A stilbene derivative, a preparation method thereof, and use thereof in preparing a medicament for treating psoriasis or ulcerative colitis

Abstract

The present invention discloses a stilbene derivative and its preparation method and its application in the preparation of a medicine for treating psoriasis or ulcerative colitis, and belongs to the field of biomedicine technology. Its structural formula is as follows: Compound II 2 and II 23 provided by the present invention target and excite AHR related pathways, promote AHR to enter the nucleus, reduce the secretion of related cytokines at the cellular level, significantly improve imiquimod-induced mouse psoriasis model to obtain mouse skin symptoms and PASI scores, compared to the lead compound benvimod, its anti-psoriatic activity is better and less toxic, and it is found that compound II 23 can effectively treat mouse ulcerative colitis, reduce inflammatory response, and improve colitis symptoms. The compounds of the present invention can be used to prepare medicines for the treatment of psoriasis and ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a stilbene derivative, a preparation method thereof, and application of the stilbene derivative in preparing a medicine for treating psoriasis or ulcerative colitis. Background Art

[0002] Psoriasis is a chronic, systemic, inflammatory skin disease associated with immune inflammation, characterized by red spots and plaques with silvery scales. It is difficult to cure and prone to recurring attacks. It commonly appears on the scalp and joints of the limbs, but in severe cases can spread throughout the body. The disease is intractable and often accompanied by multiple complications, such as hypertension, diabetes, and cardiovascular disease. Severe psoriasis not only affects patients' skin appearance and social activities, but also severely impacts their quality of life, causing significant physical and mental harm. Inadequate treatment can seriously endanger patients' lives. It can be triggered by a variety of factors, resulting in high morbidity and disability rates. Currently, treatment options for mild psoriasis include topical corticosteroids, vitamin D derivatives, calcineurin inhibitors, keratolytics, and targeted phototherapy. For severe psoriasis, four classes of biologics are used: TNF inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors, and IL-23 inhibitors. Traditional therapies are often expensive and have numerous side effects, necessitating the development of safe, effective, affordable, and readily available small molecule compounds.

[0003] Inflammatory bowel disease (IBD) is a group of conditions that includes ulcerative colitis (UC) and Crohn's disease (CD). It is characterized by chronic intestinal inflammation, often leading to mucosal ulceration and progressive loss of intestinal function. Currently, there is no effective treatment for IBD due to its complex etiology and pathophysiology, involving multiple factors including genetics, environment, epithelial, microbial, and immune factors.

[0004] Styrene compounds are composed of benzene rings connected by double bonds of ethylene. Current literature suggests that styrene skeletons have strong biopharmaceutical applications. In 2023, the FDA approved VTAMA (tapinarof, 1%) cream for the topical treatment of plaque psoriasis in adults. This is the first steroid-free topical medication and the first topical new molecular entity drug approved for the treatment of psoriasis in the United States in 25 years. Summary of the Invention

[0005] The present invention aims to provide a stilbene derivative, a preparation method thereof, and its use in the preparation of a medicament for treating psoriasis or ulcerative colitis, to address the aforementioned problems of the prior art. The present invention provides a compound having a styrene structural skeleton that exhibits excellent inhibitory activity against inflammatory skin diseases, particularly psoriasis. Compared to marketed drugs, the compound provided by the invention has a faster onset of action, better anti-psoriatic efficacy, and exhibits less toxicity.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a stilbene derivative, including II-2 and II-23, whose structural formula is as follows:

[0008]

[0009] The second technical solution of the present invention is a method for preparing the diphenylethylene derivative, comprising the following steps:

[0010] (1) Methyl 3,5-dimethoxybenzoate and 2-bromoisopropane were dissolved in 1,2-dichloroethane, and anhydrous aluminum chloride was added to carry out substitution reaction to obtain intermediate 2;

[0011] (2) adding boron tribromide to intermediate 2 to carry out a substitution reaction to obtain intermediate 3;

[0012] (3) Add N,N-diisopropylethylamine and bromomethyl methyl ether to intermediate 3 to undergo substitution reaction to obtain intermediate 4;

[0013] (4) adding lithium aluminum hydride to intermediate 4 to react and obtain intermediate 5;

[0014] (5) adding potassium carbonate and pyridinium chlorochromate to intermediate 5 for oxidation reaction to obtain intermediate 6;

[0015] (6) adding sodium hydride and phosphate to intermediate 6 to undergo substitution reaction to obtain intermediate 7 or intermediate 8;

[0016] (7) Concentrated hydrochloric acid is added to intermediate 7 or intermediate 8 to undergo a substitution reaction to obtain II-2 or II-23.

[0017] The third technical solution of the present invention is the use of the diphenylethylene derivative in the preparation of a drug for treating psoriasis or ulcerative colitis.

[0018] Based on the above technical solution, the present invention has the following technical effects:

[0019] (1) The stilbene skeleton derivatives II-2 and II-23 provided by the present invention can significantly inhibit the occurrence of psoriasis symptoms. II-23 can alleviate the occurrence of ulcerative colitis symptoms and treat ulcerative colitis in mice to a certain extent.

[0020] (2) The distyrene skeleton derivatives II-2 and II-23 provided by the present invention are prepared from methyl 3,5-dimethoxybenzoate as the starting material, and are sequentially prepared by a seven-step reaction including Friedel-Crafts alkylation, demethylation, methoxymethyl reprotection, lithium aluminum hydride reduction, PCC oxidation, Horner–Wadsworth–Emmons reaction, and re-deprotection reaction. The final yield of the product is 23%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 A and B indicate that compounds II-2 and II-23 can activate the AHR and promote its nuclear translocation. C indicates that compounds II-2 and II-23 can activate the AHR downstream pathway and promote the expression of CYP1A1 and CYP1B1. II-2 is comparable to the positive drug, while II-23 is more effective than the positive drug. D indicates that compounds II-2 and II-23 can reduce the expression of CCL5, CCL20, IL-6, IL-8, S100A9, TLR4, TNF-α, and TNFR1 in HaCaT cells.

[0023] Figure 2 A shows PCA analysis demonstrating group homogeneity. B shows the number of differentially expressed genes between groups. C and D show volcano plots of differentially expressed genes between groups. E shows a GO enrichment map. F shows a KEGG enrichment map. G shows a heatmap of differentially expressed genes. AHR downstream pathways are activated.

[0024] Figure 3 As the number of days of administration increases, imiquimod can gradually induce psoriasis symptoms on the surface of the mouse skin, manifested as redness, swelling, scaling, and thickening. Compounds II-2 and II-23 can significantly inhibit the occurrence of psoriasis symptoms.

[0025] Figure 4Body weight changes revealed a gradual decrease in the model group, while compounds II-2 and II-23 maintained the weight gain of the mice. Scoring across multiple dimensions revealed that compounds II-2 and II-23 inhibited the onset and progression of imiquimod-induced psoriasis on the backs of mice. Mild psoriasis symptoms appeared in the model group starting on the second day of treatment, gradually worsening. Compounds II-2 and II-23 only began to show mild symptoms on the third day and were able to inhibit symptom progression. Compared to the control group, imiquimod induced changes in mRNA levels of inflammatory factors in the skin tissue of psoriatic mice. Compounds II-2 and II-23 at varying concentrations showed some improvement. Among them, A is the weight change of different groups, B is the PASI score record of different groups, C is the skin thickness record of different groups, D is the comprehensive score of different groups, E is the effect of 2 and 23 on the mRNA level of CD206, F is the effect of 2 and 23 on the mRNA level of CD36, G is the effect of 2 and 23 on the mRNA level of IL-18, and H is the effect of 2 and 23 on the mRNA level of MCP-1.

[0026] Figure 5 As can be seen from the changes in colon length, weight, and DAI scores of mice, compound II-23 can alleviate the shortened colon and weight loss caused by DSS in mice, and the overall bloody stools in mice are also improved. HE results show that compound II-23 can improve the colon ulcers caused by DSS and inhibit the symptoms of ulcerative colitis in mice to a certain extent, showing therapeutic effects on ulcerative colitis. Among them, A is an image of the colon, B is the colon length record of different groups, C is the colon HE staining of different groups, D is the weight record of different groups, and E is the DAI score of different groups. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0033] The present invention provides a stilbene derivative, including II-2 and II-23, whose structural formula is as follows:

[0034]

[0035] The present invention also provides a method for preparing the stilbene derivative, comprising the following steps:

[0036] (1) Methyl 3,5-dimethoxybenzoate and 2-bromoisopropane were dissolved in 1,2-dichloroethane, and anhydrous aluminum chloride was added to carry out substitution reaction to obtain intermediate 2;

[0037] (2) adding boron tribromide to intermediate 2 to carry out a substitution reaction to obtain intermediate 3;

[0038] (3) Add N,N-diisopropylethylamine and bromomethyl methyl ether to intermediate 3 to undergo substitution reaction to obtain intermediate 4;

[0039] (4) adding lithium aluminum hydride to intermediate 4 to react and obtain intermediate 5;

[0040] (5) adding potassium carbonate and pyridinium chlorochromate to intermediate 5 for oxidation reaction to obtain intermediate 6;

[0041] (6) adding sodium hydride and phosphate to intermediate 6 to undergo substitution reaction to obtain intermediate 7 or intermediate 8;

[0042] (7) Concentrated hydrochloric acid is added to intermediate 7 or intermediate 8 to undergo a substitution reaction to obtain II-2 or II-23.

[0043] In some specific embodiments, in step (1), the molar ratio of 2-bromoisopropane to methyl 3,5-dimethoxybenzoate is (1.1-1.3):1; the molar ratio of anhydrous aluminum chloride to methyl 3,5-dimethoxybenzoate is (1.05-1.2):1; and the substitution reaction is carried out at 85-95° C. for 5-8 hours.

[0044] In some specific embodiments, in step (2), the molar ratio of boron tribromide to intermediate 2 is (4.0-7.0):1; and the reaction conditions of the substitution reaction are: reaction in an inert gas at 25-35° C. for 10-15 hours.

[0045] In some specific embodiments, in step (3), the molar ratio of N,N-diisopropylethylamine, bromomethyl methyl ether and intermediate 3 is (10-15):(10-15):1; and the conditions of the substitution reaction are: reaction at 25-35°C for 10-15 hours.

[0046] In some specific embodiments, in step (4), the molar ratio of the intermediate 4 to lithium aluminum hydride is (1-3):1; and the reaction conditions are: reaction in an inert gas at 0°C for 30 minutes.

[0047] In some specific embodiments, in step (5), the molar ratio of potassium carbonate, pyridinium chlorochromate and intermediate 5 is (0.5-0.7):(2-2.5):1; and the oxidation reaction conditions are: reaction at 25-35°C for 2-4h.

[0048] In some specific embodiments, in step (6), the molar ratio of sodium hydride, phosphate and intermediate 6 is (1.2-1.5):(1.2-1.5):1; the conditions of the substitution reaction are: reaction at 25-35°C for 6-10 hours; the phosphate is diethyl (4-methoxybenzyl)phosphonate or diethyl (4-methylbenzyl)phosphonate.

[0049] In some specific embodiments, in step (7), the molar volume ratio of the concentrated hydrochloric acid to the intermediate 7 or intermediate 8 is 1 mL: (1-1.2) mol; and the conditions of the substitution reaction are: reaction at 25-35° C. for 2-4 h.

[0050] The embodiments of the present invention also provide use of the stilbene derivative in preparing a drug for treating psoriasis or ulcerative colitis.

[0051] The compounds II-2 and II-23 provided by the present invention target and excite AHR-related pathways, promote AHR nuclear entry, reduce related cytokines at the cellular level, and significantly improve the skin symptoms and PASI scores of mice in the imiquimod-induced mouse psoriasis model. Compared with the lead compound benvimod, its anti-psoriatic activity is better and its toxicity is lower. It was found that compound II-23 can effectively treat ulcerative colitis in mice, reduce inflammatory response, and has lower toxicity, adding indications that are not available in the lead compound benvimod. The compounds of the present invention can be used to prepare drugs for the treatment of skin diseases such as psoriasis and atopic dermatitis, as well as ulcerative colitis.

[0052] Example 1

[0053] A stilbene derivative, the preparation process of which is as follows:

[0054]

[0055] The preparation method comprises the following steps:

[0056] Step 1: Methyl 3,5-dimethoxybenzoate (2 mmol, 1.0 equivalent) was dissolved in 10 ml of 1,2-dichloroethane. 2-Bromopropane (2.4 mmol, 1.2 equivalent) and aluminum chloride (2.4 mmol, 1.2 equivalent) were added sequentially at room temperature. The mixture was transferred to a 90°C oil bath and heated under reflux. After reacting for 6 hours, it was cooled to room temperature and quenched with 30 ml of saturated sodium bicarbonate. A flocculent precipitate was produced. 30 ml of dilute hydrochloric acid was added to clarify the solution. The solution was extracted three times with ethyl acetate. The organic phases were combined, washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by column chromatography (PE:EA=200:1) to obtain intermediate 2.

[0057] Step 2: Intermediate 2 (1.0 equivalent) was dissolved in ultra-dry dichloromethane. After argon replacement three times, boron tribromide (1 M in DCM, 5 equivalents) was added dropwise at -78°C. After the addition was complete, the temperature was gradually raised to room temperature. After reacting overnight, the reaction mixture was cooled to -78°C and quenched by adding anhydrous methanol. The sample was directly mixed and separated by column chromatography (PE / EA = 5 / 1) to obtain Intermediate 3.

[0058] Step 3: Dissolve intermediate 3 (1.0 equivalent) in ultra-dry dichloromethane. After argon replacement three times, add N,N-diisopropylethylamine (15 equivalents) at 0°C, and slowly add bromomethyl methyl ether (15 equivalents) dropwise. After reacting for 1 hour, slowly warm to room temperature and continue reacting for 10 hours. After completion of the reaction, monitor the reaction by TLC, wash with saturated sodium bicarbonate twice, saturated sodium chloride twice, dry over anhydrous sodium sulfate, spin dry, and separate by column chromatography (PE / EA = 20 / 1) to obtain intermediate 4.

[0059] Step 4: Intermediate 4 (1.0 equivalent) was dissolved in anhydrous ether, and the atmosphere was replaced with argon three times. Lithium aluminum tetrahydride (2.5 M in THF, 0.5 equivalent) was added dropwise at 0°C and allowed to react for 30 minutes. 1.0 equivalent of water, 1.0 equivalent of 15% sodium hydroxide solution, and 3.0 equivalents of water were added sequentially. The mixture was brought to room temperature and stirred for 15 minutes before filtration through celite. The filtrate was washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and spun down to yield Intermediate 5.

[0060] Step 5: Intermediate 5 (1.0 equivalent) was dissolved in ultra-dry dichloromethane, and potassium carbonate (0.5 equivalent) and pyridinium chlorochromate (2.2 equivalents) were added sequentially at 0°C. After reacting at room temperature for 2.5 hours, ether was added to quench the reaction, filtered through celite, and separated by column chromatography (PE / EA = 20 / 1) to obtain intermediate 6.

[0061] Step 6: Dissolve diethyl (4-methylbenzyl)phosphonate (1.2 equivalents) in tetrahydrofuran and replace the atmosphere with argon three times. Add sodium hydride (60% in paraffin oil, 1.5 equivalents) at 0°C and react for 30 minutes. Then add intermediate 6 (1.0 equivalents) and transfer to room temperature for 2 hours. Quench the reaction by adding water at 0°C, extract three times with ethyl acetate, combine the organic phases, wash three times with saturated sodium chloride, dry over anhydrous sodium sulfate, spin dry, and separate by column chromatography (PE / EA = 20 / 1) to obtain intermediate 7.

[0062] Step 7: Dissolve intermediate 7 in 10 ml of anhydrous methanol, add 1 ml of concentrated hydrochloric acid, and react at room temperature for two hours. Spin dry and separate by column chromatography (PE / EA=10 / 1) to obtain compound II-2.

[0063] II-2: 1 H NMR(600MHz,Chloroform-d)δ7.36(d,J=8.0Hz,2H),7.15(d,J=7.9Hz,

[0064] 2H), 6.95 (d, J = 16.2Hz, 1H), 6.85 (d, J = 16.3Hz, 1H), 6.48 (s, 2H), 4.80 (s, 2H), 3.44 (hept, J = 7.1Hz, 1H), 2.35 (s, 3H), 1.38 (s, 3H), 1.36 (s, 3H).

[0065] Example 2

[0066] A stilbene derivative, the preparation process of which is as follows:

[0067]

[0068] Steps 1 to 5 are the same as in Example 1.

[0069] Step 6: Dissolve diethyl (4-methoxybenzyl)phosphonate (1.2 equiv) in tetrahydrofuran and replace the atmosphere with argon three times. Add sodium hydride (60% in paraffin oil, 1.5 equiv) at 0°C and react for 30 minutes before adding intermediate 6 (1.0 equiv). Transfer the mixture to room temperature and react for 2 hours. Quench the reaction by adding water at 0°C, extract three times with ethyl acetate, combine the organic phases, wash three times with saturated sodium chloride, dry over anhydrous sodium sulfate, spin dry, and separate by column chromatography (PE / EA = 20 / 1) to obtain intermediate 8.

[0070] Step 7: Dissolve intermediate 8 in 10 ml of anhydrous methanol, add 1 ml of concentrated hydrochloric acid, and react at room temperature for two hours. Spin dry and separate by column chromatography (PE / EA=10 / 1) to obtain compound II-23.

[0071] II-23: 1 H NMR (400MHz, CDCl3) δ7.39 (d, J = 8.6 Hz, 2H), 6.91 (d, J = 16.2 Hz, 1H),

[0072] 6.87(d,J=8.6Hz,2H),6.75(d,J=16.3Hz,1H),6.45(s,2H),4.78(s,2H),3.80(s,3H),3.42(p,J=7.1Hz,1H),1.36(s,3H),1.34(s,3H).

[0073] Example 3

[0074] Using HaCaT cells transfected with knockout lentivirus, the AHR transcription level in the cells was measured before and after administration to evaluate the AhR agonist activity of compounds II-2, II-23, and tapinarof. The results are shown in Table 1.

[0075] Table 1 AhR agonist activity

[0076]

[0077] As can be seen from the data in Table 1, the EC50 of II-2 and II-23 provided by the present invention are significantly lower than that of the positive drugs, indicating that they have stronger activity in stimulating AhR.

[0078] Example 4

[0079] 1. Place a sterile cell slide of appropriate size in a six-well cell culture plate (1 slide / well), collect HaCaT cells in good growth state and dilute them to a density of 1×10 6A cell suspension of 100 cells / mL was seeded into six-well plates and cultured in an incubator for 24 hours. After 24 hours of compound administration, the supernatant was removed. The culture medium was discarded and the plates were washed three times with PBS. Fixation was performed with 4% paraformaldehyde for 20 minutes, followed by three washes with PBS. A circle was drawn with a histochemical pen to prevent loss of incubation solution during subsequent steps, followed by a wash with PBS. Blocking was performed with 5% BSA (Biofroxx, 4240GR250) for 2 hours, followed by a dropwise addition of the appropriate amount of primary antibody (AHR, 1:1000, CellSignaling Technology, USA) and incubation at 4°C overnight. Rewarming was performed, followed by three 5-minute washes with PBS. A secondary antibody (FITC-conjugated anti-rabbit) of the appropriate species was added within the circle, incubated in a 37°C waterbath in the dark for 40 minutes, and then washed three times with PBS for 5 minutes each. Nuclear staining with DAPI (Sigma, D8417-1MG) was added, followed by a dropwise addition of DAPI for 20-30 minutes at room temperature in the dark, followed by a wash with PBS. The slides were mounted with anti-fluorescence quenching mounting medium (Sigma, V900155-25G) and stored in a dark box at 4°C. The slides were observed and photographed under a microscope (OLYMPUS, IX51).

[0080] The experimental results showed that after administration, the green fluorescence of AhR migrated into the nucleus, indicating that the compound activated AhR to a certain extent and promoted its entry into the nucleus.

[0081] 2qRT-PCR was used to detect changes in the levels of inflammatory factor mRNA in cells and skin tissues

[0082] (1) Extraction of cellular RNA: HaCaT cells were evenly plated in six-well plates according to the experimental groups and cultured. Cells from different groups were collected into 1.5 mL centrifuge tubes, washed 2-3 times with pre-cooled PBS, and 1 mL of Trizol was added for 5 minutes. 0.2 mL of chloroform was added and gently inverted to mix. The tubes were allowed to stand at room temperature for 10 minutes and centrifuged at 10,000 g for 15 minutes at 4°C. The upper aqueous phase was carefully transferred to another centrifuge tube and the same volume of isopropanol as the upper aqueous phase was added to precipitate RNA. After gentle mixing, the tubes were allowed to stand at room temperature for 5 minutes and centrifuged for 10 minutes (at the same speed as before). The RNA precipitate at the bottom was retained and 1 mL of 75% ethanol prepared with DEPC water was added to wash the residual isopropanol solution. After centrifugation, the ethanol solution was discarded and the RNA was dried at room temperature or under vacuum for 10 minutes. The precipitate (RNA) was dissolved with DEPC water (50 μL / well). The RNA concentration was determined and the tubes were stored in a -80°C refrigerator.

[0083] (2) Extraction of animal RNA: Collect skin lesion cells (HaCaT) into 1.5 mL centrifuge tubes, add 1 mL of Trizol and grinding beads, and homogenize in a homogenizer. After homogenization, add 0.2 mL of chloroform and gently invert to mix. Let it stand at room temperature for 10 minutes, centrifuge at 10,000 g for 15 minutes at 4°C, carefully transfer the upper aqueous phase to another centrifuge tube, add the same volume of isopropanol as the upper aqueous phase to precipitate RNA, gently mix, let it stand at room temperature for 5 minutes, centrifuge for 10 minutes (same speed as before), retain the RNA precipitate at the bottom, add 1 mL of 75% ethanol prepared with DEPC water to wash the residual isopropanol solution, centrifuge and discard the ethanol solution. Dry the RNA at room temperature or under vacuum for 10 minutes, and dissolve the precipitate (RNA) with DEPC water (50 μL / well). Measure the RNA concentration and store in a -80°C refrigerator.

[0084] (3) Reverse transcription: Take an enzyme-free EP tube in a clean bench, add 2 μg RNA and 4 μL reverse transcription 5× Mix, then add DEPC water to the total volume to 20 μL, and react in a metal bath at 25°C for 5 min, 42°C for 40 min, and 75°C for 5 min.

[0085] (4) PCR amplification: Quantitative RT-PCR (qRT-PCR) was performed using a 10 μL system, including 2 μL sample DNA, 2 μL DEPC water, 1 μL corresponding primers, and 5 μL qPCR Mix. The PCR instrument ABIQuantStudio 5 (Thermo Fisher Scientific, USA) was used, and the temperature gradient was set to 95°C (5 min) → 95°C (10 s) → 55-60°C (20 s) → 72°C (30 s). The cDNA amplification cycle was repeated 40 times.

[0086] The results showed that after administration of compounds 2 and 23, the expression of CCL5, CCL20, IL-6, IL-8, S100A9, TLR4, TNF-α, and TNFR1 in cells was inhibited, and compound 23 had better inhibitory activity against CCL20, S100A9, and TNF-α than tapinarof. These results indicate that compounds 2 and 23 can block inflammatory factors. Compared with IMQ treatment, compound 2 reduced the levels of CD206 and MCP-1 in animal lesional tissue and showed similar effects to tapinarof treatment. In addition to these factors, compound 23 also reduced the levels of CD36 and IL-18 compared with IMQ treatment.

[0087] 3. Drug administration methods for psoriasis animal modeling

[0088] After one week of adaptive breeding in an SPF barrier environment, 8-week-old Balb / C male mice were shaved of their mid-back hair with a mouse shaver and randomly divided into five groups: blank control group (Con), imiquimod model group (IMQ), positive drug group (tapinarof), II-2 group (1%), and II-23 group (1%).

[0089] Mice in the imiquimod model group, positive drug group, II-2 group, and II-23 group were treated with 62.5 mg of imiquimod for 7 days. At the same time, mice in the positive drug group were given tapinarof (1%), mice in the II-2 group were given II-2 (1%), and mice in the II-23 group were given II-23 (1%) for skin application. When the model group mice reached the standard of weight loss, psoriasis on the skin, and skin lesion proliferation, and the PASI (Psoriasis Area and Severity Index) score continued to increase, the psoriasis-like mouse model was successfully established. The blank control group did not receive any treatment. The dynamic changes of the skin lesions on the back of the mice were observed and recorded before daily administration.

[0090] PASI score:

[0091] Scoring was based on the severity of psoriasis in mice, including the degree of scaling, erythema, and thickening of the dorsal lesions (0: no lesions; 1: mild lesions; 2: moderate lesions; 3: severe lesions; 4: very severe lesions). Three mice were assigned to each group, and scores were recorded daily before dosing.

[0092] The results showed that compounds II-2 and II-23 inhibited the onset and exacerbation of IMQ-induced psoriasis symptoms on the backs of mice, as evidenced by a reduction in the severity of erythema and skin scaling, particularly in the case of compound 23, which did not appear until the fifth day. Compound 23 also showed minimal toxicity, with no changes in body weight.

[0093] 4. Drug administration method for ulcerative colitis animal model

[0094] Eight-week-old Balb / C male mice were acclimated for one week in an SPF-grade barrier environment and then given 2.8% dextran sulfate sodium salt (DSS) solution in drinking water ad libitum for one week. Three days after free drinking water, compound groups were given 20 mg / kg II-23 or 20 mg / kg Tapinarof by gavage for seven consecutive days. Body weights were recorded and blood in stool was observed. The blank group had free access to distilled water throughout the treatment and was given sodium carboxymethylcellulose by gavage. The DSS group received 2.8% DSS in drinking water and sodium carboxymethylcellulose by gavage for the first seven days. The compound 23 group received 2.8% DSS in drinking water and compound 23 (presumably compound 23) diluted in sodium carboxymethylcellulose by gavage for the first seven days. The compound Tap group received 2.8% DSS in drinking water and compound 20 mg / kg Tapinarof (presumably compound 23) diluted in sodium carboxymethylcellulose by gavage for the first seven days.

[0095] HE staining to detect pathological changes in colon tissue

[0096] A small portion of mouse colon tissue was fixed with 4% paraformaldehyde. Embed the slices in paraffin. The specific operation is as follows: first, dehydrate the slices from low concentration to high concentration, that is, soak the slices in xylene for 20 minutes, repeat this process, pour out the xylene, add anhydrous ethanol and soak for 5 minutes, repeat this process, then add 75% alcohol gradient dewaxing for 5 minutes, and then wash with water. Then perform hematoxylin staining: the hydrated slices are stained with hematoxylin dye for cell nuclei for a few minutes to more than ten minutes.

[0097] The sections are then washed with an acid solution, such as 1% hydrochloric acid, to remove excess dye and undergo differentiation. Finally, they are treated with an alkaline solution to reverse blueing, rendering the cell nuclei blue. Eosin staining is then performed: After hematoxylin staining, the sections are then stained with eosin to stain the cytoplasm. The paraffin sections are dehydrated with a gradient of alcohol (85% ethanol → 95% ethanol) and placed in eosin stain for several minutes. Finally, dehydration and mounting are performed: the paraffin sections are soaked in anhydrous ethanol for several minutes and discarded. Then, they are soaked in a clearing agent (usually xylene) for several minutes and repeated. The sections are then allowed to dry naturally and photographed using a fluorescence microscope. In these images, the cell nuclei are stained blue and the cytoplasm red.

[0098] Results showed that the compound group showed significantly greater colon length and some weight recovery compared to the DSS group. HE staining revealed significant ulceration in the DSS group, while the compound group alleviated these ulcers, potentially curing ulcerative colitis. The Tapinarof group showed no significant improvement in weight or colon length compared to the model group, and significant ulceration was still evident in HE staining.

[0099] In summary, the present invention uses methyl 3,5-dihydroxymethylbenzoate as the starting material, introduces a group at the carbon-4 position via a Friedel-Crafts alkylation reaction or a Suzuki reaction, then demethylates the hydroxyl group and protects it with a methoxymethyl group. The ester group is then reduced and oxidized to produce the key intermediate aldehyde. The aldehyde reacts with diethyl phosphonate via a Horner–Wadsworth–Emmons reaction to produce the intermediate. Finally, the methoxymethyl group is removed under acidic conditions to yield the final products II-2 and II-23.

[0100] Compounds II-2 and II-23 can effectively inhibit the occurrence and development of psoriasis and alleviate the symptoms of psoriasis. The activity of II-2 and II-23 is stronger than that of tapinarof. II-23 has a certain effect in treating ulcerative colitis. The compounds of the present invention can be used to prepare drugs for treating psoriasis or ulcerative colitis.

[0101] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A stilbene derivative, characterized in that: II-2 and II-23, whose structural formulas are as follows: 。 2. The method for preparing a stilbene derivative according to claim 1, wherein: The following steps are involved: (1) Methyl 3,5-dimethoxybenzoate and 2-bromoisopropane were dissolved in 1,2-dichloroethane, and anhydrous aluminum chloride was added to carry out substitution reaction to obtain intermediate 2; (2) Adding boron tribromide to intermediate 2 to undergo substitution reaction to obtain intermediate 3; (3) Add N,N-diisopropylethylamine and bromomethyl methyl ether to intermediate 3 to undergo substitution reaction to obtain intermediate 4; (4) Add lithium aluminum hydride to intermediate 4 to obtain intermediate 5; (5) Add potassium carbonate and pyridinium chlorochromate to intermediate 5 for oxidation reaction to obtain intermediate 6; (6) Sodium hydride and phosphate are added to intermediate 6 to undergo substitution reaction to obtain intermediate 7 or intermediate 8; (7) Concentrated hydrochloric acid is added to intermediate 7 or intermediate 8 to undergo a substitution reaction to obtain II-2 or II-23.

3. The preparation method according to claim 2, characterized in that In step (1), the molar ratio of 2-bromoisopropane to methyl 3,5-dimethoxybenzoate is (1.1-1.3):1; the molar ratio of anhydrous aluminum chloride to methyl 3,5-dimethoxybenzoate is (1.05-1.2):1; and the substitution reaction is carried out at 85-95°C for 5-8 hours.

4. The preparation method according to claim 2, characterized in that In step (2), the molar ratio of boron tribromide to intermediate 2 is (4.0-7.0):1; the reaction conditions of the substitution reaction are: reaction in an inert gas at 25-35°C for 10-15 hours.

5. The preparation method according to claim 2, characterized in that In step (3), the molar ratio of N,N-diisopropylethylamine, bromomethyl methyl ether and intermediate 3 is (10-15): (10-15): 1; the conditions of the substitution reaction are: 25 ~ 35 ° C for 10 ~ 15 hours.

6. The preparation method according to claim 2, characterized in that In step (4), the molar ratio of the intermediate 4 to lithium aluminum hydride is (1-3):1; the reaction conditions are: reaction in an inert gas at 0°C for 30 minutes.

7. The preparation method according to claim 2, characterized in that In step (5), the molar ratio of potassium carbonate, pyridinium chlorochromate and intermediate 5 is (0.5-0.7): (2-2.5): 1; the oxidation reaction is carried out at 25-35°C for 2-4 hours.

8. The preparation method according to claim 2, characterized in that In step (6), the molar ratio of sodium hydride, phosphate ester and intermediate 6 is (1.2-1.5):(1.2-1.5):1; the conditions of the substitution reaction are: reaction at 25-35°C for 6-10 hours; the phosphate ester is (4-methoxybenzyl) diethyl phosphonate or (4-methylbenzyl) diethyl phosphonate.

9. The preparation method according to claim 2, characterized in that In step (7), the molar volume ratio of concentrated hydrochloric acid to intermediate 7 or intermediate 8 is 1 mL: (1-1.2) mol; the conditions of the substitution reaction are: reaction at 25 ~ 35 ° C for 2 ~ 4 h.

10. Use of the stilbene derivative according to claim 1 in the preparation of a medicament for treating psoriasis or ulcerative colitis.

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