Double-target inhibitor with anti-psoriasis activity and application thereof

Abstract

The invention discloses a double-target inhibitor with anti-psoriasis activity, and the structural general formula of the double-target inhibitor is shown in the specification. The double-target inhibitor with anti-psoriasis activity provided by the invention has good efficacy on in-vitro anti-psoriasis related tests and in-vivo imiquimod induced mouse models; the compound B5e shows better anti-psoriasis activity than their parent drugs and combined administration of the parent drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine preparation, and in particular relates to a dual-target inhibitor with anti-psoriasis activity and application thereof. BACKGROUND

[0002] Psoriasis is a chronic skin disease mediated by immunity, and its main features are excessive proliferation of keratinocytes, dilation of blood vessels in the dermis, and inflammatory infiltration of leukocytes. Among them, the most common type is psoriasis vulgaris, which accounts for 90% of all cases. Common symptoms include skin itching, red patches, dryness, scales, and even rupture and bleeding. The cause of psoriasis is still unclear, and it is currently believed to be the result of the interaction of external pathogen infection, genetic factors, and immune effects.

[0003] Psoriasis directly affects the physical health and quality of life of patients, leading to changes in social life and psychological state. Choosing the right treatment method is crucial to improving the quality of life of psoriasis patients. There are more and more studies on the use of drugs for routine treatment, aiming to reduce epidermal hyperplasia and inflammatory symptoms. Traditional topical drugs for treating psoriasis, such as corticosteroids, calcineurin inhibitors, vitamin D analogues, anthralins, and tar, are often limited in their application due to their poor efficacy, side effects, and poor tolerance. Therefore, developing new psoriasis inhibitors with strong immune tolerance and fewer adverse reactions has become an urgent problem and research hotspot in clinical practice.

[0004] Patent application CN119552059A discloses a sesquiterpene compound, its preparation method and application in the preparation of a drug for treating psoriasis, including compounds 1-4. This invention provides a new molecular template for the development of new psoriasis treatment drugs, and provides a new idea for further developing new treatment strategies with immune regulation.

[0005] Therefore, finding new compounds that can treat psoriasis is currently a difficult point. SUMMARY

[0006] The purpose of the present application is to provide a dual-target inhibitor with anti-psoriasis activity.

[0007] Another purpose of the present application is to provide the use of the dual-target inhibitor with anti-psoriasis activity in the preparation of a drug for treating psoriasis.

[0008] In order to achieve the above purpose, the technical solution adopted by the present application is as follows:

[0009] In a first aspect of the present application, a dual-target inhibitor with anti-psoriasis activity is provided, and the structure general formula is as follows:

[0010]

[0011] wherein,

[0012] R1 is selected from the group consisting of hydrogen, C1-C30 alkoxy (more preferably C1-C20 alkoxy, and more preferably C1-C10 alkoxy);

[0013] R2 is selected from the group consisting of hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0014] L is selected from -(CH2)n-;

[0015] n is selected from an integer from 0 to 30, more preferably an integer from 0 to 20, and most preferably an integer from 0 to 10.

[0016] More preferably, in the dual-target inhibitor with anti-psoriasis activity,

[0017] R1 is selected from the group consisting of hydrogen, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, t-butoxy, n-pentoxy, n-hexyloxy;

[0018] R2 is selected from the group consisting of hydrogen, fluorine, chlorine, bromine;

[0019] L is selected from -(CH2)n-;

[0020] n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0021] Most preferably, the dual-target inhibitor with anti-psoriasis activity has a structure selected from one of the following structures:

[0022]

[0023] In a second aspect of the present application, there is provided a use of the dual-target inhibitor with anti-psoriasis activity or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating psoriasis.

[0024] In a third aspect of the present application, there is provided a use of the dual-target inhibitor with anti-psoriasis activity or a pharmaceutically acceptable salt thereof in the manufacture of a JAK / HDAC dual-target inhibitor.

[0025] In a fourth aspect of the present application, there is provided a pharmaceutical preparation made from the dual-target inhibitor with anti-psoriasis activity or a pharmaceutically acceptable salt thereof and a medically acceptable excipient.

[0026] In a fifth aspect of the present application, there is provided a pharmaceutical composition consisting of the dual-target inhibitor with anti-psoriasis activity or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and other drugs for treating psoriasis.

[0027] The medicinal salt is an acid addition salt formed by a dual-target inhibitor with anti-psoriasis activity and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0028] In a sixth aspect, the present invention provides the use of the dual-target inhibitor having anti-psoriasis activity or a pharmaceutical salt thereof in the preparation of an anti-inflammatory medicament.

[0029] The compounds prepared in this invention exhibit good in vitro inhibitory activity against JAK and HDAC enzymes. In vitro experiments demonstrating inhibition of NO release from RAW264.7 cells, inhibition of TNF-α-induced HaCat cell proliferation, and anti-imiquimod-induced psoriasis in mice showed that compound B5e possesses good anti-inflammatory and anti-psoriatic activity, superior to its parent compounds (CYT387, SAHA) and superior to their combined administration group (CYT387+SAHA). Therefore, it can be used as a dual-target inhibitor of JAK / HDAC.

[0030] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0031] Among the compounds provided by this invention that possess anti-psoriasis activity and dual JAK / HDAC inhibitory activity, compound B5e exhibits significant inhibitory activity against JAK family enzymes (JAK1, JAK2, JAK3), with an IC50 value of [missing information]. 50 The values ​​were 219 nM, 397 nM, and 417 nM, respectively. It also exhibits inhibitory effects on HDAC family enzymes, with IC50 values ​​for HDAC1, HDAC2, HDAC3, HDAC6, and HDAC8. 50 The values ​​were 5 nM, 2.8 nM, 5.4 nM, 2.9 nM, and 266 nM, respectively. At a concentration of 0.4 μM, the compound significantly inhibited the release of nitric oxide (NO) from LPS-induced human macrophages and showed good efficacy in in vitro anti-psoriasis-related experiments and in vivo imiquimod-induced mouse models. Its anti-psoriasis activity was superior to that of the parent drug alone and in combination with the parent drug.

[0032] The compound provided by this invention, possessing anti-psoriatic activity and dual-target inhibition, is a dual-target inhibitor simultaneously targeting JAK and HDAC. It enhances therapeutic efficacy by multi-dimensionally regulating psoriasis-related inflammatory responses and epidermal cell proliferation. Compared to single-target inhibitors, the dual-target inhibitor provided by this invention may require a lower dose while achieving equivalent or superior efficacy, thereby reducing the risk of side effects and improving patient adherence. Long-term use of single-target inhibitors may lead to drug tolerance, while the JAK / HDAC dual-target inhibitor, through multi-pathway action, may reduce the occurrence of tolerance.

[0033] The compounds with anti-psoriasis activity and dual-target inhibition provided by this invention offer a new strategy for in-depth research and development of novel structural types of anti-psoriasis drugs. Attached Figure Description

[0034] Figure 1 This is a schematic diagram showing the effects of compound B5e on acetylated H3, H4, and phosphorylated STAT3 as determined by Western blotting.

[0035] Figure 2 This is a schematic diagram of the results of a study on the anti-psoriasis activity of compound B5e as a topical drug (cream) on the back skin of mice.

[0036] Figure 3 This is a schematic diagram illustrating the research results on the anti-psoriasis activity of compound B5e in topical medication (cream).

[0037] Figure 4 This is a schematic diagram of the mouse spleen results from a study on the anti-psoriasis activity of compound B5e when applied topically as a cream.

[0038] Figure 5 This is a schematic diagram showing the ratio of compound B5e to acetylated H3 and GAPDH.

[0039] Figure 6 This is a schematic diagram showing the ratio of compound B5e to acetylated H4 and GAPDH.

[0040] Figure 7 This is a schematic diagram showing the ratio of phosphorylated STAT3 to GAPDH tested by compound B5e. Detailed Implementation

[0041] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0042] In this invention, 1,4-Dioxane is 1,4-dioxane, EDCI is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, HOBT is 1-hydroxybenzotriazole, DMF is N,N-dimethylformamide, DCM is dichloromethane, MeOH is methanol, and THF is tetrahydrofuran.

[0043] The preparation route of the dual-target inhibitor W6a-6e with anti-psoriasis activity of the present invention is as follows:

[0044]

[0045] Example 1

[0046] Step 1: Under a nitrogen atmosphere, ethyl 5-bromoindole-2-carboxylate (1.000 g, 3.746 mmol), pinacol diboronate (1.903 g, 7.492 mmol), and C... 34 H 28 Cl2FeP2Pd([1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride) (0.110 g, 0.150 mmol), potassium acetate (1.791 g, 8.616 mmol), and 20 mL of 1,4-dioxane ultradry solvent were mixed and stirred overnight at 110 °C. At the end of the reaction, the reaction was quenched with saturated brine, extracted with ethyl acetate, and the organic phase was dried and purified by silica gel chromatography (petroleum ether:ethyl acetate = 4:1) to give a brown solid, compound W1, in 90.5% yield.

[0047]

[0048] In the second step, compound W1 (0.500 g, 1.586 mmol), 2,4-dichloropyrimidine (0.591 g, 3.965 mmol), and 15 mL of 1,4-dioxane and 5 mL of water were added. Tetraphenylphosphine palladium (0.459 g, 0.397 mmol) and cesium carbonate (1.292 g, 3.965 mmol) were added, and the mixture was sonicated until the substrate was uniformly dispersed. The mixture was stirred at 110 °C for 3 h. After the reaction was completed, the temperature was lowered to room temperature, and saturated saline solution was added. The mixture was extracted with ethyl acetate, dried, and purified by silica gel chromatography (dichloromethane:methanol = 100:1) to obtain a white solid, namely compound W2, with a yield of 78.6%.

[0049]

[0050] In the third step, compound W2 (0.100 g, 0.332 mmol), 1-methyl-1H-pyrazole-4-amine (0.035 g, 0.365 mmol), X-PHOS (2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl) (0.031 g, 0.066 mmol), Pd2(dba)3 (0.061 g, 0.066 mmol), and cesium carbonate (0.270 g, 0.830 mmol) were added to 20 mL of 1,4-dioxane. The reaction solution was placed under a nitrogen atmosphere and stirred at 110 °C for 12 h. After the reaction was completed, the reaction solution was poured into saturated brine, extracted with ethyl acetate, dried, and purified by silica gel chromatography (dichloromethane:methanol = 50:1) to obtain a yellow solid, namely compound W3, with a yield of 65.9%.

[0051]

[0052] In the fourth step, compound W3 (0.5 g, 1.381 mmol) and LiOH (0.108 g, 4.451 mmol) were added to a 20 mL mixture of methanol and water in a volume ratio of 3:1. The reaction mixture was stirred at room temperature for 1 h. After the reaction was complete, the methanol was evaporated, water was added, and the pH was adjusted to 3 with a 1 mol / L hydrochloric acid solution. The solid precipitated out, was washed with water, and dried to obtain a yellow solid, which is compound W4, with a yield of 93.2%.

[0053]

[0054] In the fifth step, compound W4 (0.150 g, 0.449 mmol) was added to 10 mL of DMF, along with TEA (0.136 g, 1.347 mmol), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate) (0.199 g, 0.449 mmol), and methyl 3-aminopropionate hydrochloride (0.125 g, 0.898 mmol). The reaction was carried out at room temperature for 4 h. After the reaction was completed, 2 mL of water was added to quench the reaction, followed by extraction with ethyl acetate. The organic phase was dried and purified by silica gel chromatography (dichloromethane:methanol = 50:1) to obtain a yellow solid, namely compound W5a.

[0055] Step 6: Dissolve hydroxylamine hydrochloride (4.670 g, 67 mmol) in 24 mL of methanol. Under ice bath conditions, add 12 mL of a methanol solution of potassium hydroxide (5.610 g, 100 mmol). Stir at room temperature for 0.5 h. After the reaction is complete, filter the solution. The filtrate obtained is the freshly prepared hydroxylamine methanol solution.

[0056] Compound W5a (0.083 g, 0.200 mmol) was added to 15 mL of freshly prepared hydroxylamine methanol solution and stirred at room temperature for 3–4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solution was evaporated to dryness under reduced pressure, 5 mL of water was added, and acetic acid was added dropwise to adjust the pH of the solution to 7. A yellow precipitate was formed. The precipitate was filtered, washed, and dried to obtain compound W6a, with a yield of 65.3%.

[0057]

[0058] Example 2

[0059] Prepared according to the method in step 5 of Example 1, replacing methyl 3-aminopropionate hydrochloride in step 5 with methyl 4-aminobutyrate hydrochloride (0.138 g, 0.898 mmol), yielding a yellow solid, namely compound W5b.

[0060] Prepared according to the method in step 6 of Example 1, a yellow solid, namely compound W6b, was obtained with a yield of 58.4%.

[0061]

[0062] Example 3

[0063] Prepared according to the method in step 5 of Example 1, replacing methyl 3-aminopropionate hydrochloride in step 5 with methyl 5-aminovalerate hydrochloride (0.151 g, 0.898 mmol), to obtain a yellow solid, namely compound W5c.

[0064] Prepared according to the method in step 6 of Example 1, a yellow solid, namely compound W6c, was obtained with a yield of 62.3%.

[0065]

[0066] Example 4

[0067] Prepared according to the method in step 5 of Example 1, replacing methyl 3-aminopropionate hydrochloride in step 5 with methyl 6-aminohexanoate hydrochloride (0.163 g, 0.898 mmol), yielding a yellow solid, namely compound W5d.

[0068] Prepared according to step 6 of Example 1, a yellow solid, namely compound W6d, was obtained with a yield of 63.4%.

[0069]

[0070] Example 5

[0071] Prepared according to the method in step 5 of Example 1, replacing methyl 3-aminopropionate hydrochloride in step 5 with methyl 7-aminoheptanoate hydrochloride (0.176 g, 0.898 mmol), to obtain a yellow solid, namely compound W5e.

[0072] Prepared according to step 6 of Example 1, a yellow solid, namely compound W6e, was obtained with a yield of 63.9%.

[0073]

[0074] The preparation route of the dual-target inhibitor A6b-6e with anti-psoriasis activity of the present invention is as follows:

[0075]

[0076] Example 6

[0077] Preparation method of compound A2:

[0078] Compound A1 (0.500 g, 1.586 mmol) and 2,4-dichloropyrimidine (0.591 g, 3.965 mmol) were added to a round-bottom flask containing 15 mL of 1,4-dioxane and 5 mL of water. Tetraphenylphosphine palladium (0.459 g, 0.397 mmol) and cesium carbonate (1.292 g, 3.965 mmol) were added. The mixture was sonicated until the substrate was uniformly dispersed. The mixture was stirred at 110 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, and saturated brine was added. The mixture was extracted with ethyl acetate, dried, and purified by silica gel chromatography (dichloromethane:methanol = 100:1) to give a white solid, compound A2, with a yield of 78.6%.

[0079]

[0080] Under a nitrogen atmosphere, compound A2 (0.100 g, 0.332 mmol), 3-methoxy-1-methyl-1H-pyrazole-4-amine (0.046 g, 0.365 mmol), X-PHOS (0.031 g, 0.066 mmol), Pd2(dba)3 (0.061 g, 0.066 mmol), and cesium carbonate (0.270 g, 0.830 mmol) were added to 20 mL of 1,4-dioxane and stirred at 110 °C for 12 h. After the reaction was complete, the reaction solution was poured into saturated brine, extracted with ethyl acetate, dried, and purified by silica gel chromatography (dichloromethane:methanol = 50:1) to give a yellow solid, compound A3, in 63.2% yield.

[0081]

[0082] Compound A3 (0.5 g, 1.275 mmol) and LiOH (0.108 g, 4.451 mmol) were added to a 20 mL mixture of methanol and water in a 3:1 mass ratio. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the methanol was evaporated, water was added, and the pH was adjusted to 3 with a 1 mol / L hydrochloric acid solution. A solid precipitated out. The filter cake was washed with water and dried to obtain a yellow solid, namely compound A4, with a yield of 93.6%.

[0083]

[0084] Compound A4 (0.150 g, 0.412 mmol) was added to 10 mL of DMF, followed by TEA (0.125 g, 1.236 mmol), BOP (0.182 g, 0.412 mmol), and methyl 4-aminobutyrate hydrochloride (0.126 g, 0.824 mmol). The reaction was carried out at room temperature for 4 h. After the reaction was completed, 2 mL of water was added to quench the reaction. The mixture was extracted with ethyl acetate, dried, and purified by silica gel chromatography (dichloromethane:methanol = 50:1) to give a yellow solid, namely compound A5b, with a yield of 73.9%.

[0085]

[0086] Compound A5b (0.092 g, 0.200 mmol) was added to 15 mL of freshly prepared hydroxylamine methanol solution and stirred at room temperature for 3–4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solution was evaporated to dryness under reduced pressure, 5 mL of water was added, and acetic acid was added dropwise to adjust the pH of the solution to 7. A yellow precipitate was formed. The precipitate was filtered, washed, and dried to obtain a yellow solid, namely compound A6b, with a yield of 63.8%.

[0087]

[0088] Example 7

[0089] Prepared according to the method in Example 6, replacing 4-aminobutyric acid methyl ester hydrochloride with 5-aminovalerate methyl ester hydrochloride (0.138 g, 0.824 mmol), yielding a yellow solid, namely compound A5c, with a yield of 64.3%.

[0090]

[0091] Prepared according to the method in Example 6, a yellow solid, namely compound A6c, was obtained with a yield of 63.5%.

[0092]

[0093] Example 8

[0094] Prepared according to the method in Example 6, 4-aminobutyric acid methyl ester hydrochloride was replaced with 6-aminohexanoate methyl ester hydrochloride (0.150 g, 0.824 mmol) to obtain a yellow solid, namely compound A5d, with a yield of 67.9%.

[0095]

[0096] Prepared according to the method in Example 6, a yellow solid, namely compound A6d, was obtained with a yield of 63.4%.

[0097]

[0098] Example 9

[0099] Prepared according to the method in Example 6, 4-aminobutyric acid methyl ester hydrochloride was replaced with 7-aminoheptanoic acid methyl ester hydrochloride (0.161 g, 0.824 mmol) to obtain a yellow solid, namely compound A5e.

[0100] Prepared according to the method in Example 6, a yellow solid, namely compound A6e, was obtained with a yield of 60.3%.

[0101]

[0102] The preparation route of the dual-target inhibitor B5a-5h with anti-psoriasis activity of the present invention is as follows:

[0103]

[0104] Example 10

[0105] Under a nitrogen atmosphere, compound W1 (0.500 g, 1.586 mmol) and 2,4-dichloro-5-fluoropyrimidine (0.662 g, 3.965 mmol) were added to 15 mL of 1,4-dioxane and 5 mL of water. Tetraphenylphosphine palladium (0.459 g, 0.397 mmol) and cesium carbonate (1.292 g, 3.965 mmol) were added, and the mixture was sonicated until the substrate was uniformly dispersed. The mixture was stirred at 110 °C for 3 h. After the reaction was completed, the temperature was lowered to room temperature, saturated brine was added, and the mixture was extracted with ethyl acetate. The organic phase was dried and purified by silica gel chromatography (dichloromethane:methanol = 100:1) to give a white solid, compound B1, with a yield of 79.3%.

[0106]

[0107] Under a nitrogen atmosphere, compound B1 (0.100 g, 0.313 mmol), 3-methoxy-1-methyl-1H-pyrazole-4-amine (0.044 g, 0.344 mmol), X-PHOS (0.030 g, 0.062 mmol), Pd2(dba)3 (0.056 g, 0.062 mmol), and cesium carbonate (0.255 g, 0.783 mmol) were added to 20 mL of 1,4-dioxane and stirred at 110 °C for 12 h. After the reaction was complete, the reaction solution was poured into saturated brine, extracted with ethyl acetate, dried, and purified by silica gel chromatography (dichloromethane:methanol = 20:1) to give a yellow solid, compound B2, in 65.7% yield.

[0108]

[0109] Compound B2 (0.5 g, 1.219 mmol) and LiOH (0.108 g, 4.451 mmol) were added to a 20 mL mixture of methanol and water in a 3:1 mass ratio. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the methanol was evaporated, water was added, and the pH was adjusted to 3 with a 1 mol / L hydrochloric acid solution. A solid precipitated out. The filter cake was washed with water and dried to obtain a yellow solid, namely compound B3, with a yield of 98.3%.

[0110]

[0111] Compound B3 (0.150 g, 0.393 mmol) was added to 10 mL of DMF, followed by TEA (0.119 g, 1.179 mmol), BOP (0.174 g, 0.393 mmol), and methyl 3-aminopropionate hydrochloride (0.110 g, 0.786 mmol). The reaction was carried out at room temperature for 4 h. After the reaction was completed, 2 mL of water was added to quench the reaction. The mixture was extracted with ethyl acetate, and the organic phase was dried and purified by silica gel chromatography (dichloromethane:methanol = 20:1) to give a yellow solid, namely compound B4a, with a yield of 67.9%.

[0112]

[0113] Compound B4a (0.093 g, 0.200 mmol) was added to 15 mL of freshly prepared hydroxylamine methanol solution and stirred at room temperature for 3–4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solution was evaporated to dryness under reduced pressure, 5 mL of water was added, and acetic acid was added dropwise to adjust the pH of the solution to 7. A yellow precipitate was formed. The precipitate was filtered, washed, and dried to obtain a yellow solid, namely compound B5a, with a yield of 56.8%.

[0114]

[0115] Example 11

[0116] Prepared according to the method in Example 10, methyl 3-aminopropionate hydrochloride was replaced with methyl 4-aminobutyrate hydrochloride (0.121 g, 0.786 mmol) to obtain a yellow solid, namely compound B4b, with a yield of 65.6%.

[0117]

[0118] Prepared according to the method in Example 10, a yellow solid, namely compound B5b, was obtained with a yield of 56.8%.

[0119]

[0120] Example 12

[0121] Prepared according to the method in Example 10, methyl 3-aminopropionate hydrochloride was replaced with methyl 5-aminovalerate hydrochloride (0.132 g, 0.786 mmol) to obtain a yellow solid, namely compound B4c, with a yield of 69.5%.

[0122]

[0123] Prepared according to the method in Example 10, a yellow solid, namely compound B5c, was obtained with a yield of 53.6%.

[0124]

[0125] Example 13

[0126] Prepared according to the method in Example 10, methyl 3-aminopropionate hydrochloride was replaced with methyl 6-aminohexanoate hydrochloride (0.143 g, 0.786 mmol) to obtain a yellow solid, namely compound B4d, with a yield of 73.4%.

[0127]

[0128] Prepared according to the method in Example 10, a yellow solid, namely compound B5d, was obtained with a yield of 60.2%.

[0129]

[0130] Example 14

[0131] Prepared according to the method in Example 10, methyl 3-aminopropionate hydrochloride was replaced with methyl 7-aminoheptanoate hydrochloride (0.154 g, 0.786 mmol) to obtain a yellow solid, namely compound B4e, with a yield of 75.3%.

[0132]

[0133] Prepared according to the method in Example 10, a yellow solid, namely compound B5e, was obtained with a yield of 56.8%.

[0134]

[0135] Example 15

[0136] Prepared according to the method in Example 10, methyl 3-aminopropionate hydrochloride was replaced with methyl 8-aminooctanoate hydrochloride (0.165 g, 0.786 mmol) to obtain a yellow solid, namely compound B4h, with a yield of 80.6%.

[0137]

[0138] Prepared according to the method in Example 10, a yellow solid, namely compound B5h, was obtained with a yield of 60.2%.

[0139]

[0140] Example 16

[0141] The compounds prepared in the embodiments of the present invention were tested for their inhibitory activity against JAK and HDAC targets.

[0142] The first step was to test the compound's inhibitory activity against JAK.

[0143] (i) Compound preparation: The compound was prepared into a 10 μM solution in PBS. 40 µL of the compound solution was pipetted into an Echo plate. 200 nL of the solution was transferred from the Echo plate to a 384-well plate using an ECHO650. The 384-well plate was then labeled as the reaction plate for this experiment.

[0144] (ii) Lance Ultra Assay test experiment:

[0145] (a) Preparation of kinase buffer: Add 50 mM HEPES, 10 mM MgCl2, 0.01% BSA, 0.01% Tween-20, 0.01% Triton X-100 and 2 mM DTT at pH=7.5 to PBS buffer and sonicate to dissolve.

[0146] (b) Kinase response and termination:

[0147] 1) Add the kinase to freshly prepared kinase buffer to form an enzyme solution.

[0148] 2) Transfer 10 μL of the above enzyme solution to the reaction wells of a 384-well plate, add kinase buffer as a negative control, centrifuge at 1000 rpm for 1 minute in a normal speed centrifuge, and then incubate at room temperature for about 10 minutes.

[0149] 3) Prepare substrate solution: Add substrate and reagents such as ATP to kinase buffer.

[0150] 4) Add 10 μL of the freshly prepared substrate solution to a 384-well plate, centrifuge at 1000 rpm for 1 minute in a normal speed centrifuge, and then incubate at room temperature for 30 minutes.

[0151] 5) Prepare the kinase termination reaction solution (containing antibody) in advance. Add 20 μL of the termination reaction solution to each well, centrifuge at 1000 rpm for 1 minute, and finally incubate the 384-well plate at room temperature for 60 minutes.

[0152] (c) Data reading: Fluorescence values ​​were measured on an Envision 2104 Multilabel Reader instrument.

[0153] (d) Data computation (IC) 50 ):

[0154] 1) Obtain the numerical ratio of fluorescence readings (Lance signal ratio (665nm / 615nm)).

[0155] 2) Convert the obtained data into a suppression percentage using a formula.

[0156]

[0157] “min” represents the reading of the control wells without enzyme; “max” represents the reading of the control wells with DMSO added.

[0158] Import the data into MS Excel and use XLFit Excel add-in version 5.4.0.8 to perform curve fitting. The fitting formula is as follows:

[0159]

[0160] The second step is to test the compound's HDAC inhibition activity.

[0161] (i) Preparation of kinase buffer: Add 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mg / mL BSA, and 50 mM Tris-HCl (pH=8) to PBS buffer, sonicate to dissolve, and store at 4°C for later use.

[0162] (ii) Preparation of test compounds: Prepare a 10 mM solution of the test compound with cell-grade DMSO and store it in a -20°C freezer.

[0163] (iii) Determination of the HDAC1 enzyme activity of the compound:

[0164] (a) Equilibrate the 96-well black microplate to room temperature to reduce experimental error.

[0165] (b) Dilute the prepared 10 mM drug solution (three-fold dilution) to obtain different drug concentration gradients of 100 μM, 33 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM and 0.003 μM for later use.

[0166] (c) Add 11 μL of HDAC1 enzyme to 400 μL of kinase buffer, shake well, and prepare a buffer containing 0.2 μg / mL HDAC enzyme. Add 35 μL of the prepared enzyme buffer to wells 1-11 of a black 96-well plate that has been equilibrated to room temperature.

[0167] (d) Pipette 5 μL of drug solutions of different concentrations and add them sequentially to the black 96-well plate (wells 1-10). Well 11 serves as a negative control, with 5 μL of kinase buffer added. Well 12 serves as a blank control, with 40 μL of kinase buffer added.

[0168] (e) Add 5 μL of 100 μM HDAC1 substrate (Ac-Leu-Gly-Lys(Tfa)-AMC) and 5 μL of 0.5 mg / mL trypsin to all wells of a black 96-well plate. Incubate at 37°C in the dark for 30 minutes and then measure the OD value (excitation wavelength: 360 nm, emission wavelength: 460 nm).

[0169] (f) Calculate the HDAC1 enzyme activity inhibition rate of the target compound:

[0170]

[0171] In GraphPad software, the IC50 of the compound was determined by fitting a curve of inhibition rate versus compound concentration. 50 value.

[0172] Using the Lance Ultra Assay, at Km ATP, the inhibition rates of the compounds (100 nM) prepared in the embodiments of the present invention against JAK1 and JAK2, as well as the inhibitory activity of compound B5e against other JAK isoforms, were tested. Using the Fluorescent-based HDAC activity assay, with SAHA as a standard control, the inhibitory activity of the compounds prepared in the embodiments of the present invention against HDAC1, as well as the inhibitory activity of compound B5e against other HDAC isoforms, were tested.

[0173] The inhibitory activity of the compounds against JAK1 and JAK2 is shown in Table 1. Among them, the inhibitory activities of compounds W6a, W6b, W6c, W6d and W6e prepared in this invention against JAK1 and JAK2 are all greater than 60%.

[0174] Table 1

[0175]

[0176] Determining the inhibitory activity of compounds against HDAC is crucial for identifying potential HDAC inhibitors. HDAC inhibitors play a vital role not only in cancer treatment but also in the treatment of other diseases, such as inflammatory diseases. This assay reveals how compounds function within cells, including their effects on gene expression and cellular metabolism. The efficacy of a compound may be related to the degree of HDAC inhibition; therefore, determining the inhibitory activity helps predict its potential therapeutic effect. Furthermore, analyzing the relationship between compound structure and its HDAC inhibitory effect helps optimize drug design. Overall, determining the inhibitory activity of compounds against HDAC has significant scientific and clinical implications for drug development, disease treatment, and advancements in basic biology. JAK / HDAC dual-target inhibitors all exhibited inhibitory activity against HDAC1, but the activities varied considerably. The results are shown in Table 2. Compounds W6c, W6e, B5e, and B5h all demonstrated excellent inhibitory activity against HDAC1 (inhibition rates of W6c and W6e against HDAC1 <30 nM, and inhibition rates of B5e and B5h against HDAC1 <10 nM).

[0177] Table 2

[0178]

[0179] Based on the inhibitory activities of compounds against JAK and HDAC, compound B5e with good activity was selected to evaluate the selectivity of different JAK and HDAC isoforms. The inhibitory activity of compound B5e against JAK (JAK1, 2, 3, and TYK2) was determined, and the results are shown in Table 3. The results show that compound B5e has strong inhibitory activity against JAK1–3 (IC50). 50 Range: 219~417 nM).

[0180] Table 3

[0181]

[0182] The HDAC subtype selectivity evaluation results are shown in Table 4. The data in the table show that compound B5e exhibits significant inhibitory effects on HDAC1, HDAC2, HDAC3, and HDAC6 (IC50). 50 (Range: 2.8~266 nM), compound B5e exhibits strong inhibitory activity against HDAC8 (IC50). 50 =266 nM)

[0183] Table 4

[0184]

[0185] Example 17

[0186] In vitro TNF-α-induced inhibition experiment of the compound prepared in this invention on HaCaT cells

[0187] (i) Preparation of test drug solutions: The test drug was prepared into a 10 mM stock solution using cell-grade DMSO as the solvent and stored in a freezer at -20 °C. Before the experiment, the stock solution was removed and allowed to thaw at room temperature, then diluted with complete DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin mixed antibiotics, and 89% DMEM basal medium. The concentrations of the diluted drug solutions were 100 μM, 33.33 μM, 11.11 μM, 3.70 μM, 1.23 μM, 0.41 μM, 0.14 μM, 0.05 μM, 0.015 μM, and 0.0051 μM, and were stored in a freezer at 4 °C for later use.

[0188] (ii) Cell culture: Take the frozen HaCaT cells from liquid nitrogen, place them in a 37℃ water bath for 1-2 minutes, add 3 mL of DMEM complete medium and transfer them to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 2 mL of DMEM complete medium to disperse the cells evenly, and finally transfer them to a cell culture dish and culture them in a CO2 incubator (5% CO2, 37 ℃) for 24 h. All cells used in the experiment were in the logarithmic growth phase.

[0189] (iii) CCK-8 assay to test the anti-psoriasis activity of the target compound:

[0190] (a) Cell plating: HaCaT cells in the logarithmic growth phase were collected and prepared into a concentration of 3 × 10⁻⁶ cells using DMEM complete medium containing 10% fetal bovine serum. 4 Single-cell suspension at 100 μL / mL. Dispense this suspension into 96-well plates, adding 100 μL to each well (range 2 to 11 wells). Fill the edge wells of the plate with 100 μL of PBS buffer. Then, incubate the 96-well plates in a CO2 incubator for 24 hours.

[0191] (b) TNF-α induction: Prepare DMEM complete medium containing TNF-α (100 ng / mL) in advance. Take out the 96-well plate from the CO2 incubator, aspirate the supernatant, add 100 μL of DMEM complete medium containing TNF-α to each well (except column 11), and incubate in the CO2 incubator for 24 h.

[0192] (c) Drug treatment: Prepare the required drug concentration gradient in advance, take out the 96-well plate, aspirate the supernatant, add 100 μL of DMEM complete culture medium (containing drug) to each well, set up triplet wells for each concentration, and incubate in a CO2 incubator for 48 h.

[0193] (d) Measurement of OD 450 Cell supernatant (OD) was prepared by dissolving 10% CCK-8 in DMEM basal medium. The cell supernatant and PBS buffer were removed from the 96-well plate, and 100 μL of DMEM basal medium (containing 10% CCK-8) was added to each well. The plate was then incubated in a CO2 incubator for 0.5 to 1 h. The absorbance (OD) of each well was measured at 450 nm using a microplate detector. 450 The inhibitory rate of the drug and its IC50 value for treating psoriasis were calculated according to the formula. 50 The value is calculated by GraphPad.

[0194]

[0195] Table 5

[0196]

[0197] The structural formulas of CYT387 and SAHA are shown below:

[0198]

[0199] The experimental results are shown in Table 5. Compounds A6e and B5e have certain anti-TNF-α-induced HaCaT cell proliferation activity (IC50). 50 <10 μM), among which compound B5e is a compound with good in vitro TNF-α-induced HaCaT cell inhibitory activity, which is basically consistent with the in vitro HDAC1 inhibitory activity results. The antiproliferative activity is 0.78 μM, which is superior to its parent drug and its combination (SAHA IC). 50 =2.21 μM, CYT387 IC 50 =12.24μM, SAHA+CYT387 IC 50 =1.98 μM).

[0200] Example 18

[0201] Experiments on the inhibition of NO release from macrophages by the compounds prepared in this invention.

[0202] Compound treatment: Compound B5e was prepared into a 10 mM solution using cell-grade DMSO. A total of 6 groups were set up (blank group, model group, B5e group, SAHA group, CYT387 group, SAHA+CYT387 group).

[0203] (i) Cell culture: The culture method is the same as in Example 17.

[0204] (ii) Cell plating: RAW264.7 cells in logarithmic growth phase were used to prepare single-cell suspensions of 4 × 10⁴ cells using DMEM complete culture medium containing 10% fetal bovine serum. 5 Cells / mL, add the above suspension to a 24-well plate and incubate in a CO2 cell culture incubator for 24 h.

[0205] (iii) LPS and drug treatment: Lipopolysaccharide (LPS) was prepared into a 1 mg / mL solution using cell-grade DMSO. The prepared LPS was diluted to a 2 μg / mL solution using DMEM complete medium. The cell supernatant in the 24-well plate was discarded. The drug was diluted into different concentration gradients using DMEM complete medium (containing 2 μg / mL LPS) and added to the 24-well plate (a blank control group and a negative control group were set up, and each concentration was set up in triplicate). The plate was then incubated in an incubator for 24 h.

[0206] (iv) Determination of NO content: Aspirate the cell supernatant from the 24-well plate and centrifuge. Collect the supernatant and determine the NO content using the Griess kit.

[0207] (a) Dilution of NO standard solution: used for fitting standard curve.

[0208] (b) Griess method: Add 50 μL of the cell supernatant and standard to each well of a 96-well plate. First, add 50 μL of reagent A to each well and incubate at 37 °C for 10 minutes. Then, add 50 μL of reagent B to each well and incubate at 37 °C for 10 minutes. Shake the 96-well plate to mix the solutions in each well, and measure the OD value of each well at 540 nm using a microplate reader.

[0209] Data processing: Based on the OD value of the standard, a standard curve is fitted, and the NO content of each well is calculated from the standard curve.

[0210] The experimental results are shown in Table 6:

[0211] Table 6

[0212]

[0213] The data in the table show that compound B5e exhibits excellent ability to inhibit LPS-induced NO release from macrophages, has a certain anti-inflammatory effect, and is superior to its parent drug and its combination (SAHA, CYT387, SAHA+CYT387).

[0214] The NO release concentration in the model group compared to the blank group was 73.31 μmol / L × 10⁻⁶. -2 The significant increase in NO levels indicates that the model was successfully established. All tested compounds and positive control drugs effectively inhibited NO release from human macrophages, and this inhibition was concentration-dependent.

[0215] Example 19

[0216] Western Blot method was used to test the effects of the compounds prepared in this invention on signaling pathways.

[0217] (1) Cell culture and drug preparation: Same as in Example 18.

[0218] (2) Cell plating: HaCaT cells in the logarithmic growth phase were used to prepare a single-cell suspension of 4×10⁴ cells using DMEM complete culture medium. 5 Cells / mL: Add the above cell suspension to 6-well plates (2 mL per well) and incubate in a cell culture incubator for 24 h.

[0219] (3) TNF-α and drug treatment: Discard the supernatant in the 6-well plate, add 2 mL of DMEM complete medium (containing TNF-α) to each well, and then incubate in an incubator for 24 h. Discard the supernatant in the 6-well plate, add 2 mL of DMEM complete medium (containing different concentrations of drugs) to each well, and then incubate in an incubator for 24 h.

[0220] (4) Extraction and quantification of cell proteins: Discard the supernatant in the 6-well plate, wash three times with 2 mL of PBS buffer, extract cell proteins with RIPA high strength lysis buffer, and quantify the extracted cell proteins using the BCA protein quantification kit.

[0221] (5) Electrophoresis, transfer and blocking: Separate an equal amount of protein (40 μg) by SDS-PAGE, transfer the separated protein to a PVDF membrane using a transfer apparatus (7-9 minutes), wash the PVDF membrane three times with TBST, and block with 5% BSA blocking buffer for 1.5-2 h.

[0222] (6) Antibody incubation and development: Discard the blocking buffer, wash the PVDF membrane three times with TBST, add the first antibody, and incubate at 4°C for 12 h. Recover the first antibody, wash the PVDF membrane three times with TBST, add the second antibody, and incubate at room temperature for 2 h. Recover the second antibody, wash the PVDF membrane three times with TBST, and finally obtain the target band using an infrared fluorescence scanning imaging system (GAPDH as a standardized internal control).

[0223] (7) Data analysis: Image J software was used to perform grayscale analysis on the obtained target strips, and GraphPad software was used to process and plot the obtained data.

[0224] Figure 1 This is a schematic diagram showing the effect of compound B5e on acetylation of H3, H4, and phosphorylation of STAT3 using Western blotting. The diagram shows that compound B5e inhibits STAT3 phosphorylation in a concentration-dependent manner.

[0225] Figure 5 This is a schematic diagram illustrating the ratio of acetylated H3 to GAPDH tested by compound B5e. The diagram shows that compound B5e increases the level of acetylated histone H3 in a dose-dependent manner, indicating that compound B5e inhibits the HDAC pathway intracellularly.

[0226] Figure 6This is a schematic diagram illustrating the ratio of acetylated H4 to GAPDH tested by compound B5e. The diagram shows that compound B5e increases the level of acetylated histone H4 in a dose-dependent manner, indicating that compound B5e inhibits the HDAC pathway intracellularly.

[0227] Figure 7 This is a schematic diagram illustrating the ratio of phosphorylated STAT3 to GAPDH tested by compound B5e. Compound B5e can increase the level of phosphorylated signal transducer and transcription activator STAT3, indicating that compound B5e inhibits the JAK-STAT pathway in cells.

[0228] The above results indicate that compound B5e inhibits the JAK and HDAC signaling pathways in cells.

[0229] Example 20

[0230] Experiments on imiquimod-induced psoriasis mouse model of the compound prepared in this invention

[0231] (1) Procurement and Adaptation of Mice: Male SPF-grade BALB / c mice weighing approximately 18-20 g and around 6 weeks old were procured before the experiment. Before the experiment, the mice were allowed to eat standard animal feed and drink filtered tap water freely in the animal room for 7 days. The temperature in the animal room was controlled at 22-26℃ and the humidity was controlled at 40%-70%.

[0232] (2) Mouse experimental treatment: The day before the formal start of the experiment, the hair on the back of the mice was shaved off and hair removal cream was used to expose the skin on the back.

[0233] (a) Study on the anti-psoriasis activity of compound B5e after topical skin administration

[0234] 1) Preparation of creams (containing medications):

[0235] Weigh out the corresponding proportions of polyoxyethylene (21) stearyl alcohol ether, liquid paraffin, ethylparaben, polyoxyethylene (2) stearyl alcohol ether, glyceryl monostearate, cetyl alcohol: octadecyl alcohol (mass ratio of 3:7) according to the amounts in Table 7, heat to 80℃ and keep warm to obtain phase A.

[0236] Glycerin was added to water and heated to 80°C and kept warm to obtain phase B.

[0237] Phase A is slowly added to Phase B while stirring and mixing. After Phase A is completely added, the mixture is homogenized for 3-5 minutes using a high-pressure homogenizer. When the temperature of the cream drops to 60°C, compound B5e is added. After high-pressure homogenization, different creams are obtained.

[0238] Table 7

[0239]

[0240] 2) Experimental Treatment: The experiment was divided into 6 groups (control group, normal group, B5e group, SAHA group, CYT387 group, SHAH+CYT387 group, with a drug concentration of 0.5% in all groups). At the beginning of the experiment, imiquimod cream was evenly applied to the bare back area of ​​the mice once a day. After 4 hours, a prepared cream (containing the drug) was evenly applied to the back of the mice. The mice's diet and skin changes were observed and recorded daily, and the weight of each mouse was also recorded. This model required continuous drug administration for 7 days. After the experiment, the bare skin areas of the mice were photographed and recorded. Then, the mice were dissected, and the bare back skin and spleen of the mice were removed. The spleen of the dissected mice was weighed and photographed, and the skin of the mice was stained with hematoxylin and eosin (HE).

[0241] (b) Study on the anti-psoriasis activity of compound B5e after oral administration

[0242] 1) Drug preparation: Weigh the corresponding compound and add 5% cell-grade DMSO, 10% Solutol HS-15 and saline.

[0243] 2) Experimental treatment: The experiment was divided into 6 groups (control group, normal group, B5e group, SAHA group, CYT387 group, SHAH+CYT387 group, and the drug concentration in all groups was 20 mg / kg).

[0244] Experimental results are as follows Figures 2-4 As shown, Figure 2 This is a schematic diagram of the results of a study on the anti-psoriasis activity of compound B5e (cream) on the back skin of mice. As shown in the diagram, the skin on the backs of mice in the normal group was smooth, without erythema or scaling. The skin on the backs of mice in the control group was red, with almost all lesions covered in scales, showing obvious redness, swelling, and wrinkling. The lesions were thickened and protruded, indicating an extremely severe type, demonstrating the successful construction of the in vivo experimental model. The skin on the backs of mice in the positive control groups (SAHA group, CYT387 group, and SAHA+CYT387 group) was red, with some lesions covered in scales, mainly fine scales, slightly raised above the normal skin surface, indicating poor anti-psoriasis activity. The B5e group had a color similar to normal skin, with wrinkled skin. These results indicate that the in vivo anti-psoriasis activity of compound B5e is superior to that of the positive control group and its combination therapy.

[0245] Figure 3This is a schematic diagram illustrating the anti-psoriasis activity of compound B5e as a topical medication (cream). In the diagram, A is a schematic diagram of HE staining of mouse dorsal skin, and B is a schematic diagram of the thickness of acanthotic cells in mouse dorsal skin. The results show that the thickness of acanthotic cells in the model group was significantly higher than that in the control group, proving the successful establishment of the experimental model. Compound B5e exhibited the best activity in the experimental group, significantly reducing the thickness of acanthotic cells in the dorsal skin of psoriatic mice, indicating that compound B5e possesses good anti-psoriasis activity. Figure 4 This is a schematic diagram of the results of a mouse spleen study on the anti-psoriasis activity of compound B5e topical application (cream). The results show that the spleens of mice in the blank group were smaller, while the spleens of mice in the control group were significantly enlarged, indicating significant inflammation in the model mice. The spleen volume of the compound B5e group was significantly reduced, indicating that it has an inhibitory effect on splenomegaly in mice, suggesting that compound B5e has good anti-inflammatory activity.

[0246] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A dual-target inhibitor with anti-psoriasis activity, characterized in that, The general structural formula is as follows: ； in, R1 is selected from hydrogen and C1~C30 alkoxy groups; R2 is selected from hydrogen or halogens; L is selected from -(CH2)n-; n is an integer selected from 0 to 30.

2. The dual-target inhibitor with anti-psoriasis activity according to claim 1, characterized in that, in, R1 is selected from hydrogen and C1~C20 alkoxy groups; R2 is selected from hydrogen or halogens; L is selected from -(CH2)n-; n is an integer selected from 0 to 20.

3. The dual-target inhibitor with anti-psoriasis activity according to claim 2, characterized in that, in, R1 is selected from hydrogen and C1~C10 alkoxy groups; R2 is selected from hydrogen or halogens; L is selected from -(CH2)n-; n is an integer selected from 0 to 10.

4. The dual-target inhibitor with anti-psoriasis activity according to claim 3, characterized in that, Among the dual-target inhibitors with anti-psoriasis activity, R1 is selected from hydrogen, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, and n-hexoxy. R2 is selected from hydrogen, fluorine, chlorine, and bromine; L is selected from -(CH2)n-; n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

5. The dual-target inhibitor with anti-psoriasis activity according to claim 4, characterized in that, The structure of the dual-target inhibitor with anti-psoriasis activity is selected from one of the following structures: 。 6. The use of a dual-target inhibitor with anti-psoriasis activity as described in any one of claims 1 to 5, or a pharmaceutical salt thereof, in the preparation of a medicament for treating psoriasis.

7. The use of a dual-target inhibitor with anti-psoriasis activity as described in any one of claims 1 to 5, or a pharmaceutical salt thereof, in the preparation of a JAK / HDAC dual-target inhibitor.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation is made from a dual-target inhibitor with anti-psoriasis activity as described in any one of claims 1 to 5, or a pharmaceutical salt thereof, and medically acceptable excipients.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a dual-target inhibitor with anti-psoriasis activity as described in any one of claims 1 to 5, a pharmaceutically acceptable carrier, and other pharmaceutically acceptable drugs for treating psoriasis.

10. The use of a dual-target inhibitor with anti-psoriasis activity as described in any one of claims 1 to 5, or a pharmaceutical salt thereof, in the preparation of an anti-inflammatory medicament.

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