CN109810071B - A miRNA biosynthesis inhibitor - Google Patents

Abstract

The present invention provides a compound represented by formula I, or its conformational isomer, or its optical isomer, or its pharmaceutically acceptable salt. It can tightly bind to related binding proteins in the process of miRNA biosynthesis, and can effectively inhibit the synthesis of miRNA‑21. The active compound prepared by the invention can be used as an miRNA-21 inhibitor, and further as a potential drug for treating malignant tumors.

Description

A miRNA biosynthesis inhibitor

Technical Field

The invention belongs to the field of chemical medicine, and specifically relates to a miRNA biosynthesis inhibitor.

Background Art

Cancer has become the leading cause of death in Chinese cities and the second leading cause of death in rural areas. Although my country has prioritized the development of anticancer drugs, there are currently two main difficulties in the research of anticancer drugs: First, there are too few drug targets: there are only about 300 targets of more than 2,000 drugs currently on the market, mainly distributed in seven gene families such as hormone receptors and GPCRs. Second, the lack of understanding of the occurrence of the disease and the protein signaling network has led to the failure of clinical drug development and high costs.

miRNA has important functions in the body, and abnormal expression of miRNA can lead to the occurrence of many diseases. Therefore, miRNA has become a new drug target and diagnostic marker that has received increasing attention in cancer treatment in recent years. TRBP has diverse biological functions and is related to the proliferation, differentiation and migration of various malignant tumor cells. In animal cells, TRBP acts as a Dicer partner. TRBP can affect the malignant metastasis of cancer cells by affecting the maturation of miRNA mediated by Dicer and Ago2.

However, the inhibitory effects of the existing miRNA inhibitors are far from meeting clinical requirements. Therefore, it is necessary to further study the inhibitory mechanism of compounds on miRNA and develop new inhibitors with better inhibitory effects on the generation of miRNA in tumor treatment.

Summary of the invention

The object of the present invention is to provide a compound that effectively inhibits the biosynthesis of miRNA-21.

The present invention provides a compound represented by formula I, or a conformational isomer thereof, or an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

in:

X is selected from O and S; Y is selected from N;

R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently selected from hydrogen, substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, cyano, nitro, substituted or unsubstituted phenyl, and -COOR ₉ ;

or, R ₁ , R ₂ , R ₅ are each independently selected from hydrogen, substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, cyano, nitro, substituted or unsubstituted phenyl, -COOR ₉ , R ₃ , R ₄ together with the two carbon atoms to which they are connected form a benzene ring; the substituent is selected from halogen, C1-4 alkyl, hydroxyl, carboxyl, nitro, amino, carboxyl, C ₂ -C ₄ alkenyl, C ₁ -C ₄ alkyl, C ₂ -C ₄ alkynyl, carbocyclic group, heterocyclic group;

--COOR₉、-CONHR₁₀，其中，R₉、R₁0各自独立地选自C1-2烷基、

其中L₀、L₁、L₂、L₃各自独立地选自1-4个亚甲基，R₁₁选自卤素或羟基；R ₆ and R ₈ are each independently selected from hydrogen, hydroxy, halogen, C1-4 alkyl, C3-6 cycloalkane, phenyl,

--COOR ₉ , -CONHR ₁₀ , wherein R ₉ , R ₁ 0 are each independently selected from C1-2 alkyl,

wherein L ₀ , L ₁ , L ₂ , and L ₃ are each independently selected from 1 to 4 methylene groups, and R ₁₁ is selected from halogen or hydroxyl;

When X is O, Y is N, R ₁ , R ₂ , R ₄ , and R ₅ are hydrogen, R ₃ is methoxy, R ₆ is methyl, and R ₈

When it is -COOR ₉ , R ₉ is not ethyl.

Further,

X is selected from O and S; Y is selected from N;

R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently selected from hydrogen, substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, and substituted or unsubstituted phenyl; or, R ₁ , R ₂ , and R ₅ are each independently selected from hydrogen, substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, and substituted or unsubstituted phenyl, and R ₃ and R ₄ together with the two carbon atoms to which they are connected form a benzene ring; the substituent is selected from halogen;

其中L₀选自1-4个亚甲基。R ₆ and R ₈ are each independently selected from C1-2 alkyl, -COOR ₉ , wherein R ₉ is selected from C1-2 alkyl,

Wherein L ₀ is selected from 1-4 methylene groups.

Further,

X is selected from O; Y is selected from N;

R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently selected from hydrogen, substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, and substituted or unsubstituted phenyl; the substituent is selected from halogen;

其中L₀选自2个亚甲基。R ₆ and R ₈ are each independently selected from C1-2 alkyl, -COOR ₉ , wherein R ₉ is selected from C1-2 alkyl,

Wherein L ₀ is selected from 2 methylene groups.

Further,

The structure of the compound is shown in Formula I-1:

in:

_R6 is selected from C1-2 alkyl;

其中L₀选自2个亚甲基； _R7 is selected from C1-2 alkyl,

Wherein L ₀ is selected from 2 methylene groups;

_R3 is selected from methyl, methoxy, phenyl, halogen, preferably methyl, methoxy, phenyl, chlorine, bromine.

Further,

X is selected from S; Y is selected from N;

R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently selected from hydrogen, substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, and substituted or unsubstituted phenyl; or, R ₁ , R ₂ , and R ₅ are each independently selected from hydrogen, substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, and substituted or unsubstituted phenyl, and R ₃ and R ₄ together with the two carbon atoms to which they are connected form a benzene ring; the substituent is selected from halogen;

其中L₀选自1-4个亚甲基。R ₆ and R ₈ are each independently selected from C1-2 alkyl, -COOR ₉ , wherein R ₉ is selected from C1-2 alkyl,

Wherein L ₀ is selected from 1-4 methylene groups.

Further,

X is selected from S; Y is selected from N;

R ₁ , R ₂ , R ₄ , and R ₅ are hydrogen, and R ₃ is selected from substituted or unsubstituted C1-2 alkyl, substituted or unsubstituted C1-2 alkoxy, halogen, and substituted or unsubstituted phenyl, wherein the substituent is selected from halogen; or, R ₁ , R ₂ , and R ₅ are hydrogen, and R ₃ , R ₄ and the two carbon atoms to which they are connected together form a benzene ring;

L₀为2个亚甲基。R ₆ and R ₈ are each independently selected from C1-2 alkyl, -COOR ₉ , wherein R ₉ is selected from C1-2 alkyl,

L ₀ is 2 methylene groups.

Further,

Its structure is as shown in Formula I-2:

in:

L₀为2个亚甲基；Among R ₆ and R ₈ , one is C1-2 alkyl and the other is -COOR ₉ , wherein R 9 is selected from C1-2 alkyl,

L ₀ is 2 methylene groups;

R ₃ is selected from halogenated or unhalogenated C1-2 alkyl, C1-2 alkoxy, halogen, phenyl, preferably trifluoromethyl, methoxy, ethoxy, methyl; R ₄ is selected from hydrogen, methyl; or, R ₃ and R ₄ together with the two carbon atoms to which they are connected form a benzene ring.

Further,

The compound is:

The present invention also provides use of the above compound or a pharmaceutically acceptable salt thereof as a miRNA-21 biosynthesis inhibitor.

Furthermore, the inhibitor is a drug for treating tumors, preferably a drug for treating malignant tumors.

The present invention provides a compound shown in Formula I or a pharmaceutically acceptable salt thereof, which can tightly bind to the relevant binding protein in the biosynthesis process of miRNA and effectively inhibit the synthesis of miRNA-21. The active compound prepared by the present invention can be used as a miRNA-21 inhibitor and further as a potential drug for treating malignant tumors.

"Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

The minimum and maximum carbon atom content of the hydrocarbon group is indicated by the prefix, for example, C1-4 alkyl indicates any alkyl group containing 1 to 4 carbon atoms.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the relative fluorescence intensity of the compound prepared by the present invention and the reference compound 3b in the activity screening experiment.

FIG2 shows the relative fluorescence intensity of some active compounds of the present invention and the reference compound 3b.

FIG. 3 shows the results of the Hela-luciferase-miRNA-21 cytotoxic activity detection test.

Figure 4 is a 2D (left) and 3D (right) diagram of the docking of the compound of the present invention with the Dicer-TRBP molecule. The colors in the 2D diagram represent: the amino acid residues are surrounded by colored halos, indicating the solvent accessible surface area of the interacting residues; wherein the colors corresponding to the numbers represent: (1): amino acid residues that form alkyl-π interactions with the alkyl group of the compound; (2): amino acid residues that form intermolecular van der Waals interactions with the compound; (3): amino acid residues that form intermolecular hydrogen bonds with the compound; (4): amino acid residues that form intermolecular C-H hydrogen bonds with the compound; (5): amino acid residues that form Cation-π interactions with the compound; (6): amino acid residues that form intermolecular Halogen-H (Cl, Br, I) hydrogen bonds with the compound; (7): amino acid residues that form intermolecular π-π conjugated interactions with the compound.

Figure 5 is a 2D (left) and 3D (right) diagram of the docking of the compound of the present invention with the TRBP (dsRBD2) molecule. The colors in the 2D diagram represent: the amino acid residues are surrounded by colored halos, indicating the solvent accessible surface area of the interacting residues; wherein the colors corresponding to the numbers represent: (1): amino acid residues that form alkyl-π interactions with the alkyl group of the compound; (2): amino acid residues that form intermolecular van der Waals interactions with the compound; (3): amino acid residues that form intermolecular hydrogen bonds with the compound; (4): amino acid residues that form intermolecular C-H hydrogen bonds with the compound; (5): amino acid residues that form Cation-π interactions with the compound; (6): amino acid residues that form intermolecular Halogen-H (Cl, Br, I) hydrogen bonds with the compound; (7): amino acid residues that form intermolecular π-π conjugated interactions with the compound.

FIG. 6 is a diagram of a cell screening model.

DETAILED DESCRIPTION

The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

Example 1. Synthesis of the compounds of the present invention

Ethyl 2-(4-methoxyphenyl)-4-methylthiazole-5-carboxylate (T2): 4-methoxybenzamide (152.2 mg, 1 mmol), Lawesson's reagent (485.4 mg, 1.2 mmol) and THF (20 mL) were added to a 25 mL round-bottom flask. After reacting at room temperature for 4 h, Lawesson's reagent and solvent were removed, and the intermediate thiobenzamide was obtained by column chromatography separation and purification, and then ethyl 3-bromo-2-oxobutyrate (209 mg, 1 mmol) was refluxed at 70°C for 4 h in ethanol (10 mL) as solvent. After the reaction was completed, the solvent was removed by treatment, and compound T2 was obtained by column chromatography separation and purification. Yield 92%, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝3:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.91(d, J＝8.8Hz, 2H), 6.96(d, J＝8.8Hz, 2H), 4.37(q, J＝7.1Hz, 2H), 3.86(s, 3H), 2.76(s, 3H), 1.68(s, 1H), 1.40(t, J＝7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ169.9, 162.4, 161.9, 161.0, 128.4, 125.9, 120.9, 114.4, 61.1, 55.5, 17.6, 14.4; HR-ESI-MS (positive mode)m/z:278.0851[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₃ S:278.0851), m/z:300.0668[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₃ SNa:300.0670).

Ethyl 2-(4-methoxyphenyl)-5-methyl-1H-imidazole-4-carboxylate (T3): In a 25 mL round-bottom flask, ethoxycarbonyl methylene triphenylphosphine (CEMTPP) (1044.2 mg, 3 mmol), acetyl chloride (474.5 mg, 6 mmol), N,N-diisopropylethylamine (DIPEA) (516.9 mg, 4 mmol), and dichloromethane (DCM) (20 mL) were added and reacted for 1 h to obtain 3-triphenylphosphine ethyl acetoacetate. Oxone (614.7 mg, 1 mmol) was then added to THF for oxidation. After 4 h of reaction, the intermediate ethyl 2,3-dioxobutyrate was obtained. The intermediate was then reacted with 4-methoxybenzaldehyde (272.3 mg, 2 mmol) in acetic acid solvent (10 mL) at 100°C for 12 h. After the reaction was completed, the solvent was removed by treatment and the compound T3 was obtained by separation and purification by column chromatography. Yield 69%, white solid, developing system PE/EA10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz,CDCl ₃ )7.84(d,J＝8.8Hz,2H),6.94(d,J＝8.8Hz,2H),4.38(q,J＝7.1Hz,2H),3.84(s,3H),2.54(s,3H),1.39(t,J＝7.1Hz); ¹³ C NMR (100MHz,CDCl ₃ )δ160.8,127.3,121.8,114.3,60.6,55.4,14.5; HR-ESI-MS (positive mode)m/z:261.1236[M+H] ⁺ (Calcd for C ₁₄ H ₁₇ N ₂ O ₃ :261.1239), m/z:283.1052[M+Na] ⁺ (Calcd for C ₁₄ H ₁₆ N ₂ O ₃ Na:283.1059).

2-hydroxyethyl2-(4-methoxyphenyl)-5-methyloxazole-4-carboxylate (T4): Compound 3b (ethyl 5-methyl-2-(4-methoxyphenyl)-4-oxazolecarboxylate, 261.3 mg, 1 mmol) and EtOH/10% NaOH = 1:1 (20 mL) were added to a 25 mL round-bottom flask, and the mixture was reacted at 70°C for 1 hour, and then the pH was adjusted to between 1 and 2 with concentrated hydrochloric acid, extracted with dichloromethane, and the organic phase was concentrated to obtain intermediate 3, which was directly used for the next step without further purification. 20 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (383.4 mg, 2 mmol), 4-dimethylaminopyridine (DMAP) (24.4 mg, 0.2 mmol), and ethylene glycol (124.1 mg, 2 mmol) were added to intermediate 3, and the reaction system was heated to 80°C and refluxed for 6 h. After the reaction was completed, 20 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined and concentrated, and then separated and purified by column chromatography (petroleum ether/ethyl acetate/methanol=10/1/0.01) to obtain compound T4. The yield was 77%, and the product was a white solid. The developing system was PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400 MHz, CDCl ₃ ) 7.97 (d, J=8.9 Hz, 2H), 6.97 (d, J=8.9 Hz, 2H), 4.46 (t, J=9.2 Hz, 2H), 3.97 (t, J=9.2 Hz, 2H), 3.86 (s, 3H), 2.67 (s, 3H), 2.21 (s, 1H); ¹³ C NMR (100 MHz, CDCl ₃ )δ162.6,161.8,159.9,156.3,128.3,128.0,119.0,114.3,66.8,61.0,55.4,12.1; HR-ESI-MS(positive mode)m/z:278.1023[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₅ :278.1028 ),m/z:300.0839[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₅ Na:300.0848).

2-hydroxyethyl2-(4-methoxyphenyl)-4-methylthiazole-5-carboxylate (T5): Compound T5 was prepared from T2 by referring to the synthesis method of T4. Yield 80%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.91 (d, J=8.8Hz, 2H), 6.96 (d, J=8.9Hz, 2H), 4.44 (t, J=4.6Hz, 2H), 3.94 (q, J=4.1Hz, 2H), 3.86 (s, 3H), 2.76 (s, 3H), 1.67 (s, 1H); ¹³ C NMR (100MHz, CDCl ₃ )δ170.3,162.6,162.1,161.7,128.5,125.7,120.1,114.4,66.7,61.3,55.5,17.6; HR-ESI-MS(positive mode)m/z:294.0796[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₄ S: 294.080 0),m/z:316.0612[M+Na] ⁺ (Calcd forC ₁₄ H ₁₅ NO ₄ SNa:316.0619).

Ethyl 2-(4-methoxyphenyl)-4-methyloxazole-5-carboxylate (T6): In a 25 mL round-bottom flask, 4-methoxybenzamide (152.2 mg, 1 mmol), ethyl 2-chloroacetoacetate (329.2 mg, 2 mmol), and solvent ethanol (20 mL) were added and reacted in a sealed tube at 100 °C for 24 h to obtain compound T6. Yield 87%, compound T6, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝3:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.06 (d, J＝9.0Hz, 2H), 6.97 (d, J＝9.0Hz, 2H), 4.42 (q, J＝7.2Hz, 2H), 3.86 (s, 3H), 2.51 (s, 3H), 1.42 (t, J＝7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ162.4, 162.3, 159.0, 147.1, 137.0, 129.0, 119.1, 114.3, 61.0, 55.4, 14.4, 13.5; HR-ESI-MS (positive mode)m/z:262.1072[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₄ :262.1079),m/z:284.0895[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₄ Na:284.0899).

Ethyl 2-(4-methoxyphenyl)-4-methylthiazole-5-carboxylate (T7): 4-methoxybenzamide (152.2 mg, 1 mmol), Lawesson's reagent (485.4 mg, 1.2 mmol) and THF (20 mL) were added to a 25 mL round-bottom flask and reacted at room temperature for 4 h. The Lawesson's reagent and the solvent were removed and separated and purified by column chromatography to obtain the intermediate thiobenzamide. The mixture was then refluxed at 70 ° C with ethyl 2-chloroacetoacetate (329.2 mg, 2 mmol) in ethanol (20 mL) for 6 hours. After the reaction was completed, the solvent was removed and separated and purified by column chromatography to obtain compound T7. Yield 92%, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝3:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.88 (d, J＝8.8Hz, 2H), 6.95 (d, J＝8.8Hz, 2H), 4.46 (q, J＝7.1Hz, 2H), 3.85 (s, 3H), 2.78 (s, 3H), 1.46 (t, J＝7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ163.7, 162.7, 161.3, 143.7, 142.0, 128.2, 125.9, 114.2, 61.1, 55.4, 14.4, 13.3; HR-ESI-MS (positive mode)m/z:278.0850[M+H] ⁺ (Calcdfor C ₁₄ H ₁₆ NO ₃ S:278.0851),m/z:300.0670[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₃ SNa:300.0670).

Ethyl 2-(4-methoxyphenyl)-4-methyl-1H-imidazole-5-carboxylate (T8): DL-alanine (540 mg, 6 mmol), 10% NaOH solvent (10 mL, pH = 11-12), and 4-methoxybenzoyl chloride (1026 mg, 6 mmol) were added to a 25 mL round-bottom flask, and the reaction was carried out at room temperature. After the reaction was completed by monitoring by TLC, the reaction raw materials and solvent were removed to obtain a crude product. EDCI (223 mg, 1 mmol) and DCM solvent (20 mL) were added, and the mixture was stirred at room temperature for 1 hour. Tributylphosphine (202.3 mg, 0.5 mmol) and ethyl cyanoformate (79 mg, 0.8 mmol) were added, and the reaction was monitored by TLC. After the reaction was completed, the solvent and raw materials were removed by post-treatment, and compound T8 was obtained by separation and purification by column chromatography. Yield 64%, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz, CD ₃ OD) 7.88 (d, J＝8.8 Hz, 2H), 7.03 (d, J＝8.8 Hz, 2H), 4.39 (q, J＝7.1 Hz, 2H), 3.86 (s, 3H), 2.56 (s, 3H), 1.43 (t, J＝7.0 Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ160.8, 127.3, 121.8, 114.3, 60.6, 55.4, 29.7, 29.4, 14.5; HR-ESI-MS (positive mode) m/z: 261.1246 [M+H] ⁺ (Calcd for C ₁₄ H ₁₇ N ₂ O ₃ :261.1239),m/z:283.1063[M+Na] ⁺ (Calcd for C ₁₄ H ₁₆ N ₂ O ₃ Na:283.1059).

2-hydroxyethyl 2-(4-methoxyphenyl)-4-methyloxazole-5-carboxylate (T9): The synthesis of compound T9 was based on the synthesis method of T4. Yield 81%, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 8.06 (d, J=9.0Hz, 2H), 6.98 (d, J=9.0Hz, 2H), 4.49 (t, J=4.6Hz, 2H), 3.97 (t, J=4.6Hz, 2H), 3.88 (s, 3H), 2.53 (s, 3H), 1.69 (s, 1H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.7,162.4,159.1,148.0,136.5,129.1,118.9,114.3,66.5,61.3,55.5,13.6; HR-ESI-MS(positive mode)m/z:278.1017[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₅ :278.1028 ),m/z:300.0832[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₄ Na:300.0848).

2-hydroxyethyl 2-(4-methoxyphenyl)-4-methylthiazole-5-carboxylate (T10): Compound T10 was prepared from T7 by referring to the synthesis method of T4. Yield 84%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.81 (d, J=8.8Hz, 2H), 6.94 (d, J=8.8Hz, 2H), 4.45 (t, J=4.4Hz, 2H), 3.96 (t, J=4.4Hz, 2H), 3.85 (s, 3H), 2.76 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ164.1,162.6,161.5,144.7,141.2,128.2,125.5,114.3,67.0,60.9,55.4,13.3; HR-ESI-MS(positive mode)m/z:294.0800[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₄ S: 294.080 0),m/z:316.0618[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₄ SNa: 316.0619).

Ethyl 5-methyl-2-(pyridin-4-yl)oxazole-4-carboxylate (T11): The synthesis of compound T11 was based on the synthesis method of 3b. Yield 61%, white solid, developing system PE/EA10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz,CDCl ₃ )8.74(d,J＝6.1Hz,2H),7.92(d,J＝6.2Hz,2H),4.46(q,J＝7.1Hz,2H),2.73(s,3H),1.44(t,J＝7.1Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ162.0,157.4,157.3,150.6,133.5,129.5,120.1,61.3,14.4,12.3; HR-ESI-MS (positive mode)m/z:233.0920[M+H] ⁺ (Calcd for C ₁₁ H ₁₂ NO ₅ :233.0926), m/z:255.0740[M+Na] ⁺ (Calcd for C ₁₁ H ₁₁ NO ₅ Na:255.0746).

Ethyl 5-methyl-2-(pyridin-2-yl)oxazole-4-carboxylate (T12): The synthesis of compound T12 was based on the synthesis method of 3b. Yield 67%, white solid, developing system PE/EA 10:1 (Rf = 0.5, PE/EA = 5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.71 (d, J = 4.2 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.83-7.79 (m, 1H), 7.39-7.36 (m, 1H), 4.44 (q, J = 7.2 Hz, 2H), 2.74 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.2,158.3,157.5,149.9,145.4,137.0,129.1,125.1,122.5,61.2,14.4,12.4; HR-ESI-MS (positive mode)m/z:233.0925[M+H] ⁺ (Calcd for C ₁₁ H ₁₂ NO ₅ :233.092 6),m/z:255.0749[M+Na] ⁺ (Calcd for C ₁₁ H ₁₁ NO ₅ Na:255.0746).

2-hydroxyethyl 5-methyl-2-(pyridin-2-yl)oxazole-4-carboxylate (T13): Compound T13 was prepared from T12 by referring to the synthesis method of T4. Yield: 60%, white solid, developing system: PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.70 (dd, J=1.1Hz, 1H), 7.46 (dd, J=1.1Hz, 1H), 7.12 (q, J=3.7Hz, 1H), 4.46 (t, J=4.6Hz, 2H), 3.97 (t, J=4.6Hz, 2H), 3.21 (s, 1H), 2.66 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.3,156.3,155.9,129.1,128.7,128.6,128.2,128.0,66.8,60.9,12.1; HR-ESI-MS(positive mode)m/z:247.1772[MH] ^- (Calcd for C ₁₂ H ₁₁ N ₂ O ₄ :247.0719),m/ z:271.0686[M+Na] ⁺ (Calcd for C ₁₂ H ₁₂ N ₂ O ₄ Na:271.0695).

Ethyl 2-(furan-2-yl)-5-methyloxazole-4-carboxylate (T14): The synthesis of compound T14 was based on the synthesis method of 3b. Yield 66%, colorless liquid, developing system PE/EA10:1 (Rf＝0.5, PE/EA＝5:1), ¹ HNMR (400MHz,CDCl ₃ )7.54(dd,J＝0.6Hz,1H),7.08(dd,J＝0.5Hz,1H),6.53(q,J＝1.8Hz,1H),4.42(q,J＝7.1Hz,2H),2.68(s,3H),1.41(t,J＝7.1Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ162.2,155.7,152.3,144.7,142.1,128.6,112.3,111.9,61.1,14.4,12.1; HR-ESI-MS (positive mode)m/z:222.0763[M+H] ⁺ (Calcd for C ₁₁ H ₁₂ NO ₄ :222.0766),m/z:244.0590[M+Na] ⁺ (Calcd for C ₁₁ H ₁₁ NO ₄ Na:244.0586).

2-hydroxyethyl 2-(furan-2-yl)-5-methyloxazole-4-carboxylate (T15): The synthesis route of compound T15 is based on T14 and is obtained by referring to the synthesis method of T4. Yield 60%, colorless liquid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.57 (dd, J＝0.6Hz, 1H), 7.06 (dd, J＝0.5Hz, 1H), 6.54 (q, J＝1.8Hz, 1H), 4.45 (t, J＝4.6Hz, 2H), 3.97 (t, J＝4.7Hz, 2H), 3.59 (s, 1H), 2.66 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.2,156.2,152.3,144.9,141.8,128.1,112.6,112.0,66.8,60.7,12.0; HR-ESI-MS (positive mode)m/z:238.0718[M+H] ⁺ (Calcd for C ₁₁ H ₁₂ NO ₅ : 238.0715), m/z :260.0538[M+Na] ⁺ (Calcd forC ₁₁ H ₁₁ NO ₅ Na:260.0535).

Ethyl 5-methyl-2-(thiophen-2-yl)oxazole-4-carboxylate (T16): The synthesis of compound T16 was based on the synthesis method of 3b. Yield 70%, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.72 (dd, J＝1.2Hz, 1H), 7.44 (dd, J＝1.2Hz, 1H), 7.11 (q, J＝3.7Hz, 1H), 4.43 (q, J＝7.1Hz, 2H), 2.67 (s, 3H), 1.42 (t, J＝7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ162.3, 155.9, 155.7, 129.0, 128.8, 128.7, 128.5, 127.9, 61.1, 14.4, 12.2; HR-ESI-MS (positive mode)m/z:238.0545[MH] ^- (Calcd for C ₁₁ H ₁₂ NO ₃ S:238.0538),m/z:260.0359[M+Na] ⁺ (Calcd for C ₁₁ H ₁₁ NO ₃ SNa:260.0357).

2-hydroxyethyl 5-methyl-2-(thiophen-2-yl)oxazole-4-carboxylate (T17): The synthesis route of compound T17 is based on T16 and is obtained by referring to the synthesis method of T4. Yield 74%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.70 (dd, J＝1.2Hz, 1H), 7.46 (dd, J＝1.2Hz, 1H), 7.12 (q, J＝3.7Hz, 1H), 4.45 (t, J＝4.6Hz, 2H), 3.97 (t, J＝4.6Hz, 2H), 3.11 (s, 1H), 2.66 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.3,156.3,155.9,129.1,128.7,128.6,128.2,128.0,66.8,60.9,12.1; HR-ESI-MS(positivemode)m/z:254.0465[MH] ^- (Calcd for C ₁₁ H ₁₂ NO ₄ S: 254.0487),m/z: 276.0288[M+Na] ⁺ (Calcd for C ₁₁ H ₁₁ NO ₄ SNa: 276.0306).

Ethyl 5-methyl-2-phenyloxazole-4-carboxylate (T18): The synthesis of compound T18 was based on the synthesis method of 3b. Yield 64%, white solid, developing system PE/EA 10:1 (Rf＝0.6, PE/EA＝3:1), ¹ H NMR (400MHz,CDCl ₃ )8.06-8.03(m,3H),7.74(d,J＝2.6Hz,2H),4.43(q,J＝7.1Hz,2H),2.68(s,3H),1.41(t,J＝7.1Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ162.5,159.7,156.2,130.7,128.8,128.7,126.6,61.1,14.4,12.2; HR-ESI-MS (positive mode)m/z:232.0965[M+H] ⁺ (Calcdfor C ₁₃ H ₁₄ NO ₃ :232.0974), m/z:254.0787[M+Na] ⁺ (Calcd for C ₁₃ H ₁₃ NO ₃ Na: 254.0793).

2-hydroxyethyl 5-methyl-2-phenyloxazole-4-carboxylate(T19): The synthesis of compound T19 refers to the synthesis method of T4. Yield 61%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 8.02-8.00 (m, 2H), 7.46-7.45 (m, 3H), 4.45 (t, J＝4.5, 2H), 3.97 (t, J＝4.6, 2H), 2.69 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ162.4, 159.8, 156.8, 131.0, 128.9, 128.3, 126.5, 126.3, 66.8, 60.9, 12.1; HR-ESI-MS (positive mode)m/z:248.0917[M+H] ⁺ (Calcd for C ₁₃ H ₁₄ NO ₄ :248.0923),m/z:270.0723[M+Na] ⁺ (Calcd for C ₁₃ H ₁₃ NO ₄ Na:270.0742).

Ethyl 2-(2-methoxyphenyl)-5-methyloxazole-4-carboxylate (T20): The synthesis of compound T20 was based on the synthesis method of 3b. Yield 69%, colorless liquid, developing system PE/EA 10:1 (Rf=0.5, PE/EA=5:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.93 (dd, J=1.7Hz, 1H), 7.42-7.38 (m, 1H), 7.01-6.97 (m, 2H), 4.41 (q, J=7.1Hz, 2H), 3.91 (s, 3H), 2.68 (s, 3H), 1.40 (t, J=7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.6,158.3,157.7,156.0,132.1,130.7,128.5,120.5,115.8,111.7,69.9,55.9,14.4,12.2; HR-ESI-MS(positive mode)m/z:262.1082[M+H] ⁺ (Calcd forC ₁₄ H ₁₆ NO ₄ :262.1079), m/z:284.0920[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₄ Na:284.0899).

2-hydroxyethyl 2-(2-methoxyphenyl)-5-methyloxazole-4-carboxylate (T21): The synthesis of compound T21 was based on the synthesis method of T4. Yield 66%, colorless liquid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.90 (dd, J=1.7Hz, 1H), 7.46-7.41 (m, 1H), 7.04-7.00 (m, 2H), 4.43 (t, J=4.5Hz, 2H), 3.93-3.91 (m, 5H), 3.33 (s, 1H), 2.68 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.6,158.3,157.7,156.6,132.3,130.5,128.0,120.6,115.5,111.8,66.7,60.8,55.9,12.1; HR-ESI-MS(positive mode)m/z:278.1027[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₅ :278.1028), m/z:300.0848[M+Na] ⁺ (Calcd forC ₁₄ H ₁₅ NO ₅ Na:300.0848).

Ethyl 2-(3-methoxyphenyl)-5-methyloxazole-4-carboxylate (T22): The synthesis of compound T22 was based on the synthesis method of 3b. Yield 70%, white solid, developing system PE/EA 10:1 (Rf=0.5, PE/EA=5:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.63 (d, J=7.2Hz, 1H), 7.58 (t, J=2.4Hz, 1H), 7.34 (t, J=8.0Hz, 1H), 6.99 (dd, J=2.5Hz, 1H), 4.43 (q, J=7.1Hz, 2H), 3.85 (s, 3H), 2.68 (s, 3H), 1.42 (t, J=7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.5,159.8,159.6,156.2,129.8,128.8,127.8,119.0,117.5,111.0,61.1,55.5,14.4,12.3; HR-ESI-MS(positive mode)m/z:262.1073[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₄ :262.1079), m/z:284.0899[M+Na] ⁺ (Calcd forC ₁₄ H ₁₅ NO ₄ Na:284.0899).

2-hydroxyethyl 2-(3-methoxyphenyl)-5-methyloxazole-4-carboxylate(T23): The synthesis of compound T23 refers to the synthesis method of T4. Yield 73%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.61(d, J＝7.2Hz,1H), 7.55(t, J＝2.4Hz,1H), 7.37(t, J＝8.0Hz,1H), 7.02(dd, J＝2.5Hz,1H), 4.47(t, J＝4.6Hz,2H), 3.98(t, J＝4.6Hz,2H), 3.86(s,3H), 3.27(s,1H), 2.66(s,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ162.5,159.9,159.6,156.7,130.0,128.3,127.5,119.0,117.5,111.1,66.8,60.9,55.5,12.2; HR-ESI-MS(positive mode)m/z:278.1017[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₅ :278.1028), m/z:300.0841[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₅ Na:300.0848).

Ethyl 5-methyl-2-(p-tolyl)oxazole-4-carboxylate (T24): The synthesis of compound T24 was based on the synthesis method of 3b. Yield 76%, colorless liquid, developing system PE/EA 10:1 (Rf=0.5, PE/EA=5:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.99 (d, J=8.2Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.47 (q, J=7.1Hz, 2H), 2.71 (s, 3H), 2.41 (s, 3H), 1.45 (t, J=7.2Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.6,159.9,155.9,141.1,129.4,128.7,126.6,123.9,61.0,21.5,14.4,12.2; HR-ESI-MS(positivemode)m/z:246.1122[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₃ :246.1130 ),m/z:268.0949[M+Na] ⁺ (Calcdfor C ₁₄ H ₁₅ NO ₃ Na: 268.0950).

2-hydroxyethyl 5-methyl-2-(p-tolyl)oxazole-4-carboxylate (T25): The synthesis of compound T25 was based on the synthesis method of T4. Yield 71%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.92 (d, J=8.2Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 4.48 (t, J=4.6Hz, 2H), 3.99 (t, J=4.7Hz, 2H), 3.89 (s, 1H), 2.66 (s, 3H), 2.41 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.5,159.9,156.4,141.4,129.5,128.1,126.5,123,6,66.7,60.8,21.6,12.1; HR-ESI-MS(positive mode)m/z:262.1082[M+H] ⁺ (Calcd forC ₁₄ H ₁₆ NO ₄ :261.1079 ),m/z:284.0905[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₄ Na:284.0899).

Ethyl 2-(4-(fluorooxy)phenyl)-5-methyloxazole-4-carboxylate(T26): The synthesis route of compound T26 refers to the synthesis method of 3b. Yield 80%, white solid, developing system PE/EA10:1 (Rf=0.5, PE/EA=5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.13 (d, J=8.8Hz, 2H), 7.31 (d, J=9.2Hz, 2H), 4.47 (q, J=7.2Hz, 2H), 2.72 (s, 3H), 1.45 (t, J=7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ162.3, 158.4, 156.5, 150.9 (d, J=1.9Hz), 129.0, 128.3, 125.2, 124.2 121.6,121.0,119.1,61.1,14.4,12.2; HR-ESI-MS(positive mode)m/z:316.0792[M+H] ⁺ (Calcd for C ₁₄ H ₁₃ F ₃ NO ₄ :316.0797), m/z:338.0612[M+Na] ⁺ (Calcd for C ₁₄ H ₁₂ F ₃ NO ₄ Na:338.0616).

2-hydroxyethyl5-methyl-2-(4-(trifluoromethoxy)phenyl)oxazole-4-carboxylate (T27): The synthesis of compound T27 was based on the synthesis method of T4. Yield 84%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 8.08 (d, J＝8.9Hz, 2H), 7.32 (d, J＝8.1Hz, 2H), 4.49 (t, J＝4.6Hz, 2H), 4.00 (t, J＝4.7Hz, 2H), 3.47 (s, 1H), 2.70 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.3,158.5,157.0,151.0(d,J＝1.9Hz),128.5,128.2,124.9,121.6,121.1,119.0,66.7,60.9,12.1; HR-ESI-MS(positive mode)m/z:332.0740[M+H] ⁺ (Calcd for C _{1 4} H ₁₃ F ₃ NO ₅ :332.0746), m/z: 354.0563[M+Na] ⁺ (Calcd for C ₁₄ H ₁₂ F ₃ NO ₅ Na: 354.0565).

Ethyl 2-(4-fluorophenyl)-5-methyloxazole-4-carboxylate (T28): The synthesis of compound T28 was based on the synthesis method of 3b. Yield 83%, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.07-8.04 (m, 2H), 7.15-7.11 (m, 2H), 4.44 (q, J＝7.1Hz, 2H), 2.69 (s, 3H), 1.43 (t, J＝7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ165.5, 163.0, 162.4, 158.8, 156.2, 128.8 (d, J＝8.8Hz), 123.0 (d, J＝3.4Hz), 116.1 (d, J＝22.1Hz), 61.1, 14.4, 12.2; HR-ESI-MS (positive mode)m/z:250.0877[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ FNO ₃ :250.0879),m/z:272.0698[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ FNO ₃ Na:272.0699).

2-hydroxyethyl 2-(4-fluorophenyl)-5-methyloxazole-4-carboxylate (T29): The synthesis of compound T29 was based on the synthesis method of T4. Yield 86%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 8.02-7.97 (m, 2H), 7.16-7.10 (m, 2H), 4.45 (t, J=4.6Hz, 2H), 3.96 (t, J=4.7Hz, 2H), 3.71 (s, 1H), 2.64 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ165.6,163.1,162.3,158.9,156.7,128.8(d,J＝8.8Hz),128.3,122.7(d,J＝3.4Hz),116.2(d,J＝22.3Hz),66.7,60.8,12.1; HR-ESI-MS(positive mode)m/z:266.082 9[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ FNO ₄ :266.0829),m/z:288.0651[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ FNO ₄ Na:288.0648).

Ethyl 2-(4-chlorophenyl)-5-methyloxazole-4-carboxylate (T30): The synthesis of compound T30 refers to the synthesis method of 3b. Yield 80%, white solid, developing system PE/EA10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz,CDCl ₃ )8.01-7.97(m,2H),7.43-7.40(m,2H),4.44(q,J＝7.1Hz,2H),2.69(s,3H),1.42(t,J＝7.1Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ162.3,158.7,156.4,136.9,129.1,127.9,125.1,61.1,14.4,12.2; HR-ESI-MS (positive mode)m/z:266.0580[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ ClNO ₃ :266.0584), m/z:288.0409[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ ClNO ₃ Na: 288.0403).

2-hydroxyethyl 2-(4-chlorophenyl)-5-methyloxazole-4-carboxylate(T31): The synthesis of compound T31 refers to the synthesis method of T4. Yield 87%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.95 (d, J＝8.6Hz, 2H), 7.43 (d, J＝8.6Hz, 2H), 4.47 (t, J＝4.5Hz, 2H), 3.97 (t, J＝4.6Hz, 2H), 3.20 (s, 1H), 2.67 (s, 3H); ¹³ CNMR (100MHz, CDCl ₃ ) δ162.3, 158.8, 156.9, 137.2, 129.2, 128.5, 127.8, 124.8, 66.8, 60.9, 12.2; HR-ESI-MS (positive mode)m/z:282.0532[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ ClNO ₄ :282.0533),m/z:304.0348[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ ClNO ₄ Na:304.0353).

Ethyl 2-(4-bromophenyl)-5-methyloxazole-4-carboxylate (T32): The synthesis of compound T32 was based on the synthesis method of 3b. Yield 90%, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz,CDCl ₃ )7.92(d,J＝8.6Hz,2H),7.58(d,J＝8.6Hz,2H),4.43(q,J＝7.2Hz,2H),2.68(s,3H),1.42(t,J＝7.1Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ162.3,158.8,156.4,132.0,130.0,128.0,125.5,125.3,61.1,14.4,12.3; HR-ESI-MS (positive mode)m/z:310.0087[M+H] ⁺ (Calcd for C ₁₃ H _13b rNO ₃ :310.0079), m/z:331.9902[M+Na] ⁺ (Calcd forC ₁₃ H ₁₂ BrNO ₃ Na:331.9898).

2-hydroxyethyl 2-(4-bromophenyl)-5-methyloxazole-4-carboxylate(T33): The synthesis of compound T33 refers to the synthesis method of T4. Yield 87%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.90 (d, J＝8.6Hz, 2H), 7.60 (d, J＝8.5Hz, 2H), 4.48 (t, J＝4.5Hz, 2H), 3.97 (t, J＝4.7Hz, 2H), 3.03 (s, 1H), 2.68 (s, 3H); ¹³ CNMR (100MHz, CDCl ₃ ) δ162.4, 158.9, 157.0, 132.2, 128.5, 128.0, 125.5, 125.3, 66.8, 61.0, 12.2; HR-ESI-MS (positive mode)m/z:326.0027[M+H] ⁺ (Calcd for C ₁₃ H _13b rNO ₃ :326.0028), m/z:347.9847[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ BrNO ₃ Na:347.9847).

Ethyl 2-([1,1'-biphenyl]-4-yl)-5-methyloxazole-4-carboxylate (T34): The synthesis of compound T34 was based on the synthesis method of 3b. Yield 87%, white solid, developing system PE/EA 10:1 (Rf=0.5, PE/EA=5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.15 (d, J=8.3 Hz, 2H), 7.69 (d, J=8.3 Hz, 2H), 7.64 (d, J=7.3 Hz, 2H), 7.48 (t, J=7.2 Hz, 2H), 7.40 (t, J=7.3 Hz, 1H), 4.46 (q, J=7.1 Hz, 2H), 2.72 (s, 3H), 1.45 (t, J=7.1 Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.5,159.5,156.2,143.4,140.0,128.9,128.0,127.4,127.1,127.1,125.5,61.1,14.4,12.3; HR-ESI-MS(positivemode)m/z:308.1284[M+H] ⁺ (Calcd for C ₁₉ H _{1 8} NO ₃ :308.1287), m/z:330.1097[M+Na] ⁺ (Calcdfor C ₁₉ H ₁₇ NO ₃ Na:330.1106).

2-hydroxyethyl 2-([1,1'-biphenyl]-4-yl)-5-methyloxazole-4-carboxylate(T35): The synthesis of compound T35 refers to the synthesis method of T4. Yield 87%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 8.09 (d, J＝8.4Hz, 2H), 7.69 (d, J＝8.7Hz, 2H), 7.64 (d, J＝7.2Hz, 2H), 7.48 (t, J＝7.2Hz, 2H), 7.40 (t, J＝7.3Hz, 1H), 4.49 (t, J＝4.5Hz, 2H), 4.00 (t, J＝4.6Hz, 2H), 3.20 (s, 1H), 2.69 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.5,159.6,156.8,143.6,139.9,129.0,128.4,128.0,127.5,127.1,125.2,66.8,61.0,12.2; HR-ESI-MS(positive mode)m/z:324.1242[M+H] ⁺ (Calcd forC ₁₉ H _{1 8} NO ₄ :324.1236), m/z:346.1058[M+Na] ⁺ (Calcd for C ₁₉ H ₁₇ NO ₄ Na:346.1055).

Ethyl2-(3,5-difluorophenyl)-5-methyloxazole-4-carboxylate (T36): The synthesis of compound T36 was based on the synthesis method of 3b. Yield 87%, white solid, developing system PE/EA 10:1 (Rf = 0.5, PE/EA = 5:1), ^{1 H} NMR (400MHz, CDCl ₃ ) 7.61-7.59 (m, 2H), 6.93-6.88 (m, 1H), 4.46 (q, J = 7.1Hz, 2H), 2.71 (s, 3H), 1.44 (t, J = 7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ164.5(d,J＝12.6Hz),162.1,162.0(d,J＝12.4Hz),155.9,129.4(t,J＝13.8Hz),109.8(d,J＝11.8Hz),109.7(d,J＝11.8Hz),106.4(t,J＝25.4Hz),61.3,1 4.4,12.3; HR-ESI-MS(positive mode)m/z:268.0773[M+H] ⁺ (Calcd for C ₁₃ H ₁₂ F ₂ NO ₃ :268.0785), m/z:290.0592[M+Na] ⁺ (Calcd for C ₁₃ H ₁₁ F ₂ NO ₃ Na:290.0605).

2-hydroxyethyl 2-(3,5-difluorophenyl)-5-methyloxazole-4-carboxylate (T37): The synthesis of compound T37 was based on the synthesis method of T4. Yield 86%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.58 (m, 2H), 6.94 (m, H), 4.49 (t, J＝4.6Hz, 2H), 3.98 (t, J＝4.7Hz, 2H), 2.71 (s, 3H), 2.55 (s, 1H); ¹³ C NMR (100MHz, CDCl ₃ )δ164.5(d,J＝12.6Hz),162.3,162.0(d,J＝12.4Hz),157.4,129.2(t,J＝13.8Hz),109.8(d,J＝11.8Hz),109.6(d,J＝11.8Hz),106.3(t,J＝25.4Hz),66.8,6 1.1,12.2; HR-ESI-MS(positive mode)m/z:284.0727[M+H] ⁺ (Calcd for C ₁₃ H ₁₂ F ₂ NO ₄ :284.0734), m/z:306.0549[M+Na] ⁺ (Calcd for C ₁₃ H ₁₁ F ₂ NO ₄ Na:306.0554).

Ethyl 2-(3,5-bis(trifluoromethyl)phenyl)-5-methyloxazole-4-carboxylate (T38): The synthesis of compound T38 was based on the synthesis method of 3b. Yield 89%, white solid, developing system PE/EA 10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.53 (s, 2H), 7.95 (s, 1H), 4.47 (q, J＝7.1Hz, 2H), 2.75 (s, 3H), 1.45 (t, J＝7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ161.9,157.4,156.8,133.0(q,J＝34.0Hz),129.5,128.6,127.0,126.6(d,J＝3.1Hz),124.3,124.0(q,J＝7.4Hz),121.5,118.8,61.4,14.4,12.3; HR- ESI-MS (positive mode)m/z:368.0703[M+H] ⁺ (Calcd for C ₁₅ H ₁₂ F ₆ NO ₃ :368.0721), m/z:390.0532[M+Na] ⁺ (Calcd for C ₁₅ H ₁₁ F ₆ NO ₃ Na:390.0541).

2-hydroxyethyl 2-(3,5-bis(trifluoromethyl)phenyl)-5-methyloxazole-4-carboxylate(T39): The synthesis of compound T39 was based on the synthesis method of T4. Yield 83%, white solid, developing system PE/EA/MeOH1:1:0.01(Rf＝0.5,PE/EA/MeOH＝1:1:0.01), ¹ H NMR(400MHz,CDCl ₃ )8.49(s,2H),7.96(s,1H),4.50(t,J＝4.5Hz,2H),3.99(t,J＝4.6Hz,2H),2.75(s,3H),2.52(s,1H); ¹³ C NMR(100MHz,CDCl ₃ )δ162.0,158.0,156.9,133.1(q,J＝34.0Hz),129.1,128.4,126.9,126.5(d,J＝3.2Hz),124.2(q,J＝7.6Hz),121.5,118.8,66.9,60.9,12.3; HR-ESI-MS (positive mode )m/z:384.0667[M+H] ⁺ (Calcd for C ₁₅ H ₁₂ F ₆ NO ₄ :384.0671),m/z:406.0487[M+Na] ⁺ (Calcd for C ₁₅ H ₁₁ F ₆ NO ₄ Na:406.0490).

Ethyl 5-ethyl-2-(4-methoxyphenyl)oxazole-4-carboxylate (T40): The synthesis of compound T40 was based on the synthesis method of 3b. Yield 94%, white solid, developing system PE/EA 10:1 (Rf=0.5, PE/EA=5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.01 (d, J=8.9Hz, 2H), 6.96 (d, J=8.9Hz, 2H), 4.43 (q, J=7.1Hz, 2H), 3.84 (s, 3H), 3.13 (q, J=7.7Hz, 2H), 1.42 (t, J=7.1Hz, 3H), 1.34 (t, J=7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.6,161.6,160.5,159.7,128.3,127.7,119.5,114.1,61.0,55.4,19.8,14.4,12.2; HR-ESI-MS(positive mode)m/z:276.1221[M+H] ⁺ (Calcd forC ₁₅ H ₁₈ NO ₄ :276 .1236),m/z:298.1038[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₄ Na:298.1055).

2-hydroxyethyl 5-ethyl-2-(4-methoxyphenyl)oxazole-4-carboxylate(T41): The synthesis of compound T41 refers to the synthesis method of T4. Yield 91%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.96 (d, J=8.7 Hz, 2H), 6.95 (d, J=8.7 Hz, 2H), 4.45 (t, J=4.3 Hz, 2H), 3.96 (t, J=4.5 Hz, 2H), 3.84 (s, 3H), 3.10 (q, J=7.6 Hz, 2H), 1.32 (t, J=7.6 Hz, 3H), 1.24 (s, 1H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.5,161.7,161.0,159.8,128.3,127.2,119.2,114.2,66.6,60.8,55.4,19.7,12.1; HR-ESI-MS(positive mode)m/z:292.1174[M+H] ⁺ (Calcd for C ₁₅ H ₁₈ NO ₅ :292 .1185),m/z:314.1002[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₅ Na:314.1004).

Ethyl 2-(4-methoxyphenyl)-5-propyloxazole-4-carboxylate (T42): The synthesis of compound T42 was based on the synthesis method of 3b. Yield 89%, white solid, developing system PE/EA 10:1 (Rf = 0.5, PE/EA = 5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.01 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 4.43 (q, J = 7.1 Hz, 2H), 3.84 (t, J = 7.4 Hz, 2H), 1.81-1.72 (m, 2H), 1.42 (t, J = 7.1 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.6,161.6,159.8,159.4,128.3,119.5,114.1,60.9,55.4,28.0,21.4,14.4,13.7; HR-ESI-MS(positive mode)m/z:290.1382[M+H] ⁺ (Calcd for C ₁₆ H ₂₀ NO ₄ :290. 1392),m/z:312.1202[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₄ Na:312.1212).

2-hydroxyethyl 2-(4-methoxyphenyl)-5-propyloxazole-4-carboxylate(T43): The synthesis of compound T43 refers to the synthesis method of T4. Yield 87%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.98 (d, J=8.8Hz, 2H), 6.97 (d, J=8.8Hz, 2H), 4.46 (t, J=4.5Hz, 2H), 3.97 (t, J=4.7Hz, 2H), 3.85 (s, 3H), 3.10 (s, 1H), 3.07 (t, J=7.4Hz, 2H), 1.81-1.72 (m, 2H), 1.03 (t, J=7.4Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.7,161.7,160.0,159.9,128.3,127.8,119.2,114.2,66.7,61.0,55.4,27.9,21.3,13.7; HR-ESI-MS(positive mode)m/z:306.1341[M+H] ⁺ (Calcd for C ₁₆ H ₂₀ NO ₅ :306.1347), m/z:328.1153[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₅ Na:328.1161).

Ethyl 5-isopropyl-2-(4-methoxyphenyl)oxazole-4-carboxylate (T44): The synthesis of compound T44 was based on the synthesis method of 3b. Yield 89%, white solid, developing system PE/EA 10:1 (Rf = 0.5, PE/EA = 5:1), ¹ HNMR (400MHz, CDCl ₃ ) 8.02 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 4.44 (q, J = 7.1 Hz, 2H), 3.85 (s, 3H), 3.84 (q, 7.0 Hz, 1H), 1.43 (t, J = 7.1 Hz, 3H), 1.36 (d, J = 7.0 Hz, 6H); ¹³ CNMR (100MHz, CDCl ₃ )δ163.7,162.6,161.6,159.5,128.3,126.7,119.5,114.1,60.9,55.4,26.2,20.8,14.4; HR-ESI-MS(positive mode)m/z:290.1380[M+H] ⁺ (Calcd forC ₁₆ H ₂₀ NO ₄ :290 .1392),m/z:312.1197[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₄ Na:312.1212).

2-hydroxyethyl 5-isopropyl-2-(4-methoxyphenyl)oxazole-4-carboxylate(T45): The synthesis of compound T45 refers to the synthesis method of T4. Yield 87%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.97 (d, J=8.8Hz, 2H), 6.96 (d, J=8.8Hz, 2H), 4.45 (t, J=4.5Hz, 2H), 3.96 (t, J=4.7Hz, 2H), 3.84 (s, 3H), 3.83 (q, J=7Hz, 1H), 3.67 (s, 1H), 1.34 (d, J=7.0Hz, 6H); ¹³ C NMR (100MHz, CDCl ₃ )δ164.3,162.6,161.7,159.6,128.3,126.1,119.2,114.2,66.7,60.8,55.4,26.2,20.7; HR-ESI-MS(positive mode)m/z:306.1337[M+H] ⁺ (Calcd for C ₁₆ H ₂₀ NO ₅ :306 .1341),m/z:328.1153[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₅ Na:328.1161).

Ethyl 5-(tert-butyl)-2-(4-methoxyphenyl)oxazole-4-carboxylate (T46): The synthesis of compound T46 was based on the synthesis method of 3b. Yield 85%, white solid, developed system Compound wt-3-7 was obtained as a white solid in 62% yield; isolated by column chromatography PE/EA10:1 (Rf＝0.5, PE/EA＝5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.00 (d, J＝8.8Hz, 2H), 6.96 (d, J＝8.8Hz, 2H), 4.45 (q, J＝7.1Hz, 2H), 3.86 (s, 3H), 1.50 (s, 9H), 1.44 (t, J＝7.1Hz, 3H); ¹³ CNMR (100MHz, CDCl ₃ )δ165.1,162.6,161.5,157.9,128.3,127.3,119.5,114.1,61.2,55.4,33.5,28.3,14.4; HR-ESI-MS(positive mode)m/z:304.1550[M+H] ⁺ (Calcd for C ₁₇ H ₂₂ NO ₄ :306 .1341),m/z:326.1374[M+Na] ⁺ (Calcd for C ₁₇ H ₂₁ NO ₄ Na:326.1368).

2-hydroxyethyl 5-(tert-butyl)-2-(4-methoxyphenyl)oxazole-4-carboxylate (T47): The synthesis of compound T47 was based on the synthesis method of T4. Yield 88%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.97 (d, J=8.8Hz, 2H), 6.98 (d, J=8.8Hz, 2H), 4.46 (t, J=4.4Hz, 2H), 3.97 (t, J=4.6Hz, 2H), 3.86 (s, 3H), 2.36 (s, 1H), 1.50 (s, 9H); ¹³ C NMR (100MHz, CDCl ₃ )δ166.0,162.5,161.7,158.0,128.2,126.7,119.2,114.3,67.1,61.0,55.4,33.5,28.1; HR-ESI-MS(positive mode)m/z:320.1501[M+H] ⁺ (Calcd for C ₁₇ H ₂₂ NO ₅ :320 .1498), m/z:342.1322[M+Na] ⁺ (Calcd forC ₁₇ H ₂₁ NO ₅ Na:342.1317).

Ethyl 5-cyclopropyl-2-(4-methoxyphenyl)oxazole-4-carboxylate (T48): The synthesis of compound T48 was based on the synthesis method of 3b. Yield 88%, white solid, developing system PE/EA 10:1 (Rf = 0.5, PE/EA = 5:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.93 (d, J = 8.9 Hz, 2H), 6.93 (d, J = d, J = 8.9 Hz, 2H), 4.45 (q, J = 7.1 Hz, 2H), 3.83 (s, 3H), 2.83-2.76 (m, 1H), 1.43 (t, J = 7.1 Hz, 3H), 1.18-1.15 (m, 4H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.9,161.5,160.3,158.3,128.2,128.0,119.4,114.1,60.9,55.4,14.5,9.2,8.1; HR-ESI-MS(positive mode)m/z:288.1244[M+H] ⁺ (Calcd forC ₁₆ H ₁₈ NO ₄ :288.1 236), m/z:310.1059[M+Na] ⁺ (Calcd for C ₁₆ H ₁₇ NO ₄ Na:310.1055).

2-hydroxyethyl 5-cyclopropyl-2-(4-methoxyphenyl)oxazole-4-carboxylate(T49): The synthesis of compound T49 refers to the synthesis method of T4. Yield 86%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.84 (d, J=8.6Hz, 2H), 6.91 (d, J=8.6Hz, 2H), 4.45 (t, J=4.5Hz, 2H), 4.27 (s, 1H), 3.96 (t, J=4.5Hz, 2H), 3.81 (s, 3H), 2.75-2.71 (m, 1H), 1.13-1.09 (m, 4H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.8,161.6,160.8,158.3,128.1,127.5,119.0,114.2,66.6,60.8,55.4,9.4,8.0; HR-ESI-MS(positivemode)m/z:304.1179[M+H] ⁺ (Calcd for C ₁₆ H ₁₈ NO ₅ :304.1 185), m/z:326.0999[M+Na] ⁺ (Calcdfor C ₁₆ H ₁₇ NO ₅ Na: 326.1004).

Ethyl 2-(4-methoxyphenyl)-5-phenyloxazole-4-carboxylate (T50): The synthesis of compound T50 was based on the synthesis method of 3b. Yield 92%, white solid, developing system PE/EA 10:1 (Rf=0.5, PE/EA=5:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.10-8.06 (m, 4H), 7.50-7.42 (m, 3H), 6.98 (d, J=8.9Hz, 2H), 4.47 (q, J=7.1Hz, 2H), 3.84 (s, 3H), 1.43 (t, J=7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.4,161.9,159.9,154.6,130.1,128.6,128.5,128.4,128.1,127.3,119.1,114.2,61.4,55.4,14.3; HR-ESI-MS(positive mode)m/z:324.1237[M+H] ⁺ (Calcd for C ₁₉ H ₁₈ NO ₄ :324.1236), m/z:346.1060[M+Na] ⁺ (Calcd for C ₁₉ H ₁₇ NO ₄ Na:346.1055).

2-hydroxyethyl 2-(4-methoxyphenyl)-5-phenyloxazole-4-carboxylate(T51): The synthesis of compound T51 refers to the synthesis method of T4. Yield 91%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 8.04 (dd, J=4.0Hz, 2.2Hz, 2H), 7.97 (d, J=8.8Hz, 2H), 7.46 (dd, J=0.9Hz, 2.3Hz, 3H), 6.92 (d, J=8.9Hz, 2H), 4.47 (t, J=4.4Hz, 2H), 3.98 (t, J=4.6Hz, 3H), 3.81 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.2,162.0,159.9,154.8,130.3,128.5,128.4,128.4,127.5,126.9,118.7,114.3,67.2,60.7,55.4; HR-ESI-MS(positive mode)m/z:340.1175[M+H] ⁺ (Calcd for C ₁₉ H ₁₈ NO ₅ :340.1185), m/z:362.0995[M+Na] ⁺ (Calcd for C ₁₉ H ₁₇ NO ₅ Na:362.1004).

4-hydroxybutyl2-(4-methoxyphenyl)-5-methyloxazole-4-carboxylate (T52): Compound 3b (261.3 mg, 1 mmol) and EtOH/10% NaOH = 1:1 (20 mL) were added to a 25 mL round-bottom flask, and the mixture was reacted at 70°C for 1 h. The pH was adjusted to between 1 and 2 with concentrated hydrochloric acid, and the mixture was extracted with dichloromethane. The organic phase was concentrated to obtain the intermediate compound 3 without further purification. 20 mL of dichloromethane, dicyclohexylcarbodiimide (DCC) (412.6 mg, 2 mmol), 4-dimethylaminopyridine (DMAP) (24.4 mg, 0.2 mmol), and alcohol (2 mmol) were added to the intermediate 3, and the reaction system was heated to 80°C and refluxed for 6 hours. After the reaction was completed, 20 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined and concentrated, and then separated and purified by column chromatography (petroleum ether/ethyl acetate/methanol=10/1/0.01) to obtain compound T52. Yield 82%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 8.00 (d, J=9.0Hz, 2H), 6.96 (d, J=9.0Hz, 2H), 4.41 (t, J=6.5Hz, 2H), 3.85 (s, 3H), 3.74 (t, J=6.3Hz, 2H) 2.67 (s, 3H), 1.93-1.86 (m, 3H), 1.75-1.69 (m, 2H); ¹³ C NMR (100MHz, CDCl ₃ )δ162.5,161.7,159.8,155.6,128.4,128.3,119.3,114.2,64.8,62.2,55.4,29.4,25.2,12.2; HR-ESI-MS(positive mode)m/z:306.1341[M+H] ⁺ (Calcd for C ₁₆ H ₂₀ NO ₄ :306.1333),m/z:328.1155[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₄ Na:328.1161).

N-(2-hydroxyethyl)-2-(4-methoxyphenyl)-5-methyloxazole-4-carboxamide (T53): The synthesis method of compound T53 was similar to that of T52. Yield 71%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf＝0.5, PE/EA/MeOH＝1:1:0.01), ¹ H NMR (400MHz, CDCl ₃ ) 7.91 (d, J＝8.9Hz, 2H), 7.47 (s, 1H), 6.96 (d, J＝8.9Hz, 2H), 3.85 (s, 3H), 3.83 (t, J＝4.8Hz, 2H), 3.61 (q, J＝5.6Hz, 2H), 2.67 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ163.4,161.6,158.8,152.6,129.8,128.0,119.5,114.3,62.6,55.4,42.2,11.8; HR-ESI-MS(positive mode)m/z:277.1189[M+H] ⁺ (Calcdfor C ₁₄ H ₁₇ N ₂ O ₄ :277.1 188),m/z:299.1011[M+Na] ⁺ (Calcd for C ₁₄ H ₁₆ N ₂ O ₄ Na:299.1008).

2-(2-hydroxyethoxy)ethyl 2-(4-methoxyphenyl)-5-methyloxazole-4-carboxylate(T54): The synthesis of compound T54 refers to the synthesis method of T52. Yield 80%, white solid, developing system PE/EA/MeOH 1:1:0.01 (Rf=0.5, PE/EA/MeOH=1:1:0.01), ¹ H NMR (400 MHz, CDCl ₃ ) 7.97 (d, J=8.9 Hz, 2H), 6.93 (d, J=8.9 Hz, 2H), 4.47 (t, J=4.7 Hz, 2H), 3.83-3.81 (m, 5H), 3.75 (t, J=4.2 Hz, 2H), 3.64 (t, J=4.9 Hz, 2H), 3.05 (s, 1H), 2.64 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ )δ162.4,161.7,159.8,155.9,128.3,128.2,119.2,114.2,72.6,68.8,63.7,61.6,55.4,12.1; HR-ESI-MS(positive mode)m/z:322.1295[M+H] ⁺ (Calcd for C ₁₆ H ₂₀ NO ₆ :322.1291),m/z:344.1114[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₆ Na:344.1110).

5-chloro-2-(4-methoxyphenyl)-6-methylpyrimidin-4-ol(T55): Under nitrogen protection, 4-methoxybenzamidine (330.4 mg, 2 mmol), NaOH (374 mg, 2 mmol) and methanol (20 mL) were added to a 25 mL round-bottom flask and reacted at 60 °C for 2 h. After the reaction was completed, the solvent was removed and the compound T55 was obtained by separation and purification by column chromatography. Yield 54%, white solid, developing system PE/EA4:1 (Rf＝0.5, PE/EA＝2:1), ¹ H NMR (400 MHz, DMSO-d ₆ ) 13.01 (s, 1H), 8.11 (d, J＝8.8 Hz, 2H), 7.09 (d, J＝8.9 Hz, 2H), 3.85 (s, 3H), 2.42 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ162.7, 130.2, 114.6, 56.0, 48.0, 33.8, 25.8, 24.9, 22.6; HR-ESI-MS (positive mode) m/z: 251.0588 [M+H] ⁺ (Calcd for C ₁₂ H ₁₂ ClN ₂ O ₂ :251.0587), m/z:273.0403[M+Na] ⁺ (Calcd for C ₁₄ H ₁₆ N ₂ O ₃ Na:273.0407).

2-(chloromethyl)-5-phenyl-1,3,4-oxadiazole (T56): In a 25 mL round-bottom flask, benzoyl hydrazide (272.3 mg, 2 mmol), phosphorus oxychloride (306.7 mg, 2 mmol) and chloroacetic acid (20 mL) were added and refluxed to prepare T57. Yield 96%, white solid, developing system PE/EA1:5 (Rf＝0.5, PE/EA＝1:2), ¹ H NMR (400MHz,CDCl ₃ )8.05(d,J＝6.9Hz,2H),7.56-7.47(m,3H),4.77(s,2H); ¹³ C NMR (150MHz,CDCl ₃ )δ166.0,162.2,132.2,129.1,127.1,123.3,33.0; HR-ESI-MS (positive mode)m/z:195.0315[M+H] ⁺ (Calcd for C ₉ H ₈ N ₂ OCl:195.0325),m/z:217.0139[M+Na] ⁺ (Calcd for C ₉ H ₇ N ₂ OClNa:217.0145).

2-(2-chloroethyl)-5-phenyl-1,3,4-oxadiazole(T57): The synthesis of compound T57 refers to the synthesis method of T56. Yield 95%, white solid, developing system PE/EA1:5 (Rf＝0.5, PE/EA＝1:2), ¹ H NMR (400MHz,CDCl ₃ )8.04(d,J＝7.3Hz,2H),7.55-7.48(m,3H),3.97(t,J＝6.9Hz,2H),3.43(t,J＝6.9Hz,2H); ¹³ C NMR (150MHz,CDCl ₃ )δ165.1,163.5,131.8,129.1,126.9,123.7,39.6,29.2; HR-ESI-MS (positive mode)m/z:209.0470[M+H] ⁺ (Calcd for C ₁₀ H ₁₀ N ₂ OCl:209.0482), m/z:231.0290[M+Na] ⁺ (Calcd for C ₁₀ H ₉ N ₂ OClNa: 231.0301).

2-phenylthiazole(S1): The synthesis of compound S1 refers to T7. Yield 97%, colorless liquid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz,CDCl ₃ )7.99-7.96(m,2H),7.88(d,J＝3.2Hz,1H),7.46-7.41(m,3H),7.33-7.31(m,1H); ¹³ C NMR (100MHz,CDCl ₃ )δ168.5,143.6,133.5,130.1,129.0,126.6,118.9; HR-ESI-MS (positive mode)m/z:162.0365[M+H] ⁺ (Calcd for C ₉ H ₈ NS:162.0377),m/z:184.0199[M+Na] ⁺ (Calcd for C ₉ H ₇ NSNa:184.0197).

Ethyl 2-(4-methoxyphenyl)thiazole-5-carboxylate(S2): The synthesis of compound S2 can be referred to T7. Yield 91%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.06 (s, 1H), 7.93 (d, J＝8.8Hz, 2H), 6.93 (d, J＝8.8Hz, 2H), 4.44 (q, J＝7.1Hz, 2H), 3.82 (s, 3H), 1.41 (t, J＝7.1Hz, 3H); ¹³ C NMR (150MHz, CDCl ₃ ) δ168.7, 161.6, 161.5, 147.8, 128.5, 126.2, 125.8, 114.3, 61.4, 55.4, 14.3; HR-ESI-MS (positive mode)m/z:264.0689[M+H] ⁺ (Calcd for C ₁₃ H ₁₄ NO ₃ S:264.0694),m/z:286.0509[M+Na] ⁺ (Calcd for C ₁₃ H ₁₃ NO ₃ SNa:286.0514).

2-hydroxyethyl2-(4-methoxyphenyl)thiazole-5-carboxylate (S3): Synthesis of compound S3 with reference to T10. Yield 88%. White solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.12 (s, 1H), 7.91 (d, J=8.8Hz, 2H), 6.96 (d, J=8.8Hz, 2H), 4.47 (t, J=4.5Hz, 2H), 3.95 (t, J=4.6Hz, 2H), 3.85 (s, 3H), 3.38 (s, 1H); ¹³ C NMR (150MHz, CDCl ₃ )δ169.1,161.8,161.6,147.2,128.6,127.0,126.5,125.5,114.4,114.3,67.1,60.9,55.4,52.4,29.7; HR-ESI-MS(positive mode)m/z:280.0634[M+H] ⁺ (Calcd for C _{1 3} H ₁₄ NO ₄ S: 280.0644), m/z: 302.0451[M+Na] ⁺ (Calcd for C ₁₃ H ₁₃ NO ₄ SNa: 302.0463).

(2-(4-methoxyphenyl)-4-methylthiazol-5-yl)methanol (S4): Using T7 as the starting material, the reaction was carried out in lithium aluminum hydride in THF solvent under nitrogen protection at 0°C for 4 to 6 hours to obtain compound S4. Yield 85%, white solid, developing system PE/EA1:1 (Rf＝0.5, PE/EA＝1:1), ¹ H NMR (400MHz,CDCl ₃ )7.82(d,J＝8.8Hz,2H),6.93(d,J＝8.8Hz,2H),4.78(s,2H),3.83(s,3H),2.39(s,3H),2.24(s,1H); ¹³ C NMR (100MHz,CDCl ₃ )δ166.2,161.1,150.1,130.1,127.9,126.5,114.2,55.8,55.4,15.1; HR-ESI-MS (positive mode)m/z:236.0751[M+H] ⁺ (Calcd for C ₁₂ H ₁₄ NO ₂ S:236.0745),m/z:258.0569[M+Na] ⁺ (Calcd for C ₁₂ H ₁₃ NO ₂ SNa:258.0565).

Diethyl 2-(4-methoxyphenyl)thiazole-4,5-dicarboxylate (S5): The synthesis of compound S5 was carried out with diethyl chlorooxaloacetate as the starting material, and the synthesis method of T7 was referred to. Yield 78%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz, CDCl ₃ )7.94 (d, J＝8.9Hz, 2H), 6.96 (d, J＝8.9Hz, 2H), 4.50 (q, J＝7.2Hz, 2H), 4.39 (q, J＝7.2Hz, 2H), 3.87 (s, 3H), 1.44 (t, J＝7.2Hz, 3H), 1.40 (t, J＝7.2Hz, 3H); ¹³ C NMR (150MHz, CDCl ₃ )δ171.4,163.4,162.4,160.3,150.9,128.8,127.0,125.1,114.5,62.3,62.1,55.5,14.1,14.0; HR-ESI-MS(positive mode)m/z:336.0916[M+H] ⁺ (Calcd for C ₁₆ H _{18 5} S:336.0906),m/z:358.0734[M+Na] ⁺ (Calcd for C ₁₆ H ₁₇ NO ₅ SNa:358.0725).

Ethyl 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate(S6): The synthesis of compound S6 was referenced to T7. Yield 87%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz,CDCl ₃ )7.78(d,J＝8.5Hz,2H),7.32(d,J＝8.5Hz,2H),4.32(q,J＝7.1Hz,2H),2.70(s,3H),1.36(t,J＝7.1Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ168.1,161.9,161.0,136.9,131.3,129.1,127.8,122.0,61.2,17.5,14.3; HR-ESI-MS (positive mode)m/z:282.0360[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ ClNO ₂ S: 282.0356), m/z: 304.0182[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ ClNO ₂ SNa: 304.0175).

2-hydroxyethyl 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate (S7): Synthesis of compound S7 was based on reference T10. Yield 88%, white solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.81 (d, J=8.5Hz, 2H), 7.37 (d, J=8.5Hz, 2H), 4.40 (t, J=4.6Hz, 2H), 3.92 (t, J=4.6Hz, 2H), 2.79 (s, 1H), 2.72 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ168.9,162.2,161.7,137.2,131.1,129.3,128.0,121.4,66.9,61.0,17.5; HR-ESI-MS(positivemode)m/z:298.0308[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ ClNO ₃ S: 298.0305),m /z:320.0132[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ ClNO ₃ SNa:320.0124).

Ethyl 2-(4-bromophenyl)-4-methylthiazole-5-carboxylate(S8): The synthesis of compound S8 was based on reference T7. Yield 98%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz,CDCl ₃ )7.79(d,J＝8.5Hz,2H),7.55(d,J＝8.5Hz,2H),4.37(q,J＝7.1Hz,2H),2.76(s,3H),1.40(t,J＝7.1Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ168.3,162.1,161.1,132.2,131.8,128.1,125.4,122.1,61.3,17.5,14.4; HR-ESI-MS (positive mode)m/z:325.9831[M+H] ⁺ (Calcd for C ₁₃ H _13b rNO ₂ S: 325.9850), m/z: 347.9676[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ BrNO ₂ SNa: 347.9670).

2-hydroxyethyl 2-(4-bromophenyl)-4-methylthiazole-5-carboxylate (S9): Synthesis of compound S9 was based on reference T10. Yield 94%, white solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.82 (d, J=8.5Hz, 2H), 7.59 (d, J=8.5Hz, 2H), 4.44 (t, J=4.6Hz, 2H), 3.95 (t, J=4.7Hz, 2H), 2.77 (s, 3H), 2.01 (s, 1H); ¹³ C NMR (100MHz, CDCl ₃ )δ168.9,162.3,161.8,132.3,131.7,128.2,125.6,121.4,66.9,61.2,17.6; HR-ESI-MS(positivemode)m/z:341.9807[M+H] ⁺ (Calcd for C ₁₃ H _13b rNO ₃ S: 341.9800), m/z:363.9624[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ BrNO ₃ SNa: 363.9619).

Ethyl 4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carboxylate (S10): The synthesis of compound S10 was referenced to T7. Yield 87%, white solid, developing system PE/EA20:1 (Rf=0.5, PE/EA=10:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.03 (d, J=8.1Hz, 2H), 7.67 (d, J=8.2Hz, 2H), 4.36 (q, J=7.1Hz, 2H), 2.76 (s, 3H), 1.39 (t, J=7.2Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ167.6,161.9(d,J＝74.1Hz),136.0,132.9(q,J＝32.7Hz),127.0,126.0(d,J＝3.7Hz),125.1,122.9,122.4,61.4,17.5,14.3; HR-ESI-MS (positive mode) m/z: 316.06 00[M+H] ⁺ (Calcd for C ₁₄ H ₁₃ F ₃ NO ₂ S: 316.0619), m/z: 338.0425 [M+Na] ⁺ (Calcd for C ₁₄ H ₁₂ F ₃ NO ₂ SNa: 338.0439).

2-hydroxyethyl4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carboxylate (S11): Synthesis of compound S11 was based on reference T10. Yield 86%, white solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.91 (d, J=8.2Hz, 2H), 7.59 (d, J=8.3Hz, 2H), 4.38 (t, J=4.6Hz, 2H), 3.90 (t, J=4.8Hz, 2H), 3.38 (s, 1H), 2.68 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ : 332.0560[M+H] ⁺ (Calcd for C 14 H 13 F 3 NO 3 S: 332.0568), m/ _{z: 354.0386[M+Na] + (Calcd for C 14 H 12 F 3} ^NO _{3 SNa : 354.0388 )} .

Ethyl 4-methyl-2-(4-nitrophenyl)thiazole-5-carboxylate(S12): The synthesis of compound S12 was referred to T7. Yield 83%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz,CDCl ₃ )8.31(d,J＝8.9Hz,2H),8.14(d,J＝8.9Hz,2H),4.40(q,J＝7.1Hz,2H),2.79(s,3H),1.41(t,J＝7.1Hz,3H); ¹³ C NMR (150MHz,CDCl ₃ )δ166.4,161.8,161.5,149.0,138.4,127.5,124.3,124.0,61.6,17.5,14.3; HR-ESI-MS (positive mode)m/z:293.0586[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ N ₂ O ₄ S: 293.0596), m/z: 315.0418[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ N ₂ O ₄ SNa: 315.0415).

2-hydroxyethyl4-methyl-2-(4-nitrophenyl)thiazole-5-carboxylate(S13): The synthesis of compound S13 was referred to T10. Yield 79%, white solid, developing system PE/EA1:1 (Rf＝0.5, PE/EA＝1:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.32 (d, J＝8.9Hz, 2H), 8.14 (d, J＝8.9Hz, 2H), 4.48 (t, J＝4.6Hz, 2H), 3.98 (t, J＝4.7Hz, 2H), 2.81 (s, 3H), 1.85 (s, 1H); ¹³ C NMR (150MHz, CDCl ₃ ) δ166.8, 162.2, 162.0, 149.1, 138.2, 127.5, 124.4, 123.2, 67.0, 61.2, 17.6; HR-ESI-MS (positive mode)m/z:309.0531[M+H] ⁺ (Calcd for C ₁₃ H ₁₃ N ₂ O ₅ S:309.0545),m/z:331.0352[M+Na] ⁺ (Calcd for C ₁₃ H ₁₂ N ₂ O ₅ SNa:331.0365).

Ethyl 2-(4-isocyanophenyl)-4-methylthiazole-5-carboxylate(S14): The synthesis of compound S14 was referred to T7. Yield 88%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ HNMR (400MHz,CDCl ₃ )8.07(d,J＝8.4Hz,2H),7.74(d,J＝8.4Hz,2H),4.39(q,J＝7.1Hz,2H),2.78(s,3H),1.40(t,J＝7.2Hz,3H); ¹³ C NMR (100MHz,CDCl ₃ )δ166.9,161.9,161.4,136.7,132.8,127.2,123.6,118.2,114.1,61.6,17.5,14.3; HR-ESI-MS (positive mode)m/z:273.0692[M+H] ⁺ (Calcd for C ₁₄ H ₁₃ N ₂ O ₂ S: 273.0698), m/z: 295.0505[M+Na] ⁺ (Calcd for C ₁₄ H ₁₂ N ₂ O ₂ SNa: 295.0517).

2-hydroxyethyl 2-(4-((2-hydroxyethoxy)carbonyl)phenyl)-4-methylthiazole-5-carboxylate (S15): 4-cyano substituted derivative S14 was heated at 70°C in 10% NaOH solvent for 1 h, and detected by TLC. After the reaction was completed, concentrated HCl was used to adjust Ph to 1-2, and chloroform was extracted and concentrated; then 20 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (383.4 mg, 2 mmol), 4-dimethylaminopyridine (DMAP) (24.4 mg, 0.2 mmol), and ethylene glycol (124.1 mg, 2 mmol) were added, and the reaction system was heated to 80°C and refluxed for 6 hours. After the reaction was completed, 20 mL of water was added to quench the reaction, and dichloromethane was extracted. The organic phases were combined and concentrated, and then separated and purified by column chromatography to obtain compound S15. Yield 77%, white solid, developing system PE/EA1:1 (Rf＝0.5, PE/EA＝1:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.14 (d, J＝8.4Hz, 2H), 8.05 (d, J＝8.4Hz, 2H), 4.51 (t, J＝4.6Hz, 2H), 4.47 (t, J＝4.6Hz, 2H), 4.00 (t, J＝4.6Hz, 2H), 3.97 (t, J＝4.6Hz, 2H), 2.81 (s, 3H), 1.86 (s, 2H); ¹³ C NMR (150MHz, CDCl ₃ )δ168.6,166.1,162.2,162.0,136.8,131.9,130.4,129.7,126.8,67.0,66.9,61.4,61.2,17.6; HR-ESI-MS(positive mode)m/z:352.0847[M+H] ⁺ (Calcd for C ₁₆ H _{18 6} S:352.0855),m/z:374.0657[M+Na] ⁺ (Calcd forC ₁₆ H ₁₇ NO ₆ SNa:374.0674).

Ethyl 4-methyl-2-(naphthalen-2-yl)thiazole-5-carboxylate (S16): Synthesis of compound S16 was based on reference T7. Yield 80%, white solid, developing system PE/EA20:1 (Rf=0.5, PE/EA=10:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.44 (s, 1H) 8.01 (dd, J=1.8Hz, 1.8Hz, 1H), 7.90-7.81 (m, 3H), 7.53-7.48 (m, 2H), 4.38 (q, J=7.2Hz, 2H), 2.81 (s, 3H), 1.41 (t, J=7.1Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ169.9,162.3,161.1,134.5,133.1,130.2,128.9,128.8,127.9,127.5,126.9,126.6,123.8,121.9,61.3,17.6,14.4; HR-ESI-MS (positive mode) m/z: 298.0894 [M+H] ⁺ (Calcd for C ₁₇ H ₁₆ NO ₂ S: 298.0902), m/z: 320.0706 [M+Na] ⁺ (Calcd for C ₁₇ H ₁₅ NO ₂ SNa: 320.0721).

2-hydroxyethyl 4-methyl-2-(naphthalen-2-yl)thiazole-5-carboxylate (S17): Synthesis of compound S17 was based on reference T10. Yield 83%, white solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 8.41 (s, 1H), 7.96 (dd, J=1.6Hz, 1.6Hz, 1H), 7.89-7.80 (m, 3H), 7.54-7.48 (m, 2H), 4.43 (t, J=4.5Hz, 2H), 3.95 (t, J=4.7Hz, 2H), 2.79 (s, 3H), 2.55 (s, 1H); ¹³ C NMR (100MHz, CDCl ₃ )δ170.4,162.4,161.8,134.6,133.1,130.0,129.0,128.9,127.9,127.6,127.0,126.7,123.7,121.1,66.9,61.1,17.6; HR-ESI-MS (positive mode) m/z: 314.0846 [M+H] ⁺ (Calcd for C ₁₇ H ₁₆ NO ₃ S: 314.0851), m/z: 336.0664 [M+Na] ⁺ (Calcd for C ₁₇ H ₁₅ NO ₃ SNa: 336.0670).

Ethyl 2-(3,5-dichlorophenyl)-4-methylthiazole-5-carboxylate(S18): The synthesis of compound S18 was referred to T7. Yield 90%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ HNMR (400MHz,CDCl ₃ )7.82(d,J＝1.8Hz,2H),7.42(t,J＝1.8Hz,1H),4.38(q,J＝7.1Hz,2H),2.76(s,3H),1.40(t,J＝7.1Hz,3H); ¹³ C NMR (150MHz,CDCl ₃ )δ166.3,161.8,161.1,135.8,135.5,130.5,125.0,123.1,61.5,17.4,14.3; HR-ESI-MS (positive mode)m/z:315.9970[M+H] ⁺ (Calcd for C ₁₃ H ₁₂ Cl ₂ NO ₂ S: 315.9966), m/z: 337.9777[M+Na] ⁺ (Calcd for C ₁₃ H ₁₁ Cl ₂ NO ₂ SNa: 337.9785).

2-hydroxyethyl 2-(3,5-dichlorophenyl)-4-methylthiazole-5-carboxylate(S19): The synthesis of compound S19 was based on the reference T10. Yield 88%, white solid, developing system PE/EA1:1 (Rf＝0.5, PE/EA＝1:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.85 (d, J＝1.8Hz, 2H), 7.45 (t, J＝1.8Hz, 1H), 4.46 (t, J＝4.6Hz, 2H), 3.96 (t, J＝4.7Hz, 2H), 2.79 (s, 3H), 1.81 (s, 1H); ¹³ C NMR (150MHz, CDCl ₃ ) δ166.7, 162.1, 161.9, 135.9, 135.4, 130.7, 125.1, 122.3, 66.9, 61.2, 17.5; HR-ESI-MS (positive mode)m/z:331.9906[M+H] ⁺ (Calcd for C ₁₃ H ₁₂ Cl ₂ NO ₃ S: 331.9915), m/z: 353.9720[M+Na] ⁺ (Calcd for C ₁₃ H ₁₁ Cl ₂ NO ₃ SNa: 353.9734).

Ethyl 2-(4-ethoxyphenyl)-4-methylthiazole-5-carboxylate (S20): Synthesis of compound S20 was referenced to T7. Yield 89%, white solid, developing system PE/EA20:1 (Rf=0.5, PE/EA=10:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.90 (d, J=8.8Hz, 2H), 6.94 (d, J=8.8Hz, 2H), 4.37 (q, J=7.1Hz, 2H), 4.11 (q, J=7.1Hz, 2H), 2.76 (s, 3H), 1.46 (t, J=7.0Hz, 3H), 1.40 (t, J=7.0Hz, 3H); ¹³ C NMR (150MHz, CDCl ₃ )δ169.9,162.4,161.4,160.9,128.4,125.7,120.8,114.9,63.7,61.1,17.5,14.7,14.3; HR-ESI-MS(positive mode)m/z:292.1003[M+H] ⁺ (Calcd for C ₁₅ H ₁₈ NO ₃ S: 29 2.1007), m/z:314.0816[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₃ SNa: 314.0827).

2-hydroxyethyl2-(4-ethoxyphenyl)-4-methylthiazole-5-carboxylate (S21): Synthesis of compound S21 was based on reference T10. Yield 90%, white solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.90 (d, J=8.8Hz, 2H), 6.95 (d, J=8.8Hz, 2H), 4.44 (t, J=4.6Hz, 2H), 4.12 (q, J=7.0Hz, 2H), 3.95 (t, J=4.7Hz, 2H), 2.76 (s, 3H), 2.10 (s, 1H), 1.46 (t, J=7.0Hz, 3H); ¹³ CNMR (150MHz, CDCl ₃ )δ170.4,162.6,161.7,161.5,128.5,125.5,120.0,114.9,66.7,63.7,61.3,17.6,14.7; HR-ESI-MS(positive mode)m/z:308.0955[M+H] ⁺ (Calcd for C ₁₅ H ₁₈ NO ₄ S:30 8.0957), m/z:330.0770[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₄ SNa: 330.0776).

Ethyl 4-methhyl-2-(m-tolyl)thiazole-5-carboxylate (S22): Synthesis of compound S22 was based on reference T7. Yield 86%, white solid, developed by PE/EA20:1 (Rf=0.5, PE/EA=10:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.76 (s, 1H), 7.71 (d, J=7.6 Hz, 1H), 7.29 (t, J=7.6 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 4.34 (q, J=7.1 Hz, 2H), 2.76 (s, 3H), 2.37 (s, 3H), 1.38 (t, J=7.1 Hz, 3H); ¹³ CNMR (100MHz, CDCl ₃ )δ170.0,162.2,160.9,138.8,132.8,131.7,128.8,127.2,124.0,121.6,61.1,21.3,17.5,14.3; HR-ESI-MS(positive mode)m/z:262.0900[M+H] ⁺ (Calcdfor C ₁₄ H ₁₆ NO ₂ S: 262.0902), m/z: 284.0718[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₂ SNa: 284.0721).

2-hydroxyethhyl 4-methyl-2-(m-tolyl)thiazole-5-carboxylate (S23): Synthesis of compound S23 was based on reference T10. Yield 84%, white solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.75 (s, 1H), 7.71 (d, J=7.4Hz, 1H), 7.34-7.28 (m, 2H), 4.42 (t, J=4.4Hz, 2H), 3.94 (t, J=4.7Hz, 2H), 3.01 (s, 1H), 2.76 (s, 3H), 2.40 (s, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ170.7,162.4,161.5,138.9,132.6,129.0,127.3,124.1,121.0,66.8,61.0,21.3,17.5; HR-ESI-MS(positive mode)m/z:278.0845[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₃ S:2 78.0851), m/z:300.0661[M+Na] ⁺ (Calcd for C ₁₄ H ₁₅ NO ₃ SNa: 300.0670).

Ethyyl 2-(3,4-dimethylphenyl)-4-methylthiazole-5-carboxylate (S24): Synthesis of compound S24 was based on reference T7. Yield 88%, white solid, developing system PE/EA20:1 (Rf=0.5, PE/EA=10:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.73 (s, 1H), 7.66 (dd, J=1.6Hz, 1.6Hz, 1H), 7.17 (d, J=7.8Hz, 1H), 4.36 (q, J=7.1Hz, 2H), 2.76 (s, 3H), 2.29 (d, J=7.5Hz, 6H), 1.39 (t, J=6.4Hz, 3H); ¹³ CNMR (100MHz, CDCl ₃ )δ170.3,162.4,160.9,140.2,137.4,130.6,130.3,127.7,124.4,121.2,61.4,19.9,19.7,17.6,14.4; HR-ESI-MS(positive mode)m/z:276.1046[M+H] ⁺ (Calcd for C _{1 5} H ₁₈ NO ₂ S: 276.1058), m/z: 298.0863[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₂ SNa: 298.0878).

2-hyddroxyethyl 2-(3,4-dimethylphenyl)-4-methylthiazole-5-carboxylate (S25): Synthesis of compound S25 was based on reference T10. Yield 82%, white solid, developing system PE/EA1:1 (Rf=0.5, PE/EA=1:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.54 (s, 1H), 7.48 (d, J=7.6Hz, 1H), 7.04 (d, J=7.8Hz, 1H), 4.30 (t, J=5.9Hz, 2H), 3.92 (s, 1H), 3.84 (t, J=4.5Hz, 2H), 2.63 (s, 3H), 2.18 (d, J=6.9Hz, 6H); ¹³ C NMR (100MHz, CDCl ₃ )δ170.8,162.3,161.2,140.4,137.4,130.2,127.7,124.3,120.6,66.7,60.6,19.8,19.7,17.4; HR-ESI-MS(positive mode)m/z:292.1001[M+H] ⁺ (Calcd for C ₁₅ H ₁₈ NO ₃ S: 292.1007), m/z: 314.0817[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₃ SNa: 314.0827).

Ethyl 2-(3,5-dimethoxyphenyl)-4-methylthiazole-5-carboxylate(S26): The synthesis of compound S26 was referred to T7. Yield 84%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz, CDCl ₃ ) 7.11 (d, J＝2.2Hz, 2H), 6.56 (t, J＝2.2Hz, 2H), 4.37 (q, J＝7.1Hz, 2H), 3.85 (s, 6H), 2.77 (s, 3H), 1.40 (t, J＝7.1Hz, 3H); ¹³ C NMR (150MHz, CDCl ₃ ) δ169.7, 162.2, 161.2, 160.8, 134.7, 121.9, 104.7, 103.5, 61.2, 55.6, 17.5, 14.3; HR-ESI-MS (positive mode)m/z:308.0951[M+H] ⁺ (Calcd for C ₁₅ H ₁₈ NO ₄ S:308.0957),m/z:330.0769[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₄ SNa:330.0776).

2-hydroxyethyl 2-(3,5-dimethoxyphenyl)-4-methylthiazole-5-carboxylate(S27): The synthesis of compound S27 was based on reference T10. Yield 78%, white solid, developing system PE/EA1:1 (Rf＝0.5, PE/EA＝1:1), ¹ HNMR (400MHz,CDCl ₃ )7.08(d,J＝2.2Hz,2H),6.55(t,J＝2.2Hz,1H),4.42(t,J＝4.6Hz,2H),3.94(t,J＝4.6Hz,2H),3.84(s,6H),2.76(s,3H),2.36(s,1H); ¹³ C NMR (150MHz,CDCl ₃ )δ170.2,162.4,161.5,161.2,134.4,121.2,104.7,103.7,66.8,61.1,55.6,17.5; HR-ESI-MS (positive mode)m/z:324.0894[M+H] ⁺ (Calcd for C ₁₅ H ₁₈ NO ₅ S:324.0906),m/z:346.0707[M+Na] ⁺ (Calcd for C ₁₅ H ₁₇ NO ₅ SNa:346.0725).

Ethyl 4-methyl-2-(3,4,5-trimethoxyphenyl)thiazole-5-carboxylate(S28): The synthesis of compound S28 was referred to T7. Yield 79%, white solid, developing system PE/EA20:1 (Rf＝0.5, PE/EA＝10:1), ¹ H NMR (400MHz,CDCl ₃ )7.17(s,2H),4.35(q,J＝7.1Hz,2H),3.92(s,6H),3.88(s,3H),2.74(s,3H),1.38(t,J＝7.1Hz,3H); ¹³ C NMR (150MHz,CDCl ₃ )δ169.6,162.2,160.9,153.6,140.7,128.4,121.5,104.0,61.2,60.9,56.3,17.5,14.3; HR-ESI-MS (positive mode)m/z:338.1048[M+H] ⁺ (Calcd for C ₁₆ H ₂₀ NO ₅ S: 338.1062), m/z: 360.0868[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₅ SNa: 360.0882).

2-hydroxyethyl 4-methyl-2-(3,4,5-trimethoxyphenyl)thiazole-5-carboxylate(S29): The synthesis of compound S29 was referred to T10. Yield 83%, white solid, developing system PE/EA1:1 (Rf＝0.5, PE/EA＝1:1), ¹ HNMR (400MHz,CDCl ₃ )7.18(s,2H),4.44(t,J＝4.6Hz,2H),3.95-3.93(m,8H),3.90(s,3H),2.77(s,3H),2.12(s,1H); ¹³ C NMR (150MHz,CDCl ₃ )δ170.1,162.4,161.6,153.6,140.9,128.2,120.8,104.1,66.8,61.2,61.0,56.3,17.6; HR-ESI-MS (positive mode)m/z:354.1003[M+H] ⁺ (Calcd for C ₁₆ H ₂₀ NO ₆ S:354.1011),m/z:376.0821[M+Na] ⁺ (Calcd for C ₁₆ H ₁₉ NO ₆ SNa:376.0831).

2-((5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)oxy)ethyl2-(4-methoxyphenyl)-5-methyloxazole-4-carboxylate (T58): D-biotin (244.3 mg, 1 mmol), compound 3b (261.3 mg, 1 mmol), dicyclohexylcarbodiimide (DCC) (412.6 mg, 2 mmol), 4-dimethylaminopyridine (DMAP) (24.4 mg, 0.2 mmol) and DMF (20 mL) were added to a 25 mL round-bottom flask and stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed and the mixture was separated and purified by column chromatography to obtain compound T56. Yield 49%, white solid, developing system DCM/EtOH 30:1 (Rf=0.5, DCM/EtOH=20:1), ¹ H NMR (600 MHz, DMSO-d ₆ ) 7.90 (d, J=8.8 Hz, 2H), 7.08 (d, J=8.8 Hz, 2H), 5.55 (d, J=7.9 Hz, 2H), 4.89 (t, J=5.6 Hz, 1H), 4.26 (t, J=4.9 Hz, 2H), 3.82 (s, 3H), 3.69 (q, J=4.7 Hz, 2H), 2.64 (s, 3H), 1.71-1.69 (m, 4H), 1.61-1.58 (m, 4H), 1.50-1.47 (m, 2H); ¹³ C NMR (150 MHz, DMSO-d 6 ) ₆ )δ162.2,161.8,159.2,157.1,156.4,128.4,128.3,119.2,115.1,66.5,59.5,55.9,48.0,33.8,25.8,24.9,12.4; HR-ESI-MS (positive mode) m/z:504.1804 [M+H] ⁺ (Calcd for C ₂₄ H ₃₀ N ₃ O ₇ S:504.1810), m/z:526.1622 [M+Na] ⁺ (Calcd for C ₂₄ H ₂₉ N ₃ O ₇ SNa:526.1624). Comparative Example 1, Synthesis of Comparative Compound 3b

Ethyl 2-(4-methoxyphenyl)-5-methyloxazole-4-carboxylate (3b): 4-methoxybenzylamine (274.4 mg, 2 mmol) and DMF (10 mL) were added to a 25 mL round-bottom flask, followed by I ₂ (304.6 mg, 1.2 mmol), ethyl acetoacetate (130.1 mg, 1 mmol), TBHP (180.2 mg, 2 mmol), and copper acetate (39.9 mg, 0.2 mmol); the reaction system was stirred at room temperature for 4 hours. After the reaction was stopped, 10 mL of water was added to quench the reaction, saturated sodium thiosulfate (20 mL) was added to remove unreacted iodine, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate and concentrated, and the crude product was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10/1) to obtain the target compound 3b. Yield 87%, white solid, developing system PE/EA10:1 (Rf=0.6, PE/EA=3:1), ¹ H NMR (400 MHz, CDCl ₃ ) 8.00 (d, J=9.0 Hz, 2H), 6.96 (d, J=9.0 Hz, 2H), 4.43 (q, J=7.2 Hz, 2H), 3.85 (s, 3H), 2.67 (s, 3H), 1.42 (t, J=7.1 Hz, 2H); ¹³ C NMR (100 MHz, CDCl ₃ )δ162.6,161.6,159.8,155.6,128.6,128.3,119.4,114.1,61.0,55.4,14.4,12.2; HR-ESI-MS(positivemode)m/z:262.1068[M+H] ⁺ (Calcd for C ₁₄ H ₁₆ NO ₄ :262.1079 ), m/z:284.0890[M+Na] ⁺ (Calcdfor C ₁₄ H ₁₅ NO ₄ Na: 284.0899).

The following experimental examples demonstrate the beneficial effects of the compounds prepared by the present invention.

Experimental Example 1: Analysis of the inhibitory activity of compounds on miRNA-21

1. Experimental methods:

(1) Experimental materials and instruments

a. Main reagents: DMEM/high glucose medium, PBS buffer, EDTA-0.25% trypsin, penicillin-streptomycin (double antibody), BIOMYC-3Antibiotic Solution, etc. were purchased from Hyclone; fetal bovine serum was purchased from Gibco; Coenzyme Asodium salt hydrate, D-Luciferin sodium salt were purchased from Sigma; DMSO, G418 were purchased from Amresco.

b. Main instruments: _CO2 constant temperature cell culture incubator, clean bench, full wavelength scanning multifunctional reader, cell operation table, optical inverted microscope, pressure steam sterilizer, micro centrifuge, electronic constant temperature water bath, horizontal shaker, etc.

(2) Establishment of cell model

A cell screening model for miRNA-21 small molecule inhibitors was constructed using the luciferase reporter gene: the compound promoted (or inhibited) the expression of miRNA-21 in the cell, increased (or decreased) the complementary pairing of the transfected miRNA-21 complementary sequence, and thus inhibited (or enhanced) the expression of luciferase, as shown in Figure 6.

(3) Cell culture

Take out the frozen HeLa-luciferase-miRNA-21 cells from the liquid nitrogen tank and put them into a water bath for rapid thawing. Absorb the cell suspension into a culture dish containing the prepared culture medium (DMEM high glucose cell culture medium + 10% fetal bovine serum + 1% penicillin streptomycin + 1% BIOMYC-3Antibiotic Solution + G418 (300 μg/mL)), and then place it in a 37°C cell culture incubator with 5% _CO2 .

When the cell density reaches 80-90%, pour out the cell culture medium, wash the cells twice with PBS buffer, pour out PBS, add 1mL of 0.25% trypsin containing EDTA, and place in a cell culture incubator at 37°C for digestion for 3-5min. After digestion is observed to be complete, add fresh cell culture medium, gently blow repeatedly with a pipette to blow the cells off the wall to form a cell suspension, take 100ul 1×10 ⁴ cells and evenly inoculate them into a 96-well plate, place in a 5% CO ₂ , 37°C cell culture incubator and culture overnight. The outer wells of the 96-well plate are not used for compound screening to exclude the influence of edge effects.

(4) Detection of luciferase reporter gene

After the 96-well plate cells were treated with the corresponding drugs for 24 hours, the supernatant culture medium was removed and washed with PBS buffer, and then 50 μL of cell lysis buffer was added and shaken on a shaker for 15-20 minutes, and then 50 μL of lysis solution was taken to the black 96-well plate to be tested, and the pre-thawed luciferase substrate was added to the sample to be tested (50 μL/well), and the fluorescence value of each well was detected by a multi-function reader.

(5) Activity screening

The Hela-miRNA-21 stably transfected cell line was revived and subcultured; the logarithmic phase cells were inoculated in a 96-well plate (1×10 ⁴ ), and the active compounds (the initial screening concentration of all compounds was 10 μM) were added in the clean bench after the cells adhered to the wall. Three replicate wells were made for each compound, and the 96-well plate was placed in a cell culture incubator for 24 hours. Then the fluorescence signal intensity of the cell lysate in each well was measured: RFS (Relative Fluorescence Signal). If the relative fluorescence intensity was greater than the previously verified miRNA-21 inhibitor 3b compound, the next step of the experiment was carried out. (RFS＝RFS sample/RFS control)

(6) Cytotoxic activity detection

AM-Blue cell proliferation and activity detection kit was used to determine the effects of compounds on the viability of Hela-luciferase-miRNA-21 cell lines.

The detection principle of this kit: The main component of SunBio Am-Blue is an oxidized indigo blue indicator, which will generate a stable pink fluorescent substance after being reduced. The excitation wavelength of this fluorescent substance is between 530-560nm, and the emission wavelength is 590nM. The proliferating cells are in a reduced state inside the cells compared to the outside of the cells. The SunBio Am-Blue reagent has good water solubility and is easy to enter the cells. It is eventually reduced to a pink fluorescent substance in the mitochondria, and then released outside the cells and dissolved in the culture medium, changing the culture medium from non-fluorescent indigo blue to fluorescent pink. The cell proliferation is analyzed by detecting the changes in absorbance or fluorescence intensity using an ordinary spectrophotometer or fluorescence photometer. 100 μL of 1×104 cells of Hela-luciferase-miRNA-21 stably transfected cells were inoculated in a 96-well plate, and 8 concentration gradients were set: 0, 0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM. After 24 hours of compound treatment, the fluorescence value was measured, and then the EC ₅₀ value of the screened compound was calculated using GraphPad Prism5 software.

2. Experimental results:

The relative fluorescence intensity of the compounds prepared by the present invention in the activity screening experiment is shown in Figures 1 and 2, with compound 3b as a control.

As can be seen from Figures 1 and 2, compounds T24, T30, T32, T35, T40, T2, T10, S11, S17, S20, S21, and S25 prepared by the present invention have obvious inhibitory activity on miRNA-21 biosynthesis, and the inhibitory activity is higher than that of 3b.

We further conducted a Hela-luciferase-miRNA-21 cytotoxic activity test on the above-mentioned highly active compounds. According to Figure 3, the _EC50 of S21 reached 0.093 μM, and its activity was increased by 15.3 times compared with 3b ( _EC50 = 1.38 μM). Similarly, the _EC50 of compounds T24, T30, T32, T35, T40, T2, T10, S11, S17, S20, and S25 were also lower than that of compound 3b, and they had higher activity than compound 3b.

Experimental Example 2. Affinity analysis of the docking of compounds with TRBP-related binding proteins during miRNA biosynthesis 1. Experimental methods:

In our previous study, we conducted molecular docking of seven reported TRBP-related binding proteins in the biosynthesis process of miRNA (from the PDB database, four from mammals, two from Drosophila genes and one from plants) with compound 3b. The results showed that only two protein crystal models from mammals (4WYQ: Dicer-TRBP and 3LLH: TRBP (dsRBD2)) could form an active pocket with 3b.

In animal cells, TRBP acts as a Dicer partner. TRBP can affect the malignant metastasis of cancer cells by affecting the maturation of miRNA mediated by Dicer and Ago2. TRBP has been shown to be indispensable in the RNA interference (RNAi) process. TRBP interacts with Dicer or Ago2 and combines with dsRNA to form an induced RISC complex (RNAinduced silencing complex) to exert gene silencing function and can process Pre-miRNA to form mature miRNA.

In order to further study the inhibitory effect of the compounds of the present invention on miRNA, we performed molecular affinity docking on a computer with the two protein crystal models and the highly active small molecule inhibitors obtained through the above-mentioned activity experiment screening.

(1) Target protein and instrument information

The receptor proteins read in: 4WYQ (Dicer-TRBP interface), 3LLH (dsRBD2 structure of TRBP), 5N8M (structure of TRBP dsRBD 1 and 2 complexes bound to 19 bp siRNA (complex A)), 5N8L (structure of TRBP dsRBD 1 and 2 complexes bound to 19 bp siRNA (complex B)), 3ADL (TRBP2 structure (plant origin)), 5NPA (Drosophila dsRBD2 structure), 5NPG (Drosophila dsRBD1 structure);

The receptor proteins are from the PDB database: https://www.rcsb.org/.

Software platform: Discovery Studio 4.5.

(2) Experimental operation

1) Import the protein receptor downloaded from the PDB database into the molecular window, assign CHARMM positions to the protein receptor, add hydrogen, remove water molecules, correct unreasonable conformations, etc.

2) Import small molecules in another window, name and sort the small molecules, assign ChARMM positions, and perform energy minimization operations.

3) Select possible active sites in the protein receptor based on the cavity, generate 10 groups in total, and take the first group of sites as the docking active pocket.

4) The CDOCKER module under the receptor-ligand interaction module was used for semi-flexible docking between the receptor and the small molecule. The maximum number of conformation sets generated for a single molecule was 10, and the maximum number of steps in molecular dynamics was 1000.

5) After the docking is completed, all docked conformations are scored and evaluated, and the one with the highest score is selected as the optimal conformation for further observation.

2. Experimental results

The results of molecular docking of the active compound prepared by the present invention with the protein crystal of Dicer-TRBP are shown in FIG4 . The results of molecular docking of the active compound prepared by the present invention with the protein crystal of TRBP (dsRBD2) are shown in FIG5 .

The docking energy calculation structure of the compound prepared by the present invention and the two proteins is shown in Table 1. From the binding degree and conformational binding energy of the small molecule and the amino acid residues on the protein, the number of amino acid residues on the Dicer-TRBP protein that can bind to the inhibitor is relatively large, and the docking energy of the optimal conformation is almost 1 times lower than that of TRBP (dsRBD2) (Table 1), so in the Dicer-TRBP interface protein model, the affinity is much higher than that of TRBP (dsRBD2). It can be seen from the computer-assisted molecular docking energy calculation that the more active the compound, the lower its optimal conformational energy value, indicating that the compound is more tightly bound to the target protein.

As can be seen from Table 1, the optimal conformational energy values of compounds T10, S21, and S25 docked with 4WYQ protein are lower than those of compound 3b with 4WYQ protein; the optimal conformational energy values of compounds T10, T32, S21, and S25 docked with 3LLH protein are lower than those of compound 3b with 3LLH protein. In other words, the above compounds bind more tightly to the corresponding target proteins than 3b.

Table 1 Docking energy ^a of the active compounds prepared by the present invention and proteins

^a The optimal conformation energy value; ^b The total energy value evaluation function is negative, the larger the value, the smaller the energy.

Small molecules can inhibit the biosynthesis of miRNA-21 by docking with TRBP-related binding proteins, thereby achieving the effect of inhibiting the occurrence of malignant tumors and blocking the migration of malignant tumors. Compared with the control compound 3b, the compound prepared by the present invention has a tighter affinity binding ability with TRBP-related binding proteins and has a stronger inhibitory effect on the biosynthesis of miRNA-21. Therefore, the compound of the present invention has a more excellent therapeutic effect on malignant tumors.

In summary, the present invention provides a compound represented by formula I or a pharmaceutically acceptable salt thereof, which can tightly bind to the relevant binding protein in the biosynthesis process of miRNA and can effectively inhibit the synthesis of miRNA-21. The active compound prepared by the present invention can be used as a miRNA-21 inhibitor and further as a potential drug for treating malignant tumors.

Claims (4)

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: the compound is:

2. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a miRNA-21 biosynthesis inhibitor. 3. The use according to claim 2, characterized in that the inhibitor is a drug for treating tumors. 4. The use according to claim 3, characterized in that the inhibitor is a drug for treating malignant tumors.

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