Compound taking 7-denitrified guanine as skeleton, pharmaceutical composition and application

Abstract

The invention provides a compound taking 7-denitrified guanine as a skeleton, a pharmaceutical composition and application, and belongs to the field of chemical medicines. The invention prepares a compound as shown in a formula I, or a salt thereof, or a stereoisomer thereof. The compound provided by the invention can selectively inhibit non-receptor tyrosine kinase JAK3, almost has no inhibition effect on other kinases in the same family, such as JAK1, JAK2 and TYK2, has a selection coefficient up to 9000 times or more, and shows good selectivity and inhibition effect; meanwhile, the compound provided by the invention can effectively inhibit phosphorylation of a downstream channel STAT. The compound disclosed by the invention can be used for preparing medicines for treating rheumatoid arthritis and has a wide application prospect. Formula I

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine, and in particular relates to a compound with 7-deazaguanine as a skeleton, a pharmaceutical composition and uses thereof. Background Art

[0002] The Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway has broad functions, participating in numerous important biological processes, including cell proliferation, differentiation, apoptosis, and immune regulation. Inhibiting Janus kinases (JAKs) can prevent the development of hematologic diseases, rheumatoid arthritis, and related blood cancers. The JAK family currently consists of four members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). JAK3 is primarily expressed in the bone marrow, lymphatic system, endothelial cells, and vascular smooth muscle cells, while the other three members are expressed in virtually all tissues.

[0003] To date, several non-covalent JAK3 inhibitors based on 7-deazaadenine have been launched on the market or are in clinical trials, including tofacitinib, bactinib, and ruxolitinib.

[0004] However, these compounds, which act in a non-covalent manner, typically exhibit only moderate selectivity for individual JAK family members. When inhibiting JAK3, they can also exert off-target activity against other kinases, compromising their safety and efficacy. This is largely due to the high similarity of the adenosine triphosphate (ATP) pocket across the protein kinase family, making the design of truly isoform-selective JAK inhibitors difficult.

[0005] Furthermore, the selective JAK3 inhibitor (NIBR3049) exhibits less potent blockade of the STAT5 signaling pathway than the JAK1 / 3 inhibitor (tofacitinib). JAK1 predominates over JAK3 in γC cytokine signaling. Researchers have found that the lack of efficacy of NIBR3049 at cellular ATP concentrations is due to insufficient inhibitory potency in cells, rather than selectivity for JAK3. Therefore, the development of highly isoform-selective JAK3 inhibitors with sufficient cellular potency is still needed.

[0006] Patent document CN111848631B discloses a pyrrolo[2,3-d]pyrimidine derivative targeting EGFR mutations, with the structure: However, the compound had poor inhibitory activity against JAK3 kinase (IC 50 is 2.2 nM), which needs to be further improved. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a compound with 7-deazaguanine as the skeleton, a pharmaceutical composition and use.

[0008] The present invention provides a compound represented by formula I, or a salt thereof, or a stereoisomer thereof: Formula I in, R1 is selected from halogen; R2 is selected from a benzene ring, R3 is selected from hydrogen, or R2 and R3 together form a piperidine ring; R4 is selected from COR a , R a Selected from C2~C4 alkenyl; R5 is selected from C1~C5 alkyl.

[0009] Furthermore, the compound is one of the following compounds: .

[0010] The present invention also provides use of the above-mentioned compound, or a salt thereof, or a stereoisomer thereof in the preparation of a non-receptor tyrosine kinase inhibitor.

[0011] Furthermore, the non-receptor tyrosine kinase is JAK3.

[0012] Furthermore, the non-receptor tyrosine kinase inhibitor is a drug for treating severe combined immunodeficiency, autoimmune diseases, hematological malignancies, allergic diseases or skin diseases.

[0013] Furthermore, the autoimmune diseases include rheumatoid arthritis, psoriasis and psoriatic arthritis, and inflammatory bowel disease.

[0014] Furthermore, the hematological malignancies include T-cell leukemia / lymphoma and natural killer cell (NK cell) lymphoproliferative diseases.

[0015] Furthermore, the skin diseases include asthma and atopic dermatitis.

[0016] The present invention also provides a pharmaceutical composition, which is a preparation prepared by using the above-mentioned compound, or its salt, or its stereoisomer as an active ingredient and adding pharmaceutically acceptable excipients.

[0017] The compounds and derivatives provided herein can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0018] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be given the meaning that a person skilled in the art would give them based on the disclosure and context.

[0019] In the present invention, "halogen" is fluorine, chlorine, bromine or iodine.

[0020] An "alkyl" group is a hydrocarbon group formed by removing a hydrogen atom from an alkane molecule, such as methyl -CH3, ethyl -CH3CH2, etc. C1-C5 alkyl refers to a straight or branched hydrocarbon chain containing one to six carbon atoms.

[0021] "C2-C4 alkenyl" refers to an alkenyl group containing two to four carbon atoms.

[0022] In the present invention, "room temperature" is 25±5°C; "overnight" is 12±2h.

[0023] The present invention has achieved the following beneficial effects: (1) This invention successfully constructed a library of selective covalent JAK3 kinase inhibitors based on 7-deazaguanine by combining computer-aided design with empirical screening. Three series of 12 candidate molecules were obtained by optimizing the synthesis pathway through retrosynthetic analysis.

[0024] (2) Compound 18a exhibited excellent JAK3 inhibitory activity (IC 50 = 0.52 nM) and has family kinase selectivity (JAK3 / JAK1 >9000-fold).

[0025] (3) In vitro experiments confirmed that 18a can effectively block interleukin (IL)-2 / IL-4-induced phosphorylation of the JAK-STAT pathway, alleviate arthritis symptoms in a collagen-induced arthritis (CIA) mouse model, and has relatively ideal pharmacokinetic characteristics. Therefore, the research data show that compound 18a, as a selective JAK3 inhibitor, is a promising compound for the treatment of autoimmune diseases and provides an important reference for the design of covalent drugs targeting cysteine ​​residues.

[0026] (4) Compounds disclosed in patent document CN111848631B (IC 50 2.2 nM), the compound 18a of the present invention has stronger inhibitory activity against JAK3 kinase, IC 50 Up to 0.52 nM.

[0027] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0028] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the hydrogen spectrum of compound 18a.

[0030] Figure 2 is the hydrogen spectrum of compound 18b.

[0031] Figure 3 This is the hydrogen spectrum of compound 18c.

[0032] Figure 4 This is the hydrogen spectrum of compound 18d.

[0033] Figure 5 is the hydrogen spectrum of compound 20a.

[0034] Figure 6 is the hydrogen spectrum of compound 20b.

[0035] Figure 7 is the hydrogen spectrum of compound 20c.

[0036] Figure 8 is the hydrogen spectrum of compound 20d.

[0037] Figure 9 is the hydrogen spectrum of compound 20e.

[0038] Figure 10 This is the hydrogen spectrum of compound 20f.

[0039] Figure 11 is the hydrogen spectrum of compound 26a.

[0040] Figure 12 is the hydrogen spectrum of compound 26b.

[0041] Figure 13 Compound 18a inhibits the JAK3-dependent signaling pathway.

[0042] Figure 14 The figure shows the weight change trend of mice.

[0043] Figure 15 A) H&E staining images of the blank control group; B) H&E staining images of the drug-treated group (200 mg / kg) (magnification 20×, scale bar = 20 μm).

[0044] Figure 16 The lateral and frontal views of the left hind limb of mice: A) blank group; B) model group; C) drug A group; D) drug B group; E) drug C group; F) drug D group-1; G) drug D group-2; H) line graph of the average rheumatoid arthritis score of each group; I) line graph of the average body weight of each rheumatoid arthritis group. DETAILED DESCRIPTION

[0045] Unless otherwise specified, the raw materials and equipment used in the embodiments of the present invention are all known products and were obtained by purchasing commercial products. The main reagents are shown in Table 1.

[0046] Table 1 Reagents for some experiments of the present invention Main instruments: (1) Mass spectrometer: Q-TOF spectrometer, ESI ionization source, Bruker, Germany; (2) Nuclear magnetic resonance instrument: AV II-400 MHz, AV II-600 MHz or AV II-800 MHz, TMS as internal standard, Bruker, Germany.

[0047] Example 1. Synthesis of Compound 4 The synthetic route of compound 4 is as follows: The specific reaction process of compound 4 refers to Example 2 of patent document CN111848631B (ie, compound 20 in patent document CN111848631B).

[0048] Example 2. Synthesis of Compound 8 The synthetic route of compound 8 is as follows: The specific reaction process of Compound 8a and Compound 8e can be found in Example 1 of Patent Document CN111848631B (i.e., Compounds 15 and 18 in Patent Document CN111848631B). Compound 8c is commercially available.

[0049] Compound 7b: A mixture of 2-fluoro-5-nitrotoluene (1.55 g, 10 mmol) and N-methylpiperazine (100 mmol, 11 ml) was refluxed at 80°C. After the reaction, the mixture was allowed to cool to room temperature and diluted with water. The resulting solid was filtered, washed with water, and dried to obtain the product as a brown solid with a yield of over 90%.

[0050] Compound 8b: Compound 7b (1.18 g) and palladium on carbon (0.3 g) were dissolved in 50 mL of isopropanol, mixed, and heated to reflux. Hydrazine hydrate (3 mL dissolved in 10 mL of isopropanol) was added dropwise to the reflux solution. The reaction was continued. After cooling, the reaction mixture was filtered. The filtrate was concentrated under reduced pressure and dried to obtain the product as a brown solid with a yield of over 90%.

[0051] Compound 7d: A mixed solution of 4-fluoro-3-ynyl-nitrobenzene (1.65 g, 10 mmol) and N-methylpiperazine (11.5 ml, 104 mmol) was heated to 90°C for at least 6 hours. The mixture was allowed to cool to room temperature and diluted with water. The resulting solid was filtered, washed with water, and dried to obtain the product as a brown solid with a yield of over 90%.

[0052] Compound 8d: Dissolve compound 7d (2.2 g) and palladium on carbon (0.48 g) in 80 mL of isopropanol, mix, and heat under reflux. Add 4.8 mL of hydrazine hydrate dissolved in 20 mL of isopropanol dropwise to the reflux solution and continue the reaction. After cooling, filter the reaction mixture, concentrate the filtrate under reduced pressure, and dry to obtain the product as a brown solid with a yield of over 90%.

[0053] Example 3. Preparation of Compound 18a The synthetic route of compound 18a is as follows: 1. Preparation of compound 17a The specific reaction process of compound 17a refers to Example 8 of patent document CN111848631B (i.e., compound 9 in patent document CN111848631B).

[0054] 2. Preparation of compound 18a Compound 17a (250 mg, 0.35 mmol) was dissolved in 16 mL of methanol and stirred at room temperature for 5 minutes. A NaOH solution (176 μL of 0.4 g of sodium hydroxide dissolved in 1 mL of purified water, 10 mmol) was added dropwise to the reactor and stirred for 1 hour until the reaction was complete. The pH was adjusted to neutral with saturated sodium bicarbonate solution. The reaction solution was spin-dried, extracted, concentrated, and dried. The crude product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 5), concentrated under reduced pressure, and dried to yield 102 mg of a brown solid product in a 49% yield.

[0055] Characterization data of compound 18a ( Figure 1 ): HRMS (ESI+) m / z: calcd for C 26 H 27 IN7O2 + :596.1265 [M+H] + ; Found 596.1271 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.63 (d, J =2.3 Hz, 1H, NH), 10.53 (s, 1H, NH), 9.11 (s, 1H, NH), 8.09 (d, J = 2.5 Hz, 1H,ArH), 7.74–7.49 (m, 3H, ArH), 7.42 (t, J = 8.1 Hz, 1H, ArH), 7.09 (dd, J = 3.5,2.2 Hz, 1H, ArH), 6.98 (ddd, J = 8.0, 2.4, 1.0 Hz, 1H, ArH), 6.86 (d, J = 8.8 Hz,1H, ArH), 6.50 (dd, J = 17.0, 10.1 Hz, 1H, Acrylamide CH), 6.29–6.20 (m, 2H,ArH, Acrylamide CH), 5.75 (dd,J = 10.1, 2.0 Hz, 1H, Acrylamide CH), 2.80 (t, J =4.8 Hz, 4H, Piperazine H), 2.48 (s, 4H, Piperazine H), 2.23 (s, 3H, CH3). 13 CNMR (101 MHz, DMSO) δ 163.33, 161.82, 155.05, 154.74, 153.13, 146.04, 140.39,138.49, 131.80, 129.83, 128.49, 127.07, 122.03, 120.30, 119.10, 116.74,116.11, 112.78, 98.71, 98.33, 98.19, 54.97, 52.12, 45.78. Example 4. Preparation of Compounds 18b, 18c, and 18d The synthetic routes of compounds 18b, 18c, and 18d are as follows: 1. Preparation of compounds 17b, 17c, and 17d Compound 17b: To a round-bottom flask, add tert-butanol (30 mL), compound 10 (428 mg, 1 mmol), and compound 8b (184.64 mg, 0.9 mmol). Stir the reaction mixture at 360 rpm for 5-10 minutes. Add potassium carbonate (276 mg, 2 mmol), Pd(dba) (18 mg, 0.02 mmol), and XPHOS (19 mg, 0.04 mmol). Place the reaction mixture in an oil bath (110°C) and reflux under nitrogen for 3-3.5 hours. Monitor the reaction by TLC (developing solvent: CH2Cl2 / CH3OH = 100 / 5). Cool the reaction mixture to room temperature, spin dry, extract, dry, concentrate, and separate by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). Concentrate under reduced pressure and dry to obtain 328 mg of the product as a white solid in an approximately 61% yield. HRMS (ESI+) m / z: calculated for C 33 H 40 N7O4 + : 598.3136 [M+H] + ; Found598.3145 [M+H] + . 1H NMR (400 MHz, Chloroform- d ) δ 7.72 (s, 1H, 1H, NH), 7.60 –7.51 (m, 2H, NH, ArH), 7.36 (t, J = 8.1 Hz, 1H, ArH), 7.30 (d, J = 7.0 Hz, 2H,ArH), 7.05 – 6.95 (m, 2H, ArH), 6.93 (s, 1H, ArH), 6.91 – 6.85 (m, 1H, ArH),6.41 (dd, J = 16.9, 1.5 Hz, 1H, Acrylamide CH), 6.35 (d, J = 3.7 Hz, 1H, ArH),6.26 (dd, J = 16.8, 10.1 Hz, 1H, Acrylamide CH), 6.09 (s, 2H, CH2), 5.74 (dd, J =10.1, 1.5 Hz, 1H, Acrylamide CH), 2.90 (t, J = 4.8 Hz, 4H, Piperazine H), 2.63(s, 4H, Piperazine H), 2.40 (s, 3H, CH3), 2.19 (s, 3H, CH3), 1.18 (s, 9H, t -BuH). 13 C NMR (101 MHz, CDCl3) δ 178.46, 162.72, 155.74, 155.44, 153.60,145.62, 139.18, 135.93, 133.31, 131.21, 129.90, 128.22, 124.23, 121.15,119.61, 118.16, 116.88, 100.66, 99.55, 66.49, 55.68, 51.74, 45.95, 39.02,29.84, 27.69, 27.11, 17.92, 1.16, 0.14. Compound 17c: To a round-bottom flask, add tert-butanol (30 mL), compound 10 (428 mg, 1 mmol), and compound 8c (194.52 mg, 0.9 mmol). Stir the reaction mixture at 360 rpm for 5-10 minutes. Add potassium carbonate (276 mg, 2 mmol), Pd(dba) (18 mg, 0.02 mmol), and XPHOS (19 mg, 0.04 mmol). Place the reaction mixture in an oil bath (110°C) and reflux under nitrogen for 3-3.5 hours. Monitor the reaction by TLC (developing solvent: CH2Cl2 / CH3OH = 100 / 5). Cool the reaction mixture to room temperature, spin dry, extract, dry, concentrate, and separate by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). Concentrate under reduced pressure and dry to obtain 405 mg of the product as a white solid in an approximately 74% yield. HRMS (ESI+) m / z: calculated for C 33 H 37 N8O4 + : 609.2932 [M+H] + ; Found609.2936 [M+H] + . 1 H NMR (400 MHz, Chloroform- d ) δ 7.95 (s, 1H, NH), 7.90 (s, 1H, NH), 7.81 (d, J = 2.6 Hz, 1H, ArH), 7.42–7.32 (m, 2H, ArH), 7.32–7.26 (m,1H, ArH), 7.10 (s, 1H, ArH), 7.04 (d, J = 3.7 Hz, 1H, ArH), 6.96 (ddd, J = 8.1,2.4, 1.1 Hz, 1H, ArH), 6.86 (d, J = 8.9 Hz, 1H, ArH), 6.45 (d, J = 3.7 Hz, 1H,ArH), 6.41 (dd, J = 16.9, 1.7 Hz, 1H, Acrylamide CH), 6.31 (dd, J = 16.9, 9.9 Hz,1H, Acrylamide CH), 6.08 (s, 2H, CH2), 5.74 (dd, J= 9.9, 1.7 Hz, 1H,Acrylamide CH), 3.17 (t, J = 4.8 Hz, 4H, Piperazine H), 2.78 (t, J = 4.7 Hz, 4H,Piperazine H), 2.47 (s, 3H, CH3), 1.18 (s, 9H, t -BuH). 13 C NMR (101 MHz, CDCl3)δ 178.42, 163.86, 163.01, 155.03, 153.35, 149.71, 139.18, 135.65, 131.31,129.77, 128.03, 124.75, 124.08, 122.87, 119.56, 118.38, 118.06, 117.66,114.68, 107.04, 100.72, 100.08, 66.31, 55.10, 51.58, 45.70, 39.01, 29.82,27.09, 1.15, 0.13. Compound 17d: To a round-bottom flask, add tert-butanol (50 mL), compound 10 (802 mg, 1.87 mmol), and compound 8d (403 mg, 1.87 mmol). Stir the reaction mixture at 360 rpm for 5-10 minutes. Add potassium carbonate (517 mg, 3.75 mmol), Pd(dba) (68 mg, 0.075 mmol), and XPHOS (70 mg, 0.15 mmol). Place the reaction mixture in an oil bath (110°C) and reflux under nitrogen for 3-3.5 hours. Monitor the reaction by TLC (developing solvent: CH2Cl2 / CH3OH = 100 / 5). Cool the reaction mixture to room temperature, spin dry, extract, dry, concentrate, and separate by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). Concentrate under reduced pressure and dry to obtain 613 mg of the product as a white solid in an approximately 54% yield. HRMS (ESI+) m / z: calculated for C 34 H 38 N7O4 + : 608.2980 [M+H] + ;Found 608.2988 [M+H] + . 1 H NMR (400 MHz, Chloroform- d) δ 8.04 (s, 1H, NH), 7.61(d, J J = 2.7 Hz, 1H, ArH), 7.56 (d, J J = 9.0 Hz, 2H, ArH, NH), 7.44 (dd, J J = 8.9, 2.7Hz, 1H, ArH), 7.32 (t, J J = 8.1 Hz, 1H, ArH), 7.07 (s, 1H, ArH), 7.00–6.91 (m,2H, ArH), 6.77 (d, J J = 8.9 Hz, 1H, ArH), 6.39 (dd, J J = 16.9, 1.7 Hz, 1H,Acrylamide CH), 6.33 (d, J J = 3.7 Hz, 1H, ArH), 6.28 (dd, J J = 16.9, 9.9 Hz, 1H,Acrylamide CH), 6.07 (s, 2H, CH2), 5.70 (dd, J J = 9.9, 1.7 Hz, 1H, AcrylamideCH), 3.31 (s, 1H, Alkyne H), 3.13 (s, 4H, Piperazine H), 2.62 (s, 4H,Piperazine H), 2.36 (s, 3H, CH3), 1.17 (s, 9H, t -BuH). 13 ​​​​Compound 18b: Place the reaction apparatus under vacuum, add methanol (10 mL) and compound 17b (122 mg, 0.2 mmol), and stir. Add NaOH solution (1 mL of 0.4 g sodium hydroxide dissolved in 10 mL of purified water, 10 mmol). Continue stirring until the reaction is complete. Adjust the pH to neutral with saturated sodium bicarbonate solution. The reaction mixture is spin-dried, extracted, concentrated, and dried. The crude product is separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 8), concentrated under reduced pressure, and dried to yield 84 mg of a white solid product in an approximately 87% yield. Figure 2 , HRMS (ESI+) m / z: calcdfor C 27 H 30 N7O2 + : 484.2455 [M+H] + ; Found 484.2476 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6)δ 11.49 (d, J = 2.3 Hz, 1H, NH), 10.30 (s, 1H, NH), 8.87 (d, J = 3.5 Hz, 1H, NH),7.64 (t, J = 2.2 Hz, 1H, ArH), 7.58 (td, J = 8.1, 2.1 Hz, 1H, ArH), 7.46–7.36 (m,1H, ArH), 7.40–7.30 (m, 2H, ArH), 7.06 (dd, J = 3.6, 2.2 Hz, 1H, ArH), 7.02–6.91 (m, 1H, ArH), 6.78 (t, J = 7.8 Hz, 1H, ArH), 6.42 (dd, J = 16.9, 10.1 Hz,1H, Acrylamide CH), 6.30–6.20 (m, 2H, Acrylamide CH, ArH), 5.80–5.72 (m, 1H,Acrylamide CH), 2.72 (q, J= 5.2 Hz, 4H, Piperazine H), 2.43 (s, 4H, PiperazineH), 2.22 (s, 3H, CH3), 2.07 (d, J = 3.9 Hz, 3H, CH3). 13 C NMR (101 MHz, DMSO) δ163.28, 161.81, 155.36, 155.31, 153.25, 144.73, 140.26, 136.44, 131.69,131.66, 129.83, 127.26, 121.70, 120.80, 118.55, 116.88, 116.69, 115.94,112.83, 98.31, 98.25, 68.10, 57.94, 55.29, 51.62, 45.89, 17.54. Compound 18c: Place the reaction apparatus under vacuum, add methanol (10 mL) and compound 15k (122 mg, 0.2 mmol), and stir. Add NaOH solution (1 mL of 0.4 g sodium hydroxide dissolved in 10 mL of purified water, 10 mmol). Continue stirring until the reaction is complete. Adjust the pH to neutral with saturated sodium bicarbonate solution. The reaction mixture is spin-dried, extracted, concentrated, and dried. The crude product is separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 8), concentrated under reduced pressure, and dried to yield 86 mg of a white solid product in an approximately 87% yield. Figure 3 , HRMS (ESI+) m / z: calcdfor C 27 H 27 N8O2 + : 495.2251 [M+H] + ; Found 495.2256 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6)δ 11.67 (d, J = 2.3 Hz, 1H, NH), 10.36 (s, 1H, NH), 9.29 (d, J = 3.8 Hz, 1H, NH),8.04 (d, J = 10.1 Hz, 1H, ArH), 7.74 (dd, J = 9.1, 2.7 Hz, 1H, ArH), 7.67 (t,J =2.2 Hz, 1H, ArH), 7.61 – 7.54 (m, 1H, ArH), 7.42 (dq, J = 13.7, 8.7, 8.2 Hz,1H, ArH), 7.14 – 7.08 (m, 1H, ArH), 7.03 – 6.92 (m, 2H, ArH), 6.44 (dd, J =17.0, 10.1 Hz, 1H, Acrylamide CH), 6.33 – 6.18 (m, 2H, Acrylamide CH, ArH), 5.81 – 5.71 (m, 1H, Acrylamide CH), 3.01 (q, J = 4.9 Hz, 4H, Piperazine H), 2.56 – 2.51 (m, 4H, Piperazine H), 2.26 (s, 3H, CH3). 13 C NMR (101 MHz, DMSO) δ163.32, 161.88, 154.98, 154.64, 153.07, 148.80, 140.30, 136.23, 131.72,129.89, 127.22, 124.12, 122.17, 119.41, 118.22, 116.82, 116.21, 112.73,105.41, 98.85, 98.39, 68.09, 57.94, 54.65, 51.45, 45.57. Compound 18d: Place the reaction apparatus under vacuum, add methanol (20 mL) and compound 8m (735 mg, 1.21 mmol), and stir. Add 3 mL of NaOH solution (0.4 g of sodium hydroxide dissolved in 5 mL of purified water, 10 mmol) dropwise to the reaction mixture. Continue stirring until the reaction is complete. Adjust the pH to neutral with saturated sodium bicarbonate solution. The reaction mixture is spin-dried, extracted, concentrated, and dried. The crude product is separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 8), concentrated under reduced pressure, and dried to yield 292 mg of a white solid product in approximately 49% yield. Figure 4 ,HRMS (ESI+) m / z:calcd for C 28 H 28 N7O2 + : 494.2299 [M+H] +; Found 494.2310 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.62 (d, J = 2.2 Hz, 1H, NH), 10.60 (s, 1H, NH), 9.04 (s, 1H, NH), 7.70 (dt, J = 4.6, 2.3 Hz, 2H, ArH), 7.66 – 7.55 (m, 2H, ArH), 7.41 (t, J = 8.1Hz, 1H, ArH), 7.06 (dd, J = 3.6, 2.0 Hz, 1H, ArH), 6.98 (ddd, J = 8.1, 2.4, 0.9Hz, 1H, ArH), 6.74 (d, J = 8.9 Hz, 1H, ArH), 6.52 (dd, J = 17.0, 10.2 Hz, 1H,Acrylamide CH), 6.29 – 6.18 (m, 2H Acrylamide CH, ArH), 5.74 (dd, J = 10.2, 2.0Hz, 1H, Acrylamide CH)), 4.26 (s, 1H, Alkyne H), 2.97 (s, 4H, Piperazine H), 2.43 (d, J = 8.4 Hz, 4H, Piperazine H), 2.21 (s, 3H, CH3).. 13 C NMR (101 MHz, DMSO) δ 163.35, 161.83, 155.15, 155.04, 153.15, 148.09, 140.39, 135.52,131.85, 129.78, 127.01, 123.51, 121.82, 120.18, 117.85, 116.72, 116.07,115.25, 112.75, 98.52, 98.32, 84.63, 82.51, 54.90, 50.94, 45.82. Example 8. Modification and synthesis of covalent target heads The modification and synthesis routes of the covalent target head are as follows: 1. Preparation of Compound 19 For the specific reaction process of compound 19a and compound 19b, please refer to Examples 3 and 4 of patent document CN111848631B (i.e., compound 23 and compound 26 in patent document CN111848631B).

[0056] 2. Preparation of Compound 20 Compound 20a: Compound 11b (0.22 g, 0.5 mmol), N , N -Diisopropylethylamine (0.17 mL, 1 mmol) and tetrahydrofuran (25 mL) were added to the reactor. When the internal temperature of the reactor reached -3°C, a solution of acryloyl chloride in tetrahydrofuran (96 μL, 1 mmol) was added dropwise over 1 hour. During the addition, the temperature was maintained between -5°C and 0°C. The pH was adjusted with sodium bicarbonate solution, the mixture was concentrated, and the product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was concentrated under reduced pressure and dried to obtain 206 mg of a white solid product with a yield of approximately 82%. Figure 5 , HRMS(ESI+) m / z: calcdfor C 27 H 28 FN7O2 + : 502.2361 [M+H] + ; Found 502.2355 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.59 (t, J = 2.2 Hz, 1H, NH), 10.10 (s, 1H, NH), 9.13 (s, 1H, NH), 7.65(t, J = 2.2 Hz, 1H, ArH), 7.63 – 7.51 (m, 2H, ArH), 7.39 (t, J = 8.1 Hz, 1H,ArH), 7.23 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 7.09 (dd, J = 3.6, 2.2 Hz, 1H, ArH), 7.00 – 6.93 (m, 1H, ArH), 6.85 – 6.73 (m, 2H, Alkene CH, ArH), 6.27 (dd, J=3.5, 1.9 Hz, 1H, Alkene CH), 2.88 (t, J = 4.7 Hz, 4H, Piperazine H), 2.44 (t, J =4.8 Hz, 4H, Piperazine H), 2.21 (s, 3H, CH3), 1.84 (s, 3H, CH3). 13 C NMR (101MHz, DMSO) δ 163.85, 162.01, 155.23, 154.96, 153.22, 140.69, 136.70, 132.97,129.95, 125.90, 123.50, 122.12, 119.05, 116.70, 116.11, 114.09, 112.82,106.50, 98.70, 54.85, 50.51, 50.48, 45.83, 39.94, 17.67. Compound 20b: Compound 11c (0.23 g, 0.5 mmol), N , N -Diisopropylethylamine (0.17 mL, 1 mmol) and tetrahydrofuran (25 mL) were added to the reactor. When the internal temperature of the reactor reached -3°C, a solution of acryloyl chloride in tetrahydrofuran (96 μL, 1 mmol) was added dropwise over 1 hour. During the addition, the temperature was maintained between -5°C and 0°C. The pH was adjusted with sodium bicarbonate solution, the mixture was concentrated, and the product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was concentrated under reduced pressure and dried to obtain 217 mg of a white solid product with a yield of approximately 84%. Figure 6 , HRMS(ESI+) m / z: calcdfor C 27 H 28 ClN7O2 + : 518.2066 [M+H] + ; Found 518.2025 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.61 (t, J = 2.2 Hz, 1H, NH), 10.77 (s, 1H, NH), 9.15 (s, 1H, NH), 7.76(d, J= 2.5 Hz, 1H, ArH), 7.57 – 7.50 (m, 2H, ArH), 7.48 – 7.36 (m, 2H, ArH), 7.10 (dd, J = 3.6, 2.2 Hz, 1H, ArH), 7.03 – 6.95 (m, 1H, ArH), 6.91 (d, J = 8.9Hz, 1H, ArH), 6.29 (dd, J = 3.5, 1.9 Hz, 1H, Alkene CH), 2.87 (t, J = 4.8 Hz, 4H,Piperazine H), 2.47 (s, 4H, Piperazine H), 2.23 (s, 3H, CH3), 2.04 (s, 3H,CH3). 13 C NMR (101 MHz, DMSO) δ 161.96, 155.22, 154.89, 153.13, 150.83, 142.01,140.00, 137.60, 130.13, 127.75, 122.31, 120.52, 119.57, 117.75, 117.42,116.44, 113.07, 98.80, 98.48, 84.85, 75.90, 54.87, 50.94, 45.60, 3.35. Compound 20c: Compound 11b (0.23 g, 0.5 mmol), N , N -Diisopropylethylamine (0.17 mL, 1 mmol) and tetrahydrofuran (25 mL) were added to the reactor; when the internal temperature of the reactor reached -3°C, a tetrahydrofuran solution of acryloyl chloride (125 μL, 1 mmol) was added dropwise over 1 hour; during the addition, the temperature was maintained between -5°C and 0°C; the pH was adjusted with sodium bicarbonate solution, the mixture was concentrated, and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrated under reduced pressure, and dried to obtain 219 mg of a white solid product with a yield of approximately 85%. Figure 7 , HRMS(ESI+) m / z: calcdfor C 28 H 30 FN7O2 + : 516.2518 [M+H] + ; Found 516.2522 [M+H] + .1 H NMR (400 MHz, DMSO- d 6) δ 11.59 (t, J = 2.2 Hz, 1H, NH), 10.12 (s, 1H, NH), 9.13 (s, 1H, NH), 7.65(t, J = 2.2 Hz, 1H, ArH), 7.62 – 7.51 (m, 2H, ArH), 7.40 (t, J = 8.1 Hz, 1H,ArH), 7.23 (dd, J = 8.9, 2.5 Hz, 1H, ArH), 7.00 – 6.93 (m, 1H, ArH), 6.93 –6.74 (m, 2H, ArH), 6.27 (dd, J = 3.5, 1.9 Hz, 1H, Alkene CH), 6.09 (dt, J = 15.3,1.7 Hz, 1H, Alkene CH), 3.33 (s, 3H, CH3), 2.88 (t, J = 4.8 Hz, 4H, PiperazineH), 2.44 (t, J = 4.8 Hz, 4H, Piperazine H), 2.26 – 2.15 (m, 5H, CH 3, CH2). 13 C NMR(101 MHz, DMSO) δ 164.05, 162.06, 155.28, 154.99, 153.70, 153.26, 146.80,140.75, 136.86, 133.09, 130.01, 123.54, 122.19, 119.11, 116.75, 116.15,114.15, 112.87, 106.27, 98.73, 54.87, 50.51, 50.49, 45.83, 24.69, 12.47. Compound 20d: Compound 11c (0.23 g, 0.5 mmol), N , N-Diisopropylethylamine (0.17 mL, 1 mmol) and tetrahydrofuran (25 mL) were added to the reactor; when the internal temperature of the reactor reached -3°C, a solution of acryloyl chloride in tetrahydrofuran (125 μL, 1 mmol) was added dropwise for more than 1 hour; during the addition, the temperature was maintained between -5°C and 0°C; the pH was adjusted with sodium bicarbonate solution, and the product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3) without concentration, concentrated under reduced pressure, and dried to obtain 223 mg of a white solid product with a yield of approximately 84%. Figure 8 , HRMS (ESI+) m / z:calcd for C 28 H 30 ClN7O2 + : 532.2222 [M+H] + ; Found 532.2254 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.60 (t, J = 2.2 Hz, 1H, NH), 10.12 (s, 1H, NH), 9.15 (s, 1H, NH), 7.76 (d, J = 2.5 Hz, 1H, ArH), 7.64 (t, J = 2.2 Hz, 1H, ArH), 7.56 (dd, J = 8.2,2.0 Hz, 1H, ArH), 7.47 (dd, J = 8.9, 2.6 Hz, 1H, ArH), 7.40 (t, J = 8.1 Hz, 1H,ArH), 7.10 (dd, J = 3.6, 2.2 Hz, 1H, ArH), 7.00 – 6.92 (m, 1H, ArH), 6.95 –6.80 (m, 2H, ArH), 6.28 (dd, J = 3.6, 1.9 Hz, 1H, Alkene CH), 6.09 (dt, J = 15.3,1.7 Hz, 1H, Alkene CH), 3.33 (s, 3H, CH3), 2.86 (q, J= 4.7 Hz, 4H, PiperazineH), 2.46 (s, 4H, Piperazine H), 2.23 (s, 3H, CH3), 2.28 – 2.14 (m, 2H, CH2). 13 C NMR (101 MHz, DMSO) δ 164.03, 162.05, 155.23, 154.94, 153.23, 146.80,142.08, 140.76, 137.59, 130.04, 127.73, 123.52, 122.26, 120.52, 119.58,117.76, 116.68, 116.22, 112.83, 98.83, 98.52, 54.96, 51.06, 45.76, 24.67,12.45. Compound 20e: Compound 11b (0.23 g, 0.5 mmol), N , N -Diisopropylethylamine (0.17 mL, 1 mmol) and tetrahydrofuran (25 mL) were added to the reactor. When the internal temperature of the reactor reached -3°C, a solution of acryloyl chloride in tetrahydrofuran (86 μL, 1 mmol) was added dropwise over 1 hour. During the addition, the temperature was maintained between -5°C and 0°C. The pH was adjusted with sodium bicarbonate solution, the mixture was concentrated, and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was concentrated under reduced pressure and dried to obtain 202 mg of a white solid with a yield of approximately 81%. Figure 9 , HRMS(ESI+) m / z: calcdfor C 27 H 26 FN7O2 + : 500.2205 [M+H] + ; Found 500.2212 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.59 (d, J = 2.2 Hz, 1H, NH), 10.76 (s, 1H, NH), 9.13 (s, 1H, NH), 7.62– 7.52 (m, 2H, ArH), 7.56 – 7.49 (m, 1H, ArH), 7.40 (t, J = 8.1 Hz, 1H, ArH),7.22 (dd, J= 8.7, 2.5 Hz, 1H, ArH), 7.09 (dd, J = 3.6, 2.3 Hz, 1H, ArH), 7.03 –6.96 (m, 1H, ArH), 6.80 (t, J = 9.4 Hz, 1H, ArH), 6.28 (dd, J = 3.6, 1.9 Hz, 1H,ArH), 2.90 (t, J = 4.8 Hz, 4H, Piperazine H), 2.48 (d, J = 4.9 Hz, 4H, PiperazineH), 2.24 (s, 3H, CH3), 2.04 (s, 3H, CH3). 13 C NMR (101 MHz, DMSO) δ 161.93,156.07, 155.23, 154.92, 153.67, 153.13, 150.80, 139.96, 136.72, 133.03,130.06, 122.19, 119.05, 117.45, 116.32, 114.11, 113.09, 106.24, 98.66, 84.80,75.88, 54.81, 50.44, 45.73, 3.34. Compound 20f: Compound 11c (0.23 g, 0.5 mmol), N , N -Diisopropylethylamine (0.17 mL, 1 mmol) and tetrahydrofuran (25 mL) were added to the reactor. When the internal temperature of the reactor reached -3°C, a solution of acryloyl chloride in tetrahydrofuran (86 μL, 1 mmol) was added dropwise over 1 hour. During the addition, the temperature was maintained between -5°C and 0°C. The pH was adjusted with sodium bicarbonate solution, the mixture was concentrated, and the product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was concentrated under reduced pressure and dried to obtain 206 mg of a white solid product with a yield of approximately 80%. Figure 10 , HRMS (ESI+) m / z:calcd for C 27 H 26 ClN7O2 + : 516.1909 [M+H] + ; Found 516.1931 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6) δ 11.60 (d, J = 2.3 Hz, 1H, NH), 10.10 (s, 1H, NH), 9.15 (s, 1H, NH), 7.77 (d, J = 2.5 Hz, 1H, ArH), 7.64 (t, J = 2.2 Hz, 1H, ArH), 7.55 (dd, J = 8.0,2.0 Hz, 1H, ArH), 7.47 (dd, J = 8.8, 2.6 Hz, 1H, ArH), 7.39 (t, J = 8.1 Hz, 1H,ArH), 7.10 (dd, J = 3.6, 2.2 Hz, 1H, ArH), 6.95 (dd, J = 8.0, 2.3 Hz, 1H, ArH),6.90 (d, J = 8.8 Hz, 1H, ArH), 6.86 – 6.66 (m, 1H, ArH), 6.28 (dd, J = 3.6, 1.9Hz, 1H, ArH), 6.16 – 6.06 (m, 1H, ArH), 2.85 (t, J = 4.8 Hz, 4H, Piperazine H), 2.46 (s, 4H, Piperazine H), 2.23 (s, 3H, CH3), 1.85 (dd, J = 6.9, 1.7 Hz, 3H,CH3). 13 C NMR (101 MHz, DMSO) δ 163.82, 162.00, 155.18, 154.90, 153.20, 142.05,140.71, 140.59, 137.55, 129.98, 127.69, 125.89, 122.20, 120.47, 119.54,117.71, 116.62, 116.16, 112.77, 98.80, 98.48, 54.95, 51.07, 45.78, 17.66. Example 9. Synthesis of Compounds 26a and 26b The synthetic routes of compounds 26a and 26b are as follows: 1. Route 1 (meta-modification): (1) Preparation of compound 21 Compound 21: Compound 1 (13.2 mmol, 2.47 g) was dissolved in 50 mL of anhydrous tetrahydrofuran, and sodium hydride (13.8 mmol, 552 mg, 60% dispersion in mineral oil) was slowly added. The mixture was stirred at room temperature for 0.5 hours. The mixture was then cooled again to -10°C. 2-(Trimethylsilyl)ethoxymethyl chloride (13.9 mmol, 2.46 mL) dissolved in 10 mL of anhydrous tetrahydrofuran was gradually added to the mixture, and stirring continued for 30 minutes. Aqueous ammonium chloride was then added to quench the reaction, and the organic solvent was evaporated under reduced pressure. Dichloromethane was added for extraction. The organic phase was separated, washed with saturated brine, and dried over anhydrous sodium sulfate. The dried organic phase was concentrated under reduced pressure and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 30 / 1) to yield the compound as a white solid in approximately 98% yield.

[0057] (2) Preparation of compound 23a Compound 23a: Compound 21 (1.12 g, 3.53 mmol) and compound 22a (781 mg, 3.89 mmol) were dissolved in 60 mL of anhydrous tetrahydrofuran and cooled to -20°C. Potassium tert-butoxide (3.53 mmol, 396 mg) was slowly added, and stirring was continued for 0.5 hour. The reaction was quenched with aqueous ammonium chloride, and the solvent was removed by distillation under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate, ratios ranging from 50:1 to 10:1) to obtain compound 25a. The yield was approximately 84% as an oil. HRMS (ESI+) m / z: calculated for C 22 H 36 ClN4O4Si + : 483.2189 [M+H] + ; Found 483.2185 [M+H] + . (3) Preparation of compound 24a Compound 24a: To a round-bottom flask, tert-butanol (100 mL), compound 23a (3.34 g, 6.93 mmol), and compound 8f (1.32 g, 6.3 mmol) were added sequentially. The reaction mixture was stirred at 360 rpm for 5-10 minutes. Potassium carbonate (1.91 g, 13.86 mmol), Pd(dba) (128 mg, 0.14 mmol), and XPHOS (133 mg, 0.28 mmol) were added to the reaction solution. The reaction mixture was placed in an oil bath (110°C) and refluxed under nitrogen for 5 hours. Completion of the reaction was monitored by TLC (developing solvent: CH2Cl2 / CH3OH = 10 / 1). The reaction solution was cooled to room temperature, dried, extracted, concentrated, and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was concentrated under reduced pressure and dried to obtain 3.59 g of a white solid with a yield of approximately 87%. HRMS(ESI+) m / z: calculated for C 33 H 51 FN7O4Si + : 656.3750 [M+H] + ;Found 656.3749 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 9.27 (s, 1H, NH), 7.81 (dd, J = 15.7, 2.4 Hz, 1H, ArH), 7.47 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 7.14 (d, J = 3.6Hz, 1H, ArH), 6.94 (dd, J = 10.1, 8.8 Hz, 1H, ArH), 6.32 (d, J = 3.5 Hz, 1H,ArH), 5.47 (q, J = 11.1 Hz, 2H, CH2), 3.52 (t, J = 8.1 Hz, 2H, CH2), 3.34 (s, 9H), 2.93 (t, J = 4.8 Hz, 4H, Piperazine H), 2.45 (t, J = 4.8 Hz, 4H, Piperazine H),2.21 (s, 3H, CH3), 1.89 (d,J = 47.6 Hz, 2H, CH2), 1.57 – 1.30 (m, 2H, CH2), 1.11 – 0.93 (m, 6H, CH2×3), 0.91 – 0.80 (m, 2H), -0.12 (s, 9H, t -BuH). 13 C NMR(101 MHz, DMSO) δ 161.68, 155.98, 155.12, 153.80, 153.58, 136.62, 133.11,133.02, 124.09, 119.19, 114.00, 106.45, 106.19, 99.02, 98.70, 72.22, 68.81,65.27, 54.82, 50.51, 50.48, 45.81, 27.55, 17.09. (4) Preparation of compound 25a Compound 25a: Compound 24a (3.77 g, 5.75 mmol) was dissolved in 52 mL of dichloromethane and 52 mL of trifluoroacetic acid and stirred at room temperature for 4 hours. The solution was concentrated under reduced pressure and then dissolved in a mixture of 10 mL of ethanol and 5 drops of water. Potassium carbonate (3.97 g, 29 mmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was filtered and concentrated under reduced pressure. The mixture was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 5 / 1), concentrated under reduced pressure, and dried to obtain 1.15 g of the deprotected amino compound as a white solid in an approximately 47% yield. HRMS (ESI+) m / z: calculated for C 22 H 29 FN7O + : 426.2412 [M+H] + ;Found 426.2413 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.53 (t, J = 2.1 Hz, 1H, NH), 9.22 (s, 1H, NH), 7.87 (dd, J = 15.5, 2.4 Hz, 1H, ArH), 7.40 (dd, J = 8.7, 2.4Hz, 1H, ArH), 7.08 – 6.88 (m, 2H, ArH), 6.39 (dd, J= 3.5, 1.9 Hz, 1H, ArH),5.48 (p, J = 2.6 Hz, 1H, CH), 3.50 – 3.41 (m, 2H, CH2), 3.11 (dd, J = 6.6, 3.3Hz, 4H, Piperazine H), 3.08 (s, 4H, Piperazine H), 2.66 (s, 3H, CH3), 2.16 –1.36 (m, 6H, CH2×3). 13 C NMR (101 MHz, DMSO) δ 160.81, 158.48, 158.17, 156.12,154.71, 154.17, 153.72, 137.60, 121.02, 119.71, 118.75, 115.77, 114.03,106.34, 106.08, 98.75, 98.61, 66.58, 53.40, 48.60, 45.71, 43.04, 26.51. (5) Preparation of compound 26a Compound 26a: Compound 25a (0.547 g, 1.29 mmol) was dissolved in 25 mL of tetrahydrofuran and stirred at 0°C. N , N 1-Diisopropylethylamine (0.42 mL, 2.58 mmol) was added, followed by acryloyl chloride (0.21 mL, 2.58 mmol). The reaction was stirred at 0°C for 10 minutes. The solution was concentrated under reduced pressure and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 10 / 1). The product was concentrated under reduced pressure and dried to obtain 365 mg of a white solid with a yield of approximately 59%. Figure 11 , HRMS(ESI+) m / z: calcd for C 25 H 31 FN7O2 + : 480.2518 [M+H] + ; Found 480.2517 [M+H] + . 1 H NMR (400 MHz, DMSO- d6) δ 11.46 (s, 1H, NH), 9.13 (s, 1H, NH), 7.91 – 7.74 (m, 1H, ArH), 7.45 – 7.30 (m, 1H, ArH), 7.02 – 6.78 (m, 2H, ArH), 6.63 (dd, J =16.7, 10.5 Hz, 1H, ArH), 6.24 – 5.93 (m, 2H, , Acrylamide CH), 5.77 – 5.44(m, 1H, Acrylamide CH), 5.29 (dq, J = 6.6, 3.4 Hz, 1H, CH), 4.18 – 3.36 (m, 4H, CH2×2), 2.93 (t, J = 4.7 Hz, 4H, Piperazine H), 2.45 (t, J = 4.8 Hz, 4H,Piperazine H), 2.21 (s, 3H, CH3), 2.18 – 1.38 (m, 4H, CH2×2). 13 C NMR (101MHz, DMSO) δ 165.02, 161.29, 156.04, 154.87, 154.07, 153.63, 136.81, 132.87,128.42, 126.71, 120.79, 119.21, 114.01, 106.37, 106.11, 98.54, 69.13, 54.82,50.50, 45.81, 41.90, 28.76, 21.33. 2. Route 2 (counterpoint modification): (1) Preparation of compound 23b Compound 23b: Dissolve compound 21 (8.68 g, 27.4 mmol) and compound 22b (6.06 g, 30.11 mmol) in 200 mL of anhydrous tetrahydrofuran and cool to -20°C. Slowly add potassium tert-butoxide (30.11 mmol, 3.38 g) and continue stirring for 0.5 hour. Quench the reaction with aqueous ammonium chloride solution, remove the solvent by distillation under reduced pressure, and purify the crude product by column chromatography (petroleum ether / ethyl acetate, ratios ranging from 50:1 to 10:1) to obtain compound 23b. The yield is approximately 89% as an oil. HRMS (ESI+) m / z: calculated for C 22 H36 ClN4O4Si + : 483.2189 [M+H] + ; Found 483.2190 [M+H] + . (2) Preparation of compound 24b Compound 24b: To a round-bottom flask, tert-butanol (100 mL), compound 23b (3.4 g, 7.05 mmol), and compound 8f (1.34 g, 6.41 mmol) were added sequentially. The reaction mixture was stirred at 360 rpm for 5-10 minutes. Potassium carbonate (1.93 g, 14 mmol), Pd(dba) (128 mg, 0.14 mmol), and XPHOS (133 mg, 0.28 mmol) were added to the reaction solution. The reaction mixture was placed in an oil bath (110°C) and refluxed under nitrogen for 5 hours. Completion of the reaction was monitored by TLC (developing solvent: CH2Cl2 / CH3OH = 10 / 1). The reaction solution was cooled to room temperature, dried, extracted, concentrated, and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was concentrated under reduced pressure and dried to obtain 2.18 g of a white solid in an approximately 86% yield. HRMS(ESI+) m / z: calculated for C 33 H 51 FN7O4Si + : 656.3750 [M+H] + ; Found 656.3750 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 9.30 (s, 1H, NH), 7.83(dd, J = 15.8, 2.4 Hz, 1H, ArH), 7.45 (dd, J = 8.8, 2.4 Hz, 1H, ArH), 7.15 (d, J =3.6 Hz, 1H, ArH), 6.95 (dd, J = 10.0, 8.8 Hz, 1H) , ArH, 6.38 (d, J = 3.6 Hz, 1H,ArH), 5.46 (s, 2H, CH2), 5.41 (dq, J = 8.3, 4.3 Hz, 1H, CH, Acrylamide CH),3.76 (dt,J = 13.4, 4.9 Hz, 2H, CH2), 3.52 (dd, J = 8.8, 7.4 Hz, 2H, CH2), 3.21(s, 2H, CH2), 2.93 (t, J = 4.7 Hz, 4H, Piperazine H), 2.45 (t, J = 4.8 Hz, 4H,Piperazine H), 2.21 (s, 3H, CH3), 2.04 (dq, J = 8.8, 4.0 Hz, 2H, CH2), 1.65(dtd, J = 12.8, 8.8, 3.9 Hz, 2H, CH2), 1.42 (s, 9H), 0.89–0.78 (m, 2H, CH2), -0.13 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 161.53, 155.97, 155.12, 153.89, 153.86,153.57, 136.68, 136.57, 133.14, 133.05, 124.19, 119.23, 119.18, 114.07,106.48, 106.22, 99.07, 98.62, 78.82, 72.23, 70.78, 65.34, 54.81, 50.51,50.48, 45.81, 30.56, 28.08, 17.06, -1.50. (2) Preparation of compound 25b Compound 25b: Compound 24b (0.5 mmol, 328 mg) was dissolved in 4.5 mL of dichloromethane and 4.5 mL of trifluoroacetic acid and stirred at room temperature for 4 hours. The solution was concentrated under reduced pressure and then dissolved in a mixture of 10 mL of ethanol and 5 drops of water. Potassium carbonate (345 mg, 2.5 mmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was filtered and concentrated under reduced pressure. The mixture was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 5 / 1), concentrated under reduced pressure, and dried to obtain 111 mg of the deprotected amino compound as a white solid in an approximately 52% yield. HRMS (ESI+) m / z: calculated for C 22 H 29 FN7O + : 426.2412 [M+H] +;Found 426.2410 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.50 (s, 1H, NH), 9.19 (s,1H, NH), 8.91 (s, 1H, NH), 7.87 (dd, J = 15.7, 2.3 Hz, 1H, ArH), 7.36 (dd, J =8.7, 1.9 Hz, 1H, ArH), 7.10 – 6.90 (m, 2H, ArH), 6.34 (dd, J = 3.4, 1.9 Hz, 1H,ArH), 5.41 (tt, J = 7.5, 3.3 Hz, 1H, CH), 3.37 – 3.33 (m, 2H, CH2), 3.18 – 3.11(m, 2H, CH2), 3.04 (s, 4H, Piperazine H), 2.82 (s, 4H, Piperazine H), 2.48(s, 3H, CH3), 2.30 – 2.18 (m, 2H, CH2), 1.98 (ddt, J = 17.2, 8.2, 3.7 Hz, 2H,CH2). 13 C NMR (101 MHz, DMSO) δ 161.06, 158.39, 158.09, 156.09, 154.81, 154.18,153.68, 137.34, 121.05, 119.53, 118.76, 115.78, 114.04, 106.35, 106.09,98.57, 98.27, 67.77, 54.00, 49.45, 40.92, 27.49. (3) Preparation of compound 26b Compound 26b: Compound 25b (0.52 g, 1.22 mmol) was dissolved in 25 mL of tetrahydrofuran and stirred at 0 °C. N , N1-Diisopropylethylamine (0.4 mL, 2.44 mmol) was added, followed by acryloyl chloride (0.2 mL, 2.44 mmol). The reaction was stirred at 0°C for 10 minutes. The solution was concentrated under reduced pressure and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 10 / 1). The product was concentrated under reduced pressure and dried to obtain 362 mg of a white solid with a yield of approximately 62%. Figure 12 , HRMS(ESI+) m / z: calcd for C 25 H 31 FN7O2 + : 480.2518 [M+H] + ; Found 480.2516 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 11.44 (t, J = 2.2 Hz, 1H, NH), 9.15 (s, 1H, NH), 7.86 (dd, J = 15.8, 2.4 Hz, 1H, ArH), 7.44 – 7.25 (m, 1H, ArH), 7.01 – 6.80 (m,3H, ArH), 6.30 (dd, J = 3.5, 1.9 Hz, 1H, Acrylamide CH), 6.12 (dd, J = 16.7, 2.4Hz, 1H, Acrylamide CH), 5.69 (dd, J = 10.5, 2.5 Hz, 1H, Acrylamide CH), 5.46(tt, J = 8.1, 3.8 Hz, 1H, CH), 4.13 – 3.85 (m, 2H, CH2), 3.48 (dd, J = 15.8, 6.2Hz, 2H, CH2), 2.93 (t, J = 4.7 Hz, 4H, Piperazine H), 2.46 (t, J = 4.7 Hz, 4H,Piperazine H), 2.22 (s, 3H, CH3, CH3), 2.09 (s, 2H, CH2), 1.72 (d, J = 13.2 Hz,2H, CH2). 13C NMR (101 MHz, DMSO) δ 164.34, 161.35, 156.04, 154.91, 154.09, 153.64, 136.98, 132.94, 128.46, 127.23, 120.77, 119.23, 113.97, 106.33,98.60, 70.38, 54.83, 50.53, 50.50, 45.81, 42.59, 31.48. The beneficial effects of the present invention are demonstrated by experimental examples below.

[0058] Experimental Example 1: Kinase inhibition assay 1. Experimental methods (1) Mobility shift assay a. Solution Preparation: Prepare kinase buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35) and stop solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) in the appropriate proportions.

[0059] b. The test compound is diluted in a gradient concentration.

[0060] c. Reaction system setup: Dissolve the target kinase in 1× kinase buffer to prepare a 2.5× enzyme solution. Aliquot 10 μL into a 384-well plate (add only buffer to the negative control wells) and equilibrate at room temperature for 10 minutes.

[0061] d. Dissolve the FAM-labeled peptide and ATP in buffer to prepare a 2.5x substrate solution. Add 10 μL to the reaction well and incubate at 28°C for 60 minutes. Terminate the reaction by adding 30 μL of stop solution.

[0062] e. Data acquisition and processing: The peptide phosphorylation conversion rate was determined using the Caliper EZ Reader II. The inhibition rate was calculated using the enzyme-free wells (min) and DMSO control wells (max) as the benchmarks: Inhibition rate = (max-sample conversion rate) / (max-min) × 100 IC was fitted using a four-parameter model using XLFit software (v5.4.0.8). 50 value: Y = Bottom+(Top-Bottom) / (1+(IC 50 / X)^HillSlope) (2) Lantha Screen Assay detection reaction a. Buffer Preparation: Prepare 1× kinase reaction buffer containing 50 mM HEPES (pH 7.5) and 0.0015% Brij-35 surfactant.

[0063] b. The test compound is diluted in a gradient concentration.

[0064] c. Enzymatic Reaction System Construction and Termination: Mix the target kinase with 1× buffer to prepare a 2× enzyme working solution. Add 5 μL of this solution to each well of a 384-well plate (replace the negative control wells with an equal volume of buffer). Incubate at room temperature for 10 minutes. Then, add an equal volume of 2× substrate working solution containing Fluorescein-PolyGT substrate and ATP to the reaction system. After 30 minutes of reaction at room temperature, add 10 μL of 2× stop solution containing the specific antibody and EDTA to terminate the reaction. Incubate at room temperature for 60 minutes to complete antigen-antibody binding.

[0065] d. Fluorescence signal detection: Time-resolved fluorescence detection was performed using the Envision 2014 multi-label analyzer. The fluorescence intensity ratio (Lantha signal) of the dual emission channels at 520 nm and 495 nm was recorded under an excitation wavelength of 340 nm.

[0066] e. Data analysis method: Calculate the inhibition rate according to the formula Inhibition rate = (max-Lantha signal) / (max-min) × 100 (Note: min is the control for the non-enzyme reaction system, and max is the control for the DMSO solvent) The dose-effect curve was fitted using the four-parameter equation: Y = Bottom + (Top-Bottom) / (1 + (IC 50 / X)^HillSlope) f. Data processing flow: After the experimental data were sorted by Excel, nonlinear regression analysis was performed using the XLFit 5.4.0.8 plug-in to calculate the half-maximal inhibitory concentration (IC 50 ) and related pharmacodynamic parameters.

[0067] 2. Experimental results The inhibitory activities of the compounds synthesized in the present invention on four members of the JAK kinase family (JAK1, JAK2, JAK3, TYK2) and Bruton's tyrosine kinase (BTK) containing cysteine ​​were screened using kinase inhibition assays.

[0068] Table 2 Single concentration (5 nM) kinase inhibition assay of compounds against JAK3 and BTK (% inhibition) Initially, the compounds were screened for inhibitory activity against JAK3 and BTK at a specific compound concentration (5 nM). Results showed that before the covalent head was attached, the backbone compounds exhibited some inhibitory activity against JAK3 but little inhibitory activity against BTK. Addition of a protecting group at position 9 significantly reduced the inhibitory activity against both JAK3 and BTK, with the acetylene-modified compounds showing improved activity and selectivity. Following a series of modifications on the benzene ring, the compounds exhibited strong inhibitory activity against JAK3, with compounds 18a and 18d exhibiting strong selectivity for both JAK3 and BTK (Table 2). After modification and replacement of the covalent head, the compounds exhibited weaker inhibitory activity against the tested kinases. Compounds subjected to reversible covalent modification retained some activity against BTK but showed little inhibitory activity against JAK3.

[0069] IC 50 The test results showed that compound 18a had a potent inhibitory activity against JAK3, with an IC of 50 The IC value is 0.52 nM at 1 mM ATP concentration (close to the intracellular concentration). 50 The value can also reach 1.29 nM. At the same time, the preferred compound 18a exhibits excellent selectivity for the same family kinases, with the selectivity coefficient between JAK1 and JAK3 exceeding 9000 times, and the selectivity for other kinases in the same family also exceeding 500 times (IC 50 ( TYK2 ) / IC 50 ( JAK3 ) = 529). In addition, the IC of compound 18a against TEC family kinase BTK 50 The values ​​were 11.49 nM, respectively. Compared to JAK3, the selectivity coefficient was approximately 22-fold. Preliminary kinase assay results indicate that the preferred compound 18a is an active and selective JAK3 inhibitor, exhibiting good potency and selectivity in inhibiting JAK3 phosphorylation under biochemical conditions.

[0070] Table 3 Inhibitory activity of preferred compounds against different kinases (IC 50 / nM) Experimental Example 2: Signaling Pathway Verification (Immunoblotting Analysis) 1. Experimental methods hPBMCs were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 μg / mL) (37°C, 5% CO2). Cells were plated in 24-well plates (5 × 10 cells per well).5 Cells were treated with the preferred compound for 1 hour, followed by stimulation with various recombinant human cytokines (IL-2, IL-4). After stimulation, cells were harvested and lysed using radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, and 50 mM phenylmethylsulfonyl fluoride) along with complete protease and phosphatase inhibitors. Protein lysates were then quantified using the bicinchoninic acid assay. Equal amounts of total protein were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with STAT5 (1:1000), p-STAT5 (1:1000), p-STAT6 (1:1000), and tubulin (antibodies at 1:10,000), followed by incubation with horseradish peroxidase-conjugated secondary antibodies at 1:2000. Bands were visualized using enhanced chemiluminescence reagent from Merck Millipore and quantified using ImageJ software. Protein expression was normalized to tubulin.

[0071] 2. Experimental results Each JAK isoform specifically responds to a distinct set of cytokine receptors. When these receptors bind to the corresponding cytokines, the JAK protein begins to exert its signaling function. Typically, JAKs transmit signals as heterodimers (e.g., JAK1 / JAK3) or homodimers (e.g., JAK2 / JAK2). The JAK3 isoform is expressed exclusively in hematopoietic cells and is exclusively involved in mediating signaling for cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which play an essential role in lymphocyte activation, function maintenance, and proliferation.

[0072] In order to further explore the selective inhibitory effect of the preferred compound 18a on different JAK subtypes, the present invention used multiple cytokines to stimulate human peripheral blood mononuclear cells (PBMCs) and evaluated the inhibitory effect of JAK3 by detecting the expression levels of p-STAT5 (related to IL-2 stimulation) or p-STAT6 (related to IL-4 stimulation). Experimental results ( Figure 13 ) showed that the preferred compound 18a had good inhibitory activity on the phosphorylation process of STAT5 (stimulated by IL-2) and STAT6 (stimulated by IL-4) induced by γC cytokine stimulation, but had no inhibitory effect on the phosphorylation process of STAT-3 induced by IL-6 stimulation, demonstrating the good activity and selectivity of the preferred compound at the cellular level Experimental Example 3: Acute toxicity test 1. Experimental methods A single-dose limit study was conducted according to the OECD Chemical Test Guide No. 425 acute oral toxicity test method. Preliminary studies included administration of 1 g / kg, 500 mg / kg, 200 mg / kg, and 150 mg / kg.

[0073] Kunming mice (8 weeks old) that had been acclimated for one week were randomly divided into two groups of three. The test compound was completely dissolved in saline (0.2 ml, 5%-10% DMSO, 10% castor oil, and 80% saline) using DMSO and castor oil as solubilizers. The test compound was intraperitoneally injected into the first group of mice at the appropriate dose. The second group of mice was then intraperitoneally injected with a mixture of saline, DMSO, and castor oil in the same volume and ratio. Following administration, the mice were monitored for 24 hours for immediate death, possible signs of poisoning, and the nature of death. Subsequently, the mice were observed for an additional 14 days for delayed toxic effects. Diet, water intake, body weight, and survival were recorded. Afterwards, the mice were euthanized. Organs (heart, liver, spleen, lung, and kidney) were sectioned and stained with hematoxylin-eosin (H&E) for toxicity analysis.

[0074] 2. Experimental results In order to further evaluate the drugability and clinical value of the preferred compound 18a, the present invention conducted a preliminary safety assessment. The acute toxicity characteristics of the preferred compound 18a were evaluated by intraperitoneal injection of a certain dose of the preferred compound 18a (ranging from 1 g / kg to 200 mg / kg) into Kunming mice (n = 3). After administration, the abnormal behavior and mortality of the mice were monitored in the first 4 hours, the next 24 hours and the next 14 days. During the monitoring period, no acute toxic effects were observed. Biochemical parameters were tested weekly during the experimental period. These results showed that at the time points tested, drug treatment had no significant effect on the body weight of the mice compared with the control group ( Figure 14 ).

[0075] In addition, no significant macroscopic or pathological changes were found in the liver, heart, lung, spleen, and kidney tissues by H&E staining ( Figure 15 These data demonstrate the safety of the preferred compound 18a.

[0076] Experimental Example 4: Pharmacokinetic Experiment 1. Experimental methods BALB / c mice were intravenously administered (3.0 mg kg -1 ), intraperitoneal administration (30.0 mg·kg -1 ) and oral administration (30.0 mg·kg-1 ) The pharmacokinetics of the preferred compound 18a were investigated.

[0077] 1) Sample preparation Stock solution (100 mg / mL): Weigh 30 mg of compound 18a and dissolve it in 300 μL DMSO. Intravenous solution (0.3 mg / mL): Take 3 μL of stock solution, add 10 μL of 30% HS15 solution, 10 μL of PEG300, and 977 μL of normal saline, and mix well. Intraperitoneal / oral solution (3.0 mg / mL): Take 30 μL of stock solution, add 30 μL of 30% HS15 solution, 30 μL of PEG300, and 910 μL of normal saline, and mix well.

[0078] 2) Sample collection Nine male BALB / c mice (Chengdu Dashuo Experimental Animal Co., Ltd., license number: SCXK (Chuan) 2020-030) were randomly divided into three groups, and three mice were treated with 3.0 mg·kg -1 Intravenous administration: 30.0 mg·kg -1 Intraperitoneal administration: 30.0 mg·kg -1 After oral administration, approximately 0.02 mL of blood was collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after administration. 5 µL of whole blood was collected, treated with a precipitant, sonicated, and centrifuged for analysis.

[0079] 3) Sample measurement a. Preparation of standard solutions: Quantitatively pipette 1 mg mL⁻¹ of the stock solution and prepare working solutions of the standard compound at concentrations of 30, 100, 300, 1000, 3000, 10,000, and 30,000 ng mL⁻¹ by gradient dilution with methanol. b. Preparation of internal standard working solution: Accurately measure 10 µg mL⁻¹ of the suberoylanilide hydroxamic acid (SAHA) stock solution and dilute it accurately with acetonitrile to obtain a 20 ng mL⁻¹ internal standard working solution. c. Preparation of chromatographic system solutions: Mobile phase A: aqueous solution containing 0.1% (v / v) formic acid; mobile phase B: chromatographic-grade acetonitrile; needle rinse: a 90:10 methanol-water mixture, v / v. d. Standard Curve Construction Process: 9 μL of blank matrix whole blood was added to each 1.5 mL centrifuge tube. 1 μL of the gradient standard working solution was precisely added to create a series of samples with a theoretical concentration range of 3–3000 ng mL⁻¹. 200 μL of acetonitrile precipitant containing the internal standard was added, and the mixture was vortexed for 10 seconds, ultrasonically extracted for 10 seconds, and centrifuged at 13,000 × g for 10 minutes. The supernatant was collected and injected into the sample for analysis. e. Biological Sample Pretreatment: 5 μL of the collected whole blood sample was immediately aliquoted into a centrifuge tube. 100 μL of acetonitrile protein precipitant containing the internal standard was added. Vortex, ultrasonicate, and centrifuge were performed according to the standard curve preparation method to obtain the sample solution. f. Chromatography-Mass Spectrometry Analysis Conditions: LC Parameters: ACQUITY UPLC® BEH C18 Column (1.7 μm, 2.1 × 50 mm), column oven maintained at 35°C, autosampler temperature at 15°C. The mobile phase gradient is shown in Table 4, with a flow rate of 0.7 mL·min⁻¹, an equilibrium time of 0.6 min, and an injection volume of 1 μL.

[0080] 4) Mass spectrometry conditions: Electrospray ionization (ESI+) was used with a source temperature of 500°C, a capillary voltage of 5.5 kV, and a declustering voltage of 100 V. Target and internal standard characteristic ion pairs were detected in multiple reaction monitoring (MRM) mode. The specific parameters are as follows.

[0081] Table 4 Liquid phase conditions for test compounds and internal standard SAHA Table 5 Mass spectrometry parameters of test compound 18a and internal standard SAHA 2. Experimental results To evaluate the pharmacokinetic properties of the preferred compound 18a, preliminary pharmacokinetic analysis was performed in mice after intravenous, intraperitoneal, and oral administration. As shown in Tables 6 to 11, 18a exhibited reasonable pharmacokinetic characteristics at an oral dose of 30 mg / kg.1 / 2 The intraperitoneal dose of 30 mg / kg also showed reasonable pharmacokinetic characteristics, t 1 / 2 Oral bioavailability (F po ) = (932.57*3.0) / (466.33*30.0)*100% = 20.00%. Intraperitoneal bioavailability (F IP ) = (3774.80 * 3.0) / (466.33 * 30.0) * 100% = 80.95%. Overall, the selective JAK3 inhibitor 18a demonstrated favorable safety and pharmacokinetic properties both in vitro and in vivo. Intraperitoneal administration demonstrated better absorption than oral administration, worthy of further research and development.

[0082] Table 6 Measured ct data of compounds after oral administration (30.0 mg / kg) (concentration: ng mL -1 ) Table 7 Measured ct data of compounds administered intraperitoneally (30.0 mg / kg) (concentration: ng mL -1 ) Table 8 Measured ct data of compounds after intravenous administration (3.0 mg / kg) (concentration: ng mL -1 ) Table 9 Results of oral administration (30.0 mg / kg) of compounds processed by DAS 2.0 pharmacokinetic software Table 10 Results of intraperitoneal administration (30.0 mg / kg) of compounds processed using DAS 2.0 pharmacokinetic software Table 11 Results of intravenous administration (3.0 mg / kg) of the compound processed by DAS 2.0 pharmacokinetic software Experimental Example 5: In vivo toxicity study of preferred compounds 1. Experimental methods 1) Experimental animals and groups DBA / 1 mice (male, 8 weeks old, 48 mice) were randomly divided into six groups: control group, no treatment, 8 mice; model group, 8 mice induced with rheumatoid arthritis model; drug group A, 8 mice induced with rheumatoid arthritis model and intraperitoneal injection of drug 18a (30 mg / kg); drug group B, 8 mice induced with rheumatoid arthritis model and intraperitoneal injection of drug 18a (3 mg / kg); drug group C, 8 mice induced with rheumatoid arthritis model and intraperitoneal injection of tofacitinib (3 mg / kg); drug group D, 4 mice induced with rheumatoid arthritis model and oral administration of drug 18a (30 mg / kg and 10 mg / kg); 2) Experimental Procedure a. Adaptive feeding for one week; b. Preparation of emulsified collagen: Dissolve type II collagen in 3.3 mL of 0.1 M glacial acetic acid to a concentration of 3 mg / mL. Add 3.3 mL of complete Freund's adjuvant or incomplete Freund's adjuvant to the collagen in three separate doses for emulsification. Perform the entire emulsification process on ice until the collagen is emulsified until no oil disperses when dropped on water, forming an oil-in-water structure. All reagents were prepared the day before immunization. c. Primary immunization: 3 μL of type II collagen-emulsified collagen in complete Freund's adjuvant was injected into a unilateral hind limb. d. Booster immunization: 21 days after the primary immunization, a second booster immunization was performed, with 3 μL of type II collagen-emulsified collagen in incomplete Freund's adjuvant injected into a unilateral hind limb. e. After model establishment, mice in group 3 were treated once daily for 3 consecutive weeks, starting 27 days after the primary immunization. f. During the treatment period, arthritis scores were assessed every three days for each mouse in all groups except the control group: Grade 0, normal (no swelling); Grade 1, mild but distinct redness and swelling of the ankle or wrist, or marked redness and swelling limited to one or more fingers; Grade 2, moderate redness and swelling of the ankle or wrist; Grade 3, severe redness and swelling of the entire paw, including the fingers; and Grade 4, the limb with the most inflammation, involving multiple joints. Each limb was scored on a scale of 0 to 4. All mice were weighed once a week.

[0083] 2. Experimental results To evaluate the therapeutic potential of the preferred compound 18a for rheumatoid arthritis, the collagen-induced arthritis (CIA) model of DBA / 1 mice was used to evaluate the effect of 18a on rheumatoid arthritis, an immune system disease. Two doses of 18a (30, 3 mg / kg) were injected intraperitoneally and two doses of 18a (30, 10 mg / kg) were administered orally over a 3-week period. The therapeutic effect was confirmed on day 21. Figure 16AB). Compared with vehicle-treated mice, oral administration of 18a (30 mg / kg) and intraperitoneal administration of 18a (30 mg / kg) partially prevented the progression of arthritis ( Figure 16 C, Figure 16 F), which was also confirmed in terms of clinical scores ( Figure 16 GI). As a selective JAK3 inhibitor, the present invention compared the efficacy of 18a and tofacitinib at the same dose of 3 mg / kg (intraperitoneal) in a CIA mouse model ( Figure 16 D, Figure 16 E). The results showed that they had almost the same therapeutic effects after 3 weeks of treatment.

[0084] In summary, the present invention provides a compound, pharmaceutical composition, and use thereof, based on a 7-deazaguanine backbone. The compound selectively inhibits the non-receptor tyrosine kinase JAK3, while exhibiting little inhibitory effect on other kinases in the same family, such as JAK1, JAK2, and TYK2. The selectivity coefficient reaches over 9,000-fold, demonstrating excellent selectivity and inhibitory efficacy. Furthermore, the compound effectively inhibits phosphorylation of the downstream STAT pathway. The compound can be used to prepare a drug for the treatment of rheumatoid arthritis and has broad application prospects.

Claims

1. A compound represented by formula I, or a salt thereof, or a stereoisomer thereof: Formula I in, R1 is selected from halogen; R2 is selected from a benzene ring, R3 is selected from hydrogen, or R2 and R3 together form a piperidine ring; R4 is selected from COR a , R a Selected from C2~C4 alkenyl; R5 is selected from C1~C5 alkyl.

2. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, characterized in that: The compound is one of the following compounds: 。 3. Use of the compound according to claim 1 or 2, or a salt thereof, or a stereoisomer thereof in the preparation of a JAK3 inhibitor.

4. The use according to claim 3, characterized in that The JAK3 inhibitor is a drug for treating severe combined immunodeficiency, autoimmune diseases, hematological malignancies, allergic diseases or skin diseases.

5. The use according to claim 4, characterized in that The autoimmune diseases include rheumatoid arthritis, psoriasis and psoriatic arthritis, and inflammatory bowel disease.

6. The use according to claim 4, characterized in that The hematological malignancies include T-cell leukemia / lymphoma and natural killer cell lymphoproliferative diseases.

7. The use according to claim 4, characterized in that The skin diseases include asthma and atopic dermatitis.

8. A pharmaceutical composition, characterized in that The preparation is prepared by using the compound according to claim 1 or 2, or its salt, or its stereoisomer as the active ingredient and adding pharmaceutically acceptable excipients.

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