Medical use of tropic acid and its derivatives in the preparation of drugs for treating diseases related to immunity and inflammation

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the use of tropic acid and its derivatives in the preparation of drugs for treating immune and inflammation-related diseases, and to drugs for treating immune and inflammation-related diseases. The tropic acid and its derivatives are compounds of formulas I-IV or pharmaceutically acceptable salts thereof, as well as solvent compounds, enantiomers, diastereomers, tautomers, or mixtures thereof in any proportion, including racemic mixtures, of the compounds of formulas I-IV or pharmaceutically acceptable salts thereof. Animal studies show that tropic acid and its derivatives have broad-spectrum immunomodulatory, anti-inflammatory, and analgesic effects, significantly improving pathological indicators in numerous animal models of immune and inflammation-related diseases. They can regulate abnormal immune responses towards normalcy and exert good anti-inflammatory and analgesic effects, making them suitable for patients with different disease degrees and types of immune and inflammation-related diseases, and thus have industrial applications.

Description

[0001] This application is a divisional application of the invention patent application filed on March 8, 2023, with application number 202310217736.3 and invention title "Pharmaceutical use of tropinic acid and its derivatives in the preparation of drugs for the treatment of immune and inflammatory diseases". Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, and in particular relates to the use of tropinic acid and its derivatives in the preparation of drugs for the treatment of immune and inflammatory diseases, as well as drugs for the treatment of immune and inflammatory diseases. Background Technology

[0003] Immunological diseases are a large category of diseases characterized by localized or systemic abnormal inflammatory immune responses, mainly including hypersensitivity reactions, immunodeficiency diseases, and autoimmune diseases. Type I hypersensitivity reactions include penicillin allergy, drug-induced rashes, seasonal, pollen, or dust-induced allergic rhinitis, pharyngitis, conjunctivitis, bronchial asthma, eczema, and urticaria; Type II hypersensitivity reactions include neonatal hemolytic reactions, drug-induced hemolytic anemia, and aplastic anemia; Type III hypersensitivity reactions include glomerulonephritis; and Type IV hypersensitivity reactions include tuberculosis and syphilis. Infection-related diseases include bronchitis or pneumonia, gastroenteritis, endometritis, otitis media, tonsillitis, boils, sinusitis, abscesses or granulomas, sepsis, septicemia, myocarditis, meningitis, osteoarthritis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, proctitis, paronychia, and folliculitis. Autoimmune diseases include 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, and autoimmune thyroiditis. It is estimated that the incidence of autoimmune diseases is increasing year by year, with approximately 7.6% to 9.4% of the global population suffering from various types of autoimmune diseases. These diseases are difficult to cure, and most patients require long-term or even lifelong medication. Some diseases, such as lupus nephropathy, are extremely dangerous, severely impacting patients' quality of life and threatening their lives. Approximately 50 million Americans (about one-fifth of the total population) suffer from autoimmune diseases, of which about 75% are women. Autoimmune diseases have become the third leading cause of chronic disease after cardiovascular disease and cancer. Although there is currently no definitive incidence data in China, the patient population is increasing annually.

[0004] Treatment of autoimmune diseases has two main goals: symptom relief and functional maintenance, and slowing the progression of tissue damage. Currently, drugs for treating autoimmune diseases are mainly classified into nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), disease-modifying antirheumatic drugs (DMARDs), biologics, and natural medicines. NSAIDs are commonly used to treat autoimmune diseases, effectively reducing clinical symptoms and signs and eliminating local inflammatory responses. However, these drugs cannot control disease progression, and common adverse reactions include central nervous system symptoms, cardiovascular damage, gastrointestinal symptoms, hematopoietic system changes, liver and kidney dysfunction, asthma, and drug eruptions. SAIDs have strong anti-inflammatory and immunosuppressive effects, preventing inflammatory cells from accumulating at the site of inflammation, inhibiting the release of inflammatory factors, and suppressing the proliferation and secretion of TB lymphocytes. These drugs have many adverse reactions, and relapse is common after discontinuation. Currently, they are often used in combination with other immunosuppressants in clinical practice. DMARDs are widely used in the treatment of autoimmune diseases such as chronic kidney disease, transplant rejection, and tumors. Although the chemical structures and mechanisms of action of traditional DMARDs are not entirely the same, their clinical pharmacodynamic characteristics are similar: slow onset of action, with symptoms and signs gradually alleviating after several weeks or months of use. Long-term continuous use can achieve relatively stable efficacy. The main adverse reactions include gastrointestinal reactions, bone marrow suppression, infection, and liver and kidney damage. Biologics exert their therapeutic effects by blocking key inflammatory cytokines or cell surface molecules, such as monoclonal antibodies targeting IL-1, IL-6, TNF-α, and IL-17, anti-CD20 monoclonal antibodies, B lymphocyte-stimulating factor (BAFF) inhibitors, T-cell inhibitors, integrin monoclonal antibodies, and selective adhesion molecule inhibitors. Most of these drugs are in the clinical trial stage, and a few are already on the market. They have many and serious adverse reactions, and some drugs have been banned due to serious adverse reactions. Natural drugs used to treat immune diseases include glycosides and alkaloids. Glycosides include total glucosides of paeony, total glucosides of ginseng, total glucosides of gynostemma pentaphyllum, astragaloside A, total glucosides of triptolide, and total saponins of Panax notoginseng. Alkaloids include sinomenine, total alkaloids of aconite, sophoridine, and triptolide. These drugs have fewer adverse reactions and mostly have anti-inflammatory, analgesic, and immunosuppressive effects, but their clinical treatment is not highly targeted and the efficacy is not ideal. With the deepening understanding of the pathological mechanisms of immune diseases and the discovery of new drug targets, in addition to NSAIDs, SAIDs, and traditional DMARDs, targeted small molecule drugs such as tofacitinib, baricitinib, upatacitinib, and filgotinib have also been developed and applied clinically to treat inflammatory and immune diseases. These drugs have definite efficacy, but they also have adverse reactions such as gastrointestinal symptoms, immunosuppression, bone marrow suppression, infection, and new tumor formation. Therefore, developing small molecule drugs with immunomodulatory and anti-inflammatory effects without impairing the body's physiological functions is the main strategy and direction for treating immune and inflammation-related diseases.

[0005] In summary, the patient population with immune and inflammatory diseases is large. Although there are many types of drugs or methods for treatment, their efficacy is limited, often requiring long-term, repeated, or even lifelong treatment. Some drugs may achieve temporary relief, but the short-term relapse rate is high. Most drugs are often limited by their inherent toxicity and selectivity, and various systemic and local adverse reactions are unavoidable. To address these issues, there is an urgent need to develop therapeutic drugs that are fast-acting, have short treatment courses, require small dosages, have few adverse reactions, low relapse rates, are inexpensive, and are easy to use. Simultaneously, the development of drug formulations such as topical, oral, and injectable formulations should be considered to suit patients with different disease severities and types. This invention, through extensive animal experiments, screened and clarified the effectiveness of tropic acid (DL-TropicAcid, also known as 2-phenyl-3-hydroxypropionic acid) and its derivatives in treating immune and inflammatory diseases. Tropic acid is an intermediate in the synthesis of atropine, and the raw material is inexpensive. Currently, there are no reports on the prevention and treatment of immune and inflammatory diseases using tropic acid and its derivatives. Summary of the Invention

[0006] This invention provides, in one aspect, the use of tropinic acid and its derivatives, pharmaceutically acceptable salts thereof, solvent compounds, enantiomers, diastereomers, tautomers, or mixtures thereof in any proportion thereof in the preparation of a formulation for the prevention and / or treatment of immune and inflammatory-related diseases, wherein the tropinic acid and its derivatives have the structure shown in Formula A:

[0007]

[0008] R1-R5 are each independently selected from -H, -OH or C1-C6 alkoxy groups.

[0009] In one implementation, R1-R5 are each independently selected from -H or -OH.

[0010] In one implementation, R1 and R5 are selected from -H, and R2-R4 are each independently selected from -H or -OH.

[0011] In one embodiment, R1 and R5 are selected from -H, two or three of R2-R4 are selected from -OH, and the rest are selected from -H.

[0012] In one implementation, at least two of R1-R5 are selected from -OH.

[0013] In one implementation, two or three of R1-R5 are selected from -OH.

[0014] In one implementation, two or three of R2-R4 are selected from -OH.

[0015] In one embodiment, the tropinic acid and its derivatives are selected from compounds represented by formulas I-IV:

[0016]

[0017] Wherein, Formula I is DL-Tropic Acid, Formula II is 4-Hydroxy-α-(hydroxymethyl)benzeneacetic acid, Formula III is 3,4-Dihydroxy-α-(hydroxymethyl)benzeneacetic acid, and Formula IV is 3,4,5-Trihydroxy-α-(hydroxymethyl)benzeneacetic acid.

[0018] The tropinic acid and its derivatives described in this invention have immunomodulatory, anti-inflammatory and analgesic effects. They have a significant effect on improving the pathological indicators of many animal models of immune and inflammation-related diseases, can regulate abnormal immune responses to normal, and exert good anti-inflammatory and analgesic effects.

[0019] In one implementation, the immune and inflammation-related diseases are selected from one or more of the following: allergic rhinitis, bronchitis, bronchial asthma, pharyngitis, conjunctivitis, eczema, urticaria, neonatal hemolytic reaction, hemolytic anemia, aplastic anemia, nephritis, tuberculosis, syphilis, pneumonia (including COVID-19), gastroenteritis, endometritis, otitis media, sepsis, septicemia, myocarditis, meningitis, tonsillitis, sinusitis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, proctitis, 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.

[0020] A second aspect of the present invention provides a medicament for the prevention and / or treatment of immune and inflammation-related diseases, the medicament comprising tropinic acid and its derivatives, pharmaceutically acceptable salts thereof, solvent compounds, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion thereof.

[0021] The drug provided by this invention for the prevention and / or treatment of immune and inflammatory-related diseases has broad-spectrum immunomodulatory, anti-inflammatory and analgesic effects, and is highly effective.

[0022] In one embodiment, tropinic acid and its derivatives, pharmaceutically acceptable salts thereof, solvent compounds, enantiomers, diastereomers, tautomers, or mixtures thereof in any proportion thereof serve as the active ingredient in the medicament of the present invention. Preferably, it serves as the principal active ingredient; more preferably, it serves as the sole active ingredient.

[0023] In the above-mentioned uses and drugs, tropinic acid and its derivatives, pharmaceutically acceptable salts, solvent compounds, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion can be prepared with pharmaceutically acceptable carriers or excipients into drug dosage forms for external, oral or injectable administration.

[0024] Therefore, in this invention, the drug can be a topical drug, an oral drug, or an injectable drug.

[0025] In this invention, the drug may contain a pharmaceutically acceptable carrier or excipient. The drug can be formulated into various conventional solid, liquid, or semi-solid dosage forms, such as granules, tablets, or capsules; liquid dosage forms such as sprays and injections; and semi-solid dosage forms such as creams. In one aspect, the dosage form of the drug may be: powder, tablet, coated tablet, granule, capsule, solution, emulsion, suspension, injection, spray, nasal spray, aerosol, powder spray, lotion, liniment, ointment, plaster, paste, gel, patch, etc.

[0026] In this invention, the term "pharmaceutically acceptable carrier or excipient" includes any and all solvents, cosolvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drug stabilizers, binders, excipients, diluents, flow aids, granulators, disintegrants, thickeners, viscous agents, lubricants, anti-caking agents, humectants, wetting agents, chelating agents, plasticizers, dyes, flavoring agents, etc., and combinations thereof, as is well known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 19th Ed. Mack Printing Company, 1995; Shanghai Pharmaceutical Industry Research Institute et al., Pharmaceutical Excipient Application Technology (Second Edition), China Medical Science and Technology Press, 2002; Comparative Handbook of Pharmaceutical Excipient Standards of Various Countries, Volumes 1-3, compiled by the National Pharmacopoeia Commission, China Medical Science and Technology Press, 2016; Handbook of Pharmaceutical Excipients, RC Raymond C. Rowe, Paul J Sheskey, Paul J Weller (eds.), translated by Zheng Junmin, Chemical Industry Press, 2005, etc.). Except for carriers and excipients incompatible with the active ingredient, any conventional carriers and excipients may be considered in therapeutic or pharmaceutical compositions.

[0027] For example, as a solid dosage form, the pharmaceutically acceptable carrier or excipient may include at least one of the following: (a) fillers such as starch, corn starch, modified starch, compressible starch, lactose, lactose monohydrate, microcrystalline cellulose, cyclodextrin, sorbitol, mannitol, calcium phosphate, amino acids, etc.; (b) binders such as starch paste, gelatinized starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, low-substituted hydroxypropyl cellulose, polyvinylpyrrolidone, gelatin, alginate, etc.; (c) humectants such as glycerin; (d) disintegrating agents. Disintegrants, such as dry starch, modified starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crospovidone, crospovidone carboxymethyl cellulose sodium, microcrystalline cellulose, effervescent disintegrants, crospovidone polyvinylpyrrolidone, etc.; (e) solution inhibitors, such as paraffin; (f) absorption promoters, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) absorbents, such as kaolin and bentonite; (i) lubricants, such as talc, stearic acid, magnesium or calcium stearate, micronized silica gel, hydrogenated castor oil and solid polyethylene glycol, polyethylene glycol 4000-20000, magnesium dodecyl sulfate, etc.

[0028] In this invention, the drug is applicable to humans or other warm-blooded animals. When applicable to humans, the preferred dosage of tropinic acid and its derivatives, either alone or in combination, is 1 mg / kg·d to 50 mg / kg·d, more preferably 10 mg / kg·d to 20 mg / kg·d. The therapeutically effective amount of the compound or pharmaceutical composition depends on the individual's species, weight, age, individual condition, the disease being treated, or its severity. Physicians, clinicians, or veterinarians with common skills can easily determine the effective amount of each active ingredient required for the prevention, treatment, or inhibition of disease progression.

[0029] The present invention also provides a compound of formula A, a pharmaceutically acceptable salt thereof, a solvent compound, an enantiomer, a diastereomer, a tautomer, or a mixture thereof in any proportion:

[0030]

[0031] Among them, R1-R5 are each independently selected from -H or -OH;

[0032] The condition is that at least two of R1-R5 are selected from -OH.

[0033] In one implementation, two or three of R1-R5 are selected from -OH.

[0034] In one implementation, two or three of R2-R4 are selected from -OH.

[0035] In one embodiment, the compound is selected from the compounds shown in Formulas III-IV:

[0036]

[0037] Pharmaceutically acceptable salts of the compounds of this invention include their base addition salts and acid addition salts. Preferably, the base addition salts are selected from sodium, potassium, calcium, lithium, magnesium, zinc, ammonium, tetramethylammonium, tetraethylammonium, triethylamine, trimethylammonium, ethylamine, diethanolamine, arginine, or lysine salts; the acid addition salts are selected from organic acid salts such as acetate, aspartate, benzoate, benzenesulfonate, citrate, ethanedisulfonate, ethanesulfonate, formate, fumarate, gluconate, glucuronate, lactate, malate, trifluoroacetate, and maleate, as well as inorganic acid salts such as hydrochloride, hydrobromide, hydrogen sulfate, nitrate, and phosphate. The free form of the compounds of this invention can be converted into the corresponding salt form; and vice versa. The free or salt form and solvated form of the compounds of this invention can be converted into the non-solvated free or salt form of the corresponding compounds; and vice versa.

[0038] The compounds of the present invention also comprise their solvated forms, which refer to associative compounds formed by one or more solvent molecules with the compounds of the present invention. Solvents forming solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0039] The compounds of the present invention can exist as isomers and mixtures thereof; for example, tautomers, optical isomers, enantiomers, and diastereomers. The compounds of the present invention may, for example, contain an asymmetric carbon atom, and therefore can exist as enantiomers or diastereomers and mixtures thereof, for example, as racemates. The compounds of the present invention can exist in (R)-, (S)-, or (R, S)- configurations, preferably in the (R)- or (S)- configuration at specific positions in the compound.

[0040] The present invention has the following advantages and effects compared with the prior art:

[0041] (1) This invention is the first to discover that tropinic acid and its derivatives can significantly improve pathological indicators in animal models of immune and inflammation-related diseases;

[0042] (2) Tropical acid and its derivatives can regulate abnormal immune responses to normalization and exert good anti-inflammatory and analgesic effects.

[0043] (3) Tropical acid and its derivatives are the main components for treating immune and inflammatory diseases. Compared with existing drugs, they have the advantages of low toxicity, fast onset of action, short course of treatment, small dosage, low recurrence rate and convenient use. They also take into account the dosage forms such as external use, oral and injection, and can be adapted to patients with different disease degrees and different types of immune and inflammatory diseases.

[0044] (4) The tropinic acid derivative compounds 3 and 4 obtained through structural modification have better and more significant therapeutic effects than tropinic acid in the treatment of immune and inflammation-related diseases.

[0045] (5) Tropical acid and its derivatives used in this invention are easy to obtain and synthesize, inexpensive, stable, easy to store and transport, and suitable for industrial application. Attached Figure Description

[0046] Figure 1 Compound III 1 H-NMR spectrum

[0047] Figure 2 Compound III 13 C-NMR spectrum

[0048] Figure 3 Compounds of formula IV 1 H-NMR spectrum

[0049] Figure 4 Compounds of formula IV 13 C-NMR spectrum

[0050] Figure 5 Effects of tropinic acid and its derivatives on liver histopathology in a mouse model of autoimmune hepatitis (x200)

[0051] Figure 6 Effects of tropinic acid and its derivatives on spleen histopathology in a mouse model of autoimmune hepatitis (x200)

[0052] Figure 7 Effects of tropinic acid and its derivatives on lung tissue pathology in a mouse model of COVID-19 cold-dampness epidemic (x200)

[0053] Figure 8 Comparative observation of the morphological differences in ankle joints and paws of rats before and after treatment in each group.

[0054] Figure 9 Comparison of trends in joint index and swelling in rats of different groups (n=6, ). Detailed Implementation

[0055] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention. The described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Example 1: Preparation of compound III (3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid)

[0057] 1. Synthesis process

[0058] Under ice-water bath conditions, 1.96 g (10 mmol, 1.0 eq.) of 3,4-dimethoxyphenylacetic acid was placed in a 100 mL Schlenk flask. Under nitrogen protection, 20 mL of anhydrous DCM was added, and 1.8 mL (25 mmol, 2.5 eq.) of thionyl chloride was slowly added dropwise while maintaining low temperature. The system was then transferred to room temperature and stirred for 2 h. The solvent and excess thionyl chloride were removed under reduced pressure at 40 °C. Subsequently, 20 mL of methanol was added to the system and stirred overnight at room temperature. The solvent was removed by distillation under reduced pressure to obtain 1.50 g of methyl 3,4-dimethoxyphenylacetic acid.

[0059] At room temperature, 1.0 g (4.76 mmol, 1.0 eq.) of methyl 3,4-dimethoxyphenylacetate was placed in a 100 mL Schlenk flask, along with 0.171 g (5.71 mmol, 1.2 eq.) of paraformaldehyde and 5 mL of DMSO. Under nitrogen protection, 0.0257 g (0.476 mmol, 10 mol%) of sodium methoxide was added to the system, and the mixture was stirred overnight at room temperature. For post-treatment, the reaction mixture was transferred to 100 mL of water, and the aqueous layer was extracted with EtOAc 3 × 20 mL. The organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column chromatography to obtain 0.90 g of methyl 3,4-dimethoxy-α-(hydroxymethyl)phenylacetate.

[0060] 0.6 g (2.5 mmol, 1.0 eq.) of methyl 3,4-dimethoxy-α-(hydroxymethyl)phenylacetic acid was placed in a 20 mL Schlenk flask. 2 mL of 40% hydrogen bromide solution was slowly added to the system at room temperature, and the mixture was heated under reflux overnight. For post-treatment, the reaction mixture was transferred to 50 mL of water, and the aqueous layer was extracted with DCM 3 × 20 mL. The organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column chromatography to obtain 0.35 g of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid.

[0061] 2. Structural identification

[0062] 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid is a white crystalline powder, readily soluble in methanol and soluble in water. 1 H-NMR (400MHz, CD3OD) see Figure 1 δ H (ppm)3.63(2H,m,CH2),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, CD3OD) see Figure 2 δ C (ppm)55.9(CH),65.2(CH2),114.3(CH),116.4(CH),123.9(CH),127.2(C),145.3(C),146.0(C),176.2(C=O).

[0063] Example 2: Preparation of compound IV (3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid)

[0064] 1. Synthesis process

[0065] Under ice-water bath conditions, 2.26 g (10 mmol, 1.0 eq.) of 3,4,5-trimethoxyphenylacetic acid was placed in a 100 mL Schlenk flask. Under nitrogen protection, 20 mL of anhydrous DCM was added, and 1.8 mL (25 mmol, 2.5 eq.) of thionyl chloride was slowly added dropwise while maintaining low temperature. The system was then transferred to room temperature and stirred for 2 h. The solvent and excess thionyl chloride were removed under reduced pressure at 40 °C. Subsequently, 20 mL of methanol was added to the system and stirred overnight at room temperature. The solvent was removed by distillation under reduced pressure to obtain 1.65 g of methyl 3,4,5-trimethoxyphenylacetic acid.

[0066] At room temperature, 1.5 g (6.25 mmol, 1.0 eq.) of methyl 3,4,5-trimethoxyphenylacetate was placed in a 100 mL Schlenk flask, along with 0.224 g (7.50 mmol, 1.2 eq.) of paraformaldehyde and 5 mL of DMSO. Under nitrogen protection, 0.0338 g (0.625 mmol, 10 mol%) of sodium methoxide was added to the system, and the mixture was stirred overnight at room temperature. For post-treatment, the reaction mixture was transferred to 100 mL of water, and the aqueous layer was extracted with EtOAc 3 × 20 mL. The organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column chromatography to obtain 1.17 g of methyl 3,4,5-trimethoxy-α-(hydroxymethyl)phenylacetate.

[0067] 1.0 g (3.70 mmol, 1.0 eq.) of methyl 3,4,5-trimethoxy-α-(hydroxymethyl)phenylacetic acid was placed in a 20 mL Schlenk flask. 2 mL of 40% hydrogen bromide solution was slowly added to the system at room temperature, and the mixture was heated under reflux overnight. For post-treatment, the reaction mixture was transferred to 50 mL of water, and the aqueous layer was extracted with DCM 3 × 20 mL. The organic phases were combined, washed with water, dried, concentrated under reduced pressure, and purified by column chromatography to obtain 0.69 g of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid.

[0068] 2. Structural identification

[0069] 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid is a white crystalline powder, readily soluble in methanol and soluble in water. 1 H-NMR (400MHz, CD3OD) see Figure 3 δ H (ppm)3.63(2H,m,CH2),3.99(1H,m,CH),6.10(2H,s,Ph-H); 13 C-NMR (400MHz, CD3OD) see Figure 4 δ C(ppm)55.9(CH),65.2(CH2),106.8(2×CH),131.0(C),131.3(C),146.2(2×C),176.2(C=O).

[0070] Example 3: Study on the effects of tropinic acid and its derivatives on an immunosuppressed mouse model

[0071] 1. Animal grouping, model establishment, and drug administration

[0072] One hundred and ten male KM mice were randomly divided into 11 groups of 10 mice each: a blank control group, a model group, a positive drug group, a high-dose troponic acid group (A), a low-dose troponic acid group (B), a high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), a low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), a high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), a low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), a high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), and a low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (H). Except for the blank control group, which received no treatment, all other experimental mice were intraperitoneally injected with cyclophosphamide 80 mg / kg / day for 1–3 days at the start of the experiment to establish an immunosuppressed mouse model. All groups were administered the medication simultaneously, twice daily. See Table 1 for details of the grouping and administration regimens. The treatment lasted for 14 consecutive days.

[0073] Table 1. Animal Grouping and Drug Administration Information (n=10)

[0074]

[0075]

[0076] 2. Indicator Testing

[0077] Mice were fasted for 12 hours after the last administration of the drug, then anesthetized with intraperitoneal injection of 10% chloral hydrate. They were weighed, dissected, and blood was collected from the heart. Immediately afterward, the spleen and thymus were separated. The organs were blotted dry with filter paper and weighed again. The spleen index and thymus index were calculated as follows: organ index = organ wet weight (g) / body weight (g) × 100%. Mouse blood was collected in centrifuge tubes and centrifuged at 2800 rpm for 10 min at low temperature (4℃). Serum was collected and stored at -20℃ for later use. The levels and activities of IL-4, IL-10, IFN-γ, TNF-α, and IgG in the serum were determined using an enzyme-linked immunosorbent assay (ELISA) kit.

[0078] 3. Experimental Results and Discussion

[0079] Spleen and thymus indices directly reflect the strength of the body's immune function. Compared with the blank control group, the thymus and spleen indices in the model group were significantly decreased (P<0.01). After two weeks of treatment with tropic acid and its derivatives, the spleen and thymus indices in all dose groups were significantly increased (P<0.05 or P<0.01), showing a dose-dependent effect. The high-dose group increased the spleen and thymus indices and then normalized them. This indicates that tropic acid and its derivatives can significantly improve the immune organ indices of cyclophosphamide-induced immunosuppressed mice. IL-4 has immunomodulatory effects on B cells, T cells, mast cells, macrophages, and stem cells, and can induce the production of IgG and IgE. Compared with the model group, the serum IL-4 levels in mice in all dose groups of tropic acid and its derivatives were increased (P<0.01), and the IL-4 level in the high-dose group increased and then normalized. IL-10 inhibits NK cell activity and interferes with the production of cytokines by NK cells and macrophages. Compared with the model group, the serum IL-10 level in mice in the tropic acid and its derivative groups was significantly reduced (P<0.05 or P<0.01), and the high-dose group reduced IL-4 levels and brought them closer to normal. Compared with the model group, the TNF-α level in the tropic acid and its derivative groups was significantly increased (P<0.05 or P<0.01). Therefore, tropic acid and its derivatives can stimulate TNF-α secretion, and TNF-α secretion induces enhanced macrophage activity and killing function, enabling macrophages to promote the body's immune response. Compared with the model group, the serum IgG concentration in mice in the tropic acid and its derivative groups was significantly increased (P<0.05 or P<0.01), and the high-dose group increased IgG levels and brought them closer to normal. There was no significant difference in the therapeutic effect between compounds 1 and 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 showed better therapeutic effects (P<0.05 or P<0.01). The effects of tropic acid and its derivatives on organ indices and serum immune parameters in an immunosuppressed mouse model are shown in Table 2. In summary, tropic acid and its derivatives have a certain regulatory effect on cyclophosphamide-induced immunosuppression.

[0080] Table 2. Effects of tropic acid and its derivatives on organ indices and serum immune markers in an immunosuppressed mouse model (n=10, )

[0081]

[0082] Note: Compared with the blank control group, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0083] Example 4: Study on the effect of tropinic acid and its derivatives on the proliferation of splenic lymphocytes

[0084] 1. Cell model preparation, grouping, and drug administration

[0085] Mice were euthanized by cervical dislocation, sterilized with 75% ethanol, and their spleens were removed under aseptic conditions. Splenic lymphocytes were prepared, resuspended in RPMI-1640 medium, and the cell concentration was adjusted to 1×10⁻⁶ cells after counting. 7 Cells / ml. Add 100 μL of cell suspension to each well of a 96-well plate. Add mitogen ConA to each well to a final concentration of 5 μg / ml. Add 100 μL of tropic acid or its derivatives to each well, including high-dose tropic acid (A), low-dose tropic acid (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid (F), high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid (G), and low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid (H). Add 100 μL of the blank control group. RPMI-1640 culture medium was used, and 100 μL of RPMI-1640 culture medium containing cyclophosphamide was added to the positive control. Each group was divided into 3 replicates. Grouping and drug administration information are detailed in Table 3. After culturing at 37℃ and 5% CO2 for 44 hours, 10 μL of 5 mg / mL MTT was added to each well, and the culture was continued for another 4 hours.

[0086] Table 3. Cell grouping and drug administration information (n=9)

[0087]

[0088]

[0089] 2. Indicator Testing

[0090] After cell culture, the cells were centrifuged at 1000 rpm, the supernatant was discarded, and 200 μL of DMSO was added to each well. The cells were then shaken for 10 min to dissolve the cells. The cells were then placed in a microplate reader, and the OD value was measured at a wavelength of 492 nm. The experiment was performed in triplicate. Spleen lymphocyte proliferation inhibition rate = (mean OD value of ConA model control group - mean OD value of drug treatment group) / mean OD value of ConA model control group.

[0091] 3. Experimental Results and Discussion

[0092] As shown in Table 4, none of the tropinic acid and its derivative groups exhibited cytotoxicity, and all showed varying degrees of immunosuppressive activity against ConA-induced spleen cell proliferation. Compared with the model group, the OD values ​​of each tropinic acid and its derivative group were significantly reduced (P<0.01), with the high-dose group showing an inhibition rate of 39.8%–44.5%. There was no significant difference in the effects of compounds 1 and 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 showed more significant effects (P<0.01).

[0093] Table 4. Effects of tropinic acid and its derivatives on the proliferation of mouse spleen lymphocytes (n=9, )

[0094] Grouping Drug concentration (μg / mL) OD value Inhibition rate (%) 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

[0095] Note: Compared with the model group, **P<0.01.

[0096] Example 5: Study on the effects of tropinic acid and its derivatives on delayed hypersensitivity reactions

[0097] 1. Animal grouping, model establishment, and drug administration

[0098] One hundred and ten male KM mice (6-7 weeks old, 22.0±2.0g) were randomly divided into 11 groups of 10 mice each: blank control group, model group, positive control group, high-dose troponic acid group (A), low-dose troponic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), and low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (H). On the first day of drug administration, a 1.0cm×2.0cm area of ​​hair was removed from the abdomen of each mouse in each group, and 50μL of the drug was applied. 1% 2,4-dinitrofluorobenzene (DNFB) (dissolved in acetone-olive oil, acetone:olive oil = 3:1) was applied to the abdomen of the normal group in 50 μL of acetone-olive oil (3:1). Administration was performed on the day of sensitization, twice daily for 7 consecutive days, with a second application on the second day for enhanced sensitization. All groups were administered medication synchronously. See Table 5 for grouping and administration details.

[0099] Table 5. Animal Grouping and Drug Administration Information (n=10)

[0100] Grouping Dosing regimen blank Administer 0.2 mL of normal saline by gavage. Model Administer 0.2 mL of normal saline + 50 μL of 1% DNFB by gavage. Positive drug Dexamethasone 20 mg / kg / day + 1% DNFB 50 μL A Tropine 40 mg / kg / day by gavage + 50 μL of 1% DNFB B Tropine 20 mg / kg / day by gavage + 50 μL of 1% DNFB C Administer 40 mg / kg / day of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 50 μL of 1% DNFB. D Administer 20 mg / kg / day of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 50 μL of 1% DNFB. E Administer 40 mg / kg / day of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 50 μL of 1% DNFB. F Administer 20 mg / kg / day of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 50 μL of 1% DNFB. G Administer by gavage 40 mg / kg / day of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid + 50 μL of 1% DNFB. H Administer 20 mg / kg / day of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 50 μL of 1% DNFB.

[0101] 2. Indicator Testing

[0102] Thirty minutes after the last administration, 10 μL of 1% DNFB was applied to both the anterior and posterior surfaces of the right ear of each mouse. Twenty-four hours later, the mice were weighed. After anesthesia with an intraperitoneal injection of 10% chloral hydrate (0.1 mL / 10 g), the eyeballs were enucleated to collect blood, and the mice were euthanized by cervical dislocation. Both ears were cut off, and circular ear pieces were punched at the same location using an 8 mm punch, and accurately weighed. The blood was centrifuged, and the supernatant was collected. The serum IFN-γ content was determined according to an enzyme-linked immunosorbent assay (ELISA) kit. Ear swelling degree = right ear weight - left ear weight; swelling inhibition rate = (average swelling degree of the model group - average swelling degree of the drug-treated group) / average swelling degree of the model group × 100%.

[0103] 3. Experimental Results and Discussion

[0104] Delayed-type hypersensitivity (DHT) is a T-cell-dependent immune response model, a Th1-mediated allergic reaction. Th1 cells primarily secrete INF-γ, participating in cellular immunity and the occurrence of DHT inflammation. Its main characteristic is the appearance of a delayed-type inflammatory response at the antigen attack site in the sensitized organism. DNCB is a hapten; when diluted and applied to the abdominal skin, it binds to skin proteins to form a complete antigen, thereby stimulating T lymphocytes to proliferate into sensitized lymphocytes. Seven days later, it is applied to the ear to induce a local DHT reaction. As shown in Table 6, compared with the model group, tropic acid and its derivatives significantly reduced ear swelling in mice with DHT (P<0.05 or P<0.01) and significantly inhibited the increase of serum INF-γ levels in mice with DHT in a dose-dependent manner. High-dose groups could normalize mouse ear and serum INF-γ levels. There was no significant difference in the effects of compounds 1 and 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 showed better effects (P<0.05). This indicates that tropinic acid and its derivatives have a regulatory effect on delayed-type hypersensitivity reactions, and inhibiting the increase of serum INF-γ levels may be their mechanism of action.

[0105] Table 6. Effects of tropinic acid and its derivatives on delayed hypersensitivity in mice (n=10, )

[0106] Grouping Ear swelling degree (mg) Ear swelling suppression rate (%) IFN-γ (μg / mL) blank 1.68±0.25 — 152.58±13.87 Model <![CDATA[10.56±2.63 ## ]]> — <![CDATA[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*

[0107] Note: Comparison with the blank control group. ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0108] Example 6: Study on the anti-inflammatory effects of tropinic acid and its derivatives

[0109] 1. Animal grouping, model establishment, and drug administration

[0110] One hundred and ten KM mice (6-7 weeks old, 22.0±2.0g), half male and half female, were randomly divided into 11 groups of 10 mice each: blank control group, model group, positive drug group, high-dose troponic acid group (A), low-dose troponic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), and high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E). The mice were divided into four groups: a low-dose group (F) of 3,4,5-dihydroxy-α-(hydroxymethyl)phenylacetic acid, a high-dose group (G) of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid, and a low-dose group (H) of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid. All groups were administered the drugs simultaneously. The grouping and administration regimens are detailed in Table 7. The drugs were administered twice daily by gavage for 7 consecutive days. One hour after the last administration, except for the normal control group, 40 μL of xylene was evenly applied to both sides of the right ear of the mice in the other groups to induce inflammation.

[0111] Table 7. Animal Grouping and Drug Administration Information (n=10)

[0112]

[0113]

[0114] 2. Indicator Testing

[0115] One hour after inducing inflammation, the animals were anesthetized with an intraperitoneal injection of 10% chloral hydrate, euthanized by cervical dislocation, and both ears were cut off along the auricle edge. Circular ear pieces were then made at the same location in both ears using a manual 8mm ear punch. The ear pieces were precisely weighed, and the degree of ear swelling and the swelling inhibition rate were calculated. Ear swelling degree = Right ear mass - Left ear mass; Swelling inhibition rate = (Average swelling degree of the model group - Average swelling degree of the treatment group) / Average swelling degree of the model group × 100%.

[0116] 3. Experimental Results and Discussion

[0117] As shown in Table 8, compared with the model control group, tropic acid and its derivatives significantly reduced the degree of xylene-induced ear swelling in mice (P<0.01), with the high-dose groups of tropic acid and its derivatives showing a swelling inhibition rate of 70.34%–74.31%. There was no significant difference in swelling inhibition effect between compounds 1 and 2. Compared with compounds 1 and 2, both high- and low-dose groups of compounds 3 and 4 showed better swelling inhibition effects. In summary, these results indicate that tropic acid and its derivatives have significant anti-inflammatory effects on the xylene-induced mouse ear swelling model.

[0118] Table 8. Effects of tropinic acid and its derivatives on xylene-induced ear swelling in mice (n=10, )

[0119]

[0120]

[0121] Note: Compared with the blank control group, ## P<0.01; compared with the model group, **P<0.01.

[0122] Example 7: Study on the analgesic effect of tropinic acid and its derivatives

[0123] 1. Animal grouping, model establishment, and drug administration

[0124] One hundred KM mice (5-6 weeks old, 22.0±2.0g), half male and half female, were randomly divided into 10 groups of 10 mice each: blank control group, model group, positive drug group, high-dose troponic acid group (A), low-dose troponic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), and 3, The mice in the low-dose group (F), high-dose group (G), and low-dose group (H) of 4-dihydroxy-α-(hydroxymethyl)phenylacetic acid were administered the drugs simultaneously, twice a day, for 7 consecutive days by gavage. The grouping and administration regimens are detailed in Table 9. Except for the normal control group, the mice in the other groups were intraperitoneally injected with 0.6% 10 mL / kg glacial acetic acid 2 hours after the last administration to establish a pain model.

[0125] Table 9. Animal Grouping and Drug Administration Information (n=10)

[0126] Grouping Dosing regimen Model Administer 0.2 mL of normal saline by gavage and inject 0.6% glacial acetic acid at 10 mL / kg via intraperitoneal injection. Positive drug Aspirin enteric-coated tablets 400 mg / kg / day + intraperitoneal injection of 0.6% glacial acetic acid 10 mL / kg A Tropine 40 mg / kg / day by gavage + 0.6% glacial acetic acid 10 mL / kg by intraperitoneal injection B Tropine 20 mg / kg / day by gavage + 0.6% glacial acetic acid 10 mL / kg by intraperitoneal injection C Administer 40 mg / kg / day of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 10 mL / kg of 0.6% glacial acetic acid by intraperitoneal injection. D Administer 20 mg / kg / day of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 10 mL / kg of 0.6% glacial acetic acid by intraperitoneal injection. E Administer 40 mg / kg of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid by gavage, plus 10 mL / kg of 0.6% glacial acetic acid via intraperitoneal injection. F Administer 20 mg / kg / day of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 10 mL / kg of 0.6% glacial acetic acid by intraperitoneal injection. G Administer 40 mg / kg / day of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 10 mL / kg of 0.6% glacial acetic acid by intraperitoneal injection. H Administer 20 mg / kg / day of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid by gavage + 10 mL / kg of 0.6% glacial acetic acid by intraperitoneal injection.

[0127] 2. Indicator Testing

[0128] After establishing the pain model, the latency time for the writhing response in mice and the number of writhing movements in each group within 20 minutes were observed and recorded, and the analgesia rate was calculated. Analgesia rate = (average number of writhing movements in the model group - average number of writhing movements in the drug treatment group) / average number of writhing movements in the model control group × 100%.

[0129] 3. Experimental Results and Discussion

[0130] As shown in Table 10, compared with the model control group, tropinic acid and its derivatives significantly prolonged the latency time of acetic acid-induced writhing response in mice (P<0.05 or P<0.01) and significantly reduced the number of writhing movements (P<0.01). The analgesic rate in the high-dose groups of tropinic acid and its derivatives reached 55.26–58.79%. There was no significant difference in analgesic effect between compounds 1 and 2. Compounds 3 and 4 showed better analgesic effects compared to compounds 1 and 2. Therefore, tropinic acid and its derivatives have significant peripheral analgesic effects.

[0131] Table 10. Effects of tropinic acid and its derivatives on acetic acid-induced writhing response in mice (n=10, )

[0132] Grouping Latency (s) Number of twists Analgesia 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

[0133] Note: Compared with the model group, *P<0.05, **P<0.01.

[0134] Example 8: Study on the effects of tropinic acid and its derivatives on a mouse model of autoimmune hepatitis

[0135] 1. Animal modeling, grouping, and drug administration

[0136] Liver tissue was collected from normal male KM mice (6-7 weeks old, 22.0±2.0g). Physiological saline was added at a ratio of 1:9 (g / mL), and the mixture was thoroughly homogenized using a tissue homogenizer (-4℃). The homogenate was then centrifuged for 10 minutes (2500 rpm, 4℃), and the supernatant was collected to obtain the syngeneic liver antigen. The syngeneic liver antigen was added to complete Freund's adjuvant at a ratio of 1:1 (v / v), mixed thoroughly, and emulsified to obtain the immunomodulator (prepared immediately before use).

[0137] One hundred and ten male KM mice (6-7 weeks old, 22±2g) were randomly divided into 11 groups of 10 mice each: blank control group, model group, positive drug group, high-dose troponic acid group (A), low-dose troponic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), and low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G). Except for the blank control group, all mice in the other groups received an intraperitoneal injection of 1 mL of the immunizing agent per mouse for the first immunization. A second immunization was performed 7 days later to obtain a mouse model of autoimmune hepatitis. The blank control group mice were injected intraperitoneally with 1 mL of physiological saline per mouse at the same time. The drugs were administered by gavage starting on the day the model was established, twice a day for 14 consecutive days. The grouping and administration regimens are detailed in Table 11.

[0138] Table 11. Animal Grouping and Drug Administration Information (n=10)

[0139]

[0140]

[0141] 2. Indicator Testing

[0142] After the last administration, mice were fasted for 12 hours. Mice in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.1 mL / 10 g), then dissected. Blood was collected from the heart, perfused with physiological saline, and fixed with paraformaldehyde. Liver and spleen tissues were collected and fixed in 4% paraformaldehyde for 24 hours. Blood was centrifuged for 10 minutes (3000 rpm, 4℃), and serum was collected. The activities of liver function-related indicators such as lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), as well as the content of total bilirubin (TBIL), were measured according to the kit instructions. Liver and spleen tissues were paraffin-embedded, cut into 4 μm thick sections, routinely stained with hematoxylin and eosin (HE), mounted, and observed under a light microscope for histopathological changes.

[0143] 3. Experimental Results and Discussion

[0144] Liver function tests: As shown in Table 12, the serum LDH, ALT, and AST activities were elevated in the model group mice, and the TBIL content was significantly increased (P<0.01). When liver tissue is damaged or hepatocytes die, the serum LDH, ALT, and AST activities increase, while the TBIL content is an elevated sensitive indicator of liver injury, directly reflecting the degree of hepatocyte damage and necrosis, as well as the liver's detoxification and metabolic functions. Compared with the model group, after 2 weeks of administration of tropic acid and its derivatives, the serum LDH, ALT, AST activities, and TBIL content all decreased (P<0.05 or P<0.01), and this was dose-dependent. There was no significant difference in the therapeutic effect between compound 1 and compound 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 had better therapeutic effects (P<0.05). Therefore, tropic acid and its derivatives can improve liver function in mice with autoimmune hepatitis.

[0145] Table 12. Effects of tropinic acid and its derivatives on liver function in a mouse model of autoimmune hepatitis (n=10, )

[0146]

[0147]

[0148] Note: Compared with the blank control group, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0149] Histopathological examination: such as Figure 5As shown, the livers of model group mice exhibited extensive infiltration of inflammatory factors, blurred cell boundaries, hepatocyte edema and degeneration, nuclear pyknosis, partial cellular lysis and necrosis, and vacuolation caused by fatty degeneration. After administration of tropinic acid and its derivatives, some of the above pathological indicators showed significant improvement. In particular, regenerated hepatocytes repairing liver damage were observed around necrotic hepatocytes, characterized by large cell volume, large and deeply stained nuclei, and mostly binucleated. Figure 6 As shown, in the spleen of model mice, follicular proliferative lesions related to immune activation appeared, leading to an increase and enlargement of germinal centers. The ratio of red pulp to white pulp area decreased, and the number of foamy macrophages in both the white and red pulp increased. This is attributed to a large-scale lymphocyte transformation in the spleen following intraperitoneal injection of immunizing agents, with B cells proliferating and transforming into plasma cells. The expansion of splenic follicular growth centers and the presence of numerous plasma cells increased antibody production and enhanced humoral immunity. Plasma was present in the blood vessels of the red pulp, and the proliferating macrophages destroyed the plasma and erythrocytes in the red pulp arterioles, resulting in massive congestion of the splenic cords within the red pulp. After administration of tropic acid and its derivatives, the above-mentioned immune activation pathological indicators significantly improved. This suggests that tropic acid and its derivatives may regulate immune activity by enhancing antigen recognition, thereby playing a therapeutic role in autoimmune hepatitis.

[0150] Example 9: Study on the effects of tropinic acid and its derivatives on a mouse model of COVID-19 cold-dampness epidemic

[0151] 1. Animal modeling, grouping, and drug administration

[0152] One hundred and ten male KM mice (6-7 weeks old, 22.0±2.0 mm) were randomly divided into 11 groups of 10 mice each: blank control group, model group, positive drug group, high-dose tropinic acid group (A), low-dose tropinic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), and low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G). Each group was administered the drug twice daily for seven consecutive days. Three days after administration, except for the blank control group, all other groups of mice were intraperitoneally injected with 5 mg / kg lipopolysaccharide saline solution, and then placed in an artificial climate chamber to induce a cold and damp stimulation model at a temperature of 4.0±2.0℃ and a humidity of 90.0±3.0% for 8 hours a day for 4 consecutive days, thus creating a mouse model of COVID-19 cold and dampness epidemic. See Table 13 for grouping and administration details.

[0153] Table 13. Animal Grouping and Drug Administration Information (n=10)

[0154] Grouping Dosing regimen blank Administer 0.2 mL of normal saline by gavage. Model 0.2 mL of normal saline was administered by gavage, followed by an intraperitoneal injection of 5 mg / kg of lipopolysaccharide. Positive drug Dexamethasone 2 mg / kg / day by gavage + lipopolysaccharide 5 mg / kg by intraperitoneal injection A Tropine 40 mg / kg / day by gavage + lipopolysaccharide 5 mg / kg by intraperitoneal injection B Tropine 20 mg / kg / day by gavage + lipopolysaccharide 5 mg / kg by intraperitoneal injection C 40 mg / kg of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid administered by gavage, plus 5 mg / kg of lipopolysaccharide administered intraperitoneally. D 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid 20 mg / kg / day by gavage + lipopolysaccharide 5 mg / kg by intraperitoneal injection E 40 mg / kg of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid was administered by gavage daily, plus 5 mg / kg of lipopolysaccharide administered intraperitoneally. F 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid 20 mg / kg / day by gavage + lipopolysaccharide 5 mg / kg by intraperitoneal injection G 40 mg / kg of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid was administered by gavage, plus 5 mg / kg of lipopolysaccharide was injected intraperitoneally. H 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid 20 mg / kg / day by gavage + lipopolysaccharide 5 mg / kg by intraperitoneal injection

[0155] 2. Indicator Testing

[0156] After the last administration, the mice were fasted for 12 hours, anesthetized by intraperitoneal injection of 10% chloral hydrate (0.1 mL / 10 g), perfused with physiological saline, and lung tissue was harvested. A portion was flash-frozen in liquid nitrogen and stored at -80°C for later use, while another portion was fixed in 4% paraformaldehyde for later use. 50 mg of the frozen mouse tissue was homogenized with 800 μL of PBS, centrifuged at 2800 rpm at 4°C for 10 min, and serum was collected. The levels of inflammatory factors IL-6, IL-10, IFN-γ, and TNF-α in the lung tissue were measured using an enzyme-linked immunosorbent assay (ELISA) kit. The fixed lung tissue was embedded in paraffin, sectioned into 4 μm thick sections, routinely stained with hematoxylin and eosin (HE), mounted, and observed under a light microscope for histopathological changes.

[0157] 3. Experimental Results and Discussion

[0158] Inflammatory factor detection: As shown in Table 14, compared with the blank control group, the levels of TNF-α, IFN-γ, and IL-6 in the lung tissue of the model group mice were significantly increased (P<0.05 or P<0.01), and the level of IL-10 was significantly decreased (P<0.01). After 7 days of treatment with tropic acid and its derivatives, the levels of TNF-α, IFN-γ, and IL-6 were significantly decreased (P<0.05 or P<0.01), and the level of IL-10 was significantly increased (P<0.05 or P<0.01), showing a dose-dependent effect. The high-dose group reduced lung tissue inflammatory factors and tended to normalize. There was no significant difference in the therapeutic effect between compound 1 and compound 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 had better therapeutic effects (P<0.05). Therefore, tropic acid and its derivatives can improve the inflammatory response in a mouse model of COVID-19.

[0159] Table 14. Effects of tropic acid and its derivatives on inflammatory factors in lung tissue of a mouse model of COVID-19 cold-dampness epidemic (n=10, )

[0160]

[0161]

[0162] Note: Compared with the blank control group, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0163] Histopathological examination: such as Figure 7As shown, the lung tissue structure of the normal group mice was intact, without exudate, and the alveolar septa were normal; while the alveolar scaffold of the model group mice collapsed, the lung tissue structure was disordered, the alveolar septa were significantly thickened, and the interstitial edema and inflammatory infiltration of the lung tissue were severe. After administration of tropic acid and its derivatives, some of the above-mentioned pathological indicators were significantly improved. There was no significant difference in the therapeutic effect between compounds 1 and 2. Compared with compounds 1 and 2, compounds 3 and 4 had better therapeutic effects. This indicates that tropic acid and its derivatives may exert a therapeutic effect on the COVID-19 cold-dampness epidemic mouse model by regulating immunity and inhibiting the inflammatory response.

[0164] Example 10: Study on the effects of tropinic acid and its derivatives on a mouse model of aplastic anemia

[0165] 1. Animal modeling, grouping, and drug administration

[0166] Sixty-six male SD rats (7-8 weeks old, 200.0±20.0g) were randomly divided into 11 groups of 6 rats each: blank control group, model group, positive drug group, high-dose tropinic acid group (A), low-dose tropinic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), and 3,4,5- In the high-dose group (G) of trihydroxy-α-(hydroxymethyl)phenylacetic acid and the low-dose group (G) of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid, except for the blank control group, all other groups received subcutaneous injections of 20 mg / kg of 2% acetylphenylhydrazine (Ap) saline solution on days 1 and 4. Two hours after the acetylphenylhydrazine injection on day 4, and intraperitoneally from days 5 to 7, 20 mg / kg of cyclophosphamide (Cy) saline solution was injected to establish an animal model of aplastic anemia. All rats in each treatment group received tail vein injection once daily for 14 consecutive days. See Table 15 for details of the grouping and administration regimens.

[0167] Table 15. Animal Grouping and Drug Administration Information (n=6)

[0168]

[0169]

[0170] 2. Indicator Testing

[0171] One hour after the last administration of medication to rats, the rats were anesthetized via intraperitoneal injection of 10% chloral hydrate. Blood was collected from the heart in anticoagulant tubes for later use. The thymus and spleen were weighed. A hemolytic agent was added to the whole blood in the anticoagulant tubes, and a fully automated hematology analyzer was used to detect changes in peripheral blood cell counts, mainly including changes in four indicators: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), and platelet count (PLT). The spleen index and thymus index were calculated as follows: organ index = organ wet weight (g) / body weight (g) × 100%.

[0172] 3. Experimental Results and Discussion

[0173] As shown in Table 16, compared with the normal group, the levels of WBC, RBC, HGB, and PLT in the model group were significantly decreased (P<0.01). Compared with the model group, after 14 days of treatment with tropic acid and its derivatives, the levels of all indicators increased (P<0.05 or P<0.01), showing a dose-dependent effect. Compared with the normal group, the spleen index in the model group was significantly increased, and the thymus index was significantly decreased (P<0.01). After 14 days of treatment with tropic acid and its derivatives, the spleen index and thymus index tended to normalize (P<0.05 or P<0.01). There was no significant difference in the therapeutic effect between compound 1 and compound 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 had better therapeutic effects (P<0.05 or P<0.01). Therefore, tropic acid and its derivatives can improve aplastic anemia in rat models through their immunomodulatory effects, thereby restoring the function of hematopoietic stem cells.

[0174] Table 16. Effects of tropinic acid and its derivatives on immune organ indices and peripheral blood counts in an aplastic anemia model (n=6, )

[0175]

[0176]

[0177] Note: Compared with the blank control group, ## P<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0178] Example 11: Study on the effects of tropinic acid and its derivatives on a rat model of rheumatoid arthritis

[0179] 1. Animal modeling, grouping, and drug administration

[0180] Preparation of immunoemulsifiers: 7 ml of bovine type II collagen (CII) solution was placed in a small beaker and magnetically stirred at 1500 rpm at low temperature. 7 ml of complete Freund's adjuvant (CFA) solution was slowly added to the CII solution. After all the CFA solution was added, stirring continued for approximately 30 minutes until the emulsion did not disperse when dropped onto water, thus obtaining the primary immunoemulsifier. Secondary immunoemulsifiers were obtained by replacing CFA with incomplete Freund's adjuvant (IFA) using the same preparation method. All immunoemulsifiers were prepared immediately before use.

[0181] Preparation of a rheumatoid arthritis (CIA) model: One hundred male SD rats (7-8 weeks old, 200.0±20.0g) were used. 0.2ml of the initial immunization emulsion was injected subcutaneously at the base of the tail. Eight days after the initial immunization, 0.1ml of the secondary immunization emulsion was injected subcutaneously at the base of the tail to complete a booster immunization. The blank control group received saline injection using the same method. Fourteen days after the booster immunization, the Arthritis Index (AI) was assessed using the following scoring rules: 0 points for no swelling or erythema; 1 point for erythema and mild swelling at the ankle joint; 2 points for erythema and mild swelling at the ankle to metatarsophalangeal or metacarpophalangeal joints; 3 points for erythema and moderate swelling at the ankle to metatarsophalangeal or metacarpophalangeal joints; and 4 points for erythema and severe swelling at the ankle to toe joints. The sum of the scores for both paws was used as the joint score for each rat. An AI score ≥4 indicated successful model establishment, and animals without signs of joint swelling were removed.

[0182] Grouping and administration: Six blank control rats and 60 CIA model rats were randomly divided into 10 groups of 6 rats each: model group, positive drug group, high-dose tropinic acid group (A), low-dose tropinic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), and low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G). Each group was simultaneously administered the corresponding drug by gavage twice daily for 4 consecutive weeks. The grouping and administration regimens are detailed in Table 17.

[0183] Table 17. Animal Grouping and Drug Administration Information (n=6)

[0184]

[0185]

[0186] 2. Indicator Testing

[0187] After treatment, the morphological differences in the ankle joint and paw of rats before and after treatment were compared among the groups. The arthritis status of rats in each group was scored weekly using the AI ​​scoring method described above. The volume change below the ankle joint was simultaneously measured using the paw volume displacement method, and the degree of joint swelling was calculated. Joint swelling degree = (volume after modeling - volume before modeling) / volume before modeling × 100%.

[0188] 3. Experimental Results and Discussion

[0189] like Figure 8 As shown, after modeling, rats exhibited a significant increase in metatarsal volume. In all model groups, rats showed joint swelling, stiff and curled paws, and were unable to walk normally. After 4 weeks of treatment with tropic acid and its derivatives, the low-dose group showed mild joint swelling, while the swelling subsided and the rats in the other treatment groups were in good condition. Compounds 1 and 2 showed no significant difference in therapeutic effect. Compared to compounds 1 and 2, compounds 3 and 4 showed better therapeutic effects. Figure 9 As shown, compared with the model group, the AI ​​scores of rats treated with tropic acid and its derivatives gradually decreased, and the differences became statistically significant starting from week 3 of treatment (P<0.05 or P<0.01). Compared with the model group, after 2 weeks of treatment with tropic acid and its derivatives, the joint swelling of rats began to decrease, and after 4 weeks of treatment, the joint swelling of rats decreased significantly. There was no significant difference in the treatment effect of compound 1 compared with compound 2 at any week (P>0.05). Compared with compounds 1 and 2, after 3 weeks of treatment, the high-dose groups of compounds 3 and 4 showed more significant reductions in AI scores and joint swelling (P<0.05), indicating better therapeutic effects. In conclusion, tropic acid and its derivatives have significant therapeutic effects on a rat model of rheumatoid arthritis.

[0190] Example 11: Study on the effects of tropinic acid and its derivatives on a mouse model of allergic rhinitis

[0191] 1. Animal modeling, grouping, and drug administration

[0192] One hundred and ten male BABL / c mice (5-6 weeks old, 20.0 ± 2.0 g) were randomly divided into 11 groups of 10 mice each: blank control group, model group, positive drug group, high-dose troponic acid group (A), low-dose troponic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), and low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G). Except for the blank control group, mice in all other groups were intraperitoneally injected with a suspension of sensitizing agent (containing 0.50 mg / mL) on days 1, 7, and 14 of the experiment. OVA and 0.50 mg / mL Al(OH)3) were administered to each mouse at 200 μL for basal sensitization. From day 15 to 28, each group was administered the corresponding drug via gavage. From day 22 to 28, a 4% OVA solution was instilled into the nasal cavity of each mouse for challenge, at 20 μL per nasal cavity, thus establishing an allergic rhinitis model. The blank control group received the same volume of physiological saline during both the basal sensitization and challenge phases. See Table 18 for grouping and drug administration details.

[0193] Table 18. Animal Grouping and Dosing Information (n=10)

[0194] Grouping Dosing regimen blank Administer 0.2 mL of normal saline by gavage. Model Basic sensitization + gavage administration of 0.2 mL of normal saline + challenge Positive drug Basic sensitization + oral dexamethasone 20 mg / kg / day + challenge A Basic sensitization + oral troponin 40 mg / kg / day + challenge B Basic sensitization + oral troponin 20 mg / kg / day + challenge C Basic sensitization + oral gavage of 40 mg / kg / day of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid + challenge D Basic sensitization + oral gavage of 20 mg / kg / day of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid + challenge E Basic sensitization + oral gavage of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid 40 mg / kg / day + challenge F Basic sensitization + oral gavage of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid 20 mg / kg / day + challenge G Basic sensitization + oral gavage of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid 40 mg / kg / day + challenge H Basic sensitization + oral gavage of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid 20 mg / kg / day + challenge

[0195] 2. Indicator Testing

[0196] After the last nasal stimulation, the number of times mice in each group scratched their noses and sneezed within 30 minutes was observed and recorded.

[0197] 3. Experimental Results and Discussion

[0198] As shown in Table 19, compared with the control group, the number of sneezes and nose scratches in the model group mice was significantly increased (P<0.01), indicating that the allergic rhinitis model was successfully established. Compared with the model group, tropic acid and its derivatives significantly reduced the number of sneezes (P<0.01) and nose scratches (P<0.05 or P<0.01) in mice in a dose-dependent manner. There was no significant difference in therapeutic effect between compound 1 and compound 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 had better therapeutic effects (P<0.05). This indicates that tropic acid and its derivatives have significant therapeutic effects on the allergic rhinitis mouse model.

[0199] Table 19. Effects of tropic acid and its derivatives on the number of nose scratching and sneezing in a mouse model of allergic rhinitis (n=10, )

[0200]

[0201]

[0202] Note: Compared with the blank control group, ## P<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0203] Example 12: Study on the effects of tropinic acid and its derivatives on a rat model of membranous nephritis

[0204] 1. Animal modeling, grouping, and drug administration

[0205] Replication of the membranous glomerulonephritis model: 100 mg of cationized bovine serum albumin (C-BSA) was dissolved in 15 mL of physiological saline, mixed with an equal volume of incomplete Freund's adjuvant, and thoroughly emulsified. One hundred male SD rats (7–8 weeks old, 200±20 g) were used. 1 mL of the emulsifier was injected subcutaneously at multiple points in the bilateral axillae and groin of each rat, once every other day for a total of 3 times, to complete the pre-immunization. One week after pre-immunization, each rat was injected intravenously with C-BSA physiological saline solution at a dose of 16 mg / kg, 3 times a week for 4 consecutive weeks, to complete the formal immunization. 24-hour urine samples were collected from rats using metabolic cages for urinary protein detection. A 24-hour urinary protein (24h UPro) level >20 mg was considered a successful replication of the membranous glomerulonephritis model, and rats with failed model replication were removed. The blank control group rats were injected with an equal volume of physiological saline simultaneously using the same method.

[0206] Grouping and administration: Six rats were used as the blank control group and 60 rats were used as the membranous nephritis model. The membranous nephritis model rats were randomly divided into 10 groups of 6 rats each. The groups were: model group, positive drug group, high-dose tropinic acid group (A), low-dose tropinic acid group (B), high-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (C), low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (D), high-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (E), low-dose 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), high-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), and low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G). Each group was simultaneously administered the corresponding drug by gavage twice a day for 4 consecutive weeks. The grouping and administration regimens are detailed in Table 11.

[0207] Table 20. Animal Grouping and Drug Administration Information (n=6)

[0208]

[0209]

[0210] 2. Indicator Testing

[0211] General observation: After the last administration, the therapeutic effect of the drug was evaluated by comparing and observing the hair, mental state, weight, food intake, and stool and urination of rats in each group.

[0212] Quantitative detection of urinary protein and detection of serum biochemistry: 24-hour urine of rats was collected and the concentration of urinary protein was measured using a rat ELISA kit; rats were anesthetized and serum was collected, and the levels of serum total protein (TP), serum albumin (Alb), serum creatinine (Scr), and serum blood urea nitrogen (BUN) were measured using a fully automated biochemical analyzer.

[0213] 3. Experimental Results and Discussion

[0214] General observation: In the normal group, rats showed normal activity, quick reflexes, glossy fur, normal appetite, and increased body weight. In the model group, rats exhibited poor mental state and hair luster, hair loss, decreased food intake, increased urine output, loose stools, and subcutaneous edema in the abdomen of some rats. The tropinic acid and its derivatives groups showed significant improvements in mental state, fur, appetite, and bowel movements.

[0215] Quantitative detection of urinary protein and serum biochemistry: As shown in Table 21, compared with the normal group, the model group rats showed significantly elevated proteinuria, serum Scr and BUN (P<0.01), and significantly decreased serum TP and Alb levels (P<0.01). After 4 weeks of treatment with tropic acid and its derivatives, the 24-hour urinary protein levels in rats were significantly reduced (P<0.01), and serum TP and Alb levels were significantly increased (P<0.05 or P<0.01), and tended to normalize. Compared with the normal group, the model group showed significantly elevated serum Scr and BUN levels (P<0.01). After 4 weeks of treatment with tropic acid and its derivatives, serum Scr and BUN levels decreased to varying degrees (P<0.05 or P<0.01), and tended to normalize. There was no significant difference in treatment effect between compound 1 and compound 2 (P>0.05). Compared with compounds 1 and 2, compounds 3 and 4 showed better treatment effects (P<0.05). Therefore, tropinic acid and its derivatives have the effect of improving or restoring renal function in rat models of membranous nephritis.

[0216] Table 21. Effects of tropinic acid and its derivatives on renal function in a rat model of membranous nephritis (n=6, )

[0217]

[0218]

[0219] Note: Compared with the blank control group, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0220] Example 13: Preparation of tropinic acid and its derivative tablets

[0221] The prescriptions are shown in Table 2. Take the active pharmaceutical ingredient (tropic acid) and excipients (lactose, microcrystalline cellulose, polyvinylpyrrolidone, croscarmellose sodium, microcrystalline silica, magnesium stearate, and purified water) according to prescription 1. Add tropic acid, lactose, microcrystalline cellulose, and polyvinylpyrrolidone to a wet granulator, using purified water as a wetting agent for wet granulation. After wet granulation, drying, and dry granulation, dry granules are obtained. Add croscarmellose sodium, microcrystalline silica, and magnesium stearate to the dry granules and mix thoroughly. Compress the thoroughly mixed material into tablets using a tableting machine to obtain tropic acid tablets. Take the active pharmaceutical ingredient and excipients from prescriptions 2, 3, and 4 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid tablets, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid tablets, and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid tablets using the same process as above.

[0222] Table 22. Formulation of Tropine Acid and its Derivatives Tablets

[0223] Material Name Prescription 1 (g) Prescription 2 (g) Prescription 3 (g) Prescription 4 (g) Tropine 200 — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 200 — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 200 — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 200 lactose 980 980 980 980 microcrystalline cellulose 700 700 700 700 Polyvinylpyrrolidone 160 160 160 160 Cross-linked carboxymethyl cellulose sodium 80 80 80 80 Micronized silica 20 20 20 20 magnesium stearate 20 20 20 20 Purified water 340 340 340 340

[0224] Example 14: Preparation of tropinic acid and its derivatives coated tablets

[0225] The prescription is shown in Table 3. Take the active pharmaceutical ingredient (tropic acid) and excipients (lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium lauryl sulfate, sodium carboxymethyl starch, micronized silica gel, magnesium stearate, film coating premix, and purified water) according to prescription 5. Add sodium lauryl sulfate to purified water and stir until dissolved. Add tropic acid, lactose, microcrystalline cellulose, and hydroxypropyl methylcellulose to a wet granulator. Use an aqueous solution of sodium lauryl sulfate as a wetting agent for wet granulation. After wet granulation, drying, and dry granulation, dry granules are obtained. Add sodium carboxymethyl starch, micronized silica gel, and magnesium stearate to the dry granules and mix thoroughly. Compress the thoroughly mixed material using a tableting machine to obtain uncoated tropic acid tablets. Add the film coating premix to purified water and stir continuously for more than 1 hour to obtain a film coating solution. Then, coat the obtained uncoated tablets in a coating machine to obtain tropic acid coated tablets. Take the active pharmaceutical ingredients and excipients from prescriptions 6, 7, and 8 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid coated tablets, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid coated tablets, and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid coated tablets respectively using the same process as described above.

[0226] Table 23. Formulations for Tropine Acid and its Derivatives Coated Tablets

[0227] Material Name Prescription 5g Prescription 6g Prescription 7g Prescription 8g Tropine 100 — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 100 — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 100 — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 100 lactose 960 960 960 960 microcrystalline cellulose 600 600 600 600 Hydroxypropyl methylcellulose 200 200 200 200 Sodium dodecyl sulfate 20 20 20 20 Sodium carboxymethyl starch 80 80 80 80 Micronized silica 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

[0228] Example 15: Preparation of Tropical Acid and its Derivatives Spray I

[0229] The prescription is shown in Table 4. According to prescription 9, take the active pharmaceutical ingredient (tropic acid) and excipients (polyvinylpyrrolidone K30, propylene glycol monooctanoate, ethylparaben, poloxamer, di-tert-butyl-p-cresol (BHT), 1N sodium hydroxide solution, ethanol, and water). Add the active pharmaceutical ingredient, polyvinylpyrrolidone K30, propylene glycol monooctanoate, ethylparaben, and BHT to ethanol and stir until completely dissolved. Dissolve poloxamer in an appropriate amount of water. Then add the poloxamer aqueous solution to the ethanol solution of the above mixture, and adjust the pH to the range of 3-8 with 1N sodium hydroxide solution. Add water to a final volume of 200 ml to obtain the spray solution. Fill the solution into spray bottles to obtain tropic acid spray I. Take the active pharmaceutical ingredients and excipients of prescriptions 10, 11 and 12 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid spray I, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid spray I and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid spray I respectively according to the same process method as above.

[0230] Table 24. Formulation of Tropine Acid and its Derivatives Spray I

[0231] Material Name Prescription 9 Prescription 10 Prescription 11 Prescription 12 Tropine 4.0g — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 4.0g — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 4.0g — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 4.0g Polyvinylpyrrolidone K30 8.0g 8.0g 8.0g 8.0g Propylene glycol monooctanoate 15.0g 15.0g 15.0g 15.0g Ethylparaben 0.2g 0.2g 0.2g 0.2g Polosham 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 Appropriate amount Appropriate amount Appropriate amount Appropriate amount ethanol 80g 80g 80g 80g water Add to 200ml Add to 200ml Add to 200ml Add to 200ml

[0232] Example 16: Preparation of Tropical Acid and its Derivatives Spray II

[0233] The prescriptions are shown in Table 5. According to prescription 13, take the active pharmaceutical ingredient (tropic acid) and excipients (hydroxypropyl cellulose, propylene glycol monooctanoate, ethylparaben, ethanol, and water). Add the active pharmaceutical ingredient, hydroxypropyl cellulose, propylene glycol monooctanoate, and ethylparaben to ethanol, stir until completely dissolved, and add water to a final volume of 200 ml to obtain the spray solution. Fill the solution into spray bottles to obtain tropic acid spray II. Take the active pharmaceutical ingredients and excipients from prescriptions 14, 15, and 16 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid spray II, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid spray II, and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid spray II respectively using the same process as described above.

[0234] Table 25. Formulation of Tropine Acid and its Derivatives Spray II

[0235] Material Name Prescription 13 Prescription 14 Prescription 15 Prescription 16 Tropine 2.0g — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 2.0g — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 2.0g — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 2.0g Hydroxypropyl cellulose 6g 6g 6g 6g Propylene glycol monooctanoate 20g 20g 20g 20g Ethylparaben 0.2g 0.2g 0.2g 0.2g ethanol 100g 100g 100g 100g water Add to 200ml Add to 200ml Add to 200ml Add to 200ml

[0236] Example 17: Preparation of Tropical Acid and its Derivatives Injection I

[0237] The prescriptions are shown in Table 6. According to prescription 17, take the active pharmaceutical ingredient (tropic acid) and excipients (sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, poloxamer, sodium bisulfite, and water for injection). Dissolve the active pharmaceutical ingredient and poloxamer in water for injection. Then add sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium bisulfite to the above solution and dissolve them. Finally, add water to 100 mL to obtain the injection solution. Fill the above injection solution into ampoules or vials of the appropriate volume to obtain tropic acid injection I. Take the active pharmaceutical ingredients and excipients from prescriptions 18, 19, and 20 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid injection I, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid injection I, and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid injection I respectively, following the same process as above.

[0238] Table 26. Formulation of Tropine Acid and its Derivatives for Injection I

[0239] Material Name Prescription 17 Prescription 18 Prescription 19 Prescription 20 Tropine 1.0g — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 1.0g — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 1.0g — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 1.0g Sodium chloride 0.85g 0.85g 0.85g 0.85g Sodium hydrogen phosphate 0.21g 0.21g 0.21g 0.21g Sodium dihydrogen phosphate 1.6mg 1.6mg 1.6mg 1.6mg Polosham 0.5g 0.5g 0.5g 0.5g Sodium bisulfite 0.1g 0.1g 0.1g 0.1g Water for Injection Add to 100mL Add to 100mL Add to 100mL Add to 100mL

[0240] Example 18: Preparation of Tropical Acid and its Derivatives Injection II

[0241] The prescriptions are shown in Table 7. According to prescription 21, take the active pharmaceutical ingredient (tropic acid) and excipients (sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween 80, and water for injection). Dissolve the active pharmaceutical ingredient and Tween 80 in water for injection. Then, add sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate to the above solution and dissolve them. Finally, add water to 100 mL to obtain the injection solution. Fill the above injection solution into ampoules or vials of the appropriate volume to obtain tropic acid injection II. Take the active pharmaceutical ingredients and excipients from prescriptions 22, 23, and 24 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid injection II, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid injection II, and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid injection II respectively using the same process as above.

[0242] Table 27. Formulation of Tropine Acid and its Derivatives Injection II

[0243] Material Name Prescription 21 Prescription 22 Prescription 23 Prescription 24 Tropine 0.5g — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 0.5g — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 0.5g — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 0.5g Sodium chloride 0.85g 0.85g 0.85g 0.85g Sodium hydrogen phosphate 0.21g 0.21g 0.21g 0.21g Sodium dihydrogen phosphate 1.6mg 1.6mg 1.6mg 1.6mg Twain 80 0.3g 0.3g 0.3g 0.3g Water for Injection Add to 100ml Add to 100ml Add to 100ml Add to 100ml

[0244] Example 19: Preparation of Tropical Acid and its Derivatives Cream I

[0245] The prescription is shown in Table 8. Take the active pharmaceutical ingredient (tropinic acid) and excipients (white petrolatum, cetyl alcohol, Tween 80, diethylene glycol monoethyl ether, ethylparaben, BHT, propylene glycol, citric acid / sodium citrate and water) according to prescription 25. The preparation process is as follows: (1) Dissolving the active pharmaceutical ingredient: Weigh propylene glycol, add the active pharmaceutical ingredient, and stir to dissolve at 40-50℃; (2) Preparing the oil phase: Weigh white petrolatum, cetyl alcohol, ethylparaben and BHT, heat to 60-80℃, stir to dissolve, and add the dissolved active pharmaceutical ingredient. Add the raw materials slowly, continue stirring, mix evenly, and set aside; (3) Aqueous phase preparation: Weigh purified water, add Tween 80 and diethylene glycol monoethyl ether, heat to 60-80℃, adjust the pH value with citric acid / sodium citrate, stir to dissolve and set aside; (4) Emulsification: Slowly add the aqueous phase to the oil phase, keep at 70℃, homogenize, and continue stirring for more than 30 minutes; (5) Ointment formation: Cool down, stop heating, continue stirring, gradually cool to room temperature and cool to form an ointment, and fill into containers. Tropine acid cream I is obtained. Take the raw materials and excipients of prescriptions 26, 27 and 28 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid cream I, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid cream I and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid cream I respectively according to the same process method as above.

[0246] Table 28. Formulation of Tropine Acid and its Derivatives Cream I

[0247]

[0248]

[0249] Example 20: Preparation of Tropical Acid and its Derivatives Cream II

[0250] The prescription is shown in Table 9. Take the active pharmaceutical ingredient (tropinic acid) and excipients (white petrolatum, cetyl alcohol, poloxamer 407, propylene glycol monooctanoate, sodium benzoate, BHA, propylene glycol, glacial acetic acid / sodium acetate, and water) according to prescription 29. The preparation process is as follows: (1) Dissolving the active pharmaceutical ingredient: Weigh propylene glycol, add the active pharmaceutical ingredient, and stir to dissolve at 40-50℃; (2) Preparing the oil phase: Weigh white petrolatum, cetyl alcohol, propylene glycol monooctanoate, and BHA, heat to 60-80℃, stir to dissolve, and then... (2) Add the raw materials of the solution slowly, continue stirring, mix evenly, and set aside; (3) Aqueous phase preparation: Weigh purified water, add poloxamer 407 and sodium benzoate, heat to 60-80℃, adjust the pH value with glacial acetic acid / sodium acetate, stir to dissolve and set aside; (4) Emulsification: Slowly add the aqueous phase to the oil phase, keep at 70℃, homogenize, and continue stirring for more than 30 minutes; (5) Ointment formation: Cool down, stop heating, continue stirring, gradually cool to room temperature and cool to form an ointment, and fill into containers. Tropine acid cream II is obtained. Take the raw materials and excipients of prescriptions 30, 31 and 32 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid cream I, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid cream I and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid cream II according to the same process method as above.

[0251] Table 29. Formulation of Tropine Acid and its Derivatives Cream II

[0252] Material Name Prescription 29 Prescription 30 Prescription 31 Prescription 32 Tropine 8g — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 8g — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 8g — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 8g Vaseline 20g 20g 20g 20g cetyl alcohol 40g 40g 40g 40g Polosham 407 10g 10g 10g 10g Propylene glycol monooctanoate 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 Appropriate amount Appropriate amount Appropriate amount Appropriate amount water Add to 200g Add to 200g Add to 200g Add to 200g

[0253] Example 21: Preparation of nasal formulation I of tropinic acid and its derivatives

[0254] The prescriptions are shown in Table 30. According to prescription 33, take the active pharmaceutical ingredient (tropic acid) and excipients (sorbitol, citric acid / sodium citrate, sodium benzoate, and purified water). The preparation process is as follows: Dissolve sorbitol in purified water, then add the active pharmaceutical ingredient and sodium benzoate to the above solution and dissolve them. Adjust the pH to the range of 4.0–6.5 with citric acid / sodium citrate, and add purified water to 100 ml to obtain the nasal preparation solution. Finally, fill the solution into dropper bottles or spray bottles to obtain tropic acid nasal preparation I. Take the active pharmaceutical ingredients and excipients from prescriptions 34, 35, and 36 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid cream I, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid cream I, and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid nasal preparation I respectively using the same process as above.

[0255] Table 30. Formulations of Tropine Acid and its Derivatives for Nasal Use (I)

[0256] Material Name Prescription 33 Prescription 34 Prescription 35 Prescription 36 Tropine 0.5g — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 0.5g — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 0.5g — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 0.5g Sorbitol 4.8g 4.8g 4.8g 4.8g Citric acid / Sodium citrate Appropriate amount Appropriate amount Appropriate amount Appropriate amount Sodium benzoate 0.15g 0.15g 0.15g 0.15g Purified water Add to 100mL Add to 100mL Add to 100mL Add to 100mL

[0257] Example 22: Preparation of Tropical Acid and its Derivatives Nasal Formulation II

[0258] The prescription is shown in Table 31. Take the active pharmaceutical ingredient (tropic acid) and excipients (sodium chloride, hydroxypropyl methylcellulose, Tween 80, sodium metabisulfite, sodium EDTA, 1N sodium hydroxide solution, benzalkonium chloride, and purified water) according to prescription 37. The preparation process is as follows: Add hydroxypropyl methylcellulose to purified water and stir continuously until completely dissolved. Add sodium chloride and Tween 80 to the hydroxypropyl methylcellulose solution and dissolve. Then add the active pharmaceutical ingredient, sodium metabisulfite, sodium EDTA, and benzalkonium chloride to the above solution and dissolve. Adjust the pH value to the range of 4.5–6.5 with 1N sodium hydroxide solution. Add purified water to 100 ml to obtain the nasal preparation solution. Finally, fill the solution into dropper bottles or spray bottles to obtain tropic acid nasal preparation II. Take the active pharmaceutical ingredients and excipients from prescriptions 38, 39 and 40 respectively, and prepare 4-hydroxy-α-(hydroxymethyl)phenylacetic acid cream I, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid cream I and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid nasal preparation II according to the same process as described above.

[0259] Table 31. Formulations for Tropine Acid and its Derivatives Nasal Preparations II

[0260] Material Name Prescription 37 Prescription 38 Prescription 39 Prescription 40 Tropine 2g — — — 4-Hydroxy-α-(hydroxymethyl)phenylacetic acid — 2g — — 3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid — — 2g — 3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid — — — 2g Sodium chloride 0.8g 0.8g 0.8g 0.8g Hydroxypropyl methylcellulose 3g 3g 3g 3g Twain 80 0.3g 0.3g 0.3g 0.3g Sodium metabisulfite 0.2g 0.2g 0.2g 0.2g Sodium ethylenediaminetetraacetate 0.2g 0.2g 0.2g 0.2g 1N sodium hydroxide solution Appropriate amount Appropriate amount Appropriate amount Appropriate amount benzalkonium chloride 0.02g 0.02g 0.02g 0.02g Purified water Add to 100mL Add to 100mL Add to 100mL Add to 100mL

[0261] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. Use of tropinic acid and its derivatives, and pharmaceutically acceptable salts thereof, in the preparation of medicaments for the prevention and / or treatment of immune and inflammatory diseases, wherein said tropinic acid and its derivatives are selected from the following formula I Compounds shown in Formula IV: The immune and inflammation-related diseases mentioned are selected from: rheumatoid arthritis.

2. The use according to claim 1, characterized in that, The medication is a topical, oral, or injectable medication.

3. The use according to claim 1, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

4. The use according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

5. The use according to claim 1, characterized in that, The drug is in solid, liquid, or semi-solid dosage form.

6. The use according to claim 1, characterized in that, The dosage forms of the medicine are: powder, tablet, granule, capsule, injection, spray, aerosol, powder mist, lotion, liniment, ointment, plaster, and patch.

7. The use according to claim 1, characterized in that, The dosage forms of the drug are: coated tablets, solutions, emulsions, suspensions, pastes, and gels.

8. The use according to claim 1, characterized in that, The dosage form of the drug is: nasal preparation.

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