阅读说明：本技术 溴酚-吡唑啉化合物在治疗禽冠状病毒疾病中的应用 (Application of bromophenol-pyrazoline compound in treating avian 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禽类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鸡传染性支气管炎病毒(IBV)和鸭冠状病毒(DCV)的复制。在攻毒实验和临床实验中,溴酚-吡唑啉类化合物能够治疗鸡传染性支气管炎和鸭冠状病毒性肠炎,极大的提高病禽的存活率,可用于制备治疗鸡传染性支气管炎和鸭冠状病毒性肠炎等禽类冠状病毒疾病的药物。(The invention belongs to the technical field of medicines, and particularly relates to an application of bromophenol-pyrazoline compounds in treatment of avian coronavirus diseases. The research of the invention finds that the bromophenol-pyrazoline compounds can inhibit the replication of Infectious Bronchitis Virus (IBV) and Duck Coronavirus (DCV). In toxicity attacking experiments and clinical experiments, the bromophenol-pyrazoline compounds can treat infectious bronchitis of chickens and coronavirus enteritis of ducks, greatly improve the survival rate of sick birds, and can be used for preparing medicines for treating avian coronavirus diseases such as the infectious bronchitis of chickens and the coronavirus enteritis of ducks.)

1. The application of bromophenol-pyrazoline compounds in preparing medicine for treating avian 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 selected from H, F, Cl, Br and NO₂、OH、CH₃、C₂H₅、OCH₃、OC₂H₅And isopropyl, isobutyl, tert-butyl, and tert-pentyl.

2. The use according to claim 1, wherein the avian coronavirus disease is infectious bronchitis of chicken and coronavirus enteritis of duck caused by Infectious Bronchitis Virus (IBV), coronavirus of Duck (DCV).

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 treatment of coronavirus diseases of chickens and ducks.

Background

Infectious Bronchitis (IB) is an acute, highly contagious respiratory disease that occurs only in chickens, caused by Infectious Bronchitis Virus (IBV). The disease was first discovered in 1930 in Dakota continent of the United states, and the disease was reported in 50 s of 20 th century in China. Clinically, it usually shows stretching neck, breathing, cough, sneezing, Ronchi of trachea and mouth, sticky nasal fluid, and more tears with a death rate of 20-80%. IBV is mainly transmitted through air, has high infectivity and is often attacked by the diseases of the whole chicken house at the same time. The latent period of the disease is very short, and clinical symptoms can be shown within 36-48 hours after infection generally. Mainly affecting the respiratory system and the urogenital system of the chickens, and causing respiratory tract inflammation, kidney injury, egg production and egg quality reduction of the chickens. IB is one of important epidemic diseases which affect the rapid development of poultry industry in China, and causes great economic loss to the poultry industry. At present, no specific therapeutic drug for IB is available, and vaccination with live attenuated vaccine or inactivated vaccine is reported to be used for prevention. However, because of the need of multiple vaccinations and the existence of numerous variant serotypes of IBV, the prevention effect is not obvious, and the wide use of the vaccine is limited.

Duck coronavirus enteritis is an acute infectious disease caused by infection of Duck Coronavirus (DCV). The clinical manifestations mainly include diarrhea, acute onset and rapid transmission. The incidence of the ducks before and after 20 days of age is high, the ducks start to be attacked by a few ducks, the death peak appears after 1-2 days, and the incidence and the death rate reach 80-100%. The diseased chick shrinks head, encircles back, is aversive to cold, has half-closed eyes and has mucous secretion. The excrement begins to be excreted, then the severe diarrhea is caused, the color of the white or yellow-green beak skin changes from yellow to purple after the disease occurs, and the beak skin peels off and breaks. Some of them show neurological symptoms, such as kicking, straightening or bending of the head backward with both feet in a star-like shape, and can promote death by slight expulsion or stress. So far, no specific treatment medicine for treating duck coronavirus enteritis mainly depends on active immunization of vaccines, but the prevention and treatment effects are not obvious.

The bromophenol-pyrazoline compounds are compounds which are designed by earlier research and development of the inventor and have the function of interfering the replication of 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 the treatment of the avian coronavirus diseases has very important significance.

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 compounds can inhibit the replication of avian coronavirus (such as IBV, DCV and the like); in clinical tests, the compound preparation can treat the avian coronavirus diseases (such as infectious bronchitis of chicken, duck coronavirus enteritis and the like), greatly improve the survival rate of infected young birds, and can be used for preparing the medicines for treating the avian coronavirus diseases.

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

the bromophenol-pyrazoline compounds provided by the invention can be used for preparing medicines for treating avian coronavirus diseases such as infectious bronchitis of chicken and enteritis of duck coronavirus.

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.

The invention has the beneficial effects that:

the invention discovers for the first time that bromophenol-pyrazoline compounds can inhibit the replication of avian coronavirus IBV and DCV; clinical tests prove that the bromophenol-pyrazoline compounds can treat avian coronavirus diseases caused by IBV, DCV and the like, and the survival rate of infected young birds is greatly improved.

Detailed Description

In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to 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, avian coronavirus diseases cause serious economic losses to the chicken and duck breeding industry, but no effective therapeutic drugs for avian coronavirus diseases exist at present.

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).

Coronaviruses are divided into 4 genera of coronaviruses, alpha, beta, gamma and delta coronaviruses, respectively. Although coronaviruses are widely diverse and exist, they generally have the highest affinity for receptors in their natural hosts and have a pronounced species specificity for infected hosts. Porcine Epidemic Diarrhea Virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV) belong to the genus alphacoronavirus. Human coronavirus SARS-COV, novel coronavirus (SARS-COV2) and middle east respiratory syndrome virus (MERS) belong to the genus beta coronavirus. The invention relates to Avian Infectious Bronchitis Virus (IBV) and Duck Coronavirus (DCV), which belong to Avian coronavirus (Avian coronavirus) of gamma coronavirus. Some strains of mammalian coronaviruses have serological cross-reactivity with each other, but are quite different from avian coronaviruses. At present, the phenomenon of qua-species infection of human coronavirus, pig coronavirus and poultry coronavirus is not found.

The avian coronavirus IBV and DCV, the porcine coronavirus and the human coronavirus belong to different genera of coronavirus, and have low homology, distant relationship, greatly different virus structures and no cross-species infectivity. Therefore, avian coronavirus IBV and DCV are different types of viruses from human coronavirus and porcine coronavirus, and it is difficult to predict the effect of the substances capable of inhibiting the human novel coronavirus and porcine coronavirus on the avian 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 can inhibit the replication of avian coronavirus IBV and DCV; clinical tests prove that the bromophenol-pyrazoline compounds can treat infectious bronchitis of chicken and coronavirus enteritis of ducks caused by IBV, DCV and the like, and the survival rate of sick poultries 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-pyrazolines interfere with replication of Infectious Bronchitis Virus (IBV) in chicken embryos

This example evaluates whether bromophenol-pyrazolines prevent IBV replication in chicken embryos infected with clinical isolate IBV.

The method comprises the following specific operations:

the 110 chick embryos are randomly divided into 11 groups, namely a normal control group, a toxicity counteracting control group, a traditional Chinese medicine administration group and a compound 1-8 administration toxicity counteracting group, wherein each group comprises 10 chick embryos. The normal control group was inoculated with 0.1mL of sterilized physiological saline per allantoic cavity. Control group for counteracting toxic substance, Chinese medicinal administration group, and compound 1-8 administration group, first 10⁵EID₅₀0.1mL, and then 0.25mg/0.1mL of compound diluted with sterilized normal saline is administered according to 0.1 mL/one sterilized normal saline, 0.1 mL/one Chinese medicinal original solution diluted with sterilized normal saline, and 0.25mg/0.1 mL/one compound diluted with sterilized normal saline. Wherein the Chinese medicinal liquid is prepared from flos Lonicerae, fructus forsythiae, folium Isatidis, radix Isatidis, semen Armeniacae amarum, Bulbus Fritillariae Thunbergii, Scutellariae radix, etc.

After counteracting toxic substance, the eggs are irradiated for 2 times every day, and the observation is continuously carried out for 8 days. Recording and observing the death number and death time of each group of chick embryos; and observing whether the dead chick embryos have characteristic changes such as growth obstruction, embryo body atrophy (in a pellet shape), amnion thickening, yolk sac shrinkage, allantoic fluid increase and the like. And (5) counting the number of dead embryos and the number of the embryos with development resistance of each group after the observation period is finished, and calculating the normal protection rate of the embryos.

The experimental results are as follows:

after all chick embryos are injected and dosed through an allantoic cavity and inoculated with virus allantoic fluid, the rest chick embryos survive until the observation period is finished for 8 days, except that a part of chick embryos die within 24 hours in a part of groups. The effective embryo number, the development obstruction condition, the obstruction embryo number and the development normal protection rate of each group of embryos are observed and counted, and the details are shown in table 1. All embryos survived and developed normally in the normal control group. All the surviving embryos of the control toxicity attacking group have lesions of different degrees of stunted development, curling, embryo body atrophy, amnion thickening, yolk sac shrinking, epidermal edema and the like. The embryo development obstruction number of the traditional Chinese medicine administration group is reduced, and the protection rate is 44%. The normal development protection rate of 8 administration groups is higher than 50%, and the pathological change degree of the development-hindered embryo is light. The results show that the bromophenol-pyrazoline compounds can inhibit the replication of IBV and have strong antitoxic effect, wherein the compound 7 has the strongest IBV replication inhibition activity.

TABLE 1 Effect of bromophenol-pyrazoline on the development of IBV-infected chick embryos

Example 2: challenge test for Compound 7 in treating Infectious Bronchitis Virus (IBV) infected chickens

This example evaluates whether compound 7 can treat infected chickens with IBV in an IBV challenge experiment.

(1) Determination of challenge dose

15 chickens of 20 days old were randomly divided into 3 groups, and IBV was added dropwise to the eyes of the nose at a concentration of 0.1, 0.2, or 0.3 ml. The drug is attacked for 48 hours, 2 patients are attacked in the 0.1ml group, and all the patients are attacked in the 0.2ml group and the 0.3ml group, and the drug attacking dose is determined to be 0.2 ml/patient.

(2) Operation of counteracting toxic substances

150 chickens at 20 days of age were randomly divided into 5 groups of 30 chickens. Group 1 was a blank control group: no toxic action and no medication; group 2 was the challenge control group: only counteract toxic pathogen, not take medicine; group 3 was a low dose group, administered at a dose of 1 mg/tube; group 4 is a medium dose group, and the dose is 2 mg/dose; group 5 was a high dose group, and 4 mg/tube was administered.

Each chicken is added with 0.2ml of the mixed solution of the bacteria and the toxin through the nose. The test chicken starts to take the drug after 48 hours of toxin counteracting, the drug taking mode is drenching, 2 times a day, and the drug taking lasts for 3 days. And continuously observing for 10 days, recording symptoms and death conditions, performing autopsy on dead chickens, and calculating the cure rate, morbidity and mortality at the end of the test.

(3) Test results

After 2 days of toxicity attack, the control group of toxicity attack started to develop diseases, which are manifested by lassitude, hair disorder, slow standing, thin feces, mouth-opening respiration, accompanied by rales, sneezing, lacrimation and other symptoms, and death occurs after 4 days. The cesarean examination of dead chickens can lead to the swelling of the face of the sick chickens, the mucous secretion in nasal cavities and the inflammation and edema of eye and conjunctiva; the trachea has mucus exudation and the air sac is turbid. The morbidity of the experimental drug group is obviously lower than that of the challenge control group, and the test result is shown in table 2.

The test result shows that: the high-dose group does not cause death of sick chickens, and the cure rate is 100 percent; 1 death in the medium-dose group, and the cure rate is 95 percent; the low dose group died 2, with a cure rate of 90%. The mortality rate of 3 administration groups is lower than that of a control group by more than 30 percent, which indicates that the bromophenol-pyrazoline compound can be used for treating respiratory diseases caused by IBV.

TABLE 2 challenge test of Compound 7 in the treatment of IBV infected chickens

Example 3: clinical trial of Compound 7 in treating Infectious Bronchitis Virus (IBV) naturally infected chickens

This example evaluates whether Compound 7 can treat sick chickens naturally infected with IBV in a clinical trial.

(1) Clinical symptoms of sick chicken

The sick chicken have the disadvantages of lassitude, disordered hair, stiff standing, thin excrement, cough, asthma, tracheal rale, nasal mucus, moist eyes and reduced ingestion. Nasal tract and tracheal mucosa congestion, bronchiectasis, serous exudate, kidney enlargement and pallor can be seen in pathological autopsy. Collecting intratracheal mucus, treating, inoculating to chick embryo allantoic cavity, culturing for 2 days, collecting allantoic fluid, and detecting with chicken infectious bronchitis antibody ELISA detection kit to prove IBV infection.

(2) Clinical trial operations

IBV infected chickens within 30 days of age were selected and divided into 3 groups, which were respectively untreated group, Chinese medicinal treatment group, and compound 7 treatment group. The Chinese medicinal treatment group is prepared from herba Taraxaci, herba Violae, Coptidis rhizoma, and radix Isatidis by decocting and pouring in water for 2 times per day for 3 days. Compound 7 was administered at a dose of 2.5 mg/dose 2 times a day in a form of glucose solution. The glucose solution with the same dosage as that of the compound 7 treatment group is administered by drenching in an untreated group and a traditional Chinese medicine treatment group. During the treatment period, the feed is normally fed.

(3) The clinical test results are as follows:

the results of the clinical treatment of the infectious bronchitis chickens are shown in table 3, and it can be seen that: the compound 7 has obvious effect on treating the infectious bronchitis chickens, and the death rate is far lower than that of other 2 groups. Untreated group, 16 deaths, mortality 57.1%; 7 deaths of the Chinese medicine treatment group are achieved, and the death rate is 43.8%; compound 7 treatment group, 5 deaths, mortality rate 3.7%. After ten days, the test chicken recovers the normal spirit and appetite, the rale disappears, the cough disappears, the weight rises, and no lesion is dissected, so that the test chicken is judged to be effective. The effective rate of the compound 7 treatment group is 94.8%, which indicates that the compound 7 can effectively treat infectious bronchitis chicken naturally infected by IBV.

Table 3: summary of clinical treatment data of chicken with infectious bronchitis

Note 1: the test is observed for 10 days, and the death rate is calculated according to the death number of the sick chickens at the test end point.

Example 4: duck Coronavirus (DCV) infection treatment challenge test of compound 7 for ducks

This example evaluates whether compound 7 can treat diseased ducks infected with DCV in a DCV challenge experiment.

(1) Determination of challenge dose

15 ducks of 20 days old are taken, randomly divided into 3 groups, and 0.5ml, 1.0ml and 1.5ml of DCV are respectively irrigated. The drug is attacked for 48 hours, 4 patients are attacked in the 0.5ml group, and all the patients are attacked in the 1.0ml group and the 1.5ml group, and the drug attacking dose is determined to be 1.0 ml/patient.

(2) Operation of counteracting toxic substances

150 ducks aged 20 days are randomly divided into 5 groups, and each group contains 30 ducks. Group 1 was a blank control group: no toxic action and no medication; group 2 was the challenge control group: only counteract toxic pathogen, not take medicine; group 3 was a low dose group, administered at a dose of 1 mg/tube; group 4 is a medium dose group, and the dose is 2 mg/dose; group 5 was a high dose group, and 4 mg/tube was administered.

The mixed solution of the bacteria and the toxin is 1.0ml for each drench. The test duck starts to take the drug after 24 hours of toxin counteracting, the drug administration mode is drenching, 2 times a day, and the drug administration lasts for 3 days. Continuously observing for 10 days, recording symptoms and death conditions, carrying out autopsy on dead ducks, and calculating the cure rate, the morbidity and the mortality after the experiment is finished.

(3) Test results

After 1 day of toxin counteracting, the duck in the control group begins to get ill, and the symptoms of mental retardation, appetite reduction, neck, bow and waist necking, cold intolerance are shown; diarrhea, yellow-green or white watery stool, containing mucus. Death occurred after 2 days, the mouth shell changed from light yellow to light purple before death, and the epithelium of the mouth shell fell off. The diseased duck is necropsied, so that the duodenum of the diseased duck is congested and is red, and blood mucus exists in the diseased duck; the intestinal mucosa is sloughed off, the intestinal wall is ulcerated, and the rectum and cloaca are congested and edematous. The morbidity of the experimental drug group is obviously lower than that of the challenge control group, and the test result is shown in table 4.

The test result shows that: 1 death in the high-dose group, and the cure rate is 96.7 percent; 3 deaths in the medium-dose group, and the cure rate is 90%; the low dose group died 5, and the cure rate was 83.3%. The mortality rate of 3 administration groups is more than 70 percent lower than that of a control group, which indicates that the bromophenol-pyrazoline compound can be used for treating duck coronavirus diarrhea caused by DCV.

TABLE 4 Duck challenge test for treatment of DCV infection with Compound 7

Example 5: clinical trial of Compound 7 for treating Duck Coronavirus (DCV) infected Duck

This example evaluates whether compound 7 can treat diseased ducks naturally infected with DCV in a clinical trial.

(1) Clinical symptoms of sick duck

The sick ducks aged 20 days are in mental retardation, reduced appetite, reduced activity, reduced body temperature, neck and waist necking, intolerance of cold and sticky eye secretion; diarrhea, yellow-green or white watery dilute manure, containing mucus; continuously whistling and stacking; the shell of the mouth before the death of the duck is changed from light yellow to light purple, and the epithelium of the mouth shell falls off and is broken. The pathological changes of intestinal tracts of diseased ducks are obvious through dissection, the pathological changes of duodenum are most obvious, mesenteric vessels expand, are congested and bleed, the appearance of the whole duodenum is red, purple or purplish red, and blood mucus is filled in the duodenum; the intestinal mucosa is sloughed off, the intestinal wall is ulcerated, and the rectum and cloaca are congested and edematous. Collecting diseased duck tissues, homogenizing, taking supernatant, and detecting by ELISA to prove that the duck is infected by DCV.

(2) Mode of administration

90 DCV infected ducks within 20 days of age are selected and randomly divided into 3 groups, namely an untreated group, a traditional Chinese medicine treatment group and a compound 7 treatment group. The Chinese medicinal treatment group is prepared from herba Taraxaci, herba Violae, Coptidis rhizoma, and radix Isatidis by decocting and pouring in water for 2 times per day for 3 days. Compound 7 was administered at a dose of 2.5 mg/dose 2 times a day in a form of glucose solution. The glucose solution with the same dosage as that of the compound 7 treatment group is administered by drenching in an untreated group and a traditional Chinese medicine treatment group. During the treatment period, the feed is normally fed.

(3) The clinical test results are as follows:

the clinical treatment results of DCV infected ducks are shown in Table 5, the effect of the compound 7 on treating ducks suffering from coronavirus enteritis is obvious, the death rate is far lower than that of other 2 groups, and the effective rate is 100%. Untreated group, 26 deaths, mortality 86.7%; 19 deaths were obtained in the Chinese herbal treatment group, with a mortality rate of 63.3%; compound 7 treatment group, 0 deaths, 0% mortality. After ten days, the tested ducks recover from the spirit and the appetite; feces are normal and no obvious mucus is effective; the dissected mesentery is judged to be effective if no obvious congestion exists. The compound 7 treatment group is treated for 3 days, after 7 days of recovery, the feeding, movement and excretion of all the tested ducks are recovered to be normal, the dissected pathological tissues are better recovered, and the effective rate is 100%. The above results indicate that compound 7 is capable of treating duck coronavirus enteritis, which is naturally infected with DCV.

Table 5: data summarization of clinical treatment data of ducks suffering from duck coronavirus enteritis

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.