JAK-STAT3 degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a JAK-STAT3 degradation agent and a preparation method and application thereof.The structure of the degradation agent is shown in the formula I. The compound can degrade JAK and STAT3 protein at the same time through the molecular glue effect, and compared with existing JAK-STAT3 inhibitors such as tofacitinib or ruxotinib, the degradation agent can directly down-regulate an IL6-JAK-STAT3 signal channel, and the degradation effect is good. According to the present invention, the compound can effectively inhibit the phosphorylation of STAT3, block the proliferation, the survival, the angiogenesis, the invasion, the metastasis and other processes of cancer cells, and reduce the levels of JAK1, JAK2, JAK3, STAT3 and pSTAT3, such that the compound has strong pathway inhibition activity compared with the traditional inhibitor.

Description

Technical Field

[0001] The present invention relates to a JAK-STAT3 degradation agent, and also relates to a preparation method and application of the degradation agent. Background Art

[0002] The JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway is an important signal transduction pathway in cells. Under the stimulation of various extracellular signals such as cytokines, the receptors on the cell surface bind to the corresponding ligands, resulting in the activation of the JAK kinase coupled to the receptor. The activated JAK kinase phosphorylates the STAT protein, and the phosphorylated STAT protein forms a dimer and enters the cell nucleus, regulating the transcription of target genes and participating in various biological processes such as cell proliferation, differentiation, apoptosis, and immune regulation.

[0003] STAT3 is an important member of the STAT family and plays a key role in a variety of cell functions. Under normal physiological conditions, the activation of STAT3 is strictly regulated. However, in many pathological conditions, such as tumorigenesis, inflammatory response, and autoimmune diseases, STAT3 will be abnormally activated. STAT3 is continuously activated in a variety of tumor cells. It can upregulate the expression of anti-apoptotic proteins (such as Bcl-xL, Mcl-1, etc.) and inhibit tumor cell apoptosis. At the same time, it can also promote the proliferation of tumor cells and promote the cell cycle process by regulating the expression of cell cycle-related proteins (such as Cyclin D1, etc.). In addition, STAT3 activation is also related to the migration, invasion and angiogenesis of tumor cells. It can induce the expression of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). In inflammatory response, STAT3 participates in regulating the production of inflammatory cytokines (such as IL-6, IL-10, etc.). In autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, abnormal activation of STAT3 can lead to abnormal activation of immune cells and the production of autoantibodies, exacerbating the disease process.

[0004] In the prior art, inhibitors for the JAK-STAT3 signaling pathway have certain limitations, such as the inability to completely block the activation of the signaling pathway, or the development of drug resistance after long-term use. In addition, traditional inhibitors usually only inhibit the activity of STAT3, but cannot fundamentally reduce the protein level of STAT3. Tofacitinib inhibits the JAK-STAT3 signaling pathway, which not only weakens the inflammatory response but also affects the normal function of the immune system. The risk of infection after taking the drug increases, and common infections include upper respiratory tract infections, nasopharyngitis, urinary tract infections, etc. Ruxolitinib may cause hematological adverse reactions, such as leukopenia, neutropenia, anemia, and thrombocytopenia, which will lead to decreased resistance in patients and increase the risk of bleeding and infection. Therefore, it is urgent to develop new JAK-STAT3 degraders. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a JAK-STAT3 degrader, and also to provide a preparation method and application of the above-mentioned degrader.

[0006] Technical solution: The present invention discloses a JAK-STAT3 degrader, the structure of which is shown in Formula I:

[0007]

[0008] Wherein, R1 is selected from methyl or isopropyl;

[0009] R2 is selected from:

[0010] Wherein, the degradation agent is any one of the following compounds:

[0011]

[0012]

[0013]

[0014] The degradation agent also includes pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates or polycrystals thereof.

[0015] The preparation method of the above-mentioned JAK-STAT3 degrader comprises the following steps: dissolving one mole of the compound of formula (II) and two moles of the compound of formula (III) in ethanol, slowly dropping an aqueous solution of NaOH under an ice bath, stirring at room temperature after the dropping is complete, filtering after the reaction is complete, and drying to obtain the JAK-STAT3 degrader;

[0016]

[0017] The molar ratio of the compound of formula II to the compound of formula III is 1:2, the NaOH aqueous solution is a 20% NaOH aqueous solution, and the stirring time is 3 hours.

[0018] The present invention also discloses a pharmaceutical composition, comprising a compound of general formula I or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polycrystal thereof and a pharmaceutically acceptable carrier or excipient.

[0019] The above-mentioned JAK-STAT3 degrader can be used in the preparation of drugs for inhibiting JAK kinase activity and JAK-STAT3 signaling pathway.

[0020] The above-mentioned JAK-STAT3 degrading agent can also be used in the preparation of anti-tumor drugs.

[0021] Among them, the application is that the JAK-STAT3 degrader cuts off the abnormal activation of the JAK-STAT3 signaling pathway, blocks signal transmission, and regulates cell biological behavior.

[0022] Principle of the invention: The JAK-STAT3 small molecule degrader of the present invention can fundamentally block the abnormal activation of the JAK-STAT3 signaling pathway, thereby more effectively inhibiting signal transmission and significantly enhancing the therapeutic effect on related diseases. The degrader directly degrades key proteins such as JAK1, JAK2, JAK3, STAT3 and its phosphorylated form pSTAT3 through a molecular glue mechanism, blocks the activation and conduction of the signaling pathway, and rapidly reduces the active components in the signaling pathway, thereby inhibiting signal transmission at the molecular level. At the cellular level, it regulates the signaling pathway, affects the biological behaviors of cells such as proliferation, differentiation, migration and apoptosis, reduces cell proliferation and survival, induces cell apoptosis, and inhibits abnormal cells such as tumor cells. At the physiological level, the degrader regulates physiological processes such as the body's immune response and inflammatory response, such as regulating the function of immune cells, inhibiting the production of inflammatory factors, and producing a therapeutic effect on immune-related diseases or inflammatory diseases. Although existing traditional small molecule inhibitors such as tofacitinib or ruxolitinib can inhibit the activity of JAK1, JAK2 and JAK3, they do not have protein degradation function. At present, most of the degraders targeting the JAK-STAT3 signaling pathway are heterobifunctional molecules, while the direction of small molecule degraders is still blank. This series of compounds, as the first small molecule degraders for the JAK-STAT3 signaling pathway, achieves catalytic protein degradation through a molecular glue mechanism. Compared with traditional small molecule inhibitors, it shows higher efficiency and lower dosage requirements, thereby significantly improving the effect of tumor treatment.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The JAK-STAT3 small molecule degrader of the present invention, as the first small molecule degrader of the JAK-STAT3 signaling pathway, can catalytically degrade key proteins such as JAK1, JAK2, JAK3, STAT3 and its phosphorylated form pSTAT3 through a molecular glue mechanism, thereby blocking the signal transmission of the pathway, regulating cell biological behavior, and reducing the development of drug resistance; (2) The preparation method is relatively simple, the conditions are mild and the reaction is rapid, and the target compound can be obtained by simple treatment after only three hours of reaction at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the structural formula of the JAK-STAT3 degrader of the present invention;

[0025] Figure 2 The graphs are the effects of compounds 11 and 27 of the present invention on the viability of MV-4-11, K562, MOLM13 and THP1 hematological tumor cells;

[0026] Figure 3 This is a diagram showing the effect of the compounds of the present invention on the JAK2-STAT3 signaling pathway in HEL cells;

[0027] Figure 4 This is a diagram showing the effects of compounds 11 and 27 of the present invention on the JAK-STAT pathway in K562 cells. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0029] Identification, characterization and purity determination methods of compounds:

[0030] NMR: Compounds of the Invention 1 H-NMR spectra were measured using a Bruker nuclear magnetic resonance spectrometer (300 MHz) and a Bruker nuclear magnetic resonance spectrometer (500 MHz). 13 The C-NMR spectrum was measured using a Bruker nuclear magnetic resonance spectrometer (300 MHz). Chemical shifts (δ) are expressed in ppm using tetramethylsilane as an internal standard (0.00 ppm). 1 H-NMR notation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of a doublet, dt = doublet of a triplet. Coupling constants, when given, are given in Hz.

[0031] MS: The mass spectrum of the compound of the present invention was measured by Shimadzu's LC / MS-8050 triple quadrupole mass spectrometer, the ionization mode was ESI, and the molecular weight was accurate to one decimal place.

[0032] HRMS: The high-resolution mass spectra of the compounds of the present invention are measured by LC / MS mass spectrometers from Shimadzu and Agilent, using Q-TOF as the testing method, and the molecular weight is accurate to four decimal places.

[0033] Purity determination: The purity of the compound of the present invention was determined by Shimadzu's LC-20AT high performance liquid chromatograph. The test conditions were: Agilent XDS-C18 chromatographic column (4.6 mm*250 mm), mobile phase was methanol: water (containing 0.1% formic acid) = 70:30, the test compound sample was a chromatographic methanol solution with a concentration of about 1 mg / mL, the injection volume was 10 μL, the measurement wavelength was 254 nm, and the detection time was 10 min. The purity was measured using retention time (t R ) and area ratio (%), accurate to two decimal places.

[0034] Example 1

[0035] Preparation of compound 1

[0036] 1-Methyl-3,5-di(2-methylbenzylidene)piperidin-4-one (Compound 1)

[0037]

[0038] Compound 1 was prepared according to the general method of compound synthesis:

[0039] Dissolve N-methyl-4-piperidone (113 mg, 1 mmol) and 2-methylbenzaldehyde (240 mg, 2 mmol) in ethanol in a round-bottom flask, slowly drop 0.5 ml of 20% NaOH aqueous solution under ice bath, stir at room temperature for 3 h after the addition is complete. After TLC detection, filter and dry to obtain a yellow solid product (201 mg, 63%).

[0040] 1 H NMR(300MHz,Chloroform-d)δ8.07(s,2H),7.35-7.31(m,4H),7.29(dt,J=6.1,2.8H z,2H),7.23(dd,J=6.8,1.4Hz,2H),3.68(d,J=1.7Hz,4H),2.44(s,6H).HR-MS:[M+H] + =318.1818.

[0041] Example 2

[0042] Preparation of compound 2

[0043] 1-Methyl-3,5-bis(3-methylbenzylidene)piperidin-4-one (Compound 2)

[0044]

[0045] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (195 mg, 61%).

[0046] 1 H NMR(300MHz,Chloroform-d)δ8.11(s,2H),7.36-7.30(m,4H),7.19(dt,J=6.1,2.8H z,2H),7.41(dd,J=6.8,1.4Hz,2H),3.62(d,J=1.7Hz,4H),2.33(s,6H).HR-MS:[M+H] + =318.1818.

[0047] Example 3

[0048] Preparation of compound 3

[0049] 1-Methyl-3,5-bis(4-methylbenzylidene)piperidin-4-one (Compound 3)

[0050]

[0051] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (218 mg, 68%).

[0052] 1 H NMR(300MHz,Chloroform-d)δ7.85(s,2H),7.36(d,J=8.1Hz,4H),7.28(d,J=8.0Hz,4H),3.82(d,J=1.8Hz,4H),2.52(s,3H),2.44(s,6H).HR-MS:[M+H] + =318.1815.

[0053] Example 4

[0054] Preparation of compound 4

[0055] 3,5-Bis(2-ethylbenzylidene)-1-methylpiperidin-4-one (Compound 4)

[0056]

[0057] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (190 mg, 55%).

[0058] 1 H NMR(300MHz,Chloroform-d)δ8.11(s,2H),7.39-7.28(m,6H),7.20(dd,J=7.6,1.5Hz,2H), 3.66(d,J=1.7Hz,4H),2.81-2.74(m,4H),2.40(s,3H),1.26(t,J=7.5Hz,6H).HR-MS:[M+H] + =346.2132.

[0059] Example 5

[0060] Preparation of compound 5

[0061] 3,5-Bis(4-ethylbenzylidene)-1-methylpiperidin-4-one (Compound 5)

[0062]

[0063] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (183 mg, 53%).

[0064] 1 H NMR(300MHz,Chloroform-d)δ7.85(s,2H),7.38(d,J=8.2Hz,4H),7.30(d,J=8.2Hz,4H),3. 82(d,J=1.8Hz,4H),2.73(d,J=7.6Hz,4H),2.51(s,3H),1.31(t,J=7.6Hz,6H).HR-MS:[M+H] + =346.2126.

[0065] Example 6

[0066] Preparation of compound 6

[0067] 3,5-Bis(4-isopropylbenzylidene)-1-methylpiperidin-4-one (Compound 6)

[0068]

[0069] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (190 mg, 50%).

[0070] 1H NMR(300MHz,Chloroform-d)δ7.86(s,2H),7.41(d,J=8.2Hz,4H),7.34(d,J=8.3Hz,4H),3. 84(d,J=1.8Hz,4H),3.04-2.95(m,2H),2.53(s,3H),1.34(s,6H),1.32(s,6H).HR-MS:[M+H] + =374.2439.

[0071] Example 7

[0072] Preparation of compound 7

[0073] 3,5-Bis(2-methoxybenzylidene)-1-methylpiperidin-4-one (Compound 7)

[0074]

[0075] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (211 mg, 60%).

[0076] 1 H NMR(300MHz,Chloroform-d)δ8.12(s,2H),7.42-7.36(m,2H),7.25(dd,J=7.6,1.7Hz, 2H),7.04-6.95(m,4H),3.90(s,6H),3.72(d,J=1.8Hz,4H),2.43(s,3H).HR-MS:[M+H] + =350.1713.

[0077] Example 8

[0078] Preparation of compound 8

[0079] 3,5-Bis(3-methoxybenzylidene)-1-methylpiperidin-4-one (Compound 8)

[0080]

[0081] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (186 mg, 53%).

[0082] 1H NMR(300MHz,Chloroform-d)δ7.82(d,J=1.8Hz,2H),7.41-7.35(m,2H),7.06-7.02(m, 2H),6.98-6.95(m,4H),3.88(s,6H),3.80(d,J=1.9Hz,4H),2.50(s,3H).HR-MS:[M+H] + =350.1712.

[0083] Example 9

[0084] Preparation of compound 9

[0085] 3,5-Bis(4-methoxybenzylidene)-1-methylpiperidin-4-one (Compound 9)

[0086]

[0087] The preparation method was the same as that of Example 1, and the obtained product was a dark yellow solid (193 mg, 55%).

[0088] 1 H NMR (300MHz, Chloroform-d) δ7.82 (s, 2H), 7.44-7.40 (m, 4H), 7.01-6.97 (m, 4H), 3.90 (s, 6H), 3.81 (d, J = 1.8Hz, 4H), 2.53 (s, 3H). HR-MS: [M+H] + =350.1722.

[0089] Example 10

[0090] Preparation of compound 10

[0091] 3,5-Bis(2-fluorobenzylidene)-1-methylpiperidin-4-one (Compound 10)

[0092]

[0093] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (230 mg, 70%).

[0094] 1 H NMR(300MHz,Chloroform-d)δ7.94(d,J=2.1Hz,2H),7.42-7.30(m,4H),7.24(dd,J= 7.5,1.2Hz,2H),7.18-7.11(m,2H),3.69(d,J=1.7Hz,4H),2.45(s,3H).HR-MS:[M+H] + =326.1363.

[0095] Embodiment 11

[0096] Preparation of compound 11

[0097] 3,5-Bis(3-fluorobenzylidene)-1-methylpiperidin-4-one (Compound 11)

[0098]

[0099] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (184 mg, 56%).

[0100] 1 H NMR(300MHz,Chloroform-d)δ7.78(d,J=2.0Hz,2H),7.48-7.40(m,2H),7.24-7.20(m,2H),7 .14(d,J=1.4Hz,2H),7.11(q,J=2.2Hz,2H),3.78(d,J=2.0Hz,4H),2.52(s,3H).HR-MS:[M+H] + =326.1356.

[0101] Example 12

[0102] Preparation of compound 12

[0103] 3,5-Bis(4-fluorobenzylidene)-1-methylpiperidin-4-one (Compound 12)

[0104]

[0105] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (216 mg, 66%).

[0106] 1 H NMR(300MHz,Chloroform-d)δ7.79(s,2H),7.46-7.41(m,4H),7.20-7.13(m,4H), 3.88(d,J=1.7Hz,4H),2.98(p,J=6.5Hz,1H),1.11(d,J=6.5Hz,6H).HR-MS:[M+H] + =326.1358.

[0107] Embodiment 13

[0108] Preparation of compound 13

[0109] 3,5-Bis(2-chlorobenzylidene)-1-methylpiperidin-4-one (Compound 13)

[0110]

[0111] The preparation method was the same as that of Example 1, and the obtained product was a dark yellow solid (190 mg, 53%).

[0112] 1 H NMR(300MHz,Chloroform-d)δ8.05(d,J=1.7Hz,2H),7.52-7.47(m,2H),7.35-7 .33(m,2H),7.30-7.26(m,2H),3.65(d,J=1.8Hz,4H),2.41(s,3H).HR-MS:[M+H] + =358.0766.

[0113] Embodiment 14

[0114] Preparation of compound 14

[0115] 3,5-Bis(3-chlorobenzylidene)-1-methylpiperidin-4-one (Compound 14)

[0116]

[0117] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (234 mg, 65%).

[0118] 1 H NMR(300MHz,Chloroform-d)δ8.05(d,J=1.7Hz,2H),7.41-7.38(m,4H),7.50-7.46(m,4H),3.88(d,J=1.8Hz,4H),2.41(s,3H).HR-MS:[M+H] + =358.0766.

[0119] Embodiment 15

[0120] Preparation of compound 15

[0121] 3,5-Bis(4-chlorobenzylidene)-1-methylpiperidin-4-one (Compound 15)

[0122]

[0123] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (197 mg, 55%).

[0124] 1H NMR (300MHz, Chloroform-d) δ7.79 (s, 2H), 7.48-7.41 (m, 4H), 7.37 (d, J = 8.4Hz, 4H), 3.77 (d, J = 1.9Hz, 4H), 2.51 (d, J = 1.1Hz, 3H). HR-MS: [M+H] + =358.0767.

[0125] Example 16

[0126] Preparation of compound 16

[0127] 3,5-Bis(2-bromobenzylidene)-1-methylpiperidin-4-one (Compound 16)

[0128]

[0129] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (167 mg, 38%).

[0130] 1 H NMR(300MHz,Chloroform-d)δ7.99(s,2H),7.70(dd,J=8.4,1.3Hz,2H),7.39(td,J=7.5, 1.3Hz, 2H), 7.28 (dd, J=4.3, 2.7Hz, 3H), 3.64 (d, J=1.8Hz, 4H), 2.41 (s, 3H). HR-MS: [M+H] + =445.9742.

[0131] Embodiment 17

[0132] Preparation of compound 17

[0133] 3,5-Bis(3-bromobenzylidene)-1-methylpiperidin-4-one (Compound 17)

[0134]

[0135] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (138 mg, 31%).

[0136] 1 H NMR(300MHz,Chloroform-d)δ7.77(d,J=1.9Hz,2H),7.58-7.53(m,4H),7.37-7.33(m,4H),3.78(d,J=1.9Hz,4H),2.52(s,3H).HR-MS:[M+H] + =445.9739.

[0137] Embodiment 18

[0138] Preparation of compound 18

[0139] 3,5-Bis(4-bromobenzylidene)-1-methylpiperidin-4-one (Compound 18)

[0140]

[0141] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (191 mg, 43%).

[0142] 1 H NMR (300MHz, Chloroform-d) δ7.79 (s, 2H), 7.65-7.60 (m, 4H), 7.32 (d, J = 8.3Hz, 4H), 3.78 (d, J = 1.8Hz, 4H), 2.53 (s, 3H). HR-MS: [M+H] + =445.9741. Example 19

[0143] Preparation of compound 19

[0144] 3,5-Bis(3-cyanobenzylidene)-1-methylpiperidin-4-one (Compound 19)

[0145]

[0146] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (177 mg, 52%).

[0147] 1 H NMR(300MHz,Chloroform-d)δ7.78(s,2H),7.69(q,J=2.3Hz,4H),7.66-7.57(m,4H),3.77(d,J=1.9Hz,4H),2.54(s,3H).HR-MS:[M+H] + =340.1409.Example 20

[0148] Preparation of compound 20

[0149] 3,5-Bis(4-cyanobenzylidene)-1-methylpiperidin-4-one (Compound 20)

[0150]

[0151] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (125 mg, 37%).

[0152] 1H NMR(300MHz,Chloroform-d)δ7.81(s,2H),7.79-7.74(m,4H),7.52(d,J=8.2Hz,4H),3.78(d,J=2.0Hz,4H),2.52(s,3H).HR-MS:[M+H] + =340.1405.Example 21

[0153] Preparation of compound 21

[0154] 1-Methyl-3,5-bis(3-nitrobenzylidene)piperidin-4-one (Compound 21)

[0155]

[0156] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (156 mg, 41%).

[0157] 1 H NMR(300MHz,Chloroform-d)δ8.28(dd,J=4.6,2.4Hz,4H),7.86(s,2H),7.76(d,J =7.7Hz,2H),7.70-7.64(m,2H),3.83(d,J=1.8Hz,4H),2.54(s,3H).HR-MS:[M+H] + =380.1202.

[0158] Embodiment 22

[0159] Preparation of compound 22

[0160] 1-Methyl-3,5-bis(4-(trifluoromethyl)benzylidene)piperidin-4-one (Compound 22)

[0161]

[0162] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (147 mg, 35%).

[0163] 1 H NMR(300MHz,Chloroform-d)δ7.81(s,2H),7.75-7.71(m,4H),7.64

[0164] -7.62(d,J=8.2Hz,4H),3.88(d,J=2.0Hz,4H),2.50(s,3H).HR-MS:[M+H] + =426.1248.

[0165] Embodiment 23

[0166] Preparation of compound 23

[0167] 1-Isopropyl-3,5-bis(2-methylbenzylidene)piperidin-4-one (Compound 23)

[0168]

[0169] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (188 mg, 54%).

[0170] 1 H NMR(300MHz,Chloroform-d)δ8.02(s,2H),7.32-7.29(m,4H),7.29-

[0171] 7.24(m,2H),7.24-7.19(m,2H),3.77(d,J=1.6Hz,4H),2.88(h,J=6.5Hz,1H),2.42(s,6H),1.02(s,3H),1.00(s,3H).HR-MS:[M+H] + =346.2126.

[0172] Embodiment 24

[0173] Preparation of compound 24

[0174] 1-Isopropyl-3,5-bis(3-methylbenzylidene)piperidin-4-one (Compound 24)

[0175]

[0176] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (167 mg, 58%).

[0177] 1 H NMR(300MHz,Chloroform-d)δ7.82(s,2H),7.35(d,J=7.5Hz,2H),7.25(d,J=8.6Hz,6H),3. 93(d,J=1.7Hz,4H),3.01-2.93(m,1H),2.44(s,6H),1.11(s,3H),1.09(s,3H).HR-MS:[M+H] + =346.2125.

[0178] Embodiment 25

[0179] Preparation of compound 25

[0180] 1-Isopropyl-3,5-bis(4-methylbenzylidene)piperidin-4-one (Compound 25)

[0181]

[0182] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (199 mg, 58%).

[0183] 1 H NMR(300MHz,Chloroform-d)δ7.82(s,2H),7.36(d,J=8.2Hz,4H),7.28(d,J=8.0Hz,4H),3. 93(d,J=1.8Hz,4H),2.98(p,J=6.5Hz,1H),2.44(s,6H),1.11(d,J=6.5Hz,6H).HR-MS:[M+H] + =346.2128.

[0184] Embodiment 26

[0185] Preparation of compound 26

[0186] 1-Isopropyl-3,5-bis(2-ethylbenzylidene)piperidin-4-one (Compound 26)

[0187]

[0188] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (163 mg, 44%).

[0189] 1 H NMR(300MHz,Chloroform-d)δ8.08(s,2H),7.39-7.28(m,6H),7.21(dd,J=7.6,1.4Hz,2H),3.78(d,J=1.5Hz,4 H),2.87(q,J=6.5Hz,1H),2.75(t,J=7.6Hz,4H),1.25(t,J=7.6Hz,6H),1.00(s,3H),0.98(s,3H).HR-MS:[M+H] + =374.2441.

[0191] Embodiment 27

[0192] Preparation of compound 27

[0193] 1-Isopropyl-3,5-bis(4-ethylbenzylidene)piperidin-4-one (Compound 27)

[0194]

[0195] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (173 mg, 46%).

[0196] 1 H NMR(300MHz,Chloroform-d)δ7.83(s,2H),7.42(s,4H),7.31(d,J=8.0Hz,4H),3.94(d,J=1.6Hz,4H),3 .00(p,J=6.5Hz,1H),2.73(t,J=7.6Hz,4H),1.32(t,J=7.6Hz,6H),1.13(d,J=6.5Hz,6H).HR-MS:[M+H] + =374.2435. Example 28

[0197] Preparation of Compound 28

[0198] 1-Isopropyl-3,5-bis(4-isopropylbenzylidene)piperidin-4-one (Compound 28)

[0199]

[0200] The preparation method was the same as that of Example 1, and the obtained product was a dark yellow solid (192 mg, 48%).

[0201] 1 H NMR(300MHz,Chloroform-d)δ7.83(s,2H),7.42(d,J=8.2Hz,4H),7.34(d,J=8.3Hz,4H),3.94(d,J =1.7Hz,4H),3.05-2.96(m,3H),1.34(s,6H),1.32(s,6H),1.15(s,3H),1.13(s,3H).HR-MS:[M+H] + =402.2752.

[0202] Embodiment 29

[0203] Preparation of compound 29

[0204] 1-Isopropyl-3,5-bis(2-methoxybenzylidene)piperidin-4-one (Compound 29)

[0205]

[0206] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (237 mg, 63%).

[0207] 1H NMR(300MHz,Chloroform-d)δ8.09(s,2H),7.41-7.35(m,2H),7.28-

[0208] 7.24(m,2H),7.05-6.95(m,4H),3.90(s,6H),3.84(d,J=1.7Hz,4H),2.92(p,J=6.5Hz,1H),1.04(s,3H),1.02(s,3H).HR-MS:[M+H] + =378.2026.

[0209] Embodiment 30

[0210] Preparation of compound 30

[0211] 1-Isopropyl-3,5-bis(3-methoxybenzylidene)piperidin-4-one (Compound 30)

[0212]

[0213] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (158 mg, 42%).

[0214] 1 H NMR(300MHz,Chloroform-d)δ7.80(s,2H),7.39(td,J=7.7,0.9Hz,2H),7.05(d,J=7.6Hz,2H),6.99-6.95 (m,4H),3.92(d,J=1.7Hz,4H),3.89(s,6H),2.98(p,J=6.5Hz,1H),1.12(s,3H),1.10(s,3H).HR-MS:[M+H] + =378.2025. Example 31

[0215] Preparation of compound 31

[0216] 1-Isopropyl-3,5-bis(4-methoxybenzylidene)piperidin-4-one (Compound 31)

[0217]

[0218] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (206 mg, 55%).

[0219] 1H NMR(300MHz,Chloroform-d)δ7.80(s,2H),7.45-7.40(m,4H),7.02-6.97(m,4H),3.92(d, J=1.7Hz,4H),3.90(s,6H),2.98(q,J=6.5Hz,1H),1.14(s,3H),1.12(s,3H).HR-MS:[M+H] + =378.2023.

[0220] Embodiment 32

[0221] Preparation of compound 32

[0222] 1-Isopropyl-3,5-bis(2-fluorobenzylidene)piperidin-4-one (Compound 32)

[0223]

[0224] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (195 mg, 55%).

[0225] 1 H NMR(300MHz,Chloroform-d)δ7.92(d,J=1.9Hz,2H),7.40-7.34(m,3H),7.27-7.22(m,2H),7. 18-7.14(m,2H),3.84-3.79(m,4H),2.94(p,J=6.5Hz,1H),1.05(d,J=6.5Hz,7H).HR-MS:[M+H] + =354.1670.

[0226] Embodiment 33

[0227] Preparation of compound 33

[0228] 1-Isopropyl-3,5-bis(3-fluorobenzylidene)piperidin-4-one (Compound 33)

[0229]

[0230] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (170 mg, 48%).

[0231] 1H NMR(300MHz,Chloroform-d)δ7.77(s,2H),7.45(tdd,J=7.2,5.5,2.5Hz,2H),7.23(d,J=7.8Hz,2H),7.12(t d,J=8.0,7.1,1.1Hz,4H),3.89(d,J=1.8Hz,4H),3.04-2.95(m,1H),1.13(s,3H),1.11(s,3H).HR-MS:[M+H] + =354.1667.Example 34

[0232] Preparation of compound 34

[0233] 1-Isopropyl-3,5-bis(4-fluorobenzylidene)piperidin-4-one (Compound 34)

[0234]

[0235] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (193 mg, 55%).

[0236] 1 H NMR(300MHz,Chloroform-d)δ7.79(s,2H),7.46-7.41(m,4H),7.20-7.13(m,4H), 3.88(d,J=1.7Hz,4H),2.98(p,J=6.5Hz,1H),1.11(d,J=6.5Hz,6H).HR-MS:[M+H] + =354.1668.

[0237] Embodiment 35

[0238] Preparation of compound 35

[0239] 1-Isopropyl-3,5-bis(2-chlorobenzylidene)piperidin-4-one (Compound 35)

[0240]

[0241] The preparation method was the same as that of Example 1, and the obtained product was a dark yellow solid (210 mg, 54%).

[0242] 1 H NMR(300MHz,Chloroform-d)δ7.72(s,2H),7.44(m,2H),7.37-7.34(m,4H),7.52(m,2H),3.88(d,J=1.8Hz,4H),1.12(s,6H).HR-MS:[M+H] + =386.1072.

[0244] Embodiment 36

[0245] Preparation of compound 36

[0246] 1-Isopropyl-3,5-bis(3-chlorobenzylidene)piperidin-4-one (Compound 36)

[0247]

[0248] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (191 mg, 49%).

[0249] 1 H NMR(300MHz,Chloroform-d)δ7.75(s,2H),7.41(d,J=6.0Hz,4H),7.34-7.31(m,4H),3.88(d,J=1.8Hz,4H),1.13(s,6H).HR-MS:[M+H] + =386.1072. Example 37

[0250] Preparation of compound 37

[0251] 1-Isopropyl-3,5-bis(4-chlorobenzylidene)piperidin-4-one (Compound 37)

[0252]

[0253] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (198 mg, 51%).

[0254] 1 H NMR(300MHz,Chloroform-d)δ7.77(s,2H),7.47-7.42(m,4H),7.39-7.35(m,4H),3 .87(d,J=1.8Hz,4H),2.96(h,J=6.6Hz,1H),1.11(s,3H),1.09(s,3H).HR-MS:[M+H] + =386.1076.

[0255] Embodiment 38

[0256] Preparation of compound 38

[0257] 1-Isopropyl-3,5-bis(2-bromobenzylidene)piperidin-4-one (Compound 38)

[0258]

[0259] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (158 mg, 33%).

[0260] 1 H NMR(300MHz,Chloroform-d)δ7.99(s,2H),7.74-7.70(m,2H),7.43-7.38(m,2H),7.31( s,2H),3.79(d,J=1.7Hz,4H),2.92(p,J=6.5Hz,1H),1.02(d,J=6.5Hz,6H).HR-MS:[M+H] + =476.0033.

[0261] Embodiment 39

[0262] Preparation of compound 39

[0263] 1-Isopropyl-3,5-bis(3-bromobenzylidene)piperidin-4-one (Compound 39)

[0264]

[0265] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (176 mg, 37%).

[0266] 1 H NMR(300MHz,Chloroform-d)δ7.74(d,J=2.1Hz,2H),7.59-7.53(m,4H),7.38-7.34(m,4 H),3.88(d,J=1.8Hz,4H),2.99(p,J=6.5Hz,1H),1.12(s,3H),1.10(s,3H).HR-MS:[M+H] + =476.0038.

[0267] Embodiment 40

[0268] Preparation of Compound 40

[0269] 1-Isopropyl-3,5-bis(4-bromobenzylidene)piperidin-4-one (Compound 40)

[0270]

[0271] The preparation method was the same as that of Example 1, and the obtained product was a light yellow solid (184 mg, 39%).

[0272] 1H NMR(300MHz,Chloroform-d)δ7.75(s,2H),7.63-7.58(m,4H),7.30(d,J=8.5Hz,4H ),3.86(d,J=1.8Hz,4H),2.97(p,J=6.6Hz,1H),1.10(d,J=6.5Hz,6H).HR-MS:[M+H] + =476.0036.

[0273] Embodiment 41

[0274] Preparation of compound 41

[0275] 1-Isopropyl-3,5-bis(4-cyanobenzylidene)piperidin-4-one (Compound 41)

[0276]

[0277] The preparation method was the same as that of Example 1, and the product was obtained as a yellow solid (183 mg, 50%).

[0278] 1 H NMR(300MHz,Chloroform-d)δ7.80(s,2H),7.74(d,J=8.1Hz,4H),7.54(d,J=8.1Hz,4H ),3.89(d,J=1.8Hz,4H),2.97(q,J=6.5Hz,1H),1.11(s,3H),1.09(s,3H).HR-MS:[M+H] + =368.1718.

[0279] Embodiment 42

[0280] Preparation of Compound 42

[0281] 1-Isopropyl-3,5-bis(3-nitrobenzylidene)piperidin-4-one (Compound 42)

[0282]

[0283] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (218 mg, 53%).

[0284] 1H NMR(300MHz,Chloroform-d)δ8.28(d,J=8.5Hz,4H),7.84(s,2H),7.76(d,J=7.6Hz,2H),7. 68(t,J=7.8Hz,2H),3.92(s,4H),3.00(p,J=6.6Hz,1H),1.12(d,J=6.5Hz,6H).HR-MS:[M+H] + =408.1515.

[0285] Embodiment 43

[0286] Preparation of compound 43

[0287] 1-Isopropyl-3,5-bis(4-(trifluoromethyl)benzylidene)piperidin-4-one (Compound 43)

[0288]

[0289] The preparation method was the same as that of Example 1, and the obtained product was a yellow solid (222 mg, 49%).

[0290] 1 H NMR(300MHz,Chloroform-d)δ7.83(s,2H),7.74(d,J=8.1Hz,4H),7.54(d,J=8.1Hz,4H ),3.89(d,J=1.8Hz,4H),2.97(q,J=6.5Hz,1H),1.11(s,3H),1.09(s,3H).HR-MS:[M+H] + =454.1561.

[0291] Embodiment 44

[0292] Inhibitory activity of STAT3 reporter gene:

[0293] The inhibitory activity of the compound on the STAT3 gene in THP1 cells was detected by the luciferase method. The method is as follows: 4*104 THP1 cells in the logarithmic growth phase were seeded in a 96-well plate, and different concentrations of compounds were added. The DMSO group was used as a blank control. 10μg / mL STAT3 reporter gene lysate was added and cultured with lipofectamine 2000 (Invitrogen) complex for 24h. QUANTI-Luc was used TM 10 μL of cell culture supernatant was added to a 96-well plate, and then 50 μL of QUANTI-Luc was added. TM The reagents were read using a multifunctional microplate reader (Thermo).

[0294] Relative luciferase activity calculation method: cells treated with lipofectamine 2000 were used as blank control, and cells treated with lipofectamine 2000:STAT3 complex were used as negative control.

[0295] Relative luciferase activity = (RLU - RLU blackcontrol) / (RLU negativecontrol - RLU blackcontrol), RLU represents the original luciferase value.

[0296] Table 1. Inhibition rate of the degradation agent compounds of the present invention

[0297]

[0298]

[0299] As shown in Table 1, the inhibitory activity of the compounds on the STAT3 gene in THP1 cells shows that the derivatives substituted with electron-withdrawing groups (halogen, nitro, nitrile and trifluoromethyl) on the aromatic group are more active than the derivatives substituted with electron-donating groups (alkyl, methoxy); when the substituents on the aromatic ring are the same, the derivatives substituted with methyl on the nitrogen atom of piperidone are slightly better than the isopropyl substitution. The inhibitory effect of compounds 19 and 20 on the STAT3 gene at a concentration of 1 μM can reach more than 98%, which has exceeded the existing small molecule STAT3 inhibitor Stattic (IC 50 =5.1 μM).

[0300] Embodiment 45

[0301] Tumor cell inhibitory activity of the degrader compounds of the present invention:

[0302] Cell Counting Kit-8 (CCK-8) operation steps:

[0303] Prepare cell suspension and count cells.

[0304] Inoculate the cell suspension in a 96-well plate, about 100 μL per well, and repeat the same sample three times. Place the culture plate in an incubator for a period of time (37°C, 5% CO2), and it takes about 2-4 hours for the cells to adhere to the wall.

[0305] Add 10 μL of CCK-8 solution to each well and gently shake the plate to help mix. Note that bubbles should be avoided during the addition process.

[0306] The culture plate was placed in an incubator and incubated for 4 h, and the absorbance (OD) at 450 nm was measured using an enzyme-labeled instrument.

[0307] The vitality calculation formula is as follows:

[0308] Cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] × 100%

[0309] A (drug added): OD value of the wells with cells, CCK-8 solution and drug solution

[0310] A (0 drug addition): OD value of the well with cells, CCK-8 solution but no drug solution

[0311] A (blank): OD value of the well without cells

[0312] Representative derivatives were selected from different substituents, and the CCK8 method was used to test the inhibitory effect of diaryl piperidone derivatives on the activity of different tumor cells.

[0313] Table 2. Inhibitory activity of compounds on human erythroleukemia HEL cells

[0314]

[0315]

[0316]

[0317] Table 3. Inhibitory activity of compounds on lung adenocarcinoma A549 cells

[0318]

[0319]

[0320] As shown in Table 2 and Table 3, the compounds of the present invention have strong inhibitory activity against tumor cells and have a certain broad-spectrum anti-tumor activity. Among them, the inhibitory activity of human erythroleukemia HEL cells (Table 2) is better than that of lung adenocarcinoma A549 cells (Table 3). The anti-tumor activity of existing JAK-STAT3 small molecule inhibitors is basically at the micromolar level. The IC of this series of compounds on human erythroleukemia HEL cells is 50 The highest concentration can reach 504nM, which has surpassed the existing compounds. The IC 50 At around 1.7 μM, it is on par with existing compounds.

[0321] Embodiment 46

[0322] Effects of the compounds of the present invention on the activity of hematological tumor cells:

[0323] The effects of compound 11 (high activity) and compound 27 (low activity) on the viability of hematological tumor cells such as MV-4-11, K562, MOLM13 and THP1 were tested by CCK8 method.

[0324] Table 4. Effects of compounds on the viability of hematological tumor cells

[0325]

[0326] like Figure 2 As shown in Table 4, compound 11 has a strong inhibitory effect on all tested hematological tumor cell lines, and its IC 50 The values ​​were all around 1 μM, indicating that it has a wide range of antitumor activity; while the inhibitory effect of compound 27 was relatively weak, especially in the K562 cell line, with an IC 50 The value was only 48.81 μM.

[0327] Embodiment 47

[0328] Western blotting assay to detect the effects of compounds on the JAK2-STAT3 signaling pathway:

[0329] The steps of the Western blotting experiment are as follows:

[0330] First, mix and wash the cells with 1*PBS (Gibco, 10010023), aspirate, add weak RIPA lysis buffer (100μL) (Biyuntian, P0013) to lyse the cells, the lysis time is 30 minutes, centrifuge at 13000rpm and 4℃ for 15 minutes, take 80μL of supernatant and add 20μL of loading buffer (Biyuntian, P0015), heat at 100℃ for 10 minutes, cool on ice, load 10μL on 10% SDS-PAGE gel, run at 60V constant voltage on Bio-Rad protein electrophoresis instrument until the marker is separated, then switch to 120V and run to the bottom. After electrophoresis, remove the gel and cut the PVDF membrane of appropriate size according to the size. The order from white board to black board is filter paper, PVDF membrane, gel, filter paper and black gauze. Wet transfer was performed for 2 h to transfer proteins to PVDF membrane (PerkinElmer, Northwalk, CT, USA). After the transfer, the PVDF membrane was immersed in Ponceau staining solution for 2-5 min, rinsed with distilled water, the target band was cut, and the Ponceau was washed off with 1*TBST (Biyuntian, ST671). Blocked with blocking buffer (1*TBST containing 5% w / v skim milk powder) at 37°C for 1 h. After blocking, wash three times with 15 ml 1*TBST, 5 minutes each time. The primary antibody was placed in 10 mL primary antibody dilution buffer (1*TBST containing 5% skim milk powder, to prepare 20 ml, add 1.0 g skim milk powder to 20 ml 1*TBST, and then mix thoroughly) according to the appropriate dilution recommended in the product manual, incubated overnight at 4°C and gently shaken from time to time, and washed three times with 1*TBST, 10 minutes each time. Then the membrane was incubated with the color developing solution (SignalFireTMECLReagent#6883) at room temperature for 1 minute, the excess solution was poured off (the membrane remained wet) and exposed.

[0331] The compounds of the present invention were selected to test the effects on HEL cells ( Figure 3 ) and K562 cells ( Figure 4 ) in JAK2-STAT3 signaling pathway. The experimental results showed that fluorine-substituted compound 11, nitro-substituted compound 21, and nitrile-substituted compound 41 could down-regulate the levels of JAK2, JAK3, STAT3, and pSTAT3, and could almost completely degrade them at a concentration of 10 μM; while alkyl-substituted compound 27 had a lower effect on the levels of JAK2, JAK3, STAT3, and pSTAT3, and had a weak degradation effect at a concentration of 10 μM. This result was consistent with the results of the cell viability test.

[0332] Therefore, the compounds of the present invention can simultaneously degrade JAK and STAT3 proteins through molecular glue action, and directly downregulate the IL6-JAK-STAT3 signaling pathway, more effectively inhibit the phosphorylation of STAT3, block the proliferation, survival, angiogenesis, invasion and metastasis of cancer cells, downregulate the levels of JAK1, JAK2, JAK3, STAT3 and pSTAT3, and have stronger pathway inhibition activity than traditional inhibitors, and have great application prospects.

Claims

1. A JAK-STAT3 degrading agent, characterized in that: The structure of the degradation agent is shown in Formula I: Wherein, R1 is selected from methyl or isopropyl; R2 is selected from:

2. The degradation agent according to claim 1, characterized in that: The degradation agent is any one of the following compounds:

3. The degradation agent according to claim 1, characterized in that The degradation agent also includes pharmaceutically acceptable salts, enantiomers, stereoisomers, hydrates, solvates or polymorphs thereof.

4. A method for preparing the JAK-STAT3 degrading agent according to claim 1, characterized in that: The following steps are involved: The compound of formula II and the compound of formula III are dissolved in ethanol, and an aqueous solution of NaOH is slowly added dropwise under an ice bath. After the addition is complete, the mixture is stirred at room temperature. After the reaction is complete, the mixture is filtered and dried to obtain a JAK-STAT3 degrader; 5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a compound of formula I or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof and a pharmaceutically acceptable carrier or excipient.

6. Use of the JAK-STAT3 degrader according to claim 1 in the preparation of a drug for inhibiting JAK kinase activity.

7. Use of the JAK-STAT3 degrader according to claim 1 in the preparation of anti-tumor drugs.

8. The use according to claim 6 or 7, characterized in that: The application is that the JAK-STAT3 degrader cuts off the abnormal activation of the JAK-STAT3 signaling pathway, blocks signal transmission, and regulates cell biological behavior.

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