阅读说明：本技术 溴酚-吡唑啉化合物在治疗猫冠状病毒疾病中的应用 (Application of bromophenol-pyrazoline compounds in treatment of feline coronavirus diseases ) 是由 史大永 李祥乾 王鑫 许凤 于 2021-10-13 设计创作，主要内容包括：本发明属于医药技术领域,具体涉及溴酚-吡唑啉类化合物在治疗猫冠状病毒疾病中的应用。本发明研究发现溴酚-吡唑啉类化合物能够抑制抑制猫传染性腹膜炎病毒主蛋白酶(FIPV Mpro)的活性,干扰细胞中猫传染性腹膜炎病毒(FIPV)的复制。在临床实验中,溴酚-吡唑啉类化合物能够治疗FIPV自然感染的猫传染性腹膜炎,极大的提高病猫的存活率,可用于制备治疗猫传染性腹膜炎的药物。(The invention belongs to the technical field of medicines, and particularly relates to an application of bromophenol-pyrazoline compounds in treatment of feline coronavirus diseases. The research of the invention finds that the bromophenol-pyrazoline compound can inhibit the activity of main protease (FIPV Mpro) of the feline infectious peritonitis virus and interfere the replication of the Feline Infectious Peritonitis Virus (FIPV) in cells. In clinical experiments, the bromophenol-pyrazoline compounds can treat feline infectious peritonitis naturally infected by FIPV, greatly improve the survival rate of sick cats, and can be used for preparing medicaments for treating feline infectious peritonitis.)

1. The application of bromophenol-pyrazoline compounds in preparing medicaments for preventing/treating feline coronavirus diseases; the bromophenol-pyrazoline compound has a general structural formula shown as the following formula I:

wherein: r₁、R₂、R₃、R₄、R₅Are respectively selected from H, F, Br, OH and OCH₃Any one of (a);

R₆selected from H, CHO, COCH₃、COCH₂CH₃、COCH₂CH₂CH₃、COOCH₃、COOCH₂CH₃、Ph、CH₂Ph、CONH₂、CSNH₂OrAny one of (a);

R₇、R₈、R₉are respectively selected from H, F, Cl, Br and NO₂、OH、CH₃、OCH₃OrAny one of (a);

the above-mentionedR in (1)₁₀、R₁₁、R₁₂Are respectively provided withSelected from H, F, Cl, Br, NO₂、OH、CH₃、C₂H₅、OCH₃、OC₂H₅And isopropyl, isobutyl, tert-butyl, and tert-pentyl.

2. The use according to claim 1, wherein the feline coronavirus disease is feline infectious peritonitis caused by Feline Infectious Peritonitis Virus (FIPV).

Technical Field

The invention belongs to the technical field of medicines, and relates to a new application of bromophenol-pyrazoline compounds, in particular to an application of bromophenol-pyrazoline compounds in prevention/treatment of feline coronavirus diseases.

Background

Feline Coronavirus (FCV) belongs to the order Nidovirales of the family Coronaviridae, and is an unfragmented single-stranded positive-strand RNA virus with an envelope. Feline Enterocoronavirus (FECV), the most common FCV, is ubiquitous in cats throughout the world and can be transmitted through the fecal orifice, but is not itself a serious pathogen. Approximately 5% of felines with persistent FECV infection produce the highly lethal mutant Feline Infectious Peritonitis Virus (FIPV). FECV loses the ability to infect healthy cats after mutation to FIPV, but pathogenicity and lethality are greatly increased.

FIPV is capable of sustained replication in mononuclear macrophages and is spread throughout the body as mononuclear macrophages circulate in the body, causing an over-stimulation of the host cat's immune system, resulting in morbidity and mortality. Common sites of disease for infectious peritonitis (FIP) are serosa, kidney, mesenteric lymph nodes, brain, eye, liver, spleen and lung, and clinical symptoms include ascites, dyspnea, uveitis, ataxia, refractory fever, lethargy, anorexia, jaundice and weight loss.

FIP is considered as an 'incurable disease' in the kingdom of cats, and if the FIP is not treated in time, the sick cats will die about two months generally, and the fatality rate is close to 100%. In the united states and some european countries there is an FCoV vaccine which is administered intranasally against temperature sensitive mutant strains of FCoV, but the efficacy of which is controversial. Because of the antibody-dependent enhancement observed during the experiment, more FIP-affected cats were observed after vaccination than in the control group.

For FIP, there is currently no simple, definitive treatment. The supporting therapy of symptomatic medication is adopted for treatment, and the treatment idea is to help the cat body to overcome the disease autonomously by inhibiting virus replication, inhibiting inflammatory reaction, stimulating the immune system and reducing the risk of re-infection. A developing nucleoside analog (GS-441524) was found to be useful in the treatment of naturally infected FIP by directly interfering with the viral encoded replication process. After 4 weeks of continuous treatment with 4mg/kg GS-441524, about 60% of FIP cats were relieved of the disease. Although GS-441524 can be used for treating FIP, the price is high (1.32 ten thousand per month), the cure rate is not high, the diseases are easy to relapse, and the development of an oral and efficient new targeted FIP medicament is of great significance.

The bromophenol-pyrazoline compounds are compounds which are designed by earlier research and development of the inventor and have the activity of inhibiting novel human coronavirus, and have wide application prospects in preparation of medicaments for treating human coronavirus pneumonia. Subsequent researches find that the Porcine Epidemic Diarrhea Virus (PEDV) and the porcine transmissible gastroenteritis virus (TGEV) also have a therapeutic effect. Therefore, the bromophenol-pyrazoline compounds are compounds with great medicinal potential, and further discover new medicinal effects of the bromophenol-pyrazoline compounds, and particularly have very important significance in preventing/treating feline coronavirus diseases.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a novel medical application of bromophenol-pyrazoline compounds.

The research of the invention finds that the bromophenol-pyrazoline compound can inhibit the replication of Feline Infectious Peritonitis Virus (FIPV); in clinical tests, the traditional Chinese medicine composition can treat feline infectious peritonitis, greatly improves the survival rate of sick cats, and can be used for preparing medicines for treating feline coronavirus diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of bromophenol-pyrazoline compounds in preparation of drugs for treating feline coronavirus diseases.

In the application, the bromophenol-pyrazoline compounds have a general structural formula shown as the following formula I:

wherein: r₁、R₂、R₃、R₄、R₅Are respectively selected from H, F, Br, OH and OCH₃Any one of (a);

R₆selected from H, CHO, COCH₃、COCH₂CH₃、COCH₂CH₂CH₃、COOCH₃、COOCH₂CH₃、Ph、CH₂Ph、CONH₂、CSNH₂OrAny one of (a);

R₇、R₈、R₉are respectively selected from H, F, Cl, Br and NO₂、OH、CH₃、OCH₃OrAny one of (a);

the above-mentionedR in (1)₁₀、R₁₁、R₁₂Are respectively selected from H, F, Cl, Br and NO₂、OH、CH₃、C₂H₅、OCH₃、OC₂H₅And isopropyl, isobutyl, tert-butyl, and tert-pentyl.

In the above use, the feline coronavirus disease is Feline Infectious Peritonitis (FIP) caused by Feline Infectious Peritonitis Virus (FIPV).

The invention has the beneficial effects that:

the invention discovers for the first time that bromophenol-pyrazoline compounds can inhibit the replication of Feline Infectious Peritonitis Virus (FIPV); clinical tests prove that the bromophenol-pyrazoline compounds can treat Feline Infectious Peritonitis (FIP) caused by FIPV and the like, greatly improve the survival rate of sick cats, and can be used for preparing medicaments for treating FIP.

Drawings

FIG. 1 shows the results of the present invention for compounds 1-8 on FIPV M^proThe result of the measurement of inhibitory activity of (3). Determination of different concentrations of Compound vs FIPVM^proEffect of Activity, half maximal Inhibitory Concentration (IC) determined by non-Linear regression₅₀) A value;

FIG. 2 is a graph showing the results of assays for inhibition of FIPV replication in cells by compounds 1-8 provided in the examples of the present invention, and the effect of different concentrations of compounds on viral RNA replication was evaluated.

Detailed Description

In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. The preferred embodiments of the present invention are given in the detailed description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, Feline Infectious Peritonitis Virus (FIPV) causes feline infectious peritonitis with a very high mortality rate, but no effective therapeutic agent against feline infectious peritonitis is currently available.

Aiming at a new coronavirus pneumonia epidemic situation outbreak at the end of 2019, the inventor develops a bromophenol-pyrazoline compound capable of efficiently inhibiting a novel coronavirus (COVID-19), and the bromophenol-pyrazoline compound has the effect of treating coronavirus pneumonia (see Chinese patent publication No. CN111848516A specifically). Subsequent researches find that the bromophenol-pyrazoline compounds can treat porcine epidemic diarrhea and porcine transmissible gastroenteritis virus caused by porcine coronavirus PEDV and TGEV (see Chinese patent publication No. CN112472698A for details).

Although FIPV belongs to coronaviruses with COVID-19, PEDV and TGEV, it has a large difference in host, virus properties, infected cell types, diseased tissues, onset symptoms, etc.:

(1) the host is different. FIPV has low homology with COVID-19, PEDV and TGEV, has a distant relationship, has a greatly different virus structure and does not have cross-species infectivity with each other. The phenomenon of cross-species infection of human coronavirus, porcine coronavirus and feline coronavirus is not found at present;

(2) the virus characteristics are different. FIPV is a variant of Feline Enteric Coronavirus (FECV), and FIPV in sick cats is not infectious. Different from the situation, COVID-19, PEDV and TGEV have stronger infectivity;

(3) infected cells vary in kind. FIPV infects monocytes in cats, causing an overactive systemic immune system. COVID-19 infects lung cells of human, PEDV and TGEV infect intestinal epithelial cells of pig;

(4) the diseased tissue is different. FIPV can cause lesions in various tissues and organs of cats, such as serosa, kidney, mesenteric lymph nodes, brain, eyes, liver, spleen, and lung. COVID-19 induces pneumonia in humans, PEDV and TGEV induce enteritis;

(5) the onset symptoms are different. FIPV is associated with a number of disease symptoms, including ascites, dyspnea, uveitis, ataxia, refractory fever, lethargy, anorexia, jaundice, and weight loss. COVID-19 symptoms include fever, cough, chest distress, and dyspnea. The symptom of PEDV and TGEV is diarrhea.

Therefore, feline coronavirus FIPV and human coronavirus covv-19, porcine coronavirus PEDV and TGEV belong to different types of viruses, and it is difficult to predict the effect and effect of the feline coronavirus FIPV on the novel human coronavirus and the porcine coronavirus.

In order to further expand the medicinal field of the bromophenol-pyrazoline compounds, the invention carries out a great deal of attempts, and results show that the bromophenol-pyrazoline compounds have better inhibitory activity to feline coronavirus and can inhibit the replication of FIPV; clinical tests prove that the bromophenol-pyrazoline compounds can treat feline infectious peritonitis caused by FIPV, and the survival rate of sick cats is greatly improved, so that the invention is provided.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available.

Wherein: the structural formulae of exemplary compounds 1-8 are shown below:

the synthesis of compounds 1-8 is described in the inventor's patent application CN 111848516A.

Example 1: bromophenol-pyrazoline compound for main protease FIPVM of feline infectious peritonitis^proDetermination of inhibitory Activity

(1)FIPVM^proExpression purification of

Mixing FIPVM^proThe gene is inserted into the P GEX-6P-1 vector by the conventional technical means of PCR, enzyme digestion, connection and the like. The constructed vector was transformed into E.coli BL21(DE3) competent cells, and cultured in LB containing 100mg/mL of ampicillin at 37 ℃ until the OD600 of the cells reached 0.6. Protein expression was induced at 16 ℃ for 16-18 hours with 0.5mM IPTG. The collected cells were suspended in a cell suspension buffer (140mM NaCl, 10mM Na)₂HPO₄，2.7mM KCl，1.8mM KH₂PO₄1mM DTT, 10% glycerol, pH 7.3) and disrupting the cells at 4 ℃ under high pressure. Centrifuging to remove thallus debris, and enriching GST and FIPV M by GST affinity medium^proThe fusion protein of (1), PPase excision FIPV M^proGST tag at N-terminus. Cut off tagged FIPV M^proFurther purifying with Hi Trap Q (GE) and Superdex7510/300(GE) to obtain FIPVM with purity higher than 95%^pro。

(2)FIPVM^proEnzyme activity measuring method

FIPV M^proThe activity of (2) was determined using 50mM Tris-HCl pH 7.3 buffer, FIPV M^proThe concentration of (A) is 1. mu.M, the fluorogenic substrate is MCA-AVLQSGFR-Lys (Dnp) -Lys NH2 (coronavirus main protease fluorogenic substrate), the concentration is 40. mu.M, the excitation wavelength is 320nm, and the detection wavelength is 405 nm. The concentration gradient of the compounds 1-8 was 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1. mu.M, 0.5. mu.M. The inhibitory effect of compounds 1-8 on FIPV major protease was determined separately.

(3) Compound pair FIPVM^proResults of measurement of inhibitory Activity

As shown in figure 1, bromophenol-pyrazoline compounds (1-8) are shown to be applied to FIPVM^proThe protease shows obvious inhibition effect and has good application prospect in treating feline infectious peritonitis.

Example 2: bromophenol-pyrazoline compounds interfere with FIPV virus replication in cells

To further confirm the in vitro enzymatic inhibition results, the present invention evaluated whether bromophenol-pyrazolines could prevent FIPV virus replication in clinical wild-type FIPV infected feline kidney Cells (CRFK) cells.

The method comprises the following specific operations:

at 37 ℃ with 5% CO₂In the cell culture chamber, CRFK cells were cultured with DMEM containing 8% fetal bovine serum. When the cells grow to the density of about 80%, pretreating with 5 mu M bromophenol-pyrazoline compounds to be detected for 1h, and then inoculating CRFK cells to a FIPV wild strain (MOI of 0.1). CRFK cells inoculated without compound were also set up as controls. After 24h, cell supernatants were collected and qRT-PCR analysis was performed on vRNA in the supernatants.

The experimental results are as follows:

as shown in FIG. 2, compounds 1-8 at 5. mu.M concentration all showed strong ability to interfere with FIPV virus replication in wild-type FIPV-infected CRFK cells, with compound 5 having the strongest ability to inhibit FIPV virus replication.

Example 3: clinical trial of Compound 5 for treatment of feline infectious peritonitis

(1) Detection of infectious peritonitis disease cats

Serology or morphology cannot distinguish between feline enterocoronavirus and feline infectious peritonitis virus. According to the methods of disease history, epidemiology, clinical symptoms, laboratory diagnosis, imaging examination, histopathological examination and the like, the sick cat is comprehensively diagnosed after other diseases are eliminated. Feline infectious peritonitis virus infection is identified when the sick cat gradually loses weight, has anorexia, increases body temperature, has mild non-regenerative anemia in blood, has lymphopenia, increases serum globulin concentration and has a significantly reduced white globulin ratio relative to normal cats.

(2) Clinical trial grouping and treatment protocol

30 FIPV-infected cats were classified into wet FIP group (17) and dry FIP group (13) depending on whether there were significant symptoms of pleural, peritoneal fluid, dyspnea, etc. (fibropleurisy and peritonitis). The wet FIP group (17) was randomly divided into a control group (3), a GS-441524 group (6), and a Compound 5 group (8). The dry FIP group (13) was randomly divided into a control group (2), a GS-441524 group (5), and a Compound 5 group (6).

As shown in Table 1, all FIPV infected cats were treated according to their symptoms. Wet and dry GS-441524 groups: 4.0mg/kg of the drug was administered by subcutaneous injection once every 24 hours; wet and dry compound 5 group: 3mg/kg, 1 time a day, the administration mode is glucose solution.

Table 1: experimental grouping and treatment scheme for FIPV infected cats

Rectal temperature was measured at least once daily while observing body weight, respiration, appetite and activity. Arranging a specially-assigned person to feed, and cleaning and sterilizing the edible utensils every day. Feeding the appetizing cats with conventional cat food, and matching with high-protein pet cans as nutrition supplements; for cats with poor appetite, the nutrition is supplemented by adopting interventional nutrition supplementing modes such as nasal feeding and intravenous injection, so as to ensure the nutrition intake of the test cats. The amount and shape of the defecation and urination were also observed. And performing blood routine index, serum biochemical index and abdominal B-ultrasonic examination according to actual conditions.

(3) Results of clinical trials

The effective case is defined as the case with good physical conditions such as appetite, weight, respiration, body temperature and the like, 2 times (more than 1 week interval) of routine blood and serum biochemical detection indexes when 4 weeks of treatment are finished.

The clinical trial results are shown in table 2, and after 1 day of treatment, the compound 5 treatment group started eating normal cat food and gradually decreased body temperature. After 7 days of treatment, body temperature returned to normal and body weight increased. Ascites, dyspnea, jaundice, lymph nodes and other symptoms disappear. Some cats began to have significant regression of ocular lesions within 48 hours and the aqueous humor became clear. Compared with the positive medicine GS-441524 group, the compound 5 treatment group has faster effect, less death and higher effective rate. The above results demonstrate that compound 5 is able to treat FIPV infected feline infectious peritonitis.

Table 2: results of clinical trials on FIPV-infected cats

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.