Pde4 degrading agents having therapeutic utility in psoriasis and uses thereof

Abstract

This invention discloses a class of PDE4 degrading agents or their pharmaceutical salts for treating psoriasis, with a general structural formula selected from one of the following structures: The PDE4 degrading agents for treating psoriasis provided by this invention offer a new strategy for in-depth research and development of novel structural types of anti-psoriasis drugs. The compounds of this invention exhibit excellent in vitro degradation activity against PDE4 protein.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a class of PDE4 degrading agents for treating psoriasis and their applications. Background Technology

[0002] Psoriasis, also known as cowhide lichen, is a common chronic immune-mediated skin condition characterized primarily by skin redness and scaling. 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. This not only places a heavy physical burden on patients but also negatively impacts their mental health. Therefore, there is an urgent clinical need for highly effective medications to treat psoriasis.

[0003] Currently, treatments for psoriasis can be broadly categorized into topical therapy, phototherapy, and systemic therapy; however, each method has its limitations. With a deeper understanding of the pathological mechanisms of psoriasis, treatment options for... kinase ( Research on the STAT pathway, a signal transduction and transcription activator pathway, has spurred the development of... Small molecule inhibitors are a novel strategy for treating psoriasis. Examples include tofacitinib and baricitinib. Novel psoriasis therapies, represented by inhibitors, have demonstrated excellent efficacy and safety. These drugs can precisely target... Inhibiting key nodes in the STAT pathway reduces the production and release of crucial 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 treating psoriasis. HDACs affect 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 abnormal proliferation of epidermal cells, improving the skin condition of patients; they can also regulate the differentiation and function of immune cells, reducing 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.

[0004] PROTAC (Proteolysis-Targeting Chimera) technology is an emerging strategy for targeted protein degradation. It utilizes a small molecule chimera to simultaneously recruit the target protein and an E3 ubiquitin ligase, inducing ubiquitination of the target protein, which is then degraded by the proteasome. This process achieves catalytic degradation of the target protein, unlike the one-to-one structural inhibition model of traditional small molecule inhibitors. PROTAC technology has been widely applied in oncology, autoimmune diseases, and neurodegenerative diseases, becoming a hot topic in next-generation drug development. Summary of the Invention

[0005] The first objective of this invention is to provide a class of PDE4 degrading agents that have therapeutic effects on psoriasis.

[0006] A second object of the present invention is to provide the use of the PDE4 degrading agent for treating psoriasis in the preparation of a medicament for treating psoriasis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a class of PDE4 degrading agents for treating psoriasis or pharmaceutical salts thereof, the general structural formula being selected from one of the following structures:

[0009] ;

[0010] in,

[0011] L1 is selected from ;

[0012] n1 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0013] m1 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0014] L2 is selected from ;

[0015] n2 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0016] m2 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0017] L3 is selected from ;

[0018] n3 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0019] m3 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0020] L4 is selected from ;

[0021] n4 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0022] m4 is selected from positive integers from 1 to 20 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0023] Most preferably, the structure of the PDE4 degrading agent for treating psoriasis is selected from one of the following structures:

[0024] ; ; ; .

[0025] The medicinal salt is an acid addition salt formed by a PDE4 degrading agent for treating psoriasis 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, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid.

[0026] A second aspect of the present invention provides the use of the PDE4 degrading agent for treating psoriasis or a pharmaceutical salt thereof in the preparation of a medicament for treating psoriasis.

[0027] A third aspect of the present invention provides the use of the PDE4 degrading agent for treating psoriasis or a pharmaceutical salt thereof in the preparation of the PDE4 degrading agent.

[0028] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0029] The PDE4 degrading agent for treating psoriasis provided by this invention offers a new strategy for in-depth research and development of novel structural types of anti-psoriasis drugs. The compounds of this invention exhibit excellent in vitro degradative activity against PDE4 protein.

[0030] The PDE4 degrading agent with anti-psoriasis activity provided by this invention has good efficacy in various in vitro anti-psoriasis-related tests and in vivo imiquimod-induced mouse models. Compounds EPCD and EPCF showed better anti-psoriasis activity than the positive control drug E6005. Attached Figure Description

[0031] Figure 1This is a schematic diagram showing the results of the target compound degrading the PDE4D activity in A549 cells at a single concentration (3.3 μM).

[0032] Figure 2 A schematic diagram showing the results of the concentration gradient descent of compound 4BCC-F to degrade the PDE4D activity.

[0033] Figure 3 A schematic diagram showing the results of degrading the PDE4D activity by the concentration gradients of compounds EPVD and EPCF.

[0034] Figure 4 This is a schematic diagram showing the results of the concentration gradient descent of compound EPCD to degrade the activity of PDE4D.

[0035] Figure 5 This is a schematic diagram of the HE staining results of mouse dorsal skin in a study of the anti-psoriasis activity of compound EPCD and compound EPCF as topical medications (creams).

[0036] Figure 6 This is a schematic diagram of the mouse spleen results from a study on the anti-psoriasis activity of compounds EPCD and EPCF as topical medications (creams). 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, TCFH is tetramethylchlorourea hexafluorophosphate, NMI is N-methylimidazole, HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, DMF is N,N-dimethylformamide, DCM is dichloromethane, MeOH is methanol, THF is tetrahydrofuran, TEA is triethylamine, TFA is trifluoroacetic acid, and iso-Pro-OH is isopropanol.

[0039]

[0040] Example 1

[0041]

[0042] 2,4-Dichloro-6,7-dimethoxyquinazoline (1.00 g, 3.86 mmol) was mixed with 3-aminophenylboronic acid (0.190 g, 1.39 mmol), cesium carbonate (1.26 g, 3.86 mmol), and tetrakis(triphenylphosphine)palladium (0.178 g, 0.15 mmol) in a 100 mL two-necked flask. 18 mL of 1,4-dioxane was added, and the mixture was reacted under nitrogen protection for 5 min. Then, 6 mL of water was added (1,4-dioxane to water volume ratio 3:1), and the mixture was refluxed at 100 °C for 2.5 h. After the reaction, the solvent was evaporated under reduced pressure, and the mixture was extracted with 60 mL of water and 30 mL of ethyl acetate until no target compound was detected in the aqueous phase. The organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether:ethyl acetate (1:1) as the mobile phase to give compound EP1 as a white solid in 70.2% yield.

[0043]

[0044] Compound EP1 (0.14 g, 0.45 mmol) was dissolved in a high-temperature, pressure-resistant sealed bottle containing 1.35 mL of a mixed solvent consisting of THF and isopropanol in a 2:1 volume ratio. Methylamine solution (0.89 mL) was then added, and the reaction mixture was stirred at 130 °C for 4–6 h. After the reaction was complete, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol (50:1) as the mobile phase to give compound EP2 as a white solid in 63.2% yield.

[0045]

[0046] Compound EP2 (1.000 g, 3.22 mmol) and monomethyl terephthalate (0.580 g, 3.22 mmol) were dissolved in 30 mL of acetonitrile. Then, catalysts TCFH (1.083 g, 3.86 mmol) and NMI (0.556 g, 6.77 mmol) were added, and the mixture was sonicated and stirred overnight at room temperature. After the reaction was complete, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EP3 as a white solid in 83.6% yield.

[0047] Compound EP3 (1.0 g, 2.12 mmol) and lithium hydroxide (0.152 g, 6.36 mmol) were added to a round-bottom flask containing 15 mL of a mixed solvent, which was prepared by methanol and water in a volume ratio of 4:1. The reaction solution was stirred at room temperature for 1 h. After the reaction was completed, the methanol in the flask was evaporated, an appropriate amount of water was added, and the pH was adjusted to 3 with a 1 mol / L hydrochloric acid aqueous solution. A solid precipitated out. After washing with water and drying, a yellow solid, namely compound EP4, was obtained. The yield was 96.3%.

[0048] Compound EP4 (0.100 g, 0.218 mmol) and BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate) (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((2-aminoethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.137 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPCB, a white solid with a yield of 50.3%.

[0049]

[0050] Example 2

[0051]

[0052] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((3-aminopropyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.143 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate and concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPCC as a white solid in 55.3% yield.

[0053]

[0054] Example 3

[0055]

[0056] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((4-aminobutyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.150 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate and concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPCD as a white solid in 59.3% yield.

[0057]

[0058] Example 4

[0059]

[0060] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.156 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate and concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPCE as a white solid with a yield of 63.2%.

[0061]

[0062] Example 5

[0063]

[0064] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((5-aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.162 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate and concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPCF as a white solid in 64.6% yield.

[0065]

[0066] Example 6

[0067]

[0068] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.163 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate and concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPCA as a white solid in 53.5% yield.

[0069]

[0070] Example 7

[0071]

[0072] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.188 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPCB, a white solid with a yield of 55.2%.

[0073]

[0074] Example 8

[0075]

[0076] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.201 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPCC as a white solid in 60.7% yield.

[0077]

[0078] Example 9

[0079]

[0080] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, 4-((14-amino-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.221 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPCD as a white solid in 61.3% yield.

[0081]

[0082] Example 10

[0083]

[0084] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VL2 (0.225 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPVB as a white solid in 70.3% yield.

[0085]

[0086] Example 11

[0087]

[0088] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VL3 (0.231 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPVC as a yellow solid in 72.5% yield.

[0089]

[0090] Example 12

[0091]

[0092] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VL4 (0.236 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPVD as a yellow solid in 71.9% yield.

[0093]

[0094] Example 13

[0095]

[0096] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VL5 (0.243 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPVE as a yellow solid in 70.5% yield.

[0097]

[0098] Example 14

[0099]

[0100] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VL6 (0.249 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EPVF as a yellow solid in 75.3% yield.

[0101]

[0102] Example 15

[0103]

[0104] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VOL1 (0.238 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPVA as a yellow solid in 63.8% yield.

[0105]

[0106] Example 16

[0107]

[0108] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VOL2 (0.257 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPVB as a yellow solid in 65.9% yield.

[0109]

[0110] Example 17

[0111]

[0112] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VOL3 (0.276 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPVC as a yellow solid in 63.7% yield.

[0113]

[0114] Example 18

[0115]

[0116] Compound EP4 (0.100 g, 0.218 mmol) and BOP (0.096 g, 0.218 mmol) were added to a round-bottom flask and stirred at room temperature for 15 min. Then, VOL4 (0.295 g, 0.436 mmol) was added, followed by TEA (0.061 g, 0.654 mmol). The mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound EOPVD as a yellow solid in 66.8% yield.

[0117]

[0118]

[0119] Example 19

[0120]

[0121] Compound 2,3,4-trimethoxybenzoic acid (1.00 g, 4.72 mmol) and potassium carbonate (1.956 g, 14.15 mmol) were added to a round-bottom flask containing 15 mL of DMF, followed by methyl iodoform (0.676 g, 4.76 mmol). The mixture was reacted overnight at 50 °C. After the reaction was complete, the reaction was quenched, the mixture was extracted with ethyl acetate, the organic phase was concentrated, and the residue was purified by column chromatography using petroleum ether:ethyl acetate = 10:1 as the mobile phase to give compound HP1 as a white solid in 95.6% yield.

[0122] Compound HP1 (0.500 g, 2.21 mmol) and anhydrous aluminum chloride (0.883 g, 6.64 mmol) were added to a round-bottom flask and reacted at 10 °C for 5 min. Then, 20 mL of dichloromethane was added, and the reaction was carried out at 15 °C for 3 h. After the reaction was completed, the reaction was quenched, and the organic phase was extracted with dichloromethane and concentrated. The residue was purified by column chromatography using petroleum ether:ethyl acetate = 5:1 as the mobile phase to give compound HP2 as a white solid in 50.2% yield.

[0123]

[0124] Compound HP2 (0.500 g, 2.36 mmol) and TBSCI (tert-butyldimethylchlorosilane) (1.06 g, 7.08 mmol) were added to a round-bottom flask, followed by imidazole (0.32 g, 4.72 mmol) and 8 mL of DMF. The mixture was reacted at room temperature for 3 h. After the reaction was complete, the reaction was quenched, and the mixture was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using petroleum ether:ethyl acetate = 10:1 as the mobile phase to give compound HP3 as a white solid in 92.6% yield.

[0125] Compound HP3 (0.500 g, 1.76 mmol), lithium bis(trimethylsilyl)amino (1.76 g, 10.56 mmol), and 3,5-dichloro-4-methylpyridine (0.285 g, 1.76 mmol) were added to a round-bottom flask containing 20 mL THF and reacted at 0 °C for 3 h. After the reaction was complete, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using petroleum ether:ethyl acetate = 4:1 as the mobile phase to give compound HP4 as a white solid in 43.5% yield.

[0126] Compound HP4 (0.500 g, 1.21 mmol) and TBAF (tetrabutylammonium fluoride) (0.947 g, 3.63 mmol) were added to a round-bottom flask containing 20 mL THF and reacted at room temperature for 2 h. After the reaction was complete, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 2:1 as the mobile phase to give compound HP5 as a white solid in 70.2% yield.

[0127]

[0128] Compound HP5 (0.500 g, 1.47 mmol) and ethyl bromoacetate (0.49 g, 2.94 mmol) were added to a round-bottom flask containing 10 mL of DMF, followed by potassium carbonate (0.609 g, 4.41 mmol). The mixture was reacted overnight at 95 °C. After the reaction was complete, the reaction was quenched, the mixture was extracted with ethyl acetate, the organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 4:1 as the mobile phase to give compound HP6 as a white solid in 69.5% yield.

[0129]

[0130] Compound HP6 (0.500 g, 1.25 mmol) and lithium hydroxide (0.089 g, 3.75 mmol) were added to a round-bottom flask containing 15 mL of a mixed solvent, which was prepared by methanol and water in a volume ratio of 4:1. The reaction mixture was stirred at room temperature for 1 h. After the reaction was completed, the methanol in the flask was evaporated to dryness, and an appropriate amount of water was added. The pH was adjusted to 3 with a 1 mol / L hydrochloric acid aqueous solution, and a solid precipitated out. After washing with water and drying, a yellow solid, namely compound HP7, was obtained. Yield: 96.3%.

[0131] Compound HP7 (0.100 g, 0.251 mmol) and HATU (0.114 g, 0.301 mmol) were added to a round-bottom flask containing 5 mL DMF and stirred at room temperature for 30 min. Then, DIPEA (0.971 g, 0.753 mmol) and 4-((4-aminobutyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.173 g, 0.502 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound PPD, a light green solid, in 60.5% yield.

[0132]

[0133] Example 20

[0134]

[0135] Compound HP7 (0.100 g, 0.251 mmol) and HATU (0.114 g, 0.301 mmol) were added to a round-bottom flask containing 5 mL DMF and stirred at room temperature for 30 min. Then, DIPEA (0.971 g, 0.753 mmol) and 4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.180 g, 0.502 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound PPE, a light green solid, in 62.1% yield.

[0136]

[0137] Example 21

[0138]

[0139] Compound HP7 (0.100 g, 0.251 mmol) and HATU (0.114 g, 0.301 mmol) were added to a round-bottom flask containing 5 mL DMF and stirred at room temperature for 30 min. Then, DIPEA (0.971 g, 0.753 mmol) and 4-((5-aminohexyl)amino)-2-(2,6-dioxoperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.187 g, 0.502 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound PPF, a light green solid, in 66.2% yield.

[0140]

[0141] Example 22

[0142]

[0143] Compound HP7 (0.100 g, 0.251 mmol) and HATU (0.114 g, 0.301 mmol) were added to a round-bottom flask containing 5 mL DMF and stirred at room temperature for 30 min. Then, DIPEA (0.971 g, 0.753 mmol) and 4-((2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.181 g, 0.502 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound POPA, a light green solid, in 53.2% yield.

[0144]

[0145] Example 23

[0146]

[0147] Compound HP7 (0.100 g, 0.251 mmol) and HATU (0.114 g, 0.301 mmol) were added to a round-bottom flask containing 5 mL DMF and stirred at room temperature for 30 min. Then, DIPEA (0.971 g, 0.753 mmol) and 4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.203 g, 0.502 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound POPB, a light green solid, in 56.5% yield.

[0148]

[0149] Example 24

[0150]

[0151] Compound HP7 (0.100 g, 0.251 mmol) and HATU (0.114 g, 0.301 mmol) were added to a round-bottom flask containing 5 mL DMF and stirred at room temperature for 30 min. Then, DIPEA (0.971 g, 0.753 mmol) and 4-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.225 g, 0.502 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound POPC, a light green solid, in 58.1% yield.

[0152]

[0153] Example 25

[0154]

[0155] Compound HP7 (0.100 g, 0.251 mmol) and HATU (0.114 g, 0.301 mmol) were added to a round-bottom flask containing 5 mL DMF and stirred at room temperature for 30 min. Then, DIPEA (0.971 g, 0.753 mmol) and 4-((14-amino-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.247 g, 0.502 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to give compound POPD, a light green solid, in 56.4% yield.

[0156]

[0157]

[0158] Example 26

[0159]

[0160] 2,4-Dichloro-6,7-dihydrothieno[3,2-d]pyrimidine (0.476 g, 2.29 mmol) and (1-aminocyclobutyl)methanol hydrochloride (0.364 g, 2.65 mmol) were added to a round-bottom flask containing 10 mL of acetonitrile, followed by the addition of TEA (0.929 g, 9.18 mmol). Air was removed from the flask under nitrogen, and the mixture was stirred at 75 °C for 20 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate and concentrated. The residue was purified by column chromatography using petroleum ether:ethyl acetate = 2:1 as the mobile phase to give compound 4B1 as a white solid in 70.2% yield.

[0161] S-Naphthol (0.656 g, 2.29 mmol), 10 mL DCM, tetraisopropyl titanate (0.650 g, 2.29 mmol), and 2 mL water were added to a reaction flask under nitrogen protection. The reaction solution temperature was maintained at 20–25 °C to obtain a first reactor containing a titanium catalyst. Compound 4B1 (0.500 g, 1.85 mmol) was added to a reaction flask containing 10 mL dichloromethane. The first reactor containing the titanium catalyst was then added to the above reaction flask. The mixture was stirred at room temperature for 1 h. Then, tert-butyl hydroperoxide (0.261 g, 2.91 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 50:1 as the mobile phase to obtain compound 4B2, a white solid with a yield of 53.1%.

[0162] Methyl 2-chloropyrimidin-5-carboxylate (1.00 g, 5.88 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)cyclohex-3-enylcarboxylate (1.811 g, 5.88 mmol) were added to a reaction flask containing 20 mL of a mixed solvent consisting of acetonitrile and isopropanol in a 1:1 volume ratio. Then, 5 mL of a 25% potassium phosphate aqueous solution was added, followed by dichlorobis[di-tert-butyl-(4-dimethylaminophenyl)phosphine]palladium(II) (0.416 g, 0.588 mmol). Under nitrogen protection, the mixture was stirred at 78 °C for 3 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using petroleum ether:ethyl acetate in a 4:1 mobile phase to give compound 4C1 as a white solid in 76.8% yield.

[0163]

[0164] Compound 4C1 (0.500 g, 1.57 mmol) was added to a reaction flask containing 10 mL of methanol, followed by Raney nickel (0.461 g, 7.85 mmol). The mixture was stirred at room temperature for 3 h under a hydrogen atmosphere. After the reaction was completed, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography with petroleum ether:ethyl acetate = 2:1 as the mobile phase to give compound 4C2 as a white solid with a yield of 92.1%. 1 H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 2H), 4.00 (d, J = 13.1Hz, 2H), 3.02 (tt, J = 11.6, 3.7 Hz, 1H), 2.87 (s, 2H), 1.98 – 1.86 (m, 2H),1.67 – 1.51 (m, 2H), 1.40 (s, 9H).

[0165] Compound 4C2 (0.500 g, 1.56 mmol) was added to a reaction flask containing 10 mL of dichloromethane, followed by 2 mL of TFA. The mixture was stirred at room temperature for 1 h. After the reaction was completed, the reaction solution was concentrated to obtain compound 4C3, a white solid, with a yield of 91.9%.

[0166] Compound 4C3 (0.500 g, 2.26 mmol) was added to a reaction flask containing 10 mL of NMP, followed by compound 4B2 (0.415 g, 1.88 mmol) and DIPEA (0.728 g, 5.64 mmol). Under nitrogen protection, the mixture was stirred overnight at 95 °C. After the reaction was complete, the reaction was quenched, and the mixture was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using petroleum ether:ethyl acetate = 1:1 as the mobile phase to give compound 4CA as a yellow solid with a yield of 73.1%.

[0167] Compound 4CA (0.500 g, 1.06 mmol) was added to a reaction flask containing 20 mL of a mixed solvent, which was prepared by methanol and water in a 1:1 volume ratio. Lithium hydroxide (0.076 g, 3.18 mmol) was then added, and the mixture was stirred overnight at room temperature. After the reaction was completed, the methanol in the flask was evaporated to dryness, and an appropriate amount of water was added. The pH was adjusted to 3 with a 1 mol / L hydrochloric acid aqueous solution, and a solid precipitated out. After washing with water and drying, a yellow solid, namely compound 4CB, was obtained, with a yield of 96.1%.

[0168] Compound 4CB (0.100 g, 0.218 mmol) and HATU (0.094 g, 0.262 mmol) were added to a round-bottom flask containing 5 mL of LDM. The mixture was stirred at room temperature for 30 min. Then, DIPEA (0.084 g, 0.654 mmol) and 4-((2-aminoethyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.138 g, 0.436 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 10:1 as the mobile phase to give compound 4BCB as a yellow solid with a yield of 65.1%.

[0169]

[0170] Example 27

[0171]

[0172] Compound 4CB (0.100 g, 0.218 mmol) and HATU (0.094 g, 0.262 mmol) were added to a round-bottom flask containing 5 mL of LDMF and stirred at room temperature for 30 min. Then, DIPEA (0.084 g, 0.654 mmol) and 4-((3-aminopropyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.143 g, 0.436 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 10:1 as the mobile phase to give compound 4BCC as a yellow solid with a yield of 69.1%.

[0173]

[0174] Example 28

[0175]

[0176] Compound 4CB (0.100 g, 0.218 mmol) and HATU (0.094 g, 0.262 mmol) were added to a round-bottom flask containing 5 mL of LDMF and stirred at room temperature for 30 min. Then, DIPEA (0.084 g, 0.654 mmol) and 4-((4-aminobutyl)amino)-2-(2,6-dioxo-piperidin-3-yl)isoindole-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.150 g, 0.436 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 10:1 as the mobile phase to give compound 4BCD as a yellow solid with a yield of 73.2%.

[0177]

[0178] Example 29

[0179]

[0180] Compound 4CB (0.100 g, 0.218 mmol) and HATU (0.094 g, 0.262 mmol) were added to a round-bottom flask containing 5 mL of LDMF and stirred at room temperature for 30 min. Then, DIPEA (0.084 g, 0.654 mmol) and 4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.156 g, 0.436 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction was quenched, extracted with ethyl acetate, and the organic phase was concentrated. The residue was purified by column chromatography using dichloromethane:methanol = 10:1 as the mobile phase to give compound 4BCE as a yellow solid with a yield of 69.5%.

[0181]

[0182] Example 30

[0183]

[0184] Compound 4CB (0.100 g, 0.218 mmol) and HATU (0.094 g, 0.262 mmol) were added to a round-bottom flask containing 5 mL of LDM. The mixture was stirred at room temperature for 30 min. Then, DIPEA (0.084 g, 0.654 mmol) and 4-((5-aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Shanghai Bied Pharmaceutical Technology Co., Ltd.) (0.162 g, 0.436 mmol) were added, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction was quenched, and the mixture was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by column chromatography using dichloromethane:methanol = 10:1 as the mobile phase to give compound 4BCF as a yellow solid with a yield of 68.9%.

[0185]

[0186] Example 31

[0187] The compounds prepared in the embodiments of this invention exhibit degradation activity against PDE4D protein in human lung cancer (A549) cells.

[0188] Cell line: Human lung cancer (A549) cells.

[0189] Experimental materials and instruments: DMEM medium, fetal bovine serum (10%), horse serum (5%), 1% (v / v) penicillin-streptomycin solution, phosphate-buffered saline (PBS), TBST, RIPA lysis buffer (strong), protease inhibitor, phosphatase inhibitor, BCA protein quantification kit, protein loading buffer, SDS-PAGE gel rapid preparation kit, polyvinylidene fluoride membrane (PVDF), electrophoresis buffer, rapid transfer buffer, microplate reader, microscope, CO2 incubator, centrifuge, ChemDoc high-sensitivity chemiluminescence imaging system (Bio-Rad), constant temperature water bath, electrophoresis apparatus, rapid transfer apparatus (Bio-Rad), power supply; PDE4D primary antibody (rabbit antibody, Abcam, #ab171749, 1:500), GAPDH primary antibody (rabbit antibody, Affinity, #AF7021, 1:10000), fluorescent secondary antibody (rabbit antibody, Abcam, #ab216777, 1:5000).

[0190] Experimental method: A549 cells were collected by digestion, and cells in the logarithmic growth phase were divided into groups of 4 × 10⁻⁶ cells. 5Cells were added to 6-well plates at a density of 100 cells / well, with 2 mL of complete DMEM medium added to each well. The plates were incubated for 24 h. After 24 h, the supernatant was discarded, and 2 mL of DMEM medium containing the compound was added to each well. Incubation continued (adjusting the concentration and method of addition according to different experimental requirements). After termination of incubation, the supernatant was aspirated, and the cells were gently washed three times with 4°C PBS. 50–100 μL of lysis buffer was added to each well, and the cells were incubated on ice for 15 min to lyse. The cells were then lysed and placed in 1.5 mL EP tubes. The cell suspension was collected and vortexed three times, then centrifuged at 4°C and 12000 rpm for 15 min. 2 μL of the supernatant was collected, and the protein concentration was measured using a microplate reader. A certain volume of the supernatant was taken, Loading Buffer was added, and the mixture was vortexed. The protein was then denatured at 100°C for 15 min in a metal bath. The loading amount was calculated based on the measured protein concentration and loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) for electrophoresis (70 V, 2 h). After complete protein separation, the protein was transferred to a PVDF membrane at a constant voltage of 25 V for 8 min and incubated with rapid blocking buffer at room temperature for 30 min. After blocking, the membrane was washed three times with TBST buffer for 5 min each time, and then incubated overnight at 4 °C with PDE4D rabbit monoclonal antibody (1:500 dilution). After recovering the primary antibody, the membrane was washed three more times with TBST. Subsequently, it was incubated with chemiluminescent secondary antibody at room temperature for 2 h. The secondary antibody was recovered, and the membrane was washed three times with TBST. Finally, the membrane was placed in an imager for chemiluminescence signal detection and exposure imaging.

[0191] Experimental results are as follows Figure 1 As shown, Figure 1 This is a schematic diagram showing the results of the target compound degrading PDE4D activity in A549 cells at a single concentration (3.3 μM). From Figure 1 As can be seen, compounds EPCD, EPCF and EPVD exhibit degradation activity at a concentration of 3.3 μM.

[0192] The degradation activities of compounds EPCD, EPCF, EPVD, and the 4BC series compounds are as follows: Figures 2-4 As shown, Figure 2 A schematic diagram showing the results of the concentration gradient descent of compound 4BCC-F to degrade the PDE4D activity. Figure 3 A schematic diagram showing the results of degrading the PDE4D activity by the concentration gradients of compounds EPVD and EPCF. Figure 4 This is a schematic diagram showing the results of the concentration gradient descent of compound EPCD to degrade the activity of PDE4D.

[0193] To more comprehensively test the degradation activities of EPCD, EPCF, EPVD, and the 4BC series compounds, a concentration gradient assay was used to determine their degradation activity. The results showed that compound 4BCF exhibited the highest degradation activity (DC). 50 The value was 14.16 ± 1.57 nM, representing the degradation DC of EPVD. 50 The value was 275.3 ± 66.9 nM, indicating the degradation DC value of EPCF. 50 The value is 2 ± 0.195 nM, the degradation DC of 4 BCC. 50 The value is 400±80 nM, and the degradation DC of EPCD is... 50 The value was 28.56 ± 1.97 nM, indicating that these compounds all possess nanomolar-level degradation activity, with EPCD, EPCF, and 4BCF exhibiting the best degradation activity.

[0194] Example 32

[0195] The compounds prepared in the embodiments of the present invention were tested for their PDE4D target inhibition activity.

[0196] PDE4D protein, 3H-cGMP, and different concentrations of the compound were incubated at room temperature for 15 minutes, and then the reaction was terminated with 0.2 M ZnSO4 and 0.2 M Ba(OH)2. After high-speed centrifugation, the supernatant was mixed with scintillation solution, and the unhydrolyzed substrate value was measured using a PerkinElmer 2910 Liquid 708 scintillation counter. The PDE4 values ​​of the compound were tested at different concentrations and repeated three times.

[0197] The inhibitory activity of the compounds against PDE4D is shown in Table 1. The compounds exhibited inhibition rates of over 97% against PDE4D at concentrations of 1 μM and 10 μM, indicating strong inhibitory activity. At a concentration of 100 nM, the compounds showed inhibition rates exceeding 50% for EPCD and EOPCD, making them the compounds with the best inhibitory activity.

[0198] Table 1

[0199]

[0200] Example 33

[0201] In vitro TNF-α-induced inhibition of human keratinocyte HaCaT cells by the compounds of this invention.

[0202] 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 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 diluted drug solutions were prepared at concentrations of 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 stored at 4°C for later use.

[0203] Cell culture: Frozen HaCaT cells were removed from liquid nitrogen and placed in a 37°C water bath for 1-2 minutes. 3 mL of DMEM complete medium was added and transferred to a 15 mL centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 2 mL of DMEM complete medium was added to disperse the cells evenly. Finally, the cells were transferred to cell culture dishes and cultured in a CO2 incubator (5% CO2, 37°C) for 24 h. All cells used in the experiment were in the logarithmic growth phase.

[0204] CCK-8 assay for the anti-psoriasis activity of compounds:

[0205] Cell plating: HaCaT cells in the logarithmic growth phase were collected and plated using DMEM complete medium containing 10% fetal bovine serum to prepare a concentration of 3×10⁻⁶ cells / mL. 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.

[0206] 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.

[0207] 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.

[0208] OD450 determination: Prepare DMEM basal culture medium containing 10% CCK-8. Remove the 96-well plate, discard the cell supernatant and PBS buffer, add 100 μL of DMEM basal culture medium (containing 10% CCK-8) to each well, and incubate in a CO2 incubator for 0.5 to 1 h. Measure the absorbance (OD450) of each well at 450 nm using a microplate detector, and calculate the drug's inhibition rate and IC50 against psoriasis according to the formula. 50 The value is calculated by GraphPad.

[0209] Inhibition rate (%) = (OD450 value of control wells - OD450 value of experimental wells) / OD450 value of control wells - OD450 value of blank wells × 100

[0210] Table 2

[0211]

[0212] The structural formulas for BI1015550 and E6005 are shown below:

[0213]

[0214] The experimental results are shown in Table 2. Among them, compound EPCD is the compound with good inhibitory activity against TNF-α induced by antibody in vitro (IC50). 50 (0.26 μM), which is superior to two PDE4 inhibitor positive control drugs, BI1015550 and E6005. These results indicate that compound EPCD has superior in vitro anti-psoriatic activity compared to the positive control drugs.

[0215] Example 34

[0216] Experiments on imiquimod-induced psoriasis mouse model of the compound prepared in this invention

[0217] (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%.

[0218] (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.

[0219] (3) Study on the anti-psoriasis activity of compound EPCD and compound EPCF when administered topically to the skin.

[0220] 1) Preparation of creams (containing medications):

[0221] 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 3:7) according to the amounts in Table 3, heat to 80℃ and keep warm to obtain phase A.

[0222] Glycerin was added to water and heated to 80°C and kept warm to obtain phase B.

[0223] Phase A is slowly added to Phase B while stirring. 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, compounds EPCD and EPCF are added separately. After high-pressure homogenization, different creams are obtained.

[0224] Table 3

[0225]

[0226] 2) Experimental Treatment: The experiment was divided into the following groups (control group, normal group, model group, compound EPCD group, compound EPCF group, E6005 group, with drug concentrations of 0.5% and 1% 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 recorded. This model required continuous drug administration for 7 days. After the experiment, the bare skin of the mice was photographed and recorded. Then, the mice were dissected, and the bare skin on the back of the mice and the spleens were removed. The spleens were weighed and photographed, and the skin of the mice was stained with hematoxylin and eosin (HE).

[0227] Experimental results are as follows Figures 5-6 As shown, Figure 5 This is a schematic diagram of HE staining results of mouse dorsal skin in a study of the anti-psoriasis activity of topical medications (cream) of compounds EPCD and EPCF. The results show that the thickness of spinous cells in the skin of the model group mice was significantly higher than that in the control group, proving the successful establishment of the experimental model. Compound EPCF exhibited the best activity in the experimental group, significantly reducing the thickness of spinous cells in the dorsal skin of psoriatic mice, indicating that compound EPCF has good anti-psoriasis activity. Figure 6This is a schematic diagram of the spleen results in mice used to study the anti-psoriasis activity of compounds EPCD and EPCF (topical application as a cream). The results show that the spleens of mice in the control group were smaller, while those in the control group showed significant splenomegaly, indicating significant inflammation in the model mice. The spleen volume in the EPCF group was significantly reduced, indicating that EPCF has an inhibitory effect on splenomegaly in mice, demonstrating that EPCF has good anti-inflammatory activity.

[0228] 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 PDE4 degrading agents or their pharmaceutical salts for treating psoriasis, characterized in that, The general structural formula is selected from one of the following structures: ； in, L1 is selected from ; n1 is selected from positive integers from 1 to 20; m1 is selected from positive integers from 1 to 20; L2 is selected from ; n2 is selected from positive integers from 1 to 20; m2 is selected from positive integers from 1 to 20; L3 is selected from ; n3 is selected from positive integers from 1 to 20; m3 is selected from positive integers from 1 to 20; L4 is selected from ; n4 is selected from positive integers from 1 to 20; m4 is selected from positive integers from 1 to 20.

2. The PDE4 degrading agent for treating psoriasis according to claim 1, or its pharmaceutical salt, is characterized in that, n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; m1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

3. The PDE4 degrading agent for treating psoriasis or its pharmaceutical salt according to claim 1, characterized in that, n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; m2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

4. The PDE4 degrading agent for treating psoriasis according to claim 1, or its pharmaceutical salt, is characterized in that, n3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

5. The PDE4 degrading agent for treating psoriasis according to claim 1, or its pharmaceutical salt, is characterized in that, n4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; m4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

6. The PDE4 degrading agent for treating psoriasis according to claim 1, or its pharmaceutical salt, is characterized in that, The structure of the PDE4 degrading agent for treating psoriasis is selected from one of the following structures: ； ； ； 。 7. The PDE4 degrading agent for treating psoriasis according to claim 1, or its pharmaceutical salt, is characterized in that, The medicinal salt is an acid addition salt formed by a PDE4 degrading agent for treating psoriasis 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, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid.

8. The use of a PDE4 degrading agent for treating psoriasis as described in any one of claims 1 to 7, or a pharmaceutical salt thereof, in the preparation of a medicament for treating psoriasis.

9. The use of a PDE4 degrading agent for treating psoriasis as described in any one of claims 1 to 7, or a pharmaceutical salt thereof, in the preparation of a PDE4 degrading agent.

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