Application of pyrimidine derivative as coxsackie virus CVB3 inhibitor

Abstract

The invention belongs to the technical field of antiviral drugs, and relates to application of a pyrimidine derivative as a coxsackie virus CVB3 inhibitor. The anti-CVB3 activity research experiments of several pyrimidine derivatives show that the pyrimidine derivatives inhibit the cytopathic effect (CPE) generated by CVB3 on host cells Hep-2, enhance the cell survival rate and reduce the progeny virus yield; the inhibitory activity of the pyrimidine derivative on CVB3 shows that the pyrimidine derivative has application potential in preparation of anti-CVB3 virus drugs.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug technology, specifically to the application of pyrimidine derivatives as inhibitors of Coxsackievirus CVB3. Background Technology

[0002] Coxsackievirus B3 (CVB3) is a single-stranded, positive-sense small RNA virus belonging to the genus Enterovirus, family Picornaviridae, and the genus Enterovirus. CVB3 is transmitted through the digestive and respiratory tracts, and infection leads to diseases such as viral myocarditis (VMC) and pancreatitis. It has been reported that approximately 20% of myocarditis patients and 10% of pancreatitis patients are associated with CVB3 infection. However, effective treatments are still lacking in clinical practice; therefore, the development of novel drugs for treating CVB3 infection is extremely important.

[0003] As a cornerstone of drug development, pyrimidine compounds have demonstrated broad application potential. In recent years, scientists have developed a series of novel pyrimidine derivatives through structural design and synthetic optimization. These compounds exhibit significant pharmacological activity in the fields of antitumor, antibacterial, and antiviral activity. Studies have found that some pyrimidine derivatives can precisely target the proliferation mechanism of tumor cells, inhibiting their growth and inducing apoptosis; simultaneously, their antibacterial spectrum covers a variety of drug-resistant strains, exhibiting broad-spectrum antibacterial capabilities. In the antiviral field, pyrimidine derivatives inhibit influenza viruses and HIV by blocking key stages of the viral life cycle, such as replication and invasion. Currently, research in this field continues to heat up, and the continuous emergence of new derivatives provides more possibilities for clinical applications.

[0004] The high variability of viral diseases poses a significant challenge to existing therapies, but the structural plasticity of pyrimidine rings offers a unique advantage in addressing this issue. By introducing diverse substituents onto the pyrimidine ring, researchers can modulate its physicochemical properties and biological activities, thereby designing specific inhibitors against different viral strains. This strategy not only broadens the development pathway for antiviral drugs but also provides innovative ideas for solving the problems of drug resistance and side effects of existing drugs. Pyrimidine derivatives hold promise for significantly reducing toxicity while maintaining high antiviral activity, providing a safer and more effective option for the treatment of future viral diseases.

[0005] Therefore, developing pyrimidine compounds with novel structures for the treatment of Coxsackievirus CVB3 is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the main objective of this invention is to provide pyrimidine derivatives as inhibitors of Coxsackievirus CVB3. Through experimental studies on the anti-CVB3 activity of several pyrimidine derivatives, this invention demonstrates that pyrimidine derivatives have an inhibitory effect on CVB3 virus: they can inhibit the cytopathic effect (CPE) produced by CVB3 on host cells Hep-2, enhance cell viability, and reduce progeny virus yield. The pyrimidine derivatives of this invention exhibit good anti-CVB3 virus activity and can be applied to the preparation of anti-CVB3 virus drugs.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: The structural formula of the pyrimidine derivative is shown in Formula I below:

[0008] Among them, R 1 It is one of Cl and CF3; R 2 For H, CH3, Et, , , , , , , , , , , One of them; R 2 for , , , , , , , One of them.

[0009] Furthermore, the pyrimidine derivative has anti-Coxsackievirus B3 (CVB3) activity.

[0010] Furthermore, the pyrimidine derivative exhibited an 80% inhibition rate against CVB3 in hep-2 cells, and its therapeutic index was superior to that of the positive control drug ribavirin.

[0011] Furthermore, the application also includes the use of derivatives of Formula I and / or pharmaceutically acceptable salts thereof in the preparation of Coxsackievirus CVB3 inhibitor drugs.

[0012] The present invention also provides a medicament for preparing a Coxsackievirus CVB3 inhibitor, comprising a derivative of Formula I or a pharmaceutically acceptable salt thereof.

[0013] Furthermore, the drug uses a derivative of Formula I or a pharmaceutically acceptable salt thereof as the sole or primary active ingredient.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients and carriers.

[0015] Furthermore, the pharmaceutical preparation is any one of granules, tablets, pills, capsules, injections, or dispersants.

[0016] The polysubstituted pyrimidine derivatives and their pharmaceutically acceptable salts of the present invention have potential inhibitory activity against enterovirus 71 (EV71), Coxsackievirus group B (CVB3), adenovirus (ADV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpes simplex virus (HSV), influenza virus (A), respiratory syncytial virus (RSV), Newcastle disease virus (NDV), human adenovirus 3 (AD3), vesicular stomatitis virus (VS), rotavirus (RV), and cytomegalovirus (CMV), making these compounds suitable as active ingredients in antiviral drugs.

[0017] The beneficial effects of this invention are as follows: 1. This invention develops a novel class of pyrimidine derivatives with good antiviral activity. These compounds can effectively inhibit the cytopathic effect (CPE) produced by CVB3 in host cells Hep-2 and enhance cell survival.

[0018] 2. The pyrimidine derivatives of the present invention are expected to be further developed into drugs for the effective treatment of CVB3 infection, which is of profound significance.

[0019] 3. This invention designs pyrimidine derivatives to achieve different inhibitory activities against the virus CVB3; as resistance to existing antiviral drugs increases, these compounds can be used as alternative products to prepare active ingredients for novel antiviral drugs. Attached Figure Description

[0020] The present invention will be further described below with reference to the accompanying drawings: Figure 1 This diagram illustrates the inhibitory effect of the pyrimidine derivatives of the present invention on CVB3. Figure 2 This diagram illustrates the inhibitory effect of the pyrimidine derivative of the present invention on the yield of CVB3 progeny viruses. Figure 3 This diagram illustrates the inhibitory effect of the pyrimidine derivatives of the present invention on CPE induced by CVB3 in hep-2 cells. Detailed Implementation

[0021] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The provided embodiments are merely illustrative of the method of the present invention and do not limit the remaining content disclosed herein in any way. The chemical reagents and pharmaceuticals used in the present invention are all commercially available conventional chemicals.

[0022] The pyrimidine derivatives of this invention were prepared in the laboratory, and the structures of representative compounds of the pyrimidine derivatives are shown in Table 1: Table 1. List of representative compound structures

[0023] The following examples use compounds from Table 1 for testing.

[0024] Example 1: Antiviral activity Experimental Methods: This invention uses the Therapeutic Index (SI) as an evaluation index to measure the inhibitory efficacy of drugs against viruses. The higher the therapeutic index, the greater the antiviral potential; among them, CC 50 The half-maximal toxic concentration (MCC) refers to the drug concentration that causes 50% toxicity in cells; EC 50 The half-maximal inhibitory concentration (MCI) refers to the concentration of a drug that effectively inhibits the virus by 50%.

[0025] 1. Toxicity of the compound to host cell hep-2 Hep-2 cells were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator until a confluent monolayer was formed. The cell culture medium was then discarded, and cell maintenance medium containing different concentrations (400 μM, 200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM) of the target compound was added for further culture. Untreated control wells (ribavirin) were included. After 48 hours, cell morphology was visually observed and recorded under a microscope for different drug concentrations. The absorbance at 490 nm was measured using the MTT assay, and cell viability was calculated. The median toxic concentration (CMC) of each compound was calculated using SPSS 11.5 software. 50value.

[0026] The formula for calculating cell viability is: .

[0027] 2. Inhibitory activity of the compound against CVB3 Hep-2 cells were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator until a confluent monolayer was formed. The cell culture medium was then discarded, and cell maintenance medium containing 100 mg TCID50 (TCID50: tissue culture infective dose) of CVB3 virus was added. The cells were incubated at 37°C in a 5% CO2 incubator for 1.5 h for adsorption. The virus infection medium was then discarded, and the cells were washed three times with PBS. Cell maintenance medium containing different concentrations (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.15 μM) of the compound was added, and the cells were cultured further. Virus control wells and cell control wells were set up. After 48 h of culture, when approximately 90% cytopathic effect (CPE) appeared in the virus control wells, the cytopathic effect in the drug group was observed under a microscope. The virus inhibition rate was determined by the MTT assay, and the half-maximal inhibitory concentration (EC50) of the drug was calculated using SPSS 11.5 software. 50 .

[0028] The formula for calculating the virus inhibition rate is: .

[0029] 3. Experimental Results: Combining the MTT assay for cell viability and cytopathic effect analysis, the activity of representative compounds against CVB3 was evaluated, and the results are as follows: Figure 1 As shown in Table 2.

[0030] The inhibitory effect of compounds on CVB3 virus, such as Figure 1 As shown in the figure. The results indicate that some of the target compounds exhibited significant inhibitory activity against CVB3 (hep-2 cells) at an initial screening concentration of 100 μM, and compounds 5a, 5b, 7e, 7g, and 7h showed better inhibition rates than the positive control drug Ribavirin, demonstrating superior antiviral activity.

[0031] Table 2. Cytotoxicity and anti-CVB3 activity of the compounds

[0032] Note: a EC 50 - This refers to the half-maximal inhibitory concentration, which is the drug concentration that effectively inhibits the virus by 50%. b :CC 50- The half-maximal toxicity concentration refers to the drug concentration that causes 50% drug toxicity in cells.c SI - Therapeutic Index of a Drug, SI = CC 50 / EC 50 ; d Inhibition rate below 50%; e Data: The average of three independent experiments; f Ribavirin - Control drug.

[0033] To investigate the activity of the compounds, the inventors conducted inhibitory activity measurements at different concentrations and obtained the corresponding EC values ​​for the compounds. 50 CC 50 The values ​​and SI indexes are shown in Table 2. As can be seen from Table 2, compounds 5b and 5d showed significantly better or comparable antiviral activity against CVB3 in hep-2 cells compared to the control Ribavirin, demonstrating substantial research value and application potential.

[0034] Example 2: Test on the inhibitory effect of the compound on progeny virus yield To further clarify the antiviral activity of pyrimidine compounds and provide basic research data for the development of such compounds, this invention selects some representative compounds from Table 2 to investigate their inhibitory effect on progeny virus yield.

[0035] 1. Test content: The inhibitory effect of the compound on the production of CVB3 progeny virus was investigated after hep-2 cells were infected with CVB3.

[0036] 2. Test methods: Hep-2 cells in logarithmic growth phase were seeded into 24-well plates, and after reaching a confluent monolayer, 100 TCID were applied. 50 CVB3-infected cells were incubated at 37°C for 1.5 h, then the virus solution was removed, the cells were washed three times with PBS, and cell maintenance medium containing 100 μM of the compound was added. After 48 h, cells and supernatant were collected, and the cells were lysed by three freeze-thaw cycles at -20°C and 37°C. TCID45 was then used. 50 The method was used to determine the CVB3 virus titer.

[0037] 3. Experimental Results: like Figure 2 As shown, the viral titer of hep-2 cells treated with the representative compound decreased significantly compared to the virus control group, indicating that the compound has a strong inhibitory effect on the production of CVB3 progeny virus and can significantly inhibit the replication of CVB3 virus in hep-2 cells, decreasing by 4.05 log and 3.1 log respectively compared to the virus control.

[0038] Representative compounds inhibit the CPE effect induced by CVB3 in hep-2 cells, such as Figure 3As shown, CVB3-infected Hep-2 cells become rounded, and no cells detach from the cell plate. Figure 3 The compound treatment showed a significant inhibitory effect on the corresponding pathological effects.

[0039] In summary, some pyrimidine derivatives exhibit certain inhibitory activity against CVB3 and have a high therapeutic index. The inhibitory effects of pyrimidine compounds on CVB3 include inhibiting the Hep-2 cytopathic effect induced by CVB3 virus, enhancing cell survival rate, and inhibiting viral replication and proliferation within cells, indicating that pyrimidine derivatives have the potential to prepare anti-CVB3 virus drugs.

[0040] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. Use of a pyrimidine derivative for the manufacture of a pharmaceutical for the inhibition of coxsackie virus CVB3, characterized in that, The structural formula of the pyrimidine derivative is shown as formula I: wherein R is one of Cl, CF3 1 is one of Cl, CF3 R 2 H, CH3, Et, , , , , , , , , , , one of H, CH3, Et, R 2 For , , , , , , , one of.

2. Use according to claim 1, characterized in that, The pyrimidine derivative has anti-covb3 virus activity.

3. Use according to claim 1, characterized in that, Some of the pyrimidine derivatives have an inhibition rate of 80% on CVB3 in hep-2 cells, and the therapeutic index is better than that of the positive control drug ribavirin.

4. The use according to claim 1, characterized in that The use of the derivative shown as formula I in claim 1 and / or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting coxsackie virus CVB3.

5. A medicine for the preparation of a coxsackie virus CVB3 inhibitor, characterized in that, The derivative shown as formula I in claim 1 or a pharmaceutically acceptable salt thereof.

6. The medicament according to claim 5, characterized in that, The drug takes the derivative shown as formula I in claim 1 or a pharmaceutically acceptable salt thereof as the only or main active ingredient.

7. The medicament according to claim 5, characterized by, The drug further comprises pharmaceutically acceptable adjuvants and carriers.

8. The medicament according to claim 1, characterized by, The drug preparation is any one of granules, tablets, pills, capsules, injections or dispersants.

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