CN116919949A

CN116919949A - Application of 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparation of anti-novel coronavirus drugs

Info

Publication number: CN116919949A
Application number: CN202311111865.0A
Authority: CN
Inventors: 司书毅; 张晶; 李妍; 周雯雯; 尤宝庆; 郑怡凡; 李东升; 刘超; 陈明华; 许艳妮; 王晨吟; 樊秀勇; 巫晔翔; 李顺旺
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2023-08-31
Filing date: 2023-08-31
Publication date: 2023-10-24
Anticipated expiration: 2043-08-31
Also published as: CN116919949B

Abstract

The invention provides an application of 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparing an anti-novel coronavirus drug, belonging to the technical field of medicines. 2-amino-5-phenyl thiophene-3-carboxylic acid amide (hereinafter referred to as compound IMB 63-8G) can inhibit the activity of novel coronavirus main protease, which suggests that the novel coronavirus has potential application value for resisting novel coronaviruses; the compound has good inhibition activity on HCoV-OC43 of beta genus, the selection index is 39, which is far higher than that of positive drug ribavirin, and the potential application value of the compound in the aspect of treating diseases caused by HCoV-OC43 is suggested.

Description

Application of 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparation of anti-novel coronavirus drugs

Technical Field

The invention relates to the field of medicines, in particular to application of 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparation of anti-novel coronavirus medicines.

Background

Based on the phylogenetic relationship and genomic structure, the World Health Organization (WHO) officially names the new coronavirus as severe acute respiratory syndrome coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus 2), abbreviated as SARS-CoV-2. Vaccines and drugs are important means for preventing and treating epidemic situations, but most of the currently developed vaccines against coronaviruses take Spike protein (Spike) of SARS-CoV-2 as a target point ^[4] Because the Spike protein is highly variable, the small molecule antiviral drug has the advantages of being aimed at targets which are not easy to mutate, being more stable, being easy to be orally taken, having low cost and simple administration mode and storage condition.

The main protease (Mpro), also known as 3C-like protease (3 CLpro), has important biological functions in regulating the replication of novel coronavirus RNA, responsible for the posttranslational processing of a number of functional proteins ^[5,6] The method comprises the steps of carrying out a first treatment on the surface of the Mpro evolves to a high degree in pathogenic coronaviruses ^[7] Mpro of all CoVs share the same substrate binding region ^[8] The method comprises the steps of carrying out a first treatment on the surface of the The human gene does not exist in the homolog of the Mpro, and the currently known human protease does not overlap with the substrate domain of the Mpro, thereby reducing the possibility of side effects of the Mpro-targeted drug ^[9] . Thus, mpro is considered as one of the ideal targets for the development of novel broad-spectrum anti-coronavirus drugs.

Disclosure of Invention

In order to solve the problems, the invention provides application of 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparing anti-novel coronavirus medicines, wherein the 2-amino-5-phenyl thiophene-3-carboxylic acid amide (hereinafter referred to as a compound IMB 63-8G) has good Mpro inhibitory activity and beta HCoV-OC43 inhibitory activity, which indicates potential application value of the anti-coronavirus.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparing an anti-novel coronavirus drug.

The invention also provides application of the 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparing a novel coronavirus main protease activity inhibitor.

Preferably, the structural formula of the 2-amino-5-phenylthiophene-3-carboxylic acid amide is shown as formula (I):

preferably, the novel coronavirus comprises HCoV-OC43 of the genus beta.

2-amino-5-phenylthiophene-3-carboxylic acid amide:

(1) An inhibitor which binds to and inhibits the function of a specific region of a novel coronavirus main protease, wherein the specific region of the novel coronavirus main protease is a structural region consisting of His41, met49 and Gly143 in the tertiary structure of the novel coronavirus main protease;

(2) Inhibiting the activity of a novel coronavirus main protease;

(3) Non-covalent binding to a novel coronavirus main protease;

(4) The surface plasmon resonance and the novel coronavirus main protease show a fast-binding and fast-dissociation mode;

(5) Inhibiting the activity of the low pathogenic coronavirus HCoV-OC43.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a synthetic route for the compound IMB 63-8G;

FIG. 2 is a graph showing the SARS-CoV-2 Mpro inhibitory activity of Compound IMB63-8G, A is the SARS-CoV-2 Mpro inhibitory activity of Compound IMB63-8G on a fluorescence resonance energy transfer model; b is the determination of the inhibition activity of the compound IMB63-8G on SARS-CoV-2 Mpro on a fluorescence polarization model;

FIG. 3 is a molecular docking of compound IMB63-8G with the active site of SARS-CoV-2 Mpro;

FIG. 4 shows the affinity assay of the compound IMB63-8G for SARS-CoV-2 Mpro;

FIG. 5 shows the detection of the binding pattern of the compound IMB63-8G to SARS-CoV-2 Mpro.

Detailed Description

preferably, the novel coronavirus comprises HCoV-OC43 of the genus beta.

The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.

Example 1

Synthesis route of Compound IMB63-8G

(1) In a 100mL round bottom flask, 2.0g of Compound I was added, dissolved with 30mL of methanol, added with 4.2g EDCI,0.02g DMAP, stirred overnight at room temperature, after TLC detection of the completion of the reaction, most of the anhydrous methanol was distilled off under reduced pressure, distilled water was added, dichloromethane was extracted 3 times, the organic phases were combined, anhydrous sodium sulfate was dried overnight, then anhydrous sodium sulfate solid was filtered off, the organic phase was distilled off under reduced pressure, and 1.67g of pale yellow oily Compound II was obtained by weighing. The yield thereof was found to be 76%.

(2) 100mL three-necked flask, dissolving 0.75g NaBH in 30mL dry methanol, stirring and dissolving under ice water bath condition, slowly dropwise adding 1.5g II methanol solution 20mL into a reaction flask by using a dropping funnel under ice water bath condition, after the completion of the dropwise addition, reacting for 45min at room temperature, adding water into the reaction solution to quench the reaction, evaporating most of methanol under reduced pressure, adding distilled water, extracting with dichloromethane for 3 times, merging organic phases, drying anhydrous sodium sulfate overnight, filtering anhydrous sodium sulfate solid, evaporating the organic phase under reduced pressure, weighing to obtain 0.8g colorless oily compound III. The yield thereof was found to be 71%.

(3) A100 mL round bottom flask was taken, 2.2g of Compound III was added, 50mL of dichloromethane was added, stirring was performed to dissolve, then 7.76g of PCC solid was added to the reaction solution, and 0.2g was addedThe molecular sieve accelerates the reaction progress, stirring overnight at room temperature, after the reaction, filtering molecular sieve powder in the reaction liquid, extracting the organic phase 3 times, combining the organic layers, drying over night with anhydrous sodium sulfate, then filtering anhydrous sodium sulfate solid, and evaporating the organic phase under reduced pressure to obtain 1.43g colorless oily compound IV. The yield thereof was found to be 62%.

(4) Taking a microwave reaction tank, respectively adding 0.8g of compound IV,0.55g of compound V and 0.2g of sulfur into the reaction tank, then adding 3mL of morpholine and 15mL of ethanol, then reacting for 1h under the microwave condition of 70 ℃, closing a reaction instrument after the reaction is finished, transferring the reaction solution into an eggplant type bottle, evaporating the reaction solution under reduced pressure, mixing a crude reactant silica gel sample, and performing column chromatography (CH ₂ Cl ₂ : meoh=20: 1) And separating and purifying to finally obtain 0.8G of compound VI (IMB 63-8G for short). The yield thereof was found to be 55%. The structural formulas of compounds I, II, III, IV, V and VI are shown in figure 1.

Example 2

Assay of SARS-CoV-2 Mpro inhibitory Activity of Compound IMB63-8G

The activity assay uses Fluorescence Polarization (FP) and Fluorescence Resonance Energy Transfer (FRET) models.

Fluorescence Polarization (FP) measurement principle:

the principle that the polarization of fluorescent molecules is closely related to the rotation speed of fluorescent molecules when excited is mainly based on. When a fluorescent molecule is excited by plane polarized light, if the fluorescent molecule remains stationary when excited, the emitted light will lie in the same plane of polarization; if the fluorescent molecules remain in a rotated state when excited, the emitted light will lie in a different plane of polarization than the excitation light. If the fluorescein is excited with vertically polarized light, the intensity of the emitted light can be detected in both the vertical and horizontal planes of polarization (the degree to which the emitted light deviates from the vertical plane to the horizontal plane is related to the mobility of the fluorescein-labeled molecules). If the molecular weight is larger, the rotation speed of fluorescent molecules is slower when excited, and the polarization degree of emitted light is higher; if the molecular weight is small, the rotation speed of the fluorescent molecules when excited is high, and the emitted light will be depolarized with respect to the excitation light plane. Therefore, the fluorescence polarization high throughput screening method to be established in the present study will use MCA-substrate ((MCA-AVLQSFFR-Lys (Dnp) -Lys-NH2; MW:1517.7; purity > 95%; λex/λem:320/405nm, available from Gill Biochemical Co., ltd.) as a simulated substrate for Mpro to conduct an activity assay of a small molecule inhibitor targeting a novel coronavirus.

The measuring method comprises the following steps:

(1) 400nM SARS-CoV-2 Mpro solution was added sequentially to 96-well half-bottom blackboard at 29. Mu.L/well, and then 1. Mu.L/well of compound with final concentration of 200. Mu.M, 100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M, 1.5625. Mu.M was added as Negative Control (Negative Control,0% inhibition) to 1. Mu.L DMSO well to Positive Control (Positive Control,100% inhibition) containing only 30. Mu.LFP buffer (10 mM Tris, 50mM NaCl, 1mM EDTA, 1mM DTT pH 8.0). Mixing, slowly shaking at room temperature, and incubating for 35-40min.

(2) 60nM FITC-Probe was added to each of the above-mentioned reaction wells in a 96-well half-bottom blackboard sequentially at 20. Mu.L/well, and after mixing, it was slowly shaken at room temperature and incubated for 20-25min in the absence of light.

(3) And then adding 300nMAvidin reaction solution at 10 mu L/hole, and incubating for 5-8 min at room temperature in a dark place.

(4) The inhibition ratio of the test compound was calculated using the following equation:

and determining IC of Compound IMB63-8G by GraphPad Primer 5 fit inhibition curve (FIG. 2A) ₅₀ 36.65.+ -. 7.95. Mu.M, can inhibit the activity of Mpro in vitro.

Principle of Fluorescence Resonance Energy Transfer (FRET) measurement:

the emission spectrum of the donor fluorescent molecule and the absorption spectrum of the acceptor fluorescent molecule are overlapped to a certain extent, and when the distance between the emission spectrum and the absorption spectrum is smaller than 10nm, the phenomenon that fluorescence energy is transferred from the donor to the acceptor can be observed, namely the excitation energy of the donor fluorescent molecule induces the acceptor molecule to emit fluorescence, and meanwhile, the fluorescence intensity of the donor molecule is attenuated. The present study uses the quenched FRET principle, a group of donors and acceptors is selected to be 7-methoxycoumarin-4-acetic acid (MCA) and 2, 4-dinitrophenol (Dnp), MCA is a fluorescent group, dnp is a quenching group, and the quenching groups are respectively connected to the N end and the C end of an Mpro substrate polypeptide to be used as FRET substrates, when the FRET substrates are hydrolyzed, the MCA-AVLQ fragment loses the FRET effect, and fluorescence is generated at 405 nm; if the compound is capable of inhibiting the hydrolysis of Mpro, no fluorescent signal will be generated at 405 nm.

The measuring method comprises the following steps:

(1) HEPES buffer containing 0.8. Mu.m SARS-CoV-2 Mpro was added to full black half-bottom 96 well plates at 24. Mu.L/well, and then 1. Mu.L/well was added to compound at final concentration of 200. Mu.m, 100. Mu.m, 50. Mu.m, 25. Mu.m, 12.5. Mu.m, 6.25. Mu.m, 3.125. Mu.m, 1.5625. Mu.m, and incubated for 30min at room temperature. 1. Mu.L DMSO well was added as a Negative Control (0% inhibition) and wells containing only 25. Mu.L HEPES buffer (10 mM Tris, 50mM NaCl, 1mM EDTA, 1mM DTT pH 8.0) were used as Positive controls (100% inhibition).

(2) 10. Mu.M FRET substrate was added at 25. Mu.L/well, incubation was continued for 5min at room temperature, and RFU was detected with a multifunctional microplate reader.

(3) The inhibition ratio of the test compound was calculated using the following equation:

IC determination of Compound IMB63-8G by GraphPad Primer 5 fit inhibition Curve (B in FIG. 2) ₅₀ 16.27.+ -. 0.62. Mu.M, can inhibit the activity of Mpro in vitro.

Example 3

Molecular docking of compound IMB63-8G to SARS-CoV-2 Mpro and SARS-CoV-2 Mpro active site

Was performed using BIOVIA Discovery Studio 2018R2 (Accelrys, san Diego, USA) software.

The measurement principle is as follows:

molecular docking (Molecular Docking) is a process of mutual recognition between ligands and receptors by energy and geometric matching, and mainly includes electrostatic action, hydrogen bonding action, hydrophobic action, van der waals force, etc. In recent years, with rapid development of computer-aided drug design technology, molecular docking technology has become one of the important methods for structure-based drug design and computer-aided drug design.

The measuring method comprises the following steps:

extraction of Mpro from protein database (PDB: 7D 3I) molecular modeling docking was performed, water molecules and cofactors were removed from the protein using BIOVIA Discovery Studio 2018R2 software, and possible binding pockets in Mpro were defined according to literature. After determining the potential hydrogen bond interactions between the catalytic site of Mpro and the compound, the docking results were treated and optimized using the C-dock program.

The SARS-CoV-2 Mpro domain isConsists of His41 and Cys 145 amino acids, it can be seen from FIG. 3 that IMB63-8G forms a hydrogen bond with His41 of MproAnd pi-pi interaction +.>Formation of pi-sulphur interactions with Met 49->Formation of carbon-hydrogen bond with Gly143>

Example 4

Affinity detection of Compound IMB63-8G with SARS-CoV-2 Mpro

Using Surface Plasmon Resonance (SPR).

The measurement principle is as follows:

SPR is a biosensing analysis technology which can construct biomolecular interactions by utilizing traditional optical phenomena and utilizing light to generate evanescent waves in different media to generate resonance with plasma waves so as to detect the interaction condition between ligands and analytes on a biosensing chip. The SPR biosensor has polarized light source, one layer of gold film coated onto the surface of the sensor chip, and one kind of biological molecule (target molecule) fixed onto the surface of the gold film and the molecule interacting with the biological molecule dissolved in the solution to flow through the surface of the chip. In the process of dissociation of the protein on the gold film chip and the molecules in the flow path, the resonance angle (namely SPR angle) changes along with the dissociation, and the detector detects the change, and the binding constant Ka, dissociation constant Kd or affinity constant KD between the molecules can be obtained according to the change curve graph analysis.

The measuring method comprises the following steps:

(1) Protein coats the chip surface: 153mg EDC and 23mg NHS were each dissolved in 2mL deionized water and filtered through a 0.22. Mu.M filter. EDC and NHS1:1 are mixed and injected in a sample way, the flow rate is set to 10 mu L/min, and the injection is carried out for 7min, so that the chip activation is completed. Protein was diluted to 80. Mu.g/mL with sodium acetate solution at the appropriate pH, protein immobilization was performed in the left channel, the flow rate was set at 10. Mu.L/min, and injection was performed for 7min, with the right channel serving as a blank. When the protein is immobilized to an ideal signal, 1M ethanolamine (pH 8.5) is injected for 8min to block the site of the surface of the chip, which is not bound with the protein.

(2) Binding detection of the compound: the compounds were diluted with 5% DMSO in PBST buffer at final concentrations (6.25, 12.5, 25, 50, 100, 200. Mu.M) and the loading procedure was combined for 1.5min, allowed to spontaneously dissociate for 5.5min, and set at a flow rate of 25. Mu.L/min. The binding of the compounds to the immobilized proteins on the chip was observed at various concentrations.

(3) And (3) data processing: the equilibrium dissociation constant (KD) of the compound and the protein was calculated using Trace drug software.

As shown in FIG. 4, IMB63-8G is capable of undergoing a rapid binding and dissociation, K, with recombinant Mpro in a dose-dependent manner _D The value was 1.06X10 ^-4 M. Small molecule protein-bound K _D The value is generally 10 ^-3 To 10 ^-6 M, therefore, IMB63-8G has a relatively strong affinity with the Mpro protein.

Example 5

Studies of the binding pattern of the Compound IMB63-8G and SARS-CoV-2 Mpro

Performed using HPLC-Q-TOF-MS.

The measurement principle is as follows:

the UPLC-Q-TOF-MS realizes perfect combination of an ultra-high performance liquid chromatography system and high quality high resolution mass spectrum, and the molecular weight can be accurately determined by using a small particle filler (1.7 mu m) as a stationary phase, and the molecular composition can be accurately estimated, and the accuracy is less than 3ppm.

The measuring method comprises the following steps:

(1) Setting SARS-CoV-2 Mpro protein control group and sample group, the sample group incubates the new crown Mpro protein sample expressed in vitro (5 mu M) with the compound IMB63-8G (final concentration 500 mu M) for 30min at room temperature, the protein control group is that 5 mu M protein is added into 20 mu L TBS buffer solution, after incubation for 30min at room temperature, the sample group enters HPLC-Q-TOF-MS for loading detection.

(2) The system parameters were set as follows: electrospray voltage 3.5KV,Fragmentor 175, scan range is set to one level,600-2400 (m/z), sheath gas temperature 325 ℃, gas flow rate 5L/min, nebulizer 35psig, data acquisition mode selection profile. The chromatographic column is 300 SB-C8%3.5 microns), mobile phase a was a 0.1% formic acid aqueous solution, organic mobile phase B was a 0.1% formic acid-acetonitrile solution, and the flow rate was set at 0.2mL/min.

The gradient elution procedure was set as follows:

TABLE 1 gradient elution procedure

Time (min)	0	2	8	12	16	17.1	20
								A(％)	98	98	50	2	2	98	98
B(％)	2	2	50	98	98	2	2

(3) After HPLC separation, mass spectrometry analysis is carried out, a profile map of 600-2400M/Z is acquired, and Deconvolute (MS) is used: the protein software performs deconvolution, the deconvolution algorithm is set to be the maximum entropy, and the mass range is 10000-80000Da.

After incubation of IMB63-8G with SARS-CoV-2 Mpro for 30min at room temperature, the molecular weight was detected by HPLC-Q-TOF-MS, and the results are shown in FIG. 5, after incubation of IMB63-8G with protein, the molecular weight was unchanged from that of the original protein, indicating no generation of covalent products, and its binding to Mpro was all covalent.

Example 6

Activity assay of Compound IMB63-8G on Low pathogenic coronaviruses

The detection of inhibitory activity was performed on HCoV-OC43, which is co-located with the novel coronavirus as genus beta.

The measurement principle is as follows:

the degree of cytopathic effect (CPE) of the sample-inhibited virus was determined using H460 cells as the viral host.

The measuring method comprises the following steps:

(1) H460 cells were cultured to give rise toThe cells were inoculated in 96-well plates, 5% CO ₂ Culturing at 35 ℃;

(2) Culturing for 24 hr, and culturing with 100TCID ₅₀ Is incubated at 35 ℃ for 2 hours, and the virus supernatant is discarded, and simultaneously, the culture medium containing 2 percent FBS and containing different dilutions of the compound IMB63-8G and positive control ribavirin is addedA maintenance liquid;

(3) Simultaneously setting cell control hole and virus control hole, 5% CO ₂ Culturing at 35 ℃;

(4) Observing CPE of each group of cells when the virus control group lesion cells account for more than 75% of the monolayer cells, and calculating half-Toxic Concentration (TC) of the compound on the cells by using a Reed-Muench method ₅₀ ) Cytotoxicity was examined, and half inhibitory concentration (IC ₅₀ ) The inhibitory activity against viruses was examined.

The compound IMB63-8G has better inhibiting activity on HCoV-OC43, and IC ₅₀ The value was 1.71. Mu.g/mL (7.83. Mu.M) and the selection index was 39, as shown in Table 2, which was far higher than the positive drug ribavirin.

TABLE 2 inhibition of HCoV-OC43 by IMB63-8G

Compounds	TC ₅₀ (μg/mL)	IC ₅₀ (μg/mL)	SI
				IMB63-8G	66.67	1.71	39.0
RBV	77.61	8.62	9.0

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Reference to the literature

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Claims

The application of 2-amino-5-phenyl thiophene-3-carboxylic acid amide in preparing anti-novel coronavirus medicines.
Use of 2-amino-5-phenylthiophene-3-carboxylic acid amide for the preparation of an inhibitor for inhibiting the activity of a novel coronavirus main protease.
3. The use according to claim 1 or 2, wherein the structural formula of the 2-amino-5-phenylthiophene-3-carboxylic acid amide is shown in formula (I):
4. the use according to claim 1 or 2, wherein the novel coronavirus comprises HCoV-OC43 of the genus β.