WO2024183784A1 - Pharmaceutical use of tropic acid and derivatives thereof in preparation of drug for treating immune- and inflammation-related diseases - Google Patents

Abstract

The present invention belongs to the technical field of medicines, and particularly relates to the use of tropic acid and derivatives thereof in the preparation of a drug for treating immune- and inflammation-related diseases, and a drug for treating immune- and inflammation-related diseases. The tropic acid and derivatives thereof are compounds of formulae I-IV or pharmaceutically acceptable salts thereof, and solvates, esters, enantiomers, diastereomers and tautomers of said compounds or pharmaceutically acceptable salts thereof, or mixtures of any proportion of same, including racemic mixtures. Animal tests show that tropic acid and the derivatives thereof have broad-spectrum immunoregulation, anti-inflammatory and analgesic effects, have obvious improvement effects on pathologic indices of a plurality of immune- and inflammation-related disease animal models, can regulate abnormal immune responses towards being normal, and achieve good anti-inflammatory and analgesic effects. The present invention can adapt to patients suffering from different types of immune- and inflammation-related diseases having different disease levels and is industrially applicable.

Description

Medical use of tropic acid and its derivatives in the preparation of drugs for treating immune and inflammatory related diseases

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Chinese application No. 2023102177363 filed on March 8, 2023. The application No. 2023102177363 is hereby incorporated by reference in its entirety.

Technical Field

The present invention belongs to the field of medical technology, and in particular relates to the use of tropic acid and its derivatives in preparing drugs for treating immune and inflammatory related diseases, and drugs for treating immune and inflammatory related diseases.

Background Art

Immune diseases are a large category of diseases characterized by local or systemic abnormal inflammatory immune responses, mainly including hypersensitivity reactions, immunodeficiency diseases and autoimmune diseases. Involving type I hypersensitivity reactions such as penicillin allergic reactions, drug-induced drug eruptions, allergic rhinitis, pharyngitis, conjunctivitis, bronchial asthma, eczema and urticaria caused by seasons, pollen or dust; type II hypersensitivity reactions such as neonatal hemolytic reactions, drug-induced hemolytic anemia and aplastic anemia; type III hypersensitivity reactions such as glomerulonephritis. Type IV hypersensitivity reactions such as tuberculosis and syphilis. Infection-related bronchitis or pneumonia, gastroenteritis, endometritis, otitis media, tonsillitis, furunculosis, sinusitis, abscesses or granulomas, sepsis, septicemia, myocarditis, meningitis, osteoarthritis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, anorectalitis, 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 immune diseases is increasing year by year, and about 7.6% to 9.4% of the world's population suffers from various types of autoimmune diseases. The disease is difficult to cure, and most patients need to take medication for a long time or even for life. Some diseases are serious, such as lupus nephropathy, which seriously affects the quality of life of patients and threatens their life safety. About 50 million Americans (about 1/5 of the total population) suffer from autoimmune diseases, of which about 75% are women. Immune diseases have become the third largest chronic disease after cardiovascular disease and cancer. Although there is no exact incidence data in China, the number of patients is increasing year by year.

The treatment of immune diseases includes two goals: one is symptom relief and function maintenance, and the other is to delay the process of tissue damage. At present, drugs for the treatment of immune diseases are mainly divided into non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), disease-modifying antirheumatic drugs (DMARDs), biological agents and natural medicines. NSAIDs are commonly used drugs for the treatment of autoimmune diseases. They can effectively alleviate the clinical symptoms and signs of patients and eliminate local inflammatory reactions. However, this type of drug cannot control the progression of the disease. Its common adverse reactions include central nervous system symptoms, cardiovascular damage, gastrointestinal symptoms, hematopoietic system changes, liver and kidney dysfunction, asthma and skin drug eruptions. SAIDs have strong anti-inflammatory and immunosuppressive effects, preventing inflammatory cells from gathering at the site of inflammation, inhibiting the release of inflammatory factors, and inhibiting the proliferation and secretion of TB lymphocytes. This type of drug has many adverse reactions and will relapse after discontinuation of the drug. At present, it is mostly 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 structure and mechanism of action of traditional DMARDs are different, their clinical pharmacodynamic characteristics are similar, that is, they are slow to take effect. After a few weeks or months of medication, symptoms and signs gradually alleviate. Long-term continuous medication can achieve a relatively stable therapeutic effect. The main adverse reactions include gastrointestinal reactions, bone marrow suppression, infection, and liver and kidney damage. Biological agents 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 have been marketed. There are many and serious adverse reactions, and some drugs are banned due to serious adverse reactions. Natural medicines used to treat immune diseases include glycosides and alkaloids. Glycosides include total glycosides of white peony, total ginsenosides of ginseng, total glycosides of gynostemma pentaphyllum, astragaloside I, total glycosides of tripterygium wilfordii and total saponins of Panax notoginseng, and alkaloids include sinomenine, total alkaloids of aconite, sophoracarpine and tripterygium wilfordii. These drugs have fewer adverse reactions and most have anti-inflammatory, analgesic and immunosuppressive effects, but they are not very targeted in clinical treatment of diseases and have poor results. With the in-depth elucidation 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 in clinical practice for the treatment of inflammatory immune diseases. These drugs have definite efficacy, but they also have adverse reactions such as gastrointestinal symptoms, immunosuppression, bone marrow suppression, infection, and new tumors. Therefore, the development of small molecule drugs with immunomodulatory and anti-inflammatory effects that do not damage the body's physiological functions is the main strategy and direction for the treatment of immune and inflammatory related diseases.

It is not difficult to see that immunity and inflammation involve many diseases, and the etiology and pathogenesis are complex and diverse. There are many types of existing therapeutic drugs and methods, and the short-term efficacy is acceptable, but these drugs are often subject to their inherent toxicity and selectivity. They are often accompanied by serious adverse reactions during the treatment process, especially for chronic diseases that require long-term medication. The performance is poor, and the short-term recurrence rate is high after drug withdrawal, and the high cost of treatment brings a heavy burden to the patient's family. Therefore, it is imperative to develop immune and inflammatory disease treatment drugs with small adverse reactions, fast onset, small dosage, short course of treatment, low recurrence rate, convenient use and low price. In view of the differences in the degree of disease and the patient population, it is also necessary to pay attention to the research and development of different drug dosage forms such as external use, oral administration and injection. The present invention has been studied in vivo and in vitro in the early stage, and it has been clarified that tropic acid (DL-TropicAcid, also known as 2-phenyl-3-hydroxypropionic acid) and its derivatives are effective in immune and inflammatory related diseases, as well as safety that is unmatched by other similar drugs. The raw material of tropic acid is cheap, and the preparation cost of its derivatives is low. It can be prepared into various drug dosage forms such as topical, oral and injectable. It is an excellent drug for the prevention and treatment of immune and inflammatory related diseases.

Summary of the invention

In one aspect, the present invention provides the use of tropic acid and its derivatives, pharmaceutically acceptable salts, esters, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any proportions in the preparation of a drug for preventing and/or treating immune and inflammatory related diseases, wherein the tropic acid and its derivatives have a structure shown in the following formula A:

Wherein, R ₁ -R ₅ are each independently selected from -H, -OH or C1-C6 alkoxy.

In one embodiment, R ₁ -R ₅ are each independently selected from -H or -OH.

In one embodiment, _R1 and _R5 are selected from -H, and _R2 to _R4 are each independently selected from -H or -OH.

In one embodiment, R ₁ and R ₅ are selected from -H, two or three of R ₂ to R ₄ are selected from -OH, and the rest are selected from -H.

In one embodiment, at least two of R ₁ -R ₅ are selected from -OH.

In one embodiment, two or three of R ₁ -R ₅ are selected from -OH.

In one embodiment, two or three of R ₂ -R ₄ are selected from -OH.

In one embodiment, the tropic acid and its derivatives are selected from the compounds represented by the following formula I to formula IV:

Among them, formula I is tropic acid (DL-Tropic Acid), formula II is 4-hydroxy-α-(hydroxymethyl)benzeneacetic acid (4-Hydroxy-α-(hydroxymethyl)benzeneacetic acid), formula III is 3,4-dihydroxy-α-(hydroxymethyl)benzeneacetic acid (3,4-Dihydroxy-α-(hydroxymethyl)benzeneacetic acid) and formula IV is 3,4,5-trihydroxy-α-(hydroxymethyl)benzeneacetic acid (3,4,5-Trihydroxy-α-(hydroxymethyl)benzeneacetic acid).

The tropic acid and its derivatives described in the present invention have immunomodulatory, anti-inflammatory and analgesic effects, have a significant improvement effect on the pathological indicators of many animal models of immune and inflammatory diseases, can regulate abnormal immune responses to normal, and exert good anti-inflammatory and analgesic effects.

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

The second aspect of the present invention provides a drug for preventing and/or treating immune and inflammatory diseases, which contains tropic acid and its derivatives, and pharmaceutically acceptable salts, esters, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion.

The drug for preventing and/or treating immune and inflammation-related diseases provided by the present invention has broad-spectrum immunomodulatory, anti-inflammatory and analgesic effects and has significant effects.

In one embodiment, the tropic acid and its derivatives, pharmaceutically acceptable salts, esters, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion are used as active ingredients in the medicine of the present invention. Preferably, they are used as the main active ingredient; more preferably, they are used as the only active ingredient.

In the above-mentioned uses and medicines, tropic acid and its derivatives, pharmaceutically acceptable salts, esters, solvates, enantiomers, diastereomers, tautomers or mixtures in any proportions thereof can be prepared with pharmaceutically acceptable carriers or excipients into pharmaceutical dosage forms for external use, oral administration or injection.

Therefore, in the present invention, the drug can be an external medicine, an oral medicine or an injectable medicine.

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

In the present invention, the term "pharmaceutically acceptable carrier or excipient" includes any and all solvents, co-solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (such as antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, adhesives, excipients, diluents, glidants, granulating agents, disintegrants, thickeners, viscosity enhancers, lubricants, anti-caking agents, humectants, wetting agents, chelating agents, plasticizers, dyes, flavoring agents, etc. and combinations thereof, which are well known to those skilled in the art (for example, see Remington’s Pharmaceutical Sciences). , 19th Ed. Mack Printing Company, 1995; Shanghai Pharmaceutical Industry Research Institute, etc., Application Technology of Pharmaceutical Excipients (Second Edition), China Pharmaceutical Science and Technology Press, 2002; Comparative Manual of Pharmaceutical Excipients Standards of Various Countries 1-3, compiled by the National Pharmacopoeia Committee, China Pharmaceutical Science and Technology Press, 2016; Handbook of Pharmaceutical Excipients, edited by Raymond C Rowe, Paul J Sheskey, and Paul J Weller, translated by Zheng Junmin, Chemical Industry Press, 2005, etc.). Except for carriers and excipients that are incompatible with the active ingredients, any conventional carriers and excipients are considered for use in therapeutic or pharmaceutical compositions.

For example, in a solid dosage form, the pharmaceutically acceptable carrier or excipient may include at least one of the following: (a) a filler such as starch, corn starch, modified starch, compressible starch, lactose, lactose monohydrate, microcrystalline cellulose, cyclodextrin, sorbitol, mannitol, calcium phosphate, amino acid, etc.; (b) a binder such as starch slurry, gelatinized starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, polyvinyl pyrrolidone, gelatin, alginate, etc.; (c) a humectant such as glycerol; (d) a disintegrant such as glycerin; (e) disintegrants, such as dry starch, modified starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl cellulose, cross-linked sodium carboxymethyl cellulose, microcrystalline cellulose, effervescent disintegrants, cross-linked polyvinyl pyrrolidone, etc.; (e) solution retardants, such as paraffin; (f) absorption accelerators, 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 lauryl sulfate, etc.

In the present invention, the drug can be applied to humans or other warm-blooded animals. When the applicable object is a human, the single or mixed dosage of tropic acid and its derivatives is preferably 1 mg/kg·d to 50 mg/kg.d, and more preferably 20 mg/kg·d to 40 mg/kg.d. The therapeutically effective amount of the compound or pharmaceutical composition depends on the species, weight, age and individual condition of the individual, the disease being treated or its severity. A physician, clinician or veterinarian with common skills can easily determine the effective amount of each active ingredient required to prevent, treat or inhibit the development of the disease.

The present invention also provides a compound represented by formula A, its pharmaceutically acceptable salts, esters, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion:

wherein R ₁ -R ₅ are each independently selected from -H or -OH;

Provided that at least two of R ₁ to R ₅ are selected from -OH.

In one embodiment, two or three of R ₁ -R ₅ are selected from -OH.

In one embodiment, two or three of R ₂ -R ₄ are selected from -OH.

In one embodiment, the compound is selected from the following compounds represented by formula III to formula IV:

The pharmaceutically acceptable salts of the compounds of the present invention include base addition salts and acid addition salts thereof. Preferably, the base addition salt is selected from sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, zinc salt, ammonium salt, tetramethylammonium salt, tetraethylammonium salt, triethylamine salt, trimethylammonium salt, ethylamine salt, diethanolamine salt, arginine salt or lysine salt; the acid addition salt is selected from organic acid salts such as acetate, aspartate, benzoate, benzenesulfonate, citrate, ethanedisulfonate, ethanesulfonate, formate, fumarate, gluconate, glucuronate, lactate, malate, trifluoroacetate, maleate, and inorganic acid salts such as hydrochloride, hydrobromide, bisulfate, nitrate, phosphate. The compounds of the present invention in free form can be converted into corresponding compounds in salt form; and vice versa. The compounds of the present invention in free form or salt form and solvate form can be converted into corresponding compounds in free form or salt form in non-solvate form; and vice versa.

The compounds of the present invention also include their solvate forms, which refer to the association formed by one or more solvent molecules and the compounds of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol.

The compounds of the present invention may exist in the form of isomers and mixtures thereof; for example, tautomers, optical isomers, enantiomers, diastereomers. The compounds of the present invention may, for example, contain asymmetric carbon atoms and may therefore exist in the form of enantiomers or diastereomers and mixtures thereof, for example, in the form of racemates. The compounds of the present invention may exist in (R)-, (S)- or (R, S)-configuration, preferably in the (R)- or (S)-configuration at a specific position of the compound.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention first discovered that tropic acid and its derivatives can significantly improve the pathological indicators of animal models of immune and inflammatory diseases;

(2) Tropical acid and its derivatives can regulate abnormal immune responses to normal and exert good anti-inflammatory and analgesic effects

(3) Tropical acid and its derivatives, as the main ingredients for treating immune and inflammatory diseases, have the advantages of low toxicity, rapid onset of action, short course of treatment, small dosage, low recurrence rate and convenience of use compared with existing drugs. They also have the advantages of topical, oral and injection dosage forms, and can be used by patients with different disease severity and different types of immune and inflammatory diseases.

(4) The structurally modified tropic acid derivatives 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid have more significant therapeutic effects than tropic acid in the treatment of immune and inflammatory diseases;

(5) The tropic acid and tropic acid derivatives used in the present invention are easy to obtain and synthesize, low in price, stable in nature, easy to store and transport, and suitable for industrial application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. ¹ H-NMR spectrum of the compound of formula III

Figure 2. ¹³ C-NMR spectrum of the compound of formula III

Figure 3. ¹ H-NMR spectrum of compound IV

Figure 4. ¹³ C-NMR spectrum of the compound of formula IV

Figure 5. Effects of tropic acid and its derivatives on liver tissue pathology in an autoimmune hepatitis mouse model (x200)

Figure 6. Effects of tropic acid and its derivatives on spleen pathology in an autoimmune hepatitis mouse model (x200)

Figure 7. Effects of tropic acid and its derivatives on lung tissue pathology in the COVID-19 cold-dampness epidemic mouse model (x200)

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

Figure 9. Comparison of the change trend of joint index and swelling degree of rats in each group (n=6, ).

DETAILED DESCRIPTION

The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Preparation of the compound of formula III (3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid)

1. Synthesis process

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 bottle. Under nitrogen protection, 20 mL of anhydrous DCM was added. 1.8 mL (25 mmol, 2.5 eq.) of thionyl chloride was slowly added dropwise while maintaining low temperature. The system was 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 at room temperature overnight. The solvent was removed by distillation under reduced pressure to obtain 1.50 g of methyl 3,4-dimethoxyphenylacetate.

At room temperature, 1.0 g (4.76 mmol, 1.0 eq.) of compound 3,4-dimethoxyphenylacetic acid methyl ester was placed in a 100 mL Schlenk bottle, and 0.171 g (5.71 mmol, 1.2 eq.) of paraformaldehyde and 5 mL of DMSO were added. Under nitrogen protection, 0.0257 g (0.476 mmol, 10 mol%) of catalytic sodium methoxide was added to the system, and the system was stirred at room temperature overnight. Post-treatment: The reaction was transferred to 100 mL of water, and the aqueous layer was extracted with EtOAc 3×20 mL. After the organic phases were combined, they were washed with water and dried, concentrated under reduced pressure, and purified by column separation to obtain 0.90 g of 3,4-dimethoxy-α-(hydroxymethyl)phenylacetic acid methyl ester.

Take 0.6g (2.5mmol, 1.0eq.) of methyl 3,4-dimethoxy-α-(hydroxymethyl)phenylacetate in a 20mL Schlenk bottle, slowly add 2mL of hydrogen bromide solution (40%) to the system at room temperature, and heat under reflux to react overnight. After treatment, transfer the reaction system to 50mL of water, extract the water layer with DCM 3×20mL, combine the organic phases, wash with water, dry, concentrate under reduced pressure, and purify by column separation to obtain 0.35g of 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid.

2. Structural Elucidation

3,4-Dihydroxy-α-(hydroxymethyl)phenylacetic acid is a white crystalline powder, which is easily soluble in methanol and soluble in water. ¹ H-NMR (400 MHz, CD ₃ OD) is shown in Figure 1 , δ _H (ppm) 3.63 (2H, m, CH ₂ ), 3.99 (1H, m, CH), 6.50 (1H, dd, J＝2.0, 8.0 Hz, Ph-H), 7.04 (1H, d, J＝8.0 Hz, Ph-H), 7.09 (1H, d, J＝2.0 Hz, Ph-H); ¹³ C-NMR (400 MHz, CD ₃ OD) is shown in Figure 2 , δ _C (ppm) 55.9 (CH), 65.2 (CH ₂ ), 114.3 (CH), 116.4 (CH), 123.9 (CH), 127.2 (C), 145.3 (C), 146.0 (C), 176.2 (C＝O).

Example 2: Preparation of the compound of formula IV (3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid)

1. Synthesis process

Under ice-water bath conditions, take 2.26g (10mmol, 1.0eq.) of 3,4,5-trimethoxyphenylacetic acid in a 100mL Schlenk bottle, add 20mL of anhydrous DCM under nitrogen protection, slowly add 1.8mL (25mmol, 2.5eq) of dithionyl chloride dropwise while keeping the temperature low, transfer the system to room temperature, and stir for 2h. Remove the solvent and excess thionyl chloride under reduced pressure at 40℃. Then add 20mL of methanol to the system and stir at room temperature overnight, and remove the solvent under reduced pressure to obtain 1.65g of methyl 3,4,5-trimethoxyphenylacetate.

At room temperature, 1.5 g (6.25 mmol, 1.0 eq.) of compound 3,4,5-trimethoxyphenylacetic acid methyl ester was placed in a 100 mL Schlenk bottle, and 0.224 g (7.50 mmol, 1.2 eq.) of paraformaldehyde and 5 mL of DMSO were added. Under nitrogen protection, 0.0338 g (0.625 mmol, 10 mol%) of catalytic sodium methoxide was added to the system, and the system was stirred at room temperature overnight. Post-treatment: The reaction was transferred to 100 mL of water, and the aqueous layer was extracted with EtOAc 3×20 mL. After the organic phases were combined, they were washed with water and dried, concentrated under reduced pressure, and purified by column separation to obtain 1.17 g of 3,4,5-trimethoxy-α-(hydroxymethyl)phenylacetic acid methyl ester.

Take 1.0g (3.70mmol, 1.0eq.) of methyl 3,4,5-trimethoxy-α-(hydroxymethyl)phenylacetate in a 20mL Schlenk bottle, slowly add 2mL of hydrogen bromide solution (40%) to the system at room temperature, and heat under reflux to react overnight. After treatment, transfer the reaction system to 50mL of water, extract the water layer with DCM 3×20mL, combine the organic phases, wash with water, dry, concentrate under reduced pressure, and purify by column separation to obtain 0.69g of 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid.

2. Structural Elucidation

3,4,5-Trihydroxy-α-(hydroxymethyl)phenylacetic acid is a white crystalline powder, which is easily soluble in methanol and soluble in water. ¹ H-NMR (400 MHz, CD ₃ OD) is shown in Figure 3, δ _H (ppm) 3.63 (2H, m, CH ₂ ), 3.99 (1H, m, CH), 6.10 (2H, s, Ph-H); ¹³ C-NMR (400 MHz, CD ₃ OD) is shown in Figure 4, δ _C (ppm) 55.9 (CH), 65.2 (CH ₂ ), 106.8 (2×CH), 131.0 (C), 131.3 (C), 146.2 (2×C), 176.2 (C＝O).

Example 3: Study on the effect of tropic acid and its derivatives on immunosuppressive mouse model

1. Animal grouping, modeling and drug administration

A total of 110 male KM mice were randomly divided into 11 groups, with 10 mice in each group, namely blank control group, model group, positive drug group, high-dose tropic acid group (A), low-dose tropic 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). Except for the blank control group mice, which did not receive any treatment, the mice in the other groups were intraperitoneally injected with cyclophosphamide 80 mg/kg.d at the beginning of the experiment 1 to 3 days to establish the immunosuppressive mouse model. Each group was given medication at the same time, twice a day. The grouping and medication schedule are detailed in Table 1. The treatment lasted for 14 days.

Table 1. Animal grouping and dosing information (n=10)

2. Index detection

After the last administration, mice were fasted for 12 hours, anesthetized by intraperitoneal injection of 10% chloral hydrate, weighed, dissected, and the spleen and thymus were immediately separated after blood was taken from the heart. The blood stains on the surface of the organs were absorbed with filter paper and weighed. The spleen index and thymus index were calculated. The organ index = organ wet weight (g)/body weight (g) × 100%. The blood of the mice was taken into a centrifuge tube and centrifuged at 2800r/min for 10 minutes at low temperature (4°C), and the serum was collected and stored at -20°C for 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 kit.

3. Experimental results and discussion

The spleen and thymus index can directly reflect the strength of the body's immune function. Compared with the blank control group, the thymus and spleen indexes of the model group decreased significantly (P<0.01). After two weeks of treatment with tropic acid and its derivatives, the spleen and thymus indexes of each dose group increased significantly (P<0.05 or P<0.01), and were dose-dependent. The high-dose group increased the spleen and thymus index and tended to normal, indicating that tropic acid and its derivatives can significantly improve the immune organ index of cyclophosphamide-induced immunosuppressive mice. IL-4 has an immunomodulatory effect on B cells, T cells, mast cells, macrophages and proliferative cells, and can induce the production of IgG and Ig E. Compared with the model group, the serum IL-4 content of mice in each dose group of tropic acid and its derivatives increased (P<0.01), and the IL-4 level in the high-dose group increased and tended to normal. IL-10 can inhibit NK cell activity and interfere with the production of cytokines by NK cells and macrophages. Compared with the model group, the serum IL-10 content of mice in the tropic acid and its derivatives group was significantly reduced (P<0.05 or P<0.01), and the high-dose group reduced the IL-4 level and tended to normal. Compared with the model group, the level of TNF-α in the tropic acid and its derivatives group was significantly increased (P<0.05 or P<0.01). Therefore, tropic acid and its derivatives can stimulate the secretion of TNF-α, which can induce the enhancement of macrophage activity and killing function after secretion, so that macrophages promote the body's immune response. Compared with the model group, the serum IgG mass concentration of mice in the tropic acid and its derivatives group was significantly increased (P<0.05 or P<0.01), and the high-dose group increased the IgG level and tended to normal. Under the same dose conditions, there was no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05), while in terms of the effects on IL-4, IL-10, TNF-α and IgG levels, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid (P<0.05). The effects of tropic acid and its derivatives on organ indexes and serum immune indicators in the immunosuppressive mouse model are shown in Table 2. In summary, tropic acid and its derivatives have a certain regulatory effect on the suppression of immune function caused by cyclophosphamide.

Table 2. Effects of tropic acid and its derivatives on organ indexes and serum immune indexes in immunosuppressive mouse models (n=10, )

Note: Compared with the blank control group, ^## P<0.01; compared with the model group, *P<0.05, **P<0.01; under the same dose condition, compared with tropic acid, ^△ P<0.05.

Example 4: Study on the effect of tropic acid and its derivatives on spleen lymphocyte proliferation

1. Cell model preparation, grouping and drug administration

After the mice were killed by cervical dislocation, they were disinfected with 75% ethanol, and the spleen was removed under sterile conditions to prepare spleen lymphocytes. The cells were resuspended in RPMI-1640 culture medium and the cell concentration was adjusted to 1×10 ⁷ cells/ml. 100 μL of the cell suspension was added to a 96-well plate. Mitogen ConA was added to each well to make the final concentration of 5 μg/ml. 100 μL of tropic acid and its derivatives were added to each well, including tropic acid high-dose group (A), tropic acid low-dose group (B), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (C), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (D), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (E), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (F), 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (H). The blank control group was added with 100 μL of RPMI-1640 culture medium, and the positive control was added with 100 μL of RPMI-1640 culture medium containing cyclophosphamide. Three replicate wells were set for each group. The grouping and dosing information are shown in Table 3. The cells were incubated at 37°C and 5% CO ₂ After 44 hours of culture, add 10 μL of 5 mg/mL MTT to each well and continue culturing for 4 hours.

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

2. Index detection

After the cell culture was completed, the cells were centrifuged at 1000 r/min, the supernatant was discarded, 200 μL of DMSO was added to each well and oscillated for 10 min to dissolve, and the cells were placed in an ELISA reader to measure the OD value at a wavelength of 492 nm. The experiment was performed three times in parallel. Splenic lymphocyte proliferation inhibition rate = (ConA model control average OD value - drug group average OD value) / ConA model control average OD value.

3. Experimental results and discussion

As shown in Table 4, all tropic acid and its derivative groups had no cytotoxicity and had different degrees of immunosuppressive effect on ConA-induced spleen cell proliferation. Compared with the model group, the OD values of all tropic acid and its derivative groups were significantly reduced (P<0.01), and the inhibition rate of the high-dose group could reach 39.8% to 44.5%. Under the same dose conditions, there was no significant difference between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05), while 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid (P<0.01). This shows that tropic acid and its derivatives have a certain inhibitory effect on spleen lymphocyte proliferation.

Table 4. Effects of tropic acid and its derivatives on proliferation of mouse spleen lymphocytes (n=9, )

Note: Compared with the model group, **P<0.011; under the same dose condition, compared with tropic acid, ^△△ P<0.01.

Example 5: Study on the effect of tropic acid and its derivatives on delayed hypersensitivity reactions

1. Animal grouping, modeling and drug administration

A total of 110 male KM mice (6-7 weeks, 22.0±2.0g) were randomly divided into 11 groups, with 10 mice in each group, namely blank control group, model group, positive drug group, high-dose tropic acid group (A), low-dose tropic 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-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (H), on the first day of administration, the abdomen of each group of mice was dehaired about 1.0cm×2.0cm, and 50μL 1% 2,4-dinitrofluorobenzene (DNFB) (dissolved in acetone-olive oil, acetone: olive oil = 3:1) was applied to the abdomen, and 50μL acetone-olive oil (3:1) was applied to the abdomen of the normal group. The drug was administered on the day of sensitization for 7 consecutive days, twice a day, and applied again on the second day of administration to enhance sensitization. Each group was administered synchronously, and the grouping and administration scheme are detailed in Table 5.

Table 5. Animal grouping and dosing information (n=10)

2. Index detection

30 minutes after the last administration, 10 μL of 1% DNFB was applied to the front and back of the right ear of each mouse. After 24 hours, the mice were weighed and anesthetized by intraperitoneal injection of 10% chloral hydrate (0.1 mL/10 g). The mice were killed by removing the eyeballs and taking blood. The ears were cut off and round ear pieces were punched in the same position with an 8 mm puncher and weighed accurately. The blood was centrifuged and the supernatant was taken. The IFN-γ content in the serum was determined according to the enzyme-linked immunosorbent assay kit. Ear swelling degree = right ear mass - left ear mass; swelling inhibition rate = (average swelling degree of the model group - average swelling degree of the drug group) / average swelling degree of the model group × 100%.

3. Experimental results and discussion

Delayed hypersensitivity is a T cell-dependent immune response model and a Th1 cell-mediated allergic reaction. Th1 mainly secretes INF-γ and participates in the occurrence of cellular immunity and delayed hypersensitivity inflammation. Its main feature is that the sensitized body has a delayed inflammatory response at the site of antigen attack. DNCB is a hapten. After dilution, it is applied to the abdominal skin and combined with skin proteins to form a complete antigen, which stimulates T lymphocytes to proliferate into sensitized lymphocytes. After 7 days, it is applied to the ear to produce a local delayed hypersensitivity reaction. As shown in Table 6, compared with the model group, the tropic acid and its derivative groups significantly reduced the ear swelling of delayed hypersensitivity mice (P<0.05 or P<0.01), and significantly inhibited the increase of serum INF-γ content in delayed hypersensitivity mice (P<0.05 or P<0.01), which was dose-dependent. The high-dose group can make the mouse ears and serum INF-γ tend to normal. Under the same dose conditions, there was no significant difference between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05). Compared with the high-dose groups, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid (P<0.05). This shows that tropic acid and its derivatives have a regulatory effect on delayed hypersensitivity reactions, and inhibiting the increase in serum INF-γ content may be its mechanism of action.

Table 6. Effects of tropic acid and its derivatives on delayed hypersensitivity reactions in mice (n=10, )


Note: Compared with the blank control group, ^## P<0.01; compared with the model group, *P<0.05, **P<0.01; under the same dose condition, compared with tropic acid, ^△ P<0.05.

Example 6: Study on the anti-inflammatory effect of tropic acid and its derivatives

1. Animal grouping, modeling and drug administration

A total of 110 KM mice (6-7 weeks, 22.0±2.0g) were randomly divided into 11 groups, with 10 mice in each group, namely blank control group, model group, positive drug group, high-dose tropic acid group (A), low-dose tropic 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 ), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (F), 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (H). Each group was administered with drugs at the same time. The grouping and dosing schedule are detailed in Table 7. The drugs were administered twice a day for 7 consecutive days by gavage. One hour after the last administration, 40 μL of xylene was evenly applied on both sides of the right ear of mice in all groups except the normal control group to induce inflammation.

Table 7. Animal grouping and dosing information (n=10)

2. Index detection

One hour after the inflammation, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and then killed by dislocation of the neck. The ears were cut along the edge of the auricle, and circular ear pieces were punched in the same part of the ears with a manual 8mm ear puncher. The ears were weighed accurately to calculate the ear swelling degree and swelling inhibition rate. Ear swelling degree = right ear mass - left ear mass; swelling inhibition rate = (average swelling degree of the model group - average swelling degree of the drug group) / average swelling degree of the model group × 100%.

3. Experimental results and discussion

As shown in Table 8, compared with the model control group, the tropic acid and its derivatives groups can significantly reduce the degree of ear swelling of mice induced by xylene (P<0.01), among which the swelling inhibition rate of the high-dose group of tropic acid and its derivatives can reach 70.34% to 74.31%. Under the same dose conditions, there is no significant difference in the swelling inhibition effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid, while 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid have better swelling inhibition effects than tropic acid. In summary, the results show that tropic acid and its derivatives have obvious anti-inflammatory effects on the xylene-induced mouse ear swelling model.

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


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

Example 7: Study on the analgesic effect of tropic acid and its derivatives

1. Animal grouping, modeling and drug administration

A total of 100 KM mice (5-6 weeks, 22.0±2.0g), half male and half female, were randomly divided into 10 groups, with 10 mice in each group, namely blank control group, model group, positive drug group, high-dose tropic acid group (A), low-dose tropic 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), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid group (F), and low-dose 4-hydroxy-α-(hydroxymethyl)phenylacetic acid group (G). The 4-dihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (F), the 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and the 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (H) were administered with drugs at the same time, twice a day, for 7 consecutive days. The grouping and dosing schedule are shown in Table 9. Except for the normal control group, the mice in the other groups were intraperitoneally injected with 0.6% 10mL/kg glacial acetic acid 2h after the last administration to establish the pain model.

Table 9. Animal grouping and dosing information (n=10)

2. Index detection

After the pain model was established, the latency time of the mice to writhe and the number of writhing times of each group of mice within 20 minutes were observed and recorded, and the analgesic rate was calculated. Analgesic rate = (average number of writhing times in the model group - average number of writhing times in the drug administration group) / average number of writhing times in the model control group × 100%.

3. Experimental results and discussion

As shown in Table 10, compared with the model control group, the tropic acid and its derivatives group can significantly prolong the latency time of the acetic acid-induced writhing reaction in mice (P<0.05 or P<0.01), and significantly reduce the number of writhing times (P<0.01), among which the analgesic rate of the high-dose group of tropic acid and its derivatives can reach 55.26-58.79%. Under the same dose conditions, there is no significant difference in the analgesic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid, while 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid have better analgesic effects than tropic acid. Therefore, tropic acid and its derivatives have obvious peripheral analgesic effects.

Table 10. Effects of tropic acid and its derivatives on acetic acid-induced writhing reaction in mice (n=10, )


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

Example 8: Study on the effect of tropic acid and its derivatives on autoimmune hepatitis mouse model

1. Animal modeling, grouping and drug administration

Take the liver tissue of normal male KM mice (6-7 weeks, 22.0±2.0g), add physiological saline at a ratio of 1:9 (g/mL), grind it thoroughly (-4℃) into a tissue homogenate with a tissue grinder, centrifuge it for 10 minutes (2500rpm, 4℃), take the supernatant, and obtain the isogenic liver antigen. Add the isogenic liver antigen to complete Freund's adjuvant at a ratio of 1:1 (v/v), mix well, and fully emulsify it to obtain the immune agent (prepared before use).

110 male KM mice (6-7 weeks, 22±2g) were randomly divided into 11 groups, 10 mice in each group, including blank control group, model group, positive drug group, tropic acid high-dose group (A), tropic acid low-dose group (B), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (C), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (D), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (E), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (F), 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (G). Except for the blank control group, the mice in other groups were intraperitoneally injected with 1 mL/mouse of the immunizing agent to complete the first immunization. The second immunization was performed 7 days later to obtain the autoimmune hepatitis mouse model. At the same time, mice in the blank control group were intraperitoneally injected with 1 mL of normal saline. The mice were given oral administration starting from the day of modeling, twice a day, for 14 consecutive days. The grouping and dosing schedule are detailed in Table 11.

Table 11. Animal grouping and dosing information (n=10)

2. Index detection

After the last administration, the mice were fasted for 12 hours, and each group of mice was anesthetized by intraperitoneal injection of 10% chloral hydrate (0.1mL/10g). The mice were dissected, blood was taken from the heart, and saline was perfused and fixed with paraformaldehyde. The liver and spleen tissues were taken and fixed in 4% paraformaldehyde for 24 hours. The blood was centrifuged for 10 minutes (3000rpm, 4℃), and serum was taken. The activities of serum liver function-related indicators lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the content of total bilirubin (TBIL) were detected according to the instructions of the kit; the liver and spleen tissues were paraffin-embedded, cut into 4μm thick sections, routine HE staining, and sealed. The histopathological changes were observed under a light microscope.

3. Experimental results and discussion

Liver function test: As shown in Table 12, the activities of LDH, ALT, and AST in the serum of mice in the model group increased, and the TBIL content increased significantly (P<0.01). When liver tissue is damaged or liver cells are necrotic, the activities of LDH, ALT, and AST in serum increase, and the increase in TBIL content is a sensitive indicator of liver damage, which directly reflects the degree of liver cell damage and necrosis, and the detoxification and metabolic function of the liver. Compared with the model group, after 2 weeks of administration of tropic acid and its derivatives, the activities of LDH, ALT, AST, and TBIL in serum decreased (P<0.05 or P<0.01), and were dose-dependent. Under the same dose conditions, there was no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05); compared between high-dose groups, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid (P<0.05). Therefore, tropic acid and its derivatives can improve liver function in autoimmune hepatitis model mice.

Table 12. Effects of tropic acid and its derivatives on liver function in autoimmune hepatitis mouse model (n=10, )

Note: Compared with the blank control group, ^## P<0.01; compared with the model group, *P<0.05, **P<0.01; under the same dose condition, compared with tropic acid, ^△ P<0.05.

Histopathological examination: As shown in Figure 5, a large number of inflammatory factors infiltrated the liver of the model group mice, with blurred cell boundaries, edema and degeneration of hepatocytes, condensed nuclei, lytic necrosis of some cells, and vacuolization caused by fatty degeneration of cells. After administration of tropic acid and its derivatives, some of the above pathological indicators were significantly improved, especially the regeneration of hepatocytes around necrotic hepatocytes to repair liver damage, which were characterized by large cell size, large and darkly stained nuclei, and most of them had binuclei. As shown in Figure 6, in the spleen of the model group mice, follicular hyperplastic lesions related to immune activation appeared, and then the germinal centers increased and became larger. It can be seen that the area ratio of red pulp to white pulp decreased, and the foamy macrophages in the white pulp and red pulp increased. After the intraperitoneal injection of the immune agent, a large number of lymphocyte transformation occurred in the spleen, B cells proliferated and transformed into plasma cells, and the spleen follicle growth center expanded and a large number of plasma cells appeared, which increased antibody production and enhanced humoral immunity; plasma existed in the blood vessels in the red pulp, and the proliferated macrophages destroyed the plasma red blood cells in the red pulp arterioles, resulting in a large amount of congestion of the splenic cords in the red pulp. After the administration of tropic acid and its derivatives, the above-mentioned immune activation pathological indicators were significantly improved. It shows that tropic acid and its derivatives may regulate immune activity by improving the function of recognizing antigens, and play a role in treating autoimmune hepatitis.

Example 9: Study on the effect of tropic acid and its derivatives on the COVID-19 cold-dampness epidemic mouse model

1. Animal modeling, grouping and drug administration

A total of 110 male KM mice (6-7 weeks, 22.0±2.0) were randomly divided into 11 groups, with 10 mice in each group, namely blank control group, model group, positive drug group, tropic acid high-dose group (A), tropic acid low-dose group (B), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (C), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (D), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (E), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (F), 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (G). Each group was treated with drugs twice a day for 7 consecutive days. After 3 days of administration, mice in all groups except the blank control group were intraperitoneally injected with 5 mg/kg lipopolysaccharide saline solution, and then placed in an artificial climate box to control the temperature at 4.0±2.0℃ and the humidity at 90.0±3.0% for cold and dampness stimulation modeling for 8 hours a day for 4 consecutive days to create a new coronary pneumonia cold and dampness epidemic mouse model. The grouping and dosing regimen are detailed in Table 13.

Table 13. Animal grouping and dosing information (n=10)

2. Index detection

After the last administration, fast for 12 hours, anesthetize with 10% chloral hydrate intraperitoneal injection (0.1mL/10g), perfuse with normal saline, take lung tissue, freeze part of it in liquid nitrogen, store it in a -80℃ refrigerator for later use, and fix part of it in 4% paraformaldehyde for later use. Take 50mg of frozen mouse tissue, add 800μL PBS for homogenization, centrifuge at 2800r/min (4℃) for 10min, collect serum, and use enzyme-linked immunosorbent assay kit to determine the levels of inflammatory factors IL-6, IL-10, IFN-γ and TNF-α in lung tissue. Take the fixed lung tissue, embed it in paraffin, cut 4μm thick sections, perform routine HE staining, seal the sections, and observe the histopathological changes under light microscope.

3. Experimental results and discussion

Detection of inflammatory factors: 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 mice in the model group 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), and they were dose-dependent. The high-dose group reduced the inflammatory factors in the lung tissue and returned to normal. Under the same dose conditions, there was no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05); compared between high-dose groups, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid (P<0.05). Therefore, tropic acid and its derivatives can improve the inflammatory response of the COVID-19 cold-dampness epidemic mouse model.

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

Note: Compared with the blank control group, ^## P<0.01; compared with the model group, *P<0.05, **P<0.01; under the same dose condition, compared with tropic acid, ^△ P<0.05.

Histopathological examination: As shown in Figure 7, the lung tissue structure of the mice in the normal group was intact, without exudate, and the alveolar septa were normal; while the alveolar scaffolds of the mice in the model group 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 serious. After the administration of tropic acid and its derivatives, some of the above pathological indicators were significantly improved. Under the same dose conditions, there was no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid, while 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid. It shows that tropic acid and its derivatives may play a therapeutic role in the mouse model of COVID-19 cold and dampness by regulating immunity and inhibiting inflammatory response.

Example 10: Study on the effect of tropic acid and its derivatives on aplastic anemia rat model

1. Animal modeling, grouping and drug administration

Sixty-six male SD rats (7-8 weeks, 200.0±20.0g) were randomly divided into 11 groups, with 6 rats in each group, namely blank control group, model group, positive drug group, high-dose tropic acid group (A), low-dose tropic 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), 3,4,5- The high-dose trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G) and the low-dose 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid group (G), except the rats in the blank control group, were subcutaneously injected with 2% acetylphenylhydrazine (Ap) saline solution 20 mg.kg on the 1st and 4th days, and cyclophosphamide (Cy) saline solution 20 mg.kg was injected intraperitoneally 2 hours after acetylphenylhydrazine injection on the 4th day, and on the 5th to 7th days, to establish the animal model of aplastic anemia. The rats in each drug-treated group were injected by tail vein, once a day, for 14 consecutive days. The grouping and drug administration scheme are detailed in Table 15.

Table 15. Animal grouping and dosing information (n=6)

2. Index detection

One hour after the last administration of the drug, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and blood was collected from the heart and placed in an anticoagulant tube for later use. The thymus and spleen were weighed. A hemolytic agent was added to the whole blood in the anticoagulant tube, and the number of peripheral blood cells was detected using a fully automatic blood cell analyzer, mainly including changes in four indicators such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB) and platelet count (PLT) in peripheral blood. The spleen index and thymus index were calculated, and the organ index = organ wet weight (g)/body weight (g) × 100%.

3. Experimental results and discussion

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 various indicators increased (P<0.05 or P<0.01), and were dose-dependent. Compared with the normal group, the spleen index of 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 normal (P<0.05 or P<0.01). Under the same dose conditions, there was no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05), while in terms of the effects on the levels of WBC, RBC and HGB, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid (P<0.05). Therefore, tropic acid and its derivatives can improve aplastic anemia in rat models through their immunomodulatory effects, thereby restoring their hematopoietic stem cell function.

Table 16. Effects of tropic acid and its derivatives on immune organ indexes and peripheral blood counts in aplastic anemia models (n=6, )

Note: Compared with the blank control group, ^## P<0.01; compared with the model control group, *P<0.05, **P<0.01; under the same dose conditions,
Compared with tropic acid, ^△ P<0.05.

Example 11: Study on the effect of tropic acid and its derivatives on rheumatoid arthritis model rats

1. Animal modeling, grouping and drug administration

Preparation of immunoemulsifier: Take 7 ml of bovine type II collagen (CII) solution and place it in a small beaker, stir it with a magnetic stirrer at 1500 r/min at low temperature, take 7 ml of complete Freund's adjuvant (CFA) solution, slowly add it to the CII solution, and continue stirring for about 30 minutes after the CFA solution is completely added, until the emulsion drops on the water do not disperse, and obtain the primary immunoemulsifier. Replace CFA with incomplete Freund's adjuvant (IFA) and use the same preparation method to obtain the secondary immunoemulsifier. All immunoemulsifiers are prepared before use.

Preparation of rheumatoid arthritis model (CIA): 100 male SD rats (7-8 weeks, 200.0±20.0g) were subcutaneously injected with 0.2ml of primary immune emulsion at the base of the rat's tail. On the 8th day after the primary immunization, 0.1ml of secondary immune emulsion was subcutaneously injected at the base of the rat's tail to complete the booster immunization. The blank control group was injected with normal saline in the same way. The arthritis index (AI) was scored 14 days after the booster immunization. The scoring rules are as follows: 0 points for no swelling and erythema; 1 point for erythema and mild swelling of the ankle joint; 2 points for erythema and mild swelling from the ankle joint to the metatarsal joint or the palm joint; 3 points for erythema and moderate swelling from the ankle joint to the metatarsophalangeal joint or the palm joint; 4 points for erythema and severe swelling from the ankle joint to the toe joint. The sum of the scores of the two paws was used as the joint score of each rat. An AI score of ≥4 indicated that the model was successfully constructed, and the animals without signs of joint swelling were removed.

Grouping and administration: Six rats in the blank control group and 60 CIA model rats were randomly divided into 10 groups, with 6 rats in each group, namely model group, positive drug group, tropic acid high-dose group (A), tropic acid low-dose group (B), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (C), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (D), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (E), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (F), 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (G). Each group was gavaged with the corresponding drugs at the same time, twice a day, for 4 consecutive weeks. The grouping and administration scheme are shown in Table 17.

Table 17. Animal grouping and dosing information (n=6)

2. Index detection

After the treatment, the morphological differences of the ankle joints and paws of each group of rats before and after treatment were compared and observed. According to the above AI scoring method, the arthritis condition of each group of rats was scored once a week. The volume changes of the ankle joints and below of the rats were synchronously measured by the foot volume displacement method, and the degree of joint swelling was calculated. Joint swelling degree = (volume after modeling - volume before modeling) / volume before modeling × 100%.

3. Experimental results and discussion

As shown in Figure 8, after modeling, the volume of the plantar toe of the rats increased significantly. The joints of the rats in each modeling group were red and swollen, and the paws were stiff and curled up, and they could not walk normally. After 4 weeks of treatment with tropic acid and its derivatives, the joints of the rats in the low-dose group were slightly red and swollen, and the joints of the rats in the other treatment groups were red and swollen and in good condition. Under the same dose conditions, there was no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid, while 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid. As shown in Figure 9, compared with the model group, the AI score of the tropic acid and its derivatives treatment group gradually decreased, and the difference was statistically significant from the third week of treatment (P<0.05 or P<0.01); compared with the model group, the swelling of the rat joints began to decrease after 2 weeks of treatment with tropic acid and its derivatives, and the swelling of the rat joints decreased significantly after 4 weeks of administration. Under the same dose conditions, there was no significant difference in the treatment effect of 4-hydroxy-α-(hydroxymethyl)phenylacetic acid compared with tropic acid at each week (P>0.05); after 3 weeks of administration, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid significantly reduced the AI score and joint swelling of rats compared with tropic acid (P<0.05), and had better therapeutic effect. In summary, tropic acid and its derivatives have obvious therapeutic effects on rat models of rheumatoid arthritis.

Example 11: Study on the effect of tropic acid and its derivatives on the mouse model of allergic rhinitis

1. Animal modeling, grouping and drug administration

One hundred and ten male BABL/c mice (5-6 weeks, 20.0±2.0g) were randomly divided into 11 groups, with 10 mice in each group, namely blank control group, model group, positive drug group, high-dose tropic acid group (A), low-dose tropic 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 mice in the blank control group, the mice in other groups were intraperitoneally injected with a suspension sensitizer (containing 0.50mg/mL OVA and 0.50mg/mLAl(OH) ₃ ) were administered to each mouse at 200uL for basic sensitization; the corresponding drugs were administered to each group from day 15 to day 28; on day 22 to day 28 of the experiment, 4% OVA solution was dripped into the nasal cavity of the mice for stimulation, 20μL per nasal cavity, to establish the allergic rhinitis model, and the blank control group was given the same volume of normal saline during the basic sensitization and stimulation stages. The grouping and dosing schedule are detailed in Table 18.

Table 18. Animal grouping and dosing information (n=10)

2. Index detection

After the last nasal drop challenge, the number of nose scratching and sneezing of mice in each group was observed and recorded within 30 minutes.

3. Experimental results and discussion

As shown in Table 19, compared with the blank group, the number of sneezing and scratching of the mice in the model group increased significantly (P<0.01), indicating that the allergic rhinitis model was successfully established. Compared with the model group, the tropic acid and its derivatives group can significantly reduce the number of sneezing (P<0.01) and scratching of the mice (P<0.05 or P<0.01), and it is dose-dependent. Under the same dose conditions, there is no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05), while 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid have better therapeutic effects than tropic acid (P<0.05). It shows that tropic acid and its derivatives have obvious therapeutic effects on the mouse model of allergic rhinitis.

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

Note: Compared with the blank control group, ^## P<0.01; compared with the model control group, *P<0.05, **P<0.01; under the same dose conditions,
Compared with tropic acid, ^△ P<0.05.

Example 12: Study on the effect of tropic acid and its derivatives on membranous nephritis model rats

1. Animal modeling, grouping and drug administration

Replication of membranous nephritis model: Take 100 mg of cationic bovine serum albumin (C-BSA) and dissolve it in 15 mL of normal saline, mix it with an equal volume of incomplete Freund's adjuvant and fully emulsify it. Take 100 male SD rats (7-8 weeks, 200±20g), take 1 mL of emulsifier, and make multiple subcutaneous injections in the bilateral armpits and groin of each rat, once every other day, for a total of 3 times to complete pre-immunization. One week after pre-immunization, each rat was injected with 16 mg/kg C-BSA normal saline solution through the tail vein, 3 times a week, for 4 consecutive weeks to complete formal immunization. Use metabolic cages to collect rats' 24h urine for urine protein detection. The 24h urine protein (24h UPro) amount >20 mg is considered to be a successful replication of the membranous nephritis model, and rats with failed model replication are removed. Rats in the blank control group were synchronously injected with an equal volume of normal saline in the same way.

Grouping and administration: 6 rats in the blank control group and 60 rats with membranous nephritis model were randomly divided into 10 groups, with 6 rats in each group, namely model group, positive drug group, tropic acid high-dose group (A), tropic acid low-dose group (B), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (C), 4-hydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (D), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (E), 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (F), 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid high-dose group (G) and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid low-dose group (G). Each group was gavaged with the corresponding drugs at the same time, twice a day, for 4 consecutive weeks. The grouping and administration scheme are shown in Table 11.

Table 20. Animal grouping and dosing information (n=6)

2. Index detection

General condition observation: After the last administration, the hair, mental state, body weight, food intake, stool and feces of each group of rats were compared and observed to evaluate the therapeutic effect of the drug.

Quantitative analysis of urine protein and serum biochemistry: 24-hour urine of rats was collected, and the urine protein concentration was determined using a rat ELISA kit. The rats were anesthetized, serum was collected, and the levels of serum total protein (TP), serum albumin (Alb), serum creatinine (Scr), serum urea nitrogen (BUN) and other indicators were determined using an automatic biochemical analyzer.

3. Experimental results and discussion

General condition observation: General condition of rats in each group: rats in the normal group had normal activities, sensitive reactions, shiny fur, normal diet, and increased body weight; rats in the model group had poor spirits and hair gloss, hair loss, reduced food intake, increased urine volume, loose stools, and subcutaneous edema in the abdomen of some rats. The mental state, hair, diet, and bowel movements of rats in the tropic acid and its derivatives group were significantly improved.

Quantitative detection of urine protein and serum biochemistry: As shown in Table 21, compared with the normal group, the rats in the model group had significantly increased proteinuria, serum Scr and BUN (P<0.01), and the serum TP and Alb levels were significantly decreased (P<0.01); after 4 weeks of treatment with tropic acid and its derivatives, the 24-hour urine protein of the rats was significantly decreased (P<0.01), and the serum TP and Alb levels were significantly increased (P<0.05 or P<0.01), and tended to normal; compared with the normal group, the serum Scr and BUN levels of the model group were significantly increased (P<0.01), and after 4 weeks of treatment with tropic acid and its derivatives, the serum Scr and BUN levels were decreased to varying degrees (P<0.05 or P<0.01), and tended to normal. Under the same dose conditions, there was no significant difference in the therapeutic effect between 4-hydroxy-α-(hydroxymethyl)phenylacetic acid and tropic acid (P>0.05), while in terms of the effects on 24h Upro, TP, Alb, BUN and other levels, 3,4-dihydroxy-α-(hydroxymethyl)phenylacetic acid and 3,4,5-trihydroxy-α-(hydroxymethyl)phenylacetic acid had better therapeutic effects than tropic acid (P<0.05). Therefore, tropic acid and its derivatives have the effect of improving or restoring renal function in rat models of membranous nephritis.

Table 21. Effects of tropic acid and its derivatives on renal function in rat models of membranous nephritis (n=6, )

Note: Compared with the blank control group, ^## P<0.01; compared with the model group, *P<0.05, **P<0.01; under the same dose condition, compared with tropic acid, ^△ P<0.05.

Example 13: Preparation of Tropicic Acid and Its Derivatives Tablets

The prescription is shown in Table 2. According to prescription 1, the raw material (tropic acid) and excipients (lactose, microcrystalline cellulose, polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, micropowder silica gel, magnesium stearate and purified water) are taken, and tropic acid, lactose, microcrystalline cellulose and polyvinyl pyrrolidone are added to a wet granulator, and wet granulation is performed using purified water as a wetting agent. After wet granulation, drying and dry granulation, dry granules are obtained, cross-linked sodium carboxymethyl cellulose, micropowder silica gel and magnesium stearate are added to the dry granules and mixed evenly, and the mixed material is compressed into tablets using a tablet press to obtain tropic acid tablets. The raw materials and excipients of prescriptions 2, 3 and 4 are taken respectively, and 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 are prepared respectively according to the same process as above.

Table 22. Tropical acid and its derivatives tablet formulations

Example 14: Preparation of coated tablets of tropic acid and its derivatives

The prescription is shown in Table 3. According to prescription 5, the raw material (tropic acid) and excipients (lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium lauryl sulfate, sodium carboxymethyl starch, micropowder silica gel, magnesium stearate, film coating premix and purified water) are taken, sodium lauryl sulfate is added to purified water and stirred to dissolve, tropic acid, lactose, microcrystalline cellulose and hydroxypropyl methylcellulose are added to a wet granulator, and an aqueous solution of sodium lauryl sulfate is used as a wetting agent for wet granulation. After wet granulation, drying and dry granulation, dry granules are obtained, sodium carboxymethyl starch, micropowder silica gel and magnesium stearate are added to the dry granules and mixed evenly, and the mixed material is compressed into tablets using a tablet press to obtain tropic acid plain tablets. The film coating machine premix is added to purified water, and stirring is continued for more than 1 hour to obtain a film coating solution, and then the obtained plain tablets are coated in a coating machine to obtain tropic acid coated tablets. The raw materials and excipients of prescriptions 6, 7 and 8 were taken respectively, and 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 were prepared respectively according to the same process as above.

Table 23. Formulation of coated tablets of tropic acid and its derivatives

Example 15: Preparation of Tropicic Acid and Its Derivatives Spray I

The prescription is shown in Table 4. According to prescription 9, the raw material (tropic acid) and excipients (polyvinyl pyrrolidone K30, propylene glycol monocaprylate, ethyl hydroxybenzoate, poloxamer, di-tert-butyl-p-cresol (BHT), 1N sodium hydroxide solution, ethanol and water) are taken, and the raw material, polyvinyl pyrrolidone K30, propylene glycol monocaprylate, ethyl hydroxybenzoate and BHT are added to ethanol and stirred to completely dissolve; poloxamer is added to an appropriate amount of water to dissolve; then the poloxamer aqueous solution is added to the alcohol solution of the above-mentioned mixed materials, and the pH value is adjusted to a range of 3 to 8 with 1N sodium hydroxide solution; water is added to 200 ml to obtain a spray solution; the solution is filled into a spray bottle to obtain tropic acid spray I. The raw materials and excipients of prescriptions 10, 11 and 12 were taken respectively, and 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 were prepared respectively according to the same process as above.

Table 24. Tropical acid and its derivatives spray I prescription

Example 16: Preparation of Tropicic Acid and Its Derivatives Spray II

The prescription is shown in Table 5. According to prescription 13, take the raw material (tropic acid) and excipients (hydroxypropyl cellulose, propylene glycol monocaprylate, ethyl hydroxybenzoate, ethanol, water), add the raw material, hydroxypropyl cellulose, propylene glycol monocaprylate, and ethyl hydroxybenzoate to ethanol, stir to completely dissolve, add water to 200 ml, and obtain the spray solution; fill the solution into a spray bottle to obtain tropic acid spray II. Take the raw materials and excipients of 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 according to the same process as above.

Table 25. Prescription of Tropical Acid and Its Derivatives Spray II

Example 17: Preparation of Tropicic Acid and Its Derivatives Injection I

The prescription is shown in Table 6. According to prescription 17, the raw material (tropic acid) and excipients (sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, poloxamer, sodium bisulfite and water for injection) are taken, the raw material and poloxamer are added to the water for injection to dissolve, and then sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium bisulfite are added to the above solution to dissolve, and finally water is added to 100mL to obtain the injection solution; the above injection solution is filled into an ampoule or vial of corresponding volume to obtain tropic acid injection I. The raw materials and excipients of prescriptions 18, 19 and 20 are taken respectively, and 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 are prepared respectively according to the same process as above.

Table 26. Prescription of Tropical Acid and Its Derivatives Injection I

Example 18: Preparation of Tropicic Acid and Its Derivatives Injection II

The prescription is shown in Table 7. Take the raw material (tropic acid) and excipients (sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween 80, water for injection) according to prescription 21, add the raw material and Tween 80 into water for injection to dissolve, then add sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate into the above solution to dissolve, and finally add water to 100mL to obtain the injection solution; fill the above injection solution into an ampoule or vial of corresponding volume to obtain tropic acid injection II. Take the raw materials and excipients of 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 according to the same process as above.

Table 27. Prescription of Tropical Acid and Its Derivatives Injection II

Example 19: Preparation of Tropicic Acid and Its Derivatives Cream I

The prescription is shown in Table 8. According to prescription 25, the raw material drug (tropic acid) and excipients (white vaseline, hexadecanol, Tween 80, diethylene glycol monoethyl ether, ethyl hydroxybenzoate, BHT, propylene glycol, citric acid/sodium citrate and water) were taken, and the preparation process was as follows: (1) Dissolution of the raw material drug: Weigh propylene glycol, add the raw material drug, and stir to dissolve at 40-50°C: (2) Preparation of oil phase: Weigh white vaseline, hexadecanol, ethyl hydroxybenzoate and BHT, heat to 60-80°C, stir to dissolve, and mix the dissolved raw material Add the raw materials slowly, continue to stir, mix evenly, and set aside; (3) Preparation of aqueous phase: weigh purified water, add Tween 80 and diethylene glycol monoethyl ether, heat to 60-80°C, adjust the pH value with citric acid/sodium citrate, stir and dissolve for later use; (4) Emulsification: slowly add the aqueous phase to the oil phase, maintain 70°C, homogenize, and continue to stir for more than 30 minutes; (5) Paste formation: cool down, stop heating, continue to stir, gradually cool to room temperature and cool into paste, and fill. Tropical 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 according to the same process as above.

Table 28. Tropical acid and its derivatives cream I prescription

Example 20: Preparation of Tropicic Acid and Its Derivatives Cream II

The prescription is shown in Table 9. According to prescription 29, the raw material drug (tropic acid) and excipients (white vaseline, hexadecanol, poloxamer 407, propylene glycol monocaprylate, sodium benzoate, BHA, propylene glycol, glacial acetic acid/sodium acetate and water) were taken, and the preparation process was as follows: (1) Dissolution of the raw material drug: weigh propylene glycol, add the raw material drug, and stir to dissolve at 40-50°C; (2) Preparation of oil phase: weigh white vaseline, hexadecanol, propylene glycol monocaprylate and BHA, heat to 60-80°C, stir to dissolve, and then add the solution to the mixture. Slowly add the dissolved raw materials, continue to stir, mix evenly, and set aside; (3) Preparation of aqueous phase: weigh purified water, add poloxamer 407 and sodium benzoate, heat to 60-80°C, adjust the pH value with glacial acetic acid/sodium acetate, stir and dissolve for later use; (4) Emulsification: slowly add the aqueous phase to the oil phase, maintain 70°C, homogenize, and continue to stir for more than 30 minutes; (5) Paste formation: cool down, stop heating, continue to stir, gradually cool to room temperature and cool into paste, and fill. Tropical 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 as above.

Table 29. Tropical acid and its derivatives cream II prescription

Example 21: Preparation of nasal preparation I of tropic acid and its derivatives

The prescription is shown in Table 30. According to prescription 33, the raw material (tropic acid) and excipients (sorbitol, citric acid/sodium citrate, sodium benzoate and purified water) are taken, and the preparation process is as follows: add sorbitol to purified water to dissolve, then add the raw material and sodium benzoate to the above solution to dissolve, adjust the pH value to the range of 4.0 to 6.5 with citric acid/sodium citrate, add purified water to 100 ml, and obtain the nasal preparation solution, and finally fill the solution into a dropper bottle or spray bottle to obtain tropic acid nasal preparation I. Take the raw materials and excipients of 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 according to the same process as above.

Table 30. Prescription of nasal preparations of tropic acid and its derivatives I

Example 22: Preparation of nasal preparation II of tropic acid and its derivatives

The prescription is shown in Table 31. According to prescription 37, the raw material drug (tropic acid) and excipients (sodium chloride, hydroxypropyl methylcellulose, Tween 80, sodium metabisulfite, sodium edetate, 1N sodium hydroxide solution, benzalkonium chloride and purified water) are taken. The preparation process is as follows: add hydroxypropyl methylcellulose to purified water and continue stirring to completely dissolve it. Add sodium chloride and Tween 80 to the hydroxypropyl methylcellulose solution to dissolve it. Then add the raw material drug, sodium metabisulfite, sodium edetate and benzalkonium chloride to the above solution to dissolve it. Use 1N sodium hydroxide solution to adjust the pH value to within the range of 4.5 to 6.5, and add purified water to 100 ml to obtain a nasal preparation solution. Finally, fill the solution into a dropper bottle or a spray bottle to obtain tropic acid nasal preparation II. The raw materials and excipients of prescriptions 38, 39 and 40 were taken respectively, and 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 were prepared respectively according to the same process as above.

Table 31. Prescription of nasal preparations of tropic acid and its derivatives II

The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims (10)

1. Use of tropic acid and its derivatives, pharmaceutically acceptable salts, solvent compounds, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion in the preparation of drugs for preventing and/or treating immune and inflammatory related diseases, wherein the tropic acid and its derivatives have the structure shown in the following formula A:
   

   Wherein, R ₁ -R ₅ are each independently selected from -H, -OH or C1-C6 alkoxy.
2. The use according to claim 1, characterized in that R ₁ to R ₅ are each independently selected from -H or -OH.
3. The use according to claim 1, characterized in that the tropic acid and its derivatives are selected from the compounds represented by the following formula I to formula IV:
   
4. A drug for preventing and/or treating immune and inflammatory diseases, comprising tropic acid and its derivatives as described in any one of claims 1 to 3, and pharmaceutically acceptable salts, esters, solvates, enantiomers, diastereomers, tautomers or mixtures thereof in any proportion.
5. The use according to any one of claims 1 to 3 or the medicine according to claim 4, characterized in that the medicine is an external medicine, an oral medicine or an injectable medicine.
6. The use according to any one of claims 1 to 3 or the medicine according to claim 4, characterized in that the medicine further comprises a pharmaceutically acceptable carrier or excipient.
7. The use according to any one of claims 1 to 3 or the medicine according to claim 4, characterized in that the medicine is in a solid dosage form, a liquid dosage form or a semisolid dosage form, and preferably, the dosage form of the medicine is: powder, tablet, coated tablet, granule, capsule, solution, emulsion, suspension, injection, spray, nasal agent, aerosol, powder spray, lotion, liniment, ointment, plaster, paste, gel, patch.
8. The use according to any one of claims 1 to 3, or the medicament according to any one of claims 4 to 7, characterized in that the immune and inflammation-related diseases include: allergic rhinitis, bronchitis, bronchial asthma, pharyngitis, conjunctivitis, eczema, urticaria, eczema, neonatal hemolytic reaction, hemolytic anemia, aplastic anemia, nephritis, tuberculosis, syphilis, pneumonia (including COVID-19), gastroenteritis, endometritis, otitis media, sepsis, sepsis, myocarditis, meningitis, tonsillitis, sinusitis, pleurisy, cholecystitis, osteomyelitis, prostatitis, urethritis, cystitis, anorectalitis, 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. One or more of the following.
9. A compound represented by formula A, its pharmaceutically acceptable salt, ester, solvate, enantiomer, diastereomer, tautomer or a mixture thereof in any ratio:
   

   wherein R ₁ -R ₅ are each independently selected from -H or -OH;

   Provided that at least two of R ₁ -R ₅ are selected from -OH;

   Preferably, two or three of R ₁ to R ₅ are selected from -OH.
10. The compound according to claim 9, its pharmaceutically acceptable salt, ester, solvate, enantiomer, diastereomer, tautomer or a mixture thereof in any proportion, characterized in that the compound is selected from the compounds represented by the following formula III-IV: