Polyol derivative for antagonizing and inhibiting TRPV4-rhoa target and use thereof

Abstract

Disclosed in the present invention are a compound represented by formula I, or a pharmaceutically acceptable salt thereof or an ester thereof, a prodrug thereof, an optical isomer thereof, a stereoisomer thereof, or a solvate thereof as an antagonist or an inhibitor for antagonizing and inhibiting a TRPV4-RhoA target, and use thereof in treating TRPV4-RhoA target-related diseases. The class of compounds exhibits an excellent effect of antagonizing TRPV4 and inhibiting RhoA activity, thereby laying a brand-new substance foundation for developing drugs for treating TRPV4-RhoA target-related diseases.

Description

Polyol derivatives antagonizing and inhibiting TRPV4-RhoA target and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry. Specifically, the present application relates to novel polyol derivatives and their application as derivatives antagonizing and inhibiting TRPV4-RhoA target in the preparation of drugs for related diseases. BACKGROUND

[0002] TRPV4 (transient receptor potential vanilloid 4) is a non-selective cation channel, widely distributed in various tissues and cell types, involved in the regulation of mechanical force, osmotic pressure, temperature and inflammation, and other physiological and pathological processes. TRPV4 antagonists show therapeutic potential in various diseases by inhibiting the excessive activation of the channel. In the past 10 years, it has been confirmed that it is widely present in vascular smooth muscle, endothelial cells, alveolar epithelial cells and bronchus, and plays an important role in the pathophysiological mechanism of lung diseases. The main indications include: respiratory diseases such as pulmonary edema, chronic obstructive pulmonary disease (COPD), asthma, etc.; pain and inflammation such as osteoarthritis, neuropathic pain, visceral pain (such as irritable bowel syndrome), etc.; nervous system diseases such as spinal cord injury / cerebral edema, neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.); cardiovascular diseases such as heart failure, hypertension, stroke, atrial fibrillation, etc.; urinary system diseases such as overactive bladder, etc.; skin diseases such as eczema, itching, etc. and eye diseases such as glaucoma, regulation of intraocular pressure and retinal nerve protection, etc. TRPV4 is expressed in various tissues, including the cardiovascular system, kidney, lung and nervous system. Studies have found that TRPV4 plays an important role in the pathophysiological process of heart failure (heart failure).

[0003] At present, TRPV4 antagonists have not been approved for marketing, and most indications are still in the preclinical or early clinical research stage, but their potential in multiple system diseases is worth attention.

[0004] RhoA (Ras homolog family member A) is a small GTPase that regulates a variety of physiological processes (such as cytoskeleton reorganization, migration, proliferation, etc.) in cell signaling. Its abnormal expression or activation is associated with a variety of diseases. The main indications for RhoA or its signaling pathway currently include: cardiovascular diseases such as hypertension, atherosclerosis, heart failure, etc.; cancers such as breast cancer, gastric cancer, colorectal cancer, melanoma, etc.; nervous system diseases such as neurodegenerative diseases (such as Alzheimer's disease, amyotrophic lateral sclerosis); spinal cord injury and autoimmune and inflammation, etc.; other potential indications such as fibrotic diseases (such as pulmonary fibrosis, liver fibrosis); regulation of osteoclast function in osteoporosis. RhoA signaling pathway promotes the progression of heart failure by promoting myocardial hypertrophy, fibrosis, oxidative stress and vascular dysfunction. Drugs targeting this pathway (especially ROCK) show therapeutic potential and may become one of the targets for precise treatment of heart failure in the future.

[0005] The TRPV4-RhoA-RhoGDI1 axis is a molecular pathway involved in mechanical signal transduction and cytoskeleton regulation, mainly participating in processes such as cell mechanosensing, migration, and morphological maintenance. The composition and function of the axis: TRPV4 calcium channel, which can be activated by mechanical force, osmotic pressure, temperature (warm) and lipid mediators (such as arachidonic acid metabolites). After activation, it leads to Ca 2+ influx, triggering downstream signals. RhoA switches between active and inactive states through GTP / GDP cycles (GTP-bound state is active). There is a bidirectional regulatory relationship between TRPV4 and RhoA. TRPV4 binds to non-active RhoA-GDP, thereby inhibiting RhoA activation; RhoA itself can also inhibit channel activity by binding to TRPV4. RhoGDI1 (Rho GDP Dissociation Inhibitor 1) inhibits the activity of RhoA by binding and isolating RhoA-GDP in the cytoplasm, preventing its membrane localization and activation.

[0006] The team found that a new polyol compound can selectively inhibit TRPV4 channel activity, and the cryo-EM structure of hTRPV4-compound 1-R was resolved. The data showed that TRPV4 channel in the TRPV4-compound 1-R-RhoA complex was in a closed state, and it could stabilize RhoA in the GDP-bound inactive state. The RhoA-compound 1-R complex crystal was also obtained, and the binding of RhoA further prompted the closure of the TRPV4 ion channel. This is to further explore the effect of compound 1-R on the interaction between TRPV4 and RhoA, and the PLA (proximity ligation assay) experiment was carried out in VSMC (vascular smooth muscle cells). The results showed that the derivatives such as compound 1-R can significantly promote the binding of TRPV4 and RhoA.

[0007] Therefore, the new polyol compound can inhibit the activity of RhoA through the above mechanism. RhoA can mediate the contraction response of vascular smooth muscle by activating downstream effector ROCK (Rho-associated coiled coil protein kinase). Among them, the phosphorylation level of MLC (myosin light chain) determines the degree of VSMC contraction, and MLCK can mediate the phosphorylation process of MLC, and MLCP mediates the dephosphorylation process of MLC. The results show that the new polyol compound can inhibit VSMC contraction by blocking the RhoA-ROCK pathway, achieve blood pressure reduction, and improve heart failure.

[0008] Heart failure (Heart Failure) is a complex clinical syndrome caused by the progression of various heart diseases to a severe stage, and its main features are left ventricular and / or right ventricular dysfunction and changes in neural and humoral regulation, often accompanied by dyspnea, fluid retention, reduced exercise tolerance, and significantly shortened survival time. Patients with underlying heart disease are more likely to develop heart failure. Heart failure has complex physiological and pathological mechanisms, and multiple neural and humoral mechanisms play an important role.

[0009] Heart failure is difficult to cure, and the treatment goal is to prevent and delay the development of heart failure, relieve clinical symptoms, improve quality of life, and improve long-term prognosis. Global multinational pharmaceutical companies and biotechnology companies have developed innovative drug projects targeting different targets and mechanisms for many years, and have recently entered the harvest period. However, in clinical practice, adverse reactions and high prices of drugs lead to poor patient compliance, and it is imperative to develop safe and efficient anti-heart failure drugs.

[0010] TRPV4 (Transient Receptor Potential Vanilloid Subtype 4) is a non-selective cation channel that mainly participates in the regulation of calcium ions (Ca 2+ ) and sodium ions (Na +TRPV4 is a Ca2+-permeable nonselective cation channel activated by intracellular Ca2+ and intracellular osmotic pressure. It is expressed in a variety of tissues, including the cardiovascular system, kidney, lung and nervous system. In recent years, studies have found that TRPV4 plays an important role in the pathophysiological process of heart failure,

[0011] Lipopolysaccharide (LPS) is a common inducer of acute lung injury (ALI), and is often used to induce the establishment of an ALI model. Pulmonary edema and a large amount of inflammatory exudation are one of the main pathophysiological characteristics. Studies have shown that blocking the TRPV4 signal can reduce the inflammatory level of LPS-induced ALI, and has a protective effect on ALI mice, including improving the pathological characteristics of the lung, reducing pulmonary edema and inflammation; therefore, TRPV4 may become a new target for the treatment of ALI in the future.

[0012] Therefore, the development of drugs that antagonize TRPV4 and inhibit RhoA has great clinical significance and application prospects. SUMMARY

[0013] The purpose of the present application is to provide polyol derivatives as TRPV4 antagonists.

[0014] The purpose of the present application is to provide polyol derivatives as TRPV4-RhoA target-related antagonists and inhibitors.

[0015] Another purpose of the present application is to disclose the mechanism of the above-mentioned compounds antagonizing TRPV4-RhoA.

[0016] Another purpose of the present application is to provide the use of the above-mentioned compounds in the preparation of drugs for treating TRPV4-RhoA target-related diseases or antagonizing TRPV4-RhoA targets.

[0017] In a first aspect, the present application provides a compound represented by Formula I, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer or solvate thereof, for use in the preparation of a TRPV4-RhoA target inhibitor or antagonist,

[0018] In the formula,

[0019] A is selected from a C6-C10 aromatic ring or a carbocyclic ring, or a 5-6 membered heterocyclic ring or aromatic heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O or S;

[0020] R1and R4are each independently selected from the group consisting of H, D, OH, carbonyl, optionally substituted C1-C10alkyl, optionally substituted C1-C10alkoxy, optionally substituted phenoxy, optionally substituted benzyloxy, optionally substituted NH2, NHCH3, NHCOCH3, halogen, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted aminoacetoxy B can be a variety of amino acid forming groups, sulfonato or sulfonate ester, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0021] R2and R5are each independently selected from the group consisting of H, D, OH, carbonyl, hydroxymethyl, optionally substituted C1-C10alkyl, optionally substituted C1-C10alkoxy, optionally substituted benzyloxy, optionally substituted phosphate, optionally substituted NH2, NHCH3, NHCOCH3, optionally substituted C1-C10carbonyloxy, optionally substituted benzoyloxy, optionally substituted aminoacetoxy B can be a variety of amino acid forming groups, COOH, COOCH3, CONH2, sulfonato or sulfonate ester, halogen, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0022] or

[0023] R1and R2or R4and R5are linked to form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), a cyclic carbonate, a cyclic phosphate or an optionally substituted cyclic borate;

[0024] R3is selected from the group consisting of H, D, optionally substituted C1-C10alkyl, D substituted C1-C10alkyl, optionally substituted C3-C10cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 heteroatoms selected from O, S or N, optionally substituted C2-C10alkenyl, optionally substituted C2-C10cycloalkenyl, cyano, hydroxyl, optionally substituted C1-C10alkoxy, optionally substituted C1-C10alkylthio, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted C1-C10acyl, hydroxycarbonyl, optionally substituted C1-C10alkylcarbonyloxy, nitro, optionally substituted amino, optionally substituted C1-C10alkylamino, optionally substituted C1-C10alkylcarbonylamino, halogen, optionally substituted C2-6alkynyl, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0025] or

[0026] two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen or nitrogen), an optionally substituted C5-C10aromatic ring or an optionally substituted C5-C10heteroaromatic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen or nitrogen);

[0027] m is an integer from 1 to 5;

[0028] hydrogen in the structure of formula I is optionally replaced by deuterium.

[0029] In a specific embodiment, the compound of formula I is a compound of formula II,

[0030] wherein,

[0031] X1, X2, X3are each independently selected from CH, N, S, O or absent (preferably CH or N);

[0032] R1, R4are each independently selected from H, D, OH, optionally substituted C1-C10alkoxy, optionally substituted phenoxy, optionally substituted benzyloxy, optionally substituted NH2, NHCH3, NHCOCH3, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing amino, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0033] R2and R5are each independently selected from H, D, OH, hydroxymethyl, optionally substituted C1-C10alkyl, optionally substituted C1-C10alkoxy, optionally substituted benzyloxy, NH2, NHCH3, NHCOCH3, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing amino, COOH, COOCH3, CONH2, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0034] R1, R2may form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen);

[0035] R3is selected from the group consisting of H, D, optionally substituted C1-C10alkyl, D- substituted C1-C10alkyl, optionally substituted C3-C10cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 heteroatoms selected from O, S or N, optionally substituted C1-C10alkylalkenyl, hydroxyl, optionally substituted C1-C10alkoxy, optionally substituted C1-C10alkylthio, optionally substituted C1-C10alkylcarbonyloxy, formyl, hydroxyformyl, optionally substituted C1-C10alkylcarbonyloxy, nitro, optionally substituted amino, optionally substituted C1-C10alkylamino, optionally substituted C1-C10alkylcarbonylamino, halogen, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0036] or optionally substituted C3-C6carbocyclic ring or optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen or nitrogen formed by two adjacent R3substituents;

[0037] m is an integer from 1 to 5;

[0038] hydrogen in the structure of formula II is optionally replaced by deuterium.

[0039] In a particular embodiment, the compound of formula I is a compound of formula III

[0040] wherein,

[0041] R1, R4are each independently selected from the group consisting of H, D, OH, optionally substituted C1-C10alkoxy, optionally substituted phenoxy, optionally substituted benzyloxy, optionally substituted NH2, NHCH3, NHCOCH3, optionally substituted C1-C10carbonyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing group, optionally substituted aminoacetoxy containing group, B can be various groups forming amino acids), optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0042] R2and R5are each independently selected from the group consisting of H, D, hydroxymethyl, optionally substituted C1-C10alkyl, OH, optionally substituted C1-C10alkoxy, optionally substituted benzyloxy, NH2, NHCH3, NHCOCH3, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing group, optionally substituted aminoacetoxy containing group, B can be various groups forming amino acids), COOH, COOCH3, CONH2, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0043] R1, R2may form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur, preferably oxygen;

[0044] R3is selected from the group consisting of H, D, optionally substituted C1-C10alkyl, D- substituted C1-C10alkyl, optionally substituted C3-C10cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 heteroatoms selected from O, S or N, optionally substituted C2-C10alkylalkenyl, hydroxyl, optionally substituted C1-C10alkoxy, optionally substituted C1-C10alkylthio, optionally substituted C1-C10alkylcarbonyloxy, formyl, hydroxyformyl, optionally substituted C1-C10alkylcarbonyloxy, nitro, amino, optionally substituted C1-C10alkylamino, optionally substituted C1-C10alkylcarbonylamino, fluorine, chlorine, bromine, optionally substituted monosaccharide, disaccharide or polysaccharide group;

[0045] Alternatively, two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 oxygen;

[0046] m is an integer from 1 to 5;

[0047] The hydrogens in the structure of Formula III are optionally replaced by deuterium.

[0048] In a particular embodiment, the compound is of the formula IV,

[0049] R1is selected from the group consisting of H, D, OH, NH2, optionally substituted C1-C3alkoxy, optionally substituted C1-C3alkylcarbonyloxy, optionally substituted carbamoyloxy, optionally substituted aminoacetoxy B can be a variety of groups that form amino acids), optionally substituted monosaccharide group;

[0050] R2is selected from the group consisting of OH, NH2, COOH, COONH2, COONHCH3, optionally substituted C1-C3alkoxycarbonyl, optionally substituted C1-C3alkylcarbonyloxy, optionally substituted carbamoyloxy, optionally substituted aminoacetoxy B can be a variety of groups that form amino acids), optionally substituted monosaccharide group;

[0051] R3is selected from the group consisting of H, D, optionally substituted C1-C5alkyl, D- substituted C3-C5cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 O, optionally substituted C2-C10alkylalkenyl, hydroxyl, optionally substituted C1-C5alkoxy, optionally substituted C1-C3alkylthio, optionally substituted C1-C3alkylcarbonyloxy, formyl, optionally substituted C1-C3alkylcarbonyl, hydroxyformyl, nitro, amino, optionally substituted C1-C3alkylamino, optionally substituted C1-C3alkylcarbonylamino, fluorine, chlorine, bromine;

[0052] or two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen, or sulfur (preferably oxygen or nitrogen);

[0053] m is an integer from 1 to 3;

[0054] hydrogen in the structure of Formula IV is optionally replaced by deuterium.

[0055] In preferred embodiments, "optionally substituted" means that the group modified by this term is substituted with C1-C6alkyl, C2-C6alkenyl, C1-C6alkoxy, hydroxy, halogen, amino, nitro, acetyl.

[0056] In specific embodiments, the compound is

[0057] A is selected from phenyl;

[0058] R1and R4are each independently selected from H, D, OH, optionally substituted C1-C6alkoxy;

[0059] R2and R5are each independently selected from H, D, OH, optionally substituted C1-C6alkoxy, optionally substituted C1-C6formyloxy;

[0060] or

[0061] R1and R2or R4and R5are joined to form an optionally substituted 3-6 membered ring containing 0, 1, or 2 heteroatoms selected from oxygen, nitrogen, or sulfur (preferably oxygen), cyclic carbonates;

[0062] R3is selected from H, D, optionally substituted C1-C6alkyl, D-substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C1-C6acyl;

[0063] or,

[0064] two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen, or sulfur (preferably oxygen or nitrogen);

[0065] m is an integer from 1 to 3;

[0066] hydrogen in the structure of Formula IV is optionally replaced by deuterium.

[0067] In specific embodiments, the compound is

[0068] Preferably, the compound is

[0069] Most preferably, the compound is a compound selected from the group consisting of:

[0070] In a preferred embodiment, the TRPV4-RhoA target inhibitor or antagonist is a drug for preventing or treating a TRPV4-RhoA target related disease.

[0071] In a specific embodiment, the TRPV4-RhoA target related disease includes, but is not limited to, the following indications: heart failure, hypertension, stroke, atrial fibrillation, etc., pneumonia, acute lung injury, pulmonary edema, chronic obstructive pulmonary disease (COPD), asthma, pain and inflammation such as osteoarthritis, neuropathic pain, visceral pain (such as irritable bowel syndrome), etc., neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.), urinary system diseases such as overactive bladder, etc., skin diseases such as eczema, itching, etc., eye diseases such as glaucoma, regulation of intraocular pressure and retinal nerve protection, etc.; preferably heart failure, hypertension, pain, lung disease and Parkinson's disease.

[0072] In a second aspect, the present application provides a compound of formula I, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer or solvate thereof,

[0073] wherein,

[0074] A is selected from an optionally substituted C6-C10 aromatic ring or a 5-6 membered aromatic heterocycle containing 1, 2 or 3 heteroatoms selected from N, O or S;

[0075] R1and R4are each independently selected from H, D, OH, NH2, a carbonyl group (one of R1or R4), an optionally substituted C1-C10alkyl group (preferably C1-C6alkyl), an optionally substituted C1-C10alkoxy group (preferably C1-C6alkoxy), an optionally substituted aminoacetyloxy group (R1or R4), B can be various groups forming amino acids), a sulfonato group;

[0076] R2and R5are each independently selected from H, D, OH, COOH, a sulfonato group, an optionally substituted aminoacetyloxy group (R2or R5), B can be various groups forming amino acids), an optionally substituted NH2, an optionally substituted C1-C10alkyl group (preferably C1-C6alkyl), an optionally substituted C1-C10alkoxy group (preferably C1-C6alkoxy), a phosphate group;

[0077] or,

[0078] R1and R2or R4and R5are joined to form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), a cyclic carbonate, a cyclic phosphonate or an optionally substituted cyclic boronate;

[0079] R3is selected from H, D, optionally substituted C1-C10alkyl (preferably C1-C6alkyl) or alkenyl, optionally substituted C1-C10alkyl (preferably C1-C6alkyl) formyl, optionally substituted C1-C10alkyl (preferably C1-C6alkyl), D substituted C1-C10alkyl, halogen, optionally substituted amino, optionally substituted C3-C10cycloalkyl, optionally substituted C2-6alkynyl, cyano;

[0080] or,

[0081] two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), an optionally substituted C5-C10aromatic ring or an optionally substituted C5-C10heteroaromatic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen);

[0082] m is an integer from 1 to 5;

[0083] hydrogen in the structure of Formula I is optionally replaced by deuterium.

[0084] In a particular embodiment, in the formula,

[0085] A is selected from a C6-C10aromatic ring or a 5-6 membered aromatic heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O or S;

[0086] R1and R4are each independently selected from H, D, OH, NH2, halogen, carbonyl;

[0087] R2and R5are each independently selected from H, D, OH, COOH, optionally substituted sulfonyloxy, optionally substituted NH2, halogen, optionally substituted phosphate;

[0088] or

[0089] R1and R2or R4and R5are joined to form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), a cyclic carbonate, a cyclic phosphonate or an optionally substituted cyclic boronate;

[0090] R3is selected from H, D, optionally substituted C1-C10 alkyl (preferably C1-C6 alkyl) alkenyl, formyl, optionally substituted C1-C10 alkyl (preferably C1-C6 alkyl) formyl, optionally substituted C1-C10 alkyl (preferably C1-C6 alkyl), D substituted C1-C10 alkyl, halogen, optionally substituted C3-C10 cycloalkyl, optionally substituted C2-6 alkynyl;

[0091] or,

[0092] two adjacent R3substituents form an optionally substituted C3-C6 carbocyclic ring or an optionally substituted C3-C6 heterocyclic ring containing 1 or 2 oxygen, an optionally substituted C5-C10 aromatic ring or an optionally substituted C5-C10 heteroaromatic ring containing 1 or 2 oxygen;

[0093] m is an integer from 1 to 5;

[0094] hydrogen in the structure of Formula I is optionally replaced by deuterium.

[0095] In a particular embodiment, the compound is:

[0096] Preferably, the compound is:

[0097] Most preferably, the compound is

[0098] In a third aspect, the present application provides a pharmaceutical composition comprising a compound of the second aspect, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer, or solvate thereof, and a pharmaceutically acceptable carrier or excipient.

[0099] In a fourth aspect, the present application provides a method of treating a TRPV4-RhoA target-related disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the second aspect or a pharmaceutical composition of the third aspect.

[0100] In a preferred embodiment, the subject is a mammal, including but not limited to a human, a pet, a racing animal, a livestock, etc.

[0101] In a preferred embodiment, the subject is a human.

[0102] In a preferred embodiment, the TRPV4-RhoA target site inhibitor or antagonist is a drug for preventing or treating a TRPV4-RhoA target site related disease.

[0103] In a preferred embodiment, the TRPV4-RhoA target site related disease is a pulmonary disease (acute lung injury, asthma, pulmonary fibrosis, cough and chronic obstructive pulmonary disease), a gastrointestinal disease (Crohn's disease and colitis), a bladder disease (including overactive bladder and interstitial cystitis), an eye disease (glaucoma and diabetic retinopathy), brain edema, or a disease such as pruritus; preferably a pulmonary disease; more preferably acute lung injury.

[0104] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0105] Figure 1 shows the results of a respiratory function detection experiment;

[0106] Figure 2 shows that the compound 1-R of the present application increases the survival rate of doxorubicin-induced heart failure mice;

[0107] Figure 3 shows that treatment with the compound (1-S, 1-R) of the present application improves the ultrasound indicators (EF-Ejection fraction, FS-Fractional shortening, HR-Heart rate) of doxorubicin-induced heart failure mice;

[0108] Figure 4 shows that treatment with the compound (1-S, 1-R) of the present application reduces the content of serum cardiac injury markers (CK-creatine kinase, HBDH-hydroxybutyrate dehydrogenase, LDH-lactate dehydrogenase) in mice;

[0109] Figure 5 shows that the compound 1-R of the present application improves myocardial fibrosis in doxorubicin-induced heart failure mice;

[0110] Figure 6 shows that the compound 1-R of the present application improves the ultrasound indicators of mice;

[0111] Figure 7 shows that the compound 1-R of the present application improves myocardial fibrosis and cell hypertrophy in heart failure mice;

[0112] Figure 8 shows that the compound 1-R of the present application increases the LDH level in heart failure mice after myocardial infarction;

[0113] FIG9 shows that compound 1-R of the present invention increases the survival rate of rats with heart failure 21 days after myocardial infarction;

[0114] FIG10 shows that compound 1-R of the present invention improves ultrasound indicators in rats with heart failure;

[0115] FIG11 shows that compound 1-R of the present invention improves myocardial fibrosis and cell hypertrophy in rats with heart failure;

[0116] FIG12 shows that compound 1-R of the present invention increases the LDH level in mice with heart failure after myocardial infarction;

[0117] FIG13 shows that compound 1-R of the present invention inhibits the nuclear translocation of transcriptional cofactor MRTF-A in cells;

[0118] FIG14 shows that compound 1-R of the present invention inhibits the expression of cardiomyocyte hypertrophy marker MyHC and cellular RhoA activity;

[0119] FIG15 shows that compound 1-R of the present invention inhibits the expression of collagen, a marker of myocardial fibrosis in mice;

[0120] FIG16 is a schematic diagram showing the mechanism of action of the compounds of the present invention;

[0121] FIG17 shows the anti-fibrotic shrinkage test results of compounds 2, 6, 13, 17, 27, 37, 42, 46, 59, 66, 69, 88, and 103. DETAILED DESCRIPTION

[0122] After extensive and in-depth research, the inventors unexpectedly discovered a series of compounds with excellent inhibitory activity against the TRPV4-RhoA target and therapeutic effects on related diseases. They also clarified the antagonistic mechanism of these compounds in the intracellular region of the TRPV4-RhoA target channel. This has important theoretical significance and practical application value for understanding its physiological and pathological functions and for the development of new drugs targeting the TRPV4-RhoA target channel. This is the basis for the completion of the present invention.

[0123] Definition of terms

[0124] The terms used herein with respect to the groups, substituents or structures of the compounds have the same meanings as understood by those skilled in the art. For the sake of clarity, the terms used in this specification are defined as follows.

[0125] In this document, "a", "an", "a kind" or "a category" refers to the plural form including the objects it modifies, that is, "a", "an", "a kind" or "a category" refers to at least one / kind / category or one / kind / category or more than one / kind / category.

[0126] In this article, the form "C1-n " means that the group has 1-n carbon atoms, for example, "C 1-10 " means that the group has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms; similarly, "C6-C10" means that the group has 6, 7, 8, 9 or 10 carbon atoms. At the same time, the description of the range of carbon atoms herein also includes sub-ranges therein. For example, when 1-10 carbon atoms are mentioned herein, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, and 1-3 carbon atoms are also included.

[0127] The term "alkyl" as used herein has the same meaning as commonly understood by those skilled in the art, and refers to various saturated or unsaturated, linear, side-chain, or cyclic hydrocarbon groups. For example, the alkyl group described herein refers to a lower alkyl group having 1-10 carbon atoms; preferably, a lower alkyl group having 1-8 carbon atoms; and more preferably, a lower alkyl group having 1-6 carbon atoms. In specific embodiments, the alkyl group described herein includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, and the like.

[0128] As used herein, the terms "aryl" and "aromatic ring" have the same meaning as commonly understood by those skilled in the art, and refer to a cyclic conjugated aromatic system. For example, the term "C6-C10 aryl" refers to an aromatic ring group having 6 to 10 carbon atoms, such as phenyl and naphthyl, that does not contain heteroatoms in the ring. The term "heteroaryl" as used herein refers to a cyclic conjugated aromatic system that contains one or more heteroatoms, such as N, O, or S, in the ring; for example, pyridyl and pyrazinyl.

[0129] The term "aminoacetyl" as used herein has the conventional meaning as understood by those of ordinary skill in the art, i.e., an acetyl group substituted with an amino group. In a specific embodiment, the aminoacetyl group is As shown, wherein R is various amino acid substituents.

[0130] As used herein, the terms "heterocyclyl" and "heterocycle" have the same meaning and refer to a saturated or partially unsaturated non-aromatic cyclic group, including a monocyclic, fused, spirocyclic or bridged ring, wherein the heterocycle has at least one heteroatom selected from O, S or N as a ring member. For example, a "5- or 6-membered heterocyclyl" refers to a saturated or unsaturated 5- or 6-membered cycloalkyl group containing 1 to 3 heteroatoms selected from oxygen, sulfur or nitrogen in the ring, such as dioxolanyl.

[0131] As used herein, the term "halogen" refers to F, Cl, Br or I.

[0132] Based on the teachings herein and the knowledge in the art, those skilled in the art will appreciate that the compounds according to the application and the various substituents defined above can be further substituted, for example, with C1-6alkyl, C1-6alkoxy, halogen, nitro, amino, phenyl, hydroxy, and the like, provided that the intended substituent combination is stable or chemically feasible.

[0133] As used herein, the term "substituted" means that one or more hydrogen atoms on a particular group are replaced with a particular substituent. The particular substituents can be those described above or the specific substituents appearing in the embodiments. Thus, in the present application, the substituents in the general formula (I) or (II) can each independently be the corresponding group in the specific compounds in the embodiments; that is, the present application includes combinations of the substituents in the general formula (I) or (II) described above and combinations of the substituents shown in the general formula (I) or (II) with other specific substituents appearing in the embodiments.

[0134] Unless otherwise specified, a particular substituted group can have a particular substituent at any available substitutable position of the group, and the substituents can be the same or different at each occurrence. A cyclic substituent, for example, a heterocyclyl group, can be attached to another ring, for example, a cycloalkyl group, to form a spiro bicyclic ring system, for example, two rings sharing a common carbon atom. The substituents include, but are not limited to: C 1-8 alkyl, C 2-8 alkenyl, C 2-8 alkynyl, C 3-8 cycloalkyl, 3- to 12-membered heterocyclyl, aryl, heteroaryl, halogen, hydroxy, carboxyl (-COOH), C 1-8 aldehyde, C 2-10 acyl, C 2-10 ester, amino, C 1-8 alkoxy, nitro, cyano, thiol, amino, and the like. In specific embodiments, a hydrogen atom on an alkyl and aryl group is replaced with an amino, halogen, or other group to become a group belonging to each of the above definitions.

[0135] For convenience and in accordance with common understanding, the terms "any substituted," "optionally substituted," or "substituted or unsubstituted" apply only to positions that can be substituted, and do not include those positions that are chemically impossible to substitute.

[0136] As used herein, the term "independently selected from" means that the multiple groups modified by the term can be selected independently within the stated range. For example, when it is stated herein that "R1and R4are each independently selected from H, OH, carbonyl," it is equivalent to disclose that "R1is selected from H, OH, or carbonyl" and "R4is selected from H, OH, or carbonyl."

[0137] Compounds of the invention

[0138] The present invention provides a series of novel compounds with excellent inhibitory activity against TRPV4-RhoA target and therapeutic effect on related diseases, which have different mechanisms from existing therapeutic drugs.

[0139] In a specific embodiment, the present invention provides a compound of formula I or II, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer or solvate thereof,

[0140] Each substituent in the general formula is as described above.

[0141] In a preferred embodiment, the compound of formula I is a compound of formula II,

[0142] Each substituent in the formula is as described above.

[0143] In a preferred embodiment, the compound of formula I is a compound of formula III,

[0144] Each substituent in the formula is as described above.

[0145] In a preferred embodiment, the compound of formula I is a compound of formula IV,

[0146] Each substituent in the formula is as described above.

[0147] In a specific embodiment, the preferred compound of the present invention is the following compound:

[0148] On the basis of the compounds of the present application, a person skilled in the art can make pharmaceutically acceptable salts or esters, prodrugs, optical isomers, stereoisomers or solvates thereof. For example, the compounds of the present application can be reacted with inorganic or organic acids to form conventional pharmaceutically acceptable salts. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid and phosphoric acid, etc., and the organic acids include various amino acids, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid, etc.; or the compounds of the present application can be reacted with inorganic bases to form sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt; or with organic bases to form methylamine salt, ethylamine salt or ethanolamine salt.

[0149] Since there are chiral carbon atoms in the compounds of the present application, the optical isomers or stereoisomers obtained by resolving the compounds of the present application also fall within the protection scope of the present application.

[0150] Based on the teachings herein, a person skilled in the art will understand that the compounds provided by the present application for treating heart failure and related diseases should possess various properties inherent to drugs, such as therapeutic activity, drugability, pharmacokinetic activity, etc., and the compounds of the present application should also possess acceptable toxicity. Therefore, all the pharmaceutically relevant activities of the compounds of the present application are implicitly disclosed herein, and a person skilled in the art can detect these properties by using methods known in the art.

[0151] On the basis of the compounds of the present application or pharmaceutically acceptable salts or esters, prodrugs, optical isomers, stereoisomers or solvates thereof, the present application also provides pharmaceutical compositions comprising the compounds of the present application, which optionally comprise pharmaceutically acceptable excipients.

[0152] In a specific embodiment, the pharmaceutical compositions of the present application comprise a safe and effective amount of the compounds of the present application or pharmaceutically acceptable salts thereof and pharmaceutically acceptable excipients or carriers. The "safe and effective amount" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects.

[0153] "Pharmaceutically acceptable excipient or carrier" means one or more compatible solid or liquid filler or gel materials, which are suitable for human use, and which are nontoxic to the subject in amounts necessary to deliver the compounds of the application. "Compatible" means that the components of the composition are capable of being commingled with the compounds of the application, and with each other, without any component adversely affecting the efficacy of the other. Examples of suitable pharmaceutically acceptable carriers are water, salt, buffers, carbohydrates, starches, sugars, flavorants, binders, excipients, stabilizers, and the like. Examples of suitable pharmaceutically acceptable carriers include celluloses, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, and the like; gelatin; talc; solid lubricants, such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as soybean oil, sesame oil, peanut oil, olive oil, and the like; polyols, such as propylene glycol, glycerin, mannitol, sorbitol, and the like; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; stabilizers; antioxidants; preservatives; pyrogen-free water, and the like. ) ; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; stabilizers; antioxidants; preservatives; pyrogen-free water, and the like.

[0154] The mode of administration of the compounds or pharmaceutical compositions of the present application is not narrowly critical and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0155] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with such other ingredients as binders, (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) absorbents, such as kaolin and bentonite clay; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form can also comprise buffering agents.

[0156] Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared with coatings and shells, such as enteric coatings and other coatings and shells well known in the art. They can contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0157] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, and the like, or combinations thereof.

[0158] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0159] Suspensions, in addition to the active compounds, can contain suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar-agar, or combinations thereof.

[0160] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

[0161] Dosage forms of the compounds of the present application for topical administration include ointments, powders, sprays, and inhalers. The active compound is admixed under sterile conditions with physiologically acceptable carriers and any preservatives, buffers, or propellants as can be required.

[0162] The compounds of the present application can be administered alone or in combination with other pharmaceutically acceptable compounds. When the pharmaceutical compositions of the present application are used, a safe and effective amount of the compound of the present application is administered to a mammal (e.g., human) in need of treatment, wherein the dosage is administered in an amount that is pharmaceutically considered to be an effective amount. The compounds of the present application and the pharmaceutical compositions can be administered by oral, nasal, dermal, pulmonary, or gastrointestinal routes. Most preferably, the administration is oral, in a single dose or in divided doses. Regardless of the route of administration, the optimum dose for an individual will depend on the particular treatment indicated and can be varied over time. Typically, treatment is initiated with small dosages that are less than the optimum dose, with the dose being increased if necessary, until the optimum effect is achieved. The specific dose employed will depend upon the route of administration, the condition being treated, and the health of the patient. In a specific embodiment, the compounds of the present application are preferably in a form suitable for aerosol administration.

[0163] TRPV4-Related Diseases

[0164] In the present text, the term "TRPV4-related disease" refers to various diseases in which TRPV4 is involved in the pathogenesis, such as various diseases in which TRPV4 is involved in the pathogenesis. In a specific embodiment, the TRPV4-related disease is a respiratory disease such as pulmonary edema, chronic obstructive pulmonary disease (COPD), asthma, etc.; pain and inflammation such as osteoarthritis, neuropathic pain, visceral pain (such as irritable bowel syndrome), etc.; nervous system diseases such as spinal cord injury / cerebral edema, neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.); cardiovascular diseases such as heart failure, hypertension, stroke, atrial fibrillation, etc.; urinary system diseases such as overactive bladder, etc.; skin diseases such as eczema, pruritus, etc. and eye diseases such as glaucoma, regulation of intraocular pressure and retinal nerve protection, etc. brain edema, or pruritus, etc. disease; preferably a lung disease; more preferably acute lung injury.

[0165] RhoA-related diseases cardiovascular diseases such as hypertension, atherosclerosis, heart failure, etc.; cancers such as breast cancer, gastric cancer, colorectal cancer, melanoma, etc.; nervous system diseases such as neurodegenerative diseases (such as Alzheimer's disease, amyotrophic lateral sclerosis); spinal cord injury and autoimmune and inflammation, etc.; other potential indications such as fibrotic diseases (such as pulmonary fibrosis, liver fibrosis); regulation of osteoclast function in osteoporosis.

[0166] Heart failure and related diseases

[0167] In the present text, the terms "heart failure" and "cardiac failure" have the same meaning, both refer to the fact that due to the impairment of the systolic and / or diastolic function of the heart, the heart is not able to sufficiently expel the venous return volume, leading to the accumulation of blood in the venous system and the insufficient perfusion of the arterial system, thus causing a syndrome of circulatory disorders of the heart. This syndrome of circulatory disorders of the heart is mainly manifested as pulmonary congestion and venous congestion. Heart failure is not an independent disease, but rather the end stage of the development of heart disease. The vast majority of heart failure begins with left heart failure.

[0168] Almost all cardiovascular diseases eventually lead to the occurrence of heart failure. Myocardial infarction, cardiomyopathy, excessive hemodynamic load, myocardial damage caused by inflammation, etc. can all cause changes in myocardial structure and function, eventually leading to decreased ventricular pumping and / or filling function. In addition to this, other factors can induce the occurrence of heart failure. Common causes of heart failure include, for example, infection, drug toxicity, inappropriate activity, and emotion, etc.

[0169] According to the urgency of the occurrence of heart failure, it can be clinically divided into acute heart failure and chronic heart failure. According to the site of the occurrence of heart failure, it can be divided into left heart, right heart and whole heart failure. It can also be divided into systolic or diastolic heart failure.

[0170] In specific embodiments, the heart failure and its related diseases include various etiological heart failure related diseases, including but not limited to various clinical treatment drug-induced (or caused) heart failure, heart infarction, coronary heart disease, etc. caused heart failure (ventricular remodeling), respiratory dysfunction, nocturnal sleep apnea syndrome, chronic hypoxic damage of the brain and body.

[0171] Advantages of the present application:

[0172] 1. The present application provides a series of novel compounds with excellent inhibitory activity on the TRPV4-RhoA target and therapeutic effect on related diseases; and

[0173] 2. The compounds of the present application lay a new material foundation for the development of new TRPV4-RhoA target inhibitors or antagonists and therapeutic drugs for related diseases.

[0174] The technical solutions of the present application are further described below in combination with specific implementation cases, but the following implementation cases do not constitute a limitation on the present application, and all various application methods according to the principles and technical means of the present application belong to the scope of the present application. The experimental methods not specified in the following examples are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. Unless otherwise specified, percentages and parts are calculated by weight.

[0175] Examples

[0176] Some known compounds can be purchased.

[0177] The compounds of the present application can be prepared according to conventional routes or methods, or can be obtained according to the methods or routes described herein.

[0178] Example 1, synthesis of a series of compounds

[0179] 1. Synthesis of 3-ethylphenylglycol (compound 1)

[0180] Synthesis of compound 1.3

[0181] Magnesium chips (2.2 g, 90 mmol) were placed in a 250 mL three-necked reaction flask, and one iodine particle was added. Compound 1.1 (15 g, 82 mmol) was dissolved in 120 mL of anhydrous tetrahydrofuran, and the tetrahydrofuran solution of compound 1 was dropped into the reaction flask containing magnesium chips through a constant pressure dropping funnel at a rate of 10 mL. An electric hair dryer was used to initiate the reaction. After the reaction started, the remaining 110 mL of tetrahydrofuran solution of compound 1.1 was slowly dropped in. After the dropping was completed, the reaction was refluxed for 3.5 hours, and finally a tetrahydrofuran solution of compound 1.2 was obtained.

[0182] A solution of compound 1.2 in tetrahydrofuran was added dropwise to a solution of benzyloxyacetaldehyde (12 g, 80 mmol) in 80 mL of anhydrous tetrahydrofuran under ice bath, after the end of the dropwise addition, the reaction was allowed to warm to room temperature for 1 hour, then saturated aqueous ammonium chloride solution was added to quench the reaction. Finally, tetrahydrofuran was removed and extracted with ethyl acetate, dried over anhydrous sodium sulfate and purified by column (PE:EA = 10:1) to give the target compound 12.4 g, the yield of two steps was 59.6%.

[0183] 1 H NMR (400 MHz, DMSO): δ 7.36-7.15 (m, 8H), 7.09 (d, J = 7.6 Hz, 1H), 5.38 (d, J = 4.4 Hz, 1H), 4.74-4.70 (m, 1H), 4.51 (s, 2H), 3.53-3.44 (m, 2H), 2.61 (q, J = 7.6, 2H), 1.19 (t, J = 7.6, 3H). LC-MS: 255.30 (M-H) - .

[0184] Synthesis of compound 1 (including compound 1 racemate, R-form of compound 1 1-R, S-form of compound 1 1-S)

[0185] Compound 1.3 (12.4 g, 48.4 mmol) was dissolved in ethanol 70 mL and 10% palladium on carbon 2.43 g was added, the air was replaced with H2and stirred at 38°C for 3 hours. After the end of the reaction, the solvent was removed and purified by column (PE:EA = 5:1) to give the target compound 6.42 g, the yield was 79%.

[0186] R-form of compound 1, S-form of compound 1 were obtained by chiral preparation.

[0187] 1 H NMR (400 MHz, CDCl3): δ 7.28-7.12 (m, 4H), 4.78 (dd, J1= 8.0 Hz, J2= 3.6 Hz, 1H), 3.74-3.62 (m, 2H), 2.64 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). LC-MS: 165.10 (M-H) - .

[0188] 2、Synthesis of compound 2

[0189] Synthesis of compound 2.6

[0190] Magnesium turnings (0.17 g, 7 mmol) were placed in a 50 mL three-necked flask, and one iodine crystal was added. Compound 2.4 (1 g, 6.4 mmol) was dissolved in 12 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution of compound 4 was added dropwise to the flask at a constant rate using a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 10 mL of the tetrahydrofuran solution of compound 2.4 was slowly added dropwise. After the addition was completed, the reaction was refluxed for 3.5 hours, and a tetrahydrofuran solution of compound 2.5 was finally obtained.

[0191] The tetrahydrofuran solution of compound 2.5 was added dropwise to a solution of benzyloxyacetaldehyde (0.97 g, 6.4 mmol) in 8 mL of anhydrous tetrahydrofuran at an ice bath, and after the addition was completed, the reaction was allowed to warm to room temperature for 1 hour. Then, saturated aqueous ammonium chloride solution was added to quench the reaction. Finally, tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain 0.98 g of the target compound, with a two-step yield of 67.6%.

[0192] 1 H NMR (400 MHz, DMSO): δ 7.37-7.22 (m, 10H), 5.41 (d, J = 4.0 Hz, 1H), 4.78-4.74 (m, 1H), 4.51 (s, 2H), 3.51-3.48 (m, 2H). LC-MS: 227.30 (M-H) - .

[0193] Synthesis of compound 2

[0194] Compound 2.6 (0.9 g, 4 mmol) was dissolved in 10 mL of ethanol, and 0.2 g of 10% palladium-carbon was added. After replacing the air with H2, the reaction was stirred at 38°C for 3 hours. After the reaction was completed, the solvent was removed, and the product was purified by column chromatography (PE:EA = 5:1) to obtain 0.24 g of the target compound, with a yield of 45%.

[0195] 1 H NMR (400 MHz, DMSO): δ 7.34-7.28 (m, 4H), 7.24-7.20 (m, 1H), 5.24 (d, J = 4.4 Hz, 1H), 4.74 (t, J = 6 Hz, 1H), 4.55 (q, J = 6 Hz, 1H), 3.43 (t, J = 6 Hz, 2H). LC-MS: 137.10 (M-H) - .

[0196] 3. Synthesis of compound 3

[0197] Synthesis of compound 3.9

[0198] Magnesium turnings (0.33 g, 13.8 mmol) were placed in a 100 mL three-necked flask, and one iodine crystal was added. Compound 3.7 (2 g, 11.7 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and the tetrahydrofuran solution of compound 3.7 was added dropwise into the flask at a rate of 3 mL per hour using a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 17 mL of the tetrahydrofuran solution of compound 7 was added dropwise slowly. After the addition was completed, the reaction was refluxed for 3.5 hours. Finally, a tetrahydrofuran solution of compound 3.8 was obtained.

[0199] The tetrahydrofuran solution of compound 3.8 was added dropwise into a solution of benzyloxyacetaldehyde (1.74 g, 11.6 mmol) in 14 mL of anhydrous tetrahydrofuran at an ice bath. After the addition was completed, the reaction was allowed to warm to room temperature for 1 hour, and then quenched by adding saturated aqueous ammonium chloride solution. Finally, the tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to give the target compound 0.95 g in a two-step yield of 34%.

[0200] 1 H NMR (400 MHz, DMSO): δ 7.34-7.04 (m, 9H), 5.34 (d, J = 4.0 Hz, 1H), 4.73-4.69 (m, 1H), 4.51 (s, 2H), 3.52-3.43 (m, 2H), 2.28 (s, 3H). LC-MS: 241.30 (M-H) - .

[0201] Synthesis of compound 3

[0202] Compound 3.9 (0.9 g, 3.8 mmol) was dissolved in 10 mL of ethanol, and 10% palladium on carbon (0.2 g) was added. The air was replaced with H2, and the reaction was stirred at 38°C for 3 hours. After the reaction was completed, the solvent was removed, and the product was purified by column chromatography (PE:EA = 5:1) to give the target compound 0.28 g in a yield of 50%.

[0203] 1 H NMR (400 MHz, CDCl3): δ 7.26-7.09 (m, 4H), 4.76 (dd, J1= 8.0 Hz, J2= 3.6 Hz, 1H), 3.72-3.59 (m, 2H), 3.09 (s, 2H), 2.34 (s, 3H). LC-MS: 151.10 (M-H) - .

[0204] 4、Synthesis of compound 4

[0205] Synthesis of compound 4.12

[0206] Magnesium turnings (0.31 g, 12.9 mmol) were placed in a 100 mL three-necked flask, and one iodine crystal was added. Compound 4.10 (2 g, 10.9 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution of compound 10 was added dropwise to the reaction flask at a rate of 3 mL / min using a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 17 mL of the tetrahydrofuran solution of compound 4.10 was added dropwise. After the addition was completed, the reaction was refluxed for 3.5 hours, and a tetrahydrofuran solution of compound 11 was obtained.

[0207] A tetrahydrofuran solution of compound 4.11 was added dropwise to a solution of benzyloxyacetaldehyde (1.62 g, 10.8 mmol) in 14 mL of anhydrous tetrahydrofuran at an ice bath temperature. After the addition was completed, the reaction was allowed to warm to room temperature for 1 hour, and then quenched by adding saturated aqueous ammonium chloride solution. Finally, the tetrahydrofuran was removed, and the reaction was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain 1 g of the target compound with a two-step yield of 36%.

[0208] 1 H NMR (400 MHz, DMSO): δ 7.35-7.14 (m, 9H), 5.33 (d, J = 4.0 Hz, 1H), 4.74-4.70 (m, 1H), 4.51 (s, 2H), 3.52-3.43 (m, 2H), 2.57 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). LC-MS: 255.30 (M-H) - .

[0209] Synthesis of compound 4

[0210] Compound 4.12 (0.9 g, 3.5 mmol) was dissolved in 10 mL of ethanol, and 0.2 g of 10% palladium-carbon was added. The air was replaced with H2, and the reaction was stirred at 38°C for 3 hours. After the reaction was completed, the solvent was removed, and the reaction was purified by column chromatography (PE:EA = 5:1) to obtain 0.24 g of the target compound with a yield of 37%.

[0211] 1 H NMR (400 MHz, CDCl3): δ 7.20-7.10 (m, 4H), 4.72 (dd, J1= 8.0 Hz, J2= 3.6 Hz, 1H), 3.66-3.55 (m, 2H), 2.59 (q, J = 7.6 Hz, 2H), 2.46 (s, 2H), 1.15 (t, J = 7.6 Hz, 3H). LC-MS: 165.10 (M-H) - .

[0212] 5、Synthesis of compound 5

[0213] Synthesis of compound 5.15

[0214] Magnesium turnings (0.31 g, 12.9 mmol) were placed in a 100 mL three-necked flask, and one iodine crystal was added. Compound 5.13 (2 g, 10.9 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and the tetrahydrofuran solution of compound 5.13 was added dropwise to the flask at a rate of 3 mL per minute using a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 17 mL of the tetrahydrofuran solution of compound 5.13 was added dropwise slowly. After the addition was completed, the reaction was refluxed for 3.5 hours. Finally, a tetrahydrofuran solution of compound 5.14 was obtained.

[0215] The tetrahydrofuran solution of compound 5.14 was added dropwise to a solution of benzyloxyacetaldehyde (1.62 g, 10.8 mmol) in 14 mL of anhydrous tetrahydrofuran at an ice bath. After the addition was completed, the reaction was allowed to warm to room temperature for 1 hour, and then quenched by adding saturated aqueous ammonium chloride solution. Finally, the tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to give the target compound 1.4 g in a two-step yield of 50%.

[0216] 1 H NMR (400 MHz, DMSO): δ 7.44-7.42 (m, 1H), 7.35-7.25 (m, 5H), 7.19-7.12 (m, 3H), 5.33 (d, J = 4.4 Hz, 1H), 5.01-4.97 (m, 1H), 4.55-4.47 (m, 2H), 3.51-3.40 (m, 2H), 2.66 (m, 2H), 1.15 (t, J = 7.6, 3H). LC-MS: 255.30 (M-H) - .

[0217] Synthesis of compound 5

[0218] Compound 5.15 (1.2 g, 4.7 mmol) was dissolved in 15 mL of ethanol, and 10% palladium-carbon (0.3 g) was added. The air was replaced with H2, and the reaction was stirred at 38°C for 3 hours. After the reaction was completed, the solvent was removed, and the product was purified by column chromatography (PE:EA = 5:1) to give the target compound 0.43 g in a yield of 47%.

[0219] 1 H NMR (400 MHz, DMSO): δ 7.41-7.14 (m, 4H), 5.14 (d, J = 4.0 Hz, 1H), 4.81-4.76 (m, 2H), 3.37 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). LC-MS: 165.10 (M-H)- .

[0220] 6. Synthesis of compound 6

[0221] Synthesis of compound 6.18

[0222] Magnesium turnings (0.31 g, 12.9 mmol) were placed in a 100 mL three-necked flask, and one grain of iodine was added. Compound 6.16 (2 g, 8.5 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and the tetrahydrofuran solution of compound 6.16 was added dropwise to the flask at a rate of 3 mL per minute using a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 17 mL of the tetrahydrofuran solution of compound 6.16 was added dropwise slowly. After the addition was completed, the reaction was refluxed for 3.5 hours. Finally, a tetrahydrofuran solution of compound 6.17 was obtained.

[0223] The tetrahydrofuran solution of compound 6.17 was added dropwise to a solution of benzyloxyacetaldehyde (1.28 g, 8.5 mmol) in 14 mL of anhydrous tetrahydrofuran at an ice bath. After the addition was completed, the reaction was allowed to react at room temperature for 1 hour, and then quenched by adding saturated aqueous ammonium chloride solution. Finally, the tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain the target compound 0.9 g, with a two-step yield of 32%.

[0224] 1 H NMR (400 MHz, DMSO): δ 7.38-7.02 (m, 9H), 5.33 (d, J = 4.4 Hz, 1H), 4.75-4.70 (m, 1H), 4.47 (s, 2H), 3.56-3.49 (m, 2H). LC-MS: 307.30 (M-H) - .

[0225] Synthesis of compound 6

[0226] Compound 6.18 (0.85 g, 2.8 mmol) was dissolved in 10 mL of ethanol, and 10% palladium-carbon (0.23 g) was added. The air was replaced with H2, and the reaction was stirred at 38°C for 3 hours. After the reaction was completed, the solvent was removed, and the product was purified by column chromatography (PE:EA = 5:1) to obtain the target compound 0.2 g, with a yield of 33%.

[0227] 1H NMR (400 MHz, DMSO): δ 7.35-7.29 (m, 1H), 7.19-7.13 (m, 2H), 7.02-6.98 (m, 1H), 5.34 (d, J = 4 Hz, 1H), 4.70 (t, J = 6 Hz, 1H), 4.51 (q, J = 5.6 Hz), 3.40 (t, J = 6 Hz, 2H). LC-MS: 217.10 (M-H) - .

[0228] 7. Synthesis of compound 7

[0229] Synthesis of compound 7.21

[0230] Magnesium chips (0.31 g, 12.9 mmol) were placed in a 100 mL three-necked flask, and one iodine crystal was added. Compound 7.19 (2 g, 11.7 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution of compound 19 was added dropwise to the flask through a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 7.17 mL of tetrahydrofuran solution of compound 7.19 was slowly added dropwise. After the addition was completed, the reaction was refluxed for 3.5 hours, and finally a tetrahydrofuran solution of compound 7.20 was obtained.

[0231] The tetrahydrofuran solution of compound 7.20 was added dropwise to a solution of benzyloxyacetaldehyde (1.76 g, 11.7 mmol) in 14 mL of anhydrous tetrahydrofuran under ice bath. After the addition was completed, the reaction was allowed to react at room temperature for 1 hour, and then saturated aqueous ammonium chloride solution was added to quench the reaction. Finally, tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain 1.32 g of the target compound, with a two-step yield of 47.2%.

[0232] 1 H NMR (400 MHz, DMSO): δ 7.44-7.14 (m, 9H), 5.36 (d, J = 4.4 Hz, 1H), 4.71-4.67 (m, 1H), 4.50 (s, 2H), 3.50-3.41 (m, 2H). LC-MS: 245.30 (M-H) -

[0233] Synthesis of compound 7

[0234] Compound 7.21 (1.3 g, 5.4 mmol) was dissolved in 10 mL of ethanol, and 10% palladium-carbon (0.23 g) was added. The air was replaced with H2, and the mixture was stirred at 38°C for 3 hours. After the reaction was completed, the solvent was removed, and the product was purified by column chromatography (PE:EA = 5:1) to obtain 0.2 g of the target compound, with a yield of 33%.

[0235] 1 H NMR (400 MHz, DMSO): δ 7.37-7.31 (m, 1H), 7.18-7.12 (m, 2H), 7.06-7.01 (m, 1H), 5.39 (d, J = 4.4 Hz, 1H), 4.77 (t, J = 5.6 Hz, 1H), 4.56 (q, J = 5.6 Hz), 3.43 (t, J = 6 Hz, 2H). LC-MS: 155.10 (M-H) - .

[0236] 8. Synthesis of compound 8

[0237] Synthesis of compound 8.24

[0238] Magnesium chips (0.57 g, 22.2 mmol) were placed in a 100 mL three-necked flask, and one iodine crystal was added. Compound 8.22 (4 g, 21.5 mmol) was dissolved in 40 mL of anhydrous tetrahydrofuran, and the tetrahydrofuran solution of compound 8.22 was added dropwise to the flask at a rate of 5 mL per minute using a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 35 mL of the tetrahydrofuran solution of compound 8.22 was added dropwise slowly. After the addition was completed, the reaction was refluxed for 3.5 hours, and finally a tetrahydrofuran solution of compound 8.23 was obtained.

[0239] The tetrahydrofuran solution of compound 8.23 was added dropwise to a solution of benzyloxyacetaldehyde (3.21 g, 21.5 mmol) in 30 mL of anhydrous tetrahydrofuran at an ice bath. After the addition was completed, the reaction was allowed to warm to room temperature for 1 hour, and then quenched by adding saturated aqueous ammonium chloride solution. Finally, the tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain 4 g of the target compound, with a two-step yield of 63.6%.

[0240] 1 H NMR (400 MHz, DMSO): δ 7.35-7.14 (m, 9H), 5.33 (d, J = 4.0 Hz, 1H), 4.74-4.70 (m, 1H), 4.51 (s, 2H), 3.73 (s, 3H), 3.52-3.43 (m, 2H). LC-MS: 257.30 (M-H) - .

[0241] Synthesis of compound 8

[0242] Compound 8.24 (3.5 g, 13.6 mmol) was dissolved in ethanol 30 mL and 10% palladium on carbon 0.69 g was added, the air was replaced by H2and stirred at 38 °C for 3 hours. After the reaction was completed, the solvent was removed and purified by column (PE:EA = 5:1) to obtain the target compound 1.42 g with a yield of 62.3%.

[0243] 1 H NMR (400 MHz, DMSO): δ 7.23 (t, J = 8 Hz, 1H), 6.91 (d, J = 7.6 Hz, 2H), 6.80-6.77 (m, 1H), 5.24 (d, J = 4.4 Hz, 1H), 4.73 (t, J = 5.6 Hz, 1H), 4.53 (q, J = 4.8 Hz, 1H), 3.73 (s, 3H), 3.43-3.36 (m, 2H). LC-MS: 167.10 (M-H) - .

[0244] 9. Synthesis of compound 9

[0245] Synthesis of compound 9.26

[0246] P-isopropylaniline (4 g, 29.6 mmol) was dissolved in dichloromethane (40 ml) and NBS (5.8 g, 32.6 mmol) was added in portions to the dichloromethane solution under ice bath, then the reaction temperature was raised to room temperature and the reaction was completed after 1 hour. After purification, the product was obtained 4.6 g with a yield of 74%.

[0247] 1 H NMR (400 MHz, CDCl3): δ 7.28 (d, J = 1.6 Hz, 1H), 6.98 (dd, J1= 1.6 Hz, J2= 1.6 Hz, 1H), 6.71 (d, J = 8 Hz, 1H), 3.76 (s, 2H), 2.79 (m, 1H), 1.20 (d, J = 6.8 Hz, 6H). LC-MS: 214.10 (M+H) + .

[0248] Synthesis of compound 9.27

[0249] Compound 9.26 (2 g, 9.3 mmol) was dissolved in a mixture of acetic acid (10 mL), water (4 mL) and concentrated HCl (1 mL), and then a solution of NaNO2(0.78 g, 11.2 mmol) in water (2 mL) was added dropwise to the mixture at ice bath. After the reaction was carried out for half an hour, the resulting reaction mixture was added dropwise to a 50% aqueous solution of H3PO2(12 mL) at ice bath, and then the reaction was continued for 8 hours after the dropwise addition was completed, and then the reaction temperature was raised to 25 °C for 3 days. After the reaction was completed, the product was purified by column chromatography. The yield of the product was 1.4 g, and the yield was 75.3%.

[0250] 1 H NMR (400 MHz, DMSO): δ 7.42 (s, 1H), 7.37-7.34 (m, 1H), 7.25 (d, J = 4.8 Hz, 2H), 1.20 (d, J = 6.8 Hz, 6H). LC-MS: 199.20 (M+H) + .

[0251] Synthesis of compound 9.29

[0252] Magnesium chips (0.17 g, 7.1 mmol) were placed in a 100 mL three-necked flask, and one iodine was added. Compound 9.27 (1.3 g, 7.5 mmol) was dissolved in anhydrous tetrahydrofuran 12 mL, and the tetrahydrofuran solution of compound 9.27 was added dropwise to the flask at a constant pressure dropwise funnel 2 mL, and the reaction was initiated by heating with an electric hair dryer. After the reaction started, the remaining 10 mL of tetrahydrofuran solution of compound 9.27 was slowly added dropwise. After the dropwise addition was completed, the reaction was refluxed for 3.5 hours, and finally a tetrahydrofuran solution of compound 9.28 was obtained.

[0253] The tetrahydrofuran solution of compound 9.28 was added dropwise to a solution of benzyloxyacetaldehyde (0.98 g, 7.1 mmol) in anhydrous tetrahydrofuran 10 mL at ice bath, and after the dropwise addition was completed, the reaction was carried out at room temperature for 1 hour, and then saturated aqueous ammonium chloride solution was added to quench the reaction. Finally, tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain 1 g of the target compound, with a two-step yield of 56%.

[0254] 1 H NMR (400 MHz, DMSO): δ 7.42 (s, 1H), 7.37-7.34 (m, 1H), 7.25 (d, J = 4.8 Hz, 2H), 1.20 (d, J = 6.8 Hz, 6H). LC-MS: 199.20 (M+H) + .

[0251] Synthesis of compound 9.29

[0252] Magnesium chips (0.17 g, 7.1 mmol) were placed in a 100 mL three-necked flask, and one iodine was added. Compound 9.27 (1.3 g, 7.5 mmol) was dissolved in anhydrous tetrahydrofuran 12 mL, and the tetrahydrofuran solution of compound 9.27 was added dropwise to the flask at a constant pressure dropwise funnel 2 mL, and the reaction was initiated by heating with an electric hair dryer. After the reaction started, the remaining 10 mL of tetrahydrofuran solution of compound 9.27 was slowly added dropwise. After the dropwise addition was completed, the reaction was refluxed for 3.5 hours, and finally a tetrahydrofuran solution of compound 9.28 was obtained.

[0253] The tetrahydrofuran solution of compound 9.28 was added dropwise to a solution of benzyloxyacetaldehyde (0.98 g, 7.1 mmol) in anhydrous tetrahydrofuran 10 mL at ice bath, and after the dropwise addition was completed, the reaction was carried out at room temperature for 1 hour, and then saturated aqueous ammonium chloride solution was added to quench the reaction. Finally, tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain 1 g of the target compound, with a two-step yield of 56%.

[0254] 1 H NMR (400 MHz, DMSO): δ 7.42 (s, 1H), 7.37-7.34 (m, 1H), 7.25 (d, J = 4.8 Hz, 2H), 1.20 (d, J = 6.8 Hz, 6H). LC-MS: 199.20 (M+H) + .

[0251] Synthesis of compound 9.29

[0252] Magnesium chips (0.17 g, 7.1 mmol) were placed in a 100 mL three-necked flask, and one iodine was added. Compound 9.27 (1.3 g, 7.5 mmol) was dissolved in anhydrous tetrahydrofuran 12 mL, and the tetrahydrofuran solution of compound 9.27 was added dropwise to the flask at a constant pressure dropwise funnel 2 mL, and the reaction was initiated by heating with an electric hair dryer. After the reaction started, the remaining 10 mL of tetrahydrofuran solution of compound 9.27 was slowly added dropwise. After the dropwise addition was completed, the reaction was refluxed for 3.5 hours, and finally a tetrahydrofuran solution of compound 9.28 was obtained.+ .

[0255] Synthesis of compound 9

[0256] Compound 9.28 (1 g, 3.6 mmol) was dissolved in ethanol 10 mL, and 10% palladium on carbon 0.19 g was added, replaced with air, added H2, stirred at 38 °C for 3 hours. After the reaction was completed, the solvent was removed, and the target compound 0.51 g was obtained by column purification (PE:EA = 5:1) with a yield of 80%.

[0257] 1 H NMR (400 MHz, CDC13): δ 7.19-7.04 (m, 4H), 4.69 (d, J = 4.0 Hz, 1H), 3.64-3.52 (m, 2H), 3.22 (s, 2H), 2.83-2.76 (m, 1H), 1.15 (d, J = 7.6 Hz, 6H). LC-MS: 179.10 (M-H) - .

[0258] 10. Synthesis of compound 10

[0259] Synthesis of compound 10.30 (protection of carbonyl group)

[0260] Take p-bromophenylacetone (5 g, 25.4 mmol) in a 100 mL three-necked flask, add toluene 60 mL as solvent, add ethylene glycol (10 g, 161.3 mmol), p-toluenesulfonic acid (1 g, 5 mmol), and heat to 130 °C, reflux water for 16 h. After the reaction was completed, add brine and stir, separate the layers, take the organic phase, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain compound 3 g with a yield of 48%.

[0261] 1 H NMR (400 MHz, CDC13): δ 7.49-7.44 (m, 2H), δ 7.37-7.34 (m, 2H), 4.05 (m, 2H), 3.77-3.73 (m, 2H), 1.62 (s, 3H). LC-MS: 242.10 (M-H) - .

[0262] Synthesis of compound 10.32

[0263] Magnesium turnings (0.31 g, 12.9 mmol) were placed in a 100 mL three-necked flask, and one iodine crystal was added. The malondicarboxylic acid bromide (2.5 g, 10.4 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran, and the tetrahydrofuran solution of the malondicarboxylic acid bromide was added dropwise to the reaction flask at a rate of 3 mL per minute using a constant pressure dropping funnel. The reaction was initiated by heating with an electric hairdryer. After the reaction started, the remaining 17 mL of tetrahydrofuran solution was slowly added dropwise. After the addition was completed, the reaction was refluxed for 3.5 hours. Finally, a tetrahydrofuran solution of the Grignard reagent was obtained.

[0264] The tetrahydrofuran solution of the malondicarboxylic acid bromide was added dropwise to a solution of the benzylidene acetaldehyde (1.5 g, 10 mmol) in 14 mL of anhydrous tetrahydrofuran at an ice bath. After the addition was completed, the reaction was allowed to warm to room temperature for 1 hour, and then quenched by adding saturated aqueous ammonium chloride solution. Finally, the tetrahydrofuran was removed, and the product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA = 10:1) to obtain 0.8 g of the target compound, with a two-step yield of 25%.

[0265] 1 H NMR (400 MHz, CDCl3): δ 7.44-7.29 (m, 10H), 4.90 (dd, J = 8.8 Hz, 3.2 Hz, 1H), 4.57 (q, J = 12 Hz, 2H), 3.98 (t, J = 6 Hz, 2H), 3.71 (t, J = 6 Hz, 2H), 3.60 (dd, J = 8 Hz, 3.2 Hz), 3.39 (t, J = 8 Hz, 1H), 1.62 (s, 3H). LC-MS: 313.20 (M-H) - .

[0266] Synthesis of compound 10.33 (debenzylation reaction)

[0267] The compound (0.75 g, 2.4 mmol) was dissolved in 10 mL of ethanol, and 10% palladium-carbon (0.23 g) was added. The air was replaced with H2, and the reaction was stirred at 38°C for 3 hours. After the reaction was completed, the solvent was removed, and the product was purified by column chromatography (PE:EA = 2:1) to obtain 0.2 g of the target compound, with a yield of 29%.

[0268] 1 H NMR (400 MHz, DMSO): δ 7.35-7.29 (m, 4H), 5.23 (d, J = 4 Hz, 1H), 4.74 (t, J = 6 Hz, 1H), 4.54-4.49 (m, 1H), 3.98-3.94 (m, 2H), 3.43 (t, J = 6.8 Hz, 2H), 1.53 (s, 3H). LC-MS: 223.10 (M-H) - .

[0269] Synthesis of compound 10

[0270] To compound 10.33 (0.18 g, 0.8 mmol) was added ethanol 10 mL, hydrochloric acid 5 drops, and the reaction was carried out at 50 °C for 15 h. Water was added and the organic phase was extracted with DCM, dried over anhydrous sodium sulfate and chromatographed on a column to obtain 0.06 g, with a yield of 40%.

[0271] 1 H NMR (400 MHz, DMSO): δ 7.91 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 5.42 (d, J = 4 Hz, 1H), 4.80 (t, J = 6 Hz, 1H), 4.63 (q, J = 5.6 Hz, 1H), 3.46 (t, J = 6 Hz, 2H), 2.56 (s, 3H). LC-MS: 181.10 (M+H) + .

[0272] 11. Synthesis of compound 11 (D)

[0273] 1. Synthesis of 3-vinylphenyloxirane (B)

[0274] Potassium tert-butoxide (2.214 g, 0.02 mol), THF (2 ml), DMSO (12 ml) were added dropwise to a three-necked flask containing trivinylbenzaldehyde (A, 1.215 g, 0.009 mol), trimethylsulfonium iodide (2.118 g, 0.011 mol), THF (8 ml), DMSO (15 ml) under nitrogen protection, and the reaction was carried out at 12 h in an ice bath. The progress of the reaction was monitored by TLC during the reaction. After the reaction was completed, it was extracted with dichloromethane, the organic layer was dried over anhydrous sodium sulfate, and finally separated by column chromatography (PE:EA = 150:1) to obtain 3-vinylphenyloxirane (B) 0.755 g (56%).

[0275] 1 H NMR (DMSO, 400 MHz): δ 7.43-7.21 (m, 4H), 6.77 (dd, J = 10.8 17.7 Hz, 1H,), 5.83 (d, J = 17.7 Hz, 1H), 5.29 (d, J = 10.8 Hz, 1H), 3.93 (dd, J = 2.7, 4.0 Hz, 1H), 3.13 (dd, J = 4.0, 5.5 Hz, 1H,), 2.88 (q, J = 2.7 Hz, 1H).

[0276] 2. Synthesis of 3-(1-hydroxy-2-benzyloxyethyl)styrene (C)

[0277] Benzonic acid (1.22 g, 10.0 mmol), tetrabutylammonium iodide (0.369 g, 1.0 mmol), 3-vinylphenyl oxirane (B, 1.46 g, 10.0 mmol) were dissolved in 5 mL of DMF under nitrogen atmosphere and the reaction mixture was heated at 100 °C for 12 h. The progress of the reaction was monitored by TLC. The reaction mixture was extracted with dichloromethane and the organic layer was dried over anhydrous sodium sulfate. The product was isolated by column chromatography (PE:EA = 10:1) to give 3-(1-hydroxy-2-benzyloxyethyl)styrene (C) 0.818 g (30.47 %).

[0278] 1 H NMR (DMSO, 400 MHz): δ 7.33-7.95 (m, 9H), 6.78 (dd, J = 10.9, 17.6 Hz, 1H), 5.85 (d, J = 17.6 Hz, 1H), 5.78 (d, J = 4.6 Hz, 1H), 5.27 (d, J = 10.9, 1H), 4.96 (q, J = 4.6, 1H), 4.34 (d, J = 5.7 Hz, 2H).

[0279] Synthesis of compound 11 (D)

[0280] 3-(1-hydroxy-2-benzyloxyethyl)styrene (C, 0.20 g, 0.745 mmol), sodium hydroxide (0.043 g, 1.08 mmol) were dissolved in 4 mL of methanol and the reaction mixture was refluxed for 2 h. The progress of the reaction was monitored by TLC. The reaction mixture was extracted with dichloromethane and the organic layer was dried over anhydrous sodium sulfate. The product was isolated by column chromatography (PE:EA = 1:1) to give 3-(1,2-dihydroxyethyl)styrene (D) 0.07 g (57.2 %).

[0281] 1 H NMR (CDC13, 400 MHz): δ 7.12-7.29 (m, 4H), 6.65 (dd, J = 11.1, 17.7 Hz, 1H), 5.69 (d, J = 17.7 Hz, 1H), 5.19 (d, J = 11.1 Hz, 1H), 4.71 (dd, J = 3.3, 8.3 Hz, 1H), 3.65 (m, 2H), 3.07 (s, 2H). MS (EI): 164.1.

[0282] 12. Synthesis of compound 12

[0283] Compound 1 (1.66 g, 0.01 mol) was dissolved in DCM 7 mL, 1 drop of concentrated sulfuric acid was added, and the reaction was started after slight heating. After the reaction was completed, 20 mL of solvent was added, washed with sodium bicarbonate solution, dried and concentrated, and purified by column (PE:EA = 10:1) to obtain 2.00 g of the target compound 2 with a yield of 80%.

[0284] 1 H NMR (400 MHz, CDCl3): δ 7.21-7.00 (m, 4H), 4.75 (dd, J1= 8.0 Hz, J2= 3.6 Hz, 1H), 3.72-3.60 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 2.21 (s, 3H), 2.10 (s, 3H), 1.25 (t, J = 7.6 Hz, 3H). LC-MS: 251.17 (M+H) + .

[0285] 13. Synthesis of 1-(3-cyclopropylphenyl)-1,2-ethanediol (Compound 13)

[0286] Synthesis of 1-(3-cyclopropylphenyl)ethanone

[0287] Weigh 3-bromoacetophenone (5 g, 25.12 mmol), cyclopropylboronic acid (3.25 g, 37.68 mmol), anhydrous potassium phosphate (16 g, 75.36 mmol) and tricyclohexylphosphine (1.41 g, 5.02 mmol) into a 100 ml round bottom flask, dissolve with 20 mL of toluene / water (v:v = 100:1), add palladium acetate (563 mg, 2.52 mmol), heat to 100°C under nitrogen protection for 20 hours, and track the reaction progress by TLC. After the reaction is completed, the system is cooled to room temperature, washed with water, extracted with ethyl acetate 3 times, the organic layer is combined and dried with anhydrous sodium sulfate, and the solvent is removed under reduced pressure. Purify by silica gel column (PE:EA = 100:1) to obtain 3.6 g of light yellow oil with a yield of 89%.

[0288] 1 H NMR (400 MHz, DMSO) δ 7.74-7.69 (m, 1H), 7.64 (t, J = 1.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.34-7.30 (m, 1H), 2.56 (s, 3H), 2.01 (tt, J = 8.4, 5.1 Hz, 1H), 1.03-0.95 (m, 2H), 0.75-0.70 (m, 2H).

[0289] Synthesis of 3-cyclopropylbenzoic acid

[0290] Copper bromide (6.03 g, 26.98 mmol) was taken in EA to make a suspension and heated to reflux with stirring and 1-(3-cyclopropylphenyl)ethanone (3.6 g, 22.49 mmol) in EA was added drop wise using a constant pressure dropping funnel. The reaction was monitored by TLC. After completion of the reaction, it was filtered through celite pad and the cake was washed with EA. The mother liquor was evaporated under vacuum and the residue was taken in EA and washed with water three times. The organic layer was dried over anhydrous sodium sulphate and evaporated under vacuum. The residue was taken in 20 mL of methanol and 5 g of sodium formate was added and the reaction was heated to reflux for 12 h. The reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure and the residue was taken in EA and washed with water three times. The organic layer was dried over anhydrous sodium sulphate and evaporated under vacuum. The residue was purified by silica gel column (PE:EA = 50:1) to get 1.2 g of yellowish oil, 30% yield.

[0291] 1 H NMR (400 MHz, DMSO) δ 7.73 - 7.63 (m, 1H), 7.61 (dd, J = 3.9, 2.4 Hz, 1H), 7.39 (dd, J = 14.6, 6.9 Hz, 1H), 7.34 - 7.28 (m, 1H), 5.03 (t, J = 5.5 Hz, 1H), 4.78 (d, J = 3.6 Hz, 1H), 2.04 - 1.95 (m, 1H), 1.02 - 0.94 (m, 2H), 0.75 - 0.66 (m, 2H).

[0292] Synthesis of 1-(3-cyclopropylphenyl)-1,2-ethanediol

[0293] 3-cyclopropylbenzoic acid (750 mg, 4.26 mmol) was taken in anhydrous THF and sodium borohydride (242 mg, 6.39 mmol) dissolved in THF was added drop wise slowly under ice bath condition. The reaction was monitored by TLC. After completion of the reaction, saturated ammonium chloride solution was added drop wise slowly to quench the reaction and extracted with DCM three times. The organic layer was dried over anhydrous sodium sulphate and evaporated under vacuum. The residue was purified by silica gel column (PE:EA = 2:1) to get 180 mg of white solid, 24% yield.

[0294] 1H NMR (400 MHz, DMSO) δ 7.16 (t, J = 7.6 Hz, 1H), 7.10 - 7.03 (m, 2H), 6.91 (d, J = 7.6 Hz, 1H), 5.15 (d, J = 3.9 Hz, 1H), 4.67 (t, J = 5.1 Hz, 1H), 4.48 (dd, J = 9.5, 5.7 Hz, 1H), 3.40 (t, J = 5.5 Hz, 2H), 1.93 - 1.85 (m, 1H), 0.95 - 0.88 (m, 2H), 0.67 - 0.59 (m, 2H). LC-MS: m / z: 177.20 (M-H) - ,t R :7.537 min.

[0295] The following compounds were all synthesized following the reference and similar routes as above:

[0296] 14. Synthesis of compound 1-(3-ethylphenyl)-2-hydroxyethan-1-one (Compound 14)

[0297] The compound was a colorless oil in 36.7% yield.

[0298] 1 H NMR (400 MHz, DMSO) δ 7.80 - 7.71 (m, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.46 - 7.40 (m, 1H), 5.04 (t, J = 5.4 Hz, 1H), 4.79 (d, J = 4.9 Hz, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.23 - 1.16 (m, 3H).

[0299] 15. Synthesis of compound 1-(3-methoxyphenyl)-1,2-ethanediol (Compound 15)

[0300] The compound was a pale yellow solid in 50% yield.

[0301] 1 H NMR (400 MHz, DMSO) δ 7.24 - 7.17 (m, 1H), 6.90 (d, J = 7.4 Hz, 2H), 6.82 - 6.75 (m, 1H), 4.50 (t, J = 5.9 Hz, 1H), 3.73 (s, 3H), 3.42 (dd, J = 8.9, 4.6 Hz, 2H). LC-MS: m / z: 167.15 (M-H) - .t R :6.105 min.

[0302] 16. Synthesis of compound 1-(2,3-dihydro-5-benzofuranyl)-1,2-ethanediol (Compound 16)

[0303] The compound was a yellowish solid with a yield of 44%.

[0304] 1 H NMR (400 MHz, DMSO) δ 7.17 (s, 1H), 7.01 (dd, J = 8.1, 1.0 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 5.05 (d, J = 4.1 Hz, 1H), 4.63 (t, J = 5.8 Hz, 1H), 4.48 (t, J = 8.7 Hz, 2H), 4.46 - 4.41 (m, 1H), 3.40 - 3.34 (m, 2H), 3.13 (t, J = 8.7 Hz, 2H). LC-MS: m / z: 179.15 (M-H) - ,t R :5.922 min.

[0305] 17. Synthesis of compound 1-(2,3-dihydro-1,4-benzodiazepin-6-yl)-1,2-ethanediol (Compound 17)

[0306] The compound was a yellow oil with a yield of 37.7%.

[0307] 1 H NMR (400 MHz, DMSO) δ 6.80 (s, 1H), 6.76 (s, 2H), 5.09 (d, J = 4.3 Hz, 1H), 4.64 (t, J = 5.8 Hz, 1H), 4.40 (dd, J = 10.4, 5.9 Hz, 1H), 4.20 (s, 4H), 3.37 (d, J = 5.9 Hz, 2H). LC-MS: m / z: 195.15 (M-H) - ,t R :5.776 min.

[0308] 18. Synthesis of compound 1-(1,3-benzoxadiazol-5-yl)-1,2-ethanediol (Compound 18)

[0309] The compound was a yellow solid with a yield of 40%.

[0310] 1H NMR (400 MHz, DMSO) δ 6.86 (d, J = 8.9 Hz, 1H), 6.83 (d, J = 7.9 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 5.96 (d, J = 1.8 Hz, 2H), 5.16 (d, J = 3.5 Hz, 1H), 4.66 (s, 1H), 4.44 (d, J = 2.6 Hz, 1H), 3.37 (d, J = 4.4 Hz, 2H). LC-MS: m / z: 181.15 (M-H) - ,t R :5.803 min.

[0311] 19. Synthesis of compound 1-(4-hydroxyphenyl)-1,2-ethanediol (Compound 19)

[0312] The compound was a white solid in 42% yield.

[0313] 1 H NMR (400 MHz, DMSO) δ 9.20 (s, 1H), 7.11 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.5 Hz, 2H), 5.01 (d, J = 4.1 Hz, 1H), 4.61 (t, J = 5.8 Hz, 1H), 4.42 (dd, J = 10.4, 5.7 Hz, 1H), 3.39 - 3.35 (m, 2H). LC-MS: m / z: 153.15 (M-H) - ,t R :2.187 min.

[0314] 20. Synthesis of compound 1-(3,4-dimethoxy)-1,2-ethanediol (Compound 20)

[0315] The compound was a white solid in 45% yield.

[0316] 1 H NMR (400 MHz, DMSO) δ 6.92 (d, J = 1.5 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.83 (dd, J = 8.2, 1.6 Hz, 1H), 5.12 (d, J = 4.1 Hz, 1H), 4.64 (t, J = 5.8 Hz, 1H), 4.46 (dd, J = 10.3, 5.9 Hz, 1H), 3.72 (d, J = 6.0 Hz, 6H), 3.39 (t, J = 5.9 Hz, 2H). LC-MS: m / z: 197.20 (M-H) - ,t R :5.529 min.

[0317] 21. Synthesis of compound 1 -(4-methoxyphenyl)propane- 1,2-diol (Compound 21)

[0318] The compound was a white solid with a yield of 49.0%.

[0319] 1 H NMR (400 MHz, DMSO-d6) δ 7.03 (dd, J = 148.6, 8.7 Hz, 4H), 5.02 (d, J = 4.3 Hz, 1H), 4.38 (d, J = 5.4 Hz, 1H), 4.28 (t, J = 4.8 Hz, 1H), 3.72 (s, 3H), 3.60 (q, J = 5.5 Hz, 1H), 0.97 (d, J = 6.3 Hz, 3H).

[0320] 22. Synthesis of compound 1 -(2-methoxyphenyl)propane- 1,2-diol (Compound 22)

[0321] The compound was a white solid with a yield of 16.5%.

[0322] 1 H NMR (400 MHz, DMSO-d6) δ 7.03 (dd, J = 148.6, 8.7 Hz, 4H), 5.02 (d, J = 4.3 Hz, 1H), 4.38 (d, J = 5.4 Hz, 1H), 4.28 (t, J = 4.8 Hz, 1H), 3.72 (s, 3H), 3.60 (q, J = 5.5 Hz, 1H), 0.97 (d, J = 6.3 Hz, 3H).

[0323] 23. Synthesis of compound 1 -(4-methoxyphenyl)propane- 1,2-diol (Compound 23)

[0324] The compound was a white solid with a yield of 6.5%.

[0325] 1 H NMR (400 MHz, DMSO-d6) δ 7.03 (dd, J = 148.6, 8.7 Hz, 4H), 5.02 (d, J = 4.3 Hz, 1H), 4.38 (d, J = 5.4 Hz, 1H), 4.28 (t, J = 4.8 Hz, 1H), 3.72 (s, 3H), 3.60 (q, J = 5.5 Hz, 1H), 0.97 (d, J = 6.3 Hz, 3H).

[0326] 24. Synthesis of compound 1-(4-bromophenyl)ethane-1,2-diol (compound 24)

[0327] The compound was a white solid with a yield of 6.3%.

[0328] 1 H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 5.34 (d, J = 4.3 Hz, 1H), 4.75 (t, J = 5.8 Hz, 1H), 4.51 (dt, J = 6.5, 4.8 Hz, 1H), 3.39 (tt, J = 11.4, 5.7 Hz, 2H).

[0329] 25. Synthesis of compound 1-(4-hydroxy-2-methoxyphenyl)ethane-1,2-diol (compound 25)

[0330] The compound was a light brown oily liquid with a yield of 33%.

[0331] 1 H NMR (400 MHz, DMSO-d6) δ 9.28 (s, 1H), 7.13 (d, J = 7.9 Hz, 1H), 6.40 - 6.29 (m, 3H), 4.85 (d, J = 4.0 Hz, 1H), 4.81 - 4.68 (m, 2H), 4.59 (t, J = 5.6 Hz, 1H), 3.70 (s, 3H). LCMS: calculated [M-H] - C9H 11 O4 - = 183.07, found: 183.10.

[0332] 26. Synthesis of compound 1-(3-hydroxyphenyl)ethane-1,2-diol (compound 26)

[0333] The compound was a light brown oily liquid with a yield of 35%.

[0334] 1H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 1H), 7.08 (t, J = 7.8 Hz, 1H), 6.76 (s, 1H), 6.73 (d, J = 7.6 Hz, 1H), 6.61 (dd, J = 8.0, 2.0 Hz, 1H), 5.17 (d, J = 4.0 Hz, 1H), 4.71 (t, J = 5.8 Hz, 1H), 4.44 (dd, J = 10.6, 5.1 Hz, 1H), 3.38 (t, J = 5.3 Hz, 2H). LCMS: calculated [M-H] - C8H9O3 - = 153.06, found: 153.10.

[0335] 27. Synthesis of compound 1-phenylpropane-1,2-diol (Compound 27)

[0336] The compound was a white solid with a yield of 67%.

[0337] 1 H NMR (400 MHz, DMSO-d6) δ 7.35 - 7.17 (m, 5H), 5.20 - 5.11 (m, 1H), 4.64 - 4.44 (m, 1H), 4.37 - 4.26 (m, 1H), 3.70 - 3.61 (m, 1H), 1.05 - 0.79 (m, 3H). LCMS: calculated [M-H] - C9H 11 O2 - = 151.08, found: 151.15.

[0338] 28. Synthesis of compound 1-(3-ethynylphenyl)ethane-1,2-diol (Compound 28)

[0339] The compound was a brown solid with a yield of 25%.

[0340] 1 H NMR (400 MHz, DMSO-d6) δ 7.43 (s, 1H), 7.38 - 7.31 (m, 3H), 5.32 (d, J = 4.3 Hz, 1H), 4.74 (t, J = 5.6 Hz, 1H), 4.52 (dd, J = 10.6, 5.4 Hz, 1H), 4.14 (s, 1H), 3.46 - 3.38 (m, 2H). LCMS: calculated [M-H] - C 10 H9O2 - = 161.06, found: 161.15.

[0341] 29. Synthesis of compound 1-(3-(tert-butyl)phenyl)ethane-1,2-diol (Compound 29)

[0342] The compound was a light yellow solid in 27% yield.

[0343] 1 H NMR (400 MHz, DMSO-d6) δ 7.35 (s, 1H), 7.27 - 7.19 (m, 2H), 7.12 (d, J = 6.9 Hz, 1H), 5.18 (d, J = 4.2 Hz, 1H), 4.69 (t, J = 5.8 Hz, 1H), 4.51 (dd, J = 10.3, 5.9 Hz, 1H), 3.41 (t, J = 5.9 Hz, 2H), 1.27 (s, 9H). LCMS: calculated [M-H] - C 12 H 17 O2 - = 193.12, found: 193.20.

[0344] 30. Structure of compound 1-(3-hydroxy-4-methoxyphenyl)ethane-1,2-diol (Compound 30)

[0345] The compound was a light yellow solid in 30% yield.

[0346] 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.76 (d, J = 1.5 Hz, 1H), 6.68 (dd, J = 8.1, 1.5 Hz, 1H), 5.04 (d, J = 4.0 Hz, 1H), 4.62 (t, J = 5.8 Hz, 1H), 4.38 (dd, J = 10.0, 5.7 Hz, 1H), 3.72 (s, 3H), 3.34 (t, J = 5.9 Hz, 2H). LCMS: calculated [M-H] - C9H 11 O4 - = 183.07, found: 183.15.

[0347] 31. Synthesis of compound 1-(1,3-diethyl-2-hydroxy)-1,2-ethanediol (Compound 31)

[0348] Synthesis of ethyl 2-(2,4-diethyl-3-hydroxyphenyl)-2-oxoacetate

[0349] Weigh 2,6-diethylphenol (1 g, 6.67 mmol) into a 100 mL single necked round bottom flask, dissolve in 20 mL 1,2-dichloroethane, slowly add anhydrous aluminum trichloride (2.7 g, 19.99 mmol) at 0 °C, stir for 10 min, slowly add oxalyl chloride monoethyl ester (1 g, 7.34 mmol), react for 10 min at 0 °C. TLC spot plate track the reaction progress. After the reaction is completed, slowly pour the reaction solution into ice water, extract with DCM / H2O for 3 times, combine the organic phase, dry over anhydrous sodium sulfate, and rotary evaporate. The product is a white solid, no need of purification, yield 90%.

[0350] 1 H NMR (400 MHz, DMSO) δ 9.69 (s, 1H), 7.54 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 2.65 (q, J = 7.5 Hz, 4H), 1.32 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.5 Hz, 6H).

[0351] Synthesis of 1-(2,4-diethyl-3-hydroxyphenyl)-1,2-ethanediol

[0352] Dissolve ethyl 2-(2,4-diethyl-3-hydroxyphenyl)-2-oxoacetate (627 mg, 2.52 mmol) in 10 mL methanol, slowly add sodium borohydride (426.9 mg, 11.29 mmol) in batches under ice bath stirring, react for one hour at 0 °C. TLC spot plate track the reaction progress. After the reaction is completed, add saturated aqueous ammonium chloride solution dropwise to quench, extract with EA / H2O for 3 times, combine the organic phase, dry over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure. Recrystallize to obtain the product as a white solid, yield 60%.

[0353] 1 H NMR (400 MHz, DMSO) δ 7.91 (s, 1H), 6.86 (s, 2H), 4.94 (d, J = 4.0 Hz, 1H), 4.57 (t, J = 5.8 Hz, 1H), 4.38 (dd, J = 10.1, 5.9 Hz, 1H), 3.36 (t, J = 6.0 Hz, 2H), 2.55 (q, J = 7.5 Hz, 4H), 1.11 (t, J = 7.5 Hz, 6H). LC-MS: m / z: 209.20 (M-H) - ,t R :6.736 min.

[0354] The following compounds are all obtained according to a similar synthetic route as 1-(2,4-diethyl-3-hydroxyphenyl)-1,2-ethanediol (73):

[0355] 32. Synthesis of compound 1-(3,4-dihydro-1-benzopyran-6-yl)-1,2-ethanediol (Compound 32)

[0356] The compound was a white solid in 25% yield.

[0357] 1 H NMR (400 MHz, DMSO) δ 7.02 - 6.95 (m, 2H), 6.68 - 6.61 (m, 1H), 5.04 (d, J = 3.7 Hz, 1H), 4.64 (t, J = 5.2 Hz, 1H), 4.40 (d, J = 3.0 Hz, 1H), 4.17 - 3.97 (m, 2H), 3.40 - 3.35 (m, 2H), 2.71 (t, J = 6.4 Hz, 2H), 1.97 - 1.80 (m, 2H). LC-MS: m / z: 193.20 (M-H) - ,t R : 6.462 min.

[0358] 33. Synthesis of compound 1-(4-hydroxy-3-methoxy)-1,2-ethanediol (Compound 33)

[0359] The compound was a pale yellow oil in 30% yield.

[0360] 1 H NMR (400 MHz, DMSO) δ 8.75 (s, 1H), 6.87 (s, 1H), 6.69 (s, 2H), 5.04 (d, J = 4.0 Hz, 1H), 4.61 (t, J = 5.7 Hz, 1H), 4.41 (dd, J = 10.2, 5.9 Hz, 1H), 3.74 (s, 3H), 3.37 (t, J = 6.0 Hz, 2H). LC-MS: m / z: 183.15 (M-H) - ,t R : 2.213 min.

[0361] 34. Synthesis of compound 1-(5,6,7,8-tetrahydronaphthalen-2-yl)-1,2-ethanediol (Compound 34)

[0362] The compound was a white solid in 55% yield.

[0363] 1H NMR (400 MHz, DMSO) δ 7.00 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 7.7 Hz, 1H), 5.09 (d, J = 4.2 Hz, 1H), 4.66 (t, J = 5.8 Hz, 1H), 4.43 (dd, J = 10.2, 5.9 Hz, 1H), 3.39 - 3.35 (m, 2H), 2.68 (d, J = 5.2 Hz, 4H), 1.76 - 1.66 (m, 4H). LC-MS: m / z: 191.20 (M-H) - ,t R :8.760 min.

[0364] 35. Synthesis of compound 1-(3-ethyl-4-methylphenyl)-1,2-ethanediol (Compound 35)

[0365] The compound was a light yellow oil in 60% yield.

[0366] 1 H NMR (400 MHz, DMSO) δ 7.26 - 6.80 (m, 3H), 5.15 - 5.04 (m, 1H), 4.74 - 4.64 (m, 1H), 4.49 - 4.40 (m, 1H), 3.40 - 3.35 (m, 2H), 2.55 (dt, J = 12.5, 7.3 Hz, 2H), 2.28 - 2.19 (m, 3H), 1.13 (tt, J = 7.5, 4.9 Hz, 3H). LC-MS: m / z: 179.20 (M-H) - ,t R :8.718 min.

[0367] 36. Synthesis of compound 1-(2,3-dihydro-1H-indan-5-yl)-1,2-ethanediol (Compound 36)

[0368] The compound was a white solid in 65% yield.

[0369] 1 H NMR (400 MHz, DMSO) δ 7.17 (s, 1H), 7.13 (d, J = 7.7 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 5.10 (d, J = 4.1 Hz, 1H), 4.65 (t, J = 5.8 Hz, 1H), 4.48 (dd, J = 10.3, 5.7 Hz, 1H), 3.38 (t, J = 5.9 Hz, 2H), 2.81 (dd, J = 12.1, 7.1 Hz, 4H), 1.99 (p, J = 7.4 Hz, 2H). LC-MS: m / z: 177.20 (M-H)- ,t R :8.265 min.

[0370] 37. Synthesis of compound 1-(3-ethyl-4-hydroxyphenyl)-1,2-ethanediol (Compound 37)

[0371] The compound was a white powder with a yield of 53%.

[0372] 1 H NMR (400 MHz, DMSO) δ 9.06 (s, 1H), 7.00 (d, J = 1.7 Hz, 1H), 6.92 (dd, J = 8.1, 2.0 Hz, 1H), 6.69 (d, J = 8.1 Hz, 1H), 4.97 (d, J = 4.0 Hz, 1H), 4.59 (t, J = 5.8 Hz, 1H), 4.39 (dd, J = 10.1, 5.9 Hz, 1H), 3.37 (dd, J = 8.0, 3.9 Hz, 2H), 2.54 - 2.50 (m, 2H), 1.11 (t, J = 7.5 Hz, 3H). LC-MS: m / z: 181.20 (M-H) - ,t R :5.947 min.

[0373] 38. Synthesis of compound 3-(3-ethylphenyl)tetrahydrofuran-3-ol (Compound 38)

[0374] In a 100 mL oven-dried reaction flask, 3-bromoethylbenzene (200 mg, 1.08 mmol), magnesium ribbon, 2 pieces of iodine, 20 mL of anhydrous tetrahydrofuran, room temperature reaction, argon protection, after the preparation of Grignard reagent, 2-2H-furanone (112 mg, 1.30 mmol) was dissolved in anhydrous THF and added dropwise to the prepared Grignard reagent, room temperature reaction, TLC detection of complete reaction, saturated ammonium chloride quenching reaction, silica gel column separation and purification, to get 80 mg of colorless oil, yield 38.5%.

[0375] 1H NMR (400 MHz, CDC13) δ 7.31 (s, 1H), 7.26 (dd, J = 6.8, 4.8 Hz, 2H), 7.15 - 7.07 (m, 1H), 4.79 - 4.67 (m, 1H), 4.21 - 4.13 (m, 1H), 4.08 (td, J = 8.6, 3.4 Hz, 1H), 3.98 - 3.85 (m, 2H), 2.70 (s, 1H), 2.65 (q, J = 7.6 Hz, 2H), 2.39 (dt, J = 13.0, 9.1 Hz, 1H), 2.28 - 2.18 (m, 1H), 1.23 (t, J = 7.6 Hz, 3H).

[0376] 39. Synthesis of the compound l-(5-ethylthiophen-3-yl)ethane-l,2-diol (Compound 39)

[0377] Synthesis of 4-bromo-2-ethylthiophene

[0378] Thiophene ethanone (2.05 g, 10 mmol) was dissolved in 80 mL of dry THF and sodium borohydride (1.89 g, 50 mmol) was added portionwise in an ice bath, followed by the addition of anhydrous aluminium trichloride (3.66 g, 27.5 mmol) in the last portion. The reaction was refluxed at 80 °C under nitrogen protection. The progress of the reaction was monitored by TLC (eluent: heptane) and the reaction was complete after 2 h. The reaction was quenched by the careful addition of 30 mL of water and the THF was removed by evaporation. The residue was extracted with dichloromethane and the residue was purified by column chromatography (eluent: heptane) after conventional work-up to give the reduced product as a pure compound 0.98 g in 51.3% yield.

[0379] Synthesis of (5-ethylthiophen-3-yl)magnesium bromide

[0380] Magnesium turnings (31 mg, 1.3 mmol) and a small piece of iodine were added to a reaction flask and the flask was flushed with nitrogen. Dry THF (0.5 mL) was added and the solution was followed by the addition of the above product (191 mg, 1 mmol) in THF (1.5 mL). The reaction was initiated by warming to 35 °C and the dropwise addition of the bromide in THF was continued while the reaction was maintained at a gentle boil. After the addition was complete, the reaction was continued at 35 °C for 1 h and was then cooled to room temperature for use.

[0381] Synthesis of ethyl 2-(5-ethylthiophen-3-yl)-2-oxoacetate

[0382] Add diethyl oxalate (0.41 mL, 3 mmol) and 2 mL THF into the reaction flask, cool to -78 °C under nitrogen protection, slowly drop the prepared format reagent in step one, after dropping, keep the reaction at -78 °C for 2 h, quench the reaction with 1 mL saturated ammonium chloride, extract with EA, conventional post-treatment, purify the crude product by column chromatography (PE / EA = 200:1 to 100:1), obtain 180 mg of pure product, yield 85.7%.

[0383] Synthesis of 1-(5-ethylthiophen-3-yl)ethane-1,2-diol

[0384] Add the product above (170 mg, 0.8 mmol) and 4 mL methanol into the reaction flask, after mixing evenly, add sodium borohydride (91 mg, 2.4 mmol) in batches under ice bath, after adding, move to room temperature. Monitor the reaction progress by TLC (dichloromethane / methanol = 25:1), obtain 30 mg of pure product, yield 22.2%.

[0385] 1 H NMR (400 MHz, CDCl3) δ 6.98 (s, 1H), 6.72 (s, 1H), 4.77 (dd, J1= 7.6 Hz, J1= 2.8 Hz, 1H), 3.74 (m, 1H), 3.66 (m, 1H), 3.16 (s, 2H), 2.79 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H).

[0386] 40. Synthesis of deuterated compound 3-ethylphenylglycol (compound 40)

[0387] Weigh 3-ethylacetophenone (5.00 g, 33.7 mmol) into a 50 mL three-necked flask, dilute with 25 mL 1,4-dioxane, 25 mL methanol, and protect with nitrogen. Stir for 10 min under ice bath. Use a needle tube to suck liquid bromine (5.39 g, 33.7 mmol) dissolved in 25 mL methanol, slowly drop into the reaction liquid under ice bath with a dropping funnel, after dropping, continue stirring under ice bath, monitor the reaction progress by TLC. After 5 h, the reaction is complete. Quench the reaction by adding an appropriate amount of anhydrous sodium sulfite solution. Rotary evaporation to remove the solvent. Extract the reaction liquid with ethyl acetate and water, wash the extract with ethyl acetate and saturated sodium chloride solution, and dry with anhydrous sodium sulfate. Rotary evaporation of the obtained product, obtain 6.15 g of crude product.

[0388] Compound A (1.08 g, 4.76 mmol), sodium formate (0.65 g, 9.56 mmol) were weighed into a 100 mL flask and dissolved in 10 mL of methanol. The reaction was heated to 80 °C and monitored by TLC. The reaction was complete after 4.5 hours. The solvent was removed by rotary evaporation. The reaction was extracted with ethyl acetate and water. The organic layer was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The product was obtained by rotary evaporation. The crude product was 0.75 g. The crude yield was 95.96% (with impurities).

[0389] Compound B (0.10 g, 0.61 mmol) was weighed into a 50 mL flask and dissolved in 10 mL of methanol. Sodium borodeuteride (0.013 g, 0.31 mmol) was added to the reaction in portions while stirring in an ice bath. The ice bath was removed and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC. The solvent was removed by rotary evaporation. The reaction was extracted with ethyl acetate and water. The organic layer was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The product was obtained by rotary evaporation. The product was mixed with silica gel and a small amount of dichloromethane. The sample was loaded onto a column by dry loading. The column was eluted with petroleum ether: ethyl acetate = 4: 1. The product was obtained by rotary evaporation. The yield was 0.02 g. The yield was 19.63%.

[0390] 1 H NMR (400 MHz, DMSO-d6) δ 7.21 (t, J = 7.5 Hz, 1H), 7.17 (s, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 5.14 (s, 1H), 4.67 (t, J = 5.8 Hz, 1H), 3.40 (d, J = 5.8 Hz, 2H), 2.58 (q, J = 7.5 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LCMS [M-H]": 166.20

[0391] 41. Synthesis of deuterated compound 3-ethylphenylglycol (Compound 41)

[0392] Compound B (0.10 g, 0.61 mmol) was weighed into a 50 mL flask and dissolved in 10 mL of methanol. Sodium borodeuteride (0.013 g, 0.31 mmol) was added to the reaction in portions while stirring in an ice bath. The ice bath was removed and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC. The solvent was removed by rotary evaporation. The reaction was extracted with ethyl acetate and water. The organic layer was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The product was obtained by rotary evaporation. The product was mixed with silica gel and a small amount of dichloromethane. The sample was loaded onto a column by dry loading. The column was eluted with petroleum ether: ethyl acetate = 4: 1. The product was obtained by rotary evaporation. The yield was 0.02 g. The yield was 19.63%.

[0393] Crude yield = 67.08% (with impurities)

[0394] Compound A (1.30 g, 5.72 mmol) was weighed into a 50 mL flask, 26 mL of toluene, 1.95 mL of deuterium water and potassium carbonate (0.20 g, 1.45 mmol) were added, and heated to 87 °C to reflux. After 4 hours, the water layer was separated at 40-50 °C, and 1.95 mL of deuterium water and potassium carbonate (0.20 g, 1.45 mmol) were added, and the reaction was continued. After 8 hours, the operation was repeated once, and after 12 hours, the reaction was completed. The water layer was separated, and the water layer was washed with ethyl acetate. The solvent was removed by rotary evaporation. The reaction solution was extracted with ethyl acetate and water, and the extract was washed with ethyl acetate and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The product was obtained by rotary evaporation, and the crude product was 1.01 g. Crude yield = 77.11% (with impurities)

[0395] Compound A (1.00 g, 4.36 mmol), sodium formate (0.60 g, 8.82 mmol), and methanol 5 mL were weighed into a 50 mL flask, heated to 80 °C to reflux, and the reaction progress was monitored by thin layer chromatography. After 4.5 hours, the reaction was completed. The solvent was removed by rotary evaporation. The reaction solution was extracted with ethyl acetate and water, and the extract was washed with ethyl acetate and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The product was obtained by rotary evaporation, and the crude product was 0.72 g. Crude yield = 99.35% (with impurities)

[0396] Compound C (1.00 g, 6.02 mmol) was weighed into a 50 mL flask, diluted with 5 mL of methanol, and stirred in an ice bath for 10 min. Sodium borohydride (0.23 g, 6.02 mmol) was added to the reaction solution in portions under ice bath, and after addition was completed, the ice bath was removed and the reaction was carried out at room temperature. The reaction progress was monitored by thin layer chromatography. The solvent was removed by rotary evaporation, and the reaction solution was extracted with ethyl acetate and water, and the extract was washed with ethyl acetate and saturated sodium chloride solution, and dried over anhydrous sodium sulfate, and rotary evaporation was performed. An appropriate amount of dichloromethane and silica gel were added, mixed, and then rotary evaporation was performed to prepare the sample. Dry column loading, dry loading, and column chromatography were performed with petroleum ether: ethyl acetate = 4:1. The product was obtained by rotary evaporation, and the yield was 0.21 g. Yield = 20.74%

[0397] 1 H NMR (600 MHz, Chloroform-d) δ 7.35 (d, J = 7.6 Hz, 1H), 7.33 (s, 1H), 7.24 - 7.20 (m, 2H), 4.85 (s, 1H), 3.81 (dd, J = 11.4, 3.5 Hz, 1H), 2.76 (s, 2H), 2.72 (q, J = 7.6 Hz, 2H), 1.31 (t, J = 7.6 Hz, 3H). LCMS [M+H]+: 169.1

[0398] 42. Synthesis process of compounds 42-44

[0399] Synthesis of 1-(3-(1-hydroxy-1-deutero-ethyl)phenyl)-1-deutero-ethanol (2)

[0400] 1,3-Diacetylbenzene (3.00 g, 18.50 mmol) was weighed into a 100 mL single-necked flask and dissolved in 20 mL of methanol, and stirred at 0 °C. Sodium borodeuteride (0.39 g, 9.29 mmol) was weighed into another flask and added slowly to the reaction solution. After the addition was completed, the solution was stirred at room temperature for 3 h. The solvent was removed by rotary evaporation, and the reaction was extracted with ethyl acetate. The extract was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1, v / v) to give 2.83 g of colorless crystalline solid in 91.1% yield.

[0401] 1 H NMR (400 MHz, DMSO-d6) δ 7.32 (t, J = 1.5 Hz, 1H), 7.26 - 7.15 (m, 3H), 5.09 (s, 2H), 1.30 (s, 6H). MS

[0402] Synthesis of 1,3-bis(1-dideuteroethyl)benzene (3a)

[0403] 1-(3-(1-hydroxy-1-deutero-ethyl)phenyl)-1-deutero-ethanol (12.00 g, 71.33 mmol) was weighed into a 250 mL two-necked flask and dissolved in 50 mL of deuterated ethanol. 10% palladium-carbon (2.00 g, 25%) was added to the reaction solution, and the solution was replaced with deuterium gas three times. 1 mL of deuterated hydrochloric acid was taken with a needle tube and added slowly to the reaction solution, which was stirred at room temperature overnight. The reaction solution was filtered with celite, and the filtrate was extracted with dichloromethane and washed with saturated sodium chloride solution. The extract was dried over anhydrous sodium sulfate. The dichloromethane was removed by rotary evaporation at 40 °C. The crude product was obtained as a colorless transparent liquid in 8.20 g in 83.3% yield.

[0404] 1 H NMR (400 MHz, DMSO-d6) δ 7.17 (t, J = 7.5 Hz, 1H), 7.03 (s, 1H), 7.00 (d, J = 7.5 Hz, 2H), 1.15 (s, 6H). MS

[0405] Synthesis of 1,3-bis(1-deutero-ethyl)benzene (3b)

[0406] Take 1-(3-(1-hydroxy-1-deuterio-ethyl)phenyl)-1-deuterio-ethanol (1.00 g, 5.94 mmol) into a 100 mL two-necked flask, dissolve in 5 mL dichloromethane, and stir at 0 °C. Take triethylsilane (4.90 g, 42.14 mmol) and slowly add to the reaction solution under nitrogen protection. Take 10 mL needle tube to take boron trifluoride etherate (5.90 g, 41.57 mmol), and slowly drop into the reaction solution at 0 °C. After dropping, slowly warm to room temperature and stir overnight. Quench with saturated sodium bicarbonate solution until no more bubbles are generated. Extract the reaction solution with dichloromethane and water, wash the extract with dichloromethane and saturated sodium chloride solution, and dry over anhydrous sodium sulfate. Remove dichloromethane by rotary evaporation at 40 °C. Obtain 0.50 g of colorless transparent liquid as a crude product, with a yield of 61.7%.

[0407] 1 H NMR (400 MHz, Chloroform-d) δ 7.12 (t, J = 7.6 Hz, 1H), 6.97 - 6.90 (m, 3H), 2.53 (q, J = 7.6 Hz, 2H), 1.14 (d, J = 7.6 Hz, 6H). MS

[0408] Synthesis of 1-(3-(1-bisdeuterioethyl)phenyl)ethanone (4a)

[0409] Take 1,3-di(1-dideuterioethyl)benzene (8.20 g, 59.31 mmol), N-hydroxyphthalimide (3.20 g, 19.62 mmol) into a 100 mL three-necked flask, dissolve in 60 mL acetonitrile, and replace with oxygen three times. Heat to 40 °C. Take 10 mL needle tube to take tert-butyl nitrite (6.10 g, 59.15 mmol), and slowly drop into the reaction solution. The reaction solution gradually turns orange. After dropping, warm to 55 °C and react for 2 hours. Remove the solvent by rotary evaporation. Add a small amount of dichloromethane, and a white solid precipitates. Add diatomite and filter, and dry the filtrate. Purify the crude product by silica gel column chromatography (petroleum ether / dichloromethane = 10:1, v / v) to obtain 2.80 g of yellow liquid, with a yield of 31.4%.

[0410] 1 H NMR (400 MHz, DMSO-d6) δ 7.78 (m, 2H), 7.49 (d, J = 7.7 Hz, 1H), 7.43 (t, J = 7.4 Hz, 1H), 2.57 (s, 3H), 1.19 (s, 3H).

[0411] Synthesis of 1-(3-(1-deuterioethyl)phenyl)ethanone (4b)

[0412] Synthesis procedure was the same as 4a. Yellow liquid was obtained in 29.9% yield.

[0413] 1 H NMR (400 MHz, DMSO-d6) δ 7.76-7.80 (m, 2H), 7.41-7.51 (m, 2H), 2.66 (q, J = 7.6 Hz, 1H), 2.57 (s, 3H), 1.20 (d, J = 7.6 Hz, 3H).

[0414] Synthesis of 1-(3-(1-ethenyl)phenyl)ethanone (15a)

[0415] Synthesis procedure was the same as 4a. Yellow liquid was obtained in 35.3% yield.

[0416] 1 H NMR (600 MHz, DMSO-d6) δ 7.79-7.76 (m, 2H), 7.48 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 2.68 (q, J = 7.6 Hz, 2H), 2.57 (s, 3H), 1.20 (t, J = 7.6 Hz, 3H).

[0417] Synthesis of 1-(3-(1-ethenyl)phenyl)ethanone (15a)

[0418] 1-(3-(1-ethenyl)phenyl)ethanone (2.80 g, 18.64 mmol), p-toluenesulfonic acid (1.66 g, 9.64 mmol) were weighed into a 100 mL single-necked flask and dissolved in 20 mL of acetonitrile. The solution was stirred at 0 °C. N-bromosuccinimide (3.64 g, 20.45 mmol) was slowly added to the reaction solution, which was then allowed to react at 40 °C. After 3 h, the reaction was complete. The reaction was quenched by adding an appropriate amount of water. The solvent was removed by rotary evaporation. The reaction solution was extracted with ethyl acetate and water. The extract was washed with ethyl acetate and saturated sodium chloride solution and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether / dichloromethane = 10:1, v / v) to obtain 2.12 g of yellow liquid in 50.1% yield.

[0419] Synthesis of 1-(3-(1-ethenyl)phenyl)ethanone (15a)

[0420] Synthesis procedure was the same as 5a. Yellow liquid was obtained in 35.3% yield.

[0421] Synthesis of 1-(3-(1-ethenyl)phenyl)ethanone (15a)

[0422] Synthesis procedure was the same as 5a, to give yellow liquid, which was used in the next step without purification.

[0423] Synthesis of 1-(3-(1-dideuterioethyl)phenyl)-2-hydroxyethanone (6a)

[0424] 1-(3-(1-dideuterioethyl)phenyl)-2-hydroxyethanone (6a) was synthesized by the same procedure as 5a to give yellow liquid, which was used in the next step without purification.

[0425] 1 H NMR (400 MHz, Chloroform-d) δ 7.77 (s, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 4.87 (s, 2H), 1.20 (s, 3H).

[0426] Synthesis of 1-(3-(1-dideuterioethyl)phenyl)-2-hydroxyethanone (6a)

[0427] Synthesis procedure was the same as 6a to give yellow liquid with 45.1% yield.

[0428] 1 H NMR (400 MHz, DMSO-d6) δ 7.72 - 7.77 (m, 2H), 7.50 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 4.79 (s, 2H), 2.66 (q, J = 7.6 Hz, 1H), 1.19 (d, J = 7.6 Hz, 3H).

[0429] Synthesis of 1-(3-ethylphenyl)-2-hydroxyethanone (17c)

[0430] Synthesis procedure was the same as 6a to give yellow liquid with 56.3% yield.

[0431] 1H NMR (400 MHz, Chloroform-d) δ 7.76 (s, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 4.87 (s, 2H), 2.72 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H).

[0432] Synthesis of 1-(3-(1-d2-ethyl)phenyl)-ethanediol (compound 42)

[0433] Weigh 1-(3-(1-d2-ethyl)phenyl)-2-hydroxyethanone (0.93 g, 5.60 mmol) in a 50 mL single-necked flask, dissolve in 10 mL of methanol, and stir at 0 °C. Weigh sodium borohydride (0.11 g, 2.89 mmol) and slowly add it to the reaction solution in an ice bath. After adding, react at room temperature. Remove the solvent by rotary evaporation, extract the reaction solution with ethyl acetate and water, wash the extract with ethyl acetate and saturated sodium chloride solution, and dry with anhydrous sodium sulfate. Purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1, v / v) to obtain 0.77 g of colorless solid, with a yield of 81.4%.

[0434] 1 H NMR (400 MHz, DMSO-d6) δ 7.21 (t, J = 7.5 Hz, 1H), 7.17 (s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 5.15 (d, J = 4.2 Hz, 1H), 4.66 (t, J = 5.8 Hz, 1H), 4.50 (q, J = 5.8 Hz, 1H), 3.42 (t, J = 5.9 Hz, 2H), 1.16 (s, 3H). 13 C NMR (151 MHz, DMSO) δ 143.90, 143.59, 128.25, 126.68, 126.19, 124.16, 74.41, 68.02, 28.26, 28.13, 28.00, 27.87, 27.75, 16.00. HRMS (ESI): exact mass calcd for C 10 H 12 D2O2[M+Na] + , 191.1015; found 191.1017. HPLC purity: 95.5%, retention time = 7.642 min.

[0435] Synthesis of 1-(3-(1-deuterioethyl)phenyl)-ethanediol (compound 43)

[0436] Synthetic procedure similar to TJH1, afforded colorless solid in 80.1% yield.

[0437] 1 H NMR (400 MHz, DMSO-d6) δ 7.20 (t, J = 7.5 Hz, 1H), 7.16 (s, 1H), 7.12 (d, J = 7.5 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 5.17 (d, J = 4.1 Hz, 1H), 4.68 (t, J = 5.8 Hz, 1H), 4.49 (q, J = 5.6 Hz, 1H), 3.40 (t, J = 5.9 Hz, 2H), 2.56 (q, J = 7.6 Hz, 1H), 1.16 (d, J = 7.6 Hz, 3H). 13 C NMR (151 MHz, DMSO) δ 143.90, 143.61, 128.25, 126.68, 126.18, 124.16, 74.41, 68.02, 28.48, 28.36, 28.23, 16.08. HRMS (ESI): exact mass calcd for C 10 H 13 DO2[M+Na] + , 190.0952; found 190.0954. HPLC purity: 97.2%, retention time = 7.625 min.

[0438] Synthesis of 1-(3-ethylphenyl)-1-deutero-ethanediol (Compound 44)

[0439] Synthetic procedure similar to TJH1, afforded colorless solid in 79.8% yield.

[0440] 1 H NMR (400 MHz, DMSO-d6) δ 7.20 (t, J = 7.5 Hz, 1H), 7.16 (s, 1H), 7.12 (d, J = 7.5 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 5.17 (d, J = 4.1 Hz, 1H), 4.68 (t, J = 5.8 Hz, 1H), 4.49 (q, J = 5.6 Hz, 1H), 3.40 (t, J = 5.9 Hz, 2H), 2.56 (q, J = 7.6 Hz, 1H), 1.16 (d, J = 7.6 Hz, 3H). - .

[0441] 43. Synthesis of compounds 45-49

[0442] Synthesis of 1-(3-(1-ethylphenyl)-ethanediol (7)

[0443] Weigh 1-(3-ethylphenyl)-2-hydroxyethanone (0.87 g, 5.30 mmol) into a 50 mL single-neck flask, dissolve with 10 mL of methanol, and stir at 0 °C. Weigh sodium borohydride (0.22 g, 5.30 mmol) into the reaction solution under ice bath, and add it slowly. After the addition, allow the reaction to proceed at room temperature. Remove the solvent by rotary evaporation, extract the reaction solution with ethyl acetate and water, wash the extract with ethyl acetate and saturated sodium chloride solution, and dry it with anhydrous sodium sulfate. Purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1, v / v) to obtain 0.75 g of colorless solid with a yield of 85.2%.

[0444] 1 H NMR (400 MHz, DMSO-d6) δ 7.21 (t, J = 7.5 Hz, 1H), 7.17 (s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 5.18 (d, J = 4.2 Hz, 1H), 4.69 (t, J = 5.8 Hz, 1H), 4.50 (q, J = 5.9 Hz, 1H), 3.41 (t, J = 5.9 Hz, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H).

[0445] Synthesis of 1-(3-ethylphenyl)ethane-1,2-dioate (Compound 45)

[0446] Weigh 1-(3-ethylphenyl)-ethanediol (0.50, 3.01 mmol) into a 50 mL single-neck flask, dissolve with 5 mL of dichloromethane, and stir at room temperature. Weigh 4-dimethylaminopyridine (0.04 g, 0.33 mmol) and acetic anhydride (1.23 g, 12.05 mmol) into the reaction solution, and allow the reaction to proceed at room temperature. Remove the solvent by rotary evaporation, extract the reaction solution with ethyl acetate and water, wash the extract with ethyl acetate and saturated sodium chloride solution, and dry it with anhydrous sodium sulfate. Purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 25:1, v / v) to obtain 0.61 g of colorless clear liquid with a yield of 81.0%.

[0447] 1H NMR (400 MHz, Chloroform-d) δ 7.27 (d, J = 8.6 Hz, 1H), 7.20 - 7.15 (m, 3H), 6.00 (dd, J = 7.9, 4.1 Hz, 1H), 4.35 - 4.27 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 2.12 (s, 3H), 2.06 (s, 3H), 1.24 (t, J = 7.6 Hz, 3H).

[0448] Synthesis of 4-(3-ethylphenyl)-1,3-dioxolan-2-one (Compound 46)

[0449] Weigh 1-(3-ethylphenyl)-ethanediol (0.50, 3.01 mmol) in a 50 mL single neck flask, add 5 mL of toluene to dissolve. Weigh bis(2-pyridyl) carbonate (1.30 g, 6.01 mmol) into the reaction solution, and warm to 60 °C to react. Remove the solvent by rotary evaporation, extract the reaction solution with ethyl acetate and water, wash the extract with ethyl acetate and saturated sodium chloride solution, and dry with anhydrous sodium sulfate. Purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1, v / v) to obtain 0.38 g of colorless clear liquid, with a yield of 65.7%.

[0450] 1 H NMR (400 MHz, Chloroform-d) δ 7.27 (d, J = 8.6 Hz, 1H), 7.20 - 7.15 (m, 3H), 6.00 (dd, J = 7.9, 4.1 Hz, 1H), 4.35 - 4.27 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 2.12 (s, 3H), 2.06 (s, 3H), 1.24 (t, J = 7.6 Hz, 3H).

[0451] Synthesis of 4-(3-ethylphenyl)-2,2-dimethyl-1,3-dioxolane (Compound 47)

[0452] Into a 50 mL flask, 1-(3-ethylphenyl)-ethanediol (0.50, 3.01 mmol) was weighed and dissolved in 3 mL of tetrahydrofuran. Iron chloride (0.17 g, 1.50 mmol) and 3 mL of acetone were added to the reaction solution, and the reaction was carried out at room temperature. The reaction solution was brown. The solvent was removed by rotary evaporation at room temperature, and the reaction solution was extracted with dichloromethane and water. The extract was washed with dichloromethane and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the dichloromethane was removed by rotary evaporation at room temperature. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1, v / v) to obtain a yellowish liquid of 0.52 g with a yield of 83.7%.

[0453] 1 H NMR (400 MHz, Chloroform-d) δ 7.27 (t, J = 7.5 Hz, 1H), 7.20 (s, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.14 (d, J = 7.4 Hz, 1H), 5.05 (dd, J = 8.0, 6.3 Hz, 1H), 4.29 (dd, J = 8.1, 6.2 Hz, 1H), 3.71 (t, J = 8.2 Hz, 1H), 2.65 (q, J = 7.6 Hz, 2H), 1.55 (s, 3H), 1.49 (s, 3H), 1.24 (t, J = 7.6 Hz, 3H).

[0454] Synthesis of 4-(3-ethylphenyl)-2-methyl-1,3-dioxolane (Compound 48)

[0455] Into a 50 mL flask, 1-(3-ethylphenyl)-ethanediol (0.50, 3.01 mmol) was weighed and dissolved in 3 mL of tetrahydrofuran. Iron chloride (0.17 g, 1.50 mmol) and 3 mL of acetone were added to the reaction solution, and the reaction was carried out at room temperature. The reaction solution was brown. The solvent was removed by rotary evaporation at room temperature, and the reaction solution was extracted with dichloromethane and water. The extract was washed with dichloromethane and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the dichloromethane was removed by rotary evaporation at room temperature. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1, v / v) to obtain a yellowish liquid of 0.52 g with a yield of 83.7%.

[0456] 1H NMR (400 MHz, Chloroform-d) δ 7.30 - 7.25 (m, 1H), 7.20 (s, 1H), 7.18 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 5.23 (q, J = 4.8 Hz, 1H), 5.00 (t, J = 6.8 Hz, 1H), 4.18 (t, J = 7.5 Hz, 1H), 3.79 (dd, J = 7.8, 6.4 Hz, 1H), 2.66 (q, J = 7.6 Hz, 2H), 1.53 (d, J = 4.8 Hz, 3H), 1.24 (t, J = 7.6 Hz, 3H).

[0457] Synthesis of 2-(3-ethylphenyl)oxirane (Compound 49)

[0458] Weigh 2-bromo-l-(3-ethylphenyl)ethan-l-one (0.50, 2.20 mmol) in a 50 mL single-necked flask, dissolve in 5 mL of anhydrous methanol, and stir at 0 °C. Weigh sodium borohydride (0.08 g, 2.11 mmol) into the reaction solution, and after adding it, remove the ice bath and react at room temperature. Monitor the reaction progress by spotting on a plate. After the raw material is completely reacted, weigh potassium carbonate (0.60 g, 4.40 mmol) into the reaction solution, and stir at room temperature overnight. Remove the solvent by rotary evaporation, extract the reaction solution with dichloromethane and water, wash the extract with dichloromethane and saturated sodium chloride solution, and dry with anhydrous sodium sulfate. Purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 100:1, v / v) to obtain 0.11 g of a colorless liquid with a yield of 33.7%.

[0459] 1 H NMR (400 MHz, Chloroform-d) δ 7.26 (t, J = 7.4 Hz, 1H), 7.15 - 7.09 (m, 3H), 3.84 (dd, J = 3.9, 2.7 Hz, 1H), 3.13 (dd, J = 5.5, 4.1 Hz, 1H), 2.80 (dd, J = 5.5, 2.6 Hz, 1H), 2.65 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H).

[0460] (R)-2-(3-ethylphenyl)-2-hydroxyethyl-L-valinate (Compound 50)

[0461] Step 1 (R)-2-(3-ethylphenyl)-2-hydroxyethyl (tert-butoxycarbonyl)-L-valinate (50-1)

[0462] To a 100 mL single necked round bottom flask was added (R)-1-(3- ethylphenyl)ethane-1,2-diol (8.00 g, 48.0 mmol), (tert-butoxycarbonyl)-L- valine (2.10 g, 9.60 mmol) and 4-dimethylaminopyridine (0.23 g, 1.92 mmol) sequentially, followed by 40 mL of dichloromethane as solvent. After cooling to 0 °C in an ice-salt bath, dicyclohexylcarbodiimide (3.90 g, 19.0 mmol) was added. After stirring for 2 h in an ice-bath, the reaction was allowed to warm to room temperature and stirred for 2 h. The progress of the reaction was monitored by thin layer chromatography. After the reaction was completed, the reaction mixture was filtered, concentrated, extracted with ethyl acetate, and the organic layers were combined and washed sequentially with 1 M hydrochloric acid, water, saturated sodium bicarbonate, water, saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to give a light yellow oily liquid. The product was purified by flash column chromatography on silica gel (petroleum ether / ethyl acetate = 30:4) to give the corresponding yellow oily liquid (R)-2-(3-ethylphenyl)-2-hydroxyethyl (tert-butoxycarbonyl)-L- valinate 1.20 g in 34.0% yield.

[0463] LC-MS: m / z: 366.3 (M+H) + .

[0464] Step 2 (R)-2-(3-ethylphenyl)-2-hydroxyethyl-L-valinate (50)

[0465] To a 50 mL single necked round bottom flask was added (R)-2-(3- ethylphenyl)-2-hydroxyethyl (tert-butoxycarbonyl)-L-valinate (1.80 g, 4.90 mmol) and ethyl acetate hydrochloride 20 ml as solvent. After stirring for 3 h at room temperature, the progress of the reaction was monitored by thin layer chromatography. After the reaction was completed, the reaction mixture was concentrated, extracted with ethyl acetate, and the organic layers were combined and washed sequentially with saturated sodium bicarbonate, water, saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to give a light yellow oily liquid. The product was purified by flash column chromatography on silica gel (ethyl acetate / methanol = 10:1) to give the corresponding white oily liquid (R)-2-(3-ethylphenyl)-2-hydroxyethyl-L-valinate 0.97 g in 75.0% yield.

[0466] 1H NMR (400MHz, DMSO-d6) δ7.29-7.08(m,4H),5.69-5.51(m,1H),4.74(d,J=6.0Hz,1H),4.17-3.99(m,1H),3.70-3.52(m,2H),3.22(d,J=5.0 Hz,1H),3.09(d,J=5.1Hz,1H),2.60(d,J=7.5Hz,2H),2.06-1.62(m,1H),1.17(q,J=7.1Hz,3H),0.94-0.54(m,6H).LC-MS:m / z:266.3(M+H) + .

[0467] (R)-2-((L-valyl)oxy)-2-(3-ethylphenyl)valine ethyl ester (Compound 51)

[0468] Step 1: (R)-2-(((tert-Butoxycarbonyl)-L-valyl)oxy)-2-(3-ethylphenyl)ethyl(tert-Butoxycarbonyl)valine (51-1)

[0469] To a 25 mL single-necked round-bottom flask were added (R)-1-(3-ethylphenyl)ethane-1,2-diol (0.50 g, 3.00 mmol), (tert-butyloxycarbonyl)-L-valine (4.60 g, 21.0 mmol), and 4-dimethylaminopyridine (0.60 g, 4.80 mmol), followed by 5 mL of dichloromethane as the solvent. After cooling to 0°C in an ice-salt bath, dicyclohexylcarbodiimide (8.70 g, 42.0 mmol) was added. The mixture was stirred in an ice bath for 2 h, then warmed to room temperature and stirred for 2 h. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, the reaction solution was filtered, concentrated, and extracted with ethyl acetate. The combined organic layers were washed sequentially with 1 M hydrochloric acid, water, saturated sodium bicarbonate, water, and saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent, yielding a pale yellow oil. The product was separated and purified by flash silica gel column chromatography (petroleum ether / ethyl acetate = 30:4) to obtain 1.01 g of the corresponding yellow oily liquid (R)-2-(((tert-butoxycarbonyl)-L-valyl)oxy)-2-(3-ethylphenyl)ethyl(tert-butoxycarbonyl)valine in a yield of 60.0%.

[0470] LC-MS: m / z: 565.4 (M+H) + .

[0471] Step 2 (R)-2-((L-valyl)oxy)-2-(3-ethylphenyl)valine ethyl ester (51)

[0472] In a 50 mL single necked round bottom flask was charged with (R)-2-(((tert- butoxycarbonyl)-L-valyl)oxy)-2-(3-ethylphenyl)ethyl (tert-butoxycarbonyl) valinate (1.00 g, 1.77 mmol) and hydrochloric acid in ethyl acetate 20 ml as solvent. The reaction was stirred at room temperature for 3 h, the progress of the reaction was monitored by thin layer chromatography. After completion of the reaction, the reaction was concentrated, extracted with ethyl acetate, the organic layers were combined and washed successively with saturated sodium bicarbonate, water, saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain a pale yellow oily liquid. The product was purified by flash column chromatography on silica gel (ethyl acetate / methanol = 10:1) to obtain the corresponding white oily liquid (R)-2-((L-valyl)oxy)-2-(3- ethylphenyl) valine ethyl ester 0.45 g, yield 70.0%.

[0473] 1 H NMR (400 MHz, DMSO-d6) δ 7.32 - 7.13 (m, 4H), 5.91 (dd, J = 7.5, 3.7 Hz, 1H), 4.38 (dd, J = 11.8, 3.7 Hz, 1H), 4.27 (dd, J = 11.8, 7.5 Hz, 1H), 3.17 (d, J = 5.2 Hz, 1H), 3.09 (d, J = 5.1 Hz, 1H), 2.90 (d, J = 20.1 Hz, 4H), 2.59 (q, J = 7.6 Hz, 2H), 1.85 (ddd, J = 45.9, 13.0, 6.5 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H), 0.83 (t, J = 6.5 Hz, 6H), 0.77 (d, J = 6.8 Hz, 3H), 0.70 (d, J = 6.8 Hz, 3H). LC-MS: m / z: 365.3 (M+H) + .

[0474] (R)-2-(3-ethylphenyl)-2-hydroxyethyl octanoate (52)

[0475] Synthetic procedure:

[0476] Into a 100 mL single necked round bottom flask, (R)-1-(3- ethylphenyl)ethane-1,2-diol (5.00 g, 30.0 mmol), n-octanoic acid (0.86 g, 6.00 mmol) and 4-dimethylaminopyridine (0.14 g, 1.20 mmol) were added sequentially, followed by 50 mL of dichloromethane as solvent. After cooling to 0 °C in an ice-salt bath, dicyclohexylcarbodiimide (2.50 g, 12.0 mmol) was added. After stirring for 2 h in an ice-bath, the bath was removed and the reaction was stirred for 2 h at room temperature. The progress of the reaction was monitored by thin layer chromatography. After the reaction was complete, the reaction mixture was filtered, concentrated, extracted with ethyl acetate, the organic layers were combined and washed sequentially with 1 M hydrochloric acid, water, saturated sodium bicarbonate, water, saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to give a light yellow oily liquid. The corresponding yellow oily liquid, (R)-2-(3- ethylphenyl)-2-hydroxyethyloctanoate, 0.45 g, 26.0% yield, was isolated by flash column chromatography on silica gel (petroleum ether / ethyl acetate = 30:4).

[0477] 1 H NMR (400 MHz, DMSO-d6) δ 7.33 - 7.03 (m, 4H), 5.73 - 5.42 (m, 1H), 4.84 - 4.64 (m, 1H), 4.06 (q, J = 6.7 Hz, 2H), 2.61 (q, J = 7.5 Hz, 2H), 2.27 (t, J = 7.5 Hz, 2H), 1.48 (s, 2H), 1.34 - 1.07 (m, 11H), 0.86 (d, J = 6.8 Hz, 3H).

[0478] Compounds 53-66 were synthesized using the following method:

[0479] Synthetic Route One

[0480] Reagents and conditions: (a) Pd(dppf)Cl2, K2CO3, 85 °C, N2; (b) ADmixβ, t-BuOH, H2O, room temperature.

[0481] Synthetic Route Two

[0482] Reagents and conditions: (c) Ag2CO3, AgOTf, DCM, room temperature; (d) K2CO3, MeOH / DCM, room temperature;

[0483] Synthesis of (R)-1-(3-cyclopropylphenyl)ethane-1,2-diol (Compound 53)

[0484] Step 1: Synthesis of 1-cyclopropyl-3-vinylbenzene (Compound 53)

[0485] To a 10 mL Schlenk tube was added 1-bromo-3-cyclopropylbenzene (1.01 mmol, 200 mg), potassium vinyltrifluoroborate (1.21 mmol, 162 mg), Dppf palladium dichloride (0.10 mmol, 73 mg), and potassium carbonate (2.02 mmol, 279 mg) sequentially, 4 mL of a mixture of dioxane and water (6:1) as solvent, nitrogen protection to remove oxygen, 85 °C reaction for 2 hours, TLC detection of reaction progress. After the reaction was completed, the dioxane was removed by concentration, extracted with ethyl acetate and water three times, the organic phase was concentrated and column chromatography (pure PE) to give the intermediate 1-cyclopropyl-3-vinylbenzene (transparent oil, 108 mg, yield 74.1%).

[0486] 1 H NMR (400 MHz, Chloroform-d) δ 7.23 (s, 1H), 7.22 - 7.19 (m, 2H), 7.12 (d, J = 2.1 Hz, 1H), 6.99 - 6.91 (m, 1H), 5.73 (dd, J = 17.6, 1.0 Hz, 1H), 5.22 (dd, J = 10.9, 0.9 Hz, 1H), 1.89 (tt, J = 8.4, 5.1 Hz, 1H), 1.00 - 0.92 (m, 2H), 0.70 (dt, J = 6.6, 4.6 Hz, 2H).

[0487] Step 2: Synthesis of (R)-1-(3-cyclopropylphenyl)ethane-1,2-diol (Compound 53)

[0488] To a 50 mL single-necked flask was added 1-cyclopropyl-3-vinylbenzene (0.56 mmol, 80 mg), AD-mix-β (0.34 mmol, 261 mg) was added under ice bath conditions, 1:1 tert-butanol and water as solvent, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, it was extracted with ethyl acetate and water three times, the organic phase was concentrated and column chromatography (PE:EA = 5:1) to give (R)-1-(2-ethylpyridin-4-yl)ethane-1,2-diol (white solid, 40 mg, yield 40.0%).

[0489] 1H NMR (600 MHz, Chloroform-d) δ 7.14 (t, J = 7.6 Hz, 1H), 7.02 (dt, J = 7.6, 1.4 Hz, 1H), 6.99 (t, J = 1.8 Hz, 1H), 6.89 (dt, J = 7.8, 1.4 Hz, 1H), 4.68 (dd, J = 8.5, 3.3 Hz, 1H), 3.77 - 3.61 (m, 1H), 3.55 (dd, J = 11.4, 8.4 Hz, 1H), 3.25 - 2.71 (m, 2H), 1.80 (tt, J = 8.4, 5.1 Hz, 1H), 0.89 - 0.85 (m, 2H), 0.60 (dt, J = 6.7, 4.7 Hz, 2H). LC-MS: Calculated for C 11 H 14 NO2[M+NH4] + : 196.10, found 196.15. ee value: 97.1%.

[0490] The following compounds (54-66) were synthesized following the procedures described above for steps a-b:

[0491] Synthesis of (R)-1-(2-ethylpyridin-4-yl)ethane-1,2-diol (Compound 54)

[0492] 1 H NMR (600 MHz, Chloroform-d) δ 8.30 (d, J = 5.1 Hz, 1H), 7.12 (d, J = 1.6 Hz, 1H), 7.03 (dd, J = 5.3, 1.7 Hz, 1H), 5.00 (d, J = 11.9 Hz, 1H), 4.73 (dd, J = 7.8, 3.5 Hz, 1H), 3.74 (dd, J = 11.4, 3.5 Hz, 1H), 3.56 (dd, J = 11.3, 7.7 Hz, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). LC-MS: Calculated for C9H 13 NO2[M+H] + : 168.09, found 168.1. ee value: 93.0%.

[0493] Synthesis of (R)-1-(3-isopropylphenyl)ethane-1,2-diol (Compound 55)

[0494] 1H NMR (600 MHz, Chloroform-d) δ 7.28 (t, J = 7.5 Hz, 1H), 7.21 (d, J = 1.8 Hz, 1H), 7.20 - 7.14 (m, 2H), 4.93 - 4.68 (m, 1H), 3.93 - 3.51 (m, 2H), 2.90 (hept, J = 6.9 Hz, 1H), 2.52 (s, 2H), 1.25 (s, 3H), 1.24 (s, 3H). LC-MS: Calculated for C 11 H 14 NO2[M-H] - : 179.12, found 179.1. ee value: 92.5%.

[0495] Synthesis of (R)-1-(3-(trifluoromethyl)phenyl)ethane-1,2-diol (Compound 56)

[0496] 1 H NMR (600 MHz, Chloroform-d) δ 7.47 (s, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H), 4.69 (dd, J = 8.4, 3.1 Hz, 1H), 3.59 (dd, J = 11.8, 3.1 Hz, 1H), 3.45 (dd, J = 11.6, 8.5 Hz, 1H). LC-MS: Calculated for C9H9F3O2 [M + COOH] - : 251.06, found 251.1. ee value: 94.3%.

[0497] (R)-1-(3-methoxyphenyl)ethane-1,2-diol (Compound 57)

[0498] 1 H NMR (400 MHz, DMSO-d6) δ 7.21 (t, J = 8.0 Hz, 1H), 6.93 - 6.88 (m, 2H), 6.81 - 6.76 (m, 1H), 5.22 (d, J = 4.3 Hz, 1H), 4.70 (t, J = 5.8 Hz, 1H), 4.52 (dt, J = 6.7, 4.8 Hz, 1H), 3.74 (s, 3H), 3.46 - 3.39 (m, 2H). LC-MS: Calculated for C9H 12 O3 [M + COOH] - : 213.08, found 213.10. ee value: 97.2%.

[0499] (R)-1-(3-(1,1-difluoroethyl)phenyl)ethan-1,2-diol (Compound 58)

[0500] 1 H NMR (600 MHz, DMSO-d6) δ 7.53 (s, 1H), 7.45 (q, J = 4.2 Hz, 1H), 7.42 (d, J = 4.6 Hz, 2H), 5.35 (d, J = 4.4 Hz, 1H), 4.75 (td, J = 5.8, 1.7 Hz, 1H), 4.62 - 4.55 (m, 1H), 3.45 (dtd, J = 11.2, 5.8, 3.1 Hz, 2H), 1.95 (t, J = 18.8 Hz, 3H). LC-MS: Calculated for C 10 H 12 F2O2[M+COOH] - : 247.08, found 247.10. ee value: 94.0%.

[0501] (R)-1-(3-fluorophenyl)ethan-1,2-diol (Compound 59)

[0502] 1 H NMR (500 MHz, Chloroform-d) δ 7.32 (td, J = 8.1, 6.0 Hz, 1H), 7.16 - 7.05 (m, 2H), 7.04 - 6.95 (m, 1H), 4.81 (dd, J = 8.3, 3.2 Hz, 1H), 3.76 (dd, J = 11.6, 3.2 Hz, 1H), 3.62 (dd, J = 11.4, 8.1 Hz, 1H). LC-MS: Calculated for C8H9FO2 [M+COOH] - : 201.06, found 201.05. ee value: 92.3%.

[0503] (R)-1-(2-chloro-5-(trifluoromethyl)phenyl)ethan-1,2-diol (Compound 60)

[0504] 1 H NMR (600 MHz, Chloroform-d) δ 7.95 - 7.89 (m, 1H), 7.54 - 7.45 (m, 2H), 5.27 (dd, J = 7.7, 3.1 Hz, 1H), 3.95 (dd, J = 11.4, 3.1 Hz, 1H), 3.56 (dd, J = 11.3, 7.8 Hz, 1H). LC-MS: Calculated for C9H8ClF3O2 [M+COOH] -:285.02, found 285.0. ee value: 82.9%.

[0505] (R)-1-(4-chloro-3-(trifluoromethyl)phenyl)ethan-1,2-diol (Compound 61)

[0506] 1 H NMR (600 MHz, Chloroform-d) δ 7.86 (s, 2H), 7.82 (s, 1H), 4.97 (dd, J = 7.8, 3.4 Hz, 1H), 3.87 (dd, J = 11.2, 3.3 Hz, 1H), 3.66 (dd, J = 11.2, 7.8 Hz, 1H). LC-MS: Calculated for C9H8F6O2 [M+COOH] 245.05, found 245.00. - :285.02, found 285.0. ee value: 82.9%.

[0507] (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethan-1,2-diol (Compound 62)

[0508] 1 H NMR (600 MHz, Chloroform-d) δ 7.86 (s, 2H), 7.82 (s, 1H), 4.97 (dd, J = 7.8, 3.4 Hz, 1H), 3.87 (dd, J = 11.2, 3.3 Hz, 1H), 3.66 (dd, J = 11.2, 7.8 Hz, 1H). LC-MS: Calculated for C9H8F6O2 [M+COOH] 245.05, found 245.00. 10 H8F6O2 [M+COOH] - :319.04, found 319.00.

[0509] (R)-1-(2-fluoro-5-(trifluoromethyl)phenyl)ethan-1,2-diol (Compound 63)

[0510] 1 H NMR (600 MHz, Chloroform-d) δ 7.82 (dd, J = 6.6, 2.4 Hz, 1H), 7.53 (ddd, J = 7.8, 4.7, 2.4 Hz, 1H), 7.11 (t, J = 9.1 Hz, 1H), 5.14 (dd, J = 8.1, 3.0 Hz, 1H), 3.83 (dd, J = 11.5, 3.0 Hz, 1H), 3.60 (dd, J = 11.5, 8.0 Hz, 1H). LC-MS: Calculated for C9H8F4O2 [M+COOH] 225.05, found 225.00. - :269.04, found 269.00. ee value: 94.0%.

[0511] (R)-1-(3-Fluoro-5-(trifluoromethyl)phenyl)ethan-1,2-diol (Compound 64)

[0512] 1 H NMR (600 MHz, Chloroform-d) δ 7.42 (s, 1H), 7.29 (dt, J = 9.1, 1.9 Hz, 1H), 7.25 (dt, J = 8.4, 2.0 Hz, 1H), 4.86 (dd, J = 8.0, 3.4 Hz, 1H), 3.80 (dd, J = 11.4, 3.4 Hz, 1H), 3.61 (dd, J = 11.4, 8.0 Hz, 1H). LC-MS: Calcd for C9H8F4O2 [M+COOH] 229.05, found 229.10. ee value: 80.6%. -

[0513] (R)-1-(3-Ethyl-4-fluorophenyl)ethan-1,2-diol (Compound 65)

[0514] 1 H NMR (600 MHz, DMSO-d6) δ 7.24 (dd, J = 7.7, 2.2 Hz, 1H), 7.17 (ddd, J = 7.9, 5.1, 2.2 Hz, 1H), 7.04 (dd, J = 10.2, 8.4 Hz, 1H), 5.22 (d, J = 4.2 Hz, 1H), 4.69 (t, J = 5.8 Hz, 1H), 4.52 - 4.46 (m, 1H), 3.44 - 3.36 (m, 2H), 2.60 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). LC-MS: Calcd for C 10 H 13 FO2 [M+COOH] - : 229.09, found 229.10. ee value: 80.6%.

[0515] (R)-1-(3-Ethyl-2-methylphenyl)ethan-1,2-diol (Compound 66)

[0516] 1 ​H NMR (600 MHz, Chloroform-d) δ 7.36 (d, J = 7.6 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.13 - 7.09 (m, 1H), 5.13 (dd, J = 8.6, 3.0 Hz, 1H), 3.73 (dd, J = 11.5, 3.0 Hz, 1H), 3.60 (dd, J = 11.5, 8.5 Hz, 1H), 2.74 (d, J = 2.6 Hz, 3H), 2.65 (q, J = 7.5 Hz, 2H), 2.26 (s, 3H). LC-MS: Calculated for C 11 H 16 O2[M+H] + : 181.12, found 181.15.

[0517] Synthetic route of compounds 67-69

[0518] The specific synthesis of steps c-d above is as follows:

[0519] Step 1. Synthesis of (2S,3S,4R,5S)-2-(acetyloxymethyl)-6-((R)-2-(3- ethylphenyl)-2-hydroxyethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Compound 67)

[0520] Into a 100 mL single necked round bottom flask was added (R)-1-(3- ethylphenyl)ethane-1,2-diol (0.80 mmol, 133 mg), bromosugar (0.80 mmol, 397 mg), Ag2CO3(0.48 mmol, 132 mg), silver trifluoromethanesulfonate (0.48 mmol, 123 mg) successively, TLC was used to monitor the reaction progress after stirring for 24 h. After the reaction was completed, filtration was performed and the solvent was removed by rotary evaporation. Column chromatography (PE:EA = 5:1) was used to isolate the corresponding intermediate (2S,3S,4R,5S)-2-(acetyloxymethyl)-6-((R)-2-(3- ethylphenyl)-2-hydroxyethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (119 mg, yield 30.0%).

[0521] Synthesis of (3S,4R,5R,6S)-2-((R)-2-(3-ethylphenyl)-2-hydroxyethoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (Compound 67)

[0522] To a 100 mL single round bottom flask was added (2S,3S,4R,5S)-2- (acetyloxymethyl)-6-((R)-2-(3-ethylphenyl)-2-hydroxyethoxy)tetrahydro-2H- pyran-3,4,5-triyl triacetate (0.24 mmol, 119 mg), potassium carbonate (0.24 mmol, 33 mg), dichloro / methanol (1 : 1) as solvent, TLC was used to monitor the progress of the reaction. After the reaction was completed, it was concentrated by rotary evaporation, and separation was performed using a reverse column (35 mg, yield 45.0%).

[0523] 1 H NMR (400 MHz, DMSO-d6) δ 7.37 - 6.94 (m, 4H), 5.38 - 4.39 (m, 6H), 4.12 (m, 1H), 3.81 - 3.42 (m, 4H), 3.23 - 2.84 (m, 4H), 2.59 (q, J = 7.6 Hz, 2H), 1.18 (td, J = 7.6, 2.3 Hz, 3H). LC-MS: Calculated for C 16 H 24 O7[M+NH4] + : 346.18, found 346.20. ee value: 82.2%.

[0524] The following compounds (68-69) were synthesized according to the procedures described above in steps c-d:

[0525] (2R,3R,4R,5S)-6-((R)-2-(3-ethylphenyl)-2-hydroxyethoxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-carboxylic acid (Compound 68)

[0526] 1 H NMR (400 MHz, DMSO-d6) δ 7.28 - 7.05 (m, 4H), 6.72 (s, 1H), 5.73 (d, J = 3.6 Hz, 1H), 5.20 - 4.92 (m, 2H), 4.84 - 4.61 (m, 1H), 4.32 - 4.05 (m, 1H), 3.75 - 3.56 (m, 2H), 3.27 (dd, J = 14.8, 8.9 Hz, 1H), 3.21 - 3.08 (m, 3H), 3.02 (td, J = 8.1, 3.7 Hz, 1H), 2.59 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS: Calculated for C 16 H 22 O8[M+NH4] + : 360.16, found 360.20.

[0527] (2S,3R,4S,5R,6R)-2-(((2S,4S,5S)-6-((R)-2-(3-ethylphenyl)-2-hydroxyethoxy)-4,5- dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-6-(hydroxymethyl)tetrahydro- 2H-pyran-3,4,5-triol (Compound 69)

[0528] 1 H NMR (500 MHz, DMSO-d6) d 7.29 - 7.05 (m, 4H), 5.24 - 5.16 (m, 2H), 5.07 - 4.94 (m, 2H), 4.81 - 4.55 (m, 4H), 4.38 - 4.21 (m, 2H), 3.85 - 3.51 (m, 5H), 3.47 - 3.38 (m, 1H), 3.34 - 3.12 (m, 6H), 3.12 - 2.94 (m, 3H), 2.59 (q, J = 7.6 Hz, 2H), 1.18 (td, J = 7.6, 1.6 Hz, 3H). LC-MS: calculated for C 22 H 34 O 12 [M-H] - : 489.20. Found [M-H] - : 489.30.

[0529] Synthesis of (R)-2-(3-ethylphenyl)-2-hydroxyethyl sodium sulfate (Compound 70)

[0530] Into a 250 mL single-necked round-bottom flask, (R)-1-(3-ethylphenyl)ethane-1,2-diol (30.10 mmol, 5.00 g), sulfur trioxide pyridine (30.10 mmol, 4.79 g) and acetonitrile (100 mL) were added in turn, and the reaction was carried out at 60 °C, with TLC monitoring of the reaction progress. After the reaction was completed, an appropriate amount of hydrochloric acid was added to generate pyridine hydrochloride, which was extracted with EA and water. The water layer was pyridine hydrochloride and part of the acidic product, and the EA layer was spin-dried and column chromatographed (DCM:MeOH = 20:1). The column chromatography product was combined with the water layer, and NaOH was added to make it alkaline. EA was added to extract the pyridine, and the water layer was the product. The water was spin-dried, and column chromatography was carried out (DCM:MeOH = 20:1) to obtain the target product Compound 70 (white solid, 1.78 g, yield 22%).

[0531] 1H NMR (500 MHz, Deuterium Oxide) δ 7.42 - 7.08 (m, 4H), 4.93 (dd, J = 7.6, 4.1 Hz, 1H), 4.16 - 3.98 (m, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.13 (t, J = 7.6 Hz, 3H). LC-MS: m / z calcd for C 10 H 13 NaO5S[M-H-Na] - : 245.06, found 245.10.

[0532] Synthesis of Sodium (2-(3-ethylphenyl)-2-oxoethoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate (Compound 71)

[0533] Into a 250 mL single necked round bottom flask was added 1 (18 mmol, 3 g), bromosugar (54 mmol, 21.7 g), Ag2CO3(36 mmol, 15 g), dichloromethane (100 mL), stirred for 24 h, TLC monitored the progress of the reaction. After completion of the reaction, filtered, concentrated by rotary evaporation to remove the solvent, to get yellow oily liquid. Column chromatography (PE:EA = 5:1) separation to get the corresponding intermediate 3 (yellow oily liquid, 4.5 g, yield 51%).

[0534] Into a 100 mL single round bottom flask was added intermediate 3 (9 mmol, 4.5 g), potassium carbonate (9 mmol, 1.29 g), dichloromethanol (1:1) 50 ml, TLC monitored the progress of the reaction. After completion of the reaction, concentrated by rotary evaporation, wet loaded on column (DCM:MeOH = 10:1) separation to get light yellow honeycomb solid (1.2 g, yield 37%)

[0535] Into a 50 mL single necked round bottom flask was added intermediate 4 (3.4 mmol, 1.2 g), sodium hydroxide (3.4 mmol, 135 mg) TLC monitored the progress of the reaction. After completion of the reaction, concentrated, reversed phase flash column (H2O:MeOH) gradient elution, to get the target product 71 (0.7 g, yield 60%) white solid.

[0536] 1H NMR (500 MHz, Deuterium Oxide) δ 7.72 - 7.04 (m, 4H), 4.48 (d, J = 7.8 Hz, 1H), 3.66 (dt, J = 9.0, 4.5 Hz, 1H), 3.53 - 3.44 (m, 2H), 3.40 (td, J = 7.3, 2.5 Hz, 1H), 2.55 (q, J = 7.6 Hz, 2H), 1.09 (t, J = 7.6 Hz, 3H). LC-MS: m / z calc. for C 16 H 20 O8[M+NH4] + : 358.16, found 358.1. [M-H] - : 339.10.

[0537] (R)-1-(3-ethylphenyl)-2-hydroxyethylsulfate sodium salt (synthesis of compound 72)

[0538] Into a 250 mL single necked round bottom flask was added 72-A (3 g, 18.3 mmol) was dissolved in DCM (100 mL), (Ac)20 (3.4 mL, 36.6 mmol), pyridine (2.9 mL, 36.6 mmol) was added under ice bath, after 12 h of stirring at room temperature, the reaction progress was monitored by TLC. After completion of the reaction, the solvent was removed by concentration, EA, H20 was used to extract the layers, the organic phase was concentrated and column chromatography was used to isolate the corresponding intermediate 72-B (yellow oily liquid, 3 g, yield 79.7%).

[0539] LC-MS (ESI): m / z: not responsive

[0540] 1 H NMR (500 MHz, Chloroform-d) δ 7.77 - 7.69 (m, 2H), 7.44 (dt, J = 7.8, 1.5 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 5.33 (s, 2H), 2.71 (q, J = 7.6 Hz, 2H), 2.23 (s, 3H), 1.26 (t, J = 7.6 Hz, 3H).

[0541] Into a 100 mL three-necked round-bottom flask, was added (R)-2-methyl-CBS-oxazaborolidine (403 mg, 1.4 mmol), 1 M borane in tetrahydrofuran (17 mL, 17 mmol), anhydrous tetrahydrofuran (20 mL), nitrogen was purged for three times, stirred in ice bath for 30 min, added the starting material 72-B (3 g, 14 mmol) dissolved in 15 mL of anhydrous tetrahydrofuran, stirred in ice bath for 10 min, then warmed to room temperature, stirred for 1 h, TLC monitored the reaction progress. After the reaction was completed, 5 mL of water was slowly added, stirred for 30 min, concentrated the tetrahydrofuran, extracted with ethyl acetate (100 mL), combined the organic layers and washed with saturated NaCl solution (50 mL), concentrated by rotary evaporation to remove the solvent, purified by column chromatography to obtain 72-C (yellowish oil liquid, 652 mg, yield 21%).

[0542] LC-MS (ESI): m / z: not responsive

[0543] 1 H NMR (500 MHz, Chloroform-d) δ 7.29 (t, J = 7.5 Hz, 1H), 7.24 - 7.18 (m, 2H), 7.16 (d, J = 7.6 Hz, 1H), 4.94 (dd, J = 8.7, 3.2 Hz, 1H), 4.28 (dd, J = 11.6, 3.2 Hz, 1H), 4.16 (dd, J = 11.6, 8.6 Hz, 1H), 2.66 (q, J = 7.6 Hz, 2H), 2.11 (s, 3H), 1.24 (t, J = 7.6 Hz, 3H).

[0544] Into a 250 mL single-necked round-bottom flask, was added 72-C (570 mg, 2.74 mmol), sulfur trioxide pyridine (480 mg, 3.01 mmol) and acetonitrile (100 mL), reacted at 60 °C, TLC monitored the reaction progress. After the reaction was completed, no treatment was made, and the crude was directly used in the next step.

[0545] LC-MS (ESI): m / z: 287.1 [M-H] -

[0546] Into a 50 mL single-necked round-bottom flask, was added the solution of intermediate 72L-D (the solution of the previous step) dissolved in methanol, 1 M sodium hydroxide (13.7 mL, 13.7 mmol) was added, TLC monitored the reaction progress. After the reaction was completed, reverse column chromatography was used to obtain the white solid target product 72 (120 mg, yield 16%).

[0547] LC-MS (ESI): m / z: 245.1 [M-H] -

[0548] 1 H NMR (500 MHz, DMSO-d6) δ 7.32 - 6.96 (m, 4H), 5.39 (d, J = 13.4 Hz, 1H), 4.67 (d, J = 24.3 Hz, 1H), 3.81 - 3.72 (m, 2H), 2.73 - 2.54 (m, 2H), 1.36 - 1.03 (m, 3H).

[0549] 13 C NMR (126 MHz, DMSO) δ 143.8, 143.1, 128.4, 126.9, 126.2, 124.2, 71.9, 40.5, 40.3, 40.1, 40.0, 39.8, 39.6, 39.5, 28.7, 16.1.

[0550] Synthesis of (2S, 3S, 4S, 5R)-6-((R)-1-(3-ethylphenyl)-2-hydroxyethoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid sodium salt (Compound 73)

[0551] Into a 250 mL single necked round bottom flask was added 73-A (4.5 g, 27 mmol) was dissolved in DCM (100 mL), (Ac)20 (5.2 mL, 54 mmol), pyridine (4.4 mL, 54 mmol) was added under ice bath, after 12 h of stirring at room temperature, TLC was used to monitor the reaction progress. After the reaction was completed, the solvent was removed by concentration, EA, H20 was used to extract the layer, the organic phase was concentrated, column chromatography was used to isolate the corresponding intermediate 73-B (yellow oily liquid, 4.56 g, yield 80.8%).

[0552] LC-MS (ESI): m / z: not responsive

[0553] 1 H NMR (500 MHz, Chloroform-d) δ 7.77 - 7.69 (m, 2H), 7.44 (dt, J = 7.8, 1.5 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 5.33 (s, 2H), 2.71 (q, J = 7.6 Hz, 2H), 2.23 (s, 3H), 1.26 (t, J = 7.6 Hz, 3H).

[0554] Into a 100 mL three-necked round bottom flask, (R)-2-methyl-CBS-oxazaborolidine (613 mg, 2.2 mmol), 1 M borane tetrahydrofuran solution (26 mL, 26 mmol), anhydrous tetrahydrofuran (20 mL), nitrogen was purged for three times, stirred for 30 min in ice bath, added the starting material 73-B (4.56 g, 22 mmol) dissolved in 15 mL of anhydrous tetrahydrofuran, stirred for 10 min in ice bath, then raised to room temperature, stirred for 1 h, TLC monitored the reaction progress. After the reaction was completed, 5 mL of water was slowly added, stirred for 30 min, concentrated tetrahydrofuran, extracted with ethyl acetate (100 mL), combined organic layers and washed with saturated NaCl solution (50 mL), concentrated by rotary evaporation to remove the solvent, purified by column chromatography to obtain 73-C (yellowish oil, 2.7 g, yield 58.6%).

[0555] LC-MS (ESI): m / z: not responsive

[0556] 1 H NMR (500 MHz, Chloroform-d) δ 7.29 (t, J = 7.5 Hz, 1H), 7.24 - 7.18 (m, 2H), 7.16 (d, J = 7.6 Hz, 1H), 4.94 (dd, J = 8.7, 3.2 Hz, 1H), 4.28 (dd, J = 11.6, 3.2 Hz, 1H), 4.16 (dd, J = 11.6, 8.6 Hz, 1H), 2.66 (q, J = 7.6 Hz, 2H), 2.11 (s, 3H), 1.24 (t, J = 7.6 Hz, 3H).

[0557] Into a 250 mL single-necked round bottom flask, 73-C (1.3 g, 6.3 mmol), bromosugar (6.2 g, 15.6 mmol), Ag2CO3(4.3 g, 15.6 mmol), dichloromethane (100 mL), stirred for 24 h, TLC monitored the reaction progress. After the reaction was completed, filtered, concentrated by rotary evaporation to remove the solvent to obtain yellow oil. Separated by column chromatography (PE:EA = 5:1) to obtain the corresponding intermediate 73-D (yellow oil, 1.115 g, yield 34%).

[0558] LC-MS (ESI): m / z: 542.1 [M+NH3] + .

[0559] Into a 50 mL single necked round bottom flask, intermediate 73-D (1.115 g, 2.1 mmol) was dissolved in methanol, 1 M sodium hydroxide (10.6 mL, 10.6 mmol) was added, TLC was used to monitor the progress of the reaction. After the completion of the reaction, reverse column chromatography gave the target product 73 (240 mg, yield 31%) as a white solid.

[0560] LC-MS (ESI): m / z: 341.2 [M-H] - .

[0561] 1 H NMR (500 MHz, Deuterium Oxide) δ 7.44 - 7.34 (m, 2H), 7.34 - 7.26 (m, 2H), 5.04 (dd, J = 7.7, 4.0 Hz, 1H), 4.28 (d, J = 7.7 Hz, 1H), 3.84 (dd, J = 11.9, 7.7 Hz, 1H), 3.76 (dd, J = 11.9, 4.0 Hz, 1H), 3.61 - 3.50 (m, 2H), 3.43 (dd, J = 9.5, 7.7 Hz, 1H), 3.37 (t, J = 9.0 Hz, 1H), 2.69 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H).

[0562] 13 C NMR (126 MHz, D20) δ 175.9, 145.3, 137.5, 128.7, 128.1, 127.1, 125.0, 99.1, 79.9, 75.7, 75.4, 72.9, 71.9, 65.4, 28.2, 14.9.

[0563] Synthesis of 2-fluoro-4-((4-hydroxy-4-(hydroxymethyl)pyrrolidin-3- yl)oxy)benzonitrile (Compound 74)

[0564] Into a three necked flask, trimethylsulfonium iodide (21.6 mmol, 4.4 g) was dissolved in anhydrous tetrahydrofuran, under nitrogen protection, the temperature was lowered to -20 °C, n-butyllithium (1.6 M in n-hexane) (20 mmol, 12.5 mL) was added dropwise and the temperature was kept at -20 °C, after 30 min, 74-1 dissolved in anhydrous tetrahydrofuran was added dropwise, after 30 min, the reaction was continued at room temperature for 2 h, TLC detection (molybdenum phosphate coloration) showed that the reaction was completed, 2 times the volume of methyl tert-butyl ether was added to the reaction solvent, 1 times the volume of water was added to extract, the organic layer was concentrated, column chromatography (petroleum ether: ethyl acetate = 4: 1) was used for purification, and intermediate 74-2 (700 mg) was obtained as a light yellow oil in a yield of 67.3%.

[0565] 10. Synthesis of intermediate 74-3

[0566] Into a round bottom flask, 74-2 (2.2 mmol, 438 mg), 2-fluoro-4-hydroxybenzonitrile (2.2 mmol, 302 mg), and triphenylphosphine (3.3 mmol, 865 mg) were dissolved in anhydrous THF. DIAD (3.3 mmol, 667 mg) was added under ice bath. After half an hour, TLC test showed the reaction was complete. The reaction mixture was concentrated and extracted with ethyl acetate and water. The organic layer was concentrated and purified by column chromatography to give 74-3 (560 mg, 80% yield) as a white solid.

[0567] Into a 100 mL single neck flask, 74-3 (1.57 mmol, 500 mg) was dissolved in ethyl acetate. AD-mix-β (1176 mg) was added under ice bath. The reaction was stirred at room temperature overnight. The reaction mixture was extracted with ethyl acetate and water three times. The organic layer was concentrated and purified by column chromatography to give 74-4 (418 mg, 80% yield) as a white solid.

[0568] Into a round bottom flask, 74-4 was dissolved in ethyl acetate. 4M hydrochloric acid in dioxane (10 eq) was added. The reaction was stirred at room temperature overnight. TLC test showed the reaction was complete. The reaction mixture was concentrated and filtered. The filter cake was washed with ethyl acetate to give 74 (260 mg, 67% yield) as a white solid.

[0569] 1 H NMR (500 MHz, Deuterium Oxide) δ 7.64 (t, J = 8.1 Hz, 1H), 6.91 (ddd, J = 19.7, 10.0, 2.4 Hz, 2H), 4.94 (d, J = 3.7 Hz, 1H), 3.93 - 3.63 (m, 3H), 3.58 - 3.32 (m, 3H).

[0570] Synthesis of 3-(hydroxymethyl)-4-phenoxypyrrolidin-3-ol (Compound 75)

[0571] Synthesis method is the same as 74

[0572] 1H NMR (500 MHz, DMSO-de) δ 9.70 (s, 2H), 7.48 - 7.17 (m, 2H), 7.16 - 6.68 (m, 3H), 5.50 (s, 1H), 5.36 (t, J = 5.6 Hz, 1H), 4.82 (t, J = 6.6 Hz, 1H), 3.68 (dt, J = 11.8, 5.5 Hz, 1H), 3.47 (t, J = 4.7 Hz, 2H), 3.30 (d, J = 12.0 Hz, 1H), 3.22 - 3.06 (m, 2H).

[0573] Synthesis of N-(3-(difluoromethoxy)-4-((2,3-dihydroxypropyl)amino)methyl)phenyl)-1-(4- fluorophenyl)-3-methyl-1H-pyrazole-4-carboxamide (Compound 76)

[0574] Ethyl 3-methyl-1H-pyrazole-4-carboxylate (1 g, 6.4 mmol), cuprous iodide (370 mg, 1.92 mmol) and potassium carbonate (1793 mg, 12.8 mmol) were mixed in toluene (50 mL), vacuumed, and purged with nitrogen. 1-Fluoro-4-iodobenzene (1.58 g, 7.04 mmol) and N1,N2-dimethylethane-1,2-diamine (0.34 g, 3.8 mmol) were added, vacuumed and purged with nitrogen, and stirred at 110 °C for 4 hours. The reaction mixture was allowed to cool and diluted with EtOAc (150 mL). The mixture was washed with dilute aqueous ammonium hydroxide solution (2*80 mL), water (100 mL) and brine (30 mL), then dried over Na2S04. After filtration and concentration to dryness under reduced pressure, column chromatography gave 76C (pale yellow solid, 315 mg, 45%).

[0575] LC-MS (ESI): m / z: 249.1 [M+H] + .

[0576] 1 H NMR (500 MHz, Chloroform-d) δ 8.27 (s, 1H), 7.67 - 7.59 (m, 2H), 7.18 - 7.10 (m, 2H), 4.31 (q, J = 7.2 Hz, 2H), 2.54 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H).

[0577] Into a 50 mL single necked round bottom flask was added intermediate 76C (315 mg, 1.28 mmol), 1 M sodium hydroxide (8.7 mL, 3.84 mmol) in a 1:1 solution of methanol and tetrahydrofuran, TLC monitored the progress of the reaction. After completion of the reaction, reverse column chromatography gave white solid 76D (269 mg, yield 96%)

[0578] LC-MS (ESI): m / z: 221.2 [M+H] + .

[0579] 1 H NMR (500 MHz, DMSO-d6) δ 8.88 (s, 1H), 7.95 - 7.87 (m, 2H), 7.34 (t, J = 8.8 Hz, 2H), 2.43 (s, 3H).

[0580] Into a 100 mL single necked round bottom flask was added intermediate 76E (1 g, 5 mmol), potassium hydroxide (2.85 g, 50 mmol) in a 1:1 solution of acetonitrile and water, bromofluoromethylphosphonic acid diethyl ester (2.7 g, 10 mmol) was added slowly under ice bath, after 2 hours TLC monitored the progress of the reaction. After completion of the reaction, the organic phase was concentrated, 2N hydrochloric acid was added to adjust the pH, then extracted with EA, the organic phase was concentrated and column chromatography gave white solid 76F (1084 mg, yield 91%)

[0581] LC-MS (ESI): m / z: 232.1 [M-H] - .

[0582] 1 H NMR (500 MHz, Chloroform-d) δ 10.45 (s, 1H), 8.46 - 7.94 (m, 3H), 6.80 (t, J = 71.3 Hz, 1H).

[0583] Into a 100 mL single necked round bottom flask was added intermediate 76F (1084 mg, 4.65 mmol) in methanol, sulfurous dichloride (0.84 mL, 13.9 mmol) was added slowly under ice bath, after 2 hours of reaction at 80 degree Celsius, TLC monitored the progress of the reaction. After completion of the reaction, concentrated, washed with saturated sodium bicarbonate, extracted with EA, the organic phase was concentrated and column chromatography gave yellow oily liquid 76G (515 mg, yield 44.8%)

[0584] LC-MS (ESI): m / z: not responsive

[0585] 1H NMR (500 MHz, Chloroform-d) δ 8.16 (dt, J = 8.5, 1.8 Hz, 1H), 8.13 (s, 1H), 8.05 (dd, J = 8.5, 1.3 Hz, 1H), 6.65 (td, J = 73.0, 1.3 Hz, 1H), 3.97 (d, J = 1.3 Hz, 3H).

[0586] In 100 mL single necked round bottom flask was taken intermediate 76G (500 mg, 2.02 mmol) dissolved in 4:1 solution of ethanol and water, iron powder (566 mg, 10.1 mmol), ammonium chloride (1072 mg, 20.2 mmol) was added, reaction was monitored by TLC after 3 h at 80 °C. After completion of reaction, filtered hot, washed with water, extracted in EA, concentrated organic phase, column chromatography gave 76H as white solid (440 mg, yield 100%).

[0587] 1 H NMR (500 MHz, Chloroform-d) δ 7.78 (d, J = 8.5 Hz, 1H), 6.53 (t, J = 75.3 Hz, 1H), 6.51 (dd, J = 8.6, 2.3 Hz, 1H), 6.46 (dt, J = 2.2, 1.0 Hz, 1H), 3.85 (s, 3H).

[0588] 76D (2642 mg, 12 mmol) was dissolved in dry DCM (50 mL), thionyl chloride (2.23 mL, 30 mmol) was added, 2d DMF, reaction was carried out at room temperature for 12 h, after concentration to remove the smell of thionyl chloride, it was dissolved in DCM (50 mL), 76H (1.5 g, 6.9 mmol) was added, reaction was carried out at room temperature for 5 h, concentrated, diluted in AcOEt (50 mL), washed with HCl (1 N, 30 mL), purified by column chromatography, impure (NMR not done) crude was taken to next step.

[0589] LC-MS (ESI): m / z: 432.1 [M-H] - .

[0590] 76I (3.7 g, 8.8 mmol) was dissolved in dry THF (50 mL), lithium borohydride (1.92, 88 mmol) was added, reaction was carried out at room temperature for 12 h, after monitoring completion of reaction, reaction was quenched by adding water, extracted in EA, concentrated, column chromatography gave 76J as white solid (2.278 g, yield 66.8%).

[0591] LC-MS (ESI): m / z: 392.2 [M+H] +.

[0592] 1 H NMR (500 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.85 (s, 1H), 7.62 - 7.56 (m, 2H), 7.54 (d, J = 2.0 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.23 (dd, J = 8.3, 2.0 Hz, 1H), 7.17 - 7.08 (m, 2H), 6.54 (t, J = 73.9 Hz, 1H), 4.69 - 4.64 (m, 2H), 2.59 (s, 3H).

[0593] Dissolve 76J (2.278 g, 5.8 mmol) in anhydrous DCM, slowly add phosphorus tribromide (0.28 mL, 2.9 mmol) under ice bath, after 10 min, move to room temperature for 30 min, monitor, reaction is completed, slowly add water to quench the reaction. Extract, concentrate the organic layer, column chromatography to get white solid 76K (577 mg, yield 29%).

[0594] LC-MS (ESI): m / z: 454.0 [M+H] + .

[0595] 1 H NMR (500 MHz, DMSO-d6) δ 10.05 (d, J = 63.0 Hz, 1H), 9.11 (d, J = 6.3 Hz, 1H), 7.83 (ddd, J = 8.9, 4.7, 1.9 Hz, 2H), 7.80 - 7.67 (m, 1H), 7.58 (dt, J = 8.5, 2.1 Hz, 1H), 7.49 (dd, J = 32.2, 8.5 Hz, 1H), 7.43 - 7.37 (m, 2H), 7.29 - 6.95 (m, 1H), 4.58 (d, J = 82.0 Hz, 2H), 2.47 (s, 3H).

[0596] In a 50 mL round-bottom flask, add 76K (130 mg, 0.28 mmol), 3-amino-1,2- propanediol (26 mg, 0.28 mmol), potassium carbonate (99 mg, 7 mmol), dissolve in DMF, react at 80°C for 6 h, monitor, reaction is completed, add EA, H2O extraction, concentrate the organic layer, column chromatography to get the final product 76 light yellow solid (25 mg, yield 19%).

[0597] LC-MS (ESI): m / z: 465.1 [M+H] + .

[0598] 1 H NMR (500 MHz, Methanol-d4) δ 8.89 (s, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.81 (dd, J = 9.0, 4.5 Hz, 2H), 7.62 (dd, J = 8.3, 2.1 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 8.6 Hz, 2H), 7.00 (t, J = 73.2 Hz, 1H), 4.21 (s, 2H), 3.94 (dd, J = 9.0, 4.2 Hz, 1H), 3.59 (qd, J = 11.3, 5.1 Hz, 2H), 3.13 (dd, J = 12.7, 3.3 Hz, 1H), 2.98 (dd, J = 12.8, 9.0 Hz, 1H), 2.57 (s, 3H).

[0599] Synthesis of (3-(difluoromethoxy)-4-((3,4-dihydroxybutyl)amino)methyl)phenyl)-1-(4- fluorophenyl)-3-methyl-1H-pyrazole-4-carboxamide (Compound 77)

[0600] The synthesis method of 77-1 is the same as 76K, add 77-1 (187 mg, 0.41 mmol), 4- amino-1,2-butanediol (43 mg, 0.41 mmol), potassium carbonate (170 mg, 1.2 mmol) in a 50 mL round-bottom flask, dissolved in DMF, reacted at 80 °C for 6 h, monitored, reacted, added EA, H2O extraction, concentrated the organic layer and column chromatography to get the final product 77 light yellow solid (25 mg, yield 13%).

[0601] LC-MS (ESI): m / z: 479.1 [M+H] + .

[0602] 1 H NMR (500 MHz, Methanol-d4) δ 8.97 (s, 1H), 7.93 (s, 1H), 7.80 (dd, J = 8.9, 4.7 Hz, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.26 (t, J = 8.5 Hz, 2H), 7.02 (t, J = 73.0 Hz, 1H), 4.26 (s, 2H), 3.79 (dq, J = 9.0, 4.8 Hz, 1H), 3.51 (d, J = 5.2 Hz, 2H), 3.25 (tp, J = 12.9, 6.9, 6.1 Hz, 2H), 2.54 (s, 3H), 1.84 (dq, J = 15.5, 7.8 Hz, 1H), 1.77 - 1.52 (m, 1H).

[0603] 2-(3-ethylphenoxy)-1-(hydroxymethyl)cyclopentan-1-ol (Compound 78)

[0604] In a 500 mL three-necked flask, trimethylsulfonium iodide (132 mmol, 27 g) was dissolved in 100 mL of anhydrous tetrahydrofuran under nitrogen protection, the temperature was reduced to -20 °C, n-butyllithium (1.6 N n-hexane solution) (122 mmol, 77 mL) was slowly added and the temperature was maintained at -20 °C, after 30 min, 78A (33 mmol, 2.8 g) dissolved in 50 mL of anhydrous tetrahydrofuran was slowly added, after 30 min, the reaction was continued at room temperature for 2 h, TLC detection (phosphomolybdic acid coloration) showed that the reaction was complete, 2 times the volume of methyl tert-butyl ether was added to the reaction solvent, 1 times the volume of water was extracted, the organic layer was concentrated and purified by column chromatography to obtain intermediate 78B (pale yellow oily liquid, 450 mg, yield 14%).

[0605] In a 100 mL two-necked flask, 78B (3.21 mmol, 321 mg), triethylphenol (1.63 mmol, 200 mg), tributylphosphine (3.21 mmol, 662.3 mg), ADDM (3.21 mmol, 838.9 mg) were added in turn, nitrogen was replaced for three times, 50 mL of anhydrous tetrahydrofuran was added, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction liquid was concentrated, extracted with EA and H2O three times, the organic phase was concentrated, and the crude was subjected to the next step (transparent colorless oily liquid, 67 mg, 10%).

[0606] In a 100 mL single-necked flask, 78C (0.32 mmol, 67 mg) was added, AD-mix-β (0.6 mmol, 45.5 mg) was added under ice bath conditions, 1:1 tert-butyl alcohol and water were used as solvents, and the reaction was carried out at room temperature overnight. After the reaction was completed, EA and H2O were extracted three times, the organic phase was concentrated, and column chromatography was used to separate the final product 78 (transparent colorless oily liquid, 15 mg, 20%).

[0607] 1H NMR (500 MHz, Chloroform-d) δ 7.18 (t, J = 7.8 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 6.77 - 6.66 (m, 2H), 4.52 (dd, J = 5.6, 2.6 Hz, 1H), 4.03 (dd, J = 11.4, 1.3 Hz, 1H), 3.62 (d, J = 11.4 Hz, 1H), 2.62 (q, J = 7.6 Hz, 2H), 2.28 - 2.17 (m, 1H), 1.93 - 1.63 (m, 5H), 1.23 (t, J = 7.6 Hz, 3H). LC-MS (ESI): m / z: 316.1 [M+NH3] + .

[0608] Synthesis of 1-(3-ethylphenyl)-3-methylurea (Compound 79)

[0609] Into a 100 mL single necked flask was added 79A (0.68 mmol, 100 mg), methylamine hydrochloride (0.81 mmol, 55 mg) was added in an ice bath, triethylamine (0.81 mmol, 0.1 mL) was added, anhydrous tetrahydrofuran was used as solvent, and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, the reaction solution was concentrated, extracted with EA and H2O three times, the organic phase was concentrated, and column chromatography was used to separate the end product 79 (white solid, 89 mg, 73.5%).

[0610] 1 H NMR (500 MHz, Chloroform-d) δ 7.47 (d, J = 33.7 Hz, 1H), 7.22 - 7.06 (m, 3H), 6.89 (d, J = 7.4 Hz, 1H), 5.47 (d, J = 95.5 Hz, 1H), 2.75 (d, J = 2.7 Hz, 3H), 2.58 (q, J = 7.7 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H).

[0611] 13 C NMR (126 MHz, CDCl3) δ 157.5, 145.4, 138.8, 129.0, 123.0, 120.3, 118.1, 28.8, 26.8, 15.4.

[0612] LC-MS (ESI): m / z: 179.1 [M+H] + .

[0613] Synthesis of (R) acrylate-2-(3-ethylphenyl)-2-hydroxyethyl ester (Compound 80)

[0614] Into a 100 mL single necked flask, 80A (1.20 mmol, 200 mg) was taken and dissolved in 20 mL of NMP, acryloyl chloride (3.2 mmol, 0.2 mL), triethylamine (2.6 mmol, 0.4 mL) was added under ice bath and the reaction was allowed to proceed overnight, TLC was used to monitor the progress of the reaction. After completion of the reaction, EA and H2O was used to extract the reaction mixture three times, the organic layer was concentrated and column chromatography was used to isolate the final product 80 (colorless transparent liquid, 55 mg, 20%).

[0615] 1 H NMR (500 MHz, Chloroform-d) δ 7.29 (t, J = 7.5 Hz, 1H), 7.24 (d, J = 1.8 Hz, 1H), 7.21 (dt, J = 7.7, 1.6 Hz, 1H), 7.16 (dt, J = 7.5, 1.5 Hz, 1H), 6.46 (dd, J = 17.3, 1.3 Hz, 1H), 6.18 (dd, J = 17.3, 10.4 Hz, 1H), 5.88 (dd, J = 10.5, 1.3 Hz, 1H), 4.98 (dd, J = 8.5, 3.2 Hz, 1H), 4.36 (dd, J = 11.6, 3.3 Hz, 1H), 4.25 (dd, J = 11.6, 8.5 Hz, 1H), 2.66 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H).

[0616] 13 C NMR (126 MHz, CDC13) δ 166.3, 144.7, 139.7, 131.5, 128.6, 128.0, 127.9, 125.7, 123.5, 72.5, 69.5, 28.9, 15.6.

[0617] Synthesis of (R)-1-(4-amino-3-ethylphenyl)ethan-1,2-diol (Compound 81)

[0618] Into a 250 mL single necked round bottom flask, 81A (10 mmol, 2.01 g) was taken and dissolved in THF (100 mL), (Boc)20 (11 mmol, 2.41 g) was added under ice bath and the reaction was allowed to proceed for 12 h at room temperature, TLC was used to monitor the progress of the reaction. After completion of the reaction, the solvent was removed by concentration, EA, H2O was used to extract the reaction mixture and the organic layer was concentrated and column chromatography was used to isolate the corresponding intermediate 81B (white solid, 1.81 g, 60%). 1H NMR (500 MHz, Chloroform-d) δ 7.81 - 7.68 (m, 1H), 7.43 - 7.29 (m, 2H), 6.26 (s, 1H), 2.57 (q, J = 7.5 Hz, 2H), 1.54 (s, 9H), 1.26 (t, J = 7.6 Hz, 3H). LC-MS (ESI): m / z: 299.0 [M-H] - .

[0619] Into a 250 mL two-necked flask, 81B (3.33 mmol, 1000 mg), potassium ethylene trifluoroborate (3.99 mmol, 535 mg), DPPF palladium dichloride (0.33 mmol, 244 mg) and potassium carbonate (6.66 mmol, 920 mg) were added successively, 84 mL of a mixture of dioxane and water (6:1) as solvent, replaced with nitrogen for 3 times, reacted at 85 °C for 2 h, TLC was used to monitor the reaction progress. After the reaction was completed, the dioxane was removed by rotary evaporation, EA and H2O were used to extract for 3 times, the organic phase was concentrated, and column chromatography was used to separate to obtain the corresponding intermediate 81C (light yellow solid, 400 mg, 58%).

[0620] 1 H NMR (500 MHz, Chloroform-d) δ 7.78 (d, J = 8.2 Hz, 1H), 7.30 - 7.18 (m, 2H), 6.66 (dd, J = 17.6, 10.9 Hz, 1H), 6.30 (s, 1H), 5.67 (dd, J = 17.7, 1.0 Hz, 1H), 5.22 - 5.09 (m, 1H), 2.58 (q, J = 7.6 Hz, 2H), 1.53 (s, 9H), 1.25 (t, J = 7.6 Hz, 3H). LC-MS (ESI): m / z: 246.1 [M-H] - .

[0621] Into a 100 mL single-necked flask, 81C (1.55 mmol, 383 mg) and AD-mix-β (0.73 mmol, 563 mg) were added successively under ice bath condition, 1:1 tert-butanol and water were used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, EA and H2O were used to extract for 3 times, the organic phase was concentrated, and column chromatography was used to separate to obtain the corresponding intermediate 81D (light yellow liquid, 416 mg, yield 96%).

[0622] 1H NMR (500 MHz, Chloroform-d) δ 7.73 (d, J = 8.2 Hz, 1H), 7.16 (d, J = 7.3 Hz, 2H), 6.29 (s, 1H), 4.74 (dt, J = 8.1, 3.0 Hz, 1H), 3.72 - 3.59 (m, 2H), 2.58 (q, J = 7.6 Hz, 2H), 1.51 (s, 9H), 1.23 (t, J = 7.5 Hz, 3H). LC-MS (ESI): m / z: 280.2 [M-H] - .

[0623] Into a 100 mL round-bottom flask, was placed 81D (1.48 mmol, 418 mg) dissolved in EA, 4N hydrochloric acid in dioxane (14.8 mmol, 3.7 mL) was added, and the reaction was allowed to proceed at room temperature overnight. TLC detection showed that the reaction was complete. The reaction was concentrated, dissolved in EA, and the pH was adjusted to >9 with a saturated Na2CO3 solution. The EA layer was concentrated, and column chromatography was used to isolate the final product 81 (orange-yellow oily liquid, 80 mg, 29%).

[0624] 1 H NMR (500 MHz, Chloroform-d) δ 7.01 - 6.88 (m, 2H), 6.66 (d, J = 8.0 Hz, 1H), 4.19 (dd, J = 8.8, 3.8 Hz, 1H), 3.67 (dd, J = 11.6, 8.8 Hz, 1H), 3.56 (dd, J = 11.6, 3.8 Hz, 1H), 3.27 (s, 2H), 2.51 (q, J = 7.5 Hz, 2H), 1.25 (t, J = 7.5 Hz, 3H).

[0625] 13 C NMR (126 MHz, CDCl3) δ 144.1, 128.1, 127.1, 125.6, 115.3, 84.5, 67.4, 56.5, 24.0, 12.9.

[0626] Synthesis of (R)-4-(3-(1,2-dihydroxyethyl)benzyl)-2-fluorobenzonitrile (Compound 82)

[0627] Into a 100 mL round-bottom flask, was placed 82A (34.9 mmol, 6530 mg) dissolved in DCM, and phosphorus tribromide (17.4 mmol, 1.63 mL) was slowly added dropwise under ice bath. The reaction was allowed to proceed at room temperature overnight. TLC detection showed that the reaction was complete. The reaction was quenched with water and extracted. Several layers were concentrated, and column chromatography was used to isolate 82B (colorless transparent liquid, 8300 mg, 95%).

[0628] Into a 250 mL two-necked flask, 82B (10.75 mmol, 2000 mg), potassium ethylene trifluoroborate (2.9 mmol, 1730 mg), DPPF palladium dichloride (1.08 mmol, 780 mg) and potassium carbonate (21.5 mmol, 2.97 mg) were added successively, 140 mL of a mixture of dioxane and water (6:1) as solvent, replaced by nitrogen for 3 times, reacted at 85 °C for 2 h, TLC was used to detect the reaction progress. After the reaction was completed, the dioxane was removed by rotary evaporation, EA and H2O were used to extract for 3 times, the organic phase was concentrated, and column chromatography was used to separate to obtain the corresponding intermediate 82C (colorless transparent liquid, 300 mg, 14%).

[0629] Into a 50 mL two-necked flask, 82C (1.01 mmol, 200 mg), 4-cyano-3-fluorobenzenboronic acid (1.55 mmol, 251 mg), palladium acetate (0.1 mmol, 23 mg), triphenylphosphine (0.2 mmol, 53 mg) and potassium phosphate (2.02 mmol, 431 mg) were added successively, 20 mL of toluene as solvent, replaced by nitrogen for 3 times, reacted at 95 °C for 3 h, TLC was used to detect the reaction progress. After the reaction was completed, the solvent was removed by concentration, EA and H2O were used to extract for 3 times, the organic phase was concentrated, and column chromatography was used to separate to obtain the corresponding intermediate 82D (yellow oily liquid, 77 mg, 32%).

[0630] Into a 100 mL single-necked flask, 82D (0.32 mmol, 77 mg) and AD-mix-β (0.15 mmol, 52.3 mg) were added successively under ice bath condition, 1:1 tert-butanol and water as solvent, and reacted at room temperature overnight. After the reaction was completed, EA and H2O were used to extract for 3 times, the organic phase was concentrated, and column chromatography was used to separate to obtain the final product 82 (white solid, 35 mg, 40%).

[0631] 1 H NMR (500 MHz, Methanol-d4) δ 8.04 - 7.91 (m, 2H), 7.52 (t, J = 1.8 Hz, 1H), 7.48 - 7.36 (m, 3H), 7.32 (dt, J = 7.5, 1.5 Hz, 1H), 3.89 (dq, J = 7.9, 5.1 Hz, 1H), 3.53 (qd, J = 11.1, 5.4 Hz, 2H), 2.94 (dd, J = 13.8, 5.1 Hz, 1H), 2.76 (dd, J = 13.8, 7.9 Hz, 1H).

[0632] 13C NMR (126 MHz, Methanol-d4) δ 162.37 (d, J = 256.5 Hz), 139.97, 138.74 (d, J = 3.6 Hz), 137.77, 133.80 (d, J = 8.4 Hz), 131.56, 129.16, 128.73, 127.85, 124.46, 116.44 (d, J = 19.8 Hz), 113.42, 101.13 (d, J = 15.7 Hz), 72.91, 65.23, 39.42.

[0633] LC-MS (ESI): m / z: 316.1 [M+COOH] - .

[0634] Synthesis of (3-((R)-1,2-dihydroxyethyl)phenyl)(8-(5-(trifluoromethyl)pyrimidin-2-yl)- 3,8-diazabicyclo[3.2.1]oct-3-yl)methanone (Compound 83)

[0635] Into a round bottom flask, add 83-1 (5.5 mmol, 1 g), dissolve another material (5.5 mmol, 1.16 g) in 20 mL NMP, slowly add DIPEA (13.7 mmol, 2.4 mL), react at 80 °C for 1 h, TLC test reaction complete, concentrated in ethyl acetate, water extraction, concentrated organic layer, crude into the next step, get light yellow solid 83-2, 2.55 g.

[0636] Into a round bottom flask, add 83-2, dissolve in ethyl acetate, add 4M hydrochloric acid dioxane solution (10 eq), react at room temperature overnight, TLC test reaction complete, concentrated, crude into the next step, get intermediate 83-3, 1.7 g yellow solid.

[0637] Into a round bottom flask, add 83-3 (3.4 mmol, 1 g), add solvent DMF, add o-vinylbenzoic acid (3.4 mmol, 0.51 g) HATU (4 mmol, 1.5 g), DIPEA (3.6 mmol, 2.4 mL) in turn, react at room temperature for 2 h, TLC test reaction complete, ethyl acetate water extraction, concentrated organic layer, column chromatography get light yellow solid intermediate 83-4 (1.3 g), yield 98%.

[0638] Into a 100 mL single neck flask, 83-4 (3.34 mmol, 1.3 g) was added, AD-mix-β (1.91 g) was added under ice bath condition, t-butanol and water (1 : 1) was used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, it was extracted with ethyl acetate and water for three times, the organic phase was combined and concentrated by rotary evaporation, and column chromatography (pure ethyl acetate) was used to obtain the target product 83 (white solid, 520 mg, yield 37.6%).

[0639] 1 H NMR (500 MHz, DMSO-d6) δ 8.75 (s, 2H), 7.45 - 7.33 (m, 3H), 7.26 (dt, J = 7.2, 1.7 Hz, 1H), 5.32 (d, J = 4.3 Hz, 1H), 4.88 (s, 1H), 4.72 (q, J = 7.5, 6.7 Hz, 2H), 4.57 (q, J = 5.3 Hz, 1H), 4.45 (d, J = 13.0 Hz, 1H), 3.45 (t, J = 5.4 Hz, 4H), 3.03 (d, J = 13.0 Hz, 1H), 2.08 - 1.60 (m, 4H).

[0640] Synthesis of (1R)-1-(3-((2,7-diazaspiro[4.4]nonan-2-yl)methyl)phenyl)ethane-1,2-diol (Compound 84)

[0641] Into a 500 mL two neck flask, 84A (63.9 mmol, 11.83 g) was added, potassium ethylene trifluoroborate (76.7 mmol, 10.28 g), DPPF palladium dichloride (1.9 mmol, 1.4 g) and potassium carbonate (127.8 mmol, 17.67 g) were added in sequence, a mixture of dioxane and water (6: 1) 210 mL was used as solvent, replaced with nitrogen for 3 times, and the reaction was carried out at 85°C for 2h, and TLC was used to detect the reaction progress. After the reaction was completed, the dioxane was removed by rotary evaporation, EA and H2O were used to extract for three times, the organic phase was concentrated, and column chromatography was used to separate to obtain the corresponding intermediate 84B (light yellow oily liquid, 7.28 g, 87%).

[0642] Into a 250 mL round bottom flask, 84B (2.15 mmol, 280 mg) was added, 2-Boc-2,7-diaza-spiro[4.4]nonane (2.56 mmol, 580 mg), NaBH(OAc)3 (2.68 mmol, 570 mg), acetic acid 0.1 mL, DCM 100 mL was used as solvent, and the reaction was carried out at room temperature for 2h, and TLC was used to detect the reaction progress. After the reaction was completed, saturated sodium bicarbonate solution 30 mL was used to quench the reaction, the organic phase was concentrated by rotary evaporation after the extraction and separation of layers, and column chromatography was used to separate to obtain the corresponding intermediate 84C (yellow oily liquid, 428 mg, 58%).

[0643] LC-MS (ESI): m / z: 343.2 [M+H] + .

[0644] Into a 100 mL single neck flask was added 84C (1.25 mmol, 428 mg), AD-mix-β (0.8 mmol, 629 mg) was added under ice bath condition, 1:1 t-butanol and water as solvent, and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, EA and H2O were used to extract three times, the organic phase was concentrated, and column chromatography was used to isolate the intermediate 84D (brown oily liquid, 320 mg, yield 68%). LC-MS (ESI): m / z: 377.2 [M+H] + .

[0645] Into a 100 mL round bottom flask was added 84D (0.61 mmol, 230 mg), EA was used for dissolution, 4N hydrochloric acid dioxane solution (8 mmol, 2 mL) was added, and the reaction was allowed to proceed at room temperature overnight. TLC was used to detect that the reaction was complete, and the reaction was concentrated and column chromatography was used to isolate the final product 84 (yellow solid, 140 mg, 74%).

[0646] 1 H NMR (500 MHz, DMSO-d6) δ 7.55 (dt, J = 10.3, 5.3 Hz, 2H), 7.43 - 7.32 (m, 2H), 4.56 (t, J = 5.9 Hz, 1H), 4.45 - 4.22 (m, 2H), 3.52 - 3.40 (m, 4H), 3.34 - 3.18 (m, 6H), 3.16 (s, 1H), 2.33 - 1.88 (m, 3H).

[0647] 13 C NMR (126 MHz, DMSO-d6) δ 73.53, 67.32, 59.25 (dd, J = 38.9, 8.5 Hz), 57.34 (d, J = 10.0 Hz), 53.25, 52.52 - 52.36 (m), 47.67 (d, J = 8.3 Hz), 43.56 (d, J = 12.5 Hz), 35.12 (d, J = 50.3 Hz), 33.58 (d, J = 15.1 Hz).

[0648] LC-MS (ESI): m / z: 277.1 [M+H] + .

[0649] Synthesis of (R)-1-(3-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)phenyl)ethane-1,2-diol (Compound 85)

[0650] Into a 500 mL two-necked flask, 85A (7.75 mmol, 1.42 g), 1,4-dioxa-spiro[4,5]dec-7-ene-8-boronic acid pinacol ester (10.08 mmol, 2.68 g), DPPF palladium dichloride (0.6 mmol, 0.45 g) and sodium carbonate (19.4 mmol, 2.06 g) were added successively, 200 mL of a mixture of DMF and water (4:1) as solvent, replaced with nitrogen for 3 times, reacted at 102 °C for 2 h, and the reaction progress was detected by TLC. After the reaction was completed, EA and H2O were added for extraction three times, the organic phase was concentrated, and column chromatography was used for separation to obtain the corresponding intermediate 85B (yellowish transparent liquid, 1189 mg, 63.5%). + .

[0651] Into a 100 mL single-necked flask, 85B (2.93 mmol, 710 mg) and AD-mix-β (1.33 mmol, 1043 mg) were added successively under ice bath conditions, 1:1 tert-butanol and water were used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, EA and H2O were added for extraction three times, the organic phase was concentrated, and column chromatography was used for separation to obtain the final product 85 (yellowish viscous oily liquid, 300 mg, 37%).

[0652] 1 H NMR (500 MHz, Chloroform-d) δ 7.38 (t, J = 1.9 Hz, 1H), 7.34-7.26 (m, 2H), 7.20 (dt, J = 7.3, 1.7 Hz, 1H), 5.98 (tt, J = 3.9, 1.6 Hz, 1H), 4.78 (dd, J = 8.2, 3.5 Hz, 1H), 4.01 (s, 4H), 3.75-3.58 (m, 2H), 2.65 (ddd, J = 6.9, 4.7, 2.4 Hz, 2H), 2.49-2.39 (m, 2H), 1.91 (t, J = 6.5 Hz, 2H).

[0653] 13 C NMR (126 MHz, CDCl3) δ 141.8, 140.5, 136.1, 128.4, 124.9, 124.6, 123.0, 122.0, 107.7, 74.8, 68.1, 64.5, 36.1, 31.3, 26.8.

[0654] LC-MS (ESI): m / z: 277.1 [M+H] + .

[0655] Synthesis of (1R)-1-(3-(8-oxa-3-azabicyclo[3.2.1]oct-3-yl)methyl)phenyl)ethane-1,2-diol (Compound 86)

[0656] Into a 500 mL two-necked flask, was added 86A (63.9 mmol, 11.83 g), potassium ethylene trifluoroborate (76.7 mmol, 10.28 g), DPPF palladium dichloride (1.9 mmol, 1.4 g) and potassium carbonate (127.8 mmol, 17.67 g) sequentially, 210 mL of a mixture of dioxane and water (6:1) as solvent, nitrogen was replaced for 3 times, 85 °C for 2 h, TLC was used to monitor the reaction progress. After the reaction was completed, the dioxane was removed by rotary evaporation, EA and H2O were used to extract three times, the organic phase was concentrated, and column chromatography was used to separate to obtain the corresponding intermediate 86B (yellowish oil liquid, 7.28 g, 87%).

[0657] Into a 100 mL round-bottom flask, was added 86B (1.85 mmol, 245 mg), 8-oxa-3-azabicyclo[3.2.1]octane (3.71 mmol, 420 mg), NaBH(OAc)3 (2.32 mmol, 492 mg), acetic acid 0.1 mL, DCM 100 mL as solvent, and the mixture was stirred at room temperature for 2 h. TLC was used to monitor the reaction progress. After the reaction was completed, 30 mL of saturated sodium bicarbonate solution was added to quench the reaction, and the organic phase was concentrated by rotary evaporation. Column chromatography was used to separate to obtain the corresponding intermediate 86C (transparent oil liquid, 279 mg, 66%). + .

[0658] Into a 100 mL single-necked flask, was added 86C (1.21 mmol, 279 mg), AD-mix-β (0.52 mmol, 410 mg) was added under ice bath conditions, 1:1 tert-butanol and water as solvent, and the mixture was stirred at room temperature overnight. After the reaction was completed, EA and H2O were used to extract three times, the organic phase was concentrated, and column chromatography was used to separate to obtain the final product 86 (transparent colorless oil liquid, 140 mg, 44%)

[0659] 1H NMR (500 MHz, Chloroform-d) δ 7.28 - 7.13 (m, 4H), 4.74 (dd, J = 8.3, 3.4 Hz, 1H), 4.19 (dt, J = 4.5, 2.4 Hz, 2H), 3.69 (dd, J = 11.3, 3.5 Hz, 1H), 3.59 (dd, J = 11.3, 8.3 Hz, 1H), 3.40 (s, 2H), 2.46 (d, J = 11.0 Hz, 2H), 2.27 (dt, J = 11.1, 2.4 Hz, 2H), 1.91 (t, J = 6.1 Hz, 2H), 1.78 (dh, J = 10.0, 5.2, 4.4 Hz, 2H).

[0660] 13 C NMR (126 MHz, CDC13) δ 140.6, 139.0, 128.4, 128.3, 126.4, 124.7, 74.9, 74.7, 74.0, 68.1, 67.9, 62.1, 58.5, 58.4, 28.6.

[0661] LC-MS (ESI): m / z: 264.1 [M+H] + .

[0662] Synthesis of (R)-1-(6-ethylpyridin-2-yl)ethane-1,2-diol (Compound 87)

[0663] Step 1 Synthesis of 2-ethyl-6-vinylpyridine

[0664] Into a 100 mL Schlenk tube was added V133-1 (800 mg, 4.28 mmol), potassium vinyltrifluoroborate (692 mg, 5.16 mmol), DPPF palladium dichloride (320 mg, 0.43 mmol) and potassium carbonate (1.18 g, 8.56 mmol) sequentially, 20 mL of a mixture of dioxane and water (6:1) as solvent, nitrogen protection to remove oxygen, 85 °C reaction for 2 hours, TLC detection of reaction progress. After the reaction was completed, the dioxane was first removed by rotary evaporation, extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and separated and purified using an automatic column machine to obtain the intermediate V133-2 (colorless transparent oil, 400 mg, yield 70.4%).

[0665] Step 2 Synthesis of (R)-1-(6-ethylpyridin-2-yl)ethane-1,2-diol (V133)

[0666] In a 100 mL single neck flask, V133-2 (100 mg, 0.75 mmol) was added, AD-mix-β (1.10 g, 1.41 mmol) was added under ice bath condition, t-butyl alcohol and water were used as solvent in 1:1 ratio, and the reaction was carried out at room temperature overnight. After the reaction was completed, it was extracted with ethyl acetate and water for three times, the organic phase was combined and rotary evaporated, and separated and purified using automatic column machine to obtain V133-3 (white solid, 66 mg, yield 52.6%, ee value 93.2%).

[0667] 1 H NMR (600 MHz, DMSO-d6) δ 7.67 (t, J = 7.7 Hz, 1H), 7.30 (d, J = 7.7 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 5.34 (d, J = 4.9 Hz, 1H), 4.71 (t, J = 5.9 Hz, 1H), 4.56 (dt, J = 6.9, 4.4 Hz, 1H), 3.73 - 3.63 (m, 1H), 3.51 - 3.43 (m, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H).

[0668] 13 C NMR (151 MHz, DMSO-d6) δ 161.64, 161.34, 136.75, 120.15, 117.86, 74.90, 66.46, 30.49, 13.80.

[0669] HRMS (ESI) (m / z): [M+H] + calcd for C9H 14 NO2 168.1019; found 168.1012. HPLC Purity: 96.79%, retention time: 2.584 min.

[0670] Synthesis of (R)-1-(2-ethylpyridin-4-yl)ethane-1,2-diol (Compound 88)

[0671] Step 1 Synthesis of 2-ethyl-4-vinylpyridine

[0672] To a 100 mL Schlenk tube was added 33-1 (1.00 g, 5.37 mmol), potassium ethylene trifluoroborate (863 mg, 6.44 mmol), DPPF palladium dichloride (393 mg, 0.54 mmol) and potassium carbonate (1.48 g, 10.74 mmol) sequentially, 18 mL of a mixture of dioxane and water (6:1) as solvent, nitrogen protection to remove oxygen, 85 °C for 2 hours, TLC detection of reaction progress. After the reaction was completed, the dioxane was removed by rotary evaporation, extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, column chromatography (PE:EA = 10:1) to give intermediate 33-2 (transparent oil, 538 mg, yield 75.2%).

[0673] Step 2 Synthesis of (R)-1-(2-ethylpyridin-4-yl)ethan-1,2-diol (V33)

[0674] To a 100 mL single-necked flask was added 33-2, AD-mix-β (1.90 g, 2.44 mmol) was added under ice bath conditions, 1:1 tert-butanol and water as solvent, room temperature reaction overnight. After the reaction was completed, extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, column chromatography (PE:EA = 1:1) to give 33-3 (white solid, 236 mg, yield 35.0%, ee value 93.0%).

[0675] 1 H NMR (600 MHz, Chloroform-d) δ 8.30 (d, J = 5.1 Hz, 1H), 7.12 (s, 1H), 7.03 (dd, J = 5.3, 1.7 Hz, 1H), 4.73 (dd, J = 7.8, 3.5 Hz, 1H), 3.74 (dd, J = 11.4, 3.5 Hz, 1H), 3.56 (dd, J = 11.3, 7.7 Hz, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H).

[0676] 13 C NMR (151 MHz, Chloroform-d) δ 162.58, 149.51, 147.86, 118.40, 117.46, 72.28, 66.41, 30.18, 12.90.

[0677] HRMS (ESI) (m / z): [M+H] + calcd for C9H 14 NO2 168.1019; found 168.1005. HPLC Purity: 96.53%, retention time: 2.258 min.

[0678] Synthesis of (R)-1-(6-ethylpyrimidin-4-yl)ethan-1,2-diol (Compound 89)

[0679] Synthesis of 4-ethyl-6-vinylpyrimidine

[0680] Into a 10 mL Schlenk tube was added V197-1 (100 mg, 0.70 mmol), potassium vinyltrifluoroborate (113 mg, 0.84 mmol), DPPF palladium dichloride (0.07 mmol, 51 mg) and potassium carbonate (193 mg, 1.40 mmol) sequentially, 3 mL of a mixture of dioxane and water (6:1) as solvent, nitrogen protection to remove oxygen, 85 °C for 2 hours, TLC detection of reaction progress. After the reaction was completed, the dioxane was first removed by rotary evaporation, extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and separated and purified using an automatic column machine to obtain the intermediate V197-2 (colorless transparent oil, 226 mg, the product is volatile, not too dry, not calculated yield, directly into the next step).

[0681] Synthesis of (R)-1-(6-ethylpyrimidin-4-yl)ethan-1,2-diol (Compound 89)

[0682] Into a 100 mL single-necked flask was added V193-2 (226 mg, 1.68 mmol), AD-mix-β (2.47 g, 3.17 mmol) was added under ice bath conditions, 1:1 tert-butanol and water as solvent, room temperature reaction overnight. After the reaction was completed, extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and separated and purified using an automatic column machine to obtain V197-3 (brown oily liquid, 56 mg, yield 19.8%, ee value 94.9%).

[0683] 1 H NMR (500 MHz, DMSO-d6) δ 8.96 (d, J = 1.3 Hz, 1H), 7.47 (s, 1H), 5.57 (d, J = 2.4 Hz, 1H), 4.75 (t, J = 5.9 Hz, 1H), 4.57 - 4.49 (m, 1H), 3.77 - 3.67 (m, 1H), 3.58 - 3.48 (m, 1H), 2.75 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 3.5 Hz, 3H).

[0684] 13 C NMR (151 MHz, Chloroform-d) δ 172.67, 168.33, 157.45, 116.45, 73.30, 66.39, 30.96, 12.67. HRMS (ESI) (m / z): [M+H]+ calcd for C8H 13 N2O2 169.0972; found 169.1007. HPLC Purity: 95.07%, retention time: 2.044 min.

[0685] Synthesis of (R)-1-(5-ethylpyridin-3-yl)ethane-1,2-diol (Compound 90)

[0686] Synthesis of 3-ethyl-5-vinylpyridine

[0687] Into a 100 mL Schlenk tube was added V125-1 (800 mg, 4.28 mmol), potassium vinyltrifluoroborate (692 mg, 5.16 mmol), DPPF palladium dichloride (320 mg, 0.43 mmol) and potassium carbonate (1.18 g, 8.56 mmol) sequentially, 20 mL of a mixture of dioxane and water (6:1) as solvent, nitrogen protection to remove oxygen, 85 °C for 2 hours, TLC detection of reaction progress. After the reaction was completed, the dioxane was first removed by rotary evaporation, extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and separated and purified using an automatic column machine to obtain intermediate V125-2 (transparent yellow oil, 350 mg, yield 61.6%).

[0688] Synthesis of (R)-1-(5-ethylpyridin-3-yl)ethane-1,2-diol (Compound 90)

[0689] Into a 100 mL single-neck flask was added V125-2 (100 mg, 0.75 mmol), AD-mix-β (1.10 g, 1.41 mmol) was added under ice bath conditions, 1:1 tert-butanol and water as solvent, room temperature reaction overnight. After the reaction was completed, extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and separated and purified using an automatic column machine to obtain V125-3 (white solid, 66 mg, yield 52.6%, ee value 93.9%).

[0690] 1 H NMR (500 MHz, DMSO-d6) δ 8.35 - 8.34 (m, 1H), 8.32 - 8.30 (m, 1H), 7.60 - 7.53 (m, 1H), 5.37 (d, J = 4.4 Hz, 1H), 4.78 (t, J = 5.7 Hz, 1H), 4.62 - 4.52 (m, 1H), 3.55 - 3.47 (m, 1H), 3.47 - 3.41 (m, 1H), 2.62 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H).

[0691] 13 C NMR (126 MHz, DMSO-d6) 5 148.13, 146.06, 138.69 (d, J = 14.5 Hz), 133.62, 72.13, 67.36, 25.74, 15.81. HRMS (ESI) (m / z): [M+H] + calcd for C9H 14 NO2 168.1019; found 168.1002. HPLC Purity: 97.15%, retention time: 2.820 min.

[0692] Synthesis of (R)-1-(3,4-difluorophenyl)-1,2-ethanediol (Compound 91)

[0693] Into a 100 mL single necked flask was added V27-1 (1.00 g, 7.14 mmol), AD-mix-β (10.50 g, 13.48 mmol) was added under ice bath condition, 1:1 tert-butanol and water as solvent, reaction at room temperature overnight. After reaction was completed, extracted with ethyl acetate and water for three times, combined organic phase was rotary evaporated, separated and purified using automatic column machine, V125-3 (brown oily liquid, 1.18 g, yield 95.0%, ee value 93.6%) was obtained.

[0694] 1 H NMR (500 MHz, DMSO-d6) δ 7.44 - 7.27 (m, 2H), 7.24 - 7.12 (m, 1H), 5.41 (d, J = 4.5 Hz, 1H), 4.75 (t, J = 5.8 Hz, 1H), 4.54 (q, J = 5.4 Hz, 1H), 3.51 - 3.38 (m, 2H).

[0695] 13 C NMR (126 MHz, DMSO-d6) 5 148.13, 146.06, 138.69 (d, J = 14.5 Hz), 133.62, 72.13, 67.36, 25.74, 15.81. HRMS (ESI) (m / z): [M+H]

[0696] Synthesis of (R)-1-(2,5-bis(trifluoromethyl)phenyl)ethane-1,2-diol (Compound 92)

[0697] Step 1 Synthesis of 1,4-bis(trifluoromethyl)-2-vinylbenzene

[0698] Into a 100 mL Schlenk tube, 120-1 (2.00 g, 6.83 mmol), potassium vinyltrifluoroborate (1.10 g, 8.19 mmol), DPPF palladium dichloride (150 mg, 0.20 mmol) and potassium carbonate (1.89 g, 13.66 mmol) were added in sequence, 20 mL of a mixture of dioxane and water (6:1) was used as solvent, nitrogen was used to protect the system from oxygen, the reaction was carried out at 85 °C for 2 hours, TLC was used to monitor the reaction progress. After the reaction was completed, the dioxane was removed by rotary evaporation, and the product was extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and column chromatography (PE:EA = 20:1) was used to obtain the intermediate 120-2 (pale yellow oil, 529 mg, yield 32.2%).

[0699] Step 2 Synthesis of (R)-1-(2,5-bis(trifluoromethyl)phenyl)ethane-1,2-diol (V120)

[0700] Into a 100 mL single-necked flask, 120-2 (529 mg, 2.20 mmol) was added, AD-mix-β (2.52 g, 3.23 mmol) was added under ice bath conditions, 1:1 tert-butanol and water were used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, the product was extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and column chromatography (PE:EA = 1:1) was used to obtain 120-3 (white solid powder, 30 mg, yield 5.0%, ee value 92.1%).

[0701] 1 H NMR (500 MHz, DMSO-d6) δ 8.08 (s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 5.84 (d, J = 4.5 Hz, 1H), 5.03 - 4.85 (m, 2H), 3.58 - 3.47 (m, 1H), 3.47 - 3.38 (m, 1H).

[0702] 13C NMR (151 MHz, Chloroform-d) δ 140.77, 134.36 (q, J = 33.1, 31.8 Hz), 130.52 (q, J = 32.6 Hz), 126.44 (d, J = 5.2 Hz), 125.63 (d, J = 6.0 Hz), 124.95 (d, J = 3.0 Hz), 124.30 (d, J = 34.1 Hz), 122.49 (d, J = 32.1 Hz), 70.07, 67.65. HPLC Purity: 100.00%, retention time: 6.203 min.

[0703] Synthesis of (R)-1-(3-methyl-5-(trifluoromethyl)phenyl)ethan-1,2-diol (Compound 93)

[0704] Synthesis of 1-(3-methyl-5-(trifluoromethyl)phenyl)ethan-1-one (Compound 94)

[0705] Into a 100 mL Schlenk tube, 105-1 (1.00 g, 4.18 mmol), potassium ethylene trifluoroborate (672 mg, 5.02 mmol), DPPF palladium dichloride (306 mg, 0.42 mmol) and potassium carbonate (1.20 g, 8.36 mmol) were added in sequence, 16 mL of a mixture of dioxane and water (6:1) was used as solvent, nitrogen was used to protect the system from oxygen, the reaction was carried out at 85 °C for 2 hours, TLC was used to monitor the reaction progress. After the reaction was completed, the dioxane was removed by rotary evaporation, and the product was extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and column chromatography (pure PE) was used to obtain the intermediate 103-2 (transparent oil, 572 mg, yield 73.5%).

[0706] Synthesis of (R)-1-(3-methyl-5-(trifluoromethyl)phenyl)ethan-1,2-diol (Compound 93)

[0707] Into a 100 mL single-neck flask, 105-2 (572 mg, 3.07 mmol) was added, AD-mix-β (1.40 g, 1.80 mmol) was added under ice bath conditions, 1:1 tert-butanol and water were used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, the product was extracted with ethyl acetate and water three times, the organic phase was combined and rotary evaporated, and column chromatography (PE:EA = 1:1) was used to obtain 105-3 (white solid, 11 mg, yield 1.6%, ee value 98.93%).

[0708] 1H NMR (400 MHz, DMSO-d6) δ 7.48 (s, 1H), 7.45 (s, 1H), 7.40 (s, 1H), 5.44 (d, J = 4.4 Hz, 1H), 4.78 (t, J = 5.7 Hz, 1H), 4.60 (q, J = 5.4 Hz, 1H), 3.52 - 3.47 (m, 1H), 3.47 - 3.41 (m, 1H), 2.39 (s, 3H).

[0709] 13 C NMR (151 MHz, DMSO-d6) δ 145.46, 138.92, 131.54, 129.09 (q, J = 31.2 Hz), 124.96 (q, J = 272.8 Hz), 124.31 (q, J = 2.2 Hz), 120.47 (q, J = 3.8 Hz), 73.48, 67.50, 21.25. HPLC Purity: 100.00%, retention time: 5.712 min.

[0710] Synthesis of 2-(3-ethylphenyl)butane-2,3-diol (Compound 94)

[0711] Step 1 Synthesis of (Z)-1-bromo-3-(2-buten-2-yl)benzene (V10-2)

[0712] Into a 100 ml flask, ethyltriphenylphosphonium bromide (2.24 g, 6.03 mmol) was added, nitrogen protection to remove oxygen, injected into anhydrous tetrahydrofuran solution (15 mL), low temperature (-10 ℃) stirring, added anhydrous t-BuOK solution (6.03 mL 1M, 6.03 mmol), the reaction was 1 h, after the reaction was completed, V10-1 (1.00 g, 5.02 mmol) was added, and the reaction was carried out at room temperature for 2 h. TLC was used to detect the progress of the reaction. After the reaction was completed, the organic phase was extracted with EA and H2O three times, concentrated and separated and purified using an automatic column machine to obtain V10-2 (yellow oily liquid, 1.04 g, yield 98.2%).

[0713] Step 2 Synthesis of (Z)-1-(2-buten-2-yl)-3-ethylbenzene (V10-3)

[0714] Into a 100 ml_ two-necked flask, V10-2 (1.04 g, 4.93 mmol), DPPF palladium dichloride (360.5 mg, 0.49 mmol), Cs2C03(9.64 g, 29.58 mmol) were charged under nitrogen protection, triethyl boron (5.92 mL 1 M, 5.92 mmol) was injected, anhydrous tetrahydrofuran was used as solvent, reaction was carried out at 55 °C, TLC was used to monitor the reaction progress. After the reaction was completed, it was extracted with EA and H20 three times, the organic phase was concentrated and combined, and was separated and purified using an automatic column machine to obtain V10-3 (light yellow oily liquid, 744 mg, yield 94.3%).

[0715] Synthesis of 2-(3-ethylphenyl)butane-2,3-diol (V10)

[0716] Into a 100 ml_ single-necked flask, V10-3 (744 mg, 4.64 mmol) was added, AD-mix-β (6.82 g, 8.76 mmol) was added under ice bath conditions, 1:1 t-BuOH and H20 were used as solvent, and reaction was carried out at room temperature overnight. After the reaction was completed, it was extracted with EA and H20 three times, the organic phase was concentrated and combined, and was separated and purified by column chromatography (PE:EA = 1:1) to obtain V10-4 (colorless oily liquid, 610 mg, yield 67.6%).

[0717] 1 H NMR (500 MHz, DMSO-d6) δ 7.34 - 7.15 (m, 3H), 7.02 (t, J = 6.4 Hz, 1H), 4.66 (d, J = 61.6 Hz, 1H), 4.44 (dd, J = 26.0, 5.5 Hz, 1H), 3.70 - 3.59 (m, 1H), 2.59 (q, J = 7.6 Hz, 2H), 1.41 (d, J = 3.4 Hz, 3H), 1.18 (t, J = 7.6 Hz, 3H), 0.83 (t, J = 6.1 Hz, 3H).

[0718] 13 C NMR (126 MHz, DMSO-d6) δ 148.12, 143.09, 127.80, 125.61, 125.43, 123.39, 75.77, 73.30, 28.89, 26.53, 18.15, 16.21. HPLC Purity: 96.96%, retention time: 5.989 min.

[0719] Synthesis of (R)-2-(2-ethylpyridin-4-yl)propane-1,2-diol (Compound 95)

[0720] Step 1 Synthesis of 2-bromo-4-(l-propen-2-yl)pyridine (V66-2)

[0721] Into a 100 mL Schlenk tube, V66-1 (2.00 g, 7.04 mmol), isopropenylboronic acid pinacol ester (657 mg, 3.91 mmol), DPPF palladium dichloride (143 mg, 0.20 mmol) and Cs2CO3(3.83 g, 11.74 mmol) were sequentially added, a mixture of DOX and H2O (6:1) 21 mL was used as solvent, nitrogen was used to protect the system from oxygen, the reaction was carried out at 85 °C for 2 hours, TLC was used to monitor the reaction progress. After the reaction was completed, the organic phase was extracted with EA and H2O three times, concentrated and purified by column chromatography (PE:EA = 20:1) to obtain the intermediate V66-2 (yellow oily liquid, 629 mg, yield 45.2%).

[0722] Step 2 Synthesis of 2-ethyl-4-(l-propen-2-yl)pyridine (V66-3)

[0723] Into a 250 mL two-necked flask, V66-2 (629 mg, 3.18 mmol), DPPF palladium dichloride (234 mg, 0.32 mmol), Cs2CO3(6.22 g, 19.08 mmol) were added, and the system was protected by nitrogen, triethyl boron (3.81 mL 1M, 3.81 mmol) was injected, anhydrous tetrahydrofuran was used as solvent, the reaction was carried out at 55 °C, TLC was used to monitor the reaction progress. After the reaction was completed, the organic phase was extracted with EA and H2O three times, concentrated and purified by automatic column separation to obtain V66-3 (pale yellow oily liquid, 278 mg, yield 59.4%).

[0724] Step 3 Synthesis of (R)-2-(2-ethylpyridin-4-yl)propane-1,2-diol (V66)

[0725] Into a 100 mL single-necked flask, V66-3 (278 mg, 1.89 mmol) was added, AD-mix-β (1.90 g, 2.78 mmol) was added under ice bath conditions, 1:1 t-BuOH and H2O were used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, the organic phase was extracted with EA and H2O three times, concentrated and purified by column chromatography (PE:EA = 1:1) to obtain V66-4 (colorless oily liquid, 47 mg, yield 13.7%, ee value 99.6%).

[0726] 1H NMR (500 MHz, DMSO-d6) δ 8.36 (d, J = 5.2 Hz, 1H), 7.31 (s, 1H), 7.23 (dd, J = 5.2, 1.7 Hz, 1H), 5.07 (d, J = 2.3 Hz, 1H), 4.77 (t, J = 5.9 Hz, 1H), 3.49 - 3.37 (m, 2H), 2.73 (q, J = 7.6 Hz, 2H), 1.37 (s, 3H), 1.22 (t, J = 7.6 Hz, 3H).

[0727] 13 C NMR (126 MHz, DMSO-d6) δ 161.98, 156.50, 148.21, 118.97, 118.38, 73.32, 69.91, 30.76, 25.64, 13.85. HRMS (ESI) (m / z): [M+H] + calcd for C 10 H 16 NO2 182.1176; found 182.1180. HPLC Purity: 95.30%, retention time: 2.721 min.

[0728] Synthesis of (R)-2-(3-ethylphenyl)propane-1,2-diol (Compound 96)

[0729] Step 1 Synthesis of 1-ethyl-3-(1-propen-2-yl)benzene (V68-2)

[0730] Into a 100 ml flask was added triphenylphosphine iodomethane (3.27 g, 8.10 mmol), nitrogen protection to remove oxygen, injected into anhydrous tetrahydrofuran solution (20 mL), low temperature (-10 ℃) stirring, added anhydrous t-BuOK solution (8.10 mL 1M, 8.10 mmol), the reaction was 1h, after the reaction was completed, V68-1 (1.00 g, 6.75 mmol) was added, and the reaction was carried out at room temperature for 2h. TLC was used to detect the progress of the reaction. After the reaction was completed, the organic phase was extracted with EA and H2O three times, concentrated and purified by automatic column separation to obtain V68-2 (light yellow solid, 987 mg, not purified directly into the next step reaction).

[0731] Step 2 Synthesis of (R)-2-(3-ethylphenyl)propane-1,2-diol (V68)

[0732] Into a 100 mL single necked flask, V68-2 (987 mg, 6.75 mmol) was added, AD-mix-β (9.92 g, 12.74 mmol) was added under ice bath condition, t-BuOH and H2O were used as solvent in 1:1 ratio, the reaction was carried out at room temperature overnight. After the reaction was completed, it was extracted with EA and H2O for three times, the organic phase was combined and concentrated, column chromatography (PE:EA = 1:1) was used for separation and purification, V68-3 (yellow oily liquid, 1.05 g, yield 86.4%, ee value 93.2%) was obtained.

[0733] 1 H NMR (500 MHz, DMSO-d6) δ 7.30 (s, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H), 4.79 (s, 1H), 4.62 (t, J = 5.9 Hz, 1H), 3.39 (d, J = 5.9 Hz, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.38 (s, 3H), 1.18 (t, J = 7.6 Hz, 3H).

[0734] 13 C NMR (126 MHz, DMSO-d6) δ 147.41, 142.75, 127.46, 125.39, 124.92, 122.86, 73.64, 70.49, 28.42, 26.12, 15.71. HPLC Purity: 96.04%, retention time: 5.374 min.

[0735] Synthesis of (R)-1-(2-ethyl-3-fluorophenyl)ethane-1,2-diol (Compound 97)

[0736] Synthesis of 2-bromo-1-fluoro-3-vinylbenzene (V191-2)

[0737] Into a 250 mL two necked flask, triphenylphosphine iodomethane (4.30 g, 10.64 mmol) was added, nitrogen was used to protect the flask from oxygen, anhydrous THF solution (50 mL) was injected, anhydrous t-BuOK solution (12.77 mL 1M, 12.77 mmol) was added under ice bath condition, the reaction was carried out at low temperature (-10 °C) for 1 h, V191-1 (1.80 g, 8.87 mmol) was added, the reaction was carried out at room temperature for 2 h, TLC was used to monitor the reaction progress. After the reaction was completed, it was extracted with EA and H2O for three times, the organic phase was combined and concentrated, automatic column machine was used for separation and purification, V191-2 (yellow oily liquid, 921 mg, yield 51.6%) was obtained.

[0738] Step 2 Synthesis of 2-ethyl-l-fluoro-3-vinylbenzene (V191-3)

[0739] Into a 250 mL two-necked flask was placed V191-2 (921 mg, 4.58 mmol), DPPF palladium dichloride (336 mg, 0.46 mmol), Cs2C03(8.96 g, 27.48 mmol), under nitrogen protection, injected triethyl boron (5.50 mL 1 M, 5.50 mmol), used anhydrous tetrahydrofuran as solvent, reacted at 55 °C, TLC was used to monitor the reaction progress. After the reaction was completed, extracted with EA and H20 three times, combined and concentrated the organic phase, separated and purified using automatic column machine, obtained V191-3 (colorless oily liquid, 300 mg, yield 43.7%).

[0740] Step 3 Synthesis of (R)-l-(2-ethyl-3-fluorophenyl)ethane-l,2-diol (V191)

[0741] Into a 100 mL single-necked flask was placed V191-3 (300 mg, 2.00 mmol), AD-mix-β (2.94 g, 3.77 mmol) was added under ice bath conditions, 1:1 t-BuOH and H20 were used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, extracted with EA and H20 three times, combined and concentrated the organic phase, column chromatography (PE:EA = 1:1), obtained V191-4 (white solid powder, 242 mg, yield 65.7%, ee value 98.4%).

[0742] 1 H NMR (500 MHz, DMSO-d6) δ 7.26 (dd, J = 7.8, 1.4 Hz, 1H), 7.23 - 7.17 (m, 1H), 7.05 - 6.97 (m, 1H), 5.28 (d, J = 4.3 Hz, 1H), 4.83 - 4.75 (m, 2H), 3.48 - 3.37 (m, 2H), 2.67 (qd, J = 7.6, 2.0 Hz, 2H), 1.13 (t, J = 7.5 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 160.31 (d, J = 241.5 Hz), 143.61 (d, J = 3.8 Hz), 127.95 (d, J = 15.2 Hz), 126.87 (d, J = 9.0 Hz), 122.46 (d, J = 2.9 Hz), 113.30 (d, J = 22.9 Hz), 70.08 (d, J = 2.8 Hz), 67.12, 17.68 (d, J = 4.5 Hz), 14.92. HPLC Purity: 97.65%, retention time: 5.023 min.

[0743] Synthesis of (R)-3-(3-ethylphenoxy)propane-1,2-diol (Compound 98)

[0744] In 100 ml single neck flask was added 4-a (500 mg, 4.09 mmol), (R)-3-chloro-1,2-propanediol (905 mg, 8.19 mmol) and anhydrous K2CO3 (4.53 g, 32.76 mmol), injected 20 mL of anhydrous DMF solution, the reaction was stirred at 100 °C. After the reaction was completed, it was extracted with EA and H2O three times, the organic phase was combined, after adding anhydrous Na2SO4 to remove water, rotary evaporation was concentrated, column chromatography (DCM:MeOH = 10:1) was used to separate the product, and compound 4 (pale yellow oily liquid, 420 mg, yield 52.3%) was obtained.

[0745] 1 H NMR (600 MHz, Chloroform-d) δ 7.20 (t, J = 7.9 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.77 (s, 1H), 6.73 (dd, J = 8.3, 2.1 Hz, 1H), 4.13 - 4.07 (m, 1H), 4.04 (d, J = 0.9 Hz, 1H), 4.03 (d, J = 2.7 Hz, 1H), 3.84 (dd, J = 11.5, 3.7 Hz, 1H), 3.75 (dd, J = 11.5, 5.6 Hz, 1H), 2.66 (s, 2H), 2.62 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). 13 C NMR (151 MHz, Chloroform-d) δ 158.49, 146.13, 120.98, 111.52, 70.49, 69.08, 63.74, 28.90, 15.50. HPLC Purity: 95.52%, retention time: 5.419 min.

[0746] Synthesis of (R)-3-((3-ethylbenzyl)amino)propane-1,2-diol (Compound 99)

[0747] In a 100 mL single neck flask, 6A (500 mg, 3.73 mmol), (R)-3-amino-1,2- propanediol (407 mg, 4.47 mmol) were added, 20 ml solvent (DCM:MeOH = 3:1) was injected, after 30 min, pH was adjusted to 4-6, sodium cyanoborohydride (234 mg, 3.73 mmol,) was added, the reaction was carried out at room temperature overnight, TLC was used to monitor the reaction progress. After the reaction was completed, an appropriate amount of water was added, dichloromethane was used for extraction, the organic phase was combined, anhydrous Na2SO4 was added to remove water, and then rotary evaporation was used for concentration, column chromatography (DCM:MeOH = 10:1) was used to separate the product, and 6B (colorless oily liquid, 322 mg, yield 41.2%) was obtained.

[0748] 1 H NMR (500 MHz, Chloroform-d) δ 7.31 (s, 1H), 7.28 (s, 1H), 7.27 (s, 1H), 7.22-7.17 (m, 1H), 4.17 (s, 2H), 4.07 (s, 1H), 3.69-3.61 (m, 1H), 3.56-3.45 (m, 1H), 3.13-2.95 (m, 2H), 2.63 (q, J = 7.5 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 145.64, 129.85, 129.65, 129.47, 129.35, 127.40, 67.21, 64.04, 51.96, 49.38, 28.60, 15.41. HPLC Purity: 97.87%, retention time: 3.690 min.

[0749] Synthesis of 4-((3-ethylbenzyl)amino)-1,2-butanediol (Compound 100)

[0750] In a 50 mL single neck flask, 5A (128 mg, 0.95 mmol), 4-aminobutane-1,2-diol (100 mg, 0.95 mmol) were added, 10 ml solvent (DCM:MeOH = 3:1) was injected, after 30 min, pH was adjusted to 4-6, sodium cyanoborohydride (90 mg, 1.43 mmol,) was added, the reaction was carried out at room temperature overnight, TLC was used to monitor the reaction progress. After the reaction was completed, an appropriate amount of water was added, dichloromethane was used for extraction, the organic phase was combined, anhydrous Na2SO4 was added to remove water, and then rotary evaporation was used for concentration, column chromatography (DCM:MeOH = 10:1) was used to separate the product, and 5B (yellow oily liquid, 105 mg, yield 49.5%) was obtained.

[0751] 1H NMR (400 MHz, Methanol-d4) δ 7.36 (t, J = 7.5 Hz, 1H), 7.32 (s, 1H), 7.29 - 7.27 (m, 1H), 7.30 - 7.23 (m, 1H), 4.14 (d, J = 2.2 Hz, 2H), 3.83 - 3.65 (m, 1H), 3.53 - 3.45 (m, 2H), 3.23 - 3.05 (m, 2H), 2.69 (q, J = 7.6 Hz, 2H), 1.96 - 1.87 (m, 1H), 1.86 - 1.69 (m, 1H), 1.25 (t, J = 7.6 Hz, 3H). 13 C NMR (151 MHz, Methanol-d4) δ 146.72, 133.51, 130.25, 130.15, 129.92, 127.93, 66.88, 52.55, 46.44, 30.66, 29.68, 16.04. HPLC Purity: 95.03%, retention time: 3.800 min.

[0752] Synthesis of (3R,4R,5S,6S)-2-((R)-2-(3-ethylphenyl)-2-hydroxyethoxy)-6- methyltetrahydro-2H-pyran-3,4,5-triol (Compound 101)

[0753] Step 1: Synthesis of (3R,4R,5S,6S)-2-((R)-2-(3-ethylphenyl)-2- hydroxyethoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triol triacetate

[0754] (R)-1-(3-ethylphenyl)ethane-1,2-diol (75 mg, 0.45 mmol) was dissolved in dichloromethane (5 mL), (3R,4R,5S,6S)-2-bromo-6-methyltetrahydro-2H-pyran-3,4,5-triol triacetate (317.7 mg, 0.9 mmol), silver carbonate (66 mg, 0.24 mmol) and silver trifluoromethanesulfonate (61 mg, 0.24 mmol) were added sequentially and the reaction was monitored after stirring at room temperature for 12 h. After completion of the reaction, it was filtered, rinsed with dichloromethane, concentrated under vacuum at 30 °C and purified by column chromatography to obtain the intermediate (110 mg, yield: 55.7%).

[0755] Step 2: Synthesis of (3R,4R,5R,6S)-2-((R)-2-(3-ethylphenyl)-2- hydroxyethoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triol

[0756] Intermediate (3R,4R,5S,6S)-2-((R)-2-(3-ethylphenyl)-2-hydroxyethoxy)-6- methyltetrahydro-2H-pyran-3,4,5-triol triacetate (100 mg, 0.23 mmol) was taken in dichloromethane:methanol (1:1) mixture ~ 5 mL to make it completely dissolved, K2CO3(622 mg, 4.5 mmol) was added, stirred for 15 min, reaction was monitored. After completion of reaction, it was filtered, the obtained solution was concentrated under reduced pressure and purified by column chromatography to get white sticky crystals (18 mg, yield: 25.0%).

[0757] 1 H NMR (500 MHz, CD3OD) δ 7.30-7.12 (m, 4H), 4.85-4.81 (m, 4H), 4.48-4.70 (m, 1H), 3.94-3.83 (m, 2H), 3.80-3.68 (m, 2H), 3.62-3.51 (m, 2H), 3.44-3.36 (m, 1H), 2.66 (q, J = 7.6 Hz, 2H), 1.25 (m, 6H). LC-MS: calculated for C 16 H 24 O6[M+NH4] + : 330.40, found: 330.2.

[0758] Synthesis of (3R,4R,5R,6S)-2-(3-ethylphenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran- 3,4,5-triol (Compound 102)

[0759] Step 1: Synthesis of catalyst Ag-NHCs

[0760] To a 250 mL round bottom flask, 1-benzyl-3-methylimidazolium chloride (4.44 g, 21.27 mmol) and silver oxide (2.96 g, 12.76 mmol) were taken and stirred in 80 mL of dichloromethane for 24 h at room temperature in dark condition, consumption of Ag2O was observed to confirm completion of reaction. After completion of reaction, the mixture was filtered through celite and washed with dichloromethane 3 times. The organic layer was concentrated in vacuum to get crude product which was again dissolved in dichloromethane to form near saturated solution, appropriate amount of hexane was added to get white crystals (4.3 g, yield: 75.0%).

[0761] Step 2: Synthesis of (2S,3S,4R,5R)-2-(acetoxymethyl)-6-(3-ethylphenoxy)tetrahydro-2H- pyran-3,4,5-triol triacetate

[0762] In a 100 mL reaction flask, catalyst Ag-NHCs (700 mg, 1.55 mmol), (2S,3S,4R,5R)-2-(acetyloxymethyl)-6-bromo-tetrahydro-2H-pyran-3,4,5-triol triacetate (1.4 g, 3.40 mmol) and m-ethylphenol (250 mg, 2.05 mmol) were added in sequence, with appropriate amount of dichloromethane as solvent, and reacted for about 4 h at room temperature in the dark. After the reaction was completed, it was filtered, the filtrate was concentrated, and purified using column chromatography (PE:EA = 5:2). The intermediate (2S,3S,4R,5R)-2-(acetyloxymethyl)-6-(3- ethylphenoxy)tetrahydro-2H-pyran-3,4,5-triol triacetate (640 mg, yield: 68.8%) was obtained.

[0763] Step 3: Synthesis of (3R,4R,5R,6S)-2-(3-ethylphenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0764] The synthesis method refers to the synthesis of (3R,4R,5R,6S)-2-((R)-2-(3- ethylphenyl)-2-hydroxyethoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triol, yield: 37.1%.

[0765] 1 H NMR (500 MHz, CD3OD) δ 7.19 (t, J = 7.8 Hz, 1H), 6.97-6.96 (m, 1H), 6.93-6.91 (m, 1H), 6.88-6.86 (m, 1H), 4.92-4.91 (m, 4H), 3.92-3.89 (m, 1H), 3.73-3.70 (m, 1H), 3.49-3.39 (m, 5H), 2.67 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). LC-MS: calculated C 14 H 20 O6[M+NH4] + : 302.35, found: 302.1.

[0766] Synthesis of (3R,4R,5R,6S)-2-(2,3-dihydroxy-3-(3-(trifluoromethyl)phenyl)propyl)-6- methyltetrahydro-2H-pyran-3,4,5-triol (Compound 103)

[0767] Step 1: Synthesis of (3S,4R,5S,6S)-2-allyl-6-methyl-tetrahydro-2H-pyran-3,4,5-triol triacetate

[0768] In a 100 mL two necked flask was charged with a-L-rhamnose (2 g, 6.0 mmol), anhydrous acetonitrile 25 mL, trimethylallylsilane (2.4 mL, 10.5 mmol), cooled to 0 °C in a low temperature bath and trifluoromethylsilane (1.9 mL, 9.9 mmol) was added. The reaction mixture was stirred for 0.5 h and then stirred at room temperature for 8-12 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was slowly poured into saturated NaHC03(aq) and stirred for 15 min. The reaction mixture was extracted with dichloromethane (3x). The organic layer was washed with saturated brine, dried over anhydrous Na2S04and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA = 4:1) to afford the intermediate (1.4 g, yield: 73.3%) as a pale yellow syrup.

[0769] Step 2: Synthesis of (2S,3S,4R,5S)-2-methyl-6-((E)-3-(3-(trifluoromethyl)phenyl)propenyl)tetrahydro-2H-pyran-3,4,5-triol triacetate

[0770] Step 2: Synthesis of (2S,3S,4R,5S)-2-methyl-6-((E)-3-(3-(trifluoromethyl)phenyl)propenyl)tetrahydro-2H-pyran-3,4,5-triol triacetate

[0771] Step 3: Synthesis of (3S,4R,5S,6S)-2-(2,3-dihydroxy-3-(3-(trifluoromethyl)phenyl)propyl)-6-methyltetrahydro-2H-pyran-3,4,5-triol triacetate

[0772] The intermediate from previous step (950 mg, 2.1 mmol) was completely dissolved in a mixture of t-butanol / water (1:1), and the components of Supper-AD-Mix-a (K2Os02(OH)4(7.4 mg, 0.02 mmol), (DHQ)2PHAL (156 mg, 0.2 mmol), K2C03(871 mg, 6.3 mmol) and K3Fe(CN)6(2.07 g, 6.3 mmol)) were added sequentially, and the mixture was made up to volume with t-butanol / water (1:1) and stirred at room temperature overnight. The reaction was monitored by TLC and LCMS. The reaction mixture was used as such for the next step without any further purification.

[0773] Step 4: Synthesis of (3R,4R,5R,6S)-2-(2,3-dihydroxy-3-(3-(trifluoromethyl)phenyl)propyl)-6- methyltetrahydro-2H-pyran-3,4,5-triol

[0774] If enough (3S,4R,5S,6S)-2-(2,3-dihydroxy-3-(3-(trifluoromethyl)phenyl)propyl)-6- methyltetrahydro-2H-pyran-3,4,5-triol triacetate is generated, the temperature is increased to 50 °C and heated for about 1 h until the solid is dissolved, 5 mL of methanol is added to quench the reaction, at this time a large amount of yellow solid precipitates, filtered, washed with methanol, the filtrate is concentrated under reduced pressure to obtain the crude product as a yellow syrup. Purification by column chromatography (DCM:MeOH = 3:1) gradient to obtain a white sticky substance (175 mg, yield: 22.8%).

[0775] 1 H NMR (500 MHz, CD3OD) δ 7.73 (s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.60-7.53 (m, 2H), 4.62 (d, J = 5.6 Hz, 1H), 4.10-4.07 (m, 1H), 3.88-3.85 (m, 1H), 3.69 (t, J = 2.9 Hz, 1H), 3.46 (dd, J = 8.9, 3.3 Hz, 1H), 3.37-3.32 (m, 5H), 3.21-3.15 (m, 1H), 1.87-1.82 (m, 1H), 1.35-1.24 (m, 2H), 1.21 (d, J = 6.2 Hz, 3H). LC-MS: Calculated for C 16 H 21 F3O6[M+H] + : 366.33, found: 366.1.

[0776] Synthesis of (2R,3R,4S,5S,6R)-2-(((2R,3S,4R,5R)-6-(1,2-dihydroxy-2-(3-(trifluoromethyl)phenyl)ethyl)- 4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H- pyran-3,4,5-triol (Compound 104)

[0777] Step 1: Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,3R,4S,5R)-4,5-diacetoxy-2- (acetoxymethyl)-6-bromotetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol triacetate

[0778] β-D-maltose octaacetate (3 g, 4.42 mmol) was dissolved in dichloromethane (20 mL) and the reaction flask was transferred to a low temperature reaction bath at 0 °C. HBr (6 mL, 33% in AcOH) was added slowly using a syringe with a long needle. After stirring for 15 min, the reaction flask was placed in a room temperature bath and stirred for 12 h. After the reaction was complete, saturated NaHC03(aq) was added until the pH was neutral. After stirring for 10 min, an appropriate amount of brine was added and the mixture was extracted with dichloromethane three times. The organic layer was washed with saturated NaHC03(aq) and NaCl (aq), the organic layer was separated and dried over anhydrous Na2S04, and concentrated in vacuo to give a white sticky solid (3.05 g, yield: 98.6%).

[0779] Step 2: Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((((2R,3R,4S,5S)-4,5- diacetoxy-2-(acetoxymethyl)-6-((E)-3-(trifluoromethyl)styryl)tetrahydro-2H-pyran-3- yl)oxy)tetrahydro-2H-pyran-3,4,5-triol triacetate

[0780] To the reaction flask was added the bromomaltose intermediate from the previous step (2.85 g, 4.07 mmol), m-trifluoromethylstyrene (0.5 mL, 3.37 mmol), K2C03(0.93 g, 6.74 mmol), Pd(Xantphos)Cl2(127 mg, 0.17 mmol), and Xantphos (117 mg, 0.20 mmol) sequentially under nitrogen protection. Then, triethylamine (0.7 mL, 5.05 mmol) and toluene (10 mL) were added. The resulting reaction solution was stirred at room temperature for 10 min, and then placed under irradiation from an LED blue light lamp (445 nm) at room temperature for 48 h. After the reaction was complete, the mixture was filtered, washed with dichloromethane, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (PE / EA = 4: 1 to 1: 1) to give a white foamy solid (1.35 g, yield: 50.4%).

[0781] Steps 3 and 4: Synthesis of (2R,3R,4S,5R,6R)-2-(acetyloxymethyl)-6-((((2R,3R,4S,5S)-4,5-diacetyloxy-2-(acetyloxymethyl)-6-(1,2-dihydroxy-2-(3-(trifluoromethyl)phenyl)ethyl)tetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol triacetate and (2R,3R,4S,5S,6R)-2-(((2R,3S,4R,5R)-6-(1,2-dihydroxy-2-(3-(trifluoromethyl)phenyl)ethyl)-4,5-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0782] Synthetic Procedure reference 3. Steps 3 and 4 for (3R,4R,5R,6S)-2-(2,3-dihydroxy-3-(3-(trifluoromethyl)phenyl)propyl)-6-methyltetrahydro-2H-pyran-3,4,5-triol. Yield: 22.4%.

[0783] 1 H NMR (500 MHz, CD3OD) δ 7.76 - 7.69 (m, 1H), 7.59 - 7.52 (m, 2H), 7.46 - 7.43 (m, 1H), 5.19 - 5.14 (m, 2H), 4.85 - 4.65 (m, 4H), 4.33 - 4.26 (m, 1H), 4.18 - 4.10 (m, 2H), 4.07 - 4.01 (m, 1H), 3.95 - 3.80 (m, 5H), 3.77 - 3.75 (m, 1H), 3.70 - 3.63 (m, 5H), 3.59 - 3.53 (m, 1H), 3.50 - 3.47 (m, 1H), 3.45 - 3.42 (m, 1H), 2.96 - 2.86 (m, 1H). LC-MS: Calculated for C 21 H 29 F3O 12 [M+NH4] + : 548.49, found: 548.2.

[0784] Synthesis of (5R,6S)-2-((3-ethylphenyl)(hydroxy)methyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (Compound 105)

[0785] Step 1 : Synthesis of ((2S,3R)-3-acetyloxy-6-hydroxy-3,6-dihydro-2H-pyran-2-yl)methyl acetate

[0786] A 500 mL single neck flask was wrapped in tin foil to protect from light, 20 mL of deionized water was added, and after boiling (2S,3R,4S)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4- diene diacetate (1 g, 1.8 mmol) was added along with THF (1 mL). Stirring was continued in the dark for about 1 h, and the reaction was monitored by TLC. After the reaction was complete, the water was removed by concentration under reduced pressure to give a yellow oil.

[0787] Step 2: Synthesis of ((2S,3R)-3-acetoxy-6-((tert-butoxycarbonyl)oxy)-3,6- dihydro-2H-pyran-2-yl)methyl acetate

[0788] The yellow oil from the previous step was dissolved in dichloromethane (20 mL), and di-tert-butyl dicarbonate (456 μL, 2.0 mmol), triethylamine (278 μL, 2.0 mmol), and DMAP (11 mg, 0.09 mmol) were added sequentially. After the reaction was complete, as monitored by TLC, the solvent was removed by concentration under reduced pressure. The crude product was purified by column chromatography (PE / EA = 3: 1) to give ((2S,3R)-3-acetoxy-6-((tert-butoxycarbonyl)oxy)-3,6-dihydro-2H-pyran-2- yl)methyl acetate (462 mg, two-step yield: 77.8%).

[0789] Step 3: Synthesis of ((2S,3R)-3-acetoxy-6-(((3-ethylphenyl)(hydroxy)methyl)-3,6- dihydro-2H-pyran-2-yl)methyl acetate

[0790] Copper(I) chloride (22.5 mg, 0.23 mmol), 1,3-bis(2,4,6-trimethylphenyl)imidazolium chloride (L1) (78 mg, 0.23 mmol), sodium tert-butoxide (0.87 g, 9.1 mmol), and Molecular sieves (1.5 g) were placed in a two-neck flask with a magnetic stir bar, and the flask was flushed with nitrogen. Tetrahydrofuran (0.4 mL) was added to the flask, and the mixture was stirred at 25 °C for 10 min to prepare an active catalyst. Bis(pinacolato)diboron (2.31 g, 9.1 mmol) and ((2S,3R)-3-acetoxy-6-((tert-butoxycarbonyl)oxy)-3,6-dihydro-2H-pyran-2- yl)methyl acetate (1.5 g, 4.55 mmol) were dissolved in 15 mL of tetrahydrofuran, and the mixture was transferred into the flask containing the catalyst under a nitrogen atmosphere. The mixture was stirred at room temperature overnight to produce an intermediate.

[0791] To the reaction mixture was added 3-ethylbenzaldehyde (610 mg, 4.55 mmol) and the temperature was raised to 50 °C and stirred for 12 h. The reaction was quenched by the addition of 10% triethanolamine in dichloromethane (1 mL) and after stirring for 10 min, the mixture was filtered through celite, rinsing with dichloromethane and the resulting filtrate was concentrated under reduced pressure and purified by column chromatography to give intermediate ((2S,3R)-3-acetoxy-6-(((3-ethylphenyl)(hydroxy)methyl)-3,6-dihydro-2H-pyran-2-yl)methyl acetate (840 mg, 53.0% yield).

[0792] Synthesis of ((2S,3R)-3-acetoxy-6-((3-ethylphenyl)(hydroxy)methyl)-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate and (5R,6S)-2-((3-ethylphenyl)(hydroxy)methyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0793] Synthesis Method reference 3. Step 3 and 4 of (3R,4R,5R,6S)-2-(2,3-dihydroxy-3-(3-(trifluoromethyl)phenyl)propyl)-6-methyltetrahydro-2H-pyran-3,4,5-triol. Two step yield: 8.3%.

[0794] 1 H NMR (500 MHz, CD3OD) δ 7.33-7.17 (m, 4H), 4.97-4.91 (m, 1H), 4.86-4.87 (m, 4H), 4.14-4.13 (m, 1H), 4.00-3.98 (m, 1H), 3.77-3.63 (m, 3H), 3.52-3.48 (m, 2H), 3.30 (s, 1H), 2.67 (q, J = 7.5 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). LC-MS: Calculated for C 15 H 22 O6[M+NH4] + : 316.37, found: 316.2.

[0795] Synthesis of (5R,6S)-2-(hydroxy(quinolin-3-yl)methyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (Compound 106)

[0796] Synthesis Method reference 5. Synthesis of (5R,6S)-2-((3-ethylphenyl)(hydroxy)methyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol. Yield: 12.7%.

[0797] 1H NMR (500 MHz, CD3OD) δ 8.55 (d, J = 3.9 Hz, 1H), 8.16-8.13 (m, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.58-7.57 (m, 2H), 7.43 (dd, J = 7.6, 2.2 Hz, 1H), 5.22-5.19 (m, 1H), 4.82-4.83 (m, 5H), 3.94-3.87 (m, 3H), 3.49-3.43 (m, 2H), 3.26-3.30 (m, 1H), 3.11-3.07 (m, 1H). LC-MS: calculated for C 16 H 19 NO6[M+H] + : 322.34, found: 322.1.

[0798] Synthesis of (3R,4R,5S,6R)-2-(3-((R)-1,2-dihydroxyethyl)phenyl)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (Compound 107)

[0799] Step 1: Synthesis of (3R,4S,5R,6R)-3,4,5-tri((trimethylsilyl)oxy)-6- ((trimethylsilyl)oxymethyl)-2-(3-vinylphenyl)tetrahydro-2H-pyran-2-ol

[0800] Under nitrogen, 3-bromo-styrene (1 mL, 7.68 mmol) and 2,3,4,6-tetra-O- trimethylsilyl-D-gluconolactone (4.8 g, 10.28 mmol) were dissolved in a mixture of tetrahydrofuran / toluene (1:2) and placed in a -78 °C cold bath. n-BuLi (2.5 mol / L, 3 mL, 7.5 mmol) was added slowly with a long needle and the reaction was stirred at -78 °C for about 2 h. After completion of the reaction, the reaction was allowed to warm to room temperature and ice water was added. The reaction was extracted with ethyl acetate three times and the organic layer was dried over sodium sulfate. The crude product was used directly in the next step without purification.

[0801] Step 2: Synthesis of (2R,3S,4R,5R)-2-(hydroxymethyl)-6-(3-vinylphenyl)tetrahydro- 2H-pyran-3,4,5-triol

[0802] To triethylsilane (2.3 mL, 14.44 mmol) was added a suitable amount of acetonitrile, cooled to -40 °C in a low temperature bath, then boron trifluoride etherate (1.3 mL, 10.83 mmol) was slowly added, followed by (3R,4S,5R,6R)-3,4,5-tris((trimethylsilyl)oxy)-6- ((trimethylsilyl)oxymethyl)-2-(3-vinylphenyl)tetrahydro-2H-pyran-2-ol dissolved in dichloromethane. The reaction was slowly allowed to warm to room temperature. After the reaction was complete, sodium bicarbonate was added as a solid (no water) and the reaction was adjusted to neutral pH, washed with dichloromethane, filtered, and the filtrate was concentrated to a yellow gummy material.

[0803] Step 3: Synthesis of (2R,3R,4R,5S)-2-(acetyloxymethyl)-6-(3-vinylphenyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate

[0804] The intermediate from the previous step (730 mg, 2.74 mmol) was dissolved in a suitable amount of dichloromethane and cooled to 0 °C. Dry pyridine (0.2 mL) and acetic anhydride (1.6 mL, 16.4 mmol) were added sequentially, and the reaction was stirred at room temperature for about 6 h. After the reaction was complete, the reaction was quenched with 10% NaHC03solution and extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous Na2S04, and the solvent was removed under reduced pressure. Column chromatography gave (2R,3R,4R,5S)-2-(acetyloxymethyl)-6-(3-vinylphenyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (1.12 g, yield: 94.1%).

[0805] Steps 4 and 5: Synthesis of (2R,3R,4R,5S)-2-(acetyloxymethyl)-6-(3-((R)-1,2- dihydroxyethyl)phenyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate and (3R,4R,5S,6R)-2-(3- ((R)-1,2-dihydroxyethyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0806] Synthesis was carried out according to the procedure described in Step 3 and 4 of the synthesis of (3R,4R,5R,6S)-2-(2,3-dihydroxy-3-(3-(trifluoromethyl)phenyl)propyl)-6- methyltetrahydro-2H-pyran-3,4,5-triol. Two step yield: 15.3%.

[0807] 1H NMR (500 MHz, CD3OD) δ 7.60-7.57 (m, 2H), 7.44 (dd, J = 9.25, 2.7 Hz, 1H), 7.10-7.09 (m, 1H), 4.86-4.81 (m, 6H), 4.74-4.70 (m, 1H), 3.92-3.86 (m, 5H), 3.67-3.62 (m, 2H), 3.45-3.39 (m, 2H). LC-MS: Calculated for C 14 H 20 O7[M+NH4] + : 318.35, found: 318.2.

[0808] Synthesis of (3R,4R,5S,6R)-2-(3-((R)-1,2-dihydroxyethyl)phenyl)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (Compound 108)

[0809] Synthetic procedure refers to the synthesis of (3R,4R,5S,6R)-2-(3-((R)-1,2- dihydroxyethyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (Compound 108) (Yield: 10.4%).

[0810] 1 H NMR (500 MHz, CD3OD) δ 7.53-7.09 (m, 4H), 4.86-4.80 (m, 4H), 4.64-4.85 (m, 1H), 4.17-3.77 (m, 3H), 3.73-3.52 (m, 2H), 3.40-3.37 (m, 1H), 2.66 (q, J = 7.7 Hz, 2H), 1.24 (t, J = 7.5 Hz, 3H). LC-MS: Calculated for C 14 H 20 O5[M+NH4] + : 286.35, found: 286.2.

[0811] Synthesis of (R)-1-(3-(morpholinomethyl)phenyl)ethane-1,2-diol (Compound 109)

[0812] Into a 500 mL two-necked flask, 122A (10.8 mmol, 2.00 g), potassium ethylene trifluoroborate (12.96 mmol, 1.74 g), DPPF palladium dichloride (1.08 mmol, 0.8 g) and potassium carbonate (21.6 mmol, 3.00 g) were added successively, 140 mL of a mixture of dioxane and water (6:1) was used as solvent, nitrogen was replaced for 3 times, the reaction was carried out at 85 °C for 2 h, and TLC was used to detect the progress of the reaction. After the reaction was completed, dioxane was removed by rotary evaporation, EA and H2O were used to extract three times, the organic phase was concentrated, and column chromatography was used to separate to obtain the corresponding intermediate 122B (yellowish oil liquid, 1.42 g, 78%).

[0813] Into a 100 mL round-bottom flask, 122B (8.61 mmol, 1120 mg), morpholine (17.22 mmol, 1.5 mL), NaBH(OAc)3 (10.76 mmol, 2281 mg), acetic acid 0.2 mL were added successively, 100 mL of DCM was used as solvent, the reaction was carried out at room temperature for 2 h, and TLC was used to detect the progress of the reaction. After the reaction was completed, 30 mL of saturated sodium bicarbonate solution was used to quench the reaction, the organic phase was concentrated by rotary evaporation after extraction and separation, and column chromatography was used to separate to obtain the corresponding intermediate 122C (yellow oil liquid, 1269 mg, 72%). + .

[0814] Into a 100 mL single-necked flask, 122C (6.15 mmol, 1.25 g) was added, AD-mix-β (9.0 mmol, 6996 mg) was added under ice bath conditions, 1:1 tert-butanol and water were used as solvent, and the reaction was carried out at room temperature overnight. After the reaction was completed, EA and H2O were used to extract three times, the organic phase was concentrated, and column chromatography was used to separate to obtain the final product 122 (white solid, 570 mg, 45.6%)

[0815] LC-MS (ESI): m / z: 238.1 [M+H] + .

[0816] 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 4.5 Hz, 1H), 8.41 (dt, J = 7.2, 3.6 Hz, 1H), 8.14 (dt, J = 6.1, 3.6 Hz, 1H), 7.93 (d, J = 9.1 Hz, 1H), 5.22 (d, J = 4.2 Hz, 1H), 4.71 (t, J = 5.8 Hz, 1H), 4.53 (q, J = 5.6 Hz, 1H), 3.86 (s, 2H), 3.57 (t, J = 4.6 Hz, 2H), 3.44 (s, 1H), 3.42 (t, J = 5.8 Hz, 1H), 2.71 (s, 2H), 2.35 (d, J = 4.6 Hz, 2H).

[0817] 13 C NMR (151 MHz, DMSO-d6) δ 144.39, 143.84, 128.10, 127.33, 125.46, 121.99, 74.28, 66.65, 63.08, 53.65, 49.64, 25.02, 12.16.

[0818] Synthesis of (R)-1-(4-ethylpyridin-2-yl)ethan-1,2-diol (Compound 110)

[0819] Step 1 Synthesis of 4-bromo-2-vinylpyridine (V42-2)

[0820] Into a 250 ml two-necked flask was placed methyltriphenylphosphonium iodide (12.90 mmol, 5.22 g), the flask was protected by nitrogen to remove oxygen, injected anhydrous tetrahydrofuran solution (50 mL), potassium tert-butoxide (15.48 mmol, 15.48 mL 1M) was added under low temperature (-10 ℃) and stirring, the reaction was carried out for 1 h, after the reaction was completed, V42-1 (10.75 mmol, 2.00 g) was added, the reaction was carried out for 2 h at room temperature, the reaction progress was detected by TLC. After the reaction was completed, it was extracted with ethyl acetate and water for three times, the organic phase was combined and rotary evaporated, and was separated and purified by automatic column machine to give V42-2 (white solid, 196 mg, yield 19.9%). 1 H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 5.3 Hz, 1H), 7.82 (d, J = 1.8 Hz, 1H), 7.56 (dd, J = 5.3, 1.9 Hz, 1H), 6.80 (dd, J = 17.4, 10.8 Hz, 1H), 6.32 (dd, J = 17.4, 1.5 Hz, 1H), 5.55 (dd, J = 10.8, 1.5 Hz, 1H).

[0821] Step 2 Synthesis of 4-ethyl-2-vinylpyridine (V42-3)

[0822] Into a 250 mL two-necked flask, V42-2 (1.07 mmol, 196 mg), DPPF palladium dichloride (0.11 mmol, 81 mg), cesium carbonate (6.39 mmol, 2.08 g), nitrogen protection, injection of triethyl boron (1.28 mmol, 1.28 mL 1M), anhydrous tetrahydrofuran as solvent, 55 °C reaction, TLC detection of reaction progress. After the reaction is completed, extract with ethyl acetate and water three times, combine the organic phase and rotary evaporation, use automatic column separation and purification, get V42-3 (yellow transparent oil, 60 mg, yield 42.1%). 1 H NMR (400 MHz, Chloroform-d) δ 8.45 (d, J = 5.1 Hz, 1H), 7.19 (d, J = 1.6 Hz, 1H), 7.01 (dd, J = 5.1, 1.7 Hz, 1H), 6.81 (dd, J = 17.5, 10.8 Hz, 1H), 6.20 (dd, J = 17.5, 1.3 Hz, 1H), 5.47 (dd, J = 10.8, 1.3 Hz, 1H), 2.65 (q, J = 7.6 Hz, 2H), 1.26 (t, J = 7.6 Hz, 3H).

[0823] Synthesis of (R)-1-(4-ethylpyridin-2-yl)ethane-1,2-diol (V42)

[0824] Into a 100 mL single-necked flask, V42-3 (0.45 mmol, 60 mg), AD-mix-β (0.85 mmol, 0.66 g) was added under ice bath conditions, 1:1 tert-butyl alcohol and water as solvent, room temperature reaction overnight. After the reaction is completed, extract with ethyl acetate and water three times, combine the organic phase and rotary evaporation, column chromatography (PE:EA = 1:1), get V42-4 (white solid, 20 mg, yield 26.6%).

[0825] 1 H NMR (500 MHz, Chloroform-d) δ 8.40 (s, 1H), 7.20 (s, 1H), 7.08 (d, J = 4.8 Hz, 1H), 4.81 (t, J = 4.6 Hz, 1H), 4.00 - 3.87 (m, 1H), 3.85 - 3.69 (m, 1H), 2.67 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H).

[0826] Synthesis of 1-(3-ethyl-2-methylphenyl)-2-hydroxyethanone (Compound 111)

[0827] Step 1 Synthesis of 2-bromo-l-(3-ethyl-2-methylphenyl)ethanone (V57-2)

[0828] Into a 50 ml single necked flask, 57-1 (0.62 mmol, 100 mg), p-toluenesulfonic acid (0.31 mmol, 115 mg) and appropriate amount of acetonitrile were added under ice-bath, stirred to dissolve, N-bromosuccinimide was added slowly into the reaction solution, 40 °C reaction, TLC detection of reaction progress. When the reaction was completed, sodium sulfite and N-bromosuccinimide were used to prevent the formation of bromine solution. Extracted with ethyl acetate and water three times, combined organic phase was added to saturated brine, the organic phase was dried over anhydrous sodium sulfate and rotary evaporated to give intermediate 57-2 (yellow oil), the product was directly used in the next step.

[0829] Step 2 Synthesis of l-(3-ethyl-2-methylphenyl)-2-hydroxyethanone

[0830] Into a 50 ml single necked flask, 57-2 (0.62 mmol, 150 mg) generated in the previous step, sodium formate (3.52 mmol, 240 mg) were added, appropriate amount of methanol was added to dissolve, heated to reflux at 80 °C, TLC detection of reaction progress. When the reaction was completed, extracted with ethyl acetate and water three times, combined organic phase was added to saturated brine, the organic phase was dried over anhydrous sodium sulfate and rotary evaporated, column chromatography (PE:EA = 5: 1) to give 57-3 (pale yellow solid, 45 mg, yield 40.7%).

[0831] 1 H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 7.4 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 5.17 (t, J = 6.0 Hz, 1H), 4.52 (d, J = 6.0 Hz, 2H), 2.64 (q, J = 7.5 Hz, 2H), 2.24 (s, 3H), 1.14 (t, J = 7.5 Hz, 3H). 13 C NMR (151 MHz, Chloroform-d) δ 203.27, 144.53, 136.00, 135.14, 132.40, 125.76, 125.45, 67.32, 26.71, 15.81, 14.51

[0832] Synthesis of (S)-3-(3-ethylphenoxy)propane-l,2-diol (Compound 112)

[0833] In a 100 ml single neck flask, 4-a (500 mg, 4.09 mmol), (S)-3-chloro-1,2-propanediol (905 mg, 8.19 mmol) and anhydrous potassium carbonate (4.53 g, 32.76 mmol) were added, 20 mL of anhydrous DMF was injected, and the reaction was stirred at 100°C. After the reaction was completed, it was extracted with ethyl acetate and water three times, the organic phase was combined, and after adding anhydrous sodium sulfate to remove water, it was concentrated by rotary evaporation, and the product was separated by column chromatography (DCM:MeOH = 10:1) to obtain compound 4S (yellow transparent oil, 276 mg, yield 34.4%).

[0834] 1H NMR (600 MHz, Methanol-d4) δ 7.15 (t, J = 7.8 Hz, 1H), 6.82 - 6.76 (m, 2H), 6.76 - 6.72 (m, 1H), 4.06 - 4.00 (m, 1H), 3.99 - 3.91 (m, 2H), 3.72 - 3.66 (m, 1H), 3.66 - 3.61 (m, 1H), 2.60 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H).

[0835] 13C NMR (151 MHz, Methanol-d4) δ 160.49, 147.12, 130.30, 121.45, 115.22, 112.70, 71.86, 70.18, 64.23, 29.90, 16.13. HPLC Purity: 92.63%, retention time: 3.813 min

[0836] ee value test method: The ee value analysis of all final compounds was carried out on Agilent 1260 infinity series, using CHIRALCEL OD-H, 4.6 mm x 250 mm, with a flow rate of 0.5 mL / min, and 90% n-hexane / 10% ethanol as the solvent.

[0837] Experimental Example 2. Cryo-EM electron microscopy experiment obtains TRPV4- compound 1-R complex structure

[0838] In this study, we obtained a high-resolution cryo-EM structure of human TRPV4 with a novel selective inhibitor compound 1-R (PDB ID: 8W82, resolution ) The structure captures TRPV4 in a "Closed" conformation. We found that compound 1-R is located in a novel binding pocket of TRPV4. Through structure comparison, long-range molecular dynamics simulation and single / multiple point mutation experiments, we elucidated the unique allosteric gating mechanism of compound 1-R.

[0839] Materials and Methods

[0840] 1.1 Expression and purification of MBP-TRPV4 protein

[0841] Human TRPV4 protein (amino acids: 148 to 787) was cloned into pEGBacMam vector. The resulting protein contains a maltose binding protein tag at its N-terminus. P4 baculovirus was produced in Bac-to-Bac baculovirus expression system (Invitrogen) Sf9 cells. HEK293F cells were infected with 10% (v / v) P4 baculovirus at a density of 2.0-3.0 x 10 6 cells / ml to express the protein at 37°C. After 12 to 18 hours, 10 mM sodium butyrate was added and the temperature was reduced to 30°C. Cells were collected within 72 hours post-transfection and frozen at -80°C.

[0842] Prior to solubilization, cells were suspended in a buffer containing 50 mM Hepes, 150 mM NaCl, pH 7.5, and EDTA-free protease inhibitor cocktail, and cell membranes were disrupted by dounce homogenization. Lysed cell membranes were collected by ultracentrifugation at 40,000 rpm for 60 minutes, and the pellet was resuspended and homogenized by dounce in a buffer containing 1.0% (wt / vol) N-dodecyl- -D-maltopyranoside (DDM; Anatrace), 0.1% (wt / vol) Cholesterol hemisuccinate (CHS; Anatrace), 50 mM Hepes, pH 7.5, 150 mM NaCl, pH 7.5, EDTA-free protease inhibitor cocktail. Protein solubilization was performed for 3 hours at 4°C, followed by ultracentrifugation at 40,000 rpm for 60 minutes. After centrifugation, the soluble supernatant was incubated with maltose resin (Bio Labs) for 4 hours at 4°C. The resin was eluted with 50 mM Hepes, 150 mM NaCl, 0.05% (wt / vol) GDN (Anatrace). The protein was eluted with six column volumes of 50 mM Hepes, 150 mM NaCl, 0.05% (wt / vol) GDN, and 40 mM maltose. To remove the MBP tag on TRPV4, recombinant human rhinovirus (HRV 3C) protease was added, and the reaction was left overnight at 4°C. Finally, the protein was further purified by a Superose 6 gel exclusion column (Cytiva) in 50 mM Hepes, 150 mM NaCl, pH 7.5, 0.05% (w / v) GDN after concentration to 1.0 mL using a 100 kDa molecular weight cut-off concentrator (Millipore). Peak fractions were collected and concentrated to 5 mg / mL for preparation of Cryo-EM samples.

[0843] 1.2 Cryo-EM analysis of TRPV4-Compound 1-R complex

[0844] 18.3 microliters of purified hTRPV4 protein at a concentration of 5.5 mg / mL was incubated with 2.0 microliters of Compound 1-R at a concentration of 0.25 mg / mL at a 1:20 molar ratio on ice for 40 minutes in order to proceed to the next step of centrifugation (16200 g, 4°C, 5 minutes). Then, the TRPV4-Compound 1-R complex sample was used to prepare Cryo-EM grids.

[0845] 4 pL of protein sample of TRPV4-Compound 1-R complex was added to glow-discharged 300 mesh grids (Quantifoil Au R1.2 / 1.3), blotted for 3.0 seconds and 3 times with filter paper. Subsequently, rapid freezing was performed using liquid ethane at 8°C and 100% humidity using a Thermo Fisher Vitrobot Mark IV. Cryo-EM grids were stored in liquid nitrogen before data collection. Cryo-EM micrographs were collected using a 300 kV Thermo Fisher Titan Krios G3i electron microscope, equipped with a K3 direct-detection camera and a BioContinuum energy filter (GIF, slit width of 20 eV). These micrographs were collected at a calibrated magnification of 64,000x, with a pixel size of 0.86 A at the specimen level, in super-resolution mode. A total of 15,646 micrographs were collected, with a cumulative electron dose of Stacked in 32 frames, with a defocus range of -1.0 pm to -2.0 pm. Exposure time and dose rate were 2.6 seconds and approximately 22 e-pixel-1s-1, respectively.

[0846] Beam-induced motion correction of frame stacks was performed using MotionCorr2. Contrast transfer function (CTF) parameters were determined by CTFFIND4. Further data processing was performed on the co-selected 15,646 good micrographs using CryoSPARC. These micrographs were screened and suboptimal data were removed, leaving 13,043 micrographs. In CryoSPARC, automatic particle picking was performed using the blob picker and template picker programs, followed by 3 rounds of 2D classification without reference. Next, 1,751,252 particles were selected from good 2D classes and subjected to 3 rounds of multi-reference 3D classification using models generated from traditional 3D classification. From each round of multi-reference 3D classification, a converged 3D class of TRPV4 features was selected and duplicate particles were removed. The final heterogeneity refinement was performed, selecting 1,040,133 particles from the 3D class showing the highest resolution features, and performing one round of 3D refinement with C4 symmetry, resulting in a global resolution of at the FSC = 0.143 gold standard Fourier shell correlation criterion. Local resolution was then calculated on the final map.

[0847] The model of the TRPV4-Compound 1-R complex was built by fitting the AlphaFold2-predicted complex structure to the density map in UCSF Chimera, followed by manual modeling of the complex in COOT and structure optimization in PHENIX.

[0848] Results and Discussion

[0849] In this study, we obtained a high-resolution cryo-EM structure of human TRPV4 with a novel selective inhibitor, Compound 1-R (PDB ID: 8W82, resolution ). From the 2D class averages, the high-quality cryo-EM data collected clearly showed well-resolved secondary structure elements. The four-fold rotational symmetry (C4) 3D reconstruction resulted in the TRPV4-Compound 1-R EM structure. For each subunit in the TRPV4-Compound 1-R homotetramer, we built an accurate model of 148-787 residues, excluding the S5-P region (residues 639-654), which is a loop that could not be clearly resolved in the cryo-EM density.

[0850] Based on this density map, we constructed a three-dimensional structural model of TRPV4-Compound 1-R, revealing that TRPV4 adopts a typical tetrameric assembly of TRP channels, consisting of a central transmembrane domain (TMD) flanked by extracellular and intracellular domains. Within the TMD, there are six transmembrane helices (S1-S6), with the TRP helices oriented parallel to the membrane, and several adjacent helices in the elbow and pore regions. S1-S4 form an α-helical VSLD bundle, while S5-S6 extend outward from the S1-S4 bundle and interact with adjacent monomers to form the pore domain through domain exchange. The adjacent intracellular skirt domain is composed of ankyrin repeat domains (ARDs), an N-terminal helical region, and a C-terminal β-sheet coiled domain. ARDs oligomerize with the β-pleated regions of adjacent monomers, including one at the C-terminus and two in the linker domain between the ARD and S1. The overall structure of TRPV4-compound 1-R is similar to the previously reported TRPV4-4α-PDD structure.

[0851] Compound 1-R binding induces a significant conformational rearrangement within the pore, leading to a conformational closure of the ion channel. Compared to the “open” structure of TRPV4 (PDB:7AA5), in our TRPV4-compound 1-R structure, the lower gate region of the ion permeation pathway has significant changes in both pore shape and electrostatic potential. The S6 helix rotates clockwise, and the gating M718 shifts toward the central pore, resulting in a decrease in pore diameter. Notably, M718 rotates nearly 100° and extends into the channel lumen, forming a hydrophobic seal whose diameter changes from the open state to the Significantly reduced to closed state Therefore, in the closed state, the pore radius at M718 decreases and is shorter than the narrowest region (I715) in the open state ( Compare ), effectively preventing the passage of cations. Therefore, Cyro-EM captured the TRPV4-compound 1-R structure in a "Closed" state.

[0852] Within the transmembrane helical region, there are four well-defined "C"-shaped densities, comparable in size to the compound 1-R molecule. Compound 1-R is wedged in the cleft between these helices, and the ligand matches well with the shape of this independent density. This is because we refer to the ligands with hydroxyl and phenylethyl cores as "tails" and "heads," respectively, with the hydrophobic "head" pointing toward the pore domain. From the high-resolution density map, it can be seen that the binding of compound 1-R is stabilized by extensive hydrogen bonding and salt bridge contacts, as well as hydrophobic interactions. Within the binding pocket, polar residues play an important role in attracting the hydrophilic "tail" of compound 1-R by forming hydrogen bonds and salt bridge interactions. Hydrophobic residues help stabilize the phenylethyl group ("head").

[0853] Example 3: Point mutation experiment to verify the binding site (mutation of key amino acid residues to verify binding)

[0854] Electrophysiological detection analysis:

[0855] Electrophysiological detection of hTRPV4 was performed using stable cell lines. These stable cell lines were established based on the HEK293 standard cell line, which was provided by ICE Bioscience (Beijing, China).

[0856] Electrophysiological detection of hTRPV4 mutations was performed using transiently transfected HEK-293T cells. The full-length sequence of hTRPV4 cDNA (NM_021625.5) was amplified using HEK293 cDNA as a template. The cDNA of hTRPV4 was subcloned into the pcDNA3.1 vector (Invitrogen). All hTRPV4 mutants were generated by site-directed mutagenesis. All mutant plasmids used in this study were verified by DNA sequencing.

[0857] Whole-cell patch-clamp recordings were performed at room temperature using a HEKA EPC10 amplifier and PatchMaster software (HEKA Harvard, Holliston, Church Hill, TN, USA). Currents were filtered at 2 kHz and sampled at 10 kHz. For ramp recordings, the pipette solution contained 50 mM CsCl, 10 mM NaCl, 10 mM HEPES, 60 mM CsF, and 20 mM EGTA (buffered with CsOH to pH 7.2), and the bath solution contained 140 mM NaCl, 3.5 mM KCl, 1 mM MgCl2·6H2O, 2 mM CaCl2·2H2O, 10 mM D-Glucose, 10 mM HEPES, 1.25 mM NaH2PO4·2H2O (buffered with NaOH solution to pH 7.4). Cells were held at 0 mV, and then ramped from -100 mV to +100 mV every 5 seconds.

[0858] Dose-response curves were fitted using the Hill equation Y = 1 / (1 + 10^((LogIC 50 -X)*HillSlope)), and IC 50 Calculations and curve fitting were performed using GraphPad Prism software.

[0859] All recordings were performed at room temperature, and all compounds were pumped into the bath at the same rate. All agonists were purchased from Sigma-Aldrich.

[0860] The results of the point mutation test on TRPV4 are shown in Table 1. Compound 1-R inhibits TRPV4 with an IC 50 The value is 2.27 μM.

[0861] Table 1: Electrophysiological results of TRPV4 point mutation experiments

[0862] Discussion:

[0863] In this study, by mutating the amino acids around the binding site of compound 1-R, it can be seen that the binding of compound 1-R to TRPV4 has changed significantly, thus verifying the binding site of compound 1-R to TRPV4.

[0864] Example 4: Study on the antagonistic activity of compound 1-R derivatives on TRPV4

[0865] The in vitro antagonistic effect experiment of the compound provided by the present application on TRPV4 is carried out as follows, and all compounds are determined for antagonistic activity at a concentration of 5 μM.

[0866] In order to determine the inhibitory activity of 112 compounds, lead compounds and positive drug HC067047 on calcium flow caused by GSK101 activating TRPV4 in HEK-293T cells.

[0867] I. Experimental principle:

[0868] Calcium 6 is a cell-permeable fluorescent dye that enters cells in the form of acetyl methyl ester (AM) and is retained after hydrolysis by intracellular esterases, ensuring that the dye is distributed in the cytoplasm and specifically binds to free calcium ions (Ca2+). When combined with Ca2+, its fluorescence intensity increases significantly (usually at an excitation wavelength of 488 nm and an emission wavelength of 525 nm), with fast response speed, suitable for dynamic detection.

[0869] FLIPR is a microplate-based fluorescence imager that can simultaneously detect real-time fluorescence signals of cells in multiple wells. Excitation and detection: excite Calcium 6 by a specific wavelength (such as 488 nm laser) and continuously record the change in emission fluorescence intensity, with a time resolution of seconds, which can continuously monitor the calcium transient caused by stimulation (such as drugs, ligands), and generate a time-fluorescence intensity curve.

[0870] II. Experimental steps:

[0871] 1. Inoculate HEK-293T cells in a 10 cm cell culture dish, and when the adhesion is about 80%, transfect 8000 ng of TRPV4 overexpression plasmid for 24 h;

[0872] 2. Count cells and dilute to 5000 cells per well in 25μl volume into 384 well plates and allow to adhere;

[0873] 3. Add 25μl dye and incubate for 2h in incubator;

[0874] 4. Dilute 112 compounds of interest to 5mM in DMSO and dilute again in HHBS buffer to a final concentration of 5μM for 30min;

[0875] 5. Dilute GSK101 to 10mM in DMSO and dilute again in HHBS to a final concentration of 5.04μM in a new clear 384 well plate.

[0876] 6. After baseline measurement by Flipr, pipette GSK101 into cells by robotic arm and measure the change in fluorescence intensity over time.

[0877] 7. Select the peak value of the calcium signal curve as the detection standard for drug inhibition effect (n=3).

[0878] Table 2: Antagonistic activity of aryl vicinal diols on TRPV4 (5μM inhibition of calcium signal, 1-550 for decrease of calcium signal after agonism is (+), 551-1100 for decrease of calcium signal after agonism is (++), 1101-1650 for decrease of calcium signal after agonism is (+++), 1651-2200 for decrease of calcium signal after agonism is (++++), 2201-2750 for decrease of calcium signal after agonism is (+++++), NS for Not Significant means that the compound has no significant inhibition on calcium signal)

[0879] Example 4: Selectivity experiment of compound 1-R on TRP family

[0880] Electrophysiological detection analysis:

[0881] hTRPV4, hTRPV1, hTRPA1 and hTRPM8 were detected by electrophysiology using stable cell lines. These stable cell lines were established based on HEK293 standard cell line, which was provided by ICE Bioscience (Beijing, China).

[0882] The rest of the experimental procedures are the same as Example 2.

[0883] IC of compound Compound 1-R inhibiting TRPV4 50 The IC values of the compound 1-R to other proteins of the TRP family (TRPV1, TRPA1, TRPV3 and hTRPM8) are all greater than 100 μM, showing good family selectivity. 50 The IC values of the compound 1-R to other proteins of the TRP family (TRPV1, TRPA1, TRPV3 and hTRPM8) are all greater than 100 μM, showing good family selectivity.

[0884] Discussion:

[0885] In this study, through electrophysiological in vitro experiments on other proteins of the TRP family (TRPV1, TRPA1, TRPV3 and hTRPM8), it was found that the compound 1-R is a TRPV4 selective antagonist.

[0886] Example 5: Treatment effect of compound 1-R on lipopolysaccharide (LPS) induced lung inflammation

[0887] I) Grouping:

[0888] 1. Lipopolysaccharide (LPS) induced lung inflammation model

[0889] 1) Control group NT;

[0890] 2) Operation group LPS;

[0891] 3) Operation group + positive drug group (Nintedanib)

[0892] 4) Operation group + positive drug group (Pirfenidone)

[0893] 5) Operation group + to-be-tested drug 1

[0894] 6) Operation group + to-be-tested drug 10

[0895] 7) Operation group + to-be-tested drug 50

[0896] II) Purchase of mice

[0897] Requirements: SPF level C57 male mice, 6-8 weeks old;

[0898] Number: 60;

[0899] Purchasing company: Shanghai Slek Experimental Animal Co., Ltd.;

[0900] III) Purchase of reagents

[0901] 1. Lipopolysaccharide (LPS)

[0902] 1) Total amount: 10 mg / kg per mouse, mouse weight about 25 g, a total of 50 mice, a total of 12.5 mg

[0903] IV) Construction of model

[0904] 1. Model construction method:

[0905] 1) Lipopolysaccharide (LPS) induced lung inflammation model

[0906] Lipopolysaccharide (LPS) induction, intraperitoneal injection of LPS solution at 5 mg / kg body weight three times.

[0907] 2. Administration method:

[0908] After modeling for two weeks, administration by water feeding method was started, and then administration was continued for two weeks.

[0909] C57 male mice were intraperitoneally injected with lipopolysaccharide solution at a dose of 5 mg / kg three times to induce a lung inflammation model, and after modeling for two weeks, administration by water feeding method was started, and administration was continued for two weeks. The groups were divided into control group, surgery group, surgery group + nintedanib positive drug group, surgery group + compound 1R 1 mg / kg group, surgery group + compound 1R 10 mg / kg group, and surgery group + compound 1R 50 mg / kg group. The experimental results show that the positive drug and the three dose treatment groups of compound 1R have significant differences in the index of enhanced expiratory pause (Penh) compared with the surgery group, and the treatment effects are comparable; the three dose treatment groups of compound 1R have significant differences in the index of minute ventilation (MV) compared with the surgery group.

[0910] Example 6. Evaluation of the activity of the compound in heart failure (animal experiment)

[0911] 6.1, Compound 1-R (1R) and Compound 1-S (1S) against doxorubicin (Doxorubicinone, CAS No: 24385-10-2, referred to as DOX) induced heart failure experiment

[0912] 70 six to eight week old C57BL / 6 male mice were divided into two groups, namely: Ctrl group (8) and DOX group (62), wherein DOX was purchased from MCE (item number: CS-1239).

[0913] Ctrl group: intraperitoneal injection of 100 μL of normal saline once a week for 4 consecutive weeks.

[0914] DOX group: intraperitoneal injection of DOX solution at 5 mg / kg once a week for 4 consecutive weeks, with a total drug dose of 20 mg / kg, 100 μL of DOX solution per mouse each time. Four days after the fourth injection of doxorubicin, the mouse echocardiogram was detected using a portable digital color ultrasound diagnostic instrument (LAB version, Feiyinuo Technology (Suzhou) Co., Ltd., model VINNO 6LAB) to determine the mouse heart failure modeling condition (2-4 days after 4 weeks of modeling, 2 mice died), and then the successfully modeled mice were randomly divided into 6 groups, each group of 10 mice, and administered by gavage, respectively:

[0915] DOX model group (gavage with normal saline every day)

[0916] LCZ696 (Cas: 936623-90-4) drug 1 group (gavage with 60 mg / kg twice a day)

[0917] 1-R drug dose low, medium, high 3 groups (gavage with 1, 10, 50 mg / kg twice a day)

[0918] 1-S drug 1 group (gavage with 50 mg / kg twice a day).

[0919] Gavage for about 2 weeks, observe the state of the mouse (poor state needs to prepare to kill the mouse, actually give 12 days), use portable digital color ultrasonic diagnostic instrument to record the echocardiogram of the mouse.

[0920] Kill the mouse and take the tissue sample

[0921] I. Sample collection

[0922] a) After weighing, take the eyeball to collect blood, centrifuge to collect serum. Mix 50 μl of serum with 100 μl of sterile 1xPBS for biochemical analysis to detect CK (creatine kinase), HBDH (hydroxybutyrate dehydrogenase), LDH (lactate dehydrogenase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline aminotransferase), and store the rest of the serum at -80°C.

[0923] b) Take the whole liver and spleen and store at -80°C. Take one fixed paraffin section of the kidney and freeze one.

[0924] c) Weigh the heart, cut it transversely, take the middle part to fix, dehydrate it through the automatic tissue dehydrator (Leica, HistoCore Pearl), embed it in paraffin with the paraffin embedding machine (Lecia, HistoCore Arcadia), and slice it with the manual rotary slicer (RM2235), with a slice thickness of 5 μm. Take two 15 mg to extract RNA (400 μl trizol) and protein (500 μl lysis), respectively, and store the rest at -80°C.

[0925] d) Heart paraffin section with Sirius red staining to detect cardiac fibrosis.

[0926] The prepared heart paraffin section was deparaffinated through the following steps: xylene 10 min - xylene 10 min - 100% ethanol 5 min - 100% ethanol 5 min - 95% ethanol 5 min - 90% ethanol 5 min - 80% ethanol 5 min - tap water 5 min - distilled water 5 min. The tissue was dyed with Sirius red dye for 1 h, and then PBS was washed 3 times for 5 min each time to remove the excess dye, and then the cell nucleus was dyed with hematoxylin, washed with water to return to blue, dehydrated: 80% ethanol 5 min - 90% ethanol 5 min - 95% ethanol 5 min - 100% ethanol 5 min - 100% ethanol 5 min - xylene 5 min - xylene 5 min - neutral resin mounting. After air-drying, photographing and counting the area ratio of the Sirius red staining positive area were performed.

[0927] II. Experimental results

[0928] 1. Drug treatment increases the survival rate of adriamycin-induced heart failure mice (LCZ696, 1-S, 1-R)

[0929] The results are shown in Figure 2: DOX model group (6 survived for 12 days); LCZ696 drug 1 group (9 survived for 12 days); 1-R drug low, medium and high dose groups (7, 6 and 9 survived for 12 days, respectively); 1-S drug 1 group (6 survived for 12 days).

[0930] 1-R can significantly increase the survival rate of adriamycin-induced heart failure mice, especially at a high dose (50 mg / kg), which is the same as the positive drug; 1-S has no obvious effect on heart failure mice. Adriamycin-induced heart failure significantly reduces the body weight of mice.

[0931] 2. Drug treatment improves the ultrasound indicators of adriamycin-induced heart failure mice (EF-Ejection fraction, FS-Fractional shortening, HR-Heart rate)

[0932] The results are shown in Figure 3: Compound 1-S and 1-R can antagonize the decrease in ejection fraction and fractional shortening of mice caused by adriamycin, i.e., improve the heart function of mice.

[0933] 3. Drug treatment reduces the content of serum cardiac injury markers in mice (CK-creatine kinase, HBDH-hydroxybutyrate dehydrogenase, LDH-lactate dehydrogenase)

[0934] Results are shown in Figure 4. Compound 1-S and 1-R can reduce the content of creatine kinase, lactate dehydrogenase and hydroxybutyric acid dehydrogenase in serum of heart failure mice.

[0935] 4. Drug treatment improves morphology of mouse myocardial cells and cardiac fibrosis

[0936] Results are shown in Figure 5. Compound 1-S and 1-R can inhibit adriamycin-induced myocardial fibrosis in mice. (Sirius red can stain collagen fibers into red)

[0937] III. Conclusion

[0938] Compound 1-R significantly improves adriamycin-induced heart failure in multiple aspects.

[0939] 6.2. Evaluation of activity of series of compounds in treatment of adriamycin-induced heart failure (experimental reference example 6.1)

[0940] Table 1. DOX model group (intragastrically administered physiological saline every day); LCZ696 drug 1 group (intragastrically administered 60 mg / kg twice a day); series of compound drug dose low, medium and high 3 groups (intragastrically administered 1, 10 and 50 mg / kg twice a day)

[0941] Example 7. Evaluation of compound 1-R on heart failure mice after myocardial infarction

[0942] Evaluation of the efficacy of compound 1-R on the experimental model of heart failure induced by ligation of left anterior descending coronary artery (LAD) after myocardial infarction.

[0943] I. Experimental method:

[0944] 1. Grouping: 5-6 week old ICR male mice

[0945] Ligation of left anterior descending coronary artery was performed to establish the model of heart failure after myocardial infarction. Then the model mice were randomly divided into 5 groups, 10-11 mice in each group, which were:

[0946] Model group (11 mice)

[0947] LCZ696 (Cas: 936623-90-4) group (intragastrically administered 66 mg / kg once a day) (10 mice)

[0948] Compound 1-R drug dose low (11 mice), medium (11 mice) and high (9 mice) 3 groups (intragastrically administered 3, 10 and 33 mg / kg once a day)

[0949] Gavage administration for 7 days, observe the state of the mice, and record the echocardiogram of the mice using a portable digital color ultrasonic diagnostic instrument.

[0950] Tissue samples were taken from the rats.

[0951] 1.2 Heart function detection: After the end of administration, isoflurane anesthesia was used, and a small animal ultrasound imaging system (Vevo™ 2100, Visual Sonics, Canada) was used to detect the long axis and short axis, detect the left ventricular internal dimension at systole (LVID; s), left ventricular internal dimension at diastole (LVID; d), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV), and other indicators. The fractional shortening (FS) value was calculated according to the formula for calculating the left ventricular short axis shortening rate, which is FS (%) = (LVID; d-LVID; s) / LVID; d x 100%. The ejection fraction (EF) value was calculated according to the formula for calculating the ejection fraction; the formula is EF (%) = (LVEDV-LVESV) / LVEDV x 100%; finally, the FS value and the EF value were used to evaluate the cardiac systolic and diastolic function.

[0952] 1.3 Serum myocardial injury marker detection: (1) The abdominal aorta was weighed and blood was taken, and the serum was collected by centrifugation. 50 μL of serum was mixed with 100 μL of sterile 1x PBS for biochemical analysis, and an automatic biochemical analyzer (HITACH17080, Hitachi, Japan) was used to detect NT-proBNP (N-Terminal Pro-Brain Natriuretic Peptide), LDH (lactate dehydrogenase), and the rest of the serum was stored at -80°C. (2) The heart was weighed, and the heart was cut transversely, and the middle part was fixed to make sections, and two 15 mg were taken to extract RNA (400 μl trizol) and protein (500 μl lysis), respectively, and the remaining part was stored at -80°C. (3) HE staining of heart paraffin sections was used to detect heart injury, mason staining was used to detect heart fibrosis, and WGA staining was used to detect myocardial cell hypertrophy.

[0953] 1.4 Myocardial tissue HE staining

[0954] (1) Paraffin section deparaffinization and hydration: xylene I immersion for 20 min, xylene II again for 20 min, gradient hydration from anhydrous ethanol to 50% alcohol, then flowing pure water washing for 10 min, then 0.01 mol / L PBS slow washing for 5 min, repeated 3 times;

[0955] (2) Hematoxylin staining of cell nucleus: after the section is immersed in Harris hematoxylin dye for 8 min, pure water is used for washing after staining, then the section is immersed in 1% hydrochloric acid alcohol, differentiation is performed after washing with pure water, then the section is immersed in 0.6% ammonia water for blue returning, and finally pure water is used for washing.

[0956] (3) Eosin staining of cytoplasm: the section is immersed in eosin dye for 1-3 min.

[0957] (4) Dehydration and mounting: the section is sequentially dehydrated and transparentized, gradient dehydration is performed through 70% alcohol to anhydrous ethanol, then the section is immersed in xylene I and xylene II for 15 min, transparentization is performed, the section is dried in a fume hood, and then neutral balsam is used for mounting;

[0958] (5) Image acquisition: microscopic examination is performed under a microscope, image acquisition and analysis are performed.

[0959] 1.5 Myocardial tissue Masson staining for detection of myocardial fibrosis

[0960] (1) Paraffin section deparaffinization: xylene I immersion for 20 min, xylene II again for 20 min, gradient hydration from anhydrous ethanol to 50% alcohol, then flowing pure water washing for 10 min, then 0.01 mol / L PBS slow washing for 5 min, repeated 3 times;

[0961] (2) The section is immersed in Masson A solution at room temperature for overnight, and then flowing pure water washing is performed for 10 min. After completion, the next step is performed;

[0962] (3) The section washed with pure water is immersed in a dye solution mixed by Masson B dye and Masson C dye at an equal ratio, the immersion time is 1 min, after completion, flowing pure water washing is performed for 10 min, then 1% hydrochloric acid alcohol is used for section differentiation, and then flowing pure water washing is performed;

[0963] (4) The section is immersed in Masson D dye, the immersion time is 6 min, and then flowing pure water washing is performed for 10 min after completion;

[0964] (5) The section after completion of washing is immersed in Masson E dye, the immersion time is 1 min. After completion of this process, no washing is performed;

[0965] (6) After the slices are slightly drained, they are directly immersed in Masson F solution for dyeing, and the immersion and dyeing time is 30 s;

[0966] (7) Then the slices are rinsed and differentiated by 1% glacial acetic acid. The slices are dehydrated by immersion in anhydrous ethanol A and anhydrous ethanol B.

[0967] (8) Transparent mounting: the slices are immersed in anhydrous ethanol C for 5 min, the slices are transparently immersed in xylene for 5 min, and the slices are mounted with neutral balsam.

[0968] (9) Microscopic examination, image acquisition and analysis are performed under a microscope.

[0969] 1.6 Myocardial tissue WGA staining for detecting myocardial cell hypertrophy

[0970] (1) Paraffin section deparaffination: xylene I is soaked for 20 min, xylene II is soaked again for 20 min, hydration is performed by gradient immersion in anhydrous ethanol to 50% alcohol, and then pure water is used for washing for 10 min, and 0.01 mol / L PBS is used for slow washing for 5 min, which is repeated for 3 times;

[0971] (2) Antigen repair: the 10x stock solution of antigen repair solution is mixed with distilled water at a ratio of 1:20 to prepare the antigen repair solution. The prepared antigen repair solution is preheated in a microwave oven at high fire for 8 min, then slowly immersed in the slices, and then repaired at low fire for 6 min, and then naturally cooled in an ice water mixture, and then washed with flowing pure water for 3-5 times;

[0972] (3) Staining: the washed slices are slightly shaken and dried, an immunohistochemical pen is used to draw a circle around the myocardial tissue to prevent the loss of the antibody. After the circle is drawn, the diluted WGA dye solution is added in the circle, and then incubated in a constant temperature box at 37°C for 30 min in the dark, and then washed with 0.01 mol / L PBS for 5 min, which is repeated for 3 times;

[0973] (4) DAPI re-staining of cell nucleus: the washed slices are slightly shaken and dried, and the DAPI dye solution is added in the circle, and then incubated at room temperature in the dark for 10 min, and then washed with 0.01 mol / L PBS for 5 min, which is repeated for 3 times;

[0974] (5) Mounting: the washed slices are slightly shaken and dried, and then mounted with an anti-fluorescence quenching mounting agent to reduce fluorescence quenching;

[0975] (6) Microscopic examination and photography: microscopic examination is performed under a fluorescence microscope, and images are collected for analysis.

[0976] II. Experimental results

[0977] 1. Compound 1-R improves heart function of heart failure mice

[0978] Results are shown in Figure 6. The model group had 9 survivors; the LCZ696 positive drug group had 10 survivors; the compound 1-R drug dose low, medium and high groups had 10, 11 and 9 survivors, respectively. That is, the model group had 82% survival; the LCZ696 positive drug group had 100% survival; the compound 1-R drug dose low, medium and high groups had 91%, 100% and 100% survival, respectively. Compound 1-R can antagonize the decline in ejection fraction (EF) and fractional shortening (FS) of mice induced by the model; that is, it improves the cardiac function of mice.

[0979] 2. Results are shown in Figure 7. Compound 1-R can antagonize myocardial fibrosis and myocardial cell hypertrophy in mice induced by the model.

[0980] 3. Compound 1-R improves serum indicators of heart failure mice

[0981] Results are shown in Figure 8. Compound 1-R can antagonize the serum LDH level of mice induced by the model; that is, it improves heart failure in mice.

[0982] Example 8, Evaluation of Compound 1-R on Heart Failure Rats after Myocardial Infarction

[0983] Evaluation of the efficacy of compound 1-R on the experimental model of heart failure induced by coronary left anterior descending branch ligation (LAD) after myocardial infarction.

[0984] I. Experimental method

[0985] Grouping: 6-7 week old male rats were subjected to coronary left anterior descending branch ligation to establish a heart failure model after myocardial infarction, and then the model rats were randomly divided into 5 groups, namely:

[0986] Model group (9 rats)

[0987] LCZ696 (Cas: 936623-90-4) group (8 rats) (1 time per day, 46.2 mg / kg by gavage)

[0988] Compound 1-R drug dose low (9 rats), medium (9 rats), and high (10 rats) groups (1 time per day, 2.1, 7, and 23.1 mg / kg by gavage)

[0989] Gavage for 21 days, observe the state of the rats, and record the echocardiogram of the rats using a portable digital color ultrasonic diagnostic instrument.

[0990] Kill rats and take tissue samples.

[0991] II. Experimental results

[0992] Results are shown in Figure 9, 6 rats survived in the model group; 6 rats survived in the LCZ696 positive drug group; 8 rats, 7 rats, and 8 rats survived in the low, medium, and high dose groups of Compound 1-R, respectively. That is, 67% of the rats survived in the model group; 75% of the rats survived in the LCZ696 positive drug group; 89%, 78%, and 80% of the rats survived in the low, medium, and high dose groups of Compound 1-R, respectively. Compound 1-R significantly improved the survival rate of heart failure rats.

[0993] Compound 1-R improves heart function and myocardial remodeling in heart failure rats

[0994] Results are shown in Figure 10, Compound 1-R can antagonize the decrease in the ejection fraction EF and fractional shortening FS of rats induced by the model; that is, it improves the cardiac function of rats. In addition, as shown in Figure 11, Compound 1-R can antagonize myocardial fibrosis and myocardial cell hypertrophy in rats induced by the model.

[0995] Compound 1-R improves serum indicators in heart failure rats

[0996] Results are shown in Figure 12, Compound 1-R can antagonize the serum LDH level in rats induced by the model; that is, it improves heart failure in rats.

[0997] III. Mechanism and Conclusion

[0998] Compound 1-R can significantly increase the survival rate of rats after left anterior descending coronary artery ligation (LAD) induced myocardial infarction and significantly improve heart failure, which is achieved by regulating RhoA activity to regulate the nuclear translocation of MRTF-A (Figures 13 and 14), collagen and metalloproteinase MMP2 expression, thereby regulating myocardial hypertrophy and fibrosis (Figures 15 and 16).

[0999] Example 9. Anti-myocardial fibrosis test using in vitro 3D myocardial tissue model.

[1000] 9.1 Establishment of 3D myocardial tissue model

[1001] Three types of primary cells (myocardial cells, fibroblasts, macrophages) are mixed in a ratio of 1:1:1 and co-cultured with collagen to establish a 3D myocardial tissue model.

[1002] 9.1.1 Extraction of primary myocardial cells and fibroblasts

[1003] 1) Preparation: Sterilize the surgical instruments in advance and dry them. Lay out the surgical drape on the clean bench, and arrange the relevant consumables such as pipettes, tips, beakers, centrifuge tubes, culture dishes, and ultraviolet sterilization for 15 minutes;

[1004] 2) Freeze the suckling rats: Take 1-3 day old SD rat suckling rats and place them in a self-sealing bag. Cut the breathing port and place it in a -20°C freezer for 30 minutes;

[1005] 3) Disinfection: Soak the pups in 75% alcohol for 1 min;

[1006] 4) Core extraction: Fix the pups in prone position in hand, cut open the thoracic cavity under sterile condition to expose the heart, and quickly extract the heart;

[1007] 5) Washing: Place the heart in a petri dish containing PBS, cut off the atrium, auricle, and pericardium, and repeatedly rinse the blood pump;

[1008] 6) Cutting the tissue: Cut the ventricle into 0.5 mm2 pieces and collect them in a 15 mL centrifuge tube;

[1009] 7) Digestion with a digestion solution: Prepare a 0.15% papain and 0.1% trypsin digestion solution and add it to the centrifuge tube containing the tissue pieces;

[1010] 8) Digestion in a water bath: Place the 15 mL centrifuge tube in a 37°C water bath for 30 min;

[1011] 9) Digestion in an incubator: Take the centrifuge tube out of the water bath, gently shake it, wipe the liquid off the tube wall, spray 75% alcohol, and place it in a 37°C cell incubator for digestion;

[1012] 10) Blow-off digestion: Take the centrifuge tube out of the incubator and place it on the clean bench, and use a 5 mL pipette to blow off the cells;

[1013] 11) Terminate the digestion: Transfer the upper liquid of the centrifuge tube to a new 15 mL centrifuge tube, and add complete medium containing 10% FBS to terminate the digestion;

[1014] 12) Filtration: Filter the digestion solution through a 70 μm cell sieve into a 50 mL centrifuge tube;

[1015] 13) Repeat digestion: Repeat steps 7-11 of the method for the remaining undigested tissue pieces until there are no obvious tissue pieces;

[1016] 14) Centrifugation: Centrifuge at 4°C, 1000 rpm, for 10 min, and discard the supernatant;

[1017] 15) Plating: Add an appropriate amount of complete medium containing 10% FBS to resuspend the cells, and plate them in a 10 cm cell culture dish, and then place them in a 37°C incubator for 90 min of adhesion;

[1018] 16) Isolate the cells: Gently shake the culture dish and observe the adhesion of the primary fibroblasts on the bottom. Transfer the liquid in the culture dish (containing primary myocardial cells) to a new culture dish for further culture. The primary fibroblasts that have adhered to the original dish need to be supplemented with an appropriate amount of complete medium containing 10% FBS;

[1019] 17) Medium change: change the medium of primary cardiomyocytes and primary fibroblasts after 24 hours;

[1020] 18) Note: primary cardiomyocytes and primary fibroblasts are separated by differential adhesion method; primary cardiomyocytes cannot be subcultured, and primary fibroblasts cannot be frozen and recovered, and need to be used immediately.

[1021] 9.1.2. Extraction of primary bone marrow-derived macrophages

[1022] 1) Preparation: sterilize the surgical instruments in advance and dry them. Lay the surgical drape on the clean bench, and arrange the relevant consumables such as range transfer pipettes, suction heads, beakers, centrifuge tubes, culture dishes, and ultraviolet sterilization for 15 minutes;

[1023] 2) Freeze the suckling rats: take 10-day-old SD rat suckling rats and place them in a self-sealing bag. Cut the breathing port and place it in a -20°C freezer for 30 minutes;

[1024] 3) Disinfect: soak the suckling rats in 75% alcohol for 1 minute;

[1025] 4) Separate the femur: hold the suckling rat in a prone position, gradually cut the skin and muscle layer of the hind limbs, gently rotate the femur inward to rotate the greater trochanter out of the acetabulum, and ensure that the femur is not cut;

[1026] 5) Squeeze the bone marrow: peel off the muscle attached to the femur and tibia, expose the complete and smooth bone, and squeeze the bone marrow from bottom to top with straight forceps. After the bone marrow is squeezed out, move it to the prepared complete culture medium containing 15% FBS;

[1027] 6) Lyse red blood cells: add an appropriate amount of red blood cell lysis solution and lyse at 4°C for 5 minutes;

[1028] 7) Filter: filter the lysed bone marrow through a 40μm cell sieve;

[1029] 8) Centrifuge: centrifuge at 4°C, 1000 rpm for 5 minutes, and discard the supernatant;

[1030] 9) Plate: add complete medium containing 15% FBS to the cells to form a uniform cell suspension. Plate in a 10cm cell culture dish. Then place it in a 37°C incubator for 1 hour;

[1031] 10) Separate: discard the adherent cells, collect the cell suspension in the dish and move it to a new cell culture dish for subsequent experiments;

[1032] 11) Medium change: change the medium of the cells after 48 hours, and perform subsequent experiments after 4 days;

[1033] 12) Note: The complete medium containing 15% FBS used for cell culture contains M-CSF at a concentration of 10 pg / mL for activating macrophages. The primary macrophages used in the experiment are the first generation of bone marrow-derived macrophages, which are not passaged.

[1034] 9.1.3 Rat tail collagen extraction

[1035] 1) Cut the rat tail: cut the tail of the SD rat from the root;

[1036] 2) Disinfect: immerse the tail in 70% alcohol for more than 5 min;

[1037] 3) Separate the tendon: cut the rat tail skin with a blade and remove it, and take out the silver tendon with straight scissors and straight forceps;

[1038] 4) Wash the tendon: wash the tendon in PBS for 3 times;

[1039] 5) Sterilize: transfer the tendon to 70% alcohol for more than 1 h;

[1040] 6) Dry: take out the tendon and pat it dry on the water-absorbing paper, and remove the residual alcohol as much as possible;

[1041] 7) Dissolve the tendon: transfer the tendon to a sterile bottle with magnetic beads, and add 75 mL / tail of 0.1% ice acetic acid;

[1042] 8) Stir: place the sterile bottle on a magnetic stirrer, stir at 4°C and 1000 rpm for 2 days;

[1043] 9) Check: check the solution state regularly, and add 0.1% ice acetic acid as appropriate if the viscosity is too high to cause stirring difficulty;

[1044] 10) Centrifuge: centrifuge the viscous solution obtained at 4°C and 16000 rcf for 90 min to obtain a collagen stock solution;

[1045] 11) Store at 4°C.

[1046] 9.1.4 Co-culture of cells combined with collagen

[1047] 1) Preparation: prepare the rat collagen stock solution, sterile distilled water, sterile 1N NaOH solution, PBS, and primary cell suspension on ice;

[1048] 2) Volume of each solution: the required cell volume is 1 / 10 of the total volume; the required collagen volume is calculated according to formula (1); the required 1N NaOH volume is 0.023 times the collagen volume; the remaining volume is supplemented with sterile distilled water; V IIII = (1)

[1049] 3) Suspension: In 1.5 mL centrifuge tube, suspend sterile 1N NaOH solution, sterile distilled water, collagen solution, after forming a uniform solution, quickly add the same amount of counted primary myocardial cells, primary fibroblasts, primary bone marrow-derived macrophages and mix well;

[1050] 4) Plating: Plate the mixed cell suspension on a 96-well plate, 20 μL per well;

[1051] 5) Culture: Place the 96-well plate in a 37°C incubator for 1 h to promote gel formation. Change the liquid every 24 h.

[1052] Compound

[1053] 9.2 Compound cytotoxicity test

[1054] All compounds were tested for 3D myocardial tissue progressive cytotoxicity.

[1055] 1) Cell survival rate less than 50% is considered to be toxic to the drug;

[1056] 2) Cell model used: mouse primary myocardial cells-primary cardiac fibroblasts-primary bone marrow-derived macrophages-collagen co-culture tissue model;

[1057] 3) Dose concentration and time: 20 μM, 24 h;

[1058] 4) 20 μM of 104 compounds were cultured with myocardial organoids alone for 24 h;

[1059] 5) Discard the supernatant, add CCK8 working solution (DMEM:CCK8=9:1), after 2 h, detect the cell absorbance at wavelength 450 nm and calculate.

[1060] 9.3 Anti-myocardial fibrosis activity test

[1061] Cell proliferation inhibition experiment and cell contraction inhibition experiment were performed on compounds without cytotoxicity, and the results of the two experiments were basically consistent. A series of compounds were found to have anti-myocardial fibrosis activity.

[1062] 9.3.1 Anti-fibrosis proliferation test

[1063] 1) Cell model used: mouse primary myocardial cells-primary cardiac fibroblasts-primary bone marrow-derived macrophages-collagen co-culture tissue model;

[1064] 2) Dose concentration and time: 20 μM, 24 h;

[1065] 3) The model group and the administration group were placed in a 37°C constant-temperature three-gas incubator (95% N2, 5% CO2) for 8 h, and the normal group was placed in a normal-oxygen incubator for culture. The 20 μM compound was administered to the myocardial organoid culture for 24 h;

[1066] 4) The supernatant was discarded, CCK8 working solution (DMEM:CCK8 = 9:1) was added, and after 2 h, the cell absorbance was detected at a wavelength of 450 nm and calculated. The experimental results are shown in the following table.

[1067] 9.3.2 Anti-fibrotic contraction test

[1068] 1) The cell model used: mouse primary myocardial cells-primary cardiac fibroblasts-primary bone marrow-derived macrophages-collagen co-culture tissue model;

[1069] 2) Administration concentration and time: 20 μM, 24 h;

[1070] 3) The model group and the administration group were placed in a 37°C constant-temperature three-gas incubator (95% N2, 5% CO2) for 8 h, and the normal group was placed in a normal-oxygen incubator for culture. The 20 μM compound was administered to the myocardial organoid culture for 24 h;

[1071] 4) The supernatant was discarded, CCK8 working solution (DMEM:CCK8 = 9:1) was added, and after 2 h, the cell absorbance was detected at a wavelength of 450 nm and calculated. The experimental results are shown in the following table.

[1072] The experimental results are shown in FIG. 17: wherein the 3D tissue produces a spherical contraction under hypoxic conditions, the Control group is the size of the myocardial organoid cell ball cultured under normal conditions, and the model group is the size of the myocardial organoid cell ball under hypoxic conditions. The cell ball of the myocardial organoid model under hypoxic conditions will produce contraction, and the model group and the administration group are cultured under hypoxic conditions. When the cell ball volume of the administration group is greater than that of the model group, it is considered to have the ability to inhibit the contraction of the cell ball and to have the effect of anti-myocardial fibrosis.

[1073] 9.4 Anti-myocardial injury marker (CK-MB, LDH) and inflammatory factor (IL-1β, IL-6) detection

[1074] The anti-fibrotic activity evaluation of the less cytotoxic compound was carried out, including myocardial injury marker (CK-MB, LDH); inflammatory factor (IL-1β, IL-6) detection, and it was found that the series of compounds had different degrees of inhibition on LDH, IL-1β, and IL-6.

[1075] 9.4.1 Anti-myocardial injury CK-MB, LDH test

[1076] 1) Cell model used: mouse primary cardiomyocytes-primary cardiac fibroblasts-primary bone marrow-derived macrophages-collagen co-culture tissue model;

[1077] 2) Dose and time: 20 μM, 24 h;

[1078] 3) Model group and drug administration group were placed in a constant temperature three-gas incubator (95% N2, 5% CO2) at 37°C for 8 h, and the normal group was placed in a normal oxygen incubator. 20 μM of 104 compounds were administered to the myocardial organoids for 24 h;

[1079] 4) Collect the supernatant of each sample, centrifuge at 300g, and aspirate the supernatant for subsequent experiments. Restore the Elisa test box to room temperature and perform Elisa test (take CK-MB as an example);

[1080] 5) Standard dilution: prepare 6 test tubes, and label them 0-5 in turn. Add 150 μL standard diluent to each test tube, then add 150 μL standard solution to test tube 1, and mix well. Take 150 μL solution from test tube 1 and add it to test tube 2, and mix gently. Therefore, the concentration of each test tube is 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, and 0 ng / mL, respectively;

[1081] 6) Sample addition: divide into standard wells, blank wells and sample wells. Add 50 μL of standard of each concentration to the standard wells. The blank wells do not add samples, but add 10 μL of biotinylated antibody. Add 40 μL of sample and 10 μL of biotinylated antibody to the sample wells, and shake gently without touching the well wall;

[1082] 7) Add enzyme: add 50 μL of HRP conjugate to each well except the blank wells;

[1083] 8) Incubation: seal the microplate and incubate at 37°C for 30 minutes;

[1084] 9) Prepare the washing buffer: dilute the 30-fold concentrated washing buffer with 30 times distilled water;

[1085] 10) Washing: remove the seal of the microplate, discard the liquid, and pat the microplate dry with a paper towel, then add 300 μL of buffer to each well, wait for 30 seconds, discard the liquid, and repeat 5 times, and pat dry;

[1086] 11) Color development reaction: add 50 μL of color developing solution A and 50 μL of color developing solution B to each well. Shake gently, avoid light, and incubate for 10 minutes;

[1087] 12) Reaction stop: add 50 μL of stop solution to each well, and the color changes from blue to yellow, indicating that the reaction has stopped;

[1088] 13) Analysis: zeroed based on blank wells, OD values measured within 15 minutes after reaction stopped;

[1089] 14) LDH test same as method 9, except standard dilution concentration gradient was: 12 ng / mL, 6 ng / mL, 3 ng / mL, 1.5 ng / mL, 0.75 ng / mL, 0 ng / mL, and the rest of the steps were consistent.

[1090] 9.4.2 Anti-inflammatory IL-1β, IL-6 test

[1091] 1) Cell model used: mouse primary cardiomyocytes-primary cardiac fibroblasts-primary bone marrow-derived macrophages-collagen co-culture tissue model

[1092] 2) Dose concentration and time: 20 μM, 24 h

[1093] 3) Model group and drug administration group were placed in a constant temperature three-gas incubator (95% N2, 5% CO2) at 37°C for 8 h, and the normal group was placed in a normal oxygen incubator. 20 μM of 104 compounds were administered to the myocardial organoid culture for 24 h.

[1094] 4) Collect the supernatant of each sample, centrifuge at 300g, and aspirate the supernatant for subsequent experiments. Restore the Elisa test box to room temperature and perform Elisa test (take IL-1β as an example).

[1095] 5) Standard dilution: prepare 6 test tubes, and label them 0-5 in turn. Add 150 μL standard dilution solution to each test tube, then add 150 μL standard solution to test tube 1 and mix well. Take 150 μL solution from test tube 1 and add it to test tube 2, and mix gently. Therefore, the concentration of each test tube is 960 pg / mL, 480 pg / mL, 240 pg / mL, 120 pg / mL, 60 pg / mL, and 0 pg / mL, respectively.

[1096] 6) Sample addition: divide into standard wells, blank wells and sample wells. Add 50 μL of each concentration of standard to the standard wells. No sample is added to the blank wells, but 10 μL of biotinylated antibody is added. Add 40 μL of sample and 10 μL of biotinylated antibody to the sample wells, and shake gently without touching the well wall.

[1097] 7) Add enzyme: add 50 μL of HRP conjugate reagent to each well except the blank wells.

[1098] 8) Incubation: seal the microplate and incubate at 37°C for 30 minutes.

[1099] 9) Prepare the washing buffer: dilute the 30-fold concentrated washing buffer with 30 times distilled water.

[1100] 10) Wash: Uncover the microplate seal, discard the liquid, and pat the microplate dry with absorbent paper. Add 300 μL of buffer to each well, wait 30 seconds, discard the liquid, and repeat 5 times. Pat dry.

[1101] 11) Color development: Add 50 μL of color developing solution A and 50 μL of color developing solution B to each well. Gently shake and incubate for 10 minutes in the dark.

[1102] 12) Reaction stop: Add 50 μL of stop solution to each well. The color will change from blue to yellow, indicating that the reaction has stopped.

[1103] 13) Analysis: Zero the instrument based on the blank wells. Measure the OD within 15 minutes after the reaction has stopped.

[1104] 14) IL-6 test: The procedure is the same as in 10, except that the standard dilution concentration gradient is: 480 pg / mL, 240 pg / mL, 120 pg / mL, 60 pg / mL, 30 pg / mL, 0 pg / mL. The results are shown in the following table:

[1105] Table 2. General table of compounds evaluated for anti-cardiac fibrosis activity

[1106] Except for some compounds that showed strong cytotoxicity, most of the compounds showed good inhibition of cardiac fibroblast proliferation.

[1107] All documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed formulation or method, or fragment thereof, can be implemented alone or in combination with one another. It is therefore evident that there is a combination of features that can be used to advantage. It is also evident that such features can be combined in different embodiments. It is the intention of the inventors to embrace all such modifications and alternatives.

Claims

1. Use of a compound of Formula I, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer, or solvate thereof, in the manufacture of a TRPV4-RhoA target antagonist or inhibitor, wherein A is selected from a C6-C10 aromatic or carbocyclic ring, or a 5-6 membered heterocyclic or arylheterocyclic ring containing 1, 2, or 3 heteroatoms selected from N, O, or S; R1and R4are each independently selected from the group consisting of H, D, OH, carbonyl, optionally substituted C1-C10alkyl, optionally substituted C1-C10alkoxy, optionally substituted phenoxy, optionally substituted benzyloxy, optionally substituted NH2, NHCH3, NHCOCH3, halogen, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted aminoacetoxy, and B can be various groups forming amino acids), sulfonic acid group ( or a sulfonate ester), an optionally substituted monosaccharide, disaccharide, or polysaccharide group; R2and R5are each independently selected from the group consisting of H, D, OH, carbonyl, hydroxymethyl, optionally substituted C1-C10alkyl, optionally substituted C1-C10alkoxy, optionally substituted benzyloxy, optionally substituted phosphate, optionally substituted NH2, NHCH3, NHCOCH3, optionally substituted C1-C10formyloxy, optionally substituted benzoyloxy, optionally substituted aminoacetyloxy, B can be various amino acid forming groups), COOH, COOCH3, CONH2, sulfonic acid group ( or a sulfonate ester), an optionally substituted monosaccharide, disaccharide, or polysaccharide group; or R1and R2or R4and R5are joined to form an optionally substituted 3-6 membered ring containing 0, 1, or 2 heteroatoms selected from oxygen, nitrogen, or sulfur (preferably oxygen), a cyclic carbonic acid lactone, a cyclic phosphoric acid lactone, or an optionally substituted cyclic boronic acid lactone; R3is selected from H, D, optionally substituted C1-C10alkyl, D-substituted C1-C10alkyl, optionally substituted C3-C10cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 heteroatoms selected from O, S, or N, optionally substituted C2-C10alkenyl, optionally substituted C2-C10cycloalkenyl, cyano, hydroxyl, optionally substituted C1-C10alkoxy, optionally substituted C1-C10alkylthio, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted C1-C10acyl, hydroxycarbonyl, optionally substituted C1-C10alkylcarbonyloxy, nitro, optionally substituted amino, optionally substituted C1-C10alkylamino, optionally substituted C1-C10alkylcarbonylamino, halogen, optionally substituted C2-6alkynyl, optionally substituted monosaccharide, disaccharide, or polysaccharide group; or, two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen, or sulfur (preferably oxygen or nitrogen), an optionally substituted C5-C10aromatic ring or an optionally substituted C5-C10heteroaromatic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen, or sulfur (preferably oxygen or nitrogen); m is an integer from 1-5; hydrogen in the structure of formula I is optionally replaced by deuterium.

2. Use according to claim 1, characterized in that, The compound of formula I is a compound of formula II, wherein X1, X2, X3are each independently selected from CH, N, S, O, or absent (preferably CH or N); R1, R4are each independently selected from H, D, OH, optionally substituted C1-C10alkoxy, optionally substituted phenoxy, optionally substituted benzyloxy, optionally substituted NH2, NHCH3, NHCOCH3, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing amino, optionally substituted monosaccharide, disaccharide, or polysaccharide group; R2and R5are each independently selected from H, D, OH, hydroxymethyl, optionally substituted C1-C10alkyl, optionally substituted C1-C10alkoxy, optionally substituted benzyloxy, NH2, NHCH3, NHCOCH3, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing amino, COOH, COOCH3, CONH2, optionally substituted monosaccharide, disaccharide, or polysaccharide group; R1, R2may form an optionally substituted 3-6 membered ring containing 0, 1, or 2 heteroatoms selected from oxygen, nitrogen, or sulfur (preferably oxygen); R3is selected from the group consisting of H, D, optionally substituted C1-C10alkyl, D- substituted C1-C10alkyl, optionally substituted C3-C10cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 heteroatoms selected from O, S or N, optionally substituted C2-C10alkenyl, hydroxyl, optionally substituted C1-C10alkoxy, optionally substituted C1-C10alkylthio, optionally substituted C1-C10alkylcarbonyloxy, formyl, hydroxylcarbonyl, optionally substituted C1-C10alkylcarbonyloxy, nitro, optionally substituted amino, optionally substituted C1-C10alkylamino, optionally substituted C1-C10alkylcarbonylamino, halogen, optionally substituted monosaccharide, disaccharide or polysaccharide group; or two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 oxygen heteroatoms; m is an integer from 1 to 5; hydrogen in the structure of Formula II is optionally replaced by deuterium.

3. Use according to claim 1, characterized in that, The compound of formula I is a compound of formula III wherein, R1, R4are each independently selected from the group consisting of H, D, OH, optionally substituted C1-C10alkoxy, optionally substituted phenoxy, optionally substituted benzyloxy, optionally substituted NH2, NHCH3, NHCOCH3, optionally substituted C1-C10formyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing amino, optionally substituted aminoacetoxy B can be a variety of groups that form amino acids, optionally substituted monosaccharide, disaccharide or polysaccharide group; R2and R5are each independently selected from the group consisting of H, D, hydroxymethyl, optionally substituted C1-C10alkyl, OH, optionally substituted C1-C10alkoxy, optionally substituted benzyloxy, NH2, NHCH3, NHCOCH3, optionally substituted C1-C10alkylcarbonyloxy, optionally substituted benzoyloxy, optionally substituted carbamoyloxy containing amino, optionally substituted aminoacetoxy B can be a variety of groups that form amino acids, COOH, COOCH3, CONH2, optionally substituted monosaccharide, disaccharide or polysaccharide group; R1, R2may form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen); R3is selected from the group consisting of H, D, optionally substituted C1-C10alkyl, D- substituted C1-C10alkyl, optionally substituted C3-C10cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 heteroatoms selected from O, S or N, optionally substituted C2-C10alkenyl, hydroxyl, optionally substituted C1-C10alkoxy, optionally substituted C1-C10alkylthio, optionally substituted C1-C10alkylcarbonyloxy, formyl, hydroxylcarbonyl, optionally substituted C1-C10alkylcarbonyloxy, nitro, amino, optionally substituted C1-C10alkylamino, optionally substituted C1-C10alkylcarbonylamino, fluorine, chlorine, bromine, optionally substituted monosaccharide, disaccharide or polysaccharide group; or two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 oxygen heteroatoms; m is an integer from 1 to 5; hydrogen in the structure of Formula III is optionally replaced by deuterium.

4. The use according to claim 1, characterized in that, The compound is shown as formula IV, R1is selected from H, D, OH, NH2, optionally substituted C1-C3alkoxy, optionally substituted C1-C3alkylformyloxy, optionally substituted carbamoyloxy containing, optionally substituted aminoacetoxy B can be a variety of groups that form amino acids, optionally substituted monosaccharide group; R2is selected from the group consisting of OH, NH2, COOH, COONH2, COONHCH3, optionally substituted C1-C3alkyloxycarbonyl, optionally substituted C1-C3alkylcarbonyloxy, optionally substituted carbamoyloxy, optionally substituted aminoacetyloxy B can be a variety of groups that form amino acids, optionally substituted monosaccharide group; R3is selected from the group consisting of H, D, optionally substituted C1-C5alkyl, D- substituted C3-C5cycloalkyl, optionally substituted 3-6 membered ring containing 1 or 2 O, optionally substituted C2-C10alkenyl, hydroxyl, optionally substituted C1-C5alkoxy, optionally substituted C1-C3alkylthio, optionally substituted C1-C3alkylcarbonyloxy, formyl, optionally substituted C1-C3alkylcarbonyl, hydroxylcarbonyl, nitro, amino, optionally substituted C1-C3alkylamino, optionally substituted C1-C3alkylcarbonylamino, fluorine, chlorine, bromine; or two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen or nitrogen); m is an integer from 1 to 3; the hydrogens in the structure of Formula IV are optionally replaced by deuterium.

5. The use according to claim 1, wherein, wherein, A is selected from phenyl; R1and R4are each independently selected from H, D, OH, optionally substituted C1-C6alkoxy; R2and R5are each independently selected from H, D, OH, optionally substituted C1-C6alkoxy, optionally substituted C1-C6formyloxy; or R1and R2or R4and R5are linked to form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), cyclic carbonates; R3is selected from H, D, optionally substituted C1-C6alkyl, D-substituted C1-C6alkyl, optionally substituted C2-C6alkylalkenyl, optionally substituted C1-C6acyl; or, two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen or nitrogen); m is an integer from 1 to 3; the hydrogens in the structure of Formula IV are optionally replaced by deuterium.

6. Use of the following compound, or a salt or ester, prodrug, optical isomer or solvate thereof, in the manufacture of a medicament for preventing or treating a disease related to the TRPV4-RhoA target: Preferably, the compound is Most preferably, the compound is a compound selected from the group consisting of:

7. Use according to any one of claims 1 to 6, characterized in that, The TRPV4-RhoA target related diseases include, but are not limited to, the following indications: heart failure, hypertension, stroke, atrial fibrillation, etc., pneumonia, acute lung injury, pulmonary edema, chronic obstructive pulmonary disease (COPD), asthma, pain and inflammation such as osteoarthritis, neuropathic pain, visceral pain (such as irritable bowel syndrome), etc., neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.), urinary system diseases such as overactive bladder, etc., skin diseases such as eczema, itching, etc., eye diseases such as glaucoma, regulation of intraocular pressure and retinal nerve protection, etc.; preferably heart failure, hypertension, pain, lung disease and Parkinson's disease.

8. A compound of Formula I, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer, or solvate thereof, ###00002### I wherein, A is selected from optionally substituted C6-C10aromatic ring or 5-6 membered aromatic heterocycle containing 1, 2 or 3 heteroatoms selected from N, O or S; R1 and R4 are independently selected from H, D, OH, NH2, carbonyl (one of R1 or R4), optionally substituted C1-C10 alkyl (preferably C1-C6 alkyl), optionally substituted C1-C10 alkoxy (preferably C1-C6 alkoxy), optionally substituted aminoacetoxy ( B can be various groups forming amino acids), sulfoxy; R2and R5are each independently selected from H, D, OH, COOH, sulfonato, optionally substituted aminoacetyloxy, B can be various groups forming amino acids), optionally substituted NH2, optionally substituted C1-C10alkyl (preferably C1-C6alkyl), optionally substituted C1-C10alkoxy (preferably C1-C6alkoxy), phosphate; or, R1and R2or R4and R5are linked to form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), cyclic carbonates, phosphoric acid lactones or optionally substituted cyclic boronic acid lactones; R3is selected from H, D, optionally substituted C1-C10alkyl (preferably C1-C6alkyl) or alkenyl, optionally substituted C1-C10alkyl (preferably C1-C6alkyl) formyl, optionally substituted C1-C10alkyl (preferably C1-C6alkyl), D-substituted C1-C10alkyl, halogen, optionally substituted amino, optionally substituted C3-C10cycloalkyl, optionally substituted C2-6alkynyl, cyano; or, two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), an optionally substituted C5-C10aromatic ring or an optionally substituted C5-C10heteroaromatic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen); m is an integer from 1 to 5; hydrogen in the structure of Formula I is optionally replaced by deuterium.

9. The compound of claim 8, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer, or solvate thereof, wherein, A is selected from a C6-C10aromatic ring or a 5-6 membered aromatic heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O or S; R1and R4are each independently selected from H, D, OH, NH2, halogen, carbonyl; R2and R5are each independently selected from H, D, OH, COOH, optionally substituted sulfonyloxy, optionally substituted NH2, halogen, optionally substituted phosphate; or R1and R2or R4and R5are joined to form an optionally substituted 3-6 membered ring containing 0, 1 or 2 heteroatoms selected from oxygen, nitrogen or sulfur (preferably oxygen), a cyclic carbonic acid lactone, a cyclic phosphoric acid lactone or an optionally substituted cyclic boronic acid lactone; R3is selected from H, D, optionally substituted C1-C10alkyl (preferably C1-C6alkyl) alkenyl, formyl, optionally substituted C1-C10alkyl (preferably C1-C6alkyl) formyl, optionally substituted C1-C10alkyl (preferably C1-C6alkyl), D substituted C1-C10alkyl, halogen, optionally substituted C3-C10cycloalkyl, optionally substituted C2-6alkynyl; or, two adjacent R3substituents form an optionally substituted C3-C6carbocyclic ring or an optionally substituted C3-C6heterocyclic ring containing 1 or 2 oxygen, an optionally substituted C5-C10aromatic ring or an optionally substituted C5-C10heteroaromatic ring containing 1 or 2 oxygen; m is an integer from 1 to 5; hydrogen in the structure of Formula I is optionally replaced by deuterium.

10. The following compounds, or salts or esters, prodrugs, optical isomers or solvates thereof: Preferably, the compound is: Most preferably, the compound is 11. A pharmaceutical composition comprising the compound of any one of claims 8-10, or a pharmaceutically acceptable salt or ester, prodrug, optical isomer, stereoisomer, or solvate thereof, and a pharmaceutically acceptable carrier or excipient.

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