Biphenyl 3cl protease inhibitors and methods of making and using the same

Abstract

The present application relates to a kind of containing biphenyl 3CL protease inhibitor and its preparation method and application, inhibitor has the structure shown in formula I: preparation method specifically is: (1) methyl p-hydroxyphenylpropionate is dissolved in dichloromethane, bromine is added dropwise by nucleophilic substitution reaction and obtains 3-(3-bromo-4-hydroxyphenyl) propionic acid in formula II intermediate.(2)3-(3-bromo-4-hydroxyphenyl) propionic acid is obtained by coupling reaction in the presence of sodium persulfate and ferrous sulfate heptahydrate and obtains formula III intermediate.(3)the Suzuki reaction occurs in the catalysis of palladium acetate under formula III intermediate and substituted phenylboronic acid and occurs intermediate IV.(4)formula IV intermediate and sodium hydroxide carry out hydrolysis reaction and obtain final product I.Compared with prior art, the compound of the present application is novel, has good 3CL protease inhibitory activity, and can be used for preparing the drug for inhibiting 3CL protease activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biphenyl-based 3CL protease inhibitor, its preparation method, and its application. Background Technology

[0002] Patent CN202111181570.1 discloses the application of a 9,10-dihydrophenanthrene compound in the preparation of a coronavirus 3CL protease inhibitor. The 9,10-dihydrophenanthrene compound described in this invention exhibits good inhibitory activity against the coronavirus 3CL protease. Specifically, the IC50 of the 9,10-dihydrophenanthrene compound having the structure shown in Formula I... 50 The values ​​are in the range of 1-70 μM; in particular, the IC50 values ​​of 9,10-dihydrophenanthrene compounds having the structure shown in Formula II-C are... 50 The values ​​can reach the range of 1.5-6 μM. Patent application CN202310311466.2 discloses a bis(phenylvinyl) compound in the preparation of coronavirus 3CL protease inhibitors and its use in the preparation of drugs for the prevention and / or treatment of diseases caused by coronaviruses. The bis(phenylvinyl) compound discovered in this invention has the effect of inhibiting the activity of coronavirus 3CL protease, and has a low half-maximal inhibitory concentration (IC50). 50 The values ​​are all below 30 μM, with the smallest being 6.75 μM.

[0003] It can be seen that although the above-mentioned existing technologies have a good inhibitory effect on 3CL protease activity, there is still room for further improvement. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art by providing a biphenyl 3CL protease inhibitor that is readily available, has a simple synthesis method, and exhibits good inhibitory activity, as well as its preparation method and application.

[0005] The objective of this invention can be achieved through the following technical solution: a biphenyl-based 3CL protease inhibitor, which has the structure shown in Formula I:

[0006] Ⅰ

[0007] Wherein, R is selected from any of the following structural formulas:

[0008] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

[0009] The above structures are in the middle This indicates that the R group is attached to the benzene ring.

[0010] Furthermore, R is... , , , , , , or , , Any one of them.

[0011] Furthermore, the inhibitor has one of the following chemical structural formulas:

[0012] .

[0013] This invention also provides a method for preparing a biphenyl 3CL protease inhibitor, the method comprising the following steps:

[0014] (1) Methyl p-hydroxyphenylpropionate was dissolved in dichloromethane, and bromine was added dropwise to obtain intermediate 3-(3-bromo-4-hydroxyphenyl)propionic acid of formula II via a nucleophilic substitution reaction;

[0015] (2) 3-(3-bromo-4-hydroxyphenyl)propionic acid was coupled in the presence of sodium persulfate and ferrous sulfate heptahydrate to give intermediate III;

[0016] (3) Take intermediate III and substituted phenylboronic acid and react them with palladium acetate as catalyst and diisopropylamine as acid binder to undergo the Suzuki reaction to obtain intermediate IV;

[0017] (4) The intermediate of formula IV undergoes a hydrolysis reaction with sodium hydroxide to obtain the final product I;

[0018] The preparation of biphenyl-based 3CL protease inhibitors is as follows:

[0019] .

[0020] Further, in step (1), the nucleophilic substitution reaction is carried out at a temperature of -5℃ to 5℃ for 8-12 h. After the reaction is completed, the reaction is quenched with water and then extracted with ethyl acetate. The resulting organic phase is dried with anhydrous sodium sulfate and column chromatography is used to obtain intermediate of formula II.

[0021] Further, in step (2), the coupling reaction is carried out at a temperature of 85-100℃ for 18-24 h. After the reaction is completed, the mixture is extracted with ethyl acetate, the organic phase is dried with anhydrous sodium sulfate, and column chromatography is used to obtain intermediate of formula III.

[0022] Further, in step (3), the temperature of the Suzuki reaction is -80℃ to 100℃ and the time is 14-48 h. After the Suzuki reaction, the reaction system is post-processed by the following method: the reaction system is taken out and cooled, and then the pH is adjusted to 6-7 with dilute hydrochloric acid. Then, it is extracted with ethyl acetate and water, the organic phase is dried with anhydrous sodium sulfate, and the intermediate of formula IV is obtained by column chromatography.

[0023] Further, in step (4), the hydrolysis reaction temperature is 80-90℃ and the time is 2-3 h. After the hydrolysis reaction, the following method is used for post-treatment: the reaction system is taken out and cooled, hydrochloric acid is added dropwise in an ice bath, and after the solid precipitates, it is filtered and dried to obtain the inhibitor shown in Formula I.

[0024] The present invention also provides an application of the biphenyl 3CL protease inhibitor, which is used in the preparation of a drug that inhibits the activity of 3CL protease.

[0025] The inhibitor's inhibitory effect on 3CL protease has an IC50 value. 50 The value was as low as 0.15 μM, and its inhibitory activity was superior to that of the positive control drug Ebselen.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) This invention provides a 3CL protease inhibitor with a novel backbone structure, which contains a biphenyl structure and exhibits good 3CL protease inhibitory effect, IC50. 50 The IC50 value can be as low as 0.15 μM, which can be used to prepare drugs that inhibit the activity of 3CL protease. The biphenyl-based 3CL protease inhibitors described in this invention have an IC50 value as low as 0.15 μM. 50The values ​​were all below 4.2 μM, and the IC50 values ​​of the three compounds with the best inhibitory activity were all below 4.2 μM. 50 The values ​​were 0.15 uM, 0.43 uM, and 0.63 uM, respectively, and the inhibitory activity was significantly better than that of existing technologies. It can be used to prevent or treat infections caused by coronaviruses and to prepare drugs for the prevention or treatment of diseases caused by coronaviruses.

[0028] (2) The present invention uses methyl p-hydroxyphenylpropionate as raw material and obtains the product by nucleophilic substitution reaction, coupling reaction, Suzuki reaction and hydrolysis reaction. The synthesis method is simple, the raw materials are readily available, and the production cost is greatly reduced. Attached Figure Description

[0029] Figure 1 The inhibition activity curve of the 3CL protease inhibitor in Example 1.

[0030] Figure 2 This is a diagram illustrating the docking pattern between Example 1 and the SAS-CoV-2 3CL protease. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0032] All raw materials used in this invention are commercially available products.

[0033] The biphenyl-based 3CL protease inhibitor of this invention is prepared by the following method:

[0034] (1) Methyl p-hydroxyphenylpropionate was dissolved in dichloromethane, and bromine was added dropwise at -5℃ to carry out a nucleophilic substitution reaction for 8-12 h. After the reaction was completed, the solution was quenched with water and then extracted with ethyl acetate. The resulting organic phase was dried with anhydrous sodium sulfate and column chromatography was used to obtain intermediate 3-(3-bromo-4-hydroxyphenyl)propionic acid of formula II. The ratio of bromine to methyl p-hydroxyphenylpropionate was (10-15) mmol: (8-12) mmol.

[0035] (2) The 3-(3-bromo-4-hydroxyphenyl)propionic acid obtained in step (1) is coupled in the presence of sodium persulfate and ferrous sulfate heptahydrate. The coupling reaction temperature is 85-100℃, preferably 90℃. The reaction time is 18-24 h. After the reaction is completed, the mixture is extracted with ethyl acetate, the organic phase is dried with anhydrous sodium sulfate, and column chromatography is used to obtain intermediate of formula III. The ratio of intermediate II, sodium persulfate and ferrous sulfate heptahydrate is (3-8) mmol: (6-16) mmol: (0.5-1) mmol, preferably 1 mmol: 2 mmol: 0.5 mmol.

[0036] (3) The intermediate of formula III obtained in step (2) is reacted with substituted phenylboronic acid in the presence of palladium acetate as a catalyst and diisopropylamine as an acid-binding agent to undergo the Suzuki reaction. The reaction system is placed at a temperature of 60-100℃, preferably 90℃, and the reaction time is 14-48 h, preferably 36 h. After the Suzuki reaction, post-treatment is performed. The specific process is as follows: the reaction system is taken out and cooled, and then acidified to pH=6-7 with dilute hydrochloric acid. Then, it is extracted with ethyl acetate and water, the organic phase is dried with anhydrous sodium sulfate, and intermediate IV is obtained by column chromatography. The amount of intermediate of formula III and phenylboronic acid added is (5-8) mmol:(10-20) mmol. The ratio of substituted phenylboronic acid, palladium acetate and diisopropylamine added is 3 mmol:2 mmol:0.02 mmol.

[0037] (4) The intermediate of formula IV obtained in step (3) is subjected to hydrolysis reaction with sodium hydroxide. The hydrolysis reaction temperature is 80-90℃ and the time is 2-3 h. After the hydrolysis reaction, the following post-processing method is used: the reaction system is taken out and cooled, dilute hydrochloric acid is added dropwise in an ice bath, and after the solid precipitates, it is filtered and dried to obtain the final product I. The concentration of the sodium hydroxide aqueous solution is 3M and the concentration of the dilute hydrochloric acid is 2M.

[0038] The equations for the above preparation method are shown below:

[0039]

[0040] The biphenyl 3CL protease inhibitors obtained by the above method have the structure shown in Formula I:

[0041] I

[0042] Wherein, R is selected from any of the following structural formulas:

[0043] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0044] Preferably, R is , , , , , , or , , Any one of these 10 structures.

[0045] This inhibitor can be used in the preparation of drugs that can inhibit the activity of 3CL protease.

[0046] Example 1

[0047] A method for preparing a biphenyl-based 3CL protease inhibitor, the structural formula of which is shown below:

[0048]

[0049] The specific synthesis steps are as follows:

[0050] (1) Accurately weigh 0.90 g (5 mmol) of methyl p-hydroxyphenylpropionate and dissolve it in 6 mL of dichloromethane in a 250 mL round-bottom flask under nitrogen protection. Stir in an ice bath. Then add 0.25 mL (5 mmol) of bromine dropwise using a 1 mL disposable syringe. After the addition is complete, stir the mixture at 0 °C for 24 hours. After the reaction is complete, quench the reaction with water and extract with ethyl acetate. Dry the resulting organic phase with anhydrous sodium sulfate. Column chromatography yields intermediate of formula II.

[0051] (2) Accurately weigh 1.29 g (5 mmol) of intermediate II and add 150 mL of water, 0.6 g of sodium persulfate and 0.034 g of ferrous sulfate heptahydrate to a 250 mL round bottom flask. React at 90 °C for 24 h. After the system is cooled to room temperature, extract with ethyl acetate. Dry the organic phase with anhydrous sodium sulfate and obtain intermediate III by column chromatography.

[0052] (3) Accurately weigh 0.51 g (1 mmol) of intermediate III and 0.57 g (3 mmol) of 4-(trifluoromethyl)phenylboronic acid into a 100 mL test tube, add 2 mL of water, 280 μL (2 mmol) of diisopropylamine, and 4.6 mg (2.0 mol%) of palladium acetate. Incubate at 90 °C for 24 hours. After the reaction is complete, remove the reaction system, cool it, and adjust the pH to 6-7 with dilute hydrochloric acid. Then extract with ethyl acetate and water, dry the organic phase with anhydrous sodium sulfate, and obtain intermediate IV by column chromatography.

[0053] (4) Accurately weigh 0.65 g (1 mmol) of the formula IV intermediate obtained in step (3) into a 50 mL round-bottom flask, add 3 mL of 3M sodium hydroxide aqueous solution, and stir until the solid dissolves. Then, add 2M hydrochloric acid solution dropwise at 0℃ until the pH is 3-5. After the solid has completely precipitated, filter, take the filter cake and dry it to obtain the inhibitor with the above-mentioned structure.

[0054] The inhibitors prepared were tested for their activity against 3CL protease:

[0055] 1. Experimental instruments and materials

[0056] Multifunctional fluorescent microplate reader, SP-Max 3500FL, Shanghai Flash Spectrum Biotechnology Co., Ltd.;

[0057] Clean bench;

[0058] Bond A3Pipette manual single-channel adjustable pipette, 0.5-10 µL, 20-200 µL, 100-1000 µL Titan Technology;

[0059] 96-well plate (black), sterilized, Corning;

[0060] The novel coronavirus 3CL protease inhibitor screening kit used was purchased from Beyotime Biotechnology Co., Ltd. The novel coronavirus 3CL protease inhibitor screening kit (lot No. 072822230228) (containing buffer, fluorescent substrate, active 3CL protease, positive control drug Ebselen), DMSO.

[0061] 2. Experimental Methods

[0062] The positive control drug and the target compound (i.e., 3,3'-(2',2''-dihydroxy-4,4'-bis(trifluoromethyl)-[1,1'-:3',1''-:3'-,1''-tetramethylphenyl]-5',5''-diyl)dipropionic acid prepared in Example 1 above) were dissolved in DMSO to prepare an initial concentration of 1000 µM / L. It was then serially diluted to seven concentration gradients: 100 µM / L, 20 µM / L, 4 µM / L, 0.8 µM / L, 0.16 µM / L, 0.032 µM / L, and 0.0064 µM / L. Three sets of each concentration gradient were prepared sequentially.

[0063] 2.1 Test Method

[0064] The test sample and the positive control drug Ebselen were diluted with DMSO to the required concentration gradient for later use. The 3CL protease was diluted according to the formula to serve as the Assay Reagent.

[0065] During the experiment, three replicates were set up for each concentration. 93 μL of Assay Buffer was added to the blank control wells, and 93 μL of Assay Reagent was added to the other wells.

[0066] Subsequently, 5 μL of DMSO sample solvent was added to each well of the blank control group and the enzyme activity control group, and 5 μL of the sample to be tested was added to each well of the sample group.

[0067] 2.2 Detection

[0068] Place the black 96-well plate with the added sample into the Sp-Max 3500FL multi-functional microplate reader, shake to mix, incubate at 37 °C for 10 min, and then read the sample's autofluorescence value FU0.

[0069] b. After incubation, place the 96-well plate in a low-temperature ice environment. Quickly add 2 μL of Substrate to each well, place it in a Sp-Max 3500FL multi-functional microplate reader, shake to mix, and incubate at 37 °C for about 10 min. After the values ​​stabilize, read the fluorescence values ​​of each well.

[0070] c. Repeat the above steps to perform 3 sets of parallel experiments.

[0071] Organize the fluorescence data, take the average value, and calculate the compound's effect on 3CL. pro Inhibition rate, the formula for which is as follows: Inhibition rate (%) = [FU enzyme activity control - (FU sample - FU0)] / (FU enzyme activity control - FU blank control) × 100%

[0072] Note: The first well in the 96-well plate is used as the blank group. No sample is added, but 10 μL of DMSO solution is added.

[0073] Calculate the average inhibition rate of the sample at each gradient concentration in each parallel experiment, and then fit the corresponding IC50 using Origin. 50 value.

[0074] 2.3 Experimental Results

[0075] Specifically, it is 3,3'-(2',2''-dihydroxy-4,4'-bis(trifluoromethyl)-[1,1'-:3',1''-:3'-,1''-tetramethylphenyl]-5',5''-diyl)dipropionic acid (L3), a yellow solid with a yield of 50%, a melting point of 187.5~198.1 °C, and a purity of 95.90%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.19 (s, 2H), 8.61 (s, 2H), 7.86 – 7.80 (m, 8H), 7.26 – 7.21 (m, 4H), 2.90 (t, J = 7.6 Hz, 4H), 2.63 (t, J = 7.6 Hz, 4H). 13 C NMR (100 MHz, DMSO- d 6) δ 174.51, 149.69, 143.72, 133.52, 132.30,130.72, 130.39, 129.33, 128.33, 127.87, 127.56, 126.38, 125.41, 125.38,125.34, 125.30, 36.02, 30.11. HRMS (ESI) calcd for C 32 H 24 F6O6[M+Na] + :641.1369;Found: 641.1360. Its IC 50 The value was 0.15 μM, indicating that its inhibitory activity was superior to that of the positive control drug ebuselenium (IC50). 50 = 2.04 μM).

[0076] The positive control drug and the target compound were prepared into a mixed solution with an initial concentration of 100 µM / L using DMSO solution. These two mixed solutions were then serially diluted to create six concentration gradients: 100 µM / L, 20 µM / L, 4 µM / L, 0.8 µM / L, 0.16 µM / L, 0.032 µM / L, and 0.0064 µM / L. Three sets of each concentration gradient were prepared sequentially. ① The test sample and the positive inhibitor Ebselen were diluted with DMSO to the required concentration gradient for later use. 3CL pro ① Dilute according to the specified ratio to prepare AssayReagent. ② During the experiment, set up three replicates for each concentration. Add 93 μL of Assay Buffer to the blank control wells and 93 μL of Assay Reagent to the remaining wells. Subsequently, add 5 μL of DMSO sample solvent to each well of the blank control and enzyme activity control groups, and add 5 μL of the sample to be tested to each well of the sample group. ③ Place the black 96-well plate with the added samples in a Sp-Max 3500FL multi-functional microplate reader, shake to mix, and incubate at 37 °C for 10 min before reading the sample autofluorescence value FU0. ④ After incubation, place the 96-well plate in a low-temperature ice environment. Quickly add 2 μL of Substrate to each well, place it in a Sp-Max 3500FL multi-functional microplate reader, shake to mix, and incubate at 37 °C for about 10 min. After the values ​​stabilize, read the fluorescence value of each well. Perform the experiment in parallel three times. Calculate the average inhibition rate of the sample at each gradient concentration in each parallel experiment. ⑤ Organize the fluorescence data, take the average value, and calculate the inhibition rate of the compound against 3CL protease. The inhibition rate formula is as follows: Inhibition rate (%) = [FU enzyme activity control - (FU sample - FU0)] / (FU enzyme activity control - FU blank control) × 100%. Then, use Origin to fit the corresponding IC50 value. 50 The inhibition rate of each sample was calculated, and then the corresponding IC50 was obtained by fitting the data using Origin. 50 value.

[0077] Figure 1 The figure shows the inhibitory activity curve of the 3CL protease inhibitor in Example 1. As can be seen from the figure, the curve exhibits a good inverted S-shape, and the IC50 of Example 1 is [missing information]. 50 The value was 0.15 μmol, indicating that the compound has good inhibitory activity against 3CL protease.

[0078] Figure 2This is a diagram illustrating the docking pattern of Example 1 with the SAS-CoV-2 3CL protease. The diagram shows that the compound from Example 1 effectively occupies the active cavity. It forms hydrogen bonds with five amino acids of the 3CL protease (Tyr54, Arg188, Ser144, Gly143, Asn119) and hydrophobically interacts with the catalytic dichotomy (Cys145, His41). These factors likely contribute to its good activity.

[0079] Example 2

[0080] A method for preparing a biphenyl-based 3CL protease inhibitor, wherein (4-chlorophenyl)boronic acid is used as a raw material in step three, and the rest is the same as in Example 1. The structure of the prepared inhibitor is as follows:

[0081] 3,3'-(4,4''-dichloro-2',2''-dihydroxy-[1,1':3',1'':3'',1''-tetramethylphenyl]-5',5''-diyl)dipropionic acid

[0082]

[0083] The obtained white solid product was tested using the same method as in Example 1: yield 45%, melting point: 172.9~182.1 °C, purity 98.70%, IC50. 50 The value is 4.14 μM.

[0084] 1 H NMR (400 MHz, DMSO- d 6) δ 12.22 (s, 2H), 8.53 (s, 2H), 7.64 (dd, J =8.6, 3.4 Hz, 4H), 7.53 – 7.49 (m, 4H), 7.21 – 7.18 (m, 4H), 2.88 (d, J = 6.2Hz, 4H), 2.65 (d, J = 7.6 Hz, 4H). 13 C NMR (100 MHz, DMSO- d 6) δ 174.49,149.41, 138.22, 133.55, 132.05, 131.78, 130.22, 129.62, 128.52, 36.12, 30.18.HRMS (ESI) calcd for C 30 H 24 Cl2O6[M+Na] +Found: 573.0842; Found: 573.0837.

[0085] Example 3

[0086] A method for preparing a biphenyl-based 3CL protease inhibitor, wherein (4-methoxyphenyl)boronic acid is used as a raw material in step three, and the rest is the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0087] 3,3'-(2',2''-dihydroxy-4,4'-dimethoxy-[1,1':3',1':3'-tetramethylphenyl]-5',5''-diyl)dipropionic acid:

[0088]

[0089] The obtained white solid product was tested using the same method as in Example 1: yield 40%, melting point: 169.5~178.1 °C, purity 97.50%, IC50. 50 The value is 1.68 μM.

[0090] 1 H NMR (400 MHz, DMSO- d 6) δ 8.11 (d, J = 207.9 Hz, 2H), 7.52 (d, J =8.4 Hz, 4H), 7.19 – 7.07 (m, 4H), 7.01 (d, J = 8.4 Hz, 4H), 3.81 (s, 6H), 2.86 (t, J = 7.6 Hz, 4H), 2.60 (t, J = 7.6 Hz, 4H). 13 C NMR (100 MHz, DMSO- d 6)δ 174.51, 158.78, 149.12, 133.49, 131.55, 131.04, 130.88, 130.65, 130.10,128.69, 114.05, 55.64, 36.10, 30.21. HRMS (ESI) calcd for C 30 H 30 O8[M+Na] + Found: 565.1832; Found: 565.1839.

[0091] Example 4

[0092] A method for preparing a biphenyl-based 3CL protease inhibitor, wherein 2-(3-methoxyphenyl)ethane-1-amine is used as a raw material in step one, and the rest is the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0093] 3,3'-(3,3''-,4,4''-tetrafluoro-2'-,2''-dihydroxy-[1,1':3'-,1''-tetramethylphenyl]-5',5''-diyl)dipropionic acid:

[0094]

[0095] The obtained white solid product was tested using the same method as in Example 1: yield 46%, melting point: 182.5~193.1 °C, purity 96.80%, IC 50 The value is 1.11 μM.

[0096] 1 H NMR (400 MHz, Methanol- d 4) δ 7.51 (ddt, J = 11.9, 6.2, 3.0 Hz, 2H),7.41 – 7.24 (m, 4H), 7.22 – 7.11 (m, 4H), 2.95 (t, J = 7.4 Hz, 4H), 2.67 (q, J = 7.7 Hz, 4H). 13 C NMR (100 MHz, Methanol- d 4) δ 175.67 (d, J = 3.0 Hz), 148.95 (d, J = 5.0 Hz), 136.17, 133.46, 133.24, 131.24 (d, J = 3.7 Hz),129.96, 128.60, 127.49 (d, J = 3.4 Hz), 125.77 (dd, J = 6.2, 3.3 Hz), 118.36,118.19, 116.51 (d, J = 17.1 Hz), 35.59 (d, J = 2.5 Hz), 29.83 (d, J = 11.0Hz). HRMS (ESI) calcd for C 30 H 22F4O6[M+Na] + Found: 577.1244; Found: 577.1236.

[0097] Example 5

[0098] A method for preparing a biphenyl-based 3CL protease inhibitor, wherein p-tolylboronic acid is used as a raw material in step three, and the rest is the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0099] 3,3'-(2',2''-dihydroxy-4,4'-dimethyl-[1,1'-:3',1''-:3'-,1''-tetramethylphenyl]-5',5''-diyl)dipropionic acid:

[0100]

[0101] The obtained white solid product was tested using the same method as in Example 1: yield 47%, melting point: 140.15~149.8 °C, purity 98.30%, IC50. 50 The value is 1.33 μM.

[0102] 1 H NMR (400 MHz, DMSO- d 6) δ 12.13 (s, 2H), 8.39 (s, 2H), 7.48 (d, J =7.7 Hz, 4H), 7.26 (d, J = 7.7 Hz, 4H), 7.17 – 7.10 (m, 4H), 2.87 (t, J = 7.6Hz, 4H), 2.61 (t, J = 7.6 Hz, 4H), 2.37 (s, 6H). 13 C NMR (100 MHz, DMSO- d 6) δ174.50, 149.21, 136.39, 133.49, 131.14, 130.90, 130.20, 129.82, 129.14,128.68, 36.10, 30.21, 21.28. HRMS (ESI) calcd for C 30 H 30 O6[M+Na] + Found: 533.1934; Found: 533.1940.

[0103] Example 6

[0104] A method for preparing a biphenyl-based 3CL protease inhibitor, wherein phenylboronic acid is used as a raw material in step three, and methyl quinoline-6-carboxylate is used as a raw material in step two, with the remaining steps being the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0105] 3,3'-(2',2''-dihydroxy-[1,1':3',1'':3'',1''-tetraphenyl]-5',5''-diyl)dipropionic acid:

[0106]

[0107] The obtained yellow solid product was tested using the same method as in Example 1: yield 39%, melting point: 102.1~118.2 °C, purity 97.00%, IC50. 50 The value is 3.08 μM.

[0108] 1 H NMR (400 MHz, DMSO- d 6) δ 12.12 (s, 2H), 8.44 (s, 2H), 7.59 (d, J =7.5 Hz, 4H), 7.45 (t, J = 7.6 Hz, 4H), 7.35 (t, J = 7.4 Hz, 2H), 7.19 – 7.13(m, 4H), 2.87 (t, J = 7.6 Hz, 4H), 2.61 (t, J = 7.6 Hz, 4H). 13 C NMR (100 MHz, DMSO- d 6) δ 174.50, 149.24, 139.39, 133.52, 131.39, 131.00, 130.35, 129.96,128.65, 128.53, 127.23, 36.07, 30.19. HRMS (ESI) calcd for C 30 H 26 O6[M+Na] + :505.1621; Found: 505.1616.

[0109] Example 7

[0110] A method for preparing a biphenyl-based 3CL protease inhibitor, the structural formula of which is shown below, uses 2-(3,4-dimethoxyphenyl)ethane-1-amine as a starting material in step one and 3,4-dichlorophenylboronic acid as a starting material in step three, with the remainder being the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0111] 3,3'-(3,3''-,4,4''-tetrachloro-2'-,2''-dihydroxy-[1,1':3'-,1''-tetramethylphenyl]-5',5''-diyl)dipropionic acid:

[0112]

[0113] The obtained white solid product was tested using the same method as in Example 1: yield 37%, melting point: 181.3~192.1 °C, purity 96.30%, IC50. 50 The value is 2.90 μM.

[0114] 1 H NMR (400 MHz, DMSO- d 6) δ 8.53 (s, 2H), 7.87 (s, 2H), 7.72 – 7.56 (m, 4H), 7.18 (d, J = 28.9 Hz, 4H), 3.24 – 3.17 (m, 2H), 2.89 (s, 4H), 2.72(d, J = 4.0 Hz, 4H). 13 C NMR (100 MHz, DMSO- d 6) δ 173.39, 149.73, 140.01,133.04, 132.23, 131.77, 131.14, 130.68, 130.23, 130.19, 129.90, 128.13,128.07, 35.62, 29.97. HRMS (ESI) calcd for C 30 H 22 Cl4O6[M+Na] + : 643.0037; Found: 643.0036.

[0115] Example 8

[0116] A method for preparing a biphenyl-based 3CL protease inhibitor, the structural formula of which is shown below, wherein p-fluorophenylboronic acid is used as a raw material in step three, and the rest is the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0117] 3,3'-(2',2''-dihydroxy-4,4'-difluoro-[1,1':3',1':3'-tetramethylphenyl]-5',5''-diyl)dipropionic acid:

[0118]

[0119] The obtained yellow solid product was tested using the same method as in Example 1: yield 40%, melting point: 171.3~182.1 °C, purity 96.30%, IC50. 50 The value is 3.59 μM.

[0120] 1 H NMR (400 MHz, DMSO- d 6) δ 7.59 (dd, J = 8.5, 5.6 Hz, 4H), 7.25 (t, J = 8.8 Hz, 4H), 7.13 (d, J = 7.2 Hz, 4H), 2.84 (t, J = 7.6 Hz, 4H), 2.59 (t, J = 7.6 Hz, 4H). 13 C NMR (101 MHz, DMSO- d 6) δ 174.52, 162.98, 160.56, 149.23,135.60, 133.49, 131.89, 131.81, 131.44, 130.26, 129.88, 128.51, 115.42,115.21, 36.07, 30.11. HRMS (ESI) calcd for C 30 H 24 O6F2[M+Na] + : 541.1433; Found:541.1434.

[0121] Example 9

[0122] A method for preparing a biphenyl-based 3CL protease inhibitor, the structural formula of which is shown below, is described. In step one, 3,4-dichlorophenylmethylamine is used as a raw material, and in step three, 3-chloro-4-fluorophenylboronic acid is used as a raw material; the remaining steps are the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0123] 3,3'-(2',2''-dihydroxy-3,3-dichloro-4,4-difluoro-[1,1':3',1':3'-tetramethylphenyl]-5',5''-diyl)dipropionic acid:

[0124]

[0125] The obtained gray solid product was tested using the same method as in Example 1: yield 38%, melting point: 168.3~172.1 °C, purity 96.30%, IC50. 50 The value is 0.43 μM.

[0126] 1 H NMR (400 MHz, DMSO- d 6) δ 8.47 (s, 2H), 7.78 (dd, J = 7.4, 2.1 Hz,2H), 7.59 – 7.55 (m, 2H), 7.48 (t, J = 9.0 Hz, 2H), 7.18 (d, J = 2.2 Hz, 2H), 7.13 (d, J = 2.3 Hz, 2H), 2.84 (t, J = 7.6 Hz, 4H), 2.59 (t, J = 7.6 Hz, 4H). 13 C NMR (100 MHz, DMSO- d 6) δ 174.54, 149.50, 137.05, 133.37, 131.98, 131.82,130.54, 130.27, 128.28, 128.11, 117.09, 116.88, 35.99, 30.06. HRMS (ESI)calcd for C 30 H 22 O6F2Cl2[M+K] + Found: 625.0393; Found: 625.0396.

[0127] Example 10

[0128] A method for preparing a biphenyl-based 3CL protease inhibitor, wherein 2-(3-fluorophenyl)ethane-1-amine is used as a raw material in step one, and 4-fluoro-3-methylphenylboronic acid is used as a raw material in step three, with the remaining steps being the same as in Example 1. The structure of the obtained inhibitor is as follows:

[0129] 3,3'-(2',2''-dihydroxy-3,3-dimethyl-4,4-difluoro-[1,1':3',1':3'-tetramethylphenyl]-5',5''-diyl)dipropionic acid:

[0130]

[0131] The obtained gray solid product was tested using the same method as in Example 1: yield 89%, melting point: 185.3~190.2 °C, purity 96.30%, IC50. 50 The value is 0.63 μM.

[0132] 1 H NMR (400 MHz, DMSO- d 6) δ 12.10 (s, 2H), 8.41 (s, 2H), 7.47 (d, J =7.6 Hz, 2H), 7.40 (t, J = 6.7 Hz, 2H), 7.18 (d, J = 9.0 Hz, 2H), 7.12 (d, J =7.1 Hz, 4H), 2.85 (t, J = 7.7 Hz, 4H), 2.59 (t, J = 7.8 Hz, 4H), 2.30 (s, 6H). 13 C NMR (100 MHz, DMSO- d 6) δ 174.54, 149.21, 135.33, 133.41, 133.14,131.35, 130.26, 129.98, 129.15, 128.48, 124.20, 115.09, 114.87, 36.07, 30.13,14.75. HRMS (ESI) calcd for C 32 H 28 O6F2[M+Na] + Found: 569.1746; Found: 569.1747. The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention's technical solution shall still fall within the protection scope of the present invention's technical solution.

Claims

1. A biphenyl 3CL protease inhibitor, characterized in that, The inhibitor has one of the following chemical structural formulas: 。 2. A method for preparing a biphenyl 3CL protease inhibitor as described in claim 1, characterized in that, The method comprises the following steps: (1) dissolving methyl-p-hydroxyphenylpropionate in dichloromethane, and adding bromine dropwise to obtain 3-(3-bromo-4-hydroxyphenyl) propionic acid of formula II through nucleophilic substitution reaction; (2) 3-(3-bromo-4-hydroxyphenyl) propionic acid is obtained through coupling reaction in the presence of sodium persulfate and ferrous sulfate heptahydrate; (3) the intermediate of formula III is subjected to Suzuki reaction with substituted phenylboronic acid in the presence of palladium acetate as a catalyst and diisopropylamine as an acid binding agent to obtain intermediate IV; (4) the intermediate of formula IV is subjected to hydrolysis reaction with sodium hydroxide to obtain the final product I; The preparation of the 3CL protease inhibitor containing biphenyl is as follows: 。 3. The method for preparing a biphenyl 3CL protease inhibitor according to claim 2, characterized in that, In step (1), the temperature of the nucleophilic substitution reaction is -5-5 DEG C, and the time is 8-12 h; after the reaction is completed, water is used for quenching, and then ethyl acetate is used for extraction; the obtained organic phase is dried with anhydrous sodium sulfate, and column chromatography is used to obtain the intermediate of formula II.

4. The method for preparing a biphenyl 3CL protease inhibitor according to claim 2, characterized in that, In step (2), the temperature of the coupling reaction is 85-100 DEG C, and the time is 18-24 h; after the reaction is completed, ethyl acetate is used for extraction, and the organic phase is dried with anhydrous sodium sulfate; column chromatography is used to obtain the intermediate of formula III.

5. The method for preparing a biphenyl 3CL protease inhibitor according to claim 2, characterized in that, In step (3), the temperature of the Suzuki reaction is 80-100 DEG C, and the time is 14-48 h; after the Suzuki reaction, the following method is used for post-treatment: the reaction system is taken out and cooled, and then diluted hydrochloric acid is used to adjust the pH to 6-7; then ethyl acetate and water are used for extraction, the organic phase is dried with anhydrous sodium sulfate, and column chromatography is used to obtain the intermediate of formula IV.

6. The method for preparing a biphenyl 3CL protease inhibitor according to claim 2, characterized in that, In step (4), the temperature of the hydrolysis reaction is 80-90 DEG C, and the time is 2-3 h; after the hydrolysis reaction, the following method is used for post-treatment: the reaction system is taken out and cooled, and then hydrochloric acid is added dropwise in an ice bath; after the solid is precipitated, filtration is carried out, and then drying is carried out to obtain the inhibitor of formula I.

7. An application of the biphenyl 3CL protease inhibitor as described in claim 1, characterized in that, The inhibitor is applied to the preparation of a medicine for inhibiting the activity of 3CL protease.

8. The use of a biphenyl 3CL protease inhibitor according to claim 7, characterized in that, The inhibitor has an inhibition IC50 value for 3CL protease as low as 0.15 μM. 50 The inhibitor has an inhibition IC50 value for 3CL protease as low as 0.15 μM.

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