WO2022194136A1 - 二苯烷类化合物及其制备方法、药物组合物和用途 - Google Patents

二苯烷类化合物及其制备方法、药物组合物和用途 Download PDF

Info

Publication number
WO2022194136A1
WO2022194136A1 PCT/CN2022/080897 CN2022080897W WO2022194136A1 WO 2022194136 A1 WO2022194136 A1 WO 2022194136A1 CN 2022080897 W CN2022080897 W CN 2022080897W WO 2022194136 A1 WO2022194136 A1 WO 2022194136A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
arh
present
substituted
diseases
Prior art date
Application number
PCT/CN2022/080897
Other languages
English (en)
French (fr)
Inventor
秦海林
邓安珺
张雪倩
徐令文
闫征
李志宏
Original Assignee
中国医学科学院药物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202110274881.6A external-priority patent/CN115073447B/zh
Priority claimed from CN202110276109.8A external-priority patent/CN115068473A/zh
Application filed by 中国医学科学院药物研究所 filed Critical 中国医学科学院药物研究所
Publication of WO2022194136A1 publication Critical patent/WO2022194136A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
    • C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a class of berberine-type alkaloid quaternary ammonium salt compounds whose solubility properties are more suitable for pharmaceutical use, a preparation method thereof, a pharmaceutical composition containing such compounds, and such compounds and pharmaceutical combinations thereof The use of substances to prepare medicines.
  • the natural berberine quaternary ammonium salt type compound is a kind of 5,6-dihydro-7 isoquinolino[3,2-a]isoquinoline with different substituents on the benzene ring whose counter anion is chloride anion.
  • -Onium ion quaternary ammonium salt type compound is a kind of "dream molecule” or “dream compound” that is very recognized and concerned in the field of medicinal chemistry.
  • these natural berberine-type quaternary ammonium salt compounds with chloride anion without any biological activity as the counter anion also have a significant defect, that is, the physical and chemical properties of these compounds are not suitable for drug development.
  • Some of these compounds are poorly soluble and unsuitable for administration by most routes of administration, including the vascular system, and enteral administration suffers from significant poor bioavailability.
  • the bioavailability was only 1.87%, 0.82% and 0.52% after oral administration to experimental mice at doses of 30 mg/kg, 75 mg/kg and 150 mg/kg, respectively.
  • some compounds are too soluble in solvents, and it is desirable to reduce the absorption in the small intestine while increasing the concentration of the drug in the colon (that is, by increasing the selective distribution of the drug to the intestinal tissue).
  • Interleukin-6 is a biochemical component of interleukin and is a hormone-like glycoprotein.
  • IL-6 was first discovered in 1980 as a B lymphocyte differentiation factor and was named ⁇ 2 interferon. Since then, it has gone through the research process of being discovered and named as various cytokines, and it was finally named IL-6 after the genes of these cytokines were cloned.
  • hIL-6 The gene is located on chromosome 7.
  • the three-dimensional structure of IL-6 was determined by X-ray crystallography analysis. It is composed of four helical bundles A-D with a topological structure, and there is one outside the four helical bundles. Small helix-E helix, in which the A helix and the B helix are in the same direction, while the C helix and the D helix are in the opposite direction to the former two.
  • IL-6 is a Th-2 type potent pleiotropic immunoregulatory cytokine, a variety of cells such as macrophages, dendritic cells, lymphocytes, mast cells, fibroblasts, monocytes, vascular endothelial cells , glomerular mesangial cells and some tumor cells, etc. and various mechanisms can produce IL-6.
  • IL-6 has several normal physiological functions, such as stimulating acute-phase protein production and inducing acute-phase responses to combat or eliminate pathogens. However, its abnormal over-secretion through various mechanisms is an important phenotype of the body's acute inflammatory response and the pathological activity of autoimmune diseases, and it is the most important cell to determine the disease severity in patients with specific and systemic inflammatory response syndromes.
  • IL-6 pathological excess secretion of IL-6 can reflect the severity of the body's inflammatory response, autoimmune response and various associated diseases. Inflammation and autoimmune diseases play key roles in a variety of other diseases, including blood cell production, tumorigenesis, and viral infections. First, inflammation plays a key role in adverse cardiovascular events, and the phenotype of IL-6 levels is positively correlated with the Gensini score of coronary artery disease, is closely related to disease severity, and is associated with atherosclerosis, coronary heart disease, and anemia Cardiovascular and blood system diseases such as IL-6 are closely related; IL-6 also participates in the pathological process of gestational hypertension through a variety of ways.
  • IL-6 is an important regulator of the progression of most tumor diseases. Serum or tissue IL-6 levels are often elevated in some tumor disease patients, and high IL-6 expression predicts poor clinical outcomes.
  • the phenotype of high IL-6 expression is closely related to respiratory diseases. For example, the level of IL-6 in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis is significantly increased; in patients with allergic asthma, the alveolar macrophages The content of IL-6 in the cell culture supernatant was also significantly increased; at the same time, the phenotype of high IL-6 expression was also positively correlated with dyspnea in patients with pneumonia. From a pathophysiological point of view, this represented the intensity of the pulmonary inflammatory response.
  • IL-6 in the lungs and serum was found to act as an anti-viral agent.
  • Coronavirus is a common pathogen that causes respiratory diseases, which can lead to a significant increase in cytokines such as IL-6, resulting in a cytokine storm and recruiting immune response cells in the lungs.
  • 2019 novel coronavirus disease 2019 novel coronavirus (2019 novel coronavirus or SARS-CoV2)
  • 2019 novel coronavirus 2019 novel coronavirus
  • SARS-CoV2 2019 novel coronavirus
  • IL-6 is abundantly produced by T lymphocytes and monocytes following infection by the 2019-nCoV virus, and inflammation progresses rapidly and exerts an immune-destructive effect (autoimmunity), resulting in severe pulmonary dysfunction
  • severe acute respiratory syndrome outbreak in early 2003 SARS (Severe acute respiratory syndrome, SARS) SARS-associated coronavirus (Severe acute respiratory syndrome-associated coronavirus, SARS-CoV) infection can lead to a phenotype of significantly elevated IL-6, through the production of cytokine storm, immune recruitment in the lungs Responder cells, etc., cause acute respiratory distress syndrome (ARDS), and even pulmonary fibrosis in the later stage.
  • ARDS acute respiratory distress syndrome
  • the clinical classification of patients with SARS-CoV2 virus infection was divided into mild, common, severe and critical.
  • the serum inflammatory cytokines were detected, and it was found that there were statistically significant differences in the expression level of IL-6 among the various types.
  • the level of IL-6 in patients with severe SARS-CoV2 virus pneumonia was significantly higher than that in mild patients, indicating that high IL-6 expression is an important phenotype and link for SARS-CoV2 and its variant strains to induce cytokine storm in the body.
  • coronavirus-induced IL-6 high expression phenotype plays an important role in the pathogenesis of coronavirus-infected pneumonia, or is closely related to disease severity in the development of SARS-CoV2 viral pneumonia Important phenotypes, especially in inflammation and fever.
  • the phenotype of high IL-6 expression is also associated with kidney diseases, such as patients with idiopathic glomerulonephritis (IgAN), which have significantly higher levels of IL-6 in urine and serum.
  • IgAN idiopathic glomerulonephritis
  • the receptor for IL-6 is IL-6R on the cell surface.
  • IL-6R ⁇ and gp130 The binding of IL-6 to the receptor triggers the dimerization of IL-6R ⁇ and gp130, resulting in the activation of downstream signaling pathways, including JAK/STAT, Ras/MAPK, and PI3K-PKB/AKT signaling pathways.
  • Ras(Rho)/MAPK/GATA3/IL-6 signaling mechanism is one of the important signaling mechanisms for abnormal over-secretion of IL-6. Therefore, the high expression phenotype of IL-6 can be used as a target for the treatment of inflammatory diseases, febrile diseases, cardiovascular and cerebrovascular and blood system diseases, tumor diseases, primary glomerulonephritis, viral infections and their complications. , for drug development, testing and application.
  • Nitric oxide is a ubiquitous signaling molecule in the body, which can be produced by three nitric oxide synthases (NOS), including endothelial NOS (eNOS), mainly distributed in vascular endothelial cells, mainly distributed in vascular endothelial cells.
  • NOS nitric oxide synthases
  • eNOS endothelial NOS
  • iNOS inducible NOS
  • cNOS constitutive NOS
  • NO synthesized by eNOS is involved in the regulation of blood pressure and blood flow
  • NO generated by nNOS is involved in brain development as a neurotransmitter. learning and memory process.
  • iNOS is usually silent, but can be induced by inflammatory cytokines, lipopolysaccharide, etc., resulting in a large or even excessive amount of NO, and it persists for a relatively long time.
  • NO plays an important physiological role and produces a variety of pathological effects in the body through different mechanisms; its physiological regulation has important effects on supporting organisms. Many physiological processes in the body are crucial, such as immune regulation; but in some pathological conditions, such as various inflammations, cancers, etc., both NO and iNOS are overexpressed. For example, in primary lung cancer, NO and iNOS are significantly overexpressed. expression, while excessive upregulation of iNOS is associated with reduced survival in breast cancer patients, et al.
  • Autoimmune disease is one of the most common intractable diseases in clinical practice. It is the attack of the human body's immune system on its own tissues without distinguishing between "self” and "non-self” in the presence of patients. clear. Autoimmune diseases can be divided into two categories: organ-specific autoimmune diseases and systemic autoimmune diseases. Usually, a large number of inflammatory cells accumulate in the lesion site, and the content of NO in the body fluid of the patient's lesion site is also significantly excessively high. Due to the content of NO in their blood, and the content of NO in the blood of patients with autoimmune diseases is itself higher than the content of NO in the blood of normal people, causing swelling and pain.
  • autoimmune diseases In addition to the focal site, autoimmune diseases, especially systemic autoimmune diseases, can affect any system in the body, complicating multiple diseases, often also involving the formation of autoantibodies.
  • autoimmune diseases can affect any system in the body and can be complicated by a variety of diseases. Inflammation is central to the pathology of autoimmune diseases, but it also plays an important role in the development of cardiovascular disease.
  • the physiological function of lymphocytes and their influence on the blood system in the pathological state of autoimmune diseases, the formation and characteristics of cardiovascular and cerebrovascular and blood system diseases in the pathological state of autoimmune diseases can all be used in rheumatoid arthritis. described as an example.
  • Rheumatoid arthritis is one of the most common clinical autoimmune diseases.
  • the symptoms are mainly inflammatory arthritic lesions, and its onset is chronic and systemic.
  • a large number of inflammatory cells including lymphocytes (T cells and B cells), monocytes/macrophages, etc., accumulate in the inflammatory synovium and synovial fluid, resulting in swelling, pain and Stiffness, like other autoimmune diseases, often involves the formation of autoantibodies, such as rheumatoid factor (RF) or anti-citrullinated protein antibodies.
  • RF rheumatoid factor
  • rheumatoid arthritis As a chronic systemic inflammatory disease, persistent inflammation results in articular cartilage and bone destruction, deformity, and loss of function in all affected joints, especially the small joints of the hands and feet. Moreover, as a systemic autoimmune inflammatory disease, rheumatoid arthritis can affect any system of the body in addition to joints, and is complicated by lung diseases, malignant tumors and depression. As mentioned above, inflammation is a central part of the immunopathology of rheumatoid arthritis, which also plays an important role in the development of cardiovascular disease.
  • Lymphocytes are important cells in the body's immune response function under physiological conditions, and have the functions of resisting infection, monitoring and removing foreign substances (including cancerous tissue and necrotic tissue), and protecting the body's stable state; but under the stimulation of certain pathological factors , These physiological functions are out of control, leading to hyperfunction of lymphocytes, producing negative regulators of hematopoiesis, inhibiting and damaging the formation of bone marrow clones in patients
  • Normal tissues form various autoimmune inflammatory diseases and blood system diseases, such as anemia.
  • Anemia is mainly manifested by the body's inability to make enough hemoglobin to transport oxygen to the various tissues of the body.
  • Anemia is also one of the most common extra-articular symptoms of rheumatoid arthritis, and the most common type of anemia is anemia of chronic disease, the pathogenesis of which is not fully understood; therefore, patients with rheumatoid arthritis are at risk for most types of cardiovascular disease For example, inflammation is strongly associated with developing atherosclerosis, which leads to ischemic heart disease and leads to fibrotic myocardial damage that causes diastolic dysfunction.
  • the content of NO in the synovial fluid of patients with rheumatoid arthritis is significantly higher than that in the blood of patients, although the content of NO in the blood of patients with rheumatoid arthritis itself is higher than that in the blood of normal people.
  • cardiovascular disease mortality in patients with rheumatoid arthritis is approximately 50% higher than in the general population, and cardiovascular disease appears to occur at a younger age in patients with rheumatoid arthritis than in the general population .
  • Rheumatoid arthritis also has the characteristics of repeated attacks and high disability rate.
  • the disability rate of patients in the course of 1 to 5 years, 5 to 10 years, 10 to 15 years and ⁇ 15 years is 18.6%, 43.5%, 48.1% and 61.3%, that is, with the prolongation of the disease course, the incidence of disability and functional limitation increases, which seriously affects the patients' quality of life and work ability, and brings a heavy burden to the family and society.
  • Rheumatoid arthritis can occur at any age, and its incidence and prevalence vary with different regions and times. Epidemiological surveys show that its global incidence is 0.5% to 1%, and the incidence in mainland China It was 0.42%, and the incidence was significantly higher in people over 65 years old. In 2005, the incidence of rheumatoid arthritis in the United States was 0.409%, the prevalence was about 0.72%, and the average age of onset was about 55.6 years old. The incidence of new cases in female patients was 69% more than that in males. There are also surveys that show that the ratio of male to female is about 1:4.
  • Rheumatoid arthritis cannot be cured so far, and its clinical treatment principles are early treatment, standardized treatment, regular monitoring and follow-up.
  • the clinical drugs for the treatment of rheumatoid arthritis are mainly classified into non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), glucocorticoids and biological agents.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs disease-modifying anti-rheumatic drugs
  • glucocorticoids glucocorticoids
  • Methotrexate is an anti-rheumatic drug that improves the condition and is an anchor drug for the treatment of rheumatoid arthritis
  • the average usage rate of methotrexate in European and American countries reaches 83%, which is much higher than that of other drugs.
  • the usage rate is lower than that of European and American countries, at 55.9%.
  • methotrexate alone or in combination with other traditional synthetic DMARDs, which can achieve disease remission or low disease activity treatment goals, but cannot achieve radical cure.
  • the adverse reactions of methotrexate are positively related to the dose, and only when the methotrexate is used in small doses ( ⁇ 10 mg/week) can the adverse reactions be mild and the long-term tolerance be better.
  • the efficacy and safety of leflunomide and sulfasalazine alone are comparable to those of methotrexate, but the side effects of sulfasalazine increasing IL-6 should be noted.
  • the international usage rate of leflunomide is among the highest in methotrexate, After sulfasalazine and hydroxychloroquine, it is 21%; but the usage rate in China is second only to methotrexate at 45.9%, and the usage rate in some areas even exceeds that of methotrexate.
  • the use rate of sulfasalazine in Chinese rheumatoid arthritis patients is only 4.4%, which is much lower than 43% in other countries, and most of them are combined with other traditional synthetic DMARDs.
  • Hydroxychloroquine is used by 30.4% in China and slightly higher internationally. The use of hydroxychloroquine in China is dominated by combination drugs, accounting for 95%.
  • Combination medication is required for those who still fail to meet compliance with standard single-drug therapy such as methotrexate, leflunomide or sulfasalazine.
  • standard single-drug therapy such as methotrexate, leflunomide or sulfasalazine.
  • glucocorticoids are needed, but the purpose is to control symptoms quickly, and glucocorticoids cannot be used alone or in high doses for a long time. Otherwise, glucocorticoids will produce Serious side effects.
  • cancers malignant tumors
  • cancers are major diseases that seriously endanger human health.
  • the prevention, mitigation and treatment of various human cancers is still a very difficult scientific research work that must be faced in the field of medical scientific research; Efficient and safe drug treatment of cancer is one of the ideal means. Therefore, it is of great significance in the field of medical technology to find specific chemical drugs that have efficient therapeutic effects on various malignant tumors and have low toxicity or even no toxicity.
  • scientists have discovered some anti-tumor drugs with certain curative effects. It is these drugs that prolong the average survival period of children with acute leukemia from 2 to 3 months in the past to more than 5 years, and make the life of many advanced tumor patients significantly extend.
  • the invention unexpectedly finds that the physical and chemical properties of the natural berberine alkaloid quaternary ammonium salt type compound are improved, and on this basis, biological evaluation is carried out.
  • the present invention is based on the medical field and clinical treatment of immunomodulator drugs, anti-autoimmune disease drugs, anti-viral infection and its complication drugs, anti-inflammatory and antipyretic drugs, anti-cardiovascular and cerebrovascular and blood system disease drugs and anti-tumor disease drugs. It also includes the concept of synergistic antiviral, antipyretic and anti-inflammatory multiple drug effects in the treatment of viral infections and their complications, combined with IL-6 levels, NO levels, iNOS levels and IgG antibody levels in clinical diseases.
  • the compounds of the present invention were synthesized, evaluated on the pharmacological model to inhibit the levels of IL-6, NO, iNOS, IgG antibody levels, and evaluated the efficacy of inhibiting SARS-CoV2 virus RNA replication in in vitro experiments, Evaluation of the inhibitory effect on tumor cell growth, evaluation of the effect on the hemoglobin level of model animals in animal models, evaluation of antipyretic effect on fever pathological model animals, evaluation of the improvement effect on the severity of arthritis symptoms in rheumatoid arthritis model animals As well as the animal experimental evaluation of anti-tumor effect, it also includes the comparison of the pharmacological effects and physicochemical properties of the compound of the present invention and the berberine chloride type alkaloid quaternary ammonium salt compound substrate.
  • the physicochemical properties and pharmacological effects of the compounds of the present invention are obviously superior to those of the berberine chloride type alkaloid quaternary ammonium salt compound substrates.
  • a class of drugs is used in the preparation of prevention, mitigation and/or treatment of anemia, viral infections and their complications, inflammatory diseases, febrile diseases, and autoimmune diseases.
  • Compounds with significant application value in medicines for diseases such as , tumor diseases, etc.
  • the technical problem solved by the present invention is to provide a kind of picolinate or substituted picolinate or 2-methyl-N 1 -oxypyrazine-5-carboxylate as a balance by means of chemical synthesis and drug screening.
  • the solubility in water and alcohol-water mixed solvent is significantly better than that of berberine chloride type alkaloid quaternary ammonium salt substrate from the aspect of specific drug development, and has significant prevention, mitigation and/or treatment by balancing the immune function of the body
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides a berberine-type alkaloid picolinic acid-type quaternary ammonium salt compound, which is represented by the general formula A and is represented by the general formula I, as the compound of the present invention, and which is significantly improved in solubility and is soluble, and the same
  • the soluble berberine-type alkaloid 2-methyl-N 1 -oxypyrazine-5-carboxylic acid quaternary ammonium salt compound represented by the general formula I' as the compound of the present invention contained in the general formula A is included.
  • the second aspect of the present invention provides berberine-type alkaloid picolinic acid quaternary ammonium salt compounds and berberine-type alkaloid 2-methyl-N 1 -oxide pyrazine as shown in general formula I and general formula I' -The preparation method of 5-formic acid quaternary ammonium salt compound.
  • the third aspect of the present invention provides berberine-type alkaloid picolinic acid quaternary ammonium salt compounds and berberine-type alkaloid 2-methyl-N 1 -oxide pyrazine as shown in general formula I and general formula I' -Product composition of 5-formic acid quaternary ammonium salt compound, the product is selected from medicines.
  • a fourth aspect of the present invention provides applications of the compounds and pharmaceutical compositions of the present invention in the preparation of pharmaceutical products Use, including in the preparation of immunomodulator drugs with the activities of promoting the proliferation of immune effector cells, inhibiting/reducing the excessive production of pathological NO in biological organisms, and excessively increasing the levels of IL-6 and autoantibodies, and based on independent applications, also Including the application in the preparation of medicines for preventing, alleviating and/or treating diseases that cause the pathological secretion of IL-6 in biological organisms to increase excessively, and in the preparation of drugs for preventing, alleviating and/or treating the excessive increase in pathological secretion of NO in biological organisms.
  • the fifth aspect of the present invention provides the compounds and pharmaceutical compositions of the present invention in the preparation of preventing, alleviating and/or treating cardiovascular and cerebrovascular and blood system diseases, autoimmune diseases, viral infectious diseases and their complications, inflammation caused by various causes The application of medicines for diseases, fever and tumor diseases.
  • autoimmune diseases include rheumatoid arthritis
  • cardiovascular and cerebrovascular and blood system diseases include hypohemoglobinemia anemia
  • viral infections and their complications include coronavirus infections and their complications
  • coronavirus infections coronavirus infections and their complications
  • coronavirus infections include breast cancer, colorectal cancer and lung cancer.
  • the first aspect of the present invention provides a soluble berberine-type alkaloid quaternary ammonium salt compound represented by general formula A:
  • COO - is selected from the 2-position or 3-position or 4-position or 5-position or 6-position substituted on the pyridine ring;
  • R is independently selected from hydrogen, amino, substituted or unsubstituted hydroxy, alkyl, halogen;
  • R is monosubstituted or polysubstituted
  • R When R is mono-substituted, it is coordinated with the position of COO- to form a di-substituted pyridine ring, and R can be selected from the 3-position or 4-position or 5-position or 6-position or 2-position of the pyridine ring;
  • R When R is multi-substituted, it is selected from di-substituted or tri-substituted or tetra-substituted, and forms various substitution patterns given by mathematical enumeration in coordination with COO-;
  • R is selected from the methyl group substituted at the 2-position of the pyrazine ring, and COO- is selected from the substituted at the 5 - position of the pyrazine ring;
  • R 2 and R 3 are each independently selected from H, substituted or unsubstituted hydroxy, or R 2 and R 3 are connected to form a hydrocarbylene dioxy;
  • R 9 , R 10 , R 11 are each independently selected from H, substituted or unsubstituted hydroxy, or R 9 and R 10 are linked to form a hydrocarbylene dioxy and R 11 is independently selected from H, substituted or unsubstituted hydroxy , or R 10 and R 11 are connected to form a hydrocarbylene dioxy group and R 9 is independently selected from H, substituted or unsubstituted hydroxy;
  • the substituents in the substituted or unsubstituted hydroxyl are selected from methyl and ethyl; the alkyl is selected from methyl and ethyl; the halogen is selected from fluorine, chlorine and bromine; Hydrocarbyldioxy is selected from methylenedioxy.
  • the compound is a soluble berberine-type alkaloid picolinic acid quaternary ammonium salt compound shown in the general formula I, and the general chemical structure is shown in the following formula I:
  • COO- is selected from the 2 - position or 3-position or 4-position or 5-position or 6-position substituted on the pyridine ring;
  • R is independently selected from hydrogen (H), amino, hydroxyl, methoxy, ethoxy , methyl, ethyl, fluorine (F), chlorine (Cl), bromine (Br);
  • R is monosubstituted or polysubstituted
  • R When R is monosubstituted, it is coordinated with the position of COO- to form R:COO - disubstituted, and R can be selected from the 3-position or the 4-position or the 5-position or the 6-position or the 2 - position of the pyridine ring;
  • R When R is multi-substituted, it is selected from di-, tri- or tetra-substituted, and is coordinated with COO- to form various substitution patterns given by mathematical enumeration;
  • R 2 and R 3 are each independently selected from H, substituted or unsubstituted OH, or R 2 and R 3 are connected to form a hydrocarbylene dioxy group; further, the substituted or unsubstituted hydroxyl groups described in R 2 and R 3 The substituents in are selected from methyl group and ethyl group; the hydrocarbylene dioxy groups described in R 2 and R 3 are selected from methylene dioxy groups.
  • R 9 , R 10 , R 11 are each independently selected from H, substituted or unsubstituted OH, or R 9 is linked to R 10 to form a hydrocarbylene dioxy and R 11 is independently selected from H, substituted or unsubstituted OH , or R 10 is linked to R 11 as an alkylene dioxy and R 9 is independently selected from H, substituted or unsubstituted OH.
  • the substituents in the substituted or unsubstituted OH described in R 9 , R 10 , and R 11 are selected from methyl and ethyl; the hydrocarbylene dioxy groups described in R 9 , R 10 , and R 11 is selected from methylenedioxy.
  • the compound is a berberine-type alkaloid 2-methyl-N 1 -oxypyrazine-5-carboxylic acid quaternary ammonium salt compound represented by the general formula I', and the general chemical structure is shown in the following formula I' Show:
  • R 2 and R 3 are each independently selected from H, substituted or unsubstituted OH, or R 2 and R 3 are connected to form a hydrocarbylene dioxy;
  • R 9 , R 10 , R 11 are each independently selected from H, substituted or unsubstituted OH, or R 9 is linked to R 10 to form a hydrocarbylene dioxy and R 11 is independently selected from H, substituted or unsubstituted OH , or R 10 and R 11 are linked to become hydrocarbylene dioxy and R 9 is independently selected from H, substituted or unsubstituted OH;
  • the substituents in the substituted or unsubstituted OH are selected from methyl and ethyl; the hydrocarbylenedioxy is selected from methylenedioxy.
  • the most preferred soluble berberine-type alkaloid quaternary ammonium salt compound of the present invention is selected from compound 1-47 and compound 1'-5' in the following compound group:
  • the second aspect of the present invention provides a preparation method of the soluble berberine-type alkaloid quaternary ammonium salt compound of the present invention.
  • the soluble berberine-type alkaloid quaternary ammonium salt compound can be synthesized by the following synthetic route general formula (see the examples for specific synthesis conditions):
  • Synthesis steps (a) The berberine-type alkaloid quaternary ammonium salt compound is reacted with acetone and an aqueous sodium hydroxide solution to obtain a solid 8-acetone dihydroberberine-type compound. (b) the obtained solid 8-acetone dihydroberberine-type compound is then heated with a picolinic acid compound or 2-methyl-N 1 -oxypyrazine-5-carboxylic acid in a mixed solvent of tetrahydrofuran and water The following reaction is carried out, and the reaction mixture is filtered to obtain the soluble berberine-type alkaloid quaternary ammonium salt compound of the present invention.
  • the third aspect of the present invention provides a pharmaceutical composition using the soluble berberine-type alkaloid quaternary ammonium salt compound described in 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. Any dosage form suitable for human or animal use can be prepared by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants.
  • the content of the compound of the present invention in its pharmaceutical composition is usually 0.1-99.9% (W/W).
  • the compound of the present invention or the pharmaceutical composition containing the compound of the present invention can be administered in unit dosage form, and the route of administration can be mainly gastrointestinal administration, such as oral administration, enteral administration, and the like.
  • parenteral administration forms are also acceptable, such as intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, as well as nasal, oral mucosa, ocular, Lung and respiratory tract, vaginal administration, application to skin, etc.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O/W type, W/O type and double emulsion), suspensions, injections (including water injection, powder injection and infusion), eye drops solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, drop pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Cream, gel, paste, etc.
  • the compounds of the present invention can be prepared into common preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
  • diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • wetting agent can be water, ethanol, Isopropyl alcohol, etc.
  • the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin pulp, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl Methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.
  • disintegrants can be dry starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate
  • the tablets can also be further prepared as coated tablets, such as sugar-coated, film-coated, enteric-coated, or bilayer and multi-layer tablets.
  • the compound of the present invention can be mixed with diluent and glidant, and the mixture can be directly placed in a hard capsule or soft capsule; the compound of the present invention can also be mixed with diluent, binder, The disintegrant is made into granules or pellets and placed in hard or soft capsules.
  • the various diluents, binders, wetting agents, disintegrants, and glidants used to prepare tablets of the compounds of the present invention can also be used to prepare capsules of the compounds of the present invention.
  • solubilizers can be poloxamer, lecithin, hydroxypropyl- ⁇ -cyclodextrin, etc.
  • pH adjuster can be phosphate, acetate, hydrochloride, sodium hydroxide, etc.
  • osmotic pressure adjustment The agent may be sodium chloride, mannitol, dextrose, phosphate, acetate, and the like.
  • mannitol, glucose, etc. can also be added as proppant.
  • colorants preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparations if there are special needs in terms of dosage forms.
  • the compounds (drugs) or pharmaceutical compositions of the present invention can be administered and used by any known administration methods and application modes.
  • the administration (application) or the dosage (use) of the compound or pharmaceutical composition of the present invention is based on the cardiovascular and cerebrovascular and blood system diseases, autoimmune diseases, and viral infectious diseases caused by various causes to be prevented, alleviated and/or treated.
  • the severity of its complications, inflammatory diseases, febrile diseases, tumor diseases, individual conditions of patients or animals, administration (application) routes and dosage forms can vary widely.
  • a suitable daily dosage range of the compounds of the present invention is 0.001-500 mg/kg (dose/body weight), preferably 0.1-150 mg/kg (dose/body weight), more preferably 1-100 mg/kg (dose/body weight) ), most preferably 1-50 mg/kg (dose/body weight).
  • the above-mentioned doses may be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the progression of treatment and the administration (use) regimen including the use of other therapeutic (application) means.
  • compositions of the present invention may be administered alone, or in combination with other therapeutic drugs or against Concomitant use of symptomatic drugs.
  • the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.
  • the fourth aspect of the present invention provides the application of the compounds and pharmaceutical compositions of the present invention in the preparation of pharmaceutical products, including inhibiting/reducing the excessive production of pathological NO in the body, over-expression of iNOS, and excessive levels of IL-6 and autoantibodies.
  • the fourth aspect of the present invention is proposed based on the results of specific pharmacological experiments.
  • the compounds of the present invention have a significant immunomodulatory effect.
  • the compound of the present invention has a significant inhibitory effect on the pathological overproduction of NO in a cellular inflammation model.
  • High-level expression shows significant inhibitory effect, shows significant growth inhibitory effect on tumor cells, has the pharmacological effect of significantly reducing IL-6 levels in a pharmacological model of pathologically high IL-6 levels, and shows a significant reduction in pathologically high IgG levels
  • Antibody levels Pharmacological effects of IgG antibody levels in serum of model animals.
  • the fifth aspect of the present invention provides the compounds and pharmaceutical compositions of the present invention in the preparation of preventing, alleviating and/or treating cardiovascular and cerebrovascular and blood system diseases, autoimmune diseases, viral infectious diseases and their complications, inflammation caused by various causes The application of medicines for diseases, fever and tumor diseases.
  • autoimmune diseases include rheumatoid arthritis
  • cardiovascular and cerebrovascular and blood system diseases include hypohemoglobinemia anemia
  • viral infections and their complications include coronavirus infections and their complications
  • coronavirus infections coronavirus infections and their complications
  • coronavirus infections Its complications include the 2019 novel coronavirus SARS-CoV2 infection and its complications
  • tumor diseases include breast cancer, colorectal cancer, and lung cancer.
  • the fifth aspect of the present invention is also proposed based on the results of specific pharmacological experiments.
  • the compounds of the present invention can dose-dependently inhibit the replication of 2019 novel coronavirus SARS-CoV2 RNA, dose-dependently and selectively inhibit human lung cancer cell A549 cells and human colon cancer cells
  • the growth of HCT-116 cells significantly reduces the body temperature of pathological fever model animals, significantly increases the content of hemoglobin in peripheral blood of model animals, significantly reduces the severity of rheumatoid arthritis symptoms in model animals, and has significant effects on tumor animal models. therapeutic effect. .
  • preferred autoimmune diseases include rheumatoid arthritis, cardiovascular and cerebrovascular and blood system diseases include hypohemoglobinemia anemia, and tumor diseases include breast cancer and colorectal cancer and lung cancer, viral infections including coronavirus and its complications, especially the 2019 new SARS-CoV2 and its variants and its complications.
  • the compound of the present invention has a unique pharmacological mechanism of balancing the immune function of the body. In pharmacological experiments, it has a significant inhibitory effect on the pathological overproduction of NO and the pathological high-level expression of iNOS in a cellular inflammation model. All of the above have the pharmacological effect of significantly reducing the expression of IL-6 in the pharmacological model of pathologically high IL-6 level, and also showed the pharmacological effect of significantly reducing the level of IgG antibody in the serum of the model animal with high pathologically high IgG antibody level.
  • the compound of the present invention is a compound formed by the combination of two structural units in the form of ionic bonds.
  • the data show that compared with the natural berberine-type alkaloid quaternary ammonium salt compound with the inactive chloride anion as the counter anion, the physicochemical properties are obtained. Significant improvement, with unique activity characteristics.
  • the compounds of the present invention as IL-6 signaling mechanism inhibitors and viral RNA synthesis-related process inhibitors acting on the Rho(Ras)/MAPK/GATA3/IL-6 signaling pathway, can also inhibit the 2019 novel coronavirus SARS in a dose-dependent manner - CoV2 replicates, dose-dependently and selectively inhibits the growth of tumor cells such as human lung cancer cell A549 and human colon cancer cell HCT-116, significantly reduces the body temperature of fever model animals, and significantly increases the hemoglobin level in peripheral blood of model animals.
  • the compounds of the present invention can be used for the preparation of immunomodulator drug products, the preparation of drug products for preventing, alleviating and/or treating diseases causing excessive increase in the pathological secretion of IL-6 by biological organisms, and the preparation of preventing, alleviating and/or treating biological
  • cardiovascular and cerebrovascular and blood system diseases include hypohemoglobinemia anemia caused by various causes, autoimmune diseases include rheumatoid arthritis, tumor diseases include breast cancer, colorectal cancer and lung cancer, and coronavirus infection. Its complications include the 2019 novel coronavirus SARS-CoV2 and its variants and its complications.
  • the compounds of the present invention showed significant safety in toxicological experiments.
  • the pharmacological effects and physicochemical properties of the compounds of the present invention are significantly better than those of berberine chloride type alkaloid quaternary ammonium salt compound substrates, including berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, Coptis quaternary ammonium salt, quaternary ammonium chloride isoberberine, quaternary ammonium chloride isoberberine.
  • the compound of the present invention Compared with the natural berberine chloride type alkaloid quaternary ammonium salt substrate, the compound of the present invention has more characteristics in solubility in water and alcohol-water mixed solvent, and is more suitable for drug development.
  • the above-mentioned properties of the compounds of the present invention have significant pharmaceutical preparation value, and can be prepared into various pharmaceutical dosage forms including ordinary oral dosage forms, gastric administration forms, and injection dosage forms for medicinal purposes.
  • the specific structure of the compound of the present invention is that picolinic acid or substituted picolinic acid group or 2-methyl-N 1 -oxypyrazine-5-carboxylate is the acid group balance anion unit, and 5,6-dihydrodibenzo[ a,g]Quilazine-7-cationic quaternary ammonium cations are base-balanced cationic units, and the two form soluble berberine-type alkaloid quaternary ammonium salt compounds.
  • the synthesis and biological activity evaluation of functional organic compounds is a scientific research practice.
  • the practice process has always contained new discoveries in various senses, constantly injecting new connotations into scientific progress.
  • the present invention obtains the compound of the present invention in the process of in-depth medicinal chemistry research on berberine compounds, and confirms its structure, synthesis method, physicochemical properties and biological significance.
  • the synthetic route of the compound of the present invention is simple, efficient and friendly.
  • the stability test results of these compounds by NMR test method show that the compounds of the present invention are not only stable in physical and chemical properties in solid state, but also extremely stable in structure even when placed in solution.
  • the compounds of the present invention exist in the form of ion pairs or ion clusters, and have a specific arrangement, rather than a mixture.
  • the solubility test experiment proves that the compound of the present invention has significantly improved quaternary ammonium salts in the solvent compared with the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate with inactive chloride anion as the counter anion.
  • Solubility especially by using specific counter anions in the compounds of the present invention, to obtain compounds that are more suitable for specific medicinal purposes and can be selectively used, that is, according to specific medicinal purposes, if it is necessary to improve bioavailability, it can be applied
  • the compound of the present invention with improved solubility can be used if it is necessary to reduce the absorption of the drug in the intestinal tract and improve the selective distribution of the drug in the intestinal tissue.
  • the main feature of the water solubility of the compounds of the present invention is that the water solubility is significantly improved;
  • the compound of the present invention can be dissolved in an amount greater than or significantly greater than that of the compound per milliliter of water.
  • the corresponding berberine chloride type alkaloid quaternary ammonium salt substrate for example, the amount of the compounds 1-10 of the present invention that can be dissolved in each milliliter of water is 11.5mg, >300mg, 7.5mg, 45mg, 1.1mg, 15mg, respectively , 50mg, 1.5mg, 30mg and 1.38mg; while as berberine alkaloid quaternary ammonium salt substrates, berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, coptisine chloride quaternary ammonium salt
  • the amount of quaternary ammonium chloride, isoberberine quaternary ammonium chloride and isoberberine quaternary ammonium chloride that can be dissolved in each milliliter of water in parallel determinations are 2mg, 21mg, ⁇ 1mg, ⁇ 1mg, ⁇ 1mg, respectively. However, for a
  • the solubility of the compound of the present invention in the mixed solvent of ethanol and water is also significantly improved; measured at an ambient temperature of 25°C ⁇ 2°C Solubility, most of the compounds of the present invention that can be dissolved in a mixed solvent of 95% ethanol and water per milliliter are significantly improved.
  • the amounts of compounds of the present invention that can be dissolved in 95% ethanol per milliliter are 60mg, 46mg, 7.7mg, 109mg, 26mg, 38mg, 45mg, 5mg, 22mg and 137mg; while berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, coptisine chloride quaternary ammonium salt, chloride
  • the amounts that can be dissolved in parallel assays of isoplastine quaternary ammonium salt and isoberberine chloride quaternary ammonium salt per milliliter of water are ⁇ 3 mg, 15 mg, ⁇ 1 mg, ⁇ 1 mg, ⁇ 1 mg, respectively.
  • these compounds with reduced solubility can be mainly distributed in intestinal tissue, so they are more suitable for the development of drugs for the treatment of intestinal diseases, such as compound 24 and compound 40.
  • Macrophage is an important immune effector cell located in the body tissue, and participates in non-specific defense (innate immunity) and specific defense (adaptive or cellular immunity) in the body, and plays a role in defense, surveillance, regulation and antigenic
  • innate immunity non-specific defense
  • specific defense adaptive or cellular immunity
  • the extremely important immune functions such as presentation have a very important relationship with the balance of the body's immune system and play an important role in the host's immune response.
  • the main function of macrophages is to phagocytose (ie phagocytose and digest) cellular debris and pathogens in the form of fixed or free cells, and to activate lymphocytes or other immune cells to respond to pathogens.
  • Macrophages also have the function of rebuilding tissues, repairing damaged cells, and removing apoptotic cells.
  • the compound of the present invention has no inhibitory effect on the growth of macrophages or has the effect of promoting the proliferation of macrophages, indicating that it has the effect of balancing the immune function of the body.
  • the compound of the present invention was intervened at a concentration of 10 ⁇ M or a series of concentration of 10 ⁇ M-0.01 ⁇ M or 20 ⁇ M-0.625 ⁇ M, the inhibitory effect on the growth of normal RAW264.7 cells was not obvious, indicating that the toxicity of the compound was not strong.
  • the compound of the present invention was treated with a series of action concentrations of 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.01 ⁇ M and 0.001 ⁇ M for 96 hours, and the median inhibitory concentration IC 50 value of the inhibitory effect on the growth of human embryonic lung fibroblast HELF cells was greater than 10 ⁇ M, the inhibitory effect on the growth of mouse embryonic fibroblast NIH3T3 cells is also weak, and the IC50 value of the inhibitory effect is greater than 10 ⁇ M.
  • the compound of the present invention was incubated with normal human embryonic kidney epithelial cell 293T cells for 72 hours at a concentration of 10 ⁇ M.
  • the compound of the present invention had no obvious toxicity to normal human embryonic kidney epithelial cell 293T cells, except for berberine pyridine-3-carboxylic acid. Except that the survival rate of cells intervened with quaternary ammonium salt (1) was 98.5%, the survival rate of normal 293T cells treated with other compounds of the present invention were all up to 100% (see experimental example).
  • the compounds of the present invention do not show obvious growth inhibitory effect on other normal cells.
  • the compound of the present invention did not show obvious growth inhibitory effect on Vero E6 cells that were not infected with virus (see experimental example).
  • the compound of the present invention showed a variety of important and prominent pharmacological effects in pharmacological experiments.
  • the compounds of the present invention have the pharmacological effect of significantly reducing IL-6 levels in a pharmacological model of pathologically high IL-6 expression, including significant reductions in pathological IL-6 in a pharmacological model both at the cellular level and at the animal level overexpressed pharmacological effects.
  • the inhibitory effect of the compound of the present invention on the high expression of IL-6 still showed a statistically significant effect intensity at an effect concentration of 0.625 ⁇ M under the condition of pre-incubation for 2 hours.
  • the compound 7 of the present invention was tested at 200 mg/kg, 100 mg/kg and 50 mg/kg in the high-dose group/middle-dose group/low-dose group
  • the IL-6 levels in the serum of the model animals were significantly reduced under the action doses, to 16.8596pg/ml, 17.7957pg/ml and 21.6338pg/ml, respectively, and there were statistically significant differences compared with the model group.
  • the compounds of the present invention have the activity of significantly inhibiting NO overproduction in the LPS-stimulated RAW264.7 cell inflammation model.
  • the compound of the present invention can also show statistically significant inhibitory activity on NO production in the LPS-stimulated RAW264.7 cell inflammation model at the concentration of 0.625 ⁇ M (or even lower).
  • the inhibitory effect of the compound of the present invention under the action concentration of the pharmacological experiment is obviously stronger than that of the corresponding berberine chloride type alkaloid quaternary ammonium salt in the same action concentration range.
  • the activity of the compounds of the present invention in significantly inhibiting the production of NO in the LPS-stimulated RAW264.7 cell inflammation model is related to the activity of significantly inhibiting the high expression of iNOS, and, in parallel experiments, the activity of the compounds of the present invention in inhibiting the high expression of iNOS is higher than that of the corresponding chlorine Berberine-type alkaloid quaternary ammonium salts and monomeric acids corresponding to the counter anions of the compounds of the present invention have significantly stronger inhibitory effects.
  • the compounds of the present invention have the pharmacological effect of significantly reducing the serum IgG antibody level of model animals in the pharmacological model of pathologically high IgG antibody level.
  • the compound of the present invention has a very obvious effect on reducing the IgG antibody level in the serum of model animals, and shows a unique dose-dependence; Under the pathological state that the IgG antibody level in the serum of the animals in the model group reached 22.98350 units/ml, the tested compound 7 of the present invention was tested at 200 mg/kg, 100 mg/kg and 50 mg/kg high dose group/medium dose group/low dose group doses
  • the IgG antibody levels in the serum of the model animals can be reduced to 20.91259 units/ml, 17.97435 units/ml and 16.53063 units/ml, respectively, and the low-dose group and the middle-dose group are statistically significantly different from the model group.
  • the compounds of the present invention reduce the reduction in model animals Pharmacological effects of anti-chicken type II collagen IgG antibody concentrations in serum suggest that it has a significant effect on autoimmune diseases. Combined with the effects of the compounds of the present invention on immune effector cells, the results found in the above experiments show that the compounds of the present invention have significant immunomodulatory effects, and can be used to prepare immunomodulatory drugs for preventing, relieving and/or treating the secretion of IL- 6.
  • compositions for diseases with pathologically excessive increase preparation of pharmaceutical products for preventing, alleviating and/or treating diseases causing excessively high expression of iNOS and pathologically excessively increasing NO secretion in living organisms, preparation for preventing, alleviating and/or treating diseases caused by Pharmaceutical products for diseases in which the organism forms a pathological excess of autoantibodies.
  • the compounds of the present invention can play a role in the treatment of diseases by balancing the immune function of the body, and can significantly reduce the excessive secretion of IL-6 through the pharmacological effect of significantly reducing the level of excessive secretion of IL-6.
  • Related diseases have a beneficial therapeutic effect, and can play a beneficial therapeutic role in related diseases that cause excessive secretion of NO by the biological body by significantly reducing the pharmacological effect of excessive NO levels, and can significantly reduce the excessive levels of autoantibodies.
  • Pharmacological effects play a beneficial role in the treatment of related diseases that cause the body to form an excessive amount of autoantibodies.
  • the compounds of the present invention have beneficial therapeutic effects on viral infectious diseases and their complications, inflammatory diseases, febrile diseases, cardiovascular system and blood system diseases, autoimmune diseases, and some tumor diseases, etc.
  • the compounds of the present invention can dose-dependently inhibit the replication of SARS-CoV2 virus RNA in the Vero E6 cell model.
  • the compounds of the present invention have no inhibitory effect on the cytotoxicity induced by SARS-CoV2 on Vero E6 cells, and are effective against SARS-CoV2.
  • Compounds with significant application prospects in drug research and development of viral pneumonia diseases, that is, such compounds are safe and effective.
  • the tested compounds of the present invention at the concentration of 10 ⁇ M, the compounds 1, 3, 4, 6-10, etc.
  • the tested compounds 6, 8 and 3 inhibited SARS-CoV2 viral RNA levels with EC50 values of 3.037 ⁇ M, 3.767 ⁇ M and 7.859 ⁇ M, respectively.
  • the compound of the present invention reduces the pharmacological effect of the high concentration of anti-chicken type II collagen IgG antibody in the serum of the model animal, the pharmacological effect of the compound of the present invention to reduce the excessive secretion level of IL-6 in the serum of the model animal, and significantly inhibits the SARS-CoV2 virus.
  • the compounds of the present invention have a significant therapeutic effect on SARS-CoV2 virus infection with positive anti-pathogen IgG antibody in serum and inflammatory cytokine storm in the body.
  • the body temperature (change) of the model animals measured at different time points after administration and the temperature difference between the measured body temperature at different time periods and the The difference ⁇ T (°C) of basal body temperature was used as an index to evaluate the antipyretic effect of the compound of the present invention.
  • the results show that the compound of the present invention has a significant antipyretic effect, and the antipyretic effect is characterized by a longer duration of action than the positive drug. .
  • each dosage group of the compound of the present invention also has a significant increase in hemoglobin, indicating that the compound of the present invention has a significant effect on treating anemia.
  • each dose group of the tested compounds of the present invention significantly reduced the joint inflammation index score level.
  • the joint inflammation index score of the low/medium/high dose group was 1.73 respectively. ⁇ 0.68***, 2.67 ⁇ 0.80*** and 3.58 ⁇ 0.66***, compared with the joint inflammation index score of 6.92 ⁇ 0.84 in the model group, all were significantly lower, and all had extremely significant statistical differences.
  • the compound of the present invention selectively has a significant inhibitory effect on the growth of human colorectal cancer HCT-116 cells and human lung cancer A549 cells.
  • the significant reduction of IL-6 level and the inhibitory effect of the compounds of the present invention on the signaling pathway of Ras mutation/MAPK activation/IL-6 oversecretion indicate that the compounds of the present invention have beneficial therapeutic effects on tumor diseases.
  • the compounds of the present invention show significant anti-cancer effects on breast cancer in the 4T1 breast cancer cell mouse transplanted tumor animal model experiments. Parallel experiments with berberine chloride quaternary ammonium salt substrates were carried out to control the activity, and the activity of the active compound of the present invention to inhibit the growth of tumor cell lines was significantly improved.
  • the compounds of the present invention can be used for the preparation of prevention, alleviation and/or treatment of anemia, inflammatory disease, fever, colorectal cancer, Pharmaceutical products for lung cancer, breast cancer, rheumatoid arthritis and coronavirus infections and their complications.
  • the compound of the present invention showed the strongest pharmacological activity in the low-dose group of 50 mg/kg in the treatment of rheumatoid arthritis, And the middle dose group of 100 mg/kg was better than the high dose group of 200 mg/kg. It can be shown that the compounds of the present invention have a unique pharmacological mechanism of action. This is a selective marker of pharmacological action, and its mechanism of action needs to be further studied
  • the compounds of the present invention have significant medicinal efficacy, safety and quality controllability, and can be used in the preparation of immunomodulatory drugs, and in the preparation of prevention, alleviation and/or treatment of pathological excess secretion of IL-6 caused by biological organisms.
  • Drug products for diseases with high levels, drug products for diseases caused by pathological excess of NO secretion by biological organisms, and drug products for diseases caused by pathological excess of autoantibodies in biological organisms, and preparations for prevention, mitigation and/or treatment The application prospect of drug products for inflammatory diseases, febrile diseases, tumor diseases, autoimmune diseases, cardiovascular and cerebrovascular and blood system diseases, and viral infectious diseases and their complications caused by various reasons is very significant.
  • the compounds of the present invention are used in the preparation of prevention, alleviation and/or treatment of anemia, inflammatory diseases, febrile diseases, colorectal cancer, lung cancer, breast cancer, rheumatoid arthritis diseases and pathological excess secretion of IL-6 levels in living organisms It has significant application value in the novel coronavirus SARS-CoV2 infection and its complications due to the increased formation of inflammatory cytokine storm and pathological excess of IgG antibody levels.
  • Figure 1 The overall animal test administration process for chicken type II collagen-induced arthritis (CIA) in mice.
  • CIA collagen-induced arthritis
  • Figure 2 The effect of compound 7 of the present invention on the level of IL-6 in the serum of mouse chicken type II collagen-induced arthritis model mice.
  • Figure 3 The effect of compound 7 of the present invention on the hemoglobin content of mouse chicken type II collagen-induced arthritis model mice.
  • Figure 4 The effects of different administration groups of the present invention on the joint inflammation index score of mice.
  • n 12; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Fig. 6 is a graph of changes in body temperature of rats in the pharmacodynamic evaluation of the antipyretic effect of compound 7 of the present invention.
  • n 12 in each group; # is compared with the blank control group, ### p ⁇ 0.001; * is compared with the model group, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 7 The inhibitory effect of the compounds of the present invention on the high expression of iNOS in the RAW264.7 cell inflammation model induced by LPS stimulation.
  • the compound numbers correspond to the specific compound numbers in the content of the present invention.
  • Example 12 Synthesis and structural identification data of compound 12 of the present invention
  • Example 17 Synthesis and structural identification data of compound 17 of the present invention
  • Example 26 Synthesis and structural identification data of compound 26 of the present invention
  • Example 28 Synthesis and structural identification data of compound 28 of the present invention
  • Example 33 Synthesis and structural identification data of compound 33 of the present invention
  • Example 36 Synthesis and structural identification data of compound 36 of the present invention
  • Example 37 Synthesis and structural identification data of compound 37 of the present invention
  • Example 40 Synthesis and structural identification data of compound 40 of the present invention
  • Example 41 Synthesis and structural identification data of compound 41 of the present invention
  • Example 1' the synthesis and structure identification data of the compound 1' of the present invention
  • Experimental example 1 Experimental example of water solubility detection of the compound of the present invention
  • Experimental example 2 Experimental example of the solubility detection of the compound of the present invention in 95% ethanol solvent
  • Experimental Example 3 Evaluation experiment of the effect of the compounds of the present invention on the growth of mouse mononuclear macrophage RAW264.7 cells cultured in vitro
  • Compound preparation The compound was prepared into a compound stock solution of 1 ⁇ 10 -2 mol/L (0.01M) with DMSO. When the cell experiment was performed, the compound was prepared with 10% FBS and double antibody (streptomycin 100 ⁇ g/L). mL, penicillin 100U/mL) in DMEM high glucose medium and diluted to the initial solution of the desired concentration.
  • the compound initial solution was taken and diluted with DMEM high glucose medium containing 10% FBS and double antibody (streptomycin 100 ⁇ g/mL, penicillin 100 U/mL) to a concentration of 200 ⁇ M for administration.
  • This compound administration solution was added to a 96-well plate containing 100 ⁇ l of primary passage cell suspension, 100 ⁇ l per well, and the set concentration gradient of the compound series of the present invention was realized by the doubling dilution method, that is, 100 ⁇ M-0.390625 ⁇ M, each 6 replicate wells were set for each administration concentration.
  • a normal cell control group and a blank control group were set.
  • the normal cell control group contained cells cultured by the same operation and the same amount of drug dissolving medium, but did not contain the compound of the present invention; the blank control group was the same as the normal control group except that it did not contain cells. same. After 24 hours of incubation in the incubator, 10 ⁇ L of CCK-8 solution was added to each well, and after 2 hours of incubation in the incubator, the optical density (OD) value was measured at 450 nm with a microplate reader (result 1 experiment).
  • a normal cell control group and a blank control group were set.
  • 10 ⁇ L of CCK-8 solution was added to each well, and after 2 hours of incubation in the incubator, the optical density (OD) value was measured at 450 nm with a microplate reader (Experiment in Table 3).
  • the set dosing concentration or serial dosing concentration of the compound of the present invention is prepared, and 6 duplicate wells are set for each dosing concentration.
  • a normal cell control group and a blank control group were set.
  • 10 ⁇ L of CCK-8 solution was added to each well, and after 2 hours of incubation in the incubator, the optical density (OD) value was measured at 450 nm with a microplate reader (Table 4, Table 5 experiments).
  • the cell viability after the first passage of the cultured cells treated with the compound 1 and compound 8 of the present invention at a series concentration of 100 ⁇ M-0.390625 ⁇ M is 82.13 ⁇ 0.5% except that the compound 1 is at a concentration of 100 ⁇ M.
  • the rest are greater than 100%.
  • the series of compounds of the present invention have no cytotoxicity to multi-passage RAW264.7 cells at the concentration of 0.01 ⁇ M-10 ⁇ M, and have a certain effect of promoting proliferation.
  • the cell viability and IC 50 values of each assay experiment are shown in Table 3.
  • the series of compounds of the present invention have no cytotoxicity or little cytotoxicity to normal RAW264.7 cells under the action concentration of 10 ⁇ M, and some compounds have a certain effect of promoting proliferation.
  • the cell viability values of each assay experiment are shown in Table 4.
  • Compounds 18 and 19 of the present invention have no cytotoxicity to normal RAW264.7 cells at the concentration of 20 ⁇ M-0.625 ⁇ M, and have a certain pro-proliferation effect.
  • the cell viability values of each assay experiment are shown in Table 5.
  • the series of compounds of the present invention have no inhibitory effect on the growth of normal mouse mononuclear macrophage RAW264.7 cells cultured in vitro, and have the activity of promoting the proliferation of RAW264.7 cells.
  • Experimental Example 4 The cytotoxicity evaluation experiment of the compounds of the present invention on normal human embryonic kidney epithelial cells 293T cells cultured in vitro.
  • Preparation of the compound of the present invention Dissolve the compound of the present invention in DMSO to prepare a stock solution of 1 ⁇ 10 -2 mol/L (0.01M), and then dilute it with cell culture medium to 1 ⁇ 10 -2 mol/L (0.01M) for cell experiments. 5 mol/L (ie 10 ⁇ M) working solution concentration.
  • mice normal human embryonic kidney epithelial cells 293T cells grown in vitro to 90% confluence according to the cell instructions were digested with 0.25% trypsin/0.1 EDTA, and the cell suspension was prepared in cell culture medium. The concentration of the cell suspension was adjusted and seeded in a 96-well cell culture plate with 100 ⁇ l of cell suspension per well, and the cell density was 2 ⁇ 10 3 cells/ml. The peripheral wells of each 96-well plate were filled with PBS to prevent "edge effects”. Incubate the cell culture plate in a 5% CO 2 /37°C incubator until the cells grow completely attached (overnight).
  • Experimental Example 5 The cytotoxicity evaluation experiment of the compounds of the present invention on normal human embryonic lung fibroblast HELF cells and mouse embryonic fibroblast NIH3T3 cells cultured in vitro.
  • Preparation of the compound of the present invention Dissolve the compound of the present invention with DMSO to prepare a stock solution of 1 ⁇ 10 -2 mol/L (0.01M), and when performing cell experiments, dilute it with cell culture medium to a concentration of 20 ⁇ M initial solution.
  • This compound series concentration gradient solution was added to the 96-well plate, 100 ⁇ l per well, and then 100 ⁇ l was sucked and discarded after pipetting to obtain a series of concentration gradients with concentrations of 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.01 ⁇ M and 0.001 ⁇ M, respectively.
  • Three duplicate wells were set for each administration concentration, and a normal cell control group and a blank control group were set at the same time. After 96h incubation in the incubator, the OD value of each compound was detected by MTT method, and the inhibition rate was calculated using the following formula:
  • Cell growth inhibition rate (%) ⁇ [(OD value of control well-OD value of blank well)-(OD value of administration well-OD value of blank well)]/[(OD value of control well-OD value of blank well] ⁇ 100%
  • the compounds of the present invention have no obvious toxicity to the growth of human embryonic lung fibroblasts HELF cells and mouse embryonic fibroblasts NIH3T3 cells, and are suitable for downstream activity screening experiments.
  • the Griess method was used to detect the effect of the compounds of the present invention on the NO secretion in the LPS-stimulated RAW264.7 cell inflammation model.
  • the 96-well plate was set up into experimental groups, including groups given the compounds of the present invention, LPS stimulation model group, normal cell control group and blank group.
  • groups given the compounds of the present invention LPS stimulation model group
  • normal cell control group normal cell control group
  • blank group normal cell control group
  • each experiment was set with different working concentrations according to the specific experiment, and the total volume of each well was still controlled to be 100 ⁇ l.
  • the experimental results showed that the compounds of the present invention could dose-dependently inhibit the production of NO in the RAW 264.7 cell inflammation model induced by LPS stimulation after intervention treatment by the compounds of the present invention (Tables 7-14).
  • the compound of the present invention has a significantly better inhibitory effect on NO production in the RAW 264.7 cell inflammation model induced by LPS stimulation than the berberine chloride type alkaloid quaternary ammonium salt substrate.
  • the Griess method was used to detect the effect of the compounds of the present invention on the NO secretion in the LPS-stimulated RAW264.7 cell inflammation model.
  • the cell density of the solution was 6 ⁇ 10 5 cells/mL, and it was seeded in a 96-well cell culture plate, and the amount of cell suspension seeded in each well was 100 ⁇ l.
  • the cell culture plate was placed in a 5% CO 2 /37°C incubator and cultured until the cells were completely adherent and grown (overnight), and it was observed that the cells were in good shape.
  • the 96-well plate was set up with experimental groups, including each administration of the compound of the present invention group, LPS stimulation model group, normal cell control group and blank group.
  • experimental groups including each administration of the compound of the present invention group, LPS stimulation model group, normal cell control group and blank group.
  • the experimental operation method see "Experimental Example 3, the 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"
  • each cell suspension containing the compound of the present invention was prepared by the half-dilution method, or each containing the compound of the present invention was prepared at the set administration concentration of the compound of the present invention.
  • the intervention experiment cell suspension, and control the total volume of each well is still 100 ⁇ l.
  • Inhibition rate% ⁇ [(NO amount in model group-NO amount in control group)-(NO amount in administration group-NO amount in control group)]/[(NO amount in model group-NO amount in control group] ⁇ 100%
  • the ELISA method was used to detect the effect of the compounds of the present invention on the pathological excess secretion of IL-6 in the RAW264.7 cell inflammation model induced by LPS stimulation.
  • the cell supernatant sample of the LPS stimulation-induced RAW 264.7 cell inflammation model was prepared according to the operation procedure of "Experimental Example of Evaluating the Inhibitory Effect of NO Pathological Hyperproduction in the RAW 264.7 Cell Inflammation Model Induced by LPS Stimulation, and added to the mouse IL -6
  • the wells of the microtiter plate containing the target protein-specific monoclonal capture antibody provided in the ELISA kit operate according to the operating procedures provided in the kit instructions. content was measured.
  • Experimental Example 9 Experimental example of the inhibitory effect of the compound of the present invention on tumor cell growth.
  • the experimental results show that the compounds of the present invention can dose-dependently and selectively inhibit the growth of human colon cancer HCT-116 cells and human lung cancer A549 cells.
  • Each compound of the present invention was prepared into a stock solution with a concentration of 0.02M with DMSO. When the cell experiment was performed, it was diluted with a cell culture medium (1640 medium containing 10% serum) into an initial solution with a concentration of 20 ⁇ M.
  • cell culture medium for initial cell culture, prepare cell suspension, count cells, inoculate cell suspension (100 ⁇ l) on 96-well cell culture plates, and incubate in 5% CO 2 , 37°C cell incubator until cells follow the instructions for cells The operation of fully adherent growth.
  • the initial compound solution was taken and diluted with cell culture medium into a series of concentration gradient solutions with concentrations of 20 ⁇ M, 2 ⁇ M and 0.2 ⁇ M, respectively. Add this compound serial concentration gradient solution to a 96-well cell culture plate, 100 ⁇ l per well, pipette evenly, and then remove 100 ⁇ l from each well to obtain a series of concentration gradients with concentrations of 10 ⁇ M, 1 ⁇ M, and 0.1 ⁇ M, respectively.
  • Cell growth inhibition rate (%) ⁇ [(OD value of control well-OD value of blank well)-(OD value of administration well-OD value of blank well)]/[(OD value of control well-OD value of blank well] ⁇ 100%
  • the series of compounds of the present invention have obvious cytostatic activity against HCT-116 cells.
  • the IC 50 values of cell growth inhibition were 1.286 ⁇ M, 1.195 ⁇ M, 2.248 ⁇ M, 2.637 ⁇ M, 0.594 ⁇ M and 2.462 ⁇ M; in parallel experiments, berberine quaternary ammonium chloride, berberine quaternary ammonium chloride
  • the IC50 values for the growth inhibition of HCT-116 cells by salt and isopantine chloride quaternary ammonium salt were 4.496 ⁇ M, 2.087 ⁇ M and 2.512 ⁇ M, respectively.
  • the compound of the present invention was prepared into a 0.02M compound stock solution with DMSO, and when the cell experiment was performed, it was diluted with a cell culture medium to obtain an initial solution with a concentration of 20 ⁇ M.
  • cell culture medium for initial cell culture, prepare cell suspension, count cells, inoculate cell suspension (100 ⁇ l) on 96-well cell culture plates, and incubate in 5% CO 2 , 37°C cell incubator until cells follow the instructions for cells The operation of fully adherent growth.
  • the initial solution of the compound of the present invention was taken and diluted with cell culture medium to obtain a series of concentration gradient solutions with concentrations of 20 ⁇ M, 2 ⁇ M and 0.2 ⁇ M, respectively.
  • This compound series concentration gradient solution was added to the 96-well cell plate, 100 ⁇ l per well, pipetted evenly, and then 100 ⁇ l was removed from each well to obtain a series of concentration gradients with concentrations of 10 ⁇ M, 1 ⁇ M and 0.1 ⁇ M, respectively.
  • the compounds of the present invention have obvious cell growth inhibitory activity on A549 cells within the 96h time period determined by the experiment.
  • IC 50 values were 3.114 ⁇ M, 2.319 ⁇ M, 4.378 ⁇ M, 3.497 ⁇ M and 0.919 ⁇ M, respectively; in parallel experiments, the ICs of berberine chloride quaternary ammonium chloride and coptisine quaternary ammonium chloride on the growth inhibition of A549 cells
  • the 50 values were >10 ⁇ M and 6.540 ⁇ M, respectively.
  • the compounds of the present invention can be used for the preparation of immunomodulator medicines, the preparation of medicines for the prevention, mitigation and/or treatment of diseases that cause pathologically excessive secretion of IL-6 in biological bodies, and the preparation of medicines for the prevention, mitigation and/or treatment of biological
  • the use of the identified compounds of the present invention for the preparation of immunomodulator medicines and the preparation of medicines for preventing, relieving and/or treating diseases that cause pathologically elevated NO secretion in living organisms has been proved in the aforementioned experimental examples, as follows:
  • the compound of the present invention is used to prepare pharmaceutical products for preventing, alleviating and/or treating diseases that cause pathologically excessive secretion of IL-6 in biological organisms, and preparing to prevent, alleviate and/or treat pathological increases in the formation of autoantibodies caused by biological organisms
  • Drug products for the prevention, mitigation and/or treatment of inflammatory diseases, febrile diseases, cardiovascular and cerebrovascular and blood system diseases, autoimmune diseases, and tumor diseases are prepared to demonstrate experimental examples. At the same time, these experimental examples further illustrate that the compounds of the present invention have an effect on equilibrium The important biological significance of the body's immune response, therefore, can be used to prepare immunomodulator drugs.
  • Typical symptoms of rheumatoid arthritis include joint inflammation, markedly elevated serum IL-6 levels, and formation of pathological autoantibodies; anemia is one of the most common extra-articular symptoms of rheumatoid arthritis. Therefore, the present invention adopts an animal model of rheumatoid arthritis to evaluate the efficacy of the compound of the present invention in reducing the level of pathologically high IL-6, increasing the level of hemoglobin and reducing the level of pathological autoantibodies at the animal level, and at the same time using the compound of the present invention
  • the effects on the rheumatoid arthritis inflammatory degree index score (referred to as joint inflammation index score), the incidence of joint inflammation and the thickness of the sole of the foot of the model animals were used as evaluation indicators to evaluate the curative effect of the compounds of the present invention on rheumatoid arthritis.
  • the application of the compounds of the present invention to antitumor drugs was evaluated by using a mouse 4T1 cell subcutaneously transplanted tumor
  • Experimental Example 10 Experimental Example of Efficacy Evaluation of the Compounds of the Present Invention in Reducing the High-Expression Phenotype of IL-6 Level in Model Animals
  • the tested compound of the present invention was compound 7, and the positive drug was methotrexate.
  • Other reagents include chicken type II collagen, complete Freund's adjuvant, incomplete Freund's adjuvant, phosphate buffered saline (PBS).
  • the compound 7 of the present invention was prepared by using 0.5% CMC-Na aqueous solution as a solvent to prepare a working solution for the administration of the compound of the present invention
  • the positive control drug was prepared by using 0.5% CMC-Na aqueous solution as a solvent to prepare a concentration of Appropriate positive control drug administration working solution.
  • the administration volume was 1ml/100g (administration working solution volume/animal body weight) dose, and it was administered to experimental DBA/1 mice by gavage.
  • the DBA/1 mice were raised in the experimental animal room in an environment that allowed free drinking water.
  • the room temperature of the experimental animal room was 22°C-25°C, the relative humidity was 55-65%, and the light cycle was 12h/12h. Animals were first acclimated for one week after purchase.
  • the initial immunization of the animals was recorded as day 0.
  • the method was to inject 0.1 ml of collagen and complete Freund's adjuvant emulsifier prepared by ultrasound at multiple points at 2-3 cm at the base of the tail of the animal at a volume ratio of 1:1.
  • the blank control group was injected with 0.1 ml of normal saline.
  • Arthritis was then induced on day 21 after primary immunization by boosting with collagen and incomplete adjuvant by subcutaneous injection of 0.05 ml of a 1 : 1 volumetric emulsion of the sonicated emulsion at multiple points in the tail avoiding the site of primary immunization.
  • any ankle joint, Swelling of the soles of the feet and toes is regarded as the onset of the mouse arthritis model.
  • mice were randomly divided into blank control group (normal control group), model group, positive drug control group and low/medium/high dose administration groups of the tested compounds of the present invention, a total of 6 groups.
  • the low/medium/high doses of the compounds of the present invention were set as 50 mg/kg, 100 mg/kg and 200 mg/kg (compound amount/animal body weight) in each administration group, and the administration frequency and route of administration were set as 1 time/day , p.o.
  • the administration dose, administration frequency and administration route of the positive drug methotrexate were set at 1 mg/kg (compound amount/animal body weight), 2 times/week, p.o.
  • the administration started from the 18th day after the second immunization of the mouse model, and continued administration for 4 weeks until the symptoms of arthritis in the administration group were significantly reduced compared with the model group, and there was a significant difference in the joint inflammation index score. Discontinue dosing.
  • the blank control group and the model group were given the blank vehicle by gavage, the test compound group was given the corresponding dose of the present compound working solution by gavage, and the positive control group was given the corresponding dose of the positive control working solution by gavage.
  • mice were anesthetized, the eyeballs were removed, and blood samples from each group of animals were collected through the orbits, anticoagulated, and anticoagulated.
  • Mouse IL-6 ELISA Kit was used to detect the inflammatory cytokine IL- 6 levels of changes, and compare the experimental results of different groups through statistical processing.
  • the IL-6 levels in the serum of each detected mouse in the model group increased significantly, and there was a statistical difference, indicating that the modeling was successful; compared with the model group, the methotrexate positive drug group, the compound 7 of the present invention
  • the serum IL-6 levels of the mice in the low/medium/high dose administration groups were significantly decreased, and there were statistical differences.
  • the compound 7 low/medium/high dose administration group of the present invention has a significantly better inhibitory effect on the serum IL-6 level of model mice than the administration group in which the positive drug is administered according to specific characteristics. The results are shown in Table 20 and Figure 2.
  • Table 20 The effect of tested compound 7 of the present invention on the concentration of IL-6 in the serum of model mice (pg/mL).
  • the experimental results show that the tested compounds of the present invention have a very significant effect on the level of IL-6 in the serum of model mice. the inhibitory effect.
  • Experimental Example 11 Experimental Example of Efficacy Evaluation of the Compound of the Invention to Increase Hemoglobin Content in Model Animals
  • mice in each group were anesthetized at the end of the experiment, and their eyes were removed for blood. After anticoagulation, the anticoagulant was taken for detection by routine blood test.
  • the test results showed that the average value of the hemoglobin content of the mice in the blank control group was 147.29 ⁇ 5.06 (g/L); compared with the blank control group, the hemoglobin content of the animals in the experimental model group was significantly lower, with an average value of 136.33 ⁇ 4.27 ( g/L); compared with the model group, each dose group of the tested compounds of the present invention significantly increased the hemoglobin content.
  • each dose group of the compound of the present invention also has a significant increase in hemoglobin, indicating that the compound of the present invention has a significant effect of increasing hemoglobin.
  • the average hemoglobin content of the positive drug group administered according to the specific characteristics of the positive drug was 160.92 ⁇ 7.07 (g/L).
  • the experimental results show that the tested compounds of the present invention can significantly increase the hemoglobin content of the model mice.
  • the thickness of the two hind paws of the mice was measured with a vernier caliper while the joint inflammation index was measured every other day.
  • mice were anesthetized, and blood samples were collected from the orbits of animals in each group. After anticoagulation, the content of chicken type II collagen antibody in serum was detected by ELISA.
  • the tested compounds of the present invention can significantly improve the joint inflammation index score in mice
  • the joint inflammation index score was calculated and evaluated every other day for the degree of arthritis symptoms in the limbs and joints of the mouse chicken type II collagen-induced arthritis model mice.
  • the experimental results showed that, compared with the model group, from the 4th day after the start of the experimental administration (that is, the 42nd day after the start of the primary immunization) to the end of the experiment, the compound 7 of the present invention was administered at a dose of 100 mg/kg in the middle-dose group.
  • the joint inflammation index score was always lower than that of the positive drug methotrexate, and from the 4th day to the 18th day after the start of experimental administration, the joint inflammation index score of the low-dose group with a dose of 50 mg/kg was always the same as that of the positive drug.
  • Drug methotrexate was comparable, and from day 18, joint inflammation in the low-dose group
  • the index score was always lower than that of the positive drug methotrexate, and also lower than that of the middle-dose group (ie, the efficacy was better than that of the middle-dose group) until the end point of the experiment.
  • the joint inflammation index score of the high-dose group of the compound 7 of the present invention administered at a dose of 200 mg/kg was also significantly lower, and from the 20th day after the start of administration, it was comparable to the positive drug methotrexate.
  • the therapeutic effect of pyridine is also comparable.
  • 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 was different from the joint inflammation index score of the model group of 6.92 ⁇ Compared with 0.84, both were significantly lower, and both had extremely significant differences.
  • the three dose groups of compound 7 of the present invention, low/medium/high all have the effect of significantly improving the joint inflammation condition of model animals.
  • the improvement effect of the dose group on the condition of the model animals is weaker in the lower dose group, but better than that in the high dose group.
  • This unique dose-response relationship further indicates the unique mechanism of action of the compounds of the present invention.
  • the specific data and graphical results of the compounds of the present invention significantly improving the joint inflammation index score in mice are shown in Table 22 and FIG. 4 below.
  • Table 22 The effect of different administration groups of the present invention on the joint inflammation index score of experimental mice chicken type II collagen-induced arthritis model mice.
  • n 12; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the tested compounds of the present invention have a significant inhibitory effect on the incidence of joint inflammation in experimental animals
  • the incidence data of joint inflammation in each group of animals were obtained by statistical analysis of the incidence of joint inflammation in mouse chicken type II collagen-induced arthritis model mice during the process of drug administration to model animals.
  • the incidence of joint inflammation in each group of mice during the entire treatment cycle is shown in Table 23.
  • the incidence of joint inflammation in the model group was 100%
  • the incidence of joint inflammation in the methotrexate-positive group was 100%. 75%
  • the incidence rates of joint inflammation in the low/medium/high dose groups of Compound 7 of the present invention were 45%/58%/83%, respectively.
  • the compound of the present invention has obvious inhibitory effect on the incidence of joint inflammation in mice, and can significantly improve the condition of model animal joint inflammation, especially the low-dose group has the most obvious improvement effect on the condition of model animals, and the middle-dose group has the most obvious improvement effect on the condition of model animals.
  • the improvement effect of the lower dose group was weak, but better than that of the high dose group, and still significantly better than the positive drug group.
  • the incidence data of joint inflammation further illustrate the unique mechanism of action of the compounds of the present invention.
  • the low-dose group of the compound of the present invention showed an obvious effect on the 46th day after modeling (ie, the 9th day after administration), and there was a significant difference compared with the model group; and with the prolongation of the treatment process, the low-dose group's The effect gradually exceeded that of the middle-dose group; from the 56th day after modeling (ie, the 19th day after administration), the effect of the low/middle-dose group exceeded that of the positive drug group.
  • the high-dose group of the compound of the present invention also showed an obvious improvement effect on the paw thickness of mouse chicken type II collagen-induced arthritis model mice, and the effect was comparable to that of the positive drug group.
  • Model animal paw thickness data further illustrate the unique mechanism of action of the compounds of the present invention.
  • n 12; compared with model group, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Autoimmune diseases such as rheumatoid arthritis are chronic diseases caused by tissue damage due to the production of autoantibodies.
  • This study investigated the effect of the tested compound 7 of the present invention on the level of autoantibodies in mouse chicken type II collagen-induced arthritis model mice.
  • the serum of the tested mice was diluted with the serum diluent prepared in the kit at a ratio of 1:1000, and the expression of anti-chicken type II collagen antibodies in the diluted serum was detected by ELISA. The results are shown in Table 25 and Figure 5.
  • the concentration of anti-chicken type II collagen IgG antibody in the serum of the mice in the model group was significantly increased; compared with the model group, the positive drug methotrexate group and the compound 7 of the present invention were lower/lower
  • the concentration of anti-chicken type II collagen IgG antibody in the serum of mouse chicken type II collagen-induced arthritis model mice in the middle-dose group was significantly reduced.
  • the positive drug group had anti-chicken type II collagen IgG antibody.
  • the reducing effect of the compound 7 low-dose group of the present invention on the level of anti-chicken type II collagen IgG antibody level is better than the experimental group administered according to the specific characteristics of the positive drug, and there is a very significant difference compared with the model group.
  • the effect of the compound 7 of the present invention in the middle-dose group is slightly lower than that of the low-dose group, but it is also better than the positive drug group, and there is a significant difference compared with the model group.
  • the effect of the compound 7 high-dose group of the present invention was not statistically different from that of the model group, the trend of reducing the anti-chicken type II collagen IgG antibody was also very obvious.
  • Table 25 The effect of compound 7 of the present invention on the level of anti-chicken type II collagen IgG antibody in the serum of mouse chicken type II collagen-induced arthritis model mice.
  • the compound of the present invention has obvious anti-rheumatoid arthritis effect on experimental animals, wherein, within the dose range selected in the experiment, the low/medium/high dose of compound 7 of the present invention all showed significant anti-rheumatoid arthritis effects.
  • obvious effect mainly in the improvement of the joint inflammation index score, the incidence of joint inflammation and the thickness of the sole of the mouse in the mouse chicken type II collagen-induced arthritis model, that is, the joint inflammation in the model mouse is significantly improved. of clinical symptoms, significantly reduced the incidence of joint inflammation in model animals, Significantly reduced the paw thickness of model animals.
  • the hemoglobin content in the blood of the mouse chicken type II collagen-induced arthritis model animals treated with the compound 7 of the present invention is significantly increased, which not only further indicates that the compound of the present invention has an anti-rheumatoid arthritis effect, but also It is shown that the compound of the present invention also has a significant intervention effect on cardiovascular system diseases, and can be used for the treatment of cardiovascular system diseases including anemia. It needs to be further emphasized that, within the dose range selected in the experiment, on the whole, the low-dose group in this experiment has the strongest anti-rheumatoid arthritis effect, and the effect of the middle-dose group is weaker than that of the low-dose group, but stronger than that of the low-dose group.
  • the high-dose group showed an unconventional dose-response relationship.
  • the compounds of the present invention possess unique mechanisms of action, including differing sensitivities to their targets of action.
  • Animals male SD rats with a body weight ranging from 180 to 200 g.
  • the tested compound of the present invention was Compound 7, and the administered dose was set at 100 mg/kg.
  • the positive drug is compound acetaminophen tablets (also known as Sanlitong), the specifications are acetaminophen 250mg/isoproantipyrine 150mg/caffeine anhydrous 50mg, and the dosage is set at 45mg/kg.
  • the reagent for inducing the rat fever model is yeast; the vehicle for preparing the yeast solution is 0.9% sodium chloride injection.
  • the compound of the present invention and the positive drug were prepared by using 0.5% CMC-Na aqueous solution as a solvent to prepare a working solution, which was administered by gavage at a dose of 1ml/100g (working solution/animal body weight).
  • the rectal temperature was measured 2 times every morning and recorded as the body temperature of the experimental animal.
  • the time interval of the measurement was 30 min.
  • the average value of the measured body temperature was taken as the basal body temperature, while the rats were adapted to the temperature measurement operation.
  • rats with a basal body temperature of 36°C to 38°C and a body temperature fluctuation ⁇ 0.6°C were selected to enter the experiment, and the rats were fasted 12 hours before the experiment.
  • 20% yeast suspension prepared with 0.9% sodium chloride injection as solvent was subcutaneously injected into the back of the rat, and the dose volume was 10ml/kg (yeast suspension/rat body weight) to establish rat fever.
  • Rats in the blank control group were injected with the corresponding volume of solvent (Note: The procedures of all animal experiments were strictly implemented in accordance with the guidelines of the experimental animal ethics committee, and were approved by the ethics committee).
  • mice Five hours after the rats were subcutaneously injected with 20% yeast suspension, the rats whose body temperature rose between 1.0°C and 3.0°C were selected to enter the experiment. Including the animals subcutaneously injected with the corresponding volume vehicle, the experimental animals were randomly divided into blank control group (normal control group), model group, positive drug group and test compound 7 administration group of the present invention, a total of 4 groups, with 12 experimental animals in each group. mouse. During administration, the experimental animals of the tested compound of the present invention and the positive control group were respectively given the corresponding tested compound of the present invention CMC-Na administration working solution or the positive control drug CMC-Na administration according to the dosage.
  • blank control group normal control group
  • model group positive drug group
  • test compound 7 administration group of the present invention a total of 4 groups, with 12 experimental animals in each group. mouse.
  • the experimental animals of the tested compound of the present invention and the positive control group were respectively given the corresponding tested compound of the present invention CMC-Na administration working solution or the positive control drug CMC-Na administration
  • the blank control group and the model control group were given the same volume of blank solvent by gavage, the gavage volume was 10ml/kg (yeast suspension/rat body weight), at 0h, 1h, 2h, 4h, 6h after administration
  • the rectal temperature of the rats was measured and recorded.
  • the administration frequency and administration route of the compound group of the present invention and the positive drug group were set to 1 time/day, p.o.
  • the rectal temperature of rats was measured and recorded at 0h, 1h, 2h, 4h, and 6h after administration; the difference ⁇ T (°C) between the measured body temperature and the basal body temperature in each time period was calculated and recorded.
  • the antipyretic effects of the compounds of the present invention were determined by comparing with the corresponding data of the normal control group and the model group.
  • the average basal body temperature of rats in each group was between 37.55°C and 37.88°C, with a small difference between the average values.
  • the body temperature of the blank control group was kept between 37.34°C and 37.83°C.
  • the body temperature of each modeling group rose to between 39.60 °C and 39.75 °C, and the rise in each group was basically the same.
  • the body temperature of the positive drug Sanlitong administration group decreased significantly, and the body temperature of the compound 7 administration group of the present invention decreased significantly.
  • the body temperature of the positive drug group has begun to rise.
  • the compound 7 administration group of the present invention is different from the positive drug.
  • the body temperature further decreased 7 hours after modeling (2 hours after administration), and the difference in body temperature decrease was more obvious compared with the previous observation point, and there was a very significant difference compared with the model group; 9 hours after modeling (administration) 4 hours), the body temperature of the positive drug group continued to rise significantly, and the recovery speed was accelerated, and there was no statistical difference compared with the model group; although the body temperature of the compound 7 group of the present invention also began to rise, but the recovery rate was significantly smaller than that of the positive drug.
  • n 12 in each group; # is the comparison with the blank control group, ### p ⁇ 0.001; * is the comparison with the model group, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the compound of the present invention can be displayed more intuitively by calculating and comparing the difference between the body temperature of the rats in each time period after the intervention of the compound of the present invention and the basal body temperature of the rat recorded before administration (the difference is referred to as the difference in body temperature).
  • the effect of the intervention on the body temperature of the yeast-induced fever model in rats is shown in Table 27.
  • the average body temperature of rats in each group increased by 1.74°C to 1.95°C, and the model was stable and the trend was consistent.
  • the body temperature difference of the positive medicine Sanlitong group was 0.23 ⁇ 0.77 *** , which was close to the basal body temperature, and there was a significant difference compared with the model group, indicating that the positive medicine was effective and the modeling was successful;
  • Compound 7 of the present invention The range of body temperature difference in the group decreased, but there was no statistical difference compared with the model group. The situation changed 2 hours after administration.
  • the range of body temperature difference in the positive drug Sanlitong group was significantly increased compared with the previous observation point, but the difference in body temperature still remained extremely significant compared with the model group, indicating that the positive drug still has a solution to the problem.
  • Thermal effect but the antipyretic effect began to diminish 2 hours after administration.
  • the range of body temperature difference in the compound 7 groups of the present invention was further reduced 2 hours after administration, and the difference was more obvious than the previous observation point.
  • the antipyretic effect continued to increase 2 hours after administration, and the effect was more pronounced.
  • the range of the body temperature difference in the positive medicine Sanlitong group further increased significantly, reaching 2.05 ⁇ 0.52, and there was no statistical difference compared with the model group; the range of the body temperature difference in the compound 7 groups of the present invention also increased. , but the range of the body temperature difference in the compound 7 group of the present invention was significantly smaller than that of the positive drug group, which was 1.58 ⁇ 0.83.
  • the difference in body temperature between the positive drug group and the compound administration group of the present invention further increased, and the positive drug group reached 2.46 ⁇ 0.53, which had exceeded the model group, and had no statistical significance compared with the model group; the present invention
  • the body temperature difference in the compound 7 group was 1.86 ⁇ 0.68, which was smaller than the body temperature difference in the positive drug group; although compared with the model group, the body temperature difference in the compound 7 group of the present invention also had no significant statistical difference, but the body temperature in the compound 7 group of the present invention was not statistically significant.
  • the magnitude of the difference is significantly smaller than the model group and the positive drug group, that is, although there is no significant difference compared with the model group, the antipyretic effect of the compound of the present invention lasts longer, and the trend of longer duration is more obvious than that of the positive drug group.
  • n 12 in each group; # is compared with the blank control group, ### p ⁇ 0.001; * is compared with the model group, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the compound of the present invention has a significant antipyretic effect, and the antipyretic effect has a longer duration than the positive drugs.
  • Experimental example 14 Experimental example for the determination of the efficacy of the compound of the present invention against 2019 novel coronavirus (SARS-CoV2)
  • Class II biological safety cabinet CO2 incubator; inverted microscope; 96-well cell culture plate.
  • SARS-CoV2 2019-nCoV virus (titer 8 ⁇ 10 5 TCID50/mL); DMEM basal medium; Vero E6 cells; fetal bovine serum; penicillin-streptomycin dual antibody; 0.25% trypsin-EDTA; qPCR experimental reagents; TRIzol; cell culture grade DMSO.
  • the compound of the present invention was first dissolved in DMSO as a solvent to prepare a stock solution of the compound of the present invention at 1 ⁇ 10 -2 mol/L (0.01M). When performing cell experiments, dilute with cell culture medium to the working solution of the desired concentration.
  • the experimental data were statistically analyzed using GraphPad Prism version 5.0 software.
  • Cell seeding Take Vero-E6 cells that are in good growth state and in logarithmic growth phase according to the cell instructions, aspirate and discard the culture medium, digest the cells with trypsin, prepare a cell suspension, and the cell count is 1 ⁇ 10 6 cells/ml ; Take 4 ml of the above cells, add 6 ml of cell culture medium, prepare a cell suspension with a cell density of 4 ⁇ 10 5 cells/ml, and inoculate it on a 96-well cell culture plate (100 ⁇ l of cell suspension per well, the number of cells is 1 ⁇ 10 4 ); cultured in a 5%CO 2 /37°C cell incubator until the cells grew completely adherent.
  • micearoscopic cytotoxicity assay The stock solution of the compound of the present invention was diluted to a working solution of the corresponding concentration range using complete medium.
  • the present invention observes the toxic effects of the compounds of the present invention on cells at the concentration of 20 ⁇ M, 10 ⁇ M and 5 ⁇ M.
  • Half-value toxicity concentration (CC 50 ) detection experiment The stock solution of the compound of the present invention was diluted to the corresponding concentration of working solution using complete medium.
  • the present invention is at The cytotoxicity detection of the compounds of the present invention was carried out under a series of concentration gradients, and the CC 50 value was calculated.
  • Intervention treatment of the cells by the compounds of the present invention Aspirate and discard the original medium of the cells, add the diluted medium containing the compounds of the present invention at the corresponding concentration, the total volume of each well is 100 ⁇ l, place in a 37°C cell incubator, and continue to culture for 48 hours .
  • a normal cell control group and a blank control group were set at the same time, and the normal cell control group contained cells cultured by the same operation and the same amount of drug dissolving medium, but did not contain the compound of the present invention; the blank control group was the same as normal cells except that it did not contain cells.
  • the control group was the same.
  • the cell growth inhibition rate that is, the cytotoxicity of the compounds of the present invention, was judged according to the experimental results.
  • the formula for calculating the cell growth inhibition rate is:
  • Cell growth inhibition rate (%) ⁇ [(OD value of control well-OD value of blank well)-(OD value of administration well-OD value of blank well)]/[(OD value of control well-OD value of blank well] ⁇ 100%
  • Cell seeding Take Vero-E6 cells that are in good growth state and in logarithmic growth phase according to the cell instructions, aspirate and discard the culture medium, digest the cells with trypsin, prepare a cell suspension, and the cell count is 1 ⁇ 10 6 cells/ml ; Take 4 ml of the above cells, add 6 ml of culture medium, prepare a cell suspension with a cell density of 4 ⁇ 10 5 cells/ml, and inoculate it on a 96-well cell culture plate (100 ⁇ l of cell suspension per well, the number of cells is 1 ⁇ 10 4 ); cultured in a 5%CO 2 /37°C cell culture incubator until the cells grew completely adherent.
  • Dilution of the compound of the present invention The stock solution of the compound of the present invention was diluted with DMEM maintenance medium (2% FBS) to the working solution of the corresponding concentration.
  • the compounds of the invention were administered at concentrations of 10 ⁇ M or 20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 0 ⁇ M or 40 ⁇ M, 20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 0 ⁇ M in different experiments.
  • Pretreatment of the cells with the compound of the present invention Before SARS-CoV2 virus infection, the prepared working solution of the compound of the present invention was used to perform administration intervention operations at the action concentration of the compound of the present invention set in the experiment, and the cells were pretreated for 1 hour. .
  • the cell maintenance medium in this experiment was DMEM (containing 2% FBS). Three replicate wells were set up for each test, and the corresponding concentration of DMSO was used as a negative control, and Remdesivir was used as a positive control.
  • SARS-CoV2 virus dilution Take 200 ⁇ l of SARS-CoV2 virus, add it to 25ml of medium, mix well, and dilute SARS-CoV2 virus to 100TCID 50 /0.05mL.
  • the compound of the present invention + SARS-CoV2 virus-treated cells when SARS-CoV2 virus is infected, discard the cell culture supernatant, add 50 ⁇ l of the working solution of the compound of the present invention with 2 times the concentration of the compound of the present invention to each well, and at the same time, remove the cells Except for the control, drop 50 ⁇ l of SARS-CoV2 virus dilution solution vertically into each well, each The total well volume is 100 ⁇ l.
  • the detection primer is (5′ ⁇ 3′):
  • SARS-CoV-2-N-R CAGACATTTTGCTCTCAAGCTG
  • the compound of the present invention has no obvious toxicity to normal Vero E6 cells
  • the compounds of the present invention used in this experiment have no obvious cytotoxicity to experimental cells at the concentration of 20 ⁇ M, 10 ⁇ M and 5 ⁇ M.
  • the cell growth inhibition rates of the compound 3' of the present invention under the serial concentration gradients of 320 ⁇ M, 160 ⁇ M, 80 ⁇ M, 40 ⁇ M, 20 ⁇ M, 10 ⁇ M, 5 ⁇ M and 0 ⁇ M were 78.71%, 27.77%, 8.51%, 1.98%, 1.93%, 3.14%, 1.96%, 1.44%, the experimental result of cytotoxic CC 50 detection was 311.8 ⁇ M.
  • the compound of the present invention has a significant inhibitory effect on the level of viral RNA after the experimental cells are infected with SARS-CoV2 virus
  • the compound of the present invention was tested at the concentration used in the experiment (10 ⁇ M or 20 ⁇ M, 10 ⁇ M, 5 ⁇ M or 40 ⁇ M, 20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M) and at In the case of only treating the model cells for 1 hour, it has a clear and significant inhibitory effect on the SARS-CoV2 virus.
  • the inhibition rates of the compounds 7 and 8 of the present invention on the inhibitory effect of the SARS-CoV2 virus RNA are at 5 ⁇ M, 10 ⁇ M, and 20 ⁇ M.
  • the concentration can reach 62.99%, 93.13%, 94.42% and 68.08%, 89.10%, 91.74%, respectively.
  • Table 29 shows the effects of the compounds of the present invention on the level of viral RNA after the experimental cells are infected with SARS-CoV2 virus and the EC 50 values.
  • the berberine chloride compounds detected by the present invention include berberine chloride quaternary ammonium salt, palmatine chloride quaternary ammonium salt, coptisine chlorination quaternary ammonium salt, and isoplastine chloride quaternary ammonium salt and isoberberine chloride quaternary ammonium salt, neither can effectively reduce the level of SARS-CoV2 virus RNA.
  • the compounds of the present invention can dose-dependently inhibit the replication of SARS-CoV2 virus RNA in the Vero E6 cell infection model of SARS-CoV2 virus.
  • the detected compounds of the present invention 1, 3, 4, 6-10, 1'-3', and 5' have an effect on the level of viral RNA after the experimental cells are infected with the SARS-CoV2 virus.
  • the inhibition rates were 28.63%, 65.66%, 32.95%, 93.13%, 31.00%, 89.10%, 29.62%, 17.45%, 61.37%, 65.22%, 32.01%, 57.57%, respectively.
  • RAW264.7 cells were routinely cultured in vitro, made into a cell suspension, counted, adjusted to a cell concentration of 1.0 ⁇ 10 6 cells/mL, inoculated into a 6-well plate, 2 mL per well, placed in a 6-well plate. Cultured in an incubator at 37°C and 5% CO 2 , and after 24 hours, serum-free medium was added for starvation treatment.
  • the experimental groups were set as follows, including normal cell control group, LPS stimulation model group, specific concentration of the compound group of the present invention, and chlorine-treated cells.
  • the negative ion is the natural quaternary ammonium chloride group of coptisine chloride and the organic acid group corresponding to the counter anion of the compound of the present invention.
  • the BCA protein concentration determination kit first configure the BCA working solution. Subsequently, the BSA protein standard (5 mg/mL) was diluted with PBS to an initial concentration of 0.5 mg/mL, followed by a series of gradient concentrations (0.4, 0.3, 0.2, 0.15, 0.1, 0.05, 0 mg/mL), added to 96 In the well plate, 20 ⁇ L per well; Dilute the sample 20 times with PBS, add to the well, 20 ⁇ L per well; add 200 ⁇ L BCA working solution to each standard and sample well, incubate at 37 °C for 30 min, and measure the absorbance at 562 nm The protein content of each group was calculated according to the protein standard curve, and 4 ⁇ protein loading buffer and PBS were added to dilute the protein concentration of each group to 5 mg/mL. Boil at 100°C for 10 min to denature the protein, cool to room temperature and store at -80°C for later use.
  • the protein concentration take the same amount of protein for Western-blot detection: prepare 5% stacking gel and 10% separating gel according to standard SDS-PAGE method. In each group, an equal amount of protein, 50 ⁇ g each, was added to the channel, and electrophoresis was started after the sample was loaded. After electrophoresis, it was transferred to PVDF membrane by wet transfer method. The PVDF membrane was taken out and placed in 5% nonfat milk powder to block for 1 h at room temperature on a shaker. After the blocking was completed, the cells were washed with TBST for 3 ⁇ 10 min, incubated with the primary antibody (diluted 1:1000 with 5% BSA), and placed on a shaker at 4°C overnight. Elute with TBST for 3 ⁇ 10 min, incubate with secondary antibody (diluted 1:5000 in 5% nonfat milk powder), incubate with shaking at room temperature for 1 h, and elute with TBST for 3 ⁇ 10 min.
  • chemiluminometer for imaging: under the condition that the developer solution is configured in advance, open the development software to pre-cool. After the end, take out the PVDF membrane, add developer to evenly wet the membrane surface, run the software, select the appropriate exposure time to collect images, and use Image J software for grayscale analysis.
  • the compound of the present invention has a significant inhibitory effect on the high expression of iNOS at the cellular protein level. Compared with the corresponding natural berberine chloride quaternary ammonium salt and the organic acid corresponding to the counter anion of the compound of the present invention, the effect is stronger. For details, see Figure 7.
  • Experimental example 16 an experimental example of pharmacodynamic evaluation of the compound of the present invention in treating breast cancer
  • the experimental animals were female BALB/C mice with a weight range of 20 ⁇ 2g.
  • the protocols of all animal experiments were strictly implemented in accordance with the guidelines of the experimental animal ethics committee and approved by the ethics committee.
  • the experimental mice were raised in the experimental animal room in an environment that allowed free drinking water.
  • the room temperature of the experimental animal room was 22°C-25°C, the relative humidity was 55-65%, and the light cycle was 12h/12h.
  • the experiment was divided into a 4T1 subcutaneous transplanted tumor triple-negative breast cancer model group and the compound group of the present invention, with 8 animals in each group.
  • the tested compound of the present invention was Compound 8.
  • the test compound was formulated into a 4 mg/ml% 1 ⁇ PBS suspension for intragastric administration to experimental animals, and the administered dose of the test compound was 40 mg/kg (dose/animal body weight).
  • the administration volume is 1ml/100g (administration working solution volume/animal body weight) and the dose is administered according to the specific animal body weight.
  • the 4T1 cells cultured to the logarithmic growth phase in vitro were collected, the supernatant was discarded and washed twice with sterile PBS.
  • the cells were digested with sterile trypsin, and after the digestion was completed, the cells were terminated with RPMI1640 medium (10% FBS), and the cells were gently pipetted with a pipette to prepare a single-cell suspension.
  • the cell suspension was transferred to a 15ml sterile centrifuge tube and centrifuged at 1500rpm for 10min (4°C).
  • the cells were gently resuspended with sterile PBS and prepared to the corresponding concentration, and 1 ⁇ 10 7 cells/cell were inoculated into experimental animals subcutaneously for 4T1 subcutaneous transplantation tumor culture.
  • the animal was sacrificed by dislocation, sterilized with 75% EtOH, and the tumor tissue was isolated with sterile surgical instruments and reconstituted into a sterile homogenizer.
  • Cell suspension, and inoculated into experimental animals subcutaneously according to 1 ⁇ 10 7 cells/animal to conduct 4T1 subcutaneous transplanted tumor animal model modeling, grouping, and normal feeding.
  • the model group was given the corresponding amount of 1 ⁇ PBS without compound according to body weight, and the compound group of the present invention was given the 1 ⁇ PBS suspension of the present compound 8, once a day.
  • the experiment was administered for a total of 26 days.
  • the tumor volume of the experimental animal was measured and recorded periodically from the time when the outline of the experimental animal's tumor was clearly visible, and the animal was sacrificed by dislocation after the end of the experiment.
  • the subcutaneous tumor tissue of the animal was isolated, taken out and weighed, and the tumor inhibition rate of the compound of the present invention was calculated with reference to the tumor load of the animal in the model group.
  • the efficacy of the compounds against breast cancer was evaluated by evaluation indicators such as tumor inhibition rate after the experiment.
  • the compound 8 of the present invention has obvious anti-breast cancer activity.
  • the average tumor size of the 8 animals in the model group measured by vernier calipers was 19.54mm/15.04mm (length/width), while the compound 8
  • the average tumor size of 8 animals in 8 groups was 13.95mm/12.0mm (length/width), indicating that the compound had an inhibitory effect on tumor growth.
  • the average tumor burden of the model group was 2.139 g/a
  • the average tumor load of compound 8 was 1.094 g/a
  • the tumor inhibition rate was 48.85%.
  • the animals in this experiment showed a good tolerance to the test compounds, which was manifested by a phenotype in which the body weight of the animals showed a slight upward trend.
  • the normal cell control group contained cells cultured by the same operation and the same amount of drug dissolving medium, but did not contain the compound of the present invention; the blank control group was the same as the normal control group except that it did not contain cells. same.
  • 10 ⁇ L of CCK-8 solution was added to each well, and after 2 hours of incubation in the incubator, the optical density (OD) value was measured at 450 nm with a microplate reader. Survival was calculated using the following formula:
  • Experimental Example 18 The cytotoxicity evaluation experiment of the compound 2' of the present invention on normal human embryonic lung fibroblast HELF cells and mouse embryonic fibroblast NIH3T3 cells cultured in vitro.
  • This compound series concentration gradient solution was added to the 96-well plate, 100 ⁇ l per well, pipetting evenly, and then 100 ⁇ l were sucked and discarded to obtain a series of concentration gradients with concentrations of 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.01 ⁇ M, and 0.001 ⁇ M, respectively.
  • Three replicate wells were set for each administration concentration, and a normal cell control group and a blank control group were set at the same time. After 96h incubation in the incubator, the OD value of each compound was detected by MTT method, and the inhibition rate was calculated using the following formula:
  • Cell growth inhibition rate (%) ⁇ [(OD value of control well-OD value of blank well)-(OD value of administration well-OD value of blank well)]/[(OD value of control well-OD value of blank well] ⁇ 100%
  • the compound 2' of the present invention has no obvious toxicity to the growth of human embryonic lung fibroblasts HELF cells and mouse embryonic fibroblasts NIH3T3 cells, and is suitable for downstream activity screening experiments.
  • the Griess method was used to detect the effect of the compounds of the present invention on the NO secretion in the LPS-stimulated RAW264.7 cell inflammation model.
  • the 96-well plate was set up into experimental groups, including groups given the compounds of the present invention, LPS stimulation model group, normal cell control group and blank group. According to this experiment, in each group given the compound of the present invention, according to the experimental operation method (see “Experiment 3, the effect of the present compound on the growth of mouse mononuclear macrophage RAW264.7 cells cultured in vitro" ) to prepare each intervention experiment cell suspension containing the compound of the present invention, and control the total volume of each well to still be 100 ⁇ l. After the 96-well plate was pre-incubated in a cell culture incubator for 2 hours, LPS was added to each of the compound groups of the present invention and the model group for stimulation, and the amount of LPS added was 1 ⁇ g/mL.
  • Experimental example 20 Experimental example of the efficacy evaluation of the compound 2' of the present invention in reducing the phenotype of IL-6 pathological excess secretion level in the RAW 264.7 cell inflammation model induced by LPS stimulation
  • the ELISA method was used to detect the effect of the compounds of the present invention on the phenotype of IL-6 pathological excess secretion in the RAW264.7 cell inflammation model induced by LPS stimulation.
  • the cell supernatant sample of the LPS stimulation-induced RAW 264.7 cell inflammation model was prepared according to the operation procedure of "Experimental Example of Evaluating the Inhibitory Effect of NO Pathological Hyperproduction in the RAW 264.7 Cell Inflammation Model Induced by LPS Stimulation, and added to the mouse IL -6
  • the wells of the microtiter plate containing the target protein-specific monoclonal capture antibody provided in the ELISA kit operate according to the operating procedures provided in the kit instructions. content was measured.
  • Experimental example 21 Experimental example of the efficacy evaluation of the compound 2' of the present invention in reducing the level of IL-6 in model animals
  • Experimental example 22 Experimental example of evaluating the influence of the tested compound 2' of the present invention on autoantibodies in model mice
  • the experimental operation of this experiment is the same as that in "(4)
  • the test compound of the present invention has the same effect on the mouse chicken type II collagen-induced arthritis model in "Experimental Example 12: Evaluation of the anti-rheumatoid arthritis pharmacodynamics of the compound of the present invention” Effects of Autoantibody Levels in Mice”.
  • the experimental results are shown in Table 36. It can be seen from the results that compared with the model group, the anti-chicken type II collagen IgG antibody concentration in the serum of the mice in the positive drug methotrexate group and the compound 2' administration group of the present invention was significantly reduced, and the compound 2' administration of the present invention was significantly lower.
  • the reducing effect of the anti-chicken type II collagen IgG antibody level in the drug group was better than that in the experimental group administered according to the specific characteristics of the positive drug, and there was a very significant difference compared with the model group.
  • Table 36 The effect of the compound 2' of the present invention on the level of anti-chicken type II collagen IgG antibody in the serum of mouse chicken type II collagen-induced arthritis model mice.
  • Experimental Example 23 Experimental Example of Efficacy Evaluation of Compound 2' of the Present Invention in Raising Hemoglobin Content in Model Animals
  • the compound 2' administration group of the present invention also has a significant increase in hemoglobin, indicating that the compound of the present invention has a significant effect of increasing hemoglobin.
  • the average hemoglobin content of the positive drug group was 160.92 ⁇ 7.07(g/L).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Obesity (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Urology & Nephrology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明属于医药技术领域，公开了可溶性小璧碱型生物碱季铉盐化合物及其制备药物的用途。具体而言，本发明公开了如通式A所示且可以进一步如通式I和I，具体所示的化合物，其药物组合物，制备方法和用途。所述的本发明化合物具有显著的通过抑制/降低生物机体病理性NO、iNOS、IL-6和自身抗体水平升高的活性以及抑制SARS-C0V2病毒RNA复制的活性而治疗心脑血管和血液系统疾病、炎症病、发热病、肿瘤病、自身免疫性疾病和病毒感染病及其并发症的活性。本发明化合物的理化性质和药理作用强度优于对照药及有关化合物，能用于制备预防、缓解和/或治疗心脑血管和血液系统疾病、炎症病、发热病、肿瘤病、自身免疫性疾病和病毒感染病及其并发症的药物。

Description

可溶性小檗碱型生物碱季铵盐及其制备药物的用途 技术领域
本发明属于医药技术领域，具体涉及一类溶解性能更适合制药用途的小檗碱型生物碱季铵盐化合物、其制备方法、含有该类化合物的药物组合物，以及该类化合物和其药物组合物制备药物的用途。
背景技术
天然小檗碱季铵盐型化合物是一类平衡阴离子为氯负离子的苯环上连有不同取代基的异喹啉并[3,2-a]异喹啉之5,6-二氢-7-鎓离子季铵盐型化合物，是药物化学领域非常认可和关注的一类“梦分子”或“梦化合物”。然而，这些以没有任何生物活性的氯负离子为平衡阴离子的天然小檗碱型季铵盐化合物同时又具有显著的缺陷，就是这些化合物的理化性能不适合开发药物。这类化合物中有些溶解性很差，不适合经包括血管系统在内的大多数给药途径给药，而经肠道给药又存在明显的生物利用度差的不足。例如，对于氯化黄连碱季铵盐，实验小鼠以30mg/kg、75mg/kg和150mg/kg的剂量口服给药后，生物利用度仅分别为1.87％、0.82％和0.52％。但当考虑治疗肠道疾病时，有些化合物在溶剂中的溶解度又过大，对于希望通过减小在小肠部位吸收同时增加在结肠部位药物浓度的方式(即通过增加药物选择性分布于肠组织的方式)而选择性作用于肠道部位疾病的药物而言，又需要调节其理化性质，使其在溶剂中的溶解度降低。例如，对于氯化巴马汀季铵盐，由于其溶解性相对较好，在开发治疗肠道疾病的药物时，就需要减小其溶解性。天然小檗碱季铵盐型化合物的溶解性和药代动力学性质需要通过药物化学的方式进行改善，根据具体治疗的疾病的特点，一些化合物需要提高溶解性，而另一些化合物又需要降低溶解性，这样才能更好地应用于药物开发。
白细胞介素-6(interleukin-6,IL-6)属白细胞介素类生物化学成分，是一种激素类糖蛋白。IL-6于1980年最早作为B淋巴细胞分化因子被发现并被命名为β2干扰素。其后，又经历了作为各种细胞因子而不断地被发现和被命名的研究过程，直到将这些细胞因子的基因克隆出来后，才最终将其命名为IL-6。人IL-6 基因定位于第七号染色体上，1997年，IL-6的三维结构经X射线晶体衍射分析得以确定，是由A-D四个呈拓扑结构的螺旋束组成，并在四个螺旋束之外有一个小螺旋-E螺旋，其中A螺旋和B螺旋呈同一走向，而C螺旋和D螺旋与前两者趋向相反。
IL-6是一种Th-2型强效多向性免疫调节细胞因子，多种细胞如巨噬细胞、树突状细胞、淋巴细胞、肥大细胞、成纤维细胞、单核细胞、血管内皮细胞、肾小球系膜细胞及若干肿瘤细胞等以及多种机制都可产生IL-6。IL-6有其多种正常的生理功能，比如，刺激急性期蛋白生成并诱导急性期反应以对抗或清除病原体。但是，其通过各种机制形成异常过度分泌又是机体急性炎症反应和自身免疫性疾病的病理活动的重要的表型，是确定特异性和系统性炎症反应综合征患者疾病严重程度最重要的细胞因子之一，也就是说IL-6病理性过量分泌的水平可以反映机体炎症反应、自身免疫性反应及其各种关联疾病的严重程度。而炎症和自身免疫性疾病又在多种其他疾病中扮演着关键的角色，包括血细胞生成、肿瘤发生以及病毒感染等。首先，炎症在心血管不良事件中扮演着关键的角色，IL-6水平表型与冠状动脉病变Gensini评分呈正相关，与病情严重程度有密切关系，并与动脉粥样硬化症、冠心病和贫血症等心血管和血液系统疾病均密切相关；IL-6还通过多种途径参与妊娠期高血压疾病病理过程。其次，IL-6是多数肿瘤病进展的重要调节因子，一些肿瘤病患者血清或组织IL-6水平经常升高，IL-6高表达预示着不良的临床结局。第三，IL-6高表达表型与呼吸系统疾病有密切的关系，如特发性肺纤维化患者支气管肺泡灌洗液中IL-6水平明显升高；过敏性哮喘患者，其肺泡巨噬细胞培养上清液中IL-6含量也明显增高；同时IL-6高表达表型也与肺炎患者呼吸困难表现呈正相关，从病理生理学的角度来看，这代表了肺部炎症反应的强度，并与呼吸系统疾病的全身性严重程度有关。多种病毒感染病具有突出的IL-6高表达表型，例如在感染包括2009年H1N1大流行性流感在内的流感病毒的患者中，发现肺和血清中IL-6作为针对病毒感染病的一个重要的保护机制的水平显著升高的表型。冠状病毒是常见的引起呼吸道疾病的病原体，会导致IL-6等细胞因子的显著升高，产生细胞因子风暴，在肺部募集免疫应答细胞。例如，2019年底至今在全球范围内爆发的2019新冠状病毒病(Coronavirus disease of 2019,COVID-19)2019新型冠状病毒(2019 novel coronavirus或SARS-CoV2，也 称为2019-nCoV病毒)感染后T淋巴细胞和单核细胞大量产生IL-6，炎症进展迅速，并发挥免疫破坏作用(自身免疫)，导致严重肺功能障碍；2003年初爆发的严重急性呼吸综合症(Severe acute respiratory syndrome,SARS)SARS相关冠状病毒(Severe acute respiratory syndrome-associated coronavirus,SARS-CoV)感染会导致IL-6显著升高的表型，通过产生细胞因子风暴、在肺部募集免疫应答细胞等造成急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)，甚至在后期出现肺纤维化。SARS-CoV2病毒感染患者的临床分型分为轻型、普通型、重型和危重型，对其血清中炎性细胞因子进行检测，发现各型之间IL-6在表达水平上差异有统计学意义，重症SARS-CoV2病毒肺炎患者的IL-6水平明显高于轻症患者，表明IL-6高表达是SARS-CoV2及其变异株感染诱发机体产生细胞因子风暴的重要表型和环节。综合实验数据表明冠状病毒诱导的IL-6高表达表型在冠状病毒感染的肺炎发病机制中起重要作用，或者说是在SARS-CoV2病毒性肺炎病的发展过程中与病情严重程度密切关联的重要表型，尤其是在炎症和发热方面。此外，IL-6高表达表型还与肾脏疾病有关，如原发性肾小球肾炎(IgAN)患者尿及血清中IL-6均显著增高。IL-6的受体是细胞表面的IL-6R。IL-6与受体结合后引发IL-6Rα与gp130的二聚化，导致下游信号通路被激活，包括JAK/STAT、Ras/MAPK以及PI3K-PKB/AKT信号通路。Ras(Rho)/MAPK/GATA3/IL-6信号机制是IL-6异常过度分泌的重要的信号机制之一。因此，可以将IL-6高表达表型作为治疗炎症病、发热病、心脑血管和血液系统疾病、肿瘤病、原发性肾小球肾炎、病毒感染病及其并发症等疾病的靶点，进行药物的开发、试验及应用。
一氧化氮(NO)是一种机体内普遍存在的信号分子，其可以由三种一氧化氮合酶(NOS)产生，包括主要分布在血管内皮细胞中的内皮型NOS(eNOS)、主要分布在神经组织中的神经元型NOS(nNOS)和主要分布在巨噬细胞、肥大细胞、中性粒细胞和肿瘤细胞等中的诱导型NOS(iNOS)。广义上，eNOS和nNOS又合称为构成型NOS(cNOS)，其产生相对少量的NO，其中，eNOS合成的NO参与血压和血流的调节，nNOS产生的NO作为神经递质参与脑发育、学习和记忆过程。iNOS通常呈沉默状态，但能被炎性细胞因子、脂多糖等诱导，产生大量甚至过量大量的NO，而且持续存在的时间相对较长。NO通过不同的机制在机体内既发挥重要的生理作用又产生多种病理作用；其生理性调节对支持生物有 机体中很多生理过程至关重要，如免疫调节等；但在一些病理条件下，如各种炎症、癌症等，NO和iNOS均过量表达，例如，在原发性肺癌中NO及iNOS显著过量高表达，而iNOS的过量上调与乳腺癌患者的生存率降低相关联，等。
自身免疫性疾病是目前临床上最常见的难治性疾病之一，是在病患的情况下人体免疫系统不对“自我”与“非我”进行分辨而对自身组织的攻击，其病因还不清楚。自身免疫性疾病可分为两大类：器官特异性自身免疫病和系统性自身免疫病，通常在病灶部位均聚集过量大量的炎细胞，患者病灶部位的体液中的NO的含量也明显过量高于其血中NO的含量，并且自身免疫性疾病患者血中的NO含量本身就高于正常人血中NO的含量，导致肿胀和疼痛。除了病灶部位，自身免疫性疾病，特别是系统性自身免疫性疾病，还可以影响身体的任何系统，并发多种疾病，通常也涉及自身抗体的形成。除了病灶部位，自身免疫性疾病还可以影响身体的任何系统，并发多种疾病。自身免疫性疾病的病理学核心是炎症，但其在心血管疾病的发展中也起着重要作用。淋巴细胞的生理功能及其在自身免疫性疾病的病理状态下对血液系统的影响、自身免疫性疾病病理状态下的心脑血管和血液系统疾病的形成及其特点都可以用类风湿性关节炎作为范例予以说明。
类风湿性关节炎(Rheumatoid arthritis,RA)是临床上最常见的自身免疫性疾病之一，症状特点主要是炎症性关节炎性病变，其发病具有慢性和全身性特征，病因不明，基本病理表现为滑膜炎和关节腔内血管翳形成，炎性滑膜及滑液中聚集了大量炎细胞，包括淋巴细胞(T细胞和B细胞)、单核/巨噬细胞等，导致肿胀、疼痛和僵硬，如同其他自身免疫性疾病一样，通常也涉及自身抗体的形成，如类风湿因子(RF)或抗瓜氨酸蛋白抗体。作为慢性全身性炎症性疾病，持续的炎症会导致所有受累关节的关节软骨和骨破坏损伤、畸形和功能丧失，尤其是手足部小关节。并且，类风湿性关节炎作为一种系统性自身免疫性炎症性疾病，除了关节外，还可以影响身体的任何系统，并发肺部疾病、恶性肿瘤及抑郁症。如上所述，类风湿性关节炎免疫病理学的核心部分就是炎症，其在心血管疾病的发展中同样也起着重要作用。淋巴细胞在生理状态下是机体免疫应答功能的重要细胞，具有抵御感染、监视并清除异源性物质(包括癌变组织及坏死组织)、保护机体稳定状态的功能；但在某些病理因子刺激下，这些生理功能失控，导致淋巴细胞功能亢进，产生造血负调控因子，对患者骨髓克隆的形成有抑制作用并损伤 正常组织，形成各类自身免疫性炎症性疾病和血液系统疾病，如贫血等。贫血症主要表现为身体无法制造足够的血红蛋白，不能将氧输送到身体各个组织。贫血也是类风湿性关节炎症最常见的关节外症状之一，最常见的贫血类型是慢性病性贫血，其发病机制未完全明确；因此，类风湿性关节炎患者患大多数类型心血管疾病的风险都会增加，例如，炎症与患动脉粥样硬化有密切的关联，动脉粥样硬化导致缺血性心脏病，导致引起舒张机能障碍的纤维化性心肌损伤。类风湿性关节炎患者关节液中NO的含量就明显高于患者血中NO的含量，尽管类风湿性关节炎患者血液中NO的含量本身高于正常人血液中NO的含量。总的来说，类风湿性关节炎患者的心血管疾病死亡率比普通人群增加了约50％，并且与普通人群相比，类风湿性关节炎患者的心血管疾病似乎发生在更年轻的年龄。类风湿性关节炎还具有反复发作、致残率高的特点，患者在病程1～5年、5～10年、10～15年及≥15年的致残率分别为18.6％、43.5％、48.1％和61.3％，即随着病程的延长，残疾及功能受限发生率升高，严重影响患者的生活质量和工作能力，给家庭和社会带来沉重的负担。类风湿性关节炎可发生于任何年龄段，其发病率和患病率随不同地区和时间而有所不同，流行病学调查显示其全球发病率为0.5％～1％，中国大陆地区发病率为0.42％，65岁以上人群发病率显著升高。2005年美国类风湿性关节炎发病率是0.409％，患病率约0.72％，平均患病年龄约55.6岁，女性患者以新发病例69％的发病率多于男性。也有调查表明男女患病比例约为1:4。
类风湿性关节炎至今无法根治，其临床治疗原则为早期治疗、规范治疗、定期监测与随访。目前，临床上治疗类风湿性关节炎的药物主要分类为非甾体抗炎药(NSAIDs)、改善病情的抗风湿药(DMARDs)、糖皮质激素和生物制剂。甲氨蝶呤是改善病情的抗风湿药，是类风湿性关节炎治疗的锚定药；甲氨蝶呤在欧美国家的平均使用率达到83％，远高于其他药物，中国甲氨蝶呤的使用率低于欧美国家，为55.9％。一般情况下，2/3的类风湿性关节炎患者单用甲氨蝶呤，或与其他传统合成DMARDs联用，可以达到疾病缓解或低疾病活动度治疗目标，但无法达到根治。甲氨蝶呤的不良反应与剂量呈正相关，只有在小剂量使用甲氨蝶呤(≤10mg/周)时才可以达到不良反应轻和长期耐受性较好的情况。对有甲氨蝶呤禁忌者，来氟米特和柳氮磺吡啶单用的疗效和安全性与甲氨蝶呤相当，但需注意柳氮磺吡啶升高IL-6的副作用。国际上来氟米特的使用率位于甲氨蝶呤、 柳氮磺吡啶、羟氯喹之后，为21％；但在中国的使用率仅次于甲氨蝶呤，为45.9％，在部分地区的使用率甚至超过甲氨蝶呤。中国类风湿性关节炎患者使用柳氮磺吡啶治疗在使用率方面仅为4.4％，远低于其他国家的43％，且绝大部分为联用其他传统合成DMARDs。羟氯喹在中国的使用率为30.4％，在国际上的使用率则略高。中国羟氯喹的使用以联合用药为主，占95％。经甲氨蝶呤、来氟米特或柳氮磺吡啶等单药规范治疗仍未满足依从性者，就需要联合用药。对于中/高疾病活动度的类风湿性关节炎患者需要传统合成DMARDs联合糖皮质激素治疗，但目的只是快速控制症状，不能单用或长期大剂量使用糖皮质激素，否则，糖皮质激素会产生严重的毒副作用。
虽然目前临床上对类风湿性关节炎有上述多种治疗方案，但所有方案的依从性均不能满足医者和患者的期望，更不能彻底治愈该病，因此，本质上属于姑息疗法，导致当前该病对患者的日常生活、工作能力以及与健康相关的生活质量产生严重的负面影响，致残率高，并增加了死亡率。此外，所有的抗类风湿性关节炎药物均存在一定的毒副作用，例如，绝大多数NSAIDs的毒副作用是对消化系统的损害，TNF-α阻断剂有发生严重感染的风险。这突出表明需要进一步加强抗类风湿性关节炎治疗法的研究，包括具有优效性的新的生物药和小分子化疗药物的发现。
各种恶性肿瘤(癌症)都是严重危害人类健康的重大疾病，目前对人类各种癌症的预防、缓解及治疗仍是医药科学研究领域必须面对的一项十分艰巨的科研工作；显然能用高效且安全的药物治疗癌症是比较理想的手段之一。因此，寻找对各种恶性肿瘤具有高效的治疗作用并具有低毒性甚至无毒性特点的特异性化学药物在医药技术领域具有重要的意义。目前科学家已发现了一些有一定疗效的抗肿瘤药物，正是这些药物使急性白血病患儿的平均生存期由过去的2～3个月延长到5年以上，使不少晚期肿瘤病人的生命明显延长。当然，这并不意味着抗肿瘤药物的研究不再需要发展，相反，目前它面临严峻的挑战，这就是多数常见实体瘤如肺癌、肝癌、结直肠癌、乳腺癌及胰腺癌等还缺乏有效的治疗药物，不少抗肿瘤药物在临床应用过程中产生耐药性和严重毒性。也就是说，截止目前，还没有特别高疗效低毒性的抗癌药物问世。因此，在目前恶性肿瘤发病呈明显上升趋势的情况下(见文献：杨玲，等.中国2000年至2005年恶性肿瘤发病死亡的估计与预测，中国卫生统计，2005，22(4)：218-221，231)寻找高疗效低毒 性的抗癌新药一直是药物研究领域的重要课题。
本发明在性能研究中意外地发现改善了天然小檗碱型生物碱季铵盐型化合物的理化性能，并在此基础上开展了生物学评价。本发明根据医学界和临床上对免疫调节剂药物、抗自身免疫性疾病药物、抗病毒感染及其并发症药物、抗炎解热药物、抗心脑血管和血液系统疾病药物以及抗肿瘤疾病药物的客观需求，也包括协同抗病毒、解热和抗炎的多重药效作用治疗病毒性感染病及其并发症的理念，结合IL-6水平、NO水平、iNOS水平和IgG抗体水平等在临床疾病及其治疗方面的作用，对本发明化合物开展了合成、在药理模型上对IL-6、NO、iNOS、IgG抗体水平抑制效力的评价、在体外实验中抑制SARS-CoV2病毒RNA复制的效力评价、对肿瘤细胞生长的抑制作用评价、在动物模型上对模型动物血红蛋白水平影响的评价、对发热病理模型动物的解热作用评价、对类风湿性关节炎模型动物关节炎症状严重程度的改善作用评价以及抗肿瘤作用动物实验评价，还包括本发明化合物与氯化小檗碱型生物碱季铵盐类化合物底物的药理效应和理化性质的比较。结果表明，本发明化合物在药理实验中显示出显著且独特的剂量依赖性地降低药理模型中IL-6、NO、iNOS和IgG水平的效力和药理学效应，在细胞实验中可以剂量依赖性地通过抑制冠状病毒RNA复制的机制而抑制SARS-CoV2病毒复制、剂量依赖性地且选择性地抑制肿瘤细胞的生长；在动物实验中能显著降低酵母致大鼠发热模型动物的体温、显著升高鸡II型胶原蛋白诱导的病理模型动物外周血中血红蛋白的含量、显著减轻模型动物类风湿性关节炎的症状严重程度、对肿瘤动物模型具有显著的治疗作用。本发明化合物的理化性质和药理效应明显优于氯化小檗碱型生物碱季铵盐类化合物底物。通过大量实质性的研究工作，包括对化合物的安全性评价，明确了一类在制备预防、缓解和/或治疗贫血病、病毒感染病及其并发症、炎症病、发热病、自身免疫性疾病、肿瘤病等病症药物中具有显著应用价值的化合物。
发明内容
本发明解决的技术问题是借助化学合成和药物筛选等手段提供一类以各种吡啶甲酸根或取代吡啶甲酸根类酸根或2-甲基-N1-氧化吡嗪-5-甲酸根为平衡阴离子、以苯环上连有不同取代基的异喹啉并[3,2-a]异喹啉之5,6-二氢-7-鎓离子季铵盐型阳离子为碱基平衡阳离子的在水和醇水混合溶剂中的溶解性能从具体的 药物开发方面考虑显著优于氯化小檗碱型生物碱季铵盐底物且具有显著的通过平衡机体免疫功能而预防、缓解和/或治疗引起生物机体分泌/形成IL-6和/或NO和/或/IgG抗体病理性升高的相关疾病以及预防、缓解和/或治疗各种原因引起的低血红蛋白血症性贫血病、自身免疫性疾病、病毒感染病及其并发症、炎症病、发热病、肿瘤病的活性化合物，即当X选自C时则Y选自H、当X选自N+时则Y选自O-的通式A所示的可溶性小檗碱型生物碱季铵盐化合物。
Figure PCTCN2022080897-desc-000001-original
为解决上述技术问题，本发明提供了如下技术方案：
本发明第一方面提供了包含在通式A中的如通式I所示的作为本发明化合物的溶解性能显著改善的且属于可溶性的小檗碱型生物碱吡啶甲酸类季铵盐化合物和同样包含在通式A中的如通式I'所示的作为本发明化合物的可溶性小檗碱型生物碱2-甲基-N1-氧化吡嗪-5-甲酸季铵盐化合物。
Figure PCTCN2022080897-desc-000002-original
本发明第二方面提供了如通式I和通式I'所示的小檗碱型生物碱吡啶甲酸类季铵盐化合物和小檗碱型生物碱2-甲基-N1-氧化吡嗪-5-甲酸季铵盐化合物的制备方法。
本发明第三方面提供了如通式I和通式I'所示的小檗碱型生物碱吡啶甲酸类季铵盐化合物和小檗碱型生物碱2-甲基-N1-氧化吡嗪-5-甲酸季铵盐化合物的产品组合物，所述的产品选自药物。
本发明第四方面提供了本发明化合物和药物组合物在制备药物产品中的应 用，包括在制备具有促免疫效应细胞增殖、抑制/降低生物机体病理性NO过量生成、IL-6和自身抗体水平过量升高活性的免疫调节剂药物中的应用，并基于独立的应用，也包括在制备预防、缓解和/或治疗引起生物机体病理性分泌IL-6过量升高的疾病的药物中的应用，在制备预防、缓解和/或治疗引起生物机体病理性分泌NO过量升高的疾病的药物中的应用和在制备预防、缓解和/或治疗引起生物机体病理性形成自身抗体过量升高的疾病的药物中的应用，在制备预防、缓解和/或治疗需要通过抑制病毒RNA达到治疗效果的疾病的药物中的应用。
本发明第五方面提供了本发明化合物和药物组合物在制备预防、缓解和/或治疗各种原因引起的心脑血管和血液系统疾病、自身免疫性疾病、病毒感染病及其并发症、炎症病、发热病、肿瘤病的药物中的应用。其中，自身免疫性疾病包括类风湿性关节炎，心脑血管和血液系统疾病包括低血红蛋白血症性贫血病，病毒感染病及其并发症包括冠状病毒感染病及其并发症，冠状病毒感染病及其并发症包括2019新型冠状病毒SARS-CoV2及其变异株感染病及其并发症，肿瘤病包括乳腺癌、结直肠癌和肺癌。
具体而言，本发明第一方面提供了通式A所示的可溶性小檗碱型生物碱季铵盐化合物：
Figure PCTCN2022080897-desc-000003-original
当X选自C时则Y选自H，当X选自N+时则Y选自O-；
当X选自C并且Y选自H时，
COO-选自取代于吡啶环的2位或3位或4位或5位或6位；
R独立地选自氢、氨基、取代或未取代的羟基、烷基、卤素；
R为单取代或多取代；
R为单取代时，与COO-的位置相协调地形成吡啶环的二取代，R可选自吡啶环的3位或4位或5位或6位或2位；
R为多取代时选自二取代或三取代或四取代，并与COO-相协调地形成按数学列举法给出的各种取代模式；
当X选自N+并且Y选自O-时，
R选自取代于吡嗪环2位的甲基，COO-选自取代于吡嗪环的5位；
R2、R3各自独立地选自H，取代或未取代的羟基，或R2与R3连接成为亚烃基二氧基；
R9、R10、R11各自独立地选自H、取代或未取代的羟基，或者R9与R10连接成为亚烃基二氧基而R11独立地选自H、取代或未取代的羟基，或者R10与R11连接成为亚烃基二氧基而R9独立地选自H、取代或未取代的羟基；
所述的取代或未取代的羟基中的取代基选自甲基、乙基；所述的烷基选自甲基、乙基；所述的卤素选自氟、氯、溴；所述的亚烃基二氧基选自亚甲二氧基。
优选的，所述的化合物为通式I所示的可溶性小檗碱型生物碱吡啶甲酸类季铵盐化合物，化学结构通式如下式I所示：
Figure PCTCN2022080897-desc-000004-original
在式I中，COO-选自取代于吡啶环的2位或3位或4位或5位或6位；R独立地选自氢(H)、氨基、羟基、甲氧基、乙氧基、甲基、乙基、氟(F)、氯(Cl)、溴(Br)；
R为单取代或多取代；
R为单取代时，与COO-的位置相协调形成R:COO-二取代，R可选自吡啶环的3位或4位或5位或6位或2位；
R为多取代时选自二取代或三取代或四取代，并与COO-相协调形成按数学列举法给出的各种取代模式；
R2、R3各自独立地选自H、取代或未取代OH，或R2与R3连接成为亚烃基二氧基；进一步的，R2、R3中所述的取代或未取代的羟基中的取代基选自甲基和乙基；R2、R3中所述的亚烃基二氧基选自亚甲二氧基。
R9、R10、R11各自独立地选自H、取代或未取代的OH，或者R9与R10连接成为亚烃基二氧基而R11独立地选自H、取代或未取代的OH，或者R10与R11连接成为亚烃基二氧基而R9独立地选自H、取代或未取代的OH。进一步的， R9、R10、R11中所述的取代或未取代的OH中的取代基选自甲基和乙基；R9、R10、R11中所述的亚烃基二氧基选自亚甲二氧基。
优选的，所述的化合物为通式I'所示的小檗碱型生物碱2-甲基-N1-氧化吡嗪-5-甲酸季铵盐化合物，化学结构通式如下式I'所示：
Figure PCTCN2022080897-desc-000005-original
在式I'中，R2、R3各自独立地选自H，取代或未取代的OH，或R2与R3连接成为亚烃基二氧基；
R9、R10、R11各自独立地选自H、取代或未取代的OH，或者R9与R10连接成为亚烃基二氧基而R11独立地选自H、取代或未取代的OH，或者R10与R11连接成为亚烃基二氧基而R9独立地选自H、取代或未取代的OH；
所述的取代或未取代的OH中的取代基选自甲基、乙基；所述的亚烃基二氧基选自亚甲二氧基。
本发明最优选的可溶性小檗碱型生物碱季铵盐化合物选自如下化合物群组中化合物1-47和化合物1'-5'：
Figure PCTCN2022080897-desc-000006-original
Figure PCTCN2022080897-desc-000007-original
Figure PCTCN2022080897-desc-000008-original
Figure PCTCN2022080897-desc-000009-original
Figure PCTCN2022080897-desc-000010-original
本发明第二方面提供了本发明可溶性小檗碱型生物碱季铵盐化合物的制备方法。
所述的可溶性小檗碱型生物碱季铵盐类化合物可通过如下合成路线通式合成(具体合成条件见实施例)：
Figure PCTCN2022080897-desc-000011-original
合成步骤：(a)将小檗碱型生物碱季铵盐类化合物与丙酮和氢氧化钠水溶液反应得到固体8-丙酮基二氢小檗碱型化合物。(b)所得固体8-丙酮基二氢小檗碱型化合物再与吡啶甲酸类化合物或2-甲基-N1-氧化吡嗪-5-甲酸在四氢呋喃与水的混合溶剂中在加热的条件下反应，经对反应混合液过滤，得本发明可溶性小檗碱型生物碱季铵盐化合物。
本发明第三方面提供了以本发明第一方面所述可溶性小檗碱型生物碱季铵盐化合物作为活性成份的药物组合物。鉴于本发明化合物的适合多种给药途径的 理化性质，这些药物组合物可根据公知的药物组合物的制备方法进行制备。可通过将本发明化合物与一种或多种药学领域上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。本发明化合物在其药物组合物中的含量通常为0.1-99.9％(W/W)。
本发明化合物或含有本发明化合物的药物组合物可以单位剂量形式给药，给药途径可主要为消化道给药，如口服给药、肠道给药，等。但鉴于一些本发明化合物的理化性质适合非肠道给药，因此经非肠道给药的形式也可接受，如静脉注射、肌肉注射、皮下注射、腹腔注射，以及鼻腔、口腔粘膜、眼、肺和呼吸道、阴道给药，涂布于皮肤，等等。其中，在治疗贫血症、类风湿性关节炎病、病毒感染病及其并发症、炎症病、发热病、肿瘤病的应用中，其突出的优势是既可以制成普通口服制剂形式如普通片剂和普通胶囊剂经口服直接给药而无需特殊处理(使用非常方便)的剂型，也可以制成其他剂型制剂，包括注射剂，而采用其他各种给药途径和方式。
口服给药或其他途径给药也可以采用其他给药剂型，包括采用新技术制备的各种液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括O/W型、W/O型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、乳膏型、凝胶剂、糊剂等。
本发明化合物可以制成普通制剂，也可制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将本发明化合物制成片剂，可以广泛使用相关领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；润湿剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙 基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将本发明化合物与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中；也可将本发明化合物先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明化合物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明化合物的胶囊剂。
为将本发明化合物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量药学领域常用的增溶剂、助溶剂、pH调节剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调节剂可以是磷酸盐、醋酸盐、盐酸盐、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如果在剂型方面有特殊需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其它添加剂。
为达到药用目的，增强治疗效果，本发明的化合物(药物)或药物组合物可用任何公知的给药方法和应用方式给药和使用。
本发明化合物或药物组合物的给药(应用)或用药(使用)剂量依据所要预防、缓解和/或治疗的各种原因引起的心脑血管和血液系统疾病、自身免疫性疾病、病毒感染病及其并发症、炎症病、发热病、肿瘤病的严重程度，患者或动物的个体情况，给药(应用)途径和剂型等可以有大范围的变化。一般来讲，本发明化合物的每天的合适剂量范围为0.001-500mg/kg(剂量/体重)，优选为0.1-150mg/kg(剂量/体重)，更优选为1-100mg/kg(剂量/体重)，最优选为1-50mg/kg(剂量/体重)。上述剂量可以一个剂量单位或分成几个剂量单位给药，这取决于医生的临床经验和治疗的进展以及包括运用其它治疗(应用)手段的给药(使用)方案。
本发明的化合物或产品(药物)组合物可单独服用，或与其他治疗药物或对 症药物合并使用。当本发明的化合物与其它治疗药物存在协同作用时，应根据实际情况调整它的剂量。
本发明第四方面提供了本发明化合物和药物组合物在制备药物产品中的应用，包括在制备具有抑制/降低机体病理性NO过量生成、iNOS过高水平表达、IL-6和自身抗体水平过量升高活性的免疫调节剂药物中的应用，在制备具有抑制病毒RNA复制活性的抗病毒药物中的应用，并基于独立的应用，也包括在制备预防、缓解和/或治疗引起生物机体病理性分泌IL-6过量升高的疾病的药物中的应用，在制备预防、缓解和/或治疗引起生物机体病理性分泌NO过量升高的疾病的药物中的应用和在制备预防、缓解和/或治疗引起生物机体病理性形成自身抗体过量的疾病的药物中的应用。本发明的第四方面是基于具体的药理实验结果提出的，本发明化合物具有显著的免疫调节作用，在药理实验中，对细胞炎症模型NO的病理性过量生成显示明显的抑制作用，对iNOS过高水平表达显示出显著的抑制作用、对肿瘤细胞显示出显著的生长抑制作用、具有显著降低病理性高IL-6水平药理模型中IL-6水平的药理学效应，显示显著降低病理性高IgG抗体水平模型动物血清中IgG抗体水平的药理学效应。
本发明第五方面提供了本发明化合物和药物组合物在制备预防、缓解和/或治疗各种原因引起的心脑血管和血液系统疾病、自身免疫性疾病、病毒感染病及其并发症、炎症病、发热病、肿瘤病的药物中的应用。其中，自身免疫性疾病包括类风湿性关节炎，心脑血管和血液系统疾病包括低血红蛋白血症性贫血病，病毒感染病及其并发症包括冠状病毒感染病及其并发症，冠状病毒感染病及其并发症包括2019新型冠状病毒SARS-CoV2感染病及其并发症，肿瘤病包括乳腺癌、结直肠癌和肺癌。本发明的第五方面同样是基于具体的药理实验结果提出的。除了上述药理实验结果，在药理实验中，本发明化合物还可以剂量依赖性地抑制2019新型冠状病毒SARS-CoV2RNA的复制、剂量依赖性地且选择性地抑制人肺癌细胞A549细胞和人结肠癌细胞HCT-116细胞的生长，显著降低病理性发热模型动物的体温，显著升高模型动物外周血中血红蛋白的含量、显著减轻模型动物的类风湿性关节炎症状的严重程度、对肿瘤动物模型具有显著的治疗作用。。
在上述本发明的第四和第五方面，优选的自身免疫性疾病包括类风湿性关节炎，心脑血管和血液系统疾病包括低血红蛋白血症性贫血病，肿瘤病包括乳腺癌、结直肠癌和肺癌，病毒感染病包括冠状病毒感染病及其并发症，特别是2019新 型冠状病毒SARS-CoV2及其变异株感染病及其并发症。
有益技术效果
本发明化合物具有独特的平衡机体免疫功能的药理作用机制，在药理实验中，对细胞炎症模型NO的病理性过量生成和iNOS的病理性高水平表达显示显著的抑制作用，在细胞水平和动物水平上均具有显著降低病理性高IL-6水平药理模型中IL-6表达量的药理学效应，还显示显著降低病理性高IgG抗体水平模型动物血清中IgG抗体水平的药理学效应。本发明化合物是由两部分结构单元以离子键形式结合后形成的化合物，数据表明与以无生物活性的氯负离子为平衡阴离子的天然小檗碱型生物碱季铵盐类化合物比较，理化性质获得了明显的改善，具有独特的活性作用特点。本发明化合物作为作用于Rho(Ras)/MAPK/GATA3/IL-6信号传导通路的IL-6信号机制抑制剂和病毒RNA合成相关过程抑制剂，还可以剂量依赖性地抑制2019新型冠状病毒SARS-CoV2的复制、剂量依赖性地且选择性地抑制人肺癌细胞A549和人结肠癌细胞HCT-116等肿瘤细胞的生长，显著降低发热模型动物的体温、显著升高模型动物外周血中血红蛋白的含量、显著减轻类风湿性关节炎模型动物的类风湿性关节炎症状的严重程度、显著抑制肿瘤动物模型的肿瘤负荷。因此，本发明化合物可用于制备免疫调节剂药物产品，制备预防、缓解和/或治疗引起生物机体病理性分泌IL-6过量升高的疾病的药物产品，制备预防、缓解和/或治疗引起生物机体病理性分泌NO过量升高的疾病的药物产品，制备预防、缓解和/或治疗引起生物机体形成病理性自身抗体升高的疾病的药物产品。所述的本发明化合物在制备预防、缓解和/或治疗上述相关疾病的药物产品中的应用包括了预防、缓解和/或治疗心脑血管和血液系统疾病、炎症病、发热病、肿瘤病、自身免疫性疾病和冠状病毒感染病及其并发症的应用。其中，心脑血管和血液系统疾病包括各种原因引起的低血红蛋白血症性贫血病，自身免疫性疾病包括类风湿性关节炎，肿瘤病包括乳腺癌、结直肠癌和肺癌，冠状病毒感染病及其并发症包括了2019新型冠状病毒SARS-CoV2及其变异株感染病及其并发症。本发明化合物在毒理学实验中显示出显著的安全性。本发明化合物的药理效应和理化性质均显著优于氯化小檗碱型生物碱季铵盐类化合物底物，包括氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐、氯化异小檗碱季铵盐。与天然氯化小檗碱型生物碱季铵盐类底物比较， 本发明化合物在水和醇水混合溶剂中的溶解性更具有特点，更适合药物开发。本发明化合物的上述性能具有显著的制备药物价值，可以制成包括普通口服剂型经胃给药形式以及注射剂型形式在内的各种药用剂型而发挥药物用途。本发明化合物的具体结构是以吡啶甲酸或取代吡啶甲酸类酸根或2-甲基-N1-氧化吡嗪-5-甲酸根为酸根平衡阴离子单元，以5,6-二氢二苯并[a,g]喹嗪-7-阳离子型季铵类阳离子为碱基平衡阳离子单元，二者形成可溶性小檗碱型生物碱季铵盐化合物。
开展功能有机化合物的合成及生物活性评价是一项科研实践活动，其重要目的是通过具体的科学实验在有机化学和药学领域培养专业人才，并推动相关学科的不断进步；同时这一具体的科研实践过程一直蕴藏着各种意义上的新发现，不断地为科学进步注入新的内涵。本发明正是在开展小檗碱类化合物深入的药物化学研究的过程中，获得了本发明化合物，并确证了其结构、合成方法、理化性质以及生物学意义。
本发明化合物的合成路线具有简单、高效、友好的特点。采用NMR测试手段对这些化合物开展的稳定性检测结果表明，本发明化合物不仅在固态下理化性质稳定，即便在溶液中放置时其结构也极其稳定。当然，根据有机化学的理论，在溶液中本发明化合物以离子对或离子簇形式存在，具有特定的排列方式，而非混合物形式。在理化性能方面，溶解性能测试实验证明本发明化合物与对应的以无生物活性的氯负离子为平衡阴离子的氯化小檗碱型生物碱季铵盐类底物比较，在溶剂中具有明显改善的溶解性能，特别是通过采用本发明化合物中的特定的平衡阴离子，获得了更适合根据具体药用目的而可以选择性采用的化合物，即根据具体药用，如果需要提高生物利用度，则可以应用溶解性能得到提高的本发明化合物，而如果需要减少药物在肠道的吸收，提高药物在肠组织中的选择性分布，则可以应用溶解性能降低的本发明化合物。因此对于规模化制备符合制药通用技术规范的本发明可溶性小檗碱型生物碱季铵盐化合物活性分子实体并在显著改善生物利用度或者选择性分布于肠组织、寻找新的药理活性、提高药物作用强度等各个方面具有显著的实用价值。
与对应的氯化小檗碱型生物碱季铵盐底物对比，本发明化合物的水溶性的主要特征是获得了明显改善；在25℃±2℃的环境温度下测定水溶性，每毫升水中可以溶解本发明化合物的量多数大于或显著大于每毫升水中可以溶解的对 应的氯化小檗碱型生物碱季铵盐底物，例如，在每毫升水中可以溶解的本发明化合物1-10的量分别是11.5mg、>300mg、7.5mg、45mg、1.1mg、15mg、50mg、1.5mg、30mg和1.38mg；而作为小檗碱型生物碱季铵盐底物的氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐和氯化异小檗碱季铵盐在平行的测定中在每毫升水中可以被溶解的量分别是2mg、21mg、<1mg、<1mg、<1mg。但是，对于个别几个以本发明特定的阴离子为平衡阴离子的化合物而言，其溶解性能又明显下降，这些化合物又可应用于特征的疾病治疗，如肠道局部疾病。
与对应的氯化小檗碱型生物碱季铵盐类底物对比，本发明化合物在乙醇水混合溶剂中的溶解性同样主要是获得了显著改善；在25℃±2℃的环境温度下测定溶解性，在每毫升95％乙醇水混合溶剂中可以被溶解的本发明化合物的量大部分均得到显著改善，如在每毫升95％乙醇中可以溶解的本发明化合物1-10的量分别是60mg、46mg、7.7mg、109mg、26mg、38mg、45mg、5mg、22mg和137mg；而氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐和氯化异小檗碱季铵盐在平行的测定中在每毫升水中可以被溶解的量分别是～3mg、15mg、<1mg、<1mg、<1mg。
同时，本发明化合物中，也有一些溶解性能降低的化合物，这些溶解性降低的化合物因为可以主要分布在肠组织中，因此更适合开发治疗肠道疾病的药物，例如化合物24和化合物40.
在功能有机化合物的合成和生物学功能评价的科学研究中，有关化合物显示一定的安全性是深入开展生物学研究的必要前提；当然，这也是药物发现和研制过程中首先需要开展的常规生物学实验之一。将化合物与各种正常细胞进行共孵化培养，考察对正常细胞生长的影响情况，如果实验结果表明经有关化合物干预处理的正常细胞存活率高(即化合物对细胞生长的抑制率低)，则可以初步判断化合物具有较好的生物安全性，适合于在生物学模型上开展深入的生物活性研究，也为进一步开展药物研究奠定了基础。本发明分别采用CCK-8法和MTT法对本发明化合物对正常细胞的细胞生长情况的影响进行了广泛的检测。在考察本发明化合物对体外培养初传代和多传代正常免疫效应细胞小鼠单核巨噬细胞RAW264.7的生长情况的影响实验中，采用培养细胞在本发明化合物系列作用浓 度下经24h时长干预处理后的细胞存活率评价对细胞生长情况的影响，结果表明，经本发明化合物在系列作用浓度下干预处理后，本发明化合物完全没有或基本没有出现对正常细胞生长的抑制作用，而且在一定的作用浓度下，一些化合物还对初传代或多传代正常RAW264.7细胞具有一定的促增殖作用。巨噬细胞是一种位于机体组织内的重要的免疫效应细胞，在体内参与非特异性防卫(先天性免疫)和特异性防卫(适应性免疫或细胞免疫)，发挥着防御、监视、调节以及抗原呈递等极其重要的免疫作用，与机体的免疫系统平衡有非常重要的相关性，在宿主的免疫应答过程中具有重要作用。巨噬细胞的主要功能是以固定细胞或游离细胞的形式对细胞残片及病原体进行噬菌作用(即吞噬以及消化)，并激活淋巴球或其他免疫细胞，令其对病原体作出反应。巨噬细胞还具有重新构建组织，修复损伤细胞，清除凋亡细胞的功能。本发明化合物对巨噬细胞的生长没有抑制作用或具有促巨噬细胞增殖的作用，表明其具有平衡机体免疫功能的作用。经本发明化合物在10μM的作用浓度或10μM-0.01μM或20μM-0.625μM的系列作用浓度下干预处理后，对正常RAW264.7细胞生长的抑制作用不明显，表明化合物的毒性作用不强。本发明化合物在10μM、1μM、0.1μM、0.01μM和0.001μM的系列作用浓度下经96h时长干预处理培养细胞，对人胚肺成纤维细胞HELF细胞生长的抑制作用的半数抑制浓度IC50值大于10μM，对小鼠胚胎成纤维细胞NIH3T3细胞生长的抑制作用也很弱，抑制作用的IC50值大于10μM。本发明化合物在10μM的作用浓度下与正常人胚肾上皮细胞293T细胞共孵育72h，结果表明，本发明化合物对正常人胚肾上皮细胞293T细胞没有明显毒性，除小檗碱吡啶-3-甲酸季铵盐(1)干预的细胞的存活率为98.5％外，经其他所考察的本发明化合物干预处理的正常293T细胞的存活率均达100％(见实验例)。
此外，在体外实验中，本发明化合物对其他多种正常细胞也不显示明显的生长抑制作用。如在抗病毒活性评价实验中，本发明化合物对未感染病毒的Vero E6细胞不显示明显的生长抑制作用(见实验例)。
通过进一步开展各化合物的生物活性评价，发现了本发明化合物在药理实验中显示出多种重要且作用强度突出的药理学效应。首先，本发明化合物具有显著降低病理性高IL-6表达量药理模型中IL-6水平的药理学效应，包括在细胞水平和在动物水平上均具有突出的降低药理模型中病理性IL-6水平过量高表达的 药理学效应。在细胞实验中，本发明化合物对IL-6高表达的抑制作用在预孵育2小时的情况下在0.625μM的作用浓度下仍然显示统计学极显著性作用强度。在采用鸡II型胶原蛋白诱导的病理性高IL-6水平药理模型开展的本发明化合物的生物活性评价实验中，本发明化合物降低模型动物血清IL-6水平的作用非常明显。在模型组动物血清中IL-6水平达69.8443pg/ml的病理状态下，受试本发明化合物7在200mg/kg、100mg/kg和50mg/kg的高剂量组/中剂量组/低剂量组作用剂量下均可将模型动物血清中IL-6水平显著降低，分别降低到了16.8596pg/ml、17.7957pg/ml和21.6338pg/ml，并且均与模型组比较统计学上具有显著性差异。第二，本发明化合物具有显著抑制LPS刺激的RAW264.7细胞炎症模型中NO过量生成的活性。实验中发现，本发明化合物可以在0.625μM(甚至更低)的作用浓度下对LPS刺激的RAW264.7细胞炎症模型中NO的生成也显示出统计学极显著性抑制活性。本发明化合物在药理实验的作用浓度下的抑制作用比对应的氯化小檗碱型生物碱季铵盐的在相同作用浓度范围的抑制作用明显更强。这表明了本发明化合物的抗炎作用具有独特的作用机制，这种独特的作用机制与本发明化合物独特的结构特征密切关联；本发明化合物的结构特征体现在由两部分结构单元组成，这两部分结构单元在体内肠道可以解离，解离后的两部分结构单元均分别具有各自的作用特点，作用靶点相互协同作用，有待深入研究。本发明化合物显著抑制LPS刺激的RAW264.7细胞炎症模型中NO生成的活性与其显著抑制iNOS高表达的活性相关，并且，在平行的实验中，本发明化合物抑制iNOS高表达的活性比对应的氯化小檗碱型生物碱季铵盐以及对应于本发明化合物平衡阴离子的单体酸的抑制作用明显更强。第三，本发明化合物具有显著降低病理性高IgG抗体水平药理模型中模型动物血清IgG抗体水平的药理学效应。在采用鸡II型胶原蛋白诱导的病理性高IgG抗体水平药理模型开展的生物活性评价实验中，本发明化合物降低模型动物血清中IgG抗体水平的作用非常明显，且呈现独特的剂量依赖性；在模型组动物血清中IgG抗体水平达22.98350单位/ml的病理状态下，受试本发明化合物7在200mg/kg、100mg/kg和50mg/kg的高剂量组/中剂量组/低剂量组作用剂量下可将模型动物血清中IgG抗体水平分别降低到20.91259单位/ml、17.97435单位/ml和16.53063单位/ml，并且低剂量组和中剂量组与模型组比较统计学上具有极显著性差异。本发明化合物降低模型动物 血清中抗鸡II型胶原IgG抗体浓度的药理作用表明其对自身免疫性疾病具有显著的疗效。结合本发明化合物对免疫效应细胞的影响，以上实验发现的结果表明，本发明化合物具有显著的免疫调节作用，可用于制备免疫调节剂药物，制备预防、缓解和/或治疗引起生物机体分泌IL-6病理性过量升高的疾病的药物产品，制备预防、缓解和/或治疗引起生物机体iNOS过量高表达和分泌NO病理性过量升高的疾病的药物产品，制备预防、缓解和/或治疗引起生物机体形成自身抗体病理性过量升高的疾病的药物产品。在这些产品中，本发明化合物可以通过平衡机体免疫功能的作用而发挥治疗疾病的作用，可以通过显著降低IL-6过量分泌水平的药理学作用而对引起生物机体分泌IL-6过量升高的相关疾病发挥有益的治疗作用，可以通过显著降低NO过高水平的药理学作用而对引起生物机体分泌NO过量升高的相关疾病发挥有益的治疗作用，可以通过显著降低过高的自身抗体水平的药理学作用而对引起生物机体形成自身抗体过量升高的相关疾病发挥有益的治疗作用。例如，对于病毒感染性疾病及其并发症、炎症性疾病、发热病、心血管系统和血液系统疾病、自身免疫性疾病，以及部分肿瘤病，等，本发明化合物均具有有益的治疗作用。
本发明化合物在Vero E6细胞模型中可剂量依赖性地抑制SARS-CoV2病毒RNA复制，但是，本发明化合物对在Vero E6细胞上SARS-CoV2诱导的细胞毒性没有抑制作用，是在抗SARS-CoV2病毒感染性肺炎疾病的药物研发中具有显著应用前景的化合物，即该类化合物具有安全且有效的特点。其中，在所检测本发明化合物中，在10μM的作用浓度下，本发明化合物1、3、4、6-10等对实验细胞感染SARS-CoV2病毒后病毒RNA水平的抑制率分别为28.63％、65.66％、32.95％、93.13％、31.00％、89.10％、29.62％和17.45％。所检测的化合物6、8和3抑制SARS-CoV2病毒RNA水平的EC50值分别为3.037μM、3.767μM和7.859μM。同时，将本发明化合物降低模型动物血清中抗鸡II型胶原IgG抗体过高浓度的药理作用、本发明化合物降低模型动物血清中IL-6过量分泌水平的药理作用以及显著地抑制SARS-CoV2病毒RNA复制的效力相结合，表明本发明化合物对血清中抗病原体IgG抗体呈阳性的以及机体形成炎性细胞因子风暴的SARS-CoV2病毒感染具有显著的治疗作用。在其他相关的动物实验中，在对酵母致大鼠发热模型的解热作用药效学评价中，以给药后的不同时间点测量的模 型动物体温(变化)以及不同时间段所测体温与基础体温的差值ΔT(℃)为指标评价本发明化合物的解热作用，结果表明，本发明化合物具有显著的解热作用，且解热作用的特点是比阳性药具有更长的作用持续时间。在鸡II型胶原蛋白诱导的DBA/1小鼠炎症模型上，通过考察经本发明化合物治疗后的模型动物血红蛋白水平、类风湿性关节炎关节炎症指数评分水平、关节炎发病率水平以及脚掌厚度水平的变化，表明了本发明化合物具有显著的治疗贫血症和类风湿性关节炎的活性。其中，在血红蛋白的检测方面，与正常小鼠比较，本发明所开展实验中的模型组动物的血红蛋白含量明显降低，数值为136.33±4.27(g/L)；与模型组比较，受试本发明化合物各剂量组对血红蛋白含量均有明显升高作用，低/中/高剂量组的血红蛋白含量分别为180.18±4.45(g/L)、182.75±2.22(g/L)和182.75±2.42(g/L)。特别是与正常对照组比较，本发明化合物各剂量组对血红蛋白也均有明显升高作用，表明本发明化合物具有显著的治疗贫血病的作用。在关节炎症指数评分水平的检测中，受试本发明化合物各剂量组对关节炎症指数评分水平均有明显降低作用，在实验终止时，低/中/高剂量组的关节炎症指数评分分别为1.73±0.68***、2.67±0.80***和3.58±0.66***，与模型组的关节炎症指数评分6.92±0.84相比，均显著降低，且均具有极显著性统计学差异。在关节炎发病率水平以及脚掌厚度水平变化的检测中，全部得到了与关节炎症指数评分水平检测实验中所获得的“对治疗类风湿性关节炎具有显著效果”完全相同的结果，都表明本发明化合物具有显著的抗类风湿性关节炎活性。
此外，在采用MTT法检测化合物对肿瘤细胞生长的抑制作用中发现，本发明化合物选择性地对人结直肠癌HCT-116细胞和人肺癌A549细胞的生长具有显著的抑制作用，结合本发明化合物显著的降低IL-6水平的效力以及本发明化合物对Ras突变/MAPK激活/IL-6过量分泌这一信号通路的抑制作用，表明，本发明化合物对肿瘤病具有有益的治疗作用。本发明化合物在4T1乳腺癌细胞小鼠移植瘤动物模型实验中对乳腺癌显示出显著的抗癌效果。与氯化小檗碱季铵盐类底物进行平行试验并对照活性，本发明活性化合物抑制肿瘤细胞株生长的活性获得了明显的提高。
因此，结合上述生物学重要性的评价可以得出结论，本发明化合物可用于制备预防、缓解和/或治疗各种原因引起的贫血病、炎症病、发热病、结直肠癌、 肺癌、乳腺癌、类风湿性关节炎病和冠状病毒感染病及其并发症的药物产品。
需要进一步强调的是，在鸡II型胶原蛋白诱导的DBA/1小鼠炎症模型上，本发明化合物表现出了在治疗类风湿性关节炎方面50mg/kg的低剂量组的药理活性最强，且100mg/kg的中剂量组优于200mg/kg的高剂量组的情况。可以表明，本发明化合物具有独特的药理作用机制。这是一种药理作用具有选择性的标志，其作用机制有待深入研究
综上所述，本发明化合物具有显著的药用有效性、安全性和质量可控性，在制备免疫调节剂药物，制备预防、缓解和/或治疗引起生物机体分泌IL-6病理性过量升高的疾病的药物产品、引起生物机体分泌NO病理性过量升高的疾病的药物产品以及引起生物机体形成自身抗体病理性过量升高的疾病的药物产品方面，以及制备预防、缓解和/或治疗各种原因引起的炎症病、发热病、肿瘤病、自身免疫性疾病、心脑血管和血液系统疾病以及病毒感染病及其并发症的药物产品方面的应用前景非常显著。特别是本发明化合物在制备预防、缓解和/或治疗贫血症、炎症病、发热病、结直肠癌、肺癌、乳腺癌、类风湿性关节炎病和引起生物机体分泌IL-6水平病理性过量升高形成炎性细胞因子风暴和IgG抗体水平病理性过量升高的新型冠状病毒SARS-CoV2感染病及其并发症药物产品中具有显著的应用价值。
附图说明
图1、小鼠鸡II型胶原蛋白诱导性关节炎(CIA)整体动物试验给药流程。
图2、本发明化合物7对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠血清中IL-6水平的影响。
注：数据以X±SEM表示；#为与正常对照组比，###p<0.001；*为与模型组比较，**p<0.01，***p<0.001。
图3、本发明化合物7对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠血红蛋白含量的影响。
注：数据以X±SEM表示；与模型组比较，***p<0.001。
图4、本发明不同给药组对小鼠关节炎症指数评分的影响。
注：n＝12；与模型组比较，*p<0.05，**p<0.01，***p<0.001。
图5、本发明化合物7对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠血清中的抗鸡II型胶原IgG抗体的影响
注：与模型组比较，*p<0.05，**p<0.01，***p<0.001。
图6、本发明化合物7解热作用药效学评价实验大鼠体温变化图。
注：各组n＝12；#为与空白对照组比较，###p<0.001；*为与模型组比较，**p<0.01,***p<0.001。
图7：本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中iNOS高表达的抑制作用作用。
注：与模型组比较，*p<0.05。
具体实施方式
本发明的具体实施方式不以任意方式限制本发明。
在本发明化合物的制备工艺及结构鉴定数据中，化合物编号与本发明内容中的具体化合物编号相对应。
一、本发明化合物制备实施例
实施例1：本发明化合物1的合成及结构鉴定数据
称取4g小檗碱季铵盐底物于反应瓶中，加入丙酮9.6ml溶剂搅拌均匀，随后逐滴加入5N氢氧化钠水溶液24ml，30℃搅拌反应至原料反应完全；将反应混合液抽滤，并水洗滤饼至中性，干燥得到固体8-丙酮基二氢小檗碱3.5g。
称取吡啶-3-甲酸(35mg，0.28mmol)于反应瓶中，加入2.5ml四氢呋喃/水(v/v＝24:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物1黄色固体108mg，收率92.70％。1H-NMR(400MHz,DMSO-d6)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 2O),4.94(t,J＝6.4Hz,2H,NCH 2CH2),4.09(s,3H,ArOCH3),4.07(s,3H,ArOCH3),3.20(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(150MHz,CD3OD)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-C6H4NO2]+(calc.for C20H18NO4,336.12303)。
实施例2：本发明化合物2的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取吡啶-3-甲酸(33mg，0.27mmol)于反应瓶中，加入2ml四氢呋喃/水(v/v＝24:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg， 0.24mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物2黄色固体104mg，收率96.3％。1H-NMR(400MHz,DMSO-d6)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 2CH2),4.10(s,3H,ArOCH3),4.07(s,3H,ArOCH3),3.94(s,3H,ArOCH3),3.87(s,3H,ArOCH3),3.23(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(150MHz,DMSO-d6)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-C6H4NO2]+(calc.for C21H22NO4,352.15433)。
实施例3：本发明化合物3的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取吡啶-3-甲酸(66mg，0.54mmol)于反应瓶中，加入4ml四氢呋喃/水(v/v＝24:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(100mg，0.26mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物3黄色固体89mg，收率75.9％。1H-NMR(400MHz,DMSO-d6)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 2O),6.17(s,2H,OCH 2O),4.88(t,J＝6.4Hz,2H,NCH 2CH2),3.20(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(150MHz,DMSO-d6)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-C6H4NO2]+(calc.for C19H14NO4,320.09173)。
实施例4：本发明化合物4的合成及结构鉴定数据
8-丙酮基二氢异黄连碱的合成(略)。
称取吡啶-3-甲酸(36mg，0.29mmol)于反应瓶中，加入2.5ml四氢呋喃/水(v/v＝24:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢异黄连碱(100mg，0.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物4黄色固体104mg，收率88.7％。1H-NMR(400MHz,DMSO-d6)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 2O),6.17(s,2H,OCH 2O),4.77(t,J＝6.4Hz,2H,NCH 2CH2),3.19(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(100MHz,DMSO-d6)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-C6H4NO2]+(calc.for C19H14NO4,320.09173)。
实施例5：本发明化合物5的合成及结构鉴定数据
8-丙酮基二氢异小檗碱的合成(略)。
称取氯化异小檗碱季铵盐(500mg，1.34mmol)于反应瓶中，加入5N氢氧化钠水溶液(3ml)，随后逐滴加入丙酮(1ml，13.5mmol)，室温搅拌反应4h，原料反应完全。将反应液抽滤，并水洗滤饼至中性,得到淡黄色固体8-丙酮基二氢异小檗碱,产物不经纯化直接用于下步反应。称取吡啶-3-甲酸(160mg，1.30mmol)于反应瓶中，加入21ml四氢呋喃/水(v/v＝24:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢异小檗碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物5黄色固体490mg，收率79.54％。1H-NMR(400MHz,Methanol-d4)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 2O),4.79(t,J＝6.4Hz,2H,NCH 2CH2),4.12(s,3H,ArOCH3),4.05(s,3H,ArOCH3),3.23(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(150MHz,CD3OD)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-C6H4NO2]+(calc.for C20H18NO4,336.12303)。
实施例6：本发明化合物6的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取5-氟吡啶-3-甲酸(39mg，0.28mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,2ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物6黄色固体110mg，收率91.08％。1H-NMR(400MHz,DMSO-d6)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 2O),4.94(t,J＝6.4Hz,2H,NCH 2CH2),4.09(s,3H,ArOCH3),4.07(s,3H,ArOCH3),3.21(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(100MHz,DMSO-d6)δ:164.8,159.0(d,1JCF＝251.6Hz,1C,C-5′),150.3,149.8,147.6,146.6(d,4JCF＝3.3Hz,1C,C-2′),145.4,143.6,138.6,137.4,136.6(d,2JCF＝23.0Hz,1C,C-4′),132.9,130.6,126.7,123.5,122.2(d,2JCF＝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-C6H3FNO2]+(calc.for C20H18NO4,336.12303)。
实施例7：本发明化合物7的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取5-氟吡啶-3-甲酸(38mg，0.27mmol)于反应瓶中，加入2ml四氢呋喃/水(v/v＝24:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg，0.24mmol)，加热反应，至原料反应完全，停止加热，将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物7黄色固体119mg，收率98.92％。1H-NMR(400MHz,DMSO-d6)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 2CH2),4.10(s,3H,ArOCH3),4.07(s,3H,ArOCH3),3.94(s,3H,ArOCH3),3.88(s,3H,ArOCH3),3.23(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(100MHz,DMSO-d6)δ:164.8,159.0(d,1JCF＝251.6Hz,1C,C-5′),151.5,150.2,148.7,146.6(d,4JCF＝3.3Hz,1C,C-2′),145.4,143.6,138.7,137.7,136.6(d,2JCF＝23.1Hz,1C,C-4′),133.1,128.6,126.7,123.4,122.2(d,2JCF＝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-C6H3FNO2]+(calc.for C21H22NO4,352.15433)。
实施例8：本发明化合物8的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取5-氟吡啶-3-甲酸(41mg，0.29mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,3ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(100mg，0.26mmol)，加热反应，至原料反应完全，停止加热，将反应混合液冷至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物8黄色固体96mg，收率78.69％。1H-NMR(400MHz,DMSO-d6)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 2O),6.17(s,2H,OCH 2O),4.88(t,J＝6.4Hz,2H,NCH 2CH2),3.20(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(150MHz,DMSO-d6)δ:164.8,159.0(d,1JCF＝251.7Hz,1C,C-5′),149.7,147.6,147.0,146.6(d,4JCF＝3.3Hz,1C,C-2′),144.5,143.8,138.1,136.81(d,2JCF＝22.9Hz,1C,C-4′),136.77,132.3,130.5,122.2(d,2JCF＝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-C6H3FNO2]+(calc.for C19H14NO4,320.09173)。
实施例9：本发明化合物9的合成及结构鉴定数据
8-丙酮基二氢异黄连碱的合成(略)。
称取5-氟吡啶-3-甲酸(41mg，0.29mmol)于反应瓶中，加入2.5ml四氢呋喃/水(v/v＝24:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢异黄连碱(100mg，0.26mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物9黄色固体97mg，收率79.51％。1H-NMR(400MHz,DMSO-d6)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 2O),6.17(s,2H,OCH 2O),4.77(t,J＝6.4Hz,2H,NCH 2CH2),3.19(t,J＝6.4Hz,2H,NCH2CH 2)。13C-NMR(100MHz,DMSO-d6)δ:165.0,159.0(d,1JCF＝251.5Hz,1C,C-5′),155.9,150.9,150.0,147.6,146.6(d,4JCF＝3.3Hz,1C,C-2′),145.9,138.7,138.6,138.3,136.8(d,2JCF＝23.0Hz,1C,C-4′),130.9,123.5,122.3(d,2JCF＝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-C6H3FNO2]+(calc.for C19H14NO4,320.09173)。
实施例10：本发明化合物10的合成及结构鉴定数据
8-丙酮基二氢异小檗碱的合成(略)。
称取5-氟吡啶-3-甲酸(604mg，4.19mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,52ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢异小檗碱(1.5g，3.81mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物10黄色固体68mg，收率54.09％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),4.78(t,J＝6.4Hz,2H,NCH2CH2),4.07(s,3H,ArOCH3),4.00(s,3H,ArOCH3),3.20(t,J＝6.4Hz,2H,NCH2CH2)。13C-NMR(150MHz,CD3OD)δ:170.8,161.0(d,1JCF＝253.3Hz,1C,C-5′),160.1,154.7,152.2,149.9,147.4(d,4JCF＝3.6Hz,1C,C-2′),146.2,140.4,139.4(d,2JCF＝24.3Hz,1C,C-4′),139.0,137.2,131.9,124.8(d,2JCF＝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-C6H3FNO2]+(calcd for C20H18NO4,336.12303)。
实施例11：本发明化合物11的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取5-甲基吡啶-3-甲酸(261mg，1.91mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,14ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(500mg，1.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物11黄色固体558mg，收率93.00％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),4.93(t,J＝6.0Hz,2H,NCH 2CH2),4.10(s,3H,ArOCH3),4.07(s,3H,ArOCH3),3.21(t,J＝6.0Hz,2H,NCH2CH 2),2.26(s,3H,Me)。
实施例12：本发明化合物12的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取5-甲基吡啶-3-甲酸(184mg，1.34mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,8ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(500mg，1.22mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物12黄色固体456mg，收率76.38％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2),2.26(s,3H,Me)。
实施例13：本发明化合物13的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取5-甲基吡啶-3-甲酸(109mg，0.80mmol)于反应瓶中，加入四氢呋喃 /水(v/v＝24:1,12ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物13黄色固体218mg，收率90.08％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.88(t,J＝6.0Hz,2H,NCH 2CH2),3.20(t,J＝6.0Hz,2H,NCH2CH 2),2.26(s,3H,Me)。
实施例14：本发明化合物14的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取6-甲基吡啶-3-甲酸(261mg，1.91mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,14ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(500mg，1.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物14黄色固体487mg，收率81.17％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.94(t,J＝6.0Hz,2H,NCH 2CH2),4.09(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH2CH 2),2.42(s,3H,Me)。
实施例15：本发明化合物15的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取6-甲基吡啶-3-甲酸(184mg，1.34mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,8ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(500mg，1.22mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物15黄色固体448mg，收率75.04％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2),2.41(s,3H,Me)。
实施例16：本发明化合物16的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取6-甲基吡啶-3-甲酸(109mg，0.80mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,12ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物16黄色固体178mg，收率73.55％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.88(t,J＝6.0Hz,2H,NCH 2CH2),3.20(t,J＝6.0Hz,2H,NCH2CH 2),2.42(s,3H,Me)。
实施例17：本发明化合物17的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-氯吡啶-3-甲酸(80mg，0.51mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,4ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物17黄色固体95mg，收率75.2％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.94(t,J＝5.5Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝5.5Hz,2H,NCH2CH 2)。
实施例18：本发明化合物18的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-氯吡啶-3-甲酸(48mg，0.31mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,4ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物18黄色固体88mg，收率70.4％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例19：本发明化合物19的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-氯吡啶-3-甲酸(124mg，0.79mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,7ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(100mg，0.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物19黄色固体87mg，收率69.0％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.88(t,J＝6.0Hz,2H,NCH 2CH2),3.20(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例20：本发明化合物20的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-氯-6-甲基吡啶-3-甲酸(327mg，1.91mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,16ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(500mg，1.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物20黄色固体584mg，收率90.68％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.93(t,J＝6.5Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH2CH 2),2.36(s,3H,Me)。
实施例21：本发明化合物21的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-氯-6-甲基吡啶-3-甲酸(230mg，1.34mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,8ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(500mg，1.22mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物21黄色固体581mg，收率91.07％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2), 2.36(s,3H,Me)。
实施例22：本发明化合物22的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-氯-6-甲基吡啶-3-甲酸(136mg，0.80mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,12ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物22黄色固体214mg，收率82.31％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.89(t,J＝6.5Hz,2H,NCH 2CH2),3.20(t,J＝6.5Hz,2H,NCH2CH 2),2.36(s,3H,Me)。
实施例23：本发明化合物23的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取4-氨基吡啶-3-甲酸(24mg，0.17mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,4ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.17mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物23黄色固体20mg，收率16.5％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.93(t,J＝6.5Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH2CH 2)。
实施例24：本发明化合物24的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取4-氨基吡啶-3-甲酸(42mg，0.3mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,5ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物24黄色固体43mg，收率35.5％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例25：本发明化合物25的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取6-溴吡啶-3-甲酸(385mg，1.91mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,16ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(500mg，1.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物25黄色固体607mg，收率88.87％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),4.94(t,J＝6.0Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例26：本发明化合物26的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取6-溴吡啶-3-甲酸(370mg，1.83mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,14ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(500mg，1.22mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物26黄色固体623mg，收率92.16％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例27：本发明化合物27的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取6-溴吡啶-3-甲酸(161mg，0.80mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,12ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物27黄色固体215mg，收率77.90％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.89(t,J＝6.0Hz,2H,NCH 2CH2),3.20(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例28：本发明化合物28的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取5-溴吡啶-3-甲酸(80mg，0.40mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,4ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物28黄色固体82mg，收率60.7％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),4.93(t,J＝6.0Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例29：本发明化合物29的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取5-溴吡啶-3-甲酸(118mg，0.58mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,11ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(200mg，0.49mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物29黄色固体191mg，收率70.7％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例30：本发明化合物30的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-氟吡啶-3-甲酸(43mg，0.31mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,5ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物30黄色固体55mg，收率45.5％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.93(t,J＝6.5Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝5.2Hz,2H,NCH2CH 2).
实施例31：本发明化合物31的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-氟吡啶-3-甲酸(69mg，0.49mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,3ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg，0.24mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物31黄色固体99mg，收率83.9％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例32：本发明化合物32的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-氟吡啶-3-甲酸(112mg，0.79mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,7ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(100mg，0.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物32黄色固体72mg，收率59.0％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.88(t,J＝6.5Hz,2H,NCH 2CH2),3.20(t,J＝6.5Hz,2H,NCH2CH 2).
实施例33：本发明化合物33的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-氟吡啶-4-甲酸(54mg，0.38mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,5ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物33黄色固体75mg，收率61.5％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.93(t,J＝6.5Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH2CH 2)。
实施例34：本发明化合物34的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-氟吡啶-4-甲酸(52mg，0.37mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,4ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg，0.24mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物34黄色固体79mg，收率66％。1H-NMR(400MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例35：本发明化合物35的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-氟异烟酸(112mg，0.79mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,5ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(100mg，0.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物35黄色固体84mg，收率69.0％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.88(t,J＝6.4Hz,2H,NCH 2CH2),3.20(t,J＝6.4Hz,2H,NCH2CH 2)。
实施例36：本发明化合物36的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-氯吡啶-4-甲酸(300mg，1.91mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,16ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(500mg，1.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物36黄色固体511mg，收率81.76％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.94(t,J＝6.5Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.5Hz,2H,NCH2CH 2)。
实施例37：本发明化合物37的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-氯吡啶-4-甲酸(212mg，1.34mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,12ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(500mg，1.22mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物37黄色固体554mg，收率89.21％。1H-NMR(400MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例38：本发明化合物38的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-氯吡啶-4-甲酸(125mg，0.80mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,12ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物38黄色固体202mg，收率79.84％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.88(t,J＝6.5Hz,2H,NCH 2CH2),3.20(t,J＝6.5Hz,2H,NCH2CH 2)。
实施例39：本发明化合物39的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-氨基吡啶-4-甲酸(29mg，0.21mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,4ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100mg，0.25mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物39黄色固体23mg，收率19.3％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),5.83(s,2H,NH2),4.93(t,J＝6.0Hz,2H,NCH 2CH2),4.10(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例40：本发明化合物40的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-氨基吡啶-4-甲酸(42mg，0.30mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,7ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg，0.24mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物40黄色固体84mg，收率70.6％。1H-NMR(500MHz,DMSO-d6)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,NH2),4.95(t,J＝6.5Hz,2H,NCH 2CH2),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,NCH2CH 2)。
实施例41：本发明化合物41的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-氨基吡啶-4-甲酸(24mg，0.17mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,7ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(100mg，0.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物41黄色固体20mg，收率16.5％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),6.18(s,2H,OCH2O),6.09(s,2H,NH2),4.88(t,J＝6.0Hz,2H,NCH 2CH2),3.20(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例42：本发明化合物42的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-吡啶甲酸(235mg，1.91mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,16ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(500mg，1.27mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物42黄 色固体521mg，收率89.37％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.96(t,J＝6.0Hz,2H,NCH 2CH2),4.08(s,3H,OMe),4.07(s,3H,OMe),3.20(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例43：本发明化合物43的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-吡啶甲酸(225mg，1.83mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,14ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(500mg，1.22mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物43黄色固体611mg，收率98.0％。1H-NMR(500MHz,DMSO-d6)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 2CH2),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,NCH2CH 2)。
实施例44：本发明化合物44的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-吡啶甲酸(136mg，0.80mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,12ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物44黄色固体185mg，收率79.06％。1H-NMR(400MHz,DMSO-d6)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,OCH2O),6.17(s,2H,OCH2O),4.90(t,J＝6.0Hz,2H,NCH 2CH2),3.20(t,J＝6.0Hz,2H,NCH2CH 2)。
实施例45：本发明化合物45的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取6-甲基-2-吡啶甲酸(70mg，0.51mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,8ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(100 mg，0.26mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物45黄色固体87mg，收率71.9％。1H-NMR(500MHz,DMSO-d6)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,OCH2O),4.94(t,J＝6.0Hz,2H,NCH 2CH2),4.09(s,3H,OMe),4.07(s,3H,OMe),3.21(t,J＝6.0Hz,2H,NCH2CH 2),2.39(s,3H,Me)。
实施例46：本发明化合物46的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取6-甲基-2-吡啶甲酸(41mg，0.30mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,8ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(100mg，0.24mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物46黄色固体91mg，收率76.5％。1H-NMR(400MHz,DMSO-d6)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 2CH2),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,NCH2CH 2),2.40(s,3H,Me)。
实施例47：本发明化合物47的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取6-甲基-2-吡啶甲酸(218mg，1.59mmol)于反应瓶中，加入四氢呋喃/水(v/v＝24:1,18ml)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(500mg，1.32mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，滤饼用四氢呋喃/水(v/v＝24:1)的混合溶剂洗涤三次，得化合物47黄色固体392mg，收率64.8％。1H NMR(400MHz,DMSO-d6)δ10.01(s,1H),8.97(s,1H),8.03(d,J＝8.4Hz,1H),7.83(d,J＝8.4Hz,1H),7.80(s,1H),7.51(br s,1H),7.50(br s,1H),7.08(s,1H),7.04–7.01(br,1H),6.53(s,2H),6.17(s,2H),4.90(t,J＝6.2Hz,2H),3.19(t,J＝6.2Hz,2H),2.39(s,3H)。
实施例1'、本发明化合物1'的合成及结构鉴定数据
8-丙酮基二氢小檗碱的合成(略)。
称取2-甲基-5-羧基-N1-氧化吡嗪(440mg，2.80mmol)于反应瓶中，加入40ml四氢呋喃/水(v/v＝9:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢小檗碱(1.0g，2.54mmol)，加热反应，至原料反应完全，停止加热。将反应混合 液冷却至室温后过滤，得化合物1'黄色固体1.1g，收率88.41％。1H-NMR(400MHz,DMSO-d6)δ：9.90(s,1H,ArH),8.94(s,1H,ArH),8.44(s,1H,ArH),8.36(s,1H,ArH),8.20(d,J＝9.2Hz,1H,ArH),8.00(d,J＝9.2Hz,1H,ArH),7.80(s,1H,ArH),7.09(s,1H,ArH),6.17(s,2H,OCH 2O),4.94(t,J＝6.4Hz,2H,NCH 2CH2),4.09(s,3H,ArOCH3),4.07(s,3H,ArOCH3),3.21(t,J＝6.4Hz,2H,NCH2CH 2),2.29(s,3H,ArCH3)。13C-NMR(150MHz,DMSO-d6)δ:163.8,155.7,150.3,149.7,147.6,145.9,145.4,143.5,141.6,137.4,132.9,132,4,130.6,126.6,123.4,121.3,120.3,120.1,108.3,105.4,102.0,61.8,56.9,55.1,26.2,13.6.HR-ESI-MS(pos.):336.12302[M-C6H5N2O3]+(calc.for C20H18NO4,336.12303)。
实施例2'、本发明化合物2'的合成及结构鉴定数据
8-丙酮基二氢巴马汀的合成(略)。
称取2-甲基-5-羧基-N1-氧化吡嗪(768mg，4.88mmol)于反应瓶中，加入40ml四氢呋喃/水(v/v＝9:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢巴马汀(1.0g，2.44mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，得化合物2'黄色固体944mg，收率76.50％。1H-NMR(400MHz,DMSO-d6)δ：9.90(s,1H,ArH),9.03(s,1H,ArH),8.44(s,1H,ArH),8.37(s,1H,ArH),8.21(d,J＝9.2Hz,1H,ArH),8.03(d,J＝9.2Hz,1H,ArH),7.72(s,1H,ArH),7.10(s,1H,ArH),4.96(t,J＝6.4Hz,2H,NCH 2CH2),4.10(s,3H,ArOCH3),4.07(s,3H,ArOCH3),3.94(s,3H,ArOCH3),3.87(s,3H,ArOCH3),3.23(t,J＝6.4Hz,2H,NCH2CH 2),2.29(s,3H,ArCH3)。13C-NMR(150MHz,DMSO-d6)δ:163.9,155.6,151.5,150.2,148.7,146.0,145.4,143.6,141.7,137.7,133.1,132.5,128.6,126.8,123.4,121.3,119.8,118.9,111.3,108.8,61.8,57.0,56.1,55.8,55.3,25.9,13.7.HR-ESI-MS(pos.):352.15430[M-C6H5N2O3]+(calc.for C21H22NO4,352.15433)。
实施例3'、本发明化合物3'的合成及结构鉴定数据
8-丙酮基二氢黄连碱的合成(略)。
称取2-甲基-5-羧基-N1-氧化吡嗪(92mg，0.58mmol)于反应瓶中，加入10ml四氢呋喃/水(v/v＝9:1)的混合溶剂，搅拌均匀后，加入8-丙酮基二氢黄连碱(200mg，0.53mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，得化合物3'黄色固体226mg，收率90.08％。1H-NMR(400MHz,DMSO-d6)δ：9.96(s,1H,ArH),8.95(s,1H,ArH),8.44(s,1H,ArH),8.37(s,1H,ArH),8.03(d,J＝8.8Hz,1H,ArH),7.82(d,J＝8.8Hz,1H,ArH),7.79(s,1H,ArH),7.08(s,1H,ArH),6.53(s,2H,OCH 2O),6.17(s,2H,OCH 2O),4.89(t,J＝6.4Hz,2H,NCH 2CH2),3.20(t,J＝6.4Hz,2H,NCH2CH 2),2.29(s,3H,ArCH3)。13C-NMR(150MHz,DMSO-d6)δ:164.0,155.1,149.7,147.6,147.0,146.1,144.5, 143.8,141.9,136.8,132.6,132.3,130.5,121.7,121.0,120.9,120.4,111.6,108.4,105.3,104.4,102.0,55.1,26.2,13.7.HR-ESI-MS(pos.):320.09177[M-C6H5N2O3]+(calc.for C19H14NO4,320.09173)。
实施例4'、本发明化合物4'的合成及结构鉴定数据
8-丙酮基二氢异黄连碱的合成(略)。
称取2-甲基-5-羧基-N1-氧化吡嗪(458mg，2.90mmol)于反应瓶中，加入40ml四氢呋喃/水(v/v＝9:1)的混合溶剂，搅拌均匀后，加8-丙酮基二氢异黄连碱碱(1.0g，2.65mmol)，加热反应，至原料反应完全，停止加热。将反应混合液冷却至室温后过滤，得化合物4'黄色固体1.084g，收率86.37％。1H-NMR(400MHz,DMSO-d6)δ：9.56(s,1H,ArH),8.75(s,1H,ArH),8.45(s,1H,ArH),8.37(s,1H,ArH),7.75(s,1H,ArH),7.73(s,1H,ArH),7.53(s,1H,ArH),7.10(s,1H,ArH),6.42(s,2H,OCH 2O),6.17(s,2H,OCH 2O),4.76(t,J＝6.4Hz,2H,NCH 2CH2),3.19(t,J＝6.4Hz,2H,NCH2CH 2),2.30(s,3H,ArCH3)。13C-NMR(150MHz,DMSO-d6)δ:163.8,155.8,155.6,150.8,149.9,147.5,146.0,145.8,141.7,138.6,138.5,132.4,130.7,123.4,120.2,118.8,108.4,105.3,103.8,103.6,102.5,102.0,54.3,26.3,13.6.HR-ESI-MS(pos.):320.09180[M-C6H5N2O3]+(calc.for C19H14NO4,320.09173)。
实施例5'、本发明化合物5'的合成及结构鉴定数据
称取氯化异小檗碱季铵盐(500mg，1.34mmol)于反应瓶中，加入5N氢氧化钠水溶液(3ml)，随后逐滴加入丙酮(1ml，13.5mmol)，室温搅拌反应4h，原料反应完全。将反应液抽滤，得到淡黄色固体8-丙酮基二氢异小檗碱,产物不经纯化直接用于下步反应。
称取2-甲基-5-羧基-N1-氧化吡嗪(205mg，1.33mmol)于反应瓶中，加入四氢呋喃/水(V/V＝9:1,25ml)的混合溶剂，搅拌混匀；再于室温搅拌下加入上述未经纯化的产物；在加热的条件下进行反应，至原料反应完全；停止加热。将反应混合液冷却至室温后过滤，得化合物5'黄色固体530mg，收率80.55％。1H-NMR(400MHz,CD3OD)δ：9.31(s,1H,ArH),8.67(s,1H,ArH),8.53(s,1H,ArH),8.52(s,1H,ArH),7.61(s,1H,ArH),7.59(s,1H,ArH),7.57(s,1H,ArH),6.94(s,1H,ArH),6.10(s,2H,OCH 2O),4.79(t,J＝6.4Hz,2H,NCH 2CH2),4.12(s,3H,ArOCH3),4.05(s,3H,ArOCH3),3.23(t,J＝6.4Hz,2H,NCH2CH 2),2.44(s,3H,ArCH3)。13C-NMR(100MHz,CD3OD)δ：168.6,160.0,154.6,153.5,152.2,149.8,148.1,146.2,145.9,140.4,138.9,134.92,131.9,124.2,121.9,119.5,109.4,107.3,106.5,106.4,103.7,57.5,57.1,56.3,28.3,14.4.HR-ESI-MS(pos.):336.12222[M-C6H5N2O3]+(calc.for C20H18NO4,336.12303)。
二、本发明化合物的溶解性检测实验例
实验例1：本发明化合物的水溶性检测实验例
分别称取一定量的本发明各化合物以及作为小檗碱型生物碱季铵盐底物的氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐和氯化异小檗碱季铵盐，置于25℃±2℃一定量的纯净水溶剂中，每隔5分钟强力振摇30秒，观察30分钟内的溶解情况，如无目视可见的溶质颗粒时，即视为完全溶解。
实验结果：每毫升纯净水溶解本发明化合物的量(所有测定的本发明化合物均经重结晶纯化，并经采用放大量验证)见表1。而作为小檗碱型生物碱季铵盐底物的氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐和氯化异小檗碱季铵盐在平行的测定中每毫升水中可以溶解的量分别是2mg、21mg、<1mg、<1mg、<1mg。
表1.本发明化合物在水中的溶解度(mg/ml,25℃±2℃)
Figure PCTCN2022080897-desc-000012-original
实验例2：本发明化合物在95％乙醇溶剂中溶解性检测实验例
分别称取一定量的经重结晶纯化的本发明化合物和作为小檗碱型生物碱季铵盐底物的氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐和氯化异小檗碱季铵盐，置于25℃±2℃一定量的95％乙醇溶剂中，每隔5分钟强力振摇30秒，观察30分钟内的溶解情况，如无目视可见的溶 质颗粒时，即视为完全溶解。
实验结果：每毫升95％乙醇溶剂中溶解氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐和氯化异小檗碱季铵盐的量分别是3mg、15mg、<1mg、<1mg、<1mg。本发明化合物的95％乙醇溶解度数据见表2。
表2.本发明化合物在95％乙醇中的溶解度(mg/ml,25℃±2℃)
Figure PCTCN2022080897-desc-000013-original
三、本发明化合物对正常细胞生长的影响情况评价实验例
实验例3：本发明化合物对体外培养的小鼠单核巨噬细胞RAW264.7细胞的生长情况影响评价实验
(1)化合物的配制：用DMSO将化合物配制成1×10-2mol/L(0.01M)的化合物储备液，待进行细胞实验时，用含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基稀释成所需浓度的初始液。
(2)实验方法(CCK-8法)：按照细胞说明书，将RAW264.7细胞在含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基中培养，培养条件为37℃/5％CO2，待细胞融合至80％后，按体积比1:3的稀释比对细胞进行传代培养。
(3)取生长状态良好且处于对数生长期的RAW264.7细胞，弃去原培养基，加入适当含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高 糖培养基轻轻吹打，制成细胞悬液并调整细胞悬液的细胞个数为1×105个/mL，接种于96孔细胞培养板，每孔100μl细胞悬液。每块96孔板四周边孔均用PBS填充，以防止“边缘效应”。将细胞培养板置于5％CO2、37℃培养箱中培养至细胞完全贴壁生长(过夜)。
(4)取化合物初始液，用含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基稀释成浓度为200μM的给药液。将此化合物给药液加入至含有100μl初传代细胞悬液的96孔板中，每孔100μl，采用倍半比稀释法实现设置的本发明化合物系列工作液浓度梯度，即100μM-0.390625μM，每个给药浓度设置6个复孔。同时设置正常细胞对照组和空白对照组，正常细胞对照组含相同操作培养的细胞和相同量的药物溶解介质，但不含本发明化合物；空白对照组除不含细胞外，其他与正常对照组相同。于培养箱中孵育24h后，每孔中加入10μL CCK-8溶液，于培养箱内孵育2h后，用酶标仪于450nm处测定光密度(OD)值(结果①实验)。
(5)取化合物初始液，用含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基稀释成浓度分别为20μM、2μM、0.2μM和0.02μM的系列浓度梯度溶液。将此化合物系列浓度梯度溶液分别加入至每孔含有100μl多传代细胞悬液的96孔板中，每孔100μl，吹打均匀后分别再吸弃100μl，得到作用浓度分别为10μM、1μM、0.1μM和0.01μM的系列浓度梯度。每个给药浓度设置6个复孔。同时设置正常细胞对照组和空白对照组。于培养箱中处理24h后，每孔中加入10μL CCK-8溶液，于培养箱内孵育2h后，用酶标仪于450nm处测定光密度(OD)值(表3实验)。
(6)按上述“(4)”和“(5)”的实验操作，制成设定的本发明化合物给药浓度或系列给药浓度，每个给药浓度设置6个复孔。同时设置正常细胞对照组和空白对照组。于培养箱中处理24h后，每孔中加入10μL CCK-8溶液，于培养箱内孵育2h后，用酶标仪于450nm处测定光密度(OD)值(表4、表5实验)。
利用以下公式计算细胞存活率：
细胞存活率(％)＝[(给药孔OD值-空白孔OD值)/(对照孔OD值-空白孔OD值)]×100％
(7)结果：
①在实验测定的时间范围内，培养初传代细胞经本发明化合物1和化合物8在100μM-0.390625μM的系列作用浓度下处理后的细胞存活率除化合物1在100μM作用浓度下是82.13±0.5％外，其余均大于100％。
②在实验测定的时间范围内，本发明系列化合物在0.01μM-10μM的作用浓度下对于多传代RAW264.7细胞无细胞毒性，并具有一定的促增殖作用。各测定实验细胞存活率及IC50值见表3。
③本发明系列化合物在10μM的作用浓度下对于正常RAW264.7细胞无细胞毒性或细胞毒性很小，一些化合物并具有一定的促增殖作用。各测定实验细胞存活率值见表4。
④本发明化合物18和19在20μM-0.625μM的作用浓度下对于正常RAW264.7细胞无细胞毒性，并具有一定的促增殖作用。各测定实验细胞存活率值见表5。
实验结果使用GraphPad Prism version 5.0软件进行分析。
表3 本发明化合物及氯化巴马汀季铵盐和氯化黄连碱季铵盐处理正常RAW264.7细胞后的细胞存活率①
Figure PCTCN2022080897-desc-000014-original
注：①具体实验结果与细胞生长状态存在一定的关系；正常组存活率为100％，与正常组比较，*P＜0.05，**P＜0.01，***P＜0.001。
表4本发明化合物在10μM作用浓度下处理正常RAW264.7细胞后的细胞存活率
Figure PCTCN2022080897-desc-000015-original
Figure PCTCN2022080897-desc-000016-original
表5.本发明化合物18和19在20μM-0.625μM的系列作用浓度下处理正常RAW264.7细胞后的细胞存活率
Figure PCTCN2022080897-desc-000017-original
(4)结论：本发明系列化合物对体外培养正常小鼠单核巨噬细胞RAW264.7细胞的生长无抑制作用，并具有促RAW264.7细胞增殖活性。
实验例4：本发明化合物在体外培养的正常人胚肾上皮细胞293T细胞上的细胞毒理学评价实验。
(1)本发明化合物的配制：用DMSO将本发明化合物溶解配制成1×10-2mol/L(0.01M)的储备液，待进行细胞实验时，用细胞培养基稀释成1×10-5mol/L(即10μM)的工作液浓度。
(2)实验方法(MTT法)：将按细胞说明书体外培养生长至90％汇合状态的正常人胚肾上皮细胞293T细胞以0.25％胰酶/0.1EDTA消化后，用细胞培养基 制成细胞悬液并调整细胞悬液浓度，接种于96孔细胞培养板，每孔100μl细胞悬液，细胞密度为2×103个/ml。每块96孔板四周边孔均用PBS填充，以防止“边缘效应”。将细胞培养板置于5％CO2/37℃培养箱培养至细胞完全贴壁生长(过夜)。吸弃孔中原培养基上清液，每孔加入100μl本发明化合物工作液，将细胞培养板继续置于培养箱中孵育72h。向每孔中加入10μl浓度为5mg/ml的MTT溶液，培养4h后终止培养，小心吸去孔内培养基，每孔加入100μl DMSO，置摇床上低速振荡10min，使结晶物充分溶解，用酶标仪测定各孔在490nm的OD值。实验中对每个测试设置3个重复孔，同时设立正常对照和空白对照；正常对照组是不含本发明化合物，但含相同操作培养的细胞、相同量的培养基、药物溶解介质、MTT和DMSO的孔，空白对照组除不含细胞外，其他与正常对照组相同。利用以下公式计算存活率：
细胞存活率(％)＝[(给药孔OD值-空白孔OD值)/(对照孔OD值-空白孔OD值)]×100％
(3)结果：在实验测定的时间范围内，1×10-5mol/L的本发明系列化合物对于293T正常细胞系细胞均无明显的细胞毒性，除化合物1的细胞存活率为98.5％以及化合物1'的细胞存活率为89.7％以外，其他所考察的本发明化合物2-9和2'-4'的细胞存活率均大于100％，统计学上检测无显著性差异。
实验例5：本发明化合物在体外培养的正常人胚肺成纤维细胞HELF细胞和小鼠胚胎成纤维细胞NIH3T3细胞上的细胞毒理学评价实验。
(1)本发明化合物的配制：用DMSO将本发明化合物溶解配制成1×10-2mol/L(0.01M)的储备液，待进行细胞实验时，用细胞培养基稀释成浓度为20μM的初始液。
(2)实验方法(MTT法)：按细胞说明进行细胞初培养、消化、制备细胞悬液、细胞计数、在96孔细胞培养板上接种细胞悬液(100μl)、于5％CO2/37℃细胞培养箱中培养至细胞完全贴壁生长的操作。取化合物初始液，用细胞培养基稀释成浓度分别为20μM、2μM、0.2μM、0.02μM和0.002μM的系列浓度梯度溶液。将此化合物系列浓度梯度溶液分别加入至96孔板中，每孔100μl，吹打后分别再吸弃100μl，得到作用浓度分别为10μM、1μM、0.1μM、0.01μM和0.001μM的系列浓度梯度，每个给药浓度设置3个复孔，同时设置正常细胞对照组和空白对照组。于培养箱中孵育96h后，通过MTT法检测各化合物的OD值， 利用以下公式计算抑制率：
细胞生长抑制率(％)＝{[(对照孔OD值-空白孔OD值)-(给药孔OD值-空白孔OD值)]/[(对照孔OD值-空白孔OD值]}×100％
实验结果使用GraphPad Prism version 5.0软件进行分析。
(3)结果：在实验测定的96h时长范围内，本发明系列化合物对于人胚肺成纤维细胞HELF细胞和小鼠胚胎成纤维细胞NIH3T3细胞均无明显的细胞毒性，各测定实验细胞生长抑制率以及各化合物IC50值结果见表6。
表6 本发明化合物对正常HELF和NIH3T3实验细胞的毒性检测结果
Figure PCTCN2022080897-desc-000018-original
(4)结论：本发明系列化合物对人胚肺成纤维细胞HELF细胞和小鼠胚胎成纤维细胞NIH3T3细胞的生长无明显毒性，适于进行下游活性筛选实验。
四、本发明化合物在细胞水平上的生物活性评价实验例
实验例6：本发明化合物对LPS刺激诱导的RAW 264.7细胞炎症模型中NO病理性过高产生的抑制效力评价实验例
本实验采用Griess法检测本发明化合物对LPS刺激的RAW264.7细胞炎症模型中NO分泌量的影响。
取采用含FBS及双抗(链霉素和青霉素)的DMEM高糖培养基培养的生长状态良好且处于对数生长期的RAW264.7细胞，加入适量培养基轻轻吹打，制成细胞悬液并调整细胞悬液的细胞密度为6×105个/mL，接种于96孔细胞培养板，每孔接种细胞悬液的量为100μl。将细胞培养板置于5％CO2/37℃培养箱中培养至细胞完全贴壁生长(过夜)，观察可见细胞形态良好。对96孔板设置实验分组，包括各给予本发明化合物组、LPS刺激模型组、正常细胞对照组和空白组。按此实验分组在各给予本发明化合物组中按给药实验操作方法(见“实验例3、本发明化合物对体外培养的小鼠单核巨噬细胞RAW264.7细胞的生长情况影响评价实验”)制成各含本发明化合物的干预实验细胞悬液，每次实验均根据具体实验设置不同的工作浓度，并控制每孔总体积仍为100μl。将96孔板在细胞培养箱中预孵育2h后，再在各给予本发明化合物组和模型组中加入LPS刺激，LPS的加入量为1μg/mL。将96孔板置于细胞培养箱中继续培养，24小时后，分别收集各孔中上清液50μl，加入到新的96孔板孔中，按一氧化氮检测试剂盒说明书中设定的操作程序操作，加入50μL Griess A，1min后，加入50μL Griess B，继续反应15min，然后在酶标仪上540nm处测定OD值，在预先用NaNO2建立标准曲线的基础上，计算样品中亚硝酸钠的浓度，折算各组细胞培养液中NO的含量。
实验结果表明，经各本发明化合物干预处理后，本发明化合物可以剂量依赖性地抑制LPS刺激诱导的RAW 264.7细胞炎症模型中NO的产生(表7-14)。本发明化合物对LPS刺激诱导的RAW 264.7细胞炎症模型中NO的产生的抑制作用明显优于氯化小檗碱型生物碱季铵盐类底物。
表7.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中NO分泌量的影响
Figure PCTCN2022080897-desc-000019-original
Figure PCTCN2022080897-desc-000020-original
a～0。与正常组比较，###P＜0.001；与模型组比较，***P＜0.001
表8.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中NO分泌量的影响
Figure PCTCN2022080897-desc-000021-original
与正常组比较，###P＜0.001；与模型组比，***P＜0.001
表9.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型NO分泌量的影响
Figure PCTCN2022080897-desc-000022-original
与正常组比较，###P＜0.001；与模型组比，***P＜0.001
表10.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型NO分泌量的影响
Figure PCTCN2022080897-desc-000023-original
与正常组比较，###P＜0.001；与模型组比，**P＜0.01
表11.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型NO分泌量的影响注
Figure PCTCN2022080897-desc-000024-original
注与正常组比较，###P＜0.001；与模型组比较，*P＜0.05，**P＜0.01,***P＜0.001
表12.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中NO分泌量的影响注
Figure PCTCN2022080897-desc-000025-original
注与正常组比较，###P＜0.001；与模型组比较，*P＜0.05，**P＜0.01,***P＜0.001
表13.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型NO分泌量的影响注
Figure PCTCN2022080897-desc-000026-original
Figure PCTCN2022080897-desc-000027-original
注与正常组比较，###P＜0.001；与模型组比较，**P＜0.01,***P＜0.001。
表14.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型NO分泌量的影响注
Figure PCTCN2022080897-desc-000028-original
注与正常组比较，###P＜0.001；与模型组比较，**P＜0.01,***P＜0.001。
实验例7：本发明化合物对LPS刺激诱导的RAW 264.7细胞炎症模型中NO病理性过高产生的抑制效力评价实验例
本实验采用Griess法检测本发明化合物对LPS刺激的RAW264.7细胞炎症模型中NO分泌量的影响。
取采用含FBS及链霉素和青霉素的DMEM高糖培养基培养的生长状态良好且处于对数生长期的RAW264.7细胞，加入适量培养基轻轻吹打，制成细胞悬液并调整细胞悬液的细胞密度为6×105个/mL，接种于96孔细胞培养板，每孔接种细胞悬液的量为100μl。将细胞培养板置于5％CO2/37℃培养箱中培养至细胞完全贴壁生长(过夜)，观察可见细胞形态良好。对96孔板设置实验分组，包括各 给予本发明化合物组、LPS刺激模型组、正常细胞对照组和空白组。按此实验分组在给予本发明化合物组中按给药实验操作方法(见“实验例3、本发明化合物对体外培养的小鼠单核巨噬细胞RAW264.7细胞的生长情况影响评价实验”)以设定的本发明化合物工作浓度为起始浓度采用倍半比稀释法制成各含本发明化合物的干预实验细胞悬液，或以设定的本发明化合物给药浓度制成各含本发明化合物的干预实验细胞悬液，并控制每孔总体积仍为100μl。将96孔板在细胞培养箱中预孵育2h后，再在各给予本发明化合物组和模型组中加入LPS刺激，LPS的加入量为1μg/mL。将96孔板置于细胞培养箱中继续培养，24小时后，分别收集各孔中上清液50μl，加入到新的96孔板孔中，按一氧化氮检测试剂盒说明书提供的实验操作程序操作，加入50μL Griess A，1min后，加入50μL Griess B，继续反应15min，然后在酶标仪上540nm处测定OD值，在预先用NaNO2建立标准曲线的基础上，计算样品中亚硝酸钠的浓度，折算各组细胞培养液中NO的含量。按下式计算对NO分泌量的抑制率：
抑制率％＝{[(模型组NO量-对照组NO量)-(给药组NO量-对照组NO量)]/[(模型组NO量值-对照组NO量]}×100％
实验结果表明，经各本发明化合物干预处理后，可以剂量依赖性地抑制NO的产生(表15)。
表15.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中NO分泌量的影响注
Figure PCTCN2022080897-desc-000029-original
注与正常组比较，###P＜0.001；与模型组比较，**P＜0.01,***P＜0.001。
实验例8、本发明化合物降低LPS刺激诱导的RAW 264.7细胞炎症模型中IL-6病理性过量分泌水平表型的效力评价实验例
本实验采用ELISA法检测本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6病理性过量分泌的影响。
按小鼠IL-6酶联免疫吸附检测试剂盒中说明书提供的实验操作程序所设定的样品量，根据实验所测定本发明化合物的细胞毒性等数据，取按“实验例6、本发明化合物对LPS刺激诱导的RAW 264.7细胞炎症模型中NO病理性过高产生的抑制效力评价实验例”的操作程序制备的LPS刺激诱导的RAW 264.7细胞炎症模型的细胞上清液样品，加入到小鼠IL-6酶联免疫吸附检测试剂盒中提供的含靶蛋白特异单克隆捕获抗体的酶标板孔中，按试剂盒说明书所提供的操作程序操作，对各孔细胞培养上清液中IL-6的含量进行测定。
由表16-19的实验结果数据可见，以正常对照组中IL-6的表达量为参考，模型组中的IL-6的表达量明显升高，与正常对照组比较统计学上具有极显著差异性。经各本发明化合物处理后，LPS刺激诱导的RAW264.7细胞炎症模型中IL-6的表达量显著下降，并且与模型组相比，各本发明化合物处理组对IL-6的表达量的抑制作用均具有显著性差异，并显著强于相应的小檗碱型生物碱季铵盐底物。
表16.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6表达量的影响注
Figure PCTCN2022080897-desc-000030-original
注与正常组比较，###P＜0.001；与模型组比较，**P＜0.01,***P＜0.001；与氯化巴马汀季铵盐组比较，&P＜0.05。
表17.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6表达量的影响注
Figure PCTCN2022080897-desc-000031-original
Figure PCTCN2022080897-desc-000032-original
注与正常组比较，###P＜0.001；与模型组比较，*P＜0.05，**P＜0.01,***P＜0.001。
表18.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6表达量的影响注
Figure PCTCN2022080897-desc-000033-original
注与正常组比较，###P＜0.001；与模型组比较，***P＜0.001。
表19.本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6表达量的影响注
Figure PCTCN2022080897-desc-000034-original
Figure PCTCN2022080897-desc-000035-original
注与正常组比较，###P＜0.001；与模型组比较，*P＜0.05，**P＜0.01,***P＜0.001。
实验例9：本发明化合物对肿瘤细胞生长的抑制作用实验例。
实验结果表明，本发明化合物可以剂量依赖性地且选择性地抑制人结肠癌HCT-116细胞和人肺癌A549细胞的生长。
(1)本发明化合物对HCT-116细胞生长的抑制作用
用DMSO将本发明各化合物配制成0.02M浓度的储备液，待进行细胞实验时，用细胞培养基(含10％血清的1640培养基)稀释成浓度为20μM的初始液。
按细胞说明用细胞培养基进行细胞初培养、制备细胞悬液、细胞计数、在96孔细胞培养板上接种细胞悬液(100μl)、于5％CO2、37℃细胞培养箱中培养至细胞完全贴壁生长的操作。取化合物初始液，用细胞培养基稀释成浓度分别为20μM、2μM和0.2μM的系列浓度梯度溶液。将此化合物系列浓度梯度溶液分别加入至96孔细胞培养板中，每孔100μl，吹打均匀，然后分别再从每孔中吸弃100μl，得到作用浓度分别为10μM、1μM和0.1μM的系列浓度梯度，每个给药浓度设置3个复孔，同时设置正常细胞对照组和空白对照组。于培养箱中孵育96h后，通过MTT法检测各化合物在570nm波长的OD值，利用以下公式计算抑制率：
细胞生长抑制率(％)＝{[(对照孔OD值-空白孔OD值)-(给药孔OD值-空白孔OD值)]/[(对照孔OD值-空白孔OD值]}×100％
在实验测定的96h时长范围内，本发明系列化合物对于HCT-116细胞有明显的细胞生长抑制活性，所检测的本发明化合物6、8、10、1'、3'和4'对HCT-116细胞的生长抑制作用的IC50值分别为1.286μM、1.195μM、2.248μM、2.637μM、0.594μM和2.462μM；在平行的实验中，氯化小檗碱季铵盐、氯化黄连碱季铵盐和氯化异黄连碱季铵盐对HCT-116细胞的生长抑制作用的IC50值分别为 4.496μM、2.087μM和2.512μM。
实验结果使用GraphPad Prism version 5.0软件进行分析。
(2)本发明化合物对A549细胞生长的抑制作用
用DMSO将本发明化合物配制成0.02M的化合物储备液，待进行细胞实验时，用细胞培养基稀释成浓度为20μM的初始液。
按细胞说明用细胞培养基进行细胞初培养、制备细胞悬液、细胞计数、在96孔细胞培养板上接种细胞悬液(100μl)、于5％CO2、37℃细胞培养箱中培养至细胞完全贴壁生长的操作。取本发明化合物初始液，用细胞培养基稀释成浓度分别为20μM、2μM和0.2μM的系列浓度梯度溶液。将此化合物系列浓度梯度溶液分别加入至96孔细胞板中，每孔100μl，吹打均匀，然后分别再从每孔中吸弃100μl，得到作用浓度分别为10μM、1μM和0.1μM的系列浓度梯度，每个给药浓度设置3个复孔，同时设置正常细胞对照组和空白对照组。于培养箱中孵育96h后，通过MTT法检测各化合物在570nm波长的OD值，并计算抑制率。
在实验测定的96h时长范围内，本发明系列化合物对于A549细胞有明显的细胞生长抑制活性，所检测的本发明化合物6、8、1'、3'和4'对A549细胞的生长抑制作用的IC50值分别为3.114μM、2.319μM 4.378μM、3.497μM和0.919μM；在平行的实验中，氯化小檗碱季铵盐和氯化黄连碱季铵盐对A549细的生长抑制作用的IC50值分别为＞10μM和6.540μM。
五、本发明化合物在动物水平上的生物活性评价实验例
本发明化合物可用于制备免疫调节剂药物，制备用于预防、缓解和/或治疗引起生物机体分泌IL-6病理性过量升高的疾病的药物，制备用于预防、缓解和/或治疗引起生物机体分泌NO病理性过量升高的疾病的药物，制备用于预防、缓解和/或治疗引起生物机体形成自身抗体病理性升高的疾病的药物。关于所确定的本发明化合物可用于制备免疫调节剂药物的用途和制备预防、缓解和/或治疗引起生物机体分泌NO病理性升高的疾病的药物的用途已在前述有关实验例予以证明，以下将对本发明化合物用于制备预防、缓解和/或治疗引起生物机体分泌IL-6病理性过量升高的疾病的药物产品，制备预防、缓解和/或治疗引起生物机体形成自身抗体病理性升高的疾病的药物产品，制备预防、缓解和/或治疗炎症病、发热病、心脑血管和血液系统疾病、自身免疫性疾病、肿瘤病的药物产品进行实验例的展示。同时，这些实验例进一步补充说明了本发明化合物对于平衡 机体免疫反应的重要生物学意义，因此，可用于制备免疫调节剂药物。
类风湿性关节炎的典型症状包括关节炎症、血清中IL-6水平显著升高、病理性自身抗体的形成；贫血是类风湿性关节炎最常见的关节外症状之一。因此，本发明采用类风湿性关节炎动物模型对本发明化合物在动物水平上降低病理性高IL-6含量、升高血红蛋白含量和降低病理性自身抗体水平的效力进行评价，并同时采用本发明化合物对模型动物类风湿性关节炎炎症程度指数评分(简称关节炎症指数评分)、关节炎症发病率和脚掌厚度的影响为评价指标，评价本发明化合物对类风湿性关节炎的疗效。对本发明化合物应用于抗肿瘤药物的用途，采用小鼠4T1细胞皮下移植瘤三阴性乳腺癌模型进行评价。
实验例10：本发明化合物降低模型动物IL-6水平高表达表型的效力评价实验例
1、实验材料
(1)实验动物：
7-8周龄DBA/1小鼠，雄性，体重18-20g。
(2)受试本发明化合物和试剂：
受试本发明化合物为化合物7，阳性药为甲氨蝶呤。其他试剂包括鸡II型胶原蛋白，完全弗氏佐剂，不完全弗氏佐剂，磷酸缓冲盐溶液(PBS)。
根据实验动物体重，将本发明化合物7采用0.5％CMC-Na水溶液为溶媒进行配制得浓度适当的本发明化合物给药工作液，将阳性对照药采用0.5％CMC-Na水溶液为溶媒进行配制得浓度适当的阳性对照药给药工作液。给药容量均按1ml/100g(给药工作液体积/动物体重)剂量，灌胃给药于实验DBA/1小鼠。
2、试验方法
(1)动物模型的制备
在允许自由饮水的环境下将DBA/1小鼠饲养于实验动物房，实验动物房的室温为22℃～25℃，相对湿度为55～65％，光照周期为12h/12h。购置动物后首先进行一周的适应性饲养。
将对动物的初次免疫记录为第0天，方法是于动物的尾根部2～3cm处皮内多点注射0.1ml胶原与完全弗氏佐剂按体积比1:1经超声配制的乳化剂，空白对照组注射0.1ml生理盐水。然后在初次免疫后的第21天通过用胶原和不完全佐剂加强免疫诱发关节炎，方法是于尾部皮下避开初次免疫部位多点注射0.05ml按体积比1:1经超声配制的乳剂。在初次免疫的第4周后小鼠四肢中任一踝关节、 脚掌以及脚趾出现肿胀视为小鼠关节炎模型发病。
(2)动物分组及给药
本实验将动物随机分组为空白对照组(正常对照组)、模型组、阳性药对照组和受试本发明化合物低/中/高剂量各给药组，共6组。本发明化合物低/中/高剂量各给药组给药剂量设置为50mg/kg、100mg/kg和200mg/kg(化合物量/动物体重)，给药次数和给药途径设置为1次/日，p.o.。阳性药甲氨蝶呤的给药剂量、给药次数和给药途径设置为1mg/kg(化合物量/动物体重)，2次/周，p.o.。
如图1所示，从小鼠造模第二次免疫后的第18天开始给药，连续给药4周至给药组与模型组比较关节炎症状明显减轻，关节炎症指数评分具有显著性差异后终止给药。空白对照组和模型组灌胃给予空白溶媒，受试本发明化合物组灌胃给予相应剂量本发明化合物给药工作液，阳性对照药组灌胃给予相应剂量的阳性对照药给药工作液。
(3)评价指标
在实验终点，将小鼠麻醉，摘取眼球，经眼眶收集各组动物的血样，抗凝，取抗凝血，采用Mouse IL-6 ELISA Kit法检测小鼠血清中的炎性细胞因子IL-6水平的变化情况，并通过统计学处理对不同组别的实验结果进行比较。
(4)数据处理与分析
使用GraphPad Prism version 5.0软件进行分析并作图呈现。
3、实验结果
与空白组相比，模型组各检测小鼠血清中IL-6水平显著上升，且具有统计学差异，说明造模成功；与模型组相比，甲氨蝶呤阳性药组、本发明化合物7低/中/高剂量给药组各检测小鼠血清中的IL-6水平均显著下降，且均具有统计学差异。本发明化合物7低/中/高剂量给药组对模型小鼠血清中IL-6水平的抑制作用显著优于阳性药按具体特性给药的给药组。结果如表20和图2所示。
表20.受试本发明化合物7对模型小鼠血清中IL-6浓度的影响(pg/mL)。
Figure PCTCN2022080897-desc-000036-original
注：与正常对照组比，###p<0.001；与模型组比较，**p<0.01，***p<0.001。
实验结果表明，受试本发明化合物对模型小鼠血清中IL-6的水平具有极显 著的抑制作用。
实验例11：本发明化合物升高模型动物血红蛋白含量的效力评价实验例
在“实验例10：本发明化合物降低模型动物IL-6水平的效力评价实验例”实验中，将各组小鼠于实验终点进行麻醉并摘眼球取血，将所取血液用EDTA-二钾抗凝后取抗凝血按血常规检测法进行检测。检测结果表明，本实验空白对照组小鼠血红蛋白含量的平均值为147.29±5.06(g/L)；与空白对照组比较，本实验模型组动物的血红蛋白含量明显降低，平均数值为136.33±4.27(g/L)；与模型组比较，受试本发明化合物各剂量组对血红蛋白含量均有明显升高作用，本发明化合物7低/中/高剂量给药组的血红蛋白含量的平均值分别为180.18±4.45(g/L)***、182.75±2.22(g/L)***和182.75±2.42(g/L)***，与模型组比较均具有极显著性差异(与模型组比较，***p<0.001，见图3)。特别是与空白对照组比较，本发明化合物各剂量组对血红蛋白也均有明显升高作用，表明本发明化合物具有显著的升高血红蛋白的作用。按照阳性药具体特性给药的阳性药组的血红蛋白含量平均值为160.92±7.07(g/L)。
实验结果表明，受试本发明化合物对模型小鼠血红蛋白含量具有极显著的升高作用。
实验例12：本发明化合物抗类风湿性关节炎药效学作用评价
(1)评价指标
在“实验例10：本发明化合物降低模型动物IL-6水平的效力评价实验例”实验中，继续采用类风湿性关节炎症指数评分测算、关节炎症发病率统计、模型小鼠的脚掌厚度检测和对模型小鼠自身抗体的影响检测，结合对模型动物IL-6水平和血红蛋白水平的影响的检测实验例，评价本发明化合物的抗类风湿性关节炎药效作用。
在评价实验中，从加强免疫后的第8天开始至实验终点，每隔天对各组小鼠关节进行关节炎症指数评分测算评价。每只小鼠四肢评分相加总评分为小鼠关节炎症指数评分，类风湿性关节炎症指数评分与疾病严重程度的关系见表21。
表21.类风湿性关节炎症指数评分的测算标准
Figure PCTCN2022080897-desc-000037-original
Figure PCTCN2022080897-desc-000038-original
从加强免疫后的第8天开始至实验终点，在每隔天对小鼠进行关节炎症指数评分测算的同时记录每组小鼠关节炎症发病的小鼠数量，进行发病率的统计。
从加强免疫后的第8天至实验终点，在每隔天对小鼠进行关节炎症指数评分测算的同时利用游标卡尺对小鼠两个后脚掌进行厚度的测量。
在实验终点，将小鼠麻醉，经眼眶取血收集各组动物的血样，抗凝后，采用ELISA方法检测血清中鸡II型胶原蛋白抗体含量。
(2)数据处理与分析
实验数据以X±SEM表示。连续型数据的比较采用方差分析法继以Duncan’s检验；离散型数据采用非参数检验中的Kruscal-Wallis方差分析和中位数检验。实验结果使用GraphPad Prism version 5.0软件进行分析并作图呈现。
3、实验结果
(1)受试本发明化合物能明显改善小鼠关节炎症指数评分
从造模后第28天开始，每隔天对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠四肢及关节的关节炎症状程度进行关节炎症指数评分测算与评价。
实验结果表明，造模后第21天动物开始出现继发病变，第28天更加明显，表现为以多发性关节炎症为特征的慢性周身性炎症，肉眼可见四肢均有不同程度的关节红肿变形，在非致炎侧的足肿胀症状也很明显，累及踝关节以及整个足趾，于足趾关节间和前足趾出现关节肿大，并伴有耳部红斑、尾部结节等。实验于造模后第38天开始给药。实验结果显示，与模型组相比，从实验给药开始后的第4天(即初次免疫开始后的第42天)至实验终点，本发明化合物7给药剂量为100mg/kg的中剂量组的关节炎症指数评分始终低于阳性药甲氨蝶呤，而从实验给药开始后的第4天至第18天，给药剂量为50mg/kg的低剂量组的关节炎症指数评分始终与阳性药甲氨蝶呤相当，并且从第18天开始，低剂量组的关节炎症 指数评分始终低于阳性药甲氨蝶呤，而且也低于中剂量组(即疗效优于中剂量组)，直至实验终点。与模型组相比，本发明化合物7的给药剂量为200mg/kg的高剂量组的关节炎症指数评分也显著较低，并且从给药开始后的第20天开始，与阳性药甲氨蝶呤的治疗效果也相当。在实验终止时，低/中/高剂量组的关节炎症指数评分分别为1.73±0.68***、2.67±0.80***和3.58±0.66***，与模型组的关节炎症指数评分6.92±0.84相比，均显著降低，且均具有极显著性差异。综合分析，本发明化合物7低/中/高三个剂量组均有明显改善模型动物关节炎症病情的作用，其中，随着治疗期的延长，低剂量组对模型动物病情的改善作用最明显，中剂量组对模型动物病情的改善作用较低剂量组弱，但优于高剂量组，这一独特的量效关系进一步表明了本发明化合物独特的作用机制。本发明化合物明显改善小鼠关节炎症指数评分的具体数据和图示结果如下表22和图4所示。
表22.本发明不同给药组对实验小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠关节炎症指数评分的影响.
Figure PCTCN2022080897-desc-000039-original
注：n＝12；与模型组比较，*p<0.05，**p<0.01，***p<0.001。
(2)受试本发明化合物对实验动物关节炎症发病率具有显著的抑制作用
本实验在对模型动物给药治疗过程中通过对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠关节炎症发病率进行统计分析，获得了各组动物关节炎症的发病率数据。整个治疗周期中各组小鼠关节炎症发病率情况如表23所示。截止到开始给药后的第28天(即初次免疫造模后的第66天)，模型组动物关节炎症的发病率为100％，甲氨蝶呤阳性药组小鼠关节炎症的发病率为75％，本发明化合物7低/中/高各剂量组小鼠关节炎症的发病率分别为45％/58％/83％。因此，本发明化合物对小鼠关节炎症发病率有明显的抑制作用，可以显著改善模型动物关节炎症的病情，特别是低剂量组对模型动物病情的改善作用最明显，中剂量组对模型动物病情的改善作用较低剂量组弱，但优于高剂量组，且仍显著优于阳性药组。关节炎症的发病率数据进一步说明了本发明化合物的独特的作用机制。
表23.本发明化合物对实验动物关节炎症发病率的影响
Figure PCTCN2022080897-desc-000040-original
注：n＝12。
(3)受试本发明化合物对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠的脚掌厚 度的影响
本实验在整个给药治疗过程中，利用微量游标卡尺对各组小鼠脚掌的厚度进行了测量，并进行了统计分析。各组小鼠脚掌厚度的动态变化数据见表24。由结果可见，小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠踝关节明显肿胀，受试本发明化合物有明显抑制踝关节肿胀的作用，其中，本发明化合物中剂量组在造模后第40天(即给药后第3天)即表现出了明显的作用，与模型组比较有显著性差异，效果优于阳性药组。本发明化合物低剂量组在造模后第46天(即给药后第9天)表现出了明显的作用，与模型组比较有显著性差异；并且随着治疗过程的延长，低剂量组的作用逐渐超过了中剂量组；从造模后第56天(即给药后第19天)开始，低/中剂量组的作用均超过了阳性药组。本发明化合物高剂量组对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠的脚掌厚度也显示出明显的改善作用，并与阳性药组的作用相当。模型动物脚掌厚度数据进一步说明了本发明化合物的独特的作用机制。
表24.本发明化合物对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠的脚掌厚度(mm)的影响
Figure PCTCN2022080897-desc-000041-original
Figure PCTCN2022080897-desc-000042-original
注解：n＝12；与模型组比较，*p<0.05，**p<0.01，***p<0.001。
(4)受试本发明化合物对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠自身抗体水平的影响
类风湿性关节炎等自身免疫性疾病均是由于自身抗体的产生而损伤组织引起的一类慢性疾病。本研究考察了受试本发明化合物7对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠自身抗体水平的影响。将受试小鼠的血清利用试剂盒中制定的血清稀释液，以1:1000的比例进行稀释，利用ELISA法检测稀释后的血清中抗鸡II型胶原抗体表达情况，结果数据见表25和图5。由结果可见，与空白对照组相比，模型组小鼠血清中的抗鸡II型胶原IgG抗体浓度显著升高；与模型组相比，阳性药甲氨蝶呤组、本发明化合物7低/中剂量组的小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠血清中的抗鸡II型胶原IgG抗体浓度均显著降低，其中，与模型组比较，阳性药组对抗鸡II型胶原IgG抗体水平的降低作用具有显著性差异，而本发明化合物7低剂量组对抗鸡II型胶原IgG抗体水平的降低作用优于按照阳性药具体特性给药的实验组，与模型组比较具有极显著性差异，本发明化合物7中剂量组的作用略低于低剂量组，但也优于阳性药组，与模型组比较具有显著性差异。虽然本发明化合物7高剂量组的作用与模型组比较没有统计学差异，但其对抗鸡II型胶原IgG抗体的降低作用的趋势也非常明显。
表25.本发明化合物7对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠血清中的抗鸡II型胶原IgG抗体水平的影响。
Figure PCTCN2022080897-desc-000043-original
注：#为与空白对照组比较，###p<0.001；*为与模型组比较，*p<0.05，**p<0.01，***p<0.001。
根据以上实验结果，本发明化合物对实验动物具有明显的抗类风湿性关节炎的作用，其中，在实验选定的剂量范围内，本发明化合物7的低/中/高剂量均表现出显著的或明显的作用，主要表现在对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠关节炎症指数评分、关节炎症发病率和脚掌厚度具有明显的改善作用，即显著改善了模型小鼠关节炎症的临床症状、显著降低了模型动物关节炎症发病率、 显著降低了模型动物的脚掌厚度。
此外，结合本发明化合物在细胞水平上的各个实验以及在动物水平上的生物活性评价实验，即经本发明化合物7干预治疗后的小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠血清中IL-6的含量显著降低，同时对IgG抗体的产生也有显著的抑制作用，均进一步表明本发明化合物具有显著的抗自身免疫性疾病的活性，并具有平衡机体免疫功能的作用。由于类风湿性关节炎在心血管系统疾病的发展中也起着重要作用，常造成包括贫血症等在内的血液系统功能异常，因此，结合本发明化合物在动物水平上的生物活性评价实验例，即经本发明化合物7治疗后的小鼠鸡II型胶原蛋白诱导性关节炎模型动物的血液中的血红蛋白含量显著升高，不仅进一步表明本发明化合物具有抗类风湿性关节炎的作用，同时也表明本发明化合物对于心血管系统疾病也具有显著的干预作用，可用于包括贫血症在内的心血管系统疾病的治疗。需要进一步强调的是，在实验选定的剂量范围内，总体上，本次实验的低剂量组的抗类风湿性关节炎作用最强，中剂量组的作用比低剂量组弱，但强于高剂量组，呈现非常规的量效关系。后期，需要进一步考察本发明化合物更低剂量的作用，以便获得钟形量效关系曲线，同时开展作用机制的研究。但是，可以推测，本发明化合物具有独特的作用机制，包括对于其作用靶标具有的不同的敏感性。
实验例13：本发明化合物对酵母致大鼠发热模型的解热作用药效学评价实验例
1、试验材料
(1)动物：体重在180-200g范围的雄性SD大鼠。
(2)受试本发明化合物和试剂：
受试本发明化合物为化合物7，给予剂量设定为100mg/kg。阳性药为复方对乙酰氨基酚片(又名散利痛)，规格为对乙酰氨基酚250mg/异丙安替比林150mg/无水咖啡因50mg，给药剂量设定为45mg/kg。诱导大鼠发热模型试剂为酵母；配制酵母溶液的溶媒为0.9％氯化钠注射液。
本发明化合物和阳性药均采用0.5％CMC-Na水溶液为溶媒进行配制得工作液，按1ml/100g(工作液/动物体重)剂量灌胃给药。
(3)试验耗材：
电子体温测量仪，型号为MC-246。体重计，型号为H21201A。大鼠灌胃针，2.5ml注射器。
2、试验方法
(1)酵母致大鼠发热模型的诱导
将SD大鼠在实验动物房进行适应性饲养3天后，每日上午测量肛温2次并记录为实验动物体温，测定的时长间隔为30min，取平均体温，并连续测定2天，取两天所测体温的平均值作为基础体温，同时使大鼠适应测温操作。实验时，选取基础体温为36℃～38℃且体温波动<0.6℃的大鼠进入实验，实验前12h对大鼠禁食。造模时于大鼠背部皮下注射以0.9％氯化钠注射液为溶媒配成的20％酵母混悬液，剂量容积为10ml/kg(酵母混悬液/大鼠体重)，建立大鼠发热模型。空白对照组大鼠注射对应容积溶媒(注：所有动物实验的规程均按照实验动物伦理委员会指导方针严格执行，并通过伦理委员会审批)。
(2)动物分组与给药
在大鼠皮下注射20％酵母混悬液5h后，选取体温上升1.0℃～3.0℃之间的大鼠进入实验。包括皮下仅注射对应容积溶媒的动物，将实验动物随机分为空白对照组(正常对照组)、模型组、阳性药组和受试本发明化合物7给药组共4组，每组12只实验鼠。给药时，受试本发明化合物组和阳性对照组实验动物分别灌胃给予按给药剂量配制的相应的受试本发明化合物CMC-Na给药工作液或阳性对照药CMC-Na给药工作液，空白对照组和模型对照组均灌胃给予等体积的空白溶媒，灌胃体积为10ml/kg(酵母混悬液/大鼠体重)，于给药后0h、1h、2h、4h、6h分别测量大鼠肛温并记录。本发明化合物组和阳性药组给药次数和给药途径均设置为1次/日，p.o.。
(3)评价指标
于给药后的0h、1h、2h、4h、6h分别测量大鼠肛温并记录；计算各时间段所测体温与基础体温的差值ΔT(℃)并记录。通过与正常对照组和模型组的相应数据进行比较，确定本发明化合物的解热作用。
(4)数据处理与分析
实验数据以Mean±SEM表示。连续型数据的比较采用方差分析法(ANOVA)继以Duncan’s检验；离散型数据采用非参数检验中的Kruscal-Wallis ANOVA and Median Test检验。实验结果使用GraphPad Prism version 8.0软件进行统计学分析并作图呈现。
3、试验结果
根据给药前测定记录的基础体温，各组大鼠基础体温平均值在37.55℃～37.88℃之间，平均值相差较小。在整个实验过程中，空白对照组的体温始终保持在37.34℃～37.83℃之间。造模5小时后，各造模组体温上升至39.60℃～39.75℃之间，各组上升情况基本一致。在造模后6小时(给药1小时)，与模型组比较，阳性药散利痛给药组体温出现了极显著性下降，本发明化合物7给药组体温出现显著性下降。在造模后7小时(给药2小时)，阳性药组体温已开始出现回升，当然，相比于模型组，依旧保持极显著性差异；本发明化合物7给药组与阳性药不同，在造模后7小时(给药2小时)后体温进一步出现下降，与上个观测点比较体温下降差异更加明显，且与模型组比较具有了极显著性差异；在造模后9小时(给药4小时)，阳性药组的体温继续显著回升，且回升速度加快，与模型组比较统计学上已经没有差异性；本发明化合物7组的体温虽然也开始出现回升，但回升幅度明显小于阳性药组，且与模型组比较仍具有显著性差异，表明仍具有显著性解热作用；在造模后11小时(给药6小时)，各给药组的体温均回升至模型组水平，但化合物7组的体温低于阳性药组。模型组最高体温平台期自造模后5小时维持到最后一个体温检查点，且模型组动物自给药组动物给药后每个时间点体温与空白对照组均具有显著性差异，说明造模成功(模型组体温走势也与文献中报道相同)，模型稳定。阳性药散利痛组实验结果符合其药理效果和临床认知。本实验具体记录的体温变化数据如表26和图6所示(括号外为按照造模所计算时间，括号内为按照给药所计算时间)。
表26.本发明化合物7在动物实验中解热作用药效学评价实验体温统计表
Figure PCTCN2022080897-desc-000044-original
注：各组n＝12；#为与空白对照组比较，###p<0.001；*为与模型组比较，**p<0.01，***p<0.001。
通过计算比较给于本发明化合物干预后各时间段的大鼠体温与给药前记录的大鼠基础体温的差值(该差值简称体温差值)可以更加直观展示本发明化合物 干预后对于酵母致大鼠发热模型动物体温的影响，如表27所示。
在造模5小时后且开始给于本发明化合物前，各组大鼠体温平均上升1.74℃～1.95℃，模型稳定，趋势一致。给药1小时后，阳性药散利痛组体温差值为0.23±0.77***，接近基础体温，并且与模型组比较具有显著性差异，表明阳性药有效且造模成功；本发明化合物7组体温差值幅度有确切减小，但是与模型组比较还无统计学差异。在给药2小时后情况出现变化，阳性药散利痛组体温差值幅度较上个观测点明显增大，但体温差值与模型组比较依旧保持极显著性差异，表明阳性药依然有解热作用，但解热作用在给药后2小时已经开始出现减弱情况。与阳性药不同，本发明化合物7组体温差值幅度在给药后2小时则进一步减小，较上个观测点差异更加明显，与模型组比较已具有极显著性差异，表明本发明化合物的解热作用在给药后2小时仍然继续增强，并且作用更加显著。给药4小时后，阳性药散利痛组体温差值幅度进一步显著增大，达到2.05±0.52，与模型组比较已没有统计学差异；本发明化合物7组体温差值幅度也出现了增大，但本发明化合物7组体温差值幅度已明显小于阳性药组，为1.58±0.83。给药6小时后，阳性药组和本发明化合物给药组体温差值幅度均进一步增大，阳性药组达到2.46±0.53，已超过模型组，且与模型组比较无统计学意义；本发明化合物7组体温差值幅度为1.86±0.68，小于阳性药组的体温差值；虽然与模型组比较，本发明化合物7组体温差值也没有显著性统计学差异，但本发明化合物7组体温差值幅度明显小于模型组、小于阳性药组，即，虽然与模型组比较没有显著性差异，但本发明化合物的解热作用持续时间更长，持续时间长的趋势较阳性药组更加明显。
表27.本发明化合物7解热作用药效学评价实验大鼠体温差值变化统计表
Figure PCTCN2022080897-desc-000045-original
注：各组n＝12；#为与空白对照组比较，###p<0.001；*为与模型组比较，**p<0.01,***p<0.001。
综上所述，本发明化合物具有显著的解热作用，且解热作用比阳性药具有更长的持续时间。
六、本发明化合物抗病毒药效学作用评价
实验例14：本发明化合物抗2019新型冠状病毒(SARS-CoV2)效力测定实验例
1、设备与试剂
II级生物安全柜；CO2培养箱；倒置显微镜；96孔细胞培养板。
SARS-CoV2(即2019-nCoV)病毒(滴度8×105TCID50/mL)；DMEM基础培养基；Vero E6细胞；胎牛血清；青霉素-链霉素双抗；0.25％胰酶-EDTA；qPCR实验试剂；TRIzol；细胞培养级DMSO。
2、本发明化合物的配制
以DMSO为溶媒将本发明化合物首先溶解配制成1×10-2mol/L(0.01M)的本发明化合物储备液。待进行细胞实验时，用细胞培养基稀释成所需浓度的工作液。
3、数据分析
实验数据使用GraphPad Prism version 5.0软件进行统计学分析。
4、实验方法
(1)本发明化合物在体外培养的Vero E6细胞上的细胞毒理学评价实验。
1)接种细胞：按细胞说明取生长状态良好且处于对数生长期的Vero-E6细胞，吸弃培养液，用胰酶消化细胞，制备细胞悬液，细胞计数为1×106个/ml；取上述细胞4ml，加入细胞培养基6ml，制备得到细胞密度为4×105个/ml的细胞悬液，接种到96孔细胞培养板上(每孔100μl细胞悬液，细胞个数为1×104个)；于5％CO2/37℃细胞培养箱中培养至细胞完全贴壁生长。
2)本发明化合物的稀释：
显微镜观察细胞毒性实验：使用完全培养基将本发明化合物储备液稀释至相应浓度范围的工作液。
本发明观察了本发明化合物在20μM、10μM、5μM的作用浓度下对细胞的毒性作用。
半数毒性浓度(CC50)检测实验：使用完全培养基将本发明化合物储备液稀释至相应浓度的工作液。
本发明在320μM、160μM、80μM、40μM、20μM、10μM、5μM、0或320μM、160μM、80μM、40μM、20μM、10μM、5μM、2.5μM、1.25μM、0.625μM、0.31μM、0.1625μM、0的系列浓度梯度下开展本发明化合物的细胞毒性检测，计算CC50值。
3)本发明化合物对细胞的干预处理：吸弃细胞原始培养基，加入稀释好的含相应浓度本发明化合物的培养基，每孔总体积100μl，置于37℃细胞培养箱，继续培养48小时。实验中同时设置正常细胞对照组和空白对照组，正常细胞对照组含相同操作培养的细胞和相同量的药物溶解介质，但不含本发明化合物；空白对照组除不含细胞外，其他与正常对照组相同。
4)弃去培养基上清液，每孔加入100μl含有10％CCK-8的无血清培养基，在培养箱内孵育4小时后，用酶标仪测定在450nm处的OD值。根据实验结果判断细胞生长抑制率，即本发明化合物的细胞毒性。细胞生长抑制率的计算公式为：
细胞生长抑制率(％)＝{[(对照孔OD值-空白孔OD值)-(给药孔OD值-空白孔OD值)]/[(对照孔OD值-空白孔OD值]}×100％
(2)本发明化合物抗SARS-CoV2病毒效力评价
1)接种细胞：按细胞说明取生长状态良好且处于对数生长期的Vero-E6细胞，吸弃培养液，用胰酶消化细胞，制备细胞悬液，细胞计数为1×106个/ml；取上述细胞4ml，加入培养基6ml，制备得到细胞密度为4×105个/ml的细胞悬液，接种到96孔细胞培养板上(每孔100μl细胞悬液，细胞个数为1×104个)；于5％CO2/37℃细胞培养箱中培养至细胞完全贴壁生长。
2)本发明化合物的稀释：将本发明化合物储备液以DMEM维持培养基(2％FBS)稀释至相应浓度的工作液。
在不同的实验中，本发明化合物的作用浓度为10μM或者20μM、10μM、5μM、0μM或者40μM、20μM、10μM、5μM、2.5μM、0μM。
3)本发明化合物预处理细胞：在SARS-CoV2病毒感染前，用配置好的本发明化合物工作液按实验设定的本发明化合物作用浓度进行给药干预操作，对细胞进行时长1小时预处理。本实验细胞维持液为DMEM(含2％FBS)。每检测设置3个复孔，以相应浓度的DMSO作为阴性对照，以Remdesivir作为阳性对照。
4)SARS-CoV2病毒稀释：取SARS-CoV2病毒200μl，加入到25ml培养基中，混匀，将SARS-CoV2病毒稀释至100TCID50/0.05mL。
5)本发明化合物+SARS-CoV2病毒处理细胞：SARS-CoV2病毒感染时，弃去细胞培养上清液，每孔加入2倍本发明化合物作用浓度的本发明化合物工作液50μl，同时，除细胞对照外，向每孔垂直悬滴SARS-CoV2病毒稀释液50μl，每 孔总体积100μl。
6)将上述含有本发明化合物和SARS-CoV2病毒混合液的96孔细胞培养板置于5％CO2/37℃细胞培养箱中培养1小时，弃去含本发明化合物和SARS-CoV2病毒的混合液，每孔加入100μl维持培养基，置于细胞培养箱中，继续培养48小时。
7)48小时后收集细胞上清，加入到TRIzol中裂解，提取RNA，进行RT-qPCR定量检测。
检测引物为(5′→3′)：
SARS-CoV-2-N-F:GGGGAACTTCTCCTGCTAGAAT
SARS-CoV-2-N-R:CAGACATTTTGCTCTCAAGCTG
5、实验结果
(1)本发明化合物对正常Vero E6细胞无明显毒性
通过显微镜观测，本实验采用的本发明化合物在20μM、10μM和5μM作用浓度下对实验细胞均无明显细胞毒性。
对本发明化合物3、6、7、8的细胞毒性CC50检测的实验结果见表28。
表28.本发明化合物对正常Vero E6实验细胞的毒性检测结果
Figure PCTCN2022080897-desc-000046-original
Figure PCTCN2022080897-desc-000047-original
对本发明化合物3'在320μM、160μM、80μM、40μM、20μM、10μM、5μM、0μM的系列浓度梯度下开展的细胞毒性检测的细胞生长抑制率分别为78.71％、27.77％、8.51％、1.98％、1.93％、3.14％、1.96％、1.44％，细胞毒性CC50检测的实验结果为311.8μM。
结论：本发明系列化合物对正常Vero E6实验细胞无明显毒性。
(2)本发明化合物对实验细胞感染SARS-CoV2病毒后病毒RNA水平有显著抑制作用
以本发明化合物对实验细胞SARS-CoV2病毒RNA水平的影响为指标，本发明化合物在实验采用的作用浓度下(10μM或20μM、10μM、5μM或40μM、20μM、10μM、5μM、2.5μM)以及在仅处理模型细胞1小时的情况下，对SARS-CoV2病毒具有明确且显著的抑制作用，其中，本发明化合物7和8对SARS-CoV2病毒RNA抑制作用的抑制率在5μM、10μM、20μM的作用浓度下可以分别达到62.99％、93.13％、94.42％和68.08％、89.10％、91.74％。本发明化合物对实验细胞感染SARS-CoV2病毒后病毒RNA水平的影响和EC50值见表29。
表29.本发明化合物对实验细胞感染SARS-CoV2病毒后病毒RNA水平的影响及EC50值
Figure PCTCN2022080897-desc-000048-original
同时，本发明所检测的氯化小檗碱类化合物，包括氯化小檗碱季铵盐、氯化巴马汀季铵盐、氯化黄连碱季铵盐、氯化异黄连碱季铵盐和氯化异小檗碱季铵盐，均不能有效减低SARS-CoV2病毒RNA水平。
(3)结论
根据上述实验结果，本发明化合物在Vero E6细胞感染SARS-CoV2病毒模型中可剂量依赖性地抑制SARS-CoV2病毒RNA复制。其中，在本发明化合物为10μM的作用浓度下，所检测的本发明化合物1、3、4、6-10、1'-3'、5'对实验细胞感染SARS-CoV2病毒后病毒RNA水平的抑制率分别为28.63％、65.66％、32.95％、93.13％、31.00％、89.10％、29.62％、17.45％、61.37％、65.22％、32.01％、57.57％。化合物6、8、3和3'抑制SARS-CoV2病毒RNA水平的EC50值分别为3.037μM、3.767μM、7.859μM和9.69μM。4个化合物的SI值分别为59.53、63.02、31.94、32.18。说明本发明化合物在抑制病毒RNA复制水平上具有显著的抗SARS-CoV2病毒的活性。
七、本发明化合物进一步的抗炎作用评价
实验例15：本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中iNOS 高表达的抑制作用评价实验例
1、材料和方法
(1)细胞：小鼠单核巨噬细胞RAW264.7细胞
(2)实验方法：体外常规培养RAW264.7细胞，经制成细胞悬液、细胞计数，调整细胞浓度为1.0×106个/mL后，接种于6孔板中，每孔2mL，置于37℃、5％CO2的培养箱中培养，24h后，加入无血清培养基饥饿处理，设置实验分组如下，包括正常细胞对照组、LPS刺激模型组、特定浓度的本发明化合物组、以氯负离子为平衡阴离子的天然氯化黄连碱季铵盐组以及与本发明化合物平衡阴离子对应的有机酸组。加入各浓度化合物预孵育2h后，再加入1μg/mL的LPS刺激，置于培养箱中继续培养16h。弃除上清液，加入1mL PBS，制悬液，转移至2mL管中，离心，PBS洗两次，每管加入预冷60μL裂解液(含蛋白酶抑制剂)，于冰上裂解30min，离心，将上清转移至0.5mL离心管中，置于冰上备用。
按照BCA蛋白浓度测定试剂盒说明书操作，首先配置BCA工作液。随后，将BSA蛋白标准品(5mg/mL)用PBS稀释成初始浓度0.5mg/mL，随后稀释成系列梯度浓度(0.4，0.3，0.2，0.15，0.1，0.05，0mg/mL)，加入至96孔板中，每孔20μL；将样品用PBS稀释20倍，加入至孔中，每孔20μL；取200μL BCA工作液加入至各标准品和样品孔中，37℃孵育30min，于562nm处检测吸光度值，根据蛋白标准曲线计算出各组蛋白含量，加入4×蛋白上样缓冲液和PBS，将各组蛋白浓度稀释至5mg/mL。于100℃煮沸10min使蛋白变性，冷却至室温后于-80℃保存备用。
根据蛋白浓度取等量蛋白进行Western-blot检测：按标准SDS-PAGE方法配制5％浓缩胶及10％分离胶。各组取等量蛋白各50μg加入孔道中，上样完毕后开始电泳。电泳结束后按湿转法转移至PVDF膜上。取出PVDF膜，放入5％脱脂奶粉中室温摇床封闭1h。封闭完成后，TBST洗脱3×10min，孵育一抗(用5％BSA稀释1:1000)，于4℃摇床上过夜。TBST洗脱3×10min，孵育二抗(5％脱脂奶粉稀释1:5000)，室温摇床孵育1h，TBST洗脱3×10min。
采用化学发光仪呈像：在提前配置显影液的条件下，打开显影软件预冷。结束后，取出PVDF膜，加入显影液均匀润湿膜表面，运行软件，选取适当的曝光时间采集图像，采用Image J软件进行灰度分析。
本发明化合物在细胞蛋白水平对iNOS的高表达具有显著的抑制作用，与对应的天然氯化小檗碱季铵盐以及与本发明化合物平衡阴离子对应的有机酸比较，作用更强，具体数据见图7。
八、本发明化合物的抗肿瘤动物实验
实验例16，本发明化合物治疗乳腺癌的药效学评价实验例
本实验例分别对本发明化合物8和化合物34进行了治疗乳腺癌的药效学评价
1、本发明化合物8的动物实验
(1)实验动物
本实验动物为体重范围为20±2g的雌性BALB/C小鼠。所有动物实验的规程均按照实验动物伦理委员会指导方针严格执行，并通过伦理委员会审批。在允许自由饮水的环境下将实验小鼠饲养于实验动物房，实验动物房的室温为22℃～25℃，相对湿度为55～65％，光照周期为12h/12h。
(2)实验分组
本实验分为4T1皮下移植瘤三阴性乳腺癌模型组和本发明化合物组，每组8只动物。
(3)受试本发明化合物和给药剂量
受试本发明化合物为化合物8。将受试化合物配制成4mg/ml％的1×PBS混悬液用于进行实验动物灌胃给药，受试化合物的给药剂量为40mg/kg(剂量/动物体重)。给药容量均按1ml/100g(给药工作液体积/动物体重)剂量根据具体动物体重进行给药。
(4)实验
取体外培养至对数生长期的4T1细胞，将上清弃掉并用无菌的PBS清洗2次。用无菌的胰酶对细胞进行消化，待消化好后用RPMI1640培养基(10％FBS)进行终止并用移液器轻轻地将细胞进行吹打制备成单细胞悬液。将细胞悬液移至15ml无菌的离心管中1500rpm离心10min(4℃)。用无菌的PBS轻柔地重悬细胞，并制备成相应的浓度，按1×107细胞/只，接种于实验动物皮下进行4T1皮下移植瘤培植。移植瘤在动物体内生长良好后并达到一定大小后，将动物脱臼处死，用75％EtOH对动物进行消毒，用无菌的手术器械分离肿瘤组织并用无菌 的匀浆器将肿瘤组织重新制备成细胞悬液，并按照1×107细胞/只接种于实验动物皮下进行4T1皮下移植瘤动物模型造模、分组、正常饲养。自造模后第2天开始给药，模型组按体重给予相应量的不含化合物的1×PBS，本发明化合物组给予本发明化合物8的1×PBS混悬液，每天一次。本实验给药时长共26天。
(4)本发明化合物抗三阴性乳腺癌的药效
在整个实验过程中从明显可视见实验动物肿瘤轮廓后开始测量并定期记录肿瘤体积，实验结束后将动物脱臼处死。将动物皮下的肿瘤组织进行分离取出并称重，以模型组动物肿瘤负荷为参考计算本发明化合物抑瘤率。采用实验结束后的抑瘤率等评价指标评价化合物抗乳腺癌的药效作用。
(5)实验结果
本发明化合物8具有明显的抗乳腺癌活性，在最后一次给药后，采用游标卡尺测量的模型组的8只动物的瘤体的平均大小数据为19.54mm/15.04mm(长/宽)，而化合物8组的8只动物的瘤体的平均大小数据为13.95mm/12.0mm(长/宽)，说明化合物对肿瘤的生长起到了抑制作用。实验结束后，模型组的肿瘤负荷平均值是2.139g/只，化合物8的肿瘤负荷平均值是1.094g/只，抑瘤率为48.85％。
2、本发明化合物34的动物实验
本实验操作完全与上述化合物8的实验相同，只是，每组动物是7只，给药时间为17天(至提交专利申请日，实验还在进行中)，通过体外测量发现给药组的瘤体积显著性地小于对照组的瘤体积。采用游标卡尺测量的模型组的7只动物的瘤体的平均大小数据为14.96mm/9.43mm(长/宽)，而化合物34组的7只动物的瘤体的平均大小数据为11.98mm/8.6mm(长/宽)，说明化合物对肿瘤的生长起到了抑制作用。
此外，本实验中动物显示了对受试化合物良好的耐受，表现为动物的体重呈现水平略微上升趋势的表型。通过对肺重量的统计发现肺脏在给药后出现的差异提示给药组可能抑制了肺转移结节的形成。
九、与本发明相关的其他生物活性评价实验例
(一)本发明化合物对正常细胞生长的影响情况评价实验例
实验例17、本发明化合物2'对体外培养的小鼠单核巨噬细胞RAW264.7细胞的生长情况影响评价实验。
(1)化合物2'的配制：用DMSO将化合物配制成1×10-2mol/L(0.01M)的化合物储备液，待进行细胞实验时，用含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基稀释成浓度为20μM的初始液。
(2)实验方法(CCK-8法)：按照细胞说明书，将RAW264.7细胞在含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基中培养，培养条件为37℃/5％CO2，待细胞融合至80％后，按体积比1:3的稀释比对细胞进行传代培养。
取生长状态良好且处于对数生长期的RAW264.7细胞，弃去原培养基，加入适当含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基轻轻吹打，制成细胞悬液并调整细胞悬液的细胞个数为1×105个/mL，接种于96孔细胞培养板，每孔100μl细胞悬液。每块96孔板四周边孔均用PBS填充，以防止“边缘效应”。将细胞培养板置于5％CO2、37℃培养箱中培养至细胞完全贴壁生长(过夜)。
取化合物初始液，用含有10％FBS及双抗(链霉素100μg/mL、青霉素100U/mL)的DMEM高糖培养基稀释成浓度分别为20μM、2μM、0.2μM和0.02μM的系列浓度梯度溶液。将此化合物系列浓度梯度溶液分别加入至含有100μl细胞悬液的96孔板中，每孔100μl，吹打均匀后分别再吸弃100μl，得到作用浓度分别为10μM、1μM、0.1μM和0.01μM的系列浓度梯度。每个给药浓度设置6个复孔。同时设置正常细胞对照组和空白对照组，正常细胞对照组含相同操作培养的细胞和相同量的药物溶解介质，但不含本发明化合物；空白对照组除不含细胞外，其他与正常对照组相同。于培养箱中处理24h后，每孔中加入10μL CCK-8溶液，于培养箱内孵育2h后，用酶标仪于450nm处测定光密度(OD)值。利用以下公式计算存活率：
细胞存活率(％)＝[(给药孔OD值-空白孔OD值)/(对照孔OD值-空白孔OD值)]×100％
(3)结果：在实验测定的时间范围内，本发明化合物2'对于RAW264.7细胞无细胞毒性，并具有一定的促增殖作用。各测定实验细胞存活率及IC50值见表30。
实验结果使用GraphPad Prism version 5.0软件进行分析。
表30 本发明化合物2'处理正常RAW264.7细胞后的细胞存活率及IC50值
Figure PCTCN2022080897-desc-000049-original
实验例18、本发明化合物2'在体外培养的正常人胚肺成纤维细胞HELF细胞和小鼠胚胎成纤维细胞NIH3T3细胞上的细胞毒理学评价实验。
(1)本发明化合物2'的配制：用DMSO将本发明化合物2'溶解配制成1×10-2mol/L(0.01M)的储备液，待进行细胞实验时，用细胞培养基稀释成浓度为20μM的初始液。
(2)实验方法(MTT法)：按细胞说明进行细胞初培养、消化、制备细胞悬液、细胞计数、在96孔细胞培养板上接种细胞悬液(100μl)、于5％CO2/37℃细胞培养箱中培养至细胞完全贴壁生长的操作。取化合物初始液，用细胞培养基稀释成浓度分别为20μM、2μM、0.2μM、0.02μM和0.002μM的系列浓度梯度溶液。将此化合物系列浓度梯度溶液分别加入至96孔板中，每孔100μl，吹打均匀后分别再吸弃100μl，得到作用浓度分别为10μM、1μM、0.1μM、0.01μM和0.001μM的系列浓度梯度，每个给药浓度设置3个复孔，同时设置正常细胞对照组和空白对照组。于培养箱中孵育96h后，通过MTT法检测各化合物的OD值，利用以下公式计算抑制率：
细胞生长抑制率(％)＝{[(对照孔OD值-空白孔OD值)-(给药孔OD值-空白孔OD值)]/[(对照孔OD值-空白孔OD值]}×100％
实验结果使用GraphPad Prism version 5.0软件进行分析。
(3)结果：在实验测定的96h时长范围内，本发明化合物2'对于人胚肺成纤维细胞HELF细胞和小鼠胚胎成纤维细胞NIH3T3细胞无明显的细胞毒性，测定的实验细胞生长抑制率以及化合物IC50值结果见表31。
表31 本发明化合物2'对正常HELF和NIH3T3实验细胞的毒性检测结果
Figure PCTCN2022080897-desc-000050-original
Figure PCTCN2022080897-desc-000051-original
(4)结论：本发明化合物2'对人胚肺成纤维细胞HELF细胞和小鼠胚胎成纤维细胞NIH3T3细胞的生长无明显毒性，适于进行下游活性筛选实验。
(二)本发明化合物在细胞水平上的生物活性评价实验例
实验例19：本发明化合物2'和3'对LPS刺激诱导的RAW 264.7细胞炎症模型中NO病理性过量产生的抑制效力评价实验例
本实验采用Griess法检测本发明化合物对LPS刺激的RAW264.7细胞炎症模型中NO分泌量的影响。
取采用含FBS及双抗(链霉素和青霉素)的DMEM高糖培养基培养的生长状态良好且处于对数生长期的RAW264.7细胞，加入适量培养基轻轻吹打，制成细胞悬液并调整细胞悬液的细胞密度为6×105个/mL，接种于96孔细胞培养板，每孔接种细胞悬液的量为100μl。将细胞培养板置于5％CO2/37℃培养箱中培养至细胞完全贴壁生长(过夜)，观察可见细胞形态良好。对96孔板设置实验分组，包括各给予本发明化合物组、LPS刺激模型组、正常细胞对照组和空白组。按此实验分组在各给予本发明化合物组中按给药实验操作方法(见“实验例3、本发明化合物对体外培养的小鼠单核巨噬细胞RAW264.7细胞的生长情况影响评价实验”)制成各含本发明化合物的干预实验细胞悬液，并控制每孔总体积仍为100μl。将96孔板在细胞培养箱中预孵育2h后，再在各给予本发明化合物组和模型组中加入LPS刺激，LPS的加入量为1μg/mL。将96孔板置于细胞培养箱中继续培养，24小时后，分别收集各孔中上清液50μl，加入到新的96孔板孔中，按一氧化氮检测试剂盒说明书中设定的操作程序操作，加入50μL Griess A，1min后，加入50μL Griess B，继续反应15min，然后在酶标仪上540nm处测定OD值，在预先用NaNO2建立标准曲线的基础上，计算样品中亚硝酸钠的浓度，折算各组细胞培养液中NO的含量(见表32)。
实验结果表明，经本发明化合物2'和3'干预处理后，本发明化合物可以剂量依赖性地抑制LPS刺激诱导的RAW 264.7细胞炎症模型中病理性过量的NO的产生(表32)。
表32.本发明化合物2'和3'对LPS刺激诱导的RAW264.7细胞炎症模型NO分泌 量的影响
Figure PCTCN2022080897-desc-000052-original
与正常组比较，###P＜0.001；与模型组比较，***P＜0.001
实验例20、本发明化合物2'降低LPS刺激诱导的RAW 264.7细胞炎症模型IL-6病理性过量分泌水平表型的效力评价实验例
本实验采用ELISA法检测本发明化合物对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6病理性过量分泌量表型的影响。
按小鼠IL-6酶联免疫吸附检测试剂盒中说明书提供的实验操作程序所设定的样品量，根据实验所测定本发明化合物的细胞毒性等数据，取按“实验例6、本发明化合物对LPS刺激诱导的RAW 264.7细胞炎症模型中NO病理性过高产生的抑制效力评价实验例”的操作程序制备的LPS刺激诱导的RAW 264.7细胞炎症模型的细胞上清液样品，加入到小鼠IL-6酶联免疫吸附检测试剂盒中提供的含靶蛋白特异单克隆捕获抗体的酶标板孔中，按试剂盒说明书所提供的操作程序操作，对各孔细胞培养上清液中IL-6的含量进行测定。
由表33和表34的两次重复实验结果数据可见，以正常对照组中IL-6的表达量为参考，模型组中的IL-6的表达量明显升高，与正常对照组比较统计学上具有极显著差异性。经本发明化合物2'处理后，LPS刺激诱导的RAW264.7细胞炎症模型中IL-6的表达量显著下降，并且与模型组相比，本发明化合物2'处理组对IL-6的表达量的抑制作用具有显著性差异，并强于相应的小檗碱型生物碱季铵盐底物。
表33.本发明化合物2'对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6分泌量的影响注
Figure PCTCN2022080897-desc-000053-original
注与正常组比较，###P＜0.001；与模型组比较，**P＜0.01。
表34.本发明化合物2'对LPS刺激诱导的RAW264.7细胞炎症模型中IL-6分泌量的影响注
Figure PCTCN2022080897-desc-000054-original
注与正常组比较，###P＜0.001；与模型组比较，***P＜0.001。
(三)本发明化合物在动物水平上的生物活性评价实验例
实验例21、本发明化合物2'降低模型动物IL-6水平的效力评价实验例
本实验的实验操作同“实验例10：本发明化合物降低模型动物IL-6水平的效力评价实验例”。与模型组相比，甲氨蝶呤阳性药组、本发明化合物2'给药组各检测小鼠血清中的IL-6水平均显著下降，且均具有统计学差异。本发明化合物2'给药组对模型小鼠血清中IL-6水平的抑制作用优于按照阳性药具体特性给药的实验组。结果如表35所示。
表35.受试本发明化合物对模型小鼠血清中IL-6浓度的影响(pg/mL)。
Figure PCTCN2022080897-desc-000055-original
注：与正常对照组比，###p<0.001；与模型组比较，**p<0.01，***p<0.001。
实验例22、受试本发明化合物2'对模型小鼠自身抗体的影响评价实验例
本实验的实验操作同“实验例12：本发明化合物抗类风湿性关节炎药效学作用评价”中的“(4)受试本发明化合物对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠自身抗体水平的影响”。实验结果见表36。由结果可见，与模型组相比，阳性药甲氨蝶呤组、本发明化合物2'给药组的小鼠血清中的抗鸡II型胶原IgG抗体浓度均显著降低，本发明化合物2'给药组对抗鸡II型胶原IgG抗体水平的降低作用优于按照阳性药具体特性给药的实验组，与模型组比较具有极显著性差异。
表36.本发明化合物2'对小鼠鸡II型胶原蛋白诱导性关节炎模型小鼠血清中的抗鸡II型胶原IgG抗体水平的影响。
Figure PCTCN2022080897-desc-000056-original
注：数据以X±SEM表示，n＝4；与模型组比较，*p<0.05，***p<0.001。
实验例23、本发明化合物2'升高模型动物血红蛋白含量的效力评价实验例
本实验的实验操作同“实验例11：本发明化合物升高模型动物血红蛋白含量的效力评价实验例”。检测结果表明，本实验空白对照组小鼠血红蛋白含量的平均值为147.29±5.06(g/L)；与空白对照组比较，本实验模型组动物的血红蛋白含量明显降低，平均数值为136.33±4.27(g/L)；与模型组比较，受试本发明化合物2'组对血红蛋白含量有明显升高作用，本发明化合物2'给药组的血红蛋白含量的平均值为181.17±3.47***(g/L)，与模型组比较具有极显著性差异(数据以X±SEM表示；与模型组比较，***p<0.001)。特别是与空白对照组比较，本发明化合物2'给药组对血红蛋白也有明显升高作用，表明本发明化合物具有显著的升高血红蛋白的作用。阳性药组的血红蛋白含量平均值为160.92±7.07(g/L)。
实验结果表明，受试本发明化合物2'对模型小鼠血红蛋白含量具有极显著的升高作用。

Claims (18)

  1. 通式A所示的可溶性小檗碱型生物碱季铵盐化合物：
    Figure PCTCN2022080897-clms-000001-original
    当X选自C时则Y选自H，当X选自N+时则Y选自O-；
    当X选自C并且Y选自H时，
    COO-选自取代于吡啶环的2位或3位或4位或5位或6位；
    R独立地选自氢、氨基、取代或未取代的羟基、烷基、卤素；
    R为单取代或多取代；
    R为单取代时，与COO-的位置相协调地形成吡啶环的二取代，R可选自吡啶环的3位或4位或5位或6位或2位；
    R为多取代时选自二取代或三取代或四取代，并与COO-相协调地形成按数学列举法给出的各种取代模式；
    当X选自N+并且Y选自O-时，
    R选自取代于吡嗪环2位的甲基，COO-选自取代于吡嗪环的5位；
    R2、R3各自独立地选自H，取代或未取代的羟基，或R2与R3连接成为亚烃基二氧基；
    R9、R10、R11各自独立地选自H、取代或未取代的羟基，或者R9与R10连接成为亚烃基二氧基而R11独立地选自H、取代或未取代的羟基，或者R10与R11连接成为亚烃基二氧基而R9独立地选自H、取代或未取代的羟基；
    所述的取代或未取代的羟基中的取代基选自甲基、乙基；所述的烷基选自甲基、乙基；所述的卤素选自氟、氯、溴；所述的亚烃基二氧基选自亚甲二氧基。
  2. 根据权利要求1的化合物，其特征在于，所述的化合物为通式I所示的小檗碱型生物碱吡啶甲酸类季铵盐化合物：
    Figure PCTCN2022080897-clms-000002-original
    COO-选自取代于吡啶环的2位或3位或4位或5位或6位；
    R独立地选自氢、氨基、取代或未取代的羟基、烷基、卤素；
    R为单取代或多取代；
    R为单取代时，与COO-的位置相协调地形成吡啶环的二取代，R可选自吡啶环的3位或4位或5位或6位或2位；
    R为多取代时选自二取代或三取代或四取代，并与COO-相协调地形成按数学列举法给出的各种取代模式；
    R2、R3各自独立地选自H，取代或未取代的OH，或R2与R3连接成为亚烃基二氧基；
    R9、R10、R11各自独立地选自H、取代或未取代的OH，或者R9与R10连接成为亚烃基二氧基而R11独立地选自H、取代或未取代的OH，或者R10与R11连接成为亚烃基二氧基而R9独立地选自H、取代或未取代的OH；
    所述的取代或未取代的羟基中的取代基选自甲基、乙基；所述的烷基选自甲基、乙基；所述的卤素选自氟、氯、溴；所述的亚烃基二氧基选自亚甲二氧基。
  3. 根据权利要求1的化合物，其特征在于，所述的化合物为通式I'所示的小檗碱型生物碱2-甲基-N1-氧化吡嗪-5-甲酸季铵盐化合物：
    Figure PCTCN2022080897-clms-000003-original
    R2、R3各自独立地选自H，取代或未取代的OH，或R2与R3连接成为亚烃基二氧基；
    R9、R10、R11各自独立地选自H、取代或未取代的OH，或者R9与R10连接 成为亚烃基二氧基而R11独立地选自H、取代或未取代的OH，或者R10与R11连接成为亚烃基二氧基而R9独立地选自H、取代或未取代的OH；
    所述的取代或未取代的OH中的取代基选自甲基、乙基；所述的亚烃基二氧基选自亚甲二氧基。
  4. 根据权利要求1的化合物，其特征在于，所述的化合物选自如下化合物群组：
    Figure PCTCN2022080897-clms-000004-original
    Figure PCTCN2022080897-clms-000005-original
    Figure PCTCN2022080897-clms-000006-original
    Figure PCTCN2022080897-clms-000007-original
  5. 一种制备权利要求1的化合物的方法，其特征在于，所述的方法如下：将小檗碱型生物碱季铵盐类化合物与丙酮和氢氧化钠的水溶液反应，所得固体8-丙酮基二氢小檗碱型化合物再与吡啶甲酸类化合物或2-甲基-N1-氧化吡嗪-5-甲酸在四氢呋喃与水的混合溶剂中在加热的条件下反应，经对反应混合液过滤，得小檗碱型生物碱吡啶甲酸类季铵盐化合物或小檗碱型生物碱2-甲基-N1-氧化吡嗪-5-甲酸季铵盐化合物。
  6. 一种药物组合物，其特征在于，含有有效剂量的权利要求1的化合物和药学上可接受的药用载体或赋形剂。
  7. 权利要求1所述的化合物或权利要求6所述的药物组合物在制备具有促 免疫效应细胞增殖、抑制病理状态的高NO分泌、降低生物机体病理状态的高白细胞介素-6水平和高自身抗体水平活性的免疫调节剂药物中的应用。
  8. 权利要求1所述的化合物或权利要求6所述的药物组合物在制备预防、缓解和/或治疗引起生物机体分泌白细胞介素-6升高的疾病的药物中的应用。
  9. 权利要求1所述的化合物或权利要求6所述的药物组合物在制备预防、缓解和/或治疗引起生物机体分泌NO升高的疾病的药物中的应用。
  10. 权利要求1所述的化合物或权利要求6所述的药物组合物在制备预防、缓解和/或治疗引起生物机体形成自身抗体升高的疾病的药物中的应用。
  11. 权利要求1所述的化合物或权利要求6所述的药物组合物在制备预防、缓解和/或治疗各种原因引起的心脑血管和血液系统疾病、炎症病、发热病、肿瘤病、自身免疫性疾病的药物中的应用。
  12. 权利要求1的化合物或权利要求6的药物组合物在制备预防、缓解和/或治疗各种原因引起的病毒感染病及其所致并发症、呼吸系统疾病的药物中的应用。
  13. 根据权利要求11的应用，其特征在于，所述的自身免疫性疾病包括类风湿性关节炎；所述的心脑血管和血液系统疾病包括低血红蛋白血症性贫血病、动脉粥样硬化症：所述的肿瘤病包括结直肠癌、肺癌。
  14. 根据权利要求12的应用，其特征在于，所述的病毒感染病包括冠状病毒感染病。
  15. 根据权利要求14的应用，其特征在于，所述的冠状病毒感染病包括2019新型冠状病毒SARS-CoV2感染病。
  16. 根据权利要求12的应用，其特征在于，所述的病毒感染病所致并发症包括病毒感染病引起的呼吸道病变和全身性病变。
  17. 根据权利要求16的应用，其特征在于，所述的病毒感染病所致并发呼吸道病变和全身性病变包括冠状病毒感染病引起的呼吸道病变和全身性病变。
  18. 根据权利要求17的应用，其特征在于，所述的冠状病毒感染病所致并发呼吸道病变和全身性病变包括2019新型冠状病毒SARS-CoV2感染病引起的呼吸道病变和全身性病变。
PCT/CN2022/080897 2021-03-15 2022-04-13 二苯烷类化合物及其制备方法、药物组合物和用途 WO2022194136A1 (zh)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN202110274881.6A CN115073447B (zh) 2021-03-15 小檗碱型吡啶甲酸类季铵盐化合物及其制备药物的用途
CN202110274881.6 2021-03-15
CN202110274862.3 2021-03-15
CN202110276109.8A CN115068473A (zh) 2021-03-15 2021-03-15 小檗碱型吡啶甲酸类季铵盐化合物抗病毒感染病的用途
CN202110274862.3A CN115073446B (zh) 2021-03-15 小檗碱型生物碱氧化吡嗪甲酸季铵盐及其制备药物的用途
CN202110276109.8 2021-03-15

Publications (1)

Publication Number Publication Date
WO2022194136A1 true WO2022194136A1 (zh) 2022-09-22

Family

ID=83321680

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/080897 WO2022194136A1 (zh) 2021-03-15 2022-04-13 二苯烷类化合物及其制备方法、药物组合物和用途

Country Status (1)

Country Link
WO (1) WO2022194136A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117672411A (zh) * 2023-12-05 2024-03-08 首都医科大学附属北京世纪坛医院 一种中药成分的Hormesis效应研究方法及中药成分小檗碱的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1759834A (zh) * 2004-09-17 2006-04-19 中国医学科学院医药生物技术研究所 黄连素或其与辛伐他汀联合在制备用于预防或治疗与血脂有关疾病或症状的产品中用途
CN103919773A (zh) * 2013-12-20 2014-07-16 中国药科大学 9-去甲基小檗碱在制备降血脂药物中的应用
CN103919775A (zh) * 2013-12-20 2014-07-16 中国药科大学 9-去甲基小檗碱在制备降血糖药物中的应用
CN103989677A (zh) * 2013-12-20 2014-08-20 中国药科大学 去亚甲基小檗碱在制备降血糖药物中的应用
CN105732609A (zh) * 2016-01-30 2016-07-06 合肥华方医药科技有限公司 一种烟酸小檗碱偶合物制备方法及其医药用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1759834A (zh) * 2004-09-17 2006-04-19 中国医学科学院医药生物技术研究所 黄连素或其与辛伐他汀联合在制备用于预防或治疗与血脂有关疾病或症状的产品中用途
CN103919773A (zh) * 2013-12-20 2014-07-16 中国药科大学 9-去甲基小檗碱在制备降血脂药物中的应用
CN103919775A (zh) * 2013-12-20 2014-07-16 中国药科大学 9-去甲基小檗碱在制备降血糖药物中的应用
CN103989677A (zh) * 2013-12-20 2014-08-20 中国药科大学 去亚甲基小檗碱在制备降血糖药物中的应用
CN105732609A (zh) * 2016-01-30 2016-07-06 合肥华方医药科技有限公司 一种烟酸小檗碱偶合物制备方法及其医药用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHAIKH, A. ET AL.: "Metabolite profiling of arginase inhibitor activity guided fraction of Ficus religiosa leaves by LC–HRMS", BIOMEDICAL CHROMATOGRAPHY, vol. 34, no. 12, 13 August 2020 (2020-08-13), XP071551082, ISSN: 0269-3879, DOI: 10.1002/bmc.4966 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117672411A (zh) * 2023-12-05 2024-03-08 首都医科大学附属北京世纪坛医院 一种中药成分的Hormesis效应研究方法及中药成分小檗碱的应用
CN117672411B (zh) * 2023-12-05 2024-05-14 首都医科大学附属北京世纪坛医院 一种中药成分的Hormesis效应研究方法及中药成分小檗碱的应用

Similar Documents

Publication Publication Date Title
WO2017202276A1 (zh) 苯醚类衍生物、及其制法和药物组合物与用途
WO2020177744A1 (zh) 水杨酸类小檗碱型生物碱季铵盐及其制备药物的用途
EA027782B1 (ru) Ингибитор киназ
JP2020519597A (ja) 全身性インスリン抵抗性障害の治療用化合物およびその使用
CN113773300B (zh) 磺酰胺类化合物、其制备方法及用途
JP2024050645A (ja) ヘテロアリールで置換されたピラゾール化合物及びその医薬用途
WO2022194136A1 (zh) 二苯烷类化合物及其制备方法、药物组合物和用途
CN107613967A (zh) 用于预防和/或治疗胃肠道的炎症疾病的宿主防御蛋白（hdp）模拟物
CN114956977B (zh) 一种联苯类化合物、药物组合物及其制备方法和应用
JP2022533740A (ja) メチル基及びトリフルオロメチル基を含む二置換スルファミド系選択的ｂｃｌ－２阻害剤
EP2975023A1 (en) Guanidinobenzoic acid ester compound
WO2020238179A1 (zh) 作为选择性jak2抑制剂的吡咯并嘧啶类化合物、其合成方法及用途
CN112442033A (zh) Sting通路调节剂及其用途
CN113583007B (zh) 一种吡咯并嘧啶类btk抑制剂及其制备方法与应用
CN115073447B (zh) 小檗碱型吡啶甲酸类季铵盐化合物及其制备药物的用途
CN115073446B (zh) 小檗碱型生物碱氧化吡嗪甲酸季铵盐及其制备药物的用途
WO2021197396A1 (zh) 氘代氧化苯砷化合物及其应用
CN102245606B (zh) 新化合物
CN115073447A (zh) 小檗碱型吡啶甲酸类季铵盐化合物及其制备药物的用途
CN115073446A (zh) 小檗碱型生物碱氧化吡嗪甲酸季铵盐及其制备药物的用途
CN115068473A (zh) 小檗碱型吡啶甲酸类季铵盐化合物抗病毒感染病的用途
JP2022517396A (ja) Ｅｇｆｒ阻害剤の塩、結晶形及びその製造方法
CN117510489A (zh) 溶解型黄连碱型生物碱季铵盐及其制备药物的用途
WO2024000615A1 (zh) 一种酪氨酸蛋白激酶抑制剂及其用途
WO2024061172A1 (zh) 一种脯氨酰羟化酶抑制剂及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22770489

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22770489

Country of ref document: EP

Kind code of ref document: A1