S1PR4 modulator compounds for myopathies treatment and uses thereof

Abstract

The invention relates to a compound as shown in a formula I and application of the compound as an S1PR regulator, in particular to application of a selective S1PR4 agonist in myopathy treatment. The compound can be used for treating related diseases mediated by S1PR4 and a mutant thereof.

Description

S1PR4 modulator compounds for the treatment of myopathy and their applications 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 myopathy. Background Art

[0002] Muscular dystrophy is a progressive muscle disease caused by genetic mutations and characterized by chronic oxidative stress and inflammation. Clinically, it manifests as progressive muscle weakness that affects the limbs, throat, and facial muscles to varying degrees. Based on the onset of disease, it can be divided into congenital and late-onset muscular dystrophy. Generally, congenital muscular dystrophy presents with obvious weakness at birth or in the first few months of life. In contrast, late-onset muscular dystrophy does not manifest until patients achieve independent walking. Muscular dystrophy has been found to cause multiple mutations in proteins involved in skeletal muscle development and maintenance. These include laminin α2, fukutin, and selenoprotein N1. These proteins play important roles in prenatal skeletal muscle development and function, and mutations can lead to congenital muscular dystrophy. Mutations in proteins such as dystrophin, emerin, and lamin A / C, which are involved in skeletal muscle maintenance, can also lead to late-onset muscular dystrophy.

[0003] Current treatment strategies for muscular dystrophy primarily rely on gene therapy and anti-inflammatory therapies. Gene therapy utilizes small molecules or antisense oligonucleotides to inhibit nonsense mutations that cause abnormal transcription termination or induce skipping of specific exons, leading to restoration of the reading frame and production of dystrophin. Alternatively, adeno-associated viruses (AAVs) can be used as vectors to introduce a normal, wild-type gene expressing dystrophin. However, gene therapy is limited by immune responses to the vectors and limited vector packaging size. Furthermore, gene therapy is only effective for patients with specific pathogenic gene variants and is not suitable for all patients with muscular dystrophy. Inflammation is a secondary consequence of the gene variant, and suppressing inflammation has the advantage of being applicable to all subtypes of muscular dystrophy, regardless of the gene variant. Glucocorticoids have been used to ameliorate inflammation levels to treat muscular dystrophy, but their effectiveness is limited and they have significant side effects, necessitating the identification of new inflammatory targets.

[0004] Sphingosine-1-phosphate receptors (S1PRs) belong to the GPCR family and have five subtypes, S1PR1-5, which mediate diverse physiological processes regulated by their endogenous ligand, sphingosine 1-phosphate (S1P). S1PR1 regulates the trafficking of lymphocytes from secondary lymphoid organs to the blood and lymphatic system and is an important and emerging drug target for the treatment of various autoimmune diseases. S1PR4 is associated with the migration and differentiation of immune cells, the migration of skeletal muscle precursor cells, platelet regeneration, and TGFβ1-mediated inhibition of skeletal muscle cell apoptosis. Targeting S1PR4 has promising therapeutic applications in autoimmune diseases, cancer, atherosclerosis, and diseases caused by accelerated platelet aggregation. Furthermore, targeted regulation of S1PR4 also has potential applications in muscle diseases caused by S1PR4 and its mutants.

[0005] Currently, research on myopathies is insufficient. The root causes of different types of myopathies are related to related gene mutations. Therefore, to find drugs to treat myopathies, we must first understand and confirm the target that causes each type of myopathy, and then discover therapeutic drugs based on the mutant protein.

[0006] Therefore, studying myopathy targets and developing therapeutic drugs for them has important clinical significance and application prospects for the treatment of muscle diseases and autoimmune diseases.

[0007] Summary of the Invention

[0008] The object of the present invention is to provide relevant compounds as S1PR modulators, in particular S1PR4 agonists.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising the above compound.

[0010] 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.

[0011] In a first aspect, the present invention provides a compound represented by Formula I, or an optical isomer or a pharmaceutically acceptable salt thereof:

[0012] 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 1-3 heteroatoms selected from N or O;

[0013] R is independently selected from the following groups: hydrogen, halogen, cyano, substituted or unsubstituted C1-C 10alkyl (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;

[0014] R 1 Selected from the group consisting of 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 1-2 heteroatoms selected from N, O or S, or a substituted or unsubstituted C5-C8 aromatic heterocyclic group;

[0015] n is selected from an integer of 1 to 3;

[0016] R 2 Select from the following groups:

[0017] m is selected from an integer of 1 to 4;

[0018] q is selected from an integer of 1-2.

[0019] In a preferred embodiment, the compound is not the following:

[0020] In a specific embodiment, the compound is represented by the following formula II:

[0021] Where,

[0022] R 1 Selected from the group consisting of hydrogen, halogen (preferably F, Cl or Br), cyano, substituted or unsubstituted C1-C5 alkoxy (preferably methoxy), substituted or unsubstituted C1-C 10Alkyl (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 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C5-C8 aromatic heterocyclic group;

[0023] R 2 Select from the following groups:

[0024] X is selected from: C or N;

[0025] 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;

[0026] 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;

[0027] 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.

[0028] In a specific embodiment, the compound is represented by the following formula III:

[0029] Where,

[0030] R 1 Selected from the group consisting of 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 1-2 heteroatoms selected from N, O or S, substituted or unsubstituted C5-C8 aromatic heterocyclic group;

[0031] R 2 Select from the following groups:

[0032] 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;

[0033] 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;

[0034] 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.

[0035] In a specific embodiment, the present invention provides a compound selected from the group consisting of:

[0036] In a specific embodiment, in Formula III,

[0037] 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);

[0038] R 2 for:

[0039] R 3 and R 5 Independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6 alkyl (preferably methyl, ethyl or propyl), halogen (preferably F);

[0040] R 4 Selected from the following groups: 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.

[0041] In a specific embodiment, the present invention provides a compound selected from the group consisting of:

[0042] In a specific embodiment, in Formula III,

[0043] 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);

[0044] R 2 for:

[0045] 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);

[0046] R 4 Selected from the following groups: 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.

[0047] In a specific embodiment, the present invention provides a compound selected from the group consisting of:

[0048] 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.

[0049] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound according to the first aspect, or an optical isomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0050] 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.

[0051] In a preferred embodiment, the pharmaceutical composition is used as an S1PR4 agonist or an S1PR4 mutant agonist.

[0052] In a preferred embodiment, the pharmaceutical composition is used to treat or prevent S1PR4-mediated diseases.

[0053] In a preferred embodiment, the S1PR4-mediated disease comprises a myopathy.

[0054] In a preferred embodiment, the myopathy includes but is not limited to idiopathic inflammatory myopathy, infectious myopathy, drug-induced myopathy, toxic myopathy, hereditary myopathy, and the like.

[0055] In a preferred embodiment, the myopathy includes but is not limited to Duchenne muscular dystrophy, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, distal muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, Emory muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, etc.

[0056] In a preferred embodiment, the myopathy includes but is not limited to sepsis-induced myopathy or sepsis-acquired muscle weakness, especially atrophy of the gastrocnemius and tibialis anterior muscles.

[0057] In a third aspect, the present invention provides use of the compound described in the first aspect, or an optical isomer or 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.

[0058] In a preferred embodiment, the S1PR modulator is an S1PR4 agonist or an S1PR4 mutant agonist.

[0059] In a preferred embodiment, the S1PR4 agonist or S1PR4 mutant agonist is a drug for treating or preventing S1PR4-mediated diseases.

[0060] In a preferred embodiment, the S1PR4-mediated disease comprises a myopathy.

[0061] In a preferred embodiment, the myopathy includes but is not limited to idiopathic inflammatory myopathy, infectious myopathy, drug-induced myopathy, toxic myopathy, hereditary myopathy, and the like.

[0062] In a preferred embodiment, the myopathy includes but is not limited to Duchenne muscular dystrophy, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, distal muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, Emory muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, etc.

[0063] In a fourth aspect, the present invention provides a method for regulating S1PR in a subject, 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.

[0064] 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.

[0065] In a preferred embodiment, the method is used to treat or prevent a disease mediated by S1PR4.

[0066] In a preferred embodiment, the S1PR4-mediated disease comprises a myopathy.

[0067] In a preferred embodiment, the myopathy includes but is not limited to idiopathic inflammatory myopathy, infectious myopathy, drug-induced myopathy, toxic myopathy, hereditary myopathy, and the like.

[0068] In a preferred embodiment, the myopathy includes but is not limited to Duchenne muscular dystrophy, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, distal muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, Emory muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, etc.

[0069] In a fifth aspect, the present invention provides the compound of the first aspect, or an optical isomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect, for use in treating or preventing S1PR4-mediated diseases.

[0070] In a preferred embodiment, the S1PR4-mediated disease comprises a myopathy.

[0071] In a preferred embodiment, the myopathy includes but is not limited to idiopathic inflammatory myopathy, infectious myopathy, drug-induced myopathy, toxic myopathy, hereditary myopathy, and the like.

[0072] In a preferred embodiment, the myopathy includes but is not limited to Duchenne muscular dystrophy, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, distal muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, Emory muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, etc.

[0073] In a sixth aspect, the present invention provides a drug for treating or preventing S1PR4-mediated diseases, comprising the compound of the first aspect, or an optical isomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect.

[0074] In a preferred embodiment, the S1PR4-mediated disease comprises a myopathy.

[0075] In a preferred embodiment, the myopathy includes but is not limited to idiopathic inflammatory myopathy, infectious myopathy, drug-induced myopathy, toxic myopathy, hereditary myopathy, and the like.

[0076] In a preferred embodiment, the myopathy includes but is not limited to Duchenne muscular dystrophy, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, distal muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, Emory muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, etc.

[0077] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 shows the identification of S1PR4 R79C and NDUFA10 R217W mutations in a late-onset muscular dystrophy pedigree; wherein, (a) a Chinese pedigree diagram containing four generations. Patients are marked with black fill, individuals with mild symptoms are marked with black squares, males are represented by squares and females are represented by circles, and the first and second generations are already sick. The symbols under each member indicate the mutation carrying status of S1PR4 R79C (green) and NDUFA10 R217W (purple), respectively; (b) S1PR4 R79C is located in ICL1 (intracellular loop 1); (c) Sanger sequencing verification of S1PR4 R79C; (d) NDUFA10 R217W is located in the deoxynucleoside kinase domain (dNK); and (e) Sanger sequencing verification of NDUFA10 R217W;

[0079] Figure 2 shows the test results of paw grip strength, serum creatine kinase levels, and muscle fibrosis in S1PR4 mutant mice; among them, the paw grip strength of S1PR4 mutant mice is no different from that of normal mice (Figure a), the serum creatine kinase level of S1PR4 mutant mice is no different from that of WT (Figure b), and muscle fibrosis is not seen in the muscles of S1PR4 mutant mice (HE staining and picrosirius red staining), indicating that the S1PR4 mutation does not cause muscular dystrophy.

[0080] Figure 3 shows the test results of paw grip strength, serum creatine kinase levels, and muscle fibrosis in NDUFA10 mutant mice; among them, the paw grip strength of NDUFA10 mutant mice was no different from that of normal mice (Figure a), the serum creatine kinase level of NDUFA10 mutant mice was no different from that of WT (Figure b), and muscle fibrosis was not observed in the muscles of NDUFA10 mutant mice (HE staining and picrosirius red staining), indicating that NDUFA10 mutation does not lead to muscular dystrophy.

[0081] Figure 4 shows the results of testing for paw grip strength, serum creatine kinase levels, and muscle nuclear fibers and collagen deposition in S1PR4 / NDUFA10 mutant mice; starting at 5 months of age, the paw grip strength of S1PR4 / NDUFA10 mutant mice was significantly reduced compared with normal mice (Figure a), while the serum creatine kinase level in S1PR4 / NDUFA10 mutant mice was significantly increased (Figure b). Muscles of S1PR4 / NDUFA10 mutant mice showed nuclear fibers and collagen deposition, indicating muscle fibrosis (HE and picrosirius red staining), which can be diagnosed as muscular dystrophy.

[0082] Figure 5 shows the recovery effect of compound 30 of the present invention after inducing S1PR4 internalization;

[0083] Figure 6 shows the changes in body weight of double mutant mice after drug administration;

[0084] Figure 7 shows the changes in grip strength of double mutant mice after drug administration;

[0085] Figure 8 shows that the serum creatine kinase level in the double mutant mice was significantly increased compared to that in healthy mice; after drug administration, the creatine kinase level was significantly reduced, while the prednisolone-administered group had no reducing effect;

[0086] Figure 9 is a representative image of the central nucleated fibers in the muscle after drug administration;

[0087] Figure 10 shows the results of the test on the number of nucleated fibers in the middle of the muscle after drug administration;

[0088] FIG11 is a representative graph of collagen area after drug administration;

[0089] FIG12 is a statistical diagram of collagen area after drug administration;

[0090] Figure 13 is a representative immunofluorescence image of macrophages after drug administration;

[0091] FIG14 is a statistical diagram of immunofluorescence of macrophages after drug administration;

[0092] FIG15 shows the changes in body weight of Duchenne muscular dystrophy model mice after drug administration;

[0093] FIG16 shows the changes in grip strength of Duchenne muscular dystrophy model mice after drug administration;

[0094] FIG17 shows the changes of creatine kinase in Duchenne muscular dystrophy model mice;

[0095] Figure 18 is a representative image of the central nucleated fibers in the muscle after drug administration;

[0096] Figure 19 shows the results of the test on the number of centrally nucleated fibers in muscles after drug administration;

[0097] FIG20 is a representative graph of collagen area after drug administration;

[0098] FIG21 is a statistical diagram of collagen area after drug administration;

[0099] Figure 22 is a representative image of immunofluorescence of macrophages after drug administration;

[0100] FIG23 is a statistical diagram of immunofluorescence of macrophages after drug administration;

[0101] Figure 24 shows the effects of compound 30 of the present invention on the tibialis and gastrocnemius muscles in the LPS sepsis model;

[0102] Figure 25 shows that after the Duchenne muscular dystrophy model mice were treated with the drug, there was no significant difference in body weight changes between the drug-treated groups compared with the model group;

[0103] Figure 26 shows that after 42 days of treatment with Compound 67 3 mg / kg and Vamorolone 20 mg / kg, the grip strength of mice in each treatment group increased significantly compared with the model group, indicating a good therapeutic effect;

[0104] Figure 27 shows that on day 42 after administration of compound 67 3 mg / kg and vamorolone 20 mg / kg, the rotarod time of each administration group increased compared with the model group, indicating a good therapeutic effect. In addition, the increase in grip strength was more significant in the compound 67 3 mg / kg group than in the vamorolone 20 mg / kg group.

[0105] Figure 28 is a representative image of intermediate nucleated fibers after drug administration;

[0106] Figure 29 shows the nucleated fiber statistics after drug administration;

[0107] FIG30 is a representative diagram of collagen area after drug administration;

[0108] Figure 31 shows the collagen area statistics after drug administration;

[0109] Figure 32 is a representative graph of the number of F4 / 80 macrophage markers (muscle inflammation) after drug administration;

[0110] Figure 33 shows the statistics of the number of F4 / 80 macrophage markers (muscle inflammation) after drug administration;

[0111] FIG34 shows the serum creatine kinase statistics after drug administration. DETAILED DESCRIPTION

[0112] After extensive and in-depth research, the inventors found that S1PR4 agonists have therapeutic effects on certain myopathies 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 their structures were characterized. This series of compounds were tested for activity at the cellular level, and a group of compounds with S1PR4 agonist activity were obtained. These compounds can modulate S1P receptor activity, among which the EC of S1PR4 agonist activity is 50The value reaches nM level, which has a good targeting effect on S1PR4. On this basis, the present invention was completed.

[0113] Definition of terms

[0114] Some of the groups involved in this article are defined as follows:

[0115] As used herein, "alkyl" refers to a saturated, branched, straight, or cyclic alkyl group having a carbon chain length of 1-10 carbon atoms. Preferred alkyl groups include those having 1-5, 1-2, 1-6, 1-4, or 3-8 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and heptyl. An alkyl group may be substituted with one or more substituents, such as halogen or haloalkyl. For example, an alkyl group may be substituted with 1-4 fluorine atoms, or an alkyl group may be substituted with a fluoroalkyl.

[0116] As used herein, "alkenyl" generally refers to a monovalent hydrocarbon radical having at least one double bond, typically containing 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, and may be straight or branched. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.

[0117] As used herein, "ester group" generally refers to a carboxylic acid derivative having at least one ester group, typically containing 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms, and may be straight 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.

[0118] As used herein, "hydroxyl" refers to a branched or straight-chain alcohol having a carbon chain length of 1-10 carbon atoms, typically containing 1-10 carbon atoms, preferably containing 1-6 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.

[0119] Herein, "amido" refers to a group of the 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.

[0120] 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 group consisting of 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.

[0121] As used 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, furyl, thienyl, pyrrolyl, pyrrolidinyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, pyranyl, pyridyl, pyrimidinyl, pyrazinyl, piperidinyl, morpholinyl, and the like.

[0122] 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 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 and the like. The aromatic heterocyclic group may be optionally substituted with 1-5 (e.g., 1, 2, 3, 4 or 5) substituents selected from the group consisting of 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 group.

[0123] As used herein, "alkoxy" refers to an oxy group substituted with an alkyl group. Preferred alkoxy groups are those having 1-6 carbon atoms in length, and more preferably those having 1-3 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 with one or more substituents, such as halogen or haloalkyl. For example, an alkoxy group may be an alkyl group substituted with 1-4 fluorine atoms, or an alkyl group may be an alkyl group substituted with a fluoroalkyl group.

[0124] As used herein, "halogen" refers to fluorine, chlorine, bromine or iodine.

[0125] Herein, "optionally substituted" means that the substituent it modifies can be optionally substituted with 1-5 (eg, 1, 2, 3, 4 or 5) substituents selected from the group consisting of 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 group.

[0126] Compounds of the present invention

[0127] The inventors discovered that males and females in one family began displaying a muscular dystrophy phenotype at age 50, with symptoms becoming more severe with age, demonstrating strong familial hereditary characteristics. Whole-genome sequencing and other studies of this family revealed that mutations in NDUFA10 and S1PR4 are potential causative genes for muscular dystrophy.

[0128] Given that there are currently no clinically available drugs targeting S1PR4, the S1P receptor modulators currently on the market and under development primarily target S1PR1 and S1PR5. The present inventors have designed and synthesized a series of structurally novel compounds that are selective for different S1PR subtypes. Therefore, the compounds of the present invention have different regulatory effects on different S1PR subtypes; in other words, the compounds of the present invention are S1PR modulators. In specific embodiments, the compounds of the present invention are S1PR4 agonists. In preferred embodiments, the compounds of the present invention are selective for S1PR4 and S1PR1; that is, they have an agonistic effect on S1PR4 but not on S1PR1.

[0129] 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,

[0130] Where A, R, and R 1 、n、R 2 , m, q are as described above.

[0131] In a preferred embodiment, the compounds of the present invention do not include the following compounds:

[0132] In a preferred embodiment, the compound of the present invention is represented by the following formula II:

[0133] Where R 1 、R 2 , X, R3 、R 4 、R 5 As mentioned above.

[0134] In a preferred embodiment, the compound of the present invention is represented by the following formula III:

[0135] Where R 1 、R 2 、R 3 、R 4 、R 5 As mentioned above.

[0136] Specifically, the present invention provides a compound selected from the group consisting of:

[0137] The following compounds are preferred:

[0138] More preferred are the following compounds:

[0139] Based on the compound of the present invention, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0140] Examples of pharmaceutically acceptable salts of the compounds of the present 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 salts formed with inorganic and organic bases such as sodium hydroxide, tris(hydroxymethyl)aminomethane (TRIS, tromethamine), and N-methylglucamine.

[0141] The pharmaceutical compositions of the present invention can be formulated into dosage forms suitable for various routes of administration, including but not limited to forms formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal or topical administration, for the treatment of tumors and other diseases. The dosage is the amount of drug that effectively improves or eliminates one or more symptoms. For the treatment of a specific disease, an effective amount is an amount of drug 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 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.

[0142] 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. Among these mammals, the most important one is human.

[0143] The compounds of the present invention or their pharmaceutical compositions can be used to prepare S1PR modulators. In a specific embodiment, the S1PR modulator 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 myopathies or autoimmune diseases. In a specific embodiment, the muscle diseases mediated by S1PR4 (including its mutants) are idiopathic inflammatory myopathies, infectious myopathies, drug-induced myopathies, toxic myopathies, hereditary myopathies, etc., specifically including (but not limited to) Duchenne muscular dystrophy, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, distal muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, Emory muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0144] The pharmaceutical preparations of the present invention can be manufactured in known manners. For example, they can be manufactured by conventional mixing, granulation, tableting, dissolution, or freeze-drying processes. For oral preparations, solid excipients and the active compound can be combined and the mixture can be optionally ground. After adding appropriate amounts of adjuvants, if desired or necessary, the granular mixture can be processed to obtain tablets or lozenge cores.

[0145] Suitable excipients include, 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 pastes including corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, disintegrants may be added, 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. Auxiliary agents include, in particular, flow conditioners and lubricants, for example, silica, talc, stearates such as magnesium calcium stearate, stearic acid, or polyethylene glycol. If necessary, the tablet cores may be provided with a suitable coating resistant to gastric juices. For this purpose, a concentrated sugar solution may be used. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and / or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. To prepare a coating resistant to gastric juices, suitable cellulose solutions, such as cellulose acetate phthalate or hydroxypropylmethylcellulose phthalate, can be used. Dyes or pigments can be added to the coating of tablets or lozenge cores, for example, for identification or to characterize the combination of active ingredient doses.

[0146] Based on the above compounds and pharmaceutical compositions, the present invention further provides a method for treating S1PR4-mediated diseases, which comprises administering the compound or pharmaceutical composition of the present invention to a subject in need thereof.

[0147] The method of administration includes, but is not limited to, various methods known in the art, which can be determined based on the patient's actual situation, including, but not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal, or topical administration.

[0148] Given that the compounds of the present invention or their pharmaceutical compositions can be used to prepare S1PR modulators, the present invention also provides methods for modulating S1PR in a subject, comprising administering an effective amount of a compound of the present invention, or an optical isomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a subject in need thereof. In a specific embodiment, the method is a method for stimulating S1PR4 in the subject; more preferably, the method stimulating S1PR4 in the subject without stimulating S1PR1.

[0149] Advantages of the present invention:

[0150] 1. The compound provided by the present invention is a novel S1PR modulator, particularly an S1PR4 selective receptor modulator;

[0151] 2. The compounds provided by the present invention have excellent biological activity against S1PR4 and S1PR4 mutant proteins;

[0152] 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.

[0153] The technical solutions of the present invention are further described below with reference to specific examples. However, the following examples do not constitute a limitation of the present invention. All various application methods adopted in accordance with the principles and technical means of the present invention are within the scope of the present invention. Experimental methods in the following examples, where specific conditions are not specified, are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0154] Materials and Methods

[0155] The synthesis of the compound of the present invention is as follows:

[0156] Example 1

[0157] Synthesis of compounds

[0158] Synthesis of 4-(Bromomethyl)-2-methyl-1,1'-biphenyl

[0159] 3-Methyl-4-phenylbenzoic acid (2.00 g, 9.40 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran and stirred under an ice bath. 1.0 M borane tetrahydrofuran solution (14 mL, 14.20 mmol) was added dropwise. After the dropwise addition, stirring was resumed at room temperature. The reaction was complete after 2 hours, and water was added dropwise for quenching, followed by extraction with dichloromethane. Wash with saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1.50 g of a colorless oil with a yield of 80.6%. The product was used directly in the next step without further purification. LC-MS (ESI): m / z: 199.1 (M+H) + .

[0160] 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-cooling. After addition, stir at room temperature. The reaction is complete after 0.5 hour. Quench with water, extract with dichloromethane, and wash with saturated brine. The combined organic phases are dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 1.80 g of a colorless oil (91.0% yield).

[0161] 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) + .

[0162] Synthesis of Ethyl (E)-N-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)acetylcarbamate

[0163] 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 complete, 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 complete, the mixture was returned to room temperature and stirred, and the reaction progress was monitored by TLC. After completion of the reaction, 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. The mixture was passed through a silica gel column to obtain 821 mg of an oily, colorless liquid with a yield of 74.4%.

[0164] 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).

[0165] Synthesis of (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethan-1-one O-((2-methyl-1,1'-biphenyl]-4-yl)methyl)oxime

[0166] Dissolve (E)-N-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)acetylcarbamate (200 mg, 0.70 mmol) in 5 mL of methanol. Add 4.0 M hydrogen chloride in dioxane (0.4 mL, 0.30 mmol) dropwise to the reaction solution. After stirring for 0.5 hour, triethylamine was added dropwise to adjust the pH to between 4 and 6. Then, 1-(3-ethyl-4-(hydroxymethyl)phenyl)ethanone (125 mg, 0.70 mmol) was dissolved in 2 mL of methanol and added dropwise to the reaction system. Monitor the reaction progress by TLC. After completion, 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 through a silica gel column to obtain 140 mg of an oily, colorless liquid with a yield of 53.6%.

[0167] 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).

[0168] Synthesis of (E)-2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)iminoethyl)benzaldehyde

[0169] (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. 2-Iodobenzoic acid (IBX) oxidant (84 mg, 0.30 mmol) was added. The mixture was stirred at room temperature for approximately 2 hours. The reaction progress was monitored by TLC. After completion, 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 yield 99 mg of an oily, colorless liquid (89.1% yield).

[0170] 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).

[0171] 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)

[0172] (E)-2-Ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)iminoethyl)benzaldehyde (74 mg, 0.20 mmol) was dissolved in 5 mL of methanol and 1 drop of acetic acid was added, followed by the addition of S-pyrrolidine-3-carboxylic acid (35 mg, 0.30 mmol). After stirring at room temperature for 3 minutes, sodium cyanoborohydride (19 mg, 0.30 mmol) was added. The reaction progress was monitored by TLC. After the reaction was complete, the reaction system was distilled under reduced pressure, and water and dichloromethane were added for extraction. The organic phases were washed with saturated brine, and the combined organic phases were dried over anhydrous sodium sulfate and concentrated on a silica gel column to obtain 29 mg of a white solid with a yield of 30.8%.

[0173] 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] + calcd for C 30 H 34 N2O3, 471.2648; found: 471.2650. HPLC Purity: 98.16%, retention time: 1.52min.

[0174] The synthetic route of representative compound 13 is as follows:

[0175] Synthesis of Ethyl (E)-N-((3-(Trifluoromethyl)benzyl)oxy)acetylcarbamate

[0176] Ethyl acetohydroxamate (865 mg, 8.40 mmol) was dissolved in 10 mL of tetrahydrofuran solution and then placed in an ice bath. 1.0 M potassium tert-butoxide tetrahydrofuran solution (17 mL, 16.70 mmol) was added dropwise to the reaction system. After the addition was complete, stirring was continued in an ice bath for 0.5 hours. 3-(Trifluoromethyl)benzyl bromide (1.99 g, 8.40 mmol) was dissolved in 5 mL of tetrahydrofuran and then added dropwise to the reaction system. After the addition was complete, 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 over a silica gel column to give 1.81 g of an oily colorless liquid with a yield of 82.6%.

[0177] 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).

[0178] Synthesis of (E)-1-(3-ethyl-4-hydroxymethyl)phenyl)ethane-1-one O-(3-(trifluoromethyl)benzyl)oxime

[0179] Dissolve (E)-N-((3-(trifluoromethyl)benzyl)oxy)acetylcarbamate (157 mg, 0.60 mmol) in 5 mL of methanol. Add 0.4 mL of 4.0 M hydrogen chloride dioxane solution 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 (107 mg, 0.60 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 on a silica gel column to obtain 150 mg of an oily colorless liquid with a yield of 71.1%.

[0180] 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).

[0181] Synthesis of (E)-2-ethyl-4-(1-(((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzaldehyde

[0182] (E)-1-(3-ethyl-4-hydroxymethyl)phenyl)ethane-1-one O-(3-(trifluoromethyl)benzyl)oxime (211 mg, 0.60 mmol) was dissolved in 3 mL of DMSO. IBX oxidant (308 mg, 1.10 mmol) was added. The mixture was stirred at room temperature for approximately 2 hours. The reaction progress was monitored by TLC. After completion, 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 on a silica gel column to obtain 126 mg of an oily, colorless liquid (yield 60.0%).

[0183] 1 H 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).

[0184] Synthesis of (E)-1-(2-ethyl-4-(1-((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 13)

[0185] (E)-2-Ethyl-4-(1-(((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzaldehyde (105 mg, 0.30 mmol) was dissolved in 5 mL of methanol and 1 drop of acetic acid was added, followed by the addition of 3-azetidinecarboxylic acid (61 mg, 0.60 mmol). After stirring at room temperature for 3 minutes, sodium cyanoborohydride (38 mg, 0.60 mmol) was added. The reaction progress was monitored by TLC. 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 on a silica gel column to give 78 mg of an oily substance with a yield of 60.0%.

[0186] 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). 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] + calcd for C 23 H 25 F3N2O3, 435.1896; found: 435.1897. HPLC Purity: 99.79%, retention time: 1.17min.

[0187] The synthetic route of representative compound 16 is shown below:

[0188] Synthesis of 5-(Bromomethyl)-2-isopropoxybenzonitrile

[0189] A 2.0 M solution of oxalyl chloride in dichloromethane (14.6 mL, 29.20 mmol) was added to 3-cyano-4-(isopropoxy)benzoic acid (2.01 g, 9.80 mmol) 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 (923 mg, 4.50 mmol) was dissolved in 10 mL of tetrahydrofuran and cooled to 0°C. Sodium borohydride (414 mg, 11.20 mmol) was added, followed by 2 mL of methanol. 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 1.0 M hydrochloric acid. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated to give 1.12 g of a colorless oil (59.9% yield).

[0190] 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).

[0191] 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. After addition, stir at room temperature. The reaction is complete after 0.5 hour. Quench with water, extract with dichloromethane, and wash with saturated brine. The combined organic phases are dried over anhydrous sodium sulfate and concentrated to yield 1.19 g of a colorless oil (90.5% yield).

[0192] 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).

[0193] Synthesis of Ethyl-N-((3-cyano-4-(isopropoxy)benzyl)oxy)acetylcarbamate

[0194] Ethyl acetohydroxamate (402mg, 3.90mmol) was dissolved in 10mL of 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 after completion of the addition, stirring was continued under an ice bath for 0.5 hours. 3-cyano-4-isopropoxybenzyl bromide (987mg, 3.90mmol) was dissolved in 5mL of tetrahydrofuran and then added dropwise to the reaction system. After completion of the addition, the mixture was returned to room temperature and stirred, and the reaction progress was monitored by TLC. After completion of the reaction, 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 give 619mg of an oily colorless liquid with a yield of 57.5%.

[0195] 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).

[0196] Synthesis of (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one-O-(3-cyano-4-(isopropoxy)benzyl)oxime

[0197] 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. 1-(3-ethyl-4-(hydroxymethyl)phenyl)ethanone (90 mg, 0.50 mmol) was then 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 on a silica gel column to give 127 mg of an oily colorless liquid with a yield of 69.3%.

[0198] 1H 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).

[0199] Synthesis of (E)-5-((((1-(3-ethyl-4-formylphenyl)ethylidene)amino)oxy)methyl)-2-isopropoxybenzonitrile

[0200] (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one-O-(3-cyano-4-(isopropoxy)benzyl)oxime (147 mg, 0.40 mmol) was dissolved in 4 mL of DMSO and IBX oxidant (225 mg, 0.80 mmol) was added. Stirring was carried out at room temperature for approximately 2 hours, and the reaction progress was monitored by TLC. After completion, the reaction was quenched with water, extracted with dichloromethane, and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated on a silica gel column to yield 119 mg of an oily, colorless liquid (81.2% yield).

[0201] 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).

[0202] Synthesis of (E)-1-(2-ethyl-4-(1-((3-cyano-4-(isopropoxy)benzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 16)

[0203] (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. The reaction progress was monitored by TLC. After the reaction was complete, the reaction system was distilled under reduced pressure, extracted with water and dichloromethane, and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated on a silica gel column to obtain 70 mg of a light-colored solid with a yield of 52.3%.

[0204] 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] + calcd for C 26 H 31 N3O4, 450.2396; found: 450.2395. HPLC Purity: 96.46%, retention time: 1.02min.

[0205] The synthetic route of compound 17 is as follows:

[0206] Synthesis of Ethyl (E)-N-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)acetylcarbamate

[0207] Ethyl acetohydroxamate (196mg, 1.90mmol) was dissolved in 5mL tetrahydrofuran solution and then placed under an ice bath. 1.0M potassium tert-butoxide tetrahydrofuran solution (4mL, 3.90mmol) was added dropwise to the reaction system, and the addition was completed and continued to stir under an ice bath for 0.5 hour. 4-chloromethyl-1-cyclopentyl-2-trifluoromethylbenzene (498mg, 1.90mmol) was dissolved in 3mL tetrahydrofuran and then added dropwise to the reaction system. The reaction was stirred at room temperature after the addition was completed, 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 concentrated over a silica gel column with anhydrous sodium sulfate drying to give 532mg of an oily colorless liquid, with a yield of 85.1%.

[0208] 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).

[0209] Synthesis of (E)-1-(3-ethyl-4-(hydroxymethyl)phenyl)ethane-1-one-O-(4-cyclopentyl-3-(trifluoromethyl)benzyl)oxime

[0210] Ethyl (E)-N-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)acetylcarbamate (461 mg, 1.40 mmol) was dissolved in 5 mL of methanol. 4.0 M hydrogen chloride dioxane solution (1.0 mL, 0.70 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. 1-(3-ethyl-4-(hydroxymethyl)phenyl)ethanone (267 mg, 1.50 mmol) was then 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 on a silica gel column to give 489 mg of an oily colorless liquid with a yield of 83.4%.

[0211] 1H 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).

[0212] Synthesis of (E)-4-(1-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzaldehyde

[0213] (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. IBX oxidant (952 mg, 2.40 mmol) was added, and the mixture was stirred at room temperature for approximately 2 hours. The reaction progress was monitored by TLC. After completion, 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 yield 302 mg of an oily, colorless liquid, yielding 60.3%.

[0214] 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).

[0215] Synthesis of (E)-1-(4-(1-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 17)

[0216] (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, 1 drop of acetic acid was added, and then 3-azetidinecarboxylic acid (242 mg, 2.40 mmol) was added. After stirring at room temperature for 3 minutes, sodium cyanoborohydride (113 mg, 1.80 mmol) was added. The reaction progress was monitored by TLC. 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 on a silica gel column to obtain 182 mg of a light-colored solid with a yield of 30.2%.

[0217] 1 H 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] + calcd for C 28 H 33 F3N2O3,503.2522; found:503.2523.HPLC Purity:96.46%,retention time:2.28min.

[0218] The reaction conditions for other compounds are similar to those of the above compounds.

[0219] (E)-1-(4-(1-((4-cyclohexylbenzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 1)

[0220] 1 H 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) + .

[0221] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 2)

[0222] 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) + .

[0223] (E)-1-(4-(1-((4-cyclohexylbenzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 3)

[0224] 1 H 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) + .

[0225] 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)

[0226] 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.

[0227] 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)

[0228] 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

[0229] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyloxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 6)

[0230] 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) + .

[0231] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)piperidine-3-carboxylic acid (Compound 7)

[0232] 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) + .

[0233] (E)-4-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)amino)butanoic acid (Compound 8)

[0234] 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) + .

[0235] (E)-3-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)amino)cyclohexane-1-carboxylic acid (Compound 9)

[0236] 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 CNMR(151MHz,MeOD)δ182.91,155.03,146.14,136.31,135.59,131.60,128.55,128.47,128.10,125.97,124.84,1 24.79,74.73,55.54,49.13,45.59,40.06,34.19,31.24,29.43,26.60,25.70,11.40.LC-MS(ESI):m / z:531.2(M+H) + .

[0237] (E)-3-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)benzyl)amino)propanoic acid (Compound 10)

[0238] 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=11.1Hz,3H),1.48(ddd,J=33.8,22.3,10.1Hz,5H). 13 C NMR(151MHz,Methanol-d4)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) + .

[0239] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-fluorobenzyl)azetidine-3-carboxylic acid (Compound 11)

[0240] 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] + calcd for C 27 H 30 F4N2O3,507.2271; found:507.2270.HPLC Purity:91.98%,retention time:2.26min.

[0241] Structure of (E)-1-(2-bromo-4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 12)

[0242] 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] + calcd for C 27 H 30 BrF3N2O3,567.1470; found:567.1473.HPLC Purity:90.25%,retention time:2.87min.

[0243] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-methylbenzyl)azetidine-3-carboxylic acid (Compound 18)

[0244] White solid, yield 40.6%, melting point: 155.1-155.6℃. 1H 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.

[0245] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-methoxybenzyl)azetidine-3-carboxylic acid (Compound 27)

[0246] White solid, yield 40.6%, melting point: 145.2-145.6℃. 1H NMR(400MHz,CD3OD)δ7.66(s,1H),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] + calcd for C 28 H 33 F3N2O4,519.2471; found:519.2473.HPLC Purity:96.28%,retention time:1.27min.

[0247] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-(trifluoromethyl)benzyl)azetidine-3-carboxylic acid (Compound 19)

[0248] White solid, yield 40.6%, melting point: 135.6-138.9℃. 1 H 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). 13C 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] + calcd for C 28 H 30 F6N2O3,557.2239; found:557.2237.HPLC Purity:97.16%,retention time:2.56min.

[0249] Structure of (E)-1-(4-(1-(([1,1'-biphenyl]-4-ylmethoxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 20)

[0250] White solid, yield 40.6%, melting point: 120.3-121.9℃. 1 H 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] + calcd for C 28 H 30 N2O3, 443.2335; found: 443.2337. HPLC Purity: 98.03%, retention time: 1.30min.

[0251] Structure of (E)-1-(4-(1-((4-cyclopentyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 17)

[0252] Light-colored solid, yield 30.2%, melting point: 131.7-133.2°C. 1 H 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] + calcd for C 28 H 33 F3N2O3,503.2522; found:503.2523.HPLC Purity:96.46%,retention time:2.28min.

[0253] Structure of (E)-1-(4-(1-((4-cyclohexyl-3-methylbenzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 24)

[0254] Yellow solid, yield 33.3%, melting point: 135.8-136.3℃. 1 H 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] + calcd for C 29 H 38 N2O3, 463.2961; found: 463.2962. HPLC Purity: 98.19%, retention time: 2.39min.

[0255] (E)-3-((4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)amino)propanoic acid (Compound 25)

[0256] 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) + .

[0257] (E)-1-(4-(1-((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)pyrrolidine-3-carboxylic acid (Compound 26)

[0258] 1 H 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). 13C 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) + .

[0259] Structure of (E)-1-(2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 22)

[0260] White solid, yield 40.6%, melting point: 154.2-154.8℃. 1 H NMR (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] + calcd for C 29 H 32N2O3, 457.2492; found: 457.2493. HPLC Purity: 91.98%, retention time: 2.30min.

[0261] Structure of (E)-1-(2-ethyl-4-(1-((2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 21)

[0262] White solid, yield 40.6%, melting point: 155.5-155.9℃. 1 H NMR(400MHz,CD3OD)δ7.69(d,J=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] + calcd for C 29 H 29 F3N2O3,511.2209; found:511.2211.HPLC Purity:97.71%,retention time:1.56min.

[0263] Structure of (E)-1-(2-ethyl-4-(1-((3-(trifluoromethyl)phenyl)oxy)imino)ethyl)benzyl)azetidine-3-carboxylic acid (Compound 13)

[0264] Pale yellow oil, yield 60.0%. 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). 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] + calcd for C 23 H 25 F3N2O3, 435.1896; found: 435.1897. HPLC Purity: 99.79%, retention time: 1.17min.

[0265] Structure of (E)-1-(2-ethyl-4-(1-((4-methyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 14)

[0266] Pale yellow oil, yield 21.3%. 1H 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). 13 C 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] + calcd for C 24 H 27 F3N2O3,449.2052; found:449.2050.HPLC Purity:99.32%,retention time:1.35min.

[0267] Structure of (E)-1-(2-ethyl-4-(1-((naphthalen-2-ylmethoxy)imino)ethyl)azetidine-3-carboxylic acid (Compound 15)

[0268] 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). 13C 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] + calcd for C 26 H 28 N2O3, 417.2179; found: 417.2179. HPLC Purity: 98.77%, retention time: 1.24min.

[0269] 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)

[0270] 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 CNMR(151MHz,CD3OD)δ178.90,154.68,142.58,136.56,136.32,136.01,134.99,134.56,128.69,128.62,128.61,12 6.00,125.26,123.45,75.81,58.35,57.83,36.29,29.01,28.78,25.11,23.06,14.25,11.51.HRMS(ESI)(m / z):[M+H] + calcd for C 26 H32 N2O3, 421.2492; found: 421.2492. HPLC Purity: 98.54%, retention time: 1.48min.

[0271] Structure of (E)-1-(4-(1-(((3-cyano-4-isopropoxybenzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (Compound 16)

[0272] 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] + calcd for C 26 H 31 N3O4, 450.2396; found: 450.2395. HPLC Purity: 96.46%, retention time: 1.02min.

[0273] 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)

[0274] Gray-brown solid, yield 30.1%, melting point: 144.3-146.4°C. 1 H 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 C NMR (151MHz, CD3OD) δ178.58,154.38,143.61,141.73,141.47,137.53,137.03,134.92,130.99,130.41,129.86,129.34,128.79,127 .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] + calcd for C 30 H 34 N2O3, 471.2648; found: 471.2647. HPLC Purity: 96.16%, retention time: 1.66min.

[0275] 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)

[0276] White solid, yield 14.9%, melting point: 149.6-150.6℃. 1H NMR(400MHz,CD3OD)δ7.60(s,1H),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] + calcd for C 30 H 34 N2O3, 471.2648; found: 471.2650. HPLC Purity: 97.51%, retention time: 1.45min.

[0277] 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)

[0278] 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] + calcd for C 30 H 34 N2O3, 471.2648; found: 471.2650. HPLC Purity: 98.16%, retention time: 1.52min.

[0279] Structure of (E)-3-((2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)amine)propanoic acid (Compound 31)

[0280] White solid, yield 22.4%, melting point: 149.6-149.8℃. 1H NMR (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] + calcd for C 28 H 32 N2O3, 445.2491; found: 445.2490. HPLC Purity: 99.29%, retention time: 1.24min.

[0281] Structure of (E)-4-((2-ethyl-4-(1-((2-methyl-[1,1'-biphenyl]-4-yl)methoxy)imino)ethyl)benzyl)aminobutyric acid (Compound 32)

[0282] White solid, yield 13.2%, melting point: 133.3-136.3℃. 1 H 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). 13C 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] + calcd for C 29 H 34 N2O3, 459.2648; found: 459.2647. HPLC Purity: 98.69%, retention time: 1.34min.

[0283] 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)

[0284] Pale yellow oil, yield 15.3%. 1 H NMR (400MHz, CD3OD) δ7.61 (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] + calcd for C 31 H 36N2O3, 485.2804; found: 485.2802. HPLC Purity: 97.56%, retention time: 1.42min.

[0285] 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)

[0286] 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). 13 C 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.

[0287] 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)

[0288] Pale yellow solid, yield 18.7%. 1H 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 CNMR(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.

[0289] 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)

[0290] Pale yellow solid, yield 21.2%. 1H 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.

[0291] 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)

[0292] 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.

[0293] 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)

[0294] 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.

[0295] 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)

[0296] 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.

[0297] 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)

[0298] 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 C NMR (151MHz, CD3OD) δ177.95,154.26,143.69,138.10,137.85,136.50,134.86,131.45,130.44,130.23,129.63,128.60,126.6 6,125.64,123.96,121.92,75.66,56.60,54.49,53.58,43.61,37.44,27.73,25.22,20.08,14.53,11.38.MS(ESI)(m / z):[M+H] + 475.30.

[0299] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(thien-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 41)

[0300] 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.

[0301] 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)

[0302] 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.

[0303] 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)

[0304] 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.

[0305] 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)

[0306] 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.

[0307] 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)

[0308] 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)δ178.08,165.70,159.25,154.41,143.72,138.64,137.45,130.52,13 0.46,129.72,128.36,126.66,125.58,123.98,115.90,75.51,56.54,54.47,53.59,43.60, 27.73,25.24,18.47,14.55,11.43,9.83,8.99.MS(ESI)(m / z):[M+H] + 490.30.

[0309] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 46)

[0310] 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 C NMR (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.

[0311] 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)

[0312] 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.

[0313] 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)

[0314] 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) δ178.01,153.87,149.09,143.66,137.93,131.74,130.47,129.79,128.56,126.61,126.49,12 3.93,115.53,75.99,56.52,54.41,53.55,50.82,43.67,27.75,25.24,24.30,19.25,14.57,11.40.MS(ESI)(m / z): [M+H] + 464.30.

[0315] 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)

[0316] 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.

[0317] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-morpholinylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 50)

[0318] 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.

[0319] 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)

[0320] 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.

[0321] 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)

[0322] 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.

[0323] 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)

[0324] 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.

[0325] 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)

[0326] 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.

[0327] 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)

[0328] 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 C NMR (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.

[0329] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(thiophen-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 56)

[0330] 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 C NMR (126MHz, CD3OD) δ154.35,143.75,141.83,137.95,136.87,135.39,130.47,128.38,126.72,125.90,125.85 ,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

[0331] 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)

[0332] 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.

[0333] 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)

[0334] 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.

[0335] 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)

[0336] 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.

[0337] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-cyclohexyl-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 60)

[0338] 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.

[0339] 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)

[0340] 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 CNMR(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

[0341] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-cyclopentyl-3-methylbenzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 62)

[0342] 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

[0343] 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)

[0344] 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.

[0345] 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)

[0346] 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.

[0347] 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)

[0348] 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

[0349] 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)

[0350] 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.

[0351] 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)

[0352] 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.

[0353] 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)

[0354] 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.

[0355] 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)

[0356] 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

[0357] Structure of (S,E)-1-(2-ethyl-4-(1-(((4-(pyrimidin-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 70)

[0358] 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

[0359] 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)

[0360] 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

[0361] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-methyl-4-(thien-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 72)

[0362] 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). 13 C 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

[0363] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(thiophen-2-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 73)

[0364] 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

[0365] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(isothiazol-4-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 74)

[0366] Pale yellow solid, yield 11.6%. 1H 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

[0367] Structure of (S,E)-1-(2-ethyl-4-(1-((4-(isothiazol-5-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 75)

[0368] 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

[0369] 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)

[0370] 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

[0371] 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)

[0372] 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

[0373] 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)

[0374] 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

[0375] 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)

[0376] 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

[0377] 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)

[0378] 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(t d,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 C NMR (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

[0379] 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)

[0380] 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

[0381] 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)

[0382] 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

[0383] 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)

[0384] 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

[0385] 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)

[0386] 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

[0387] Structure of (S,E)-1-(2-ethyl-4-(1-(((3-fluoro-4-(thien-3-yl)benzyl)oxy)imino)ethyl)benzyl)pyrrolidine-3-carboxylic acid (Compound 85)

[0388] Pale yellow solid, yield 26.5%. 1H 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

[0389] 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)

[0390] Pale yellow solid, yield 26.9%. 1H NMR(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,2 H),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 CNMR(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

[0391] 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)

[0392] 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,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

[0393] 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)

[0394] 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

[0395] 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)

[0396] 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)δ154.62,143.69,140.08,138.24,138.17,132.34,132.04,130.43,128.28, 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):[M+H] + 477.20

[0397] 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)

[0398] White solid, yield 28.8%. 1 H 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). 13C 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

[0399] 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)

[0400] 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). 13 C NMR(126MHz,Methanol-d4)δ176.26,160.27,158.28,154.47,152.86,143.53,141.29,141.22,139.45,137.66,136.37,131.94,1 31.64,130.32,129.57,129.51,126.66,125.50,123.98,75.33,57.18,54.93,34.86,25.16,19.04,14.45,11.39.MS(m / z):[M+H] + 477.20

[0401] 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)

[0402] 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

[0403] 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)

[0404] White solid, yield 70.1%. 1H 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)δ177.94,160.40,160.38,158.43,158.40,154.73,151.6 6,143.71,141.43,141.35,137.54,132.19,131.88,130.49,130.34,127.45,127.41 ,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

[0405] 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)

[0406] White solid, yield 71.5%. 1H 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

[0407] 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)

[0408] 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.

[0409] Example 2. Relationship between S1PR4 and NDUFA10 mutations and muscular dystrophy

[0410] Muscular dystrophy is a progressive muscle disease caused by genetic mutations and characterized by chronic oxidative stress and inflammation. Clinically, it manifests as progressive muscle weakness that affects the limbs, throat, and facial muscles to varying degrees. Based on the onset of the disease, it can be divided into congenital muscular dystrophy and late-onset muscular dystrophy. Generally, congenital muscular dystrophy presents with obvious muscle weakness at birth or in the first few months of life. In contrast, late-onset muscular dystrophy does not manifest muscle weakness until patients achieve independent walking.

[0411] Current treatment strategies for muscular dystrophy primarily rely on gene therapy and anti-inflammatory therapies. Gene therapy utilizes small molecules or antisense oligonucleotides to inhibit nonsense mutations that cause abnormal transcription termination or induce skipping of specific exons, leading to restoration of the reading frame and production of dystrophin. Alternatively, adeno-associated viruses (AAVs) can be used as vectors to introduce a normal, wild-type gene expressing dystrophin. However, gene therapy is limited by immune responses to the vectors and limited vector packaging size. Furthermore, gene therapy is only effective for patients with specific pathogenic gene variants and is not suitable for all patients with muscular dystrophy. Inflammation is a secondary consequence of the gene variant, and suppressing inflammation has the advantage of being applicable to all subtypes of muscular dystrophy, regardless of the gene variant. Glucocorticoids have been used to ameliorate inflammation levels to treat muscular dystrophy, but their effectiveness is limited and they have significant side effects, necessitating the identification of new inflammatory targets.

[0412] In this example, the inventors conducted whole-genome sequencing on patients with muscular dystrophy and discovered that all patients had mutations in NDUFA10 and S1PR4. Using mouse models with NDUFA10 and S1PR4 mutations, they found that NDUFA10 mutations lead to mitochondrial dysfunction, while S1PR4 mutations lead to elevated inflammation. Furthermore, double-mutant mice, obtained by crossing S1PR4 and NDUFA10 mutant mice, exhibited a muscular dystrophy phenotype. Administration of an S1PR4 agonist ameliorates the muscular dystrophy phenotype while also reducing muscle inflammation and fibrosis. This suggests that S1PR4 and NDUFA10 play a crucial role in the pathogenesis of muscular dystrophy and provides a new inflammation-related target for the treatment of muscular dystrophy.

[0413] The inventors discovered that males in a family began exhibiting muscular dystrophy phenotypes starting at age 50, with the phenotype worsening with age. Whole-genome sequencing revealed that all muscular dystrophy patients harbored mutations in NDUFA10 and S1PR4. Studies have shown that NDUFA10 and S1PR4 may link oxidative stress and chronic inflammation in dystrophy. In a mouse model with NDUFA10 mutations, NDUFA10 was found to cause mitochondrial dysfunction, a phenotype that is non-sex-specific and affects both males and females. In a mouse model with S1PR4 mutations, S1PR4 mutations were found to cause elevated inflammation, a phenotype that is non-sex-specific and affects both males and females. When both NDUFA10 and S1PR4 mutations are present in mice, male mice develop a muscular dystrophy phenotype, characterized by decreased grip strength, elevated serum creatine kinase, and muscle fibrosis and inflammation compared to normal mice. Results showed that mice with either single mutation did not develop muscular dystrophy, while mice with the double mutation did. Treatment with an S1PR4 agonist can improve muscle performance and the muscular dystrophy phenotype in mice.

[0414] 1. Experimental Background

[0415] Muscular dystrophy (MD) is a group of genetic disorders characterized by progressive muscle wasting associated with chronic oxidative stress and inflammation. Characterized by progressive muscle weakness and atrophy, MD can be inherited in an autosomal recessive, autosomal dominant, or sex-linked manner.

[0416] The inventors, through genetic screening of families with muscular dystrophy, discovered mutations in the S1PR4 and NDUFA10 genes. They hypothesized that these mutations could lead to muscular dystrophy. Therefore, they constructed mouse models with S1PR4 and NDUFA10 mutations, hybridized the two mice, and generated S1PR4 / NDUFA10 double-mutant mice to test whether these two mutations could cause muscular dystrophy.

[0417] II. Association between NDUFA10 and S1PR4 mutations and myopathy

[0418] The inventors discovered that males and females in a family began to exhibit a muscular dystrophy phenotype at age 50, with the disease becoming more severe with age, demonstrating strong familial hereditary characteristics. They then performed whole-genome sequencing on this family. After bioinformatics analysis of the sequencing results, they identified 23 gene mutations shared by all patients but not their healthy spouses (see the table below). The table shows columns W and X: Column W lists the mutated gene name, and column X lists the corresponding mutation site. This is followed by the test results for each sample, with four columns of data for each sample. In the first column (marked in green), 0|1 indicates a heterozygous mutation, 1|1 indicates a homozygous mutation, and 0|0 indicates no mutation.

[0419] Through extensive research into muscular dystrophy and the functions of 23 mutant genes, two genes, NDUFA10 and S1PR4, have garnered attention. NDUFA10 is a subunit of mitochondrial respiratory chain complex I and may be involved in mitochondrial energy production. Muscle cells have a high energy demand, and mutations in S1PR4 may affect muscle cell function by reducing mitochondrial energy efficiency. S1PR4, on the other hand, is associated with immune function and may be the primary cause of altered muscle tissue traits in patients with muscular dystrophy. Therefore, the inventors first investigated NDUFA10 and S1PR4 mutations as potential pathogenic gene mutations in muscular dystrophy.

[0420] Figure 1 shows the results of target-disease association studies: Figure 1 shows the identification of S1PR4 R79C and NDUFA10 R217W mutations in a late-onset muscular dystrophy pedigree. (a) A four-generation Chinese pedigree. Affected individuals are marked with black fill, mildly symptomatic individuals are marked with black squares, males are represented by squares, and females are represented by circles. The first and second generations are already affected. The symbols below each member indicate the mutation status of S1PR4 R79C (green) and NDUFA10 R217W (purple), respectively. (b) S1PR4 R79C is located within ICL1 (intracellular loop 1). (c) Sanger sequencing verification of S1PR4 R79C. (d) NDUFA10 R217W is located within the deoxynucleoside kinase (dNK) domain. (e) Sanger sequencing verification of NDUFA10 R217W.

[0421] In the figure, the all-black areas represent confirmed patients, all of whom have heterozygous mutations in both S1PR4 and NDUFA10. Other offspring have inherited one, two, or neither mutation, conforming to Mendelian inheritance. Because the disease doesn't develop until a certain age, it's currently unclear whether the next generation will develop the disease, and a definitive diagnosis is not yet possible. However, based on their own experience, some have already experienced mild muscle symptoms. The inventors have marked one-quarter of these individuals as black for reference.

[0422] Whole-genome sequencing of families with muscular dystrophy revealed that all patients harbored mutations in NDUFA10 and S1PR4. In an NDUFA10 mutant mouse model, NDUFA10 was found to cause mitochondrial dysfunction, with no sex-selective effect, affecting both males and females. In an S1PR4 mutant mouse model, S1PR4 mutations were found to cause elevated inflammation, with no sex-selective effect, affecting both males and females. Results showed that neither single-gene mutant mouse developed muscular dystrophy. However, double-mutant mice, obtained by crossing S1PR4 mutant mice with NDUFA10 mutant mice, exhibited a muscular dystrophy phenotype, characterized by decreased grip strength in all four limbs, elevated serum creatine kinase, and muscle fibrosis and inflammation compared to normal mice. Therefore, the double-mutant mice developed muscular dystrophy.

[0423] 3. Experimental Process

[0424] 3.1 Limb Grip Strength Testing

[0425] The mouse is placed horizontally on the grip test board. Grab the mouse's tail and pull it in the opposite direction. Do it gently and do not use force. Pull the mouse's tail gradually until the mouse releases the grip force receptor. The maximum value recorded by the grip force receptor is the grip strength value. Repeat 3 times and take the average value.

[0426] 3.2 Serum creatine kinase content detection

[0427] After the administration, blood was collected from the eye socket, allowed to stand for 30 min, and centrifuged at 3000 rpm / min and 4°C for 15 min. Serum was extracted and stored in a frozen state at -20°C. The serum was tested according to the kit instructions.

[0428] 3.3 Pathological examination of muscle tissue sections

[0429] After the experiment, the tibialis anterior muscle, extensor digitorum longus muscle and diaphragm of the mice were fixed and sectioned for HE staining and picrosirius red staining to observe the degree of muscle fibrosis.

[0430] 4. Experimental Results

[0431] 4.1 S1PR4 mutations do not cause muscular dystrophy

[0432] The paw grip strength of S1PR4 mutant mice was no different from that of normal mice (Figure 2a). The serum creatine kinase levels of S1PR4 mutant mice were no different from those of WT mice (Figure 2b). Muscle fibrosis was not observed in S1PR4 mutant mice (HE and picrosirius red staining). These results indicate that the S1PR4 mutation does not cause muscular dystrophy.

[0433] 4.2 NDUFA10 mutations do not cause muscular dystrophy

[0434] The paw grip strength of NDUFA10 mutant mice was no different from that of normal mice (Figure 3a). Serum creatine kinase levels in NDUFA10 mutant mice were no different from those in WT mice (Figure 3b). Muscle fibrosis was not observed in NDUFA10 mutant mice (HE and picrosirius red staining). These results indicate that NDUFA10 mutations do not cause muscular dystrophy.

[0435] 4.3 S1PR4 / NDUFA10 mutations cause muscular dystrophy

[0436] The paw grip strength of S1PR4 / NDUFA10 mutant mice was significantly reduced compared with that of normal mice starting from 5 months of age (Figure 4a), the creatine kinase level in the serum of S1PR4 / NDUFA10 mutant mice was significantly increased (Figure 4b), and the muscles of S1PR4 / NDUFA10 mutant mice showed nucleated fibers and collagen deposition, indicating muscle fibrosis (HE staining and picrosirius red staining), which can be diagnosed as muscular dystrophy.

[0437] Combined, these experimental results indicate that double-mutant mice develop muscular dystrophy by five months of age, with significantly reduced grip strength, significantly elevated creatine kinase levels, and muscle fibrosis. Because mutations in either S1PR4 or NDUFA10 alone do not cause muscular dystrophy, while simultaneous mutations in both genes do, it is believed that treating one of these targets will hinder disease progression. Targeted drug development for S1PR4 and NDUFA10 is valuable for treating muscular dystrophy.

[0438] Example 3. Discovery of Selective S1PR4 Agonists

[0439] Based on the aforementioned mutant mouse research results—that neither single gene mutation leads to a muscular dystrophy phenotype, but double mutations do—the inventors proposed a therapeutic approach to reverse the muscular dystrophy phenotype by targeting and regulating one of the mutant proteins. Compared to NDUFA10, S1PR4 was chosen as the primary therapeutic target due to its more established research background, well-defined binding pocket, and ease of targeting. Targeting this target, the inventors developed a series of S1PR4-selective agonists.

[0440] S1PR4 agonist bioactivity assay

[0441] 1. Experimental Principle

[0442] Tango assay technology detects ligand-dependent GPCR activity through the interaction between β-arrestin and GPCR. TEV separation technology utilizes the complementation of two inactive fragments of TEV protease (NTEV, 1-118 and CTEV, 119-221) to reconstitute a functional protease. The NTEV fragment and the artificial transcription factor tetracycline transactivator (tTA) are fused to the C-terminus of the GPCR, and the CTEV fragment is fused to human β-arrestin 2. To enhance the stability of the interaction between GPCR and β-arrestin, a short sequence from the C-terminus of human arginine vasopressin receptor 2 (V2 tail) is introduced between the GPCR and NTEV. The V2 tail contains multiple GRK phosphorylation sites, which, upon phosphorylation, enhance β-arrestin binding. The CTEV fragment carries the stabilizing point mutation S219P and is truncated at amino acid 221 to remove the autoinhibitory C-terminal tail. This truncation also truncates β-arrestin 2, resulting in the deletion of the entire C-terminal tail (lacking amino acids 383-410). This truncation has a strong stimulus-dependent receptor desensitization effect, thereby improving detection sensitivity compared to wild-type β-arrestin 2. The UAS reporter construct consists of an upstream active sequence (UAS), a CMV minimal promoter, and a tTA-dependent luciferase reporter gene. Therefore, constructing Tango cells requires simultaneous transfection of these three plasmids. Upon activation of the GPCR to recruit β-arrestin, the TEV enzyme at the β-arrestin terminus cleaves tTA from the GPCR terminus. The freed tTA initiates tTA-dependent luciferase transcription, ultimately determining compound activity by measuring fluorescence intensity.

[0443] 2. Experimental Process

[0444] 1) Tango cells were digested from the culture dishes, counted, and plated onto white opaque 96-well plates at a density of 10,000 cells per well (700 μL / well) (with 60 μL of D'hanks added around the perimeter). Cultured overnight (20 hours) in a 5% CO2 incubator at 37°C. The cell density reached approximately 90% on the next day.

[0445] 2) After 20 h, 30 μL of serially diluted compound solution was added to each well, and 30 μL of F12K complete medium was added to the control group. Each group had three replicate wells.

[0446] 3) After 20 hours of drug treatment, 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 minutes at room temperature in the dark before testing.

[0447] 4) Place the plate on a microplate reader to read the relative light unit (RLU), record the fluorescence value, and process the data using Graphpad Prism software to calculate the EC of the compound. 50 .

[0448] 3. Test results:

[0449] (All compounds were tested at a final concentration of 45 μM, with 9 gradients and 5-fold dilutions. 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)

[0450] Although some S1PR4 modulators have been reported, the mechanisms of action of most of these compounds are unclear, and detailed structure-activity relationship analysis and in vivo pharmacokinetic parameters are lacking, making it impossible to evaluate their in vivo efficacy. The present inventors used the S1PR1 / 5 agonist BAF312 as a lead compound. Based on the β-arrestin recruitment activity evaluation system for S1PR1 and S1PR4, they modified the polar head and hydrophobic ends of BAF312 to synthesize and evaluate a series of compounds. Through thorough S1PR1 / 4 structure-activity relationship analysis of compound BAF312, the present inventors discovered that replacing the cyclohexyl group of BAF312 with a benzene ring and the trifluoromethyl group with a methyl group to synthesize compound 22 significantly reduced S1PR1 activity while maintaining S1PR4 activity. Subsequently, the present inventors modified the polar head region of compound 22 and found that the introduction of a chiral pyrrolidine carboxylic acid further enhanced the compound's activity against S1PR4, thus discovering the selective S1PR4 agonist 30. The test results showed that compound 30 had an EC50-like activity in recruiting β-arrestin to S1PR4. 50=9.09nM. Next, we conducted a S1PR1-5 selectivity analysis on the leading compound 30. The results showed that compared to the endogenous ligand S1P, compound 30 exhibited no agonist activity against S1PR2 / 3 / 5 targets, but exhibited nearly 10-fold selectivity for S1PR1. This further demonstrated that compound 30 is a selective S1PR4 agonist, with significantly higher agonist activity against S1PR4 than the endogenous ligand S1P. Furthermore, compound 67 exhibited superior S1PR4 selectivity and activity compared to compound 30. Compound 67 exhibited activity against S1PR4 of 1.80nM, with greater than 100-fold selectivity for S1PR1.

[0451] Selectivity of compounds 30 and 67 in S1PRs

[0452] 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 β-restin recruitment activity in PathHunter CHO-K1-S1PR3 cells, expressed as EC 50 The data are the mean ± SD of at least two independent determinations and are reported as the mean ± SD (standard deviation).

[0453] Example 4. Pharmacokinetic properties of a series of compounds tested

[0454] In the discussion of structure-activity relationships, the inventors obtained the key compound 22. Then, based on 22, the polar head of the compound was modified to a chiral pyrrolidine carboxylic acid, resulting in selective S1PR4 agonists 30 and 67. We analyzed the pharmacokinetic properties of compounds 22, 30, and 67, respectively, and found that compound 22 retained a cyclobutane polar head similar to BAF312, but exhibited dual S1PR1 and S1PR4 activity, and its bioavailability was only 23.5%, indicating poor drugability. The newly discovered compounds 30 and 67 not only exhibited excellent S1PR4 agonist activity, but also had excellent oral absorption properties, greatly improved in vivo exposure, and oral bioavailability of 119.7% and 76.21%, respectively, showing greater drugability.

[0455] Pharmacokinetic parameters of compound 22 a

[0456] Pharmacokinetic parameters of compound 30 a

[0457] a SD rats (male, 3 per group) weighing 200-220 g were used for the study.

[0458] Pharmacokinetic parameters of compound 67 a

[0459] a SD rats (male, 3 per group) weighing 200-220 g were used for the study.

[0460] Example 5. Internalization function test

[0461] S1P can cause receptor internalization, inhibiting 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 1μM concentration of S1P, BAF312 and compound 30 for 1.5 hours at 37°C, and then the expression of S1PR4 receptors on the cell surface was detected immediately or after 5 hours or 20 hours. As shown in Figure 5, BAF312, as an agonist of S1PR1 and S1PR5, cannot cause S1PR4 receptor internalization because it does not have S1PR4 agonist activity. S1P can induce S1PR4 internalization, and receptor expression is restored after 20 hours. Selective S1PR4 agonist 30 has a sustained internalization ability of S1PR4 for up to 20 hours, demonstrating that compound 30 is a strong selective S1PR4 agonist. The recovery effect of compound 30 after inducing S1PR4 internalization is shown in Figure 5.

[0462] Example 6. Improvement of muscle performance and muscular dystrophy phenotype in S1PR4-R82C / NDUFA10-R217W double mutant mice using S1PR4 agonists (Compound 30)

[0463] Based on the above-mentioned studies on the relationship between targets and myopathy and the discovery of selective S1PR4 modulators, the present inventors evaluated the in vivo efficacy of compound 30 in a muscular dystrophy disease model caused by the S1PR4-R82C / NDUFA10-R217W double mutation.

[0464] Experimental results showed that administration of S1PR4 agonists could improve the dystrophic phenotype and reduce muscle inflammation and fibrosis.

[0465] This study shows that S1PR4 and NDUFA10 mutations play an important role in the pathogenesis of muscular dystrophy, and provides new inflammation-related targets and therapeutic drug options for the treatment of muscular dystrophy.

[0466] Experimental animals and groups

[0467] Experimental animals: A total of 34 S1PR4-R82C / NDUFA10-R217W mice were divided into 6 groups as shown below.

[0468] 1. Main reagents and drugs

[0469] Positive drugs: prednisolone (1 mg / kg), BAF312 (1 mg / kg), compound 30 (0.3 / 1 / 3 mg / kg)

[0470] 2 Experimental methods

[0471] Gavage administration began on day 0, and body weight was measured daily to adjust the drug dosage. Grip strength was measured at the beginning and end of the experiment, and motor changes in the mice were observed. Limb grip strength, serum creatine kinase levels, and changes in muscle fibrosis / inflammation were measured as indicators of drug efficacy.

[0472] 2.1 Test contents after drug treatment

[0473] 2.1.1 Limb Grip Strength Test

[0474] The mouse is placed horizontally on the grip test board. Grab the mouse's tail and pull it in the opposite direction. Do it gently and do not use force. Pull the mouse's tail gradually until the mouse releases the grip force receptor. The maximum value recorded by the grip force receptor is the grip strength value. Repeat 3 times and take the average value.

[0475] 2.1.2 Serum creatine kinase content detection

[0476] After the administration, blood was collected from the eye socket, allowed to stand for 30 min, and centrifuged at 3000 rpm / min and 4°C for 15 min. Serum was extracted and stored in a frozen state at -20°C. The serum was tested according to the kit instructions.

[0477] 2.1.3 Pathological examination of muscle tissue sections

[0478] After the experiment, the tibialis anterior, extensor digitorum longus, and diaphragm muscles of the mice were fixed and sectioned for HE and picrosirius red staining to observe the degree of muscle fibrosis. The sections were immunofluorescently incubated with F4 / 80 to observe the level of muscle inflammation.

[0479] The weight changes of double mutant mice after drug administration are shown in Figure 6:

[0480] After drug treatment, the double mutant mice showed no difference in weight gain trends compared with the model group in the BAF312 1 mg / kg group and the compound 30 0.3, 1, and 3 mg / kg groups. The weight gain trend of the prednisolone 1 mg / kg group was slower than that of the other groups.

[0481] The changes in grip strength of double mutant mice after drug administration are shown in Figure 7:

[0482] After treatment, the double mutant mice were measured for grip strength at the start and end of treatment. The grip strength was calculated as the ratio of grip strength to body weight to account for the effect of body weight. Initially, the grip strength of the double mutant mice was significantly lower than that of wild-type mice. However, after 21 days of treatment, grip strength significantly increased in the 0.3 and 1 mg / kg groups of compound 30 and in the 1 mg / kg prednisolone group, demonstrating a promising therapeutic effect.

[0483] The changes in creatine kinase in double mutant mice are shown in Figure 8:

[0484] The serum creatine kinase levels in the double mutant mice were significantly elevated compared to those in healthy mice. Compound 30 (1 mg / kg, 4 mg / kg) and BAF312 (1 mg / kg) significantly reduced creatine kinase levels after treatment, while prednisolone (1 mg / kg) had no effect.

[0485] The current diagnostic standard for muscular dystrophy is muscle biopsy, which ultimately determines improvement in muscular dystrophy by observing the level of muscle fibrosis and inflammation. The degree of fibrosis can be determined by observing the number of nucleated fibers in the muscle using HE staining. Normal muscle fibers have normal spacing between them, with cell nuclei located peripherally, and no inflammatory cell infiltration. Muscles that undergo fibrosis experience hypertrophy and atrophy, resulting in varying muscle fiber sizes, rounded hypertrophic fiber contours, inward displacement of myocyte nuclei, and extensive inflammatory cell infiltration surrounding damaged cells. Regenerating muscle fibers with centrally located nuclei, which are almost completely absent in undamaged muscle, can serve as markers of prior necrosis and regeneration cycles.

[0486] Representative images of intermediate nucleated fibers in double mutant mice are shown in Figure 9 , and nucleated fiber statistics are shown in Figure 10 .

[0487] The degree of muscle fibrosis can be assessed by counting the number of centrally nucleated fibers in the muscle. Statistical results showed that the model group had the highest proportion of centrally nucleated fibers. After treatment, all treatment groups significantly reduced the number of centrally nucleated fibers. Compound 30 at 1 mg / kg reduced nucleated fibers the most, more significantly than the BAF312 1 mg / kg and prednisolone 1 mg / kg groups. This suggests that Compound 30 at 1 mg / kg is more effective than those in the BAF312 1 mg / kg and prednisolone 1 mg / kg groups. Furthermore, the reduction observed with Compound 30 at 1 mg / kg was greater than with Compound 30 at 3 mg / kg. This suggests that Compound 30 can achieve therapeutic efficacy at 1 mg / kg.

[0488] Representative graphs of collagen area in double mutant mice are shown in FIG11 ; and collagen area statistics are shown in FIG12 .

[0489] Another indicator of fibrosis severity is collagen area. Compared to healthy controls, patients with muscular dystrophy exhibit collagen deposition, resulting in a significantly increased collagen area ratio within the muscle. Statistical results showed that the model group had the highest collagen area ratio. Treatment with compound 30 (1mg / kg, 3mg / kg), BAF312 (1mg / kg), and prednisolone (1mg / kg) significantly reduced collagen deposition.

[0490] Based on the number of centrally nucleated fibers and the proportion of collagen deposition, compound 30 1 mg / kg, compound 30 3 mg / kg, BAF312 1 mg / kg, and prednisolone 1 mg / kg groups significantly improved the degree of fibrosis in double mutant mice. The therapeutic effect of compound 30 1 mg / kg was stronger than that of prednisolone 1 mg / kg, indicating that compound 30 1 mg / kg is sufficient for therapeutic effects.

[0491] Representative images of macrophage immunofluorescence of double mutant mice are shown in FIG13 ; and statistical images of macrophage immunofluorescence are shown in FIG14 .

[0492] One of the typical pathological characteristics of muscular dystrophy is muscle inflammation. After drug treatment, the double mutant mice showed a significant reduction in macrophage content and alleviated muscle inflammation in all drug treatment groups, among which the compound 30 1 mg / kg group had the largest reduction.

[0493] Taken together, these results indicate that compound 30 (1 mg / kg) significantly improved the pathological features of double-mutant mice, including reduced creatine kinase levels, decreased fibrosis, and reduced muscle inflammation, achieving a therapeutic effect. This therapeutic effect was stronger than that observed with prednisolone (1 mg / kg), a positive drug, suggesting that compound 30 may be a promising new compound for the treatment of muscular dystrophy.

[0494] Example 7. In vivo efficacy test on Duchenne muscular dystrophy (Compound 30)

[0495] To verify the potential application of compound 30 in other myopathy models, we further selected the Duchenne muscular dystrophy model for in vivo efficacy evaluation. The experimental plan and results are as follows:

[0496] 1 Experimental Materials

[0497] Experimental animals and groups

[0498] Experimental animals: A total of 35 Duchenne muscular dystrophy model mice were divided into 6 groups as shown below.

[0499] Main reagents and drugs

[0500] Positive drug prednisone (1mg / kg): Duchenne muscular dystrophy has the highest incidence among all muscular dystrophy subtypes and is also the most widely studied of all muscular dystrophy subtypes. Currently approved treatments for Duchenne muscular dystrophy include anti-inflammatory glucocorticoids and gene therapy.

[0501] 2 Experimental methods

[0502] Drug administration began via gavage on day 0, with daily body weight measurements to adjust drug dosage. Grip strength was tested every two days until the end of the experiment on day 21, and changes in the mice's grip strength were observed. Limb grip strength, serum creatine kinase levels, and changes in muscle fibrosis / inflammation were measured as indicators of drug efficacy.

[0503] 2.1 Test contents after drug treatment

[0504] 2.1.1 Limb Grip Strength Test

[0505] The mouse is placed horizontally on the grip test board. Grab the mouse's tail and pull it in the opposite direction. Do it gently and do not use force. Pull the mouse's tail gradually until the mouse releases the grip force receptor. The maximum value recorded by the grip force receptor is the grip strength value. Repeat 3 times and take the average value.

[0506] 2.1.2 Serum creatine kinase content detection

[0507] After the administration, blood was collected from the eye socket, allowed to stand for 30 min, and centrifuged at 3000 rpm / min and 4°C for 15 min. Serum was extracted and stored in a frozen state at -20°C. The serum was tested according to the kit instructions.

[0508] 2.1.3 Pathological examination of muscle tissue sections

[0509] After the experiment, the tibialis anterior, extensor digitorum longus, and diaphragm muscles of the mice were fixed and sectioned for HE and Sirius red staining to observe the degree of muscle fibrosis. The sections were immunohistochemically incubated with F4 / 80 to observe the level of muscle inflammation.

[0510] The changes in body weight of Duchenne muscular dystrophy model mice after administration are shown in FIG15 .

[0511] After drug treatment, the weight gain trends of Duchenne muscular dystrophy model mice were similar in the BAF312 1 mg / kg group, prednisolone group, and compound 30 0.3 and 1 mg / kg groups compared with the model group. The weight gain trend of compound 30 3 mg / kg was slower than that of the other groups.

[0512] The changes in grip strength of Duchenne muscular dystrophy model mice after drug administration are shown in FIG16 .

[0513] After treatment, the grip strength of Duchenne muscular dystrophy model mice was measured every two days. The ratio of grip strength to body weight was calculated to represent changes in grip strength, eliminating the influence of body weight. During the first 14 days, grip strength increased in all groups, with the rate of increase in the treatment group exceeding that of the model group. Compound 30 at 3 mg / kg showed the most significant increase, demonstrating a positive therapeutic effect. However, after 14 days, grip strength in all groups began to decline. Analysis suggests that the frequent grip strength measurements may have caused the mice to become accustomed to the grip tests and no longer resisted aggressively.

[0514] The changes in creatine kinase in Duchenne muscular dystrophy model mice are shown in Figure 17

[0515] Serum creatine kinase levels in Duchenne muscular dystrophy model mice were significantly elevated compared to healthy mice. Treatment with compound 30 at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, as well as prednisolone at 1 mg / kg, significantly reduced creatine kinase levels. BAF312 at 1 mg / kg had no effect. Furthermore, the magnitude of the reduction in the 1 mg / kg 30 group was greater than that in the 1 mg / kg prednisolone group, indicating that 1 mg / kg 30 was more effective than prednisolone.

[0516] The current gold standard for diagnosing muscular dystrophy is muscle biopsy, which ultimately determines improvement in muscular dystrophy by observing the level of muscle fibrosis and inflammation. The degree of fibrosis can be determined by observing the number of nucleated fibers in the muscle using HE staining. Normal muscle fibers have normal spacing between them, with cell nuclei located peripherally and no inflammatory cell infiltration. Muscles undergoing fibrosis experience hypertrophy and atrophy, resulting in varying muscle fiber sizes, rounded hypertrophic fiber contours, inward displacement of myocyte nuclei, and extensive inflammatory cell infiltration surrounding damaged cells. Regenerating muscle fibers with centrally located nuclei, which are almost completely absent in undamaged muscle, can serve as markers of prior necrosis and regeneration cycles.

[0517] Representative intermediate nucleated fibers of Duchenne muscular dystrophy model mice are shown in FIG18 ; and nucleated fiber statistics are shown in FIG19 .

[0518] Counting the number of centrally nucleated fibers in muscle can be used to assess the degree of muscle fibrosis. Statistical results showed that the model group had the highest proportion of centrally nucleated fibers. After treatment, the Compound 30 1mg / kg, Compound 30 3mg / kg, BAF312 1mg / kg, and Prednisolone 1mg / kg groups all significantly reduced the number of centrally nucleated fibers. However, the Compound 30 0.3mg / kg group showed no significant difference compared to the Model group. The Compound 30 1mg / kg and Compound 30 3mg / kg groups showed the greatest reduction in centrally nucleated fibers, exceeding those observed in the BAF312 1mg / kg and Prednisolone 1mg / kg groups. This suggests that Compound 30 1mg / kg and Compound 30 3mg / kg treatments are more effective than those in the BAF312 1mg / kg and Prednisolone 1mg / kg groups. Furthermore, there was no difference in treatment effect between Compound 30 1mg / kg and Compound 30 3mg / kg, indicating that Compound 30 1mg / kg is sufficient to achieve therapeutic efficacy.

[0519] The collagen area of ​​Duchenne muscular dystrophy model mice is represented in FIG20 ; the collagen area statistics are shown in FIG21 .

[0520] Another indicator of fibrosis severity is collagen area. Compared to healthy controls, patients with muscular dystrophy exhibit collagen deposition, resulting in a significant increase in the collagen area percentage in muscle. Experimental statistical results showed that the model group had the highest collagen area percentage. After treatment, the compound 30 0.3 mg / kg, 1 mg / kg, and 3 mg / kg groups, as well as the prednisolone 1 mg / kg group, significantly reduced collagen deposition. The BAF312 1 mg / kg group showed no difference from the model group, indicating that it was unable to reduce collagen deposition. Compound 30 1 mg / kg and 3 mg / kg were more effective in reducing collagen deposition than the prednisolone 1 mg / kg group, and there was no difference in the effect of compound 30 1 mg / kg and 3 mg / kg on reducing collagen deposition.

[0521] Based on the number of intermediate nucleated fibers and the proportion of collagen deposition, compound 30 (1 mg / kg, 3 mg / kg), and prednisolone (1 mg / kg) significantly improved the degree of fibrosis in Duchenne muscular dystrophy mice. BAF312 (1 mg / kg) showed less improvement in muscle fibrosis. The therapeutic effects of compound 30 (1 mg / kg, 3 mg / kg) were stronger than those of prednisolone (1 mg / kg), and there was no difference in the therapeutic effects between compound 30 (1 mg / kg, 3 mg / kg), indicating that compound 30 (1 mg / kg) is sufficient for therapeutic effects.

[0522] Representative images of macrophage immunofluorescence of Duchenne muscular dystrophy model mice are shown in FIG22 ; statistical images of macrophage immunofluorescence are shown in FIG23 .

[0523] One of the hallmark pathological features of Duchenne muscular dystrophy is muscle inflammation. Glucocorticoids, the current first-line treatment for Duchenne muscular dystrophy, work by alleviating muscle inflammation. In Duchenne muscular dystrophy model mice, treatment with Compound 30 at 1mg / kg and 3mg / kg, and prednisolone at 1g / kg, significantly reduced macrophage counts and alleviated muscle inflammation. However, BAF312 at 1mg / kg had no effect on muscle inflammation.

[0524] Taken together, these results indicate that compound 30 at doses of 1 and 3 mg / kg, along with prednisolone at 1 mg / kg, significantly improved the pathological features of the Duchenne muscular dystrophy model in mice, including reduced creatine kinase levels, decreased fibrosis, and reduced muscle inflammation, achieving a therapeutic effect. Furthermore, the therapeutic effects of compound 30 at 1 and 3 mg / kg were stronger than those of the active prednisolone 1 mg / kg group, indicating that compound 30 has the potential to become a new treatment for Duchenne muscular dystrophy and further demonstrating the research value of selective S1PR4 agonists in the treatment of myopathies.

[0525] Example 8. Pharmacodynamic evaluation of compound 30 on sepsis-induced myopathy

[0526] Sepsis-induced myopathy (SIM) is defined as a rapidly progressive muscle disorder that affects muscle electrophysiology and morphology, and can affect both respiratory and limb muscles. Approximately 40% of patients with severe sepsis develop intensive care unit-acquired weakness (ICUAW), characterized by loss of muscle mass, decreased muscle fiber size, and decreased muscle strength, ultimately leading to persistent physical impairment.

[0527] Injecting exogenous toxins into animals can establish an animal model of sepsis. In this study, intraperitoneal injection of lipopolysaccharide (LPS) was used to establish a sepsis model and to investigate the effects of compounds on the development and progression of acquired myasthenia gravis. This is a widely used method for modeling sepsis. LPS injection in mice showed increased sepsis severity, decreased forelimb grip strength, decreased compound muscle action potential (CMAP) amplitude and prolonged latency, and decreased muscle fiber diameter and cross-sectional area, indicating that a mouse model of sepsis-induced myasthenia gravis was successfully established.

[0528] Experimental methods

[0529] The lipopolysaccharide-induced skeletal muscle atrophy model was used, which can simulate the muscle atrophy caused by sepsis in the clinic.

[0530] Mice were intraperitoneally injected with LPS (1 mg / kg) once, followed by continuous intraperitoneal injections of solvent (1% DMSO dissolved in saline) or compound 30 for 14 days. The mice were weighed daily. After 14 days of LPS treatment, the mice were anesthetized and the gastrocnemius and tibialis anterior muscles were removed.

[0531] Experimental Materials

[0532] 2.1 Experimental animals and groups

[0533] 2.2 Main reagents and drugs

[0534] LPS, compound 30;

[0535] 3. Experimental Results

[0536] After a single intraperitoneal injection of LPS (1 mg / kg), 14 days after the injection, the survival rate of the control group was 100% (8 (survived) / 8 (total)), the survival rate of the LPS group was 50% (4 (survived) / 8 (total)), the survival rate of the high-dose group of compound 30 was 87.5% (7 (survived) / 8 (total)), the survival rate of the medium-dose group of compound 30 was 50% (4 (survived) / 8 (total)), and the survival rate of the low-dose group of compound 30 was 75%.

[0537] The effects of compound 30 on the tibialis anterior muscle and gastrocnemius muscle in the LPS-induced sepsis model are shown in FIG24 .

[0538] in conclusion:

[0539] After 14 days of LPS treatment, mice were anesthetized and the gastrocnemius and tibialis anterior muscles were harvested. Both the gastrocnemius and tibialis anterior muscles in the model group atrophied compared to the control group, and medium and high doses of compound 30 significantly inhibited tibialis anterior muscle atrophy.

[0540] Example 9. In vivo efficacy test of Duchenne muscular dystrophy (Compound 67) Pharmacodynamic evaluation in mdx mice

[0541] 1. Experimental Materials

[0542] 1.1 Experimental animals and grouping

[0543] Experimental animals: A total of 18 Duchenne muscular dystrophy model mice were divided into 3 groups, with 6 mice in each group.

[0544] 1.2 Main reagents and drugs

[0545] Positive drug Vamorolone (20mg / kg): Duchenne muscular dystrophy is the most common muscular dystrophy subtype and the most widely studied of all muscular dystrophy subtypes. Recently, the steroid drug Vamorolone was approved by the US FDA for the treatment of Duchenne muscular dystrophy, while also minimizing the risk of side effects.

[0546] Main instruments and equipment

[0547] Hand Grip DynamometerShanghai Xinruan Information Technology Co., Ltd.

[0548] 2. Experimental Methods

[0549] Drug administration began via gavage on day 0, with daily body weight measurements to adjust drug dosage. Grip strength and rotarod testing were performed on days 20 and 42. Grip strength, exercise duration, and changes in muscle fibrosis / inflammation were measured in all four limbs to evaluate drug efficacy.

[0550] 2.1 Test contents after drug treatment

[0551] 2.1.1 Limb Grip Strength Test

[0552] The mouse is placed horizontally on the grip test board. Grab the mouse's tail and pull it in the opposite direction. Do it gently and do not use force. Pull the mouse's tail gradually until the mouse releases the grip force receptor. The maximum value recorded by the grip force receptor is the grip strength value. Repeat 3 times and take the average value.

[0553] 2.1.2 Rotarod test

[0554] Rotarod test was performed on day 0, day 20 and day 42. The test was performed at an accelerating speed (0-16 rpm), reaching the maximum speed after 300 s, and the maximum test time was 600 s. The time until the animal fell was recorded. 7 .

[0555] 2.1.3 Pathological examination of muscle tissue sections

[0556] After the experiment, the tibialis anterior muscle, extensor digitorum longus muscle and diaphragm of the mice were fixed and sectioned for HE staining and Sirius red staining to observe the degree of muscle fibrosis.

[0557] Experimental results:

[0558] After the Duchenne muscular dystrophy model mice were treated with the drug, there was no significant difference in body weight changes between the drug treatment groups compared with the model group ( FIG25 ).

[0559] After 42 days of treatment, the grip strength of mice in all treatment groups increased significantly compared to the model group. Compound 673 mg / kg and Vamorolone 20 mg / kg significantly increased grip strength, demonstrating a good therapeutic effect (Figure 26).

[0560] Rotarod duration can be used to assess animal exercise tolerance. On day 42 of treatment, rotarod duration increased in all treatment groups compared to the model group. Grip strength increased significantly in both the Compound 67 3 mg / kg and Vamorolone 20 mg / kg groups, demonstrating a favorable therapeutic effect. The increase in grip strength was more pronounced in the Compound 67 3 mg / kg group than in the Vamorolone 20 mg / kg group (Figure 27).

[0561] The current gold standard for diagnosing muscular dystrophy is muscle biopsy, which ultimately determines improvement in muscular dystrophy by observing the level of muscle fibrosis and inflammation. The degree of fibrosis can be determined by observing the number of nucleated fibers in the muscle using HE staining. Normal muscle fibers have normal spacing between them, with peripherally located nuclei and no inflammatory cell infiltration. Muscles undergoing fibrosis experience hypertrophy and atrophy, resulting in varying muscle fiber sizes, rounded hypertrophic fibers, inward displacement of myocyte nuclei, and extensive inflammatory cell infiltration surrounding damaged cells. Regenerating muscle fibers with centrally located nuclei, which are almost completely absent in intact muscle, can serve as a marker of prior necrosis-regeneration cycles. Figure 28 shows the centrally located nucleated fibers after administration of compound 67 and vamorolone. Figure 29 shows the count of nucleated fibers after administration of compound 67 and vamorolone.

[0562] The degree of muscle fibrosis can be assessed by counting the number of centrally nucleated fibers in the muscle. Statistical results showed that the model group had the highest proportion of nucleated fibers. After treatment, Compound 67 (3 mg / kg) and Vamorolone (20 mg / kg) significantly reduced the number of centrally nucleated fibers. The Compound 67 (3 mg / kg) group showed the greatest reduction in nucleated fibers, greater than the BAF312 (1 mg / kg) and Vamorolone (20 mg / kg) groups, indicating that Compound 67 (3 mg / kg) was more effective than the Vamorolone (20 mg / kg) group. The collagen area after administration of Compound 67 and Vamorolone is shown in Figure 30.

[0563] Another indicator of fibrosis severity is collagen area. Compared to healthy subjects, patients with muscular dystrophy exhibit collagen deposition, resulting in a significant increase in the percentage of collagen area in muscle. Experimental statistical results showed that the model group had the highest percentage of collagen area. After treatment, the Compound 67 3mg / kg and Vamorolone 20mg / kg groups significantly reduced collagen deposition. The effect of Compound 67 3mg / kg on reducing collagen deposition was no different from that of Vamorolone 20mg / kg. Based on the number of intermediate nucleated fibers and the percentage of collagen deposition, the Compound 67 3mg / kg and Vamorolone 20mg / kg groups significantly improved the degree of fibrosis in Duchenne muscular dystrophy mice. The collagen area statistics after administration of Compound 67 and Vamorolone are shown in Figure 31.

[0564] One of the typical pathological characteristics of Duchenne muscular dystrophy is muscle inflammation. The current first-line drug for treating Duchenne muscular dystrophy, glucocorticoids, exerts its therapeutic effect by reducing muscle inflammation. After drug administration to Duchenne muscular dystrophy model mice, the compound 67 3mg / kg and Vamorolone 20mg / kg groups significantly reduced the content of macrophages and reduced muscle inflammation. In addition, there was no significant difference in the F4 / 80 reduction effect between the compound 67 3mg / kg and Vamorolone 20mg / kg groups, indicating that the two groups had similar effects in improving muscle inflammation. The number of F4 / 80 macrophage markers (muscle inflammation) after administration of compound 67 and Vamorolone is shown in Figure 32. The statistics of the number of F4 / 80 macrophage markers (muscle inflammation) after administration of compound 67 and Vamorolone are shown in Figure 33.

[0565] Serum creatine kinase levels in Duchenne muscular dystrophy model mice were significantly elevated compared to healthy mice. Following treatment, both the vamorolone 20 mg / kg group and the compound 67 3 mg / kg group significantly reduced creatine kinase levels. Furthermore, the compound 67 3 mg / kg group exhibited a stronger creatine kinase-lowering effect than the vamorolone 20 mg / kg group, indicating that the therapeutic effect of the compound 67 3 mg / kg group was superior to that of the vamorolone 20 mg / kg group. Serum creatine kinase levels after administration of compound 67 and vamorolone are shown in Figure 34.

[0566] Based on all the above results, compound 67 3 mg / kg and Vamorolone 20 mg / kg can significantly improve the symptoms and pathological characteristics of Duchenne muscular dystrophy model mice, including reducing fibrosis levels, alleviating muscle inflammation and lowering serum creatine kinase levels, thereby achieving a therapeutic effect.

[0567] discuss:

[0568] After extensive and in-depth research, the inventors first confirmed that S1PR4 and NDUFA10 play a crucial role in the pathogenesis of muscular dystrophy, providing novel targets for its treatment. They then designed and synthesized a series of previously unreported small-molecule S1P receptor modulators. These compounds were then tested for cellular activity, yielding a group capable of regulating S1PR4 and S1PR4 mutant proteins. In vivo pharmacokinetic parameters were determined for the most active S1PR4 agonist, demonstrating good oral absorption and promising development potential. In internalization testing, compounds 30 and 67 demonstrated sustained internalization, a capability not achieved by non-selective agonists. In the Duchenne muscular dystrophy mouse model and the S1PR4-R82C / NDUFA10-R217W mouse muscular dystrophy model, compounds 30 and 67 demonstrated significantly superior in vivo efficacy to the active drugs prednisone and BAF312, further demonstrating the significant value of targeting S1PR4 for the treatment of muscular dystrophy diseases and demonstrating that compounds 30 and 67 have a significant therapeutic effect on diseases caused by S1PR4 and S1PR4 mutations. Furthermore, in the LPS-induced sepsis model, medium and high doses of compounds 30 and 67 significantly inhibited tibialis anterior muscle atrophy.

[0569] The above work lays the foundation for the treatment of muscular dystrophy and other myopathies mediated by S1PR4 receptor.

[0570] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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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