Application of Phelligridimer A-based compounds in the preparation of JAK1-STAT1 signaling pathway inhibitors

Abstract

This invention discloses the application of Phelligridimer A-based compounds in the preparation of JAK1-STAT1 signaling pathway inhibitors. This invention reveals for the first time a novel biological function of Phelligridimer A: exerting its anti-inflammatory effect by selectively inhibiting the JAK1-STAT1 pathway within the JAK-STAT signaling pathway. Phelligridimer A exhibits strong molecular interactions with both JAK1 and STAT1. Compared to the most widely used highly selective JAK1 inhibitor globally (representative drug utpatinib), Phelligridimer A demonstrates a higher binding affinity for both JAK1 and STAT1, resulting in superior therapeutic efficacy in colitis models.

Description

Technical Field

[0001] This invention relates to the technical field of medicinal chemistry, and in particular to the application of compounds with Phelligridimer A as the parent nucleus in the preparation of JAK1-STAT1 signaling pathway inhibitors. Background Technology

[0002] The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is one of the core pathways mediating cytokine signal transduction, widely involved in various physiological and pathological processes such as immune responses, inflammatory responses, and cell proliferation and differentiation. The JAK family includes four members: JAK1, JAK2, JAK3, and TYK2; the STAT family includes seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Different JAK-STAT combinations mediate the signal transduction of different cytokines. Among them, the JAK1-STAT1 signaling pathway plays a crucial role in the transduction of type I / II interferon (IFN) and interleukin (IL)-6. JAK1 plays a pivotal role in the signal transduction of pro-inflammatory cytokines; abnormal phosphorylation and activation of JAK1 kinase can lead to persistent phosphorylation of STAT1, thereby triggering immune dysregulation, amplified inflammation, and tissue damage. Numerous studies have confirmed that abnormal and persistent activation of the JAK1-STAT1 pathway is closely related to the occurrence and development of a variety of major diseases, including inflammatory bowel disease (including ulcerative colitis and Crohn's disease), rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, atopic dermatitis, psoriasis, and other inflammatory and autoimmune diseases. Therefore, inhibiting the JAK1-STAT1 signaling pathway, especially specifically blocking the phosphorylation of JAK1 and STAT1 proteins, has become an important strategy for the treatment of the above-mentioned diseases.

[0003] Clinically used JAK1-STAT1 signaling pathway inhibitors are mainly JAK inhibitors, including (1) pan-JAK inhibitors and (2) selective JAK1 inhibitors. They indirectly inhibit STAT1 phosphorylation by inhibiting JAK1 phosphorylation, thereby blocking STAT1 from entering the cell nucleus as a nuclear transcription factor to initiate downstream gene expression. As a first-generation JAK inhibitor, pan-JAK inhibitors have low selectivity and can bind to homologous targets such as JAK1, JAK2, JAK3, and TYK2. Their broad JAK family inhibition brings serious toxicity. For example, incorrect inhibition of JAK2 can lead to thrombocytopenia and anemia, and incorrect inhibition of JAK3 can cause immunodeficiency and infection. Safety issues have become a concern. This poses a significant obstacle to the development of this class of drugs. Selective JAK1 inhibitors, as second-generation JAK inhibitors, exhibit higher inhibitory effects on JAK1 than other JAK family proteins, offering advantages of high efficacy and low toxicity, greatly improving the safety of clinical use. Currently, four selective JAK1 inhibitor drugs have been approved for marketing globally: utpatinib, abuxitinib, filogrinib, and imaxitinib. However, both pan-JAK inhibitors and selective JAK1 inhibitors currently share a pyrrolopyrimidine or pyridopyrimidine backbone, exhibiting high structural homogeneity. Moreover, this structure relies on chemical synthesis, resulting in complex, costly, and environmentally challenging preparation processes. Therefore, the development of novel selective JAK1 inhibitors remains urgent.

[0004] Phelligridimer A is a 26-membered, highly oxidized, unsaturated macrocyclic compound derived from fungi. It was first discovered in Phellinus igniarius in 2005 and has since been found in other fungi such as Sanghuangporus, Phellinus linteus, and Phellinus baumii. Patent CN114957277B discloses the role of Phelligridimer A in the prevention and treatment of stroke and neurodegenerative diseases. However, there are no reports in the prior art of this compound acting as an inhibitor of the JAK1-STAT1 signaling pathway. Summary of the Invention

[0005] The purpose of this invention is to solve the problems in the prior art by proposing the application of compounds with Phelligridimer A as the parent core in the preparation of JAK1-STAT1 signaling pathway inhibitors, which can solve the above problems.

[0006] To achieve the above objectives, this invention proposes the application of compounds with Phelligridimer A as the parent nucleus in the preparation of JAK1-STAT1 signaling pathway inhibitors.

[0007] This invention also proposes the use of Phelligridimer A-based compounds in the preparation of drugs for the prevention or treatment of diseases associated with abnormal activation of the JAK1-STAT1 signaling pathway.

[0008] Preferably, the compound with Phelligridimer A as the parent nucleus is Phelligridimer A or a pharmaceutically acceptable salt, ester, glycoside, solvate, prodrug, stereoisomer, or tautomer thereof.

[0009] Preferably, the compound with Phelligridimer A as the parent nucleus is selected from derivatives of Phelligridimer A in which the hydroxyl groups at the C-3, C-4, 3'- or 4'-position of the benzene ring are replaced by methyl, ethyl, propyl, glucosyl, galactosyl, mannose, sulfate, or phosphate groups.

[0010] Preferably, the compound with Phelligridimer A as its core works by inhibiting the autophosphorylation of JAK1 protein, thereby inhibiting the JAK1-STAT1 signaling pathway.

[0011] Preferably, the compound with Phelligridimer A as its parent nucleus can bind to the SH2 domain of STAT1, inhibiting STAT1 recruitment to the receptor and reducing STAT1 phosphorylation activation.

[0012] Preferably, the compound with Phelligridimer A as its parent nucleus specifically inhibits the JAK1-STAT1 signaling pathway, reduces the transcriptional expression of downstream genes in the JAK1-STAT1 signaling pathway, and does not directly inhibit the NF-κB inflammatory signaling pathway.

[0013] Preferably, the diseases include, but are not limited to, inflammatory bowel disease, joint and spinal autoimmune diseases, skin autoimmune diseases, and systemic autoimmune diseases.

[0014] Preferably, the disease is ulcerative colitis, Crohn's disease, microscopic colitis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, psoriasis, atopic dermatitis, discoid lupus erythematosus, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, or mixed connective tissue disease.

[0015] The beneficial effects of this invention are: This invention reveals for the first time a novel biological function of Phelligridimer A in exerting its anti-inflammatory effect by selectively inhibiting the JAK1-STAT1 pathway in the JAK-STAT signaling pathway. Currently, the parent nuclei of clinically used JAK inhibitors are all pyrrolopyrimidine or pyridopyrimidine-based artificial fused heterocyclic skeletons, with highly homogeneous structures. Phelligridimer A has a highly oxidized 26-membered macrocyclic structure, which is fundamentally different from existing JAK inhibitors in chemical space. This invention provides a novel template for the design of macrocyclic JAK inhibitors.

[0016] Phelligridimer A exhibits strong molecular interactions with both JAK1 and STAT1. Compared to the most widely used highly selective JAK1 inhibitor globally (representative drug utpatinib), Phelligridimer A has a higher binding affinity for JAK1 and STAT1, resulting in superior therapeutic efficacy in colitis models.

[0017] Phelligridimer A is a secondary metabolite of edible and medicinal Phellinus linteus, which can be prepared on a large scale under mild conditions through biomanufacturing, avoiding the technical constraints of complex chemical reactions and the environmental pollution problems caused by the use of various organic solvents.

[0018] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the chemical structure of Phelligridimer A; Figure 2 The results of Western Blot analysis showed that Phelligridimer A inhibited the phosphorylation levels of JAK and STAT family proteins in LPS-induced mouse primary macrophages. Figure 3 The results show the expression of downstream genes of the JAK1-STAT1 pathway (iNOS, IFN-γ) and downstream genes of the NF-κB inflammatory signaling pathway (IL-1β, TNF-α) in LPS-induced mouse primary macrophages by Phelligridimer A. Figure 4 This is a schematic diagram showing the binding pattern of Phelligridimer A to the functional domain of the JAK1 protein (PBD: 7TDF) phosphokinase, as displayed by molecular docking results. Figure 5 This is a diagram showing the binding pattern of Phelligridimer A to the SH2 domain of STAT1 protein (PDB: 8D3F) as displayed by molecular docking results. Figure 6 The surface plasmon resonance (SPR) bimolecular interaction response map shows the real-time binding kinetics curves and fitting results of Phelligridimer A with JAK1 and STAT1 proteins. Figure 7 The changes in body weight and the disease activity index scores on day 10 of mice in the DSS-induced mouse colitis model; Figure 8 Photographs of the colon and changes in colon length in different groups of mice in a DSS-induced mouse colitis model; Figure 9 HE-stained pathological sections and pathological scores of colon tissue from different groups of mice in a DSS-induced mouse colitis model. Detailed Implementation

[0020] Phelligridimer A has the following properties: Figure 1 The structure shown is as follows.

[0021] The compound shown in the formula has a highly oxidized 26-membered unsaturated macrocyclic structure, and its molecular formula is C2. 52 H 32 O 20 Its molecular weight is approximately 976.8 g / mol.

[0022] Phelligridimer A can selectively inhibit the JAK1-STAT1 signaling pathway, specifically as follows: It selectively inhibits JAK1 in the JAK family, but has no significant inhibitory effect on JAK2, JAK3, and TYK2.

[0023] It selectively inhibits STAT1 in the STAT family, but has no significant inhibitory effect on STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6.

[0024] It can bind to JAK1 in the phosphokinase functional domain and inhibit the phosphorylation and activation of JAK1.

[0025] It can bind to the SH2 domain of STAT1, inhibiting STAT1 recruitment to the receptor and reducing STAT1 phosphorylation activation.

[0026] It can specifically inhibit the JAK1-STAT1 signaling pathway and reduce the transcriptional expression of downstream genes (such as iNOS and IFN-γ) without directly inhibiting the NF-κB inflammatory signaling pathway.

[0027] Phelligridimer A can be used to prepare drugs for the prevention or treatment of diseases associated with abnormal activation of the JAK1-STAT1 signaling pathway, including but not limited to: (1) Inflammatory bowel disease: ulcerative colitis, Crohn's disease; (2) Autoimmune diseases of the joints and spine: rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, reactive arthritis; (3) Skin autoimmune diseases: psoriasis, atopic dermatitis, lichen planus, discoid lupus erythematosus, dermatomyositis-related skin lesions; (4) Systemic autoimmune diseases: systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, mixed connective tissue disease.

[0028] Example 1: Preparation and structural identification of Phelligridimer A (1) Strain source and identification The strain used is the medicinal fungus *Sanghuangporus vaninii*, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.41355. The accession certificate has been submitted in the patent document with publication number CN119709420A.

[0029] (2) Fermentation culture The *Sanghuangporus vaninii* strain was inoculated into liquid PDA medium and cultured at 25°C and 180 rpm for approximately 14 days. The culture solution with fully colonized mycelium was then inoculated into solid culture bags at a rate of 5% (v / v) (formula: 40% mulberry sawdust, 30% cottonseed hulls, 25% wheat bran, 2.5% white sugar, 2.5% gypsum; water was added after preparing the solid material to a moisture content of 60%). After the mycelium had fully colonized the bags, the bags were cut open and cultured at room temperature and 85% humidity for one month before the fruiting bodies of *Sanghuangporus vaninii* were collected.

[0030] (3) Extraction, separation and purification The fruiting bodies of Phelligridimer A were dried and pulverized, and extracted with 85% acetone at a material-to-liquid ratio of 1:50. The extract was concentrated by rotary evaporation and then fully extracted with ethyl acetate. After the extract was dried by rotary evaporation, column chromatography was performed on a C8 SMB high-purity silica gel column. The column was eluted with a gradient of 30%, 40%, and 60% methanol solutions containing 0.1% formic acid. The 60% methanol eluent was collected using an automatic sample collector. After thin-layer chromatography analysis, the fractions corresponding to the Phelligridimer A standard were collected and combined, and column chromatography was performed again on a C8 SMB high-purity silica gel column to obtain a high-purity monomer (HPLC purity: 98.7%).

[0031] (4) Structural identification The extracted compound was a yellow amorphous powder, readily soluble in dimethyl sulfoxide; 13C NMR (125 MHz, DMSO-d6) δ 170.9, 170.6, 163.4, 163.0, 159.5, 158.0, 147.4, 146.5, 145.8, 145.4, 142.0, 129.8, 126.3, 126.0, 118.7, 118.2, 116.9, 115.7, 113.7, 112.3, 101.6, 100.6, 99.5, 95.1, 89.8, 51.7; the above spectral data are consistent with those in the literature (Wang Y, Wang SJ, Mo SY. Phelligridimer A, a highly oxygenated and unsaturated 26-membered macrocyclic metabolite with antioxidant activity from The fungus *Phellinus igniarius*. *ChemIFNorm* 2005, 7(21), 4733-4736. reported data consistent with Phelligridimer A, therefore it was identified as Phelligridimer A, with the structure as follows: Figure 1 As shown.

[0032] Example 2: Selective inhibition of the JAK1-STAT1 pathway by Phelligridimer A Cellular assays were performed on Phelligridimer A prepared in Example 1 to evaluate its selective inhibitory effect on the JAK1-STAT1 pathway.

[0033] 1. Experimental Methods: Isolation of mouse peritoneal macrophages: C57 / B6 mice were anesthetized and euthanized by cervical dislocation. 5 mL of DMEM culture medium was injected into the peritoneal cavity of the mice, and the cavity was immersed in 75% ethanol solution for 5 min. The mice were removed, and the abdomen was cut open under aseptic conditions. Peritoneal fluid was collected using a sterile pipette, and the peritoneal cavity was irrigated with 5 mL of serum-free DMEM culture medium. The irrigated fluid was then recovered. The collected peritoneal irrigated fluid was centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. The cell pellet was washed with DMEM culture medium, and the cells were resuspended in DMEM culture medium with 10% serum. The cells were then seeded into cell culture flasks. Taking advantage of the rapid adhesion of macrophages, the cells were purified to obtain purified peritoneal macrophages.

[0034] Cell culture and treatment: 5×10 5Mouse peritoneal macrophages were seeded per well in 6-well plates and cultured at 37°C and 5% CO2 in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were divided into two treatments: the model group was treated with LPS (1 μg / mL) to induce inflammatory activation of macrophages; the drug treatment group was treated with LPS (1 μg / mL) and Phelligridimer A (50 μg / mL) was added at the same time. The cell culture time for Western blot analysis was 24 h, and the cell culture time for gene expression analysis was 4 h.

[0035] Western blotting of protein levels: Cells were washed three times with PBS, and SD-1 lysis buffer containing protease inhibitors and phosphatase inhibitors was added. Proteins were extracted using a protein extraction kit, and protein concentration was determined using a BCA protein quantification kit. After adjusting the protein concentration to the same level, sample loading was added. Prepare the sample loading solution with buffer, denature at 95℃ for 5 minutes, and perform 4-12% SDS-PAGE electrophoresis. After electrophoresis, transfer the protein to an NC membrane using a semi-dry method. Block with 5% skim milk powder / TBST at room temperature for 1 hour. Add the following primary antibodies (1:1000 dilution): rabbit anti-p-JAK1 (Tyr1022 / 1023 / 1034 / 1035, Affinity, AF2012), rabbit anti-p-JAK2 (Tyr931, Affinity, AF3024), rabbit anti-p-JAK3 (Tyr981, Affinity, AF8160), rabbit anti-p-TYK2 (Tyr433, Affinity, AF7288), rabbit anti-p-STAT1 (Tyr701, Abcam, ab307836), and rabbit anti-p-STAT2 (Tyr631, Affinity). Rabbit anti-p-STAT3 (Tyr705, Affinity, AF3293), rabbit anti-p-STAT4 (Tyr693, Affinity, AF3441), rabbit anti-p-STAT5A (Tyr694, Abcam, ab278764), rabbit anti-p-STAT5B (Tyr699, Affinity, AF3305), rabbit anti-p-STAT6 (Tyr641, Affinity, AF3301), and rabbit anti-β-actin (Proteintech, 66009-1-Ig) were incubated overnight at 4°C on a shaker. The rabbit was washed three times with TBST for 5 minutes each time. HRP-labeled secondary antibody (goat anti-rabbit IgG, 1:2000 dilution) was added, and the rabbit was incubated at room temperature for 1 hour. The rabbit was washed three times with TBST. The rabbit was then developed using ECL chemiluminescent substrate, and images were acquired using a ChemiDoc MP gel imaging system (Bio-Rad).

[0036] Gene expression detection: Total RNA was extracted and cDNA was synthesized by reverse transcription according to the kit instructions. Ct values ​​were detected on a Real-time PCR instrument, with three replicates for each sample. The PCR reaction volume was 10 μL: 5 μL TB Green Master (ROX), 3 μL sterile water, 0.75 μL each of forward and reverse primers, and 0.5 μL cDNA. The PCR temperature program was 95℃ for 30 s preheating, 95℃ for 5 s, and 62℃ for 34 s, for 40 cycles. The target genes were iNOS, IFN-γ, IL-1β, and TNF-α, and the internal reference gene was GAPDH. The relative gene expression levels were calculated using the ΔΔCt method.

[0037] 2. Results Western blot results showed that ( Figure 2 Compared with the LPS model group, the grayscale of the p-JAK1 and p-STAT1 bands in the Phelligridimer A treatment group was significantly lower than that in the LPS group, while the grayscale of the protein bands of p-JAK2, p-JAK3, p-TYK2, p-STAT2, p-STAT3, p-STAT4, p-STAT5A, p-STAT5B, p-STAT6 and the internal reference β-actin did not change significantly. This indicates that Phelligridimer A specifically inhibits the JAK1-STAT1 signaling pathway in each JAK-STAT pathway, that is, it has selective inhibitory activity on the JAK1-STAT1 pathway.

[0038] Gene expression assays showed that, compared with the LPS group, Phelligridimer A significantly inhibited the mRNA expression of STAT1 downstream target genes iNOS and IFN-γ. Figure 3 A), but it has no inhibitory effect on IL-1β and TNF-α, downstream target genes of another inflammatory signaling pathway, NF-κB. Figure 3 (B) This indicates that Phelligridimer A does not inhibit the NF-κB inflammatory signaling pathway, but selectively inhibits the JAK1-STAT1 pathway.

[0039] Example 3: Molecular docking of Phelligridimer A with JAK1 and STAT1 proteins The interaction and possible interaction mechanisms between Phelligridimer A and JAK1 and STAT1 proteins were evaluated using computer-aided virtual molecular docking technology.

[0040] 1. Experimental Methods Protein structure preparation: Crystal structures of human JAK1 (PDB ID: 7TDF) and STAT1 protein (PDB: 8D3F) were obtained from the Protein Data Bank database; the protein structures were pretreated using AutoDock Vina 1.2.2 to remove water molecules, original ligands and non-standard residues.

[0041] Ligand structure preparation: The two-dimensional structure of Phelligridimer A was drawn using ChemDraw 22.0 software and imported into Chem3D Pro software for three-dimensional conformation optimization.

[0042] Molecular docking calculations: Molecular docking was performed using AutoDock Vina 1.2.2 software; docking calculations were run, and the top 10 conformations with the best binding modes were output. The conformation with the lowest binding energy was taken as the optimal result for analysis.

[0043] 2. Results Molecular docking results showed that Phelligridimer A and JAK1 bind to the functional domain of phosphokinase through electrostatic interactions. Figure 4 The binding energy is -9.036 kcal / mol, exhibiting binding stability; Phelligridimer A binds to the SH2 domain of STAT1 through hydrogen bonding and strong electrostatic interactions. Figure 5 The hydrogen bond is located at the tyrosine residue TYR666, with a binding energy as low as -16.967 kcal / mol, indicating a highly stable binding.

[0044] It is known that the phosphokinase domain is essential for the kinase activity of JAK, and the SH2 domain is essential for the recruitment of STAT1 to the receptor. Therefore, PhA may inhibit further phosphorylation of STAT1 by inhibiting JAK kinase activity or by masking the recruitment functional domain of STAT1.

[0045] Example 4: SPR analysis of the interaction between Phelligridimer A and JAK1 and STAT1 proteins Phelligridimer A prepared in Example 1 was subjected to SPR bimolecular interaction experiments with JAK1 and STAT1 proteins to evaluate its binding ability with the two proteins.

[0046] 1. Experimental Methods Instruments and Materials: SPR experiments were performed using the Biacore 8K molecular interaction analysis system; CM7 series sensor chips, amine coupling kits, and ethanolamine hydrochloride were all purchased from Cytiva; recombinant human JAK1 and STAT1 proteins were purchased from Unimicron Biotechnology Co., Ltd.

[0047] Protein coupling: JAK1 and STAT1 proteins were diluted to 80 μg / mL with acetate buffer at pH=4 and activated on the CM7 chip via EDC / NHS to bind the proteins to the chip; Phelligridimer A was dissolved in DMSO to prepare a 10 mM stock solution and then diluted with HBS-EP+ buffer to a series of concentrations of 0.78, 1.26, 3.125, 6.25, 12.5, 25, 50, and 100 μM; the binding time was 90 seconds and the dissociation time was 100 seconds; after the experiment, the chip surface was regenerated with 10 mM glycine-HCl (pH 2.0) at a flow rate of 30 μL / min for 40 seconds to start the next binding-dissociation cycle.

[0048] Kinetic assay: Phelligridimer A was first prepared into a 10 mM stock solution with DMSO, and then diluted with HBS-EP+ buffer to a series of concentrations: 0.78, 1.26, 3.125, 6.25, 12.5, 25, 50, and 100 μM (containing 5% DMSO); the sample was loaded at a flow rate of 30 μL / min, with a binding time of 90 seconds and a dissociation time of 100 seconds; after the experiment, the chip surface was regenerated with 10 mM glycine-HCl (pH 2.0) at a flow rate of 30 μL / min for 40 seconds, and the next binding-dissociation cycle was performed.

[0049] Data Analysis: Biacore Insight Evaluation was used to perform fitting analysis on the SPR data; firstly, solvent correction was performed to subtract the volume effect of DMSO; then, a 1:1 Langmuir model was used to globally fit the response values ​​at each concentration, and the equilibrium dissociation constant (K) was calculated. D ).

[0050] 2. Results SPR analysis results showed that Phelligridimer A and immobilized JAK1 protein ( Figure 6 A) STAT1 protein ( Figure 6 B) The binding of both proteins is concentration-dependent, and the response plots exhibit typical binding-dissociation curve characteristics. The equilibrium dissociation constant K with JAK1 and STAT1 proteins was calculated using a 1:1 Langmuir model for global fitting. D The concentrations were 4.54 μM and 7.38 μM, respectively, indicating that Phelligridimer A has a high affinity for and specifically binds to JAK1 and STAT1 proteins.

[0051] Example 5: Evaluation of the therapeutic effect of Phelligridimer A on colitis with abnormal activation of the JAK1-STAT1 signaling pathway Using mice with ulcerative colitis characterized by persistent abnormal activation of the JAK1-STAT1 signaling pathway as an inflammatory disease model, the in vivo anti-inflammatory activity of Phelligridimer A prepared in Example 1 was evaluated, and its efficacy was compared with that of utpatinib, a JAK1 selective inhibitor commonly used in clinical practice for this disease, as a control drug.

[0052] 1. Experimental Methods Experimental animals and grouping: SPF-grade male C57BL / 6 mice, 7-8 weeks old, weighing 20-24 g, were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd.; the mice were housed in an SPF-grade animal room with a 12-hour light / dark cycle and free access to food and water; after one week of acclimatization, they were randomly divided into 4 groups according to their weight (n=8 / group): (1) Normal control group (Control); (2) Colitis model group (DSS); (3) Ph A treatment group (DSS+Phelligridimer A 50 mg / kg); (4) Upatinib group (DSS+Upatinib 50 mg / kg).

[0053] Establishment of colitis model and drug administration regimen: Three days before DSS administration, mice in the Ph A treatment group and utpatinib group were given 50 mg / kg of the corresponding drug by gavage, while the normal control group and model group were given the corresponding volume of aqueous solution. Three days later, except for the normal control group, 2% (w / v) sodium dextran sulfate (DSS, MW 36,000-50,000, MP Biomedicals) was added to the drinking water of the other groups for 7 consecutive days, and then DSS administration was stopped to establish an acute colitis model. On the 10th day after DSS administration, the mice were euthanized by cervical dislocation, and samples were taken by dissection. The weight of the mice was measured daily during the feeding period.

[0054] Disease Activity Index (DAI) score: On the last day of the experiment, the body weight, stool characteristics, and bloody stool of each group of mice were observed and recorded, and the DAI score was calculated according to the Cooper criteria; Disease Activity Index = (body weight loss score + stool characteristics score + bloody stool score); specific scoring criteria: body weight loss score is 0 (no decrease), 1 (1-5%), 2 (5-10%), 3 (10-20%), 4 (>20%); stool characteristics score is 0 (normal), 2 (loose), 4 (diarrhea); bloody stool score is 0 (no bleeding), 2 (occult blood positive), 4 (gross bloody stool).

[0055] HE staining and pathological scoring: Mouse colons were fixed with 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and then cut into 4 μm thick sections. After dewaxing and hydration, the nuclei were stained with hematoxylin for 5 minutes, differentiated with 1% hydrochloric acid alcohol, and the cytoplasm was stained with eosin for 3 minutes. After graded dehydration, clearing, and mounting with neutral resin, the sections were observed and photographed under an optical microscope. Histological scoring was performed according to the Dieleman scoring criteria, including: degree of inflammation (0-3 points), lesion depth (0-3 points), and degree of crypt destruction (0-4 points), with a total score of 0-10 points.

[0056] 2. Results Weight and disease activity index scores show ( Figure 7 Compared with the normal control group, the body weight of mice in the colitis model group began to decrease from day 5 after DSS administration. Compared with the model group, the rate of body weight loss in the Phelligridimer A treatment group slowed down from day 8 to day 10, and the body weight loss in the positive control drug utpatinib treatment group also slowed down from day 8 to day 10, but its average body weight on day 10 was lower than that in the Phelligridimer A treatment group. The disease activity index scores of mice on day 10 showed that the disease activity index of the colitis model group was significantly higher than that of the normal control group. Compared with the model group, the disease activity index scores of both the Phelligridimer A treatment group and the positive control drug utpatinib group were significantly lower, and the degree of reduction in the Phelligridimer A treatment group was greater than that in the utpatinib group.

[0057] The colon length results showed ( Figure 8 In the colitis model group, the colon length of mice was significantly shorter than that of the normal control group. In the Phelligridimer A treatment group, the colon length was significantly increased compared with the model group. The effect of Phelligridimer A in increasing colon length was better than that of the utpatinib group, suggesting that Phelligridimer A can significantly reduce colonic tissue edema and shorten the colon, and its effect is better than that of the utpatinib group.

[0058] HE staining showed ( Figure 9 In the normal control group, the colonic mucosa structure was intact, the crypts were neatly arranged, goblet cells were abundant, and there was no inflammatory cell infiltration. In the DSS model group, there was extensive necrosis and shedding of the mucosal epithelium, a large number of crypts were lost and deformed, goblet cells were significantly reduced, and there was extensive infiltration of neutrophils and lymphocytes in the mucosa and submucosa, with some ulcers forming. In the Phelligridimer A treatment group, the pathological damage was significantly reduced, the mucosal structure was basically intact, only a small number of scattered inflammatory cells were seen in the submucosa, and no obvious ulcers were seen. In the utpatinib treatment group, the mucosal integrity was also significantly improved, but there were still mucosal defects. The inflammatory infiltration was reduced, but some ulcers were still visible. Histological pathology scores showed ( Figure 9B) The colitis model group showed a significant increase compared to the normal group, while both the Phelligridimer A treatment group and the utpatinib treatment group showed a significant decrease compared to the colitis model group. However, the decrease in the Phelligridimer A treatment group was greater than that in the utpatinib treatment group. These results indicate that Phelligridimer A has a significant therapeutic effect on ulcerative colitis characterized by persistent abnormal activation of the JAK1-STAT1 signaling pathway, and its therapeutic effect is superior to that of the commonly used clinical drug, the JAK1 selective inhibitor utpatinib.

[0059] The above embodiments are illustrative of the present invention and are not intended to limit the present invention. Any simple modifications to the present invention are within the scope of protection of the present invention.

Claims

1. Application of Phelligridimer A-based compounds in the preparation of JAK1-STAT1 signaling pathway inhibitors.

2. Application of Phelligridimer A-based compounds in the preparation of drugs for the prevention or treatment of diseases associated with abnormal activation of the JAK1-STAT1 signaling pathway.

3. The application as described in claim 1 or 2, characterized in that: The compound with Phelligridimer A as its parent nucleus is Phelligridimer A or its pharmaceutically acceptable salt, ester, glycoside, solvate, prodrug, stereoisomer, or tautomer.

4. The application as described in claim 3, characterized in that: The compounds with Phelligridimer A as the parent nucleus are selected from derivatives of Phelligridimer A in which the hydroxyl groups at the C-3, C-4, 3'- or 4'-position of the benzene ring are replaced by methyl, ethyl, propyl, glucosyl, galactosyl, mannose, sulfate, or phosphate groups.

5. The application as described in claim 1, characterized in that: The compound with Phelligridimer A as its core works by inhibiting the autophosphorylation of the JAK1 protein, thereby inhibiting the JAK1-STAT1 signaling pathway.

6. The application as described in claim 5, characterized in that: The compound with Phelligridimer A as its parent nucleus can bind to the SH2 domain of STAT1, inhibiting STAT1 recruitment to the receptor and reducing STAT1 phosphorylation activation.

7. The application as described in claim 5, characterized in that: The compound with Phelligridimer A as its parent nucleus specifically inhibits the JAK1-STAT1 signaling pathway, reduces the transcriptional expression of downstream genes in the JAK1-STAT1 signaling pathway, and does not directly inhibit the NF-κB inflammatory signaling pathway.

8. The application as described in claim 2, characterized in that: The diseases mentioned include, but are not limited to, inflammatory bowel disease, joint and spinal autoimmune diseases, skin autoimmune diseases, and systemic autoimmune diseases.

9. The application as described in claim 7, characterized in that: The diseases mentioned are ulcerative colitis, Crohn's disease, microscopic colitis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, psoriasis, atopic dermatitis, discoid lupus erythematosus, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, or mixed connective tissue disease.

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