Stilbene derivative, and preparation method therefor and use thereof in preparation of drug for treating psoriasis or ulcerative colitis

Abstract

The present invention belongs to the technical field of biomedicine. Disclosed are a stilbene derivative, and a preparation method therefor and the use thereof in the preparation of a drug for treating psoriasis or ulcerative colitis. The structural formula of the derivative is as follows: compounds II-2 and II-23 provided in the present invention target and activate an AHR-related pathway, promote the nuclear translocation of AHR, reduce the secretion of related cytokines at the cellular level, and significantly ameliorate the skin symptoms and PASI scores of mice of an imiquimod-induced mouse psoriasis model. Compared with a lead compound benvitimod, compounds II-2 and II-23 exhibit a better anti-psoriasis activity and less toxicity. Moreover, it is found that compound II-23 can effectively treat ulcerative colitis in mice, reduce inflammatory responses, and ameliorate the symptoms of colitis. The compounds of the present invention can be used in the preparation of a drug for treating psoriasis and ulcerative colitis.

Description

A stilbene derivative, its preparation method, and its application in the preparation of drugs for treating psoriasis or ulcerative colitis. Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a stilbene derivative, its preparation method, and its application in the preparation of medicaments for treating psoriasis or ulcerative colitis. Background Technology

[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 recurrence. It commonly appears on the scalp and joints of the limbs, and in severe cases can spread throughout the body. This stubborn disease is often accompanied by complications such as hypertension, diabetes, and cardiovascular disease. Severe psoriasis not only affects the patient's appearance and social activities but also severely impacts their quality of life, causing immense psychological and physical harm, and improper treatment can seriously endanger their life. It can be triggered by various factors and has a relatively high incidence and disability rate. Currently, treatment options for mild psoriasis include topical corticosteroids, vitamin D derivatives, calcineurin inhibitors, keratolytic agents, and targeted phototherapy. For severe psoriasis, four classes of biological agents are used for treatment: TNF inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors, and IL-23 inhibitors. Traditional therapies are mostly expensive and have many side effects; there is an urgent need to develop safe, effective, inexpensive, and readily available small molecule compounds.

[0003] Inflammatory bowel disease (IBD) is a group of diseases including ulcerative colitis (UC) and Crohn's disease (CD). It is characterized by chronic intestinal inflammation, often leading to mucosal ulceration and progressive loss of bowel function. Currently, due to the complex etiology and pathophysiology of IBD, involving multiple factors such as genetics, environment, epithelium, microbiota, and immunity, there is no effective treatment.

[0004] Styrene compounds are skeletal structures with ethylene double bonds linking benzene rings. Current literature reports that styrene skeletons have strong biomedical application value. In 2023, the FDA approved VTAMA (tapinarof, 1%) cream for the topical treatment of adult plaque psoriasis. This is the first steroid-free topical drug and also the first new topical molecular entity drug approved in the United States for the treatment of psoriasis in 25 years. Summary of the Invention

[0005] The purpose of this invention is to provide a stilbene derivative, its preparation method, and its application in the preparation of drugs for treating psoriasis or ulcerative colitis, thereby addressing the problems existing in the prior art. This invention provides a compound with a stilbene structural skeleton that exhibits excellent inhibitory activity against inflammatory skin diseases, particularly psoriasis. Compared to marketed drugs, the compound provided by this invention has a faster onset of action, better anti-psoriasis effect, and exhibits lower toxicity.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention is a stilbene derivative, comprising II-2 and II-23, with the following structural formula:

[0008] The second technical solution of the present invention, the method for preparing the stilbene derivative, includes the following steps:

[0009] (1) Methyl 3,5-dimethoxybenzoate and 2-bromoisopropane were dissolved in 1,2-dichloroethane, and anhydrous aluminum trichloride was added to undergo a substitution reaction to obtain intermediate 2.

[0010] (2) Add boron tribromide to intermediate 2 to carry out a substitution reaction to obtain intermediate 3;

[0011] (3) Add N,N-diisopropylethylamine and bromomethyl methyl ether to intermediate 3 and undergo a substitution reaction to obtain intermediate 4;

[0012] (4) Add lithium aluminum hydride to intermediate 4 and react to obtain intermediate 5;

[0013] (5) Add potassium carbonate and pyridine chlorochromate to intermediate 5 and oxidize to obtain intermediate 6;

[0014] (6) Add sodium hydride and phosphate ester to intermediate 6 and undergo a substitution reaction to obtain intermediate 7 or intermediate 8;

[0015] (7) Add concentrated hydrochloric acid to intermediate 7 or intermediate 8 to undergo a substitution reaction to obtain II-2 or II-23.

[0016] The third technical solution of the present invention is the application of the stilbene derivative in the preparation of drugs for treating psoriasis or ulcerative colitis.

[0017] Based on the above technical solution, the present invention has the following technical effects:

[0018] (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.

[0019] (2) The stilbene skeleton derivatives II-2 and II-23 provided by the present invention are obtained by using methyl 3,5-dimethoxybenzoate as the starting material and sequentially through a seven-step reaction including Friedel-Crafts alkylation, demethylation protection, methoxymethyl reprotection, lithium aluminum hydride reduction, PCC oxidation, Horner–Wadsworth–Emmons reaction and reprotection reaction. The final yield of the product is 23%. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] In Figure 1, A and B show that compounds II-2 and II-23 can activate AHR and promote AHR nuclear translocation. C shows that compounds II-2 and II-23 can activate downstream pathways of AHR and promote the expression of CYP1A1 and CYP1B1. II-2 is comparable to the positive control, while II-23 is more effective than the positive control. D shows 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.

[0022] Figure 2 shows: A) PCA analysis confirming group homogeneity; B) the number of differentially expressed genes between different groups; C and D) volcano plots of differentially expressed genes between different groups; E) GO enrichment plot; F) KEGG enrichment plot; and G) a heatmap of differentially expressed genes indicating activation of downstream pathways of AHR.

[0023] Figure 3 shows that imiquimod gradually induced psoriatic symptoms on the skin surface of mice, including redness, swelling, scaling, and thickening, with increasing administration days. Compounds II-2 and II-23 significantly inhibited the development of psoriatic symptoms.

[0024] Figure 4 shows that the body weight of the model group gradually decreased, while compounds II-2 and II-23 maintained the increase in mouse body weight. Based on multi-dimensional scoring results, compounds II-2 and II-23 inhibited the occurrence and development of imiquimod-induced psoriasis symptoms on the back of mice. The model group showed mild psoriasis symptoms from the second day of administration, which gradually worsened. Compounds II-2 and II-23 only showed mild symptoms on the third day and could inhibit the deterioration of symptoms. Compared with the normal group, imiquimod caused changes in the mRNA levels of inflammatory factors in the skin tissue of psoriatic mice. Different concentrations of compounds II-2 and II-23 showed some ameliorative effects on these changes. In this table, A represents weight changes in different groups, B represents PASI scores in different groups, C represents skin thickness in different groups, D represents overall scores in different groups, E represents the effects of 2 and 23 on CD206 mRNA levels, F represents the effects of 2 and 23 on CD36 mRNA levels, G represents the effects of 2 and 23 on IL-18 mRNA levels, and H represents the effects of 2 and 23 on MCP-1 mRNA levels.

[0025] Figure 5 shows that compound II-23 can improve the shortened colon and reduced body weight induced by DSS in mice, and the overall situation of bloody stools in mice is also improved, based on changes in colon length, body weight, and DAI score. HE results show that compound II-23 can improve colonic ulceration induced by DSS, and to some extent inhibit the symptoms of ulcerative colitis in mice, indicating a therapeutic effect on ulcerative colitis. In the figures, A is an image of the colon, B is a record of colon length in different groups, C is HE staining of the colon in different groups, D is a record of body weight in different groups, and E is a DAI score in different groups. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0032] This invention provides a stilbene derivative, including II-2 and II-23, with the following structural formula:

[0033] This invention also provides a method for preparing the stilbene derivative, comprising the following steps:

[0034] (1) Methyl 3,5-dimethoxybenzoate and 2-bromoisopropane were dissolved in 1,2-dichloroethane, and anhydrous aluminum trichloride was added to undergo a substitution reaction to obtain intermediate 2.

[0035] (2) Add boron tribromide to intermediate 2 to carry out a substitution reaction to obtain intermediate 3;

[0036] (3) Add N,N-diisopropylethylamine and bromomethyl methyl ether to intermediate 3 and undergo a substitution reaction to obtain intermediate 4;

[0037] (4) Add lithium aluminum hydride to intermediate 4 and react to obtain intermediate 5;

[0038] (5) Add potassium carbonate and pyridine chlorochromate to intermediate 5 and oxidize to obtain intermediate 6;

[0039] (6) Add sodium hydride and phosphate ester to intermediate 6 and undergo a substitution reaction to obtain intermediate 7 or intermediate 8;

[0040] (7) Add concentrated hydrochloric acid to intermediate 7 or intermediate 8 to undergo a substitution reaction to obtain II-2 or II-23.

[0041] In some specific implementations, 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 trichloride 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.

[0042] In some specific implementations, in step (2), the molar ratio of boron tribromide to intermediate 2 is (4.0~7.0):1; the reaction conditions for the substitution reaction are: reaction in an inert gas at 25~35℃ for 10~15h.

[0043] In some specific implementation schemes, in step (3), the molar ratio of N,N-diisopropylethylamine, bromomethyl methyl ether and intermediate 3 is (10-15):(10-15):1; the conditions for the substitution reaction are: reaction at 25-35°C for 10-15 h.

[0044] In some specific implementation schemes, in step (4), the molar ratio of intermediate 4 to lithium aluminum hydride is (1-3):1; the reaction conditions are: reaction in an inert gas, reaction at 0°C for 30 min.

[0045] In some specific implementations, 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.

[0046] In some specific implementation schemes, 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 substitution reaction conditions are: reaction at 25-35℃ for 6-10 h; the phosphate ester is (4-methoxybenzyl)phosphonate diethyl ester or (4-methylbenzyl)phosphonate diethyl ester.

[0047] In some specific implementation schemes, in step (7), the molar volume ratio of the concentrated hydrochloric acid to intermediate 7 or intermediate 8 is 1 mL:(1-1.2) mol; the conditions for the substitution reaction are: reaction at 25-35℃ for 2-4 h.

[0048] The present invention also provides the use of the stilbene derivative in the preparation of medicaments for treating psoriasis or ulcerative colitis.

[0049] The compounds II-2 and II-23 provided by this invention target and activate the AHR-related pathway, promoting AHR nuclear translocation and reducing related cytokines at the cellular level. They significantly improve skin symptoms and PASI scores in a mouse model of imiquimod-induced psoriasis, exhibiting superior anti-psoriatic activity and lower toxicity compared to the lead compound benvitide. Compound II-23 was found to effectively treat ulcerative colitis in mice, reducing inflammatory responses with lower toxicity, adding an indication not present in the lead compound benvitide. The compounds of this invention can be used to prepare drugs for treating skin diseases such as psoriasis and atopic dermatitis, as well as ulcerative colitis.

[0050] Example 1

[0051] The preparation process of a stilbene derivative is as follows:

[0052] The preparation method includes the following steps:

[0053] 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 trichloride (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 for 6 h. After cooling to room temperature, 30 mL of saturated sodium bicarbonate was added to quench the reaction, resulting in a flocculent precipitate. 30 mL of dilute hydrochloric acid was added until the solution became clear. The mixture was extracted three times with ethyl acetate, and the organic phases were combined. The solution was 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.

[0054] Step 2: Dissolve intermediate 2 (1.0 equivalent) in ultra-dry dichloromethane, purge with argon three times, and add boron tribromide (1 M in DCM, 5 equivalent) dropwise at -78°C. After the addition is complete, gradually raise the temperature to room temperature and react overnight. Then, cool the reaction to -78°C and quench the reaction with anhydrous methanol. Directly mix the sample and separate by column chromatography (PE / EA = 5 / 1) to obtain intermediate 3.

[0055] Step 3: Dissolve intermediate 3 (1.0 equivalent) in ultra-dry dichloromethane, purge three times with argon, add N,N-diisopropylethylamine (15 equivalent) at 0°C, and slowly add bromomethyl methyl ether (15 equivalent). After reacting for 1 hour, slowly raise the temperature to room temperature and continue reacting for 10 hours. After the reaction is complete as monitored by TLC, wash twice with saturated sodium bicarbonate, twice with saturated sodium chloride, dry with anhydrous sodium sulfate, evaporate to dryness, and separate by column chromatography (PE / EA = 20 / 1) to obtain intermediate 4.

[0056] Step 4: Dissolve intermediate 4 (1.0 equivalent) in anhydrous diethyl ether, purge three times with argon, and add lithium aluminum hydride (2.5 M in THF, 0.5 equivalent) dropwise at 0°C. React at this temperature for 30 minutes. Then add 1.0 equivalent of water, 1.0 equivalent of 15% sodium hydroxide solution, and 3.0 equivalent of water sequentially. Transfer to room temperature and continue stirring for 15 minutes. Filter with diatomaceous earth. Wash the filtrate twice with saturated sodium chloride, dry with anhydrous sodium sulfate, and filter to obtain intermediate 5.

[0057] Step 5: Dissolve intermediate 5 (1.0 equivalent) in ultra-dry dichloromethane, add potassium carbonate (0.5 equivalent) and pyridinium chlorochromate (2.2 equivalent) sequentially at 0°C, react at room temperature for 2.5 hours, quench the reaction with diethyl ether, filter with diatomaceous earth, evaporate to dryness and separate by column chromatography (PE / EA = 20 / 1) to obtain intermediate 6.

[0058] Step Six: Dissolve diethyl (4-methylbenzyl)phosphonate (1.2 equivalents) in tetrahydrofuran, purging three times with argon. 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 with water at 0°C, extract three times with ethyl acetate, combine the organic phases and wash three times with saturated sodium chloride, dry with anhydrous sodium sulfate, evaporate to dryness, and separate by column chromatography (PE / EA = 20 / 1) to obtain intermediate 7.

[0059] 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. After rotary evaporation, separate by column chromatography (PE / EA = 10 / 1) to obtain compound II-2.

[0060] II-2: 1 H NMR(600MHz,Chloroform-d)δ7.36(d,J=8.0Hz,2H),7.15(d,J=7.9Hz,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).

[0061] Example 2

[0062] The preparation process of a stilbene derivative is as follows:

[0063] Steps one through five are the same as in Example 1.

[0064] Step Six: Dissolve diethyl (4-methoxybenzyl)phosphonate (1.2 equivalents) in tetrahydrofuran, purging three times with argon. 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), transfer to room temperature and react for 2 hours. Quench the reaction with water at 0°C, extract three times with ethyl acetate, combine the organic phases and wash three times with saturated sodium chloride, dry with anhydrous sodium sulfate, evaporate to dryness and separate by column chromatography (PE / EA = 20 / 1) to obtain intermediate 8.

[0065] 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. After rotary evaporation, separate by column chromatography (PE / EA = 10 / 1) to obtain compound II-23.

[0066] II-23: 1 H NMR (400MHz, CDCl3) δ7.39(d,J=8.6Hz,2H),6.91(d,J=16.2Hz,1H),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).

[0067] Example 3

[0068] The AhR agonist activity of compounds II-2, II-23, and tapinarof was evaluated by measuring the transcriptional level of AHR in cells before and after drug administration using HaCaT cells transfected with knockout lentivirus. The results are shown in Table 1.

[0069] Table 1 AhR agonist activity

[0070] As can be seen from the data in Table 1, the EC50 of II-2 and II-23 provided by this invention is significantly lower than that of the positive control drug, indicating that they have stronger AhR activating activity.

[0071] Example 4

[0072] 1. Place appropriately sized sterile cell smears into six-well cells culture plates (1 smear / well). Collect HaCaT cells in good growth condition and dilute them to a density of 1×10⁻⁶. 6Cell suspension was prepared at 1 cell / mL and seeded into 6-well plates. After culturing for 24 hours, the cells were given a cell compound and the supernatant was removed after 24 hours. The culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 20 minutes and washed three times with PBS. Circles were drawn with a histochemical pen to prevent the incubation medium from flowing away in subsequent steps, and the cells were washed with PBS. The cells were blocked with 5% BSA (Biofroxx, 4240GR250) for 2 hours, and then an appropriate amount of primary antibody (AHR, 1:1000, Cell Signaling Technology, USA) working solution was added and incubated overnight at 4°C. The cells were warmed and washed three times with PBS for 5 minutes each time. The corresponding species of secondary antibody (FITC-labeled anti-rabbit) working solution was added to the circle, and the cells were incubated in a 37°C water bath in the dark for 40 minutes, and washed three times with PBS for 5 minutes each time. The nuclei were stained with DAPI (Sigma, D8417-1MG) and incubated at room temperature in the dark for 20-30 minutes, and then washed with PBS. Mount the slides with anti-fluorescence quenching mounting medium (Sigma, V900155-25G), and then store the slides in a dark chamber at 4°C. Observe and photograph under a microscope (OLYMPUS, IX51).

[0073] Experimental results showed that after drug administration, the green fluorescence of AhR migrated into the nucleus, indicating that the compound activated AhR to some extent and promoted its entry into the nucleus.

[0074] 2qRT-PCR method for detecting changes in the levels of inflammatory factor mRNA in cells and skin tissues

[0075] (1) RNA extraction from cells: According to the experimental groups, HaCaT cells were evenly seeded in six-well plates and cultured. Cells from different groups were collected into 1.5 mL centrifuge tubes. The cells were gently washed 2-3 times with pre-cooled PBS, and 1 mL of Trizol was added for 5 min. Then, 0.2 mL of chloroform was added, and the mixture was gently inverted to mix. The mixture was allowed to stand at room temperature for 10 min, and then centrifuged at 10000g for 15 min at 4℃. 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 the RNA. The mixture was gently mixed, and the mixture was allowed to stand at room temperature for 5 min, and then centrifuged for 10 min (at the same speed as before). The bottom RNA precipitate was retained, and 1 mL of 75% ethanol (prepared with DEPC water) was added to wash away the residual isopropanol solution. After centrifugation, the ethanol solution was discarded, and the RNA was dried at room temperature or under vacuum for 10 min. The precipitate (RNA) was dissolved in DEPC water (50 μL / well). The RNA concentration was measured and stored at -80℃.

[0076] (2) Animal RNA extraction: Skin lesion cells (HaCaT) were collected into 1.5 mL centrifuge tubes, 1 mL of Trizol and a grinding bead were added, and the tubes were homogenized in a homogenizer. After homogenization, 0.2 mL of chloroform was added, and the mixture was gently inverted to mix. The mixture was allowed to stand at room temperature for 10 min, then centrifuged at 10000g for 15 min at 4℃. The upper aqueous phase was carefully transferred to another centrifuge tube, and an equal volume of isopropanol was added to precipitate the RNA. The mixture was gently mixed, allowed to stand at room temperature for 5 min, and then centrifuged for 10 min (at the same speed as before). The bottom RNA precipitate was retained, and 1 mL of 75% ethanol (prepared with DEPC water) was added to wash away the residual isopropanol solution. After centrifugation, the ethanol solution was discarded, and the RNA was dried at room temperature or under vacuum for 10 min. The precipitate (RNA) was dissolved in DEPC water (50 μL / well). The RNA concentration was determined, and the precipitate was stored at -80℃.

[0077] (3) Reverse transcription: Take an enzyme-free EP tube in a clean bench, add 2 μg RNA, 4 μL reverse transcribe 5×Mix, then add DEPC water to make up the total volume to 20 μL, react in a metal bath at 25℃ for 5 min, at 42℃ for 40 min, and at 75℃ for 5 min.

[0078] (4) PCR amplification: A 10 μL system, including 2 μL sample DNA, 2 μL DEPC water, 1 μL corresponding primers, and 5 μL qPCR Mix, was used for quantitative RT-PCR (qRT-PCR). The PCR instrument was ABI QuantStudio 5 (Thermo Fisher Scientific, USA), and the temperature gradient was set to 95℃ (5 min) → 95℃ (10 s) → 55-60℃ (20 s) → 72℃ (30 s). The cDNA amplification cycle was 40 times.

[0079] 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, while compound 23 showed superior inhibitory activity against CCL20, S100A9, and TNF-α compared to 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 skin lesions, showing 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.

[0080] 3. Drug administration methods for animal models of psoriasis

[0081] Eight-week-old male Balb / C mice were acclimatized in an SPF-grade barrier environment for one week. The hair on the midline of their backs was removed using a mouse shaver, and they were 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%).

[0082] 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. Simultaneously, mice in the positive drug group received tapinarof (1%), mice in the II-2 group received II-2 (1%), and mice in the II-23 group received II-23 (1%). When the model group mice reached the criteria of weight loss, psoriasis, 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 received no treatment. The dynamic changes in skin lesions on the backs of mice were observed and recorded before daily administration.

[0083] PASI score:

[0084] The scoring was based on the severity of psoriasis in mice, specifically the degree of scaling, erythema, and thickening of the skin lesions on the back. (0 points: no skin lesions; 1 point: mild skin lesions; 2 points: moderate skin lesions; 3 points: severe skin lesions; 4 points: extremely severe skin lesions). Three mice were assigned to each group, and the scores were recorded once a day before administration.

[0085] The results showed that compounds II-2 and II-23 inhibited the onset and exacerbation of IMQ-induced psoriasis symptoms on the back of mice, mainly manifested as a reduction in the severity of erythema and scaling, especially compound II-23, which did not show these symptoms until the 5th day. Compound II-23 also showed very low toxicity, with no change in body weight.

[0086] 4. Administration methods for animal modeling of ulcerative colitis

[0087] Eight-week-old male Balb / C mice were acclimatized in an SPF-grade barrier environment for one week, followed by free access to 2.8% sodium dextran sulfate (DSS) solution for one week. After three days of free access, the compound groups were administered either 20 mg / kg II-23 or 20 mg / kg Tapinarof via gavage for seven consecutive days. Mouse weight was recorded, and fecal hemorrhage was observed. The control group received free access to distilled water throughout the treatment and was administered sodium carboxymethyl cellulose via gavage. The DSS group received free access to 2.8% DSS for the first seven days and was administered sodium carboxymethyl cellulose via gavage. The compound 23 group received free access to 2.8% DSS for the first seven days and was administered compound II-23 (prepared with sodium carboxymethyl cellulose) at 20 mg / kg via gavage. The compound Tap group received free access to 2.8% DSS for the first seven days and was administered compound Tapinarof (prepared with sodium carboxymethyl cellulose) at 20 mg / kg via gavage.

[0088] HE staining method for detecting pathological changes in colon tissue

[0089] A small portion of mouse colon tissue was fixed in 4% paraformaldehyde. Paraffin-embedded sections were prepared using the following procedure: First, the sections were dehydrated from low to high concentrations by soaking them in xylene for 20 minutes, repeating this process once, then discarding the xylene. Next, anhydrous ethanol was added for 5 minutes, and this process was repeated once. Then, 75% ethanol was added in a gradient dewaxing process for 5 minutes, followed by washing with water. Hematoxylin staining was then performed: the hydrated sections were stained with hematoxylin for nuclear staining for several minutes to over ten minutes.

[0090] Next, excess dye is removed by acid washing, such as with a 1% hydrochloric acid solution, and differentiation is performed. Finally, an alkaline solution is used for bluing to make the cell nuclei appear blue. Then, eosin staining is performed: after hematoxylin staining, the cytoplasm is stained with eosin dye. Paraffin sections are dehydrated with a gradient of alcohols (85% ethanol → 95% ethanol) and then immersed in eosin staining solution for a few 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 a few minutes and repeated once. After allowing the sections to air dry, the tissue on the sections is photographed using a fluorescence microscope. In the images, the cell nuclei are stained blue, and the cytoplasm appears red.

[0091] The results showed that the colon length in the compound group was significantly increased compared to the DSS group, and the body weight recovered somewhat. HE staining revealed significant ulceration in the DSS group, while the compound group alleviated this ulceration, indicating that it treated ulcerative colitis to some extent. The Tapinarof group did not show significant improvement in body weight or colon length compared to the model group, and numerous ulcers were still present in HE staining.

[0092] In summary, this invention uses methyl 3,5-dihydroxymethylbenzoate as the starting material, introduces a group at the C4 position through Friedel-Crafts alkylation or Suzuki reaction, followed by demethylation and protection of the hydroxyl group with methoxymethyl groups, and then reduces and oxidizes the ester group to obtain the key intermediate aldehyde. The aldehyde reacts with diethyl phosphonate via the Horner–Wadsworth–Emmons reaction to obtain an intermediate, and finally, under acidic conditions, the methoxymethyl protection is removed to obtain the final products II-2 and II-23.

[0093] Compounds II-2 and II-23 can effectively inhibit the occurrence and development of psoriasis and alleviate its symptoms. II-2 and II-23 are more potent than tapinarof. II-23 has a certain therapeutic effect on ulcerative colitis. The compounds of this invention can be used to prepare drugs for treating psoriasis or ulcerative colitis.

[0094] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A stilbene derivative, characterized in that, Including II-2 and II-23, their structural formulas are as follows:

2. The method for preparing the stilbene derivative as described in claim 1, characterized in that, Includes the following steps: (1) Methyl 3,5-dimethoxybenzoate and 2-bromoisopropane were dissolved in 1,2-dichloroethane, and anhydrous aluminum trichloride was added to undergo a substitution reaction to obtain intermediate 2. (2) Add boron tribromide to intermediate 2 to carry out a substitution reaction to obtain intermediate 3; (3) Add N,N-diisopropylethylamine and bromomethyl methyl ether to intermediate 3 and undergo a substitution reaction to obtain intermediate 4; (4) Add lithium aluminum hydride to intermediate 4 and react to obtain intermediate 5; (5) Add potassium carbonate and pyridine chlorochromate to intermediate 5 and oxidize to obtain intermediate 6; (6) Add sodium hydride and phosphate ester to intermediate 6 and undergo a substitution reaction to obtain intermediate 7 or intermediate 8; (7) Add concentrated hydrochloric acid 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 trichloride 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 for the substitution reaction are: reaction in an inert gas atmosphere, reaction at 25~35℃ for 10~15h.

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 for the substitution reaction are: 25-35℃ for 10-15 h.

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

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 for the substitution reaction are: reaction at 25-35℃ for 6-10 h; the phosphate ester is (4-methoxybenzyl)phosphonate diethyl ester or (4-methylbenzyl)phosphonate diethyl ester.

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

10. The use of the stilbene derivative as described in claim 1 in the preparation of a medicament for treating psoriasis or ulcerative colitis.

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