Application of bromophenol-benzamide compound as 3CLpro and PLpro double-target inhibitor in treatment of porcine epidemic diarrhea virus disease

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a bromophenol-benzamide compound as a dual inhibitor of PLpro enzyme and 3CLpro enzyme in treatment of porcine epidemic diarrhea (PED) diseases. The research finds that the bromophenol-benzamide compound can simultaneously inhibit the activity of PLpro enzyme and 3CLpro enzyme in porcine epidemic diarrhea virus (PEDV) in vitro, inhibit the duplication of the PEDV at the cellular level, reduce the cytoinflammation caused by the PEDV and treat diarrhea piglets infected by the PEDV.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to the use of bromophenol-benzamide compounds as 3CL pro and PL pro dual-target inhibitors in the treatment of porcine epidemic diarrhea disease. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is highly contagious, associated with severe diseases such as diarrhea and vomiting, and increases the mortality rate of piglets (usually 100%). They can cause sporadic outbreaks (endemic) or large-scale epidemics in pig-raising countries, characterized by acute onset, high infectivity, and high lethality, causing serious economic losses to the pig-raising industry in Asia, Europe, and the Americas. Since October 2010, a series of large-scale porcine epidemic diarrhea virus (PEDV) outbreaks have also occurred in China, causing huge economic losses of up to hundreds of billions of yuan.

[0003] In 2010, the incidence of porcine epidemic diarrhea (PED) increased significantly. Through expert sampling and analysis, it was found that this porcine epidemic diarrhea virus was a mutant strain, mainly mutated in bats, and the virulence of the virus increased significantly after mutation. For the pig population, porcine epidemic diarrhea (PED) is most harmful to suckling piglets, with a mortality rate close to 100%. So far, there is no specific drug for treating PEDV on the market. Clinical prevention mainly relies on vaccination, but due to the existence of multiple mutant strains of PEDV, the effect is not obvious. For example, 15 new wild PEDV mutants were detected in diarrhea pig feces or intestinal samples collected from December 2013 to June 2015.

[0004] Currently, there is no effective drug for PEDV on the international and domestic markets. In addition to using vaccination for early prevention (there is no vaccine with a definite immune effect), only traditional Chinese medicine and antibody conservative treatment can be used. However, due to the continuous mutation of the virus strain and the increasing virulence, showing greater and greater lethality, the treatment effect of traditional Chinese medicine and antibodies is not good.

[0005] The replication of coronaviruses depends on a complex proteolytic process, and 3CL pro and PL pro play a key role in this complex process. 3CL pro is highly conserved in the coronavirus family and is an important enzyme that can cleave viral polyproteins into multiple basic non-structural proteins crucial for the viral replication cycle. 3CL pro The unique difference from human proteases makes it a promising and side-effect-free target for developing broad-spectrum anti-coronavirus therapeutic drugs. PL proIt plays a crucial role in virus replication by cleaving viral polyproteins into essential non-structural proteins and disrupting the host antiviral immune response through deubiquitination. Therefore, PL with potent antiviral replication pro inhibitors provide a superior treatment strategy against PEDV infection. The dual-target mechanism of action provides a universal solution for coping with the variation and evolution of the coronavirus family. Compared with the technical routes focusing on single targets in international similar studies, it has significant advantages in terms of drug resistance and the persistence of treatment effects, laying an important foundation for the development of a new generation of anti-coronavirus drugs.

[0006] In addition, PEDV infection can lead to severe inflammatory responses, and excessively elevated cytokines or uncontrolled inflammation can damage the intestinal structure and function. Therefore, reducing the expression of inflammatory cytokines will help prevent PEDV-induced intestinal damage. Therefore, novel anti-PEDV drugs should also possess anti-inflammatory activity to better protect against intestinal damage caused by PEDV infection.

[0007] Bromophenol-benzamide compounds are compounds with dual inhibitory activities against 3CL pro and PL pro designed and synthesized by the inventors for the first time. They can also regulate the host's excessive inflammatory response, breaking through the limitation of traditional antiviral drugs that only target pathogens, and have broad application prospects in the preparation of drugs for treating PEDV. Therefore, bromophenol-benzamide compounds are a class of compounds with great medicinal potential, and further discovery of their medicinal effects will be of great significance. Summary of the Invention

[0008] In view of the above-mentioned prior art, the purpose of the present invention is to provide a new medical use of bromophenol-benzamide compounds. The present invention has found through research that bromophenol-benzamide compounds can inhibit the activities of PL pro enzyme and 3CL pro enzyme in PEDV in vitro, inhibit the replication of PEDV at the cellular level, reduce the cell inflammation caused by PEDV, and can treat piglets infected with PEDV.

[0009] To achieve the above purpose, the present invention adopts the following technical solutions:

[0010] The present invention provides the use of bromophenol-benzamide compounds in the preparation of drugs for treating porcine coronavirus diseases.

[0011] In the above application, the bromophenol-benzamide compounds have the following general structural formula shown in Formula I:

[0012]

[0013] Wherein: R 1 、R2 Selected from any one of OH and OCH 3 respectively;

[0014] R 2 is selected from any one of them;

[0015] The porcine epidemic diarrhea disease is a disease caused by porcine epidemic diarrhea virus.

[0016] Preferably, the bromophenol-benzamide compound is any of the following compounds:

[0017]

[0018]

[0019] In the above application, the porcine epidemic diarrhea disease is gastroenteritis, diarrhea and other diseases caused by porcine epidemic diarrhea virus (PEDV).

[0020] Advantages of the present invention:

[0021] It is first discovered in the present invention that bromophenol-benzamide compounds can inhibit the activities of PL pro enzyme and 3CL pro enzyme in vitro, inhibit the replication of porcine PEDV at the cell level, can also reduce the cell inflammation caused by PEDV, and treat diarrhea piglets infected with PEDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the determination result of compound 3 provided by the embodiment of the present invention for inhibiting PEDV replication in host cells, evaluating the influence of compounds at different concentrations on viral RNA replication.

[0023] Figure 2 It is the determination result of compound 3 for inhibiting cell inflammation caused by PEDV in cells, evaluating the influence of compounds at different concentrations on the expression of cell inflammatory factors. DETAILED DESCRIPTION OF THE INVENTION

[0024] For ease of understanding of the present invention, the present invention will be described in more detail below with reference to the drawings and specific embodiments. Preferred embodiments of the present invention are given in the specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0025] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0026] As introduced in the background art section, porcine epidemic diarrhea disease has caused serious economic losses to the pig farming industry, but there is currently no effective therapeutic drug for porcine epidemic diarrhea virus.

[0027] Coronaviruses (CoVs) are a class of single-stranded RNA viruses with envelopes and are currently known to have the largest RNA genomes. They belong to the family Coronaviridae, genus Coronavirus, in the order Nidovirales. The virus can cause diseases in the respiratory, digestive, and nervous systems of animals and mainly infects humans and vertebrates.

[0028] Porcine epidemic diarrhea virus (PEDV) belongs to the genus Coronavirus and is the pathogen that causes porcine epidemic diarrhea. It can cause acute symptoms such as diarrhea, vomiting, and dehydration, and lead to an increased mortality rate of neonatal piglets.

[0029] Since 2010, the reappearance and mutation of PEDV have led to global outbreaks, having a significant economic impact on the global pig farming industry, especially in Asian countries such as China and South Korea. Unfortunately, so far, there is no specific drug for treating PEDV on the market, and clinical prevention mainly relies on vaccination. Given the high variability and strain diversity of PEDV, traditional attenuated vaccines are difficult to provide comprehensive protection. Due to the lack of effective drugs and vaccines, the mortality rate of piglets infected with PEDV is close to 100%, causing huge economic losses to the global pig farming industry.

[0030] The dual-targeting strategy can simultaneously block the virus self-replication and immune escape pathways, having significant antiviral advantages compared to single-target therapies, especially suitable for dealing with continuously mutating PEDV strains. To further develop dual-target drugs for PEDV, the present invention synthesized a series of bromophenol-benzamide compounds as dual-target inhibitors of PL pro enzyme and 3CL pro enzyme. Through antiviral activity screening, dual-target inhibitors of PL pro enzyme and 3CL pro enzyme with in vitro anti-PEDV activity were first discovered. We evaluated the antiviral efficacy and anti-inflammatory activity of compound 3 in vitro and studied its therapeutic effect on piglets infected with PEDV in vivo.

[0031] To enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0032] The test materials not specifically described in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0033] Among them: The structural formulas of Compounds 1-18 are as follows:

[0034]

[0035] Example 1: In vitro inhibition of porcine epidemic diarrhea virus (PEDV) PL pro enzyme and 3CL pro enzyme activity:

[0036] This example evaluated whether bromophenol-benzamide compounds could inhibit the activity of PEDV PL pro enzyme and 3CL pro enzyme in vitro.

[0037] Specific operation: Use a multi-channel pipette to add 100 μl of buffer (100 nM PL pro enzyme or 3CL pro enzyme, 2 μM substrate and 10 μM compound) to a 96-well black microplate. After mixing, incubate in the dark at 37 °C for 5 min. Use a multi-functional microplate reader to detect the change in fluorescence value within 30 min and calculate the inhibition rate. Set the compound concentration to 0-10 μM and determine the inhibitory effect of the compound on PL pro enzyme or 3CL pro enzyme at different concentrations, and calculate IC 50 .

[0038] Experimental results: As shown in Table 1, Compounds 1-18 at a concentration of 10 μM all showed strong inhibitory effects on PL pro enzyme or 3CL pro enzyme in vitro. Among them, Compounds 3, 6, 10, and 12 had the strongest inhibitory effects on PL pro enzyme or 3CL pro enzyme, and their IC 50 were all lower than 3 μM.

[0039] Table 1: Inhibitory activities of Compounds 1-18 on PL pro enzyme or 3CL pro enzyme (IC 50 )

[0040]

[0041] Example 2: Bromophenol-benzamide compounds interfere with the replication of PEDV in cells:

[0042] This example evaluated whether bromophenol-benzamide compounds could block virus replication in PEDV-infected Vero E6 cells.

[0043] Specific operation: The pre-seeded Vero E6 cells (5×10 4 cells / well) were pretreated with 10 μM of the test bromophenol-benzamide compound for 1 h, and then PEDV (MOI = 0.1) was added to infect for 2 h. Then, the virus-compound mixture was removed, and the cells were further cultured with fresh medium containing the bromophenol-benzamide compound. After 48 h, the cell lysate was collected, and qRT-PCR analysis was performed on the RNA in the lysate. Treatment without adding the bromophenol-benzamide compound was used as a control.

[0044] Experimental results: As shown in Table 2, in PEDV-infected Vero E6 cells, compounds 1-18 all showed strong antiviral effects, and among them, compound 3 had the strongest in vitro inhibitory activity against porcine coronavirus replication.

[0045] Table 2: Inhibitory activity of compounds 1-18 against PEDV replication (EC 50 )

[0046]

[0047] Example 3: Compound 3 inhibits the replication of PEDV in host cells IPEC-J2 in a dose-dependent manner:

[0048] This example evaluated whether compound 3 could block virus replication in PEDV-infected IPEC-J2 cells.

[0049] Specific operation: The pre-seeded Vero E6 cells (5×10 4Cells / well) were pretreated with 10 μM of the bromophenol-benzamide compound to be tested for 1 h, and then PEDV (MOI = 0.1) was added to infect them for 2 h. Then, the virus-compound mixture was removed, and the cells were further cultured with fresh medium containing the bromophenol-benzamide compound. GC376 was used as the positive control group. After 48 h, the cells were fixed with 4% paraformaldehyde for 15 min and washed 3 times. Then, they were incubated with 0.2% Triton X-100 in PBS (pH 7.2) for 10 min and blocked with PBS containing 5% BSA at room temperature for 1 h. Subsequently, the cells were incubated with the primary antibody against PEDV N protein diluted 1:1000 for 12 h. After thoroughly washing for 15 min to remove unbound antibodies, the cells were incubated with the FITC-labeled goat anti-rabbit IgG antibody diluted 1:500 for 1 h. The slides were washed for 15 min and stained with 4,6-diamidino-2-phenylindole (DAPI) to observe the cell nuclei. Finally, imaging was performed using a confocal fluorescence microscope.

[0050] Experimental results: As Figure 1 shown, in PEDV-infected IPEC-J2 cells, compound 3 could inhibit the replication of PEDV in a dose-dependent manner ( Figure 1 ).

[0051] Example 4: Compound 3 can inhibit cell inflammation induced by PEDV:

[0052] In this example, it was evaluated whether compound 3 could inhibit cell inflammation induced by PEDV in IPEC-J2 cells.

[0053] Specific operation: The pre-seeded IPEC-J2 cells (5×10 4 cells / well) were pretreated with 10 μM of the bromophenol-benzamide compound to be tested for 1 h, and then PEDV (MOI = 0.1) was added to infect them for 2 h. Then, the virus-compound mixture was removed. In the control group, 150 μL of medium was added, and in the experimental groups, medium and compound 3 were added respectively (low concentration group 1.25 μM, medium concentration group 2.5 μM, high concentration group 5 μM). Each group was set with 3 replicate wells and incubated for 48 h. After 48 h, the cell lysates of the blank group, experimental groups, and control group were taken, centrifuged, and the inflammatory factors IL-1β, TNF-α, and IL-6 were detected by ELISA. The experimental results are shown in Figure 2 .

[0054] Experimental results: As Figure 2 shown, in PEDV-infected IPEC-J2 cells, compound 3 could inhibit the inflammation induced by PEDV in a dose-dependent manner ( Figure 2 ).

[0055] Example 5: Clinical trial of compound 3 in treating piglets infected with porcine epidemic diarrhea virus

[0056] 1. PCR detection of virus infection in diseased pigs

[0057] PCR detection was performed on the tissues of diseased pigs in a Zhucheng pig farm in November 2022 to determine the type of virus they were infected with. The PCR results showed that PEDV was detected in the tissue samples of diseased pigs, while transmissible gastroenteritis virus and rotavirus were not detected.

[0058] 2. Clinical trial of compound 3 in treating pigs with epidemic diarrhea

[0059] Ten-day-old piglets with epidemic diarrhea were selected and randomly divided into three groups: a control group (untreated), an experimental group (treated with compound 3), and an antibody group (intervened with lgY antibody). All individuals maintained the breast-feeding mode, and the treatment effect was continuously observed.

[0060] Compound 3 group: Administered by gavage twice a day with a glucose solution (5%), single dose 25 mg / kg

[0061] lgY antibody group: Orally administered specific chicken egg antibodies at the same frequency, with the same dose as the compound 3 group

[0062] Control group: Received gavage treatment with an equal volume of glucose solution

[0063] 3. Clinical trial results:

[0064] (1) Clinical treatment results of pigs with epidemic diarrhea

[0065] The clinical treatment results of pigs with epidemic diarrhea are shown in Table 3. It can be seen from Table 3 that compound 3 has a significant effect on treating pigs with epidemic diarrhea, with a mortality rate much lower than that of the other two groups and an effective rate of 100%. In the untreated group, 10 pigs died, with a mortality rate of 90.9%; in the treatment group with compound 3, 0 pigs died, with a mortality rate of 0% and an effective rate of 100%; in the lgY antibody group, 6 pigs died, with a mortality rate of 75% and an effective rate of 25%.

[0066] The data in Table 3 show that compound 3 has significant treatment advantages for pigs with epidemic diarrhea: ① The survival rate is 100% (0 / 11), significantly better than the untreated group (survival rate 9.1%, 1 / 11) and the lgY antibody group (survival rate 25%, 2 / 8); ② The treatment effectiveness reaches 100%, while the effective rate of the lgY antibody group is only 25%. The experiment shows that the mortality control ability of compound 3 (0%) is more than 3 times higher than that of traditional antibody therapy (75%).

[0067] Table 3: Summary of clinical trial data for epidemic diarrhea

[0068]

[0069] Note 1: 6 + 5 in parentheses indicates that the experimental subjects are placed in two columns, with the number of piglets in each column. (8 + 8 + 8 + 6 + 8 + 6 + 6) indicates that the experimental subjects are placed in 7 columns, with the number of piglets in each column.

[0070] Note 2: The experiment was observed for 72 hours. The mortality rate was calculated based on the number of dead piglets at the end of the experiment, and the effective rate was calculated based on the diarrhea score index (Table 4).

[0071] (2) Clinical treatment diarrhea score of pigs with epidemic diarrhea

[0072] As shown in Table 4, there were significant differences in the clinical outcomes of pigs in different treatment groups: the diarrhea index of the pigs in Treatment Group 3 (n = 8) decreased by more than 50% 24 hours after oral administration, basically recovered 48 hours later, and achieved a 100% cure rate 72 hours later; the mortality rate of the lgY antibody group (n = 8) reached 75% within 72 hours, and only 2 cases had symptom relief; all the pigs in the untreated group (n = 6) died within 48 hours. The data indicate that Compound 3 demonstrated significant therapeutic advantages by rapidly relieving diarrhea symptoms (taking effect in 24h) and achieving a complete cure (in 72h).

[0073] Table 4: Diarrhea score index

[0074]

[0075]

[0076] Note: (Formed feces, 0; Thick feces, unformed 1; Fecal water separation, 2; Watery feces, 3. A score of 0 or 1 is considered normal; a score of 2 or 3 is considered diarrhea.)

[0077] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Use of bromophenol-benzamide compounds in the preparation of drugs for treating porcine epidemic diarrhea; the bromophenol-benzamide compounds have the general structural formula shown in the following formula I: in: R1 and R2 are selected from OH and OCH3 respectively; R3 is selected from Any of; The porcine epidemic diarrhea disease is a disease caused by porcine epidemic diarrhea virus (PEDV).

2. The use according to claim 1, characterized in that: The bromophenol-benzamide compound is PEDV PL pro and PEDV 3CL pro Dual-target inhibitors. This class of compounds simultaneously inhibits PEDV PL pro and PEDV 3CL pro activity, thereby inhibiting the replication of PEDV.

3. The use according to claim 1, characterized in that: The porcine epidemic diarrhea disease includes: porcine gastroenteritis and / or diarrhea.

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