A compound with 7-deazaguanine as a skeleton, a pharmaceutical composition, and use

Abstract

The application provides a kind of compound with 7-deaza guanine as skeleton, pharmaceutical composition and purposes, belongs to chemical medicine field.The application prepares the compound shown in formula I, or its salt, or its stereoisomer.The compound of the application can selectively inhibit non-receptor tyrosine kinase JAK3, and has almost no inhibitory effect on other kinases in the same family, such as JAK1, JAK2 and TYK2, with a selection coefficient of more than 9000 times, showing good selectivity and inhibitory effect;Meanwhile, the compound of the application can effectively inhibit the phosphorylation of downstream pathway STAT.The compound of the application can be used for preparing a drug for treating rheumatoid arthritis, and has wide application prospect.Formula I.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical medicine, and particularly relates to a compound with 7-deaza guanine as a skeleton, a pharmaceutical composition and use. BACKGROUND

[0002] Janus kinase / signal transducer and activator of transcription pathway (JAK-STAT signaling pathway) has a wide range of functions, and is involved in cell proliferation, differentiation, apoptosis, immune regulation and many other important biological processes. By inhibiting Janus kinase (JAK), the occurrence of diseases such as blood system diseases, rheumatoid arthritis, related blood tumors and the like can be prevented. There are currently four members in the JAK family, which are JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2). Among them, JAK3 is mainly expressed in bone marrow, lymphatic system, endothelial cells and vascular smooth muscle cells, while the other three members are expressed in almost all tissues.

[0003] So far, there have been several non-covalent JAK3 inhibitors based on 7-deaza adenine on the market or in clinical research, including tofacitinib, baricitinib, ruxolitinib and the like.

[0004]

[0005] However, these compounds acting in a non-covalent mode usually only have moderate selectivity for a single JAK family member. When inhibiting JAK3, they also have off-target activity on other kinases, thereby affecting their safety and effectiveness. This is largely due to the high similarity of the adenosine triphosphate (ATP) pocket in the protein kinase family, making it difficult to design a truly subtype-selective JAK inhibitor.

[0006] In addition, the selective JAK3 inhibitor (NIBR3049) has weaker blocking of the STAT5 signaling pathway compared to the JAK1 / 3 inhibitor (tofacitinib). JAK1 is dominant over JAK3 in the signaling of γC cytokines. Researchers found that the lack of efficacy of NIBR3049 at cellular ATP concentrations was due to insufficient inhibitory potency of NIBR3049 in cells, rather than its selectivity for JAK3. Therefore, it is also necessary to develop a highly subtype-selective JAK3 inhibitor with sufficient cellular efficacy.

[0007] Patent document CN111848631B discloses a pyrrolo[2,3-d]pyrimidine derivative targeting EGFR mutation, the structure is: However, the compound has poor inhibitory activity on JAK3 kinase (IC 50 2.2 nM), which needs to be further improved. SUMMARY

[0008] To solve the problems in the prior art, the present application aims to provide a compound with 7-deazaguanine as a skeleton, a pharmaceutical composition and use.

[0009] The present application provides a compound shown in formula I, or a salt thereof, or a stereoisomer thereof:

[0010]

[0011] Formula I

[0012] wherein,

[0013] R1 is selected from halogen;

[0014] R2 is selected from a benzene ring, and R3 is selected from hydrogen, or R2 and R3 together form a piperidine ring;

[0015] R4 is selected from COR a , R a is selected from C2-C4 alkenyl;

[0016] R5 is selected from C1-C5 alkyl.

[0017] Further, the compound is one of the following compounds:

[0018] .

[0019] The present application also provides use of the above-mentioned compound, or a salt thereof, or a stereoisomer thereof in preparation of a non-receptor tyrosine kinase inhibitor.

[0020] Further, the non-receptor tyrosine kinase is JAK3.

[0021] Further, the non-receptor tyrosine kinase inhibitor is a drug for treating severe combined immunodeficiency, autoimmune diseases, hematological malignancies, allergic diseases or skin diseases.

[0022] Further, the autoimmune diseases include rheumatoid arthritis, psoriasis and psoriatic arthritis, and inflammatory bowel disease.

[0023] Further, the hematological malignancies include T-cell leukemia / lymphoma and natural killer cell (NK cell) lymphoproliferative disease.

[0024] Further, the skin disease includes asthma, atopic dermatitis.

[0025] The present application also provides a pharmaceutical composition, which is a preparation prepared by adding a pharmaceutically acceptable adjuvant to the above-mentioned compound, or a salt thereof, or a stereoisomer thereof as an active ingredient.

[0026] The compounds and derivatives provided in the present application can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstract Service, Columbus, OH) nomenclature system.

[0027] Definitions of terms used in the present application: unless otherwise specified, the initial definition of a group or term provided herein applies throughout the specification; for terms not specifically defined herein, the meaning given to them by those skilled in the art in light of the disclosure and context should be given.

[0028] In the present application, "halogen" is fluorine, chlorine, bromine or iodine.

[0029] "Alkyl" is a hydrocarbon group formed by removing one 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.

[0030] "C2-C4 alkenyl" refers to an alkenyl group containing two to four carbon atoms.

[0031] In the present application, "room temperature" is 25±5℃; "overnight" is 12±2h.

[0032] The present application has the following beneficial effects:

[0033] (1) The present application successfully constructs a selective covalent JAK3 kinase inhibitor library with 7-deazaguanine as the skeleton by combining computer-aided design with empirical screening, and obtains 12 candidate molecules in 3 series through reverse synthetic analysis to optimize the synthesis path.

[0034] (2) Compound 18a exhibits excellent JAK3 inhibitory activity (IC 50 = 0.52 nM) and selectivity of the same family kinases (JAK3 / JAK1>9000 times).

[0035] (3) In vitro experiments confirmed that 18a can effectively block the phosphorylation of the JAK-STAT pathway induced by interleukin (IL)-2 / IL-4, alleviate the symptoms of arthritis in a collagen-induced arthritis (CIA) mouse model, and has a relatively ideal pharmacokinetic characteristic. Therefore, the research data show that compound 18a as a selective JAK3 inhibitor is a preferred compound for the treatment of autoimmune diseases with research value, and also provides an important reference for the covalent drug design targeting cysteine residues.

[0036] (4) Compared with the compound disclosed in patent document CN111848631B (IC 50 2.2 nM) is stronger, and the IC 50 can reach 0.52 nM.

[0037] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above technical idea of the present application.

[0038] The above content of the present application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is the hydrogen spectrum of compound 18a.

[0040] Figure 2 is the hydrogen spectrum of compound 18b.

[0041] Figure 3 is the hydrogen spectrum of compound 18c.

[0042] Figure 4 is the hydrogen spectrum of compound 18d.

[0043] Figure 5 is the hydrogen spectrum of compound 20a.

[0044] Figure 6 is the hydrogen spectrum of compound 20b.

[0045] Figure 7 is the hydrogen spectrum of compound 20c.

[0046] Figure 8 is the hydrogen spectrum of compound 20d.

[0047] Figure 9 The hydrogen spectrum of compound 20e.

[0048] Figure 10 The hydrogen spectrum of compound 20f.

[0049] Figure 11 The hydrogen spectrum of compound 26a.

[0050] Figure 12 The hydrogen spectrum of compound 26b.

[0051] Figure 13 The inhibition of JAK3-dependent signaling pathway of compound 18a.

[0052] Figure 14 The mouse weight change trend.

[0053] Figure 15 A) blank control group H&E staining image; B) drug group (200 mg / kg) H&E staining image (magnification 20x, scale = 20 μm).

[0054] Figure 16 The mouse left hind limb side and front: 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) average score line chart of each group of rheumatoid arthritis; I) average weight line chart of each group of rheumatoid arthritis. DETAILED DESCRIPTION

[0055] Unless otherwise specified, the raw materials and equipment used in the detailed description of the present application are known products, which are obtained by purchasing commercially available products. The main reagents are shown in Table 1.

[0056] Table 1 Reagents for some experiments of the present application

[0057]

[0058] Main instruments:

[0059] (1) Mass spectrometer: Q-TOF spectrometer, ESI ion source, Germany Bruker;

[0060] (2) Nuclear magnetic resonance instrument: AV II-400MHz, AV II-600MHz or AV II-800MHz, TMS as internal standard, Germany Bruker.

[0061] Example 1, synthesis of compound 4

[0062] The synthesis route of compound 4 is as follows:

[0063]

[0064] The specific reaction process of compound 4 refers to Example 2 of the patent document CN111848631B (i.e. compound 20 in the patent document CN111848631B).

[0065] Example 2, synthesis of compound 8

[0066] The synthesis route of compound 8 is as follows:

[0067]

[0068] The specific reaction process of compound 8a and compound 8e refers to Example 1 of the patent document CN111848631B (i.e. compound 15, compound 18 in the patent document CN111848631B). Compound 8c can be purchased.

[0069] Compound 7b: A mixture solution of 2-fluoro-5-nitrotoluene (1.55 g, 10 mmol), N-methylpiperazine (100 mmol, 11 ml) was refluxed at 80°C, after the reaction was completed, it was placed at room temperature, diluted with water, the obtained solid was filtered, washed with water, and dried to obtain the product. Brown solid, yield more than 90%.

[0070] Compound 8b: Compound 7b (1.18 g) was dissolved in 50 ml of isopropanol with palladium on carbon (0.3 g), and the mixture was heated to reflux, hydrazine hydrate (3 ml dissolved in 10 ml of isopropanol) was added dropwise to the refluxing liquid, and the reaction was continued, after cooling, the reaction mixture was filtered; the filtrate was concentrated under reduced pressure and dried to obtain the product. Brown solid, yield more than 90%.

[0071] Compound 7d: A mixture solution of 4-fluoro-3-alkynyl-nitrobenzene (1.65 g, 10 mmol), N-methylpiperazine (11.5 ml, 104 mmol) was heated to 90°C, and the reaction was continued for more than 6 hours, then it was placed at room temperature, diluted with water, the obtained solid was filtered, washed with water, and dried to obtain the product. Brown solid, yield more than 90%.

[0072] Compound 8d: Compound 7d (2.2 g) was dissolved in 80 ml of isopropanol with palladium on carbon (0.48 g), and the mixture was heated to reflux, hydrazine hydrate (4.8 ml dissolved in 20 ml of isopropanol) was added dropwise to the refluxing liquid, and the reaction was continued, after cooling, the reaction mixture was filtered, the filtrate was concentrated under reduced pressure and dried to obtain the product. Brown solid, yield more than 90%.

[0073] Example 3, preparation of compound 18a

[0074] The synthetic route of compound 18a is as follows:

[0075]

[0076] 1. Preparation of compound 17a

[0077] The specific reaction process of compound 17a is referred to Example 8 of patent document CN111848631B (i.e. compound 9 in patent document CN111848631B).

[0078] 2. Preparation of compound 18a

[0079] Compound 17a (250 mg, 0.35 mmol) was dissolved with 16 mL of methanol and stirred at room temperature for 5 minutes. NaOH solution (0.4 g of sodium hydroxide was dissolved in 1 mL of purified water, 10 mmol, 176 μL was taken) was added dropwise into the reactor, and stirring was continued for 1 h to the end of the reaction. The pH was adjusted to neutral with saturated sodium bicarbonate solution, and 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 obtain 102 mg of brown solid product with a yield of 49%.

[0080] 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.

[0081] Example 4, Preparation of compounds 18b, 18c, 18d

[0082] The synthesis route of compounds 18b, 18c, 18d is as follows:

[0083]

[0084] 1. Preparation of compounds 17b, 17c, 17d

[0085] Compound 17b: In a round bottom flask, add tert-butanol (30 mL), compound 10 (428 mg, 1 mmol), compound 8b (184.64 mg, 0.9 mmol); stir the reaction mixture at 360 rpm for 5-10 min; add potassium carbonate (276 mg, 2 mmol), Pd2(dba)3(18 mg, 0.02 mmol), XPHOS (19 mg, 0.04 mmol) to the reaction solution; place the reaction in an oil bath (110 °C) and stir under reflux for 3-3.5 h under nitrogen protection; monitor the end of the reaction by TLC (eluent: CH2Cl2 / CH3OH = 100 / 5). After the reaction solution is cooled to room temperature, spin dry, extract, dry, concentrate, separate by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrate under reduced pressure, and dry to obtain 328 mg of white solid product with a yield of about 61%. HRMS (ESI+) m / z: calcd for C 33 H 40 N7O4 + : 598.3136 [M+H] + ; Found598.3145 [M+H] + . 1 H 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.

[0086] Compound 17c: In a round bottom flask, add tert-butyl alcohol (30 mL), compound 10 (428 mg, 1 mmol), compound 8c (194.52 mg, 0.9 mmol); stir the reaction mixture at 360 rpm for 5-10 min; add potassium carbonate (276 mg, 2 mmol), Pd2(dba)3(18 mg, 0.02 mmol), XPHOS (19 mg, 0.04 mmol) to the reaction solution; place the reaction in an oil bath (110 °C) and stir under reflux for 3-3.5 h under nitrogen protection; monitor the end of the reaction by TLC (eluent: CH2Cl2 / CH3OH = 100 / 5). After the reaction solution is cooled 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 white solid product with a yield of about 74%. HRMS (ESI+) m / z: calcd 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.

[0087] Compound 17d: In a round bottom flask, add tert-butanol (50 mL), compound 10 (802 mg, 1.87 mmol), compound 8d (403 mg, 1.87 mmol), stir the reaction mixture at 360 rpm for 5-10 min, add potassium carbonate (517 mg, 3.75 mmol), Pd2(dba)3(68 mg, 0.075 mmol), XPHOS (70 mg, 0.15 mmol) to the reaction solution, place the reaction in an oil bath (110 °C), stir under reflux for 3-3.5 h under nitrogen protection. TLC monitor the end of the reaction (developing agent: CH2Cl2 / CH3OH = 100 / 5). After the reaction is cooled to room temperature, spin dry, extract, dry, concentrate, silica gel column chromatography separation (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrate under reduced pressure, dry, to obtain white solid product 613 mg, yield about 54%. HRMS (ESI+) m / z: calcd 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 = 2.7 Hz, 1H, ArH), 7.56 (d, J = 9.0 Hz, 2H, ArH, NH), 7.44 (dd, J = 8.9, 2.7Hz, 1H, ArH), 7.32 (t, J = 8.1 Hz, 1H, ArH), 7.07 (s, 1H, ArH), 7.00–6.91 (m,2H, ArH), 6.77 (d, J = 8.9 Hz, 1H, ArH), 6.39 (dd, J = 16.9, 1.7 Hz, 1H,Acrylamide CH), 6.33 (d, J = 3.7 Hz, 1H, ArH), 6.28 (dd, J = 16.9, 9.9 Hz, 1H,Acrylamide CH), 6.07 (s, 2H, CH2), 5.70 (dd,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 C NMR (101 MHz, CDCl3)δ 178.47, 163.81, 162.68, 155.51, 155.27, 153.37, 149.21, 139.20, 134.81,131.27, 129.87, 128.07, 124.47, 124.30, 120.51, 118.32, 117.97, 117.04,116.14, 113.92, 100.68, 99.71, 82.35, 82.16, 66.52, 55.40, 51.42, 46.07,39.01, 29.78, 27.10, 1.14, 0.12.

[0088] 2. Preparation of compounds 18b, 18c, and 18d

[0089] Compound 18b: The reaction apparatus was placed under vacuum, and methanol (10 mL) and compound 17b (122 mg, 0.2 mmol) were added and stirred. NaOH solution (0.4 g sodium hydroxide dissolved in 10 mL purified water, 10 mmol, 1 mL) was added dropwise to the reaction solution. Stirring continued until the reaction was complete. The pH was adjusted to neutral with saturated sodium bicarbonate solution. The reaction solution was evaporated to dryness, extracted, concentrated, and dried. The crude product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 8), concentrated under reduced pressure, and dried to give 84 mg of a white solid product, with a yield of approximately 87%. 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) δ 163.28, 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.

[0090] Compound 18c: The reaction apparatus was placed under vacuum condition, methanol (10 mL) was added, compound 15k (122 mg, 0.2 mmol) was stirred; NaOH solution (0.4 g sodium hydroxide was dissolved in 10 mL purified water, 10 mmol, 1 mL was taken) was added dropwise to the above reaction solution; continue to stir until the reaction is completed; adjust the pH to neutral with saturated sodium bicarbonate solution, spin dry the reaction solution, extract, concentrate and dry. The crude product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 8), concentrated under reduced pressure and dried to obtain white solid product 86 mg, yield about 87%. Figure 3 HRMS (ESI+) m / z: calcd for 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) δ 163.32, 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.

[0091] Compound 18d: The reaction device was placed under vacuum condition, methanol (20 mL) was added, compound 8m (735 mg, 1.21 mmol) was stirred; NaOH solution (0.4 g sodium hydroxide was dissolved in 5 mL purified water, 10 mmol, 3 mL) was added dropwise to the above reaction solution; continue to stir until the reaction is completed; adjust the pH to neutral with saturated sodium bicarbonate solution, spin dry the reaction solution, extract, concentrate and dry. The crude product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 8), concentrated under reduced pressure and dried to obtain white solid product 292 mg, yield about 49%. 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.

[0092] Example 8: Modification and Synthesis of Covalent Targets

[0093] The modification and synthesis route of the covalent target is as follows:

[0094]

[0095] 1. Preparation of Compound 19

[0096] The specific reaction processes of compounds 19a and 19b are described in Examples 3 and 4 of patent document CN111848631B (i.e., compounds 23 and 26 in patent document CN111848631B).

[0097] 2. Preparation of Compound 20

[0098] Compound 20a: Compound 11b (0.22 g, 0.5 mmol), N ,N Diisopropylethylamine (0.17 mL, 1 mmol) and tetrahydrofuran (25 mL) were added into the reactor; when the internal temperature of the reactor reached -3 ℃, a tetrahydrofuran solution of acryloyl chloride (96 μL, 1 mmol) was added dropwise, and the dropwise addition took more than 1 hour; during the dropwise addition, the temperature was maintained between -5-0 ℃; the pH was adjusted with sodium bicarbonate solution, concentrated, separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrated under reduced pressure, dried, and white solid product was obtained, 206 mg, yield about 82%. Figure 5 HRMS (ESI+) m / z: calcd for 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). 13C NMR (101 MHz, 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.

[0099] Compound 20b: Compound 11c (0.23 g, 0.5 mmol) was dissolved in 5 mL of THF, and then 2- chloro-5,5-dimethyl-1,3,4-oxadiazole (0.1 g, 0.6 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the residue was separated by column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3) to give a white solid product 20b, 0.23 g, yield about 84%. 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 ℃, a tetrahydrofuran solution of acryloyl chloride (96 μL, 1 mmol) was added dropwise, and the dropwise addition took more than 1 hour; during the dropwise addition, the temperature was maintained between -5-0 ℃; the pH was adjusted with sodium bicarbonate solution, concentrated, separated by column chromatography on silica gel (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrated under reduced pressure, and dried to give a white solid product 217 mg, yield about 84%. Figure 6 HRMS (ESI+) m / z: calcd for 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.

[0100] Compound 20c: Compound 1 lb (0.23 g, 0.5 mmol) was dissolved in 5 mL of THF, and the solution was cooled to -3 °C. Diisopropyl ethylamine (0.17 mL, 1 mmol) and THF (25 mL) were added to the reactor. When the internal temperature of the reactor reached -3 °C, acryloyl chloride in THF (125 μL, 1 mmol) was added dropwise over 1 h. The temperature was maintained between -5-0 °C during the addition. The pH was adjusted with sodium bicarbonate solution, concentrated, and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was obtained as a white solid after concentration under reduced pressure and drying, 219 mg, about 85% yield. N , N - Diisopropyl ethylamine (0.17 mL, 1 mmol) and THF (25 mL) were added to the reactor. When the internal temperature of the reactor reached -3 °C, acryloyl chloride in THF (125 μL, 1 mmol) was added dropwise over 1 h. The temperature was maintained between -5-0 °C during the addition. The pH was adjusted with sodium bicarbonate solution, concentrated, and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3). The product was obtained as a white solid after concentration under reduced pressure and drying, 219 mg, about 85% yield. Figure 7 , HRMS (ESI+) m / z: calcd for 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.

[0101] Compound 20d: Compound 11c (0.23 g, 0.5 mmol), 4-(dimethylamino)-pyridine (0.07 g, 0.6 mmol), and dichloromethane (25 mL) were added to the reactor; when the internal temperature of the reactor reached 20 °C, acetic anhydride (0.1 mL, 1 mmol) was added dropwise; the temperature was maintained between 20-25 °C during the addition; after the addition was completed, the reaction was stirred for 2 hours; the reaction was quenched with a sodium bicarbonate solution; the organic phase was separated and dried over sodium sulfate; the solvent was removed under reduced pressure; the product was separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), dried under reduced pressure, and white solid product was obtained, 0.23 g, yield about 95%. 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, the dropwise addition took more than 1 hour; the temperature was maintained between -5-0 °C during the addition; the pH was adjusted with a sodium bicarbonate solution, without concentration, separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), dried under reduced pressure, and white solid product was obtained, 223 mg, yield about 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). 13C 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.

[0102] Compound 20e: Compound 11b (0.23 g, 0.5 mmol) was dissolved in 5 mL of THF, and the solution was cooled to -3 °C. Then, diisopropylethylamine (0.17 mL, 1 mmol) and THF (25 mL) were added into the reactor. When the internal temperature of the reactor reached -3 °C, the acryloyl chloride solution in THF (86 μL, 1 mmol) was added dropwise, and the dropwise addition took more than 1 h. During the dropwise addition, the temperature was maintained between -5-0 °C. The pH was adjusted with sodium bicarbonate solution, concentrated, separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrated under reduced pressure, and dried to obtain the white solid product 202 mg, with a yield of about 81%. N , N - Diisopropylethylamine (0.17 mL, 1 mmol) and THF (25 mL) were added into the reactor; when the internal temperature of the reactor reached -3 °C, the acryloyl chloride solution in THF (86 μL, 1 mmol) was added dropwise, and the dropwise addition took more than 1 h; during the dropwise addition, the temperature was maintained between -5-0 °C; the pH was adjusted with sodium bicarbonate solution, concentrated, separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrated under reduced pressure, and dried to obtain the white solid product 202 mg, with a yield of about 81%. Figure 9 HRMS (ESI+) m / z: calcd for 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.

[0103] Compound 20f: Compound 11c (0.23 g, 0.5 mmol) was dissolved in 5 mL of THF, and the solution was cooled to -3 ℃. Acryloyl chloride (86 μL, 1 mmol) was added dropwise to the solution over 1 h. The reaction mixture was stirred at -3 ℃ for 2 h. The reaction mixture was adjusted to pH 8 with NaHCO3 solution, and concentrated. The residue was purified by column chromatography on silica gel (eluent: CH2Cl2 / CH3OH = 100 / 3) to give the product as a white solid, 206 mg, yield about 80%. 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 ℃, a tetrahydrofuran solution of acryloyl chloride (86 μL, 1 mmol) was added dropwise, and the dropwise addition took more than 1 h; during the dropwise addition, the temperature was maintained between -5-0 ℃; the pH was adjusted with a sodium bicarbonate solution, concentrated, separated by column chromatography on silica gel (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrated under reduced pressure, and dried to give the product as a white solid, 206 mg, yield about 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.

[0104] Example 9, synthesis of compounds 26a, 26b

[0105] The synthesis route of compounds 26a, 26b is as follows:

[0106] 1. Route one (meta-modification):

[0107] (1) Preparation of compound 21

[0108] Compound 21 : Compound 1 (13.2 mmol, 2.47 g) was dissolved in 50 mL of dry tetrahydrofuran, and sodium hydride (60% dispersion in mineral oil, 13.8 mmol, 552 mg) was added slowly. The mixture was stirred at room temperature for 0.5 hour. After that, it was cooled again to -10 °C, and then 2-(trimethylsilyl)ethoxymethyl chloride (13.9 mmol, 2.46 mL) dissolved in 10 mL of dry tetrahydrofuran was added gradually to the above mixture, and stirring was continued for 30 minutes. Then, an aqueous solution of ammonium chloride was added to quench the reaction, and then the organic solvent was evaporated under reduced pressure. Dichloromethane was added, and extraction was performed. 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 then purified by column chromatography (eluent: petroleum ether / ethyl acetate = 30 / 1) to obtain the compound. White solid, yield about 98%.

[0109] (2) Preparation of compound 23a

[0110] Compound 23a: Compound 21 (1.12 g, 3.53 mmol) and compound 22a (781 mg, 3.89 mmol) were dissolved in 60 mL of dry tetrahydrofuran, and cooled to -20 °C. Potassium tert-butoxide (3.53 mmol, 396 mg) was added slowly, and stirring was continued for 0.5 hour. The reaction was quenched with an aqueous solution of ammonium chloride, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate, ratio from 50:1 to 10:1) to obtain compound 25a. Oil, yield about 84%. HRMS (ESI+) m / z: calcd for C 22 H 36 ClN4O4Si + : 483.2189 [M+H] + ; Found 483.2185 [M+H] + .

[0111] (3) Preparation of compound 24a

[0112] Compound 24a: In a round bottom flask, add tert-butanol (100 mL), compound 23a (3.34 g, 6.93 mmol), compound 8f (1.32 g, 6.3 mmol) successively, stir the reaction mixture at 360 rpm for 5-10 min; add potassium carbonate (1.91 g, 13.86 mmol), Pd2(dba)3(128 mg, 0.14 mmol), XPHOS (133 mg, 0.28 mmol) into the reaction solution; place the reaction in an oil bath (110 °C), reflux stir for 5 h under nitrogen protection; monitor the end of the reaction by TLC (developing agent: CH2Cl2 / CH3OH = 10 / 1). After the reaction solution is cooled to room temperature, spin dry, extract, dry, concentrate, separate by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrate under reduced pressure, dry, to obtain white solid product 3.59 g, with a yield of about 87%. HRMS (ESI+) m / z: calcd 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 =3.6 Hz, 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, CH2x3), 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.

[0113] (4) Preparation of compound 25a

[0114] Compound 25a: Compound 24a (3.77 g, 5.75 mmol) was dissolved in 52 mL of dichloromethane and 52 mL of trifluoroacetic acid, stirred at room temperature for 4 hours. The solution was concentrated under reduced pressure, 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 was stirred overnight. The reaction mixture was filtered and concentrated under reduced pressure. Silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 5 / 1) was used for separation, and the product was concentrated under reduced pressure and dried to obtain the deprotected amino compound 1.15 g, white solid, with a yield of about 47%. HRMS (ESI+) m / z: calcd 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, 2.4 Hz, 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.

[0115] (5) Preparation of compound 26a

[0116] Compound 26a: Compound 25a (0.547 g, 1.29 mmol) was dissolved in 25 mL of tetrahydrofuran and stirred at 0 °C. To the reaction system was added diisopropylethylamine (0.42 mL, 2.58 mmol) 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, separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 10 / 1), concentrated under reduced pressure, and dried to obtain 365 mg of white solid product with a yield of about 59%. N , N - diisopropylethylamine (0.42 mL, 2.58 mmol), 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, separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 10 / 1), concentrated under reduced pressure, and dried to obtain 365 mg of white solid product with a yield of about 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,CH2x2), 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, CH2x2). 13 C NMR (101 MHz, 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.

[0117] 2. Route two (para modification):

[0118]

[0119] (1) Preparation of compound 23b

[0120] Compound 23b: Compound 21 (8.68 g, 27.4 mmol) and compound 22b (6.06 g, 30.11 mmol) were dissolved in 200 mL of anhydrous tetrahydrofuran, and cooled to -20 °C. Potassium tert-butoxide (30.11 mmol, 3.38 g) was added slowly, and stirring was continued for 0.5 hour. The reaction was quenched with aqueous ammonium chloride solution, the solvent was removed by distillation under reduced pressure, and the crude product was purified by column chromatography (petroleum ether / ethyl acetate, ratio from 50:1 to 10:1) to obtain compound 23b. Oil, yield about 89%. HRMS (ESI+) m / z: calcd for C 22 H 36 ClN4O4Si + : 483.2189 [M+H] + ; Found 483.2190 [M+H] + .

[0121] (2) Preparation of compound 24b

[0122] Compound 24b: In a round bottom flask, tert-butanol (100 mL), compound 23b (3.4 g, 7.05 mmol), compound 8f (1.34 g, 6.41 mmol) were added in sequence, and the reaction mixture was stirred at 360 rpm for 5-10 minutes; potassium carbonate (1.93 g, 14 mmol), Pd2(dba)3(128 mg, 0.14 mmol), XPHOS (133 mg, 0.28 mmol) were added to the reaction solution; the reaction was placed in an oil bath (110 °C) and stirred under reflux for 5 hours under nitrogen protection; TLC monitoring showed that the reaction was completed (developing agent: CH2Cl2 / CH3OH = 10 / 1). After the reaction solution was cooled to room temperature, it was rotary evaporated, extracted, dried, concentrated, and separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 100 / 3), concentrated under reduced pressure, and dried to obtain white solid product 2.18 g, yield about 86%. HRMS (ESI+) m / z: calcd 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.

[0123] (2) Preparation of compound 25b

[0124] Compound 25b: Compound 24b (0.5 mmol, 328 mg) was dissolved in 4.5 mL of dichloromethane and 4.5 mL of trifluoroacetic acid, stirred at room temperature for 4 hours. The solution was concentrated under reduced pressure, 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 was stirred overnight. The reaction mixture was filtered and concentrated under reduced pressure. Silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 5 / 1) was used for separation, and the product was dried under reduced pressure to obtain 111 mg of the deprotected amino compound as a white solid, with a yield of about 52%. HRMS (ESI+) m / z: calcd 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). 13C 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.

[0125] (3) Preparation of compound 26b

[0126] Compound 26b: Compound 25b (0.52 g, 1.22 mmol) was dissolved in 25 mL of tetrahydrofuran and stirred at 0 °C. The following were added to the reaction system: N , N -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 min. The solution was concentrated under reduced pressure, separated by silica gel column chromatography (eluent: CH2Cl2 / CH3OH = 10 / 1), concentrated under reduced pressure, and dried to give 362 mg of white solid product, 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). 13 C 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.

[0127] The advantageous effects of the present application are demonstrated by the following experimental examples.

[0128] Experimental Example 1, Kinase Inhibition Test

[0129] 1. Experimental Method

[0130] (1) Mobility shift assay test

[0131] a. Solution preparation: Prepare the kinase buffer solution (50 mM HEPES, pH 7.5, 0.0015% Brij-35) and the termination solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) in proportion.

[0132] b. Dilute the test compound in gradient concentration for standby.

[0133] c. Reaction system construction: Dissolve the target kinase in 1x kinase buffer, prepare 2.5x enzyme solution, take 10 μL and dispense into a 384-well plate (add only buffer to the negative control well), and equilibrate at room temperature for 10 minutes.

[0134] d. Dissolve FAM-labeled polypeptide and ATP in buffer to prepare 2.5x substrate solution, take 10 μL to reaction well, incubate at 28 ℃ for 60 min. Add 30 μL stop solution to terminate the reaction.

[0135] e. Data collection and processing: use Caliper EZ Reader II to determine the phosphorylation conversion rate of polypeptide, take the enzyme-free well (min) and DMSO control well (max) as the reference, and calculate the inhibition rate according to the formula:

[0136] Inhibition rate = (max-sample conversion rate) / (max-min) x 100

[0137] Fit IC value by XLFit software (v5.4.0.8) four-parameter model: 50

[0138] Y = Bottom + (Top-Bottom) / (1+(IC 50 / X)HillSlope)

[0139] (2) Lantha Screen Assay detection reaction

[0140] a. Buffer system preparation: prepare 1x kinase reaction buffer system containing 50 mM HEPES (pH 7.5) and 0.0015% Brij-35 surfactant.

[0141] b. The test compound is diluted at gradient concentration for standby.

[0142] c. Enzymatic reaction system construction and termination: mix the target kinase with 1x buffer to prepare 2x enzyme working solution, take 5 μL to 384-well plate detection well (replace negative control well with the same volume of buffer); after incubation at room temperature for 10 min, add equal volume of 2x substrate working solution containing Fluorescein-PolyGT substrate and ATP to the reaction system; after 30 min of reaction at room temperature, add 10 μL of 2x termination solution containing specific antibody and EDTA to terminate the reaction, and incubate at room temperature for 60 min to complete the antigen-antibody binding.

[0143] d. Fluorescence signal detection: use Envision2014 multi-label analyzer for time-resolved fluorescence detection, record the fluorescence intensity ratio of 520 nm and 495 nm dual emission channels (Lantha signal) under 340 nm excitation wavelength.

[0144] e. Data analysis method: calculate the inhibition rate according to the formula

[0145] ​Inhibition rate = (max - Lantha signal) / (max - min) x 100 (Note: min is the control of enzyme-free reaction system, max is the control of DMSO solvent)

[0146] The dose-effect curve fitting was performed by four-parameter equation: Y = Bottom + (Top-Bottom) / (1 + (IC 50 / X)HillSlope)

[0147] f. Data processing procedure: After the experimental data was arranged by Excel, non-linear regression analysis was performed by using XLFit 5.4.0.8 plug-in to calculate the half-inhibitory concentration (IC 50 ) of the compound and related pharmacodynamic parameters.

[0148] 2. Experimental results

[0149] The synthesized compounds of the application were screened for inhibitory activity on four members of the JAK kinase family (JAK1, JAK2, JAK3, TYK2) and Bruton's tyrosine kinase (BTK) with cysteine using a kinase inhibition experiment.

[0150] Table 2. Single-concentration (5 nM) kinase inhibition experiment of compounds on JAK3 and BTK (% inhibition rate)

[0151]

[0152] First, by setting a specific compound concentration (5 nM), the inhibitory activity of the compound on JAK3 and BTK was screened, and the results showed that before the compound was connected with a covalent target head, the skeleton compound had certain inhibitory activity on JAK3, but almost no inhibitory activity on BTK. After connecting a protecting group at position 9, the inhibitory activity on JAK3 and BTK was significantly reduced, and the activity and selectivity of the ethynyl-modified compound were better. After a series of modifications on the benzene ring, it was found that the compounds in the series all exhibited good inhibitory activity on JAK3, and among them, compound 18a and compound 18d exhibited good selectivity on JAK3 and BTK (Table 2). After modification and replacement of the covalent target head, the inhibitory activity of the compounds in the series on the tested kinases was weak. The compound after reversible covalent modification retained some activity on BTK, but almost no activity on JAK3.

[0153] IC 50 The test results showed that compound 18a had strong inhibitory activity on JAK3, and the IC 50 value was 0.52 nM in the presence of Km ATP, and the IC 50The value can also reach 1.29 nM. Meanwhile, the preferred compound 18a exhibits excellent selectivity for its family of kinases, with a selectivity exceeding 9000-fold between JAK1 and JAK3, and selectivity exceeding 500-fold for other kinases in the same family (IC50). 50 ( TYK2 IC 50 ( JAK3 () = 529). Furthermore, compound 18a showed an IC50 retardation rate against TEC family kinase BTK. 50 The values ​​were 11.49 nM. The selectivity was approximately 22 times that of JAK3. Preliminary kinase assays indicate that compound 18a is a JAK3 inhibitor with good activity and selectivity, exhibiting excellent efficacy and selectivity in inhibiting JAK3 phosphorylation under biochemical conditions.

[0154] Table 3. Inhibitory activity of preferred compounds against different kinases (IC50) 50 / nM)

[0155]

[0156] Experiment 2: Signaling pathway verification (immunoblotting analysis)

[0157] 1. Experimental Methods

[0158] hPBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 μg / mL) at 37 °C, 5% CO2. Cells were seeded into 24-well plates (5 × 10⁶ cells per well). 5STAT5 (1 : 1000), p-STAT5 (1 : 1000), p-STAT6 (1 : 1000) and tubulin (1 : 10000 antibodies) followed by incubation with secondary antibodies conjugated with horseradish peroxidase (1 : 2000). Bands were visualized by using enhanced chemiluminescence reagent from Merck Millipore followed by quantification using ImageJ software. Protein expression was normalized to tubulin.

[0159] 2. Experimental results

[0160] Each JAK sub-type is able to respond specifically to a different set of cytokine receptors. When these receptors bind to the corresponding cytokines, the JAK proteins start to transmit the signal. Usually, JAKs transmit the signal in the form of heterodimers (e.g. JAK1 / JAK3) or homodimers (e.g. JAK2 / JAK2). Among them, JAK3 sub-type is only expressed in hematopoietic cells and only involved in mediating the signal transduction of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, etc. cytokines, which are indispensable for the activation, function maintenance and proliferation of lymphocytes.

[0161] To further investigate the selective inhibitory effect of the preferred compound 18a on different JAK sub-types, the present application uses a variety of cytokines to stimulate human peripheral blood mononuclear cells (PBMCs), and by detecting the expression level of p-STAT5 (related to IL-2 stimulation) or p-STAT6 (related to IL-4 stimulation), to evaluate the inhibitory effect of JAK3. The experimental results ( Figure 13 ) show that the preferred compound 18a has good inhibitory activity on the phosphorylation process of STAT5 (stimulated by IL-2) and STAT6 (stimulated by IL-4) caused by γC cytokine stimulation, and has no inhibition on the phosphorylation process of STAT-3 caused by IL-6 stimulation, which shows the good activity and selectivity of the preferred compound at the cellular level

[0162] Experimental Example 3, Acute toxicity test

[0163] 1. Experimental method

[0164] A single dose limit test was performed according to the OECD Chemical Test Guidelines No. 425, Acute Oral Toxicity Test Method. The pre-experiment was administered at 1 g / kg, 500 mg / kg, 200 mg / kg, 150 mg / kg, respectively.

[0165] Kunming mice (8 weeks) that were acclimated for 1 week were randomly divided into 2 groups, 3 mice in each group. The test compound was completely dissolved in physiological saline (0.2 ml, 5-10% DMSO, 10% castor oil and 80% physiological saline) using DMSO and castor oil as solubilizers. The test compound was injected intraperitoneally into the first group of mice according to the dose. Then the second group of mice was injected intraperitoneally with the same volume and proportion of physiological saline, DMSO, and castor oil mixture. After administration of the drug to the mice, they were continuously monitored for 24 hours to observe whether there was immediate death, possible signs of poisoning, and death. Then, the mice were continuously observed for another 14 days to check for delayed toxicity, and the diet, water intake, body weight, and survival were recorded. Then, the mice were euthanized. Their organs (heart, liver, spleen, lung, kidney) were taken and sliced and stained with hematoxylin-eosin (H&E) to observe and analyze the toxicity.

[0166] 2. Experimental results

[0167] To further evaluate the drugability and clinical value of the preferred compound 18a, the safety of the preferred compound 18a was preliminarily evaluated. The acute toxicity characteristics of the preferred compound 18a were evaluated by intraperitoneal injection of a certain dose of the preferred compound 18a (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 for the first 4 hours, the next 24 hours, and the following 14 days. During the monitoring period, no acute toxicity was observed. Biochemical parameters were tested every week during the experiment. These results showed that there was no significant effect of drug treatment on the body weight of the mice compared to the control group at the time points tested ( Figure 14 ).

[0168] In addition, no significant macroscopic and 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.

[0169] Experimental Example 4, Pharmacokinetic experiment

[0170] 1. Experimental method

[0171] The pharmacokinetics of the preferred compound 18a was investigated by intravenous (3.0 mg·kg -1 intraperitoneal (30.0 mg·kg -1 intraperitoneal (30.0 mg·kg -1 intraperitoneal (30.0 mg·kg

[0172] 1) Sample preparation

[0173] Stock solution (100 mg / mL): 30 mg of compound 18a was weighed and dissolved in 300 μL of DMSO; intravenous administration solution (0.3 mg / mL): 3 μL of the stock solution was taken and mixed with 10 μL of 30% HS15 solution, 10 μL of PEG300 and 977 μL of physiological saline; intraperitoneal / oral administration solution (3.0 mg / mL): 30 μL of the stock solution was taken and mixed with 30 μL of 30% HS15 solution, 30 μL of PEG300 and 910 μL of physiological saline.

[0174] 2) Sample collection

[0175] Nine male BALB / c mice (Chengdu Dashuo Experimental Animal Co., Ltd., License No.: SCXK (Chuan) 2020-030) were randomly divided into 3 groups, 3 according to 3.0 mg·kg -1 intravenous administration, 3 according to 30.0 mg·kg -1 intraperitoneal administration, and 3 according to 30.0 mg·kg -1 oral administration. About 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 taken, treated with precipitant, ultrasonicated, centrifuged and analyzed by sample injection.

[0176] 3) Sample determination

[0177] a. Preparation of standard solution: 1 mg mL⁻¹ stock solution was quantitatively pipetted, and the working solution of standard compound was prepared by methanol gradient dilution method with the concentration of 30, 100, 300, 1000, 3000, 10000 and 30000 ng mL⁻¹, respectively. b. Preparation of internal standard working solution: 10 μg mL⁻¹ suberoylanilide hydroxamic acid (SAHA) stock solution was accurately measured and precisely diluted with acetonitrile as solvent to obtain a 20 ng·mL⁻¹ internal standard working solution. c. Preparation of chromatographic system solution: mobile phase A: 0.1% (v / v) formic acid aqueous solution; mobile phase B: chromatographically pure acetonitrile; injection needle cleaning solution: methanol-water mixed solution (90:10, v / v). d. Standard curve construction process: 9 μL of blank matrix whole blood was added to a 1.5 mL centrifuge tube, and 1 μL of gradient standard working solution was accurately added to form a series of samples with theoretical concentrations of 3-3000 ng mL⁻¹. 200 μL of acetonitrile containing internal standard precipitant was added, vortexed for 10 seconds, ultrasonically extracted for 10 seconds, and the supernatant was collected after centrifugation at 13000 x g for 10 minutes for injection analysis. e. Pretreatment of biological samples: 5 μL of the collected whole blood sample was immediately transferred to a centrifuge tube, 100 μL of acetonitrile containing internal standard protein precipitant was added, and the vortex, ultrasonic and centrifugation treatment was performed according to the standard curve preparation method to obtain the test solution. f. Chromatography-mass spectrometry analysis conditions: liquid chromatography parameters: ACQUITY UPLC® BEH C18 column (1.7 μm, 2.1 x 50 mm), column oven maintained at 35 °C, autosampler temperature 15 °C. The flow phase gradient is shown in Table 4, the flow rate is 0.7 mL·min⁻¹, the equilibrium time is 0.6 minutes, and the injection volume is 1 μL.

[0178] 4) Mass spectrometry conditions: electrospray ionization source (ESI+), ion source temperature 500 °C, capillary voltage 5.5 kV, de-clustering voltage 100 V. The target and internal standard characteristic ion pairs were detected in multiple reaction monitoring (MRM) mode, and the specific parameters are as follows.

[0179] Table 4 Liquid chromatography conditions for the test compound and internal standard SAHA

[0180]

[0181] Table 5 Mass spectrometry parameters for the test compound 18a and internal standard SAHA

[0182]

[0183] 2. Experimental results

[0184] 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 6-11, 18a exhibited reasonable pharmacokinetic characteristics with a t 1 / 2 maxof 1.62 hours for the 30 mg / kg oral dose. The 30 mg / kg intraperitoneal dose also exhibited reasonable pharmacokinetic characteristics with a t 1 / 2 maxof 1.04 hours. The oral bioavailability (F po ) = (932.57*3.0) / (466.33*30.0)*100% = 20.00%. The intraperitoneal bioavailability (F IP ) = (3774.80*3.0) / (466.33*30.0)*100% = 80.95%. Overall, the selective JAK3 inhibitor 18a exhibited good safety and pharmacokinetic properties in vivo and in vitro, and was better absorbed by intraperitoneal administration than by oral administration, and is worthy of further research and development.

[0185] Table 6. Observed c-t data (concentration: ng· mL -1 ) for compound after oral administration (30.0 mg / kg)

[0186]

[0187] Table 7. Observed c-t data (concentration: ng· mL -1 ) for compound after intraperitoneal administration (30.0 mg / kg)

[0188]

[0189] Table 8. Observed c-t data (concentration: ng· mL -1 ) for compound after intravenous administration (3.0 mg / kg)

[0190]

[0191] Table 9. Results of DAS 2.0 pharmacokinetic software processing for compound after oral administration (30.0 mg / kg)

[0192]

[0193] Table 10. Results of DAS 2.0 pharmacokinetic software processing for compound after intraperitoneal administration (30.0 mg / kg)

[0194]

[0195] Table 11. Results of DAS 2.0 pharmacokinetic software processing for compound after intravenous administration (3.0 mg / kg)

[0196]

[0197] Experimental Example 5, in vivo toxicity study of preferred compounds

[0198] 1. Experimental method

[0199] 1) Experimental animals and grouping

[0200] DBA / 1 mice (male, 8 weeks old, 48) were randomly divided into six groups: control group, no treatment, 8; model group, rheumatoid arthritis model, 8; drug A group, rheumatoid arthritis model, intraperitoneal injection of drug 18a (30 mg / kg), 8; drug B group, rheumatoid arthritis model + intraperitoneal injection of drug 18a (3 mg / kg), 8; drug C group, rheumatoid arthritis model + intraperitoneal injection of drug tofacitinib (3 mg / kg), 8; drug D group, rheumatoid arthritis model + oral administration of drug 18a (30 mg / kg and 10 mg / kg), 4 each;

[0201] 2) Experimental steps

[0202] a. Adaptation for one week; b. Emulsified collagen preparation: dissolve type II collagen in 3.3 mL of 0.1M glacial acetic acid, concentration 3 mg / mL. Add 3.3 mL of complete Freund's adjuvant or incomplete Freund's adjuvant to the collagen three times to emulsify, the whole process of emulsification is carried out on ice, finally emulsify to the state of oil-in-water, the oil does not spread out when the collagen drops on water. The above reagents are prepared one day before immunization; c. Primary immunization: unilateral hind limb injection of 3 μL of emulsified collagen of type II collagen-complete Freund's adjuvant; d. Booster immunization: 21 days after primary immunization, the second booster immunization was carried out, unilateral hind limb injection of 3 μL of emulsified collagen of type II collagen-incomplete Freund's adjuvant; e. After the model is completed, start treatment on the 27th day of primary immunization, once a day, for 3 weeks; f. During the administration period, every three days, each mouse in groups other than the control group was given an arthritis score: 0, normal (no swelling); 1, mild but definite redness and swelling of the ankle joint or wrist, or only obvious redness and swelling limited to a single or multiple fingers; 2, moderate redness and swelling of the ankle or wrist; 3, severe redness and swelling of the entire paw including the fingers; 4, the most inflamed limb, involving multiple joints. The score for each limb is 0-4 points. All mice were weighed once a week.

[0203] 2. Experimental results

[0204] To evaluate the therapeutic potential of the preferred compound 18a for rheumatoid arthritis, the DBA / 1 mouse collagen-induced arthritis (CIA) model was used to evaluate the effect of 18a on the immune system disease rheumatoid arthritis. Arthritis mice were treated by intraperitoneal injection of two doses of 18a (30, 3 mg / kg) and gavage with two doses of 18a (30, 10 mg / kg) over a period of 3 weeks, and the therapeutic effect after treatment was confirmed on day 21 (Fig. 1 Figure 16 A-B). Both gavage administration of 18a (30 mg / kg) and intraperitoneal injection of 18a (30 mg / kg) partially prevented the progression of arthritis compared to vehicle-treated mice (Fig. 1 Figure 16 C, Figure 16 F), which was also confirmed in terms of clinical scores (Fig. 1 Figure 16 G-I). As a selective JAK3 inhibitor, the present application compared the efficacy of 18a and tofacitinib at the same dose of 3 mg / kg (intraperitoneal) in the CIA mouse model (Fig. 1 Figure 16 D, Figure 16 E). The results showed that they had almost the same therapeutic effect after 3 weeks of treatment.

[0205] In summary, the present application provides a compound with 7-deazaguanine as a skeleton, a pharmaceutical composition and uses. The compound of the present application can selectively inhibit non-receptor tyrosine kinase JAK3, and has almost no inhibitory effect on other kinases in the same family, such as JAK1, JAK2, TYK2, with a selectivity coefficient of more than 9000 times, showing good selectivity and inhibitory effect. At the same time, the compound of the present application can effectively inhibit the phosphorylation of the downstream pathway STAT. The compound of the present application can be used for the preparation of a drug for treating rheumatoid arthritis, and has a broad application prospect.

Claims

1. A compound of the following formula: ###0001### or a salt thereof. 。 2. Use of the compound of claim 1, or a salt thereof, for the manufacture of a JAK3 inhibitor.

3. Use according to claim 2, characterized in that, The JAK3 inhibitor is a drug for treating severe combined immunodeficiency, an autoimmune disease, a hematological malignancy, an allergic disease, or a skin disease.

4. Use according to claim 3, characterized in that, The autoimmune disease is selected from rheumatoid arthritis, psoriasis and psoriatic arthritis, an inflammatory bowel disease.

5. Use according to claim 3, characterized in that, The hematological malignancy is selected from T-cell leukemia / lymphoma, natural killer cell lymphoproliferative disease.

6. Use according to claim 3, characterized in that, The skin disease is selected from atopic dermatitis.

7. Use according to claim 3, characterized in that, The allergic disease is selected from asthma.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition is a preparation prepared by adding a pharmaceutically acceptable adjuvant to the compound of claim 1, or a salt thereof, as an active ingredient.

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