CN117298085A - Medical application of tolypic acid and derivative thereof in preparation of medicines for treating immune and inflammation related diseases - Google Patents

Medical application of tolypic acid and derivative thereof in preparation of medicines for treating immune and inflammation related diseases Download PDF

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CN117298085A
CN117298085A CN202311318843.1A CN202311318843A CN117298085A CN 117298085 A CN117298085 A CN 117298085A CN 202311318843 A CN202311318843 A CN 202311318843A CN 117298085 A CN117298085 A CN 117298085A
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acid
hydroxymethyl
alpha
group
phenylacetic acid
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王长福
谢瑞强
辛萍
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Guangdong Pharmaceutical University
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Guangdong Pharmaceutical University
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    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of tolypic acid and derivatives thereof in preparing medicines for treating immune and inflammation related diseases. The tolipomoea and the derivative thereof are compounds of the formulas I-IV or pharmaceutically acceptable salts thereof, and solvent compounds, enantiomers, diastereomers, tautomers or mixtures of any ratio thereof, including racemic mixtures, of the compounds of the formulas I-IV or pharmaceutically acceptable salts thereof. Animal experiments show that the tolipomoea and the derivatives thereof have broad-spectrum immunoregulation, anti-inflammatory and analgesic effects, have obvious improvement effect on pathological indexes of animal models of a plurality of immune and inflammation related diseases, can regulate abnormal immune response to be normal and perform good performanceThe anti-inflammatory and analgesic effects of the traditional Chinese medicine composition can be suitable for patients with different disease degrees and different types of immunity and inflammation related diseases, and can be applied to industry.

Description

Medical application of tolypic acid and derivative thereof in preparation of medicines for treating immune and inflammation related diseases
The application is a divisional application of an invention patent application of which the application date is 2023, 03 and 08, the application number is 202310217736.3, and the invention name is 'medical application of tolpinic acid and derivatives thereof in preparing medicines for treating immune and inflammation related diseases'.
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of tolypic acid and derivatives thereof in preparing medicines for treating immune and inflammation related diseases and medicines for treating immune inflammation related diseases.
Background
Immune diseases are a broad class of diseases characterized by abnormal inflammatory immune responses, either local or systemic, and mainly include hypersensitivity, immunodeficiency, autoimmune diseases, and the like. Relates to type I hypersensitivity such as penicillin anaphylactic reaction, drug eruption caused by medicine, allergic rhinitis, sphagitis, conjunctivitis, bronchial asthma, eczema, urticaria and the like caused by pollen or dust; type II hypersensitivity reactions such as neonatal hemolytic reaction, drug-induced hemolytic anemia and aplastic anemia; type III hypersensitivity reactions such as glomerulonephritis. Type IV hypersensitivity such as tuberculosis, syphilis, etc. Bronchitis or pneumonia associated with infection, gastrointestinal inflammation, endometritis, otitis media, tonsillitis, furuncle, sinusitis, abscess or granuloma, septicemia, sepsis, myocarditis, meningitis, osteoarthritis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, anorectal inflammation, paronychia, folliculitis, and the like. Autoimmune diseases such as hepatitis, systemic lupus erythematosus, spondylitis, rheumatoid arthritis, nephritis, diabetes, pancreatitis, enteritis, rheumatic heart disease, pneumonia, scleroderma, vasculitis, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, autoimmune thyroiditis, and the like. It is estimated that the onset of immune diseases is an upward trend year by year, wherein about 7.6% -9.4% of people worldwide suffer from various types of autoimmune diseases, the diseases are difficult to cure, most patients need to take medicine for a long time or even for life, and part of diseases are serious, such as lupus nephropathy, seriously affect the life quality of patients and threaten the life safety of the patients. About 5000 tens of thousands of americans (about 1/5 of the general population) suffer from autoimmune diseases, of which about 75% are females. Immune diseases have become the third greatest chronic disease in addition to cardiovascular diseases and cancers. At present, although there is no exact disease data in China, the patient population is increasing year by year.
Treatment of immune disorders involves two goals, symptomatic relief and maintenance of function, and delay of tissue damage progression. Currently, drugs for treating immune diseases are mainly classified into nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), disease-improving antirheumatic drugs (DMARDs), biological agents and natural drugs. NSAIDs are commonly used drugs for the treatment of autoimmune diseases, which are effective in alleviating clinical symptoms and signs of patients and eliminating local inflammatory reactions, but such drugs cannot control the progress of the diseases, and their common adverse reactions include central nervous system symptoms, cardiovascular damage, gastrointestinal symptoms, hematopoietic system changes, liver and kidney insufficiency, asthma, skin rash, etc. SAIDs have strong anti-inflammatory and immunosuppressive effects, prevent inflammatory cells from gathering to inflammatory parts, inhibit inflammatory factor release, inhibit TB lymphocyte proliferation and secretion, have a plurality of adverse reactions, and relapse after stopping the medicine, and are clinically used in combination with other immunosuppressants at present. DMARDs are widely applied to treatment of autoimmune disease chronic kidney disease, transplant rejection reaction, tumor and the like, and although the chemical structure and action mechanism of the traditional DMARDs are different, the clinical pharmacodynamics characteristics are similar, namely the effect is slow, symptoms and physical signs are gradually lightened after taking the medicine for weeks or months, and a relatively stable curative effect can be obtained after continuously taking the medicine for a long time, and the main adverse reactions comprise gastrointestinal tract reaction, bone marrow suppression, infection, liver and kidney injury and the like. Biological agents exert therapeutic effects by blocking critical inflammatory cytokines or cell surface molecules, such as monoclonal antibodies targeting IL-1, IL-6, TNF- α and IL-17, anti-CD 20 mab, B lymphocyte stimulating factor (BAFF) inhibitors, T cell inhibitors, integrin monoclonal antibodies, and selective adhesion molecule inhibitors, most of which are in clinical trials, few of which have been marketed for use, and few of which have a number of adverse effects and are severe, and individual drugs are disabled due to severe adverse effects. The natural medicines for treating immune diseases comprise glycosides and alkaloids, wherein the glycosides include white peony root total glycosides, ginseng total glycosides, gynostemma pentaphylla total glycosides, astragaloside, tripterygium total glycosides, notoginseng total saponins and the like, the alkaloids include sinomenine, common monkshood mother root total alkaloids, sophocarpine, tripterygium wilfordii neone and the like, and the medicines have fewer adverse reactions and have the functions of anti-inflammatory, analgesic and immunosuppression, but have weak pertinence and poor effect in clinical treatment of diseases. With the deep elucidation of the pathological mechanism of immune diseases and the discovery of new drug targets, drugs for treating inflammatory immune diseases, besides NSAIDs, SAIDs and traditional DMARDs, targeted small molecule drugs such as Tofacitinib, baricitinib, upatacitinib and Filgotinib and the like are also developed and applied to clinic, and the drugs have definite curative effects, but also have adverse reactions such as gastrointestinal symptoms, immunosuppression, myelosuppression, infection, new tumor and the like. Therefore, the development of small molecule drugs with immunoregulatory and anti-inflammatory effects that do not impair the physiological functions of the body is a major strategy and direction for the treatment of immune and inflammatory related diseases.
In summary, the patients suffering from immune and inflammation related diseases have huge groups, the treatment drugs or methods have limited types, but often need to receive treatment for a long time, repeatedly and even for a whole life, while some drugs have a curative effect, but have high short-term recurrence rate, and most drugs are often limited by inherent toxicity and selectivity of the drugs, so various adverse reactions of systems and parts are unavoidable. Aiming at the problems, the development of external, oral and injection pharmaceutical preparations is considered to be suitable for patients with different disease degrees and different types at the same time. The invention screens and confirms the effectiveness of tropinic acid (DL-TropicAcid, alias 2-phenyl-3-hydroxy propionic acid) and derivatives thereof for treating immune and inflammation related diseases through a large amount of animal experimental researches in the early stage. Torpedo acid is an intermediate for synthesizing atropine, the raw materials are low in cost, and no report on prevention and treatment of immune and inflammation related diseases by using the Torpedo acid and derivatives thereof is currently available.
Disclosure of Invention
In one aspect, the present invention provides the use of tropinic acid and its derivatives, pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, tautomers, or mixtures thereof in any ratio, for the preparation of a medicament for the prophylaxis and/or treatment of diseases associated with immune and inflammatory conditions, said tropinic acid and its derivatives having the structure shown in formula a:
Wherein R is 1 -R 5 Each independently selected from-H, -OH or C1-C6 alkoxy.
In one embodiment, R 1 -R 5 Each independently selected from-H or-OH.
In one embodiment, R 1 And R is 5 Selected from-H, R 2 -R 4 Each independently selected from-H or-OH.
In one embodiment, R 1 And R is 5 Selected from-H, R 2 -R 4 Two or three of them are selected from-OH and the others are selected from-H.
In one embodiment, R 1 -R 5 At least two of which are selected from-OH.
In one embodiment, R 1 -R 5 Two or three of which are selected from-OH.
In one embodiment, R 2 -R 4 Two or three of which are selected from-OH.
In one embodiment, the tropine acid and derivatives thereof are selected from the group consisting of compounds represented by formulas I-IV below:
wherein formula I is tolmetic Acid (DL-Tropic Acid), formula II is 4-Hydroxy-alpha- (hydroxymethyl) phenylacetic Acid (4-Hydroxy-alpha- (hydroxymethyl) benzeneacetic Acid), formula III is 3, 4-Dihydroxy-alpha- (hydroxymethyl) phenylacetic Acid (3, 4-Dihydroxy-alpha- (hydroxymethyl) benzeneacetic Acid) and formula IV is 3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic Acid (3, 4, 5-Trihydroxy-alpha- (hydroxymethyl) benzeneacetic Acid).
The tolmetin and the derivative thereof have the functions of immunoregulation, anti-inflammation and pain relief, have obvious improvement effect on pathological indexes of a plurality of animal models of immune and inflammation related diseases, can regulate abnormal immune response to be normal, and have good anti-inflammation and pain relief effects.
In one embodiment, the immune and inflammation related disorder is selected from one or more of allergic rhinitis, bronchitis, bronchial asthma, pharyngolaryngitis, conjunctivitis, eczema, urticaria, eczema, neonatal hemolytic reaction, hemolytic anemia, aplastic anemia, nephritis, tuberculosis, syphilis, pneumonia (including neocrown), gastrointestinal inflammation, endometritis, otitis media, sepsis, myocarditis, meningitis, tonsillitis, sinusitis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, anorectal inflammation, paronychia and folliculitis, osteoarthritis, hepatitis, systemic lupus erythematosus, spondylitis, rheumatoid arthritis, diabetes, pancreatitis, enteritis, rheumatic heart disease, vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, and thyroiditis.
In a second aspect, the present invention provides a prophylactic and/or therapeutic agent for immune-and inflammation-related diseases, which comprises tolypic acid and its derivatives, pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any ratio.
The medicine for preventing and/or treating immune and inflammation related diseases provided by the invention has broad-spectrum immunoregulation, anti-inflammatory and analgesic effects and obvious effects.
In one embodiment, the topiramate and its derivatives, pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any ratio, are used as active ingredient in the medicament of the invention. Preferably, it is as the main active ingredient; more preferably, it is the only active ingredient.
In the application and the medicine, the tropine acid and the derivative thereof, the pharmaceutically acceptable salt, the solvent compound, the enantiomer, the diastereoisomer, the tautomer or the mixture with any proportion thereof can be prepared into a pharmaceutical dosage form for external use, oral administration or injection with pharmaceutically acceptable carriers or auxiliary materials.
Thus, in the present invention, the drug may be an external drug, an oral drug or an injection drug.
In the present invention, the medicament may comprise a pharmaceutically acceptable carrier or adjuvant. The medicine can be made into various conventional solid dosage forms, such as granule, tablet or capsule, or semisolid dosage forms, such as spray and injection, or semisolid dosage forms, such as cream. In one aspect, the dosage form of the medicament may be: powders, tablets, coated tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, nasal preparations, aerosols, powder mists, lotions, liniments, ointments, plasters, pastes, gels, patches, and the like.
In the present invention, the term "pharmaceutically acceptable carrier or excipient" includes any and all solvents, co-solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, diluents, glidants, granulating agents, disintegrants, thickening agents, tackifiers, lubricants, anti-caking agents, humectants, wetting agents, chelating agents, plasticizers, dyes, flavoring agents, etc., and combinations thereof, which are well known to those skilled in the art (see, e.g., remington's Pharmaceutical Sciences,19th Ed.MackPrinting Company,1995; editors of Shanghai pharmaceutical industry institute, pharmaceutical excipient application techniques (second edition), chinese pharmaceutical technology publishing agency, 2002; national standard contrast manuals 1-3 volumes, national pharmacopoeia, chinese pharmaceutical technology publishing agency 2016; pharmaceutical excipients, r.c. rograd C Rowe), p.j. Scherskiki (Paul J. P. Pallese, palles et al. Chemical society, 2005, etc., national institutes of medical science, and the like, and combinations thereof. In addition to carriers and excipients that are incompatible with the active ingredient, any conventional carrier and excipient is contemplated for use in therapeutic or pharmaceutical compositions.
For example, as a solid dosage form, the pharmaceutically acceptable carrier or adjuvant may include at least one of the following: (a) Fillers such as starch, corn starch, modified starch, compressible starch, lactose monohydrate, microcrystalline cellulose, cyclodextrin, sorbitol, mannitol, calcium phosphate, amino acids, and the like; (b) Binders such as starch slurry, gelatinized starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hypromellose, methyl cellulose, ethyl cellulose, hydroxypropyl methylcellulose, low substituted hydroxypropyl cellulose, polyvinylpyrrolidone, gelatin, alginate, and the like; (c) humectants, such as glycerin; (d) Disintegrants such as dry starch, modified starch, sodium carboxymethyl starch, low-substituted hydroxypropylcellulose, crospovidone, croscarmellose sodium, microcrystalline cellulose, effervescent disintegrants, crosslinked polyvinylpyrrolidone, etc.; (e) solution retarders, such as paraffin; (f) absorption enhancers, such as quaternary ammonium compounds; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite; (i) Lubricants, for example talc, stearic acid, magnesium or calcium stearate, colloidal silica, hydrogenated castor oil and solid polyethylene glycols, polyethylene glycols 4000-20000, magnesium lauryl sulfate and the like.
In the present invention, the pharmaceutically suitable object may be a human or other warm-blooded animal. When the subject is a human, the single or mixed amount of tolterodine and its derivatives is preferably 1 mg/kg.d to 50mg/kg.d, more preferably 10 mg/kg.d to 20mg/kg.d. The therapeutically effective amount of a compound, pharmaceutical composition is dependent on the species, weight, age and condition of the individual, the disease being treated, or its severity. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of the respective active ingredients required to prevent, treat or inhibit the progression of the disease.
The invention also provides a compound shown in a formula A, pharmaceutically acceptable salts, solvent compounds, enantiomers, diastereomers, tautomers or mixtures thereof in any ratio:
wherein R is 1 -R 5 Each independently selected from-H or-OH;
provided that R 1 -R 5 At least two of which are selected from-OH.
In one embodiment, R 1 -R 5 Two or three of which are selected from-OH.
In one embodiment, R 2 -R 4 Two or three of which are selected from-OH.
In one embodiment, the compound is selected from the group consisting of compounds represented by the following formulas III-IV:
pharmaceutically acceptable salts of the compounds of the present invention include the base addition salts and acid addition salts thereof. Preferably, the base addition salt is selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, zinc, ammonium, tetramethylammonium, tetraethylammonium, triethylamine, trimethylammonium, ethylamine, diethanolamine, arginine or lysine salts; the acid addition salt is selected from organic acid salts such as acetate, aspartate, benzoate, benzenesulfonate, citrate, ethanedisulfonate, ethanesulfonate, formate, fumarate, gluconate, glucuronate, lactate, malate, trifluoroacetate, maleate and inorganic acid salts such as hydrochloride, hydrobromide, bisulfate, nitrate and phosphate. The compounds of the invention in free form can be converted into the corresponding compounds in salt form; and vice versa. The compounds of the invention in free form or in salt form and in solvate form can be converted into the corresponding compounds in free form or in salt form in non-solvate form; and vice versa.
The compounds of the present invention also include solvated forms thereof, which refer to associations formed by one or more solvent molecules with the compounds of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethylsulfoxide, ethyl acetate, acetic acid, aminoethanol.
The compounds of the present invention may exist as isomers and as mixtures thereof; such as tautomers, optical isomers, enantiomers, diastereomers. The compounds of the invention may for example comprise asymmetric carbon atoms and may therefore exist in the form of enantiomers or diastereomers and mixtures thereof, for example in the form of racemates. The compounds of the invention may exist in the (R) -, (S) -or (R, S) -configuration, preferably in the (R) -or (S) -configuration at a particular position of the compound.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention discovers that the tolypic acid and the derivative thereof can obviously improve the pathological indexes of animal models of immune and inflammation related diseases for the first time;
(2) Torpedo acid and its derivatives can regulate abnormal immune reaction to normal, and exert good anti-inflammatory and analgesic effects
(3) Compared with the existing medicines, the tolperisoic acid and the derivatives thereof have the advantages of low toxicity, quick response, short treatment course, small dosage, low recurrence rate, convenient use and the like, and take into account dosage forms such as external use, oral administration, injection and the like, thereby being applicable to patients with different disease degrees, different types of immune diseases and inflammatory diseases;
(4) The tropine acid derivative compounds 3 and 4 obtained through structural modification have better and obvious treatment effect than tropine acid in the treatment of immune and inflammation related diseases;
(5) The tropine acid and the tropine acid derivative used in the invention are easy to obtain and synthesize, low in price, stable in property, convenient to store and transport and suitable for industrial application.
Drawings
FIG. 1A compound of formula III 1 H-NMR spectrum
FIG. 2A compound of formula III 13 C-NMR spectrum
FIG. 3A compound of formula IV 1 H-NMR spectrum
FIG. 4A compound of formula IV 13 C-NMR spectrum
FIG. 5 influence of tolpinic acid and its derivatives on liver histopathology in mouse model of autoimmune hepatitis (x 200)
FIG. 6 influence of tolpinic acid and its derivatives on spleen tissue pathology in a mouse model of autoimmune hepatitis (x 200)
FIG. 7 influence of tolpinic acid and its derivatives on lung tissue pathology in New crown-cold-dampness epidemic mouse model (x 200)
FIG. 8 comparison of ankle and paw morphology differences before and after treatment for rats of each group
Fig. 9, joint index and swelling degree trend of each group of rats compared (n=6,)。
Detailed Description
The following description of the preferred embodiments of the present invention is provided in conjunction with the accompanying drawings, and it is to be understood that the preferred embodiments described herein are merely illustrative and not restrictive of the invention, and that the described embodiments are merely some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: preparation of the Compound of formula III (3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid)
1. Synthesis process
Under ice water bath conditions, 1.96g (10 mmol,1.0 eq.) of 3, 4-dimethoxyphenylacetic acid was taken in a 100mL Schlenk flask, 20mL of anhydrous DCM was added under nitrogen protection, 1.8mL (25 mmol,2.5 eq.) of thionyl chloride was slowly added dropwise while maintaining a low temperature, the system was transferred to room temperature and stirred for 2h. The solvent and excess thionyl chloride were removed under reduced pressure at 40 ℃. Subsequently, 20mL of methanol was added to the system and stirred overnight at room temperature, and the solvent was distilled off under reduced pressure to obtain 1.50g of methyl 3, 4-dimethoxyphenylacetate.
1.0g (4.76 mmol,1.0 eq.) of methyl 3, 4-dimethoxyphenylacetate was taken in a 100mL Schlenk flask at room temperature, 0.171g (5.71 mmol,1.2 eq.) of paraformaldehyde, 5mL of DMSO and 0.0257g (0.356 mmol,10 mol%) of catalytic amount of sodium methoxide were added to the system under nitrogen and the system was kept stirring at room temperature overnight. Work-up the reaction was transferred to 100mL of water, the aqueous layer was extracted with EtOAc (3X 20 mL), the organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column separation to give 0.90g of methyl 3, 4-dimethoxy- α - (hydroxymethyl) phenylacetate.
0.6g (2.5 mmol,1.0 eq.) of methyl 3, 4-dimethoxy- α - (hydroxymethyl) phenylacetate was taken in a 20mL Schlenk flask, 2mL of hydrogen bromide solution (40%) was slowly added to the system at room temperature, and the reaction was heated under reflux overnight. After-treatment the reaction was transferred to 50mL of water, the aqueous layer was extracted with DCM (3X 20 mL), the organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column separation to give 0.35g of 3, 4-dihydroxy- α - (hydroxymethyl) phenylacetic acid.
2. Structural identification
The 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid is white crystalline powder, is easy to dissolve in methanol and is soluble in water. 1 H-NMR(400MHz,CD 3 OD) see FIG. 1, delta H (ppm)3.63(2H,m,CH 2 ),3.99(1H,m,CH),6.50(1H,dd,J＝2.0,8.0Hz,Ph-H),7.04(1H,d,J＝8.0Hz,Ph-H),7.09(1H,d,J＝2.0Hz,Ph-H)； 13 C-NMR(400MHz,CD 3 OD) see FIG. 2, delta C (ppm)55.9(CH),65.2(CH 2 ),114.3(CH),116.4(CH),123.9(CH),127.2(C),145.3(C),146.0(C),176.2(C＝O)。
Example 2: preparation of the Compound of formula IV (3, 4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid)
1. Synthesis process
2.26g (10 mmol,1.0 eq.) of 3,4, 5-trimethoxyphenylacetic acid was taken in a 100mL Schlenk flask under ice water bath, 20mL of anhydrous DCM was added under nitrogen protection, 1.8mL (25 mmol,2.5 eq.) of thionyl chloride was slowly added dropwise while maintaining a low temperature, the system was transferred to room temperature and stirred for 2h. The solvent and excess thionyl chloride were removed under reduced pressure at 40 ℃. Subsequently, 20mL of methanol was added to the system and stirred overnight at room temperature, and the solvent was distilled off under reduced pressure to obtain 1.65g of methyl 3,4, 5-trimethoxyphenylacetate.
1.5g (6.25 mmol,1.0 eq.) of methyl 3,4, 5-trimethoxyphenylacetate was taken in a 100mL Schlenk flask at room temperature, 0.224g (7.50 mmol,1.2 eq.) of paraformaldehyde was added, and 5mL of DMSO was added to the system under nitrogen blanket with a catalytic amount of sodium methoxide 0.0338g (0.625 mmol,10 mol%) and the system was left to stir at room temperature overnight. Work-up the reaction was transferred to 100mL of water, the aqueous layer was extracted with EtOAc (3X 20 mL), the organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column separation to give 1.17g of methyl 3,4, 5-trimethoxy- α - (hydroxymethyl) phenylacetate.
1.0g (3.70 mmol,1.0 eq.) of methyl 3,4, 5-trimethoxy- α - (hydroxymethyl) phenylacetate was taken in a 20mL Schlenk flask, 2mL of hydrogen bromide solution (40%) was slowly added to the system at room temperature and the reaction was heated under reflux overnight. After-treatment the reaction was transferred to 50mL of water, the aqueous layer was extracted with DCM (3X 20 mL), the organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column separation to give 0.69g of 3,4, 5-trihydroxy- α - (hydroxymethyl) phenylacetic acid.
2. Structural identification
The 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid is white crystalline powder, is easy to dissolve in methanol and is soluble in water. 1 H-NMR(400MHz,CD 3 OD) see FIG. 3, delta H (ppm)3.63(2H,m,CH 2 ),3.99(1H,m,CH),6.10(2H,s,Ph-H)； 13 C-NMR(400MHz,CD 3 OD) is shown in FIG. 4, delta C (ppm)55.9(CH),65.2(CH 2 ),106.8(2×CH),131.0(C),131.3(C),146.2(2×C),176.2(C＝O)。
Example 3: research on the effect of tolypic acid and its derivatives on immunosuppressive mouse model
1. Grouping, modeling and administration of animals
Male KM mice were randomly divided into 11 groups of 10 mice each, which were respectively a blank control group, a model group, a positive drug group, a tropine high dose group (A), a tropine low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (H). Except for the blank control group mice, the other groups of test mice are injected with cyclophosphamide 80mg/kg.d intraperitoneally for 1-3 d at the beginning of the experiment to establish an immunosuppression mouse model. Each group was administered simultaneously, 2 times per day, and the grouping and dosing schedule is detailed in table 1, for 14d of continuous treatment.
Table 1. Animal group and administration information table (n=10)
2. Index detection
Mice were fasted for 12h after last dosing, anesthetized with 10% chloral hydrate by intraperitoneal injection, weighed, dissected, spleen and thymus were isolated immediately after heart blood collection, weighed after blotting the surface of the viscera with filter paper, and spleen index and thymus index were calculated as viscera index = viscera wet weight (g)/body weight (g) ×100%. The mouse blood is taken in a centrifuge tube, centrifuged for 10min at 2800r/min and low temperature (4 ℃) and serum is collected and preserved at-20 ℃ for standby. The enzyme-linked immunosorbent assay kit is used for measuring the level and activity of IL-4, IL-10, IFN-gamma, TNF-alpha and IgG in serum.
3. Experimental results and discussion
Spleen and thymus indexes can intuitively reflect the intensity of the immune function of the organism, compared with a blank control group, the thymus and spleen indexes of a model group are obviously reduced (P < 0.01), after two weeks of treatment by the tropine and the derivatives thereof, the spleen and thymus indexes of each dose group are obviously increased (P <0.05 or P < 0.01), and the spleen and thymus indexes of a high-dose group are increased and tend to be normal due to dose dependency. Indicating that the tolypic acid and the derivative thereof can obviously improve the immune organ index of cyclophosphamide induced immunosuppression mice. IL-4 has immunoregulatory effects on B cells, T cells, mast cells, macrophages and osteoblasts, and can induce the production of IgG and IgE, compared with a model group, the serum IL-4 content of mice in each dose group of tolipomoea and derivatives thereof is increased (P < 0.01), and the IL-4 level of the high dose group is increased and is normalized. IL-10 can inhibit NK cell activity, interfere NK cells and macrophages to produce cytokines, compared with a model group, the serum IL-10 content of mice in the tolipomoea and derivatives thereof is obviously reduced (P <0.05 or P < 0.01), and the IL-4 level is reduced and is normalized by a high-dose group. Compared with the model group, the level of the tropine acid and the derivative thereof is obviously increased (P <0.05 or P < 0.01), so that the tropine acid and the derivative thereof can stimulate the secretion of the TNF-alpha, and the activity and the killing function of macrophages are enhanced after the secretion of the TNF-alpha, so that the macrophages promote the immune response of organisms. Compared with the model group, the serum IgG mass concentration of the mice in the topiramate and the derivative group is obviously increased (P <0.05 or P < 0.01) compared with that in the model group, and the IgG level is increased and is normalized by the high-dose group. Compound 1 has no significant difference in therapeutic effect (P > 0.05) compared to 2. Compounds 3 and 4 have better therapeutic effect (P <0.05 or P < 0.01) than compounds 1 and 2. The effect of topiramate and its derivatives on organ index and serum immune index of immunosuppressive mouse model is shown in table 2. Taken together, tolmetin and its derivatives have a certain regulatory effect on the inhibition of immune function caused by cyclophosphamide.
Table 2. Influence of tolypic acid and its derivatives on organ index and serum immune index of immunosuppressive mouse model (n=10,)
note that: in comparison with the blank group, ## P<0.01; comparison with model group, P<0.05，**P<0.01。
Example 4: studies of the effects of tolypic acid and its derivatives on spleen lymphocyte proliferation
1. Cell model preparation, grouping and administration
After the mice are killed by cervical vertebra removal, the whole body is sterilized in 75% ethanol, spleen is taken out under aseptic condition, spleen lymphocytes are prepared, cells are resuspended in RPMI-1640 culture solution, and the cell concentration is counted and adjusted to be 1 multiplied by 10 7 And each ml. 100. Mu.L of the cell suspension was added to a 96-well plate. The final concentration of mitogen ConA is 5 μg/ml, 100 μl of tropinic acid and its derivatives are added to each well, including tropinic acid high dose group (A), tropinic acid low dose group (B), 4-hydroxy- α - (hydroxymethyl) phenylacetic acid high dose group (C), 4-hydroxy- α - (hydroxymethyl) phenylacetic acid low dose group (D), 3, 4-dihydroxy- α - (hydroxymethyl) phenylacetic acid high dose group (E), 3, 4-dihydroxy- α - (hydroxymethyl) phenylacetic acid low dose group (F), 3,4, 5-trihydroxy- α - (hydroxymethyl) phenylacetic acid high dose group (G) and 3,4, 5-trihydroxy- α - (hydroxymethyl) phenylacetic acid low dose group (H), blank group is added with 100 μl RPMI-1640 culture solution, positive control is added with 100 μl of RPMI-1640 culture solution containing cyclophosphamide, 3 compound wells are provided, grouping and administration information is detailed in Table 3, 37 ℃,5% CO 2 After 44 hours of incubation, 10. Mu.L of 5mg/mLMTT was added to each well and incubation was continued for 4 hours.
Table 3 cell grouping and administration information table (n=9)
2. Index detection
After the cell culture is finished, the cell culture is centrifuged at 1000r/min, the supernatant is discarded, 200 mu L of DMSO is added to each well and the mixture is oscillated for 10min for dissolution, and the mixture is placed in an enzyme-labeled instrument, and OD value is measured at the wavelength of 492 nm. Experiments were performed 3 times in parallel. Spleen lymphocyte proliferation inhibition rate = (ConA model control mean OD value-dosing group mean OD value)/ConA model control mean OD value.
3. Experimental results and discussion
As shown in Table 4, each tropine acid and its derivative group has no cytotoxicity, and has different degrees of immunosuppression on spleen cell proliferation caused by ConA, compared with the model group, OD value of each tropine acid and its derivative group is obviously reduced (P < 0.01), and high dose group inhibition rate can reach 39.8% -44.5%. Compound 1 had no significant difference in effect (P > 0.05) compared to 2. Compounds 3 and 4 act more significantly (P < 0.01) than compounds 1 and 2.
Table 4. Influence of tolypic acid and its derivatives on proliferation of spleen lymphocytes in mice (n=9,)
grouping Drug concentration (μg/mL) OD value Inhibition ratio (%)
Model — 0.6155±0.0076 0
Positive drug 20 0.3394±0.0087** 44.8578
A 40 0.3648±0.0105** 40.7311
B 10 0.5037±0.0093** 18.1641
C 40 0.3704±0.0084** 39.8213
D 10 0.4925±0.0113** 19.9838
E 40 0.3415±0.0072** 44.5167
F 10 0.4696±0.0116** 23.7043
G 40 0.3431±0.0096** 44.2567
H 10 0.4752±0.0108** 22.7945
Note that: p <0.01 compared to model group.
Example 5: research on the effect of tolypic acid and its derivatives on delayed hypersensitivity
1. Grouping, modeling and administration of animals
Male KM mice (6-7 weeks, 22.0+ -2.0G) were randomly divided into 11 groups, 10 groups each, respectively, of a blank control group, a model group, a positive drug group, a tropinic acid high dose group (A), a tropinic acid low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (H), and on day 1, the abdomen of each group was divided into Mao Yao 1.0.0 cm by 2.0cm size areas, 50. Mu.L of 1% 2, 4-Dinitrofluorobenzene (DNFB) (olive oil was applied to dissolve, 3:3 acetone=2 days, and the olive oil was applied continuously on day 1 day, 2 days. The groups were dosed simultaneously and the grouping and dosing schedule is detailed in table 5.
Table 5 animal group and administration information table (n=10)
Grouping Dosing regimen
Blank space Gastric lavage 0.2mL physiological saline
Model Gastric lavage 0.2mL physiological saline +1% DNFB50 μl
Positive drug Dexamethasone 20mg/kg.d+1% DNFB 50. Mu.L
A 50 mu L of DNFB with 40mg/kg.d+1% of gastric tropinic acid
B 50 mu L of DNFB with 20mg/kg.d+1% of gastric tropine
C Gastric lavage of 40mg/kg.d+1% DNFB 50. Mu.L of 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid
D Gastric lavage of 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid 20mg/kg.d+1% DNFB 50. Mu.L
E Gastric lavage of 40mg/kg.d+1% DNFB 50. Mu.L of 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid
F Gastric lavage of 20mg/kg.d+1% DNFB 50. Mu.L of 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid
G Gastric lavage of 40mg/kg.d+1% DNFB 50. Mu.L of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid
H Gastric lavage of 20mg/kg.d+1% DNFB 50. Mu.L of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid
2. Index detection
After 30min of the last administration, 10. Mu.L of 1% DNFB was applied to both the front and back of the right ear of each mouse. Weighing after 24 hours, performing anesthesia by intraperitoneal injection (0.1 mL/10 g) of 10% chloral hydrate, taking out the eyeball, taking blood, removing cervical vertebra, killing the mice, respectively cutting off ears, and accurately weighing by punching round lugs at the same position by an 8mm puncher. And centrifuging blood, taking supernatant, and measuring the IFN-gamma content in serum according to an ELISA detection kit. Ear swelling degree = right ear mass-left ear mass; swelling inhibition = (mean swelling degree of model group-mean swelling degree of dosing group)/mean swelling degree of model group x 100%.
3. Experimental results and discussion
The delayed hypersensitivity is a T-cell dependent immune response model, is a Th1 cell mediated allergic response, and takes part in cellular immunity and delayed hypersensitivity inflammation by Th1 mainly secreting INF-gamma, and is mainly characterized in that a sensitized organism has delayed inflammatory response at an antigen attack position. DNCB is a hapten, and is diluted and smeared on the abdominal skin to combine with skin proteins to form complete antigen, so that T lymphocyte proliferation Cheng Zhimin lymphocyte is stimulated, and the complete antigen is smeared on the ear after 7 days, so that delayed hypersensitivity is locally generated. As shown in Table 6, compared with the model group, the tolterone and the derivative group thereof obviously reduce the ear swelling degree (P <0.05 or P < 0.01) of the mice with delayed hypersensitivity and obviously inhibit the rise of the serum INF-gamma content (P <0.05 or P < 0.01) of the mice with delayed hypersensitivity, and the mice ear and the serum INF-gamma can be normalized by the high-dose group. Compound 1 had no significant difference in effect (P > 0.05) compared to 2. Compounds 3 and 4 act better (P < 0.05) than compounds 1 and 2. Indicating that tropine and derivatives thereof have a regulating effect on delayed hypersensitivity reactions, and inhibiting the increase of the INF-gamma content of serum is probably an action mechanism.
Table 6. Influence of tolypic acid and its derivatives on delayed hypersensitivity reactions in mice (n=10,)
grouping Ear swelling degree (mg) Ear swelling inhibition (%) IFN-γ(μg/mL)
Blank space 1.68±0.25 — 152.58±13.87
Model 10.56±2.63 ## — 187.82±14.41 ##
Positive drug 2.87±0.81** 72.83 163.37±12.15*
A 4.11±1.02** 61.08 167.22±13.03**
B 6.57±1.88* 47.23 172.12±14.11*
C 4.19±1.04** 60.32 166.94±10.23**
D 6.50±2.17* 47.88 171.14±11.18*
E 2.95±0.96** 72.09 155.24±11.20**
F 5.21±2.05** 50.65 168.19±13.51*
G 2.81±0.77** 73.41 154.26±10.54**
H 5.23±1.89** 50.48 167.20±12.71*
Note that: the comparison of the blank group was carried out, ## P<0.01; comparison with model group, P<0.05，**P<0.01。
Example 6: anti-inflammatory action research of tolmetin and derivative thereof
1. Grouping, modeling and administration of animals
The KM mice (6-7 weeks, 22.0+ -2.0G) were randomly divided into 11 groups of 10 mice each, which were a blank control group, a model group, a positive drug group, a tropinic acid high dose group (A), a tropinic acid low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (H), respectively, were simultaneously administered, and the groups and the administration schemes were detailed in Table 7, and were administered 2 times per day for 7 days, followed by gastric lavage for 1 hour, and after the last administration, the mice were uniformly and evenly coated with two-liter mice except the right side of the control group.
Table 7. Animal group and administration information Table (n=10)
2. Index detection
After 1h of inflammation, 10% chloral hydrate is used for intraperitoneal injection anesthesia, cervical removal and sacrifice, ears are cut off along the edges of auricles, round lugs are cut off at the same positions of the ears by using a manual 8mm ear-beating device, and the ear swelling degree and swelling inhibition rate are calculated by respectively precisely weighing. Ear swelling degree = right ear mass-left ear mass; swelling inhibition = (mean swelling degree of model group-mean swelling degree of dosing group)/mean swelling degree of model group x 100%.
3. Experimental results and discussion
As shown in Table 8, compared with the model control group, the tolypic acid and the derivative group thereof can obviously reduce the ear swelling degree (P < 0.01) of mice caused by dimethylbenzene, wherein the swelling inhibition rate of the tolypic acid and the derivative group with high dosage can reach 70.34% -74.31%. The difference in the swelling inhibition effect was not significant in compound 1 compared to compound 2. Both the high and low dose groups of compounds 3 and 4 have better swelling inhibition effect than compounds 1 and 2. The above results show that the tolmetic acid and the derivatives thereof have obvious anti-inflammatory effect on mice ear swelling model caused by the dimethylbenzene.
Table 8. Influence of tolipoic acid and its derivatives on mouse ear swelling with xylene (n=10,)
note that: in comparison with the blank group, ## P<0.01; comparison with model group, P<0.01。
Example 7: analgesic study of tolmetic acid and derivatives thereof
1. Grouping, modeling and administration of animals
100 KM mice (5-6 weeks, 22.0+ -2.0G) were randomly divided into 10 groups, 10 groups each, respectively, consisting of a blank control group, a model group, a positive drug group, a tropinic acid high dose group (A), a tropinic acid low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (H), were simultaneously administered, and the groups were intraperitoneally administered 2 times per day, and the groups were continuously perfused for 7 days, with a detailed grouping and administration schedule as shown in Table 9, and the remaining groups were injected with 2 kg of glacial pain in the model of 0.6% by the final mice after 2 hours.
Table 9. Animal group and administration information Table (n=10)
Grouping Dosing regimen
Model Lavage of 0.2mL physiological saline + intraperitoneal injection of 0.6%10mL/kg glacial acetic acid
Positive drug 400mg/kg.d+ enteric coated tablet of aspirin and 0.6%10mL/kg glacial acetic acid are injected intraperitoneally
A Gastric administration tropine 40mg/kg. D+ intraperitoneal injection of 0.6%10mL/kg glacial acetic acid
B Gastric tolipomoacid 20mg/kg. D+ intraperitoneal injection of 0.6%10mL/kg glacial acetic acid
C Lavage of 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid 40mg/kg. D+ intraperitoneal injection of 0.6%10mL/kg glacial acetic acid
D Lavage of 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid 20mg/kg. D+ intraperitoneal injection of 0.6%10mL/kg glacial acetic acid
E Lavage of 40mg/kg. D+ intraperitoneal injection of 4, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid 0.6%10mL/kg glacial acetic acid
F Stomach lavage3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid 20mg/kg. D+ intraperitoneal injection of 0.6%10mL/kg glacial acetic acid
G Lavage of 40mg/kg.d+ intraperitoneal injection of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid 0.6%10mL/kg glacial acetic acid
H Lavage of 20mg/kg.d+ intraperitoneal injection of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid 0.6%10mL/kg glacial acetic acid
2. Index detection
After the pain model is established, observing and recording the latency of the mice to generate torsion reaction and the torsion times of each group of mice within 20min, and calculating the analgesic rate. Analgesic rate = (mean number of twists in model group-mean number of twists in dosing group)/mean number of twists in model control group x 100%.
3. Experimental results and discussion
As shown in Table 10, compared with the model control group, the tolypic acid and the derivative thereof can obviously prolong the latency time of acetic acid-induced mouse torsion reaction (P <0.05 or P < 0.01) and obviously reduce the torsion frequency (P < 0.01), wherein the analgesic rate of the tolypic acid and the derivative thereof in the high-dose group can reach 55.26-58.79%. The difference in analgesic effect is not obvious between compounds 1 and 2. Compounds 3 and 4 have better analgesic effect than compounds 1 and 2. Thus, tropine and its derivatives have a pronounced peripheral analgesic effect.
Table 10. Influence of tolipomoea and its derivatives on acetic acid induced torsion response in mice (n=10,)
grouping Latency(s) Number of times of twisting body Analgesic Rate (%)
Model 5.49±0.88 45.50±6.31** —
Positive drug 8.64±1.03** 13.22±3.88** 70.94
A 8.55±0.92** 20.36±5.74** 55.26
B 6.42±0.75* 24.63±6.84** 45.86
C 8.37±1.10** 20.60±4.57** 54.72
D 6.56±0.84* 24.59±5.25** 45.95
E 8.87±1.06** 19.04±3.63** 58.15
F 7.15±0.97* 23.19±4.91** 49.04
G 8.94±1.12** 18.75±3.39** 58.79
H 7.21±0.94* 23.29±5.95** 48.81
Note that: p <0.05, P <0.01 compared to model group.
Example 8: research on effect of tolmetin and derivative thereof on autoimmune hepatitis mouse model
1. Animal modeling, grouping and administration
Normal male KM mice (6-7 weeks, 22.0+ -2.0 g) liver tissue was taken, physiological saline was added at a ratio of 1:9 (g/mL), and after tissue homogenization was performed by tissue grinder sufficient grinding (-4 ℃), centrifugation was performed for 10 minutes (2500 rpm,4 ℃), the supernatant was taken to obtain the syngeneic liver antigen. Adding complete Freund's adjuvant into isogenic liver antigen at ratio of 1:1 (v/v), mixing, and emulsifying to obtain immunity agent (prepared in time).
110 male KM mice (6-7 weeks, 22+ -2G) were randomly divided into 11 groups, each group being a blank control group, a model group, a positive drug group, a tropine high dose group (A), a tropine low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (G), and each group of the other mice except the blank control group mice were intraperitoneally injected with 1 mL/immunizing agent to complete the first immunization. And 7 days later, performing secondary immunization to obtain an autoimmune hepatitis mouse model. The mice of the blank group were simultaneously injected intraperitoneally with 1 mL/saline. The administration of the stomach was started on the day of molding, 2 times per day, and the treatment was continued for 14 days, and the grouping and administration schedule was shown in Table 11.
Table 11. Animal group and administration information Table (n=10)
2. Index detection
After the last administration, the mice of each group were fasted for 12 hours, anesthetized with 10% chloral hydrate by intraperitoneal injection (0.1 mL/10 g), dissected, and fixed with physiological saline perfusion and paraformaldehyde after heart blood collection, and liver and spleen tissues were collected and fixed with 4% paraformaldehyde for 24 hours. Centrifuging blood for 10 min (3000 rpm,4 ℃) and taking serum, and detecting the activity of liver function related indexes Lactate Dehydrogenase (LDH), glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) and the content of Total Bilirubin (TBIL) in the serum according to the instruction of a kit; paraffin embedding of liver and spleen tissue, slicing into 4 μm thick sections, conventional HE staining, sealing, and observing histopathological changes under a light microscope.
3. Experimental results and discussion
Liver function detection: as shown in table 12, the LDH, ALT, AST activity was increased in the serum of the mice of the model group, and the TBIL content was significantly increased (P < 0.01). When liver tissue is damaged or liver cells are necrotized, LDH, ALT and AST activities in serum are increased, and the increase of TBIL content is a sensitive index of liver damage, and directly reflects the degree of liver cell damage necrosis, detoxification and metabolic functions of liver. Compared to the model group, LDH, ALT, AST activity and TBIL content in serum decreased (P <0.05 or P < 0.01) and were dose dependent after 2 weeks of administration of tolterodine and its derivatives. Compound 1 has no significant difference in therapeutic effect (P > 0.05) compared to 2. Compounds 3 and 4 have better therapeutic effect (P < 0.05) than compounds 1 and 2. Thus, tropine and its derivatives may improve liver function in autoimmune hepatitis model mice.
Table 12. Influence of tolypic acid and its derivatives on liver function in mouse model of autoimmune hepatitis (n=10,)
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note that: in comparison with the blank group, ## P<0.01; comparison with model group, P<0.05，**P<0.01。
Histopathological detection: as shown in FIG. 5, a large amount of inflammatory factor infiltration was seen in the liver of model mice, and cavitation caused by cell boundary blurring, hepatic cell edema degeneration, cell nucleus shrinkage, partial cell lytic necrosis and cell steatosis was observed. After tolterodine and derivatives thereof are administered, the pathological indexes of the parts are obviously improved, and especially, regeneration liver cells appear around necrotic liver cells to repair liver injury, and the preparation is characterized by large cell volume, large cell nucleus staining depth and most of double nuclei. As shown in fig. 6, in the spleen of the mice in the model group, follicular proliferative lesions related to immune activation appear, and further, the increase of germinal centers is variable, and it can be seen that the area ratio of red marrow to white marrow decreases, the white marrow and foam-like macrophages in red marrow increase, and after the intraperitoneal injection of an immunizing agent, a great deal of lymphocyte transformation phenomenon occurs in the spleen, B cells proliferate and transform into plasma cells, and the growth centers of spleen follicles are enlarged to generate a great deal of plasma cells, so that the antibody generation is increased, and the humoral immunity is enhanced; plasma is present in the blood vessels in the red marrow and the proliferating macrophages destroy plasma erythrocytes in the small red marrow arteries, resulting in massive congestion of the spleen cord in the red marrow. After administration of tolterodine and its derivatives, the above-mentioned immune activating pathological index is significantly improved. It is shown that tolypic acid and its derivatives can play a role in treating autoimmune hepatitis by improving the function of recognizing antigen to regulate immune activity.
Example 9: research on action of tropinic acid and derivatives thereof on new crown cold-dampness epidemic mouse model
1. Animal modeling, grouping and administration
110 male KM mice (6-7 weeks, 22.0.+ -. 2.0) were randomly divided into 11 groups, each of which was a blank group, a model group, a positive drug group, a tropine high dose group (A), a tropine low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (G), each group was administered 2 times daily for 7 days continuously. After 3 days of administration, except for mice in a blank control group, each group of mice is injected with 5mg/kg of lipopolysaccharide physiological saline solution intraperitoneally, and then placed in an artificial climatic chamber, and cold-dampness stimulation modeling is carried out in an environment with the temperature of 4.0+/-2.0 ℃ and the humidity of 90.0+/-3.0%, 8 hours per day for 4 continuous days, so that a new crown cold-dampness epidemic mouse model is formed. The grouping and dosing schedule is detailed in table 13.
Table 13. Animal group and administration information Table (n=10)
Grouping Dosing regimen
Blank space Gastric lavage 0.2mL physiological saline
Model Lavage of 0.2mL physiological saline + intraperitoneal injection of 5mg/kg lipopolysaccharide
Positive drug 2mg/kg.d+ intraperitoneal injection of dexamethasone for 5mg/kg lipopolysaccharide
A Gastric lavage tropine acid 40mg/kg. D+ intraperitoneal injection 5mg/kg lipopolysaccharide
B Gastric lavage tropine acid 20mg/kg. D+ intraperitoneal injection 5mg/kg lipopolysaccharide
C Lavage of 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid 40mg/kg. D+ intraperitoneal injection of 5mg/kg lipopolysaccharide
D Lavage of 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid 20mg/kg. D+ intraperitoneal injection of 5mg/kg lipopolysaccharide
E Lavage of 40mg/kg.d+ intraperitoneal injection of 5mg/kg lipopolysaccharide of 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid
F Lavage of 20mg/kg.d+ intraperitoneal injection of 5mg/kg lipopolysaccharide of 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid
G Lavage of stomach 40mg/kg. D+ intraperitoneal injection of 5mg/kg lipopolysaccharide of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid
H Lavage of 20mg/kg. D+ intraperitoneal injection of 5mg/kg lipopolysaccharide of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid
2. Index detection
After the end of the last administration, the administration is fasted for 12 hours, 10% chloral hydrate is injected into the abdominal cavity (0.1 mL/10 g) for anesthesia, physiological saline is used for perfusion, lung tissues are taken, a part of liquid nitrogen is quickly frozen, the liquid nitrogen is preserved in a refrigerator at the temperature of minus 80 ℃ for standby, and the part of liquid nitrogen is placed in 4% paraformaldehyde for standby. Taking 50mg of frozen and preserved mouse tissues, adding 800 mu L of PBS for homogenization, centrifuging at 2800r/min for 10min at low temperature (4 ℃) and collecting serum, and measuring the levels of inflammatory factors IL-6, IL-10, IFN-gamma and TNF-alpha in lung tissues by using an ELISA detection kit. The fixed lung tissue is embedded by paraffin, cut into 4 mu m thick slices, stained by conventional HE, sealed and observed under a light microscope for histopathological changes.
3. Experimental results and discussion
Detecting inflammatory factors: as shown in table 14, the lung tissue TNF- α, IFN- γ, IL-6 levels were significantly elevated (P <0.05 or P < 0.01), IL-10 levels were significantly reduced (P < 0.01), TNF- α, IFN- γ, IL-6 levels were significantly reduced (P <0.05 or P < 0.01), IL-10 levels were significantly elevated (P <0.05 or P < 0.01) and the lung tissue inflammatory factor was reduced and normalized in the high dose group compared to the placebo group. Compound 1 has no significant difference in therapeutic effect (P > 0.05) compared to 2. Compounds 3 and 4 have better therapeutic effect (P < 0.05) than compounds 1 and 2. Thus, tropine and its derivatives can improve the inflammatory response of the new mouse model of crown cold-dampness epidemic.
Table 14. Influence of tolypic acid and its derivatives on lung tissue inflammatory factor of new mouse models of cold-dampness (n=10,)
note that: in comparison with the blank group, ## P<0.01; comparison with model group, P<0.05，**P<0.01。
Histopathological detection: as shown in fig. 7, the lung tissue structure of the normal group mice is complete, no effusion exists, and the alveolar space is normal; the model group mice have collapsed alveolar scaffolds, disordered pulmonary tissue structures, obvious alveolar space thickening degree, serious pulmonary interstitial edema and inflammatory infiltration, and obviously improved pathological indexes after the administration of tropine acid and derivatives thereof. There was no significant difference in therapeutic effect between compounds 1 and 2. Compounds 3 and 4 have better therapeutic effects than compounds 1 and 2. Indicating that tropine acid and derivatives thereof may exert therapeutic effects on a new mouse model of crown cold-dampness epidemic disease by modulating immunity and inhibiting inflammatory response.
Example 10: research on effect of tolypic acid and derivative thereof on mouse model of aplastic anemia
1. Animal modeling, grouping and administration
66 male SD rats (7-8 weeks, 200.0+ -20.0G) were randomly divided into 11 groups, each group being a blank control group, a model group, a positive drug group, a tropinic acid high dose group (A), a tropinic acid low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (G), wherein the rest groups except the blank control group mice were subcutaneously injected with 2% phenylhydrazine (Ap) physiological saline solution 20 mg/kg on days 1 and 4 th, and after 2h of phenylacetic acid injection, 7 th day 7 mg of cyclic amide physiological saline solution was continuously injected into the mice, and the model was continuously injected with a physiological saline solution was continuously injected into the mice on days 14 days. The grouping and dosing schedule is detailed in table 15.
Table 15. Animal group and administration information Table (n=6)
2. Index detection
After the last administration of the rats for 1 hour, 10% chloral hydrate is injected into the abdominal cavity for anesthesia, the heart is taken for standby in an anticoagulant tube, and thymus and spleen are taken as the masses. The whole blood in the anticoagulation tube is added with a hemolytic agent, and the number of peripheral blood cells is detected by adopting a full-automatic blood cell analyzer, and the method mainly comprises 4 index changes such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB) and platelet count (PLT) in the peripheral blood. Spleen index and thymus index were calculated, organ index = wet organ weight (g)/body weight (g) ×100%.
3. Experimental results and discussion
As shown in table 16, both model group WBC, RBC, HGB and PLT levels were significantly reduced (P < 0.01) compared to the normal group; levels of each indicator increased (P <0.05 or P < 0.01) and were dose dependent after 14d of treatment with topiramate and its derivatives compared to the model group. The spleen index was significantly increased and thymus index was significantly decreased (P < 0.01) in the model group compared to the normal group, and the spleen index and thymus index tended to be normal (P <0.05 or P < 0.01) after 14d of treatment with topiramate and its derivatives. Compound 1 has no significant difference in therapeutic effect (P > 0.05) compared to 2. Compounds 3 and 4 have better therapeutic effect (P <0.05 or P < 0.01) than compounds 1 and 2. Thus, tropine and its derivatives can improve aplastic anemia in a rat model by its immunomodulatory effects, thereby restoring its hematopoietic stem cell function.
Table 16. Influence of tolypic acid and its derivatives on immune organ index and peripheral blood image of aplastic anemia model (n=6,)
note that: in comparison with the blank group, ## P<0.01; compared with the model control group, P<0.05，**P<0.01。
Example 11: research on effect of tolmetin and derivative thereof on rheumatoid arthritis model rats
1. Animal modeling, grouping and administration
Preparation of the immune emulsifier: taking 7ml of bovine type II Collagen (CII) solution, placing the solution into a small beaker, magnetically stirring at a low temperature of 1500r/min, taking 7ml of Complete Freund's Adjuvant (CFA) solution, slowly adding the solution into the CII solution, and continuously stirring for about 30min after all the CFA solution is added until emulsion drops are not dispersed on water, thus obtaining the primary immune emulsifier. The CFA is replaced by Incomplete Freund's Adjuvant (IFA), and the secondary immune emulsifier is obtained by adopting the same preparation method. The immune emulsifying agent is prepared at the time of clinical use.
Preparation of rheumatoid arthritis model (CIA): 100 male SD rats (7-8 weeks, 200.0+ -20.0 g) were taken, 0.2ml of the primary immune emulsifier was injected subcutaneously into the root of the rat, 0.1ml of the secondary immune emulsifier was injected subcutaneously into the root of the rat on day 8 after the primary immunization to complete the booster immunization, and the blank control group was injected with physiological saline by the same method. Arthritis Index (AI) scores were performed 14 days after boost and the scoring rules were as follows: 0 is divided into no swelling and no erythema; 1, erythema and slight swelling of the ankle joint; 2, erythema and mild swelling from ankle to plantar or palmar; 3, erythema and moderate swelling of ankle to metatarsophalangeal or metacarpophalangeal joints; 4 are divided into erythema and severe swelling of the ankle to toe joints. The sum of the scores of the 2 paws was taken as the joint score of each rat, and an AI score of > 4 indicates that the model was successfully constructed and animals without signs of joint swelling were removed.
Grouping drug administration: the blank group of 6 rats and 60 CIA model rats were randomly divided into 10 groups of 6 rats, which were respectively a model group, a positive drug group, a tropine high dose group (a), a tropine low dose group (B), a 4-hydroxy- α - (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy- α - (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy- α - (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy- α - (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy- α - (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy- α - (hydroxymethyl) phenylacetic acid low dose group (G), each of which was simultaneously perfused with the corresponding drug 2 times daily for 4 weeks, and the continuous treatment schedule was shown in table 17.
Table 17 animal group and administration information Table (n=6)
2. Index detection
After the treatment, the morphological differences of ankle joints and paw of the rats in each group are compared and observed. The status of arthritis was scored once a week for each group of rats according to the AI scoring method described above. The volume change of the ankle joint and below of the rat was measured simultaneously by the foot volume drainage method, and the degree of joint swelling was calculated. Joint swelling degree= (post-molding volume-pre-molding volume)/pre-molding volume x 100%.
3. Experimental results and discussion
As shown in fig. 8, after the molding, the volume of the plantar toe of the rat was increased significantly, the joints of the rats of each molding group were red and swollen, the feet were stiff and contracted, the normal walking was impossible, the joints of the rats of the low dose group were slightly red and swollen after 4 weeks of treatment with tolterodine and its derivatives, and the joints of the rats of the other treatment groups were red and swollen, and the conditions were good. There was no significant difference in therapeutic effect between compounds 1 and 2. Compounds 3 and 4 have better therapeutic effects than compounds 1 and 2. As shown in fig. 9, the treatment group AI scores for topiramate and its derivatives gradually decreased compared to the model group, and the differences at the beginning of treatment week 3 were statistically significant (P <0.05 or P < 0.01); compared with the model group, the joint swelling degree of the rat starts to decrease after 2 weeks of treatment with tolterodine and the derivative thereof, and the joint swelling degree of the rat is remarkably decreased after 4 weeks of administration. Compound 1 had no significant difference in therapeutic effect for each week (P > 0.05) compared to 2. The high dose groups of compound 3 and 4 showed a more pronounced decrease in AI score and joint swelling (P < 0.05) with better therapeutic effect than compounds 1 and 2 after 3 weeks of administration. Taken together, tolmetin and its derivatives have obvious therapeutic effects on rheumatoid arthritis rat models.
Example 11: research on effect of tolmetin and derivative thereof on allergic rhinitis mouse model
1. Animal modeling, grouping and administration
Taking 110 male BABL/C mice (5-6 weeks, 20.0+ -2.0G), randomly dividing into 11 groups, each group being 10 blank control group, model group, positive drug group, tropinic acid high dose group (A), tropinic acid low dose group (B), 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (G), except the blank control group mice, the following miceEach group of mice was intraperitoneally injected with suspension sensitizer (containing 0.50mg/mL OVA and 0.50mg/mLAl (OH)) on days 1, 7, and 14 of the experiment 3 ) 200uL per mouse, basal sensitization; each group of medicines corresponding to the stomach irrigation on 15 th to 28 th days; on the 22 th to 28 th days of the experiment, the nasal cavities of the mice are instilled with 4% OVA solution for excitation, 20 mu L of each nasal cavity is used for establishing an allergic rhinitis model, and the blank control group is given with physiological saline with the same volume in the basic sensitization and excitation links. The grouping and dosing schedule is detailed in table 18.
Table 18. Animal group and administration information Table (n=10)
Grouping Dosing regimen
Blank space Gastric lavage 0.2mL physiological saline
Model Basic sensitization, gastric lavage 0.2mL physiological saline + excitation
Positive drug Basal sensitization and gastric lavage dexamethasone 20mg/kg. D+ excitation
A Basal sensitization and gastric lavage tropine acid 40mg/kg. D+ excitation
B Basal sensitization and gastric tropine acid 20mg/kg. D+ excitation
C Basal sensitization and lavage 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid40mg/kg. D+ challenge
D Basal sensitization and lavage of 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid 20mg/kg. D+ excitation
E Basal sensitization and lavage of 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid 40mg/kg. D+ excitation
F Basal sensitization and lavage of 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid 20mg/kg. D+ excitation
G Basal sensitization and lavage of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid 40mg/kg. D+ excitation
H Basal sensitization and lavage of 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid 20mg/kg. D+ excitation
2. Index detection
After the last nasal drop challenge, the number of nasal scratching and sneezing was observed and recorded for each group of mice over 30 min.
3. Experimental results and discussion
As shown in table 19, the number of sneezes and nasal deflection was significantly increased (P < 0.01) in the model group mice compared to the blank group, indicating successful modeling of allergic rhinitis model. Compared with the model group, the tropine acid and the derivative group can significantly reduce the sneeze time (P < 0.01) and the nasal deflection time (P <0.05 or P < 0.01) of mice, and are dose-dependent. Compound 1 has no significant difference in therapeutic effect (P > 0.05) compared to 2. Compounds 3 and 4 have better therapeutic effect (P < 0.05) than compounds 1 and 2. The tolperisoic acid and the derivative thereof have obvious therapeutic effect on allergic rhinitis mice models.
Table 19. Influence of tolypic acid and its derivatives on the number of sneezes in mice model of allergic rhinitis (n=10,)/>
note that: in comparison with the blank group, ## P<0.01; compared with the model control group, P<0.05，**P<0.01。
Example 12: research on effect of tolmetic acid and derivative thereof on model rats with membranous nephritis
1. Animal modeling, grouping and administration
Replication of the membranous nephritis model: 100mg of cationized bovine serum albumin (C-BSA) was dissolved in 15mL of physiological saline, and mixed with an incomplete Freund's adjuvant in equal volume and emulsified thoroughly. 100 male SD rats (7-8 weeks, 200+ -20 g) were taken, 1mL of the emulsifier was taken, and multi-point subcutaneous injections were made on the bilateral underarms and groin of each rat, 1 time every other day, 3 times in total, and the pre-immunization was completed. After preimmunization for 1 week, each rat was injected with a C-BSA physiological saline solution at 16mg/kg tail vein 3 times a week for 4 weeks continuously to complete the formal immunization. Urine protein detection was performed by collecting 24h urine from rats with metabolic cages, and an amount of 24h urine protein (24 h UPro) of >20mg was considered successful replication of the model for membranous nephritis, and the model replication-failed rats were removed. The rats of the blank group were synchronously injected with an equal volume of physiological saline in the same manner.
Grouping drug administration: the control group of 6 rats and 60 rats with membranous nephritis model were randomly divided into 10 groups, each group of 6 rats being a model group, a positive drug group, a tropinic acid high dose group (A), a tropinic acid low dose group (B), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (C), a 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (D), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (E), a 3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (F), a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid high dose group (G) and a 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid low dose group (G), each group being simultaneously perfused with the corresponding drugs 2 times per day, and continuously treated for 4 weeks, the grouping and the dosing schedule are shown in Table 11.
Table 20. Animal group and administration information Table (n=6)
2. Index detection
General condition observation: after the last administration, the hair, mental state, body weight, ingestion, stool, and the like of each group of rats were compared and observed to evaluate the therapeutic effect of the drug.
Urine protein quantification and serum biochemical detection: collecting urine of the rat for 24 hours, and measuring the urine protein concentration by using a rat ELISA kit; rats were anesthetized, serum was collected, and index levels of serum Total Protein (TP), serum albumin (Alb), serum creatinine (Scr), serum urea nitrogen (BUN) and the like were measured using a full-automatic biochemical analyzer.
3. Experimental results and discussion
General condition observation: normal rat movement of each group of rats with normal general conditions has the advantages of sensitive response, bright hair color, normal diet and increased body mass; the rats in the model group have poor mental and hair glossiness, hair loss, reduced ingestion, increased urine volume, loose stool and abdominal subcutaneous edema of individual rats. The tropenic acid and the derivatives thereof can obviously improve the mental state, hair, diet, urination and defecation of rats.
Quantitative detection of urine protein and serum biochemistry: as shown in table 21, rats in the model group showed significantly elevated proteinuria, serum Scr and BUN (P < 0.01), and significantly reduced serum TP, alb levels (P < 0.01) compared to the normal group; after 4 weeks of treatment with tolterodine and its derivatives, the urine protein levels in rats were significantly reduced (P < 0.01), serum TP and Alb levels were significantly increased (P <0.05 or P < 0.01), and tended to be normal for 24 hours; the serum Scr and BUN levels were significantly elevated (P < 0.01) in the model group compared to the normal group, and both serum Scr and BUN levels were reduced to varying degrees (P <0.05 or P < 0.01) and tended to be normal after 4 weeks of treatment with tropinic acid and its derivatives. Compound 1 has no significant difference in therapeutic effect (P > 0.05) compared to 2. Compounds 3 and 4 have better therapeutic effect (P < 0.05) than compounds 1 and 2. Thus, tropine and its derivatives have the effect of improving or restoring kidney function in a rat model of membranous nephritis.
Table 21. Influence of tolypic acid and its derivatives on kidney function in a rat model of membranous nephritis (n=6,)/>
note that: in comparison with the blank group, ## P<0.01; comparison with model group, P<0.05，**P<0.01。
Example 13: preparation of topiramate and its derivative tablet
The prescription is shown in Table 2. Taking bulk drugs (tolterodine) and auxiliary materials (lactose, microcrystalline cellulose, polyvinylpyrrolidone, crosslinked sodium carboxymethyl cellulose, micro-powder silica gel, magnesium stearate and purified water) according to a prescription 1, adding tolterodine, lactose, microcrystalline cellulose and polyvinylpyrrolidone into a wet granulating machine, carrying out wet granulation by using purified water as a wetting agent, carrying out wet granulation, drying and dry granulation to obtain dry granules, adding the crosslinked sodium carboxymethyl cellulose, the micro-powder silica gel and the magnesium stearate into the dry granules, uniformly mixing, and tabletting the uniformly mixed materials by using a tablet press to obtain the tolterodine tablet. Taking the raw materials and auxiliary materials of the prescriptions 2, 3 and 4 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid tablet, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid tablet and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid tablet respectively according to the same technological method.
Table 22 topaz and its derivatives tablet formulation
Material name Prescription 1 (g) Prescription 2 (g) Prescription 3 (g) Prescription 4 (g)
Tropine acid 200 — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 200 — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 200 —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 200
Lactose and lactose 980 980 980 980
Microcrystalline cellulose 700 700 700 700
Polyvinylpyrrolidone 160 160 160 160
Croscarmellose sodium 80 80 80 80
Micro powder silica gel 20 20 20 20
Magnesium stearate 20 20 20 20
Purified water 340 340 340 340
Example 14: preparation of tropinic acid and its derivative coated tablet
The prescription is shown in Table 3. Taking bulk drugs (tolterodine) and auxiliary materials (lactose, microcrystalline cellulose, hypromellose, sodium dodecyl sulfate, carboxymethyl starch sodium, micro powder silica gel, magnesium stearate, film coating premix and purified water) according to a prescription 5, adding the sodium dodecyl sulfate into the purified water, stirring to dissolve the components, adding tolterodine, lactose, microcrystalline cellulose and hypromellose into a wet granulator, carrying out wet granulation by using an aqueous solution of the sodium dodecyl sulfate as a wetting agent, carrying out wet granulation, drying and dry granulation to obtain dry granules, adding the sodium carboxymethyl starch, the micro powder silica gel and magnesium stearate into the dry granules, uniformly mixing, and tabletting the uniformly mixed materials by a tablet press to obtain the tablet of tolterodine. Adding the premix of the film coating machine into purified water, continuously stirring for more than 1 hour to prepare film coating liquid, and then coating the prepared plain tablets in the coating machine to obtain the topiramate coated tablets. Taking the bulk drugs and auxiliary materials of the prescriptions 6, 7 and 8 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid coated tablet, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid coated tablet and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid coated tablet respectively according to the same technological method.
TABLE 23 coated tablet formulation of tolterodine acid and its derivatives
Material name Prescription 5 (g) Prescription 6 (g) Prescription 7 (g) Prescription 8 (g)
Tropine acid 100 — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 100 — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 100 —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 100
Lactose and lactose 960 960 960 960
Microcrystalline cellulose 600 600 600 600
Hydroxypropyl methylcellulose 200 200 200 200
Sodium dodecyl sulfate 20 20 20 20
Carboxymethyl starch sodium 80 80 80 80
Micro powder silica gel 20 20 20 20
Magnesium stearate 20 20 20 20
Purified water 320 320 320 320
Film coating premix 80 80 80 80
Purified water (for film coating) 667 667 667 667
Example 15: preparation of topiramate and its derivative spray I
The prescription is shown in Table 4. Taking bulk drugs (tropinic acid) and auxiliary materials (polyvinylpyrrolidone K30, propylene glycol monocaprylate, ethylparaben, poloxamer, di-tert-butyl-p-cresol (BHT), 1N sodium hydroxide solution, ethanol and water) according to a prescription 9, adding the bulk drugs, polyvinylpyrrolidone K30, propylene glycol monocaprylate, ethylparaben and BHT into ethanol, and stirring to completely dissolve the bulk drugs; adding poloxamer into proper amount of water for dissolution; then adding the solution of the poloxamer Sha Mshui into the alcohol solution of the mixed materials, and adjusting the pH value to be in the range of 3-8 by using a 1N sodium hydroxide solution; adding water to 200ml to obtain spray solution; and filling the solution into a spray bottle to obtain the topiramate spray I. Taking the raw materials and auxiliary materials of the prescriptions 10, 11 and 12 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid spray I, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid spray I and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid spray I respectively according to the same technological method.
TABLE 24 topiramate and derivatives thereof spray I formulation
Material name Prescription 9 Prescription 10 Prescription 11 Prescription 12
Tropine acid 4.0g — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 4.0g — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 4.0g —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 4.0g
Polyvinylpyrrolidone K30 8.0g 8.0g 8.0g 8.0g
Propylene glycol monocaprylate 15.0g 15.0g 15.0g 15.0g
Hydroxy-phenyl ethyl ester 0.2g 0.2g 0.2g 0.2g
Poloxamer (poloxamer) 1.0g 1.0g 1.0g 1.0g
Di-tert-butyl-p-cresol (BHT) 0.5g 0.5g 0.5g 0.5g
1N sodium hydroxide solution Proper amount of Proper amount of Proper amount of Proper amount of
Ethanol 80g 80g 80g 80g
Water and its preparation method To 200ml To 200ml To 200ml To 200ml
Example 16: preparation of topiramate and its derivative spray II
The prescription is shown in Table 5. Taking raw materials (tropinic acid) and auxiliary materials (hydroxypropyl cellulose, propylene glycol monocaprylate, ethylparaben, ethanol and water) according to a prescription 13, adding the raw materials, the hydroxypropyl cellulose, the propylene glycol monocaprylate and the ethylparaben into the ethanol, stirring to completely dissolve the raw materials, adding water to 200ml, and obtaining a spray solution; and filling the solution into a spray bottle to obtain the topiramate spray II. Taking the raw materials and auxiliary materials of the prescriptions 14, 15 and 16 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid spray II, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid spray II and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid spray II respectively according to the same technological method.
Table 25 formulation of topiramate and its derivatives as spray II
Material name Prescription 13 Prescription 14 Prescription 15 Prescription 16
Tropine acid 2.0g — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 2.0g — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 2.0g —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 2.0g
Hydroxypropyl cellulose 6g 6g 6g 6g
Propylene glycol monocaprylate 20g 20g 20g 20g
Hydroxy-phenyl ethyl ester 0.2g 0.2g 0.2g 0.2g
Ethanol 100g 100g 100g 100g
Water and its preparation method To 200ml To 200ml To 200ml To 200ml
Example 17: preparation of tolypic acid and derivative injection I thereof
The prescription is shown in Table 6. Taking bulk drugs (tropinic acid) and auxiliary materials (sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, poloxamer, sodium hydrogen sulfite and water for injection) according to a prescription 17, adding the bulk drugs and the poloxamer into the water for injection to be dissolved, then adding the sodium chloride, the disodium hydrogen phosphate, the sodium dihydrogen phosphate and the sodium hydrogen sulfite into the solution to be dissolved, and finally adding the water to 100mL to obtain an injection solution; and filling the injection solution into ampoule bottles or penicillin bottles with corresponding volumes to obtain the tropine acid injection I. Taking the raw materials and auxiliary materials of the prescriptions 18, 19 and 20 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid injection I, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid injection I and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid injection I respectively according to the same technological method.
Table 26 Torpedo acid and its derivatives for injection I prescription
Material name Prescription 17 Prescription 18 Prescription 19 Prescription 20
Tropine acid 1.0g — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 1.0g — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 1.0g —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 1.0g
Sodium chloride 0.85g 0.85g 0.85g 0.85g
Disodium hydrogen phosphate 0.21g 0.21g 0.21g 0.21g
Sodium dihydrogen phosphate 1.6mg 1.6mg 1.6mg 1.6mg
Poloxamer (poloxamer) 0.5g 0.5g 0.5g 0.5g
Sodium bisulfite 0.1g 0.1g 0.1g 0.1g
Water for injection To 100mL To 100mL To 100mL To 100mL
Example 18: preparation of tolypic acid and derivative injection II thereof
The prescription is shown in Table 7. Taking raw materials (tropinic acid) and auxiliary materials (sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, tween 80 and water for injection) according to a prescription 21, adding the raw materials and the tween 80 into the water for injection to dissolve, then adding the sodium chloride, the disodium hydrogen phosphate and the sodium dihydrogen phosphate into the solution to dissolve, and finally adding the water to 100mL to obtain an injection solution; and filling the injection solution into ampoule bottles or penicillin bottles with corresponding volumes to obtain the tropine acid injection II. Taking the raw materials and auxiliary materials of the prescriptions 22, 23 and 24 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid injection II, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid injection II and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid injection II respectively according to the same technological method.
Table 27 Torpedo acid and its derivatives for injection II prescription
Material name Prescription 21 Prescription 22 Prescription 23 Prescription 24
Tropine acid 0.5g — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 0.5g — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 0.5g —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 0.5g
Sodium chloride 0.85g 0.85g 0.85g 0.85g
Disodium hydrogen phosphate 0.21g 0.21g 0.21g 0.21g
Sodium dihydrogen phosphate 1.6mg 1.6mg 1.6mg 1.6mg
Tween 80 0.3g 0.3g 0.3g 0.3g
Water for injection To 100ml To 100ml To 100ml To 100ml
Example 19: preparation of tolypic acid and its derivative cream I
The prescription is shown in Table 8. Taking bulk drugs (tropinic acid) and auxiliary materials (white vaseline, cetyl alcohol, tween 80, diethylene glycol monoethyl ether, ethyl hydroxybenzoate, BHT, propylene glycol, citric acid/sodium citrate and water) according to a prescription 25, and the preparation process is as follows: the method comprises the following steps of (1) dissolving bulk drugs: propylene glycol is weighed, raw materials are added, and stirring and dissolving are carried out at 40-50 ℃: (2) oil phase preparation: weighing white vaseline, cetyl alcohol, ethyl hydroxybenzoate and BHT, heating to 60-80deg.C, stirring for dissolving, slowly adding the dissolved raw materials, stirring, and mixing well; (3) aqueous phase preparation: weighing purified water, adding tween 80 and diethylene glycol monoethyl ether, heating to 60-80 ℃, adjusting the pH value by citric acid/sodium citrate, and stirring and dissolving for later use; (4) emulsification: slowly adding the water phase into the oil phase, maintaining at 70deg.C, homogenizing, and stirring for more than 30 min; (5) forming paste: cooling, stopping heating, continuing stirring, gradually cooling to room temperature, cooling to obtain paste, and packaging. Thus obtaining the tropine acid cream I. Taking the raw materials and auxiliary materials of the prescriptions 26, 27 and 28 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I respectively according to the same technological method.
Table 28 Torpedo acid and its derivative cream I prescription
Example 20: preparation of tolypic acid and its derivative cream II
The prescription is shown in Table 9. The preparation method comprises the steps of taking bulk drug (tropinic acid) and auxiliary materials (white vaseline, cetyl alcohol, poloxamer 407, propylene glycol monocaprylate, sodium benzoate, BHA, propylene glycol, glacial acetic acid/sodium acetate and water) according to a prescription 29, wherein the preparation process comprises the following steps: the method comprises the following steps of (1) dissolving bulk drugs: weighing propylene glycol, adding the raw materials, and stirring at 40-50 ℃ for dissolution; (2) oil phase preparation: weighing white vaseline, cetyl alcohol, propylene glycol monocaprylate and BHA, heating to 60-80 ℃, stirring for dissolving, slowly adding the dissolved raw materials, continuously stirring, and uniformly mixing for later use; (3) aqueous phase preparation: weighing purified water, adding poloxamer 407 and sodium benzoate, heating to 60-80 ℃, adjusting the pH value by glacial acetic acid/sodium acetate, and stirring and dissolving for later use; (4) emulsification: slowly adding the water phase into the oil phase, maintaining at 70deg.C, homogenizing, and stirring for more than 30 min; (5) forming paste: cooling, stopping heating, continuing stirring, gradually cooling to room temperature, cooling to obtain paste, and packaging. Thus obtaining the tropine acid cream II. Taking the raw materials and auxiliary materials of the prescriptions 30, 31 and 32 respectively, and preparing the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste II respectively according to the same technological method.
Table 29 Torpedo acid and its derivative cream II formulation
Material name Prescription 29 Prescription 30 Prescription 31 Prescription 32
Tropine acid 8g — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 8g — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 8g —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 8g
White vaseline 20g 20g 20g 20g
Cetyl alcohol 40g 40g 40g 40g
Poloxamer 407 10g 10g 10g 10g
Propylene glycol monocaprylate 20g 20g 20g 20g
Sodium benzoate 1g 1g 1g 1g
BHA 0.4g 0.4g 0.4g 0.4g
Propylene glycol 20g 20g 20g 20g
Glacial acetic acid/sodium acetate Proper amount of Proper amount of Proper amount of Proper amount of
Water and its preparation method To 200g To 200g To 200g To 200g
Example 21: preparation of tropine and its derivative nasal preparation I
The prescription is shown in Table 30. The preparation method comprises the steps of taking bulk drug (tropinic acid) and auxiliary materials (sorbitol, citric acid/sodium citrate, sodium benzoate and purified water) according to a prescription 33, wherein the preparation process is as follows: adding sorbitol into purified water for dissolution, then adding crude drug and sodium benzoate into the solution for dissolution, adjusting the pH value to be in the range of 4.0-6.5 by using citric acid/sodium citrate, adding purified water to 100ml to obtain a nasal preparation solution, and finally filling the solution into a dropping bottle or a spraying bottle to obtain the tropine acid nasal preparation I. The raw materials and auxiliary materials of the prescriptions 34, 35 and 36 are respectively taken, and the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid nasal preparations I are respectively prepared according to the same technological method.
TABLE 30 nasal formulation I prescription for tolterodine acid and its derivatives
Material name Prescription 33 Prescription 34 Prescription 35 Prescription 36
Tropine acid 0.5g — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 0.5g — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 0.5g —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 0.5g
Sorbitol 4.8g 4.8g 4.8g 4.8g
Citric acid/sodium citrate Proper amount of Proper amount of Proper amount of Proper amount of
Sodium benzoate 0.15g 0.15g 0.15g 0.15g
Purified water To 100mL To 100mL To 100mL To 100mL
Example 22: preparation of tropine acid and its derivative nasal preparation II
The prescription is shown in Table 31. Taking raw materials (tropinic acid) and auxiliary materials (sodium chloride, hypromellose, tween 80, sodium metabisulfite, sodium ethylenediamine tetraacetate, 1N sodium hydroxide solution, benzalkonium chloride and purified water) according to a prescription 37, and the preparation process is as follows: adding hydroxypropyl methylcellulose into purified water, continuously stirring to dissolve completely, adding sodium chloride and Tween 80 into hydroxypropyl methylcellulose solution to dissolve, then adding bulk drugs, sodium metabisulfite, sodium ethylenediamine tetraacetate and benzalkonium chloride into the solution to dissolve, adjusting the pH value to be in the range of 4.5-6.5 by using 1N sodium hydroxide solution, adding purified water to 100ml to obtain nasal preparation solution, and finally filling the solution into a dropping bottle or a spray bottle to obtain the tropine acid nasal preparation II. The raw materials and auxiliary materials of the prescriptions 38, 39 and 40 are respectively taken, and the 4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I, the 3, 4-dihydroxyl-alpha- (hydroxymethyl) phenylacetic acid emulsifiable paste I and the 3,4, 5-trihydroxy-alpha- (hydroxymethyl) phenylacetic acid nasal preparations II are respectively prepared according to the same technological method.
Table 31 formula II for nasal formulations of tolterodine and its derivatives
Material name Prescription 37 Prescription 38 Prescription 39 Prescription 40
Tropine acid 2g — — —
4-hydroxy-alpha- (hydroxymethyl) phenylacetic acid — 2g — —
3, 4-dihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — 2g —
3,4, 5-Trihydroxy-alpha- (hydroxymethyl) phenylacetic acid — — — 2g
Sodium chloride 0.8g 0.8g 0.8g 0.8g
Hydroxypropyl methylcellulose 3g 3g 3g 3g
Tween 80 0.3g 0.3g 0.3g 0.3g
Sodium metabisulfite 0.2g 0.2g 0.2g 0.2g
Ethylenediamine tetraacetic acid sodium salt 0.2g 0.2g 0.2g 0.2g
1N sodium hydroxide solution Proper amount of Proper amount of Proper amount of Proper amount of
Benzalkonium chloride 0.02g 0.02g 0.02g 0.02g
Purified water To 100mL To 100mL To 100mL To 100mL
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (9)

1. Use of tropine acid and derivatives thereof, pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any ratio, for the preparation of a medicament for the prophylaxis and/or treatment of diseases associated with immunity and inflammation, said tropine acid and derivatives thereof having the structure shown in formula a:
wherein R is 1 -R 5 Each independently selected from-H, -OH or C1-C6 alkoxy;
the immune and inflammation related diseases include: allergic rhinitis, bronchitis, bronchial asthma, sphagitis, conjunctivitis, neonatal hemolytic reaction, hemolytic anemia, aplastic anemia, nephritis, tuberculosis, syphilis, pneumonia (including neocrown), gastrointestinal inflammation, endometritis, otitis media, sepsis, myocarditis, meningitis, tonsillitis, sinusitis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, anorectal inflammation, paronychia and folliculitis, osteoarthritis, hepatitis, systemic lupus erythematosus, spondylitis, rheumatoid arthritis, diabetes, pancreatitis, enteritis, rheumatic heart disease, vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue diseases, and thyroiditis.
2. The use according to claim 1, wherein said immune and inflammatory related diseases comprise: allergic rhinitis, aplastic anemia, membranous nephritis, neocrown, autoimmune hepatitis, and rheumatoid arthritis.
3. Use according to claim 1 or 2, characterized in that R 1 -R 5 Each independently selected from-H or-OH.
4. Use according to claim 1 or 2, characterized in that the tropine acid and its derivatives are selected from the compounds of the following formulae I-IV:
5. a prophylactic and/or therapeutic agent for an immune-inflammatory related disease, which comprises the tolperisoic acid and its derivatives, pharmaceutically acceptable salts, solvent compounds, enantiomers, diastereomers, tautomers, or mixtures thereof in any ratio according to any one of claims 1 to 4;
the immune and inflammation related diseases include: allergic rhinitis, bronchitis, bronchial asthma, sphagitis, conjunctivitis, neonatal hemolytic reaction, hemolytic anemia, aplastic anemia, nephritis, tuberculosis, syphilis, pneumonia (including neocrown), gastrointestinal inflammation, endometritis, otitis media, sepsis, myocarditis, meningitis, tonsillitis, sinusitis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, anorectal inflammation, paronychia and folliculitis, osteoarthritis, hepatitis, systemic lupus erythematosus, spondylitis, rheumatoid arthritis, diabetes, pancreatitis, enteritis, rheumatic heart disease, vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue diseases, and thyroiditis.
6. The use according to claim 5, wherein said immune and inflammatory related diseases comprise: allergic rhinitis, aplastic anemia, membranous nephritis, neocrown, autoimmune hepatitis, and rheumatoid arthritis.
7. The use according to any one of claims 1-4 or the medicament according to claim 5 or 6, wherein the medicament is for external use, oral administration or injection.
8. The use according to any one of claims 1 to 4 or the medicament according to claim 5 or 6, wherein the medicament further comprises a pharmaceutically acceptable carrier or adjuvant.
9. The use according to any one of claims 1-4 or the medicament according to claim 5 or 6, wherein the medicament is in a solid, liquid or semi-solid dosage form, preferably the medicament is in a dosage form of: powders, tablets, coated tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, nasal preparations, aerosols, powder mists, lotions, liniments, ointments, plasters, pastes, gels, patches.
CN202311318843.1A 2023-03-08 2023-03-08 Medical application of tolypic acid and derivative thereof in preparation of medicines for treating immune and inflammation related diseases Pending CN117298085A (en)

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