Medical use of 7-aminocephalosporanic acid derivatives against infection with the new coronavirus

Abstract

The application discloses a medical use of a 7-aminocephalosporanic acid derivative for resisting new coronavirus infection, and a structural formula of the compound is as follows: wherein R1 is hydrogen, (Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxy imine) acetyl or (R)-2-amino-2-(4-hydroxyphenyl) acetyl; and R2 is acetyloxymethyl, ethenyl or 1-propenyl. In vitro activity test results show that the compound disclosed by the application can inhibit the binding of the spike protein of the new coronavirus SARS-CoV-2 to the receptor on the host cell membrane, and can effectively reduce the infectivity of the wild strain and various mutant strains of the new coronavirus. In vitro cytotoxicity test results show that the compound does not show cytotoxicity, and has been clinically used as an anti-inflammatory drug. The old drug is used for new use, and the compound is used for an anti-new coronavirus infection drug, thereby providing a new idea for new coronavirus epidemic prevention and control, and having a good application prospect.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the pharmaceutical use of a 7-aminocephalosporanic acid derivative that resists novel coronavirus infection; more specifically, it relates to the use of a 7-aminocephalosporanic acid derivative that can inhibit the binding of the spike protein (S protein) of the novel coronavirus (SARS-CoV-2) to the ACE2 receptor of host cells in the preparation of anti-COVID-19 drugs. Background Technology

[0002] The widespread transmission of the novel coronavirus (SARS-CoV-2) has become a major global concern. Severe acute respiratory syndrome (COVID-19) caused by the novel coronavirus infection has complex clinical symptoms and a high mortality rate, while the extremely high infectivity and variability of SARS-CoV-2 make drug research exceptionally difficult. SARS-CoV-2 has mutated in many ways, and these variants have caused widespread outbreaks of acute respiratory syndrome (COVID-19) in countries such as the UK, Brazil, and South Africa. Epidemiological investigations have shown that the delta variant, first detected in India in May 2021, has exhibited rapid spread and immune evasion during the epidemic (Alizon S, Haim-Boukobza S, Foulongne V, et al. Rapid spread of the SARS-CoV-2 Delta variant in some French regions[J]. Eurosurveillance, 2021, 26(28): 2100573). The S protein is an important surface membrane protein of SARS-CoV-2. It is the main antigen that stimulates the production of neutralizing antibodies in the human body and is also an important target for cytotoxicity against lymphocytes. The S protein contains two subunits, S1 and S2: S1 contains the receptor-binding domain (RBD) responsible for recognizing cell receptors, while S2 contains the essential elements required for membrane fusion. Studies have shown that the S protein plays a crucial role in the viral infection of host cells. Therefore, investigating how to inhibit the binding of the SARS-CoV-2 S protein to receptors or the membrane of host cells is of great significance for researching how to inhibit the invasion of the novel coronavirus into the human body and how to effectively control the epidemic.

[0003] Currently, compounds proven to inhibit the binding of the S protein to its receptor can be categorized into neutralizing antibodies and small molecule compounds. The antibody SARS2-38 targets a conserved epitope of the S protein, exhibiting high activity and a wide spectrum of activity. It can control infection in K18-hACE2 mice and hamsters. However, this neutralizing antibody shows reduced efficacy against some viral mutant strains and is currently only available through intravenous infusion or inhalation. Currently reported small molecule compounds that can inhibit the binding of the S protein to its receptor include epigallocatechin gallate (Liu J, Bodnar BH, Meng FZ, et al. Epigallocatechin gallate from Green Tea Effectively Blocks Infection of SARS-CoV-2 and New Variants by Inhibiting Spike Binding to ACE2 Receptor. 2021. DOI:10.1101 / 2021.03.17.435637), glycyrrhetinic acid (Li H, Cheng C, Li S, et al. Discovery and structural optimization of 3-O-β-chacotriosyl oleanane-type triterpenoids as potent entry inhibitors of SARS-CoV-2 virus infections. Eur J Med Chem. 2021, 215:113242), and other natural drugs, as well as the recently reported semi-synthetic antibiotic ceftazidime (Chang Dong, Lin Y, Li...). MY, et al. Ceftazidime Is a Potential Drug to Inhibit SARS-CoV-2 Infection In Vitro by Blocking Spike Protein-ACE2 Interaction[J]. SigTransduct Target Ther. 2020, 6:1671-1674). Epicatechin gallate (EGCG) in green tea possesses low toxicity, anti-inflammatory, and antioxidant properties, and has the ability to block the binding of the S protein to cells, making it a compound with potential anti-SARS-CoV-2 activity. Ceftazidime can block the binding of the S protein to angiotensin-converting enzyme 2 (ACE2) in human respiratory epithelial cells, thereby inhibiting the cellular entry of SARS-CoV-2. The above compounds have inhibitory effects on wild-type virus cell infection, but their effects are not significant against some mutant strains.Meanwhile, the immune escape phenomenon of the above compounds is greatly reduced due to the fact that the Indian mutant strain of the novel coronavirus has escaped immune response.

[0004] 7-Aminocephalosporanic acid (7-ACA) is an intermediate in the synthesis of cephalosporin antibiotics. In its production, it is prepared by bio-fermentation and chemical extraction of cephalosporin C, followed by chemical lysis or enzymatic hydrolysis of cephalosporin C. Currently, it is only used as an intermediate in the synthesis of cephalosporin antibiotics. Cefixime is an orally absorbed third-generation cephalosporin antibiotic with antibacterial activity against Gram-positive and Gram-negative bacteria, and exhibits high resistance to β-lactamase degradation (Faulkner R D, Yacobi A, Barone JS, et al. Pharmacokinetic profile of cefixime in man. The Pediatricinfectious disease journal, 1987, 6(10): 963-970.). The bactericidal effect of cefixime stems from its inhibition of bacterial cell wall synthesis, and due to the presence of vinyl groups on the cephen nucleus, it exhibits stability against acid hydrolysis. (Anacona J R, Estacio J. Synthesis and antibacterial activity of cefixime metal complexes. Transition Metal Chemistry, 2006, 31(2):227-231.) This drug is effective against Haemophilus influenzae, Moraxella catarrhalis, and penicillin-sensitive Streptococcus pneumoniae, but ineffective against Staphylococcus aureus. Cefixime has a relatively long elimination half-life (3h), and some trials have confirmed that this drug has certain clinical efficacy in patients with lower respiratory tract infections (LRTI) (Markham A, Brogden R N. Cefixime. A review of its therapeutic efficacy in lower respiratory tract infections. Drugs, 1995, 49(6):1007.).

[0005] Cefprozil is an oral second-generation cephalosporin antibiotic that exerts its antibacterial effect primarily by inhibiting bacterial cell wall biosynthesis. It is effective against Gram-positive streptococci, pneumoniae, and alactamase, as well as methicillin-sensitive Staphylococcus aureus. It can be used to treat tonsillitis, pharyngitis, and respiratory infections. (Wiseman LR, Benfield P. Cefprozil. Drugs, 1993, 45(2):295-317.) Furthermore, studies have shown that cefprozil has a certain therapeutic effect on persistent and recurrent acute otitis media in children. (Pichichero ME, Mclinn S, Aronovitz G, et al. Cefprozil treatment of persistent and recurrent acuteotitis media[J]. The Pediatric infectious disease journal, 1997, 16(5):471-478.) Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical use for compounds with a 7-aminocephalosporanic acid core. Pharmacological studies have shown that several compounds with a 7-aminocephalosporanic acid core structure disclosed in this invention can inhibit the binding of the spike protein (S protein) of wild-type and mutant SARS-CoV-2 to the ACE2 receptor on host cells, thus inhibiting the spread of the virus.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention relates to a compound having a 7-aminocephalosporanic acid core structure, and its use in the preparation of a drug that inhibits the binding of the spike protein (S protein) of the novel coronavirus (SARS-CoV-2) to the ACE2 receptor on host cells, the structure of which is shown in formula (I):

[0009] Wherein, R1 is hydrogen, (Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxyimine)acetyl or (R)-2-amino-2-(4-hydroxyphenyl)acetyl; R2 is acetoxymethyl, vinyl or 1-propenyl.

[0010] As one embodiment of the present invention, the structure of the compound having a 7-aminocephalosporanic acid core is shown in formula (II):

[0011]

[0012] In another embodiment of the present invention, the structure of the compound having a 7-aminocephalosporanic acid core is shown in formula (Ⅲ):

[0013]

[0014] In another embodiment of the present invention, the compound having 7-aminocephalosporanic acid has the structure shown in formula (Ⅳ):

[0015]

[0016] Secondly, the use of a compound having a 7-aminocephalosporanic acid core as described in structures I to VI above in the preparation of a medicament for treating SARS-CoV-2 infection.

[0017] As one embodiment of the present invention, the aforementioned novel coronavirus includes wild-type novel coronaviruses and / or mutants.

[0018] As another embodiment of the present invention, the novel coronavirus mutant includes one or more of the following: the Brazilian mutant of the novel coronavirus (P.1), the British mutant of the novel coronavirus (B.1.1.7), the D614G mutant of the novel coronavirus, and the Indian mutant of the novel coronavirus.

[0019] As another embodiment of the present invention, the Indian mutant of the novel coronavirus includes one or more of the following: the Indian mutant of the novel coronavirus B.1.617, the Indian mutant of the novel coronavirus B.1.617.2, and the Indian mutant of the novel coronavirus B.1.618.

[0020] Compared with existing technologies, this invention, based on the current research status of SARS-CoV-2 novel coronavirus both domestically and internationally, and building upon the study of the mechanism of action between the viral spike protein (S protein) and the host cell receptor ACE2, established a cell model with high ACE2 receptor expression. In the established test model, four compounds with a 7-aminocephalosporanic acid core exhibited strong inhibitory activity against the binding of the S protein to the receptor. In vitro toxicity studies showed that these compounds had no significant cytotoxic effects on host cells. These four compounds are existing clinical antibacterial drugs and can be further optimized to develop highly effective and low-toxicity topical, oral, and injectable drugs against 2019-nCoV novel coronavirus. Attached Figure Description

[0021] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0022] Figure 1A schematic diagram illustrating the activity of cefixime, ceftazidime, and epicatechin gallate in testing with wild-type SARS-CoV-2.

[0023] Figure 2 A schematic diagram illustrating the activity of cefprozil tested using wild-type SARS-CoV-2.

[0024] Figure 3 This is a schematic diagram illustrating the activity of 7-aminocephalosporanic acid tested using the wild-type SARS-CoV-2 virus. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the manufacturer.

[0026] Example 1

[0027] This embodiment evaluates the inhibitory effects of ceftazidime, epicatechin gallate, and cefixime (III) on the binding of the wild-type SARS-CoV-2 S protein to the ACE receptor of host cells.

[0028] Experimental principle:

[0029] First, a human kidney embryonic cell line, HEK293T, with high ACE2 receptor expression was established using HIV lentiviral transfection plasmids. Based on an HIV lentiviral vector and the SARS-CoV-2 spike protein (S protein), the lentiviral envelope protein VSVG was replaced with the SARS-CoV-2 S protein and co-transfected with lentiviral packaging plasmids and CMV-GFP-T2A-Luciferase plasmid into 293T cells to prepare a SARS-CoV-2 S pseudovirus. This pseudovirus expressed the SARS-CoV-2 S protein on its surface and simultaneously carried a GFP fluorescent reporter gene. The activity of the pseudovirus in infecting cells could be evaluated by observing fluorescence intensity. This pseudovirus lacked autonomous replication ability, exhibited high safety, and could be used for SARS-CoV-2 receptor drug screening. HuiLi et al. used a similar pseudovirus model to screen for SARS-CoV-2 receptor drugs, detecting the pseudovirus infection rate by measuring fluorescence intensity and selecting drugs with anti-pseudovirus activity. The antiviral activity of this drug was also confirmed in a true virus infection model, indicating that this compound could be used for the actual treatment of SARS-CoV-2 infection. The results shown by the compounds in the pseudovirus model are consistent with those in the real virus model (Li H, Cheng C, Li S, et al. Discovery and Structural Optimization of 3-O-β-Chacotriosyl Oleanane-type Triterpenoids as Potent Entry Inhibitors of SARS-CoV-2 Virus Infections[J]. European Journal of Medicinal Chemistry, 2021, 215: 113242.). This proves that the compounds screened by our pseudovirus model and their measurement results can reflect the antiviral effect of the real virus, and compounds effective against pseudoviruses can certainly be used to treat the novel coronavirus in practice.

[0030] Experimental methods:

[0031] HEK293T cells were used to study pseudovirus S protein infection. HEK293T cells were digested with trypsin and seeded at a density of 100,000 cells / mL in 100 μL per well. After 24 h, the supernatant was removed, and 92 μL of culture medium containing ceftazidime (final concentration 200 μM), cefixime (final concentration 200 μM), and epicatechin gallate (final concentration 100 μM) were added, respectively, with dimethyl sulfoxide (DMSO) at a concentration of 0.5%. The positive control group was treated with 92 μL of culture medium containing an equal volume of DMSO. Add an equal amount of pseudovirus (8 μL per well after a 20-fold dilution) to each well, mix again, and after 24 hours, aspirate the supernatant. Add 200 μL of culture medium containing the same concentration of the compound as the original wells. After another 48 hours, digest the cells separately, dilute with phosphate-buffered saline (PBS), centrifuge at 1600 rpm for 10 min, discard the supernatant, and resuspend each sample in 200 μL of PBS. Perform FITC fluorescence assay using flow cytometry to obtain the proportion of positive cells. Calculate the inhibition rate using the following formula: Inhibition rate (%) = (Infection rate of cells in the drug-free control group - Infection rate of cells in the drug-treated group) / Infection rate of cells in the drug-free control group × 100%.

[0032] Obtained through infection experiments using wild-type pseudoviruses of the novel coronavirus, such as Figure 1 Compared with the control group, the proportion of positive cells in the experimental group with added ceftazidime decreased from 33.06% to 12.12%, and its virus inhibition rate was 63.34%; the proportion of positive cells in the experimental group with added cefixime decreased from 33.06% to 9.27%, and its virus inhibition rate was 71.96%, which was 1.14 times that of ceftazidime; the proportion of positive cells in the experimental group with added epicatechin gallate (100μM) increased from 24.02% to 28.74%, but no inhibitory effect on viral infection was observed.

[0033] Table 1. Testing the activity of ceftazidime and cefixime using a pseudovirus derived from a wild-type strain of SARS-CoV-2.

[0034]

[0035] Example 2

[0036] This embodiment relates to the evaluation of the inhibitory effect of the compound (cefaprom) described in structural formula IV on the binding of the wild-type strain of novel coronavirus S protein to the ACE2 receptor of host cells.

[0037] Experimental methods:

[0038] HEK293T cells were used to study pseudovirus S protein infection. HEK293T cells were digested with trypsin and seeded at a density of 100,000 cells / mL in 100 μL per well. After 24 h, 92 μL of cefprozil (DMSO) medium (final concentration 200 μM) was added to each well, with DMSO content at 0.5%. The positive control group was treated with 92 μL of medium containing an equal volume of dimethyl sulfoxide. The mixture was then thoroughly mixed. An equal volume of pseudovirus (8 μL diluted 20-fold) was added to each well, and the mixture was stirred again. After 24 h, the supernatant was removed, and 200 μL of cefprozil medium (the same concentration as the original well) was added. After another 48 h, the cells were digested, diluted with PBS, centrifuged at 1600 rpm for 10 min, and the supernatant was removed. Each sample was resuspended in 200 μL of PBS and subjected to FITC fluorescence assay using flow cytometry to determine the proportion of positive cells.

[0039] Table 2. Cefprozil activity was tested using a pseudovirus derived from a wild-type strain of SARS-CoV-2.

[0040]

[0041] like Figure 2 The results obtained using a wild-type pseudovirus infection experiment showed that, compared with the control group, the proportion of positive cells in the experimental group with added cefprozil decreased from 31.33% to 22.89%, and the viral infection inhibition rate was 26.9%.

[0042] Example 3

[0043] This embodiment relates to the evaluation of the inhibitory effect of the compound (7-aminocephalosporanic acid) described in structural formula II on the binding of the wild-type strain of novel coronavirus S protein to the ACE2 receptor of host cells.

[0044] Experimental methods:

[0045] HEK293T cells were used to study pseudovirus S protein infection. HEK293T cells were digested with trypsin and seeded at a density of 100,000 cells / mL in 100 μL per well. After 24 h, 92 μL of 7-aminocephalosporanic acid (7-aminocephalosporanic acid) medium (400 μM final concentration) was added to each well. The positive control group received an equal volume of 92 μL of medium. The cells were then mixed. An equal volume of pseudovirus (8 μL diluted 20-fold) was added to each well, and the mixture was stirred again. After 24 h, the supernatant was removed, and 200 μL of 7-aminocephalosporanic acid-containing medium (the same concentration as the original well) was added. After another 48 h, the cells were digested, diluted with PBS, centrifuged at 1600 rpm for 10 min, and the supernatant was removed. Each sample was resuspended in 200 μL of PBS and analyzed by flow cytometry using FITC fluorescence to determine the proportion of positive cells.

[0046] Table 3. Testing 7-aminocephalosporanic acid activity using a pseudovirus derived from a wild-type strain of SARS-CoV-2.

[0047]

[0048] like Figure 3 Compared with the control group, the proportion of GFP-positive cells in the experimental group with added 7-aminocephalosporanic acid decreased from 22.63% to 15.63%, and its viral infection inhibition rate was 30.9%.

[0049] Example 4

[0050] This embodiment relates to the evaluation of the inhibitory effect of the compound described in structural formula III (cefixime) on the binding of the S protein of the novel coronavirus wild-type strain and different mutant strains to the ACE2 receptor of host cells.

[0051] Experimental methods:

[0052] HEK293T cells were digested with trypsin and seeded at a density of 100,000 cells / mL in 100 μL per well. After 24 h, 40 μL of culture medium containing different concentrations of compound III (cefixime) were added to each well (final concentrations of 400 μM, 200 μM, and 100 μM). The positive control group was treated with an equal volume of culture medium. The mixture was then incubated. An equal volume of pseudovirus (10 μL diluted 20-fold) was added to each well, and the mixture was incubated again. After 24 h, the supernatant was removed, and 50 μL of cefixime-containing culture medium of the same concentration was added. After another 48 h, the cells were digested, diluted with phosphate-buffered saline (PBS), centrifuged at 1600 rpm for 10 min, and the supernatant was removed. Each sample was resuspended in 200 μL of PBS, and the GFP fluorescence intensity was measured using flow cytometry to determine the proportion of infected positive cells.

[0053] Table 4. Cefixime activity tested using different SARS-CoV-2 mutant pseudoviruses.

[0054]

[0055] (Note: All of the above mutant pseudoviruses can be obtained by those skilled in the art based on the description in Example 1, that is, the S protein corresponding to each mutant strain is publicly available.)

[0056] In infection experiments using a pseudovirus of the wild-type SARS-CoV-2 strain, the proportion of GFP-positive cells decreased with increasing cefixime concentration: 14.84% at 100 μL, 9.27% ​​at 200 μL, and 4.40% at 400 μL. In infection experiments using a pseudovirus of the Brazilian mutant SARS-CoV-2 (P.1), the proportion of GFP-positive cells also decreased with increasing cefixime concentration: 13.24% at 100 μL, 9.88% at 200 μL, and 9.08% at 400 μL. After infection experiments using a pseudovirus of the Indian mutant SARS-CoV-2 (B.1.617), the proportion of positive cells was 18.76% at 100 μL, 18.90% at 200 μL, and 8.10% at 400 μL. In infection experiments using the Indian (B.1.617.2) mutant pseudovirus of SARS-CoV-2, the positive cell rate was 9.97% at a dose of 100 μL, 9.64% at a dose of 200 μL, and 3.22% at a dose of 400 μL. Similarly, in infection experiments using the Indian (B.1.618) mutant pseudovirus of SARS-CoV-2, the positive cell rate was 14.64% at a dose of 100 μL, 13.34% at a dose of 200 μL, and 6.56% at a dose of 400 μL. The viral inhibition rate generally increased with increasing cefixime dosage.

[0057] Example 5

[0058] This embodiment relates to the evaluation of the inhibitory effect of the compound described in structural formula IV (cefaprom) on the binding of the novel coronavirus mutant S protein to the host cell ACE2 receptor.

[0059] Experimental methods:

[0060] HEK293T cells were digested with trypsin and seeded at a density of 100,000 cells / mL in 100 μL per well. After 24 h, 40 μL of medium containing compound IV (cefaprom) (final concentration 200 μM, DMSO, 0.5%) was added to each well. An equal volume of medium was added to the positive control group, and the mixture was stirred. An equal volume of pseudovirus (10 μL diluted 20-fold) was added to each well, and the mixture was stirred again. After 24 h, the supernatant was removed, and 50 μL of cefprom-containing medium of the same concentration was added. After another 48 h, the cells were digested, diluted with phosphate-buffered saline (PBS), centrifuged at 1600 rpm for 10 min, and the supernatant was removed. Each sample was resuspended in 200 μL of PBS, and the GFP fluorescence intensity was measured using flow cytometry to determine the proportion of infected positive cells.

[0061] Table 5. Cefprozil activity was tested using different SARS-CoV-2 mutant pseudoviruses.

[0062]

[0063] Infection experiments were conducted using the D614G mutant of SARS-CoV-2, the UK mutant of SARS-CoV-2 (B.1.1.7), the Indian mutant of SARS-CoV-2 (B.1.617), the Indian mutant of SARS-CoV-2 (B.1.617.2), and the Indian mutant of SARS-CoV-2 (B.1.618). All of these mutants exhibited inhibitory activity against the virus at a cefprozil concentration of 200 μM, with inhibition rates of 9.45%, 5.27%, 54.18%, 15.26%, and 28.97%, respectively.

[0064] Example 6

[0065] This embodiment relates to the evaluation of the inhibitory effect of ceftazidime on the binding of different mutant S proteins of the novel coronavirus to the ACE2 receptor on host cells.

[0066] Experimental methods:

[0067] HEK293T cells were digested with trypsin and seeded at a density of 100,000 cells / mL in 100 μL per well. After 24 h, 40 μL of ceftazidime-containing medium (final concentrations of the compound were 100 μM, 200 μM, and 400 μM, in deionized water) were added to each well, and the mixture was homogenized. An equal volume of pseudovirus (diluted 20 times and added to each well) was added to each well, and the mixture was homogenized again. After 24 h, the supernatant was aspirated, and 50 μL of ceftazidime-containing medium of the same concentration was added. After another 48 h, the cells were digested, diluted with phosphate-buffered saline (PBS), centrifuged at 1600 rpm for 10 min, and the supernatant was discarded. Each sample was resuspended in 200 μL of PBS, and the GFP fluorescence intensity was measured by flow cytometry to obtain the proportion of infected positive cells.

[0068] Table 6. Ceftazidime activity tested using different SARS-CoV-2 mutant pseudoviruses.

[0069]

[0070] Infection experiments were conducted using the British mutant of SARS-CoV-2 (B.1.1.7), the Indian mutant of SARS-CoV-2 (B.1.617), the Indian mutant of SARS-CoV-2 (B.1.617.2), and the Indian mutant of SARS-CoV-2 (B.1.618). The viral infection inhibition rate showed an increasing trend with the increase of ceftazidime dosage.

[0071] Example 7

[0072] This embodiment relates to the evaluation of the inhibitory effect of the compound (7-aminocephalosporanic acid) described in structural formula II on the binding of different mutant S proteins of the novel coronavirus to the ACE2 receptor of host cells.

[0073] Experimental methods:

[0074] HEK293T cells were digested with trypsin and seeded at a density of 100,000 cells / mL in 100 μL per well. After 24 h, 40 μL of culture medium containing compound II (7-aminocephalosporanic acid) (final concentration of compound: 800 μM, solvent: deionized water) was added to each well. An equal volume of culture medium was added to the positive control group. The mixture was then mixed. An equal volume of pseudovirus (10 μL per well after a 20-fold dilution) was added to each well, and the mixture was mixed again. After 24 h, the supernatant was aspirated, and 50 μL of culture medium containing 7-aminocephalosporanic acid of the same concentration was added. After another 48 h, the cells were digested, diluted with phosphate-buffered saline (PBS), centrifuged at 1600 rpm for 10 min, and the supernatant was discarded. Each sample was resuspended in 200 μL of PBS, and the GFP fluorescence intensity was measured using flow cytometry to obtain the proportion of infected positive cells.

[0075] Table 7. 7-Aminocephalosporanic acid activity tested using different SARS-CoV-2 mutant pseudoviruses.

[0076]

[0077] Infection experiments were conducted using the UK mutant of SARS-CoV-2 and the Indian B.1.617 mutant of SARS-CoV-2. Both mutants showed inhibitory activity against the virus at a concentration of 800 μM of 7-aminocephalosporanic acid, with inhibition rates of 18.78% and 37.41%, respectively.

[0078] Example 8

[0079] This embodiment relates to the toxicity test of 7-aminocephalosporanic acid (II), cefixime (III), and cefprozil (IV) on HEK293T cells.

[0080] Experimental methods:

[0081] Cells were seeded at a density of 100,000 cells / mL, with 100 μL of cell suspension in each well. The medium was changed after 24 hours, and the drugs were administered. Each compound was serially diluted according to its half-maximal concentration (MCD), with cefprozil at a maximum concentration of 200 μM, cefixime at a maximum concentration of 400 μM, and 7-aminocephalosporanic acid at a maximum concentration of 800 μM, and each diluted in triplicate. Cefprozil was dissolved in DMSO (5‰ concentration), while cefixime and 7-aminocephalosporanic acid were dissolved in deionized water. The supernatant was first aspirated with a 200 μL pipette tip, and then 100 μL of culture medium was added to each well. Two positive control groups were set up: one group received 100 μL of culture medium containing an equal amount of DMSO, and the other group received 100 μL of culture medium. After 24 hours, the supernatant was aspirated, and 200 μL of culture medium containing the same concentration of the compound as the original well was added to each well. After 48 hours, 20 μL of MTT was added to each well. After 4 hours, the cells were treated, and the absorbance of each well was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = OD value of the drug group / OD value of the control group × 100%.

[0082] Table 8. Survival rate of HEK293T cells under different concentrations of compounds

[0083]

[0084]

[0085] The above results indicate that, at the tested concentrations, none of the three compounds had significant cytotoxic effects on cell growth.

[0086] In summary, the compounds disclosed in this invention can inhibit the binding of the spike protein of the novel coronavirus SARS-CoV-2 to receptors on the host cell membrane, effectively reducing the infectivity of the virus. In vitro cytotoxicity tests show that the compounds do not exhibit cytotoxicity, and they are already in clinical use as anti-inflammatory drugs. Repurposing these existing drugs for use against COVID-19 infection provides a new approach for COVID-19 prevention and control, demonstrating promising application prospects. Furthermore, the 7-aminocephalosporanic acid derivative disclosed in this invention has a well-defined structure that can be further optimized, which is of great significance for developing highly effective and low-toxicity new drugs against the novel coronavirus.

[0087] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The use of a compound having a 7-aminocephalosporanic acid core structure as shown in Formula (III) in the preparation of a medicament for treating novel coronavirus SARS-CoV-2 infection, wherein the novel coronavirus is a mutant of SARS-CoV-2 B.1.617.2; （Ⅲ）。 2. Use of a compound having a 7-aminocephalosporanic acid core structure as shown in Formula (IV) in the preparation of a medicament for treating novel coronavirus SARS-CoV-2 infection, wherein the novel coronavirus is a B.1.617 mutant of SARS-CoV-2; （Ⅳ）。

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