A quinoline compound targeting angiotensin converting enzyme 2 receptor and a preparation method and application thereof

Abstract

The application discloses a quinoline compound targeting angiotensin converting enzyme 2 receptor and a preparation method and application thereof, and belongs to the field of antiviral drugs. The structure of the quinoline compound is shown as formula 1. The compound blocks the combination of the novel coronavirus and a host by targeting ACE2 protein, so that the antiviral infection effect is achieved. The half maximal effective concentration of the compound for resisting the antiviral infection is 10 muM. The compound is chemically stable, easy to modify, and can be connected with different groups through a chemical bond to further improve the antiviral activity. The compound provides a theoretical basis for subsequent researches such as antiviral infection activity experiments, in-vivo animal experiments and toxicology experiments, and can also be used for preparing a medicine for resisting the novel coronavirus infection. The application has a good application prospect in the field of resisting the novel coronavirus infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of antiviral drugs, and particularly relates to a quinoline compound targeting angiotensin converting enzyme 2 receptor and a preparation method and application thereof. BACKGROUND

[0002] At present, various drugs targeting Spike protein, angiotensin converting enzyme 2 (ACE2), 3CL pro protein (3C-like protease), PL pro protein (papain-like protease) and other target proteins have been widely used in the treatment of COVID-19 infection. However, as the virus continues to evolve, its mortality rate has been continuously reduced, and the infectivity has been enhanced, greatly increasing the risk of COVID-19 infection. Therefore, the development of drugs against COVID-19 infection has practical application value.

[0003] Angiotensin converting enzyme 2 (ACE2) is a membrane protein located on the surface of human cells, an important member of the renin-angiotensin system, and an important system for regulating blood pressure and multiple organs, including lungs, hearts and kidneys. ACE2 protein is a target protein for COVID-19 invasion of the host and is also a hot target for drug development. In the context of the global viral pandemic, it takes a long time to find potential antiviral compounds through traditional drug development modes, so it is more suitable to develop lead compounds with antiviral effects through other ways, such as old drugs with new uses, high-throughput screening or computer-aided drug design. SUMMARY

[0004] The application aims to provide a quinoline compound targeting angiotensin converting enzyme 2 receptor. The application also aims to provide a preparation method and application of the quinoline compound.

[0005] The application achieves the above-mentioned purpose through the following technical solutions.

[0006] The quinoline compound targeting angiotensin converting enzyme 2 receptor is a quinoline small molecule compound with a modified group introduced at positions 2 and 4 of the quinoline compound, and its structural formula is shown as formula 1.

[0007]

[0008] In formula 1, R1 is selected from:

[0009] R2 is selected from:

[0010] In some preferred embodiments, the quinoline compound targeting angiotensin converting enzyme 2 receptor is DH1, DH2, as shown in the following structure:

[0011]

[0012] The preparation method of the above-mentioned quinoline compound targeting angiotensin converting enzyme 2 receptor has the following synthetic route:

[0013]

[0014] Specifically, it includes the following steps:

[0015] (1) Compound 1 is dissolved in a solvent, compound 2 is added for reaction to obtain intermediate compound 3. The solvent is preferably dichloromethane, and the reaction temperature is preferably 15-35°C.

[0016] (2) Compound 3 is dissolved in a solvent, sodium hydride is added for reaction, after the reaction is completed, methyl tert-butyl ether is extracted, and after acidification, intermediate compound 4 is obtained. The solvent is preferably tetrahydrofuran, and the reaction temperature is preferably 15-35°C.

[0017] (3) Compound 4 is dissolved in a solvent, and refluxed for reaction, and after the reaction is completed, intermediate compound 5 is obtained. The solvent is preferably phosphorus oxychloride.

[0018] (4) Compound 5 and sodium acetate are dissolved in a solvent, and refluxed for reaction, and after the reaction is completed, intermediate compound 6 is obtained. The solvent is preferably glacial acetic acid.

[0019] (5) Compound 6 is reacted with R2-NH-R' or R2-Br to obtain intermediate compound 7.

[0020] The R2-NH-R' or R2-Br is selected from:

[0021] (6) Intermediate compound 7 is reacted with R1-NH-R' or R1-OH to obtain compound 8, i.e. the quinoline compound.

[0022] The R1-NH-R' or R1-OH is selected from:

[0023] When the quinoline compound is DH1, the preparation method thereof includes the following steps:

[0024] 1) Compound 6, benzyl bromide, and a catalyst are reacted in a solvent to obtain product 1. The catalyst is preferably DIPEA, the solvent is preferably ethanol, and the reaction temperature is preferably 15-35°C.

[0025] 2) reacting product 1, piperazine, DIPEA in a solvent to obtain product 2. The solvent is preferably DMF, and the reaction temperature is preferably 40-60 DEG C.

[0026] 3) reacting product 2, benzoic acid, a catalyst in a solvent to obtain DH1. The catalyst is preferably HOBT, HBTU, DIPEA, the solvent is preferably DMF, and the reaction temperature is preferably 15-35 DEG C.

[0027] When the quinoline compound is DH2, the preparation method comprises the following steps:

[0028] 1) reacting compound 6, bromopentane, a catalyst in a solvent to obtain product 1. The catalyst is preferably DIPEA, the solvent is preferably ethanol, and the reaction temperature is preferably 15-35 DEG C.

[0029] 2) reacting product 1, piperidine hydrochloride, a catalyst in a solvent to obtain DH2. The catalyst is preferably DIPEA, the solvent is preferably DMF, and the reaction temperature is preferably 30-50 DEG C.

[0030] A pharmaceutically acceptable salt of the above-mentioned quinoline compound targeting angiotensin converting enzyme 2 receptor.

[0031] The above-mentioned quinoline compound targeting angiotensin converting enzyme 2 receptor can block the binding of viruses to hosts by targeting ACE2 protein, thereby achieving an antiviral infection effect. Therefore, the above-mentioned quinoline compound or its pharmaceutically acceptable salt can be used for preparing an ACE2 binding agent or an antiviral drug. The virus is a virus that can infect a host through ACE2. Further, the virus is a new coronavirus. The half maximal effective concentration of DH1 and DH2 in the above-mentioned quinoline compound against new coronavirus infection is 10 muM and 13.06 muM, respectively.

[0032] An antiviral drug comprising the above-mentioned quinoline compound or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

[0033] Advantages and beneficial effects of the present application:

[0034] The present application obtains a quinoline small molecule compound capable of playing an anti-new coronavirus infection role by targeting angiotensin converting enzyme 2 (ACE2) by modifying different substituents at positions 2 and 4 of the quinoline compound, wherein the IC 50 value of compound DH1 is 10 muM, and the IC 50 value of compound DH2 is 13.06 muM.

[0035] The quinoline compound has stable chemical properties, is easy to modify, can be further improved in antiviral activity by connecting different groups through chemical bonds, and can provide a theoretical basis for subsequent research on anti-viral infection activity, in-vivo animal experiments, and toxicological experiments. The application has good application prospects in the field of anti-novel coronavirus infection. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Fig. 1 is a hydrogen spectrum and carbon spectrum nuclear magnetic spectrum diagram of a quinoline small molecule compound DH1; A is a hydrogen spectrum nuclear magnetic spectrum diagram of DH1, and B is a carbon spectrum nuclear magnetic spectrum diagram of DH1.

[0037] Figure 2 Fig. 2 is a high-resolution mass spectrum diagram of the quinoline small molecule compound DH1.

[0038] Figure 3 Fig. 3 is a hydrogen spectrum and carbon spectrum nuclear magnetic spectrum diagram of a quinoline small molecule compound DH2; A is a hydrogen spectrum nuclear magnetic spectrum diagram of DH2, and B is a carbon spectrum nuclear magnetic spectrum diagram of DH2.

[0039] Figure 4 Fig. 4 is a high-resolution mass spectrum diagram of the quinoline small molecule compound DH2.

[0040] Figure 5 Fig. 5 is a result diagram of anti-novel coronavirus activity of the compounds DH1 and DH2.

[0041] Figure 6 Fig. 6 is a surface plasmon resonance (SPR) test diagram of the compounds DH1 and DH2 and ACE2 protein.

[0042] Figure 7 Fig. 7 is a surface plasmon resonance test diagram of the compounds DH1 and DH2 and spike protein (S).

[0043] Figure 8 Fig. 8 is a surface plasmon resonance test diagram of the compounds DH1 and DH2 and transmembrane serine protein 2 (TMPRSS2). DETAILED DESCRIPTION

[0044] The synthesis route of the quinoline compound targeting angiotensin converting enzyme 2 receptor provided by the application is as follows:

[0045]

[0046] The preparation method of the quinoline compound specifically includes the following steps:

[0047] (1) Compound 1 was dissolved in dichloromethane, 1.5 eq of triethylamine was added, stirred for 2 min in ice bath, then compound 2 was slowly added to the mixture, and the reaction was stirred at room temperature for 15 h. After the reaction was completed, saturated NaHCO3 solution was added to quench residual acyl chloride species, the reaction solution was extracted with dichloromethane 3 times, the organic layer was washed with saturated sodium chloride solution 3 times, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was further purified by column chromatography to obtain intermediate compound 3.

[0048] (2) Compound 3 was dissolved in super-dry tetrahydrofuran, 1.4 eq of sodium hydride was added in ice bath, and the reaction was stirred at room temperature for 16 h. After the reaction was completed, the solvent was rotary evaporated, water was added, and methyl tert-butyl ether was used to extract 3 times. The water phase was reserved, and the pH of the water phase was adjusted to 2 with dilute hydrochloric acid. The mixture was filtered and dried to obtain a light yellow solid, which was intermediate compound 4.

[0049] (3) Compound 4 was slowly added dropwise with phosphorus oxychloride in ice bath, and stirred at 120°C for 18 h. The phosphorus oxychloride was removed by distillation under reduced pressure, and quenched with ice water. The mixture was stirred for 30 min with a mixture of ethyl acetate and water, and extracted with ethyl acetate 3 times. The organic layer was washed with saturated sodium chloride solution 3 times, and the organic phase was dried over anhydrous sodium sulfate. The solvent was rotary evaporated to obtain the crude product, which was further purified by column chromatography to obtain intermediate compound 5.

[0050] (4) Compound 5 was dissolved in glacial acetic acid, and 5 eq of sodium acetate was added. The reaction was stirred at 120°C for 18 h. After the reaction was completed, the acetic acid was rotary evaporated, and the reaction solution was diluted with water, filtered and dried to obtain a white solid, which was intermediate compound 6.

[0051] (5) Intermediate compound 6 was dissolved in anhydrous ethanol or DMF, 1.5 eq of R2-NH-R’ (or R2-Br) and 5 eq of DIPEA were added, and the reaction was carried out at 20°C to 100°C for 8 to 24 h. After the reaction was completed, the reaction solution was rotary evaporated and concentrated, extracted with a mixture of ethyl acetate and water 3 times, and the organic phase was washed with saturated brine 3 times. The organic phase was dried over anhydrous sodium sulfate, rotary evaporated and concentrated, and purified by column chromatography to obtain intermediate compound 7. The specific structure of R2-NH-R’ (or R2-Br) is as follows:

[0052]

[0053] (6) a: Intermediate compound 7 was dissolved in DMF, 1.5 eq R1-NH-R' (or R1-OH) was added, 5 eq DIPEA was added, and the mixture was reacted at 20-100°C for 8-24 h. After the reaction was completed, rotary evaporation and concentration were performed, extraction was performed three times using a mixture of ethyl acetate and water, the organic phase was washed three times with saturated brine, anhydrous sodium sulfate was added for drying, rotary evaporation and concentration were performed, and column chromatography was performed to separate and purify to obtain compound 8. The specific structures of R1-NH-R' (or R1-OH) are as follows:

[0054]

[0055] b: Intermediate compound 7 was dissolved in DMF, 1.5-2.0 eq piperazine or 4-hydroxypiperidine was added, 5 eq DIPEA was added, and the mixture was reacted at 20-100°C for 8-24 h. After the reaction was completed, rotary evaporation and concentration were performed, extraction was performed three times using a mixture of ethyl acetate and water, the organic phase was washed three times with saturated brine, anhydrous sodium sulfate was added for drying, rotary evaporation and concentration were performed, and column chromatography was performed to separate and purify to obtain an intermediate compound. 1.2 eq cyclohexanecarboxylic acid or benzoic acid or phenylacetic acid, 3 eq HOBT, 3 eq HBTU, and 5 eq DIPEA were dissolved in DMF, and the mixture was stirred at room temperature for 10-30 min. Then, the intermediate compound (1.0 eq) obtained in the above step was added, and the mixture was reacted at 20-50°C for 12-18 h. After the reaction was completed, rotary evaporation and concentration were performed, extraction was performed three times using a mixture of ethyl acetate and water, the organic phase was washed three times with saturated brine, anhydrous sodium sulfate was added for drying, rotary evaporation and concentration were performed, and column chromatography was performed to separate and purify to obtain compound 8.

[0056] The quinoline compounds obtained by the above preparation method were characterized by nuclear magnetic resonance and high-resolution mass spectrometry.

[0057] The following describes the present application in detail by taking quinoline compounds DH1 and DH2 (the structures of which are shown below) as examples. It should be understood that the examples provided below are only illustrative of the present application and do not limit the remaining content disclosed by the present application in any way.

[0058]

[0059] Example 1: Synthesis and characterization of quinoline compound DH1

[0060]

[0061] (a) 1.5 eq Et3N, DCM, r.t., 15 h; (b) 1.4 eq 60% NaH, dry THF, r.t., 16 h; (c) POCl3, 110°C, 5 h; (d) 5 eq AcONa·3H2O, AcOH, 120°C, reflux, 18 h;

[0062]

[0063] (1) Compound 1 (2.00 g, 13.24 mmol) was dissolved in 15 mL of dichloromethane, 1.5 eq of triethylamine (2.003 g) was added, stirred for 2 min in an ice bath, then compound 2 (2.677 g, 17.85 mmol) was slowly added to the mixture, and the reaction was stirred at room temperature for 15 hours. After the reaction was completed, saturated NaHCO3 solution was added to quench residual acyl chloride species, and the reaction solution was extracted with dichloromethane 3 times. The organic layer was washed with saturated sodium chloride solution 3 times, and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated to obtain a crude product, which was further purified by column chromatography to obtain 3.472 g of compound 3 as a light yellow oily liquid, with a yield of 99%; 1 H NMR (400 MHz, Chloroform-d) δ 11.45 (s, 1H), 8.67 (dd, J = 8.5, 1.2 Hz, 1H), 8.02 (dd, J = 7.9, 1.7 Hz, 1H), 7.53 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.10 (td, J = 7.7, 1.2 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 3.52 (s, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 168.24, 167.57, 164.02, 140.71, 134.41, 130.79, 122.92, 120.63, 115.53, 61.61, 52.37, 52.35, 44.69, 14.01.

[0064] (2) Compound 3 (3.472 g, 13.10 mmol) was dissolved in 16 mL of super dry tetrahydrofuran, 1.4 eq of sodium hydride (0.7335 g, 18.34 mmol) was added in an ice bath, and the reaction was stirred at room temperature for 16 hours. After the reaction was completed, the solvent was evaporated, 200 mL of water was added, and methyl tert-butyl ether was extracted 3 times. The aqueous phase was reserved, and the pH of the aqueous phase was adjusted to 2 with dilute hydrochloric acid. The filtrate was dried to obtain 2.471 g of compound 4 as a light yellow white solid, with a yield of 80.9%; 1H NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 7.94 (dd, J = 8.1, 1.4 Hz, 1H), 7.62 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.20 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). 13 CNMR (101 MHz, DMSO-d6) δ 170.68, 169.20, 159.85, 140.30, 134.16, 124.59, 122.19, 115.77, 61.84, 14.51.

[0065] (3) Slowly drop compound 4 (2.471 g, 10.60 mmol) in phosphorus oxychloride at ice bath, stir at 120 °C for 18 hours, remove phosphorus oxychloride by distillation under reduced pressure, and quench phosphorus oxychloride with ice water, add 20 mL ethyl acetate and 100 mL water to the mixture and stir for 30 minutes, extract 3 times with ethyl acetate, wash the organic layer with saturated sodium chloride solution 3 times, dry the organic phase with anhydrous sodium sulfate, and spin dry the solvent to obtain the crude product, further purify the crude product by column chromatography to obtain 2.5663 g of yellow solid compound 5, yield 90%; 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (ddd, J = 8.4, 1.4, 0.7 Hz, 1H), 8.07 (ddd, J = 8.4, 1.3, 0.6 Hz, 1H), 8.01 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.87 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 4.51 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H). 13 CNMR (101 MHz, DMSO-d6) δ 163.55, 147.19, 144.69, 141.04, 133.68, 130.10, 129.09, 127.04, 124.88, 124.25, 60.22, 14.31.

[0066] (4) Dissolve compound 5 (2.516 g, 9.353 mmol) in 20 mL glacial acetic acid, add 5 eq sodium acetate (1.401 g, 46.77 mmol), and stir at reflux at 120 °C for 18 hours, after the reaction is completed, spin dry the acetic acid, dilute with water, and filter and dry to obtain 1.822 g of white solid product 6, yield 77%; 1H NMR (400 MHz, Chloroform-d) δ 12.96 (s, 1H), 7.98 (dd, J = 8.3, 1.3 Hz, 1H), 7.63 (ddd, J = 8.5, 7.1, 1.4 Hz, 1H), 7.49 (dd, J = 8.3, 1.1 Hz, 1H), 7.34 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 4.55 (q, J = 7.1 Hz, 2H), 1.48 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 163.85, 160.44, 143.40, 137.68, 132.73, 126.05, 125.66, 123.79, 117.75, 116.93, 62.38, 14.21.

[0067] (5) Intermediate compound 6 (0.5 g, 1.992 mmol) was dissolved in 25 mL of anhydrous ethanol, 1.5 eq of benzyl bromide (0.5079 g, 2.988 mmol) was added, 5 eq of DIPEA (1.287 g, 9.96 mmol) was added, and stirring was performed at room temperature for 16 h. After the reaction was completed, rotary evaporation and concentration were performed, extraction was performed three times using a mixture of ethyl acetate and water, the organic phase was washed three times with saturated brine, dried with anhydrous sodium sulfate, rotary evaporation and concentration were performed, and column chromatography was performed to isolate and purify, thereby obtaining 0.5570 g (1.633 mmol) of intermediate compound.

[0068] (6) The product of step (5) was dissolved in 25 mL of DMF, 1.5 eq of piperazine (0.2105 g, 2.450 mmol) was added, 5 eq of DIPEA (1.055 g, 8.165 mmol) was added, and stirring was performed at 50°C for 24 h. After the reaction was completed, extraction was performed using a mixture of ethyl acetate and water, the solvent was removed, the organic phase was washed three times with saturated brine, and dried with anhydrous sodium sulfate.

[0069] (7) Benzoic acid (0.2990 g, 2.450 mmol) was dissolved in 15 mL of DMF, 1.5 eq of HOBT (0.497 g, 3.675 mmol), 1.5 eq of HBTU (1.394 g, 3.675 mmol), and 2.5 eq of DIPEA (0.792 g, 6.125 mmol) were added, stirring was performed at room temperature for 0.5 h, 10 mL of DMF was added to the reaction mixture of step (6) to introduce it into a reaction flask, and stirring was performed at room temperature for 24 h. After the reaction was completed, extraction was performed using a mixture of ethyl acetate and water, the solvent was removed, the organic phase was washed three times with saturated brine, dried with anhydrous sodium sulfate, rotary evaporation and concentration were performed, and column chromatography was performed to isolate and purify, thereby obtaining 0.421 g of a yellowish solid product DH1 in a yield of 52%. The structural characterization data thereof are as follows:1 H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.47 (s, 6H), 7.42 - 7.37 (m, 3H), 7.34 (d, J = 7.2 Hz, 1H), 5.57 (s, 2H), 4.42 (q, J = 7.2 Hz, 2H), 3.89 - 3.00 (m, 8H), 1.35 (t, J = 7.1 Hz, 3H). 13 CNMR (151 MHz, Chloroform-d) δ 170.88, 167.40, 158.97, 154.97, 147.55, 137.33, 135.76, 130.60, 130.10, 129.13, 128.82, 128.51, 128.22, 127.95, 127.89, 127.86, 127.40, 127.30, 124.46, 124.01, 122.29, 113.05, 68.10, 62.28, 14.31. HRMS (TOF) m / z Calcd for C30H29N3O4 [M + H+] 496.2158; found: 496.2346.

[0070] The hydrogen spectrum, carbon spectrum nuclear magnetic spectrum of compound DH1 are as shown in Figure 1 The high resolution mass spectrum spectrum is as shown in Figure 2

[0071] Synthesis and characterization of quinoline compound DH2 in example 2

[0072]

[0073] (1) According to the method in example 1, intermediate compound 6 was obtained, compound 6 (0.5 g, 1.992 mmol) was dissolved in 25 mL of anhydrous ethanol, 1.5 eq of bromopentane (0.451 g, 2.988 mmol) was added, 5 eq of DIPEA (1.287 g, 9.96 mmol) was added, stirred at room temperature for 16 h, after the reaction was completed, rotary evaporation, concentration, extraction with a mixture of ethyl acetate and water three times, the organic phase was washed with saturated brine three times, dried over anhydrous sodium sulfate, rotary evaporation, concentration, column chromatography separation and purification, 0.4605 g (1.434 mmol) of intermediate compound was obtained.

[0074] ​(2) The product of step (1) was dissolved in 25 mL DMF, piperidine hydrochloride 0.262 g (2.151 mmol) was added, DIPEA 1158 μL (7.17 mmol) was added, 40 °C stirring for 16 h, after the reaction was complete, extraction was performed using a mixture of ethyl acetate and water, the organic phase was washed with saturated brine three times, then dried over anhydrous sodium sulfate for 1 h, rotary evaporation, concentration, column chromatography separation and purification, 0.451 g of yellow oily liquid product DH2 was obtained, the yield was 85%.

[0075] The structural characterization data of quinoline compound DH2 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.47 (s, 6H), 7.42 - 7.37 (m, 3H), 7.34 (d, J = 7.2 Hz, 1H), 5.57 (s, 2H), 4.42 (q, J = 7.2 Hz, 2H), 3.89 - 3.00 (m, 8H), 1.35 (t, J = 7.1 Hz, 3H). 13 C NMR (151 MHz, Chloroform-d) δ 168.00, 159.72, 130.12, 127.74, 124.62, 123.71, 122.57, 61.81, 52.91, 28.81, 28.46, 26.81, 24.54, 22.64, 14.41, 14.28. HRMS (TOF) m / z Calcd for C22H30N2O3 [M+H+] 371.2256; found: 371.2377.

[0076] The hydrogen spectrum, carbon spectrum nuclear magnetic spectrum chart of compound DH2 is as shown in Figure 3 The high resolution mass spectrum chart is as shown in Figure 4

[0077] Example 3 Anti-new coronavirus activity of quinoline compound

[0078] ​Anti-pseudovirus activity gradient screening: 6 μL of compound (10 mM) was added to wells A1-A11 in a 96-well plate, and then 300 μL of DM10 medium was added per well, and mixed by blowing; 200 μL of DM10 medium containing 2% DMSO was added to wells B-G in a six-row plate; 100 μL was taken from row A of the 96-well plate and added to row B, which was mixed by blowing, and then sequentially diluted. An opaque 96-well plate was prepared, and 50 μL was taken from the corresponding well of the 96-well plate and added to the opaque plate, and the wells were duplicated, 2 μL of new coronavirus pseudovirus and 23 μL of DM10 medium were added per well; row H was the control well, and 25 μL of ACE2-overexpressing BHK21 cells, 25 μL of pseudovirus, and 50 μL of DM10 medium containing 2% DMSO were added per well, and then incubated in an incubator for 16 h. The fluorescence intensity was detected by an enzyme-labeled instrument, and the IC50 of different compounds against the new coronavirus was calculated. 50 The new coronavirus pseudovirus used was a pseudovirus of the new coronavirus spike protein as the outer membrane protein, a water blister virus as the skeleton, and containing a Luc reporter gene.

[0079] The specific results of the anti-new coronavirus activity of compounds DH1 and DH2 are shown in Figure 5 , and the half maximal effective concentrations of DH1 and DH2 against new coronavirus infection were 10 μM and 13.06 μM, respectively.

[0080] Example 4: Determination of target proteins of quinoline compounds

[0081] Surface plasmon resonance (SPR) experiments were performed on compounds DH1 and DH2 with spike protein, ACE2 protein, and TMPRSS2 protein to determine the target proteins of compounds DH1 and DH2, and the specific method is as follows.

[0082] Protein coupling buffer solution: 1.0 x PBS-P+ (pH 7.4). Interaction buffer solution: 1.0 x PBS-P+ (pH 7.4), 5% (v / v) DMSO. In this experiment, the CM5 chip was coupled by the amino coupling method, and the proteins were immobilized in three channels, and the 1st channel was activated and blocked as a reference channel. The protein coupling conditions were: a concentration of about 60 μg / mL, a system of pH 5.0 sodium acetate solution, a chip activation time of 420 s, and a blocking time of 420 s. The solvent calibration solution preparation table is shown below:

[0083]

[0084] According to the solubility of the compound and the chip surface test, a series of concentrations of the compound were prepared, and the compound determination conditions were: the injection time of the compound was 120 s, and the dissociation time was 180 s. The detection results are shown in Figure 6 , 7, 8, the two compounds have the highest affinity to ACE2 protein, and the KD values are 3.25 x 10 -5 and 7.25 x 10 -7 Thus, the mechanism of quinoline compounds against SARS-CoV-2 infection is that the quinoline compounds bind to the host receptor ACE2 protein, cause changes in protein conformation, hinder the binding of ACE2 receptor to the virus spike protein, and thus achieve the effect of anti-viral infection.

Claims

1. A quinoline compound targeting angiotensin converting enzyme 2 receptor, characterized by: DH1, DH2 selected from the compounds shown in the following structures: 、 。 2. The preparation method of the quinoline compound of claim 1, characterized in that: When the quinoline compound is DH1, the preparation method comprises the following steps: 1) reacting compound 6, benzyl bromide and a catalyst in a solvent to obtain product 1; 2) reacting product 1, piperazine and DIPEA in a solvent to obtain product 2; 3) reacting product 2, benzoic acid and a catalyst in a solvent to obtain DH1; When the quinoline compound is DH2, the preparation method comprises the following steps: 1) reacting compound 6, bromopentane and a catalyst in a solvent to obtain product 1'; 2) reacting product 1', piperidine hydrochloride and a catalyst in a solvent to obtain DH2; The synthesis route of compound 6 is as follows: ； comprising the following steps: (1) dissolving compound 1 and triethylamine in a solvent, adding compound 2 for reaction to obtain intermediate compound 3; (2) dissolving compound 3 in a solvent, adding sodium hydride for reaction, after the reaction is completed, methyl tert-butyl ether is extracted, and after acidification, intermediate compound 4 is obtained; (3) dissolving compound 4 in a solvent, refluxing for reaction, and after the reaction is completed, intermediate compound 5 is obtained; (4) dissolving compound 5 and sodium acetate in a solvent, refluxing for reaction, and after the reaction is completed, intermediate compound 6 is obtained.

3. A pharmaceutically acceptable salt of the quinoline compound of claim 1.

4. Use of the quinoline compound of claim 1 or the pharmaceutically acceptable salt of claim 3 in the preparation of an ACE2 binding agent.

5. Use of the quinoline compound according to claim 1 or the pharmaceutically acceptable salt according to claim 3 in the manufacture of an antiviral drug, characterized in that: The virus is a virus that infects a host through ACE2, and the virus is a new coronavirus.

6. An ACE2 binding agent or antiviral drug, characterized in that: The virus is a new coronavirus.

7. The ACE2 binding agent or antiviral drug of claim 6, wherein: Further comprising a pharmaceutically acceptable carrier.

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