Compound with anti-psoriasis activity and double-target inhibition and application thereof

Abstract

The invention discloses a compound with anti-psoriasis activity and double-target inhibition, and the structural general formula of the compound is shown in the specification. The compound with anti-psoriasis activity and double-target inhibition provided by the invention has good efficacy on in-vitro anti-psoriasis related tests and in-vivo imiquimod induced mouse models; the compound H7e shows better anti-psoriasis activity than their parent drugs and combined administration of the parent drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a class of compounds with anti-psoriasis activity and dual-target inhibition and their applications. Background Technology

[0002] Psoriasis, also known as cowhide lichen, is a common chronic immune-mediated skin condition characterized primarily by redness and scaling of the skin. The exact cause of the disease is not fully understood, but genetic, immune abnormalities, and environmental factors are known to be involved. Unlike other diseases, psoriasis is prone to relapse, and there is currently no specific cure, making treatment challenging and placing a heavy physical burden on patients while also impacting their mental health. Therefore, there is an urgent clinical need for highly effective medications to treat psoriasis.

[0003] Currently, psoriasis treatments can be broadly categorized into topical therapy, phototherapy, and systemic therapy; however, each approach has its limitations. With a deeper understanding of the pathological mechanisms of psoriasis, research targeting the Janus kinase (JAK)-signal transduction and activating transcription factor (STAT) pathway has spurred the development of JAK small molecule inhibitors as a novel strategy for treating psoriasis. Novel psoriasis therapies, represented by JAK inhibitors such as tofacitinib and baricitinib, have demonstrated excellent efficacy and safety. These drugs can precisely target key nodes in the JAK / STAT pathway, reducing the production and release of key cytokines such as interleukin-23 (IL-23), IL-22, and interferon, thereby inhibiting abnormal proliferation of keratinocytes and infiltration of inflammatory cells. Furthermore, histone deacetylases (HDACs) are also considered potential new targets for psoriasis treatment. HDACs influence gene expression and cellular function by regulating the acetylation state of histones. Studies have shown that HDAC inhibitors can reduce the levels of inflammatory mediators in psoriatic skin lesions, such as tumor necrosis factor-α (TNF-α) and interleukin-17 (IL-17); they can also inhibit the expression of proliferation markers such as Ki-67 and PCNA in skin tissue and the abnormal proliferation of epidermal cells, improving the skin condition of patients; they can also regulate the differentiation and function of immune cells and reduce excessive immune responses. For example, they can inhibit the differentiation of Th17 cells and reduce the production of IL-17, thereby effectively alleviating the symptoms of psoriasis. Summary of the Invention

[0004] The purpose of this invention is to provide a class of compounds with anti-psoriasis activity and dual-target inhibition.

[0005] Another object of the present invention is to provide the use of the compound having anti-psoriasis activity and dual-target inhibition in the preparation of a medicament for treating psoriasis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a class of compounds with anti-psoriasis activity and dual-target inhibition, the general structural formula of which is shown below:

[0008]

[0009] in,

[0010] L is selected from one of the following structures:

[0011]

[0012] n1 is selected from integers from 0 to 20, preferably integers from 0 to 10, and most preferably 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0013] n2 is selected from integers from 0 to 20, preferably integers from 0 to 10, and most preferably 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0014] n3 is selected from integers from 0 to 20, preferably integers from 0 to 10, and most preferably 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0015] n4 is selected from integers from 0 to 20, preferably integers from 0 to 10, and most preferably 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0016] n5 is selected from integers from 0 to 20, preferably integers from 0 to 10, and most preferably 0, 1, 2, 3, 4, 5, 6, 7, 8.

[0017] Most preferably, the structure of the compound having anti-psoriasis activity and dual-target inhibition is selected from one of the following structures:

[0018]

[0019] In a second aspect, the present invention provides the use of the compound having anti-psoriasis activity and dual-target inhibition, or a pharmaceutical salt thereof, in the preparation of a medicament for treating psoriasis.

[0020] A third aspect of the present invention provides the use of the compound having anti-psoriasis activity and dual-target inhibition, or a pharmaceutical salt thereof, in the preparation of a JAK / HDAC dual-target inhibitor.

[0021] In a fourth aspect, the present invention provides a pharmaceutical formulation made from the compound having anti-psoriasis activity and dual-target inhibition, or a pharmaceutically acceptable salt thereof, and medically acceptable excipients.

[0022] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the compound having anti-psoriasis activity and dual-target inhibition, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and other pharmaceutically acceptable drugs for treating psoriasis.

[0023] The medicinal salt is an acid addition salt formed by a compound with anti-psoriasis activity and dual-target inhibition with 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.

[0024] In a sixth aspect, the present invention provides the use of the compound having anti-psoriasis activity and dual-target inhibition, or a pharmaceutical salt thereof, in the preparation of an anti-inflammatory medicament.

[0025] 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 H7e 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.

[0026] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0027] The present invention provides a compound with anti-psoriasis activity and dual-target inhibition of JAK / HDAC, wherein compound H7e exhibits significant inhibitory activity against JAK family enzymes, with an IC50 value of [missing information]. 50 The values ​​were: JAK1 8.88 nM, JAK2 2.10 nM, JAK3 24.43 nM, and TYK2 13.11 nM, respectively. Simultaneously, it also showed inhibitory effects on HDAC family enzymes, with IC50 values ​​for HDAC1, HDAC2, HDAC3, and HDAC6. 50 The values ​​were 49 nM, 5.3 nM, 14 nM, and 3.1 nM, respectively. At a concentration of 0.4 μM, compound H7e significantly inhibited LPS-induced nitric oxide (NO) release from human macrophages. It demonstrated good efficacy in in vitro anti-psoriasis studies and in an in vivo imiquimod-induced mouse model, exhibiting superior anti-psoriasis activity compared to its parent drugs and in combination with parent drugs.

[0028] 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.

[0029] 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

[0030] Figure 1 This is a schematic diagram showing the effect of compound H7e on acetylated H3, H4, and phosphorylated STAT3 as determined by Western blotting.

[0031] Figure 2 This is a schematic diagram of the results of a study on the anti-psoriasis activity of compound H7e as a topical drug (cream) on the back skin of mice.

[0032] Figure 3 This is a schematic diagram illustrating the research results on the anti-psoriasis activity of compound H7e in topical medication (cream).

[0033] Figure 4 This is a schematic diagram of the mouse spleen results from a study on the anti-psoriasis activity of compound H7e when applied topically as a cream.

[0034] Figure 5 This is a schematic diagram showing the ratio of compound H7e to acetylated H3 and GAPDH.

[0035] Figure 6 This is a schematic diagram showing the ratio of compound H7e to acetylated H4 and GAPDH.

[0036] Figure 7 This is a schematic diagram showing the ratio of phosphorylated STAT3 to GAPDH tested by compound H7e. Detailed Implementation

[0037] 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.

[0038] 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.

[0039] The preparation route of the compound with anti-psoriasis activity and dual-target inhibition of this invention is as follows:

[0040]

[0041] Example 1

[0042] In the first step, 4-methanesulfonylaminophenylboronic acid pinacol ester (0.100 g, 0.336 mmol) and 2,4-dichloropyrimidine (0.125 g, 0.841 mmol) were added to a round-bottom flask containing 15 mL of 1,4-dioxane and 5 mL of water. Tetraphenylphosphine palladium (0.389 g, 0.0336 mmol) and cesium carbonate (0.274 g, 0.841 mmol) were added, and the mixture was sonicated until the substrate was uniformly dispersed. The reaction was carried out 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, namely compound H1, with a yield of 63.2%.

[0043]

[0044] In the second step, ethyl 5-nitroindole-2-carboxylate (0.100 g, 0.427 mmol) and 10% palladium on carbon (0.023 g, 0.214 mmol) were added to a three-necked flask containing 15 mL of methanol. The reaction was carried out at room temperature for 1.5 h under the action of hydrogen. After the reaction was completed, the sample was filtered with diatomaceous earth and dried to obtain ethyl 5-aminoindole-2-carboxylate in 95% yield.

[0045] Ethyl 5-aminoindole-2-carboxylate (0.100 g, 0.352 mmol), compound H1 (0.079 g, 0.388 mmol), X-PHOS (2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl) (0.017 g, 0.035 mmol), Pd2(dba)3 (0.032 g, 0.035 mmol), and cesium carbonate (0.327 g, 0.880 mmol) were added to a round-bottom flask containing 20 mL of 1,4-dioxane. The mixture was placed under a nitrogen atmosphere and stirred at 100 °C for 12 h. After the reaction was complete, saturated brine was added, and the mixture was extracted with ethyl acetate, dried, and purified by silica gel chromatography (dichloromethane:methanol = 50:1) to give a yellow solid, compound H2, in 80.3% yield.

[0046]

[0047] In the third step, compound H2 (0.5 g, 1.108 mmol) and lithium hydroxide (0.075 g, 3.132 mmol) were added to a mixed solution containing 15 mL of methanol and water, with a volume ratio of methanol to water of 3:1. The reaction solution was stirred at room temperature for 1 h. After the reaction was completed, the methanol was evaporated, water was added, and the pH was adjusted to 3 with a 1 mol / L hydrochloric acid aqueous solution. The solid precipitated out, was washed with water and dried to obtain a yellow solid, namely compound H3, with a yield of 95.3%.

[0048]

[0049] In the fourth step, compound H3 (0.150 g, 0.355 mmol) and 10 mL of DMF were added to a 25 mL round-bottom flask. TEA (0.108 g, 1.065 mmol), BOP (benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate) (0.157 g, 0.355 mmol) and methyl 3-aminopropionate hydrochloride (0.099 g, 0.710 mmol) were added. The reaction was carried out at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, 2 mL of water was added to quench the reaction. Ethyl acetate was added for extraction, and the mixture was dried. The solution was then purified by silica gel chromatography (dichloromethane:methanol = 50:1) to obtain a yellow solid, namely compound H6a, with a yield of 62.1%.

[0050]

[0051] Fifth step: Dissolve hydroxylamine hydrochloride (4.670 g, 67 mmol) in 24 mL of methanol, add 12 mL of potassium hydroxide (5.610 g, 100 mmol) methanol solution under ice bath conditions, stir at room temperature for 0.5 h, filter after the reaction is complete, and the filtrate is the freshly prepared hydroxylamine methanol solution.

[0052] Compound H6a (0.102 g, 0.200 mmol) was placed in a round-bottom flask with 15 mL of hydroxylamine methanol solution and stirred at room temperature for 3–4 h. The mixture was monitored by TLC. After the reaction was complete, the mixture was evaporated to dryness under reduced pressure. 5 mL of water was added, followed by the addition of acetic acid to adjust the pH of the solution to 7. A yellow precipitate was formed. The precipitate was filtered, dried, and compound H7a was obtained with a yield of 56.2%.

[0053]

[0054] Example 2

[0055] Synthesis of compound H7b

[0056] Prepared according to the fourth step of compound H7a, replacing methyl 3-aminopropionate hydrochloride with methyl 4-aminobutyrate hydrochloride (0.109 g, 0.710 mmol) to obtain a yellow solid, namely compound H6b, with a yield of 69.3%.

[0057]

[0058] Compound H7b was prepared according to the fifth step of the preparation method in compound H7a, with a yield of 53.4%.

[0059]

[0060] Example 3

[0061] Synthesis of compound H7c

[0062] The compound H7a was prepared according to the fourth step of the preparation method. The methyl 3-aminopropionic acid hydrochloride in the fourth step was replaced with methyl 5-aminovalerate hydrochloride (0.119 g, 0.710 mmol) to obtain a yellow solid, namely compound H6c, with a yield of 70.2%.

[0063]

[0064] The compound was prepared according to step 5 of the preparation method in compound H7a, and a yellow solid, namely compound H7c, was obtained with a yield of 49.3%.

[0065]

[0066] Example 4

[0067] Synthesis of compound H7d

[0068] Prepared according to the fourth step of compound H7a, replacing methyl 3-aminopropionate hydrochloride with methyl 6-aminohexanoate hydrochloride (0.129 g, 0.710 mmol) to obtain a yellow solid, namely compound H6d, with a yield of 65.9%.

[0069]

[0070] The compound was prepared according to step 5 of the preparation method in compound H7a, and a yellow solid, namely compound H7d, was obtained with a yield of 51.3%.

[0071]

[0072] Example 5

[0073] Synthesis of compound H7e

[0074] Prepared according to the fourth step of compound H7a, replacing methyl 3-aminopropionic acid hydrochloride with methyl 7-aminoheptanoate hydrochloride (0.139 g, 0.710 mmol) to obtain a yellow solid, namely compound H6e, with a yield of 55.3%.

[0075]

[0076] The compound was prepared according to step 5 of the preparation method for compound H7a, yielding a yellow solid, namely compound H7e, with a yield of 49.5%.

[0077]

[0078] Example 6

[0079] Synthesis of compound H7g

[0080] Compound H3 (0.150 g, 0.355 mmol) and 10 mL of THF were added to a round-bottom flask, followed by HOBT (0.072 g, 0.533 mmol) and EDCI (0.102 g, 0.533 mmol). The mixture was stirred for 30 minutes, then DIPEA (0.137 g, 1.065 mmol) and methyl 4-piperidincarnate (0.076 g, 0.533 mmol) were added. The mixture was reacted at room temperature for 12 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. The solution was purified by silica gel chromatography (dichloromethane:methanol = 50:1) to give a yellow solid, namely compound H6 g, with a yield of 72.4%.

[0081]

[0082] The compound was prepared according to step 5 of the preparation method in compound H7a, and a yellow solid, namely compound H7g, was obtained with a yield of 56.9%.

[0083]

[0084] Example 7

[0085] Synthesis of compound H7h

[0086] The compound H6g was prepared by replacing methyl 4-piperidinecarboxylate with methyl aziridine-3-carboxylate hydrochloride (0.081 g, 0.533 mmol) to obtain a yellow solid, namely compound H6h, with a yield of 72.3%.

[0087]

[0088] The compound was prepared according to step 5 of the preparation method in compound H7a, and a yellow solid, namely compound H7h, was obtained with a yield of 40.3%.

[0089]

[0090] Example 8

[0091] Synthesis of compound H7x

[0092] The compound H7a was prepared according to the fourth step of the preparation method. The methyl 3-aminopropionate hydrochloride in the fourth step was replaced with methyl 4-aminomethylbenzoate hydrochloride (0.143 g, 0.710 mmol) to obtain a yellow solid, namely compound H6x, with a yield of 65.3%.

[0093]

[0094] The compound was prepared according to step 5 of the preparation method for compound H7a, and a yellow solid, namely compound H7x, was obtained with a yield of 69.3%.

[0095]

[0096] Example 9

[0097] Synthesis of compound H7p

[0098] Prepared according to the fourth step of compound H7a, replacing methyl 3-aminopropionate hydrochloride in the fourth step with methyl 3-aminomethylbenzoate hydrochloride (0.143 g, 0.710 mmol), to obtain a yellow solid, namely compound H6p, with a yield of 65.9%.

[0099]

[0100] The compound was prepared according to step 5 of the preparation method in compound H7a, and a yellow solid, namely compound H7p, was obtained with a yield of 50.6%.

[0101]

[0102] Example 10

[0103] Synthesis of compound H7q

[0104] Prepared according to the fourth step of compound H7a, replacing methyl 3-aminopropionate hydrochloride in the fourth step with methyl 4-aminomethylphenylacetate (0.127 g, 0.710 mmol), to obtain a yellow solid, namely compound H6q, with a yield of 60.3%.

[0105]

[0106] The compound was prepared according to step 5 of the preparation method in compound H7a, and a yellow solid, namely compound H7q, was obtained with a yield of 53.6%.

[0107]

[0108] Example 11

[0109] The compounds prepared in the embodiments of the present invention were tested for their inhibitory activity against JAK and HDAC targets.

[0110] The first step was to test the compound's inhibitory activity against JAK.

[0111] (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.

[0112] (ii) Lance Ultra Assay test experiment:

[0113] (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.

[0114] (b) Kinase response and termination:

[0115] 1) Add the kinase to freshly prepared kinase buffer to form an enzyme solution.

[0116] 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.

[0117] 3) Prepare substrate solution: Add substrate and reagents such as ATP to kinase buffer.

[0118] 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.

[0119] 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.

[0120] (c) Data reading: Fluorescence values ​​were measured on an Envision 2104 Multilabel Reader instrument.

[0121] (d) Data computation (IC) 50 ):

[0122] 1) Obtain the numerical ratio of fluorescence readings (Lance signal ratio (665nm / 615nm)).

[0123] 2) Convert the obtained data into a suppression percentage using a formula.

[0124]

[0125] “min” represents the reading of the control wells without enzyme; “max” represents the reading of the control wells with DMSO added.

[0126] 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:

[0127]

[0128] The second step is to test the compound's HDAC inhibition activity.

[0129] (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.

[0130] (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.

[0131] (iii) Determination of the HDAC1 enzyme activity of the compound:

[0132] (a) Equilibrate the 96-well black microplate to room temperature to reduce experimental error.

[0133] (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.

[0134] (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.

[0135] (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.

[0136] (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).

[0137] (f) Calculate the HDAC1 enzyme activity inhibition rate of the target compound:

[0138]

[0139] In GraphPad software, the IC50 of the compound was determined by fitting a curve of inhibition rate versus compound concentration. 50 value.

[0140] 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 H7e 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 H7e against other HDAC isoforms, were tested.

[0141] The inhibitory activity of the compounds against JAK1 and JAK2 is shown in Table 1. Among them, the compounds H7a, H7b, H7c, H7d, H7e, H7g, H7h, H7x, H7p, and H7q prepared in this invention all showed inhibitory activity greater than 90% against JAK1 and JAK2.

[0142] Table 1

[0143]

[0144] 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 H7d, H7e, H7p, and H7q all demonstrated excellent inhibitory activity against HDAC1 (inhibition rates of HDAC1 for compounds H7d, H7e, H7p, and H7q were all <100 nM). Structure-activity relationship studies showed that isohydroxamic acid structures exhibited excellent inhibition of HDAC1 activity, especially when the carbon chain length reached 5-6 carbon atoms (compounds H7d and H7e), where the inhibitory activity reached its optimal state (IC50). 50 (27 nM and 49 nM, respectively).

[0145] Table 2

[0146]

[0147] Based on the inhibitory activities of compounds against JAK and HDAC, the highly active compound H7e was selected to evaluate the selectivity of different JAK and HDAC isoforms. The inhibitory activity of compound H7e against JAK (JAK1, 2, 3, and TYK2) was determined, and the results are shown in Table 3. The results show that compound H7e has a potent inhibitory effect on all JAK isoforms (IC50, 100 mg / L). 50 Range: 2.1~24.43 nM).

[0148] Table 3

[0149]

[0150] The HDAC subtype selectivity evaluation results are shown in Table 4. The data in the table show that compound H7e exhibits significant inhibitory effects on HDAC1, HDAC2, HDAC3, and HDAC6 (IC50). 50 Range: 3.1~49 nM). Furthermore, compound H7e showed almost no inhibitory effect on HDAC8 (IC50). 50 > 1000 nM).

[0151] Table 4

[0152]

[0153] Example 12

[0154] In vitro TNF-α-induced inhibition experiment of the compound prepared in this invention on HaCaT cells

[0155] (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.

[0156] (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.

[0157] (iii) CCK-8 assay to test the anti-psoriasis activity of the target compound:

[0158] (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.

[0159] (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.

[0160] (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.

[0161] (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.

[0162]

[0163] Table 5

[0164]

[0165] The structural formulas of CYT387 and SAHA are shown below:

[0166]

[0167] The experimental results are shown in Table 5. Compounds H7d and H7e have certain anti-TNF-α-induced HaCaT cell proliferation activity (IC50). 50 <10 μM), among which, compound H7e 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 1.21 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).

[0168] Example 13

[0169] Experiments on the inhibition of NO release from macrophages by the compounds prepared in this invention.

[0170] Compound treatment: Compound H7e was prepared into a 10 mM solution using cell-grade DMSO. A total of 6 groups were set up (blank group, model group, H7e group, SAHA group, CYT387 group, SAHA+CYT387 group).

[0171] (i) Cell culture: The culture method is the same as in Example 12.

[0172] (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.

[0173] (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.

[0174] (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.

[0175] (a) Dilution of NO standard solution: used for fitting standard curve.

[0176] (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.

[0177] 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.

[0178] The experimental results are shown in Table 6:

[0179] Table 6

[0180]

[0181] The data in the table show that compound H7e exhibits excellent ability to inhibit LPS-induced NO release from macrophages and has a certain anti-inflammatory effect, which is superior to its parent drug and its combination (SAHA, CYT387, SAHA+CYT387).

[0182] 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 successful model establishment. All tested compounds and positive control drugs effectively inhibited NO release from human macrophages, and this inhibition was concentration-dependent. Notably, compound H7e at a concentration of 0.4 μM showed particularly significant inhibition of NO release from human macrophages, with an effect almost equivalent to the NO release level in the untreated control group, and superior to the SAHA, CYT387, and SAHA+CYT387 groups.

[0183] Example 14

[0184] Western Blot method was used to test the effects of the compounds prepared in this invention on signaling pathways.

[0185] (1) Cell culture and drug preparation: Same as in Example 13.

[0186] (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.

[0187] (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.

[0188] (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.

[0189] (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.

[0190] (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).

[0191] (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.

[0192] Figure 1 This is a schematic diagram illustrating the effects of compound H7e on acetylation of H3 and H4, and phosphorylation of STAT3, as determined by Western blotting. The figure shows that compound H7e effectively inhibits the deacetylation of histones H3 and H4, and this inhibition exhibits a significant concentration-dependent effect. Compound H7e also inhibits STAT3 phosphorylation in a concentration-dependent manner. These results indicate that compound H7e inhibits the JAK and HDAC signaling pathways within cells.

[0193] Figure 5This is a schematic diagram illustrating the ratio of acetylated H3 to GAPDH tested by compound H7e. The diagram shows that compound H7e increases the level of acetylated histone H3 in a dose-dependent manner, indicating that compound H7e inhibits the HDAC pathway intracellularly.

[0194] Figure 6 This is a schematic diagram illustrating the ratio of acetylated H4 to GAPDH tested by compound H7e. The diagram shows that compound H7e increases the level of acetylated histone H4 in a dose-dependent manner, indicating that compound H7e inhibits the HDAC pathway intracellularly.

[0195] Figure 7 This is a schematic diagram showing the ratio of phosphorylated STAT3 to GAPDH tested by compound H7e. Compound H7e increases the level of phosphorylated signal transducer and transcription activator STAT3 in a dose-dependent manner, indicating that compound H7e inhibits the JAK-STAT pathway in cells.

[0196] Example 15

[0197] Experiments on imiquimod-induced psoriasis mouse model of the compound prepared in this invention

[0198] (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%.

[0199] (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.

[0200] (a) Study on the anti-psoriasis activity of compound H7e after topical skin administration

[0201] 1) Preparation of creams (containing medications):

[0202] 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.

[0203] Glycerin was added to water and heated to 80°C and kept warm to obtain phase B.

[0204] 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 H7e is added. After high-pressure homogenization, different creams are obtained.

[0205] Table 7

[0206]

[0207] 2) Experimental Treatment: The experiment was divided into 6 groups (control group, normal group, H7e 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).

[0208] (b) Study on the anti-psoriasis activity of compound H7e after oral administration

[0209] 1) Drug preparation: Weigh the corresponding compound and add 5% cell-grade DMSO, 10% Solutol HS-15 and saline.

[0210] 2) Experimental treatment: The experiment was divided into 6 groups (control group, normal group, H7e group, SAHA group, CYT387 group, SHAH+CYT387 group, and the drug concentration in all groups was 20 mg / kg).

[0211] Experimental results are as follows Figures 2-4 As shown, Figure 2This is a schematic diagram of the results of a study on the anti-psoriasis activity of compound H7e (cream) on the back skin of mice. As shown in the diagram, the skin on the backs of mice in the control group was smooth, without erythema or scaling. The skin on the backs of mice in the model group was red, with almost all lesions covered in scales, showing obvious redness, swelling, and wrinkling. The lesions were thickened and protruding, 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, CYT387, and SAHA+CYT387) was red, with some lesions covered in scales, mainly fine scales, slightly raised above the normal skin surface, indicating poor anti-psoriasis activity. The skin on the backs of mice in the H7e group was smooth, without erythema or scaling, and the skin color was similar to that of the control group, exhibiting normal skin color. These results indicate that the in vivo anti-psoriasis activity of compound H7e is superior to that of the positive control drugs and their combination therapy.

[0212] Figure 3 This is a schematic diagram illustrating the anti-psoriasis activity of compound H7e as a topical medication (cream). In the diagram, A shows HE staining of mouse dorsal skin, and B shows the thickness of acanthosis cells in the mouse dorsal skin. The results show that the thickness of acanthosis cells in the model group was significantly higher than that in the control group, proving the successful establishment of the experimental model. Compound H7e exhibited the best activity in the experimental group, significantly reducing the thickness of acanthosis cells in the dorsal skin of psoriatic mice, indicating that compound H7e 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 H7e 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 that there was significant inflammation in the model mice. The spleen volume of the compound H7e group was significantly reduced, indicating that it has an inhibitory effect on splenomegaly in mice, suggesting that compound H7e has good anti-inflammatory activity.

[0213] 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 class of compounds possessing anti-psoriasis activity and dual-target inhibition, characterized in that, The general structural formula is as follows: ； in, L is selected from one of the following structures: ； n1 is selected from integers from 0 to 20; n2 is selected from integers between 0 and 20; n3 is selected from integers between 0 and 20; n4 is selected from integers between 0 and 20; n5 is selected from integers from 0 to 20.

2. The compound with anti-psoriasis activity and dual-target inhibition according to claim 1, characterized in that, n1 is an integer selected from 0 to 10.

3. The compound with anti-psoriasis activity and dual-target inhibition according to claim 1, characterized in that, n2 is selected from integers between 0 and 10; Alternatively, n3 is selected from integers from 0 to 10.

4. The compound with anti-psoriasis activity and dual-target inhibition according to claim 1, characterized in that, n4 is selected from integers between 0 and 10; Alternatively, n5 is selected from integers from 0 to 10.

5. The compound with anti-psoriasis activity and dual-target inhibition according to claim 1, characterized in that, The structure of the compound having anti-psoriasis activity and dual-target inhibition is selected from one of the following structures: 。 6. The use of a compound having anti-psoriasis activity and dual-target inhibition 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 compound with anti-psoriasis activity and dual-target inhibition 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 the compound with anti-psoriasis activity and dual-target inhibition as described in any one of claims 1 to 5, or its pharmaceutical salt and medically acceptable excipients.

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

10. The use of a compound having anti-psoriasis activity and dual-target inhibition 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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