CN115073447A - Berberine type pyridine carboxylic acid quaternary ammonium salt compound and application thereof in preparing medicines - Google Patents

Berberine type pyridine carboxylic acid quaternary ammonium salt compound and application thereof in preparing medicines Download PDF

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CN115073447A
CN115073447A CN202110274881.6A CN202110274881A CN115073447A CN 115073447 A CN115073447 A CN 115073447A CN 202110274881 A CN202110274881 A CN 202110274881A CN 115073447 A CN115073447 A CN 115073447A
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秦海林
邓安珺
张雪倩
徐令文
李志宏
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Abstract

The invention belongs to the technical field of medicines, and discloses a berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound and a preparation method thereofThe use of a medicament. Specifically, the invention discloses a compound shown as a general formula I, a pharmaceutical composition, a preparation method and application thereof. The compound has obvious activity of treating cardiovascular and cerebrovascular diseases, blood system diseases, inflammatory diseases, fever diseases, tumor diseases, autoimmune diseases, coronavirus infection diseases and complications thereof by balancing the immune function of an organism. The compound of the invention has better physicochemical property and pharmacological action intensity than a reference medicament and related compounds, and can be used for preparing medicaments for preventing, relieving and/or treating cardiovascular and cerebrovascular diseases and blood system diseases, inflammatory diseases, fever diseases, tumor diseases, autoimmune diseases, virus infection diseases and complications thereof.

Description

Berberine type pyridine carboxylic acid quaternary ammonium salt compound 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 pyridine carboxylic acid quaternary ammonium salt compound, a preparation method thereof, a pharmaceutical composition containing the compound, and application of the compound and the pharmaceutical composition thereof in preparing medicines.
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, the gene of various cytokines has been discovered and named after a research process, and it was not finally named as IL-6 until the genes of the cytokines have been cloned. The human IL-6 gene maps to 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, with a minor helix-E helix outside the four helix bundles, where 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 relation with respiratory system diseases, such as IL-6 level in bronchoalveolar lavage fluid of idiopathic pulmonary fibrosis patients is obviously increased; 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 manifestation of dyspnea in patients with pneumonia, which represents the intensity of the inflammatory response in the lungs from a pathophysiological point of view 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) which has exploded worldwide by 2019 so far, T lymphocytes and monocytes produce IL-6 in large quantities after infection with new 2019 Coronavirus (2019novel Coronavirus or SARS-CoV-2, also known as 2019-nCoV virus), accelerate inflammation progression, and exert immune destruction, resulting in severe pulmonary 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 IL-6 in urine and serum of patients with primary glomerulonephritis (IgAN) is increased remarkably. The receptor for IL-6 is IL-6R on the cell surface. IL-6 binding to the receptor initiates dimerization of IL-6R α with gp130, resulting in activation of downstream signaling pathways, including the JAK/STAT, Ras, and PI3K signaling pathways. Therefore, the IL-6 can be used as a target for treating diseases such as inflammatory diseases, fever diseases, cardiovascular and cerebrovascular diseases, blood system diseases, tumor diseases, viral infection diseases, complications thereof and the like, and is developed, tested and applied.
Autoimmune diseases are one of the most common clinically intractable diseases at present, the causes are not completely clear, and a large number of inflammatory cells, including lymphocytes (T cells and B cells), are usually accumulated at the focal site; the level of NO in the body fluid of the lesion of a patient is also significantly higher than the level of NO in the blood, and the level of NO in the blood of autoimmune disease patients is itself higher than that of normal humans, leading to swelling and pain, often also involving the formation of autoantibodies. In addition to the focal site, autoimmune diseases can affect any system of the body and are complicated by 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. The physiological function of lymphocytes and their influence on the blood system in the pathological state of autoimmune diseases, the formation of cardiovascular and cerebrovascular diseases and blood system diseases in the pathological state of autoimmune diseases and their characteristics can be exemplified by rheumatoid arthritis.
Rheumatoid Arthritis (RA) is one of the most common autoimmune diseases in clinic, and symptoms are mainly characterized by inflammatory arthritic lesions, the onset of which has chronic and systemic characteristics, the etiology is unknown, the basic pathology is manifested by synovitis and pannus formation in joint cavities, and a large number of inflammatory cells, including lymphocytes (T cells and B cells), monocytes/macrophages and the like, are accumulated in inflammatory synovium and synovial fluid, causing swelling, pain and stiffness, and like other autoimmune diseases, the formation of autoantibodies, such as Rheumatoid Factor (RF) or anti-citrulline protein antibodies, is also commonly involved. As a chronic systemic inflammatory disease, persistent inflammation can lead to cartilage and bone destruction injuries, deformities and loss of function in all affected joints, especially in the small joints of the hands and feet. Also, rheumatoid arthritis, as a systemic autoimmune inflammatory disease, may affect any system of the body in addition to the joints, and may be accompanied by lung diseases, malignant tumors, and depression. As mentioned above, the central part of the immunopathology of rheumatoid arthritis is inflammation, which 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. Anemia is also one of the most common extra-articular symptoms of rheumatoid arthritis, the most common type of anemia is anemia of chronic disease, the pathogenesis of which is not completely clear; thus, rheumatoid arthritis patients are at increased risk for 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. The content of NO in the joint fluid of the rheumatoid arthritis patient is obviously higher than that in the blood of the patient, although the content of NO in the blood of the rheumatoid arthritis patient is higher than that of the NO in the blood of a normal person. Overall, cardiovascular disease mortality in rheumatoid arthritis patients increased by about 50% over the general population, and cardiovascular disease appeared to occur at a younger age than in the general population. The rheumatoid arthritis also has the characteristics of repeated attack and high disability rate, the disability rates of patients in 1-5 years, 5-10 years, 10-15 years and more than or equal to 15 years are respectively 18.6%, 43.5%, 48.1% and 61.3%, namely the disability and function limitation incidence rate is increased along with the extension of the disease course, the life quality and the working capacity of the patients are seriously influenced, and heavy burden is brought to families and society. The rheumatoid arthritis can occur in any age group, the incidence and the prevalence rate of the rheumatoid arthritis vary with different regions and time, and epidemiological investigation shows that the global incidence rate is 0.5-1%, the incidence rate of the rheumatoid arthritis in Chinese continental areas is 0.42%, and the incidence rate of people over 65 years old is obviously increased. The incidence of rheumatoid arthritis in the united states was 0.409% in 2005, the prevalence was about 0.72%, the mean age of the disease was about 55.6 years, and the incidence of 69% of new cases in female patients was greater than in males. There are also investigations that show that the proportion of disease between men and women is about 1: 4.
Rheumatoid arthritis can not be cured radically so far, and the clinical treatment principle of the rheumatoid arthritis is early treatment, standard treatment, regular monitoring and follow-up visit. Currently, drugs for clinically treating rheumatoid arthritis are mainly classified into non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), glucocorticoids, and biologicals. Methotrexate is an antirheumatic drug for improving the state of illness, and is an anchoring drug for treating rheumatoid arthritis; the average usage rate of the methotrexate in Europe and America countries reaches 83%, which is much higher than that of other medicines, and the usage rate of the methotrexate in China is 55.9% lower than that in Europe and America countries. Generally, 2/3 patients with rheumatoid arthritis can achieve disease remission or low disease activity treatment goals, but cannot achieve radical treatment, by using methotrexate alone or in combination with other traditional synthetic DMARDs. The adverse reaction of the methotrexate is positively correlated with the dosage, and the conditions of light adverse reaction and better long-term tolerance can be achieved only when the methotrexate is used in small dosage (less than or equal to 10 mg/week). For those with methotrexate contraindications, the efficacy and safety of leflunomide and sulfasalazine alone are comparable to methotrexate, but attention should be paid to the side effects of sulfasalazine on IL-6 elevation. The international usage rate of leflunomide is 21% after methotrexate, sulfasalazine and hydroxychloroquine; however, the usage rate in China is only 45.9% after methotrexate, and the usage rate in partial regions even exceeds the usage rate of methotrexate. The use rate of Chinese rheumatoid arthritis patients treated by sulfasalazine is only 4.4 percent, which is far lower than that of other countries, and most of the Chinese rheumatoid arthritis patients are combined with other traditional synthetic DMARDs. The usage rate of the hydroxychloroquine in China is 30.4%, and the usage rate is slightly higher internationally. The use of the hydroxychloroquine in China is mainly combined, and accounts for 95 percent. Combination is needed for patients who have not met compliance with single-drug standard treatment such as methotrexate, leflunomide or sulfasalazine. Traditional synthetic DMARDs in combination with glucocorticoid therapy are required for rheumatoid arthritis patients with moderate/high disease activity, but the aim is only to control symptoms rapidly and to use glucocorticoids alone or in large doses for a long time, otherwise the glucocorticoids would cause severe toxic side effects.
Although there are currently many clinical treatment regimens for rheumatoid arthritis, compliance with all the regimens does not meet the expectations of the medical and patient, and even completely cures the disease, and thus is essentially a palliative therapy, resulting in the current disease having a severe negative impact on the patient's daily life, working ability, and health-related quality of life, a high disability rate, and an increased mortality rate. In addition, all anti-rheumatoid arthritis drugs have certain toxic side effects, for example, most NSAIDs have toxic side effects to damage the digestive system, and TNF- α blockers have the risk of serious infection. Therefore, the current treatment strategy for rheumatoid arthritis aims to achieve the aim of disease alleviation or low disease activity, namely palliative therapy or standard treatment, and finally aims to control the disease condition, reduce the disability rate and improve the life quality of patients. This highlights the need for further intensive research into anti-rheumatoid arthritis therapies, including the discovery of new biochemical drugs with superior efficacy.
The invention carries out synthesis, evaluation of inhibition efficacy of IL-6, NO and IgG antibody level on a pharmacological model, evaluation of influence condition on growth of in vitro cultured normal immune effector cells (normal mouse mononuclear macrophage RAW264.7) on cell level, evaluation of inhibition efficacy of SARS-CoV-2 virus replication in vitro experiment, and application of the compound in clinical experiment, The method comprises the following steps of evaluating the inhibition effect on the growth of tumor cells, evaluating the influence of the hemoglobin level of a model animal on an animal model, evaluating the antipyretic effect of a fever pathology model animal and evaluating the improvement effect on the severity of the inflammatory joint of a rheumatoid arthritis model animal, and also comprises the comparison of the pharmacological effect and the physicochemical property of the compound of the invention and the substrate of the berberine alkaloid quaternary ammonium salt chloride compound. The results show that the compound of the invention shows obvious efficacy and pharmacological effects of dose-dependently reducing IL-6, NO and IgG levels in a pharmacological model in pharmacological experiments, and shows obvious proliferation promoting effect on immune effector cells; can inhibit coronavirus replication in a cell experiment in a dose-dependent manner, and inhibit the growth of tumor cells in a dose-dependent manner and selectively; in animal experiments, the temperature of a rat fever model animal caused by yeast can be obviously reduced, the content of hemoglobin in peripheral blood of a pathological model animal induced by chicken type II collagen can be obviously reduced, and the severity of symptoms of rheumatoid arthritis of the model animal can be obviously relieved. The physicochemical property and pharmacological effect of the compound of the invention are obviously superior to those of the berberine chloride type alkaloid quaternary ammonium salt compound substrate. Through a large amount of substantial research works including the safety evaluation of the compounds, the compounds with significant application value in the preparation of medicines for preventing, relieving and/or treating anemia, virus infection diseases and complications thereof, inflammatory diseases, fever diseases, autoimmune diseases, neoplastic diseases and other diseases are defined.
Disclosure of Invention
The invention provides a method for preventing, relieving and/or treating related diseases causing the secretion/formation of IL-6 and/or NO and/or IgG antibody increase of organism and related diseases causing the hypohemoglobinemia anemia and autoimmune diseases caused by various reasons by balancing the immunity of the organism, which takes various picolinate radicals or substituted picolinate radicals as balancing anions and berberine type alkaloid quaternary ammonium cations as basic balancing cations and has better solubility in a mixed solvent of water and alcohol and water than a chlorinated berberine type alkaloid quaternary ammonium salt substrate by means of chemical synthesis, drug screening and the like, active compounds for treating virus infection and its complications, inflammation, fever and tumor, i.e. berberine type alkaloid pyridine formic acid quaternary ammonium salt compound shown in general formula I.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a berberine type alkaloid pyridine formic acid quaternary ammonium salt compound as shown in a general formula I.
The second aspect of the invention provides a preparation method of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound shown as the general formula I.
The third aspect of the invention provides a product composition of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound shown as the general formula I, wherein the product is selected from medicines.
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 a living organism, 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 level of IL-6 pathologically secreted in a living organism, the use in the manufacture of medicaments for preventing, alleviating and/or treating diseases that cause an increase in the level of pathological NO secreted in a living organism, and the use in the manufacture of medicaments for preventing, alleviating and/or treating diseases that cause a pathological autoantibody formation in a living organism.
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 diseases, viral infections and their complications, inflammatory diseases, febrile diseases, neoplastic diseases, all caused by various 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 chemical structural general formula of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound shown in the general formula I provided by the first aspect of the invention is shown in the following formula I:
Figure BDA0002976196020000071
in the formula I, COO - Is selected from 2 position or 3 position or 4 position or 5 position or 6 position substituted on pyridine ring; r is independently selected from hydrogen (H), amino, hydroxyl, methoxy, ethoxy, methyl, ethyl, fluorine (F), chlorine (Cl), bromine (Br);
r is mono-or polysubstituted;
when R is monosubstituted, with COO - Form R to COO - Disubstituted, R can be selected from 3 or 4 or 5 or 6 or 2 positions of the pyridine ring;
r is selected from di-substituted, tri-substituted or tetra-substituted when being polysubstituted, and is reacted with COO - Forming various substitution modes according to a mathematical enumeration method in a coordinated manner;
R 2 、R 3 each independently selected from H, substituted or unsubstituted OH, or R 2 And R 3 Linked as an alkylenedioxy group; further, R 2 、R 3 The substituents in the substituted or unsubstituted hydroxyl group in (1) are selected from methyl and ethyl; 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 substituents in said substituted or unsubstituted OH are 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 pyridine carboxylic acid quaternary ammonium salt compound of the invention is selected from the compounds 1-46 in the following compound group:
Figure BDA0002976196020000081
Figure BDA0002976196020000091
Figure BDA0002976196020000101
Figure BDA0002976196020000111
the second aspect of the invention provides a preparation method of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound.
The berberine type alkaloid picolinic acid quaternary ammonium salt compound can be synthesized by the following general formula (the specific synthesis conditions are shown in the examples):
Figure BDA0002976196020000112
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 type compound reacts with picolinic acid type compounds in a mixed solvent of tetrahydrofuran and water under the condition of heating, and the reaction mixed liquid is filtered to obtain the berberine type alkaloid picolinic acid type quaternary ammonium salt compound.
In a third aspect, the present invention provides a pharmaceutical composition comprising the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound of the first aspect of the present invention as an active ingredient. These pharmaceutical compositions can be prepared according to known methods for preparing pharmaceutical compositions. The compounds of the invention may be formulated into any dosage form suitable for human or animal use by combining them with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants. 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 administration primarily to the digestive tract, e.g., orally, enterally, etc. However, in view of the excellent physicochemical properties of the compounds of the present invention, forms of parenteral administration are also acceptable, such as intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, and administration to the nasal cavity, oral mucosa, eye, lung and respiratory tract, vaginal application, application to the skin, and the like. The application of treating anemia, rheumatoid arthritis, viral infection and complications thereof, inflammation, fever and tumor has the outstanding advantages that the traditional Chinese medicine composition can be prepared into common oral preparation forms such as common tablets and common capsules, and can be directly taken orally without special treatment (very convenient use), and can also be prepared into other preparation forms including injections, and other administration routes and modes are adopted.
Other dosage forms for oral or other 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.
In order to prepare the compound of the present invention into tablets, various excipients well known in the related art, including diluents, binders, wetting agents, disintegrants, lubricants, glidants, can 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 may 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 can 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 severity of cardiovascular and cerebrovascular and hematological diseases, autoimmune diseases, viral infectious diseases and complications thereof, inflammatory diseases, febrile diseases, neoplastic diseases, and the individual condition of the patient or animal, the route and dosage form of administration, etc., which are caused by various causes to be prevented, alleviated and/or treated. Generally, a suitable daily dosage range of the compounds of the invention is 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 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. 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 diseases, viral infections and their complications, inflammatory diseases, febrile diseases, neoplastic diseases, all caused by various 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 disclosed by the invention can also inhibit 2019novel coronavirus SARS-CoV-2 from replicating in a dose-dependent manner, inhibit the growth of human lung cancer cell A549 cell and human colon cancer cell HCT-116 cell in a dose-dependent manner and selectively, remarkably reduce the body temperature of a pathologic fever model animal, remarkably increase the content of hemoglobin in peripheral blood of the model animal and remarkably relieve the severity of rheumatoid arthritis symptoms of the model animal.
In the above fourth and fifth aspects of the present invention, preferred autoimmune diseases include rheumatoid arthritis, cardiovascular and cerebrovascular 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 immune function of an organism, shows a remarkable proliferation promoting effect on immune effector cells and a remarkable inhibiting effect on the generation of NO of 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 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 the pathological high IgG antibody level model animal. The compound is formed by combining two structural units in an ionic bond form, the physicochemical properties of the natural berberine type alkaloid quaternary ammonium salt compound are obviously improved, and the compound has unique action characteristics, comprises an obvious inhibiting effect on NO generation in an RAW264.7 cell inflammation model stimulated by LPS, and the inhibiting effect on NO generation in an initial passage RAW264.7 cell inflammation model stimulated by LPS is not a conventional linear dose dependence relationship but a unique irregular dose dependence relationship; the unique dose-effect relationship is also shown in the corresponding animal experiment. The compound serving as an IL-6 inhibitor can inhibit the replication of 2019novel coronavirus SARS-CoV-2 in a dose-dependent manner, inhibit the growth of human lung cancer cells A549 and human colon cancer cells HCT-116 in a dose-dependent manner and selectively, remarkably reduce the body temperature of a fever model animal, remarkably increase the content of hemoglobin in peripheral blood of the model animal and remarkably relieve the severity of rheumatoid arthritis symptoms of a rheumatoid arthritis model animal. Therefore, the compound can be used for preparing an immunomodulator drug product, preparing a drug product for preventing, relieving and/or treating diseases causing the increase of IL-6 secretion of a living organism, preparing a drug product for preventing, relieving and/or treating diseases causing the increase of NO secretion of the living organism and preparing a drug product for preventing, relieving and/or treating diseases causing the increase of autoantibodies formed in the living organism. The application of the compound in preparing the medicine product for preventing, relieving and/or treating the related diseases comprises the application of preventing, relieving and/or treating cardiovascular and cerebrovascular diseases and blood system diseases, inflammatory diseases, fever diseases, tumor diseases, autoimmune diseases, coronavirus infection diseases and complications thereof. The cardiovascular and cerebrovascular diseases and the blood system diseases comprise hypohemoglobinemia anemia caused by various reasons, autoimmune diseases comprise rheumatoid arthritis, and neoplastic diseases comprise colorectal cancer and lung cancer. 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 berberine chloride type alkaloid quaternary ammonium salt compound substrates, including 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. Compared with the natural berberine chloride type alkaloid quaternary ammonium salt substrate, the solubility of the compound in a mixed solvent of water and alcohol water is obviously improved. The compound has obvious medicine preparing value and may be prepared into various kinds of medicine preparation including common orally taken preparation form and injection form for medicinal use. The specific structure of the compound is that the pyridine carboxylic acid or substituted pyridine carboxylic acid radical is taken as an acid radical balancing anion unit, the 5, 6-dihydrodibenzo [ a, g ] quinolizine-7-cationic quaternary ammonium cation is taken as a basic group balancing cation unit, and the two form the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound.
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 synthesis 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 stability detection results of the compounds by adopting an NMR test means show that the compounds of the invention have stable physicochemical properties in a solid state, and have extremely stable structures even when the compounds are placed in a 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 compound has obvious practical value for large-scale preparation of active molecular entities of the berberine type alkaloid picolinic acid type quaternary ammonium salt compound, which accords with general pharmaceutical technical specifications, searching for new pharmacological activity and improving the action strength of a medicament.
Compared with the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate, the water solubility of the compound is obviously improved; water solubility, measured at ambient temperature of 25 ℃ ± 2 ℃, is such that the amount of all of the compounds of the invention that can be dissolved in water per ml is greater than or significantly greater than the corresponding berberine chloride-type alkaloid quaternary ammonium salt substrate that can be dissolved in water per ml, e.g. the amounts of compounds 1-10 of the invention that can be dissolved in water per ml are 11.5mg, 800mg, 7.5mg, 45mg, 1.1mg, 15mg, 50mg, 1.5mg, 30mg and 1.38mg, respectively; and the 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 parallel measurement can be respectively 2mg, 21mg, <1mg and <1mg in each milliliter of water.
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 1-46 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 experimental examples 1 and 2 of the invention.
In the scientific research of the synthesis and biological function evaluation of functional organic compounds, the necessary premise for deeply developing biological research is that related compounds show certain safety; 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 of mouse mononuclear macrophage RAW264.7 of a normal immune effector cell cultured in vitro in an initial passage (a few generations of initial passage), the influence on the growth of the cell is evaluated by the cell survival rate of the cultured cell after the intervention treatment for 24 hours under the series action concentration of the compound, and the result shows that the compound intervenes and treats the cell under the series action concentration of 100 mu M-0.390625 mu M, and then the compound is subjected to the intervention treatmentThe compound has no inhibition effect on the growth of the primary passage normal cells, and has obvious proliferation promoting effect on the primary passage normal RAW264.7 cells under certain action concentration. As a prognosis of the inventive compound 8 stem, the survival of normal primary passage RAW264.7 cells was 107.42 ± 2.76%, 139.70 ± 4.92%, 152.41 ± 8.44%, 152.18 ± 4.74%, 147.56 ± 4.46%, 133.53 ± 2.64%, 121.74 ± 7.50%, 117.94 ± 7.79%, 107.15 ± 2.08% (survival of normal group was 100%, P <0.05 + P < 0.01.001.001.01%) at serial concentrations of action of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M, 1.5625 μ M, 0.78125 μ M and 0.390625 μ M, respectively. Macrophages are important immune effector cells located in body tissues, participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vivo, play an extremely important role in immunity such as defense, surveillance, regulation, antigen presentation, and the like, have very important relevance to the balance of the immune system of the body, and play 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 has obvious effect of promoting macrophage proliferation, and shows that the compound has the effect of balancing the immune function of an 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 inhibition effect isIC for use 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, and the survival rate of the cells interfered by other investigated compounds of the invention reaches 100% except that the survival rate of the cells interfered by berberine pyridine-3-quaternary ammonium formate (1) is 98.5% (see the experimental example).
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 present invention have pharmacological effects of significantly reducing IL-6 levels in pharmacological models of pathologically high IL-6 expression levels, including pharmacological effects that significantly reduce IL-6 levels in pharmacological models both at the cellular level and at the animal level. 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 pathological conditions in which the IL-6 level in the serum of model animals reaches 69.8443pg/ml, the compound 7 of the invention can reduce the IL-6 level in the serum of model animals to 16.8596pg/ml, 17.7957pg/ml and 21.6338pg/ml respectively at the action dose of 200mg/kg, 100mg/kg and 50mg/kg of high dose group/medium dose group/low dose group, and has statistically significant difference compared with the model group. 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 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M and 0.625 μ M, but also showed unique dose-dependent inhibitory activity at the concentrations of the series of effects of 10 μ M, 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, 0.001220703125 μ M and 0.0006103515625 μ M, with the inhibitory rates being 47.36%, 31.92%, 36.50%, 39.70%, 13.58%, 24.94%, 27.03%, 31.42%, 36.69%, -3.72%, 17.89%, 31.54%, 84%, 51.04%, 84%, 8945% and a significantly lower inhibitory rate at the concentrations of 8236 μ M, i.e. 0.0006103515625 μ M. The unique mechanism of action of the compounds of the present invention is manifested in a number of ways, one of which is in the inhibition of NO secretion in the initial passage RAW264.7 cell inflammatory model stimulated by LPS. The inhibition effect of the compound of the invention under the higher action concentration of 10 mu M-1.25 mu M generally adopted in pharmacological experiments is obviously stronger than that of the corresponding berberine chloride type alkaloid quaternary ammonium salt in the action concentration range, particularly the inhibition effect of the compound of the invention on the NO secretion in an LPS-stimulated initial passage RAW264.7 cell inflammation model under the unconventionally lower action concentration of 0.001220703125 mu M and 0.0006103515625 mu M is stronger than that of the corresponding berberine chloride type alkaloid quaternary ammonium salt under the obviously higher action concentration, for example, the inhibition rate of the palmatine chloride quaternary ammonium salt on the NO secretion under the action concentration of 6.25-5 mu M is less than 20 percent in the result of multiple experiments, and the inhibition rate of the compound of the invention under the same action concentration is obviously lower. 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 very obvious effect of reducing the IgG antibody level in the serum of a model animal and presents unique dose dependence; in a pathological state in which the serum level of IgG antibodies in the model animal reaches 22.98350 units/ml, the compound 7 of the present invention can reduce the serum level of IgG antibodies in the model animal to 20.91259 units/ml, 17.97435 units/ml and 16.53063 units/ml at the high dose group/medium dose group/low dose group action dose of 200mg/kg, 100mg/kg and 50mg/kg, respectively, and the low dose group and the medium dose group have a statistically significant difference from the model group. 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 immunoregulation 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, 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 1,3, 4, 6-10 of the invention on the virus RNA level after experimental cells are infected with SARS-CoV-2 virus are respectively 28.63%, 65.66%, 32.95%, 93.13%, 31.00%, 89.10%, 29.62% and 17.45%. Compounds 6, 8 and 3 EC for inhibition of SARS-CoV-2 viral RNA levels 50 The values were 3.037. mu.M, 3.767. mu.M and 7.859. mu.M, respectively. Meanwhile, 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, the pharmacological action of the compound for reducing the IL-6 level in the 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 positive to the anti-SARS-CoV-2 IgG antibody in the serum. In other related animal experiments, in the pharmacodynamic evaluation of the antipyretic effect of a yeast-induced rat fever model, the body temperature (change) of a model animal measured at different time points after administration and the difference delta T (DEG C) between the body temperature measured at different time periods and the basal body temperature are taken as indexes to evaluate the antipyretic effect of the compound, and the result shows that the compound has remarkable antipyretic effect, and the antipyretic effect is characterized by longer action duration than that of a positive medicine. On a DBA/1 mouse inflammation model induced by chicken type II collagen, changes of hemoglobin level, rheumatoid arthritis index score level, arthritis incidence level and sole thickness level of a model animal treated by the compound are considered, and the compound is shown to have obvious activity of treating anemia and rheumatoid arthritis. Wherein, in the aspect of hemoglobin detection, compared with normal mice, the blood of the model group animals in the experiment carried out by the inventionThe content of the hemoglobin is obviously reduced, and the value is 136.33 +/-4.27 (g/L); compared with the model group, each dose group of the tested compound of the invention has obvious effect of increasing the hemoglobin content, and the hemoglobin contents of the low/medium/high dose groups are 180.18 +/-4.45 (g/L), 182.75 +/-2.22 (g/L) and 182.75 +/-2.42 (g/L), respectively. Particularly, compared with a normal control group, each dosage group of the compound has obvious effect of increasing hemoglobin, and the compound has obvious effect of treating anemia. In the detection of the level of the joint inflammation index score, each dose group of the compound of the invention tested had a significant reduction in the mean of the joint inflammation index score, and at the end of the experiment, the joint inflammation index scores of the low/medium/high dose groups were 1.73 ± 0.68 ×, 2.67 ± 0.80 ×, and 3.58 ± 0.66 ×, respectively, which were all significantly reduced compared to the joint inflammation index score of the model group of 6.92 ± 0.84, and all had a very significant statistical difference. In the detection of the change of the incidence level of arthritis and the thickness level of the sole, the results which are completely the same as the results of 'remarkable effect on treating rheumatoid arthritis' obtained in the detection experiment of the joint inflammation index score level are obtained, and the results show that the compound has remarkable activity on resisting the rheumatoid arthritis.
In addition, in the detection of the inhibition effect of the compound on the growth of tumor cells by adopting an MTT method, the compound selectively has obvious inhibition effect on the growth of human colorectal cancer HCT-116 cells and human lung cancer A549 cells, and the combination of the obvious effect of reducing the IL-6 level of the compound shows that the compound has beneficial treatment effect on the tumor diseases. 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 line growth inhibition activity, and the compound has no inhibition activity on the growth of human liver cancer cells HepG2 and human stomach cancer cells BGC-823.
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 showed the strongest pharmacological activity in the low dose group and better in the medium dose group than in the high dose group in the treatment of rheumatoid arthritis in the chicken type II collagen-induced model of inflammation in DBA/1 mice. In combination with the unique dose-dependent inhibition of NO production in the initial passage RAW264.7 cell inflammation model stimulated by LPS found in cell experiments, it can be shown that the compounds of the present invention have a unique pharmacological mechanism of action. This is a selective marker for pharmacological effects.
In conclusion, the compound has remarkable medicinal effectiveness, safety and quality controllability, and has very remarkable application prospects 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 organisms and medicine products causing the increase of autoantibodies formed by organisms, and preparing medicine products for preventing, relieving and/or treating inflammatory diseases, febrile diseases, neoplastic diseases, autoimmune diseases, cardiovascular and cerebrovascular diseases, blood system diseases, virus infection diseases and complications thereof caused by various reasons. In particular, the compound has significant application value in preparing medicine products for preventing, relieving and/or treating anemia, inflammatory diseases, fever diseases, colorectal cancer, lung cancer, rheumatoid arthritis, and novel coronavirus SARS-CoV-2 infectious diseases and complications thereof, wherein the infection causes the increase of the level of IL-6 secreted by organisms and the increase of the level of IgG antibodies.
Drawings
FIG. 1 shows a whole animal experiment and administration process of mouse chicken type II collagen-induced arthritis (CIA).
FIG. 2 shows the effect of Compound 7 of the present invention on the level of IL-6 in serum of a mouse chicken type II collagen-induced arthritis model mouse.
Note: data are expressed as X ± SEM; # in comparison with the normal control group, ### p<0.001; is represented byModel group comparison<0.01，***p<0.001。
FIG. 3 shows the effect of Compound 7 of the present invention on the hemoglobin content of a mouse chicken type II collagen-induced arthritis model mouse.
Note: data are expressed as X ± SEM; compared to model groups, p < 0.001.
Figure 4, effect of different administration groups of the invention on mouse joint inflammation index scores.
Note: n is 12; compared to the model group, p <0.05, p <0.01, p < 0.001.
FIG. 5 shows the effect of Compound 7 of the present invention on IgG anti-collagen II antibody in serum of mouse model mouse with chicken type II collagen-induced arthritis
Note: compared to the model group, p <0.05, p <0.01, p < 0.001.
FIG. 6 is a body temperature change chart of rats for pharmacodynamic evaluation of antipyretic action of Compound 7 of the present invention.
Note: each group n is 12; # in order to compare with the blank control group, ### p<0.001; is compared with the model group<0.01,***p<0.001。
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 of the present invention, the compound number corresponds to the specific compound number in the present invention.
First, examples of production of the Compound of the present invention
Example 1: synthesis and Structure identification data for 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.
Pyridine-3-carboxylic acid (35mg, 0.28mmol) was weighed into a reaction flask, and 2.5ml of tetrahydrofuran/water (v/v ═ 24:1) was addedMixing the solvents, stirring, adding 8-acetonyl dihydroberberine (100mg, 0.25mmol), heating for reaction, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v-24: 1) to obtain 108mg of compound 1 as a yellow solid with a yield of 92.70%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),8.94(s,1H,ArH),8.90(dd,J＝2.0,0.8Hz,1H,ArH),8.41(dd,J＝4.8,2.0Hz,1H,ArH),8.21(d,J＝9.2Hz,1H,ArH),8.04(ddd,J＝7.6,2.0,2.0Hz,1H,ArH),8.00(d,J＝9.2Hz,1H,ArH),7.80(s,1H,ArH),7.23(ddd,J＝7.6,4.8,0.8Hz,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 ),3.20(t,J＝6.4Hz,2H,NCH 2 CH 2 )。 13 C-NMR(150MHz,CD 3 OD)δ:172.3,152.2,152.0,151.1,150.9,149.9,146.4,145.8,139.7,138.7,135.3,135.2,131.9,128.1,124.6,124.5,123.3,121.9,121.5,109.4,106.6,103.7,62.6,57.7,57.2,28.2。HR-ESI-MS(pos.):336.12311[M-C 6 H 4 NO 2 ] + (calc.for C 20 H 18 NO 4 ,336.12303)。
Example 2: synthesis and Structure identification data for Compound 2 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Pyridine-3-carboxylic acid (33mg, 0.27mmol) is weighed into a reaction flask, 2ml of mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) is added, after uniform stirring, 8-acetonyl dihydropalmatine (100mg, 0.24mmol) is added, the reaction is heated until the reaction of the raw materials is complete, and the heating is stopped. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 104mg of compound 2 as a yellow solid in a yield of 96.3%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),9.05(s,1H,ArH),8.91(dd,J＝2.0,0.8Hz,1H,ArH),8.40(dd,J＝4.8,2.0Hz,1H,ArH),8.21(d,J＝9.2Hz,1H,ArH),8.05(ddd,J＝7.6,2.0,2.0Hz,1H,ArH),8.04(d,J＝9.2Hz,1H,A H),7.77(s,1H,ArH),7.23(ddd,J＝7.6,4.8,0.8Hz,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 )。 13 C-NMR(150MHz,DMSO-d 6 )δ:166.3,151.4,150.4,150.1,148.6(2×C),145.4,143.5,137.6,136.1,135.9,133.0,128.5,126.6,123.3,122.3,121.2,119.8,118.8,111.2,108.7,61.8,56.9,56.0,55.7,55.2,25.9。HR-ESI-MS(pos.):352.15375[M-C 6 H 4 NO 2 ] + (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 pyridine-3-formic acid (66mg, 0.54mmol) into a reaction bottle, adding 4ml of mixed solvent of tetrahydrofuran/water (v/v ═ 24:1), stirring uniformly, adding 8-acetonyldihydrocoptisine (100mg, 0.26mmol), heating for reaction until the raw material reaction is complete, and stopping heating. The reaction mixture was cooled to room temperature, and then filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 89mg of compound 3 as a yellow solid with a yield of 75.9%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.96(s,1H,ArH),8.96(s,1H,ArH),8.91(dd,J＝2.0,0.8Hz,1H,ArH),8.42(dd,J＝4.8,2.0Hz,1H,ArH),8.05(ddd,J＝7.6,2.0,2.0Hz,1H,ArH),8.03(d,J＝8.4Hz,1H,ArH),7.82(d,J＝8.4Hz,1H,ArH),7.79(s,1H,ArH),7.27(ddd,J＝7.6,4.8,0.8Hz,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.88(t,J＝6.4Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.4Hz,2H,NCH 2 CH 2 )。 13 C-NMR(150MHz,DMSO-d 6 )δ:166.3,150.4,149.6,148.9,147.6,146.9,144.5,143.7,136.7,135.9,135.5,132.2,130.4,122.3,121.6,120.9,120.8,120.4,111.5,108.3,105.2,104.4,102.0,54.9,26.2。HR-ESI-MS(pos.):320.09174[M-C 6 H 4 NO 2 ] + (calc.for C 19 H 14 NO 4 ,320.09173)。
Example 4: synthesis and Structure identification data for Compound 4 of the invention
Synthesis of 8-acetonyldihydroisomerizine (Ex.).
Pyridine-3-carboxylic acid (36mg, 0.29mmol) was weighed into a reaction flask, 2.5ml of a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) was added, and after stirring uniformly, 8-acetonyldihydroisomerizine (100mg, 0.27mmol) 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, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 104mg of compound 4 as a yellow solid in 88.7% yield. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.61(s,1H,ArH),8.92(dd,J＝2.0,0.8Hz,1H,ArH),8.76(s,1H,ArH),8.42(dd,J＝4.8,2.0Hz,1H,ArH),8.06(ddd,J＝7.6,2.0,2.0Hz,1H,ArH),7.75(s,1H,ArH),7.74(s,1H,ArH),7.53(s,1H,ArH),7.24(ddd,J＝7.6,4.8,0.8Hz,1H,ArH),7.09(s,1H,ArH),6.42(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.77(t,J＝6.4Hz,2H,NCH 2 CH 2 ),3.19(t,J＝6.4Hz,2H,NCH 2 CH 2 )。 13 C-NMR(100MHz,DMSO-d 6 )δ:166.5,155.8,150.7,150.4,149.8,148.7,147.5,145.9,138.6,138.5,136.0,135.9,130.7,123.5,122.3,120.2,118.8,108.3,105.3,103.8,103.7,102.5,102.0,54.2,26.3。HR-ESI-MS(pos.):320.09137[M-C 6 H 4 NO 2 ] + (calc.for C 19 H 14 NO 4 ,320.09173)。
Example 5: synthesis and Structure identification data for Compound 5 of the invention
Synthesis of 8-acetonyldihydroisomerizine (omitted).
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, washing a filter cake to be neutral by water to obtain a light yellow solid 8-acetonyl dihydro-iso-berberine, and directly using the product for the next reaction without purification. Pyridine-3-carboxylic acid (160mg, 1.30mmol) was weighed into a reaction flask, and 21ml of a tetrahydrofuran/water (v/v ═ 24:1) mixed solution was addedStirring, adding 8-acetonyl dihydroisoberberine (200mg, 0.53mmol), heating for reaction, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 490mg of compound 5 as a yellow solid, in 79.54% yield. 1 H-NMR(400MHz,Methanol-d 4 )δ：9.30(s,1H,ArH),9.03(br s,1H,ArH),8.52(s,1H,ArH),8.51(br d,J＝4.8Hz,1H,ArH),8.28(ddd,J＝8.0,2.0,2.0Hz,1H,ArH),7.62(s,1H,ArH),7.60(s,1H,ArH),7.58(s,1H,ArH),7.41(dd,J＝8.0,4.8Hz,1H,ArH),6.95(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 )。 13 C-NMR(150MHz,CD 3 OD)δ：172.3,160.1,154.7,152.2,151.1,151.0,149.9,146.2,140.4,139.0,138.7,135.2,131.9,124.6,124.2,121.9,119.5,109.4,107.3,106.5,106.4,103.7,57.5,57.1,56.3,28.3。HR-ESI-MS(pos.):336.12225[M-C 6 H 4 NO 2 ] + (calc.for C 20 H 18 NO 4 ,336.12303)。
Example 6: synthesis and Structure identification data for Compound 6 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 5-fluoropyridine-3-formic acid (39mg, 0.28mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,2ml), stirring uniformly, adding 8-acetonyldihydroberberine (100mg, 0.25mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, and then filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 110mg of compound 6 as a yellow solid in a yield of 91.08%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),8.94(s,1H,ArH),8.76(dd,J＝2.0,1.6Hz,1H,ArH),8.40(d,J＝3.2Hz,1H,ArH),8.20(d,J＝9.2Hz,1H,ArH),8.00(d,J＝9.2Hz,1H,ArH),7.80(s,1H,ArH),7.77(ddd,J＝9.6,3.2,1.6Hz,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 )。 13 C-NMR(100MHz,DMSO-d 6 )δ:164.8,159.0(d, 1 J CF ＝251.6Hz,1C,C-5′),150.3,149.8,147.6,146.6(d, 4 J CF ＝3.3Hz,1C,C-2′),145.4,143.6,138.6,137.4,136.6(d, 2 J CF ＝23.0Hz,1C,C-4′),132.9,130.6,126.7,123.5,122.2(d, 2 J CF ＝16.5Hz,1C,C-6′),121.4,120.4,120.2,108.4,105.4,102.0,61.9,57.0,55.1,26.3。HR-ESI-MS(pos.):336.12308[M-C 6 H 3 FNO 2 ] + (calc.for C 20 H 18 NO 4 ,336.12303)。
Example 7: synthesis and Structure identification data for Compound 7 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 5-fluoropyridine-3-carboxylic acid (38mg, 0.27mmol), adding 2ml of mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) into a reaction bottle, stirring uniformly, adding 8-acetonyl dihydropalmatine (100mg, 0.24mmol), heating for reaction until the raw materials react completely, stopping heating, cooling the reaction mixture to room temperature, filtering, and washing a filter cake three times with the mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 119mg of a compound 7 yellow solid, wherein the yield is 98.92%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),9.03(s,1H,ArH),8.77(dd,J＝2.0,1.2Hz,1H,ArH),8.41(d,J＝2.8Hz,1H,ArH),8.21(d,J＝9.2Hz,1H,ArH),8.03(d,J＝9.2Hz,1H,ArH),7.78(ddd,J＝9.6,2.8,1.2Hz,1H,ArH),7.72(s,1H,ArH),7.10(s,1H,ArH),4.95(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 ),3.88(s,3H,ArOCH 3 ),3.23(t,J＝6.4Hz,2H,NCH 2 CH 2 )。 13 C-NMR(100MHz,DMSO-d 6 )δ:164.8,159.0(d, 1 J CF ＝251.6Hz,1C,C-5′),151.5,150.2,148.7,146.6(d, 4 J CF ＝3.3Hz,1C,C-2′),145.4,143.6,138.7,137.7,136.6(d, 2 J CF ＝23.1Hz,1C,C-4′),133.1,128.6,126.7,123.4,122.2(d, 2 J CF ＝16.5Hz,1C,C-6′),121.3,119.9,118.9,111.3,108.7,61.9,57.0,56.1,55.8,55.3,26.0。HR-ESI-MS(pos.):352.15433[M-C 6 H 3 FNO 2 ] + (calc.for C 21 H 22 NO 4 ,352.15433)。
Example 8: synthesis and Structure identification data for Compound 8 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 5-fluoropyridine-3-carboxylic acid (41mg, 0.29mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,3ml), stirring uniformly, adding 8-acetonyl dihydrocoptisine (100mg, 0.26mmol), heating for reaction until the raw materials react completely, stopping heating, cooling the reaction mixture to room temperature, filtering, and washing a filter cake for three times by using the mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 96mg of a compound 8 yellow solid, wherein the yield is 78.69%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.95(s,1H,ArH),8.95(s,1H,ArH),8.76(dd,J＝2.0,1.2Hz,1H,ArH),8.41(d,J＝2.8Hz,1H,ArH),8.03(d,J＝8.4Hz,1H,ArH),7.82(d,J＝8.4Hz,1H,ArH),7.79(s,1H,ArH),7.78(ddd,J＝10.0,2.8,1.2Hz,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.88(t,J＝6.4Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.4Hz,2H,NCH 2 CH 2 )。 13 C-NMR(150MHz,DMSO-d 6 )δ:164.8,159.0(d, 1 J CF ＝251.7Hz,1C,C-5′),149.7,147.6,147.0,146.6(d, 4 J CF ＝3.3Hz,1C,C-2′),144.5,143.8,138.1,136.81(d, 2 J CF ＝22.9Hz,1C,C-4′),136.77,132.3,130.5,122.2(d, 2 J CF ＝16.8Hz,1C,C-6′),121.7,121.0,120.9,120.4,111.6,108.3,105.3,104.4,102.0,55.0,26.2。HR-ESI-MS(pos.):320.09183[M-C 6 H 3 FNO 2 ] + (calc.for C 19 H 14 NO 4 ,320.09173)。
Example 9: synthesis and Structure identification data for Compound 9 of the invention
Synthesis of 8-acetonyldihydroisomerizine (Ex.).
Weighing 5-fluoropyridine-3-carboxylic acid (41mg, 0.29mmol) into a reaction flask, adding 2.5ml of a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1), stirring uniformly, adding 8-acetonyldihydroisomerizine (100mg, 0.26mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 97mg of compound 9 as a yellow solid in a yield of 79.51%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.59(s,1H,ArH),8.77(dd,J＝2.0,1.6Hz,1H,ArH),8.77(s,1H,ArH),8.41(d,J＝2.8Hz,1H,ArH),7.79(ddd,J＝9.6,2.8,1.6Hz,1H,ArH),7.75(s,1H,ArH),7.73(s,1H,ArH),7.53(s,1H,ArH),7.09(s,1H,ArH),6.42(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.77(t,J＝6.4Hz,2H,NCH 2 CH 2 ),3.19(t,J＝6.4Hz,2H,NCH 2 CH 2 )。 13 C-NMR(100MHz,DMSO-d 6 )δ:165.0,159.0(d, 1 J CF ＝251.5Hz,1C,C-5′),155.9,150.9,150.0,147.6,146.6(d, 4 J CF ＝3.3Hz,1C,C-2′),145.9,138.7,138.6,138.3,136.8(d, 2 J CF ＝23.0Hz,1C,C-4′),130.9,123.5,122.3(d, 2 J CF ＝16.6Hz,1C,C-6′),120.3,118.9,108.5,105.4,103.9,103.8,102.6,102.1,54.4,26.4。HR-ESI-MS(pos.):320.09174[M-C 6 H 3 FNO 2 ] + (calc.for C 19 H 14 NO 4 ,320.09173)。
Example 10: synthesis and Structure identification data for Compound 10 of the invention
Synthesis of 8-acetonyldihydroisomerizine (omitted).
Weighing 5-fluoropyridine-3-formic acid (604mg, 4.19mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,52ml), stirring uniformly, adding 8-acetonyldihydroisomerizine (1.5g, 3.81mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 68mg of compound 10 as a yellow solid with a yield of 54.09%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.57(s,1H,ArH),8.77(s,2×H,ArH),8.41(d,J＝3.2Hz,1H,ArH),7.78(ddd,J＝9.6,3.2,1.6Hz,1H,ArH),7.74(s,1H,ArH),7.73(s,1H,ArH),7.58(s,1H,ArH),7.10(s,1H,ArH),6.18(s,2H,OCH 2 O),4.78(t,J＝6.4Hz,2H,NCH 2 CH 2 ),4.07(s,3H,ArOCH 3 ),4.00(s,3H,ArOCH 3 ),3.20(t,J＝6.4Hz,2H,NCH 2 CH 2 )。 13 C-NMR(150MHz,CD 3 OD)δ:170.8,161.0(d, 1 J CF ＝253.3Hz,1C,C-5′),160.1,154.7,152.2,149.9,147.4(d, 4 J CF ＝3.6Hz,1C,C-2′),146.2,140.4,139.4(d, 2 J CF ＝24.3Hz,1C,C-4′),139.0,137.2,131.9,124.8(d, 2 J CF ＝18.0Hz,1C,C-6′),124.2,121.9,119.5,109.4,107.3,106.5,106.4,103.7,57.5,57.1,56.3,28.3。(+)HRESI-MS(m/z):336.12241[M-C 6 H 3 FNO 2 ] + (calcd for C 20 H 18 NO 4 ,336.12303)。
Example 11: synthesis and Structure identification data for Compound 11 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 5-methylpyridine-3-formic acid (261mg, 1.91mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,14ml), stirring uniformly, adding 8-acetonyldihydroberberine (500mg, 1.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 558mg of compound 11 as a yellow solid in 93.00% yield. 1 H-NMR(400MHz,DMSO-d 6 )δ:9.89(s,1H,ArH),8.94(s,1H,ArH),8.69(d,J＝1.6Hz,1H,ArH),8.23(d,J＝2.0Hz,1H,ArH),8.20(d,J＝9.2Hz,1H,ArH),8.00(d,J＝9.2Hz,1H,ArH),7.86(m,1H,ArH),7.80(s,1H,ArH),7.09(s,1H,ArH),6.18(s,2H,OCH 2 O),4.93(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,ArOCH 3 ),4.07(s,3H,ArOCH 3 ),3.21(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.26(s,3H,Me)。
Example 12: synthesis and Structure identification data for Compound 12 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 5-methylpyridine-3-formic acid (184mg, 1.34mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,8ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (500mg, 1.22mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 456mg of compound 12 as a yellow solid in a yield of 76.38%. 1 H-NMR(500MHz,DMSO-d 6 )δ:9.90(s,1H,ArH),9.04(s,1H,ArH),8.70(brs,1H,ArH),8.24(brs,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),7.86(brs,1H,ArH),7.72(s,1H,ArH),7.10(s,1H,ArH),4.96(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.26(s,3H,Me)。
Example 13: synthesis and Structure characterization data for Compound 13 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 5-methylpyridine-3-formic acid (109mg, 0.80mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,12ml), stirring uniformly, adding 8-acetonyldihydrocoptisine (200mg, 0.53mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v-24: 1) to obtain 218mg of compound 13 as a yellow solid with a yield of 90.08%. 1 H-NMR(500MHz,DMSO-d 6 )δ:9.96(s,1H,ArH),8.96(s,1H,ArH),8.70(brs,1H,ArH),8.25(brs,1H,ArH),8.03(d,J＝8.5Hz,1H,ArH),7.87(brs,1H,ArH),7.82(d,J＝8.5Hz,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),4.88(t,J＝6.0Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.26(s,3H,Me)。
Example 14: synthesis and Structure identification data for Compound 14 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 6-methylpyridine-3-formic acid (261mg, 1.91mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,14ml), stirring uniformly, adding 8-acetonyldihydroberberine (500mg, 1.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 487mg of compound 14 as a yellow solid in 81.17% yield. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),8.95(s,1H,ArH),8.78(d,J＝2.0Hz,1H,ArH),8.20(d,J＝9.2Hz,1H,ArH),8.00(d,J＝9.2Hz,1H,ArH),7.94(dd,J＝8.0,2.0Hz,1H,ArH),7.80(s,1H,ArH),7.09(s,1H,ArH),7.08(d,J＝8.0Hz,1H,ArH),6.17(s,2H,OCH 2 O),4.94(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.09(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.42(s,3H,Me)。
Example 15: synthesis and Structure identification data for Compound 15 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 6-methylpyridine-3-formic acid (184mg, 1.34mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,8ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (500mg, 1.22mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v-24: 1) to obtain 448mg of compound 15 as a yellow solid with a yield of 75.04%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),9.04(s,1H,ArH),8.78(d,J＝2.0Hz,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),7.93(dd,J＝8.0,2.0Hz,1H,ArH),7.73(s,1H,ArH),7.10(s,1H,ArH),7.07(d,J＝8.0Hz,1H,ArH),4.96(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.41(s,3H,Me)。
Example 16: synthesis and Structure identification data for Compound 16 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 6-methylpyridine-3-formic acid (109mg, 0.80mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,12ml), stirring uniformly, adding 8-acetonyldihydrocoptisine (200mg, 0.53mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, and then filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 178mg of compound 16 as a yellow solid with a yield of 73.55%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.96(s,1H,ArH),8.97(s,1H,ArH),8.79(brs,1H,ArH),8.03(d,J＝8.5Hz,1H,ArH),7.95(brd,J＝7.5Hz,1H,ArH),7.83(d,J＝8.5Hz,1H,ArH),7.79(s,1H,ArH),7.09(d,J＝7.5Hz,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.88(t,J＝6.0Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.42(s,3H,Me)。
Example 17: synthesis and Structure identification data for Compound 17 of the invention
Synthesis of 8-acetonyl dihydroberberine (not shown).
Weighing 2-chloropyridine-3-formic acid (80mg, 0.51mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,4ml), stirring uniformly, adding 8-acetonyldihydroberberine (100mg, 0.25mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 95mg of compound 17 as a yellow solid in a yield of 75.2%. 1 H-NMR(500MHz,DMSO-d 6 )δ:9.89(s,1H,ArH),8.94(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.11(d,J＝3.5Hz,1H,ArH),8.00(d,J＝9.0Hz,1H,ArH),7.80(s,1H,ArH),7.60(d,J＝7.5Hz,1H,ArH),7.21(dd,J＝7.5,3.5Hz,1H,ArH),7.09(s,1H,ArH),6.17(s,2H,OCH 2 O),4.94(t,J＝5.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝5.5Hz,2H,NCH 2 CH 2 )。
Example 18: synthesis and Structure identification data for Compound 18 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 2-chloropyridine-3-formic acid (48mg, 0.31mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,4ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (100mg, 0.25mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 88mg of compound 18 as a yellow solid in a yield of 70.4%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.02(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.12(dd,J＝5.0,Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),7.72(s,1H,ArH),7.61(d,J＝7.5Hz,1H,ArH),7.21(dd,J＝7.5,5.0Hz,1H,ArH),7.10(s,1H,ArH),4.95(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(t,s,OMe),3.23(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 19: synthesis and Structure identification data for Compound 19 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 2-chloropyridine-3-formic acid (124mg, 0.79mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,7ml), stirring uniformly, adding 8-acetonyl dihydrocoptisine (100mg, 0.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 87mg of compound 19 as a yellow solid in a yield of 69.0%. 1 H-NMR(400MHz,DMSO-d 6 )δ:9.95(s,1H,ArH),8.94(s,1H,ArH),8.15(dd,J＝4.8,2.0Hz,1H,ArH),8.02(d,J＝8.8Hz,1H,ArH),7.81(d,J＝8.8Hz,1H,ArH),7.78(s,1H,ArH),7.65(dd,J＝7.2,2.0Hz,1H,ArH),7.24(dd,J＝7.2,4.8Hz,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.88(t,J＝6.0Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 20: synthesis and Structure identification data for Compound 20 of the invention
Synthesis of 8-acetonyl dihydroberberine (not shown).
Weighing 2-chloro-6-methylpyridine-3-formic acid (327mg, 1.91mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,16ml), stirring uniformly, adding 8-acetonyldihydroberberine (500mg, 1.27mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 584mg of the compound 20 as a yellow solid in 90.68% yield. 1 H-NMR(500MHz,DMSO-d 6 )δ:9.89(s,1H,ArH),8.94(s,1H,ArH),8.20(d,J＝9.0Hz,1H,ArH),8.00(d,J＝9.0Hz,1H,ArH),7.80(s,1H,ArH),7.53(d,J＝7.5Hz,1H,ArH),7.09(s,1H,ArH),7.05(d,J＝7.5Hz,1H,ArH),6.18(s,2H,OCH 2 O),4.93(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH 2 CH 2 ),2.36(s,3H,Me)。
Example 21: synthesis and Structure characterization data for Compound 21 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 2-chloro-6-methylpyridine-3-carboxylic acid (230mg, 1.34mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,8ml), stirring uniformly, adding 8-acetonyldihydropalmatine (500mg, 1.22mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 581mg of a compound 21 as a yellow solid, with a yield of 91.07%. 1 H-NMR(500MHz,DMSO-d 6 )δ:9.89(s,1H,ArH),9.03(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),7.72(s,1H,ArH),7.54(d,J＝7.5Hz,1H,ArH),7.10(s,1H,ArH),7.05(d,J＝7.5Hz,1H,ArH),4.95(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.36(s,3H,Me)。
Example 22: synthesis and Structure identification data for Compound 22 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 2-chloro-6-methylpyridine-3-formic acid (136mg, 0.80mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,12ml), stirring uniformly, adding 8-acetonyldihydrocoptisine (200mg, 0.53mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 214mg of compound 22 as a yellow solid in a yield of 82.31%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.96(s,1H,ArH),8.96(s,1H,ArH),8.03(d,J＝8.5Hz,1H,ArH),7.82(d,J＝8.5Hz,1H,ArH),7.79(s,1H,ArH),7.54(d,J＝7.5Hz,1H,ArH),7.08(s,1H,ArH),7.05(d,J＝7.5Hz,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.89(t,J＝6.5Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.5Hz,2H,NCH 2 CH 2 ),2.36(s,3H,Me)。
Example 23: synthesis and Structure identification data for Compound 23 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 4-aminopyridine-3-formic acid (24mg, 0.17mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,4ml), stirring uniformly, adding 8-acetonyldihydroberberine (100mg, 0.17mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 20mg of compound 23 as a yellow solid in 16.5% yield. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),8.94(s,1H,ArH),8.55(s,1H,ArH),8.20(d,J＝9.0Hz,1H,ArH),8.00(d,J＝9.0Hz,1H,ArH),7.92(d,J＝6.0Hz,1H,ArH),7.80(s,1H,ArH),7.09(s,1H,ArH),6.58(d,J＝6.0Hz,1H,ArH),6.18(s,2H,OCH 2 O),4.93(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 24: synthesis and Structure identification data for Compound 24 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 4-aminopyridine-3-formic acid (42mg, 0.3mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,5ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (100mg, 0.25mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 43mg of compound 24 as a yellow solid in a yield of 35.5%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.02(s,1H,ArH),8.54(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),7.86(d,J＝6.0Hz,1H,ArH),7.72(s,1H,ArH),7.10(s,1H,ArH),6.47(d,J＝6.0Hz,1H,ArH),4.95(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 25: synthesis and Structure identification data for Compound 25 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 6-bromopyridine-3-formic acid (385mg, 1.91mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v is 24:1,16ml), stirring uniformly, adding 8-acetonyldihydroberberine (500mg, 1.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v-24: 1) to obtain 607mg of a compound 25 as a yellow solid with a yield of 88.87%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),8.94(s,1H,ArH),8.64(d,J＝2.4Hz,1H,ArH),8.21(d,J＝9.2Hz,1H,ArH),8.01(d,J＝9.2Hz,1H,ArH),7.98(dd,J＝8.0,2.4Hz,1H,ArH),7.80(s,1H,ArH),7.47(d,J＝8.0Hz,1H,ArH),7.09(s,1H,ArH),6.18(s,2H,OCH 2 O),4.94(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 26: synthesis and Structure identification data for Compound 26 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 6-bromopyridine-3-carboxylic acid (370mg, 1.83mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,14ml), stirring uniformly, adding 8-acetonyldihydropalmatin (500mg, 1.22mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v-24: 1) to obtain 623mg of compound 26 as a yellow solid with a yield of 92.16%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.03(s,1H,ArH),8.64(d,J＝2.0Hz,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),7.98(dd,J＝8.0,2.0Hz,1H,ArH),7.72(s,1H,ArH),7.46(d,J＝8.0Hz,1H,ArH),7.10(s,1H,ArH),4.96(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 27: synthesis and Structure identification data for Compound 27 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 6-bromopyridine-3-formic acid (161mg, 0.80mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,12ml), stirring uniformly, adding 8-acetonyldihydrocoptisine (200mg, 0.53mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 215mg of compound 27 as a yellow solid with a yield of 77.90%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.96(s,1H,ArH),8.96(s,1H,ArH),8.64(dd,J＝2.0,0.8Hz,1H,ArH),8.03(d,J＝8.8Hz,1H,ArH),7.98(dd,J＝8.0,2.0Hz,1H,ArH),7.83(d,J＝8.8Hz,1H,ArH),7.80(s,1H,ArH),7.47(dd,J＝8.0,0.8Hz,1H,ArH),7.08(s,1H,ArH),6.54(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.89(t,J＝6.0Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 28: synthesis and Structure identification data for Compound 28 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 5-bromopyridine-3-formic acid (80mg, 0.40mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,4ml), stirring uniformly, adding 8-acetonyldihydroberberine (100mg, 0.25mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, and then filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1), whereby 82mg of compound 28 was obtained as a yellow solid in a yield of 60.7%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),8.94(s,1H,ArH),8.84(d,J＝1.6Hz,1H,ArH),8.54(d,J＝2.4Hz,1H,ArH),8.20(d,J＝9.2Hz,1H,ArH),8.17(dd,J＝2.4,1.6Hz,1H,ArH),8.00(d,J＝9.2Hz,1H,ArH),7.80(s,1H,ArH),7.09(s,1H,ArH),6.18(s,2H,OCH 2 O),4.93(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 29: synthesis and Structure identification data for Compound 29 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 5-bromopyridine-3-carboxylic acid (118mg, 0.58mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,11ml), stirring uniformly, adding 8-acetonyldihydropalmatin (200mg, 0.49mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 191mg of a compound 29 as a yellow solid in a yield of 70.7%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.02(s,1H,ArH),8.85(brs,1H,ArH),8.54(brs,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.18(brs,1H,ArH),8.02(d,J＝9.0Hz,1H,ArH),7.72(s,1H,ArH),7.10(s,1H,ArH),4.95(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 30: synthesis and Structure identification data for Compound 30 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 2-fluoropyridine-3-formic acid (43mg, 0.31mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,5ml), stirring uniformly, adding 8-acetonyldihydroberberine (100mg, 0.25mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, and then filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 55mg of a compound 30 as a yellow solid, with a yield of 45.5%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),8.94(s,1H,ArH),8.20(d,J＝9.0Hz,1H,ArH),8.00(d,J＝9.0Hz,1H,ArH),7.99(m,1H,ArH),7.91(m,1H,ArH),7.80(s,1H,ArH),7.16(m,1H,ArH),7.09(s,1H,ArH),6.18(s,2H,OCH 2 O),4.93(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝5.2Hz,2H,NCH 2 CH 2 ).
Example 31: synthesis and Structure identification data for Compound 31 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 2-fluoropyridine-3-carboxylic acid (69mg, 0.49mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,3ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (100mg, 0.24mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 99mg of a compound 31 as a yellow solid in 83.9% yield. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.03(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),8.00(m,1H,ArH),7.93(m,1H,ArH),7.72(s,1H,ArH),7.16(m,1H,ArH),7.10(s,1H,ArH),4.95(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 32: synthesis and Structure identification data for Compound 32 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 2-fluoropyridine-3-formic acid (112mg, 0.79mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,7ml), stirring uniformly, adding 8-acetonyl dihydrocoptisine (100mg, 0.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 72mg of a compound 32 as a yellow solid in a yield of 59.0%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.95(s,1H,ArH),8.95(s,1H,ArH),8.03(d,J＝8.8Hz,1H,ArH),8.01(ddd,J＝4.8,2.0,1.2Hz,1H,ArH),7.94(ddd,J＝10.0,7.2,2.0Hz,1H,ArH),7.82(t,J＝8.8Hz,1H,ArH),7.79(s,1H,ArH),7.17(ddd,J＝7.2,4.8,2.0Hz,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.88(t,J＝6.5Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.5Hz,2H,NCH 2 CH 2 ).
Example 33: synthesis and Structure identification data for Compound 33 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 2-fluoropyridine-4-formic acid (54mg, 0.38mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,5ml), stirring uniformly, adding 8-acetonyl dihydroberberine (100mg, 0.25mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 75mg of compound 33 as a yellow solid in a yield of 61.5%. 1 H-NMR(500MHz,DMSO-d 6 )δ:9.89(s,1H,ArH),9.34(s,1H,ArH),8.20(d,J＝9.0Hz,1H,ArH),8.09(brd,J＝5.0Hz,1H,ArH),7.99(d,J＝9.0Hz,1H,ArH),7.80(s,1H,ArH),7.56(m,1H,ArH),7.23(m,1H,ArH),7.09(s,1H,ArH),6.18(s,2H,OCH 2 O),4.93(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 34: synthesis and Structure identification data for Compound 34 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 2-fluoropyridine-4-formic acid (52mg, 0.37mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,4ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (100mg, 0.24mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 79mg of compound 34 as a yellow solid in 66% yield. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.02(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.09(ddd,J＝5.2,0.8,0.8Hz,1H,ArH),8.02(d,J＝9.0Hz,1H,ArH),7.72(s,1H,ArH),7.56(ddd,J＝4.8,2.8,1.2Hz,1H,ArH),7.23(m,1H,ArH),7.10(s,1H,ArH),4.95(t,J＝6.4Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.4Hz,2H,NCH 2 CH 2 )。
Example 35: synthesis and Structure identification data for Compound 35 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 2-fluoroisonicotinic acid (112mg, 0.79mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,5ml), stirring uniformly, adding 8-acetonyl dihydrocoptisine (100mg, 0.27mmol), heating for reaction until the raw material reaction is complete, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 84mg of a compound 35 as a yellow solid in a yield of 69.0%. 1 H-NMR(400MHz,DMSO-d 6 )δ:9.95(s,1H,ArH),8.95(s,1H,ArH),8.09(ddd,J＝4.8,0.8,0.8Hz,1H,ArH),8.03(d,J＝8.8Hz,1H,ArH),7.82(d,J＝8.8Hz,1H,ArH),7.79(s,1H,ArH),7.56(ddd,J＝4.8,2.8,1.2Hz,1H,ArH),7.23(m,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.88(t,J＝6.4Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.4Hz,2H,NCH 2 CH 2 )。
Example 36: synthesis and Structure identification data for Compound 36 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 2-chloropyridine-4-formic acid (300mg, 1.91mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,16ml), stirring uniformly, adding 8-acetonyldihydroberberine (500mg, 1.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 511mg of compound 36 as a yellow solid in a yield of 81.76%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),8.94(s,1H,ArH),8.28(d,J＝5.0Hz,1H,ArH),8.20(d,J＝9.0Hz,1H,ArH),7.99(d,J＝9.0Hz,1H,ArH),7.80(s,1H,ArH),7.60(m,2H,ArH),7.09(s,1H,ArH),6.18(s,2H,OCH 2 O),4.94(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 37: synthesis and Structure identification data for Compound 37 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 2-chloropyridine-4-carboxylic acid (212mg, 1.34mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,12ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (500mg, 1.22mmol), heating for reaction until the raw materials completely react, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 554mg of compound 37 as a yellow solid in 89.21% yield. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.02(s,1H,ArH),8.28(d,J＝5.2Hz,1H,ArH),8.21(d,J＝9.2Hz,1H,ArH),8.03(d,J＝9.2Hz,1H,ArH),7.72(s,1H,ArH),7.60(m,2H,ArH),7.10(s,1H,ArH),4.95(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 38: synthesis and Structure identification data for Compound 38 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 2-chloropyridine-4-formic acid (125mg, 0.80mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,12ml), stirring uniformly, adding 8-acetonyl dihydrocoptisine (200mg, 0.53mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v-24: 1) to obtain 202mg of a compound 38 as a yellow solid in a yield of 79.84%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.95(s,1H,ArH),8.95(s,1H,ArH),8.28(d,J＝5.0Hz,1H,ArH),8.03(d,J＝8.5Hz,1H,ArH),7.82(d,J＝8.5Hz,1H,ArH),7.79(s,1H,ArH),7.60(m,2H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.88(t,J＝6.5Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 39: synthesis and Structure identification data for Compound 39 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 2-aminopyridine-4-formic acid (29mg, 0.21mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,4ml), stirring uniformly, adding 8-acetonyldihydroberberine (100mg, 0.25mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 23mg of the compound 39 as a yellow solid in 19.3% yield. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),8.94(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),7.99(d,J＝9.0Hz,1H,ArH),7.85(d,J＝5.2Hz,1H,ArH),7.80(s,1H,ArH),7.09(s,1H,ArH),6.87(br,1H,ArH),6.82(dd,J＝5.2,1.6Hz,1H,ArH),6.18(s,2H,OCH 2 O),5.83(s,2H,NH 2 ),4.93(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 40: synthesis and Structure identification data for Compound 40 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 2-aminopyridine-4-formic acid (42mg, 0.30mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,7ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (100mg, 0.24mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to obtain 84mg of compound 40 as a yellow solid in a yield of 70.6%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.89(s,1H,ArH),9.02(s,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.03(d,J＝9.0Hz,1H,ArH),7.87(d,J＝5.0Hz,1H,ArH),7.72(s,1H,ArH),7.10(s,1H,ArH),6.88(brs,1H,ArH),6.83(brd,J＝5.0Hz,1H,ArH),5.87(s,2H,NH 2 ),4.95(t,J＝6.5Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.5Hz,2H,NCH 2 CH 2 )。
Example 41: synthesis and Structure identification data for Compound 41 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 2-aminopyridine-4-formic acid (24mg, 0.17mmol) into a reaction flask, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,7ml), stirring uniformly, adding 8-acetonyl dihydrocoptisine (100mg, 0.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, and then filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 20mg of compound 41 as a yellow solid in 16.5% yield. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.96(s,1H,ArH),8.96(s,1H,ArH),8.04(d,J＝8.8Hz,1H,ArH),7.96(d,J＝5.2Hz,1H,ArH),7.82(d,J＝8.8Hz,1H,ArH),7.79(s,1H,ArH),7.08(s,1H,ArH),6.91(brs,1H,ArH),6.85(brd,J＝5.2Hz,1H,ArH),6.53(s,2H,OCH 2 O),6.18(s,2H,OCH 2 O),6.09(s,2H,NH 2 ),4.88(t,J＝6.0Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 42: synthesis and Structure identification data for Compound 42 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 2-picolinic acid (235mg, 1.91mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,16ml), stirring uniformly, adding 8-acetonyl dihydroberberine (500mg, 1.27mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 521mg of compound 42 as a yellow solid in a yield of 89.37%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.93(s,1H,ArH),8.96(s,1H,ArH),8.39(br,1H,ArH),8.19(d,J＝9.0Hz,1H,ArH),8.00(d,J＝9.0Hz,1H,ArH),7.81(s,1H,ArH),7.70(d,J＝7.5Hz,1H,ArH),7.61(dd,J＝7.5,7.5Hz,1H,ArH),7.15(br,1H,ArH),7.09(s,1H,ArH),6.17(s,2H,OCH 2 O),4.96(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.08(s,3H,OMe),4.07(s,3H,OMe),3.20(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 43: synthesis and Structure identification data for Compound 43 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 2-picolinic acid (225mg, 1.83mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,14ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (500mg, 1.22mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 611mg of the compound 43 as a yellow solid in 98.0% yield. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.91(s,1H,ArH),9.05(s,1H,ArH),8.39(br,1H,ArH),8.21(d,J＝9.0Hz,1H,ArH),8.04(d,J＝9.0Hz,1H,ArH),7.73(s,1H,ArH),7.70(d,J＝7.5Hz,1H,ArH),7.61(dd,J＝7.5,7.5Hz,1H,ArH),7.15(br,1H,ArH),7.10(s,1H,ArH),4.96(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.07(s,3H,OMe),3.94(s,3H,OMe),3.87(s,3H,OMe),3.23(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 44: synthesis and Structure identification data for Compound 44 of the invention
Synthesis of 8-acetonyl dihydrocoptisine (omitted).
Weighing 2-picolinic acid (136mg, 0.80mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,12ml), 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, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v: 24:1) to obtain 185mg of a compound 44 as a yellow solid in a yield of 79.06%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.99(s,1H,ArH),8.96(s,1H,ArH),8.40(d,1H,J＝4.4Hz,ArH),8.02(d,J＝8.8Hz,1H,ArH),7.82(d,J＝8.8Hz,1H,ArH),7.80(s,1H,ArH),7.71(d,J＝7.5Hz,1H,ArH),7.62(ddd,J＝7.5,7.5,2.0Hz,1H,ArH),7.17(m,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2 O),6.17(s,2H,OCH 2 O),4.90(t,J＝6.0Hz,2H,NCH 2 CH 2 ),3.20(t,J＝6.0Hz,2H,NCH 2 CH 2 )。
Example 45: synthesis and Structure identification data for Compound 45 of the invention
Synthesis of 8-acetonyldihydroberberine (abbreviated).
Weighing 6-methyl-2-picolinic acid (70mg, 0.51mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,8ml), stirring uniformly, adding 8-acetonyldihydroberberine (100mg, 0.26mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1) to give 87mg of the compound 45 as a yellow solid with a yield of 71.9%. 1 H-NMR(500MHz,DMSO-d 6 )δ：9.91(s,1H,ArH),8.94(s,1H,ArH),8.20(d,1H,J＝9.0Hz,ArH),8.00(d,J＝9.0Hz,1H,ArH),7.80(s,1H,ArH),7.48(m,2H,ArH),7.09(s,1H,ArH),7.01(m,1H,ArH),6.17(s,2H,OCH 2 O),4.94(t,J＝6.0Hz,2H,NCH 2 CH 2 ),4.09(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH 2 CH 2 ),2.39(s,3H,Me)。
Example 46: synthesis and Structure identification data for Compound 46 of the invention
Synthesis of 8-acetonyldihydroplatine (omitted).
Weighing 6-methyl-2-picolinic acid (41mg, 0.30mmol) into a reaction bottle, adding a mixed solvent of tetrahydrofuran/water (v/v ═ 24:1,8ml), stirring uniformly, adding 8-acetonyl dihydropalmatine (100mg, 0.24mmol), heating for reaction until the raw materials react completely, and stopping heating. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed three times with a mixed solvent of tetrahydrofuran/water (v/v 24:1) to obtain 91mg of compound 46 as a yellow solid in a yield of 76.5%. 1 H-NMR(400MHz,DMSO-d 6 )δ：9.90(s,1H,ArH),9.03(s,1H,ArH),8.21(d,1H,J＝9.2Hz,ArH),8.03(d,J＝9.2Hz,1H,ArH),7.72(s,1H,ArH),7.507(d,J＝5.2Hz,1H,ArH),7.506(d,J＝4.0Hz,1H,ArH),7.10(s,1H,ArH),7.03(dd,J＝5.2,4.0Hz,1H,ArH),4.95(t,J＝6.4Hz,2H,NCH 2 CH 2 ),4.10(s,3H,OMe),4.08(s,3H,OMe),3.94(s,3H,OMe),3.88(s,3H,OMe),3.23(t,J＝6.4Hz,2H,NCH 2 CH 2 ),2.40(s,3H,Me)。
Second, Experimental example for solubility detection of Compound of the present invention
Experimental example 1: experimental example for measuring Water solubility 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 the temperature of 25 +/-2 ℃, and the amount of the compound dissolved in purified water per milliliter is 11.5mg of berberine pyridine-3-formic acid quaternary ammonium salt (1), 800mg of palmatine pyridine-3-formic acid quaternary ammonium salt (2), 7.5mg of coptisine pyridine-3-formic acid quaternary ammonium salt (3), 45mg of isoberberine pyridine-3-formic acid quaternary ammonium salt (4), 1.1mg of isoberberine pyridine-3-formic acid quaternary ammonium salt (5), 15mg of berberine 5-fluoropyridine-3-formic acid quaternary ammonium salt (6), 50mg of palmatine 5-fluoropyridine-3-formic acid quaternary ammonium salt (7), 1.5mg of coptisine 5-fluoropyridine-3-formic acid quaternary ammonium salt (8), 30mg of isoflavidine 5-fluoropyridine-3-formic acid quaternary ammonium salt (9), Isoberberine 5-fluoropyridine-3-carboxylic acid quaternary ammonium salt (10)1.38 mg. 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.
According to the solubility of each berberine chloride type alkaloid quaternary ammonium salt substrate, 4mg of each compound 1, 6, 11, 14, 17, 20, 23, 25, 28, 30, 33, 36, 39, 42, 45, 2mg of each compound, 7, 12, 15, 18, 21, 24, 26, 29, 31, 34, 37, 40, 43, 46, 42mg of each compound, 3, 4, 5, 8, 9, 10, 13, 16, 19, 22, 27, 32, 35, 38, 41, 44 are weighed respectively, placed in 1.8ml of pure water solvent at the temperature of 25 +/-2 ℃, shaken vigorously for 30 seconds every 5 minutes, and the dissolution within 30 minutes is observed, if no visible solute particles exist, the dissolution is considered to be complete. The experimental result shows that the amount of the compound 1, 6, 11, 14, 17, 20, 23, 25, 28, 30, 33, 36, 39, 42 and 45 dissolved in each milliliter of the purified water solvent is respectively larger than the amount of the corresponding berberine chloride quaternary ammonium salt substrate dissolved in each milliliter of the purified water solvent, the amount of the compound 2, 7, 12, 15, 18, 21, 24, 26, 29, 31, 34, 37, 40, 43 and 46 dissolved in each milliliter of the purified water solvent is respectively larger than the amount of the corresponding palmatine chloride quaternary ammonium salt substrate dissolved in each milliliter of the purified water solvent, the amount of the compound 3, 4, 5, 8, 9, 10, 13, 16, 19, 22, 27, 32, 35, 38, 41 and 44 dissolved in each milliliter of the purified water solvent is respectively more than the amount of the corresponding berberine type alkaloid quaternary ammonium salt chloride substrate dissolved in each milliliter of the purified water.
Experimental example 2: experimental example for detecting solubility of the 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 chloridized berberine type alkaloid quaternary ammonium salt substrate, 22mg of each compound 1, 6, 11, 14, 17, 20, 23, 25, 28, 30, 33, 36, 39, 42, 45, 2mg of each compound, 7, 12, 15, 18, 21, 24, 26, 29, 31, 34, 37, 40, 43, 46, 30mg of each compound, 3, 4, 5, 8, 9, 10, 13, 16, 19, 22, 27, 32, 35, 38, 41, 44 are weighed respectively, placed in 1.8ml of 95% ethanol solvent at the temperature of 25 ℃ plus or minus 2 ℃, shaken vigorously for 30 seconds every 5 minutes, and the dissolution condition within 30 minutes is observed, if no solute particles are visible, the dissolution is considered to be complete. The experimental results show that the amount of the compounds 1, 6, 11, 14, 17, 20, 23, 25, 28, 30, 33, 36, 39, 42 and 45 which can be dissolved in 95% ethanol per milliliter is respectively larger than the amount of the corresponding berberine chloride quaternary ammonium salt substrate which can be dissolved in 95% ethanol per milliliter, the amount of the compounds 2, 7, 12, 15, 18, 21, 24, 26, 29, 31, 34, 37, 40, 43 and 46 which can be dissolved in 95% ethanol per milliliter is respectively larger than the amount of the corresponding palmatine chloride quaternary ammonium salt substrate which can be dissolved in water per milliliter, the amounts of the compounds 3, 4, 5, 8, 9, 10, 13, 16, 19, 22, 27, 32, 35, 38, 41, 44 of the present invention that can be dissolved per ml of 95% ethanol solvent are also each greater than the amount of the corresponding berberine chloride-type alkaloid quaternary ammonium salt substrate that can be dissolved per ml of 95% ethanol.
Third, evaluation of the Effect of the Compounds of the present invention on the growth of Normal cells
Experimental example 3: evaluation experiment for influence of compound on growth condition of mouse mononuclear 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 the initial solution of the required concentration in DMEM high-sugar medium containing 10% FBS and double antibody (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%, subculturing the cells according to the dilution ratio of 1:3 in volume ratio.
(3) 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).
(4) The compound starting solution was diluted to a 200. mu.M solution in DMEM high-glucose medium containing 10% FBS and diabodies (streptomycin 100. mu.g/mL, penicillin 100U/mL). The compound administration solution is added into a 96-well plate containing 100. mu.l of the primary passage cell suspension, 100. mu.l of each well, and the concentration gradient of the compound series working solution of the present invention, i.e., 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.5625. mu.M, 0.78125. mu.M, 0.390625. mu.M, is set by the half-ratio dilution method, and 6 duplicate wells are set for each administration 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 incubation in an incubator for 24h, 10. mu.L of CCK-8 solution was added to each well, and after incubation in an incubator for 2h, the Optical Density (OD) value was measured at 450nm using a microplate reader (results of first experiment).
(5) 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 serial concentration gradient solutions 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 mul of multi-passage cell suspension per well, each well is 100 mul, and 100 mul is respectively sucked away after being blown evenly, so that series concentration gradients with the action concentrations of 10 mul, 1 mul, 0.1 mul and 0.01 mul are obtained. 6 duplicate wells were set for each dosing concentration. A normal cell control group and a blank control group were set simultaneously. After 24h 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 (Table 1 experiment).
Cell viability was calculated using the following formula:
cell survival (%) < 100%
(3) As a result:
(vi) within the time range determined by the experiment, the survival of cells cultured from the primary passage cells treated with compound 1 and compound 8 at serial concentrations of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M, 1.5625 μ M, 0.78125 μ M, 0.390625 μ M was 82.13 ± 0.5%, 117.21 ± 1.15%, 133.08 ± 1.49%, 143.46 ± 10.3%, 129.05 ± 2.61%, 119.76 ± 7.29%, 107.02 ± 3.44%, 111.8 ± 2.30%, 119.02 ± 1.84%, 107.42 ± 2.76%, 139.70 ± 4.92%, 152.41 ± 8.44%, 368 ± 38%, 360.7%, 360.7.7%, 360% and 360.7%, +/-8 ± 38%.
② in the time range of experimental determination, the series of compounds of the invention has no cytotoxicity to multi-passage RAW264.7 cells under the action concentration of 0.01-10 μ M 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 survival after treatment of Normal RAW264.7 cells with the Compound of the present invention Table 1 cell survival after treatment of Normal RAW264.7 cells with the Berberine-type alkaloid picolinic acid quaternary ammonium salt Compound ①
Figure BDA0002976196020000431
Figure BDA0002976196020000441
Note: ① the specific experimental result has a certain relation with the cell growth state; ② the survival rate of the normal group was 100%, P <0.05, P <0.01, P <0.001 compared to the normal group.
(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: the compound of the invention is used for cell toxicological evaluation experiments on the 293T cells of normal human embryonic kidney epithelial cells cultured in vitro.
(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): normal human embryonic kidney epithelial cells 293T cells grown to 90% confluence in vitro culture according to cell instructions were treated with 0.25% pancreatin/0.1 EDAfter TA digestion, cell suspensions were prepared from cell culture media and cell suspension concentrations were adjusted and plated on 96-well cell culture plates at a cell density of 2X 10 with 100. mu.l cell suspension per well 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, 1 × 10 -5 The mol/L of the compounds of the series of the invention have no obvious cytotoxicity to 293T normal cell line cells, except that the cell survival rate of the compound 1 is 98.5%, the cell survival rates of other investigated compounds of the invention are all more than 100%, and no significant difference is detected statistically.
Experimental example 5: the compound of the invention is used in the evaluation experiment of the cytotoxicity on HELF cells of normal human embryonic lung fibroblasts and NIH3T3 cells of mouse embryonic fibroblasts cultured in vitro.
(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) of the stock solution was diluted with the cell culture medium to a concentration of 20. mu.M as the starting solution for the cell experiments.
(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, 5% CO 2 The operation of 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 that series concentration gradients with the action concentrations of 10 mu M, 1 mu M, 0.1 mu M, 0.01 mu M and 0.001 mu M are respectively obtained, 3 multiple wells are arranged for each administration concentration, and a normal cell control group and a blank control group are simultaneously arranged. 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%
The results of the experiment were analyzed using GraphPad Prism version 5.0 software.
(3) As a result: within the time range of 96h determined by experiments, the series of compounds have no obvious cytotoxicity on HELF cells of human embryonic lung fibroblasts and NIH3T3 cells of mouse embryonic fibroblasts, and the growth inhibition rate of experimental cells and the IC of each compound are determined 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
Figure BDA0002976196020000451
Figure BDA0002976196020000461
(4) And (4) conclusion: the series of compounds of the invention have no obvious toxicity on the growth of human embryonic lung fibroblast HELF cells and mouse embryonic fibroblast NIH3T3 cells, and are 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 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. 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 converted into the content of NO in the cell culture solution of each group (see tables 3-9).
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 (tables 3-9). The compound of the invention has obviously better inhibiting effect on NO generation in an RAW264.7 cell inflammation model induced by LPS stimulation than a berberine chloride type alkaloid quaternary ammonium salt substrate.
TABLE 3 Effect of the Compounds of the present invention on the amount of NO secretion in a model of inflammation of RAW264.7 cells induced by LPS stimulation
Figure BDA0002976196020000471
a -0. In comparison with the normal group, ### p is less than 0.001; comparison with model group, P <0.001
TABLE 4 influence of the Compounds of the invention on the amount of NO secretion in a model of inflammation of RAW264.7 cells induced by LPS stimulation
Figure BDA0002976196020000472
In comparison with the normal group, ### p is less than 0.001; p <0.001 in comparison with model group
TABLE 5 Effect of the Compounds of the present invention on NO secretion in RAW264.7 cell inflammation model induced by LPS stimulation
Figure BDA0002976196020000481
In comparison with the normal group, ### p is less than 0.001; p <0.001 in comparison with model group
TABLE 6 influence of the Compounds of the present invention on the amount of NO secretion induced by LPS stimulation in a model of inflammation of RAW264.7 cells
Figure BDA0002976196020000482
And normalThe comparison of the groups is carried out, ### p is less than 0.001; p <0.001 in comparison with model group
TABLE 7 influence of the Compounds of the invention on the NO secretion in the model RAW264.7 cell inflammation induced by LPS stimulation Note that
Figure BDA0002976196020000483
Note that In comparison with the normal group, ### p is less than 0.001; comparing with model group, P <0.05, P <0.01, P <0.001
TABLE 8 influence of the Compounds of the invention on the amount of NO secretion in a model of inflammation of RAW264.7 cells induced by LPS stimulation Note that
Figure BDA0002976196020000491
Note that In comparison with the normal group, ### p is less than 0.001; comparing with model group, P <0.05, P <0.01, P <0.001
TABLE 9 Effect of Compounds of the present invention on NO secretion in RAW264.7 cell inflammation model induced by LPS stimulation Note that
Figure BDA0002976196020000492
Note that In comparison with the normal group, ### p is less than 0.001; in comparison with the set of models, ** P＜0.01, *** 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
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, streptomycin and penicillin and have good growth state and are in logarithmic growth phase, and adding a proper amount of culture mediumLightly beating nutrient medium to obtain cell suspension, and adjusting cell density of the cell suspension to 6 × 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 by preparing each intervening experimental cell suspension containing the compound of the present invention by a half-ratio dilution method using a working concentration of the compound of the present invention of 10. mu.M as an initial concentration, and controlling the total volume per well to be still 100. mu.l. 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 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 the standard curve, the concentration of sodium nitrite in the sample was calculated and converted to the NO content in the cell culture fluid of each group (see Table 10). 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 10). 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 10). 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 10 Effect of Compounds of the invention on NO secretion in the initial passage RAW264.7 cell inflammation model induced by LPS stimulation
Figure BDA0002976196020000501
Figure BDA0002976196020000511
a 0. In comparison with the normal group, ### p is less than 0.001; comparing with model group, 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-6ELISA 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-6ELISA 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 11 and 12. As can be seen from the experimental data in tables 11 and 12, the expression level of IL-6 in the model group was significantly increased with reference to the expression level of IL-6 in the normal control group, and was statistically significantly different from that in the normal control group. After the compounds are 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 inhibition effect of each compound treatment group on the expression level of IL-6 has obvious difference and is obviously stronger than that of a corresponding berberine type alkaloid quaternary ammonium salt substrate.
TABLE 11 influence of the Compounds of the present invention on the expression level of IL-6 in a model of inflammation of RAW264.7 cells induced by LPS stimulation Note that
Figure BDA0002976196020000512
Figure BDA0002976196020000521
Note that In comparison with the normal group, ### 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。
TABLE 12 influence of the Compounds of the invention on the expression level of IL-6 in a model of inflammation of RAW264.7 cells induced by LPS stimulation Note that
Figure BDA0002976196020000522
Note that 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 is less than 0.001; compared with the palmatine chloride quaternary ammonium salt group, & P＜0.05。
experimental example 9: experimental examples of the inhibitory effect of the compounds 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 and human lung cancer A549 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 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-well cell culture plate, each well is 100 mu l, then 100 mu l is respectively absorbed and discarded from each well, so as to obtain series concentration gradients with the action concentration of 10 mu M, 1 mu M and 0.1 mu M, 3 multiple 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 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%
The series of compounds of the invention showed significant cytostatic activity against HCT-116 cells over a period of 96h as measured experimentally, and the IC of the growth inhibition of HCT-116 cells by compounds 6, 8 and 10 of the invention was determined 50 Values were 1.286 μ M, 1.195 μ M and 2.248 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.
The results of the experiment were analyzed using GraphPad Prism version 5.0 software.
(2) Inhibition of A549 cell growth by 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-well cell plate, each well is 100 mu l, then 100 mu l is respectively absorbed and discarded from each well, so as to obtain series concentration gradients with the action concentration of 10 mu M, 1 mu M and 0.1 mu M, 3 multiple 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 of each compound at a wavelength of 570nm was measured by the 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 6 and 8 of the invention on the A549 cells 50 Values of 3.114 μ M and 2.319 μ M, respectively; in parallel experiments, IC of berberine chloride quaternary ammonium salt and coptisine chloride quaternary ammonium salt on growth inhibition of A549 fine 50 Values were > 10. mu.M and 6.540. 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 immunomodulator drugs and the use for preparing drugs for preventing, alleviating and/or treating diseases causing the increase of NO secreted by a living organism are proved or partially proved in the aforementioned relevant experimental examples, and the compound of the present invention is used for preparing drug products for preventing, alleviating and/or treating diseases causing the increase of IL-6 secreted by a living organism, preparing drug products for preventing, alleviating and/or treating diseases causing the increase of autoantibodies formed by a living organism, and preparing drug products for preventing, alleviating and/or treating inflammatory diseases, febrile diseases, cardiovascular and cerebrovascular diseases, blood system diseases and autoimmune diseases are shown in the experimental examples. 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.
Typical symptoms of rheumatoid arthritis include joint inflammation, significantly elevated levels of IL-6 in serum, pathological autoantibody formation; anemia is one of the most common extra-articular symptoms of rheumatoid arthritis. Therefore, the efficacy of the compound in reducing the pathological high IL-6 content, increasing the hemoglobin content and reducing the pathological autoantibody level on the animal level is evaluated by adopting a rheumatoid arthritis animal model, and the effect of the compound on the rheumatoid arthritis degree index score (joint inflammation index score for short), the joint inflammation incidence and the sole thickness of a model animal is used as evaluation indexes to evaluate the curative effect of the compound on the rheumatoid arthritis.
Experimental example 10: evaluation of efficacy of the Compound of the present invention in reducing IL-6 level in model animal Experimental example 1, Experimental 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 7, 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 7 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 volume was 1ml/100g (volume of working solution/body weight of animal) and administered to experimental DBA/1 mice by intragastric administration.
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. Arthritis 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 of any of the ankle, sole and toes of the mice in the limbs after the 4 th week of primary immunization was considered as the onset of a mouse arthritis model.
(2) Animal grouping and administration
In this experiment, animals were randomly divided into 6 groups, i.e., a blank control group (normal control group), a model group, a positive drug control group, and low/medium/high dose test compounds of the present invention. The administration dose of each administration group of the compound of the present invention at low/medium/high dose was set to 50mg/kg, 100mg/kg and 200mg/kg (amount of compound/body weight of animal), and the number of administrations and 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..
As shown in fig. 1, administration was started on day 18 after the second immunization from mouse molding, and continued for 4 weeks until the administration group had significantly reduced arthritis symptoms compared with the model group, and administration was terminated after significant difference in joint inflammation index score. The blank control group and the model group are administered with blank menstruum by intragastric gavage, the tested compound group is administered with the compound administration working solution with corresponding dose by intragastric gavage, and the positive control medicine group is administered with the positive control medicine administration working solution with corresponding dose by intragastric gavage.
(3) Evaluation index
At the end of the experiment, the mice are anesthetized, the eyeballs are picked up, blood samples of all groups of animals are collected through the eyesockets, anticoagulation is carried out, anticoagulation blood is taken, the change condition of the level of the inflammatory cytokine IL-6 in the serum of the mice is detected by adopting a Mouse IL-6ELISA Kit method, and the experimental results of different groups are compared 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 has statistical difference, which indicates that the modeling is successful; compared with a model group, the level of IL-6 in serum of each mouse detected by a methotrexate positive medicine group and a compound 7 low/medium/high dose administration group is obviously reduced, and has statistical difference. The compound 7 low/medium/high dose administration group of the invention has obviously better inhibition effect on IL-6 level in serum of a model mouse than a positive drug. The results are shown in table 13 and fig. 2.
TABLE 13 Effect of Compound 7 of the invention on IL-6 concentration in serum of model mice tested (pg/mL).
Figure BDA0002976196020000551
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 remarkable inhibitory effect on the level of IL-6 in serum of a model mouse.
Experimental example 11: experimental example for evaluating efficacy of compound of the present invention in increasing hemoglobin content in model animal
In "Experimental example 10: efficacy evaluation test example "in the experiment of the compound for reducing IL-6 level of model animals, each group of mice was anesthetized at the end of the experiment, the eyeballs were removed, blood was taken, the taken blood was anticoagulated with EDTA-dipotassium, and the anticoagulated blood was detected by a conventional blood detection method. 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 hemoglobin content of each dose group of the compound of the invention tested had a significant increase in comparison to the model group, and the mean values of the hemoglobin content of the low/medium/high dose group of compound 7 of the invention were 180.18 ± 4.45(g/L), 182.75 ± 2.22(g/L) and 182.75 ± 2.42(g/L), respectively, with a very significant difference in comparison to the model group (p <0.001 in comparison to the model group, see fig. 3). Particularly, compared with a blank control group, each dosage group of the compound 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.
Experimental example 12: evaluation of pharmacological effects of the compound of the invention on rheumatoid arthritis (1) evaluation index
In "Experimental example 10: in the experiment, the evaluation experiment example of the efficacy of the compound for reducing the IL-6 level of the model animal continuously adopts the evaluation and calculation of rheumatoid arthritis index score, the statistics of the incidence rate of arthritis, the sole thickness detection of the model mouse and the detection of the influence on the autoantibody of the model mouse, and combines the detection experiment example of the influence on the IL-6 level and the hemoglobin level of the model animal to evaluate the drug efficacy of the compound for resisting rheumatoid arthritis.
In the evaluation experiment, joint inflammation index scoring, calculating and evaluating are carried out on joints of each group of mice every other day from 8 days after the boosting to the end point of the experiment. The total score of the limb scores of each mouse is added to the mouse joint inflammation index score, and the relationship between the rheumatoid joint inflammation index score and the disease severity is shown in table 14.
TABLE 14 evaluation criteria for rheumatoid arthritis index scores
Figure BDA0002976196020000561
Figure BDA0002976196020000571
From 8 days after the boosting immunization to the experimental end point, the mice are subjected to joint inflammation index scoring measurement every other day, the number of mice suffering from joint inflammation of each group of mice is recorded, and the incidence rate is counted.
From day 8 after the boosting to the end of the experiment, the thickness of the two hind soles of the mice was measured with a vernier caliper while the mice were scored for the arthritic index every other day.
At the end of the experiment, the mice were anesthetized, blood samples of each group of animals were collected by orbital bleeding, and after anticoagulation, the content of chicken type II collagen antibody in serum was detected by ELISA.
(2) Data processing and analysis
Experimental data are presented as X ± SEM. The comparison of the continuous data was performed using analysis of variance followed by Duncan's test; the discrete data were analyzed by Kruscal-Wallis analysis of variance and median among nonparametric tests. The results of the experiment were analyzed using GraphPad Prism version 5.0 software and presented graphically.
3. Results of the experiment
(1) The tested compound of the invention can obviously improve the mouse joint inflammation index score
And (3) from the 28 th day after model building, performing joint inflammation index scoring, calculation and evaluation on the arthritis symptom degrees of the limbs and joints of the mouse chicken type II collagen induced arthritis model mouse every other day.
Experimental results show that the animals begin to have secondary lesions on the 21 st day after the model building, the lesions are more obvious on the 28 th day, the chronic systemic inflammation characterized by multiple arthritis is shown, the limbs can be seen by naked eyes to have red swelling and deformation of joints with different degrees, the foot swelling symptom on the non-inflammation side is also obvious, ankle joints and the whole toes are involved, the joint swelling appears between the toe joints and the front toes, and ear erythema, tail nodules and the like are accompanied. The experiment was started on day 38 after molding. The experimental results showed that, compared to the model group, the joint inflammation index score of the medium dose group administered with compound 7 of the present invention at a dose of 100mg/kg was consistently lower than that of the positive drug methotrexate from day 4 after the start of experimental administration (i.e., day 42 after the start of primary immunization) to the end of the experiment, whereas the joint inflammation index score of the low dose group administered with a dose of 50mg/kg was consistently comparable to that of the positive drug methotrexate from day 4 to day 18 after the start of experimental administration, and the joint inflammation index score of the low dose group was consistently lower than that of the positive drug methotrexate and also lower than that of the medium dose group (i.e., the efficacy was superior to that of the medium dose group) from day 18 until the end of the experiment. The high dose group administered with compound 7 of the present invention at a dose of 200mg/kg also had a significantly lower joint inflammation index score than the model group, and the therapeutic effect was comparable to that of the positive drug methotrexate from day 20 after the start of administration. At the end of the experiment, the low/medium/high dose groups had joint inflammation index scores of 1.73 ± 0.68, 2.67 ± 0.80 and 3.58 ± 0.66, respectively, which were all significantly reduced compared to the joint inflammation index score of 6.92 ± 0.84 in the model group, with all very significant differences. Comprehensive analysis shows that the low/medium/high dosage groups of the compound 7 have the effect of obviously improving the disease condition of joint inflammation of model animals, wherein the effect of improving the disease condition of the model animals is most obvious in the low dosage group along with the prolonging of the treatment period, the effect of improving the disease condition of the model animals in the medium dosage group is weaker in the lower dosage group but better than that in the high dosage group, and the unique dose-effect relationship further shows the unique action mechanism of the compound. Specific data and graphical results of significant improvement in mouse joint inflammation index scores for compounds of the invention are shown in table 15 below and figure 4.
Table 15. effect of different administration groups of the present invention on the joint inflammation index score of experimental mouse chicken type II collagen-induced arthritis model mouse.
Figure BDA0002976196020000581
Note: n-12; compared to the model group, p <0.05, p <0.01, p < 0.001.
(2) The tested compound of the invention has obvious inhibition effect on the incidence rate of arthritis of experimental animals
In the experiment, the incidence rate data of the arthritis of each group of animals is obtained by carrying out statistical analysis on the incidence rate of the arthritis of the mouse chicken II type collagen induced arthritis model mouse in the administration treatment process of the model animals. The incidence of inflammation in the joints of the mice in each group throughout the treatment cycle is shown in Table 16. By day 28 after the start of administration (i.e., day 66 after primary immunization), the incidence of arthritis in the model animals was 100%, the incidence of arthritis in the mice as the methotrexate-positive drug was 75%, and the incidence of arthritis in the mice as the low/medium/high dose group of compound 7 of the present invention was 45%/58%/83%, respectively. Therefore, the compound has obvious inhibiting effect on the incidence rate of the joint inflammation of the mouse, can obviously improve the disease condition of the joint inflammation of the model animal, particularly has the most obvious effect on improving the disease condition of the model animal by using a low-dose group, has weak effect on improving the disease condition of the model animal by using a medium-dose group, is superior to a high-dose group and is still significantly superior to a positive medicine group. The incidence data of joint inflammation further illustrate the unique mechanism of action of the compounds of the present invention.
TABLE 16 Effect of Compounds of the invention on the incidence of arthritis in laboratory animals
Figure BDA0002976196020000591
Note: n is 12.
(3) Effect of the Compound of the present invention on the thickness of the sole of mouse Chicken model mouse of type II collagen-induced arthritis
In the whole drug administration treatment process, the thickness of the soles of the mice of each group is measured by using a vernier caliper, and statistical analysis is carried out. The dynamic data of the sole thickness of each group of mice are shown in Table 17. The results show that the ankle joint of a mouse chicken type II collagen-induced arthritis model mouse is obviously swollen, and the tested compound has the effect of obviously inhibiting the ankle joint swelling, wherein the dosage group in the compound shows an obvious effect on the 40 th day (namely the 3 rd day after administration) after the model is made, and has obvious difference compared with the model group, and the effect is better than that of a positive medicine group. The compound low-dose group shows obvious effect at 46 days after model building (namely 9 days after administration), and has significant difference compared with a model group; and the effect of the low dose group gradually exceeded the medium dose group as the course of treatment was prolonged; the low/medium dose groups all exceeded the positive group by day 56 after molding (i.e., day 19 after dosing). The compound high-dose group also shows obvious improvement effect on the thickness of the sole of a mouse chicken II type collagen induced arthritis model mouse, and the effect is equivalent to that of a positive medicine group. The sole thickness data of the model animals further illustrate the unique mechanism of action of the compounds of the present invention.
TABLE 17 Effect of Compounds of the present invention on paw thickness (mm) of mouse Chicken type II collagen-induced arthritis model mice
Figure BDA0002976196020000601
Figure BDA0002976196020000611
And annotating: n is 12; compared to the model group, p <0.05, p <0.01, p < 0.001.
(4) Effect of the tested Compounds of the present invention on the level of autoantibodies in mouse Chicken type II collagen-induced arthritis model mice
Autoimmune diseases such as rheumatoid arthritis are chronic diseases caused by tissue damage due to autoantibody production. This study examined the effect of tested compound 7 of the invention on levels of mouse autoantibodies in a mouse chicken type II collagen-induced arthritis model mouse. 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, and the data of the results are shown in table 18 and fig. 5. 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 antibody in the serum of the mouse chicken type II collagen induced arthritis model mouse of the positive medicine methotrexate group and the compound 7 low/medium dose group are both obviously reduced, wherein compared with the model group, the positive group has obvious difference on the reduction action of the anti-chicken type II collagen IgG antibody level, while the reduction action of the compound 7 low dose group is better than that of the positive medicine and has extremely obvious difference compared with the model group, the action of the compound 7 medium dose group is slightly lower than that of the low dose group but is also better than that of the positive medicine, and compared with the model group, the effect of the compound 7 medium dose group is obviously different. Although the effect of the high dose group of compound 7 of the present invention was not statistically different from that of the model group, the tendency of its decreasing effect against IgG antibodies of chicken type II collagen was also very significant.
TABLE 18 Effect of Compound 7 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.
Figure BDA0002976196020000612
Note: # in order to compare with the blank control group, ### p<0.001; is compared with the model group<0.05，**p<0.01，***p<0.001。
According to the experimental results, the compound has obvious effect of resisting rheumatoid arthritis on experimental animals, wherein in the dose range selected by experiments, the low/medium/high dose of the compound 7 shows obvious or obvious effect, and the effect is mainly shown in that the compound has obvious improvement effect on the joint inflammation index score, the joint inflammation incidence rate and the sole thickness of a mouse chicken type II collagen induced arthritis model mouse, namely the clinical symptoms of the joint inflammation of the model mouse are obviously improved, the joint inflammation incidence rate of the model animal is obviously reduced, and the sole thickness of the model animal is obviously reduced.
In addition, by combining various experiments of the compound of the invention on a cell level and biological activity evaluation experiments on an animal level, namely the compound 7 of the invention is intervened and treated to obviously reduce the content of IL-6 in the serum of a mouse chicken type II collagen induced arthritis model mouse, and has obvious inhibiting effect on the generation of IgG antibodies, the compound of the invention is further shown to have obvious activity of resisting autoimmune diseases and has the effect of balancing the immune function of the organism. Since rheumatoid arthritis plays an important role in the development of cardiovascular system diseases and often causes blood system dysfunction including anemia and the like, the compound of the invention has a significant increase in hemoglobin content in blood of mouse chicken type II collagen induced arthritis model animals treated by the compound 7 of the invention, which is combined with the experimental examples of biological activity evaluation on animal level, and not only further shows that the compound of the invention has an anti-rheumatoid arthritis effect, but also shows that the compound of the invention has a significant intervention effect on cardiovascular system diseases, and can be used for treating cardiovascular system diseases including anemia. It is further emphasized that, within the dose range selected in the experiment, the low dose group of the experiment has the strongest effect on rheumatoid arthritis, and the medium dose group has weaker effect than the low dose group but stronger effect than the high dose group, and shows unconventional dose-effect relationship.
Experimental example 13: pharmacodynamic evaluation Experimental example 1 of antipyretic Effect of the Compound of the present invention on Yeast-induced rat fever model, test Material
(1) Animals: male SD rats weighing 180-200 g.
(2) Test compounds and reagents of the invention:
the compound of the invention tested was compound 7, administered at a dose set at 100 mg/kg. The positive drug is compound acetaminophen tablet (also called sanlitong), the specification is 250mg of acetaminophen/150 mg of isopropyl antipyrine/50 mg of anhydrous caffeine, and the administration dose is set to be 45 mg/kg. The reagent for inducing the rat fever model is yeast; the solvent for preparing the yeast solution is 0.9 percent sodium chloride injection.
The compound and the positive drug are prepared into working solution by adopting 0.5 percent CMC-Na aqueous solution as a solvent, and the compound and the positive drug are administrated by gastric gavage according to the dosage of 1ml/100g (working solution/animal body weight).
(3) Test consumables:
the electronic body temperature measuring instrument is MC-246. The scale is H21203, model No. 21201A. Rat gavage needle, 2.5ml syringe.
2. Test method
(1) Induction of Yeast-induced rat fever model
After 3 days of adaptive breeding of SD rats in an experimental animal room, anal temperature is measured 2 times in the morning and recorded as the body temperature of the experimental animals, the measurement time interval is 30min, the average body temperature is taken, 2 days of continuous measurement are carried out, the average value of the body temperatures measured on two days is taken as the basal body temperature, and meanwhile, the rats are adaptive to temperature measurement operation. During the experiment, rats with the basal body temperature of 36-38 ℃ and the body temperature fluctuation of less than 0.6 ℃ are selected to enter the experiment, and the rats are fasted for 12 hours before the experiment. When in molding, the 20% yeast suspension prepared by taking 0.9% sodium chloride injection as a solvent is injected subcutaneously at the back of a rat, the dosage volume is 10ml/kg (yeast suspension/weight of the rat), and a rat fever model is established. Rats in the blank control group were injected with the corresponding volume of vehicle (note: the protocol for all animal experiments was strictly performed according to the guidelines of the ethical committee on laboratory animals and approved by the ethical committee).
(2) Animal grouping and administration
After injecting 20% yeast suspension into rat subcutaneously for 5h, selecting rat with body temperature rising 1.0-3.0 deg.C for experiment. The test method comprises the following steps of injecting animals with corresponding volume of menstruum subcutaneously, randomly dividing experimental animals into 4 groups including a blank control group (a normal control group), a model group, a positive medicine group and a tested compound 7 administration group, and each group comprises 12 experimental mice. When in administration, experimental animals of a tested compound group and a positive control group are respectively administered with corresponding tested compound CMC-Na administration working solution or positive control drug CMC-Na administration working solution prepared according to administration dosage through intragastric administration, a blank control group and a model control group are both administered with an isovolumetric blank solvent through intragastric administration, the intragastric administration volume is 10ml/kg (yeast suspension/rat body weight), and the anal temperature of a rat is respectively measured and recorded at 0h, 1h, 2h, 4h and 6h after administration. The administration times and administration routes of the compound group and the positive medicine group are set to be 1 time/day and p.o..
(3) Evaluation index
Respectively measuring and recording the anal temperature of the rat at 0h, 1h, 2h, 4h and 6h after administration; and calculating and recording the difference value delta T (DEG C) between the body temperature measured in each time period and the basal body temperature. The antipyretic effect of the compounds of the present invention was determined by comparison with the corresponding data of the normal control group and the model group.
(4) Data processing and analysis
The experimental data are expressed as Mean ± SEM. Continuous data comparisons were performed using analysis of variance (ANOVA) followed by Duncan's test; the discretized data were tested by Krusacal-Wallis ANOVA and media Test among non-parametric tests. The results of the experiment were statistically analyzed and presented graphically using GraphPad Prism version 8.0 software.
3. Test results
The mean basal body temperatures of rats in each group were between 37.55 ℃ and 37.88 ℃ with a small difference in the mean values, based on basal body temperatures recorded from pre-dose measurements. The body temperature of the blank was maintained between 37.34 ℃ and 37.83 ℃ throughout the experiment. After 5 hours of molding, the temperature of each molding module body rises to 39.60-39.75 ℃, and the rising conditions of each group are basically consistent. Compared with the model group, the body temperature of the positive medicine powder pain-relieving administration group is remarkably reduced 6 hours after the model is made (1 hour after the administration), and the body temperature of the compound 7 administration group is remarkably reduced. At 7 hours after the model building (2 hours after the drug administration), the body temperature of the positive drug group begins to rise again, and of course, the body temperature still keeps very significant difference compared with the model group; the compound 7 administration group of the invention is different from the positive drug, the body temperature further drops after 7 hours (2 hours) after the model is made, the difference of the body temperature drop is more obvious compared with the last observation point, and the difference is very significant compared with the model group; after 9 hours (4 hours of drug administration) after the model is made, the body temperature of the positive drug group continues to rise significantly, the rising speed is accelerated, and the difference is statistically absent compared with the model group; although the body temperature of the compound 7 group of the invention begins to rise again, the rising amplitude is obviously smaller than that of the positive medicine group, and the compound still has significant difference compared with the model group, which shows that the compound still has significant antipyretic effect; at 11 hours after molding (6 hours after administration), the body temperature of each administration group rose back to the level of the model group, but the body temperature of the compound 7 group was lower than that of the positive drug group. The model group is maintained to the last body temperature check point 5 hours after the highest body temperature platform period self-molding, and the body temperature of the animal in the model group at each time point after the animal in the drug administration group is administrated has significant difference with that of a blank control group, which indicates that the molding is successful (the body temperature trend of the model group is also the same as that reported in the literature), and the model is stable. The positive medicine powder pain-relieving group experiment result accords with the pharmacological effect and clinical cognition. The body temperature change data recorded for this experiment are shown in table 19 and figure 6 (time calculated in accordance with molding out of parentheses and time calculated in accordance with administration in parentheses).
TABLE 19 Experimental clinical thermometry of pharmacodynamic evaluation of antipyretic effect of Compound 7 of the present invention in animal experiments
Figure BDA0002976196020000641
Note: each group n is 12; # in order to compare with the blank control group, ### p<0.001; is compared to model groups<0.01，***p<0.001。
The influence of the compound dry prognosis of the invention on the body temperature of the yeast induced rat fever model animal can be more intuitively demonstrated by calculating and comparing the difference between the body temperature of the rat given to the compound of the invention in each time period after the dry prognosis and the basal body temperature of the rat recorded before the administration (the difference is simply called body temperature difference), as shown in table 20.
After 5 hours of molding and before the compound of the invention is fed, the body temperature of rats in each group averagely rises 1.74-1.95 ℃, the model is stable, and the trend is consistent. After 1 hour of administration, the positive powder is used for alleviating painThe temperature difference value is 0.23 +/-0.77 *** The temperature is close to the basal body temperature, and the significant difference is obtained compared with the model group, which indicates that the positive medicine is effective and the molding is successful; the amplitude of the temperature difference value of the compound 7 group body is reduced definitely, but has no statistical difference compared with the model group. The situation changes after 2 hours of administration, the range of the positive powder pain-relieving group temperature difference value is obviously increased compared with the upper observation point, but the difference of the body temperature difference value and the model group still keeps very significant difference, which shows that the positive powder pain-relieving group still has the antipyretic effect, but the antipyretic effect begins to weaken after 2 hours of administration. Different from the positive drug, the temperature difference value amplitude of the compound 7 group body is further reduced after 2 hours of administration, the difference is more obvious compared with the difference of the upper observation point, and the difference is very obvious compared with the model group, which shows that the antipyretic effect of the compound is still continuously enhanced after 2 hours of administration, and the effect is more obvious. After 4 hours of administration, the temperature difference value of the positive powder pain-relieving group body is further obviously increased to reach 2.05 +/-0.52, and no statistical difference exists when compared with a model group; the temperature difference value amplitude of the compound 7 group body of the invention is also increased, but the temperature difference value amplitude of the compound 7 group body of the invention is obviously smaller than that of the positive medicine group, and is 1.58 +/-0.83. After the administration is carried out for 6 hours, the body temperature difference amplitude of the positive medicine group and the compound administration group is further increased, the positive medicine group reaches 2.46 +/-0.53 and exceeds the model group, and the statistical significance is avoided compared with the model group; the temperature difference value amplitude of the compound 7 group body is 1.86 +/-0.68 and is smaller than the temperature difference value of the positive medicine group; although the body temperature difference value of the compound 7 group of the invention has no significant statistical difference compared with the model group, the body temperature difference value of the compound 7 group of the invention has a range which is obviously smaller than that of the model group and is smaller than that of the positive drug group, namely, although the body temperature difference value of the compound 7 group of the invention has no significant difference compared with the model group, the antipyretic effect of the compound of the invention has longer duration and the trend of the long duration is more obvious than that of the positive drug group.
TABLE 20 statistical table of body temperature difference values of rats for evaluation of antipyretic pharmacodynamics of compound 7 of the present invention
Figure BDA0002976196020000651
Note: each group n is 12; # in order to compare with the blank control group, ### p<0.001; is compared with the model group<0.01,***p<0.001。
In conclusion, the compound of the invention has significant antipyretic effect, and the antipyretic effect has longer duration than that of the positive medicine.
Sixthly, evaluation of antiviral pharmacodynamic action of the compound
Experimental example 14: 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 Cell suspension per ml, seeded on 96-well cell culture plates (per cell culture plate)Well 100. mu.l of cell suspension, cell number 1X 10 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 observed the toxic effect of the compounds of the present invention on cells at the action concentrations of 20. mu.M, 10. mu.M, and 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 test of the compound of the invention is carried out under the series concentration gradient of 320 mu M, 160 mu M, 80 mu M, 40 mu M, 20 mu M, 10 mu M, 5 mu M, 0 or 320 mu M, 160 mu M, 80 mu M, 40 mu M, 20 mu M, 10 mu M, 5 mu M, 2.5 mu M, 1.25 mu M, 0.625 mu M, 0.31 mu M, 0.1625 mu M and 0, 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:
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%
(2) Evaluation of anti-SARS-CoV-2 Virus efficacy of Compounds of the invention
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 culture medium to obtain the cell with the 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 hrThe mixture containing the compound of the present invention and SARS-CoV-2 virus was discarded, and 100. mu.l of the maintenance medium was added to each well, and the mixture was placed in a cell incubator and cultured for another 48 hours.
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.
Cytotoxicity CC for Compounds 3, 6, 7, 8 of the invention 50 The results of the test are shown in Table 21.
TABLE 21 toxicity test results of the Compounds of the invention on Normal Vero E test cells
Figure BDA0002976196020000681
Figure BDA0002976196020000691
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
The compound has clear and significant inhibitory effect on SARS-CoV-2 virus under the action concentration adopted in the experiment (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, 2.5 mu M) and under the condition of only treating model cells for 1 hour by taking the influence of the compound on the RNA level of SARS-CoV-2 virus as an index, wherein the compounds 7 and 8 have clear and significant inhibitory effect on SARSThe inhibition rate of the RNA inhibition of the-CoV-2 virus can reach 62.99%, 93.13%, 94.42% and 68.08%, 89.10% and 91.74% respectively at the action concentration 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 22.
TABLE 22 Effect of Compounds of the invention on RNA levels of SARS-CoV-2 Virus infection in test cells and EC 50 Value of
Figure BDA0002976196020000692
Figure BDA0002976196020000701
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 1,3, 4, 6-10 of the invention on the virus RNA level after experimental cells are infected with SARS-CoV-2 virus are respectively 28.63%, 65.66%, 32.95%, 93.13%, 31.00%, 89.10%, 29.62% and 17.45%. Compounds 6, 8 and 3 EC for inhibition of SARS-CoV-2 viral RNA levels 50 The values were 3.037. mu.M, 3.767. mu.M and 7.859. mu.M, respectively. The SI values for the 3 compounds were 59.53, 63.02, 31.94, respectively. Thus, the compound of the invention has obvious activity of resisting SARS-CoV-2 virus.

Claims (10)

1. A berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound shown in a general formula I:
Figure FDA0002976196010000011
COO - is selected from 2 position or 3 position or 4 position or 5 position or 6 position substituted on pyridine ring;
r is independently selected from hydrogen, amino, substituted or unsubstituted hydroxyl, alkyl, halogen;
r is mono-or polysubstituted;
when R is monosubstituted, with COO - In coordination with the position(s) to form a disubstituted pyridine ring, R may be selected from the 3-or 4-or 5-or 6-or 2-position of the pyridine ring;
r is selected from di-substituted, tri-substituted or tetra-substituted when being polysubstituted, and is reacted with COO - Forming various substitution patterns given by a mathematical enumeration method in a coordinated manner;
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 alkyl is selected from methyl and ethyl; the halogen is selected from fluorine, chlorine and bromine; the alkylenedioxy is selected from methylenedioxy.
2. The berberine type alkaloid picolinic acid based quaternary ammonium salt compound according to claim 1, wherein the compound is selected from the group consisting of:
Figure FDA0002976196010000021
Figure FDA0002976196010000031
Figure FDA0002976196010000041
Figure FDA0002976196010000051
3. a process for the preparation of the quaternary ammonium salt of berberine type alkaloid pyridine carboxylic acid according to any of the claims 1-2, characterized in that the process comprises the following steps: reacting berberine type alkaloid quaternary ammonium salt compound with water solution of acetone and sodium hydroxide, reacting the obtained solid 8-acetonyldihydroberberine type compound with picolinic acid compound in mixed solvent of tetrahydrofuran and water under heating condition, and filtering the reaction mixed solution to obtain berberine type alkaloid picolinic acid quaternary ammonium salt compound.
4. A pharmaceutical composition comprising an effective amount of the quaternary ammonium salt of berberine type alkaloid pyridine carboxylic acid according to any one of claims 1-2 and a pharmaceutically acceptable carrier or excipient.
5. Use of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound according to any one of claims 1-2 or the pharmaceutical composition according to claim 4 for preparing an immunomodulator medicament with activities of promoting immune effector cell proliferation, inhibiting high NO secretion of pathological states, reducing high interleukin-6 level and increasing high autoantibody level of pathological states of organism.
6. Use of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound according to any one of claims 1-2 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for preventing, alleviating and/or treating a disease causing an increase of interleukin-6 secretion in a living organism.
7. Use of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound according to any one of claims 1-2 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for preventing, alleviating and/or treating diseases causing the increase of NO secreted by a living organism.
8. Use of the berberine type alkaloid, quaternary ammonium picolinate compound according to any one of claims 1-2 or the pharmaceutical composition according to claim 4, for the preparation of a medicament for the prevention, alleviation and/or treatment of a disease causing an increase in the formation of autoantibodies by a living organism.
9. Use of the berberine type alkaloid pyridine carboxylic acid quaternary ammonium salt compound according to any one of claims 1-2 or the pharmaceutical composition according to claim 4 for the preparation of a medicament for preventing, alleviating and/or treating cardiovascular and cerebrovascular diseases and blood system diseases, inflammatory diseases, febrile diseases, neoplastic diseases and autoimmune diseases caused by various reasons.
10. The use according to claim 9, wherein said autoimmune disease comprises rheumatoid arthritis; the cardiovascular and cerebrovascular diseases and blood system diseases comprise hypohemoglobinemia anemia and atherosclerosis: the tumor diseases comprise colorectal cancer and lung cancer.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117327063A (en) * 2023-12-01 2024-01-02 中国医学科学院医药生物技术研究所 Antibacterial medicament and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103919773A (en) * 2013-12-20 2014-07-16 中国药科大学 Application of 9-demethylberberine in preparation of hypolipidemic drug
CN103919775A (en) * 2013-12-20 2014-07-16 中国药科大学 Application of 9-demethylberberine in preparation of hpyerglycemic drug
CN103989677A (en) * 2013-12-20 2014-08-20 中国药科大学 Use of demethyleneberberine in preparation of blood sugar-reduction drug
CN105732609A (en) * 2016-01-30 2016-07-06 合肥华方医药科技有限公司 Preparation method and medical application of berberine hydrochloride conjugate

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103919773A (en) * 2013-12-20 2014-07-16 中国药科大学 Application of 9-demethylberberine in preparation of hypolipidemic drug
CN103919775A (en) * 2013-12-20 2014-07-16 中国药科大学 Application of 9-demethylberberine in preparation of hpyerglycemic drug
CN103989677A (en) * 2013-12-20 2014-08-20 中国药科大学 Use of demethyleneberberine in preparation of blood sugar-reduction drug
CN105732609A (en) * 2016-01-30 2016-07-06 合肥华方医药科技有限公司 Preparation method and medical application of berberine hydrochloride conjugate

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117327063A (en) * 2023-12-01 2024-01-02 中国医学科学院医药生物技术研究所 Antibacterial medicament and application thereof
CN117327063B (en) * 2023-12-01 2024-03-15 中国医学科学院医药生物技术研究所 Antibacterial medicament and application thereof

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