CN115073446A

CN115073446A - Berberine type alkaloid oxidized pyrazine formic acid quaternary ammonium salt and application thereof in preparing medicines

Info

Publication number: CN115073446A
Application number: CN202110274862.3A
Authority: CN
Inventors: 秦海林; 邓安珺; 张雪倩; 李志宏; 徐令文
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2022-09-20

Abstract

The invention belongs to the technical field of medicines, and discloses a berberine type alkaloid oxidized pyrazine formic acid quaternary ammonium salt and application thereof in preparing a medicine. In particular to berberine type alkaloid 2-methyl-N shown as a general formula I ¹ -pyrazine oxide-5-formic acid quaternary ammonium salt compound and application thereof in preparing medicaments. The compound of the invention has better physicochemical property and pharmacological action strength than a reference drug and related compounds, and can be used for preparing drugs for preventing, relieving and/or treating cardiovascular and cerebrovascular diseases, blood system diseases, inflammatory diseases, fever diseases, tumor diseases, respiratory system diseases, autoimmune diseases, coronavirus infection diseases and complications thereof.

Description

Berberine type alkaloid oxidized pyrazine formic acid quaternary ammonium salt and application thereof in preparing medicines

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a berberine type alkaloid 2-methyl-N ¹ -pyrazine oxide-5-formic acid quaternary ammonium salt compounds, preparation methods thereof, pharmaceutical compositions and uses thereof in preparing medicaments.

Background

Interleukin-6 (IL-6) belongs to interleukin biochemical components and is a hormone glycoprotein with molecular weight of 22-28 KD. IL-6 was first discovered in 1980 as a B lymphocyte differentiation factor and was designated as interferon-beta. Thereafter, it has been subjected to a research process in which various cytokines are continuously discovered and named, and it is not until the genes of these cytokines are cloned and then they are finally named as IL-6. The human IL-6 gene is localized on chromosome seven, and in 1997, the three-dimensional structure of IL-6 was determined by X-ray crystallography and consists of four topological helix bundles A-D, and a minor helix-E helix outside the four helix bundles, in which the A and B helices run in the same direction, and the C and D helices run in opposite directions.

IL-6 is a proinflammatory pleiotropic cytokine, and various cells such as macrophages, lymphocytes, fibroblasts, monocytes, vascular endothelial cells, mesangial cells, a plurality of tumor cells and the like can produce IL-6, and the abnormality and the excessive secretion of the IL-6 play various roles in the acute inflammatory response of an organism and the pathological activity of autoimmune diseases, and are one of the most important cytokines for determining the severity of the diseases of patients with systemic inflammatory response syndrome. Inflammation plays a key role in a variety of other diseases, including hematopoiesis, tumorigenesis, and immune responses. Firstly, inflammation plays a key role in cardiovascular adverse events, and the IL-6 level is positively correlated with the Gensini score of coronary artery lesion, has close relation with the severity of the condition and is closely correlated with cardiovascular and blood system diseases such as atherosclerosis, coronary heart disease, anemia and the like; IL-6 is involved in the pathological process of hypertensive disorders of pregnancy through a variety of pathways. Secondly, IL-6 is an important regulator of tumor progression, and serum IL-6 levels are often elevated in some tumor patients, suggesting poor clinical outcome. Thirdly, IL-6 has close relationship with respiratory system diseases, such as obvious increase of IL-6 level in bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis; the content of IL-6 in the culture supernatant of alveolar macrophage of patients with allergic asthma is obviously increased; IL-6 is also positively associated with the manifestations of dyspnea in patients with pneumonia, which from a pathophysiological point of view represents the intensity of the inflammatory response in the lungs and is associated with the systemic severity of respiratory disease. Coronaviruses are common pathogens causing respiratory diseases, and can cause the remarkable increase of proinflammatory cytokines such as IL-6 and the like, generate cytokine storms and recruit immune response cells in the lung. For example, new 2019 Coronavirus (Coronavirus disease of 2019, COVID-19)2019, which has been outbreaked in the world by the end of 2019, 2019 new Coronavirus (2019novel Coronavirus or SARS-CoV-2, also called 2019-nCoV), produces IL-6 in a large amount in T lymphocytes and monocytes after infection, accelerates the progress of inflammation, and exerts immune destruction, resulting in severe lung dysfunction; SARS-associated coronavirus (SARS-CoV) infection, which was a Severe Acute Respiratory Syndrome (SARS) outbreak in the early 2003, causes a significant increase in IL-6, Acute Respiratory Distress Syndrome (ARDS) by the production of cytokine storm, recruitment of immunoresponsive cells in the lung, and the like, and even pulmonary fibrosis in later stages. The SARS-CoV-2 virus infected patient is divided into normal type, heavy type and critical type, and the inflammatory cell factor in the serum is detected, and the difference of IL-6 in expression level between the types is statistically significant, and the IL-6 level of SARS-CoV-2 virus pneumonia patient is obviously higher than that of patient with mild disease, so that IL-6 may be an important link for SARS-CoV-2 infection to induce organism to produce cell factor storm. Comprehensive experimental data indicate that coronavirus-induced IL-6 plays an important role in the pathogenesis of pneumonia of coronavirus infection, particularly in inflammation and fever. In addition, IL-6 is also associated with kidney diseases, such as significant increases in IL-6 in urine and serum of patients with primary glomerulonephritis (IgAN). The receptor for IL-6 is IL-6R on the cell surface. IL-6 binding to the receptor initiates dimerization of IL-6 Ra with gp130, leading to activation of downstream signaling pathways, including the JAK/STAT, Ras, and PI3K signaling pathways. Therefore, the IL-6 can be used as a target spot for treating diseases such as inflammatory diseases, fever diseases, cardiovascular and cerebrovascular diseases, blood system diseases, tumor diseases, viral infection diseases and complications thereof, respiratory system diseases, primary glomerulonephritis and the like, and can be used for development, test and application of medicaments.

Autoimmune diseases are one of the most common clinical refractory diseases at present, the causes are not completely clear, a large number of inflammatory cells are usually accumulated at the focus, the content of NO in body fluid of the focus of a patient is also obviously higher than that in blood of the patient, and the content of NO in the blood of the patient with the autoimmune diseases is higher than that in the blood of a normal person, so that swelling and pain are caused, and the formation of autoantibodies is usually involved. In addition to focal sites, autoimmune diseases can affect any system of the body and are associated with a variety of diseases. The pathological core of autoimmune diseases is inflammation, but it also plays an important role in the development of cardiovascular diseases. Lymphocytes are important cells of the immune response function of the organism in a physiological state, and have the functions of resisting infection, monitoring and eliminating heterogeneous substances (including cancerated tissues and necrotic tissues) and protecting the stable state of the organism; under the stimulation of some pathological factors, the physiological functions are out of control, so that lymphocyte hyperfunction is caused, hematopoietic negative regulatory factors are generated, the formation of bone marrow clone of a patient is inhibited, normal tissues are damaged, and various autoimmune inflammatory diseases and blood system diseases, such as anemia and the like, are formed. Anemia is primarily manifested by the body's inability to produce sufficient hemoglobin to deliver oxygen to various tissues of the body. The most common type of anemia is anemia of chronic nature, the pathogenesis of which is not completely clear; thus, patients with autoimmune diseases are at increased risk of most types of cardiovascular disease, for example, inflammation is closely associated with atherosclerosis, which leads to ischemic heart disease, and fibrotic myocardial damage leading to diastolic dysfunction. Autoimmune diseases also have the characteristic of repeated attacks, which seriously affect the life quality and the working capacity of patients and bring heavy burden to families and society. Autoimmune diseases can not be cured radically so far, and the clinical treatment principle of the autoimmune diseases is early treatment, standard treatment, regular monitoring and follow-up. The autoimmune diseases include rheumatoid arthritis, lupus erythematosus, multiple sclerosis, and ulcerative colitis.

Although there are many clinical treatment schemes for autoimmune diseases at present, the compliance of all the schemes can not meet the expectations of medical personnel and patients, and even can not achieve complete cure, resulting in serious negative effects of the current autoimmune diseases on the daily life, the working ability and the health-related quality of life of patients. Therefore, current treatment strategies for autoimmune diseases are aimed at achieving disease remission or low disease activity, i.e. palliative or standard treatment, with the ultimate goal of controlling the disease state and improving the quality of life of the patient. This highlights the need for further enhanced research into anti-autoimmune disease therapies, including the discovery of new biochemical drugs with superior efficacy.

At the end of the last century, few antiviral drugs have been developed, and only a few have been approved worldwide. However, in recent years, a variety of factors have brought to the recognition of the urgent need of the medical community for new effective therapies against viral infectious diseases, including the deep understanding of the severity of viral diseases, the deep understanding of the characteristics of the viral life cycle (including the adsorption, fusion and propagation of viruses to host cells, etc.), and the discovery and demonstration of some targets for the treatment and intervention of viral diseases, among others. In particular, the increasing prevalence of chronic viral infectious diseases and the emergence of new highly pathogenic infectious viruses make both chronic viral infectious diseases and highly pathogenic infectious viral infectious diseases a major medical problem in the world, and intervention and treatment for various viral infectious diseases are required in the medical field. For example, infection with Hepatitis B (Hepatitis B) virus (HBV) and Acquired Immune Deficiency Syndrome (AIDS) Human Immunodeficiency Virus (HIV) infection, as well as early 2003 outbreak of SARS-CoV infection, late 2012 9 outbreak of Middle East Respiratory Syndrome (MERS) Middle east respiratory syndrome coronavirus (MERS) infection and late 2019 worldwide outbreak of SARS-CoV infection, MERS-CoV infection, and the like, lack of specific therapies all seriously affect the patient's daily life, working ability and quality of life related to health and the normal lives of the Human society, causing huge rhythms and losses in Human society. Although vaccines against some viruses have been developed and effective vaccines have or may kill some important viral pathogens, to date, vaccine strategies have been poorly or ineffectively effective against many viral infectious diseases or have resulted in serious adverse events (such as the occurrence of death of the subject from influenza vaccine injections and the occurrence of death of the subject from SARS-CoV-2 vaccine injections and the occurrence of SARS-CoV-2 tests) resulting from vaccine use, and thus vaccine strategies have not eliminated the need for effective chemotherapeutic agents, particularly specific chemotherapeutic agents. In terms of drug discovery, admittedly, since the beginning of this century, several antiviral drugs have been approved by the drug Authority of several international countries for marketing, but most of them are used for the treatment of HIV infection and some are used for the treatment of other viral infections. Therefore, we still lack effective treatment methods for many viral infectious diseases, or the existing treatment methods are in need of improvement in effectiveness and compliance. For example, the international medical community does not consider human coronavirus infections as a difficult-to-treat viral infectious disease before the outbreak of viral diseases such as SARS-CoV, MERS-CoV and SARS-CoV-2 infection, and some of the human coronavirus infections that have been reported are thought to cause only mild upper respiratory diseases, which can be treated by vaccination or other antiviral therapies. However, with the outbreak of the above-mentioned 3 highly pathogenic coronavirus infectious diseases, the recognition and treatment of coronavirus infectious diseases have changed significantly. The three coronaviruses are newly discovered human highly pathogenic coronaviruses and all develop into interpersonal transmission; the three pathogens can cause serious acute upper respiratory tract infection in human, and the serious pathogens can cause viral infectious pneumonia in the lower respiratory tract and cause systemic inflammatory reaction. General clinical symptoms and pathological features of SARS, MERS and COVID-19 are similar, and the most typical early clinical symptoms are fever accompanied by hypodynamia, headache, pharyngalgia, muscular soreness, dry cough and the like; some critically ill patients develop severe pulmonary inflammation, manifested by severe systemic symptoms of high fever, chest distress, dyspnea, and the like, and rapidly cause pulmonary edema, ARDS, or multiple organ failure and death. Viral infection is the cause and viral inflammation is the disorder. At present, no specific medicine exists for pneumonia caused by invasion of the three coronaviruses into an organism, and the pneumonia is mainly resisted by an immune system of the organism, so that a situation that a temporarily established treatment scheme is adopted in clinical treatment under the condition that no clearly available medicine exists, and the treatment compliance cannot meet the requirements of medical personnel and patients occurs. This highlights the need for further intensive research into antiviral therapeutics, including the discovery of biochemical drugs. In particular, it is of undeniable importance to find active compounds acting synergistically with the antiviral and anti-inflammatory effects, according to the etiology of viral infections and the etiological and pathological features of viral inflammations.

According to the objective requirements of the anti-virus infectious disease medicine, the anti-autoimmune disease medicine, the anti-cardiovascular and cerebrovascular and blood system disease medicine, the anti-respiratory system disease medicine and the anti-tumor medicine, the invention also includes the idea of treating virus infection venereal disease and complications thereof by the synergistic anti-virus and anti-inflammatory dual drug effect, and the compound has the effect of promoting IL-6 to clinically treat diseases and the treatment aspect thereofThe compound of the invention is synthesized, the inhibition effect on IL-6 level on a high IL-6 pharmacological model is evaluated, the influence condition on the growth of in vitro culture normal RAW264.7 cells is evaluated on a cell level, and the inhibition effect on NO generation of a cell inflammation model is evaluated in vitro experiments, the method comprises the following steps of evaluating the effect of reducing the IgG antibody level in serum of a model animal on a high IgG antibody pharmacological model against autoimmune diseases, evaluating the effect of inhibiting 2019novel coronavirus SARS-CoV-2 replication in an in vitro experiment, evaluating the inhibition effect on the growth of tumor cells in the in vitro experiment, evaluating the influence on the hemoglobin level of the model animal on the high hemoglobin pharmacological model, and comparing the pharmacological effect and the physicochemical property of the compound with the substrate of the berberine chloride type alkaloid quaternary ammonium salt compound. The result shows that in pharmacological experiments, the compound of the invention shows the obvious effect of reducing the IL-6 level in a high IL-6 pharmacological model, has obvious proliferation promoting effect on normal RAW264.7 cells, can inhibit NO generation of a RAW264.7 cell inflammation model stimulated by LPS in a dose-dependent manner, and has the effect of obviously reducing the IgG antibody level in the serum of an animal with a high IgG antibody model. In cell experiments, the compound can inhibit coronavirus replication in a dose-dependent manner, inhibit the growth of tumor cells in a dose-dependent and selective manner, and remarkably reduce the content of hemoglobin in peripheral blood of a hyperphaemia pathological model animal. The pharmacological effect and the physicochemical property of the compound are obviously superior to those of a berberine chloride type alkaloid quaternary ammonium salt compound substrate. Through a large amount of substantial research work including the safety evaluation of the compound, the berberine type alkaloid 2-methyl-N related to the invention is clarified ¹ The-pyrazine oxide-5-formic acid quaternary ammonium salt compound is a compound with significant application value in preparing medicaments for preventing, relieving and/or treating cardiovascular and cerebrovascular diseases and blood system diseases, virus infection diseases and complications thereof, inflammatory diseases, fever diseases, autoimmune diseases, tumor diseases, respiratory system diseases and other diseases.

Disclosure of Invention

The invention solves the technical problem of providing a new type of medicine by means of chemical synthesis, medicine screening and the likeWith 2-methyl-N ¹ The dissolving performance of the unit taking oxidized pyrazine-5-formate as a balanced anion unit and taking berberine type alkaloid quaternary ammonium cation as a basic group balanced cation unit in a mixed solvent of water and alcohol water is better than that of a chlorinated berberine type alkaloid quaternary ammonium salt substrate, and the unit has obvious functions of preventing, relieving and/or treating related diseases causing the increase of IL-6 and/or NO and/or IgG antibody secreted/by organism and preventing, relieving and/or treating virus infection diseases caused by various reasons and complications, autoimmune diseases, cardiovascular and cerebrovascular diseases and blood system diseases caused by the virus infection diseases, an active compound for treating inflammation, fever, tumor, respiratory system diseases, and primary glomerulonephritis, which is berberine type alkaloid 2-methyl-N shown in formula I. ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt compounds.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, the present invention provides berberine type alkaloids as compounds of the invention, 2-methyl-N, as shown in general formula I ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt compounds.

In a second aspect, the invention provides berberine type alkaloids 2-methyl-N as shown in general formula I ¹ A preparation method of-pyrazine oxide-5-formic acid quaternary ammonium salt compound.

In a third aspect, the invention provides berberine type alkaloids as shown in general formula I, 2-methyl-N ¹ -a product composition of a pyrazine-5-carboxylic acid quaternary ammonium salt compound, said product being selected from the group of pharmaceuticals.

In a fourth aspect, the invention provides the use of the compounds and pharmaceutical compositions of the invention in the manufacture of pharmaceutical products, including the use in the manufacture of immunomodulator drugs having the activities of promoting immune effector cell proliferation, inhibiting/reducing pathological NO production in the body, and increasing IL-6 and autoantibody levels, and on an independent basis, also including the use in the manufacture of medicaments for preventing, alleviating and/or treating diseases that cause an increase in the pathological secretion of IL-6 from the body, the use in the manufacture of medicaments for preventing, alleviating and/or treating diseases that cause an increase in the pathological secretion of NO from the body, and the use in the manufacture of medicaments for preventing, alleviating and/or treating diseases that cause the pathological formation of autoantibodies from the body.

In a fifth aspect, the invention provides the use of the compounds and pharmaceutical compositions of the invention in the manufacture of a medicament for the prevention, alleviation and/or treatment of cardiovascular and cerebrovascular and hematological disorders, autoimmune disorders, viral infections and their complications, respiratory disorders, inflammatory diseases, febrile diseases, neoplastic diseases, primary glomerulonephritis disorders, all of which are caused by a variety of causes. The autoimmune diseases comprise rheumatoid arthritis, cardiovascular and cerebrovascular diseases and blood system diseases comprise hypohemoglobinemia anemia, viral infections and complications thereof comprise coronavirus infections and complications thereof, the coronavirus infections and complications thereof comprise 2019novel coronavirus SARS-CoV-2 infections and complications thereof, and the tumor diseases comprise colorectal cancer, liver cancer and lung cancer.

Specifically, the invention provides the berberine type alkaloid 2-methyl-N shown as the general formula I in the first aspect ¹ The chemical structural general formula of the pyrazine-5-formic acid quaternary ammonium salt compound is shown as the following formula I:

in the formula I, R ₂ 、R ₃ Each independently selected from H, substituted or unsubstituted OH, or R ₂ And R ₃ (ii) linked to an alkylenedioxy group; further, R ₂ 、R ₃ Wherein the substituent of said substituted or unsubstituted hydroxyl group is selected from the group consisting of methyl and ethyl, and R ₂ 、R ₃ The alkylenedioxy group in (1) is selected from methylenedioxy.

R ₉ 、R ₁₀ 、R ₁₁ Each independently selected from H, substituted or unsubstituted OH, or R ₉ And R ₁₀ Linked as alkylenedioxy and R ₁₁ Independently selected from H, substituted or unsubstituted OH, or R ₁₀ And R ₁₁ Linked as alkylenedioxy and R ₉ Independently selected from H, substituted or unsubstituted OH. Further, R ₉ 、R ₁₀ 、R ₁₁ The substituent in the substituted or unsubstituted OH mentioned in (1) is selected from methyl and ethyl, R ₉ 、R ₁₀ 、R ₁₁ The alkylenedioxy group in (1) is selected from methylenedioxy.

The most preferred berberine type alkaloid of the invention is 2-methyl-N ¹ The pyrazine-5-carboxylic acid quaternary ammonium salt compound is preferably selected from the following compound groups, namely compound 1-5:

in a second aspect, the invention provides the berberine type alkaloids of the invention 2-methyl-N ¹ A preparation method of-pyrazine oxide-5-formic acid quaternary ammonium salt compounds.

The berberine type alkaloid 2-methyl-N ¹ The compound of-pyrazine-5-carboxylic acid quaternary ammonium salt can be synthesized by the following general synthetic route (see the examples for specific synthetic conditions):

the synthesis steps are as follows: (a) reacting berberine type alkaloid quaternary ammonium salt compound with acetone and sodium hydroxide water solution to obtain solid 8-acetonyl dihydroberberine type compound. (b) The obtained solid 8-acetonyl dihydroberberine compound is mixed with 2-methyl-5-carboxyl-N ¹ Reacting pyrazine oxide in a mixed solvent of tetrahydrofuran and water under a heating condition, and filtering the reaction mixed solution to obtain the berberine type alkaloid 2-methyl-N ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt compounds.

In a third aspect, the invention provides the berberine type alkaloids of the first aspect of the invention 2-methyl-N ¹ A pharmaceutical composition containing as an active ingredient a compound of pyrazine-5-carboxylic acid quaternary ammonium salt. These pharmaceutical compositions can be prepared according to known methods for preparing pharmaceutical compositions. Can be prepared by mixing the compound of the invention with one or more pharmaceutically acceptable solid or liquidExcipients and/or adjuvants, and making into any dosage form suitable for human or animal use. The content of the compound of the present invention in the pharmaceutical composition thereof is usually 0.1 to 99.9% (W/W).

The compounds of the present invention or pharmaceutical compositions containing the compounds of the present invention may be administered in unit dosage form by routes of administration primarily through the digestive tract, such as oral administration, enteral administration, and the like. However, the compounds of the present invention are excellent in physicochemical properties, and can be administered parenterally in a form acceptable and also can be the main administration route. Such as intravenous, intramuscular, subcutaneous, intraperitoneal, and nasal, oromucosal, ophthalmic, pulmonary and respiratory, vaginal administration, application to the skin, and the like. The traditional Chinese medicine composition can be prepared into common oral preparation forms such as common tablets and common capsules for direct oral administration without special treatment (very convenient use), and can also be prepared into other preparation forms including injections for other various administration routes and modes.

Other dosage forms for oral administration or other routes of administration may also be employed, including various liquid, solid or semi-solid dosage forms prepared using novel techniques. The liquid dosage forms can be solution (including true solution and colloidal solution), emulsion (including O/W type, W/O type and multiple emulsion), suspension, injection (including water injection, powder injection and infusion), eye drop, nose drop, lotion and liniment; the solid dosage form can be tablet (including common tablet, enteric coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (including hard capsule, soft capsule, and enteric coated capsule), granule, powder, pellet, dripping pill, suppository, pellicle, patch, aerosol (powder), spray, etc.; semisolid dosage forms can be ointments, creams, gels, pastes, and the like.

The compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various particle drug delivery systems.

For tableting the compound of the present invention, various excipients well known in the related art, including diluents, binders, wetting agents, disintegrants, lubricants, glidants, may be widely used. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder can be starch slurry, dextrin, syrup, Mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

To encapsulate the administration unit, the compounds of the invention can be mixed with diluents, glidants and the mixture placed directly into hard or soft capsules; or mixing the compound of the invention with diluent, adhesive and disintegrating agent to prepare granules or pellets, and then placing the granules or pellets into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, glidants used to prepare the compound tablets of the present invention may also be used to prepare capsules of the compound of the present invention.

In order to prepare the compound of the invention into injection, water, ethanol, isopropanol, propylene glycol or the mixture of the water, the ethanol, the isopropanol and the propylene glycol can be used as a solvent, and a proper amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator which are commonly used in the pharmaceutical field can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloride, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol and glucose can be added as proppant for preparing lyophilized powder for injection.

In addition, if the dosage form requires special, can also be added to the pharmaceutical preparation of coloring agent, preservatives, spices, flavoring agent or other additives.

For pharmaceutical purposes, to enhance therapeutic effect, the compounds (drugs) or pharmaceutical compositions of the present invention can be administered and used by any of the well-known methods of administration and application.

The administration (application) or administration (use) dosage of the compound or the pharmaceutical composition of the present invention may vary widely depending on the cardiovascular and cerebrovascular and hematological diseases, autoimmune diseases, viral infectious diseases and complications thereof, respiratory diseases, inflammatory diseases, febrile diseases, severity of neoplastic diseases, individual condition of the patient or animal, administration (application) route and dosage form, etc., which are caused by various causes to be prevented, alleviated and/or treated. Generally, suitable daily dosages of the compounds of the present invention range from 0.001 to 500mg/kg (dose/body weight), preferably from 0.1 to 150mg/kg (dose/body weight), more preferably from 1 to 100mg/kg (dose/body weight), most preferably from 1 to 50mg/kg (dose/body weight). The above-mentioned dose may be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the progress of the treatment and the administration (use) regimen including the use of other therapeutic (application) means.

The compounds or product (pharmaceutical) compositions of the invention may be administered alone or in combination with other therapeutic or symptomatic agents. When the compound of the present invention is used in a synergistic manner with other therapeutic agents, the dosage thereof should be adjusted according to the actual circumstances.

In a fourth aspect, the invention provides the use of the compounds and pharmaceutical compositions of the invention in the manufacture of a medicament, including the use in the manufacture of a medicament for an immunomodulator having the activities of promoting immune effector cell proliferation, inhibiting/reducing pathological NO production in the body, and increasing IL-6 and autoantibody levels, and on an independent basis, also including the use in the manufacture of a medicament for preventing, alleviating and/or treating a disease that causes an increase in the pathological secretion of IL-6 from the body, the use in the manufacture of a medicament for preventing, alleviating and/or treating a disease that causes an increase in the pathological secretion of NO from the body, and the use in the manufacture of a medicament for preventing, alleviating and/or treating a disease that causes the pathological formation of autoantibodies from the body. The fourth aspect of the present invention is based on the results of specific pharmacological experiments, wherein the compound of the present invention has significant immunoregulatory activity, and in the pharmacological experiments, the compound exhibits significant proliferation-promoting activity on immune effector cells, a certain inhibitory activity on the pathological production of cellular inflammation model NO, a pharmacological effect of significantly reducing the level of IL-6 in a pathological high IL-6 level pharmacological model, and a pharmacological effect of significantly reducing the level of IgG antibodies in the serum of a pathological high IgG antibody level model animal.

In a fifth aspect, the invention provides the use of the compounds and pharmaceutical compositions of the invention in the manufacture of a medicament for the prevention, alleviation and/or treatment of cardiovascular and cerebrovascular and hematological disorders, autoimmune disorders, viral infections and their complications, respiratory disorders, inflammatory diseases, febrile diseases, neoplastic diseases, all caused by any of a variety of causes. The autoimmune diseases comprise rheumatoid arthritis, cardiovascular and cerebrovascular diseases and blood system diseases comprise hypohemoglobinemia anemia, viral infections and complications thereof comprise coronavirus infections and complications thereof, the coronavirus infections and complications thereof comprise 2019novel coronavirus SARS-CoV-2 infections and complications thereof, and neoplastic diseases comprise colorectal cancer and lung cancer. The fifth aspect of the present invention is proposed based on the results of specific pharmacological experiments. In addition to the results of the pharmacological experiments, in the pharmacological experiments, the compound can also inhibit 2019novel coronavirus SARS-CoV-2 replication in a dose-dependent manner, inhibit the growth of human lung cancer cell A549 cell, human liver cancer cell HepG2 cell and human colon cancer cell HCT-116 cell in a dose-dependent manner and selectively, remarkably increase the content of hemoglobin in peripheral blood of model animals, and remarkably reduce the severity of rheumatoid arthritis symptoms of the model animals.

In the above fourth and fifth aspects of the present invention, preferred autoimmune diseases include rheumatoid arthritis, cardiovascular and cerebrovascular diseases and hematological diseases including hypohemoglobinemia anemia, neoplastic diseases including colorectal cancer and lung cancer, viral infectious diseases including coronavirus infectious diseases, particularly 2019novel coronavirus SARS-CoV-2 infectious disease and complications thereof.

Advantageous technical effects

The compound has a unique pharmacological action mechanism for balancing the body immune function, shows a remarkable proliferation promoting effect on immune effector cells and a remarkable inhibiting effect on the generation of NO in a cell inflammation model in a pharmacological experiment, has a pharmacological effect of remarkably reducing the expression quantity of IL-6 in a pathological high IL-6 level pharmacological model on both a cell level and an animal level, and also shows a pharmacological effect of remarkably reducing the level of IgG antibodies in the serum of an animal model with a pathological high IgG antibody level. The compound is formed by combining two active structural units in an ionic bond form, has unique action characteristics, and comprises a unique irregular dose dependence relationship instead of a conventional linear dose dependence relationship on the inhibition effect of NO generation in an LPS-stimulated initial passage RAW264.7 cell inflammation model; the unique dose-effect relationship is also shown in the corresponding animal experiments. The compound can inhibit the replication of 2019novel coronaviruses (namely 2019novel coronavirus or SARS-CoV-2 or 2019-nCoV) in a dose-dependent manner, inhibit the growth of human lung cancer cells A549, human colon cancer cells HCT-116 and human liver cancer cells HepG2 in a dose-dependent manner and obviously increase the content of hemoglobin in peripheral blood of model animals. Therefore, the compound can be used for preparing immunomodulator medicines, preparing medicine products for preventing, relieving and/or treating diseases causing the increase of IL-6 secretion of a living organism, preparing medicine products for preventing, relieving and/or treating diseases causing the increase of NO secretion of the living organism and preparing medicine products for preventing, relieving and/or treating diseases causing the increase of autoantibodies formed in the living organism. The application of the compound of the invention in preparing the medicine product for preventing, relieving and/or treating the related diseases comprises preventing, relieving and/or treating cardiovascular and cerebrovascular diseases and blood system diseases and autoimmunitySexual disease, respiratory system disease, inflammation disease, fever disease, tumor disease, coronavirus infection disease and complications thereof. The cardiovascular and cerebrovascular diseases and blood system diseases comprise hypohemoglobinemia anemia caused by various reasons, the autoimmune diseases comprise rheumatoid arthritis, the neoplastic diseases comprise colorectal cancer, lung cancer and liver cancer, and the coronavirus infectious diseases comprise 2019novel coronavirus SARS-CoV-2 infectious diseases. The compounds of the invention show significant safety in toxicological experiments. The pharmacological effect and physicochemical property of the compound are obviously superior to those of a chloridized berberine type alkaloid quaternary ammonium salt compound substrate; the effect of the compound of the invention for inhibiting NO secretion is better than that of a chloridized berberine type alkaloid quaternary ammonium salt substrate; compared with the berberine chloride type alkaloid quaternary ammonium salt substrate, the solubility of the compound of the invention in a mixed solvent of water and alcohol water is obviously improved. The above properties of the compound of the present invention have obvious pharmaceutical preparation value, and can be prepared into various pharmaceutical dosage forms including common oral dosage forms for transgastric administration and injection dosage forms for pharmaceutical use. The specific structure of the compound of the invention is 2-methyl-N ¹ -pyrazine oxide-5-formate as acid radical balancing anion unit, with 5, 6-dihydrodibenzo [ a, g]Quinolizine-7-cationic quaternary ammonium cation is base balance cation unit, and the quinolizine-7-cationic quaternary ammonium cation and the base balance cation unit form berberine type alkaloid 2-methyl-N by ionic bond ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt compounds. The compound can be used for preparing medicine products for preventing, relieving and/or treating coronavirus infection diseases and complications thereof, inflammatory diseases, fever diseases, respiratory diseases, tumor diseases, autoimmune diseases and anemia caused by various etiological factors.

The development of the synthesis of functional organic compounds and the evaluation of biological activity is a scientific research practice activity, and the important aim is to culture professional talents in the fields of organic chemistry and pharmacy through specific scientific experiments and promote the continuous progress of related subjects; meanwhile, the specific scientific research practice process always contains new findings in various meanings, and new connotations are continuously injected for scientific progress. The invention is obtained in the process of carrying out deep medicinal chemical research on the berberine compounds, and confirms the structure, the synthetic method, the physicochemical property and the biological significance of the berberine compounds.

The synthetic route of the compound has the characteristics of simplicity, high efficiency and friendliness. The results of stability tests carried out on these compounds by NMR measurement means show that the compounds of the present invention are not only stable in physical and chemical properties in the solid state, but also extremely stable in structure even when placed in solution. Of course, according to the theory of organic chemistry, the compounds of the present invention exist in solution as ion pairs or ion clusters, with a specific arrangement, rather than as a mixture. In the aspect of physical and chemical properties, a solubility test experiment proves that the compound has obviously improved solubility in a solvent compared with a corresponding berberine chloride type alkaloid quaternary ammonium salt substrate, so that the 2-methyl-N-berberine alkaloid meeting the general pharmaceutical technical specification for large-scale preparation ¹ The pyrazine oxide-5-formic acid quaternary ammonium salt compound active molecular entity has obvious practical value in searching new pharmacological activity and improving the action strength of the medicine.

Compared with the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate, the water solubility of the compound is obviously improved; measuring water solubility at 25 ℃ + -2 ℃, wherein the amount of the compound capable of dissolving the invention in each milliliter of water is respectively larger or significantly larger than the amount of the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate capable of dissolving in each milliliter of water, for example, the amount of the compound 1-5 capable of dissolving the invention in each milliliter of water is 342mg, 425mg, 5mg, 42mg and 3.3m respectively; and the soluble amounts of berberine quaternary ammonium chloride, palmatine quaternary ammonium chloride, coptisine quaternary ammonium chloride, isoflavine quaternary ammonium chloride and isoberberine quaternary ammonium chloride which are used as the substrates of the berberine type alkaloid quaternary ammonium salt in each milliliter of water in parallel measurement are 2mg, 21mg, 1mg and 1mg respectively.

Compared with the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate, the solubility of the compound in an ethanol-water mixed solvent is obviously improved; the solubility is measured at the ambient temperature of 25 +/-2 ℃, the amount of the compounds 1-5 of the invention which can be dissolved in 95 percent ethanol-water mixed solvent per milliliter is respectively larger than the amount of the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate which can be dissolved in 95 percent ethanol per milliliter, and the specific data are shown in the examples 1 and 2 of the invention.

In the scientific research of the synthesis of functional organic compounds and the evaluation of biological functions, the related compounds show certain safety which is a necessary prerequisite for the deep development of biological research; of course, this is also the first routine biological experiment to be performed in the drug discovery and development process. The compound and various normal cells are incubated and cultured together, the influence on the growth of the normal cells is investigated, if the experimental result shows that the survival rate of the normal cells subjected to the intervention treatment of the related compound is high (namely the inhibition rate of the compound on the growth of the cells is low), the compound can be preliminarily judged to have better biological safety, and the method is suitable for carrying out deep biological activity research on a biological model and lays a foundation for further carrying out pharmaceutical research. The invention respectively adopts a CCK-8 method and an MTT method to widely detect the influence of the compound on the cell growth condition of normal cells. In an experiment for investigating the influence of the compound on the growth condition of mouse mononuclear macrophage RAW264.7 with normal initial passage (passage of several generations) in vitro culture, the influence on the cell growth condition is evaluated by the cell survival rate of cultured cells subjected to intervention treatment for 24 hours under the serial action concentration of the compound, and the result shows that after the intervention treatment of the compound under the serial action concentration of 10 mu M-0.01 mu M, the compound not only has no inhibition effect on the growth of normal cells of the initial passage, but also has an obvious proliferation promoting effect on the normal RAW264.7 cells of the initial passage. The survival of normal primary passage RAW264.7 cells at serial effect concentrations of 10. mu.M, 1. mu.M, 0.1. mu.M and 0.01. mu.M, as interfered with by Compound 2 of the present invention, was 109.49. + -. 7.3%, 108.54. + -. 6.61%, 110.25. + -. 5.78% and 109.38. + -. 5.1%, respectively. Macrophages are important immune effector cells located in body tissues, participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vivo, and exert defense, surveillance, regulation, antigen presentation, and the likeThe important immune function has very important correlation with the balance of the immune system of the body and plays an important role in the immune response process of a host. The primary function of macrophages is to phagocytose (i.e., phagocytose and digest) cellular debris and pathogens in the form of fixed or free cells, and to activate lymphocytes or other immune cells in response to pathogens. Macrophages also have the function of reconstructing tissues, repairing damaged cells, and eliminating apoptotic cells. The compound of the invention has obvious effect of promoting macrophage proliferation, which shows that the compound has the effect of balancing the immune function of the organism. After the intervention treatment of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound under the series action concentration of 10 mu M-0.01 mu M, the inhibition effect of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound on the growth of multi-passage normal RAW264.7 cells is not obvious, which shows that the toxicity effect of the compound is not strong. The half inhibitory concentration IC of the inhibition effect of the compound on the HELF cell growth of human embryonic lung fibroblasts is obtained by intervening and treating cultured cells for 96 hours under the series of action concentrations of 10 mu M, 1 mu M, 0.1 mu M, 0.01 mu M and 0.001 mu M ₅₀ The value is more than 10 mu M, the inhibition effect on the growth of mouse embryo fibroblast NIH3T3 cells is weak, and the IC of the inhibition effect ₅₀ The value is greater than 10. mu.M. The compound of the invention is incubated with 293T cells of normal human embryonic kidney epithelial cells for 72h under the action concentration of 10 mu M, and the result shows that the compound of the invention has no obvious toxicity to the 293T cells of normal human embryonic kidney epithelial cells, except berberine 2-methyl-N ¹ The survival rate of the cells treated by the quaternary ammonium salt of pyrazine-5-carboxylic acid (1) is 89.7%, and the survival rate of the normal 293T cells subjected to other intervention of the compounds 2-4 of the invention is 100% (see experimental examples).

In addition, the compounds of the present invention also did not show significant growth inhibitory effects on a variety of other normal cells in vitro. As in the antiviral activity evaluation test, the compounds of the present invention showed no significant growth inhibitory effect on Vero E6 cells not infected with virus (see experimental examples).

By further performing the evaluation of the biological activity of each compound, the compound of the invention is found to show various important pharmacological effects with outstanding action intensity in pharmacological experiments. First, the compounds of the invention have a pharmacological effect of significantly reducing IL-6 levels in a pharmacological model of pathologically high IL-6 levels. In a bioactivity evaluation experiment of the compound, which is carried out by adopting a chicken II type collagen-induced pathological high IL-6 level pharmacological model, the compound has a very obvious effect of reducing the serum IL-6 level of a model animal and shows dose dependence. In a pathological state that the IL-6 level in the serum of the model animals reaches 69.8443pg/ml, the compound 2 can reduce the IL-6 level in the serum of the model animals to 25.0039pg/ml at the action dose of 50mg/kg, and has a very significant difference compared with the model animals in statistics. Second, the compounds of the present invention have significant inhibitory activity against NO production in the LPS-stimulated RAW264.7 cell inflammation model, and have a unique mechanism of action. It was found experimentally that compound 2 of the present invention not only showed inhibitory activity against NO production in the LPS-stimulated primary passage RAW264.7 cell inflammation model at the concentrations commonly used for pharmacological experiments of 5 μ M, 2.5 μ M and 1.25 μ M, but also showed unique dose-dependent inhibitory activity at the concentrations of the series of effects of 5 μ M, 2.5 μ M, 1.25 μ M, 0.625 μ M, 0.3125 μ M, 0.15625 μ M, 0.078125 μ M, 0.0390625 μ M, 0.01953125 μ M, 0.009765625 μ M, 0.0048828125 μ M, 0.00244240625 μ M and 0.001220703125 μ M, at inhibition rates of 56.43%, 31.59%, 4.05%, 11.04%, 35.28%, 9.29%, 19.82%, -0.99%, 15.23%, 9.80%, 9.01%, 36.33% and 27.81%, respectively, i.e. at lower concentrations of the effects of 0.00244240625 μ M and 0.001220703125 μ M, also at higher inhibition rates. The unique action mechanism of the compound is embodied in a plurality of aspects, one of the action mechanisms is in the aspect of inhibiting the NO secretion amount in an LPS-stimulated initial-passage RAW264.7 cell inflammation model, the inhibiting action of the compound under the higher action concentration of 10 mu M-1.25 mu M generally adopted by pharmacological experiments is obviously stronger than the inhibiting action of the corresponding berberine chloride type alkaloid quaternary ammonium salt in the action concentration range, particularly the inhibiting action of the compound under the unconventionally and obviously lower action concentrations of 0.00244240625 mu M and 0.001220703125 mu M on the NO secretion amount in the LPS-stimulated initial-passage RAW264.7 cell inflammation model is stronger than the inhibiting action of the corresponding berberine chloride type alkaloid quaternary ammonium salt under the obviously higher action concentration, for example, the inhibiting rate of the palmatine chloride on the NO secretion amount under the action concentration of 6.25-5 mu M is less than 20 percent in the result of a plurality of experiments, the inhibition rate of the compound is obviously lower than that of the compound under the same action concentration. That is, the compounds of the present invention inhibited NO production dose-dependently after each intervention treatment, but the inhibitory effect of the compounds of the present invention on NO production in the initial passage RAW264.7 cell inflammation model induced by LPS stimulation was not a conventional linear dose-dependent relationship, but rather a unique irregular dose-dependence. This indicates that the anti-inflammatory action of the compounds of the present invention has a unique mechanism of action that is closely linked to the unique structural characteristics of the compounds of the present invention; the structural characteristics of the compound are represented by two structural units, the two structural units can be dissociated in intestinal tracts in vivo, the dissociated two structural units respectively have respective action characteristics, different action targets and mutual synergistic action, and the compound needs to be studied deeply. Third, the compounds of the invention have a pharmacological effect of significantly reducing serum IgG antibody levels in a model animal in a pharmacological model of pathologically high IgG antibody levels. In a bioactivity evaluation experiment carried out by a pharmacological model of chicken II type collagen-induced pathological high IgG antibody level, the compound has an obvious effect of reducing the IgG antibody level in the serum of a model animal; in a pathological state that the IgG antibody level in the blood serum of the model animals reaches 22.98350 units/ml, the compound 2 can reduce the IgG antibody level in the blood serum of the model animals to 16.37524 units/ml at the action dose of 50mg/kg, and has very significant difference in statistics compared with the model animals. The pharmacological action of the compound for reducing the concentration of the anti-chicken II type collagen IgG antibody in the serum of a model animal shows that the compound has obvious curative effect on autoimmune diseases. The results of the experiments show that the compound has obvious immune balance effect, can be used for preparing immunomodulator medicines, preparing medicine products for preventing, relieving and/or treating diseases causing the increase of IL-6 secretion of organisms, preparing medicine products for preventing, relieving and/or treating diseases causing the increase of NO secretion of the organisms and preparing medicine products for preventing, relieving and/or treating diseases causing the increase of autoantibodies formed by the organisms. Among these products, the compound of the present invention can exert a therapeutic effect on diseases by balancing the immune function of the body, can exert a beneficial therapeutic effect on diseases associated with an increase in the secretion of IL-6 from the body by remarkably reducing the pharmacological effect of the level of IL-6, can exert a beneficial therapeutic effect on diseases associated with an increase in the secretion of NO from the body by remarkably reducing the pharmacological effect of the level of NO, and can exert a beneficial therapeutic effect on diseases associated with an increase in the formation of autoantibodies from the body by remarkably reducing the pharmacological effect of the level of autoantibodies. For example, the compounds of the present invention have beneficial therapeutic effects on viral infectious diseases and their complications, inflammatory diseases, febrile diseases, cardiovascular and hematological diseases, autoimmune diseases, respiratory diseases, and partial neoplastic diseases.

The compound can inhibit SARS-CoV-2 virus replication in a Vero E6 cell model in a dose-dependent manner, wherein in the tested compound, under the action concentration of 10 mu M, the inhibition rates of the compounds 1-5 of the invention on the virus RNA level after experimental cells are infected with SARS-CoV-2 virus are respectively 61.37%, 65.22%, 32.01%, 35.44% and 57.57%. Compound 3 EC for inhibiting SARS-CoV-2 viral RNA levels ₅₀ The value was 9.69. mu.M. Meanwhile, the pharmacological action of the compound for reducing the concentration of IgG antibody in serum of a model animal, the pharmacological action of the compound for reducing the IL-6 level in serum of the model animal and the effect of obviously inhibiting the replication of SARS-CoV-2 virus are combined, which shows that the compound has obvious treatment effect on SARS-CoV-2 virus infection with positive IgG antibody in serum and obviously increased IL-6 level.

In addition, the compound has obvious inhibition effect on the growth of human colorectal cancer HCT-116 cells, human liver cancer HepG2 cells and human lung cancer A549 cells selectively in the inhibition effect of the compound on the growth of tumor cells detected by an MTT method, and the compound has beneficial treatment effect on the tumor diseases by combining the obvious effect of reducing the level of IL-6. The activity of the active compound for inhibiting the growth of tumor cell strains is obviously improved by carrying out parallel tests with a berberine chloride quaternary ammonium salt substrate and contrasting the activity. The compound has selective tumor cell strain growth inhibition activity, and the compound has no inhibition activity on human gastric cancer cell BGC-823 cell growth.

Therefore, in combination with the above evaluation of biological importance, the compounds of the present invention can be used for preparing pharmaceutical products for preventing, alleviating and/or treating anemia, inflammatory diseases, febrile diseases, colorectal cancer, lung cancer, rheumatoid arthritis and coronavirus infection and complications thereof caused by various reasons.

It is further emphasized that the compounds of the present invention, in combination with the unique dose-dependent inhibition of NO production found in cellular experiments in the initial passage RAW264.7 cell inflammatory model stimulated by LPS, are judged to have a unique pharmacological mechanism of action. This is a selective marker for pharmacological effects.

In conclusion, the compound has obvious medicinal effectiveness, safety and quality controllability, and has very obvious application prospect in the aspects of preparing immunomodulator medicines, preparing medicine products for preventing, relieving and/or treating diseases causing the increase of IL-6 secretion of organisms, medicine products causing the increase of NO secretion of the organisms and medicine products causing the diseases causing the increase of autoantibodies formed by the organisms, and preparing medicine products for preventing, relieving and/or treating inflammatory diseases, fever diseases, tumor diseases, autoimmune diseases, cardiovascular and cerebrovascular diseases, blood system diseases, respiratory system diseases, viral infection diseases and complications thereof caused by various reasons. In particular, the compound of the invention has significant application value in preparing novel coronavirus SARS-CoV-2 infectious diseases and complications thereof, which cause the increase of the level of IL-6 secreted by organisms and the increase of the level of IgG antibodies.

Detailed Description

The specific embodiments of the present invention do not limit the present invention in any way.

In the preparation process and the structure identification data of the compound, the compound number corresponds to the specific compound number in the content of the invention.

First, examples of production of the Compound of the present invention

Example 1 Synthesis and Structure identification data of Compound 1 of the invention

Weighing 4g of berberine quaternary ammonium salt substrate into a reaction bottle, adding 9.6ml of acetone solvent, stirring uniformly, then dropwise adding 24ml of 5N sodium hydroxide aqueous solution, and stirring at 30 ℃ for reaction until the raw materials react completely; and (3) carrying out suction filtration on the reaction mixed solution, washing a filter cake to be neutral, and drying to obtain 3.5g of solid 8-acetonyl dihydroberberine.

Weighing 2-methyl-5-carboxy-N ¹ Adding pyrazine oxide (440mg, 2.80mmol) into a reaction flask, adding 40ml of mixed solvent of tetrahydrofuran/water (v/v ═ 9:1), stirring uniformly, adding 8-acetonyldihydroberberine (1.0g, 2.54mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature and then filtered to obtain 1.1g of compound 1 as a yellow solid with a yield of 88.41%. ¹ H-NMR(400MHz,DMSO-d ₆ )δ：9.90(s,1H,ArH),8.94(s,1H,ArH),8.44(s,1H,ArH),8.36(s,1H,ArH),8.20(d,J＝9.2Hz,1H,ArH),8.00(d,J＝9.2Hz,1H,ArH),7.80(s,1H,ArH),7.09(s,1H,ArH),6.17(s,2H,OCH ₂ O),4.94(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),4.09(s,3H,ArOCH ₃ ),4.07(s,3H,ArOCH ₃ ),3.21(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),2.29(s,3H,ArCH ₃ )。 ¹³ C-NMR(150MHz,DMSO-d ₆ )δ:163.8,155.7,150.3,149.7,147.6,145.9,145.4,143.5,141.6,137.4,132.9,132,4,130.6,126.6,123.4,121.3,120.3,120.1,108.3,105.4,102.0,61.8,56.9,55.1,26.2,13.6.HR-ESI-MS(pos.):336.12302[M-C ₆ H ₅ N ₂ O ₃ ] ⁺ (calc.for C ₂₀ H ₁₈ NO ₄ ,336.12303)。

Example 2 Synthesis and Structure identification data of Compound 2 of the invention

Synthesis of 8-acetonyldihydroplatine (omitted).

Weighing 2-methyl-5-carboxy-N ¹ Adding pyrazine oxide (768mg, 4.88mmol) into a reaction flask, adding 40ml of mixed solvent of tetrahydrofuran/water (v/v ═ 9:1), stirring uniformly, adding 8-acetonyl dihydropalmatine (1.0g, 2.44mmol), heating for reaction until the reaction of the raw materials is complete, and stopping heating. The reaction mixture was cooled to room temperature and filtered to obtain 944mg of compound 2 as a yellow solid with a yield of 76.50%. ¹ H-NMR(400MHz,DMSO-d ₆ )δ：9.90(s,1H,ArH),9.03(s,1H,ArH),8.44(s,1H,ArH),8.37(s,1H,ArH),8.21(d,J＝9.2Hz,1H,ArH),8.03(d,J＝9.2Hz,1H,ArH),7.72(s,1H,ArH),7.10(s,1H,ArH),4.96(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),4.10(s,3H,ArOCH ₃ ),4.07(s,3H,ArOCH ₃ ),3.94(s,3H,ArOCH ₃ ),3.87(s,3H,ArOCH ₃ ),3.23(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),2.29(s,3H,ArCH ₃ )。 ¹³ C-NMR(150MHz,DMSO-d ₆ )δ:163.9,155.6,151.5,150.2,148.7,146.0,145.4,143.6,141.7,137.7,133.1,132.5,128.6,126.8,123.4,121.3,119.8,118.9,111.3,108.8,61.8,57.0,56.1,55.8,55.3,25.9,13.7.HR-ESI-MS(pos.):352.15430[M-C ₆ H ₅ N ₂ O ₃ ] ⁺ (calc.for C ₂₁ H ₂₂ NO ₄ ,352.15433)。

Example 3 Synthesis and Structure identification data of Compound 3 of the invention

Synthesis of 8-acetonyl dihydrocoptisine (omitted).

Weighing 2-methyl-5-carboxy-N ¹ Adding pyrazine oxide (92mg, 0.58mmol) into a reaction flask, adding 10ml of tetrahydrofuran/water (v/v ═ 9:1) mixed solvent, stirring uniformly, adding 8-acetonyl dihydrocoptisine (200mg, 0.53mmol), heating for reaction until the raw material reaction is complete, and stopping heating. The reaction mixture was cooled to room temperature and then filtered to obtain 226mg of compound 3 as a yellow solid with a yield of 90.08%. ¹ H-NMR(400MHz,DMSO-d ₆ )δ：9.96(s,1H,ArH),8.95(s,1H,ArH),8.44(s,1H,ArH),8.37(s,1H,ArH),8.03(d,J＝8.8Hz,1H,ArH),7.82(d,J＝8.8Hz,1H,ArH),7.79(s,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH ₂ O),6.17(s,2H,OCH ₂ O),4.89(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),3.20(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),2.29(s,3H,ArCH ₃ )。 ¹³ C-NMR(150MHz,DMSO-d ₆ )δ:164.0,155.1,149.7,147.6,147.0,146.1,144.5,143.8,141.9,136.8,132.6,132.3,130.5,121.7,121.0,120.9,120.4,111.6,108.4,105.3,104.4,102.0,55.1,26.2,13.7.HR-ESI-MS(pos.):320.09177[M-C ₆ H ₅ N ₂ O ₃ ] ⁺ (calc.for C ₁₉ H ₁₄ NO ₄ ,320.09173)。

Example 4 Synthesis and Structure identification data of Compound 4 of the invention

Synthesis of 8-acetonyldihydroisomerizine (Ex.).

Weighing 2-methyl-5-carboxy-N ¹ Pyrazine oxide (458mg, 2.90mmol) was put in a reaction flask, 40ml of tetrahydrofuran/water (v/v ═ 9:1) mixed solvent was added, after stirring uniformly, 8-acetonyldihydroisoxanthine base (1.0g, 2.65mmol) was added, and the reaction was heated until the reaction of the starting materials was completed, and then the heating was stopped. The reaction mixture was cooled to room temperature and filtered to obtain 1.084g of compound 4 as a yellow solid in 86.37% yield. ¹ H-NMR(400MHz,DMSO-d ₆ )δ：9.56(s,1H,ArH),8.75(s,1H,ArH),8.45(s,1H,ArH),8.37(s,1H,ArH),7.75(s,1H,ArH),7.73(s,1H,ArH),7.53(s,1H,ArH),7.10(s,1H,ArH),6.42(s,2H,OCH ₂ O),6.17(s,2H,OCH ₂ O),4.76(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),3.19(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),2.30(s,3H,ArCH ₃ )。 ¹³ C-NMR(150MHz,DMSO-d ₆ )δ:163.8,155.8,155.6,150.8,149.9,147.5,146.0,145.8,141.7,138.6,138.5,132.4,130.7,123.4,120.2,118.8,108.4,105.3,103.8,103.6,102.5,102.0,54.3,26.3,13.6.HR-ESI-MS(pos.):320.09180[M-C ₆ H ₅ N ₂ O ₃ ] ⁺ (calc.for C ₁₉ H ₁₄ NO ₄ ,320.09173)。

Example 5 Synthesis and Structure identification data of Compound 5 of the invention

Weighing isoberberine chloride quaternary ammonium salt (500mg, 1.34mmol) into a reaction bottle, adding 5N sodium hydroxide aqueous solution (3ml), then dropwise adding acetone (1ml, 13.5mmol), stirring at room temperature for 4h, and completely reacting the raw materials. And (3) carrying out suction filtration on the reaction liquid to obtain light yellow solid 8-acetonyl dihydro-iso-berberine, and directly using the product in the next reaction without purification.

Weighing 2-methyl-5-carboxy-N ¹ Adding pyrazine oxide (205mg, 1.33mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (V/V ═ 9:1,25ml), and uniformly stirring; adding the unpurified product at room temperature under stirring; carrying out reaction under the condition of heating until the raw materials are completely reacted; the heating was stopped. The reaction mixture was cooled to room temperature and then filtered to obtain 530mg of compound 5 as a yellow solid with a yield of 80.55%. ¹ H-NMR(400MHz,CD ₃ OD)δ：9.31(s,1H,ArH),8.67(s,1H,ArH),8.53(s,1H,ArH),8.52(s,1H,ArH),7.61(s,1H,ArH),7.59(s,1H,ArH),7.57(s,1H,ArH),6.94(s,1H,ArH),6.10(s,2H,OCH ₂ O),4.79(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),4.12(s,3H,ArOCH ₃ ),4.05(s,3H,ArOCH ₃ ),3.23(t,J＝6.4Hz,2H,NCH ₂ CH ₂ ),2.44(s,3H,ArCH ₃ )。 ¹³ C-NMR(100MHz,CD ₃ OD)δ：168.6,160.0,154.6,153.5,152.2,149.8,148.1,146.2,145.9,140.4,138.9,134.92,131.9,124.2,121.9,119.5,109.4,107.3,106.5,106.4,103.7,57.5,57.1,56.3,28.3,14.4.HR-ESI-MS(pos.):336.12222[M-C ₆ H ₅ N ₂ O ₃ ] ⁺ (calc.for C ₂₀ H ₁₈ NO ₄ ,336.12303)。

Second, Experimental example for solubility detection of Compound of the present invention

Experimental example 1, Experimental example for Water solubility detection of Compound of the present invention

Weighing a certain amount of each compound of the invention and berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, berberine chloride quaternary ammonium salt, isoflavine chloride quaternary ammonium salt and isoberberine chloride quaternary ammonium salt which are used as berberine type alkaloid quaternary ammonium salt substrates respectively, placing the compounds in a certain amount of pure water solvent at 25 +/-2 ℃, shaking strongly for 30 seconds every 5 minutes, and observing the dissolution condition within 30 minutes, wherein the compounds are considered to be completely dissolved if no visible solute particles exist.

The experimental results are as follows: the solubility is measured at 25 ℃ +/-2 ℃, and the amount of berberine 2-methyl-N dissolved in each milliliter of purified water is ¹ Pyrazine oxide-5-carboxylic acid Quaternary ammonium salt (1)342mg, palmatine 2-methyl-N ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt (2)425mg coptisine 2-methyl-N ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt (3)5mg, isoxadrine 2-methyl-N ¹ 42mg of (4) Quaternary ammonium salt of (5) -Oxyrazine-Carboxylic acid, Isoberberine 2-methyl-N ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt (5)3.3 mg. The soluble amounts of berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, berberine chloride quaternary ammonium salt, isoberberine chloride quaternary ammonium salt and isoberberine chloride quaternary ammonium salt which are used as berberine type alkaloid quaternary ammonium salt substrates in each milliliter of purified water in parallel measurement are respectively 2mg, 21mg,<1mg、<1mg、<1mg。

Experimental example 2 solubility test of Compound of the present invention in 95% ethanol solvent

Weighing a certain amount of berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, berberine chloride quaternary ammonium salt, isoberberine chloride quaternary ammonium salt and isoberberine chloride quaternary ammonium salt which are used as berberine type alkaloid quaternary ammonium salt substrates respectively, placing the weighed materials in a certain amount of 95% ethanol solvent at 25 +/-2 ℃, shaking strongly for 30 seconds every 5 minutes, and observing the dissolution condition within 30 minutes, wherein if no visible solute particles exist, the materials are considered to be completely dissolved.

The experimental results are as follows: the solubility was measured at 25 ℃. + -. 2 ℃ and the amounts of berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, coptisine chloride quaternary ammonium salt, isoberberine chloride quaternary ammonium salt and isoberberine chloride quaternary ammonium salt dissolved in 95% ethanol solvent per ml were 11mg, 15mg, <1mg and <1mg, respectively.

According to the solubility of each berberine chloride type alkaloid quaternary ammonium salt substrate, 22mg, 30mg, 2mg and 2mg of the compound 1-5 are respectively weighed, the compound is placed in 1.8ml of 95% ethanol solvent at the temperature of 25 +/-2 ℃, strong shaking is carried out for 30 seconds every 5 minutes, the dissolution condition within 30 minutes is observed, and if no visible solute particles exist, the compound is considered to be completely dissolved. Experimental results show that the amount of the compounds 1-5 dissolved in 95% ethanol per milliliter is respectively more than the amount of the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate dissolved in 95% ethanol per milliliter.

Third, evaluation of the Effect of the Compounds of the present invention on the growth of Normal cells

Experimental example 3 evaluation of the Effect of the compounds of the present invention on the growth of mouse monocyte macrophage RAW264.7 cells cultured in vitro.

(1) Preparation of the compound: compounds were formulated in DMSO to 1X 10 ^-2 mol/L (0.01M) compound stock solution, when cell experiments are to be carried out, is diluted to 20. mu.M starting solution in DMEM high-glucose medium containing 10% FBS and diabodies (streptomycin 100. mu.g/mL, penicillin 100U/mL).

(2) Experimental method (CCK-8 method): RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS and diabodies (streptomycin 100. mu.g/mL, penicillin 100U/mL) at 37 ℃/5% CO according to the cell instructions ₂ And after the cells are fused to 80%, carrying out subculture on the cells according to the dilution ratio of 1:3 in volume ratio.

Taking RAW264.7 cells with good growth state and in logarithmic growth phase, discarding original culture medium, adding DMEM high-sugar medium containing 10% FBS and double antibodies (streptomycin 100 μ g/mL, penicillin 100U/mL), gently blowing and beating to obtain cell suspension, and adjusting cell number of the cell suspension to 1 × 10 ⁵ one/mL, seeded in 96-well cell culture plates, 100. mu.l cell suspension per well. The wells around each 96-well plate were filled with PBS to prevent "edge effects". Place the cell culture plate in 5% CO ₂ And cultured in an incubator at 37 ℃ until the cells grow completely adherent (overnight).

The compound starting solution was diluted with DMEM high-glucose medium containing 10% FBS and diabodies (streptomycin 100. mu.g/mL, penicillin 100U/mL) to a series of concentration gradients of 20. mu.M, 2. mu.M, 0.2. mu.M and 0.02. mu.M, respectively. The compound series concentration gradient solution is respectively added into a 96-well plate containing 100 mu l of cell suspension, each well is 100 mu l, and 100 mu l is respectively absorbed after being evenly blown, so as to obtain the series concentration gradient with the action concentration of 10 mu M, 1 mu M, 0.1 mu M and 0.01 mu M respectively. 6 duplicate wells were set for each dosing concentration. Setting a normal cell control group and a blank control group at the same time, wherein the normal cell control group contains cells cultured by the same operation and the same amount of drug dissolution medium, but does not contain the compound of the invention; the blank control group was identical to the normal control group except that it contained no cells. After 24h of treatment in the incubator, 10. mu.L of CCK-8 solution was added to each well, incubated in the incubator for 2h, and the Optical Density (OD) was measured at 450nm using a microplate reader. The survival rate was calculated using the following formula:

cell viability (%) (administered well OD value-blank well OD value)/(control well OD value-blank well OD value) x 100%

(3) As a result: within the time range determined by experiments, the compound has no cytotoxicity on RAW264.7 cells and has certain proliferation promoting effect. Each assay cell viability and IC ₅₀ The values are shown in Table 1.

The results of the experiment were analyzed using GraphPad Prism version 5.0 software.

TABLE 1 cell viability and IC following treatment of Normal RAW264.7 cells with Compounds of the invention ₅₀ Value of

(4) And (4) conclusion: the series of compounds of the invention have no inhibition effect on the growth of RAW264.7 cells of normal mouse mononuclear macrophages cultured in vitro and have the activity of promoting the proliferation of RAW264.7 cells.

Experimental example 4 evaluation of cytotoxicity of the Compound of the present invention on 293T cells, which were cultured in vitro, were performed on normal human embryonic kidney epithelial cells.

(1) Preparation of the compound of the invention: the compound of the invention is dissolved and prepared into 1X 10 by DMSO ^-2 mol/L (0.01M) stock solution, to be diluted to 1X 10 with cell culture medium for cell experiments ^-5 Working solution concentration of mol/L (i.e., 10. mu.M).

(2) Experimental method (MTT method): will pressCell instruction Normal human embryonic Kidney epithelial cells 293T cells grown to 90% confluency were cultured in vitro and digested with 0.25% trypsin/0.1 EDTA, then made into cell suspension with cell culture Medium and adjusted for cell suspension concentration, and inoculated into 96-well cell culture plates with 100. mu.l cell suspension per well and cell density of 2X 10 ³ And (4) respectively. The wells around each 96-well plate were filled with PBS to prevent "edge effects. Place the cell culture plate in 5% CO ₂ Incubate at 37 ℃ until cells grow fully adherent (overnight). And (3) absorbing and removing the supernatant of the original culture medium in the hole, adding 100 mu l of the compound working solution of the invention into each hole, and continuously placing the cell culture plate in an incubator for incubation for 72 hours. Mu.l of 5mg/ml MTT solution was added to each well, the culture was terminated after 4 hours, the medium in each well was carefully aspirated, 100. mu.l DMSO was added to each well, the mixture was placed on a shaker and shaken at a low speed for 10 minutes to dissolve the crystals sufficiently, and the OD at 490nm was measured in each well using a microplate reader. In the experiment, 3 repeated holes are set for each test, and a normal control and a blank control are set simultaneously; the normal control group is a well containing cells cultured in the same procedure, the same amount of culture medium, drug dissolution medium, MTT and DMSO, but not containing the compound of the present invention, and the blank control group is identical to the normal control group except that it does not contain cells. The survival rate was calculated using the following formula:

cell survival (%) < 100%

(3) As a result: within the time range of experimental determination, the series of compounds of the invention evaluated have no obvious cytotoxicity to 293T normal cell line cells at the action concentration of 10 μ M, except that the cell survival rate of the compound 1 is 89.7%, the cell survival rates of other compounds 2-4 of the invention are all determined to be more than 100%, and no significant difference is detected statistically.

Experimental example 5. the compounds of the present invention were used in the evaluation of cytotoxicity on HELF cells cultured in vitro in normal human embryonic lung fibroblasts and NIH3T3 cells in mouse embryonic fibroblasts.

(1) Preparation of the compound of the invention: the compound of the invention is dissolved and prepared into 1X 10 by DMSO ^-2 mol/L (0.01M) stock solution to be refinedFor the cell experiments, the cells were diluted to a starting solution concentration of 20. mu.M with cell culture medium.

(2) Experimental method (MTT method): primary cell culture, digestion, preparation of cell suspension, cell counting, seeding of cell suspension (100. mu.l) in 96-well cell culture plates, in 5% CO ₂ And culturing in a 37 ℃ cell culture box until the cells grow completely adherent. The compound starting solution was diluted with cell culture medium to a series of concentration gradient solutions of 20. mu.M, 2. mu.M, 0.2. mu.M, 0.02. mu.M and 0.02. mu.M, respectively. The compound series concentration gradient solution is respectively added into a 96-well plate, each well is 100 mu l, then 100 mu l is respectively absorbed and discarded, so as to obtain series concentration gradients with the action concentration of 10 mu M, 1 mu M, 0.1 mu M, 0.01 mu M and 0.001 mu M, wherein 3 duplicate wells are arranged for each administration concentration, and a normal cell control group and a blank control group are arranged at the same time. After incubation for 96h in an incubator, the OD value of each compound was measured by MTT method, and the inhibition rate was calculated using the following formula:

cell growth inhibition (%) { [ (control well OD value-blank well OD value) - (administration well OD value-blank well OD value) ]/[ (control well OD value-blank well OD value ] } × 100%

(3) As a result: within the time range of 96h determined by experiments, the compound has no obvious cytotoxicity on human embryonic lung fibroblast HELF cells and mouse embryonic fibroblast NIH3T3 cells, and the determined experimental cell growth inhibition rate and compound IC ₅₀ The results are shown in Table 2.

TABLE 2 toxicity test results of the compounds of the present invention on normal HELF and NIH3T3 test cells

(4) And (4) conclusion: the compound has no obvious toxicity on the growth of human embryonic lung fibroblast HELF cells and mouse embryonic fibroblast NIH3T3 cells, and is suitable for downstream activity screening experiments.

Fourth, evaluation of biological Activity of the Compound of the present invention at cellular level

Experimental example 6: experimental example for evaluating the inhibitory Effect of the Compound of the present invention on NO production in RAW264.7 cell inflammation model induced by LPS stimulation

In the experiment, a Griess method is adopted to detect the influence of the compound on the NO secretion amount in an RAW264.7 cell inflammation model stimulated by LPS.

Taking RAW264.7 cells which are cultured by adopting a DMEM high-sugar medium containing FBS and double antibiotics (streptomycin and penicillin) and have good growth state and are in logarithmic growth phase, adding a proper amount of the medium, slightly blowing and beating to prepare cell suspension, and adjusting the cell density of the cell suspension to be 6 x 10 ⁵ one/mL, seeded in 96-well cell culture plates, with a cell suspension amount of 100. mu.l per well. Place the cell culture plate in 5% CO ₂ The cells were cultured in a 37 ℃ incubator until they grew completely adherent (overnight), and good cell morphology was observed. Experimental groups were set up on a 96-well plate, including a group to which the compound of the present invention was administered, a LPS stimulation model group, a normal cell control group, and a blank group, respectively. The intervention experiment cell suspensions of the compounds are prepared according to the operation method of the administration experiment (see 'experiment example 3, the experiment for evaluating the influence of the compounds of the invention on the growth condition of mouse mononuclear macrophage RAW264.7 cells cultured in vitro') in each group of the compounds of the invention according to the experiment group, different working concentrations are set according to specific experiments in each experiment, and the total volume of each hole is still controlled to be 100 mul. After pre-incubation of the 96-well plate in a cell culture incubator for 2 hours, LPS was added to each of the compound group and the model group in an amount of 1. mu.g/mL for stimulation. Placing the 96-well plate in a cell culture box for continuous culture, collecting 50 μ L of supernatant in each well after 24 hours, adding into a new 96-well plate, adding 50 μ L of Griess A according to the operation procedure set in the instruction of the nitric oxide detection kit, adding 50 μ L of Griess B after 1min, continuing to react for 15min, measuring OD value at 540nm on a microplate reader, and using NaNO in advance ₂ On the basis of establishing a standard curve, the concentration of sodium nitrite in the sample is calculated, and the content of NO in the cell culture solution of each group is converted (see table 3).

The experimental results show that the compounds of the present invention can dose-dependently inhibit the production of NO in the model of RAW264.7 cell inflammation induced by LPS stimulation after the intervention treatment with each of the compounds of the present invention (table 3).

TABLE 3 Effect of the Compounds of the present invention on the amount of NO secretion in RAW264.7 cell inflammation model induced by LPS stimulation

In comparison with the normal group, ^### p is less than 0.001; in comparison with the set of models, ^*** P＜0.001

experimental example 7: experimental example for evaluating the inhibitory Effect of the Compound of the present invention on NO production in RAW264.7 cell inflammation model induced by LPS stimulation

Taking RAW264.7 cells which are cultured by a DMEM high-sugar medium containing FBS, streptomycin and penicillin and have good growth state and logarithmic growth phase, adding a proper amount of the medium, slightly blowing and beating to prepare cell suspension, and adjusting the cell density of the cell suspension to be 6 x 10 ⁵ one/mL, seeded in 96-well cell culture plates, with a cell suspension amount of 100. mu.l per well. Place the cell culture plate in 5% CO ₂ The cells were cultured in a 37 ℃ incubator until they grew adherent to the wall (overnight) and were observed to have good morphology. Experimental groups were set up on a 96-well plate, including a group to which the compound of the present invention was administered, a LPS stimulation model group, a normal cell control group, and a blank group, respectively. According to the experimental grouping, in the group given the compound of the present invention, the operation method of the administration experiment (see "experimental example 3, evaluation experiment of the effect of the compound of the present invention on the growth of mouse mononuclear macrophage RAW264.7 cells cultured in vitro") was carried out to prepare each intervening experimental cell suspension containing the compound of the present invention by the half-fold dilution method using the working concentration of the compound of the present invention of 5.0. mu.M as the starting concentration, and the total volume per well was controlled to be still 100. mu.l. After pre-incubation of 96-well plates in a cell culture incubator for 2 hours, LPS stimulation was added to each of the compound group and the model group to which the present invention was administered, and LPS was addedThe amount was 1. mu.g/mL. Placing the 96-well plate in a cell culture box for continuous culture, collecting 50 μ L of supernatant in each well after 24 hours, adding into a new 96-well plate, adding 50 μ L of Griess A according to the experimental operation procedure provided by the instruction of the nitric oxide detection kit, adding 50 μ L of Griess B after 1min, continuing to react for 15min, measuring OD value at 540nm on a microplate reader, and using NaNO in advance ₂ On the basis of establishing a standard curve, the concentration of sodium nitrite in the sample is calculated, and the content of NO in the cell culture solution of each group is converted (see table 4). The inhibition of NO secretion was calculated as follows:

inhibition { [ (model NO amount-control NO amount) - (administration NO amount-control NO amount) ]/[ (model NO amount-control NO amount ] } × 100%

The results of the experiments show that the dose-dependent inhibition of NO production is possible after the intervention treatment with the respective compounds of the invention (Table 4). The inhibitory effect of the compounds of the present invention on NO production in the initial passage RAW264.7 cell inflammation model induced by LPS stimulation is not a conventional linear dose-dependent relationship, but rather a unique irregular dose-dependence (table 4). This suggests that the pharmacological actions of the compounds of the present invention have unique mechanisms of action and are in need of further study.

TABLE 4 Effect of Compounds of the invention on NO secretion in LPS stimulated Primary passage RAW264.7 cell inflammation model

^a 0. In comparison with the normal group, ^### p is less than 0.001; in comparison with the set of models, ^* P＜0.05， ^** P＜0.01； ^*** P＜0.001。

experimental example 8 evaluation of efficacy of the Compound of the present invention in reducing IL-6 secretion level in RAW264.7 cell inflammation model induced by LPS stimulation

The experiment adopts an ELISA method to detect the influence of the compound on the IL-6 secretion amount in an RAW264.7 cell inflammation model induced by LPS stimulation.

According to the sample amount set by the experimental operation program provided by the specification in the mouse IL-6 ELISA kit, according to the cytotoxicity and other data of the compound determined by the experiment, the cell supernatant sample of the RAW264.7 cell inflammation model induced by LPS stimulation prepared according to the operation program of 'Experimental example 6, the inhibition efficacy evaluation experiment example of the compound of the invention on NO production in the RAW264.7 cell inflammation model induced by LPS stimulation' is added into the ELISA plate hole containing the target protein specific monoclonal capture antibody provided by the mouse IL-6 ELISA kit, and the IL-6 content in the cell culture supernatant of each hole is determined according to the operation program provided by the kit specification.

The results are shown in tables 5 and 6. As can be seen from the experimental data in tables 5 and 6, the expression level of IL-6 in the model group is significantly increased with reference to the expression level of IL-6 in the normal control group, and has a statistically significant difference compared with the normal control group. After the compound is treated, the expression level of IL-6 in an RAW264.7 cell inflammation model induced by LPS stimulation is obviously reduced, and compared with a model group, the compound 2 treatment group has obvious difference on the inhibition effect of the expression level of IL-6 and is obviously stronger than a corresponding berberine type alkaloid quaternary ammonium salt substrate.

TABLE 5 Effect of the Compounds of the present invention on IL-6 secretion in a model of inflammation of RAW264.7 cells induced by LPS stimulation ^{Note that}

^{Note that} In comparison with the normal group, ^### p is less than 0.001; p < 0.01 compared to model group.

TABLE 6 influence of the Compounds of the invention on the amount of IL-6 secretion in a model of inflammation of RAW264.7 cells induced by LPS stimulation ^{Note that}

^{Injection bottle} And normal groupIn comparison, the comparison result is obtained, ^### p is less than 0.001; in comparison with the set of models, ^** P＜0.01, ^*** p is less than 0.001; compared with the palmatine chloride quaternary ammonium salt group, ^& P＜0.05。

experimental example 9 and Experimental example of the inhibitory effect of the Compound of the present invention on the growth of tumor cells.

The experimental result shows that the compound can inhibit the growth of human colon cancer HCT-116 cells, human lung cancer A549 cells and human liver cancer Hepg2 cells in a dose-dependent and selective manner.

(1) Inhibition of HCT-116 cell growth by compounds of the invention

Each compound of the present invention was prepared in DMSO as a stock solution at a concentration of 0.02M, and when cell experiments were performed, it was diluted with a cell culture medium (1640 medium containing 10% serum) to a starting solution at a concentration of 20. mu.M.

Primary cell culture with cell culture Medium according to cell instructions, preparation of cell suspension, cell counting, seeding of cell suspension (100. mu.l) on 96-well cell culture plates, in 5% CO ₂ And culturing in a 37 ℃ cell culture box until the cells grow completely adherent. The compound starting solution was diluted with cell culture medium to a series of concentration gradient solutions of 20. mu.M, 2. mu.M and 0.2. mu.M, respectively. The compound series concentration gradient solution is respectively added into a 96-hole cell culture plate, each hole is 100 mu l, 100 mu l is respectively sucked from each hole after the compound series concentration gradient solution is evenly blown and beaten, the series concentration gradients of the compound of the invention with the action concentrations of 10 mu M, 1 mu M and 0.1 mu M are obtained, 3 compound holes are arranged for each administration concentration, and a normal cell control group and a blank control group are simultaneously arranged. After incubation in an incubator for 96h, the OD value at 570nm of each compound was measured by MTT method, and the inhibition ratio was calculated using the following formula:

The compounds of the series of the invention have significant cytostatic activity on HCT-116 cells over a period of 96h determined experimentally, and the compounds 1, 3 and 4 of the invention tested have growth inhibitory effects on HCT-116 cellsIC ₅₀ Values were 2.637 μ M, 0.594 μ M and 2.462 μ M, respectively; in parallel experiments, IC of growth inhibition of HCT-116 cells by berberine chloride, berberine chloride and isoxadrine chloride ₅₀ The values were 4.496. mu.M, 2.087. mu.M and 2.512. mu.M, respectively.

(2) Inhibition of A549 cell growth by the compounds of the invention

The compounds of the present invention were prepared in DMSO as a 0.02M stock solution of the compounds, and diluted with a cell culture medium to a concentration of 20. mu.M as a starting solution for cell experiments.

Primary cell culture with cell culture Medium according to cell instructions, preparation of cell suspension, cell counting, seeding of cell suspension (100. mu.l) on 96-well cell culture plates, in 5% CO ₂ And culturing in a 37 ℃ cell culture box until the cells grow completely adherent. The initial solution of the compound of the present invention was diluted with the cell culture medium to a series of concentration gradient solutions having concentrations of 20. mu.M, 2. mu.M and 0.2. mu.M, respectively. The compound series concentration gradient solution is respectively added into a 96-hole cell plate, each hole is 100 mu l, 100 mu l is respectively sucked from each hole after being blown evenly, so as to obtain series concentration gradients with the action concentrations of 10 mu M, 1 mu M and 0.1 mu M, 3 compound holes are arranged for each administration concentration, and a normal cell control group and a blank control group are arranged at the same time. After incubation in an incubator for 96h, the OD value at 570nm of each compound was measured by MTT method, and the inhibition rate was calculated. Within the length range of 96h determined by experiments, the compounds of the invention have obvious cell growth inhibition activity on A549 cells, and the detected IC of the growth inhibition effect of the compounds 1, 3 and 4 of the invention on the A549 cells ₅₀ The values were 4.378. mu.M, 3.497. mu.M and 0.919. mu.M, respectively. In parallel experiments, IC of berberine chloride quaternary ammonium salt and berberine chloride quaternary ammonium salt on growth inhibition of A549 cells ₅₀ Values were > 10. mu.M and 6.540. mu.M, respectively.

(3) Inhibition of HepG2 cell growth by Compounds of the invention

Each compound of the present invention was prepared as a stock solution at a concentration of 0.02M in DMSO, and when cell experiments were performed, it was diluted with a cell culture medium (1640 medium containing 10% serum) to a starting solution at a concentration of 20. mu.M.

Primary cell culture with cell culture Medium according to cell instructions, preparation of cell suspension, cell counting, seeding of cell suspension (100. mu.l) on 96-well cell culture plates, in 5% CO ₂ And culturing in a 37 ℃ cell culture box until the cells grow completely adherent. The compound starting solution was diluted with cell culture medium to a series of concentration gradient solutions of 20. mu.M, 2. mu.M and 0.2. mu.M, respectively. The compound series concentration gradient solution is respectively added into a 96-hole cell culture plate, each hole is 100 mu l, after being blown evenly, the compound series concentration gradient solution is respectively sucked and discarded from each hole by 100 mu l, so as to obtain series concentration gradients with the action concentrations of 10 mu M, 1 mu M and 0.1 mu M, 3 compound holes are arranged for each administration concentration, and a normal cell control group and a blank control group are arranged at the same time. After incubation for 96h in an incubator, the OD value of each compound at a wavelength of 570nm was measured by MTT method, and the inhibition rate was calculated using the following formula:

cell growth inhibition ratio (%) { [ (control well OD value-blank well OD value) - (administration well OD value-blank well OD value) ]/[ (control well OD value-blank well OD value ] } × 100%

IC of the growth inhibition of HepG2 cells by the compounds 3 and 4 according to the invention, determined over a period of 96h ₅₀ Values of 2.768 μ M and 1.313 μ M, respectively; in parallel experiments, IC of berberine chloride quaternary ammonium salt, coptisine chloride quaternary ammonium salt and isoflavine chloride quaternary ammonium salt on growth inhibition of Hepg2 cells ₅₀ Values were > 10. mu.M, 9.562. mu.M and 4.475. mu.M, respectively.

Fifth, evaluation of biological Activity of the Compound of the present invention at animal level

The compound can be used for preparing immunomodulator medicines, medicines for preventing, relieving and/or treating diseases causing the increase of IL-6 secretion of a biological body, medicines for preventing, relieving and/or treating diseases causing the increase of NO secretion of the biological body and medicines for preventing, relieving and/or treating diseases causing the increase of autoantibodies formed by the biological body. The use of the compound of the present invention for preparing an immunomodulator medicament and the use for preparing a medicament for preventing, alleviating and/or treating diseases causing an increase in NO secretion of a living organism are demonstrated in the aforementioned experimental examples or partially demonstrated in the foregoing, and the compound of the present invention is used for preparing a medicament for preventing, alleviating and/or treating diseases causing an increase in IL-6 secretion of a living organism, preparing a medicament for preventing, alleviating and/or treating diseases causing an increase in autoantibody formation of a living organism, preparing a medicament for preventing, alleviating and/or treating cardiovascular and cerebrovascular diseases and blood system diseases, and preparing a medicament for preventing, alleviating and/or treating autoimmune diseases. Meanwhile, the experimental examples further complement and explain the important biological significance of the compound of the invention on balancing the immune response of the organism, so the compound can be used for preparing immunomodulator medicines. The biological functions of the compound are considered comprehensively, and the application value of the compound in preparing the medicine for preventing, relieving and/or treating viral infection diseases caused by various reasons and complications thereof, autoimmune diseases, cardiovascular and cerebrovascular diseases, blood system diseases, inflammatory diseases, fever diseases, neoplastic diseases, respiratory system diseases and primary glomerulonephritis diseases is proved. Experimental example 10 evaluation of efficacy of the Compound of the present invention in reducing IL-6 level in model animal Experimental example 1 and test Material

(1) Experimental animals:

7-8 weeks old DBA/1 mouse, male, 18-20g body weight.

(2) Test compounds and reagents of the invention:

the tested compound of the invention is compound 2, and the positive drug is methotrexate. Other agents include chicken type II collagen, complete freund's adjuvant, incomplete freund's adjuvant, Phosphate Buffered Saline (PBS).

According to the weight of the experimental animal, the compound 2 of the invention is prepared by adopting 0.5% CMC-Na aqueous solution as a solvent to prepare the compound administration working solution with proper concentration, and the positive control medicament is prepared by adopting 0.5% CMC-Na aqueous solution as a solvent to prepare the positive control medicament administration working solution with proper concentration. The dose volumes were 1ml/100g (volume of working solution administered/body weight of animal) and administered intragastrically to experimental DBA/1 mice.

2. Test method

(1) Preparation of animal models

Feeding DBA/1 mice into an experimental animal room in an environment allowing free drinking water, wherein the room temperature of the experimental animal room is 22-25 ℃, the relative humidity is 55-65%, and the illumination period is 12h/12 h. Animals were first acclimatized for one week after purchase.

The initial immunization of the animals is recorded as the 0 th day, and the method comprises the steps of injecting 0.1ml of collagen and an emulsifying agent prepared by complete Freund's adjuvant through ultrasonic treatment according to the volume ratio of 1:1 into the root 2-3 cm of the tail of the animals at multiple points in the skin, and injecting 0.1ml of normal saline into a blank control group. Inflammation was then induced on day 21 after the primary immunization by boosting with collagen and incomplete adjuvant by injecting 0.05ml of ultrasound formulated emulsion at multiple points under the tail subcutaneous avoiding the primary immunization site in a volume ratio of 1: 1. The development of swelling in any of the ankle, sole and toe of the mice after the 4 th week of primary immunization was considered to be the onset of an inflammatory model in mice.

(2) Animal grouping and administration

In this experiment, animals were randomly divided into 4 groups, i.e., a blank control group (normal control group), a model group, a positive drug control group, and a test group to which the compound 2 of the present invention was administered. The administration dose of the compound administration group of the present invention was set to 100mg/kg (amount of compound/body weight of animal), and the number of administrations and the administration route were set to 1 time/day, p.o.. The dose, frequency and route of administration of the positive drug methotrexate were set to 1mg/kg (amount of compound/body weight of animal), 2 times per week, p.o..

The administration was started on day 18 after the second immunization from the mouse model, and was terminated after 4 weeks of continuous administration until the degree of inflammation was significantly reduced in the administration group as compared with the model group. A blank solvent is administered to the blank control group and the model group by intragastric administration, the compound 2 group tested by the invention is administered with the compound administration working solution with corresponding dose by intragastric administration, and the positive control medicine group is administered with the positive control medicine administration working solution with corresponding dose by intragastric administration.

(3) Evaluation index

At the end of the experiment, anesthetizing the Mouse, picking up eyeballs, collecting blood samples of animals of each group through eyesockets, anticoagulating, taking anticoagulation, detecting the change condition of the IL-6 level of inflammatory cytokines in the serum of the Mouse by adopting a Mouse IL-6 ELISA Kit method, detecting the expression condition of anti-chicken type II collagen antibodies in the serum by adopting an ELISA method, evaluating the change condition of the hemoglobin level by adopting a conventional blood detection experiment, and comparing the experiment results of different groups through statistical treatment.

(4) Data processing and analysis

Analysis was performed using GraphPad Prism version 5.0 software and presented graphically.

3. Results of the experiment

Compared with the blank group, the IL-6 level in the serum of each detected mouse in the model group is obviously increased, and the statistical difference is provided, which indicates that the model building is successful; compared with the model group, the methotrexate positive medicine group and the compound 2 administration group of the invention have obviously reduced IL-6 level in the serum of each detected mouse, and have statistical difference. The compound 2 administration group of the invention has better inhibition effect on IL-6 level in serum of a model mouse than a positive drug. The results are shown in Table 7.

TABLE 7 Effect of the compounds of the invention tested on IL-6 concentration in serum of model mice (pg/mL).

Note: compared with the normal control group, the composition has the advantages that, ^### p<0.001; comparing with model group<0.01，***p<0.001。

Experimental results show that the tested compound has extremely obvious inhibition effect on the level of IL-6 in serum of a model mouse.

Experimental example 11 evaluation of Effect of test Compound of the present invention on model mouse autoantibodies

Autoimmune diseases are a type of chronic disease caused by the damage of tissues due to the production of autoantibodies. This study examined the effect of tested compound 2 of the invention on model mouse autoantibodies. The serum of the test mouse was diluted at a ratio of 1:1000 using a serum diluent prepared in the kit, and the expression of the anti-chicken type II collagen antibody in the diluted serum was detected by ELISA, the data of which are shown in table 8. As seen from the results, the concentration of the anti-chicken type II collagen IgG antibody in the serum of the model group mouse is obviously increased compared with that of the blank control group; compared with the model group, the concentrations of anti-chicken type II collagen IgG antibodies in the serum of mice of the positive methotrexate group and the compound 2 administration group are obviously reduced, wherein the positive drug group has obvious difference on the reduction effect of the anti-chicken type II collagen IgG antibody level compared with the model group, while the compound 2 administration group has better reduction effect on the anti-chicken type II collagen IgG antibody level than the positive drug group and has extremely obvious difference compared with the model group.

TABLE 8 Effect of Compound 2 of the present invention on IgG antibody levels against chicken type II collagen in serum from mouse chicken type II collagen-induced arthritis model mice.

Note: data are expressed as X ± SEM, n ═ 4; compared to the model group, p <0.05, p < 0.001.

Experimental example 12 evaluation of Effect of the Compound of the present invention in elevating hemoglobin content in model animal

In "Experimental example 10: efficacy evaluation of the Compounds of the invention for reduction of IL-6 levels in model animals Experimental example in the experiment, anticoagulation of each group was determined by a conventional blood assay. The detection result shows that the average value of the hemoglobin content of the blank control group mouse in the experiment is 147.29 +/-5.06 (g/L); compared with a blank control group, the hemoglobin content of the animals of the experimental model group is obviously reduced, and the average value is 136.33 +/-4.27 (g/L); the test group of compounds of the invention showed a significant increase in hemoglobin content compared to the model group, and the mean value of hemoglobin content of the compound 2 administered group of the invention was 181.17 ± 3.47 × g/L, with very significant differences compared to the model group (data expressed as X ± SEM; p <0.001 compared to the model group). Particularly, compared with a blank control group, the compound administration group has obvious effect of increasing hemoglobin, and the compound has obvious effect of increasing hemoglobin. The mean value of the hemoglobin content of the positive drug group is 160.92 +/-7.07 (g/L).

The experimental result shows that the tested compound has extremely obvious effect of increasing the hemoglobin content of a model mouse.

Sixthly, evaluation of antiviral pharmacodynamic action of the compound

Experimental example 13, Experimental example for efficacy measurement of the Compound of the present invention against 2019novel coronavirus (SARS-CoV-2)

1. Apparatus and reagents

A class II biosafety cabinet; CO 2 ₂ An incubator; inverting the microscope; 96-well cell culture plates.

SARS-CoV-2 (i.e., 2019-nCoV) virus (titer 8X 10) ⁵ TCID 50/mL); DMEM basal medium; vero E6 cells; fetal bovine serum; penicillin-streptomycin double resistance; 0.25% pancreatin-EDTA; qPCR experimental reagents; TRIzol; cell culture grade DMSO.

2. Preparation of the Compounds of the invention

The compound of the invention is firstly dissolved and prepared into 1 × 10 by taking DMSO as a solvent ^-2 mol/L (0.01M) of a stock solution of a compound of the invention. When cell experiments are carried out, the cell culture medium is diluted into working solution with required concentration.

3. Data analysis

Experimental data were statistically analyzed using GraphPad Prism version 5.0 software.

4. Experimental methods

(1) The compound of the invention is used in the evaluation experiment of the cytotoxicity on Vero E cells cultured in vitro.

1) Inoculating cells: Vero-E cells which are in good growth state and in logarithmic growth phase are taken according to cell specifications, the culture solution is sucked and removed, the cells are digested by pancreatin, a cell suspension is prepared, and the cell count is 1 multiplied by 10 ⁶ Per ml; taking 4ml of the above cells, adding 6ml of cell culture medium to obtain the final product with cell density of 4 × 10 ⁵ One/ml of the cell suspension was inoculated into a 96-well cell culture plate (100. mu.l of the cell suspension per well, 1X 10 cells in number ⁴ One); at 5% CO ₂ Culturing in a 37 ℃ cell culture box until the cells grow completely adherent.

2) Dilution of the compounds of the invention:

microscopic observation cytotoxicity experiments: complete medium was used to dilute the compound stock solution of the invention to the working solution in the corresponding concentration range.

The present invention observes the toxic effects of the compounds of the present invention on cells at action concentrations of 20. mu.M, 10. mu.M, 5. mu.M.

Half toxic concentration (CC) ₅₀ ) Detection experiment: the compound stock solution of the present invention was diluted to the corresponding concentration of working solution using complete medium.

The cytotoxicity detection of the compound is carried out under the series concentration gradients of 320 mu M, 160 mu M, 80 mu M, 40 mu M, 20 mu M, 10 mu M, 5 mu M and 0 mu M, and the CC is calculated ₅₀ The value is obtained.

3) Intervention treatment of cells with the compounds of the invention: the original cell culture medium was aspirated, the diluted medium containing the compound of the present invention at the corresponding concentration was added in a total volume of 100. mu.l per well, and the mixture was placed in a cell culture chamber at 37 ℃ and cultured for another 48 hours. A normal cell control group and a blank control group are simultaneously arranged in the experiment, the normal cell control group contains cells cultured by the same operation and the same amount of drug dissolution medium, but does not contain the compound of the invention; the blank control group was identical to the normal control group except that it contained no cells.

4) The medium supernatant was discarded, 100. mu.l of serum-free medium containing 10% CCK-8 was added to each well, and after incubation in an incubator for 4 hours, the OD at 450nm was measured using a microplate reader. The cell growth inhibition rate, i.e., the cytotoxicity of the compound of the present invention, was judged from the experimental results. The formula for calculating the inhibition rate of cell growth is as follows:

(2) Evaluation of anti-SARS-CoV-2 Virus efficacy of Compounds of the invention

1) Inoculating cells: taking Vero-E cells which are in good growth state and are in logarithmic growth phase according to cell specifications, sucking and discarding culture solution, digesting the cells with pancreatin, preparing cell suspension, and counting the cells1×10 ⁶ Per ml; taking 4ml of the above cells, adding 6ml of culture medium to obtain a cell with a density of 4 × 10 ⁵ One/ml of the cell suspension was inoculated into a 96-well cell culture plate (100. mu.l of the cell suspension per well, 1X 10 cells in number ⁴ One); at 5% CO ₂ Culturing in a 37 ℃ cell culture box until the cells grow completely adherent.

(2) Dilution of the compounds of the invention: the compound stock solution of the present invention was diluted with DMEM maintenance medium (2% FBS) to a working solution of a corresponding concentration.

In different experiments, the compounds of the invention act at concentrations of 10. mu.M or 20. mu.M, 10. mu.M, 5. mu.M, 0. mu.M or 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 0. mu.M.

(3) Compounds of the invention pre-treat cells: before SARS-CoV-2 virus infection, the prepared compound working solution is used for carrying out administration intervention operation according to the action concentration of the compound set by the experiment, and the cells are pretreated for 1 hour. The cell maintenance solution for this experiment was DMEM (containing 2% FBS). Each assay was set up in 3 duplicate wells with DMSO at the corresponding concentration as a negative control and Remdesivir as a positive control. (4) Dilution of SARS-CoV-2 virus: adding 200 μ l of SARS-CoV-2 virus into 25ml of culture medium, mixing, diluting SARS-CoV-2 virus to 100TCID ₅₀ /0.05mL。

(5) The compound of the invention + SARS-CoV-2 virus treated cells: when SARS-CoV-2 virus is infected, cell culture supernatant is discarded, 50. mu.l of working solution of the compound of the present invention with 2 times of the concentration of the compound of the present invention is added into each well, and 50. mu.l of SARS-CoV-2 virus diluent is vertically dripped into each well except for cell control, wherein the total volume of each well is 100. mu.l.

(6) The above 96-well cell culture plate containing the mixture of the compound of the present invention and SARS-CoV-2 virus was placed in 5% CO ₂ Culturing at 37 deg.C for 1 hr, discarding the mixture containing the compound of the present invention and SARS-CoV-2 virus, adding 100. mu.l of maintenance medium to each well, placing in a cell culture box, and culturing for another 48 hr.

(7) And collecting cell supernatant after 48 hours, adding the cell supernatant into TRIzol for cracking, extracting RNA, and carrying out RT-qPCR quantitative detection.

The detection primer was (5 '→ 3'):

SARS-CoV-2-N-F:GGGGAACTTCTCCTGCTAGAAT

SARS-CoV-2-N-R:CAGACATTTTGCTCTCAAGCTG

5. results of the experiment

(1) The compound has no obvious toxicity to normal Vero E cells

The compound of the invention has no obvious cytotoxicity to experimental cells under the action concentration of 20 mu M, 10 mu M and 5 mu M through microscopic observation.

The cell growth inhibition rates of the cytotoxicity tests of the compound 3 of the invention under the series of concentration gradients of 320. mu.M, 160. mu.M, 80. mu.M, 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M and 0. mu.M are 78.71%, 27.77%, 8.51%, 1.98%, 1.93%, 3.14%, 1.96% and 1.44%, respectively, and the cytotoxicity CC is CC ₅₀ The assay result was 311.8. mu.M.

And (4) conclusion: the series of compounds of the invention have no obvious toxicity to normal Vero E experimental cells.

(2) The compound of the invention has obvious inhibition effect on the level of virus RNA after experimental cells are infected with SARS-CoV-2 virus

By taking the influence of the compound on the RNA level of SARS-CoV-2 virus as an index, the compound has definite and significant inhibitory action on SARS-CoV-2 virus under the action concentration adopted in experiments (10 mu M or 20 mu M, 10 mu M, 5 mu M or 40 mu M, 20 mu M, 10 mu M, 5 mu M and 2.5 mu M) and under the condition of only treating model cells for 1 hour, wherein the inhibitory rates of the compounds 7 and 8 on the RNA inhibitory action of SARS-CoV-2 virus can respectively reach 62.99%, 93.13%, 94.42% and 68.08%, 89.10% and 91.74% under the action concentrations of 5 mu M, 10 mu M and 20 mu M. Effect of Compounds of the invention on viral RNA levels and EC following infection of Experimental cells with SARS-CoV-2 Virus ₅₀ The values are shown in Table 9.

TABLE 9 Effect of Compounds of the invention on RNA levels of SARS-CoV-2 Virus infection in test cells and EC ₅₀ Value of

Meanwhile, the berberine chloride compounds detected by the invention comprise berberine quaternary ammonium chloride salts, palmatine quaternary ammonium chloride salts, berberine quaternary ammonium chloride salts, isoberberine chloride quaternary ammonium salts and isoberberine chloride quaternary ammonium salts, which can not effectively reduce the RNA level of SARS-CoV-2 virus.

(3) Conclusion

According to the above experimental results, the compound of the present invention can inhibit SARS-CoV-2 virus replication dose-dependently in a model of Vero E6 cell infection with SARS-CoV-2 virus. Wherein, under the action concentration of 10 μ M of the compound, the inhibition rates of the tested compounds 1-5 of the invention on the virus RNA level of experimental cells infected with SARS-CoV-2 virus are respectively 61.37%, 65.22%, 32.01%, 35.44% and 57.57%. Compound 3 EC for inhibiting SARS-CoV-2 viral RNA levels ₅₀ The value was 9.68. mu.M. Compound 3 had an SI value of 32.18. Thus, the compound of the invention has obvious activity of resisting SARS-CoV-2 virus.

Claims

1. Berberine type alkaloid 2-methyl-N shown in general formula I ¹ -pyrazine-5-carboxylate quaternary ammonium compounds:

R ₂ 、R ₃ each independently selected from H, substituted or unsubstituted OH, or R ₂ And R ₃ Linked as an alkylenedioxy group;

R ₉ 、R ₁₀ 、R ₁₁ each independently selected from H, substituted or unsubstituted OH, or R ₉ And R ₁₀ Linked as alkylenedioxy and R ₁₁ Independently selected from H, substituted or unsubstituted OH, or R ₁₀ And R ₁₁ Linked as alkylenedioxy and R ₉ Independently selected from H, substituted or unsubstituted OH;

the substituent of the substituted or unsubstituted hydroxyl is selected from methyl and ethyl; the alkylenedioxy is selected from methylenedioxy.

2. The berberine type alkaloid 2-methyl-N according to claim 1 ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt compounds, characterized in that said compounds are selected from the following group of compounds:

3. preparation of berberine type alkaloid 2-methyl-N ¹ -a process for the oxidation of pyrazine-5-carboxylic acid quaternary ammonium salt compounds, characterized in that said process is as follows: reacting berberine type alkaloid quaternary ammonium salt compound with acetone and sodium hydroxide water solution to obtain solid 8-acetonyl dihydroberberine type compound, and reacting with 2-methyl-5-carboxyl-N ¹ Reacting pyrazine oxide in a mixed solvent of tetrahydrofuran and water under a heating condition, and filtering the reaction mixed solution to obtain the berberine type alkaloid 2-methyl-N ¹ -pyrazine oxide-5-carboxylic acid quaternary ammonium salt compounds.

4. A pharmaceutical composition characterized by comprising an effective amount of the berberine type alkaloid 2-methyl-N according to any one of claims 1-2 ¹ -a pyrazine oxide-5-carboxylic acid quaternary ammonium salt compound and a pharmaceutically acceptable pharmaceutical carrier or excipient.

5. The berberine type alkaloid of any one of claims 1-2, 2-methyl-N ¹ -use of a pyrazine oxide-5-carboxylic acid quaternary ammonium salt compound or a pharmaceutical composition according to claim 4 for the preparation of an immunomodulator medicament having activity in promoting immune effector cell proliferation, inhibiting high NO secretion in pathological states, reducing high interleukin-6 levels and high autoantibody levels in pathological states of a biological body.

6. The berberine type alkaloid of any one of claims 1-2, 2-methyl-N ¹ -use of a pyrazine oxide-5-carboxylic acid quaternary ammonium salt compound or a pharmaceutical composition according to claim 4 for the preparation of a medicament for the prevention, alleviation and/or treatment of disorders associated with an increased secretion of interleukin-6 by a living organism.

7. The berberine type alkaloid of any one of claims 1-2, 2-methyl-N ¹ -use of a pyrazine oxide-5-carboxylic acid quaternary ammonium salt compound or a pharmaceutical composition according to claim 4 for the preparation of a medicament for the prevention, alleviation and/or treatment of diseases causing an increase in NO secretion by a living organism.

8. The berberine type alkaloid of any one of claims 1-2, 2-methyl-N ¹ -use of a pyrazine oxide-5-carboxylic acid quaternary ammonium salt compound or a pharmaceutical composition according to claim 4 for the preparation of a medicament for the prevention, alleviation and/or treatment of diseases causing an increase in the formation of autoantibodies by a living organism.

9. The berberine type alkaloid of any one of claims 1-2, 2-methyl-N ¹ -oxidative pyrazine-5-carboxylic acid quaternary ammonium salt compound or the pharmaceutical composition of claim 4 for preparing a medicament for preventing, alleviating and/or treating viral infections caused by various reasons and complications thereof, autoimmune diseases, cardiovascular and cerebrovascular diseases and blood system diseases, inflammatory diseases, febrile diseases, neoplastic diseases, respiratory diseases and primary glomerulonephritis diseases.

10. The use of claim 9, wherein said viral infection comprises a coronavirus infection; the autoimmune diseases comprise rheumatoid arthritis, lupus erythematosus, multiple sclerosis and ulcerative colitis; the neoplastic diseases comprise colorectal cancer, liver cancer and lung cancer; the cardiovascular and cerebrovascular diseases and blood system diseases comprise hypohemoglobinemia anemia, atherosclerosis, coronary heart disease, hypertension, ischemic heart disease, diastolic dysfunction fibrotic myocardial damage; the respiratory system diseases comprise pneumonia and allergic asthma.

11. The use according to claim 10, wherein said coronavirus infection comprises a 2019novel coronavirus SARS-CoV-2 infection.