CN112315959A - Application of porphyrin compound in preparation of anti-coronavirus drugs - Google Patents

Abstract

The invention discloses an application of porphyrin compounds or pharmaceutically acceptable salts thereof in preparing anti-coronavirus medicines. Research experiments on the anti-coronavirus activity of a plurality of porphyrin compounds show that the porphyrin compounds have higher inhibitory activity on coronavirus and have obvious inhibitory effect on coronavirus at low concentration; low toxicity, no adverse effect on cells due to effective concentration, and can be used as novel coronavirus inhibitor for preparing anti-coronavirus medicines.

Description

Application of porphyrin compound in preparation of anti-coronavirus drugs

Technical Field

The invention relates to the field of medicines, in particular to the technical field of antiviral medicines, and specifically relates to an application of porphyrin compounds in preparation of anti-coronavirus medicines.

Background

Coronaviruses (CoVs) are a class of spherical positive-stranded RNA viruses with an envelope. They are widely present in humans and other mammals and can cause diseases of multiple systems, such as respiratory, intestinal, liver and nerves. Since its discovery, coronavirus has caused 3 major outbreaks of fatal infectious diseases in this century, constituting a great threat to human health.

A global infectious pneumonia epidemic caused by a novel coronavirus (SARS-CoV-2) infection is outbreaked in 12 months in 2019, and hundreds of countries around the world are related at present. This is also the 7 th coronavirus that has been discovered to date that can infect humans. According to the analysis of the phylogenetic relationship of this coronavirus, SARS-CoV-2 has about 79% homology with Severe acute respiratory syndrome coronavirus (SARS-CoV) and about 50% homology with Middle East respiratory syndrome coronavirus (MERS-CoV), and the lethality rates of these two viruses are 10% and 37%, respectively. SARS-CoV-2 also has the characteristics of rapid propagation speed, wide range and the like, can cause fatal pneumonia, and poses serious threat to the world.

However, until now, no specific antiviral drug that directly inhibits SARS-CoV-2 has been found, and the current treatment scheme for SARS-CoV-2 mainly aims at relieving symptoms, reducing complications, preventing secondary infection, and supporting organ functions. Therefore, in the face of the increasing serious harm of SARS-CoV-2 and the lack of specific antiviral drugs, it is very important to find and develop new antiviral active compounds and study their antiviral mechanisms in order to combat the epidemic situation.

However, compounds having anti-coronavirus activity have not been found in the prior art, and thus, it is necessary to provide a compound having anti-coronavirus activity, particularly anti-novel coronavirus activity.

Disclosure of Invention

The invention aims to develop a new high-efficiency and low-toxicity active component for resisting SARS-CoV-2 aiming at the defects of the prior art. The invention discovers that porphyrin compounds have higher inhibitory activity to SARS-CoV-2 through numerous screening experiments and verification of a large number of biological experiments, has obvious inhibitory effect to SARS-CoV-2 at low concentration, has low toxicity and no adverse effect on cells due to effective concentration, and can be used as a novel coronavirus inhibitor. Based on the discovery, the invention provides an application of porphyrin compounds in preparation of anti-coronavirus medicines.

The invention aims to provide an application of porphyrin compounds or pharmaceutically acceptable salts thereof in preparing anti-coronavirus medicines, wherein the structural formula of the porphyrin compounds is as follows:

wherein R is₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃Independently or simultaneously selected from the following groups:

preferably, the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus.

Further, the coronavirus is a novel coronavirus.

Further, the porphyrin compound is TMPyP4 or NMM, and the porphyrin compound is preferably TMPyP 4.

Wherein, the structural formula of TMPyP4 is shown as the following formula 2:

the structural formula of NMM is shown in formula 3 below:

further, the application of the porphyrin compound or the pharmaceutically acceptable salt thereof in preparing the anti-coronavirus medicine comprises the drug combination of different porphyrin compounds or the pharmaceutically acceptable salts thereof.

The second purpose of the invention is an anti-coronavirus drug which comprises a porphyrin compound or pharmaceutically acceptable salt thereof.

Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials and carriers.

Further, the pharmaceutical preparation of the medicine is granules, tablets, pills, capsules, injection or suppositories.

The invention has the beneficial effects that:

1. the porphyrin compound has obvious inhibition effect on coronavirus, especially novel coronavirus (SARS-CoV-2) at low concentration;

2. the porphyrin compound is low in toxicity and has no adverse effect on cells under effective action concentration;

3. the porphyrin compound is soluble in water at effective concentration, is not easy to degrade, has high stability, can be used as a novel coronavirus inhibitor, and is easy to prepare a novel coronavirus resistant medicinal preparation.

Drawings

FIG. 1 is a graph showing the cytotoxicity of TMPyP4 and NMM and the effect against SARS-CoV-2.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: cell and virus culture

The test cells were Vero E6(ATCC CRL-1586), the medium was DMEM (supplemented with 2% fetal bovine serum, 100IU/ml penicillin and 100. mu.g/ml streptomycin), and the cells were incubated in CO₂Constant temperature incubator (37 ℃, 5% CO)₂) Culturing in medium. The SARS-CoV-2 strain (nCoV-2019BetacoV/Wuhan/WIV04/2019) was obtained from the National viral resources library of the Wuhan Institute of Virus, National Institute of Sciences (National Virus Resource, Wuhan Institute of Virology, Chinese Academy of Sciences).

Example 2: preparation of Compound solutions

According to the design of experimental dosage, TMPyP4 and NMM are accurately weighed in a 1.5ml centrifuge tube respectively, and a certain amount of deionized water and dimethyl sulfoxide (DMSO) are added respectively to prepare a mother solution with the concentration of 20mM, and the mother solution is fully dissolved. For the experiments, the samples were diluted to the designed final concentrations and blanked with deionized water and DMSO, respectively.

Example 3: cytotoxicity assays

In this example, cell counting kit-8(CCK8) (China, Byunnan) was used to evaluate the cytotoxicity of compounds against Vero E6 cells. Vero E6 cells were cultured at 5X 10³Density seeding of cells/wellsIn 96-well cell culture plates, and in CO₂Constant temperature incubator (37 ℃, 5% CO)₂) And culturing for 24 hours. Then, after exposing the cells to different concentrations of TMPyP4(0, 7.8125, 15.625, 31.25, 62.5, 125 and 500 μ M) and NMM (0, 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 μ M) for 24 hours, the medium in the wells was discarded, and fresh DMEM medium containing 10% CCK-8 solution was added and incubated at 37 ℃ for 1.5 hours. Finally, the absorbance (OD) at 450nm was monitored using a microplate reader (SynergyH1, BioTek, USA)₄₅₀Value).

Half toxic concentrations of TMPyP4 and NMM to cells were calculated, respectively (Medium cytoxic concentration, CC 50).

The cytotoxicity of these two compounds was measured using CCK8 cytotoxicity assay kit, and the results of the cytotoxicity of TMPyP4 are shown in fig. 1B, which indicates that the CC50 (50% cytotoxic concentration) of TMPyP4 is higher than 1000 μ M (fig. 1B), and the results of the cytotoxicity of NMM are shown in fig. 1D, which indicates that the CC50 (50% cytotoxic concentration) of NMM is 40.5 μ M (fig. 1D).

Example 4: antiviral assays

Vero E6 cells were cultured at 1X 10⁵cells/well were plated at 24-well cell culture plates and various concentrations (0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10. mu.M) of compounds were added to infect cells with SARS-CoV-2 at 0.005 MOI. After 24 hours post-infection, total viral RNA was extracted from the supernatant of each well, and the yield of virus was determined by quantitative real-time fluorescent PCR (qRT-PCR) to calculate the virus inhibition rate. Specifically, primers ORF1ab-F (5'-ccctgtgggttttacacttaa-3') and ORF1ab-R (5'-acgattgtgcatcagctga-3') with probe 5'-FAM-tggttgacctacacaggtgccatca-BHQ1-3' were used to detect the expression level of ORF1 ab.

Vero-E6 cells infected with SARS-CoV-2 were treated with different concentrations of TMPyP4 and NMM, respectively, to determine the antiviral activity of TMPyP4 and NMM against SARS-CoV-2. Specifically, primers targeting ORF1ab were designed and the viral RNA after compound treatment was quantitatively analyzed by qRT-PCR. The results of the antiviral activity of TMPyP4 against SARS-CoV-2 are shown in FIG. 1A, and the resistance of NMM against SARS-CoV-2The results of viral activity are shown in FIG. 1C, and show that both compounds exhibit dose-dependent SARS-CoV-2 inhibitory activity in infected cells, EC for TMPyP4 and NMM₅₀(half effective concentration) was 1.21. mu.M and 0.32. mu.M, respectively.

The results of example 3 were combined (CC of TMPyP 4)₅₀(50% cytotoxic concentration) above 1000. mu.M (FIG. 1B), whereas CC for NMM₅₀40.5. mu.M (FIG. 1D)), it can be seen that the selectivity index (SI, CC50/EC50) of Vero E6 cells for TMPyP4 and NMM is higher than 844 and 126, respectively, thus indicating that the two compounds have good safety.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (9)

1. The application of porphyrin compounds or pharmaceutically acceptable salts thereof in preparing anti-coronavirus medicines is as follows:

wherein R is₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃Independently or simultaneously selected from the following groups:

2. the use according to claim 1, wherein the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus.

3. Use according to claim 1, wherein the coronavirus is a novel coronavirus.

4. The use according to claim 1, wherein the porphyrin-like compound is TMPyP4 or NMM.

5. The use according to claim 1, wherein the porphyrin-based compound is TMPyP 4.

6. The use according to claim 1, characterized in that it comprises a combination of drugs of different porphyrins or pharmaceutically acceptable salts thereof.

7. An anti-coronavirus drug, which is characterized by comprising a porphyrin compound or a pharmaceutically acceptable salt thereof.

8. The medicament of claim 7, further comprising pharmaceutically acceptable excipients and carriers.

9. The medicament of claim 7, wherein the pharmaceutical formulation is a granule, tablet, pill, capsule, injection or suppository.

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