Stilbene derivative, preparation method thereof and application of stilbene derivative in preparation of medicine for treating psoriasis or ulcerative colitis

Abstract

The invention discloses a stilbene derivative, a preparation method thereof and application of the stilbene derivative in preparation of drugs for treating psoriasis or ulcerative colitis, and belongs to the technical field of biological medicines. The compounds II-2 and II-23 provided by the invention are used for targeted excitation of AHR related pathways, promotion of AHR nucleation, reduction of secretion of related cytokines at a cellular level, and substantial improvement of mouse skin symptoms and PASI scores of an imiquimod-induced mouse psoriasis model, compared with a lead compound benvimod, the compounds II-2 and II-23 have better psoriasis resistance activity and lower toxicity, and can be used for preparation of drugs for treating psoriasis. Meanwhile, the compound II-23 is found to be capable of effectively treating mouse ulcerative colitis, reducing inflammatory response and improving colitis symptoms. The compound can be used for preparing medicines for treating 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 and 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 on the skin and plaques with silver scales. It is not easy to cure and is prone to recurrence. It is common on the patient's scalp and limb joints, and can be widespread on the skin of the whole body in severe cases. This disease is stubborn and difficult to treat, and is accompanied by multiple complications such as hypertension, diabetes, and cardiovascular disease. Severe psoriasis not only affects the patient's skin appearance and social activities, but also seriously affects the patient's quality of life, causing great harm to the patient's body and mind, and improper treatment can seriously endanger the patient's life. It can be induced by a variety of factors, and the incidence and disability rates are relatively high. At present, for patients with mild psoriasis, treatment options include topical corticosteroids, vitamin D derivatives, calcineurin inhibitors, keratolytic agents and targeted phototherapy. For patients with severe psoriasis, the four types of biological agents used for treatment are TNF inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors and IL-23 inhibitors. Most traditional therapies are expensive and have many side effects. There is an urgent need to develop safe, effective, cheap and easily available small molecule compounds.

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

[0004] Styrene compounds are skeletons of benzene ring structures connected by ethylene double bonds. 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 plaque psoriasis in adults. This is the first steroid-free topical drug 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 purpose of the present invention is to provide a stilbene derivative and a preparation method thereof and use thereof in the preparation of a drug for treating psoriasis or ulcerative colitis, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a compound having a styrene structural skeleton, which exhibits good inhibitory activity against inflammatory skin diseases, especially psoriasis, etc. Compared with the listed drugs, the compound provided by the invention has a faster onset of action, better anti-psoriasis effect, 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, and its 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) dissolving methyl 3,5-dimethoxybenzoate and 2-bromoisopropane in 1,2-dichloroethane, adding anhydrous aluminum chloride, and performing 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) adding 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 to obtain intermediate 5;

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

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

[0016] (7) Concentrated hydrochloric acid is added to intermediate 7 or intermediate 8 to carry out 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 obtained by a seven-step reaction including a Friedel-Crafts alkylation reaction, a demethylation protection, a methoxymethyl re-protection, a lithium aluminum hydride reduction, a PCC oxidation, and then a Horner–Wadsworth–Emmons reaction and a re-deprotection reaction, with a final yield of 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0022] Figure 1 A and B are compounds II-2 and II-23, which can activate AHR and promote the nuclear entry of AHR. C is compounds II-2 and II-23, which can activate the downstream pathway of AHR and promote the expression of CYP1A1 and CYP1B1. II-2 is equivalent to positive drugs, and II-23 is better than positive drugs. D is compounds II-2 and II-23, which can reduce the expression of CCL5, CCL20, IL-6, IL-8, S100A9, TLR4, TNF-α and TNFR1 in HaCaT cells.

[0023] Figure 2 A is PCA analysis that proves the homogeneity of the groups. B is the number of differentially expressed genes between different groups. C and D are volcano plots of differentially expressed genes between different groups. E is a GO enrichment map. F is a KEGG enrichment map. G is a differentially expressed gene heat map. The downstream pathway of AHR is activated.

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

[0025] Figure 4From the changes in body weight, it can be seen that the body weight of the model group gradually decreased, and compounds II-2 and II-23 could maintain the increase in the weight of mice; from the scoring results of multiple dimensions, compounds II-2 and II-23 could inhibit the occurrence and development of psoriasis symptoms on the back of mice caused by imiquimod. The model group had mild psoriasis symptoms from the second day of administration, and gradually worsened. Compounds II-2 and II-23 began to show mild symptoms on the third day and could inhibit the deterioration of symptoms. Compared with the normal group, imiquimod can cause changes in the mRNA levels of inflammatory factors in the skin tissue of psoriasis mice. Different concentrations of compounds II-2 and II-23 have certain improvements. 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 From the length of the mouse colon, the degree of weight change and the DAI score, it can be seen that compound II-23 can improve the shortening of the mouse colon and the weight loss caused by DSS, and the overall situation of blood in the mouse stool has also improved. HE results show that compound II-23 can improve the colon ulcer caused by DSS, inhibit the symptoms of ulcerative colitis in mice to a certain extent, and has the effect of treating ulcerative colitis. Among them, A is the apparent picture of the colon, B is the record of the colon length of different groups, C is the colon HE staining of different groups, D is the record of the weight 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

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

[0033] The embodiment of 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) dissolving methyl 3,5-dimethoxybenzoate and 2-bromoisopropane in 1,2-dichloroethane, adding anhydrous aluminum chloride, and performing 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) adding 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 to obtain intermediate 5;

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

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

[0042] (7) Concentrated hydrochloric acid is added to intermediate 7 or intermediate 8 to carry out 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 5-8h.

[0044] In some specific embodiments, 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: reacting in an inert gas at 25-35°C for 10-15h.

[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 10-15h.

[0046] In some specific embodiments, in step (4), the molar ratio of the intermediate 4 to lithium aluminum hydride is (1-3):1; the reaction conditions are: reacting 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; the conditions of the oxidation reaction are: react 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: reacting at 25-35° C. for 2-4 h.

[0050] The embodiment of the present invention also provides the 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 less toxic. It was found that compound II-23 can effectively treat ulcerative colitis in mice, reduce inflammatory responses, and has less 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 treating skin diseases such as psoriasis and atopic dermatitis and 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 (2mmol, 1.0 equivalent) was dissolved in 10ml 1,2-dichloroethane, 2-bromopropane (2.4mmol, 1.2 equivalent) and aluminum chloride (2.4mmol, 1.2 equivalent) were added in sequence at room temperature, and the mixture was transferred to a 90°C oil bath and heated under reflux. After reacting for 6h, the mixture was cooled to room temperature, and 30ml of saturated sodium bicarbonate was added to quench the reaction. A flocculent precipitate was generated, and 30ml of dilute hydrochloric acid was added to clarify the solution. The mixture was extracted with ethyl acetate three times, the organic phases were combined, washed with saturated sodium chloride three times, dried over anhydrous sodium sulfate, and separated by column chromatography (PE:EA=200:1) after spin drying to obtain intermediate 2.

[0057] Step 2: Dissolve intermediate 2 (1.0 equivalent) in ultra-dry dichloromethane. After argon replacement three times, add boron tribromide (1M in DCM, 5 equivalents) dropwise at -78°C. After the addition is complete, gradually warm the temperature to room temperature. After reacting overnight, cool the reaction to -78°C, add anhydrous methanol to quench the reaction, and directly mix the sample for column chromatography separation (PE / EA=5 / 1) to obtain intermediate 3.

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

[0059] Step 4: Dissolve the intermediate 4 (1.0 equivalent) in anhydrous ether, replace the argon three times, add lithium aluminum tetrahydride (2.5M in THF, 0.5 equivalent) dropwise at 0°C, and react at this temperature for 30 minutes. Add 1.0 equivalent of water, 1.0 equivalent of 15% sodium hydroxide solution, and 3.0 equivalent of water in sequence, transfer to room temperature and continue stirring for 15 minutes, then filter through diatomaceous earth, wash the filtrate twice with saturated sodium chloride, dry over anhydrous sodium sulfate, filter and spin dry to obtain the intermediate 5.

[0060] Step 5: Dissolve intermediate 5 (1.0 equivalent) in ultra-dry dichloromethane, add potassium carbonate (0.5 equivalent) and pyridinium chlorochromate (2.2 equivalent) in sequence at 0°C, react at room temperature for 2.5 hours, add ether to quench the reaction, filter with celite, spin dry and separate by column chromatography (PE / EA=20 / 1) to obtain intermediate 6.

[0061] Step 6: Dissolve (4-methylbenzyl) diethyl phosphonate (1.2 equivalents) in tetrahydrofuran and replace 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. Add water at 0°C to quench the reaction, extract three times with ethyl acetate, combine the organic phases and 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 the intermediate 7 in 10 ml of anhydrous methanol, add 1 ml of concentrated hydrochloric acid, 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 equivalents) in tetrahydrofuran and replace 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. Add water at 0°C to quench the reaction, extract three times with ethyl acetate, combine the organic phases and 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 the intermediate 8 in 10 ml of anhydrous methanol, add 1 ml of concentrated hydrochloric acid, 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 transcription level of AHR in 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] It can be seen from the data in Table 1 that the EC50 of II-2 and II-23 provided by the present invention is significantly lower than that of the positive drug, indicating that their activity in stimulating AhR is stronger.

[0078] Example 4

[0079] 1 Place a sterile cell slide of appropriate size in a six-well cell culture plate (1 slide / well), collect and dilute the HaCaT cells with good growth status to a density of 1×10 6Cell suspension of 1000 cells / mL was then inoculated into a six-well plate and placed in an incubator for 24 hours. After 24 hours of cell culture, the supernatant was removed after the cell compound was given. The culture medium was poured off and washed three times with PBS. 4% paraformaldehyde was fixed for 20 minutes and washed three times with PBS. A circle was drawn with a histochemical pen to prevent the incubation solution from flowing away in the following process, and washed with PBS. After blocking with 5% BSA (Biofroxx, 4240GR250) for 2 hours, an appropriate amount of primary antibody (AHR, 1:1000, CellSignaling Technology, USA) working solution was added dropwise and incubated at 4°C overnight. Rewarmed, washed with PBS three times, 5 minutes each time. The corresponding species of secondary antibody (FITC-labeled anti-rabbit) working solution was added dropwise in the circle, incubated in a 37°C water bath in the dark for 40 minutes, and washed with PBS three times, 5 minutes each time. DAPI (Sigma, D8417-1MG) was added dropwise to stain the nucleus, incubated in the dark at room temperature for 20 to 30 minutes, and washed with PBS. The slides were sealed with anti-fluorescence quenching sealing agent (Sigma, V900155-25G), and then 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 detection of changes in mRNA levels of inflammatory factors 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 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 minutes. 0.2 mL of chloroform was added, and the mixture was gently inverted and mixed. The mixture was allowed to stand at room temperature for 10 minutes, and centrifuged at 10000 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 mixture was allowed to stand at room temperature for 5 minutes, and centrifuged for 10 minutes (same speed as before). The RNA precipitate at the bottom was retained, and 1 mL of ethanol with a volume fraction of 75% prepared by 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 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 Trizol and grinding beads and place in a homogenizer for homogenization. After homogenization, add 0.2 mL of chloroform and gently mix by inverting. Let stand at room temperature for 10 min, centrifuge at 4°C and 10,000 g for 15 min, carefully transfer the upper aqueous phase to another centrifuge tube, add isopropanol of the same volume as the upper aqueous phase to precipitate RNA, gently mix, let stand at room temperature for 5 min, centrifuge for 10 min (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, pour off the ethanol solution after centrifugation, dry the RNA at room temperature or under vacuum for 10 min, 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, 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: 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 ABI QuantStudio 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 taking 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 show that compounds 2 and 23 can block inflammatory factors. Compared with IMQ treatment, compound 2 can reduce the levels of CD206 and MCP-1 in animal lesional tissues and show 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 feeding in an SPF barrier environment, 8-week-old Balb / C male mice were shaved of the midline hair on their backs with a mouse shaver and randomly divided into 5 groups: blank control group (Con), imiquimod model group (IMQ), positive drug group (tapinarof), II-2 group (1%), and II-23 group (1%).

[0089] The mice in the imiquimod model group, the positive drug group, the II-2 group, and the II-23 group were treated with 62.5 mg imiquimod for 7 days. At the same time, the mice in the positive drug group were given tapinarof (1%), the II-2 group was given II-2 (1%), and the II-23 group was given II-23 (1%) for skin application. When the mice in the model group reached the standard of weight loss, psoriasis on the skin, and skin lesion hyperplasia, the PASI (psoriasis area and severity index) score continued to increase, and the psoriasis-like mouse model was successfully established. The blank control group was not treated in any way. 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, i.e., the degree of scaling, erythema, and thickening of the back lesions (0 points: no lesions; 1 point: mild lesions; 2 points: moderate lesions; 3 points: severe lesions; 4 points: extremely severe lesions). Each group had three mice, and the score was recorded once a day before drug administration.

[0092] The results showed that compounds II-2 and II-23 inhibited the onset and deterioration of IMQ-induced psoriasis symptoms on the back of mice, which was mainly manifested by the reduction of the severity of erythema and skin scaling, especially compound 23, which did not show such symptoms until the 5th day. Compound 23 also showed little toxicity, with no change in body weight.

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

[0094] After one week of adaptive feeding in an SPF barrier environment, 8-week-old Balb / C male mice were given 2.8% dextran sulfate sodium salt (DSS) aqueous solution for one week of free drinking water. After 3 days of free drinking water, the compound group was given 20 mg / kg Ⅱ-23 or 20 mg / kg Tapinarof for 7 consecutive days of oral administration. The weight of the mice was recorded and the blood in the stool of the mice was observed. The blank group was given free drinking water and sodium carboxymethyl cellulose by oral administration during the whole process; the DSS group was given 2.8% DSS in free drinking water and sodium carboxymethyl cellulose by oral administration for the first seven days; the compound 23 group was given 2.8% DSS in free drinking water and compound 20 mg / kg Ⅱ-23 prepared with sodium carboxymethyl cellulose by oral administration for the first seven days; the compound Tap group was given 2.8% DSS in free drinking water and compound 20 mg / kg Tapinarof prepared with sodium carboxymethyl cellulose by oral administration 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. The sections were embedded in paraffin and the specific operation was as follows: first, the sections were dehydrated from low concentration to high concentration, that is, the sections were soaked in xylene for 20 minutes, and then the xylene was poured out and soaked in anhydrous ethanol for 5 minutes. After repeating the process, 75% alcohol gradient dewaxing was performed for 5 minutes, and then water was added to wash. Then hematoxylin staining was performed: the hydrated sections were stained with hematoxylin dye for nuclei for a few minutes to more than ten minutes.

[0097] Then, use acid washing, such as 1% hydrochloric acid solution, to remove excess dye and perform differentiation operation, and finally use alkaline solution to return blue treatment to make the cell nucleus appear blue. Then perform eosin staining: After the hematoxylin staining, use eosin dye to stain the cytoplasm part, dehydrate the paraffin sections with gradient alcohol (85% ethanol → 95% ethanol), and put them in eosin dye solution for staining for about a few minutes. Finally, dehydrate and seal the sections for observation: Soak the paraffin sections in anhydrous ethanol for several minutes and then discard them, then soak them in a transparent agent (usually xylene) for a few minutes and repeat this once, let the sections dry naturally, and take pictures of the tissue on the sections with a fluorescence microscope. In the picture, the cell nucleus is dyed blue and the cytoplasm is red.

[0098] The results showed that the colon length of the compound group was significantly longer than that of the DSS group, and the body weight was restored. HE results showed that the DSS group had obvious ulcers, and the compound group alleviated the ulcers, which treated ulcerative colitis to a certain extent. The body weight and colon length of the Tapinarof group did not show significant improvement compared with the model group, and a large number of ulcers were still found in HE staining.

[0099] In summary, the present invention uses 3,5-dihydroxymethylbenzoic acid methyl ester as a starting material, introduces a group at the carbon four position through Friedel-Crafts alkylation reaction or Suzuki reaction, then demethylates and uses methoxymethyl to protect the hydroxyl group, and then reduces and oxidizes the ester group to obtain the key intermediate aldehyde. The aldehyde and diethyl phosphonate are reacted by Horner–Wadsworth–Emmons reaction to obtain an intermediate, and finally the methoxymethyl protection is removed under acidic conditions to obtain 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 activities of II-2 and II-23 are stronger than 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 clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A stilbene derivative, characterized in that: Including II-2 and II-23, the structural formula is as follows:

2. The method for preparing a stilbene derivative according to claim 1, characterized in that: The following steps are involved: (1) dissolving methyl 3,5-dimethoxybenzoate and 2-bromoisopropane in 1,2-dichloroethane, adding anhydrous aluminum chloride, and performing substitution reaction to obtain intermediate 2; (2) adding boron tribromide to intermediate 2 to carry out a substitution reaction to obtain intermediate 3; (3) adding N,N-diisopropylethylamine and bromomethyl methyl ether to intermediate 3 to carry out substitution reaction to obtain intermediate 4; (4) adding lithium aluminum hydride to intermediate 4 to react to obtain intermediate 5; (5) adding potassium carbonate and pyridinium chlorochromate to intermediate 5 to undergo oxidation reaction to obtain intermediate 6; (6) adding sodium hydride and phosphate to intermediate 6 to carry out substitution reaction to obtain intermediate 7 or intermediate 8; (7) Concentrated hydrochloric acid is added to intermediate 7 or intermediate 8 to carry out 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: reacting 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 10-15h.

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: reacting 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 conditions of the oxidation reaction are: reacting at 25-35°C for 2-4h.

8. The preparation method according to claim 2, characterized in that: 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.

9. The preparation method according to claim 2, characterized in that: 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: reacting 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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