(s, 2H),4.31(s,2H),4.10(dd,J=21.9,12.7Hz,2H),4.02(t,J=8.4Hz,2H),3.35 (s,3H),2.78-2.70(m,2H),1.49-1.36(m,9H).LC-MS(ESI):m / z:431.1(M+H) + .

[0292] (E)-1-(4-(1-((4-cyclohexylbenzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 3)

[0293] 1H NMR(400MHz, DMSO)δ8.36(s,1H),8.04(dt,J=28.8,6.4Hz,4H),7.71–7.46(m,4H),4.38-4.33(m,2H),4.10-4.01(m,2H),3.59-3.55(m,2 H),2.85–2.76(m,2H),2.79(q,J=7.5Hz,2H),2.57–2.45(m,1H),1.24-1.21(m,3H),1.20(t,J=7.5Hz,3H).LC-MS(ESI):m / z:449.1(M+H) + .

[0294] Structure of (E)-1-(2-ethyl-4-(1-(((4-(5-fluoropyrazin-2-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 4)

[0295]

[0296] Pale yellow solid, yield 18.4% 1 H NMR (500MHz, Methanol-d4) δ8.58(dd,J=8.3,1.3Hz,1H),8.37(d,J=1.5Hz,1H),7.64(d,J=1.9Hz,1 H),7.58(dd,J=8.1,1.9Hz,1H),7.49(d,J=8.1Hz,1H),7.42(d,J=7.8Hz,1H),7.40–7.35(m,2H),5. 26(s,2H),4.40(s,2H),3.53(dd,J=11.2,5.8Hz,1H),3.39–3.35(m,2H),3.28(dd,J=11.0,7.4Hz,1 H),3.15–3.06(m,1H),2.82(q,J=7.5Hz,2H),2.37(s,3H),2.34–2.22(m,5H),1.26(t,J=7.5Hz,3H). 13C NMR (126MHz, Methanol-d4) δ177.85,160.27,158.28,154.49,152.90,143.73,141.29,141.22,139.46,137.74,136.37,135.08,131.94,131.64,13 0.48,130.33,130.21,129.58,126.66,125.50,123.97,75.34,56.55,54. 38,53.52,43.60,27.72,25.23,19.04,14.55,11.40.MS(ESI)(m / z):[M+H] + 491.25.

[0297] Synthesis of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(5-hydroxypyrazin-2-yl)-benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 5)

[0298]

[0299] Yellow solid, yield 72.5%. 1 H NMR (500MHz, Methanol-d4) δ8.17(d,J=1.4Hz,1H),7.67(d,J=1.8Hz,1H),7.61(dd,J=8.1,1.9Hz, 1H),7.57(d,J=8.1Hz,1H),7.51(d,J=1.4Hz,1H),7.35(d,J=8.1Hz,2H),7.33–7.29(m,1H),5.24( s,2H),4.53(s,2H),3.70–3.57(m,2H),3.51–3.43(m,2H),3.38(q,J=8.6,8.2Hz,1H),2.85(q,J=7 .5Hz,2H),2.49–2.41(m,1H),2.38(s,3H),2.36–2.30(m,1H),2.29(s,3H),1.27(t,J=7.5Hz,3H). 13 C NMR (126MHz, Methanol-d4) δ154.34,143.95,138.49,138.17,136.24,135.20,130.78,130.24,129.10,129. 01,126.79,125.48,124.11,75.49,55.17,54.38,53.64,27.07,25.36,19.14,14.60,11.39.MS(m / z):[M+H]+ 489.20

[0300] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyloxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 6)

[0301] 1 H NMR (400MHz, Methanol-d4) δ7.70–7.63(m,3H),7.57(d,J=16.3Hz,2H),7.44(d,J =8.2Hz,2H),5.22(s,2H),3.96(d,J=5.7Hz,2H),3.35(s,2H),3.16-3.12(m,1H), 3.05-2.98(m,2H),2.95–2.82(m,2H),2.26(s,3H),2.16(d,J=8.0Hz,2H),1.82-1 .80(m,,2H),1.78-1.75(m,3H),1.58-1.45(m,6H).LC-MS(ESI):m / z:503.1(M+H) + .

[0302] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)piperidine-3-carboxylic acid (Compound 7)

[0303] 1 H NMR (400MHz, Methanol-d4) δ7.70–7.63(m,3H),7.57(d,J=16.3Hz,2H),7.44(d,J =8.2Hz,2H),5.22(s,2H),3.96(d,J=5.7Hz,2H),3.35(s,2H),3.16-3.12(m,1H), 3.05-2.98(m,2H),2.95–2.82(m,2H),2.26(s,3H),2.16(d,J=8.0Hz,2H),1.82-1 .80(m,,2H),1.78-1.75(m,3H),1.58-1.45(m,8H).LC-MS(ESI):m / z:517.2(M+H) + .

[0304] (E)-4-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)amino)butanoic acid (Compound 8)

[0305] 1H NMR (400MHz, Methanol-d4) δ7.65(d,J=8.1Hz,3H),7.58(t,J=10.4Hz,1H),7.53(d,J=8.1Hz,1H),7.41(t,J=10.5Hz,2H),5.21(s,2H),3.88( s,2H),2.91(t,J=11.2Hz,1H),2.76(t,J=7.1Hz,2H),2.30–2.17(m,5H),1.85-1.75(m,6H),1.61–1.29(m,6H).LC-MS(ESI):m / z:491.2(M+H) + .

[0306] (E)-3-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)amino)cyclohexane-1-carboxylic acid (Compound 9)

[0307] 1 H NMR(400MHz,Methanol-d4)δ7.77–7.32(m,7H),5.21(s,2H),3.91(s,2H),2.91(s,1H) ),2.68(s,1H),2.25(s,3H),2.18-2.15(m,2H),1.88-1.70(m,9H),1.65–1.33(m,8H). 13 C NMR(151MHz,MeOD)δ182.91,155.03,146.14,136.31,135.59,131.60,128.55,128.47,128.10,125.97,124.84,12 4.79,74.73,55.54,49.13,45.59,40.06,34.19,31.24,29.43,26.60,25.70,11.40.LC-MS(ESI):m / z:531.2(M+H) + .

[0308] (E)-3-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)amino)propanoic acid (Compound 10)

[0309] 1H NMR (400MHz, Methanol-d4) δ7.65(d,J=7.4Hz,3H),7.56(dd,J=24.3,8.1Hz,2H),7.39(d,J=8.0Hz,2H),5.21(s, 2H),3.90(s,2H),2.95-2.90(m,3H),2.44(t,J=6.6Hz,2H),2.25(s,3H),1.87(d,J=16.6Hz,2H),1.78(d,J=11.1

[0310] Hz, 3H), 1.48 (ddd, J = 33.8, 22.3, 10.1Hz, 5H). 13 C NMR (151MHz, Methanol-d4)

[0311] δ178.44,154.94,146.14,137.98,136.30,135.86,131.60,128.53,128.10,126.03,124.84,124. 79,74.74,51.72,45.19,40.06,35.04,34.18,26.59,25.70,11.38.LC-MS(ESI):m / z:478.2(M+H) + .

[0312] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-fluorobenzyl)azetidine-3-carboxylic acid (Compound 11)

[0313]

[0314] White solid, yield 40.6%, melting point: 144.6-144.9℃. 1 H NMR (400MHz, CDCl3) δ7.74-7.27(m,6H),5.26(s,2H),3.97(s,2H),3.80(d,J=8.2Hz,2H),3.72-3.67(m,3H),2.94-2.90( m,1H),2.27(s,3H),1.94-1.82(m,2H),1.80-1.75(m,2H),1.56-1.50(m,2H),1.45-1.40(m,4H).HRMS(ESI)(m / z):[M+H] + Calculate for C 27 H 30F4N2O3,507.2271; found:507.2270.HPLCPurity:91.98%,retention time:2.26min.

[0315] Structure of (E)-1-(2-bromo-4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 12)

[0316]

[0317] White solid, yield 40.6%, melting point: 131.6-131.9℃. 1 H NMR (400MHz, CD3OD) δ7.91(d,J=1.6Hz,1H),7.69-7.63(m,2H),7.63-7.49(m,2H),7.45(d,J=8.1Hz,1H),5.23(s,2H),4.08(s,2H),3 .87(t,J=8.6Hz,2H),3.75(t,J=8.2Hz,2H),2.93-2.86(m,2H),2.30(s,3H),1.84-1.80(m,2H),1.77-1.70(m,3H),1.60-1.34(m,5H). 13 C NMR (151MHz, CD3OD) δ178.85,154.09,147.89,146.25,138.90,131.73,129.72,129.46,128.84,128.53,128.16,127 .33,124.97,121.07,112.36,90.94,74.97,61.53,58.01,40.07,34.17,26.69,25.70,12.97.HRMS(ESI)(m / z):[M+H] + Calculate for C 27 H 30 BrF3N2O3,567.1470; found:567.1473.HPLCPurity:90.25%,retention time:2.87min.

[0318] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-methylbenzyl)azetidine-3-carboxylic acid (Compound 18)

[0319]

[0320] White solid, yield 40.6%, melting point: 155.1-155.6℃. 1 H NMR (400MHz, CD3OD) δ7.64 (d, J=5.9Hz, 1H), 7.59-7.42 (m, 4H), 7.37-7.29 (m,1H),5.20(s,2H),4.05(s,2H),3.91(t,J=8.9Hz,2H),3.77(dd,J=17.0, 8.5Hz,2H),3.60(dd,J=14.1,7.1Hz,1H),2.91(t,J=11.3Hz,1H),2.39(s, 3H),2.23(s,3H),1.88-1.80(m,2H),1.79-1.70(m,3H),1.46-1.39(m,5H). 13 C NMR (151MHz, CD3OD) δ178.49,154.91,146.15,138.90,136.99,136.37,131.61,128.89,128.52,128.11,127.91,127.31,124.88,124.8 0(q,J=273.5Hz),123.68,74.75,57.59,40.05,35.70,34.18,34.13,26.59,25.70,17.99,16.97,11.38.HRMS(ESI)(m / z):[M+H]+calcd for C 28 H 33 F3N2O3,503.2522; found:503.2523.HPLC Purity:94.29%,retention time:2.57min.

[0321] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-methoxybenzyl)azetidine-3-carboxylic acid (Compound 27)

[0322]

[0323] White solid, yield 40.6%, melting point: 145.2-145.6℃. 1H NMR (400MHz, CD3OD) δ7.66(s1H),7.64-7.49(m,3H),7.25(t,J=8.7Hz,2H),5.21(s,2H),3.84(s,3H),3.70(s,2H),3.59(d,J=8.2Hz,2H),3 .52(t,J=8.0Hz,1H),3.42(t,J=8.2Hz,2H),2.91(d,J=10.6Hz,1H),2.23(s,3H),1.92-1.83(m,2H),1.79-1.87(m,3H),1.63-1.48(m,5H). 13 C NMR (151MHz, CD3OD) δ175.87,157.76,154.59,146.26,139.82,138.93,136.16,131.75,128.55,127.30,127.01,124.98,124.80(q, J=273.5Hz),119.36,118.66,107.91,74.92,60.20,57.19,54.77,40.06,34.72,34.18,26.58,25.69,11.39.HRMS(ESI)(m / z):[M+H] + Calculate for C 28 H 33 F3N2O4,519.2471; found:519.2473.HPLC Purity:96.28%,retention time:1.27min.

[0324] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-(trifluoromethyl)benzyl)azetidine-3-carboxylic acid (Compound 19)

[0325]

[0326] White solid, yield 40.6%, melting point: 135.6-138.9℃. 1H NMR (400MHz, CD3OD) δ7.98(s,1H),7.88(d,J=8.1Hz,1H),7.69-7.51(m,4H),5.24(s,2H),3.96(s,2H),3.73(t,J=8.3Hz,2H) ,3.53(t,J=8.0Hz,2H),3.29-3.19(m,1H),2.92(t,J=11.3Hz,1H),1.86-1.80(m,2H),1.78-1.70(m,3H),1.62-1.35(m,5H). 13 C NMR (151MHz, CD3OD) δ178.45,153.61,146.30,138.92,135.99,135.83,131.79,129.85,129.41,128.52,128.15,127.29,125.06,124.77(q ,J=273.5Hz),124.22(q,J=273.6Hz),123.02,75.08,58.10,57.75,40.06,36.07,34.17,26.59,25.70,11.05.LC-MS(ESI):m / z:557.2(M+H) + .HRMS(ESI)(m / z):[M+H] + Calculate for C 28 H 30 F6N2O3,557.2239; found:557.2237.HPLC Purity:97.16%,retentiontime:2.56min.

[0327] Structure of (E)-1-(4-(1-(([1,1'-biphenyl]-4-ylmethoxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 20)

[0328]

[0329] White solid, yield 40.6%, melting point: 120.3-121.9℃. 1H NMR (400MHz, CD3OD) δ7.64-7.53(m,4H),7.49-7.39(m,6H),7.36-7.23(m,2H),5.24(s,2H),3.76(s,2H),3.67-3 .58(m,2H),3.42(t,J=8.1Hz,2H),3.29-3.15(m,1H),2.72(q,J=7.5Hz,2H),2.23(s,3H),1.22(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ179.26,155.07,142.50,140.80,140.59,137.27,135.67,135.39,129.45,128.52,128.44,128.33,126.93, 126.58,126.55,126.52,126.50,125.94,123.39,75.38,58.84,57.94,36.49,25.11,14.23,11.55.LC-MS(ESI):m / z:443.2(M+H) + .HRMS(ESI)(m / z):[M+H] + Calculate for C 28 H 30 N2O3,443.2335; found:443.2337.HPLCPurity:98.03%,retention time:1.30min.

[0330] Structure of (E)-1-(4-(1-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 17)

[0331]

[0332] Light-colored solid, yield 30.2%, melting point: 131.7-133.2°C. 1HNMR (400MHz, CD3OD) δ7.64(s,1H),7.53(dt,J=9.8,5.1Hz,4H),7.38(t,J=11.5Hz,1H),5.22(s,2H),4.25(s,2H),4.02(d,J=8.3Hz,4H),3. 43-3.33(m,1H),2.75(dd,J=15.0,7.5Hz,2H),2.29-2.14(s,3H),2.03(m,2H),1.93-1.81(m,2H),1.78-1.57(m,4H),1.20(t,J=7.1Hz,3H). 13 C NMR(151MHz,CD3OD)δ177.79,154.79,145.36,143.28,137.08,136.13,131.80,130.92,129.27,127.96,127.80(q,J=28.7Hz),1 26.46,124.78(q,J=273.5Hz),124.76,123.84,74.79,57.14,40.91,35.57,25.62,25.20,14.36,11.42.HRMS(ESI)(m / z):[M+H] + Calculate for C 28 H 33 F3N2O3,503.2522; found:503.2523.HPLC Purity:96.46%,retention time:2.28min.

[0333] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-methylbenzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 24)

[0334]

[0335] Yellow solid, yield 33.3%, melting point: 135.8-136.3℃. 1H NMR (400MHz, CD3OD) δ7.53 (s, 1H), 7.47 (dd, J = 8.0, 1.5Hz, 1H), 7.30 (d, J = 8.1Hz, 1H),7.15(d,J=8.5Hz,3H),5.11(s,2H),4.00(s,2H),3.84(t,J=8.8Hz,2H),3.69 (t,J=8.4Hz,2H),3.30-3.22(m,1H),2.82-2.60(m,3H),2.29(s,3H),2.20(s,3H) ,1.83-1.80(m,2H),1.76-1.72(m,3H),1.48-1.29(m,5H),1.21(t,J=7.8Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.12,154.47,145.22,142.85,136.54,134.88,134.61,133.03,129.89,128.89,126.20,125.88,12 4.96,123.61,75.80,57.62,57.34,39.83,35.88,33.49,26.85,26.03,25.14,18.10,14.33,11.49.HRMS(ESI)(m / z):[M+H] + Calculate for C 29 H 38 N2O3, 463.2961; found: 463.2962. HPLC Purity: 98.19%, retention time: 2.39min.

[0336] (E)-3-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)amino)propanoic acid (Compound 25)

[0337]

[0338] 1H NMR (400MHz, CD3OD) δ7.65 (s, 1H), 7.58 (dd, J=11.7, 7.9Hz, 2H), 7.51 (t, J=9.5 Hz,2H),7.46–7.35(m,1H),5.21(s,2H),4.19(s,2H),3.18(t,J=6.3Hz,2H),3.0 7(t,J=6.4Hz,1H),2.91(t,J=11.3Hz,1H),2.78(q,J=7.5Hz,2H),2.51(t,J=6. 3Hz,2H),2.24(s,3H),1.83-1.80(m,2H),1.77-1.70(m,3H),1.59–1.33(m,5H). 13 C NMR (151MHz, CD3OD) δ177.24,154.83,146.18,143.23,137.30,136.22,131.68,131.45,129.89,128.13,127.19,127.00,126.43,1 24.96,124.91,123.96,74.81,44.84,40.06,34.19,33.03,32.12,26.59,25.70,25.07,14.37,11.42.LC-MS(ESI):m / z:505.2(M+H) + .

[0339] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)pyrrolidine-3-carboxylic acid (Compound 26)

[0340]

[0341] 1H NMR (400MHz, CD3OD) δ7.65(s,1H),7.59(d,J=7.2Hz,2H),7.53(d,J=7.5Hz,2H),7.45(d,J=8.1 Hz,1H),5.22(s,2H),4.33(s,2H),3.40-3.42(m,1H),3.38–3.32(m,1H),3.25(t,J=6.9Hz,2H) ,3.11–3.00(m,1H),2.91(t,J=11.3Hz,1H),2.80(q,J=7.5Hz,2H),2.24(s,3H),1.85(d,J=11. 7Hz,2H),1.77(d,J=11.9Hz,3H),1.59–1.50(m,2H),1.46–1.31(m,3H),1.27(t,J=7.1Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.42,154.79,146.19,143.65,137.44,136.23,131.68,130.98,130.40,128.13,127.20,127.01,126.59,125.71,1 24.95,124.91,123.91,74.82,56.80,54.66,53.60,43.92,40.06,34.18,26.59,25.69,25.21,14.51,11.39.LC-MS(ESI):m / z:531.3(M+H) + .

[0342] Structure of (E)-1-(2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 22)

[0343]

[0344] White solid, yield 40.6%, melting point: 154.2-154.8℃. 1HNMR (400MHz, CD3OD) δ7.55(s,1H),7.49(d,J=8.0Hz,1H),7.38(t,J=7.3Hz,2H),7.34-7.23(m,6H),7.15(d,J=7.7Hz,1H),5.17(s,2H),4.00( s,2H),3.84(t,J=8.7Hz,2H),3.69(t,J=8.3Hz,2H),3.27-3.20(m,1H), 2.74(q,J=7.5Hz,2H),2.24(s,3H),2.23(s,3H),1.21(q,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.15,154.69,142.88,141.76,141.46,137.05,136.48,134.91,133.09,129.85,129.32,128.92,128. 79,127.78,126.51,126.21,125.39,123.63,75.61,57.61,57.33,35.89,25.15,19.26,14.34,11.52.HRMS(ESI)(m / z):[M+H] + Calculate for C 29 H 32 N2O3, 457.2492; found: 457.2493. HPLC Purity: 91.98%, retention time: 2.30min.

[0345] Structure of (E)-1-(2-ethyl-4-(1-((2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 21)

[0346]

[0347] White solid, yield 40.6%, melting point: 155.5-155.9℃. 1HNMR (400MHz, CD3OD) δ7.69 (dJ=12.3Hz, 1H), 7.56 (t, J=8.1Hz, 1H), 7.49 (s, 1H) ,7.42(d,J=8.0Hz,1H),7.26(dd,J=5.3,2.9Hz,4H),7.22(d,J=7.8Hz,1H),7.19- 7.13(m,2H),5.21(s,2H),4.11(s,2H),3.93(t,J=9.1Hz,2H),3.83(t,J=8.4Hz,2 H),3.29-3.22(m,1H),2.66(q,J=7.5Hz,2H),2.16(s,3H),1.12(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ177.22,155.11,143.30,140.77,139.59,138.22,136.91,132.05,131.31,130.97,129.29,128.64,127.87(q,J=29.8Hz ),127.46,127.37,126.45,125.32,124.23(q,J=273.8Hz),123.86,74.64,57.34,55.98,35.39,25.18,14.40,11.50.HRMS(ESI)(m / z):[M+H] + Calculate for C 29 H 29 F3N2O3,511.2209; found:511.2211.HPLC Purity:97.71%,retention time:1.56min.

[0348] Structure of (E)-1-(2-ethyl-4-(1-((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 13)

[0349]

[0350] Pale yellow oil, yield 60.0%. 1H NMR (400MHz, CD3OD) δ7.71(s,1H),7.67(d,J=7.4Hz,1H),7.62-7.51(m,3H),7.47(dd,J=8.0,1.6Hz,1H),7.31(d,J=8.0Hz,1H),5.28(s,2H),3. 98(s,2H),3.83(t,J=8.8Hz,2H),3.67(t,J=8.4Hz,2H),3.28(dd,J=15.2,6.9Hz,1H),2.73(q,J=7.5Hz,2H),2.26(s,3H),1.21(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.20,155.32,142.88,139.73,136.16,133.52,131.41,130.23(q,J=32.0Hz),128.87,128.82,12 6.17,124.32,124.31(q,J=272.0Hz),124.06,123.61,74.70,57.68,35.94,25.11,14.28,11.47.HRMS(ESI)(m / z):[M+H] + Calculate for C 23 H 25 F3N2O3, 435.1896; found: 435.1897. HPLC Purity: 99.79%, retention time: 1.17min.

[0351] Structure of (E)-1-(2-ethyl-4-(1-((4-methyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 14)

[0352]

[0353] Pale yellow oil, yield 21.3%. 1 H NMR (400MHz, CD3OD) δ7.67(s,1H),7.57(s,1H),7.52(t,J=7.9Hz,2H),7.35(dd,J=12.5,8.1Hz,2H),5.22(s,2H),4.22(s,2 H),3.97(dd,J=22.5,8.5Hz,4H),3.40-3.33(m,1H),2.76(q,J=7.5Hz,2H),2.47(s,3H),2.25(s,3H),1.22(t,J=7.5Hz,3H). 13C NMR (151MHz, CD3OD) δ177.72,155.00,143.07,136.62,136.50,135.81,132.36,131.84,131.45,129.07,128.29(q,J=30.2Hz),1 26.30,125.10,124.70(q,J=273.3Hz),123.70,74.70,57.55,56.78,35.67,25.12,17.72,14.32,11.40.HRMS(ESI)(m / z):[M+H] + Calculate for C 24 H 27 F3N2O3, 449.2052; found: 449.2050. HPLC Purity: 99.32%, retention time: 1.35min.

[0354] Structure of (E)-1-(2-ethyl-4-(1-((naphthalen-2-ylmethoxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 15)

[0355]

[0356] White solid, yield 25.9%, melting point: 131.5-131.7℃. 1 H NMR (400MHz, CD3OD) δ7.89-7.79(m,4H),7.61-7.51(m,2H),7.52-7.41(m,3H),7.35-7.18(m,1H),5.40(s,2H),3.95(s,2H) ,3.80(t,J=8.6Hz,2H),3.61(dt,J=10.7,5.9Hz,2H),3.29-3.22(m,1H),2.77-2.59(m,2H),2.27(s,3H),1.25-1.14(m,3H). 13 C NMR(151MHz,CD3OD)δ177.73,154.96,142.81,136.31,135.65,133.40,133.14,128.83,127.62,127.55,127.29,1 26.58,126.18,125.78,125.74,125.59,123.60,75.84,57.73,36.00,25.11,14.28,11.53.HRMS(ESI)(m / z):[M+H] + Calculate for C 26 H 28N2O3, 417.2179; found: 417.2179. HPLC Purity: 98.77%, retention time: 1.24min.

[0357] Structure of (E)-1-(2-ethyl-4-(1-(((5,6,7,8-tetrahydronaphthalen-2-yl)methoxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 23)

[0358]

[0359] White solid, yield 10.2%, melting point: 135.3-138.9℃. 1 H NMR (400MHz, CD3OD) δ7.50 (s, 1H), 7.44 (dd, J=8.0, 1.5Hz, 1H), 7.28 (d, J=8 .0Hz,1H),7.13-7.05(m,2H),7.00(d,J=7.7Hz,1H),5.09(s,2H),3.83(s,2H ),3.70(t,J=8.3Hz,2H),3.51(t,J=8.2Hz,2H),3.24(dd,J=16.6,8.3Hz,1H ),2.78-2.71(m,6H),2.20(s,3H),1.80-1.70(m,4H),1.23(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.90,154.68,142.58,136.56,136.32,136.01,134.99,134.56,128.69,128.62,128.61,126 .00,125.26,123.45,75.81,58.35,57.83,36.29,29.01,28.78,25.11,23.06,14.25,11.51.HRMS(ESI)(m / z):[M+H] + Calculate for C 26 H 32 N2O3, 421.2492; found: 421.2492. HPLC Purity: 98.54%, retention time: 1.48min.

[0360] Structure of (E)-1-(4-(1-(((3-cyano-4-isopropoxybenzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 16)

[0361]

[0362] Pale yellow solid, yield 52.3%, melting point: 112.6-112.9℃. 1 H NMR (400MHz, CD3OD) δ7.64 (d, J=6.7Hz, 2H), 7.56 (s, 1H), 7.50 (dd, J=8.0, 1.6Hz, 1H),7.33(d,J=8.1Hz,1H),7.16(d,J=9.4Hz,1H),5.14(s,2H),4.78-4.73(m,1H) ,4.18(s,2H),3.99(t,J=9.3Hz,2H),3.90(t,J=8.6Hz,2H),3.40-3.33(m,1H),2. 76(q,J=7.5Hz,2H),2.23(s,3H),1.38(s,3H),1.37(s,3H),1.23(t,J=7.5Hz,3H). 13 C NMR(151MHz,CD3OD)δ177.23,159.53,154.92,143.23,136.96,134.67,133.44,131.00,129.23,126.42,123.80, 116.06,113.66,101.88,74.22,71.68,57.49,56.20,35.40,25.12,20.71,14.38,11.39.HRMS(ESI)(m / z):[M+H] + calcdfor C 26 H 31 N3O4, 450.2396; found: 450.2395. HPLC Purity: 96.46%, retention time: 1.02min.

[0363] Structure of (E)-1-(2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 28)

[0364]

[0365] Gray-brown solid, yield 30.1%, melting point: 144.3-146.4°C. 1H NMR(600MHz,CD3OD)δ7.62(s,1H),7.55(dd,J=8.0,1.6Hz,1H),7.47(dd,J=14.4,6.3Hz,1H),7 .41-7.35(m,2H),7.33-7.28(m,2H),7.26(dd,J=5.0,3.2Hz,3H),7.14(d,J=7.7Hz,1H),5.20(s ,2H),4.29(s,2H),3.47-3.35(m,1H),3.34-3.30(m,2H),3.24(t,J=7.1Hz,2H),3.03-3.09(m, 1H),2.80(q,J=7.5Hz,2H),2.31-2.27(m,1H),2.25(s,3H),2.22(s,3H),1.20(t,J=7.1Hz,3H). 13 CNMR(151MHz,CD3OD)δ178.58,154.38,143.61,141.73,141.47,137.53,137.03,134.92,130.99,130.41,129.86,129.34,128.79,12 7.79,126.57,126.52,125.40,123.90,75.69,56.76,54.66,53.58,43.99,27.89,25.26,19.27,14.57,11.48.HRMS(ESI)(m / z):[M+H] + Calculate for C 30 H 34 N2O3, 471.2648; found: 471.2647. HPLC Purity: 96.16%, retention time: 1.66min.

[0366] Structure of (R,E)-1-(2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 29)

[0367]

[0368] White solid, yield 14.9%, melting point: 149.6-150.6℃. 1H NMR (400MHz, CD3OD) δ7.60(s1H),7.54(d,J=8.0Hz,1H),7.45(d,J=8.0Hz,1H),7 .38(t,J=7.3Hz,2H),7.34-7.18(m,5H),7.14(d,J=7.7Hz,1H),5.20(s,2H),4.2 6(s,2H),3.42-3.29(m,3H),3.20(t,J=7.1Hz,2H),3.09-2.97(m,1H),2.79(q,J =7.5Hz,2H),2.30-2.27(m,1H),2.24(s,3H),2.22(s,3H),1.22(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.63,154.39,143.55,141.74,141.47,137.42,137.04,134.92,131.32,130.35,129.85,129.33,128.79,127 .78,126.53,126.51,125.39,123.85,75.68,56.79,54.73,53.59,44.01,27.89,25.24,19.26,14.55,11.47.HRMS(ESI)(m / z):[M+H] + Calculate for C 30 H 34 N2O3,471.2648; found:471.2650.HPLCPurity:97.51%,retention time:1.45min.

[0369] Structure of (S,E)-1-(2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 30)

[0370]

[0371] White solid, yield 31.8%, melting point: 145.6-148.9℃. 1H NMR (400MHz, CD3OD) δ7.57 (s, 1H), 7.51 (d, J = 8.0Hz, 1H), 7.44-7.35 (m, 3H), 7. 33-7.26(m,5H),7.17(d,J=7.7Hz,1H),5.20(s,2H),4.02(s,2H),3.19(t,J=8.6 Hz,1H),3.11-3.00(m,3H),2.90(dd,J=17.2,7.9Hz,1H),2.79(q,J=7.5Hz,2H) ,2.26(s,3H),2.24(s,3H),2.15(dd,J=14.6,7.3Hz,2H),1.23(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ179.95,154.70,143.25,141.79,141.48,137.08,136.62,134.91,130.01,129.84,129.31,128.79,127.77 ,126.49,126.28,125.37,123.57,75.60,57.40,55.54,53.76,44.56,28.06,25.20,19.22,14.48,11.50.HRMS(ESI)(m / z):[M+H] + Calculate for C 30 H 34 N2O3,471.2648; found:471.2650.HPLCPurity:98.16%,retention time:1.52min.

[0372] Structure of (E)-3-((2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)amine)propanoic acid (Compound 31)

[0373]

[0374] White solid, yield 22.4%, melting point: 149.6-149.8℃. 1HNMR (400MHz, CD3OD) δ7.66 (s, 1H), 7.60 (d, J = 8.1Hz, 1H), 7.43 (dd, J = 17.6, 7.9Hz, 3H), 7.36-7.28 (m, 5H), 7.19 (d, J = 7.7Hz, 1H), 5.24 (s,2H),4.28(s,2H),3.25(t,J=6.2Hz,2H),2.83(q,J=7.5Hz,2H),2.60-2.47(m,2H),2.28(s,3H),2.25(s,3H),1.29(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ176.80,154.30,143.38,141.76,141.51,137.84,137.02,134.93,130.29,130.06,129.85,129.31,128. 78,127.78,126.55,126.52,125.39,124.05,75.71,44.64,31.36,25.08,21.94,19.22,14.38,11.40.HRMS(ESI)(m / z):[M+H] + Calculate for C 28 H 32 N2O3, 445.2491; found: 445.2490. HPLC Purity: 99.29%, retention time: 1.24min.

[0375] Structure of (E)-4-((2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)aminobutyric acid (Compound 32)

[0376]

[0377] White solid, yield 13.2%, melting point: 133.3-136.3℃. 1H NMR (400MHz, CD3OD) δ7.63(s,1H),7.58(d,J=8.1Hz,1H),7.49-7.36(m,3H),7.36-7.28(m,5H),7.17(d,J=7.7Hz,1H),5.21(s,2H),4.21(s ,2H),3.15(t,J=6.2Hz,2H),2.79(q,J=7.5Hz,2H),2.45-2.37(m,2H),2.26(s,3H),2.24(s,3H),1.90-1.85(m,2H),1.26(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ176.86,154.32,143.24,141.77,141.51,137.67,137.04,134.93,130.46,129.84,129.80,129.31,128. 78,127.78,126.52,126.48,125.38,124.04,75.70,35.68,25.21,21.93,21.44,19.22,14.36,11.40.HRMS(ESI)(m / z):[M+H] + Calculate for C 29 H 34 N2O3, 459.2648; found: 459.2647. HPLC Purity: 98.69%, retention time: 1.34min.

[0378] Structure of (E)-1-(2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)piperidine-3-carboxylic acid (Compound 33)

[0379]

[0380] Pale yellow oil, yield 15.3%. 1 H NMR (400MHz, CD3OD) δ7.67.61(s,1H),7.56(d,J=8.0Hz,1H),7.50-7.37(m,3H),7.36-7.28(m,5H),7.22-7.09(m,1H),5.23(s,2H), 4.12(s,2H),3.22-2.88(m,4H),2.83(q,J=7.5Hz,2H),2.61(s,1H),2.27(s,3H),2.25(s,3H),1.79-1.70(m,4H),1.31-1.19(m,3H).13 C NMR (151MHz, CD3OD) δ178.19,154.48,143.99,141.75,141.48,137.42,137.05,134.92,130.86,129.85,129.32,128.81,128.79, 128.77,127.79,126.51,125.39,123.77,75.67,57.43,55.07,52.96,25.22,22.28,19.25,14.48,11.48.HRMS(ESI)(m / z):[M+H] + Calculate for C 31 H 36 N2O3,485.2804; found:485.2802.HPLCPurity:97.56%,retention time:1.42min.

[0381] Structure of (S,E)-1-(2-ethyl-4-(1-(((4'-fluoro-2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 34)

[0382]

[0383] Pale yellow solid, yield 11.3%. 1 H NMR (600MHz, CD3OD) δ7.64 (d, J=1.9Hz, 1H), 7.58 (dd, J=8.1, 1.9Hz, 1H), 7.47 (d, J=8.1Hz, 1H),7.33-7.25(m,4H),7.19-7.10(m,3H),5.22(s,2H),4.42-4.34(m,2H),3.53-3.47(m,1 H),3.38-3.32(m,2H),3.29-3.24(m,1H),3.12-3.04(m,1H),2.82(q,J=7.5Hz,2H),2.32(d t,J=14.2,7.9Hz,1H),2.27(s,3H),2.24(s,3H),2.23-2.18(m,1H),1.26(t,J=7.5Hz,3H). 13C NMR (151MHz, CD3OD) δ177.91,162.81,161.19,154.29,143.70,140.39,137.84,137.27,135.04,130.62,130.56,130.44,12 9.88,129.37,114.54,114.40,75.64,56.64,54.50,53.58,43.83,27.74,25.22,19.19,14.54,11.38.MS(ESI)(m / z):[M+H] + 489.25.

[0384] Structure of (S,E)-1-(2-ethyl-4-(1-(((2-fluoro-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 35)

[0385]

[0386] Pale yellow solid, yield 18.7%. 1 H NMR(600MHz,CD3OD)δ7.63(s,1H),7.57(d,J=6.1Hz,1H),7.52(d,J=8.0Hz,2H),7.48–7.40(m,4H),7 .35(t,J=7.4Hz,1H),7.28(d,J=9.4Hz,1H),7.22(d,J=11.5Hz,1H),5.26(s,2H),4.33(d,J=4.1Hz,2H ),3.46(dd,J=11.1,5.8Hz,1H),3.34(d,J=11.2Hz,2H),3.25(t,J=9.0Hz,1H),3.07(ddd,J=14.5,8.6 ,5.8Hz,1H),2.81(q,J=7.5Hz,2H),2.29(s,4H),2.22(dq,J=13.0,6.4Hz,1H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.47,159.53,154.78,143.69,135.54,130.42,130.38,128.61,128.58,128.09,127.35,126.66,12 3.95,123.69,123.66,115.10,114.94,74.70,56.65,54.60,53.63,43.70,27.76,25.22,14.52,11.42.MS(ESI)(m / z):[M+H] +475.30.

[0387] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(furan-3-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 36)

[0388]

[0389] Pale yellow solid, yield 21.2%. 1 H NMR (600MHz, CD3OD) δ7.61(dd,J=13.5,1.8Hz,2H),7.56–7.53(m,2H),7.47(d,J=8.1Hz,1H),7.30(d,J=7.8Hz,1H),7.2 7(s,1H),7.23(dd,J=7.8,1.8Hz,1H),6.62(d,J=1.9Hz,1H),5.18(s,2H),4.44–4.28(m,2H),3.52(dd,J=11.3,5.7Hz,1 H),3.40–3.36(m,1H),3.35–3.32(m,1H),3.27(dt,J=11.1,7.4Hz,1H),3.08(ddd,J=14.4,8.8,5.7Hz,1H),2.80(q,J=7 .5Hz,2H),2.36(s,3H),2.30(dt,J=15.6,7.0Hz,1H),2.24(s,3H),2.22(dd,J=13.5,6.2Hz,1H),1.23(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ177.76,154.25,143.72,142.57,139.97,137.86,136.85,135.34,131.69,130.51,130.15, 128.74,126.66,125.53,123.98,110.90,75.65,56.45,54.32,53.53,43.55,27.70,25.24,20.03,14.56,11.41.

[0390] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyridin-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 37)

[0391]

[0392] Pale yellow solid, yield 19.2%. 1H NMR (600MHz, CD3OD) δ8.56(d,J=6.2Hz,2H),7.63(s,1H),7.56(d,J=8.1Hz,1H),7.47(d,J=8.1Hz,1H),7.40(d,J =6.2Hz,2H),7.37(s,1H),7.34(d,J=7.8Hz,1H),7.22(d,J=7.8Hz,1H),5.24(s,2H),4.42–4.33(m,2H),3.50(dd, J=11.2,5.7Hz,1H),3.38(d,J=7.8Hz,1H),3.34(s,1H),3.30–3.24(m,1H),3.11–3.04(m,1H),2.81(q,J=7.5Hz, 2H), 2.32(dd,J=15.2,7.1Hz,1H),2.28(s,3H),2.27(s,3H),2.22(dt,J=13.6,6.7Hz,1H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ177.89,154.44,150.81,148.56,143.72,138.78,138.11,137.73,134.81,130.48,130.13, 128.98,126.66,125.69,124.62,123.97,75.40,56.56,54.41,53.57,43.62,27.73,25.20,19.02,14.56,11.38.

[0393] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 38)

[0394]

[0395] Pale yellow solid, yield 19.7%. 1H NMR (600MHz, CD3OD) δ9.14(s,1H),8.78(s,2H),7.62(d,J=1.9Hz,1H),7.56(dd,J=8.1,1.9Hz,1H),7.47(d,J=8. 1Hz,1H),7.40(d,J=1.7Hz,1H),7.37(dd,J=7.8,1.8Hz,1H),7.26(d,J=7.8Hz,1H),5.24(s,2H),4.42–4.32(m,2H ),3.49(dd,J=11.2,5.8Hz,1H),3.38–3.32(m,2H),3.27(dt,J=10.8,7.3Hz,1H),3.08(ddd,J=14.4,8.7,5.8Hz,1 H),2.81(q,J=7.5Hz,2H),2.35–2.31(m,1H),2.29(s,3H),2.27(s,3H),2.25–2.19(m,1H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.06,156.57,156.36,154.49,143.72,139.33,137.69,135.64,135.61,133.41,130.46, 130.18,129.63,126.65,125.90,123.96,75.31,56.55,54.46,53.59,43.61,27.73,25.24,18.95,14.55,11.39.

[0396] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(-1-(tert-butyl)-1H-pyrazol-4-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 39)

[0397]

[0398] Pale yellow solid, yield 31.1%. 1H NMR(600MHz,CD3OD)δ7.85(d,J=0.8Hz,1H),7.65–7.61(m,2H),7.56(dd,J=8.1,1.9Hz,1H),7.45(d,J=8.1Hz,1H), 7.33(d,J=7.8Hz,1H),7.29–7.26(m,1H),7.23(dd,J=7.8,1.8Hz,1H),5.18(s,2H),4.39–4.31(m,2H),3.47(dd,J= 11.1,5.8Hz,1H),3.33(dd,J=10.8,6.1Hz,2H),3.25(d,J=10.3Hz,1H),3.07(ddd,J=14.7,8.8,5.9Hz,1H),2.80(q ,J=7.5Hz,2H),2.39(s,3H),2.33–2.26(m,1H),2.25(s,3H),2.24–2.19(m,1H),1.62(s,9H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.15,154.25,143.67,137.80,137.77,136.36,134.87,131.84,130.41,130.20,128.69,126.65,125.63,1 25.41,123.95,121.12,75.70,58.41,56.64,54.55,53.60,43.65,28.66,27.75,25.19,20.06,14.54,11.39.MS(ESI)(m / z):[M+H] + 517.30.

[0399] Structure of (S,E)-1-(2-ethyl-4-(1-((3-methyl-4-(1-methyl-1H-pyrazol-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 40)

[0400]

[0401] Pale yellow solid, yield 29.5%. 1H NMR (600MHz, CD3OD) δ7.74(s,1H),7.62(d,J=1.9Hz,1H),7.61–7.60(m,1H),7.56(dd,J=8.1,1.9Hz,1H),7.45(d,J=8.1H z,1H),7.32(d,J=7.8Hz,1H),7.28(d,J=1.8Hz,1H),7.22(dd,J=7.9,1.8Hz,1H),5.18(s,2H),4.41–4.32(m,2H),3.93(s ,3H),3.49(dd,J=11.2,5.8Hz,1H),3.35(dd,J=11.2,7.0Hz,2H),3.26(dt,J=10.8,7.3Hz,1H),3.12–3.02(m,1H),2.80( q,J=7.5Hz,2H),2.38(s,3H),2.30(ddd,J=15.7,8.4,6.2Hz,1H),2.25(s,3H),2.24–2.18(m,1H),1.24(t,J=7.5Hz,3H). 13 CNMR(151MHz,CD3OD)δ177.95,154.26,143.69,138.10,137.85,136.50,134.86,131.45,130.44,130.23,129.63,128.60,126. 66,125.64,123.96,121.92,75.66,56.60,54.49,53.58,43.61,37.44,27.73,25.22,20.08,14.53,11.38.MS(ESI)(m / z):[M+H] + 475.30.

[0402] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(thiophen-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 41)

[0403]

[0404] Pale yellow solid, yield 9.5%. 1H NMR (600MHz, CD3OD) δ7.63(d,J=1.9Hz,1H),7.56(dd,J=8.1,1.9Hz,1H),7.48–7.46(m,1H),7.43(dd,J=4.9,3.0 Hz,1H),7.29–7.27(m,1H),7.27(dd,J=3.0,1.3Hz,1H),7.26–7.22(m,2H),7.13(dd,J=5.0,1.3Hz,1H),5.19(s,2 H),4.38(dd,J=6.1,3.1Hz,2H),3.53–3.49(m,1H),3.36(dd,J=10.8,4.3Hz,2H),3.28(dd,J=10.9,7.2Hz,1H),3. 09(td,J=8.6,4.2Hz,1H),2.81–2.78(m,2H),2.31(s,4H),2.25(s,3H),2.24–2.20(m,1H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.04,154.24,143.72,141.88,137.88,137.04,136.10,135.31,130.50,129.99,129.30,128 .45,126.68,125.40,124.71,123.99,122.26,75.67,56.42,54.43,53.60,43.54,27.71,25.26,19.58,14.56,11.41.

[0405] Structure of (S,E)-1-(2-ethyl-4-(1-(((2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 42)

[0406]

[0407] Pale yellow solid, yield 31.8%. 1H NMR (600MHz, CD3OD) δ7.81(s,1H),7.66(d,J=7.8Hz,1H),7.63(d,J=1.9Hz,1H),7.56(dd,J=8.0,1.9Hz,1H),7.48( d,J=8.1Hz,1H),7.38(dd,J=5.1,1.9Hz,3H),7.34(d,J=7.8Hz,1H),7.28(dd,J=6.7,2.9Hz,2H),5.32(s,2H),4.41 –4.32(m,2H),3.49(dd,J=11.2,5.8Hz,1H),3.34(t,J=8.1Hz,2H),3.29–3.23(m,1H),3.08(ddd,J=14.4,8.7,5.9H z,1H),2.81(q,J=7.5Hz,2H),2.34–2.29(m,1H),2.29(s,3H),2.23(tt,J=13.5,6.2Hz,1H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ179.63,154.93,143.74,140.81,139.60,138.23,137.55,132.04,130.96,130.48,128.64,128.62,127.45 ,127.36,126.66,125.35,125.31,123.97,74.69,56.58,54.49,53.60,43.68,27.75,25.23,14.52,11.41.MS(ESI)(m / z):[M+H] + 525.25.

[0408] Structure of (S,E)-1-(2-ethyl-4-(1-((3-methyl-4-(1-methyl-1H-pyrrol-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 43)

[0409]

[0410] Pale yellow solid, yield 21.5%. 1H NMR (600MHz, CD3OD) δ7.61(s,1H),7.54(d,J=7.8Hz,1H),7.44(d,J=8.1Hz,1H),7.29(d,J=8.0Hz,1H),7. 20(s,1H),7.15(d,J=7.8Hz,1H),6.74(s,1H),6.64(s,1H),6.21(s,1H),5.15(s,2H),4.35(t,J=10.7Hz, 2H),3.66(s,3H),3.48(dd,J=11.3,5.7Hz,1H),3.34(s,2H),3.24(d,J=9.9Hz,1H),3.07(d,J=8.1Hz,1H) ,2.79(q,J=7.5Hz,2H),2.39(s,3H),2.30–2.24(m,1H),2.23(s,3H),2.21(s,1H),1.23(t,J=7.6Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.09,154.09,143.65,137.89,135.78,134.80,134.36,130.45,130.22,130.11,128.42,126.64,125.44,123.9 4,123.77,121.28,120.43,108.50,75.92,56.53,54.45,53.57,43.63,34.81,27.73,25.22,20.45,14.53,11.40.MS(ESI)(m / z):[M+H] + 474.25.

[0411] Structure of (S,E)-1-(2-ethyl-4-(1-((3-methyl-4-(1-methyl-1H-imidazol-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 44)

[0412]

[0413] Pale yellow solid, yield 27.1%. 1H NMR (600MHz, CD3OD) δ7.72(s,1H),7.62(s,1H),7.56(d,J=7.9Hz,1H),7.46(d,J=8.1Hz, 1H),7.38(s,1H),7.32(d,J=7.8Hz,1H),7.21(d,J=7.8Hz,1H),6.88(s,1H),5.24(s,2H), 4.34(d,J=4.2Hz,2H),3.45(s,4H),3.34(s,2H),3.26(s,1H),3.07(q,J=6.0Hz,1H),2.81 (q,J=7.6Hz,2H),2.27(s,3H),2.23(t,J=6.7Hz,2H),2.18(s,3H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.17,154.52,143.68,139.34,138.14,137.90,137.62,132.16,130.91,130.41,129.62,128.07,126.6 3,126.33,125.24,123.94,75.41,56.69,54.56,53.60,43.72,30.75,27.78,25.23,18.73,14.54,11.42.MS(ESI)(m / z):[M+H] + 475.25.

[0414] Structure of (S,E)-1-(2-ethyl-4-(1-(((3,5-dimethylisoxazol-4-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 45)

[0415]

[0416] Pale yellow solid, yield 20.7%. 1H NMR (600MHz, CD3OD) δ7.64(s,1H),7.57(d,J=8.1Hz,1H),7.48(d,J=8.1Hz,1H),7.39(s,1H),7.31(d,J=7. 8Hz,1H),7.12(dd,J=7.8,4.1Hz,1H),5.23(s,2H),4.40(d,J=13.6Hz,2H),3.50(dd,J=11.3,5.8Hz,1H),3 .36(q,J=10.0,9.1Hz,2H),3.30–3.25(m,1H),3.12–3.05(m,1H),2.82(q,J=7.5Hz,2H),2.31(dd,J=14.6, 6.8Hz,1H),2.27(s,3H),2.26–2.23(m,1H),2.21(s,3H),2.14(s,3H),2.05(s,3H),1.25(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.08,165.70,159.25,154.41,143.72,138.64,137.45,130.52,130.46,129.72,128.36,126.66,125 .58,123.98,115.90,75.51,56.54,54.47,53.59,43.60,27.73,25.24,18.47,14.55,11.43,9.83,8.99.MS(ESI)(m / z):[M+H] + 490.30.

[0417] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 46)

[0418]

[0419] Pale yellow solid, yield 50.1%. 1H NMR (600MHz, CD3OD) δ7.62(d,J=1.9Hz,1H),7.55(dd,J=8.1,1.9Hz,1H),7.47(d,J=8.1Hz,1H),7.2 5–7.19(m,2H),7.17(d,J=7.5Hz,1H),7.10(d,J=7.5Hz,1H),5.16(s,2H),4.42–4.32(m,2H),3.50( dd,J=11.3,5.9Hz,1H),3.38–3.33(m,2H),3.28(dd,J=10.9,7.5Hz,1H),3.09(ddd,J=14.6,8.5,5. 8Hz,1H),2.81(q,J=7.5Hz,2H),2.33(s,3H),2.32–2.28(m,1H),2.24(s,4H),1.24(t,J=7.5Hz,3H). 13 CNMR(151MHz,CD3OD)δ178.42,154.23,143.69,137.94,137.83,137.66,130.47,128.47,128.09,127.88,126.6 5,124.91,123.96,75.94,56.52,54.52,53.64,43.66,27.75,25.25,20.06,14.55,11.40.MS(ESI)(m / z):[M+H] + 395.25.

[0420] Structure of (S,E)-1-(2-ethyl-4-(1-(((2',4'-difluoro-2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 47)

[0421]

[0422] Pale yellow solid, yield 19.0%. 1H NMR (600MHz, CD3OD) δ7.63(s,1H),7.56(d,J=7.9Hz,1H),7.47(d,J=8.1Hz,1H),7.33(s,1H),7.28(d,J=8. 0Hz,1H),7.25(d,J=7.5Hz,1H),7.14(d,J=7.9Hz,1H),7.01(t,J=8.8Hz,2H),5.22(s,2H),4.41–4.28(m,2 H),3.48(dd,J=11.1,5.7Hz,1H),3.37–3.32(m,2H),3.25(q,J=8.0Hz,1H),3.07(t,J=7.8Hz,1H),2.80(q, J=7.5Hz,2H),2.34–2.28(m,1H),2.26(s,3H),2.22(d,J=14.8Hz,1H),2.15(s,3H),1.24(t,J=7.5Hz,3H). 13 C NMR(151MHz,CD3OD)δ177.31,162.53,160.90,159.67,158.03,153.59,142.87 ,137.32,136.91,135.57,133.46,131.48,131.44,129.64,129.00,128.62,12 5.84,124.47,123.14,110.15,109.98,102.59,102.42,102.24,74.75,55.85, 53.67,52.76,42.92,26.96,24.42,17.83,13.73,10.62.MS(ESI)(m / z):[M+H] + 507.25.

[0423] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyrrolidin-1-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 48)

[0424]

[0425] Pale yellow solid, yield 17.5%. 1H NMR (600MHz, CD3OD) δ7.61(d,J=1.9Hz,1H),7.54(dd,J=8.0,1.9Hz,1H),7.46(d,J=8.1Hz,1H),7.14(d,J=2.1Hz,1 H),7.11(dd,J=8.2,2.2Hz,1H),6.87(d,J=8.2Hz,1H),5.08(s,2H),4.36(d,J=8.0Hz,2H),3.50(dd,J=11.2,5.7Hz, 1H),3.38–3.32(m,2H),3.26(dt,J=10.9,7.3Hz,1H),3.15–3.10(m,4H),3.07(td,J=8.8,4.4Hz,1H),2.80(q,J=7. 5Hz,2H),2.33–2.30(m,1H),2.29(s,3H),2.26–2.22(m,1H),2.20(s,3H),1.94–1.88(m,4H),1.24(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.01,153.87,149.09,143.66,137.93,131.74,130.47,129.79,128.56,126.61,126.49,123. 93,115.53,75.99,56.52,54.41,53.55,50.82,43.67,27.75,25.24,24.30,19.25,14.57,11.40.MS(ESI)(m / z):[M+H] + 464.30.

[0426] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(piperidin-1-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 49)

[0427]

[0428] Pale yellow solid, yield 19.4%. 1H NMR(500MHz,CD3OD)δ7.62(d,J=1.9Hz,1H),7.55(dd,J=8.1,1.9Hz,1H),7.47(d,J=8.1Hz,1H),7.20(d,J=2.1Hz,1H), 7.17(dd,J=8.1,2.1Hz,1H),6.99(d,J=8.1Hz,1H),5.11(s,2H),4.39–4.29(m,2H),3.48(dd,J=11.1,5.8Hz,1H),3.37 (s,1H),3.35–3.31(m,1H),3.29–3.22(m,1H),3.12–3.04(m,1H),2.86–2.77(m,6H),2.32(dd,J=13.2,7.4Hz,1H),2.2 9(s,3H),2.25(d,J=12.9Hz,1H),2.23(s,3H),1.72(p,J=5.7Hz,4H),1.59(q,J=5.8,5.4Hz,2H),1.25(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.42,154.03,152.53,143.62,137.78,132.13,132.04,130.84,130.42,126.59,126.44,123.91, 118.45,75.89,56.59,54.54,53.60,53.20,43.76,27.78,26.29,25.24,24.08,16.69,14.55,11.42.MS(ESI)(m / z):[M+H] + 478.30.

[0429] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-morpholinylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 50)

[0430]

[0431] Pale yellow solid, yield 20.7%. 1H NMR (500MHz, CD3OD) δ7.64(d,J=1.8Hz,1H),7.57(dd,J=8.1,1.9Hz,1H),7.48(d,J=8.1Hz,1H),7.2 6–7.18(m,2H),7.04(dt,J=8.1,1.9Hz,1H),5.13(s,2H),4.43–4.33(m,2H),3.88–3.77(m,4H),3.55 –3.47(m,1H),3.37(dd,J=9.5,6.3Hz,2H),3.32–3.26(m,1H),3.12(tt,J=8.7,6.0Hz,1H),2.88(dq ,J=4.8,2.6Hz,4H),2.82(q,J=7.6Hz,2H),2.39–2.29(m,4H),2.28–2.20(m,4H),1.29–1.23(m,3H). 13 C NMR (126MHz, CD3OD) δ178.88,154.10,151.00,143.69,137.88,132.82,132.22,130.99,130.47,130.17,126.64,126.57,1 23.96,118.46,75.76,67.06,56.48,54.55,53.67,52.11,43.64,27.74,25.25,16.72,14.56,11.41.MS(ESI)(m / z):[M+H] + 480.30.

[0432] Structure of (S,E)-1-(2-ethyl-4-(1-(((2',3',4'-trifluoro-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 51)

[0433]

[0434] Pale yellow solid, yield 20.1%. 1H NMR(600MHz,CD3OD)δ7.63(s,1H),7.56(d,J=8.0Hz,1H),7.48(d,J=8.2Hz,1H),7.34 (s,1H),7.30(d,J=7.9Hz,1H),7.16(d,J=7.5Hz,2H),7.03(q,J=6.3Hz,1H),5.22(s,2 H),4.38(t,J=10.0Hz,2H),3.50(s,1H),3.36(d,J=8.6Hz,2H),3.28(d,J=10.5Hz,1H) ,3.09(s,1H),2.80(q,J=7.5Hz,2H),2.26(s,5H),2.16(s,3H),1.24(t,J=7.5Hz,3H). 13 C NMR(151MHz,CD3OD)δ154.43,143.70,138.70,137.73,136.31,133.12,133.11,130.48, 130.25,129.75,129.50,126.65,125.36,125.14,125.12,125.06,123.96,111.81,111. 76,111.76,75.44,56.57,56.51,56.48,54.74,54.43,53.59,53.01,43.63,43.61,43.5 6,43.45,43.30,43.11,27.74,25.24,18.57,18.55,14.53,11.42.MS(ESI)(m / z):[M+H] + 525.20.

[0435] Structure of (S,E)-1-(2-ethyl-4-(1-(((2',4',5'-trifluoro-2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 52)

[0436]

[0437] Pale yellow solid, yield 17.9%. 1H NMR (600MHz, CD3OD) δ7.62(s,1H),7.55(d,J=8.0Hz,1H),7.51(d,J=8.3Hz,1H),7.33(s, 1H),7.28(d,J=7.6Hz,1H),7.22(d,J=6.7Hz,1H),7.14(d,J=7.3Hz,2H),5.21(s,2H),4.3 8(t,J=8.9Hz,2H),3.49(s,1H),3.35(s,2H),3.29(s,1H),3.10(s,1H),2.81(d,J=7.5Hz, 2H), 2.30 (d, J = 7.4Hz, 1H), 2.25 (s, 3H), 2.23 (s, 1H), 2.16 (s, 3H), 1.23 (t, J = 7.5Hz, 3H). 13 C NMR(151MHz,CD3OD)δ155.58,154.43,153.96,150.21,148.51,147.31,145.70,14 3.71,138.63,137.70,136.34,133.18,130.53,130.33,129.70,129.49,126.64,1 25.34,123.95,118.93,118.76,105.40,105.26,105.20,105.06,75.46,56.40,54 .49,53.66,43.71,27.79,25.30,18.59,18.57,14.56,11.43.MS(ESI)(m / z):[M+H] + 525.25.

[0438] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(6-fluoropyridin-3-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 53)

[0439]

[0440] Pale yellow solid, yield 25.7%. 1H NMR (500MHz, CD3OD) δ8.12–8.08(m,1H),7.88(ddd,J=8.5,7.8,2.6Hz,1H),7.62(d,J=1.9Hz,1H),7.55(dd,J=8.0,1.9Hz,1H),7.4 7(d,J=8.1Hz,1H),7.35(d,J=1.7Hz,1H),7.31(dd,J=7.8,1.8Hz,1H),7.19(d,J=7.8Hz,1H),7.11(ddd,J=8.5,2.6,0.7Hz,1H),5.2 2(s,2H),4.39–4.31(m,2H),3.48(dd,J=11.2,5.7Hz,1H),3.36(s,1H),3.33(d,J=2.6Hz,1H),3.29–3.21(m,1H),3.08(tt,J=8.5,5 .8Hz,1H),2.80(q,J=7.5Hz,2H),2.34–2.27(m,1H),2.25(s,3H),2.24(s,3H),2.21(dd,J=13.5,6.4Hz,1H),1.23(t,J=7.6Hz,3H). 13 C NMR (126MHz, CD3OD) δ179.54,165.00,163.11,155.80,148.23,148.12,145. 08,143.92,143.86,139.83,139.04,137.56,136.94,136.90,136.84,131.8 5,131.43,130.95,128.01,127.07,125.32,110.23,109.93,76.82,57.91,5 5.84,54.97,45.08,29.14,26.63,20.46,15.94,12.80.MS(ESI)(m / z):[M+H] + 490.25.

[0441] Structure of (S,E)-1-(2-ethyl-4-(1-(((2,4'-difluoro-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 54)

[0442]

[0443] Pale yellow solid, yield 28.1%. 1H NMR (600MHz, CD3OD) δ7.62(s,1H),7.56(s,2H),7.52(dd,J=7.3,5.3Hz,2H),7.42(t,J=8.0Hz,1H ),7.27(d,J=7.9Hz,1H),7.21(d,J=11.6Hz,1H),7.15(t,J=8.8Hz,2H),5.24(s,2H),4.46–4.38(m ,2H),3.54(dd,J=11.3,6.2Hz,1H),3.44–3.37(m,2H),3.36(s,1H),3.16–3.10(m,1H),2.82(q,J =7.5Hz,2H),2.34(dd,J=14.3,7.3Hz,1H),2.28(s,3H),2.27–2.23(m,1H),1.23(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ179.36,163.26,161.63,160.25,158.62,154.74,143.80,140. 37,137.67,131.67,130.67,130.55,130.53,130.49,130.47,130.32,130.29,130.06 127.05,126.67,123.98,123.75,123.72,115.11,114.95,114.81,74.67,56 .19,54.41,53.74,43.59,27.72,25.37,14.56,11.43.MS(ESI)(m / z):[M+H] + 493.25.

[0444] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(1-methyl-1H-pyrazol-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 55)

[0445]

[0446] Pale yellow solid, yield 34.3%. 1H NMR (500MHz, CD3OD) δ7.94–7.88(m,1H),7.79(dd,J=2.7,1.2Hz,1H),7.62(q,J=1.8Hz,1H),7.56(dt,J=8.1,2.2Hz,1H), 7.52(dt,J=8.3,2.0Hz,2H),7.45(d,J=8.1Hz,1H),7.39(dd,J=8.2,1.9Hz,2H),5.20(s,2H),4.43–4.33(m,2H),3.92–3.8 7(m,3H),3.52(dd,J=11.3,5.7Hz,1H),3.37(dt,J=10.6,7.1Hz,2H),3.28(d,J=9.4Hz,1H),3.14–3.05(m,1H),2.79(dtt, J=7.6,3.9,2.0Hz,2H),2.31(dt,J=15.7,7.4Hz,1H),2.26–2.24(m,3H),2.22(dd,J=13.7,6.9Hz,1H),1.25–1.22(m,3H). 13 CNMR(126MHz,CD3OD)δ177.66,154.30,143.73,137.91,136.13,135.97,132.02,130.48,129.94,128.49,127.75,126.6 9,124.90,123.99,122.83,75.65,56.45,54.38,53.56,43.44,37.53,27.67,25.22,14.54,11.39.MS(ESI)(m / z):[M+H] + 461.30.

[0447] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(thiophen-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 56)

[0448]

[0449] Pale yellow solid, yield 19.7%. 1H NMR (500MHz, CD3OD) δ7.64 (dd, J=8.5, 2.0Hz, 3H), 7.61 (dd, J=2.8, 1.5Hz, 1H), 7.58 (dd, J=8.1, 1.9 Hz,1H),7.48–7.40(m,5H),5.23(s,2H),4.40(d,J=2.5Hz,2H),3.53(dd,J=11.3,5.8Hz,1H),3.37(d d,J=12.5,5.6Hz,2H),3.27(d,J=10.5Hz,1H),3.10(ddd,J=11.8,8.6,5.7Hz,1H),2.81(q,J=7.5Hz, 2H), 2.32(dq,J=15.8,7.9Hz,1H),2.27(s,3H),2.22(dd,J=13.4,6.7Hz,1H),1.25(t,J=7.5Hz,3H). 13 CNMR(126MHz,CD3OD)δ154.35,143.75,141.83,137.95,136.87,135.39,130.47,128.38,126.72,125.90,125.8 5,125.71,124.02,119.91,75.59,56.53,54.43,53.55,43.44,27.68,25.21,14.53,11.39.MS(ESI)(m / z):[M+H] + 463.20

[0450] Structure of (S,E)-1-(2-ethyl-4-(1-((2-fluoro-4-(thien-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 57)

[0451]

[0452] Pale yellow solid, yield 27.7%. 1H NMR(500MHz,CD3OD)δ7.67(dd,J=2.9,1.4Hz,1H),7.63(d,J=2.0Hz,1H),7.56(dd,J=8.1,1.9Hz,1H) ,7.51–7.45(m,4H),7.44(dd,J=5.1,1.4Hz,1H),7.40(dd,J=11.4,1.6Hz,1H),5.28(d,J=1.1Hz,2H) ,4.43–4.34(m,2H),3.53(dd,J=11.3,5.6Hz,1H),3.43–3.34(m,2H),3.28(dt,J=11.0,7.4Hz,1H),3 .15–3.05(m,1H),2.80(q,J=7.5Hz,2H),2.37–2.29(m,1H),2.28–2.19(m,4H),1.24(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ177.72,162.31,160.35,154.57,143.71,140.56,137. 91,137.84,137.72,131.06,130.50,130.02,126.66,126.26,125.60,123.9 7,123.49,123.37,121.53,121.51,121.01,112.54,112.35,69.22,69.19,5 6.44,54.29,53.52,43.54,27.69,25.22,14.51,11.30.MS(ESI)(m / z):[M+H] + 481.20.

[0453] Structure of (S,E)-1-(2-ethyl-4-(1-((2-fluoro-4-(1-methyl-1H-pyrazol-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 58)

[0454]

[0455] Pale yellow solid, yield 31.6%. 1H NMR (500MHz, CD3OD) δ8.00–7.93(m,1H),7.82(d,J=0.8Hz,1H),7.63(d,J=2.0Hz,1H),7.56(dd,J=8.1,1. 9Hz,1H),7.50–7.41(m,2H),7.34(dd,J=7.8,1.7Hz,1H),7.30(dd,J=11.3,1.7Hz,1H),5.26(s,2H),4.44– 4.34(m,2H),3.91(s,3H),3.53(dd,J=11.2,5.7Hz,1H),3.43–3.34(m,2H),3.28(dt,J=11.0,7.4Hz,1H), 3.10(tt,J=8.7,5.7Hz,1H),2.81(q,J=7.6Hz,2H),2.37–2.29(m,1H),2.25(s,4H),1.25(t,J=7.6Hz,3H). 13 C NMR (126MHz, CD3OD) δ177.73,162.41,160.45,154.51,143.71,137.73,136.16, 134.81,134.74,131.22,131.18,130.49,130.06,128.17,126.65,123.95,122. 64,122.52,121.79,121.77,120.55,120.52,111.53,111.35,69.24,69.22,56. 48,54.31,53.53,43.56,37.61,27.69,25.21,14.50,11.28MS(ESI)(m / z):[M+H] + 479.30.

[0456] Structure of (S,E)-1-(2-ethyl-4-(1-(((2'-fluoro-2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 59)

[0457]

[0458] Pale yellow solid, yield 33.5%. 1H NMR(500MHz,CD3OD)δ7.63(s,1H),7.56(d,J=8.0Hz,1H),7.49(d,J=8.1Hz,1H),7.38(td,J=8.9,8.2,3.6Hz,1H), 7.32(s,1H),7.28(d,J=7.7Hz,1H),7.22(d,J=4.6Hz,2H),7.15(t,J=8.0Hz,2H),5.22(s,2H),4.38–4.31(m,2H),3 .48(dd,J=11.3,6.1Hz,1H),3.35(s,2H),3.29–3.24(m,1H),3.09(ddd,J=14.7,8.6,6.1Hz,1H),2.81(q,J=7.5Hz ,2H),2.31(dd,J=14.5,7.0Hz,1H),2.27(s,3H),2.23(dd,J=13.4,6.8Hz,1H),2.16(s,3H),1.24(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ179.07,160.43,158.81,154.38,143.68,137.85,137.75 ,136.26,135.28,131.31,131.28,130.47,129.67,129.35,129.09,129.04,12 8.96,126.66,125.19,123.95,123.92,123.89,115.08,114.93,75.63,56.46, 54.60,53.70,43.65,27.75,25.28,18.68,14.54,11.44.MS(ESI)(m / z):[M+H] + 489.25.

[0459] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-cyclohexyl-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 60)

[0460]

[0461] Pale yellow solid, yield 34.5%. 1H NMR(500MHz,CD3OD)δ7.62(d,J=2.0Hz,1H),7.55(dd,J=8.1,1.9Hz,1H),7.48(d,J=8.1Hz,1H),7.20–7.13(m,3H) ,5.13(s,2H),4.42–4.30(m,2H),3.50(dd,J=11.2,5.8Hz,1H),3.36(dd,J=11.7,7.9Hz,2H),3.27(dt,J=10.9,7.3 Hz,1H),3.09(ddd,J=14.6,8.9,5.8Hz,1H),2.81(q,J=7.5Hz,2H),2.74(ddd,J=11.5,9.7,3.2Hz,1H),2.32(s,4H ),2.23(s,4H),1.91–1.82(m,2H),1.81–1.73(m,3H),1.52–1.39(m,4H),1.33–1.28(m,1H),1.25(t,J=7.6Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.14,154.05,145.25,143.65,137.82,134.85,134.63,130.45,130.24,129.88,126.62,125.87,124.97, 123.93,75.90,56.52,54.43,53.57,43.69,39.82,33.49,27.76,26.83,26.02,25.24,18.11,14.56,11.42.MS(ESI)(m / z):[M+H] + 477.30.

[0462] Structure of (S,E)-1-(2-ethyl-4-(1-((2-methyl-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 61)

[0463]

[0464] Pale yellow solid, yield 31.2%. 1H NMR (500MHz, CD3OD) δ7.60(d,J=2.0Hz,1H),7.52(dd,J=8.1,1.9Hz,1H),7.46(d,J=8.1Hz,1H),7.15(d,J=1.7Hz,1H),7.11( dd,J=7.8,1.8Hz,1H),6.97(d,J=7.7Hz,1H),5.47(td,J=3.8,2.0Hz,1H),5.11(s,2H),4.39–4.28(m,2H),3.47(dd,J=11.1, 5.7Hz,1H),3.37–3.31(m,2H),3.24(dt,J=10.9,7.3Hz,1H),3.06(tt,J=8.9,5.8Hz,1H),2.78(q,J=7.5Hz,2H),2.33–2.21( m,5H),2.20(s,3H),2.16–2.10(m,4H),1.73(tdd,J=8.2,4.9,2.6Hz,2H),1.67(qq,J=5.8,2.6Hz,2H),1.22(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.11,154.10,144.02,143.65,138.75,137.74,135.99,134.52,130.49,130.29,129.59,127.88,126.60,125.31, 125.22,123.93,75.85,56.47,54.35,53.53,43.72,29.82,27.78,25.26,24.97,22.81,21.92,18.61,14.59,11.45.MS(ESI)(m / z):[M+H] + 475.30

[0465] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-cyclopentyl-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 62)

[0466]

[0467] Pale yellow solid, yield 33.5%. 1H NMR (500MHz, CD3OD) δ7.61(d,J=1.9Hz,1H),7.54(dd,J=8.0,1.9Hz,1H),7.47(d,J=8.1Hz,1H),7.19(d,J=7.8Hz,1H),7.16(d,J= 1.9Hz,1H),7.13(d,J=1.8Hz,1H),5.11(s,2H),4.45–4.30(m,2H),3.52(dd,J=11.3,5.8Hz,1H),3.41–3.32(m,2H),3.28(dd,J=1 1.0,7.4Hz,1H),3.19(tt,J=9.2,7.5Hz,1H),3.09(ddd,J=14.6,8.8,5.8Hz,1H),2.79(q,J=7.6Hz,2H),2.31(s,4H),2.21(s,4H) ,1.99(ddddd,J=12.2,10.9,6.7,2.8,1.4Hz,2H),1.85–1.76(m,2H),1.72–1.64(m,2H),1.58–1.47(m,2H),1.23(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.02,154.03,143.86,143.70,137.92,135.45,134.86,130.52,129.89,129.76,126.65,125.83,124. 80,123.98,75.90,56.35,54.35,53.58,43.56,41.31,33.26,27.72,25.27,25.10,18.61,14.59,11.43.MS(ESI)(m / z):[M+H] + 463.30

[0468] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(cyclopent-1-en-1-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 63)

[0469]

[0470] Pale yellow solid, yield 30.4%. 1H NMR (500MHz, CD3OD) δ7.60(d,J=1.9Hz,1H),7.53(dd,J=8.1,1.9Hz,1H),7.46(d,J=8.1Hz,1H),7.18(d,J=1.7Hz, 1H),7.16–7.10(m,2H),5.73(p,J=2.2Hz,1H),5.13(s,2H),4.39–4.26(m,2H),3.48(dd,J=11.2,5.7Hz,1H),3.37– 3.32(m,1H),3.29(s,1H),3.26–3.20(m,1H),3.06(ddd,J=14.6,8.8,5.8Hz,1H),2.78(q,J=7.5Hz,2H),2.67–2.58 (m,2H),2.51(tq,J=7.2,2.4Hz,2H),2.31(s,3H),2.30–2.19(m,5H),1.97(p,J=7.5Hz,2H),1.22(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.11,154.18,143.66,143.14,137.75,137.50,136.20,135.06,130.47,130.29,130.01,128.84,127.61,126. 61,125.17,123.93,75.74,56.50,54.38,53.54,43.71,36.25,33.04,27.76,25.24,23.30,20.04,14.57,11.43.MS(ESI)(m / z):[M+H] + 461.30.

[0471] Structure of (S,E)-1-(2-ethyl-4-(1-((2,2'-difluoro-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 64)

[0472]

[0473] Pale yellow solid, yield 28.9%. 1H NMR (500MHz, CD3OD) δ7.63(d,J=1.9Hz,1H),7.57(dd,J=8.1,2.0Hz,1H),7.47(d,J=8.1Hz,1H),7.44–7.40(m,1H),7.39(d,J=2.5 Hz,1H),7.38(d,J=2.6Hz,1H),7.31(dd,J=7.9,1.6Hz,1H),7.28–7.25(m,1H),7.24(d,J=1.7Hz,1H),7.19(ddd,J=9.6,8.2,1.2H z,1H),5.28(s,2H),4.31(d,J=3.9Hz,2H),3.44(dd,J=10.6,5.7Hz,1H),3.29(d,J=9.8Hz,2H),3.23(s,1H),3.07(ddd,J=14.5,8 .6,6.0Hz,1H),2.81(q,J=7.5Hz,2H),2.31(d,J=0.8Hz,3H),2.29–2.25(m,1H),2.23(dd,J=13.6,7.0Hz,1H),1.27–1.23(m,3H). 13 C NMR (126MHz, CD3OD) δ160.79,160.67,158.83,154.90,143.63,141.17,141.11,1 37.38,131.32,131.22,130.37,129.77,129.71,126.62,123.99,123.96,123.90 ,123.32,123.30,123.15,122.70,122.57,115.27,115.10,114.64,114.46,74.6 6,56.68,54.68,53.65,43.80,27.79,25.22,14.50,11.45.MS(ESI)(m / z):[M+H] + 493.20.

[0474] Structure of (S,E)-1-(2-ethyl-4-(1-((2-fluoro-4-(6-fluoropyridin-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 65)

[0475]

[0476] Pale yellow solid, yield 20.3%. 1H NMR (500MHz, CD3OD) δ8.49–8.43(m,1H),8.23–8.16(m,1H),7.64(q,J=1.8Hz,1H),7.61(dt,J=7.8,1.8Hz,1H),7.58(dq,J=9.9,1.9H z,1H),7.49(s,1H),7.47(d,J=4.2Hz,1H),7.44(ddd,J=10.5,3.9,2.0Hz,1H),7.16(dq,J=8.5,2.4Hz,1H),5.34(d,J=2.2Hz,2H),4.4 3–4.34(m,2H),3.52(dd,J=11.2,5.8Hz,1H),3.41–3.37(m,1H),3.36–3.34(m,1H),3.28(dd,J=10.9,7.3Hz,1H),3.10(tt,J=8.9,5. 8Hz,1H),2.85–2.78(m,2H),2.37–2.29(m,1H),2.27(q,J=2.6,2.2Hz,3H),2.23(dd,J=13.5,6.7Hz,1H),1.25(tt,J=7.6,1.9Hz,3H). 13 C NMR (126MHz, CD3OD) δ162.39,154.77,145.28,145.16,143.72,140.36,1 40.29,138.32,137.59,131.37,131.33,130.46,130.34,126.66,125.13 ,125.01,123.96,122.37,113.51,113.32,109.52,109.22,69.01,56.52 ,54.43,53.58,43.59,27.71,25.21,14.50,11.28.MS(ESI)(m / z):[M+H] + 494.20

[0477] Structure of (S,E)-1-(2-ethyl-4-(1-(((6'-fluoro-[2,3'-bipyridyl]-5-yl)methoxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 66)

[0478]

[0479] Pale yellow solid, yield 17.2%. 1H NMR (500MHz, CD3OD) δ8.84–8.80(m,1H),8.72(dd,J=2.3,0.9Hz,1H),8.54(ddd,J=8.6,7.6,2.6Hz,1H),7.98(dd,J=8.1,2.2Hz,1H),7. 92(dd,J=8.1,0.9Hz,1H),7.64(d,J=2.0Hz,1H),7.58(dd,J=8.1,1.9Hz,1H),7.49(d,J=8.1Hz,1H),7.19(ddd,J=8.6,2.6,0.6Hz,1H),5 .33(s,2H),4.45–4.35(m,2H),3.53(dd,J=11.2,5.7Hz,1H),3.42–3.37(m,1H),3.37–3.34(m,1H),3.29(dd,J=11.0,7.3Hz,1H),3.11(d dt,J=11.2,8.4,5.9Hz,1H),2.82(q,J=7.5Hz,2H),2.34(dt,J=15.1,7.7Hz,1H),2.29(s,3H),2.28–2.21(m,1H),1.25(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ177.73,164.91,162.99,155.08,152.89,149.35,14 5.86,145.74,143.78,140.31,140.25,137.56,137.50,133.65,132.98,13 2.94,130.50,130.31,126.69,123.99,120.45,109.42,109.12,72.79,56. 49,54.38,53.57,43.52,27.69,25.22,14.53,11.38.MS(ESI)(m / z):[M+H] + 477.20.

[0480] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(5-fluoropyrazin-2-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 67)

[0481]

[0482] Pale yellow solid, yield 16.3%. 1H NMR (500MHz, CD3OD) δ8.59(dd,J=8.3,1.4Hz,1H),8.38(d,J=1.6Hz,1H),7.66(d,J=1.9Hz,1H),7.60(dd,J=8.1,1.9 Hz,1H),7.51(d,J=8.1Hz,1H),7.43(d,J=7.7Hz,1H),7.40(s,1H),7.38–7.35(m,1H),5.27(s,2H),4.49–4.41(m,2H ),3.58(dd,J=11.4,6.0Hz,1H),3.45(d,J=8.2Hz,1H),3.41(d,J=7.7Hz,1H),3.39–3.34(m,1H),3.20–3.12(m,1H), 2.83(q,J=7.5Hz,2H),2.38(s,3H),2.35(dd,J=9.1,6.9Hz,1H),2.30(s,3H),2.28–2.24(m,1H),1.29–1.25(m,3H). 13 C NMR (126MHz, CD3OD) δ179.74,154.46,152.91,143.82,141.30,141.23,139.46,137.93,136.38,135.09,131.95,131.65,130.58,130.3 2,129.70,129.58,126.73,125.50,124.04,75.35,56.22,54.40,53.64,43.26,27.61,25.28,19.04,14.57,11.40.MS(ESI)(m / z):[M+H] + 491.25.

[0483] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(6-fluoropyridin-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 68)

[0484]

[0485] Pale yellow solid, yield 27.6%. 1H NMR (500MHz, CD3OD) δ8.41(dt,J=2.7,0.8Hz,1H),8.16(ddd,J=8.5,7.6,2.6Hz,1H),7.63(d,J=1.9Hz,1H),7.62(d,J=1.8Hz,1H),7.61( d,J=2.0Hz,1H),7.57(dd,J=8.1,1.9Hz,1H),7.54(d,J=1.8Hz,1H),7.52(d,J=1.8Hz,1H),7.48(d,J=8.1Hz,1H),7.13(ddd,J=8.5,2.6,0 .7Hz,1H),5.28(s,2H),4.43–4.34(m,2H),3.52(dd,J=11.2,5.7Hz,1H),3.37(ddd,J=11.0,8.0,3.1Hz,2H),3.29(dt,J=11.0,7.3Hz,1H) ,3.10(tt,J=8.5,5.7Hz,1H),2.81(q,J=7.5Hz,2H),2.38–2.30(m,1H),2.28(s,3H),2.24(dt,J=13.6,6.8Hz,1H),1.24(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ177.94,163.99,162.09,154.52,145.03,144.92,1 43.73,140.27,140.21,138.46,137.72,135.77,134.78,134.74,130.48 ,130.19,128.57,126.66,126.64,123.97,109.39,109.09,75.30,56.45 ,54.36,53.56,43.56,27.71,25.24,14.55,11.42.MS(ESI)(m / z):[M+H] + 476.20.

[0486] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-fluoro-4-(6-fluoropyridin-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 69)

[0487]

[0488] Pale yellow solid, yield 29.2%. 1H NMR(500MHz,CD3OD)δ8.39(s,1H),8.14(d,J=8.5Hz,1H),7.64(d,J=4.0Hz,1H),7.60–7.55(m,1H),7.53(dd, J=8.0,2.3Hz,1H),7.47(dd,J=8.2,2.1Hz,1H),7.38–7.33(m,1H),7.32(d,J=11.2Hz,1H),7.19(dd,J=8.9,2 .9Hz,1H),5.29(d,J=4.3Hz,2H),4.41–4.30(m,2H),3.48(dd,J=10.8,5.4Hz,1H),3.37(s,1H),3.26(q,J=9. 3,8.4Hz,2H),3.09(tt,J=8.5,5.9Hz,1H),2.83(q,J=5.8,4.4Hz,2H),2.34–2.22(m,5H),1.27–1.22(m,3H). 13 C NMR (126MHz, CD3OD) δ178.47,163.95,162.05,160.52,158.55,154.93,146.85,146.82,146 .74,146.70,143.71,142.17,142.11,141.75,141.69,137.46,130.72,130.44,130.22,130 .19,129.71,126.64,123.99,123.97,123.94,123.35,123.24,115.19,115.01,109.24,108 .94,74.50,56.56,54.53,53.63,43.69,27.74,25.23,14.52,11.43..MS(ESI)(m / z):[M+H] + 494.20

[0489] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(pyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 70)

[0490]

[0491] Pale yellow solid, yield 19.8%. 1H NMR(500MHz,CD3OD)δ9.17–9.12(m,1H),9.11–9.05(m,2H),7.75(dd,J=9.5,3.5Hz, 2H),7.65–7.55(m,4H),7.47(d,J=8.1Hz,1H),5.32(d,J=4.0Hz,2H),4.33(s,2H),3 .43(dd,J=10.8,6.1Hz,1H),3.31–3.20(m,3H),3.08(q,J=7.5,7.0Hz,1H),2.88–2. 74(m,2H),2.31–2.29(m,3H),2.28–2.19(m,2H),1.25(ddd,J=9.6,4.3,2.7Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.39,156.47,154.70,154.64,143.64,139.57,137.42,134.33,133.16,130.98,130.38,12 8.74,126.71,126.58,123.88,75.16,56.68,54.58,53.59,43.81,27.79,25.22,14.53,11.43.MS(ESI)(m / z):[M+H] + 459.20

[0492] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-fluoro-4-(pyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 71)

[0493]

[0494] Pale yellow solid, yield 30.0%. 1 H NMR(600MHz,CD3OD)δ9.39–9.24(m,1H),9.14(d,J=8.3Hz,2H),7.84–7.46(m,6 H),5.45(d,J=7.7Hz,2H),4.58–4.42(m,2H),3.67–3.57(m,1H),3.53(s,1H),3. 45(s,1H),3.39(d,J=9.6Hz,1H),3.28–3.19(m,1H),2.96(q,J=7.5Hz,2H),2.4 9–2.44(m,2H),2.42(d,J=2.3Hz,1H),2.37(q,J=6.8Hz,1H),1.40–1.37(m,3H). 13C NMR (126MHz, CD3OD) δ178.30,160.69,158.72,156.78,156.20,156.16,155. 06,143.69,142.91,142.85,137.34,130.42,130.09,130.07,129.97,129.95 ,126.61,124.22,124.20,123.92,120.82,120.71,115.26,115.07,74.40,5 6.66,54.53,53.59,43.77,27.77,25.22,14.52,11.43.MS(ESI)(m / z):[M+H] + 477.20

[0495] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(thiophen-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 72)

[0496]

[0497] Pale yellow solid, yield 18.3%. 1 H NMR (500MHz, CD3OD) δ7.62(s,1H),7.54(d,J=7.8Hz,1H),7.47(d,J=8.0Hz,1H),7.39(d,J=4.6H z,1H),7.38–7.28(m,2H),7.23(d,J=7.8Hz,1H),7.14–7.01(m,2H),5.19(s,2H),4.34(d,J=6.8H z,2H),3.48(dd,J=11.2,5.8Hz,1H),3.35(s,1H),3.31(s,1H),3.25(q,J=10.5,8.7Hz,1H),3.1 3–3.02(m,1H),2.79(q,J=7.3Hz,2H),2.37(s,3H),2.24(q,J=6.5Hz,5H),1.23(t,J=7.5Hz,3H). 13C NMR (126MHz, CD3OD) δ178.28,154.41,143.66,142.51,137.70,135.65,133.55,130.47,130.21,129.96,126.81,126.62,12 6.22,125.44,124.87,123.95,75.48,56.50,54.42,53.56,43.76,27.79,25.25,20.01,14.58,11.49.MS(ESI)(m / z):[M+H] + 477.20

[0498] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(thiophen-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 73)

[0499]

[0500] Pale yellow solid, yield 21.2%. 1 H NMR (500MHz, CD3OD) δ7.64–7.59(m,3H),7.56(dd,J=8.1,2.0Hz,1H),7.47(d,J=8.1Hz,1H),7.4 3–7.39(m,2H),7.36(ddd,J=8.2,4.4,1.2Hz,2H),7.08(dd,J=5.1,3.6Hz,1H),5.22(s,2H),4.40 –4.30(m,2H),3.49(dd,J=11.2,5.8Hz,1H),3.35(d,J=7.7Hz,1H),3.32(s,1H),3.26(dt,J=10.9 ,7.3Hz,1H),3.12–3.04(m,1H),2.80(q,J=7.5Hz,2H),2.34–2.18(m,5H),1.24(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.13,154.46,143.72,143.69,137.71,137.42,133.91,130.48,130.27,128.47,127.75,126.64 ,125.28,124.49,123.95,122.92,75.43,56.49,54.41,53.56,43.70,27.76,25.25,14.55,11.46.MS(ESI)(m / z):[M+H] + 463.20

[0501] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(isothiazol-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 74)

[0502]

[0503] Pale yellow solid, yield 11.6%. 1 H NMR(500MHz,CD3OD)δ9.07(s,1H),8.86(s,1H),7.72–7.68(m,2H),7.63(d,J=1.9Hz,1H),7.57(d d,J=8.1,1.9Hz,1H),7.52(d,J=8.1Hz,1H),7.50–7.46(m,2H),5.26(s,2H),4.46–4.37(m,2H),3 .52(dd,J=11.3,6.0Hz,1H),3.44–3.39(m,1H),3.39(d,J=4.0Hz,1H),3.35(d,J=6.9Hz,1H),3.1 2(ddd,J=14.4,8.7,5.9Hz,1H),2.82(q,J=7.5Hz,2H),2.37–2.22(m,5H),1.24(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.11,155.89,154.49,143.75,143.06,139.51,138.10,137.76,131.87,130.56,130.04,12 8.57,126.66,126.46,123.98,75.38,56.36,54.37,53.59,43.65,27.77,25.29,14.57,11.44.MS(ESI)(m / z):[M+H] + 464.20

[0504] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(isothiazol-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 75)

[0505]

[0506] Pale yellow solid, yield 14.9%. 1H NMR(500MHz,CD3OD)δ8.48(d,J=1.8Hz,1H),7.72–7.66(m,2H),7.63(d,J=1.9Hz,1H),7.60(d ,J=1.8Hz,1H),7.57(dd,J=8.0,1.9Hz,1H),7.52(s,2H),7.51(s,1H),5.27(s,2H),4.46–4.3 6(m,2H),3.52(dd,J=11.3,5.9Hz,1H),3.43–3.38(m,1H),3.36(dd,J=12.2,5.6Hz,2H),3.12 (dq,J=11.3,4.4,2.9Hz,1H),2.82(q,J=7.5Hz,2H),2.38–2.23(m,5H),1.24(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ177.97,167.11,158.52,154.68,143.76,140.25,137.69,130.55,130.11,129.81,128.63,12 6.67,126.35,123.99,119.97,75.14,56.38,54.37,53.58,43.64,27.77,25.28,14.56,11.44.MS(ESI)(m / z):[M+H] + 464.20

[0507] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyrimidin-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 76)

[0508]

[0509] Pale yellow solid, yield 19.9%. 1H NMR (500MHz, CD3OD) δ9.21(d,J=1.4Hz,1H),8.83(d,J=5.3Hz,1H),7.65(dt,J=3.4,1.5Hz,2H),7.59(d d,J=8.1,1.9Hz,1H),7.49(dd,J=7.8,6.3Hz,2H),7.39(d,J=8.6Hz,2H),5.28(s,2H),4.49–4.39(m,2H ),3.56(dd,J=11.3,5.6Hz,1H),3.45–3.34(m,2H),3.30(t,J=5.6Hz,1H),3.13(p,J=7.2Hz,1H),2.83( q,J=7.5Hz,2H),2.43(s,3H),2.41–2.32(m,1H),2.30(s,3H),2.29–2.23(m,1H),1.26(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ167.30,157.63,156.90,154.54,143.78,140.16,137.83,136.66,136.11,130.53,130.43,129.94,129.4 4,126.71,125.53,124.02,121.62,75.26,56.40,54.34,53.54,29.38,27.65,25.24,19.11,14.57,11.41.MS(ESI)(m / z):[M+H] + 473.20

[0510] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(5-fluoropyrimidin-2-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 77)

[0511]

[0512] Pale yellow solid, yield 15.4%. 1H NMR (500MHz, CD3OD) δ8.82(d,J=1.9Hz,2H),7.76(d,J=7.7Hz,1H),7.64(d,J=2.0Hz,1H),7 .57(dd,J=8.1,2.0Hz,1H),7.48(d,J=8.1Hz,1H),7.35(d,J=8.9Hz,2H),5.26(s,2H),4.44 –4.34(m,2H),3.52(dd,J=11.3,5.4Hz,1H),3.41–3.33(m,2H),3.32–3.25(m,1H),3.10(s, 1H), 2.82 (q, J = 7.6Hz, 2H), 2.51 (s, 3H), 2.29 (d, J = 1.4Hz, 5H), 1.25 (td, J = 7.5, 1.7Hz, 3H). 13 C NMR (126MHz, CD3OD) δ163.34,157.57,155.48,154.51,144.66,144.50,143.73,139.67,137.77,137.07,136.48,130.50,130.46,13 0.20,130.14,126.67,125.12,123.98,75.35,56.58,54.40,53.55,29.35,27.76,25.23,19.92,14.55,11.43.MS(ESI)(m / z):[M+H] + 491.20

[0513] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyrimidin-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 78)

[0514]

[0515] Pale yellow solid, yield 21.4%. 1H NMR (500MHz, CD3OD) δ8.89(d,J=4.9Hz,2H),7.69(d,J=7.9Hz,1H),7.65(d,J=1.9Hz,1H),7.59(dd,J=8.0,1 .9Hz,1H),7.52(d,J=8.1Hz,1H),7.44(t,J=5.0Hz,1H),7.37(d,J=7.2Hz,2H),5.27(s,2H),4.47–4.39(m,2 H),3.56(dd,J=11.3,5.9Hz,1H),3.42(dt,J=11.2,7.4Hz,2H),3.35(d,J=7.0Hz,1H),3.15(td,J=8.6,4.1H z,1H),2.83(q,J=7.5Hz,2H),2.48(s,3H),2.35(dd,J=14.4,7.4Hz,1H),2.30(s,4H),1.26(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ167.16,157.00,154.46,143.78,139.72,137.86,137.42,136.88,130.58,130.37,129.95,129.87,12 6.70,125.17,124.01,119.06,75.37,56.32,54.38,53.60,43.49,27.71,25.29,19.54,14.57,11.41.MS(ESI)(m / z):[M+H] + 473.20

[0516] Structure of (S,E)-1-(2-ethyl-4-(1-(((2-fluoro-4-(2-fluoropyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 79)

[0517]

[0518] Pale yellow solid, yield 14.3%. 1H NMR(500MHz,CD3OD)δ8.98(d,J=1.4Hz,2H),7.67–7.62(m,2H),7.57(dd,J=8 .0,1.9Hz,1H),7.52(t,J=8.6Hz,3H),5.34(s,2H),4.47–4.37(m,2H),3.54( dd,J=11.4,5.8Hz,1H),3.43–3.36(m,2H),3.35(s,1H),3.18–3.09(m,1H),2 .83(q,J=7.5Hz,2H),2.39–2.29(m,1H),2.27(s,4H),1.25(t,J=7.5Hz,3H). 13 C NMR (126MHz, CD3OD) δ163.34,162.29,161.60,160.32,159.08,158.99,154.85,143.77,137.60,131.55,131.52,130.54,130.13,126.66 ,126.05,125.93,123.98,122.45,113.63,113.45,68.93,56.36,54.37,53.60,43.53,27.71,25.25,14.52,11.28.MS(ESI)(m / z):[M+H] + 495.20

[0519] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(2-fluoropyrimidin-5-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 80)

[0520]

[0521] Pale yellow solid, yield 22.2%. 1H NMR(500MHz,CD3OD))δ8.69(d,J=1.6Hz,2H),7.64(d,J=1.9Hz,1H),7.57(dd,J=8.1,1.9Hz,1H),7 .49(d,J=8.1Hz,1H),7.41(s,1H),7.38(dd,J=7.8,1.8Hz,1H),7.27(d,J=7.8Hz,1H),5.25(s,2H) ,4.42–4.34(m,2H),3.51(dd,J=11.1,5.7Hz,1H),3.35(t,J=3.5Hz,2H),3.31–3.24(m,1H),3.10( td,J=8.7,8.0,4.3Hz,1H),2.82(q,J=7.5Hz,2H),2.30(s,4H),2.28(s,4H),1.25(t,J=7.5Hz,3H). 13 CNMR(126MHz,CD3OD)δ177.99,162.77,161.05,160.69,160.59,157.18, 154.51,143.71,139.35,137.62,135.80,133.62,133.58,132.50,130.4 7,130.44,130.16,129.72,126.62,125.87,123.94,75.29,56.58,54.39 ,53.55,43.69,27.75,25.24,18.95,14.56,11.40.MS(ESI)(m / z):[M+H] + 491.20

[0522] Structure of (E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 81)

[0523]

[0524] Pale yellow solid, yield 35.6%. 1H NMR(500MHz,CD3OD)δ9.15(s,1H),8.85–8.75(m,2H),7.63(s,1H),7.56(d,J=8.1Hz,1H),7.45–7.35(m,3H),7.26(t,J=6.5Hz,1H),5.25(d, J=3.0Hz,2H),4.40(s,2H),4.16(p,J=9.5Hz,4H),3.41(p,J=8.3Hz,1H),2.80(q,J=7.5Hz,2H),2.30(s,3H),2.27(s,3H),1.27–1.22(m,3H). 13 C NMR (126MHz, CD3OD) δ176.27,156.56,156.36,154.50,143.54,139.32,137.57,135.63,133.39,130.18,129.64 ,129.54,126.62,125.90,123.97,75.30,57.16,54.94,34.92,25.19,18.97,14.46,11.41.MS(ESI)(m / z):[M+H] + 459.20

[0525] Structure of (E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)piperidine-4-carboxylic acid (Compound 82)

[0526]

[0527] Pale yellow solid, yield 30.3%. 1 H NMR(500MHz,CD3OD))δ9.15(s,1H),8.80(s,2H),7.63(d,J=1.9Hz,1H),7.56(dd,J=8.1,1.9Hz ,1H),7.47(d,J=8.1Hz,1H),7.42(s,1H),7.39(dd,J=7.8,1.7Hz,1H),7.27(d,J=7.8Hz,1H),5. 26(s,2H),4.13(s,2H),3.30–3.27(m,1H),2.90–2.78(m,4H),2.46–2.32(m,2H),2.31(s,3H), 2.29(s,3H),2.05(dd,J=14.6,4.0Hz,2H),1.90(q,J=12.8,12.2Hz,2H),1.25(t,J=7.5Hz,3H). 13C NMR (126MHz, CD3OD) δ179.59 (d, J = 2.2Hz), 156.57, 156.35, 154.63, 144.37, 139.34, 137.46, 135.63, 135.60, 133.38, 131.20, 130.18,129.63,126.58,125.90,123.73,75.28,57.31,52.14,40.79,26.66,25.39,18.97,14.65,11.45.MS(ESI)(m / z):[M+H] + 487.30

[0528] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(4-methylpyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 83)

[0529]

[0530] Pale yellow solid, yield 42.5%. 1 H NMR (500MHz, Methanol-d4) δ9.03 (s, 1H), 8.48 (s, 1H), 7.66 (d, J = 1.9 Hz, 1H), 7.59 (dd, J = 8.1, 1. 9Hz, 1H), 7.51 (d, J = 8.1Hz, 1H), 7.43 (d, J = 1.7Hz, 1H), 7.38 (dd, J = 7.8, 1.7Hz, 1H), 7.17 (d, J = 7. 8Hz,1H),5.28(s,2H),4.48–4.36(m,2H),3.53(d,J=7.7Hz,1H),3.43–3.37(m,2H),3.31(s,1H), 3.13(s,1H),2.84(q,J=7.5Hz,2H),2.33(s,4H),2.30(s,4H),2.11(s,3H),1.27(t,J=7.5Hz,3H). 13 C NMR (126MHz, Methanol-d4) δ165.58,156.48,156.26,154.49,143.76,139.12,137.77,135.87,134.85,134.16,130.53,130.18,129.8 1,129.17,126.68,125.69,124.00,75.40,56.58,54.41,53.58,48.46,27.77,25.25,20.95,18.56,14.57,11.43.MS(ESI)(m / z):[M+H]+ 487.30

[0531] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(pyridazin-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 84)

[0532]

[0533] Pale yellow solid, yield 27.3%. 1 H NMR (500MHz, CD3OD) δ9.27–9.23(m,2H),7.77(dd,J=5.3,2.4Hz,1H),7.65(d,J=1.9Hz,1H),7.58(dd,J=8.1,1.9H z,1H),7.48(d,J=8.0Hz,1H),7.45(s,1H),7.42(dd,J=7.8,1.7Hz,1H),7.34(d,J=7.8Hz,1H),5.28(s,2H),4.36(d ,J=2.5Hz,2H),3.47(dd,J=11.1,6.0Hz,1H),3.37–3.34(m,1H),3.31(d,J=7.1Hz,1H),3.27(q,J=9.5,8.4Hz,1H) ,3.09(ddd,J=12.3,8.4,5.8Hz,1H),2.83(q,J=7.5Hz,2H),2.36(s,3H),2.33–2.22(m,5H),1.27(t,J=7.5Hz,3H). 13 C NMR (151MHz, CD3OD) δ178.13,154.62,151.80,150.95,143.69,141.24,139.97,137.58,135.47,134.18,130.41,130.34,129.3 6,127.39,126.63,125.97,123.94,75.20,56.73,54.58,53.60,43.75,27.78,25.22,18.89,14.54,11.39.MS(ESI)(m / z):[M+H] + 473.30

[0534] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-fluoro-4-(thiophen-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 85)

[0535]

[0536] Pale yellow solid, yield 26.5%. 1 H NMR(500MHz,CD3OD)δ7.66(dt,J=2.8,1.4Hz,1H),7.64–7.59(m,2H),7.55(dd,J=8.1,1.9Hz,1H),7. 49–7.45(m,2H),7.44(dt,J=5.1,1.4Hz,1H),7.26–7.18(m,2H),5.23(s,2H),4.39–4.30(m,2H),3.49 (dd,J=11.1,5.8Hz,1H),3.35(d,J=9.5Hz,1H),3.32–3.30(m,1H),3.25(ddd,J=14.6,7.7,3.8Hz,1H) ,3.08(ddd,J=14.6,8.7,5.8Hz,1H),2.79(q,J=7.6Hz,2H),2.33–2.20(m,5H),1.23(t,J=7.6Hz,3H). 13 C NMR (126MHz, CD3OD) δ178.15,160.47,158.51,154.74,143.70,139.64,139.58,13 7.55,135.32,135.31,130.48,130.44,129.15,129.11,127.08,127.05,126.63,12 5.21,123.94,123.69,123.67,123.14,123.08,122.77,122.67,115.25,115.06,74 .68,56.49,54.38,53.55,43.69,27.75,25.23,14.53,11.45.MS(ESI)(m / z):[M+H] + 481.20

[0537] Structure of (S,E)-1-(2-ethyl-4-(1-(((2,6-difluoro-4-(6-fluoropyridin-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 86)

[0538]

[0539] Pale yellow solid, yield 26.9%. 1HNMR(500MHz,CD3OD)δ8.49(d,J=2.6Hz,1H),8.22(ddd,J=8.5,7.5,2.7Hz,1H),7.64(d,J=1.9Hz,1H) ,7.56(dd,J=8.1,1.9Hz,1H),7.47(d,J=8.1Hz,1H),7.35(d,J=8.3Hz,2H),7.19–7.15(m,1H),5.33(s, 2H),4.42–4.34(m,2H),3.51(dd,J=11.2,5.8Hz,1H),3.39–3.34(m,2H),3.32–3.25(m,1H),3.10(ddd, J=14.4,8.5,5.8Hz,1H),2.81(q,J=7.6Hz,2H),2.36–2.24(m,2H),2.20(s,3H),1.26(t,J=7.3Hz,3H). 13 C NMR (126MHz, CD3OD) δ172.97,165.98,164.82,164.08,162.83,156.01,146.90,146.78,145.01,141.80,141.73,140.95,138.87,131.81,127.9 3,125.25,114.64,111.05,111.03,110.84,110.74,64.06,57.88,55.90 ,55.04,45.00,29.11,26.57,20.85,15.78,12.33.MS(ESI)(m / z):[M+H] + 512.20

[0540] Structure of (S,E)-1-(2-ethyl-4-(1-(((2-fluoro-4-(5-fluoropyrazin-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 87)

[0541]

[0542] White solid, yield 78.9%. 1H NMR(500MHz,Methanol-d4)δ8.77(s,1H),8.56(s,1H),7.90–7.79(m,2H),7.65–7.59(m,2H),7.56(s,1H),7.48(d ,J=8.1Hz,1H),5.34(s,2H),4.44–4.34(m,2H),3.55–3.46(m,1H),3.36(d,J=7.7Hz,1H),3.28(dd,J=10.9,7.3Hz,

[0543] 1H),3.13–3.05(m,1H),2.81(q,J=8.6Hz,2H),2.33(dt,J=15.2,7.9Hz,1H) ,2.28(s,3H),2.23(dd,J=13.5,6.8Hz,1H),2.03(s,1H),1.28–1.21(m,3H). 13 C NMR(126MHz,Methanol-d4)δ154.84,143.72,138.45,138.38,137.57,132.58,132.27,131.05,130.46,130.30,1 26.64,123.95,121.93,113.21,113.02,69.00,56.53,54.37,53.56,43.58,27.71,25.21,11.29.MS(m / z):[M+H] + 495.20

[0544] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-fluoro-4-(5-fluoropyrazin-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 88)

[0545]

[0546] Yellow solid, yield 75.0%. 1H NMR (500MHz, Methanol-d4) δ8.67(s,1H),8.62(d,J=8.3Hz,1H),7.63(s,1H),7.56(d,J= 7.4Hz,1H),7.49(d,J=8.1Hz,1H),7.38(d,J=8.0Hz,1H),7.32(d,J=12.1Hz,1H),5.30(s, 2H),4.37(d,J=11.1Hz,2H),3.29(t,J=6.7Hz,1H),3.09(t,J=7.6Hz,1H),2.82(q,J=7.5 Hz,2H),2.31(s,3H),2.26–2.17(m,1H),2.03(s,1H),1.95(s,1H),1.24(t,J=7.5Hz,3H). 13 C NMR (126MHz, Methanol-d4) δ155.04,143.70,143.17,141.41,141.34,141.24,137.37,132.95,132.65,130.83,130.54,130. 52,130.46,126.61,123.93,123.82,115.16,114.98,74.39,56.65,54.53,53.57,43.85,25.24,14.52,11.44.MS(m / z):[M+H] + 495.20

[0547] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(5-fluoropyrazin-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 89)

[0548]

[0549] White solid, yield 76.6%. 1H NMR(500MHz,Methanol-d4)δ8.79–8.69(m,1H),8.58–8.51(m,1H),8.03(dd,J=16.8,5.3Hz,2H) ,7.63(d,J=7.0Hz,1H),7.58–7.54(m,3H),7.47(d,J=8.1Hz,1H),5.30(s,2H),4.41–4.30(m,2H ),3.50(dd,J=10.7,5.5Hz,1H),3.37(s,1H),3.28–3.20(m,1H),3.09(dd,J=10.2,4.2Hz,1H),2 .85–2.78(m,2H),2.36–2.31(m,1H),2.30(s,3H),2.24(q,J=6.9,6.4Hz,1H),1.28–1.20(m,3H). 13 C NMR(126MHz,Methanol-d4)δ154.62,143.69,140.08,138.24,138.17,132.34,132.04,130.43,128.2 8,126.65,126.44,123.94,75.24,56.57,54.45,53.58,43.66,27.73,25.22,14.53,11.42.MS(m / z):

[0550] [M+H] + 477.20

[0551] Structure of (E)-1-(2-ethyl-4-(1-(((3-methyl-4-(5-fluoropyrazin-2-yl)-benzyl)oxy)imino)ethyl)benzyl)piperidine-4-carboxylic acid (Compound 90)

[0552]

[0553] White solid, yield 28.8%. 1H NMR(500MHz, Methanol-d4)δ8.58(d,J=8.2Hz,1H),8.37(s,1H),7.60(s,1H),7.52(d, J=10.0Hz,1H),7.41(dd,J=18.7,7.9Hz,4H),5.26(s,2H),3.96(s,2H),3.18(d,J=11. 6Hz,2H),2.80(q,J=7.6Hz,2H),2.66(d,J=11.5Hz,2H),2.38(s,3H),2.33(d,J=15.1H z,1H),2.28(s,3H),2.02–1.93(m,2H),1.84(d,J=12.0Hz,2H),1.24(t,J=7.6Hz,3H). 13 C NMR(126MHz,Methanol-d4)δ158.27,154.80,144.11,141.30,139.50,136.91,136.35,131.93,131.63,130.86,1 30.33,129.57,126.42,125.50,123.52,75.26,57.89,52.46,27.35,25.33,19.05,14.60,11.48.MS(m / z):[M+H] + 505.20

[0554] Structure of (E)-1-(2-ethyl-4-(1-(((3-methyl-4-(5-fluoropyrazin-2-yl)-benzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 91)

[0555]

[0556] White solid, yield 62.5%. 1 H NMR (500MHz, Methanol-d4) δ8.58(d,J=8.3Hz,1H),8.37(s,1H),7.63(s,1H),7.56(d,J=8.0Hz,1H),7.40(dd,J=19.3,7.9Hz, 4H), 5.26 (s, 2H), 4.40 (s, 2H), 4.16 (d, J = 8.3Hz, 4H), 2.79 (q, J = 7.5Hz, 3H), 2.37 (s, 7H), 2.28 (s, 3H), 1.25 (t, J = 7.5Hz, 3H). 13C NMR(126MHz,Methanol-d4)δ176.26,160.27,158.28,154.47,152.86,143.53,141.29,141.22,139.45,137.66,136.37,131.9 4,131.64,130.32,129.57,129.51,126.66,125.50,123.98,75.33,57.18,54.93,34.86,25.16,19.04,14.45,11.39.MS(m / z):

[0557] [M+H] + 477.20

[0558] Structure of (E)-1-(2-ethyl-4-(1-(((3-methyl-4-(5-fluoropyrazin-2-yl)-benzyl)oxy)imino)ethyl)benzyl)piperidine-3-carboxylic acid (Compound 92)

[0559]

[0560] White solid, yield 83.5%. 1 H NMR(500MHz,Methanol-d4)δ8.58(d,J=7.0Hz,1H),8.37(s,1H),7.65(s,1H),7.57 (d,J=8.0Hz,1H),7.48(d,J=8.1Hz,2H),7.45–7.35(m,6H),5.26(s,5H),4.23(d,J= 3.6Hz,5H),3.37(s,4H),3.12(d,J=10.1Hz,5H),2.82(dq,J=14.9,7.3Hz,3H),2.6 5(s,2H),2.38(s,7H),2.29(s,6H),1.85(d,J=48.6Hz,11H),1.27(t,J=7.5Hz,6H). 13 C NMR(126MHz,Methanol-d4)δ160.27,158.28,154.54,152.92,144.11,141.30,141.22,139.48,137.76,136.37,135.06,131.94,1 31.64,130.98,130.33,129.58,126.60,125.51,123.93,75.33,54.53,52.79,48.46,25.20,19.04,14.45,11.41.MS(m / z):[M+H] + 505.20

[0562] Structure of (S,E)-1-(2-ethyl-4-(1-(((2-fluoro-4-(5-fluoropyrazin-2-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 93)

[0563]

[0564] White solid, yield 70.1%. 1 H NMR(500MHz,Methanol-d4)δ8.59(d,J=8.2Hz,1H),8.37(s,1H),7.63(s,1H),7.56(d,J=8.0 Hz,1H),7.49(d,J=8.1Hz,1H),7.42(t,J=7.5Hz,1H),7.25(d,J=7.9Hz,1H),5.33(s,2H),4.4 4–4.33(m,2H),3.52(dd,J=11.2,5.6Hz,1H),3.41–3.35(m,2H),3.28(dt,J=10.8,7.3Hz,1H) ,3.10(dq,J=8.7,4.3,2.9Hz,1H),2.81(q,J=7.5Hz,2H),2.27(s,8H),1.24(t,J=7.4Hz,3H). 13 C NMR (126MHz, Methanol-d4)δ

[0565] 177.94,160.40,160.38,158.43,158.40,154.73,151.66,143.71,141.43,141.35,137.54,132.19,131.88,130.49,130.34,127.45,127.41,126.63,125. 98,125.85,124.93,124.90,123.94,123.68,123.54,69.41,69.38,56.46,54 .32,53.54,43.62,27.73,25.23,14.53,11.32,10.54,10.49.MS(m / z):[M+H] + 509.19

[0566] Structure of (S,E)-1-(2-ethyl-4-(1-(((2-fluoro-4-(5-fluoropyrazin-2-yl)-5-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 94)

[0567]

[0568] White solid, yield 71.5%. 1 H NMR (500MHz, Methanol-d4) δ8.60(dd,J=8.2,1.4Hz,1H),8.41(t,J=1.5Hz,1H),7.65(d,J=1.9Hz,1H),7.58(dd,J=8. 1,1.9Hz,1H),7.48(d,J=8.1Hz,1H),7.45(d,J=7.2Hz,1H),7.25(d,J=10.3Hz,1H),5.31(s,2H),4.43–4.35(m,2H),3 .52(dd,J=11.2,5.8Hz,1H),3.39–3.35(m,2H),3.29(dd,J=12.9,5.5Hz,1H),3.11(ddd,J=14.4,8.7,5.8Hz,1H),2.8 2(q,J=7.5Hz,2H),2.35(s,3H),2.33–2.30(m,1H),2.27(s,3H),2.24(dd,J=13.7,6.5Hz,1H),1.25(d,J=7.5Hz,3H). 13 C NMR(126MHz,Methanol-d4)δ178.22,160.41,160.09,158.41,158.14,154.72,15 1.50,143.70,141.35,141.27,137.60,136.95,133.09,133.05,132.26,132.08, 131.96,130.46,130.37,126.65,125.86,125.74,123.96,116.17,115.99,69.06 ,56.50,54.48,53.62,43.57,27.71,25.21,18.32,14.50,11.27.MS(m / z):[M+H] + 509.20

[0569] Structure of (R,E)-1-(2-ethyl-4-(1-(((4-(5-fluoropyrazin-2-yl)-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 95)

[0570]

[0571] Pale yellow solid, yield 21.4%. 1H NMR (500MHz, Methanol-d4) δ8.57(dd,J=8.3,1.3Hz,1H),8.36(t,J=1.5Hz,1H),7.64(d,J=1.9Hz,1H),7.57(dd,J=8.1 ,1.9Hz,1H),7.49(d,J=8.1Hz,1H),7.41(d,J=7.8Hz,1H),7.39–7.34(m,2H),5.26(s,2H),4.44–4.34(m,2H),3.52(dd, J=11.2,5.7Hz,1H),3.37(td,J=9.1,8.2,3.3Hz,2H),3.28(dt,J=11.0,7.4Hz,1H),3.10(ddd,J=14.5,8.8,5.7Hz,1H), 2.82(q,J=7.5Hz,2H),2.37(s,3H),2.32(td,J=7.9,3.6Hz,1H),2.28(s,3H),2.27–2.22(m,1H),1.25(t,J=7.5Hz,3H). 13 C NMR(126MHz,Methanol-d4)δ177.86,160.26,158.26,154.49,152.89,152.85 ,143.73,141.28,141.21,139.46,137.71,136.37,135.07,135.06,131.94,13 1.64,130.50,130.33,130.21,129.58,126.65,125.50,123.96,75.34,56.48, 54.34,53.54,43.60,27.72,25.24,19.06,14.55,11.42.MS(ESI)(m / z):[M+H] + 491.25.

[0572] Example 2. S1PR4 agonist biological activity test

[0573] 1. Experimental Principle

[0574] Tango detection technology detects ligand-dependent GPCR activity through the interaction between β-arrestin and GPCR. TEV separation technology uses the complementation of two inactive fragments of TEV protease (NTEV, 1-118 and CTEV, 119-221) to reconstruct the functional protease. The NTEV fragment and the artificial transcription factor tetracycline transactivator (tTA) are fused to the C-terminus of GPCR, and the CTEV fragment is fused to human β-arrestin 2. In order to improve the stability of the interaction between GPCR and β-arrestin, a short sequence (V2 tail) from the C-terminus of human arginine vasopressin receptor 2 is introduced between GPCR and NTEV. V2 tail contains multiple GRK phosphorylation sites, which can enhance the binding of β-arrestin after phosphorylation. The CTEV fragment carries a stable point mutation S219P and is truncated at amino acid 221 to remove the autoinhibitory C-terminal tail. At the same time, β-arrestin 2 is truncated to delete the entire C-terminal tail (missing amino acids 383-410). The truncation has a strong stimulus-dependent receptor desensitization effect, so the sensitivity of the detection is improved compared with the wild-type β-arrestin 2. The UAS reporter structure consists of an upstream active sequence element (UAS), a CMV minimal promoter, and a tTA-dependent luciferase reporter gene. Therefore, the construction of Tango cells requires the simultaneous transfection of the above three plasmids into the cells. After GPCR is activated to recruit β-arrestin, the TEV enzyme at the end of β-arrestin removes tTA from the end of GPCR, and the free tTA will initiate tTA-dependent luciferase transcription, and the activity of the compound is finally determined by detecting the fluorescence intensity.

[0575] 2. Experimental process

[0576] 1) Digest the Tango cell lines from the culture dishes, count the cells, and plate them on white opaque 96-well plates at a density of 10,000 cells per well (add 60 μL D'hanks for four weeks), and culture them in a 5% CO2 incubator at 37°C overnight (20 hours). The cell density is about 90% on the next day.

[0577] 2) After 20 h, 30 μL of graded diluted compound solution was added to each well, and 30 μL of F12K complete medium was added to the control group, with three replicates per group;

[0578] 3) After 20 hours of drug exposure, samples were collected and cells were taken out of the incubator. TM Luciferase Assay: Add 5 μL of reagent to each well and shake gently for 10 min at room temperature in the dark before testing;

[0579] 4) Place the plate on a microplate reader to read the relative light unit (RLU), record the fluorescence value, process the data using Graphpad prism software, and calculate the EC50 of the compound.

[0580] 3. Test results:

[0581] (Compounds were tested at a final concentration of 45 μM, 9 gradients, 5-fold dilution. 1 μM BAF-312 was used as a positive control for S1PR1 and 25 μM BAF-312 was used as a positive control for S1PR4)

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593] Although some S1PR4 modulators have been reported, most of these compounds have unclear mechanisms, no detailed structure-activity relationship analysis, and lack of in vivo pharmacokinetic parameters, making it impossible to evaluate the in vivo effectiveness of these compounds. The inventors used the S1PR1 / 5 agonist BAF312 as a lead compound, and based on the β-arrestin recruitment activity evaluation system of S1PR1 and S1PR4, the polar head and hydrophobic end of BAF312 were modified to synthesize and evaluate a series of compounds. Through a thorough discussion of the S1PR1 / 4 structure-activity relationship of the compound BAF312, the inventors found that replacing the cyclohexyl group of BAF312 with a benzene ring and replacing its trifluoromethyl group with a methyl group to synthesize compound 22 can significantly reduce the activity of S1PR1, but can maintain the activity of S1PR4. Subsequently, the inventors modified the polar head region of compound 22 and found that the introduction of chiral pyrrolidine carboxylic acid further increased the activity of the compound on S1PR4, thus discovering the selective S1PR4 agonist 30. The test results showed that compound 30 had an EC-like activity in recruiting β-arrestin to S1PR4. 50 =9.09nM, then the S1PR1-5 selectivity analysis was performed on the superior compound 30. The test results showed that compared with the endogenous ligand S1P, compound 30 did not show agonist activity on the S1PR2 / 3 / 5 targets, and the selectivity for S1PR1 was nearly 10 times, further proving that compound 30 is a selective S1PR4 agonist, and the agonist activity for S1PR4 is significantly higher than that of the endogenous ligand S1P. At the same time, compound 67 showed better S1PR4 selectivity and activity than compound 30. The activity of compound 67 against S1PR4 reached 1.80nM, and the selectivity for S1PR1 was greater than 100 times.

[0594] Selectivity of compounds 30 and 67 in S1PRs

[0595]

[0596] a β-restin recruitment activity based on Tango-S1PR1-CHO-K1 and Tango-S1PR4-CHO-K1 cells, expressed as EC 50 Values ​​are mean ± SD. b FLIPR assays were performed on S1PR2-CHO and S1PR5-CHO cell lines and expressed as EC 50 Values ​​are mean ± SD. c Based on PathHunter CHO-K1-S1PR3 cell-based β-restin recruitment activity, expressed as EC 50The data are the mean ± SD of at least two independent determinations and are reported as the mean ± SD (standard deviation).

[0597] Example 3. Pharmacokinetic properties of a series of compounds tested

[0598] In the discussion of structure-activity relationship, the inventors obtained the key compound 22, and then modified the polar head of the compound into chiral pyrrolidine carboxylic acid based on 22, and obtained the selective S1PR4 agonists 30 and 67. We analyzed the pharmacokinetic properties of compounds 22, 30 and 67 respectively, and found that compound 22 retained the cyclobutane acid polar head similar to BAF312, but showed dual activity of S1PR1 and S1PR4, and the bioavailability was only 23.5%, and the drugability was poor. The newly discovered compounds 30 and 67 not only showed excellent S1PR4 agonist activity, but also had excellent oral absorption properties, greatly improved in vivo exposure, and oral bioavailability reached 119.7% and 76.21% respectively, which had more drugability potential.

[0599] Pharmacokinetic parameters of compound 22 a

[0600]

[0601] Pharmacokinetic parameters of compound 30 a

[0602]

[0603]

[0604] a SD rats (male, 3 per group) weighing 200-220 g were used for the study.

[0605] Pharmacokinetic parameters of compound 67 a

[0606]

[0607] a SD rats (male, 3 per group) weighing 200-220 g were used for the study.

[0608] Example 4. Internalization function test

[0609] S1P can cause receptor internalization and inhibit the outflow of lymphocytes from secondary lymphoid tissues, thereby reducing the inflammatory response in the body's tissues. This is also the key biological effect of S1P target drugs in treating diseases. Therefore, the degree of receptor recovery after S1PR4 internalization by compound 30 was evaluated, and the recovery effect of compound 30 after receptor internalization was evaluated in cells with high S1PR4 expression. S1PR4-293T cells were treated with 1 μM concentration of S1P, BAF312 and compound 30 at 37°C for 1.5 hours, and then the expression of S1PR4 receptors on the cell surface was detected immediately or at intervals of 5 hours or 20 hours. Figure 1 As shown in the figure, BAF312, as an agonist of S1PR1 and S1PR5, cannot induce S1PR4 receptor internalization because it does not have S1PR4 agonist activity. S1P can induce S1PR4 internalization, and receptor expression is restored after 20 hours. The selective S1PR4 agonist 30 has a sustained internalization ability for S1PR4 for up to 20 hours, proving that compound 30 is a strong selective S1PR4 agonist. The recovery effect of compound 30 after inducing S1PR4 internalization is shown in the figure. Figure 1 shown.

[0610] Example 5. In vivo efficacy test of compound 30 in systemic lupus erythematosus (SLE)

[0611] Systemic lupus erythematosus (SLE) is a typical autoimmune disease that affects multiple organs and tissues. Its main characteristics are the production of multiple specific autoantibodies and abnormal activation of the immune system, often accompanied by abnormal coagulation and fibrinolysis. It is more common in young and middle-aged women, with an incidence rate of nearly 70 / 100,000 in my country. The etiology and pathogenesis of SLE have not yet been fully clarified. Existing studies suggest that the pathogenesis is caused by the combined effects of genes, hormones, environment and other comprehensive factors, which induce the accumulation of antigens, the production of autoantibodies, and immune overactivation, breaking the immune balance and causing disorders in self-tolerance function, thus leading to organ damage and inflammatory reactions, accompanied by abnormal immune cells. High activation of T and B lymphocytes is considered to be a key factor in the onset of autoimmune diseases. Activated T lymphocytes secrete related cytokines that react to B lymphocytes, causing them to proliferate and differentiate to produce autoantibodies, form immune complexes, and deposit in organs and tissues throughout the body, causing inflammatory reactions and immune system dysfunction.

[0612] The kidney is the most commonly affected organ in SLE, which is called lupus nephritis. The kidneys experience proliferation of capillaries, mesangial cells and matrix, infiltration of inflammatory cells, deposition of immune complexes, etc., which in turn lead to renal interstitial fibrosis, glomerular sclerosis and abnormal renal function.

[0613] There is no effective cure for SLE. Currently, the clinical treatment of SLE is still large doses of glucocorticoids. Although it can control the progression of the disease to a certain extent, it cannot cure it. Long-term medication can easily cause the body to develop drug resistance. The adverse reactions of the drugs are serious, and common complications will seriously affect the quality of life of SLE patients.

[0614] The S1PR4 agonist compound 30 designed and synthesized by the inventors has shown good therapeutic effects on a variety of inflammatory and immune-related diseases. On this basis, the inventors used lupus erythematosus MRL / lpr model mice to evaluate the efficacy of the compound. The results showed that after 8 weeks, the weight of the mice in the SLE model group increased significantly (indicating renal damage), and mice died. Compound 30, administered continuously for 8 weeks, can prevent the animals from gaining weight and reduce the mortality rate of animals in a dose-dependent manner, and its effect is better than prednisolone (such as Figure 2 shown).

[0615] The study of SLE phenotype showed that compound 30 could significantly reduce the subcutaneous nodules in SLE mice and make the nodules disappear after a period of treatment. It also significantly reduced the level of serum antinuclear antibody dsDNA, which was better than prednisolone and showed good dose dependence (such as Figure 3 shown).

[0616] Immunological studies have shown that the number of Th1 and Th17 cells in the spleen of SLE mice was significantly higher than that in the normal control group. Compound 30 can significantly reduce the number of Th1 and Th17 positive cells, the Th1 / Th2 and Th17 / Treg ratios, suggesting that its therapeutic effect is related to regulating the number and function of T lymphocytes (such as Figure 4 shown).

[0617] Considering that lupus nephritis is the most commonly damaged organ in SLE, the inventors performed HE and PAS staining on the kidneys of each group of mice. The results showed that SLE mice had severe renal interstitial inflammatory cell infiltration, mesangial cell hyperplasia and basement membrane thickening; after 8 weeks of treatment with compound 30, renal interstitial inflammatory cell infiltration was significantly reduced, and the pathological changes of mesangial cell hyperplasia and basement membrane thickening were alleviated, and the effect was better than prednisolone (such as Figure 5 shown).

[0618] In summary, compound 30 has a good efficacy against SLE, and its efficacy is better than that of the positive drug prednisolone. As a selective agonist of S1PR4, the target and mechanism of action of compound 30 are different from those of the currently used clinical therapeutic drugs. It is an immunomodulator with a new target and is expected to become a drug with a new target and new mechanism for the treatment of lupus erythematosus.

[0619] Example 6. Evaluation of the efficacy of compound 30 on mice with acute / chronic ulcerative colitis

[0620] UC is an inflammatory bowel disease (IBD) that is common in adults aged 30 to 40. The disease mainly starts in the rectum and extends to the sigmoid colon, and is prone to recurrence. The main clinical manifestations include diarrhea, abdominal pain, mucus and blood in the stool, etc. The pathological features are mainly inflammatory cell infiltration and ulcers. In severe cases, it can lead to colon cancer.

[0621] The etiology and pathogenesis of ulcerative colitis remain unclear. Persistent intestinal infection, intestinal mucosal barrier defects, abnormal intestinal mucosal immune regulation, genetics and environment are all involved in the disease process. Immune factors are recognized as an extremely important factor in the pathogenesis of IBD. As an important component of the barrier, the intestinal epithelium plays an important hub role between immune defense and immune activation, maintaining mucosal homeostasis through various mechanisms to protect the host. Under normal physiological conditions, molecular communication between epithelial cells, immune cells and interstitial cells maintains epithelial cell regeneration and mucosal healing. Various types of cells, including antigen presenting cells, helper T cells, regulatory T cells and natural killer T cells, play a vital role in the pathogenesis of UC by regulating, maintaining or inhibiting inflammation.

[0622] Commonly used drugs for the treatment of UC include 5-aminosalicylic acid, glucocorticoids and immunosuppressants. Although these drugs can effectively reduce disease activity, they all have their own limitations and potential complications. For example, glucocorticoids have serious side effects and extensive contraindications and are not suitable for long-term use, while biological agents have the risk of inducing infection and promoting tumors. In addition, with the increase in the incidence of UC in recent years, some patients have poor or no response to traditional drug treatment, resulting in prolonged illness and long-term recurrence, which has prompted people to work hard to develop new treatment methods and methods. Immunosuppressants and biological agents are currently the focus of attention. By constructing acute and chronic ulcerative colitis mouse models, the inventors evaluated the therapeutic effect of S1PR4 agonist compound 30 in the disease. The specific experimental methods and results are shown below.

[0623] 1. Experimental Methods

[0624] 1.1 Acute ulcerative colitis

[0625] The mice were weighed and their blood and stool were observed every day from Day 0 to Day 14 of the experiment, and the scores were recorded according to the following table (Table 1). The mice were randomly divided on Day 1, and the models were established from Day 1 to Day 7. Except for the Control group, each group was given 3.5% DSS water every day, and the mice were re-grouped on Day 8. The Control group and the Model group were not given any medication, but tap water; the other groups were given 30 1mg / kg, 3mg / kg, 9mg / kg, and SASP (sulfasalazine or salicyloylsulfapyridine) 500mg / kg by gavage. Blood and colon were collected on Day 15, the length of the colon was measured, and 1 to 2cm of the colon was fixed with formaldehyde.

[0626] 1.2 Chronic ulcerative colitis

[0627] BALB / c mice were adaptively fed for 7 days before the experiment. The mice were weighed and their blood and stool were observed every day from Day 7 to Day 50. The scores were recorded according to the following table (Table 1). After random grouping, except for the Control group, the other groups were given 2-3% DSS solution to drink freely from Day 7 to Day 14 to establish the model, and they drank normal water from Day 7 to Day 14. 14 days was a cycle, which was repeated 3 times, and the groups were regrouped and the drug administration began at the beginning of the last cycle; the Control group and the Model group were not given the same solvent; the other groups were given 30 1mg / kg, 3mg / kg, 9mg / kg, and SASP 500mg / kg by gavage. Blood and colon were taken on Day 45, the colon length was measured, and 1-2cm of the colon was fixed with formaldehyde.

[0628] 2. Experimental materials include:

[0629] BALB / c mice, male, 20-22g

[0630] Dextran sulfate sodium salt MW: 36000~50000 (DSS)

[0631] 30 1mg / kg, 3mg / kg, 9mg / kg, SASP (500mg / kg) observation index

[0632] 3. Therapeutic effect

[0633] 3.1 Efficacy

[0634] (1) Clinical symptom scoring: During the experiment, the mice were weighed and their blood and stool were observed every day, and the DAI score was calculated and recorded according to the following table (Table 1).

[0635]

[0636] (2) At the end of the experiment, the colon was removed, the length of the colon was measured, and the colon was weighed to calculate the colon weight index (colon weight / body weight × 100%) and colon swelling rate (colon weight / length × 100%).

[0637] 3.2 Pathological morphological changes

[0638] Colonic tissue was taken from 0.5 cm to about 1 cm from the anal verge, and the ileocervical region was taken for histopathological examination. After H&E staining, the tissue was scored under single-blind conditions to assess the level of inflammation and tissue damage in the distal colon; the score was based on the Ameho criteria and was calculated as follows:

[0639] 0 points: no abnormality found in the texture;

[0640] 1 point: mild inflammatory infiltration of mucosa and / or mucosa (mixed neutrophils), edema of the underlying layer, punctate erosion of mucosa, accompanied by capillary hyperplasia. The muscularis mucosa is intact;

[0641] 2 points: 1 point change accounts for more than 50%;

[0642] 3 points: obvious inflammatory infiltration and edema (neutrophils often dominate); usually the ulcer penetrates the muscularis mucosa to the submucosal layer. There is almost no inflammatory cell infiltration in the muscularis propria and no muscle necrosis;

[0643] 4 points: 3 changes account for more than 50%;

[0644] 5 points: large ulcer with coagulative necrosis, with a large number of neutrophil infiltration at the edge and fewer monocytes.

[0645] Necrotic tissue penetrates the muscularis propria;

[0646] 6 points: 5 changes account for more than 50% of the specimens;

[0647] 3.3 Changes in colon mucosal barrier

[0648] Occludin's barrier function can separate the top and bottom sides of the epithelial cell plasma membrane, restrict the free flow of lipids on the cell membrane, and is conducive to the formation of cell polarity, maintaining the impermeability and hardness of the intestinal mucosa, and preventing the entry of pathogenic microorganisms. Occludin's barrier function can regulate the transport of small molecules and maintain tissue homeostasis. The expression and distribution of related proteins were detected by immunohistochemistry;

[0649] 4. Experimental Results

[0650] 4.1 Observation of the efficacy of compound 30 on DSS-induced acute ulcerative colitis in mice

[0651] After 7 days of modeling in this experiment, the mice were regrouped with 8 mice in each group. Starting from the 8th day, mice died one after another. Among them, there was no death in the Control group; 2 mice died in the Model group with a survival rate of 75%; 1 mouse died in the Positive Drug SASP group with a survival rate of 87.5%; 1 mouse died in the 30 1mg / kg group with a survival rate of 87.5%; 1 mouse died in the 30 3mg / kg group with a survival rate of 87.5%; 2 mice died in the 30 9mg / kg group with a survival rate of 75%; compared with the Model group, the administration of positive drugs SASP and 30 could improve the survival rate of UC model mice. The survival curve of acute UC model mice administered with compound 30 is shown in the figure. Figure 6 shown.

[0652] 4.2 Effects of compound 30 on body weight and DAI scores in acute UC model mice

[0653] Body weight: On the 4th day of modeling, the weight of mice began to decrease, and on the 8th day, the weight generally dropped to the lowest point, and then began to rise again; Figure 7 It showed that compared with the control group, the body weight of other groups decreased significantly, indicating that the model was successfully established; compared with the model group, the body weight of the SASP and 30-3mg / kg administration groups recovered faster and the weight recovery was larger;

[0654] DAI score: DAI (Disease Activity Index) score gradually increased with the increase of modeling time, and gradually decreased after stopping modeling. Compared with the model group, the DAI score of SASP and 30 administration groups decreased faster (e.g. Figure 8 shown);

[0655] Sum up the DAI for 14 days (such as Fig. 9 As shown in the figure, compared with the model group, the 30 3 mg / kg group showed a significant decrease, indicating that 30 3 mg / kg can improve the symptoms of weight loss, bloody stools, diarrhea, etc. caused by DSS in mice.

[0656] 4.3 Effects of compound 30 on colon length, weight and swelling rate in acute UC model mice

[0657] UC patients and mouse models often show a shortened colon. Fig.10 The results showed that compared with the Control group, the colon length of the Model group mice was significantly shortened (*p<0.05). 30 1mg / kg and 3mg / kg significantly inhibited the shortening of the colon caused by DSS (30 1mg / kg, *p<0.05; 30 3mg / kg, **p<0.01, vs. Model group), which was basically consistent with the results of the disease activity index. Fig.11The figure is the colon weight, but there is no statistically significant difference. In order to exclude the factor of weight increase caused by colon lengthening, the ratio of colon weight to length is used to represent the degree of colon swelling. The results are as follows Fig.12 As shown, the ratio of colon weight to length in the Model group was significantly increased compared with that in the Control group.

[0658] 4.4 Pathological morphological changes

[0659] Histopathological examination is an important means of clinically diagnosing UC. To further confirm the effect of 30 on UC, we fixed the colon with PFA and then performed paraffin sections and observed the results with H&E staining. The hematoxylin stain in H&E staining is alkaline, which can stain the chromatin in the nucleus and the nucleic acid in the cytoplasm into purple-blue; the eosin stain is acidic, which can stain the cytoplasm and extracellular matrix into red. Through H&E staining, the pathological characteristics of UC such as structural changes in tissues, inflammation, and abscesses can be observed. The results are as follows Fig.13 As shown in the figure, the colon mucosa structure of the mice in the Control group was intact, the intestinal epithelium was intact, a large number of goblet cells were visible and arranged in an orderly manner, and no inflammatory cell infiltration was observed. After DSS modeling, the colon mucosal lamina propria and submucosal of the mice in the model group showed obvious inflammatory cell infiltration, and infiltrated into the muscular layer. The goblet cells were also significantly reduced, the mucosa became thinner, and the mucosal epithelial cells were lost, indicating that the modeling was successful. Although there was inflammatory cell infiltration in the positive drug SASP group, the degree was not as high as that in the Model group, and the goblet cells were more than the Model group and arranged more neatly. Obvious inflammatory cell infiltration and reduced goblet cells were observed in the 30 9mg / kg group. After administration of compound 30 1mg / kg and 3mg / kg, the mucosal structure was normal, the goblet cells increased and were arranged neatly, no obvious inflammatory cell infiltration was observed, and the loss of epithelial cells was also improved. Fig.14 Therefore, overall, compared with the Model group, SASP and compound 30 had a certain improvement effect on colon mucosal injury, but the protective effect of compound 30 1mg / kg and 3mg / kg was better than that of other groups.

[0660] Changes in colon mucosal barrier

[0661] The colon mucosal barrier is the most effective barrier in the intestine. Colon epithelial cells adhere to each other and to the extracellular matrix through adhesion molecules. Colon epithelial cells can be connected together through tight junctions (TJs), adhesion junctions, gap junctions, etc. Among them, TJs are the most important connection method. TJs have two functions: first, the selective barrier function, which can help maintain the selective permeability of the intestinal epithelium and only allow ions and small soluble molecules to pass through; second, the fence function. This special physiological function enables the colon mucosa to not only absorb water, nutrients and molecules required for cell metabolism, but also prevent bacteria and antigens from invading the intestinal cavity. TJs protein is at the top of the cell-cell junction complex. Changes in its expression and distribution can change the level of colon inflammation by regulating colon mucosal homeostasis and intestinal permeability. DSS can cause damage to the colon barrier in mice, but the specific mechanism is unclear. It may be that the cytotoxicity of DSS causes the destruction of TJs in the colon epithelium, and the position of TJs in the colon mucosa is also changed, making it easier for bacteria to penetrate into the intestinal tissue and produce inflammation. Occludin is an important TJ protein. Therefore, immunohistochemistry was used to detect the expression of related proteins.

[0662] The fence function of occludin can separate the apical and basal sides of the epithelial cell plasma membrane, restrict the free flow of lipids on the cell membrane, facilitate the formation of cell polarity, maintain the impermeability and hardness of the intestinal mucosa, and prevent the entry of pathogenic microorganisms. The barrier function of occludin can regulate the transport of small molecules and maintain tissue homeostasis.

[0663] The expression of occludin in colonic mucosa was determined by immunohistochemistry. Fig.15 Compared with the Control group, the expression of Occludin in the Model group was reduced, indicating that the intestinal barrier of mice was damaged after DSS modeling. After SASP and compound 30 treatment, the expression of Occludin increased. Fig.16 Western blotting was used to detect the expression of Occludin protein in the colon. Compared with the Model group, compound 30 could increase the expression of Occludin, which was basically consistent with the results of immunohistochemistry.

[0664] Observation on the efficacy of compound 30 on DSS-induced chronic ulcerative colitis in mice

[0665] 5.1 Effect of compound 30 on body weight of mice with chronic ulcerative colitis

[0666] During the modeling period, the weight of mice in the control group increased steadily; during the first modeling period, the model group was given 3% DSS to drink freely for 7 days and tap water to drink freely for 7 days. On the 4th day of modeling, the weight of mice began to generally decrease, and the weight generally dropped to the lowest point on the 8th day, and then the weight began to recover; during the second modeling period, 2% DSS was given to drink freely for 7 days, and tap water was given freely for 7 days. The weight of mice no longer increased during the 7 days of DSS administration, which was significantly lower than that of the control group, and the weight began to recover after water administration; before the third modeling, the mice in the model group were evenly divided into model, sasp-500mg / kg, 30-1mg / kg, 30-3mg / kg, and 30-9mg / kg groups according to the weight and DAI scores of the mice in the model group, with 11 mice in each group; 2.75% DSS was given to drink freely for 7 days, and tap water was given freely for 7 days. Compared with the model group, the weight loss in the drug-treated group was lighter, and the weight recovered faster after resuming drinking water;

[0667] Compared with the control group, the body weight of other groups decreased significantly, indicating that the model was successfully established; compared with the model group, the body weight of the SASP and 30 administration groups recovered faster and the weight recovery was larger; the body weight of mice was relatively stable during the 30-1mg / kg and 30-3mg / kg administration periods;

[0668] The weight change rate of the model group was significantly lower than that of the control group. The drug-treated group inhibited the weight loss of the model mice to a certain extent. The weight change rate of the 30-3mg / kg drug-treated group was significantly higher than that of the model group. Fig.17 As shown; the changes in body weight of each group after the start of drug administration are as shown Fig.18 The weight change rate of each group before and after administration is shown as Fig.19 shown.

[0669] 5.2 Effect of compound 30 on DAI scores in chronic UC model mice

[0670] DAI score: DAI (Disease Activity Index) score gradually increased with the increase of modeling time, and gradually decreased after stopping modeling. Compared with the model group, the DAI score of the SASP and 30 administration groups decreased faster;

[0671] The sum of DAI after 14 days of administration showed a significant decrease in the 30 3 mg / kg administration group compared with the model group, indicating that 30 3 mg / kg can improve DSS-induced symptoms in mice such as weight loss, bloody stools, and diarrhea.

[0672] The daily DAI scores of chronic UC models during the modeling period were as follows Fig. 20 As shown; the daily DAI scores of the chronic UC model during the administration period are as follows Fig.21As shown; the total daily DAI scores of the chronic UC model in each group during the administration period are as follows Fig. 22 shown.

[0673] 5.3 Effects of compound 30 on colon length, weight and swelling rate in chronic UC model mice

[0674] UC patients and mouse models often show a shortened colon. Fig.23 In the experiment, compared with the Control group, the colon length of the Model group mice was significantly shortened, and compound 30 3mg / kg and 9mg / kg significantly inhibited the shortening of the colon caused by DSS. Fig.24 This is a colon weight chart. In order to exclude the factor of weight increase caused by colon lengthening, the ratio of colon weight to length is used to represent the degree of colon swelling. The results are shown in Figure 2. Fig.25 As shown, the ratio of colon weight to length in the Model group was significantly increased compared with the Control group;

[0675] 5.4 Pathological changes

[0676] Histopathological examination is an important means for clinical diagnosis of UC. To further confirm the effect of 30 on UC, the inventors fixed the colon with PFA and then performed paraffin sections, and observed the results by H&E staining. The hematoxylin stain in H&E staining is alkaline, which can dye the chromatin in the nucleus and the nucleic acid in the cytoplasm into purple-blue; the eosin stain is acidic, which can dye the cytoplasm and the extracellular matrix into red. Through H&E staining, the pathological characteristics of UC such as structural changes in tissues, inflammation, and abscesses can be observed.

[0677] The results are as follows Fig.26 As shown in the figure, the colon mucosa structure of the mice in the Control group was intact, the intestinal epithelium was intact, a large number of goblet cells were visible and arranged in an orderly manner, and no inflammatory cell infiltration was observed. After chronic DSS modeling, the colon mucosal lamina propria and submucosal of the mice in the model group showed obvious inflammatory cell infiltration, and infiltrated into the muscular layer. The goblet cells were also significantly reduced, the mucosa became thinner, and the mucosal epithelial cells were lost, indicating that the modeling was successful. Although there was inflammatory cell infiltration in the positive drug SASP group, the degree was not as high as that in the Model group, and the goblet cells were more than those in the Model group, and the arrangement was also more orderly. After administration of 1mg / kg and 3mg / kg, the mucosal structure was normal, the goblet cells increased and were arranged in an orderly manner, no obvious inflammatory cell infiltration was observed, and the loss of epithelial cells was also improved. Fig. 27 The pathological scores of each group are shown in Table 1. Therefore, overall, compared with the Model group, SASP and 30 had a certain improvement effect on colon mucosal injury, but the protective effect of 30 1mg / kg and 3mg / kg was better than that of other groups.

[0678] Changes in colon mucosal barrier

[0679] The colon mucosal barrier is the most effective barrier in the intestine. Colon epithelial cells adhere to each other and to the extracellular matrix through adhesion molecules. Colon epithelial cells can be connected together through tight junctions (TJs), adhesion junctions, gap junctions, etc. Among them, TJs are the most important connection method. TJs have two functions: first, the selective barrier function, which can help maintain the selective permeability of the intestinal epithelium and only allow ions and small soluble molecules to pass through; second, the fence function. This special physiological function enables the colon mucosa to not only absorb water, nutrients and molecules required for cell metabolism, but also prevent bacteria and antigens from invading the intestinal cavity. TJs protein is at the top of the cell-cell junction complex. Changes in its expression and distribution can change the level of colon inflammation by regulating colon mucosal homeostasis and intestinal permeability. DSS can cause damage to the colon barrier in mice, but the specific mechanism is unclear. It may be that the cytotoxicity of DSS causes the destruction of TJs in the colon epithelium, and the position of TJs in the colon mucosa is also changed, making it easier for bacteria to penetrate into the intestinal tissue and produce inflammation. Occludin is an important TJ protein. Therefore, immunohistochemistry was used to detect the expression of related proteins.

[0680] The fence function of occludin can separate the apical and basal sides of the epithelial cell plasma membrane, restrict the free flow of lipids on the cell membrane, facilitate the formation of cell polarity, maintain the impermeability and hardness of the intestinal mucosa, and prevent the entry of pathogenic microorganisms. The barrier function of occludin can regulate the transport of small molecules and maintain tissue homeostasis.

[0681] The expression of occludin in colonic mucosa was determined by immunohistochemistry. Fig.28 and 29 Compared with the Control group, the expression of Occludin in the Model group was reduced, indicating that the intestinal barrier of mice was damaged after DSS modeling. After SASP and 30 treatment, the expression of Occludin increased.

[0682] Example 7. Efficacy of Compounds 30 / 38 / 53 / 67 / 77 / 80 in Acute UC Model in Mice

[0683] In addition to compound 30, the present inventors further evaluated several other S1PR4 compounds 38, 53, 67, 77, and 80, further demonstrating the application potential of S1PR4 agonists in the treatment of ulcerative colitis.

[0684] DSS was used to model mice, with the method of 2.5% DSS drinking water for 5 days + free drinking water for 2 days. Gavage was performed at the same time as modeling, and RPC1063 was used as a positive drug. The specific grouping was as follows: RPC1063 was gavaged at 1 mg / kg. The dosage of compound 30 was 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Compound 38 was 5 mg / kg, compound 53 was 1 mg / kg, compound 67 was 1 mg / kg, compound 77 was 1 mg / kg, and compound 80 was 1 mg / kg.

[0685] Experimental results (such as Fig.30 The results show that the weight loss of compound 67 was milder, and the recovery of compound 30 was concentration-dependent. The DAI score showed that compound 30 and compound 67 had better effects. In terms of colon length, compound 30 and compound 67 had better effects, and the positive drug had no obvious effect.

[0686] Example 8. Efficacy of Compound 30 in a Rat Osteoarthritis (OA) Model

[0687] 1. Animals and main reagents:

[0688] Experimental Animals:

[0689] SD rats, about 200 g, male, purchased from Shanghai Bikai Animal Co., Ltd. Adaptive feeding for about 7 days.

[0690] Experimental drug: Compound 30.

[0691] 2. Experimental Grouping:

[0692]

[0693] 3. Experimental plan:

[0694] 3.1. Modeling method:

[0695] 3.1.1. Anesthesia: Place the rat in the anesthesia box, turn on the O2 gas source and gas delivery pump, and rotate the gas adjustment knob to make the ratio of O2 and isoflurane gas 8:2. Adjust the anesthesia depth knob, first point the pointer to 3, and deeply anesthetize the rat; during the subsequent surgery, adjust the pointer to 1.5 to maintain anesthesia. Open the three-way valve to allow gas to enter the anesthesia box and the rat anesthesia mask.

[0696] 3.1.2. Needle insertion: Use 75% ethanol to disinfect the skin of the right knee of the rat, bend it 90°, touch the knee joint of the rat with your hand to find the patella, slowly feel the position of the tibial platform from the lower edge of the patella to the inner side of the joint, and insert the needle along the lateral side of the patellar ligament at a 45° angle between the needle tip of the syringe and the platform, as shown in the figure below. The control group was injected with 50μl of 0.9% sterile saline, and the other groups were injected with 50μl of sterile saline containing 3mg of MIA. After the operation was completed, the rat was removed and placed in a cage, and irradiated with an incandescent lamp to help the rat restore its body temperature until the rat woke up from anesthesia.

[0697] 3.2. Drug preparation:

[0698] The control group was given 5% DMSO + 35% PEG400 + 60% saline. The compound was dissolved in 5% dimethyl sulfoxide (DMSO), 35% polyethylene glycol 400 (PEG400) and 60% saline. The drug solution was prepared the night before administration.

[0699] 3.3. Drug administration

[0700] The rats were allowed to grow adaptively for 1 week and randomly divided into 10 groups. The control group was given 5% DMSO + 35% PEG400 + 60% saline, and the other groups were given the test compound, with the solvent being 5% DMSO + 35% PEG400 + 60% saline.

[0701] 3.4. Experimental process: as above Fig.31 shown.

[0702] 3.4.1. Rats were given drugs by gavage once a day from day 1 to day 14 of the experiment, with a gavage volume of 5 mL / kg. The control group and the model group were given 5% DMSO + 35% PEG400 + 60% saline during the entire experiment, and the other groups were given corresponding drugs.

[0703] OA Disease Activity Index Score

[0704] The rats were tested for behavior and joint pain on the 7th and 14th days of the experiment. To reduce interference factors, the test time was fixed at 1 hour after administration to prevent the different states of rats at different time periods from affecting the experimental results; the rats were allowed to adapt to the environment for 20 minutes before the start of the experiment.

[0705] (1) Observation of walking distance and number of standing in five minutes: Measure the size of the resin box, make a piece of paper of the corresponding size, and draw a 1cm×1cm grid on the paper. Seal the paper with transparent tape to prevent it from being damaged by the rat's excrement. Then place the paper with a 1cm×1cm grid in a transparent resin box to ensure that the bottom is flat. Use a video camera to record the walking process and trajectory of the rat within 5 minutes, and count the distance the rat walks and the number of times it stands.

[0706] (2) Joint pain test: Fix the rat and wait until its mood is stable. Gently bend the knee joint of the rat. If the rat does not react, it is scored as 0 points. If the rat retreats briefly and obviously or screams, it is scored as 1 point. The scores of leg retraction and screaming reflect the degree of knee joint pain of the rat. Perform joint pain test every 5 seconds for a total of 5 times.

[0707] 3.4.3. Tissue collection

[0708] On the 15th day of the experiment, the rats were anesthetized and blood was collected from their eyeballs. The serum was kept at 4°C overnight. The rats were centrifuged at 4°C and 3600rpm for 15 minutes. The serum was aspirated and stored at -80°C. The rats were killed by dislocating the neck, and the rats were cut open along the knee joints on ice to remove the fur, muscle and other tissues. Five rats were selected from each group, and the knee joints on both sides were peeled off to observe the color, roughness and integrity of the femoral condyle surface. The knee cartilage was then taken and frozen at -80°C. The joint specimens of the remaining five rats were fixed in formaldehyde fixative.

[0709] 3.4.4. Safranin-Fast Green Staining

[0710] The inventors further evaluated the pathological changes of the knee joints of OA model rats using safranin-fast green staining and OARSI scoring. Safranin is alkaline and can bind to acidic proteoglycans to dye the cartilage matrix red; fast green is acidic, and the amino acids rich in collagen fibers are alkaline, so fast green can dye the superficial cartilage and subchondral bone green. The proteoglycans in the cartilage will be released after the cartilage is damaged, resulting in the scattered arrangement of the uniformly dispersed matrix and the loss of safranin staining, so safranin can indirectly reflect the content of proteoglycans in the matrix.

[0711] OARIS Scoring

[0712] The lesions in the rat knee joints were observed under a microscope. The severity of cartilage damage was assessed using the OARIS Osteoarthritis Cartilage Histopathology Assessment System (OOCHAS) grading system (grading = 6, score = 0-24) (see table) to determine whether the drug can alleviate the severity of osteoarthritis.

[0713]

[0714]

[0715] 4. Experimental results:

[0716] 4.1. Before modeling, rats ate and drank normally, and their fur was shiny. After MIA injection modeling, when OA modeling was performed, the knee joints of rats injected with MIA in the joint cavity began to swell from the injection site, and the swelling gradually spread to the entire knee joint over time. The joints became red, swollen, hot and painful, and the rats' movements decreased. The left hind limbs flexed and limped, and the spirits were depressed. As time went on, the degree of swelling decreased.

[0717] 4.2. Pain index:

[0718] On DAY7, compared with the Model group, the pain index of rats in the diclofenac sodium-2.5 mg / kg, compound 30-0.3 mg / kg, and compound 30-3 mg / kg treatment groups were significantly reduced compared with the Model group.

[0719] On DAY14, compared with the Mode group, the pain index of rats in the compound 30-1 mg / kg and compound 30-3 mg / kg treatment groups were significantly lower than that in the Model group.

[0720] Pain index statistics Fig.32 shown.

[0721] 4.3. Distance walked and number of times standing upright in five minutes:

[0722] On DAY7, compared with the Model group, the walking distances of rats in the diclofenac sodium-2.5 mg / kg, compound 30-0.3 mg / kg, and compound 30-1 mg / kg treatment groups were significantly increased.

[0723] On DAY14, compared with the Model group, the walking distance of the rats in the Compound 30-0.3 mg / kg and Compound 30-3 mg / kg treatment groups increased significantly. The 5-minute walking distance statistics are as follows: Fig.33 shown.

[0724] On DAY7 and DAY14, there was no statistical difference in the number of upright standing times compared with the Model group. Fig.34 shown.

[0725] 4.4. Five rats were sacrificed in each group, and the knee joints were dissected to expose the femoral condyle, photographed and observed. We made an overall observation of the color, roughness and integrity of the femoral condyle surface of the rat knee joint. The results are as follows: Fig.35 As shown:

[0726] There was no hyperplasia of the synovial bursa in the joint cavity of rats in the Control group, and the cartilage surface was smooth, complete, bright in color, and without cracks; no osteophytes were formed on the joint edges.

[0727] In the Model group, the articular cartilage surface of the rats was slightly rough, partially eroded, with small cracks and a grayish color; the articular cartilage lost its color, the surface was worm-eaten, and osteophytes formed on the edges.

[0728] After treatment with diclofenac sodium and compound 30, the cartilage of the knee joints of most rats was smooth and bright, and the wear was significantly improved. Although some cartilage was slightly worm-eaten, no obvious osteophyte formation was observed. Therefore, it can be considered that compound 30 can alleviate the cartilage lesions in the knee joints of OA model rats.

[0729] Effects of compound 30 on safranin fast green staining and OARSI scores of knee joints in OA rats

[0730] from Fig.36 It can be seen that the cartilage matrix of the Control group is bright red; the superficial cartilage and subchondral bone are stained green with fast green. The cartilage structure is intact, with flat and round chondrocytes in the surface and middle layers, respectively, and chondrocyte proliferation in the deep layer. In the Model group, a small amount of safranin staining faded and partially lost staining; the surface chondrocytes became swollen, the number decreased, and the arrangement was disordered; the tide line extended irregularly, the boundary was blurred, and partly disappeared.

[0731] After treatment with diclofenac sodium and compound 30, the knee joints of OA model mice improved, safranin staining was enhanced, the number of chondrocytes increased, and the arrangement was more regular and neat.

[0732] Next, the inventors used OARIS to evaluate the severity of osteoarthritis in each group of rats. Fig.36 As shown, the score of the Model group was significantly higher than that of the Control group, and after treatment with diclofenac sodium and compound 30, the rats' knee osteoarthritis was significantly lighter than that of the Model group. The rats in the compound 30-0.3mg / kg, compound 30-1mg / kg, and compound 30-3mg / kg treatment groups were significant and could well alleviate the damage to the knee joints of OA rats. This shows that compound 30 can delay the progression of OA.

[0733] Experimental results:

[0734] The inventors established a rat OA model by intra-articular injection of MIA and studied the efficacy of compound 30 on the model rats. It was found that compound 30 could improve the walking ability of rats with joint injuries and reduce the pain of rats. When observing the lesion site, it was found that compound 30 could improve the wear, erosion and fracture of the knee cartilage in OA rats.

[0735] in conclusion:

[0736] (1) The experimental results show that the walking distance of rats in the treatment group of compound 30 increased significantly. It showed good therapeutic effect in the MIA-induced OA model of rats, and could effectively relieve the pain of rats and improve their walking state.

[0737] (2) The macroscopic observation results showed that compound 30 treatment group could effectively alleviate the damage of knee joint in OA rats.

[0738] (3) The results of pathological sections and OARSI scores showed that the compound 30 treatment group could effectively alleviate the damage to the knee joint of OA rats.

[0739] Example 9. Efficacy of Compound 67 in Rat Osteoarthritis (OA) Model

[0740] The rats were allowed to grow adaptively for 1 week and randomly divided into groups, with 10 rats in each group. The model was induced using monoiodoacetic acid (MIA). The control group was given 5% DMSO + 35% PEG400 + 60% saline, and the other groups were given the test compound, with the solvent being 5% DMSO + 35% PEG400 + 60% saline. The specific grouping of the drug group is as follows, with diclofenac sodium 2.5 mg / kg as the positive control group. Compound 67 was divided into 3 dose groups, namely 0.3 mg / kg, 1 mg / kg and 3 mg / kg.

[0741] Before modeling, the rats ate and drank normally, and their fur was shiny. After MIA injection modeling, when OA modeling was performed, the knee joints of rats injected with MIA in the joint cavity began to swell from the injection site, and the swelling gradually spread to the entire knee joint over time. The joints became red, swollen, hot and painful, and the rats' movements decreased, the left hind limbs flexed, limped, and the spirits were depressed. As time went on, the degree of swelling decreased.

[0742] The pain index of compound 67 is as follows Fig.37 shown.

[0743] On DAY7, compared with the Model group, the pain index of rats in the diclofenac sodium-2.5 mg / kg, compound 67-0.3 mg / kg, compound 67-1 mg / kg, and compound 67-3 mg / kg treatment groups were significantly reduced compared with the Model group.

[0744] On DAY14, compared with the Mode group, the pain index of rats in the compound 67-1 mg / kg and compound 67-3 mg / kg treatment groups were significantly lower than that in the Model group.

[0745] Distance walked and number of times standing upright in five minutes Fig.38 and 39 As shown:

[0746] The results of the five-minute walking distance experiment of rats showed that on DAY7, compared with the Model group, the walking distance of rats in the diclofenac sodium-2.5mg / kg and compound 67-1mg / kg treatment groups increased significantly.

[0747] On DAY14, compared with the Model group, the walking distances of rats in the compound 67-0.3 mg / kg, 67-1 mg / kg, and 67-3 mg / kg treatment groups increased significantly.

[0748] The results of the five-minute upright standing test of rats showed:

[0749] On DAY7 and DAY14, there was no statistically significant difference in the number of upright postures compared with the Model group.

[0750] Five rats were sacrificed in each group, and the knee joints were dissected to expose the femoral condyle, photographed and observed. The color, roughness and integrity of the femoral condyle surface of the rat knee joint were observed overall.

[0751] The results are as follows Fig.40 As shown: The joint bursa of rats in the Control group did not proliferate, and the cartilage surface was smooth, complete, bright in color, and without cracks; no osteophytes formed on the joint edges. In the Model group, the articular cartilage surface of rats was slightly rough, partially eroded, with small cracks and a dark color; the articular cartilage lost its color, the surface was worm-eaten, and osteophytes formed on the edges.

[0752] After treatment with diclofenac sodium and compound 67, the cartilage of the knee joints of most rats was smooth and bright, and the wear was significantly improved. Although some cartilage was slightly eroded, no obvious osteophyte formation was observed. Therefore, compound 67 can alleviate the cartilage lesions in the knee joints of OA model rats.

[0753] Effects of compound 67 on serum cytokines in OA rats Fig.41 As shown:

[0754] The level of anti-inflammatory factor IL-10 in the serum of rats in the Model group was reduced. Compared with the Model group, the level of IL-10 in the diclofenac sodium group was significantly increased, and compound 67 had a tendency to upregulate the levels of IL-10 and IL-17.

[0755] There was no difference in the level of anti-inflammatory factor IL-17 in the serum of rats in each group.

[0756] Effects of compound 67 on safranin fast green staining and OARSI scores of knee joints in OA rats Fig.42As shown: In the figure, the cartilage matrix of the Control group is bright red; the superficial cartilage and subchondral bone are stained green with fast green. The cartilage structure is intact, with flat and round chondrocytes in the surface and middle layers respectively, and chondrocyte proliferation can be seen in the deep layer. In the Model group, a small amount of safranin staining faded and partially lost staining; the surface chondrocytes became swollen, the number decreased, and the arrangement was disordered; the tide line extended irregularly, the boundary was blurred, and partly disappeared.

[0757] After treatment with diclofenac sodium and compound 67, the knee joints of OA model mice improved, safranin staining was enhanced, the number of chondrocytes increased, and the arrangement was more regular and neat.

[0758] Next, the severity of osteoarthritis in each group of rats was assessed using the OARIS score. Fig.42 As shown, the score of the Model group was significantly higher than that of the Control group, and the knee osteoarthritis of rats treated with diclofenac sodium and compound 67 was significantly lighter than that of the Model group. The rats in the compound 67-1mg / kg and 3mg / kg treatment groups had significant effects and could well alleviate the damage to the knee joints of OA rats.

[0759] The inventors established an OA model in rats by injecting MIA into the knee joint cavity and studied the efficacy of compound 67 on the model rats. It was found that compound 67 can improve the walking ability of rats with joint injuries and reduce the pain of rats. When observing the lesion site, it was found that compound 67 can improve the wear, erosion and fracture of the knee cartilage in OA rats.

[0760] in conclusion:

[0761] (1) The experimental results show that the walking distance of rats in the treatment group of compound 67 increased significantly. It showed good therapeutic effect in the MIA-induced OA model of rats, and could effectively relieve the pain of rats and improve their walking state.

[0762] (2) The results of gross observations showed that compound 67 treatment could effectively alleviate the damage to the knee joints of OA rats.

[0763] (3) The results of pathological sections and OARSI scores showed that the compound 67 treatment group could effectively alleviate the damage to the knee joint of OA rats.

[0764] (4) Elisa results showed that the compound 67 treatment group had a tendency to upregulate IL-10.

[0765] In summary, as a S1PR4 selective agonist, compound 67 has significant efficacy in the treatment of rat osteoarthritis model.

[0766] Example 10. Evaluation of the efficacy of compound 67 in systemic lupus erythematosus (SLE) MRL / Lpr model mice The efficacy of compound 67 was evaluated using systemic lupus erythematosus MRL / Lpr model mice.

[0767] The specific groups were: MPJ control group, model group, positive drug Cenerimod group 0.5 mg / kg, and compound 67 doses were divided into two groups of 1 and 3 mg / kg.

[0768] The statistical results of body weight and survival rate within 8 weeks are shown in Fig.43 .

[0769] MAU refers to urinary microalbumin, which reflects abnormal protein leakage from the kidneys. MAU determination can reflect early kidney disease and kidney damage. The results showed that after 8 weeks, the urinary microalbumin level of mice in the SLE model group increased (indicating kidney damage). Compound 67, administered continuously for 8 weeks, significantly reduced the urinary microalbumin level of mice, and its effect was better than that of the Cenerimod 0.5 mg / kg group (such as Fig.44 shown).

[0770] Anti-ds-DNA antibody (IgG) has a high sensitivity to SLE and is a diagnostic indicator and activity marker for SLE. The results showed that after 8 weeks, the level of anti-ds-DNA antibody in mice in the SLE model group increased significantly. Compound 67, administered continuously for 8 weeks, significantly reduced the level of anti-ds-DNA antibody in mice (e.g. Fig.45 shown).

[0771] Serum creatinine is a product of human muscle metabolism. When renal function is normal, most of the creatinine is filtered out from the glomerulus, so it can be used as a diagnostic and screening indicator for glomerular filtration function. When glomerular filtration function decreases, serum creatinine increases due to retention in the blood. The results showed that after 8 weeks, the serum creatinine level of mice in the SLE model group increased significantly. Compound 67, which was administered continuously for 8 weeks, significantly reduced the serum creatinine level of mice (e.g. Fig.46 shown).

[0772] Immunological studies have shown that the number of Th1 and Th17 cells in the spleen of SLE mice was significantly higher than that in the normal control group. Compound 67 can significantly reduce the number of Th1 and Th17 positive cells, the ratio of Th1 / Th2 and Th17 / Treg, suggesting that its therapeutic effect is related to regulating the number and function of T lymphocytes (such as Fig.47 shown).

[0773] Since lupus nephritis is the most commonly damaged organ in SLE, we performed HE and PAS staining on the kidneys of mice in each group. The results showed that the SLE model group had severe inflammatory cell infiltration, mesangial cell proliferation, basement membrane thickening, and capillary endothelial cell proliferation; after 8 weeks of treatment with compound 67, inflammatory cell infiltration was significantly reduced, mesangial cell proliferation, basement membrane thickening, and capillary endothelial cell proliferation were improved, and the effect was better than that of the Cenerimod 0.5 mg / kg group (such as Fig.48 shown).

[0774] In summary, compound 67 has a good efficacy against SLE and its efficacy is better than that of the positive drug Cenerimod.

[0775] Example 11. Efficacy of Compounds 30 and 67 in ankylosing spondylitis (AS) mouse model

[0776] Ankylosing spondylitis (AS) is a chronic, progressive, inflammatory disease that primarily affects the spine and sacroiliac joints and may cause pain and stiffness in these areas.

[0777] 1. Establishment of AS mouse model and drug administration

[0778] The experiment was conducted after SKG mice were adaptively raised. In addition to 5 mice as blank controls, the other 20 mice were intraperitoneally injected with Curdlan (3 mg / mouse) in the 0th and 2nd weeks of the experiment to establish the AS mouse model, and were randomly divided into 4 groups, namely the model group, the positive control group using adalimumab (10 mg / kg) and compound 30 (3 mg / kg) and compound 67 group (3 mg / kg), with 5 mice in each group. The mice in the compound 30 and 67 groups were gavaged with compounds 30 and 67 daily from week 3.3 until they were killed; the positive control group was intraperitoneally injected with adalimumab injection (the first dose in week 3.3 and the second dose in week 9). The body weight was weighed every week to adjust the dosage.

[0779] 2. Detection indicators

[0780] 2.1 Peripheral clinical symptoms

[0781] The clinical symptoms of the limbs and toes were observed, and the redness and swelling were scored, with a total of 16 points (4 points for each foot).

[0782] Scoring criteria:

[0783] 0 = No swelling

[0784] 1 = Mild swelling and redness on top of foot

[0785] 2 = Severe swelling and redness on the top of the foot

[0786] 3 = Severe swelling and redness of the wrist or ankle

[0787] 4 = Severe swelling of wrists or ankles and toes.

[0788] 2.2 Spinal joint lesions

[0789] At week 13.4 and week 14.3 of the experiment, mice were scanned by CT to obtain imaging data of their bones and joints. Image WL: 1400; WW: 2000.

[0790] Scoring criteria:

[0791] 1. Spine (2 points in total): 1 point for narrowing of the space and 2 points for complete fusion.

[0792] 2. Sacroiliac (1 point on each side, 2 points in total): 0.5 point for blurred and hardened joint surface, and 0.5 point for narrowing of the space.

[0793] 3. Spinal facet joints (1 point on each side, 2 points in total): 0.5 points for narrowing of the gap and 1 point for complete fusion.

[0794] 2.3 Statistical analysis

[0795] IBM SPSS Statistics software was used to perform analysis of variance (ANOVA) to obtain the statistical results of the data set, and P < 0.05 was considered a significant difference.

[0796] 3. Experimental results

[0797] 3.1 Peripheral clinical symptoms

[0798] AS mice began to show symptoms of peripheral inflammation in the 4th week: redness and swelling of the feet, swelling of the toes; redness and swelling of the ears; psoriasis and redness and swelling of the tail; slight hair loss and dermatitis in the nose near the eyes; redness of the ears. The symptoms of the mice in the model group gradually worsened over time. The symptoms of the compound 30 and 67 groups and the positive control (adalimumab) group were weaker than those of the model group mice. The clinical symptom scores showed that compared with the model group, the scores of the compound 30 and 67 groups and the positive control (adalimumab) group were significantly lower than those of the model group at 12 weeks (P<0.05). The results are as follows Fig.49 , 50 Compounds 30 and 67 can also significantly reduce wrist and ankle swelling (such as Fig.51 shown).

[0799] 3.2 Spinal joint lesions

[0800] CT examination results showed that the articular surface of the sacroiliac joint on one side of the model group mice was unclear and had some damage. Obvious fusion could be observed on one side of the upper and lower articular process joints, while the gap on the other side was narrowed and the articular surface was relatively blurred. There was a slight fusion phenomenon in the spine; the joint gap of the spinal articular process was narrowed. The spinal joint lesions in the compound 30 and 67 groups and the positive control (adalimumab) group were weaker than those in the model group mice, and the imaging scores were significantly lower than those in the model group (P<0.05). Fig.52 , 53 shown.

[0801] Conclusion: In the ankylosing spondylitis mouse model, compounds 30 and 67 can achieve the same therapeutic effect as the positive drug adalimumab or even surpass adalimumab in terms of reducing clinical scores and alleviating bone and joint pathological changes. This fully proves that compounds 30 and 67 have therapeutic effects on ankylosing arthritis.

[0802] Example 12: Evaluation of the efficacy of compounds 30 and 67 in a mouse psoriasis model

[0803] The Imiquimod-induced mouse psoriasis model is a commonly used model for psoriasis research. This model uses the topical immunomodulator Imiquimod (IMQ) to induce lesions similar to human psoriasis on the mouse skin.

[0804] Experimental Materials

[0805] Mice: BALB / c mice, 6-8 weeks old, female.

[0806] Imiquimod cream: 5% IMQ cream.

[0807] Experimental procedures

[0808] Prepare mice: Acclimate mice to the laboratory environment for at least one week.

[0809] The mice were randomly divided into groups, each group generally containing 5 mice. The specific grouping was the adalimumab injection group, which was used as a positive control. Compounds 30 and 67 were administered orally at 3 doses of 0.3, 1 and 3 mg / kg.

[0810] Application of Imiquimod cream (molding):

[0811] Each group used a razor to shave the hair on the back of the mice, with an area of ​​approximately 2 x 3 cm. 2 .

[0812] Apply 5% IMQ cream to the shaved area of ​​the mouse back every day, with a dose of 62.5 mg IMQ cream, for 6-7 days. After 7 days, observe the skin reactions of the mice, including erythema, scaling, and skin thickening. The specific experimental results are as follows Fig.54shown.

[0813] In terms of clinical manifestations, in the imiquimod-induced BALB / c female mouse psoriasis model, compounds 30 and 67 had significant alleviating effects at all concentrations, and were dose-dependent. Among them, compounds 30 and 67 had the best efficacy at a high dose of 3 mg / kg, and could achieve a therapeutic effect similar to that of adalimumab. The above results prove that compounds 30 and 67 have the potential to treat psoriasis.

[0814] discuss:

[0815] After extensive and in-depth research, the inventors designed and synthesized a series of small molecule compounds that are S1P receptor modulators that have not been reported in the literature. The obtained compounds were tested for activity at the cellular level, and a batch of compounds that can regulate S1PR4 and S1PR4 mutant proteins were obtained. The in vivo pharmacokinetic parameters of several S1PR4 agonists with excellent activity were determined, and it was found that they have good oral absorption properties and have certain development potential.

[0816] The inventors evaluated the invented S1PR4 agonist compounds in disease models such as ulcerative colitis, systemic lupus erythematosus, osteoarthritis, ankylosing spondylitis, etc., and showed in vivo efficacy in various autoimmune diseases, and were superior to S1PR1 agonists or antibody drugs with the same target.

[0817] The above work lays the foundation for the treatment of autoimmune diseases mediated by S1PR4 receptor.

[0818] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound represented by formula I, or an optical isomer or a pharmaceutically acceptable salt thereof: A is selected from substituted or unsubstituted C5-C8 aromatic ring (such as phenyl) or C8-C 14 A fused aromatic ring (e.g. naphthyl), a substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 3 heteroatoms selected from N or O; R is independently selected from the following group: hydrogen, halogen, cyano, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl or cycloalkenyl, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C5-C8 aryl (e.g., phenyl), substituted or unsubstituted five-membered or six-membered heterocyclic or heteroaryl containing 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, p is an integer of 1-3; R 1 Selected from the following groups: hydrogen, halogen, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C3-C8 lactone, substituted or unsubstituted C1-C 10 An amide group, a substituted or unsubstituted C5-C8 aryl group, a substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O or S, or a substituted or unsubstituted C5-C8 aromatic heterocyclic group; n is selected from an integer of 1 to 3; R 2 Select from the following group: m is selected from an integer of 1 to 4; q is selected from an integer of 1-2.

2. The compound according to claim 1, characterized in that The compound is shown in the following formula II: In the formula, R 1 Selected from the following group: hydrogen, halogen (preferably F, Cl or Br), cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C 10 Alkyl (preferably substituted or unsubstituted C1-C6 alkyl, for example, methyl, ethyl, trifluoromethyl), substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O or S, substituted or unsubstituted C5-C8 aromatic heterocyclic group; R 2 Select from the following group: X is selected from: C or N; R 3 is absent or is a substituent selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S; R 5 Selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S; R 4 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl or cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic or heteroaryl containing 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C1-C 10 an alkylformyl group, or a substituted or unsubstituted arylformyl group.

3. The compound according to claim 1, characterized in that The compound is shown in the following formula III: In the formula, R 1 Selected from the following group: hydrogen, halogen (preferably F, Cl or Br), cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C 10 Alkyl (preferably substituted or unsubstituted C1-C6 alkyl, for example, methyl, ethyl, trifluoromethyl), substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O or S, substituted or unsubstituted C5-C8 aromatic heterocyclic group; R 2 Select from the following group: R 3 A substituent selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S; R 5 Selected from the group consisting of hydrogen, cyano, halogen, substituted or unsubstituted C1-C 10 alkyl (e.g., methyl, trifluoromethyl, trifluoroethyl), substituted or unsubstituted C3-C8 cycloalkyl, cyano, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C1-C 10 Alkylformyl, substituted or unsubstituted C5-C8 arylformyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic group containing 1-2 heteroatoms selected from N, O or S; R 4 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl or cycloalkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted five-membered or six-membered heterocyclic or heteroaryl containing 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C1-C 10 an alkylformyl group, or a substituted or unsubstituted arylformyl group.

4. A compound selected from the group consisting of:

5. The compound according to claim 3, characterized in that In formula III, R 1 Selected from the following group: hydrogen, halogen (preferably F, Cl or Br), cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C6 alkyl (preferably methyl, ethyl, propyl); R 2 for: R 3 and R 5 Independently selected from the following group: hydrogen, substituted or unsubstituted C1-C6 alkyl (preferably methyl, ethyl or propyl), halogen (preferably F); R 4 Selected from the following group: substituted or unsubstituted C5-C8 aryl (preferably phenyl or F-substituted phenyl), substituted or unsubstituted five-membered or six-membered heterocyclic group or heteroaryl group containing 1-2 heteroatoms selected from N, O or S.

6. A compound selected from the group consisting of:

7. The compound according to claim 3, characterized in that In formula III, R 1 Selected from the following group: cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C6 alkyl (preferably methyl, ethyl, propyl); R 2 for: R 3 and R 5 Independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C3 alkyl (preferably methyl, ethyl or propyl), halogen (preferably F); R 4 Selected from the following group: substituted or unsubstituted C5-C8 aryl (preferably phenyl or F-substituted phenyl), substituted or unsubstituted five-membered or six-membered heteroaryl containing 1-2 heteroatoms selected from N, O or S.

8. A compound selected from the group consisting of:

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8, or an optical isomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

10. Use of the compound according to any one of claims 1 to 8, or an optical isomer or a pharmaceutically acceptable salt thereof in the preparation of an S1PR modulator. In a preferred embodiment, the S1PR modulator is an S1PR4 selective agonist or an S1PR4 mutant selective agonist.

11. A method for regulating S1PR in a subject, the method comprising the step of administering an effective amount of the compound of any one of claims 1 to 8, or an optical isomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 9 to a subject in need thereof.

12. The compound according to any one of claims 1 to 8, or an optical isomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9, for use in treating or preventing a disease mediated by S1PR4.

13. A medicament for treating or preventing a disease mediated by S1PR4, comprising the compound according to any one of claims 1 to 8, or an optical isomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9.

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